DiGuardo, Margaret

Abstract Within the field of regenerative medicine for musculoskeletal regenerative medicine, orthobiologics and cellular therapies are evolving at a rapid pace. Exuberant ideas for new treatments often outpace their scientific evidence, which may create confusion for both patients and providers on what treatments are evidence based. Orthobiologics and cellular therapies are a subset of regenerative therapies focused on treatment of musculoskeletal disease. For the field of orthobiologics to progress, it is essential that musculoskeletal providers understand the necessary components for translating novel therapies from discovery into practice. In this article, we provide an overview of both the lexicon and regulatory processes related to translation of orthobiologics and cellular therapies along with a comparison of how to translate orthobiologics and related novel cellular therapies in hopes to dispel misinformation while promoting responsible advancements and ethical practices in medicine.

AbstractBackgroundSince FDA approval in 2017, CAR‐T therapy has seen rapid clinical adoption. Shifting clinical trends have emerged with increasing utilization of CAR‐T therapies and a downward trend in HSCTs. Given the overlapping resources required for the manufacture and storage of these products, we sought to examine trends over a 6‐year period.MethodsThe apheresis patient database and Lab database were reviewed to compile a list of patients that underwent either CAR‐T or HSCT (autologous) collections, and/or received CAR‐T or autologous HSCT infusions between January 1, 2018 and December 12, 2023. This was further examined by year and disease group.ResultsThe total number of patients collecting for CAR‐T increased from 52 in 2018 to 150 in 2023 (slope = 21.97; p = .0013), accompanied by a decrease in the number of patients collecting for HSCT, from 425 in 2018 to 341 in 2023 (slope = −21.2; p = .0177). Neither total number (calculated as number of HSCT + CAR‐T) of patients collected (mean 476 + 20.4 per year), nor collection procedures (mean 972 ± 75.8 per year) changed significantly over the 6‐year period. The total number of CAR‐T infusions increased from 24 in 2018 to 111 in 2023 (slope = 17.7; p = .004), with a decrease in auto‐HSCT from 400 to 289 (slope = −28.7; p = .008). The overall number of infusions (calculated as number of HSCT + CAR‐T) did not change significantly (slope = −10.9; p = .13) over the 6‐year period.DiscussionOur findings confirm the increasing adoption of CAR‐T therapy occurring alongside a decreasing stem‐cell transplants, with stable overall resource utilization.

AbstractBackgroundFlow cytometry protocols for counting fresh CD34+ cell samples are not ideal for cryopreserved products due to cryoprotectant cytotoxicity. For cryopreserved samples, often large volumes of hypotonic solutions, which can cause cell death, are used to remove the cryoprotectant with a post‐thaw wash. We recently developed a novel multistep dilution method with subsequent flow cytometry analysis to allow for accurate and reproducible results. The previous method involved washing steps which invalidate the ability to enumerate cell recovery, and success had to be gauged solely on viability. The new method allows for assessment of total cell recovery and viable cell recovery.Study Design and MethodsApheresis products were cryopreserved in 10% DMSO at a target WBC concentration of 300 × 106/mL. Cryovials from these products were thawed at 37°C, and samples were diluted 1:2 by three additions of 1/3 sample volume using 1%‐Human Albumin in Dextran 40 (10% Low Molecular Weight Dextran in 0.9% NaCl) separated by 5 min between each addition. A 1:10 dilution was performed to obtain the correct cell concentration for flow cytometric analysis resulting in a 1:20 dilution. End WBC concentrations were ~15 × 106/mL with DMSO diluted to 0.5%.ResultsFifty‐two samples were tested with this new method. Total and viable cell recoveries were calculated based on pre‐cryopreservation data. Median total cell recoveries for CD34 and CD3 were >85%, while median viable cell recoveries were >75%.DiscussionCryopreserved samples can be reliably prepared for flow cytometric testing using a step‐wise dilution to preserve cell integrity and robust recoveries.

AbstractBackgroundStandard flow cytometry protocols for CD34+ cell enumeration designed for fresh samples are not appropriate for cryopreserved products. Special protocols have been developed to remove the cryoprotectant by quickly washing a freshly thawed sample. Exposing cells to a large volume of hypotonic solution and subsequent washing process was hypothesized to cause lab‐induced cell death. Moreover, standard gating strategies must be altered to avoid reporting falsely high viabilities.Study Design and MethodsWe developed a novel method whereby thawed samples were diluted step‐wise to 1:2 by 3 additions of 1/3 sample volume using 1% Human Albumin in Dextran 40 (10% Low Molecular Weight Dextran in 0.9% NaCl) separated by 5 min between each addition. An additional 1:10 dilution was required to obtain a desired cell concentration for flow cytometry testing resulting in a 1:20 dilution.ResultsTwenty samples were tested simultaneously in a method comparison; the new method demonstrated significant increases in mean cell viabilities for white blood cells, hematopoietic progenitor cells, and T cells as well as reduced standard deviations for each parameter.DiscussionSlow, step‐wise dilutions of freshly thawed samples of cryopreserved apheresis products to 1:20 yielded higher and more precise viability measurements compared to quickly washing samples to remove DMSO.

