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Background Stroke or Cerebrovascular Accident (CVA) is one of the leading causes of mortality in the world whether it be ischemic or hemorrhagic 1,2. There has been an association of increased incidence of stroke in patients with cancer due to multiple factors 3. Regarding hematologic malignancies, there have been studies to assess the risk of developing stroke in patients with Hodgkin lymphoma (HL), however the information on non-Hodgkin lymphoma (NHL) is limited and not well understood4. We conducted a retrospective case-control study to assess the incidence, risks, and complications and length of stay of patients with current or prior history of NHL who developed an ischemic stroke. Methods This case-control study identified 29,632 patients older than 18 years, with or without CVA and with or without active or history of Non-Hodgkin Lymphoma (NHL) who were admitted to diverse private hospitals between 2018-2022. The patients were divided into two groups, one with current or prior NHL (n=10,699) and a control group of patients (n=18,933) with similar characteristics. Patients with active hemorrhagic stroke, pregnancy, malignancies other than NHL, prior hemorrhagic or ischemic stroke, infective endocarditis, atrial fibrillation, and acute coronary syndrome on admission were excluded. We compared patient's characteristics and the risk of developing a stroke based on the presence or absence of NHL as well as the factors that increased hospital mortality. Length of stay was evaluated as a secondary outcome. Bivariate statistical analysis was used to calculate the odds ratios using RStudio 4.2.2 and JMP Pro 16.0 for all data analysis. Results Of the group with NHL 46.39 % (n=4958) were female, and 53.61% (n=5729) were male, 42% were current or former smokers (n=4497), 27.49% (n=2941) had diabetes, 44.68% (n=4780) had hypertension; 3.34% (357) of them passed away. Of the control group 57.51 % (n=10876) were female, and 42.49% (n=8036) were male, 35% were current or former smokers (n=6763), 16.43% (n=3111) had diabetes, 28.89% (n=5469) had hypertension; 0.47% (n=88) of them passed away. The mean length of stay of patients with stroke was 7.99 days in the NHL group, and 5.99 days in the control group. Regarding the admissions due to stroke, there were 214 patients in the group with NHL, and in the control group where there were 113. Patients with NHL are more likely to have a CVA compared to the control group (OR 2.39 p<0.0001 CI 1.47~3.85) Patients who have NHL are more likely to die (OR=6.08 p<0.001) CI 4.77~7.74) compared to patients without NHL from all causes. Conclusion Patients with Non-Hodgkin lymphoma have an increased risk of developing a stroke compared to patients with similar characteristics but without NHL (or other malignancies). It was also noted that patients had an increased length of stay. Other variables and characteristics of patients that were identified to be more likely to have a CVA and statistically significant for this sample are already well-known risk factors for CVA including diabetes, hypertension, and cardiovascular disease. However, this study aimed to isolate those variables to identify the risk for stroke in this unique population. Our study limitation is the broad spectrum of NHL and possible confounders between the different variants of NHL. More studies are needed to investigate this. References Navi B, Reiner A, Kamel H, et al. Risk of Arterial Thromboembolism in Patients with Cancer. J Am Coll Cardiol. 2017 Aug, 70 (8) 926-938. Turner, M., Murchie, P., Derby, S. et al. Is stroke incidence increased in survivors of adult cancers? A systematic review and meta-analysis. J Cancer Surviv 16, 1414-1448 (2022). https://doi.org/10.1007/s11764-021-01122-7 Dardiotis E, Aloizou AM, Markoula S, et al. Cancer-associated stroke: Pathophysiology, detection, and management (Review). Int J Oncol. 2019;54(3):779-796. doi:10.3892/ijo.2019.4669 Wei Y, Yang Q, Qin Q, et al. Profiling of the Risk Factors and Designing of a Model to Identify Ischemic Stroke in Patients with Non-Hodgkin Lymphoma: A Multicenter Retrospective Study. Eur Neurol. 2020;83(1):41-48. doi:10.1159/000506046

