Chelating Phosphorus-An O, C, O-Coordinating Pincer-Type Ligand Coordinating PIII and PV Centres.

The sequence of reactions of the phosphorus-containing aryllithium compound 5-t-Bu-1,3-[(P(O)(O-i-Pr)2 ]2 C6 H2 Li (ArLi) with Ph2 PCl, KMnO4 , elemental sulfur and elemental selenium, respectively, gave the aryldiphenylphosphane chalcogenides 5-t-Bu-1,3-[(P(O)(O-i-Pr)2 ]2 C6 H2 P(E)Ph2 (1, E=O; 2, E=S; 3, E=Se). Compound 1 partially hydrolysed giving [5-t-Bu-1-{(P(O)(O-i-Pr)2 }-3-{(P(O)(OH)2 }C6 H2 ]P(O)Ph2 (4). The reaction of ArLi with PhPCl2 provided the benzoxaphosphaphosphole [1(P), 3(P)-P(O)(O-i-Pr)OPPh-6-t-Bu-4-P(O)(O-i-Pr)2 ]C6 H2 P (5i) as a mixture of the two diastereomers. The oxidation of 5i with elemental sulfur gave the benzoxaphosphaphosphole sulfide [1(P), 3(P)-P(O)(O-i-Pr)OP(S)Ph-6-t-Bu-4-P(O)(O-i-Pr)2 ]C6 H2 (5) as pair of enantiomers P1(R), P3(S)/P1(S), P3(R) of the diastereomer (RS/SR)-5 (5b). The aryldiphenylphosphane 5-t-Bu-1,3-[(P(O)(O-i-Pr)2 ]2 C6 H2 PPh2 (6) was obtained from the reaction of the corresponding aryldiphenylphosphane sulfide 2 with either sodium hydride, NaH, or disodium iron tetracarbonyl, Na2 Fe(CO)4 . The oxidation of the aryldiphenylphosphane 6 with elemental iodine and subsequent hydrolysis yielded the aryldiphenyldioxaphosphorane 9-t-Bu-2,6-(OH)-4,4-Ph2 -3,5-O2 -2,6-P2 -4λ5 -P-[5.3.1.0]-undeca-1(10),7(11),8-triene (7). Both of its diastereomers, (RR/SS)-7 (7a) and (RS/SR)-7 (7b), were separated as their chloroform and i-propanol solvates, 7a⋅2CHCl3 and 7b⋅i-PrOH, respectively. DFT calculations accompanied the experimental work.

Patient-individual cancer cell lines and tissue analysis delivers no evidence of sequences from DNA viruses in colorectal cancer cells.

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.

Comparison Between Endoscopic Vacuum Therapy and Conventional Treatment for Leakage After Rectal Resection.

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.

The Water-Holding Procedure for Ensuring Postoperative Continence Prior Restoring Intestinal Continuity.

BACKGROUND: 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. METHOD: We performed a prospective pilot study to evaluate the role of anorectal manometry and the water-holding procedure as a predictor of postoperative continence prior to stoma reversal. Inclusion criteria were a successfully passed water-holding test, any type of fecal diversion and the possibility of restoring intestinal continuity. Preoperative low manometric pressure levels were not an exclusion criteria for stoma reversal. Fifty-two patients with ostomy were consecutively enrolled in this study between October 2013 and February 2016. Anorectal manometry was performed in all patients prior to stoma reversal. After stoma removal, patients were followed-up for 6 months. Postoperative incontinence was determined using the Wexner incontinence score. RESULTS: A total of 52 patients (38 males, 14 females) were included at an average age of 59 (range 33-83) years. Most frequent indications for intestinal diversion were rectal cancer surgery, IBD-related surgery, or surgery for diverticular disease. Low anterior rectal resection was performed in 17 patients (32.7%), followed by a proctocolectomy in 9 (17.3%), colectomy in 9 (17.3%), and recto-sigmoid resection in 7 patients (13.5%). Median time from stoma creation to reversal was 206 days (range 48-871 days). All patients had successfully passed the standardized water-holding test. At the same time, the majority of patients had low preoperative manometric pressure values and would normally not have been reversed at that point. The median postoperative Wexner incontinence score was at 1.5 (range 0-20), 0.5 (range 0-14), and 0 (range 0-11) at 14, 60, and 180 days after stoma reversal. Low preoperative manometric squeeze and/or resting pressure levels were not associated with a higher postoperative incontinence score at 14, 60, or 180 days after stoma reversal. CONCLUSION: A standardized water-holding test can function as an easy and reliable method before stoma reversal to predict sufficient postoperative fecal continence. In case of a sufficient water-holding test despite low manometric pressure levels, the risk for postoperative anal incontinence seems to be low. Preoperative manometric pressure levels do not appear to predict postoperative continence.

Neoadjuvant radio-chemotherapy prolongs healing of anastomotic leakage after rectal resection treated with endoscopic vacuum therapy.

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.

Human Colorectal Carcinoma Infiltrating B Lymphocytes Are Active Secretors of the Immunoglobulin Isotypes A, G, and M.

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.

Establishment, functional and genetic characterization of three novel patient-derived rectal cancer cell lines.

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.

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.

Endoscopic vacuum therapy for various defects of the upper gastrointestinal tract.

BACKGROUND: 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. METHODS: A 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. RESULTS: EVT 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%. CONCLUSION: The 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.

