ORIGINAL ARTICLE Age-dependent clinical prognostic value of histone modifications in colorectal cancer
J. GOOSSENS-BEUMER1, ANNE BENARD1, ANNEKE Q. VAN HOESEL, INES € ELIANE C. M. ZEESTRATEN, HEIN PUTTER, STEFAN BOHRINGER, GERRIT-JAN LIEFERS, HANS MORREAU, CORNELIS J. H. VAN DE VELDE, and PETER J. K. KUPPEN LEIDEN, THE NETHERLANDS

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. (Translational Research 2014;-:1–11) Abbreviations: CRC ¼ colorectal cancer; DRFS ¼ distant recurrence–free survival; DSS ¼ disease-specific survival; HR ¼ hazard ratio; H3K9Ac ¼ acetylation of histone 3 lysine 9; H3K27me3 ¼ trimethylation of histone 3 lysine 27; LRFS ¼ locoregional recurrence–free survival; OS ¼ overall survival

1 In
es J. Goossens-Beumer and Anne Benard contributed equally to this work.

From the Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands; Department of Medical Statistics and Bioinformatics, Leiden University Medical Center, Leiden, The Netherlands; Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

Reprint requests: Peter J.K. Kuppen, Department of Surgery, K6-R, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands; e-mail: [email protected]. 1931-5244/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2014.11.001

Submitted for publication July 3, 2014; revision submitted October 22, 2014; accepted for publication November 4, 2014.

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AT A GLANCE COMMENTARY Goossens-Beumer IJ, et al. Background

Expression patterns of epigenetic regulators, including histone modifications, are altered during aging and epigenetic mechanisms are involved in cancer development. We hypothesized that expression of histone modifications H3K9Ac and H3K27me3 might show an age-dependent prognostic value in colorectal cancer. Translational significance

We demonstrated that the prognostic value of the studied histone modification reverses with advancing age, with high nuclear expression associated with good clinical outcome in young adults and, in contrast, with worse clinical outcome in elderly patients. This new insight supports the hypothesis that colorectal cancer biology is different in young vs elderly patients.

INTRODUCTION

Aging, a prime risk factor for many human diseases including cancer, is accompanied by changes in transcriptional profiles and increased genomic instability. A complex and delicate interplay between genetic and epigenetic mechanisms regulates cellular function. Deregulation of these mechanisms can lead to pathologic disease, including cancer, whereby genetic aberrations influence epigenetic regulation and vice versa.1-3 Epigenetic mechanisms are tissue specific and celltype specific and are dynamic processes that function to fulfill the gene and mRNA expression requirements of a cell at specific time points. Epigenetic patterns are altered during aging,1 a phenomenon referred to as epigenetic drift, of which a gradual decrease in global DNA demethylation with advancing age is the best described example.4,5 Age-related DNA demethylation is also associated with changes in histone modification patterns.6 For example, global expressions of histone modifications, acetylation of histone 3 lysine 9(H3K9Ac)7 and trimethylation of histone 3 lysine 27 (H3K27me3),8 have been shown to decrease with age in normal cells. Histone modifications regulate chromatin structure, and thereby modulate transcription, replication, and DNA repair activities.9,10 At the level of gene expression in normal cells, H3K9Ac and H3K27me3 were, respectively, positively and negatively associated with gene expression.11,12 Histone

