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Background: MLH1 epimutation is characterised by constitutional monoallelic MLH1 promoter hypermethylation, which can cause colorectal cancer (CRC). Tumour molecular profiles of MLH1 epimutation CRCs were used to classify germline MLH1 promoter variants of uncertain significance and MLH1 methylated early-onset CRCs (EOCRCs). Genome-wide DNA methylation and somatic mutational profiles of tumours from two germline MLH1: c.-11C > T and one MLH1: c.-[28A > G; 7C > T] carriers and three MLH1 methylated EOCRCs (< 45 years) were compared with 38 reference CRCs. Methylation-sensitive droplet digital PCR (ddPCR) was used to detect mosaic MLH1 methylation in blood, normal mucosa and buccal DNA. Results: Genome-wide methylation-based Consensus Clustering identified four clusters where the tumour methylation profiles of germline MLH1: c.-11C > T carriers and MLH1 methylated EOCRCs clustered with the constitutional MLH1 epimutation CRCs but not with the sporadic MLH1 methylated CRCs. Furthermore, monoallelic MLH1 methylation and APC promoter hypermethylation in tumour were observed in both MLH1 epimutation and germline MLH1: c.-11C > T carriers and MLH1 methylated EOCRCs. Mosaic constitutional MLH1 methylation in MLH1: c.-11C > T carriers and 1 of 3 MLH1 methylated EOCRCs was identified by methylation-sensitive ddPCR. Conclusions: Mosaic MLH1 epimutation underlies the CRC aetiology in MLH1: c.-11C > T germline carriers and a subset of MLH1 methylated EOCRCs. Tumour profiling and ultra-sensitive ddPCR methylation testing can be used to identify mosaic MLH1 epimutation carriers.

Additional file 1: Table S1. Table displaying optimal cut-offs for the six tumor features determined previously (Walker et al. 2023) in the additive feature combination approach. Table S2. SLS tumors (n=13) that showed discordant MMR IHC findings between clinical diagnostic testing before study entry and testing completed internally during this study and the change in their MMR status and/or pattern of MMR protein loss. Table S3. The concordance between the final MMR IHC result and the predicted dMMR status from the additive feature combination approach overall and by tumor type. Table S4. The tumor MLH1 methylation testing completed for SLS tumors prior to entering the study showing either negative, inconclusive, or not tested results and the subsequent MLH1 methylation testing results from internal testing using MethyLight and MS-HRM assays highlighting the positive MLH1 methylation results found by this study. Table S5. Presentation of germline pathogenic variants and variants of uncertain clinical significance (VUS) identified in the MMR, MUTYH and POLE genes. Table S6. Summary of the clinicopathological features for the double somatic MMR mutation (dMMR-DS) tumors overall and by tumor type. Figure S1. Bar plots presenting the results from the additive tumor feature combination approach to assess the MMR status in the double somatic mutation cohort for A) all tumors combined and separated by B) CRC, C) EC and D) SST tissue types. Figure S2. Bar plot presenting the prevalence of pathogenic/likely pathogenic somatic mutations (including loss of heterozygosity, LOH) by subtype for the study cohort. Figure S3. Pie graphs displaying the frequency of the mutation combination type (two single somatic mutations versus a single somatic mutation with loss of heterozygosity (LOH)) as well as the type of mutation A) overall and B) separated by tissue type. Figure S4. Bar graphs presenting the site distribution in the double somatic mutation cohort across all CRCs and SSTs. Figure S5. Boxplots presenting the site distribution in the double somatic mutation cohort across all A) CRCs and B) SSTs. Significant (< 0.05) p-values are indicated for pairwise (t-test) and multigroup comparisons (Anova). Figure S6. Scatter plots presenting the PREMM5 score distribution in the test cohort for A) all tumors combined and separated by B) CRC, C) EC and D) SST tissue types. Figure S7. The distribution of tumor values for each of the six features that are included in the additive feature combination approach for determining tumor dMMR status grouped by molecular subtype and by combining sporadic dMMR groups dMMR-DS and dMMR-MLH1me into a “sporadic combined” group.

