Nuclear expression of histone deacetylases and their histone modifications predicts clinical outcome in colorectal cancer.

AIMS: Epigenetic changes are of crucial importance in cancer development and are potentially reversible; they are therefore targets of interest for anti-cancer therapy. The aim of this study was to investigate the clinical prognostic value of the histone deacetylases SIRT1, HDAC1 and HDAC2 and the histone modifications H4K16Ac and H3K56Ac in colorectal cancer. METHODS AND RESULTS: The epigenetic markers were immunohistochemically stained on tissue microarrays containing colorectal tumours (n = 254) and normal colorectal tissues (n = 50). Nuclear expression was assessed on the semi-automated Ariol system. Multivariate trend survival analyses of the combined markers showed better patient survival and less tumour recurrence when more markers showed high nuclear expression. For the combination of the histone deacetylases and H3K56Ac, the hazard ratio (HR) for overall survival (OS) was 0.82 [95% confidence interval (CI) 0.72-0.94; P = 0.005] and the HR for distant recurrence-free survival (DRFS) was 0.77 (95% CI 0.64-0.92; P = 0.003) per additional marker showing high expression. Similarly, for the combination of histone deactylases and H4K16Ac, HRs of 0.86 (95% CI 0.76-0.97; P = 0.01) for OS and 0.79 (95% CI 0.68-0.93; P = 0.006) for DRFS were observed per additional marker showing high expression. CONCLUSIONS: The studied epigenetic markers showed clinical prognostic value in colorectal cancer, both as individual markers and when combined into multimarker analyses. These results indicate that epigenetic mechanisms play an important role in colorectal carcinogenesis.

Chromatin status of apoptosis genes correlates with sensitivity to chemo-, immune- and radiation therapy in colorectal cancer cell lines.

The apoptosis pathway of programmed cell death is frequently deregulated in cancer. An intact apoptosis pathway is required for proper response to anti-cancer treatment. We investigated the chromatin status of key apoptosis genes in the apoptosis pathway in colorectal cancer cell lines in relation to apoptosis induced by chemo-, immune- or radiation therapy. Using chromatin immunoprecipitation (ChIP), we measured the presence of transcription-activating histone modifications H3Ac and H3K4me3 and silencing modifications H3K9me3 and H3K27me3 at the gene promoter regions of key apoptosis genes Bax, Bcl2, Caspase-9, Fas (CD95) and p53. Cell lines DLD1, SW620, Colo320, Caco2, Lovo and HT29 were treated with cisplatin, anti-Fas or radiation. The apoptotic response was measured by flow cytometry using propidium iodide and annexin V-FITC. The chromatin status of the apoptosis genes reflected the activation status of the intrinsic (Bax, Bcl2, Caspase-9 and p53) and extrinsic (Fas) pathways. An active intrinsic apoptotic pathway corresponded to sensitivity to cisplatin and radiation treatment of cell lines DLD1, SW620 and Colo320. An active Fas promoter corresponded to an active extrinsic apoptotic pathway in cell line DLD1. mRNA expression data correlated with the chromatin status of the apoptosis genes as measured by ChIP. In conclusion, the results presented in this study indicate that the balance between activating and silencing histone modifications, reflecting the chromatin status of apoptosis genes, can be used to predict the response of tumor cells to different anti-cancer therapies and could provide a novel target to sensitize tumors to obtain adequate treatment responses.

DNA methylation of apoptosis genes in rectal cancer predicts patient survival and tumor recurrence.

