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Abstract: We report a first-in-human clinical trial using chimeric antigen receptor (CAR) T cells targeting CD37, an antigen highly expressed in B- and T-cell malignancies. Five patients with relapsed or refractory CD37+ lymphoid malignancies were enrolled and infused with autologous CAR-37 T cells. CAR-37 T cells expanded in the peripheral blood of all patients and, at peak, comprised >94% of the total lymphocytes in 4 of 5 patients. Tumor responses were observed in 4 of 5 patients with 3 complete responses, 1 mixed response, and 1 patient whose disease progressed rapidly and with relative loss of CD37 expression. Three patients experienced prolonged and severe pancytopenia, and in 2 of these patients, efforts to ablate CAR-37 T cells, which were engineered to coexpress truncated epidermal growth factor receptor, with cetuximab were unsuccessful. Hematopoiesis was restored in these 2 patients after allogeneic hematopoietic stem cell transplantation. No other severe, nonhematopoietic toxicities occurred. We investigated the mechanisms of profound pancytopenia and did not observe activation of CAR-37 T cells in response to hematopoietic stem cells in vitro or hematotoxicity in humanized models. Patients with pancytopenia had sustained high levels of interleukin-18 (IL-18) with low levels of IL-18 binding protein in their peripheral blood. IL-18 levels were significantly higher in CAR-37-treated patients than in both cytopenic and noncytopenic cohorts of CAR-19-treated patients. In conclusion, CAR-37 T cells exhibited antitumor activity, with significant CAR expansion and cytokine production. CAR-37 T cells may be an effective therapy in hematologic malignancies as a bridge to hematopoietic stem cell transplant. This trial was registered at www.ClinicalTrials.gov as #NCT04136275., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Chimeric antigen receptor (CAR) modified T cell therapies targeting BCMA have displayed impressive activity in the treatment of multiple myeloma. There are currently two FDA licensed products, ciltacabtagene autoleucel and idecabtagene vicleucel, for treating relapsed and refractory disease. Although correlative analyses performed by product manufacturers have been reported in clinical trials, there are limited options for reliable BCMA CAR T detection assays for physicians and researchers looking to explore it as a biomarker for clinical outcome. Given the known association of CAR T cell expansion kinetics with toxicity and response, being able to quantify BCMA CAR T cells routinely and accurately in the blood of patients can serve as a valuable asset. Here, we optimized an accurate and sensitive flow cytometry test using a PE-conjugated soluble BCMA protein, with a lower limit of quantitation of 0.19% of CD3+ T cells, suitable for use as a routine assay for monitoring the frequency of BCMA CAR T cells in the blood of patients receiving either ciltacabtagene autoleucel or idecabtagene vicleucel., (© 2024 International Clinical Cytometry Society.)

CD19-directed chimerical antigen receptor T-cell (CAR-T) products have gained US Food and Drug Administration approval for systemic large B-cell lymphoma. Because of concerns about potential immune cell-associated neurotoxicity syndrome (ICANS), patients with primary central nervous system (CNS) lymphoma (PCNSL) were excluded from all pivotal CAR-T studies. We conducted a phase 1/2 clinical trial of tisagenlecleucel in a highly refractory patients with PCNSL and significant unmet medical need. Here, we present results of 12 relapsed patients with PCNSL who were treated with tisagenlecleucel and followed for a median time of 12.2 months (range, 3.64-23.5). Grade 1 cytokine release syndrome was observed in 7/12 patients (58.3%), low-grade ICANS in 5/12 (41.6%) patients, and only 1 patient experienced grade 3 ICANS. Seven of 12 patients (58.3%) demonstrated response, including a complete response in 6/12 patients (50%). There were no treatment-related deaths. Three patients had ongoing complete remission at data cutoff. Tisagenlecleucel expanded in the peripheral blood and trafficked to the CNS. Exploratory analysis identified T-cell, CAR T, and macrophage gene signatures in cerebrospinal fluid following infusion when compared with baseline. Overall, tisagenlecleucel was well tolerated and resulted in a sustained remission in 3/7 (42.9%) of initial responders. These data suggest that tisagenlecleucel is safe and effective in this highly refractory patient population. This trial was registered at www.clinicaltrials.gov as #NCT02445248., (© 2022 by The American Society of Hematology.)

