Simplified and more sensitive criteria for identifying individuals with pathogenic CDH1 variants.

BACKGROUND: Hereditary diffuse gastric cancer (HDGC) is an autosomal-dominant syndrome most often caused by pathogenic variants in CDH1. The International Gastric Cancer Linkage Consortium (IGCLC) recently updated its criteria for genetic testing. The purpose of this study was to estimate the sensitivity of IGCLC's 2020 criteria for identifying carriers of CDH1 pathogenic variants and to formulate a new set of criteria that is simpler and more sensitive. METHODS: Medical histories of 112 CDH1 mutation carriers, identified predominantly by multigene panel testing, and their 649 family members were reviewed. The percentage of subjects fulfilling the IGCLC 2015 and 2020 criteria was calculated, once without making any assumptions about unavailable pathology, and once assuming gastric cancer to be diffuse when pathology was unavailable. For comparison, we calculated the percentage of subjects who fulfilled our proposed criteria. RESULTS: When making no assumptions about missing pathology, a small (19%) and equal percentage of CDH1 mutation carriers fulfilled the IGCLC 2015 and 2020 criteria. When assuming unspecified gastric cancer to be diffuse, 45 out of 112 (40%) subjects met the 2015 criteria and 53 out of 112 (47%) met the 2020 criteria. Eighty-seven per cent (97/112) fulfilled our proposed criteria. CONCLUSION: In consecutive cases, mostly unselected for clinical criteria of HDGC, the IGCLC 2020 criteria are, at best, marginally more sensitive than previous iterations, but they are also more cumbersome. Unavailable cancer pathology reports are a real-world obstacle to their proper application. Our proposed Yale criteria both address this issue and offer significantly greater sensitivity than the IGCLC 2020 criteria.

Systems approach to enhance Lynch syndrome diagnosis through tumour testing.

BACKGROUND: Guidelines recommend universal mismatch repair (MMR) tumour testing of colorectal adenocarcinomas (CRCs) to screen for Lynch syndrome (LS). However, its implementation remains disjointed and referral for genetic testing dismal, particularly among minorities. We aimed to increase referral, cancer genetic testing and eventually LS diagnosis by developing the CLEAR LS (Closed Loop Enhanced Assessment and Referral for Lynch Syndrome) intervention, a systems approach which in the second phase was automated. METHODS: This is a cohort study of all patients diagnosed with CRC at an academic centre between 1 January 2012, when implementation of universal CRC testing began, and 31 January 2021. The original cohort spanned through 31 May 2015. Tumour testing included MMR immunohistochemistry, followed by BRAF V600E/MLH1 promoter methylation testing when indicated. The intervention included a manual phase (1 June 2015 through 31 July 2018), which systematised pathology screening and cancer genetics (CG) referral mechanisms, and an automated phase (1 August 2018 through 31 January 2021) using computer programming. RESULTS: A total of 249/1541 CRC (17.38%) had MMR loss of expression and 129 (8.37%) qualified for CG evaluation. Referral was 27.58% in the original cohort and 92.1% in the intervention (p<0.001). Patients seen by CG among referred were 27.58% in the original cohort and 74.3% in the intervention (p two-sided<0.001). The distribution of race/ethnicity among patients qualifying and referred for CG evaluation was not significantly different across cohorts. LS diagnosis increased from 0.56% (original cohort) to 1.43% (intervention). Cost per new diagnosis of LS decreased from US$173 675 to $87 960 from original cohort to intervention. CONCLUSION: Implementation of systematic case identification and referral support mechanisms significantly increased the proportion of patients undergoing genetic testing and doubled the percentage of patients diagnosed with LS with no referral differences across racial/ethnic groups.

Mutational signature profiling classifies subtypes of clinically different mismatch-repair-deficient tumours with a differential immunogenic response potential.

