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Immune thrombocytopenia (ITP) is traditionally considered an antibody-mediated disease. However, a number of features suggest alternative mechanisms of platelet destruction. In this study, we use a multi-dimensional approach to explore the role of cytotoxic CD8+ T cells in ITP. We characterised patients with ITP and compared them to age-matched controls using immunophenotyping, next-generation sequencing of T cell receptor (TCR) genes, single-cell RNA sequencing, and functional T cell and platelet assays. We found that adults with chronic ITP have increased polyfunctional, terminally differentiated effector memory CD8+ T cells (CD45RA+CD62L-) expressing intracellular interferon-g, tumour necrosis factor-a, and Granzyme B defining them as TEMRA cells. These TEMRA cells expand when the platelet count falls and show no evidence of physiological exhaustion. Deep sequencing of the T cell receptor showed expanded T cell clones in patients with ITP. T cell clones persisted over many years, were more prominent in patients with refractory disease, and expanded when the platelet count was low. Combined single-cell RNA and TCR sequencing of CD8+ T cells confirmed that the expanded clones are TEMRA cells. Using in vitro model systems, we show that CD8+ T cells from patients with ITP form aggregates with autologous platelets, release interferon-g and trigger platelet activation and apoptosis through TCR-mediated release of cytotoxic granules. These findings of clonally expanded CD8+ T cells causing platelet activation and apoptosis provide an antibody-independent mechanism of platelet destruction, indicating that targeting specific T-cell clones could be a novel therapeutic approach for patients with refractory ITP.

Background Myelodysplastic syndromes (MDS) and MDS/MPNs are heterogeneous disorders with various combinations of mutations and cytogenetic abnormalities associated with distinct clinical phenotypes, prognosis, and implications for targeted therapies. We previously demonstrated that ex vivo drug sensitivity screening (DSS) identified subgroups of MDS and MDS/MPNs with differing patterns of sensitivity to various drug classes including hypomethylating agents (HMAs), kinase inhibitors, and other small molecules. In this study, we used hierarchical clustering to identify MDS and MDS/MPN genomic subgroups in a large single-center cohort. We then examined associations between these genomic subgroups and ex vivo sensitivity to various drug classes in a cohort of patients with ex vivo DSS. Methods Patients: We identified 294 patients with MDS or MDS/MPNs who had cytogenetics and HemeSTAMP NGS panel (164 genes) performed at Stanford between June 2018 and June 2021. A separate, partially overlapping cohort of 60 patients had ex vivo DSS as described below. Genomic clusters: We used a hierarchical Dirichlet Process (HDP), incorporating mutations and cytogenetics, to identify genomic subgroups. We included pathogenic and likely pathogenic variants with VAF >2% and excluded variants of unknown significance. Ex vivo DSS: Fresh bone marrow aspirates and peripheral blood specimens were RBC-lysed and resuspended in serum-free media with cytokines (Spinner et al, Blood Adv 2020;4(12):2768-78). Samples were plated in 384-well microtiter plates and screened against a collection of up to 74 drugs and 36 drug combinations in triplicate. Specimens were treated for 72 hours and assayed using flow cytometry to assess for blast viability. Statistical analysis: An HDP model was trained on the cohort of 294 patients. To tune the hyperparameters of the model, the log-likelihood of the test data was optimized using cross validation combined with Gaussian Process Bayesian optimization. Inference using the trained model was performed on 60 patients with ex vivo DSS producing a genomic component distribution for each patient. Jensen-Shannon distance was then computed between each pair of patients using their genomic component distributions. Patients were then clustered via agglomerative clustering (average linkage and using a maximum distance cutoff of 0.5) using this distance matrix. Ex vivo sensitivity to drug classes was then compared across clusters using ANOVA on drug sensitivity per drug class averaged over each patient. Results Patient characteristics: Among all 294 patients, the median age was 73 years, 78% had MDS, 16% had CMML, and 6% had other MDS/MPNs. 45% had >5% blasts and 53% had higher risk disease with IPSS-R >3.5. 94% had at least 1 mutation or cytogenetic abnormality with a median of 2 mutations (range 0-7). Among the 60 patients with ex vivo DSS, the median age was 77 years, 82% had MDS, 18% had CMML or other MDS/MPN, 55% had >5% blasts, and 67% had higher risk disease. Genomic subgroups and clusters: An HDP model trained on all 294 patients identified 16 genomic subgroups. Applying these genomic subgroups to the 60 patients with ex vivo DSS, we identified 12 genomic clusters, of which 6 clusters were most common: cluster 0 (enriched for RUNX1/BCOR mutations, N=6), cluster 1 (enriched for TET2/SRSF2/ASXL1, N=13), cluster 3 (enriched for DNMT3A, N=8), cluster 6 (enriched for KRAS/NRAS, N=5), cluster 7 (enriched for STAG2/ASXL1, N=6), and cluster 10 (enriched for TP53/complex cytogenetics, N=5). Associations between genomic clusters and drug sensitivity: Ex vivo drug sensitivity for 60 patients, organized by genomic cluster, is shown in Figure 1A. Ex vivo sensitivity to various drug classes is shown for the most common clusters in Figure 1B. Cluster 10 (enriched for TP53/complex cytogenetics) demonstrated greater ex vivo sensitivity to proteasome inhibitors (p=0.018). In Cluster 6 (enriched for NRAS/KRAS), there was a trend towards greater ex vivo resistance to HMAs and PARP inhibitors (p=0.1 for both comparisons). Conclusions Hierarchical clustering identified distinct genomic subgroups of MDS and MDS/MPNs, which displayed differing ex vivo sensitivity to various drug classes. While the small sample size limits our analysis, these associations between genotype and drug sensitivity phenotype are hypothesis generating and have potential implications for personalized therapy in MDS and MDS/MPNs. Figure 1 Figure 1. Disclosures Spinner: Notable Labs: Honoraria. Schaffert: Notable Labs: Consultancy, Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Santaguida: Notable Labs: Consultancy, Current holder of individual stocks in a privately-held company. Kita: Notable Labs: Current Employment, Current holder of stock options in a privately-held company. Aleshin: Notable Labs: Consultancy. Greenberg: Notable Labs: Research Funding.

