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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ItemDetection of the transforming AKT1 mutation E17K in non-small cell lung cancer by high resolution melting.(Springer Science and Business Media LLC, 2008-05-16)BACKGROUND: A recurrent somatic mutation, E17K, in the pleckstrin homology domain of the AKT1 gene, has been recently described in breast, colorectal, and ovarian cancers. AKT1 is a pivotal mediator of signalling pathways involved in cell survival, proliferation and growth. The E17K mutation stimulates downstream signalling and exhibits transforming activity in vitro and in vivo. FINDINGS: We developed a sensitive high resolution melting (HRM) assay to detect the E17K mutation from formalin-fixed paraffin-embedded tumours. We screened 219 non-small cell lung cancer biopsies for the mutation using HRM analysis. Four samples were identified as HRM positive. Subsequent sequencing of those samples confirmed the E17K mutation in one of the cases. A rare single nucleotide polymorphism was detected in each of the remaining three samples. The E17K was found in one of the 14 squamous cell carcinomas. No mutations were found in 141 adenocarcinomas and 39 large cell carcinomas. CONCLUSION: The AKT1 E17K mutation is very rare in lung cancer and might be associated with tumorigenesis in squamous cell carcinoma. HRM represents a rapid cost-effective and robust screening of low frequency mutations such as AKT1 mutations in clinical samples.
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ItemA multiplex endpoint RT-PCR assay for quality assessment of RNA extracted from formalin-fixed paraffin-embedded tissues(BIOMED CENTRAL LTD, 2010-12-17)BACKGROUND: RNA extracted from formalin-fixed paraffin-embedded (FFPE) samples is chemically modified and degraded, which compromises its use in gene expression studies. Most of the current approaches for RNA quality assessment are not suitable for FFPE derived RNA. RESULTS: We have developed a single-tube multiplex endpoint RT-PCR assay specifically designed to evaluate RNA extracted from FFPE tissues for mRNA integrity and performance in reverse transcription - quantitative real-time PCR (RT-qPCR) assays. This single-tube quality control (QC) assay minimises the amount of RNA used in quality control. mRNA integrity and the suitability of RNA for RT-PCR is evaluated by the multiplex endpoint RT-PCR assay using the TBP gene mRNA as the target sequence. The RT-PCR amplicon sizes, 92, 161, 252 and 300 bp, cover a range of amplicon sizes suitable for a wide range of RT-qPCR assays. The QC assay was used to evaluate RNA prepared by two different protocols for extracting total RNA from needle microdissected FFPE breast tumour samples. The amplification products were analysed by gel electrophoresis where the spectrum of amplicon sizes indicated the level of RNA degradation and thus the suitability of the RNA for PCR. The ability of the multiplex endpoint RT-PCR QC assay to identify FFPE samples with an adequate RNA quality was validated by examining the Cq values of an RT-qPCR assay with an 87 bp amplicon. CONCLUSIONS: The multiplex endpoint RT-PCR assay is well suited for the determination of the quality of FFPE derived RNAs, to identify which RT-PCR assays they are suitable for, and is also applicable to assess non-FFPE RNA for gene expression studies. Furthermore, the assay can also be used for the evaluation of RNA extraction protocols from FFPE samples.