Deregulation of the apoptotic pathway, one of the hallmarks of tumor growth and -progression, has been shown to have prognostic value for tumor recurrence in rectal cancer. In order to develop clinically relevant biomarkers, we studied the methylation status of promoter regions of key apoptosis genes in rectal cancer patients, using methylation-sensitive restriction enzymes. DNA was extracted from fresh-frozen tumor tissues of 49 stage I-III rectal cancer patients and 10 normal rectal tissues. The results of this pilot study were validated in 88 stage III tumor tissues and 18 normal rectal tissues. We found that methylation of the intrinsic apoptotic pathway genes Apaf1, Bcl2 and p53 correlated with the apoptotic status (M30) of the tumor. Combined survival analyses of these three genes, based on the number of genes showing high methylation (all low, 1 high, 2 high or all high), showed shorter patient survival and recurrence-free periods with an increasing number of methylated markers. Multivariate analyses showed significant differences for overall survival (p = 0.01; HR = 0.28 (0.09-0.83)), cancer-specific survival (p = 0.004; HR = 0.13 (0.03-0.67)) and distant recurrence-free survival (p = 0.001; HR = 0.22(0.05-0.94)). The shortest survival was observed for patients showing low methylation of all markers, which-as was expected-correlated with high apoptosis (M30), but also with high proliferation (Ki-67). The study of epigenetic regulation of apoptosis genes provides more insight in the tumorigenic process in rectal cancer and might be helpful in further refining treatment regimens for individual patients.

Histone trimethylation at H3K4, H3K9 and H4K20 correlates with patient survival and tumor recurrence in early-stage colon cancer.

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. RESULTS: The histone methylation markers only showed prognostic value in early-stage (TNM stage I and II) colon cancer. Therefore, only this patient set (n = 121) was used for further statistical analyses. Low nuclear expression of H3K4me3, and high expression of H3K9me3 and H4K20me3 were associated with good prognosis. In combined marker analyses, the patient group showing most favorable expression (low H3K4me3, high H3K9me3 and high H4K20me3) was associated with the best prognosis. Multivariate trend analyses showed significantly increased hazard ratios (HR) for each additional marker showing unfavorable expression, as compared to the "all favorable" reference group. The HR for disease-free survival was 3.81 (1.72-8.45; p = 0.001), for locoregional recurrence-free survival 2.86 (1.59-5.13; p < 0.001) and for distant recurrence-free survival 2.94 (1.66-5.22; p < 0.001). CONCLUSIONS: Combined nuclear expression of histone modifications H3K4me3, H3K9me3 and H4K20me3 is prognostic in early-stage colon cancer. The combination of expression of the three histone modifications provides better stratification of patient groups as compared to the individual markers and provides a good risk assessment for each patient group.

High nuclear expression levels of histone-modifying enzymes LSD1, HDAC2 and SIRT1 in tumor cells correlate with decreased survival and increased relapse in breast cancer patients.

BACKGROUND: Breast 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. METHODS: Immunohistochemical 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. RESULTS: Expression 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). CONCLUSIONS: When 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.

Epigenetic control of CCR5 transcript levels in immune cells and modulation by small molecules inhibitors.

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 leukaemic 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.

Development of sporadic microsatellite instability in colorectal tumors involves hypermethylation at methylated-in-tumor loci in adenoma.

Microsatellite instability (MSI) and genomic hypermethylation of methylated-in-tumor (MINT) loci are both strong prognostic indicators in a subgroup of patients with sporadic colorectal cancer (CRC). The present study was designed to determine whether the methylation of MINT loci during the progression of adenoma to CRC is related to MSI in CRC cases. Methylation index (MI) was measured by absolute quantitative assessment of methylated alleles at seven MINT loci in primary CRC with contiguous adenomatous and normal tissues of 79 patients. Results were then validated in primary CRC tissues from an independent group of 54 patients. Increased MI of both MINT loci 1 and 31 was significantly associated with MSI in CRC and was specific for adenoma. Total MI and the number of methylated loci were threefold (P=0.02) and fivefold (P=0.004) higher, respectively, in adenomas associated with microsatellite-stable CRC versus microsatellite-unstable CRC. MINT MI was found to be correlated with mismatch repair protein expression, MSI, BRAF (V600E) mutation status, mut-L homologue 1 methylation status, and disease-specific survival in the second independent validation group of patients. MI of specific MINT loci may be prognostic indicators of colorectal adenomas that will develop into sporadic microsatellite-unstable CRCs. Increased MINT locus methylation appears to precede MSI and may have utility in defining clinical pathology in the absence of features of malignant invasive tumors.