Background: Chimeric Antigen Receptor T-cell (CAR-T) therapies were first approved by the FDA in 2017, and multiple commercial products are currently available. Hematopoietic stem cell transplantation (HSCT) is another treatment strategy often utilized for patients with advanced and/or relapsed hematologic malignancies. Both CAR-T and HSCT require significant, overlapping resources for their collection, storage, and processing. Understanding the impact of evolving standards of care in clinical practice is necessary in the planning of resource allocation and laboratory expansion. We aimed to characterize the trends of CAR-T and HSCT infusions over a five-year period. Methods: The processing lab database was reviewed to compile a list of patients that received either CAR-T or HSCT (autologous or allogeneic) infusions, between 01/01/2018 and 12/12/2022. The number of individual HSCTs and CAR-T infusions were plotted over time. We also calculated the percent contribution of both HSCT and CAR-T to the total number of products infused per year. We subsequently examined these parameters, within the specific diagnoses of diffuse large B-Cell lymphoma/large cell lymphoma (DLBCL/LCL, including double and triple hit DLBCL and High-Grade lymphoma) and multiple myeloma (MM), including MM with amyloid and MM with plasma cell leukemia/plasmacytoma. Simple linear regression was used to calculate statistical significance to determine if the slopes were non-zero, and the direction of change over time. Results: Overall, our study included 210 CAR-T infusions and 2366 HSCTs. 89/210 (42%) patients who received a CAR-T product also received HSCT between 2018 and 2022, with 85/89 receiving HSCT prior to CAR-T infusion and 4/89 receiving CAR-T prior to HSCT, including two patients who received HSCT before and after CAR-T. The median interval between the most recent HSCT and subsequent CAR-T was 37 months. DLBCL/LCL and MM were the disease indications for which CAR-T was most utilized, accounting for 87.6% of the 210 procedures. The total number of CAR-T procedures increased significantly over the five-year period, from 24 in 2018 to 74 in 2022 (Slope=13.8; p=0.02), accompanied by a marked decrease in the number of HSCT from 502 in 2018 to 393 in 2022 (Slope=-26.6; p=0.06). The overall number of infusions (calculated as number of HSCT + number of CAR-T) declined slightly, though this was not statistically significant (mean 511 + 30.6 per year; Slope=-12.8; p=0.22). When expressed as a percentage of the total number of infusions, we observed a significant increase in the proportion of CAR-T, from 4.6% in 2018 to 15.8 % in 2022, (Slope=2.98; p=0.02), and a significant decrease in the proportion of infusions that were HSCT, from 95.4% in 2018 to 84.2% in 2022 (Slope=-2.98; p=0.02). In DLBCL, we observed a slight decrease in the number of overall infusions, from 71 in 2018 to 58 in 2022 (Slope=-3.5; p=0.18) and a significant decrease in HSCT from 47 in 2018 to 26 in 2022 (Slope=-6.0; p=0.0172), while the number of CAR-T infusions remained stable (mean=27.4 + 5.3 per year; Slope=2.5; p=0.14). In MM, we observed a marked increase in CAR-T infusions, from none in 2018 to 33 in 2022 (p=0.0544), while the total number of infusions remained stable (Mean 231.8 + 16.54; slope=-3.5; p=0.58) and HSCT slightly decreased, from 228 in 2018 to 184 in 2022, though this was not statistically significant (Slope=-11.5; p=0.18). We observed a non-significant increase in the percent contribution of CAR-T to the total number of infusions performed for MM, from 0% in 2018 to 15.3% in 2022 (Slope=3.62; p=0.06) while in DLBCL we observed a significant increase, from 33.8% in 2018 to 55.2% in 2022 (slope=6.42; p=0.017). Conclusion: The findings from this study suggest a gradual trend in the clinical utilization of human cellular therapy products over time. Since many of the resources and personnel involved in the collection, storage, and processing of these products are shared, an increase in CAR-T is an important observation. These findings underscore key issues in lab management for cell manufacturing facilities when considering resource allocation and infrastructure development. More analysis is needed to determine if the decrease in HSCT and the increase in CART are related.