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Torque Teno Virus (TTV) is a single-stranded circular DNA virus which has been identified as a surrogate marker of immune competence in transplantation. In this study we investigated the dynamics of plasma TTV DNAemia in 79 adult patients undergoing chimeric antigen receptor T-cell (CAR-T) therapy for relapsed or refractory large B-cell lymphoma, also evaluating the impact of TTV on immunotoxicities, response and survival outcomes. After lymphodepleting therapy, TTV DNA load decreases slightly until reaching nadir around day 10, after which it increased steadily until reaching maximum load around day 90. TTV DNA load

ObjectivesThere is scarce information as to the durability of immune responses elicited by the Comirnaty® COVID-19 vaccine in nursing home residents. Here, we assessed SARS-CoV-2-Spike (S)-targeted antibody and functional T cell responses at around 6 months after complete vaccination.MethodsThe sample comprised 46 residents (34 females; age, 60-100 years), of whom 10 had COVID-19 prior to vaccination. Baseline (median of 17.5 days after vaccination) and follow-up (median, 195 days) plasma specimens were available for quantitation of SARS-CoV-2-S antibodies and enumeration of SARS-CoV-2-S-reactive IFN-γ CD4+ and CD8+ T cells by flow cytometry.ResultsIn total, 44/45 participants had detectable SARS-CoV-2-S antibodies at follow-up. Overall, antibody levels were found to decrease (median, 4.8 fold). Antibodies waning was more frequent (P+ T cells were detected in 33/46 and 24/46 at baseline and follow-up, respectively. The figures for CD4+ T cell counterparts were 12/46 and 30/46. Detectable SARS-CoV-2 IFN-γ CD8+ and CD4+ T cell responses at follow-up were more common in recovered (8/10 and 7/10, respectively) than in naïve residents (9/36 and 25/36, respectively). For those with detectable responses at both time points, SARS-CoV-2-S IFN-γ CD8+ T cell frequencies decreased significantly (P=0.001) over time whereas the opposite (P=0.01) was observed in CD4+ T cells.ConclusionAlmost all residents displayed detectable SARS-CoV-2-S-reactive antibodies and T cell responses, respectively, by around 6 months after complete vaccination with Comirnaty® COVID-19 vaccine, albeit generally waning in magnitude over time.

We conducted a multicenter interventional study to assess the efficacy of Therakos ECP to treat steroid-resistant graft-vs-host disease (SRes-GVHD) after allogeneic HSCT and to identify biomarkers of GVHD response. A total of 62 patients were treated for acute SRes-GVHD (n = 37) or chronic SRes-GVHD (n = 25). Median time to best response was 35 days (range, 28-85) and 90 days (range, 27-240) in acute and chronic SRes-GVHD, respectively. Overall, 27 patients (72.9%) with SRes-aGVHD responded to treatment (40.5% CR and 32.4% PR). The response rate was significantly higher in grade I-II than in grade III-IV aGVHD (100% vs 50.0%, respectively, P-value = .001). In chronic SRes-GVHD, 22 patients (88%) achieved a clinical response (24.0% CR and 64% PR). Response was higher in moderate than in severe SRes-cGVHD (100% vs 75%, P = .096). In both acute and chronic SRes-GVHD patients, the percentage of peripheral blood CD3+ CD4+ was higher and CD3+ CD8+ lower in responding than nonresponding patients. Acute SRes-GVHD responding patients presented a higher number of Treg cells (CD4+ CD25+ CD127low/- ) at day 0 (P = .028) than nonresponding patients, differences that were maintained over the observation period. Phenotypic analysis of T-cell maturation showed a trend toward reduction in TCD8 naive cells, along with an increased percentage of TCD8 Mem Efect T cells after starting ECP in responding patients. None of the studied serum cytokines displayed statistically significant changes in either acute or chronic SRes-GVHD. ECP is an effective treatment for patients with SRes-GVHD. Biomarkers could help guide decision-making on ECP treatment initiation and duration.

Objectives: We examined the relationship between peripheral blood levels of SARS-CoV-2 S1/M-reactive IFN-γ-producing CD4+ and CD8+ T cells, serum levels of biomarkers of clinical severity and mortality in critically ill COVID-19 patients. Methods: Immune responses were monitored in 71 non-consecutive patients (49 male and 22 female; median age, 65 years) by whole-blood flow cytometry (326 specimens). SARS-CoV-2 RNA loads in paired tracheal aspirates [TA] (n=147) were available from 54 patients. Serum levels of interleukin-6, ferritin, D-Dimer, lactose dehydrogenase and C-reactive protein in paired sera were known. Results: SARS-CoV-2 T cells (either CD4+, CD8+ or both) were detectable in 70 patients. SARS-CoV-2 IFN-γ CD4+ T-cell responses were documented more frequently than their CD8+ counterparts (62 vs. 56 patients) and were of greater magnitude overall. Detectable SARS-CoV-2 S1/M-reactive CD8+ and CD4+ T-cell responses were associated with higher SARS-CoV-2 RNA loads in TA. SARS-CoV-2 RNA load in TA decreased over time, irrespective of the dynamics of SARS-CoV-2-reactive CD8+ and CD4+ T cells. Conclusion: Enumeration of peripheral blood levels of SARS-CoV-2-S1/M-reactive IFN-γ CD4+ and CD8+ T cells does not predict viral clearance from the lower respiratory tract or poor clinical outcomes in critically ill COVID-19 patients.