Background: Characterization of T-cell receptor (TCR) diversity and dynamics is increasingly critical to understanding therapeutic immune responses targeting tumors. Current TCR profiling methods generally require invasive tissue biopsies that capture a single snapshot of immune activity or are limited by the sheer diversity of the circulating TCR repertoire. In theory, T-cells with the greatest turnover could best reflect pivotal immune dynamics from both circulating and tissue-derived compartments, including non-circulating tissue-resident memory T-cells (Trm). To noninvasively capture such responses in the blood, we developed and benchmarked a high-throughput TCR profiling approach using plasma, optimized for the fragmented nature of cfDNA and the non-templated nature of rearranged TCRs. We then applied this method for residual disease monitoring in mature T-cell lymphomas (TCL) without circulating disease and for characterizing immune dynamics after anti-CD19 chimeric antigen receptor (CAR19) T-cell therapy of B-cell lymphomas with axicabtagene ciloleucel. Methods: We developed SABER (Sequence Affinity capture & analysis By Enumeration of cell-free Receptors) as a technique for TCR enrichment and analysis of fragmented rearrangements shed in cfDNA and applied this method using Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq). We used SABER to profile a total of 381 samples (300 cfDNA and 81 PBMC samples) from 75 lymphoma patients and 18 healthy controls. After mapping sequencing reads (hg38) to identify candidate rearrangements within TCR loci, unique cfDNA fragments were resolved by a novel strategy to define consensus of unique molecular identifiers clustered by Levenshtein distances, followed by CDR3-anchoring for enumeration of final receptor clonotypes. SABER thus leverages information from fragmented TCRs, a critical requirement for cfDNA, to make V gene, CDR3, and J gene assignments after deduplication-mediated error-correction. We benchmarked SABER against established amplicon-based TCR-β targeted sequencing (LymphoTrack, Invivoscribe) and repertoire analysis methods (MiXCR; Bolotin et al, 2015 Nature Methods) when considering both cfDNA and PBMC samples from healthy adults and TCL patients. We assessed SABER performance for tracking clonal molecular disease in patients with mature TCLs from both cellular and cell-free circulating compartments (n=9). Malignant TCL clonotypes were identified in tumor specimens using clonoSEQ (Adaptive Biotechnologies). Finally, we evaluated TCR repertoire dynamics over time in 66 DLBCL patients after CAR19 T-cell therapy. Results: SABER demonstrated superior recovery of TCR clonotypes from cfDNA compared to both amplicon sequencing (LymphoTrack, Invivoscribe) and hybrid-capture methods when enumerating receptors using MiXCR (Fig. 1A). When applied to blood samples from TCL patients, SABER identified the malignant clonal TCR-β rearrangement in 8/9 (88.9%) cases, with significantly improved detection in cfDNA (p=0.015, Fig. 1B). Specifically, tumoral TCR clonotype was detectable only in cfDNA in 6 cases (75%), cfDNA-enriched in 1 case (12.5%), and detectable only in PBMCs in 1 case (12.5%). We applied SABER to monitor TCR repertoire dynamics in cfDNA after CAR T-cell therapy of patients with relapsed/refractory DLBCL and observed increased T-cell turnover and repertoire expansion (greater total TCR-β clonotypes) (Fig. 1C). As early as 1-week after CAR19 infusion, TCR repertoire size was significantly correlated both with cellular CAR19 T-cell levels by flow cytometry (p=0.008) as well as with retroviral CAR19 levels in cfDNA (p=2.20e-07) suggesting faithful monitoring of CAR T-cell activity (Fig. 1D). TCR repertoire size one month after infusion was significantly associated with longer progression-free survival (HR 0.246, 95% CI 0.080-0.754, p=0.014). Conclusions: SABER has a favorable profile for cfDNA TCR repertoire capture when compared to existing methods and could thus have potential broad applicability to diverse disease contexts. Given the higher abundance of lymphoma-derived TCRs in cfDNA than intact circulating leukocytes, SABER holds promise for monitoring minimal residual disease in T-cell lymphomas. This approach also holds promise for monitoring T-cell repertoire changes including after CAR T-cell therapy and for predicting therapeutic responses. Disclosures Kurtz: Genentech: Consultancy; Foresight Diagnostics: Other: Ownership; Roche: Consultancy. Kim:Corvus: Research Funding; Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Elorac: Research Funding; Forty Seven Inc: Research Funding; Galderma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Horizon Pharma: Consultancy, Research Funding; Innate Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kyowa-Kirin Pharma: Research Funding; Medivir: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; miRagen: Research Funding; Neumedicine: Consultancy, Research Funding; Portola: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Solingenix: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Trillium: Research Funding. Mackall:Lyell Immunopharma: Consultancy, Current equity holder in private company; BMS: Consultancy; Allogene: Current equity holder in publicly-traded company; Apricity Health: Consultancy, Current equity holder in private company; Nektar Therapeutics: Consultancy; NeoImmune Tech: Consultancy. Miklos:Kite-Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Adaptive Biotech: Consultancy, Other: Travel support, Research Funding; Juno-Celgene-Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Novartis: Consultancy, Other: Travel support, Research Funding; Allogene Therapeutics Inc.: Research Funding; Pharmacyclics: Consultancy, Other: Travel support, Patents & Royalties, Research Funding; Janssen: Consultancy, Other: Travel support; Miltenyi Biotec: Research Funding. Diehn:Varian Medical Systems: Research Funding; Illumina: Research Funding; Roche: Consultancy; AstraZeneca: Consultancy; RefleXion: Consultancy; BioNTech: Consultancy. Khodadoust:Seattle Genetics: Consultancy; Kyowa Kirin: Consultancy. Alizadeh:Janssen: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Chugai: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy; Pfizer: Research Funding.

