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Introduction: The presence of cfDNA in the blood is a sign of tissue damage. DNA from lung cells is rarely found in the blood of healthy subjects. We aimed to evaluate lung tissue damage among COVID19 patients and try to find out if the level of cfDNA derived from the lungs might be a predictor of disease severity. Method(s): We recruited hospitalized COVID19 patients and compared them to a control group. The control group included volunteers without current or past diagnosis of Covid19 and no history of pulmonary diseases. Blood samples for cfDNA were taken from all participants. Basic demographic and clinical information were evaluated. Six known methylation patterns typical of pulmonary origin were selected and their levels were measured in each blood sample. Statistical analysis was done to evaluate differences between the levels of cfDNA between COVID19 patients and the control group. Evaluation was done to find correlation between disease severity and cfDNA levels. Result(s): One hundred and eighteen COVID19 patients and 40 volunteers were recruited for the study. Our findings showed a significant difference of cfDNA derived from the lungs between COVID19 patients and the control group (P<0.05). The levels of cfDNA from pulmonary origin were significantly higher among patients with severe disease and were correlated with clinical variables such as mortality, need for respiratory support, chest X Ray severity and hospitalization in intensive care (P<0.05). Conclusion(s): The study demonstrated elevated levels of cfDNA among COVID19 patients. Higher levels were significantly associated with disease severity and mortality.
ABSTRACT
Background: The Pfizer/BioNTech BNT162b2 vaccine, employing mRNA technology, has been recently approved by both the FDA and EMA for the prevention of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, demonstrating a 94.6% protection rate in a phase 3 study. While this vaccine is recommended by the FDA, EBMT and ASH-ASTCT for immunosuppressed patients, data regarding protection efficacy and safety in patients undergoing immunologic cell therapy are scarce. Aims: We aimed to evaluate efficacy and toxicity of the BNT162b2 vaccine in patients that underwent hematopoietic cell transplantation and CAR-T therapy. Methods: All patients under active treatment at the long-term follow-up HCT clinic (n=124) at the Tel Aviv Sourasky Medical Center, were evaluated for immunologic recovery (CD19+, CD4+, and CD8+ cell blood levels) pre-vaccination and were recommended to receive the commercial vaccination based on the EBMT recommendations. Patients were prospectively followed for vaccination-safety profile (laboratory tests, GVHD monitoring, and symptom-based questionnaire). We evaluated the humoral immune response to vaccine, 7-14 days after the second vaccine dose, by in vitro quantitative determination of anti-SARSCoV- 2S antibodies using Elecsys. assay and cellular immune response by ELISpot, estimating IL-2 and IFN-gamma secretion in response to a pool of lyophilized SARS-COV-2 S and M peptides (PepTivator;Miltenyi). The trial was approved by the local Ethics Committee and was registered by the clinical trials network (NCT04724642). Results: From 23-Dec-2020 all sequential patients (allogeneic, n=101 and CAR-T, n=23) were assessed for eligibility based on the EBMT recommendations (Version 5.0, Feb 21, 2021). Of those, 100 patients were eligible and 79 patients (allogeneic, n=65 and CAR-T, n=14) were vaccinated per-protocol. Characteristics of patients are depicted in Table 1. Overall, the 2 vaccine doses were well tolerated. Adverse events were reported in 39% of allogeneic HCT recipients (4.6% grade ≥3) and 32% of CART recipients (7% grade ≥3). All events resolved within few days, with the exception of 1 secondary graft rejection which is still under investigation. Among the CAR-T group, 5 patients (36%) had humoral antibody response. Patients with CD19+ lymphocytes >0 had a higher likelihood to develop antibodies compared to those with B cell aplasia (67% vs. 12.5%, p=.036). Among the allogeneic HCT group - 47 patients (81%) had a humoral antibody response. Incidence of positive serology was lower in patients with concomitant high intensity immunosuppressive therapy (IST) compared to those with low intensity IST (69% vs. 94%, p=.016). Linear regressions identified that male sex (beta=-.380, p=.012) and high intensity IST (beta=-.497, p=.014) were associated with lower antibody titer, while age, months from HCT, intensity of conditioning, low CD19 cell count, and active GVHD did not predict response. Analysis of peptide induced cytokine release by ELISpot is ongoing and will be presented at the EHA meeting. Summary/Conclusion: Humoral response to the BNT162b2 mRNA COVID-19 vaccine in CAR-T patients with B cell aplasia is significantly impaired, while overall response in patients after allogeneic HCT is encouraging. Patients on concomitant high intensity IST had impaired humoral response to BNT162b2. Longer follow-up is mandatory to test persistence of antibodies, and general preventive practices should be continued until more data are available.
