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Purpose: Mutation of BRAF at the valine 600 residue occurs in approximately 10% of colorectal cancers, a group with particularly poor prognosis. The response of BRAF mutant colorectal cancer to recent targeted strategies such as anti-BRAF or combinations with MEK and EGFR inhibitors remains limited and highly heterogeneous within BRAF V600E cohorts. There is clearly an unmet need in understanding the biology of BRAF V600E colorectal cancers and potential subgroups within this population.Experimental Design: In the biggest yet reported cohort of 218 BRAF V600E with gene expression data, we performed unsupervised clustering using non-negative matrix factorization to identify gene expression–based subgroups and characterized pathway activation.Results: We found strong support for a split into two distinct groups, called BM1 and BM2. These subtypes are independent of MSI status, PI3K mutation, gender, and sidedness. Pathway analyses revealed that BM1 is characterized by KRAS/AKT pathway activation, mTOR/4EBP deregulation, and EMT whereas BM2 displays important deregulation of the cell cycle. Proteomics data validated these observations as BM1 is characterized by high phosphorylation levels of AKT and 4EBP1, and BM2 patients display high CDK1 and low cyclin D1 levels. We provide a global assessment of gene expression motifs that differentiate BRAF V600E subtypes from other colorectal cancers.Conclusions: We suggest that BRAF mutant patients should not be considered as having a unique biology and provide an in depth characterization of heterogeneous motifs that may be exploited for drug targeting. Clin Cancer Res; 23(1); 104–15. ©2016 AACR.

File containing Supplementary Tables S1-S7 and Legends to Supplementary Figures S1-S4. Supplementary Table S1. Primers for promoter methylation analysis by MSP of the 43 candidate genes. Supplementary Table S2. Primers for mRNA expression analysis by qRT-PCR for 22 genes. Supplementary Table S3. Candidate gene selection. Supplementary Table S4. Validation of expression microarrays and MBD-NGS by MSP. Supplementary Table S5. Candidate gene promoter methylation associated with patient and tumor characteristics - hospital-based series. Supplementary Table S6. Hazard ratio's with 95% confidence intervals and p-values, including model fit according to Harrell's C and AIC, for the single markers, all possible two- and three-marker panels, and the four-marker panel for both the age- and sex-adjusted models and the multivariable models in the hospital-based series. Supplementary Table S7. Hazard ratio's with 95% confidence intervals and p-values, including model fit according to Harrell's C and AIC, for the single markers, all possible two- and three-marker panels, and the four-marker panel for both the age- and sex-adjusted models and the multivariable models in the population-based series. Supplementary Figure S1. Methylation analysis of GREM1, LAD1, NEFH and NEURL by methylation-specific PCR (MSP) in the hospital- and population-based series and in the TCGA series. Supplementary Figure S2. Validation of up-regulation of gene expression after demethylation with DAC by qRT-PCR for 22 genes. Supplementary Figure S3. Cause-specific overall survival curves for candidate genes in hospital-based ccRCC series (related to Supplementary Table S5). Supplementary Figure S4. Cause-specific overall survival curves for GREM1, LAD1, NEFH, and NEURL in hospital- and population-based ccRCC series (related to Table 1 and Table 2).

Analysis of GO processes enriched in several mTOR signatures.

Supplementary Figure S2. Validation of up-regulation of gene expression after demethylation with DAC by qRT-PCR for 22 genes. Relative mRNA expression of 22 genes analyzed by qRT-PCR in ccRCC cell lines SKRC1, SKRC52 and SKRC59 treated with either mock or 5μm 5-aza-2'-deoxycytidine (DAC) for 96 hours. Expression levels were analyzed in triplo unless stated otherwise. Error bars indicate the standard error of the mean. * in duplo; ** single values due to technical failure; *** no expression in SKRC1 and SKRC59.

Purpose: The currently used prognostic models for patients with nonmetastatic clear cell renal cell carcinoma (ccRCC) are based on clinicopathologic features and might be improved by adding molecular markers. Epigenetic alterations occur frequently in ccRCC and are promising biomarkers. The aim of this study is to identify prognostic promoter methylation markers for ccRCC.Experimental Design: We integrated data generated by massive parallel sequencing of methyl-binding domain enriched DNA and microarray-based RNA expression profiling of 5-aza-2′-deoxycytidine–treated ccRCC cell lines to comprehensively characterize the ccRCC methylome. A selection of the identified methylation markers was evaluated in two independent series of primary ccRCC (n = 150 and n = 185) by methylation-specific PCR. Kaplan–Meier curves and log-rank tests were used to estimate cause-specific survival. HRs and corresponding 95% confidence intervals (CI) were assessed using Cox proportional hazard models. To assess the predictive capacity and fit of models combining several methylation markers, HarrellC statistic and the Akaike Information Criterion were used.Results: We identified four methylation markers, that is, GREM1, NEURL, LAD1, and NEFH, that individually predicted prognosis of patients with ccRCC. The four markers combined were associated with poorer survival in two independent patient series (HR, 3.64; 95% CI, 1.02–13.00 and HR, 7.54; 95% CI, 2.68–21.19). These findings were confirmed in a third series of ccRCC cases from The Cancer Genome Atlas (HR, 3.60; 95% CI, 2.02–6.40).Conclusions: A four-gene promoter methylation marker panel consisting of GREM1, NEURL, LAD1, and NEFH predicts outcome of patients with ccRCC and might be used to improve current prognostic models. Clin Cancer Res; 23(8); 2006–18. ©2016 AACR.