Cytomegalovirus (CMV) DNAemia and CMV disease have been reported as more frequent in patients undergoing haploidentical allogeneic hematopoietic stem cell transplantation (Haplo-HSCT) than in those receiving HLA-matched allografts. This could be due to impaired CMV-specific T-cell reconstitution. Here, we conducted a multicenter observational study to assess CMV pp65 and IE-1-specific T cells kinetics in patients undergoing unmanipulated Haplo-HSCT with posttransplant cyclophosphamide (PT/Cy-haplo) and compared it with patients allografted with HLA-matched donors. Plasma CMV DNA load was monitored by real-time PCR and enumeration of CMV-specific IFN-γ-producing CD8+ and CD4+ T cells was performed by flow cytometry for intracellular cytokine staining at days +30, +60, +90, and +180 after transplantation. CMV DNAemia developed in 62 patients, occurring with comparable frequency in PT/Cy-haplo and MRD/MUD recipients (P = 0.14). There were no significant differences across groups in the number of patients either displaying detectable CMV-specific CD8+ and CD4+ T-cell responses or acquiring CMV-specific T-cell levels conferring protection against subsequent infection. CMV-specific T-cell counts were comparable between groups at most time points examined, irrespective of whether CMV DNAemia occurred or not prior to monitoring. Collectively the data suggest that PT/Cy-haplo recipients may reconstitute CMV-specific T-cell immunity to the same extent as patients undergoing HLA-matched allo-HSCT.

Objectives The immunogenicity of the Comirnaty® COVID-19 vaccine is understudied in elderly people with comorbidities. SARS-CoV-2-S-targeted antibody and T cell responses following full vaccination were assessed in nursing home residents. Methods Sixty nursing home residents (44 female; age, 53-100 years), of whom 10 had previously been diagnosed of COVID-19, and 18 healthy controls (15 female; age, 27-54 years) were recruited. Pre- and post-vaccination blood specimens were available for quantitation of total antibodies binding SARS-CoV-2 S protein and enumeration of SARS-CoV-2-S-reactive IFN-γ CD4+ and CD8+ T cells by flow cytometry. Results The seroconversion rate in presumably SARS-CoV-2 naïve nursing home residents (41/43; 95.3%), was similar to controls (17/18; 94.4%). A booster effect was documented in post vaccination samples of nursing home residents with prior COVID-19. Plasma antibody levels were higher (P

Highlights • CMV DNA is frequently detected in BAL fluid specimens from allo-HSCT. • CMV DNA detection in BAL fluids is comparable across pneumonia etiologies. • CMV DNA loads in BAL fluids are comparable across pneumonia etiologies. • CMV DNA load in BAL may predict attributable-pneumonia mortality., Summary Objectives To date no definitive cut-off value for cytomegalovirus (CMV) DNA load in bronchoalveolar lavage (BAL) fluid specimens has been established to discriminate between CMV pneumonia and pulmonary CMV DNA shedding in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients. Methods The current retrospective study is aimed at assessing the range of CMV DNA loads quantified in BAL fluid specimens from allo-HSCT patients with pneumonia in which different microorganisms were causally involved. Results A total of 144 BAL specimens from 123 patients were included. CMV DNA was detected in 56 out of 144 BAL fluid specimens and the median CMV DNA load from patients in whom CMV pneumonia was unlikely or could be tentatively ruled out was 1210 (31–68, 920) IU/ml. The frequency of CMV DNA detection and median CMV DNA loads were comparable, irrespective of the attributable cause of pneumonia. Detection of CMV DNA loads in BAL fluid specimens >500 IU/ml was independently associated with pneumonia-attributable mortality. Conclusions The current study highlights the difficulty in establishing universal CMV DNA load thresholds in BAL fluid specimens for distinguishing between CMV pneumonia and pulmonary CMV DNA shedding, and suggests that the presence of CMV DNA in BAL fluid specimens beyond a certain level may have a deleterious impact on patient outcome.

ObjectivesThe immunogenicity of the BNT162b2 COVID-19 vaccine is understudied in elderly people with comorbidities. We assessed SARS-CoV-2-S-targeted antibody and T cell responses following full vaccination in nursing home residents (NHR).MethodsWe recruited 60 NHR (44 female; median age, 87.5 years), of whom 10 had previously had COVID-19, and 18 healthy controls (15 female; median age, 48.5 years). Pre- and post-vaccination blood specimens were available for quantitation of total antibodies binding RBD and enumeration of SARS-CoV-2-S-reactive IFN-γ CD4+ and CD8+ T cells by flow cytometry.ResultsThe seroconversion rate in presumably SARS-CoV-2 naïve NHR (95.3%), either with or without comorbidities, was similar to controls (94.4%). A robust booster effect was documented in NHR with prior COVID-19. Plasma antibody levels were higher in convalescent NHR than in individuals across the other two groups. A large percentage of NHR had SARS-CoV-2 S-reactive IFN-γ CD8+ and/or CD4+ T cells at baseline, in contrast to healthy controls. Either CD8+ and/or CD4+ T-cell responses were documented in all control subjects after vaccination. Contrariwise, the percentage of NHR exhibiting detectable SARS-CoV-2 IFN-γ CD8+ or CD4+ T-cell responses (or both), irrespective of their baseline SARS-CoV-2 infection status, dropped consistently after vaccination. Overall, SARS-CoV-2 IFN-γ CD8+ and CD4+ T-cell responses in NHR decreased in post-vaccination specimens.ConclusionThe BNT162b2 COVID-19 vaccine elicits robust SARS-CoV-2-S antibody responses in NHR. Nevertheless, the frequency and magnitude of detectable SARS-CoV-2 IFN-γ T-cell responses after vaccination was lower in NHR compared to controls.

