Dissecting the DNA Repair Machinery in Biological Subgroups of Childhood Acute Lymphoblastic Leukemia

Background. Aberrations in the DNA repair pathway among children with acute lymphoblastic leukemia (ALL) are largely unknown. Knowledge of such mutations may improve the understanding of tumorigenesis and direct patient care. Germline aberrations of the DNA-repair machinery are crucial for generating chromosomal instability and occurrence of acute leukemia. In order to better understand this mechanism, we addressed our research to NBS1 (Nijmegen Breakage Syndrome) gene and Fanconi Anemia/BRCA1 and BRCA2 pathway, in the attempt to identify mutations and aberrant expression, predisposing and/or cooperating with the leukemogenic process, among biological subgroups of childhood ALL.

Materials and Methods. We analyzed 48 diagnostic samples from children with ALL treated at our institution from 2000 to 2010: 11 cases with chromosome Philadelphia positive (Ph+) leukemias (8 ALL and 3 CML), 15 with t(12;21), 11 with t(1;19) positive ALL, respectively and 10 samples with B-ALL without known translocations. We also analyzed three cases of relapse. In Ph+ subgroup, we analyzed remission samples in order to detect germline mutations. We performed RT-PCR and sequencing analyses to detect mutations in NBS1 gene (exons 3-6). The status of FANCD2 and PALB2 genes was studied by a multiplex ligation-dependent probe amplification (MLPA). Samples from healthy donors (HDs) were used as wild-type control. For data elaboration, we used Coffalyser.Net software for MLPA. We performed Sybr-Green Real-Time PCR amplification of BRCA1 (exons 14-15) and BRCA2 (exons 15-16) genes, respectively, calculating the expression in patients with method 2^-ΔΔC comparing with HDs.

Results. Among the 15 cases with t(12;21) positive ALL, we found overexpression of BRCA1 and BRCA2 in 8 and 10 cases, respectively, showing a statically significant difference (p<0.0001; p<0.0048, respectively) vs HDs. We identified in 7 cases (44%) a missense variant mutation (G>C;Glu185Gln; SNP-rs1805794) in NBS1 gene. Deletions or duplications for FANCD2 and PALB2 genes were not found. In the 8 cases with Ph+ B-ALL, we detected an overexpression of BRCA1 and BRCA2 in 4 and 7, respectively, showing a statistically significant difference (p<0.0084 and p<0.0009, respectively) vs HDs. In 3 cases with CML we found the same pattern. Analysis of NBS1 showed a missense variant mutation (G>C;Glu185Gln) in 2 cases (25%) with B-ALL and in 1 patients with CML, respectively. Analyses of FANCD2 showed deletions in 3 cases with B-ALL and 2 children with CML; PALB2 resulted deleted in 2 cases with B-ALL and 2 cases with CML. Interestingly, NBS1 mutations were detected in remission samples, showing a germline genomic aberration, as well. Surprisingly, deletions of FANCD2 and PALB2 were found only in leukemic samples. In 11 cases with t(1;19) positive ALL, we observed statically significant difference in BRCA1 (p<0.0293) and BRCA2 (p<0.0705) expression vs HDs, respectively. Analyzing NBS1, we detected missense variant mutation (G>C;Glu185Gln) in 4 patients (36%); in one case we detected an NBS1 exon 4 deletion. We found FANCD2 exon-1 duplication in 3 patients; in 4 cases we detected PALB2 genomic aberrations (3 duplication and one exon-1 deletion). Among 10 cases with B-ALL without known translocations, we observed statically significant difference in BRCA1 (p<0.0031) and BRCA2 (p<0.125) expression vs HDs. We found 3 patients (30%) with a missense variant mutation (G>C;Glu185Gln) in NBS1. We also detected in 4 cases different mutations in FANCD2 and PALB2 genes (deletion in exons 9-30 and duplication in exons 1-2-29, respectively). Notably, the relapsed case within the latter subgroup, at diagnosis showed genomic aberrations in all the selected genes in association with BRCA1 and BRCA2 overexpression.

Conclusion Our findings strongly suggest that the DNA repair machinery is frequently disrupted in childhood ALL. The significance of BRCA1 and BRCA2 overexpression in all subgroups is still unclear. NBS1 showed a missense mutation in a range from 25% to 45% of children with ALL. Interestingly, these mutations were found at germline level in cases with Ph+ leukemias, suggesting a predisposition profile. Aberrations of FANCD2 and PALB2 genes were mostly detected in the subgroup of children without known translocation. Finally the role in leukemic predisposition and the clinical impact of these genomic aberrations need to be more elucidated in a larger population.