Sir Peter MacCallum Department of Oncology - Research Publications
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ItemNo Preview AvailableDNA methylation in ductal carcinoma in situ of the breast(BMC, 2013)Ductal carcinoma in situ (DCIS) is a non-obligate precursor lesion of invasive carcinoma of the breast. Current prognostic markers based on histopathological examination are unable to accurately predict which DCIS cases will progress to invasive carcinoma or recur after surgical excision. Epigenetic changes have been shown to be a significant driver of tumorigenesis, and DNA methylation of specific gene promoters provides predictive and prognostic markers in many types of cancer, including invasive breast cancer. In general, the spectrum of genes that are methylated in DCIS strongly resembles that seen in invasive ductal carcinoma. The identification of specific prognostic markers in DCIS remains elusive and awaits additional work investigating a large panel of methylatable genes by using sensitive and reproducible technologies. This review critically appraises the role of methylation in DCIS and its use as a biomarker.
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ItemA multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma(NATURE PORTFOLIO, 2013-04-15)Melanoma patients with BRAF mutations respond to treatment with vemurafenib, thus creating a need for accurate testing of BRAF mutation status. We carried out a blinded study to evaluate various BRAF mutation testing methodologies in the clinical setting. Formalin-fixed, paraffin-embedded melanoma samples were macrodissected before screening for mutations using Sanger sequencing, single-strand conformation analysis (SSCA), high resolution melting analysis (HRM) and competitive allele-specific TaqMan® PCR (CAST-PCR). Concordance of 100% was observed between the Sanger sequencing, SSCA and HRM techniques. CAST-PCR gave rapid and accurate results for the common V600E and V600K mutations, however additional assays are required to detect rarer BRAF mutation types found in 3-4% of melanomas. HRM and SSCA followed by Sanger sequencing are effective two-step strategies for the detection of BRAF mutations in the clinical setting. CAST-PCR was useful for samples with low tumour purity and may also be a cost-effective and robust method for routine diagnostics.
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ItemQuantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2 V617F mutant allele detection(BMC, 2013-04-24)BACKGROUND: The JAK2 V617F mutation is the most frequent somatic change in myeloproliferative neoplasms, making it an important tumour-specific marker for diagnostic purposes and for the detection of minimal residual disease. Sensitive quantitative assays are required for both applications, particularly for the monitoring of minimal residual disease, which requires not only high sensitivity but also very high specificity. METHODS: We developed a highly sensitive probe-free quantitative mutant-allele detection method, Quantitative Threefold Allele-Specific PCR (QuanTAS-PCR), that is performed in a closed-tube system, thus eliminating the manipulation of PCR products. QuantTAS-PCR uses a threefold approach to ensure allele-specific amplification of the mutant sequence: (i) a mutant allele-specific primer, (ii) a 3'dideoxy blocker to suppress false-positive amplification from the wild-type template and (iii) a PCR specificity enhancer, also to suppress false-positive amplification from the wild-type template. Mutant alleles were quantified relative to exon 9 of JAK2. RESULTS: We showed that the addition of the 3'dideoxy blocker suppressed but did not eliminate false-positive amplification from the wild-type template. However, the addition of the PCR specificity enhancer near eliminated false-positive amplification from the wild-type allele. Further discrimination between true and false positives was enabled by using the quantification cycle (Cq) value of a single mutant template as a cut-off point, thus enabling robust distinction between true and false positives. As 10,000 JAK2 templates were used per replicate, the assay had a sensitivity of 1/10(-4) per replicate. Greater sensitivity could be reached by increasing the number of replicates analysed. Variation in replicates when low mutant-allele templates were present necessitated the use of a statistics-based approach to estimate the load of mutant JAK2 copies. QuanTAS-PCR showed comparable quantitative results when validated against a commercial assay. CONCLUSIONS: QuanTAS-PCR is a simple, cost-efficient, closed-tube method for JAK2 V617F mutation quantification that can detect very low levels of the mutant allele, thus enabling analysis of minimal residual disease. The approach can be extended to the detection of other recurrent single nucleotide somatic changes in cancer.
