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Summary To evaluate pathological complete response (pCR, ypT0ypN0) after neoadjuvant treatment compared with non-complete response (non-CR) in patients with esophageal cancer (EC), and 393 patients were retrospectively analyzed. Survival probability was analyzed in patients with: (i) pCR vs non-CR; (ii) complete response of the primary tumor but persisting lymphatic metastases (non-CR-T0N+) and (iii) pCR and tumor-free lymphnodes exhibiting signs of postneoadjuvant regression vs. no signs of regression. (i) Median overall survival (mOS) was favorable in patients with pCR (pCR: mOS not reached vs. non-CR: 41 months, P

Background Mass spectrometry imaging (MSI) derives spatial molecular distribution maps directly from clinical tissue specimens and thus bears great potential for assisting pathologists with diagnostic decisions or personalized treatments. Unfortunately, progress in translational MSI is often hindered by insufficient quality control and lack of reproducible data analysis. Raw data and analysis scripts are rarely publicly shared. Here, we demonstrate the application of the Galaxy MSI tool set for the reproducible analysis of a urothelial carcinoma dataset. Methods Tryptic peptides were imaged in a cohort of 39 formalin-fixed, paraffin-embedded human urothelial cancer tissue cores with a MALDI-TOF/TOF device. The complete data analysis was performed in a fully transparent and reproducible manner on the European Galaxy Server. Annotations of tumor and stroma were performed by a pathologist and transferred to the MSI data to allow for supervised classifications of tumor vs. stroma tissue areas as well as for muscle-infiltrating and non-muscle infiltrating urothelial carcinomas. For putative peptide identifications, m/z features were matched to the MSiMass list. Results Rigorous quality control in combination with careful pre-processing enabled reduction of m/z shifts and intensity batch effects. High classification accuracy was found for both, tumor vs. stroma and muscle-infiltrating vs. non-muscle infiltrating urothelial tumors. Some of the most discriminative m/z features for each condition could be assigned a putative identity: stromal tissue was characterized by collagen peptides and tumor tissue by histone peptides. Immunohistochemistry confirmed an increased histone H2A abundance in the tumor compared to the stroma tissues. The muscle-infiltration status was distinguished via MSI by peptides from intermediate filaments such as cytokeratin 7 in non-muscle infiltrating carcinomas and vimentin in muscle-infiltrating urothelial carcinomas, which was confirmed by immunohistochemistry. To make the study fully reproducible and to advocate the criteria of FAIR (findability, accessibility, interoperability, and reusability) research data, we share the raw data, spectra annotations as well as all Galaxy histories and workflows. Data are available via ProteomeXchange with identifier PXD026459 and Galaxy results via https://github.com/foellmelanie/Bladder_MSI_Manuscript_Galaxy_links. Conclusion Here, we show that translational MSI data analysis in a fully transparent and reproducible manner is possible and we would like to encourage the community to join our efforts.

Background/aim The presence of circulating tumor cells (CTC) has been reported to have an impact on prognosis in different tumor entities. Little is known about CTC morphology and heterogeneity. Patients and methods In a multicenter setting, pre-therapeutic peripheral blood specimens were drawn from patients with non-metastatic esophageal adenocarcinoma (EAC). CTCs were captured by size-based filtration (ScreenCell®), subsequently Giemsa-stained and evaluated by two trained readers. The isolated cells were categorized in groups based on morphologic criteria. Results Small and large single CTCs, as well as CTC-clusters, were observed in 69.2% (n=81) of the 117 specimens; small CTCs were observed most frequently (59%; n=69), followed by large CTCs (40%; n=47) and circulating cancer-associated macrophage-like cells (CAMLs; 34.2%, n=40). Clusters were rather rare (12%; n=14). CTC/CAML were heterogeneous in the cohort, but also within one specimen. Neither the presence of the CTC subtypes/CAMLs nor the exact cell count were associated with the primary clinical TNM stage. Conclusion Morphologically heterogenic CTCs and CAMLs are present in patients with non-metastatic, non-pretreated EAC.

An overwhelming number of proteomics software tools and algorithms have been published for different steps of Data Independent Acquisition analysis of clinical samples. Nonetheless, there is still a lack of comprehensive benchmark studies evaluating which combinations of those isolated components perform best.Here, we used 92 lymph nodes from distinct patients to create a unique benchmark dataset representing real-world inter-individual heterogeneity. The publicly available dataset comprises 118 LC-MS/MS runs with > 12 million MS2 spectra and allowed us to objectively evaluate how well different combinations of spectral libraries, DIA software, sparsity reduction, normalization and statistical tests can detect differentially abundant proteins, while also taking sample size into account.Evaluation of 2 million data analysis workflows showed that a gas phase fractionation refined spectral library in combination with DIA-NN and Significance Analysis of Microarrays reliably detected differentially abundant proteins. Furthermore, DIA-NN and Spectronaut robustly avoided the false detection of truly absent proteins.*KF and EB share first authorship. CK and OS share last authorship.

Numerous software tools exist for data-independent acquisition (DIA) analysis of clinical samples, necessitating their comprehensive benchmarking. We present a benchmark dataset comprising real-world inter-patient heterogeneity, which we use for in-depth benchmarking of DIA data analysis workflows for clinical settings. Combining spectral libraries, DIA software, sparsity reduction, normalization, and statistical tests results in 1428 distinct data analysis workflows, which we evaluate based on their ability to correctly identify differentially abundant proteins. From our dataset, we derive bootstrap datasets of varying sample sizes and use the whole range of bootstrap datasets to robustly evaluate each workflow. We find that all DIA software suites benefit from using a gas-phase fractionated spectral library, irrespective of the library refinement used. Gas-phase fractionation-based libraries perform best against two out of three reference protein lists. Among all investigated statistical tests non-parametric permutation-based statistical tests consistently perform best.

