Effects of extended transport on cryopreserved allogeneic hematopoietic progenitor cell (HPC) product quality and optimal methods to assess HPC stability.

BACKGROUND: Since the beginning of the COVID-19 pandemic, cryopreservation of hematopoietic progenitor cell (HPC) products has been increasingly used to ensure allogeneic donor graft availability prior to recipient conditioning for transplantation. However, in addition to variables such as graft transport duration and storage conditions, the cryopreservation process itself may adversely affect graft quality. Furthermore, the optimal methods to assess graft quality have not yet been determined. STUDY DESIGN AND METHODS: A retrospective review was performed on all cryopreserved HPCs processed and thawed at our facility from 2007 to 2020, including both those collected onsite and by the National Marrow Donor Program (NMDP). HPC viability studies were also performed on fresh products, retention vials, and corresponding final thawed products by staining for 7-AAD (flow cytometry), AO/PI (Cellometer), and trypan blue (manual microscopy). Comparisons were made using the Mann-Whitney test. RESULTS: For HPC products collected by apheresis (HPC(A)), pre-cryopreservation and post-thaw viabilities, as well as total nucleated cell recoveries were lower for products collected by the NMDP compared to those collected onsite. However, there were no differences seen in CD34+ cell recoveries. Greater variation in viability testing was observed using image-based assays compared to flow-based assays, and on cryo-thawed versus fresh samples. No significant differences were observed between viability measurements obtained on retention vials versus corresponding final thawed product bags. DISCUSSION: Our studies suggest extended transport may contribute to lower post-thaw viabilities, but without affecting CD34+ cell recoveries. To assess HPC viability prior to thaw, testing of retention vials offers predictive utility, particularly when automated analyzers are used.

Longitudinal transcriptional analysis of peripheral blood leukocytes in COVID-19 convalescent donors.

BACKGROUND: SARS-CoV2 can induce a strong host immune response. Many studies have evaluated antibody response following SARS-CoV2 infections. This study investigated the immune response and T cell receptor diversity in people who had recovered from SARS-CoV2 infection (COVID-19). METHODS: Using the nCounter platform, we compared transcriptomic profiles of 162 COVID-19 convalescent donors (CCD) and 40 healthy donors (HD). 69 of the 162 CCDs had two or more time points sampled. RESULTS: After eliminating the effects of demographic factors, we found extensive differential gene expression up to 241 days into the convalescent period. The differentially expressed genes were involved in several pathways, including virus-host interaction, interleukin and JAK-STAT signaling, T-cell co-stimulation, and immune exhaustion. A subset of 21 CCD samples was found to be highly "perturbed," characterized by overexpression of PLAU, IL1B, NFKB1, PLEK, LCP2, IRF3, MTOR, IL18BP, RACK1, TGFB1, and others. In addition, one of the clusters, P1 (n = 8) CCD samples, showed enhanced TCR diversity in 7 VJ pairs (TRAV9.1_TCRVA_014.1, TRBV6.8_TCRVB_016.1, TRAV7_TCRVA_008.1, TRGV9_ENST00000444775.1, TRAV18_TCRVA_026.1, TRGV4_ENST00000390345.1, TRAV11_TCRVA_017.1). Multiplexed cytokine analysis revealed anomalies in SCF, SCGF-b, and MCP-1 expression in this subset. CONCLUSIONS: Persistent alterations in inflammatory pathways and T-cell activation/exhaustion markers for months after active infection may help shed light on the pathophysiology of a prolonged post-viral syndrome observed following recovery from COVID-19 infection. Future studies may inform the ability to identify druggable targets involving these pathways to mitigate the long-term effects of COVID-19 infection. TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT04360278 Registered April 24, 2020.

Incorporating the entity of under-transfusion into hemovigilance monitoring: Documenting cases due to lack of inventory.

BACKGROUND: Under-transfusion is an underreported entity within most hospitals and hemovigilance systems. While critical blood shortages are being reported more frequently, without incident codes to document instances of under-transfusion due to lack of inventory, estimating its impact on patient care as it relates to hemotherapy (HT) has hampered our ability to assess and inform strategic initiatives to combat inventory issues as well as prepare for future blood supply threats. STUDY DESIGN AND METHOD: An 11-member working group of the AABB (Association for the Advancement of Blood and Biotherapies) Hemovigilance Committee was formed in October 2020 to study the topic of under-transfusion including its potential causes and clinical expressions. The group was also charged with proposing simple definition/incident codes to be used by hemovigilance systems to document such instances. RESULTS: The working group proposed four simple incident codes under the new process code-No Blood (NB)-that can be used by hemovigilance systems to appropriately document instances of under-transfusion due to lack of inventory. The codes were described as: (1) NB 01-Inventory less than usual level due to supplier shortage; (2) NB 02-Demand for blood product exceeding usual inventory levels; (3) NB 03-Substitution with incompatible/inappropriate units; and (4) NB 04-Suboptimal dose/no transfusion given. CONCLUSION: The adoption of these codes within hemovigilance systems globally would assist in recognition and reporting instances of under-transfusion due to inventory, thus supporting development of better collection strategies, inventory management techniques as well as effective policies to improve blood safety and availability.

