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Hyperleukocytosis, most commonly defined as a white blood cell (WBC) count greater than 100,000/μL, is an emergency in acute leukemia, possibly resulting in life-threatening microvascular obstruction, or leukostasis, leading to neurologic (CNS hemorrhage, thrombosis) or pulmonary (respiratory distress, hypoxia) complications. The underlying mechanisms remain poorly understood and are canonically attributed to blood hyperviscosity secondary to high WBC count and abnormal biophysical properties of leukemia cells themselves (leukemia immunophenotype, increased cell size, adhesion, and stiffness). Leukapheresis is a commonly-used therapy for rapid cytoreduction in symptomatic patients, but the procedure carries risk and existing guidelines are supported by scant evidence. Interestingly, despite hematocrit(Hct)/hemoglobin(Hgb) levels being major drivers of blood viscosity due to the high prevalence of circulating red cells (RBCs), how Hct/Hgb mediates hyperviscosity in acute leukemia is unknown. This is clinically important as Hct/Hgb often decrease as leukemic cell counts rise, and acute leukemia patients with anemia are often transfused. While sickle cell disease guidelines advise using a target post transfusion Hct of 30% to minimize iatrogenic hyperviscosity and its morbid complications, no guidelines have been established for acute leukemia. As such, can RBC transfusion actually increase leukostasis risk in acute leukemia? To explore this question requires new biophysical tools as the complexity of blood viscosity increases substantially at the microvascular level as the physical properties of the cells themselves become the major determinants of resistance to blood flow. To that end, we developed "microvasculature-on-a-chip" devices that recapitulate microvascular biophysical and hemodynamic conditions to investigate how the differing presentations of acute leukemia and transfusion support affect the effective blood viscosity at the microvascular level to cause "in vitro leukostasis." A multiple-vessel "multiplex" microfluidic device that operates at the appropriate size scale and mimics the microvascular geometry was designed to enable assessing accurate biophysical measurements of blood hyperviscosity. The devices were microfabricated using standard polydimethylsiloxane-based photolithography (Figure 1). Acute B-cell lymphoblastic (B-ALL, 697), acute T-cell lymphoblastic (T-ALL, Jurkat) and acute myelocytic (AML, HL60) leukemia cell lines were maintained via standard cell culture conditions. Patient samples were obtained through our institution's IRB. RBCs from healthy donors were isolated and mixed with leukemia cells to achieve target Hct/Hgb and WBC levels. Various physiologic leukemia "mixtures" were then perfused under physiologic microcirculatory flow conditions through the microvascular device and microchannels occlusion was tracked via videomicroscopy (Figure 2). Using a standard least squares multivariable linear regression with first and second order effects, microchannel size, Hct/Hgb, WBC count and leukemia cell type all showed statically significant effect on in vitro leukostasis, or microchannel occlusion over time, (all p values < 0.03) (Figure 3). Overall, severe anemia appears to be protective against in vitro leukostasis and there appears to be Hct/Hgb thresholds above which in vitro leukostasis becomes more prevalent, though this is different for B-ALL versus T-ALL. This is in contrast to AML, where severe anemia does not appear to offer protection against in vitro leukostasis as occlusion was appreciated at all Hct/Hgb levels. These data suggest when determining risk for leukostasis, WBC count and leukemia immunophenotype should not be the sole determinants. Here we show Hct/Hgb levels affect microvascular blood viscosity and risk for microvascular occlusion. These results may impact decisions regarding RBC transfusions and possibly initiation of leukapheresis in asymptomatic patients. Having a model to assess risk associated with RBC transfusions and informing clinicians when a patient might become at risk for leukostasis can have a significant impact on their clinical outcome, morbidity and mortality. Ongoing studies incorporating patient lymphoid and myeloid leukemia cells are needed to support this cell line data. Figure 1 Figure 1. Disclosures Kemp: Parthenon Therapeutics: Membership on an entity's Board of Directors or advisory committees. Lam: Sanguina, Inc.: Current holder of individual stocks in a privately-held company.

