Purpose:WRN promoter CpG island hypermethylation in colorectal cancer has been reported to increase sensitivity to irinotecan-based therapies. We aimed to characterize methylation of the WRN promoter, determine the effect of WRN promoter hypermethylation upon expression, and validate a previous report that WRN promoter hypermethylation predicts improved outcomes for patients with metastatic colorectal cancer (mCRC) treated with irinotecan-based therapy.

Experimental Design:WRN methylation status was assessed using methylation-specific PCR and bisulfite sequencing assays. WRN expression was determined using qRT-PCR and Western blotting. WRN methylation status was correlated with overall survival (OS) and progression-free survival (PFS) in 183 patients with mCRC. Among these patients, 90 received capecitabine monotherapy as first-line therapy, and 93 received capecitabine plus irinotecan (CAPIRI) therapy as part of the CAIRO phase III clinical trial.

Results:WRN mRNA and WRN protein expression levels were low in colorectal cancer cell lines and in primary colorectal cancer and were largely independent of WRN methylation status. Patients with methylated WRN colorectal cancer had a shorter OS compared with patients who had unmethylated WRN colorectal cancer (HR = 1.6; 95% confidence interval [CI], 1.2–2.2; P = 0.003). Patients with unmethylated WRN showed a significantly longer PFS when treated with CAPIRI compared with capecitabine alone (HR = 0.48; 95% CI, 0.32–0.70; P = 0.0001). In contrast, patients did not benefit from adding irinotecan to capecitabine when WRN was methylated (HR = 1.1; 95% CI, 0.69–1.77; P = 0.7).

Conclusions:WRN expression is largely independent of WRN promoter hypermethylation in colorectal cancer. Moreover, we could not validate the previous finding that WRN promoter hypermethylation predicts improved clinical outcomes of mCRC treated with irinotecan-based therapy and found instead the opposite result. Clin Cancer Res; 22(18); 4612–22. ©2016 AACR.

Translational Relevance

The current care for metastatic colorectal cancer (mCRC) includes, if clinically indicated, surgical resection of the primary tumor and/or liver metastases, together with chemotherapy (5-fluorouracil [5-FU] and oxaliplatin or irinotecan) and in some patients targeted therapy (anti-EGFR antibodies or anti-VEGF therapy). The clinical response to this regimen is variable, and it is difficult to predict who will benefit from treatment. Moreover, for most therapies, we lack accurate biomarkers to identify the optimal treatment for individual patients. DNA repair proteins, such as the Werner syndrome RECQ helicase, WRN, are promising biomarkers for predicting the response to genotoxic chemotherapy. We attempted to validate previous studies that showed WRN promoter hypermethylation predicted the response to irinotecan using an independent sample set. We did not find a clear association between aberrant WRN promoter hypermethylation and reduced WRN expression. Moreover, in contrast to earlier studies, we found an inverse correlation of WRN promoter hypermethylation with survival in patients with mCRC treated with irinotecan. Our results highlight the need for further studies to identify biomarkers that can predict the response of colorectal cancer to standard-of-care chemotherapeutic agents, including irinotecan, oxaliplatin, and 5-FU.

Colorectal cancer is among the most common cancers in the world, with an incidence of over 1.2 million and with nearly 700,000 deaths per year (1). Half of patients with colorectal cancer have or will develop distant metastases by the time of diagnosis, or shortly thereafter (2). A majority of patients with metastatic disease are not candidates for curative surgical therapy and thus receive systemic palliative therapy, most often with a fluoropyrimidine together with irinotecan or oxaliplatin (3). The more recent addition of molecularly targeted drugs, such as anti-EGFR or anti-VEGF antibodies, has further improved survival (4, 5). Colorectal cancer is a heterogeneous disease at the molecular level, and recurrent genetic and epigenetic alterations may be important drivers of clinical behavior and the response to therapy (6, 7). Despite this, we lack robust tools to select the best therapy for individual patients to reliably improve treatment outcomes.

Promoter region DNA hypermethylation has been associated with loss of expression at many genetic loci (8). Simple, reliable gene-specific assays can detect DNA hypermethylation in clinical specimens and thus could be used to help guide the selection of therapy for genes whose expression level modulates the response to clinically approved drugs (9). One association of this type was reported in 2006: hypermethylation of the Werner syndrome WRN RECQ helicase gene was linked to transcriptional silencing of WRN in colorectal cancers (10), and WRN silencing was suggested to improve treated outcomes for patients with cancer receiving irinotecan therapy (10–12).

WRN is a human RECQ helicase protein that plays critical roles in DNA replication, recombination, repair, and telomere maintenance (13, 14). The heritable loss of WRN leads to Werner syndrome, a progeroid syndrome associated with genetic instability, an elevated risk of cancer, and cellular sensitivity to DNA topoisomerase I inhibitors, such as camptothecin and irinotecan and several other important classes of chemotherapeutic drugs (15). WRN was recently identified as the top-ranked gene associated with advanced clinical stage colorectal cancer by the combined analysis of copy number alterations (CNA), methylation status, and expression, that is, WRN promoter hypermethylation, CNA/loss, and decreased expression were all associated with stage III and IV colorectal cancer (16). These provocative and potentially exciting findings suggested that methylated WRN might be a predictive marker for irinotecan sensitivity in advanced stage colorectal cancer.

In this study, we determined the methylation and expression status of WRN in colorectal cancer cell lines and primary colorectal cancer tissue samples. We also examined whether methylated WRN predicted clinical outcomes for patients enrolled in the Dutch CApecitabine, IRinotecan, and Oxaliplatin (CAIRO) study (17). We developed and validated assays to determine WRN promoter methylation status, then used these assays to determine whether WRN methylation status correlated with WRN expression at the mRNA or protein levels (10, 12), and predicted survival in patients with colorectal cancer who received irinotecan therapy (10).

Experiments were conducted at the University of Washington (UWSEA; Seattle, WA) and the VU University Medical Center (VUmc; Amsterdam, the Netherlands) using cell lines and patient samples. A brief overview of materials and methods is given below, with full sample details and methods in the Supplementary Information.

