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Background:Tumour aggressiveness might be related to the degree of main cancer hallmark acquirement of tumour cells, reflected by expression levels of specific biomarkers. We investigated the expression of Aldh1, Survivin, and EpCAM, together reflecting main cancer hallmarks, in relation to clinical outcome of colorectal cancer (CRC) patients.Methods:Immunohistochemistry was performed using a tumour tissue microarray of TNM (Tumour, Node, Metastasis)-stage I–IV CRC tissues. Single-marker expression or their combination was assessed for associations with the clinical outcome of CRC patients (N=309).Results:Increased expression of Aldh1 or Survivin, or decreased expression of EpCAM was each associated with poor clinical outcome, and was therefore identified as clinically unfavourable expression. Analyses of the combination of all three markers showed worse clinical outcome, specifically in colon cancer patients, with an increasing number of markers showing unfavourable expression. Hazard ratios ranged up to 8.3 for overall survival (P<0.001), 36.6 for disease-specific survival (P<0.001), and 27.1 for distant recurrence-free survival (P<0.001).Conclusions:Our data identified combined expression levels of Aldh1, Survivin, and EpCAM as strong independent prognostic factors, with high hazard ratios, for survival and tumour recurrence in colon cancer patients, and therefore reflect tumour aggressiveness.

Numerous changes in epigenetic mechanisms have been described in various types of tumors. In search for new biomarkers, we investigated the expression of Polycomb-group (PcG) proteins EZH2, BMI1 and SUZ12 and associated histone modification H3K27me3 in colorectal cancer. Nuclear expression of PcG proteins and histone modification H3K27me3 were immunohistochemically (IHC) stained on a tissue microarray (TMA), including 247 tumor tissues and 47 normal tissues, and scored using the semi-automated Ariol system. Tumor tissues showed higher expression of EZH2 (p = 0.05) and H3K27me3 (p<0.001) as compared to their normal counterparts. Combined marker trend analyses indicated that an increase in the number of markers showing high expression was associated with better prognosis. High expression of all four markers in the combined marker analyses was correlated with the best patient survival and the longest recurrence-free survival, with overall survival (p = 0.01, HR 0.42(0.21–0.84)), disease-free survival (p = 0.007, HR 0.23(0.08–0.67) and local recurrence-free survival (p = 0.02, HR 0.30(0.11–0.84)). In conclusion, we found that expression of PcG proteins and H3K27me3 showed prognostic value in our study cohort. Better stratification of patients was obtained by combining the expression data of the investigated biomarkers as compared to the individual markers, underlining the importance of investigating multiple markers simultaneously.

Research towards biomarkers that predict patient outcome in colorectal cancer (CRC) is rapidly expanding. However, none of these biomarkers have been recommended by the American Association of Clinical Oncology or the European Group on Tumor Markers. Current staging criteria result in substantial under-and over-treatment of CRC patients. Evasion of apoptosis, a characteristic feature of tumorigenesis, is known to correlate with patient outcome. We reviewed the literature on immunohistochemistry-based studies between 1998 and 2011 describing biomarkers in this pathway in CRC and identified 26 markers. Most frequently described were p53, Bcl-2, survivin, and the Fas and TRAILR1 receptors and their ligands. None of the studies reviewed provided sufficient support for implementing a single marker into current clinical practice. This is likely due to the complex biology of this pathway. We suggest focusing on the combination of key markers within the apoptosis pathway that together represent an ‘apoptotic tumor profile’, which better reflects the status of this pathway in a tumor.

Background: Post-translational modification of histone tails by methylation plays an important role in tumorigenesis. In this study, we investigated the nuclear expression of H3K4me3, H3K9me3 and H4K20me3 in early-stage colon cancer in relation to clinical outcome. Methods: Tumor tissue cores of 254 TNM stage I-III colorectal cancer patients were immunohistochemically stained for H3K4me3, H3K9me3 and H4K20me3 and scored using the semi-automated Ariol system. Cox proportional hazard trend analyses were performed to assess the prognostic value of the combined markers with respect to patient survival and tumor recurrence.

