Constitutional MLH1 Methylation Is a Major Contributor to Mismatch Repair-Deficient, MLH1-Methylated Colorectal Cancer in Patients Aged 55 Years and Younger.

BACKGROUND: Most mismatch repair-deficient (MMRd) colorectal cancer (CRC) cases arise sporadically, associated with somatic MLH1 methylation, whereas approximately 20% have germline mismatch repair pathogenic variants causing Lynch syndrome (LS). Universal screening of incident CRC uses presence of MLH1 methylation in MMRd tumors to exclude sporadic cases from germline testing for LS. However, this overlooks rare cases with constitutional MLH1 methylation (epimutation), a poorly recognized mechanism for LS. We aimed to assess the frequency and age distribution of constitutional MLH1 methylation among incident CRC cases with MMRd, MLH1-methylated tumors. METHODS: In retrospective population-based studies, we selected all CRC cases with MMRd, MLH1-methylated tumors, regardless of age, prior cancer, family history, or BRAF V600E status, from the Columbus-area HNPCC study (Columbus) and Ohio Colorectal Cancer Prevention Initiative (OCCPI) cohorts. Blood DNA was tested for constitutional MLH1 methylation by pyrosequencing and real-time methylation-specific PCR, then confirmed with bisulfite-sequencing. RESULTS: Results were achieved for 95 of 98 Columbus cases and all 281 OCCPI cases. Constitutional MLH1 methylation was identified in 4 of 95 (4%) Columbus cases, ages 34, 38, 52, and 74 years, and 4 of 281 (1.4%) OCCPI cases, ages 20, 34, 50, and 55 years, with 3 showing low-level mosaic methylation. Mosaicism in blood and normal colon, plus tumor loss of heterozygosity of the unmethylated allele, demonstrated causality in 1 case with sample availability. Age stratification showed high rates of constitutional MLH1 methylation among younger patients. In the Columbus and OCCPI cohorts, respectively, these rates were 67% (2 of 3) and 25% (2 of 8) of patients aged <50 years but with half of the cases missed, and 75% (3 of 4) and 23.5% (4 of 17) of patients aged ≤55 years with most cases detected. CONCLUSIONS: Although rare overall, a significant proportion of younger patients with MLH1-methylated CRC had underlying constitutional MLH1 methylation. Routine testing for this high-risk mechanism is warranted in patients aged ≤55 years for a timely and accurate molecular diagnosis that will significantly alter their clinical management while minimizing additional testing.

POLE-Mutant Colon Cancer Treated with PD-1 Blockade Showing Clearance of Circulating Tumor DNA and Prolonged Disease-Free Interval.

Colon cancer with high microsatellite instability is characterized by a high tumor mutational burden and responds well to immunotherapy. Mutations in polymerase ɛ, a DNA polymerase involved in DNA replication and repair, are also associated with an ultra-mutated phenotype. We describe a case where a patient with POLE-mutated and hypermutated recurrent colon cancer was treated with pembrolizumab. Treatment with immunotherapy in this patient also led to the clearance of circulating tumor DNA (ctDNA). ctDNA is beginning to emerge as a marker for minimal residual disease in many solid malignancies, including colon cancer. Its clearance with treatment suggests that the selection of pembrolizumab on the basis of identifying a POLE mutation on next-generation sequencing may increase disease-free survival in this patient.

The Fatty Liver, Cirrhosis, and Liver Cancer Study (TENDENCY): Protocol for a Multicenter Case-Control Study.

