Limited copy number - high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies

<p>Abstract</p> <p>Background</p> <p>Mutation detection in clinical tumour samples is challenging when the proportion of tumour cells, and thus mutant alleles, is low. The limited sensitivity of conventional sequencing necessitates the adoption of more sensitive approaches. High resolution melting (HRM) is more sensitive than sequencing but identification of the mutation is desirable, particularly when it is important to discriminate false positives due to PCR errors or template degradation from true mutations.</p> <p>We thus developed limited copy number - high resolution melting (LCN-HRM) which applies limiting dilution to HRM. Multiple replicate reactions with a limited number of target sequences per reaction allow low level mutations to be detected. The dilutions used (based on Ct values) are chosen such that mutations, if present, can be detected by the direct sequencing of amplicons with aberrant melting patterns.</p> <p>Results</p> <p>Using cell lines heterozygous for mutations, we found that the mutations were not readily detected when they comprised 10% of total alleles (20% tumour cells) by sequencing, whereas they were readily detectable at 5% total alleles by standard HRM. LCN-HRM allowed these mutations to be identified by direct sequencing of those positive reactions.</p> <p>LCN-HRM was then used to review formalin-fixed paraffin-embedded (FFPE) clinical samples showing discordant findings between sequencing and HRM for <it>KRAS </it>exon 2 and <it>EGFR </it>exons 19 and 21. Both true mutations present at low levels and sequence changes due to artefacts were detected by LCN-HRM. The use of high fidelity polymerases showed that the majority of the artefacts were derived from the damaged template rather than replication errors during amplification.</p> <p>Conclusion</p> <p>LCN-HRM bridges the sensitivity gap between HRM and sequencing and is effective in distinguishing between artefacts and true mutations.</p

Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase

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 BRAFV600 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

Detection of the transforming AKT1 mutation E17K in non-small cell lung cancer by high resolution melting

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HER3 and downstream pathways are involved in colonization of brain metastases from breast cancer

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.Ludwig Institute of Cancer ResearchNational Breast Cancer FoundationUniv Queensland, Clin Res Ctr, Brisbane, Qld 4029, AustraliaQueensland Inst Med Res, Brisbane, Qld 4006, AustraliaUniversidade Federal de São Paulo, EPM, Dept Anat Patol, BR-04024000 São Paulo, BrazilGriffith Univ, Brisbane, Qld 4011, AustraliaUniv Queensland, Ctr Magnet Resonance, Brisbane, Qld 4072, AustraliaEijkman Inst, Jakarta 10430, IndonesiaInst Nacl Canc, Dept Patol, BR-20230130 Rio de Janeiro, BrazilLab Salomao & Zoppi, Dept Patol, BR-04104000 São Paulo, BrazilCharles Univ Prague, Fac Med, Dept Pathol, Plzen 30605, Czech RepublicUniv Sydney, Inst Clin Pathol & Med Res, Sydney W Area Hlth Serv, Sydney, NSW 2145, AustraliaUniv Sydney, Westmead Millennium Inst, Sydney W Area Hlth Serv, Sydney, NSW 2145, AustraliaPeter MacCallum Canc Ctr, Dept Pathol, Melbourne, Vic 3002, AustraliaUniv Queensland, Queensland Brain Inst, Brisbane, Qld 4072, AustraliaRoyal Brisbane & Womens Hosp, Brisbane, Qld 4029, AustraliaUniversidade Federal de São Paulo, EPM, Dept Anat Patol, BR-04024000 São Paulo, BrazilWeb of Scienc

High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies

<p>Abstract</p> <p>Background</p> <p>Epithelial growth factor receptor (<it>EGFR</it>) and <it>KRAS </it>mutation status have been reported as predictive markers of tumour response to <it>EGFR </it>inhibitors. High resolution melting (HRM) analysis is an attractive screening method for the detection of both known and unknown mutations as it is rapid to set up and inexpensive to operate. However, up to now it has not been fully validated for clinical samples when formalin-fixed paraffin-embedded (FFPE) sections are the only material available for analysis as is often the case.</p> <p>Methods</p> <p>We developed HRM assays, optimised for the analysis of FFPE tissues, to detect somatic mutations in <it>EGFR </it>exons 18 to 21. We performed HRM analysis for <it>EGFR </it>and <it>KRAS </it>on DNA isolated from a panel of 200 non-small cell lung cancer (NSCLC) samples derived from FFPE tissues.</p> <p>Results</p> <p>All 73 samples that harboured <it>EGFR </it>mutations previously identified by sequencing were correctly identified by HRM, giving 100% sensitivity with 90% specificity. Twenty five samples were positive by HRM for <it>KRAS </it>exon 2 mutations. Sequencing of these 25 samples confirmed the presence of codon 12 or 13 mutations. <it>EGFR </it>and <it>KRAS </it>mutations were mutually exclusive.</p> <p>Conclusion</p> <p>This is the first extensive validation of HRM on FFPE samples using the detection of <it>EGFR </it>exons 18 to 21 mutations and <it>KRAS </it>exon 2 mutations. Our results demonstrate the utility of HRM analysis for the detection of somatic <it>EGFR </it>and <it>KRAS </it>mutations in clinical samples and for screening of samples prior to sequencing. We estimate that by using HRM as a screening method, the number of sequencing reactions needed for <it>EGFR </it>and <it>KRAS </it>mutation detection can be reduced by up to 80% and thus result in substantial time and cost savings.</p

