Gene Panel Sequencing of Primary and Relapsed Pediatric T-ALL Shows That Relapse-Specific Mutations Are Diverse and Mostly Non-Recurrent
Authors: Pechanska Paulina , Kunz Joachim , Rausch Tobias , Bandapalli Obul Reddy , Orlova Elena , Sagi Judit , Stanulla Martin , Schrappe Martin , Kirschner-Schwabe Renate , Hof Jana , Eckert Cornelia , Köhler Rolf , Muckenthaler Martina U. , Korbel Jan o. , Kulozik Andreas E 
Published: October 2019

Abstract
Precursor T-cell acute lymphoblastic leukemia (T-ALL) remains one of the major challenges of pediatric oncology, because relapses are frequently refractory to treatment and fatal. We aimed at identifying relapse specific genetic alterations by analyzing a cohort of 147 primary T-ALL patients and of 70 relapsed T-ALL patients with targeted gene panel sequencing. In addition to the analysis of single nucleotide variants (SNVs) and small insertions and deletions (InDels), we made use of the available coverage data to characterize aberrant copy number alterations (CNA).
DNA from bone marrow of these 217 pediatric T-ALL patients was analyzed by gene panel sequencing after target capture with Agilent HaloPlex. In the target capture design, exons of 324 genes were included that had been found before by whole exome sequencing to carry somatic mutations in a pilot set of relapsed T-ALL or that have been reported to be mutated in T-ALL in the literature. We did not analyze corresponding remission samples and did not discriminate between germline and somatic alterations. Only mutations with an allele frequency (AF) > 10% were considered and absence of the mutation in the 1000 Genomes variant catalogue was required. Copy number analysis based on read-depth data identified deletions (DEL) and amplifications (AMP). Direct comparison of CNAs by multiplex ligation-dependent probe amplification (MPLA) and gene panel sequencing was possible for 13 overlapping regions covering 14 genes in 185 samples. Recognition rate by coverage analysis was 98% (256/260) for biallelic alterations and 81% (92/114) for monoallelic alterations found by MLPA.
On average, gene panel sequencing identified 6.7 mutations in initial diagnosis samples (SNVs: 5.2; InDels: 1.5) and 7.9 mutations in relapse samples (SNVs: 5.9; InDels: 2). In the group of primary leukemia and relapse samples, the average AMP/DEL per patient was 8.2 (AMP: 3.2, DEL: 5.0) and 8.8 (AMP: 4.2, DEL: 4.6), respectively. 31 genes were found to be mutated and 46 deleted/amplified in 10 or more patients (see Table 1 and 2).
Table 1. Most commonly mutated genes (SNVs and InDels) Gene Total # of mutations # pts with mutation in primary T-ALL(n=147) # pts with mutation in relapsed T-ALL (n=70) NOTCH1 178 88 (60%) 38 (54%) PHF6 47 24 (16%) 16 (23%) FBXW7 42 20 (14%) 17 (24%) OBSCN 35 20 (14%) 10 (14%) DNM2 28 17 (12%) 10 (14%) PTEN 34 20 (14%) 4 (6%) XIRP2 24 19 (13%) 5 (7%) CDH23 23 11 (7%) 11 (16%) WT1 36 11 (7%) 8 (11%) NT5C2 22 1 (1%) 17 (24%)
Table 2. Most common copy number alterations Amplifications Deletions Gene primary T-ALL (n=147) relapsed T-ALL (n=64) Gene primary T-ALL (n=147) relapsed T-ALL (n=64) MYB 9 (6%) 9 (15%) CDKN2A 102 (70%) 36 (59%) MYC 11 (8%) 6 (10%) CDKN2B 83 (57%) 31 (51%) NRG1 11 (8%) 3 (5%) MLLT3 28 (19%) 5 (8%) UNC5D 11 (8%) 3 (5%) PHIP 18 (12%) 6 (10%) NCOA2 11 (8%) 3 (5%) ELOVL4 18 (12%) 5 (8%) PTK2B 11 (8%) 3 (5%) MAP3K7 17 (12%) 5 (8%) FDFT1 11 (8%) 3 (5%) CASP8AP2 17 (12%) 5 (8%) ABL1 8 (5%) 3 (5%) APC 19 (13%) 3 (5%) CNOT3 8 (5%) 3 (5%) LEF1 16 (11%) 5 (8%) SMG8 3 (2%) 7 (10%) PAX5 15 (10%) 5 (8%)
Potential novel mechanisms of oncogene activation are amplifications of PTK2B, a gene that has been found to be deregulated by fusion in Philadelphia-like BCP-ALL and that is potentially targetable by tyrosine kinase inhibitors, and of MYC, which has long been known to be a key player in T-ALL leukemogenesis and that is amplified in neuroblastoma and medulloblastoma.
Enriched in relapse, we identified mutations in NT5C2 (p=1.4E-08), TP53 (p=0.0006) and CCDC88A (p=0.01), and amplifications of a region on chr 17q represented by the genes CLTC, ABCA5, C17orf80 and SRSF2. MLLT3 deletions were enriched in primary samples (p=0.04), consistent with the observation that MLLT3 deletions confer a lower risk of relapse in patients treated on BFM protocols.
Conclusion
Gene panel sequencing emerges as a suitable tool for a comprehensive genetic characterization of pediatric T-ALL. Within the group of selected genes contained in the panel, CNA were as frequent as point mutations. Only few genes were found to be specifically altered in relapse, indicating that progression to relapse may involve diverse, non-recurrent genetic alterations.

Disclosures
No relevant conflicts of interest to declare.