DOG1 overexpression is associated with mismatch repair deficiency and BRAF mutations but unrelated to cancer progression in colorectal cancer.

INTRODUCTION: The transmembrane channel protein DOG1 (Discovered on GIST1) is normally expressed in the gastrointestinal interstitial cells of Cajal and also in gastrointestinal stroma tumors arising from these cells. However, there is also evidence for a relevant role of DOG1 expression in colorectal cancers. This study was undertaken to search for associations between DOG1 expression and colon cancer phenotype and key molecular alterations. METHODS: A tissue microarray containing samples from more than 1,800 colorectal cancer patients was analyzed by immunohistochemistry. RESULTS: DOG1 immunostaining was detected in 503 (30.2%) of 1,666 analyzable colorectal cancers and considered weak in 360 (21.6%), moderate in 78 (4.7%), and strong in 65 (3.9%). Strong DOG1 immunostaining was associated with advanced pT stage (p=0.0367) and nodal metastases (p=0.0145) but these associations were not retained in subgroups of 1,135 mismatch repair proficient and 86 mismatch repair deficient tumors. DOG1 positivity was significantly linked to several molecular tumor features including mismatch repair deficiency (p=0.0034), BRAF mutations (p<0.0001), nuclear p53 accumulation (p=0.0157), and PD-L1 expression (p=0.0199) but unrelated to KRAS mutations and the density of tumor infiltrating CD8 positive lymphocytes. CONCLUSION: Elevated DOG1 expression is frequent in colorectal cancer and significantly linked to important molecular alterations. However, DOG1 overexpression is largely unrelated to histopathological parameters of cancer aggressiveness and may thus not serve as a prognostic parameter for this tumor entity.

Mucin 5AC expression is common but unrelated to tumor progression in pancreatic adenocarcinoma.

Introduction: Mucin 5AC (MUC5AC) belongs to the family of secreted gel-forming mucins. It is physiologically expressed in some normal mucin producing epithelial cells but also in pancreatic, ovarian, and colon cancer cells. The role of MUC5AC expression in cancer is not fully understood. This study was designed to explore the role of MUC5AC for pancreatic cancer progression, its association to microsatellite instability, and its diagnostic utility. Methods: Mucin 5AC expression was studied immunohistochemically in a tissue microarray (TMA) from 532 pancreatic cancers, 61 cancers of the ampulla Vateri, six acinar cell carcinomas and 12 large sections of pancreatitis. Results: Mucin 5AC staining was interpretable in 476 of 599 (79%) arrayed cancers. Staining was completely absent in normal pancreas and pancreatitis, but frequent in pancreatic cancer. Membranous and cytoplasmic MUC5AC expression was most common in pancreatic adenocarcinomas (71% of 423), followed by carcinomas of the ampulla Vateri (43% of 47), and absent in six acinar cell carcinomas. Mucin 5AC expression was unrelated to tumor phenotype (tumor stage, tumor grade, lymph node, and distant metastasis), and microsatellite instability in ductal adenocarcinomas and carcinomas of the ampulla Vateri. Conclusion: Our study indicates that MUC5AC is an excellent biomarker for pancreatic cancer diagnosis, especially to support the sometimes-difficult diagnosis on small biopsies. Mucin 5AC expression is unrelated to pancreatic cancer aggressiveness.

DOG1 is commonly expressed in pancreatic adenocarcinoma but unrelated to cancer aggressiveness.