The hindgut theory hypothesizes a key role of differential hindgut stimulation following metabolic procedures in ameliorating diabetes mellitus. We used two strategies to remove the hindgut from intestinal continuity in order to analyze its impact on diabetes mellitus. Loop duodeno-jejunostomy (DJOS) with exclusion of one-third of total intestinal length was performed in 3 groups of 9-week-old Zucker diabetic fatty rats. In group 1, no further alteration of the intestinal tract was made. Group 2 received additional ileal exclusion (IE). Group 3 underwent additional resection of 50% of the ileum with side-to-side ileocecal anastomosis (IR). One, 2, and 4 months after surgery, fasting blood glucose levels, oral glucose tolerance tests (OGTT), and glucose-stimulated hormone analyses were conducted, and bile acid blood levels were compared. Body weight was documented weekly. In relation to DJOS, glucose control was not impaired in IR or IE. On the contrary, only IR could maintain preOP glucose values until 4 months. There were no significant weight differences between the groups. Confirming effective ileal diversion, bile acid blood levels were significantly higher in the DJOS group compared with both IR and IE (p = 0.0025 and p = 0.0047). Operative interventions had no impact on GLP-1 levels at any time point (ANOVA p > 0.05 for all). Insulin secretion was preserved in all groups. This data supports the hypothesis that the mechanisms driving amelioration of diabetes mellitus are complex and cannot be reduced to the ileum.

Simple Summary Less than 1% of all pancreatic malignancies are acinar cell carcinomas. Based on data from the German Cancer Registry Group, we performed a comparative analysis of characteristics and prognostic factors of pancreatic acinar cell carcinoma and the most common type of pancreatic cancer—pancreatic ductal adenocarcinoma. Compared to pancreatic ductal adenocarcinoma, patients with pancreatic acinar cell carcinoma were younger at the time of diagnosis and the percentage of males was higher. The prognosis of patients with pancreatic acinar carcinoma was better than that of patients with pancreatic ductal adenocarcinoma. Surgical resection was the strongest positive prognostic factor for pancreatic acinar cell carcinoma. The study shows that pancreatic acinar cell carcinoma has features distinct from pancreatic ductal adenocarcinoma. Radical resection should be advocated, whenever feasible. Abstract Background: Pancreatic acinar cell carcinoma (PACC) is a distinct type of pancreatic cancer with low prevalence. We aimed to analyze prognostic factors and survival outcome for PACC in comparison to pancreatic ductal adenocarcinoma (PDAC), based on data from the German Cancer Registry Group. Methods: Patients with PACC and PDAC were extracted from pooled data of the German clinical cancer registries (years 2000 to 2019). The distribution of demographic parameters, tumor stage and therapy modes were compared between PACC and PDAC. The Kaplan–Meier method and Cox regression analysis were used to delineate prognostic factors for PACC. Propensity score matching was used to compare survival between PACC and PDAC. Results: There were 233 (0.44%) patients with PACC out of 52,518 patients with pancreatic malignancy. Compared to PDAC, patients with PACC were younger (median age 66 versus 70, respectively, p < 0.001) and the percentage of males was higher (66.1% versus 53.3%, respectively, p < 0.001). More patients were resected with PACC than with PDAC (56.2% versus 38.9%, respectively, p < 0.001). The estimated overall median survival in PACC was 22 months (95% confidence interval 15 to 27), compared to 12 months (95% confidence interval 10 to 13) in the matched PDAC cohort (p < 0.001). Surgical resection was the strongest positive prognostic factor for PACC after adjusting for sex, age, and distant metastases (hazard ratio 0.34, 95% confidence interval 0.22 to 0.51, p < 0.001). There was no survival benefit for adjuvant therapy in PACC. Conclusions: PACC has overall better prognosis than PDAC. Surgical resection is the best therapeutic strategy for PACC and should be advocated even in advanced tumor stages.

Surgically assessed pancreatic texture has been identified as the strongest predictor of postoperative pancreatic fistula. However, texture is a subjective parameter with no proven reliability or validity. Therefore, a more objective parameter is needed. In this study, we evaluated the fibrosis level at the pancreatic neck resection margin and correlated fibrosis and all clinico-pathologic parameters collected over the course of the Pancreatogastrostomy vs Pancreatojejunostomy for RECOnstruction (RECOPANC) study.The RECOPANC trial was a multicenter randomized prospective trial of patients undergoing pancreatoduodenectomy. There were 261 hematoxylin and eosin-stained slides allocated for histopathologic analyses. Pancreatic fibrosis was scored from 0 to III (no fibrosis up to severe fibrosis) by 2 blinded independent pathologists. All variables possibly associated with POPF were entered into a generalized linear model for multivariable analysis.The fibrosis grade and pancreatic texture were scored in all 261 patients. In POPF B/C (postoperative pancreatic fistula grade B or C) patients, 71% had a soft pancreas, and fibrosis grades were distributed as follows: 48% with score 0, 28% with score I, 20% with score II, and 7% with score III, respectively. Fibrosis grading showed substantial inter-rater reliability (kappa = 0.74) and correlated positively with hard pancreatic texture (p0.05). In univariable analysis, area under the curve (AUC) for POPF B/C prediction was higher for fibrosis grade than for pancreatic texture (0.71 vs 0.59). In multivariate analysis, the following predictors were selected: sex, surgeon volume, pancreatic texture, and fibrosis grade. However, the addition of pancreatic texture only led to an incremental improvement (AUC 0.794 vs 0.819).Histologically evaluated pancreatic fibrosis is an easily applicable and highly reproducible POPF predictor and superior to surgically evaluated pancreatic texture. Future studies might use fibrosis grade for risk stratification in pancreatoduodenectomy.

