The first screen comprised 74 individuals with SPS selected based on early age at diagnosis, high numbers of serrated polyps throughout the colon and having a first-degree relative with SPS or CRC ( ...table 1 ) and consisted of WES (n=58; Agilent XT SureSelect_V4 52 Mb capture, 100 bp paired-end sequencing on a HiSeq2500 to a mean depth of 100×) and whole-genome sequencing (WGS; n=16; 150 bp paired-end reads using Illumina Hi-Seq X Ten sequencer to average 30× coverage). Table 1 SPS cases All N=295 Whole-exome sequencing N=58 Whole-genome sequencing N=16 HRM N=221 Families 287 55 12 221 Females 188 (63.7%) 36 (62.1%) 14 (87.5%) 138 (62.4%) Mean age at diagnosis+-SD (years) 48.1+-14.5 47.2+-14.4 39.6+-15.8 48.4+-14.5 Age at diagnosis range (years) 15-76 18-70 25-67 15-76 Minimum polyp count+-SD (range) 36.9+-28.4 (5-130) 50.8+-33.6 (7-130) 33.4+-21.9 (5-100) 28.4+-19.8 Colorectal cancer-affected 85 (28.8%) 24 (41.4%) 4 (25%) 57 (25.8%) HRM, high resolution melting; SPS, serrated polyposis syndrome. Table 2 Age Dx Serrated polyp count Variant rs number ESP6500si_EA PolyPhen2_HDIV SIFT FATHMM CADD_phred 18 >100 RNF43 NM_017763:exon6:c.C640G:p.L214V rs200626293 0.0003 Damaging Tolerated Tolerated 18.97 57 34 RNF43 NM_017763:exon4:c.C443G:p.A148G rs142178517 0.0001 Damaging Tolerated Tolerated 26.8 A second targeted genetic screen was performed specifically testing for the...
ObjectiveGermline pathogenic variants (PVs) in the DNA mismatch repair (MMR) genes and in the base excision repair gene MUTYH underlie hereditary colorectal cancer (CRC) and polyposis syndromes. We ...evaluated the robustness and discriminatory potential of tumour mutational signatures in CRCs for identifying germline PV carriers.DesignWhole-exome sequencing of formalin-fixed paraffin-embedded (FFPE) CRC tissue was performed on 33 MMR germline PV carriers, 12 biallelic MUTYH germline PV carriers, 25 sporadic MLH1 methylated MMR-deficient CRCs (MMRd controls) and 160 sporadic MMR-proficient CRCs (MMRp controls) and included 498 TCGA CRC tumours. COSMIC V3 single base substitution (SBS) and indel (ID) mutational signatures were assessed for their ability to differentiate CRCs that developed in carriers from non-carriers.ResultsThe combination of mutational signatures SBS18 and SBS36 contributing >30% of a CRC’s signature profile was able to discriminate biallelic MUTYH carriers from all other non-carrier control CRCs with 100% accuracy (area under the curve (AUC) 1.0). SBS18 and SBS36 were associated with specific MUTYH variants p.Gly396Asp (p=0.025) and p.Tyr179Cys (p=5×10-5), respectively. The combination of ID2 and ID7 could discriminate the 33 MMR PV carrier CRCs from the MMRp control CRCs (AUC 0.99); however, SBS and ID signatures, alone or in combination, could not provide complete discrimination (AUC 0.79) between CRCs from MMR PV carriers and sporadic MMRd controls.ConclusionAssessment of SBS and ID signatures can discriminate CRCs from biallelic MUTYH carriers and MMR PV carriers from non-carriers with high accuracy, demonstrating utility as a potential diagnostic and variant classification tool.