Cytomegalovirus (CMV) DNAemia and CMV disease have been reported as more frequent in patients undergoing haploidentical allogeneic hematopoietic stem cell transplantation (Haplo-HSCT) than in those receiving HLA-matched allografts. This could be due to impaired CMV-specific T-cell reconstitution. Here, we conducted a multicenter observational study to assess CMV pp65 and IE-1-specific T cells kinetics in patients undergoing unmanipulated Haplo-HSCT with posttransplant cyclophosphamide (PT/Cy-haplo) and compared it with patients allografted with HLA-matched donors. Plasma CMV DNA load was monitored by real-time PCR and enumeration of CMV-specific IFN-γ-producing CD8

Azacitidine (AZA) is a DNA hypomethylation agent administered in myeloid neoplasms; however, there is still a lack of established predictors of response. We studied 113 patients with myelodysplastic syndromes (n = 85) or acute myeloid leukemia (n = 28) who received AZA to assess the predictive value on response of clinical features, cytogenetics, and molecular markers. Overall, 46 patients (41%) responded to AZA. Platelet doubling after the first AZA cycle was associated with a better response (68% vs. 32% responders, P = 0.041). Co-occurrence of chromosome 7 abnormalities and 17p deletion was associated with a worse response (P = 0.039). Pre-treatment genetic mutations were detected in 98 patients (87%) and methylation of CDKN2B and DLC-1 promoters were detected in 50 (44%) and 37 patients (33%), respectively. Patients with SF3B1 mutations showed a better response to AZA (68% vs. 35% responders, P = 0.008). In contrast, subjects with mutations in transcription factors (RUNX1, SETBP1, NPM1) showed a worse response (20% vs. 47% responders, P = 0.014). DLC-1 methylation pre-treatment was associated with poor clinical features and its reduction post-treatment resulted in a better response to AZA in MDS patients (P = 0.037). In conclusion, we have identified several predictors of response to AZA that could help select the best candidates for this treatment.

Background Therapy-related acute myeloid leukemia (t-AML) develops in patients with prior exposure to cytotoxic therapies. Selection of a pre-existing TP53 mutated clone prone to acquire additional mutational events has been suggested as the main pathogenic mechanism of t-AML. Here, we report a unique case of t-AML which developed from a pre-existing DNMT3A mutated clone that persisted in the patient for more than 10 years despite treatment with intensive chemotherapy and allogeneic hematopoietic stem cell transplantation (alloHSCT). Case presentation A 42-year-old male was diagnosed with AML harboring a normal karyotype and mutations in the NPM1 (c.863_864ins, p.W288 fs*12), DNMT3A (c.2645G > A, p.R882H), and IDH1 (c.395G > A, p.R132H) genes. He achieved complete remission with intensive chemotherapy and was subsequently submitted to alloHSCT. Eleven years later, he was given chemotherapy and radiotherapy to treat a lung carcinoma. Three years later, t-AML was diagnosed; the disease had arisen from a pre-existing DNMT3A mutated patient-origin clone that had subsequently acquired a TP53 mutation and a complex karyotype. Although a second transplantation was intended, the disease was refractory to induction chemotherapy, and the patient eventually died from disease complications. We retrospectively demonstrated the persistence and post-transplantation latency of the R882H- DNMT3A mutation using a real-time PCR allele-specific analysis at different time-points during the observation period. Discussion and conclusion The present case highlights the potential clinical implications of a R882H- DNMT3A mutated clone that persisted after conventional AML treatment, including alloHSCT. It also reinforces the notion of the importance of cell non-intrinsic factors, such as the hematopoietic-stress induced by chemotherapy and radiotherapy, as drivers of clonal expansion.

To gauge the risk of delaying initiation of prophylaxis with letermovir from the time of donor infusion to prevent CMV infection in allo-HSCT recipients we investigated the clinical outcomes of CMV DNAemia episodes occurring before engraftment, and compared to that of episodes developing after engraftment (up to day +365). A total of 197 consecutive adult patients were included. Plasma CMV DNA load was monitored by real-time PCR assays [limit of detection: 31 IU/ml]. A total of 150 out of 197 patients had CMV DNAemia (cumulative incidence of 77%; 95% CI, 73-81%), and 38 out of the 197 patients developed it before engraftment (cumulative incidence, 19%; 95% CI, 10-30.3%). Nine episodes of CMV DNAemia were detected prior to the time of donor progenitor cell infusion. A greater number of post-engraftment episodes required preemptive antiviral therapy compared with pre-engraftment episodes (62.5% vs 44.7%; P = 0.05). The cellular content of the donor progenitor cell infusion and transplant characteristics of patients did not differ between patients with pre-engraftment or post-engraftment CMV DNAemia. The cumulative incidence of overall mortality by days 100 and 365, aGvHD by day 100 and relapse by day 365 were not significantly different between patients with pre-engraftment or post-engraftment CMV DNAemia.

Aim: To analyze the clinical profile and rates of stroke, major bleeding and intracranial hemorrhage of patients with atrial fibrillation (AF) treated with rivaroxaban in clinical practice. Methods: Retrospective study of AF patients anticoagulated with rivaroxaban in a Healthcare Area of Valencia, Spain. Patients started treatment with rivaroxaban from July 2012 to December 2015. Results: A total of 230 patients (mean age 76.9 ± 9.9 years; CHA2DS2-VASc 4.3 ± 1.7; HAS-BLED 1.7 ± 0.9) were included. Rates of stroke, major bleeding and intracranial bleeding were 0.4, 1.9 and 0.5 events per 100 patient-years, respectively. Conclusion: In this cohort of AF patients anticoagulated with rivaroxaban, despite patients being older and having a high thromboembolic risk, rates of stroke, major bleeding and intracranial bleeding were low.