// Sarah Lang 1 , Hauke Busch 2, 3 , Melanie Boerries 3, 4 , Tilman Brummer 3, 4, 5 , Sylvia Timme 6 , Silke Lassmann 4, 5, 6 , Klaus Aktories 1 and Gudula Schmidt 1 1 Institute for Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, Albert-Ludwigs-University, Freiburg, Germany 2 Lubeck Institute of Experimental Dermatology, Institute for Cardiogenetics, University of Lubeck, Lubeck, Germany 3 Institute of Molecular Medicine and Cell Research, Faculty of Medicine, University of Freiburg, Freiburg, Germany 4 German Cancer Consortium (DKTK), Freiburg, Germany, German Cancer Research Center (DKFZ), Heidelberg, Germany 5 Center for Biological Signalling Studies (BIOSS), University of Freiburg, Freiburg, Germany 6 Institute for Surgical Pathology, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany Correspondence to: Gudula Schmidt, email: Gudula.Schmidt@pharmakol.uni-freiburg.de Keywords: Rho GTPase, invasion, cyclooxygenase, breast cancer Received: June 14, 2017 Accepted: August 26, 2017 Published: September 16, 2017 ABSTRACT Rho GTPases are regulators of many cellular functions and are often dysregulated in cancer. However, the precise role of Rho proteins for tumor development is not well understood. In breast cancer, overexpression of RhoC is linked with poor prognosis. Here, we aim to compare the function of RhoC and its homolog family member RhoA in breast cancer progression. We established stable breast epithelial cell lines with inducible expression of RhoA and RhoC, respectively. Moreover, we made use of Rho-activating bacterial toxins (Cytotoxic Necrotizing Factors) to stimulate the endogenous pool of Rho GTPases in benign breast epithelial cells and simultaneously knocked down specific Rho proteins. Whereas activation of Rho GTPases was sufficient to induce an invasive phenotype in three-dimensional culture systems, overexpression of RhoA or RhoC were not. However, RhoC but not RhoA was required for invasion, whereas RhoA and RhoC equally regulated proliferation. We further identified downstream target genes of RhoC involved in invasion and identified PTGS2 (COX-2) being preferentially upregulated by RhoC. Consistently, the COX-2 inhibitor Celecoxib blocked the invasive phenotype induced by the Rho-activating toxins.

Background: Carcinoembryonic antigen cell adhesion molecule (CEA) is a commonly immunohistochemically used antibody in pathological routine diagnostics with an overexpression in different cancers. We aimed to examine the immunohistochemically detectable CEA level in ampullary cancer and to correlate it with clinico-pathological data. Methods: Shot-gun proteomics revealed CEA in undifferentiated ampullary cancer cell lines. Next, tumor tissue of 40 ampullary cancers of a retrospective single center cohort of 40 patients was stained immunohistochemically for CEA; CEA expression was determined and correlated with clinico-pathological data. Results: Thirty-six patient specimens were included in statistical analysis. CEA expression and lymph node ratio (LNR) were the only independent predictors of overall survival in multivariate analysis. Conclusion: To our knowledge, cell line and patient cohorts are the largest and characterized cohorts examined for CEA so far. Hereby, CEA expression in ampullary cancer cells permits an estimation of outcome and suggests an opportunity for individualized CEA-directed therapy. Further trials with larger cohorts are needed to verify our results and to integrate CEA immunohistochemistry into clinical routine.

In this retrospective study, we analyzed the association between tumor budding and perineural invasion as well as their prognostic role in pancreatic ductal adenocarcinoma. A total of N = 119 patients resected for pancreatic ductal carcinoma from 1996 to 2015 were included. Clinical and standard histopathological parameters were retrieved from the patient’s records. One representative hematoxylin and eosin section from the tumor region was examined for perineural invasion and tumor budding using light microscopy. Tumor budding was assessed independently using two different methods: in the first approach, the number of buds was counted over three fields of 0.237 mm2 at 40-fold magnification; in the second approach, tumor budding was quantified according to the recommendation of the International Tumor Budding Consensus Conference (ITBCC) over a field of 0.785 mm2 at 20-fold magnification. Linear and logistic regression was applied to delineate association between perineural invasion, tumor budding, and other parameters; Kaplan-Meier and Cox regression were used in the survival analysis. Regardless of the quantification approach, high tumor budding was a significant negative prognostic factor in the univariable Cox regression (> 5 buds/0.237 mm2, hazard ratio (HR) 1.66, 95% confidence interval (CI) 1.06–2.61, p = 0.027; ≥ 10 buds/0.785 mm2, HR 1.68, 95% CI 1.07–2.64, p = 0.024). In the multivariable model adjusting for stage and standard histopathological parameters, lymph vessel invasion (HR = 2.43, 95% CI 1.47–4.03, p = 0.001) and tumor budding > 5 buds/0.237 mm2 (HR = 1.70, 95% CI 1.07–2.7, p = 0.026) were independent negative prognostic factors, while adjuvant therapy was a positive prognostic factor (HR = 0.54, 95% CI 0.33–0.86, p = 0.009). No significant prognostic value could be delineated for perineural invasion. In conclusion, tumor budding is an independent negative prognostic factor in pancreatic ductal adenocarcinoma associated with lymph node metastasis. The prognostic role of perineural invasion remains uncertain.

Background: Cathepsin L (Ctsl) is a cysteine protease mainly located within the endosomal/lysosomal cell compartment. High expression of Ctsl indicates poor prognosis in human breast cancer. However, the cell type-specific Ctsl functions responsible for this association remain elusive. Methods: Because constitutive Ctsl&minus, /&minus, mice develop a complex phenotype, we developed a conditional model allowing for cell type-specific inactivation of Ctsl in mammary epithelium or myeloid cells in the transgenic mouse mammary tumor virus (MMTV)-polyoma middle T (PyMT) breast cancer model. Results: Ctsl ablation in mammary epithelial cells resulted in delayed initiation and end-stage of cancers. The latter displayed large dead cell areas. Inducible in vitro deletion of Ctsl in MMTV-PyMT-derived breast cancer cells revealed expansion of the acidic cell compartment, alteration of intracellular amino acid levels, and impaired mTOR signaling. In consequence, Ctsl-deficient cells exhibited slow growth rates and high apoptosis susceptibility. In contrast to Ctsl-deficient mammary epithelium, selective knockout of Ctsl in myeloid cells had no effects on primary tumors, but promoted lung metastasis formation. Conclusions: Our cell type-specific in vivo analysis provides strong evidence for a cancer cell-intrinsic, tumor-promoting role of Ctsl in primary breast cancer, whereas metastasis is negatively regulated by Ctsl expressed by bone marrow-derived cells.

