Medicine (RMH) - Research Publications
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ItemInherited BRCA1 and RNF43 pathogenic variants in a familial colorectal cancer type X family(SPRINGER, 2024-03)Genetic susceptibility to familial colorectal cancer (CRC), including for individuals classified as Familial Colorectal Cancer Type X (FCCTX), remains poorly understood. We describe a multi-generation CRC-affected family segregating pathogenic variants in both BRCA1, a gene associated with breast and ovarian cancer and RNF43, a gene associated with Serrated Polyposis Syndrome (SPS). A single family out of 105 families meeting the criteria for FCCTX (Amsterdam I family history criteria with mismatch repair (MMR)-proficient CRCs) recruited to the Australasian Colorectal Cancer Family Registry (ACCFR; 1998-2008) that underwent whole exome sequencing (WES), was selected for further testing. CRC and polyp tissue from four carriers were molecularly characterized including a single CRC that underwent WES to determine tumor mutational signatures and loss of heterozygosity (LOH) events. Ten carriers of a germline pathogenic variant BRCA1:c.2681_2682delAA p.Lys894ThrfsTer8 and eight carriers of a germline pathogenic variant RNF43:c.988 C > T p.Arg330Ter were identified in this family. Seven members carried both variants, four of which developed CRC. A single carrier of the RNF43 variant met the 2019 World Health Organization (WHO2019) criteria for SPS, developing a BRAF p.V600 wildtype CRC. Loss of the wildtype allele for both BRCA1 and RNF43 variants was observed in three CRC tumors while a LOH event across chromosome 17q encompassing both genes was observed in a CRC. Tumor mutational signature analysis identified the homologous recombination deficiency (HRD)-associated COSMIC signatures SBS3 and ID6 in a CRC for a carrier of both variants. Our findings show digenic inheritance of pathogenic variants in BRCA1 and RNF43 segregating with CRC in a FCCTX family. LOH and evidence of BRCA1-associated HRD supports the importance of both these tumor suppressor genes in CRC tumorigenesis.
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ItemIntratumoral presence of the genotoxic gut bacteria pks+ E. coli, Enterotoxigenic Bacteroides fragilis, and Fusobacterium nucleatum and their association with clinicopathological and molecular features of colorectal cancer(Springer Nature, 2024)Background: This study aimed to investigate clinicopathological and molecular tumour features associated with intratumoral pks+ Escherichia coli (pks+E.coli+), pks+E.coli- (non-E.coli bacteria harbouring the pks island), Enterotoxigenic Bacteroides fragilis (ETBF) and Fusobacterium nucleatum (F. nucleatum). Methods: We screened 1697 tumour-derived DNA samples from the Australasian Colorectal Cancer Family Registry, Melbourne Collaborative Cohort Study and the ANGELS study using targeted PCR. Results: Pks+E.coli+ was associated with male sex (P < 0.01) and APC:c.835-8 A > G somatic mutation (P = 0.03). The association between pks+E.coli+ and APC:c.835-8 A > G was specific to early-onset CRCs (diagnosed<45years, P = 0.02). The APC:c.835-A > G was not associated with pks+E.coli- (P = 0.36). F. nucleatum was associated with DNA mismatch repair deficiency (MMRd), BRAF:c.1799T>A p.V600E mutation, CpG island methylator phenotype, proximal tumour location, and high levels of tumour infiltrating lymphocytes (Ps < 0.01). In the stratified analysis by MMRd subgroups, F. nucleatum was associated with Lynch syndrome, MLH1 methylated and double MMR somatic mutated MMRd subgroups (Ps < 0.01). Conclusion: Intratumoral pks+E.coli+ but not pks+E.coli- are associated with CRCs harbouring the APC:c.835-8 A > G somatic mutation, suggesting that this mutation is specifically related to DNA damage from colibactin-producing E.coli exposures. F. nucleatum was associated with both hereditary and sporadic MMRd subtypes, suggesting the MMRd tumour microenvironment is important for F. nucleatum colonisation irrespective of its cause.
