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Introduction Considerable evidences have demonstrated that the growth of malignant tumors must be dependent upon angiogenesis, the formation of new capillary blood vessels from pre-existing vasculature (1-4), and certain evidences have also showed that cancer metastasis is dependent on angiogenesis (5, 6). Once a nest of cancer cells reaches a certain size (1–2 mm in diameter), it must develop a blood supply in order to grow larger. Diffusion is no longer adequate to supply the cells with oxygen and nutrients and to take waste away (2, 4, 7). A wide variety of angiogenesis factors have been identified by using modern biotechnology throughout the past decades (8, 9). Here are a few examples: angiopoietin-1 (10), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) (8, 9). On the contrary, inhibition of angiogenesis can slow down tumor growth and even result in tumor regression. Meanwhile, various angiogenesis inhibitors (AIs) have been discovered and are being studied under clinical trials in the advanced patients with cancers including gastrointestinal cancer (11-13).

or (VEGF) (8, 9). On the contrary, inhibition of angiogenesis can slow down tumor growth and even result in tumor regression. Meanwhile, various angiogenesis inhibitors (AIs) have been discovered and are being studied under clinical trials in the advanced patients with cancers including gastrointestinal cancer (11-13). Clinical trials of antiangiogenesis therapy in gastric cancer So far, at least 21 clinical trials of antiangiogenesis therapy are being conducted in gastric cancer in 8 countries (Table 1). These trials are multi-centered and randomly controlled, most of them are in phase III clinical studies. Bevacizumab (Avastin) is widely used in these trials combined either chemotherapy or other therapies. The response rate and overall survival are encouraging, with time to disease progression improved over historical controls by 75% (13). In addition, new molecules such as Sunitinib (Sutent) and mTOR inhibitor Temsirolimus are most likely promising AIs in treating gastric cancer. Once these studies have been completed, optimization of clinically active anti-angiogenic agents will need to be further refined in order to determine where they best fit in gastric cancer, either as single agents or in combination with classical anticancer therapies. Finally, the use of these new agents may in the future encompass every aspect of cancer management, not only from palliative to curative treatment but also in the prevention of cancer.

rder to determine where they best fit in gastric cancer, either as single agents or in combination with classical anticancer therapies. Finally, the use of these new agents may in the future encompass every aspect of cancer management, not only from palliative to curative treatment but also in the prevention of cancer. Table 1 Clinical trials of antiangiogenesis therapy in gastric cancers source (from http://clinicaltrials.gov) 1. NCT00780494: Phase II of Capecitabine, Carboplatin & Bevacizumab for Gastroesophageal Junction & Gastric Carcinoma. Stanford University and Genentech, USA 2. NCT00394433: Docetaxel, Cisplatin, Irinotecan and Bevacizumab (TPCA) in Metastatic Esophageal and Gastric Cancer Dana-Farber Cancer Institute, Brigham and Women's Hospital, Massachusetts General Hospital, and Genentech, USA 3. NCT00403468: Combination Chemotherapy and Bevacizumab in Treating Patients With Recurrent, Unresectable, or Metastatic Gastric Cancer, Gastroesophageal Junction Cancer, or Esophageal Cancer. Memorial Sloan-Kettering Cancer Center and National Cancer Institute (NCI), USA 4. NCT00673673: FOLFOX With Bevacizumab in Metastatic or Unresectable Gastroesophageal and Gastric Cancer Yale University and Genentech, USA 5. NCT00555672: Study Of Sunitinib In Combination With Cisplatin And 5-Fluorouracil In Patients With Advanced Gastric Cancer. Pfizer, USA 6. NCT00555620: Study Of Sunitinib In Combination With Cisplatin/Capecitabine Or Oxaliplatin/Capecitabine In Patients With Advanced Gastric Cancer. Pfizer, USA 7. NCT00553696: Study Of Sunitinib With S-1 And Cisplatin For Gastric Cancer Pfizer, USA 8. NCT00524186: Sunitinib, Irinotecan, Fluorouracil, and Leucovorin In Treating Patients With Advanced Stomach Cancer or Gastroesophageal Cancer. Roswell Park Cancer Institute and National Cancer Institute (NCI), USA 9. NCT00450203: Combination Chemotherapy With or Without Bevacizumab in Treating Patients With Previously Untreated Stomach Cancer or Gastroesophageal Junction Cancer That Can Be Removed by Surgery. National Cancer Institute (NCI), USA 10. NCT00217581: Bevacizumab, Oxaliplatin, and Docetaxel in Treating Patients With Locally Advanced Unresectable or Metastatic Stomach or Gastroesophageal Junction Cancer. Barbara Ann Karmanos Cancer Institute and National Cancer Institute (NCI), USA 11. NCT00350753: Avastin and Tarceva for Upper Gastrointestinal Cancers. Rigshospitalet, Denmark 12.

xaliplatin, and Docetaxel in Treating Patients With Locally Advanced Unresectable or Metastatic Stomach or Gastroesophageal Junction Cancer. Barbara Ann Karmanos Cancer Institute and National Cancer Institute (NCI), USA 11. NCT00350753: Avastin and Tarceva for Upper Gastrointestinal Cancers. Rigshospitalet, Denmark 12. NCT00548548: A Study of Bevacizumab in Combination With Capecitabine and Cisplatin as First-Line Therapy in Patients With Advanced Gastric Cancer. Genentech, Hoffmann-La, Roche and Chugai, USA 13. NCT00178698: Hyperthermia/Thermal Therapy With Chemotherapy to Treat Inoperable or Metastatic Tumors. The University of Texas Health Science Center, Houston, USA 14. NCT00447330: Xelox (Xeloda + Oxaliplatin) and Avastin for Metastatic Esophagogastric Adenocarcinoma. Duke University, Hoffmann-La Roche, Sanofi-Aventis, and Genentech, USA 15. NCT00737438: Pre-Operative Chemotherapy Plus Bevacizumab With Early Salvage Therapy Based on PET Assessment of Response in Patients With Locally Advanced But Resectable Gastric and GEJ Adenocarcinoma.Memorial Sloan-Kettering Cancer Center and Genentech, USA 16. NCT00390416: Study of Docetaxel, Cisplatin, and Fluorouracil (Modified DCF) With Bevacizumab in Patients With Unresectable or Metastatic Gastroesophageal Adenocarcinoma.Memorial Sloan-Kettering Cancer Center, Sanofi-Aventis, Genentech, USA 17. NCT00172627: Association and Mechanism Between Cyclooxygenase-2 and Interleukin-6 in Gastric Cancer. National Taiwan University Hospital, Taiwan 18. NCT00565370: Phase I/II XP+Sorafenib in Advanced Gastric Cancer. Asan Medical Center, Asian Stomach cancer Investigator Association will join in the phase II portion, Asia 19. NCT00595972: Epirubicin Cisplatin and 5-FU Combined With Endostar in Patients With Advanced or Metastatic Gastric Cancer. Fudan University, China 20. NCT00570531: Phase II Trial for Patients With Loco-Regional Esophageal Carcinoma..University of Michigan Cancer Center and Genentech, USA 21. NCT00703625: Phase I Study of Docetaxel and Temsirolimus in Resistant Solid Malignancies. Washington University School of Medicine, USA FDA-approved antiangiogenic agents in cancer treatment Anti-angiogenesis studies have led to the development of new agents targeting either angiogenic factors, endothelial cells or other components of the tumor neovasculature (14-22). Many angiogenesis inhibitors have already entered clinical trials in cancer patients.

FDA-approved antiangiogenic agents in cancer treatment Anti-angiogenesis studies have led to the development of new agents targeting either angiogenic factors, endothelial cells or other components of the tumor neovasculature (14-22). Many angiogenesis inhibitors have already entered clinical trials in cancer patients. Anti-angiogenesis is a targeted therapy using drugs or other substances to prevent tumors from creating new blood vessels, so to stop the growth of tumor. Both natural and synthetic anti-angiogenesis inhibitors are being studied, although many of these drugs are still available only in clinical trials, the first anti-angiogenic drug, Bevacizumab (Avastin), was approved by the Food and Drug Administration (FDA) in 2004, for use in the treatment of metastatic colon cancer. Up to now, there have been several angiogenesis inhibitors approved by FDA in cancer treatment (Table 2). These agents, which can interrupt critical cell signaling pathways involved in tumor angiogenesis and growth, consist of three major categories: (a) monoclonal antibodies directed against specific proangiogenic growth factors and/or their receptors; (b) small molecule tyrosine kinase inhibitors (TKIs) of multiple proangiogenic growth factor receptors; and (c) inhibitors of mTOR (mammalian target of rapamycin) represent a smaller category of antiangiogenic therapies with one currently approved agent. In addition, at least two other approved angiogenic agents may indirectly inhibit angiogenesis through mechanisms that are not completely understood.

h factor receptors; and (c) inhibitors of mTOR (mammalian target of rapamycin) represent a smaller category of antiangiogenic therapies with one currently approved agent. In addition, at least two other approved angiogenic agents may indirectly inhibit angiogenesis through mechanisms that are not completely understood. Table 2 FDA-approved angiogenesis inhibitors in oncology 1. Monoclonal antibodies: Bevacizumab (Avastin), Genentech, Description: A humanized monoclonal antibody that binds biologically active forms of vascular endothelial growth factor (VEGF) and prevents its interaction with VEGF receptors (VEGFR-1 and VEGFR-2), thereby inhibiting endothelial cell proliferation and angiogenesis. Approved indications: Metastatic colorectal cancer (mCRC), non-small cell lung cancer (NSCLC), advanced breast cancer. Cetuximab (Erbitux), Bristol-Myers Squibb, ImClone Description: A humanized monoclonal antibody that binds biologically active forms of vascular endothelial growth factor (VEGF) and prevents its interaction with VEGF receptors (VEGFR-1 and VEGFR-2), thereby inhibiting endothelial cell proliferation and angiogenesis. Approved indications: Metastatic colorectal cancer, head and neck cancer. 2. Small molecule tyrosine kinase inhibitors: Sorafenib (Nexavar), Bayer Onyx Description: Small molecule TK inhibitor of of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-ß, and Raf-1. Approved indications: Advanced renal cell carcinoma, advanced hepatocellular carcinoma.

Cetuximab (Erbitux), Bristol-Myers Squibb, ImClone Description: A humanized monoclonal antibody that binds biologically active forms of vascular endothelial growth factor (VEGF) and prevents its interaction with VEGF receptors (VEGFR-1 and VEGFR-2), thereby inhibiting endothelial cell proliferation and angiogenesis. Approved indications: Metastatic colorectal cancer, head and neck cancer. 2. Small molecule tyrosine kinase inhibitors: Sorafenib (Nexavar), Bayer Onyx Description: Small molecule TK inhibitor of of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-ß, and Raf-1. Approved indications: Advanced renal cell carcinoma, advanced hepatocellular carcinoma. Sunitinib (Sutent), Pfizer Description: Small molecule TK inhibitor of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR- ß, and RET. Approved indications: Advanced renal cell carcinoma, and gastrointestinal stromal tumor (GIST). 3. Inhibitors of mTOR: Temsirolimus (Torisel™), Wyeth Description: A small molecule inhibitor of mTOR (mammalian target of rapamycin), part of the PI3 kinase/AKT pathway involved in tumor cell proliferation and angiogenesis. Approved indications: Advanced renal cell carcinoma. Other angiogenic inhibitors: Bortezomib (Velcade®), Millennium, Description: A proteasome inhibitor that disrupts signaling of the cancer cell, leading to cell death and tumor regression. Bortezomib may have indirect antiangiogenic properties, although the mechanisms are unclear. Approved indications: Multiple myeloma, mantle cell lymphoma (MCL).

Bortezomib (Velcade®), Millennium, Description: A proteasome inhibitor that disrupts signaling of the cancer cell, leading to cell death and tumor regression. Bortezomib may have indirect antiangiogenic properties, although the mechanisms are unclear. Approved indications: Multiple myeloma, mantle cell lymphoma (MCL). Thalidomide (Thalomid®), Celgene Description: Possesses immunomodulatory, anti-inflammatory, and antiangiogenic properties, although the precise mechanisms of action are not fully understood. Approved indications: Multiple myeloma Mechanisms of anti-angiogenesis drugs Anti-angiogenic drugs do not directly attack cancer cells, but target the blood supply necessary for survival and growth of the tumor. In this way, they prevent new tumors formaiton, and cause existing tumors to shrink. VEGF is one of the most important proteins in tumor angiogenesis. This protein is not made in large amounts by normal cells, but some cancer cells secrete it into the area around them. VEGF then attaches to a VEGF receptor (VEGFR) on the surface of nearby endothelial cells and then switch signals to begin growth and formation of new blood vessels. Many of the anti-angiogenesis drugs used today attack the VEGF pathway. Bevacizumab (Avastin) was the first drug targeted at new blood vessels approved by FDA for use against cancer. This monoclonal antibody is a synthetic version of an immune system protein that binds to VEGF and blocks it from reaching the VEGF receptor (23).

he anti-angiogenesis drugs used today attack the VEGF pathway. Bevacizumab (Avastin) was the first drug targeted at new blood vessels approved by FDA for use against cancer. This monoclonal antibody is a synthetic version of an immune system protein that binds to VEGF and blocks it from reaching the VEGF receptor (23). Other drugs, such as sunitinib (Sutent) and sorafenib (Nexavar), are small molecules that attach to the VEGF receptor itself, preventing it from being activated (24, 25). Some other drugs used to treat cancer, such as thalidomide and lenalidomide (Revlimid), are also known to affect blood vessel growth. However, they work against cancer in other ways also (26-30). Drugs that target other blood vessel pathways are now being tested. Some drugs already used to treat cancer have been found to affect blood vessel growth, too. For example, low dose chemotherapy may prevent tumor growth without causing severe side effects that higher doses would. Some studies suggest the drugs may be effective because they can stop the growth of endothelial cells (28, 31).

drugs already used to treat cancer have been found to affect blood vessel growth, too. For example, low dose chemotherapy may prevent tumor growth without causing severe side effects that higher doses would. Some studies suggest the drugs may be effective because they can stop the growth of endothelial cells (28, 31). Features of anti-angiogenesis treatment different from traditional chemotherapy In recent decades, an effort to find drugs without potential severe side effects of chemotherapy has led to the discovery of new drugs that target the tumor itself while sparing normal cells of the body. Traditional chemotherapy drugs work by attacking cells which divide rapidly in the body. Unfortunately, some normal cells, such as those in the mouth mucosa or the digestive tract, also divide rapidly, but they do not differentiate into malignant cells. So these drugs can lead to the side effects including mouth sores, nausea and diarrhea. Anti-angiogenesis drugs do not target normal cells, so in many cases, side effects are milder. However, they may have their own side effects, because many drugs are still being evaluated in the clinical trials, it is not yet known whether side effects will be similar for all drugs in this class. Future directions of anti-angiogenesis study Finding new anti-angiogenic drugs More and more new drugs are being developed to target angiogenesis, and some of these new drugs will also target the tumor cells.

Features of anti-angiogenesis treatment different from traditional chemotherapy In recent decades, an effort to find drugs without potential severe side effects of chemotherapy has led to the discovery of new drugs that target the tumor itself while sparing normal cells of the body. Traditional chemotherapy drugs work by attacking cells which divide rapidly in the body. Unfortunately, some normal cells, such as those in the mouth mucosa or the digestive tract, also divide rapidly, but they do not differentiate into malignant cells. So these drugs can lead to the side effects including mouth sores, nausea and diarrhea. Anti-angiogenesis drugs do not target normal cells, so in many cases, side effects are milder. However, they may have their own side effects, because many drugs are still being evaluated in the clinical trials, it is not yet known whether side effects will be similar for all drugs in this class. Future directions of anti-angiogenesis study Finding new anti-angiogenic drugs More and more new drugs are being developed to target angiogenesis, and some of these new drugs will also target the tumor cells. Vascular targeting agents (VTAs) are a related group of drugs that may prove to be important in treating cancer (32, 33). Anti-angiogenesis drugs may stop new blood vessels formation, but is there a way to attack tumor blood vessels that have already formed? Some differences have been found between normal blood vessels in the body and those that nourish tumors. Some new drugs may be able to exploit these differences, attacking tumor blood vessels only but sparing normal blood vessels. Several VDAs are now being studied in clinical trials. Early studies have shown that these drugs seem to work best on the inner parts of tumors. This may mean they will work well when combined with other treatments that are more likely to work on the surface of the tumor, such as chemotherapy.

ing normal blood vessels. Several VDAs are now being studied in clinical trials. Early studies have shown that these drugs seem to work best on the inner parts of tumors. This may mean they will work well when combined with other treatments that are more likely to work on the surface of the tumor, such as chemotherapy. Combining anti-angiogenesis drugs It is now clear that tumors can make and release many molecules capable of stimulating angiogenesis. Targeting only one of these chemicals may not be sufficient to induce blockade of tumor angiogenesis, However, combination of drugs that attack different targets may prove to be more effective. Studies on combination of these drugs are under way.

can make and release many molecules capable of stimulating angiogenesis. Targeting only one of these chemicals may not be sufficient to induce blockade of tumor angiogenesis, However, combination of drugs that attack different targets may prove to be more effective. Studies on combination of these drugs are under way. Combining angiogenesis inhibitors with other therapies Anti-angiogenesis drugs tend to have milder side effects that are different from other cancer treatments. This makes the idea of combining them with other types of treatment very appealing. Combination of these drugs with chemotherapy drugs, radiation therapy, or other new types of targeted therapies has indicated promising. In the clinical trials, there are currently lots of anti-angiogenesis drugs being studied. Some of these drugs are being tested as single agents, while others are being used in combination with other treatments. Several anti-angiogenesis drugs are now used in the treatment of cancer, and others will be put into use in the near future. Because these are still new drugs, many questions about them remain open. For example, are they most effective used alone or with other treatments? What is the best way of administration? what is the appropriate dosage? These and other important questions are now being studied in clinical trials.

se in the near future. Because these are still new drugs, many questions about them remain open. For example, are they most effective used alone or with other treatments? What is the best way of administration? what is the appropriate dosage? These and other important questions are now being studied in clinical trials. There is some reason to believe that standard chemotherapy drugs and anti-angiogenesis drugs may work well in combination (34). In early clinical trials of bevacizumab (Avastin), it was determined that bevacizumab alone did not help cancer patients survive longer. In subsequent studies, however, it was found that, when combined with other chemotherapy drugs, it was more effective. Using metronomic chemotherapy as anti-angiogenesis approach Most chemotherapy drugs were designed to attack cancer cells directly, but some of them may be useful as anti-angiogenic agents, too. When they are given at low doses over a longer period of time (versus high doses usually given at regular intervals), they seem to work without causing major side effects. This approach is known as metronomic chemotherapy. Some evidences suggest that the chemotherapy, when used this way, may be acting on tumor blood vessels. Several studies are now undergoing to test the value of metronomic chemotherapy, either alone or combined with anti-angiogenic drugs (31, 32).

ng major side effects. This approach is known as metronomic chemotherapy. Some evidences suggest that the chemotherapy, when used this way, may be acting on tumor blood vessels. Several studies are now undergoing to test the value of metronomic chemotherapy, either alone or combined with anti-angiogenic drugs (31, 32). Concluding remark Since so many potent angiogenesis inhibitors are being tested in the clinical trials worldwide, it is optimistic and promising that AIs will be selected as candidates for standard therapy of cancers including gastrointestinal cancers. Conflicts of Interest The authors declare no conflict of interests.

Introduction From the ancient time, the Chinese herb schisandra chinensis has been used as bactericidal agent in some Asian countries(1). From time to time, it is indicated in cases of chronic cough and dyspnea, diarrhea, night sweats, wasting disorders, irritability, palpitations, dream-disturbed sleep and insomnia(2).

Introduction From the ancient time, the Chinese herb schisandra chinensis has been used as bactericidal agent in some Asian countries(1). From time to time, it is indicated in cases of chronic cough and dyspnea, diarrhea, night sweats, wasting disorders, irritability, palpitations, dream-disturbed sleep and insomnia(2). The main active constituents of schisandra chinensis related to liver disease therapy are schisandrin B and schisandrin C. Diphenyl dimethyl bicarboxylate (DDB) is a synthesized intermediate derivative of schizandrin C. DDB is commonly used as adjuvant hepatoprotectant in the treatment of chronic viral hepatitis and other liver diseases with different etiologies in China(3) and some other Asian countries(4-6). Only in recent decades, the world began to understand its pharmacological possibilities and clinical applications(7). In recent years, people are enthusiastic to excavate its new therapeutic potentials in the treatment of liver diseases, and indeed, new pharmacological links to the liver diseases have been found. Along with these progresses, there are also certain controversies about this synthetic compound. In the past, people usually used it as an agent for improving liver function and lowering serum alanine aminotransferase (ALT) in viral hepatitis and in liver injuries induced by various chemicals and drugs(8-10). However, people recently started to investigate its effect in anti-virus, treating fatty liver, and anti-malignancy. Is schisandra chinensis born to be a completely liver herb? Can DDB play a principal role in the treatment of viral hepatitis and other liver damages? In this concise review, we focus on the current findings of DDB in the treatment of liver disease, and discuss the novel possibilities of DDB for further roles in liver disease therapy.

inensis born to be a completely liver herb? Can DDB play a principal role in the treatment of viral hepatitis and other liver damages? In this concise review, we focus on the current findings of DDB in the treatment of liver disease, and discuss the novel possibilities of DDB for further roles in liver disease therapy. Compound characteristics Schisandra chinensis is a deciduous woody climbing vine about eight meters long which produces red spherical fruit. Because the whole fruit (including seeds) is said to have a salty taste, the skin and pulp are sweet and sour, and the kernels are pungent and bitter, therefore its name in Chinese is “Wu Wei Zi” denoting “five flavors”(Fig. 1a). Schisandra fruit contains dibenzocyclooctene lignans (about 2% by weight), with the main constituents being schisandrin, schisandrin A, schisandrin B, schisandrin C, g-schisandrin (the racemic form of schisandrin B), gomisin A and gomisin N(11). Among these constituents, the schisandrin C and schisandrin B are most investigated for their therapeutical potentials for liver diseases. Figure 1 The schisandra chinensis, and the molecular structures of diphenyl dimethyl bicarboxylate (DDB), schisandrin B (Sch B).

Schisandra fruit contains dibenzocyclooctene lignans (about 2% by weight), with the main constituents being schisandrin, schisandrin A, schisandrin B, schisandrin C, g-schisandrin (the racemic form of schisandrin B), gomisin A and gomisin N(11). Among these constituents, the schisandrin C and schisandrin B are most investigated for their therapeutical potentials for liver diseases. Figure 1 The schisandra chinensis, and the molecular structures of diphenyl dimethyl bicarboxylate (DDB), schisandrin B (Sch B). Diphenyl dimethyl bicarboxylate (DDB), or full name dimethyl-4,4’-bimethyloxy-5,6,5’,6’-dimethylene-dioxydiphenyl-2,2’-bicarboxylate (DDB) (Fig. 1b), is a synthesized intermediate derivative of schizandrin C, an active component isolated from the fructus schizandrae(1, 12). Schisandrin B (Sch B) is another active dibenzocyclooctadiene derivative isolated from the fruit of schisandra chinensis. Compared with Sch B, DDB lacks the cyclooctadiene ring (Fig 1c). Based on the molecular structure, there are other acronyms for DDB used in the literatures, biphenyl dimethyl-dicarboxylate (BDD)(13, 14), diphenyl dimethyl dicarboxylate (PMC)(15-17). DDB for liver disease therapy DDB is used in the treatment of chronic viral hepatitis for improving liver functions, DDB and Sch B could reduce serum ALT activity in animal models and in humans, it has also antioxidant action for scavenging free radicals and inhibiting lipid peroxidation reaction in in vitro systems(18-20).

or liver disease therapy DDB is used in the treatment of chronic viral hepatitis for improving liver functions, DDB and Sch B could reduce serum ALT activity in animal models and in humans, it has also antioxidant action for scavenging free radicals and inhibiting lipid peroxidation reaction in in vitro systems(18-20). Improving liver function Clinical study showed that DDB is effective in lowering the serum ALT in patients with chronic viral hepatitis(8) and in protecting against carbon tetrachloride (CCl4), D-galactosamine, and thioacetamide induced hepatic damage(21, 22). In a controlled trial of chronic viral hepatitis, patients who received schisandra extract showed normalized serum ALT levels after 4 weeks, after withdrawal of schisandra treatment ALT remained at normal levels, improvement of other liver function parameters was less pronounced(11). Schisandra was effective in relieving symptoms of sleeplessness, fatigue, abdominal tension and diarrhea(11). This was confirmed by another randomized controlled clinical trial which showed DDB significantly improved liver functions in patients with hepatitis B, by lowering serum ALT, bilirubin, a-fetoprotein, and alleviating symptoms(8). Other researchers found that DDB can effectively normalizes elevated ALT levels in patients with chronic liver diseases of different etiologies, the aspartate aminotransferase (AST), gamma-glutamyl transferase, and glutamate dehydrogenase levels are not affected, however, after DDB treatment, ALT relapsed in all patients within 2–6 weeks(23).

that DDB can effectively normalizes elevated ALT levels in patients with chronic liver diseases of different etiologies, the aspartate aminotransferase (AST), gamma-glutamyl transferase, and glutamate dehydrogenase levels are not affected, however, after DDB treatment, ALT relapsed in all patients within 2–6 weeks(23). DDB can improve liver functions of hepatitis B, lower blood bilirubin and a-fetoprotein and alleviate symptoms of patients(4, 5, 24). During the administration of DDB, side effects have not been described previously(23). The serum AST, ALT are commonly used enzymatic markers in assessment of liver functions(25). In hepatocelluar damage, these enzymes which normally locate in the cytosol are released into the blood circulation. Measurements of these enzymes in blood help evaluate the extent and type of hepatocellular damage(26). In the in vitro liver cell injury experiments, cultured hepatocytes treated with DDB had significant decrease of cellular ALT, indicating that the DDB affects the synthesis and/or degradation of ALT in liver cells(12, 23, 27, 28). It is considered that the normalization of only ALT by DDB treatment does not indicate therapeutic efficacy, therefore DDB is not recommended for the routine treatment of chronic liver diseases(23), especially in the anti-hepatitis B therapy during which the ALT levels is only a monitoring marker of anti-viral efficacy.

t is considered that the normalization of only ALT by DDB treatment does not indicate therapeutic efficacy, therefore DDB is not recommended for the routine treatment of chronic liver diseases(23), especially in the anti-hepatitis B therapy during which the ALT levels is only a monitoring marker of anti-viral efficacy. Anti-HBV In the treatment of viral hepatitis, DDB is mostly indicated as an adjuvant hepatoprotectant. However, recent studies showed it may have effect of anti-HBV. DDB can stimulate Jak/Stat signaling, and induce the expression of interferon alpha (IFN-α) stimulated genes, most notably 6-16 and ISG12(29). When DDB is administered combined with amantadine, it can directly inhibit IFN- α signaling-mediated replication of HBV in infected hepatocytes, this represents a novel treatment potential for chronic hepatitis B(29). However, in another study, patients with chronic hepatitis C, B, or steatohepatitis, with persistently elevated ALT were treated with DDB, ALT can rapidly normalized in most of the patients and remained normal during treatment. But there was no significant effect on the hepatitis B virus DNA level. Histological examination revealed no improvement on the grade and stage of liver disease(23). Treatment of chemicals or drugs induced hepatic injury DDB is effective in treating or preventing chronic hepatitis induced by chemicals or drug poisoning(22), hepatoprotective effects of DDB were reported against a variety of toxicants(8, 30, 31).

However, in another study, patients with chronic hepatitis C, B, or steatohepatitis, with persistently elevated ALT were treated with DDB, ALT can rapidly normalized in most of the patients and remained normal during treatment. But there was no significant effect on the hepatitis B virus DNA level. Histological examination revealed no improvement on the grade and stage of liver disease(23). Treatment of chemicals or drugs induced hepatic injury DDB is effective in treating or preventing chronic hepatitis induced by chemicals or drug poisoning(22), hepatoprotective effects of DDB were reported against a variety of toxicants(8, 30, 31). It was found that DDB could protect against carbon tetrachloride (CCl4) induced liver damage in mice without obvious side effects(32). DDB also alleviates liver injury induced by D-galactosamine, thioacetamide and prednisolone in animal models(12, 15, 22). DDB inhibits tamoxifen-induced hepatic injury, by reducing the oxidative stress, such as lipid peroxidation, and enhancing the antioxidant enzyme activities. DDB can mediate its biochemical effects through the enhancement of the antioxidant enzyme activities and reduced glutathione level as well as decreasing the level of lipid peroxides(7).

induced hepatic injury, by reducing the oxidative stress, such as lipid peroxidation, and enhancing the antioxidant enzyme activities. DDB can mediate its biochemical effects through the enhancement of the antioxidant enzyme activities and reduced glutathione level as well as decreasing the level of lipid peroxides(7). In the concanavalin A (Con A) induced liver injury mice, DDB significantly inhibites the elevation of serum ALT, TBIL and total bile acid levels, but it has no effect on AST(3). The early DNA fragmentation and cell apoptosis was observed in the Con A hepatitis model, the DNA fragmentation and cell apoptosis was considered induced in part by tumor necrosis factor-α (TNF-α)(33), DDB strongly down-regulated expression of TNF-α mRNA(3), this was evidenced by the marked decrease of serum TNF-α level(3). These results indicate that DDB could directly protect hepatocyte DNA from oxidative damage, and inhibit TNF-α mRNA expression in liver tissue, thus prevent the liver damages induced by Con A(3). DDB is hepatoprotective against erythromycin toxicity in rats. Oral daily administration of toxic dose of erythromycin stearate (100 mg/kg body weight) was given to male rats for fourteen days to induce hepatotoxicity. The results of DDB treatment showed that DDB (100 mg/kg body weight) significantly prevented the erythromycin stearate induced liver damage. The biochemical parameters(ALT, AST, TBIL, total lipids and cholesterol) and histology improved compared with ursodesoxycholic acid or Silymarin, as reference drugs(34).

toxicity. The results of DDB treatment showed that DDB (100 mg/kg body weight) significantly prevented the erythromycin stearate induced liver damage. The biochemical parameters(ALT, AST, TBIL, total lipids and cholesterol) and histology improved compared with ursodesoxycholic acid or Silymarin, as reference drugs(34). The mechanisms of DDB hepatoprotection might involve the follows. The first, DDB has hepatoprotective effect and functions as a potent antioxidant agent when it is used in the treatment of viral and chemically induced hepatitis(27, 28, 35). The second, pharmacological study shows that DDB increases liver protein and glycogen synthesis and has an inducing effect on the cytochrome P-450 enzyme system(36), therefore DDB has the effects of anti-toxic, anti-carcinogenic and anti-mutagenic effects(36). The third, DDB may protect hepatocytes by stimulating the hepatic mitochondrial reduced glutathione (GSH) antioxidant system via activation of GSH related enzyme, this is evidenced by the increased tissue GSH(37). GSH plays an important role in numerous cellular functions, including DNA synthesis, regulation of cytosolic Ca2+ homeostasis, and detoxification of reactive oxygen species(38-40). Deficiency of cellular GSH increases prooxidant production, and enhances apoptosis(10, 41). GSH works with the antioxidant enzymes, such as Se-glutathione peroxidase, glutathione S-transferases, and glutathione reductase, in combating reactive oxygen species and maintaining cellular glutathione status, in this process, the maintenance of mitochondrial glutathione status was critical for cell survival(42, 43).

GSH works with the antioxidant enzymes, such as Se-glutathione peroxidase, glutathione S-transferases, and glutathione reductase, in combating reactive oxygen species and maintaining cellular glutathione status, in this process, the maintenance of mitochondrial glutathione status was critical for cell survival(42, 43). Immunological modulation When mice were administered 20% ethanol alone, splenic plaque forming cells and hemaglutination titers to sheep red blood cells, and the secondary IgG antibody response to bovine serum albumin were decreased, however, they restored to normal level after DDB treatment. The elevations of serum ALT and total protein levels caused by ethanol were also reduced to normal by the treatment of DDB. The lowered serum albumin and albumin: globulin ratio were also increased to normal level. These findings indicate that DDB has a protective effect against ethanol induced humoral immunosuppression(16, 44). DDB also can restore the cellular immune functions suppressed by CCl4 or ketoconazole in mice(15, 17), indicated by the natural killer cells and phagocytic activity were significantly augmented(45). These results showed DDB restores and prevents the immune functions depression by hepatotoxicity agents.

Immunological modulation When mice were administered 20% ethanol alone, splenic plaque forming cells and hemaglutination titers to sheep red blood cells, and the secondary IgG antibody response to bovine serum albumin were decreased, however, they restored to normal level after DDB treatment. The elevations of serum ALT and total protein levels caused by ethanol were also reduced to normal by the treatment of DDB. The lowered serum albumin and albumin: globulin ratio were also increased to normal level. These findings indicate that DDB has a protective effect against ethanol induced humoral immunosuppression(16, 44). DDB also can restore the cellular immune functions suppressed by CCl4 or ketoconazole in mice(15, 17), indicated by the natural killer cells and phagocytic activity were significantly augmented(45). These results showed DDB restores and prevents the immune functions depression by hepatotoxicity agents. Treatment of malignancy DDB has anticancer activity and inhibits malignancy differentiation effects on cancer cells(46). In an in vitro study, DDB was confirmed to have multidrug resistance chemosensitizing effect on a panel of cancer cell lines. DDB at nontoxic concentrations partly reversed the resistance to vincristine, doxorubicin, paclitaxel in acquired multidrug resistance breast carcinoma MCF-7/Adr cells, KBv200 and intrinsic multidrug resistance human hepatocarcinoma Bel(7402) cells, confirmed by increasing the intracellular accumulation of doxorubicin and inhibited surface P-gp expression in MCF-7/Adr cells. Also DDB promoted doxorubicin-induced apoptosis of Bel(7402) cells by enhancing caspase-3 activation. These results indicate that DDB has multidrug resistance reversal activity by inhibiting P-gp when used combined with cancer drugs (47).

mulation of doxorubicin and inhibited surface P-gp expression in MCF-7/Adr cells. Also DDB promoted doxorubicin-induced apoptosis of Bel(7402) cells by enhancing caspase-3 activation. These results indicate that DDB has multidrug resistance reversal activity by inhibiting P-gp when used combined with cancer drugs (47). DDB can prevent malignant transformation of WB-F344 rat liver epithelial cells induced by 3-methylcholanthrene and 12-O-tetradecanoyl phorbol 13-acetate. This showed DDB has a potential chemopreventive effect on hepato-carcinogenesis induced by carcinogens in vitro(48). Schisandrin B versus DDB In addition to DDB, schisandrin B (Sch B) is another main active component from the schisandra chinensis. It was indicated that the Sch B pretreatment independently enhances glutathione antioxidant status (mtGAS) and induces heat shock protein (HSP) 25/70 (HSP 25/70) production, particularly under conditions of oxidative stress, therefore protecting against CCl4 hepatotoxicity in mice(49) and against TNF-α induced hepatic apoptosis in D-galactosamine-sensitized mice(50), whereas the DDB did not have this effect(49). Sch B protects against CCl4 induced hepatotoxicity(37), myocardical ischemia/reperfusion injury and brain oxidative damage in rodents(51, 52). These effects are considered attributing to the enhancement of cellular glutathione antioxidant status(37, 51, 52), particularly in the mitochondrion(53). However, the DDB did not stimulate mitochondrial glutathione status nor did protect CCl4 induced hepatotoxicity in mice(28).

ain oxidative damage in rodents(51, 52). These effects are considered attributing to the enhancement of cellular glutathione antioxidant status(37, 51, 52), particularly in the mitochondrion(53). However, the DDB did not stimulate mitochondrial glutathione status nor did protect CCl4 induced hepatotoxicity in mice(28). In another study, pretreating mice with Sch B or DDB significantly lowers the CCl4 induced serum ALT in mice, with the inhibitory effect of Sch B being much more potent. Sch B, but not DDB, could decrease the serum sorbital dehydrogenase (SDH) activity. The lowering of serum SDH activity, indicative of hepatoprotection against CCl4 toxicity, by Sch B treatment was associated with an enhancement in hepatic mitochondrial glutathione redox status as well as an increase in mitochondrial glutathione reductase (mtGRD) activity in CCl4-treated mice. Again, DDB pretreatment, though enhancing both hepatic mitochondrial glutathione redox status and mtGRD activity in control animals, did not produce any beneficial effect in CCl4 treated mice. These differences in hepatoprotective action against CCl4 toxicity between Sch B and DDB might therefore be related to their ability to maintain hepatic mitochondrial glutathione redox status under oxidative stress condition(28). All these apparent differences and discrepancies regarding to the efficacy of DDB from the aforementioned might also result from the different research protocols, differences of individual laboratories, or even the uncertainties of DDB effects under different scenarios.

In another study, pretreating mice with Sch B or DDB significantly lowers the CCl4 induced serum ALT in mice, with the inhibitory effect of Sch B being much more potent. Sch B, but not DDB, could decrease the serum sorbital dehydrogenase (SDH) activity. The lowering of serum SDH activity, indicative of hepatoprotection against CCl4 toxicity, by Sch B treatment was associated with an enhancement in hepatic mitochondrial glutathione redox status as well as an increase in mitochondrial glutathione reductase (mtGRD) activity in CCl4-treated mice. Again, DDB pretreatment, though enhancing both hepatic mitochondrial glutathione redox status and mtGRD activity in control animals, did not produce any beneficial effect in CCl4 treated mice. These differences in hepatoprotective action against CCl4 toxicity between Sch B and DDB might therefore be related to their ability to maintain hepatic mitochondrial glutathione redox status under oxidative stress condition(28). All these apparent differences and discrepancies regarding to the efficacy of DDB from the aforementioned might also result from the different research protocols, differences of individual laboratories, or even the uncertainties of DDB effects under different scenarios. The fact that Sch B and DDB may share the same efficacy in the treatment of hepatitis is not surprising, since both compounds are derived from the fruits of schisandra chinensis with the similar molecular structure.

All these apparent differences and discrepancies regarding to the efficacy of DDB from the aforementioned might also result from the different research protocols, differences of individual laboratories, or even the uncertainties of DDB effects under different scenarios. The fact that Sch B and DDB may share the same efficacy in the treatment of hepatitis is not surprising, since both compounds are derived from the fruits of schisandra chinensis with the similar molecular structure. Conclusions The leakage of hepatic enzymes such as ALT and AST is commonly used as an indirect index of hepatocellular damage. Numerous studies demonstrated that DDB could lower the blood ALT, it even may not affect the AST levels. Now, the controversy of DDB in treating viral hepatitis is whether DDB can improve liver histology, and whether DDB has any anti-viral effect. Solely lowering blood ALT without improving liver histology is considered no substantial treatment efficacy; furthermore, ALT normalization sometimes occurs only during the DDB treatment period. Normalization of ALT may comfort patients temporarily, and sometimes may lessen the patients’ anxieties. However, in this regard, patients may misunderstand or be misled by this superficial effect, any claims of treatment effect judged by the solely ALT decrease are unethical. Another concern is that during antiviral agent is being used, such as the interferon, nucleotides, the fluctuation of serum ALT is the assessment marker of antiviral efficacy, in these cases, ALT lowering drugs as DDB are not recommended.

, any claims of treatment effect judged by the solely ALT decrease are unethical. Another concern is that during antiviral agent is being used, such as the interferon, nucleotides, the fluctuation of serum ALT is the assessment marker of antiviral efficacy, in these cases, ALT lowering drugs as DDB are not recommended. There are still too much unknowns about the DDB effects, more randomized controlled trials should be carried out to ascertain these effects. Prior to the full elucidation of the total panel of DDB therapeutical potentials, we should bear in mind that a single drug (herb or compound derived) can not have unlimited therapeutical potentials, viral hepatitis, as a complicated clinical setting, can not be cured by a single drug. Conflicts of Interest The authors declare no conflict of interests. Abbreviations DDBdimethyl diphenyl bicarboxylate Sch Bschisandrin B ALTalanine aminotransferase ASTaspartate aminotransferase TBILtotal bilirubin CCl4carbon tetrachloride GSHreduced glutathione mtGRDmitochondrial glutathione reductase SDHsorbitol dehydrogenase

adenocarcinoma element. The higher proportion of small cell carcinoma suggests that the sqaumous cell carcinoma element and adenocarcinoma element were differentiated from the small cell carcinoma element. Otherwise, this esophageal tumor arises from totipotent stem cell of the esophagus, as suggested by Ho et al [2]. Most of esophageal tumors with multiple differentiation is small cell carcinoma [1-12], although basaloid cell squamous cell carcinoma also shows multiple differentiation [13]. The cellular origin of small cell carcinoma is unknown. In the review of the English literature on multiple differentiation of esophageal cancers, Maru et al [1] reported that a combination of small cell carcinoma and adenocarcinoma was seen in 15/40 cases, and a combination of small cell carcinonoma and squamous cell carcinoma in 1/40 cases. Ho et al [2] reported that a combination of small cell carcinoma and squamous cell carcinoma or adenocarcinoma was seen in 3/4 cases. Sasajima et al [3] demonstrated one case of esophageal carcinoma showing multiple differentiation into oat cell carcinoma, adenoid cystic carcinoma, adenocarcinoma, and squamous cell carcinoma. Reyes et al [4] identified that small cell carcinoma coexisted with squamous cell carcinoma in 4/16 cases. Reid et al [5] showed a case of combination of small cell carcinoma and squamous cell carcinoma. Yun et al [6] identified squamous differentiation in small cell carcinoma in 2/21 cases. Medgyesy et al [7] found a combination of small cell carcinoma and adenocarcinoma in 1/8 case, and a combination of small cell carcinoma and squamous cell carcinoma in 1/8 case. Wu et al [10] reported that small cell carcinoma with squamous cell carcinoma was found in 3/9 cases. Yamamoto et al [11] demonstrated a combination of small cell carcinoma and squamous cell carcinoma was seen in 3/6 cases. Takubo et al [12] found a combination of small cell carcinoma and squamous cell carcinoma in 11/21 cases, and a combination of small cell carcinoma and mucoepidermid carcinoma in 1/21 cases. Finally, Cho et al [14] identified a combination of basaloid squamous cell carcinoma and squamous cell carcinoma in 8/18, a combination of basaloid squamous cell carcinoma and adenocarcinoma in 3/18 cases, a combination of basaloid squamous cell carcinoma and small cell carcinoma in 2/18 cases. The review of esophageal carcinoma with multiple differentiation is mostly limited to two differentiation.

Introduction Patients presenting with renal stones are fully assessed for an underlying metabolic disorder but this is not established practice for patients with gallstones. Evidence from over 30 years ago showed that over half of patients with gallstones would have a lipid disorder[1]. This would increase their risk of developing coronary heart disease and stroke [2-8]. The current national service framework for coronary artery disease recommends a cholesterol level < 5 mmol/L and low-density lipoprotein (LDL) < 3 mmol/L in patients with 30% risk of developing coronary heart disease over ten years[9]. In the United Kingdom around 5.5 million people have gallstones and over 50, 000 cholecystectomies are performed each year suggesting there is a large population at risk[10]. Recent European studies have shown that hypertriglyceridaemia and low levels of high-density lipoprotein cholesterol (HDL), in addition to hypercholestrolaemia, are common findings[11, 12], which in turn are considered risk factors for coronary artery disease and stroke[13-15]. There is no current up to date UK data on which to base our current practice. This study retrospectively investigates the frequency of lipid disorders in patients who had undergone cholecystectomy and prospectively on patients with cholelithiasis to identity the proportion at risk of coronary heart disease or stroke. Methods An initial retrospective study of patient who had a cholecystectomy was performed with a subsequent prospective analysis of patients with proven cholelithiasis.

There is no current up to date UK data on which to base our current practice. This study retrospectively investigates the frequency of lipid disorders in patients who had undergone cholecystectomy and prospectively on patients with cholelithiasis to identity the proportion at risk of coronary heart disease or stroke. Methods An initial retrospective study of patient who had a cholecystectomy was performed with a subsequent prospective analysis of patients with proven cholelithiasis. We obtained the details of all patients who had a cholecystectomy at the North West Wales NHS Trust from April 2003 to May 2006 inclusive from Patient Information and Management System (PIMS). The available data was cross-referenced with the biochemical and histological database (Telepath) to identify those patients who had a cholecystectomy and a lipid profile. We have previously validated the hospital PIMS in surgical patients and demonstrated an error rate of about 5%[16]. The initial analysis was done using the hospital number but a second check was made for General Practitioners (GP) requests using the date of birth. The basic lipid profile reported by our laboratory was cholesterol and triglycerides. HDL and LDL were analysed only if requested and if the sample was fasting.

ut 5%[16]. The initial analysis was done using the hospital number but a second check was made for General Practitioners (GP) requests using the date of birth. The basic lipid profile reported by our laboratory was cholesterol and triglycerides. HDL and LDL were analysed only if requested and if the sample was fasting. Our hospital does not have a radiological database and we therefore, could not retrospectively identify patients with gallstones on ultrasound scan. There was a high incidence of lipid abnormality in the retrospective study but the percentage of patients who had a full lipid profile was low. We then conducted a prospective study of patients with proven gallstones referred to a single Upper Gastro-intestinal surgeon either as an outpatient or as an emergency to give a more accurate assessment of the lipid abnormalities. We analysed the patient demographics, the serum cholesterol, triglycerides, HDL cholesterol and lLDL cholesterol. The source of the request and whether it was done pre-operatively were recorded. Chi-square was used to analyse the differences between the sexes. The study was reviewed and cleared by the North West Wales Ethical committee board at Bangor. Results Retrospective study Of the 715 cholecystectomies which were performed, 536 (75%) were females and 179 were (25 %) males. The median age of the females was 55 years (range 15-90) and 61 years (29-88) for males.

We analysed the patient demographics, the serum cholesterol, triglycerides, HDL cholesterol and lLDL cholesterol. The source of the request and whether it was done pre-operatively were recorded. Chi-square was used to analyse the differences between the sexes. The study was reviewed and cleared by the North West Wales Ethical committee board at Bangor. Results Retrospective study Of the 715 cholecystectomies which were performed, 536 (75%) were females and 179 were (25 %) males. The median age of the females was 55 years (range 15-90) and 61 years (29-88) for males. Only 194/536 (36.2%) of women and 66/179 (36.9%) of men had a full lipid profile including HDL and LDL. A partial lipid profile (cholesterol and triglyceride) was done in 119/536 (21.8%) of women and 57/179 (31.8%) of men. 76.4% of women and 70.7% of men had one or more abnormalities. The lipid profiles were done pre-operatively in 247/313 (78.9%) of women and 111/123 (90.2%) of men. This was done by the GP in 334/436 (76.6%). Using the current NSF guidelines, 211/313 (67.4%) of women and 66/123 (53.7%) of men had a cholesterol > 5 mmol/L, and 121/194 (62.4%) of women and 37/66 (56.1%) of men had an LDL >3 mmol/L. Full details are shown in table 1.

Only 194/536 (36.2%) of women and 66/179 (36.9%) of men had a full lipid profile including HDL and LDL. A partial lipid profile (cholesterol and triglyceride) was done in 119/536 (21.8%) of women and 57/179 (31.8%) of men. 76.4% of women and 70.7% of men had one or more abnormalities. The lipid profiles were done pre-operatively in 247/313 (78.9%) of women and 111/123 (90.2%) of men. This was done by the GP in 334/436 (76.6%). Using the current NSF guidelines, 211/313 (67.4%) of women and 66/123 (53.7%) of men had a cholesterol > 5 mmol/L, and 121/194 (62.4%) of women and 37/66 (56.1%) of men had an LDL >3 mmol/L. Full details are shown in table 1. Table 1 Retrospective study of lipid profiles in patients who have had a cholecystectomy Lipids (abnormal range) Females Males P value * Cholesterol (> 5.0 mmol/L) 211/313 (67.4%) 66/ 123 (53.7%) ≤ 0.01 Triglycerides (> 1.92 mmol/L) 108/313 (34.5%) 50/ 123 (40.7%) ns HDL cholesterol (< 1.00 mmol/L) 9/199 (4.5%) 13/72 (18.1%) ≤ 0.001 LDL cholesterol (> 3.00 mmol/L) 121/194 (62.4%) 37/66 (56.1%) ns * Chi square with 1 degree of freedom. ns = not significant. Prospective study The 129 patients with cholelithiasis were studied, of which 102 (79%) were females and 27 (21%) were males. The median age of females was 49 (range 18-88) and 59 (range 26-77) for males. 91.1% (93/102) of women and 96.3% (26/27) of men had a full lipid profile including HDL and LDL. Most of the tests were done in the hospital if the patients were fasted or a request sent to the GP.

(79%) were females and 27 (21%) were males. The median age of females was 49 (range 18-88) and 59 (range 26-77) for males. 91.1% (93/102) of women and 96.3% (26/27) of men had a full lipid profile including HDL and LDL. Most of the tests were done in the hospital if the patients were fasted or a request sent to the GP. Overall 81.4% of women and 70.4 % of men had abnormal lipid profiles. 66/102 (64.7%) of women and 18/27 (66.7%) of men had a cholesterol of > 5mmol/L and 52/93 (55.9%) of women and 17/26 (65.4%) of men had an LDL of > 3 mmol/L. Full details of the lipid abnormalities are shown in the table 2. Table 2 Prospective study of lipid profiles in patients with cholelithiasis Lipids (abnormal range) Females Males P value * Cholesterol (> 5.0 mmol/L) 66/102 (64.7 %) 18/27 (66.7 %) ns Triglycerides (> 1.92 mmol/L) 37/102 (36.3 %) 7/27 (25.9 %) ns HDL cholesterol (< 1.00 mmol/L) 9/93 (9.7 %) 4/26 (15.4 %) ns LDL cholesterol (> 3.00 mmol/L) 52 / 93 (55.9 %) 17 / 26 (65.4 %) ns * Chi square with 1 degree of freedom. ns = not significant. Discussion Unlike patients with renal stones, only a third of patients who had a cholecystectomy had a full lipid profile to identify an underlying metabolic disorder. These were done mostly by their GP’s. Although the initial retrospective study showed that this was abnormal in over 76% of women and 70% of men, this increased to 81% in women when studied prospectively. Hypercholesterolaemia was the most common abnormality in both sexes followed by a raised LDL cholesterol.

disorder. These were done mostly by their GP’s. Although the initial retrospective study showed that this was abnormal in over 76% of women and 70% of men, this increased to 81% in women when studied prospectively. Hypercholesterolaemia was the most common abnormality in both sexes followed by a raised LDL cholesterol. Hypercholesterolemia was statistically more significant among women in the retrospective group but this was not the case in the prospective group, probably because of fewer cases. A quarter of males and third of females also had hypertriglyceridaemia, an increasingly recognised risk factor for heart disease and diabetes[7, 17]. Men have a statistically significant lower HDL than women but this is less frequent, this was again observed only in the retrospective group. We were unable to retrospectively analyse patients with cholelithiasis in this study, as our radiological database cannot provide this information. We overcame this problem by prospectively studying patients with gallstones although some did not have a complete profile. This would underestimate the true incidence. Although this study has only considered abnormal lipids, patients with gallstones are more likely to have additional factors for coronary heart disease, such as obesity, which would increase their risk. Using the current NSF guidelines for coronary heart disease it is likely that a large proportion of patients with gallstones should have treatment for their hyperlipidaemia in the presence of other risk factors.

ely to have additional factors for coronary heart disease, such as obesity, which would increase their risk. Using the current NSF guidelines for coronary heart disease it is likely that a large proportion of patients with gallstones should have treatment for their hyperlipidaemia in the presence of other risk factors. From the UK cholecystectomy data, around 25,000 women and 6250 men are at an increased risk of developing coronary artery disease and stroke if not managed according to the NSF guidelines. If this risk is applied to all patients with gallstone disease, over 2.7 million women and nearly 690,000 men are at risk. This study highlights one of the major weaknesses of current practice, namely that gallstones are regarded only as a surgical condition needing cholecystectomy. They should now be regarded as a marker of an underlying metabolic disorder and investigated and treated accordingly. Further studies of patients with gallstones detected on ultrasound will help establish the true incidence of the lipid abnormalities although this study shows it is already very high. We recommend that all patients who have cholelithiasis should now have a full lipid profile, including HDL and LDL, as a routine part of their clinical assessment. Meinir Williams, medical secretary and Eddie Williams, IT data coordinator. The authors declare no competing interest in this study.

Introduction Cancer is an epigenetic disease due to aberrant epigenetic patterns in the development of human cell, especially in abnormal DNA methylation. Transcriptional silencing of tumor-suppressor genes by CpG methylation may contribute to tumorigenesis. Moreover, the potential way is to drug-induced reactivation of methylation silenced tumor suppressor genes. 5-Aza-2’-deoxycytidine (5-Aza-CdR) inhibits DNA methylation and often is used in vitro to induce the re-expression of genes putatively silenced by promoter methylation(1). 5-Aza-CdR substituted for cytosine during replication and is recognized by DNA methyltransferases (DNMTs)(2). This treatment ultimately depletes cellular stores of DNMTs and results in widespread genomic hypomethylation(3). In addition to reactivation of methylation-silenced tumor-suppressor genes, 5-aza-Cyd and 5-aza-dCyd are highly toxic in cultured cells(4, 5) and animals(6, 7).

ognized by DNA methyltransferases (DNMTs)(2). This treatment ultimately depletes cellular stores of DNMTs and results in widespread genomic hypomethylation(3). In addition to reactivation of methylation-silenced tumor-suppressor genes, 5-aza-Cyd and 5-aza-dCyd are highly toxic in cultured cells(4, 5) and animals(6, 7). DNA methylation of CpG dinucleotides is known to be mediated by at least three DNMTs, including DNMT3A, DNMT3B, and DNMT1. DNMT3A and DNMT3B are de novo methyltransferases that initiate the methylation process, whereas DNMT1, the maintenance methyltransferase, directs methylation of the newly synthesized strand complimentary to the hemimethylated DNA(8). Studies suggested that over-expression of DNMT1 and /or DNMT3B involved in tumorigenesis and development of most cancers(9-12), including hepatocellular carcinoma(13). The DNMT1 siRNA treatment led to a partial removal of DNA methylation from inactive promoter CpG islands, and restored the expression of tumor suppressor genes(14). Gene targeting experiments have shown that DNMT3B plays an important role in the hypermethylation of CpG islands in human cancers(15). Rhee(16) demonstrated that somatic cell knockouts of both DNMT3B and DNMT1 genes led to demethylation and re-expression of tumor suppressor genes in a colon cancer cell line. We used a combination of genetic (siRNA) and pharmacological (5-aza-2'-deoxycytidine) inhibitors of DNMTs to study the contribution of the DNMT isotypes to cancer-cell methylation. In the current study, we investigated the effects of 5-aza-2'-deoxycytidine alone and suppression of DNMT1 or DNMT3B on human hepatoma cell lines. We try to explore the possibility of treatment strategy to hepatocellular carcinoma (HCC) by modifying the aberrant methylation.

he DNMT isotypes to cancer-cell methylation. In the current study, we investigated the effects of 5-aza-2'-deoxycytidine alone and suppression of DNMT1 or DNMT3B on human hepatoma cell lines. We try to explore the possibility of treatment strategy to hepatocellular carcinoma (HCC) by modifying the aberrant methylation. Materials and Methods Cell Culture and 5-Aza-CdR Treatment The human hapetocellular carcinoma cell line SMMC-7721 was obtained from the Cell Bank (Shanghai, China). Cell lines were maintained in RPIM1640 medium supplemented with 12% heat-inactivate NBS, 100 units/ml of penicillin, and 100 units /ml of streptomycin. Cells were cultured at 75 ml flask and treated in the next day with 100 uM 5-Aza-CdR (Sigma Chemical Co., St. Louis, MO) up to 4 days. The cultured medium was changed 3 days after treatment, and total RNA was extracted at day 3 from exponentially growing cultures. Preparation of siRNA of DNMT, a vector-based siRNA construct and transfection of DMMT RNAi construct to hepatocellular carcinoma cell line SMMC-7721 SiRNAs targeting DNMT1 or DNMT3B were designed and prepared as previous described(17, 18). The siRNA sequences against DNMT1 were designed as sense and antisense oligonucleotides corresponding to nucleotide position 2,620-2,638 of human DNMT1 (GenBank accession No. NM001379.1). The sequence of DNMT3B siRNA corresponds to nucleotide position 470-488 of DNMT3B (GenBank accession no. AF331857), there was no homology with other human genes was found by scanning the GenBank of NCBI using these siRNA.

orresponding to nucleotide position 2,620-2,638 of human DNMT1 (GenBank accession No. NM001379.1). The sequence of DNMT3B siRNA corresponds to nucleotide position 470-488 of DNMT3B (GenBank accession no. AF331857), there was no homology with other human genes was found by scanning the GenBank of NCBI using these siRNA. The human hepatocellular carcinoma cell line SMMC-7721 (No. TCHu13 Cell Bank Shanghai, China) was maintained by serial passage in RPMI 1640 (Life Technologies, Inc., Rockville, MD) containing 10% heat-inactivated new born bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin, and incubated at 37°C, 5% CO2, 95% air using the standard tissue culture incubators. One day before transfection, cells were seeded in order that they were 30–50% confluent the next day. Cells were transfected with 1.5ug of DNMT1 or DNMT3B siRNA (pMT1 or pMT3B) construct using transfectamine transfection reagent (Invitrogen) reduced serum medium at 37°C in a 5% CO2 atmosphere for 5 h. The medium was removed and replaced with fresh RPMI 1640 supplemented with 20% new born bovine serum. Control cells were treated with transfectamine alone or with pSUPER-GFP plasmid. Cells were grown and selectively cultured in 0.4 mg/ml Genetincin (life technologies) for two months after the initial transfection. The cells stably harboring targeting vector were monitored with GFP expression. SMMC-7721 cells were transfected with pMT1 named as 7721-MT1 cell lines, with pMT3B named as 7721-MT3B cell lines.

ere grown and selectively cultured in 0.4 mg/ml Genetincin (life technologies) for two months after the initial transfection. The cells stably harboring targeting vector were monitored with GFP expression. SMMC-7721 cells were transfected with pMT1 named as 7721-MT1 cell lines, with pMT3B named as 7721-MT3B cell lines. RNA extraction and synthesis of cDNA first strand Total RNA was extracted using TRIZOL (Gibco BRL, Life Technologies Inc.,) according to the manufacturer’s protocol. RNA was resuspended in DEPC treated water and quality verified by agarose gel electrophoresis stained by EB. Total RNA was reverse transcribed using 2 µg of RNA and oligo-dT(18) (Life Technologies, Inc.) in a 13.5-ul reaction. The oligo-dT-RNA mixture was placed at 70°C for 2 min and then rapidly cooled to 0°C. Then, the Superscript II reverse transcriptase and its MIX (Life Technologies, Inc.) was supplied into the oligo-dT-RNA mixture followed by 42°C for 60 min, and 90°C for 5 min and then rapidly cooled to 0°C. cDNA microarrary The 14K cDNA microarrary used in this study were obtained through the Shanghai BioChip Co., Ltd. The 14K slides contained human genes together with 10 positive and 6 negative controls. The spotting patterns and the complete annotated list of cDNAs are available at the following web site: http://www.shbiochip.com. The 5’ends of the genes up-regulated in this manner were analyzed in the BLAST database to determine whether they have promoter CpG islands.

ogether with 10 positive and 6 negative controls. The spotting patterns and the complete annotated list of cDNAs are available at the following web site: http://www.shbiochip.com. The 5’ends of the genes up-regulated in this manner were analyzed in the BLAST database to determine whether they have promoter CpG islands. Results 5-Aza-CdR induced genes expression in global changes We first assessed the global changes in gene expression induced by 5-Aza-CdR in the liver tumor cell line exposed to 100uM 5-Aza-CdR for 4 days. We analyzed those genes that were up- or down-regulated by more than 2.5-fold 4 days after drug treatment and categorized the genes as to whether or not they are known to be in some pathways. The 5’ ends of the genes up-regulated in this manner were analyzed in the BLAST database to determine whether they might have promoter CpG islands. The data showed that only 70% up-regulated genes in the hepatocellular carcinoma cell line definitely contained 5’ CpG islands. SiRNA of DNMT1 or DNMT3B constructs induced genes expression in global changes not only in those containing CpG island in the promoter regions We next assessed the global changes in gene expression induced by siRNA of DNMT1 constructs or siRNA of DNMT3B constructs in hepetocellular carcinoma cell lines SMMC-7721. There is some information indicating that genes of non-CpG island promoter up-regulates expression induced by knockdown DNMT1 or DNMT3B, therefore, the mechanism by which the knockdown of DNMT1 or DNMT3B activated genes without CpG islands remains unclear.

3B constructs in hepetocellular carcinoma cell lines SMMC-7721. There is some information indicating that genes of non-CpG island promoter up-regulates expression induced by knockdown DNMT1 or DNMT3B, therefore, the mechanism by which the knockdown of DNMT1 or DNMT3B activated genes without CpG islands remains unclear. SiRNA of DNMT3B induces more genes identical to induced by 5-Aza-CdR than siRNA of DNMT1 in hepatocellular carcinoma cells We compared those genes that both were up-regulated by DNMT1 siRNA and 5-Aza-CdR treatment, and also compared up-regulated genes both were induced by DNMT3B siRNA and 5-Aza-CdR treatment. A total of 9 genes were found to be over expressed by more than two-fold induced by DNMT1 siRNA and 5-Aza-CdR in SMMC-7721 (Table 1). The 5’ ends of the genes up-regulated in this manner were analyzed in the BLAST database to determine whether they have promoter CpG islands. The data showed that almost all of up-regulated genes contained 5’ CpG islands except KRT14. We next focused our attention on those genes that induced by DNMT3B siRNA with demethylation drug. A total of 30 genes were found to be over expressed by more than two-fold induced by DNMT3B siRNA and 5-Aza-CdR in SMMC-7721. The 5’ ends of the genes up-regulated in this manner were analyzed in the BLAST database to determine whether they have promoter CpG islands. The data showed that 76.6% up-regulated genes conjectural contained 5' CpG islands (Table 2). Hierarchical clustering of gene expression in untreated and treated cells is shown in Fig. 1. As expected, some similarities in gene expression were seen between untreated cell by DNMT3B RNAi and 5-Aza-CdR.

omoter CpG islands. The data showed that 76.6% up-regulated genes conjectural contained 5' CpG islands (Table 2). Hierarchical clustering of gene expression in untreated and treated cells is shown in Fig. 1. As expected, some similarities in gene expression were seen between untreated cell by DNMT3B RNAi and 5-Aza-CdR. Table 1 Genes altered more than 2-fold after 5-Aza-CdR and pMT1 treatment in the hepatocellular carcinoma SMMC-7721 cell line. GenBank_ID Gene_Symbol 5’CpG island (CGI)a Function Regulation of transcription NM_182917 EIF4G1 CGI regulation of translational initiation Signal transduction NM_002733 PRKAG1 CGI regulates adipose differentiation NM_004583 RAB5C CGI signal transduction, regulation Others NM_016219 MAN1B1 CGI hydrolase activity NM_017883 WDR13 CGI mRNA processing NM_000526 KRT14 None epidermis development NM_004860 FXR2 CGI insulin receptor binding NM_005993 TBCD CGI integrin-mediated signaling pathway NM_000034 ALDOA CGI RNA binding a Based on a PubMed search; CGI, 5’ CpG island; None, no known 5’ CpG island. Table 2 Genes altered by more than 2.5-fold after pMT3B and 5-Aza-CdR treatment in the hepatocellular carcinoma cell line SMMC-7721 GenBank_ID Symbol 5 CpG island (CGI)a Function Cell differentiation NM_006096 NDRG1 CGI cell differentiation Cell proliferation NM_005557 KRT16 NO cell proliferation NM_013402 FADS1 CGI regulation of cell differentiation Regulation of transcription NM_003926 MBD3 CGI regulation of transcription NM_005253 FOSL2 CGI regulation of transcription

Table 2 Genes altered by more than 2.5-fold after pMT3B and 5-Aza-CdR treatment in the hepatocellular carcinoma cell line SMMC-7721 GenBank_ID Symbol 5 CpG island (CGI)a Function Cell differentiation NM_006096 NDRG1 CGI cell differentiation Cell proliferation NM_005557 KRT16 NO cell proliferation NM_013402 FADS1 CGI regulation of cell differentiation Regulation of transcription NM_003926 MBD3 CGI regulation of transcription NM_005253 FOSL2 CGI regulation of transcription Signal transduction NM_138769 ARHT2 CGI signal transduction NM_004218 RAB11B CGI signal transduction NM_145173 DIRAS1 CGI signal transduction Others NM_017510 HSGP25L2G CGI intracellular protein transport NM_006700 FLN29 CGI nucleic acid binding NM_001956 EDN2 NO protein kinase C activation NM_057089 AP1S1 CGI intracellular protein transport NM_020196 XAB2 CGI DNA repair NM_006412 AGPAT2 CGI metabolism NM_001074 UGT2B7 NO metabolism NM_006247 PPP5C CGI regulation of I-kappaB kinase/NF-kappaB cascade NM_002273 KRT8 NO cytoskeleton organization and biogenesis NM_006445 PRPF8 CGI nuclear mRNA splicing, via spliceosome NM_002233 KCNA4 CGI potassium ion transport NM_004204 PIGQ CGI carbohydrate metabolism NM_004818 DDX23 CGI protein kinase C activation NM_017458 MVP NO response to drug NM_032038 SPINL CGI tetracycline transport NM_020230 PPAN CGI RNA splicing NM_001897 CSPG4 CGI cell motility NM_021979 HSPA2 NO male meiosis NM_018110 DOK4 CGI insulin receptor binding NM_144568 C14orf9 CGI hydrolase activity a Based on a PubMed search; CGI, 5’ CpG island; None, no known 5’ CpG island.

response to drug NM_032038 SPINL CGI tetracycline transport NM_020230 PPAN CGI RNA splicing NM_001897 CSPG4 CGI cell motility NM_021979 HSPA2 NO male meiosis NM_018110 DOK4 CGI insulin receptor binding NM_144568 C14orf9 CGI hydrolase activity a Based on a PubMed search; CGI, 5’ CpG island; None, no known 5’ CpG island. Figure 1 Hierarchical clustering based on gene expression data from SMMC-7721 DNMT3B unsuppressed and suppressed cells, and SMMC-7721 5-Aza-CdR untreated and treated cells.

response to drug NM_032038 SPINL CGI tetracycline transport NM_020230 PPAN CGI RNA splicing NM_001897 CSPG4 CGI cell motility NM_021979 HSPA2 NO male meiosis NM_018110 DOK4 CGI insulin receptor binding NM_144568 C14orf9 CGI hydrolase activity a Based on a PubMed search; CGI, 5’ CpG island; None, no known 5’ CpG island. Figure 1 Hierarchical clustering based on gene expression data from SMMC-7721 DNMT3B unsuppressed and suppressed cells, and SMMC-7721 5-Aza-CdR untreated and treated cells. Discussion Epigenetic mechanisms including DNA methylation and histone modifications are one of the most important means of gene expression regulation. More and more evidences indicated that epigenetic modifications have a crucial role in cancer development. Epigenetic therapy is also a potential therapy because epigenetic defects are thought to be more easily reversible with pharmacological intervention(19, 20). Aberrant DNA hypermethylation, a prevalent alteration in most tumor playing a key role in human carcinogenesis, involved in global hypomethylation(21-23), specific gene hypermethylation(24-26) and loss of imprinting (LOI)(27, 28). DNMTs are responsible for setting up and maintenance of DNA methylation in biological process and thought to be involved in tumorigenesis. Therefore DNMTs theoretically serve as a reasonable target for anti-tumor therapy. In preclinical work, DNMT inhibitors have reversed the growth of cancer cell lines and demonstrated antineoplastic effects in animal models, including prolongation of survival(29, 30). One such agent, 5-aza-2’-deoxycytidine (5-aza-CdR), is a potent inhibitor of genomic and promoter-specific DNA methylation(25). However, there also are limitations of epigenetic therapeutic agents for prevention of tumorigenesis due to lack of specificity, these result in accelerated tumor progression(31, 32) and drug toxicity(33, 34). Moreover, it is necessary to find a better way to correct epigenetic abnormity in tumor, especially in DNA methylation.

there also are limitations of epigenetic therapeutic agents for prevention of tumorigenesis due to lack of specificity, these result in accelerated tumor progression(31, 32) and drug toxicity(33, 34). Moreover, it is necessary to find a better way to correct epigenetic abnormity in tumor, especially in DNA methylation. Evidences showed that DNMT1 and DNMT3B cooperatively maintain DNA methylation and gene silencing in human cancer cells(16, 35). DNMT3B may not only silence genes by several mechanisms including direct DNA methylation or recruitment of proteins that modify chromatin, but also play an important role in transformation(36, 37). DNMT3B over-expression may be involved in the suppression or lower expression of p14ARF and p16INK4a observed in esophageal squamous cell carcinoma(38). The DNMT1 or DNMT 3B can be considered as a target for epigenetic therapy in human tumor. Antisense oligonucleotides are short, defined sequences of nucleotides that are complementary to mRNAs and hybridize with them and make them inactive, thereby blocking translation. Antisense oligonucleotides that are complementary to mRNA for human DNMT1 are undergoing preclinical(39) as well as clinical(40) trials. RNA interference (RNAi) is a natural mechanism in organisms in resistance to virus invasion and inhibition of transposon mobility by double stranded RNA (dsRNA).

blocking translation. Antisense oligonucleotides that are complementary to mRNA for human DNMT1 are undergoing preclinical(39) as well as clinical(40) trials. RNA interference (RNAi) is a natural mechanism in organisms in resistance to virus invasion and inhibition of transposon mobility by double stranded RNA (dsRNA). Latest study shows that 21-25 nt small interference RNA (siRNA) can mediate specific gene silencing in mammal cells(41). Being effective and highly specific, RNAi probably becomes a novel technique in knocking gene down and plays important roles in gene function study and gene therapy of diseases(42). In this study, we try to knock down the expression of DNMT1 or DNMT3B via siRNA to explore the mechanism of DNMTs involved in hepatoma therapy. An advantage of using high-throughput oligonucleotide microarray data from treated cell lines is to enable us to provide a conservative estimate of the number of genes directly affected by aberrant methylation in hepatocellular carcinoma cell line. There are different induced genes by suppression of DNMTs among DNMT1 siRNA, DNMT3B siRNA and demehylation drug. Compared with inhibition of DNMT1, DNMT3B siRNA induced more tumor-related genes identical to that of demehylation drug 5-aza-2’-deoxycytidine.

by aberrant methylation in hepatocellular carcinoma cell line. There are different induced genes by suppression of DNMTs among DNMT1 siRNA, DNMT3B siRNA and demehylation drug. Compared with inhibition of DNMT1, DNMT3B siRNA induced more tumor-related genes identical to that of demehylation drug 5-aza-2’-deoxycytidine. Approximate 33.3% (10/30) genes were reported to be involved in tumorigenesis, such as FADS1, MBD3, MVP, DIRAS1 and so on. Among them, there are 76.6% up-regulated genes conjectural contained 5’ CpG islands. These data indicated a new strategy in therapy of hepatocellular carcinoma through suppression of DNMT3B rather than treatment with demethylation agent. Because it has not been reported that DNMT3B are highly toxic in cultured cells and animals. Recently, frequent epigenetic changes such as DNA hypo- and hypermethylation and altered methylation pattern have been observed in hepatocellular carcinoma(43, 44). In fact, over-expression of DNMT3b4 is involved in human hepatocarcinogenesis, even at the precancerous stages by affecting the expression of specific genes(45). An increase in the DNMT3B mRNA levels in hepatocellular carcinoma relative to their non-cancerous tissues may be a predictor of poor survival(46). Over expression of a splice variant of DNMT 3B, DNMT3b4, is associated with DNA hypomethylation on pericentromeric satellite regions during human hepatocarcinogenesis(47). One of the novel findings of our present study is that DNMT3B siRNA could induce more genes identical to demethylation agent in hepatocellular cancer. Although these results need to be extended in larger clinical studies, this may serve as a potential new approach for hepatocellular carcinoma.

hepatocarcinogenesis(47). One of the novel findings of our present study is that DNMT3B siRNA could induce more genes identical to demethylation agent in hepatocellular cancer. Although these results need to be extended in larger clinical studies, this may serve as a potential new approach for hepatocellular carcinoma. This work was supported by grants of the National Natural Science Foundation of China (NO. 30470950). Thanks Dr. Peng Hou from department of Biomedical Engineering of Southeast University for supplying the CpG island through the BLAST. Thanks for the Shanghai gene chip company of China to supplying the analysis of gene expression. The authors declare no commercial associations or conflict of interest related to this article.

Introduction Gastric cancer is one of the most common malignancies, it mostly occurs in gastric antrum. The advanced gastric antral cancer often results in obstruction of the gastric pylorus, namely the gastric outlet obstruction. Though the surgical treatment may relieve the obstruction, usually the patients with advanced cancer are emaciated and in poor general condition, hence surgery is contraindicated. The gastrointestinal decompression, parenteral nutrition, and percutaneous enteral nutrition contribute little to improving the patient’s life quality. The placement of expandable metallic stent is a relatively novel approach used in recent years for relieving esophageal and gastric cardia stricture (1, 2), however, there are very limited reports on application of this approach in the management of gastric malignant outlet obstruction. In addition, the maneuvers for metallic stent placement in gastric malignant outlet obstruction are quite different from that of esophageal stent placement. The techniques of esophageal stent placement can not be adopted completely in the gastric outlet stent placement. In the past two years, we successfully performed metallic stent placement for gastric malignant outlet obstruction in 9 patients, in this report, we summarized the efficacy and our technical experiences.

t. The techniques of esophageal stent placement can not be adopted completely in the gastric outlet stent placement. In the past two years, we successfully performed metallic stent placement for gastric malignant outlet obstruction in 9 patients, in this report, we summarized the efficacy and our technical experiences. Materials and Methods Patients All 9 patients were from our hospital from April 2006 to August 2007, these patients were confirmed by endoscopy and histology as malignant pylorus obstruction resulted from progressive gastric pylorus cancer, the patients’ characteristics are shown in table 1. Table 1 Baseline characteristic of the 9 patients Clinical characteristics No. of patients Sex (Male/female) 4/5 Age (year, mean) 46-88 (75) Cancer sites Antrum 2 Antrum-pylorus invasion cancer 5 Gastric corpus, antrum and pylorus invasion cancer 2 Histology Adenocarcinoma 4 Gastric corpus adenosquamous carcinoma 1 Signet ring cell carcinoma 4 Stoler grade(3) III 1 IV 8 Extra-gastric Metastasis 4 Ascites 1 All 9 patients had one or more of the following conditions, inoperable; refusal to surgical treatment; severe deteriorating; irresponsive to short-term parenteral nutrition therapy; or with severe cardio-pulmonary diseases contradictive to laparotomy.

ma 4 Stoler grade(3) III 1 IV 8 Extra-gastric Metastasis 4 Ascites 1 All 9 patients had one or more of the following conditions, inoperable; refusal to surgical treatment; severe deteriorating; irresponsive to short-term parenteral nutrition therapy; or with severe cardio-pulmonary diseases contradictive to laparotomy. Instruments Olympus XQ-240 electronic endoscope was used. Stents were shape-memory MTN-shape titanium-nickel alloy intestinal stents (Micro-tech Co., Nanjing, China). Tailored design of pylorus stent was as follows, the length and width of the pylorus and antrum malignant stricture were first measured, the proximal end of the stent was designed as grand funnel-shape or plate-shpe, the distal end was designed as spherical shape (Fig. 1). The diameter of the stent was 20 - 22 mm, the stent length was 30 - 40 mm longer than the pylorus or antral stricture. The stent introducers were either, (1) dispensable luminal stent introducer MTN-CR-(4-6)/(1000-1400)-L (Micro-tech Co., Nanjing), diameter of stent introducer 4-8 mm, length 140 mm. The Savary-Gilliard dilator with a guidewire in diameter of 0.38 mm was used; (2) the duodenal stent introducer (Micro-Tech Co., Nanjing), this introducer can be inserted through the endoscope biopsy channel in diameter of with 3.2 mm or 4.2 mm. Balloon dilator with diameter of 2 cm (Fig. 2). Figure 1 Stents used in this study. a, Duodenal stent; b, pylorus stent. Figure 2 Stent delivery system. (a), stent delivery system; (b), the front tip of the delivery system; (c), the handler of the delivery system.

