TP53, ETV6 and RUNX1 Germline Variants in Patients Developing Secondary Neoplasms after Treatment for Childhood Acute Lymphoblastic Leukemia

Introduction: Today most of the children treated for acute lymphoblastic leukemia (ALL) can be cured by the application of intensive combination chemotherapy regimens. However, up to 10% develop a secondary malignancy (SMN) after undergoing ALL treatment with cure rates being often dismal. Thus, strategies are needed for an early identification of patients at risk for SMN development, an improved understanding of the underlying pathobiology and, ideally, the development of preventive actions. Analyzing three candidates associated with predisposition to neoplastic diseases - TP53, ETV6 and RUNX1 - we aimed to determine the frequency of single nucleotide variants (SNV) in these genes in a cohort of SMN patients.

Methods: Using targeted sequencing, we analyzed a cohort of childhood ALL patients with SMN after treatment on one of seven consecutive ALL-BFM protocols - ALL-BFM 79 to AIEOP-BFM ALL 2000.In the observation period from 1984 to 2008, 168 SMN patients were identified. The median follow-up for the entire patient cohort was 10.6 years as of April, 2013. With the exception of ALL-BFM 79, treatment was stratified into 3 branches, mainly according to the initial leukemic cell load, adverse genetic aberrations and treatment response. DNA isolated from remission bone marrow smears was analyzed employing two different multiplex PCR-based Ion AmpliSeq™ Panels (Life Technologies, Darmstadt, Germany) according to the manufacturer's instructions. The first panel, covering all coding exons of TP53, was employed in 49 patients with adequate material available for analysis. The second panel, interrogating the complete coding regions of ETV6 and RUNX1, was applied in 38 patients with sufficient material available after TP53 analysis. SPSS (IBM Deutschland GmbH, Ehningen, Germany) was used for computerized calculations.

Results: In our TP53 analyses, only 1/49 (2%) genotyped SMN patients carried a heterozygous non-synonymous SNV within the coding region. This rare missense variant, p.Asn235Ser (rs144340710), was detected in a patient developing a small round cell sarcoma after treatment for ALL. The in silico scores from Polyphen and Sift algorithms indicated a benign effect for this change. Regarding ETV6, 3/38 (8%) patients carried a heterozygous non-synonymous SNV; two of them developed a hematologic and one patient a solid SMN. One frameshift insertion (p.Ile48Glyfs*2) and a missense variant (p.Leu79Pro) were observed in exon 2, part of the pointed domain. A further missense variant (p.Arg399Gly) was detected in exon 7 within the C-terminal DNA-binding ETS domain. Although ETV6 p.Arg399 is a hotspot mutation site for recurrent somatic mutations in malignancies, none of these variants was recorded in the databases dbSNP, the 1000Genomes Project, the NHLBI GO Exome Sequencing Project, Exome Aggregation Consortium or the Catalogue of Somatic Mutations in Cancer. For both missense variants in silico scores from Sift and Polyphen algorithms predicted a probably damaging effect. Molecular modeling of p.Arg399Gly suggests that the change might directly modulate the DNA binding qualities of the ETS domain. Furthermore, 2/38 patients (5%) had coding variants in RUNX1, both developed a hematologic SMN. One patient carried a nonsense insertion (Leu253Argfs*3). A second patient had a missense SNV (p.Leu56Ser, rs111527738) within the N-terminal RUNT domain, with a probably damaging effect (Polyphen). The described ETV6 and RUNX1 SNV were mutually exclusive. Compared to published data on childhood ALL patients (T. Moriyama et al. Lancet Oncol. 2015 and J. Zhang et al. N Engl J Med. 2015), we found approximately 10-fold and 4-fold higher frequencies of ETV6 and RUNX1 SNV, respectively, in patients developing a SMN after ALL treatment on ALL-BFM protocols.

Conclusions: The low frequency of TP53 SNV in our case series suggests that alterations in this frequently mutated tumor suppressor gene may not play a major role in the pathobiology of SMN associated with the treatment of ALL. However, our findings in ETV6 and RUNX1 analyses suggest that genetic variation in these genes may be involved in the development of SMN after undergoing ALL treatment and warrant additional studies employing appropriate control groups for further clarification.