EGFR and KRAS Quality Assurance Schemes in Pathology

Generating Normative Data for Molecular Predictive Marker Analysis in Targeted Therapy

Erik Thunnissen; Judith V M G Bovée; Hans Bruinsma; Adriaan J C van den Brule; Winand Dinjens; Daniëlle A M Heideman; Els Meulemans; Petra Nederlof; Carel van Noesel; Clemens F M Prinsen; Karen Scheidel; Peter M van de Ven; Roel de Weger; Ed Schuuring; Marjolijn Ligtenberg

Authors and Disclosures

Abstract and Introduction
Materials and Methods
Results
Discussion
Conclusion

Abstract and Introduction

Abstract

Introduction The aim of this study was to compare the reproducibility of epidermal growth factor receptor (EGFR) immunohistochemistry (IHC), EGFR gene amplification analysis, and EGFR and KRAS mutation analysis among different laboratories performing routine diagnostic analyses in pathology in The Netherlands, and to generate normative data.
Methods In 2008, IHC, in-situ hybridisation (ISH) for EGFR, and mutation analysis for EGFR and KRAS were tested. Tissue microarray sections were distributed for IHC and ISH, and tissue sections and isolated DNA with known mutations were distributed for mutation analysis. In 2009, ISH and mutation analysis were evaluated. False-negative and false-positive results were defined as different from the consensus, and sensitivity and specificity were estimated.
Results In 2008, eight laboratories participated in the IHC ring study. In only 4/17 cases (23%) a consensus score of ≥75% was reached, indicating that this analysis was not sufficiently reliable to be applied in clinical practice. For EGFR ISH, and EGFR and KRAS mutation analysis, an interpretable result (success rate) was obtained in ≥97% of the cases, with mean sensitivity ≥96% and specificity ≥95%. For small sample proficiency testing, a norm was established defining outlier laboratories with unsatisfactory performance.
Conclusions The result of EGFR IHC is not a suitable criterion for reliably selecting patients for anti-EGFR treatment. In contrast, molecular diagnostic methods for EGFR and KRAS mutation detection and EGFR ISH may be reliably performed with high accuracy, allowing treatment decisions for lung cancer.

Introduction

Recently, an empirical treatment approach with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) showed spectacular responses in a subset of patients with advanced non-small cell lung cancer (NSCLC).^[1–4] In those patients, EGFR mutations and DNA amplifications were detected, and several studies have since been performed on the adjuvant treatment of NSCLC with EGFR TKIs. For prediction of response to EGFR TKI treatment, EGFR gene copy number,^[5] EGFR protein expression, EGFR mutation^{[4 6]} and KRAS mutation are informative.^{[5 7–9]} However, in those studies, biomarker analysis was performed in central laboratories.
Before the biomarkers can be used to select patients for this novel type of treatment, it is necessary to evaluate the reproducibility of testing by different laboratories. Recently, recommendations for the use of EGFR molecular assays have been reported, and these include guidelines for tissue storage, handling and processing.^[10] In addition, recommendations for the standardisation of molecular assays have been discussed. For mutation analysis, sequencing has been the procedure of choice, although the development of more rapid and sensitive techniques is awaited. For gene copy number changes, fluorescence in-situ hybridisation (FISH) analysis was preferred, as true amplification of the EGFR gene in NSCLC is less frequent as polysomy. Since then, high correlation between EGFR FISH and chromogenic in-situ hybridisation (CISH) analysis has been shown.^{[11 12]} For colorectal cancer, a norm for mutation analysis has been published recently,^[13] and the required level of minimum performance was based on arbitrary grounds. For lung cancer, suggestions for a norm in mutation and amplification analysis have not been published.
Proficiency testing for molecular diagnostic pathology has been performed in The Netherlands for more than a decade.^[14] In addition to intralaboratory validation of any novel molecular assay for diagnostic purposes, the performance of molecular testing in different laboratories needs to be assessed before using new molecular markers in daily healthcare procedures. Therefore, in 2008 and 2009, EGFR and KRAS analysis was evaluated nationwide. In two ring studies, the reproducibility was evaluated and a performance level for mutation and amplification analysis obtained.

full-text:
EGFR and KRAS Quality Assurance Schemes in Pathology: Abstract and Introduction