modification H3K9Ac was associated with, but not exclusively found on, the more actively transcribed genes.13 In contrast, H3K27me3 was especially associated with subtelomeric regions and to a lesser extent with silent genes.13 However, H3K9Ac was also found on subtelomeric regions.13 Gene associated agerelated increase in DNA methylation at CpG islands was associated with a bivalent promoter status containing both repressive H3K27me3 and activating modification like H3K9Ac.14 Regulation of both H3K9Ac15,16 and H3K27me317 was described to be critical for normal aging. In cancer cells, an accelerated genome-wide increase in CpG-island methylation and genome-wide decrease in nonisland CpG methylation with advancing patient age have been frequently reported.5,18,19 Seventy percent to 90% of the CpG sites with reported ageassociated expression in normal tissue show significantly increased methylation in several cancer types.6 Therefore, it was suggested in the literature that an accelerated and uncontrolled aging process could lead to tumor formation.20 Studies showing that early inhibition of DNA methylation can prevent tumorigenesis in mice endorse this hypothesis.21 In this study, we hypothesized that there might be a prognostic relation between the age of patients with cancer at surgery and the pattern of epigenetic deregulation in their primary tumors. Using immunohistochemistry, we studied nuclear expression of activating histone modification H3K9Ac and silencing histone modification H3K27me3, which were described to exhibit strong age-related expression in normal cells and their regulation to be critical for normal aging. We assessed the association of expression of these histone modifications in tumor cells of patients with TNM stage I–III colorectal cancer (CRC) with clinical outcome in relation to age at surgery. MATERIALS AND METHODS Study cohort. The study population consisted of 254 patients with CRC with TNM stage I–III primary tumors and has been described previously.22 Information of covariate data included age at surgery, gender, TNM stage, tumor location, tumor diameter, microsatellite stability status, history of cancer, adjuvant treatment, tumor recurrence, and the occurrence of a new primary tumor in the follow-up period. The follow-up period was right censored in October 2011 or ended earlier because of death or loss to follow-up. Research was performed according to the principles of the Declaration of Helsinki. Patient record information was anonymized and deidentified before analysis according to the National Ethical Guidelines (‘‘Code

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for Proper Secondary Use of Human Tissue,’’ Dutch Federation of Medical Scientific Societies) and approved by the Medical Ethical Committee of the Leiden University Medical Center. This study was performed according to the REMARK guidelines (NCI-EORTC).23 Assessment of marker expression. Tissue microarrays were constructed previously.24 Tissue microarray sections were immunohistochemically stained with primary antibodies against H3K9Ac (ab61231; Abcam, Cambridge, UK) and H3K27me3 (ab6002; Abcam) using a previously described standard protocol.25 Nuclear expression, defined as the percentage of tumor cells showing positive nuclear staining, was determined for H3K9Ac and H3K27me3 using the semiautomated Ariol system (Leica Microsystems, Wetzlar, Germany) according to the manufacturer’s recommendations and as described previously.22 The mean percentage of positive cells of the 3 cores per patient was used for survival analysis. Linear regression analysis including an age-interaction term was used to determine statistical significance of the relation between the expression of histone modifications, stratified by survival event status, and age at surgery. Patients younger than 65 years (range from 30) at surgery are referred to as ‘‘young adults,’’ and patients of 65 years and older (range up to 91) are referred to as ‘‘elderly.’’ Survival analyses. Data were analyzed using the statistical package SPSS 20.0 for Windows (SPSS Inc, Chicago, Illinois). Analyses were performed for a single-marker expression as continuous variables and for the combination of the 2 markers as categorized variables in a trend survival analyses. For combination of H3K9Ac and H3K27me3 expressions, patients were dichotomized per histone modification. On the basis of the skewed distributions of percentages of positive cells, median expression was used as cutoff values for dichotomization for each of the histone modifications separately. On the basis of the results of the individual marker analyses, patients were subsequently grouped as follows: below-median expression of both markers (group 1), above-median expression of 1 of the 2 markers (group 2), and abovemedian expression of both markers (group 3). The relationship between single-marker expression and established clinicopathologic factors was investigated using an independent t test for dichotomized factors, an ANOVA for categorical variables, or a linear regression for continuous variables. The relationship between combined-marker expression and established clinicopathologic factors was investigated using the Pearson c2 test. The Cox proportional hazard model was used to analyze the association between the marker expression

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and patient survival, including an age-interaction term. Overall survival (OS) was defined as the time from surgery until death by any cause. Diseasespecific survival (DSS) was defined as the time from surgery until death by CRC. Locoregional recurrence– free survival (LRRFS) was defined as the time from surgery until the diagnosis of a locoregional recurrence or death by cancer. Distant recurrence–free survival (DRFS) was defined as the time from surgery until the diagnosis of a distant recurrence or death by cancer. Cumulative incidence curves were calculated for DSS, accounting for death because of other causes.26 All important nonsubjective covariates for CRC, as described in the section on the study cohort, were included in multivariate analyses irrespective of the statistical significance. Both the occurrence of a new primary tumor in the follow-up and adjuvant treatment in the follow-up were entered as time-dependent covariates. Differences in clinical outcome between patient groups are presented as hazard ratios (HRs). All statistical tests were 2-tailed and P values ,0.05 were considered statistically significant.

