Purpose: In neuroblastoma, activating ALK receptor tyrosine kinase point mutations play a major role in oncogenesis. We explored the potential occurrence of ALK mutations at a subclonal level using targeted deep sequencing.Experimental Design: In a clinically representative series of 276 diagnostic neuroblastoma samples, exons 23 and 25 of the ALK gene, containing the F1174 and R1275 mutation hotspots, respectively, were resequenced with an extremely high depth of coverage.Results: At the F1174 hotspot (exon 23), mutations were observed in 15 of 277 samples (range of fraction of mutated allele per sample: 0.562%-40.409%). At the R1275 hotspot (exon 25), ALK mutations were detected in 12 of 276 samples (range of fraction of mutated allele: 0.811%-73.001%). Altogether, subclonal events with a mutated allele fraction below 20% were observed in 15/27 ALK-mutated samples. The presence of an ALK mutation was associated with poorer 5-year overall survival (OS: 75% vs. 57%, P ¼ 0.0212 log-rank test), with a strong correlation between F1174 ALK mutations and MYCN amplification being observed.Conclusions: In this series, deep sequencing allows the detection of F1174 and R1275 ALK mutational events at diagnosis in 10% of cases, with subclonal events in more than half of these, which would have gone undetected by Sanger sequencing. These findings are of clinical importance given the potential role of ALK mutations in clonal evolution and relapse. These findings also demonstrate the importance of deep sequencing techniques for the identification of patients especially when considering targeted therapy.
2 Dozens of recurrent chromosomal rearrangements have been described in acute leukaemia, where they often result in the fusion of two genes and in the expression of hybrid proteins.These genetic markers often delineate distinct clinical entities, and have been incorporated into the World Health Organisation classification. Today, their evaluation at diagnosis thus provides crucial information for risk stratification, treatment decisions and residual disease evaluation 1,2 .Conventional cytogenetics is usually regarded as the gold standard method for the detection of these rearrangements. It is often considered mandatory at diagnosis but it requires a high level of expertise, is time consuming, and is not suitable for the detection of many cryptic rearrangements 3,4 . Complementary approaches such as fluorescent in situ hybridisation (FISH) and RT-PCR are thus often needed 5 , but the cost of a comprehensive analysis can rapidly become prohibitive. Many markers that may provide important clinical information are therefore never tested, lowering the chances for the patients to benefit from the optimal treatments. To address this issue, we have developed a rapid and parsimonious ligationdependant RT-PCR amplification assay (LD-RTPCR) 6 that allows for the detection of dozens of gene rearrangements (Figure 1).We created a mix of 153 LD-RTPCR probes to target more than 50 recurrent fusion genes and three NPM1 mutations ( Figure 1B and C). The procedure is detailed in supplemental methods, and representative results are provided in supplemental figure 1. As shown in supplemental figure 2A, the results inform on the identity of the two partner genes, but also on the localisation of the breakpoint, guiding the choice of an appropriate assay for residual disease evaluation. The analysis of serial dilutions showed that approximately 3.10 3 copies of transcripts are sufficient for the identification of the two partners (Supplemental figure 2B).To validate this method, we applied it to a retrospective cohort of 540 patients treated in our institutions ( Figure 2 and supplemental table 2). Sixty eight rearrangements were detected in the 180 childhood ALL cases we tested. Fifty two (76.5%) had been identified using RT-PCR at diagnosis and 20 (29.4%) using cytogenetics. Sixty seven of these rearrangements (98.5%) were detected using LD-RTPCR. As expected in this cohort, the most common were the ETV6-RUNX1 fusion (33 cases), rearrangements of the MLL gene (8 cases, four with AFF1, two with MLLT1, one with AFF4 and one with MLLT3) 7 , and the BCR-ABL1 (six cases), TCF3-PBX1 (five cases) and STIL-TAL1 fusions (four cases) 2 . The LD-RTPCR assay only failed to detect one t(4;11)(q21;q23), which was identified at diagnosis using cytogenetics but which could not be confirmed using RT-PCR. The breakpoint of this rearrangement may thus be unusual, or involve genes other than MLL and AFF1. It also validated four junctions which © 2015 Macmillan Publishers Limited. All rights reserved.3 were detected using cytogenetics, but which had not...
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