BACKGROUND: Hepatocellular cancer (HCC) is associated with high mortality, and early diagnosis leads to better survival. Patients with cirrhosis, especially due to nonalcoholic fatty liver disease and viral hepatitis, are at higher risk of developing HCC and form the main screening group. The current screening methods for HCC (6-monthly screening with serum alpha fetoprotein and ultrasound liver) have low sensitivity; hence, there is a need for better screening markers for HCC. OBJECTIVE: Our study, TENDENCY, aims to validate the novel screening markers (methylated septin 9, urinary volatile organic compounds, and urinary peptides) for HCC diagnosis and study these noninvasive biomarkers in liver disease. METHODS: This is a multicenter, nested case-control study, which involves comparing the plasma levels of methylated septin 9 between confirmed HCC cases and patients with cirrhosis (control group). It also includes the comparison of urine samples for the detection of HCC-specific volatile organic compounds and peptides. Based on the findings of a pilot study carried out at University Hospital Coventry & Warwickshire, we estimated our sample size to be 308 (n=88, 29% patients with HCC; n=220, 71% patients with cirrhosis). Urine and plasma samples will be collected from all participants and will be frozen at -80 °C until the end of recruitment. Gas chromatography-mass spectrometry will be used for urinary volatile organic compounds detection, and capillary electrophoresis-mass spectrometry will be used for urinary peptide identification. Real-time polymerase chain reaction will be used for the qualitative detection of plasma methylated septin 9. The study will be monitored by the Research and Development department at University Hospital Coventry & Warwickshire. RESULTS: The recruitment stage was completed in March 2023. The TENDENCY study is currently in the analysis stage, which is expected to finish by November 2023. CONCLUSIONS: There is lack of effective screening tests for hepatocellular cancer despite higher mortality rates. The application of more sensitive plasma and urinary biomarkers for hepatocellular cancer screening in clinical practice will allow us to detect the disease at earlier stages and hence, overall, improve HCC outcomes. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/44264.

MLH1-methylated endometrial cancer under 60 years of age as the "sentinel" cancer in female carriers of high-risk constitutional MLH1 epimutation.

OBJECTIVE: Universal screening of endometrial carcinoma (EC) for mismatch repair deficiency (MMRd) and Lynch syndrome uses presence of MLH1 methylation to omit common sporadic cases from follow-up germline testing. However, this overlooks rare cases with high-risk constitutional MLH1 methylation (epimutation), a poorly-recognized mechanism that predisposes to Lynch-type cancers with MLH1 methylation. We aimed to determine the role and frequency of constitutional MLH1 methylation among EC cases with MMRd, MLH1-methylated tumors. METHODS: We screened blood for constitutional MLH1 methylation using pyrosequencing and real-time methylation-specific PCR in patients with MMRd, MLH1-methylated EC ascertained from (i) cancer clinics (n = 4, <60 years), and (ii) two population-based cohorts; "Columbus-area" (n = 68, all ages) and "Ohio Colorectal Cancer Prevention Initiative (OCCPI)" (n = 24, <60 years). RESULTS: Constitutional MLH1 methylation was identified in three out of four patients diagnosed between 36 and 59 years from cancer clinics. Two had mono-/hemiallelic epimutation (∼50% alleles methylated). One with multiple primaries had low-level mosaicism in normal tissues and somatic "second-hits" affecting the unmethylated allele in all tumors, demonstrating causation. In the population-based cohorts, all 68 cases from the Columbus-area cohort were negative and low-level mosaic constitutional MLH1 methylation was identified in one patient aged 36 years out of 24 from the OCCPI cohort, representing one of six (∼17%) patients <50 years and one of 45 patients (∼2%) <60 years in the combined cohorts. EC was the first/dual-first cancer in three patients with underlying constitutional MLH1 methylation. CONCLUSIONS: A correct diagnosis at first presentation of cancer is important as it will significantly alter clinical management. Screening for constitutional MLH1 methylation is warranted in patients with early-onset EC or synchronous/metachronous tumors (any age) displaying MLH1 methylation.

Methylated Septin9 (mSEPT9): A promising blood-based biomarker for the detection and screening of early-onset colorectal cancer.

Early-onset colorectal cancer (EOCRC), defined as a diagnosis under age 50, is an emerging public health burden. As many of these individuals fall outside of screening guidelines, the development of a minimally invasive, accurate screening modality for this population is warranted. We evaluated the FDA-approved blood-based biomarker methylated Septin9 (mSEPT9) test as screening tool for EOCRC. EOCRC plasma, healthy plasma, and serum-free conditioned media from cancer cell lines was collected. Cell-free DNA (cfDNA) was isolated and bisulfite converted for use in the assay. mSEPT9 and ACTB measured using Epi proColon® V2.0. EOCRC plasma was collected at Massachusetts General Hospital (2005-2019) and controls were collected at the National Institutes of Health and by ZenBio Inc. (prior to 2019). Twenty-seven EOCRC cases, 48 healthy controls <50 years old, and 39 healthy controls ≥50 years old were included in this study. mSEPT9 was detected more frequently in EOCRC cases (88.9%) compared to healthy controls age <50 (4.2%) and ≥50 (15.4%), respectively (p<0.001). The sensitivity, specificity, positive predictive value, and negative predictive values of the mSEPT9 assay to detect EOCRC was 90.8% (95% CI: 84.7-96.9%), 88.9% (95% CI: 77.0-100.0%), 96.3% (95% CI: 92.3-100.0%), and 75.0% (95% CI 60.0-90.0%), respectively, compared to all healthy controls. mSEPT9 cfDNA level was an independent predictor of survival (p=0.02). mSEPT9 is a sensitive and specific biomarker for EOCRC detection. These results suggest that mSEPT9 may be useful in the detection of EOCRC, providing a minimally invasive method for screening in this growing population of CRC patients.