BACKGROUND: Mismatch repair (MMR) deficiency is the hallmark of tumours from Lynch syndrome (LS), sporadic MLH1 hypermethylated and Lynch-like syndrome (LLS), but there is a lack of understanding of the variability in their mutational profiles based on clinical phenotypes. The aim of this study was to perform a molecular characterisation to identify novel features that can impact tumour behaviour and clinical management. METHODS: We tested 105 MMR-deficient colorectal cancer tumours (25 LS, 35 LLS and 45 sporadic) for global exome microsatellite instability, cancer mutational signatures, mutational spectrum and neoepitope load. RESULTS: Fifty-three percent of tumours showed high contribution of MMR-deficient mutational signatures, high level of global exome microsatellite instability, loss of MLH1/PMS2 protein expression and included sporadic tumours. Thirty-one percent of tumours showed weaker features of MMR deficiency, 62% lost MSH2/MSH6 expression and included 60% of LS and 44% of LLS tumours. Remarkably, 9% of all tumours lacked global exome microsatellite instability. Lastly, HLA-B07:02 could be triggering the neoantigen presentation in tumours that show the strongest contribution of MMR-deficient tumours. CONCLUSIONS: Next-generation sequencing approaches allow for a granular molecular characterisation of MMR-deficient tumours, which can be essential to properly diagnose and treat patients with these tumours in the setting of personalised medicine.

NCCN Guidelines® Insights: Genetic/Familial High-Risk Assessment: Colorectal, Version 1.2021.

Identifying individuals with hereditary syndromes allows for timely cancer surveillance, opportunities for risk reduction, and syndrome-specific management. Establishing criteria for hereditary cancer risk assessment allows for the identification of individuals who are carriers of pathogenic genetic variants. The NCCN Guidelines for Genetic/Familial High-Risk Assessment: Colorectal provides recommendations for the assessment and management of patients at risk for or diagnosed with high-risk colorectal cancer syndromes. The NCCN Genetic/Familial High-Risk Assessment: Colorectal panel meets annually to evaluate and update their recommendations based on their clinical expertise and new scientific data. These NCCN Guidelines Insights focus on familial adenomatous polyposis (FAP)/attenuated familial adenomatous polyposis (AFAP) syndrome and considerations for management of duodenal neoplasia.

Decreased copy-neutral loss of heterozygosity in African American colorectal cancers.

Despite improvements over the past 20 years, African Americans continue to have the highest incidence and mortality rates of colorectal cancer (CRC) in the United States. While previous studies have found that copy number variations (CNVs) occur at similar frequency in African American and White CRCs, copy-neutral loss of heterozygosity (cnLOH) has not been investigated. In the present study, we used publicly available data from The Cancer Genome Atlas (TCGA) as well as data from an African American CRC cohort, the Chicago Colorectal Cancer Consortium (CCCC), to compare frequencies of CNVs and cnLOH events in CRCs in the two racial groups. Using genotype microarray data, we analyzed large-scale CNV and cnLOH events from 166 microsatellite stable CRCs-31 and 39 African American CRCs from TCGA and the CCCC, respectively, and 96 White CRCs from TCGA. As reported previously, the frequencies of CNVs were similar between African American and White CRCs; however, there was a significantly lower frequency of cnLOH events in African American CRCs compared to White CRCs, even after adjusting for demographic and clinical covariates. Although larger differences for chromosome 18 were observed, a lower frequency of cnLOH events in African American CRCs was observed for nearly all chromosomes. These results suggest that mechanistic differences, including differences in the frequency of cnLOH, could contribute to clinicopathological disparities between African Americans and Whites. Additionally, we observed a previously uncharacterized phenomenon we refer to as small interstitial cnLOH, in which segments of chromosomes from 1 to 5 Mb long were affected by cnLOH.

AGA White Paper: Roadmap for the Future of Colorectal Cancer Screening in the United States.

The American Gastroenterological Association's Center for Gastrointestinal Innovation and Technology convened a consensus conference in December 2018, entitled, "Colorectal Cancer Screening and Surveillance: Role of Emerging Technology and Innovation to Improve Outcomes." The goal of the conference, which attracted more than 60 experts in screening and related disciplines, including the authors, was to envision a future in which colorectal cancer (CRC) screening and surveillance are optimized, and to identify barriers to achieving that future. This White Paper originates from that meeting and delineates the priorities and steps needed to improve CRC outcomes, with the goal of minimizing CRC morbidity and mortality. A one-size-fits-all approach to CRC screening has not and is unlikely to result in increased screening uptake or desired outcomes owing to barriers stemming from behavioral, cultural, and socioeconomic causes, especially when combined with inefficiencies in deployment of screening technologies. Overcoming these barriers will require the following: efficient utilization of multiple screening modalities to achieve increased uptake; continued development of noninvasive screening tests, with iterative reassessments of how best to integrate new technologies; and improved personal risk assessment to better risk-stratify patients for appropriate screening testing paradigms.

NCCN Guidelines Insights: Colorectal Cancer Screening, Version 2.2020.

