Jihoon Eric Joo

The germline MLH1 c.-42 C > T (rs41285097) promoter variant has been identified in cases with MLH1-deficient colorectal or endometrial cancers but remains a variant of uncertain significance. Genetic testing identified two new MLH1 c.-42 C > T index cases from Australia and the USA. Clinicopathologic and molecular characterisation of tumour and non-neoplastic tissues was performed to investigate the potential mechanism of pathogenesis of this variant. The male Australian proband developed MLH1-deficient, BRAF p.V600 wildtype, CIMP-negative colon cancer at 61 years. MLH1 monoallelic methylation and a somatic pathogenic mutation, MLH1 c.1122_1126dup p.Asp376Valfs*27, were identified in his tumour. Droplet digital PCR (ddPCR) detected mosaic MLH1 methylation in normal colonic mucosa adjacent to the cancer (3.7%) with lower levels in blood (0.07%) and saliva (0.09%). The USA proband developed MLH1-deficient endometrial cancer at 38 years with MLH1 monoallelic methylation and loss-of-heterozygosity of the wildtype c.-42 C allele. No evidence of MLH1 methylation was found by ddPCR in normal tissues. MLH1 c.-42 C > T heterozygous relatives from both families had either no or extremely low levels of MLH1 methylation within blood or saliva. Allelic expression from MLH1 c.-42T was reduced to 70% relative to the wild-type allele in saliva from three heterozygotes. Three additional pedigrees were identified from the Colon Cancer Family Registry. Evaluation of combined multifactorial data from pooled informative index cases supports reclassification of this variant as “likely pathogenic” according to current ACMG/AMP mismatch repair gene-specific guidelines, though with likely reduced penetrance and/or modified phenotype. These findings highlight the clinical importance of identifying MLH1 c.-42 C > T to inform cancer risk management.

Adenomatous polyposis syndromes are hereditary conditions characterised by the development of multiple adenomas in the gastrointestinal tract, particularly in the colon and rectum, significantly increasing the risk of colorectal cancer and, in some cases, extra-colonic malignancies. These syndromes are caused by germline pathogenic variants (PVs) in genes involved in Wnt signalling and DNA repair. The main autosomal dominant adenomatous polyposis syndromes include familial adenomatous polyposis (FAP) and polymerase proofreading-associated polyposis (PPAP), caused by germline PVs in APC and the POLE and POLD1 genes, respectively. Autosomal recessive syndromes include those caused by biallelic PVs in the DNA mismatch repair genes MLH1, MSH2, MSH6, PMS2, MSH3 and probably MLH3, and in the base excision repair genes MUTYH, NTHL1 and MBD4. This review provides an in-depth discussion of the genetic and molecular mechanisms underlying hereditary adenomatous polyposis syndromes, their clinical presentations, tumour mutational signatures, and emerging approaches for the treatment of the associated cancers. Considerations for genetic testing are described, including post-zygotic mosaicism, non-coding PVs, the interpretation of variants of unknown significance and cancer risks associated with monoallelic variants in the recessive genes. Despite advances in genetic testing and the recent identification of new adenomatous polyposis genes, many cases of multiple adenomas remain genetically unexplained. Non-genetic factors, including environmental risk factors, prior oncologic treatments, and bacterial genotoxins colonising the intestine - particularly colibactin-producing Escherichia coli - have emerged as alternative pathogenic mechanisms.

