ALBERT

Description: BACKGROUND: Chemotherapy and radiation therapy-induced oral mucositis (OM) causes significant morbidity including pain, infection, poor nutrition, and rarely, upper airway compromise. Alopecia is rated as one of the top causes of distress from cancer therapy. Fahl et al. showed that alopecia was prevented in animals by applying topical epinephrine or norepinephrine prior to radiation or chemotherapy. They also demonstrated that animals receiving oro-topical phenylephrine (PhE) prior to radiation had improved weight gain and decreased inflammation of the oral mucosa. Vasoconstrictive techniques (i.e., cryotherapy) have shown modest success, but compliance can be challenging and most modalities have not been rigorously tested. Concern has been expressed that vasoconstrictive methods to prevent alopecia and OM may decrease chemotherapy delivery to tissues, thereby creating a sanctuary site for untreated cancer cells. The primary objective of this study was to compare survival of mice with leukemia who were given topical vasoconstrictors prior to receiving chemotherapy to animals who did not receive vasoconstrictors. METHODS B6D2F1 mice received 10^4 L1210 cells (mouse leukemia strain) via retro-orbital injection. Group A received L1210 cells with no further treatment. All other groups received a single intra-peritoneal (IP) 90 mg dose of cyclophosphamide (sub-curative) 24 hours after injection with L1210 cells. Group B did not receive a vasoconstrictor. Groups C through G had their fur clipped and were given increasing doses of topical epinephrine (epi) 15 minutes prior to cyclophosphamide. Groups H through M were given increasing doses of oro-topical phenylephrine 15 minutes prior to cyclophosphamide. All mice were followed until date of death. P-values and hazard ratios were calculated comparing median survival of groups C-M with Group B (see table) RESULTS Differences in median survival between Group B mice and all other mice who received cyclophosphamide and oro-topical phenylephrine or topical epinephrine were not statistically significant. Table. Group # IV L1210 Cells IP Cytoxan (ug/gm bw) Topical Vasoconstrictor Treatment (topical Epinephrine or Oral-Topical Phenylephrine) # mice Female B6D2F1 Median Survival (days post-L1210 injection) P-value (all compared to B) Hazard Ratio with 95% confidence interval A 104 - - 16 9

Description: Introduction Systemic mastocytosis (SM) represents a heterogeneous group of disorders characterized by accumulation of neoplastic mast cells (MCs) in one or more organ systems. Patients with SM present with a broad range of symptoms resulting from excessive mast cell mediator release, especially histamine, that frequently overlap with those of allergic disease. Episodes of life-threatening anaphylaxis are a recognized feature of SM. Here at St. Michael's Hospital we currently follow more than 50 SM patients, the largest patient cohort in Canada. Omalizumab is a subcutaneously administered monoclonal antibody which acts on circulating IgE, reducing binding to the high-affinity IgE receptor (FCεR1) on mast cells, thereby reducing the potential reactivity of these cells. At St. Michael's Hospital, omalizumab is used as an add-on, off-label therapy in SM patients at risk for recurrent anaphylaxis. The efficacy of omalizumab treatment for SM patients remains unclear. Typically, highly symptomatic patients who are refractory to all other medication are candidates for omalizumab therapy. Objectives Our primary objective was to describe the response to treatment by omalizumab in patients with SM in a tertiary care centre. Our secondary objective was to compare the markers of disease in SM patients between those who were non-responsive versus responsive to omalizumab. The clinical and biological markers to be studied are symptoms and tryptase levels. Methods This is an observational, retrospective study (n=6) of SM patients treated with omalizumab at St. Michael's Hospital between January, 2014 and June, 2018. Electronic medical records were reviewed for mastocytosis treatment, symptom progression and tryptase levels, if available. All patients included in the study were diagnosed with SM according to the 2016 WHO criteria by undergoing a bone marrow biopsy. A baseline was established 2-5 months pre omalizumab exposure, as well as two follow-ups, each ranging from 2-8 months post omalizumab exposure (av. 4.7 