Copy number alterations and allelic ratio in relation to recurrence of rectal cancer

BACKGROUND: In rectal cancer, total mesorectal excision surgery combined with preoperative (chemo)radiotherapy reduces local recurrence rates but does not improve overall patient survival, a result that may be due to the harmful side effects and/or co-morbidity of preoperative treatment. New biomarkers are needed to facilitate identification of rectal cancer patients at high risk for local recurrent disease. This would allow for preoperative (chemo)radiotherapy to be restricted to high-risk patients, thereby reducing overtreatment and allowing personalized treatment protocols. We analyzed genome-wide DNA copy number (CN) and allelic alterations in 112 tumors from preoperatively untreated rectal cancer patients. Sixty-six patients with local and/or distant recurrent disease were compared to matched controls without recurrence. Results were validated in a second cohort of tumors from 95 matched rectal cancer patients. Additionally, we performed a meta-analysis that included 42 studies reporting on CN alterations in colorectal cancer and compared results to our own data. RESULTS: The genomic profiles in our study were comparable to other rectal cancer studies. Results of the meta-analysis supported the hypothesis that colon cancer and rectal cancer may be distinct disease entities. In our discovery patient study cohort, allelic retention of chromosome 7 was significantly associated with local recurrent disease. Data from the validation cohort were supportive, albeit not statistically significant, of this finding. CONCLUSIONS: We showed that retention of heterozygosity on chromosome 7 may be associated with local recurrence in rectal cancer. Further research is warranted to elucidate the mechanisms and effect of retention of chromosome 7 on the development of local recurrent disease in rectal cancer. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1550-0) contains supplementary material, which is available to authorized users

Prognostic Value of Polycomb Proteins EZH2, BMI1 and SUZ12 and Histone Modification H3K27me3 in Colorectal Cancer

<div><p>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.</p></div

Univariate and multivariate survival analyses individual markers.

<p>Shown are the results of the univariate and multivariate analyses of all individual markers, with all p-values and hazard ratios (HR) and their 95% confidence intervals (95% CI). The "low expression" group was used as the reference group. Covariates included in all multivariate analyses were age at operation, gender, TNM tumor stage, tumor location, tumor size, microsatellite stability (MSS) status, tumor in the follow up and adjuvant therapy. OS  =  overall survival, DSS  =  disease-specific survival, LRRFS  =  locoregional recurrence-free survival, DRFS  =  distant recurrence- free survival. Significant p-values are indicated in <b>bold</b>, p-values showing a trend (0.05≤0.1) in <i>Italic</i>.</p><p>Univariate and multivariate survival analyses individual markers.</p

Identification of positive and negative tumor cell nuclei by the Ariol system.

<p>The Ariol system trainer overlay shows correct identification of positive (indicated by yellow dots) and negative (blue dots) nuclei in tumor cores. TMA slides were scanned using a 20x magnification. Shown for all markers are positively stained tumor cores (<i>top row</i>) and negative tumor cores (<i>bottom row</i>). The Ariol system was trained to identify positive and negative cells for each marker individually.</p

Univariate and multivariate analyses combined markers.

<p>Results of Cox proportional hazard univariate and multivariate analyses are shown for combined markers EZH2, BMI1, SUZ12 and H3K27me3, with p-values and hazard ratios with their 95% confidence intervals. Patient groups were made based on the number of markers showing high expression: all low (group 1), one, two or three high (groups 2–4) and all high (group 5). Group 1 was used as the reference group. Covariates included in all multivariate analyses were age at operation, gender, TNM tumor stage, tumor location, tumor size, microsatellite stability (MSS) status, tumor in the follow up and adjuvant therapy. Significant values are shown in <b>bold</b>, p-values showing a trend (between 0.5 and 1.0) in <i>Italic</i>.</p><p>Univariate and multivariate analyses combined markers.</p

Differences in nuclear expression between normal and tumor tissues of individual markers.

<p>(A) Displayed are differences in nuclear expression, measured as the percentage of positively stained nuclei, between normal and tumor tissues (n = 47). Boxplots show the median and range of expression of each of the individual markers in normal (N) and tumor (T) samples. The median percentages of positive nuclei are given for each of the markers. P-values represent statistical differences between normal and tumor samples, calculated using the Wilcoxon signed rank test. (B) Histograms show the difference in expression between tumor and paired normal tissues (y-axis) for each of the individual patients (x-axis). Differences in expression were calculated as follows: expression difference  =  expression in tumor tissue – expression in normal tissue. Negative values indicate higher expression in normal tissues, positive values indicate higher expression in tumor tissues.</p

Trend analyses and hazard ratios of combined marker groups.

<p>Patient groups were made based on the number of markers showing high expression: all low (group 1), one high (group 2), two high (group 3), three high (group 4) and all high (group 5). (A) Univariate and multivariate Cox regression trend analyses were performed using the combined marker groups as continuous variables. Hazard ratios per unit increase of each of the patient groups were plotted (B) and listed (C) for both the univariate and multivariate Cox regression trend analyses using the combined marker groups as categorical variables. The numbers of patients in the individual patient groups were: group 1 (n = 28), group 2 (n = 59), group 3 (n = 55), group 4 (n = 74) and group 5 (n = 31).</p

Kaplan-Meier and cumulative incidence curves of the combined markers.

<p>Combined marker (EZH2, BMI1, SUZ12 and H3K27me3) expression groups were divided into three patient groups: group 1, groups 2–4 and group 5. The numbers of patients in the individual patient groups were: group 1 (n = 28), group 2 (n = 59), group 3 (n = 55), group 4 (n = 74) and group 5 (n = 31). Kaplan-Meier curves were made for overall survival (A) and cumulative incidence curves are shown for disease-free survival (B), locoregional recurrence-free survival (C) and distant recurrence-free survival (D). 5-year survival rates are given for each patient group. Tables below the curves indicate the numbers at risk (#) per group for the different time points.</p

Patient characteristics of the study cohort.

<p>Patient characteristics are shown for both the study cohort (n = 247) and the complete colorectal cancer series (n = 408). Patient selection was based on availability of FFPE tissues and available data for all four studied markers. The study cohort selection was representative for the entire colorectal cancer series. P-values represent the results of Student’s t-tests. For TNM stage, only tumor stage I-III of the complete patient cohort were compared to the patients in the study cohort.</p><p>Patient characteristics of the study cohort.</p