Search

To characterize the clinical, pathological, and imaging features of DCIS occult on conventional imaging diagnosed on MRI-guided biopsy associated with increased risk of invasive disease on surgical excision. All consecutive patients with MRI-detected DCIS occult on conventional imaging between January 2009 and December 2018 were included. Women were divided into two groups based on final pathology: Pure DCIS or DCIS with invasive component. Clinical, imaging, and pathological risk factors for upgrade to invasion were evaluated. Of 50 patients who met the inclusion criteria, 12 (24%) were upgraded to invasive malignancy in the final pathology. The only parameters that showed statistically significant association with upgrade were related to kinetic characteristics: 53% of patients with the combination of fast early upstroke and either plateau or washout curve were upgraded, compared to 12% of women without this combination (p = 0.006). The sensitivity of combined kinetic features for predicting upgrade was 67% (95% CI 35–90%), specificity was 84% (CI 95% 68–94%), positive predictive value was 57% (CI 95% 37–75%), negative predictive value was 89% (CI 95% 77–95%), and OR was 78% (64–88%). Kinetic characteristics show the strongest association with upgrade to invasion in DCIS occult on mammogram and US. Larger studies should be encouraged to consolidate our findings, which may have implication for treatment planning.

Endometrial carcinoma is one of the prototypical malignancies associated with Lynch syndrome, an inherited cancer syndrome most commonly caused by germline mutations in DNA mismatch repair (MMR) genes, although rare alternative mechanisms also exist. In this report, we describe a patient first diagnosed with colorectal cancer at age 33, then vulvar squamous cell carcinoma, facial sebaceous adenoma/sebaceoma, and finally endometrial carcinoma at age 52. All tumors were MLH1/PMS2-deficient by immunohistochemistry, and MLH1 promoter methylation was identified in the endometrial cancer. Germline MLH1 testing was negative for pathogenic variants, but she was subsequently diagnosed with Lynch syndrome secondary to a germline monoallelic constitutional epimutation of the MLH1 promoter. Identification of patients displaying a Lynch syndrome phenotype but lacking germline MMR mutations is important to avoid delays in the diagnosis of Lynch syndrome as well as the initiation of appropriate cancer screening and genetic counseling.

Immunohistochemical (IHC) evaluation of DNA mismatch repair proteins (MMR) has become routine practice for Lynch syndrome screening and/or part of diagnostic evaluation in endometrial cancer. Approximately 20% to 30% of endometrial carcinomas demonstrate microsatellite instability due to defective DNA MMR. Vast majority of MLH1/PMS2-deficient tumors are sporadic and show MLH1 promoter methylation. MLH1 methylation testing by quantitative polymerase chain reaction-based technique is time, labor, and tissue intensive with an average institutional turnaround time of 2 weeks. MLH1 and EPM2AIP1 genes share a common promoter whose methylation has been shown to affect both genes. We assessed whether IHC for EPM2AIP1 in combination with MMR proteins can serve as surrogate marker for MLH1 promoter methylation status. We performed a retrospective review of all MLH1/PMS2-deficient endometrial carcinomas that underwent MLH1 promoter methylation testing from January 1 to September 31, 2020, at our institution. Microscopic slides were reviewed and EMP2AIP1 IHC was performed. The results were correlated with MLH1 promoter methylation status (percent methylated rate). A total of 119 cases were identified and successfully tested. Nuclear EPM2AIP1 protein expression was observed in benign endometrial cells and myometrial smooth muscle cells. Loss of nuclear EPM2AIP1 staining was identified in 90/110 (81.8%) methylated tumors with additional 14/110 (12.7%) cases showing aberrant staining patterns. Only 6/110 (5.5%) tumors demonstrated intact EPM2AIP1 nuclear expression in presence of MLH1 promoter methylation. EMP2AIP1 IHC is concordant with MLH1 promoter methylation results in 95% of endometrial carcinomas (94.5% sensitivity, 98.1% positive predictive value) and shows promise as a surrogate marker for methylation testing.

