Identifying primary and secondary MLH1 epimutation carriers displaying low-level constitutional MLH1 methylation using droplet digital PCR and genome-wide DNA methylation profiling of colorectal cancers.

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.

A tumor focused approach to resolving the etiology of DNA mismatch repair deficient tumors classified as suspected Lynch syndrome.

Routine screening of tumors for DNA mismatch repair (MMR) deficiency (dMMR) in colorectal (CRC), endometrial (EC) and sebaceous skin (SST) tumors leads to a significant proportion of unresolved cases classified as suspected Lynch syndrome (SLS). SLS cases (n = 135) were recruited from Family Cancer Clinics across Australia and New Zealand. Targeted panel sequencing was performed on tumor (n = 137; 80×CRCs, 33×ECs and 24xSSTs) and matched blood-derived DNA to assess for microsatellite instability status, tumor mutation burden, COSMIC tumor mutational signatures and to identify germline and somatic MMR gene variants. MMR immunohistochemistry (IHC) and MLH1 promoter methylation were repeated. In total, 86.9% of the 137 SLS tumors could be resolved into established subtypes. For 22.6% of these resolved SLS cases, primary MLH1 epimutations (2.2%) as well as previously undetected germline MMR pathogenic variants (1.5%), tumor MLH1 methylation (13.1%) or false positive dMMR IHC (5.8%) results were identified. Double somatic MMR gene mutations were the major cause of dMMR identified across each tumor type (73.9% of resolved cases, 64.2% overall, 70% of CRC, 45.5% of ECs and 70.8% of SSTs). The unresolved SLS tumors (13.1%) comprised tumors with only a single somatic (7.3%) or no somatic (5.8%) MMR gene mutations. A tumor-focused testing approach reclassified 86.9% of SLS into Lynch syndrome, sporadic dMMR or MMR-proficient cases. These findings support the incorporation of tumor sequencing and alternate MLH1 methylation assays into clinical diagnostics to reduce the number of SLS patients and provide more appropriate surveillance and screening recommendations.

A tumor focused approach to resolving the etiology of DNA mismatch repair deficient tumors classified as suspected Lynch syndrome.

Routine screening of tumors for DNA mismatch repair (MMR) deficiency (dMMR) in colorectal (CRC), endometrial (EC) and sebaceous skin (SST) tumors leads to a significant proportion of unresolved cases classified as suspected Lynch syndrome (SLS). SLS cases (n=135) were recruited from Family Cancer Clinics across Australia and New Zealand. Targeted panel sequencing was performed on tumor (n=137; 80xCRCs, 33xECs and 24xSSTs) and matched blood-derived DNA to assess for microsatellite instability status, tumor mutation burden, COSMIC tumor mutational signatures and to identify germline and somatic MMR gene variants. MMR immunohistochemistry (IHC) and MLH1 promoter methylation were repeated. In total, 86.9% of the 137 SLS tumors could be resolved into established subtypes. For 22.6% of these resolved SLS cases, primary MLH1 epimutations (2.2%) as well as previously undetected germline MMR pathogenic variants (1.5%), tumor MLH1 methylation (13.1%) or false positive dMMR IHC (5.8%) results were identified. Double somatic MMR gene mutations were the major cause of dMMR identified across each tumor type (73.9% of resolved cases, 64.2% overall, 70% of CRC, 45.5% of ECs and 70.8% of SSTs). The unresolved SLS tumors (13.1%) comprised tumors with only a single somatic (7.3%) or no somatic (5.8%) MMR gene mutations. A tumor-focused testing approach reclassified 86.9% of SLS into Lynch syndrome, sporadic dMMR or MMR-proficient cases. These findings support the incorporation of tumor sequencing and alternate MLH1 methylation assays into clinical diagnostics to reduce the number of SLS patients and provide more appropriate surveillance and screening recommendations.

Heterogeneity in the psychosocial and behavioral responses associated with a diagnosis of suspected Lynch syndrome in women with endometrial cancer.

BACKGROUND: A suspected Lynch syndrome (SLS) diagnosis is made when a tumor exhibits DNA mismatch repair deficiency but cannot be definitively assigned to an inherited or non-inherited etiology. This diagnosis poses challenges for healthcare professionals, patients, and their families in managing future cancer risks and clinical care. METHODS: This qualitative study aimed to explore the psychosocial and behavioral responses of endometrial cancer (EC) patients receiving a SLS diagnosis (EC-SLS). Semi-structured telephone interviews were conducted with 15 EC-SLS women, transcribed, and thematically analyzed. RESULTS: Most who interpreted their result as negative for Lynch syndrome (LS) believed they were at population-level risk of cancer and felt happy and relieved. Many participants who interpreted their result as inconclusive/not definitive for LS were confused about their cancer risk and experienced negative emotions of anger and frustration. Despite variation in colorectal cancer screening recommendations reported by participants, most adhered to the advice given. Almost all participants communicated their genetic test result to immediate family members; however, communication of family cancer risk management advice was more limited due to most participants reporting not receiving family screening advice. A family history of cancer and a professional healthcare background influenced participants' engagement in regular cancer screening. CONCLUSION: These findings highlight variability in the psychosocial and behavioral responses associated with EC-SLS, providing insight into how healthcare professionals can optimally manage and support such individuals.

"Left in limbo": Exploring how patients with colorectal cancer interpret and respond to a suspected Lynch syndrome diagnosis.

BACKGROUND: A diagnosis of suspected Lynch syndrome (SLS) is given when a tumour displays characteristics consistent with Lynch syndrome (LS), but no germline pathogenic variant is identified. This inconclusive diagnosis results in uncertainty around appropriate cancer risk management. This qualitative study explored how patients with CRC interpret and respond to an SLS diagnosis. METHODS: Semi-structured telephone interviews were conducted with 15 patients with CRC who received an SLS diagnosis, recruited from cancer genetics services across Australia. Interviews were transcribed verbatim and analysed using thematic analysis. Participant responses were compared with appointment summary letters from cancer genetics services. RESULTS: Participants' interpretations of genetic test results were found to vary widely. While this variation often aligned with variation in interpretations by cancer genetics services, participants also had difficulties with the complexity and recall of genetic test results. Participants had a range of psychological responses to the uncertainty that their results presented, from relief to disappointment and doubt. Cancer risk perceptions also varied widely, with participants' interpretations of their genetic test results just one of several influencing factors. Despite this variability, almost all participants adhered to cancer risk management advice, although different participants received different advice. All participants also communicated any cancer risk management advice to first-degree relatives, motivated by protecting them, but information communicated was not always consistent with advice received. CONCLUSIONS: Our study findings highlight the variability in patients' interpretations of their diagnosis, cancer risk management and family communication when a diagnosis of SLS is received, and provide novel insights into how healthcare professionals can better support patients with SLS.