The Genomic Landscape of Childhood T-Lineage Acute Lymphoblastic Leukemia
Authors: Liu Yu , Easton John , Shao Ying , Wilkinson Mark , Edmonson Michael , Ma Xiaotu , Smith Malcolm A. , Rusch Michael , Auvil Jaime Guidry , Gerhard Daniela S. , Relling Mary V. , Winick Naomi J. , Raetz Elizabeth , Devidas Meenakshi , Willman Cheryl L. , Harvey Richard C. , Carroll William L. , Dunsmore Kimberly P. , Winter Stuart S. , Wood Brent L , Downing James R. , Loh Mignon L. , Hunger Stephen P , Zhang Jinghui , Mullighan Charles G. 
Published: October 2019

Abstract
Comprehensive studies examining the genomic landscape of T-lineage ALL are lacking, but are important to identify all oncogenic drivers. Here we report sequencing of 264 T-ALL consecutive cases treated on the Children's Oncology Group AALL0434 clinical trial. Whole exome sequencing, copy number analysis using exome and single nucleotide polymorphism array analysis of tumor and remission DNA, and RNA-sequencing of tumor RNA were performed. Cases with immunophenotypic data (N=189) included 19 early T-cell precursor (ETP) cases, 24 near-ETP (with normal CD5 expression) and 146 Non-ETP cases. Median exomic coverage was 89% (72%-96%) of exons with at least 20-fold coverage.
We identified 4657 non-synonymous clonal and subclonal somatic mutations (3926 single nucleotide variants (SNV) and 731 insertion-deletion mutations; indels) in 3030 genes, with a mean of 17.6 per case (range 1-50). 176 potential driver genes were identified statistical analysis or by known pathogenic role in cancer. These included NOTCH1 (n=194, 73%), FBXW7 (n=64, 24%), PHF6 (n=50, 19%), PTEN (n=37, 14%), USP7 (n=32, 12%), DNM2 (n=29, 11%) and BCL11B (n=27, 10%). New mutations in T-ALL included CCND3 (n=15, 6%), MYB (n=13, 5%), CTCF (n=13, 5%), MED12 (n=7, 3%), USP9X (n=7, 3%), SMARCA4 (n=7, 3%) and CREBBP (n=6, 2%). In addition to MYB amplification, we identified missense mutations and in-frame protein insertions at the N-terminus of MYB, with a hotspot at codon 14 in a region of six acidic residues in an otherwise hydrophilic N-terminal tail. These mutations resulted in a disordered region that is predicted to affect nuclear localization. The MYB mutations detected were enriched in non-ETP cases (n=13; 8 non-ETP, 1 near-ETP, 4 unknown). Other genes enriched in non-ETP cases included RPL10, CNOT3, MYCN and DDX3X. MED12 mutations were more common in ETP ALL.
Sub-clonal mutations (mutant allele fraction of less than 30%) were identified in 111 of 176 driver genes in 198 (75%) cases including NOTCH1 (n=94), FBXW7 (n=29) and PTEN (n=17) indicating that sub-clonal evolution is a hallmark of T-ALL. In addition, multiple mutations in individual genes were commonly observed in single cases. For example, up to 3 different somatic NOTCH1 mutations were detected in each of 9 patients, with 2 different NOTCH1 mutations in 49 cases.
Integration of sequence mutations with copy number aberration data showed the following pathways to be most frequently mutated: cell cycle/tumor suppression (N=225; CDKN2A/B (n=206), CDKN1B (n=35), RB1 (n=28)); NOTCH1/FBXW7 (n=212), PI3K-AKT (n=130), JAK-STAT (n=99) and Ras (n=51). Mutations in the PI3K-AKT, JAK-STAT and Ras signaling pathways were mutually exclusive. We identified a high frequency of mutations in transcriptional regulators in 222 cases, including 108 cases with mutations in a core regulatory complex comprising TAL1 (n=51), MYB (n=45) RUNX1 (n=18) and GATA3 (n=13). In 90 of these 108 cases (83%), only a single mutation was present in any of the four genes, consistent with a central role of this complex in leukemogenesis. Epigenetic alterations were identified in 178 cases, including PHF6 (n=63), SMARCA4 (n=23), KDM6A (n=22) and EZH2 (n=18), and new deletions and mutations in KMT2A (MLL; n=11).
Interim analysis of transcriptome sequencing data of 126 T-ALL cases detected fusions in 61 (48%) samples, which could be separated into two categories. One weres in-frame fusions resulting in a chimeric protein. The most frequent included MLLT10 fusions (PICALM-MLLT10 (n=3), DDX3X-MLLT10 (n=2) and NAP1L1-MLLT10 (n=1)), KMT2A fusions (KMT2A-MLLT1 (n=4), MLLT6-KMT2A (n=1) and MKT2A-MLLT4 (n=1)), as well as internal tandem duplication mutations involving FLT3 (n=6; 3 ETP, 1 near-ETP, 1 non-ETP, 1 unknown) and NOTCH1 (n=2). We also identified novel fusions including ETV6-CTNNB1 and STMN1-SPI1 (n=1 each). The other category contains rearrangement-driven aberrant expression, including rearrangements in TLX1 (n=11), TLX3 (n=4), TAL1 (n=2), and TAL2 (n=3). Moreover, we found a novel TAL2 transcript in all the 3 cases with TAL2 rearrangement, hijacking a new exon 6kb upstream of the canonical TAL2 transcription start site and extending approximate 3.5kb downstream.
These findings provide the first comprehensive landscape of genomic alterations in T-ALL and have provided new insights into the genes and pathways mutated in this disease, their interaction, and the nature of clonal heterogeneity in T-ALL.

Disclosures
Hunger: Spectrum Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Sigma Tau: Consultancy; Merck: Equity Ownership. Mullighan:Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria; Loxo Oncology: Research Funding.