Integrated mutation, copy number and expression profiling in resectable non-small cell lung cancer

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to identify critical genes involved in non-small cell lung cancer (NSCLC) pathogenesis that may lead to a more complete understanding of this disease and identify novel molecular targets for use in the development of more effective therapies.</p> <p>Methods</p> <p>Both transcriptional and genomic profiling were performed on 69 resected NSCLC specimens and results correlated with mutational analyses and clinical data to identify genetic alterations associated with groups of interest.</p> <p>Results</p> <p>Combined analyses identified specific patterns of genetic alteration associated with adenocarcinoma vs. squamous differentiation; <it>KRAS </it>mutation; <it>TP53 </it>mutation, metastatic potential and disease recurrence and survival. Amplification of 3q was associated with mutations in <it>TP53 </it>in adenocarcinoma. A prognostic signature for disease recurrence, reflecting <it>KRAS </it>pathway activation, was validated in an independent test set.</p> <p>Conclusions</p> <p>These results may provide the first steps in identifying new predictive biomarkers and targets for novel therapies, thus improving outcomes for patients with this deadly disease.</p

Evaluation of high resolution melting methodology for biomarker studies in non-small cell lung cancer

© 2011 Dr. Hongdo DoThe adoption of molecularly targeted therapies in non-small cell lung cancer (NSCLC) has demonstrated the clinical efficacy of treating individual patients based on tumour molecular defects, emphasising the necessity of accurate and reliable methodology for molecular biomarker studies. High resolution melting (HRM) is a fast, sensitive, and cost-effective methodology that can screen both genetic and epigenetic biomarkers. Using HRM-based assays developed as part of this thesis, testing for a range of genetic and epigenetic biomarkers that have a predictive role for various therapies in NSCLC has been studied with a focus on the problems associated with diagnostic material. Mutational profiling of seven selected genes in the EGFR signalling pathway (EGFR, HER2, KRAS, BRAF, PIK3CA, and AKT1/3) was performed in a panel of 200 NSCLC samples that had been sent for diagnostic EGFR testing. Gain-of-function mutations were detected in 54% of the NSCLC samples. All 75 EGFR mutations that were previously identified by Sanger sequencing were detected by HRM analysis. Forty nine per cent of the NSCLC tumours harboured either EGFR exon 18-21 mutations (36.5%) or KRAS codon 12 and 13 mutations (12.5%). Somatic PIK3CA (3.5%), BRAF (3%), and AKT1 (0.5%) mutations were less frequently detected. EGFR, KRAS, and BRAF mutations were mutually exclusive whereas concomitant PIK3CA and EGFR mutations were detected in four NSCLC tumours. Limited copy number (LCN)-HRM was developed for screening and detection of low levels of mutations present below the sensitivity of Sanger sequencing as the limited sensitivity of this method is a particular problem in lung cancer diagnostics. The analytic sensitivity of HRM (5%) was shown to be greater than of Sanger sequencing (10%), demonstrating that low levels of mutations (5-10%) was detectable by HRM only. When clinical samples showing the discordant results between Sanger sequencing and HRM were tested by LCN-HRM, both low levels of true mutations and PCR artefacts were detected. Template-mediated PCR artefacts, mainly transitional changes (G>A and C>T), were prevalent in DNA extracted from formalin-fixed paraffin-embedded tissues. STK11 mutations were screened by HRM in a panel of tumours from 195 NSCLC patients who were surgically treated in combination with platinum-based chemotherapies. A total of 27 non-synonymous STK11 mutations (14%) were detected, including 9 nonsense, 5 frame-shift, 6 missense, and 7 splice site variants. The majority of STK11 mutations (93%) were detected in the kinase domain. STK11 mutations were significantly associated with worse progression free survival and overall survival of the NSCLC patients. The frequency of promoter region methylation in two sets of genes, DNA repair genes and selected genes previously associated with recurrence after surgery, was examined in 56 N1 tumours using methylation sensitive (MS)-HRM. DNA methylation was not detected in BRCA1, MLH1, and XPC, contrary to previous reports in the literature. Methylation was detected at a low frequency in ERCC1 (2%) and RAD23B (2%). Modest levels of MGMT methylation was seen in more individuals (12.5%) and was significantly associated with the ‘T’ allele of the MGMT promoter rs16906252 SNP (P<0.0001). A high frequency of methylation in the APC, CDH13, RASSF1A, and p16 genes was confirmed in NSCLC tumours, giving an overall methylation frequency of 25%, 50%, 32%, and 25% respectively. A strong association between KRAS mutation and CDH13 methylation was also detected. In conclusion, HRM is a sensitive and accurate methodology that can be used in a range of molecular diagnostic tests for genetic and epigenetic biomarkers. When used in combination with sequencing, it is suitable for routine diagnostic testing for molecularly tailored therapies for NSCLC patients