Background: PTT and anti-Xa activity are used for monitoring unfractionated heparin (UFH) and dosage adjustment in hospitalized patients. There is no consensus on how to best use these tests, including whether to rely on one over the other or how to best combine their use. Outcome data supporting the use of any approach are lacking. Given the frequency of heparin-associated adverse events, hospital readmissions, and mortality, this critical knowledge gap impedes efforts to improve patient safety and optimize quality of care. A pilot retrospective analysis cohort of hospitalized patients on UFH (n=539) revealed significant discordance between the two measures. Patients with at least two consecutively high PTT to anti-Xa values had increased 21-day major bleeding (9% vs 3%; p = 0.0316) and 30-day mortality (14% dead vs 5% dead at 30 days; p = 0.0202) compared with patients with consistently concordant values. As a follow-up study to validate and extend these observations, EHR data was extracted from adult inpatients treated with UFH anticoagulation over a 7-year period from 2011-2017. Methods: Our study population included patients who received paired aPTT and anti-Xa lab tests at Stanford Hospital between 1/1/2011 to 12/31/2017. We extracted all labs, diagnoses codes, procedure codes, medications, demographics, and mortality information. In addition to the directly extracted features we computationally calculated the Charlson comorbidity score and also defined if a paired test was part of a sequential set of paired tests within a hospital stay. Using R and Python we converted the data extract into a data frame for downstream analysis. All statistical analysis was completed in R (version 3.3.3, R foundation for statistical computing). For classification and regression tree analysis (CART) all covariates prior to the simultaneous lab draw were used. Results: 9467 patients with 56,775 paired PTT and anti-Xa values were included. In patients treated with UFH infusions, the PTT and anti-Xa were highly discordant (r2 = 0.356) with only 6.8% of values falling into the defined therapeutic range for both PTT (50-100sec) and anti-Xa (0.3-0.7U/mL) (Figure 1). For subsequent analysis we focus on the second paired-test obtained for a patient while on UFH and an anti-Xa < 0.2. As the discordance between anti-Xa and PTT increased, the 30 day all-cause mortality was 23.2% and bleeding risk was 8.5% (P141.7 and anti-Xa < 0.33 (p152.5, regardless of other factors. Laboratory values significantly correlating with increased severity of PTT and anti-Xa discordance included total bilirubin (trending from 0.6 to 1.1, P Conclusions: Patients treated with UFH with PTT above therapeutic range >152.5 sec) were at increased risk for early mortality (2d) Patients who were treated with UFH and exhibited discordance in PTT and anti-Xa measurements (in the form of higher PTT than expected for an anti -Xa value) were at increased risk of mortality (30d and 1 year) and bleeding (30d). Based on these results, we propose that PTT/anti-Xa discordance in patients on UFH is a highly significant marker for increased risk of bleeding and death. Laboratory correlates included increase in total bilirubin and AST, which may impact heparin metabolism, and a decrease in platelet count. This exaggerated anticoagulant effect for a given heparin concentration may represent a defect in homeostatic balance increasing anticoagulation risk. These data have implications for patient management. A PTT significantly greater than expected for a given anti-Xa result is a predictor for increased mortality and bleeding risk, providing an opportunity for revisiting risk-benefit estimates for anticoagulation. If anticoagulation is continued, CART analysis in this large cohort suggests that the heparin infusion in such cases might better be titrated to the PTT therapeutic range rather than to anti-Xa levels. Figure 1 Disclosures No relevant conflicts of interest to declare.

