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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.

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.

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.

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.

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.

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.

When should preemptive antiviral therapy for active CMV infection be withdrawn from allogeneic stem cell transplant recipients?

It is uncertain whether monitoring plasma ganciclovir (GCV) levels is useful in predicting cytomegalovirus (CMV) DNAemia clearance in preemptively treated allogeneic stem cell transplant recipients. In this observational study, including 13 episodes of CMV DNAemia treated with intravenous (i.v.) GCV or oral valganciclovir, we showed that monitoring trough plasma GCV levels does not reliably predict response to therapy. Rather, immunological monitoring (pp65 and immediate-early [IE]-1-specific gamma interferon [IFN-γ]-producing CD8 + T cells) appeared to perform better for this purpose.

Objective Previous studies on monitoring of post-transplant cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) are limited by single-centre designs and disparate risk categories. We aimed to assess the clinical value of a regular monitoring strategy in a large multicentre cohort of intermediate-risk kidney transplant (KT) recipients. Methods We recruited 124 CMV-seropositive KT recipients with no T-cell-depleting induction pre-emptively managed at four Spanish institutions. CMV-specific interferon-γ-producing CD4+ and CD8+ T cells were counted through the first post-transplant year by intracellular cytokine staining after stimulation with pp65 and immediate early-1 peptides (mean of six measurements per patient). The primary outcome was the occurrence of any CMV event (asymptomatic infection and/or disease). Optimal cut-off values for CMV-specific T cells were calculated at baseline and day 15. Results Twelve-month cumulative incidence of CMV infection and/or disease was 47.6%. Patients with pre-transplant CMV-specific CD8+ T-cell count Conclusions Monitoring for CMV-specific CMI in intermediate-risk KT recipients must be regular to reflect dynamic changes in overall immunosuppression and individual susceptibility. The early assessment at post-transplant day 15 remains particularly informative.

Patients with essential thrombocythemia (ET) and polycythemia vera (PV) have an increased incidence of acute myeloid leukemia and new nonhematologic malignancies compared with the general population. However, information on the factors determining the risk for such complications is limited. In the present study, we investigated whether constitutional genetic variations in DNA repair predispose to leukemic transformation and new nonmyeloid neoplasias in patients with ET and PV. Case-control studies for predisposition to both types of malignancies were nested in a cohort of 422 subjects diagnosed with ET or PV during the period 1973-2010 in several institutions in Spain. A total of 64 incidence cases of leukemia and 50 cases of primary nonmyeloid cancers were accrued. At conditional regression analysis, the Gln/Gln genotype in the XPD codon 751 showed the strongest association with both leukemic transformation (odds ratio [OR] = 4.9; 95% confidence interval [95% CI], 2.0-12) and development of nonmyeloid malignancies (OR = 4.2; 95% CI, 1.5-12). Additional predictive factors were exposure to cytoreductive agents for leukemic transformation (OR = 3.5; 95% CI, 2.0-6.2) and age for nonmyeloid malignancies (OR = 2.0; 95% CI, 1.4-2.8). These findings provide further evidence about the contribution of inherited genetic variations to the pathogenesis and clinical course of myeloproliferative neoplasms.

We report that in a population of allogeneic stem cell transplant recipients, determination of the viral doubling time (dt) of the cytomegalovirus (CMV) DNA plasma load predicted the eventual need for inception of preemptive antiviral therapy, whereas the level of the initial plasma CMV DNA load did not. The data thus indicated that determination of the dt of CMV DNA may be useful in the therapeutic management of CMV infection in this clinical setting.

Standardized criteria of response to treatment and a unified definition of resistance/intolerance to hydroxycarbamide (HC) in essential thrombocythaemia (ET) have been proposed by the European LeukaemiaNet (ELN). We have retrospectively evaluated such criteria in 166 ET patients treated with HC for a median of 4·5 years. Overall, 134 patients achieved a complete clinicohaematological response (CR) and 25 a partial response. Thirty-three patients met at least one of the ELN criteria defining resistance (n = 15) or intolerance (n = 21) to HC. Fifteen cases developed anaemia with thrombocytosis, which was associated with a high incidence of myelofibrosis and death from any cause. Other definitions of resistance were less useful. Factors determining the thrombotic risk were a history of prior thrombosis and a baseline leucocyte count >10 × 10⁹/ l. Of note, patients achieving a CR, even if sustained during the entire follow-up, did not benefit from a lower incidence of thrombosis or an improved survival. In conclusion, most ET patients respond to HC, but the achievement of response, as defined by the ELN, does not correlate with the patients' outcome. The best discriminating ELN criterion of resistance to HC was the detection of anaemia, which also identified a subgroup of patients with poor prognosis.