Neoadjuvant systemic therapy is becoming more commonly used in patients with earlier stages of breast cancer. To assess tumour response to neoadjuvant chemotherapy, pathological evaluation is the gold standard. Depending on the treatment response, the pathological examination of these specimens can be quite challenging. However, a uniform approach to evaluate post-neoadjuvant-treated breast specimens has been lacking. Furthermore, there is no single universally accepted or endorsed classification system for assessing treatment response in this setting. Recent initiatives have attempted to create a standardised protocol for evaluation of post-neoadjuvant breast specimens. This review outlines the necessary information that should be collected prior to macroscopic examination of these specimens, the recommended and most pragmatic approach to tissue sampling for microscopic examination, describes the macroscopic and microscopic features of post-therapy breast specimens, summarises two commonly used systems for classifying treatment response and outlines the critical variables that should be included in the final pathology report.

Advances in digital image analysis have the potential to transform the practice of breast pathology. In the near future, a move to a digital workflow offers improvements in efficiency. Coupled with artificial intelligence (AI), digital pathology can assist pathologist interpretation, automate time-consuming tasks, and discover novel morphologic patterns. Opportunities for digital enhancements abound in breast pathology, from increasing reproducibility in grading and biomarker interpretation, to discovering features that correlate with patient outcome and treatment. Our objective is to review the most recent developments in digital pathology with clear impact to breast pathology practice. Although breast pathologists currently undertake limited adoption of digital methods, the field is rapidly evolving. Care is needed to validate emerging technologies for effective patient care.

Colorectal cancers (CRCs) undergo distinct genetic and epigenetic alterations. Expression of mutL homolog 1 (MLH1), a mismatch repair gene that corrects DNA replication errors, is lost in up to 15% of sporadic tumours due to mutation or, more commonly, due to DNA methylation of its promoter CpG island. A single nucleotide polymorphism (SNP) in the CpG island of MLH1 (MLH1-93G>A or rs1800734) is associated with CpG island hypermethylation and decreased MLH1 expression in CRC tumours. Further, in peripheral blood mononuclear cell (PBMC) DNA of both CRC cases and non-cancer controls, the variant allele of rs1800734 is associated with hypomethylation at the MLH1 shore, a region upstream of its CpG island that is less dense in CpG sites. To determine whether this genotype-epigenotype association is present in other tissue types, including colorectal tumours, we assessed DNA methylation in matched normal colorectal tissue, tumour, and PBMC DNA from 349 population-based CRC cases recruited from the Ontario Familial Colorectal Cancer Registry. Using the semi-quantitative real-time PCR-based MethyLight assay, MLH1 shore methylation was significantly higher in tumour tissue than normal colon or PBMCs (P

Background Colorectal cancers (CRCs) undergo distinct genetic and epigenetic alterations. Expression of mutL homolog 1 (MLH1), a mismatch repair gene that corrects DNA replication errors, is lost in up to 15% of sporadic tumours due to mutation or, more commonly, due to DNA methylation of its promoter CpG island. A single nucleotide polymorphism (SNP) in the CpG island of MLH1 (MLH1-93G>A or rs1800734) is associated with CpG island hypermethylation and decreased MLH1 expression in CRC tumours. Further, in peripheral blood mononuclear cell (PBMC) DNA of both CRC cases and non-cancer controls, the variant allele of rs1800734 is associated with hypomethylation at the MLH1 shore, a region upstream of its CpG island that is less dense in CpG sites. Results To determine whether this genotype-epigenotype association is present in other tissue types, including colorectal tumours, we assessed DNA methylation in matched normal colorectal tissue, tumour, and PBMC DNA from 349 population-based CRC cases recruited from the Ontario Familial Colorectal Cancer Registry. Using the semi-quantitative real-time PCR-based MethyLight assay, MLH1 shore methylation was significantly higher in tumour tissue than normal colon or PBMCs (P

