Michael Gock

Establishment, Characterization and Chemosensitivity of Three Mismatch Repair Deficient Cell Lines from Sporadic and Inherited Colorectal Carcinomas

Background Colorectal cancer (CRC) represents a morphologic and molecular heterogenic disease. This heterogeneity substantially impairs drug effectiveness and prognosis. The subtype of mismatch repair deficient (MMR-D) CRCs, accounting for about 15% of all cases, shows particular differential responses up to resistance towards currently approved cytostatic drugs. Pre-clinical in vitro models representing molecular features of MMR-D tumors are thus mandatory for identifying biomarkers that finally help to predict responses towards new cytostatic drugs. Here, we describe the successful establishment and characterization of three patient-derived MMR-D cell lines (HROC24, HROC87, and HROC113) along with their corresponding xenografts. Methodology MMR-D cell lines (HROC24, HROC87, and HROC113) were established from a total of ten clinicopathological well-defined MMR-D cases (120 CRC cases in total). Cells were comprehensively characterized by phenotype, morphology, growth kinetics, invasiveness, and molecular profile. Additionally, response to clinically relevant chemotherapeutics was examined in vitro and in vivo. Principal Findings Two MMR-D lines showing CIMP-H derived from sporadic CRC (HROC24: K-raswt, B-rafmut, HROC87: K-raswt, B-rafmut), whereas the HROC113 cell line (K-rasmut, B-rafwt) was HNPCC-associated. A diploid DNA-status could be verified by flow cytometry and SNP Array analysis. All cell lines were characterized as epithelial (EpCAM+) tumor cells, showing surface tumor marker expression (CEACAM+). MHC-class II was inducible by Interferon-γ stimulation. Growth kinetics as well as invasive potential was quite heterogeneous between individual lines. Besides, MMR-D cell lines exhibited distinct responsiveness towards chemotherapeutics, even when comparing in vitro and in vivo sensitivity. Conclusions These newly established and well-characterized, low-passage MMR-D cell lines provide a useful tool for future investigations on the biological characteristics of MMR-D CRCs, both of sporadic and hereditary origin. Additionally, matched patient-derived immune cells allow for comparative genetic studies.

Impact of portal branch ligation on tissue regeneration, microcirculatory response and microarchitecture in portal blood-deprived and undeprived liver tissue

Partial ligation of portal branches leads to atrophy of the deprived lobes and hypertrophy of the intact lobes. In this study we investigated the microcirculatory response and their consequences on tissue regeneration after left-sided portal branch ligation (PBL) in Sprague-Dawley rats. At day 1 and 3 after PBL the hepatic microcirculation was assessed by intravital microscopy (IVM). In addition histological, immunohistochemical and biochemical techniques were used to determine alterations of hepatic microarchitecture. IVM analysis of the microcirculation of the ligated hepatic lobes revealed significant alterations with a reduction in sinusoidal perfusion rate, a decrease of red blood cell velocity, an increase of sinusoidal diameter and a marked reduction in shear stress at days 1 and 3 after PBL. On the contrary, the non-ligated lobes presented with higher blood flow velocities, marked sinusoidal vasoconstriction and thus, shear stress elevation. In consequence, ligated liver lobes exhibited marked cell apoptosis and necrosis, being accompanied by massive intrahepatic leukocyte accumulation and a ~30% weight loss. The non-ligated liver tissue showed marked PCNA expression and thereby completely compensated weight loss. Beside full restoration of liver mass, sinusoidal blood flow was comparable in ligated and non-ligated lobes as well as in sham-treated controls. This study shows that the liver aims at constant tissue mass and blood flow, most probably for maintenance of adequate clearance function. In addition, it supports the hypothesis that shear stress plays a pivotal role in triggering liver hypertrophy in the non-ligated lobes.