BACKGROUND: DOG1 (ANO1; TMEM16A) is a voltage-gated calcium-activated chloride and bicarbonate channel. DOG1 is physiologically expressed in Cajal cells, where it plays an important role in regulating intestinal motility and its expression is a diagnostic hallmark of gastrointestinal stromal tumors (GIST). Data on a possible role of DOG1 in pancreatic cancer are rare and controversial. The aim of our study was to clarify the prevalence of DOG1 expression in pancreatic cancer and to study its association with parameters of cancer aggressiveness. METHODS: DOG1 expression was analyzed by immunohistochemistry in 599 pancreatic cancers in a tissue microarray format and in 12 cases of pancreatitis on large tissue sections. RESULTS: DOG1 expression was always absent in normal pancreas but a focal weak expression was seen in four of 12 cases of pancreatitis. DOG1 expression was, however, common in pancreatic cancer. Membranous and cytoplasmic DOG1 expression in tumor cells was highest in pancreatic ductal adenocarcinomas (61% of 444 interpretable cases), followed by cancers of the ampulla Vateri (43% of 51 interpretable cases), and absent in 6 acinus cell carcinomas. DOG1 expression in tumor associated stroma cells was seen in 76 of 444 (17%) pancreatic ductal adenocarcinomas and in seven of 51 (14%) cancers of the ampulla Vateri. Both tumoral and stromal DOG1 expression were unrelated to tumor stage, grade, lymph node and distant metastasis, mismatch repair protein deficiency and the density of CD8 positive cytotoxic T-lymphocytes in the subgroups of ductal adenocarcinomas and cancers of ampulla Vateri. Overall, the results of our study indicate that DOG1 may represent a potential biomarker for pancreatic cancer diagnosis and a putative therapeutic target in pancreatic cancer. However, DOG1 expression is unrelated to pancreatic cancer aggressiveness.

High density of cytotoxic T-lymphocytes is linked to tumoral PD-L1 expression regardless of the mismatch repair status in colorectal cancer.

BACKGROUND: Immune checkpoint-inhibitors targeting the PD-1/PD-L1 system are FDA approved in microsatellite instable (MSI) or mismatch repair deficient (dMMR) colorectal cancer (CRC). PD-L1 expression is tightly linked to features connected to immune checkpoint inhibitor response, but studies on large subsets of cancers analyzing the correlation between different status of MSI/dMMR, tumor infiltrating lymphocytes and PD-L1 expression are still lacking. METHODS: More than 1800 CRC were analyzed for PD-L1 by immunohistochemistry in a tissue microarray format. Data were compared to MMR, the number of intratumoral CD8+ cytotoxic T-cells, and adverse clinico-pathological parameters. Different cutoff levels for defining PD-L1 positivity in tumor cells (1%, 5%, 10%, and 50%) yielded comparable results. RESULTS: At a cutoff level of 5%, PD-L1 positivity was seen in 5.1% of tumors. PD-L1 was more often positive in dMMR (18.6%) than in MMR proficient (pMMR) cancers (4.1%; p < 0.0001). The number of intratumoral CD8+ lymphocytes was strikingly higher in PD-L1 positive (939.5 ± 118.2) than in PD-L1 negative cancers (310.5 ± 24.8). A higher number of intratumoral CD8+ lymphocytes was found in dMMR CRC (PD-L1 positive: 1999.7 ± 322.0; PD-L1 negative: 398.6 ± 128.0; p < 0.0001) compared to pMMR CRC (PD-L1 positive: 793.2 ± 124.8; PD-L1 negative: 297.2 ± 24.2; p < 0.0001). In dMMR and pMMR CRC, PD-L1 expression in tumor cells was unrelated to tumor stage, lymph node status or lymphatic/venous invasion. PD-L1 positivity in tumor associated immune cells was seen in 47.5% of cases and was significantly linked to high numbers of tumor infiltrating CD8+, low tumor stage, and absence of lymph node metastasis and lymphatic/venous invasion (p < 0.0001 each). CONCLUSION: The data support the previously suggested fact that PD-L1 expression in tumor cells is driven by extensive cytotoxic T-cell infiltration in highly immunogenic dMMR and pMMR CRC. Frequent and intense PD-L1 expression in tumor cells of dMMR CRC may contribute to the high response rates of dMMR CRC to immune checkpoint-inhibitors.

Mesothelin is Commonly Expressed in Pancreatic Adenocarcinoma but Unrelated to Cancer Aggressiveness.

Data on Mesothelin (MSLN) expression in human normal and cancerous tissues is controversial. We employed immunohistochemistry (IHC) on a tissue microarray (TMA) from 599 pancreatic cancers and 12 large tissue sections of pancreatitis. MSLN expression was highest in pancreatic adenocarcinomas (89%) and adenocarcinomas of the ampulla Vateri (79%), infrequent in pancreatitis and absent in 6 acinus cell carcinomas and normal pancreas. MSLN expression was unrelated to pathological tumor stage, grade, metastasis, and tumor-infiltrating CD8+ lymphocytes. In conclusion, pancreatic cancer may be ideally suited for putative anti- MSLN therapies, and MSLN may represent a suitable biomarker for pancreatic cancer diagnosis, especially on small biopsies.

