Abstract Current cancer therapies are directed at molecular markers or dominant pathways present in the bulk of neoplastic cells. However, recent studies have identified many genetically distinct subclones co-existing within a single neoplasm. In over 50% of patients with relapsed acute lymphoblastic leukemia (ALL), the genetic clones present at relapse are not the dominant clone present at diagnosis, but have evolved from a minor or ancestral clone (Mullighan et al., Science, 2008). Previous work has shown that this subclonal diversity in B-ALL exists at the level of the leukemia-initiating cells (L-IC) capable of generating patient derived xenografts (Notta et al., Nature, 2011). In order to investigate the functional consequences of genetic clonal evolution during disease progression, we performed in-depth genomic and functional analysis of 14 paired diagnosis/relapse samples from adult and pediatric B-ALL patients of varying cytogenetics. Patient samples were subjected to whole exome sequencing (WES), SNP analysis and RNA sequencing. Diagnosis-specific, relapse-specific, and shared variants at both clonal and subclonal frequencies were identified. Limiting dilution analysis by transplantation of CD19+ leukemic blasts into 870 immune-deficient mice (xenografts) identified no significant trend in enrichment in L-IC frequency between paired patient samples with a median frequency of 1 in 2691. Despite similar frequencies of L-IC, functional differences within identically sourced patient xenografts were observed, including increased leukemic dissemination of relapse cells to distal sites such as the central nervous system (CNS), differences in engraftment levels and differences in immunophenotypes. Targeted-sequencing and copy number analysis of the xenografts, in comparison to the patient sample from which they were derived, has uncovered clonal variation and the unequivocal identification of minor subclones ancestral to the relapse in xenografts transplanted with the diagnostic sample from 8 patients. Some of these subclones are rare and were not captured through standard WES analysis of the patient samples, highlighting the value of xenografting to functionally identify and viably isolate subclones for further study. Interrogation of the therapeutic responses of the ‘relapse-like’ diagnosis subclones in secondary xenografts displayed differential resistance to standard chemotherapeutic agents (vincristine and L-asparaginase) pre-existing in the patient diagnosis samples prior to treatment. Furthermore, investigation of different sites of leukemic infiltration in the xenografts provided evidence of distinct clonal selection in the CNS, a known site of disease relapse, in comparison to the bone marrow. Using this data we can begin to draw the evolutionary paths to relapse. We have shown evidence that minor subclones at diagnosis, ancestral to the relapsing clone, possess functional advantages over other diagnostic clones. Overall, this work provides a substantial advance in connecting genetic diversity to functional consequences, thereby furthering our understanding of the heterogeneity identified in B-ALL and its contributions to therapy failure and disease recurrence. Citation Format: Stephanie M. Dobson, Robert Vanner, Esmé Waanders, Olga I. Gan, Jessica McLeod, Ildiko Grandal, Debbie Payne-Turner, Michael Edmonson, Zhaohui Gu, Xioatu Ma, Yiping Fan, Pankaj Gupta, Sagi Abelson, Michael Rusch, Ying Shao, Scott Olsen, Geoffrey Neale, John Easton, Cynthia J. Guidos, Jayne S. Danska, Jinghui Zhang, Mark D. Minden, Charles G. Mullighan, John E. Dick. Evolving functional heterogeneity in B-acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-341.
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Sep 27, 2018
Anna L Mcnaughton + 7
Open Access