months). The Brown Anaphylaxis score was used to capture severity of anaphylaxis. Mild (1), moderate (2), and severe (3) scores were associated with cutaneous manifestations, systemic (GI, respiratory, cardiovascular) involvement and systemic (hypoxia, hypotension, neurological compromise) collapse, respectively. Results Our study consisted of 4 females and 2 males, with an average age of 49 years old [IQR 36-74]. All 6 patients were diagnosed with indolent SM, the more moderate of the six SM subtypes. In every system, except for respiratory, it appears that symptoms decreased once therapy began. From baseline to first follow-up: all three patients who were experiencing systemic symptoms, three of the six manifesting cutaneous symptoms, and two of the three with cardiovascular involvement, responded fully to treatment. At second follow-up, patient 1 presented to clinic asymptomatically. Overall, 100% of patients responded to treatment with responses ranging from 17% to 100% improvement of mastocytosis-related symptoms. The grading of anaphylaxis severity reported three of the six patients improving from scores of 3 (severe) to 1 (mild). The other three patients remained at scores of 2. Patients 1 and 2 (only patients with available tryptase levels at both baseline and follow-up) saw a decrease in tryptase level from 134 to 84.1 and 11.4 to 8.3, respectively. Conclusions Omalizumab appears to be an effective therapy for patients with SM with anaphylaxis and reduces tryptase levels. It should be readily considered in the management of this population. Next steps include following these patients prospectively to better capture the efficacy of omalizumab within this population. Disclosures No conflicts of interest to declare Disclosures No relevant conflicts of interest to declare.

Description: Chimeric antigen receptor (CAR) redirected T cells can induce remission in highly refractory leukemia and lymphoma subjects. Despite the potential of this emerging therapeutic modality, treatment with CD19 CAR T cells is not uniformly effective for remission induction, and remissions can be short-lived in a significant proportion of treated subjects. Here, we analyzed 43 pediatric and young adult subjects participating in the phase 1 trial (NCT02028455) and correlated their outcomes with in vivo performances of SCRI-CAR19v1(a CD19 specific CAR T cell product), as well as starting material (SM) T cell repertoire- and final cell product (FP) -intrinsic attributes. Subjects were allocated to the dysfunctional response group defined as early treatment failure (no remission or early disease progression after remission while still having CAR engraftment) (n=5) and the functional response group defined as those who obtained an MRD-negative remission that was sustained beyond 63 days (n=37). We found the magnitude of absolute CAR T cell engraftment area under the curve (AUC) was attenuated in the dysfunctional response group (AUC 150.3, range 0.54-752.8, Mann-Whitney p=0.0033) as compared to the functional response group (median AUC 1309, range 5.23-9537). The absolute number of CD8+CAR+ cells and CD4+CAR+ cells at peak engraftment was also significantly higher in the functional response versus the dysfunctional response. The phenotype of the CAR+ cells was analyzed at peak engraftment by multiparameter flow cytometry. CAR+ CD8+ cells from both groups had similar frequencies of PD-1+ cells, whereas the dysfunctional response group showed a significantly higher frequency of LAG-3+ T cells, both in the CAR+CD8+ cells and the CAR+CD4+ cells. A similar trend was seen with the expression of TIM-3. In order to assess whether T cell intrinsic factors contributed to therapeutic failure in these subjects, we studied T cell repertoire status in apheresis products and final expanded CAR T cell products. Several reports have shown that using SM rich in terminally differentiated cells result in CD19 CAR-T cell products having limited replicative capacity and attenuated ability to transition to long lived memory cells . When comparing apheresis derived SM from the functional and dysfunctional response subject groups by flow cytometry for markers associated with functional exhaustion (LAG-3, TIM-3, PD-1), we observed a significantly higher percentage of CD8+ T cells expressing PD-1 and LAG-3 in the dysfunctional response group compared to the functional response subjects (p=0.0266 and p=0.0052). We