Elevated MUC5AC expression is associated with mismatch repair deficiency and proximal tumor location but not with cancer progression in colon cancer.

Mucin 5AC (MUC5AC) is a secreted gel-forming mucin expressed by several epithelia. In the colon, MUC5AC is expressed in scattered normal epithelial cells but can be abundant in colorectal cancers. To clarify the relationship of MUC5AC expression with parameters of tumor aggressiveness and mismatch repair deficiency (dMMR) in colorectal cancer, a tissue microarray containing 1812 colorectal cancers was analyzed by immunohistochemistry. MUC5AC expression was found in 261 (15.7%) of 1,667 analyzable colorectal cancers. MUC5AC expression strongly depended on the tumor location and gradually decreased from proximal (27.4% of cecum cancers) to distal (10.6% of rectal cancers; p < 0.0001). MUC5AC expression was also strongly linked to dMMR. dMMR was found in 21.3% of 169 cancers with MUC5AC positivity but in only 4.6% of 1051 cancers without detectable MUC5AC expression (p < 0.0001). A multivariate analysis showed that dMMR status and tumor localization predicted MUC5AC expression independently (p < 0.0001 each). MUC5AC expression was unrelated to pT and pN status. This also applied to the subgroups of 1136 proficient MMR (pMMR) and of 84 dMMR cancers. The results of our study show a strong association of MUC5AC expression with proximal and dMMR colorectal cancers. However, MUC5AC expression is unrelated to colon cancer aggressiveness.

Case report: acute abdominal pain in a 37-year-old patient and the consequences for his family.

BACKGROUND: Hereditary diffuse gastric cancer is a rare condition that accounts for approximately 1-3% of all gastric cancer cases. Due to its rapid and invasive growth pattern, it is associated with a very poor prognosis. As a result, comprehensive genetic testing is imperative in patients who meet the current testing criteria in order to identify relatives at risk. This case report illustrates the substantial benefit of genetic testing in the family of a patient diagnosed with hereditary diffuse gastric cancer. CASE PRESENTATION: A 37-year-old patient was admitted to the emergency department with acute abdominal pain. Following explorative laparoscopy, locally advanced diffuse gastric cancer was diagnosed. The indication for genetic testing of CDH1 was given due to the patient's young age. A germline mutation in CDH1 was identified in the index patient. As a result, several family members underwent genetic testing. The patient's father, brother and one aunt were identified as carriers of the familial CDH1 mutation and subsequently received gastrectomy. In both the father and the aunt, histology of the surgical specimen revealed a diffuse growing adenocarcinoma after an unremarkable preoperative gastroscopy. CONCLUSION: Awareness and recognition of a potential hereditary diffuse gastric cancer can provide a substantial health benefit not only for the patient but especially for affected family members.

MMR Deficiency is Homogeneous in Pancreatic Carcinoma and Associated with High Density of Cd8-Positive Lymphocytes.

BACKGROUND: Microsatellite instability (MSI) has emerged as a predictive biomarker for immune checkpoint inhibitor therapy. Cancer heterogeneity represents a potential obstacle for the analysis of predicitive biomarkers. MSI has been reported in pancreatic cancer, but data on the possible extent of intratumoral heterogeneity are lacking. METHODS: To study MSI heterogeneity in pancreatic cancer, a tissue microarray (TMA) comprising 597 tumors was screened by immunohistochemistry with antibodies for the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6. RESULTS: In six suspicious cases, large section immunohistochemistry and microsatellite analysis (Bethesda panel) resulted in the identification of 4 (0.8%) validated MSI cases out of 480 interpretable pancreatic ductal adenocarcinomas. MSI was absent in 55 adenocarcinomas of the ampulla of Vater and 7 acinar cell carcinomas. MMR deficiency always involved MSH6 loss, in three cases with additional loss of MSH2 expression. Three cancers were MSI-high and one case with isolated MSH6 loss was MSS in PCR analysis. The analysis of 44 cancer-containing tumor blocks revealed that the loss of MMR protein expression was always homogeneous in affected tumors. Automated digital image analysis of CD8 immunostaining demonstrated markedly higher CD8 + tumor infiltrating lymphocytes in tumors with (mean = 685, median = 626) than without (mean = 227; median = 124) MMR deficiency (p < 0.0001), suggesting a role of MSI for immune response. CONCLUSIONS: Our data suggest that MSI occurs early in a small subset of ductal adenocarcinomas of the pancreas and that immunohistochemical MMR analysis on limited biopsy or cytology material may be sufficient to estimate MMR status of the entire cancer mass.