Background Next-generation sequencing (NGS) panels have created an unprecedented opportunity to interrogate a broad array of variants in a multiplex fashion. Few data have demonstrated how NGS testing impacts diagnosis and treatment decisions. In this retrospective analysis, we evaluated the clinical application of NGS myeloid panels (MP) in patients (pts) evaluated by physicians in the Stanford Division of Hematology. Methods The study was approved by the Stanford University IRB. The cohort consisted of 1,015 pts ≥18 years of age (median 66; range 18-96 years; 51% female). A total of 1,213 MPs obtained from peripheral blood (n=568) or bone marrow (n=645) from March 2017 to June 2018 were analyzed at Stanford (n=761) using the 54-gene TruSight® Myeloid Sequencing Panel (Illumina, San Diego, CA) or Oregon Health & Science University (n=452) using the GeneTrails® Hematologic Malignancies 76-Gene Panel. Electronic medical records were reviewed from t-3 to t+9 months from when MPs were obtained to assess physician reasoning for MP acquisition, documentation of results, and how results were clinically applied. We defined three categories of MP acquisition: 1) diagnostic clarification, 2) prognostication and/or management guidance, or 3) minimal residual disease (MRD) monitoring. We analyzed changes in clinical management, including addition of targeted or non-targeted therapeutics, clinical trial eligibility, or other practice recommendations. Results Of the 1,213 MPs, 882 (73%) demonstrated at least one pathogenic/ likely pathogenic variant (median 2; range 1-8). Median turn-around-time was 18 days (range 7-31) and average cost was $2,600. Of all MPs, 462 (38%) were obtained for diagnostic clarification, 732 (60%) for prognostication / management guidance in pts with known myeloid (n=701) or lymphoid (n=31) neoplasms, and 19 (2%) for MRD monitoring, although the assay was not designed for this indication. MPs were ordered to clarify a diagnosis for the following reasons: unexplained cytopenia(s) (n=199), molecular profiling for a suspected hematolymphoid neoplasm (n=156), unexplained -cytosis (n=86), and testing for other lab abnormalities (e.g. elevated serum tryptase, paraproteinemia) (n=18), or signs (e.g. splenomegaly, splenic vein thrombosis)(n=3). A pathogenic/likely pathogenic variant was found in 294 (64%) pts, confirming or establishing the presence of a myeloid (n=266) or lymphoid neoplasm (n=7) or resulting in a diagnosis of CHIP (n=9) or CCUS (n=12) in pts who did not meet 2016 World Health Organization diagnostic criteria for a hematolymphoid neoplasm (Fig. 1). Of the 732 MPs ordered for prognostication/ management guidance, 272 MPs (37%) were obtained in the initial workup of non-APL AML pts. The frequency of favorable (21%), intermediate (55%), and adverse risk (24%) genetics according to the European LeukemiaNet stratification in non-APL AML is shown in Figure 2a, which also denotes the frequency of favorable (9%) or adverse risk (70%) variants in MDS, and adverse risk variants in MF (52%), MDS/MPN (68%), and advanced systemic mastocytosis (44%). Among MPs obtained for prognostication/ management guidance, 163 (22%) led to a modification in clinical practice, divided between 132 (18%) which led the physician to change therapy (e.g. FDA-approved targeted therapy, clinical trial, or FDA-approved therapy, such as hypomethylating agents in MDS) and 31 (4%) resulted in a non-therapeutic change (e.g. expedited HSCT referral or more frequent follow-up)(Fig. 2b). 87 pts with a myeloid neoplasm had 184 repeat MPs for relapsed/refractory disease (n=52), transformation to higher-risk MDS or AML (n=45) or progressive cytopenias (n=87). Among these MPs, 38 (21%) identified a new pathogenic/likely pathogenic variant of which 29% (n=11) led to either a) initiation of targeted therapy with enasidenib in relapsed IDH2+ AML (n=3) or midostaurin for secondary FLT3+ AML from MDS (n=3), or b) consideration for a clinical trial with a splicing modulator for MDS characterized by a splicing variant (n=5). Conclusion In our academic hematology practice, two-thirds of MPs ordered for diagnostic clarification identified a pathogenic/likely pathogenetic variant that helped to confirm or establish a new diagnosis of a hematolymphoid neoplasm, CHIP, or CCUS. In addition, approximately 20% of MPs ordered for prognostication/ management guidance led to a change in clinical practice. Disclosures Gotlib: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Blueprint Medicines: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Promedior: Research Funding; Pharmacyclics: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Allakos: Honoraria, Membership on an entity's Board of Directors or advisory committees; Deceiphera: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Castleman disease is a rare lymphoproliferative disorder known to represent at least four distinct clinico-pathologic sub-types. Large advancements in our clinical and histopathologic description of these diverse diseases have been made, resulting in subtyping based on degree of nodal involvement (unicentric versus multicentric) and according to viral infection (human herpes virus 8 - HHV8 and Human Immunodeficiency Virus - HIV). However, we continue to lack a foundational understanding of the biologic mechanisms driving at least a subset of patients with these diseases. In recent years, significant molecular and genetic abnormalities that may be associated with this disease have been described; yet we are still unclear as to a unified vision of what loci and genes are involved in at least a subset of cases of Castleman disease. In our study we performed a meta-analysis of all cases (unicentric and multicentric) of Castleman disease described in literature to date, and correlate cytogenetic, molecular and genetic abnormalities with disease subtypes, phenotypes and clinical outcomes. We further performed targeted DNA deep sequencing and RNA-sequencing on a cohort of Castleman Disease cases. Our results demonstrate that genetic abnormalities in the MAPK-pathways, PI3K-pathway and Interleukin signaling are frequent in unicentric Castleman disease, whereas abnormalities in DNA methylation are seen more commonly in idiopathic multicentric Castleman disease, along with abnormalities in MAPK-pathways. We conclude that UCD and iMCD may share similar pathways of MAPK pathogenesis but can also be distinguished by cytokine and other DNA modification pathways that differ. Disclosures Ohgami: Agilent technologies: Other: received support/funding.

Background: Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by increased platelet destruction and impaired platelet production. Abnormal T cell immunity plays a critical role in the pathogenesis of ITP. Analysis of T cell receptor (TCR) repertoire can identify specific T cell population proliferations in response to antigenic stimulation. No systematic next-generation sequencing (NGS)-based TCR repertoire characterization of pediatric ITP patients has been reported to date. We herein report our comprehensive analysis of TCR-β repertoires of peripheral blood samples from a cohort of pediatric patients with ITP. Method: Pediatric patients from Lucile Packard Children's Hospital (Stanford Children's Health) with newly diagnosed ITP (n=11), chronic ITP (n=8), resolved from ITP (n=3), and healthy individuals (n=4) were consented to be included in the study. Total RNA was extracted from peripheral blood samples of the study subjects and reverse transcribed into cDNA. Completely rearranged TRB gene locus was amplified and used for library preparation, which were subsequently sequenced on the Illumina MiSeq platform. The sequencing reads were analyzed with IMGT (ImMunoGeneTics)/HighV-QUEST tools. Further analysis was performed to evaluate TRB repertoire diversity, V-J gene segment usage, clonotype sharing among patient samples, clonality pattern, predominant clonotype frequency changes in different disease state, and sequence similarity among expanded clonotypes. Result: The normalized clonotype counts and the calculated TRB repertoire diversity indices (Shannon and Simpson indices) did not reveal significant difference among newly diagnosed ITP, chronic ITP and healthy control groups. In terms of the V-J gene segment usage among the top 200 clonotypes (as defined by CDR3 amino acid sequences), newly diagnosed ITP group used TRBV12-3 significantly more frequently, and TRBV6-4 significantly less frequently, compared to the healthy control group. The means of overlap coefficient, reflective of TRB clonotype sharing, for newly diagnosed ITP, chronic ITP and healthy control groups are 0.0045 (range 0.005-0.008), 0.0057 (range 0-0.016), 0.0041(range 0.004-0.005), respectively. Chronic ITP patients had significantly more overlap in TRB clonotypes than healthy donors. No common or "public" clonotypes shared within each group were identified. The nucleotide sequence and CDR3 amino acid sequence of dominant TRB clonotypes in ITP patients were compared, and the percent similarity ranged from 30.18% to 88.26% for the former, and from 16.9% to 36.04% for the latter. Conclusion: We performed comprehensive analysis of TCR repertoire of peripheral blood samples from pediatric ITP patients by next-generation sequencing. TCR repertoire diversity at different disease states was analyzed. Differences in V-J gene segment usage and clonotype sharing among patients were observed. These findings provide unique insights into the potential pathophysiology of T cell immunity in patients with ITP. Disclosures No relevant conflicts of interest to declare.