Cell lines and tissue samples

Two independent collections of cultured colorectal cancer–derived cell lines were investigated. The adenoma cell line AAC1 and colorectal cancer cell lines RKO, LoVo, SW480, LS174T, AAC1/SB10, HCT116, SW48, FET, VACO400, VACO411, and VACO5 were cultured at UWSEA. The UWSEA lines were authenticated by DNA fingerprint analysis prior to use (IDEXX/Radil Bioresearch). Colorectal cancer cell lines Colo205, Colo320, HCT116, HCT15, HT29, LIM1863, LS174T, LS513, RKO, SW480, and SW1398 were cultured at VUmc and authenticated by array comparative genomic hybridization (aCGH, 244 k Agilent oligonucleotide platform) at VUmc. The patterns of chromosomal changes observed were in concordance to the previously described chromosomal changes in these cell lines (18, 19). Twenty-six fresh frozen primary colorectal cancer tissues with matched fresh frozen normal colon tissue and 21 formalin-fixed paraffin-embedded (FFPE) normal colon tissues from cancer-free patients were collected and studied following IRB-approved protocols and in accordance with the ethical regulations of the corresponding institutions (UWSEA and VUmc). The samples used at UWSEA were provided by the Cooperative Human Tissue Network. Collection, storage, and use of patient-derived tissue and data from VUmc were performed in accordance with the Code for Proper Secondary Use of Human Tissue in the Netherlands (20).

Tissue samples from the CAIRO clinical trial

In the CAIRO study, patients with colorectal cancer with metastatic disease were randomized between sequential treatment (capecitabine followed upon disease progression by irinotecan, then oxaliplatin plus capecitabine [CAPOX]) or combination therapy with irinotecan plus capecitabine (CAPIRI) followed by CAPOX (17). The primary endpoint of the study was overall survival (OS). DNA was isolated from FFPE tissue of surgically resected primary tumors from 183 patients who participated in the CAIRO study. Of these 183 patients, 93 received CAPIRI as first-line therapy, while 90 received first-line capecitabine monotherapy. From the 90 patients who received first-line capecitabine monotherapy, 52 received more than 2 cycles of second-line irinotecan. These samples were selected to match stratification factors in the original study for the subgroup of patients that underwent primary tumor resection, that is, resection status, World Health Organization (WHO) performance status, predominant localization of metastases, previous adjuvant therapy, and serum lactate dehydrogenase (LDH) levels. Samples were also selected on the basis of a high proportion of tumor cells in sections (at least 70%). A large proportion of these samples overlap with samples described in ref. 21.

WRN methylation analyses

WRN methylation status was assessed by two different methylation-specific PCR (MSP) assays together with bisulfite sequencing (see Supplementary Methods for additional detail). A WRN 5′ region from −31 bp to +128 relative to the transcription start site (TSS), hereafter referred to as region 1, was analyzed by a gel-based MSP assay. Region 2, located at −410 to −331 bp upstream of the WRN TSS was analyzed with a quantitative MSP assay. Bisulfite sequencing was performed for the region −193 bp to +157 bp that encompassed the TSS and overlapped with the locations of the WRN MSP primer pairs described in the study by Agrelo and colleagues (10) and an independent set of WRN MSP primers reported by Ogino and colleagues (22).

WRN expression analyses

RNA expression analyses were performed by qRT-PCR assays using TaqMan Gene Expression Assays from Applied Biosystems for WRN (Hs00172155_m1), β-2 micoglobulin (B2M, Hs00984230_m1), and β-glucuronidase (GusB, Hs99999908_m1). Protein expression analyses were performed by Western blotting, using mAbs for WRN (W0393, Sigma) and β-actin (13E5, Cell Signaling Technology).

TCGA data

WRN DNA methylation (Illumina Infinium HM27 bead array; HM27) and mRNA expression (Agilent microarray) data from 223 colorectal cancer tumors from The Cancer Genome Atlas (TCGA) Colorectal Cancer project (23) were obtained via cBioPortal (http://www.cbioportal.org; data downloaded on March 2, 2014; ref. 24). When data from more than one probe per gene are available from the methylation assay, cBioPortal uses methylation data from the probe with the strongest negative correlation between the methylation signal and mRNA gene expression.

Statistical analyses

Student t test was used to compare WRN expression levels in HCT116 and Colo205 before and after 5-aza-2-deoxycytidine (DAC) and/or trichostatin A (TSA) treatment. Pearson correlation analysis was used to measure the correlation between WRN methylation and mRNA expression levels.

Progression-free survival (PFS) for first-line treatment was calculated from the date of randomization to the date of first observed disease progression or death after first-line treatment. OS was measured from the date of randomization to the date of death due to cancer. Other causes of death were censored. The prognostic or predictive value of WRN methylation status was assessed by a Kaplan–Meier survival analysis and log-rank test.

A Cox proportional hazard regression model was used to estimate HRs and 95% confidence intervals (95% CI). A multivariate Cox regression model was used to assess and adjust for important prognostic variables, including age, gender, serum LDH, WHO performance status, previous adjuvant therapy, and location of metastases. Multivariate Cox regression analysis was also used to assess and adjust for possible prognostic variables microsatellite instability (MSI) status, BRAF mutational status, and mucinous differentiation, for which information was available on a subset of the samples (136/183; refs. 25, 26). Results were considered significant when P values were ≤ 0.05.

WRN methylation and expression status in colon cancer cell lines

To accurately detect and quantify WRN promoter methylation in colorectal cancer samples, we independently developed and cross-validated MSP primer sets and assays in both laboratories (UWSEA and VUmc) for two WRN regions adjacent to and overlapping the TSS at base pair position +1: region 1 (−31 bp to +128 bp) and region 2 (−410 to −331 bp; Fig. 1A).

Figure 1.