Circulating tumor DNA dynamics and response to immunotherapy in colorectal cancer.

Circulating tumor DNA (ctDNA) is increasingly being investigated as a tool to detect minimal residual disease in resected, stage I-III colorectal cancer. Recent ctDNA studies have indicated that detection of ctDNA following surgery for resectable colorectal cancer confers a significantly higher risk of recurrence than those with negative ctDNA postoperatively. In those with postoperative ctDNA positivity, clearance of minimal residual disease with adjuvant chemotherapy is a positive prognostic indicator. Lastly, ctDNA has demonstrated superior sensitivity to the conventional blood tumor marker carcinoembryonic antigen (CEA) and can offer median lead times of up to 11 months for radiographic detection of recurrence during the surveillance of resected, stage I-III colorectal cancer. In metastatic colorectal cancer (mCRC), there is growing evidence to suggest that plasma ctDNA can be used to monitor tumor response to conventional chemotherapy as well. The present case series demonstrated that plasma ctDNA is a predictor of tumor response to immunotherapy in patients with mCRC that are microsatellite stable or microsatellite instability high. Plasma ctDNA could serve as a dynamic marker of immunotherapy response even in colorectal tumors that were CEA non-producers. Overall, these findings add to ongoing efforts to establish the role of plasma ctDNA in monitoring response to immunotherapy in CRC.

A systematic review and meta-analysis: the diagnostic accuracy of methylated SEPTIN9 for the detection of hepatocellular carcinoma and the clinical evaluation of its use in combination with other surveillance modalities.

BACKGROUND: Hepatocellular carcinoma (HCC) lacks a suitable biomarker for minimally-invasive disease detection. Methylated SEPTIN9 (mSEPT9) is an emerging liquid biopsy test. We aimed to investigate recent studies that applied mSEPT9 for HCC diagnosis. Furthermore, we evaluated the combinations of other surveillance modalities for the detection of HCC. METHODS: A systematic review was performed on the diagnostic accuracy of mSEPT9 for the detection of HCC. Using a bivariate model, the pooled sensitivity and specificity were calculated. Additionally, Fagan's nomograms were used to calculate the pre-test and post-test probabilities of HCC for various combinations of surveillance modalities. RESULTS: Six full texts were included in the meta-analysis. The pooled sensitivity and specificity of mSEPT9 for the detection of HCC, were 0.80 (95% CI, 0.67-0.89) and 0.90 (95% CI, 0.84-0.94). The area under the receiver operating curve was 0.92. The probability of having HCC for the combinations of mSEPT9+ ultrasound scan (USS) and mSEPT9+ Alpha fetoprotein (AFP) were 0.7% and 1.2% respectively if both tests were negative (in a population with 10% HCC prevalence). The combination of USS and AFP would miss relatively fewer cancers for 1000 patients in comparison to other combinations of two surveillance modalities. CONCLUSION: Test combinations have superior performance for the detection of HCC than any individual test. mSEPT9 has shown promise in the detection of HCC with higher estimates of performance accuracy. mSEPT9 has potential for use as an HCC surveillance modality in adjunct with other tests to improve detection rates. However, cost effectiveness of this approach needs further evaluation.

Large Cancer Pedigree Involving Multiple Cancer Genes including Likely Digenic MSH2 and MSH6 Lynch Syndrome (LS) and an Instance of Recombinational Rescue from LS.