Poor adherence to best practices, insufficient training, and pressure to produce data quickly may lead to publications of suboptimal biomedical research flow cytometry data, which contributes to the body of irreproducible research findings. In addition, documentation of compliance with best flow cytometry practices for submission, visualization, and publication of flow cytometry data is currently endorsed by very few scientific journals, which is particularly concerning as numerous peer-reviewed flow cytometry publications emphasize instrumentation, experimental design, and data analysis as important sources of variability. Guidelines and resources for adequate reporting, annotation and deposition of flow cytometry experiments are provided by MIFlowCyt and the FlowRepository database, and comprehensive expert recommendations covering principles and techniques of field-specific flow cytometry applications have been published. To facilitate the integration of quality-defining parameters into manuscript and grant submission and publication requirements across biomedical fields that rely on the use of flow-cytometry-based techniques, a single comprehensive yet easily and universally applicable document is needed. To produce such a list of gold-standard parameters that assess whether a research flow cytometry experiment has been planned, conducted, interpreted, and reported at the highest standard, a new initiative defining the minimum set of standards a robust and rigorous research flow experiment must fulfill (MiSet RFC Standards) was proposed at CYTO 2019. MiSet RFC Standards will integrate and simplify existing resources to provide a universal benchmark a flow cytometry experiment can easily be measured against. The goal of MiSET RFC Standards is its integration into peer-review and publication procedures through partnership with stakeholders, journals and publishers in biomedical and translational research. This article introduces the aims and anticipated timeline and discusses strategies for interdisciplinary consensus and implementation. A single-resource broadly applicable guideline will harmonize standards across different fields of biomedical research and lead to publication of more robust research findings. © 2019 International Society for Advancement of Cytometry., (© 2019 International Society for Advancement of Cytometry.)

Novel techniques have been developed to image cells at cellular and subcellular levels. They allow images to be analyzed with ultra-high resolution, in 2D and/or 3D. Several of these tools have been tested on cytology specimens demonstrating emerging applications that are likely to change the field of cytopathology. This review covers several of these advanced imaging methods. The use of optical coherence tomography to perform optical biopsies during endoscopic ultrasound procedures or visualize cells within effusion samples is discussed. The potential for quantitative phase microscopy to accurately screen Pap test slides or resolve indeterminate diagnoses in urine cytology is reviewed. The article also examines the application of 3D cytology using LuCED for lung cancer detection in sputum samples and the feasibility of imaging flow and mass cytometry to measure multiple biomarkers at the single cell level. Although these novel technologies have great potential, further research is necessary to validate their routine use in cytopathology practice., (© 2018 Wiley Periodicals, Inc.)

Purpose: Treatment of malignant and nonmalignant hematologic diseases with hematopoietic stem-cell transplantation (HSCT) was first described almost 60 years ago, and its use has expanded significantly over the last 20 years. Whereas HSCT has become the standard of care for many patients in developed countries, the significant economic investment, infrastructure, and health care provider training that are required to provide such a service have prohibited it from being widely adopted, particularly in developing countries., Methods: Over the past two decades, however, efforts to bring HSCT to the developing world have increased, and several institutions have described their efforts to establish such a program. We aim to provide an overview of the current challenges and applications of HSCT in developing countries as well as to describe our experience in developing an HSCT program at Dhaka Medical College and Hospital in Bangladesh via a partnership with health care providers at Massachusetts General Hospital., Results and Conclusion: We discuss key steps of the program, including the formation of a collaborative partnership, infrastructure development, human resource capacity building, and financial considerations.

