Establishment of a Patient-Derived Acute B Lymphoblastic Leukemia (B-ALL)Ex Vivo and In Vivoplatform for Evaluation of Novel Therapeutic Agents

B-cell Acute Lymphoblastic Leukemia (B-ALL)is a hematological malignancy with malignant transformation and proliferation of B-Cell progenitors in bone marrow, blood and extramedullary sites. It is more prevalent in children and adolescent and is one of the top leading cause of death related to malignancies in this population. Majority of young patients fare well with common therapeutic agents and hematopoietic stem cell transplant, but a subpopulation is chemo-resistant and has a high relapse rate. Besides, prognosis in adult B-ALL patient is worse than younger individuals. Thus, there is an increasing need for development of novel and targeted therapeutic strategies to improve outcome in elderly and resistant individual. Lack of suitablein vitroculture conditions for primary B-ALL cells severely impairs their experimental accessibility and evaluation of new therapeutics. To corroborate theex vivostudies in a more natural microenvironment there is an unmet necessity of a robust preclinical patient derived xenograft model for B-ALL.

We have established and optimized a short-term culture system that supports survival of primary B-ALL cellsex vivofor evaluation and/or screening of candidate agents. Our B-ALL bank comprise of patient-derived specimens with complete cytogenetics information including Philadelphia translocation positive and negative samples and surface marker expression. Different medias, supplements and culture conditions were evaluated to select conditions that support the survival and maintenance of primary B-ALL specimens. Cell growth/viability was assessed using Cell titer-Glo assay. Out of 13 primary B-ALL models studied 5 models proliferated in the optimized media and 5 remain stable and viable till day 3. Phenotypic characterization by flow cytometry showed that all the models stably expressed CD19 and CD20 throughout the assay. To verify the applicability of the 3-day ex vivo system for drug testing, models were tested for sensitivity towards chemotherapeutic drugs doxorubicin, vincristine, imatinib and cytarabine. Heterogeneous dose dependent responses were observed across the models (IC₅₀ 17nM to 0.3 µM doxorubicin), (IC₅₀ 3 nM to 79 nM vincristine) indicating a range of relative sensitivity to B-ALL models. Interestingly, BCR-ABL-ve models were resistant to imatinib and BCR-ABL+ve sensitive, when treated with small molecule inhibitor imatinib.

Next, to develop patient derived xenograft preclinical B-ALL models for drug efficacy study , sub lethally irradiated NCG mice were transplanted intravenously with two million PBMC from B-ALL patients(n=14). 100% engraftment(n=5/model P1) was observed in all the models, but the latency of engraftment(>3%) in peripheral blood varied from 15 days to 3.5 months depending on the aggressiveness of the models. The time of passage varied from 40 to 120 days at P1. Mice were sacrificed when 10-20% of hCD45+ve cells were observed in peripheral blood by flow cytometry. 30-95% of the bone marrow was occupied by hCD45+, hCD19+ cells. Splenomegaly was observed and infiltration of 40-98% of B-ALL cells was detected in spleen. Engrafted cells were lymphoid-restricted and matched the immunophenotype of the corresponding patient. On serial transplantation in secondary NCG mice (n=15/model P2,P3), these models showed similar engraftment pattern and immunophenotype characteristic.

Thus , here we show that theex vivoculture condition optimized at Champions supports growth and proliferation of patient derived B-ALL specimen and is a good platform for screening new therapeutic drugs for B-ALL . We also demonstrate that NCG mice supports the engraftment and development of B-ALL disease initiating and leukemic cells in vivo, allowing to create a robust passagable patient derived preclinical xenograft model that recapitulates the disease and probably could be used for drug efficacy study.