Metabolomics analysis of biofluids is increasingly being recognized as a useful tool for the diagnosis and management of a number of infectious diseases. Here we showed that plasma metabolomics profiling by untargeted 1H nuclear magnetic resonance may allow the anticipation of the occurrence of cytomegalovirus (CMV) DNAemia in allogeneic stem cell transplant. For this purpose, key discriminatory metabolites were total glutathione, taurine, methylamine, trimethylamine N-oxide and lactate, all of which were upregulated in patients eventually developing CMV DNAemia. The overall classification accuracy (predictability) of the projection to latent structure discriminant analysis (PLS-DA) model in cross-validation technical replicates was 73 %. Increased levels of alanine, lactate and total fatty acids, and a shift in the fatty acid profile towards unsaturated species, were observed in patients with detectable CMV DNA in plasma. The classification accuracy of this PLS-DA model in cross-validation technical replicates was 81 %. Plasma metabolomics profiling may prove useful for identifying patients at highest risk for CMV DNAemia thus allowing early inception of antiviral therapy.

Pre-emptive antiviral therapy for active CMV infection in adult allo-SCT patients guided by plasma CMV DNAemia quantitation using a real-time PCR assay: clinical experience at a single center

The performance of the QuantiFERON-cytomegalovirus (CMV) assay was compared to that of a flow cytometry intracellular cytokine staining (ICS) method for the detection of CMV-specific gamma interferon (IFN-γ)-producing CD8+T-cell responses in allogeneic stem cell transplant (allo-SCT) recipients and for estimations of their magnitude and functionality. A total of 90 whole-blood specimens from 23 allo-SCT recipients was analyzed by both methods. Overall, the percentage of specimens that yielded concordant results by both methods was 68.8% (κ = 0.691; 95% confidence interval [CI], 0.548 to 0.835), and the sensitivity of the QuantiFERON-CMV assay for the detection of positive IFN-γ T-cell responses (>0.2 IU/ml), taking the ICS method as the reference, was 76.3%. The magnitude of IFN-γ-producing CD8+T-cell responses to CMV-specific peptides measured with the QuantiFERON-CMV assay correlated significantly (σ = 0.695;P= +T cells and dual-functional (IFN-γ/tumor necrosis factor alpha [TNF-α] [σ = 0.652;P= P= P= >0.001]) CMV-specific CD8+T-cell responses, as quantitated by ICS. In summary, the data indicated that the QuantiFERON-CMV assay is less sensitive than the ICS method for the detection of CMV-specific IFN-γ-producing CD8+T-cell responses in the allo-SCT setting. Nevertheless, it allowed the estimation of the total and polyfunctional CMV-specific IFN-γ-producing CD8+T-cell responses in specimens that tested positive by both methods.