Figure S1. Principal component analysis and hierarchical clustering of the 218 BRAF mutant patient cohort;Figure S2: Gene-wise and patient-wise biclustering heatmap of the 218 BRAF mutant patient cohort and comparison of BRAF mutant subtypes obtained with and without adjusting for microsatellite instability status;Figure S3: Geneset enrichment analysis (GSEA) of BM subtypes for the four most enriched signature of the Hallmark collection and gene overlap between signatures and BM-specific genes;Figure S4: Patient-level single sample geneset enrichment analysis (ssGSEA) between BM subtypes, double BRAF wild-type KRAS wild-type (WT2) and KRAS mutant patients for the hallmark signature collection;Figure S5: Patient-averaged single sample geneset enrichment analysis (ssGSEA) between BM subtypes, double BRAF wild-type KRAS wild-type (WT2) and KRAS mutant patients for the hallmark signature collection;Figure S6: Gene overlap between selected sets of gene signatures used in this study;Figure S7: Immune landscape of the BM subtypes;Figure S8: Methylation analysis of BM subtypes using TCGA data;Figure S9: Survival and clinical characteristics of the BRAF mutant patients enrolled in the survival analysis;Figure S10: Performance of the generalized model used to classify patients into BM1 and BM2 subtypes;Figure S11: Drug responses of colorectal cancer cell lines classified into BM1 and BM2.

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Table S1: Definitions of mutation calls used in this study;Table S2: List of genes with non-zero coefficients obtained by the classifier used to categorize gene expression profiles into BM1 and BM2.

This file contains the supplementary methods that either describe the methodological part related to supplementary figures or the detailed aspects for the following methods: clustering, pathway analysis, survival, methylation and proteomic analyses, and cell line drug response analyses. The file also includes the legends for all supplementary data (Figures and Tables).

Purpose: Colorectal cancer (CRC) is a common cause of death worldwide. Tumor-node-metastasis-system stage is currently used to guide therapy decisions but lacks precision. Prognostic biomarkers are needed to refine stratification of patients for chemotherapy but validated biomarkers are not yet available. Recently, a SNP in a lethal-7 (let-7) miRNA complementary site (LCS6) in the KRAS 3′untranslated region was suggested to affect survival in metastatic CRC. Effects in early-stage CRC are however unknown. We studied KRAS-LCS6 genotype, hypothesizing that it might identify early-stage cases with a poor prognosis, and could potentially be used in therapy decision-making.Experimental Design: We studied 409 early stage, 182 stage III, and 69 stage IV cases, and 1,886 subcohort members from the Netherlands Cohort Study. KRAS-LCS6 genotype was assessed with TaqMan PCR. Kaplan–Meier analyses or Cox regression were used to assess associations between genotype and CRC risk or cause-specific survival.Results: Early-stage cases with the KRAS-LCS6 variant had a lower CRC risk (incidence-rate ratio 0.68; 95% CI: 0.49–0.94) and a better survival (log-rank P = 0.038; HR 0.46; 95% CI: 0.18–1.14). In patients with KRAS-mutated CRC carrying the KRAS-LCS6 variant, the better outcome was enhanced as no patients died of CRC (log-rank P = 0.017). In advanced patients, no clear association between genotype and CRC risk or survival was observed.Conclusions: Our results indicate that early-stage CRC cases with the KRAS-LCS6 variant have a better outcome. In advanced disease, the better outcome no longer exists. For early-stage patients, KRAS-LCS6 genotype combined with KRAS mutations merits validation as a prognostic biomarker and consideration in therapy decision-making. Clin Cancer Res; 17(24); 7723–31. ©2011 AACR.

Biomarker-guided treatment for patients with colon cancer is needed. We tested ABCG2 and topoisomerase 1 (TOP1) mRNA expression as predictive biomarkers for irinotecan benefit in the PETACC-3 patient cohort. The present study included 580 patients with mRNA expression data from Stage III colon cancer samples from the PETACC-3 study, which randomized the patients to Fluorouracil/leucovorin (5FUL) +/&minus, irinotecan. The primary end-points were recurrence free survival (RFS) and overall survival (OS). Patients were divided into one group with high ABCG2 expression (above median) and low TOP-1 expression (below 75 percentile) (&ldquo, resistant&rdquo, ) (n = 216) and another group including all other combinations of these two genes (&ldquo, sensitive&rdquo, ) (n = 364). The rationale for the cut-offs were based on the distribution of expression levels in the PETACC-3 Stage II set of patients, where ABCG2 was unimodal and TOP1 was bimodal with a high expression level mode in the top quarter of the patients. Cox proportional hazards regression was used to estimate the hazard ratios and the association between variables and end-points and log-rank tests to assess the statistical significance of differences in survival between groups. Kaplan-Meier estimates of the survival functions were used for visualization and estimation of survival rates at specific time points. Significant differences were found for both RFS (Hazard ratio (HR): 0.63 (0.44&ndash, 0.92), p = 0.016) and OS (HR: 0.60 (0.39&ndash, 0.93), p = 0.02) between the two biomarker groups when the patients received FOLFIRI (5FUL+irinotecan). Considering only the Microsatellite Stable (MSS) and Microsatellite Instability-Low (MSI-L) patients (n = 470), the differences were even more pronounced. In contrast, no significant differences were observed between the groups when patients received 5FUL alone. This study shows that the combination of ABCG2 and TOP1 gene expression significantly divided the Stage III colon cancer patients into two groups regarding benefit from adjuvant treatment with FOLFIRI but not 5FUL.