also observed a higher frequency of CD4+ cells expressing PD-1 in the dysfunctional group. There was no difference in the frequency of cells expressing CD45RA, CD45RO, CCR7, CD27, or in the frequency of cells expressing TNF-α, IFN-γ or IL-2 in response to CD3/CD28 stimulation in both CD4 and CD8 SM T cells between the groups. Lastly, using classification and regression tree analysis, subjects could be classified by the frequency of SM CD8+ T cells expressing LAG-3 and the frequency of SM CD8+ T cells capable of secreting TNF-α upon CD3/CD28 bead activation (r2=0.636). Subjects with fewer than 0.745% SM CD8+ T cells expressing LAG-3 were all in the functional response group (n=26/43). Subjects with equal or more than 0.745% SM CD8+ T cells expressing LAG-3 (n=16) could be further sub-divided into two groups: subjects with equal or more than 25.283% of CD8+ T cells expressing TNF-α were also functional responders (n=8/8), while subjects with fewer than 25.283% of CD8+ T cells expressing TNF-α were in majority in the dysfunctional response group (n=5/8). Of the three subjects from the functional response group with high frequencies of LAG3+ cells and low frequencies of TNF- α -secreting cells, all three had short duration BCA and relapsed within 6 months. The combination of elevated frequency of cells expressing LAG-3 and a reduced capacity to secrete cytokines in response to stimulation potential serve as biomarkers for patients with perturbated T cell repertoires that generate CAR T cell products with attenuated anti-leukemic potency. The parameters identified herein, should they be validated in larger trial cohorts, have the potential to prospectively identify patients at risk for initial therapeutic failure thus requiring alternative therapies from those who have an excellent prognosis. Disclosures Li: Juno Therapeutics: Employment, Equity Ownership. Jensen:Juno Therapeutics, Inc.: Consultancy, Patents & Royalties, Research Funding.

Description: INTRODUCTION Bleeding from thrombocytopathy is a common complication of advanced chronic lymphocytic leukaemia (CLL). In addition to disease-related thrombocytopenia, the presence of the CLL clone and/or therapeutic interventions may further impair platelet function. In particular, the BTK inhibitors ibrutinib and acalabrutinib are known to inhibit platelet glycoprotein VI (GPVI)-mediated platelet aggregation. We compared platelet function and markers of GPVI activation between untreated CLL patients, ibrutinib-treated CLL patients and healthy controls, and studied the in vitro effects of ibrutinib and acalabrutinib on clinically utilised platelet function assays to assess their impact on GPVI-mediated as well as non-GPVI-mediated platelet activation pathways. METHODS Blood samples from 17 healthy volunteers and 8 untreated CLL patients were spiked with vehicle or comparable plasma concentrations of ibrutinib (0.3µM, 1.0µM) and acalabrutinib (1.8µM, 6.0µM) attainable during the treatment of CLL. Additional samples were obtained from 5 CLL patients undergoing ibrutinib treatment. Platelet function was evaluated using whole blood multiple electrode aggregometry (MEA - Multiplate®) and light transmission aggregometry (LTA - AggRAM®) in response to varying concentrations of aggregation-inducing reagents (collagen, CRP-XL, ADP, TRAP, ristocetin, arachidonic acid, and adrenaline). Shear-induced platelet adhesion was assessed using PFA-100®. Soluble GPVI plasma levels were assessed by ELISA. RESULTS In the absence of treatment, CLL patients exhibited significant platelet defects on whole-blood platelet function analyses in response to various agonists including ADP, ristocetin, TRAP and collagen (MEA) and prolongation of PFA-100® collagen/epinephrine closure time. This impairment was not replicated in assays using platelet-rich plasma (LTA). Ibrutinib-treated CLL patients demonstrated an additive impairment of platelet function, especially in regards to collagen-mediated activation by MEA or PFA-100®. There was no significant difference in soluble GPVI levels between normal, untreated or ibrutinib treated CLL patients. Addition of clinically-attainable concentrations of ibrutinib and acalabrutinib in vitro produced similar concentration-dependent inhibition of platelet function in healthy controls, with inhibition of aggregation evident in response to various agonists including collagen, CRP-XL, ristocetin and ADP but not