Instruments Olympus XQ-240 electronic endoscope was used. Stents were shape-memory MTN-shape titanium-nickel alloy intestinal stents (Micro-tech Co., Nanjing, China). Tailored design of pylorus stent was as follows, the length and width of the pylorus and antrum malignant stricture were first measured, the proximal end of the stent was designed as grand funnel-shape or plate-shpe, the distal end was designed as spherical shape (Fig. 1). The diameter of the stent was 20 - 22 mm, the stent length was 30 - 40 mm longer than the pylorus or antral stricture. The stent introducers were either, (1) dispensable luminal stent introducer MTN-CR-(4-6)/(1000-1400)-L (Micro-tech Co., Nanjing), diameter of stent introducer 4-8 mm, length 140 mm. The Savary-Gilliard dilator with a guidewire in diameter of 0.38 mm was used; (2) the duodenal stent introducer (Micro-Tech Co., Nanjing), this introducer can be inserted through the endoscope biopsy channel in diameter of with 3.2 mm or 4.2 mm. Balloon dilator with diameter of 2 cm (Fig. 2). Figure 1 Stents used in this study. a, Duodenal stent; b, pylorus stent. Figure 2 Stent delivery system. (a), stent delivery system; (b), the front tip of the delivery system; (c), the handler of the delivery system. Procedures Pre-procedural preparations were as routine gastrointestinal endoscopy, the patient lied in left, endoscope was inserted and the esophagus and stomach were examined. Two apporaches of stent placement were employed.

Figure 2 Stent delivery system. (a), stent delivery system; (b), the front tip of the delivery system; (c), the handler of the delivery system. Procedures Pre-procedural preparations were as routine gastrointestinal endoscopy, the patient lied in left, endoscope was inserted and the esophagus and stomach were examined. Two apporaches of stent placement were employed. (1) Stent placement under guidance of endoscopy (7 patients) The obstructed pylorus was observed as the endoscope advanced into the gastric antrum. The endoscope was passed into the stricture and reached the middle or lower part of the descending duodenum. The length of stricture and the distance between the proximal demarcation of the stricture and the central incisor tooth were delineated. A metallic guidewire was inserted through the biopsy channel of the endoscope. The guidewire was immobilized and the endoscope was withdrawn. A introducer mounted with stent was advanced through the guidewire to the malignant stricture in gastric antrum or to the pylorus. Next, the endoscope was re-inserted below the stent introducer and rotated 90 degrees rightwards, and the stent introducer was advanced. The rotation of the endoscope made the stent introducer above clear away the gastric greater curvature, therefore the stent introducer could reach the upper part of the descending duodenum along the gastric lesser curvature. When the stent was confirmed in the appropriate location of the obstruction, then it was released.

rotation of the endoscope made the stent introducer above clear away the gastric greater curvature, therefore the stent introducer could reach the upper part of the descending duodenum along the gastric lesser curvature. When the stent was confirmed in the appropriate location of the obstruction, then it was released. (2) Duodenal stent placement through the endoscopic biopsy tunnel (2 patients) Because our endoscope is not a therapeutic endoscopy, therefore the duodenal stent introducer can not pass through the biopsy channel. So in vitro, we first inserted a zebra guidewire throughout the biopsy channel and then into the lumen of a duodenal stent introducer mounted with stent, by doing so, the endoscope and the duodenal stent introducer were connected. The next, the guidewire was tightened, the whole assembly was inserted into the descending part of the duodenum, the zebra guidewire was pulled out, followed by withdrawal of endoscope, then the stent was released. If the stent location was inappropriate, it could be adjusted before the endoscope’s pulling out. The endoscope was only pulled out when the stent was confirmed expanded properly. In one patient, because the endoscope could not pass through the stricture, pre-dilation with a 2 cm balloon was performed, and then the stent was placed through the guidewire.

ate, it could be adjusted before the endoscope’s pulling out. The endoscope was only pulled out when the stent was confirmed expanded properly. In one patient, because the endoscope could not pass through the stricture, pre-dilation with a 2 cm balloon was performed, and then the stent was placed through the guidewire. Efficacy evaluation The gastric outlet malignant stricture was divided into 5 grades(3), grade 0, alimentation of regular food; grade I, soft food; grade II, semiliquid food; grade III, liquid food; IV, without any food intake. The efficacy of endoscopic stent placement for pylorus obstruction was evaluated as follows, (1) complete response (CR), symptoms disappear or decrease to grade I and maintain longer than 1 month; (2) partial response (PR), stricture significantly improves and reaches grade II and may last less than 1 month; (3) minor reponse (MR), stricture improves to grade III, may last 4 weeks; (4) no response (NR): stricture does not change, stricture is at grade IV, patient deteriorates or dies.

intain longer than 1 month; (2) partial response (PR), stricture significantly improves and reaches grade II and may last less than 1 month; (3) minor reponse (MR), stricture improves to grade III, may last 4 weeks; (4) no response (NR): stricture does not change, stricture is at grade IV, patient deteriorates or dies. Results Efficacy of the stent placement Of the 9 patients, 3 patients were placed with 2 cm x 10 cm stents, 6 with 2 cm x 8 cm stents. Two patients were placed with specially designed stents, 6 patients with duodenal stents, and 1 with esophageal stent. The placed stents expanded about 90% 24 - 48 h after placement confirmed by fluoroscopy. Patients resumed food intake 24 h after stent placement. The efficacy of stent placement is shown in table 2. There were no gastrointestinal perforation and massive hemorrhage occurred. A typical patient with pylorus stent placement was shown in Figure 3. Figure 3 Expandable metallic stent placement for malignant gastric outlet obstruction under endoscopy in a male patient (46 years old, progressive gastric pylorus cancer). (a), stricture of the pylorus; (b), upper part of the pylorus stent; (c), middle part of the pylorus stent; (d), lower part of the pylorum stent.

Results Efficacy of the stent placement Of the 9 patients, 3 patients were placed with 2 cm x 10 cm stents, 6 with 2 cm x 8 cm stents. Two patients were placed with specially designed stents, 6 patients with duodenal stents, and 1 with esophageal stent. The placed stents expanded about 90% 24 - 48 h after placement confirmed by fluoroscopy. Patients resumed food intake 24 h after stent placement. The efficacy of stent placement is shown in table 2. There were no gastrointestinal perforation and massive hemorrhage occurred. A typical patient with pylorus stent placement was shown in Figure 3. Figure 3 Expandable metallic stent placement for malignant gastric outlet obstruction under endoscopy in a male patient (46 years old, progressive gastric pylorus cancer). (a), stricture of the pylorus; (b), upper part of the pylorus stent; (c), middle part of the pylorus stent; (d), lower part of the pylorum stent. Table 2 Efficacy of stents placement Observations No. of patients Post-procedural obstruction I 1 II 3 III 4 IV 1 Efficacy CR 4 PR 1 MR 1 NR 3 Complications Vomiting, nausea 6 Upper abdominal pain 4 Melena 2 Enteric infection 1 Jaundice 1 Electrolytes disorder 1 Mean stent potency 53.4 day Survival 10-15 day 3 1-2 mo 2 >3 mo 4 CR, complete response; PR, partial response; MR, minor reponse; NR, no response.

tion I 1 II 3 III 4 IV 1 Efficacy CR 4 PR 1 MR 1 NR 3 Complications Vomiting, nausea 6 Upper abdominal pain 4 Melena 2 Enteric infection 1 Jaundice 1 Electrolytes disorder 1 Mean stent potency 53.4 day Survival 10-15 day 3 1-2 mo 2 >3 mo 4 CR, complete response; PR, partial response; MR, minor reponse; NR, no response. Procedural complications Post procedural jaundice and electrolytes disorder occurred in one patient and this patient died 10 days after the procedure. Two patients with nausea and vomiting were given gastrointestinal decompression, approximately 1800 ml was drained daily, these two patients deteriorated quickly with intractable electrolytes disorder 15 days after stent placement, then the patients refused further treatment. Vomiting and nausea occurred in a patient with gastric antral cancer invaded the gastric corpus, a second stent was placed in this patient. All patients were followed up for 3 months, there were no stent migration, removal and occlusion (Table 2). Discussion The incidence of gastric cancer in China is very high, a quite number of patients are in progressive stage with extra-gastric metastasis and are inoperable. In addition, some elderly patients can not be operated due to the severe cardiac-pulmonary disorders and diabetes mellitus. Therefore, in all of these patients, only the conservative treatments are considered.

ry high, a quite number of patients are in progressive stage with extra-gastric metastasis and are inoperable. In addition, some elderly patients can not be operated due to the severe cardiac-pulmonary disorders and diabetes mellitus. Therefore, in all of these patients, only the conservative treatments are considered. For some relapsed patients post surgery, jejunostomy is sometimes performed for nutritional sustaining, these patients can not take alimentation orally, thus leading to a poor life quality for the discomfort of carrying a feeding tube. There have been reports about using memory alloy stent to treat gastrointestinal obstruction(4). Janusdhowski(5), pinto(6) and Nevitt(1) firstly reported gastric and duodenal stent placements. Scott-Mackie(7) et al modified the stent delivery system by employing stiff guidewire and specially designed stent introducer, consequently, the successful rate of internal duodenal stent placement perorally increased, this established the stent placement method for a distant gastrointestinal tract site.

nt placements. Scott-Mackie(7) et al modified the stent delivery system by employing stiff guidewire and specially designed stent introducer, consequently, the successful rate of internal duodenal stent placement perorally increased, this established the stent placement method for a distant gastrointestinal tract site. Due to the long and tortuous route from mouth to duodenum, and the storage and buffering effect of stomach, when patients are aware of symptoms, the malignant gastric outlet obstruction is usually very severe; moreover, the intestinal route becomes more tortuous, all these changes make it harder to place the internal stent. In addition, the insufficient length of stent delivering system and the anatomic variation of patients make the stent placement more difficult. There have been studies on the modification of the stent delivery system in order to solve these problems, Feretis(2) et al introduced the stent with aid of a stabilizing overtube in the treatment of gastric malignant obstruction, the stent was supported as it was being advanced through the stricture, this prevented the delivery system from folding inside stomach. In recent years, the techniques of expandable metallic stent (EMS) placement originally used in the interventional cardiovascular has been adopted in the treatment of gastroduodenal malignant obstruction, its pliable delivery system has the merits of easily passing through the tortuous gastrointestinal tract, without the disadvantages of covered or uncovered EMS. Currently, there are two approaches for the gastrointestinal stent placement.

In recent years, the techniques of expandable metallic stent (EMS) placement originally used in the interventional cardiovascular has been adopted in the treatment of gastroduodenal malignant obstruction, its pliable delivery system has the merits of easily passing through the tortuous gastrointestinal tract, without the disadvantages of covered or uncovered EMS. Currently, there are two approaches for the gastrointestinal stent placement. Firstly, stent is placed via anatomical entrance or the surgical fistula under fluoroscopy. For the complete strictures, the guidewire can not be introduced through endoscope, the thermal modalities, such as microwave or heating pole via endoscopy under guidance of fluoscopy, were used to produce a small tunnel, then the guidewire and stent were introduced. When the stent was deployed, the expansion of the stent can be adjusted through internal dilation by a balloon catheter or through changing stent location. Secondly, stent placement under endoscopy. By this approach, the range and location of strictures can be observed precisely, therefore the appropriate size and shape of the stent can be selected. In addition, when procedural related hemorrhage, stent migration or perforation occur, prompt and proper measures can be taken under endoscopy.

cement under endoscopy. By this approach, the range and location of strictures can be observed precisely, therefore the appropriate size and shape of the stent can be selected. In addition, when procedural related hemorrhage, stent migration or perforation occur, prompt and proper measures can be taken under endoscopy. In this report, we performed stent placement successfully in 9 patients with malignant gastric outlet obstruction, the patients alimentation and quality of life were significantly improved, the total efficacy rate was 66.7%, the significant efficacy was achieved in the 6 patients with gastric antrum cancer or antrum-pylorus invasion cancer. However, in two patients with gastric corpus-antrum-pylorus invasion cancer, with peritoneal metastasis and marked acites, stent placement did not yield satisfactory results. We speculated that the efficacy is closely associated with the stent location and range of lesions. From our 9 patients reported herein, we summarized our technical experiences as follows.

In this report, we performed stent placement successfully in 9 patients with malignant gastric outlet obstruction, the patients alimentation and quality of life were significantly improved, the total efficacy rate was 66.7%, the significant efficacy was achieved in the 6 patients with gastric antrum cancer or antrum-pylorus invasion cancer. However, in two patients with gastric corpus-antrum-pylorus invasion cancer, with peritoneal metastasis and marked acites, stent placement did not yield satisfactory results. We speculated that the efficacy is closely associated with the stent location and range of lesions. From our 9 patients reported herein, we summarized our technical experiences as follows. Firstly, the size of stent and introducer should be selected according to the distance between the stricture and the central incisor tooth, these stents and introducers can be specially designed by the manufactures. Because the malignant stricture of the gastric outlet is distant from the incisor tooth, and the dilated proximal lumen, the guidwire and the stent delivery system tend to fold inside the stomach due to lacking of proper support, the delivery system with guidewire usually can not be advanced when reaching somewhere in the greater curvature. To overcome this difficulty, we advanced endoscope under the stent delivery system, when the endoscope reached the gastric fundus and corpus, we rotated the endoscope 90 degrees rightwards, then the stent delivery system was further advanced, due to the rotation of the endoscope, the stent delivery system can enter duodenum along the lesser curvature rather than the greater curvature.

delivery system, when the endoscope reached the gastric fundus and corpus, we rotated the endoscope 90 degrees rightwards, then the stent delivery system was further advanced, due to the rotation of the endoscope, the stent delivery system can enter duodenum along the lesser curvature rather than the greater curvature. Secondly, pre-dilation with balloon catheter was performed in severe stricture. In one patient, due to the severe stricture of the gastric outlet, the endoscope could not be passed through the stricture, and the guidewire could not be placed. In this case, we performed pre-dilation with a 2 cm balloon, then inserted the endoscope and the guidewire, finally the stent was deployed with aid of guidewire. Thirdly, the sent release site should be lower rather than higher. The stent can be adjusted upwards easily by pulling the stent, whereas the placed stent is hard to be adjusted downwards, if this occurs, the stent must be retrieved and placed again.

Secondly, pre-dilation with balloon catheter was performed in severe stricture. In one patient, due to the severe stricture of the gastric outlet, the endoscope could not be passed through the stricture, and the guidewire could not be placed. In this case, we performed pre-dilation with a 2 cm balloon, then inserted the endoscope and the guidewire, finally the stent was deployed with aid of guidewire. Thirdly, the sent release site should be lower rather than higher. The stent can be adjusted upwards easily by pulling the stent, whereas the placed stent is hard to be adjusted downwards, if this occurs, the stent must be retrieved and placed again. Fourthly, the indications, contradictions and complications of stent placement in the malignant stricture of gastric outlet should be emphasized. In our 9 patients, the most common complications were nausea, vomiting, upper gastrointestinal hemorrhage, upper abdominal pain. Six patents complicated with post-precedural nausea and vomiting, these symptoms spontaneously relieved 2 - 3 days after stent placement, of these 6 patients, 2 patients with gastric corpus or antrum invasion cancer (one patient was 85 years old, the other was with peritoneal metastasis and abundant ascites) had significant nausea and vomiting, gastrointestinal decompression was performed and approximate 1800 ml liquid was drained daily, we speculated that this was due to the extensive lesion of gastric cancer, gastrointestinal dysfunction, gastric juice over-excretion, reduced gastric peristalsis, and loss of anti-reflux function of pylorus, all these factors lead to the reflux of intestinal contents. Therefore, we concluded that it is contradicted to stent placement in patients with gastric corpus, antrum and pylorus invasion cancer, with peritoneal cavity metastasis and ascites. In one patient, the upper abdominal pain and persistent jaundice occurred, this was because the distal end of stent suppressed the duodenal ampulla and blocked bililary drainage, in this case, the stent should be adjusted upwards, however the patient refused further interventional treatment. The suitable indications should be further studied in more patients.

and persistent jaundice occurred, this was because the distal end of stent suppressed the duodenal ampulla and blocked bililary drainage, in this case, the stent should be adjusted upwards, however the patient refused further interventional treatment. The suitable indications should be further studied in more patients. We performed stent placement successfully in the first attempt in all 9 patients, we concluded that the stent placement under endoscopy is simple, direct, without exposure of X-ray, time-saving, and less traumatic, however, it needs precise localization and adequate maneuvers. Clinical data with more patients should be studied to clarify the indications and improve efficacy, and to prevent severe complications. Acknowledgement The authors declare no conflict of interest or commercial affiliation.

Despite significant advances in systemic therapies, pancreatic cancer remains an often incurable disease; the need for additional therapeutic strategies is hoped to be answered by the way of a better understanding of cellular processes involved in pancreatic cancer. The tyrosine kinase epidermal growth factor receptor (EGF-R) and its ligands are expressed in pancreatic cancer tissues, and this coexpression seems to be correlated with tumour progression [1]. Due to drug costs and to the fear of side effects, it was proposed to select subgroups of patients whose tumours express EGF-R for clinical trial with EGF-R inhibitors [2-4]. In the present work, we investigated the EGF-R expression in 13 patients undergoing pancreatic resection for cancer, including 7 males and 6 females, with middle age 68.2 years; nodal metastases were found in 6 cases, microscopic vascular infiltration in 10 cases and perineural infiltration in 11 cases. The average survival time was 16.9 months (range 10-28 months).

xpression in 13 patients undergoing pancreatic resection for cancer, including 7 males and 6 females, with middle age 68.2 years; nodal metastases were found in 6 cases, microscopic vascular infiltration in 10 cases and perineural infiltration in 11 cases. The average survival time was 16.9 months (range 10-28 months). Two different methods of investigation have been used to target the EGF-R: immunohistochemistry (IHC), aimed to evaluate EGF-R protein expression, and fluorescence in situ hybridization (FISH), aimed to evaluate EGF-R gene amplification. IHC was done with the DAKO® kit, employed with little differences from the manufacturer’s recommendations. Slides were deparaffinized and rehydrated in graded solutions of ethanol and distilled water. Endogenous peroxidase was blocked, followed by sequential application of EGF-R primary antibody for 60 minutes and of NovoLink Polymer for 30 minutes (NovoLink Polymer Detection System®, Novocastra Laboratories, Newcastle, UK). The immunoprecipitate was visualized by treatment with chromogen for 10 minutes and counterstained by hematoxylin. The expression was evaluated by two observers, following the scoring system suggested by the FDA guidelines (3+, complete and intense membrane staining of >10% tumor cells; 2+, complete but moderate staining of >10% cells; 1+, weak and incomplete staining in >10% cells; 0, no membrane staining, or staining in <10% cells). For the FISH technique, formalin-fixed, paraffin-embedded tissues were cut at 3 mm, mounted on charged slides, and dried. Slides were submitted to enzymatic digestion with 0.005% pepsine (Roche, Germany) for 30 minutes, followed by washing in phosphate buffer saline and fixing by a 10 minutes passage in methanol and acetic acid (3:1 ratio). Fluorochrome-marked DNA probes were employed. The probe for EGFR (Her1/c-erbB; chromosome 7p12) was marked with Spectrum Orange (Vysis Olympus - Milan, Italy), while the centromeric one alpha-satellite, used to look at the centromer of the chromosome 7 (7p11.1-q11.1), was marked with Spectrum Green (Kit PathVysion, Vysis Olympus - Milan, Italy). Target DNA and the probe do pair at the annealing temperature of 37°C overnight in a dark room; after the post-hybridization washing by the detergent NP40 pH 7.00 (Nonidet 40 - Vysis Olympus - Milan, Italy), and coloration by 4,6-diamidino-2-phenylindale (DAPI), the fluorescent microscope (Nikon Optiphot-2) is finally employed for the evaluation.

pair at the annealing temperature of 37°C overnight in a dark room; after the post-hybridization washing by the detergent NP40 pH 7.00 (Nonidet 40 - Vysis Olympus - Milan, Italy), and coloration by 4,6-diamidino-2-phenylindale (DAPI), the fluorescent microscope (Nikon Optiphot-2) is finally employed for the evaluation. The gene is considered amplified when the ratio between the number of gene signals and its centromer is greater than 2. So, in a cell with a normal number of EGF-R gene copies and of the relative centromer, we will observe respectively 2 orange spots and 2 green spots. EGF-R protein was expressed by IHC in 7 out of 13 cases, both independent observers concording; in 2 cases the expression was graded +2/+3, in the remaining 5 it was +1. EGF-R gene was amplified in no case. Thus, a mismatch between HIC and FISH was evident from our analysis in almost 7 cases. EGF-R protein expression was evident in 53.8% of cases, and the EGF-R gene was amplified in 0% of cases.

rvers concording; in 2 cases the expression was graded +2/+3, in the remaining 5 it was +1. EGF-R gene was amplified in no case. Thus, a mismatch between HIC and FISH was evident from our analysis in almost 7 cases. EGF-R protein expression was evident in 53.8% of cases, and the EGF-R gene was amplified in 0% of cases. IHC is a technique that, despite its low cost and simple concept, may give divergent results in different series, mainly because of different sensitivity and specificity of the commercially available antibodies, the different systems of tissue processing, the lack of a universal standard of interpretation and the observers’ subjectivity in assessing the results. These problems are confirmed in the present study, as in 8/13 cases a different immunohistochemical score was attributed in the same case by two observers (despite using the same technique and the same microscope). However, this should not explain the absence of correlation between the expression of the protein and the gene amplification in the 7 cases in which the protein was shown to be expressed; a possible explanation may be that other factors, different from the gene amplification, such as genetic mutations or alternative signalling pathways [4], are involved in the receptor activation. This hypothesis introduces some doubts in the field of EGF-R targeted genetic therapy of pancreatic cancer; the reported 0% amplification gene rate seriously questions the more obvious mechanism of EGF involvement in the carcinogenic process.

alternative signalling pathways [4], are involved in the receptor activation. This hypothesis introduces some doubts in the field of EGF-R targeted genetic therapy of pancreatic cancer; the reported 0% amplification gene rate seriously questions the more obvious mechanism of EGF involvement in the carcinogenic process. Further studies are needed to better understand the role of EGF-R in pancreatic cancer. By now, targeted therapies based on EGF-R inhibitors such as Erlotinib should not be decided only on the basis of IHC detection of an over expressed EGF-R. Acknowledgement The authors declare no conflict of interests

Introduction Chylous ascites is defined as the accumulation of triglyceride-rich, milk-like peritoneal fluid in the abdominal cavity. Most chylous disorders are rare and commonly due to secondary conditions. Once secondary causes are excluded, primary chylous disorders are often suspected. Primary chylous disorders represent a diagnostic and therapeutic challenge; they can be congenital or present later in life. In this article we report a refractory case of primary chyloperitoneum treated with sclerotherapy and a lymphovenous shunt. Case Report A 34- year- old woman from the north of Mexico presented nonspecific abdominal complaints one month before she developed new-onset tense ascites. Her medical history included a hemithyroidectomy 12 years earlier due to a papillary thyroid cancer, an abdominal myomectomy 4 years ago, and a cesarean section done 4 months prior to admission to her community hospital. An abdominal paracentesis was done, revealing a milky fluid. Diagnostic study was extensive, but etiology for chylous ascites could not be established even after laparoscopic examination. One month later she was referred to our institution for diagnostic and treatment.

ma and squamous cell carcinoma in 8/18, a combination of basaloid squamous cell carcinoma and adenocarcinoma in 3/18 cases, a combination of basaloid squamous cell carcinoma and small cell carcinoma in 2/18 cases. The review of esophageal carcinoma with multiple differentiation is mostly limited to two differentiation. Esophageal carcinoma with more than three differentiation as seen in the present study, is extremely rare. In summary, the author presented an extremely rare case of esophageal carcinoma with triplicate differentiation (squamous cell carcinoma, small cell carcinoma, and adenocarcinoma. The author speculates that the tumor is basically small cell carcinoma with squamous and adenocarcinomatous differentiation, or that the present esophageal carcinoma arises from totipotent stem cell of the esophagus. Acknowledgements The author declares no conflicts of interest related to this report.

nths prior to admission to her community hospital. An abdominal paracentesis was done, revealing a milky fluid. Diagnostic study was extensive, but etiology for chylous ascites could not be established even after laparoscopic examination. One month later she was referred to our institution for diagnostic and treatment. Laboratory workup showed normal complete blood count (CBC), liver transaminases, and urinalysis; serum albumin of 1.8g/dL, ascites-triglyceride concentration of 2291mg/dL, serum-ascites albumin gradient (SAAG) <1.1g/dL, and negative ascitic cytology and cultures. Abdominal ultrasound (US), computed tomography (CT), and magnetic resonance (MRI) showed ascites and a hepatic hemangioma (Fig.1). Chest CT was normal, and the patient was negative for Mantoux test and adenosine deaminase (ADA) in ascitic fluid. Biopsy specimens from her referral hospital showed chronic peritonitis and dilated lymphatics (Fig.2). The patient was started on a low-fat diet supplemented with medium-chain triglycerides (MCT) and abdominal paracentesis as needed (10Lt/week). Figure 1 MRI showing massive ascites and a hepatic hamangioma. Figure 2 Peritoneum showing dilated lymphatics with normal CD34 positive endothelium.

Laboratory workup showed normal complete blood count (CBC), liver transaminases, and urinalysis; serum albumin of 1.8g/dL, ascites-triglyceride concentration of 2291mg/dL, serum-ascites albumin gradient (SAAG) <1.1g/dL, and negative ascitic cytology and cultures. Abdominal ultrasound (US), computed tomography (CT), and magnetic resonance (MRI) showed ascites and a hepatic hemangioma (Fig.1). Chest CT was normal, and the patient was negative for Mantoux test and adenosine deaminase (ADA) in ascitic fluid. Biopsy specimens from her referral hospital showed chronic peritonitis and dilated lymphatics (Fig.2). The patient was started on a low-fat diet supplemented with medium-chain triglycerides (MCT) and abdominal paracentesis as needed (10Lt/week). Figure 1 MRI showing massive ascites and a hepatic hamangioma. Figure 2 Peritoneum showing dilated lymphatics with normal CD34 positive endothelium. Initial treatment did not prove useful; therefore, total parenteral nutrition (TPN) and octreotide acetate depot (20mg IM) were started. After a four-week period of intensive diagnostic study, no clear etiology for the chylous disorder was established. Lymphatic anatomy was evaluated with a whole-body lymphangioscintigraphy (LAS) that showed normal tracer distribution. Surgical exploration was planned to clarify the etiology and offer operative management.

er a four-week period of intensive diagnostic study, no clear etiology for the chylous disorder was established. Lymphatic anatomy was evaluated with a whole-body lymphangioscintigraphy (LAS) that showed normal tracer distribution. Surgical exploration was planned to clarify the etiology and offer operative management. Sudan Black B (5 grams) plus a fat-rich diet were administered eight hours before surgery. Laparoscopic examination did not prove useful, so it was converted to open exploration. Lymphangiectasias and a lymphatic leak from retroperitoneum were saw at rectouterine pouch (pouch of Douglas). Primary closure with sutures and fibrin glue was done (Fig.3). Fourteen days after surgery vaginal discharge was noted (200 ml/day). Colposcopic exploration showed copious cervical discharge similar to the ascitic fluid. A new surgical approach was planned. Figure 3 Intraoperative picture showing lymphangiectasia and a lymphatic leak (chylous effusion is stained with Sudan Black B).

Sudan Black B (5 grams) plus a fat-rich diet were administered eight hours before surgery. Laparoscopic examination did not prove useful, so it was converted to open exploration. Lymphangiectasias and a lymphatic leak from retroperitoneum were saw at rectouterine pouch (pouch of Douglas). Primary closure with sutures and fibrin glue was done (Fig.3). Fourteen days after surgery vaginal discharge was noted (200 ml/day). Colposcopic exploration showed copious cervical discharge similar to the ascitic fluid. A new surgical approach was planned. Figure 3 Intraoperative picture showing lymphangiectasia and a lymphatic leak (chylous effusion is stained with Sudan Black B). Prior to resection of retroperitoneal, mesenteric, and pelvic lymphatics, sclerotherapy of dilated vessels was done. Iodine (60 ml) and sterile talc (4 grams) were diluted in 100 ml of normal saline and irrigated directly to provoke obstructive lymphangitis. A side to side lymphovenous shunt to the left ovarian vein was made from a large lymphatic channel (4 mm) located below the renal arteries. Postoperative evolution was favorable, but six months later the patient recurred with non-tense chylous ascites. A third surgery revealed thrombosis of the lymphovenous shunt, with no evidence of large lymphatic channels. Sclerotherapy with iodine and talc was performed again. The patient has been without evidence of ascites for eight months.

was favorable, but six months later the patient recurred with non-tense chylous ascites. A third surgery revealed thrombosis of the lymphovenous shunt, with no evidence of large lymphatic channels. Sclerotherapy with iodine and talc was performed again. The patient has been without evidence of ascites for eight months. Discussion Chylous ascites is a rare disorder. At large reference centers, its incidence represents around one in 20,000 admissions over a 20-year period(1). The incidence is increasing, perhaps because of more aggressive retroperitoneal surgery treatments and longer survival for cancer patients(1, 2). Diagnostic paracentesis is most important in any patient with ascites. The serum-ascites albumin gradient (SAAG) calculation is the first diagnostic approach and a useful portal pressure prognosticator(3). Chylous ascites is suspected by gross analysis, but definitive diagnosis is established by measuring ascites-triglyceride concentration, which is usually above 110-200 mg/dL(1-4). After confirmation of a chylous disorder, most important step in clinical practice is to look for its etiology. In Western countries approximately 75% are malignancy or cirrhosis related. Lymphomas account for 33-50% of all cases; breast, colon, and ovarian cancer are also associated(1). Infectious diseases such as tuberculosis and filariasis are common causes in developing countries(2).

cal practice is to look for its etiology. In Western countries approximately 75% are malignancy or cirrhosis related. Lymphomas account for 33-50% of all cases; breast, colon, and ovarian cancer are also associated(1). Infectious diseases such as tuberculosis and filariasis are common causes in developing countries(2). Treatment for chylous ascites starts with dietary modifications. High protein, low-fat and MCT supplementation successfully resolves lymphatic leaks in up to 50% of cases(4, 5). If no improvement is noticed, second line therapies (TPN, octreotide) should be started. Resolution rates for these therapies had been reported from 60 to 100%(4, 6). First and second line therapies failed for this patient. LAS was performed in order to define lymphatic anatomy and the site of the lymphatic leak, but it did not prove useful. Even after a thorough investigation no signs of cancer, cirrhosis or infection were demonstrated. Less common causes of chylous ascites include congenital, inflammatory, traumatic, and postoperative disorders. Postoperative chylous effusions result from injury to the thoracic duct, cisterna chyli or its intestinal tributaries. It can occur from a few days after surgery (lymphatic disruption) up to several months later (adhesions)(1). No case has been reported associated to cesarean section(7).

First and second line therapies failed for this patient. LAS was performed in order to define lymphatic anatomy and the site of the lymphatic leak, but it did not prove useful. Even after a thorough investigation no signs of cancer, cirrhosis or infection were demonstrated. Less common causes of chylous ascites include congenital, inflammatory, traumatic, and postoperative disorders. Postoperative chylous effusions result from injury to the thoracic duct, cisterna chyli or its intestinal tributaries. It can occur from a few days after surgery (lymphatic disruption) up to several months later (adhesions)(1). No case has been reported associated to cesarean section(7). Next step in diagnosis and treatment is surgical exploration. Preparation included a fat-rich diet and a lipophilic dye to depict lymphatic anatomy(8, 9). Surgery main findings were a lymphatic leak and dilated vessels; therefore, the most probable diagnosis is a primary chylous disorder. Primary chylous disorders are rare, and most of them are caused by congenital lymphangiectasia. They can be accompanied with obstruction, agenesis, hypoplasia, disruption or dysplasias(5, 8). Less frequent causes include the Yellow nail syndrome and Lymphangioleiomyomatosis(1, 2).

Next step in diagnosis and treatment is surgical exploration. Preparation included a fat-rich diet and a lipophilic dye to depict lymphatic anatomy(8, 9). Surgery main findings were a lymphatic leak and dilated vessels; therefore, the most probable diagnosis is a primary chylous disorder. Primary chylous disorders are rare, and most of them are caused by congenital lymphangiectasia. They can be accompanied with obstruction, agenesis, hypoplasia, disruption or dysplasias(5, 8). Less frequent causes include the Yellow nail syndrome and Lymphangioleiomyomatosis(1, 2). During the first surgery, primary closure of lymphatic leak was attempted(9). Success for this therapy has been described in about 80% of cases (8), but chylocolporrhea demonstrated that the problem persisted. A multidisciplinary team discussed the remaining therapeutic alternatives, and a second surgical approach was planned. New surgery objectives were sclerotherapy, resection of lymphangiectasic tissue, and a derivative resolution(10). Lymphovenous and peritoneovenous (Denver) shunts were both considered(10, 11), but lymphatic characteristics made feasible a lymphovenous shunt. Postoperative evolution was favorable, but after 6 months the problem recurred. A new surgery was done, no dilated lymphatics were observed and retroperitoneal sclerotherapy was performed. Eight months later, the patient is free of symptoms, but there is a great concern about a new recurrence.

ble a lymphovenous shunt. Postoperative evolution was favorable, but after 6 months the problem recurred. A new surgery was done, no dilated lymphatics were observed and retroperitoneal sclerotherapy was performed. Eight months later, the patient is free of symptoms, but there is a great concern about a new recurrence. Conclusions: Primary chylous disorders are rare and complex conditions that represent a diagnostic and therapeutic challenge. Almost half of these patients will require surgery, and their outcome significantly depend on the medical experience and technology available(5, 8, 10). Proper follow up is important, because one third of patients with primary chylous ascites will recur even when proper treatment has been instituted(5). Acknowledgement The authors declare no commercial associations or conflict of interest related to this article.

Introduction Up to now, orthotropic liver transplantation (OLT) is still the most effective treatment of a variety of life-threatening liver diseases, however, due to the shortage of donors, the number of patients who can benefit from this modality is very limited(1, 2). In recent years, cell based therapies have been investigated as alternatives to whole liver transplantation. There are some advantages of cellular transplantation over whole liver transplant. A single donor liver could be shared by multiple patients; it is less invasive, procedures are safer and easier to manage, multiple treatments can be carried out on a single patient(3); only 1-5 percent of the total hepatocytes mass transplantation could correct a variety of metabolic disorders(1); the hepatocytes can be manipulated in vitro easily, such as genetic manipulation(3, 4). This brief review summarizes some highlights in the cellular transplantation in the treatment of liver diseases, such as the source of cells, routes of transplantation, and indications of transplantation. Potential clinical applications of cellular transplantation for liver diseases Bridging patients with liver failure to whole liver transplantation This is to improve the patient’s condition and extend short-term survival until a donor liver becomes available or until the patient’s own liver recovers(3, 5, 6). Previous clinical observations using liver cell transplantation for the hepatic failure showed that the patients’ condition was transiently improved and thus successfully bridged to OLT(5, 7).

dition and extend short-term survival until a donor liver becomes available or until the patient’s own liver recovers(3, 5, 6). Previous clinical observations using liver cell transplantation for the hepatic failure showed that the patients’ condition was transiently improved and thus successfully bridged to OLT(5, 7). Correct of inborn errors of metabolism The studies of cellular transplantations for inborn errors of metabolism have been conducted both in animal models(8-14) and in humans(15-18). These animal models with genetic liver defects include Gunn rat, which lacks hepatic bilirubin UDP-glucuronosyl transferase actificty (UGT1A1), and the Nagase analbuminemic rat model(19, 20). Allogeneic hepatocytes transplantation was used as a treatment for inherited glycogen storage type 1a(21) and the crigler-Najjar syndrome type I(16). Liver failure and cirrhosis When patients with acute liver failure were transplanted allogeneic hepatocytes, clinical improvement was observed though the donor hepatocytes constitute approximately 1% to 10% of the liver mass. However, there was no survival improvement in the extremely sick patients(6).

Correct of inborn errors of metabolism The studies of cellular transplantations for inborn errors of metabolism have been conducted both in animal models(8-14) and in humans(15-18). These animal models with genetic liver defects include Gunn rat, which lacks hepatic bilirubin UDP-glucuronosyl transferase actificty (UGT1A1), and the Nagase analbuminemic rat model(19, 20). Allogeneic hepatocytes transplantation was used as a treatment for inherited glycogen storage type 1a(21) and the crigler-Najjar syndrome type I(16). Liver failure and cirrhosis When patients with acute liver failure were transplanted allogeneic hepatocytes, clinical improvement was observed though the donor hepatocytes constitute approximately 1% to 10% of the liver mass. However, there was no survival improvement in the extremely sick patients(6). The causes of liver disease often involve exposure to extrinsic agents, especially drugs or environmental toxins, which induce acute to subacute hepatocellular disease(3). The thearapeutic significance of hepatocytes transplantation is likely to be disease context-dependent. In the D-galactosamine model, there was increased survival after transplantation of hepatocytes subcellular fractions, culture supernatants or even bone marrow cells, these results indicate that the early therapeutic effect requires only cell-derived factors(22-24) for the first 3-5 days after induction of acute liver failure. After this period, the transplanted hepatocytes will repopulate and proliferate with reorganization to restore liver function, and reproduce fully normal hepatic tissue, this process needs several weeks or months(3), thus, it is of significance for the treatment of chronic liver disease, such as liver cirrhosis(25).

e. After this period, the transplanted hepatocytes will repopulate and proliferate with reorganization to restore liver function, and reproduce fully normal hepatic tissue, this process needs several weeks or months(3), thus, it is of significance for the treatment of chronic liver disease, such as liver cirrhosis(25). Types of cells for transplantation in liver diseases Primary hepatocytes The adult primary hepatocytes were used commonly in experimental and pre-clinical studies(26). These cells include allogeneic, xenogeneic or autologus primary isolated hepatocytes. Primary xenogenic hepatocytes seems to be an unlimited source of hepatocytes and can solve the human donor shortage(27-30). When the porcine hepatocytes transplantated into a rat cirrhotic model, it can correct liver function(29). However, using xenogenic hepatocytes transplantation may incur some concerns, such as xenozoonoses, xenoantifenicity(31), and immunorejection, host versus graft reaction(32).

solve the human donor shortage(27-30). When the porcine hepatocytes transplantated into a rat cirrhotic model, it can correct liver function(29). However, using xenogenic hepatocytes transplantation may incur some concerns, such as xenozoonoses, xenoantifenicity(31), and immunorejection, host versus graft reaction(32). Immortalized cell lines The immortalized heaptocytes are derived from gene transfer(32-48) in vitro. The conditionally immortalized rat hepatocytes by transduction using the simian virus 40 T antigen(SV40 Tag) can lower the risk of transplanted hepatocyte malignant transformation(49), because after excision of the SV40Tag, these immortalized hepatocytes can stop growing and the differentiated characteristics are enhanced(50). These conditionally immortalized cell lines transplantation in rats with end-stage liver failure can provide life-supporting liver-specific metabolic function(49). When immortalized heaptocytes isolated from P19ARF null mice were intrasplenically transplanted into Fas-injured liver of SCID mice (severe combined immunodeficiency), the cells participated the liver repopulating and regenerating process, and were capable of generating hepatic progenitor cells during liver restoration(51).

immortalized heaptocytes isolated from P19ARF null mice were intrasplenically transplanted into Fas-injured liver of SCID mice (severe combined immunodeficiency), the cells participated the liver repopulating and regenerating process, and were capable of generating hepatic progenitor cells during liver restoration(51). Hepatocyte-like cells derived from adult bone marrow stem cells Previous studies showed that bone marrow cells contains a subpopulation of hepatocytes-orientated stem cells expressing a-AFP, c-met, CD34 and c-kit(31, 52, 53). These cells in vitro and in vivo can differentiated into hepatocytes(54-56) with the capacity of reversing liver failure(56), and synthesizing albumin in the lethally irradiated mice(57). Hepatocyte-like cells derived from embryonic stem cells The embryonic stem cells can differentiate into hepatocytes in vitro(58-60) and in vivo(61, 62). The embryonic stem cells could differentiate into hepatocytes under specific culture conditions. Mouse embryonic stem cells were able to grow and showed morphology consistent with typical mature hepatocytes and expressed hepatocytes specific genes, when these cells transplanted into the carbon tetrachloride-injured mouse liver, they were able to incorporated into liver tissue and rescued mice from hepatic injury(63). The embryonic stem cells derived hepatocytes have been found to be effective in a liver failure mouse model induced by diphtheria toxin(64).

genes, when these cells transplanted into the carbon tetrachloride-injured mouse liver, they were able to incorporated into liver tissue and rescued mice from hepatic injury(63). The embryonic stem cells derived hepatocytes have been found to be effective in a liver failure mouse model induced by diphtheria toxin(64). Routes of cell transplantation Intraperitoneal injection The peritoneal cavity is a commonly used site for cell implantation(65). The peritoneal cavity has a relatively large space into which a remarkable number of cells can be transplanted. Intrahepatic transplantion The intrahepatic transplantation of normal hepatocytes can prevent Wilson's disease in these LEC rats(66). In another study, the intrahepatic transplantation of human hepatocytes in immunodeficient, fumarylacetoacetate hydrolase-deficient (fah(-/-) mice, three months after transplantation, about 20% of the mouse liver was observed repopulated by human hepatocytes, and sustained expression of lentiviral vector transduced gene(67). Portal veinous infusion Hepatocytes can be seeded into the liver by portal vein infusion and the transplanted hepatocytes can integrate into the liver cords, leaving the hepatic architecture intact(68). Hepatocytes engrafted in the liver have the benefits of exposure to the portal nutrients, contacting with other hepatocytes and nonparenchymal cells, thus have the ability to secrete bile into the indigenous biliary system (33).

epatocytes can integrate into the liver cords, leaving the hepatic architecture intact(68). Hepatocytes engrafted in the liver have the benefits of exposure to the portal nutrients, contacting with other hepatocytes and nonparenchymal cells, thus have the ability to secrete bile into the indigenous biliary system (33). Intrasplenic infusion Intrasplenic transplantation is another route of cell delivery for the liver diseases. Previous studies showed that intrasplenic transplantation of hepatocytes increased survival of the totally hepatectomized rats(69). The intrasplenic transplantation of human fetal immortal hepatocytes could prolong survival of 90% hepatectomized rats(70). After massive hepatectomy, such as in 90% PH, or under the situation that the diseased liver is not a suitable site for cell transplantation, the intrasplenic cell transplantation may become an ideal alternative. Hepatocytes can be injected into the splenic pulp from which most cells translocate to the liver through the splenic vein, a small fraction of the cells engraft within the spleen and can develop into bile cannuliculae, sinusoidal structures, and endothelial cells, producing a similar structure to the liver(71, 72).

ve. Hepatocytes can be injected into the splenic pulp from which most cells translocate to the liver through the splenic vein, a small fraction of the cells engraft within the spleen and can develop into bile cannuliculae, sinusoidal structures, and endothelial cells, producing a similar structure to the liver(71, 72). Storage and availability of cells Isolated hepatocytes can be stored in a cryopreserved hepatocytes bank. Since human liver cells are sensitive to damage due to the freezing-thawing procedure, reliable procedures for long-term cryopreservation of human hepatocytes need to be investigated(1). The major concern of using heaptocytes is the lack of donor availability, in addition, the cryopreserved liver cells have not yet been shown to engraft as well as fresh hepatocytes(73). Immunorejection To solve the shortage of donor livers, researchers turn to the cells from animal sources, the animal source seems to be an unlimited supply. However, the xeno-transplantation can cause host immunorejection and the immunosuppressive agents must be used(10, 74).

Storage and availability of cells Isolated hepatocytes can be stored in a cryopreserved hepatocytes bank. Since human liver cells are sensitive to damage due to the freezing-thawing procedure, reliable procedures for long-term cryopreservation of human hepatocytes need to be investigated(1). The major concern of using heaptocytes is the lack of donor availability, in addition, the cryopreserved liver cells have not yet been shown to engraft as well as fresh hepatocytes(73). Immunorejection To solve the shortage of donor livers, researchers turn to the cells from animal sources, the animal source seems to be an unlimited supply. However, the xeno-transplantation can cause host immunorejection and the immunosuppressive agents must be used(10, 74). Considerations for future studies Though some positive results obtained in the cell based therapy for liver disease either in animal or clinical studies, there are, however, still some important theoretical and practical issues that need to be addressed. Firstly, whatever transplantation route employed, the cells engraftment and cell efficacy are crucial points(75), the functions of transplanted cells are closely related to the engraftment rate. The use of better quality cells and regeneration stimulus can improve the engraftment(76, 77). Secondly, if stem cell derived hepatocytes are used, these cells may have potential of malignant transformation. Thirdly, except the autologus or syngeneous cell sources, the allogeneic or xenogeneic cell transplantation needs co-administration of immunosuppressive agents which may aggravate or contraindicate the patients’ illness.

stem cell derived hepatocytes are used, these cells may have potential of malignant transformation. Thirdly, except the autologus or syngeneous cell sources, the allogeneic or xenogeneic cell transplantation needs co-administration of immunosuppressive agents which may aggravate or contraindicate the patients’ illness. Conclusions Cellular therapy is a promising approach for the liver diseases, although there are a number of issues unsolved. With the cellular and molecular biology advancement, it is hopeful that cell transplantation will be developed into a standard therapy for liver failure or inborn metabolic diseases in the future. Conflicts of Interest The authors declare no commercial associations or conflict of interest related to this article.

Introduction Many kinds of benign pathologic lesions occur in the esophagus. They include esophageal atresia, heterotopic gastric mucosa, heterotopic pancreatic tissue, diverticula, esophageal cyst, achalasia, Lye stricture, reflex esophagitis, Barrett’s esophagus, herpes simplex esophagistis, cytomegalovirus esophagitis, eosiphophilic esophagitis, Crohn’s disease, candidiasis, leiomyoma, glycogenic acanthosis, amyloidosis, squamous papilloma, hyperplastic polyp, and granular cell tumor [1, 2]. Recent advances in endoscopy have made it possible that these esophageal benign lesions are biopsied and diagnosed correctly. In the present study, the authors reviewed 931 archival cases of the esophageal biopsies in search for benign tumors of the esophagus. Materials and Methods The authors retrospectively reviewed consecutive 931 cases of esophageal biopsy specimens in the last 15 years in the pathology laboratory in our hospital. The ages of the patients ranged from 12 years to 95 years with a mean of 53 years. Male to female ratio was 547:384. Clinical and endoscopic records were also reviewed.

authors retrospectively reviewed consecutive 931 cases of esophageal biopsy specimens in the last 15 years in the pathology laboratory in our hospital. The ages of the patients ranged from 12 years to 95 years with a mean of 53 years. Male to female ratio was 547:384. Clinical and endoscopic records were also reviewed. Histochemical stainings including PAS and alcian blue were employed in appropriate cases. In appropriated cases, an immunohistochimical study was performed, using Dako Envision method (Dako Corp., Glostrup, Denmark), as previously described [3, 4]. The antibodies employed were anti-cytokeratin (AE1/3, Dako), anti-cytokeratin (polyclonal wide, Dako), anti-p53 protein (DO-7, Dako), anti-Ki-67 antigen (MIB-1, Dako), neuron-specific enolase (BBS/NC/VI-H14, Dako), CD34 (QBEND10, Dako), vimentin (Vim 3B4, Dako), desmin (D33, Dako), α-smooth muscle actin (1A4, Dako), S100 protein (polyclonal, Dako) KIT (polyclonal, Dako), and PDGFRA (Santa Cruz, CA, USA). Results Forty-one cases (4.4%) of squamous papilloma, 4 cases (0.4%) of granular cell tumor, 3 cases (0.3%) of leiomyoima, and 1 case (0.1%) tubular adenoma were identified.

Histochemical stainings including PAS and alcian blue were employed in appropriate cases. In appropriated cases, an immunohistochimical study was performed, using Dako Envision method (Dako Corp., Glostrup, Denmark), as previously described [3, 4]. The antibodies employed were anti-cytokeratin (AE1/3, Dako), anti-cytokeratin (polyclonal wide, Dako), anti-p53 protein (DO-7, Dako), anti-Ki-67 antigen (MIB-1, Dako), neuron-specific enolase (BBS/NC/VI-H14, Dako), CD34 (QBEND10, Dako), vimentin (Vim 3B4, Dako), desmin (D33, Dako), α-smooth muscle actin (1A4, Dako), S100 protein (polyclonal, Dako) KIT (polyclonal, Dako), and PDGFRA (Santa Cruz, CA, USA). Results Forty-one cases (4.4%) of squamous papilloma, 4 cases (0.4%) of granular cell tumor, 3 cases (0.3%) of leiomyoima, and 1 case (0.1%) tubular adenoma were identified. In the 41 cases of squamous papilloma, the age ranged from 35 years to 81 years with a mean of 51 years. Male to female ratio was 25:16. The presenting symptoms were asymptomatic in 32 cases, nausea in 4 cases, chest burn in 3 cases, and dysphasia in 2 cases. The locations of the 41 cases of squamous papilloma were the cervical esophagus in 6 cases, the proximal esophagus in 12 cases, the middle esophagus in 11 cases, and the distal esophagus in 12 cases. Squamous papilloma was endoscopically recognized as small polypoid tumor. The size of squamous pailloma ranged from 2 mm to 10 mm. Histologically, squamous papilloma was characterized in papillary proliferation of mature squamous epithelium (Fig 1). No koilocytosis was identified in this series. The squamous papilloma was immunohistochemically positive for various cytokeratins and negative for vimentin and other antigens examined.

m 2 mm to 10 mm. Histologically, squamous papilloma was characterized in papillary proliferation of mature squamous epithelium (Fig 1). No koilocytosis was identified in this series. The squamous papilloma was immunohistochemically positive for various cytokeratins and negative for vimentin and other antigens examined. Figure 1 Squamous papilloma of the esophagus. Papillary proliferation of mature squamous epithelium is seen. HE, x20 In the 4 cases of granular cell tumor, the ages of the patients were 36, 45, 67 and 78 years, and male to female ratio was 1:3. Two cases complained of dysphagia and two cases were asymptomatic. They were located in the proximal esophagus in 3 cases, and in the middle esophagus in 1 case. Granular cell tumor was endoscopically recognized by an elevated small lesion. The size ranged from 4 mm to 12 mm. Histologically, granular cell tumors were characterized by a proliferation of large cells with acidphilic granular cytoplasm (Fig 2a). Hyaline globules positive with PAS stains were recognized in many areas (Fig 2b). The granular cell tumors were immunohistochemically positive for vimentin, S100 protein (Fig 2c), and neuron-specific enolase (Figure 2d), and negative for cytokeratins and other antigens examined.

acidphilic granular cytoplasm (Fig 2a). Hyaline globules positive with PAS stains were recognized in many areas (Fig 2b). The granular cell tumors were immunohistochemically positive for vimentin, S100 protein (Fig 2c), and neuron-specific enolase (Figure 2d), and negative for cytokeratins and other antigens examined. Figure 2 Granular cell tumor of the esophagus. A: Histologic features. Large cells with acidophilic granular cytoplasm are seen. Many hyaline globules are also seen. HE, x200 B: The cytoplasm and hyaline globules are positive with PAS stain. PAS, x200 C: The tumor cells are positive for S100 protein. Immunostaining, x200 D: The tumor cells are positive for neuron-specific enolase. Immunostaining, x200. In the 1 case of tubular adenoma, the patient was 46 years old man. He was asymptomatic. The tubular adenoma was located in the distal esophagus. It was endoscopically a slightly elevated lesion. Histologically, it was composed of adenomatous proliferation (Fig 3a) reminiscent of gastric or colonic adenoma. Interestingly, it was associated with heterotopic gastric mucosa. Immunohistochemically, it was faintly positive for p53 protein (Fig 3b). The Ki-67 labeling was 26% (Fig 3c). Figure 3 Tubular adenoma of the esophagus. A: adenomatous tubules are seen. HE, x200 B: The tumor cells are very focally positive for p53 protein. Immunostaining, x100 C: The Ki-67 labeling is 26%. Immunostaining (MIB1), x200

In the 1 case of tubular adenoma, the patient was 46 years old man. He was asymptomatic. The tubular adenoma was located in the distal esophagus. It was endoscopically a slightly elevated lesion. Histologically, it was composed of adenomatous proliferation (Fig 3a) reminiscent of gastric or colonic adenoma. Interestingly, it was associated with heterotopic gastric mucosa. Immunohistochemically, it was faintly positive for p53 protein (Fig 3b). The Ki-67 labeling was 26% (Fig 3c). Figure 3 Tubular adenoma of the esophagus. A: adenomatous tubules are seen. HE, x200 B: The tumor cells are very focally positive for p53 protein. Immunostaining, x100 C: The Ki-67 labeling is 26%. Immunostaining (MIB1), x200 In the 3 cases of leiomyoma, the ages were 34, 45, and 85 years. Male to female ratio was 1:2. The leiomyoma were located in the cervical esophagus in 1 case and in the proximal esophagus in 2 cases. It was endoscopically recognized as a submucosal tumor. The sizes were 4mm, 6mm, and 12mm. Histologically, leiomyoma was characterized by well defined nodule (Fig 4a), composed of spindle smooth muscles (Figure 4b). No atypia or mitotic figures were identified. The leiomyoma was immunohistochemically positive for vimentin, α-smooth muscle actin, and desmin (1 case), but negative for cytokeratins, CD34, KIT, and PDGFRA. Figure 4 Leiomyoma of the esophagus. A: Low power view shows well-define nodule composed of acidophilic spindle cells. HE, x40 B: The tumor is composed of spindle cells considered as smooth muscle cells. HE, x200

In the 3 cases of leiomyoma, the ages were 34, 45, and 85 years. Male to female ratio was 1:2. The leiomyoma were located in the cervical esophagus in 1 case and in the proximal esophagus in 2 cases. It was endoscopically recognized as a submucosal tumor. The sizes were 4mm, 6mm, and 12mm. Histologically, leiomyoma was characterized by well defined nodule (Fig 4a), composed of spindle smooth muscles (Figure 4b). No atypia or mitotic figures were identified. The leiomyoma was immunohistochemically positive for vimentin, α-smooth muscle actin, and desmin (1 case), but negative for cytokeratins, CD34, KIT, and PDGFRA. Figure 4 Leiomyoma of the esophagus. A: Low power view shows well-define nodule composed of acidophilic spindle cells. HE, x40 B: The tumor is composed of spindle cells considered as smooth muscle cells. HE, x200 Discussion Benign tumors of the esophagus are rare, and most of them are not clinically important lesions. However, differential diagnosis is important. In addition, large benign tumors may obstruct the esophageal lumen, and treatment was difficult in such cases.

Figure 4 Leiomyoma of the esophagus. A: Low power view shows well-define nodule composed of acidophilic spindle cells. HE, x40 B: The tumor is composed of spindle cells considered as smooth muscle cells. HE, x200 Discussion Benign tumors of the esophagus are rare, and most of them are not clinically important lesions. However, differential diagnosis is important. In addition, large benign tumors may obstruct the esophageal lumen, and treatment was difficult in such cases. Squamous papilloma of the esophagus is a rare lesion. Most of the previously reported study was case reports and studies of small series [5-9]. The squamous papilloma of the esophagus does not transform into squamous cell carcinoma. In the present study, the frequency was 4.4%, and the size was small. The hot debate is whether or not the squamous cell carcinoma is caused by human papilloma virus. Some researchers insisted the association of squamous papilloma and human papilloma virus [5, 7, 8], but others denied the association [6]. In the present study, although no viral examination was performed, koilocytosis as seen in uterine cervix neoplasm and condyloma acuminatum was not recognized.

man papilloma virus. Some researchers insisted the association of squamous papilloma and human papilloma virus [5, 7, 8], but others denied the association [6]. In the present study, although no viral examination was performed, koilocytosis as seen in uterine cervix neoplasm and condyloma acuminatum was not recognized. Granular cell tumor of the esophagus is extremely rare. Only several case reports or studies of small series have been published [10-12]. Its important point is differential diagnosis. In general, acidophilic granular cytoplasm and immunoreactions for vimentin, S100 protein, and neuron-specific enolase are important points in this tumor. The present 4 cases fulfilled the diagnostic criteria of granular cell tumor. The frequency in the present study was 0.4%. Tubular adenoma of the esophagus is extremely rare. Only a few case reports are present in the literature [13]. In the present study, it is very interesting that the tubular adenoma was associated with heterotopic gastric mcuosa [14, 15]. Adenocarcinoma very infrequently develops in the heterotopic gastric mucosa [16, 17]. However, adenoma developing in heterotopic gastric mucosa has not been reported, to the author’s best knowledge. In the present study, it frequency was 0.1%.

Tubular adenoma of the esophagus is extremely rare. Only a few case reports are present in the literature [13]. In the present study, it is very interesting that the tubular adenoma was associated with heterotopic gastric mcuosa [14, 15]. Adenocarcinoma very infrequently develops in the heterotopic gastric mucosa [16, 17]. However, adenoma developing in heterotopic gastric mucosa has not been reported, to the author’s best knowledge. In the present study, it frequency was 0.1%. Three leiomyomas were recognized in the present study. Esophageal leiomyoma is rare but the most common mesenchymal tumor. In the literature, several case reports are recorded [18, 19]. Large leiomyoma may obstruct the esophageal lumen, causing serious problems. The most important points of esophageal leiomyoma are differential diagnosis from leiomyosarcoma and gastrointestinal stromal tumor (GIST). In making a diagnosis of leiomyoma, mitotic counts, cellular atypia, and necrosis were required. In the present case, no atypia or mitotic figures were present. Leiomyoma must be distinguished from GIST. In making a diagnosis of GIST, immunohistochemical demonstration of KIT and/or CD 34 is mandatory [20, 21]. In addition, genetic analysis of KIT and PDGFRA genes may be required in KIT-negative GIST cases. The present cases were negative for KIT, CD34, and PDGFRA. Therefore the present cases are true leiomyomas. Acknowledgement The author declares no commercial associations or conflict of interests related to this article.

Introduction Parvovirus B19, a member of the family Parvoviridae, subfamily Parvovirinae, genus Erythrovirus, is a small, single-stranded DNA virus with approximately 5,000 nucleotides that codes for two major structural or capsid proteins, VP1 and VP2, and one nonstructural protein, NS1. B19 virus has been implicated in a wide spectrum of illnesses, which are transmitted via blood products or through aerosolized droplets and fomite contamination. B19 causes erythema infectiosum (Fifth disease), which has been suggested as one of the causes of acute non-A, non-E hepatitis in children infection [1]. Parvovirus B19 infection in adults often manifests as arthropathy, particularly in females [2]. B19 infection is also associated with transient aplastic crisis by destroying the erythroid precursor pool in patients with chronic hemolytic anemia, such as sickle cell disease or hereditary spherocytosis [3]. In immunocompromised patients, symptomatic B19 infection can persist for months or even years [4]. On the other hand, asymptomatic infection has been reported to occur in about one-quarter of immunocompetent adults [5]. Most infections occur during childhood, resulting in anti-B19 IgG prevalence of about 60% at the age of 12 years [6]. Uncommon manifestations of B19 infection include dermatological, hematological, vascular, and neurological presentations [7]. Liver involvements in B19 infection are uncommon, especially in adult patients [8]. Herein, we report an adult case of parvovirus B19 infection with acute liver failure and myelosuppression.

6]. Uncommon manifestations of B19 infection include dermatological, hematological, vascular, and neurological presentations [7]. Liver involvements in B19 infection are uncommon, especially in adult patients [8]. Herein, we report an adult case of parvovirus B19 infection with acute liver failure and myelosuppression. Case Report A 34-year-old female, without any positive medical history, reported malaise and fever of 23 days, and jaundice of 1 week. In addition to sporadic doses of paracetamol and penicillin, she had not received any other treatment before admission. Examination upon admission revealed slight hepatomegaly and splenomegaly. Maculopapules were found in cervical and thoracic areas. However, no anemia, palpable cervical lymph nodes or other remarkable physical signs were observed. Blood alanine aminotransferase (ALT) was 307 U/L (normal range, NR, 0-40 U/L); aspartate aminotransferase (AST), 444 U/L (NR, 0-30 U/L); gamma-glutamyl transferase (GGT), 97 U/L (NR, 0-50 U/L); alkaline phosphatase (ALP), 142 U/L (NR, 45-132U/L); total bilirubin (TBIL), 195.70 µmol/L (NR, 3.0-22.0 µmol/L); direct bilirubin (DB), 183.80 µmol/L (NR, 0.0-5.0 µmol/L); total protein (TP), 54.4 g/L (NR, 60-80 g/L); albumin (ALB), 26.9 g/L (NR, 35-50 g/L), indirect bilirubin and globulin were normal. White blood cell (WBC) count was 3.75 x 109 /L (84.2% neutrophils, 11.5% lymphocytes, 4% monocytes, 0.3% eosinophils, 0% basophlis), and red blood cell (RBC), hemoglobin and platelet counts were within normal limits. Thrombin time was 32.2 seconds (NR, 14-21 sec); prothrombin time (PT), 22.8 sec (NR, 11-14 sec); international normalized ratio (INR) 2.35; activated partial thromboplastin time (APTT), 36.1 second (NR, 25-36 second); fibrinogen, 0.86 g/L (NR, 2-4 g/L). Erythrocyte sedimentation rate (ESR) was normal. Antinuclear antibodies and rheumatoid factor were negative. Blood culture was negative.

22.8 sec (NR, 11-14 sec); international normalized ratio (INR) 2.35; activated partial thromboplastin time (APTT), 36.1 second (NR, 25-36 second); fibrinogen, 0.86 g/L (NR, 2-4 g/L). Erythrocyte sedimentation rate (ESR) was normal. Antinuclear antibodies and rheumatoid factor were negative. Blood culture was negative. Abdominal echography revealed homogeneous enlargement of spleen, but revealed the enlargement of the only right lobe of liver. Chest and abdominal computed tomography (CT) scan showed pachynsis of right pleura, interstitial change of lobus inferior pulmonis, and lower density of liver parenchyma and splenomegaly (Fig. 1). Serology was negative for typhus; typhoid fever; tuberculosis; syphilis; human syncytial virus; hepatitis A, B, C, D, and E virus; human immunodeficiency virus; cytomegalovirus; and Epstein-Barr virus. The patient was given antibiotics and adjuvant hepatic treatments for 1 week. The fever and maculopapules subsided, but the jaundice and liver function test still unimproved, anemia existed. Blood chemistry showed ALT 236 U/L, AST 124U/L, GGT 106 U/L, TBIL 530.6 µmol/L, DB 376.6 µmol/L. WBC count, 0.25 x 109 /L (4% neutrophils, 84% lymphocytes, 12% monocytes, 0% eosinophils, 0% basophlis); RBC count, 3.37 x 1012 /L; hemoglobin 87 g/L; platelet 10 x 109 /L. The bone marrow aspirate showed lack of red blood cell and platelet. Bone marrow biopsy revealed decrease in all three hemopoeitic precursors, no megakaryocytes were found, mild hyperplasia of fibrous tissue with invasion of neutrophil was detected, no apparent pathogen and malignancy was noticed (Fig. 2). IgM and IgG antibodies against parvovirus B19 were positive. The patient was treated with gamma globulin, liver function and bone marrow test results improved. In a follow-up visit 3 weeks after, patient was asymptomatic, the clinical blood test was normal, and liver function was normalized.

nancy was noticed (Fig. 2). IgM and IgG antibodies against parvovirus B19 were positive. The patient was treated with gamma globulin, liver function and bone marrow test results improved. In a follow-up visit 3 weeks after, patient was asymptomatic, the clinical blood test was normal, and liver function was normalized. Figure 1 Abdominal computed tomography (CT). CT scan showed lower density of liver parenchyma and splenomegaly. Figure 2 Bone marrow findings on admission. The decrease in all three hemopoietic precursors, no megakaryocytes, with mild hyperplasia of fibrous tissue with invasion of neutrophils. No apparent pathogen and malignancy were noticed. Discussion In this report, we describe a 34-year-old female patient, without obvious underlying disease, developed acute liver failure and myelosuppression following B19 virus infection. Parvovirus has been associated with acute hepatic failure, especially in children [9, 10], patients with parvovirus associated liver failure seems to do better than those secondary to other etiology [11]. However, cases of liver failure in adults due to parvovirus infection are rarely reported. Our case, as well as others reported in adults [12, 13], suggests that liver involvement in parvovirus B19 infection appears less severe than that in pediatric patients.

s to do better than those secondary to other etiology [11]. However, cases of liver failure in adults due to parvovirus infection are rarely reported. Our case, as well as others reported in adults [12, 13], suggests that liver involvement in parvovirus B19 infection appears less severe than that in pediatric patients. The pathophysiology of hepatic lesion in B19 infection is still unclear. Some have proposed an immunologically mediated mechanism and others have postulated a direct viral cytopathic effect on hepatocytes [1]. Basic research has demonstrated that B19 virus is capable of infecting and inducing apoptosis in both primary hepatocytes and a liver-derived cell line [14]. B19 virus induced apoptosis may proceed through a caspase 3-dependent pathway, but not the caspase 8 activity. The effect of B19 virus on HepG2 cells is a result of an apoptotic pathway initiated within the infected cell rather than as a result of an exogenous signal of a tumor necrosis factor (TNF) receptor family member [14]. A candidate molecule for affecting apoptosis in HepG2 cells is the B19 virus nonstructural protein NS1, which is a multifunctional protein with endonuclease, helicase, nucleotide triphosphate binding, and transactivating activities. B19 virus causes apoptosis of hepatocytes through NS1 expression. NS1 induces apoptosis in HepG2 cells independently of the presence of full-length viral genomic DNA or other viral proteins, this confirms that NS1 is sufficient to induce apoptosis [15]. NS1 transfection induces activation of caspases 3 and 9, these caspases are necessary for optimal induction of apoptosis. Involvement of the caspase 9 pathway is typical of apoptosis induced by internal stress, in contrast to apoptosis induced by TNF-α [15].

his confirms that NS1 is sufficient to induce apoptosis [15]. NS1 transfection induces activation of caspases 3 and 9, these caspases are necessary for optimal induction of apoptosis. Involvement of the caspase 9 pathway is typical of apoptosis induced by internal stress, in contrast to apoptosis induced by TNF-α [15]. The anemia and myelosuppression could be secondary to parvovirus B19 infection acquired during the course of the disease. This is possible due to the marked trophism of parvovirus to the erythroid precursor [1, 16]. The most plausible explanation for the pathogenesis of myelosuppression is that some immune-mediated mechanisms induce a disturbance in the bone marrow and lead to decreased hematopoiesis. The B19 virus is well demonstrated to infect the erythroid progenitor cells and is associated with aplastic crisis in patients with chronic hemolysis [17]. The in vitro and in vivo analysis showed that the B19 virus infects mainly the erythroid progenitor cells, while other progenitor cells may also be affected by inefficient viral replication [18]. The NS1 both induces production of TNF-α and sensitizes cells to killing by TNF-α in erythroid cells [15].

c hemolysis [17]. The in vitro and in vivo analysis showed that the B19 virus infects mainly the erythroid progenitor cells, while other progenitor cells may also be affected by inefficient viral replication [18]. The NS1 both induces production of TNF-α and sensitizes cells to killing by TNF-α in erythroid cells [15]. Acute liver failure with myelosuppression due to parvovirus B19 infection is extremely rare in adult patients, although there are isolated cases reported on the presentation with severe hemophagocytosis and acute hepatitis associated with parvovirus infection in children [10]. We believe that parvovirus B19 infection should be considered a possible cause of liver injury, the antibody of B19 virus should be routinely tested in patients with liver dysfunction of unclear etiology. Acknowledgements The authors declare no commercial associations or conflicts of interest related to this article.

Introduction Esophageal carcinomas with multiple differentiation are very rare in the English literature [1-12]. Most of them are associated with small cell carcinoma of the esophagus. Basaloid sauamous cell carcinoma of the esophagus is also known to display multiple differentiation [13]. The author herein reports a very rare case of esophageal carcinoma with triplicate differentiation into squamous cell carcinoma, small cell carcinoma, and adenocarcinoma. Case report A 78-year-old man was admitted to our hospital because of dysphagia. An endoscopic examination revealed a polypoid tumor (3 x 4 x 3 cm) in the distal esophagus, and biopsy was taken. The biopsy showed a tumor composed of moderately differentiated squamous cell carcinoma (Fig. 1a), small cell carcinoma (Fig. 1b), and adenocarcinoma (Fig. 1c). The proportions of them were 40% in squamous cell carcinoma, 50% in small cell carcinoma, and 10% in adenocarcinoma. The squamous cell carcinoma element showed keratinization and intercellular bridge (Fig. 1a). The small cell carcinoma element was composed of malignant small cells with hyperchromatic nuclei, scant cytoplasm, finely granular chromatin, molded nuclei, and absent or inconspicuous nucleoli (Fig. 1b). The adenocarcinoma element was composed of malignant cells with tubular formations (Fig. 1c) with neutral and acidic mucins. There were gradual transitions among them (Fig. 1c, d).

ells with hyperchromatic nuclei, scant cytoplasm, finely granular chromatin, molded nuclei, and absent or inconspicuous nucleoli (Fig. 1b). The adenocarcinoma element was composed of malignant cells with tubular formations (Fig. 1c) with neutral and acidic mucins. There were gradual transitions among them (Fig. 1c, d). Figure 1 (a) Histology of squamous cell carcinoma element of the esophageal tumor. Keratinization is seen. HE, x 200. (b) Small cell carcinoma element of the esophageal carcinoma. The tumor cells shows characteristic morphologies of small cell carcinoma. HE, x 200. (c) Adenocarcinoma element of the esophageal carcinoma. Tubular formations are seen. There is a gradual transition between adenocarcinoma element and small cell carcinoma element. HE, x 200. (d) Transitional zone between squamous cell carcinoma element and small cell carcinoma element of the esophageal carcinoma. HE, x 100.

denocarcinoma element of the esophageal carcinoma. Tubular formations are seen. There is a gradual transition between adenocarcinoma element and small cell carcinoma element. HE, x 200. (d) Transitional zone between squamous cell carcinoma element and small cell carcinoma element of the esophageal carcinoma. HE, x 100. An immunohistochemical study was performed by Dako Envision method (Dako Corp, Glostrup, Denmark), as previously described [14-17]. Immunohistochemically, the squamous cell carcinoma component was positive for pancytokeratins (AE1/AE3, polyclonal wide, Dako), high-molecular weight cytokeratin (34βE12, Dako) (Fig. 2a), cytokeratin (CK) 7, CK8, CK18, CK19, CK20, and p53 protein (Fig. 2b). The Ki-67 labeling (MIB1, Dako) was 43%. It was negative for CEA (Dako), CD56 (Dako), chromogranin (Dako), neuron-specific enolase (Dako), synaptophysin (polyclonal, Dako), vimentin, and KIT (polyclonal, Dako). The small cell carcinoma component was positive for pancytokeratins, CK7, CK8, CK18, p53 protein, synaptophysin (Fig. 2c), CD56, and KIT. The Ki-67 labeling was 95% (Fig. 2d). It was negative for high-molecular weight cytokeratin, CK19, CK20, neuron-specific enolase, CEA, vimentin, and chromogranin. The adenocarcinoma component was positive for pancytokeratins, CK7, CK8, CK18, CK19, p53 protein, and CEA. The Ki-67 labeling was 52%. It was negative for high molecular weight cytokeratin, CK 20, neuron-specific enolase, CD56, chromogranin, synaptophysin, vimentin, and KIT.

ic enolase, CEA, vimentin, and chromogranin. The adenocarcinoma component was positive for pancytokeratins, CK7, CK8, CK18, CK19, p53 protein, and CEA. The Ki-67 labeling was 52%. It was negative for high molecular weight cytokeratin, CK 20, neuron-specific enolase, CD56, chromogranin, synaptophysin, vimentin, and KIT. Figure 2 (a) High molecular weight cytokeratin is positive in the squamous cell carcinoma element. Immunostaining (AE1/3), x 200. (b) p53 expression in squamous cell carcinoma element of the esophageal tumor. Immunostaining, x 200. (c) Synaptophysin is positive in the small cell carcinoma element of the esophageal tumor cells. Immunostaining, x 200. (d) Ki-67 labeling in the small cell carcinoma element of the esophageal tumor. The labeling is 95%. Immunostaining, x 200. The patient was pathologically diagnosed as an esophageal carcinoma with triple differentiation (squamous cell carcinoma, small cell carcinoma, and adenocarcinoma). The patient was treated by radiation (50 Gray) and cisplatin-based chemotherapy, but died of systemic metastasis 13 months after the initial presentation

Figure 2 (a) High molecular weight cytokeratin is positive in the squamous cell carcinoma element. Immunostaining (AE1/3), x 200. (b) p53 expression in squamous cell carcinoma element of the esophageal tumor. Immunostaining, x 200. (c) Synaptophysin is positive in the small cell carcinoma element of the esophageal tumor cells. Immunostaining, x 200. (d) Ki-67 labeling in the small cell carcinoma element of the esophageal tumor. The labeling is 95%. Immunostaining, x 200. The patient was pathologically diagnosed as an esophageal carcinoma with triple differentiation (squamous cell carcinoma, small cell carcinoma, and adenocarcinoma). The patient was treated by radiation (50 Gray) and cisplatin-based chemotherapy, but died of systemic metastasis 13 months after the initial presentation Discussion Histologically, the present esophageal tumor was composed of three elements. The pathology and immunohistochemistry were characteristic of each tumor. The squamous cell carcinoma component was characterized by keratinization, intercellular bridge and high-molecular weight cytokeratin, the small cell carcinoma component by characterized cellular morphologies and neuroendocrine antigens, and adenocarcinoma by tubular formations, mucin and CEA. Therefore, the diagnosis of the present tumor is correct.

onent was characterized by keratinization, intercellular bridge and high-molecular weight cytokeratin, the small cell carcinoma component by characterized cellular morphologies and neuroendocrine antigens, and adenocarcinoma by tubular formations, mucin and CEA. Therefore, the diagnosis of the present tumor is correct. Histologically, there were gradual transitions among the three elements. The proportions of these three elements were 40% in squamous cell carcinoma element, 50% in small cell carcinoma element, and 10% in adenocarcinoma element. The higher proportion of small cell carcinoma suggests that the sqaumous cell carcinoma element and adenocarcinoma element were differentiated from the small cell carcinoma element. Otherwise, this esophageal tumor arises from totipotent stem cell of the esophagus, as suggested by Ho et al [2].

Introduction Ectopic varices that are not esophagogastric are located predominantly in the duodenum, jejunum, ileum, colon, rectum, or enterostomy stoma [1]. Bleeding from ileal varices, which is rare in patients with portal hypertension, is generally massive and life threatening. Diagnosis of ruptured ileal varices and control of bleeding are difficult. Endoscopic injection sclerotherapy (EIS) is a standard procedure for treatment of esophageal varices [2]. More recently, endoscopic variceal ligation (EVL) has been applied widely in the treatment of esophageal varices [3]. Although balloon-occluded retrograde transvenous obliteration (B-RTO) is a new interventional modality for gastric fundic varices [4], a definitive treatment for bleeding ileal varices has not been established. Here we present a case of ruptured ileal varices controlled successfully using B-RTO. Case Report A 55-year-old man with hepatitis B virus antigen-positive liver cirrhosis was admitted to our hospital in October 2007 with anal bleeding. At 36 years of age, he underwent an operation of the ileocecum to remove a benign colonic tumor. At 46 year of age (in 1998), he first received EIS for treatment of esophageal varices and then underwent EIS in 1999 and 2003 for recurrent esophageal varices.

s admitted to our hospital in October 2007 with anal bleeding. At 36 years of age, he underwent an operation of the ileocecum to remove a benign colonic tumor. At 46 year of age (in 1998), he first received EIS for treatment of esophageal varices and then underwent EIS in 1999 and 2003 for recurrent esophageal varices. At the time of admission, the patient’s blood pressure was 120/62 mmHg, pulse 98 beats/min and regular, and body temperature 36.5°C. He had anemic conjunctivae, but there was no scleral icterus. The abdomen was soft and flat, there were no obvious abdominal masses. Bleeding was massive and recurrent, and blood transfusions were required. The fibergastroscopic examination on admission revealed small, red color-negative esophageal varices. Laboratory findings were: red blood cell count 209 x 104 /mm3 (normal:353 - 466 x 104 /mm3), hemoglobin 5.6g/dL (10.6 - 14.4 g/dL), white blood cell count 6900/mm3 (3000 - 7800/mm3), platelet count 13.2 x 104 /mm3 (13.8 - 30.9 x 104 /mm3), serum albumin 2.6 g/mL (4.0 - 5.2 g/mL), total bilirubin 0.6 mg/mL (0.2 - 1.2 mg/mL), glutamic oxaliacetic transaminase 14 IU/L (8 - 38 IU/L), glutamic pyruvic transaminase 18 IU/L (4 - 44 IU/L), alkaline phosphatase 184 IU/L (104 - 338 IU/L), blood urea nitrogen 22.9 mg/dL (7.0 - 24.0 mg/dL), and creatine 0.9 mg/dL (0.4 - 0.9 mg/dL). Prothrombin time was 54% (90 -140%) and serological assays for markers of hepatitis B viruses were positive. All test results for tumor markers were within the normal range.