Despite the complexity of endometrial cancer (EC) tumor biology, treatment decisions are still mainly based on the post-surgical International Federation of Gynecology and Obstetrics (FIGO) stage. Prediction models considering more prognostic factors may represent a better risk assessment than FIGO stage alone. We tested the Memorial Sloan Kettering Cancer Center (MSKCC) nomogram for the prediction of overall survival (OS) in a German EC population. Overall, 454 EC patients (322 type I and 132 type II) who received primary surgical treatment at our department between 1991 and 2011 were included in the analysis with a dataset of 68 covariates. Predicted OS was calculated using the online MSKCC nomogram and compared with the observed survival in our population. To estimate the discriminatory power, the concordance probabilities were calculated using the concordance probability estimate (CPE). Receiver operating characteristic curves were created and the area under the curve (AUC) values compared between predicted and actual OS. After a mean follow-up of 183 months, 211 patients were reported dead (47%). Mean OS for all stages was 101 months (standard deviation 66.7 months). The 2009 FIGO system showed an AUC value of 0.6 and a CPE of 0.63, while the 3-year OS prediction of the MSKCC nomogram showed an AUC value of 0.8 and a CPE of 0.77. This external validation of the MSKCC nomogram showed better discrimination and calibration values than the conventional FIGO classification system. The nomogram was externally validated and can serve as a tool for better risk-adapted treatment decisions and patient stratification, e.g. in clinical trials.

Esophageal cancers are highly aggressive tumors with poor prognosis despite some recent advances in surgical and radiochemotherapy treatment options. This study addressed the feasibility of drugs targeting epigenetic modifiers in esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) cells. We tested inhibition of histone deacetylases (HDACs) by SAHA, MS-275, and FK228, inhibition of DNA methyltransferases by Azacytidine (AZA) and Decitabine (DAC), and the effect of combination treatment using both types of drugs. The drug targets, HDAC1/2/3 and DNMT1, were expressed in normal esophageal epithelium and tumor cells of ESCC or EAC tissue specimens, as well as in non-neoplastic esophageal epithelial (Het-1A), ESCC (OE21, Kyse-270, Kyse-410), and EAC (OE33, SK-GT-4) cell lines. In vitro, HDAC activity, histone acetylation, and p21 expression were similarly affected in non-neoplastic, ESCC, and EAC cell lines post inhibitor treatment. Combined MS-275/AZA treatment, however, selectively targeted esophageal cancer cell lines by inducing DNA damage, cell viability loss, and apoptosis, and by decreasing cell migration. Non-neoplastic Het-1A cells were protected against HDACi (MS-275)/AZA treatment. RNA transcriptome analyses post MS-275 and/or AZA treatment identified novel regulated candidate genes (up: BCL6, Hes2; down: FAIM, MLKL), which were specifically associated with the treatment responses of esophageal cancer cells. In summary, combined HDACi/AZA treatment is efficient and selective for the targeting of esophageal cancer cells, despite similar target expression of normal and esophageal cancer epithelium, in vitro and in human esophageal carcinomas. The precise mechanisms of action of treatment responses involve novel candidate genes regulated by HDACi/AZA in esophageal cancer cells. Together, targeting of epigenetic modifiers in esophageal cancers may represent a potential future therapeutic approach.

Background and Aim Pancreatic ductal adenocarcinoma (PDAC) is characterized by aggressive biology and poor prognosis even after resection. Long-term survival is very rare and cannot be reliably predicted. Experimental data suggest an important role of epithelial-mesenchymal transition (EMT) in invasion and metastasis of PDAC. Tumor budding is regarded as the morphological correlate of local invasion and cancer cell dissemination. The aim of this study was to evaluate the biological and prognostic implications of EMT and tumor budding in PDAC of the pancreatic head. Methods Patients were identified from a prospectively maintained database, and baseline, operative, histopathological, and follow-up data were extracted. Serial tissue slices stained for Pan-Cytokeratin served for analysis of tumor budding, and E-Cadherin, Beta-Catenin, and Vimentin staining for analysis of EMT. Baseline, operative, standard pathology, and immunohistochemical parameters were evaluated for prediction of long-term survival (≥ 30 months) in uni- and multivariate analysis. Results Intra- and intertumoral patterns of EMT marker expression and tumor budding provide evidence of partial EMT induction at the tumor–host interface. Lymph node ratio and E-Cadherin expression in tumor buds were independent predictors of long-term survival in multivariate analysis. Conclusions Detailed immunohistochemical assessment confirms a relationship between EMT and tumor budding at the tumor–host interface. A small group of patients with favorable prognosis can be identified by combined assessment of lymph node ratio and EMT in tumor buds.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. Circulating tumor cells (CTC) in the blood are hypothesized as the means of systemic tumor spread. Blood obtained from healthy donors and patients with PDAC was therefore subject to size-based CTC-isolation. We additionally compared Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in pancreatic CTC and corresponding tumors, and evaluated their significance as prognostic markers. Samples from 68 individuals (58 PDAC patients, 10 healthy donors) were analyzed; CTCs were present in patients with UICC stage IA-IV tumors and none of the controls (p 0.001). Patients with3 CTC/ml had a trend for worse median overall survival (OS) than patients with 0.3–3 CTC/ml (P = 0.12). Surprisingly, CTCs harbored various KRAS mutations in codon 12 and 13. Patients with a KRAS G12V mutation in their CTC (n = 14) had a trend to better median OS (24.5 months) compared to patients with other (10 months), or no detectable KRAS mutations (8 months; P = 0.04). KRAS mutations in CTC and corresponding tumor were discordant in 11 of 26 “tumor-CTC-pairs” (42%), while 15 (58%) had a matching mutation; survival was similar in both groups (P = 0.36). Genetic characterization, including mutations such as KRAS, may prove useful for prognosis and understanding of tumor biology.

