A defunctioning stoma can become necessary in a relevant number of patients undergoing gastrointestinal surgery. As a matter of course, patients seek an early closure of the stoma. However, preoperative management of these patients varies and the prediction of continence after stoma removal can become challenging. Patients might be fully continent despite low manometric pressures and vice versa. An easy and reliable way to predict continence after stoma reversal would improve patients’ management and outcome. Although frequently performed in various surgical centers in Germany, there is no published data on the water-holding test. Hence, this is the first study evaluating the role of the test in clinical practice.

Background Anastomotic leakage after rectal resection represents a severe complication for the patient and requires an early and appropriate management. Endoscopic vacuum therapy (EVT) has become the treatment of choice for anastomotic leakage after rectal resection in several institutions in Germany, and commercially available systems are currently distributed in approximately 30 countries worldwide. However, there is no evidence that EVT is superior to any other treatment for anastomotic leakage after rectal resection. Methods Twenty-one patients treated with EVT for anastomotic leakage after rectal resection were retrospectively compared to a historical cohort of 41 patients that received conventional treatment. Primary endpoints were death, treatment success and long-term preservation of intestinal continuity. Secondary endpoints were length of hospital stay and duration of treatment. Results There was no difference in mortality ( p = 0.624). The intention-to-treat analysis showed a significantly higher success rate of EVT compared to conventional treatment (95.2% vs. 65.9%, p = 0.011). EVT was associated with preservation of intestinal continuity in a significant higher percentage of patients than patients undergoing conventional treatment (86.7% vs. 37.5%, p = 0.001). Conventional treatment tended to a shorter length of hospital stay (31.1 vs. 42.2 days, p = 0.066) but with no difference in overall duration of treatment. Time until closing of a diverting stoma did not differ between groups (10.2 months in the EVT group vs. 9.4 months in the conventional treatment group, p = 0.721). Conclusion According to this retrospective study, conventional therapy and EVT are both options for the treatment of anastomotic leakage after rectal resection. EVT might be more effective in terms of definite healing and preservation of intestinal continuity.

Background Several DNA viruses are highly suspicious to have oncogenic effects in humans. This study investigates the presence of potentially oncogenic viruses such as SV40, JCV, BKV and EBV in patient-derived colorectal carcinoma (CRC) cells typifying all molecular subtypes of CRC.Methods Sample material (gDNA and cDNA) of a total of 49 patient-individual CRC cell lines and corresponding primary material from 11 patients, including normal, tumor-derived and metastasis-derived tissue were analyzed for sequences of SV40, JVC, BKV and EBV using endpoint PCR. In addition, the susceptibility of CRC cells to JCV and BKV was examined using a long-term cultivation approach of patient-individual cells in the presence of viruses.Results No virus-specific sequences could be detected in all specimens. Likewise, no morphological changes were observed and no evidence for viral infection or integration could be provided after long term CRC cell cultivation in presence of viral particles.Conclusions In summary, the presented data suggest that there is no direct correlation between tumorigenesis and viral load and consequently no evidence for a functional role of the DNA viruses included into this analysis in CRC development.

Background Several DNA viruses are highly suspicious to have oncogenic effects in humans. This study investigates the presence of potentially oncogenic viruses such as SV40, JCV, BKV and EBV in patient-derived colorectal carcinoma (CRC) cells typifying all molecular subtypes of CRC. Methods Sample material (gDNA and cDNA) of a total of 49 patient-individual CRC cell lines and corresponding primary material from 11 patients, including normal, tumor-derived and metastasis-derived tissue were analyzed for sequences of SV40, JVC, BKV and EBV using endpoint PCR. In addition, the susceptibility of CRC cells to JCV and BKV was examined using a long-term cultivation approach of patient-individual cells in the presence of viruses. Results No virus-specific sequences could be detected in all specimens. Likewise, no morphological changes were observed and no evidence for viral infection or integration could be provided after long term CRC cell cultivation in presence of viral particles. Conclusions In summary, the presented data suggest that there is no direct correlation between tumorigenesis and viral load and consequently no evidence for a functional role of the DNA viruses included into this analysis in CRC development.