RESULTS Age-related expression of histone markers by survival event stratification. Nuclear expression, defined as the

number of positively stained tumor cells, of histone modifications H3K9Ac and H3K27me3 was assessed using the semiautomated Ariol system. Expression of H3K9Ac and H3K27me3 was not correlated to any of the standard clinicopathologic parameters (Supplementary File 1), including age at surgery. This indicates that, in contrast to normal tissues as reported in the literature, in tumor cells no direct relation between age and nuclear expression of the studied histone modifications was observed. However, agerelated expression patterns were observed in tumor cells when stratifying for survival event status. H3K9Ac and H3K27me3 showed comparable agerelated nuclear expression patterns when stratified for survival event status (Fig 1, A). Decrease in the expression with advancing age was observed in the patient group that were alive at the end of the followup (no-event group) and, in contrast, increase in the expression with advancing age was observed in patients presenting with a recurrence or death in follow-up (event group). The statistical significance of these opposing expression patterns was confirmed using the linear regression analysis (Supplementary File S2). The expression difference between the noevent group and the event group was calculated by subtracting the mean expression of the patients in the event group from the mean expression of patients in

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Fig 1. Age-related differences in the expression of histone modifications related to death or tumor recurrence. (A) Expression of H3K9Ac and H3K27me3 in tumor cells are shown per age group (x-axis) for DSS (H3K9Ac) or DRFS (H3K27me3). Patients are stratified according to their survival event status (no event in green vs event in red), with ‘‘alive vs death by CRC’’ for DSS and ‘‘alive vs distant recurrence or death by cancer’’ for DRFS. Mean expression per stratified age group is indicated by blue horizontal lines. Trend-lines showing increase or decrease in nuclear expression with advancing age are included (colored arrows). The y-axis represents the percentage of cells with positively stained nuclei per histone modification. Tables below (A) give the number of patients (N) in the no-event (green) and the event (red) group per age category. (B) The difference in median nuclear expression of H3K9Ac or H3K27me3 between tumors of patients without a survival-related event (no-event group) vs patients with a survival-related event (event group) is calculated by the formula ‘‘mean expression of the no-event group 2 mean expression of the event group’’ (y-axis) and is depicted as solid dots per age category (x-axis). A decreasing trend line (blue) implicates that this difference decreases with age, indicating an agerelated tumor expression in the individual survival event status groups. CRC, colorectal cancer; DR, distant recurrence; DRFS; distant recurrence–free survival; DSS, disease-specific survival; N, number of patients; H3K9Ac, acetylation of histone 3 lysine 9; H3K27me3, trimethylation of histone 3 lysine 27. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.

the no-event group for different age-categories (Fig 1, B). The difference in the mean expression decreased expectedly with advancing age and crossed the y 5 0 line at an age between 60 and 70 years indicating a reversal at approximately that age. Thus, both H3K9Ac and H3K27me3 expressions in tumor cells showed age-related expression patterns that were

opposite between patients who were alive after the follow-up period and patients presenting with a recurrence or death by cancer. Age-dependent prognostic value of individual marker expression. The prognostic value of nuclear expression

of H3K9Ac or H3K27me3 was assessed in patients with CRC using the Cox Proportional hazard model