The NCCN Guidelines for Colorectal Cancer (CRC) Screening describe various colorectal screening modalities as well as recommended screening schedules for patients at average or increased risk of developing sporadic CRC. They are intended to aid physicians with clinical decision-making regarding CRC screening for patients without defined genetic syndromes. These NCCN Guidelines Insights focus on select recent updates to the NCCN Guidelines, including a section on primary and secondary CRC prevention, and provide context for the panel's recommendations regarding the age to initiate screening in average risk individuals and follow-up for low-risk adenomas.

Clinical features and cancer risk in families with pathogenic CDH1 variants irrespective of clinical criteria.

BACKGROUND: The clinical phenotype of CDH1 pathogenic variant carriers has mostly been studied in families that fulfil criteria of hereditary diffuse gastric cancer (HDGC). We aimed at determining cancer phenotype and cancer risk estimation among families with CDH1 pathogenic variants not selected by HDGC clinical criteria. METHODS: Patients were all consecutively identified CDH1 pathogenic variant carriers from a clinical laboratory tested with multigene panel testing and from an academic cancer genetics programme. Clinical and demographic features, cancer phenotypes and genotype-phenotype correlations were determined among CDH1 families. Age-specific cumulative cancer risks (penetrance) were calculated based on 38 families with available pedigrees. RESULTS: Within the 113 CDH1 pathogenic variant probands and 476 relatives, 113 had gastric cancer, 177 breast cancer and 196 other cancers. Mean age at diagnosis was 47 for gastric and 54 for breast cancer. Forty-six per cent fulfilled criteria of HDGC. While 36% of families had both gastric and breast cancers, 36% had breast but no gastric cancers and 16% had gastric but not breast cancers. Cumulative risk of cancer by age 80 was 37.2% for gastric and 42.9% for breast cancer. CONCLUSION: In unselected CDH1 pathogenic variant carrier families, gastric cancer risks were lower and age at diagnosis higher than previously reported in families pre-selected for HDGC criteria. A substantial proportion of families did not present with any gastric cancers and their cancers were limited to breast. Thus, clinical criteria for CDH1 testing should be widened, including breast cancer families only, and a consideration for delayed prophylactic gastrectomy/surveillance should be evaluated.

NCCN Guidelines Insights: Genetic/Familial High-Risk Assessment: Colorectal, Version 2.2019.

Identifying individuals with hereditary syndromes allows for improved cancer surveillance, risk reduction, and optimized management. Establishing criteria for assessment allows for the identification of individuals who are carriers of pathogenic genetic variants. The NCCN Guidelines for Genetic/Familial High-Risk Assessment: Colorectal provide recommendations for the assessment and management of patients with high-risk colorectal cancer syndromes. These NCCN Guidelines Insights focus on criteria for the evaluation of Lynch syndrome and considerations for use of multigene testing in the assessment of hereditary colorectal cancer syndromes.

Lack of APC somatic mutation is associated with early-onset colorectal cancer in African Americans.

African Americans (AAs) have higher incidence and mortality rates of colorectal cancer (CRC) compared with other US populations. They present with more right-sided, microsatellite stable disease and are diagnosed at earlier ages compared with non-Hispanic Whites (NHWs). To gain insight into these trends, we conducted exome sequencing (n = 45), copy number (n = 33) and methylation analysis (n = 11) of microsatellite stable AA CRCs. Results were compared with data from The Cancer Genome Atlas (TCGA). Two of the 45 tumors contained POLE mutations. In the remaining 43 tumors, only 27 (63%) contained loss-of-function mutations in APC compared with 80% of TCGA NHW CRCs. APC-mutation-negative CRCs were associated with an earlier onset of CRC (P = 0.01). They were also associated with lower overall mutation burden, fewer copy number variants and a DNA methylation signature that was distinct from the CpG island methylator phenotype characterized in microsatellite unstable disease. Three of the APC-mutation-negative CRCs had loss-of-function mutations in BCL9L. Mutations in driver genes identified by TCGA exome analysis were less frequent in AA CRC cases than TCGA NHWs. Genes that regulate the WNT signaling pathway, including SOX9, GATA6, TET1, GLIS1 and FAT1, were differentially hypermethylated in APC-mutation-negative CRCs, suggesting a novel mechanism for cancer development in these tumors. In summary, we have identified a subtype of CRC that is associated with younger age of diagnosis, lack of APC mutation, microsatellite and chromosome stability, lower mutation burden and distinctive methylation changes.