The aim of this study was to calculate the validity parameters of the Digene Hybrid Capture 2 (HC2) high-risk human papillomavirus DNA test with and without cytology in the follow-up examinations after laser treatment of the transformation zone or large loop excision of the transformation zone (LLETZ) for cervical intraepithelial neoplasia (CIN).We performed a standardized follow-up examination in 113 postlaser and 153 post-LLETZ patients in our colposcopy clinic. Routine cytology, HC2 tests, and colposcopically-guided cervical biopsies were performed and sensitivity, specificity, and positive and negative predictive values were calculated using the histological cervical biopsy result as the criterion standard.After a median follow-up time of 25.5 months, the overall posttreatment recurrence/persistence rate of CIN 2 or higher (CIN 2+) was 24% after laser and 12.4% after Post-LLETZ treatment. Hybrid Capture 2 alone had a sensitivity/NPV of 70/88% in post-laser and 70/93% in post-LLETZ patients. Cytology alone had a sensitivity/NPV for CIN 2+ of 48/84% in post-laser and 58/91% in post-LLETZ patients. Combined testing of HC2 with cytology had a sensitivity/NPV of 81/92% in postlaser and 88/95% in post-LLETZ patients.In this test of cure study, combined testing of cytology with HC2 resulted in a high sensitivity and NPV. Hybrid Capture 2 and cytology-negative women may safely return to routine recall. Cytology alone is not an adequate follow-up strategy in postlaser patients.

Background/Aim: The pro-angiogenic Cyr61 protein has been associated with tumorigenesis and cancer progression in different gynecological carcinomas. In this study, we evaluated the potential impact and clinical relevance of Cyr61 expression in patients with primary non-metastatic cervical cancer (CC). Patients and Methods: Cyr61 expression was assessed in tissue specimen of 48 patients with primary CC by immunohistochemical analysis. Expression levels were scored and correlated to clinico-pathological factors and outcome data. Results: High Cyr61 expression levels were present in 54.2% of CC tissues. Associations with histological grade (p=0.030), depth of tumor invasion (p=0.007) and GOG score (p=0.027) were observed. Patients who overexpressed Cyr61 displayed an increased death rate (30.8% vs. 18.2%) and a decreased 5-year-survival (76.9% vs. 86.4%). Conclusion: Our data indicate a potential functional impact of Cyr61 in development and the progression of CC. The definite tumor-relevant function (suppressive/promoting) of Cyr61 in CC and the prognostic relevance of Cyr61 overexpression has to be evaluated in larger cohorts.

Purpose: Proliferation and pre-malignancy are typical features of DCIS. The expression of PHH3 as a marker for proliferation and LDH 5 as an indicator for oxygen independent energy metabolism was investigated in order to assess their potential for an improved characterisation of these lesions. Methods: Archived tissue blocks and clinical records of 130 patients with DCIS. Immunohistochemistry for PHH3, LDH5, estrogen receptor (ER), progesterone receptor (PR), human EGF receptor 2 (HER2). Silverstein nuclear grading. Chi Square test for all factors. Results: Percentage of positive patients was 69% for PHH3, 94% for LDH5, 76% for ER, 67% for PR, and 21% for HER2. Significant correlation was seen between PHH3 and LDH5 expression. No correlation could be found for any of the other comparisons. Conclusion: This is the first description of PHH3 and LDH5 in DCIS. The biological significance of PHH3 remains to be determined in a larger set of patients. LDH5 may be a useful diagnostic marker in the future. Positive HER2 receptors were considerably less frequent than previously reported., Diagnostic Pathology, Vol 3 No 1 (2017): 2017

Cancer cell invasion takes place at the cancer-host interface and is a prerequisite for distant metastasis. The relationships between current biological and clinical concepts such as cell migration modes, tumour budding and epithelial-mesenchymal transition (EMT) remains unclear in several aspects, especially for the 'real' situation in human cancer. We developed a novel method that provides exact three-dimensional (3D) information on both microscopic morphology and gene expression, over a virtually unlimited spatial range, by reconstruction from serial immunostained tissue slices. Quantitative 3D assessment of tumour budding at the cancer-host interface in human pancreatic, colorectal, lung and breast adenocarcinoma suggests collective cell migration as the mechanism of cancer cell invasion, while single cancer cell migration seems to be virtually absent. Budding tumour cells display a shift towards spindle-like as well as a rounded morphology. This is associated with decreased E-cadherin staining intensity and a shift from membranous to cytoplasmic staining, as well as increased nuclear ZEB1 expression.

Clinical data indicate that laparoscopic surgery has a beneficial effect on intestinal wound healing and is associated with a lower incidence of anastomotic leakage. This observation is based on weak evidence, and little is known about the impact of intraoperative parameters during laparoscopic surgery, e.g., temperature and humidity.A small-bowel anastomosis was formed in rats inside an incubator, in an environment of stable humidity and temperature. Three groups of ten Wistar rats were operated: a control group (G1) in an open surgical environment and two groups (G2 and G3) in the incubator at a humidity of 60 % and a temperature of 30 and 37 °C (G2 and G3, respectively). After 4 days, bursting pressure and hydroxyproline concentration of the anastomosis were analyzed. The tissue was histologically examined. Serum levels of C-reactive-protein (CRP) were measured.No significant changes were seen in the evaluation of anastomotic stability. Bursting pressure was very similar among the groups. Hydroxyproline concentration in G3 (36.3 μg/g) was lower by trend (p = 0.072) than in G1 (51.7 μg/g) and G2 (46.4 μg/g). The histological evaluation showed similar results regarding necrosis, inflammatory cells, edema, and epithelization for all groups. G3 (2.56) showed a distinctly worse score for submucosal bridging (p = 0.061) than G1 (1.68). A highly significant increase (p = 0.008) in CRP was detected in G3 (598.96 ng/ml) compared to G1 (439.49 ng/ml) and G2 (460 ng/ml).A combination of high temperature and humidity during surgery induces an increased systemic inflammatory response and seems to be attenuating the early regeneration process in the anastomotic tissue.

It has been shown that crystalloid fluid-overload promotes anastomotic instability. As physiologic anastomotic healing requires the sequential infiltration of different cells, we hypothesized this to be altered by liberal fluid regimes and performed a histomorphological analysis.36 Wistar rats were randomized into 4 groups (n=8-10 rats/group) and treated with either liberal (+) or restrictive (-) perioperative crystalline (Jonosteril = Cry) or colloidal fluid (Voluven = Col). Anastomotic samples were obtained on postoperative day 4, routinely stained and histophathologically reviewed. Anastomotic healing was assessed using a semiquantitative score, assessing inflammatory cells, anastomotic repair and collagenase activity.Overall, the crystalloid overload group (Cry (+)) showed the worst healing score (P0.01). A substantial increase of lymphocytes and macrophages was found in this group compared to the other three (P0.01). Both groups that received colloidal fluid (Col (+) and Col (-)) as well as the group that received restricted crystalloid fluid resuscitation (Cry (-)) had better intestinal healing. Collagenase activity was significantly higher in the Cry (+) group.Intraoperative infusion of high-volume crystalloid fluid leads to a pathological anastomotic inflammatory response with a marked infiltration of leukocytes and macrophages resulting in accelerated collagenolysis.

