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Evaluation of NGS and RT-PCR methods for ALK rearrangement in European NSCLC patients: results from the ETOP Lungscape Project


Abstract

INTRODUCTION: The reported prevalence of ALK rearrangement in NSCLC ranges from 2%-7%. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proven to be a reproducible and sensitive technique. Reverse transcriptase-polymerase chain reaction (RT-PCR) has also been advocated and most recently the advent of targeted Next-Generation Sequencing (NGS) for ALK and other fusions has become possible. This study compares ALK evaluation with all 4 techniques in resected NSCLC from the large ETOP Lungscape cohort.
METHODS: 96 cases from the ETOP Lungscape iBiobank, with any ALK immunoreactivity were examined by FISH, central RT-PCR and NGS. H-score>120 defines IHC-positivity. RNA was extracted from the same formalin-fixed, paraffin-embedded tissues. For RT-PCR, primers covered the most frequent ALK translocations. For NGS, the Oncomine™ Solid Tumour Fusion Transcript Kit was used. The concordance was assessed using the Cohen's kappa coefficient (two-sided alpha≤5%).
RESULTS: NGS provided results for 77 out of the 95 cases tested (81.1%), while RT-PCR for 77 out of 96 (80.2%). Concordance occurred in 55 cases out of the 60 cases tested with all 4 methods (43 ALK-negative, 12 ALK-positive). Using ALK co-positivity for IHC and FISH as gold standard, we derive sensitivity for RT-PCR/NGS 70.0%/85.0%, with specificity 87.1%/79.0%. Combining either RT-PCR or NGS with IHC, the sensitivity remains the same, while the specificity increases to 88.7% and 83.9% respectively.
CONCLUSION: NGS evaluation with the Oncomine™ Solid Tumour Fusion transcript kit and RT-PCR proves to have high sensitivity and specificity advocating their use in routine practice. For maximal sensitivity and specificity, ALK status should be assessed using two techniques and a third one in discordant cases. We therefore propose a customizable testing algorithm. These findings significantly influence existing testing paradigms and have clear clinical and economic impact.

Abstract

INTRODUCTION: The reported prevalence of ALK rearrangement in NSCLC ranges from 2%-7%. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proven to be a reproducible and sensitive technique. Reverse transcriptase-polymerase chain reaction (RT-PCR) has also been advocated and most recently the advent of targeted Next-Generation Sequencing (NGS) for ALK and other fusions has become possible. This study compares ALK evaluation with all 4 techniques in resected NSCLC from the large ETOP Lungscape cohort.
METHODS: 96 cases from the ETOP Lungscape iBiobank, with any ALK immunoreactivity were examined by FISH, central RT-PCR and NGS. H-score>120 defines IHC-positivity. RNA was extracted from the same formalin-fixed, paraffin-embedded tissues. For RT-PCR, primers covered the most frequent ALK translocations. For NGS, the Oncomine™ Solid Tumour Fusion Transcript Kit was used. The concordance was assessed using the Cohen's kappa coefficient (two-sided alpha≤5%).
RESULTS: NGS provided results for 77 out of the 95 cases tested (81.1%), while RT-PCR for 77 out of 96 (80.2%). Concordance occurred in 55 cases out of the 60 cases tested with all 4 methods (43 ALK-negative, 12 ALK-positive). Using ALK co-positivity for IHC and FISH as gold standard, we derive sensitivity for RT-PCR/NGS 70.0%/85.0%, with specificity 87.1%/79.0%. Combining either RT-PCR or NGS with IHC, the sensitivity remains the same, while the specificity increases to 88.7% and 83.9% respectively.
CONCLUSION: NGS evaluation with the Oncomine™ Solid Tumour Fusion transcript kit and RT-PCR proves to have high sensitivity and specificity advocating their use in routine practice. For maximal sensitivity and specificity, ALK status should be assessed using two techniques and a third one in discordant cases. We therefore propose a customizable testing algorithm. These findings significantly influence existing testing paradigms and have clear clinical and economic impact.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Institute of Pathology and Molecular Pathology
04 Faculty of Medicine > University Hospital Zurich > Clinic for Thoracic Surgery
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:2018
Deposited On:12 Dec 2017 16:25
Last Modified:20 Feb 2018 02:02
Publisher:Elsevier
ISSN:1556-0864
OA Status:Closed
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1016/j.jtho.2017.11.117
PubMed ID:29191776

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