Lynch syndrome (LS), caused by heterozygous pathogenic variants affecting one of the mismatch repair (MMR) genes (MSH2, MLH1, MSH6, PMS2), confers moderate to high risks for colorectal, endometrial, and other cancers. We describe a four-generation, 13-branched pedigree in which multiple LS branches carry the MSH2 pathogenic variant c.2006G>T (p.Gly669Val), one branch has this and an additional novel MSH6 variant c.3936_4001+8dup (intronic), and other non-LS branches carry variants within other cancer-relevant genes (NBN, MC1R, PTPRJ). Both MSH2 c.2006G>T and MSH6 c.3936_4001+8dup caused aberrant RNA splicing in carriers, including out-of-frame exon-skipping, providing functional evidence of their pathogenicity. MSH2 and MSH6 are co-located on Chr2p21, but the two variants segregated independently (mapped in trans) within the digenic branch, with carriers of either or both variants. Thus, MSH2 c.2006G>T and MSH6 c.3936_4001+8dup independently confer LS with differing cancer risks among family members in the same branch. Carriers of both variants have near 100% risk of transmitting either one to offspring. Nevertheless, a female carrier of both variants did not transmit either to one son, due to a germline recombination within the intervening region. Genetic diagnosis, risk stratification, and counseling for cancer and inheritance were highly individualized in this family. The finding of multiple cancer-associated variants in this pedigree illustrates a need to consider offering multicancer gene panel testing, as opposed to targeted cascade testing, as additional cancer variants may be uncovered in relatives.

Clinical Applications of Minimal Residual Disease Assessments by Tumor-Informed and Tumor-Uninformed Circulating Tumor DNA in Colorectal Cancer.

Emerging data suggest that circulating tumor DNA (ctDNA) can detect colorectal cancer (CRC)-specific signals across both non-metastatic and metastatic settings. With the development of multiple platforms, including tumor-informed and tumor-agnostic ctDNA assays and demonstration of their provocative analytic performance to detect minimal residual disease, there are now ongoing, phase III randomized clinical trials to evaluate their role in the management paradigm of CRC. In this review, we highlight landmark studies that have formed the basis for ongoing studies on the clinically applicability of plasma ctDNA assays in resected, stage I-III CRC and metastatic CRC. We discuss clinical settings by which ctDNA may have the most immediate impact in routine clinical practice. These include the potential for ctDNA to (1) guide surveillance and intensification or de-intensification strategies of adjuvant therapy in resected, stage I-III CRC, (2) predict treatment response to neoadjuvant therapy in locally advanced rectal cancer inclusive of total neoadjuvant therapy (TNT), and (3) predict response to systemic and surgical therapies in metastatic disease. We end by considering clinical variables that can influence our ability to reliably interpret ctDNA dynamics in the clinic.

Role of methylated septin 9 as an adjunct diagnostic and prognostic biomarker in hepatocellular carcinoma.

BACKGROUND: Methylated septin 9 (mSEPT9) has a role in hepatocarcinogenesis. We evaluated mSEPT9 performance in patients with hepatocellular carcinoma (HCC) and those at risk of HCC METHODS: Using Epi-proColon® V2.0 assay adapted for 1 mL plasma, we investigated mSEPT9 sensitivity, specificity, associations with influential covariates and relation to death. RESULTS: Of 141 participants included, 136 had liver disease, 38 with HCC (mean-age 71 years) and 103 without HCC (mean-age 56.8 years), with further five without liver disease. 41 patients died (23 HCC) by the end of the study follow-up period. In HCC, mSEPT9 sensitivity and specificity were 89.47% (CI:75.20%-97.06%) and 81.55% (CI:72.70%-88.51%), whilst alpha fetoprotein (AFP) sensitivity and specificity were 50% (CI:33.38%-66.62%) and 97.09% (CI:91.72%-99.40%), respectively. Age-adjusted logistic regression showed mSEPT9 was associated with age, body mass index, HCC, liver cirrhosis, AFP, platelets, neutrophil-to-lymphocyte-ratio, albumin-bilirubin grade and fibrosis-4 index (p < 0.05). Odds for HCC patients to have positive mSEPT9 were 27.4 times more than those without HCC. Time-to-death was associated with mSEPT9 positivity (p < 0.05). Kaplan-Meier curves showed higher HCC survival with mSEPT9 compared to AFP. CONCLUSIONS: The mSEPT9 offers potential diagnostic and prognostic biomarker for HCC. After adjusting for age, mSEPT9 remained associated with liver function, liver fibrosis and inflammatory surrogate markers.

Development of a DNA Methylation-Based Diagnostic Signature to Distinguish Benign Oncocytoma From Renal Cell Carcinoma.