Additional file 1: Supplementary Table 1. Female Sub-analysis investigating the association between SPS and characteristics/ lifestyle factors. Supplementary Table 2. Female Sub-analysis investigating the association between WHO criteria I and characteristics/ lifestyle factors. Supplementary Table 3. Female Sub-analysis investigating the association between WHO criteria III and characteristics/ lifestyle factors. Supplementary Table 4. Female Sub-analysis investigating the association between both WHO criteria I and III and characteristics/ lifestyle factors. Supplementary Table 5. Female Sub-analysis investigating the association between CRC and characteristics/ lifestyle factors. Supplementary Table 6. Female Sub-analysis investigating the association between SPS patients with no CRC and characteristics/ lifestyle factors

BACKGROUND: Current clinical practice guidelines for carriers of pathogenic variants of DNA mismatch repair genes (Lynch syndrome) are based on the average age-specific cumulative risk (penetrance) of colorectal cancer for all carriers of pathogenic variants in the same gene. We aimed to estimate how much penetrance varies between carriers of pathogenic variants in the same gene by sex and continent of residence of the carrier. METHODS: We studied 79,809 relatives from 5,255 families, of at least three relatives, in which at least one was a confirmed carrier of a pathogenic or likely pathogenic variant in a mismatch repair gene (1,829 MLH1, 2,179 MSH2, 798 MSH6, 449 PMS2), recruited in 15 countries from North America, Europe and Australasia by the collaborative centres of the International Mismatch Repair Consortium. We used modified segregation analysis conditioned on ascertainment to estimate the average penetrance and modelled unmeasured polygenic factors to estimate the variation in penetrance of colorectal cancer. The existence of familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers was tested using a Wald p-value for the null hypothesis that the polygenic standard deviation is zero. FINDINGS: There was strong evidence of the existence of familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers (pT variant. The variation was more prominent for MLH1 and MSH2 variant carriers; depending on gene, sex, and continent, with 7–56% of carriers having a risk of colorectal cancer to age 80 of less than 20%, and 9–44% having a risk of more than 80%, while only 10–19% had a risk of 40–60%. INTERPRETATION: Our study findings highlight the important role of risk modifiers, which could lead to personalised risk assessment for precision prevention and early detection of colorectal cancer for Lynch syndrome.

The influence of lifestyle factors on survival following a diagnosis of colorectal cancer (CRC) is not well established. We examined associations between lifestyle factors measured before diagnosis and CRC survival. The Melbourne Collaborative Cohort Study collected data on alcohol intake, cigarette smoking and physical activity, and body measurements at baseline (1990-1994) and wave 2 (2003-2007). We included participants diagnosed to 31 August 2015 with incident stages I-III CRC within 10-years post exposure assessment. Information on tumor characteristics and vital status was obtained. Tumor DNA was tested for microsatellite instability (MSI) and somatic mutations in oncogenes BRAF (V600E) and KRAS. We estimated hazard ratios (HRs) for associations between lifestyle factors and overall and CRC-specific mortality using Cox regression. Of 724 eligible CRC cases, 339 died (170 from CRC) during follow-up (average 9.0 years). Exercise (non-occupational/leisure-time) was associated with higher CRC-specific survival for stage II (HR = 0.25, 95% CI: 0.10-0.60) but not stages I/III disease (p for interaction = 0.01), and possibly for colon and KRAS wild-type tumors. Waist circumference was inversely associated with CRC-specific survival (HR = 1.25 per 10 cm increment, 95% CI: 1.08-1.44), independent of stage, anatomic site and tumor molecular status. Cigarette smoking was associated with lower overall survival, with suggestive evidence of worse survival for BRAF mutated CRC, but not with CRC-specific survival. Alcohol intake was not associated with survival. Survival did not differ by MSI status. We have identified pre-diagnostic predictors of survival following CRC that may have clinical and public health relevance.