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ItemTargeted-capture massively-parallel sequencing enables robust detection of clinically informative mutations from formalin-fixed tumours(NATURE PORTFOLIO, 2013-12-13)Massively parallel sequencing offers the ability to interrogate a tumour biopsy for multiple mutational changes. For clinical samples, methodologies must enable maximal extraction of available sequence information from formalin-fixed and paraffin-embedded (FFPE) material. We assessed the use of targeted capture for mutation detection in FFPE DNA. The capture probes targeted the coding region of all known kinase genes and selected oncogenes and tumour suppressor genes. Seven melanoma cell lines and matching FFPE xenograft DNAs were sequenced. An informatics pipeline was developed to identify variants and contaminating mouse reads. Concordance of 100% was observed between unfixed and formalin-fixed for reported COSMIC variants including BRAF V600E. mutations in genes not conventionally screened including ERBB4, ATM, STK11 and CDKN2A were readily detected. All regions were adequately covered with independent reads regardless of GC content. This study indicates that hybridisation capture is a robust approach for massively parallel sequencing of FFPE samples.
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ItemPIK3CA mutations are frequently observed in BRCAX but not BRCA2-associated male breast cancer(BMC, 2013)INTRODUCTION: Although a substantial proportion of male breast cancers (MBCs) are hereditary, the molecular pathways that are activated are unknown. We therefore examined the frequency and clinicopathological associations of the PIK3CA/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) pathways and their regulatory genes in familial MBC. METHODS: High resolution melting analysis and confirmatory sequencing was used to determine the presence of somatic mutations in PIK3CA (exon 9 and 20), AKT1 (exon 4), KRAS (exon 2) and BRAF (exon 15) genes in 57 familial MBCs. Further analysis of the PIK3CA/mTOR pathway was performed using immunohistochemistry for the pAKT1, pS6 and p4EBP1 biomarkers. RESULTS: PIK3CA somatic mutations were identified in 10.5% (6 of 57) of cases; there were no AKT1, KRAS or BRAF somatic mutations. PIK3CA mutations were significantly more frequent in cancers from BRCAX patients (17.2%, 5/29) than BRCA2 (0%, 0/25) carriers (P = 0.030). Two BRCAX patients had an E547K mutation which has only been reported in one female breast cancer previously. PIK3CA mutation was significantly correlated with positive pS6 (83.3% vs. 32.0%, P = 0.024) and negative p4EBP1 (100% vs. 38.0%, P = 0.006) expression, but not pAKT expression. Expression of nuclear p4EBP1 correlated with BRCA2 mutation carrier status (68.0% vs. 38.7%, P = 0.035). CONCLUSIONS: Somatic PIK3CA mutation is present in familial male breast cancer but absent in BRCA2 carriers. The presence of two of the extremely rare E547K PIK3CA mutations in our cohort may have specific relevance in MBCs. Further study of PIK3CA in MBCs, and in particular BRCAX patients, may contribute to further establishing the relevance of specific PIK3CA mutations in MBC aetiology and in the identification of particular patient groups most likely to benefit from therapeutic targeting with the novel PIK3CA inhibitors that are currently in development.