Background: Colorectal cancers (CRCs) from people with biallelic germline likely pathogenic/pathogenic variants in MUTYH or NTHL1 exhibit specific single base substitution (SBS) mutational signatures, namely combined SBS18 and SBS36 (SBS18+SBS36), and SBS30, respectively. The aim was to determine if adenomas from biallelic cases demonstrated these mutational signatures at diagnostic levels. Methods: Whole-exome sequencing of FFPE tissue and matched blood-derived DNA was performed on 9 adenomas and 15 CRCs from 13 biallelic MUTYH cases, on 7 adenomas and 2 CRCs from 5 biallelic NTHL1 cases and on 27 adenomas and 26 CRCs from 46 non-hereditary (sporadic) participants. All samples were assessed for COSMIC v3.2 SBS mutational signatures. Results: In biallelic MUTYH cases, SBS18+SBS36 signature proportions in adenomas (mean±standard deviation, 65.6%±29.6%) were not significantly different to those observed in CRCs (76.2%±20.5%, p-value =0.37), but were significantly higher compared with non-hereditary adenomas (7.6%±7.0%, p-value =3.4x10 ^-4 ). Similarly, in biallelic NTHL1 cases, SBS30 signature proportions in adenomas (74.5%±9.4%) were similar to those in CRCs (78.8%±2.4%) but significantly higher compared with non-hereditary adenomas (2.8%±3.6%, p-value =5.1x10 ^-7 ). Additionally, a compound heterozygote with the c.1187G>A p.(Gly396Asp) pathogenic variant and the c.533G>C p.(Gly178Ala) variant of unknown significance (VUS) in MUTYH demonstrated high levels of SBS18+SBS36 in four adenomas and one CRC, providing evidence for reclassification of the VUS to pathogenic. Conclusions: SBS18+SBS36 and SBS30 were enriched in adenomas at comparable proportions observed in CRCs from biallelic MUTYH and biallelic NTHL1 cases, respectively. Therefore, testing adenomas may improve the identification of biallelic cases and facilitate variant classification, ultimately enabling opportunities for CRC prevention.

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.

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.

Carriers of germline biallelic pathogenic variants in the MUTYH gene have a high risk of colorectal cancer. We test 5649 colorectal cancers to evaluate the discriminatory potential of a tumor mutational signature specific to MUTYH for identifying biallelic carriers and classifying variants of uncertain clinical significance (VUS). Using a tumor and matched germline targeted multi-gene panel approach, our classifier identifies all biallelic MUTYH carriers and all known non-carriers in an independent test set of 3019 colorectal cancers (accuracy = 100% (95% confidence interval 99.87–100%)). All monoallelic MUTYH carriers are classified with the non-MUTYH carriers. The classifier provides evidence for a pathogenic classification for two VUS and a benign classification for five VUS. Somatic hotspot mutations KRAS p.G12C and PIK3CA p.Q546K are associated with colorectal cancers from biallelic MUTYH carriers compared with non-carriers (p = 2 × 10⁻²³ and p = 6 × 10⁻¹¹, respectively). Here, we demonstrate the potential application of mutational signatures to tumor sequencing workflows to improve the identification of biallelic MUTYH carriers.

Genetic variants in FOXO3 are associated with longevity. Here, we assessed whether blood DNA methylation at FOXO3 was associated with cancer risk, survival, and mortality. We used data from eight prospective case–control studies of breast (n = 409 cases), colorectal (n = 835), gastric (n = 170), kidney (n = 143), lung (n = 332), prostate (n = 869), and urothelial (n = 428) cancer and B-cell lymphoma (n = 438). Case–control pairs were matched on age, sex, country of birth, and smoking (lung cancer study). Conditional logistic regression was used to assess associations between cancer risk and methylation at 45 CpGs of FOXO3 included on the HumanMethylation450 assay. Mixed-effects Cox models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for associations with cancer survival (total n = 2286 deaths). Additionally, using data from 1088 older participants, we assessed associations of FOXO3 methylation with overall and cause-specific mortality (n = 354 deaths). Methylation at a CpG in the first exon region of FOXO3 (6:108882981) was associated with gastric cancer survival (HR = 2.39, 95% CI: 1.60–3.56, p = 1.9 × 10⁻⁵). Methylation at three CpGs in TSS1500 and gene body was associated with lung cancer survival (p < 6.1 × 10⁻⁵). We found no evidence of associations of FOXO3 methylation with cancer risk and mortality. Our findings may contribute to understanding the implication of FOXO3 in longevity.