PURPOSE: A challenge in the diagnosis of renal cell carcinoma (RCC) is to distinguish chromophobe RCC (chRCC) from benign renal oncocytoma, because these tumor types are histologically and morphologically similar, yet they require different clinical management. Molecular biomarkers could provide a way of distinguishing oncocytoma from chRCC, which could prevent unnecessary treatment of oncocytoma. Such biomarkers could also be applied to preoperative biopsy specimens such as needle core biopsy specimens, to avoid unnecessary surgery of oncocytoma. METHODS: We profiled DNA methylation in fresh-frozen oncocytoma and chRCC tumors and adjacent normal tissue and used machine learning to identify a signature of differentially methylated cytosine-phosphate-guanine sites (CpGs) that robustly distinguish oncocytoma from chRCC. RESULTS: Unsupervised clustering of Stanford and preexisting RCC data from The Cancer Genome Atlas (TCGA) revealed that of all RCC subtypes, oncocytoma is most similar to chRCC. Unexpectedly, however, oncocytoma features more extensive, overall abnormal methylation than does chRCC. We identified 79 CpGs with large methylation differences between oncocytoma and chRCC. A diagnostic model trained on 30 CpGs could distinguish oncocytoma from chRCC in 10-fold cross-validation (area under the receiver operating curve [AUC], 0.96 (95% CI, 0.88 to 1.00)) and could distinguish TCGA chRCCs from an independent set of oncocytomas from a previous study (AUC, 0.87). This signature also separated oncocytoma from other RCC subtypes and normal tissue, revealing it as a standalone diagnostic biomarker for oncocytoma. CONCLUSION: This CpG signature could be developed as a clinical biomarker to support differential diagnosis of oncocytoma and chRCC in surgical samples. With improved biopsy techniques, this signature could be applied to preoperative biopsy specimens.

Validation of a genomic classifier for prediction of metastasis and prostate cancer-specific mortality in African-American men following radical prostatectomy in an equal access healthcare setting.

BACKGROUND: The Decipher 22-gene genomic classifier (GC) may help in post-radical prostatectomy (RP) decision making given its superior prognostic performance over clinicopathologic variables alone. However, most studies evaluating the GC have had a modest representation of African-American men (AAM). We evaluated the GC within a large Veteran Affairs cohort and compared its performance to CAPRA-S for predicting outcomes in AAM and non-AAM after RP. METHODS: GC scores were generated for 548 prostate cancer (PC) patients, who underwent RP at the Durham Veteran Affairs Medical Center between 1989 and 2016. This was a clinically high-risk cohort and was selected to have either pT3a, positive margins, seminal vesicle invasion, or received post-RP radiotherapy. Multivariable Cox models and survival C-indices were used to compare the performance of GC and CAPRA-S for predicting the risk of metastasis and PC-specific mortality (PCSM). RESULTS: Median follow-up was 9 years, during which 37 developed metastasis and 20 died from PC. Overall, 55% (n = 301) of patients were AAM. In multivariable analyses, GC (high vs. intermediate and intermediate vs. low) was a significant predictor of metastasis in all men (all p < 0.001). Consistent with prior studies, relative to CAPRA-S, GC had a higher C-index for 5-year metastasis (0.78 vs. 0.72) and 10-year PCSM (0.85 vs. 0.81). There was a suggestion GC was a stronger predictor in AAM than non-AAM. Specifically, the 5-year metastasis risk C-index was 0.86 in AAM vs. 0.69 in non-AAM and the 10-year PCSM risk C-index was 0.91 in AAM vs. 0.78 in non-AAM. However, the test for interaction of race and the performance of the GC in the Cox model was not significant for either metastasis or PCSM (both p ≥ 0.3). CONCLUSIONS: GC was a very strong predictor of poor outcome and performed well in both AAM and non-AAM. Our data support the use of GC for risk stratification in AAM post-RP. While our data suggest that GC may actually work better in AAM, given the limited number of events, further validation is needed.

Highly sensitive MLH1 methylation analysis in blood identifies a cancer patient with low-level mosaic MLH1 epimutation.