Tumor testing of colorectal cancers (CRC) for mismatch repair (MMR) deficiency is an effective approach to identify carriers of germline MMR gene mutation (Lynch syndrome). The aim of this study was to identify MMR gene mutation carriers in two cohorts of population-based CRC utilizing a combination of tumor and germline testing approaches.Colorectal cancers from 813 patients diagnosed with CRC 60 years of age from the Australasian Colorectal Cancer Family Registry (ACCFR) and from 826 patients from the Melbourne Collaborative Cohort Study (MCCS) were tested for MMR protein expression using immunohistochemistry, microsatellite instability (MSI), BRAFOf the 813 ACCFR probands, 90 probands demonstrated tumor MMR deficiency (11.1%), and 42 had a MMR gene germline mutation (5.2%). For the MCCS, MMR deficiency was identified in the tumors of 103 probands (12.5%) and seven had a germline mutation (0.8%). All the mutation carriers were diagnosed prior to 70 years of age. Probands with a MMR-deficient CRC without MLH1 methylation and a gene mutation were considered Lynch-like and comprised 41.1% and 25.2% of the MMR-deficient CRCs for the ACCFR and MCCS, respectively.Identification of MMR gene mutation carriers in Australian CRC-affected patients is optimized by immunohistochemistry screening of CRC diagnosed before 70 years of age. A significant proportion of MMR-deficient CRCs will have unknown etiology (Lynch-like) proving problematic for clinical management.

The CpG Island Methylator Phenotype (CIMP) is a major molecular pathway in colorectal cancer (CRC). Approximately 25% to 60% of CIMP tumors are microsatellite unstable (MSI-H) due to DNA hypermethylation of the MLH1 gene promoter. Our aim was to determine if the distributions of clinicopathologic factors in CIMP-positive tumors with MLH1 DNA methylation differed from those in CIMP-positive tumors without DNA methylation of MLH1.

Germline mutations in the DNA base excision repair gene MUTYH are known to increase a carrier’s risk of colorectal cancer. However, the risks of other (extracolonic) cancers for MUTYH mutation carriers are not well defined. We identified 266 probands (91% Caucasians) with a MUTYH mutation (41 biallelic and 225 monoallelic) from the Colon Cancer Family Registry. Mutation status, sex, age, and histories of cancer from their 1,903 first- and 3,255 second-degree relatives, were analysed using modified segregation analysis conditioned on the ascertainment criteria. Compared with incidences for the general population, hazard ratios (HRs) (95% confidence intervals [CIs]) for biallelic MUTYH mutation carriers were: urinary bladder cancer, 19(3.7–97); and ovarian cancer, 17(2.4–115). The HRs (95%CI) for monoallelic MUTYH mutation carriers were: gastric cancer, 9.3(6.7–13); hepatobiliary cancer, 4.5(2.7–7.5); endometrial cancer, 2.1(1.1–3.9); and breast cancer, 1.4(1.0–2.0). There was no evidence for an increased risk of cancers at the other sites examined (brain, pancreas, kidney or prostate). Based on the USA population incidences, the estimated cumulative risks (95%CI) to age 70 years for biallelic mutation carriers were: bladder cancer, 25%(5%–77%) for males and 8%(2%–33%) for females; and ovarian cancer, 14%(2%–65%). The cumulative risks (95%CI) for monoallelic mutation carriers were: gastric cancer, 5%(4%–7%) for males and 2.3%(1.7%–3.3%) for females; hepatobiliary cancer, 3%(2%–5%) for males and 1.4%(0.8%–2.3%) for females; endometrial cancer, 3%(2%–6%); and breast cancer 11%(8%–16%). These unbiased estimates of both relative and absolute risks of extracolonic cancers for people, mostly Caucasians, with MUTYH mutations will be important for their clinical management.