U/L), alkaline phosphatase 184 IU/L (104 - 338 IU/L), blood urea nitrogen 22.9 mg/dL (7.0 - 24.0 mg/dL), and creatine 0.9 mg/dL (0.4 - 0.9 mg/dL). Prothrombin time was 54% (90 -140%) and serological assays for markers of hepatitis B viruses were positive. All test results for tumor markers were within the normal range. Although colonoscopy revealed blood retention in the entire colon, no bleeding lesions were found (Fig. 1). Because computed tomography (CT) images of vessels in the ileum showed connection to vessels in the right testicular vein (Fig. 2), we suspected ileal varices to be the most probable cause of bleeding. We immediately performed double balloon enteroscopy, but failed to find any site of bleeding owing to the difficulty of fiberscope insertion with sever adhesion. Subsequent interventional radiology involving retrograde transvenous venography using balloon catheter from right subclavian vein revealed ileal varices communicating with the right testicular vein (efferent vein) (Fig. 3a). Angiography revealed that the superior mesenteric vein branch was the afferent vein for the ileal varices. Figure 1 Colonoscopy revealed blood retention in the entire colon, but no bleeding lesion was detected. Figure 2 Computed tomography images of a vessel in the ileum (arrow) and its connection to the right testicular vein (arrowhead).

Although colonoscopy revealed blood retention in the entire colon, no bleeding lesions were found (Fig. 1). Because computed tomography (CT) images of vessels in the ileum showed connection to vessels in the right testicular vein (Fig. 2), we suspected ileal varices to be the most probable cause of bleeding. We immediately performed double balloon enteroscopy, but failed to find any site of bleeding owing to the difficulty of fiberscope insertion with sever adhesion. Subsequent interventional radiology involving retrograde transvenous venography using balloon catheter from right subclavian vein revealed ileal varices communicating with the right testicular vein (efferent vein) (Fig. 3a). Angiography revealed that the superior mesenteric vein branch was the afferent vein for the ileal varices. Figure 1 Colonoscopy revealed blood retention in the entire colon, but no bleeding lesion was detected. Figure 2 Computed tomography images of a vessel in the ileum (arrow) and its connection to the right testicular vein (arrowhead). Figure 3 (a) Ileal varices (arrowhead) communicating with the right testicular vein (arrow) were found using retrograde transvenous venography. (b) Balloon occluded retrograde transvenous obliteration for ileal varices (arrowhead) was performed via the efferent vein of the varices.

Figure 2 Computed tomography images of a vessel in the ileum (arrow) and its connection to the right testicular vein (arrowhead). Figure 3 (a) Ileal varices (arrowhead) communicating with the right testicular vein (arrow) were found using retrograde transvenous venography. (b) Balloon occluded retrograde transvenous obliteration for ileal varices (arrowhead) was performed via the efferent vein of the varices. We performed B-RTO via the efferent vein of the varices. Because of the insufficient varicealography of ileal varices caused by rapid washout of the contrast medium, we performed microcoil embolization using 0.014-inch steel coils for the distal right testicular vein and injected 18 ml of 5% ethanolamine oleate (EO) containing iopamidol (Fig. 3b). There were no post-operative complications. After B-RTO, venography on the next day could not distinguish the ileal varices or the afferent vein (Fig. 4), and ileal vessel images before B-RTO disappeared after the treatment under CT (Fig. 5). One year after treatment, the patient had experienced no further bleeding. Figure 4 Following balloon occluded retrograde transvenous obliteration, both ileal varices (arrow) and the afferent vein were not visible by venography the next day. Figure 5 (a) Computed tomography image of ileal vessels before balloon occluded retrograde transvenous obliteration. (b) The ileal vessels are no longer visible following balloon occluded retrograde transvenous obliteration.

Figure 4 Following balloon occluded retrograde transvenous obliteration, both ileal varices (arrow) and the afferent vein were not visible by venography the next day. Figure 5 (a) Computed tomography image of ileal vessels before balloon occluded retrograde transvenous obliteration. (b) The ileal vessels are no longer visible following balloon occluded retrograde transvenous obliteration. Discussion Portal hypertension can involve either reopening of collapsed embryonic channels or a reversed in flow within existing adult veins [5]. Whereas esophagogastric varices are the most common complication in patients with portal hypertension, ectopic varices defined by large portosystemic venous collaterals occurring anywhere in gastrointestinal tract except in the esophagogastric region are less common and account for between 1% and 5% of all variceal bleeding [6, 7]. Several cases of bleeding ileal varices have been reported [8-16].

h portal hypertension, ectopic varices defined by large portosystemic venous collaterals occurring anywhere in gastrointestinal tract except in the esophagogastric region are less common and account for between 1% and 5% of all variceal bleeding [6, 7]. Several cases of bleeding ileal varices have been reported [8-16]. Ectopic varices have been reported to occur at numerous sites, including 18% in the jejunum or ileum, 17% in the duodenum, 14% in the colon, 8% in the rectum, and 9% in the peritoneum [1]. Most bleeding jejunal and ileal varices, generally detected previous intra-abdominal surgery, are serious due to the difficulty of early diagnosis. In our case, the patient's risk factors included portal hypertension due to liver cirrhosis, EIS for esophageal varices, and previous surgery. Collaterals formation within adhesions from a previous surgery is the usual mechanism for the development of ectopic varices [1]. Adhesions tend to bring the parietal surface of the viscera in contact with the abdominal wall, and portal hypertension results in the formation of varices below intestinal mucosa. Double balloon enteroscopy has gained worldwide acceptance as an endoscopic technique that can be used safely and effectively to provide complete examination of the small bowel, offer therapeutic intervention, and favorably affect clinical outcomes [17-19]. In this case, we immediately performed double balloon enteroscopy to find any site of bleeding, but failed to diagnose owing to the difficulty of fiberscope insertion with sever adhesion.

to provide complete examination of the small bowel, offer therapeutic intervention, and favorably affect clinical outcomes [17-19]. In this case, we immediately performed double balloon enteroscopy to find any site of bleeding, but failed to diagnose owing to the difficulty of fiberscope insertion with sever adhesion. Surgical approaches such as segmental resection and ligation generally control bleeding from ileal varices successfully [12, 13, 20, 21]. In patients with poor condition, interventional radiologic treatment such as insertion of a transjugular intrahepatic portosystemic shunt (TIPS) for ileal varices have been performed as a non-operative treatment option [7, 14, 16]. Although TIPS is a relatively safe and effective means of decompressing the portal pressure, it may not prove effective in patients with severe liver atrophy or complications such as encephalopathy and cerebral embolization. Because B-RTO can obliterate not only varices but also the afferent veins and efferent veins, it is practical for treating ileal varices [22], as described here. In the future, interventional radiologic treatments such as B-RTO may also be applied as therapy for patients in poor condition. In summary, ileal varices should be considered in patients that have undergone previous surgery and display lower gastrointestinal bleeding and portal hypertension as well as endoscopic findings that do not reveal any bleeding point. Acknowledgements The authors declare no commercial associations or conflict of interests related to this article.

Introduction Anticoagulants and antiplatelets may potentiate gastrointestinal (GI) bleeding due to medication, thromboembolism related to interruption of medication for endoscopic procedure, and bleeding after endoscopic procedure. A recent international survey has shown that there is a wide variation between the Eastern and Western endoscopists in managing anticoagulants and antiplatelets during the periendoscopic period [1]. This might be based on the differences in education, guidelines, genetics, environments, risk factors (diabetes mellitus, hypertension, hyperlipidemia, and obesity), prevalence of cardiovascular attack (stroke, deep vein thrombosis, and ischemic heart disease), effectiveness and side effects of antiplatelets or anticoagulants. For example, Eastern endoscopists frequently experience severe bleeding while following Western guidelines (ie, for a polypectomy with aspirin medication or a biopsy with warfarin medication) [2-5], and thus recent Eastern guideline recommends to withdraw either antiplatelets or anticoagulants even for a biopsy [6, 7]. Unfortunately, little is known about rationales on such differences between the East and West in managing antiplatelets or anticoagulants for GI endoscopy. The aim of this review is to better understand the differences between the East and West by analyzing the: (1) East and West differences in endoscopists’ conception, (2) East and West differences in complications such as hemorrhage and embolism, and (3) East and West differences in drug metabolism.

Introduction Anticoagulants and antiplatelets may potentiate gastrointestinal (GI) bleeding due to medication, thromboembolism related to interruption of medication for endoscopic procedure, and bleeding after endoscopic procedure. A recent international survey has shown that there is a wide variation between the Eastern and Western endoscopists in managing anticoagulants and antiplatelets during the periendoscopic period [1]. This might be based on the differences in education, guidelines, genetics, environments, risk factors (diabetes mellitus, hypertension, hyperlipidemia, and obesity), prevalence of cardiovascular attack (stroke, deep vein thrombosis, and ischemic heart disease), effectiveness and side effects of antiplatelets or anticoagulants. For example, Eastern endoscopists frequently experience severe bleeding while following Western guidelines (ie, for a polypectomy with aspirin medication or a biopsy with warfarin medication) [2-5], and thus recent Eastern guideline recommends to withdraw either antiplatelets or anticoagulants even for a biopsy [6, 7]. Unfortunately, little is known about rationales on such differences between the East and West in managing antiplatelets or anticoagulants for GI endoscopy. The aim of this review is to better understand the differences between the East and West by analyzing the: (1) East and West differences in endoscopists’ conception, (2) East and West differences in complications such as hemorrhage and embolism, and (3) East and West differences in drug metabolism. Differences in endoscopists’ conception between the East and West Given the paucity of available data on different practice patterns between the Eastern and Western endoscopists, I and my colleagues performed an international survey to the GI endoscopists in Eastern (Korea, Japan, China, India, Thailand, Singapore, Malaysia, and Philippines) and Western (United States and Canada) countries [1]. It appeared that Eastern endoscopists do not typically perform an endoscopic biopsy while their patients are on warfarin therapy and do not perform a polypectomy while their patients are taking aspirin. In addition, there was a delay (1 to 3 days later) on restarting medications after an endoscopic biopsy or a polypectomy only in the Easternists. This occurred despite published Western guidelines that suggest it is safe to perform an endoscopic biopsy during warfarin therapy or a polypectomy while on aspirin medication [2-5], and despite a published paper stated that patients treated by the American Society of Gastrointestinal Endoscopy guidelines had 0% rate of thrombosis [8]. Through our survey [1], we could find that Eastern endoscopists believe it to be dangerous to follow Western guidelines because of an increased risk of bleeding in Asian patients. It seems that personal experience is a more powerful driver of practice than the published literature for the GI endoscopists.

rombosis [8]. Through our survey [1], we could find that Eastern endoscopists believe it to be dangerous to follow Western guidelines because of an increased risk of bleeding in Asian patients. It seems that personal experience is a more powerful driver of practice than the published literature for the GI endoscopists. Management of drugs for diagnostic endoscopy Most Eastern and Western endoscopists continued all medications when performing diagnostic endoscopy without a biopsy, and restarted it on the same day after the procedure [1]. After an endoscopic biopsy, the Eastern endoscopists tended to restart medications 1 to 3 days later, except for nonsteroidal anti-inflammatory drugs (NSAIDs) restarting on the same day, whereas the Western endoscopists restarted all medications on the same day. A recent European guideline makes the differences more definite by recommending warfarin and clopidogrel to be continued [5], while Japanese guidelines recommend cessation of aspirin and clopidogrel for low-risk procedures [6].

on the same day, whereas the Western endoscopists restarted all medications on the same day. A recent European guideline makes the differences more definite by recommending warfarin and clopidogrel to be continued [5], while Japanese guidelines recommend cessation of aspirin and clopidogrel for low-risk procedures [6]. Management of drugs for therapeutic endoscopy Most Eastern and Western endoscopists withdrew all medications other than NSAIDs for a polypectomy, and restarted 1 to 3 days after a polypectomy [1]. Main difference between the East and West was found on managing aspirin for a polypectomy. Eastern endoscopists tended to withdraw aspirin for more than 7 days before a polypectomy, whereas Western endoscopists performed a polypectomy without withdrawing aspirin medication. In addition, Eastern endoscopists restarted aspirin 1 to 3 days after a polypectomy, whereas Western endoscopists restarting it on the same day [1].

scopists tended to withdraw aspirin for more than 7 days before a polypectomy, whereas Western endoscopists performed a polypectomy without withdrawing aspirin medication. In addition, Eastern endoscopists restarted aspirin 1 to 3 days after a polypectomy, whereas Western endoscopists restarting it on the same day [1]. Publications on managing antiplatelets and/or antiplatelets for endoscopy Great differences between the East and West were found in published articles on managing anticoagulants and antiplatelets for GI endoscopy (Table 1) [1, 2, 4-6, 8-15]. To explain this discrepancy, differences in effectiveness and side effects of antiplatelets and anticoagulants between the East and West should be discussed with the differences in complications such as bleeding and thromboembolism [16]. Although it is difficult to conclude the superiority, it should be reminded that life threatening bleeding is rare when compared to the risk of thromboembolism. Bleeding usually result in less morbidity and can be managed by the endoscopists, whereas major thromboembolic events during discontinuation of drugs often lead to death or permanent disability [7, 8, 17]. Cultural difference should be also considered for such difference because it might be speculated that Western gastroenterologists would limit anticoagulant abstinence for their patients, because of more litigious nature of medicine in Western societies.

n of drugs often lead to death or permanent disability [7, 8, 17]. Cultural difference should be also considered for such difference because it might be speculated that Western gastroenterologists would limit anticoagulant abstinence for their patients, because of more litigious nature of medicine in Western societies. Table 1 Published papers on managing antiplatelets and/or anticoagulants for GI endoscopy (in recent order) Country (author, year) Key Messages Japan (Fujishiro M, 2009) [8] • Aspirin, ticlopidine, and ethyl icosapentate were stopped 7 days before the procedures by most of the endoscopists, whereas warfarin was stopped 4 days before the procedures. • With regard to the drug discontinuation before the procedures, no major differences were observed between a biopsy (low-risk procedure) and endoscopic mucosal resection (high-risk procedure). • In case of higher risk of thromboembolism during the cessation of drugs, most of the endoscopists never performed a biopsy or endoscopic mucosal resection. Korea (Lee SY, 2008) [1] • Eastern endoscopists do not typically perform an endoscopic biopsy while their patients are on warfarin therapy. • Eastern endoscopists restarted medications later (1 to 3 days) than Western endoscopists after a biopsy (same day). • Eastern endoscopists do not perform a polypectomy while their patients are taking aspirin. They withdrew aspirin for more than 7 days before a polypectomy, and restarted it 1 to 3 days after a polypectomy. UK (Veitch A, 2008) [5] • Low-risk procedures can be performed during warfarin or clopidogrel intake. • High-risk procedures should be done after stopping warfarin for 5 days (INR < 1.5), and substituted with LMWH in high-risk conditions. • In low-risk conditions, high-risk procedures can be performed after stopping clopidogrel for 7 days. Aspirin should be considered during the cessation of clopidogrel in such conditions. • In high-risk conditions, continuing aspirin and stopping clopidogrel for 7 days should be considered, if 12 months have passed drug-eluting coronary stent insertion. UK (Goel A, 2007) [10] • With regard to warfarin, 26% of the endoscopists stopped for EGD, whereas 48.7% stopped for colonoscopy. • During warfarin intake, 21% took biopsies as usual, while 54% performed after checking the INR. • Warfarin was usually stopped for 3 days. Preferred INR was below 2.0 during the endoscopic procedure.

2007) [10] • With regard to warfarin, 26% of the endoscopists stopped for EGD, whereas 48.7% stopped for colonoscopy. • During warfarin intake, 21% took biopsies as usual, while 54% performed after checking the INR. • Warfarin was usually stopped for 3 days. Preferred INR was below 2.0 during the endoscopic procedure. Israel (Kimchi NA, 2007) [11] • Aspirin can be continued for diagnostic EGD, but should be stopped 5 to 7 days before colonoscopy, spincterotomy, esophageal dilation, endoscopic ultrasound-guided biopsy, or drainage. • Aspirin use should be avoided for 2 weeks after polypectomy, and 10 days after spincterotomy. However, when the cardiovascular risk is high, aspirin should be resumed within a week after the procedure. • NSAIDs should be stopped 8 hours before any endoscopy, and be resumed 7 to 14 days after a high-risk procedure. • Clopidogrel should be stopped 5 days before colonoscopy. US (Makar GA, 2006) [12] • For low-risk procedure (e.g. diagnostic endoscopy and colonoscopy without polypectomy), there is no need to discontinue or adjust anticoagulation. • For high-risk procedures (e.g. polypectomy and biliary sphincterotomy), an individual approach is required. This approach includes stopping oral anticoagulant therapy with or without the administration of unfractionated heparin or LMWH during which the patient’s INR is in the subtherapeutic range. • Antiplatelet therapy can be withheld for high risk procedures, but there is insufficient evidence to indicate that bleeding risk is impacted. France (Naploen B, 2006) [4] • Low-risk procedures can be performed during medications. • High-risk procedures and transnasal endoscopy should be performed after the cessation of drugs. • Colon polypectomy and endoscopic sphincterotomy could be performed without cessation of aspirin or NSAIDs. Japan (Ogoshi K, 2005) [6] • Warfarin should be stopped 3 to 4 days before all kinds of endoscopic procedure. INR less than 1.5 is preferred during the procedure. • Aspirin should be stopped 3 days before low-risk procedures, and stopped 7 days before high-risk procedures. • Ticlopidine should be stopped 5 days before low-risk procedures, and stop 10 to 14 days before high-risk procedures. • In case of dual antiplatelet intake (aspirin and ticlopidine), both antiplatelets should be stopped 7 days before low-risk procedures. Germany (Mosler P, 2004) [13] • Antiplatelets were usually continued before elective procedures including diagnostic EGD, colonoscopy, and ERCP.

o 14 days before high-risk procedures. • In case of dual antiplatelet intake (aspirin and ticlopidine), both antiplatelets should be stopped 7 days before low-risk procedures. Germany (Mosler P, 2004) [13] • Antiplatelets were usually continued before elective procedures including diagnostic EGD, colonoscopy, and ERCP. • Aspirin and clopidogrel were more frequently stopped than NSAIDs prior to any endoscopic procedure. • More than 80% stopped aspirin and clopidogrel before elective therapeutic endoscopic procedures. • Warfarin was usually discontinued 3-5 days prior to the procedure, and was substituted with LMWH. Belgium (Hittelet A, 2003) [14] • It is not necessary to discontinue aspirin or NSAIDs for endoscopic procedures, when used in standard doses. • It is not necessary to adjust anticoagulation for low-risk procedures, such as EGD, colonoscopy, ERCP with biopsy or stent insertion (without sphincterotomy). • For ticlopidine or clopidogrel, it is prudent to discontinue 7 to 10 days for high-risk procedures. • Warfarin should be discontinued 3 to 5 days before high-risk procedures. US (Eisen G, 2002) [2] • Any endoscopic procedure may be performed in patients taking aspirin or NSAIDs in the absence of pre-existing bleeding disorders. • Discontinuation of oral anticoagulation is needed for high-risk endoscopic procedures. • Consider a heparin window only for patients with high thromboembolic risk. • Resumption of warfarin is generally recommended at the night of the procedure except for sphincterotomy. US (Kadakia SC, 1996) [15] • Physicians stopped aspirin and NSAIDs more frequently before colonoscopy and ERCP than before EGD. • With aspirin or NSAIDs intake, cold biopsy or hot biopsy during EGD or colonoscopy was performed, but sphincterotomy was not. • Optimal cessation period of aspirin or NSAIDs was less than 10 days. • Warfarin was resumed immediately after diagnostic endoscopy, whereas 7 days of cessation period was observed after therapeutic endoscopy. Note: INR, international normalized ratio; LMWH, low molecular weight heparin; EGD, esophagogastroduodenoscopy; ERCP, endoscopic retrograde cholangiopancreaticography; NSAIDs, nonsteroidal anti-inflammatory drugs.

y after diagnostic endoscopy, whereas 7 days of cessation period was observed after therapeutic endoscopy. Note: INR, international normalized ratio; LMWH, low molecular weight heparin; EGD, esophagogastroduodenoscopy; ERCP, endoscopic retrograde cholangiopancreaticography; NSAIDs, nonsteroidal anti-inflammatory drugs. Taken as a whole, racial differences in susceptibility to complications have led to establish different guidelines between countries, especially between the East and West. A large, prospective, randomized, and double-blinded clinical study might be a solution, but it is very dangerous and unethical to perform such a prospective study in patients on antiplatelets or anticoagulants. Therefore, opinion of the experts or review papers should be an alternative proposal. Differences in complications between the Easterners and Westerners Complications in patients on anticoagulation and/or antiplatelet medications can be divided into three categories: (1) spontaneous GI bleeding from medication; (2) GI bleeding related to an endoscopic procedure; and (3) thromboembolism from cessation of medications. Embolism High risks for thromboembolisms are valvular heart disease with atrial fibrillation, mechanical valve with past history of thromboembolism, and mechanical valve in mitral area [2]. Of these risk factors, atrial fibrillation is most dangerous factor that increases thromboembolic risk with older age more than 80 year-old, past or family history of cerebrovascular embolism, hypertension, congestive heart failure, diabetes mellitus, or hyperlipidemia [18].

sm, and mechanical valve in mitral area [2]. Of these risk factors, atrial fibrillation is most dangerous factor that increases thromboembolic risk with older age more than 80 year-old, past or family history of cerebrovascular embolism, hypertension, congestive heart failure, diabetes mellitus, or hyperlipidemia [18]. In Western studies, it has been reported that acute coronary syndrome is not rare within one month of aspirin withdrawal [19, 20]. In contrary, it seems that Asians are more susceptible to cerebral stroke than Caucasians, whereas Caucasians are more susceptible to other cardiovascular attacks including deep vein thrombosis (Table 2) [7, 8, 17, 21-23]. Therefore, it should be emphasized again that thromboembolism affecting Westerners may be more likely to be of the cardiovascular variety, while that of Easterners may be more likely to be of the cerebrovascular variety. It should be stressed again to Eastern endoscopists, because cerebral infarction during discontinuation of the anticoagulants or antiplatelets often leads to permanent disability or death as observed in previous Eastern reports [7, 8, 17].

hile that of Easterners may be more likely to be of the cerebrovascular variety. It should be stressed again to Eastern endoscopists, because cerebral infarction during discontinuation of the anticoagulants or antiplatelets often leads to permanent disability or death as observed in previous Eastern reports [7, 8, 17]. Table 2 Reports on embolism that happened during the cessation of antiplatelets and/or anticoagulants Country (author, year) Frequency Type of embolism East Korea (Lee SY, 2006) [17] Six of 81 (7.4%) endoscopists have experienced embolism during past one year. 5 cerebral infarction 1 mesenteric infarction Japan (Ishizawa T, 2006) [7] Seven of 81 (8.6%) endoscopists have experienced embolism during past three years. 5 cerebral infarction 2 myocardial infarction Japan (Fujishiro M, 2009) [8] Three of 13 (23.1%) endoscopists have experienced embolism during their career. 2 cerebral infarction 1 mesenteric infarction West US (Dunn A, 2003) [21] Twenty nine of 1868 (1.6%) patients undergoing dental or orthopedic procedures, or cataract surgery have experienced thromboembolic events. 21 cardiovascular embolism including peripheral arterial thromboembolism 7 cerebral infarction 1 unspecified US (Garcia D, 2008) [22] Seven of 1024 (0.7%) patients showed embolism during the study period. 3 cerebral infarction 2 pulmonary embolism 1 deep vein thrombosis 1 mesenteric infarction US (Kuwada SK, 1996) [23] One of 27 (3.7%) patients showed embolism during the study period. 1 peripheral arterial thromboembolism According to recent data on death rates from ischemic heart disease and stroke among nations, the incidence of stroke in Korea and Japan is higher than that of ischemic heart disease [24], despite rapid increasing rate of cardiovascular disease in Asian countries since 1985 [25]. However, the rise in risk factors due to westernized life style in Japan did not result in an increase in coronary heart disease mortality and morbidity like in US, even in the post-World War II cohorts [25]. The ratio of ischemic heart disease to stroke is highly different between the East and West, although the prevalence of thromboembolism and their subtypes are getting similar throughout the world [24]. Therefore, we can speculate that genetic factor might be involved in addition to environmental factor such as westernized dietary habits (higher fat intake, lower polyunsaturated/saturated ration, and less omega-3 fatty acids).

valence of thromboembolism and their subtypes are getting similar throughout the world [24]. Therefore, we can speculate that genetic factor might be involved in addition to environmental factor such as westernized dietary habits (higher fat intake, lower polyunsaturated/saturated ration, and less omega-3 fatty acids). Such an ethic difference suggests that there might be some powerful and important genetic factors in Asians, and identification of such factors would clearly have great importance for prevention of cardiovascular diseases. Bleeding Endoscopy-induced bleeding can classified into high-risk procedures (bleeding rate > 1%) and low-risk procedures (bleeding rate < 1%) [2]. High-risk bleeding procedures include tissue resections such as polypectomy, mucosectomy, anpullectomy, endoscopic submucosal dissection, sphincterotomy, bougiennation, pneumatic or balloon dilation, metal stent insertion, percutaneous endoscopic gastrostomy, endoscopic ultrasonograph guided fine needle aspiration, laser ablation, photocoagulation, hemostatic procedures, and variceal ligation. According to the Western guidelines [2-5], these procedures require cessation of drug, while low-risk procedures do not. Notably, transnasal esophagogastroduodenoscopy (EGD) should be managed as high-risk procedures because of epistaxis, whereas biliary or pancreatic stenting should be managed as low-risk procedures because of their relative safety [4].

[2-5], these procedures require cessation of drug, while low-risk procedures do not. Notably, transnasal esophagogastroduodenoscopy (EGD) should be managed as high-risk procedures because of epistaxis, whereas biliary or pancreatic stenting should be managed as low-risk procedures because of their relative safety [4]. In contrast to the outcomes of thromboembolic events from withdrawing antiplatelets and anticoagulants, hemorrhagic complications from endoscopic procedures are less likely to result in death or permanent disability. However, despite all these publications from the West, the risk of procedure-related bleeding is still heavily weighted toward the risk of a thromboembolism in the East [1]. For example, Eastern endoscopists do not follow Western guidelines (ie, for a polypectomy with aspirin medication or a biopsy with warfarin medication) because it may be responsible for patients experiencing massive bleeding [1, 6-8, 17].

g is still heavily weighted toward the risk of a thromboembolism in the East [1]. For example, Eastern endoscopists do not follow Western guidelines (ie, for a polypectomy with aspirin medication or a biopsy with warfarin medication) because it may be responsible for patients experiencing massive bleeding [1, 6-8, 17]. Anticoagulants are not by themselves ulcerogenic, but associated with a greater risk of upper GI bleeding because of an exacerbation of pre-existing lesions in the GI tract associated with NSAIDs, aspirin, orHelicobacter pylori infection [26]. Predictors of bleeding include past history of GI bleeding or ulcer disease, higher intensity of anticoagulation, old age more than 65 year-old, combination therapy (anticoagulants with antiplatelets), and presence of comorbid conditions like chronic renal failure, congestive heart failure, diabetes mellitus, or alcoholic liver disease [27]. Highest bleeding risk happens with warfarin followed by aspirin, NSAIDs, ticlopidine, clopidogrel, dipyridamole), and cyclooxygenase-2 (COX-2) inhibitor [28]. Of endoscopic findings, peptic ulcer is the most common, followed by hemorrhagic gastritis, and esophagitis [29].

etes mellitus, or alcoholic liver disease [27]. Highest bleeding risk happens with warfarin followed by aspirin, NSAIDs, ticlopidine, clopidogrel, dipyridamole), and cyclooxygenase-2 (COX-2) inhibitor [28]. Of endoscopic findings, peptic ulcer is the most common, followed by hemorrhagic gastritis, and esophagitis [29]. In case of GI bleeding, complete or partial reversal of anticoagulation is undertaken based on the balance of risks between bleeding and thromboembolism. Early endoscopy can reveal lesions requiring endoscopic hemostasis, which can be performed in the setting of low-intensity anticoagulation [29, 30]. For the hemostasis of GI bleeding during warfarin intake, partial reversal of anticoagulation to international normalized ratio (INR) 1.5 to 2.5 with fresh frozen plasma can allow for successful endoscopic hemostatic therapy [30]. Fresh frozen plasma should be used instead of vitamin K, because vitamin K infusion often leads to thromboembolic complication after an urgent infusion [23]. Thrombin spraying or hemoclipping could also be considered with dried human blood coagulant IX factor complex 500 unit to normalize the prolongated INR [31]. Platelet concentrate transfusion should be considered in case of antiplatelet intake, and protamine sulfate injection should be considered in case of heparin infusion.

bin spraying or hemoclipping could also be considered with dried human blood coagulant IX factor complex 500 unit to normalize the prolongated INR [31]. Platelet concentrate transfusion should be considered in case of antiplatelet intake, and protamine sulfate injection should be considered in case of heparin infusion. Decision for discontinuation of drugs in the setting of GI bleeding must be made on an individual basis, based upon potential thrombotic and hemorrhagic risks. It seems prudent to briefly discontinue the drugs until lack of rebleeding, because hemodynamic instability and hemostatic changes induced by acute GI bleeding may further increase the risk of thrombosis without medications [32]. In case of longer period of cessation, low molecular weighted heparin (LMWH) should be considered instead of warfarin [33]. When it is difficult to stop aspirin, proton pump inhibitor (PPI), misoprostol, or COX-2 inhibitor should be considered [34]. In patients with a past history of peptic disease or bleeding from an acid-related lesion, PPI and H. pylori eradication should be considered to reduce the risk of upper GI bleeding even with antiplatelet intake [32, 35].

p aspirin, proton pump inhibitor (PPI), misoprostol, or COX-2 inhibitor should be considered [34]. In patients with a past history of peptic disease or bleeding from an acid-related lesion, PPI and H. pylori eradication should be considered to reduce the risk of upper GI bleeding even with antiplatelet intake [32, 35]. Differences in drug metabolism between the Easterners and Westerners Medications that may potentiate GI bleeding has become more widespread these days (Table 3). These drugs are classified into (1) antiplatelets such as glycoprotein IIb/IIIa inhibitors, adenosine diphosphate receptor antagonist, prostaglandin analogue, COX inhibitors, etc; (2) anticoagulants such as vitamin K antagonists, direct thrombin II inhibitors, direct factor Xa inhibitors, heparin groups, glycosaminoglycans, etc; (3) thrombolytic drugs/fibrinolytics; and (4) others such as non-medicinal.

tors, adenosine diphosphate receptor antagonist, prostaglandin analogue, COX inhibitors, etc; (2) anticoagulants such as vitamin K antagonists, direct thrombin II inhibitors, direct factor Xa inhibitors, heparin groups, glycosaminoglycans, etc; (3) thrombolytic drugs/fibrinolytics; and (4) others such as non-medicinal. Table 3 Medications that may potentiate GI bleeding (in alphabetical order) Drug Half life Time of action Mechanism of action Abciximab 0.7 hours (in alpha), 10 hours (in beta) 0.5-2.5 hours Nonspecific antagonist for glycoprotein IIb/IIIa receptor. Anagrelide hydrochloride 76 hours Long Reduction in platelet production resulting from a decrease in megakaryocyte. Aspirin 0.25-19 hours (depends on dose) 0.5-5 hours Irreversibly acetylates and inactivates cyclooxygenase, and thereby inhibits platelet production of thromboxane A2. Beraprost sodium 1 hour 0.5 hour Reversibly exacerbates adenylcyclase activation (reversible within 8 hours). Clopidogrel 7-8 hours 2 hours Same with ticlopidine, but has less side effects such as severe neutropenia and thrombotic thrombocytopenic purpura than ticlopidine. Cilostazol 11-13 hours 3-6 hours Inhibition of phosphoestrase (reversible within 48 hours). Dilazep dihydrochloride 4 hours 0.5-1 hour Reversibly inhibits phosphoestrase. Dipyridamole 1.7 hours 2-3 hours Reversibly inhibits phosphoestrase and inhibits uptake of adenosine. Ethyl icosapentate < 24 hours Long Irreversibly inhibits thromboxane A2 production. Heparin 0.5-2.5 hours Immediately Activates antithrombin III, accerelates the rate of inhibiting clotting enzymes, particulary thrombin and factor Xa. Ifenprodil tartate 1.4 hour Short Inhibits binding to serotonin 5HT2 receptor. Nonsteroidal anti-inflammatory agents < 24 hours 0.5 hours Reversibly inhibit platelet cycloxygenase. Ozagrel sodium 1.5 hour Short Inhibits enzymatic synthesis of thromboxane. Sarpogrelate hydrochloride 0.7 hour 1.5 hour Reversibly inhibits binding to serotonin 5HT2 receptor as a selective antagonist. Ticlopidine 12.6 hours 6 hours Irreversibly inhibits the binding of adenosine diphosphate to platelet cell-surface adenosine diphosphate (P2) receptor, and the subsequent ADP-mediated activation of the glycoprotein IIb/IIIa receptor. Tirofiban 1.5-3 hours 0.1 hour Specific antagonist for glycoprotein IIb/IIIa receptor. Trapidil 2-4 hours 0.5-2 hours Reversibly inhibits phosphoestrase, reversibly inhibits thromboxane A2 production.

adenosine diphosphate (P2) receptor, and the subsequent ADP-mediated activation of the glycoprotein IIb/IIIa receptor. Tirofiban 1.5-3 hours 0.1 hour Specific antagonist for glycoprotein IIb/IIIa receptor. Trapidil 2-4 hours 0.5-2 hours Reversibly inhibits phosphoestrase, reversibly inhibits thromboxane A2 production. Triflusal 0.5 hour 24 hours Inhibits platelet arachidonic acid metabolism. Warfarin 36-42 hours 72-96 hours Prohibit the synthesis of Vitamin K dependent coagulation factor (II, VII, IX, X) in the liver Vitamin K is used as an antagonist. Informations obtained at http://www.rxlist.com, http://kimsonline.co.kr, and http://www.druginfo.co.kr. Warfarin A wide variation exists in managing warfarin before an endoscopic procedure, since the management of perioperative anticoagulation therapy for patients having a high risk of thromboembolism who are receiving long-term oral anticoagulant therapy is still uncertain. The prevalent approach is to discontinue oral anticoagulation therapy and initiate heparin therapy (Figure 1) [36]. Another potential strategy is to continue oral anticoagulation therapy with a temporary adjustment of warfarin intensity to a preprocedural INR. For example, a Western study showed a very low risk of bleeding when clips are applied immediately after polypectomy in anticoagulated patients at high risk of thrombosis [37]. It should be emphasized that recommended INR in the West is below 2.0 [2, 37, 38], while that of the East is below 1.5 for an endoscopic procedure [6].

ple, a Western study showed a very low risk of bleeding when clips are applied immediately after polypectomy in anticoagulated patients at high risk of thrombosis [37]. It should be emphasized that recommended INR in the West is below 2.0 [2, 37, 38], while that of the East is below 1.5 for an endoscopic procedure [6]. Figure 1 Recommendations on managing anticoagulants for high-risk patients undergoing high-risk procedures. (a) Warfarin substituted with intravenous heparin infusion. Warfarin should be stopped 3-5 days before the procedure, and be substituted with unfractionized heparin during the cessation period. (b) Warfarin substituted with subcutaneous heparin injection. Subcutaneous low molecular weighted heparin (LMWH) can be used instead of unfractionized heparin. Risk of thromboembolic complications must be carefully weighed against the increased risk of bleeding by maintaining anticoagulation.

ring the cessation period. (b) Warfarin substituted with subcutaneous heparin injection. Subcutaneous low molecular weighted heparin (LMWH) can be used instead of unfractionized heparin. Risk of thromboembolic complications must be carefully weighed against the increased risk of bleeding by maintaining anticoagulation. An appropriate prothrombin time-INR seems to be lower in the Easternist than in the Westernist. INR values between 1.6 and 2.6 seem optimal to prevent ischemic or haemorrhagic events in Japanese, because bleeding complications during warfarin medication seems to be higher in Japanese [39]. An INR of 2.0 to 3.0 in Caucasians is equal to an INR of 1.6 to 2.8 in Japanese when checked by the thrombo test. It has been also reported that lower intensity warfarin (INR 1.5 to 2.1) treatment may be safer for the secondary prevention of stroke in Japanese patients with nonvalvular atrial fibrillation with presumed cardioembolic transient ischemic attack or stroke within the previous 6 months, than conventional-intensity (INR 2.2 to 3.5) warfarin treatment, because major haemorrhagic complications are avoided [40]. In this multicenter, prospective, randomized trial, optimal intensity of warfarin therapy for secondary prevention of stroke in Japanese patients with nonvalvular atrial fibrillation was INR 1.5 to 2.1, showing a lesser major hemorrhagic complication than conventional INR 2.2 to 3.5 without difference in thromboembolic complication.

enter, prospective, randomized trial, optimal intensity of warfarin therapy for secondary prevention of stroke in Japanese patients with nonvalvular atrial fibrillation was INR 1.5 to 2.1, showing a lesser major hemorrhagic complication than conventional INR 2.2 to 3.5 without difference in thromboembolic complication. Such differences seem to come from the difference in warfarin metabolism between the Easterners and Westerners. Japanese patients receiving warfarin therapy had a significantly greater body weight-normalised plasma unbound clearance of S-warfarin than white patients [41, 42]. Caucasian and Japanese patients who carried CYP2C9 variants possessed a lower unbound oral clearance for S-warfarin (decreased metabolic activity), thereby required a smaller daily dose of the drug [42]. In addition, Japanese possessing the wild-type promoter and coding sequences had significantly greater CYP2C9 activity than white patients with the corresponding genotype [43]. Recently, a pharmacological dose algorithm for warfarin that uses genotypes from two genes (VKORC1 and CYP2C9) and clinical variables was developed to predict the stable therapeutic dose [42]. Those carrying the VKORC1 1173C/C wild-type allele needed higher unbound concentrations of S-warfarin to achieve a therapeutic anticoagulation response (reduced sensitivity), and a greater daily dose was required [42]. Apart from the genetic variability in determining the dose of warfarin, diet might play a role in bleeding time and in response to warfarin. Patients with high fish diets eat a lot of omega-3 fatty acid which inhibits thromboxane production. In addition, patients with low vitamin K level would require smaller dose of warfarin. Recently, an algorithm was developed for estimating the appropriate warfarin dose that is based on both clinical and genetic data from a broad population base [44].

t a lot of omega-3 fatty acid which inhibits thromboxane production. In addition, patients with low vitamin K level would require smaller dose of warfarin. Recently, an algorithm was developed for estimating the appropriate warfarin dose that is based on both clinical and genetic data from a broad population base [44]. Heparin Heparin should be stopped 4 to 6 hours before the procedure and restarted 2 to 6 hours later after the procedure if there is no evidence of bleeding (Figure 1) [36]. LMWH consisted of smaller fragments of heparin, has replaced heparin because of its advantages such as a better bioavailability and longer half-life after subcutaneous injection, dose-independent clearance, predictable anticoagulant response, lower risk of heparin-induced thrombocytopenia, and osteoporosis [45]. In addition, LMWH has been demonstrated to cause less bleeding that unfractionized heparin because it binds to platelets, and thus is less likely to interfere with the interaction between platelets and the vessel wall [45]. Moreover, an advantage of LMWH over unfractionated heparin is that perioperative conversion from warfarin therapy with LMWH can be carried out in the outpatient. However, lack of a readily available laboratory test for monitoring is a great disadvantage, since endoscopists cannot be certain of the amount of anticoagulant effect present at the time of procedure. Since antifactor Xa activity may persist up to 12 hours after a single subcutaneous injection, the best test to monitor activity is an antifactor Xa assay, but this takes a long process time [3, 45]. LMWH is only partially reversible by protamine sulfate, and there is no proven evidence in the safety and effect for GI endoscopy [46, 47].

activity may persist up to 12 hours after a single subcutaneous injection, the best test to monitor activity is an antifactor Xa assay, but this takes a long process time [3, 45]. LMWH is only partially reversible by protamine sulfate, and there is no proven evidence in the safety and effect for GI endoscopy [46, 47]. Aspirin There are Western reports showing that the administration of aspirin at standard dosages does not significantly increase the risk of bleeding after an endoscopic biopsy, colon polypectomy, or endoscopic sphincterotomy [48-52]. However, aspirin is known to cause a significant GI bleeding after endoscopic procedures in the Easternists, and thus recommended to stop not only for polypectomy but even for a biopsy [6-8]. A retrospective study from Honkong is the only Asian study that encouraged polypectomy without cessation of aspirin [50]. Of their 1657 subjects who underwent colon polypectomies, 127 subjects were taking aspirin, and 4.72% (6/127) revealed post-polypectomy bleeding. Notably, another study from Hongkong showed that aspirin therapy increased the risk of postsphincterotomy bleeding [53]. They reported that withholding aspirin for one week before endoscopic sphincterotomy did not seem to decrease the risk of post-sphincterotomy bleeding, because aspirin induces a long-lasting functional defect in platelets. These opposite results from Hongkong made problem unsolved, because the risk of postpolypectomy bleeding (1.4%) is known to be identical to that of postpolypectomy bleeding (1.4%) [54, 55]. Therefore, it is not strange that most of the Eastern endoscopists stop aspirin for a polypectomy [1].

tional defect in platelets. These opposite results from Hongkong made problem unsolved, because the risk of postpolypectomy bleeding (1.4%) is known to be identical to that of postpolypectomy bleeding (1.4%) [54, 55]. Therefore, it is not strange that most of the Eastern endoscopists stop aspirin for a polypectomy [1]. Actual effects of aspirin are likely to differ by race and ethnicity. Although Western reports showed that aspirin does not affect GI bleeding time [48], significant prolongation was noted in colonic bleeding time after aspirin ingestion in Easternists [56]. Indeed, the threshold of antiplatelet therapy for Asian populations is considered to be 2 to 5 times higher than those for the US population, because of higher risks of hemorrhagic complications [57]. In addition, it has been reported that prevalences of gastrodeuodenal mucosal injury and bleeding are higher in Japanese receiving antiplatelet agents [58]. The higher estimated incidence of major GI bleeding might be related to the higher prevalence of H. pylori infection in this population. In a study case-control study of 695 consecutive users of low-dose aspirin with upper GI bleeding, H. pylori infection was identified as an independent risk factor of upper GI bleeding [59]. Besides, prophylaxis with a PPI effectively prevents recurrent upper GI bleeding with low-dose aspirin, despite failure of H. pylori eradication and concomitant use of NSAIDs [32]. Taken altogether, recommended dose of aspirin should be smaller in the Easternists because low-dose aspirin (75-150 mg) is effective, and because high-dose aspirin (> 100-200 mg) produces double rate of bleeding compared with low-dose aspirin (75-100 mg) per day [24, 57].

eradication and concomitant use of NSAIDs [32]. Taken altogether, recommended dose of aspirin should be smaller in the Easternists because low-dose aspirin (75-150 mg) is effective, and because high-dose aspirin (> 100-200 mg) produces double rate of bleeding compared with low-dose aspirin (75-100 mg) per day [24, 57]. Notably, there were few Western studies that showed bleeding risks during aspirin intake. Minor bleeding occurred after biopsy or polypectomy at EGD or colonoscopy in 20 of 320 (6.3%) patients who had recently consumed aspirin or NSAIDs compared with 8 of 374 (2.1%) patients who had not [49]. Even in low-dose aspirin, major right sided colonic hemorrhage occurred after hot biopsy of diminutive colonic polyps [60]. Among elderly patients, the odd ratios of bleeding with daily doses of aspirin of 75, 150, and 300 mg were 2.3, 3.2, and 3.9, respectively [61]. In this study, the use of low-dose aspirin was associated with a two to fourfold greater risk of upper GI event, which is not reduced by the use of buffered or enteric-coated preparations [32, 61].

, the odd ratios of bleeding with daily doses of aspirin of 75, 150, and 300 mg were 2.3, 3.2, and 3.9, respectively [61]. In this study, the use of low-dose aspirin was associated with a two to fourfold greater risk of upper GI event, which is not reduced by the use of buffered or enteric-coated preparations [32, 61]. Non-aspirin NSAIDs Both the Eastern and Western endoscopists do not stop NSAIDs for endoscopic procedure [1], NSAIDs does not appear to prolong mucosal bleeding time [62], and does not increase the incidence of major bleeding [49]. Among non-aspirin-NSAIDs, aceclofenac had the lowest risk of upper GI bleeding, whereas ketorolac had the highest [63]. In this study, rofecoxib increased the risk of upper GI bleeding, whereas celecoxib, paracetamol or concomitant use of a PPI with an NSAID presented no increased risk. However, when combined with low-dose aspirin, the differences between non-selective NSAIDs and coxibs tend to disappear [63]. An apparent interaction was found between low-dose aspirin and use of non-aspirin-NSAIDs, coxibs or thienopyridines, which increased risk of upper GI bleeding. Treatment with either non-aspirin antiplatelet or cardioprotective aspirin has a similar risk of upper GI bleeding [32, 63].

and coxibs tend to disappear [63]. An apparent interaction was found between low-dose aspirin and use of non-aspirin-NSAIDs, coxibs or thienopyridines, which increased risk of upper GI bleeding. Treatment with either non-aspirin antiplatelet or cardioprotective aspirin has a similar risk of upper GI bleeding [32, 63]. Platelet cell-surface adenosine diphosphate receptor antagonist (ticlopidine, clopidogrel, dipyridamole) Platelet cell-surface adenosine diphosphate receptor antagonist includes thinopyridines (ticlopidine and clopidogrel) and dipyridamole. Ticlopidine and clopidogrel are recommended to be ceased 7 to 10 days before the procedure [3, 14]. In a recent European publication, clopidogrel is recommended to be ceased only for the high-risk procedures [5]. Besides, Japanese studies recommends 3 days of cessation period for aspirin, 5 days for ticlopidine, and 7 days for dual aspirin and ticlopidine therapy even for a biopsy [6, 64, 65]. Notably, dipyridamole does not increase bleeding risk, even with a dual antiplatelet therapy with aspirin [66].

igh-risk procedures [5]. Besides, Japanese studies recommends 3 days of cessation period for aspirin, 5 days for ticlopidine, and 7 days for dual aspirin and ticlopidine therapy even for a biopsy [6, 64, 65]. Notably, dipyridamole does not increase bleeding risk, even with a dual antiplatelet therapy with aspirin [66]. Ticlopidine are more likely to induce endoscopic evidence of mucosal damage than patients taking aspirin or NSAIDs [28]. Besides, substitution of clopidogrel for aspirin is not recommended to reduce the risk of recurrent ulcer bleeding in high-risk patients, because their anti-angiogenic effects may impair healing of gastric erosions or small ulcerations that develop from other medications or H. pylori infection [32]. In summary, to minimize GI bleeding, history of ulcer and other GI risk factors should be assessed first, followed by test for H. pylori and treat if infected. PPI reduces the bleeding risk in most cases [32, 63].

of gastric erosions or small ulcerations that develop from other medications or H. pylori infection [32]. In summary, to minimize GI bleeding, history of ulcer and other GI risk factors should be assessed first, followed by test for H. pylori and treat if infected. PPI reduces the bleeding risk in most cases [32, 63]. Glycoprotein IIb/IIIa receptor antagonist (etifibatide, abciximab, tirofiban) Glycoprotein IIb/IIIa receptor antagonists are intravenously administered drugs given as a bolus followed by a continous infusion in acute coronary syndrome. There is no guideline for glycoprotein IIb/IIIa receptor antagonists, and it seems that a considerable number of endoscopists are unaware of these drugs [1]. Platelet functions are known to be recovered within 4 hours after the cessation of tirofiban and eptifibatide, whereas 24 hours are required for abciximab [12]. The antiplatelet effects can be partially reversed by platelet infusions or desmopressin (DDAVP) [3].

le number of endoscopists are unaware of these drugs [1]. Platelet functions are known to be recovered within 4 hours after the cessation of tirofiban and eptifibatide, whereas 24 hours are required for abciximab [12]. The antiplatelet effects can be partially reversed by platelet infusions or desmopressin (DDAVP) [3]. Collaboration between the endoscopist and physicians prescribing drugs Collaboration between the endoscopists and other engaged physicians (cardiologists, neurologists, surgeons, or general practitioners) are important to balance the risks of bleeding and thrombosis when deciding the cessation of antiplatelets and/or anticoagulants. Such collaboration will help endoscopists to make decision more appropriately by minimizing cardiovascular concerns. However, it seems that only 61.9% of Eastern endoscopists and 56.0% of Western endoscopists usually consult with the referring physician who prescribed the medications [1]. Reasons why many endoscopists do not consult primary care physicians about management of antiplatelet and/or anticoagulation medications might be related to (1) patient factors; (2) endoscopist factor; and (3) external factor [67]. First, some people take aspirin simply for cardioprotective reasons without significant disease, and thus endoscopists do not consult primary physicians in such situations. Second, endoscopists usually do not perform high-risk endoscopic procedures on patients taking warfarin except in emergency situations. That is, endoscopists usually defer elective procedures in high-risk patients. Third, there is no appropriate guideline that covers the exact situation in each patient. Personal experience seems to be more powerful driver of practice than published literature as noted in our survey [1].

except in emergency situations. That is, endoscopists usually defer elective procedures in high-risk patients. Third, there is no appropriate guideline that covers the exact situation in each patient. Personal experience seems to be more powerful driver of practice than published literature as noted in our survey [1]. Not knowing the importance of collaboration and measuring the risks/benefits in each individual patient on a case-by-case basis, it would be very difficult to estimate the risks. A schema weighting the risk of bleeding and thromboembolism might be suggested (Figure 2) considering the differences between the East and West (Table 4).

he importance of collaboration and measuring the risks/benefits in each individual patient on a case-by-case basis, it would be very difficult to estimate the risks. A schema weighting the risk of bleeding and thromboembolism might be suggested (Figure 2) considering the differences between the East and West (Table 4). Figure 2 Recommendation on managing antiplatelets for GI endoscopy by risk stratification. For low-risk procedures, GI endoscopy could be performed without discontinuing the antiplatelets. If the patient is Easternist, taking dual therapy with anticoagulants or other antiplatelets, has a history of GI bleeding or ulcer disease, H. pylori infection, or other conditions that increase the risk of bleeding, cessation of drugs should be considered (marked as “Step up” with an arrow in the schema). For high-risk procedures with more than 1% of bleeding complication rate, cessation of antiplatelets should be considered at least 1 week before the procedure, and restarted when there is no evidence of bleeding. If the patient is Westernist, has atrial fibrillation, venous thromboembolism, valvular heart disease, mechanical valve, ischemic heart disease, coronary artery stents, past history of thromboembolism, or other conditions that increase the risk of thromboembolism, shorter cessation of antiplatelets should be considered (marked as “Step down” with an arrow in the schema). The degree of stepping up and down should be decided according to the number and severity of risk factors written above.

history of thromboembolism, or other conditions that increase the risk of thromboembolism, shorter cessation of antiplatelets should be considered (marked as “Step down” with an arrow in the schema). The degree of stepping up and down should be decided according to the number and severity of risk factors written above. Table 4 Summary on major differences between the East and West East West Risk of embolism Lower than the Westerners. Common form is cerebrovascular variety that may lead to death or disability. Higher than the Easterners. Common form is cardiovascular variety including deep vein thrombosis. Risk of bleeding Higher than the Westeners due to different drug metabolism (greater body weight-normalized plasma unbound clearance of drug) and higher rate of H. pylori infection. Lower than the Easterners. Tolerates well with low-risk endoscopic procedures during antiplatelet and/or anticoagulant medications. Managing warfarin Lower international normalized ratio value (1.6-2.6) than the Westerners are appropriate for prophylaxis of thromboembolism. Tolerates well with low-risk procedures (endoscopic biopsy) without significant bleeding. Managing aspirin Lower dose is recommended than the Westerners due to higher risk of bleeding. Tolerates well with few high-risk procedures (endoscopic sphincterotomy and colon polypectomy) without significant bleeding. Conclusions There are significant differences of opinion and practice patterns between Eastern and Western endoscopists based on significant differences on embolism, bleeding, and drug metabolism. The ratio of cerebral infarction to cardiovascular thromboembolism is different, and the recommended doses of antiplatelet and anticoagulant are smaller in the East due to different drug metabolism. The risk/benefit ratio of antiplatelets and anticoagulants may differ across population, and thus higher bleeding tendency due to higher concentration of drugs in Easterners should be considered.

erent, and the recommended doses of antiplatelet and anticoagulant are smaller in the East due to different drug metabolism. The risk/benefit ratio of antiplatelets and anticoagulants may differ across population, and thus higher bleeding tendency due to higher concentration of drugs in Easterners should be considered. Increased use of anticoagulants and antiplatelets will keep this as an argument not only to the GI endoscopists but also to the physicians prescribing these drugs. Both the concerns of endoscopists who want to stop medications to lessen the risk of hemorrhage, and the concerns of prescribing physicians who want to continue medications because of a fear of thromboembolic events, should always be considered based on patient’s individual characteristics such as a racial difference. Being aware of such wide variation between the East and West will be helpful in weighing the risk of bleeding and thromboembolism, when managing antiplatelets and anticoagulants for GI endoscopy. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (KRF 2008-531-E00030). The author declares no conflict of interests related to this article.

Wireless Capsule Endoscopy (WCE) is a recently developed noninvasive technique for imaging of small bowel pathologies. In 1995, Gavriel Idan first presented the idea of WCE to Applitec Ltd. It was improved by “Given Imaging Research and Development” in Israel in 1999. Later it was approved in Western countries in 2001. WCE is a swallowable wireless mini-camera for getting images of the gastrointestinal (GI) mucosa [1-3]. The system has an external receiving antenna with attached portable hard disc drive (data recorder) and customized PC Workstation. Data recorder records the views for 8 hours. During this period patient can continue working. At the end of this period, data is downloaded to the computer, and doctor analyses the results, generally, this takes 1 hour. Widely accepted indications of WCE are as follows [1-3], obscure bleeding; iron deficiency anemia; crohn disease; abdominal pain; polyposis coli; celiac disease; Small bowel tumors. In some selected patients, it can be used to monitor the small bowel in renal/bone marrow transplanted patients, in management of graft versus-host disease, monitoring deleterious effects of drugs (Nonsteroid anti-inflammatory drugs), after small bowel transplantation and in chronic refractory pouchitis [4-6].

Widely accepted indications of WCE are as follows [1-3], obscure bleeding; iron deficiency anemia; crohn disease; abdominal pain; polyposis coli; celiac disease; Small bowel tumors. In some selected patients, it can be used to monitor the small bowel in renal/bone marrow transplanted patients, in management of graft versus-host disease, monitoring deleterious effects of drugs (Nonsteroid anti-inflammatory drugs), after small bowel transplantation and in chronic refractory pouchitis [4-6]. Although capsule may retain safely for a long time in a small bowel [7], obstruction must be excluded before CE with small bowel radiography or computerized tomography (CT) with oral contrast when indicated. Retained capsule is the indication for surgery, but nowadays it can be taken out by double balloon enteroscopy (DBE) (Fig. 1a-c). Retained capsule also can be a positive sign in the malign obstruction for surgery. All these findings show that retained capsule which is the main complication of CE, is not accepted as a complication nowadays. In our experience, to exclude obstructive lesions, we prefer CT enteroclysis. Small bowel radiography can miss obstruction. In our cases, capsule retained in 4 out of 86 patients, one was out after one week, two underwent operation (one for multiple strictures, one for adenocancer), and one was taken out by DBE. All of these patients had small bowel radiogram which was reported normal. We performed CE, in spite of the high risk retention in only one patient who had multiple strictures. After CE retention, we should wait at least two weeks. CT enteroclysis is not only to show obstructive lesions but also to help exclude other pathologies in abdomen which can cause external impression, such as extraintestinal tumors, lympadenopathy, lymphoma.

k retention in only one patient who had multiple strictures. After CE retention, we should wait at least two weeks. CT enteroclysis is not only to show obstructive lesions but also to help exclude other pathologies in abdomen which can cause external impression, such as extraintestinal tumors, lympadenopathy, lymphoma. Figure 1 Capsule endoscopic view of a 31 years old patient diagnosed of diaphragm diseased caused by NSAID. (a), capsule retained in narrowed ulcer area. (b, c), taking out of retained capsule by DBE. Contraindications of WCE are as follows. The absolute contraindications are Stricture, obstruction, fistulas, widespread Crohn disease, swallowing problems, pseudoobstruction, Motility problems. The relative contraindications are pregnancy, long term use of NSAIDs, large diverticula, Zenker diverticula, gastroparesia, history of surgical operations. Bowel preparation is important for the success of procedure. Good preparation is also important for good visualization. The diagnostic yield of CE depends on the quality of visualization of the small-bowel wall and complete passage through the small bowel. Although one multicenter randomized controlled trial including 129 patients did not support the recommendation of small-bowel preparation with oral NaP for CE exploration in patients with occult gastrointestinal bleeding [8]. In most studies, bowel preparation offers better visualization than overnight fasting alone and is associated with fewer disturbances due to intraluminal turbid fluid. Because of this, bowel preparation is recommended before capsule endoscopy [9-12].

CE exploration in patients with occult gastrointestinal bleeding [8]. In most studies, bowel preparation offers better visualization than overnight fasting alone and is associated with fewer disturbances due to intraluminal turbid fluid. Because of this, bowel preparation is recommended before capsule endoscopy [9-12]. Currently, obscure GI bleeding is the most common indication of CE. The diagnostic yield of CE has been reported to be 38% to 93% and most common lesions in patients with obscure bleeding are as follows [13-16], angioectasia, tumor, varices, diverticula and ulcer. CE has a high diagnostic yield (91.9%) in patients with overt active bleeding and the most common findings were in order of angiodysplasia, ulcer, polyp, tumor [17]. Hartmann et al [18] showed that CE (74.4%) has same diagnostic value with intraoperative enteroscopy (72.3%). They evaluated forty-seven consecutive patients with obscure GI bleeding (11 with ongoing overt bleeding, 24 with previous overt bleeding, and 12 with obscure-occult bleeding) from German gastroenterology centers. In comparison to intraoperative enteroscopy (IOE), the sensitivity, specificity, positive and negative predictive values of capsule endoscopy were 95%, 75%, 95% and 86%, respectively. Diagnostic yield was 100% in overt bleeding by CE.

bleeding, and 12 with obscure-occult bleeding) from German gastroenterology centers. In comparison to intraoperative enteroscopy (IOE), the sensitivity, specificity, positive and negative predictive values of capsule endoscopy were 95%, 75%, 95% and 86%, respectively. Diagnostic yield was 100% in overt bleeding by CE. The suspected blood indicator (SBI) feature of CE was developed for rapid screening of intestinal lesions with bleeding potential. However, it is not useful in routine practice. Buscaglia et al [19] showed that the performance characteristics of the currently available SBI feature in CE are suboptimal and insufficient to screen for lesions with bleeding potential. The sensitivity and positive predictive value were low for actively bleeding lesions (58.3% and 70%, respectively). The sensitivity was 58.3% and 41.3% for obscure GI bleeding and anemia, respectively. CE is also useful for the long-term outcome of obscure GI bleeding. Lai et al [20] followed 49 patients for a median of 19 months (12-31 months), 63.3% of these patients were capsule positive (possible bleeding lesions were detected) and 36.7% were capsule negative. Rebleeding rate was 32.7% in the first year [17]. They showed that the patients with obscure GI bleeding and negative CE had a very low rebleeding rate. CE is accepted as the first line technique in patients with obscure bleeding in American Gastroenterological Association algorithm (Fig. 2) [21]. Figure 2 Algorithm for the diagnosis and management of obscure GI bleeding (Adapted from AGA algorithm) [21].

CE is also useful for the long-term outcome of obscure GI bleeding. Lai et al [20] followed 49 patients for a median of 19 months (12-31 months), 63.3% of these patients were capsule positive (possible bleeding lesions were detected) and 36.7% were capsule negative. Rebleeding rate was 32.7% in the first year [17]. They showed that the patients with obscure GI bleeding and negative CE had a very low rebleeding rate. CE is accepted as the first line technique in patients with obscure bleeding in American Gastroenterological Association algorithm (Fig. 2) [21]. Figure 2 Algorithm for the diagnosis and management of obscure GI bleeding (Adapted from AGA algorithm) [21]. Clinical use of CE in inflammatory bowel disease is limited. Diagnosis of Crohn disease (CD) is based on clinical, endoscopic, radiologic, histologic and biochemical tests. Sometimes diagnosis can be a problem and CE may be useful for diagnosis in non-stricturing small bowel CD. Capsule retention rate is approximately 1.5% in patients with suspected CD and 5-13% in patients with diagnosed CD. Although Triester et al [22] showed that CE is superior to alternative imaging methods for diagnosing non-stricturing small bowel CD in their meta-analysis of 16 studies, it should be remembered that CE shows only the presence of ulcers. The other causes, such as NSAIDs (Fig. 3) and infections, must be excluded before making a diagnosis of Crohn disease. However, in clinical practice, radiographic studies should be performed before CE to exclude obstruction. Therefore, very few patients with CD need CE for diagnosis. In the follow-up period, CE may also be useful in CD patients. It is an effective, safe and well-tolerated method for detecting lesions after surgery (recurrence of CD). It may be helpful for the management of treatment [23]. CE should be considered in ulcerative colitis patients with atypical clinical features [24].

. In the follow-up period, CE may also be useful in CD patients. It is an effective, safe and well-tolerated method for detecting lesions after surgery (recurrence of CD). It may be helpful for the management of treatment [23]. CE should be considered in ulcerative colitis patients with atypical clinical features [24]. Figure 3 A 61 years old man diagnosed with obscure GI bleeding caused by chronic use of NSAID, showing diaphragm ulcer. CE has a low yield for evaluation of abdominal pain or diarrhea and cannot be recommended as a first-line test without further studies in patients with only symptom of chronic abdominal pain [25]. May et al [26] evaluated 50 patients with chronic abdominal pain and “plus” symptoms (weight loss, inflammation markers, anemia, or suspected mid-GI bleeding, diarrhea) in a prospective multicenter trial. The additional symptoms or signs of inflammation were associated with the highest diagnostic yield (odds ratio 3.2). They concluded that strict patient selection on the basis of additional symptoms or signs is the key for increasing the yield of CE in patients with chronic abdominal pain and inflammation. GI polyposis syndromes are defined as the presence of multiple polypoid lesions in the GI system. Most of these syndromes are inherited and associated with increased risk of cancer. Endoscopic and intraoperative resection of polyps is recommended. CE is an alternative imaging technique for surveillance in patients with hereditary polyposis. CE has high diagnostic yield for small polyps (< 15 mm), but larger polyps can be missed [27].

omes are inherited and associated with increased risk of cancer. Endoscopic and intraoperative resection of polyps is recommended. CE is an alternative imaging technique for surveillance in patients with hereditary polyposis. CE has high diagnostic yield for small polyps (< 15 mm), but larger polyps can be missed [27]. Serology and biopsy is important for the diagnosis of celiac disease and CE is unlikely to replace completely duodenal biopsies, but it produces high quality images of small intestine. It may be useful in assessing patients with celiac disease, and may show entire small bowel and complications of celiac disease (ulcerative jejunoileitis and lymphoma) [28]. Biopsy is not ideal as the gold standard for diagnosis of celiac disease. Mucosal lesions may be patchy thus missed by biopsy and gastroscopy. Rondonotti et al [29] evaluated the potential of videocapsule endoscopy in assessing the severity and extent of mucosal changes in patients with suspected celiac disease. They reported that celiac disease has a high sensitivity (87.5%) and specificity (90.9%) for the detection of villous atrophy in patients with suspected celiac disease. CE may be an option to recognize villous atrophy in patients with a positive endomysial antibody test who are unwilling, or unable to have a gastroscopy [30].

ported that celiac disease has a high sensitivity (87.5%) and specificity (90.9%) for the detection of villous atrophy in patients with suspected celiac disease. CE may be an option to recognize villous atrophy in patients with a positive endomysial antibody test who are unwilling, or unable to have a gastroscopy [30]. Small bowel malignencies are uncommon and account for less than 6% of all the GI neoplasms [31]. Detection rate has been limited by the inability to endoscopically examine the entire small intestine. Now, it is able to diagnose small bowel malignancies by CE and double balloon enteroscopy (DBE). Most common lesions of small bowel detected by DBE are adenocarcinoma, gastrointestinal stromal tumor, neuroendocrine tumor, lymphoma, cavernous hemangioma, lipoma and hamartoma [32]. Percentages of these lesions are variable in different series. CE has some potential limitations when used to diagnose small bowel tumors, these limitations are inability to provide histological confirmation, differantiation between malign and benign lesion, missed polypoid lesions [33, 34]. Ross et al [32] evaluated 183 patients presented obscure GI bleeding by DBE. A small bowel mass lesion was detected in 18 patients, 15 of them had prior CE, and mass lesion was identified in 5 patients by CE, but it failed to identify all four cases of primary SB adenocarcinoma. Pasha et al [35] reported a meta-analysis of 11 studies that compares CE and DBE. Summary of the study are shown in figure 4. They found similar diagnostic capability for both.

f them had prior CE, and mass lesion was identified in 5 patients by CE, but it failed to identify all four cases of primary SB adenocarcinoma. Pasha et al [35] reported a meta-analysis of 11 studies that compares CE and DBE. Summary of the study are shown in figure 4. They found similar diagnostic capability for both. Figure 4 Comparison of CE and DBE (with kindly permission of author’s) [35] In another study, it was found that an initial DBE is a cost effective approach for patients with obscure bleeding. But they concluded that capsule directed DBE may be associated with better long-term outcomes because of the potential for fewer complications [36]. In conclusion, bowel preparation before capsule endoscopy is recommended for good visualization (therefore with higher diagnostic yield); the main indication for CE is obscure GI bleeding; in selected cases, CE can be useful for the other indications; large polypoid lesions may be missed by CE; diagnostic capabilities of CE and DBE are similar; CE is a good complemantary method for DBE. Acknowledgements The authors declare no conflict of interests related to this article.

Introduction Hepatocellular carcinoma (HCC) is one of the most common cancers with an incidence of 4 to 15 per 100,000 in Western countries, compared with 120 per 100,000 in Asia and Africa [1, 2]. Although advanced research in the clinical study of HCC has been made, the prognosis remain poor, HCC is one of the leading causes of worldwide cancer mortality, with an estimated number of 1 million annually deaths and a 5-year survival rate of less than 5% [3, 4]. Notably, men are about three to five times more likely to develop HCC than women [5]. Hormonal factors are underlining the hepatocarcinogenesis. Sex disparity characteristic in HCC could be attributed by both sex hormone pathways, with distinct role in each sex [6]. The increased activity of estrogens in female patients, which might protect them form carcinogenesis process [6, 7]. Therefore, up-regulation of the androgen pathways in male patients is considered to accelerate liver carcinogenesis [6]. Recently, it was demonstrated that estrogens can protect hepatocytes from malignant transformation via down-regulation of the secretion of IL-6 from Kupffer cells, a critical process in the diethylnitrosamine-induced HCC in mouse model [5, 7]. Chronic infections with the hepatitis B virus (HBV) and the hepatitis C virus (HCV) have been involved in about 80% of cases worldwide of HCC; also environmental risk factors, including alcohol abuse or dietary intake of food contaminant like aflatoxin B1, were proven to be significant [8].

Introduction Hepatocellular carcinoma (HCC) is one of the most common cancers with an incidence of 4 to 15 per 100,000 in Western countries, compared with 120 per 100,000 in Asia and Africa [1, 2]. Although advanced research in the clinical study of HCC has been made, the prognosis remain poor, HCC is one of the leading causes of worldwide cancer mortality, with an estimated number of 1 million annually deaths and a 5-year survival rate of less than 5% [3, 4]. Notably, men are about three to five times more likely to develop HCC than women [5]. Hormonal factors are underlining the hepatocarcinogenesis. Sex disparity characteristic in HCC could be attributed by both sex hormone pathways, with distinct role in each sex [6]. The increased activity of estrogens in female patients, which might protect them form carcinogenesis process [6, 7]. Therefore, up-regulation of the androgen pathways in male patients is considered to accelerate liver carcinogenesis [6]. Recently, it was demonstrated that estrogens can protect hepatocytes from malignant transformation via down-regulation of the secretion of IL-6 from Kupffer cells, a critical process in the diethylnitrosamine-induced HCC in mouse model [5, 7]. Chronic infections with the hepatitis B virus (HBV) and the hepatitis C virus (HCV) have been involved in about 80% of cases worldwide of HCC; also environmental risk factors, including alcohol abuse or dietary intake of food contaminant like aflatoxin B1, were proven to be significant [8]. HCC is usually diagnosed at an advanced stage resulting in limited therapeutic options and poor prognosis. The identification of prognostic biomarkers is an important issue since such markers could facilitate early detection of HCC. Furthermore, such biomarkers could display potential therapeutic targets for HCC.

Chronic infections with the hepatitis B virus (HBV) and the hepatitis C virus (HCV) have been involved in about 80% of cases worldwide of HCC; also environmental risk factors, including alcohol abuse or dietary intake of food contaminant like aflatoxin B1, were proven to be significant [8]. HCC is usually diagnosed at an advanced stage resulting in limited therapeutic options and poor prognosis. The identification of prognostic biomarkers is an important issue since such markers could facilitate early detection of HCC. Furthermore, such biomarkers could display potential therapeutic targets for HCC. Tumorigenesis As for most types of cancer, hepatocarcinogenesis is a multi-step process involving different genetic alterations that ultimately lead to malignant transformation of the hepatocyte [1, 3]. A malignant cell phenotype is initiated when mutant hepatocytes produce mitogens that activate cellular receptors and intracellular signalling pathways. Genetic and molecular abnormalities generally associated with viral infection or due to the inflammatory condition represent an early step in hepatocarcinogenesis [9]. HCC tumorigenesis includes alteration in cellular proliferation markers, cell cycle regulators, suppressor genes, oncogenes and their receptors, apoptosis related factors as well as modification of genes involved in angiogenesis and immune response as shown in Figure 1. However specific and molecular pathways that play pivotal role in the liver tumour development were not fully identified [4, 10-12]. Figure 1 Molecular pathways involved in HCC tumorigenesis

HCC tumorigenesis includes alteration in cellular proliferation markers, cell cycle regulators, suppressor genes, oncogenes and their receptors, apoptosis related factors as well as modification of genes involved in angiogenesis and immune response as shown in Figure 1. However specific and molecular pathways that play pivotal role in the liver tumour development were not fully identified [4, 10-12]. Figure 1 Molecular pathways involved in HCC tumorigenesis Epigenetics alteration pathways Epigenetic alteration pathways may be a consequence of the normal aging process, persistent viral infection, and chronic inflammation. The epigenetic pathways are characterized by three main mechanisms: DNA hypermethylation leading to gene inactivation, DNA hypomethylation causing genomic instability, histone modifications affecting chromatin conformation [9, 13-15]. DNA methylation is the most frequent epigenetic alteration seen in mammalian genome, and it frequently mediates transcriptional repression [13]. DNA methylation occurs in different stages of liver disease (noncirrhosis, cirrhosis and HCC) [11]. It was observed that persistent viral infection, particularly HCV accelerates age-related methylation in the liver, suggesting the role in the pathogenesis of HCC [15]. Recent study demonstrated that both DNA hypomethilation and CpG hypermethilation are the dominant event during HCC development and progression [9, 11].

It was observed that persistent viral infection, particularly HCV accelerates age-related methylation in the liver, suggesting the role in the pathogenesis of HCC [15]. Recent study demonstrated that both DNA hypomethilation and CpG hypermethilation are the dominant event during HCC development and progression [9, 11]. Therefore, epigenetic changes may serve as indicator or biomarker for screening of patients with an increased risk for HCC [14]. Therapeutic strategies being able to modify the methylation status or multikinase inhibitors of liver cancer cells and to target tumor suppressor genes may be highly beneficial in the treatment of human HCC [13, 16, 17]. Tumor suppressor genes Tumor suppressor genes represent genes that are likely to play a role negatively regulating cell growth. Loss or inactivations of these genes are associated with malignancy and carcinogenesis process. Apart from deletions and mutations, growing evidence has indicated that epigenetic alterations are implicated in inactivation of tumor suppressor genes [13, 18].

t are likely to play a role negatively regulating cell growth. Loss or inactivations of these genes are associated with malignancy and carcinogenesis process. Apart from deletions and mutations, growing evidence has indicated that epigenetic alterations are implicated in inactivation of tumor suppressor genes [13, 18]. Like in other cancer types, DNA methylations of the tumour suppressor genes have been reported in HCC [13]. A correlation between hypermethylation expression and down-regulation of E-cadherin was reported in HCC. Decreased E-cadherin expression is correlated with epithelial-to-mesenchymal transition and metastasis and poor prognosis [13]. In addition, reduced or loss of E-adhering expression is mainly caused by aberrant CpG hypermethylation with a detectable frequency from 33% to 67%. Another study [19] described that loss of E-cadherin was closely associated with loss of heterozygosity of E-cadherin [14]. More in human HCC tissue was registered correlation among reactive species, E-cadherin regulation [13], Ras upstream inducer or downstream effectors [19].

h a detectable frequency from 33% to 67%. Another study [19] described that loss of E-cadherin was closely associated with loss of heterozygosity of E-cadherin [14]. More in human HCC tissue was registered correlation among reactive species, E-cadherin regulation [13], Ras upstream inducer or downstream effectors [19]. Deletion at 17p and alterations of the p53 gene at 17p13 are common genetic changes reported in human cancers. The tumor suppressor gene p53 encodes a 53-kD nuclear phosphoprotein that acts as a transcription factor. The major functions of the gene are blockage of cell cycle progression in response to DNA damage, and mediation of DNA repair or apoptosis [18, 20, 21]. Mutations in the p53 gene abrogated its normal functions, leading to genomic instability and loss of growth control, p53 overexpression may be involved in determining the differentiation and the proliferative activity in many cancers including HCC [21-24]. Determination of p53 antigen and anti-p53 antibodies was proved to have a sensitivity of 41.1% in the diagnosis of HCC, and the over-expression of p53 in the serum or liver tissues of HCC patients prefigures the poorer prognosis and a shorter survival time [24].

iferative activity in many cancers including HCC [21-24]. Determination of p53 antigen and anti-p53 antibodies was proved to have a sensitivity of 41.1% in the diagnosis of HCC, and the over-expression of p53 in the serum or liver tissues of HCC patients prefigures the poorer prognosis and a shorter survival time [24]. Cell cycle regulators Disruption of the G1/S and G2/M check points leads to uncontrolled cell growth, resulting in the development and progression of cancers [25-27]. Overexpression of cyclin A, cyclin D, and cyclin E have been found correlate with the tumor relapse of human HCC, and are independent predictive markers for their recurrence and prognosis [27]. Liver cyclin proteins in general and cyclin D1 in particular may be considered as potential target for preventive and therapeutic strategies liked with the progression of the disease and culminating with HCC [25]. Amplification of the cyclin D1 gene and its overexpression was associated with aggressive forms of HCC [25, 26]. Experimental evidence also shows that cyclin D1 expression is sufficient to promote cell cycle progression in the absence of mitogen factor. In another in vitro study protein levels and activities of cyclin D1, E, Cdk4, cyclin A and Wee1 increased proportionally with the development of HCC, especially in the transition process from chronic hepatitis to HCC. Cdk6 and Cdk7 activities remained unchanged in the process from normal liver to HCC. These data suggest that the increase in Cdc2 kinase may play a role in the process from normal liver to chronic hepatitis, whereas the predominant increase in cyclin D1, Cdk4, cyclin E, cyclin A, and Wee1 suggests involvement not only in the process from normal liver to chronic hepatitis, but also during transition into HCC [27].

suggest that the increase in Cdc2 kinase may play a role in the process from normal liver to chronic hepatitis, whereas the predominant increase in cyclin D1, Cdk4, cyclin E, cyclin A, and Wee1 suggests involvement not only in the process from normal liver to chronic hepatitis, but also during transition into HCC [27]. The p27 protein is a member of cyclin/cyclin-dependent kinase inhibitors involved in cell-cycle progression. p27 also promotes cell migration in metastatic HCC cells through the regulation of RhoA activity [28]. Reduced p27 expression correlates with poor prognosis meanwhile high p27 expression, correlated with prolonged survival, used as prognostic parameter for HCC [11, 13]. Recent findings demonstrate p73 accumulation in HCC, suggesting that p73 plays a role in the malignant phenotype. p73 expression status is related to prognosis of HCC patients [26, 27, 29]. Oncogenes and their receptors Proto-oncogenes encode a wide range of proteins products involved in the control of cell proliferation and differentiation, including growth factors, growth factors receptors, components of signal transduction pathways and transcription factors [30]. Aberrations of many oncogens were identified in HCC being associated with poor prognosis [12, 23].

range of proteins products involved in the control of cell proliferation and differentiation, including growth factors, growth factors receptors, components of signal transduction pathways and transcription factors [30]. Aberrations of many oncogens were identified in HCC being associated with poor prognosis [12, 23]. The overexpression of epidermal growth factor (EGF) and epidermal growth factor receptors (EGFR) were observed in HCC [25, 31], being associated with late-stage disease, increased cell proliferation and degree of tumor differentiation [21]. EGFR can be considered as a marker for predicting the metastasis and recurrence of HCC [32, 33]. Because of high prevalence of EGFR overexpression in HCC, inhibitors of EGF-EGFR pathways are potential therapeutic agents [31, 33]. Studies of EGFR inhibitors in vitro, phase I or phase II studies were encouraging in HCC therapy [34, 35].

idered as a marker for predicting the metastasis and recurrence of HCC [32, 33]. Because of high prevalence of EGFR overexpression in HCC, inhibitors of EGF-EGFR pathways are potential therapeutic agents [31, 33]. Studies of EGFR inhibitors in vitro, phase I or phase II studies were encouraging in HCC therapy [34, 35]. Transforming growth factor b1 (TGF-β1) is a potent growth factor inhibitor for most epithelial cells, involved in cell proliferation, migration, and differentiation and in response to injury [23]. TGF-β1 in human HCC cells displays significant intracellular expression, by autocrine stimulatory mechanism [2, 36]. It has been reported that TGF-β1 and TGF-β1 mRNA levels were significantly higher in the serum of patients with HCC compared to patients with non-malignant chronic liver diseases, the sensitivity was 89.5% and the specificity was 94% [36, 37]. It was also reported that TGF-β1 levels might increased in patients with cirrhosis, owing to decrease hepatic clearance. In addition, this biomarker is up-regulate in extrahepatic tumors, wound healing, angiogenesis and fibrosis, indicating the lack of disease specificity [37].