There is evidence suggesting that cytomegalovirus (CMV) reactivation following allogeneic hematopoietic stem cell transplantation (allo-HSCT) may be associated with a lower relapse incidence in acu...

It has been recently shown that RAS mutations, which occur in 11-38% of Chronic Myelomonocytic Leukemia (CMML), do not only act via RAS/MEK/ERK signaling, but contribute to the disease through NLRP3 inflammasome activation (Hamarsheh, Nat Comm 2020). Despite a therapeutic approach based on NLRP3/IL1β axis blockade, as bring to a stem cell transplantation (SCT) has been proposed, data on the efficacy of IL1β inhibitors in hematopoietic neoplasms is limited. A 55 year old man with previous autoinflammatory episodes (constrictive pericarditis) was diagnosed on September 2020 of CMML-1 KRAS G12D (Inter-2). Due to worsening (orchiepidedymitis, pneumonitis, cellulitis), and the impossibility of performing an SCT at that time, on December 02 2020 he started anakinra (a IL1β receptor antagonist) with good response. Due to new episodes of autoinflammation, anakinra was discontinued (12 April 2021) with severe clinical worsening (heart failure) and no response to diuretic/corticosteroid. After anakinra was restarted (04 May 2021), a progressive improvement was seen, allowing a successful pericardiectomy before an SCT. We obtained blood samples from this patient (at different times) and plasma and whole blood samples from 11 and 5 other CMML KRAS mut patients, respectively. We also included CMML patients without KRAS mutations (KRAS wt) (n=8), with sepsis (n=5) and healthy individuals (n=9). Plasma levels of 15 inflammatory cytokines associated with NLRP3 inflammasome and NFkB pathways were measured using a customized MILLIPLEX ® kit. The inflammasome marker activation assays were conducted as previously published (Martínez García JJ, Nature Comm 2019). Compared to healthy controls, KRAS wt CMML patients did not show differences in any cytokine tested, except IL6, while KRAS mut patients showed significantly higher levels of IL1α, IL1ra, IL18, IL12p40 (associated with NLRP3 inflammasome), IL6, IL8 (associated with NFkB pathway) and M-CSF (Fig. 1A B). Compared to KRAS wt CMML patients, those with KRAS mut showed higher levels of cytokines associated with both the NLRP3 and NFkB pathways, reaching statistical significance for those related with NLRP3 inflammasome. We also observed changes in inflammasome related cytokines before and after anakinra (Table 1). This cytokine profile in the plasma made us analyze the oligomerization of ASC as a marker of inflammasome activation in monocytes of KRAS mut CMML. We found that in all cases of KRAS mut CMML patients around 30 to 80% of monocytes presented oligomers of ASC measured by the time of flight assay, while in healthy donors and KRAS wt CMML patients, ASC oligomerization occurred upon NLRP3 inflammasome activation with lipopolysaccharide (LPS) + ATP or Pyrin inflammasome activation with LPS and Clostridium difficile B toxin (TcdB) (Fig. 2A). Ex vivo activation of PBMCs from KRAS mut CMML patients showed that despite the high percentage of cells with ASC oligomers, very low levels of IL1b released from these cells, even when NLRP3 was activated with LPS+ATP (Fig. 2B), suggesting that this inflammasome is activated in vivo and could not be further activated ex vivo. As control, Pyrin inflammasome activation in PBMCs from KRAS mut CMML was able to induce IL1b release similarly to healthy controls (Fig. 2B). We then found that anakinra treatment of the KRAS mut CMML patient followed in this study, resulted in a decrease of the percentage of monocytes with basal active inflammasomes (Fig. 2C). A little ex vivo activation of the NLRP3 inflammasome was obtained when cells were treated with LPS+ATP, while Pyrin inflammasome was activated at normal levels after LPS+TcdB treatment (Fig. 2D). The inflammasome basal activation increased in the monocytes of the KRAS mut CMML patient after anakinra withdraw and during clinical deterioration and restarting anakinra (second arrow) decreased the basal percentage of monocytes with ASC oligomers (Fig. 2C). Since ASC oligomers are associated to pyroptosis via caspase 1 activation and gasdermin D processing, we then analyzed pyroptotic markers in the plasma of the patient during the time. ASC was increased when monocytes presented elevated percentage of ASC oligomers (Fig. 2E), suggesting that ASC detection could be a promising biomarker. Overall, we show that, in vivo, the NLRP3 inflammasome activation of KRAS mut CMML patients may revert with IL1β blockers. ASC could identify those candidates to receive this therapy. PI18/00316 Figure 1 Figure 1. Disclosures Jerez: Novartis: Consultancy; BMS: Consultancy; GILEAD: Research Funding. Bellosillo: Thermofisher Scientific: Consultancy, Speakers Bureau; Roche: Research Funding, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau. Hernández-Rivas: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ferrer Marin: Cty: Research Funding; Incyte: Consultancy, Research Funding; Novartis: Speakers Bureau.