Motivation Whole genome expression profiling of large cohorts of different types of cancer led to the identification of distinct molecular subcategories (subtypes) that may partially explain the observed inter-tumoral heterogeneity. This is also the case of colorectal cancer (CRC) where several such categorizations have been proposed. Despite recent developments, the problem of subtype definition and recognition remains open, one of the causes being the intrinsic heterogeneity of each tumor, which is difficult to estimate from gene expression profiles. However, one of the observations of these studies indicates that there may be links between the dominant tumor morphology characteristics and the molecular subtypes. Benefiting from a large collection of CRC samples, comprising both gene expression and histopathology images, we investigated the possibility of building image-based classifiers able to predict the molecular subtypes. We employed deep convolutional neural networks for extracting local descriptors which were then used for constructing a dictionary-based representation of each tumor sample. A set of support vector machine classifiers were trained to solve different binary decision problems, their combined outputs being used to predict one of the five molecular subtypes. Results A hierarchical decomposition of the multi-class problem was obtained with an overall accuracy of 0.84 (95%CI=0.79–0.88). The predictions from the image-based classifier showed significant prognostic value similar to their molecular counterparts. Availability and Implementation Source code used for the image analysis is freely available from https://github.com/higex/qpath. Supplementary information Supplementary data are available at Bioinformatics online.

Colorectal cancer is among the most frequent of human cancers with an estimated 1.4 million new cases of CRC occurred worldwide in 2012. Most colorectal cancers are sporadic, but about 20% of colorectal cancers arise in the context of a familial syndrome or as a complication of inflammatory bowel disease, and these cancers have distinct characteristics. Colorectal cancers develop in different molecular patterns, which are known as the chromosomal instability (CIN), microsatellite instability (MIN) and CpG island methylator phenotype (CIMP) pathways. Results of recent molecular profiling studies have clearly established molecular and genetic heterogeneity of colorectal cancer beyond these established categories, which has resulted in consensus molecular subtypes. About 40% of colorectal cancers are KRAS mutated and these will not respond to EGFR blocking therapies. Microsatellite instable colorectal cancers have gained significant interest recently as they epitomize a category of hypermutating immunogenic tumors, which respond well to immunotherapy through blocking of the PD1 immune-checkpoint.

Anal cancers are relatively uncommon at around 2% of large bowel cancers. Squamous cell carcinoma is the most frequent. Squamous cell carcinomas (including the basal cell variant) are associated with high risk Human Papilloma Virus (HPV type 16) infection and occur most frequently in high risk groups such as males with male sexual partners, immunodeficient patients due to HIV or to immunosuppressive therapy and women with a history of HPV associated cervical squamous intra-epithelial lesions. Anal intraepithelial neoplasia is a precursor. Verrucous carcinoma is also known as giant condyloma acuminatum and represents low malignant squamous cell carcinoma, associated with low risk HPV types (6 and 11). Rare primary tumors of the anal canal are extramammary Paget's disease and anal melanoma.

Telomerase activity, not detectable in somatic cells but frequently activated during carcinogenesis, confers immortality to tumors. Mechanisms governing expression of the catalytic subunit hTERT, the limiting factor for telomerase activity, still remain unclear. We previously proposed a model in which the binding of the transcription factor CTCF to the two first exons of hTERT results in transcriptional inhibition in normal cells. This inhibition is abrogated, however, by methylation of CTCF binding sites in 85% of tumors. Here, we showed that hTERT was unmethylated in testicular and ovarian tumors and in derivative cell lines. We demonstrated that CTCF and its paralogue, BORIS/CTCFL, were both present in the nucleus of the same cancer cells and bound to the first exon of hTERT in vivo. Moreover, exogenous BORIS expression in normal BORIS-negative cells was sufficient to activate hTERT transcription with an increasing number of cell passages. Thus, expression of BORIS was sufficient to allow hTERT transcription in normal cells and to counteract the inhibitory effect of CTCF in testicular and ovarian tumor cells. These results define an important contribution of BORIS to immortalization during tumorigenesis.