Mucinous differentiation is associated with both CpG island methylator phenotype and microsatellite instability in colorectal cancer. The mucinous phenotype derives from abundant expression of the ...colonic goblet cell mucin, MUC2, and de novo expression of gastric foveolar mucin, MUC5AC. We, therefore, investigated the protein expression levels of MUC2 and MUC5AC, as well as MUC5B and MUC6, in molecular subtypes of colorectal cancer. Seven-hundred and twenty-two incident colorectal carcinomas occurring in 702 participants of the Melbourne Collaborative Cohort Study were characterized for methylator status, MLH1 methylation, somatic BRAF and KRAS mutations, microsatellite-instability status, MLH1, MSH2, MSH6, and PMS2 mismatch repair, and p53 protein expression, and their histopathology was reviewed. Protein expression levels of MUC2, MUC5AC, MUC5B, MUC6, and the putative mucin regulator CDX2 were compared with molecular and clinicopathological features of colorectal cancers using odds ratios and corresponding 95% confidence intervals. MUC2 overexpression (>25% positive tumor cells) was observed in 33% colorectal cancers, MUC5B expression in 53%, and de novo MUC5AC and MUC6 expression in 50% and 39%, respectively. Co-expression of two or more of the mucins was commonly observed. Expression of MUC2, MUC5AC and MUC6 was strongly associated with features associated with tumorigenesis via the serrated neoplasia pathway, including methylator positivity, somatic BRAF p.V600E mutation, and mismatch repair deficiency, as well as proximal location, poor differentiation, lymphocytic response, and increased T stage (all P<0.001). Overexpression was observed in tumors with and without mucinous differentiation. There were inverse associations between expression of all four mucins and p53 overexpression. CDX2 expression was inversely associated with MUC2, MUC5AC and MUC6 expression. Our results suggest that, in methylator-positive tumors, mucin genes on chromosome 11p15.5 region undergo increased expression via mechanisms other than direct regulation by CDX2.
KRAS-mutated carcinomas comprise 35–40% of all colorectal carcinomas but little is known about their characteristics. The aim of this study was to examine the pathological and molecular features of ...KRAS-mutated colorectal carcinomas and to compare them with other carcinoma subgroups. KRAS mutation testing was performed in 776 incident tumors from the Melbourne Collaborative Cohort Study. O6-methylguanine DNA methyltransferase (MGMT) status was assessed using both immunohistochemistry and MethyLight techniques. Microsatellite instability (MSI) phenotype and BRAF V600E mutation status were derived from earlier studies. Mutation in KRAS codon 12 or codon 13 was present in 28% of colorectal carcinomas. Compared with KRAS wild-type carcinomas, KRAS-mutated carcinomas were more frequently observed in contiguity with a residual polyp (38 vs 21%; P<0.001), demonstrated mucinous differentiation (46 vs 31%; P=0.001) and were associated with different MSI status (P<0.001) and with MGMT methylation (47 vs 21%; P=0.001). Compared with tumors demonstrating neither BRAF nor KRAS mutation, KRAS-mutated carcinomas showed more frequent location in the proximal colon (41 vs 27%; P=0.001), mucinous differentiation (46 vs 25%; P<0.001), presence of a contiguous polyp (38 vs 22%; P<0.001), MGMT methylation (47 vs 26%; P=0.01) and loss of MGMT immunohistochemical expression (27 vs 19%; P=0.02). KRAS-mutated carcinomas were distributed in a bimodal pattern along the proximal–distal axis of the colorectum. Compared with male subjects, female subjects were more likely to have KRAS-mutated carcinoma in the transverse colon and descending colon (39 vs 15%; P=0.02). No difference in overall survival was observed in patients according to their tumor KRAS mutation status. In summary, KRAS-mutated carcinomas frequently develop in contiguity with a residual polyp and show molecular features distinct from other colorectal carcinomas, in particular from tumors with neither BRAF nor KRAS mutation.
