Context-dependent interpretation of the prognostic value of BRAF and KRAS mutations in colorectal cancer.

BACKGROUND: The mutation status of the BRAF and KRAS genes has been proposed as prognostic biomarker in colorectal cancer. Of them, only the BRAF V600E mutation has been validated independently as prognostic for overall survival and survival after relapse, while the prognostic value of KRAS mutation is still unclear. We investigated the prognostic value of BRAF and KRAS mutations in various contexts defined by stratifications of the patient population. METHODS: We retrospectively analyzed a cohort of patients with stage II and III colorectal cancer from the PETACC-3 clinical trial (N = 1,423), by assessing the prognostic value of the BRAF and KRAS mutations in subpopulations defined by all possible combinations of the following clinico-pathological variables: T stage, N stage, tumor site, tumor grade and microsatellite instability status. In each such subpopulation, the prognostic value was assessed by log rank test for three endpoints: overall survival, relapse-free survival, and survival after relapse. The significance level was set to 0.01 for Bonferroni-adjusted p-values, and a second threshold for a trend towards statistical significance was set at 0.05 for unadjusted p-values. The significance of the interactions was tested by Wald test, with significance level of 0.05. RESULTS: In stage II-III colorectal cancer, BRAF mutation was confirmed a marker of poor survival only in subpopulations involving microsatellite stable and left-sided tumors, with higher effects than in the whole population. There was no evidence for prognostic value in microsatellite instable or right-sided tumor groups. We found that BRAF was also prognostic for relapse-free survival in some subpopulations. We found no evidence that KRAS mutations had prognostic value, although a trend was observed in some stratifications. We also show evidence of heterogeneity in survival of patients with BRAF V600E mutation. CONCLUSIONS: The BRAF mutation represents an additional risk factor only in some subpopulations of colorectal cancers, in others having limited prognostic value. However, in the subpopulations where it is prognostic, it represents a marker of much higher risk than previously considered. KRAS mutation status does not seem to represent a strong prognostic variable.

BORIS/CTCFL-mediated transcriptional regulation of the hTERT telomerase gene in testicular and ovarian tumor cells.

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.

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 moderate-severe (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.

PAX5 activates the transcription of the human telomerase reverse transcriptase gene in B cells.

Telomerase is an RNA-dependent DNA polymerase that synthesizes telomeric DNA. Its activity is not detectable in most somatic cells but it is reactivated during tumorigenesis. In most cancers, the combination of hTERT hypermethylation and hypomethylation of a short promoter region is permissive for low-level hTERT transcription. Activated and malignant lymphocytes express high telomerase activity, through a mechanism that seems methylation-independent. The aim of this study was to determine which mechanism is involved in the enhanced expression of hTERT in lymphoid cells. Our data confirm that in B cells, some T cell lymphomas and non-neoplastic lymph nodes, the hTERT promoter is unmethylated. Binding sites for the B cell-specific transcription factor PAX5 were identified downstream of the ATG translational start site through EMSA and ChIP experiments. ChIP assays indicated that the transcriptional activation of hTERT by PAX5 does not involve repression of CTCF binding. In a B cell lymphoma cell line, siRNA-induced knockdown of PAX5 expression repressed hTERT transcription. Moreover, ectopic expression of PAX5 in a telomerase-negative normal fibroblast cell line was found to be sufficient to activate hTERT expression. These data show that activation of hTERT in telomerase-positive B cells is due to a methylation-independent mechanism in which PAX5 plays an important role.

Unison or cacophony: postgraduate training in pathology in Europe.

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.

[Predictive biomarkers in colorectal cancer]. / Les biomarqueurs prédictifs dans le cancer colorectal.

While for many years the diagnosis and therapy of colon cancer did not change drastically, recently new drugs (irinotecan and oxaliplatin, used in adjuvant or neo-adjuvant approaches) and even more recently the introduction of therapies targeting the epidermal growth factor receptor (EGFR) through the monoclonal antibodies cetuximab and panitumumab, are revolutionizing the field. The finding that only patients with a tumor with a wild type (non mutated) KRAS gene respond to anti-EGFR therapy has also affected the way pathologists address colorectal cancer. Molecular analysis of the KRAS gene has become almost a routine in a very short period of time. Pathologists will have to be prepared for a new era: from standard morphology based diagnostic procedures to the prediction of response to therapy using molecular tools.

An agarose matrix facilitates sectioning of tissue microarray blocks.