Abstract 3128 Background: follicular lymphoma is an incurable, long-lasting disease with an heterogeneous outcome. Several prognostic systems have been proposed, and recently a new one, the FLIPI2 score based on five parameters has been published. However, in order to confirm its prognostic utility, further studies at other centers are highly recommendable. Aim: to validate the new FLIPI2 score in independent series of follicular lymphoma patients diagnosed at our institution between February 1990 and July 2010. Patients and methods. We considered 180 patients consecutively diagnosed with follicular diagnosis in the period described and from whom all variables required were available. The variables included were: beta2microglobulin higher than the upper normal value, longest diameter of the largest involved node longer than 6 cm, bone marrow infiltration, hemoglobin level lower than 120 g/L and age older than 60 years (one point if present). Three risk groups were identified: low risk (0 points), intermediate risk (1 -2) and high risk (3 or more) Progression-free survival was measured from date of treatment until date of progression or death from any cause. Continuous variables were summarized as median and range, categorical variables reported as counts, and PFS and OS carried out using the Kaplan-Meier method and curves compared by the log-rank test. Results: median age was 55 years (range, 24 to 77), male sex 92 (51%), Ann Arbor Stage I-II: 32(18%), III-IV: 143 (82%), age > 60 y 70 (39%), Hb < 120 g/L 38 (21%), β2microglobulin > UNV: 45 (25%), LDH > UNV: 34 (19%), bone marrow infiltration 82 (48%), longer diameter of the largest involved node > 6 cm 64 (36%). 47 patients (26%) received rituximab-containing regimens and 124 received conventional chemotherapy regimens (pre-rituximab era). Median follow-up of the series was 66.9 months (range,1.3-221). Using the FLIPI score (n=162) 58 patients (36%) were in the low risk group, 54 (33%) were in the intermediate group and 50 (31%) in the high risk group. Using the FLIPI2 (n=180) 36 patients (20%) were in the low risk group, 103 (57%) in the intermediate group and 41 (23%) in the high risk group. According to FLIPI 5y- PFS rate was 79% for the low risk group, 63% for the intermediate group and 32% for the high risk group, p < 0.001. According to FLIPI2 score, 5y-PFS rate was 82% for the low risk, 54% for the intermediate and 43% for the high risk groups, p=0.017. Concerning OS, applying the FLIPI, 5y-OS rate for the low, intermediate and high risk groups were 94%m 84% and 64%, respectively, p=0.003. Using the FLIPI2, 5y-OS for the low, intermediate and high risk groups were 96%, 80% and 67% respectively, p=0.006. Conclusions: in our experience the FLIPI2 score is a reproducible prognostic index in patients with follicular lymphoma although the FLIPI score seems to discriminate better between groups than the FLIPI2 score. Disclosures: No relevant conflicts of interest to declare.

Abstract 4086 Definition of resistance/intolerance to hydroxyurea (HU) in essential thrombocythemia (ET) has been proposed by the European LeukemiaNet (ELN) however its clinical utilility has not been validated yet. We have retrospectively evaluated such criteria in 166 ET patients treated with HU for a median of 4.5 years. Response to HU treatment was categorized using the ELN criteria. The ELN definitions of resistance/intolerance to HU required the fulfillment of at least one of the following criteria: platelet count greater than 600 × 109/L after 3 months of at least 2 g/day of HU (2.5 g/day in patients with a body weight over 80 kg); platelet count greater than 400 × 109/L and leukocytes less than 2.5 × 109/L or hemoglobin (Hb) less than 100 g/L at any dose of HU; presence of leg ulcers or other unacceptable mucocutaneous manifestations at any dose of HU; HU-related fever. Survival and time-to-event curves were estimated using the Kaplan-Meier method.Variab les attaining a significant level at the univariate analysis were included in a Cox proportional hazards model. Overall, 134 patients achieved a complete clinicohematologic response (CR) and 25 a partial response. Thirty-three patients met at least one of the ELN criteria defining resistance (n=15) or intolerance (n=21) to HU. Fifteen cases developed anemia with thrombocytosis. Other definitions of resistance were less useful. When compared with the others, resistant patients were more likely to display hyperproliferative features at ET diagnosis, such as higher levels of leukocytes (p= 0.05), platelets (p=0.004) and serum LDH (p=0.02). Eleven patients developed leg ulcers leading to a permanent discontinuation of the drug in 8 cases. No distinctive clinical profile could be ascribed to patients exhibiting leg ulcers, with the exception of a high prevalence of cardiovascular risk factors. Other unacceptable mucocutaneous manifestations occurred in 9 patients. Hematologic and mucocutaneous complications were unrelated, with only two patients presenting both types of toxicities. With a median follow-up from ET diagnosis of 7 years (range: 0.5–23), 38 patients (23%) had died, resulting in a survival probability of 65% at 10 years from HU start. The risk of death from any cause was increased by 6.2-fold (95%CI: 2.3–16.7, P0.001). In conclusion, the best discriminating ELN criterion of resistance to HU is based on the detection of anemia. Moreover, such criterion is particularly useful since it also identifies a subset of ET patients with a poor prognosis. Disclosures: No relevant conflicts of interest to declare.