ABSTRACT
We studied 17,576 members of 166 MLH1 and 224 MSH2 mutation‐carrying families from the Colon Cancer Family Registry. Average cumulative risks of colorectal cancer (CRC), endometrial cancer ...(EC), and other cancers for carriers were estimated using modified segregation analysis conditioned on ascertainment criteria. Heterogeneity in risks was investigated using a polygenic risk modifier. Average CRC cumulative risks at the age of 70 years (95% confidence intervals) for MLH1 and MSH2 mutation carriers, respectively, were estimated to be 34% (25%–50%) and 47% (36%–60%) for male carriers and 36% (25%–51%) and 37% (27%–50%) for female carriers. Corresponding EC risks were 18% (9.1%–34%) and 30% (18%–45%). A high level of CRC risk heterogeneity was observed (P < 0.001), with cumulative risks at the age of 70 years estimated to follow U‐shaped distributions. For example, 17% of male MSH2 mutation carriers have estimated lifetime risks of 0%–10% and 18% have risks of 90%–100%. Therefore, average risks are similar for the two genes but there is so much individual variation about the average that large proportions of carriers have either very low or very high lifetime cancer risks. Our estimates of CRC and EC cumulative risks for MLH1 and MSH2 mutation carriers are the most precise currently available.
Our estimates of colorectal and endometrial cancer risks for MLH1 and MSH2 mutation carriers are the most precise currently available. Average risks are similar for the two genes but there is so much individual variation about the average that a sizeable proportion of carries are almost certain to develop cancer whereas another sizeable proportion only have population‐level risks.
We report an autosomal recessive, multi-organ tumor predisposition syndrome, caused by bi-allelic loss-of-function germline variants in the base excision repair (BER) gene MBD4. We identified five ...individuals with bi-allelic MBD4 variants within four families and these individuals had a personal and/or family history of adenomatous colorectal polyposis, acute myeloid leukemia, and uveal melanoma. MBD4 encodes a glycosylase involved in repair of G:T mismatches resulting from deamination of 5′-methylcytosine. The colorectal adenomas from MBD4-deficient individuals showed a mutator phenotype attributable to mutational signature SBS1, consistent with the function of MBD4. MBD4-deficient polyps harbored somatic mutations in similar driver genes to sporadic colorectal tumors, although AMER1 mutations were more common and KRAS mutations less frequent. Our findings expand the role of BER deficiencies in tumor predisposition. Inclusion of MBD4 in genetic testing for polyposis and multi-tumor phenotypes is warranted to improve disease management.
Background Preventive programs for individuals who have high lifetime risks of colorectal cancer may reduce disease morbidity and mortality. Thus, it is important to identify the factors that are ...associated with hereditary colorectal cancer and to monitor the effects of tailored surveillance. In particular, patients with Lynch syndrome, hereditary nonpolyposis colorectal cancer (HNPCC), have an increased risk to develop colorectal cancer at an early age. The syndrome is explained by germline mutations in DNA mismatch repair (MMR) genes, and there is a need for diagnostic tools to preselect patients for genetic testing to diagnose those with HNPCC. Methods Patients (n = 112) from 285 families who were counseled between 1990 and 2005 at a clinic for patients at high risk for HNPCC were selected for screening to detect mutations in MMR genes MLH1, MSH2, MSH6, and PMS2 based on family history, microsatellite instability (MSI), and immunohistochemical analysis of MMR protein expression. Tumors were also screened for BRAF V600E mutations; patients with the mutation were considered as non-HNPCC. Results Among the 112 patients who were selected for screening, 69 had germline MMR mutations (58 pathogenic and 11 of unknown biologic relevance). Sixteen of the 69 mutations (23%) were missense mutations. Among patients with MSI-positive tumors, pathogenic MMR mutations were found in 38 of 43 (88%) of patients in families who met Amsterdam criteria and in 13 of 22 (59%) of patients in families who did not. Among patients with MSI-negative tumors, pathogenic MMR mutations were found in 5 of 17 (29%) of families meeting Amsterdam criteria and in 1 of 30 (3%) of non-Amsterdam families with one patient younger than age 50 years. In three patients with MSI-negative tumors who had pathogenic mutations in MLH1 or MSH6, immunohistochemistry showed loss of the mutated protein. Conclusion Our findings suggest that missense MMR gene mutations are common in HNPCC and that germline MMR mutations are also found in patients with MSI-negative tumors.
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).
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.
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).
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.