WRN promoter region methylation analysis in cell lines. A, top, WRN promoter region CpG island and primer locations genomic coordinates (GC), CpG density, and positions are shown. Each vertical bar in the bottom panel represents the presence of a CpG dinucleotide. Black horizontal bars indicate regions amplified by newly designed and validated MSP primer pairs (region 1 and region 2), the region amplified by the original primer pair described by Agrelo and colleagues (10) and by Ogino and colleagues (22), and the region targeted for bisulfite sequencing (BS). This figure was created using MethPrimer (39). B, methylation analysis of region 1 (see Fig. 1A) in the adenoma cell line AAC1 and in colon cancer cell lines (M, methylated; U, unmethylated). DNA from peripheral blood lymphocytes (PBL) was used as an unmethylated control. H2O and DNA from SssI methylase-treated DNA from the colorectal cancer cell line SW48 were used, respectively, as "no template" and "methylated template" controls. C, quantitative methylation analysis of WRN promoter region 2 in the same colon cancer cell lines as shown in Fig. 1B. D, sodium bisulfite sequencing results of WRN gene promoter on cell lines HCT116 and SW480 in the region depicted in Fig. 1A. Each row represents an individual cloned allele, and each circle indicates a CpG dinucleotide. Black circle, methylated CpG site; white circle, unmethylated CpG site; no circle, not determined.

Figure 1.

WRN promoter region methylation analysis in cell lines. A, top, WRN promoter region CpG island and primer locations genomic coordinates (GC), CpG density, and positions are shown. Each vertical bar in the bottom panel represents the presence of a CpG dinucleotide. Black horizontal bars indicate regions amplified by newly designed and validated MSP primer pairs (region 1 and region 2), the region amplified by the original primer pair described by Agrelo and colleagues (10) and by Ogino and colleagues (22), and the region targeted for bisulfite sequencing (BS). This figure was created using MethPrimer (39). B, methylation analysis of region 1 (see Fig. 1A) in the adenoma cell line AAC1 and in colon cancer cell lines (M, methylated; U, unmethylated). DNA from peripheral blood lymphocytes (PBL) was used as an unmethylated control. H2O and DNA from SssI methylase-treated DNA from the colorectal cancer cell line SW48 were used, respectively, as "no template" and "methylated template" controls. C, quantitative methylation analysis of WRN promoter region 2 in the same colon cancer cell lines as shown in Fig. 1B. D, sodium bisulfite sequencing results of WRN gene promoter on cell lines HCT116 and SW480 in the region depicted in Fig. 1A. Each row represents an individual cloned allele, and each circle indicates a CpG dinucleotide. Black circle, methylated CpG site; white circle, unmethylated CpG site; no circle, not determined.

Close modal

WRN methylation status in region 1 was assessed in 11 colon cancer cell lines (SW480, Vaco411, AAC1/SB10, Vaco400 LS174T, LoVo, HCT116, Vaco5, FET, RKO, and SW48) and 1 adenoma cell line (AAC1) from UWSEA. Seven of 11 colon cancer cell lines (64%) had region 1–methylated WRN (Fig. 1B), whereas the adenoma cell line was unmethylated. There was no association between WRN region 1 methylation and MSI and/or CpG island methylator phenotype (Supplementary Materials and Methods; Supplementary Table S1).

WRN methylation status in region 2 was successfully evaluated in 10 colon cancer cell lines (SW480, Vaco411, Vaco400, LS174T, LoVo, HCT116, Vaco5, FET, RKO, and SW48; UWSEA) and was comparable with region 1 methylation status within a cell line (Fig. 1C). Bisulfite sequencing of cells lines with methylated (HCT116) or unmethylated WRN (SW480) was performed to confirm the methylation status of both regions and validate the MSP results using an orthogonal assay (Fig. 1D). We assessed WRN region 2 methylation status in a second, overlapping series of colon cancer cell lines (Colo205, Colo320, HCT116, HCT15, HT29, LIM1863, LS174T, LS513, RKO, SW480, and SW1398, SW48 and Caco2; VUmc). These analyses revealed that 10 of 13 cell lines, or 77%, were WRN region 2 methylated (Fig. 2B).

Figure 2.

WRN expression analysis in cell lines. A,WRN mRNA (top) and protein (bottom) expression in colorectal cancer cell lines in relation to methylation status in WRN promoter region 1 (bottom). Error bars represent SDs across triplicate independent experiments, in which WRN mRNA was normalized to mRNA expression of the reference gene GUSB (top) and for protein expression β-actin (bottom). Methylation status of WRN promoter region 1 is indicated below each pair of immunoblots (M, methylated; U, unmethylated). B,WRN mRNA expression level in relation to methylation status of WRN promoter region 2. Error bars represent SDs of mean expression values of two independent experiments. Methylation status of WRN promoter region 2 is indicated below each cell line designation. C,WRN mRNA expression analysis of Colo205 (left) and HCT116 (right) with and without DAC or DAC/TSA treatment. Bars, mean in two independent experiments; error bars, SDs. Expression was quantified relative to mRNA expression levels of B2M. *, P = 0.001.

Figure 2.

WRN expression analysis in cell lines. A,WRN mRNA (top) and protein (bottom) expression in colorectal cancer cell lines in relation to methylation status in WRN promoter region 1 (bottom). Error bars represent SDs across triplicate independent experiments, in which WRN mRNA was normalized to mRNA expression of the reference gene GUSB (top) and for protein expression β-actin (bottom). Methylation status of WRN promoter region 1 is indicated below each pair of immunoblots (M, methylated; U, unmethylated). B,WRN mRNA expression level in relation to methylation status of WRN promoter region 2. Error bars represent SDs of mean expression values of two independent experiments. Methylation status of WRN promoter region 2 is indicated below each cell line designation. C,WRN mRNA expression analysis of Colo205 (left) and HCT116 (right) with and without DAC or DAC/TSA treatment. Bars, mean in two independent experiments; error bars, SDs. Expression was quantified relative to mRNA expression levels of B2M. *, P = 0.001.