Background Myelodysplastic syndrome (MDS) patients who are refractory to hypomethylating agents (HMAs) have a poor prognosis with median survival Methods Study design: We performed a prospective feasibility study in 21 patients with HMA-refractory MDS enrolled at Stanford University from April 2018 through March 2019. All patients had a baseline bone marrow (BM) biopsy with BM aspirate and peripheral blood (PB) samples sent for mutation testing (596-gene panel, Tempus, Chicago, IL) and ex vivo DSS (Notable Labs, Foster City, CA). Ex vivo DSS: BM aspirate and PB specimens were RBC-lysed and re-suspended in serum-free media with cytokines. Samples were plated in 384-well microtiter plates and screened against FDA-approved and investigational drugs (up to 76) and drug combinations in triplicate. Specimens were treated for 72 hours and assayed using high-throughput, multi-parametic flow cytometry for cytotoxicity and differentiation (Blood 2016;128:5206). In silico CBM: Genomic data were input into a computational biology model (Cell Works Group, San Jose, CA) to generate protein network maps for each patient. Mathematical modeling of MDS cell proliferation or inhibition was simulated for each patient and used to calculate drug efficacy scores for numerous agents (Leuk Res 2017;52:1-7). Study endpoints: Once the gene panel, ex vivo DSS, and in silico CBM results were available, we (M.A.S., A.A., J.Z., P.L.G.) met for a molecular tumor board (MTB) to review the data and provide personalized treatment recommendations for each patient. The primary endpoint was the feasibility of providing personalized recommendations within an actionable timeframe (≤30 days). Secondary endpoints included concordance between the ex vivo and in silico assays and the accuracy of our MTB recommendations in predicting clinical responses in vivo. Results The median age of the patients was 76 years (range 55-87) and 71% were male. Seventeen patients had MDS, 3 had an MDS/MPN disorder, and 1 patient had progressed to AML. 76% had higher risk disease by IPSS-R, 57% had excess blasts, and 52% had adverse cytogenetics or mutations. Patients had a median of 2 pathogenic mutations (range 0-6) with the most common including TET2, ASXL1, STAG2, DNMT3A, RUNX1, and SRSF2. The median turnaround time for results of the gene panel, ex vivo DSS, and in silico CBM were 14.5, 15, and 20 days, respectively. The median turnaround time to our MTB was 27 days (range 20-32 days). MTB recommendations varied widely among patients and encompassed various drug classes including targeted therapies (venetoclax, sorafenib, lenalidomide, ruxolitinib, midostaurin, everolimus), cytotoxic agents (cytarabine, fludarabine), differentiative agents (calcitriol, ATRA), HMAs, and androgens (danazol) as single agents or in combination. The ex vivo and in silico assays were highly concordant, particularly in predicting sensitivity to HMAs and venetoclax. Eight patients received treatment per our MTB recommendations. Of these 8 patients, 6 (75%) responded to the recommended therapy and 2 (25%) had stable disease. Two responding patients were bridged to allogeneic hematopoietic cell transplantation (HCT). The remaining patients elected for best supportive care (N=5), hospice (N=3), other approved therapies (N=3), a clinical trial (N=1), or allogeneic HCT without bridging therapy (N=1). Conclusions We demonstrate the feasibility of a novel precision medicine approach for HMA-refractory MDS patients combining mutation data, ex vivo DSS, and in silico CBM to guide clinical therapeutic decisions within an actionable timeframe. Personalized treatment recommendations accurately predicted clinical responses in vivo and enabled some patients to be bridged to allogeneic HCT. Randomized prospective trials are needed to determine whether this approach may improve outcomes for patients with HMA-refractory MDS. Disclosures Aleshin: Notable Labs: Consultancy. Santaguida:Notable Labs: Employment. Schaffert:Notable Labs: Employment. Abbasi:Cell Works Group, Inc.: Employment. Patterson:Notable Labs: Employment. Heiser:Notable Labs: Employment. Greenberg:Notable Labs: Research Funding; Celgene: Research Funding; Genentech: Research Funding; H3 Biotech: Research Funding; Aprea: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees.

Pediatric immune thrombocytopenia (ITP) is usually self-limited. However, approximately 20% of children develop chronic ITP, which can be associated with significant morbidity because of long-term immunosuppression and splenectomy in refractory cases. To explore the molecular mechanism of chronic ITP compared with acute ITP, we studied 63 pediatric patients with ITP. Gene expression analysis of whole blood revealed distinct signatures for acute and chronic ITP. Oxidative stress–related pathways were among the most significant chronic ITP-associated pathways. Overexpression of VNN1, an oxidative stress sensor in epithelial cells, was most strongly associated with progression to chronic ITP. Studies of normal persons demonstrated VNN1 expression in a variety of blood cells. Exposure of blood mononuclear cells to oxidative stress inducers elicited dramatic up-regulation of VNN1 and down-regulation of PPARγ, indicating a role for VNN1 as a peripheral blood oxidative stress sensor. Assessment of redox state by tandem mass spectrometry demonstrated statistically significant lower glutathione ratios in patients with ITP versus healthy controls; lower glutathione ratios were also seen in untreated patients with ITP compared with recently treated patients. Our work demonstrates distinct patterns of gene expression in acute and chronic ITP and implicates oxidative stress pathways in the pathogenesis of chronic pediatric ITP.