BACKGROUND:Neoadjuvant radiochemotherapy (nRCT) is an important component in the treatment of advanced rectal cancer. Endoscopic vacuum therapy (EVT) has become the treatment of choice for anastomotic leakage after rectal resection in many institutions in Germany. Published case series report on average success and stoma reversal rates of more than 80%. However, so far, there is no distinct report on the potential influence of nRCT on EVT. METHODS:A total of 11 patients treated with EVT for anastomotic leakage after nRCT and rectal resection were retrospectively compared with a cohort of eight patients with rectal anastomotic leakage without neoadjuvant treatment. Primary endpoints were death, treatment success, and long-term preservation of intestinal continuity. Secondary endpoint was the duration of treatment. Statistical analysis was performed using Statistical Package for Social Science (SPSS) version 23.0. RESULTS:There was no difference in mortality (0%), success rate (90.9% versus 100%, p = 0.381), or long-term preservation of continuity (63.6% versus 62.5%, p = 0.960). After nRCT, patients showed a significant longer duration of EVT (31.1 days versus 15.9 days, p = 0.040) which was associated with a significantly higher number of sponge applications (9.6 versus 5.0, p = 0.042). CONCLUSIONS:In our analysis, EVT showed success in over 90% of patients with anastomotic leakage after rectal resection for colorectal cancer, regardless of neoadjuvant treatment. However, in case of anastomotic leakage, nRCT seems to be associated with the need for a significant longer duration of EVT.

Despite the importance of tumor infiltrating B cells (TiBc) in immunological circuits, their functional role is scarcely investigated. Here, we analyzed immunoglobulin (Ig) secretion of the subtypes IgA, IgG, and IgM of TiBc from freshly resected primary and secondary colorectal carcinomas (CRC) by FluoroSpot (n = 30 CRC) directly ex vivo. High, intermediate, and low secretion was observed in 33%, 37%, and 30% of the tumors for IgA; in 10%, 27%, and 63% for IgG; and in 21%, 36%, and 50% for IgM, respectively. These ex vivo data validate our previous findings: Most TiBc present in the CRC microenvironment are functional since they produce and actively secrete Ig (IgA > IgG > IgM). Of note, the presence of major histocompatibility complex (MHC) class II expressing cells in the tumor micromilieu only correlated with IgG secretion (p = 0.0004). Supporting recent findings in several other tumor entities, TiBc in CRC thus likely can contribute to tumor control in a dual role of sole antigen-presentation and additionally anti-tumoral Ig-production.

AIM:To establish patient-individual tumor models of rectal cancer for analyses of novel biomarkers, individual response prediction and individual therapy regimens.METHODS:Establishment of cell lines was conducted by direct in vitro culturing and in vivo xenografting with subsequent in vitro culturing. Cell lines were in-depth characterized concerning morphological features, invasive and migratory behavior, phenotype, molecular profile including mutational analysis, protein expression, and confirmation of origin by DNA fingerprint. Assessment of chemosensitivity towards an extensive range of current chemotherapeutic drugs and of radiosensitivity was performed including analysis of a combined radio- and chemotherapeutic treatment. In addition, glucose metabolism was assessed with 18F-fluorodeoxyglucose (FDG) and proliferation with 18F-fluorothymidine.RESULTS:We describe the establishment of ultra-low passage rectal cancer cell lines of three patients suffering from rectal cancer. Two cell lines (HROC126, HROC284Met) were established directly from tumor specimens while HROC239 T0 M1 was established subsequent to xenografting of the tumor. Molecular analysis classified all three cell lines as CIMP-0/ non-MSI-H (sporadic standard) type. Mutational analysis revealed following mutational profiles: HROC126: APCwt , TP53wt , KRASwt , BRAFwt , PTENwt ; HROC239 T0 M1: APCmut , P53wt , KRASmut , BRAFwt , PTENmut and HROC284Met: APCwt , P53mut , KRASmut , BRAFwt , PTENmut . All cell lines could be characterized as epithelial (EpCAM+) tumor cells with equivalent morphologic features and comparable growth kinetics. The cell lines displayed a heterogeneous response toward chemotherapy, radiotherapy and their combined application. HROC126 showed a highly radio-resistant phenotype and HROC284Met was more susceptible to a combined radiochemotherapy than HROC126 and HROC239 T0 M1. Analysis of 18F-FDG uptake displayed a markedly reduced FDG uptake of all three cell lines after combined radiochemotherapy.CONCLUSION:These newly established and in-depth characterized ultra-low passage rectal cancer cell lines provide a useful instrument for analysis of biological characteristics of rectal cancer.

