Ex Vivo High-Throughput Flow Cytometry Screening Identifies Subsets of Responders to Differentiation Agents in Individual AML Patient Samples

Abstract

Background: Prognoses for acute promyelocytic leukemia (APL) patients improved drastically upon the introduction of differentiation therapy with all-trans-retinoic acid (ATRA) in combination with conventional chemotherapy. Unfortunately, this therapeutic approach has not translated to other genetic subtypes of acute myeloid leukemia (AML) where patients demonstrate marked heterogeneity to differentiating agents. To provide improved detection of drug-induced differentiation in AML patients, we have developed a high-throughput, flow cytometry-based personalized medicine platform. Methods: Total white blood cells were isolated from each patient sample by red cell lysis, plated in serum-free media in 384-well format and incubated with drugs for 3 days. Viable cells remaining after each drug treatment were identified and quantified using cell surface marker expression, cell membrane integrity, and morphology (FSC/SSC) to determine the compound's efficacy and specificity against the blast population. Changes in cell surface marker expression and shifts in morphology indicative of blast differentiation were also evaluated with each compound. As a control for ex vivo differentiation, two APL patient samples were treated ex vivo with ATRA and we observed the blasts gaining CD66b expression indicating granulocytic differentiation. Results: A refractory AML patient was identified whose leukemic blasts exhibited a strong differentiating response to dexamethasone treatment ex vivo. This resulted in loss of CD34 expression (a marker of immature blast cells), gain of CD163 expression (a marker of monocytic/macrophage maturation) and a significant change in cellular size and granularity. After being enrolled in a clinical trial (REB: 13-6962-C) the patient was treated based on the assay for 1 week (40 mg/day) with dexamethasone. Post-treatment samples from the peripheral blood and bone marrow of the patient exhibited the same morphological and cell surface marker changes predicted by the ex vivo assay. The CD163+ cells in the patient also gained additional markers of myeloid differentiation (CD11b, CD14, CD16). After additional cytarabine and fludarabine treatment, the patient remains in remission 4 months post-treatment. Conclusions: Following this initial study, we have continued to identify subgroups of both AML and Myelodysplastic Syndrome patients where blasts differentiate in response to dexamethasone, calcitriol, ATRA or other known differentiating agents using unique cell surface markers of monocytic and myeloid maturation. Flow cytometry expression changes correlated with changes in morphology as observed by May-Grunwald Giemsa staining. In the patient described above this included an increase in cytoplasm and vacuoles consistent with monocytic/macrophage differentiation, which positively correlates with CD163 expression. We aim to apply our assay towards the identification of subgroups of AML patients who respond to differentiation therapies and develop clinical trials to combine differentiating agents with chemotherapy. This approach has the potential to extend the clinical success of APL differentiation therapy to AML patients. Disclosures Prashad: Notable Labs: Employment, Equity Ownership. Western:Notable Labs: Consultancy. Biondi:Notable Labs: Employment. Shah:Notable Labs: Employment. Liu:Notable Labs: Employment, Equity Ownership. Nguyen:Notable Labs: Employment, Equity Ownership. Warnock:Notable Labs: Employment, Equity Ownership. Quinzio:Notable Labs: Employment, Equity Ownership. De Silva:Notable Labs: Employment, Equity Ownership. Schimmer:Novartis: Honoraria. Heiser:Notable Labs: Employment, Equity Ownership.

Ex vivo drug screening defines novel drug sensitivity patterns for informing personalized therapy in myeloid neoplasms

“…Newer fully automated techniques that use high-throughput flow cytometry have improved upon older methods and can now identify potentially active drugs in refractory AML patients. [18][19][20] Multiple studies in patients with refractory acute leukemia or multiply relapsed lymphomas have demonstrated the ability of ex vivo DSS to predict clinical therapeutic responses in vivo. [21][22][23] We report our experience using a novel, fully automated ex vivo DSS platform in 54 patients with newly diagnosed or treatmentrefractory myeloid neoplasms (predominantly MDSs) to evaluate sensitivity to a large panel of US Food and Drug Administrationapproved or investigational drugs and drug combinations.…”

Tumor Genomic Profiling andEx VivoDrug Sensitivity Testing for Pediatric Leukemia and Lymphoma Patients

“…Although multiple studies have demonstrated the utility of these tests, even when they are done they may not have immediate actionability, as we also observed in this cohort where most of the studies did not result in recommendations that were applied by the clinicians. [10][11][12][13]24,27 Although current levels of implementation are low, as experience with these methodologies increase, we anticipate that our institutions and collaborative groups will use the information provided by ex vivo drug sensitivity testing and/or genomic profiling assays upfront for patients in the future to help guide treatment. Correspondence.…”

“…[6][7][8][9] However, results of these tests may or may not be implemented in the care of patients. [10][11][12] The primary objective of this case series is to describe the application and utility of tumor genomic profiling and functional ex vivo drug sensitivity screening in pediatric patients with leukemia and lymphoma, with a focus on the population with relapsed and refractory malignancies.…”

Cancer Prognosis According to Parthanatos Features

“…With a general trend towards whole transcriptome RNA sequencing (Arindrarto et al, 2020) and other high-throughput biomarker discovery (Bai et al, 2013;Drenberg et al, 2019;Prashad et al, 2016;Saad et al, 2018), the profiling of genes such as PARP1 that is associated with cytarabineinduced parthanatos may provide a practical means for patient stratification. Indeed, PARP-1 is known to play a central role in parthanatos, DNA damage repair, and hematopoietic differentiation (Gil-Kulik et al, 2020;Hsieh et al, 2017).…”