arachidonic acid or TRAP. Ibrutinib also impaired aggregation in response to epinephrine, and caused selective prolongation of the PFA-100® collagen/epinephrine closure time, an effect not observed with acalabrutinib. MEA appears more sensitive and reproducible than LTA to describe the various inhibitory effects on platelet aggregation. Similar concentration-dependent inhibition of platelet function was observed by adding ibrutinib and acalabrutinib in vitro to blood samples from untreated CLL patients. CONCLUSIONS CLL is associated with a broad platelet function defect, which can be exacerbated by BTK inhibitors. Acalabrutinib induces a platelet function defect similar but less potent to that observed with ibrutinib, with the exception of shear-induced platelet adhesion (PFA-100®) which was only abnormal with ibrutinib. Routine platelet function assays are capable of quantifying BTK inhibitor-induced platelet dysfunction in CLL patients, with the most sensitive and reproducible measure being collagen-induced aggregation by MEA. There was no evidence for BTK-dependent platelet GPVI cleavage. Whole-blood platelet function assays may have utility in managing CLL patients presenting with bleeding or requiring urgent surgery during therapy with BTK inhibitors. Disclosures McGregor: Pfizer: Other: Conference travel support; Bristol-Myers Squibb: Other: Conference travel support; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria. Baker:CSL Behring: Research Funding; Biogen Idec: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Research Funding; Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support, Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support; Roche: Other: Conference travel support; Novo Nordisk: Other: Conference travel support.

Description: Background: Marginal zone lymphoma (MZL) is an incurable but heterogeneous disorder for which there is no standard of care treatment in the relapsed/refractory (R/R) setting. The BTK inhibitor, ibrutinib (IB) is well tolerated with an overall response rate (ORR) of 48% as monotherapy in R/R MZL but rarely complete remission (Noy et al, Blood, 2017). The BCL-2 inhibitor, venetoclax (VEN) has also been evaluated in a small number of R/R MZL patients (pts) with evidence of activity and tolerability (Davids et al, JCO, 2017). We sought to evaluate the efficacy and safety of combination IB and VEN in pts with MZL based upon the rationale of (1) distinct mechanisms of action, (2) activity as monotherapies in MZL, and (3) acceptable non-overlapping toxicity profiles. We report preliminary results of the MZL cohort of an investigator-initiated phase II trial of combination IB and Ven (NCT02471391). Methods: Pts with MZL by WHO 2008 criteria who were R/R or treatment naïve but considered inappropriate for treatment with chemotherapy were enrolled. Pts commenced treatment with IB monotherapy at a dose of 560mg per day. After 4 weeks, VEN was added to IB treatment, in weekly step-wise dose escalation over 6 weeks to a target dose of 800mg per day in the initial cohort (n = 4), amended to 400mg per day, due to a reported IB-VEN drug interaction in a similar trial (NCT02910583). The primary endpoint was the complete remission rate (CRR) at 16 weeks. The secondary endpoints were to determine ORR and CRR, to determine minimal residual disease (MRD) elimination rates, to describe progression free survival (PFS), overall survival, duration of response, time to progression and frequency and severity of adverse events. Investigator-assessed response assessments, based on IWG criteria (Cheson et al, JCO, 2007), were performed with CT at 4, 16, 28, 40 and 56 weeks, PET/CT at weeks 16 and 56, and bone marrow (BM) aspirate and trephine, and MRD assessments by flow cytometry at all time-points. Results: Fourteen of 15 planned pts were enrolled at the time of data cut-off (May 2019), of whom 12 had reached the week 16 primary endpoint or discontinued and are reported here. Two remain on study for