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with and without inclusion of an age-interaction term (age 3 expression). A significant age-interaction term indicates that the relation between the level of nuclear expression and age at surgery is different between the no-event and the event group. Univariate and multivariate survival analyses of the individual markers without an age-interaction term showed no significant prognostic value. HRs for OS were 0.93 (0.78–1.12; P 5 0.45) for H3K9Ac and 0.97 (0.803– 1.18; P 5 .79) for H3K27me3. As shown previously, nuclear expression of H3K9Ac and H3K27me3 showed age-related expression when stratifying for survival-related event status. We therefore assessed whether this relation translated into an age-dependent prognostic value in patients with CRC using an age-interaction term. Univariate and multivariate survival analyses, with individual histone modifications as continuous variables, indicated significant age-dependent prognostic values for clinical outcome in CRC (Table I). Univariate and multivariate survival analyses of H3K9Ac expression, including an interaction between the expression and age at surgery, showed significant correlations for DSS, LRRFS, and DRFS. For H3K27me3 expression, significant univariate and multivariate correlations with age were identified for LRRFS and DRFS. As indicated by a statistically significant interaction term, association of nuclear expression with clinical outcome changes with advancing age (Fig 2, A). In young adult patients (younger than 65 years) above-median expression is associated with good clinical outcome and below-median expression is associated with worse clinical outcome. In contrast, in elderly patients (65 years or older), above-median expression is associated with worse clinical outcome and below-median expression is associated with good clinical outcome. The plotted relation between HR and age at surgery showed that HRs increased with age and crossed the HR 5 1 line between the age of 65 and 70 years (Fig 3). This indicated that association of nuclear expression with clinical outcome reverses at approximately that age. In summary, nuclear expression of H3K9Ac or H3K27me3 was identified in multivariate survival analyses as age-dependent prognostic factors for clinical outcome in patients with CRC. Impact of combined-marker expression on clinical outcome. Combination of biomarkers might increase

their clinical discriminative and prognostic value. We therefore assessed the relation of the combination of H3K9Ac and H3K27me3 expression levels with clinical outcome. Median expression levels of tumor cells positive for H3K9Ac and H3K27me3 were 93.2% and 92.9%, respectively. For combined-marker expression, patients were dichotomized per histone modification based on median expression and subsequently grouped

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into 3 groups: below-median expression of both markers (group 1), above-median expression of 1 of the 2 markers (group 2), and above-median expression of both markers (group 3). Combined-marker expression was not correlated to standard clinicopathologic parameters except for microsatellite stability status (Supplementary File 3), which showed a higher number of microsatellite stable tumors when an increasing number of histone modifications showed above-median nuclear expression. Multivariate survival trend analyses of combinedmarker expression that included an interaction of combined-marker expression with age at surgery showed a significant association for OS, DSS, LRRFS, and DRFS (Table II). This indicated combined-marker expression as an independent prognostic factor, except for age at surgery, for clinical outcome in patients with CRC. As indicated by a statistically significant interaction term, association of combined nuclear expression with clinical outcome changes with advancing age. In young adult patients, a higher number of histone modifications showing above-median expression are associated with good clinical outcome. In contrast, in elderly patients, a lower number of histone modifications showing above-median expression are associated with good clinical outcome (Fig 2, B). For combined-marker expression, the relation between HR and age at surgery was plotted for OS, DSS, LRRFS, and DRFS (Fig 3). HRs for combined-marker expression increased with age and crossed the HR 5 1 line between the age of 65–70 years, indicating that association of combined expression of histone modifications with clinical outcome reverses at approximately that age. The linear relation of the HR as a function of combined-marker expression and age at surgery is generally steeper (higher HRs) in combined-marker expression compared with the individual histone modifications, indicating that their combination results in better stratification and prognostication. In summary, combined-marker expression of H3K9Ac and H3K27me3 was identified in multivariate analyses as an age-dependent prognostic factor for clinical outcome in patients with CRC, which shows a reversal in prognostic value at the age of approximately 65 years. DISCUSSION

Results in this study showed an age-dependent prognostic value for histone modifications H3K9Ac and H3K27me3 and their combination in CRC. Other agedependent prognostic biomarkers have been identified previously with prognostic effects that either increase or decrease with age.27-32 To our best knowledge, we are the first to show age-dependent prognostication of

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Table I. Results of univariate and multivariate survival analyses of single-marker expression OS Single-marker expression

H3K9Ac expression Univariate H3K9Ac (per 10% increase) Age at surgery (per 10% increase) Interaction (H3K9Ac 3 age) Multivariate H3K9Ac (per 10% increase) Age at surgery (per 10% increase) Interaction (H3K9Ac 3 age) H3K27me3 expression Univariate H3K27me3 (per 10% increase) Age at surgery (per 10% increase) Interaction (H3K27me3 3 age) Multivariate H3K27me3 (per 10% increase) Age at surgery (per 10% increase) Interaction (H3K27me3 3 age)