Establishment of a cell processing laboratory to support hematopoietic stem cell transplantation and chimeric antigen receptor (CAR)-T cell therapy.

Cell processing laboratories are an important part of cancer treatment centers. Cell processing laboratories began by supporting hematopoietic stem cell (HSC) transplantation programs. These laboratories adapted closed bag systems, centrifuges, sterile connecting devices and other equipment used in transfusion services/blood banks to remove red blood cells and plasma from marrow and peripheral blood stem cells products. The success of cellular cancer immunotherapies such as Chimeric Antigen Receptor (CAR) T-cells has increased the importance of cell processing laboratories. Since many of the diseases successfully treated by CAR T-cell therapy are also treated by HSC transplantation and since HSC transplantation teams are well suited to manage patients treated with CAR T-cells, many cell processing laboratories have begun to produce CAR T-cells. The methods that have been used to process HSCs have been modified for T-cell enrichment, culture, stimulation, transduction and expansion for CAR T-cell production. While processing laboratories are well suited to manufacture CAR T-cells and other cellular therapies, producing these therapies is challenging. The manufacture of cellular therapies requires specialized facilities which are costly to build and maintain. The supplies and reagents, especially vectors, can also be expensive. Finally, highly skilled staff are required. The use of automated equipment for cell production may reduce labor requirements and the cost of facilities. The steps used to produce CAR T-cells are reviewed, as well as various strategies for establishing a laboratory to manufacture these cells.

Loss of FOXA1 Drives Sexually Dimorphic Changes in Urothelial Differentiation and Is an Independent Predictor of Poor Prognosis in Bladder Cancer.

We previously found loss of forkhead box A1 (FOXA1) expression to be associated with aggressive urothelial carcinoma of the bladder, as well as increased tumor proliferation and invasion. These initial findings were substantiated by The Cancer Genome Atlas, which identified FOXA1 mutations in a subset of bladder cancers. However, the prognostic significance of FOXA1 inactivation and the effect of FOXA1 loss on urothelial differentiation remain unknown. Application of a univariate analysis (log-rank) and a multivariate Cox proportional hazards regression model revealed that loss of FOXA1 expression is an independent predictor of decreased overall survival. An ubiquitin Cre-driven system ablating Foxa1 expression in urothelium of adult mice resulted in sex-specific histologic alterations, with male mice developing urothelial hyperplasia and female mice developing keratinizing squamous metaplasia. Microarray analysis confirmed these findings and revealed a significant increase in cytokeratin 14 expression in the urothelium of the female Foxa1 knockout mouse and an increase in the expression of a number of genes normally associated with keratinocyte differentiation. IHC confirmed increased cytokeratin 14 expression in female bladders and additionally revealed enrichment of cytokeratin 14-positive basal cells in the hyperplastic urothelial mucosa in male Foxa1 knockout mice. Analysis of human tumor specimens confirmed a significant relationship between loss of FOXA1 and increased cytokeratin 14 expression.

Improving CAR T cell therapy by optimizing critical quality attributes.

Whether as a cure or bridge to transplant, chimeric antigen receptor (CAR)-T cell therapies have shown dramatic outcomes for the treatment of hematologic malignancies, and particularly relapsed/refractory B cell leukemia and lymphoma. However, these therapies are not effective for all patients, and are not without toxicities. The challenge now is to optimize these products and their manufacture. The manufacturing process is complex and subject to numerous variabilities at each step. These variabilities can affect the critical quality attributes of the final product, and this can ultimately impact clinical outcomes. This review will focus on optimizing the manufacturing variables that can impact the safety, purity, potency, consistency and durability of CAR-T cells.

Advances in gene therapy for hematologic disease and considerations for transfusion medicine.

As the list of regulatory agency-approved gene therapies grows, these products are now in the therapeutic spotlight with the potential to cure or dramatically alleviate several benign and malignant hematologic diseases. The mechanisms for gene manipulation are diverse, and include the use of a variety of cell sources and both viral vector- and nuclease-based targeted approaches. Gene editing has also reached the realm of blood component therapy and testing, where cultured products are being developed to improve transfusion support for individuals with rare blood types. In this review, we summarize the milestones in the development of gene therapies for hematologic diseases, mechanisms for gene manipulation, and implications for transfusion medicine and blood centers as these therapies continue to advance and grow.

Advances in T-cell Immunotherapies.

