Clinical Pathology - Research Publications
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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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ItemNo Preview AvailableDeciphering colorectal cancer genetics through multi-omic analysis of 100,204 cases and 154,587 controls of European and east Asian ancestries(NATURE PORTFOLIO, 2023-01)Colorectal cancer (CRC) is a leading cause of mortality worldwide. We conducted a genome-wide association study meta-analysis of 100,204 CRC cases and 154,587 controls of European and east Asian ancestry, identifying 205 independent risk associations, of which 50 were unreported. We performed integrative genomic, transcriptomic and methylomic analyses across large bowel mucosa and other tissues. Transcriptome- and methylome-wide association studies revealed an additional 53 risk associations. We identified 155 high-confidence effector genes functionally linked to CRC risk, many of which had no previously established role in CRC. These have multiple different functions and specifically indicate that variation in normal colorectal homeostasis, proliferation, cell adhesion, migration, immunity and microbial interactions determines CRC risk. Crosstissue analyses indicated that over a third of effector genes most probably act outside the colonic mucosa. Our findings provide insights into colorectal oncogenesis and highlight potential targets across tissues for new CRC treatment and chemoprevention strategies.
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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.
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ItemColorectal cancer incidences in Lynch syndrome: a comparison of results from the prospective lynch syndrome database and the international mismatch repair consortium(BMC, 2022-10-01)OBJECTIVE: To compare colorectal cancer (CRC) incidences in carriers of pathogenic variants of the MMR genes in the PLSD and IMRC cohorts, of which only the former included mandatory colonoscopy surveillance for all participants. METHODS: CRC incidences were calculated in an intervention group comprising a cohort of confirmed carriers of pathogenic or likely pathogenic variants in mismatch repair genes (path_MMR) followed prospectively by the Prospective Lynch Syndrome Database (PLSD). All had colonoscopy surveillance, with polypectomy when polyps were identified. Comparison was made with a retrospective cohort reported by the International Mismatch Repair Consortium (IMRC). This comprised confirmed and inferred path_MMR carriers who were first- or second-degree relatives of Lynch syndrome probands. RESULTS: In the PLSD, 8,153 subjects had follow-up colonoscopy surveillance for a total of 67,604 years and 578 carriers had CRC diagnosed. Average cumulative incidences of CRC in path_MLH1 carriers at 70 years of age were 52% in males and 41% in females; for path_MSH2 50% and 39%; for path_MSH6 13% and 17% and for path_PMS2 11% and 8%. In contrast, in the IMRC cohort, corresponding cumulative incidences were 40% and 27%; 34% and 23%; 16% and 8% and 7% and 6%. Comparing just the European carriers in the two series gave similar findings. Numbers in the PLSD series did not allow comparisons of carriers from other continents separately. Cumulative incidences at 25 years were < 1% in all retrospective groups. CONCLUSIONS: Prospectively observed CRC incidences (PLSD) in path_MLH1 and path_MSH2 carriers undergoing colonoscopy surveillance and polypectomy were higher than in the retrospective (IMRC) series, and were not reduced in path_MSH6 carriers. These findings were the opposite to those expected. CRC point incidence before 50 years of age was reduced in path_PMS2 carriers subjected to colonoscopy, but not significantly so.
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ItemDNA methylation-based signature of CD8+tumor-infiltrating lymphocytes enables evaluation of immune response and prognosis in colorectal cancer(BMJ PUBLISHING GROUP, 2021-09)BACKGROUND: Tumor-infiltrating lymphocytes (TILs), especially CD8+ TILs, can be used for predicting immunotherapy responsiveness and survival outcome. However, the evaluation of CD8+ TILs currently relies on histopathological methodology with high variability. We therefore aimed to develop a DNA methylation signature for CD8+ TILs (CD8+ MeTIL) that could evaluate immune response and prognosis in colorectal cancer (CRC). METHODS: A CD8+ MeTIL signature score was constructed by using CD8+ T cell-specific differentially methylated positions (DMPs) that were identified from Illumina EPIC methylation arrays. Immune cells, colon epithelial cells, and two CRC cohorts (n=282 and 335) were used to develop a PCR-based assay for quantitative analysis of DNA methylation at single-base resolution (QASM) to determine CD8 + MeTIL signature score. RESULTS: Three CD8+ T cell-specific DMPs were identified to construct the CD8+ MeTIL signature score, which showed a dramatic discriminability between CD8+ T cells and other cells. The QASM assay we developed for CD8+ MeTIL markers could measure CD8+ TILs distributions in a fully quantitative, accurate, and simple manner. The CD8+ MeTIL score determined by QASM assay showed a strong association with histopathology-based CD8+ TIL counts and a gene expression-based immune marker. Furthermore, the low CD8+ MeTIL score (enriched CD8+ TILs) was associated with MSI-H tumors and predicted better survival in CRC cohorts. CONCLUSIONS: This study developed a quantitative DNA methylation-based signature that was reliable to evaluate CD8+ TILs and prognosis in CRC. This approach has the potential to be a tool for investigations on CD8+ TILs and a biomarker for therapeutic approaches, including immunotherapy.