AIM To establish cell line and patient-derived xenograft (PDX) models for neuroendocrine carcinomas (NEC) which is highly desirable for gaining insight into tumor development as well as preclinical research including biomarker testing and drug response prediction. METHODS Cell line establishment was conducted from direct in vitro culturing of colonic NEC tissue (HROC57). A PDX could also successfully be established from vitally frozen tumor samples. Morphological features, invasive and migratory behavior of the HROC57 cells as well as expression of neuroendocrine markers were vastly analyzed. Phenotypic analysis was done by microscopy and multicolor flow cytometry. The extensive molecular-pathological profiling included mutation analysis, assessment of chromosomal and microsatellite instability; and in addition, fingerprinting (i.e., STR analysis) was performed from the cell line in direct comparison to primary patient-derived tissues and the PDX model established. Drug responsiveness was examined for a panel of chemotherapeutics in clinical use for the treatment of solid cancers. RESULTS The established cell line HROC57 showed distinct morphological and molecular features of a poorly differentiated large-cell NEC with KI-67 > 50%. Molecular-pathological analysis revealed a CpG island promoter methylation positive cell line with microsatellite instability being absent. The following mutation profile was observed: KRAS (wt), BRAF (mut). A high sensitivity to etoposide, cisplatin and 5-FU could be demonstrated while it was more resistant towards rapamycin. CONCLUSION We successfully established and characterized a novel patient-derived NEC cell line in parallel to a PDX model as a useful tool for further analysis of the biological characteristics and for development of novel diagnostic and therapeutic options for NEC.

Postoperative, iatrogenic or spontaneous upper gastrointestinal defects result in significant morbidity and mortality of the patients. In the last few years, endoscopic vacuum therapy (EVT) has been recognized as a new promising method for repairing upper gastrointestinal defects of different etiology. However, probably due to insufficient data and no commercially available system for EVT of the upper gastrointestinal tract, until the end of 2014, covering of esophageal defects with self-expanding metal stents (SEMS) were still the mainstay of endoscopic therapy. The aim of this article is to review the data available about EVT for various upper gastrointestinal defects.

Purpose/Background: Ileal pouch-anal anastomosis (IPAA) is associated with a high risk of hospital readmission within 30 days of discharge.Risk factors for readmission are poorly understood and reasons for readmission have not been well studied.We sought to evaluate the incidence of readmission at 30 days and identify any modifiable perioperative factors for potential future intervention.Methods/Interventions: A retrospective review of all patients undergoing either total proctocolectomy with IPAA (two stage) or completion proctectomy with IPAA (three stage) for chronic ulcerative colitis (CUC) at our institution between January 2002 and July 2015 was performed.Baseline demographics, perioperative clinical and surgical variables, readmissions, readmission diagnoses, and management of readmission were recorded.Summary, univariate, and multivariate analyses were performed.Results/ Outcomes: A total of 911 patients underwent either two or three stage IPAA during the study period.185 patients (20.3%) required readmission within 30 days with 166 (90%) requiring one readmission and 19 (10%) requiring two readmissions.This resulted in 204 readmissions with 237 diagnoses.The leading etiologies for readmission included small bowel obstruction (n = 55, 23.2%), pelvic sepsis (n = 43, 18.1%), dehydration (n=42, 17.7%), and venous thromboembolism (VTE) (n=31, 13.1%).65.4% (n=119) of readmissions were managed medically, 19.2% (n = 35) required radiologic intervention, and 14.8% (n= 27) required operative intervention.The mean (SD) readmission length of stay (LOS) was 5.9 days (±4.2) for a mean (SD) total LOS of 12.7 days (±5.8) compared to a mean (SD) LOS of 7.1 days (±4.1) for those not readmitted.On univariate analysis, younger age (readmitted mean [95% CI] = 36.2years [34.3-38.1]versus not readmitted mean [95% CI] = 38.5 years [37.4-39.4],p = .03)and female sex (readmitted n = 87 [47%] versus not readmitted n = 281 [38.9%], p = .04)had a significantly increased risk of readmission.No other perioperative risk factors reached significance.On multivariate analysis, neither patient age (OR = 1.01; 95% CI, 1.0-1.03,p = 0.06) nor female sex (OR = 1.33; 95% CI, 0.96-1.85;p = 0.08) were independent predictors of readmission.Conclusion/Discussion: Readmission rates following IPAA for CUC remain high.No independent risk factors for readmission were identified in our cohort.Etiologies of readmission that may be preventable include dehydration and VTE.Future areas of quality improvement could focus on education regarding stoma output and VTE prophylaxis to decrease readmission rates following IPAA. Mo1627