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ItemThe expression of the ubiquitin ligase SIAH2 (seven in absentia homolog 2) is mediated through gene copy number in breast cancer and is associated with a basal-like phenotype and p53 expression(BIOMED CENTRAL LTD, 2011)INTRODUCTION: The seven in absentia homolog 2 (SIAH2) protein plays a significant role in the hypoxic response by regulating the abundance of hypoxia-inducible factor-α; however, its role in breast carcinoma is unclear. We investigated the frequency and expression pattern of SIAH2 in two independent cohorts of sporadic breast cancers. METHODS: Immunohistochemical evaluation of SIAH2protein expression was conducted in normal breast tissues and in tissue microarrays comprising ductal carcinoma in situ (DCIS) and a cohort of invasive breast carcinomas. Correlation analysis was performed between SIAH2 and clinicopathological variables and intrinsic breast cancer subgroups and validated in a cohort of 293 invasive ductal carcinomas. Promoter methylation, gene copy number and mRNA expression of SIAH2 were determined in a panel of basal-like tumors and cell lines. RESULTS: There was a significant increase in nuclear SIAH2 expression from normal breast tissues through to DCIS and progression to invasive cancers. A significant inverse correlation was apparent between SIAH2 and estrogen receptor and progesterone receptor and a positive association with tumor grade, HER2, p53 and an intrinsic basal-like subtype. Logistic regression analysis confirmed the significant positive association between SIAH2 expression and the basal-like phenotype. No SIAH2 promoter methylation was identified, yet there was a significant correlation between SIAH2 mRNA and gene copy number. SIAH2-positive tumors were associated with a shorter relapse-free survival in univariate but not multivariate analysis. CONCLUSIONS: SIAH2 expression is upregulated in basal-like breast cancers via copy number changes and/or transcriptional activation by p53 and is likely to be partly responsible for the enhanced hypoxic drive through abrogation of the prolyl hydroxylases.
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ItemA high-throughput protocol for mutation scanning of the BRCA1 and BRCA2 genes(BIOMED CENTRAL LTD, 2011-06-24)BACKGROUND: Detection of mutations by DNA sequencing can be facilitated by scanning methods to identify amplicons which may have mutations. Current scanning methods used for the detection of germline sequence variants are laborious as they require post-PCR manipulation. High resolution melting (HRM) is a cost-effective rapid screening strategy, which readily detects heterozygous variants by melting curve analysis of PCR products. It is well suited to screening genes such as BRCA1 and BRCA2 as germline pathogenic mutations in these genes are always heterozygous. METHODS: Assays for the analysis of all coding regions and intron-exon boundaries of BRCA1 and BRCA2 were designed, and optimised. A final set of 94 assays which ran under identical amplification conditions were chosen for BRCA1 (36) and BRCA2 (58). Significant attention was placed on primer design to enable reproducible detection of mutations within the amplicon while minimising unnecessary detection of polymorphisms. Deoxyinosine residues were incorporated into primers that overlay intronic polymorphisms. Multiple 384 well plates were used to facilitate high throughput. RESULTS: 169 BRCA1 and 239 BRCA2 known sequence variants were used to test the amplicons. We also performed an extensive blinded validation of the protocol with 384 separate patient DNAs. All heterozygous variants were detected with the optimised assays. CONCLUSIONS: This is the first HRM approach to screen the entire coding region of the BRCA1 and BRCA2 genes using one set of reaction conditions in a multi plate 384 well format using specifically designed primers. The parallel screening of a relatively large number of samples enables better detection of sequence variants. HRM has the advantages of decreasing the necessary sequencing by more than 90%. This markedly reduced cost of sequencing will result in BRCA1 and BRCA2 mutation testing becoming accessible to individuals who currently do not undergo mutation testing because of the significant costs involved.
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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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ItemNo evidence for promoter region methylation of the succinate dehydrogenase and fumarate hydratase tumour suppressor genes in breast cancer.(Springer Science and Business Media LLC, 2009-09-25)BACKGROUND: Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are tricarboxylic acid (TCA) cycle enzymes that are also known to act as tumour suppressor genes. Increased succinate or fumarate levels as a consequence of SDH and FH deficiency inhibit hypoxia inducible factor-1alpha (HIF-1alpha) prolyl hydroxylases leading to sustained HIF-1alpha expression in tumours. Since HIF-1alpha is frequently expressed in breast carcinomas, DNA methylation at the promoter regions of the SDHA, SDHB, SDHC and SDHD and FH genes was evaluated as a possible mechanism in silencing of SDH and FH expression in breast carcinomas. FINDINGS: No DNA methylation was identified in the promoter regions of the SDHA, SDHB, SDHC, SDHD and FH genes in 72 breast carcinomas and 10 breast cancer cell lines using methylation-sensitive high resolution melting which detects both homogeneous and heterogeneous methylation. CONCLUSION: These results show that inactivation via DNA methylation of the promoter CpG islands of SDH and FH is unlikely to play a major role in sporadic breast carcinomas.