Contains fulltext : 177917pub.pdf (Publisher’s version ) (Closed access) Tumor budding is a well-established independent prognostic factor in colorectal cancer but a standardized method for its assessment has been lacking. The primary aim of the International Tumor Budding Consensus Conference (ITBCC) was to reach agreement on an international, evidence-based standardized scoring system for tumor budding in colorectal cancer. The ITBCC included nine sessions with presentations, a pre-meeting survey and an e-book covering the key publications on tumor budding in colorectal cancer. The 'Grading of Recommendation Assessment, Development and Evaluation' method was used to determine the strength of recommendations and quality of evidence. The following 10 statements achieved consensus: tumor budding is defined as a single tumor cell or a cell cluster consisting of four tumor cells or less (22/22, 100%). Tumor budding is an independent predictor of lymph node metastases in pT1 colorectal cancer (23/23, 100%). Tumor budding is an independent predictor of survival in stage II colorectal cancer (23/23, 100%). Tumor budding should be taken into account along with other clinicopathological features in a multidisciplinary setting (23/23, 100%). Tumor budding is counted on H&E (19/22, 86%). Intratumoral budding exists in colorectal cancer and has been shown to be related to lymph node metastasis (22/22, 100%). Tumor budding is assessed in one hotspot (in a field measuring 0.785 mm2) at the invasive front (22/22, 100%). A three-tier system should be used along with the budding count in order to facilitate risk stratification in colorectal cancer (23/23, 100%). Tumor budding and tumor grade are not the same (23/23, 100%). Tumor budding should be included in guidelines/protocols for colorectal cancer reporting (23/23, 100%). Members of the ITBCC were able to reach strong consensus on a single international, evidence-based method for tumor budding assessment and reporting. It is proposed that this method be incorporated into colorectal cancer guidelines/protocols and staging systems.

Purpose: The currently used prognostic models for patients with nonmetastatic clear cell renal cell carcinoma (ccRCC) are based on clinicopathologic features and might be improved by adding molecular markers. Epigenetic alterations occur frequently in ccRCC and are promising biomarkers. The aim of this study is to identify prognostic promoter methylation markers for ccRCC. Experimental Design: We integrated data generated by massive parallel sequencing of methyl-binding domain enriched DNA and microarray-based RNA expression profiling of 5-aza-2′-deoxycytidine–treated ccRCC cell lines to comprehensively characterize the ccRCC methylome. A selection of the identified methylation markers was evaluated in two independent series of primary ccRCC (n = 150 and n = 185) by methylation-specific PCR. Kaplan–Meier curves and log-rank tests were used to estimate cause-specific survival. HRs and corresponding 95% confidence intervals (CI) were assessed using Cox proportional hazard models. To assess the predictive capacity and fit of models combining several methylation markers, HarrellC statistic and the Akaike Information Criterion were used. Results: We identified four methylation markers, that is, GREM1, NEURL, LAD1, and NEFH, that individually predicted prognosis of patients with ccRCC. The four markers combined were associated with poorer survival in two independent patient series (HR, 3.64; 95% CI, 1.02–13.00 and HR, 7.54; 95% CI, 2.68–21.19). These findings were confirmed in a third series of ccRCC cases from The Cancer Genome Atlas (HR, 3.60; 95% CI, 2.02–6.40). Conclusions: A four-gene promoter methylation marker panel consisting of GREM1, NEURL, LAD1, and NEFH predicts outcome of patients with ccRCC and might be used to improve current prognostic models. Clin Cancer Res; 23(8); 2006–18. ©2016 AACR.

In this article, the authors review the current status of biomarker research in the adjuvant treatment of colon cancer, drawing on their experiences and considering future strategies for biomarker discovery in the postgenomic era., The number of agents that are potentially effective in the adjuvant treatment of locally advanced resectable colon cancer is increasing. Consequently, it is important to ascertain which subgroups of patients will benefit from a specific treatment. Despite more than two decades of research into the molecular genetics of colon cancer, there is a lack of prognostic and predictive molecular biomarkers with proven utility in this setting. A secondary objective of the Pan European Trials in Adjuvant Colon Cancer-3 trial, which compared irinotecan in combination with 5-fluorouracil and leucovorin in the postoperative treatment of stage III and stage II colon cancer patients, was to undertake a translational research study to assess a panel of putative prognostic and predictive markers in a large colon cancer patient cohort. The Cancer and Leukemia Group B 89803 trial, in a similar design, also investigated the use of prognostic and predictive biomarkers in this setting. In this article, the authors, who are coinvestigators from these trials and performed similar investigations of biomarker discovery in the adjuvant treatment of colon cancer, review the current status of biomarker research in this field, drawing on their experiences and considering future strategies for biomarker discovery in the postgenomic era.

Molecular subtypes have been recently derived for various types of cancer, in an attempt to characterize the intertumoral heterogeneity. In this work we explore the possibility of constructing predictors for molecular subtypes based on histopathology images. For this, we introduce a novel 2-level bag-of-features method and we apply it to a collection of colorectal cancer samples. The resulting image features capture some relevant tumor morphology patterns and led to a classifier performing similarly to one constructed from features annotated by an expert pathologist. The significance of our results extends beyond subtype prediction since they demonstrate a possible approach to multimodal (histopathology and molecular) data mining and biomarker identification.