Description: Introduction: Geriatric assessment (GA) is a multidimensional evaluation of patient health and function that may detect impairments not identified as part of routine care, predict treatment-related morbidity and mortality, and inform treatment plans. Given evidence of these benefits, the National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology (NCCN Guidelines®) recommend GA for older candidates of hematopoietic stem cell transplantation (HCT). However, both older and younger HCT candidates will often receive multiple rounds of chemotherapy before HCT, leading to functional impairments in all age groups. Furthermore, HCT patients often experience a significant gap between when they are first evaluated and actually proceed to transplant (e.g., while a donor search is conducted), creating an opportunity to identify impairments and optimize function prior to transplant. Methods: To address this opportunity, we created a clinical pre-HCT optimization program (C-POP) to evaluate physical function, cognitive function, nutritional status, and mental health in all adults who were deemed potential candidates for allogeneic HCT by a HCT physician. We applied this standard of care program to all adult candidates for HCT, regardless of age, with the goal of identifying functional impairments and then referring patients to services to optimize those impairments prior to HCT. We defined impairments using validated measures and compared results to established norms or scoring, controlling for age and gender where appropriate (e.g., the cut-off for six-minute walk distance was adjusted for age, gender, height, and weight, while the cut-off for falls was any fall regardless of characteristics). Patients with impairments were referred to the appropriate supportive care (e.g., physical function impairment -〉 referral to physical therapy). Results were prospectively analyzed at new patient evaluation (NPE), which was the first time the patient met a HCT physician and sign-off, which occurred within a week before starting transplant. While the program is ongoing, we present here the results of patients evaluated between October 16th, 2017 and July 1st, 2019. Patients are divided into three pre-specified age groups: =60 years old, with results compared using a chi-squared test. Results: We evaluated 115 patients: 21 (18%) =60 years). There were no differences between the age groups in other demographics (gender, race, and ethnicity). At NPE, 93 (81%) met criteria for at least 1 impairment in physical function, cognitive function, nutritional status, or mental health; 62 (54%) met criteria for impairments in 2 or more areas. Surprisingly, patients =60 years (36/54, 67%) (p=0.03). Of those 115 patients, 52 (45%) proceeded to HCT, including 12 (57%) =60 years (p=0.75); of those patients who have not proceeded to HCT, 40 (35%) will never proceed to HCT (e.g., deemed not a candidate after functional evaluation or died of disease prior to HCT) while 23 (20%) are still awaiting HCT (e.g., donor search ongoing). Patients who proceeded to HCT were less likely to have mental health impairments (2/52, 4% vs. 9/40, 23%, p=0.006). Of the 52 who were seen at new patient evaluation and proceeded to transplant, 40 (77%) were seen at sign off. Of those who had impairments at NPE, 12/23 (52%) improved their physical function to normal limits, 4/9 (44%) improved their cognitive function, and 9/13 (69%) improved their nutritional status by the time of sign-off (of those who were seen at sign-off, none had mental health impairments at NPE). Discussion: These results demonstrate that younger as well as older candidates for HCT exhibit a high degree of functional impairment. However, this impairment could be amenable to improvement prior to HCT. These findings support application of GA to all HCT candidates regardless of age. We will investigate the effect of referred interventions (e.g., physical therapy, seeing a dietician) in improving functional impairments in future studies, as well as look at the effect of these findings on HCT outcomes. Disclosures Wiggins: Incyte, Inc.: Speakers Bureau. Gasparetto:Janssen: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; Celgene: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; BMS: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed . Rizzieri:Millennium: Speakers Bureau; Novartis: Consultancy; AbbVie: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Incyte: Consultancy, Speakers Bureau; TEVA: Consultancy; Spectrum: Consultancy; Kite Pharma: Consultancy; Gilead Sciences: Consultancy, Speakers Bureau; Seattle Genetics: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau. Horwitz:Abbvie Inc: Membership on an entity's Board of Directors or advisory committees. Sung:Novartis: Research Funding; Merck: Research Funding; Seres: Research Funding.

