Elsevier

Lung Cancer

Volume 76, Issue 3, June 2012, Pages 309-315
Lung Cancer

Histologic subtypes, immunohistochemistry, FISH or molecular screening for the accurate diagnosis of ALK-rearrangement in lung cancer: A comprehensive study of Caucasian non-smokers

https://doi.org/10.1016/j.lungcan.2011.11.004Get rights and content

Abstract

EML4-ALK adenocarcinomas constitute a new molecular subgroup of lung tumours that respond very well to crizotinib, an ALK inhibitor. However, the diagnosis of ALK rearrangement in lung cancer is challenging. The aim of this study was to compare the diagnostic accuracy of five different methods in a series of 20 EGFRwt/wt lung adenocarcinomas from non- or light- smokers. Multiplex RT-PCR was considered as gold standard and identified four ALK-rearranged tumours among the 20 tested tumours. qRT-PCR got an interpretability rate of 100% and accurately typed all 20 tumours. qRT-PCR from corresponding formalin-fixed paraffin-embedded (FFPE) specimens got an interpretability rate of 65%. Out of the four previously identified ALK-rearranged cases, three were interpretable and two were retrieved using FFPE qRT-PCR. ALK break-apart FISH got an interpretability rate of 60% and accurately typed all of the twelve remaining cases. Anti-ALK immunohistochemistry (IHC) accurately typed all twenty tumours using a cut-off value of strong staining of 100% tumour cells. The 16 non ALK-rearranged tumours got no/light staining in 13 cases, and a moderate staining of 80–100% tumour cells in 3 cases. We then analysed four solid signet-ring lung adenocarcinomas. FFPE qRT-PCR, FISH and immunohistochemistry were concordant in three cases, with positive and negative results in respectively one and two cases. The fourth case, which was positive by FISH and immunohistochemistry but negative by RT-PCR, was shown to have a non-EML4-ALK ALK-rearrangement. As various factors such as RNA quality, fixation quality and type of ALK rearrangement may impede ALK screening, we propose a combined FISH/molecular biology diagnostic algorithm in which anti-ALK immunohistochemistry is used as a pre-screening step.

Introduction

In 2007, anaplastic lymphoma kinase (ALK) was shown to be involved in the oncogenesis [1], [2] of a small subset (∼4%) [3] of lung carcinomas. In these tumours, a small inversion within the short arm of chromosome 2 leads to an open reading frame that links the first exons of EML4 to the 3′ part of ALK (from exon 20) [2]. EML4-ALK tumours, which are now considered as a new molecular subgroup of lung tumours, are almost exclusively adenocarcinomas, without mutation of EGFR or KRAS, and preferentially arise in non-smokers [3]. Crizotinib, a small orally dispensable ALK inhibitor, is currently tested in a phase III clinical trial and the preliminary results of the phase II trial evidenced a surprising 90% disease control rate in ALK-rearranged lung tumours [4]. Nevertheless, if crizotinib confirms its high therapeutic efficiency, it is likely that its use will be restricted to tumours with a proven ALK rearrangement.

However, assessing ALK rearrangement in lung tumours remains a diagnostic challenge [3]. In the literature, reverse transcriptase polymerase chain reaction (RT-PCR) of the EML4-ALK transcripts [5] appeared to be a sensitive and specific tool. However, thirteen variants of EML4-ALK have been already described according to the break point on EML4 (from exon 2 to exon 20) [3]. Furthermore, beside EML4, TFG [1] and KIF5B [6], [7] have also been described to be fused to ALK in rare cases. Thus, RT-PCR has to be multiplexed, i.e. several forward primers on EML4 (± on other fusion partners) are required in order to target all of the potential fusion variants of ALK [5], [6]. As some EML4-ALK RT-PCR amplicons size more than 1000 bp, the diagnostic relevance of RT-PCR directly relies on the quality of the extracted RNA, thus requiring a proper cryopreservation of tumour samples. Nevertheless, in routine, such specimens are not always available for studies. ALK break-apart fluorescent in situ hybridization (FISH) [8], [9], [10], [11], [12] has the advantage to be performable on formalin-fixed paraffin-embedded (FFPE) tumour specimens. However, as the ALK rearrangement in lung tumours is usually intra-chromosomal (small 2p inversion), split signals (that indicate ALK-rearranged alleles) can be subtle [8], [10] and difficult to recognize for untrained pathologists. As another FFPE-suitable diagnostic tool, anti-ALK immunohistochemistry should allow a rapid pre-screening of cases that require further ALK testing by RT-PCR or FISH. Unfortunately, anti-ALK immunohistochemistry gives poor results with the antibody clone ALK1 [6], [8], [10], [12], [13], which is the reference clone for ALK screening in lymphoma [14]. More reliable results have however been obtained, but required specific conditions [6], [10], such as very high antibody titres, unusual signal amplification methods, or using not commercially available antibody clones [12].

As a comprehensive analysis of the various diagnostic methods was lacking in the literature, we aimed to compare five different methods for the diagnosis of ALK rearrangement in lung tumours, in order to propose a diagnostic procedure that could be used in daily practice.

Section snippets

Patients

We studied a series of 159 surgically resected lung adenocarcinomas with available frozen material, which had been collected between 2003 and 2006 by the pathology department of the Georges Pompidou European hospital (study approved by the Comité de Protection des Personnes Ile de France II, #CCP 2008-136). All the tumours had been tested (HB) for mutation of the EGFR (exons 18–21), KRAS (codons 12, 13 and 61), TP53 (exons 4–9), ERBB2 (exons 18–21), PIK3CA (exons 10 and 21), STK11 (exons 1–9)

Patients description

In order to increase the ratio of ALK-rearranged cases among the tested tumours, we narrowed a series of 159 surgically resected lung adenocarcinomas to tumours without EGFR mutation from non- or light (<10 pack year) smoking patients. Four of the 24 tumours that met these criteria were excluded for insufficiently available frozen material. Clinical, pathological and molecular data of the 20 studied tumours are presented in Table 1. None had mutation of the PIK3CA, STK11 and PXN genes.

Multiplex RT-PCR and sequencing from frozen material

We first

Discussion

In our series of 20 selected lung adenocarcinomas, we identified four ALK-rearranged tumours, which is a quite similar ratio to what have been observed in other selected series (∼14%) [10], [11]. As previously described, ALK rearrangement appeared to be linked to an absence of concomitant KRAS mutation [3] and to either a mucinous cribriform pattern [13], [16] or to a solid signet-ring cell pattern [10], [16], [17]. We studied four other lung adenocarcinomas with this latter pathological

Conflict of interest statement

The Repeat-Free Poseidon ON ALK/EML4 t(2;2);inv(2) fusion probes set was a gift from Kreatech (Amsterdam, The Netherlands).

Acknowledgement

We thank the National Cancer Institute (INCA) for general funding.

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