Detecting anaplastic lymphoma kinase (ALK) gene rearrangements with next-generation sequencing remains a reliable approach in patients with non-small-cell lung cancer

Abstract

Next-generation sequencing (NGS) is rapidly becoming routine in clinical oncology practice to identify therapeutic biomarkers, including gene rearrangements in anaplastic lymphoma kinase (ALK). Our study investigated the concordance of ALK positivity evaluated by DNA-based NGS with orthogonal ALK testing methods such as fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and RNA-based NGS (RNA-NGS). Thirty-eight patients with lung adenocarcinoma who were detected with ALK rearrangements using DNA-NGS and also had adequate tissue samples submitted for FISH, IHC, and RNA-NGS, were included in this study. Of the 38 patients, RNA samples from 3 patients failed quality control for RNA-NGS. The concordance of ALK positivity was calculated relative to DNA-NGS results. The concordance rates were 97.1% (34/35) for RNA-NGS, 94.7% (36/38) for IHC, and 97.4% (37/38) for FISH. DNA-NGS detected single ALK rearrangements in 14 (35.0%) patients and complex ALK rearrangements in 26 (65.0%). RNA-NGS detected only single transcripts of the primary ALK fusions. A novel LANCL1-ALK (L7:A20) detected using DNA-NGS was detected as EML4-ALK (E13:A20) transcripts using RNA-NGS. Interestingly, patients with single ALK rearrangements were more likely to be detected with atypical isolated red signals (p < 0.001), while patients with complex ALK rearrangements were more likely to be detected with atypical split red and green signals less than 2 signal diameters apart (p < 0.001). Our study highlights the reliability of NGS in the accurate detection of specific ALK fusion variants and concomitant mutations that are crucial for individualized treatment decisions in patients with lung cancer.

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https://doi.org/10.1007/s00428-022-03339-y
ORIGINAL ARTICLE
Detecting anaplastic lymphoma kinase (ALK) gene rearrangements
withnext‑generation sequencing remains areliable approach
inpatients withnon‑small‑cell lung cancer
YingDing1· ChangSun2· WeiSu2· ChenMiao1· XiaoHe1· Jin‑SongWang3· Zhi‑HongZhang1
Received: 5 January 2022 / Revised: 12 April 2022 / Accepted: 12 May 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022
Abstract
Next-generation sequencing (NGS) is rapidly becoming routine in clinical oncology practice to identify therapeutic biomark-
ers, including gene rearrangements in anaplastic lymphoma kinase (ALK). Our study investigated the concordance of ALK
positivity evaluated by DNA-based NGS with orthogonal ALK testing methods such as fluorescence insitu hybridization
(FISH), immunohistochemistry (IHC), and RNA-based NGS (RNA-NGS). Thirty-eight patients with lung adenocarcinoma
who were detected with ALK rearrangements using DNA-NGS and also had adequate tissue samples submitted for FISH,
IHC, and RNA-NGS, were included in this study. Of the 38 patients, RNA samples from 3 patients failed quality control
for RNA-NGS. The concordance of ALK positivity was calculated relative to DNA-NGS results. The concordance rates
were 97.1% (34/35) for RNA-NGS, 94.7% (36/38) for IHC, and 97.4% (37/38) for FISH. DNA-NGS detected single ALK
rearrangements in 14 (35.0%) patients and complex ALK rearrangements in 26 (65.0%). RNA-NGS detected only single
transcripts of the primary ALK fusions. A novel LANCL1-ALK (L7:A20) detected using DNA-NGS was detected as EML4-
ALK (E13:A20) transcripts using RNA-NGS. Interestingly, patients with single ALK rearrangements were more likely to be
detected with atypical isolated red signals (p < 0.001), while patients with complex ALK rearrangements were more likely
to be detected with atypical split red and green signals less than 2 signal diameters apart (p < 0.001). Our study highlights
the reliability of NGS in the accurate detection of specific ALK fusion variants and concomitant mutations that are crucial
for individualized treatment decisions in patients with lung cancer.
Keywords ALK rearrangement· FISH· NGS· NSCLC
Abbreviations
ALK
Anaplastic lymphoma kinase
EML4
Echinoderm microtubule-associated protein-
like 4
FDA
Food and Drug Administration
FFPE Formalin-fixed, paraffin-embedded
FISH
Fluorescence insitu hybridization
IHC Immunohistochemistry
NGS Next-generation sequencing
NSCLC
Non-small-cell lung cancer
qRT-PCR
Quantitative reverse transcriptase-polymerase
chain reaction
TKI
Tyrosine kinase inhibitor
Introduction
Between 2 and 7% of unselected non-small-cell lung cancer
(NSCLC) harbor chromosomal translocations involving the
anaplastic lymphoma kinase (ALK) gene [1–5]. A majority
of ALK-rearranged NSCLCs harbor gene fusion between the
5′ portion of the echinoderm microtubule-associated protein-
like 4 (EML4) gene and the 3′ portion of the ALK gene that
contains its tyrosine kinase domain [1]. EML4-ALK fusion
results from the paracentric inversion in the short arm of
chromosome 2 (between 2p21 and 2p23) and leads to the
constitutive activation of downstream signaling pathways
that promote cell proliferation [1]. At least 15 variants of
* Zhi-Hong Zhang
zhangzh@njmu.edu.cn
1 Department ofPathology, the First Affiliated Hospital
ofNanjing Medical University, Nanjing210029, China
2 The First School ofClinical Medicine, Nanjing Medical
University, Nanjing210029, China
3 Department ofPathology, Nanjing First Hospital, Nanjing
Medical University, Nanjing210000, China
/ Published online: 28 May 2022
Virchows Archiv (2022) 481:405–419
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