Hongdo Do

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

BackgroundEpithelial growth factor receptor (EGFR) and KRAS mutation status have been reported as predictive markers of tumour response to EGFR 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.MethodsWe developed HRM assays, optimised for the analysis of FFPE tissues, to detect somatic mutations in EGFR exons 18 to 21. We performed HRM analysis for EGFR and KRAS on DNA isolated from a panel of 200 non-small cell lung cancer (NSCLC) samples derived from FFPE tissues.ResultsAll 73 samples that harboured EGFR mutations previously identified by sequencing were correctly identified by HRM, giving 100% sensitivity with 90% specificity. Twenty five samples were positive by HRM for KRAS exon 2 mutations. Sequencing of these 25 samples confirmed the presence of codon 12 or 13 mutations. EGFR and KRAS mutations were mutually exclusive.ConclusionThis is the first extensive validation of HRM on FFPE samples using the detection of EGFR exons 18 to 21 mutations and KRAS exon 2 mutations. Our results demonstrate the utility of HRM analysis for the detection of somatic EGFR and KRAS 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 EGFR and KRAS mutation detection can be reduced by up to 80% and thus result in substantial time and cost savings.

Sequence Artifacts in DNA from Formalin-Fixed Tissues: Causes and Strategies for Minimization

BACKGROUND Precision medicine is dependent on identifying actionable mutations in tumors. Accurate detection of mutations is often problematic in formalin-fixed paraffin-embedded (FFPE) tissues. DNA extracted from formalin-fixed tissues is fragmented and also contains DNA lesions that are the sources of sequence artifacts. Sequence artifacts can be difficult to distinguish from true mutations, especially in the context of tumor heterogeneity, and are an increasing interpretive problem in this era of massively parallel sequencing. Understanding of the sources of sequence artifacts in FFPE tissues and implementation of preventative strategies are critical to improve the accurate detection of actionable mutations. CONTENT This mini-review focuses on DNA template lesions in FFPE tissues as the source of sequence artifacts in molecular analysis. In particular, fragmentation, base modification (including uracil and thymine deriving from cytosine deamination), and abasic sites are discussed as indirect or direct sources of sequence artifacts. We discuss strategies that can be implemented to minimize sequence artifacts and to distinguish true mutations from sequence artifacts. These strategies are applicable for the detection of actionable mutations in both single amplicon and massively parallel amplicon sequencing approaches. SUMMARY Because FFPE tissues are usually the only available material for DNA analysis, it is important to maximize the accurate informational content from FFPE DNA. Careful consideration of each step in the work flow is needed to minimize sequence artifacts. In addition, validation of actionable mutations either by appropriate experimental design or by orthogonal methods should be considered.

DNA methylation biomarkers in cancer: progress towards clinical implementation.

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.

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

IntroductionMetastases 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.MethodsWe 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.ResultsMost 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.ConclusionsDeregulated 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.

Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines

We have investigated the potential for the p16‐cyclin D‐CDK4/6‐retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three‐quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma‐specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16INK4A) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance.

Reducing Sequence Artifacts in Amplicon-Based Massively Parallel Sequencing of Formalin-Fixed Paraffin-Embedded DNA by Enzymatic Depletion of Uracil-Containing Templates

BACKGROUND Formalin-fixed, paraffin-embedded (FFPE) tissues are routinely used for detecting mutational biomarkers in patients with cancer. A previous intractable challenge with FFPE DNA in genetic testing has been the high number of artifactual single-nucleotide changes (SNCs), particularly for the detection of low-level mutations. Pretreatment of FFPE DNA with uracil-DNA glycosylase (UDG) can markedly reduce these C:G>T:A SNCs with a small panel of amplicons. This procedure has implications for massively parallel sequencing approaches to mutation detection from DNA. We investigated whether sequence artifacts were problematic in amplicon-based massively parallel sequencing and what effect UDG pretreatment had on reducing these artifacts. METHODS We amplified selected amplicons from lung cancer FFPE DNAs using the TruSeq Cancer Panel. SNCs occurring at a frequency <10% were considered most likely to represent sequence artifacts and were enumerated for both UDG-treated and -untreated DNAs. RESULTS Massively parallel sequencing of FFPE DNA samples showed multiple SNCs, predominantly C:G>T:A changes, with a significant proportion occurring above the background sequencing error (defined as 1%). UDG pretreatment markedly reduced C:G>T:A SNCs without affecting the detection of true somatic mutations. However, C:G>T:A changes within CpG dinucleotides were often resistant to the UDG treatment as a consequence of 5-methyl cytosine being deaminated to thymine rather than uracil. CONCLUSIONS UDG pretreatment greatly facilitates the accurate discrimination of mutations in FFPE samples by use of amplicon-based approaches. This is particularly important when working with samples with low tumor purity or for the assessment of mutational heterogeneity in tumors.

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

BackgroundMutation 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.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.ResultsUsing 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.LCN-HRM was then used to review formalin-fixed paraffin-embedded (FFPE) clinical samples showing discordant findings between sequencing and HRM for KRAS exon 2 and EGFR 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.ConclusionLCN-HRM bridges the sensitivity gap between HRM and sequencing and is effective in distinguishing between artefacts and true mutations.

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

BackgroundA 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.FindingsWe 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.ConclusionThe 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.

Genomic Classification of Serous Ovarian Cancer with Adjacent Borderline Differentiates RAS Pathway and TP53-Mutant Tumors and Identifies NRAS as an Oncogenic Driver

Purpose: Low-grade serous ovarian carcinomas (LGSC) are Ras pathway-mutated, TP53 wild-type, and frequently associated with borderline tumors. Patients with LGSCs respond poorly to platinum-based chemotherapy and may benefit from pathway-targeted agents. High-grade serous carcinomas (HGSC) are TP53-mutated and are thought to be rarely associated with borderline tumors. We sought to determine whether borderline histology associated with grade 2 or 3 carcinoma was an indicator of Ras mutation, and we explored the molecular relationship between coexisting invasive and borderline histologies. Experimental Design: We reviewed >1,200 patients and identified 102 serous carcinomas with adjacent borderline regions for analyses, including candidate mutation screening, copy number, and gene expression profiling. Results: We found a similar frequency of low, moderate, and high-grade carcinomas with coexisting borderline histology. BRAF/KRAS alterations were common in LGSC; however, we also found recurrent NRAS mutations. Whereas borderline tumors harbored BRAF/KRAS mutations, NRAS mutations were restricted to carcinomas, representing the first example of a Ras oncogene with an obligatory association with invasive serous cancer. Coexisting borderline and invasive components showed nearly identical genomic profiles. Grade 2 cases with coexisting borderline included tumors with molecular features of LGSC, whereas others were typical of HGSC. However, all grade 3 carcinomas with coexisting borderline histology were molecularly indistinguishable from typical HGSC. Conclusion: Our findings suggest that NRAS is an oncogenic driver in serous ovarian tumors. We demonstrate that borderline histology is an unreliable predictor of Ras pathway aberration and underscore an important role for molecular classification in identifying patients that may benefit from targeted agents. Clin Cancer Res; 20(24); 6618–30. ©2014 AACR.

A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma

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.