Endoscopic vacuum therapy for various defects of the upper gastrointestinal tract

BackgroundPostoperative, 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.MethodsA selective literature search was conducted in Medline and PubMed (2007–2016), taking into account all the published case series and case reports reporting on the use of EVT in the management of upper gastrointestinal defects.ResultsEVT works through intracorporal application of negative pressure at the defect zone with an electronic controlled vacuum device along a polyurethane sponge drainage. This results in closure of the esophageal defect and internal drainage of the septic focus, simultaneously. Compared to stenting, EVT enables regular viewing of wound conditions with control of the septic focus and adjustment of therapy. Moreover, endoscopical negative pressure is applicable in all esophageal regions (cricopharygeal, tubular, gastroesophageal junction) and in anastomotic anatomic variants. EVT can be used solely as a definite treatment or as a complimentary therapy combined with operative revision. In total, there are published data of more than 200 patients with upper gastrointestinal defects treated with EVT, showing succes rates from 70–100%.ConclusionThe available data indicate that EVT is feasible, safe and effective with good short-term and long-term clinical outcomes in the damage control of upper GI-tract leaks. Still, a prospective multi-center study has to be conducted to proof the definite benefit of EVT for patients with esophageal defects.

Intensified neoadjuvant radiochemotherapy for rectal cancer enhances surgical complications

BackgroundNeoadjuvant radiochemotherapy has proven superior to adjuvant treatment in reducing the rate of local recurrence without impairing cancer related survival or the incidence of distant metastases. The present study aimed at addressing the effects of an intensified protocol of neoadjuvant treatment on the development of postoperative complications.MethodsA total of 387 patients underwent oncological resection for rectal cancer in our institution between January 2000 and December 2009. 106 patients received an intensified radiochemotherapy. Perioperative morbidity and mortality were analyzed retrospectively with special attention on complication rates after intensified radio-chemotherapy. Therefore, for each patient subjected to neoadjuvant treatment a patient without neoadjuvant treatment was matched in the following order for tumor height, discontinuous resection/exstirpation, T-category of the TNM-system, dividing stoma and UICC stage.ResultsOf all patients operated for rectal cancer, 27.4% received an intensified neoadjuvant treatment. Tumor location in the matched patients were in the lower third (55.2%), middle third (41.0%) and upper third (3.8%) of the rectum. Postoperatively, surgical morbidity was higher after intensified neoadjuvant treatment. In the subgroup with low anterior resection (LAR) the anastomosis leakage rate was higher (26.6% vs. 9.7%) and in the subgroup of patients with rectal exstirpations the perineal wound infection rate was increased (42.2% vs. 18.8%) after intensified radiochemotherapy.ConclusionsIn rectal cancer the decision for an intensified neoadjuvant treatment comes along with an increase of anastomotic leakage and perineal wound infection. Quality of life is often reduced considerably and has to be balanced against the potential benefit of intensifying neoadjuvant radiochemotherapy.

Establishment and characterization of cell lines from chromosomal instable colorectal cancer

AIM To generate novel tumor models for preclinical validation of biomarkers that allow drug response prediction and individual therapeutic decisions. METHODS Cell 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. RESULTS Herein 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. CONCLUSION We successfully established and characterized novel ultra-low passage patient-derived CRC models as useful instruments for analyzing biological characteristics associated with the CIN(+) phenotype.

Expression of young HERV-H loci in the course of colorectal carcinoma and correlation with molecular subtypes

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.

Is the lymph node ratio superior to the Union for International Cancer Control (UICC) TNM system in prognosis of colon cancer

BackgroundDecision 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.MethodsA 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.ResultsFor 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.ConclusionsThere 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.

Establishment, functional and genetic characterization of a colon derived large cell neuroendocrine carcinoma cell line

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.

478 Surgical Endoscopic Vacuum Therapy for Defects of the Upper GI Tract

S A T A b st ra ct s 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 >10th 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 [IQR 11-20] vs median postoperative score 0 [IQR 0-3]; p<0.0001). The resting LES pressure (median preoperatively 27 [IQR 19-36] vs median postoperatively 11 [IQR 8-14]; p<0.001) and IRP (median preoperatively 27.4 [IQR 20.4-35] 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.

568 Endoscopic Vacuum Therapy in Colorectal Surgery

Introduction 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.