Exome Sequencing of Late Recurrence T-Cell Acute Lymphoblastic Leukemia in Children Confirms Second Leukemia and Exposes Predisposition Candidate Genes

Abstract 755

Second hematologic malignancies in non-syndromic children without a pronounced family history for cancer may be mistaken for relapses or therapy-related malignancies. Recently, we characterized diagnosis and presumed relapse samples of 22 patients with very late disease recurrences (>2.5 years), and identified 8 patients with leukemic presentations that were fully discordant at the level of TCR-rearrangements and DNA copy number aberrations (J Clin Oncol 2011; 29:1643-9). One of these patients showed a germline deletion comprising the recombination activating genes RAG1 and RAG2, and regulatory sequences of LMO2, genes frequently affected somatically in T-ALL, suggesting a genetic predisposition to leukemia. In the current study, we performed exome sequencing to assess whether consecutive leukemic presentations in such patients are indeed fully discordant, also at the sequence level, and to identify candidate pathogenic germline variants that point at a genetic predisposition.

We sequenced the exomes in samples obtained from 2 consecutive leukemic presentations, and intermittent complete remissions, from 2 patients with very late disease recurrences (>2.5 years) and discordant leukemic presentations. We found on average 26,600 variants per exome. Recurrent variants recorded in the dbSNP and/or 1000 Genomes databases, or those present in our in-house database (>300 exomes) were excluded, resulting in an average of 989 private variants per exome. We divided these variants into 3 groups (i) somatic variants shared between the consecutive leukemic samples but not detected in remission (ii) somatic variants present in only one of the leukemic samples and (iii) germline variants present in the remission samples of the patients.

All candidate somatic variants shared between two consecutive leukemic samples were re-sequenced by Sanger sequencing and were shown to be either present in all three samples, and thus originally missed in the remission sample, or falsely detected in one or more leukemic samples. Therefore, we conclude that in both patients no somatic variants were shared between the first and second leukemic presentations, which confirms that these patients suffered from clonally unrelated second T-ALLs.

From all somatic variants present in only one of the leukemic samples, we focused on variants in exons or splice junction sites. We found 4 nonsense mutations, 9 frame-shift mutations, 12 in-frame in/dels and 7 non-synonymous missense variants with a high interspecies conservation score (PhyloP>3.0), mostly affecting genes implicated in oncogenesis like PTEN, TET3, CDKN2C, CD109, and GLRX2. Each leukemic sample harbored 2–11 of these putative deleterious variants.

In the germline of the two patients, we identified 314 and 190 non-synonymous unknown variants in exons or splice junction sites, respectively. Among these were 12 nonsense mutations, 7 canonical splice-site mutations, 20 frame-shift mutations, 11 in-frame in/dels and 143 non-synonymous missense variants at highly conserved positions (PhyloP>3.0). Filtering of these variants for known T-ALL associated genes resulted in several interesting novel candidate predisposing genes such as, among others, RANBP17 and HOXC13. Sequencing of the entire RANBP17 open reading frame in a cohort of 24 sporadic T-ALL samples revealed that this gene was somatically affected in one of them.

In conclusion, we confirmed by exome sequencing that consecutive leukemic presentations in patients with late T-ALL recurrences may be fully discordant and thus represent independent leukemia occurrences, most likely caused by predisposing germline abnormalities.