BackgroundBreast cancer is a heterogeneous disease with a highly variable clinical outcome in which both genetic and epigenetic changes have critical roles. We investigated tumor expression levels of histone-modifying enzymes LSD1, HDAC2 and SIRT1 in relation with patient survival and tumor relapse in a retrospective cohort of 460 breast cancer patients. Additionally, we correlated expression levels with tumor differentiation and tumor cell proliferation.MethodsImmunohistochemical staining for LSD1, HDAC2 and SIRT1 was performed on tissue microarrays of tumor and corresponding normal formalin-fixed paraffin-embedded tissues from breast cancer patients. Median nuclear expression levels in tumor tissues were used to divide the patients into low and high expression categories. In combined expression analyses, patients were divided into four subgroups: 1, all enzymes below-median; 2, one enzyme above-median; 3, two enzymes above-median; 4, all three enzymes above-median. The Cox proportional hazard model was used for univariate and multivariate survival analyses. The Pearson Chi-square method was used to assess correlation of combined expression levels with tumor cell proliferation and tumor differentiation.ResultsExpression of LSD1 and SIRT1, but not of HDAC2, was significantly increased in tumor tissues compared to their normal counterparts (both p < 0.001). Multivariate survival analyses identified SIRT1 as independent prognostic factor for relapse-free survival (RFS) with a hazard ratio (HR) of 1.34 (95% CI = 1.04-1.74, p = 0.02). For overall survival (OS), no significant differences were found when the individual enzymes were analyzed. Analyses of combined expression levels of the three histone-modifying enzymes correlated with OS (p = 0.03) and RFS (p = 0.006) with a HR of respectively 1.49 (95% CI = 1.07-2.08) and 1.68 (95% CI = 1.16-2.44) in multivariate analyses and were also related to tumor differentiation (p < 0.001) and tumor cell proliferation (p = 0.002).ConclusionsWhen the combined expression levels were analyzed, high expression of LSD1, HDAC2 and SIRT1 showed shorter patient survival time and shorter time to tumor relapse and correlated with poor tumor differentiation and a high level of tumor cell proliferation. Expression of these histone-modifying enzymes might therefore be involved in breast cancer pathogenesis.

Background:We evaluated the clinical prognostic value of methylation of two non-coding repeat sequences, long interspersed element 1 (LINE-1) and Alu, in rectal tumour tissues. In addition to DNA methylation, expression of histone modifications H3K27me3 and H3K9Ac was studied in this patient cohort.Methods:LINE-1 and Alu methylation were assessed in DNA extracted from formalin-fixed paraffin-embedded tissues. A pilot (30 tumour and 25 normal tissues) and validation study (189 tumour and 53 normal tissues) were performed. Histone modifications H3K27me3 and H3K9Ac were immunohistochemically stained on tissue microarrays of the study cohort.Results:In early-stage rectal cancer (stage I-II), hypomethylation of LINE-1 was an independent clinical prognostic factor, showing shorter patient survival (P=0.014; HR: 4.6) and a higher chance of tumour recurrence (P=0.001; HR: 9.6). Alu methylation did not show any significant correlation with clinical parameters, suggesting an active role of LINE-1 in tumour development. Expression of H3K27me3 (silencing gene expression) and H3K9Ac (activating gene expression) in relation to methylation status of LINE-1 and Alu supported this specific role of LINE-1 methylation.Conclusion:The epigenetic status of LINE-1, but not of Alu, is prognostic in rectal cancer, indicating an active role for LINE-1 in determining clinical outcome.

Previously, we have shown that CCR5 transcription is regulated by CREB-1. However, the ubiquitous pattern of CREB-1 expression suggests the involvement of an additional level of transcriptional control in the cell type-specific expression of CCR5. In this study we show that epigenetic changes (i.e. DNA methylation and histone modifications) within the context of the CCR5 P1 promoter region correlate with transcript levels of CCR5 in healthy and in malignant CD4+ T lymphocytes as well as in CD14+ monocytes. In normal naïve T cells and CD14+ monocytes the CCR5 P1 promoter resembles a bivalent chromatin state, with both repressive and permissive histone methylation and acetylation marks. The CCR5 expressing CD14+ monocytes however show much higher levels of acetylated histone H3 (AcH3) compared to the non–CCR5-expressing naïve T cells. Combined with a highly methylated promoter in CD14+ monocytes, this indicates a dominant role for AcH3 in CCR5 transcription. We also show that pharmacological interference in the epigenetic repressive mechanisms that account for the lack of CCR5 transcription in T leukemic cell lines results in an increase in CREB-1 association with CCR5 P1 chromatin. Furthermore RNA polymerase II was also recruited into CCR5 P1 chromatin resulting in CCR5 re-expression. Together, these data indicate that epigenetic modifications of DNA, and of histones, contribute to the control of CCR5 transcription in immune effector cells.