Close modal

Cell lines that carried methylated WRN expressed relatively high levels of WRN as assessed by WRN mRNA qRT-PCR (Fig. 2A and B). There was either no or a slightly positive correlation between WRN region 2 methylation and expression level in two different groups of colorectal cancer cell lines: SW480, Vaco411, Vaco400 LS174T, LoVo, HCT116, Vaco5, FET, RKO, SW48 (UWSEA; Pearson correlation of 0.32; P = 0.3); and Colo205, Colo320, HCT116, HCT15, HT29, LIM1863, LS174T, LS513, RKO, SW480, and SW1398, SW48 and Caco2 (VUmc; Pearson correlation of 0.68; P = 0.04). Consistent with these results, treatment of the methylated colorectal cancer cell lines HCT116 and Colo205 with the demethylating agent DAC and/or TSA either did not change or resulted in decreased WRN mRNA expression (Fig. 2C). Western blot analysis of WRN protein expression as a function of region 1 and 2 promoter methylation in colorectal cancer cell lines in the UWSEA collection further emphasized the lack of correlation between WRN promoter hypermethylation and mRNA and protein expression (Fig. 2A and B).

WRN methylation and expression status in colorectal cancer tissues

To determine whether there was a more consistent relationship between WRN methylation status and expression in primary tumor samples, we analyzed WRN methylation status and expression in primary colorectal cancer samples and in adjacent normal colon mucosa. We detected region 1 methylation in 33% (7/21) of primary colorectal cancers, but in none of the paired normal mucosa samples tested (N = 12). Region 2 methylation was detected in 45% (9/20) of primary colorectal cancers and in 1 of 20 matched normal mucosa samples (Fig. 3A). Methylation status was largely concordant between the two regions: all samples that showed methylation in region 1 were also region 2 methylated. Only two cases showed an unmethylated region 1 and a methylated region 2. Bisulfite sequencing of a subset of these samples (8 colorectal cancers and 2 normal mucosa samples) confirmed the results of MSP assays (data not shown). A second analysis of region 2 methylation using an independent series of primary colorectal cancers (N = 183 from the CAIRO series, see next section) and normal colon mucosa samples (N = 21, VUmc) revealed WRN promoter hypermethylation in 40% (74/183) of the primary colorectal cancers and very low or absent WRN methylation level in normal colon mucosa.

Figure 3.

WRN promoter region methylation and expression analyses in colorectal cancer and matched normal colon tissues. A,WRN methylation levels in colorectal cancer tumor tissues (black bars) and matched normal colon tissues (gray bars). Bars, mean expression of duplicate measurements in one experiment (M, methylated; U, unmethylated). A sample was considered methylated when the Ct ratio exceeded the threshold of 0.03, which was set based on an analysis of normal colon samples (N = 21), which all had values below this threshold. B,WRN mRNA expression versus a GUSB control in the same colorectal cancer tumor (black bars) and matched normal colon (gray bars) samples shown in A. Bars, mean expression of triplicate measurements in one experiment.

Figure 3.

WRN promoter region methylation and expression analyses in colorectal cancer and matched normal colon tissues. A,WRN methylation levels in colorectal cancer tumor tissues (black bars) and matched normal colon tissues (gray bars). Bars, mean expression of duplicate measurements in one experiment (M, methylated; U, unmethylated). A sample was considered methylated when the Ct ratio exceeded the threshold of 0.03, which was set based on an analysis of normal colon samples (N = 21), which all had values below this threshold. B,WRN mRNA expression versus a GUSB control in the same colorectal cancer tumor (black bars) and matched normal colon (gray bars) samples shown in A. Bars, mean expression of triplicate measurements in one experiment.

Close modal

In our first series of colon tissues, WRN mRNA expression was higher in primary colorectal cancer versus matched normal mucosa samples in 10 of 20 patients (50%), lower in 6 samples (6/20 or 30%), and equivalent in the remaining 4 samples (20%; Fig. 3B). No association was observed between WRN region 1 or 2 hypermethylation and mRNA expression (region 2: Pearson correlation 0.14; P = 0.4). WRN protein expression could not be detected by Western blot analysis in 10 of 20 (50%) paired primary colorectal cancer/normal mucosa samples (data not shown). An independent assessment of WRN methylation status and mRNA expression in 223 colorectal cancers included in the TCGA Colorectal Cancer Project (see TCGA database at www.cBioportal.org; ref. 24) did not reveal a negative correlation between WRN methylation level and mRNA expression (Pearson correlation of 0.1; P = 0.03; Supplementary Fig. S1).

Relationship of WRN methylation to clinical outcome

To determine whether there is a relationship between WRN promoter hypermethylation and treatment outcomes, we assessed the correlation between WRN promoter methylation status and survival in patients who participated in the CAIRO study (17). OS did not differ between the two treatment arms in the original study population or in the subset included in this analysis. Patient characteristics, such as age, sex, performance status, predominant localization of metastases, previous adjuvant therapy, and serum LDH level, were comparable between the two treatment arms in the subset included in this analysis (Supplementary Table S2). Thus, we pooled patients from the two treatment arms to evaluate the association of WRN promoter methylation status and OS.

The cohort of 183 patients included a total of 160 death events. The group of 109 patients with unmethylated WRN had 91 death events, and the group of 74 patients with methylated WRN had 69 death events. Patients with methylated WRN colorectal cancer had shorter OS compared with patients with unmethylated WRN (median OS of 407 vs. 610 days for methylated vs. unmethylated WRN, respectively (HR = 1.6; 95% CI, 1.2–2.2; P = 0.003; Fig. 4A). This was observed for patients in the sequential treatment arm (median OS of 405 vs. 589 days; HR = 1.5; 95% CI, 1.0–2.4; P = 0.05), as well as in the combination treatment arm (median OS of 410 vs. 680 days for methylated vs. unmethylated WRN, respectively; HR = 1.7; 95% CI, 1.1–2.7; P = 0.02; compare Fig. 4B and C). However, in the sequential treatment arm, a negative effect of WRN promoter hypermethylation on outcome was observed only for patients who received irinotecan during their treatment course (n = 55; median OS of 567 vs. 646 days for methylated vs. unmethylated WRN, respectively; HR = 1.9; 95% CI, 1.1–3.5; P = 0.03; Fig. 4D). This effect was not observed in patients who did not receive irinotecan (n = 37; median OS of 320 vs. 326 days for methylated vs. unmethylated WRN, respectively; HR = 1.0; 95% CI, 0.5–2.0; P = 1.0; Fig. 4E).

Figure 4.