DSS

LRRFS

DRFS

HR

95% CI

P

HR

95% CI

P

HR

95% CI

P

HR

95% CI

P

0.42 0.64 1.13

0.11–1.62 0.12–3.58 0.93–1.36

0.21 0.61 0.22

0.06 0.03 1.54

0.01–0.30 0.00–0.26 1.21–1.97

0.001 0.002 0.001

0.14 0.10 1.34

0.04–0.53 0.02–0.61 1.09–1.64

0.004 0.012 0.005

0.10 0.06 1.42

0.02–0.43 0.01–0.43 1.14–1.77

0.002 0.005 0.002

0.35 0.57 1.14

0.09–1.32 0.11–3.07 0.95–1.38

0.12 0.51 0.16

0.03 0.01 1.73

0.01–0.17 0.00–0.13 1.31–2.28

0.000 0.000 0.000

0.10 0.07 1.39

0.02–0.41 0.01–0.50 1.11–1.73

0.001 0.008 0.004

0.07 0.04 1.49

0.01–0.30 0.01–0.31 1.18–1.89

0.000 0.002 0.001

0.48 0.76 1.10

0.11–2.06 0.11–5.06 0.90–1.36

0.32 0.78 0.35

0.15 0.13 1.29

0.03–0.82 0.01–1.34 1.00–1.68

0.028 0.09 0.050

0.15 0.11 1.32

0.03–0.65 0.01–0.85 1.05–1.66

0.012 0.034 0.018

0.11 0.08 1.37

0.02–0.53 0.01–0.67 1.08–1.75

0.006 0.020 0.009

0.36 0.53 1.15

0.09–1.52 0.08–3.59 0.93–1.42

0.17 0.52 0.19

0.24 0.28 1.20

0.04–1.37 0.03–2.95 0.92–1.56

0.11 0.29 0.18

0.16 0.13 1.29

0.04–0.77 0.02–1.10 1.02–1.64

0.022 0.06 0.035

0.09 0.07 1.40

0.02–0.48 0.01–0.62 1.09–1.80

0.005 0.017 0.008

Abbreviations: CI, confidence interval; DRFS, distant recurrence–free survival; DSS, disease-specific survival; HR, hazard ratio; LRRFS, locoregional recurrence–free survival; OS, overall survival; H3K9Ac, acetylation of histone 3 lysine 9; H3K27me3, trimethylation of histone 3 lysine 27. Shown are the data from univariate and multivariate survival analyses of single-marker expression of histone modifications. The survival analyses included an age 3 expression interaction term (interaction). The inclusion of the age-interaction term is to model the reversal of the prognostic value of the single-marker expression with advancing age. Other covariates included in the multivariate survival analyses are listed in the Materials and methods section. HRs are given per 10% increase in nuclear expression. Significant associations are indicated in bold.

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Fig 2. Incidence curves illustrating the reversal of clinical prognostic value of histone modification expression level with advancing age. Cumulative incidence curves were plotted to illustrate the reversal of prognostic value with advancing age. The y-axis represents the cumulative incidence, accounting for death because of other causes. The x-axis represents time in years since surgery. (A) The cumulative incidence curves for H3K9Ac representing DSS were plotted for 3 different age groups to illustrate the age-dependent clinical prognostic value of single markers. Patients were dichotomized based on median nuclear expression, with below-median expression (light blue line) and above-median expression (dark blue line). (B) The cumulative incidence curves representing DSS were plotted for the combination of H3K9Ac and H3K27me3, with patients divided into 3 groups: all below-median expression in group 1 (light blue line), below-median expression of one marker in group 2 (slightly dark blue line), and all above-median expression in group 3 (dark blue line). DSS, disease-specific survival; N, number of patients; H3K9Ac, acetylation of histone 3 lysine 9; H3K27me3, trimethylation of histone 3 lysine 27. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.

