Diverse Mechanisms of Therapy Resistance in Relapsed Childhood B-cell Acute Lymphoblastic Leukemia (B-ALL): a Report from the Children’s Oncology Group

Despite high cure rates of >90% for newly diagnosed childhood B-ALL, outcomes remain poor for relapsed patients. Prior genomics studies have revealed 12 genes with mutations enriched at relapse. However, the resistance mechanisms for >50% of relapses remain elusive. Here we performed WGS and RNA sequencing on initial diagnostic, relapse, and remission specimens from 183 patients enrolled in the AALL1331 trial of the Children’s Oncology Group. In this cohort, 96 cases did not harbor a known resistance mutation. These cases were enriched in several common subtypes, including those with favorable outcomes (hyperdiploid ALL, ETV6::RUNX1, and DUX4) and those with less favorable outcomes (Ph-Like, iAMP21). By analyzing tumor mutation burdens coupled with ancestral drivers such as fusions, we uncovered 5 cases where the relapses were a novel clone unrelated to the clone at diagnosis, indicative of secondary B-ALL. In the remaining 178 truly relapsed cases, we discovered four mechanisms of therapy resistance. First, a collaborating mutation can be acquired and confer resistance in either a subtype-dependent manner (NSD2 in TCF3::PBX1 (P=1.2×10-6) or IKZF1 in Ph-Like (P=1.7×10-22) and DUX4 (P=0.02) subtypes), or in a pan-subtype manner (the previously reported NT5C2/PRPS1/PRPS2 genes). Second, amplification of the fusion allele of ETV6::RUNX1 can confer elevated resistance. In the AALL1331 cohort, 26% (out of 31) of relapsed ETV6::RUNX1 tumors harbor an amplification of the fusion allele, as compared to 13% of the original 31 diagnostic tumors (P=0.12). We also validated this trend in a recently published cohort of diagnostic samples enriched for eventual relapse (MP2PRT, Cheng et al, JCO 2024) at a frequency of 10% in patients experiencing a subsequent relapse and 8% in those who remained in long term remission. Third, fusion isoforms formed from different introns can confer elevated resistance. In AALL1331, 19% of ETV6::RUNX1 patients have isoform E5-E4 in their diagnostic and relapse tumors, compared with the expected frequency of 4% (P=0.007) if E5-E4 had comparable selection pressure as isoform E5-E3. In the MP2PRT cohort, E5-E4 is observed in 20% of those who went on to relapse as compared to 11% (P=0.01) of patients who did not relapse. Fourth, the total number of chromosomes in hyperdiploid ALL can confer elevated resistance. In AALL1331, 41% (out of 29) of hyperdiploid ALL cases had ≤52 chromosomes, as compared to 13% (out of 403) in the MP2PRT cohort (P=2×10-4). Concordantly, in the MP2PRT cohort, hyperdiploid ALL with ≤52 chromosomes had an eventual relapse rate of 43% compared to 26% for patients with 53 chromosomes, versus ∼17% when there are ≥54 chromosomes. Together, these mechanisms explained 72% of relapses in our cohort, suggesting risk prediction may benefit from incorporating these newly discovered mutations. Xiaotu Ma, Yuan Feng, Hanxia Li, Ying Shao, Heather Mulder, Pandurang Kolekar, Yanling Liu, Quang Tran, Zhikai Liang, Li Fan, Scott Foy, Ti-Cheng Chang, Wenana Chen, Stanley Pounds, Gang Wu, Charles Mullighan, William L. Carroll, Jinghui Zhang, John Easton, Patrick Brown, Mignon L. Loh. Diverse mechanisms of therapy resistance in relapsed childhood B-cell acute lymphoblastic leukemia (B-ALL): a report from the Children’s Oncology Group [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1205.