Effect of date of drug marketing on disproportionality measures in pharmacovigilance: the example of suicide with SSRIs using data from the UK MHRA.

BACKGROUND: Warnings concerning an increased risk of suicide in patients treated with selective serotonin reuptake inhibitors (SSRIs) re-emerged in early 2003, culminating in the broadcast of a television programme in the UK. In the following months, cumulated proportional reporting ratios showed that the most recently marketed drug, escitalopram, had a much higher proportion of reports of suicide to other adverse drug reactions (ADRs) than the other drugs in the class. OBJECTIVE: To study the reporting patterns over time concerning suicide with the six SSRIs marketed in the UK as of March 2003 and their potential effect on disproportionality signal detection. METHODS: Monthly cumulated numbers of reports were obtained from the UK Medicines and Healthcare products Regulatory Agency (MHRA), from the time of the first marketing of the drugs concerned and monthly for the 2 months prior to and the 9 months following the broadcast of the television programme (broadcast date: 11 May 2003), and the monthly ratio of suicide to other reports was computed for each SSRI individually and for all SSRIs combined. RESULTS: Of the six SSRIs studied, five (citalopram, paroxetine, fluoxetine, sertraline and venlafaxine) had been marketed for several years and escitalopram for only a few months. At the end of the analysis period, 1.42% (4/281) of all ADR reports for escitalopram were of suicide versus 0.58% for the other five drugs combined (146/25 197). For all SSRIs combined, suicide represented 0.5% (123/24 315) of reports before the broadcast of the television programme, and increased to 2.3% (27/1163) following the programme. For escitalopram, suicide represented 1.1% (1/89) of all ADR reports before the television programme and 1.6% (3/192) afterwards. For the five other drugs combined, suicide represented 0.5% (122/24 226) of ADR reports before the television programme and 2.5% (24/971) afterwards (varying from 1.4% to 4.7% for the various drugs). The post-programme events represented 68% of all reports and 75% of suicides for escitalopram, whereas for older drugs they represented 3.6% of reports and 13% of suicides. CONCLUSION: For older drugs, the events reported during the high-reporting post-television programme period were diluted by years of low reporting. For escitalopram, although the television programme had little absolute impact on the number of reports, because the drug had been on the market for such a short period of time, a large relative effect was observed. Differential effects related to time on market on cumulated reporting of adverse drug reactions should be taken into account when analysing spontaneous reporting databases with automated signal generation methods after an alert has changed the spontaneous reporting patterns. Proper use of measures of disproportionality requires thorough knowledge of potential biases and careful analysis of reporting patterns. We found no obvious differences between SSRIs once these were taken into account.

Age-dependent clinical prognostic value of histone modifications in colorectal cancer.

Aging is one of the prime risk factors for the development of cancer. Expression patterns of epigenetic regulators, including histone modification levels, are altered during aging of normal cells, a phenomenon referred to as epigenetic drift. Furthermore, it is known that epigenetic mechanisms are involved in the development of cancer. We hypothesized that expression of histone modifications, acetylation of histone 3 lysine 9 (H3K9Ac) and trimethylation of histone 3 lysine 27 (H3K27me3), with reported normal age-related expression patterns might show an age-dependent prognostic value in colorectal cancer (CRC). To quantify expression, we performed immunohistochemical staining of these histone modifications on a tissue microarray containing colorectal tissues of the 254 patients with TNM stage I-III CRC. Stratification of patients according to survival status revealed age-related tumor expression patterns of both H3K9Ac and H3K27me3. Decreased expression with advancing age was observed in patients who were alive after follow-up (no-event group), whereas increased expression with advancing age was observed in patients who presented with a recurrence or death in follow-up (event group). These opposite expression patterns translated into an age-dependent prognostic value in CRC for the individual histone modifications and their combination. The prognostic value reverses with advancing age, high nuclear expression associated with good clinical outcome in young adults, and, in contrast, with worse clinical outcome in elderly patients. In conclusion, for the first time, we demonstrated prognostic impact of epigenetic biomarkers that reverses with advancing age. This new insight supports the hypothesis that CRC biology is different in young vs elderly patients and emphasizes the importance of focusing on age-related effects in CRC.