an epigenetic biomarker that shows a reversal in impact on clinical outcome. This means that in elderly patients high nuclear expression of the same histone modification was associated with worse clinical outcome, whereas in young adults high nuclear expression was associated with good clinical outcome. Similar phenomena where the effect of a condition or level of gene or protein expression that changes from beneficial to harmful or vice versa over time have been described previously. For example, an age-dependent biomarker

signature of 12 serum proteins for autism spectrum disorders (ASDs) was identified by Ramsey et al.33 By comparing expression between patients with ASD and their unaffected siblings in relation to age, the expression of these proteins was found to show an opposite expression pattern with advancing age, thereby indicating an age-dependent effect on ASD. In the same way, changes in epigenetic regulation might have different effects on younger patients compared with elderly patients.

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Fig 3. Relation between HR and age at surgery for OS, DSS, LRRFS, and DRFS. Solid lines ( ), statistically significant in multivariate survival analyses; dashed lines ( ), not significant in multivariate survival analyses. Single-marker expression: the linear relation between HR and age at surgery (x-axis) for single-marker expression of H3K9Ac and H3K27me3. HRs represent the risk of event per 10% increase in nuclear expression. HRs (y-axis) ,1 indicate that below-median expression is associated with worse clinical outcome (less events), whereas HRs .1 indicate that above-median expression is associated with worse clinical outcome. Combined-marker expression: the linear relation between HR and age at surgery (x-axis) is shown for combined-marker expression. HRs represent the hazard per increase in one additional histone modification showing above-median expression. HRs (y-axis) ,1 indicate that a lower number of histone modifications showing above-median expression are associated with worse clinical outcome, whereas HRs .1 indicate that a higher number of histone modifications showing above-median expression are associated with worse clinical outcome. DRFS, distant recurrence–free survival; DSS, disease-specific survival; HR, hazard ratio; LRRFS, locoregional recurrence–free survival; OS, overall survival; H3K9Ac, acetylation of histone 3 lysine 9; H3K27me3, trimethylation of histone 3 lysine 27.

When considering their role in gene expression,11,12 activating modification H3K9Ac and silencing modification H3K27me3 would be expected to show opposite effects on clinical outcome. However, only a small portion of the genome is dedicated to proteincoding genes.13 With our focus on global levels of these histone modifications, we show that the global or genome-wide expression of H3K9Ac and H3K27me3 in cancer had similar effects on clinical outcome and

were age-dependent (see individual marker analyses). In addition to global levels, histone modifications at individual genes are being studied.34-36 Because of the described global age-related changes in DNA methylation and specific histone modifications in normal cells in the literature, we intentionally set out to study global levels of H3K9Ac and H3K27me3 in tumor cells. In normal tissue, a slight gradual decrease in global expression of both histone modifications with

Abbreviations: CI, confidence interval; DRFS, distant recurrence–free survival; DSS, disease-specific survival; HR, hazard ratio; LRRFS, locoregional recurrence–free survival; OS, overall survival. Shown are the data from univariate and multivariate survival trend analyses of combined-marker expression of histone modifications H3K9Ac and H3K27me3. The survival analyses included an age 3 expression interaction term (interaction). The inclusion of the age-interaction term is to model the reversal of the prognostic value of the combined-marker expression with advancing age. Other covariates included in the multivariate survival analyses are listed in the Materials and methods section. Patients were dichotomized per histone modification and grouped into 3 groups: below-median expression of both markers (group 1), above-median expression of 1 of the 2 markers (group 2), and above-median expression of both markers (group 3). HRs are given per increase in one additional histone modification showing above-median expression. Significant associations are indicated in bold.

