Samples were obtained from Envoi Specialist Pathology (Envoi) Brisbane, Australia, over a six-year period and are part of two previously published series [10, 11]. Envoi Specialist Pathology is a community based specialist gastroenterology practice. These series include polyps and cancers removed both endoscopically and surgically. Tissue from Envoi was embedded in formalin fixed paraffin embedded (FFPE) blocks, with DNA extracted using chelex, as previously reported . Cancers were obtained in a fresh state from patients undergoing surgery at the Royal Brisbane and Women’s Hospital, Brisbane, Australia, and from FFPE blocks at Envoi. Fresh samples were extracted using salt precipitation  and FFPE samples were extracted using chelex. For the control cohort, blood samples were taken from consenting patients who presented to gastroenterology clinics in Brisbane for investigation of symptoms and in whom subsequent colonoscopy showed no polyps or cancer.
Each sample was review by independently by two expert pathologists. Criteria for the diagnosis of a traditional serrated adenoma can be found in Bettington et al., 2015 . Criteria for the diagnosis of a dysplastic sessile serrated adenoma can be found in Bettington et al., 2017 .”
BRAF and CIMP analysis
The BRAF V600E mutation was assessed in each sample using allelic discrimination as previously reported . We assessed CIMP status using a methylation specific PCR with a marker panel consisting of NEUROG1, SOCS1, CACNAIG, IGF2 and RUNX3 as reported by Weisenberger and colleagues . To avoid the potential confounding of MLH1 loss secondary to Lynch Syndrome, only polyps and cancers bearing the BRAFV600E mutation were included. BRAF mutation has previously been shown to be an excellent marker of somatic MLH1 loss due to promoter hypermethylatioon .
MLH1 methylation and immunohistochemical analysis
For SSAD,TSA and cancer cohorts, MLH1 methylation was determined by bisulfite conversion, followed by methylation specific qPCR as previously reported . MLH1 protein expression was assessed by immunohistochemistry using previously reported methods , staining patterns were analyzed by an experienced gastrointestinal pathologist (MB).
SNP genotyping analysis
MLH1–93 genotypes were determine by high resolution melt analysis using 2.4 mM MgCl2, 0.24 mM dNTP, 0.24uM forward primer (5-‘TGACTGGCATTCAAGCTGTC-3’), 0.24uM reverse primer (5’-TTCAGCCAATCACCTCAGTG-3′), 0.24uM SYTO9, 1X DNA polymerase GoBuffer (Promega, Wisconsin USA), 1 unit GoTaq DNA Polymerase (Promega, Wisconsin USA) and 1 ng template DNA. The PCR thermal conditions were 95 °C for 120 s; 40 cycles of: 94 °C for 30s, 60 °C for 30s, 72 °C for 45 s followed by 95 °C for 300 s, 50 °C for 120 s and high resolution melt from 75 °C to 87 °C ramping by 0.2 °C / step) and consequent high resolution melt profile analysis. High resolution melt profile was confirmed using Sanger sequencing (Forward primer: 5’ TCTGCTCCTATTGGCTGGAT3’, Reverse primer: 5’ CCCTCCGTACCAGTTCTCAA3’).
Statistical analysis was carried out in GraphPad Prism 7. For categorical variables, a χ2 test was used for contingencies >2 × 2, with Fishers Exact test used for 2 × 2 contingencies. For percentage of methylated reference comparisons, a Mann-Whittey-U test was used. The null hypothesis was rejected at p < 0.05.
The study was approved by the QIMR Berghofer Medical Research Institute Human Research Ethics Committee and the Royal Brisbane and Women’s Hospital Ethics Committee. All participants gave informed written consent prior to participation in this study.