Genetic testing for hereditary prostate cancer: Current status and limitations.

A significant proportion of prostate cancer diagnoses may be associated with a strong hereditary component. Men who have multiple single-gene polymorphisms and a family history of prostate cancer have a significantly greater risk of developing prostate cancer. Numerous single-gene alterations have been confirmed to increase the risk of prostate cancer. These include breast cancer genes 1 and 2 (BRCA1 and BRCA2, respectively), mutL homolog 1 (MLH1), mutS homologs 2 and 6 (MSH2 and MSH6, respectively), postmeiotic segregation increased 2 (PMS2), homeobox B13 (HOXB13), checkpoint kinase 2 (CHEK2), nibrin (NBN), BRCA1-interacting protein C-terminal helicase 1 (BRIP1), and ataxia telangiectasia mutated (ATM). Currently, there are no uniform guidelines on the definition of hereditary prostate cancer and genetic testing. With the advent of next-generation sequencing, which is capable of testing multiple genes simultaneously, and the approval of olaparib for BRCA1/BRCA2 or ATM-mutated, metastatic, castrate-resistant prostate cancer, it is being recognized that the results of genetic testing have an impact on therapeutic strategies. In this review, the authors examine the role of genetic counseling and testing, the challenges of insurance coverage for testing, the available germline and somatic testing panels, and the complexity of each testing method and its implications. Cancer 2018. © 2018 American Cancer Society.

NCCN Guidelines Insights: Colorectal Cancer Screening, Version 1.2018.

The NCCN Guidelines for Colorectal Cancer (CRC) Screening outline various screening modalities as well as recommended screening strategies for individuals at average or increased-risk of developing sporadic CRC. The NCCN panel meets at least annually to review comments from reviewers within their institutions, examine relevant data, and reevaluate and update their recommendations. These NCCN Guidelines Insights summarize 2018 updates to the NCCN Guidelines, with a primary focus on modalities used to screen individuals at average-risk for CRC.

Race-dependent association of sulfidogenic bacteria with colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) incidence is higher in African Americans (AAs) compared with non-Hispanic whites (NHWs). A diet high in animal protein and fat is an environmental risk factor for CRC development. The intestinal microbiota is postulated to modulate the effects of diet in promoting or preventing CRC. Hydrogen sulfide, produced by autochthonous sulfidogenic bacteria, triggers proinflammatory pathways and hyperproliferation, and is genotoxic. We hypothesised that sulfidogenic bacterial abundance in colonic mucosa may be an environmental CRC risk factor that distinguishes AA and NHW. DESIGN: Colonic biopsies from uninvolved or healthy mucosa from CRC cases and tumour-free controls were collected prospectively from five medical centres in Chicago for association studies. Sulfidogenic bacterial abundance in uninvolved colonic mucosa of AA and NHW CRC cases was compared with normal mucosa of AA and NHW controls. In addition, 16S rDNA sequencing was performed in AA cases and controls. Correlations were examined among bacterial targets, race, disease status and dietary intake. RESULTS: AAs harboured a greater abundance of sulfidogenic bacteria compared with NHWs regardless of disease status. Bilophila wadsworthia-specific dsrA was more abundant in AA cases than controls. Linear discriminant analysis of 16S rRNA gene sequences revealed five sulfidogenic genera that were more abundant in AA cases. Fat and protein intake and daily servings of meat were significantly higher in AAs compared with NHWs, and multiple dietary components correlated with a higher abundance of sulfidogenic bacteria. CONCLUSIONS: These results implicate sulfidogenic bacteria as a potential environmental risk factor contributing to CRC development in AAs.

NCCN Guidelines Insights: Genetic/Familial High-Risk Assessment: Colorectal, Version 3.2017.

The NCCN Guidelines for Genetic/Familial High-Risk Assessment: Colorectal provide recommendations for the management of patients with high-risk syndromes associated with an increased risk of colorectal cancer (CRC). The NCCN Panel for Genetic/Familial High-Risk Assessment: Colorectal meets at least annually to assess comments from reviewers within their institutions, examine relevant data, and reevaluate and update their recommendations. These NCCN Guidelines Insights focus on genes newly associated with CRC risk on multigene panels, the associated evidence, and currently recommended management strategies.

Association of a let-7 miRNA binding region of TGFBR1 with hereditary mismatch repair proficient colorectal cancer (MSS HNPCC).