In any profession, medical or not, assurance of professional standards and of the quality of the end-product or the service rendered has become an essential requirement. This holds true also for the practice of pathology. It is the intention to cover in this issue of Virchows Archiv a variety of relevant aspects of quality assurance, directly pertaining to the practice of pathology, and also subjects indirectly related to pathology practice. This includes not only quality assurance in the laboratories (including issues in basic histology, immunohistochemistry, and molecular pathology) but also the question on how the quality of diagnostic practice at the microscope can be monitored, quality assurance in underand postgraduate education and continuous professional development in pathology, sense and non-sense in laboratory accreditation, communication in pathology including the role of structured standard reporting, the role of the autopsy in quality of care assurance, quality assurance in postgraduate education, and last but not least quality assurance in pathology research. Aims and scope of this journal pretend to emphasize “evidence based” approaches. The subjects discussed in this issue, however, to a large extent express “what we think” or “what we are convinced of” as factual supporting evidence is often lacking, for want of studies or because of the nature of the subject. The papers therefore are mostly position papers, intended to stimulate reflection and discussion rather than providing practical solutions. In the first paper in the series (DOI 10.1007/s00428-0151838-0) Clark describes how Lean management, an approach developed by the Toyota automobile company which creates value for the end-user by continuously improving operational effectiveness and removing waste, can be adopted in histopathology. It has been applied in departments throughout the world to simultaneously improve quality (reducing errors and shortening turnaround times) and lower costs (by increasing efficiency). The paper describes the key concepts and how these were adapted to histopathology, using a case study of Lean implementation and evidence from the literature. It discusses the benefits, limitations, and pitfalls encountered when implementing Lean management systems. The paper notably emphasizes the need for a change of mindset in the process of Lean implementation. The process goes way beyond introduction of a particular management system; it requires sustained specific people management skills in a working environment emphasizing continuous improvement. It requires significant efforts and means but its potential benefits are far reaching. One of the established core techniques in diagnostic pathology is immunohistochemistry. In spite of the more than 30 years of its steady growth into automated immunostaining aswe know it today, it remains a method beset with pitfalls, in terms of the quality of the reagents and of the analyte, of the protocols used and of the final interpretation of the obtained staining result. This has become even more important in the era of targeted therapy. Vyberg and Nielsen. (DOI 10.1007/s00428-015-1829-1) report on the experience gained with Nordic Immunohistochemical Quality Control (NordiQC), an international academic proficiency testing primarily aimed at assessing quality of the analytical phases of immunohistochemistry. A substantial proportion of the analyses performed (between 20 and 30 %) * Fred T. Bosman fred.bosman@citycable.ch

Vreuls C P H, Van Den Broek M A, Winstanley A, Koek G H, Wisse E, Dejong C H, Olde Damink S W M, Bosman F T & Driessen A ?(2012) Histopathology similar to 61, 314318 Hepatic sinusoidal obstruction syndrome (SOS) reduces the effect of oxaliplatin in colorectal liver metastases Aims: Oxaliplatin is an important chemotherapeutic agent used to reduce hepatic colorectal metastases, resulting in tumour reduction and permitting surgical resection. This treatment has significant side effects, as oxaliplatin can induce sinusoidal obstruction syndrome (SOS) in the non-tumour-bearing liver, resulting in increased morbidity. We hypothesized that SOS might impede hepatic perfusion, thereby interfering with the tumour environment and attenuate the response to the chemotherapy. Methods and results: From the prospective database of the Maastricht University Medical Centre we collected 50 patients with hepatic colorectal carcinoma metastases. All patients received neo-adjuvant oxaliplatin followed by partial hepatectomy. Metastases and non-tumour-bearing liver were studied histopathologically. Thirty-two of 50 (64%) patients showed SOS lesions, classified as mild (26%) and moderatesevere (38%). The response to treatment, as expressed in the tumour regression grade (TRG), was grade 1 (10%); grade 2 (14%); grade 3 (28%); grade 4 (32%) and grade 5 (16%). Statistical analysis showed that a higher grade of SOS was associated with a higher grade of TRG (P = 0.016). Conclusion: Developing SOS is associated with a lower tumour response to neo-adjuvant oxaliplatin treatment. Hepatic hypoperfusion due to sinusoidal obstruction syndrome might induce hepatic hypoxia, diminishing the response to chemotherapy.

Purpose Our purpose was development and assessment of a BRAF-mutant gene expression signature for colon cancer (CC) and the study of its prognostic implications. Materials and Methods A set of 668 stage II and III CC samples from the PETACC-3 (Pan-European Trails in Alimentary Tract Cancers) clinical trial were used to assess differential gene expression between c.1799T>A (p.V600E) BRAF mutant and non-BRAF, non-KRAS mutant cancers (double wild type) and to construct a gene expression–based classifier for detecting BRAF mutant samples with high sensitivity. The classifier was validated in independent data sets, and survival rates were compared between classifier positive and negative tumors. Results A 64 gene-based classifier was developed with 96% sensitivity and 86% specificity for detecting BRAF mutant tumors in PETACC-3 and independent samples. A subpopulation of BRAF wild-type patients (30% of KRAS mutants, 13% of double wild type) showed a gene expression pattern and had poor overall survival and survival after relapse, similar to those observed in BRAF-mutant patients. Thus they form a distinct prognostic subgroup within their mutation class. Conclusion A characteristic pattern of gene expression is associated with and accurately predicts BRAF mutation status and, in addition, identifies a population of BRAF mutated-like KRAS mutants and double wild-type patients with similarly poor prognosis. This suggests a common biology between these tumors and provides a novel classification tool for cancers, adding prognostic and biologic information that is not captured by the mutation status alone. These results may guide therapeutic strategies for this patient segment and may help in population stratification for clinical trials.