In this issue of Blood, Wu et al describe a novel dominant negative loss-of-function mutation (BLVRB S111L) in a heme catabolic pathway deregulating reactive oxygen species (ROS) and associated with thrombocytosis.1

To the editor: In contrast to the familial predisposition observed in somatically acquired myeloproliferative neoplasms (low penetrance, clonal hematopoiesis), the hereditary thrombocythemias (HT) are characterized by Mendelian inheritance, high penetrance, and polyclonal hematopoiesis, and appear

Background: Although some studies have validated the 2001 WHO classification of acute myeloid leukemia (AML), including the importance of multilineage dysplasia, others have suggested that multilineage dysplasia correlates with unfavorable cytogenetics but has no independent impact on prognosis. In 2008, the revised WHO classification system has expanded this category into “AML with myelodysplasia-related changes” (AML-MRC) that now includes 1) AML arising from myelodysplastic syndrome (MDS), 2) AML with MDS-related cytogenetic abnormalities, and 3) AML with multilineage dysplasia. An individual case may fall into this category by meeting any of the criteria. The goal of the current study is to clinically characterize this newly defined AML-MRC subgroup. Methods: One-hundred consecutive AML patients diagnosed at Stanford University Hospital between 2005 and 2007 with adequate material for mutation analysis were studied. Cases were classified using the 2008 WHO criteria. Diagnostic cytogenetic findings were reviewed and patients were stratified into risk groups using Southwest Oncology Group criteria. Available flow cytometry immunophenotyping results were reviewed and all samples were tested for NPM, FLT3 (ITD and D835) and CEBPA mutations. Clinical parameters including hemogram data at time of diagnosis were reviewed. Clinical follow-up including overall survival (OS), progression free survival (PFS) and complete remission (CR) rates were retrospectively determined. Kaplan-Meier methods and univariate and multivariate Cox proportional hazards regression analysis were used to compare the clinical data. Results: The cases included 57 males and 43 females with a median age of 56 (range 17–81). Cytogenetic risk-group stratification resulted in 9 patients with favorable, 65 with intermediate and 19 with unfavorable risk status. Using the 2008 WHO criteria, there were 48 AML-MRC, 40 AML not otherwise specified (AML-NOS), 9 AML with either t(8;21), inv(16) or t(15;17), and 3 therapy related AMLs. Overall, 26 patients had a NPM1 mutation (16 of which were FLT3 mutated), 25 had FLT3-ITD, 8 had FLT3-D835 and 9 had a CEBPA mutation (3 of which were FLT3 mutated). Compared to AML-NOS, patients with AML-MRC were significantly older (59 vs 51 years, p=0.014) and presented with lower hemoglobin (9 vs 11.2 g/dL, p=0.044), lower platelets (47 vs 54 K/uL, p=0.059), unfavorable cytogenetics (14/46 vs 3/36, p=0.014) and exhibited a decreased frequency of CEBPA mutation (0/46 vs 7/40, p=0.001) as compared to AML-NOS. Based on the flow cytometry immunophenotyping, the blasts from patients with AML-MRC more frequently expressed CD14 compared to AML-NOS (10/46 vs 4/36, p=0.048). Clinical outcome data showed that patients with AML-MRC had a significantly worse OS, PFS and CR compared to AML-NOS (Figure, all p 60), FLT3-ITD and AML-MRC status as significant predictors of worse OS with the following respective hazard ratios: 2.82 (95% CI, 1.52–5.26), 2.11 (1.01–4.42), 1.98 (1.01–3.90), 1.92 (1.01–3.65). Conclusion: The newly defined WHO category of AML-MRC exhibits a significantly worse clinical outcome compared to AML-NOS and is predictive of worse overall survival in the multivariate analysis of AML patients, independent of age or cytogenetic risk group. These findings support the clinical, morphologic and cytogenetic criteria for this 2008 WHO AML category. Figure Figure

The CD31 monoclonal antibody, LYP21, binds to the CD31 domain 6 and inhibits the human mixed-lymphocyte reaction (MLR) in a specific and dose-dependent fashion. A synthetic CD31 peptide based on human CD31 epitope (amino acids 551 to 574) recognized by LYP21 is equally effective in inhibiting the MLR. In this study, we used the murine homolog of CD31 peptide 551 to 574 and a control peptide to study the role of CD31 molecule on T-cell activation. In vitro, CD31 peptide inhibited the MLR across several major and minor histocompatibility differences in a specific and dose-dependent fashion, similar to the results observed in the human system. Maximal inhibition was achieved at a dose of 200 μg/mL. In the cytotoxic T-lymphocyte (CTL) assay, CD31 peptide inhibited CTL responses by 97%. To study the in vivo effect of this peptide, graft-versus-host disease (GVHD) across minor histocompatibility barriers was induced in the B10.D2 (H-2d) → BALB/c (H-2d) model. BALB/c recipients received CD31 peptide (100 μg/d), or phosphate-buffered saline (PBS), or control peptide (100 μg/d) intraperitoneally (IP) for the first 5 weeks. CD31 peptide delayed onset of graft-versus-host disease and significantly increased long-term survival. Twelve of 14 mice receiving CD31 peptide survived more than 100 days after transplantation, as compared with none of 10 mice receiving PBS and none of five mice receiving control peptide (P = .0001). Long-term engraftment of allogeneic bone marrow was documented in all transplanted mice by polymerase chain reaction (PCR) analysis of microsatellite region in the interleukin (IL)-1β gene. Our data suggest that the CD31 molecule has an important functional role in T-cell activation in vitro and in vivo.

Steroid refractory gastrointestinal (GI) acute graft-versus-host disease (aGVHD) is a major cause of mortality in hematopoietic stem cell transplantation (HCT) without immune markers to establish a diagnosis or guide therapy. We found that T-cell receptor β (TCRβ) complementarity-determining region 3 repertoire sequencing reveals patterns that could eventually serve as a disease biomarker of T-cell alloreactivity in aGVHD. We identified T-cell clones in GI biopsies in a heterogeneous group of 15 allogeneic HCT patients with GI aGVHD symptoms. Seven steroid-refractory aGVHD patients showed a more conserved TCRβ clonal structure between different biopsy sites in the GI tract than 8 primary therapy–responsive patients. Tracking GI clones identified longitudinally at endoscopy in the blood also revealed an increased clonal expansion in patients with steroid-refractory disease. Immune repertoire sequencing-based methods could enable a novel personalized way to guide diagnosis and therapy in diseases where T-cell activity is a major determinant.

