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Generali D.,Azienda Instituti Ospitalieri Of Cremona | Symmans W.F.,University of Houston | Berruti A.,University of Turin | Fox S.B.,Molecular Pathology Research and Development Laboratory
Journal of the National Cancer Institute - Monographs | Year: 2011

The adoption of personalized medicine has led to the search for prognostic and predictive markers that can be applied to individual patients to give optimal information for their clinical management. We have used samples from randomized clinical trials of hormonal and chemotherapy to identify relevant markers of sensitivity and resistance using a neoadjuvant approach by linking expression of a panel of proteins involved in growth factor receptor signaling, angiogenesis, estrogen receptor signaling, and hypoxia to individual patient response. We evaluated samples from randomized clinical trials of epirubicin with or without tamoxifen, and letrozole with or without metronomic cyclophosphamide, to study chemotherapy, hormonal therapy, and antiangiogenic effects. We present a proof of principle of this approach in identifying several key pathways that are associated with clinical and pathological response. Thus, we have shown that the hypoxia-inducible factor (HIF) pathway, mitogen activated protein kinase, and phosphorylated estrogen receptor-a can identify patients who are likely to respond to hormonal therapy and that HIF signaling is also a marker of resistance for anthracycline-based chemotherapy. To redress the role of HIF, we then evaluated samples from a randomized control trial of an anthracycline chemotherapy with and without erythropoietin. These studies demonstrate that the approach of using primary systemic therapy in breast can identify markers of response and potentially targets for rationale design of new therapies. © The Author 2011. Published by Oxford University Press. All rights reserved.

Candiloro I.L.,Molecular Pathology Research and Development Laboratory
Epigenetics : official journal of the DNA Methylation Society | Year: 2011

Heterogeneous DNA methylation leads to difficulties in accurate detection and quantification of methylation. Methylation-sensitive high resolution melting (MS-HRM) is unique among regularly used methods for DNA methylation analysis in that heterogeneous methylation can be readily identified, although not quantified, by inspection of the melting curves. Bisulfite pyrosequencing has been used to estimate the level of heterogeneous methylation by quantifying methylation levels present at individual CpG dinucleotides. Sequentially combining the two methodologies using MS-HRM to screen the amplification products prior to bisulfite pyrosequencing would be advantageous. This would not only replace the quality control step using agarose gel analysis prior to the pyrosequencing step but would also provide important qualitative information in its own right. We chose to analyze DAPK1 as it is an important tumor suppressor gene frequently heterogeneously methylated in a number of malignancies, including chronic lymphocytic leukemia (CLL). A region of the DAPK1 promoter was analyzed in ten CLL samples by MS-HRM. By using a biotinylated primer, bisulfite pyrosequencing could be used to directly analyze the samples. MS-HRM revealed the presence of various extents of heterogeneous DAPK1 methylation in all CLL samples. Further analysis of the biotinylated MS-HRM products by bisulfite pyrosequencing provided quantitative information for each CpG dinucleotide analyzed, and confirmed the presence of heterogeneous DNA methylation. Whereas each method could be used individually, MS-HRM and bisulfite pyrosequencing provided complementary information for the assessment of heterogeneous methylation.

Do H.,Molecular Pathology Research and Development Laboratory | Do H.,University of Melbourne | Dobrovic A.,Molecular Pathology Research and Development Laboratory | Dobrovic A.,University of Melbourne
Oncotarget | Year: 2012

