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Werner syndrome is a premature aging syndrome characterized by accelerated aging and early onset of cancer and other diseases. WRN (the Werner syndrome gene) and its product (DNA helicase1) have been shown to be important in maintenance of telomere structures,2 and initiation of DNA damage response after telomere disruption.3 Normal telomere function is an important cellular mechanism against aging and manifestations of Werner syndrome.4, 5, 6 WRN can interact with p53,7, 8 and replication protein A1,9 the latter of which is required for stabilization of single-stranded DNA during DNA replication.10 Considering the importance of the WRN helicase in the maintenance of telomere function, WRN likely acts as a ‘caretaker’ tumor suppressor gene for genome integrity. Promoter methylation and gene silence of WRN have been shown in cell lines from colon cancer, breast cancer and leukemia.11 Restoration of WRN expression causes reduced colony formation and inhibition of tumor growth in a xenograft model, confirming tumor suppressor property of WRN.11 WRN promoter methylation may predict good survival among colorectal cancer patients treated by irinotecan,11 which is a topoisomerase inhibitor.

Transcriptional inactivation by cytosine methylation at promoter CpG islands of tumor suppressor genes is an important mechanism in human carcinogenesis.12, 13, 14, 15 A number of tumor suppressor genes are silenced by promoter methylation in colorectal cancers.12, 13 A subset of colorectal cancers exhibit widespread promoter CpG island methylation, which is referred to as the CpG island methylator phenotype (CIMP).16 CIMP-high colorectal tumors have a distinct clinical, pathologic, and molecular profile, such as associations with proximal tumor location, female sex, poor differentiation, microsatellite instability (MSI), and high BRAF and low TP53 mutation rates.17, 18, 19, 20 In addition, mucinous colorectal carcinomas frequently show the CIMP and MSI phenotypes.20, 21, 22, 23 However, the mechanism of mucinous differentiation is poorly understood.

Werner syndrome patients have been known to demonstrate elevated levels of hyaluronic acid in serum and urine.24, 25, 26, 27 WRN-deficient cells exhibit abnormal metabolism of glycosaminoglycan,28, 29, 30, 31 and in particular, excretion of glycosaminoglycan is increased from WRN-deficient cells.32 Thus, we hypothesized that WRN promoter methylation and gene silencing might, at least in part, explain excessive mucin secretion in a subset of colorectal cancers with CIMP and/or MSI.

In this study, using quantitative DNA methylation analysis (MethyLight technology) and a large number of population-based colorectal cancers, we examined the relationship between WRN promoter methylation, CIMP, MSI and mucinous features. We have shown that WRN methylation is correlated with mucinous differentiation independent of CIMP and MSI, thus providing a possible explanation to the enigmatic association between mucin and CIMP/MSI.

Materials and methods

Study Group

We utilized the databases of two large prospective cohort studies; the Nurses' Health Study (N=121 700 women followed since 1976),33 and the Health Professional Follow-up Study (N=51 500 men followed since 1986).34 Informed consent was obtained from all participants prior to inclusion in the cohorts. A subset of the cohort participants developed colorectal cancers during prospective follow-up. Thus, these colorectal cancers represented population-based, relatively unbiased samples (compared to retrospective or single-hospital-based samples). Previous studies on Nurses' Health Study and Health Professionals Follow-up Study have described baseline characteristics of cohort participants and incident colorectal cancer cases, and confirmed that our colorectal cancer cases were well representative as a population-based sample.33, 34 Clinical features of each colorectal cancer case were obtained by chart review. We collected paraffin-embedded tissue blocks from hospitals where cohort participants with colorectal cancers had undergone resections of primary tumors. We excluded cases if adequate paraffin-embedded tumor tissue was not available at the time of the study. As a result, a total of 903 colorectal cancer cases (405 from men's cohort and 498 from women's cohort) were included. Among our cohort studies, there was no significant difference in demographic features between cases with tissue available and those without available tissue.35 Many of the cases have been previously characterized for status of CIMP, MSI, KRAS and BRAF.23 However, no tumor has been examined for WRN methylation in our previous studies. Tissue collection and analyses were approved by the Dana–Farber Cancer Institute and Brigham and Women's Hospital Institutional Review Boards.