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ItemHER3 and downstream pathways are involved in colonization of brain metastases from breast cancer(BMC, 2010)INTRODUCTION: Metastases to the brain from breast cancer have a high mortality, and basal-like breast cancers have a propensity for brain metastases. However, the mechanisms that allow cells to colonize the brain are unclear. METHODS: We used morphology, immunohistochemistry, gene expression and somatic mutation profiling to analyze 39 matched pairs of primary breast cancers and brain metastases, 22 unmatched brain metastases of breast cancer, 11 non-breast brain metastases and 6 autopsy cases of patients with breast cancer metastases to multiple sites, including the brain. RESULTS: Most brain metastases were triple negative and basal-like. The brain metastases over-expressed one or more members of the HER family and in particular HER3 was significantly over-expressed relative to matched primary tumors. Brain metastases from breast and other primary sites, and metastases to multiple organs in the autopsied cases, also contained somatic mutations in EGFR, HRAS, KRAS, NRAS or PIK3CA. This paralleled the frequent activation of AKT and MAPK pathways. In particular, activation of the MAPK pathway was increased in the brain metastases compared to the primary tumors. CONCLUSIONS: Deregulated HER family receptors, particularly HER3, and their downstream pathways are implicated in colonization of brain metastasis. The need for HER family receptors to dimerize for activation suggests that tumors may be susceptible to combinations of anti-HER family inhibitors, and may even be effective in the absence of HER2 amplification (that is, in triple negative/basal cancers). However, the presence of activating mutations in PIK3CA, HRAS, KRAS and NRAS suggests the necessity for also specifically targeting downstream molecules.
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ItemRapid detection of carriers with BRCA1 and BRCA2 mutations using high resolution melting analysis(BIOMED CENTRAL LTD, 2008-02-25)BACKGROUND: Germline inactivating mutations in BRCA1 and BRCA2 underlie a major proportion of the inherited predisposition to breast and ovarian cancer. These mutations are usually detected by DNA sequencing. Cost-effective and rapid methods to screen for these mutations would enable the extension of mutation testing to a broader population. High resolution melting (HRM) analysis is a rapid screening methodology with very low false negative rates. We therefore evaluated the use of HRM as a mutation scanning tool using, as a proof of principle, the three recurrent BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population in addition to other mutations that occur in the same regions. METHODS: We designed PCR amplicons for HRM scanning of BRCA1 exons 2 and 20 (carrying the founder mutations185delAG and 5382insC respectively) and the part of the BRCA2 exon 11 carrying the 6174delT founder mutation. The analysis was performed on an HRM-enabled real time PCR machine. RESULTS: We tested DNA from the peripheral blood of 29 individuals heterozygous for known mutations. All the Ashkenazi founder mutations were readily identified. Other mutations in each region that were also readily detected included the recently identified Greek founder mutation 5331G>A in exon 20 of BRCA1. Each mutation had a reproducible melting profile. CONCLUSION: HRM is a simple and rapid scanning method for known and unknown BRCA1 and BRCA2 germline mutations that can dramatically reduce the amount of sequencing required and reduce the turnaround time for mutation screening and testing. In some cases, such as tracking mutations through pedigrees, sequencing may only be necessary to confirm positive results. This methodology will allow for the economical screening of founder mutations not only in people of Ashkenazi Jewish ancestry but also in other populations with founder mutations such as Central and Eastern Europeans (BRCA1 5382insC) and Greek Europeans (BRCA1 5331G>A).