Sir Peter MacCallum Department of Oncology - Research Publications
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ItemReply to the Baader-Meinhof phenomenon in ductal carcinoma in situ of the breast(WILEY-BLACKWELL, 2016-09)
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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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ItemMethylation profiling of ductal carcinoma in situ and its relationship to histopathological features(BMC, 2014)INTRODUCTION: DNA methylation is a well-studied biomarker in invasive breast cancer, but its role in ductal carcinoma in situ (DCIS) is less well characterized. The aims of this study are to assess the methylation profile in DCIS for a panel of well-characterized genes that are frequently methylated in breast cancer, to investigate the relationship of methylation with pathological features, and to perform a proof-of-principle study to evaluate the practicality of methylation as a biomarker in diagnostic DCIS material. METHODS: Promoter CpG island methylation for a panel of 11 breast cancer-related genes was performed by methylation-sensitive high resolution melting (MS-HRM). Formalin-fixed, paraffin-embedded (FFPE) biopsies from 72 samples of pure DCIS (DCIS occurring in the absence of synchronous invasive carcinoma), 10 samples of mixed DCIS (DCIS adjacent to invasive carcinoma), and 18 samples of normal breast epithelium adjacent to a DCIS lesion were micro-dissected prior to DNA extraction. RESULTS: Methylation was seen for all the tested genes except BRCA1. RASSF1A was the most frequently methylated gene (90% of DCIS samples) and its methylation was associated with comedo necrosis (p = 0.018). Cluster analysis based on the methylation profile revealed four groups, the highly methylated cluster being significantly associated with high nuclear grade, HER2 amplification, negative estrogen receptor (ER) α status, and negative progesterone receptor (PgR) status, (p = 0.038, p = 0.018, p <0.001, p = 0.001, respectively). Methylation of APC (p = 0.017), CDH13 (p = 0.017), and RARβ (p <0.001) was associated with negative ERα status. Methylation of CDH13 (p <0.001), and RARβ (p = 0.001) was associated with negative PgR status. Methylation of APC (p = 0.013) and CDH13 (p = 0.026) was associated with high nuclear grade. Methylation of CDH13 (p = 0.009), and RARβ (p = 0.042) was associated with HER2-amplification. CONCLUSIONS: DNA methylation can be assessed in FFPE-derived samples using suitable methodologies. Methylation of a panel of genes that are known to be methylated in invasive breast cancer was able to classify DCIS into distinct groups and was differentially associated with phenotypic features in DCIS.
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ItemSequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing(BMC, 2014-05-13)BACKGROUND: Clinical specimens undergoing diagnostic molecular pathology testing are fixed in formalin due to the necessity for detailed morphological assessment. However, formalin fixation can cause major issues with molecular testing, as it causes DNA damage such as fragmentation and non-reproducible sequencing artefacts after PCR amplification. In the context of massively parallel sequencing (MPS), distinguishing true low frequency variants from sequencing artefacts remains challenging. The prevalence of formalin-induced DNA damage and its impact on molecular testing and clinical genomics remains poorly understood. METHODS: The Cancer 2015 study is a population-based cancer cohort used to assess the feasibility of mutational screening using MPS in cancer patients from Victoria, Australia. While blocks were formalin-fixed and paraffin-embedded in different anatomical pathology laboratories, they were centrally extracted for DNA utilising the same protocol, and run through the same MPS platform (Illumina TruSeq Amplicon Cancer Panel). The sequencing artefacts in the 1-10% and the 10-25% allele frequency ranges were assessed in 488 formalin-fixed tumours from the pilot phase of the Cancer 2015 cohort. All blocks were less than 2.5 years of age (mean 93 days). RESULTS: Consistent with the signature of DNA damage due to formalin fixation, many formalin-fixed samples displayed disproportionate levels of C>T/G>A changes in the 1-10% allele frequency range. Artefacts were less apparent in the 10-25% allele frequency range. Significantly, changes were inversely correlated with coverage indicating high levels of sequencing artefacts were associated with samples with low amounts of available amplifiable template due to fragmentation. The degree of fragmentation and sequencing artefacts differed between blocks sourced from different anatomical pathology laboratories. In a limited validation of potentially actionable low frequency mutations, a NRAS G12D mutation in a melanoma was shown to be a false positive. CONCLUSIONS: These findings indicate that DNA damage following formalin fixation remains a major challenge in laboratories working with MPS. Methodologies that assess, minimise or remove formalin-induced DNA damaged templates as part of MPS protocols will aid in the interpretation of genomic results leading to better patient outcomes.
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ItemTargeting Mdmx to treat breast cancers with wild-type p53(NATURE PUBLISHING GROUP, 2015-07)The function of the tumor suppressor p53 is universally compromised in cancers. It is the most frequently mutated gene in human cancers (reviewed). In cases where p53 is not mutated, alternative regulatory pathways inactivate its tumor suppressive functions. This is primarily achieved through elevation in the expression of the key inhibitors of p53: Mdm2 or Mdmx (also called Mdm4) (reviewed). In breast cancer (BrCa), the frequency of p53 mutations varies markedly between the different subtypes, with basal-like BrCas bearing a high frequency of p53 mutations, whereas luminal BrCas generally express wild-type (wt) p53. Here we show that Mdmx is unexpectedly highly expressed in normal breast epithelial cells and its expression is further elevated in most luminal BrCas, whereas p53 expression is generally low, consistent with wt p53 status. Inducible knockdown (KD) of Mdmx in luminal BrCa MCF-7 cells impedes the growth of these cells in culture, in a p53-dependent manner. Importantly, KD of Mdmx in orthotopic xenograft transplants resulted in growth inhibition associated with prolonged survival, both in a preventative model and also in a treatment model. Growth impediment in response to Mdmx KD was associated with cellular senescence. The growth inhibitory capacity of Mdmx KD was recapitulated in an additional luminal BrCa cell line MPE600, which expresses wt p53. Further, the growth inhibitory capacity of Mdmx KD was also demonstrated in the wt p53 basal-like cell line SKBR7 line. These results identify Mdmx growth dependency in wt p53 expressing BrCas, across a range of subtypes. Based on our findings, we propose that Mdmx targeting is an attractive strategy for treating BrCas harboring wt p53.