5604
Background: In the single arm phase 2 PHAEDRA trial, MMR deficiency (dMMR) was predictive of response to durvalumab (1500mg IV Q4W), with an objective tumor response rate (OTR; defined by ...iRECIST) of 47% in dMMR compared with 3% in MMR-proficient (pMMR) advanced endometrial cancer (AEC). This substudy of the PHAEDRA trial investigates MMR molecular subtypes and other genomic tumor features and their correlation with treatment outcomes. Methods: Testing was performed to determine molecular subtypes of dMMR, including germline pathogenic variants in DNA MMR genes (Lynch syndrome), somatic biallelic MMR gene inactivation through somatic mutation and somatic hypermethylation of the MLH1 gene promoter. DNA from formalin-fixed paraffin-embedded tumor tissue and matched peripheral blood was available from 41/71 (25 dMMR, 16 pMMR) participants for testing on a custom capture-based 298-gene targeted panel (2.005Mb) including the MMR genes and key somatic AEC driver genes. The derived tumor genomic features included microsatellite instability (MSI), tumor mutational burden (TMB), COSMIC v.3.2 tumor mutational signatures and insertion/deletion (Indel) somatic mutation count. Results: Of the 71 patients recruited, 35 were dMMR and 36 were pMMR. Median follow-up was 44 vs 52 months in dMMR vs pMMR participants, respectively. The dMMR molecular subtypes were 4 (11.4%) Lynch syndrome, 4 (11.4%) somatic MMR mutation, 25 (71.4%) MLH1 methylated and 2 (5.7%) dMMR-uncategorised. The OTR rate was 100% (4/4; 95%CI 40-100%) for Lynch, 75% (3/4; 95%CI 22-99%) for somatic MMR mutations and 40% (10/25; 95%CI 22-61%) for MLH1 methylated groups. The median TMB (assessed in 41/71) was higher in those with a confirmed radiological response (37, IQR:26-50) vs non-responders (16, IQR:9-25; p < 0.001). Within the MLH1 methylated group, TMB was also higher in responders vs non-responders (40 v 21; p = 0.03). Somatic mutations in KRAS, PTEN, PIK3CA, ARID1A and TP53 were not associated with OTR rate. Conclusions: dMMR- MLH1 methylated AEC demonstrated greater heterogeneity in OTR to single agent durvalumab than the dMMR-Lynch and dMMR-somatic MMR mutation molecular subtypes. Higher TMB was seen in responders, and specifically within dMMR- MLH1 methylated responders, compared to non-responders. Clinical trial information: ANZGOG1601.
Ethanol in alcoholic beverages is a causative agent for colorectal cancer. Colorectal cancer is a biologically heterogeneous disease, and molecular subtypes defined by the presence of somatic ...mutations in BRAF and KRAS are known to exist. We examined associations between lifetime alcohol intake and molecular and anatomic subtypes of colorectal cancer. We calculated usual alcohol intake for 10-year periods from age 20 using recalled frequency and quantity of beverage-specific consumption for 38,149 participants aged 40-69 years from the Melbourne Collaborative Cohort Study. Cox regression was performed to derive hazard ratios (HRs) and 95% confidence intervals (CIs) for the association between lifetime alcohol intake and colorectal cancer risk. Heterogeneity in the HRs across subtypes of colorectal cancer was assessed. A positive dose-dependent association between lifetime alcohol intake and overall colorectal cancer risk (mean follow-up=14.6 years; n=596 colon and n=326 rectal cancer) was observed (HR=1.08, 95% CI: 1.04-1.12 per 10 g/day increment). The risk was greater for rectal than colon cancer (p(homogeneity)=0.02). Alcohol intake was associated with increased risks of KRAS+ (HR=1.07, 95% CI: 1.00-1.15) and BRAF-/KRAS- (HR=1.05, 95% CI: 1.00-1.11) but not BRAF+ tumors (HR=0.89, 95% CI: 0.78-1.01; p(homogeneity)=0.01). Alcohol intake is associated with an increased risk of KRAS+ and BRAF-/KRAS- tumors originating via specific molecular pathways including the traditional adenoma-carcinoma pathway but not with BRAF+ tumors originating via the serrated pathway. Therefore, limiting alcohol intake from a young age might reduce colorectal cancer originating via the traditional adenoma-carcinoma pathway.
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