Abstract
Acute Lymphoblastic leukemia (ALL) is a malignancy of bone marrow. Accumulation of mutations in lymphoid progenitor cells give rise to either B-ALL or T-ALL. Treatment for ALL has improved in recent years, yet relapse of the disease and development of resistance are observed in patients. Lack of suitable and robust in vitro and in vivo drug testing platforms for primary ALL cells along with the lack of rapid development of novel therapeutics drugs encompassing the heterogeneity of the disease contribute to the delay in approved patient treatments.
We have developed a short-term culture system that supports the survival of primary B-ALL and T-ALL cells. Our ALL bank includes patient-derived specimens with complete cytogenetics and surface marker expression information. Different culture conditions were evaluated to select conditions that support the survival and maintenance of primary B-ALL and T-ALL specimens. Cell growth/viability was assessed using the Cell Titer-Glo ® assay. Primary B-ALL cells survived in the optimized media for 3 days and a heterogenous dose dependent response was observed across the models to chemotherapeutic drugs doxorubicin, vincristine, imatinib and cytarabine. BCR-ABl - B-ALL patient samples were found to be resistant to imatinib in contrast to BCR-ABL + samples which were sensitive to imatinib. Similarly, culture conditions optimized for T-ALL primary cells supported the survival until day 6 and displayed a diverse response to standard of care drugs like venetoclax, imatinib, vincristine, cytarabine and methotrexate, reflecting the heterogeneity of the patient derived specimens. Immunophenotypic characterization of ALL cells grown in culture displayed retention of the B and T cells surface marker expression.
Further, a patient derived pre-clinical xenograft model was developed in NCG mice to study in vivo ALL drug efficacy. 100% engraftment was observed for B-ALL primary cells, with latency of engraftment (>3%) in peripheral blood varying from 15 days to 3.5 months. 30-90% of the bone marrow cells were occupied by human CD45 cells. Infiltration of human B-ALL cells were observed in the spleen causing splenomegaly. 8 out of the 14 models having high penetrance were passaged until P3. Flow analysis at each passage demonstrated surface marker expression displaying low divergence from the primary samples. Additionally, evaluation of ex vivo drug response from B-ALL PDX splenocytes was largely concordant with the primary specimen ex vivo data in three of the models evaluated. In an in vivo drug efficacy study administration of venetoclax, CHOP and R-CHOP inhibited the proliferation of B-ALL cells. Significant reduction of B-ALL cells was observed while on treatment with Venetoclax. At termination of the study, up to 80% reduction of human B-ALL cells was observed in whole blood, bone marrow, and spleen after treatment with CHOP and R-CHOP in comparison to the vehicle cohort.
Similarly, patient derived T-ALL pre-clinical xenograft model development is in progress. Thus, we have developed a robust in vitro drug testing platform for B-ALL and T-ALL to evaluate drug efficacy. We also demonstrate that NCG mice support the growth and proliferation of primary B-ALL cells and have successfully developed an in vivo platform that will facilitate the testing of clinically relevant chemotherapeutic drugs for ALL.
Disclosures
No relevant conflicts of interest to declare.
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