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ItemAppraisal of the technologies and review of the genomic landscape of ductal carcinoma in situ of the breast(BMC, 2015-06-16)Ductal carcinoma in situ is a biologically diverse entity. Whereas some lesions are cured by local surgical excision, others recur as in situ disease or progress to invasive carcinoma with subsequent potential for metastatic spread. Reliable prognostic biomarkers are therefore desirable for appropriate clinical management but remain elusive. In common with invasive breast cancer, ductal carcinoma in situ exhibits many genomic changes, predominantly copy number alterations. Although studies have revealed the genomic heterogeneity within individual ductal carcinoma in situ lesions and the association of certain copy number alterations with nuclear grade, none of the genomic changes defined so far is consistently associated with invasive transformation or recurrence risk in pure ductal carcinoma in situ. This article will review the current landscape of genomic alterations in ductal carcinoma in situ and their potential as prognostic biomarkers together with the technologies used to define these.
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ItemBreast Tissue Composition and Immunophenotype and Its Relationship with Mammographic Density in Women at High Risk of Breast Cancer(PUBLIC LIBRARY SCIENCE, 2015-06-25)AIM: To investigate the cellular and immunophenotypic basis of mammographic density in women at high risk of breast cancer. METHODS: Mammograms and targeted breast biopsies were accrued from 24 women at high risk of breast cancer. Mammographic density was classified into Wolfe categories and ranked by increasing density. The histological composition and immunophenotypic profile were quantified from digitized haematoxylin and eosin-stained and immunohistochemically-stained (ERα, ERβ, PgR, HER2, Ki-67, and CD31) slides and correlated to mammographic density. RESULTS: Increasing mammographic density was significantly correlated with increased fibrous stroma proportion (rs (22) = 0.5226, p = 0.0088) and significantly inversely associated with adipose tissue proportion (rs (22) = -0.5409, p = 0.0064). Contrary to previous reports, stromal expression of ERα was common (19/20 cases, 95%). There was significantly higher stromal PgR expression in mammographically-dense breasts (p=0.026). CONCLUSIONS: The proportion of stroma and fat underlies mammographic density in women at high risk of breast cancer. Increased expression of PgR in the stroma of mammographically dense breasts and frequent and unexpected presence of stromal ERα expression raises the possibility that hormone receptor expression in breast stroma may have a role in mediating the effects of exogenous hormonal therapy on mammographic density.
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ItemBRCA2 carriers with male breast cancer show elevated tumour methylation(BIOMED CENTRAL LTD, 2017-09-11)BACKGROUND: Male breast cancer (MBC) represents a poorly characterised group of tumours, the management of which is largely based on practices established for female breast cancer. However, recent studies demonstrate biological and molecular differences likely to impact on tumour behaviour and therefore patient outcome. The aim of this study was to investigate methylation of a panel of commonly methylated breast cancer genes in familial MBCs. METHODS: 60 tumours from 3 BRCA1 and 25 BRCA2 male mutation carriers and 32 males from BRCAX families were assessed for promoter methylation by methylation-sensitive high resolution melting in a panel of 10 genes (RASSF1A, TWIST1, APC, WIF1, MAL, RARβ, CDH1, RUNX3, FOXC1 and GSTP1). An average methylation index (AMI) was calculated for each case comprising the average of the methylation of the 10 genes tested as an indicator of overall tumour promoter region methylation. Promoter hypermethylation and AMI were correlated with BRCA carrier mutation status and clinicopathological parameters including tumour stage, grade, histological subtype and disease specific survival. RESULTS: Tumours arising in BRCA2 mutation carriers showed significantly higher methylation of candidate genes, than those arising in non-BRCA2 familial MBCs (average AMI 23.6 vs 16.6, p = 0.01, 45% of genes hypermethylated vs 34%, p < 0.01). RARβ methylation and AMI-high status were significantly associated with tumour size (p = 0.01 and p = 0.02 respectively), RUNX3 methylation with invasive carcinoma of no special type (94% vs 69%, p = 0.046) and RASSF1A methylation with coexistence of high grade ductal carcinoma in situ (33% vs 6%, p = 0.02). Cluster analysis showed MBCs arising in BRCA2 mutation carriers were characterised by RASSF1A, WIF1, RARβ and GTSP1 methylation (p = 0.02) whereas methylation in BRCAX tumours showed no clear clustering to particular genes. TWIST1 methylation (p = 0.001) and AMI (p = 0.01) were prognostic for disease specific survival. CONCLUSIONS: Increased methylation defines a subset of familial MBC and with AMI may be a useful prognostic marker. Methylation might be predictive of response to novel therapeutics that are currently under investigation in other cancer types.