High ALK mRNA expression has a negative prognostic significance in rhabdomyosarcoma

Abstract

Background: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in cancer, but its clinical and functional importance remain controversial. Mutation or amplification of ALK, as well as its expression levels assessed by conventional immunohistochemistry methods, has been linked to prognosis in cancer, although with potential bias because of the semi-quantitative approaches. Herein, we measured ALK mRNA expression in rhabdomyosarcoma (RMS) and determined its clinical impact on patients' stratification and outcome.

Methods: Specimens were obtained from RMS patients and cell lines, and ALK expression was analysed by quantitative RT-PCR, western blotting, IHC, and copy number analysis.

Results: High ALK mRNA expression was detected in the vast majority of PAX3/7-FOXO1-positive tumours, whereas PAX3/7-FOXO1-negative RMS displayed considerably lower amounts of both mRNA and protein. Notably, ALK mRNA distinguished unfavourable PAX3/7-FOXO1-positive tumours from PAX3/7-FOXO1-negative RMS (P<0.0001), and also correlated with larger tumour size (P<0.05) and advanced clinical stage (P<0.01), independently of fusion gene status. High ALK mRNA levels were of prognostic relevance by Cox univariate regression analysis and correlated with increased risk of relapse (P=0.001) and survival (P=0.01), whereas by multivariate analysis elevated ALK mRNA expression resulted a negative prognostic marker when clinical stage was not included.

Conclusion: Quantitative assessment of ALK mRNA expression helps to improve risk stratification of RMS patients and identifies tumours with adverse biological characteristics and aggressive behaviour.

Figure 1

Expression analysis of ALK in RMS cell lines. (A) Expression of ALK mRNA in tumour cell lines, using primers spanning the extracellular region (ALK-1) and the intracytoplasmic portion (ALK-2) of ALK gene. β2-Microglobulin (B2M) was used as housekeeping gene. RMS-t, PAX3/7-FOXO1-positive RMS; RMS-not, PAX3/7-FOXO1-negative RMS; NB, neuroblastoma; SKM, fetal skeletal muscle; ALCL, anaplastic large cell lymphoma. (B) Relative ALK mRNA expression levels in RMS-t and RMS-not cell lines measured by qRT–PCR. LAN5 and SKM were included as positive and negative controls, respectively. Distribution of data is represented by box plot analysis (dashed box). *P<0.05. (C) ALK protein expression in RMS-t and -not cell lines by western blotting, using β-actin as protein-loading control. (D) Total (α-ALK, upper panel) and phosphorylated (α-pALK, lower panel) ALK proteins detected in RMS-t and RMS-not cell lines after immunoprecipitation (IP:ALK). LAN5 and KARSPAS-299 (K299) cells were used as positive controls for ALK and NPM-ALK expression, respectively. LAN5 total lysates were used as input (Tot.lys.). Asterisk marks non-specific bands in RMS and NB cells. (E) ALK tyrosine phosphorylation in RH30 and RD cell lines exposed to pleiotrophin (PTN) or ALK agonist antibody (mAb) in the presence (+) or absence (−) of ALK inhibitor crizotinib. Total and phosphorylated ALK and ERK1/2 proteins were detected by western blotting. γ-Tubulin was included as loading control.

Figure 2

ALK mRNA and protein expression in RMS tumour specimens. (A) ALK protein expression analysis in PAX3/7-FOXO1-negative (ARMS-not, a; ERMS, b) and -positive (ARMS-t, c and d) RMS tumours, by immunohistochemistry (left) and western blotting (right). (B) Point plot analysis of ALK mRNA relative expression in PAX3/7-FOXO1-positive (ARMS-t) and -negative (ARMS-not) ARMS, and in ERMS tumours by qRT–PCR. P-values were calculated using the Student's t-test. ***P<0.001. (C) ALK gene amplification analysis in RMS tumours and cell lines, normalised for RNA Polymerase IID expression (Pol IID). Graph shows true ALK amplification in normal cells (open circles), tumour cell lines (RMS, open triangles; NB1, closed triangle), and RMS specimens (closed squares). Dotted line represents cutoff for definition of gene amplification (gene copy number ⩾4).

Figure 3

Correlation between ALK mRNA expression levels and clinicopathological parameters in RMS tumours. ALK mRNA levels, assessed by qRT–PCR and normalised to GAPDH housekeeping gene, were compared among groups classified by tumour size, staging, or age. *P<0.05; **P<0.01.

Figure 4

Receiver operating characteristic curve (ROC) analysis. ROCs showing the sensitivity and specificity of ALK mRNA as a parameter to classify RMS patients (n=71) on the basis of the fusion gene status (PAX-FOXO1, RMS-t vs RMS–not; ΔΔCT=555) (A) or the risk of failure (EVENT, NO vs YES; ΔΔCT=200) (B). P-values, AUC, and 95% CIs were computed by using SAS statistical programme. Abbreviations: AUC= area under the curve; CI, confidence interval.

Figure 5

Prognostic significance of ALK gene expression. Kaplan–Meier and log-rank analysis for progression-free (PFS) and overall survival (OS) of RMS patients (n=71) based on specific quantitative ALK mRNA cutoff value (ALK low=ΔΔCT<200; ALK high=ΔΔCT⩾200) identify two subgroups of patients with significantly different outcome (PFS, P=0.001; OS, P=0.01).