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

Abstract

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.

Keywords: Real-time reverse-transcription polymerase chain reaction (RT-qPCR); heregulin; non/small cell lung cancer; validation.

Figure 1.

PCR efficiency and linearity for HRG and 3 housekeeping genes: (A) HRG^a,b (B) HMBS^b, (C) EIF2B1^b, and (D) IPO8^b. Ct values as a function of input template. Ct indicates cycle threshold. ^aThe HRG figure represents a 7-point curve analysis due to no amplification seen in lowest point of 0.01 ng RNA. ^bEach point represents the mean Ct across 6 runs.

Figure 2.

Unimodal HRG distribution in NSCLC commercial samples using the validated RT-qPCR assay. Ct indicates cycle threshold; HRG, heregulin. *One sample with ΔCt = −7.2 excluded from the analysis.