Treatment of pediatric acute lymphoblastic leukemia (ALL) has seen dramatic improvements over the last several decades, leading to survival rates of over 90% for specific groups. However, for patients with relapsed/refractory disease, achieving complete remission remains a significant challenge. To further improve treatment outcomes for these patients, improved functional screens are needed to better match high risk leukemia patients with novel therapeutic strategies. Current functional screens are limited by the large patient samples required, particularly for combination drug screening and evaluation of higher order drug interactions. To that end, this work aims to develop a microfluidic multi-drug and multi-dose chemosensitivity assay to directly test candidate combinations of therapeutics in patient derived ALL cells. This will allow for analysis of response to combinations of 3 small molecule drugs while minimizing required patient sample and reducing experimental workload. Once validated, this assay can be used to identify biomarkers of response to novel combination regimens in pediatric ALL, and ultimately, could be used to prospectively guide therapy in newly diagnosed patients. Specifically, this is accomplished by generating stable, overlapping concentration gradients of small molecule drugs across 3D cultures of leukemia cells with and without human bone marrow derived mesenchymal stem cells (Fig 1A). We have demonstrated the ability to generate and maintain these gradients over 48 hours, and have shown that experimentally generated concentration gradients closely match steady state computational fluid dynamics (CFD) model predictions (Fig 1B). Moreover, exposing a T-ALL cell line (Jurkats) to a single gradient of cytarabine results in a gradient in cell viability. In drug treated devices, increasing drug dose led to decreasing averaged viability, while no appreciable difference was seen in vehicle control devices (Fig 1C). A commonly used 3 drug combination, Daunorubicin, Vincristine, and Prednisolone, was then used to validate the utility of the device for evaluating multi-dose drug combinations. Here, Jurkats were exposed to superimposed concentration gradients of Daunorubicin, Vincristine, and Prednisolone for 48 hours, after which cell viability as a function of concentration was examined. Specifically, a ternary contour map of the average viability across the device was generated, clearly depicting the range of response across the device (Fig 2B). Previous results have demonstrated Jurkats' sensitivity to Daunorubicin as well as Vincristine and relative resistance to Prednisolone, and these results are closely recapitulated in the device. Previous reports in literature have also demonstrated potential for antagonism between anthracyclines and vinca alkaloids, specifically between Doxorubicin and Vincristine in Jurkats (Ehrhardt, 2011). Our results also suggest that some amount of antagonism may exist between Daunorubicin and Vincristine in Jurkats at these concentration ranges tested, evidenced by the high average viability in regions of high vincristine concentrations and mid-range daunorubicin concentrations. Finally, we then used this system to evaluate response to drug combinations in a test patient sample. Specifically, combinations of Vincristine, Prednisolone, and Daunorubicin, as well as combinations of Nelarabine, Etoposide, and Cytoxan were evaluated in a standard risk B-ALL bone marrow aspirate sample. Drug treated devices resulted in significantly reduced viability relative to the vehicle control, with a differential in response observed between the 2 combinations tested (Fig 3A). Moreover, at these drug concentration ranges, it appears that combinations of Etoposide and Ara-G are particularly ineffective relative to other combinations in this sample (Fig 3B). Future studies will work to improve the baseline viability of banked samples in the device, and include testing of fresh leukemia samples as well. We are also working to develop methods to use data collected from these devices to identify synergistic or antagonistic drug combinations, and in the future, can correlate this with clinical outcome metrics as well as biomarkers of response. Tissue samples were provided by the Children's Healthcare of Atlanta Pediatric Bio-Repository. Other investigators may have received specimens from the same subjects. Figure 1 Figure 1. Disclosures Lam: Sanguina, Inc.: Current holder of individual stocks in a privately-held company.

Background: Sickle cell disease (SCD) is a group of genetic disorders in which sickle hemoglobin polymerizes under deoxygenation, altering red blood cell (RBC) morphology and behavior. The properties of sickle RBCs contribute to increased viscosity of blood and occurrence of vaso-occlusions, a major aspect of SCD pathophysiology. Voxelotor is a novel FDA-approved treatment for SCD which modulates hemoglobin O 2 affinity, and while its known mechanism inhibits sickle polymerization, the impact on other aspects of SCD pathophysiology remain unknown. Thus, despite the new treatment option, highly variable clinical manifestation continues to be a hallmark of sickle cell and there is consequently a need to optimize the use of current therapies based on patient-specific factors. In this work, we leverage datasets generated from a unique microfluidic assay that measures blood flow behavior under varying oxygen tension in conjunction with novel statistical approaches to model and assess sources of variability in sickle blood flow response to voxelotor. Methods: RBCs from patients with SCD (n=28) were treated with voxelotor at 500 uM concentration. Treated samples and untreated controls were perfused through a microfluidic platform that dynamically modulates oxygen tension and measures flow velocity (Wood et al, 2012; Valdez et al, 2019). The area between curves (ABC) of the normalized velocity across the range of oxygen tension between treated and control conditions was calculated to quantify the effect of voxelotor for each sample (figure 1). A paired t-test was used to assess the difference in response between treated and untreated samples. Where available, clinical data including the hemoglobin fractions and complete blood count (CBC) were collected for each sample as predictor variables, and partial least squares regression (PLSR) modeling was used to assess the correlation of predictors and responses. Results: Voxelotor increased the velocity ABC from untreated to treated conditions (p Conclusions: Our analysis quantified patient-specific differences in the blood flow response to voxelotor, showing a wide variability in response despite treatment by the same drug concentration. Genotype-specific multivariable models that take into account easily measurable clinical variables such as the CBC have the potential to explain the variability in patient response to voxelotor treatment. In HbSC samples, the WBC, platelet, and reticulocyte counts were highly correlated and strong predictors of response to voxelotor, which may point to markers of hemolysis and inflammation being useful in determining patients that can be optimally treated with this drug. In HbSS, response to voxelotor was mainly inversely correlated with HbA levels, which is a surrogate marker for blood transfusions, indicating that the effect of voxelotor is lessened for patients who are receiving transfusions. However, the low R2Y of this model highlights the clinical variability in this SCD genotype and consequent need for additional biomarkers of disease severity. In conclusion, our hybrid experimental-computational approach is able to identify clinical factors that highly impact the response of patient blood samples to treatment with voxelotor for HbSC patients, and highlights the need for precision therapy recommendations in SCD. Figure 1 Figure 1. Disclosures Lam: Sanguina, Inc.: Current holder of individual stocks in a privately-held company. Kemp: Parthenon Therapeutics: Membership on an entity's Board of Directors or advisory committees.