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Background: Chimeric antigen receptor T cells (CAR-T) cells targeting CD19, demonstrate highly effective anti-tumor response in Diffuse Large B Cell Lymphoma (DLBCL). However, can result in significant side effects such as prolong neutropenia and infections. The incidence of infections in the setting of real world data hasn't been completely identified. Aims: To perform a comprehensive analysis of infection rate and profile in the first month after CAR-T cells in a cohort of infirm patients treated with commercially available CAR-T cells. Methods: This is a retrospective, single center study conducted in the Bone marrow transplant unit, Tel Aviv, analyzing the infection rate in consecutive patients with DLBCL who were treated with commercially available axicabtagene ciloleucel or tisagenlecleucel. Following a local protocol, all patients were treated with prophylaxis with ciprofloxacin and fluconazole when neutrophil counts decreased below 0.5∗103/μl. Acyclovir was administered at conditioning initiation. Microbiology and clinical documented infections (MDI and CDI, respectively) were defined according to the ECIL guidelines. Following white blood cell recovery, patients carried out weekly full blood count, and monthly CMV and HHV-6. A logistic regression was performed for the association between baseline characteristics and documented infections. Results: From June 2019 to December 2020, we included 60 consecutive patients with DLBCL treated with axicabtagene ciloleucel (n=16, 27%) or tisagenlecleucel (n=44, 73%). The median age was 69.3 (range, 19.8-85.2) years and ECOG-Performance status(PS) was 2-3 in most patients (58%). Broad spectrum antibiotics was administered to patients experiencing neutropenic fever (n=53, 88%). Overall infections were noted in 27/60 patients (45%).Bacterial infections were seen in 16 patients (27%) and included CDI in 7 (Pneumonia, n=5;periodontal infection, n=1 and cellulitis, n=1) and MDI in 9 patients (Gram negative rod bacteremia (GNR), n= 5;Gram positive cocci(GPC) bacteremia, n= 3;Both GNR and GPC, n=1). Viral infection was described in 14 patients (23%). The most common viral infection was CMV reactivation (n=10, 17%) leading to initiation of anti-CMV treatment in 6 among these patients. None had CMV disease. HHV-6 was positive in 3 patients (5%) with one of these patients presenting with acute encephalitis. Other viral infections reported were RSV (n=2)COVID-19 (n=1). No fungal infection was documented. Incidence of documented infections was higher in patients with CRS/ICANS compared with patients without [23/44 (52%) vs 4/16 (25%), p=0.005]. While CRP levels predicted documented infections (p= 0.041), ferritin blood levels did not (p=0.130). In univariate analysis, ICANS (OR= 4.5, p = 0.018) was associated with higher incidence of bacterial infection while there was a trend for lower incidence of bacterial infections in patients with chemo-sensitive disease to bridging therapy (OR= 0.375, p= 0.074)(Table 1). Age or ECOG-PS were not associated with increased risk of bacterial infection. Patients with documented infection had an increase in hospitalization days compared to those without documented infection (26.44 vs 21.7 days, p= 0.085). Summary/Conclusion: Infections were common in the first month following CAR-T cell administration, however were not increased in elderly patients or those presenting with poorer PS. Patients refractory to bridging therapy and ICANS should be monitored cautiously for the occurrence of infections. CMV monitoring should also be considered.
ABSTRACT
INTRODUCTION: Testing for active SARS-CoV-2 infection is a fundamental tool in the public health measures taken to control the COVID-19 pandemic. Because of the overwhelming use of SARS-CoV-2 reverse transcription (RT)-PCR tests worldwide, the availability of test kits has become a major bottleneck and the need to increase testing throughput is rising. We aim to overcome these challenges by pooling samples together, and performing RNA extraction and RT-PCR in pools. METHODS: We tested the efficiency and sensitivity of pooling strategies for RNA extraction and RT-PCR detection of SARS-CoV-2. We tested 184 samples both individually and in pools to estimate the effects of pooling. We further implemented Dorfman pooling with a pool size of eight samples in large-scale clinical tests. RESULTS: We demonstrated pooling strategies that increase testing throughput while maintaining high sensitivity. A comparison of 184 samples tested individually and in pools of eight samples showed that test results were not significantly affected. Implementing the eight-sample Dorfman pooling to test 26 576 samples from asymptomatic individuals, we identified 31 (0.12%) SARS-CoV-2 positive samples, achieving a 7.3-fold increase in throughput. DISCUSSION: Pooling approaches for SARS-CoV-2 testing allow a drastic increase in throughput while maintaining clinical sensitivity. We report the successful large-scale pooled screening of asymptomatic populations.