Intraluminal therapy used in the gastrointestinal (GI) tract was first shown for anastomotic leaks after rectal resection. Since a few years vacuum sponge therapy is increasingly being recognized as a new promising method for repairing upper GI defects of different etiology. The principles of vacuum-assisted closure (VAC) therapy remain the same no matter of localization: Continuous or intermittent suction and drainage decrease bacterial contamination, secretion, and local edema. At the same time, perfusion and granulation is promoted. However, data for endoscopic vacuum therapy (EVT) of the upper intestinal tract are still scarce and consist of only a few case reports and small series with low number of patients.

Background. For development of individualized treatment on a routine basis, transfer of patients’ tumor tissue in a xenograft model (i.e., generation of patient-derived xenografts (PDX)) is desirable for molecular, biochemical, or functional analyses. Drawbacks are dissatisfactory tumor take rates, the necessity of fast tumor tissue processing, and extensive logistics demanding teamwork of surgeons, pathologists, and laboratory researchers. Methods. The take rates of ten colorectal cancer (CRC) tissue samples in immunodeficient mice were compared after direct cryopreservation and after a 24 h cooling period at 4°C prior to cryopreservation. Additionally, the effect of simultaneous Matrigel application on the take rates was investigated. Beside take rates, tumor growth characteristics and cell culture success were analyzed. Results. Tumor takes of CRC tissue samples were significantly improved after Matrigel application (8 versus 15 takes, p=0.04). As expected, they diminished furthermore after 24 h cooling. Application of Matrigel could counteract this decrease significantly (2 versus 7 takes, p=0.03). Cumulative take rate after cryopreservation was satisfactory (70%). Conclusion. Matrigel application after 24 h delay in tissue processing facilitates CRC PDX model development. These data help developing strategies for individualized tumor therapies in the context of multicenter clinical studies and for basic research on primary patient tumors.

Endoscopic vacuum therapy (EVT) has been established in Germany for the treatment of anastomotic leakage after rectal resection. Continuous or intermittent suction and drainage decrease bacterial contamination, secretion, and local edema promoting perfusion and granulation at the same time. However, data for use and long-term results of EVT in colorectal surgery are still scarce and are often limited by short-term follow-up.