Background and aims Colorectal cancer (CRC) is the second most frequent cancer in developed countries. Newfoundland has the highest incidence of CRC in Canada and the highest rate of familial CRC yet reported in the world. To determine the impact of mutations in known CRC susceptibility genes and the contribution of the known pathways to the development of hereditary CRC, an incident cohort of 750 patients with CRC (708 different families) from the Newfoundland population was studied. Methods Microsatellite instability (MSI) testing was performed on tumours, together with immunohistochemistry analysis for mismatch repair (MMR) genes. Where indicated, DNA sequencing and multiplex ligation-dependent probe amplifications of MMR genes and APC was undertaken. DNA from all patients was screened for MUTYH mutations. The presence of the BRAF variant, p.V600E, and of MLH1 promoter methylation was also tested in tumours. Results 4.6% of patients fulfilled the Amsterdam criteria (AC), and an additional 44.6% fulfilled the revised Bethesda criteria. MSI-high (MSI-H) was observed in 10.7% (n=78) of 732 tumours. In 3.6% (n=27) of patients, CRC was attributed to 12 different inherited mutations in six known CRC-related genes associated with chromosomal instability or MSI pathways. Seven patients (0.9%) carried a mutation in APC or biallelic mutations in MUTYH . Of 20 patients (2.7%) with mutations in MMR genes, 14 (70%) had one of two MSH2 founder mutations. 17 of 28 (61%) AC families did not have a genetic cause identified, of which 15 kindreds fulfilled the criteria for familial CRC type X (FCCTX). Conclusions Founder mutations accounted for only 2.1% of cases and this was insufficient to explain the high rate of familial CRC. Many of the families classified as FCCTX may have highly penetrant mutations segregating in a Mendelian-like manner. These families will be important for identifying additional CRC susceptibility loci.

ApoE single nucleotide polymorphisms (SNPs) Cys112Arg (Epsilon-4), and Arg158Cys (Epsilon-2) have been implicated in cardiovascular and Alzheimer's disease, but their role in colorectal cancer (CRC) has not been extensively studied. We investigated whether ApoE polymorphisms alone or in combination with dietary factors selectively contribute to mismatch-repair (MMR) proficient (microsatellite stable/low or MSS/L) vs deficient (microsatellite unstable or MSI-H) CRCs. We carried out a case–control study with 906 CRC cases and 911 unaffected controls to examine the associations between ApoE polymorphisms and dietary factors and assessed their contribution to MSS/L and MSI-H CRCs. We used unconditional logistic regression to evaluate the associations between ApoE SNPs, tumour MSI status, and dietary factors after adjusting for age and sex. All statistical tests were two-sided. No significant differences in ApoE genotype frequencies were observed between CRC cases and unaffected controls. We observed that increased dietary intake of total fat, saturated fat, cholesterol, and red meat was significantly associated with CRC. Among non-ApoE4 carriers, 2–4 and >4 red meat servings/week were associated with developing MSS/L CRC (OR=1.51, 95% CI 1.10–2.07 and OR=1.80, 95% CI 1.30–2.48, respectively), whereas among ApoE4 allele carriers, four or more red meat servings/week were associated with MSI-H CRC (OR=4.62, 95% CI 1.20–17.77) when compared with the controls. ApoE isoforms modulate the risk of MSI-H and MSS/L CRCs among high red meat consumers.

The most important indicator of colorectal cancer (CRC) risk is the presence of family history of the disease. Inherited genetic changes, such as single nucleotide polymorphisms, in key candidate genes may contribute to CRC risk. We investigated whether promoter polymorphisms in DNA mismatch repair (MMR) genes MSH2 and MSH6 are associated with the risk of CRC. We genotyped 929 CRC patients and 1098 control subjects from Ontario, and 467 patients and 344 controls from Newfoundland and Labrador, for two promoter polymorphisms in the MMR genes MSH2 and MSH6 using the fluorogenic 5' nuclease assay. We used unconditional logistic regression to evaluate the association between each polymorphism and CRC after adjusting for age and sex. The associations between polymorphisms and tumor clinicopathological features were evaluated with a Pearson's chi-squared test or Fisher's exact test. All statistical tests were two sided. We observed strong associations between the MSH2 -118T>C polymorphism and family history of CRC based on the Amsterdam criteria I (P = 0.005) and Amsterdam criteria I and II (P = 0.036) among cases from Ontario. This association was especially evident among female CRC patients in Ontario (for Amsterdam criteria I, and I and II combined, P = 0.003 and P = 0.0001, respectively). The MSH2 -118T>C polymorphism was associated with strong family history of CRC in Ontario patients.