.5% and the specificity was 94% [36, 37]. It was also reported that TGF-β1 levels might increased in patients with cirrhosis, owing to decrease hepatic clearance. In addition, this biomarker is up-regulate in extrahepatic tumors, wound healing, angiogenesis and fibrosis, indicating the lack of disease specificity [37]. Hepatoma-derived growth factor (HDGF) is the original member of the HDGF family of proteins, which contain a well-conserved N-terminal amino acid sequence and nuclear localization signals (NLSs) in gene-specific regions other than the hath region and is essential for the induction of cell growth activity [36]. HDGF was more abundantly expressed in HCC than in the tumor free adjacent liver tissues in human and murine samples [36, 38]. An increased level HDGF in well-differentiated HCC compared with poorly or undifferentiated subtypes can have a potential prognostic utility for disease free and overall survival with HCC [36, 39, 40]. HDGF is an index of tumor multiplicity, being a sign of poor prognosis in HCC [36, 38, 39]. Hepatocyte growth factor (HGF) is present specifically in cancerous tissues, suggesting a correlation between this serine protease and hepatic malignancies [41]. Patients with inoperable HCC had higher levels of serum HGF than the healthy controls. Higher serum HGF levels for HCC patients are suggestive for a poor prognostic, indicating the active roles in disease progression [42].

ous tissues, suggesting a correlation between this serine protease and hepatic malignancies [41]. Patients with inoperable HCC had higher levels of serum HGF than the healthy controls. Higher serum HGF levels for HCC patients are suggestive for a poor prognostic, indicating the active roles in disease progression [42]. Insulin-like-growth factor II (IGF-II) has been implicated in the pathogenesis of neoplasm of different tissues, including liver. This growth factor is believed to exert its effect during cellular proliferation [43]. A positive relationship of IGF-II levels and HCC progression was found in liver tissues and serum on experimental rats [44] and a limited number of studies on humans. Deregulation of c-myc gene expression was frequently observed in experimentally induced HCC in rodents, as well as in primary human liver tumors [23]. Disease-free survival in patients with c-myc amplification is significantly shorter than in those without amplification [23]. Other nuclear oncogenes overexpressed are c-Ki-ras, c-Ha-ras, c-fos, c-fms and b-catenin with important role of HCC malignant phenotype [10, 30, 45, 46]. Apoptosis Apoptosis is a key mechanism causing cell death and organ diseases, failure of apoptosis is now understood to contribute to the development of human malignancies. Apoptosis rarely occurs in normal livers but increases in HCC, indicating that bcl-2 and bcl-xL expression play important role in regulating the apoptosis of normal liver and HCC. The relationship between bcl-2 related genes and HCC is still unclear [47, 48].

tood to contribute to the development of human malignancies. Apoptosis rarely occurs in normal livers but increases in HCC, indicating that bcl-2 and bcl-xL expression play important role in regulating the apoptosis of normal liver and HCC. The relationship between bcl-2 related genes and HCC is still unclear [47, 48]. The expression of Fas and Fas ligand (Fas L) is associated with the prognosis of cancer patients. Fas expression level is significantly decreased in poorly differentiated HCC and of a large size tumor while Fas L expression in carcinoma cells is observed exclusively in moderately or poorly differentiated cases [26]. Sensitivity to TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and the lysosomal pathway of cell death are features of cancer cells. Lysosomal permeabilization contributes to TRAIL-induced apoptosis of HCC cells and suggests that cFLIPL cytoprotection is, in part, due to p42/44 MAPK-dependent inhibition of lysosomal breakdown [48, 49]. Regulating action of apoptotic genes will deepen the understanding about the growth and development of HCC and offer valuable information to genetic therapy of HCC, thus enhancing the sensibility to radiotherapy and chemotherapy [47].

The expression of Fas and Fas ligand (Fas L) is associated with the prognosis of cancer patients. Fas expression level is significantly decreased in poorly differentiated HCC and of a large size tumor while Fas L expression in carcinoma cells is observed exclusively in moderately or poorly differentiated cases [26]. Sensitivity to TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and the lysosomal pathway of cell death are features of cancer cells. Lysosomal permeabilization contributes to TRAIL-induced apoptosis of HCC cells and suggests that cFLIPL cytoprotection is, in part, due to p42/44 MAPK-dependent inhibition of lysosomal breakdown [48, 49]. Regulating action of apoptotic genes will deepen the understanding about the growth and development of HCC and offer valuable information to genetic therapy of HCC, thus enhancing the sensibility to radiotherapy and chemotherapy [47]. Angiogenesis Angiogenesis is a multistep process, physiological angiogenesis occurs during liver regeneration, leading to the formation of new blood vessel from pre-existing vasculature, meanwhile pathological angiogenesis occurs in HCC [4, 50]. Angiogenesis makes significant contribution to tumor growth, invasiveness, and metastatic potential of HCC. Differentially expressed angiogenesis genes and proteins were identified including,)platelet-derived growth factor receptor (PDGFR) vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), matrix metalloproteinases (MMPs) and its inhibitors (TIMPs), angiopoitin-1 (Ang-1) angiopoitin-2 (Ang-1) have been evaluated and found to be related to HCC tumorigenesis and prognosis [50, 51].

telet-derived growth factor receptor (PDGFR) vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), matrix metalloproteinases (MMPs) and its inhibitors (TIMPs), angiopoitin-1 (Ang-1) angiopoitin-2 (Ang-1) have been evaluated and found to be related to HCC tumorigenesis and prognosis [50, 51]. PDGFR is overexpressed in endothelium of highly metastatic HCC [52]. The role of PDGFR in tumor angiogenesis is not clear; the correlation between its expression level and metastatic potential of HCC warrants further investigation [52, 53]. Over-expression of bFGF and their receptors is thought to be involved in carcinogenesis and, to clarify its clinical significance, the study of its blood level in cancer patients is important [44]. VEGF is considered one of the most important factors involved in tumor-associated angiogenesis, involved in neovascularization [51], development and/or progression of HCC, being associated with a poor survival [50, 51, 53]. HCC patients with level of serum and tissue VEGF overexpression have a lower survival rate [24]. In another study was demonstrated for the first time that retinoblastoma protein (pRb2/p130) is inversely correlated with VEGF expression and tumor aggressiveness. VEGF together with pRb2/p130 may act a diagnostic or prognostic indicator in HCC [24, 53, 54].

d tissue VEGF overexpression have a lower survival rate [24]. In another study was demonstrated for the first time that retinoblastoma protein (pRb2/p130) is inversely correlated with VEGF expression and tumor aggressiveness. VEGF together with pRb2/p130 may act a diagnostic or prognostic indicator in HCC [24, 53, 54]. HCC tumour spread is partly dependent on neoangiogenesis [17, 54]. VEGF is considered one of the most important factors involved in neoangiogenesis, development and/or progression of HCC, being associated with a poor survival [50, 51, 53]. HCC patients with serum and tissue VEGF overexpression have a lower survival rate [24]. Oral multikinase inhibitor (e.g. Sorafenib, sunitinib) of the VEGF, PDGFR of downstream intracellular serine/threonine, is used with success in HCC therapy [17, 54]. Ang-1 and its antagonist, Ang-2, are ligands that regulate the Tie2 receptor. The Ang-2 gene is upregulated in the hypervascular type of HCC, being investigated in HCC [55]. Dominant Ang-2 expression against Ang-1 through Tie2 receptor in the presence of VEGF plays a critical role in initiating early neovascularization and transformation of noncancerous liver to HCC [53, 54, 55].

Tie2 receptor. The Ang-2 gene is upregulated in the hypervascular type of HCC, being investigated in HCC [55]. Dominant Ang-2 expression against Ang-1 through Tie2 receptor in the presence of VEGF plays a critical role in initiating early neovascularization and transformation of noncancerous liver to HCC [53, 54, 55]. MMPs are proteins relate to the growth and infiltration of cancer cells. Expression of MMPs and TIMPs were investigated in vitro and surgically resected HCC tissues [56]. A high expression was observed for MMP-2, MMP-9, MT1-MMP and TIMP-2. Expression of MMP-7, MT2-MMP and TIMP-1 was found at a low frequency and a low amount in both cells and tissues. MMPs and TIMPs are involved in the progression of HCC [56].

and TIMPs were investigated in vitro and surgically resected HCC tissues [56]. A high expression was observed for MMP-2, MMP-9, MT1-MMP and TIMP-2. Expression of MMP-7, MT2-MMP and TIMP-1 was found at a low frequency and a low amount in both cells and tissues. MMPs and TIMPs are involved in the progression of HCC [56]. Immune surveillance The inflammatory immune response of the host to viral antigens induces hepatocyte damage, which is followed by the regeneration of hepatocytes and the development of fibrosis and cirrhosis-important features in the pathogenesis of HCC [5, 7, 57]. Studies have demonstrated that cytokines can affect a multitude of cellular pathways. Although their normal function may be to keep the tumorigenic cascade at bay, disease states such as the presence of hepatitis viruses can alter cytokines to function as protumorigenic factors. These immune-related proteins are not only affecters of transformation and primary tumor phases, but they have also been shown to play a role in downstream pathways related to tumor progression, such as angiogenesis. Despite the known associations between cytokines and HCC, the full portrait of their mechanistic roles in this disease remains to be elucidated [7].

fecters of transformation and primary tumor phases, but they have also been shown to play a role in downstream pathways related to tumor progression, such as angiogenesis. Despite the known associations between cytokines and HCC, the full portrait of their mechanistic roles in this disease remains to be elucidated [7]. Mounting evidence indicates the involvement of cytokines in hepatocarcinogenesis [6, 57]. Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a critical role in normal hepatic growth and liver regeneration following a reduction in hepatic mass. Concentrations of IL-6 in serum are increased in situations of chronic liver inflammation including alcoholic hepatitis, HBV and HCV infections, and steatohepatitis, conditions that may lead to development of HCC [7].

a critical role in normal hepatic growth and liver regeneration following a reduction in hepatic mass. Concentrations of IL-6 in serum are increased in situations of chronic liver inflammation including alcoholic hepatitis, HBV and HCV infections, and steatohepatitis, conditions that may lead to development of HCC [7]. Serum levels of IL-6 and IL-10 are frequently elevated in patients with HCC but not in benign liver disease or non-HCC tumors [58, 59]. IL-6 and IL-10 may help identify a subset of HCC patients may serve as complementary tumor markers in these patients [7, 57, 60, 61]. These studies suggest that increases in IL-10 and perhaps other Th2 cytokines correlate with progression of HCC [57, 59, 60]. Proinflammatory IL-1β was elevated in HCC patients compared with healthy individuals [28, 57]. Serum IL-15 was higher in HCC indicating the degree of liver inflammation [62], TNF-α expression was elevated in HCC patients, especially those with recurrence. In addition, the levels of the TNF-α Rs (TNFαRI and TNFα RII) were higher in HCC patients [63]. In other studies, TNF-α level was lower in HCC tumor tissue versus the tissue surrounding the tumor and in HCC patients versus healthy individuals [57]. IFN-α was not detected in HCC, and was concluded that IFN-α may not play a large role in liver inflammation. The proinflammatory cytokines IL-12 and IL-2 are also increased in HCC. Th1 cytokines are mainly up-regulated in HCC [57].

us the tissue surrounding the tumor and in HCC patients versus healthy individuals [57]. IFN-α was not detected in HCC, and was concluded that IFN-α may not play a large role in liver inflammation. The proinflammatory cytokines IL-12 and IL-2 are also increased in HCC. Th1 cytokines are mainly up-regulated in HCC [57]. Cox-2 is an isoform of cyclooxygenase, which is the key enzyme converting arachidonic acid to prostaglandins. Overexpression of Cox-2 affects many mechanisms involved in carcinogenesis, such as angiogenesis, inhibition of apoptosis, invasion and metastasis [64]. It has been shown that Cox-2 induces angiogenesis, which in turn aids tumor growth, invasion and metastasis. It was found positive correlations between Cox-2 and iNOS expression in HCV-positive HCC and this could be partially attributable to modulation of angiogenesis by Cox-2 [64, 65]. Overexpression of Cox-2 is generally higher in well-differentiated HCC compared with less-differentiated HCC or histological normal liver, with implication in the early stages of hepatocarcinogenesis [65], and increased expression of Cox-2 being significantly associated with shorter disease-free survival in patients with HCC. Cox-2 inhibitors might be effective in prevention of both cancer development and disease progression of HCC [64, 65]. Experimental studies on animal models with liver cancer have shown that both selective and non-selective Cox-2 inhibitors exert chemopreventive as well as therapeutic effects. However, the key mechanism by which Cox-2 inhibitors affect HCC cell growth isn’t yet fully understood [49, 50].

se progression of HCC [64, 65]. Experimental studies on animal models with liver cancer have shown that both selective and non-selective Cox-2 inhibitors exert chemopreventive as well as therapeutic effects. However, the key mechanism by which Cox-2 inhibitors affect HCC cell growth isn’t yet fully understood [49, 50]. Novel serum biomarker candidate for the enhanced detection of HCC The overall increase in the incidence of HCC warrants efforts to prevent and treat more efficiently this disease. Therefore, early diagnosis, based on serum markers and the development of novel systemic therapies for advanced disease are very important [2, 4, 26]. Alpha-fetoprotein (AFP) is the only biomarker used for HCC diagnosis, however, its use in the early detection of HCC is limited, especially because about one-third parts of patients with HCC have normal levels of serum AFP [66]. Serum AFP is a marker that has low sensitivity and high specificity [8, 24, 67]. Other candidate markers are isoforms of AFP like lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) and monosialylated AFP (msAFP) [68].

ally because about one-third parts of patients with HCC have normal levels of serum AFP [66]. Serum AFP is a marker that has low sensitivity and high specificity [8, 24, 67]. Other candidate markers are isoforms of AFP like lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) and monosialylated AFP (msAFP) [68]. In a recent study serum transglutaminase-2 was presented as a novel histological/serologic candidate in HCC, especially for the individuals with normal serum AFP [64]. Determination of serum IGF-II in parallel with AFP as marker increases the diagnostic accuracy and sensitivity. IGF-II may be used as complementary tumor marker to discriminate HCC from cirrhosis [56]. Circulating interleukin-6 can be considered a promising tumor marker for HCC. In particular, the diagnostic value is significantly increased when is combined with other serum markers [69]. Glypican-3 (GPC3), a heparin-sulfate-proteoglycan was suggested to be serologic markers of HCC [70], results reached by three independent laboratories. Furthermore, due to the lack of correlation between serologic concentrations of GPC3 and AFP in HCC patients, the simultaneous use of both markers significantly increases the sensitivity of the test [71, 72]. Plasma levels of lipids, lipoproteins and apolipoproteins in HCC patients may reflect the status of hepatic cellular impairments, and decreased serum levels of cholesterol and apoAI may indicate a poor prognosis in HCC [73].

Glypican-3 (GPC3), a heparin-sulfate-proteoglycan was suggested to be serologic markers of HCC [70], results reached by three independent laboratories. Furthermore, due to the lack of correlation between serologic concentrations of GPC3 and AFP in HCC patients, the simultaneous use of both markers significantly increases the sensitivity of the test [71, 72]. Plasma levels of lipids, lipoproteins and apolipoproteins in HCC patients may reflect the status of hepatic cellular impairments, and decreased serum levels of cholesterol and apoAI may indicate a poor prognosis in HCC [73]. Des-γ-carboxyprothrombin (DCP) is an abnormal prothrombin that lacks γ- carboxylation of its glutamine residue. DCP is unable to bind calcium ion that is essential for its conformational transition and functional activity. DCP may differentiate HCC from non-malignant liver diseases, it can also identify HCC at an earlier stage [24]. Hepatoma specific gamma-glutamyl transferase (HS-GGT) was expressed in the development of HCC. The abnormal alteration of serum HS-GGT level is a sensitive tumor marker for HCC diagnosis, differentiation or prognostic marker, and the overexpression of GGT in HCC may be related to the hypomethylational status of GGT genes [42]. Some tumor markers, such as human cervical cancer oncogene and human telomerase reverse transcriptase mRNA, have also been indicated to have higher accuracies than AFP [24].

osis, differentiation or prognostic marker, and the overexpression of GGT in HCC may be related to the hypomethylational status of GGT genes [42]. Some tumor markers, such as human cervical cancer oncogene and human telomerase reverse transcriptase mRNA, have also been indicated to have higher accuracies than AFP [24]. Human telomerase reverse transcriptase (hTERT) mRNA has been reported to be detectable in the serum of patients with different cancer type. The expression of hTERT mRNA in the serum of HCC patients is significantly higher than that in the serum of healthy adults with non-malignant hepatopathy. It has been reported that the sensitivity and specificity of hTERT mRNA in detecting HCC are 88.2% and 70.0%, respectively, indicating that the expression of serum hTERT mRNA is associated with the serum concentration of AFP, tumor size, and tumor differentiation degree, being a indicator of poor prognosis for HCC patients [24]. Squamous cell carcinoma antigen (SCCA), serine protease inhibitor, is physiologically expressed in the skin and other squamous epithelial cells. SCCA levels were significantly elevated in HCC patients, compared to patients with cirrhosis only or normal subjects [74]. SCCA is also over-expressed in serum from patients with HCC. SCCA was proved to have high sensitivity and low specificity [37].

ologically expressed in the skin and other squamous epithelial cells. SCCA levels were significantly elevated in HCC patients, compared to patients with cirrhosis only or normal subjects [74]. SCCA is also over-expressed in serum from patients with HCC. SCCA was proved to have high sensitivity and low specificity [37]. Golgi protein 73 (GP73, also known as Golph2) is a resident Golgi-specific membrane protein expressed by biliary epithelial cells in normal liver. GP73 is up-regulated in HCC. Measurement of serum GP73 based on immunoblots revealed a sensitivity and specificity of 69% and 75%, respectively [8, 37]. Some other serum markers, such as: alpha-L-fucosidase, TGF-1, TNF-a, VEGF, IL-8, HGF are useful for diagnosis but none of these markers has been validated for clinical use [8, 24, 67]. The limitation of developing HCC markers is probably due to heterogeneity assay methods and result reporting, limited analysis of demographics and causes of liver disease as covariates in the expression of these biomarkers. In addition, these molecules need to be validated and cost-effectiveness especially those markers proposed as diagnostic or prognostic role [8]. Further studies are need to confirm the roles and to validate these biomarkers.

f demographics and causes of liver disease as covariates in the expression of these biomarkers. In addition, these molecules need to be validated and cost-effectiveness especially those markers proposed as diagnostic or prognostic role [8]. Further studies are need to confirm the roles and to validate these biomarkers. Conclusions In recent years many biological factors have shown association with the invasiveness of HCC in addition to their prognostic significance. These suggest new targeted biological therapy for HCC, antiangiogenic therapy using specific inhibitors for expressed angiogenesis genes or their receptors, methylation status of tumors suppressor genes and oncogenes. Other targets are serum proteins involved in inflammatory processes or apoptotic pathways. The success in HCC treatment is influenced by early detection. Clearly, new biochemical markers are needed for HCC screening. AFP in simultaneous combination with other serum markers could enhance the sensitivity for HCC detection. The possibility of distinguishing cirrhosis as a high-risk group for HCC offers a hope for the early detection of HCC. The prognostic factors or diagnostic biomarkers of plasma or serum are important trends that deserve attention. Developing new blood biomarkers will help develop more effective therapeutic strategy targeting key signalling. These novel markers should be easily measurable, reproducible and minimally invasive and need to be validated in large-scale studies in clinical practice.

or serum are important trends that deserve attention. Developing new blood biomarkers will help develop more effective therapeutic strategy targeting key signalling. These novel markers should be easily measurable, reproducible and minimally invasive and need to be validated in large-scale studies in clinical practice. We gratefully acknowledge members of Functional Genomics and Experimental Pathology from Cancer Institute “I Chiricuta” Cluj-Napoca for helpful comments and suggestions. Conflict of interest The authors have no conflict of interest in relation to this article.

Introduction As many as 1.4 million persons in the United States and 2.2 million persons in Europe suffer from inflammatory bowel disease (IBD). The strongest environmental factors identified are cigarette smoking and appendectomy, both being risk factors for Crohn’s disease (CD), protective factors for ulcerative colitis (UC) [1]. We will outline the errors made in the management of the condition as well as the complications that arise from the condition and its treatment. We have reviewed the diagnostic and therapeutic modailties already approved and accepted. Though there many newer medications and agents being investigated, these are not within the scope of our discussion.

in the management of the condition as well as the complications that arise from the condition and its treatment. We have reviewed the diagnostic and therapeutic modailties already approved and accepted. Though there many newer medications and agents being investigated, these are not within the scope of our discussion. Diagnosis Serology Diagnosis of IBD is made after a thorough history and physical, endoscopy, reviewing the histology since successful treatment is achieved through correct diagnosis. We would like to comment on the difficulty encountered with serologies. The diagnostic gain of a test is minimal when the pre-test probability of disease is very high or very low, and the gain from diagnostic testing is maximal when the pre-test probability of disease is somewhere in the middle. The same can be said of the serological assays for IBD. The most commonly used antibodies are deoxyribonuclease (DNase)-sensitive perinuclear antineutrophil cytoplasmic antibody (DNase-sensitive pANCA), IgA and IgG antibodies to Saccharomyces cerevisiae (IgA and IgG ASCA), and antibody to Escherichia coli outer membrane porin (anti-OmpC). Serological assays should play a limited role in the diagnosis of IBD. These serological assays may be helpful when the results of the appropriate evaluation are inconclusive [2]. Although the test characteristics in regards to sensitivity and specificity are reasonably good for the most comprehensive serologic panel for IBD, the serologic tests should not be considered a diagnostic tool, and treatment for IBD should not be initiated solely on the results of serologic testing [3].

onclusive [2]. Although the test characteristics in regards to sensitivity and specificity are reasonably good for the most comprehensive serologic panel for IBD, the serologic tests should not be considered a diagnostic tool, and treatment for IBD should not be initiated solely on the results of serologic testing [3]. As pointed out by Dubinsky, the titers may also be used for prognostication in certain cases. The presence of certain markers, such as ASCA, is an indication for a high risk of postsurgi­cal CD or fistulization of the pouch, and the presence of multiple antibodies indicates more aggressive disease course. Endoscopy Although conventionally it is accepted that rectal sparing or patchy involvement should increase suspicions of CD, there are circumstances where patchiness can be observed in UC. This may be seen in patients who have received prior local or systemic therapy. Rectal sparing usually occurs if the patient has applied topical enemas [4]. “Skip lesions” seen on colonoscopy may be secondary to partial healing in ulcerative colitis. “Cobblestoning” is a result of ulceration and healing and is seen in both UC and CD. Therefore, it is important to avoid placing too much emphasis on the traditionally described findings. Capsule endoscopy is useful in bleeding and small bowel assessment, but poses a few problems in patients with CD, potential for capsule getting stuck, frequency of nonspecific aphthous ulcers in patients taking NSAIDs. MR enterography and CT enterography have been cited as imaging modalities with comparable efficacy.

Endoscopy Although conventionally it is accepted that rectal sparing or patchy involvement should increase suspicions of CD, there are circumstances where patchiness can be observed in UC. This may be seen in patients who have received prior local or systemic therapy. Rectal sparing usually occurs if the patient has applied topical enemas [4]. “Skip lesions” seen on colonoscopy may be secondary to partial healing in ulcerative colitis. “Cobblestoning” is a result of ulceration and healing and is seen in both UC and CD. Therefore, it is important to avoid placing too much emphasis on the traditionally described findings. Capsule endoscopy is useful in bleeding and small bowel assessment, but poses a few problems in patients with CD, potential for capsule getting stuck, frequency of nonspecific aphthous ulcers in patients taking NSAIDs. MR enterography and CT enterography have been cited as imaging modalities with comparable efficacy. Patients initially diagnosed with either UC or CD may demonstrate with time additional features which may support or be against the initial diagnosis. In a study from Norway out of the 527 patients initially diagnosed with UC, 88% had their diagnosis confirmed on follow up in 1-2 years. 91% of 228 patients with CD had their diagnosis confirmed in the same follow-up period. 36 patients were diagnosed originally to have Indeterminate Colitis. On follow- up, 33% of these were re-classified as UC and 17% as CD. The study illustrates the importance of the re-evaluation of the initial diagnosis as up to 10%, both among patients with UC and CD, were reclassified at follow up [5]. This fact was also demonstrated by Langevin et al in a study involving 96 patients with an initial diagnosis of ulcerative proctitis, 14% of them developed features of CD in 29 months of follow-up [6]. Given this finding, it is not unreasonable that a patient be given a provisional diagnosis with the caveat that it may change and the clinician should follow up and assess for accuracy of initial diagnosis.

an initial diagnosis of ulcerative proctitis, 14% of them developed features of CD in 29 months of follow-up [6]. Given this finding, it is not unreasonable that a patient be given a provisional diagnosis with the caveat that it may change and the clinician should follow up and assess for accuracy of initial diagnosis. It is also important to avoid confusion with other conditions which can cause similar symptoms and endoscopy findings. The endoscopic appearances of the mucosa and the histologic changes in infective and inflammatory colitis may be virtually indistinguishable [7]. A third of patients presenting with mucoid bloody diarrhea and suspected IBD have an infective etiology. Patients with IBD have the propensity for bacterial superinfection [8]. The most common enteric pathogens implicated are Campylobacter, Salmonella, Shigella, Amoeba and Clostridium difficile. In the majority of cases the history, presentation in addition to serological tests and stool cultures help in the differentiation between infective colitides and IBD. Typical examples of other conditions that mimic IBD are ischemic colitis, diverticular colitis. Medical therapy Table 1 is a list of medications approved for the treatment of IBD.

It is also important to avoid confusion with other conditions which can cause similar symptoms and endoscopy findings. The endoscopic appearances of the mucosa and the histologic changes in infective and inflammatory colitis may be virtually indistinguishable [7]. A third of patients presenting with mucoid bloody diarrhea and suspected IBD have an infective etiology. Patients with IBD have the propensity for bacterial superinfection [8]. The most common enteric pathogens implicated are Campylobacter, Salmonella, Shigella, Amoeba and Clostridium difficile. In the majority of cases the history, presentation in addition to serological tests and stool cultures help in the differentiation between infective colitides and IBD. Typical examples of other conditions that mimic IBD are ischemic colitis, diverticular colitis. Medical therapy Table 1 is a list of medications approved for the treatment of IBD. Table 1 Medications approved for treatment of IBD Class Examples Indications Sulfasalazine and 5-amino salicylates Azulfidine-Olsalazine, Asacol, Pentasa, Balsalazide Mild to moderate UC and CD Corticosteroids Hydrocortisone, Prednisone, Budesonide UC and CD Immunosuppressives Azathioprine, 6-Mercaptopurine, Methotrexate Evidence for CD > UC. MTX-no role in UC Anti-TNFα Antibody Infliximab, Adalimumab, Certolizumab pegol Severe UC (Infliximab)/ all 3 for CD Antibiotics Metronidazole, Trimethoprim-sulfamethoxazole, Ciprofloxacin, Clarithromycin, Ancillary in treatment of IBD Glucocorticoids Topical Mild cases, especially when only the distal colon is involved, respond well to topical therapy with or without adjunctive oral therapy [9]. Non-systemic steroids such as oral and rectal budesonide for ileal and right-sided CD and distal UC respectively are also effective in mild-moderate disease [10]. Treatment with budesonide enema in active distal ulcerative colitis was comparable to treatment with conventional prednisolone enema. A prolongation of the treatment time from 4 to 8 weeks doubled the clinical remission rate in both groups. However, budesonide may be preferable to prednisolone since it causes less systemic effects as reflected by a lack of plasma cortisol suppression.

was comparable to treatment with conventional prednisolone enema. A prolongation of the treatment time from 4 to 8 weeks doubled the clinical remission rate in both groups. However, budesonide may be preferable to prednisolone since it causes less systemic effects as reflected by a lack of plasma cortisol suppression. Oral In cases with CD, mild to moderate ileal disease responds well to oral Budesonide [11]. For mild to moderate UC, a dose-response effect for prednisone 20-60 mg/d has been reported, but doses greater than 60 mg/d confer no additional benefit [12]. In patients with severe UC, there is a concern for relapse when using a low dose of prednisone. Similarly using very high doses or for a prolonged period exposes the patient to avoidable side effects which span nearly all the organ systems. If the patient is treated several times with glucocorticoids, even if topical, physicians must start monitoring them for side effects such as glucose intolerance, gastric irritation, cataracts, increased risk of fracture [13]. In patients with severe UC, it is important to use doses of glucocorticoids equivalent to hydrocortisone 100 mg intravenous every 8 hours. If there is no response in 3-7 days, consider steroid resistance, since the management depends on quick diagnosis and carefully ruling out other confounding conditions [14]. Involving surgery early on is very important in order to avoid colonic perforation which in some cases can prove to be fatal.

enous every 8 hours. If there is no response in 3-7 days, consider steroid resistance, since the management depends on quick diagnosis and carefully ruling out other confounding conditions [14]. Involving surgery early on is very important in order to avoid colonic perforation which in some cases can prove to be fatal. It may also be appropriate to perform flexible sigmoidoscopy to rule out opportunistic infections such as cytomegalovirus (CMV) colitis [15]. If CMV colitis is ruled out and the patient is no better, consider treatment with intravenous cyclosporine/ infliximab/ or surgery. Sulfasalazine and 5-amino salicylates Rectal 5-ASA has been found to be superior to steroid enemas in the management of distal ulcerative colitis [16]. Oral 5-ASA compounds, including olsalazine, sulfasalazine mesalamine, balsalazide are effective in inducing remissions in active ulcerative colitis although side effects are significantly higher with sulfasalazine [17, 18]. Sulfasalazine is effective for the treatment of Crohn's colitis, but is less useful in patients with active ileitis. This diminished response probably reflects the need for colonic bacteria to cleave the drug to release the active 5-ASA moiety [19].

ugh side effects are significantly higher with sulfasalazine [17, 18]. Sulfasalazine is effective for the treatment of Crohn's colitis, but is less useful in patients with active ileitis. This diminished response probably reflects the need for colonic bacteria to cleave the drug to release the active 5-ASA moiety [19]. If a little is good, more is better. There is evidence for dose dependence in 5-ASA therapy for oral preparations, with optimal effects being observed at 4.8g/ day of mesalamine as compared with 2.4 g/day.The latter works well in mild cases while higher doses are utilized in moderate/extensive disease. Both doses of mesalamine had similar saftey profiles and both were well tolerated [20, 21]. Adequate attention should be paid to patients’ level of compliance with the prescribed medicine regime. Nonadherence with medication increases the risk of clinical relapse among patients with quiescent ulcerative colitis. In a study by Kane et al, it was found that patients who were not adherent with medications had more than a fivefold greater risk of recurrence than compliant patients (hazard ratio = 5.5; 95% confidence interval: 2.3 to 13; p < 0.001) [22].

the risk of clinical relapse among patients with quiescent ulcerative colitis. In a study by Kane et al, it was found that patients who were not adherent with medications had more than a fivefold greater risk of recurrence than compliant patients (hazard ratio = 5.5; 95% confidence interval: 2.3 to 13; p < 0.001) [22]. Immunomodulator therapy 6-Mercaptopurine (6-MP) and its prodrug azathioprine (AZA) are thiopurine analogues and are immuno-modulatory agents. Of the AZA compound, 88% is converted via nonenzymatic process to 6-MP. AZA or 6-MP should be considered in UC patients who are refractory to maximal doses of 5-ASA medications and require oral corticosteroids to control symptoms. Both agents are used as “steroid sparing” medicines. As a general rule, consider using these drugs in patients who require four or more months of continuous corticosteroid therapy to control symptoms and/or three or more flare-ups in a given year that require steroids to achieve remission. In the pediatric study by Markowitz and colleagues, only 4% of patients of the 6-MP group required another course of steroids within 540 days after being weaned off of prednisone, clearly in contrast to the 57% of pediatric CD patients receiving placebo with a need to restart prednisone within 360 days (P < 0.0001) [23].

In the pediatric study by Markowitz and colleagues, only 4% of patients of the 6-MP group required another course of steroids within 540 days after being weaned off of prednisone, clearly in contrast to the 57% of pediatric CD patients receiving placebo with a need to restart prednisone within 360 days (P < 0.0001) [23]. AZA and 6-MP are also effective for maintaining remission for many years in patients with CD whose remission was initially achieved with these drugs [24]. There is also expanding evidence that AZA is effective as a post-operative maintenance therapy [25]. The risk of infection with these medication ranges between 0.3%-7.4% and includes herpes viruses, human papilloma virus and upper respiratory infections [26]. Increased risk of hematologic malignancies has also been associated with prolonged leucopenia in IBD patients on 6-MP, and EBV-positive lymphomas have also been found more frequently in patients exposed to 6-MP or AZA [27, 28].

AZA and 6-MP are also effective for maintaining remission for many years in patients with CD whose remission was initially achieved with these drugs [24]. There is also expanding evidence that AZA is effective as a post-operative maintenance therapy [25]. The risk of infection with these medication ranges between 0.3%-7.4% and includes herpes viruses, human papilloma virus and upper respiratory infections [26]. Increased risk of hematologic malignancies has also been associated with prolonged leucopenia in IBD patients on 6-MP, and EBV-positive lymphomas have also been found more frequently in patients exposed to 6-MP or AZA [27, 28]. One in 300 individuals lacks Thiopurine methyltransferase (TPMT) and 11% of the population is heterzygous with intermediate activity. This enzyme is essential for metabolism of the purine analogs. It is important to check TPMT enzyme activity and monitor the CBC monthly to avoid severe myelosuppression. Patients with low enzyme levels are at risk but can be treated with careful dosing titration. Patients with absent enzyme should not be treated with these drugs [29]. It is important to follow up on regular bloodwork monitoring (full blood count, liver panel) after starting antimetabolites and judiciously use these in patients not responding to adequate weight based dosing.

reated with careful dosing titration. Patients with absent enzyme should not be treated with these drugs [29]. It is important to follow up on regular bloodwork monitoring (full blood count, liver panel) after starting antimetabolites and judiciously use these in patients not responding to adequate weight based dosing. Monoclonal antibodies The monoclonal antibodies currently being used in IBD are infliximab, adalimumab, certolizumab pegol. These agents mediate pro-inflammatory processes central to the pathogenesis of IBD. Adalimumab and certolizumab pegol have an advantage of subcutaneous administration rather than intravenous administration required for infliximab. The safety record with infliximab and adalimumab is longer than with certolizumab pegol. Only infliximab is approved for UC. All three agents are approved for CD. We are not discussing Natalizumab in our paper. Patients with moderate-to-severe active ulcerative colitis treated with infliximab were more likely to have a clinical response than were those receiving placebo as shown in two randomized placebo controlled trials in ACT1 and ACT2 [30]. Patients treated with moderate to severe Crohn’s disease treated with infliximab also had significantly higher remission rates as demonstarated by the landmark ACCENT I and ACCENT II trials.

a clinical response than were those receiving placebo as shown in two randomized placebo controlled trials in ACT1 and ACT2 [30]. Patients treated with moderate to severe Crohn’s disease treated with infliximab also had significantly higher remission rates as demonstarated by the landmark ACCENT I and ACCENT II trials. The preliminary data from a comparison of top-down versus step-up therapy were presented in which combined immunosuppression was compared with a conventional step-up approach in 129 CD patients with a CD activity index of at least 200[31]. Patients had to be steroid nave and had not been exposed previously to infliximab or antimetabolites. The step-up algorithm included induction of remission with corticosteroids, followed by repeat courses of steroids and azathioprine in the case of new exacerbations, and eventually infliximab if these therapeutic interventions failed. Mucosal healing was found in 73% of patients assigned to top-down treatment versus 30% in the conventional group at 24 months.

of remission with corticosteroids, followed by repeat courses of steroids and azathioprine in the case of new exacerbations, and eventually infliximab if these therapeutic interventions failed. Mucosal healing was found in 73% of patients assigned to top-down treatment versus 30% in the conventional group at 24 months. The FDA published a summary of 47 cases of HF associated with infliximab reported to the Adverse Events Response System (AERS) through January, 2002. In a patient who develops heart failure while on a TNF inhibitor, a drug-induced cause should be suspected and the medication should be discontinued. TNF inhibitors have rarely been associated with the development or exacerbation of neurologic disorders associated with demyelination, such as multiple sclerosis. However, the true nature of this association has not been established. Anti-TNF therapy should be discontinued immediately in any patient with suspected demyelination.

tors have rarely been associated with the development or exacerbation of neurologic disorders associated with demyelination, such as multiple sclerosis. However, the true nature of this association has not been established. Anti-TNF therapy should be discontinued immediately in any patient with suspected demyelination. Before starting monoclonal antibodies it is important to screen for tuberculosis with PPD testing and chest X-ray, understanding that anergy is common in IBD. It should be stressed that patients are exposed several opportunistic infections including pulmonary and systemic fungal infections, and that their immune defense mecchanisms are compromised. There is also a concern for exacerbation of latent infections such as hepatitis B and increased occurrence of non Hodgkin’s lymphoma [32]. The increased lymphoma rates observed with anti-TNF therapy may reflect channeling bias, whereby patients with the highest risk of lymphoma preferentially receive anti-TNF therapy. Current data are insufficient to establish a causal relationship between anti-TNF and the development of lymphoma. It is also important to stress maintenance to improve outcomes and avoid/decrease antibody formation.

eling bias, whereby patients with the highest risk of lymphoma preferentially receive anti-TNF therapy. Current data are insufficient to establish a causal relationship between anti-TNF and the development of lymphoma. It is also important to stress maintenance to improve outcomes and avoid/decrease antibody formation. It has been shown that risk for squamous cell cancers increases with global immunosuppression [33]. A trend of elevated risk for cervical cancer with IBD and IBD medications was observed, but it was not statistically significant. Regular cervical cancer screening for women with IBD has been recommended [34]. It is especially important then, for patients on chronic immunosuppressive therapy to undero regular dermatologic examinations. The FDA published a summary of 47 cases of heart failure (HF) associated with infliximab reported to the Adverse Events Response System (AERS) through January, 2002. In a patient who develops HF while on a TNF inhibitor, a drug-induced cause should be suspected and the medication should be discontinued. TNF inhibitors have rarely been associated with the development or exacerbation of neurologic disorders associated with demyelination, such as multiple sclerosis. However, the true nature of this association has not been established. Anti-TNF therapy should be discontinued immediately in any patient with suspected demyelination.

tors have rarely been associated with the development or exacerbation of neurologic disorders associated with demyelination, such as multiple sclerosis. However, the true nature of this association has not been established. Anti-TNF therapy should be discontinued immediately in any patient with suspected demyelination. Antibiotics Mild perianal disease in Crohn’s may respond to antibioics. The most closely studied antibiotic for treatment of CD has been mertronidazole, this has effects similar to sulfasalaine and has superior efficacy as compared with placebo in mild to moderate disease [35]. Antibiotics are useful in UC in the setting of complications like pouchitis either alone or in combination with ciprofloxacin [36]. Other than this, there is no role for antibiotics in UC. Table 2 shows a list of major side effects of medications used for treatment of IBD.

with placebo in mild to moderate disease [35]. Antibiotics are useful in UC in the setting of complications like pouchitis either alone or in combination with ciprofloxacin [36]. Other than this, there is no role for antibiotics in UC. Table 2 shows a list of major side effects of medications used for treatment of IBD. Table 2 Major side effects of medicines used for treatment of IBD Sulfasalazine and 5-ASA compounds Hypersensitivity, sperm abnormalities, blood dyscrasias Corticosteroids Adrenal insufficiency, hyperglycemia, edema, osteonecrosis, cataracts myopathy, peptic ulcer disease, hypokalemia, osteoporosis, euphoria, psychosis, altered cell mediated immunity Azathioprine/ Methotrexate Blood dyscrasia, drug induced hepatitisand pancreatitis. AZA implied in T cell lymphoma, MTX in Hodgkin’s lymphoma Metronidazole Seizures, peripheral neuropathy, disulfiram reaction with alcohol TNF–Alpha inhibitors Anaphylaxis, superinfections, chest pain or rash, risk of reactivation of tuberculosis, rare occurrence of multifocal leucoencephalopathy Colon cancer surveillance Are we waiting too long? In an attempt to detect precancerous dysplasia and asymptomatic cancers in patients with IBD, the major gastroenterology societies recommend initiating colonoscopic surveillance beginning 8-10 years after the onset of disease in pancolitis and after 15-20 years for left-sided colitis, immediately and annually with primary sclerosing cholangitis (PSC) diagnosis.

sia and asymptomatic cancers in patients with IBD, the major gastroenterology societies recommend initiating colonoscopic surveillance beginning 8-10 years after the onset of disease in pancolitis and after 15-20 years for left-sided colitis, immediately and annually with primary sclerosing cholangitis (PSC) diagnosis. There is data from Ullman et al which shows low grade dysplasia confirmed by a second expert IBD pathologist has high risk of progression and surveillance may not detect progression before metastatic spread. In a study presented by Lutgens and colleagues which identified patients diagnosed with IBD-associated colorectal cancer, of 104 patients diagnosed with IBD and colorectal cancer, 26% developed colorectal cancer before the start of surveillance. Although these data suggest that the diagnosis of colorectal cancer may be delayed or missed using current surveillance guidelines, it is not clear what the proper time point is for initiating surveillance, as every time point will reveal patients who develop cancer prior to the initiation of surveillance [37]. The internist and gastroenterologist taking care of the IBD patients should consider all the risk factors for colorectal cancer in ulcerative colitis including the duration and extent of colitis, PSC, family history of colon cancer, development of dysplasia, endoscopic appearance, and severity of inflammation at surveillance colonoscopy [38, 39].

rologist taking care of the IBD patients should consider all the risk factors for colorectal cancer in ulcerative colitis including the duration and extent of colitis, PSC, family history of colon cancer, development of dysplasia, endoscopic appearance, and severity of inflammation at surveillance colonoscopy [38, 39]. Chromoendoscopy may eventually help to better define additional areas of biopsy. The use of chromoendoscopy for surveillance in IBD is not currently the standard of care; however, these studies add to a growing body of literature suggesting that this technique may improve the detection of dysplasia in IBD [40]. Colon cancer risk for Crohn's colitis is the same as UC [41]. The relative risk cancers of the small intestine is increased in Crohn’s, however small bowel cancers are rare with estimated ranges from 5.7- 7.5 cases per million [42]. In addition, a meta-analysis demonstrated an increased risk of small bowel, colon, extraintestinal cancers, and lymphoma in patients with CD [43]. Polypoid adenomas can be followed if complete polypectomy is performed and there is no dyplasia in adjacent flat mucosa. Figure 1 shows the suggested management of dyplasia. Figure 1 The suggested management of dyplasia in IBD

Colon cancer risk for Crohn's colitis is the same as UC [41]. The relative risk cancers of the small intestine is increased in Crohn’s, however small bowel cancers are rare with estimated ranges from 5.7- 7.5 cases per million [42]. In addition, a meta-analysis demonstrated an increased risk of small bowel, colon, extraintestinal cancers, and lymphoma in patients with CD [43]. Polypoid adenomas can be followed if complete polypectomy is performed and there is no dyplasia in adjacent flat mucosa. Figure 1 shows the suggested management of dyplasia. Figure 1 The suggested management of dyplasia in IBD The detection of precancerous dysplasia in IBD can be challenging because lesions can be flat, subtle, or difficult to detect on conventional surveillance colonoscopy [44]. Furthermore, few physicians take the required 32 biopsies in the colon needed to detect flat dysplasia [45]. Finally, meta-analysis of 5-ASA chemoprevention trials done by Rubin and Lashner shows a favorable role of this medicine in the prevention of cancer and dysplasia. The role of folate in chemoprevention has also been studies in murine models but needs to be confirmed in human intervention trials. In a patient diagnosed with IBD, flare up is in the differential diagnosis of abdominal pain bouts (Table 3).

The detection of precancerous dysplasia in IBD can be challenging because lesions can be flat, subtle, or difficult to detect on conventional surveillance colonoscopy [44]. Furthermore, few physicians take the required 32 biopsies in the colon needed to detect flat dysplasia [45]. Finally, meta-analysis of 5-ASA chemoprevention trials done by Rubin and Lashner shows a favorable role of this medicine in the prevention of cancer and dysplasia. The role of folate in chemoprevention has also been studies in murine models but needs to be confirmed in human intervention trials. In a patient diagnosed with IBD, flare up is in the differential diagnosis of abdominal pain bouts (Table 3). Table 3 Common causes of non- flare pain and diarrhea in IBD 1. Bile acid diarrhea 2. Increased NSAID use 3. Short gut syndrome 4.Infectious 5. Ischemic 6. Irritable bowel syndrome Toxic megacolon It is important correctly identify and treat toxic megacolon. The diagnosis is based upon the history/physical and finding an enlarged dilated colon accompanied by severe systemic toxicity. The transverse or right colon is usually the most dilated, frequently greater than 6 cm and occasionally up to 15 cm on supine films. Repositioning of the patient results in redistribution of air in the colon and therefore location of air is not as important as the degree of dilatation. Anemia related to blood loss leukocytosis with a left shift and electrolyte disturbances are common and should be corrected.

occasionally up to 15 cm on supine films. Repositioning of the patient results in redistribution of air in the colon and therefore location of air is not as important as the degree of dilatation. Anemia related to blood loss leukocytosis with a left shift and electrolyte disturbances are common and should be corrected. All antimotility agents, opiates, and anticholinergics should be discontinued. High dose glucocorticoids should be initiated as soon as diagnosis is made and they do not increase the risk of perforation [46]. Broad spectrum antibiotics are started to minimize any septic complications. Parenteral nutrition is of limited value [47], Sulfasalazine and 5-ASA compounds have no role in patients with toxic megacolon due to IBD and should be initiated only after resolution of the attack. Free perforation, massive hemorrhage, increasing transfusion requirements, worsening signs of toxicity, and progression of colonic dilatation are absolute indications for surgery. In addition, most surgical studies recommend colectomy if there is persistent colonic distention after 48 to 72 hour [48]. Surgical treatment Table 4 shows the indications for surgery in IBD. Surgical treatment is indicated in IBD if there is no response to optimal medical management and for dysplastic lesions. An honest discussion of the risks and benefits of the proposed operation with the patient and family is mandatory to avoid the frustrations stemming from unrealistic expectations. In patients who will receive an ostomy, a consultation with a stomal therapist is useful for selecting the ostomy site.

dysplastic lesions. An honest discussion of the risks and benefits of the proposed operation with the patient and family is mandatory to avoid the frustrations stemming from unrealistic expectations. In patients who will receive an ostomy, a consultation with a stomal therapist is useful for selecting the ostomy site. Table 4 Indications for surgery in IBD Crohn’s disease Ulcerative colitis 1. Intra-abdominal/ perianal abcess 1. Dysplasia complicating long standing UC 2. Complex fistulae 2. Recurrent, frequent relapses with poor quality of life despite optimal therapy 3. Mechanical complications like fibrotic strictures 3. Fulminant UC unreponsive to medical therapy 4. Fulminant CD unreponsive to medical therapy In order to provide adequate pre-operative care it is important to optimise the medical status, such as correcting anemia, addressing fluids/electrolytes. Most immunosuppressive therapy can be discontinued prior to sugery except glucocorticoids which should be tapered after surgery. Continuation of immunosuppressive therapy may be desirable in some patients with CD to prevent postoperative recurrence.

cal status, such as correcting anemia, addressing fluids/electrolytes. Most immunosuppressive therapy can be discontinued prior to sugery except glucocorticoids which should be tapered after surgery. Continuation of immunosuppressive therapy may be desirable in some patients with CD to prevent postoperative recurrence. In UC, the usual procedures performed are proctocolectomy with ileostomy or colectomy with ileal pouch-anal canal anastomosis (IPAA). Segmental resections are sometimes performed for limited areas of Crohn's colitis but are inappropriate for patients with ulcerative colitis because of the risk of recurrent active inflammation or cancer developing in the remaining colon. Ileal pouch-anal canal anastomosis is usually avoided in patients with CD because of poorer functional outcomes and a higher failure rate. The most frequent early complications are bowel obstruction, bleeding, pelvic and wound sepsis, transient urinary dysfunction, and dehydration due to high output from the ostomy. These can be addressed without re-operation. Late complications include stricture of the anastomosis, anal fistula and abscess, poor postoperative anorectal function, reduced fertility and pouchitis.

n, bleeding, pelvic and wound sepsis, transient urinary dysfunction, and dehydration due to high output from the ostomy. These can be addressed without re-operation. Late complications include stricture of the anastomosis, anal fistula and abscess, poor postoperative anorectal function, reduced fertility and pouchitis. The only absolute contraindication to IPAA is anal sphincter dysfunction. Other contraindication includes suspected CD. The need for pelvic radiation also is a contraindication to pelvic reservoir construction. The diagnosis of UC must be certain before an ileal pouch reservoir is created in a patient with inflammatory bowel disease. It is important to avoid pouch surgery or total proctcolectomy for severe colitis. It may be important to discuss with the surgeon about avoiding total proctocolectomy for severe colitis and to leave all options open [49]. This is because of concerns for UC recurrence in pouch, infections of pouch, wound dehiscence made possible during a fulminant attack in which the patient's condition is compromised, with a potentially poor nutritional status, low albumin level, low hematocrit level, and complications of high-dose corticosteroid use.

. This is because of concerns for UC recurrence in pouch, infections of pouch, wound dehiscence made possible during a fulminant attack in which the patient's condition is compromised, with a potentially poor nutritional status, low albumin level, low hematocrit level, and complications of high-dose corticosteroid use. CD is often complicated by fibrostenotic strictures that can be located within the whole gastrointestinal tract. Strictures can remain clinically asymptomatic over years until the intraluminal caliber causes obstruction. However, it is often difficult to differentiate between an inflammatory or fibrostenotic stricture. Ultrasound and MRI with the possibility to visualize mucosal blood flow are helpful in differential diagnosis. MR enterography is useful in distinguishing fixed strictures from inflammatory strictures at least in prelim studies. Before initiating surgical interventions, many clinicians try at least one attempt of medical treatment for strictures suggested to have an inflammatory component. Corticosteroids are most commonly used in this clinical situation. Fibrostenotic strictures will not respond to medical therapy. Endoscopic ballon dilatations, stricturoplasty or resections are required [50], remembering that there is lack of data and long-term follow-up in endoscopic dilation of strictures and that malignancy may cause stricture.

only used in this clinical situation. Fibrostenotic strictures will not respond to medical therapy. Endoscopic ballon dilatations, stricturoplasty or resections are required [50], remembering that there is lack of data and long-term follow-up in endoscopic dilation of strictures and that malignancy may cause stricture. Intra-abdominal abscess may need surgery even if it can be initially treated with percutaneous drainage with or without antibiotics. Patients who undergo surgical management are significantly less likely to develop a recurrent abscess compared to those who have been managed with antibiotics, 12 versus 56 percent [51]. Sutherland showed that the risk of reoperation 5 year after surgery in patients with CD was approximately 20% in nonsmokers and 36% in smokers. These figures increased to 41% and 70%, respectively, at 10 year. Female smokers and those with small bowel disease were at greatest risk. The effect of smoking can be reduced by long-term immunosuppression [52]. It is also important to identify the patients who are at high risk for recurrence of post-operative recurrence of CD. Even if macroscopically involved intestine is removed, the disease usually recurs proximal to, and at, the anastomosis. This often leads to the recurrent need for treatment of active disease, complications, and reoperation [53].

entify the patients who are at high risk for recurrence of post-operative recurrence of CD. Even if macroscopically involved intestine is removed, the disease usually recurs proximal to, and at, the anastomosis. This often leads to the recurrent need for treatment of active disease, complications, and reoperation [53]. Patients with an ileocolic anastomosis are known to have higher recurrence rates than those with a colectomy and end-ileostomy [54]. Yet another predictor of recurrence is disease behavior. Repeat resection after the primary operation for perforating disease occurred in half the time, on average, compared to those with nonperforating disease. Time to first reoperation was 4.7 years in the perforating group compared with 8.8 years in the nonperforating [55].

her predictor of recurrence is disease behavior. Repeat resection after the primary operation for perforating disease occurred in half the time, on average, compared to those with nonperforating disease. Time to first reoperation was 4.7 years in the perforating group compared with 8.8 years in the nonperforating [55]. It is important to identify clinical recurrence of the disease versus endoscopic recurrence. Rutgeerts and colleagues described endoscopic scoring instrument for clinical use in patients who underwent complete surgical resection, comparing endoscopic remission versus clinical remission versus the need for recurrent surgery. These researchers found that patients with grade III and IV endoscopic recurrence in the neoterminal ileum were at increased risk for a clinical recurrence. In high-risk patients, it would seem reasonable to implement prophylactic therapy using metronidazole initially later on immunomodulators. There is evidence that AZA is effective as a post-operative maintenance therapy though it is limited [25]. Landmark trial by Regueiro showed that administration of infliximab after intestinal resective surgery was effective at preventing endoscopic and histologic recurrence of CD. In summary, there are many advances made in the management of IBD. With just a few precautions, health care providers could optimize the care and stall complications of the disease and its therapy.

Introduction Torsion of the omentum is a rare pathology, and its clinical presentation mimics acute appendicitis [1]. The first case of torsion of the omentum was described by Bush in 1896. By 1908 approximately 112 cases had been described [2], and in 1991 Coppo gathered data on nearly 150 cases [3, 4]. The first cases of omentum pathology at the Hospital General Regional 36, at the Instituto Mexicano del Seguro Social in Puebla, Mexico, were reported in 1998 [5] and 2004 [6]. By 2001, slightly fewer than 300 cases had been reported, 85% of them in the adult population and the remaining 15% in the pediatric population, almost all diagnosed as acute appendicitis and discovered during an exploratory laparotomy [1].

no del Seguro Social in Puebla, Mexico, were reported in 1998 [5] and 2004 [6]. By 2001, slightly fewer than 300 cases had been reported, 85% of them in the adult population and the remaining 15% in the pediatric population, almost all diagnosed as acute appendicitis and discovered during an exploratory laparotomy [1]. In children, 0.05 to 0.1% of cases are diagnosed during a laparotomy for acute appendicitis[1, 7]. The condition is more common in males, with a ratio of 2-5.1 [1, 8], in the third and fourth decades of life, but it can occur at any age[5, 6]. It can be primary or idiopathic, when no underlying cause or associated factors are found; or secondary, when the cause is identified [1, 9-11]. Primary torsion is less common than secondary [1]. Longer or more swollen than normal omentum, internal hernias, inflammatory pathologies of other organs such as acute cholecystitis, pancreatitis, and adnexitis, tumors, and postsurgical adhesions are causes of secondary torsion of the omentum [1]. Its etiology remains associated with the predisposing factors such as sex, obesity, sudden strong increase in intra-abdominal pressure brought on by coughing or violent exercise, traumas, autonomics (large pedicle), larger or more twisted than normal epiploic blood vessels, accelerated peristalsis, surgical adherences, or some acute process in an intracavitary organ that causes the migration of a segment of the omentum to the affected site, resulting in torsion[5, 6, 9]. Torsion of the omentum presents with light abdominal pain that is similar to acute appendicitis or less similar to any other abdominal surgical pathology. However, the evolution of symptoms and clinical signs is slower and less intense, which creates a delay in the time it takes for the patient to seek medical assistance[10, 11]. The diagnosis is frequently made during exploratory laparotomy [1, 5, 6]. However, ultrasound and computed axial tomography are useful tools for making a preoperative diagnosis [12-16]. Torsion of the omentum is considered a cause of right lower quadrant pain (RLQP) and acute abdomen [5].

ek medical assistance[10, 11]. The diagnosis is frequently made during exploratory laparotomy [1, 5, 6]. However, ultrasound and computed axial tomography are useful tools for making a preoperative diagnosis [12-16]. Torsion of the omentum is considered a cause of right lower quadrant pain (RLQP) and acute abdomen [5]. When omental torsion is present, edema and the inflammatory process make the clinical presentation to progress to necrosis of the twisted segment, more frequently on the right side due to the length and characteristics of the greater omentum [8, 11]. The treatment consists in the removal of the affected segment either by means of laparoscopy or laparotomy, with excellent results [17-20]. The aim of this paper is to present the clinical characteristics of patients with torsion of the omentum, treatment, and evolution. Methods An observational and retrospective review was conducted of all cases of torsion of the omentum in a second level medical facility in Puebla, Mexico. The study included all patients who were operated on for torsion of the omentum, between January 1, 1998 and December 31, 2008. Volume 4-30-27/90 (Surgical Record) was consulted to locate clinical records for the study. The study variables were age, sex, body mass index (BMI), time of evolution from the onset of symptoms to surgical intervention, preoperative and postoperative diagnosis and evolution. The BMI was interpreted according to Quetelet´s Index: 18 to 25 healthy; above 25 overweight; above 30 somewhat obese; and above 40 morbidly obese. Descriptive statistical analysis was employed.

ime of evolution from the onset of symptoms to surgical intervention, preoperative and postoperative diagnosis and evolution. The BMI was interpreted according to Quetelet´s Index: 18 to 25 healthy; above 25 overweight; above 30 somewhat obese; and above 40 morbidly obese. Descriptive statistical analysis was employed. Results A total of 112,830 surgical procedures (January 1, 1998 to December 31, 2007) were performed (source: Unique Information System database (UISD) in the Puebla State Regional Office of the Instituto Mexicano del Seguro Social, of these, 11 patients had torsion of the omentum. Seven (63.63%) were women and 4 (36.36%) were men, with a median age of 33 years (20 to 58); hyperthermia was present in 4 (36.36%) patients; the average BMI was 29.06 kg/m2 (SD 2.76); 2 patients (18.18%) had a BMI < 24.91 kg/m2 and 9 (81.81%) had a BMI > 25kg/m2; the average duration of clinical presentation of symptoms was 6.54 (SD 3.47) days. The signs and symptoms of the patients are shown in Table 1. The predisposing factors, cause of lesion, and surgery performed of the cases are shown in Table 2. No patients’ abdomens were examined by means of ultrasound or computed axial tomography.

duration of clinical presentation of symptoms was 6.54 (SD 3.47) days. The signs and symptoms of the patients are shown in Table 1. The predisposing factors, cause of lesion, and surgery performed of the cases are shown in Table 2. No patients’ abdomens were examined by means of ultrasound or computed axial tomography. Table 1 Signs and symptoms of patients with torsion of the omentum Signs and symptoms of patients with torsion of the omentum n= 11 % Constipation 3 27.27 Malaise 3 27.27 Fever (> 38.2 °C) 4 36.36 Vomiting 4 36.36 March impairment 4 36.36 Abdominal distention 6 54.54 Pain 11 100 Table 2 Clinical Characteristics of patients with torsion of the omentum n Sex Age (Years) BMI Previous Surgery Duration (Days) Location of pain Cause of lesion 1 F 20 28.84 Caesarian 6 RLQ X 2 F 33 32.02 None 4 RLQ X 3 F 58 29.74 Appendect. 2 RUQ CCC 4 M 26 30.22 None 12 RLQ X 5 F 32 30.26 None 8 RLQ TROC 6 M 41 29.49 Appendect. 5 RLQ X 7 F 33 31.98 None 9 RLQ X 8 F 23 23.33 None 12 RLQ X 9 F 41 30.85 Appendect 2 RLQ X 10 M 36 32.81 None 5 RLQ X 11 M 28 30.47 Appendect 7 RLQ X BMI= body mass index, RLQ= right lower quadrant, RUQ= right upper quadrant, X= none, CCC= chronic calculous cholecystitis, TROC= twisted right ovarian cyst. In 7 (63.63%) patients the preoperative diagnosis was acute appendicitis and the postoperative was confirmed in all cases by histopathologic study as torsion of the omentum. In a year follow up, none presented any complication.

Table 1 Signs and symptoms of patients with torsion of the omentum Signs and symptoms of patients with torsion of the omentum n= 11 % Constipation 3 27.27 Malaise 3 27.27 Fever (> 38.2 °C) 4 36.36 Vomiting 4 36.36 March impairment 4 36.36 Abdominal distention 6 54.54 Pain 11 100 Table 2 Clinical Characteristics of patients with torsion of the omentum n Sex Age (Years) BMI Previous Surgery Duration (Days) Location of pain Cause of lesion 1 F 20 28.84 Caesarian 6 RLQ X 2 F 33 32.02 None 4 RLQ X 3 F 58 29.74 Appendect. 2 RUQ CCC 4 M 26 30.22 None 12 RLQ X 5 F 32 30.26 None 8 RLQ TROC 6 M 41 29.49 Appendect. 5 RLQ X 7 F 33 31.98 None 9 RLQ X 8 F 23 23.33 None 12 RLQ X 9 F 41 30.85 Appendect 2 RLQ X 10 M 36 32.81 None 5 RLQ X 11 M 28 30.47 Appendect 7 RLQ X BMI= body mass index, RLQ= right lower quadrant, RUQ= right upper quadrant, X= none, CCC= chronic calculous cholecystitis, TROC= twisted right ovarian cyst. In 7 (63.63%) patients the preoperative diagnosis was acute appendicitis and the postoperative was confirmed in all cases by histopathologic study as torsion of the omentum. In a year follow up, none presented any complication. Discussion Torsion of the omentum is a rare cause of acute abdomen. Its presentation mimics acute appendicitis and other pathologies that cause acute abdomen. In this study group, females were more commonly affected, although most authors report predominance in men [1, 4, 8], and in some groups no predominance by sex is mentioned [1]. The reported incidence ranges between 0.16 and 0.37. The duration of the clinical presentation of symptoms in this group is greater than that of acute appendicitis, probably due to the fact that the intensity of symptoms is less. This concurs with reports from other groups [1].

no predominance by sex is mentioned [1]. The reported incidence ranges between 0.16 and 0.37. The duration of the clinical presentation of symptoms in this group is greater than that of acute appendicitis, probably due to the fact that the intensity of symptoms is less. This concurs with reports from other groups [1]. Pain is the predominant symptom, and its location depends on the affected site of the omentum, in literature it is reported in 100% of the cases, rarely occurs in the upper right quadrant of the abdomen and is continuous. The hyperthermia that was present in 36.36% of patients was less than 38.2° C, which does not concur with information from some authors who report temperature elevations up to 39.5°C [1]. Previous surgery was a predisposing factor in 5 (45.45%) of the patients with a BMI greater than 25. 7 (63.63%) patients had a BMI greater than 30, which coincides with data in world literature that identifies obesity as a predisposing factor for torsion of the omentum [1, 5, 6, 9]. In 2 (18.18%) patients the cause of the torsion was identified: one (9.09%) with an intensifying chronic calculous cholecystitis that caused pain in the upper right quadrant of the abdomen and one (9.09%) with a twisted right ovarian cyst. The acute inflammatory process in these organs caused the omentum to migrate, resulting in its torsion.

the cause of the torsion was identified: one (9.09%) with an intensifying chronic calculous cholecystitis that caused pain in the upper right quadrant of the abdomen and one (9.09%) with a twisted right ovarian cyst. The acute inflammatory process in these organs caused the omentum to migrate, resulting in its torsion. As in other study groups, all the patients underwent surgery for acute abdomen. The definitive diagnosis was obtained by means of exploratory laparotomy. In the cases where there was no preceding appendectomy, the preoperative diagnosis was probable acute appendicitis due to the similarity of symptoms. As in the majority of published studies, no preoperative diagnosis was made in any of the cases in this group [21]. The imaging data from simple x-rays of the abdomen showed the presence of a fixed, air-filled small bowel loop in 6 (54.54%) patients, an indication that suggested surgical acute abdomen. Although some authors report that therapeutic diagnostic laparoscopy can be very useful for diagnosing and managing segmental torsion of the greater omentum [15, 17-20], the majority of cases reported in the literature have been diagnosed by laparotomy.

4%) patients, an indication that suggested surgical acute abdomen. Although some authors report that therapeutic diagnostic laparoscopy can be very useful for diagnosing and managing segmental torsion of the greater omentum [15, 17-20], the majority of cases reported in the literature have been diagnosed by laparotomy. Treatment consists of removing the affected segment of the greater omentum, as well as managing concomitant pathology when it exists [16, 18, 19]. Conservative management in patients without associated complications has also been reported [22]. In this study group, treatment by means of laparotomy achieved good results without complications. However, treatment by laparoscopy can achieve a better aesthetic result and lessen the time of hospitalization [19, 20]. In conclusion, torsion of the omentum is a rare pathology. Its clinical presentation is similar to acute appendicitis or to any other cause of surgical acute abdomen. It has a tendency to present in obese people. Pain and abdominal distention are the predominant symptoms. For the most part, the condition is idiopathic, which makes it difficult to identify the cause in up to 33.3% of cases. Preoperative clinical diagnosis is difficult, and it must depend on complementary studies when they are available. Management of the condition is surgical, partial omentectomy and treatment of the original cause of the torsion. The evolution is good when correct treatment is applied, even when delayed.

33.3% of cases. Preoperative clinical diagnosis is difficult, and it must depend on complementary studies when they are available. Management of the condition is surgical, partial omentectomy and treatment of the original cause of the torsion. The evolution is good when correct treatment is applied, even when delayed. We thank Kathryn J Hurlbert for her collaboration in the translation of this paper. This work was presented in the X Congreso IBEROLATINOAMERICANO de cirugía, “Cirugía 2008” from Dec 2nd to Dec 5th 2008 in La Habana, Cuba. There was no financial support received for this work from any person or institution. Conflict of interest: None.

Introduction In clinical settings, early total enteral nutrition (TEN) is known to reduce the postoperative complication and infection rate as well as duration of postoperative stay compared with total parenteral nutrition (TPN) in a variety of critical conditions: postoperative recovery [1-3], abdominal trauma [4, 5], pancreatitis [6], burn [7-9]. To support the superiority of TEN to TPN obtained as clinical evidences, rodent’s models demonstrated a couple of mechanisms that TEN reduced cytokine production after operation or burn [10, 11], TPN increased apoptosis in the small bowel mucosa [12] and bacterial and endotoxin translocation [13-15]. However, these evidence using rodents models were fragmented and not comprehensive. Therefore, we aimed to compare effects of TEN and TPN on wound healing by measuring a variety of parameters using burned rat model.

PN increased apoptosis in the small bowel mucosa [12] and bacterial and endotoxin translocation [13-15]. However, these evidence using rodents models were fragmented and not comprehensive. Therefore, we aimed to compare effects of TEN and TPN on wound healing by measuring a variety of parameters using burned rat model. Materials and Methods Treatment of animals All studies were carried out in compliance with the institutional guidelines of animal experiments at Jikei University School of Medicine. Male Sprague-Dawley rats at 11 weeks (Charles River Japan Inc., Yokohama, Japan) were fed with CRF-1: standard meal for rats (Oriental Co., Tokyo, Japan). After making sure rats’ condition healthy by spending 7 days, either intragastric root for TEN or intravenous root for TPN was obtained under anesthesia induced by administrating 40 mg/kg of pentobarbital intraperitoneally. For TEN group, a polyvinyl catheter (5 Fr) (Japan Sharwood, Tokyo, Japan) was inserted into intragastric space of the rat and the other end of catheter was guided subcutaneously to the back, and then connected with joint pipe with stainless steel by opened abdominal operation. For TPN group, opened abdominal operation alone was performed.

yvinyl catheter (5 Fr) (Japan Sharwood, Tokyo, Japan) was inserted into intragastric space of the rat and the other end of catheter was guided subcutaneously to the back, and then connected with joint pipe with stainless steel by opened abdominal operation. For TPN group, opened abdominal operation alone was performed. One week after abdominal operation, burns were made on the center of rats’ back under anesthesia by intraperitoneally administrating pentobarbital. Briefly, after shaving the back, 42 rats had burn of 15 mm in diameter by pressing an electrical soldering iron at 200 °C for 30 seconds. Two days after making burn, debridement was performed for skin of wound tissue under anesthesia with pentobarbital. Three days after making burn, absorbent cotton covered with OpSite Wound (Smith and Nephew Inc., Florida, USA) was put on wound tissue to absorb exudative solution. Wound healing was leave opened between 7 and 28 days after making burn.

idement was performed for skin of wound tissue under anesthesia with pentobarbital. Three days after making burn, absorbent cotton covered with OpSite Wound (Smith and Nephew Inc., Florida, USA) was put on wound tissue to absorb exudative solution. Wound healing was leave opened between 7 and 28 days after making burn. At the day of making burn (defined as day 0), either TEN or TPN was started. For TEN, the other end of joint pipe was connected to a swivel via fixing spring with polyethylene tube through harness on the back of rat. For TPN, a small skin incision was made in the inguinal region of the rat to insert a silicon catheter (inner diameter: 0.5 mm, outer diameter: 1.0 mm; Kaneka Medix, Tokyo, Japan) into the right femoral vein to reach vena cava inferior. The other end of the catheter was guided subcutaneously to the posterior aspect of the neck, and then connected to a swivel via harness on the back of rat. The skin wound for insertion of the catheter was closed by a stitch of 3-0 silk. Rats of TEN group had incision at inguinal region alone.

reach vena cava inferior. The other end of the catheter was guided subcutaneously to the posterior aspect of the neck, and then connected to a swivel via harness on the back of rat. The skin wound for insertion of the catheter was closed by a stitch of 3-0 silk. Rats of TEN group had incision at inguinal region alone. Either TEN or TPN, nutrition was continuously infused through polyethylene tube connecting to the other side of swivel for 24 hours per day using perista pump (Watoson-Marlow 502S; Nikkisou Co. Ltd., Tokyo, Japan) without peroral feeding. Rats belonging to either TEN or TPN group were administrated nutrition 60 kcal/day from day 0 to day 1 and 80 kcal/day from day 2 to day 28. To confirm normal range of parameters, control rats (N = 12) not treated with burn or operation were fed with the same calorie during the same period of time. Components of nutrients used for TEN: Twinline® (Otsuka Pharmaceutical Co., Ltd.; Tokyo, Japan), and for TPN: Unicaliq® (N) (Termo Co., Tokyo, Japan) + Multamin® (Sankyo Co. Ltd., Tokyo, Japan) + Mineralin® (Takeda Pharmaceutical Co. Ltd., Tokyo, Japan) + Intrafat® (20%) (Takeda Pharmaceutical Co. Ltd., Tokyo, Japan) + 20%(W/W) Choline chloride (Wako Pure Chemical Ind., Osaka, Japan), solutions were shown in Table 1. These solutions were made up in clean bench. As a control group, rats without operation nor treatment for burn were fed CRF-1 for the same calorie during the same experimental period to know normal ranges of serum/urine parameters for nutrition and inflammation.

cal Ind., Osaka, Japan), solutions were shown in Table 1. These solutions were made up in clean bench. As a control group, rats without operation nor treatment for burn were fed CRF-1 for the same calorie during the same experimental period to know normal ranges of serum/urine parameters for nutrition and inflammation. Table 1 Components of nutrients used for TEN or TPN (/100 kcal) Component TEN*1 TPN*2 Carbohydrate 14.68 g 20.59 g Lipid LCT 0.812 g 0.374 g MCT 1.968 g 0 g Amino acid 4.05 g 3.53 g Total nitrate 0.6 g 0.55 g Volume 100 ml 120 ml Calorie/N 176 182 Calorie of non protein 83.7 kcal 85.9 kcal Calorie of non protein/N 140 157 *1: Twinline® (Otsuka co. ltd, Tokyo, Japan) *2: Unicaliq® (N) + Multamin®+ Mineralin® + Intrafat® (20%) + 20% Chorine chloride At final day of observation, opened abdominal operation was performed under anesthesia with intraperitoneally administrated pentobarbital for blood sampling from portal vein and vena cava inferior. After sacrificed with total bleeding, spleen was resected for weight measure. Outcome measures Degree of wound healing was estimated by area of wound at day 0 of making burn and day 28 of sacrificed using Image analysis software (IPAP-WIN: Sumika Technoservice Co., Osaka, Japan) for measuring area. Body weights were measured at day 0 and day 28.

