Abstract 4876: Low frequent ALK hotspot mutations in neuroblastoma tumors detected by ultra deep sequencing: Implications for ALK inhibitor treatment
Authors: Fransson Susanne M. , Javanmardi Niloufar , Sjoberg Rose-Marie , Kogner Per , Martinsson Tommy 
Published: July 2017

Abstract
Background The pediatric cancer neuroblastoma (NB) is heterogeneous in terms of both genotype and clinical behavior. NB patients show complex patterns of genetic abnormalities, which may include amplification, translocation or oncogenic mutations of ALK kinase in sporadic and familial cases. Three hot spot residues (F1174, F1245, and R1275) localized within the kinase domain of ALK, accounts for 85% of mutant ALK in NB. In this study we used ultra deep sequencing for sensitive detection of ALK mutations.
Methods To detect the sub-clonal ALK mutations in 105 neuroblastoma tumors that can remain undetected using Sanger chemistry, we used two different approaches; hybridization-based Haloplex kit (Agilent) and PCR-based amplicon sequencing, both sequenced on the MiSeq Illumina platform. Coverage, uniformity and variant calling ability showed similar performance between both methods.
Findings ALK mutations were detected in 14/105 patients (13%) whereof six with allele frequency below 20%. At the I1171 locus an ALK mutation was detected in one case with variant allele frequency as low as 2.6%, the lowest level detected in our cohort. Mutations at the F1174 hotspot were observed in nine cases: six cases with F1174L, while the remaining three showed F1174C, F1174S and F1174I substitutions, with mutated allele fractions ranging from 14% to 60%. Interestingly the patient harboring F1174S mutation with 58% frequency, in parallel contained a small sub-clone of F1174I mutation with 8% frequency of the mutated allele. F1245-codon alterations were detected in three cases: two F1245I mutations and one F1245C mutation, with frequencies of 14%, 51% and 52%, respectively. A single case showed a L1240V variant with a mutated allele fraction at 57%. Sanger sequencing of all ALK mutated samples could confirm ALK status for all samples with variant allele fraction above 15%. Because of the limits of detection, 4 out of 6 samples with a mutated allele fraction below 15% would have gone undetected relying on Sanger sequencing. Our results fail to reveal a distinct mutation spectrum in relation to neuroblastoma genomic subtypes. Moreover, a variety of neuroblastic tumours, from benign ganglioneuroma to very aggressive metastatic neuroblastoma, could be observed in the context of ALK mutation.
Interpretation These results indicate that, in neuroblastoma, ALK mutations can be present at sub-clonal level with the possibility of subsequent clonal expansion at the time of progression. Tyrosine kinase inhibitors have become the gold standard therapy in treating ALK positive NBs, making diagnostic high sensitive detection of ALK mutations a necessary step in identifying optimal treatment modalities. This study is of utmost importance in clinical practice, highlighting the potential of NGS and the importance of serial samplings for therapeutic decisions.
Citation Format: Susanne M. Fransson, Niloufar Javanmardi, Rose-Marie Sjoberg, Per Kogner, Tommy Martinsson. Low frequent ALK hotspot mutations in neuroblastoma tumors detected by ultra deep sequencing: Implications for ALK inhibitor treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4876. doi:10.1158/1538-7445.AM2017-4876