OS in patients with metastatic colorectal cancer with unmethylated or methylated WRN promoter regions. OS of patients with colorectal cancer with unmethylated (solid lines, U) or methylated (dashed lines, M) WRN promoter regions in response to sequential and combination treatment arms combined [A; sequential or combined capecitabine (CAP) and irinotecan (IRI), followed by CAPOX]; in the sequential treatment arm alone (B; first-line capecitabine, second-line irinotecan, third-line CAPOX); in the combination treatment arm alone (C; first-line CAPIRI, second-line CAPOX); in the subset of patients who received (D) or did not receive (E) irinotecan in the sequential treatment arm. HR (methylated WRN vs. unmethylated WRN).

Figure 4.

OS in patients with metastatic colorectal cancer with unmethylated or methylated WRN promoter regions. OS of patients with colorectal cancer with unmethylated (solid lines, U) or methylated (dashed lines, M) WRN promoter regions in response to sequential and combination treatment arms combined [A; sequential or combined capecitabine (CAP) and irinotecan (IRI), followed by CAPOX]; in the sequential treatment arm alone (B; first-line capecitabine, second-line irinotecan, third-line CAPOX); in the combination treatment arm alone (C; first-line CAPIRI, second-line CAPOX); in the subset of patients who received (D) or did not receive (E) irinotecan in the sequential treatment arm. HR (methylated WRN vs. unmethylated WRN).

Close modal

We next determined whether WRN methylation status had predictive value for irinotecan-treated outcomes by assessing the relationship between WRN methylation status and response to CAPIRI. Patients with unmethylated WRN showed significantly longer PFS when treated with CAPIRI compared with capecitabine alone, as was expected from the results of the original CAIRO trial (median PFS of 272 vs. 164 days for CAPIRI vs. capecitabine, respectively; HR = 0.48; 95% CI, 0.32–0.70; P = 0.0001; Fig. 5A; ref. 17). However, patients with methylated WRN did not benefit from CAPIRI therapy (median PFS of 211 vs. 190 days for CAPIRI vs. capecitabine, respectively; HR = 1.1; 95% CI, 0.69–1.77; P = 0.7; Fig. 5B). The same trend was observed for patients receiving second-line irinotecan monotherapy in the sequential treatment arm, although the number of patients was small (Supplementary Fig. S2).

Figure 5.

PFS in mCRC patients treated with capecitabine (CAP; solid lines) or CAPIRI (dashed lines) as a function of WRN promoter region methylation. PFS is shown for colorectal cancers with unmethylated (A) or methylated (B) WRN promoter regions. HR (CAPIRI vs. capecitabine).

Figure 5.

PFS in mCRC patients treated with capecitabine (CAP; solid lines) or CAPIRI (dashed lines) as a function of WRN promoter region methylation. PFS is shown for colorectal cancers with unmethylated (A) or methylated (B) WRN promoter regions. HR (CAPIRI vs. capecitabine).

Close modal

Multivariate Cox regression analysis showed significant interaction effects between treatment arm and WRN methylation status, even after adjusting for potentially confounding factors, including age, gender, serum LDH, WHO performance status, previous adjuvant therapy, predominant location of metastasis, MSI status, BRAF mutational status, and mucinous differentiation (Table 1 and Supplementary Table S4).

Table 1.

Multivariate Cox regression analysis showing the relationship between different covariates, including an interaction between WRN methylation and treatment arm, and PFS

CovariateHR (95% CI)Pa
Treatment arm 0.47 (0.32–0.70) 0.0002 
WRN methylation status 0.36 (0.14–0.98) 0.05 
Previous adjuvant therapy 1.65 (1.09–2.50) 0.02 
Serum LDH 1.52 (1.07–2.15) 0.02 
WHO performance status 1.18 (0.89–1.56) 0.26 
Gender 0.74 (0.52–1.04) 0.09 
Age 0.99 (0.98–1.01) 0.51 
Location of metastases 1.10 (0.85–1.43) 0.45 
Interaction of treatment arm and WRN methylation status 2.11 (1.13–3.92) 0.02 
CovariateHR (95% CI)Pa
Treatment arm 0.47 (0.32–0.70) 0.0002 
WRN methylation status 0.36 (0.14–0.98) 0.05 
Previous adjuvant therapy 1.65 (1.09–2.50) 0.02 
Serum LDH 1.52 (1.07–2.15) 0.02 
WHO performance status 1.18 (0.89–1.56) 0.26 
Gender 0.74 (0.52–1.04) 0.09 
Age 0.99 (0.98–1.01) 0.51 
Location of metastases 1.10 (0.85–1.43) 0.45 
Interaction of treatment arm and WRN methylation status 2.11 (1.13–3.92) 0.02 

NOTE: Analysis based on 182 samples, of which 179 events (one observation deleted due to missingness).

Abbreviation: WRN, Werner gene.

aWald test.

DNA repair proteins such as the RECQ helicase WRN are promising biomarkers for predicting the response to genotoxic chemotherapy. In this study, we aimed to validate the reported association between WRN promoter hypermethylation and transcriptional silencing and determine the predictive value of WRN promoter hypermethylation for increased sensitivity to irinotecan-based therapy in patients with colorectal cancer (10).

We developed and used two new sets of MSP primers to reliably assess WRN methylation status in both colorectal cancer and normal colon tissue. Methylation status was also analyzed by bisulfite sequencing of a region overlapping the WRN TSS. Our new MSP primer pairs and bisulfite sequencing assay covered the regions analyzed in previous reports (Fig. 1A; refs. 10, 22) and proved more reliable in our hands than the originally reported primer pair for WRN MSP assays (10). Despite using these newly developed and well-validated methylation-specific reagents, we found no consistent association between WRN promoter hypermethylation and WRN expression at the mRNA or protein level. Moreover, we found that WRN promoter hypermethylation was associated with reduced, as opposed to the previously reported increased, OS in patients with colorectal cancer with metastases who received irinotecan (10). PFS improved only when irinotecan was added to capecitabine in the presence of unmethylated WRN, which was not expected from the results of the original CAIRO trial (17).

