P348: genomic characterization of relapsed/refractory b-cell acute lymphoblastic leukemia pediatric patients undergoing car-t treatment in a single center

Background: Chimeric Antigen Receptor T cells (CAR-T) have revolutionized the treatment of pediatric patients with relapsed/refractory B-cell acute lymphoblastic leukemia (R/R-ALL), with a significant improvement in survival. However, a significant proportion of patients relapse after CAR-T. Little is known regarding the factors associated with failure to CAR-T therapy. The identification of such prognostic factors may help to better select patients who will benefit from this therapy. Further genetic characterization at the time of indication of CAR T-cell therapy might help as a predictive factor to identify patients at higher risk of CAR-T cell failure. Aims: We aimed to identify genomic alterations with prognostic relevance at CAR-T cell therapy indication in R/R-ALL pediatric patients in a single center. Methods: The study included 28 R/R-ALL pediatric patients treated with CAR-T at Hospital Sant Joan de Déu from 2016 to 2022. To obtain the karyotype G-banding technique was performed. For identification of fusion-genes and mutations, we sequenced bone marrow samples before CAR-T therapy using a targeted-Next Generation Sequencing panel. Only mutations with VAF>10% were reported, except for TP53, as high- and low-VAF mutations in this gene have been previously reported to be associated with prognosis in CAR-T cell patients in lymphoma and leukemia. Results: We studied twenty-eight pediatric R/R-ALL patients treated with CAR-T in a single center. Globally, at the time of CAR-T cell indication, considering karyotype, DNA mutations and fusion genes, 85% of patients (24/28) showed at least one genetic alteration (Fig 1). Regarding karyotype, 8 patients were classified as high hyperdiploid karyotype (51 to 67 chromosomes), 5 as hyperdiploid (47-50 chromosomes), 1 as composite karyotype, and 14 presented a normal karyotype. Regarding fusions, we observed five different rearrangements in 9/28 patients, all of them previously described and with potential clinical impact: ETV6::RUNX1 (n=3), P2RY8::CRLF2 (n=3), and KMT2A::AFF1 (n=1), BCR::ABL1 (n=1), and PAX5::NOL4L (n=1) rearrangement. Twenty-three patients (82%) had at least one mutation. The mean number of mutations per patient was 1.7 (range 1 to 6 mutations per sample). The most commonly mutated genes were TP53 (8/23, 34.8%) and KRAS (7/23, 30.4%). One of the patients with a TP53 mutation had a VAF<10%. NRAS and PRPS1 were mutated in three patients; MSH6, NT5C2, PTPN11, ASXL1, JAK1, and FLT3 were mutated in two patients, and ABL1, IKZF1, ASXL2, JAK2, NF1, CDKN2A, and PTEN were mutated in one patient each. Interestingly, the patient with mutated ABL1 presented two different mutations in this gene with a VAF>10%. Overall, a high rate of pathogenic variants in genes involved in signaling pathways and driver genes were observed, all of them involving hotspot regions and previously reported in pediatric acute leukemia.Figure 1: Genetic abnormalities found at the time of indication of CAR-T cell therapy: mutations (blue), fusion genes (purple) and the main findings in the karyotype (green, yellow, pink, grey) are depicted. Summary/Conclusion: Our results showed a heterogeneous genomic landscape in those patients with R/R-ALL at the time of CAR-T cell therapy indication. Most alterations involved apoptosis and cell cycle regulation, kinase activation, and cell proliferation, and have been described as therapeutic targets. A thorough biological characterization of R/R-ALL patients may help understand the impact of genetic alterations in the era of immunotherapy, and predict the risk of relapse after CAR-T cell therapy. Keywords: CAR-T, Relapsed acute lymphoblastic leukemia, Genomics, Pediatric