The purpose of this study was to identify novel colorectal cancer (CRC)-causing alleles in unexplained familial CRC cases. In order to do so, coding regions in five candidate genes (MGMT, AXIN2, CTNNB1, TGFBR1 and TGFBR2) were sequenced in 11 unrelated microsatellite-stable hereditary non-polyposis CRC (MSS HNPCC) cases. Selected genetic variants were genotyped in a discovery set of 27 MSS HNPCC cases and 85 controls. One genetic variant, rs67687202, in TGFBR1 emerged as significant (P = 0.002), and it was genotyped in a replication set of 87 additional MSS HNPCC-like cases and 338 controls where it was also significantly associated with MSS HNPCC cases (P = 0.041). In the combined genotype data, rs67687202 was associated with a moderate increase in CRC risk (OR = 1.68; 95% CI = 1.13-2.50; P = 0.010). We tested a highly correlated SNP rs868 in 723 non-familial CRC cases compared with 629 controls, and it was not significantly associated with CRC risk (P = 0.370). rs868 is contained in a let-7 miRNA binding site in the 3'UTR of TGFBR1, which might provide a functional basis for the association in MSS HNPCC. In luciferase assays, the risk-associated allele for rs868 was associated with half the luciferase expression in the presence of miRNA let-7b-5p compared with protective allele, suggesting more binding of let-7b-5p and less TGFBR1 expression. Thus, rs868 potentially is a CRC risk-causing allele. Our results support the concept that rs868 is associated with lower TGFBR1 expression thereby increasing CRC risk.

Mutation spectrum and risk of colorectal cancer in African American families with Lynch syndrome.

BACKGROUND & AIMS: African Americans (AAs) have the highest incidence of and mortality resulting from colorectal cancer (CRC) in the United States. Few data are available on genetic and nongenetic risk factors for CRC among AAs. Little is known about cancer risks and mutations in mismatch repair (MMR) genes in AAs with the most common inherited CRC condition, Lynch syndrome. We aimed to characterize phenotype, mutation spectrum, and risk of CRC in AAs with Lynch syndrome. METHODS: We performed a retrospective study of AAs with mutations in MMR genes (MLH1, MSH2, MSH6, and PMS2) using databases from 13 US referral centers. We analyzed data on personal and family histories of cancer. Modified segregation analysis conditioned on ascertainment criteria was used to estimate age- and sex-specific CRC cumulative risk, studying members of the mutation-carrying families. RESULTS: We identified 51 AA families with deleterious mutations that disrupt function of the MMR gene product: 31 in MLH1 (61%), 11 in MSH2 (21%), 3 in MSH6 (6%), and 6 in PMS2 (12%); 8 mutations were detected in more than 1 individual, and 11 have not been previously reported. In the 920 members of the 51 families with deleterious mutations, the cumulative risks of CRC at 80 years of age were estimated to be 36.2% (95% confidence interval [CI], 10.5%-83.9%) for men and 29.7% (95% CI, 8.31%-76.1%) for women. CRC risk was significantly higher among individuals with mutations in MLH1 or MSH2 (hazard ratio, 13.9; 95% CI, 3.44-56.5). CONCLUSIONS: We estimate the cumulative risk for CRC in AAs with MMR gene mutations to be similar to that of individuals of European descent with Lynch syndrome. Two-thirds of mutations were found in MLH1, some of which were found in multiple individuals and some that have not been previously reported. Differences in mutation spectrum are likely to reflect the genetic diversity of this population.

Prevalence of MLH1 constitutional epimutations as a cause of Lynch syndrome in unselected versus selected consecutive series of patients with colorectal cancer.

BACKGROUND: The prevalence of MLH1 constitutional epimutations in the general population is unknown. We sought to analyse the prevalence of MLH1 constitutional epimutations in unselected and selected series of patients with colorectal cancer (CRC). METHODS: Patients with diagnoses of CRC (n=2123) were included in the unselected group. For comparison, a group of 847 selected patients with CRC who fulfilled the revised Bethesda guidelines (rBG) were also included. Somatic and constitutional MLH1 methylation was assayed via methylation-specific multiplex ligation-dependent probe amplification of cases lacking MLH1 expression. Germline alterations in mismatch-repair (MMR) genes were assessed via Sanger sequencing and methylation-specific multiplex ligation-dependent probe amplification. RESULTS: Loss of MLH1 expression occurred in 5.5% of the unselected series and 12.5% of the selected series (p<0.0001). No constitutional epimutations in MLH1 were detected in the unselected population (0/62); five cases from the selected series were positive for MLH1 epimutations (15.6%, 5/32; p=0.004). CONCLUSIONS: Our results suggest a negligible prevalence of MLH1 constitutional epimutations in unselected cases of CRC. Therefore, MLH1 constitutional epimutation analysis should be conducted only for patients who fulfil the rBG and who lack MLH1 expression with methylated MLH1.