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ItemNonequivalent Gene Expression and Copy Number Alterations in High-Grade Serous Ovarian Cancers with BRCA1 and BRCA2 Mutations(AMER ASSOC CANCER RESEARCH, 2013-07-01)PURPOSE: High-grade serous carcinoma (HGSC) accounts for the majority of epithelial ovarian cancer deaths. Genomic and functional data suggest that approximately half of unselected HGSC have disruption of the BRCA pathway and defects in homologous recombination repair (HRR). Pathway disruption is regarded as imparting a BRCAness phenotype. We explored the molecular changes in HGSC arising in association with specific BRCA1/BRCA2 somatic or germline mutations and in those with BRCA1 DNA promoter methylation. EXPERIMENTAL DESIGN: We describe gene expression and copy number analysis of two large cohorts of HGSC in which both germline and somatic inactivation of HRR has been measured. RESULTS: BRCA1 disruptions were associated with the C2 (immunoreactive) molecular subtype of HGSC, characterized by intense intratumoral T-cell infiltration. We derived and validated a predictor of BRCA1 mutation or methylation status, but could not distinguish BRCA2 from wild-type tumors. DNA copy number analysis showed that cases with BRCA1 mutation were significantly associated with amplification both at 8q24 (frequencies: BRCA1 tumors 50%, BRCA2 tumors 32%, and wild-type tumors 9%) and regions of the X-chromosome specifically dysregulated in basal-like breast cancer (BLBC; BRCA1 62%, BRCA2 34%, and wild-type 35%). Tumors associated with BRCA1/BRCA2 mutations shared a negative association with amplification at 19p13 (BRCA1 0%, BRCA2 3%, and wild-type 20%) and 19q12 (BRCA1 6%, BRCA2 3%, and wild-type 29%). CONCLUSION: The molecular differences between tumors associated with BRCA1 compared with BRCA2 mutations are in accord with emerging clinical and pathologic data and support a growing appreciation of the relationship between HGSC and BLBC.
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ItemNo evidence for PALB2 methylation in high-grade serous ovarian cancer(BIOMED CENTRAL LTD, 2013-04-12)BACKGROUND: High-grade serous ovarian cancers are a distinct histological subtype of ovarian cancer often characterised by a dysfunctional BRCA/Fanconi anaemia (BRCA/FA) pathway, which is critical to the homologous recombination DNA repair machinery. An impaired BRCA/FA pathway sensitises tumours to the treatment with DNA cross-linking agents and to PARP inhibitors. The vast majority of inactivating mutations in the BRCA/FA pathway are in the BRCA1 and BRCA2 genes and occur predominantly in high-grade serous cancer. Another member of the BRCA/FA pathway, PALB2 (FANCN), was reported to have been inactivated by DNA methylation in some sporadic ovarian cancers. We therefore sought to investigate the role of PALB2 methylation in high-grade serous ovarian cancers. FINDING: PALB2 methylation was investigated in 92 high-grade serous ovarian cancer samples using methylation-sensitive high-resolution melting analysis. DNA methylation of PALB2 was not detected in any of the ovarian cancer samples investigated. CONCLUSION: Epigenetic silencing by DNA methylation of PALB2 is not a common event in high-grade serous ovarian cancers.
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ItemHigh Frequency of Germline TP53 Mutations in a Prospective Adult-Onset Sarcoma Cohort(PUBLIC LIBRARY SCIENCE, 2013-07-22)Sarcomas are a key feature of Li-Fraumeni and related syndromes (LFS/LFL), associated with germline TP53 mutations. Current penetrance estimates for TP53 mutations are subject to significant ascertainment bias. The International Sarcoma Kindred Study is a clinic-based, prospective cohort of adult-onset sarcoma cases, without regard to family history. The entire cohort was screened for mutations in TP53 using high-resolution melting analysis and Sanger sequencing, and multiplex-ligation-dependent probe amplification and targeted massively parallel sequencing for copy number changes. Pathogenic TP53 mutations were detected in blood DNA of 20/559 sarcoma probands (3.6%); 17 were germline and 3 appeared to be somatically acquired. Of the germline carriers, one appeared to be mosaic, detectable in the tumor and blood, but not epithelial tissues. Germline mutation carriers were more likely to have multiple cancers (47% vs 15% for non-carriers, P = 3.0×10(-3)), and earlier cancer onset (33 vs 48 years, P = 1.19×10(-3)). The median survival of mutation carriers following first cancer diagnosis was not significantly different from non-carriers. Only 10/17 (59%) pedigrees met classical or Chompret criteria for LFS. In summary, germline TP53 mutations are not rare in adult patients with sarcoma, with implications for screening, surveillance, treatment and genetic counselling of carriers and family members.