Cell therapies have become an important part of clinical hematology and oncology. Cell therapy laboratories were first established in academic health centers to process ABO-incompatible marrow grafts. These laboratories now produce a wide variety of cell and gene therapies. Some of the most widely used and clinically important cell therapies are T-cell immunotherapies. These therapies include donor lymphocyte infusions, tumor-infiltrating lymphocytes, T-cell receptor-engineered T cells, chimeric antigen receptor T cells, and virus-specific T cells. The clinical application and methods used to manufacture these adoptive cell therapies are reviewed.

Robust Selections of Various Hematopoietic Cell Fractions on the CliniMACS Plus Instrument.

Cell separation technologies play a vital role in the graft engineering of hematopoietic cellular fractions, particularly with the rapid expansion of the field of cellular therapeutics. The CliniMACS Plus Instrument (Miltenyi Biotec) utilizes immunomagnetic techniques to isolate hematopoietic progenitor cells (HPCs), T cells, NK cells, and monocytes. These products are ultimately used for HPC transplantation and for the manufacture of adoptive immunotherapies. We evaluated the viable cell recovery and cell purity of selections and depletions performed on the CliniMACS Plus over a 10-year period at our facility, specifically assessing for the isolation of CD34+, CD4+, CD3+/CD56+, CD4+/CD8+, and CD25+ cells. Additionally, patient- and instrument-related factors affecting these parameters were examined. Viable cell recovery ranged from 32.3 ± 10.2% to 65.4 ± 15.4%, and was the highest for CD34+ selections. Cell purity ranged from 86.3 ± 7.2% to 99.0 ± 1.1%, and was the highest for CD4+ selections. Undesired cell fractions demonstrated a range of 1.2 ± 0.45 to 5.1 ± 0.4 log reductions. Red cell depletions averaged 2.12 ± 0.68 logs, while platelets were reduced by an average of 4.01 ± 1.57 logs. Donor characteristics did not impact viable cell recovery or cell purity for CD34+ or CD4+ cell enrichments; however, these were affected by manufacturing variables, including tubing size, bead quantity, and whether preselection platelet washes were performed. Our data demonstrate the efficient recovery of hematopoietic cellular fractions on the CliniMACS Plus that may be optimized by adjusting manufacturing variables.

Programmed death-ligand 1 (PD-L1) is expressed in a significant number of the uterine cervical carcinomas.

BACKGROUND: The programmed death-1/programmed death-ligand-1 (PD-1/PD-L1) immune regulatory axis has emerged as a promising new target for cancer therapeutics, with lasting responses seen in the treatment of metastatic renal and lung carcinomas, as well as melanomas. As tumor surface expression of PD-L1 has been found to correlate with objective responses to anti-PD-L1 immunotherapies, we investigated the expression of PD-L1 in human cervical tumors and provide an adopted scoring system for the systematic evaluation of PD-L1 staining. METHODS: Immunohistochemical staining for PD-L1 expression was performed on a tissue microarray of 101 normal and neoplastic cervical tissues. Neoplastic cores were divided into three groups: squamous cell carcinoma, adenosquamous carcinoma, and endocervical adenocarcinoma. PD-L1 expression was scored based on an adopted scoring system accounting to percentage and intensity of positivity, and results provided alongside available clinical and demographic data. RESULTS: Overall, PD-L1 was positive in 32 of 93 (34.4%) cervical carcinomas. Subcategorically, PD-L1 was positive in 28 of 74 (37.8%) squamous cell carcinomas, two of seven (28.6%) adenosquamous carcinomas, and two of 12 (16.7%) endocervical adenocarcinomas. It was negative in six benign cervical tissues. CONCLUSIONS: This study shows a significant expression of PD-L1 in 34.4% of cervical carcinomas and no expression of PD-L1 in benign cervical tissues. These findings suggest a role for further investigation of anti-PD-L1/PD-1 immunotherapies in the treatment of PD-L1-positive cervical tumors. In addition, our adopted scoring system will facilitate more systematic correlations between tumor reactivity and response to treatment.

Application of Immunohistochemistry in the Diagnosis of Pulmonary and Pleural Neoplasms.

CONTEXT: - A vast majority of neoplasms arising from lung or pleura are initially diagnosed based on the histologic evaluation of small transbronchial, endobronchial, or needle core biopsies. Although most diagnoses can be determined by morphology alone, immunohistochemistry can be a valuable diagnostic tool in the workup of problematic cases. OBJECTIVE: - To provide a practical approach in the interpretation and immunohistochemical selection of lung/pleura-based neoplasms obtained from small biopsy samples. DATA SOURCES: - A literature review of previously published articles and the personal experience of the authors were used in this review article. CONCLUSION: - Immunohistochemistry is a useful diagnostic tool in the workup of small biopsies from the lung and pleura sampled by small biopsy techniques.