Daniel D Buchanan,1,2 Christophe Rosty,1,3,4 Mark Clendenning,1 Amanda B Spurdle,5 Aung Ko Win2 1Oncogenomics Group, Genetic Epidemiology Laboratory, Department of Pathology, The University of Melbourne, Parkville, VIC, Australia; 2Centre for Epidemiology and Biostatistics, Melbourne School of Population and Global Health, The University of Melbourne, Parkville, VIC, Australia; 3Envoi Specialist Pathologists, Herston, QLD, Australia; 4School of Medicine, University of Queensland, Herston, QLD, Australia; 5Molecular Cancer Epidemiology Laboratory, Genetics and Computational Biology Division, QIMR Berghofer Medical Research Institute, Herston, QLD, AustraliaAbstract: Carriers of a germline mutation in one of the DNA mismatch repair (MMR) genes have a high risk of developing numerous different cancers, predominantly colorectal cancer and endometrial cancer (known as Lynch syndrome). MMR gene mutation carriers develop tumors with MMR deficiency identified by tumor microsatellite instability or immunohistochemical loss of MMR protein expression. Tumor MMR deficiency is used to identify individuals most likely to carry an MMR gene mutation. However, MMR deficiency can also result from somatic inactivation, most commonly methylation of the MLH1 gene promoter. As tumor MMR testing of all incident colorectal and endometrial cancers (universal screening) is becoming increasingly adopted, a growing clinical problem is emerging for individuals who have tumors that show MMR deficiency who are subsequently found not to carry an MMR gene mutation after genetic testing using the current diagnostic approaches (Sanger sequencing and multiplex ligation-dependent probe amplification) and who also show no evidence of MLH1 methylation. The inability to determine the underlying cause of tumor MMR deficiency in these "Lynch-like" or "suspected Lynch syndrome" cases has significant implications on the clinical management of these individuals and their relatives. When the data from published studies are combined, 59% (95% confidence interval [CI]: 55% to 64%) of colorectal cancers and 52% (95% CI: 41% to 62%) of endometrial cancers with MMR deficiency were identified as suspected Lynch syndrome. Recent studies estimated that colorectal cancer risk for relatives of suspected Lynch syndrome cases is lower than for relatives of those with MMR gene mutations, but higher than for relatives of those with tumor MMR deficiency resulting from methylation of the MLH1 gene promoter. The cause of tumor MMR deficiency in suspected Lynch syndrome cases is likely due to either unidentified germline MMR gene mutations, somatic cell mosaicism, or biallelic somatic inactivation. Determining the underlying cause of tumor MMR deficiency in suspected Lynch syndrome cases is likely to reshape the current triaging schemes used to identify germline MMR gene mutations in cancer-affected individuals and their relatives.Keywords: Lynch syndrome, cancer risk, screening, somatic mutation, germline mutation

We studied 2332 individuals with monoallelic mutations in MUTYH among 9504 relatives of 264 colorectal cancer (CRC) cases with a MUTYH mutation. We estimated CRC risks, through 70 y of age, of 7.2% for male carriers of monoallelic mutations (95% confidence interval [CI], 4.6%–11.3%) and 5.6% for female carriers of monoallelic mutations (95% CI, 3.6%–8.8%), irrespective of family history. For monoallelic MUTYH mutation carriers with a first-degree relative with CRC, diagnosed by 50 y of age who does not have the MUTYH mutation, risks of CRC were 12.5% for men and (95% CI, 8.6%–17.7%) and 10% for women (95% CI, 6.7%–14.4%). Risks of CRC for carriers of monoallelic mutations in MUTYH with a first-degree relative with CRC are sufficiently high to warrant more intensive screening than for the general population.

The question of whether prostate cancer is part of the Lynch syndrome spectrum of tumors is unresolved. We investigated the mismatch repair (MMR) status and pathologic features of prostate cancers diagnosed in MMR gene mutation carriers. Prostate cancers (mean age at diagnosis = 62 ± SD = 8 years) from 32 MMR mutation carriers (23 MSH2, 5 MLH1 and 4 MSH6) enrolled in the Australasian, Mayo Clinic and Ontario sites of the Colon Cancer Family Registry were examined for clinico-pathologic features and MMR-deficiency (immunohistochemical loss of MMR protein expression and high levels of microsatellite instability; MSI-H). Tumor MMR-deficiency was observed for 22 cases [69 %; 95 % confidence interval (CI) 50–83 %], with the highest prevalence of MMR-deficiency in tumors from MSH2 mutation carriers (19/23, 83 %) compared with MLH1 and MSH6 carriers combined (3/9, 33 %; p = 0.01). MMR-deficient tumors had increased levels of tumor infiltrating lymphocytes compared with tumors without MMR-deficiency (p = 0.04). Under the assumption that tumour MMR-deficiency occurred only because the cancer was caused by the germline mutation, mutation carriers are at 3.2-fold (95 % CI 2.0–6.3) increased risk of prostate cancer, and when assessed by gene, the relative risk was greatest for MSH2 carriers (5.8, 95 % CI 2.6–20.9). Prostate cancer was the first or only diagnosed tumor in 37 % of carriers. MMR gene mutation carriers have at least a twofold or greater increased risk of developing MMR-deficient prostate cancer where the risk is highest for MSH2 mutation carriers. MMR IHC screening of prostate cancers will aid in identifying MMR gene mutation carriers.