Strong epidemiologic evidence suggests an association between Alzheimer disease (AD) and type 2 diabetes. To determine if amyloid beta (A$\beta$) and hyperphosphorylated tau occurs in type 2 diabetes, pancreas tissues from 21 autopsy cases (10 type 2 diabetes and 11 controls) were analyzed. APP and tau mRNAs were identified in human pancreas and in cultured insulinoma beta cells (INS-1) by RT-PCR. Prominent APP and tau bands were detected by Western blotting in pancreatic extracts. Aggregated A$\beta$, hyperphosphorylated tau, ubiquitin, apolipoprotein E, apolipoprotein(a), IB1/JIP-1 and JNK1 were detected in Langerhans islets in type 2 diabetic patients. A$\beta$ was co-localized with amylin in islet amyloid deposits. In situ beta sheet formation of islet amyloid deposits was shown by infrared microspectroscopy (SIRMS). LPS increased APP in non-neuronal cells as well. We conclude that A$\beta$ deposits and hyperphosphorylated tau are also associated with type 2 diabetes, highlighting common pathogenetic features in neurodegenerative disorders, including AD and type 2 diabetes and suggesting that A deposits and hyperphosphorylated tau may also occur in other organs than the brain.

In the beginning, colorectal cancer (CRC) was a simple disease. It was considered a single entity, be it with different faces.1 Also, the precursor lesions of CRC were known, and the adenoma–carcinoma sequence as a model for the stepwise progression from an adenoma to an invasive CRC became a paradigm for similar lesions in other organs. Even the underlying molecular genetic alterations driving the consecutive steps of the adenoma–carcinoma were clarified.2 And so CRC was conceived to be the result of the accumulation of specific genetic mutations, at least partly due to chromosomal instability, with activated oncogenes and inactivated tumour suppressor genes causing uncontrollable cell growth. Loss of function of the APC tumour suppressor gene, the gatekeeper of the colorectum, was the initiating genetic event of adenoma formation, and familial adenomatous polyposis, caused by a germline mutation in APC , was perceived as the hereditary counterpart of sporadic CRC in the general population.3 Soon, however, the above picture became a bit more complicated, when a second form of familial CRC was discovered: the Lynch syndrome.4 The Lynch syndrome is caused by a germline defect in one of the mismatch repair genes ( MLH1 , MSH2 , MSH6 , PMS1 and PMS2 ) resulting in microsatellite instability. This new pathway leading to CRC was paralleled by characteristic clinicopathological features.5 Since then, through advances in endoscopical technology resulting in high-resolution views of the colonic mucosa, as well as advances in molecular genetic technology, the picture has evolved of CRC arising through a variety of pathways that can be defined at the molecular level, each accompanied by more or less specific morphology.6 The conventional adenoma no longer has the monopoly of being at the origin of …

With the free movement of people in the European Union, medical mobility has increased significantly. This is notably the case for disciplines for which shortage of well-trained staff has occurred. Pathology is among those specialties and effectively the discipline is confronted with a striking increase in mobility among trainees and qualified specialists. The presumption underlying unlimited mobility is that the competencies of the medical specialists in the European countries are more or less equal, including significant similarities in the postgraduate training programs. In order to assess whether reality corresponds with this presumption, we conducted a survey of the content and practice requirements of the curricula in the EU and affiliated countries. The results indicate a striking heterogeneity in the training program content and practice requirements. To name a few elements: duration of the training program varied between 4 and 6 years; the number of autopsies required varied between none at all and 300; the number of biopsies required varied between none at all and 15,000. We conclude that harmonization of training outcomes in Europe is a goal that needs to be pursued. This will be difficult to reach through harmonization of training programs, as these are co-determined by political, cultural, and administrative factors, difficult to influence. Harmonization might be attained by defining the general and specific competencies at the end of training and subsequent testing them through a test to which all trainees in Europe are subjected.

L’œsophage de Barrett est une complication du reflux gastro-œsophagien qui se caracterise par une metaplasie intestinale de l’œsophage distal. Dans un certain pourcentage de cas, la persistance de l’irritation est responsable du developpement d’une dysplasie epitheliale qui peut progresser par la suite vers un adenocarcinome intramuqueux, puis invasif et finalement metastatique. Sur la base essentiellement de la morphologie, l’anatomopathologiste essaie de predire a partir des biopsies, quels patients sont susceptibles de progresser vers le cancer. Pour l’instant, ce risque est essentiellement base sur le degre de dysplasie epitheliale. Cependant, sa classification est sujette a une mauvaise reproductibilite et sa valeur predictive dans la progression du cancer est limitee. Dans ce contexte, les nouvelles approches de diagnostic moleculaire, notamment: a) la detection de dommages a l’ADN, b) le controle du cycle cellulaire et c) l’instabilite genomique sont tres prometteuses et sont brievement discutees dans cette revue. L’arrivee imminente de la typisation moleculaire devrait permettre de mieux comprendre, diagnostiquer et predire l’evolution de cette maladie.