Introduction: Cyclic thrombocytopenia (CT) is a rare disease characterized by periodic fluctuations in platelet counts. The etiology of this disease has not been fully elucidated. Here, we present molecular characterization of CT in a 53-year-old male patient who has platelet counts ranging between 1 x 109/L to >400 x 109/L during a cycle period of 40 days which has occurred over several years. Methods: Blood transcriptome profiles and plasma cytokine levels were investigated in 24 samples that were sequentially collected every 3-4 days to cover two complete cycles. Total RNA was extracted after lysing red blood cells. 3SEQ (3′-end RNA sequencing for expression quantification) was performed for transcriptome profiling. SAMSeq quantitative analyses were conducted to identify differentially expressed genes. Three screening criteria were applied to this data set to identify a panel of exclusive platelet-specific genes. Plasma TPO (thrombopoietin)/testosterone/ estradiol/CD41a/CD42b levels were detected by ELISA. A total of 62 cytokines were screened using a luminex immunoassay. MPL (TPO receptor) gene was Sanger-sequenced using blood DNA and mRNA templates. DNA was extracted from the patient's hair follicles to exam the presence of the identified MPL mutation, and in-silico predictions were conducted to evaluate the MPL mutation. The wild-type (WT) and c.1210G>A mutant MPL expression constructs were generated and transfected into Ba/F3 cells for stable MPL expression. TPO-stimulated, IL-3 independent growth of MPL-expressing Ba/F3 cells were assessed to determine the function of MPL proteins. Results: Plasma TPO levels cycled between 6 to 2745 pg/mL during the sampling period; TPO levels inversely mirrored the fluctuation of platelet counts and preceded the latter by 3-4 days. Additional plasma cytokines demonstrated either an inverse correlation (e.g. FasL, GM-CSF, and TNF-β, etc.), or well synchronized correlation (e.g. BDNF, PDGF-BB, and RANTES etc.), with platelet counts. The patient was negative for platelet autoantibodies. Transcriptome analysis revealed 977 genes with expression that positively correlated with platelet count changes. Unsupervised clustering stratified these genes into subgroups, including: i) platelet-specific genes, which precede platelet count changes by 3-4 days; ii) platelet-modulated genes, which follow platelet count changes and are regulated by coagulation factors (e.g. F2) and platelet-contained growth factors (e.g. PDGF-BB and EGF); and iii) neutrophil-specific genes, which precede platelet count changes by 7-10 days. Among platelet-specific genes, 34 genes were identified to have >50-fold induction over a cycle, and are present in the platelet transcriptome [Rowley JW, 2011]. A heat map of these exclusive platelet genes is shown in Figure 1A. A novel MPL c.1210G>A heterozygous mutation, which leads to a p.Gly404Arg substitution, was identified in both blood and hair follicle DNA samples of the patient. This substitution resides in a highly conserved site and is predicted to be deleterious. In addition, the mutation creates a novel splice site, which results in the insertion of a 36 bp intron fragment in approximately 15% of MPL transcripts. In vitro analysis confirmed that MPL c.1210G>A is a loss-of-function (LOF) variant. Ba/F3 cells that express WT MPL exhibited IL-3 independent growth in the presence of TPO; however, the Gly404Arg mutant failed to support TPO-stimulated growth (Figure 1B). Conclusion: We describe a patient with CT who exhibits fluctuations of multiple cytokines including an inverse correlation between TPO and platelet counts, and profound gene expression changes in neutrophil- and platelet-specific gene expressions which precede platelet count fluctuations. These data suggest that the cyclic megakaryopoiesis and thrombopoiesis underlies pathogenesis of the disease. Moreover, the identified novel LOF MPL allele may contribute to the disease pathogenesis. The biologic and molecular annotation of this case presents a unique opportunity to examine the transcriptional regulation of platelet homeostasis. Figure 1 (A) Heat map of 34 genes that are exclusively expressed in platelet. (B) Growth of Ba/F3 cells expressing WT or c.1210G>A MPL in IL-3 free medium containing TPO (50 ng/mL). Average viable cell numbers of duplicate experiments were plotted with SD as error bars. Figure 1. (A) Heat map of 34 genes that are exclusively expressed in platelet. (B) Growth of Ba/F3 cells expressing WT or c.1210G>A MPL in IL-3 free medium containing TPO (50 ng/mL). Average viable cell numbers of duplicate experiments were plotted with SD as error bars. Disclosures No relevant conflicts of interest to declare.

CD31 (PECAM-1) is an immunoglobulin gene superfamily cell adhesion molecule found on vascular endothelium, platelets, and leukocytes. Lymphocyte expression of CD31 is most closely associated with the CD45RA+CD8+ naive T phenotype. CD31 has recently been shown to play a role in leukocyte egress to inflammatory sites. The mechanism of CD31 adhesion remains under investigation. Several investigators have reported evidence for a heterotypic ligand. We have previously shown that CD31 is phosphorylated with cell activation, which suggests a possible role for CD31 in cell activation events. We therefore studied the effects of CD31 antibodies on in vitro assays of lymphocyte activation. One CD31 antibody, LYP21, inhibited the mixed lymphocyte reaction (MLR) in a specific and dose-dependent fashion. An LYP21 epitope was localized to the sixth Ig domain of CD31. This peptide and a scrambled control peptide were synthesized and used to study effects of this epitope on lymphocyte activation. The CD31 peptide strongly inhibited the MLR. Because CD31 is expressed on both stimulator and responder populations, stimulator peripheral blood leukocytes and responder lymphocyte populations were separately incubated with CD31 peptide or control peptide and then washed before mixing. The CD31 peptide inhibited the MLR equally when either stimulator or responder cells were preincubated with the CD31 peptide. We further sorted responder cells into CD31-high and CD31-low populations and separately incubated these subsets with peptides. The CD31 peptide strongly inhibited MLRs, regardless of level of responder-cell CD31 expression. Examination of MLR reactions involving the CD31 peptide showed dispersed small aggregates of cells, rather than the single large aggregate observed in control MLRs. The CD31 peptide did not affect activation of lymphocytes by phorbol myristate acetate (PMA) and ionomycin. These results suggest that a surface CD31-ligand interaction may have a functional role in alloimmune lymphocyte activation and identify a functionally important domain of CD31.