Histopathologic Evaluations

Hematoxylin and eosin (H&E) stained tissue sections were examined under a light microscope by one of the investigators (SO) blinded from clinical and other laboratory data. Various pathologic features were examined as described previously.36 Tumors were classified into well/moderately-differentiated (<50% solid areas); and poorly-differentiated tumors (≥50% solid areas). The extent and type (intracellular or extracellular) of mucinous component in each tumor were evaluated. In addition, tumor infiltrating lymphocytes, Crohn's-like reaction, and peritumoral lymphocytic reaction have been evaluated, and graded as absent/mild or moderate/severe.36

Genomic DNA Extraction and Whole Genome Amplification

Genomic DNA was extracted from dissected tumor tissue sections using QIAmp DNA Mini Kit (Qiagen, Valencia, CA USA) as described previously.37 Normal DNA was obtained from colonic tissue at resection margins. Whole genome amplification (WGA) of genomic DNA was performed by PCR using random 15-mer primers for subsequent MSI analysis and KRAS and BRAF sequencing.37 Previous studies by us and others showed that WGA did not significantly affect KRAS mutation detection or microsatellite analysis.37, 38

Analyses for Microsatellite Instability and 18q Loss of Heterozygosity

Methods to analyze for MSI status have been described previously.37 In addition to the recommended MSI panel consisting of D2S123, D5S346, D17S250, BAT25 and BAT26,39 we also used BAT40, D18S55, D18S56, D18S67 and D18S487 (ie, 10-marker panel).37 A ‘high degree of MSI’ (MSI-H) was defined as the presence of instability in ≥30% of the markers. A low degree of MSI (MSI-L) was defined as the presence of instability in <30% of the markers, and ‘microsatellite stable’ (MSS) tumors were defined as tumors without an unstable marker.

18q loss of heterozygosity (LOH) analysis using microsatellite markers D18S55, D18S56, D18S67 and D18S487 were performed as described previously.37 We duplicated PCR and electrophoresis in each sample to exclude allele dropouts of one of two alleles. Loss of heterozygosity at each locus was defined as 40% or greater reduction of one of two allele peaks in tumor DNA relative to normal DNA.

Sequencing of KRAS and BRAF

Methods of PCR and sequencing targeted for KRAS codons 12 and 13, and BRAF codon 600 have been described previously.37 Pyrosequencing was performed using the PSQ96 HS System (Biotage AB and Biosystems, Uppsala, Sweden) according to the manufacturer's instructions.

Real-Time PCR (MethyLight) for Quantitative DNA Methylation Analysis

Sodium bisulfite treatment on genomic DNA was performed as described previously.40 Real-time PCR to measure DNA methylation (MethyLight) was performed as described previously.41 Utilizing ABI 7300 (Applied Biosystems, Foster City, CA, USA), we examined WRN promoter and eight other CIMP-specific promoters (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1).19, 23 We have shown that these eight markers are sensitive and specific markers for CIMP diagnosis.23 COL2A1 (the collagen 2A1 gene) was used to normalize for the amount of input bisulfite-converted DNA.40 PCR primers and probe for WRN were (bisulfite-converted nucleotides are highlighted by bold face and italics): WRN-F, 5′-GTA TCG TTC GCG GCG TTT AT-3′ (Genbank No AY442327, nucleotide Nos 1827–1846); WRN-R, 5′-ACG AAA CCG ATA TCC GAA ATC A -3′ (nucleotide Nos 1887–1908); WRN-probe, 6FAM-5′-TTT TTT TTG CGG TCG TTG CGG G-3′-BHQ-1 (nucleotides 1855–1876). The WRN promoter CpG island that we examined is the one which was analyzed by Agrelo et al.11 All other primers and probes were described previously.19 The percentage of methylated reference (PMR, ie, degree of methylation) at a specific locus was calculated by dividing the GENE:COL2A1 ratio of template amounts in a sample by the GENE:COL2A1 ratio of template amounts in SssI-treated human genomic DNA (presumably fully methylated) and multiplying this value by 100.41 A PMR cutoff value of 4 (except for 6 in CRABP1 and IGF2, and 10 for WRN) was based on previously validated data.40 Precision and performance characteristics of bisulfite conversion and subsequent MethyLight assays have been previously evaluated and the assays have been validated.40 CIMP-high was defined as the presence of ≥6/8 methylated promoters, CIMP-low as 1/8–5/8 methylated promoters and CIMP-0 as the absence (0/8) of methylated promoters, according to the previously established criteria.23

Tissue Microarrays and Immunohistochemistry for p53 and p21 (CDKN1A)

Tissue microarrays were constructed as described previously,35 using the Automated Arrayer (Beecher Instruments, Sun Prairie, WI, USA). We examined two to four tumor tissue cores for each marker. A previous validation study has shown that examining two TMA cores can yield comparable results to examining whole tissue sections in more than 95% of cases.42 We examined whole tissue sections for cases in which no tissue block was available for TMA construction or results were equivocal in TMAs. Immunohistochemistry for p53 was performed as described previously.37 p53 positivity was defined as 50% or more of tumor cells with unequivocal strong nuclear staining, as this high threshold considerably improved specificity in previous studies.43, 44