At final day of observation, opened abdominal operation was performed under anesthesia with intraperitoneally administrated pentobarbital for blood sampling from portal vein and vena cava inferior. After sacrificed with total bleeding, spleen was resected for weight measure. Outcome measures Degree of wound healing was estimated by area of wound at day 0 of making burn and day 28 of sacrificed using Image analysis software (IPAP-WIN: Sumika Technoservice Co., Osaka, Japan) for measuring area. Body weights were measured at day 0 and day 28. Urine and stool were collected during day 23, day 24 and day 25. Urine was frozen and stool was freeze-dried and packaged until nitrogen measures. Urine and feces nitrogen quantity was determined daily by a chemiluminescence technique from day 0 to day 28 after burn. Nitrogen accounts = medication nitrogen – (urine nitrogen + feces nitrogen); Prices = nitrogen accounts/(medication nitrogen – feces nitrogen)

l was freeze-dried and packaged until nitrogen measures. Urine and feces nitrogen quantity was determined daily by a chemiluminescence technique from day 0 to day 28 after burn. Nitrogen accounts = medication nitrogen – (urine nitrogen + feces nitrogen); Prices = nitrogen accounts/(medication nitrogen – feces nitrogen) Serum protein levels were measured as following combinations: prolyl hydroxylase by enzyme immunoassay (EIA)-kit (Fujiyakuhin Co., Ltd. Saitama, Japan); total protein and albumin by A/G B-test WAKO (Wako Pure Chemical Ind., Osaka, Japan); transferrin by EIA-kit (Panafirm Laboratories Co. Ltd., Kumamoto, Japan); sialic acid by sialic acid measurement kit (Kyokuto Pharmaceutical Industrial Co., Ltd. Tokyo, Japan); endotoxin assay kit by toxi-color (Seikagaku Co., Tokyo, Japan); tumor necrotizing factor (TNF)-a, interleukin (IL)-6, IL-4 and IL-10 by ELISA kit (Biosource International Inc., California, USA); IL-8 by Rat GRO/CINC ELISA system (Amersham Lifescience Inc., Buckinghamshire, UK). Biochemical parameters were measured with an automatic serum analyzer (Model 7150, Hitachi Ltd., Tokyo, Japan).

necrotizing factor (TNF)-a, interleukin (IL)-6, IL-4 and IL-10 by ELISA kit (Biosource International Inc., California, USA); IL-8 by Rat GRO/CINC ELISA system (Amersham Lifescience Inc., Buckinghamshire, UK). Biochemical parameters were measured with an automatic serum analyzer (Model 7150, Hitachi Ltd., Tokyo, Japan). Statistics Significant differences among TEN, TPN and control group were estimated with Kruskal-Wallis test and defined as significant when p-value was less than 0.05. On the other hand, significant differences between two groups: TEN and TPN; TEN and control; TPN and control; were estimated with Mann-Whitney (Two-sample Wilcoxon rank-sum) test. The difference was defined significant when p-value was less than 0.016 according to Bonferroni correction. All statistical analyses were performed using STATA version 8.0 (Stata Corporation, College Station, TX, USA). Results Changes of body weight and nutrition markers Total calorie intake was not different among TEN (N = 17; 2223.0 ± 10.0 kcal), TPN (N = 15; 2246.1 ± 11.0 kcal) and control groups (N = 12; 2203.7 ± 12.0 kcal). Percent changes of body weight at day 28 divided by bodyweight at day 0 were compared among TEN, TPN, and normal groups (Fig. 1). Increase of body weight was equivalent between TEN (N = 17; 122.4 ± 7.8%) and TPN (N = 15; 126.3 ± 7.2%). While body weights of burned rats treated with either TEN or TPN were significantly less than control rats without burn (N = 12; 139.8 ± 5.1%).

ght at day 0 were compared among TEN, TPN, and normal groups (Fig. 1). Increase of body weight was equivalent between TEN (N = 17; 122.4 ± 7.8%) and TPN (N = 15; 126.3 ± 7.2%). While body weights of burned rats treated with either TEN or TPN were significantly less than control rats without burn (N = 12; 139.8 ± 5.1%). Figure 1 Comparison of bodyweight changes among TEN, TPN, and normal groups. Changes were calculated as bodyweight at day 28/ bodyweight at day 0 (%). Statistical difference was evaluated with Man Whitney U test. Nutritional markers were compared among three groups (Table 2). Although medication nitrogen quantity and feces nitrogen was least in TPN group, urine nitrogen was most. Thus, the prices were least in TPN, second in TEN, and most in control group. Serum levels of total protein, albumin and transferrin at day 28 were equivalent between TEN and TPN, whereas those of control group were significantly higher than TEN and TPN.

d feces nitrogen was least in TPN group, urine nitrogen was most. Thus, the prices were least in TPN, second in TEN, and most in control group. Serum levels of total protein, albumin and transferrin at day 28 were equivalent between TEN and TPN, whereas those of control group were significantly higher than TEN and TPN. Table 2 Effects of TEN on nutrition Biomarker TEN N = 17 TPN*2 N = 15 Control*3 N = 12 Kruskal-Wallis *1 Medication nitrogen quantity 947.8 ± 27.3 881.2 ± 15.1‡ 1515.7 ± 8.1‡ 0.0001 Urine inside nitrogen quantity 659.2 ± 63.6 755.1 ± 73.2† 625.4 ± 42.1‡ 0.0077 Feces inside nitrogen quantity 62.2 ± 30.7 22.9 ± 8.6† 365.4 ± 7.1‡ < 0.0001 Nitrogen accounts 226.3 ± 87.2 103.3 ± 80.7† 524.8 ± 34.9‡ < 0.0001 Prices 25.3 ± 9.1 11.9 ± 9.3† 45.6 ± 3.3‡ < 0.0001 Total protein (g/dl) 5.4 ± 0.3 5.3 ± 0.4 4.9 ± 0.2‡ 0.0004 Albumin (g/dl) 1.8 ± 0.1 1.8 ± 0.1 2.2 ± 0.1‡ < 0.0001 Transferrin (mg/ml) 3.19 ± 0.41 3.13 ± 0.62 3.16 ± 0.50‡ 0.0005 *1: Statistical differences were calculated based on Kruskal-Wallis equality of populations rank test. Statistical significance was defined when p-value was less than 0.05. *2: Value of TPN was compared with TEN by correcting with Bonferroni. *3: Value of control was compared with TEN.: † p < 0.016, ‡: p < 0.005.

Table 2 Effects of TEN on nutrition Biomarker TEN N = 17 TPN*2 N = 15 Control*3 N = 12 Kruskal-Wallis *1 Medication nitrogen quantity 947.8 ± 27.3 881.2 ± 15.1‡ 1515.7 ± 8.1‡ 0.0001 Urine inside nitrogen quantity 659.2 ± 63.6 755.1 ± 73.2† 625.4 ± 42.1‡ 0.0077 Feces inside nitrogen quantity 62.2 ± 30.7 22.9 ± 8.6† 365.4 ± 7.1‡ < 0.0001 Nitrogen accounts 226.3 ± 87.2 103.3 ± 80.7† 524.8 ± 34.9‡ < 0.0001 Prices 25.3 ± 9.1 11.9 ± 9.3† 45.6 ± 3.3‡ < 0.0001 Total protein (g/dl) 5.4 ± 0.3 5.3 ± 0.4 4.9 ± 0.2‡ 0.0004 Albumin (g/dl) 1.8 ± 0.1 1.8 ± 0.1 2.2 ± 0.1‡ < 0.0001 Transferrin (mg/ml) 3.19 ± 0.41 3.13 ± 0.62 3.16 ± 0.50‡ 0.0005 *1: Statistical differences were calculated based on Kruskal-Wallis equality of populations rank test. Statistical significance was defined when p-value was less than 0.05. *2: Value of TPN was compared with TEN by correcting with Bonferroni. *3: Value of control was compared with TEN.: † p < 0.016, ‡: p < 0.005. Effects of TEN on wound healing Typical wound areas of rats’ back at day 28 treated with either TEN or TPN were shown as Figure 2. Burned area was significantly smaller in rats treated with TEN (12.7 ± 1.6% of burned area at day 0) than with TPN (19.0 ± 2.3% of burned area at day 0) (Fig. 3). Serum levels of prolyl hydroxylase were significantly lower in TEN (449.4 ± 98.2 ng/ml) than TPN group (953.2 ± 611.3 ng/ml) (P = 0.0004), which were more than control (286.5 ± 41.3 ng/ml) (P = 0.0001). Figure 2 Difference of burned area at day 0 (left panel) and day 28 (right panel) between TPN (upper) and TEN (lower).

Effects of TEN on wound healing Typical wound areas of rats’ back at day 28 treated with either TEN or TPN were shown as Figure 2. Burned area was significantly smaller in rats treated with TEN (12.7 ± 1.6% of burned area at day 0) than with TPN (19.0 ± 2.3% of burned area at day 0) (Fig. 3). Serum levels of prolyl hydroxylase were significantly lower in TEN (449.4 ± 98.2 ng/ml) than TPN group (953.2 ± 611.3 ng/ml) (P = 0.0004), which were more than control (286.5 ± 41.3 ng/ml) (P = 0.0001). Figure 2 Difference of burned area at day 0 (left panel) and day 28 (right panel) between TPN (upper) and TEN (lower). Figure 3 Changes of burned area: burned area at day 28 divided by wound area at day 0 (%) in either TPN or TEN. Statistical difference was evaluated with Man Whitney U test.

Effects of TEN on wound healing Typical wound areas of rats’ back at day 28 treated with either TEN or TPN were shown as Figure 2. Burned area was significantly smaller in rats treated with TEN (12.7 ± 1.6% of burned area at day 0) than with TPN (19.0 ± 2.3% of burned area at day 0) (Fig. 3). Serum levels of prolyl hydroxylase were significantly lower in TEN (449.4 ± 98.2 ng/ml) than TPN group (953.2 ± 611.3 ng/ml) (P = 0.0004), which were more than control (286.5 ± 41.3 ng/ml) (P = 0.0001). Figure 2 Difference of burned area at day 0 (left panel) and day 28 (right panel) between TPN (upper) and TEN (lower). Figure 3 Changes of burned area: burned area at day 28 divided by wound area at day 0 (%) in either TPN or TEN. Statistical difference was evaluated with Man Whitney U test. Effects of TEN on morphology of small intestine Weight of small intestinal loop per 100 g of body weight was heavier in TEN (2.08 ± 0.22 g) and control group (2.00 ± 0.16 g) than in TPN group (1.48 ± 0.26 g), although no significant difference existed between TEN and control group (Fig. 4). Then, length of villi and depth of crypt were measured in duodenum, jejunum and ileum under hematoxylin eosin staining among TEN, TPN, and control group: Examples of hematoxylin and eosin staining of jejunum were shown (Fig. 5). Then, ratio: length of villi divided by depth of crypt in jejunum was compared among TPN, TEN and control group (Fig. 6). The ratio was significantly smaller in TPN group than in TEN (P = 0.012). Weight of small bowel per 100 g of bodyweight showed positive and linear association with anabolism of nitrogen (Fig. 7).

). Then, ratio: length of villi divided by depth of crypt in jejunum was compared among TPN, TEN and control group (Fig. 6). The ratio was significantly smaller in TPN group than in TEN (P = 0.012). Weight of small bowel per 100 g of bodyweight showed positive and linear association with anabolism of nitrogen (Fig. 7). Figure 4 Comparison of weights of small intestine at day 28 among TPN, EN and control group. Statistical difference was evaluated with Man Whitney U test. Figure 5 Hematoxylin and eosin staining of jejunum obtained from TEN (left panel) and TPN (right panel). Figure 6 Comparison of ratio: length of villi divided by depth of crypt in jejunum among TPN, EN and control group. Statistical difference was evaluated with Man Whitney U test. Figure 7 Association between weight of small bowel and prices. Line and gray area are showing linear regression and 95% confidence interval, respectively. Effects of TEN on immunological and biochemical markers Other parameters possibly related with wound healing were also compared among TEN, TPN, and control group (Table 3). Among cytokines measured in this experiment, only TNF-a (P = 0.0042) were significantly higher in TPN group than in TEN. Weights of spleen were also heavier in TPN than in TEN (P < 0.0001) or control group (P < 0.0001).

possibly related with wound healing were also compared among TEN, TPN, and control group (Table 3). Among cytokines measured in this experiment, only TNF-a (P = 0.0042) were significantly higher in TPN group than in TEN. Weights of spleen were also heavier in TPN than in TEN (P < 0.0001) or control group (P < 0.0001). Table 3 Effects of TEN on inflammation Types Biomarker TEN N = 17 TPN N = 15 Control N = 12 Kruskal-Wallis *1 Immune Sialic acid (mg/dl) 109.6 ± 12.2 125.5 ± 21.5 75.6 ± 6.8‡ < 0.0001 IL-4 (pg/ml) 5.4 ± 8.3 10.6 ± 12.7 9.7 ± 10.6 NS IL-6 (pg/ml) 2.4 ± 6.7 56.7 ± 91.6 0 ± 0 0.0061 IL-8 (pg/ml) 193.2 ± 144.0 283.5 ± 277.0 122.8 ± 46.4 NS IL-10 (pg/ml) 3.6 ± 9.8 18.2 ± 42.4 3.9 ± 13.4 NS TNF-α (pg/ml) 1.7 ± 4.2 3.9 ± 4.1‡ 1.3 ± 1.7 0.0087 Endotoxin (pg/ml) 18.5 ± 6.2 20.1 ± 6.1 5.7 ± 1.5 0.0012 Spleen (g/100g bodyweight) 0.25 ± 0.03 0.71 ± 0.40‡ 0.22 ± 0.03 < 0.0001 Liver AST (U/l) 129.0 ± 42.1 567.5 ± 1005.2*2 327.0 ± 698.6 NS ALT (U/l) 36.4 ± 9.4 92.1 ± 145.6 30.7 ± 7.5 NS Alkaliphosphatase (U/l) 360.5 ± 74.4 569.1 ± 222.8‡ 491 ± 109.4 0.0012 LAP (IU/l) 54.6 ± 1.5 70.1 ± 25.5 71.6 ± 6.1† 0.0066 Direct bilirubin (mg/dl) 0.113 ± 0.057 0.196 ± 0.117‡ 0.088 ± 0.016 0.0018 Indirect bilirubin (mg/dl) 0.015 ± 0.014 0.055 ± 0.075† 0.030 ± 0.012 NS Renal BUN (mg/dl) 18.5 ± 2.3 22.7 ± 4.5‡ 16.4 ± 15.5 < 0.0001 Cr (mg/dl) 0.46 ± 0.05 0.55 ± 0.08‡ 0.42 ± 0.05 < 0.0001 Metabolism Glucose (mg/dl) 157.3 ± 5.5 116.8 ± 32.7‡ 141.9 ± 17.5 0.0004 Triglyceride (mg/dl) 51.8 ± 19.6 23.1 ± 11.1‡ 35.8 ± 17.3† 0.0002 Total cholesterol (mg/dl) 60.8 ± 11.5 55.5 ± 10.4 54.0 ± 10.8 NS *1: Statistical differences were calculated based on Kruskal-Wallis equality of populations rank test. Statistical significance was defined when p-value was less than 0.05. *2. Two high levels of AST (2864, 2994) were included in TPN group. †: p < 0.016, ‡: p < 0.005.

erol (mg/dl) 60.8 ± 11.5 55.5 ± 10.4 54.0 ± 10.8 NS *1: Statistical differences were calculated based on Kruskal-Wallis equality of populations rank test. Statistical significance was defined when p-value was less than 0.05. *2. Two high levels of AST (2864, 2994) were included in TPN group. †: p < 0.016, ‡: p < 0.005. Both serum levels of direct (P = 0.0025), indirect bilirubin (P = 0.0041) and alkaliphosphatase (P = 0.0083) were significantly higher in TPN group than in TEN group. Moreover, BUN (P = 0.0058) and creatinine (P = 0.0021) were also higher in TPN group than in TEN group. In contrast, plasma glucose (P = 0.0015) and triglyceride levels (P = 0.0001) were lower in TPN group than in TEN group. Discussion In this study, wound healing was faster in TEN group than in TPN group, in spite of equivalent body weight changes after burn. There were few original articles to demonstrate superiority of TEN to TPN in wound healing using rat model [16-18]. Judging from data of nitrogen accounts and prices in this experiment, TEN can direct more anabolic state than TPN, although total protein, albumin, transferrin were equivalent between TPN and TEN group. In addition, blood urea nitrogen (BUN) that is one of protein metabolites was lower in TEN than in TPN in our study. TPN treated malnourished rats gained more weight with greater body fat formation than TEN group but had lower nitrogen [19]. Thus, our results and a previous report suggest that TEN may facilitate wound healing by maintaining protein anabolism more than TPN.

rotein metabolites was lower in TEN than in TPN in our study. TPN treated malnourished rats gained more weight with greater body fat formation than TEN group but had lower nitrogen [19]. Thus, our results and a previous report suggest that TEN may facilitate wound healing by maintaining protein anabolism more than TPN. Weight of small intestinal loop was heavier in TEN and control group than in TPN group, although no significant difference existed between TEN and control group. Moreover, ratio length of villi divided by depth of crypt was significantly smaller in TPN group than in TEN and control group, which may be consistent with previous reports that morphometry revealed an increased submucosal thickness while intestinal circumference markedly decreased in TPN-treated rats compared with TEN [20, 21]. TPN may keep gut little stress, conversely cause mucosal atrophy. Furthermore, these morphological changes induced by TPN were demonstrated to associate with reduced lymphocytes, increased gut permeability and enhanced bacterial translocation [22-26], which can increase risk of postoperative sepsis and postoperative morbidity/mortality [13, 27]. Small intestinal atrophy was shown to affect nitrogen metabolism to a greater extent than liver by-pass [28], which was also reconfirmed in our study that weight of small bowel showed positive linear relationship with levels of nitrate anabolism.

k of postoperative sepsis and postoperative morbidity/mortality [13, 27]. Small intestinal atrophy was shown to affect nitrogen metabolism to a greater extent than liver by-pass [28], which was also reconfirmed in our study that weight of small bowel showed positive linear relationship with levels of nitrate anabolism. Among cytokine production, serum levels of TNF-a were significantly lower in TEN than in TPN group. TPN increases the expression of TNF-a mRNA in organ tissues and systemic TNF-a production, and reduces the survival rate of rats after thermal injury, but TEN does not [11]. Thus, differences in cytokine levels between TEN and TPN in our study were consistent with previous studies. Moreover, increased weight of spleen confirmed in this study was not pointed out previously to our knowledge. Decreased stimulation to intestinal immunity may be compensated by hypertrophy of spleen at least in part.

differences in cytokine levels between TEN and TPN in our study were consistent with previous studies. Moreover, increased weight of spleen confirmed in this study was not pointed out previously to our knowledge. Decreased stimulation to intestinal immunity may be compensated by hypertrophy of spleen at least in part. Serum levels of bilirubin and alkaliphosphatase were significantly higher in TPN group than in TEN group. During TPN, hepatic concentration of the important intracellular antioxidant glutathione was reported to decrease [29]. Capacity of hepatic drug metabolism was shown to decrease in rats treated with TPN [30, 31]. Moreover, BUN and creatinine were also higher in TPN group than in TEN group. Rats given TEN after ischemic acute renal failure have improved renal function compared with rats given TPN [32]. These suggest that TEN may protect multiple organs against failure in critical conditions. In contrast, plasma glucose and triglyceride levels were lower in TPN group than in TEN group, of which meanings remain unknown. In conclusion, TEN may facilitate wound healing compared with TPN through preventing intestinal atrophy, keeping protein anabolism and suppressing inflammation. This study was supported by Grant in Aid for Scientific Research in Japan.

Introduction Budd-Chiari syndrome (BCS) is defined as chronic, progressive and congestive liver dysfunction resulting from obstruction of the outflow of the inferior vena cava (IVC) and/or hepatic veins [1, 2]. Wang et al [3] reviewed 2564 cases of BCS in China, among them, 433 were typed with short segmental occlusion of retrohepatic IVC (SSOR-IVC). Clinically, most SSOR-IVC are accompanied by various extent of obstructive lesion in intrahepatic veins, which can be further classified into 3 pathologic types: Type 1, partial intrahepatic venous obstruction; Type 2, complete intrahepatic venous obstruction with short hepatic vein dilation; and Type 3, complete intrahepatic venous obstruction without short hepatic vein dilation [4]. Medical intervention is not effective for the BCS with SSOR-IVC. The effective treatment is surgical intervention to decrease or eliminate the hypertension of IVC and portal vein (PV) [5, 6]. Type 3 intrahepatic obstruction is usually treated with mesoatrial bypass. The mainstay of the surgery for Type1 and 2 SSOR-IVC is cavoartrial bypass via the thoracolaparotomic approach [7]. However, the thoracolaparotomic approach is commonly accompanied by thoracic and pulmonary complications intra- and/or post-operation. In 2005, we developed a novel abdominal approach for suprahepatic and retrohepatic inferior vena cavocaval bypass for the treatment of type 1 or type 2 SSOR-IVC. Since then, we have performed 16 operations with the abdominal approach. This study is to summarize the abdominal approach and compare it with the conventional thoracolaparotomic approach in the treatment of the patients with type 1 or type 2 SSOR-IVC.

na cavocaval bypass for the treatment of type 1 or type 2 SSOR-IVC. Since then, we have performed 16 operations with the abdominal approach. This study is to summarize the abdominal approach and compare it with the conventional thoracolaparotomic approach in the treatment of the patients with type 1 or type 2 SSOR-IVC. Methods Clinical characteristics of patients From 2005 to 2008, 16 consecutive cases (group A) were performed suprahepatic and retrohepatic inferior vena cavocaval bypass, these patients had short segmental (< 3 cm) occlusion of IVC accompanied by type 1 or type 2 intrahepatic venous obstruction, aging from 25 to 65 years. The duration of the illness ranged from 0.5 to 32 years. Eighteen consecutive cases (group B) of same pathological type were selected for retrospective comparison, who received traditional thoracolaparotomic cavoatrial bypass from 2001 to 2004. All the patients had similar preoperative clinical symptoms and signs including upper quadrant abdominal fullness, dyspepsia, superficial varices of the tharacoabdominal wall esophageal varies and hepatosplenomegaly. The preoperative Child-Pugh scores of liver function in group A were class B (n = 11) and C (n = 5), while in group B there were 12 cases of class B and 6 cases of class C. Ascites existed in 13 cases of group A, and in 15 of group B. All class C patients in both groups were treated with liver protective drugs, diuretics, and albumin infusion. The diagnosis was confirmed by combined superior and inferior cavography and percutaneous transhepatic hepatovenography.

B and 6 cases of class C. Ascites existed in 13 cases of group A, and in 15 of group B. All class C patients in both groups were treated with liver protective drugs, diuretics, and albumin infusion. The diagnosis was confirmed by combined superior and inferior cavography and percutaneous transhepatic hepatovenography. Operation procedure The abdominal approach was performed under general anesthesia. A bilateral subcostal incision was made with an upper midline extension and removal of xiphoid process to achieve excellent exposure of the right lobe of the liver. The right lobe was mobilized to the left to fully expose the right lateral wall of retrohepatic IVC which was isolated for a length of about 8 cm from hepatic vein outlet with a mandatory protection of the short hepatic veins. The sub-phrenic IVC was carefully isolated and maximized to 2.5 – 3 cm in length for later anastomosis. This may be assisted by a careful isolation of the IVC at the entrance to the diaphragm without penetration to the pericardium. A 6 – 8 cm long, 1.6 cm in diameter artificial vessel with stent spring (Gore-Tex, USA) was prepared and beveled with 40° angle at both ends for an end-to-side anastomosis. The retrohepatic IVC distal to the occlusion lesion was longitudinally clamped with a curved vascular clamp for preparation of distal anastomosis. A 1.8 cm linear venotomy immediately distal to the occlusion lesion was made and then an end-to-side anastomosis of the graft to the IVC was performed using 4-0 prolene with interrupted and mattress sutures. The proximal end-to-side anastomosis of graft to subphrenic IVC was made in the same manner. As required, a groove on the liver surface was created with a superficial incision to facilitate the alignment of the graft vessel to both anastomosis. The clamp for the distal anastomosis was released allowing blood to fill the graft vessel and a needle puncture was made on the graft vessel to discharge the air inside. The proximal anastomosis was unclamped in a very slow manner to prevent acute right heart failure due to abrupt overload to the right heart. The patency of the bypass was confirmed by intraoperative ultrasonography in all cases.

raft vessel and a needle puncture was made on the graft vessel to discharge the air inside. The proximal anastomosis was unclamped in a very slow manner to prevent acute right heart failure due to abrupt overload to the right heart. The patency of the bypass was confirmed by intraoperative ultrasonography in all cases. The conventional thoracolaparotomic approach of cavoatrial bypass for SSOR-IVC adopted in this study was described previously by Sun et al [8]. Briefly the thoracolaparotomy was performed through a 7th-8th intercostal incision to gain access to the retroheatic region. A 12-14 cm long, 1.6 cm in diameter artificial vessel with stent spring (Gore-Tex, USA) was used for a bypass between the retrohepatic IVC distal to the occlusion lesion and the right atrium.

8]. Briefly the thoracolaparotomy was performed through a 7th-8th intercostal incision to gain access to the retroheatic region. A 12-14 cm long, 1.6 cm in diameter artificial vessel with stent spring (Gore-Tex, USA) was used for a bypass between the retrohepatic IVC distal to the occlusion lesion and the right atrium. Results Retrospective comparison of the abdominal and thoracolaparotomic approaches is illustrated in Table 1. In group A (n = 16), the mean operation time was 250 ± 28 min versus 340 ± 36 min in group B, Mean ± STDV, P < 0.05. Postoperative ICU stay was 2.8 ± 0.3 days in group A versus 5.2 ± 0.8 days in group B, Mean ± STDV, P < 0.05. All operations in group A were uneventful except one patient suffering from intraoperative acute cardiac failure due to rapid opening of the bypass which was resolved immediately through prompt management. In group A, no postoperative complication or graft vessel thrombosis was found after 10 to 55 months follow-up. In group B, one patient had intraoperative acute pericardial tamponment due to anastomotic leakage, the occurrence rate of postoperative complications was 27.8%, with three cases of pleural effusion, one pulmonary infection and one acute pericarditis, three patients had graft vessel thrombosis at 37, 42 and 58 months post-operation respectively. Table 1 Comparison of abdominal approach and conventional thoracic approach Clinical data Abdominal approach Thoracic approach Operation Time (min) 250 ± 28 340 ± 36 ICU stay(days) 2.8 ± 0.3 5.2 ± 0.8

Results Retrospective comparison of the abdominal and thoracolaparotomic approaches is illustrated in Table 1. In group A (n = 16), the mean operation time was 250 ± 28 min versus 340 ± 36 min in group B, Mean ± STDV, P < 0.05. Postoperative ICU stay was 2.8 ± 0.3 days in group A versus 5.2 ± 0.8 days in group B, Mean ± STDV, P < 0.05. All operations in group A were uneventful except one patient suffering from intraoperative acute cardiac failure due to rapid opening of the bypass which was resolved immediately through prompt management. In group A, no postoperative complication or graft vessel thrombosis was found after 10 to 55 months follow-up. In group B, one patient had intraoperative acute pericardial tamponment due to anastomotic leakage, the occurrence rate of postoperative complications was 27.8%, with three cases of pleural effusion, one pulmonary infection and one acute pericarditis, three patients had graft vessel thrombosis at 37, 42 and 58 months post-operation respectively. Table 1 Comparison of abdominal approach and conventional thoracic approach Clinical data Abdominal approach Thoracic approach Operation Time (min) 250 ± 28 340 ± 36 ICU stay(days) 2.8 ± 0.3 5.2 ± 0.8 Intra-op complications acute right heart failure, n = 1 acute pericardial tamponment, n = 1 Post-op complications 0 27.80% pleural effusion, n = 3 pulmonary infection, n = 1 pericarditis, n = 1 Post-op thrombosis 0 3

Table 1 Comparison of abdominal approach and conventional thoracic approach Clinical data Abdominal approach Thoracic approach Operation Time (min) 250 ± 28 340 ± 36 ICU stay(days) 2.8 ± 0.3 5.2 ± 0.8 Intra-op complications acute right heart failure, n = 1 acute pericardial tamponment, n = 1 Post-op complications 0 27.80% pleural effusion, n = 3 pulmonary infection, n = 1 pericarditis, n = 1 Post-op thrombosis 0 3 Discussion The first case of Budd-Chiari syndrome (BCS) was reported by George Budd in 1845, and then in 1899 Hans Chiari first reported the findings of occlusion of intrahepatic vein in BCS. The incidence is higher in the Eastern countries and South Africa with the lesions predominantly at the retrohepatic portion of IVC, while in Western countries the lesion occurred mainly in the intrahepatic veins [9-11].

and then in 1899 Hans Chiari first reported the findings of occlusion of intrahepatic vein in BCS. The incidence is higher in the Eastern countries and South Africa with the lesions predominantly at the retrohepatic portion of IVC, while in Western countries the lesion occurred mainly in the intrahepatic veins [9-11]. Budd-Chiari syndrome is characterized with portal and IVC hyertension due to obstruction of the main hepatic veins or/and supra- or retrohepatic IVC. One of the common lesions in BCS, especially among Eastern countries, is the short segmental occlusion of retrohepatic IVC (SSOR-IVC) accompanied by various obstruction of hepatic veins [12-13]. According to a recent operation review in 2564 BCS cases in China, 433 were reported to have the SSOR-IVC and treated with cavoatrial bypass [3]. The conventional surgical intervention on SSOR-IVC is cavoartrial bypass via thoracolaparotomy, and it was indicated only for type 1 and type 2 SSOR-IVC [14-17]. This thoracolaparotomic approach offers a better exposure for anastomosis of graft vessel to the IVC and the atrium, but is accompanied by postoperative complications due to the operative trauma in thoracolaparotomy [18-20]. The postoperative pericarditis may be caused by the irritation of the artificial graft [21]. In our previous thoracolaparotomic approach operations, the postoperative complication occurred in 27.8% cases. The thoracolaparotomic approach also has a reduced patency rate at long-term due to application of a longer artificial vessel (12 - 14cm). The length of the artificial graft is critical for prevention of thrombosis in the bypass because longer artifical vessel may impede the cellular seeding to form an intact endothelial layer in the graft vessel [22-24]. An experimental study found that only 4 to 5 cm of the artificial vessel to the anastomosis can be completely covered by the endothelial cells 12 weeks after operation [25-29]. According to a recent review, the patency rate of cavoatrial bypass is 90.7% at one year after operation, and reduced to 77.1% at 3 years, 61.1% at 5 years, and 50% at 10 years [25-29].

5 cm of the artificial vessel to the anastomosis can be completely covered by the endothelial cells 12 weeks after operation [25-29]. According to a recent review, the patency rate of cavoatrial bypass is 90.7% at one year after operation, and reduced to 77.1% at 3 years, 61.1% at 5 years, and 50% at 10 years [25-29]. We have developed an abdominal approach for SSOR-IVC with partial hepatic venous obstruction or complete obstruction with dilation of short hepatic vein. This procedure of suprahepatic and retrohepatic cacocaval bypass has the following advantages over the conventional thoracolaparotomic approach of cavoartrial bypass: 1), the operation is less traumatic with fewer postoperative complications; 2), Shorter artificial vessel (6 – 8 cm) is needed, this facilitates endothelial seeding to improve long term patency; 3), Void of the risk of fatal pericardial tamponment; 4), Prevention of acute pericarditis caused by pericardial irritation by the artificial vessel in the thoracolaparotomic approach. Thus, our comments and experiences on the abdominal approach include: only 2.5 - 3.0 cm of subphrenic IVC is required for the anastomosis, and it can be achieved with isolation at its entrance to diaphragm with special care for not penetrating the pericardium; a groove incision on the liver may assist the alignment of the graft vessel to both anastomoses, unclamping the proximal anastomosis is always in a very slow manner to prevent acute right heart failure.

it can be achieved with isolation at its entrance to diaphragm with special care for not penetrating the pericardium; a groove incision on the liver may assist the alignment of the graft vessel to both anastomoses, unclamping the proximal anastomosis is always in a very slow manner to prevent acute right heart failure. In conclusion, our preliminary results showed that, compared with the traditional thoracolaparoctomic approach, this novel abdominal approach is a safe and effective procedure for treatment of short segmental occlusion of inferior vena cava in Budd-Chiari syndrome with improved long term patency and reduced postoperative complications. Competing interest No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

Introduction Tumors of vermiform appendix are relatively rare conditions. More than 50% of appendiceal tumors are carcinoid tumor [1]. Adenocarcinoma of the appendix accounts for 58% of malignant appendiceal tumors [2]. The incidence of adenocarcinoma is reported to be 0.1% [2]. According to WHO, adenocarcinoma of the appendix is defined as a malignant epithelial neoplasm of the appendix with invasion beyond the muscularis mucosa [2]. The appendiceal carcinomas are classified into adenocarcinoma, mucinous adenocarcinoma, singet-ring cell carcinoma, small cell carcinoma, and undifferentiated carcinoma [2]. There are several comprehensive studies of appendiceal carcinoma [3-10]. The author reviewed 512 consecutive pathological specimens of appendectomies in last ten years in our pathology laboratory in search for appendiceal tumors. The author herein reports the results. Case reports The author reviewed 512 consecutive pathological specimens of appendectomies in last ten years in our pathology laboratory in search for appendiceal tumors. Clinical records were also reviewed. In carcinoma cases, an immunohistochemical study was performed, using Dako’s Envision methods, (Dako Corp. Glostrup, Denmark), as previously reported [11, 12]. The antibodies used were anti-p53 protein (DO-7, Dako) and anti Ki-67 antigen (MIB-1, Dako). Among the 512 appendiceal specimens, 4 cases of non-invasive adenocarcinoma were identified. Therefore, the incidence of appendiceal adenocarcinoma was 0.8% of all appendectomies. No cases of other tumors including carcinoid tumors were found.

Case reports The author reviewed 512 consecutive pathological specimens of appendectomies in last ten years in our pathology laboratory in search for appendiceal tumors. Clinical records were also reviewed. In carcinoma cases, an immunohistochemical study was performed, using Dako’s Envision methods, (Dako Corp. Glostrup, Denmark), as previously reported [11, 12]. The antibodies used were anti-p53 protein (DO-7, Dako) and anti Ki-67 antigen (MIB-1, Dako). Among the 512 appendiceal specimens, 4 cases of non-invasive adenocarcinoma were identified. Therefore, the incidence of appendiceal adenocarcinoma was 0.8% of all appendectomies. No cases of other tumors including carcinoid tumors were found. Case 1 A 48-year-old woman was admitted to our hospital because of acute right lower abdominal pain. Clinically, acute appendicitis was diagnosed, and an appendectomy was performed. Pathologically, the appendix was small and fibrotic (Fig. 1a). Papillary epithelial proliferation was recognized in the appendiceal mucosa (Fig. 1a). The tumor epithelium showed cellular atypia regarded as malignant (Fig. 1b). No invasive features were recognized (Fig. 1a). Immunohistochemically, p53 protein was positive (Fig. 1c) and Ki-67 labeling was 90% (Fig. 1d). No pseudomyxoma peritonei was recognized. The patient is healthy without metastasis and recurrence 37 months after the operation.

llular atypia regarded as malignant (Fig. 1b). No invasive features were recognized (Fig. 1a). Immunohistochemically, p53 protein was positive (Fig. 1c) and Ki-67 labeling was 90% (Fig. 1d). No pseudomyxoma peritonei was recognized. The patient is healthy without metastasis and recurrence 37 months after the operation. Figure 1 (a) Low power view of papillary adenocarcinoma of the appendix in case 1. Papillary proliferation is apparent. No invasion is seen. HE, x 20. (b) Higher power view of Figure 1A. The cellular atypia is evident. HE, x 200. (c) The tumor cells are positive for p53 protein, Immunostaining, x 200. (d) The Ki-67 labeling is 90%. Immunostaining, x 100. Case 2 An 84-year-old man was admitted to our hospital complaining of acute abdominal pain. Clinically, acute appendicitis was diagnosed, and an appendectomy was performed. Pathologically, the appendix showed acute phlegmonous appendicitis. Flat type adenocarcinoma was recognized in the mucosa (Fig. 2). The carcinoma cells showed enough cellular atypia regarded as adenocarcinoma. No invasive features were recognized. P53 protein was positive and Ki-67 labeling was 40%. The patient is healthy without metastasis and recurrence 51 months after operation. Figure 2 Flat type adenocarcinoma in case 2. HE, x 200.

Case 2 An 84-year-old man was admitted to our hospital complaining of acute abdominal pain. Clinically, acute appendicitis was diagnosed, and an appendectomy was performed. Pathologically, the appendix showed acute phlegmonous appendicitis. Flat type adenocarcinoma was recognized in the mucosa (Fig. 2). The carcinoma cells showed enough cellular atypia regarded as adenocarcinoma. No invasive features were recognized. P53 protein was positive and Ki-67 labeling was 40%. The patient is healthy without metastasis and recurrence 51 months after operation. Figure 2 Flat type adenocarcinoma in case 2. HE, x 200. Case 3 A 39-year-old man presented with chronic abdominal pain and fever. Imaging modalities including US, CT and MRI revealed cystic dilation of the appendix. Resection of appendix, terminal ileum and cecum was performed (Fig. 3a) under the clinical diagnosis of suspected appendiceal tumor. Grossly, the proximal appendix showed cystic dilation (Fig. 3a). Histologically, the cystic dilation was covered by flat type atypical epithelium with cellular atypia (Fig. 3B). The atypia was enough to be diagnosed as adenocarcinoma (Fig. 3c). No invasive features were recognized (Fig. 3b, c). P53 protein was positive and Ki-67 labeling was 50%. The patient is healthy without metastasis and recurrence 8 months after the operation. Figure 3 (a) The resected appendix shows cystic dilation (arrow) in case 3. (b) The cystic lining is adenocarcinoma cells. HE, x 100. (c) The cellular atypia is enough to be regarded as adenocarcinoma. HE, x 200.

Case 3 A 39-year-old man presented with chronic abdominal pain and fever. Imaging modalities including US, CT and MRI revealed cystic dilation of the appendix. Resection of appendix, terminal ileum and cecum was performed (Fig. 3a) under the clinical diagnosis of suspected appendiceal tumor. Grossly, the proximal appendix showed cystic dilation (Fig. 3a). Histologically, the cystic dilation was covered by flat type atypical epithelium with cellular atypia (Fig. 3B). The atypia was enough to be diagnosed as adenocarcinoma (Fig. 3c). No invasive features were recognized (Fig. 3b, c). P53 protein was positive and Ki-67 labeling was 50%. The patient is healthy without metastasis and recurrence 8 months after the operation. Figure 3 (a) The resected appendix shows cystic dilation (arrow) in case 3. (b) The cystic lining is adenocarcinoma cells. HE, x 100. (c) The cellular atypia is enough to be regarded as adenocarcinoma. HE, x 200. Case 4 An 86-year-old woman was admitted to our hospital because of acute chronic abdominal pain. Colon endoscopy revealed a tumor at the orifice of the appendectomy, and biopsies from the tumor showed atypical cells suggestive of adenocarcinoma. Therefore, resection of appendix, terminal ileum and cecum was performed under the clinical diagnosis of probable appendiceal adenocarcinoma. Grossly, the appendiceal lumen was filled with papillary epithelial proliferation and much mucus (Fig. 4a). Histologically, the papillary epithelial proliferation consisted of atypical cells regarded as adenocarcinoma (Fig. 4b). Much mucus was impacted in the lumen (Fig. 4c). No invasion was recognized (Fig. 4a, b). The diagnosis was mucinous adenocarcinoma. Immunohistochemically, p53 protein was positive and Ki-67 labeling was 60%. No pseudomyxoma peritonei was recognized. The patient is healthy without metastasis and recurrence 7 years after the operation.

impacted in the lumen (Fig. 4c). No invasion was recognized (Fig. 4a, b). The diagnosis was mucinous adenocarcinoma. Immunohistochemically, p53 protein was positive and Ki-67 labeling was 60%. No pseudomyxoma peritonei was recognized. The patient is healthy without metastasis and recurrence 7 years after the operation. Figure 4 (a) Loupe figures of appendiceal mucinous adenocarcinoma in case 4. Papillary epithelial proliferation and intraluminal mucus are evident. No invasion of tumor cells is recognized. (b) Higher power view of the mucosa of the appendix. Papillary adenocarcinoma is evident. HE, x 200. (c) The intraluminal area show adenocarcinoma cells and much mucus. HE, x 200. Discussion The most common appendiceal tumor is carcinoid tumor, followed by carcinoma [1, 2]. The present series did not contain carcinoid tumors, although 4 cases of adenocarcinoma were identified. These findings suggest that appendiceal carcinoid tumors and benign epithelial tumors are infrequent in our hospital. In the present series, the incidence of adenocarcinoma was 0.8 % of all appendectomies. In the WHO blue book [2], the incidence of adenocarcinoma is 0.1% of all appendectomies. According to the data of Marudanayagam et al [3], the incidence of carcinoid tumor, adenocarcinoma and mucinous cystadema was 0.52%, 0.39% and 0.6% of all appendectomies, respectively. The incidence of 0.8% of the present series is highest. These findings suggest that appendiceal adenocarcinoma is more prevalent in our hospital.

to the data of Marudanayagam et al [3], the incidence of carcinoid tumor, adenocarcinoma and mucinous cystadema was 0.52%, 0.39% and 0.6% of all appendectomies, respectively. The incidence of 0.8% of the present series is highest. These findings suggest that appendiceal adenocarcinoma is more prevalent in our hospital. Clinically, two cases (case 1 and case 2) of the present series showed clinical features of acute appendicitis. The other two cases (case 3 and case 4) in the present cases showed some clinical features of appendiceal tumors. In particular, imaging modalities identified abnormities of the appendix in these two cases. These findings suggest that clinicians should be aware of appendiceal carcinoma even in patients with typical clinical features of acute appendicitis. Further, imaging techniques including US, CT and MRI are essential for identification of appendiceal tumors. Pathologists also should carefully examine the appendectomies. The prognosis was good in the present series.

aware of appendiceal carcinoma even in patients with typical clinical features of acute appendicitis. Further, imaging techniques including US, CT and MRI are essential for identification of appendiceal tumors. Pathologists also should carefully examine the appendectomies. The prognosis was good in the present series. Pathologically, the appendiceal adenocarcinoma of the present series was papillary (or villous) adenocarcinoma in one case, flat type adenocarcinoma in two cases, and mucinous adenocarcinoma in one case. All the four cases were in situ adenocarcinomas without apparent invasion. These findings may indicate that the present adenocarcinomas were in early stages in the carcinomatous progression. No pseudomyxoma peritonei [8-10] was noted in the present cases, suggesting the above hypothesis. The mucinous adenocarcinoma in case 4 of the present series is classified as low-grade mucinous adenocarcinoma, according to Misdraji et al [6]. In the present series, immunoreactive p53 protein was expressed in all the 4 adenocarcinomas. Kabbani et al [7] suggested that p53 expression was found in only 1 (3%) case of the 30 appendiceal mucinous adenocarcinoma. In contrast, Yajima et al [5] showed p53 positive cells percentage was 29 % in appendiceal mucinous adenocarcinoma. Much more studies are required as to p53 gene status in appendiceal adenocarcinoma. In the present series, Ki-67 labeling ranged from 40 % to 90 %, indicating a high proliferative activity of tumor cells.

In contrast, Yajima et al [5] showed p53 positive cells percentage was 29 % in appendiceal mucinous adenocarcinoma. Much more studies are required as to p53 gene status in appendiceal adenocarcinoma. In the present series, Ki-67 labeling ranged from 40 % to 90 %, indicating a high proliferative activity of tumor cells. In summary, the present series suggest that incidence of appendiceal tumor was 0.8% of all appendectomies. All the detected 4 adenocarcinomas were non-invasive adenocarcinomas of the appendix showing variable morphologies. Conflicts of interest The author has no conflict of interest.

ancreatic ascites is controversial [11]. There are no randomized control studies regarding therapy due to the rarity of the condition. Though no consensus is established on the management, Chebli et al [12] propose that the approach depends on the endoscopic retrograde pancreatography defined pancreatic ductal anatomy. Conservative therapy includes the use of somastatin analogues [13, 14] to decrease pancreatic secretion, keeping the patient Nil Per Oral (NPO) with parenteral nutrition and repeated ascitic fluid drainage. Initial continuous octreotide infusions followed by subcutaneous injections are suggested. Long acting octreotide designed for once a month intra-muscular administration is an option. The total duration of therapy is unknown and a trial of 2 - 4 weeks is suggested with a consideration for interventional therapy if no resolution. The interventional therapy is either endoscopic or surgical. ERCP is a valuable tool in the evaluation of patients with pancreatic ascites. It delineates the anatomy, shows communication to the pseudocyst and reveals disruption of the pancreatic duct. Patients undergoing ERCP usually do better than those without ERCP [15]. Increased pressure within the pseudocysts or the pancreatic duct results in disruption of pancreatic duct, and ERCP reduces intraductal pressure by sphincterotomy or by insertion of a transpapillary stent [16]. In addition, pre-operative ERCP is recommended before surgery to decide on the approach based on the location of the leak.

Introduction Pancreatic ascites is a rare complication and should be suspected in patients with chronic alcoholism and pancreatitis presenting with ascites. The etiology is likely from a pancreatic pseudocyst leakage or due to ductal disruption. Medical treatment includes holding oral feedings, total parenteral nutrition, paracentesis and administering octreotide. Interventional therapy includes endoscopic transpapillary pancreatic duct stenting or surgery which includes cystgastrostomy, cystenterostomy or partial pancreatic resection. We present a case of pancreatic ascites in a patient with alcohol use and chronic pancreatitis. This was successfully managed with a combination of medical and interventional therapy including endoscopic pancreatic duct stenting.

r surgery which includes cystgastrostomy, cystenterostomy or partial pancreatic resection. We present a case of pancreatic ascites in a patient with alcohol use and chronic pancreatitis. This was successfully managed with a combination of medical and interventional therapy including endoscopic pancreatic duct stenting. Case report A 45 years old man presented with one month history of generalized abdominal pain and increasing abdominal distension. Three days prior to admission the pain was worsening in severity with associated nausea and constipation. The patient denied any fever, chills or gastrointestinal bleeding. His prior medical history included chronic pancreatitis, liver cirrhosis, polysubstance use including alcohol and multiple episodes of seizures. The patient had many prior admissions in our hospital for chronic pancreatitis and was diagnosed with a pancreatic pseudocyst and bacterial peritonitis in one prior admission. The patient continued to consume alcohol until the current admission despite alcohol cessation counseling multiple times on prior admissions. Physical examination was remarkable for marked abdominal distension with mild generalized tenderness and shifting dullness. Laboratory findings revealed elevated white blood cell count, abnormal liver function tests and elevated amylase and lipase levels. Contrast CT scan of abdomen revealed acute on chronic pancreatitis with extra-hepatic biliary duct and main pancreatic ductal dilatation. The previously noted pseudocyst was resolved with interval development of large amount of ascites with partial small bowel obstruction. A diagnostic and therapeutic paracentesis was performed. Ascitic fluid analysis revealed a white blood cell count of 2470 cells/mm3 with 98% granulocytes, total protein was > 3g/dL. Ascitic fluid amylase was > 20,000 IU/L which increased to 35,000 IU/L on a subsequent paracentesis. Ascitic fluid culture grew Streptococcus viridans which was treated with antibiotics for two weeks. Patient was managed conservatively including holding oral feedings and octreotide infusion but continued to have worsening ascites and required repeated therapeutic paracentesis. A diagnosis of pancreatic ascites with possible pancreatic duct dehiscence was made. An endoscopic retrograde cholangiopancreatography (ERCP) was performed, which confirmed a duct dehiscence with extravasation of the injected contrast (Fig. 1) and was treated with placement of a stent.

epeated therapeutic paracentesis. A diagnosis of pancreatic ascites with possible pancreatic duct dehiscence was made. An endoscopic retrograde cholangiopancreatography (ERCP) was performed, which confirmed a duct dehiscence with extravasation of the injected contrast (Fig. 1) and was treated with placement of a stent. Patient improved clinically and symptomatically with a repeat paracentesis showing decreased amylase levels. On a follow-up clinic visit, patient was noted to have marked improvement in abdominal distension and discomfort. Figure 1 Pancreatic duct dehiscence with extravasation of contrast.

epeated therapeutic paracentesis. A diagnosis of pancreatic ascites with possible pancreatic duct dehiscence was made. An endoscopic retrograde cholangiopancreatography (ERCP) was performed, which confirmed a duct dehiscence with extravasation of the injected contrast (Fig. 1) and was treated with placement of a stent. Patient improved clinically and symptomatically with a repeat paracentesis showing decreased amylase levels. On a follow-up clinic visit, patient was noted to have marked improvement in abdominal distension and discomfort. Figure 1 Pancreatic duct dehiscence with extravasation of contrast. Discussion Ascites is commonly seen in patients with alcoholic liver disease and is usually a consequence of portal hypertension. Pancreatic ascites is a rare diagnostic possibility in patients with chronic alcoholism and pancreatic disease. Pancreatic ascites refers to the accumulation of peritoneal fluid in the presence of pancreatic disease and is usually secondary to pancreatic pseudocysts or pancreatic duct dehiscence. This was first reported in the literature in 1953 in two cases of chronic pancreatitis associated with ascites [1]. Pancreatic ascites is an exudative ascites characterized by high amylase concentration in ascitic fluid (usually over 1000 IU/L) and protein concentration over 3 g/dl [2, 3] that differentiates it from ascites secondary to cirrhosis, tuberculosis or carcinomatosis. Rarely, the origin is indeterminate in 10% of cases [4-6]. In addition to ascites, pleural effusions were seen. Pancreatic ascites is infrequent and is reported in 3.5% of patients with chronic pancreatitis and 6 – 14% of patients with pseudocysts [7, 8].

ascites secondary to cirrhosis, tuberculosis or carcinomatosis. Rarely, the origin is indeterminate in 10% of cases [4-6]. In addition to ascites, pleural effusions were seen. Pancreatic ascites is infrequent and is reported in 3.5% of patients with chronic pancreatitis and 6 – 14% of patients with pseudocysts [7, 8]. Causes for pancreatic ascites include chronic pancreatitis, pancreatic trauma and cystic duplications of biliopancreatic ducts, ampullary stenosis, or ductal lithiasis [3]. Pancreatic ascites is more prevalent in men (male:female ratio 2:1) and between 20 - 50 years of age [3]. In children, it is uncommon and is usually caused by abdominal trauma or congenital obstruction of the pancreatic duct [9]. Acute pancreatitis can be complicated with transient ascites rich in pancreatic enzymes; however, this does not usually cause pancreatic ascites. The clinical presentation includes mild abdominal pain, weight loss and progressive ascites. Severe pain is unusual. Pancreatic ascites should be considered in the differential in patients with chronic ascites with history of alcoholism, chronic pancreatitis or abdominal trauma [10]. Therapy for pancreatic ascites is controversial [11]. There are no randomized control studies regarding therapy due to the rarity of the condition. Though no consensus is established on the management, Chebli et al [12] propose that the approach depends on the endoscopic retrograde pancreatography defined pancreatic ductal anatomy.

ssure within the pseudocysts or the pancreatic duct results in disruption of pancreatic duct, and ERCP reduces intraductal pressure by sphincterotomy or by insertion of a transpapillary stent [16]. In addition, pre-operative ERCP is recommended before surgery to decide on the approach based on the location of the leak. Surgical therapy for pancreatic ascites is usually recommended early when the etiology is due to trauma. Surgical therapy is also recommended when there is no response to conservative therapy for 3 - 4 weeks. Pseudocysts are usually treated by distal pancreatectomy when the leak is in the pancreatic tail or drained by cystogastrostomy, cystojejunostomy, or cystoduodenostomy. Internal drainage is preferred to external drainage. Fistulas in the pancreatic duct are usually drained to a Roux-en-Y jejunal loop. Recurrence rates from 50 - 64% have been reported in patients undergoing surgical intervention without ERCP [15, 17]. Mortality rates have been reported to be similar with surgical and medical therapies (15 - 25%) [18]. In this case report we successfully managed a patient with pancreatic ascites with a combination of conservative and interventional therapy including an ERCP with pancreatic ductal stenting. Our case report augments the existing data from two prior reported case series [19, 20], and this modality of management should be actively pursued in such cases. However, a larger number of patients and randomized controlled trials are necessary to confirm this. The authors declare no conflict of interest related to this report.

Introduction A large effusion in a patient with cirrhotic ascites is described as hepatic hydrothorax which is a complication of cirrhosis and found in about 4% to 6% of cirrhotic patients in the absence of primary pulmonary, pleural, or cardiac disease [1-4]. Usually, it is located in the right hemithorax, but it can also be seen on the left side, bilaterally and even in the absence of ascites [5, 6is caused by direct passage of ascitic fluid from ]. It the abdominal cavity to the pleural space through transdiaphragmatic defects [7]. Diagnosis of hydrothorax is crucial since it could lead to hazardous complications such as bacterial pleuritis or empyema [8]. Patients with a long history of stable cirrhosis and a sudden development of pleural effusions should be suspected of hepatic hydrothorax as well as other causes that have precipitated fluid collection such as malignancy and tuberculosis. Although the exact mechanism of hepatic hydrothorax is controversial, it is probably due to the ascites fluid that is transported directly into the pleural space. A number of different mechanisms have been proposed to explain the development of hepatic hydrothorax, including: hypoalbuminemia and a decrease in colloid osmotic pressure; leakage of plasma from hypertensive azygos veins; lymphatic leakage from thoracic duct; passage of fluid from peritoneal cavity to the pleural space via lymphatic channels in the diaphragm; and passage of fluid into the pleural space directly via defects in the diaphragm [1-7].

d a decrease in colloid osmotic pressure; leakage of plasma from hypertensive azygos veins; lymphatic leakage from thoracic duct; passage of fluid from peritoneal cavity to the pleural space via lymphatic channels in the diaphragm; and passage of fluid into the pleural space directly via defects in the diaphragm [1-7]. Among the different mechanisms proposed, it is mostly accepted that peritoneal fluid flows into the pleural space directly, along a pressure gradient through congenital or acquired fenestrations connecting these two spaces. The mechanism can be explained in such a way that the fenestrations in the tendinous portion of the diaphragm are covered with pleuroperitoneum and they are raised into blisters as intra abdominal pressure rises by ascites. Eventually blisters rupture and microscopic defects may appear. Positive intra abdominal pressure coupled with negative intrathoracic pressure causes a direct flow of fluid from abdominal cavity into the pleura. A pleural effusion develops if the flow exceeds the absorptive capacity of pleura [4]. In clinical practice, presence of communication between pleural and peritoneal cavities may be successfully demonstrated by peritoneal scintigraphy. In this technique, firstly, radiolabeled tracer should be simply introduced into the ascitic fluid by paracentesis. Later, scintigraphic images of abdomino-thoracic region are obtained by gamma camera. Using this technique, demonstration of the presence of a communication between abdomen and thoracic cavities can be quickly confirmed [9-11].

tly, radiolabeled tracer should be simply introduced into the ascitic fluid by paracentesis. Later, scintigraphic images of abdomino-thoracic region are obtained by gamma camera. Using this technique, demonstration of the presence of a communication between abdomen and thoracic cavities can be quickly confirmed [9-11]. With this study we presented two cases of pleural effusion in which 99mTc sulfur colloid peritoneal scintigraphy was used to detect whether there is such a passage of ascites fluid from the peritoneal cavity to the pleural space via the diaphragmatic defects. Patients Two (one hepatitis B virus and one alcohol-related) cirrhotic patients with ascites and pleural effusions were evaluated to determine the cause of fluid accumulation and were searched whether there was a communication between peritoneal and pleural cavities. In addition to clinical evidence of ascites and pleural effusion, patients were also confirmed by paracentesis and thoracentesis of fluids. Protein content of ascitic and pleural fluids was used to estimate whether the fluid was due to chronic liver disease.

was a communication between peritoneal and pleural cavities. In addition to clinical evidence of ascites and pleural effusion, patients were also confirmed by paracentesis and thoracentesis of fluids. Protein content of ascitic and pleural fluids was used to estimate whether the fluid was due to chronic liver disease. Scintigraphic analysis After aspiration of ascitic fluid, approximately 37 MBq of 99mTc sulfur colloid instilled the peritoneal cavity under sterile conditions. Patients were then asked to turn frequently from side to side for proper mixing of radiopharmaceutical into the ascitic fluid. Approximately 10 - 15 min after injection of the tracer, each patient was positioned supine under a large field-of-view single-head gamma camera (Adac Argus Epic, ADAC Laboratories, Milpitas, CA, USA), fitted with a low-energy general purpose collimator. An anterior abdominal image was acquired in a 256 x 256 byte matrix for 400 kcounts to confirm the presence of tracer in the peritoneal cavity. Thereafter, images of upper abdomen and chest in anterior and posterior positions were obtained at 30 min intervals for 6 h. Twenty-four-hour images were also acquired in the second patient (Case 2) since no activity was noticed in the chest region until 6 h. Markers placed at the costal margins and xyphoid to show anatomic orientation. Scintigraphic images were evaluated by two experienced nuclear medicine physicians for the presence or absence of radiotracer in pleural regions.

red in the second patient (Case 2) since no activity was noticed in the chest region until 6 h. Markers placed at the costal margins and xyphoid to show anatomic orientation. Scintigraphic images were evaluated by two experienced nuclear medicine physicians for the presence or absence of radiotracer in pleural regions. Case 1 A 44-year-old male patient with a diagnosis of hepatitis B virus related cirrhosis of liver for 2 years was hospitalized with dispnea due to suddenly developed ascites and right sided pleural effusion. Posteroanterior (PA) chest radiograph of the patient demonstrated a large pleural effusion evident in the right hemithorax (Fig. 1). Prompt aspiration of thoracic fluid with paracentesis was performed. The biochemical examination of fluids revealed a transudate type pleural effusion with a high albumin gradient ascites (Table 1). After injection of 99mTc sulfur colloid into the peritoneal space, rapid accumulation of radioactivity in the right pleural effusion was noted (Fig. 2), suggesting a peritoneo-pleural communication. With the treatment of ascites via paracentesis, salt restriction and diuretics ascites disappeared as well as the pleural effusion decreased to minimal levels. PA chest radiograph demonstrated the absence of the effusion (Fig. 3). Figure 1 PA chest radiograph of the case 1: a large pleural effusion was seen in the right hemithorax.

Case 1 A 44-year-old male patient with a diagnosis of hepatitis B virus related cirrhosis of liver for 2 years was hospitalized with dispnea due to suddenly developed ascites and right sided pleural effusion. Posteroanterior (PA) chest radiograph of the patient demonstrated a large pleural effusion evident in the right hemithorax (Fig. 1). Prompt aspiration of thoracic fluid with paracentesis was performed. The biochemical examination of fluids revealed a transudate type pleural effusion with a high albumin gradient ascites (Table 1). After injection of 99mTc sulfur colloid into the peritoneal space, rapid accumulation of radioactivity in the right pleural effusion was noted (Fig. 2), suggesting a peritoneo-pleural communication. With the treatment of ascites via paracentesis, salt restriction and diuretics ascites disappeared as well as the pleural effusion decreased to minimal levels. PA chest radiograph demonstrated the absence of the effusion (Fig. 3). Figure 1 PA chest radiograph of the case 1: a large pleural effusion was seen in the right hemithorax. Figure 2 Peritoneal scintigraphy of the case 1 at 3 h: at 10 min, the tracer was diffused in the abdominal region, which confirmed that it was injected properly in the peritoneal cavity; at 3 h, the tracer was seen in the right side of the thoracic region (i.e. pleural space) (arrows). This image suggested peritoneo-pleural communication and hepatic hydrothorax. Figure 3 Post-treatment PA chest radiograph of the case 1: no evidence of pleural effusion was seen.

Figure 2 Peritoneal scintigraphy of the case 1 at 3 h: at 10 min, the tracer was diffused in the abdominal region, which confirmed that it was injected properly in the peritoneal cavity; at 3 h, the tracer was seen in the right side of the thoracic region (i.e. pleural space) (arrows). This image suggested peritoneo-pleural communication and hepatic hydrothorax. Figure 3 Post-treatment PA chest radiograph of the case 1: no evidence of pleural effusion was seen. Table 1 Laboratory results of patients Case 1 Case 2 Age 44 53 Sex Male Male Side of effusion Right Left WBC 15300 3800 Hb 15.7 12.9 ESR 35 90 Serum albumin 2.9 3.1 SAAG 1.5 0.8 SPAG 1.1 0.4 INR 1.7 1.1 Leukocytes in ascites - 507 Lymphocytes in ascites - 190 ADA level in ascites 10 165 WBC: White blood cell; Hb: Hemoglobin; ESR: Eryhtrocyte sedimentation rate; SAAG: Serum ascites albumin gradient; SPAG: Serum pleural effusion albumin gradient; INR: International normalized ratio; ADA: Adenosine deaminase activity.

.1 Leukocytes in ascites - 507 Lymphocytes in ascites - 190 ADA level in ascites 10 165 WBC: White blood cell; Hb: Hemoglobin; ESR: Eryhtrocyte sedimentation rate; SAAG: Serum ascites albumin gradient; SPAG: Serum pleural effusion albumin gradient; INR: International normalized ratio; ADA: Adenosine deaminase activity. Case 2 A 53-year-old male patient with newly developed abdominal distension, ascites and left sided pleural effusion was referred. During the diagnostic work up, he was found to have alcoholic liver disease. PA chest radiograph of the patient showed, clearly, a large pleural effusion in the left hemithorax (Fig. 4). Prompt aspiration of fluids revealed an exudate type pleural effusion with a low albumin gradient ascites (Table 1). After injection of 99mTc sulfur colloid into the peritoneal space, no accumulation of radioactivity in lung region was observed up to 24 h (Fig. 5). According to the peritoneal scintigraphy, absence of peritoneo-pleural communication was considered and further diagnostic work up was initiated. Computed tomographic examination of thoracic and abdominal cavities did not yield certain diagnosis even though there was a fluid accumulation with fibrotic changes in pleural and peritoneal membranes. Broncoscopic evaluation of airways with aspiration of broncoalveolar fluid confirmed lung tuberculosis, and laparascopic evaluation of intra abdominal cavity with peritoneal biopsy confirmed peritoneal tuberculosis with final demonstration of tuberculosis bacilli itself. An anti-tuberculosis treatment was started. Post-treatment PA chest radiograph demonstrated the absence of the effusion (Fig. 6).

med lung tuberculosis, and laparascopic evaluation of intra abdominal cavity with peritoneal biopsy confirmed peritoneal tuberculosis with final demonstration of tuberculosis bacilli itself. An anti-tuberculosis treatment was started. Post-treatment PA chest radiograph demonstrated the absence of the effusion (Fig. 6). Figure 4 Pre-treatment PA chest radiograph of the case 2: a large pleural effusion evident in the left hemithorax was seen. Figure 5 Peritoneal scintigraphy of the case 2 at 24 h: at 10 min, the tracer was diffused in the abdominal region, which confirmed that it was injected in the peritoneal cavity; at 6 h and 24 h, the tracer was seen in the region of abdominal cavity, but not in the thoracic region. This image suggested no peritoneo-pleural communication in the case 2. Figure 6 Post-treatment PA chest radiograph of the case 2: no evidence of pleural effusion was seen.

Figure 5 Peritoneal scintigraphy of the case 2 at 24 h: at 10 min, the tracer was diffused in the abdominal region, which confirmed that it was injected in the peritoneal cavity; at 6 h and 24 h, the tracer was seen in the region of abdominal cavity, but not in the thoracic region. This image suggested no peritoneo-pleural communication in the case 2. Figure 6 Post-treatment PA chest radiograph of the case 2: no evidence of pleural effusion was seen. Discussion It is well known that ascites frequently forms in the setting of cirrhosis as a result of portal hypertension. Hypoalbuminemia, increased pressure in thoracic duct and azygos veins are common findings in cirrhotic patients with ascites. Furthermore, ascites can be complicated by high-output or low-output heart failure, nephrotic syndrome, malignancy, tuberculosis and some other infections such as chlamydia or coccidioidomycosis. Pancreatic or biliary ascites forms by leakage of pancreatic juice or bile into the peritoneal cavity or by a ‘chemical burn’ of the peritoneum. After abdominal surgery, especially extensive retroperitoneal dissection, lymphatics may be transected and may leak lymph for variable amounts of time [12, 13].

dioidomycosis. Pancreatic or biliary ascites forms by leakage of pancreatic juice or bile into the peritoneal cavity or by a ‘chemical burn’ of the peritoneum. After abdominal surgery, especially extensive retroperitoneal dissection, lymphatics may be transected and may leak lymph for variable amounts of time [12, 13]. In daily clinical practice, pleural effusions with ascites are not uncommonly seen. Although mainly seen secondary to alcohol-related cirrhosis of liver, they can also be seen in viral hepatitis related cirrhosis [14]. They are usually unilateral and right sided but occasionally may be bilateral or left sided. A unilateral right sided pleural effusion with ascites is usually thought to be due to passage of ascitic fluid from the abdomen to the pleural space through acquired transdiaphragmatic defects [3]. Only the demonstration of these defects may clinically provide sufficient diagnostic criteria for hepatic hydrothorax, especially when there is no other clinical evidence away from liver disease. However, a unilateral left sided pleural effusion is usually poorly associated with these transdiaphragmatic defects and frequently needs further serious investigations.

linically provide sufficient diagnostic criteria for hepatic hydrothorax, especially when there is no other clinical evidence away from liver disease. However, a unilateral left sided pleural effusion is usually poorly associated with these transdiaphragmatic defects and frequently needs further serious investigations. The transdiaphragmatic defects probably result from anatomic thinning and separation of the collagenous fibers of the tendinous portion of the diaphragm [4]. Congenital factors, high intra abdominal pressure and prolonged bed rest may contribute to the diaphragmatic thinning [15]. Another possible explanation for the formation of transdiaphragmatic holes that allow the peritoneum to rupture into pleural space may be an increase of pressure caused by Valsalva maneuvers (cough, defecation, parturition) and trauma [16]. These defects are rarer on the left side because the left diaphragm is thicker and more muscular [17].

ation for the formation of transdiaphragmatic holes that allow the peritoneum to rupture into pleural space may be an increase of pressure caused by Valsalva maneuvers (cough, defecation, parturition) and trauma [16]. These defects are rarer on the left side because the left diaphragm is thicker and more muscular [17]. In clinical evaluation, practice of ordering every conceivable body fluid test on every pleural fluid specimen is expensive and seems more confusing than helpful, especially when unexpectedly abnormal results, such as left sided pleural effusions in cirrhotic patients, are encountered. A rapid and simple model method for establishing whether there is any communication between two cavities is to inject a radioactive tracer into the peritoneal cavity. Other diagnostic modalities of transdiaphragmatic fenestration have included intraperitoneal instillation of air or contrast agents, MRI and surgical exploration by thoracoscopy [17-19]. In order to prove peritoneo-pleural communication by peritoneal scintigraphy, several radiopharmaceuticals such as 99mTc sulfur colloid, 99mTc macroaggragated albumin (MAA) and 99mTc albumin colloid have been used [9, 20]. We used 99mTc sulfur colloid because of the ready availability and low cost. This technique is very easy to perform. It is safe, cheap, minimally invasive, and it has low radiation dose to the patient. For its effectiveness and meaningful results, peritoneal scintigraphy has already been used to diagnose of hydrothorax not only in patients with cirrhosis but also in patients on continuous ambulatory peritoneal dialysis (CAPD) [21].

. It is safe, cheap, minimally invasive, and it has low radiation dose to the patient. For its effectiveness and meaningful results, peritoneal scintigraphy has already been used to diagnose of hydrothorax not only in patients with cirrhosis but also in patients on continuous ambulatory peritoneal dialysis (CAPD) [21]. Initial demonstration of the presence of this communication may play a vital role in patient management because it may not only stop further efforts to investigate other causes but also quickly start treatment. In our first patient, the pleural effusion was shown to be due to the passage of ascitic fluid across the diaphragm via the diaphragmatic defects or the lymphatic channels. In addition to clinical features and biochemical findings of ascites and pleural fluid, with the scintigraphic demonstration of transdiaphragmatic passage, patient was confirmed to have hepatic hydrothorax. In the second patient, there was not any communication seen between ascitic and pleural fluids. Lack of communication between two fluids led to separate investigations of thorax and abdomen. Invasive procedures, such as broncoscopy and laparascopic peritoneal biopsy procedures, were performed to further explore the patient. Only after the laparoscopic demonstration of tuberculous granulomas, certain diagnosis was established and anti-tuberculosis treatment was started.

investigations of thorax and abdomen. Invasive procedures, such as broncoscopy and laparascopic peritoneal biopsy procedures, were performed to further explore the patient. Only after the laparoscopic demonstration of tuberculous granulomas, certain diagnosis was established and anti-tuberculosis treatment was started. In conclusion, use of 99mTc sulfur colloid peritoneal scintigraphy to identify abnormal transdiaphragmatic communication between thoracic and abdominal cavities may play a critical role in the management of ascites with pleural effusion; it may help to start treatment quickly and, furthermore, may save healthcare resources. All authors included in this report declare that they have no financial or proprietary interest to disclose.

Introduction Despite developments in diagnostic and therapeutic modalities, gastric cancer is still the second most common cause of cancer-related death throughout the world [1, 2]. The highest incidences of gastric cancer are reported from Eastern Asia, Eastern Europe, and South America [3]. The prognosis for gastric cancer patients remains poor, especially in advanced stages of the disease. There were about 158,000 deaths from stomach cancer in Europe in 2000 [4]. According to 1999 figures of State’s Statistics Institute, death from gastric cancer was 3.22/100.000 in Turkey [5]. A very rigorous screening program that detects patients in an early stage of the disease was developed in Japan. Up to 70% of all gastric cancers are diagnosed as early cancers in the East, but the rate of gastric cancers identified as early cancers accounts for only about 15% in the West [6]. Patients with transmural advanced gastric cancer (T2N0-T3N2) make up the largest group with uncertain outcome. Although the therapeutic results in this group are variable, the identifying prognostic factors are important to establish a therapeutic strategy [7]. This retrospective study was designed with the aim to evaluate perioperative morbidity, mortality and the prognostic factors that influence survival of the patients with transmural advanced gastric carcinoma after curative surgical therapy.

fying prognostic factors are important to establish a therapeutic strategy [7]. This retrospective study was designed with the aim to evaluate perioperative morbidity, mortality and the prognostic factors that influence survival of the patients with transmural advanced gastric carcinoma after curative surgical therapy. Materials and Methods One hundred and forty-five patients with primary gastric cancer underwent gastric resection in our clinic, between January 1997 and March 2004. Fifty patients (34.5%) with transmural advanced gastric cancer were selected for this retrospective study. Hospital records of the patients were reviewed for clinicopathological findings, morbidity, mortality, and survival. The patients with synchronous distant metastases, peritoneal carcinomatosis, gastric stump carcinoma, recurrent gastric cancer, and those that received noncurative resections (R1, R2) were excluded.

udy. Hospital records of the patients were reviewed for clinicopathological findings, morbidity, mortality, and survival. The patients with synchronous distant metastases, peritoneal carcinomatosis, gastric stump carcinoma, recurrent gastric cancer, and those that received noncurative resections (R1, R2) were excluded. Preoperative work-up included upper gastrointestinal endoscopy with biopsy, abdominal ultrasound scan, and computed tomography. Diagnostic laparoscopy was selectively performed. Gastrectomy with an intent of D2 lymphadenectomy was performed as described by Nakajima and Kajitani [8]. Total gastrectomy was performed for tumors located on the proximal or middle portions of the stomach and subtotal gastrectomy for distal tumors with curative aim. Staging of the gastric cancer was made with reference to the TNM classification of malignant tumors by the International Union Against Cancer (UICC). The pN category which was established to evaluate different groups of lymph node metastases was used (pN0:0, pN1: 1-6, pN2: 7-15 and pN3: >15) [9]. The extent of lymph node dissection and lymph node ratio (the proportion of the number of positive nodes to the number of all resected nodes) are noted. The patients were put into two groups considering lymph node ratio as less than 0.2, and greater than or equal to 0.2.

d (pN0:0, pN1: 1-6, pN2: 7-15 and pN3: >15) [9]. The extent of lymph node dissection and lymph node ratio (the proportion of the number of positive nodes to the number of all resected nodes) are noted. The patients were put into two groups considering lymph node ratio as less than 0.2, and greater than or equal to 0.2. The patients were followed up at regular intervals by outpatient visits and telephone interviews. The cases that died in the early postoperative period were excluded from the survival analysis. Survival curves were estimated using the Kaplan-Meier method and differences in survival were compared by the log-rank test. Survival was calculated from the date of operation to the date of death, or to the date of last follow-up for the surviving patients. Median survival time was given with their standard error (SE). Simultaneous associations of multiple variables were performed using the Cox proportional hazards regression model to estimate the simultaneous effect on overall survival. Association of the independent prognostic factors, which were age, gender, location and size of the tumor, type of surgery, blood transfusion, depth of tumor invasion (pT), lymph node metastases (pN), stage of the disease, grading, vascular invasion, lymph vessel invasion, characteristics of the tumor according to Lauren’s classification, and lymph node ratio with patients’ survival were evaluated by using univariate analysis methods. Independent variables that showed statistical significance in the univariate analysis were then entered in the multivariate analysis. The prognostic factors were compared by hazard ratio and the 95% confidence interval (CI). Stepwise selection was used to find the most significant factors. A P value of less than 0.05 was considered statistically significant for all patients. Statistical Package for Social Sciences (SPSS 10.0, Chicago, IL, USA) for Windows (Microsoft) was used for the statistical analyses.

the 95% confidence interval (CI). Stepwise selection was used to find the most significant factors. A P value of less than 0.05 was considered statistically significant for all patients. Statistical Package for Social Sciences (SPSS 10.0, Chicago, IL, USA) for Windows (Microsoft) was used for the statistical analyses. Results Of the 50 patients, 30 (60%) were male and 20 (40%) were female, with a median age of 61 years (range 31 - 77 years). Total gastrectomy was performed in 25 patients and subtotal gastrectomy in the other 25. Histopathological examination of the resected tumors revealed that all of the cases were adenocarcinoma: 38% of them were intestinal type and 62% were diffuse type. The most common site of the primary lesion was antrum (25 cases, 50%). The majority of the tumors were pT3 (39 cases, 78%). All patients had received D2 lymphadenectomy. The median count of dissected lymph nodes was 34 ranging between 25 and 46. Distribution of the cases according to lymph node metastases was as follows: pN0:10 cases, pN1:28 cases, and pN2:12 cases. Deaths during the first postoperative month were considered surgical mortality. A total of 16 major morbidities developed in 12 patients (24%). Anastomotic leakage was observed in five cases, duodenal stump dehiscence in three cases, abdominal abscess, pneumonia, and ileus in two cases for each, hemorrhage, and myocardial infarction in one case for each. Five patients (10%) died due to these complications and they were excluded from the survival analysis.

4%). Anastomotic leakage was observed in five cases, duodenal stump dehiscence in three cases, abdominal abscess, pneumonia, and ileus in two cases for each, hemorrhage, and myocardial infarction in one case for each. Five patients (10%) died due to these complications and they were excluded from the survival analysis. Thirty-six patients received postoperative concomitant radio-chemotherapy as adjuvant treatment. All of them completed the whole course of adjuvant treatment. Nine patients did not receive adjuvant therapy due to postoperative complications (four patients), or patient’s refusal to receive radio-chemotherapy (five patients). Mean hospital stay was 13.5 days (8 - 40 days). Median follow-up was 25 months (range 2 - 74 months). During follow-up period 29 patients died of gastric cancer and 16 patients are still alive. The overall survival rate was 48% at 1 year, 31% at 3 years, and 19% at 5 years, and median survival time was 23.00 ± 2.51 months, 95% Cl (18.07 - 27.93). There was no significant difference in survival between the patients with pT2 and pT3 tumors, nor between stage III B disease and the other stages. Median survival time was 24.0 ± 2.45 months, 95% Cl (19.19 - 28.81), for the patients with a lymph node ratio less than 0.2; and 5.0 ± 2.0 months, 95% Cl (1.08 - 8.92), for those with a lymph node ratio greater or equal to 0.2 (P = 0.0012, log-rank test).

