Cargando…

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

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 (NSCL...

Descripción completa

Detalles Bibliográficos
Autores principales: Kristof, Jessica, Sakrison, Kellen, Jin, Xiaoping, Nakamaru, Kenji, Schneider, Matthias, Beckman, Robert A, Freeman, Daniel, Spittle, Cindy, Feng, Wenqin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: SAGE Publications 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391987/
https://www.ncbi.nlm.nih.gov/pubmed/28469400
http://dx.doi.org/10.1177/1177271917699850
_version_ 1783229378242543616
author Kristof, Jessica
Sakrison, Kellen
Jin, Xiaoping
Nakamaru, Kenji
Schneider, Matthias
Beckman, Robert A
Freeman, Daniel
Spittle, Cindy
Feng, Wenqin
author_facet Kristof, Jessica
Sakrison, Kellen
Jin, Xiaoping
Nakamaru, Kenji
Schneider, Matthias
Beckman, Robert A
Freeman, Daniel
Spittle, Cindy
Feng, Wenqin
author_sort Kristof, Jessica
collection PubMed
description 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.
format Online
Article
Text
id pubmed-5391987
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher SAGE Publications
record_format MEDLINE/PubMed
spelling pubmed-53919872017-05-03 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 Kristof, Jessica Sakrison, Kellen Jin, Xiaoping Nakamaru, Kenji Schneider, Matthias Beckman, Robert A Freeman, Daniel Spittle, Cindy Feng, Wenqin Biomark Insights Original Research 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. SAGE Publications 2017-03-30 /pmc/articles/PMC5391987/ /pubmed/28469400 http://dx.doi.org/10.1177/1177271917699850 Text en © The Author(s) 2017 http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (https://us.sagepub.com/en-us/nam/open-access-at-sage).
spellingShingle Original Research
Kristof, Jessica
Sakrison, Kellen
Jin, Xiaoping
Nakamaru, Kenji
Schneider, Matthias
Beckman, Robert A
Freeman, Daniel
Spittle, Cindy
Feng, Wenqin
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
title 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
title_full 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
title_fullStr 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
title_full_unstemmed 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
title_short 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
title_sort 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
topic Original Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391987/
https://www.ncbi.nlm.nih.gov/pubmed/28469400
http://dx.doi.org/10.1177/1177271917699850
work_keys_str_mv AT kristofjessica realtimereversetranscriptionquantitativepolymerasechainreactionassayisafeasiblemethodfortherelativequantificationofheregulinexpressioninnonsmallcelllungcancertissue
AT sakrisonkellen realtimereversetranscriptionquantitativepolymerasechainreactionassayisafeasiblemethodfortherelativequantificationofheregulinexpressioninnonsmallcelllungcancertissue
AT jinxiaoping realtimereversetranscriptionquantitativepolymerasechainreactionassayisafeasiblemethodfortherelativequantificationofheregulinexpressioninnonsmallcelllungcancertissue
AT nakamarukenji realtimereversetranscriptionquantitativepolymerasechainreactionassayisafeasiblemethodfortherelativequantificationofheregulinexpressioninnonsmallcelllungcancertissue
AT schneidermatthias realtimereversetranscriptionquantitativepolymerasechainreactionassayisafeasiblemethodfortherelativequantificationofheregulinexpressioninnonsmallcelllungcancertissue
AT beckmanroberta realtimereversetranscriptionquantitativepolymerasechainreactionassayisafeasiblemethodfortherelativequantificationofheregulinexpressioninnonsmallcelllungcancertissue
AT freemandaniel realtimereversetranscriptionquantitativepolymerasechainreactionassayisafeasiblemethodfortherelativequantificationofheregulinexpressioninnonsmallcelllungcancertissue
AT spittlecindy realtimereversetranscriptionquantitativepolymerasechainreactionassayisafeasiblemethodfortherelativequantificationofheregulinexpressioninnonsmallcelllungcancertissue
AT fengwenqin realtimereversetranscriptionquantitativepolymerasechainreactionassayisafeasiblemethodfortherelativequantificationofheregulinexpressioninnonsmallcelllungcancertissue