Background: Adoptive cell immunotherapies for opportunistic virus in immunocompromised patients using haploidentical memory T cells have shown to be safe and effective. Since severe cases of COVID-19 present a dysregulated immune system with T cell lymphopenia and a hyper-inflammatory state we have proposed that a similar strategy could be proven to be efficient for COVID-19 patients. This is a study protocol of an open-label, multicenter, double-arm, randomized, dose-finding phase I/II clinical trial to evaluate the feasibility, safety, tolerability, and efficacy of the administration of a single dose of allogenic SARS-CoV-2 specific memory CD45RA - T cells and Natural Killer (NK) cells in COVID-19 patients with lymphopenia and pneumonia. The aim of the study is to find efficient treatments for patients with moderate/severe COVID-19. Identification of Specific memory T cells and NK cells: i)Memory T Cells: we first determined the existence of SARS-CoV-2 specific T cells within the CD45RA - T memory cells of the blood of convalescent donors. Memory T cells can respond quickly to the infection and provide long-term immune protection to reduce the severity of the COVID-19 symptoms without inducing classically T cell alloreactivity. Also, CD45RA - memory T cells confer protection for other pathogens the donors encountered in their life. ii)NK cells: we determined the phenotype of NK cells after COVID-19 and the main characteristic of SARS-CoV-2 specific NK population in the blood of convalescent donors, as it has been shown for cytomegalovirus infections. Also, NK cells confer protection for other pathogens the donors encountered in their life. Pilot Phase I- Safety, feasibility, and dose escalation: Between September and November 2020 a phase 1, dose-escalation, single-center clinical trial was conducted to evaluate the safety and feasibility of the infusion of CD45RA - memory T cells containing SARS-CoV-2 specific T cells as adoptive cell therapy against moderate/severe cases of COVID-19. Nine participants with pneumonia and/or lymphopenia and with at least one human leukocyte antigen (HLA) match with the donor were infused. The first three subjects received the lowest dose (1x10 5 cells/kg), the next three received the intermediate dose (5x10 5 cells/kg) and the last three received the highest dose (1x10 6 cells/kg) of CD45RA - memory T cells. Clinicaltrials.gov registration: NCT04578210. Findings: All participants' clinical status measured by National Early Warning Score (NEWS) and 7-category point ordinal scales showed improvement six days after infusion. No serious adverse events were reported. Inflammatory parameters were stabilized post-infusion and the participants showed lymphocyte recovery two weeks after the procedure. Donor microchimerism was observed at least for three weeks after infusion in all patients. Interpretation: This study provides preliminary evidence supporting the idea that treatment of COVID-19 patients with moderate/severe symptoms using convalescent SARS-CoV-2 specific CD45RA - memory T cells is feasible and safe. We did not find dose-liming toxicity. The Recommended Phase 2 dose was 1x10 6 CD45RA - T cells. Phase II- Efficacy: Between January 2021 and July 2021 patients have been enrolled based on the matched with the HLA genotype of the convalescent donors and following the protocol inclusion/exclusion criteria. The primary outcome is the incidence of patient recovery at day 14, defined as normalization of fever and oxygen saturation or lymphopenia recovery. Secondary outcomes are the time to normal level of lymphocytes, the proportion of patients showing clinical improvement at day 7, time to first negative SARS-CoV-2 PCR, the incidence of treatment-related adverse events, duration of hospitalization, time to discharge, time to improvement by one category a 7-point ordinal scale or NEWS score, the proportion of patients requiring intensive care unit, and all-cause mortality. In addition, lymphocyte recovery by multiparametric flow cytometry and donor chimerism by real-time PCR in the experimental arm was monitored weekly during the first month. This study provides preliminary evidence supporting the idea that treatment of COVID-19 patients with moderate/severe symptoms using convalescent CD45RA - memory T cells is safe and feasible. The phase II clinical trial is ongoing to demonstrate efficacy. Figure 1 Figure 1. Disclosures Soria: Celgene: Other: Fees; Gilead: Other: Fees; AbbVie: Other: Fees.

Patients with postransplant lymphoproliferative disease (PTLD) who are refractory to rituximab-based regimens have extremely poor prognosis. Data is lacking in the setting of solid organ transplantation (SOT)-related PTLD treated with chimeric antigen receptor T-cell (CAR-T) therapy. Moreover, limited information is available on the influence of concomitant immunosuppressive drugs on CAR-T function. Here, we describe the clinical outcome in one PTLD patient and propose a strategy for tailoring immunosuppressive treatment and organ monitoring in patients with kidney allografts after CAR-T infusion. This report also reviews the limited published data in the setting of SOT-related PTLD treated with CAR-T, which appears to be a feasible treatment in this clinical scenario, without severe toxicity and capable of inducing sustained responses. A noteworthy finding is that in most reported cases patients underwent complete or partial discontinuation of immunosuppressive drugs, with only one documented case of allograft rejection.

ObjectivesThere is an imperative need to determine the durability of adaptive immunity to SARS-CoV-2. We enumerated SARS-CoV-2-reactive CD4+ and CD8+ T cells targeting S1 and M proteins and measured RBD-specific serum IgG over a period of 2-6 months after symptoms onset in a cohort of subjects who had recovered from severe clinical forms of COVID-19.MethodsWe recruited 58 patients (38 males and 20 females; median age, 62.5 years), who had been hospitalized with bilateral pneumonia, 60% with one or more comorbidities. IgG antibodies binding to SARS-CoV-2 RBD were measured by ELISA. SARS-CoV-2-reactive CD69+-expressing-IFNγ-producing-CD4+ and CD8+ T cells were enumerated in heparinized whole blood by flow cytometry for ICS.ResultsDetectable SARS-CoV-2-S1/M-reactive CD69+-IFN-γ CD4+ and CD8+ T cells were displayed in 17 (29.3%) and 6 (10.3%) subjects respectively, at a median of 84 days after onset of symptoms (range, 58-191 days). Concurrent comorbidities increased the risk (OR, 3.15; 95% CI, 1.03-9.61; P=0.04) of undetectable T-cell responses in models adjusted for age, sex and hospitalization ward. Twenty-one out of the 35 patients (60%) had detectable RBD-specific serum IgGs at a median of 118 days (range, 60 to 145 days) after symptoms onset. SARS-CoV-2 RBD-specific IgG serum levels were found to drop significantly over time.ConclusionA relatively limited number of subjects who developed severe forms of COVID-19 had detectable SARS-CoV-2-S1/M IFNγ CD4+ and CD8+ T cells at midterm after clinical diagnosis. Our data also indicated that serum levels of RBD-specific IgGs decline over time, becoming undetectable in some patients.