Background: Recent data from the field of autoimmune platelet antibodies in vaccine-induced immune thrombotic thrombocytopenia (VITT) suggests that antibodies with restricted clonality may be associated with more severe outcomes. Warm autoimmune hemolytic anemia (WAIHA) is an IgG-mediated red blood cell (RBC) disorder. The goal of this study was to investigate the clonality of anti-RBC IgG antibodies in WAIHA, and to assess if clonal restriction was associated with more severe disease course and outcomes. Methods: 15 patients with WAIHA were evaluated. All patients had proven IgG positivity in direct antiglobulin testing (DAT, or “Coombs” test). RBC eluates were prepared and both the patient plasma sample, as well as the RBC eluate, were subject to liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF MS). Based on the number of antibodies seen in the RBC eluate, patients were classified into mono/oligoclonal vs. polyclonal antibody WAIHA groups Assessment of response to treatment was based on international consensus criteria: complete (CR; normal Hb or Hb ≥12 g/dL, with resolution of hemolysis); partial (PR; Hb >10 but <12 g/dL or Hgb >12 g/dL without resolution of hemolysis); or no response (neither CR nor PR). Results: Eleven of the 15 patients (73%) were found to have polyclonal antibodies, while four patients (27%) had mono/oligoclonal WAIHA antibodies. Of these four patients with restricted clonality, two patients had monoclonal antibodies (Figure 1 shows one of the monoclonal WAIHA antibodies identified). There was no significant difference in demographic composition, clinical findings, or treatment received in both groups (Table 1). 66.7% (n=10) of patients were male, and the mean age (SD) was 62.5 years (24.5). Two pediatric patients, ages 10 and 16, were included. Concomitant lymphoproliferative disease was present in 46.7% of the patients. The three most common treatments were steroids (100%), rituximab (93.3%), and splenectomy (13.3%). 40.0% of patients had died at the time of this analysis with no difference between the groups. The number of treatments received were similar between the mono/oligoclonal and polyclonal groups. After the first line of treatment, 100% and 77.8% of the mono/oligoclonal and polyclonal groups respectively achieved PR or CR (P=0.31). Additionally, 100% of the mono/oligoclonal group and 54.5% of the polyclonal group were in PR or CR by their most recent CBC (P=0.10). 50.0% and 54.5% of the mono/oligoclonal group and polyclonal groups respectively relapsed at least once (P=0.25). In those who relapsed, there was an average of 1.0 relapse events in the mono/oligoclonal group compared to 1.7 relapse events in the polyclonal group (P=0.32). Conclusion: This study demonstrates, for the first time, that monoclonal and oligoclonal antibodies can mediate warm autoimmune hemolytic anemia. While these data suggest no difference in clinical outcomes or disease course between patients with mono/oligoclonal vs. polyclonal anti-RBC antibodies, a key limitation is the small sample size.

Our group has reported that transient CD19 antigen masking of leukemic cells with the CD19 monoclonal antibody (mAb), tafasitamab, resulted in reduction of CD19 chimeric antigen receptor T cell (CART19) apoptosis, enhanced CAR T proliferation, improved anti-tumor activity, diminished tumor pyroptosis, and reduced severity of cytokine release syndrome (CRS) in preclinical models. In this report, we aimed to study the mAb characteristics responsible for these observations. Specifically, we hypothesized that binding affinity of CD19 mAbs might affect the subsequent activation and apoptosis of CART19 as well as tumor cell pyroptosis in preclinical models. To test our hypothesis, we first determined the affinities of the CD19-targeting antibodies tafasitamab, HD37 and FMC63 to recombinant human CD19 protein. Tafasitamab demonstrated the strongest binding affinity among the three tested antibodies [tafasitamab ( K D: 1.0±0.2 nM), HD37 ( K D: 4.1±0.3 nM), FMC63 ( K D: 3.4±1.2 nM)]. Next, we studied CART19 apoptosis in the presence of these CD19 mAbs with different binding affinities. Here, we co-cultured CART19, CD19 + mantle cell lymphoma JeKo, and 400 μg/mL of CD19 mAb for 1 hour and measured CD3 + 7-AAD - Annexin V + T cells with flow cytometry. The number of apoptotic CART19 cells was significantly reduced by tafasitamab co-treatment compared to HD37, FMC63, or control IgG [control IgG (mean = 19.1, standard deviation (SD) = 0.8), tafasitamab (mean = 13.1, SD = 2.9), HD37 (mean = 17.8, SD = 0.8), FMC63 (mean = 18.4, SD = 1.0)]. We also performed an in vitro high mobility group box 1 (HMGB1) assay to assess if different affinities of the CD19 mAbs affect tumor cell pyroptosis triggered by CART19. Here, we co-cultured CART19, CD19 + JeKo, and 400 μg/mL of CD19 mAb for 24 hours and assessed HMGB1 on JeKo. In analogy to the apoptosis data, tafasitamab was the only mAb that prevented tumor cell pyroptosis, indicated by reduced HMGB1 expression (Fig 1A). Given these results, we aimed to study the impact of CD19 occupancy with different CD19 mAbs prior to CART19 infusion on tumor pyroptosis in vivo. We utilized a mouse model that mimics CAR