Role of the DNA Repair Machinery in Childhood B-Lineage Acute Lymphoblastic Leukemia: Aberrations of NBS1, Fancd2, Palb2 Genes and Expression of BRCA1 and BRCA2

Background. Successful treatment of B-lineageAcute Lymphoblastic Leukemia (B-ALL) in children has been related to novel biological findings and a better supportive therapy. Genomic aberrations have a major role in development of ALL and in the treatment response. Conventional treatment is based on chemotherapy, mainly acting on the structure of DNA in both leukemic and host cells. The result is a complete achievement of durable remission and/or a high incidence of toxicity. The role of DNA repair machinery in the occurrence of de novo or relapsed ALL and of high-grade toxicity is still unclear. We decide to dissect genes involved in the Fanconi Anemia (FA) pathway ( BRCA1, BRCA2, FANCD2 and PALB2), which cooperates with the Nijmegen breakage syndrome ( NBS1) gene. We evaluate the correlation of DNA repair machinery alterations with occurrence of relapse and/or with severe (III-IV grade - CTCAE) toxicity during chemotherapy treatment.

Materials and Methods.The study was approved by the Local Clinical Research Ethics Committee (Catania-1). Written informed consents were obtained from the parents of each child involved in this research. We analyzed bone marrow (BM) samples from diagnosis and remission (Dx/Rem) of 111 children with B-ALL, diagnosed and treated at the Center of Pediatric Hematology Oncology, Catania Italy, from 2000 to 2017. These children were enrolled in three consecutive protocols of Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) [AIEOP-BFM ALL 2000; AIEOP-R 2006; AIEOP BFM ALL 2009]. Biological subgroups were as follow: 16 children presenting with t(1;19); 48 with t(12;21), 9 with t(9;22)/Ph+, 5 with a KMT2A (formerly MLL)rearrangement and 33 defined as “B-Others”. Traditional PCR method was used to determinate the NBS1 mutations in exons 3-6, followed by Sanger sequencing. FANCD2 and PALB2 mutations were investigated by multiplex ligation-dependent probe amplification (MLPA). Samples from healthy donors were used as wild-type controls. A real-time PCR assay was applied to evaluate the BRCA1 and BRCA2 genes expression using the SYBR green, fluorescent dye. We used specific primers for BRCA1 exons 14-15 and BRCA2 exons 15-16, respectively. The expression of BRCA1 and BRCA2 was determined as high (H) or low (L) using the Mean and the Median of fold change (FC) as cut-off.

Results. Fifty-four patients (48%) out of 111 had more than one episode of severe toxicity during chemotherapy treatment. Twenty-one children (19%) with B-ALL out of 111 experienced the disease recurrence. The NBS1 polymorphism (rs1805794 SNP_G>C) was found in 28% of the patients enrolled in the study and showed a protective role against severe toxicity (p<0.05 ): only 10 of 54 patients, who experienced high grade toxicity, had this polymorphism. This protective effect was confirmed in the subgroup with t(12;21) positive B-ALL (p<0.05). There were no statistically significant relationships between NBS1 and relapse or between FANCD2/PALB2 aberrations and B-ALL toxicity/recurrence. At diagnosis, children with high expression of both BRCA genes ( BRCA1-H/ BRCA2-H) showed a correlationwith an increased incidence of severe toxicity:17 patients with severe toxicity out of 27 BRCA1-H/ BRCA2-H cases vs 10 out of 28 BRCA1-L/ BRCA2-L (p<0.05 ). At remission, high BRCA1 expression ( BRCA1-H) appears to be related to an increased risk of relapse, albeit not statistically significant (p=0.17) . BRCA2-H is associated with a higher incidence of recurrence than BRCA2-L (p<0.03): 8 cases with relapse out of 22 (BRCA2-H ) vs 13 out of 88 (BRCA2-L ). More importantly, we observed that cases with BRCA1-H /BRCA2-Hat remission are statistically significant associated with relapse: 3 children presented a relapseout of 6 cases with BRCA1-H/ BRCA2-H vs 8 out of 65 cases with BRCA1-L /BRCA2-L(p<0.02).

Conclusions. Our study strongly demonstrates that the NBS1 gene polymorphism (rs1805794 SNP_G>C) has a protective role against toxicity, reducing the incidence of severe grade episodes. High expression of both BRCA1 and BRCA2 genes in leukemic cells seems to be associated with a higher incidence of severe toxicity. Finally, high expression of BRCA1 and BRCA2 genes in the host cells, at remission, is strongly correlated with a higher risk of relapse. Although this study has been performed with an adequate number of children with B-ALL, it is mandatory to perform a prospective project with a larger population.