Description: Background Iron deficiency (ID) is the most common and widespread nutritional deficiency in both developing and developed countries (WHO, 2001; Mei et al., 2011). Women of child bearing age are at the highest risk, but this risk increases even more during pregnancy. The expansion of blood volume, growth of the fetus and placenta increase demand for iron to approximately 5.0mg/day by the third trimester (Met et al., 2011). Common symptoms of ID during pregnancy include fatigue, shortness of breath, and difficulty concentrating (WHO, 2001). Poor prenatal iron status is associated with diminished cognitive performance, language ability, and motor functions in the child (Tamura et al, 2002). For the mother, it is associated with risk of blood transfusion and post-partum depression. Despite international recommendations and guidelines on the management of ID in pregnancy, it remains a problem of epidemic proportions and is often unrecognized and left untreated. To increase awareness of ID, a quality improvement project, IRON Deficiency project in Pregnancy: Maternal Iron Optimization (IRON MOM) was implemented January 1st, 2017 at St. Michael's Hospital (SMH), in Toronto, Canada. Phase 1 of the project involved adapting lab requisitions and workflow in the obstetrics clinic to incorporate routine measurement of ferritin in week 12, 16 and 28 of pregnancy. As part of the IRON MOM, laboratory requisitions were modified to include ferritin as part of routine screening for all pregnant women. Objective The primary objective of this study was to assess the prevalence of ID in pregnant women consistently screened for ID after the implementation of the IRON MOM quality improvement project at a tertiary hospital in Toronto, Canada. Methods Administrative laboratory data was collected from the electronic medical record system at SMH, Toronto, Canada between January 1 and December 31, 2017. Suboptimal iron stores was defined as serum ferritin between 30-50μg/L. ID was defined as serum ferritin between 15-29μg/L, and severe ID was defined as

Description: Key Points DLBCL can be detected in the blood by immunoglobulin high-throughput sequencing (Ig-HTS) with high specificity. Although DLBCL can be detected in leukocytes or plasma by Ig-HTS, plasma has greater sensitivity and more accurately reflects disease.

Description: Sickle cell disease (SCD) is characterized by acute and repetitive vaso-occlusive crises (VOC). These crises have been hypothesized to occur when blood flow is reduced following obstruction of sickle-shaped red blood cells in the vasculature. However, it is now well established that inflammation, oxidative stress, endothelial activation and pro-coagulation in sickle cell disease patients also contribute to the formation of heterocellular aggregates that can lead to VOC (Vercellotti and Belcher, 2014). Transgenic SCD mice recapitulate the pathology of human disease in response to stimuli such as heme injection and hypoxia/reoxygenation. SCD SS Townes mice, which express human α and sickle γAβS globins, AA Townes mice expressing normal human α and normal γAβA globins and heterozygous AS mice which express only one allele of the γAβS sickle gene were used. To characterize vaso-occlusion in these mice and evaluate the efficacy of different pharmacological mechanisms, we modified the skinfold vaso-occlusion model (Kalambur et al, 2004) using fluorescent intravital microscopy to visualize blood flow occlusion following hemin injection or hypoxia/reoxygenation challenge. Dorsal skinfold chambers were implanted and24 hours post-surgery mice were injected with FITC-dextran for visualization of flowing blood vessels. Skinfold bearing mice were then subjected to hemin treatment (16 μmoles/kg) or hypoxia (7%; 1 hour)/reoxygenation (1 hour) followed by the injection of Alexa fluor 647-labeled albumin to allow quantification of occluded vessels through dual fluorescent image analysis. Following hemin injection, SS mice showed significant ~30% vaso-occlusion in comparison to AA mice with ~8%, whereas the AS mice showed an intermediate phenotype with ~20% vaso-occlusion. Hypoxia/reoxygenation challenge also resulted in significant vaso-occlusion for SS mice (~25%) whereas only 5% was observed in AA mice. Interestingly, AS mice also showed a significant amount of vaso-occlusion (~25%) similar to SS mice when challenged with hypoxia/reoxygenation. Although no sickling can be observed in an ex vivo sickling assay using AS red blood cells, an intermediate amount of free Hemoglobin (Hb) can be detected in the plasma of these mice and rolling can be observed. This suggests that these vaso-occlusive models relate more on the inflammatory and endothelial activation state and are independent of the sickling potential of the red blood cell. We then used our model with hypoxia/reoxygenation challenge to evaluate the effects of dimethyl fumarate (DMF, 15 mpk BID), an anti-P-Selectin antibody (150ug/mouse) and the covalent hemoglobin oxygen affinity modulator GBT-440 (300 mpk). As anti-inflammatory agents, DMF and Anti-P-Selectin significantly reduced vaso-occlusion in SS mice by ~60% compared to the vehicle treated mice, but GBT-440 did not inhibit vaso-occlusion at a dose where a significant reduction in p50 was observed. In conclusion, our data have shown that obstruction of blood flow in the skinfold vaso-occlusion model in SCD Townes mice reflects the vascular inflammatory state of the disease and is independent of the ex vivo capacity of red blood cell to sickle. Disclosures Sturtevant: Sanofi: Employment. Macias-Garcia:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment. van der Flier:Sanofi: Employment. Hicks:Sanofi: Employment. Demers:Sanofi: Employment.

