Pathology - Research Publications
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ItemNo Preview AvailableExpression of MUC2, MUC5AC, MUC5B, and MUC6 mucins in colorectal cancers and their association with the CpG island methylator phenotype(ELSEVIER SCIENCE INC, 2013-12)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.
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ItemNo Preview AvailableColorectal carcinomas with KRAS mutation are associated with distinctive morphological and molecular features(NATURE PUBLISHING GROUP, 2013-06)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. O(6)-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.
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ItemProspective validation of the breast cancer risk prediction model BOADICEA and a batch-mode version BOADICEACentre(NATURE PUBLISHING GROUP, 2013-09-03)BACKGROUND: Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) is a risk prediction algorithm that can be used to compute estimates of age-specific risk of breast cancer. It is uncertain whether BOADICEA performs adequately for populations outside the United Kingdom. METHODS: Using a batch mode version of BOADICEA that we developed (BOADICEACentre), we calculated the cumulative 10-year invasive breast cancer risk for 4176 Australian women of European ancestry unaffected at baseline from 1601 case and control families in the Australian Breast Cancer Family Registry. Based on 115 incident breast cancers, we investigated calibration, discrimination (using receiver-operating characteristic (ROC) curves) and accuracy at the individual level. RESULTS: The ratio of expected to observed number of breast cancers was 0.92 (95% confidence interval (CI) 0.76-1.10). The E/O ratios by subgroups of the participant's relationship to the index case and by the reported number of affected relatives ranged between 0.83 and 0.98 and all 95% CIs included 1.00. The area under the ROC curve was 0.70 (95% CI 0.66-0.75) and there was no evidence of systematic under- or over-dispersion (P=0.2). CONCLUSION: BOADICEA is well calibrated for Australian women, and had good discrimination and accuracy at the individual level.
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ItemNo Preview AvailableGermline HOXB13 p.Gly84Glu mutation and risk of colorectal cancer(ELSEVIER SCI LTD, 2013-08)INTRODUCTION: The HOXB13 pGly84Glu mutation has recently been associated with an increased risk of prostate cancer but the association of other cancer sites with this allele has not been assessed. Data has suggested that HOXB13 expression levels are decreased in colorectal cancer (CRC) cell lines indicating this gene may be involved in colorectal tumourigenesis. METHODS: To evaluate a potential association of this mutation with CRC, we genotyped the mutation in 2695 CRC cases and 4593 controls from population-based registries in Canada and Australia. RESULTS: The HOXB13 pGly84Glu mutation was more common in CRC cases than controls (0.48% vs. 0.17%, P=0.02) indicating a significant association between the HOXB13 variant and CRC risk (OR=2.8; 95%CI: 1.2-6.8). This association was attenuated but remained significant with the inclusion of previously published and publicly available genotype data. Pedigree analysis of cases and controls revealed that 7/21 HOXB13 mutation carriers had a family history of prostate cancer. DISCUSSION: This report is the first to suggest a risk of CRC associated with mutations in the HOXB13 gene. These findings require further validation but may be of importance in the screening and genetic counseling of families known to carry the HOXB13 pGly84Glu mutation.
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ItemPopulation-Based Estimate of Prostate Cancer Risk for Carriers of the HOXB13 Missense Mutation G84E(PUBLIC LIBRARY SCIENCE, 2013-02-15)The HOXB13 missense mutation G84E (rs138213197) is associated with increased risk of prostate cancer, but the current estimate of increased risk has a wide confidence interval (width of 95% confidence interval (CI) >200-fold) so the point estimate of 20-fold increased risk could be misleading. Population-based family studies can be more informative for estimating risks for rare variants, therefore, we screened for mutations in an Australian population-based series of early-onset prostate cancer cases (probands). We found that 19 of 1,384 (1.4%) probands carried the missense mutation, and of these, six (32%) had a family history of prostate cancer. We tested the 22 relatives of carriers diagnosed from 1998 to 2008 for whom we had a DNA sample, and found seven more carriers and one obligate carrier. The age-specific incidence for carriers was estimated to be, on average, 16.4 (95% CI 2.5-107.2) times that for the population over the time frame when the relatives were at risk prior to baseline. We then estimated the age and birth year- specific cumulative risk of prostate cancer (penetrance) for carriers. For example, the penetrance for an unaffected male carrier born in 1950 was 19% (95% CI 5-46%) at age 60 years, 44% (95% CI 18-74%) at age 70 years and 60% (95% CI 30-85%) at age 80 years. Our study has provided a population-based estimate of the average risk of prostate cancer for HOXB13 missense mutation G84E carriers that can be used to guide clinical practice and research. This study has also shown that the majority of hereditary prostate cancers due to the HOXB13 missense mutation are 'sporadic' in the sense that unselected cases with the missense mutation do not typically report having a family history of prostate cancer.