Background/aim Azacitidine (AZA) is a hypomethylating agent used in myeloid neoplasms, however, approximately half of patients show treatment failure or relapse. This in vitro study investigated the effect of the combination of AZA with the natural compound curcumin (CUR) in increasing its efficacy. Materials and methods We analyzed the effects of AZA plus CUR on proliferation, apoptosis, cell cycle and differentiation in myeloid leukemic cell lines (U-937, HL-60, K-562, and OCI-AML3) and bone marrow samples of patients. Results The results showed a synergy between AZA and CUR in all leukemic lines and in most leukemic samples, with a decrease in proliferation and an increase in apoptosis compared to the activity of each drug separately. In addition, AZA plus CUR showed low cytotoxicity in healthy samples. Conclusion A remarkable antioncogenic effect of the combination of AZA plus CUR was shown, providing a basis for future studies analyzing the clinical efficacy of these drugs.

Cytomegalovirus (CMV) DNAemia occurs frequently in CMV-seropositive allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients, and usually results from reactivation of latent infection established in the recipient. Predicting the occurrence of CMV DNAemia may be helpful in managing CMV infection in allo-HSCT recipients. Here, the kinetics of several inflammatory biomarkers in plasma were characterized and assessed for their potential value in anticipating the development and features of active CMV infection in allo-HSCT recipients, as documented using real-time PCR assays. The cohort consisted of 46 non-consecutive adult patients who underwent T-cell replete allo-HSCT at our center. Plasma levels of C-reactive protein (CRP), soluble tumor necrosis factor receptor type 2 (sTNF-R2), transforming growth factor-β1 (TGF-β1), and interferon-inducible protein 10 (IP-10/CXCL10) were measured in consecutive specimens obtained from conditioning either by nephelometry (CRP) or by specific immunoassays (the rest). Of the 46 patients, 22 had a first episode of CMV DNAemia at a median of 34 days after allo-HSCT (range, day 19-day 50). We found that both the TGF-β1 area under a curve (AUC) and peak levels were significantly lower in patients who subsequently developed CMV DNAemia than in patients with no CMV DNAemia. Interestingly, CRP but not TGF-β1 AUC and peak levels predicted the occurrence of CMV DNAemia episodes requiring preemptive antiviral therapy. The data presented herein suggest that kinetics of inflammatory biomarkers in plasma might be useful to anticipate post-engraftment CMV DNAemia episodes and predict the need for preemptive antiviral therapy in allo-HSCT recipients.

Summary Pathogenic interactions between bacteria and cytomegalovirus (CMV) may potentially occur early after allogeneic stem cell transplantation (Allo-SCT). This possibility nevertheless has not been investigated in depth. This was a retrospective study that included 170 consecutive patients who underwent 173 Allo-SCTs. Both bacterial infection (most of which were bacteremic) and CMV DNAemia were detected in 78 Allo-SCTs (62.9%). In total, 51 and 32 episodes of bacterial infection preceded or occurred after CMV DNAemia detection, respectively. Both events were diagnosed concurrently in four Allo-SCTs. The cumulative incidence of bacterial infection (of any type) over the study period was comparable in patients with or without a preceding episode of CMV DNAemia (P=0.321). Cox proportional hazards regression analysis failed to identify CMV DNAemia as a significant risk factor for bacterial infection. Likewise, the cumulative incidence CMV DNAemia within the study period was not significantly different in patients with or without a preceding episode of bacterial infection (P=0.189). Furthermore, the occurrence of bacterial infection within episodes of active CMV infection had no apparent impact on the kinetics of CMV DNAemia. Our data, thus, do not support the existence of a bidirectional synergistic effect between bacterial infection and active CMV infection in the Allo-SCT setting. This article is protected by copyright. All rights reserved.