Dysregulated Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling due to activation of tyrosine kinases is a common feature of myeloid malignancies. Here we report the first human disease-related mutations in the adaptor protein LNK, a negative regulator of JAK-STAT signaling, in 2 patients with JAK2 V617F–negative myeloproliferative neoplasms (MPNs). One patient exhibited a 5 base-pair deletion and missense mutation leading to a premature stop codon and loss of the pleckstrin homology (PH) and Src homology 2 (SH2) domains. A second patient had a missense mutation (E208Q) in the PH domain. BaF3-MPL cells transduced with these LNK mutants displayed augmented and sustained thrombopoietin-dependent growth and signaling. Primary samples from MPN patients bearing LNK mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34+ early progenitors were abnormally abundant in both patients. These findings indicate that JAK-STAT activation due to loss of LNK negative feedback regulation is a novel mechanism of MPN pathogenesis.

A 65 year old patient who was exposed to topical bovine thrombin during cardiac surgery developed markedly prolonged clotting times and a severe bleeding diathesis. Mixing studies with normal plasma failed to correct the clotting times. Platelet transfusions, immunosuppressive and immunomodulatory therapies were ineffective, but plasmapheresis was effective in decreasing clotting times and in the resolution of clinical bleeding events. The patient's purified IgG reacted with bovine thrombin by immunoblotting and enzyme-linked immunosorbent assay (ELISA). However, the IgG reacted minimally with human thrombin. In view of the severe bleeding, a coexisting inhibitor was sought. The patient's factor V activity was 1% of normal and was not corrected by mixing with normal plasma, demonstrating the presence of an inhibitor against factor V. The patient's IgG reacted with both bovine and human factor V. Immunoblotting localized the site of antibody binding to the light chain of activated bovine factor V. Detectable amounts of bovine factor V were found in commercial bovine thrombin preparations by ELISA. The data suggest that patients exposed to topical bovine thrombin may develop antibodies to thrombin and factor V. Anti-thrombin antibodies may mask coexisting factor V inhibitors responsible for clinical bleeding.

Background: Whole genome sequencing (WGS) identified activating CXCR4WHIM somatic mutations in nearly 30% of patients with Waldenstrom's Macroglobulinemia (WM) (Blood 123(11):1637-46). Both nonsense and frameshift CXCR4WHIM mutations occur in WM, with over 30 different types of mutations described within the regulatory carboxyl-terminal domain of CXCR4. CXCR4WHIM mutations almost always occur with activating MYD88 mutations, and impact both disease presentation and treatment outcome (Blood 123(18):2791-6; NEJM 372(15):1430-40.). The clonal architecture of CXCR4WHIM mutations relative to MYD88 mutations and their role in disease evolution remains to be clarified. Methods: We used Sanger sequencing and highly sensitive AS-PCR assays that we developed for the most common CXCR4WHIM mutations (S338X C>A and C>G) to evaluate for CXCR4WHIM mutations. In conjunction with an AS-PCR MYD88L265P assay that we previously developed (Leukemia 28(8):1698-707), we also profiled tumor samples for MYD88L265P and CXCR4S338X mutations in 164 WM, 12 IgM MGUS, 20 MZL, 32 CLL, 14 MM, 7 non-IGM MGUS patients, and 32 healthy donors. Next generation transcriptome sequencing data was also performed for validation in select cases. Results: AS-PCR detected CXCR4S338X mutations in WM and IgM MGUS patients not revealed by Sanger sequencing. By combined AS-PCR and Sanger sequencing, CXCR4WHIM mutations were identified in 44/102 (43%), 21/62 (34%), 2/12 (17%), and 1/20 (5%) untreated WM, previously treated WM, IgM MGUS and MZL patients, respectively, but not in CLL, MM, non-IGM MGUS patients or healthy donors. Cancer cell fraction analysis in WM and IgM MGUS patients showed CXCR4S338X mutations were primarily subclonal, with highly variable clonal distribution (median 35.1%, range 1.2%-97.5%; Figure 1). Sanger sequencing identified 3 patients with multiple CXCR4 mutations, which were shown to be compound heterozygous by TA cloning and sequencing of at least 40 clones. The addition of AS-PCR to the Sanger sequencing results also revealed multiple CXCR4WHIM mutations in many individual patients that included homozygous and compound heterozygous mutations that were validated by next generation sequencing that offered a median of 5,819 (range 5,217-12,235) reads that overlapped the mutated loci. Conclusions: Taken together, these findings show that CXCR4WHIM mutations are more common in WM patients than previously revealed by WGS or Sanger sequencing. Moreover, CXCR4 mutations are primarily subclonal supporting their acquisition after MYD88L265P in WM oncogenesis. The exclusive finding of frameshift and nonsense but not missense variants within the carboxyl-terminal domain of CXCR4 suggests that significant selection pressures exist for activating mutations within the WM clone. Lastly, multiple CXCR4WHIM mutations are common in WM patients indicative of targeted genomic instability within the carboxyl-terminal domain of CXCR4. Disclosures No relevant conflicts of interest to declare.

We investigated a family whose proband has a severe bleeding disorder and factor V antigenic and functional levels of 8% and less than 1% of control values, respectively. Molecular analysis of the factor V gene revealed a novel homozygous mutation in the last nucleotide of exon 10. 1701G>T causes activation of a cryptic exonic splice site in exon 10, which encodes part of the factor V heavy chain (A2 domain). This leads to the deletion of 35 nucleotides and results in a frameshift with a premature stop codon at amino acid position 498. The G1701 and corresponding Gln509 are conserved in murine, bovine, and porcine factor V and in human factor VIII. Few factor V deficiency mutations have been identified as yet. Several are present in the heterozygous form in combination with factor V Leiden (Arg506Gln). This is the first reported homozygous splice site mutation in a patient with factor V deficiency.