The aim of this study was to evaluate specific immunostaining and background staining in formalin-fixed, paraffin-embedded human tissues with the 2 most frequently used immunohistochemical detection systems, Avidin-Biotin-Peroxidase (ABC) and EnVision+. A series of fixed tissues, including breast, colon, kidney, larynx, liver, lung, ovary, pancreas, prostate, stomach, and tonsil, was used in the study. Three monoclonal antibodies, 1 against a nuclear antigen (Ki-67), 1 against a cytoplasmic antigen (cytokeratin), and 1 against a cytoplasmic and membrane-associated antigen and a polyclonal antibody against a nuclear and cytoplasmic antigen (S-100) were selected for these studies. When the ABC system was applied, immunostaining was performed with and without blocking of endogenous avidin-binding activity. The intensity of specific immunostaining and the percentage of stained cells were comparable for the 2 detection systems. The use of ABC caused widespread cytoplasmic and rare nuclear background staining in a variety of normal and tumor cells. A very strong background staining was observed in colon, gastric mucosa, liver, and kidney. Blocking avidin-binding capacity reduced background staining, but complete blocking was difficult to attain. With the EnVision+ system no background staining occurred. Given the efficiency of the detection, equal for both systems or higher with EnVision+, and the significant background problem with ABC, we advocate the routine use of the EnVision+ system.

Expression of hTERT is the major limiting factor for telomerase activity. We previously showed that methylation of the hTERT promoter is necessary for its transcription and that CTCF can repress hTERT transcription by binding to the first exon. In this study, we used electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) to show that CTCF does not bind the methylated first exon of hTERT. Treatment of telomerase-positive cells with 5-azadC led to a strong demethylation of hTERT 5'-regulatory region, reactivation of CTCF binding and downregulation of hTERT. Although complete hTERT promoter methylation was associated with full transcriptional repression, detailed mapping showed that, in telomerase-positive cells, not all the CpG sites were methylated, especially in the promoter region. Using a methylation cassette assay, selective demethylation of 110 bp within the core promoter significantly increased hTERT transcriptional activity. This study underlines the dual role of DNA methylation in hTERT transcriptional regulation. In our model, hTERT methylation prevents binding of the CTCF repressor, but partial hypomethylation of the core promoter is necessary for hTERT expression.

Communication is an essential element of good medical practice also in pathology. In contrast to technical or diagnostic skills, communication skills are not easy to define, teach, or assess. Rules almost do not exist. In this paper, which has a rather personal character and cannot be taken as a set of guidelines, important aspects of communication in pathology are explored. This includes what should be communicated to the pathologist on the pathology request form, communication between pathologists during internal (interpathologist) consultation, communication around frozen section diagnoses, modalities of communication of a final diagnosis, with whom and how critical and unexpected findings should be communicated, (in-)adequate routes of communication for pathology diagnoses, who will (or might) receive pathology reports, and what should be communicated and how in case of an error or a technical problem. An earlier more formal description of what the responsibilities are of a pathologist as communicator and as collaborator in a medical team is added in separate tables. The intention of the paper is to stimulate reflection and discussion rather than to formulate strict rules.

Current standard treatments for metastatic colorectal cancer (CRC) are based on combination regimens with one of the two chemotherapeutic drugs, irinotecan or oxaliplatin. However, drug resistance frequently limits the clinical efficacy of these therapies. In order to gain new insights into mechanisms associated with chemoresistance, and departing from three distinct CRC cell models, we generated a panel of human colorectal cancer cell lines with acquired resistance to either oxaliplatin or irinotecan. We characterized the resistant cell line variants with regards to their drug resistance profile and transcriptome, and matched our results with datasets generated from relevant clinical material to derive putative resistance biomarkers. We found that the chemoresistant cell line variants had distinctive irinotecan- or oxaliplatin-specific resistance profiles, with non-reciprocal cross-resistance. Furthermore, we could identify several new, as well as some previously described, drug resistance-associated genes for each resistant cell line variant. Each chemoresistant cell line variant acquired a unique set of changes that may represent distinct functional subtypes of chemotherapy resistance. In addition, and given the potential implications for selection of subsequent treatment, we also performed an exploratory analysis, in relevant patient cohorts, of the predictive value of each of the specific genes identified in our cellular models. publisher: Elsevier articletitle: Establishment and characterization of models of chemotherapy resistance in colorectal cancer: Towards a predictive signature of chemoresistance journaltitle: Molecular Oncology articlelink: http://dx.doi.org/10.1016/j.molonc.2015.02.008 content_type: article copyright: Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. ispartof: Molecular Oncology vol:9 issue:6 pages:1169-85 ispartof: location:United States status: published