Changes in the methylation levels of DNA from white blood cells (WBCs) are putatively associated with an elevated risk for several cancers. The aim of this study was to investigate the association between colorectal cancer (CRC) and the methylation status of three DNA repetitive elements in DNA from peripheral blood. WBC DNA from 539 CRC cases diagnosed before 60 years of age and 242 sex and age frequency-matched healthy controls from the Australasian Colorectal Cancer Family Registry were assessed for methylation across DNA repetitive elements Alu, LINE-1 and Sat2 using MethyLight. The percentage of methylated reference (PMR) of cases and controls was calculated for each marker. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using multivariable logistic regression adjusted for potential confounders. CRC cases demonstrated a significantly higher median PMR for LINE-1 (p < 0.001), Sat2 (p < 0.001) and Alu repeats (p = 0.02) when compared with controls. For each of the DNA repetitive elements, individuals with PMR values in the highest quartile were significantly more likely to have CRC compared with those in the lowest quartile (LINE-1 OR = 2.34, 95%CI = 1.48–3.70; p < 0.001, Alu OR = 1.83, 95%CI = 1.17–2.86; p = 0.01, Sat2 OR = 1.72, 95%CI = 1.10–2.71; p = 0.02). When comparing the OR for the PMR of each marker across subgroups of CRC, only the Alu marker showed a significant difference in the 5-fluoruracil treated and nodal involvement subgroups (both p = 0.002). This association between increasing methylation levels of three DNA repetitive elements in WBC DNA and early-onset CRC is novel and may represent a potential epigenetic biomarker for early CRC detection.

Lynch syndrome is an autosomal dominantly inherited disorder caused by germline mutations in DNA mismatch repair (MMR) genes. Previous studies have shown that MMR gene mutation carriers are at increased risk of colorectal, endometrial, and several other cancers following an initial diagnosis of colorectal cancer. We estimated cancer risks following an endometrial cancer diagnosis for women carrying MMR gene mutations.

Serrated polyposis has only recently been accepted as a condition which carries an increased personal and familial risk of colorectal cancer. Described over four decades ago, it remains one of the most underrecognized and poorly understood of all the intestinal polyposes. With a variety of phenotypic presentations, it is likely that serrated polyposis represents a group of diseases rather than a single entity. Further, neoplastic progression in serrated polyposis may be associated with premature aging in the normal mucosa, typified by widespread gene promoter hypermethylation. From this epigenetically altered field, arise diverse polyps and cancers which show a range of molecular features. Despite a high serrated polyp count, only one-third of colorectal cancers demonstrate a BRAF V600E mutation, the molecular hallmark of the canonical serrated pathway, suggesting that though multiple serrated polyps act as a marker of an abnormal mucosa, the majority of CRC in these patients arise within lesions other than BRAF-mutated serrated polyps.

Immunohistochemistry for DNA mismatch repair proteins is used to screen for Lynch syndrome in individuals with colorectal carcinoma (CRC). Although solitary loss of PMS2 expression is indicative of carrying a germline mutation in PMS2, previous studies reported MLH1 mutation in some cases. We determined the prevalence of MLH1 germline mutations in a large cohort of individuals with a CRC demonstrating solitary loss of PMS2 expression.

Background: Colorectal cancer (CRC) diagnosed at

The average age at diagnosis for colorectal cancer (CRC) in Australia is 69, and the age-specific incidence rises rapidly after age 50 years. The incidence has stabilized or is declining in older age groups in Australia during recent decades, possibly related to the increased uptake of screening and high-risk surveillance. In the same time frame, a rising incidence of CRC in younger adults has been well-documented in the United States. This rise in incidence in the young has not been reported from other countries that share long-term exposure to westernised urban lifestyles. Using data from the Australian Institute of Health and Welfare, we examined trends in national incidence rates for CRC under age 50 years and observed that rates in people under age 40 years have been rising for the last two decades.We further performed a review of the literature regarding CRC in young adults to outline the extent of current understanding, explore potential risk factors such as obesity, alcohol, and sedentary lifestyles, and to identify the questions remaining to be addressed. Although absolute numbers might not justify a population screening approach, the dispersal of young adults with CRC across the primary health-care system decreases probability of their recognition. Patient and physician awareness, aided by stool and emerging blood-screening tests and risk profiling tools, have the potential to aid in identification of those young adults who would most benefit from a colonoscopy through early detection of CRCs or by removal of advanced polyps. Refereed/Peer-reviewed