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ItemDNA Mismatch Repair Gene Variant Classification: Evaluating the Utility of Somatic Mutations and Mismatch Repair Deficient Colonic Crypts and Endometrial Glands(MDPI, 2023-10)Germline pathogenic variants in the DNA mismatch repair (MMR) genes (Lynch syndrome) predispose to colorectal (CRC) and endometrial (EC) cancer. Lynch syndrome specific tumor features were evaluated for their ability to support the ACMG/InSiGHT framework in classifying variants of uncertain clinical significance (VUS) in the MMR genes. Twenty-eight CRC or EC tumors from 25 VUS carriers (6xMLH1, 9xMSH2, 6xMSH6, 4xPMS2), underwent targeted tumor sequencing for the presence of microsatellite instability/MMR-deficiency (MSI-H/dMMR) status and identification of a somatic MMR mutation (second hit). Immunohistochemical testing for the presence of dMMR crypts/glands in normal tissue was also performed. The ACMG/InSiGHT framework reclassified 7/25 (28%) VUS to likely pathogenic (LP), three (12%) to benign/likely benign, and 15 (60%) VUS remained unchanged. For the seven re-classified LP variants comprising nine tumors, tumor sequencing confirmed MSI-H/dMMR (8/9, 88.9%) and a second hit (7/9, 77.8%). Of these LP reclassified variants where normal tissue was available, the presence of a dMMR crypt/gland was found in 2/4 (50%). Furthermore, a dMMR endometrial gland in a carrier of an MSH2 exon 1-6 duplication provides further support for an upgrade of this VUS to LP. Our study confirmed that identifying these Lynch syndrome features can improve MMR variant classification, enabling optimal clinical care.
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ItemNo Preview AvailableElucidating the Risk of Colorectal Cancer for Variants in Hereditary Colorectal Cancer Genes(W B SAUNDERS CO-ELSEVIER INC, 2023-10)
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ItemA mosaic pathogenic variant in MSH6 causes MSH6-deficient colorectal and endometrial cancer in a patient classified as suspected Lynch syndrome: a case report(SPRINGER, 2023-10)Germline pathogenic variants in the DNA mismatch repair (MMR) genes (Lynch syndrome) predispose to colorectal (CRC) and endometrial (EC) cancer. However, mosaic variants in the MMR genes have been rarely described. We identified a likely de novo mosaic MSH6:c.1135_1139del p.Arg379* pathogenic variant in a patient diagnosed with suspected Lynch syndrome/Lynch-like syndrome. The patient developed MSH6-deficient EC and CRC at 54 and 58 years of age, respectively, without a detectable germline MMR pathogenic variant. Multigene panel sequencing of tumor and blood-derived DNA identified an MSH6 somatic mutation (MSH6:c.1135_1139del p.Arg379*) common to both the EC and CRC, raising suspicion of mosaicism. A droplet digital polymerase chain reaction (ddPCR) assay detected the MSH6 variant at 5.34% frequency in normal colonic tissue, 3.49% in saliva and 1.64% in blood DNA, demonstrating the presence of the MSH6 variant in all three germ layers. This study highlights the utility of tumor sequencing to guide sensitive ddPCR testing to detect low-level mosaicism in the MMR genes. Further investigation of the prevalence of MMR mosaicism is needed to inform routine diagnostic approaches and genetic counselling.
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ItemNo Preview AvailableIdentifying 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(BMC, 2023-06-03)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.
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ItemNo Preview AvailableGermline mutations in WNK2 could be associated with serrated polyposis syndrome(BMJ PUBLISHING GROUP, 2023-06)BACKGROUND: Patients with serrated polyposis syndrome (SPS) have multiple and/or large serrated colonic polyps and higher risk for colorectal cancer. SPS inherited genetic basis is mostly unknown. We aimed to identify new germline predisposition factors for SPS by functionally evaluating a candidate gene and replicating it in additional SPS cohorts. METHODS: After a previous whole-exome sequencing in 39 SPS patients from 16 families (discovery cohort), we sequenced specific genes in an independent validation cohort of 211 unrelated SPS cases. Additional external replication was also available in 297 SPS cases. The WNK2 gene was disrupted in HT-29 cells by gene editing, and WNK2 variants were transfected using a lentiviral delivery system. Cells were analysed by immunoblots, real-time PCR and functional assays monitoring the mitogen-activated protein kinase (MAPK) pathway, cell cycle progression, survival and adhesion. RESULTS: We identified 2 rare germline variants in the WNK2 gene in the discovery cohort, 3 additional variants in the validation cohort and 10 other variants in the external cohorts. Variants c.2105C>T (p.Pro702Leu), c.4820C>T (p.Ala1607Val) and c.6157G>A (p.Val2053Ile) were functionally characterised, displaying higher levels of phospho-PAK1/2, phospho-ERK1/2, CCND1, clonogenic capacity and MMP2. CONCLUSION: After whole-exome sequencing in SPS cases with familial aggregation and replication of results in additional cohorts, we identified rare germline variants in the WNK2 gene. Functional studies suggested germline WNK2 variants affect protein function in the context of the MAPK pathway, a molecular hallmark in this disease.