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ItemIdentification of a BRCA2-Specific Modifier Locus at 6p24 Related to Breast Cancer Risk(PUBLIC LIBRARY SCIENCE, 2013-03)Common genetic variants contribute to the observed variation in breast cancer risk for BRCA2 mutation carriers; those known to date have all been found through population-based genome-wide association studies (GWAS). To comprehensively identify breast cancer risk modifying loci for BRCA2 mutation carriers, we conducted a deep replication of an ongoing GWAS discovery study. Using the ranked P-values of the breast cancer associations with the imputed genotype of 1.4 M SNPs, 19,029 SNPs were selected and designed for inclusion on a custom Illumina array that included a total of 211,155 SNPs as part of a multi-consortial project. DNA samples from 3,881 breast cancer affected and 4,330 unaffected BRCA2 mutation carriers from 47 studies belonging to the Consortium of Investigators of Modifiers of BRCA1/2 were genotyped and available for analysis. We replicated previously reported breast cancer susceptibility alleles in these BRCA2 mutation carriers and for several regions (including FGFR2, MAP3K1, CDKN2A/B, and PTHLH) identified SNPs that have stronger evidence of association than those previously published. We also identified a novel susceptibility allele at 6p24 that was inversely associated with risk in BRCA2 mutation carriers (rs9348512; per allele HR = 0.85, 95% CI 0.80-0.90, P = 3.9 × 10(-8)). This SNP was not associated with breast cancer risk either in the general population or in BRCA1 mutation carriers. The locus lies within a region containing TFAP2A, which encodes a transcriptional activation protein that interacts with several tumor suppressor genes. This report identifies the first breast cancer risk locus specific to a BRCA2 mutation background. This comprehensive update of novel and previously reported breast cancer susceptibility loci contributes to the establishment of a panel of SNPs that modify breast cancer risk in BRCA2 mutation carriers. This panel may have clinical utility for women with BRCA2 mutations weighing options for medical prevention of breast cancer.
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ItemEpigenetic analysis leads to identification of HNF1B as a subtype-specific susceptibility gene for ovarian cancer(NATURE RESEARCH, 2013-03)HNF1B is overexpressed in clear cell epithelial ovarian cancer, and we observed epigenetic silencing in serous epithelial ovarian cancer, leading us to hypothesize that variation in this gene differentially associates with epithelial ovarian cancer risk according to histological subtype. Here we comprehensively map variation in HNF1B with respect to epithelial ovarian cancer risk and analyse DNA methylation and expression profiles across histological subtypes. Different single-nucleotide polymorphisms associate with invasive serous (rs7405776 odds ratio (OR)=1.13, P=3.1 × 10(-10)) and clear cell (rs11651755 OR=0.77, P=1.6 × 10(-8)) epithelial ovarian cancer. Risk alleles for the serous subtype associate with higher HNF1B-promoter methylation in these tumours. Unmethylated, expressed HNF1B, primarily present in clear cell tumours, coincides with a CpG island methylator phenotype affecting numerous other promoters throughout the genome. Different variants in HNF1B associate with risk of serous and clear cell epithelial ovarian cancer; DNA methylation and expression patterns are also notably distinct between these subtypes. These findings underscore distinct mechanisms driving different epithelial ovarian cancer histological subtypes.