AIMTo generate novel tumor models for preclinical validation of biomarkers that allow drug response prediction and individual therapeutic decisions.METHODSCell line establishment was conducted by both direct in vitro culturing and in vivo xenografting followed by in vitro culturing procedure. A comprehensive characterization was subsequently performed. This included quality control, consisting of the confirmation of human and colorectal cancer (CRC) origin by DNA fingerprint and epithelial cell adhesion molecule (EpCAM) staining, as well as mycoplasma and human virus testing. Phenotypic analysis was done by light microscopy and multicolor flow cytometry. Histopathological examination (β-catenin and cytokeratin staining) was conducted in direct comparison to parental tumor tissues. Extensive molecular-pathological profiling included mutation analysis for CRC-associated driver mutations, assessment of chromosomal and microsatellite instability, and the grade of CpG island methylation. Additionally, an array-based comparative genomic hybridization analysis was performed. Drug responsiveness was assessed for a panel of classical and novel substances in clinical use for the treatment of solid cancers. Finally, tumorigenicity of the cell lines was tested by xenografting into immunocompromised nude mice.RESULTSHerein we describe the establishment of three ultra-low passage cell lines from two individual patients suffering from sporadic CRC. One cell line was derived directly from an early stage case (HROC18), whereas two cell lines could be established both direct from patient material and after xenografting from a late stage tumor (HROC32). All cell lines were free of contaminating mycoplasma and viruses. Molecular-pathological analysis allowed all cell lines to be classified as chromosomal instable (CIN(+)). They were aneuploid, with CpG island promoter methylation and microsatellite instability being absent. The following mutational profile was observed both in the cell lines and the parental tumor tissue: HROC18: APC(mut), p53(mut), K-ras(wt); HROC32: APC(wt), p53(mut), K-ras(mut). All cell lines were characterized as epithelial (EpCAM(+)) cells, showing distinct morphology and migration speed, but comparable growth kinetics. The cell lines showed different patterns of response towards clinically approved and novel drugs, with HROC18 being more resistant than HROC32 cells. Finally, in vivo tumorigenicity was demonstrated.CONCLUSIONWe successfully established and characterized novel ultra-low passage patient-derived CRC models as useful instruments for analyzing biological characteristics associated with the CIN(+) phenotype.

of esophagogastric junction (EGJ) relaxation.Indeed, the diagnosis of achalasia is established by HRM on the basis of an IRP>15mmHg and absence of normal peristalsis in the esophageal body.Our aim was to assess the effect of Heller myotomy on IRP in achalasia patients.Patients and Methods: We evaluated all consecutive patients who underwent laparoscopic Heller myotomy as first treatment from 2009-2014 and had a HRM evaluation before and after surgery.Patients who had already been treated for achalasia (with Heller myotomy, endoscopic treatment) were excluded from the study.The diagnosis of primary achalasia was established by esophageal manometry on the basis of accepted esophageal motility characteristics (i.e.absence of normal peristalsis in the esophageal body).Symptoms were collected and scored using a detailed questionnaire for dysphagia, regurgitation, and chest pain; barium swallow, endoscopy, HRM were performed, before and 6 months after surgical treatment.Treatment failure was defined as a postoperative symptom score >10 th percentile of the preoperative score (i.e.> 10).Results: 139 consecutive achalasia patients (M:F= 72:67) represented the study population.All the patients had 100% simultaneous waves but 11 had a IRP<15 mmHg.According to the HRM classification, patients were classified as having: 58 (42.3%) type I, 63 (46%) type II and 16 (11.7%)type III.At a median follow-up of 24 months, the symptom score was significantly lower after surgery (median preoperative score 18 vs median postoperative score 0 [IQR 0-3]; p<0.0001).The resting LES pressure (median preoperatively 27 vs median postoperatively 11 [IQR 8-14]; p<0.001) and IRP (median preoperatively vs median postoperatively 7.1 [IQR: 4.4-9.8];p<0.001).The surgical procedure was completed laparoscopically in all the patients.The failures of surgical treatment were 7 (5%).At univariate analysis IRP was correlated with the gender, LES basal and resting pressure, and the dysphagia score.Conclusion: This is the first study evaluating the role of IRP in achalasia and its modifications after surgery.An increased preoperative IRP directly correlated with dysphagia severity in achalasia patients.Heller myotomy was able to resolve this symptom by reducing the IRP to a value lower than 10 mmHg.

