Low Frequency ALK Hotspots Mutations In Neuroblastoma Tumours Detected By Ultra-deep Sequencing: Implications For ALK Inhibitor Treatment

Niloufar Javanmardi,Susanne Fransson,Staffan Nilsson,Tommy Martinsson,Per Kogner,Rose-Marie Sjöberg,Katarina Truvé,Anna Djos

doi:10.1038/s41598-018-37240-z

Niloufar Javanmardi, Susanne Fransson + Show 6 more

Open Access

https://doi.org/10.1038/s41598-018-37240-z

Copy DOI

Abstract

The ALK tyrosine kinase receptor is oncogenically activated in neuroblastoma. Whereas numerous ALK fusion genes have been reported in different malignancies, in neuroblastoma ALK is mainly activated through point mutations. Three hotspot residues (F1174, F1245, and R1275) account for 85% of mutant ALK seen in neuroblastoma. In a cohort of 105 Swedish neuroblastoma cases of all stages, these hotspot regions were re-sequenced (>5000X). ALK mutations were detected in 16 of 105 patients (range of variant allele fraction: 2.7–60%). Mutations at the F1174 and F1245 hotspot were observed in eleven and three cases respectively. ALK mutations were also detected at the I1171 and L1240 codons in one tumor each. No mutations were detected at R1275. Sanger sequencing could confirm ALK status for all mutated samples with variant allele fraction above 15%. Four of the samples with subclonal ALK mutation fraction below this would have gone undetected relying on Sanger sequencing only. No distinct mutation spectrum in relation to neuroblastoma tumours genomic subtypes could be detected although there was a paucity of ALK mutations among 11q-deleted tumors. As ALK mutations status opens up an excellent opportunity for application of small molecule inhibitors targeting ALK, early and sensitive detection of ALK alterations is clinically important considering its potential role in tumour progression.

Highlights

Somatically acquired ALK alterations are observed in 6–12% of sporadic cases[7,8,9]
In this study we performed ultra-deep sequencing in order to determine the frequency of ALK hotspot mutations that might be missed with conventional Sanger sequencing due to different degrees of sensitivity
At the F1174 hotspot, alterations were observed in eleven cases: seven cases harbored a mutation leading to the amino acid change F1174L, two cases with F1174I, one each of the F1174C, and F1174S substitutions, with the mutated allele fractions ranging from 14% to 60%

Summary

Introduction

Somatically acquired ALK alterations are observed in 6–12% of sporadic cases[7,8,9]. Activating point mutations are mainly seen at three “hot spot” residues; F1174 (mutated to L, S, I, C or V), F1245 (mutated to L, I, V, or C), and R1275 (mutated to Q or L), all localized within the kinase domain of ALK and together accounting for 85% of all mutant ALK in NB10. It is highly likely that these initial subclones present at diagnosis confer a selective advantage after first line treatment with chemotherapy, leading to clonal expansion and tumour relapse These data strongly suggest that precise molecular characterization of ALK mutations should be included in clinical diagnostics of NB tumours at diagnosis and continuously throughout the clinical management of the patient. To investigate this rigorously we have undertaken a study of a series of 105 NB tumours to explore ALK copy number gain as well as the frequency and type of ALK mutations that may remain undetected using Sanger-based sequencing methodology.

Methods

Results

Discussion

Conclusion