T3 tumors, nor between stage III B disease and the other stages. Median survival time was 24.0 ± 2.45 months, 95% Cl (19.19 - 28.81), for the patients with a lymph node ratio less than 0.2; and 5.0 ± 2.0 months, 95% Cl (1.08 - 8.92), for those with a lymph node ratio greater or equal to 0.2 (P = 0.0012, log-rank test). Univariate analysis was performed to evaluate the relationship between clinicopathological features and patients’ survival. Of the 14 clinical and pathological variables entered in the analysis, four were found to have significant influence. Lymph node metastases (P = 0.030), lymph vessel invasion (P = 0.001), blood transfusion (P = 0.021), and lymph node ratio (P = 0.006) were the prognostic features identified by univariate analysis. Evaluation of the prognostic factors for the cases and the results of univariate analysis are shown in Table 1. Among multiple significant prognostic factors in the univariate analysis, only one factor, lymph node ratio, proved to be independently significant in the multivariate analysis, risk ratio 4.47, 95% CI (1.168 - 17.117) (Table 2).

gnostic factors for the cases and the results of univariate analysis are shown in Table 1. Among multiple significant prognostic factors in the univariate analysis, only one factor, lymph node ratio, proved to be independently significant in the multivariate analysis, risk ratio 4.47, 95% CI (1.168 - 17.117) (Table 2). Table 1 Univariate Analysis of The Prognostic Factors In Patients With Transmural Gastric Cancer Parameters No. of patients 95% Confidence Survival rate (%) P† Median survival (month) ± SE Interval P* 1-year 3-years 5-years Age (years) NS NS ≤65 30 25.0 ± 7.25 27.88 – 48.77 52 40 20 >65 15 16.0 ± 9.02 0 – 33.67 39 13 13 Gender NS NS Male 27 21.0 ± 3.44 14.25 – 27.75 44 25 25 Female 18 27.0 ± 7.80 11.70 – 42.30 53 41 0 Tumor location NS NS Cardia 9 30.0 ± 7.28 15.73 – 44.27 49 32 0 Corpus 11 18.0 ± 3.48 11.17 – 24.83 35 35 35 Antrum 22 24.0 ± 3.03 18.07 – 29.93 55 35 35 Diffuse 3 0 0 - - - Tumor size (cm) NS NS >10 in diameter 24 24.0 ± 5.30 13.61 – 34.39 54 36 18 ≤10 in diameter 21 21.0 ± 2.28 16.53 – 25.47 43 28 28 Depth of tumor invasion NS NS pT2 11 62.0 ± 19.12 24.52 – 99.48 53 53 26 pT3 34 18.0 ± 5.38 7.45 – 28.55 46 26 18 Lymph node metastases 0.04 0.03 pN0 10 - - 78 65 65 pN1 25 21.0 ± 2.38 16.33 – 25.67 37 27 13 pN2 10 16.0 ± 6.39 3.47 – 28.53 46 11 11 Stage NS NS I B 3 - - 100 100 100 II 16 21.0 ± 5.04 11.13 – 30.87 46 37 0 III A 17 18.0 ± 4.84 8.52 – 27.48 38 25 25 III B 9 24.0 ± 6.01 12.21 – 35.79 51 13 - Grading 0.017 NS G1 6 - - 100 100 100 G2 6 21.0 ± 9.19 3.0 – 39.0 50 33 0 G3 33 18.0 ± 3.39 11.36 – 24.64 38 19 19 Type of Surgery NS NS Subtotal gastrectomy 22 24.0 ± 3.03 18.07 – 29.93 55 35 35 Total gastrectomy 23 20.0 ± 3.16 13.81 – 26.19 41 28 0 Vascular invasion NS NS Absent 26 25.0 ± 5.07 15.07 – 34.93 54 35 35 Present 19 21.0 ± 2.05 16.99 – 25.01 39 26 9 Lymph vessel invasion 0.0003 0.001 Absent 17 62.0 ± 22.96 17.0 – 107.0 80 64 22 Present 28 15.0 ± 2.55 10.01 – 19.99 29 12 12 Blood transfusions 0.0166 0.021 < 500 ml 27 27.0 ± 6.85 13.58 – 40.42 57 44 44 ≥ 500 ml 18 16.0 ± 5.63 4.97 – 27.03 32 13 4 Lauren’s classification NS NS Intestinal 16 25.0 ± 6.0 13.24 – 36.76 63 42 42 Diffuse 29 18.0 ± 3.62 10.90 – 25.10 39 25 8 Lymph node ratio 0.0012 0.006 LNR < 0.2 12 24.0 ± 2.45 19.19 – 28.81 52 34 20 LNR ≥ 0.2 33 5.0 ± 2.0 1.08 – 8.92 - - - All patients 45 23.0 ± 2.51 18.07 – 27.93 48 31 19 SE: Standard error, P *: Log-rank test, P†: Chi-square test, NS: Not significant

0 13.24 – 36.76 63 42 42 Diffuse 29 18.0 ± 3.62 10.90 – 25.10 39 25 8 Lymph node ratio 0.0012 0.006 LNR < 0.2 12 24.0 ± 2.45 19.19 – 28.81 52 34 20 LNR ≥ 0.2 33 5.0 ± 2.0 1.08 – 8.92 - - - All patients 45 23.0 ± 2.51 18.07 – 27.93 48 31 19 SE: Standard error, P *: Log-rank test, P†: Chi-square test, NS: Not significant Table 2 Multivariate Analysis of the Independent Prognostic Factors Parameters RR 95% CI P Lymph node metastases (pN1 vs pN0) 1.11 0.285 – 4.341 0.877 (pN2 vs pN0) 1.46 0.282 – 7.544 0.652 Lymph vessel invasion (Extensive vs not extensive) 1.39 0.444 – 4.379 0.559 Blood transfusions (≥ 500 ml vs < 500 ml) 1.01 0.433 – 2.362 0.979 Lymph node ratio (≥ 0.2 vs < 0.2) 4.47 1.168 – 17.117 0.029 P: Cox’s proportional hazards model. CI: Confidence Interval. RR: Relative Risk. The overall survival curve and the survival curves according to lymph node metastases, lymph vessel invasion, blood transfusions, and lymph node ratio are shown in Figure 1 - 5. Figure 1 Overall survival curve of the patients with transmural gastric carcinoma calculated by the method of Kaplan-Meier. Figure 2 Survival according to nodal involvement. The survival rate for the patients with pN2 lymph node metastases was lower than the rate for pN1 and pN0 cases. (P = 0.0400, log-rank test). Figure 3 Survival according to lymph vessel invasion. The survival rate for patients with lymph vessel invasion was lower than the rate for the patients without lymph vessel invasion. (P = 0.0003, log-rank test).

Figure 2 Survival according to nodal involvement. The survival rate for the patients with pN2 lymph node metastases was lower than the rate for pN1 and pN0 cases. (P = 0.0400, log-rank test). Figure 3 Survival according to lymph vessel invasion. The survival rate for patients with lymph vessel invasion was lower than the rate for the patients without lymph vessel invasion. (P = 0.0003, log-rank test). Figure 4 Survival according to blood transfusion. Survival rate for the patients that received blood transfusions more than or equal to 500 ml was lower than the rate for the patients that received less than 500 ml. (P = 0.0166, log-rank test). Figure 5 Survival according to lymph node ratio. Survival rate for patients with a lymph node ratio greater than or equal to 0.2 was lower then the rate for those with a lymph node ratio less than 0.2. (P = 0.0012, log-rank test). Discussion Curative surgical treatment of gastric cancer still remains difficult in the Western countries, primarily because most of the patients present with advanced disease [10]. Transmural gastric carcinomas cover a wide range between stages T2N0 and T3N2 with varying survival rates. The extent of the disease, the operative procedure, and patient selection are crucial in optimizing outcome [7]. The standard treatment policy for all potentially curable patients with gastric cancer is radical resection with extensive lymphadenectomy. However, the incidence of complications after gastrectomy increases significantly when extended lymph node dissection is added [7, 11].

ction are crucial in optimizing outcome [7]. The standard treatment policy for all potentially curable patients with gastric cancer is radical resection with extensive lymphadenectomy. However, the incidence of complications after gastrectomy increases significantly when extended lymph node dissection is added [7, 11]. Postoperative morbidity and mortality rates for patients who undergo curative gastrectomy are reported to range from 10.5 to 33% and from 2 to 11.9%, respectively [10, 12, 13]. In our series, morbidity and mortality rates were 24% and 10%, respectively. Anastomotic leakage was the most common major postoperative complication (10%). The reported incidence rates for anastomotic leakage after extended gastrectomy varies between 1% and 12.3% [14, 15]. Sasako et al reported that the recent incidence of leakage at the esophago-jejunostomy site after learning curve for stapled anastomosis is less than 1% [11]. There have been reports on minimal positive effects of adjuvant chemotherapy for gastric cancer patients [16]. Results of the large American Southwest Oncology Group (SWOG) study advocate postoperative radio-chemotherapy for T2N0-T3N2 gastric cancer patients. National Intergroup Trial (INT-116), a large study of postoperative radio-chemotherapy with 5-flourouracil/leucoverin and 5-fluorouracil/leucoverin + 45 Gy radiotherapy, showed that adjuvant radio-chemotherapy significantly improves survival [17]. Thus, adjuvant radio-chomotherapy for surgically treated gastric cancer patients is advocated by the oncology group in our hospital as in many other centers [6].

th 5-flourouracil/leucoverin and 5-fluorouracil/leucoverin + 45 Gy radiotherapy, showed that adjuvant radio-chemotherapy significantly improves survival [17]. Thus, adjuvant radio-chomotherapy for surgically treated gastric cancer patients is advocated by the oncology group in our hospital as in many other centers [6]. The prognosis for patients with transmural advanced gastric carcinoma has been improved by curative surgery [7]. Several reports have demonstrated that the significant prognostic factors are the stage of the disease, lymph node status, and the depth of penetration into the gastric wall [12, 14, 18]. Five-year survival rate after curative surgical resection ranges from 50% to 70% in patients with stage II disease and from 29% to 42% in patients with stage III disease [10, 12]. In our series, the patients with pT2 and stage IB disease had better survival than those with pT3 and the other stages, but they were not significant variables in univariate analysis. In Dhar’s [7] series 49% of the patients survived more than 7 years whereas 5-year survival rate was 19% in this study. Five-year survival rates indicate the difference between patients in the West and in the East, and that the prognosis of resectable gastric carcinoma in the West remains poor. The survival rate that we obtained in this study is comparable with those from the Western countries but lower than those from Japan. Better results of the Japanese series may be due to early diagnosis, different tumor biology, patient type, and extended lymphadenectomy.

able gastric carcinoma in the West remains poor. The survival rate that we obtained in this study is comparable with those from the Western countries but lower than those from Japan. Better results of the Japanese series may be due to early diagnosis, different tumor biology, patient type, and extended lymphadenectomy. It has been reported that the prognosis of the patients with gastric cancer is influenced most strongly by lymph node involvement. Moreover, lymphatic spread is reported to be one of the most relevant prognostic factors in advanced gastric cancer resected for cure [18, 19]. Since risk of relapse and overall survival are also highly dependent on the number of lymph node metastases, overall survival of the patients with no lymph node metastasis is about 40-80% [20]. In the present study, 5-year survival rate in relation to lymph node involvement was 65%, 13%, and 11% for pN0, pN1, and pN2, respectively and it was one of the significant prognostic factors by univariate analysis (P = 0.030). Studies that have been published during last few years emphasize the prognostic importance of lymph vessel invasion. Yokota et al found that lymphatic vessel invasion is an unfavorable prognostic factor for gastric cancer [18]. Also, von Rahden et al reported lymphatic vessel invasion as a prognostic factor in patients with resected primary adenocarcinomas of the esophagogastric junction [21]. In our study, lymphatic vessel invasion was found to be one of the prognostic factors by the univariate analysis (P = 0.001).

for gastric cancer [18]. Also, von Rahden et al reported lymphatic vessel invasion as a prognostic factor in patients with resected primary adenocarcinomas of the esophagogastric junction [21]. In our study, lymphatic vessel invasion was found to be one of the prognostic factors by the univariate analysis (P = 0.001). Multiple blood transfusions have been reported to be associated with poor survival rate, and have been determined as an independent risk factor for poor prognosis by multivariate analysis in several studies [22, 23]. In a retrospective study on 1000 patients that had undergone curative surgery for gastric cancer at the National Cancer Center Hospital in Japan from 1976 to 1981, statistical analysis revealed significant adverse influence of blood transfusions on survival [24]. In our study, blood transfusions more than 500 ml was one of the significant prognostic factors by univariate analysis (P = 0.021).

ry for gastric cancer at the National Cancer Center Hospital in Japan from 1976 to 1981, statistical analysis revealed significant adverse influence of blood transfusions on survival [24]. In our study, blood transfusions more than 500 ml was one of the significant prognostic factors by univariate analysis (P = 0.021). Lymph node ratio may give more accurate prognostic information about nodal involvement than the current pN category. Several authors have already suggested the role of lymph node ratio as a prognostic factor [14, 25, 26]. Siewert et al reported relevant prognostic factors in 1182 patients with gastric cancer undergoing R0 resection and they noted that lymph node ratio and lymph node status were the most important prognostic factors in patients with resected gastric cancer [26]. Analyzing the survival by comparing lymph node ratio against the number of involved lymph nodes, Santiago et al have demonstrated that the ratio stands out as the best prognostic factor [25]. The results of our study show that lymph node ratio was the only important predictor in patients with transmural gastric cancer (P = 0.029, RR:4.47).

l by comparing lymph node ratio against the number of involved lymph nodes, Santiago et al have demonstrated that the ratio stands out as the best prognostic factor [25]. The results of our study show that lymph node ratio was the only important predictor in patients with transmural gastric cancer (P = 0.029, RR:4.47). In conclusion, lymph node metastases, lymph vessel invasion, blood transfusions (more than or equal to 500 ml), and lymph node ratio (greater than or equal to 0.2) were found as significant prognostic factors by the univariate analysis in this study. However, lymph node ratio was the strongest predictor of survival in multivariate analysis. Our data showed that we can expect a good survival for patients with a lymph node ratio less than 0.2, when extended lymph node dissection is performed. Competing Interests The authors of this article claim to have no financial interest in any company or any of the products mentioned in this article.

Introduction Hepatocellular carcinoma is a part of the natural course of liver cirrhosis, there are about 3-10% of cirrhotic patients who are reported to develop liver cancer every year [1]. Almost 80% of HCC paitents have background of liver cirrhosis, therefore liver cirrhosis patients are in extremely high risk for HCC development [2]. It is easy to make an early diagnosis of HCC among cirrhosis patients if we can identify the risk factors relative to HCC transformation and follow up closely [3-5]. Survivin is a gene with the role of suppressing apoptosis which has been studied mostly over the past ten years [6, 7]. It was identified up-regulated in many type of cancer but not in normal tissues. In recent years, reports have mentioned that survivin was presents in some preneoplastic lesions, this can be inferred that survivin expression may appear early during malignant transition.

en studied mostly over the past ten years [6, 7]. It was identified up-regulated in many type of cancer but not in normal tissues. In recent years, reports have mentioned that survivin was presents in some preneoplastic lesions, this can be inferred that survivin expression may appear early during malignant transition. Materials and Methods Specimen collection Fresh specimens of cirrhotic were taken from 21 patients with hepatitis B related cirrhosis underwent splenectomy for portal hypertension, 17 males, 4 females, aged 33 - 62 years (36 years in average). Sixteen patients had history of gastrointestinal bleeding, 6 patients had hypersplenism. The liver function was Child-Pugh Grading A in 7 patients, Grading B in 12 patients and Grading C in 2 patients. Fresh specimens of hepatocellular carcinoma were taken from 21 patients with HCC after operation, including patients 16 males and 5 females. Five patients had tumor over 10 cm in diameter, 6 patients had multiple tumors. The normal control liver tissues came from 5 patients with angioma underwent surgery therapy. Specimens were fixed quickly by placing in the paraformaldehyde for 2h, then separated into two portions. One portion was paraffin-embedded and cut into slice routinely for the study of immunohistochemisty, other portion was preserved in the refrigerator at -70°C for extracting RNA afterward. Stocked specimens included 30 samples of cirrhosis and 30 samples of HCC which obtained from the archived paraffin block specimens in the pathology department of our hospital. The pathological sections of each sample were conducted staining with HE (hematoxylin-eosin) to make further confirmation of original diagnosis. Besides, we have made a rank classification for liver specimens of cirrhosis patients on liver fibrosis according to the standard referred to SSS [8] and for HCC specimens on the degree of cell differentiation referred to the standard of Edmondson. All works mentioned above have been done by a senior pathological professor in order to achieve accuracy.

ification for liver specimens of cirrhosis patients on liver fibrosis according to the standard referred to SSS [8] and for HCC specimens on the degree of cell differentiation referred to the standard of Edmondson. All works mentioned above have been done by a senior pathological professor in order to achieve accuracy. Survivin RNA extract Fresh liver tissues of 50-100 mg were used to extract total RNA, according the reagent instruction of Omeg Company. RNA was stored at -70°C until needed. Survivin primer design We have found survivin cDNA sequence from GenBank as following: a tgggtgcccc gacgttgccc cctgcctggc agccctttct caaggaccac cgcatctcta cattcaagaa ctggcccttc ttggagggct gcgcctgcac cccggagcgg atggccgagg ctggcttcat ccactgcccc actgagaacg agccagactt ggcccagtgt ttcttctgct tcaaggagct ggaaggctgg gagccagatg acgaccccat agaggaacat aaaaagcatt cgtccggttg cgctttcctt tctgtcaaga agcagtttga agaattaacc cttggtgaat ttttgaaact ggacagagaa agagccaaga acaaaattgc aaaggaaacc aacaataaga agaaagaatt tgaggaaact gcggagaaag tgcgccgtgc catcgagcag ctggctgcca tggattga. We designed the primer with the aid of software as follow. Upstream 5’-CCA CCG CAT CTC TAC ATT C-3’ (97-115base). Downstream 5’-CTT TCT CCG CAG TTT CCT C-3’ (422-440 base). The total length of primer was 19 bp. DNA fragment to be amplified was 344bp.

aatt tgaggaaact gcggagaaag tgcgccgtgc catcgagcag ctggctgcca tggattga. We designed the primer with the aid of software as follow. Upstream 5’-CCA CCG CAT CTC TAC ATT C-3’ (97-115base). Downstream 5’-CTT TCT CCG CAG TTT CCT C-3’ (422-440 base). The total length of primer was 19 bp. DNA fragment to be amplified was 344bp. RT-PCR for survivin RNA The total volume for cDNA synthesis was 20 µl. Including RNA template 3 µl (about 1-3 microgram), downstream primer 1 µl, RNA enzyme inhibitor 0.5 µl, M-MLV reverse transcriptase 1 µl, dNTP 1.25 µl, distilled water 13.25 µl. Reacting at 42°C for 60 min, then at 99°C for 5 min. The total volume for survivin DNA amplification was 50 µl. Including cDNA (from the production of above reaction) 10 µl, PCR buffer 5 µl, upstream primer 1 µl, distilled water 33 µl, 95°C 10min, then 1 µl Tag enzyme was added and 1 µl paraffin oil covered on the surface. The amplification process as follows: 94°C 2 min, 55°C 30s and 72°C 60s. The process is repeated for 30 times. At the last time, the reactant was kept for 5 min in order to make sure to be fully extended.

µl, distilled water 33 µl, 95°C 10min, then 1 µl Tag enzyme was added and 1 µl paraffin oil covered on the surface. The amplification process as follows: 94°C 2 min, 55°C 30s and 72°C 60s. The process is repeated for 30 times. At the last time, the reactant was kept for 5 min in order to make sure to be fully extended. Immunohistochemistry for PCNA Mouse anti-PCNA monoclonal antibody PC10 (product of MBI, MAB-0145) was bought from The Biological Technology Development Company in Fu-Zhou, China. The immunohistochemistry SP staining kit was purchased from Beijing Zhongshan Biotechnology Company. All procedures were done according to the product instruction. The PCNA positive cells were counted under the microscope (200X). The average number of 10 visual fields counted was regarded as the real quantity of PCNA positive cells of the sample. Results The survivin expression of liver tissues in cirrhosis and hepatocellular carcinoma The specific 344bp band of survivin gene was found in 11 specimens of 21 HCC patients. HE stained tissues of 10 specimens comes from presupposed HCC patients but showing survivin negative were checked out under light microscopy by a pathologist. Among them, tissues of 3 cases had not been found any cancer cell, and 1 case only was found necrotic tissue under light microscopy. That means these 4 specimens did not pertaining to cancer tissue. So we concluded the positive rate of survivin gene expression was 64.7% (11/17). Meanwhile, no positive band was found in 10 specimens of liver cirrhosis patients (Fig. 1).

ny cancer cell, and 1 case only was found necrotic tissue under light microscopy. That means these 4 specimens did not pertaining to cancer tissue. So we concluded the positive rate of survivin gene expression was 64.7% (11/17). Meanwhile, no positive band was found in 10 specimens of liver cirrhosis patients (Fig. 1). Figure 1 The result of Survivin expression (RT-PCR). 1, 6: Normal liver tissue (control); 2, 3: Liver cirrhosis tissue; 4, 5: HCC tissue; M: DNA marker. Proliferation in liver cirrhosis and hepatocellular carcinoma The results of immunohistochemistry on PCNA showed that the proliferating cell nuclear antigen was all located in the nucleus of liver cells in both liver cirrhosis and HCC specimens. The PCNA antigen was showed brown in the tissues of liver cirrhosis, PCNA positive cells gathered in a cluster or distributed at the border of regenerative nodes. In the tissues of hepatocellullar carcinoma, the PCNA positive cells scattered among the cancer nodules.

ver cells in both liver cirrhosis and HCC specimens. The PCNA antigen was showed brown in the tissues of liver cirrhosis, PCNA positive cells gathered in a cluster or distributed at the border of regenerative nodes. In the tissues of hepatocellullar carcinoma, the PCNA positive cells scattered among the cancer nodules. Comparing the results of stocked specimens of 30 liver cirrhosis with that of 30 HCC, the activity of proliferation of HCC was higher than that of liver cirrhosis. The PCNA positive score was 10.1 ± 12.3 for HCC and 2.4 ± 2.1 for liver cirrhosis, which showed significant difference, P = 0.003. In five cases of normal liver tissue, the PCNA-positive cell counts were 0.5, 1.2, 2.3, 2.0, and1.8, the mean was 1.56, which was significant lower than that of both HCC and liver cirrhosis. If we took two-fold of mean (3.12) as a value to evaluate the activity of proliferation, 6 of 30 liver cirrhosis patients with mean PCNA labeling index 5.05 ± 2.61 were considered in a high proliferative status, which was higher than that of normal liver tissue but was lower than that of HCC (10.08 ± 12.28). The rest 24 cases of liver cirrhosis with a relative low level of proliferation had the same PCNA labeling score as that of normal liver tissue (P > 0.05).

index 5.05 ± 2.61 were considered in a high proliferative status, which was higher than that of normal liver tissue but was lower than that of HCC (10.08 ± 12.28). The rest 24 cases of liver cirrhosis with a relative low level of proliferation had the same PCNA labeling score as that of normal liver tissue (P > 0.05). Relationship between proliferation and fibrosis in liver cirrhosis tissue At the same time, the fibrotic levels of 19 cases of liver cirrhosis were evaluated by reference to the SSS. No statistical relationship between scores of fibrosis and PCNA in each sample was found by correlation analysis (r = 0.337, P = 0.159), which can be inferred that liver cell proliferation had nothing to do with the degree of liver fibrosis in the liver cirrhosis. Relationship between cell proliferation and cell differentiation grade in HCC tissue According to the cell differentiation, 30 samples of HCC were graded into grade I to grade IV from high to low. The mean PCNA scores of each group were 3.7 (grade I), 9.74 (grade II), 14.36 (grade III), 15.7 (grade IV). Correlation analysis by Spearman showed that there were rank relationship of PCNA scores in each group (r = 0.74, P < 0.001), which can be inferred that the proliferative extent of liver cells in HCC tissue was relevant to their malignant degrees.

were 3.7 (grade I), 9.74 (grade II), 14.36 (grade III), 15.7 (grade IV). Correlation analysis by Spearman showed that there were rank relationship of PCNA scores in each group (r = 0.74, P < 0.001), which can be inferred that the proliferative extent of liver cells in HCC tissue was relevant to their malignant degrees. Relationship between proliferative activity and survivin gene expression in HCC tissue In 11 HCC tissues of positive survivin gene, the median PCNA labeling index was 6.8 (0.5 - 4.0). Meanwhile the median PCNA labeling index of 6 other HCC tissues of survivin gene negative was 2.1 (0.5 - 3.0). There was a significant difference between the two groups (P < 0.05), which proved that the survivin gene expression of HCC was correlation with cell proliferative activity.

labeling index was 6.8 (0.5 - 4.0). Meanwhile the median PCNA labeling index of 6 other HCC tissues of survivin gene negative was 2.1 (0.5 - 3.0). There was a significant difference between the two groups (P < 0.05), which proved that the survivin gene expression of HCC was correlation with cell proliferative activity. Discussion It is known that the biocharacteristics of liver cells playing an important role in hepatocarcinogenesis. Acceleration of proliferation and the deceleration of apoptosis in liver cells are the main reasons for the formation of hepatocellular carcinoma [9, 10]. It is also proved that there is a defection in O6-methyl guanine repair enzymes in patients with liver cirrhosis. If the damaged O6-methyl guanine in the gene sequence can not been repaired promptly, the genetic mutation will happen, which result in activation of oncogene and inactivation of anti-oncogene [11]. Clinical studies showed that the high level proliferation of liver cells in patients with liver cirrhosis is one of the most important risk factors for the formation of hepatocellular carcinoma. Donato et al [12] used the scores of PCNA as an indicator for cell proliferation, 208 patients with cirrhosis were investigated in a prospective studies for 88 ± 42 months. Fifty patients with HCC were found in the study. The average score of patients with HCC at the baseline was 3.6 ± 2.4%, while that of patients without HCC was 1.6 ± 1.5 %, which showed significant difference. To make a demonstration more clear, patients were classified into two groups according to the baseline PCNA index, 80 patients with above 2% were regarded as high risk group, while other 158 patients were regarded as low risk group. The incidence of liver cancer was 5.2% in the high risk group compared 1.1 % in the low risk group. In another study, liver tissues of 97 liver cirrhosis patients were investigated with flow cytometer by Sangiovannio et al [13]. The cell ratio of S phase in mitotic cycle of liver cells was determined. Within the period of 53 months, 12 patients of liver cirrhosis were found to become HCC, with which the cell ratio of S phase was 2.5 ± 1.6%, while other patients without HCC was 0.9% ± 0.6%. When taking the ratio of S phase above 1.8% as the high proliferation of liver cells, the HCC incidence was of 60% in patients of liver cirrhosis in the group with high proliferation, while only 4% in the group of low proliferation.

cell ratio of S phase was 2.5 ± 1.6%, while other patients without HCC was 0.9% ± 0.6%. When taking the ratio of S phase above 1.8% as the high proliferation of liver cells, the HCC incidence was of 60% in patients of liver cirrhosis in the group with high proliferation, while only 4% in the group of low proliferation. All above evidence certify that the state of liver cell proliferation in patients with liver cirrhosis have a close relation with the occurrence of hepatocarcinoma. We have investigated liver tissues of 30 patients with liver cirrhosis and 30 patients with HCC using the PC10 monoclonal antibody against PCNA by the technique of immunohistochemistry. The results showed that proliferation activity of liver cells of HCC tissue was significantly higher than that of the cirrhotic tissue. The PCNA labeling index was 10.08 and 2.38 respectively, which is identical with the conclusions of aforementioned. In our research, there were 20% liver cirrhosis patients (6 cases) with the PCNA labeling index 2 times more than normal liver tissue, even though it is lower than that of HCC patients, it is significantly higher than that of other patients with liver cirrhosis and normal control. These patients must be considered have a HCC predisposition and need to be strictly followed up in order to make an early diagnosis of HCC.

es more than normal liver tissue, even though it is lower than that of HCC patients, it is significantly higher than that of other patients with liver cirrhosis and normal control. These patients must be considered have a HCC predisposition and need to be strictly followed up in order to make an early diagnosis of HCC. We have found in the research that there is no significant correlation between the level of liver cell proliferation and the degree of liver fibrosis, which means that it is not the fibrosis of liver cirrhosis but the high cell proliferation induces the HCC occurrence.

es more than normal liver tissue, even though it is lower than that of HCC patients, it is significantly higher than that of other patients with liver cirrhosis and normal control. These patients must be considered have a HCC predisposition and need to be strictly followed up in order to make an early diagnosis of HCC. We have found in the research that there is no significant correlation between the level of liver cell proliferation and the degree of liver fibrosis, which means that it is not the fibrosis of liver cirrhosis but the high cell proliferation induces the HCC occurrence. Survivin gene is an apoptosis suppressive gene studied in recent years. It is different from other apoptosis suppressive gene, its expression is highly tissue-specific. It expresses in embryonic tissues and many kinds of tumor tissues, such as stomach, breast, colorectal, pancreatic and so on. However, it is not found in the mature normal tissues. In addition to its anti-apoptotic functions, recent studies have shown that survivin gene expression is related to enhanced activity of liver cancer cell proliferation. By the balance destruction role of proliferation and apoptosis, the survivin gene plays an important part in the course of liver cancer occurrence. With the method of immunohistochemistry, Ito et al [14] found the survivin protein in 14 of 20 HCC tissue samples, but did not find in the surrounding tissues of tumor, neither in the tissues of chronic hepatitis and cirrhosis. Using the same method, Ito et al found that 64.6% of 48 HCC patients showed survivin protein positive, which was positively correlated to the degree of low cell differentiation of liver cancer.

ssue samples, but did not find in the surrounding tissues of tumor, neither in the tissues of chronic hepatitis and cirrhosis. Using the same method, Ito et al found that 64.6% of 48 HCC patients showed survivin protein positive, which was positively correlated to the degree of low cell differentiation of liver cancer. In our research, there were 11 of 17 HCC specimens showed survivin gene expression by RT-PCR. The median PCNA labeling index of 11 survivin positive HCC specimens (9.68 ± 11.64) was significantly higher than that of other six survivin negative HCC specimens (3.27 ± 1.73). The results we obtained were consistent with that of other studies mentioned above, which demonstrated clearly that the gene expression is a gradual process and must be accompanied by increased activity of liver cell proliferation. In the process of carcinomatous change, the level of survivin gene expression gradually increased. Survivin gene expression of liver cells indicates the carcinomatous changes occurrence; this is useful in the differential diagnosis of liver lumps caused by cirrhosis and carcinoma. Ikeguchi et al [15] evaluated the survivin gene expression in 61 surrounding tissue samples of HCC, there was no sample was positive. Thereby, they presume that the survivin gene only expressed in the cirrhotic liver cells of malignant transformation. Be coincidence with above study, all 21 samples of liver cirrhosis showed survivin gene negative in our research, which means all patients with liver cirrhosis in our study, the malignant transformation had not yet taken place.

t the survivin gene only expressed in the cirrhotic liver cells of malignant transformation. Be coincidence with above study, all 21 samples of liver cirrhosis showed survivin gene negative in our research, which means all patients with liver cirrhosis in our study, the malignant transformation had not yet taken place. According to the above mentioned results we presume that the PCNA and survivin gene expression have different meanings in the HCC prevention strategy among liver cirrhosis patients. PCNA detection can be used for identifying people with predisposition to HCC, while survivin detection can be used for differentiation nodes of HCC and cirrhosis.

entioned results we presume that the PCNA and survivin gene expression have different meanings in the HCC prevention strategy among liver cirrhosis patients. PCNA detection can be used for identifying people with predisposition to HCC, while survivin detection can be used for differentiation nodes of HCC and cirrhosis. Liver cell proliferation and survivin gene expression is not only related to the occurrence of HCC, but also to the prognosis and recurrence of HCC. Among 17 cases of hepatocellular carcinoma in this study, the PCNA-positive cell count was significantly higher in 11 survivin positive tissues than that of six other survivin negative tissues (P < 0.05). Moreover, the PCNA-positive cells count of liver cancer tissues was positively correlated to the degree of cancer differentiation. The poorer the differentiation, the PCNA-positive cell count is higher. All these facts fully demonstrated that the cell proliferation has a close relationship with liver cancer development and progression. As survivin gene expression occurred in the early stage of HCC, its detection can only be used in finding patients with early HCC, but may not suitable for finding HCC high-risk group patients with liver cirrhosis. According to the above results, we concluded that it is the PCNA detection but not survivin gene can be effectively used in screening people with high-risk HCC malignant transformation among patients with liver cirrhosis.

Appendiceal endometriosis is a rare condition. Its frequency is less than 1% of patients with pelvic endometriosis [2]. Most patients present with acute appendicitis or asymptomatic [1-6]. The present case presented as an appendiceal tumor, and clinicians strongly suggested a gastrointestinal stromal tumor. Such a case appears very rare. The two biopsies failed to detect atypical glands. This is because the endometriosis did not involve appendiceal mucosa. Thus, appendiceral endometriosis should be taken into account in female patients when an appendiceal tumor is clinically found. Repeated biopsy may not be effective. In summary, the author presented a case of appendiceal endometoriosis involving lymph nodes presenting an appendiceral tumor. Conflict of interest The author has no conflict of interest.

Introduction Cervical web is a common benign cause of dysphagia. Endoscopic fracture/dilatation of the web results in resolution of the symptoms. Recurrence of cervical web is rare. Cervical web is usually associated with iron deficiency anemia. Celiac disease is a common cause of refractory iron deficiency anemia in North India. We describe a 35 year old man with recurrent cervical webs with severe iron deficiency anemia, secondary to celiac disease who responded dramatically to gluten free diet. Case Report A 35 years old man presented with symptoms of dysphagia of 5 years duration to solids which was insidious in onset and persistent. His appetite was normal but had weight loss secondary to decreased intake. There was no history suggestive of reflux disease, drug ingestion or corrosive intake. His bowel habits were normal. He underwent 4 sessions of esophageal dilations by surgeons under general anaesthesia, but the webs recurred and he was referred to gastroenterology services.

Introduction Endometriosis of the vermiform appendix is an uncommon condition [1-6]. Most patients with this disease are asymptomatic or present as acute or chronic appendicitis. Perforation of the appendix has been also reported [6]. However, appendiceal endometriosis presenting as a tumor is very rare. The author herein reports a case of appendiceal endometriosis presenting as a tumor. Case Report A 41-year-old woman complained of chronic abdominal pain. A colon endoscopy showed a tumor in the appendiceal orifice. Two biopsies of the tumor showed no remarkable changes. Imaging modalities including CT and MRI also revealed an appendiceal tumor. Resection of appendix, cecum, ascending colon, terminal ileum, and 16 lymph nodes were performed under the clinical diagnosis of gastrointestinal stromal tumor. Grossly, a tumor measuring 3 x 3 x 3 cm was recognized in the appendiceral orifice (Fig. 1a). Histologically, the tumor was endometriosis consisting of islands of endometrial glands and stroma (Fig. 1b, c). The endometriosis involved submucosa, proper muscular layer, and subserosa, sparing the mucosa. The muscular layer was very hypertrophic. Similar lesions without stroma were found in the six lymph nodes among the 16 lymph nodes dissected (Fig. 1d).

is consisting of islands of endometrial glands and stroma (Fig. 1b, c). The endometriosis involved submucosa, proper muscular layer, and subserosa, sparing the mucosa. The muscular layer was very hypertrophic. Similar lesions without stroma were found in the six lymph nodes among the 16 lymph nodes dissected (Fig. 1d). Figure 1 (a) Gross features. A tumor measuring 3 x 3 x 3 cm is recognized in the appendiceal orifice. (b) Low power view of the histology. Endometrial glands and stroma is scattered. HE, x 40. (c) High power view of the histology. The endometrial glands and stroma are evident. HE, x 200. (d) Estrogen receptor is expressed in the endometrial glands. Immunostaining, x 200. (e) Lymph node histology. Glandular structures of endometriosis or Mullerian duct remnants are recognized. An immunohistochemical study was performed by Dako envision method (Dako Corp., Glostrup, Denmark) as previously described [7, 8]. The antigens examined were anti-estrogen receptor (ER) (clone 1D5, Dako), anti-progesterone receptor (PgR) (clone 1A6, Novocastra, Newcastle upon Tyne, UK), p53 protein (clone DO-7, Dako), and Ki-67 antigen (clone MIB-1, Dako). The endometrial tissue of the appendix was positive for ER (Figure 1E) and PgR, but negative for p53. The Ki-67 labeling was very low (0.5%). The lymph node lesions showed the similar immunohistochemical findings.

vocastra, Newcastle upon Tyne, UK), p53 protein (clone DO-7, Dako), and Ki-67 antigen (clone MIB-1, Dako). The endometrial tissue of the appendix was positive for ER (Figure 1E) and PgR, but negative for p53. The Ki-67 labeling was very low (0.5%). The lymph node lesions showed the similar immunohistochemical findings. Discussion The present appendiceal tumor was relatively large. Histologically, it was composed of endometrial glands and stroma associated with muscular hypertrophy. Immunohistochecally, the tumor cells were positive for ER and PGR, and negative for p53 protein. The Ki-67 labeling was very low. Therefore, the present case is tumor-forming appendiceral endometriosis. The lymph nodes lesion also appears endometriosis, but may be Mullerian ducts remnants because no endometrial stroma was present. Appendiceal endometriosis is a rare condition. Its frequency is less than 1% of patients with pelvic endometriosis [2]. Most patients present with acute appendicitis or asymptomatic [1-6]. The present case presented as an appendiceal tumor, and clinicians strongly suggested a gastrointestinal stromal tumor. Such a case appears very rare. The two biopsies failed to detect atypical glands. This is because the endometriosis did not involve appendiceal mucosa. Thus, appendiceral endometriosis should be taken into account in female patients when an appendiceal tumor is clinically found. Repeated biopsy may not be effective. In summary, the author presented a case of appendiceal endometoriosis involving lymph nodes presenting an appendiceral tumor.

d weight loss secondary to decreased intake. There was no history suggestive of reflux disease, drug ingestion or corrosive intake. His bowel habits were normal. He underwent 4 sessions of esophageal dilations by surgeons under general anaesthesia, but the webs recurred and he was referred to gastroenterology services. On evaluation he was thin built and pale. General physical examination was unremarkable except for pallor. Barium swallow showed multiple esophageal webs in the post cricoid region (Fig. 1). He had microcytic and hypochromic anemia with a hemoglobin of 8.2g%. Stool for occult blood was negative and iron studies showed severe iron deficiency state with serum iron of 13 micrograms/dl and percent transferrin saturation of 2.7%. Upper gastro-intestinal endoscopy showed multiple cervical webs and scalloping and grooving of the proximal duodenum (Fig. 2). A possibility of celiac was considered and duodenal biopsy was done. Serum tissue transglutaminase (tTG) was elevated at 28 U/ml (normal < 10 U/ml) and the duodenal biopsy showed villous atrophy with increase in the intra-epithelial lymphocytes consistent with the diagnosis of celiac disease. He was advised gluten free diet and iron supplementation. On this treatment his dysphagia was resolved, and there was no recurrence of webs. He gained 6 kilograms of weight, and his anemia and serum iron profile also started showing improvement (hemoglobin 12g%, serum iron 53 mg/dl, and percent transferring saturation 9.8%) within six weeks of treatment. He developed a pruritic papular skin rash over extensor aspects of limbs suggestive of dermatitis herpertiformis which responded to Dapsone therapy. He has been doing well since then and is asymptomatic on 7 years of follow-up.

erum iron 53 mg/dl, and percent transferring saturation 9.8%) within six weeks of treatment. He developed a pruritic papular skin rash over extensor aspects of limbs suggestive of dermatitis herpertiformis which responded to Dapsone therapy. He has been doing well since then and is asymptomatic on 7 years of follow-up. Figure 1 Barium swallow examination showing evidence of multiple webs in the upper esophagus (arrows). Figure 2 Upper gastrointestinal endoscopic image showing a web in the upper esophagus. Discussion Most of the esophageal webs are asymptomatic and only a few cause dysphagia [1]. Esophageal webs are usually single, in the upper one third of the esophagus and respond completely to single time dilatation [2]. Recurrent cervical webs are uncommon and usually associated with iron deficiency. Other conditions associated with esophageal webs are autoimmune disorders of thyroid, pernicious anemia, graft versus host disease, rheumatoid arthritis, psoariasis and blistering diseases of the skin [3, 4]. Association of celiac disease with esophageal web has been reported in literature [5-7]. In this case there were multiple esophageal webs which were recurrent and refractory to multiple sessions of dilatation. Detection and treatment of underlying celiac disease led to long term resolution of esophageal webs and improvement in anemia.

eliac disease with esophageal web has been reported in literature [5-7]. In this case there were multiple esophageal webs which were recurrent and refractory to multiple sessions of dilatation. Detection and treatment of underlying celiac disease led to long term resolution of esophageal webs and improvement in anemia. In India, the commonest cause of iron deficiency is nutritional inadequacy and worm infestation. However, in North India, celiac disease is very important cause of refractory iron deficiency anemia [8]. All patients with refractory iron deficiency anemia should be evaluated for celiac disease especially in high incidence populations [9]. A high index of suspicion for diagnosis of celiac disease is necessary in patients with iron deficiency anemia or esophageal web. Institution of gluten free diet in these patients provides gratifying results. Competing Interest No conflicts of interest exist for the authors.

Introduction Gastrointestinal stromal tumors (GISTs), which are the most common ‘mesenchymal’ tumor of the gastrointestinal tract, are nonepithelial neoplasms that arise from the muscularis propria of the gastrointestinal tract wall. GISTs originate from interstitial Cajal cells on intestinal pacemaker cells [1, 2]. GISTs have been classified as spindle cell, epithelioid, or (occasionally) pleomorphic mesenchymal tumors of the gastrointestinal tract that express the KIT protein (CD 117, stem cell factor receptor). That protein, which is detected via immunohistochemical analysis, is the primary diagnostic criterion for a GIST [1, 2]. Tumors can develop anywhere in the gastrointestinal tract. GISTs occur most frequently in the stomach (60%) and then most often in the small bowel (30%); other sites include the colon and rectum (5%) and the esophagus (< 5%) [1-4]. GISTs account for 1% to 3% of gastric neoplasms, 20% of small bowel tumors, and 0.2% to 1% of colorectal tumors [1-4]. Although there are a few reports of ruptured GISTs, this is to our knowledge the first case in the English literature of a GIST with an accompanying abscess. Case Report A 52-year-old woman with abdominal pain and tenderness, fever, and swelling of the lower abdomen of a few days’ duration was admitted to our institution. Physical examination revealed a palpable abdominal mass.

Although there are a few reports of ruptured GISTs, this is to our knowledge the first case in the English literature of a GIST with an accompanying abscess. Case Report A 52-year-old woman with abdominal pain and tenderness, fever, and swelling of the lower abdomen of a few days’ duration was admitted to our institution. Physical examination revealed a palpable abdominal mass. A preoperative computed tomographic scan showed a huge primary calcified soft tissue tumor (18 x 13 x 7 cm) in the upper abdomen. A cystic component of the tumor and hemoperitoneum were also identified (Fig. 1). One cystic lesion (10 x 11 x 8 cm) with an air-fluid level was detected in the pelvic region (Fig. 2). Figure 1 A postcontrast computed tomographic scan showed a huge primary calcified GIST (arrows) with a cystic component. Figure 2 A postcontrast computed tomographic scan revealed a pelvic abscess with an air-fluid level. The findings at surgery confirmed that the primary GIST site was identified from the preoperative computed tomographic scan. The primary tumor had ruptured on 2 sides, and what appeared to be a cystic pelvic lesion on computed tomographic examination was a large pelvic abscess. Culture of the abscess yielded Enterococcus species.

s at surgery confirmed that the primary GIST site was identified from the preoperative computed tomographic scan. The primary tumor had ruptured on 2 sides, and what appeared to be a cystic pelvic lesion on computed tomographic examination was a large pelvic abscess. Culture of the abscess yielded Enterococcus species. The results of pathologic examination suggested that the primary lesion had originated in the serosa of the small bowel and had not invaded adjacent visceral organs. The mitotic index of the lesion was 3 mitoses per 50 high-power fields (HPFs). The tumor formed by spindle cells with round hyperchromatic nuclei and prominent nucleoli forming bundles immunohistochemically; the cells were diffusely positive for CD-117 (c kit) and focal and weak positivite for S-100; SMA, CD-34 and desmin were negative (Fig. 3). Chemotherapy with imatinib (Gleevec, Novartis, Switzerland) was initiated because of the large size of the tumor (> 5 cm), the presence of coagulative tumor necrosis, borderline mitotic activity (3/50 HPFs), and confirmed rupture of the neoplasm. Our patient gave written informed consent to radiologic examination and to participation. Figure 3 On microscopic examination the tumor showed spindle cells with round hyperchromatic nuclei and prominent nucleoli forming bundles (haemotoxylin and eosin); 200 x Inset: Immunohistochemically, the cells were diffusely positive for CD-117 (c kit), CD-117, 200 x.

The results of pathologic examination suggested that the primary lesion had originated in the serosa of the small bowel and had not invaded adjacent visceral organs. The mitotic index of the lesion was 3 mitoses per 50 high-power fields (HPFs). The tumor formed by spindle cells with round hyperchromatic nuclei and prominent nucleoli forming bundles immunohistochemically; the cells were diffusely positive for CD-117 (c kit) and focal and weak positivite for S-100; SMA, CD-34 and desmin were negative (Fig. 3). Chemotherapy with imatinib (Gleevec, Novartis, Switzerland) was initiated because of the large size of the tumor (> 5 cm), the presence of coagulative tumor necrosis, borderline mitotic activity (3/50 HPFs), and confirmed rupture of the neoplasm. Our patient gave written informed consent to radiologic examination and to participation. Figure 3 On microscopic examination the tumor showed spindle cells with round hyperchromatic nuclei and prominent nucleoli forming bundles (haemotoxylin and eosin); 200 x Inset: Immunohistochemically, the cells were diffusely positive for CD-117 (c kit), CD-117, 200 x. Discussion In the gastrointestinal tract, CD 117-positive cells (interstitial Cajal cells) are autonomic nerve-related gastrointestinal pacemaker cells that regulate intestinal motility [1, 2]. Because of the immunohistochemical and ultrastructural similarities of Cajal cells and GISTs, a histogenetic origin of GISTs from Cajal cells has been proposed [1, 2].

CD 117-positive cells (interstitial Cajal cells) are autonomic nerve-related gastrointestinal pacemaker cells that regulate intestinal motility [1, 2]. Because of the immunohistochemical and ultrastructural similarities of Cajal cells and GISTs, a histogenetic origin of GISTs from Cajal cells has been proposed [1, 2]. A few previous studies have investigated ruptured GISTs by means of several radiologic modalities such as ultrasonography and computed tomography. A literature search yielded only 9 reports of ruptured primary GISTs to date. Cegarra-Navarro et al reported 5 patients with a ruptured primary GIST and 1 patient with a ruptured metastatic GIST [5]. We did not evaluate ruptured metastatic GISTs. One of the nine patients’ tumours was small bowel location and another one was transverse mesocolon location; the others were gastric location [5-9]. Seven of the nine patients’ ruptured GISTs were spontaneous [5-9]; the others were result of the trauma [5]. To our knowledge, this case is the second published report of a spontaneously ruptured GIST in the small bowel. Eight cases of a GIST with hemoperitoneum and 1 case of a GIST with concomitant hemoperitoneum and peritonitis have been reported [5, 7-9]. Our patient demonstrated a GIST with hemoperitoneum and abscess but without peritonitis.

is case is the second published report of a spontaneously ruptured GIST in the small bowel. Eight cases of a GIST with hemoperitoneum and 1 case of a GIST with concomitant hemoperitoneum and peritonitis have been reported [5, 7-9]. Our patient demonstrated a GIST with hemoperitoneum and abscess but without peritonitis. The computed tomographic findings of ruptured GISTs (noncontrast and postcontrast images) showed huge masses of low density with exophytic growth in the peritoneal cavity and ascites. These ruptured tumors are echogenic on ultrasonography and dense on computed tomographic scans (findings that suggest hemoperitoneum). The size of 3 of the 9 previously reported GISTs was less than 10 cm [5], and the 6 remaining GISTs were larger than 10 cm [5-8]. As in most of the other cases reported, the mass found in our patient was larger than 10 cm. Seven of the previously reported GISTs exhibited low mitotic activity (less than 5 mitoses per 50 HPF) [5-9]. Two of the reported ruptured primary GISTs demonstrated a high mitotic index (20 mitoses per 50 HPF) [5-9]. As in most of the other cases reported, our patient’s tumor was characterized by borderline mitotic activity. All primary GISTs that were previously reported except 2 demonstrated low or borderline mitotic activity [5-8], but all had a large cystic and necrotic component. We may speculate that the rupture of primary GISTs involves the cystic and necrotic tumor components but not high-grade mitotic activity.

Seven of the previously reported GISTs exhibited low mitotic activity (less than 5 mitoses per 50 HPF) [5-9]. Two of the reported ruptured primary GISTs demonstrated a high mitotic index (20 mitoses per 50 HPF) [5-9]. As in most of the other cases reported, our patient’s tumor was characterized by borderline mitotic activity. All primary GISTs that were previously reported except 2 demonstrated low or borderline mitotic activity [5-8], but all had a large cystic and necrotic component. We may speculate that the rupture of primary GISTs involves the cystic and necrotic tumor components but not high-grade mitotic activity. Most of the patients with a ruptured GIST presented for urgent care and all patients underwent emergency laparotomy [5-9]. Segmental small bowel or gastric resection is standard treatment for perforated local GIST. Imatinib is being evaluated as adjuvant treatment following surgery, especially in high malignant potential cases [10]. To our knowledge, a ruptured GIST with an accompanying abscess has not been previously reported. We describe the postcontrast computed tomographic findings, surgical correction, and histopathologic findings of a ruptured primary GIST and review the clinical and radiologic follow-up. In our patient, the operation was successful, and she did well after surgery.

Introduction Pancreatic neoplasms are classified into exocrine and endocrine neoplasms [1, 2]. The majority of pancreatic neoplasm is exocrine neoplasm such as ductal adenocarcinoma. Endocrine neoplasm accounts for 1-2% of all pancreatic neoplasms [1-3]. Pancreatic endocrine neoplasms were classified into endocrine microadenoma, well-differentiated pancreatic endocrine neoplasm, poorly differentiated endocrine carcinoma, and mixed endocrine carcinoma [1, 2]. The well differentiated pancreatic endocrine neoplasms were further categorized into functional and nonfunctional ones. The author herein reports a typical but rare case of non-functioning well differentiated pancreatic endocrine carcinoma.

oorly differentiated endocrine carcinoma, and mixed endocrine carcinoma [1, 2]. The well differentiated pancreatic endocrine neoplasms were further categorized into functional and nonfunctional ones. The author herein reports a typical but rare case of non-functioning well differentiated pancreatic endocrine carcinoma. Case report A 67-year-old man was admitted to our hospital because of abdominal pain. No hormone-related symptoms were recognized. He denied a familiar history of MEN type I and Hippel-Lindau syndrome. Various imaging modalities including US, CT and MRI revealed a tumor of the pancreatic body. Distal pancreatectomy and splenectomy were performed. Grossly, a solid well-defined tumor measuring 60 x 55 x 50 mm was present in the pancreatic body (Fig. 1). Peripancreatic lymph nodes showed marked swelling suggestive of metastases (Fig. 1). Histologically, tumor cells with hyperchromatic nuclei were arranged in a trabecular patten (Fig. 2a, 2b). Clear cell change was recognized in some areas (Fig. 2c). The nuclei of tumor cells showed ‘salt and pepper’ appearances (Fig. 2b, 2c), and mitotic figures were present in 5 per 10 high power fields. Invasive features, and vascular and perineural invasions were recognized in the periphery of the tumor (Fig. 2d). No ductal element was recognized. The peripancreatic lymph nodes showed metastases. The spleen was devoid of carcinoma cells. Figure 1 Gross features of resected pancreas. A well defined tumor measuring 60 x 55 x 50 mm is recognized (left). Lymph node metastasis is also seen (left upper).

Case report A 67-year-old man was admitted to our hospital because of abdominal pain. No hormone-related symptoms were recognized. He denied a familiar history of MEN type I and Hippel-Lindau syndrome. Various imaging modalities including US, CT and MRI revealed a tumor of the pancreatic body. Distal pancreatectomy and splenectomy were performed. Grossly, a solid well-defined tumor measuring 60 x 55 x 50 mm was present in the pancreatic body (Fig. 1). Peripancreatic lymph nodes showed marked swelling suggestive of metastases (Fig. 1). Histologically, tumor cells with hyperchromatic nuclei were arranged in a trabecular patten (Fig. 2a, 2b). Clear cell change was recognized in some areas (Fig. 2c). The nuclei of tumor cells showed ‘salt and pepper’ appearances (Fig. 2b, 2c), and mitotic figures were present in 5 per 10 high power fields. Invasive features, and vascular and perineural invasions were recognized in the periphery of the tumor (Fig. 2d). No ductal element was recognized. The peripancreatic lymph nodes showed metastases. The spleen was devoid of carcinoma cells. Figure 1 Gross features of resected pancreas. A well defined tumor measuring 60 x 55 x 50 mm is recognized (left). Lymph node metastasis is also seen (left upper). Figure 2 Microscopic findings of the tumor. (a) very low power view of the tumor. Tumor cells are arranged in a trabecular pattern. HE, x20. (b) tumor cells are arranged in a trabecular pattern. The nuclei show ‘salt and pepper’ appearances. HE, x200. (c) clear cell change of tumor cells. HE, x200. (d) vascular invasion of tumor cells. HE, x100.

gs of the tumor. (a) very low power view of the tumor. Tumor cells are arranged in a trabecular pattern. HE, x20. (b) tumor cells are arranged in a trabecular pattern. The nuclei show ‘salt and pepper’ appearances. HE, x200. (c) clear cell change of tumor cells. HE, x200. (d) vascular invasion of tumor cells. HE, x100. An immunohistochemical study was performed using Dako Envision methods (Dako Corp. Glostrup, Denmark), as previously described [4, 5]. The antibododies used were anti-cytokeratin (AE1/3, Dako), anti-cytokeratin (polyclonal wide, Dako), carcinoembrionic antigen (CEA) (polyclonal, Dako), chromorgranin (DAK-A3, Dako), synaptophysin (polyclonal, Dako), neuron-specific enolase (NSE) (BBS/NC/VI-H14, Dako), CD56 (MOC-1, Dako), insulin (polyclonal, Dako), glucagon (polyclonal, Dako), gastrin (polyclonal, Dako), somatostatin (polyclonal, Dako), pancreatic polypeptide (polyclonal, Dako), and vasoactive intestinal polypeptide (polyclonal, Dako). Immunohistyochemically, tumor cells were positive for cytokeratin (Fig. 3a), synaptophysin (Fig. 3b), neuron-specific enolase, and CD56 (Fig. 3c); they were negative for chromogranin, gastrin, glucagon, somatostatin, pancreatic polypeptide, and vasoactive intestinal polypectide. Figure 3 Immunohistochemical findings of the tumor. (a) tumor cells are positive for cytokeratin. Immunostaining, x200. (b) Tumor cells are positive for synaptophysin. Immunostaining, x200. (c) Tumor cells are positive for CD56. Immunostaining, x200.

Immunohistyochemically, tumor cells were positive for cytokeratin (Fig. 3a), synaptophysin (Fig. 3b), neuron-specific enolase, and CD56 (Fig. 3c); they were negative for chromogranin, gastrin, glucagon, somatostatin, pancreatic polypeptide, and vasoactive intestinal polypectide. Figure 3 Immunohistochemical findings of the tumor. (a) tumor cells are positive for cytokeratin. Immunostaining, x200. (b) Tumor cells are positive for synaptophysin. Immunostaining, x200. (c) Tumor cells are positive for CD56. Immunostaining, x200. The pathological diagnosis was non-functioning well differentiated endocrine carcinoma of the pancreas. At the 36 months post-operative follow-up, the patient was alive with liver metastases. Discussion The present case is typical pancreatic well differentiated endocrine tumor. The trabelular arrangement and ‘salt and pepper’ nuclei of tumor cells are typical for this neoplasm. Further, the present tumor was immunohistochemically positive for neuroendocrine markers (synaptophysin, NSE and CD56), highly suggesting that the present is an endocrine tumor. The absence of ductal element indicates that the present tumor is not mixed endocrine tumor [5, 6]. Clear cell change of tumor cells, as seen in the present case, has been reported to be present in pancreatic endocrine tumors and it is due to lipid deposition [7]. In addition, the present case showed infiltrative growth, perineural invasion, vascular permeation, and metastases, indicating that the present case is malignant, namely the well differentiated pancreatic endocrine carcinoma.

n reported to be present in pancreatic endocrine tumors and it is due to lipid deposition [7]. In addition, the present case showed infiltrative growth, perineural invasion, vascular permeation, and metastases, indicating that the present case is malignant, namely the well differentiated pancreatic endocrine carcinoma. The present case clinically lacked hormone-related paraneoplasmic syndrome. In addition, the present endocrine carcinoma was immunohistochemically negative for insulin, glucagon, gastrin, pancreatic polypeptide, and vasoactive intestinal polypeptide. These observations indicate that the present case is non-functional pancreatic endocrine carcinoma. In addition, the absence of familiar history of MEN type I and Hippel-Lindau syndrome suggests that the present tumor is not familiar cancer syndrome but a sporadic non-functional endocrine carcinoma. The incidence of non-functional endocrine carcinoma is less than 1% of all pancreatic neoplasms [8]. Clinically, presenting symptoms are non-specific, such as nausea and abdominal pain, in non-functional endocrine tumor [1-3].

The present case clinically lacked hormone-related paraneoplasmic syndrome. In addition, the present endocrine carcinoma was immunohistochemically negative for insulin, glucagon, gastrin, pancreatic polypeptide, and vasoactive intestinal polypeptide. These observations indicate that the present case is non-functional pancreatic endocrine carcinoma. In addition, the absence of familiar history of MEN type I and Hippel-Lindau syndrome suggests that the present tumor is not familiar cancer syndrome but a sporadic non-functional endocrine carcinoma. The incidence of non-functional endocrine carcinoma is less than 1% of all pancreatic neoplasms [8]. Clinically, presenting symptoms are non-specific, such as nausea and abdominal pain, in non-functional endocrine tumor [1-3]. The present case was characterized by abdominal pain. This contrasts with functional endocrine tumor such as insulinoma, gastrinoma, glucagonoma, PPoma and VIPoma, which shows specific hormone-related paraneoplasmic syndrome [1-3]. The present case lacked this syndrome. The age of the patients with pancreatic endocrine tumor ranges from 40 to 60 ages, the mean being 58 years. The present case was 67 years. Male to female ratio is 1:1 [1-3]. The treatment is operation. Adjuvant chemotherapy and radiation may be useful [1-3]. The prognosis of pancreatic endocrine tumors depends on tumor size, histology, and tumor stages, but the 5-year survival after pancreatic resection is 65% and 10-year survival is 45% [8]. The present case was alive at the 36th month follow-up after operation.

The present case was characterized by abdominal pain. This contrasts with functional endocrine tumor such as insulinoma, gastrinoma, glucagonoma, PPoma and VIPoma, which shows specific hormone-related paraneoplasmic syndrome [1-3]. The present case lacked this syndrome. The age of the patients with pancreatic endocrine tumor ranges from 40 to 60 ages, the mean being 58 years. The present case was 67 years. Male to female ratio is 1:1 [1-3]. The treatment is operation. Adjuvant chemotherapy and radiation may be useful [1-3]. The prognosis of pancreatic endocrine tumors depends on tumor size, histology, and tumor stages, but the 5-year survival after pancreatic resection is 65% and 10-year survival is 45% [8]. The present case was alive at the 36th month follow-up after operation. The pathogenesis of pancreatic endocrine tumors is unknown. Accumulating studies have suggested that pancreatic endocrine tumors are derived from ductal cells or ductal stem cells [8-10]. However, in MEN type I, the pancreatic endocrine tumors arise from islets of Langerhans [1, 2]. Much more studies are required to determine the original cells of pancreatic endocrine tumors. Conflicts of Interest The author has no conflict of interest.

Introduction Acute pancreatitis (AP) is an inflammatory disease with increasing incidence worldwide. The development of systemic inflammatory response syndrome (SIRS) is one of leading events responsible for the mortality of AP. SIRS results from the excessive release of inflammatory mediators from the local tissues and results in the systemic amplification of AP, which may ultimately cause multiple organ system failure (MOF) within 24-72 hours [1-3]. Many of the systemic features of severe acute pancreatitis can be attributed to the release of proteolytic enzymes, cytotoxic and inflammatory substances, reactive oxygen species, cytokines and other mediators into the circulation and explosive activation of the systemic inflammatory response [4]. Oxidative stress is emerging as the pivotal effector of acinar cell injury in experimental AP, irrespective of the initiating agent or its route of attack [5]. Different oxygen radical species induce severe acinar cell damage in a dose and time dependent manner, which suggests that generation of reactive oxygen species (ROS) may be crucial for initiating the pathophysiologic changes of AP [6].

n experimental AP, irrespective of the initiating agent or its route of attack [5]. Different oxygen radical species induce severe acinar cell damage in a dose and time dependent manner, which suggests that generation of reactive oxygen species (ROS) may be crucial for initiating the pathophysiologic changes of AP [6]. It is well known that antioxidants are potent scavengers of free radicals and serve as inhibitors of oxidant stress related pathologies. A large number of synthetic and natural antioxidants have been demonstrated to induce beneficial effects on human health and disease prevention. However, the structure-activity relationship, bioavailability and therapeutic efficacy of the antioxidants differ extensively. Proanthocyanidins consist of a group of polyhydroxyl-flavan-3-ol (or flavan-3,4-diol) oligomers and polymers linked by carbon-carbon bonds between flavanol subunits [7]. They are the most abundant natural phenolic components [8, 9], including phenoldienones, epicatechin, epigallocatechin, epigallocatechin gallate, ferulic acid, caffeic acid, p-coumaric acid, kaempferol, quercetin, and myricetin derived from common dietary foods such as grapes, cranberries and almonds, as well as chocolate and cacao beans [10, 11]. These compounds have been reported to possess a broad spectrum of biological, pharmacological and therapeutic activities against free radicals and oxidative stress both in vitro and in vivo [10-13]. Previous studies have shown that proanthocyanidins provide significant protection against free radicals induced lipid peroxidation and DNA fragmentation in liver and brain tissue [13] and provided better protection than vitamin C, vitamin E, and b-carotene [10].

s and oxidative stress both in vitro and in vivo [10-13]. Previous studies have shown that proanthocyanidins provide significant protection against free radicals induced lipid peroxidation and DNA fragmentation in liver and brain tissue [13] and provided better protection than vitamin C, vitamin E, and b-carotene [10]. Despite the considerable works done on proanthocyanidin against free radical associated tissue injury, its effect and role in the acute pancreatitis remain to be elucidated. In the present study, therefore, we investigated the protective effect of proanthocyanidin against cerulein-induced pancreatitis and oxidative injury in rats. Materials and Methods Animals Sprague–Dawley rats of either sex (200 - 250 g) were kept in a room at a constant temperature 22 ± 1 °C with 12 h light/dark cycles and fed standard pellet chow and water ad libitum. All experimental protocols were approved by the Marmara University School of Medicine Animal Care and Use Committee.

and Methods Animals Sprague–Dawley rats of either sex (200 - 250 g) were kept in a room at a constant temperature 22 ± 1 °C with 12 h light/dark cycles and fed standard pellet chow and water ad libitum. All experimental protocols were approved by the Marmara University School of Medicine Animal Care and Use Committee. Experimental protocol Acute pancreatitis was induced by 4 subcutaneous injections of 20 µg/kg body weight of cerulein (Sigma, St. Louis, MO, USA) at hourly intervals within 4 hours. Control animals received isotonic saline. Proanthocyanidin was administered orally at a dose of 100 mg/kg per rat 15 min before first cerulein injection. The dose of proanthocyanidin was previously shown as an effective anti-inflammatory dose [14]. Six hours after cerulein or saline injections, the animals were killed by decapitation. Trunk blood was collected for the assessment of amylase, lipase, TNF-α, IL-1β. In order to evaluate the presence of oxidant injury in the pancreas tissue, samples were taken and stored at -80 °C for the determination of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) and Na+-K+-ATPase activities. Formation of reactive oxygen species in the tissue samples was monitored by using chemiluminescence (CL) technique with luminol and lucigenin probes. For histological analysis, samples of the tissues were fixed in 10% (v/v) buffered formaldehyde and prepared for routine paraffin embedding. Tissue sections (6 µm) were stained with hematoxylin and eosin and examined under a light microscope (Olympus-BH-2). An experienced histologist who was unaware of the treatment conditions performed the histological assessments.

s were fixed in 10% (v/v) buffered formaldehyde and prepared for routine paraffin embedding. Tissue sections (6 µm) were stained with hematoxylin and eosin and examined under a light microscope (Olympus-BH-2). An experienced histologist who was unaware of the treatment conditions performed the histological assessments. Biochemical analysis Plasma amylase and lipase levels were determined spectrophotometrically using an automated analyser (Olympus AU 600, Diamond Diagnostics, Holliston, MA) while tumor necrosis factor-alpha (TNF-α) and interleukin IL-1β were quantified according to the manufacturer’s instructions and guidelines using enzyme-linked immunosorbent assay (ELISA) kits (Biosource International, Nivelles, Belgium). These particular assay kits were selected because of their high degree of sensitivity, specificity, inter- and intra-assay precision and small amount of plasma sample required for conducting the assay.

ions and guidelines using enzyme-linked immunosorbent assay (ELISA) kits (Biosource International, Nivelles, Belgium). These particular assay kits were selected because of their high degree of sensitivity, specificity, inter- and intra-assay precision and small amount of plasma sample required for conducting the assay. Chemiluminescence (CL) assay To assess the contribution of reactive oxygen species in cerulein-induced pancreatic damage, luminol and lucigenin chemiluminescences were measured as indicators of radical formation. Measurements were made at room temperature using Junior LB 9509 luminometer (EG&G Berthold, Germany). Specimens were put into vials containing PBS-HEPES buffer (0.5 M PBS containing 20 mM HEPES, pH 7.2). ROS were quantified after addition of enhancers, lucigenin or luminal, for a final concentration of 0.2 mM. Luminol detects a group of reactive species, i.e. .OH, H2O2, HOCl radicals, while lucigenin is selective for O-2 [15, 16]. Counts were obtained at 1 min intervals and the results were given as the area under curve (AUC) for a counting period of 5 min. Counts was corrected for wet tissue weight and expressed as relative light units (rlu/mg tissue) [17].

e species, i.e. .OH, H2O2, HOCl radicals, while lucigenin is selective for O-2 [15, 16]. Counts were obtained at 1 min intervals and the results were given as the area under curve (AUC) for a counting period of 5 min. Counts was corrected for wet tissue weight and expressed as relative light units (rlu/mg tissue) [17]. Measurement of pancreatic malondialdehyde and glutathione levels Tissue samples were homogenized with ice-cold 150 mM KCl for the determination of MDA and GSH levels. The MDA levels were assayed for the products of lipid peroxidation by monitoring thiobarbituric acid reactive substance formation as described previously [18]. Lipid peroxidation was expressed in terms of MDA equivalents using an extinction coefficient of 1.56 x 105 M–1 cm –1 and results were expressed as nmol MDA/g tissue. GSH measurements were performed using a modification of the Ellman procedure [19]. Briefly, after centrifuged at 1200 g for 10 min, 0.5 ml of supernatant was added to 2 ml of 0.3 mol/l Na2HPO4.2H2O solution. A 0.05 ml solution of 10 mM dithiobisnitrobenzoate (disolved in 1% sodium citrate) was added and the absorbance at 412 nm was measured immediately after mixing. GSH levels were calculated using an extinction coefficient of 1.36 x 104 M–1 cm –1. Results were expressed in µmol GSH/g tissue.

3 mol/l Na2HPO4.2H2O solution. A 0.05 ml solution of 10 mM dithiobisnitrobenzoate (disolved in 1% sodium citrate) was added and the absorbance at 412 nm was measured immediately after mixing. GSH levels were calculated using an extinction coefficient of 1.36 x 104 M–1 cm –1. Results were expressed in µmol GSH/g tissue. Measurement of pancreatic myeloperoxidase activity Myeloperoxidase (MPO) is an enzyme that is found predominantly in the azurophilic granules of polymorphonuclear leukocytes (PMN). Tissue MPO activity is frequently utilized to estimate tissue PMN accumulation in inflamed tissues and correlates significantly with the number of PMN determined histochemically in tissues [20]. MPO activity was measured in tissues in a procedure similar to that documented by Hillegass et al [21]. Tissue samples were homogenized in 50 mM potassium phosphate buffer (PB, pH 6.0), and centrifuged at 41400 g (10 min); pellets were suspended in 50 mM PB containing 0.5 % hexadecyltrimethylammonium bromide (HETAB). After three freeze and thaw cycles, with sonication between cycles, the samples were centrifuged at 41400 g for 10 min. Aliquots (0.3 ml) were added to 2.3 ml of reaction mixture containing 50 mM PB, o-dianisidine, and 20 mM H2O2 solution. One unit of enzyme activity was defined as the amount of MPO present that caused a change in absorbance measured at 460 nm for 3 min. MPO activity was expressed as U/g tissue.

d at 41400 g for 10 min. Aliquots (0.3 ml) were added to 2.3 ml of reaction mixture containing 50 mM PB, o-dianisidine, and 20 mM H2O2 solution. One unit of enzyme activity was defined as the amount of MPO present that caused a change in absorbance measured at 460 nm for 3 min. MPO activity was expressed as U/g tissue. Measurement of Na+- K+ ATPase activity Measurement of Na+, K+-ATPase activity is based on the measurement of inorganic phosphate that is formed from 3 mM disodium adenosine triphosphate added to the medium during the incubation period [22]. The medium was incubated in a 37 °C water bath for 5 min with a mixture of 100 mM NaCl, 5 mM KCl, 6 mM MgCl2, 0.1 mM EDTA, 30 mM Tris HCl (pH 7.4). Following the preincubation period, Na2ATP, at a final concentration of 3 mM, was added to each tube and incubated at 37 °C for 30 min. After the incubation, the tubes were placed in an ice bath, and the reaction was stopped. Subsequently, the level of inorganic phosphate was determined in a spectrophotometer (Shimadzu, Japan) at excitation wavelength of 690 nm. The specific activity of the enzyme was expressed as mmol Pi mg-1 protein h-1. The protein concentration of the supernatant was measured by the Lowry method [23].

ction was stopped. Subsequently, the level of inorganic phosphate was determined in a spectrophotometer (Shimadzu, Japan) at excitation wavelength of 690 nm. The specific activity of the enzyme was expressed as mmol Pi mg-1 protein h-1. The protein concentration of the supernatant was measured by the Lowry method [23]. Histopathological evaluation of pancreatic damage For light microscopic analysis, samples from pancreas were fixed in 10% buffered formalin for 48 hours, dehydrated in ascending alcohol series and embedded in paraffin wax. Approximately 5 mm thick sections were stained with hematoxylin-eosin (H&E) for general morphology. Histological assessments were made with a photomicroscope (Olympus BX 51, Tokyo) by an experienced histologist who was unaware of the experimental groups. Statistics Statistical analysis was carried out using GraphPad Prism 3.0 (GraphPad Software, San Diego; CA; USA). All data were expressed as means ± SEM. Groups of data were compared with an analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests. Values of p < 0.05 were regarded as significant.

Histopathological evaluation of pancreatic damage For light microscopic analysis, samples from pancreas were fixed in 10% buffered formalin for 48 hours, dehydrated in ascending alcohol series and embedded in paraffin wax. Approximately 5 mm thick sections were stained with hematoxylin-eosin (H&E) for general morphology. Histological assessments were made with a photomicroscope (Olympus BX 51, Tokyo) by an experienced histologist who was unaware of the experimental groups. Statistics Statistical analysis was carried out using GraphPad Prism 3.0 (GraphPad Software, San Diego; CA; USA). All data were expressed as means ± SEM. Groups of data were compared with an analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests. Values of p < 0.05 were regarded as significant. Results As shown in Table 1, plasma amylase and lipase levels in the pancreatitis group were found to be significantly higher than those in control rats (p < 0.001). When proanthocyanidin was administered before cerulein injection, these elevations in serum amylase and lipase levels were significantly depressed (p < 0.01). Plasma levels of pro-inflammatory cytokines (TNF-α, IL-1β) in the pancreatitis group were significantly higher (p < 0.001) than that of the control group, while treatment of proanthocyanidin abolished these elevations significantly (p < 0.01).

in serum amylase and lipase levels were significantly depressed (p < 0.01). Plasma levels of pro-inflammatory cytokines (TNF-α, IL-1β) in the pancreatitis group were significantly higher (p < 0.001) than that of the control group, while treatment of proanthocyanidin abolished these elevations significantly (p < 0.01). Table 1 The plasma amylase, lipase, tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels in sham control group or cerulein-induced acute pancreatitis groups treated with either saline or proanthocyanidine (100 mg/kg). Each group consists of 6 animals. Control Pancreatitis Saline-treated Proanthocyanidin-treated Amylase (U/L) 646.03 ± 46.2 1202.82 ± 67.3 *** 666.80 ± 53.7 ++ Lipase (U/L) 114.92 ± 23.2 393.37 ± 38.5 *** 163.13 ± 26.7 ++ TNF-α (pg/ml) 5.37 ± 0.91 40.58 ± 8.54 *** 15.30 ± 2.76 ++ IL-1β (pg/ml) 9.52 ± 1.69 52.07 ± 5.99 *** 23.28 ± 4.99 ++ Data are the mean ± SEM of six animals. ***P < 0.001 compared to control group; ++P < 0.01, compared with the saline-treated pancreatitis group. Chemiluminescence levels in the pancreatic samples detected by both luminol and lucigenin probes showed significant increases in the vehicle-treated pancreatitis group as compared to the CL levels of the control group (p < 0.01-0.001; Fig. 1a and b). On the other hand, proanthocyanidin treatment in the pancreatitis group abolished the pancreatitis-induced increases in both lucigenin- and luminol-detected CL (p < 0.05-0.01).

nificant increases in the vehicle-treated pancreatitis group as compared to the CL levels of the control group (p < 0.01-0.001; Fig. 1a and b). On the other hand, proanthocyanidin treatment in the pancreatitis group abolished the pancreatitis-induced increases in both lucigenin- and luminol-detected CL (p < 0.05-0.01). Figure 1 (a) Luminol and (b) lucigenin chemiluminescence (CL) in the pancreatic tissues of control, saline-treated pancreatitis and proanthocyanidin-treated pancreatitis groups. Each group consists of 6 animals. rlu: relative light units. ** p < 0.01, *** p < 0.001, compared to the control group. +p < 0.05, ++p < 0.01, compared to saline-treated pancreatitis group. In accordance with these findings, levels of the major cellular antioxidant GSH in the vehicle-treated pancreatitis group was depleted (p < 0.01); however, in the proanthocyanidin treated pancreatitis group, depleted GSH stores were partially replenished with this antioxidant proanthocyanidin treatment (p < 0.01; Fig. 2 a). The MDA levels, measured as a major degradation product of lipid peroxidation in the pancreatic tissue, were found to be significantly higher in the pancreatitis group (p < 0.05) as compared to those of the control group, while treatment with proanthocyanidin abolished these elevations (p < 0.05; Fig. 2b).

