EML4-ALK rearrangement in non-small cell lung cancer and non-tumor lung tissues

Abstract

A fusion gene, echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK), with transforming activity has recently been identified in a subset of non-small cell lung cancer (NSCLC), but its pathogenetic, diagnostic, and therapeutic roles remain unclear. Both frequency and type of EML4-ALK transcripts were investigated by reverse transcription PCR in 120 frozen NSCLC specimens from Italy and Spain; non-neoplastic lung tissues taken far from the tumor were used as controls. In cases carrying the fusion transcript, we determined EML4-ALK gene and protein levels using fluorescence in situ hybridization, Western blotting, and immunoprecipitation. We also analyzed ALK protein levels in paraffin samples from 662 NSCLC specimens, including the 120 cases investigated in the molecular studies. EML4-ALK transcripts (variants 1 and 3) were detected in 9 of 120 NSCLC samples but were not specific for NSCLC since they were also found in non-cancerous lung tissues taken far from the tumor. Notably, no transcripts were detected in matching tumor samples from these patients. Fluorescence in situ hybridization analysis of cases expressing EML4-ALK transcripts showed that only a minority of cells harbored the EML4-ALK gene. None of these cases was found to express the EML4-ALK protein as examined by immunohistochemistry, Western blotting, and immunoprecipitation. The EML4-ALK transcript cannot be regarded as a specific diagnostic tool for NSCLC. Our results show therefore that the causal role and value of EML4-ALK as a therapeutic target remain to be defined.

Figure 1

EML4-ALK transcripts in NSCLC and non-tumor lung samples. A: Analysis of variant 1 (EML4 exon13-ALK exon 20) and (B) variant 3 (EML4 exon 6 – ALK exon 20) fusion transcripts in tumor and non-tumor lung samples. Genes analyzed are indicated on the right. Amplicon size in bp is indicated on the left. Case IDs are reported on top; suffix “T” indicates lung cancer samples, while suffix “N” indicates non-tumor lung samples from cancer patients. “C” is the no-template negative control of PCR. H2228 lung cancer cell line served as positive control. C: Sequence electropherogram of EML4-ALK variant 3 isoforms representing fusion of EML4 exon 6 – ALK exon 20. The fusion transcripts generated may contain a short isoform of variant 3 fusion gene (155 bp, upper panel), a long isoform (188 bp, including 33bp from intron 6 of EML4 gene, lower panel) or both (155/188 bp, middle panel).

Figure 2

Western blotting and immunoprecipitation assays on NSCLC harboring EML4-ALK fusion transcript. A: Testing of anti-ALK mAb (ALKc) in Western blot and immunoprecipitation. Left: Western blot analysis on cell lysates of Rh30, Karpas 299 and H2228 cell lines, expressing full-length ALK (about 200 kDa), NPM-ALK (80 kDa), and the short form of EML4-ALK (about 90 kDa), respectively. Right: ALKc is able to recognize and immunoprecipitate the EML4-ALK fusion protein variant 1 (about 120 kDa) from the Phoenix cells transfected with EML4-ALK fusion gene construct, variant 1 (Px_EML4-ALK). The lower molecular weight bands appearing in the membrane likely represent degradation products of the over-expressed protein. B: Western blot analysis on EML4-ALK-positive samples: a band corresponding to EML4-ALK fusion protein (variant 1, about 120 kDa, left; variant 3, about 90 kDa, right) is not detectable in either NSCLC (cases B/252T, B/5796T, B/8237T, B/9020T, 424T, 447T for variant 1; 470T for variant 3) or non-neoplastic patient samples (cases 446N and 442N for variant 3). RIPA extracts or whole cell lysates, WCL. The membrane was stripped and reblotted with anti-Hsp90 rabbit polyclonal antibody (lower panels). C: Absence of detectable EML4-ALK fusion protein in the ALK-immunoprecipitates from EML4-ALK-positive NSCLC patient samples. As positive controls for ALK-immunoprecipitation, the H2228 and the Phoenix cell line transfected with EML4-ALK fusion gene construct, variant 1 (Px_EML4-ALK) were used. As positive control, immunoprecipitation of Hsp90 from one out of six NSCLC samples was performed. The same membrane was blotted with ALKc mAb (upper) and then stripped and reblotted with anti-Hsp90 rabbit polyclonal antibody (lower). D: Same experiment as in (C) was conducted on one EML4-ALK-positive non-tumor lung tissue sample (397N).

Figure 3

FISH analysis of ALK rearrangements in NSCLC and non-tumor lung samples positive for EML4-ALK transcripts by RT-PCR. The ALK break-apart FISH probe contains two differently labeled probes on opposite sides of the breakpoint of the ALK gene. The normal ALK appears as a yellow signal, while rearranged ALK at this locus will result in separate red and green signals or only one red signal. A: FISH analysis on paraffin-embedded section of NSCLC samples 435T and 447T and on touch imprints samples of non-tumor lung tissues of cases 439N, 466N, 430N. Red arrows indicate rearranged ALK. B: FISH analysis performed with the same probe on metaphases of cell-line H2228 (positive for EML4-ALK variant 3 by RT-PCR): two normal chromosome 2 (with a co-localization of the red and a green signal) and a deletion of the 5′ ALK (with only one red signal present) are visible indicating a rearranged ALK locus. The latter is localized on an extrachromosomal element, possibly a double minute, indicated by the red arrow, which is present in more than one copy per cell in 4% of the nuclei analyzed.

Figure 4

ALK protein expression in NSCLC. A lung squamous cell carcinoma (A, magnification = original ×400) and a lung adenocarcinoma (B, magnification = original ×400) positive for EML4-ALK fusion transcript do not express the ALK protein. C: Phoenix cells transfected with EML4-ALK show strong cytoplasmic-restricted ALK positivity (magnification = original ×800). D: No staining is observed in Phoenix cells transfected with the empty vector (magnification = original ×800). E: Anaplastic large cell lymphoma carrying t(2;5)/NPM-ALK shows cytoplasmic and nucleolar positivity for ALK (arrow; magnification = original ×800). F: ALK positivity in a rhabdomyosarcoma carrying full-length ALK is mainly restricted to the cell surface (arrow; magnification = original ×800). A-F: immunostaining with anti-ALK mAb clone 5A4; the same results (not shown) were obtained with the ALK1 and ALKc mAbs; Immuno-alkaline phosphatase anti-alkaline phosphatase technique.