T toxicities. Immunocompromised NSG mice were inoculated via tail vein with 5x10 6 leukemic cells derived from patients with relapsed/refractory acute lymphoblastic leukemia. Two weeks later (day -7), tumor burden was assessed by peripheral blood sampling. Leukemic cells were defined as murine CD45 -, human CD45 + CD19 + CD20 + via flow cytometry, and volumetric absolute number was calculated. Mice were then randomized according to tumor burden to receive IgG control [Group 1 (G1)], tafasitamab (G2), HD37 (G3), or FMC63 (G4) (5 mg/kg, ip) at day -7. CRS was determined by weight loss, development of motor weakness, poor body posture, and elevated serum cytokine levels. At day 0, all mice received 3x10 6 luciferase + CART19 via tail vein. Mice were monitored with daily bioluminescent imaging (BLI) to track and quantify CART19 expansion and physical assessment to detect toxicity. BLI on day 4 revealed that CART19 expansion in the tafasitamab-treated group (G2, mean = 1x10 7, SD = 2.3x10 7) was significantly lower than in the control group (mean = 3.5x10 7, SD = 9.3x10 7), the HD37-treated group (G3, mean = 4x10 7, SD = 6.6x10 7), and the FMC63-treated group (G4, mean = 2x10 7, SD = 3.59x10 7), [G1 vs G2: 95% confidence interval (CI) 3.5x10 7 to 1.1x10 8, p <0.0001, G1 vs G3: 95% CI -7.5x10 7 to -1.1x10 8, p=0.005, G1 vs G4: 95% CI -4.5x10 7 to 1.9x10 7, p=0.71] indicating that tafasitamab pretreatment significantly reduced early activation of CART19 compared to IgG control and HD37 in this model. Mice were bled on day 4 to analyze plasma cytokine. Tafasitamab-treated mice (G2) secreted significantly less IFNγ (Fig 1B), IL10 (Mean G1: 78, G2: 2.52, G3: 12.2, G4: 13.9 pg/mL), MDC (Mean G1: 4072, G2: 445, G3: 921, G4: 1364 pg/mL), and MIP1β (Mean G1: 283, G2: 59.1, G3: 148, G4: 123 pg/mL) compared to G1, G3, and G4. Satellite mice were euthanized on day 1 and spleens were harvested. Western blot analysis of splenocytes revealed that G1, 3, and 4 showed significantly lower expression of apoptosis-inducing protein gasdermin E on leukemic cells compared to G2, indicating reduced tumor pyroptosis in G2. In summary, application of the high affinity mAb tafasitamab prior to CART19 resulted in reduced CAR T apoptosis, diminished tumor cell pyroptosis, and reduction of CRS compared to lower affinity CD19 mAbs in preclinical models.

We identified a patient with a recently described syndrome, monoclonal gammopathy of thrombotic significance (MGTS; Blood. 2023 Apr 6;141(14):1772-1776), and three additional patients with high 4Ts scores who were consistently negative in the PF4-polyanion HIT ELISA (Lifecodes PF4 IgG, Immucor). The MGTS patient, with a monoclonal IgG kappa, had recurrent thrombosis, and transient mild thrombocytopenia (nadir in the low 100s) over a period of years, and was noted to have a HIT ELISA optical density (OD) of 0.117. The three HIT patients experienced thrombocytopenia and thrombosis with ELISA ODs of 0.37, 0.18 and 0.20. All patients were positive in platelet-activation (functional) testing demonstrating PF4 dependence and inhibited with both high concentrations of heparin (100U/mL) and the FcγRIIa blocking monoclonal antibody (mAb) IV.3, consistent with the presence of anti-PF4 antibodies in the patient samples ( data not shown). Because the serologic profile of these patients suggested a novel HIT subtype characterized by negative HIT ELISA results, we undertook a functional platelet assay screen of 500 consecutive HIT ELISA-negative patients tested in our diagnostic laboratory. Research studies were approved by the Institutional Review Board of Mayo Clinic. Of the 500 patients tested, 20 (4.0%) stimulated platelet activation in the PF4-dependent P-selectin Expression Assay (PEA ≥19%; data not shown). To exclude non-specific activation that can be induced by HLA antibodies, additional testing was performed to evaluate whether platelet activation was PF4-dependent using buffer or PF4-treated platelets. Three patients demonstrated PF4-dependent activation that was inhibited by high heparin concentrations and mAb IV.3, as would be expected for HIT antibodies (ELISA-negative HIT, EN-HIT1-3; Figure 1). On retrospective review, all three patients thus identified had clinical histories and courses consistent with high clinical probability for HIT. To develop tools to study this new class of antibodies, monoclonal HIT antibodies were generated in mice after PF4/heparin immunizations and screened using a novel approach that included simultaneous testing of hybridoma supernatants in the PEA and PF4-polyanion ELISA. This strategy was designed to enabled us to identify mAbs that activated platelets (in the PEA), but that did not bind antigenic