; Fig. 2 a). The MDA levels, measured as a major degradation product of lipid peroxidation in the pancreatic tissue, were found to be significantly higher in the pancreatitis group (p < 0.05) as compared to those of the control group, while treatment with proanthocyanidin abolished these elevations (p < 0.05; Fig. 2b). Figure 2 (a) Glutathione (GSH) level and (b) malondialdehyde (MDA) level in the pancreatic tissues of control, saline-treated pancreatitis and proanthocyanidin-treated pancreatitis groups. Each group consists of 6 animals. *p < 0.05, **p < 0.01, compared to the control group. +p < 0.01, ++p < 0.001, compared to saline-treated pancreatitis group. Myeloperoxidase activity, which is accepted as an indicator of neutrophil infiltration, was significantly higher in the pancreatic tissue of the pancreatitis group treated with vehicle (p<0.01) than that of the control group (Fig. 3 a). On the other hand, proanthocyanidin treatment in the pancreatitis group significantly decreased pancreatic MPO level (p<0.05) back to the levels of the control group. The activity of Na+-K+ ATPase, indicating the functional transport capacity of the pancreatic cells, was found to be significantly decreased in the pancreatitis group as compared with control group (p < 0.01); however, proanthocyanidin treatment significantly reduced the cerulein-induced decrease in pancreatic Na+-K+ ATPase activity (p<0.05; Fig. 3b).

dicating the functional transport capacity of the pancreatic cells, was found to be significantly decreased in the pancreatitis group as compared with control group (p < 0.01); however, proanthocyanidin treatment significantly reduced the cerulein-induced decrease in pancreatic Na+-K+ ATPase activity (p<0.05; Fig. 3b). Figure 3 (a) Myeloperoxidase activity and (b) Na+-K+ ATPase activity in the pancreatic tissues of control, saline-treated pancreatitis and proanthocyanidin-treated pancreatitis groups. Each group consists of 6 animals. **p < 0.01, compared to the control group. +p < 0.05, compared to saline-treated pancreatitis group. Control pancreas tissues demonstrated a regular morphology with acinar structures and Langerhans islets (Fig. 4a).Cerulein treated tissues showed severe degeneration in acinar structures with thyroidization of acini and overall tissue vacuolization (Fig. 4b. In proanthocyanidin treated group, regeneration of acinar morphology and the loss of vacuolar structures were prominent (Fig. 4c), whereas the cytoplasm of acinar cells were still vacuolated. Figure 4 (A) Control group, regular acini (arrow) with Langerhans islets (**). (B) Cerulein treated group, severe degeneration of acini which shows thyroidization (arrowheads) with overall tissue vacuolization (arrow), note the partially intact acini (*), (C) Proanthocyanidin treated group, regenerated acini (arrow), loss of tissue vacuoles, cytoplasmic vacuolization (inset-arrowhead). HE, X200, insets X400.

treated group, severe degeneration of acini which shows thyroidization (arrowheads) with overall tissue vacuolization (arrow), note the partially intact acini (*), (C) Proanthocyanidin treated group, regenerated acini (arrow), loss of tissue vacuoles, cytoplasmic vacuolization (inset-arrowhead). HE, X200, insets X400. Discussion One of the animal models of AP is that induced by supramaximal secretagogue stimulation by the trophic agent cerulein, a cholecystokinin analogue. Cerulein can stimulate the acinar cells to synthesize large amounts of digestive zymogens and pancreatic fluid, resulting in oedematous pancreatitis characterized by interstitial oedema, leukocyte infiltration and the vacuolization of acinar cells [4, 24, 25]. As assessed by both histological and biochemical parameters, the results of the present study demonstrated that cerulein caused oxidative injury of pancreatic tissues while proanthocyanidin treatment attenuates the severity of oxidative pancreatic damage along with concomitant reductions in the serum pro-inflammatory cytokines, suggesting that proanthocyanidin has a potent anti-inflammatory and anti-oxidant effect on the inflamed pancreatic tissue.

ative injury of pancreatic tissues while proanthocyanidin treatment attenuates the severity of oxidative pancreatic damage along with concomitant reductions in the serum pro-inflammatory cytokines, suggesting that proanthocyanidin has a potent anti-inflammatory and anti-oxidant effect on the inflamed pancreatic tissue. Although the pathophysiology is not fully understood, some of the early events of AP have been characterized by a dysregulation of the production and secretion of digestive enzymes, particularly, the inhibition of pancreatic secretion and an elevation in their serum levels, the death of acinar cells, and an infiltration of inflammatory cells into the pancreas [26-29]. Indeed, in our study, the amylase and lipase blood levels, indicators of the severity of acute pancreatitis [30], were increased following cerulein injection while, proanthocyanidin treatment reduced the levels of these enzymes.

death of acinar cells, and an infiltration of inflammatory cells into the pancreas [26-29]. Indeed, in our study, the amylase and lipase blood levels, indicators of the severity of acute pancreatitis [30], were increased following cerulein injection while, proanthocyanidin treatment reduced the levels of these enzymes. The injured acinar cells release cytokines that attract neutrophils, activate platelets and the complement system. Indirectly, they act on the arachidonic acid cascade by increasing the production of thromboxane, which lowers tissue circulation by its potent platelet-aggregating and vasoconstricting effects, and by enhancing the production of leukotriene B4, which promotes the activation of leukocytes and discharges of lysosomal enzymes [31]. It is well known that severe form of acute pancreatitis is characterized by the development of systemic inflammatory response syndrome, which is reported to result in high mortality rates [32]. It has been suggested that pro-inflammatory cytokines, such as TNF-α and IL-1β are upregulated during pancreatitis [33]. These cytokines play important roles in the induction of PMN activation and infiltration and induce many local and systemic manifestations of acute pancreatitis. Recently, it has been shown that neutrophil accumulation and the cytokine content, including TNF-α and IL-1β, were increased in pancreatic injury [34]. In accordance with these findings, in the present study, the plasma levels of the pro-inflammatory cytokines TNF-α and IL-1β were significantly elevated due to cerulean injection while proanthocyanidin treatment reduced the levels of these inflammatory mediators and protected the pancreatic tissue against cerulein-induced oxidative injury.

ese findings, in the present study, the plasma levels of the pro-inflammatory cytokines TNF-α and IL-1β were significantly elevated due to cerulean injection while proanthocyanidin treatment reduced the levels of these inflammatory mediators and protected the pancreatic tissue against cerulein-induced oxidative injury. Reactive oxygen metabolites (ROM) are involved in the development of tissue injury in pancreatitis, as well as in many inflammatory diseases [35]. In the current study, we investigated the free radical generation in the pancreatic tissue using chemiluminescence, a simple and reproducible technique for demonstrating the generation of oxidants in tissue. Chemiluminescence is a general assay for the production of reactive oxygen species, while cytochrome C reduction is a specific assay for superoxide anion. The luminol probe used in this technique detects H2O2, OH-, hypochlorite, peroxynitrite and lipid peroxyl radicals, while lucigenin is selective for superoxide radical [15-17]. Since increased CL values detected by both probes were significantly decreased with proanthocyanidin treatment, it seems likely that the protective effect of proanthocyanidin on the pancreatic tissue partly involves its direct antioxidant properties.

adicals, while lucigenin is selective for superoxide radical [15-17]. Since increased CL values detected by both probes were significantly decreased with proanthocyanidin treatment, it seems likely that the protective effect of proanthocyanidin on the pancreatic tissue partly involves its direct antioxidant properties. It is shown that acinar cells produce large amounts of ROS at early stage of AP in rats [36]. Highly reactive ROS directly attacks lipids, proteins in the biological membranes and cause their dysfunction [37]. Peroxidation of lipid membranes, disintegration of cytoskeleton and intracellular compartments by ROS might lead to the disturbances of digestive and liposomal enzymes transport within the acinar cell leading to this cell damage [30]. Degradation of polyunsaturated fatty acids in cell membranes by ROS results in the destruction of membranes and formation of thiobarbituric acid reactive substances, MDA or conjugated dienes as indicator of lipid peroxidation in the course of pancreatitis [38, 39]. In parallel to the CL results, the increased lipid peroxidation in the pancreatic tissue, as demonstrated by MDA assay, was also reversed with proanthocyanidin treatment, emphasizing the antioxidant action of proanthocyanidin on the deleterious consequences of ROMs in oxidative pancreatic injury. In accordance with our results, a previous report based on an experimental hepatoxicity and neurotoxicity model has demonstrated that proanthocyanidin treatment reduced lipid peroxidation and restored the transmembrane enzymes, thereby maintained the antioxidant status of the hepatic and brain cells [13].

eatic injury. In accordance with our results, a previous report based on an experimental hepatoxicity and neurotoxicity model has demonstrated that proanthocyanidin treatment reduced lipid peroxidation and restored the transmembrane enzymes, thereby maintained the antioxidant status of the hepatic and brain cells [13]. Proanthocyanidin is an anti-inflammatory and antioxidant molecule, acting as an ROS scavenger, it promotes synthesis and accumulation of glutathione precursors [40]. Glutathione, the physiologically most important nonprotein antioxidant, is a major contributor to the intracellular reducing environment and acts as a scavenger of hydrogen peroxide and other peroxides [41]. In accordance with the previous reports, which have reported a marked and early depletion of GSH pancreatic tissue in different models of experimental pancreatitis [38, 42], we also showed GSH depletion in the pancreatic tissue. GSH plays a role in acinar stimulus-secretion coupling [43], in the maintenance of the cytoskeleton [44], and in appropriate protein folding in the endoplasmic reticulum [45]. Thus, depletion of intracellular GSH may contribute to impaired zymogen granule transport and to premature activation of pancreatic proenzymes [42]. Restoration of intracellular glutathione levels has been shown to ameliorate cerulein-induced pancreatitis in rat, suggesting that generation of reactive oxygen radicals and consequent depletion of glutathione play pivotal roles in the initiation of acute pancreatitis [39, 46]. Similarly in the present study, following cerulein injection, GSH was depleted; however, proanthocyanidin treatment restoring tissue GSH reduced the severity of pancreatitis. Thus, it is likely that proanthocyanidin increases the total amount of intracellular GSH and has an important role in the maintenance of this crucial antioxidant.

present study, following cerulein injection, GSH was depleted; however, proanthocyanidin treatment restoring tissue GSH reduced the severity of pancreatitis. Thus, it is likely that proanthocyanidin increases the total amount of intracellular GSH and has an important role in the maintenance of this crucial antioxidant. As we have also confirmed histologically in our pancreatitis model, commonly observed changes of pancreatic morphology during pancreatitis include various degrees of acinar cell damage, hemorrhage, and the recruitment of leukocytes into the damaged gland [47, 48]. MPO is an essential enzyme for normal neutrophil function, and when neutrophils are stimulated by various stimulants, MPO, as well as other tissue damaging substances, is released from the cells. Therefore, MPO is used as an index of tissue neutrophil infiltration [49]. In the present study, the cerulein-induced increase of MPO activity was significantly reduced by proanthocyanidin, suggesting that pancreatic oxidative damage involves the interaction of neutrophils, and the protective effect of proanthocyanidin on the pancreas depends on blockade of neutrophil infiltration. As activation of neutrophils might lead to the generation of reactive oxygen metabolites, the reduction in tissue neutrophil accumulation may also result in reduced lipid peroxidation and attenuated tissue injury. Proanthocyanidin treatment markedly reduced the MPO activity. Bomser et al [50] have shown that proanthocyanidin treatment reduced neutrophil infiltration. However, an exact explanation of the effects of proanthocyanidin on neutrophil activation remains unknown.

reduced lipid peroxidation and attenuated tissue injury. Proanthocyanidin treatment markedly reduced the MPO activity. Bomser et al [50] have shown that proanthocyanidin treatment reduced neutrophil infiltration. However, an exact explanation of the effects of proanthocyanidin on neutrophil activation remains unknown. Proanthocyanidins, naturally occurring compounds widely available in fruits, vegetables, nuts, seeds, flowers and bark, are a group of polyphenolic bioflavonoids diverse in chemical structure, pharmacology and characteristics. Proanthocyanidins have been reported to exhibit a wide range of biological effects including antibacterial, antiviral, anti-inflammatory, antiallergic and vasodilatory actions [51-53]. Furthermore, proanthocyanidins have been reported to inhibit lipid peroxidation, platelet aggregation and capillary permeability and fragility and to modulate the activity of enzyme including cyclooxygenase and lipooxygenase [53]. Proanthocyanidins are believed to be nontoxic. If they are absorbed and biologically active in vivo, they may prevent free radical-mediated cytotoxicity and lipid peroxidation and protect low-density lipoproteins from oxidation [13].

nd to modulate the activity of enzyme including cyclooxygenase and lipooxygenase [53]. Proanthocyanidins are believed to be nontoxic. If they are absorbed and biologically active in vivo, they may prevent free radical-mediated cytotoxicity and lipid peroxidation and protect low-density lipoproteins from oxidation [13]. Our results also indicate that cerulein impairs pancreatic Na+-K+-ATPase activity. The Na+-K+-ATPase, which is found exclusively in the pancreatic acini, plays a central role in pancreas electrolyte regulation and in the pathogenesis of pancreatic inflammation [54]. Since decreased Na+-K+-ATPase activity most likely reflects a diminished number of enzyme molecules due to a loss of Na+-K+-ATPase-containing mucosal cells [55], consequently, it also indicates severe mucosal inflammation and the loss of physiological function. Since proanthocyanidin treatment in the present study reversed cerulein-induced increment in MPO and reduction in Na+-K+-ATPase enzyme activity, it indicates that proanthocyanidin alleviates the pancreatic injury by preserving membrane structure.

cates severe mucosal inflammation and the loss of physiological function. Since proanthocyanidin treatment in the present study reversed cerulein-induced increment in MPO and reduction in Na+-K+-ATPase enzyme activity, it indicates that proanthocyanidin alleviates the pancreatic injury by preserving membrane structure. During the past years, several substances have been tested, with varying degrees of success, in experimental models of acute pancreatitis as an attempt to modify the natural history of the disease by either blocking or neutralizing one or more inflammatory mediators which are involved in the pathophysiology of the disease [4, 39]. On the basis of our data, proanthocyanidin, by preventing free radical damaging cascades, oxidant radical release and through its membrane stabilizing effects, supports the maintenance of pancreatic integrity against acute inflammatory processes. Furthermore, proanthocyanidin augments the level of the main intracellular antioxidant glutathione which preserves the total antioxidant capacity in the pancreas. In conclusion, the results of the present study suggest that proanthocyanidin may have utility in treating acute pancreatititis. Acknowledgements The authors declare no commercial associations or conflict of interests related to this article.

Introduction Patients with malignant tumors in descending duodenum, hepatopancreatic ampulla, pancreas and gastric antrum, and patients with post-operative relapsed cancers, usually have biliary obstructions in addition to gastric outlet obstruction. To those patients with old age, tumor invasion of the large vessels, distant metastasis, complex anatomical structures and intolerability to radical surgery, the palliative treatment modalities with minimal traumatic, fast recovery and satisfactory efficacy should be the priority options [1-6]. With the development of endoscopic techniques as well as the invention of intestinal stents via endoscopic biopsy channel (through the scope, TTS), the gastric outlet stenting becomes easy and convenient [7-9]. Percutaneous transhepatic biliary drainage (PTCD) with stenting in the treatment of obstructive jaundice has been widely used and has become the first choice for the palliative treatment of malignant obstructive jaundice [6-8]. In this study, we attempted to observe the palliative efficacy of the combined intestinal and biliary stenting on the malignant gastric outlet obstruction with common bile duct obstruction.

aundice has been widely used and has become the first choice for the palliative treatment of malignant obstructive jaundice [6-8]. In this study, we attempted to observe the palliative efficacy of the combined intestinal and biliary stenting on the malignant gastric outlet obstruction with common bile duct obstruction. Materials and Methods Patients All patients were from the Departments of Gastroenterology and General Surgery in Provincial Hospital Affiliated to Shandong University between January 2001 and September 2007. All patients had malignant gastric outlet obstruction and bile duct obstruction, these patients had one or more of the following, old age, weak condition, cardiopulmonary diseases, tumor invasion of the large vessel, or distant metastasis. These patients could not tolerate or refused radical surgery. Patients with severe bleeding tendency, severe ascites, or any signs intolerable to endoscopy were excluded from this treatment. A total of 32 cases were included in this study, 19 male and 13 female, average age 61.3 years, ranging from 46 to 83 years. These patients included ten cases of duodenal cancer, 6 recurred gastric antrum cancer after operations, 6 periampullary cancer, 8 pancreatic cancer, 2 lower common bile duct cancer. All patients presented with vomiting, inability to eat or other gastrointestinal obstruction symptoms and jaundice, pruritus and other biliary obstruction symptoms, total bilirubin 85 - 613 µmol/L. Preoperatively, all patients were diagnosed to have malignant gastric outlet obstruction with stenosis length between 2 - 7 cm, confirmed by endoscopy and/or upper gastrointestinal diatrizoate contrast. In addition, the ultrasound, computed tomography (CT) and/or magnetic resonance cholangiopancreatography (MRCP) were performed to examine the intra- and extra-hepatic bile duct expansion including the common bile duct obstruction, the length of common bile duct obstruction was 1.5 - 4 cm.

testinal diatrizoate contrast. In addition, the ultrasound, computed tomography (CT) and/or magnetic resonance cholangiopancreatography (MRCP) were performed to examine the intra- and extra-hepatic bile duct expansion including the common bile duct obstruction, the length of common bile duct obstruction was 1.5 - 4 cm. Materials and instruments Intestinal stenting instruments: Olympus GIF-IT260 electronic endoscopy with a 3.2 mm in diameter biopsy channel; MTN-intestinal expandable stent (Nanjing Minimal Invasive Medical Technology Co., China) with inner diameter 20 mm; zebra guiding wires with diameter of 0.035 in, length 4 m; 5 F straight catheters or multi-purpose catheters; extra-lubricity wire with diameter of 0.035 in, length of 2.6 m. Biliary stenting instruments: The biliary micro-needle system; the interior and exterior drainage catheters with diameter of 8.5 F, length of 40 cm (Cook, Bloomington, USA); 6 F catheter sheath; extra-lubricity Paramisgurnus wire; 5 F multi-purpose catheter; dilating balloon with diameter of 0.8 - 1.0 cm, length of 3 - 4 cm; hard extra-lubricity wire; MTN-biliary expandable stent (Nanjing Minimal Invasive Medical Technology Co., China), with diameter 0.8 cm, length 5 - 8 cm. Stenting procedures Preoperative preparations included obtaining of consent from patients. Vitamin K1 and calcium gluconate were administered for three days prior to stenting.

Biliary stenting instruments: The biliary micro-needle system; the interior and exterior drainage catheters with diameter of 8.5 F, length of 40 cm (Cook, Bloomington, USA); 6 F catheter sheath; extra-lubricity Paramisgurnus wire; 5 F multi-purpose catheter; dilating balloon with diameter of 0.8 - 1.0 cm, length of 3 - 4 cm; hard extra-lubricity wire; MTN-biliary expandable stent (Nanjing Minimal Invasive Medical Technology Co., China), with diameter 0.8 cm, length 5 - 8 cm. Stenting procedures Preoperative preparations included obtaining of consent from patients. Vitamin K1 and calcium gluconate were administered for three days prior to stenting. During the intestinal stenting, the routine endoscopy procedure was carried out to aspirate the gastric contents and to observe the stenosis, 40 - 60 ml of 76% diatrizoate was injected via the biopsy channel to mark the range of stenosis, based on which the appropriate length of stent was selected. If the endoscopy could not pass the stenosis, a Zebra wire was inserted first, then the stents were placed under the guidance of the wire. After the stent placement, the endoscopy and X-ray were used to examine the location and expansion of the stents; if necessary, adjustment of stent position was performed under endoscopy. If two stents were needed in one patient, the distal one must be placed first, then the proximal one.

nder the guidance of the wire. After the stent placement, the endoscopy and X-ray were used to examine the location and expansion of the stents; if necessary, adjustment of stent position was performed under endoscopy. If two stents were needed in one patient, the distal one must be placed first, then the proximal one. During the biliary stent placement, we usually used the right axillary midline between 7 and 8 intercostal as the puncture point when patients were in supine position. After successful puncture, the percutaneous transhepatic cholangiography (PTC) was performed to observe the location and the length of obstruction, after expansion with the 6 F catheter sheath, a 5 F multipurpose catheter was placed at the proximal of the stenosis first, then advanced through the stenosis to reach the intestinal stent along the wire, cholangiography was performed in order to check the location and the extent of biliary stenosis. Afterwards, the wire was replaced with a hard wire, and a dilating balloon was inserted to expand the biliary stenosis and the mesh of the intestinal stent, the biliary stents then were inserted along the wire. After biliary stenting, the interior and exterior drainage catheters with diameter of 8.5 F, length of 40 cm were placed, with the distal end located in the duodenum, the proximal in the right hepatic duct or hepatic duct, and it was connected to the exterior drainage.

biliary stents then were inserted along the wire. After biliary stenting, the interior and exterior drainage catheters with diameter of 8.5 F, length of 40 cm were placed, with the distal end located in the duodenum, the proximal in the right hepatic duct or hepatic duct, and it was connected to the exterior drainage. After the stents placement, close observations were made to jaundice, vomiting, abdominal pain, abdominal distension, vomiting blood or melena. The liquid to semi-liquid diet was given 24 h after stenting; the volume and features of drainage were recorded. Biliary flushing with 80,000 units of gentamycin in 100 ml of 0.9% NaCl solution was performed daily for three days, then the exterior drainage was closed. If the interior drainage was patent, the exterior drainage catheter was removed. Three to 7 days after the stenting, oral contrast agent imaging was performed to observe the status of the intestinal stent patency. The hematology, liver function, bilirubin, blood chemistry were ananlyzed. If jaundice ameliorated significantly, and blood bilirubin decreased more than 50% of pre-stenting level, the patients could be discharged. Follow-up was made by telephone or clinic visit.

d to observe the status of the intestinal stent patency. The hematology, liver function, bilirubin, blood chemistry were ananlyzed. If jaundice ameliorated significantly, and blood bilirubin decreased more than 50% of pre-stenting level, the patients could be discharged. Follow-up was made by telephone or clinic visit. Stent patency evaluation Two weeks after stenting, if blood total bilirubin (TBil) decreased ≥ 50% and patient could tolerate semi-liquid diet, it was considered complete response; if TBil decresed less than 50% and patient tolerated only liquid diet, it was partial response; if TBil unchanged and patient could not tolerate liquid diet, it was non response; if TBil elevated and patient could not tolerate liquid diet, and the gastrointestinal decompression was still required, it was deterioration [1-9]. Statistical analysis SPSS 13.0 statistical program was used for analyzing the data, data was expressed in Mean ± SD, inter-group comparison used Student’s t test, the P value of less than 0.05 was considered significantly different.

Stent patency evaluation Two weeks after stenting, if blood total bilirubin (TBil) decreased ≥ 50% and patient could tolerate semi-liquid diet, it was considered complete response; if TBil decresed less than 50% and patient tolerated only liquid diet, it was partial response; if TBil unchanged and patient could not tolerate liquid diet, it was non response; if TBil elevated and patient could not tolerate liquid diet, and the gastrointestinal decompression was still required, it was deterioration [1-9]. Statistical analysis SPSS 13.0 statistical program was used for analyzing the data, data was expressed in Mean ± SD, inter-group comparison used Student’s t test, the P value of less than 0.05 was considered significantly different. Results Effecacy of stenting A total of 37 intestinal stents were placed in 32 patients, single stent was placed in 27 patients; in 5 patients, due to the long duodenal stenosis, double stents were placed in each patient. A total of 32 biliary stents were placed in 32 patients. The cholangiography 3 days after stenting proved patency of both intestinal and biliary stents, the success rate of combined intestinal and biliary stenting was 100%. Figure 1a shows the extrahepatic obstructive jaundice with biliary infection before stent placement, PTC revealed common bile duct lesions accompanied by duodenal stenosis. Figure 1b shows the biliary stenting following the intestinal stent was placed, PTCD was carried out to place the biliary stent. Figure 1c shows the biliary stent across the mesh of intestinal stent, the biliary and intestinal stents were both revealed patent. Figure 2 shows the relationship between biliary stent and intestinal stent.

the biliary stenting following the intestinal stent was placed, PTCD was carried out to place the biliary stent. Figure 1c shows the biliary stent across the mesh of intestinal stent, the biliary and intestinal stents were both revealed patent. Figure 2 shows the relationship between biliary stent and intestinal stent. Figure 1 Procedure of double stenting. (a) Duodenal stenosis combined with common bile duct lesions. The arrow indicates descending duodenal stenosis, the arrow head shows the irregular contrast deficiency of lower common bile duct, which might be invaded by tumors which led to the expansion of common bile duct above the stenosis; (b) Biliary stent placement, the arrow shows duodenal stent, the arrow head shows balloon dilatation of common bile duct stenosis and the mesh of intestinal stent; (c) Biliary stent placement, the arrow shows duodenal stent, the arrow head shows biliary stent. The cholangiography showed both were patent. Figure 2 The relationship of the intestinal and biliary stents. (a) anteroposterior; (b) lateral. Short-term efficacy The obstructive symptoms in all the 32 patients improved gradually after stenting, all the patients had complete response or partial response. One week after stenting, the jaundice, itching and other symptoms ameliorated. In 21 patients, the serum TBil and direct bilirubin (DBil) decreased more than 50% one week after stenting; in all the 32 patients, the alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase ( ALP), γ-glutamyltranspeptidase enzyme (γ-GT) decreased significantly (Table 1).

ms ameliorated. In 21 patients, the serum TBil and direct bilirubin (DBil) decreased more than 50% one week after stenting; in all the 32 patients, the alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase ( ALP), γ-glutamyltranspeptidase enzyme (γ-GT) decreased significantly (Table 1). Table 1 Serum bilirubin and liver function before and 2 weeks after stenting Pre-stenting Post-stenting (2 weeks) P value N 32 32 TBil (µmol/L) 276.54 ± 72.67 116.37 ± 48.42 < 0.01 DBil (µmol/L) 152.32 ± 42.64 65.70 ± 31.93 < 0.01 ALT (U/L) 109.22 ± 37.56 46.77 ± 33.92 < 0.01 AST (U/L) 72.38 ± 13.86 34.17 ± 18.20 < 0.01 ALP (U/L) 486.35 ± 129.67 117.56 ± 93.42 < 0.01 γ-GT (U/L) 603.22 ± 145.79 247.83 ± 63.07 < 0.01 Complications In some patients, minor hemorrhage occurred around the stent during intestinal stent placement, after flushing with saline and spraying hemostat drug locally, no further bleeding occurred. There was no gastrointestinal perforation observed. All 32 patients had upper abdominal pain, nausea or other discomforts immediately after stenting, these symptoms relieved or disappeared 3 – 5 days after. If the pain was marked without perforation, painkillers could be administered. After biliary stenting, serum amylase elevated transiently in 7 patients. One patient had acute pancreatitis after stenting, and was cured by conservative treatment. Three patients had drainage blockage or infection, these were cured by repeated flushing with antibiotics. Two patients had gastric outlet restenosis in the first month, one was caused by food blockade; another was caused by outgrowth of the tumor through the stent mesh which was relieved by re-endoscopic treatment. There were no biliary leak, abdominal bleeding, drainage catheter migration, stents migration and puncture tunnel bleeding.

ad gastric outlet restenosis in the first month, one was caused by food blockade; another was caused by outgrowth of the tumor through the stent mesh which was relieved by re-endoscopic treatment. There were no biliary leak, abdominal bleeding, drainage catheter migration, stents migration and puncture tunnel bleeding. Survival Twenty eight patients were followed up, 2 patients died within 2 months after stenting; 5 died 3-4 months after stenting; 3 died 5-6 months after stenting. The longest survival was 11 months, the average survival time was 164 days.

ad gastric outlet restenosis in the first month, one was caused by food blockade; another was caused by outgrowth of the tumor through the stent mesh which was relieved by re-endoscopic treatment. There were no biliary leak, abdominal bleeding, drainage catheter migration, stents migration and puncture tunnel bleeding. Survival Twenty eight patients were followed up, 2 patients died within 2 months after stenting; 5 died 3-4 months after stenting; 3 died 5-6 months after stenting. The longest survival was 11 months, the average survival time was 164 days. Discussion Patients with malignant gastric outlet obstruction combining with bile duct obstruction may undergo palliative surgeries, such as choledochojejunostomy, gastrojejunostomy, these modalities are traumatic and have complications, therefore, they are now gradually replaced by less traumatic methods such as ERCP, PTCD, intestinal stenting [10-17]. However, some patients with lesions of antral and/or duodenal stenosis or tumor invasion of the papilla are impossible to be performed ERCP, so the PTCD plus metal expandable stent implantation could overcome these shortcomings [5-7]. The only common bile duct drainage is not effective either because of duodenum stenosis which always leads to poor drainage or because of bile reflux to the stomach that would not achieve physiological effects. In such patients, due to the biliary and gastric outlet obstruction occur simultaneously or one after another, the first is to clear the intestinal tract, then to place the intestinal tract stent, the biliary stenting can be performed at the same time as or after the intestinal stenting. If intestinal stent is needed because of gastric outlet obstruction after biliary stent placement, though the two stents may overlap or cover the papilla opening, due to the large mesh of intestinal stent, it will not affect the biliary stent drainage [1, 2, 8, 9, 11, 18].

same time as or after the intestinal stenting. If intestinal stent is needed because of gastric outlet obstruction after biliary stent placement, though the two stents may overlap or cover the papilla opening, due to the large mesh of intestinal stent, it will not affect the biliary stent drainage [1, 2, 8, 9, 11, 18]. Intestinal self-expandable metal stent implantation is one of the effective ways to treat malignant gastric outlet obstruction [9, 11, 16, 19]. Due to the long route from the mouth to gastric outlet, the stent placement is difficult. In1996, Feretis et al adopted an extended catheter to prevent the delivery system from rolling in the stomach. In 1997, Scott Mackie adopted the stiff wire and a special delivery system which greatly improved the success of stent placement [5, 6, 10, 15]. In 2003, the invention of TTS intestinal stent makes stenting easier and time saving, which is gradually accepted in clinical work [7-9].

y system from rolling in the stomach. In 1997, Scott Mackie adopted the stiff wire and a special delivery system which greatly improved the success of stent placement [5, 6, 10, 15]. In 2003, the invention of TTS intestinal stent makes stenting easier and time saving, which is gradually accepted in clinical work [7-9]. In this study, by combining fluoroscopy with endoscopy, the intestinal stenting was all successful in the first attempt. No bleeding, perforation, or other complications were observed, indicating that this method is safe and efficacious. The adequate preoperative preparation is an important factor for the success of stening, such as gastrointestinal decompression, gastric lavage and nutrition support. The injection of the contrast agent through the endoscopic biopsy channel to mark the stenosis is important for choosing appropriate stent. If the endodcopy could go cross the stenosis, the stent could be released under the surveillance of endoscopy; if not, we advanced the catheter and extra lubricity wire first, then X-ray was used to measure the range of the stenosis and to mark the both ends. The catheter must be guided by the wire and advanced to the ends as far as possible, then the hard extra lubricity wire was used in the upper jejunum in order to place the stent successfully, because the hard extra lubricity wire can not fold in the gastrointestinal tract [20-25].

he stenosis and to mark the both ends. The catheter must be guided by the wire and advanced to the ends as far as possible, then the hard extra lubricity wire was used in the upper jejunum in order to place the stent successfully, because the hard extra lubricity wire can not fold in the gastrointestinal tract [20-25]. The PTCD with stenting placement is a frequently used method for bilary reconstruction and alleviation of jaundice. In 1974, PTCD was firstly used by Molnar and Stocknm, in the late 1980s, the stent placement started in some countries. Due to the large mesh and unique weaving of the intestinal stents, the biliary stents can cross it. For the lower common bile duct obstruction, the PTCD plus stenting is simple and convenient, especially for those patients with intestinal obstruction. With the ongoing development of instruments and improved PTCD technology, its successful rate is currently close to 100% [10-12]. In intestinal obstruction, for restoration of physiological functions, the adequate interior and exterior biliary drainage are necessary. The PTCD plus stenting should be recommended for alleviating jaundice in cancer patients with gastrointestinal tract reconstruction [1, 2, 8, 19, 20, 26].

ntly close to 100% [10-12]. In intestinal obstruction, for restoration of physiological functions, the adequate interior and exterior biliary drainage are necessary. The PTCD plus stenting should be recommended for alleviating jaundice in cancer patients with gastrointestinal tract reconstruction [1, 2, 8, 19, 20, 26]. The use of the grid-type stents makes it easier to place the two stents at the same time, this avoids the suppression of the pancreatic duct openings which may result in pancreatic obstructions and pancreatitis. The patients usually feel painful when the intestinal stent mesh is being expanded, when this occur, the painkillers might be administered. The biliary stent should not exceed half of the intestinal stent lumen in order not to affect the patency of the intestinal stent. The indications of stenting placement are very important, these include: (1) the preoperative ultrasound, CT, or MRI confirmed distant metastasis and, radical surgery is impossible; (2) the patients general condition is severely deteriorated, or with severe heart or lung diseases, laparotomy surgery is impossible; (3) patients or their families refuse open surgery; (4) the malignant recurrence surrounding the anastomosis after operation, and the adjacent organs are invaded. The contraindications of the combined stent placement include massive ascites; multiple obstructions of the intestinal tract; and severe bleeding tendency.

; (3) patients or their families refuse open surgery; (4) the malignant recurrence surrounding the anastomosis after operation, and the adjacent organs are invaded. The contraindications of the combined stent placement include massive ascites; multiple obstructions of the intestinal tract; and severe bleeding tendency. In the patients with malignant obstructive jaundice, if no effective biliary drainage is carried out, the survival time is usually around 70 days, and eventually the patients die of liver failure [11, 21, 25]. In this study, the survival time we observed was 164 days in average, which was significantly longer than those without biliary drainage in others’ reports. In our study, we observed a small number of patients, the overall efficacy need further evaluated. We used the palliative approach to reconstruct biliary duct, so the short-term effect was obvious, but with the tumor growth, invasion and suppression to adjacent organs, the quality of patient life and the survival time will be affected over time. Hence, the combined chemotherapy, radiotherapy and endoscopic therapy should be considered to prolong survival. In short, the combined intestinal and biliary Stenting is a safe, minimal traumatic, effective treatment for malignant gastric outlet obstruction with common bile duct obstruction, with less complications, it is useful to improve the patient’s life quality and survival time. Acknowledgements The authors declare no conflict of interests related to this article.

Introduction Carcinoid tumors, also called neuroendocrine tumors (NET), are relatively rare in the digestive organs [1-8]. The incidence is reported to be less that 0.1% of all digestive organ tumors [1-3]. Carcinod tumors are potentially malignant tumor, but the malignant potential depends on tumor size and morphologies [3]. In general, carcinoid tumors less than 20 mm are benign, and those more than 20 mm have malignant potential [3]. The author herein reports a clinicopathology of 13 cases of carcinoid tumors obtained from 18,267 archival pathologic specimens of digestive organs. Materials and Methods The author retrospectively reviewed 18,267 pathological specimens of digestive organs pathologic specimens in the last 10 years in our pathology laboratory in search for carcinoid tumors. In carcinoid tumors, clinical and pathologic records were reviewed, and the pathologic slides were re-examined. An immunohistochemical study was performed using Dako Envision methods (Dako Corp. Glostrup, Denmark), as previously described [9, 10]. The antibododies used were anti-cytokeratin (AE1/3, Dako), anti-cytokeratin (polyclonal wide, Dako), carcinoembrionic antigen (polyclonal, Dako), chromorgranin (DAK-A3, Dako), synaptophysin (polyclonal, Dako), neuron-specific enolase (BBS/NC/VI-H14, Dako), CD56 (MOC-1, Dako), and glucagon (polyclonal, Dako). Results A total of 13 carcinoid tumors (0.07%) were found among the 18,267 digestive organ’s pathologic specimens in the last 10 years in our pathology laboratory.

An immunohistochemical study was performed using Dako Envision methods (Dako Corp. Glostrup, Denmark), as previously described [9, 10]. The antibododies used were anti-cytokeratin (AE1/3, Dako), anti-cytokeratin (polyclonal wide, Dako), carcinoembrionic antigen (polyclonal, Dako), chromorgranin (DAK-A3, Dako), synaptophysin (polyclonal, Dako), neuron-specific enolase (BBS/NC/VI-H14, Dako), CD56 (MOC-1, Dako), and glucagon (polyclonal, Dako). Results A total of 13 carcinoid tumors (0.07%) were found among the 18,267 digestive organ’s pathologic specimens in the last 10 years in our pathology laboratory. The carcinoid tumor locations were rectum in 9 cases, duodenum in 2 cases, liver in 1 case, and stomach in 1 case. The age of the patients ranged from 52 to 82 years with a mean of 63 years. Male to female ratio was 7 : 6. The presenting symptoms were abnormal pain in 3 cases and asymptomatic in 10 cases. None of the cases showed carcinoid syndrome. The diameter ranged from 5 mm to 25 mm in gastrointestinal carcinoids, and 60 mm in the hepatic carcinoid. The treatment was endoscopic mucosal resection in 10 cases and surgical resection in 3 cases. The outcome is good except for hepatic atypical carcinoid which showed metastases and died of systemic metastasis.

ome. The diameter ranged from 5 mm to 25 mm in gastrointestinal carcinoids, and 60 mm in the hepatic carcinoid. The treatment was endoscopic mucosal resection in 10 cases and surgical resection in 3 cases. The outcome is good except for hepatic atypical carcinoid which showed metastases and died of systemic metastasis. Histologically, the carcinoids were located in the submucosa (Figure 1A), and were grossly identified as submucosal tumor. Of the 13 carcinoids, 12 carcinoid tumors were typical carcinoids (Figures 1B, 1C and 1D). The liver carcinoid was an atypical carcinoid composed of atypical endocrine cells (Figure 1E). Organoid pattern was present in 12 cases. Trabecular arrangement, ribbon arrangement, rosette formation, and pseudoglandular arrangement were recognized in 12 cases, in 8 cases, in 7 cases, and in 5 cases, respectively. Immunohistochemically, tumor cells were positive for cytokeratin in 7 cases and negative for carcinoembryonic antigen in all cases. The tumor cells were positive for at least one of pan-neuroendocrine markers including chromogranin (Figure 2A), synaptophysin (Figure 2B), neuron-specific enolase (Figure 2C), CD56 (Figure 2D), and glucagon (Figure 2E). Of these, synaptophysin was positive in 11/13 (85%), neuron-specific enolase 10/13 (80%), chromogranin 8/13 (62%), CD56 6/13 (46%), and glucagon 4/13 (31%).

roendocrine markers including chromogranin (Figure 2A), synaptophysin (Figure 2B), neuron-specific enolase (Figure 2C), CD56 (Figure 2D), and glucagon (Figure 2E). Of these, synaptophysin was positive in 11/13 (85%), neuron-specific enolase 10/13 (80%), chromogranin 8/13 (62%), CD56 6/13 (46%), and glucagon 4/13 (31%). Figure 1 (a) Carcinoid is located in the submucosa. HE, x 40. (b) Carcinoid of the colon. HE, x 200. (c) Carcinoid of the duodenum. HE, x 200. (d) Carcinoid of the stomach. HE, x 200. (e) Atypical carcinoid or neuroendcrine carcinoma of the liver. HE, x 200. Figure 2 (a) Expression of chromogranin in carcinoid. Immunostaining, x 200. (b) Expression of synaptophysin in carcinoid. Immunostaining, x 200.(c) Expression of neuron-specific enolase in carcinoid. Immunostaining, x 200. (d) Expression of CD56 in carcinoid. Immunostaining, x 200. (e) Expression of glucagon in carcinoid. Immunostaining, x200. Discussion In the present series, only 13 carcinoids were found in the 18,267 pathologic specimens in the last 10 years in our pathology laboratory. The incidence was 0.07%. The incidence of carcinoids of digestive organs is reported to be less than 0.1% of all digestive organ tumors [1-3]. Therefore, the incidence of 0.07 % in the present series is compatible with other institutes [1-3]. These findings suggest that carcinoids of digestive organs are rare.

oratory. The incidence was 0.07%. The incidence of carcinoids of digestive organs is reported to be less than 0.1% of all digestive organ tumors [1-3]. Therefore, the incidence of 0.07 % in the present series is compatible with other institutes [1-3]. These findings suggest that carcinoids of digestive organs are rare. In the present series, the locations of carcinoids were rectum in 9 cases, duodenum in 2 cases, liver in 1 case, and stomach in 1 case. A study of larger series indicated that the frequency of carcinoids of digestive organs is highest in the colon, followed in order by ileum, rectum, appendix, stomach, duodenum, jejunum, pancreas, and liver. Carcinoid of the liver is very rare [11]. The locations of carcinoids in this study were somewhat different from other institutes. In the present series, the age of the patients ranged from 52 to 82 years with a mean of 63 years. Male to female ratio was 7 : 6. These findings are compatible with other institutes (1-8). The presenting symptoms were abnormal pain in 3 cases and asymptomatic in 10 cases in the present series. None of the cases showed carcinoid syndrome. In general, patients with carcinoids present with non-specific symptoms such as abdominal pain and nausea [3]. The presenting symptoms depend on locations and size of carcinoids [3]. The tumor size of the present study was small. Therefore, asymptomatic patients predominate in the present study.

wed carcinoid syndrome. In general, patients with carcinoids present with non-specific symptoms such as abdominal pain and nausea [3]. The presenting symptoms depend on locations and size of carcinoids [3]. The tumor size of the present study was small. Therefore, asymptomatic patients predominate in the present study. The prognosis of the present series is good except for the hepatic atypical carcinoid. In other institutes, five-year survival of carcinoids depends on tumor stage and tumor biologic behavior; it ranges from 60% to 98% [3]. In the present series, the treatment was endoscopic mucosal resection in 10 cases and surgical resection in 3 cases. It seems that the best choice of treatment is endoscopic resection in small carcinoids. In larger carcinoids, surgical operation may be effective. Histopathologically, all cases but the hepatic atypical carcinoid showed typical features of carcinoids, such as trabecular, ribbon, rosette, and pseudoglandular arrangements, in the present series. The liver carcinoid of the present series may be a neuroendocrine carcinoma rather than atypical carcinoid.

In the present series, the treatment was endoscopic mucosal resection in 10 cases and surgical resection in 3 cases. It seems that the best choice of treatment is endoscopic resection in small carcinoids. In larger carcinoids, surgical operation may be effective. Histopathologically, all cases but the hepatic atypical carcinoid showed typical features of carcinoids, such as trabecular, ribbon, rosette, and pseudoglandular arrangements, in the present series. The liver carcinoid of the present series may be a neuroendocrine carcinoma rather than atypical carcinoid. Immunohistochemically, tumor cells were positive for cytokeratin in 7 cases and negative for carcinoembryonic antigen in all cases in the present series, suggesting that carcinoid tumor cells may be negative for cytokeratin. In the present series, the tumor cells were positive for at least one of pan-neuroendocrine markers including chromogran, synaptophysin, neuron-specific enolase, CD56, and glucagon. Of these, synaptophysin was positive in 11/13 (85%), neuron specific enolase 10/13 (80%), chromogranin 8/13 (62%), CD56 6/13 (46%), and glucagon 4/13 (31%). These findings suggest that these pan-neuroendocrine markers are useful diagnostic tools in the carcinoid diagnosis. Acknowledgements The author has no conflict of interest.

Introduction Aflatoxins are a group of mycotoxins produced by the fungi Aspergillus flavus and Aspergillus parasiticus and are known contaminants of a variety of foods. Chronic, low dose exposure to aflatoxins is a well documented cause of liver malignancy and immunologic disturbances. Short term, high dose exposure, particularly to Aflatoxin B1 (AFB1), the most potent of these toxins, may cause acute aflatoxicosis. Aflatoxin poisoning may result in hemorrhagic liver necrosis, steatosis, bile duct proliferation and subsequent organ failure, with a mortality rate of close to 25% and may be higher [1, 2]. Aflatoxin exposure has also been suggested to contribute to the incidence of Reye and Reye-like syndromes [3]. Outbreaks of aflatoxicosis have been reported in developing nations around the world [2, 4, 5]. However, due to food manufacturing and monitoring standards, it is virtually un-documented in the developed world. We present here a case of suspected severe aflatoxicosis in a migrant Thai worker who consumed large quantities of contaminated food shipped to him from his native country.

ions around the world [2, 4, 5]. However, due to food manufacturing and monitoring standards, it is virtually un-documented in the developed world. We present here a case of suspected severe aflatoxicosis in a migrant Thai worker who consumed large quantities of contaminated food shipped to him from his native country. Case report A 28 year old, healthy Thai male, employed in Israel as an agricultural worker, presented to the emergency room with complaints of nausea, vomiting and diffuse abdominal pain starting 3 days earlier. Past medical history was unremarkable and without mention of recent illness. On presentation, the patient was well oriented in no severe distress, physical examination was notable for epigastric tenderness with guarding. Heart rate was 116 beats/minute, blood pressure 94/59 mm/Hg and a temperature of 37.4°C. The patient was admitted to the surgical department for evaluation. A chest and abdomen CT revealed marked liver enlargement with fatty change, and minimal amounts of pleural and pericardial fluid. No surgical etiology was recognized and due to laboratory findings of renal failure (Creatinine 3.88 mg/dl BUN 119.7 mg/dl), the patient was transferred to the medical department.

valuation. A chest and abdomen CT revealed marked liver enlargement with fatty change, and minimal amounts of pleural and pericardial fluid. No surgical etiology was recognized and due to laboratory findings of renal failure (Creatinine 3.88 mg/dl BUN 119.7 mg/dl), the patient was transferred to the medical department. Shortly after arrival, the patient's condition deteriorated rapidly to a state of agitation and shock and he was transferred to the intensive care unit. On arrival blood pressure measured 79/45 mm/Hg, pulse 170 beats/minute with episodes of SVT. In addition, the patient developed dyspnea, with a respiratory rate of 48 breaths/minute and anuria. Core temperature measured 33.1°C. Examination revealed marked peripheral cyanosis, distended jugular veins with cool extremities. Air entry to both lungs was reduced and palpation of the abdomen was noted for tenderness and an enlarged liver. Laboratory analysis showed lactic acidosis (pH 7.22, lactate 129 mg/dl), renal failure (creatinine 2.11 mg/dl), and rhabdomyolysis (CPK 3,205 IU/l). Liver function tests and coagulation indices were also disturbed ( total bilirubin 2.74, direct bilirubin 1.03 mg/dl, AST 1169 IU/l, ALT 890 IU/l, LDH 1743 IU/l, INR 1.7, factor V level 18%,). Erythrocyte sedimentation rate was within normal limits. Factor V levels continued to decline to a nadir of 5%. The above laboratory results, in combination with encephalopathy, indicated fulminant hepatic failure accompanied by multi-system organ failure.

/l, ALT 890 IU/l, LDH 1743 IU/l, INR 1.7, factor V level 18%,). Erythrocyte sedimentation rate was within normal limits. Factor V levels continued to decline to a nadir of 5%. The above laboratory results, in combination with encephalopathy, indicated fulminant hepatic failure accompanied by multi-system organ failure. The patient failed a trial of positive pressure ventilation and was subsequently intubated and ventilated mechanically. A Swan-Ganz catheter was inserted and fluid resuscitation started, steroids and vasopressors were given. Echocardiography was used to rule out pericardial tamponade; normal ventricular and valvular function was demonstrated. Continuous hemodiafiltration was started and switched to standard hemodialysis a week later. A tracheotomy was later performed due to the need for prolonged ventilation. Blood, urine, sputum and peritoneal fluid aspirate were cultured and gram and Ziell-Nielsen stained. Empiric antibiotic therapy was then started. Culture results were negative. Serologic testing for acute or chronic infection with viral hepatitis types A, B and C, HIV, Epstein-Barr virus, Cytomegalovirus returned negative. Further serologic testing for Ricketsia conorii, Coxiella burnetii were also negative. A thick film smear for blood-borne parasites and stool collection for ova and parasites were negative. Due to a high osmolar gap, levels of methanol, ethylene glycol and acetyl salicylic acid were checked and no traces were found.

e. Further serologic testing for Ricketsia conorii, Coxiella burnetii were also negative. A thick film smear for blood-borne parasites and stool collection for ova and parasites were negative. Due to a high osmolar gap, levels of methanol, ethylene glycol and acetyl salicylic acid were checked and no traces were found. A health bureau inspector was sent to the residence of the patient and found a stock of canned food the patient consumed almost exclusively for a period of months. Specimens were examined at the ministry of health. AFB1 was found at a level of 19.6 ppb (allowed level < 5 ppb). The patient's condition gradually improved on supportive treatment. Vasopressors were stopped and dialysis discontinued after blood pressure, renal function and CPK values returned to normal. Liver function tests improved markedly. After 21 days of mechanical ventilation the patient was weaned and the tracheotomy closed on the 36th day. The patient was returned to the medical department for observation and physiotherapy. No apparent sequels were observed other than mild residual hypertension. The patient was discharged on day 45.

proved markedly. After 21 days of mechanical ventilation the patient was weaned and the tracheotomy closed on the 36th day. The patient was returned to the medical department for observation and physiotherapy. No apparent sequels were observed other than mild residual hypertension. The patient was discharged on day 45. Discussion Disease due to aflatoxin exposure was first recognized in livestock a few decades ago with similar patterns of disease apparent in different species. Susceptibility to aflatoxin has been noted to vary considerably between species and between different individuals, including man. Of note is the higher susceptibility in the young. The level of aflatoxin exposure needed to cause acute aflatoxicosis has not, thus far, been determined in humans. Aflatoxin is recognized by the FDA as an unavoidable contaminant of food and standards have been set for its level in commercially distributed products. These standards are not applicable in the developing world where food manufacturing technology and surveillance is lacking. As a result, billions of people are exposed to uncontrolled amounts of toxin [1]. A literature search reflects this dichotomy and reports of aflatoxicosis emerge mostly during sporadic epidemics in developing countries [2, 4, 5], perhaps due to the fact that individual cases are under-diagnosed. In this case, the presumptive diagnosis of aflatoxicosis was based on the clinical picture, the finding of high levels of AFB1 in foods the patient consumed almost exclusively, and after alternate diagnoses' were sufficiently excluded.

Discussion Disease due to aflatoxin exposure was first recognized in livestock a few decades ago with similar patterns of disease apparent in different species. Susceptibility to aflatoxin has been noted to vary considerably between species and between different individuals, including man. Of note is the higher susceptibility in the young. The level of aflatoxin exposure needed to cause acute aflatoxicosis has not, thus far, been determined in humans. Aflatoxin is recognized by the FDA as an unavoidable contaminant of food and standards have been set for its level in commercially distributed products. These standards are not applicable in the developing world where food manufacturing technology and surveillance is lacking. As a result, billions of people are exposed to uncontrolled amounts of toxin [1]. A literature search reflects this dichotomy and reports of aflatoxicosis emerge mostly during sporadic epidemics in developing countries [2, 4, 5], perhaps due to the fact that individual cases are under-diagnosed. In this case, the presumptive diagnosis of aflatoxicosis was based on the clinical picture, the finding of high levels of AFB1 in foods the patient consumed almost exclusively, and after alternate diagnoses' were sufficiently excluded. The combination of fulminant hepatic failure and rhabdomyolysis should raise the suspicion of a toxic etiology. The marked discrepancy between extremely low factor V levels and only moderately disturbed liver function tests and coagulation indices may be another clue to this etiology. The full recovery without sequels from a state of multi-system organ failure after receiving intensive, yet basically supportive therapy, is again compatible with poisoning. Although the elevated levels of AFB1 in the specimens collected, were not as high as in previously reported cases [2, 4], we believe that, given the known variable susceptibility to AFB1 and the fact that the level of AFB1 in the specimen collected may be an underestimate, it may well be the cause of disease.

oisoning. Although the elevated levels of AFB1 in the specimens collected, were not as high as in previously reported cases [2, 4], we believe that, given the known variable susceptibility to AFB1 and the fact that the level of AFB1 in the specimen collected may be an underestimate, it may well be the cause of disease. We conclude that chronic exposure to moderately elevated levels of aflatoxin B1 may result in acute aflatoxicosis and fulminant hepatic failure. This case also illustrates the appearance of disease in a part of the world where it is practically unknown, due to immigration of people and the shipment of goods between the developed and developing world. This epidemiologic pattern will most likely become increasingly more prevalent and will require clinicians to consider a wider range of diagnoses in a given clinical presentation. Acknowledgement The authors declare no conflict of interest related to this report. Abbreviations AFB1aflatoxin B1 CTcomputerized tomography SVTsupra-ventricular tachycardia CPKcreatinine phosphokinase FDAFood and Drug Administration (US)

nsportal obliteration, left gastric artery embolization, and partial splenic artery embolization, were introduced. In 1986, endoscopic variceal ligation was first used by Stiegmana. These developments have remarkably improved survival of variceal bleeding. The choosing of these therapies becomes an attractive question. II. Primary Prophylaxis of Variceal Hemorrhage The first episode of variceal bleeding is associated with not only a high mortality but also a high recurrence of bleeding [2]. Hence, prevention of the first episode of hemorrhage is of vital importance. Factors related to the risk of variceal bleeding include portal pressure, endoscopic features of varices and the location of varices [2-4]. Thus, screening the development of varices may help predict the risk of bleeding. Primary prophylaxis of variceal hemorrhage involves reasonable surveillance strategies and appropriate choice of therapeutic modalities. The management of primary prophylaxis is illustrated in Figure 1. Figure 1 The management of primary prophylaxis Surveillance strategies Varix is a progressive complication of portal hypertension. Thus, surveillance strategies are vitally important since the management of esophageal varices largely depends on its natural history. Hepatic venous pressure gradient (HVPG) is a promising predicative marker of the first episode of bleeding. But its application is limited given its invasive nature. Screening endoscopic is the recommended form for surveillance [5].

tant since the management of esophageal varices largely depends on its natural history. Hepatic venous pressure gradient (HVPG) is a promising predicative marker of the first episode of bleeding. But its application is limited given its invasive nature. Screening endoscopic is the recommended form for surveillance [5]. HVPG HVPG is a reliable parameter of portal pressure given the positive correlation between variceal pressure and HVPG value [6]. The normal HVPG value is 1 - 5 mmHg. Pressure exceeding the upper threshold defines the presence of portal hypertension, regardless of clinical manifestations. HVPG ≥10 mmHg is defined as clinically significant portal hypertension, predicating the development of varices [7]. HVPG above 12 mmHg is the threshold pressure that leads to variceal rupture [2, 3, 7, 8, 9]. In other words, if HVPG can be lowered to less than 12 mmHg, bleeding does not occur. Overall, the HVPG has predictive value in the development of varices and the risk of bleeding. It is also helpful in assessing the therapeutic efficacy of β-blockers [2, 5, 10]. However, considering the invasive nature, sequential measurements of HVPG are rarely used in practice [4, 5].

Introduction Adenosquamous carcinoma of the stomach is very rare, the incidence being less than 0.5% of all stomach malignancies [1]. This disease has been sporadically reported as case reports [1-4], and comprehensive studies using large series are a few [5-7]. The author reports two cases of adenosquamous carcinoma of the stomach. Case report Case 1 An 87-year-old woman was admitted to our hospital because of nausea and vomiting. Endoscopy revealed a large type 4 (linitis plastica type) tumor in the stomach, and biopsy showed squamous cell carcinoma. Total gastrectomy, cholecystectomy, splenectomy and lymph node dissection were performed. Grossly, the stomach showed a large type 4 tumor measuring 10 x 8 x 7 cm. Histologically, the gastric tumor consisted of a mixture of adenocarcinoma (30% in area) (Fig. 1a) and squamos cell carcinoma (Fig. 1b) (70% in areas). The adenocarcinoma consisted of signet ring cell carcinoma, poorly differentiated carcinoma, and tubular adenocarcinoma. There was a gradual transition between adenocarcinoma and squamous cell carcinoma. Severe lymphovascular tumor cells permeation was recognized. The carcinoma cells invaded into the serosa. The gall bladder, lymph nodes and peritoneum showed metastases of signet ring cell adenocarcinoma. The patient’s condition deteriorated, and systemic metastasis emerged. The patient died of five months after operation.

re lymphovascular tumor cells permeation was recognized. The carcinoma cells invaded into the serosa. The gall bladder, lymph nodes and peritoneum showed metastases of signet ring cell adenocarcinoma. The patient’s condition deteriorated, and systemic metastasis emerged. The patient died of five months after operation. Figure 1 Histology of case 1. Tumor cells are composed of adenocarcinoma (poorly differentiated adenocarcinoma and signet ring cell carcinoma) element (a) and squamous cell carcinoma element (b). HE, x 200. Case 2 A 77-year-old woman was admitted to our hospital because of epigastralgia. Marked leukocytosis was present without inflammation. Endoscopic examination revealed a large type 3 tumor (ulcerative infiltrating tumor), and biopsy showed squamous cell carcinoma. Gastrectomy and lymph node dissection was performed. Pathologically, the gastric tumor measuring 6 x 5 x 7 cm, composed of a mixture of adenocarcinoma (10%) (Fig. 2a) and squamous cell carcinoma (90%) (Fig. 2b). The carcinoma invaded into subserosa. Lymphovascular permeation was present. The adenocarcinoma element consisted of signet ring cell carcinoma. Immunohistochemical study was performed using Dako Envision methods, as previously reported [8, 9]. Tumor cells were immunohistochemically positive for granulocyte-colony stimulating factor. The lymph nodes showed metastases of signet ring cell carcinoma. The patient showed systemic metastasis, and died eight months after the operation.

l study was performed using Dako Envision methods, as previously reported [8, 9]. Tumor cells were immunohistochemically positive for granulocyte-colony stimulating factor. The lymph nodes showed metastases of signet ring cell carcinoma. The patient showed systemic metastasis, and died eight months after the operation. Figure 2 Histology of case 2. Tumor cells are composed of adenocarcinoma (signet ring cell carcinoma) element (a) and squamous cell carcinoma element (b). HE, x 200. Discussion The present two tumors consisted of a mixture of adenocarcinoma and squamous cell carcinoma, and gradual transition between the two was present. Thus, the present two tumors were not collision tumors but true adenosquamous carcinomas. The biopsies of the two cases showed squamous cell carcinoma. This is because squamous cell carcinoma predominated over adenocarcinoma in area. Thus, biopsy diagnosis of squamous cell carcinoma of the stomach does not exclude adenosquamous carcinoma. Pure squamous cell carcinoma of the stomach is extremely rare.Clinically, the both patients of the present study showed a rapid clinical course and prognosis was very poor. Studies of large series also reported poor prognosis of adenosquamous carcinoma of the stomach [5, 6].

ch does not exclude adenosquamous carcinoma. Pure squamous cell carcinoma of the stomach is extremely rare.Clinically, the both patients of the present study showed a rapid clinical course and prognosis was very poor. Studies of large series also reported poor prognosis of adenosquamous carcinoma of the stomach [5, 6]. It is interesting that the present two cases showed metastases of adenocarcinoma but not squamous cell carcinoma. Thus, it seems that the biologic behaviors may be determined by adenocarcinoma element in adenosquamous carcinoma of the stomach. It is also interesting that the present case 2 was granulocyte-colony stimulating factor producing adenosquamous carcinoma. Similar case was reported only once [2]. Much more studies of such cases are required. The origin of adenosquamous carcinoma is unclear. Mori et al [4, 7] considered that adenosquamous carcinoma is derived from squamous transdifferentiation of adenocarcinoma cells, while Mingzzini et al [3] considered that adenosquamous carcinoma of the stomach are derived from totipotential undifferentiated cell of the stomach. The cellular origin of the present cases is unclear. More studies using modern techniques are required. Acknowledgement The author declares no conflict of interest related to this report.

I. Introduction Variceal hemorrhage is a frequent and lethal complication of portal hypertension. Esophageal varices are present in around 50% of cirrhotic patients [1]. Bleeding occurs in 30% - 40% of cirrhotic patients once varices have formed [2]. The first episode of variceal bleeding is associated with mortality between 17% - 57% [1], and approximately two thirds of the survivors who do not receive active treatment might suffer from recurrent episode of hemorrhage [2]. Therefore, management of varices can be categorized into three phases, primary prophylaxis (prevention of the first episode of bleeding); emergency treatment (management of acute bleeding); and secondary prophylaxis (prevention of re-bleeding). During the last decades, management of variceal hemorrhage has been well developed. In 1939, endoscopic injection sclerotheropy emerged; quinine was used as the sclerosant. In the 1970s, interventional radiology procedures, including transportal obliteration, left gastric artery embolization, and partial splenic artery embolization, were introduced. In 1986, endoscopic variceal ligation was first used by Stiegmana. These developments have remarkably improved survival of variceal bleeding. The choosing of these therapies becomes an attractive question.

2 mmHg, bleeding does not occur. Overall, the HVPG has predictive value in the development of varices and the risk of bleeding. It is also helpful in assessing the therapeutic efficacy of β-blockers [2, 5, 10]. However, considering the invasive nature, sequential measurements of HVPG are rarely used in practice [4, 5]. Endoscopy surveillance There are several non-invasive markers reported which are related to the risk of bleeding, such as platelet count, diameter of portal vessel, size of spleen, and so on. However, these markers could offer less predictive accuracy. Endoscopy is now the recommended form of screening [5]. Esophagogastroduodenoscopy (EGD), the gold standard in the diagnosis of varices, should be performed once the diagnosis of cirrhosis is established [5, 11]. The morphological features at the initial endoscopy, as well as the natural history of cirrhotic patients, determine the schedule of endoscopy surveillance. For patients with compensated cirrhosis, screening endoscopy should be repeated every 1 - 2 years in individuals with small varices [3-5, 9, 12], while it is reasonable to repeat at 2 - 3 years’ intervals in those without varices [3, 5, 9, 11, 12]. Patients with medium or large varices or red wale signs are generally considered to correlate with high risk of bleeding [13], it is suggested that this group should take non-selective β-blockers for primary prophylaxis [1, 3-5, 11-14]. Endoscopy surveillance can be avoided in these patients [5]. Once there is evidence of decompensate cirrhosis, screening endoscopies should be performed annually in order to monitor the formation and progression of varices [5, 9].

this group should take non-selective β-blockers for primary prophylaxis [1, 3-5, 11-14]. Endoscopy surveillance can be avoided in these patients [5]. Once there is evidence of decompensate cirrhosis, screening endoscopies should be performed annually in order to monitor the formation and progression of varices [5, 9]. Esophageal capsule endoscopy Esophageal capsule endoscopy (ECE) has opened a new era in variceal examination. First introduced in 2000, ECE is considered as a promising alternative to EGD for patients who are unwilling or unable to undergo EGD [5, 9, 15]. ECE is associated with minimal invasiveness which is responsible for good tolerance [15], and the good agreement with EGD has been certified [9, 15]. However, it is expensive and further studies are required to confirm its application.

ternative to EGD for patients who are unwilling or unable to undergo EGD [5, 9, 15]. ECE is associated with minimal invasiveness which is responsible for good tolerance [15], and the good agreement with EGD has been certified [9, 15]. However, it is expensive and further studies are required to confirm its application. Therapeutic strategies The non-selective β-blockers, including propranolol and nadolol, are the recommended agents for primary prophylaxis of variceal hemorrhage [8, 16]. It is believed that they can reduce portal pressure by reducing cardiac output as well as splanchnic arterial blood flow [4, 9, 17]. Thus, the selective β-blockers are considered ineffective in prevention of variceal hemorrhage if it doesn’t affect splanchnic circulation [8]. The therapy usually starts with a low dose and then increases to an optimal dose step by step [9, 16]. As discussed above, sequential measurements of HVPG are not widely used in assessing the therapeutic response of β-blockers, therefore, most clinicians empirically adjust the dosage via observing the reduction in heart rate. The optimal dosage is generally considered to be able to reduce the resting heart rate by 25%, or to reduce the resting heart rate to 55 beats per minute [9, 16]. However, the reduction in heart rate does not virtually correlate with the decrease in portal pressure [9, 16].

ge via observing the reduction in heart rate. The optimal dosage is generally considered to be able to reduce the resting heart rate by 25%, or to reduce the resting heart rate to 55 beats per minute [9, 16]. However, the reduction in heart rate does not virtually correlate with the decrease in portal pressure [9, 16]. The contraindications for non-selective β-blockers include asthma, pulmonary edema, chronic obstructive pulmonary disease, congestive heart failure, bradycardia, atrioventricular block, Raynaud’s phenomenon and poorly controlled diabetes mellitus. Nadolol is a preferred agent with fewer adverse effects [8, 16]. Considering these drawbacks, patients with small varices or without varices do not need to take non-selective β-blockers for primary prophylaxis; only patients with medium or large varices take it as the current agent to prevent the first episode of hemorrhage. The nitrates, such as isosorbide mononitrate (ISMN), could further reduce portal pressure [9, 17], however, it is now no longer used in mono-therapy, it is recommended to be used with β-blockers when patients are not adequately sensitive to β-blockers [1, 8, 16]. Moreover, approximately 30% of patients with large varices have contraindication or intolerance to β-blockers [9, 17]. Endoscopic variceal ligation (EVL) is the option of endoscopic treatment when pharmacological therapy is infeasible [1]. Endoscopic injection sclerotherapy (EIS) has no role in primary prophylaxis of variceal hemorrhage, so it is the same with transjugular intrahepatic portosystemic shunting (TIPS) and shunt surgery [1, 8, 9].

ic variceal ligation (EVL) is the option of endoscopic treatment when pharmacological therapy is infeasible [1]. Endoscopic injection sclerotherapy (EIS) has no role in primary prophylaxis of variceal hemorrhage, so it is the same with transjugular intrahepatic portosystemic shunting (TIPS) and shunt surgery [1, 8, 9]. III. Emergency Treatment of Variceal Hemorrhage The emergent management of acute variceal bleeding consists of multiple steps, initial resuscitation, emergency endoscopy for diagnosis, hemostasis and prevention of early complications (including bacterial infections and renal failure). The current therapy fails to control acute bleeding or prevent very early re-bleeding in approximately 10% - 20% of the patients [2, 5, 9], in such cases, timely justification of the therapeutic strategy is of vital importance. The emergency management of variceal hemorrhage is illustrated in Figure 2. Figure 2 Algorithm for the emergency management Initial resuscitation Variceal hemorrhage classically presents as massive upper digestive bleeding with hematemesis or melena. Hemodynamic instability is responsible for many life-threatening complications, such as shock and renal failure. Thus, initial resuscitation is quite crucial.

Figure 2 Algorithm for the emergency management Initial resuscitation Variceal hemorrhage classically presents as massive upper digestive bleeding with hematemesis or melena. Hemodynamic instability is responsible for many life-threatening complications, such as shock and renal failure. Thus, initial resuscitation is quite crucial. Hemodynamic restitution should be initiated as soon as possible [2, 18]. After the initial assessment of blood loss, volume replacement should be employed at once. A protected air way is necessary [3, 19]. Endotracheal intubation might be required given that aspiration may take place especially in patients with hepatic encephalopathy or uncontrolled massive bleeding [2, 18, 19]. Peripheral venous access need to be prepared for volume replacement [3]. A central venous line is helpful to monitor central venous pressure which is the guidance of volume resuscitation [3]. Volume replacement should be individualized according to clinical manifestation, age, cardiac function, etc [18]. Gelatin-based colloids, human albumin fractions, fresh frozen plasma and packed red blood cells (PRBC) are widely used for transfusion [9, 18, 20]. Generally, normal saline, dextrans, hydroxyethyl starch and Ringer’s lactate solution are believed to be avoided (Table 1). The transfusion of fresh frozen plasma and platelet should be employed in case of coagulopathy and significant thrombocytopenia (platelet count < 5 × 104 /ml), which are commonly found in cirrhotic patients [2, 9, 19].

ne, dextrans, hydroxyethyl starch and Ringer’s lactate solution are believed to be avoided (Table 1). The transfusion of fresh frozen plasma and platelet should be employed in case of coagulopathy and significant thrombocytopenia (platelet count < 5 × 104 /ml), which are commonly found in cirrhotic patients [2, 9, 19]. Table 1 Agents forbidden in hemodynamic restitution for variceal hemorrhage Liquid forbidden use Reasons Normal saline Worsen the formation of ascites as well as other extra-vascular fluid accumulation Dextrans Side-effect on bleeding times Hydroxyethyl starch Worsen hepatic function Ringer’s lactate solution Contraindicated in case of liver dysfunction Furthermore, most clinicians are opt to correct hypovolemia conservatively and cautiously [2, 3]. During the correction of hypovolemia, portal pressure increases 20% more rapidly than blood volume. Thus, over-expansion of plasma volume may result in a more severe increase in portal pressure and the risk of further bleeding subsequently increases [3]. In practice, volume placement is performed with the goal of maintaining hemoglobin at approximately 8 g/dL [9].

ressure increases 20% more rapidly than blood volume. Thus, over-expansion of plasma volume may result in a more severe increase in portal pressure and the risk of further bleeding subsequently increases [3]. In practice, volume placement is performed with the goal of maintaining hemoglobin at approximately 8 g/dL [9]. Antibiotic prophylaxis Bacterial infection is a serious complication of cirrhosis, especially in bleeding patients [2, 3]. It is reported that 30% - 40% of cirrhotic patients undergo bacterial infections during an episode of variceal hemorrhage or within the first week following bleeding [20]. Spontaneous peritonitis and bacteriaemia are the most common infections seen in cirrhotic patients. Infections alter systemic and splanchnic hemodynamics, worsen coagulation disorders, impair liver function and subsequently may induce variceal bleeding [21]. Altogether, bacterial infection leads to failure in controlling acute bleeding, early re-bleeding and death.

e most common infections seen in cirrhotic patients. Infections alter systemic and splanchnic hemodynamics, worsen coagulation disorders, impair liver function and subsequently may induce variceal bleeding [21]. Altogether, bacterial infection leads to failure in controlling acute bleeding, early re-bleeding and death. Prophylactic use of antibiotics has been proved to reduce the incidence of bacterial infections [2, 3, 9, 12, 20], reduce the rate of re-bleeding [9, 12], and significantly improve survival [1, 2, 8, 9, 12, 19, 20]. Therefore, antibiotic prophylaxis has been an indispensable component of the management of acute variceal hemorrhage. Norfloxacin (400 mg, twice a day) is the most conventional antibiotics [8, 9, 20]. The duration of therapy is 7 days [1, 8, 9]. It can not be used in pregnancy, lactating women and pediatric patients. The most familiar adverse reaction is hypersensitivity, especially cutaneous anaphylaxis [8]. Intravenous quinolones are applied when oral administration is not available [9]. Besides, intravenous cephalosporins, such as ceftriaxone, are used when there is advanced liver dysfunction [5]. Aminoglycosides should be avoided considering the risk of renal toxicity [9, 18].

vity, especially cutaneous anaphylaxis [8]. Intravenous quinolones are applied when oral administration is not available [9]. Besides, intravenous cephalosporins, such as ceftriaxone, are used when there is advanced liver dysfunction [5]. Aminoglycosides should be avoided considering the risk of renal toxicity [9, 18]. Hemostasis The combination of pharmacological and endoscopic therapy is recommended as the first-line management of acute variceal bleeding [2, 9]. A meta-analysis reported that the combination reduced overall and variceal re-bleeding in cirrhosis more than either therapy alone [22]. Once there is suspicion of variceal hemorrhage, vaso-active drug should be administered as soon as possible, even preceding diagnostic endoscopy [9]. Emergency endoscopy ought to be performed within 12 h after admission and endoscopic therapy should be performed simultaneously once the suspected variceal source of bleeding is identified [5]. Pharmacological therapy Pharmacological therapy plays an important role in emergency hemostasis. Vaso-active drug can temporarily decrease portal pressure and bleeding, which provides better visualization of the esophageal lumen for endoscopy. It is recommended that pharmacological therapy should last for 5 days [9]. The following we will discuss vasopressin, terlipressin, somatostatin, and octreotide.

hemostasis. Vaso-active drug can temporarily decrease portal pressure and bleeding, which provides better visualization of the esophageal lumen for endoscopy. It is recommended that pharmacological therapy should last for 5 days [9]. The following we will discuss vasopressin, terlipressin, somatostatin, and octreotide. Vasopressin (0.2 - 0.4 U/min) causes splanchnic vasoconstriction so that it reduces portal blood flow and variceal pressure [2, 19]. However, it is associated with increased risk of myocardial infarction and mesenteric ischemia. Hence, it was abandoned as a mono-therapy 25 years ago in most countries [23]. Some clinicians advocates that vasopressin in combination with nitroglycerin can be used in emergency hemostasis [2, 9]. However, the efficacy needs further studies. Terlipressin (Triglycyl-lysl-vasopressin) is a synthetic analogue of vasopressin with a longer half-life [8, 11]. It is currently given by a 2 mg bolus every 4 h and the duration should be maintained for 2 - 5 days [20]. It is as effective as EIS in emergency treatment and secondary prophylaxis of variceal bleeding [4]. It also induces ischemic complications, including myocardia ischemia, intestinal infarction, and limb ischemia [18]. However, the complications are not as severe as vasopressin [2, 19].

e maintained for 2 - 5 days [20]. It is as effective as EIS in emergency treatment and secondary prophylaxis of variceal bleeding [4]. It also induces ischemic complications, including myocardia ischemia, intestinal infarction, and limb ischemia [18]. However, the complications are not as severe as vasopressin [2, 19]. Somatostatin (a 100 µg bolus followed by infusion at 50 µg/h) is a splanchnic vasoconstrictor which can significantly reduce the HVPG, variceal pressure and azygos blood flow [19]. It achieves a reduction in HVPG by 17% without affecting systemic circulation [18]. However, the half-life of natural somatostatin is about only 3 minutes which limits its application in clinical practice [23]. Octreotide (a 50 µg bolus followed by a constant infusion at 50 µg/h) is a synthetic analogue of somatostatin with a longer half-life. It is the only drug licensed for acute variceal bleeding in the United States. Severe adverse reactions are rarely seen [2, 20]. However, the efficacy of octreotide as a mono-therapy in hemostasis is still controversial [18, 20]. It seems to be more effective when combined with endoscopic therapy [8]. Endoscopic therapy Emergency endoscopy is recommended as soon as possible after admission, especially in patients with clinically significant hemorrhage or in patients with features suggesting cirrhosis [3, 5, 9, 11, 12]. Emergency endoscopy can clearly identify the bleeding source which helps guide the following steps of management. In addition, endoscopic treatment could be performed simultaneously if necessary.

ally in patients with clinically significant hemorrhage or in patients with features suggesting cirrhosis [3, 5, 9, 11, 12]. Emergency endoscopy can clearly identify the bleeding source which helps guide the following steps of management. In addition, endoscopic treatment could be performed simultaneously if necessary. Endoscopic treatment is the cornerstone of management of variceal hemorrhage. Endoscopic modalities, consisting of endoscopic injection sclerotherapy (EIS) and endoscopic variceal ligation (EVL), achieve hemostasis in approximately 90% of cases [4, 24, 25]. Although the two modalities are comparable in eradication of varices, EIS is thought to be inferior in terms of re-bleeding rates, complications, numbers of sessions, and duration for eradication [1, 2, 4, 5, 12, 19, 20, 26, 27]. Thus, EVL is generally considered as the option of endoscopic therapy [5]. However, during acute bleeding, EVL is sometimes technically difficult given that the spurting blood and clot may obscure the field of view upon endoscopy. In this situation, EIS should be employed [2, 11, 18].