For p21 (CDKN1A/CIP1) immunohistochemistry, we incubated deparaffinized whole tissue sections in citrate buffer at high power in a microwave for 30 min (in a pressure cooker). Tissue sections were then incubated with 3% H2O2 (10 min) to block endogenous peroxidase, and then incubated with protein block (Vector Laboratories, Burlingame, CA, USA) (10 min). Primary anti-p21 antibody (Pharmingen, San Diego, CA, USA) (dilution 1:50) was applied for 30 min at room temperature. Then, biotinylated secondary multilink antibody (Biogenex, San Ramon, CA, USA) was applied (20 min), horse radish peroxidase avidin complex (Biogenex) was added and sections were visualized by DAB (30 s) and methyl-green counterstain. p21 loss was defined as less than 5% of tumor cells with nuclear staining.

Appropriate positive and negative controls were included in each run of immunohistochemistry. All immunohistochemically-stained slides were interpreted by one of the investigators (SO) blinded from any other clinical and laboratory data.

Statistical Analysis

In statistical analysis, χ2 test (or Fisher's exact test when the number in any category was less than 10) was performed for categorical data, and kappa coefficients were calculated to determine the degree of agreement between two observers, using SAS program (Version 9.1, SAS Institute, Cary, NC, USA). All P-values were two-sided, and statistical significance was set at P≤0.05.

Results

WRN Promoter Methylation and CIMP in Colorectal Cancer

Utilizing MethyLight technology, we quantified DNA methylation in WRN and a panel of eight promoters (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1). The latter eight promoters constitutes a sensitive and specific marker panel for CIMP-high.23 Among the 903 tumors, 266 (29%) were positive for WRN promoter methylation. WRN methylation was slightly more common in women (32%=159/498) than in men (26%=107/405) though the difference was not statistically significant. Sensitivity and specificity of WRN methylation for the diagnosis of CIMP-high (≥6/8 methylated promoters, not including WRN) were 89 and 81%, respectively (Table 1), indicating that WRN was a good marker for CIMP-high (slightly better than CDKN2A23). This fact also indicates that CIMP status is a confounding factor when one analyzes the relationship between WRN methylation and any clinicopathologic or molecular variables. Thus, in subsequent analyses, we stratified tumors according to WRN and CIMP status (as in Table 2 and Table 3). Because 5/8 methylated tumors show borderline features between CIMP-high and CIMP-low,23 those were excluded from further analyses.

Table 1 Sensitivity and specificity of WRN methylation for the diagnosis of CIMP-high
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Table 2 Frequencies of specific clinical and pathologic features in colorectal cancer according to WRN methylation and CIMP status
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Table 3 Frequencies of specific molecular features in colorectal cancer according to WRN methylation and CIMP status
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We also quantified WRN methylation in normal colon tissue from eight WRN-methylated tumor cases and seven WRN-unmethylated tumor cases. Only one normal sample from the eight WRN-methylated tumor cases showed WRN methylation, and no normal sample from the seven WRN-unmethylated tumor cases showed WRN methylation.

WRN Methylation is Associated with Mucinous Differentiation Independent of CIMP Status

Table 2 summarizes the relations between WRN methylation and clinical and pathologic features in colorectal cancer. The presence of any mucinous component was significantly correlated with WRN methylation in all cases (P<0.0001), in CIMP-high tumors (P=0.04), and in CIMP-low/0 tumors (P<0.0001). The frequencies of both 1–49% mucinous tumors and ≥50% mucinous tumors were higher in WRN-methylated tumors than WRN-unmethylated tumors, regardless of CIMP status (Table 2).

Correlations of WRN Methylation with Other Clinicopathologic and Molecular Features

Proximal tumor location, poor differentiation, signet ring cells, tumor infiltrating lymphocytes, Crohn's-like reaction and peritumoral lymphocytic reaction were associated with WRN methylation in all cases, but no significant correlations persisted after tumors were stratified by CIMP status (Table 2), indicating these features were associated primarily with CIMP, but not directly with WRN methylation. There was no significant correlation between WRN and age at diagnosis.

Table 3 summarizes the relations between WRN methylation and molecular alterations in colorectal cancer. WRN methylation was correlated with MSI-H, BRAF mutation, 18q loss of heterozygosity negativity, p53 negativity and intact p21 expression in all cases, but no significant relationship persisted after tumors were stratified by CIMP status.