Constitutional MLH1 methylation (epimutation) is a rare cause of Lynch syndrome. Low-level methylation (≤ 10%) has occasionally been described. This study aimed to identify low-level constitutional MLH1 epimutations and determine its causal role in patients with MLH1-hypermethylated colorectal cancer.Eighteen patients with MLH1-hypermethylated colorectal tumors in whom MLH1 methylation was previously undetected in blood by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) were screened for MLH1 methylation using highly sensitive MS-melting curve analysis (MS-MCA). Constitutional methylation was characterized by different approaches.MS-MCA identified one patient (5.6%) with low-level MLH1 methylation (~ 1%) in blood and other normal tissues, which was confirmed by clonal bisulfite sequencing in blood. The patient had developed three clonally related gastrointestinal MLH1-methylated tumor lesions at 22, 24, and 25 years of age. The methylated region in normal tissues overlapped with that reported for other carriers of constitutional MLH1 epimutations. Low-level MLH1 methylation and reduced allelic expression were linked to the same genetic haplotype, whereas the opposite allele was lost in patient's tumors. Mutation screening of MLH1 and other hereditary cancer genes was negative.Herein, a highly sensitive MS-MCA-based approach has demonstrated its utility for the identification of low-level constitutional MLH1 epigenetic mosaicism. The eventual identification and characterization of additional cases will be critical to ascertain the cancer risks associated with constitutional MLH1 epigenetic mosaicism.

Pancreatic cancer 'mismatch' in Lynch syndrome.

OBJECTIVE: Immune therapy with the PD1 inhibitor pembrolizumab has been approved to treat unresectable/metastatic solid tumours exhibiting mismatch repair (MMR) deficiency. Lynch syndrome (LS), caused by autosomal dominant germline mutations of a MMR gene, predisposes to the development of MMR-deficient cancers. We report a case of MSH2-LS with an MMR-intact pancreatic ductal adenocarcinoma (PDAC) ineligible for treatment with pembrolizumab. DESIGN: Immunohistochemistry of MMR proteins was performed in each malignancy developed in a MSH2-LS patient to determine MMR status. RESULTS: The patient carried a pathogenic MSH2 germline mutation and had a history of LS-type cancers, including endometrial carcinoma, colorectal adenocarcinoma, urothelial carcinoma of the bladder and PDAC. Three malignancies (endometrial, colorectal, urothelial) lacked MSH2 and MSH6 expression, consistent with MSH2-associated tumorigenesis. However, MSH2 and MSH6 expression were intact in the PDAC, suggesting the sporadic occurrence of the pancreatic tumour unrelated to the germline MSH2 mutation. These inconsistent MMR statuses among the tumours rendered the patient ineligible for the immunotherapy pembrolizumab. CONCLUSION: Testing for MMR protein expression is recommended for each tumour in patients with LS, especially pancreatic, as discordant results may have profound effects on treatment opportunities. To our knowledge, this is the first documented case of MMR-intact PDAC in a patient with MSH2-LS.

Methylated SEPTIN9 plasma test for colorectal cancer detection may be applicable to Lynch syndrome.

OBJECTIVE: The plasma-based methylated SEPTIN9 (mSEPT9) is a colorectal cancer (CRC) screening test for adults aged 50-75 years who are at average risk for CRC and have refused colonoscopy or faecal-based screening tests. The applicability of mSEPT9 for high-risk persons with Lynch syndrome (LS), the most common hereditary CRC condition, has not been assessed. This study sought preliminary evidence for the utility of mSEPT9 for CRC detection in LS. DESIGN: Firstly, SEPT9 methylation was measured in LS-associated CRC, advanced adenoma, and subject-matched normal colorectal mucosa tissues by pyrosequencing. Secondly, to detect mSEPT9 as circulating tumor DNA, the plasma-based mSEPT9 test was retrospectively evaluated in LS subjects using the Epi proColon 2.0 CE assay adapted for 1mL plasma using the "1/1 algorithm". LS case groups included 20 peri-surgical cases with acolonoscopy-based diagnosis of CRC (stages I-IV), 13 post-surgical metastatic CRC, and 17 pre-diagnosis cases. The control group comprised 31 cancer-free LS subjects. RESULTS: Differential hypermethylation was found in 97.3% (36/37) of primary CRC and 90.0% (18/20) of advanced adenomas, showing LS-associated neoplasia frequently produce the mSEPT9 biomarker. Sensitivity of plasma mSEPT9 to detect CRC was 70.0% (95% CI, 48%-88%)in cases with a colonoscopy-based CRC diagnosis and 92.3% (95% CI, 64%-100%) inpost-surgical metastatic cases. In pre-diagnosis cases, plasma mSEPT9 was detected within two months prior to colonoscopy-based CRC diagnosis in 3/5 cases. Specificity in controls was 100% (95% CI 89%-100%). CONCLUSION: These preliminary findings suggest mSEPT9 may demonstrate similar diagnostic performance characteristics in LS as in the average-risk population, warranting a well-powered prospective case-control study.