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ItemFamily History of Colorectal Cancer in BRAF p.V600E-Mutated Colorectal Cancer Cases(AMER ASSOC CANCER RESEARCH, 2013-05)BACKGROUND: Previous reports suggest that relatives of colorectal cancer (CRC)-affected probands carrying the BRAF p.V600E mutation are at an increased risk of CRC and extracolonic cancers (ECC). In this study, we estimated the association between a family history of either CRC or ECC and risk of CRC with a BRAF p.V600E mutation. METHODS: Population-based CRC cases (probands, ages 18-59 years at diagnosis), recruited irrespective of family cancer history, were characterized for BRAF p.V600E mutation and mismatch repair (MMR) status. ORs and 95% confidence intervals (CI) were estimated using multivariable logistic regression. RESULTS: The 690 eligible probands showed a mean age at CRC diagnosis of 46.9 ± 7.8 years, with 313 (47.9%) reporting a family history of CRC and 53 (7.7%) that were BRAF-mutated. Probands with BRAF-mutated, MMR-proficient CRCs were less likely to have a family history of CRC than probands that were BRAF wild-type (OR, 0.46; 95% CI, 0.24-0.91; P = 0.03). For probands with a BRAF-mutated CRC, the mean age at diagnosis was greater for those with a CRC-affected first- or second-degree relative (49.3 ± 6.4 years) compared with those without a family history (43.8 ± 10.2 years; P = 0.04). The older the age at diagnosis of CRC with the BRAF p.V600E mutation, the more likely these probands were to show a family history of CRC (OR, 1.09 per year of age; 95% CI, 1.00-1.18; P = 0.04). CONCLUSIONS: Probands with early-onset, BRAF-mutated, and MMR-proficient CRC were less likely to have a family history of CRC than probands that were BRAF-wild-type. IMPACT: These findings provide useful insights for cancer risk assessment in families and suggest that familial or inherited factors are more important in early-onset, BRAF-wild-type CRC.
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ItemHi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2(BMC, 2013-11-08)BACKGROUND: Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour. METHODS: Here, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run. RESULTS: We observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing. CONCLUSIONS: These results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings.
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ItemNo Preview AvailableAssociation between hypermethylation of DNA repetitive elements in white blood cell DNA and early-onset colorectal cancer(TAYLOR & FRANCIS INC, 2013-07-01)Changes in the methylation levels of DNA from white blood cells (WBCs) are putatively associated with an elevated risk for several cancers. The aim of this study was to investigate the association between colorectal cancer (CRC) and the methylation status of three DNA repetitive elements in DNA from peripheral blood. WBC DNA from 539 CRC cases diagnosed before 60 years of age and 242 sex and age frequency-matched healthy controls from the Australasian Colorectal Cancer Family Registry were assessed for methylation across DNA repetitive elements Alu, LINE-1 and Sat2 using MethyLight. The percentage of methylated reference (PMR) of cases and controls was calculated for each marker. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using multivariable logistic regression adjusted for potential confounders. CRC cases demonstrated a significantly higher median PMR for LINE-1 (p < 0.001), Sat2 (p < 0.001) and Alu repeats (p = 0.02) when compared with controls. For each of the DNA repetitive elements, individuals with PMR values in the highest quartile were significantly more likely to have CRC compared with those in the lowest quartile (LINE-1 OR = 2.34, 95%CI = 1.48-3.70; p < 0.001, Alu OR = 1.83, 95%CI = 1.17-2.86; p = 0.01, Sat2 OR = 1.72, 95%CI = 1.10-2.71; p = 0.02). When comparing the OR for the PMR of each marker across subgroups of CRC, only the Alu marker showed a significant difference in the 5-fluoruracil treated and nodal involvement subgroups (both p = 0.002). This association between increasing methylation levels of three DNA repetitive elements in WBC DNA and early-onset CRC is novel and may represent a potential epigenetic biomarker for early CRC detection.