ALK Gene Mutation, Protein Expression in Advanced Neuroblastoma

Abstract and Introduction

Abstract

Objectives: The protein ALK is targeted for therapy in neuroblastoma, and ALK mutation confers a poor prognosis. We evaluated ALK in a cohort of patients with advanced neuroblastoma diagnosed by fine-needle aspiration biopsy (FNAB).

Methods: Fifty-four cases of neuroblastoma were evaluated for ALK protein expression by immunocytochemistry and ALK gene mutation by next-generation sequencing. MYCN amplification by fluorescence in situ hybridization, International Neuroblastoma Risk Group (INRG) staging, and risk assignment was performed, and patients were managed accordingly. All parameters were correlated with overall survival (OS).

Results: ALK protein showed cytoplasmic expression in 65% cases and did not correlate with MYCN amplification (P = .35), INRG groups (P = .52), and OS (P = .2); however, ALK-positive, poorly differentiated neuroblastoma showed better prognosis (P = .02). ALK negativity was associated with poor outcome by Cox proportional hazard model (hazard ratio, 2.36). Two patients showed ALK gene F1174L mutation with 8% and 54% allele frequency and high ALK protein expression; they died of disease 1 and 17 months following diagnosis, respectively. A novel IDH1 exon 4 mutation was also detected.

Conclusions: ALK expression is a promising prognostic and predictive marker in advanced neuroblastoma that can be evaluated in cell blocks from FNAB samples along with traditional prognostic parameters. ALK gene mutation confers a poor prognosis for patients with this disease.

Introduction

Neuroblastoma is an embryonal tumor originating from the autonomic nervous system that has a heterogeneous clinical and biological spectrum.^[1] A wide range of clinical outcomes from spontaneous regression or relentless progression leads to death, despite extensive multimodal therapies.^[1–3] As per the International Neuroblastoma Risk Group (INRG) Task Force, age and stage at presentation, histologic category based on tumor differentiation and mitosis-karyorrhexis index (MKI), DNA ploidy, MYCN amplification, and chromosome 11q status are parameters required for better risk stratification into very low, low, intermediate, and high-risk groups.^[4] Patients' disease is managed according to risk groups, but the outcome for patients with high-risk neuroblastoma remains poor, with 5-year overall survival (OS) below 50%.^[4,5] Fine-needle aspiration biopsy (FNAB) is a useful technique for diagnosing neuroblastic tumors, especially advanced neuroblastoma, and can provide a rapid and accurate diagnosis wherever cytopathology expertise is available. The cytomorphologic features of neuroblastic tumors is well described.^[6–8] Furthermore, evaluation of MKI and MYCN amplification allows the application of INPC and further risk stratification, as the INRG has reported.^[9–11]

The ALK gene and corresponding protein are important players in embryonic neural development and are strongly expressed in that context.^[12] The biological role of ALK in neuroblastoma has been studied in neuroblastoma cell lines.^[13] The discovery of activating mutations in the tyrosine kinase domain of ALK as the major cause of hereditary neuroblastoma provides the first example of a pediatric cancer arising from germline mutations in an oncogene.^[14] The role of MYCN in neuroblastoma is well established, and evaluation of MYCN amplification in tissue samples is an important aspect of current risk stratification protocols, including the INRG and Children's Oncology Group algorithms for management.^[4,15] Interestingly, both genes are just 13.2 megabases apart on chromosome 2p. As a therapeutic target in primary neuroblastoma, ALK represents a major research advance in this childhood cancer.^[16] There have been a few studies on ALK protein expression in neuroblastoma by immunohistochemistry (IHC),^[17–20] but ALK's utility as an independent prognostic marker and correlation to traditional prognostic markers such as age, tumor morphology, and MYCN amplification are still not well established, and hence this was the main aim of this study. We also evaluated a subset of cases for ALK gene mutation by next-generation sequencing (NGS) to determine its frequency in our cohort of patients.