Tumor resistance to endocrine therapy triggers estrogen-independent cancer progression, which is a major obstacle to the successful treatment of hormone receptor positive breast cancer (BC). The underlying molecular mechanisms of endocrine resistance are not fully understood yet. The matricellular protein cysteine-rich angiogenic inducer 61 (Cyr61) is associated with tumor invasiveness and the induction of tumorigenesis in various malignancies in vivo and the induction of estrogen-independence and endocrine therapy resistance in BC. The present study evaluated the potential effects and clinical relevance of Cyr61 expression levels in 67 patients with primary non-metastatic BC. Immunohistochemical analysis of formalin-fixed paraffin-embedded tissue sections was performed, and the association between Cyr61 protein expression and clinicopathological factors and survival was analyzed. Cyr61 overexpression was revealed to be significantly associated with a positive estrogen receptor (ER)/progesterone receptor (PR) status (P=0.016) and to the molecular subtype of BC (P=0.039). Compared with patients without Cyr61 overexpression, patients with Cyr61 overexpression exhibited an increased recurrence rate (30.6 vs. 22.6%) and decreased long-term survival (10-year overall survival, 62.9 vs. 69.7%); however, these associations did not reach statistically significant levels in Cox regression model analysis. Similar results were identified in the subgroup analysis of patients with ER/PR positive BC. These results indicate that Cyr61 serves a role in the development of endocrine therapy resistance in BC and is thus a potential therapeutic target to overcome endocrine therapy resistance. However, additional long-term survival analyses with large patient populations are required.

Introduction: Our aim was to assess the practicability and reliability of a novel labeling regime for axillary sentinel lymph nodes (SLNs) in early breast cancer. Methods: 362 patients with early breast cancer (bilateral in 9 cases, giving a total of 371 cases) underwent intradermal radio tracer injection with simultaneous manual lymphatic drainage. SLN biopsy was performed within 24 h. For retrospective analysis, data were extracted from patient's records. Results: At least 1 SLN was detected intraoperatively in 369 cases (99.5%, range 1-9 nodes). This node was metastatic in 88 and unaffected in 281 cases. Coincidentally removed but unlabeled lymph nodes were affected in 3 cases in which the SLN was unaffected (3/153 = 2%). In all cases, on histological evaluation, tissue removed as SLN contained lymph nodes. After a period of 69.5 months (median 1.7-115.8 months), no axillary recurrences were observed in 213 patients. Conclusion: Manual lymphatic drainage is a simple technique that leads to an extremely high pick-up rate of axillary SLNs after subepidermal radio tracer injection. If unaffected, this node correctly predicts nodal-negative disease in 98% of cases studied.

Objective To compare the efficacy of the new reusable vessel-sealing device MarSeal (KLS Martin, Tuttlingen, Germany) with the conventional standard disposable LigaSure device (Covidien-Valleylab, Boulder, CO) in an in vivo animal model. Design Prospective animal study. Setting Center of Experimental Surgery at a university hospital. Animals Thirteen sheep. Intervention(s) The carotid and femoral arteries were dissected bilaterally and sealed randomly with MarSeal unilaterally and consecutively with LigaSure contralaterally in vivo. Afterward the burst pressure was determined for each artery. Main Outcome Measure(s) Sealing time, failure rate, and burst pressure. Result(s) The mean diameter of all arteries sealed did not differ significantly between the two groups (MarSeal 5.40 mm vs. LigaSure 5.35 mm). The mean sealing time was significantly shorter with use of the reusable device (MarSeal 5.2 seconds vs. LigaSure 9.1 seconds). We did not find a significant difference in sealing failure rates between the groups (MarSeal 7.0% vs. LigaSure 9.1%). In addition, analysis of mean burst pressure did not reveal a significant difference between the different devices (MarSeal 429 mm Hg vs. LigaSure 484 mm Hg). There was no significant difference with respect to lateral thermal damage (MarSeal 0.91 cm vs. LigaSure 0.93 cm). Conclusion(s) In our in vivo animal study, the new reusable MarSeal device appears to be equivalently effective for vessel sealing when compared with the conventional disposable LigaSure device.

Background: Anastomotic leakage is a major factor for morbidity in colorectal surgery. Anastomotic reinforcement with biological or synthetic materials has been claimed to be useful in preventing anastomotic leakage. Methods: We evaluated a non-cross-linked collagenous matrix Bio-Gide® (BG) for sealing colonic anastomoses in a rodent model. The animals were investigated for 4, 30 and 90 days. Macroscopic examination, histological examination and measurement of bursting pressure were performed. The anastomotic stricture rate was evaluated by radiographic contrast enema. Results: Microscopically anastomoses sealed by BG showed impaired anastomotic healing. Blood vessel ingrowth and collagen deposition were decreased without reaching significance after 4 days. The anastomotic bursting pressure was significantly decreased (p = 0.0454) in the early phase of healing. Anastomotic neovascularization was significantly decreased compared to the control group after 30 (p = 0.0058) and 90 days (p = 0.0275). Although no difference in anastomotic stricture rate was evident, the rate of intra-abdominal adhesions was significantly increased after 30 (p = 0.0124) and 90 days (p = 0.0281). Conclusion: BG failed to improve colonic anastomotic healing. Early anastomotic healing was impaired if anastomoses were reinforced with BG. BG did not affect the anastomotic stricture rate for up to 3 months; nevertheless, intra-abdominal adhesions were increased.