Relationship Between WRN Methylation and Mucinous Differentiation Persisted in Each MSI/CIMP Subtype

Because mucinous features are correlated with both MSI-H and CIMP-high, we stratified tumors into nine MSI/CIMP subtypes and examined the frequency of mucinous differentiation according to WRN methylation status (Figure 1). There was no MSI-L CIMP-high WRN-unmethylated tumor. After exclusion of MSI-L/CIMP-high, within each of the remaining eight MSI/CIMP subtypes, WRN-methylated tumors consistently exhibited higher frequencies of both ≥50% mucinous tumors and tumors with any mucinous component. Under the null hypothesis that WRN methylation was unrelated with mucinous features, the probability that WRN-methylated tumors showed a higher frequency of mucinous tumors within one MSI/CIMP group by chance would be 1/2; thus, the statistical significance level for our consistent observations in all of the eight MSI/CIMP categories was P=(1/2)8=0.004. These results implied that WRN methylation was associated with mucinous differentiation independent of MSI and CIMP.

Figure 1
figure 1

Frequencies of mucinous tumors in WRN-methylated and WRN-unmethylated colorectal cancers within each of the 9 MSI/CIMP subtypes. Note that WRN-methylated tumors show consistently higher frequencies of mucinous differentiation (both ≥50% mucinous tumors and any mucinous tumors) than WRN-unmethylated tumors (P=0.004). Abbreviations: CIMP, CpG island methylator phenotype; M, methylated; MSI, microsatellite instability; MSS, microsatellite stable; UM, unmethylated.

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Discussion

We conducted this study to examine the relationship between mucinous differentiation and promoter methylation in WRN (the Werner syndrome gene). We have found that, compared to WRN-unmethylated colorectal cancers, WRN-methylated tumors consistently show higher frequencies of both ≥50% mucinous tumors and tumors with any mucinous component, within each MSI/CIMP subtype. Thus, WRN methylation appears to be correlated with mucinous differentiation regardless of CIMP and MSI status. Considering the relation between WRN methylation and CIMP/MSI, and the link between Werner syndrome and abnormal glycosaminoglycan metabolism, our data suggest the possibility that the enigmatic association between mucinous differentiation and CIMP/MSI may be connected by WRN methylation.

We utilized quantitative DNA methylation assays (MethyLight), which is robust and can reproducibly differentiate low-level methylation from high-level methylation.40 Our resource of a large number of colorectal cancers, derived from two large prospective cohorts (relatively unbiased samples compared to retrospective or single-hospital-based samples), has enabled us to precisely estimate the frequency of colorectal cancers with a specific molecular feature (eg, WRN methylation, MSI-H, etc). The large number of samples has also provided a sufficient power to examine the relation between WRN methylation and mucinous features in rare tumor subtypes, such as MSI-H CIMP-0, MSI-L CIMP-low, etc.

The association between WRN methylation and mucinous features in colorectal cancer is intriguing, and it appears to be independent of CIMP and MSI status. The presence of mucinous component (even with a minor component) in colorectal cancer implies specific molecular pathologic features, including associations with MSI-H, BRAF mutation, KRAS mutation, p53 negativity and fatty acid synthase overexpression.37 However, pathogenetic mechanism of mucinous differentiation is poorly understood. Abnormal glycosaminoglycan metabolism is present in Werner syndrome cells,28, 29, 30, 31 and Werner syndrome patients show elevated levels of hyaluronic acid in serum and urine.24, 25, 26, 27 Thus, it is possible that abnormal glycosaminoglycan metabolism may cause mucin overproduction in colorectal cancer with WRN methylation and functional loss. This hypothesis can, at least in part, explain the well-known association between mucinous differentiation and MSI/CIMP in colorectal cancer,20, 21, 22, 23 as we have shown the positive correlations between WRN methylation and MSI/CIMP, and between WRN methylation and mucinous differentiation.

Our data also indicate that WRN methylation can serve as a good marker for the diagnosis of CIMP-high with 89% sensitivity and 81% specificity. We have previously shown that all of the eight promoters including RUNX3, CACNA1G, IGF2, MLH1, NEUROG1, CRABP1, SOCS1 and CDKN2A exhibit good sensitivity and specificity, and thus can be used as a CIMP-high diagnostic panel.23 In fact, WRN shows slightly superior performance to CDKN2A (with sensitivity 85% and specificity 81% when CDKN2A is excluded from the CIMP panel23). Thus, WRN can be included in a methylation maker panel for CIMP-high diagnosis.

In summary, WRN promoter methylation in colorectal cancer is associated with mucinous differentiation independent of MSI and CIMP status. WRN methylation may connect the enigmatic link between mucinous differentiation and MSI/CIMP. Further studies are necessary to elucidate the exact pathogenic mechanism of mucinous differentiation in colorectal cancer.

Note added in proof

Detailed methods were previously described as follows: the selection of CDKN2A, CRABP1 and MLH1 as CIMP panel markers,45 whole genome amplification and KRAS Pyrosequencing,46 BRAF Pyrosequencing,47 and p53 immunohistochemistry.48