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ItemGenome-wide association study and meta-analysis in Northern European populations replicate multiple colorectal cancer risk loci(WILEY, 2018-02-01)Genome-wide association studies have been successful in elucidating the genetic basis of colorectal cancer (CRC), but there remains unexplained variability in genetic risk. To identify new risk variants and to confirm reported associations, we conducted a genome-wide association study in 1,701 CRC cases and 14,082 cancer-free controls from the Finnish population. A total of 9,068,015 genetic variants were imputed and tested, and 30 promising variants were studied in additional 11,647 cases and 12,356 controls of European ancestry. The previously reported association between the single-nucleotide polymorphism (SNP) rs992157 (2q35) and CRC was independently replicated (p = 2.08 × 10-4 ; OR, 1.14; 95% CI, 1.06-1.23), and it was genome-wide significant in combined analysis (p = 1.50 × 10-9 ; OR, 1.12; 95% CI, 1.08-1.16). Variants at 2q35, 6p21.2, 8q23.3, 8q24.21, 10q22.3, 10q24.2, 11q13.4, 11q23.1, 14q22.2, 15q13.3, 18q21.1, 20p12.3 and 20q13.33 were associated with CRC in the Finnish population (false discovery rate < 0.1), but new risk loci were not found. These results replicate the effects of multiple loci on the risk of CRC and identify shared risk alleles between the Finnish population isolate and outbred populations.
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ItemMendelian randomisation implicates hyperlipidaemia as a risk factor for colorectal cancer(WILEY, 2017-06-15)While elevated blood cholesterol has been associated with an increased risk of colorectal cancer (CRC) in observational studies, causality is uncertain. Here we apply a Mendelian randomisation (MR) analysis to examine the potential causal relationship between lipid traits and CRC risk. We used single nucleotide polymorphisms (SNPs) associated with blood levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) as instrumental variables (IV). We calculated MR estimates for each risk factor with CRC using SNP-CRC associations from 9,254 cases and 18,386 controls. Genetically predicted higher TC was associated with an elevated risk of CRC (odds ratios (OR) per unit SD increase = 1.46, 95% confidence interval [CI]: 1.20-1.79, p = 1.68 × 10-4 ). The pooled ORs for LDL, HDL, and TG were 1.05 (95% CI: 0.92-1.18, p = 0.49), 0.94 (95% CI: 0.84-1.05, p = 0.27), and 0.98 (95% CI: 0.85-1.12, p = 0.75) respectively. A genetic risk score for 3-hydoxy-3-methylglutaryl-coenzyme A reductase (HMGCR) to mimic the effects of statin therapy was associated with a reduced CRC risk (OR = 0.69, 95% CI: 0.49-0.99, p = 0.046). This study supports a causal relationship between higher levels of TC with CRC risk, and a further rationale for implementing public health strategies to reduce the prevalence of hyperlipidaemia.
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ItemPhysical activity and the risk of colorectal cancer in Lynch syndrome(WILEY, 2018-11-01)Greater physical activity is associated with a decrease in risk of colorectal cancer for the general population; however, little is known about its relationship with colorectal cancer risk in people with Lynch syndrome, carriers of inherited pathogenic mutations in genes affecting DNA mismatch repair (MMR). We studied a cohort of 2,042 MMR gene mutations carriers (n = 807, diagnosed with colorectal cancer), from the Colon Cancer Family Registry. Self-reported physical activity in three age-periods (20-29, 30-49 and ≥50 years) was summarized as average metabolic equivalent of task hours per week (MET-hr/week) during the age-period of cancer diagnosis or censoring (near-term exposure) and across all age-periods preceding cancer diagnosis or censoring (long-term exposure). Weighted Cox regression was used to estimate the hazard ratio (HR) and 95% confidence intervals (CI) for the association between physical activity and colorectal cancer risk. Near-term physical activity was associated with a small reduction in the risk of colorectal cancer (HR ≥35 vs. <3.5 MET-hr/week, 0.71; 95% CI, 0.53-0.96). The strength and direction of associations were similar for long-term physical activity, although the associations were not nominally significant. Our results suggest that physical activity is inversely associated with the risk of colorectal cancer for people with Lynch syndrome; however, further confirmation is warranted. The potential modifying effect of physical activity on colorectal cancer risk in people with Lynch syndrome could be useful for risk prediction and support counseling advice for lifestyle modification to reduce cancer risk.