Although up to 30% of patients with colorectal cancer have a positive family history of colorectal neoplasia, few colorectal cancers can be explained by mutations in high-penetrance genes. We investigated whether polymorphisms in DNA mismatch repair genes are associated with the risk of colorectal cancer.We genotyped 929 case patients and 1098 control subjects from Ontario and 430 case patients and 275 control subjects from Newfoundland and Labrador for five polymorphisms in the mismatch repair genes MLH1 and MSH2 with the fluorogenic 5' nuclease assay. Tumor microsatellite instability (MSI) was determined with a polymerase chain reaction-based method; MSI status was assigned as high (MSI-H,or = 30% unstable markers among all markers tested), low (MSI-L,30% markers unstable), or stable (MSS, no unstable markers). We used unconditional logistic regression to evaluate the association between each polymorphism and colorectal cancer after adjusting for age and sex. The associations between polymorphisms and tumor clinicopathologic features were evaluated with a Pearson's chi-square or Fisher's exact test. All statistical tests were two-sided.We observed strong associations between the MLH1 -93GA polymorphism and MSI-H tumors among case patients from Ontario (P = .001) and Newfoundland (P = .003). When compared with the control populations, homozygosity for the MLH1 -93GA variant allele was associated with MSI-H tumors among case patients in Ontario (adjusted odds ratio [OR] = 3.23, 95% confidence interval [CI] = 1.65 to 6.30) and in Newfoundland (OR = 8.88, 95% CI = 2.33 to 33.9), as was heterozygosity among case patients in Ontario (OR = 1.84, 95% CI = 1.20 to 2.83) and in Newfoundland (OR = 2.56, 95% CI = 1.14 to 5.75). Genotype frequencies were similar among case patients with MSS and MSI-L tumors and control subjects, and the majority of homozygous variant carriers had MSS tumors. Among case patients from Ontario, an association between the MLH1 -93GA polymorphism and a strong family history of colorectal cancer (for Amsterdam criteria I and II, P = .004 and P = .02, respectively) was observed.In two patient populations, the MLH1 -93GA polymorphism was associated with an increased risk of MSI-H colorectal cancer.