Prognostic value of polycomb proteins EZH2, BMI1 and SUZ12 and histone modification H3K27me3 in colorectal cancer.

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.

The prognostic value of the apoptosis pathway in colorectal cancer: a review of the literature on biomarkers identified by immunohistochemistry.

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.

Identification of a quantitative MINT locus methylation profile predicting local regional recurrence of rectal cancer.

PURPOSE: Risk assessment for locoregional disease recurrence would be highly valuable in preoperative treatment planning for patients undergoing primary rectal tumor resection. Epigenetic aberrations such as DNA methylation have been shown to be significant prognostic biomarkers of disease outcome. In this study, we evaluated the significance of a quantitative epigenetic multimarker panel analysis of primary tumors to predict local recurrence in rectal cancer patients from a retrospective multicenter clinical trial. EXPERIMENTAL DESIGN: Primary tumors were studied from patients enrolled in the trial who underwent total mesorectal excision for rectal cancer (n=325). Methylation levels of seven methylated-in-tumor (MINT) loci were assessed by absolute quantitative assessment of methylated alleles. Unsupervised random forest clustering of quantitative MINT methylation data was used to show subclassification into groups with matching methylation profiles. RESULTS: Variable importance parameters [Gini-Index (GI)] of the clustering algorithm indicated MINT3 and MINT17 (GI, 20.2 and 20.7, respectively) to be informative for patient grouping compared with the other MINT loci (highest GI, 12.2). When using this two-biomarker panel, four different patient clusters were identified. One cluster containing 73% (184 of 251) of the patients was at significantly increased risk of local recurrence (hazard ratio, 10.23; 95% confidence interval, 1.38-75.91) in multivariate analysis, corrected for standard prognostic factors of rectal cancer. This group showed a significantly higher local recurrence probability than patients receiving preoperative radiation (P<0.0001). CONCLUSION: Quantitative epigenetic subclassification of rectal cancers has clinical utility in distinguishing tumors with increased risk for local recurrence and may help tailor treatment regimens for locoregional control.

First insights of Cr speciation in leached Portland cement using X-ray spectromicroscopy.

X-ray spectromicroscopy has been successfully applied to determine the evolution of the Cr oxidation state in Portland cement during leaching experiments. To our knowledge, this is the first study that demonstrates the possibility to study the chromium oxidoreduction phenomena in cement materials at natural Cr concentration (approximately 60 ppm) and at the micron scale. Line scans of Cr for Cr(VI) doped (2000 ppm) and undoped samples indicate that the altered layer (0-1000 microm from the surface) is characterized by a lower amount of Cr as compared to the core part, whereas an accumulation appears in the intermediate region (1000-1300 microm). This Cr-rich interface could correspond to an accumulation of ettringite (3CaO x Al2O3 x 3CaSO4 x 32H2O) as reported by previous works. This mineral exhibits the property to incorporate Cr(III) and Cr(VI) by replacement of aluminum and sulfate, respectively, in the structure. The most surprising result concerns the evolution of the Cr(VI)/Cr(tot) ratio along the line spectra, which is constant from the altered layer to the core (both for doped and undoped samples). This means thatthe same amounts of Cr(VI) and Cr(tot) are released during leaching. Even for the undoped sample, Cr(VI) was detected in the altered layer at 40 microm from the surface. This result is not in perfect agreement with literature, which usually states that Cr(VI) is mainly leached out. Although this result must be confirmed, it clearly indicates that Cr(VI) may be less mobile than predicted by models. An attempt is made to identify potential Cr(VI) fixation phases.