Background: Fundamental experimental research into intestinal anastomotic healing in rodent models will gain increasing interest in the future. Methods: The aim of this study was to describe our 5-year experience with a standardized experimental setup of small and large bowel anastomoses in a rodent model and present a basic set of assessment tools investigating anastomotic healing. Anastomotic technique, perioperative complications such as anastomotic insufficiency (AI) and obstructive ileus were in the focus. Results: During different studies with varying study patterns, 167 rat small bowel anastomoses and 120 colonic anastomoses were performed. Overall mortality was 3.6% in small bowel and 2.5% in colonic anastomoses, AI occurred in 2.9 and 4%, respectively. A postoperative obstructive ileus was seen in 3/167 small bowel anastomoses and none in the colonic group. Conclusion: When performing experimental intestinal anastomoses in a standardized operative setting and critically considering special perioperative issues, the incidence of relevant complications can be maintained at an adequately low level.

The aim of this study was to investigate if colloid infusions have different effects on intestinal anastomotic healing when compared to crystalloid infusions depending on the amount of the administered volume. Twenty-eight Wistar rats were randomly assigned to four groups receiving different amounts of either a crystalloid (Cry) or a colloid (Col) infusion solution. Animals with volume restriction (Cry (−) or Col (−)) were treated with a low and animals with volume overcharge (Cry (+) or Col (+)) with a high flow rate. All animals received an infusion for a 60-min period, while an end-to-end small bowel anastomosis was performed. At reoperation, the anastomotic bursting pressure (millimeters of mercury) was measured, as well as anastomotic hydroxyproline concentration. The presence of bowel wall edema was assessed histologically. Median bursting pressures were comparable in the Col (−) [118 mm Hg (range 113–170)], the Cry (−) [118 mm Hg (78–139)], and the Col (+) [97 mm Hg (65–152)] group. A significantly lower median bursting pressure was found in animals with crystalloid volume overload Cry (+) [73 mm Hg (60–101)]. Corresponding results were found for hydroxyproline concentration. Histology revealed submucosal edema in Cry (+) animals. In case of a fixed, high-volume load, colloids seem to have benefits on intestinal anastomotic healing when compared to crystalloid infusions.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by aggressive biology and poor prognosis even after resection. Long-term survival is very rare and cannot be reliably predicted. Experimental data suggest an important role of epithelial-mesenchymal transition (EMT) in invasion and metastasis of PDAC. Tumor budding is regarded as the morphological correlate of local invasion and cancer cell dissemination. The aim of this study was to evaluate the biological and prognostic implications of EMT and tumor budding in PDAC of the pancreatic head.Patients were identified from a prospectively maintained database, and baseline, operative, histopathological, and follow-up data were extracted. Serial tissue slices stained for Pan-Cytokeratin served for analysis of tumor budding, and E-Cadherin, Beta-Catenin, and Vimentin staining for analysis of EMT. Baseline, operative, standard pathology, and immunohistochemical parameters were evaluated for prediction of long-term survival (≥ 30 months) in uni- and multivariate analysis.Intra- and intertumoral patterns of EMT marker expression and tumor budding provide evidence of partial EMT induction at the tumor-host interface. Lymph node ratio and E-Cadherin expression in tumor buds were independent predictors of long-term survival in multivariate analysis.Detailed immunohistochemical assessment confirms a relationship between EMT and tumor budding at the tumor-host interface. A small group of patients with favorable prognosis can be identified by combined assessment of lymph node ratio and EMT in tumor buds.

Purpose FFPE (formalin fixed, paraffin embedded) tissue cohorts represent an enduring archive of clinical specimens. Proteomic analysis of FFPE tissues is gaining interest for the in-depth analysis of aberrant proteome composition. Procedures for FFPE tissue processing are standardized but there is diversity regarding the different processing systems. This work focuses on three different processing methods commonly used in large European pathology institutes. Experimental design Formalin fixed tissue specimens of different tumors were serially sliced and processed with three different processing systems (xylene, ethanol/vacuum or microwave based). After paraffin embedding, they were subjected to MS-based proteomic analysis to investigate the impact of tissue processing techniques on the quality of proteomic analysis. Results were compared with proteomic analysis of corresponding cryopreserved tissue specimens. Results All processing techniques achieved very good proteome coverage similar to the cryopreserved counterpart. Gene ontology profiles, relative protein abundances, and peptide modifications such as methionine oxidation or proteolytic truncation were highly similar for all techniques as well as for the cryopreserved samples. Conclusions and clinical relevance The results show that different processing procedures do not impede proteomic analysis as a robust and powerful approach for the identification of protein determinants and markers of disease processes and highlights the general robustness of FFPE-tissue based proteomics.

To examine bowel wall edema development in laparoscopic and open major visceral surgery. In a prospective study, 47 consecutively operated patients with gastric and pancreatic resections were included. Twenty-seven patients were operated in a conventional open procedure (open group) and 20 in a laparoscopic fashion (lap group). In all procedures, a small jejunal segment was resected during standard preparation, of which we measured the dry-wet ratio. Furthermore, HE staining was performed for measuring of bowel wall thickness and edema assessment. Mean value (±std) of dry-wet ratio was significantly lower in the open than in the lap group (0.169 ± 0.017 versus 0.179 ± 0.015; p = 0.03) with the same amount of fluid administration in both groups and a longer infusion interval during laparoscopic surgery. Subgroup analyses (only pancreatic resections) still showed similar results. Histologic examination depicted a significantly larger bowel wall thickness in the open group. Laparoscopic surgery does not seem to lead to the bowel wall edema observed to occur in open surgery regardless of the degree of intravenous fluid administration, thus supporting its use even in major visceral surgery.