Non-reproducible sequence artefacts are frequently detected in DNA from formalin-fixed and paraffin-embedded (FFPE) tissues. However, no rational strategy has been developed for reduction of sequence artefacts from FFPE DNA as the underlying causes of the artefacts are poorly understood. As cytosine deamination to uracil is a common form of DNA damage in ancient DNA, we set out to examine whether treatment of FFPE DNA with uracil-DNA glycosylase (UDG) would lead to the reduction of C>T (and G>A) sequence artefacts. Heteroduplex formation in high resolution melting (HRM)-based assays was used for the detection of sequence variants in FFPE DNA samples. A set of samples that gave false positive HRM results for screening of the E17K mutation in exon 4 of the AKT1 gene were chosen for analysis. Sequencing of these samples showed multiple non-reproducible C:G>T:A artefacts. Treatment of the FFPE DNA with UDG prior to PCR amplification led to a very marked reduction of the sequence artefacts as indicated by both HRM and sequencing analysis. Similar results were shown for the BRAF V600 region in the same sample set and EGFR exon 19 in another sample set. UDG treatment specifically suppressed the formation of artefacts in FFPE DNA as it did not affect the detection of true KRAS codon 12 and true EGFR exon 19 and 20 mutations. We conclude that uracil in FFPE DNA leads to a significant proportion of sequence artefacts. These can be minimised by a simple UDG pre-treatment, which can be readily carried out in the same tube as the PCR, immediately prior to commencing thermal cycling. HRM is a convenient way of monitoring both the degree of damage and the effectiveness of the UDG treatment. These findings have immediate and important implications for cancer diagnostics where FFPE DNA is used as the primary genetic material for mutational studies guiding personalised medicine strategies and where simple effective strategies to detect mutations are required. © Do et al.

Mikeska T.,Molecular Pathology Research and Development Laboratory | Mikeska T.,University of Melbourne | Bock C.,Austrian Academy of Sciences | Bock C.,Medical University of Vienna | And 4 more authors.
Expert Review of Molecular Diagnostics | Year: 2012

Altered DNA methylation is ubiquitous in human cancers and specific methylation changes are often correlated with clinical features. DNA methylation biomarkers, which use those specific methylation changes, provide a range of opportunities for early detection, diagnosis, prognosis, therapeutic stratification and post-therapeutic monitoring. Here we review current approaches to developing and applying DNA methylation biomarkers in cancer therapy. We discuss the obstacles that have so far limited the routine use of DNA methylation biomarkers in clinical settings and describe ways in which these obstacles can be overcome. Finally, we summarize the current state of clinical implementation for some of the most widely studied and well-validated DNA methylation biomarkers, including SEPT9, VIM, SHOX2, PITX2 and MGMT. © 2012 Expert Reviews Ltd.

Kristensen L.S.,Molecular Pathology Research and Development Laboratory | Kristensen L.S.,University of Aarhus | Raynor M.,The Queen Elizabeth Hospital | Candiloro I.,Molecular Pathology Research and Development Laboratory | And 3 more authors.
Oncotarget | Year: 2012

Epigenetic silencing by promoter methylation of genes associated with cancer initiation and progression is a hallmark of tumour cells. As a consequence, testing for DNA methylation biomarkers in plasma or other body fluids shows great promise for detection of malignancies at early stages and/or for monitoring response to treatment. However, DNA from normal leukocytes may contribute to the DNA in plasma and will affect biomarker specificity if there is any methylation in the leukocytes. DNA from 48 samples of normal peripheral blood mononuclear cells was evaluated for the presence of methylation of a panel of DNA methylation biomarkers that have been implicated in cancer. SMART-MSP, a methylation specific PCR (MSP) methodology based on real time PCR amplification, high-resolution melting and strategic primer design, enabled quantitative detection of low levels of methylated DNA. Methylation was observed in all tested mononuclear cell DNA samples for the CDH1 and HIC1 promoters and in the majority of DNA samples for the TWIST1 and DAPK1 promoters. APC and RARB promoter methylation, at a lower average level, was also detected in a substantial proportion of the DNA samples. We found no BRCA1, CDKN2A, GSTP1 and RASSF1A promoter methylation in this sample set. Several individuals had higher levels of methylation at several loci suggestive of a methylator phenotype. In conclusion, methylation of many potential DNA methylation biomarkers can be detected in normal peripheral blood mononuclear cells, and is likely to affect their specificity for detecting low level disease. However, we found no evidence of promoter methylation for other genes indicating that panels of analytically sensitive and specific methylation biomarkers in body fluids can be obtained. © Kristensen et al.

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