Transvaginal specimen removal after laparoscopic distal pancreatic resection.

PURPOSE: Transvaginal specimen removal has been introduced 20 years ago but then abandoned. With the advent of transvaginal interventions following the introduction of natural orifice transluminal endoscopic surgery, renewed interest was generated for hybrid procedures with minimal access for the intervention and use of transvaginal (TV) specimen removal. We present the first such series after laparoscopic distal pancreatectomy. METHODS: In seven subsequent women (median age 48 years) with body and tail pancreatic tumors undergoing laparoscopic distal pancreatectomy, the new method of TV specimen removal was applied. The patients' data and the technical successes as well as intra- and postprocedural complications were recorded prospectively. The patients were followed after discharge for gynecological examination. RESULTS: Specimen removal consisting of the pancreas and spleen in five and the pancreas only in two cases was technically successful; no intraoperative complications were encountered. Postoperative complications consisted of one case of intra-abdominal hemorrhage and one case of pancreatic fistula, attributable to the resection and not to TV specimen removal. Gynecological follow-up was normal in all seven patients. CONCLUSIONS: The technique of TV specimen removal is feasible and safe also after laparoscopic distal pancreatectomy. It may help to further diminish the access trauma of laparoscopic pancreatic surgery.

The EURO-NOTES clinical registry for natural orifice transluminal endoscopic surgery: a 2-year activity report.

BACKGROUND: The EURO-NOTES Clinical Registry (ECR) was established as a European database to allow the monitoring and safe introduction of Natural Orifice Transluminal Endoscopic Surgery (NOTES). The aim of this study was to analyze different techniques applied and relative results during the first 2 years of the ECR. METHODS: The ECR was designed as a voluntary database with online access. All members of the European Society for Gastrointestinal Endoscopy and the European Association for Endoscopic Surgery were requested to participate in the registry. Demographic and therapy data as well as data on the postoperative course are recorded in the ECR in an anonymous way. RESULTS: A total of 533 patients who underwent NOTES procedures were included in the study. Four different hybrid techniques for 435 cholecystectomies were described, registering postoperative complications in 2.8% of patients, addition of a single trocar in 5.3%, and conversions to laparoscopy in 0.5%. Both flexible endoscopic and rigid laparoscopic cholecystectomy techniques proved to be safe and effective with minor differences. There was a shorter operative time in the rigid laparoscopic group. Thirty-three appendectomies were reported by transgastric and transvaginal techniques, with transvaginal techniques scoring shorter operative time and hospital stay, but with a frequent need to add more trocars. Overall complications occurred in 14.7% of patients but they did not differ significantly among the different techniques. One transvaginal and 31 transanal sigmoidectomies were included for prolapse and diverticulitis, with four postoperative complications (12.5%), but none needing further treatment. Twenty peroral esophageal myotomies were included with three postoperative complications (15.0%), but none needing further treatment. CONCLUSIONS: Five years since the introduction of NOTES into clinical practice, hybrid techniques have gained considerable clinical application. Several NOTES hybrid cholecystectomy and appendectomy techniques are practicable and safe alternatives to laparoscopic procedures. Also, sigmoidectomies and peroral esophageal myotomies were described, proving feasibility and safety. Nevertheless, the real benefit of NOTES for patients still needs to be assessed.

Is the transvaginal route appropriate for intra-abdominal NOTES procedures? Experience and follow-up of 222 cases.