Wnt genes encode a large family of secreted glycoproteins with widespread roles in development and carcinogenesis. These proteins have classically been divided into canonical or non-canonical family members based on their ability to activate these distinct arms of the Wnt signalling network. While canonical Wnt ligands promote cell growth and proliferation by controlling cytoplasmic degradation and subsequently nuclear accumulation of β-catenin. Non-canonical Wnt ligands govern cell motility and polarity through the Wnt/calcium and/or planar cell polarity pathways, which act through a variety of intracellular mediators including calmodulin-dependent protein kinase II, protein kinase C, and c-Jun N-terminal kinase. Aberrant hyperactivation of the canonical Wnt pathway is a critical tumour-initiating event in the majority of colorectal cancers (CRCs); however, the status of non-canonical Wnt activity in cancer and its roles in carcinogenesis are poorly understood. These functions have mainly been examined through Wnt5a, the quintessential non-canonical Wnt ligand. Wnt5a is ubiquitously expressed in normal tissues (Ying et al, 2008), but is often dysregulated in tumours (Wang et al, 2007, 2010) and has been controversially implicated in both tumour suppression and oncogenesis in addition to being recognised asa marker of both favourable and poor outcome in primary cancer. Studies supporting the former have demonstrated that Wnt5a inhibits invasion and proliferation in thyroid cancer (Kremenevskaja et al, 2005), low expression or loss of expression is prognostically unfavourable in breast cancer (Jonsson et al, 2002; Leris et al, 2005), and hemizygous mice develop lymphomas (Liang et al, 2003). On the other hand, oncogenic roles have been identified in prostate cancer, where Wnt5a promotes cell motility (Wang et al, 2010; Yamamoto et al, 2010), melanoma, where expression associates with tumour invasion (Weeraratna et al, 2002), and hepatocellular carcinoma, where expression is associated with poorly differentiated and invasive cell lines (Yuzugullu et al, 2009). In CRC, Wnt5a expression has been associated with favourable outcome in early stage tumours (Dejmek et al, 2005), while loss of Wnt5a expression is a hallmark of CRC-derived liver metastases (Ki et al, 2007). Collectively, these seemingly conflicting findings underscore the poorly understood functional and prognostic roles of Wnt5a in cancer. Recent studies in leukaemia (Martin et al, 2010) and CRC (Ying et al, 2008; Ahn et al, 2011) have shown that a major mechanism for Wnt5a downregulation is promoter hypermethylation. Aberrant promoter hypermethylation is a hallmark of CRC that has a pivotal role in defining tumour subtypes; somatic methylation of the MLH1 promoter causes the majority of sporadic CRC tumours with microsatellite instability (MSI), and widespread CpG island methylation defines the CpG island methylator phenotype (CIMP). Importantly, these methylation-dependent subtypes stratify disease outcome; MSI tumours are clinically favourable, but respond poorly to 5-fluorouracil-based chemotherapy (Ribic et al, 2003; Carethers et al, 2004), whereas CIMP tumours have complex associations with patient outcome that may depend on MSI and/or BRAF V600E status (Kim et al, 2009; Ogino et al, 2009; Dahlin et al, 2010). Despite the importance of these epigenetic modifications in defining these CRC subtypes, the specific methylation events that contribute to subtype-specific outcome has been poorly characterised. Our previous study suggested that microsatellite stable tumours may exhibit preferential gain of non-canonical Wnt signalling through methylation of Wnt antagonists (Rawson et al, 2011). Given the importance of Wnt5a in mediating non-canonical Wnt activity and its emerging role in carcinogenesis and prognosis, we investigated the promoter methylation status of this gene in a large cohort of MSI-stratified CRCs from two distinct Canadian populations. We examined associations to patient clinicopathological features focusing on tumour subtype and patient survival in hopes that these relationships would provide further insight into subtype-specific epigenetic mediation of non-canonical Wnt signalling and would help define the prognostic role of Wnt5a methylation in CRC.

Aberrant activation of canonical Wnt signaling is a hallmark event in colorectal carcinogenesis. The Dickkopf-1 (DKK1) and Secreted Frizzled Related Protein 1 (SFRP1) genes encode extracellular inhibitors of Wnt signaling that are frequently silenced by promoter hypermethylation in colorectal cancer (CRC). These methylation events have been identified as prognostic markers of patient outcome and tumor subtype in several cancers but similar roles in CRC have not been comprehensively examined. In CRC, the microsatellite instability (MSI) subtype associates with favorable disease outcome but the molecular events that are responsible remain poorly understood. Consequently, we quantified promoter methylation status of the Wnt antagonist genes DKK1 and SFRP1 in a large population-based cohort of CRCs from Ontario (n = 549) and Newfoundland (n = 696) stratified by MSI status. We examined the association between methylation status and clinicopathological features including tumor MSI status and patient survival. DKK1 and SFRP1 were methylated in 13 and 95% of CRCs, respectively. In Ontario, DKK1 methylation was strongly associated with MSI tumors after adjustment for age, sex and tumor location [odds ratio (OR) = 13.7, 95% confidence interval (CI) = 7.8-24.2, P < 0.001]. Conversely, SFRP1 methylation was inversely associated with MSI tumors after these adjustments (OR = 0.3, 95% CI = 0.1-0.9, P = 0.009). Similar results were obtained in Newfoundland. There were no independent associations with recurrence-free survival. This is the first large study to identify associations between Wnt antagonist promoter hypermethylation and CRC MSI subtype. These events provide insight into subtype-specific epigenetic mediation of Wnt signaling in CRC.