Background: Previous studies identified B cell gene signatures and predominance of specific B cell subsets as a marker of operational tolerance after kidney transplantation. These findings suggested a role for B cells in the establishment or maintenance of tolerance. Here we analyzed B cell recovery in 4 subjects, 3 of whom achieved tolerance after combined kidney/bone marrow transplantation., Methods: Peripheral B cell subsets were examined longitudinally by flow cytometry. Immunoglobulin heavy chain repertoire analysis was performed using next-generation sequencing. Lastly, the patients' serum reactivity to HLA was assessed by Luminex., Results: B cell counts recovered approximately 1 year posttransplant except for 1 subject who experienced delayed reconstitution. This subject resumed immunosuppression for acute rejection at 10 months posttransplant and underwent preemptive retransplantation at 3 years for chronic rejection. B cell recovery was accompanied by a high frequency of CD20 + CD24CD38 transitional B cells and a diversified clonal repertoire. However, all 4 subjects showed prevalence of CD20 + CD27+ memory B cells around 6 months posttransplant when B cell counts were still low and the clonal B cell repertoire very limited. The predominance of memory B cells was also associated with high levels of somatically mutated immunoglobulin heavy chain variable sequences and transient serum reactivity to HLA., Conclusions: Our observations reveal the presence of memory B cells early posttransplant that likely escaped the preparative regimen at a time consistent with the establishment of tolerance. Further studies are warranted to characterize the functional properties of these persisting memory cells and evaluate their potential contribution to tolerance induction.

Objective: Tonsil surgical biopsy or excision is a very common procedure. However, there exist no consensus guidelines for the pathologic handling of tonsil specimens; gross and/or microscopic evaluation may be used. Diagnosis of tonsillar hematologic malignancy requires histology, immunohistochemistry and/or flow cytometry. Data regarding the utility of flow cytometry in tonsillar tissues are limited. We assessed our experience with flow cytometry for tonsil diagnosis with regard to accuracy and use patterns at a tertiary academic medical center., Methods: We retrospectively analyzed all surgically biopsied or excised tonsil specimens that underwent flow cytometry evaluation from August 2011 to March 2014. Patient clinical information, intraoperative frozen section, histology, immunohistochemistry, and flow cytometry diagnoses were recorded., Results: The study included 154 tonsil specimens from 89 females and 65 males. Patients averaged 27.4 years old (range 2-87 years); 73 were pediatric. Both histology and flow cytometry were benign for 148 patients (96.1%). Hematolymphoid malignancy was diagnosed in 6 adults by histology/immunohistochemistry: diffuse large B-cell lymphoma (2), small B-cell lymphoma (2), concomitant follicular lymphoma and histiocytic sarcoma (1), and extraosseous plasmacytoma (1). Flow cytometry identified abnormal populations in 5 of 6 cases, and detected clonal populations in 2 reactive follicular hyperplasia cases., Conclusion: Tonsillar hematolymphoid malignancy is uncommon, and flow cytometry was less accurate than histology/immunohistochemistry for its diagnosis. Despite the rarity of tonsillar lymphoma in children, nearly half of study patients were pediatric. Intraoperative frozen section diagnosis showed excellent sensitivity for malignancy, and could be used to effectively triage cases for flow cytometry evaluation.

In this study, we established a novel isotope-free approach for the detection of cell-mediated lympholysis (CML) in MHC defined peripheral blood mononuclear cells (PBMCs) using multiparameter flow and imaging cytometry. CML is an established in vitro assay to detect the presence of cytotoxic effector T-lymphocytes precursors (CTLp). Current methods employed in the identification of CTLp in the context of transplantation are based upon the quantification of chromium ((51)Cr) released from target cells. In order to adapt the assay to flow cytometry, primary porcine PBMC targets were labeled with eFluor670 and incubated with major histocompatibility complex (MHC) mismatched effector cytotoxic lymphocytes (CTLs). With this method, we were able to detect target-specific lysis that was comparable to that observed with the (51)Cr-based assay. In addition, the use of quantitative cell imaging demonstrates the presence of accessory cells involved in the cytotoxic pathway. This innovative technique improves upon the standard (51)Cr release assay by eliminating the need for radioisotopes and provides enhanced characterization of the interactions between effector and target cells. This technique has wide applicability to numerous experimental and clinical models involved with effector-cell interactions., (Copyright © 2015 Elsevier B.V. All rights reserved.)