Bei Patienten vor geplanter Kontinuitätswiederherstellung bzw. Rückverlagerung eines künstlichen Darmausganges ist die Überprüfung des Schließmuskelapparates auf einen für die Kontinenz ausreichenden Ruhe- und Kneiftonus obligat. Es werden hier verschiedene Untersuchungstechniken (Digital rektale Untersuchung, Wasserhalteversuch, Manometrie) angewandt. Einen einheitlichen Standard in Deutschland gibt es bisher nicht. Die anale Manometrie ermöglicht es, die Ruhe- und Kneifdrücke durch einen standardisierten Ablauf anhand einer Druckwerteskala zu objektivieren. Der Wasserhalteversuch ermöglicht ebenfalls eine Einschätzung der Kontinenzleistung. Zur Etablierung der klinischen Wertigkeit beider Verfahren vergleichen wir in dieser Studie einen standardisierten präoperativen Wasserhalteversuch mit der präoperativen Manometrie.

Background: Expression of the human endogenous retrovirus (HERV)-H family has been associated with colorectal carcinomas (CRC), yet no individual HERV-H locus expression has been thoroughly correlated with clinical data.Here, we characterized HERV-H reactivations in clinical CRC samples by integrating expression profiles, molecular patterns and clinical data. Expression of relevant HERV-H sequences was analyzed by qRT-PCR on two well-defined clinical cohorts (n = 139 pairs of tumor and adjacent normal colon tissue) including samples from adenomas (n = 21) and liver metastases (n = 16). Correlations with clinical and molecular data were assessed.Results: CRC specific HERV-H sequences were validated and found expressed throughout CRC disease progression. Correlations between HERV-H expression and lymph node invasion of tumor cells (p = 0.0006) as well as microsatellite instable tumors (p < 0.0001) were established. No association with regard to age, tumor localization, grading or common mutations became apparent. Interestingly, CRC expressed elements belonged to specific young HERV-H subfamilies and their 5' LTR often presented active histone marks.Conclusion: These results suggest a functional role of HERV-H sequences in colorectal carcinogenesis. The pronounced connection with microsatellite instability warrants a more detailed investigation. Thus, HERV-H sequences in addition to tumor specific mutations may represent clinically relevant, truly CRC specific markers for diagnostic, prognostic and therapeutic purposes.

Colliquative necrosis after ingestion of an alkaline solution is a very rare and life-threatening clinical picture, which in adults is mainly caused by ingestion either accidentally or with suicidal intent. In cases where severe corrosive injury with transmural necrosis has occurred, esophageal resection may be inevitable. If esophageal salvage is possible, consecutive stenosing scar formation can become a long-term complication. Therefore, timely bouginage is recommended but is also associated with an increased risk of esophageal perforation.

Background: Decision making for adjuvant chemotherapy in stage III colon cancer is based on the TNM system. It is well known that prognosis worsens with higher pN classification, and several recent studies propose superiority of the lymph node ratio (ln ratio) to the TNM system. Therefore, we compared the prognosis of ln ratio to TNM system in our stage III colon cancer patients.Methods: A total of 939 patients underwent radical surgery for colorectal cancer between January 2000 and December 2009. From this pool of patients, 142 colon cancer stage III patients were identified and taken for this analysis. Using martingale residuals, this cohort could be separated into a group with a low ln ratio and one with a high ln ratio. These groups were compared to pN1 and pN2 of the TNM system.Results: For ln ratio, the cutoff was calculated at 0.2. There was a good prognosis of disease-free and cancer-related survival for the N-category of the TNM system as well as for the lymph node ratio. There was no statistical difference between using the N-category of the TNM system and the ln ratio.Conclusions: There might not be a benefit in using the lymph node ratio rather than the N category of the TNM system as long as the number of subgroups is not increased. In our consideration, there is no need to change the N categorization of the TNM system to the ln ratio.

Einleitung: Die neoadjuvante Radiochemotherapie beim Rektumkarzinom führt zu einer Senkung der Lokalrezidivate ohne Beeinflussung des tumorabhängigen Überlebens oder der Metastasierung. Um das Langzeitüberleben zu verbessern, wurden Konzepte der Intensivierung der RCT etabliert. In dieser Arbeit soll der Einfluss der intensivierten neoadjuvanten RCT auf die Kontinenzleistung sowie die Lebensqualität im Verlauf untersucht werden.