The aim of the article is to investigate a new anastomotic technique compared with standardized intestinal anastomotic procedures.A total of 32 male Wistar rats were randomized to three groups. In the Experimental Group (n = 10), the new double 90 degrees inversely rotated anastomosis was used, in the End Group (n = 10) a single-layer end-to-end anastomosis, and in the Side Group (n = 12) a single-layer side-to-side anastomosis. All anastomoses were done using interrupted sutures. On postoperative day 4, rats were relaparotomized. Bursting pressure, hydroxyproline concentration, a semiquantitative adhesion score and two histological anastomotic healing scores (mucosal healing according to Chiu and overall anastomotic healing according to Verhofstad) were collected. Most data are presented as median (range). p0.05 was considered significant.Anastomotic insufficiency occurred only in one rat of the Side Group. Median bursting pressure in the Experimental Group was 105 mm Hg (range = 72-161 mm Hg), significantly higher in the End Group (164 mm Hg; range = 99-210 mm Hg; p = 0.021) and lower in the Side Group by trend (81 mm Hg; range = 59-122 mm Hg; p = 0.093). Hydroxyproline concentration did not differ significantly in between the groups. The adhesion score was 2.5 (range = 1-3) in the Experimental Group, 2 (range = 1-2) in the End Group, but there were significantly more adhesions in the Side Group (range = 3-4); p = 0.020 versus Experimental Group, p0.001 versus End Group. The Chiu Score showed the worst mucosal healing in the Experimental Group. The overall Verhofstad Score was significantly worse (mean = 2.032; standard deviation [SD] = 0.842) p = 0.031 and p = 0.002 in the Experimental Group, compared with the Side Group (mean = 1.729; SD = 0.682) and the End Group (mean = 1.571; SD = 0.612).The new anastomotic technique is feasible and did not show any relevant complication. Even though it was superior to the side-to-side anastomosis by trend with respect to functional stability, mucosal healing surprisingly showed the worst results. Classical end-to-end anastomosis still seems to be the best choice regarding structural and functional anastomotic stability.

The protein tyrosine kinase Ephrin type-B receptor 3 (EPHB3) counteracts tumor-cell dissemination by regulating intercellular adhesion and repulsion and acts as tumor/invasion suppressor in colorectal cancer. This protective mechanism frequently collapses at the adenoma–carcinoma transition due to EPHB3 transcriptional silencing. Here, we identify a transcriptional enhancer at the EPHB3 gene that integrates input from the intestinal stem-cell regulator achaete-scute family basic helix-loop-helix transcription factor 2 (ASCL2), Wnt/β-catenin, MAP kinase, and Notch signaling. EPHB3 enhancer activity is highly variable in colorectal carcinoma cells and precisely reflects EPHB3 expression states, suggesting that enhancer dysfunction underlies EPHB3 silencing. Interestingly, low Notch activity parallels reduced EPHB3 expression in colorectal carcinoma cell lines and poorly differentiated tumor-tissue specimens. Restoring Notch activity reestablished enhancer function and EPHB3 expression. Although essential for intestinal stem-cell maintenance and adenoma formation, Notch activity seems dispensable in colorectal carcinomas. Notch activation even promoted growth arrest and apoptosis of colorectal carcinoma cells, attenuated their self-renewal capacity in vitro, and blocked tumor growth in vivo. Higher levels of Notch activity also correlated with longer disease-free survival of colorectal cancer patients. In summary, our results uncover enhancer decommissioning as a mechanism for transcriptional silencing of the EPHB3 tumor suppressor and argue for an antitumorigenic function of Notch signaling in advanced colorectal cancer.

Purpose: This investigation focuses on the physiological characteristics of gene transcription of intestinal tissue following anastomosis formation. Methods: In eight rats, end-to-end ileo-ileal anastomoses were performed (n = 2/group). The healthy intestinal tissue resected for this operation was used as a control. On days 0, 2, 4 and 8, 10-mm perianastomotic segments were resected. Control and perianastomotic segments were examined with an Affymetrix microarray chip to assess changes in gene regulation. Microarray findings were validated using real-time PCR for selected genes. In addition to screening global gene expression, we identified genes intensely regulated during healing and also subjected our data sets to an overrepresentation analysis using the Gene Ontology (GO) and Kyoto Encyclopedia for Genes and Genomes (KEGG). Results: Compared to the control group, we observed that the number of differentially regulated genes peaked on day 2 with a total of 2,238 genes, decreasing by day 4 to 1,687 genes and to 1,407 genes by day 8. PCR validation for matrix metalloproteinases-3 and -13 showed not only identical transcription patterns but also analogous regulation intensity. When setting the cutoff of upregulation at 10-fold to identify genes likely to be relevant, the total gene count was significantly lower with 55, 45 and 37 genes on days 2, 4 and 8, respectively. A total of 947 GO subcategories were significantly overrepresented during anastomotic healing. Furthermore, 23 overrepresented KEGG pathways were identified. Conclusion: This study is the first of its kind that focuses explicitly on gene transcription during intestinal anastomotic healing under standardized conditions. Our work sets a foundation for further studies toward a more profound understanding of the physiology of anastomotic healing.

Background Pancreatic ductal adenocarcinoma (PDAC) is characterized by an aggressive biology and poor prognosis. Experimental evidence has suggested a role for the transcriptional repressor Zinc finger E-box binding homeobox 1 (ZEB1) in epithelial-mesenchymal transition, invasion, and metastasis in PDAC. ZEB1 expression has been observed in cancer cells as well as stromal fibroblasts. Our study aimed to evaluate the prognostic value of ZEB1 expression in PDAC tissue. Methods Patient baseline and follow-up data were extracted from a prospectively maintained database. After clinicopathologic re-review, serial sliced tissue slides were immunostained for ZEB1, E-cadherin, vimentin, and pan-cytokeratin. ZEB1 expression in cancer cells and adjacent stromal fibroblasts was graded separately and correlated to routine histopathologic parameters and survival after resection. Results A total of 117 cases of PDAC were included in the study. High ZEB1 expression in cancer cells and in stromal cancer-associated fibroblasts was associated with poor prognosis. There was also a trend for poor prognosis with a lymph node ratio of greater than 0.10. In line with its role as an inducer of epithelial-mesenchymal transition, ZEB1 expression in cancer cells was positively correlated with Vimentin expression and negatively with E-Cadherin expression. In multivariate analysis, stromal ZEB1 expression grade was the only independent factor of survival after resection. Conclusion Our data suggest that ZEB1 expression in cancer cells as well as in stromal fibroblasts are strong prognostic factors in PDAC. Stromal ZEB1 expression is identified for the first time as an independent predictor of survival after resection of PDAC. This observation suggests that therapies targeting ZEB1 and its downstream pathways could hit both cancer cells and supporting cancer-associated fibroblasts.