[1, 2, 4, 5, 12, 19, 20, 26, 27]. Thus, EVL is generally considered as the option of endoscopic therapy [5]. However, during acute bleeding, EVL is sometimes technically difficult given that the spurting blood and clot may obscure the field of view upon endoscopy. In this situation, EIS should be employed [2, 11, 18]. It is still questionable whether it is beneficial to combine the two modalities. Mohamed AR et al reported the combined therapy was inferior to either therapy alone with regarding to the re-bleeding rate [28]. A meta-analysis of endoscopic sequential ligation plus sclerotherapy (EVLS) suggests the sequential combination lead to less complications and a higher incidence of variceal eradication [29]. The totally different conclusions may be owing to different techniques of EIS, different timing of each modality, etc. In summary, the efficiency of the combined endoscopic therapy requires future studies.

uggests the sequential combination lead to less complications and a higher incidence of variceal eradication [29]. The totally different conclusions may be owing to different techniques of EIS, different timing of each modality, etc. In summary, the efficiency of the combined endoscopic therapy requires future studies. EIS was first reported by Grafoord in Sweden in 1939. This technique was then widely used and had been the first-line treatment for acute variceal hemorrhage before EVL emerged. The standard strategy of EIS is still unknown despite years of research. There are wide variations in sclerosants (type, concentration, volume injected each session), intervals between sessions, site of injection (paravariceal, intravariceal, or combined), etc. Sclerosants often used include sodium morrhuate, sodium tetradecyl sulphate, ethanolamine oleate, polidocanol, absolute alcohol, thrombin, cephalothin, phenol and tissue glue [4]. Until now there is no consensus on the optimal sclerosant for EIS [2, 4]. The commonly used sclerosants are listed in table 2.

mbined), etc. Sclerosants often used include sodium morrhuate, sodium tetradecyl sulphate, ethanolamine oleate, polidocanol, absolute alcohol, thrombin, cephalothin, phenol and tissue glue [4]. Until now there is no consensus on the optimal sclerosant for EIS [2, 4]. The commonly used sclerosants are listed in table 2. Table 2 Commonly used sclerosants Sclerosant Concentration Volume/site (ml) The max value of volume/session (ml) Special points Sodium morrhuate 5% 4-6 20 Commonly used in China Ethanolamine oleate 5% 2-3 25 Commonly used in China Polidocanol 1% 1-2 20 n/a Sodium tetradecyl sulphate 0.5%-1.5% 5 n/a Associated with more complications and seldom used now In spite of these differences, EIS is beneficial either in emergency hemostasis or prevention of recurrent hemorrhage. However, it is associated with a series of complications, retro-sternal chest pain, ulcer, esophageal stenosis, fever, pneumonitis, dysphagia, esophygeal perforation and bacteriemia [1, 4, 8, 16, 19]. It is noteworthy that EIS can not alleviate portal hypertension. After EIS, portal hypertension still exists, which is responsible for the recurrence of varices and hemorrhage. Thus, it is necessary to perform a long term strategy of EIS until eradication of varices, and a life-long endoscopic follow-up is recommended [30].

noteworthy that EIS can not alleviate portal hypertension. After EIS, portal hypertension still exists, which is responsible for the recurrence of varices and hemorrhage. Thus, it is necessary to perform a long term strategy of EIS until eradication of varices, and a life-long endoscopic follow-up is recommended [30]. EVL was first reported by Stiegmana in 1986. Unlike EIS, the technique of EVL is relatively identical [4]. Complications associated with EVL mainly include superficial ulcer, esophageal mucosal tears, variceal rupture, etc [1, 4, 8, 16]. Several trials have been published comparing EVL and EIS. It is reported that EVL is as effective as EIS in eradication of varices and emergent hemostasis. But EVL tends to be associated with less complications and relative lower frequency of re-bleeding [31]. A problem worthy to be pointed out is that EVL, compared to EIS, is associated with a higher incidence of recurrent varices and portal hypertensive gastropathy [32]. However, the higher incidence of recurrent varices doesn’t result in a higher risk of re-bleeding. Thus, EVL has replaced EIS in most cases and it is now the most promising choice of endoscopic therapy [27]. Repeat sessions of EVL and long-term endoscopic surveillance are also required given risk of recurrent varices and bleeding. The details will be discussed in the secondary prophylaxis of variceal bleeding.

ding. Thus, EVL has replaced EIS in most cases and it is now the most promising choice of endoscopic therapy [27]. Repeat sessions of EVL and long-term endoscopic surveillance are also required given risk of recurrent varices and bleeding. The details will be discussed in the secondary prophylaxis of variceal bleeding. Failure of the first-line therapy Even in the best condition, the current therapy seems to be ineffective in 10% - 20% patients. When the first-line treatment fails to control acute variceal bleeding, we have to immediately change our therapeutic strategies. Thus an explicit definition is quite important. The definition of failure has undergone obviously transitions during last decades. The Baveno I (1990) definition is not quite definite. Factors regarded include blood pressure, pulse, hematocrit and hemoglobin. But no accurate data was given. The time frame is 24 h. Bleeding that occurs after a 24-h interval from “time zero” (the time of first hospitalized) is defined as re-bleeding [33].

Failure of the first-line therapy Even in the best condition, the current therapy seems to be ineffective in 10% - 20% patients. When the first-line treatment fails to control acute variceal bleeding, we have to immediately change our therapeutic strategies. Thus an explicit definition is quite important. The definition of failure has undergone obviously transitions during last decades. The Baveno I (1990) definition is not quite definite. Factors regarded include blood pressure, pulse, hematocrit and hemoglobin. But no accurate data was given. The time frame is 24 h. Bleeding that occurs after a 24-h interval from “time zero” (the time of first hospitalized) is defined as re-bleeding [33]. The Baveno II (1995) and Baveno III (2000) criteria are more detailed and painstaking [11]. The definition of failure is divided into two frames. Within 6 h: any of the following factors: (a) transfusion of 4 units of blood or more, and (b) inability to achieve an increase in systolic blood pressure of 20 mmHg or to 70 mmHg or more, and/or (c) a pulse reduction to less than 100/min or a reduction of 20/min from baseline pulse rate. After 6 h: any of the following factors: (a) the occurrence of hematemesis, (b) reduction in blood pressure of more than 20 mmHg from the 6-h point, and/or (c) increase of pulse rate of more than 20/min from the 6-h point on two consecutive readings 1 h apart, (d) transfusion of 2 units of blood or more (over and above the previous transfusion) required to increase the HCT to above 27% or Hb to above 9 g/dL.

n blood pressure of more than 20 mmHg from the 6-h point, and/or (c) increase of pulse rate of more than 20/min from the 6-h point on two consecutive readings 1 h apart, (d) transfusion of 2 units of blood or more (over and above the previous transfusion) required to increase the HCT to above 27% or Hb to above 9 g/dL. The Baveno IV Consensus Conference (2005) offers a well developed definition of failure in emergent hemostasis [11]. The time frame for the acute bleeding episode is 120 h. That is, the very early re-bleeding (within 5 days) also implies the failure of emergency hemostais. Thus, the treatment of re-bleeding within 5 days becomes a part of emergency management. The therapy is considered to have failed in case whichever below occurs, (1) fresh hematemesis ≥ 2h after the standard therapy starts. And in patients with a naso-gastric tube in place, aspiration of greater than 100ml of fresh blood signifies failure; (2) a drop in the hemoglobulin value ≥ 3g if no transfusion is administered; (3) ABRI ≥ 0.75 at any time point (however, the threshold of defining failure requires further studies). ABRI (adjusted bleed requirement index) = (blood units transfused)/ [(final HCT-initial HCT) + 0.01]; (4) death. In case of failure, we have to turn to rescue therapies.

value ≥ 3g if no transfusion is administered; (3) ABRI ≥ 0.75 at any time point (however, the threshold of defining failure requires further studies). ABRI (adjusted bleed requirement index) = (blood units transfused)/ [(final HCT-initial HCT) + 0.01]; (4) death. In case of failure, we have to turn to rescue therapies. Rescue modalities The failure of the first-line therapy generally indicates a second attempt at endoscopic treatment [1, 4, 9, 12, 18, 26, 34]. However, if the second endoscopic treatment fails, salvage modalities should be employed at once [4, 9, 11, 18, 26]. Some clinicians suggest rescue therapy be taken immediately after the initial failure [3, 11, 16, 19, 35, 36]. Rescue modalities include balloon tamponade, TIPS, PTVE and shunt surgery. Balloon tamponade, including Minnesota tube and Sengstaken-Blakemore tube, is a temporary life-saving modality against fierce bleeding that cannot be controlled by current therapy. It can successfully achieve haemostasis in most cases [2]. However, once the balloon is deflated, bleeding recurs in 50% of the patients within 24 h [9, 26]. Thus, it usually works as a “bridge” to a more definite therapy, such as repeat endoscopic therapy, TIPS and shunt surgery [1, 4, 9]. A long-duration usage of balloon tamponade may result in esophageal ulcer or even peroration [2]. So it is mentioned that the duration should not exceed 24 h [5, 9]. Other balloon-associated complications are mainly vomiting and aspiration [9, 16]. Hence, air way protection as well as sedation is of importance when using balloon tamponade [26].

alloon tamponade may result in esophageal ulcer or even peroration [2]. So it is mentioned that the duration should not exceed 24 h [5, 9]. Other balloon-associated complications are mainly vomiting and aspiration [9, 16]. Hence, air way protection as well as sedation is of importance when using balloon tamponade [26]. TIPS, first reported in 1988, is expensive and invasive. It is an interventional radiologic procedure. General anesthesia is not always required. A shunt is produced between the hepatic vein and the intra-hepatic portion of the portal vein. TIPS could achieve hemostasis in most cases. Thus it is now considered as the first option of rescue therapy when the combination of vaso-active drug and endoscopic treatment fails. Unlike shunt surgery, TIPS is not contraindicated for patients awaiting liver transplantation. Patients with de-compensated cirrhosis (Child’s class B or C) are recommended to receive TIPS rather than shunt surgery. Complications include portosystemic encephalopathy, stenosis of shunt, shunt thrombosis, portal venous thrombosis, bleeding, hemolytic anemia, cardiac arrhythmias, TIPS-associated biliary fistula, liver failure and renal failure [8, 16].

(Child’s class B or C) are recommended to receive TIPS rather than shunt surgery. Complications include portosystemic encephalopathy, stenosis of shunt, shunt thrombosis, portal venous thrombosis, bleeding, hemolytic anemia, cardiac arrhythmias, TIPS-associated biliary fistula, liver failure and renal failure [8, 16]. Over the past years, Zhang et al developed a modified percutaneous transhepatic embolization of varices (PTVE) with 2-octyl cyanoacrylate (2-OCA), in which 2-OCA was injected into the whole lower esophageal and para-esophageal varices, the submucosal varices and the adventitial plexus of the cardia and fundus, this way, not only the esophageal varices but also the feeders were obliterated sufficiently to prevent variceal recurrence and improve long-term efficacy [37, 38]. In the prospective randomized controlled trial [38], cirrhotic patients with acute or recent esophageal variceal bleeding were assigned randomly to PTVE (52 patients) or EVL (50 patients) groups. With the whole lower esophageal and peri or para-esophageal varices, the submucosal varices, and the adventitial plexus of the cardia and fundus sufficiently obliterated by 2-OCA, this modified PTVE was more effective than EVL in the management of esophageal varices recurrence and rebleeding.

EVL (50 patients) groups. With the whole lower esophageal and peri or para-esophageal varices, the submucosal varices, and the adventitial plexus of the cardia and fundus sufficiently obliterated by 2-OCA, this modified PTVE was more effective than EVL in the management of esophageal varices recurrence and rebleeding. Common types of shunt surgery include spleno-renal shunt, meso-caval shunt and portal-caval shunt. Shunt surgery is reserved for patients unresponsive to the first-line therapy when TIPS is not available or an attempt at TIPS has already failed. However, it seems to be a better choice than TIPS for patients with Child’ class A. Patients awaiting liver transplantation are not appropriate candidates for shunt surgery. Common complications include infection, portosystemic encephalopathy, shunt thrombosis, hehepatorenal syndrome, liver failure, multisystem organ failure [8, 16].

ms to be a better choice than TIPS for patients with Child’ class A. Patients awaiting liver transplantation are not appropriate candidates for shunt surgery. Common complications include infection, portosystemic encephalopathy, shunt thrombosis, hehepatorenal syndrome, liver failure, multisystem organ failure [8, 16]. IV. Secondary Prophylaxis of Variceal Hemorrhage Endoscopic modalities can achieve haemostasis in approximately 90% of the patients. Whereas, after cession of acute esophageal variceal bleeding, the risk of rebleeding approaches 70% if further preventive measures are not taken [2, 4]. The risk of recurrent hemorrhage may be increased by several factors: fierce bleeding during the first episode of hemorrhage; presence of hepatic encephalopathy; severely increased portal pressure; large varices; presence of hepatoma, etc [2]. Hence, prevention of recurrent hemorrhage is of vital importance. All patients who have survived the first episode of bleeding should take preventive measures. In the last 3 decades, many therapies have been developed to prevent the occurrence of re-bleeding. It is proposed in recent practice guidelines that a combination of EVL plus non-selective β-blockers is the most promising strategy [5, 11]. However, pharmacological therapy alone is sufficient for patients responsive to β-blockers [17]. Regular surveillance also helps monitor the prognosis of varices. The secondary prophylaxis starts on day 6 after the first episode [3, 11, 12, 17]. Very early re-bleeding (within 5 days of acute bleeding) has been discussed above.

IV. Secondary Prophylaxis of Variceal Hemorrhage Endoscopic modalities can achieve haemostasis in approximately 90% of the patients. Whereas, after cession of acute esophageal variceal bleeding, the risk of rebleeding approaches 70% if further preventive measures are not taken [2, 4]. The risk of recurrent hemorrhage may be increased by several factors: fierce bleeding during the first episode of hemorrhage; presence of hepatic encephalopathy; severely increased portal pressure; large varices; presence of hepatoma, etc [2]. Hence, prevention of recurrent hemorrhage is of vital importance. All patients who have survived the first episode of bleeding should take preventive measures. In the last 3 decades, many therapies have been developed to prevent the occurrence of re-bleeding. It is proposed in recent practice guidelines that a combination of EVL plus non-selective β-blockers is the most promising strategy [5, 11]. However, pharmacological therapy alone is sufficient for patients responsive to β-blockers [17]. Regular surveillance also helps monitor the prognosis of varices. The secondary prophylaxis starts on day 6 after the first episode [3, 11, 12, 17]. Very early re-bleeding (within 5 days of acute bleeding) has been discussed above. Pharmacological therapy The efficacy of pharmacological therapy in prevention of re-bleeding has been confirmed. It is observed that patients in whom the HVPG is pharmacologically reduced to less than 12 mm Hg or a reduction in HVPG is greater than 20% from baseline are associated with a rather low re-bleeding rate [9]. Non-selective β-blockers are the most widely used agent in the secondary prophylaxis of variceal hemorrhage. It reduces the risk of recurrent hemorrhage by 40% and the mortality by 20% [26]. The mechanism has been discussed above. The addition of ISMN enhances the reduction of portal pressure but it is abandoned in monotherapy. Thus, a combination of β-blockers and ISMN is considered to be pharmacological therapy of choice. However, the combination also causes greater side effects and intolerance, which limit its application in practice [5, 17]. In summary, either the combination of β-blockers plus ISMN or β-blockers alone is beneficial. The choice of the optimal agents is virtually a “one from the two” question based on individual cases.

However, the combination also causes greater side effects and intolerance, which limit its application in practice [5, 17]. In summary, either the combination of β-blockers plus ISMN or β-blockers alone is beneficial. The choice of the optimal agents is virtually a “one from the two” question based on individual cases. Endoscopic therapy EIS EIS is confirmed to be beneficial in prevention of re-bleeding [26, 39]. Despite the inferiorities to EVL, it is still widely used all over the world. There is overwhelming consensus that EIS should be repeated until eradication. Thus, the best schedule of the long-term strategy is worth attention. Generally, initial EIS is performed at the time of diagnostic endoscopy and the second session is suggested within a week [2]. Thereafter, EIS should be repeated weekly or bi-weekly in line with current consensus [5]. Heretofore, 6 trials have been published, aiming to find the optimal schedule [40-45]. Factors taken into consideration involved duration for eradication, numbers of sessions, amount of sclerosants consumed, complications and re-bleeding rate. In 1984, Westaby D et al led a randomized control trial comparing weekly schedule and 3-week schedule. The weekly schedule was reported to be with shorter duration required for eradication but a significantly higher incidence of ulcerations [40]. Sarin’s study in 1986 showed a similar result except that the weekly schedule significantly decreased re-bleeding rate [41].

rial comparing weekly schedule and 3-week schedule. The weekly schedule was reported to be with shorter duration required for eradication but a significantly higher incidence of ulcerations [40]. Sarin’s study in 1986 showed a similar result except that the weekly schedule significantly decreased re-bleeding rate [41]. A study from Japan certified there were no significant differences between weekly and 2-week schedule in terms of most parameters except for the duration for eradication [42]. The 2-week group achieved eradication significantly earlier. Massoud’s trial offered entirely equal results [43]. There are only 2 trials focusing on intervals less than a week. Conflicting results were found. Akriviadis E proposed EIS intercalated by interval shorter than weekly was less effective and more dangerous while a study from Bombay raised the advantages of 3-day schedule with regarding to duration for eradication as well as survival [44, 45]. Utterly different techniques used in the two trials might be responsible for the collision to some extent. And limited sample capacity partially affected the outcome. In conclusion, a weekly or bi-weekly schedule of EIS is reasonable on the basis of available evidence. After eradication, endoscopy follow-up should start, it is the same with EVL. The details of surveillance endoscopy will be discussed below.

There are only 2 trials focusing on intervals less than a week. Conflicting results were found. Akriviadis E proposed EIS intercalated by interval shorter than weekly was less effective and more dangerous while a study from Bombay raised the advantages of 3-day schedule with regarding to duration for eradication as well as survival [44, 45]. Utterly different techniques used in the two trials might be responsible for the collision to some extent. And limited sample capacity partially affected the outcome. In conclusion, a weekly or bi-weekly schedule of EIS is reasonable on the basis of available evidence. After eradication, endoscopy follow-up should start, it is the same with EVL. The details of surveillance endoscopy will be discussed below. EVL EVL is the recommended form of endoscopic therapy [5]. It has been illustrated above that repeat EVL should be performed until eradication of varices. The optimal frequency of repeated EVL is still controversial. EVL is currently repeated every 7 - 14 days [5]. Gin Ho Lo raised the proposal that the schedule be modified [46]. A study from Japan in 2005 proved that EVL performed at bi-monthly intervals gained a higher total eradication rate, lower recurrence rate and lower rate of additional treatment [47]. Gavin C Harewood reported, in 2006, that a longer interval between sessions of EVL might be related to a reduced risk of re-bleeding [48]. However, evidence available now is not sufficient considering repeatability, reproducibility and sample capacities. Future studies are still required to identify the optimal schedule.

vin C Harewood reported, in 2006, that a longer interval between sessions of EVL might be related to a reduced risk of re-bleeding [48]. However, evidence available now is not sufficient considering repeatability, reproducibility and sample capacities. Future studies are still required to identify the optimal schedule. Endoscopy surveillance Considering the recurrence of varices and bleeding, endoscopy surveillance is of vital importance. Screening endoscopy is recommended to be repeated every 6 - 12months after the eradication of varices [1, 2, 4, 5, 9, 49]. The follow-up should be life-long [30]. Once there is evidence of recurrent varices, a comprehensive endoscopic therapy should be initiated again [9, 12, 49]. Clinicians in China proposed a detailed schedule of endoscopy surveillance, which is illustrated in table 3. Table 3 Schedule of endoscopy surverillance Time frame Intervals between re-endoscopy ≤ 2 years after eradication 3 - 6 months > 2 and ≤ 3 years after eradication 6 - 12 months > 3 years after eradication till death 12 months Endoscopic ultrasound (EUS) is a non-invasive technique which can provide good delineation of the cross sectional anatomy around the gastro-esophageal junction, including presence of varices, size of varices, wall thickness, presence of perforating veins, etc [25, 50]. The presence of esophageal collaterals and perorating veins are thought to be correlated with the recurrence of esophageal varices in cirrhotic patients [51-55]. Thus, EUS might help predicate the recurrence of esophageal varices.

of varices, size of varices, wall thickness, presence of perforating veins, etc [25, 50]. The presence of esophageal collaterals and perorating veins are thought to be correlated with the recurrence of esophageal varices in cirrhotic patients [51-55]. Thus, EUS might help predicate the recurrence of esophageal varices. Failure of secondary prophylaxis The secondary prophylaxis is illustrated in Figure 3. The failure of secondary prophylaxis is difficult to formulate. Jake E. J. Krige et al suggested that patients who developed life-threatening variceal hemorrhage after an adequate course of treatment should be regarded as failures of long-term therapy [49]. However, re-bleeding also requires alternative treatment options in case that a patient is receiving the combined therapy (EVL + β-blocker) [9]. Salvage modalities (TIPS, PTVE or shunt surgery) should be used in case of failure [4, 9, 11, 26, 49]. TIPS is usually the preferred therapy [9]. If necessary, liver transplantation could be taken into consideration as well [9]. It is worth notice that portacaval shunts should be avoided in candidates for transplantation [2]. Figure 3 Algorithm of the secondary prophylaxis

Failure of secondary prophylaxis The secondary prophylaxis is illustrated in Figure 3. The failure of secondary prophylaxis is difficult to formulate. Jake E. J. Krige et al suggested that patients who developed life-threatening variceal hemorrhage after an adequate course of treatment should be regarded as failures of long-term therapy [49]. However, re-bleeding also requires alternative treatment options in case that a patient is receiving the combined therapy (EVL + β-blocker) [9]. Salvage modalities (TIPS, PTVE or shunt surgery) should be used in case of failure [4, 9, 11, 26, 49]. TIPS is usually the preferred therapy [9]. If necessary, liver transplantation could be taken into consideration as well [9]. It is worth notice that portacaval shunts should be avoided in candidates for transplantation [2]. Figure 3 Algorithm of the secondary prophylaxis V. Conclusions Variceal hemorrhage is a life-threatening complication of portal hypertension. The management can be divided into 3 parts, primary prophylaxis, emergency treatment, and secondary prophylaxis. Screening endoscopy is recommended once the diagnosis of cirrhosis is established. Non-selective β-blocker, plus ISMN or not, is the first-line choice for patients with large varices. EVL is the alternative option when β-blocker is contraindicated or in-tolerated. Once there is a suspicion of variceal bleeding, hemodynamic restitution should be initiated as soon as possible. Volume replacement should be cautiously performed since overload of volume may lead to a severe increase in portal pressure and subsequently the risk of further bleeding. A 7-day course of prophylactic antibiotics decreases the incidence of re-bleeding and significantly improves survival. Norfloxacin, 400 mg twice a day, is the current option. Emergency endoscopy is recommended within 12 h after admission and endoscopic therapy should be performed once the suspected variceal source of bleeding is identified. The combination of vaso-active drug and EVL is recommended as the first-line management of acute variceal bleeding. EIS can be administered when EVL is technically infeasible. The failure of current therapy requires a second attempt at endoscopy therapy or rescue therapy. Balloon tamponade works as a bridge to more definitive measures (e.g, TIPS PTVE or shunt surgery); TIPS or PTVE is the option of salvage measures. Combination of non-selective β-blockers plus EVL is the best option of secondary prophylaxis. After the eradication of varices, a life-long follow-up upon endoscopy is administered. Moreover, newly developed techniques, such as ECE and EUS, may play an important part in the future and requires further studies.

easures. Combination of non-selective β-blockers plus EVL is the best option of secondary prophylaxis. After the eradication of varices, a life-long follow-up upon endoscopy is administered. Moreover, newly developed techniques, such as ECE and EUS, may play an important part in the future and requires further studies. Acknowledgements The authors declare no conflict of interests related to this article.

Introduction Cholangiocarcinoma (CC) is a fatal neoplasm that arises from cholangiocytes, the epithelial cells lining the bile duct liver. CC is the second most common primary liver cancer after hepatocellular cancer [1, 2]. CC occurs in approximately 2 per 100,000 people and accounts for approximately 13% of primary liver cancers [2, 3]. The prevalence of CC shows a wide geographical variety with the highest rates in Asia [1, 4] and the lowest rate in Australia [3]. Epidemiologic studies suggest an increasing incidence in the Western countries and Unites States [3, 4, 5]. According to American Society Cancer about 2,000 to 3,000 people in the United States develop bile duct cancer each year [4, 6]. The etiologies of CC remains unknown, but in most cases CC are associated with chronic biliary inflammation, cellular injury of bile ducts together with obstruction of bile flow [6, 7]. High risk-conditions in CC development are manifested thought: congenital biliary anomalies, primary sclerosing cholangitis, hepatolithiasis and hepatobiliary flukes (e.g. Clonorchis sinensis, and Opisthorchis viverrini). Other risk factors for cholangiocarcinoma include environmental toxins such as dioxin, and vinyl chloride, nitrosamines [4].

development are manifested thought: congenital biliary anomalies, primary sclerosing cholangitis, hepatolithiasis and hepatobiliary flukes (e.g. Clonorchis sinensis, and Opisthorchis viverrini). Other risk factors for cholangiocarcinoma include environmental toxins such as dioxin, and vinyl chloride, nitrosamines [4]. CC is an epithelial cancer of the biliary duct system that may originate in the liver and extrahepatic bile ducts, which may terminate at the ampulla of Vater. Based upon anatomic location, CC can be divided into three categories: (1) intrahepatic CC (20-25%), occurring in the bile ducts residing within the liver; (2) extrahepatic or perihilar CC (also known as Klatskin tumour, 50%), occurring at the confluence of the right and left hepatic ducts; and (3) distal extrahepatic bile duct cancers (20-25%) [2]. More than 90% of CCs are adenocarcinomas, with different histological variants including adenocarcinoma, papillary adenocarcinoma, intestinal-type adenocarcinoma, and mucinous adenocarcinoma [1, 2, 6]. Conventional chemotherapy and radiation therapy have not been shown to be effective in prolonging long-term survival and although photodynamic therapy has been reported to be effective as a palliative treatment, it is not curative [6]. Radical surgery is the only potentially curative treatment modality, but in most cases, the tumors are well advanced at the time of diagnosis, which results in limited treatment options [2], the impact of chemotherapy on survival remains controversial [4, 6]. The overall survival is approximately 6 months [4].

ive [6]. Radical surgery is the only potentially curative treatment modality, but in most cases, the tumors are well advanced at the time of diagnosis, which results in limited treatment options [2], the impact of chemotherapy on survival remains controversial [4, 6]. The overall survival is approximately 6 months [4]. The poor response of cholangiocarcinoma to therapy highlights the need for increased efforts in understanding the etiology and pathogenesis of this primary liver cancer [4, 8]. More new strategies should be developed that allow detecting these tumors at early stage and permit to apply radical curative modalities [2, 4]. Pathogenesis The development of cholangiocarcinoma, as with most cancers, is multifactorial process (Fig. 1). Contributers are thought to include chronic inflammatory processes that induce alteration of cellular detoxification mechanisms, induce activation of oncogenes, functional loss of tumor-suppressor genes and dysregulation of cell cycle, cell apoptotic mechanisms and angiogenesis [2, 4]. Figure 1 Relation between multiple factors behind CC, with important role in initiation and development of CC.

Pathogenesis The development of cholangiocarcinoma, as with most cancers, is multifactorial process (Fig. 1). Contributers are thought to include chronic inflammatory processes that induce alteration of cellular detoxification mechanisms, induce activation of oncogenes, functional loss of tumor-suppressor genes and dysregulation of cell cycle, cell apoptotic mechanisms and angiogenesis [2, 4]. Figure 1 Relation between multiple factors behind CC, with important role in initiation and development of CC. The persistent tissue damaging as well an enrichment or reactive oxygen and nitrogen species contribute to a cancer-prone microenvironment [7]. During inflammation, cholangiocites produce reactive oxygen species and other toxic compounds such as: nitric oxide, nitrate, nitrite, and oxygen radicals, that lead to death and regeneration of the bile ducts [7, 9]. This species are known to be DNA mutagens and are linked to malignant transformation. During this procees inflammatory processes, cholangiocites secrets mitogens that activate local cellular receptors and intracellular signalling pathways [4, 7, 9]. Inflammation The biliary tract inflammation underlines the pathogenesis in the most of the patients [3, 7, 9]. Chronic inflammation is thought to promote carcinogenesis by causing DNA damage [9], activating tissue reparative proliferation and by creating a local environment that is enriched with cytokines and other growth factors [2, 7].

liary tract inflammation underlines the pathogenesis in the most of the patients [3, 7, 9]. Chronic inflammation is thought to promote carcinogenesis by causing DNA damage [9], activating tissue reparative proliferation and by creating a local environment that is enriched with cytokines and other growth factors [2, 7]. The inflammatory cytokine interleukin-6 (IL-6) enhances tumor growth in CC by altered gene expression via autocrine mechanisms [10-12]. Interleukin-6 (IL-6)-mediated signal transducers activation is aberrantly sustained in cholangiocarcinoma cells, resulting in resistance to apoptosis [10, 11]. IL-6 expression is inversely related to cell proliferation and positively related to differentiation in CC [3, 12]. Suppressors of cytokine signalling-3 (SOCS-3) are a newly identified family of intracellular protein controlling the magnitude and/or duration of signals propagated by diverse cytokine receptors by suppressing their signal transduction process [11, 14, 15]. SOCS-3 epigenetic silencing is responsible for sustained IL-6/STAT-3 signalling in cholangiocarcinoma [10]. Recent reports suggest SOCS-3 may be silenced by epigenetic phenomenon in human cancers, namely methylation of CpG islands [10, 14]. Methylation of cytosine residues within promoter CpG islands is a well-established epigenetic process causing gene silencing. CpG island methylation is an attractive mechanism explaining sustained IL-6 signalling in human cholangiocarcinoma [10, 11, 13].

non in human cancers, namely methylation of CpG islands [10, 14]. Methylation of cytosine residues within promoter CpG islands is a well-established epigenetic process causing gene silencing. CpG island methylation is an attractive mechanism explaining sustained IL-6 signalling in human cholangiocarcinoma [10, 11, 13]. Tumor necrosis factor-a (TNF-a) is a mediator of inflammation with actions directed towards both tissue destruction and recovery. Accumulated evidence suggests that TNF-a may act as an endogenous tumor promoter in addition to its role in immune responses [16, 17]. It was observed overexpression of tumor necrosis factor receptors (TNFR) genes in CC associated with hepatolithiasis [3, 6, 16, 17]. Recently, there has been evidence for implication of chemokines migration in tumor dissemination. Among chemokines, CXC chemokine, stromal cell-derived factor-1 (SDF-1) (CXCL12), and its specific receptor CXCR4 have gained considerable interest because of their roles in carcinogenesis, being involved in the migration or invasion CC [17, 18].

n evidence for implication of chemokines migration in tumor dissemination. Among chemokines, CXC chemokine, stromal cell-derived factor-1 (SDF-1) (CXCL12), and its specific receptor CXCR4 have gained considerable interest because of their roles in carcinogenesis, being involved in the migration or invasion CC [17, 18]. Expression of cyclooxygenase-2 (Cox-2), an inducible enzyme controlling the synthesis of lipid inflammatory mediator prostaglandins, has been reported to be up-regulated in the malignant neoplastic epithelium of intrahepatic cholangionomas of both humans and experimental rodent models [17]. Additional induction of Cox-2 is mediated by bile acids, oxysterols, and iNOS [3]. Cox-2 plays an important role in the cholangiocarcinogenesis as a mediator of mitogenesis, anti-apoptosis and angiogenesis. However, the biologic function and molecular mechanisms of COX-2 in the control of cholangiocarcinoma cell growth have not been well established [18]. Genetic and molecular abnormalities associated in CC As in most of the cancers, multiple genes are involved in molecular transformation of normal functioning liver tissue to malignant cholangiocites [19]. Reactive oxygen species produced during inflammatory processes modify DNA bases and result DNA damage. This reactive species produced also alteration of key repair proteins of the DNA promoting the accumulation of potential oncogenic mutations important in the initiation and/or progression of CC [9].

olangiocites [19]. Reactive oxygen species produced during inflammatory processes modify DNA bases and result DNA damage. This reactive species produced also alteration of key repair proteins of the DNA promoting the accumulation of potential oncogenic mutations important in the initiation and/or progression of CC [9]. Genetic and epigenetic changes Genetic defects such as mutation and deletion can lead to the dysfunction of genes. Although epigenetic alteration does not change DNA sequence, it can affect the expression of genes by chemical modification including DNA methylation and histone deacetyleases [20, 21]. Recent data suggest that both genetic and epigenetic changes are required for transformation, promotion and progression of CC. DNA methylation cooperates with histone deacetyleases in inhibiting transcription [21]. Methylation of multiple tumor suppressor genes is seen in cholangiocarcinoma [22]. The methylation profile of multiple genes in cholangiocarcinoma may facilitate the distinction of cholangiocarcinoma from benign biliary epithelium in clinical settings [23]. Hypermethylation of regulatory regions called CpG islands in some tumor suppressor genes induce their inactivation [21]. Therefore, understanding the molecular events associated with the neoplastic transformation of cholangiocytes to CC may aid in the development of improved therapeutic strategies [21, 22].

DNA methylation cooperates with histone deacetyleases in inhibiting transcription [21]. Methylation of multiple tumor suppressor genes is seen in cholangiocarcinoma [22]. The methylation profile of multiple genes in cholangiocarcinoma may facilitate the distinction of cholangiocarcinoma from benign biliary epithelium in clinical settings [23]. Hypermethylation of regulatory regions called CpG islands in some tumor suppressor genes induce their inactivation [21]. Therefore, understanding the molecular events associated with the neoplastic transformation of cholangiocytes to CC may aid in the development of improved therapeutic strategies [21, 22]. Cell cycle regulators Cyclin and cyclin-dependent kinase complexes are involved in the cell cycle progression. Disruption of the G1/S and G2/M check points leads to uncontrolled cell growth, resulting in the development and progression of cancers (Fig. 2). Over-expressions of cyclins have been found correlate with the tumor relapse of human CC [2, 24]. Overexpression of cyclin B1 which acts at the G2/M phase checkpoint of the cell cycle has found in CC being associated with poor prognosis [20, 24]. Cyclin D1 is considered as oncogene and can promote progression of the cell cycle to S by cyclin D-dependent kinases (CDK4/CDK6)-mediated phosphorylation of the retinoblastoma (Rb) protein. Cyclin D1 overexpression was more frequently observed in cases of intrahepatic cholangiocarcinoma with poor or moderate differentiation and with lymph node metastasis [6]. Cyclin D1 seems to be involved in the carcinogenesis of both biliary intraepithelial neoplasia and intraductal papillary neoplasm of the bile duct [24].

in D1 overexpression was more frequently observed in cases of intrahepatic cholangiocarcinoma with poor or moderate differentiation and with lymph node metastasis [6]. Cyclin D1 seems to be involved in the carcinogenesis of both biliary intraepithelial neoplasia and intraductal papillary neoplasm of the bile duct [24]. Figure 2 Oncogenes and their receptors involved in CC. The p53 tumor suppressor gene is the most common mutated gene in human cancer, occurring in approximately 50% cancers. In CC, p53 mutation has been shown to be present in 28-61% of patients [6, 25, 27]. Located on chromosome 17p13.1, p53 is responsible for cell cycle regulation at the G1/S and G2/M checkpoints. Inactivation of p53 caused by missens mutations or interaction with oncogenic viral proteins allows progression through the cell cycle without a physiological checkpoint and resulting from a selective growth advantage for cancer cells. Alteration of p53 gene plays a key role in late-stage events of tumor pathogenesis and is associated with poor prognosis of CC [25-27], but the others show no association of protein over-expression with outcomes, however the role of p53 remains controversial. Alterations in p53 and p16INK4a are frequently detected in CC and are likely contributing to oncogenesis in the biliary tract. Point mutations in the promoter region of p16INK4a seem to represent an apparent early event associated CC [6].

The p53 tumor suppressor gene is the most common mutated gene in human cancer, occurring in approximately 50% cancers. In CC, p53 mutation has been shown to be present in 28-61% of patients [6, 25, 27]. Located on chromosome 17p13.1, p53 is responsible for cell cycle regulation at the G1/S and G2/M checkpoints. Inactivation of p53 caused by missens mutations or interaction with oncogenic viral proteins allows progression through the cell cycle without a physiological checkpoint and resulting from a selective growth advantage for cancer cells. Alteration of p53 gene plays a key role in late-stage events of tumor pathogenesis and is associated with poor prognosis of CC [25-27], but the others show no association of protein over-expression with outcomes, however the role of p53 remains controversial. Alterations in p53 and p16INK4a are frequently detected in CC and are likely contributing to oncogenesis in the biliary tract. Point mutations in the promoter region of p16INK4a seem to represent an apparent early event associated CC [6]. The retinoblastoma gene encodes a protein, Rb, which blocks transition from G1 to S phase in cell cycle being necessary for prevention of cell replication when DNA has been damaged. The correlation between Rb expression and survival of patients with CC has not been proven [10, 24].

Alterations in p53 and p16INK4a are frequently detected in CC and are likely contributing to oncogenesis in the biliary tract. Point mutations in the promoter region of p16INK4a seem to represent an apparent early event associated CC [6]. The retinoblastoma gene encodes a protein, Rb, which blocks transition from G1 to S phase in cell cycle being necessary for prevention of cell replication when DNA has been damaged. The correlation between Rb expression and survival of patients with CC has not been proven [10, 24]. DCP4 is a tumor suppressor gene involved in transforming growth factor b signalling pathway (TGF-b) [3, 30], TGF-b being a major cell proliferation inhibitor. Loss of DCP4 led to progression in the cell cycle from G1 to S phase with increasing proliferation. Mutational inactivation of DCP4/Smad4, has been found to occur more commonly in distal bile duct cancers (55-60%) than in proximal bile duct and intrahepatic tumors but there was no correlation with survival [20, 28]. It was studied the expression of transformation suppressor gene RECK (reversion-inducing-cysteine-rich protein with kazal motifs) in hilar cholangiocarcinomas and its clinical significance by using reverse transcription-polymerase reaction in 42 paraffin-embedded samples of hilar cholangiocarcinoma and 10 samples of benign bile duct diseases. The abnormal expression of RECK gene might be one of the molecular mechanisms of hilar cholangiocarcinoma metastasis [30]. Others tumor suppressor gene whose expression is modified in CC are p16, p27, p57, SMAD4, p16INK4a, p21WAF1 [4, 6, 20].

ples of hilar cholangiocarcinoma and 10 samples of benign bile duct diseases. The abnormal expression of RECK gene might be one of the molecular mechanisms of hilar cholangiocarcinoma metastasis [30]. Others tumor suppressor gene whose expression is modified in CC are p16, p27, p57, SMAD4, p16INK4a, p21WAF1 [4, 6, 20]. Oncogenes and their receptors Proto-oncogenes encode a wide range of proteins products involved in the control of cell proliferation and differentiation (Fig. 2), including growth factors, growth factors receptors, components of signal transduction pathways and transcription factors [4, 20]. In CC, like in many other malignancies, receptors of tyrosine kinase (RTK) and its ligands are overexpressed. The binding of RTK to their growth factors can induce homodimerize or heterodimerize of the receptors proteins and activate different molecules that encode and regulate cell diferentioation, cell proliferation, cell survial and angiogenesis [3, 19, 20, 29]. RTK appear to be considered as important protooncogenes for cholangiocarcinogenesis, being used as pharmaceutically targeted.

ize or heterodimerize of the receptors proteins and activate different molecules that encode and regulate cell diferentioation, cell proliferation, cell survial and angiogenesis [3, 19, 20, 29]. RTK appear to be considered as important protooncogenes for cholangiocarcinogenesis, being used as pharmaceutically targeted. The trans-membrane RTK of the epidermal growth facteor receptor (EGFR) family plays a significant role in cellular growth and proliferation signalling. Activation of EGFR and its ligands, transforming growth factor alpha (TGF-a) are hypothesized to form an autocrine growth loop and initiates a series of signal transduction cascades that include mitogen-activated protein kinase (MAPK), Akt, and other enzymes [31]. Inhibition of EGFR signaling has been shown to significantly suppress cholangiocarcinoma cell growth [3, 25, 29]. Generally, TGF-a is overexpressed in CC cells [2], more commonly in distal bile duct cancers than in more proximal bile duct and intrahepatic tumors. TGF-β1 expression is low in normal intrahepatic biliary cells, but markedly is increase in inflammatory and obstructive lesions of the bile duct. There are also reports suggesting that the TGF-β1 signaling system plays a role in carcinogenesis and cancer progression [3, 28].

proximal bile duct and intrahepatic tumors. TGF-β1 expression is low in normal intrahepatic biliary cells, but markedly is increase in inflammatory and obstructive lesions of the bile duct. There are also reports suggesting that the TGF-β1 signaling system plays a role in carcinogenesis and cancer progression [3, 28]. The c-erbB-2 proto-oncogene encodes a transmembrane protein which is homologous to the EGFR. Overexpression of c-erbB-2 protein has been reported in many human carcinomas including CC and also in noncancerous biliary proliferative lesions such as hepatolithiasis. These suggest that c-erbB-2 oncogene participates not only in cholangiocarcinogenesis but also in biliary cell proliferation in non-neoplastic conditions [29, 30]. While it now seems apparent that aberrant EGFR and/or ErbB2 expression and signaling is associated with the molecular pathogenesis of intrahepatic cholangiocarcinoma, there is still a significant gap in our knowledge as how to best exploit such alterations in terms of targeted therapies that can then be successfully translated into positive clinical outcomes.

EGFR and/or ErbB2 expression and signaling is associated with the molecular pathogenesis of intrahepatic cholangiocarcinoma, there is still a significant gap in our knowledge as how to best exploit such alterations in terms of targeted therapies that can then be successfully translated into positive clinical outcomes. c-Met is a heterodimeric tyrosine kinase receptor for HGF. It is overexpressed especially in well-differentiated CC being liked to cell invasion, angiogenesis, and tumor differentiation/proliferation [19, 33]. Hepatocyte growth factor (HGF) regulates diverse biological responses including cells proliferation. HGF is a mitogenic factor and its overexpression has found in CC [3]. HGF activates both proliferation and invasion machinery in CC cells, suggesting that HGF might promote their malignant behaviour by concomitant activation of different biological functions [33]. c-Met and HGF antibody directed therapies receptor interaction have been shown biological activity in animal models and human studies [19]. A large number of c-Met tyrosine kinases inhibitors have examined, some of this inhibitors completing Phase I trials and beginning Phase II trials in humans. Due to their side effect have a limited application in CC treatment [19].

interaction have been shown biological activity in animal models and human studies [19]. A large number of c-Met tyrosine kinases inhibitors have examined, some of this inhibitors completing Phase I trials and beginning Phase II trials in humans. Due to their side effect have a limited application in CC treatment [19]. Recently was shown that CC express estrogens receptor (ER–α and –β) directly linked with insulin-like growth factor 1 (IGF1) and IGF1-R (receptor) [21], which could represent a strategy for the management of cholangiocarcinoma. Expression of other growth factors like: platelet–derived growth factor (PDGF), and hepatoma-derived growth factor (HDGF) have shown to be altered during cholangiocarcinogenesis [2, 28]. The k-ras oncogene encodes a family of signal transduction proteins downstream of growth factor receptors. Mutations of k-ras gene activate cellular proliferation and promote cellular growth. K-ras mutations, typically at codon 12, have been reported to be less frequently detected in peripheral CC than in hilar CC. The incidence of K-ras mutations has been reported to be higher in CC patients with lymph node metastasis than in those without lymph node metastasis but no significant correlation with survival has found [4, 6, 20].

cally at codon 12, have been reported to be less frequently detected in peripheral CC than in hilar CC. The incidence of K-ras mutations has been reported to be higher in CC patients with lymph node metastasis than in those without lymph node metastasis but no significant correlation with survival has found [4, 6, 20]. Mucins The transmembrane mucins (MUC) interact by different mechanisms with RTK family receptors and can activate signal transduction. MUC are high molecular weight glycoproteins synthesised by epithelial cells in many organs which form a protective barrier at the mucosal surface or act as transmembrane proteins [25, 29]. Meanwhile the cytoplasmic domain of mucins harbors several tyrosine residues that when phosphorylated may provide critical docking sites for initiating downstream cytoplasmic signaling pathways relevant to cancer development and progression. MUC1 and MUC2 have been shown to function as intramembrane ligand and modulator for ErbB-2. More recently, MUC1 have also shown to modulate TGF-a dependent CC progression but also was reported to facilitate neoplastic development by blocking activation of the intrinsic apoptotic pathways [25, 29].

ment and progression. MUC1 and MUC2 have been shown to function as intramembrane ligand and modulator for ErbB-2. More recently, MUC1 have also shown to modulate TGF-a dependent CC progression but also was reported to facilitate neoplastic development by blocking activation of the intrinsic apoptotic pathways [25, 29]. MUC1 apomucin is frequently expressed in various subtypes of intrahepatic cholangiocarcinoma, including mass-forming and periductal infiltrating forms [6]. In contrast, MUC2, an intestinal-type mucin that is selectively expressed predominantly in well-differentiated and noninvasive mucinous-type intraductal cholangiocarcinomas with gastrointestinal differentiation, predicts a more favourable prognosis [29]. MUC2 is rarely expressed in invasive CC, but is expressed in cystadenocarcinoma [25]. MUC4 was also recently demonstrated to be an independent risk factor for poor prognosis in patients with the mass-forming type of intrahepatic cholangiocarcinoma. MUC5AC, which is a gastric-type mucin was not expressed in mass-forming cholangiocarcinoma [6, 25]. MUC6 showed a good correlation with the survival of CC patients, it may be used as prognostic marker for CC [34]. Apoptosis - a tumour promotion factor Failure of apoptosis is one of the key hallmarks of tumorogenesis. Apoptosis is regulated by two major pathways. One is the extrinsic pathway via death receptors on the cell surface and the other, the intrinsic pathway, depends on mitochondria and is initiated by cytochrome C release [6, 35, 36, 37].

r promotion factor Failure of apoptosis is one of the key hallmarks of tumorogenesis. Apoptosis is regulated by two major pathways. One is the extrinsic pathway via death receptors on the cell surface and the other, the intrinsic pathway, depends on mitochondria and is initiated by cytochrome C release [6, 35, 36, 37]. The extrinsic pathway is initiated through the stimulation of the transmembrane death receptors, belong to the tumor necrosis factor superfamily formed by six death receptors including TNF receptor 1 (TNF-R1), Fas, DR3, DR6, TNF related apoptosis-inducing ligand receptor (TRAIL-R1, TRAIL-R2). In recent years, the role of the Fas/Fas ligand (Fas/FasL) apoptotic signalling pathway in CC has been increasingly investigated [8, 35, 38]. Fas over-expression has observed in CC being associated with tumor differentiation. There are differences in the frequency of Fas expression between intrahepatic and extrahepatic bile duct cancers, which may affect tumor development by activation or deactivation of different tumor-promoting activities [6, 17, 39]. TRAIL expression was upregulated in preneoplastic disease, primary sclerosing cholangitis, human cholangiocarcinoma specimens and acts by promoting cell migration and invasion via a NF-kB-dependent pathway. Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy [20, 40].

TRAIL expression was upregulated in preneoplastic disease, primary sclerosing cholangitis, human cholangiocarcinoma specimens and acts by promoting cell migration and invasion via a NF-kB-dependent pathway. Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy [20, 40]. The intrinsic pathway involved the mitochondria and is regulated by several families including Bcl-2 protein family. There are at least 20 proteins in the Bcl-2 family divided into pro-apoptotic (Bax, Bak, Bok, Bid, Bim, Noxa, Puma, Hrk) and antiapoptotic (Bcl-2, Mcl-1, Bcl-1, A1, Bcl-xL) members [8, 36, 37]. High concentrations of Bcl-2 or Bcl-xL affect the susceptibility of a cell to the induction of apoptosis by altering the ratio of death promoters to suppressors, providing tumour cells with a survival advantage, and permitting expansion of transformed cells harbouring mutations within their genome [36, 37]. Benign and neoplastic biliary epithelium co-expresses the cell survival proteins Bcl-XL and Mcl-1, but not Bcl-2. Through inhibiting apoptosis, the Bcl-xL and Mcl-1 proteins expressed by CC may be contributing to the low efficacy of chemotherapy and radiotherapy in this disease [4, 36, 40]. The role of the Bcl-2 family in CC remains to be established.

thelium co-expresses the cell survival proteins Bcl-XL and Mcl-1, but not Bcl-2. Through inhibiting apoptosis, the Bcl-xL and Mcl-1 proteins expressed by CC may be contributing to the low efficacy of chemotherapy and radiotherapy in this disease [4, 36, 40]. The role of the Bcl-2 family in CC remains to be established. Angiogenesis, invasion and metastasis During tumor invasion, neovascularization (de novo formation of functional microvascular networks) and angiogenesis (pre-existing capillary extension) deliver nutrients and oxygen to malignant cells and help prevent the tumor mass from outgrowing the native vascular network (Fig. 3) [7, 41]. However, there are only few studies that have investigated this area in relation to the prognostic implications in CC [6, 42]. Figure 3 Cholangiocarcinoma tumor angiogenesis. Vascular endothelial growth factor (VEGF or VEGF-A), as well as of the lymphangiogenic factor VEGF-C, has been detected to be overexpressed in the cancerous epithelium of a significant percentage of human intrahepatic cholangiocarcinomas and in human cholangiocarcinoma cell lines [41, 42]. VEGF may contribute to the “angiogenic switch” and malignant phenotype in human cholangiocarcinoma [29, 41]. Several angiogenesis-related factors including angiopoietin-1, angiopoietin-2 and thrombospondin-1 have been studied in CC but no correlation was found with survival [2, 4].

olangiocarcinoma cell lines [41, 42]. VEGF may contribute to the “angiogenic switch” and malignant phenotype in human cholangiocarcinoma [29, 41]. Several angiogenesis-related factors including angiopoietin-1, angiopoietin-2 and thrombospondin-1 have been studied in CC but no correlation was found with survival [2, 4]. Both invasion and neovascularization require extracellular matrix breakdown and the subsequent migration of cells through the degraded structures [25]. Because extracellular matrix remodeling is the major activity of a family of enzymes known as matrix metalloproteinases (MMPs), these enzymes have come under investigation for their contributions to the malignant phenotype [43, 44]. Matrix metalloproteinase MMP-2, MMP-7 and MMP-9, regulated by tissue inhibitor of metalloproteinases TIMP-2, TIMP-7 and TIMP-9, respectively, play important roles in the degradation of the basement membrane during tumor invasion [25, 30, 43, 44]. The activities of MMP-2 and loss of balanced expressions of MMP-2/TIMP-2 and MMP-9/TIMP-1 are suggested as playing important roles in invasive growth related to the gross type of cholangiocarcinoma [45]. A matrix metalloproteinase inhibitor to treat unresectable cholangiocarcinoma has reported [43]. Serum biomarkers for the enhanced detection of cholangiocarcinoma CC is a fatal neoplasm and usually hard to get diagnosed in the early stage due to the unfavourable anatomic location. Therefore, early diagnosis, based on serum markers and the development of novel systemic therapies for advanced disease are very important.

m biomarkers for the enhanced detection of cholangiocarcinoma CC is a fatal neoplasm and usually hard to get diagnosed in the early stage due to the unfavourable anatomic location. Therefore, early diagnosis, based on serum markers and the development of novel systemic therapies for advanced disease are very important. Carcinoembryonic antigen (CEA) is a glycoprotein tumor marker meanwhile carbohydrate antigen 19-9 (CA19-9), is a mucin-type glycoprotein in serum being used for the diagnosis of malignancies in the stomach, colon and pancreas but also for bile duct cancers. Serum CA19-9 was proven to be superior to serum CEA in the diagnosis of cholangiocarcinoma and often considered the standard marker for pancreatic cancer and cholangiocarcinoma [46]. However, these markers are not always helpful, with sensitivity and specificity of approximately 70% and 50%, respectively [3].

cancers. Serum CA19-9 was proven to be superior to serum CEA in the diagnosis of cholangiocarcinoma and often considered the standard marker for pancreatic cancer and cholangiocarcinoma [46]. However, these markers are not always helpful, with sensitivity and specificity of approximately 70% and 50%, respectively [3]. Apolipoprotein A-II (ApoA-II) is the second most abundant protein in high-density lipoproteins (HDL) and is primarily synthesized by liver. In most studies, a constant steady-state level of ApoA-II is essential to maintain functional homeostatic regulation [20, 47]. There are an increasing number of reports suggesting a relationship between cancer susceptibility and the lipid metabolic pathway proteins, including apolipoproteins. Serum levels of ApoA-II were significantly elevated in CC but not other cancer types like oesophagus and ovarian cancers [47]. Some other serum markers, such as: bilirubin, C-reactive protein, sialic acid have been indicated to be available as supplementary markers to CEA and CA 19-9 [20, 47]. MUC1 and MUC4 have been reported as being independent diagnosis and prognostic markers for predicting poor outcomes in patients with mass-forming intrahepatic cholangiocarcinoma [29]. Serum MUC5AC may be used to enhance the diagnostic accuracy of CC [48]. Serum IL-6 concentration is proposed as novel biomarker for diagnosis of cholangiocarcinoma but also for monitoring the response to different therapies [12].

MUC1 and MUC4 have been reported as being independent diagnosis and prognostic markers for predicting poor outcomes in patients with mass-forming intrahepatic cholangiocarcinoma [29]. Serum MUC5AC may be used to enhance the diagnostic accuracy of CC [48]. Serum IL-6 concentration is proposed as novel biomarker for diagnosis of cholangiocarcinoma but also for monitoring the response to different therapies [12]. Conclusions Cholangiocarcinoma continues to be a challenging cancer that requires innovative approaches to permit early diagnosis or prevention in high-risk population. This type of cancer is increasing in its incidence worldwide and at present, there is no standard treatment for this fatal cancer. A better clarification of mechanism linking inflammation and cholangiocarcinogenesis will be beneficial in identification of molecules associated with cholangiocite inflammation, taking in account that is the main risk factor associated with this type of cancer.

nt, there is no standard treatment for this fatal cancer. A better clarification of mechanism linking inflammation and cholangiocarcinogenesis will be beneficial in identification of molecules associated with cholangiocite inflammation, taking in account that is the main risk factor associated with this type of cancer. The molecular mechanisms associated with cancer initiation and progression remains unclear. Further molecular characterization of CC may lead to earlier diagnosis, and the development of more effective therapies. In recent years, proteomics has become a widely developing technique in the field of biotechnology. A primary goal of proteomics is biomarker discovery for various human diseases, especially cancers, and plasma and serum are considered as the sources of choice in molecular diagnostics. The prognostic factors or diagnostic biomarkers of plasma or serum are important trends that deserve attention. Developing new blood biomarkers will help develop more effective therapeutic strategy targeting key signalling. In present are identified new target molecules involved in cholangiocarcinogensis that are in the research stage and are needed to be validate. One novel candidate as biomarker for early diagnosis is circulating IL-6.

Introduction Hepatocellular carcinoma (HCC) is one of the most common malignancies and the third most common cause of cancer death worldwide, with an overall five-year survival rate of approximately 5% [1]. Surgical resection and transplantation have been considered the optimal therapies for long-term control of the disease. However, most patients with HCC present with advanced disease, only 10% to 20% of patients suitable for surgical intervention due to multiple tumors, inadequate liver function, and/or involvement of vascular or major biliary structures. In many trials, transcatheter arterial chemoembolization (TACE) had been shown to improve survival compared to best supportive care in patients with unresectable HCC [2, 3]. However, its benefit is modest and limited. Therefore, it should be considered for combination with other adjuvant treatment. In recent 20 years, HCC belonging to the radiosensitive tumor had been confirmed. With the development of the new radiotherapy technology and facility, the research about brachyhtherapy, especially 125I seeds implantation therapy, has provoked more interests in the world. The purpose of this study was to evaluate the outcome and the prognostic factors of unresectable HCC patients, who failed the TACE, treated with 125I radioactive seeds implantation therapy.

facility, the research about brachyhtherapy, especially 125I seeds implantation therapy, has provoked more interests in the world. The purpose of this study was to evaluate the outcome and the prognostic factors of unresectable HCC patients, who failed the TACE, treated with 125I radioactive seeds implantation therapy. Materials and Methods Patients All patients signed the informed consent prior to their inclusion in the study, which had been reviewed by the appropriate ethics committee and had been performed in accordance with the ethical standards laid down in an appropriate version of the 1964 Declaration of Helsinki. From September 2002 to March 2006, 58 patients with unresectable HCC were referred, in Oncology Center Surgery of Nanjing First Hospital, for 125I seeds implantation. These patients had failed TACE. No patient had received prior radiotherapy for liver disease. Ten patients were excluded owing to Child-Pugh class C or Karnofsky performance status (KPS) < 50. The diagnosis of HCC was based on histological confirmation or on radiography (by CT scan as well as hepatic angiography) and a serum alpha-fetoprotein (AFP) value > 400 ng/ml. Those patients with AFP level < 400 ng/ml underwent liver biopsy for diagnosis. The judgment of TACE failure was based on tumor progression demonstrated on computerized tomography (CT) scan after several sessions. The frequency of TACE was 2-6 sessions (median 4) and time interval between the last TACE and the start of intrahepatic 125I brachytherapy was 4 weeks. Patients’ characteristics are shown in Table 1. Thirty-eight patients were male and 10 female. Mean age was 59 years, ranging from 32 to 86. KPS was 100 in 10 patients, 80 in 21 patients, and 60 in 17 patients. According to Child-Pugh classification for cirrhosis of the liver, 34 patients were in class A and 14 patients in class B. Twenty-two patients had AFP level > 400 ng/ml. Tumor size was defined as the mean of three diameters on CT scan, < 5cm in 17 patients, 5-10 cm in 18 patients, and > 10cm in 13 patients. Thirty-four patients had massive tumors, 14 patients presented single hepatic tumor. The percentage of positive HBV/HCV patient was 79.2% and 20.8%, respectively. Twenty-two patients had Okuda Stage I, 24 patients Stage II, and 2 patients Stage III. According to the AJCC staging system (6th edition), 10 patients were in Stage II (T2N0M0), 20 in Stage IIIa (T3N0M0) and 18 in Stage IIIb (T4N0M0).

tumor. The percentage of positive HBV/HCV patient was 79.2% and 20.8%, respectively. Twenty-two patients had Okuda Stage I, 24 patients Stage II, and 2 patients Stage III. According to the AJCC staging system (6th edition), 10 patients were in Stage II (T2N0M0), 20 in Stage IIIa (T3N0M0) and 18 in Stage IIIb (T4N0M0). Table 1 Patient characteristics (n = 48) Characteristic Number of patients(%) Age (mean) 32-86 years (59) Gender Male (%) 38(79.2%) Female (%) 10(20.8%) KPS 100 10(20.8%) 80 10(20.8%) 60 17(35.4%) Viral antigen HBV 38(79.2%) HCV 10(20.8%) AFP (ng/ml) > 400 22(45.8%) ≤ 400 26(54.2%) Tumor Size < 5 cm 17(35.4%) 5-10 cm 23(47.9%) > 10 cm 8(16.7%) Tumor type Single 14(29.2%) Massive 34(70.8%) Liver Child-Pugh A 34(70.8%) B 14(29.2%) Okuda stage I 22(45.8%) II 24(50%) III 2(4.2%) AJCC stage II 10(20.8%) IIIa 20(41.7%) IIIb 18(37.5%) Prior TACE (%) 2 5(10.4%) 2+ 43(89.6%) TACE median sessions 4(95%CI,4-5) Implantation brachytherapy planning A treatment-planning CT scan was performed that included a portion of the inferior chest and the entire abdomen to allow for planning of non-axial fields. The gross tumor volume (GTV) was defined as high CT value area in early phase contrast-enhanced CT images. The clinical target volume (CTV) was defined as the GTV plus 1 cm. The planning target volume (PTV) was defined as the CTV plus 0.5 cm for daily patient setup variation, and 1cm in the cranial-caudal dimension to account for the ventilatory motion of the liver. Treatment plans were designed for each patient with the high-dose region including the PTV. The postplans were evaluated with both dose-volume histogram (DVH) and the matched peripheral dose (MPD). Table 2 shows the characteristics of 125I brachytherapy. Patients were placed in a supine position with both arms raised above the head and with the head in a natural position. In order to suppress the movement of respiration, patients were immobilized using a low-density body cradle and the breathing of the patient was repressed by applying thermoplastic material on the abdomen. After target-volume determination, interstitial needles (18-gauge, stainless steel, hollow needles, 15 cm long) were inserted into the tumor, approximately 1 cm apart. CT was used to guide the placement of the needles. Precautions were taken to avoid puncture of large blood vessels (central vein and inferior vena cava). Any bleeding was stopped by application of pressure.

s (18-gauge, stainless steel, hollow needles, 15 cm long) were inserted into the tumor, approximately 1 cm apart. CT was used to guide the placement of the needles. Precautions were taken to avoid puncture of large blood vessels (central vein and inferior vena cava). Any bleeding was stopped by application of pressure. A Mick applicator (Radioactive seeds Implantation Instruments, HTA CO., LTD., China) was then sequentially attached to the distal end of each needle to place the 125I seeds (Model-6711, HTA CO., LTD., China) into the tumor, spaced approximately 1 cm apart along the needle track. A median of 60 seeds (range, 30-108) was implanted per patient, with a median activity per seed of 0.7 mCi and a median total implanted activity of 22.6 mCi (range, 10-70 mCi). The 83.3% percent of the patients had only 1 site implanted, whereas 16.7% had 2 or 3 sites implanted. The median MPD was 114Gy (range, 60-160Gy). For most implants, more than 50% of the target volume received between 90 and 120 Gy. Table 2 characteristics of 125I brachytherapy Characteristic Number of patients (%) Activity per seed 0.4 mCi 10 (20.8%) 0.7 mCi 23 (47.9%) 1.0 mCi 15 (31.3%) Number of seeds 30-40 8 (16.7%) 41-60 21 (43.8%) ≥61 18 (37.5%) Total activity 10-20 mCi 10 (20.8%) 21-30 mCi 30 (62.5%) 31-50 mCi 8 (16.7%) MPD 60-80 Gy 12 (25%) 90-120 Gy 29 (60.4%) 130-160 Gy 7 (14.6%) Number of implant sites 1 40(83.3%) 2 5(10.4%) 3 3(6.25%) mCi: millicuries; MPD: matched peripheral dose; Gy: gray.

Number of seeds 30-40 8 (16.7%) 41-60 21 (43.8%) ≥61 18 (37.5%) Total activity 10-20 mCi 10 (20.8%) 21-30 mCi 30 (62.5%) 31-50 mCi 8 (16.7%) MPD 60-80 Gy 12 (25%) 90-120 Gy 29 (60.4%) 130-160 Gy 7 (14.6%) Number of implant sites 1 40(83.3%) 2 5(10.4%) 3 3(6.25%) mCi: millicuries; MPD: matched peripheral dose; Gy: gray. During the treatment, the patients were monitored weekly with physical examination and blood chemistry evaluation. Evaluation of tumor response was based on serial CT scans. All patients had CT scans before initiation of brachytherapy and 4 weeks after completion of radiation therapy and then at 1-3 months intervals. Complete disappearance of hepatic tumor was considered as complete response (CR); decrease of less than 50% of the tumor size as partial response (PR); decrease of more than 50% of the tumor size or no change as stable of disease (SD); and progression as progressive disease (PD). Acute toxicity was evaluated weekly during the treatment and 1 month following the treatment using the Radiation Therapy Oncology Group/European Organization for Research and Treatment of Cancer (RTOG-EORTC) scale. Sub-acute or chronic toxicity was defined as occurring after 1 month. Survival was estimated from the date of diagnosis according to the Kaplan-Meier method. Log rank test was used in the analysis of prognostic factors.

herapy Oncology Group/European Organization for Research and Treatment of Cancer (RTOG-EORTC) scale. Sub-acute or chronic toxicity was defined as occurring after 1 month. Survival was estimated from the date of diagnosis according to the Kaplan-Meier method. Log rank test was used in the analysis of prognostic factors. Results All patients underwent evaluation of tumor response based on CT scan. Among 48 patients with unresectable hepatic tumors, 8 patients achieved CR and 26 patients achieved PR (Table 3). In total, an objective response was observed in 34 of 48 patients, giving a response rate of 70.8%. At the time of last follow-up in March 2006, 9 patients remained alive and 39 were dead. 16 patients (33.3%) developed intrahepatic metastasis outside the radiation field. Extrahepatic metastasis developed in 7 patients (14.6%), including 3 in lung and 4 in bone. There were no treatment-related deaths. Survival rates were evaluated in all patients from the time of diagnosis. Acute toxicity of 125I brachytherapy is summarized in Table 4. The survival rates at 1, 2 and 3 years were 75%, 45.8% and 27.1%, respectively, with a median survival time of 15.5 months (Table 5). Elevation of transaminase was seen in 5 patients (2 patients of grade 1 and 3 of grade 2), bilirubin in 3 patients (1 of grade 1 and 2 of grade 2), albumin in 5 patients (2 of grade 1 and 3 of grade 2) and alkaline phosphatase in 4 patients (2 of grade 1 and 2 of grade 2). Hematologic toxicity included thrombocytopenia in 4 patients (1 of grade 1 and 3 of grade 2), anemia in 7 patients (5 of grade 1 and 2 of grade 2) and leucocytopenia in 6 patients (3 of grade 1 and 3 of grade 2). No patient had radiation-related gastrointestinal bleeding. In the analysis of prognostic factors (Table 6), age, gender, performance status (KPS) did not influence survival significantly (P > 0.05). Tumor type had significant impact on survival rates for single and massive (logrank test, P = 0.0001, Fig. 1). Tumor size had significant impact on survival rates for < 5 cm versus 5-10 cm and versus > 10 cm (logrank test, P = 0.0000, Fig. 2). Okuda stage had significant impact on survival rates for Stage I and Stage II plus III patients (logrank test, P = 0.0011, Fig.3). AJCC stage had significant impact on survival rates for stage II and IIIa+IIIb (logrank test, P=0.0011, Fig.4). Liver Child-Pugh had significant impact on survival rates for Child-A and Child-B (logrank test, P = 0.0000, Fig. 5).

rvival rates for Stage I and Stage II plus III patients (logrank test, P = 0.0011, Fig.3). AJCC stage had significant impact on survival rates for stage II and IIIa+IIIb (logrank test, P=0.0011, Fig.4). Liver Child-Pugh had significant impact on survival rates for Child-A and Child-B (logrank test, P = 0.0000, Fig. 5). Pretreatment AFP levels had a similar impact on survival (logrank test, P = 0.0013, Fig. 6). MPD also had a significant impact on survival (logrank test, P = 0.0223, Fig. 7), indicating MPD 90-120Gy had higher survival rates than which of MPD 60-80Gy and MPD 130-160Gy. Figure 1 Tumor type, Single VS. Massive Figure 2 Tumor size, < 5 cm VS. 5-10 cm VS. >10 cm Figure 3 Okuda stage, I VS. II+III Figure 4 AJCC stage, II VS. IIIa+IIIb Figure 5 Liver Child-Pugh, A VS. B Figure 6 AFP(ng/ml), > 400 VS. ≤ 400 Figure 7 MPD, 60-80 Gy VS. 130-160 Gy VS.90-120 Gy Table 3 Response of unresectable hepatic tumor to 125I brachytherapy Response Number of patients(%) CR 8 (16.7%) PR 26 (54.2%) SD 10 (20.8%) PD 4 (8.3%) CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease

Figure 6 AFP(ng/ml), > 400 VS. ≤ 400 Figure 7 MPD, 60-80 Gy VS. 130-160 Gy VS.90-120 Gy Table 3 Response of unresectable hepatic tumor to 125I brachytherapy Response Number of patients(%) CR 8 (16.7%) PR 26 (54.2%) SD 10 (20.8%) PD 4 (8.3%) CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease Table 4 Acute toxicity of 125I brachytherapy (n = 48) Toxicity grade 1 2 3 4 Transaminase 2 3 - - Bilirubin 1 2 - - Albumin 2 3 - - Alkaline phosphatase 2 2 - - Leucopenia 3 3 - - Anemia 5 2 - - Thrombocytopenia 1 3 - - Table 5 Actuarial overall survival (n = 48) Category Number of Patients (%) Number remaining alive 9 (18.8%) Death of intrahepatic disease 16 (33.3%) Death of extrahepatic disease 7 (14.6%) Death of complications of further Treatment 10 (20.8%) Death of unrelated cause 6 (12.5%) Actuarial survival 1 year 75% 2 year 45.8% 3 year 27.1% Median survival time 15.5 months (95%CI,12.7-27) Table 6 Actuarial overall survival (n = 48) Patient group Survival rate(%) Mean survival (months) P 1-Y 2-Y 3-Y All patients 75% 45.8% 27.1% 24.2 (95% CI, 19.2-29.2) - Age(years) <50 80% 60% 40% 28.9 (95% CI, 14.7-43.1) 0.4683 ≥50 73.7% 42.1% 23.7% 22.9 (95% CI, 17.5-28.4) Gender Male 76.3% 44.7% 26.3% 22.9 (95% CI, 17.7-28.1) 0.5499 Female 70% 50% 30% 29.1 (95% CI, 13.1-45.1) KPS 0.3461 100 80% 60% 40% 28.9 (95% CI, 14.7-43.1) - 80 81% 57.1% 28.6% 26.5 (95% CI, 18.4-34.6) - 60 64.7% 23.5% 17.6% 18.5 (95% CI, 11.2-25.8) - Tumor type Single 92.9% 71.4% 64.3% 41.2 (95% CI, 29.8-52.6) 0.0001 Massive 67.6% 32.4% 11.8% 17.1 (95% CI, 13.7-20.6) Tumor Size 0.0000 <5 cm 94.1% 70.6% 52.9% 35.5 (95% CI, 25.6-45.5) 5-10 cm 82.6% 43.5% 17.4% 21.1 (95% CI, 15.6-26.7) >10 cm 12.5% 0% 0% 8.75 (95% CI, 6.9-10.6) Okuda stage I 95.4% 72.7% 45.5% 33 (95% CI, 25.3-40.7) 0.0011 II+III 57.7% 23.1% 11.5% 16.7 (95% CI, 11.3-22.1) AJCC stage II 90% 80% 70% 41.7 (95% CI, 28-55.4) 0.0011 IIIa+IIIb 71.1% 36.8% 15.8% 19.6 (95% CI, 15.1-24) Liver Child–Pugh A 85.3% 61.8% 35.3% 9.5 (95% CI, 23.4-35.5) 0.0000 B 35.7% 7.1% 7.1% 11.3 (95% CI, 6.6-16) AFP(ng/ml) > 400 59.1% 22.7% 9.1% 16.6 (95% CI, 11.1-22.1) 0.0013 ≤ 400 88.5% 65.4% 42.3% 30.6 (95% CI, 23.2-38) MPD 0.0223 60-80 Gy 66.7% 16.7% 8.3% 15.1 (95% CI, 9.2-21) 90-120 Gy 86.2% 58.6% 34.5% 29.1(95% CI, 22.3-36) 130-160 Gy 42.9% 28.6% 28.6% 20.6 (95% CI, 2-39.1) Discussion 125I brachytherapy for the treatment of prostate carcinoma has been confessed successful [4-7], however, the sample therapy for HCC is still in research stage.

60-80 Gy 66.7% 16.7% 8.3% 15.1 (95% CI, 9.2-21) 90-120 Gy 86.2% 58.6% 34.5% 29.1(95% CI, 22.3-36) 130-160 Gy 42.9% 28.6% 28.6% 20.6 (95% CI, 2-39.1) Discussion 125I brachytherapy for the treatment of prostate carcinoma has been confessed successful [4-7], however, the sample therapy for HCC is still in research stage. Because of the sensitivity of the normal liver limits the dose that can be delivered, radiotherapy has not been considered for the treatment of HCC for some time. However, 125I seeds brachytherapy is an ideal technique that combines the ability to target tumor cells under direct visualization and spare uninvolved liver parenchyma secondary to the sharp dose fall off outside of the implanted volume. Moreover, because of the liver’s natural regenerative capabilities and its ability to tolerate loss of over 75% of its tissue without ensuing failure, high dose of radiation can be delivered to restricted volumes by brachytherapy. With the property of local “conformal radiotherapy”, normal liver is spared, a potentially tumoricidal dose of radiation (much higher than whole-liver tolerance) can be administered with acceptable complications. TACE has been an effective therapeutic options for treatment of unresectable HCC [8], however, its benefit is limited. So, combination with other adjuvant treatment should be necessary. Our study of 48 patients is unique in that it is the largest series reported to date of 125I brachytherapy in the successful treatment of advanced, unresectable HCC, who had failed with TACE, and has the longer follow-up.

owever, its benefit is limited. So, combination with other adjuvant treatment should be necessary. Our study of 48 patients is unique in that it is the largest series reported to date of 125I brachytherapy in the successful treatment of advanced, unresectable HCC, who had failed with TACE, and has the longer follow-up. The prognostic factors of HCC reported in the literature include tumor size, tumor type, tumor stage, serum AFP status, etc. In our study, tumor type, tumor size, Okuda stage, AJCC stage, liver Child–Pugh, AFP level, and MPD had significant impact on survival. Other known factors were not significant. The significance of the radiation dose has been suggested in terms of induction of tumor regression as well as in overall survival. The best prescription dosage of radioactive 125I seed interstitial implantation for HCC and the best radioactivity of seed are still controversial. Ricke J, et al [9-11] thought that the mean minimal dose inside the liver tumor margin amounted to 17-18Gy (range, 10-25Gy); Zhang FJ, et al [12] described that 125I seeds of the radioactivity of 30 MBq, MPD was 100 approximately 150 Gy. These reports strongly support the importance of reasonable MPD in inducing tumor regression. In our study, the MPD had significant impact on survival. The survival rates of patients were higher, with MPD 90-120Gy, than which of less than 80Gy or more than 130Gy (χ2 = 5.22, P = 0.0223). However, it should be mentioned that the function of the non-tumorous part of the liver might be compromised owing to preexisting parenchymal disease, especially cirrhosis of the liver. Most HCC patients referred for radiotherapy present with advanced unresectable disease, usually associated with cirrhosis of the liver. In the report of the University of Michigan group, 128 patients were treated with conformal hyperfractionated RT delivered with concurrent continuous infusion hepatic arterial FUdR. Thirty-eight patients (30%) developed grade 3-4 toxicity, and 5 cases (4%) of radiation-induced liver disease (RILD) were observed. The median survival of 35 HCC patients was 15.2 months [13, 14]. In our study, no sub-acute or chronic toxicity of grade 3-4 were observed and the median survival was 15.5 months. Nevertheless, the encouraging results did not mean that the occurrence of RILD could be ignored. In our group, the patients with Child-Pugh class C or KPS < 50 were excluded.

ents was 15.2 months [13, 14]. In our study, no sub-acute or chronic toxicity of grade 3-4 were observed and the median survival was 15.5 months. Nevertheless, the encouraging results did not mean that the occurrence of RILD could be ignored. In our group, the patients with Child-Pugh class C or KPS < 50 were excluded. During the observation, there was neither treatment-related fatal hepatic toxicity nor radiation-related gastrointestinal complication, including gastroduodenal ulcer and bleeding, 34 patients obtained objective response with a response rate of 70.8%. In reports of Zhang FJ et al [12], 37 of the 45 lesions obtained CR or PR, the response rate was 82.2%, no other severe complications, such as massive hemorrhage, bile fistulae, and pancreatic fistula were seen. Using 125I brachytherapy in our study, treatment plans according to TPS were designed for each patient, in which the high-dose region encompassed the planning target volume and spared normal tissues. Our study had no treatment-related deaths.

such as massive hemorrhage, bile fistulae, and pancreatic fistula were seen. Using 125I brachytherapy in our study, treatment plans according to TPS were designed for each patient, in which the high-dose region encompassed the planning target volume and spared normal tissues. Our study had no treatment-related deaths. In conclusion, permanent 125I brachytherapy induced a substantial tumor response rate of 70.8% with survival rates at 1, 2 and 3 years of 75%, 45.8% and 27.1%, respectively, and a median survival time of 15.5 months in patients with unresectable HCC who had failed TACE. Patients with massive tumor, tumor size ≥ 5cm, Okuda stage II/III, AJCC stage III, Liver Child–Pugh B, pretreatment AFP level of >400 ng/ml, and MPD ≤ 80Gy or ≥ 130Gy had significantly shorter survival. With the property of local “conformal radiotherapy” and the advantages of minimal invasion, convenience, high performance, slight adverse effect, permanent 125I seeds implantation is a safe and effective adjuvant treatment for unresectable HCC. The complications are acceptable and can be managed with conservative treatment. Although we do not know whether there is a long-term survival benefit through the use of this treatment, permanent 125I brachytherapy seems to be an important alternative to other locally ablative techniques for this subset of patients. Further study is warranted to evaluate the survival of such patients with controlled trials. Acknowledgements The authors declare no commercial associations or conflict of interests related to this article.

Introduction Many kinds of pathologic lesions occur in the esophagus. They include esophageal atresia, heterotopic gastric mucosa, heterotopic pancreatic tissue, diverticula, esophageal cyst, achalasia, Lye stricture, reflex esophagitis, Barrett’s esophagus, dysplasia and carcinoma in Barrett’s esophagus, Herpes simplex esophagistis, cytomegalovirus esophagitis, eosiphophilic esophagitis, Crohn’s disease, candidiasis, squamous cell carcinoma, carcinoma in situ, intraepithelial neoplasm (dysplasia), sarcomatoid carcinoma, verrucous carcinoma, adenocarcinoma, adenosquamous carcinoma, mucoepidermoid carcinoma, basaloid carcinoma, small cell carcinoma, leiomyoma, leiomyosarcoma, gastrointestinal stromal tumor, carcinoid tumor, lymphoepithelioma-like carcinoma, glycogenic acanthosis, amyloidosis, squamous papilloma, hyperplastic polyp, granular cell tumor, malignant melanoma, malignant lymphoma, plasmacytoma, malignant mesenchymal tumors, and metastatic carcinoma [1, 2]. Recent advances in endoscopy have made it possible that these esophageal lesions are biopsied and diagnosed correctly. In the present study, the authors reviewed 950 archival cases of the esophageal biopsies in search for esophageal cancers.