ALK gene amplification is associated with poor prognosis in colorectal carcinoma

Abstract

Background: Recently, the anaplastic lymphoma kinase (ALK) has been found to be altered in several solid and haematological tumours. ALK gene copy number changes and mutations in colorectal cancers (CRCs) are not well characterised. We aimed to study the prevalence of ALK copy number changes, translocations, gene mutations and protein expression in 770 CRC patients, and correlate these findings with molecular and clinico-pathological data.

Methods: ALK gene copy number variations and ALK expression were evaluated by fluorescence in situ hybridisation (FISH) and immunohistochemistry, respectively.

Results: Translocations of the ALK gene were not observed; 3.4% (26 out of 756) of the CRC patients tested had an increase in ALK gene copy number either amplification or gain. Interestingly, increased ALK gene copy number alteration was associated with poor prognosis (P=0.0135) and was an independent prognostic marker in multivariate Cox proportional hazards model. The study reveals a significant impact of ALK gene copy number alterations on the outcome of patients with CRC.

Conclusion: The findings of our study highlight a potential role of targeting ALK in advanced CRCs by using ALK FISH and ALK IHC as a screening tool to detect ALK alterations. Based on these findings, a potential role of ALK inhibitor as a therapeutic agent in a subset of CRC merits further investigation.

Figure 1

Tissue microarray based immunohistochemical analysis of ALK in CRC patients. An IHC H score was assigned to each case according to the following criteria: 3+, intense, cytoplasmic and/or granular staining; 2+, moderate, smooth cytoplasmic staining; 1+, faint cytoplasmic staining; and 0, no staining and the proportion of the tumour staining for that intensity was recorded as 5% increments from a range of 0–100. (A) CRC array spot showing ALK overexpression with membranous staining. (B) A CRC array spot showing ALK overexpression with strong, granular cytoplasmic staining (3+). (C) A CRC array spot showing ALK overexpression with moderate cytoplasmic staining(2+). (D) A CRC array spot showing low cytoplasmic expression of ALK (1+). (E) Colorectal cancer tissue array spots with no cytoplasmic expression of ALK (0). (F) Normal colon tissue array spots showing no expression of ALK (0). All figures had × 10 magnified image captured with an Aperio ScanScope XT (Aperio Technologies, Vista, CA, USA; objective insets: magnified view × 40 magnified image).

Figure 2

Determination of ALK gene copy number by flourescence in situ hybridization (FISH) and qPCR in ALK-amplified and ALK-non-amplified CRC samples. A minimum of 20 cells with both the centromeric and ALK gene signals were scored to give conclusive data. (A) Colorectal cancer ALK-amplified sample hybridised with ALK break-apart probe showing six red and green fused signals (inset showing a single cell with amplification). (B) Colorectal cancer ALK-non-amplified sample hybridised with ALK break-apart probe showing normal tissue with two red and green fused signals (inset showing a single cell). (C) Colorectal cancer ALK-amplified sample hybridised with BAC probe RP11–328L16 and CEP2 probe showing two red CEP2 signals and 5–6 green signals representing the ALK gene amplification (inset showing a single cell with amplification). (D) Colorectal cancer ALK-non-amplified sample hybridised with BAC probe RP11–328L16 and CEP2 probe showing two red CEP2 signals and two green signals representing normal ALK copy number (inset showing a single cell). ( × 20/0.70 objective on an Olympus BX 51 microscope (Olympus America Inc, Center Valley, PA, USA) with the inset showing a × 40/0.85 aperture magnified view of the same for Figure 1A–C). (E) Verification of the ALK gene copy number by qRTPCR. Histogram showing ALK gene copy number obtained from normal colon samples1–4 with two normal copies of ALK gene, and samples 5–8 are amplified colon cancer samples.

Figure 3

Prognostic significance of ALK in CRC and the Kaplan–Meier survival analysis. (A) Colorectal cancer patients with FISH amplification/gain and increased copy number had reduced overall survival of 53.5% at 5 years compared with 70.9% with normal FISH (P= 0.0135). (B) In stage IV CRC, patients with FISH gain/amplification and increased copy number had reduced median overall survival of 3 months as compared with a median survival of 27 months with normal FISH (P= 0.0097).