One explanation for the differing results between our study and a previous report (10) could be the use of different methylation assays. However, this is unlikely: we designed and validated new primer sets for overlapping MSP and bisulfite sequencing assays that worked reliably and covered a 567 bp region that encompassed the TSS. These reagents reliably and accurately detected WRN promoter methylation status in both cell lines and primary tumor samples across the locations of both the originally reported (10) and an additional reported overlapping primer pair (Fig. 1A; ref. 22). Other possible reasons for the contrasting results in the current and previous report (10) encompass the lack of robust analytic tools in the previous report (10), together with the limited number of cell lines and the small size and nature of the clinical samples analyzed (10). Of note, the clinicopathologic details of the 88 patients reported in ref. 10 were not described in the original report or in the reference to this cohort included in the initial report (10). Hence, selection bias cannot be excluded.

We further corroborated our finding of no consistent relationship between WRN promoter methylation level and gene expression using data on 223 colorectal cancer samples included in the TCGA Colorectal Cancer Project, where again no correlation could be identified between WRN hypermethylation and WRN transcriptional silencing (23, 24).

To test the association between WRN methylation status and clinical outcomes, we used material from patients enrolled in the CAIRO study (the Dutch CAIRO study [17]). Our CAIRO study cohort (n = 183) was larger than the initial cohort (n = 183 vs. 88) and has been described in detail. The CAIRO study provided high-quality clinical data, which are essential to evaluate predictive biomarkers (27, 28) and to test the association between WRN methylation status and clinical outcomes. The CAIRO cohort also offered the opportunity to compare first-line capecitabine monotherapy versus CAPIRI therapy.

Despite our larger well-characterized study population, we were not able to confirm the initial observation that WRN promoter hypermethylation was associated with improved outcome in irinotecan-treated patients with metastatic colorectal cancer (10). In contrast, we observed a significantly worse outcome for irinotecan-treated patients with colorectal cancer with WRN-methylated tumors. This is similar to the outcome observed in an independent, well-described study (22) that used primer pairs targeting the same WRN region as the initial report (see Fig. 1A; ref. 10). These observations indicate that WRN promoter hypermethylation may be useful as a biomarker, to predict a worse response to irinotecan treatment.

This effect is likely to reflect as-yet unidentified covariables, as WRN promoter hypermethylation does not consistently alter WRN expression. WRN is a housekeeping gene that is expressed at comparatively low copy number (≤1,000 to 10,000 copies/cell) in many cell types (29–32). The WRN promoter region includes Sp1, RCE (retinoblastoma/TP53), AP2, and MYC E-box–binding sites, and there are experimental data showing that these binding sites and/or transcription factors can alter WRN transcription (33, 34). WRN expression is also known to be cell cycle responsive and upregulated by cellular oncogenic transformation (31), although none of these mechanisms has been shown thus far to be WRN DNA methylation dependent or modulated.

Alternatively, WRN promoter hypermethylation has been associated to MSI, CpG island methylator phenotype, BRAF mutations, and mucinous differentiation, which themselves are associated to clinical outcome in colon cancer (11, 22, 35). Information on MSI, BRAF mutational status, and mucinous differentiation available on a subset of our sample set revealed that those variables did not explain the association between WRN promoter hypermethylation and clinical outcome after treatment with irinotecan-based therapy. However, the number of samples with MSI status and/or BRAF mutation was very low (n = 6 and n = 11, respectively); hence, no hard conclusions can be drawn from these results. Future functional analyses and validation studies in large, independent, and well-annotated cohorts are needed to shed light on the role of WRN promoter hypermethylation as a determinant of the response to irinotecan-based therapy.

Our study has the following limitations. First, measurements were performed on the primary tumors, while patients were treated for their metastases, which raises the question whether intratumoral heterogeneity could play a role. Although metastases can acquire additional genomic alterations, they keep most alterations present in the primary tumor (36, 37). Furthermore, DNA methylation is usually an early event in colorectal carcinogenesis, which we suspect is true for WRN methylation as well (38).

Second, we were not able to independently analyze all cell lines at both participating institutions, although note that the subset of cells analyzed by both groups gave concordant results. This strengthens our conclusion that previous findings on the negative relationship between WRN promoter methylation level and gene expression at the mRNA or protein level could not be validated.

A final limitation of this study was the use of DNA from 183 patients and tumor tissue, which represented a subset of all patients in the CAIRO trial (17). However, this selection was representative for the subgroup of patients who underwent resection of the primary tumor in terms of clinical characteristics and survival outcome (see also ref. 21). Furthermore, the current cohort is larger than the cohort as described in ref. 10 (n = 183 vs. n = 88) and was large enough to have statistical power.

In summary, we found that the methylation status of the WRN promoter region can be reliably assessed in both colorectal cancer and normal colorectal tissue using newly developed MSP and bisulfite sequencing assays. However, there was no consistent association between WRN promoter hypermethylation and loss of WRN expression at the mRNA or protein level in colorectal cancer cell lines or tumors. Moreover, we could not validate findings from a previous study that WRN promoter hypermethylation was associated with a better response to irinotecan-based therapy and found instead that WRN promoter hypermethylation was associated with reduced OS and PFS in our well-characterized colorectal cancer patient cohort that received irinotecan-based therapy. Despite growing evidence for a role for WRN genomic alterations in colorectal cancer disease progression (16), our results indicate that WRN promoter hypermethylation does not reliably predict WRN gene expression or, as originally reported (10), improved clinical outcomes in patients with colorectal cancer treated with irinotecan-based chemotherapy regimens.

