Histologic subtypes, immunohistochemistry, FISH or molecular screening for the accurate diagnosis of ALK-rearrangement in lung cancer: A comprehensive study of Caucasian non-smokers
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.
References (21)
- et al.
Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer
Cell
(2007) - et al.
The biology and treatment of EML4-ALK non-small cell lung cancer
Eur J Cancer
(2010) - et al.
Anaplastic lymphoma kinase immunoreactivity correlates with ALK gene rearrangement and transcriptional up-regulation in non-small cell lung carcinomas
Hum Pathol
(2009) - et al.
EML4-ALK rearrangement in non-small cell lung cancer and non-tumor lung tissues
Am J Pathol
(2009) - et al.
EML4-ALK fusion is linked to histological characteristics in a subset of lung cancers
J Thorac Oncol
(2008) - et al.
Frequent ALK rearrangement and TTF-1/p63 co-expression in lung adenocarcinoma with signet-ring cell component
Lung Cancer
(2011) - et al.
Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer
Nature
(2007) - et al.
Clinical activity of the oral ALK inhibitor PF-02341066 in ALK-positive patients with non-small cell lung cancer (NSCLC). 2010 ASCO Annual Meeting Proceedings (Post-Meeting Edition)
J Clin Oncol
(2010) - et al.
Multiplex reverse transcription-PCR screening for EML4-ALK fusion transcripts
Clin Cancer Res
(2008) - et al.
A novel fusion oncokinase identified by an immunohistochemistry-based diagnostic system for ALK-positive lung cancer
Clin Cancer Res
(2009)