Linking Subclonal Genetic Diversity with Functional Heterogeneity Identifies Diagnosis Subclones Destined to Relapse
Authors: Dobson Stephanie M. , Waanders Esme , Vanner Robert , Gan Olga I , McLeod Jessica , Grandal Ildiko , Payne-Turner Debbie , Edmonson Michael , Gu Zhaohui , Ma Xiaotu , Fan Yiping , Abelson Sagi , Gupta Pankaj , Rusch Michael , Shao Ying , Shi Lei , Pounds Stanley , Olsen Scott , Neale Geoffrey A. , Easton John , Guidos Cynthia J. , Danska Jayne S. , Zhang Jinghui , Minden Mark D. , Mullighan Charles G. , Dick John E. 
Published: November 2019

Abstract
Despite significant advances in the treatment of B cell acute lymphoblastic leukemia (B-ALL), mortality rates following disease relapse remain high. Recent studies have identified many genetically distinct subclones co-existing within a single neoplasm. In over 50% of patients with relapsed 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; Anderson et al., Nature, 2011). 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 (PDX) (Notta et al., Nature, 2011). However, little is known of the functional properties of relapse driving subclones and how they contribute to disease recurrence. 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, SNP analysis and RNA sequencing. Diagnosis-specific, relapse-specific, and shared variants at both clonal and subclonal frequencies were detected. Limiting dilution analysis by transplantation of CD19+ leukemic blasts into 870 immune-deficient mice (PDX) 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 PDX 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, uncovered clonal variation and the unequivocal identification of minor subclones ancestral to the relapse in xenografts transplanted with the diagnostic sample. In 8 of the 14 patient samples, PDX at varying cell doses allowed for the selection and isolation of rare relapse driving subclones present at diagnosis ('relapse-like' diagnosis clones). In 2 of these 8 samples, as well as in 5 other patient samples, relapse specific variants were identified in the PDX that were not detected in the patient diagnosis genomic analysis at our level of detection. In secondary xenografts, comparison of the therapeutic responses of the identified 'relapse-like' diagnosis subclones against more representative diagnosis subclones displayed differential resistance to standard chemotherapeutic agents (vincristine, L-asparaginase and dexamethasone). This indicates that genetic subclones possessing varying therapeutic responses preexisted in the patient diagnostic sample. In addition to therapeutic differences, variations in cell migration were detected. This may contribute to the therapeutic evasion of the relapse driving subclones. Interestingly, 'relapse-like' diagnosis cells also displayed phenotypic plasticity generating CD19-CD33+ cells from CD19+ cells in 2 patient samples upon treatment with dexamethasone. This is suggestive that relapse driving subclones may arise from primitive cells with multilineage potential upon steroid challenge. 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 draw an evolutionary path to relapse for these patients samples. We have shown evidence that minor subclones at diagnosis, ancestral to the relapsing clone, possess functional advantages and unique properties over other diagnostic subclones prior to treatment exposure. 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.

Disclosures
No relevant conflicts of interest to declare.