W. Van Criekinge is a consultant/advisory board member for MDxHealth.

Conception and design: L.J.W. Bosch, V.V. Lao, I. Vlassenbroeck, P. Welcsh, G.A. Meijer, R.J. Monnat Jr, B. Carvalho, W.M. Grady

Development of methodology: L.J.W. Bosch, V.V. Lao, P. Snaebjornsson, G. Trooskens, I. Vlassenbroeck, S. Mongera, W. Tang, J.G. Herman, R.J. Monnat Jr

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L.J.W. Bosch, Y. Luo, V.V. Lao, P. Snaebjornsson, G. Trooskens, S. Mongera, W. Tang, P. Welcsh, M. Koopman, I.D. Nagtegaal, C.J.A. Punt, W. van Criekinge, G.A. Meijer, W.M. Grady

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L.J.W. Bosch, Y. Luo, V.V. Lao, P. Snaebjornsson, G. Trooskens, W. Tang, P. Welcsh, J.G. Herman, W. van Criekinge, G.A. Meijer, R.J. Monnat Jr, B. Carvalho, W.M. Grady

Writing, review, and/or revision of the manuscript: L.J.W. Bosch, Y. Luo, V.V. Lao, P. Snaebjornsson, G. Trooskens, I. Vlassenbroeck, J.G. Herman, M. Koopman, I.D. Nagtegaal, C.J.A. Punt, G.A. Meijer, R.J. Monnat Jr, B. Carvalho, W.M. Grady

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): P. Snaebjornsson, S. Mongera, P. Welcsh

Study supervision: G.A. Meijer, B. Carvalho, W.M. Grady

Other (also provided funding to enable the completion of this study, as noted in the manuscript): R.J. Monnat Jr

The authors thank Dr. H. van Tinteren for his critical review of the manuscript and valuable suggestions.