Introduction : Azacitidine (AZA) is a DNA hypomethylating agent used in myeloid neoplasms, however approximately half of patients show treatment failure or relapse. Last years, several studies have showed that genetic mutations may influence on response and survival of the treated patients. Other biomarkers that have traditionally been associated with the response to AZA are the recovery of the platelet count and the presence of abnormalities in the chromosome 7. Finally, the methylation dynamics of genes promoters could be a useful tool to predict the clinical response. Aim: To assess the predictive value on response to AZA of clinical features, cytogenetics, genetic mutations and the methylation dynamics of CDKN2B and DLC-1 promoters in a series of patients with myeloid neoplasms. Methods: We studied 85 patients with myelodysplastic syndromes (MDS) and 28 patients with acute myeloid leukemia (AML) who received AZA. Treatment schedules included 5-azacytidine 75 mg/m2/day for 7 days (n=62) and 5-azacytidine 75 mg/m2/day for 5 days (n=51). According to the International Working Group (IWG) 2006 criteria for MDS, responders were defined as patients achieving complete remission (CR), bone marrow CR (mCR), partial remission (PR) or stable disease with hematologic improvement (SDHI). Sixteen genes with prior implication in the pathogenesis of myeloid diseases were analyzed: NPM1, FLT3, CEBPA, TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, RUNX1, SETBP1, TP53, SF3B1, SRSF2, U2AF1 and ETNK1. For methylation assay, EZ DNA Methylation-Gold kit (Zymo Research) was employed for the bisulfite treatment of DNA. Methylation of CDKN2B and DLC-1 were qualitatively studied pre- and post-AZA with unmethylated and methylated primers and analysis of the PCR products in a QIAxcel System (Qiagen). Overall survival (OS) was measured from start of AZA treatment until the last follow-up or death from any cause. P values Results: Of the 113 patients analyzed, 46 patients (41%) showed response to AZA (median cycles=6): CR=19, mCR=6, PR=12, and SDHI=9. Stable disease and treatment failure were observed in 20 (17%) and 47 patients (42%), respectively. Analysis of clinical features showed that platelet doubling after the first AZA cycle was significantly associated with a better response (58% vs. 31% responders, P=0.041). Forty-seven patients (42%) had chromosomal abnormalities. A positive co-occurrence between del(7q)/-7 and del(17p) was found (n=6, P5%) of bone marrow blasts (63% vs. 40%, P=0.016), higher frequency of complex karyotypes (40% vs. 15%, P=0.002), a positive co-occurrence with TP53 alterations (38% vs. 16%, P=0.009) and its methylation dynamics showed a strong trend towards predict the response to AZA: 57% of patients without DLC-1 methylation or who reduced it post-treatment responded favourably to the drug and 65% of patients who maintained or increased it did not respond (P=0.053). In addition, leukemia transformation was higher in MDS patients with DLC-1 methylation post-AZA than in the remaining MDS patients (58% vs. 21%, P=0.002). Conclusion: The combination of chromosomal alterations and genetic mutations together with the monitoring of platelet count and DLC-1 methylation could contribute to give light in myeloid neoplasms to the heterogeneity observed both in the response to AZA and in patient survival. Samples and data from patients included in this study were provided by the INCLIVA Biobank (PT13/0010/0004). Disclosures Hernandez Boluda: Incyte: Other: Travel expenses paid.

The role of cytomegalovirus (CMV)-specific polyfunctional CD8+ T-cells and that of antibodies neutralizing virus epithelial infection (AbNEI) in the control of CMV DNAemia were investigated in 39 CMV-seropositive allogeneic stem-cell transplant (Allo-SCT) recipients with (n = 24) or without (n = 15) CMV DNAemia. AbNEI levels were monitored prospectively by means of a neutralization assay employing retinal epithelial cells (ARPE-19) and the recombinant CMV strain BADrUL131-Y4. Quantification of CMV-specific polyfunctional CD8+ T-cells (expressing two or three of the following markers: IFN-γγ, TNF-α and CD107a) in whole blood was performed by flow cytometry for intracellular cytokine staining. We found no differences in the dynamic pattern of AbNEI in patients with or without subsequent CMV DNAemia. Baseline and peak AbNEI titres were not predictive of the dynamics of CMV replication within episodes. No correlation was found between CMV DNA loads and AbNEI levels during episodes of CMV DNAemia (ρ = 0.09; 95 % confidence interval − 0.52 to 0.64; P = 0.78). The detection of pp65/IE-1 CMV-specific polyfunctional CD8+ T-cells was associated with low-level virus replication within subsequent episodes of CMV DNAemia. Interestingly, the presence of AbNEI titres (inverse) >4.7 log2 was predictive of the occurrence of CMV DNAemia (sensitivity, 83 %; specificity, 80 %). Our findings provide an insight to the role of humoral and cellular immunity in the control of CMV infection in an Allo-SCT setting.

Objectives Population-based studies have reported an increased incidence of skin cancer in patients with essential thrombocythemia (ET) and polycythemia vera (PV). We have examined the risk factors for non-melanoma skin cancer (NMSC) in patients diagnosed with ET or PV during 1973–2012. Methods A case–control study was performed to compare the clinical and treatment-related data of 51 ET/PV patients who had NMSC with that of 401 patients who did not. We also evaluated whether polymorphisms in 12 genes involved in DNA integrity predisposed to NMSC. Results By multivariate logistic regression analysis, risk factors for NMSC were older age (OR: 1.7, 95% CI: 1.3–2.1, P

Background The functional profile of cytomegalovirus (CMV)-specific CD8+ T cells that associate with protection from and control of CMV DNAemia in allogeneic stem cell transplant (allo-SCT) recipients remains incompletely characterized. Methods We enumerated pp65 and immediate early (IE)-1-specific CD8+ T cells expressing interferon-gamma, tumor necrosis factor-alpha, and CD107a, by flow cytometry in 94 patients at days +30 and +60 after allo-SCT. Results Fifty of 94 patients had CMV DNAemia within the first 100 days after transplant. CMV-specific CD8+ T-cell responses (of any functional type) were more likely to be detected in patients who did not display CMV DNAemia than in those who did (P = 0.04). Qualitatively, no major differences in the functional signature of CMV-specific CD8+ T cells were noted between patients who had or did not have CMV DNAemia. Patients displaying levels of polyfunctional CD8+ T cells at day +30 >0.30 cell/μL had a lower risk of CMV DNAemia (positive predictive value 76%, and negative predictive value 43%). Conclusion The presence of polyfunctional CD8+ T cells (either expressing CD107a or not) was associated with lower levels of CMV replication, and higher frequency of self-resolved episodes. The data reported further clarify the role of polyfunctional CD8+ T cells in control of CMV DNAemia in allo-SCT recipients.

