Detection of Epidermal Growth Factor Receptor (EGFR) Gene Mutation in Formalin Fixed Paraffin Embedded Tissue by Polymerase Chain Reaction-Single Strand Conformational Polymorphism (PCR-SSCP) in Non-Small Cell Lung Cancer in the Northeastern Region of Thailand

Abstract Context.—Monoclonal anti–epidermal growth factor receptor antibodies bind to the epidermal growth factor receptor and inhibit receptor kinase activity. Clinical trials have indicated that evaluation of the mutational status of KRas and BRaf is necessary to exclude patients who are resistant to the clinical benefit of anti–epidermal growth factor receptor therapy. Objective.—To develop a multiplex polymerase chain reaction–based assay for the evaluation of KRas and BRaf mutational status. Design.—A sample-saving and cost-effective, multiplex polymerase chain reaction–based assay to detect somatic mutations in KRAS exon 2 and exon 3 as well as in BRAF exon 15 was developed. The same primer pairs could be successfully used in amplification of a single DNA fragment under the same conditions. Results.—DNA isolated from 20 retrospective formalin-fixed, paraffin-embedded samples of colorectal cancer was screened for mutations using the multiplex polymerase chain reaction assay followed by dideoxy-termination sequencing. Five samples bearing mutations—p.G12D (identified twice), p.G12V, p.G12S, and p.G13D, all encoded in KRAS exon 2—were identified. Three samples were found bearing amino acid substitution p.V600E of BRaf. The detected KRas and BRaf mutations were found to be mutually exclusive. Conclusions.—A multiplex polymerase chain reaction–based amplification followed by dideoxy-termination sequencing may be used advantageously for the evaluation of KRas and BRaf mutational status from formalin-fixed, paraffin-embedded samples.

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Real-Time Reverse-Transcription Quantitative Polymerase Chain Reaction Assay Is a Feasible Method for the Relative Quantification of Heregulin Expression in Non–Small Cell Lung Cancer Tissue

Biomarker Insights ◽

10.1177/1177271917699850 ◽

2017 ◽

Vol 12 ◽

pp. 117727191769985

Author(s):

Jessica Kristof ◽

Kellen Sakrison ◽

Xiaoping Jin ◽

Kenji Nakamaru ◽

Matthias Schneider ◽

...

Keyword(s):

Quantitative Polymerase Chain Reaction ◽

Rna Extraction ◽

Qpcr Assay ◽

Small Cell Lung ◽

Chain Reaction ◽

Nsclc Tissue ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Polymerase Chain ◽

Formalin Fixed

In preclinical studies, heregulin ( HRG) expression was shown to be the most relevant predictive biomarker for response to patritumab, a fully human anti–epidermal growth factor receptor 3 monoclonal antibody. In support of a phase 2 study of erlotinib ± patritumab in non–small cell lung cancer (NSCLC), a reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay for relative quantification of HRG expression from formalin-fixed paraffin-embedded (FFPE) NSCLC tissue samples was developed and validated and described herein. Test specimens included matched FFPE normal lung and NSCLC and frozen NSCLC tissue, and HRG-positive and HRG-negative cell lines. Formalin-fixed paraffin-embedded tissue was examined for functional performance. Heregulin distribution was also analyzed across 200 NSCLC commercial samples. Applied Biosystems TaqMan Gene Expression Assays were run on the Bio-Rad CFX96 real-time PCR platform. Heregulin RT-qPCR assay specificity, PCR efficiency, PCR linearity, and reproducibility were demonstrated. The final assay parameters included the Qiagen FFPE RNA Extraction Kit for RNA extraction from FFPE NSCLC tissue, 50 ng of RNA input, and 3 reference (housekeeping) genes ( HMBS, IPO8, and EIF2B1), which had expression levels similar to HRG expression levels and were stable among FFPE NSCLC samples. Using the validated assay, unimodal HRG distribution was confirmed across 185 evaluable FFPE NSCLC commercial samples. Feasibility of an RT-qPCR assay for the quantification of HRG expression in FFPE NSCLC specimens was demonstrated.

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