0.000 0.020 0.000 0.01–0.24 0.31–0.91 1.25–2.15 0.04 0.53 1.64 0.000 0.007 0.000 0.01–0.18 0.30–0.83 1.27–2.18 0.03 0.50 1.66 0.002 0.050 0.002 0.04 0.54 1.64 0.03–0.75 0.70–1.78 1.05–1.65 0.16 1.11 1.32

0.021 0.65 0.018

0.01–0.30 0.31–1.00 1.20–2.24

0.005 0.13 0.005 0.01–0.47 0.40–1.12 1.12–1.90 0.08 0.67 1.46 0.001 0.041 0.002 0.01–0.32 0.37–0.98 1.17–1.97 0.06 0.60 1.52 0.009 0.13 0.009 0.07 0.64 1.49 0.05–1.24 0.78–2.01 0.98–1.55 0.25 1.25 1.23

0.088 0.36 0.079

0.01–0.51 0.36–1.14 1.11–2.02

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Univariate Combined marker Age at surgery (per 10% increase) Interaction (H3K27me3 3 age) Multivariate Combined marker Age at surgery (per 10% increase) Interaction (combined marker 3 age)

95% CI 95% CI HR P 95% CI Combined-marker expression

HR

95% CI

P

HR

DSS OS

Table II. Results of univariate and multivariate survival analyses of combined-marker expression

LRRFS

P

HR

DRFS

P

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advancing age is reported in the literature.7,8 This presumed normal pattern of decrease in nuclear expression of histone modifications H3K9Ac and H3K27me3 with advancing age was observed in tumors of patients who did not present with survival-related events (death or recurrent disease) in follow-up. This expression pattern might be explained by the course of telomere shortening. In normal cells, telomeres are subject to shortening with advancing age, eventually leading to senescence of cells. Telomere shortening contributes to an age-related decrease in the total number of histones available for stabilization of the human genome37 and thereby to the decrease in the number of histone modifications present within a cell with advancing age in normal cells.7,8 The pattern of increase in nuclear expression of histone modifications H3K9Ac and H3K27me3 with advancing age, which was observed in tumors of patients who were deceased or diagnosed with recurrent disease in follow-up, might be explained by the epigenetic regulation of replicative immortalization of tumor cells. Replicative immortality, one of the cancer hallmarks,38,39 is typically enabled by lengthening of telomeres and maintenance of telomere length by constitutive expression of human telomerase reverse transcriptase (hTERT), which is the catalytic subunit of telomerase. Increased acetylation of H3K9, because of the lack of histone deacetylase 2 recruitment to the hTERT proximal promoter, was found to stimulate hTERT expression and telomerase activity,15 thereby promoting tumor cell survival. The age-related decrease in H3K27me3 expression in normal cells, as reported in the literature,8 suggests loss of chromatin compaction during aging8 and thereby increase in global level of gene expression. Preserving high levels of trimethylation of H3K27 was suggested to be critical for longevity of cells.17 Increased age-related expression of H3K27me3 in patients with survival-related events could therefore be related to increased survival and longevity of tumor cells, which is in accordance with the observed increase in H3K9Ac and the expected effect on telomerase activity. We observed that tumor expression of H3K9Ac and H3K27me3 that is most divergent from the normal age-related pattern of decrease in expression results in higher HRs and worse clinical outcome. Thus, age-related increase in global expression of H3K9Ac and H3K27me3 in tumor cells seems to be associated with better tumor cell survival and longevity of tumor cells15,17 and could hence be correlated to tumor aggressiveness, which is corresponding to the identified worse clinical outcome in our study. A combination of these 2 markers, both related to cell survival and longevity, resulted in better

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prognostication for patients with CRC, indicating their synergistic effect. CONCLUSIONS

9. 10.

We identified an age-dependent prognostic effect of nuclear expression of histone modifications H3K9Ac and H3K27me3 and their combination in patients with CRC. We have demonstrated for the first time the prognostic impact of an epigenetic marker that not only changed but also reversed with advancing age. This observation supports the hypothesis that CRC biology is distinct between young adults and elderly and emphasizes the importance of focusing on age-related effects in CRC. More research is needed to elucidate specific genomic locations of the histone modification changes and mechanisms of age-dependent epigenetic effects on tumorigenesis in CRC.

11.

12.

13.

14.

15.

ACKNOWLEDGMENTS

Conflicts of Interest: All authors have read the journal’s policy on disclosure of potential conflicts of interest and have none to declare. The authors would like to thank C.M. Janssen, H. Horati, and W. de Graaf for their contributions to this study. All authors have read the journal’s authorship agreement, and the manuscript has been reviewed and approved by all authors.

16.

17. 18. 19.

20.

Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.trsl.2014.11.001.

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