Background: We previously identified an association between a mismatch repair gene, MLH1, promoter SNP (rs1800734) and microsatellite unstable (MSI-H) colorectal cancers (CRCs) in two samples. The current study expanded on this finding as we explored the genetic basis of DNA methylation in this region of chromosome 3. We hypothesized that specific polymorphisms in the MLH1 gene region predispose it to DNA methylation, resulting in the loss of MLH1 gene expression, mismatch-repair function, and consequently to genome-wide microsatellite instability. Methodology/Principal Findings: We first tested our hypothesis in one sample from Ontario (901 cases, 1,097 controls) and replicated major findings in two additional samples from Newfoundland and Labrador (479 cases, 336 controls) and from Seattle (591 cases, 629 controls). Logistic regression was used to test for association between SNPs in the region of MLH1 and CRC, MSI-H CRC, MLH1 gene expression in CRC, and DNA methylation in CRC. The association between rs1800734 and MSI-H CRCs, previously reported in Ontario and Newfoundland, was replicated in the Seattle sample. Two additional SNPs, in strong linkage disequilibrium with rs1800734, showed strong associations with MLH1 promoter methylation, loss of MLH1 protein, and MSI-H CRC in all three samples. The logistic regression model of MSI-H CRC that included MLH1-promotermethylation status and MLH1 immunohisotchemistry status fit most parsimoniously in all three samples combined. When rs1800734 was added to this model, its effect was not statistically significant (P-value =0.72 vs. 2.3610 24 when the SNP was examined alone). Conclusions/Significance: The observed association of rs1800734 with MSI-H CRC occurs through its effect on the MLH1 promoter methylation, MLH1 IHC deficiency, or both.

Defective mismatch repair leads to the microsatellite instability (MSI) phenotype of colorectal cancer (CRC). We previously showed that the MLH1-93GA promoter polymorphism is strongly associated with MSI tumours, suggesting a modifier role for this polymorphism in CRC. The MLH1 promoter is bi-directional with the EPM2AIP1 gene located on the antisense strand. In order to evaluate the functional effects of this polymorphism, we transfected a panel of CRC, endometrial cancer and non-tumourigenic cell lines with MLH1 luciferase promoter constructs. We used constructs in reverse orientation to assess the effect of this polymorphism on EPM2AIP1. The luciferase activities were compared using a two-sided Student's t-test. Electrophoretic mobility shift assays (EMSAs) were used to evaluate whether differential protein binding was responsible for the differences in promoter activity. We observed a higher level of activity with the -93G allele in all the cell lines observed; including the CRC cell line, HCT116 (P=0.002), the endometrial cancer cell line, HEC-1-A (P0.001) and the normal colonic cell line, CCD-841-CoTr (P=0.002). This polymorphism also affected EPM2AIP1 transcription with the -93A allele demonstrating higher promoter activity in the HCT116 (P=0.007) and HEC-1-A (P=0.004) cells. The EMSA results suggest that this polymorphism alters the affinity of nuclear factors that bind to this region. Our findings indicate that the -93GA polymorphism modifies the efficiency of MLH1/EPM2AIP1 transcription.