targets in the HIT ELISA. A novel mAb 431.4 that was developed using this approach, and the well-characterized HIT-like mAb KKO were evaluated for their abilities to both bind PF4-polyanion targets in ELISA, and to activate PF4-treated platelets in the PEA (Figure 2). In significant contrast to KKO, mAb 431.4 did not bind PF4/polyanion complexes, however, like KKO, strongly activated platelets in a PF4-dependent manner. Activation induced by both KKO and mAb 431.4 was inhibited by high heparin concentrations, as would be expected for HIT antibodies. In vivo murine studies using this novel mAb are pending at this time. In summary, the non-canonical diagnostic profile of HIT ELISA-negative but platelet activation-positive antibodies were found at a relatively high incidence in HIT-suspected patients (0.6%; 3 of 500 ELISA-negative patients). These findings have important diagnostic/management implications and suggest a re-evaluation of ASH's HIT management guidelines as noted in recommendations 2.7 and 2.8 of that document (“In patients with an intermediate (Recommendation 2.7) or high (Recommendation 2.8)-probability 4Ts score and a negative immunoassay, the ASH guideline panel recommends discontinuation of the non-heparin anticoagulant and resumption of heparin, if indicated ”; Blood Adv (2018) 2 (22): 3360-3392). Figure 1. A subset of ELISA-negative HIT-suspected patients harbor anti-PF4 platelet-activating antibodies. Three ELISA-negative (EN) HIT patient samples from a screen of 500 samples demonstrate PF4-dependent platelet activation and inhibition with high heparin concentration and mAb IV.3 incubation. PBS, phosphate buffered saline; IC, isotype control; HDH- high dose heparin (100U/mL). Figure 2. Murine mAb 431.4 serologically mimics pathogenic ELISA-negative HIT/MGTS antibodies. ELISA and PEA testing was performed using the “HIT-like” mAb KKO, novel mAb 431.4, or an isotype control (IC) antibody. PVS-polyvinyl sulfonate (polyanion used in the Immucor IgG ELISA assay).

Abstract Background The BioFire® FilmArray® Gastrointestinal Pathogen Panel (GIPP) tests for 22 potential pathogens in stool, including Clostridioides difficile. C. difficile nucleic acid is detected via nested polymerase chain reaction (PCR) targeting toxin A (TcdA) and/or toxin B (TcdB) genes. While PCR-based diagnostic methods are highly sensitive, they lack specificity to differentiate colonization from C. difficile infection (CDI). Mayo Clinic Arizona (MCA) uses enzyme immunoassay (EIA) testing for toxin A/B and glutamate dehydrogenase antigens with reflex to PCR for discordance as the primary algorithm for diagnosing CDI; however, GIPP PCR testing circumvents this algorithm. When used in patients with low pretest probability for CDI, clinical false positives can lead to inappropriate diagnosis and treatment of colonization. GIPA Results in the Electronic Medical Record Pre-Intervention MCA Standalone C. difficile Testing Algorithm Abbreviations – EIA, enzyme immunoassay; GDH, glutamate dehydrogenase; NAAT, nucleic acid amplification test Methods We convened a multidisciplinary quality improvement team to reduce the detection and treatment of C. difficile colonization secondary to GIPP test results. Surveys were sent to providers and nurses as part of a formal gap analysis and included questions around ordering patterns, knowledge of appropriate tests, and associated costs. Baseline data was compiled from GIPP orders in MCA from 1/1/2022-9/30/2022. Results There were 1978 GIPP orders for 1694 patients during the study period (∼7 GIPPs/day). Most orders (65%) originated from ambulatory settings. The most common positive target was C. difficile (355/723, 49%), which resulted in treatment for suspected CDI in 94.9% (337/355) of cases. Of the 49 providers who responded to the survey, 86% ordered the GIPP at least monthly. Testing for C. difficile was the 2nd most common reason for ordering the GIPP. Most providers underestimated the cost of the panel and were unfamiliar with the differences between the GIPP and EIA testing algorithm. Nurses sought clarification on stool samples suitable for GIPP testing. Following education and modifications to ordering in the electronic health record, the decision was made to remove the C. difficile target from the GIPP. Provider Understanding of C. difficile Testing Methods GIPA Results in the Electronic Medical Record Post-Intervention Conclusion Multiplex PCR panels are comprehensive but require careful implementation to facilitate diagnostic stewardship. Facilities with robust standalone C. difficile testing algorithms may consider removing the C. difficile target from the GIPP. Disclosures All Authors: No reported disclosures