Tissue microarray (TMA) is a powerful, high-throughput technique for in situ investigation of biomarkers in many tissue samples in a paraffin block by immunohistochemistry or fluorescence in situ hybridization (FISH), and has rapidly become the standard in marker studies. One of the difficult steps in the procedure is the sectioning of array blocks and mounting of sections using special slides and/or adhesive-coated tape, which demands specific experience and is time-consuming. We report an arraying method that allows melting of the receiving paraffin block and subsequent sectioning like any ordinary paraffin-embedded tissue block. The major difference from the standard microarray technique is the use of an agarose matrix in the recipient block. The agarose matrix allows melting of the paraffin without disturbing the array, resulting in perfect integration of the tissue cores. The agarose-paraffin TMA blocks limit tissue core loss during cutting, mounting, or immunohistochemical or FISH staining and better maintains the array.

Background staining of visualization systems in immunohistochemistry: comparison of the Avidin-Biotin Complex system and the EnVision+ system.

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.

CTCF binds the proximal exonic region of hTERT and inhibits its transcription.

The expression of the catalytic subunit (hTERT) represents the limiting factor for telomerase activity. Previously, we detected a transcriptional repressor effect of the proximal exonic region (first two exons) of the hTERT gene. To better understand the mechanism involved and to identify a potential repressor, we further characterized this region. The addition of the hTERT proximal exonic region downstream of the hTERT minimal promoter strongly reduced promoter transcriptional activity in all cells tested (tumor, normal and immortalized). This exonic region also significantly inhibited the transcriptional activity of the CMV and CDKN2A promoters, regardless of the cell type. Therefore, the repressor effect of hTERT exonic region is neither cell nor promoter-dependent. However, the distance between the promoter and the exonic region can modulate this repressor effect, suggesting that nucleosome positioning plays a role in transcriptional repression. We showed by electrophoretic mobility shift assay that CCCTC-binding factor (CTCF) binds to the proximal exonic region of hTERT. Chromatin immunoprecipitaion assays confirmed the binding of CTCF to this region. CTCF is bound to hTERT in cells in which hTERT is not expressed, but not in telomerase-positive ones. Moreover, the transcriptional downregulation of CTCF by RNA interference derepressed hTERT gene expression in normal telomerase-negative cells. Our results suggest that CTCF participates in key cellular mechanisms underlying immortality by regulating hTERT gene expression.

BRAF V600E Mutant Colorectal Cancer Subtypes Based on Gene Expression.

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.

A Four-Gene Promoter Methylation Marker Panel Consisting of GREM1, NEURL, LAD1, and NEFH Predicts Survival of Clear Cell Renal Cell Cancer Patients.

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.

Immunohistochemical expression of endothelial markers CD31, CD34, von Willebrand factor, and Fli-1 in normal human tissues.

Few systematic studies have been published comparing the expression and distribution of endothelial cell (EC) markers in different vascular beds in normal human tissues. We investigated by immunohistochemistry the expression of CD31, CD34, von Willebrand factor (vWF), and Fli-1 in EC of the major organs and large vessels. Tissue samples obtained from autopsies and biopsy specimens were routinely processed and stained immunohistochemically for CD31, CD34, and vWF. Biopsy material was also stained immunohistochemically for Fli-1, D2-40, and Lyve-1. The expression pattern of the markers was heterogeneous in some of the organs studied. In the kidney, fenestrated endothelium of the glomeruli strongly expressed CD31 and CD34 but was only focally positive or completely negative for vWF. Alveolar wall capillaries of the lung strongly stained for CD31 and CD34 but were usually negative for vWF. The staining intensity for vWF increased gradually with the vessel caliber in the lung. Sinusoids of the spleen and liver were diffusely positive for CD31. They were negative for CD34 in the spleen and only expressed CD34 in the periportal area in the liver. Fli-1 was expressed in all types of EC but also in lymphocytes. D2-40 stained lymphatic endothelium only. Lyve-1 immunostaining was too variable to be applied to routinely processed tissues. The expression of EC markers CD31, CD34, and vWF in the vascular tree is heterogeneous with a specific pattern for individual vessel types and different anatomic compartments of the same organ. D2-40 labels lymphatic EC only.

Monoallelic methylation of the APC promoter is altered in normal gastric mucosa associated with neoplastic lesions.

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.

Communication skills in diagnostic pathology.

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.

[How important is the clinical context for a histopathological diagnosis?]. / Diagnostic anatomopathologique: l'importance du contexte clinique.

A histopathological examination consists of two distinct phases: observation and interpretation. As a rule, macro- and microscopical examination give strong diagnostic indicators and the diagnosis is made instantaneously: the two phases are strongly interwoven. Also, the clinical context often provides essential clues as to the final diagnosis. A request for a pathology examination must hence come with clinical informations and a specific question. Yet, the pathologist has to keep in mind that the interpretation of what he sees might follow too enthusiastically the proposed clinical diagnosis. It is hence preferable for the pathologist to take note of the clinical context only after the microscopic examination has been accomplished.