The role of Natural killer (NK) cells in the control of cytomegalovirus (CMV) infection in allogeneic stem cell transplant recipients has not been precisely characterized. The current study is aimed at investigating the potential role of NK cells expressing the activating receptor NKG2C in affording protection against the development of CMV DNAemia in patients exhibiting detectable CMV-specific CD8+ T-cell responses early following transplantation. A total of 61 nonconsecutive patients were included in the study. Peripheral levels of CD56brightCD16−/low and CD56dimCD16+ NKG2C+ NK cells and CMV pp65/IE-1-specific IFN-γ-producing CD8+ T-cells were enumerated by flow cytometry at days +30 and +60 after transplant. Neither the absolute number of NKG2C+ NK cells, nor that of CD56brightCD16−/low and CD56dimCD16+ NKG2C+ NK-cell subsets at day 30 differed significantly between patients with or without subsequent CMV DNAemia. No significant correlation was found between levels of both NKG2C+ NK-cell populations and the peak CMV DNA load within subsequent episodes of CMV DNAemia. The data indicate that enumeration of NKG2C+ NK cells early after transplant is unlikely to be helpful in identifying those patients at highest risk of developing CMV DNAemia. Moreover, the data do not support a direct implication of NKG2C+ NK cells in preventing the development of CMV DNAemia. J. Med. Virol. 87:1601–1607, 2015. © 2015 Wiley Periodicals, Inc.

This is an observational-retrospective study comparing the real-world outcomes associated with posaconazole vs. itraconazole as prophylaxis treatments. Two hundred and ninety-three patient admissions attributable to 174 patients were included in the study. Patients were treated with itraconazole (n = 114 admissions; 39%) or posaconazole (n = 179; 61%). Antifungal prophylaxis failure (APF) due to treatment-related adverse events (in 34 out of 293 patient admissions; 11.6%) was more frequent in the posaconazole group (6.1% vs. 15.1%; P = 0.024). There were 9 patient admissions for episodes of APF due to probable/proven breakthrough fungal infection (primary endpoint): 6 and 3 in the itraconazole and posaconazole group, respectively (5.3% vs. 1.7%; P = 0.095). All of them were associated with invasive pulmonary aspergillosis (IPA). APF was more frequent with itraconazole (65% vs. 30%; P < 0.001), along with failure due to possible/probable/proven IPA (25% vs. 10%; P = 0.002) and overall failure by any of the three different causes of prophylaxis failure (70% vs. 38%; P < 0.001). In agreement with clinical trial data, this real-world evidence supports the use of posaconazole over itraconazole in AML or MDS patients undergoing intensive chemotherapy. This article is protected by copyright. All rights reserved.

When should preemptive antiviral therapy for active CMV infection be withdrawn from allogeneic stem cell transplant recipients?

Background We conducted a retrospective single-center study to investigate the potential impact of cytomegalovirus (CMV) DNAemia on mortality in allogeneic stem cell transplant recipients (allo-SCT). Methods A total of 151 consecutive patients who underwent T-cell replete allo-SCT were included in the study. Patients with CMV DNAemia were treated pre-emptively with antivirals upon detection of plasma CMV DNA loads >1500 IU/mL. Results At least one episode of CMV DNAemia occurred in 109 (72.2%), and 67 of these patients (61.5%) required one or more courses of antiviral therapy. The cumulative incidence of 1-year overall and non-relapse mortality (NRM) was 28.5% (95% confidence interval [CI], 18.4%-39.5%) and 23.2% (95% CI 12.81%-35.4%), respectively. The occurrence of either CMV DNAemia or CMV recurrences had no apparent effect on 1-year overall mortality and NRM; nevertheless, a trend towards an increased risk of death was seen in patients with one or more episodes of CMV DNAemia requiring antiviral therapy (hazard ratio [HR], 2.10; 95% CI, 0.96-4.61; P=0.06 for overall mortality, and HR, 2.36; 95% CI, 0.96-5.76; P=0.06 for NRM) but not in those displaying one or more self-resolving episodes. Conclusion Therefore, the data suggest that withholding pre-emptive antiviral therapy until the plasma CMV DNA load reaches 1500 IU/mL has no apparent detrimental effect on patient survival. This article is protected by copyright. All rights reserved.

Background Torque Teno virus (TTV) plasma DNA load directly correlate with the level of immunosuppresion in different clinical settings. It is uncertain whether this may be the case in allogeneic hematopoietic stem cell transplant recipients (allo-HSCT). Objectives We characterized the dynamics of TTV DNAemia in patients undergoing T-cell replete allo-SCT. Study design Retrospective single-center observational study including 72 allo-HSCT patients. Plasma TTV DNA loads were quantified before initiating the conditioning regimen and at different time-points after transplant by real-time PCR. White blood cells (WBC) and absolute lymphocyte counts (ALC) were measured by flow cytometry. Results A dramatic drop in plasma TTV DNA load was observed shortly after conditioning. The TTV DNA load increased steadily after engraftment reaching its peak at day +90 after transplant. The increase in TTV DNA load paralleled that of ALC, and was of greater magnitude in patients who developed severe (grades II–IV) acute graft vs. host disease. Conclusion Repopulation of lymphocytes early after allo-HSCT correlates with an increase of plasma TTV DNA load. Prospective studies are nevertheless needed to determine whether the kinetics of TTV DNAemia may allow inference of the degree of overall immunocompetence in these patients.