Enrichment of inflammatory bowel disease and colorectal cancer risk variants in colon expression quantitative trait loci.

BACKGROUND: Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with diseases of the colon including inflammatory bowel diseases (IBD) and colorectal cancer (CRC). However, the functional role of many of these SNPs is largely unknown and tissue-specific resources are lacking. Expression quantitative trait loci (eQTL) mapping identifies target genes of disease-associated SNPs. This study provides a comprehensive eQTL map of distal colonic samples obtained from 40 healthy African Americans and demonstrates their relevance for GWAS of colonic diseases. RESULTS: 8.4 million imputed SNPs were tested for their associations with 16,252 expression probes representing 12,363 unique genes. 1,941 significant cis-eQTL, corresponding to 122 independent signals, were identified at a false discovery rate (FDR) of 0.01. Overall, among colon cis-eQTL, there was significant enrichment for GWAS variants for IBD (Crohn's disease [CD] and ulcerative colitis [UC]) and CRC as well as type 2 diabetes and body mass index. ERAP2, ADCY3, INPP5E, UBA7, SFMBT1, NXPE1 and REXO2 were identified as target genes for IBD-associated variants. The CRC-associated eQTL rs3802842 was associated with the expression of C11orf93 (COLCA2). Enrichment of colon eQTL near transcription start sites and for active histone marks was demonstrated, and eQTL with high population differentiation were identified. CONCLUSIONS: Through the comprehensive study of eQTL in the human colon, this study identified novel target genes for IBD- and CRC-associated genetic variants. Moreover, bioinformatic characterization of colon eQTL provides a tissue-specific tool to improve understanding of biological differences in diseases between different ethnic groups.

Prevalence of somatic mutl homolog 1 promoter hypermethylation in Lynch syndrome colorectal cancer.

BACKGROUND: Colorectal cancers (CRCs) that have microsatellite instability (MSI) and mutL homolog 1 (MLH1) immunoloss are observed in 3 clinical scenarios: Lynch syndrome (LS), sporadic MSI CRC, and Lynch-like syndrome (LLS). v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutational analysis is used to differentiate LS from sporadic MSI CRC. The role of MLH1 promoter methylation status for the differential diagnosis of these clinical forms is not well established. The objectives of this study were: 1) to analyze MLH1 promoter methylation in MLH1-deficient CRCs by pyrosequencing, and 2) to assess its role in the differential diagnosis of MLH1-deficient CRCs. METHODS: In total, 165 CRCs were analyzed, including LS (n = 19), MSI BRAF-mutated CRC (n = 37), MSI BRAF wild-type CRC (n = 60), and a control group of CRCs without MSI (microsatellite stable [MSS] CRC; n = 49). MLH1 promoter methylation status was analyzed by pyrosequencing, and the ability of different strategies to identify LS was assessed. RESULTS: The average ± standard deviation methylation in LS (9% ± 7%) was significantly lower than that in MSI BRAF-mutated CRC (42% ± 17%; P < .001) and in MSI BRAF wild-type CRC (25% ± 19%; P = .002). Somatic MLH1 hypermethylation was detected in 3 patients (15.8%) with LS, in 34 patients (91.9%) with MSI BRAF-mutated CRC, and in 37 patients (61.7%) with MSI BRAF wild-type tumors. Patients with MSI BRAF wild-type, unmethylated tumors (ie, LLS) had a stronger family history of CRC than those who had tumors with MLH1 methylation (P < .05). The sensitivity for ruling out LS was 100% for BRAF analysis, 84.2% for MLH1 methylation analysis, and 84.2% for the combination of both analyses. CONCLUSIONS: Somatic MLH1 promoter methylation occurs in up to 15% of LS CRCs. Somatic BRAF analysis is the most sensitive strategy for ruling out LS. Patients who have CRCs with loss of MLH1 protein expression and neither BRAF mutation nor MLH1 methylation resemble patients with LS.