Support for these studies was provided by the NIH (RO1CA115513, P30CA15704, UO1CA152756, U54CA143862, and P01CA077852), and a Burroughs Wellcome Fund Translational Research Award for Clinician Scientist (to W.M. Grady). V.V. Lao was supported by ACS fellowship PF-11-086-01-TBG; 2T32DK007742-16; ASCRS GSRRIG; and NIH NCI F32CA1591555-01. L.J.W. Bosch was supported by Dutch Cancer Society (KWF Fellowship VU 2013-5885). Support for these studies was also provided by National Natural Science Foundation of China (81201920, 81472257; to Y. Luo). Parts of this study were performed within the framework of CTMM, the Center for Translational Molecular Medicine, DeCoDe project (grant 03O-101), and the Dutch Colorectal Cancer Group (DCCG).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
American Cancer Society
.
Global Cancer Facts & Figures 2nd Edition.
Atlanta, GA
:
American Cancer Society
; 
2011
.
p.
13
5
.
2.
DeVita
VT
 Jr
,
Lawrence
TS
,
Rosenberg
SA
.
DeVita, Hellman, Rosenberg's cancer: principles and practice of oncology,
10th Edition; 
2015
.
3.
Koopman
M
,
Punt
CJ
. 
Chemotherapy, which drugs and when
.
Eur J Cancer
2009
;
45
Suppl 1
:
50
6
.
4.
Cunningham
D
,
Atkin
W
,
Lenz
HJ
,
Lynch
HT
,
Minsky
B
,
Nordlinger
B
, et al
Colorectal cancer
.
Lancet
2010
;
375
:
1030
47
.
5.
Tol
J
,
Punt
CJ
. 
Monoclonal antibodies in the treatment of metastatic colorectal cancer: a review
.
Clin Ther
2010
;
32
:
437
53
.
6.
Hanahan
D
,
Weinberg
RA
. 
Hallmarks of cancer: the next generation
.
Cell
2011
;
144
:
646
74
.
7.
Lao
VV
,
Grady
WM
. 
Epigenetics and colorectal cancer
.
Nat Rev Gastroenterol Hepatol
2011
;
8
:
686
700
.
8.
Deaton
AM
,
Bird
A
. 
CpG islands and the regulation of transcription
.
Genes Dev
2011
;
25
:
1010
22
.
9.
Paz
MF
,
Yaya-Tur
R
,
Rojas-Marcos
I
,
Reynes
G
,
Pollan
M
,
Aguirre-Cruz
L
, et al
CpG island hypermethylation of the DNA repair enzyme methyltransferase predicts response to temozolomide in primary gliomas
.
Clin Cancer Res
2004
;
10
:
4933
8
.
10.
Agrelo
R
,
Cheng
WH
,
Setien
F
,
Ropero
S
,
Espada
J
,
Fraga
MF
, et al
Epigenetic inactivation of the premature aging Werner syndrome gene in human cancer
.
Proc Natl Acad Sci U S A
2006
;
103
:
8822
7
.
11.
Kawasaki
T
,
Ohnishi
M
,
Suemoto
Y
,
Kirkner
GJ
,
Liu
Z
,
Yamamoto
H
, et al
WRN promoter methylation possibly connects mucinous differentiation, microsatellite instability and CpG island methylator phenotype in colorectal cancer
.
Mod Pathol
2008
;
21
:
150
8
.
12.
Masuda
K
,
Banno
K
,
Yanokura
M
,
Tsuji
K
,
Kobayashi
Y
,
Kisu
I
, et al
Association of epigenetic inactivation of the WRN gene with anticancer drug sensitivity in cervical cancer cells
.
Oncol Rep
2012
;
28
:
1146
52
.
13.
Croteau
DL
,
Popuri
V
,
Opresko
PL
,
Bohr
VA
. 
Human RecQ helicases in DNA repair, recombination, and replication
.
Annu Rev Biochem
2014
;
83
:
519
52
.
14.
Sidorova
JM
,
Monnat
RJ
 Jr.
Human RECQ helicases: roles in cancer, aging, and inherited disease
.
Adv Genomics Genet
2015
;
5
:
19
33
.
15.
Mao
FJ
,
Sidorova
JM
,
Lauper
JM
,
Emond
MJ
,
Monnat
RJ
. 
The human WRN and BLM RecQ helicases differentially regulate cell proliferation and survival after chemotherapeutic DNA damage
.
Cancer Res
2010
;
70
:
6548
55
.
16.
Lee
H
,
Flaherty
P
,
Ji
HP
. 
Systematic genomic identification of colorectal cancer genes delineating advanced from early clinical stage and metastasis
.
BMC Med Genomics
2013
;
6
:
54
.
17.
Koopman
M
,
Antonini
NF
,
Douma
J
,
Wals
J
,
Honkoop
AH
,
Erdkamp
FL
, et al
Sequential versus combination chemotherapy with capecitabine, irinotecan, and oxaliplatin in advanced colorectal cancer (CAIRO): a phase III randomised controlled trial
.
Lancet
2007
;
370
:
135
42
.
18.
The Wellcome Trust Sanger Institute Cancer Genome Project
. 
The Wellcome Trust Sanger Institute Cancer Genome Project web site
; 
2011
.
Available from:
http://www.sanger.ac.uk/genetics/CGP.
19.
Hermsen
M
,
Snijders
A
,
Guervos
MA
,
Taenzer
S
,
Koerner
U
,
Baak
J
, et al
Centromeric chromosomal translocations show tissue-specific differences between squamous cell carcinomas and adenocarcinomas
.
Oncogene
2005
;
24
:
1571
9
.
20.
Dutch Federation of Biomedical Scientific Societies
.
Code for proper secondary use of human tissue in the Netherlands;
2011
.
21.
Haan
JC
,
Labots
M
,
Rausch
C
,
Koopman
M
,
Tol
J
,
Mekenkamp
LJ
, et al
Genomic landscape of metastatic colorectal cancer
.
Nat Commun
2014
;
5
:
5457
.
22.
Ogino
S
,
Meyerhardt
JA
,
Kawasaki
T
,
Clark
JW
,
Ryan
DP
,
Kulke
MH
, et al
CpG island methylation, response to combination chemotherapy, and patient survival in advanced microsatellite stable colorectal carcinoma
.
Virchows Arch
2007
;
450
:
529
37
.
23.
The Cancer Genome Atlas Research Network
. 
Comprehensive molecular characterization of human colon and rectal cancer
.
Nature
2012
;
487
:
330
7
.
24.
Cerami
E
,
Gao
J
,
Dogrusoz
U
,
Gross
BE
,
Sumer
SO
,
Aksoy
BA
, et al
The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data
.
Cancer Discov
2012
;
2
:
401
4
.
25.
Mekenkamp
LJ
,
Heesterbeek
KJ
,
Koopman
M
,
Tol
J
,
Teerenstra
S
,
Venderbosch
S
, et al
Mucinous adenocarcinomas: poor prognosis in metastatic colorectal cancer
.
Eur J Cancer
2012
;
48
:
501
9
.
26.
Venderbosch
S
,
Nagtegaal
ID
,
Maughan
TS
,
Smith
CG
,
Cheadle
JP
,
Fisher
D
, et al
Mismatch repair status and BRAF mutation status in metastatic colorectal cancer patients: a pooled analysis of the CAIRO, CAIRO2, COIN, and FOCUS studies
.
Clin Cancer Res
2014
;
20
:
5322
30
.
27.
Koopman
M
,
Venderbosch
S
,
Nagtegaal
ID
,
van Krieken
JH
,
Punt
CJ
. 
A review on the use of molecular markers of cytotoxic therapy for colorectal cancer, what have we learned?
Eur J Cancer
2009
;
45
:
1935
49
.
28.
Simon
RM
,
Paik
S
,
Hayes
DF
. 
Use of archived specimens in evaluation of prognostic and predictive biomarkers
.
J Natl Cancer Inst
2009
;
101
:
1446
52
.
29.
Beck
M
,
Schmidt
A
,
Malmstroem
J
,
Claassen
M
,
Ori
A
,
Szymborska
A
, et al
The quantitative proteome of a human cell line
.
Mol Syst Biol
2011
;
7
:
549
.
30.
Nagaraj
N
,
Wisniewski
JR
,
Geiger
T
,
Cox
J
,
Kircher
M
,
Kelso
J
, et al
Deep proteome and transcriptome mapping of a human cancer cell line
.
Mol Syst Biol
2011
;
7
:
548
.
31.
Kawabe
T
,
Tsuyama
N
,
Kitao
S
,
Nishikawa
K
,
Shimamoto
A
,
Shiratori
M
, et al
Differential regulation of human RecQ family helicases in cell transformation and cell cycle
.
Oncogene
2000
;
19
:
4764
72
.
32.
Moser
MJ
,
Kamath-Loeb
AS
,
Jacob
JE
,
Bennett
SE
,
Oshima
J
,
Monnat
RJ
 Jr.
WRN helicase expression in Werner syndrome cell lines
.
Nucleic Acids Res
2000
;
28
:
648
54
.
33.
Yamabe
Y
,
Shimamoto
A
,
Goto
M
,
Yokota
J
,
Sugawara
M
,
Furuichi
Y
. 
Sp1-mediated transcription of the Werner helicase gene is modulated by Rb and p53
.
Mol Cell Biol
1998
;
18
:
6191
200
.
34.
Grandori
C
,
Wu
KJ
,
Fernandez
P
,
Ngouenet
C
,
Grim
J
,
Clurman
BE
, et al
Werner syndrome protein limits MYC-induced cellular senescence
.
Genes Dev
2003
;
17
:
1569
74
.
35.
Ogino
S
,
Nosho
K
,
Kirkner
GJ
,
Kawasaki
T
,
Meyerhardt
JA
,
Loda
M
, et al
CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer
.
Gut
2009
;
58
:
90
6
.
36.
Knijn
N
,
Mekenkamp
LJ
,
Klomp
M
,
Vink-Borger
ME
,
Tol
J
,
Teerenstra
S
, et al
KRAS mutation analysis: a comparison between primary tumours and matched liver metastases in 305 colorectal cancer patients
.
Br J Cancer
2011
;
104
:
1020
6
.
37.
Mekenkamp
LJ
,
Haan
JC
,
Israeli
D
,
van Essen
HF
,
Dijkstra
JR
,
van
CP
, et al
Chromosomal copy number aberrations in colorectal metastases resemble their primary counterparts and differences are typically non-recurrent
.
PLoS One
2014
;
9
:
e86833
.
38.
Derks
S
,
Postma
C
,
Moerkerk
PT
,
van den Bosch
SM
,
Carvalho
B
,
Hermsen
MA
, et al
Promoter methylation precedes chromosomal alterations in colorectal cancer development
.
Cell Oncol
2006
;
28
:
247
57
.
39.
Li
LC
,
Dahiya
R
. 
MethPrimer: designing primers for methylation PCRs
.
Bioinformatics
2002
;
18
:
1427
31
.