Tumor Take Rate Optimization for Colorectal Carcinoma Patient-Derived Xenograft Models.

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 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. OBJECTIVES: Here, we aimed at analyzing the treatment spectrum and long-term outcome of EVT for defects of the lower gastrointestinal tract. METHODS: This is a retrospective single-center analysis of EVT for defects of the lower gastrointestinal tract of different etiology in 41 patients over a time period of 8 years (2007-2015) with a mean follow-up of 36 (2-89) months. RESULTS: In total, 426 polyurethane sponges were placed in lower GI defects of 41 patients (31 male, 10 female) with a median age of 70 years (range, 29-91). Most frequent indications for EVT were anastomotic leakage after rectal resection (n = 20), Hartmann's stump insufficiency (n = 12), and rectal perforation (n = 3). The median number of sponge insertions was six (range, 1-37) with a mean changing interval of 3 days (range, 1-5). Median time of therapy was 20 days. A successful vacuum therapy with local control of the septic focus was achieved in 18 of 20 patients (90 %) with anastomotic leakage after rectal resection and in nine of 12 patients with a Hartmann's stump insufficiency. In 15 of 19 (79 %) patients with a diverting stoma, take-down after successful treatment was possible. Median time to closure was 244 days (range, 152-488 days). CONCLUSION: To our knowledge, this retrospective observation of EVT application for rectal lesions represents the largest patient series in literature. EVT has earned its indication in complication management after colorectal surgery and can achieve a successful control of a local septic focus in the majority of patients.

Surgical Endoscopic Vacuum Therapy for Defects of the Upper Gastrointestinal Tract.

INTRODUCTION: 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. OBJECTIVES: Here, we present a single center experience of EVT for substantial wall defects in the upper GI tract. METHODS: Retrospective single-center analysis of EVT for various defects of the upper GI tract over a time period of 4 years (2011-2015) with a mean follow-up of 17 (2-45) months was used. If necessary, initial endoscopic sponge placement was performed in combination with open surgical revision. RESULTS: In total, 126 polyurethane sponges were placed in upper gastrointestinal defects of 21 patients with a median age of 72 years (range, 49-80). Most frequent indication for EVT was anastomotic leakage after esophageal or gastric resection (n = 11) and iatrogenic esophageal perforation (n = 8). The median number of sponge insertions was five (range, 1-14) with a mean changing interval of 3 days (range, 2-4). Median time of therapy was 15 days (range, 3-46). EVT in combination with surgery took place in nine of 21 patients (43 %). A successful vacuum therapy for upper intestinal defects with local control of the septic focus was achieved in 19 of 21 patients (90.5 %). CONCLUSION: EVT is a promising approach for postoperative, iatrogenic, or spontaneous lesions of the upper GI tract. In this series, EVT was combined with operative revision in a relevant proportion of patients.

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.

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 (ß-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.

Intensified neoadjuvant radiochemotherapy for rectal cancer enhances surgical complications.

BACKGROUND: Neoadjuvant 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. METHODS: A 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. RESULTS: Of 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. CONCLUSIONS: In 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.

Prognosis of rectal cancer patients improves with downstaging by intensified neoadjuvant radiochemotherapy - a matched pair analysis.

BACKGROUND: Neoadjuvant radiochemotherapy has been proven superior to adjuvant treatment in reducing the rate of local recurrence without impairing cancer related survival or the incidence of distant metastases in standard protocols of neoadjuvant radiochemotherapy. The present study aimed at addressing the effects of an intensified neoadjuvant radiochemotherapy on long term cancer related and disease free survival. METHODS: A total of 387 patients underwent oncologic resection for rectal cancer in our institution between January 2000 and December 2009. There were 106 patients (27.4%) who received an intensified radiochemotherapy protocol completely and without excluding criteria (study group). A matched pair analysis was performed by comparing the study group with patients undergoing primary surgery and postoperative radiochemotherapy, if necessary and possible (control group). Matching was carried out in descending order for UICC stage, R-status, tumor height, T-, N-, V-, L-, M- and G-category of the TNM-system according to the histopathological staging. Follow-up data included local recurrence rate, cancer related and disease free survival. RESULTS: In the study group histopathological work-up of the specimen revealed a treatment response in terms of tumor regression in 92.5% (98/106) of these patients. Undergoing intensified neoadjuvant RCT the actuarial cancer related and disease free survival was 67.9% and 70.4%, local recurrence was 5.7% after an observation period of 4.3 ± 2.55 years. In the control group cancer related and disease free survival was 71.7% and 82.7%, local recurrence was 4.7% after an observation period of 3.8 ± 3.05 years revealing no statistical significant difference between the two groups. Moreover, estimated 5-year results of cancer related survival (66.7% vs 67.9% (controls)), the disease free survival (66.7% vs 79.9% (controls)) as well as subgroup analysis of UICC 0-III and UICC IV patients showed no difference between the study and control group as well. CONCLUSION: In our study, intensified neoadjuvant radio-chemotherapy shows a high rate of tumor regression. The resulting inferior histopathological tumor stage shows the same long term local control and systemic tumor control as the control group with a primary more favorable tumor stage.

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

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.

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-ras(wt), B-raf(mut), HROC87: K-ras(wt), B-raf(mut)), whereas the HROC113 cell line (K-ras(mut), B-raf(wt)) 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.