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ItemNo Preview AvailableEvaluating Multiple Next-Generation Sequencing-Derived Tumor Features to Accurately Predict DNA Mismatch Repair Status(ELSEVIER SCIENCE INC, 2023-02)Identifying tumor DNA mismatch repair deficiency (dMMR) is important for precision medicine. Tumor features, individually and in combination, derived from whole-exome sequenced (WES) colorectal cancers (CRCs) and panel-sequenced CRCs, endometrial cancers (ECs), and sebaceous skin tumors (SSTs) were assessed for their accuracy in detecting dMMR. CRCs (n = 300) with WES, where mismatch repair status was determined by immunohistochemistry, were assessed for microsatellite instability (MSMuTect, MANTIS, MSIseq, and MSISensor), Catalogue of Somatic Mutations in Cancer tumor mutational signatures, and somatic mutation counts. A 10-fold cross-validation approach (100 repeats) evaluated the dMMR prediction accuracy for i) individual features, ii) Lasso statistical model, and iii) an additive feature combination approach. Panel-sequenced tumors (29 CRCs, 22 ECs, and 20 SSTs) were assessed for the top performing dMMR predicting features/models using these three approaches. For WES CRCs, 10 features provided >80% dMMR prediction accuracy, with MSMuTect, MSIseq, and MANTIS achieving ≥99% accuracy. The Lasso model achieved 98.3% accuracy. The additive feature approach, with three or more of six of MSMuTect, MANTIS, MSIseq, MSISensor, insertion-deletion count, or tumor mutational signature small insertion/deletion 2 + small insertion/deletion 7 achieved 99.7% accuracy. For the panel-sequenced tumors, the additive feature combination approach of three or more of six achieved accuracies of 100%, 95.5%, and 100% for CRCs, ECs, and SSTs, respectively. The microsatellite instability calling tools performed well in WES CRCs; however, an approach combining tumor features may improve dMMR prediction in both WES and panel-sequenced data across tissue types.
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ItemNo Preview AvailableA tumor focused approach to resolving the etiology of DNA mismatch repair deficient tumors classified as suspected Lynch syndrome(BMC, 2023-04-26)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.
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ItemBody Mass Index, sex, non-steroidal anti-inflammatory drug medications, smoking and alcohol are differentially associated with World Health Organisation criteria and colorectal cancer risk in people with Serrated Polyposis Syndrome: an Australian case-control study(BMC, 2022-11-26)OBJECTIVE: The unknown aetiology of Serrated Polyposis Syndrome (SPS) impedes risk prediction and prevention. We investigated risk factors for SPS, overall and stratified by World Health Organization (WHO)2010 clinical criteria and by colorectal cancer (CRC). METHOD: A retrospective case-control study involving a cross-sectional analysis from 350 unrelated individuals with SPS from the Genetics of Colonic Polyposis Study and 714 controls from the Australasian Colorectal Cancer Family Registry. Univariate and multivariate logistic regression modelling was used to determine the association between risk factors and SPS and risk factors associated with CRC in SPS. RESULTS: Female biological sex (odds ratio (OR) = 4.54; 95%Confidence interval (CI) = 2.77-7.45), increasing body mass index (BMI) at age 20 years (OR = 1.09; 95%CI = 1.04-1.13), hormone replacement therapy (OR = 0.44; 95%CI = 0.20.98), and increasing weekly folate intake (OR = 0.82; 95%CI = 0.75-0.90) were associated with SPS by multivariate analysis. Increasing weekly calcium intake (OR = 0.79; 95%CI = 0.64-0.97) and smoking > 10 cigarettes daily (OR = 0.45; 95%CI = 0.23-0.86) were associated with WHO criterion I only. The consumption of 1-100 g of alcohol per week (OR = 0.39; 95%CI = 0.18-0.83) was associated with WHO criterion III only. Smoking 1-5 cigarettes daily (OR = 2.35; 95%CI = 1.09-5.05), weekly non-steroidal anti-inflammatory drug (NSAIDs) intake (OR = 0.88; 95%CI = 0.78-0.99), and increased height (OR = 1.09; 95% = 1.05-1.13), were associated with SPS fulfilling both WHO criteria I and III. Moreover, weekly NSAIDs intake (OR = 0.81; 95%CI = 0.67-0.98) was associated with a reduced likelihood of CRC in SPS. CONCLUSION: We identified novel risk and potential protective factors associated with SPS, some specific for certain WHO2010 criteria. Weekly use of NSAIDs may reduce the risk of CRC in people with SPS.