Predictive factors in locally advanced rectal cancer treated with preoperative hyperfractionated and accelerated radiotherapy.

This study examines the prognostic significance of pathologic factors in patients with primary locally advanced rectal cancer treated prospectively with preoperative radiotherapy. From 1992 to 1998, 104 patients with rectal cancer of grades T3 or T4 and any N underwent preoperative radiotherapy followed by surgical resection. Survival curves were estimated according to the Kaplan-Meier method. Correlation of outcome with clinicopathologic variables (pathologic tumor and lymph node staging, histology, radial resection margin [RRM], clearance, vessel involvement, and tumor regression grade [TRG], quantitated in 5 grades) was evaluated using the Cox proportional hazards model. None of the patients achieved a histologically confirmed complete pathologic response, but 79% of the patients showed partial tumor regression (TRG2-4) and 21% did not show any tumor regression (TRG5). Among the tumors, 22% were of a mucinous type. The RRM was free of tumor in 76% of the surgical specimens. The median clearance was 2 mm. Vascular invasion was present in 37 cases (36%). In the univariate analysis, lymph node metastases, absence of tumor regression, positive RRM, and vascular invasion were correlated with adverse overall survival and disease-free survival; absence of tumor regression, positive RRM, and clearance <2 mm were correlated with local recurrences; and advanced pT stage was correlated only with disease-free survival. However, in the multivariate analysis, only lymph node metastases and RRM were independent prognostic factors for overall survival and disease-free survival, and clearance <2 mm was an independent prognostic factor for local control. Pathologic parameters remain strong determinants of local recurrence and survival in locally advanced rectal cancer, treated preoperatively with hyperfractionated and accelerated radiotherapy. We show that patients with advanced pT, positive lymph nodes, vascular invasion, positive RRM, clearance <2 mm, or absence of tumor regression are known to have poor clinical outcome.

Beta-catenin expression and its association with prognostic factors in adenocarcinoma developed in Barrett esophagus.

The majority of the adenocarcinomas arising in Barrett esophagus manifest clinically at an advanced stage and have a poor prognosis. As a result of this poor prognosis, much attention has been directed toward the exploration of markers for neoplastic progression in Barrett esophagus. The objective of the present study was to determine the expression of beta-catenin by immunohistochemical analysis in 70 adenocarcinomas developed in Barrett esophagus and to examine its relationship to various prognostic factors currently in use. Abnormal beta-catenin expression, consisting of the loss of membranous staining and the appearance of the nuclear staining, was found in 43 cases (61%). Of patients with the 43 tumors showing abnormal beta-catenin expression, 25 (58%) survived more than 1 year. In contrast, only 7 (26%) of 27 patients with tumors showing normal beta-catenin expression survived longer than 1 year. Most of the superficial (Tis-T1) tumors (83% [10/12]) exhibited abnormal beta-catenin expression compared with only 53% (31/58) in the T2-T3 group. These results suggest a possible correlation among beta-catenin expression, tumor stage, and length of survival as prognostic factors in patients with adenocarcinoma in Barrett esophagus.

Application of molecular genetics to the diagnosis of lymphoid-rich effusions: study of 95 cases with concomitant immunophenotyping.

The cytological differentiation between reactive lymphocytosis and malignant lymphoma in serous effusions is often difficult. The present study was designed to evaluate the potential contribution of molecular genetic clonality analysis to a solution to this problem. We examined the cytological specimens of 95 consecutive patients collected during a 4-yr period, including 74 pleural, 20 peritoneal, and one pericardial fluids. Cytological diagnosis in the 95 lymphocyte-rich effusions was positive for lymphoma in 20 cases, suspicious for lymphoma in 26 cases, and negative in 49 cases. The analysis by ICC was not carried out, inconclusive, or noninterpretable in 25 cases. In five cases molecular genetic analysis was hampered by technical problems. By immunocytochemistry, eight additional cases of lymphoma were detected and lineage classification was achieved in 15 of the 20 cytologically positive effusions. PCR and Southern blot analysis were used to assess B- and T-cell clonality. Monoclonality was found in 40 (42%) of the 95 effusions analyzed. One-third of the effusions with a monoclonal B-cell gene rearrangement were detected by Southern blot analysis but not by the PCR performed in parallel. The results of molecular genetic analysis were corroborated by histological findings and/or clinical evolution in 15 cases. Our results indicate that molecular genetic analysis is a useful tool in the analysis of lymphocyte-rich serous effusions.