Description: Background Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Although a majority of patients are cured with standard chemo-immunotherapy, up to 40% of DLBCL patients have refractory disease or develop relapse following R-CHOP regimen, warranting development of novel, more effective therapeutic strategies for this cohort[1]. The composition of immune cells in the tumor microenvironment (TME) and tumor PD-L1 expression have been reported to predict DLBCL outcomes, however PD1 inhibitors demonstrate response rates of less than 10%.[2-5] We hypothesize that a better characterization of spatial architecture of the tumour microenvironment (TME) in lymphoma will help explain why DLBCL has poor responses to PD1 inhibitors and guide future targeted immunotherapies for these patients. Methods Here we characterized the TME in DLBCL using imaging mass cytometry (IMC), which allows high-dimensional, single-cell and spatial analysis of FFPE tissues at sub-cellular resolution [6]. Using a panel of 32 antibodies, IMC was performed 41 tissue microarray cores from 33 DLBCL cases. IMC images were analyzed for relevant immunophenotypes, the spatial architecture of those phenotypes and compared to clinical outcomes to identify immune contexture based biomarkers. Results Phenograph was used to cluster tumor and immune cells based on phenotype. Immune cells represented 33% of the cells broken down to CD4 (36%), CD8 (30%), macrophages (26%) and TREG(8%) (Figure A).Immune cell infiltration in individual tumor samples ranged from 7% to 75% with marked heterogeneity between samples. Analysis of immune marker expression on tumor cells identified co-expression of PD-L1/CCR4/TIM3 to be highly prognostic for overall survival (p=0.003, Figure B-C) To characterize the patterns of spatial interaction in the TME, we developed an unsupervised multivariate model to construct spatial meta-clusters based on average distances from CD8 to the centroids of 5 nearest endothelial cells, TREG, CD4 T cells, macrophages, and tumor cells (Figure D). Spatial analysis revealed 11 meta-clusters for CD8 T cell interactions (Figure E). Each CD8 spatial interaction pattern is distinctive with case to case heterogeneity (Figures F). Risk assessment analyses of spatial clusters 1, 2 and 4 ("hazardous") had almost 3 times higher odds of being identified in refractory cases compared to clusters 3, 5 and 6 ("protective") (Figure G). In the "protective" spatial neighborhoods, we observed the presence of activated CD8, Th1-like CD4, and less suppressive TREGphenotypes, with opposite in "hazardous" areas (Figures H). TIM-3 expression was high both on T cells and tumor cells in the "hazardous" neighborhoods. Finally, we show that sub-setting our analysis of CD8 phenotypes based on their spatial location to other cells improved our ability to predict overall survival in the cohort. Conclusion These results are the first to demonstrate that spatial profiling of immune architecture in DLBCL is associated with clinical outcomes, and that spatial analysis of immune cells should be explored as a potential biomarker for patients treated with immunotherapies. References [1] Coiffier, B. et al.,Blood116, 2040-5 (2010). [2] Lenz, G. et al.,N. Engl. J. Med.359, 2313-2323 (2008). [3] Ansell, S. et al.,J. Clin. Oncol.19, 720-726 (2001). [4] Qiu, L., et al., BMC Cancer19, 273 (2019). [5] Kridel, R., et al., Haematologica100, 143-5 (2015). [6] Giesen, C. et al., Nat. methods |11, 417 (2014). Figure Disclosures Merchant: Agios: Speakers Bureau; Pfizer: Consultancy, Research Funding.