The challenge of standardizing CAR-T cell monitoring: A comparison of two flow-cytometry methods and correlation with qPCR technique.

Chimeric antigen receptor (CAR) T-cell therapy is a breakthrough in hematologic malignancies, such as acute B lymphoblastic leukemia (B-ALL). Monitoring this treatment is recommended, although standardized protocols have not been developed yet. This work compares two flow cytometry monitoring strategies and correlates this technique with qPCR method. CAR-T cells were detected by two different flow-cytometry protocols (A and B) in nine blood samples from one healthy donor and five B-ALL patients treated with Tisagenlecleucel (Kymriah®, USA). HIV-1 viral load allowed CAR detection by qPCR, using samples from seven healthy donors and nine B-ALL patients. CAR detection by protocol A and B did not yield statistically significant differences (1.9% vs. 11.8% CD3 + CAR+, p = 0.07). However, protocol B showed a better discrimination of the CD3 + CAR+ population. A strong correlation was observed between protocol B and qPCR (r = 0.7, p < 0.0001). CD3 + CAR+ cells were detected by flow cytometry only when HIV-1 viral load was above 104 copies/mL. In conclusion, protocol B was the most specific flow-cytometry procedure for the identification of CAR-T cells and showed a high correlation with qPCR. Further efforts are needed to achieve a standardized monitoring approach.

Predicting anti-TNF treatment response in rheumatoid arthritis: An artificial intelligence-driven model using cytokine profile and routine clinical practice parameters.

Introduction: Rheumatoid arthritis (RA) is a heterogeneous disease in which therapeutic strategies used have evolved dramatically. Despite significant progress in treatment strategies such as the development of anti-TNF drugs, it is still not possible to differentiate those patients who will respond from who will not. This can lead to effective-treatment delays and unnecessary costs. The aim of this study was to utilize a profile of the patient's characteristics, clinical parameters, immune status (cytokine profile) and artificial intelligence to assess the feasibility of developing a tool that could allow us to predict which patients will respond to treatment with anti-TNF drugs. Methods: This study included 38 patients with RA from the RA-Paz cohort. Clinical activity was measured at baseline and after 6 months of treatment. The cytokines measured before the start of anti-TNF treatment were IL-1, IL-12, IL-10, IL-2, IL-4, IFNg, TNFa, and IL-6. Statistical analyses were performed using the Wilcoxon-Rank-Sum Test and the Benjamini-Hochberg method. The predictive model viability was explored using the 5-fold cross-validation scheme in order to train the logistic regression models. Results: Statistically significant differences were found in parameters such as IL-6, IL-2, CRP and DAS-ESR. The predictive model performed to an acceptable level in correctly classifying patients (ROC-AUC 0.804167 to 0.891667), suggesting that it would be possible to develop a clinical classification tool. Conclusions: Using a combination of parameters such as IL-6, IL-2, CRP and DAS-ESR, it was possible to develop a predictive model that can acceptably discriminate between remitters and non-remitters. However, this model needs to be replicated in a larger cohort to confirm these findings.

Variants in CASP10, a diagnostic challenge: Single center experience and review of the literature.

Autoimmune lymphoproliferative syndrome is a primary immunodeficiency caused by variants in FAS-mediated apoptosis related genes and is characterized by lymphadenopathy, splenomegaly and autoimmunity. A total of six different variants in CASP10 have been described as potential causative of disease, although two of them have recently been considered polymorphisms. The high allele frequency of these variants in healthy population in addition to the broad clinical spectrum of the disease difficult the interpretation of their pathogenicity. Here, we describe the clinical and analytical findings of three new patients carrying variants in CASP10 and summarize 12 more cases from the literature. Autoimmune cytopenias, adenopathies and increment of TCRαß+CD4-CD8- cells have been the most common findings, being possibly the FAS-mediated apoptosis pathway the pathogenic mechanism of this disease. The clinical impact and the consequences of CASP10 variants are not fully elucidated, therefore the description of new cases will contribute to solve this issue.

Solutions to Avoid False Positives for Rituximab in Pre-Transplant Crossmatches.

Rituximab (anti-CD20) is commonly used as immunotherapy against B cells, in the context of pre-transplant crossmatches, where the presence of rituximab in the tested sera with donor cells can alter their results both by flow cytometry (FCXM) as complement-dependent cytotoxicity (CDCXM) giving rise to false positives. In the present study, we tested the use of an anti-rituximab monoclonal antibody (10C5, Abnova) as a method to avoid false positives in FCXM and CDCXM. We used the serum from ten patients who received therapy with rituximab, and the cells were incubated with sera treated or untreated with the 10C5 clone. In previous studies, attempts have been made to control these false positives through the use of pronase, although in these cases the alteration of Human Leukocyte Antigen (HLA) molecules has been found to be a limitation. As an alternative, we performed an assay to exclude false positives by a pre-incubation with anti-rituximab antibody (10C5) in 1:5 proportion avoiding the misinterpretation of crossmatches, particularly in patients with specific donor antibodies (DSA) without affecting the HLA molecules.