Cytoskeletal research in recent years has revolutionized cell biology and biomedicine. The cytoskeleton spans the cytoplasm and interconnects the cell nucleus with the extracellular matrix, thereby forming a structural link between molecules involved in cell communication on the one hand, and gene expression on the other. Since the cytoskeleton is involved in virtually all cellular processes, abnormalities in this essential cellular component frequently result in disease. In this introduction, the basic structure of the cytoskeleton is briefly outlined. Furthermore, the disease processes in which the cytoskeleton plays a decisive role, and which are reviewed in detail in the papers in this issue, are briefly introduced. The advances in our understanding of the cytoskeleton and its function in disease will lead to new diagnostic and therapeutic applications in the foreseeable future. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Adenomatous polyposis coli (APC) promoter hypermethylation has been reported frequently in normal gastric mucosa, but it remained to be clarified whether this occurs in every individual. In this study, methylation of the APC promoter was analyzed in histologically normal-appearing gastric mucosa samples by methylation-sensitive single-strand conformation analysis and by a methylation-sensitive dot blot assay. Epithelial cell samples were collected by microdissection from tissue sections. Equal amounts of methylated and unmethylated APC alleles were found in all gastric mucosa samples from patients without any gastric lesions (20 samples). Allele-specific methylation analysis showed that the methylation of the APC promoter was monoallelic; however, which allele was methylated depended on the cell type. Increased or decreased methylation was found in 10 of 36 (28%) normal gastric mucosa samples adjacent to a gastric or esophageal adenocarcinoma. No allelic loss was found at the APC locus. Modification of the methylation status was also found in 3 of 21 (14%) normal-appearing gastric mucosa samples adjacent to intestinal metaplasia. In contrast, all normal mucosa samples in cases with chronic gastritis but without metaplasia or dysplasia showed a monoallelic methylation pattern. Our results indicate the following: (a) In normal gastric mucosa, the APC promoter shows monoallelic methylation, which is not due to imprinting but most likely due to allelic exclusion; (b) the excluded allele differs between foveolar and glandular epithelial cells; (c) the APC methylation pattern is frequently altered in normal-appearing gastric mucosa of gastric or esophageal adenocarcinoma patients; and (d) such alterations also occur in normal gastric mucosa adjacent to intestinal metaplasia.

The second preclinical year of the medical curriculum at the Medical Faculty of the University of Lausanne in Switzerland includes nine multidisciplinary organ-system-oriented modules consisting of lectures and problem-based-learning tutorials. This study reports the experience accumulated with the evaluation of lectures during the academic years 1998–1999 and 1999–2000. The ratings of the different modules were highly variable, indicating the ability of students to have a critical view on the teaching and the teachers. There was a close to very close correlation between the ratings obtained during the two study years. The high discriminative capacity of students in evaluating the teaching and the teachers was also supported by their critical point of view expressed in free comments. Our observations stress therefore the reliability of teaching evaluations systematically performed by medical students.

Jean Paul Thiery started his lifetime dedication to Science as a chemistry student in Strasbourg. He subsequently performed his graduate studies in Giorgio Bernardi's laboratory, where he was involved in projects aiming at elucidating the physico-chemical properties of DNA. He also studied the effects of restriction enzymes and their use as a tool in the elucidation of the structure of the genome.

This analysis of DNA‐ploidy heterogeneity in advanced gastric carcinomas is consistent with the hypothesis of the emergence of a single aneuploid cell clone as a crucial mechanism in the progression from early gastric carcinoma to advanced gastric cancer. The prognostic value of DNA‐ploidy in gastric cancers has been a matter of controversy. Tumour DNA‐ploidy heterogeneity, the presence within the same tumour of multiple stemlines differing in DNA content, has been described in various tumours including gastric cancers. The occurrence of such heterogeneity has been accepted as an explanation for the divergent DNA‐ploidy results in this type of tumours. A previous study of early gastric cancers suggested that in pure diploid superficial carcinomas, genetic instability might lead to a cell clone which has undergone a ploidy shift and is more aggressive. If so, this would initially result in DNA‐ploidy heterogeneity. Proliferative dominance of the aneuploid clone could eventually evolve to a homogeneous aneuploid tumour. In order to test this hypothesis, we studied DNA‐aneuploidy and DNA‐ploidy heterogeneity in advanced gastric carcinomas. We performed DNA cytophotometry on multiple samples collected from 16 advanced gastric carcinomas and found 15 DNA‐aneuploid tumours (94%) and one diploid tumour. Multiple DNA‐stemlines were found in 4 cases (26%). Analysis of proliferative activity performed on the same samples revealed higher proliferation rate in DNA‐ploidy homogeneous tumours than in aneuploid heterogeneous tumours. Heterogeneous tumours did not overexpress p53. These results confirm that DNA‐aneuploidy is frequent in advanced gastric cancer and demonstrate that a majority of these aneuploid tumours are not DNA‐ploidy heterogeneous. Furthermore, the higher proliferative activity in homogeneous‐aneuploid carcinomas and their more frequent overexpression of p53 support the hypothesis that in gastric cancer tumour progression implies the development of a dominant and more aggressive (higher proliferative activity, p53 overexpression) aneuploid cell clone.