Gene silencing via CpG island hypermethylation contributes to colorectal cancer (CRC). We previously demonstrated that genetic variants (single nucleotide polymorphisms, or SNPs) in the MLH1 gene promoter region are associated with MLH1 CpG island hypermethylation, MLH1 protein loss, and DNA mismatch repair deficiency in tumors. Recently, CpG-rich “shore” regions were identified flanking many CpG islands which exhibiting distinct methylation profiles among different tissues and in normal versus tumor states. To explore the role of MLH1 CpG island shore methylation, we performed global methylation profiling (Illumina 450K microarrays) to investigate DNA methylation in peripheral blood cell (PBC) DNA of over 800 CRC cases and 800 controls (Savio et al., 2012). We found that MLH1 CpG island shore hypomethylation occurred in PBC DNA of both cases and controls. Intriguingly, individuals carrying variant alleles of the MLH1 promoter SNP rs1800734 incur significant hypomethylation at the MLH1 CpG island shore, located 1.5 kb upstream irrespective of disease status. Based on these observations we sought to determine whether these SNP-associated epigenetic changes were apparent in other tissues, including CRC and normal colonic tissue. To address this, we investigated MLH1 CpG island shore methylation in matched PBC, normal colon, and tumor DNA of 349 CRC cases using the real-time PCR-based technique, MethyLight. Methylation was significantly lower in PBC DNA of CRC cases with variant SNP genotypes (both heterozygous and homozygous variant carriers) and this association was also observed in normal colonic DNA. The association between genotype and epigenotype was lost in tumor DNA, as no differences in methylation were seen among different genotypes. Hypermethylation in tumor DNA compared to normal tissues (PBC and colon) was also observed. This hypermethylation and lack of association between SNP genotype and shore methylation in tumors may indicate an epigenetic switch occurring. Bisulfite sequencing of DNA from fresh frozen tumors and paired normal colonic mucosa have recapitulated these results, with hypomethylation incurred in variant SNP carriers. Hypermethylation in tumor compared to normal DNA was also observed. Bisulfite sequencing of four CRC cell lines has also revealed SNP-dependent hypermethylation at the MLH1 CpG island shore. Taken together, we have integrated methylation data from a microarray platform, sensitive real-time PCR, and bisulfite sequencing for a comprehensive and systematic analysis of DNA methylation at the CpG island shore of MLH1 in multiple tissues of CRC cases, controls, and cell lines. These results indicate that static genetic variants can modulate dynamic epigenetic regulation at the MLH1 gene region, and may play a role in colorectal tumorigenesis. Citation Format: Andrea J. Savio, Mathieu Lemire, Miralem Mrkonjic, Steven Gallinger, Brent W. Zanke, Thomas J. Hudson, Bharati Bapat. Novel insights into the genetic and epigenetic regulation of the MLH1 CpG island and shore in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1058. doi:10.1158/1538-7445.AM2015-1058

Background: Aberrant Wnt pathway activation is a vital carcinogenic event in colorectal cancer (CRC). DKK1 and SFRP1 encode extracellular inhibitors of canonical and canonical/non-canonical Wnt signaling, respectively, that are frequently silenced by promoter hypermethylation in CRC. Despite their known tumor-suppressive roles, few studies have systematically examined the prognostic/predictive significance of methylation in these genes in tumor development. Using a population-based genetic epidemiological approach, we investigated the methylation status of DKK1 and SFRP1 in a large cohort of primary CRCs and correlations to patient clinicopathological data. Methods: As part of a Canadian interdisciplinary initiative to study the genetic and environmental determinants of CRC, we accrued a large number of primary colorectal carcinoma cases diagnosed in the province of Ontario, representative of a heterogeneous population (n = 558), and in the province of Newfoundland, representative of a founder population (n = 650). We examined the methylation status of DKK1 and SFRP1 gene promoters in colorectal tumors and matched normal colon tissues using MethyLight assay, a semi-quantitative methylation detection technique. We examined correlations between methylation levels and frequency, and a comprehensive array of patient clinicopathological features such as: age, sex, tumor stage, grade, tumor MSI subtype, and clinical outcome. Statistical analysis was performed using 2-tailed Fisher's exact test, SPSS v16. Results: Respective DKK1 and SFRP1 methylation frequencies were similar in Ontario (13%, 95%) and Newfoundland (14%, 94%). Methylation was highly tumor specific. DKK1 methylation was a strong predictor of the microsatellite instability (MSI) tumor subtype in Ontario (OR=13.7 [7.8, 24.2], p < 0.001) and in Newfoundland (OR=3.9 [2.8, 5.6] p < 0.001). SFRP1 methylation was a predictor against MSI tumors in Ontario (OR=0.25 [0.15, 0.52], p < 0.001). Conclusion: Our study highlights the prognostic role of DKK1 and SFRP1 methylation related to CRC tumor subtype and suggests a novel distinction between the relative involvements of different Wnt pathways in microsatellite stable vs. unstable CRC. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B28