BACKGROUND: Since 2007, natural orifice transluminal endoscopic surgery (NOTES) has been applied in humans. We performed this prospective study to evaluate the transvaginal route in terms of risks, complication rate, and long-term side effects such as dyspareunia. METHODS: From June 2007 to September 2011, we performed 222 transvaginal hybrid NOTES procedures: 220 transvaginal cholecystectomies (TVC) and 2 transvaginal appendectomies (TVA). All patients were asked to present to our associated gynecologists within 1 week for an examination. After at least 3 months, the patients were interviewed using a standard questionnaire. RESULTS: All operations could be successfully performed in this technique except two cases, which were converted to conventional laparoscopic cholecystectomy. The only intraoperative complication was the puncture of the urine bladder. We observed two postoperative complications: one biliary fistula 3 days after TVC, and one abscess in the Douglas pouch 3 weeks after TVC. The gynecological examinations revealed no abnormalities. The interview (median postoperative time, 6 months) with a follow-up rate of 93 % revealed no pain in the pelvis, dyspareunia, or sexual dysfunction after TVC. CONCLUSIONS: The transvaginal route is appropriate for NOTES procedures; there is only a minor and acceptable rate of intra- and postoperative morbidity.

NOTES cholecystectomy: matched-pair analysis comparing the transvaginal hybrid and conventional laparoscopic techniques in a series of 216 patients.

BACKGROUND: Natural orifice transluminal endoscopic surgery (NOTES) is currently a very important topic for both gastroenterologists and surgeons. We have developed a technique of transvaginal hybrid NOTES cholecystectomy (TVC) that leaves no visible scar and is applicable to daily use. This technique is compared to the conventional laparoscopic cholecystectomy (CLC) in a matched-pair analysis. METHODS: From June 2007 until February 2009, 108 NOTES cholecystectomies were performed. For a matched-pair analysis we first selected a group of 192 female patients who had undergone CLC and who were operated on by the same group of surgeons in the same time period. Then 108 pairs who had TVC were matched according to the degree of inflammation of the gallbladder and age. We were able to contact 208 patients at least 3 months after surgery. Hence, the study analysis was performed with 100 complete pairs. RESULTS: All 200 cholecystectomies were performed successfully without conversion. The TVC procedure was significantly longer than CLC (52 vs. 35 min, p<0.001). There were no intraoperative complications in either group. There were no significant differences with respect to reoperations, wound infections, consumption of analgesic drugs, length of hospital stay, and sick leave. Seventy-five TVC and 73 CLC patients had sexual intercourse after the operation without any complaints. CONCLUSION: We present here the largest series of NOTES for cholecystectomy published to date and the first comparative study with the gold standard. The TVC technique is as successful as the CLC, it causes no more complications than CLC, especially with respect to the vaginal approach, it is more time-consuming to perform, but has an ideal cosmetic result, i.e., no visible scar.

Single-access surgery laparoscopic cholecystectomy and appendectomy.

The objective of this study was to achieve an ideal cosmetic result and minimize the access trauma to the abdominal wall. The authors developed a technique to perform cholecystectomies and appendectomies with only one incision in the umbilicus. With the upcoming idea of Natural Orifice Transluminal Endoscopic Surgery (NOTES) in the recent years and noticing the lack of feasibility of the technique for the daily routine beside the technique described by these authors, another development was raising the interest of the surgeons around the world. The single-access surgery through the umbilicus is a technique, that can be used in the daily routine and provides the best cosmetic results. Furthermore, injury of the abdominal wall is located at only one site, which might reduce the rate of trocar hernias and abdominal wall infections. Two 5.5-mm trocars were inserted through an incision at the upper edge of the umbilicus. After perforation of the abdominal wall with a stylet of a 5.5-mm trocar, a curved grasper was inserted, without the use of a trocar, into the abdominal cavity. The use of curved instruments facilitates better triangulation and instrument handling. No gas leakage was observed due to the nonexistence of a trocar. Dissection of the Calot' s triangle or appendix vessels can be done with standard instruments. The curved grasper allows retraction of the gallbladder or appendix. The specimen can be removed through the umbilical incision. The authors present a single-access surgery technique for cholecystectomies and appendectomies using curved instruments. The single-access surgery with parallel inserted curved instruments is feasible. No additional complications are related to this modification other than those known to be associated with laparoscopic surgery. This method offers an almost scarless surgery. Whether other advantages such as less trocar hernias, wound infections, and/or a faster recovery can be achieved, it has not yet to be proven.