ALBERT

Description: Fluorescent cell barcoding (FCB) is a high-throughput multiplexing technique, in which samples from one or more donors can be combined, minimizing staining variability, antibody consumption, and decreasing sample volumes needed. In this study, we optimized FCB technique for routine application in immunophenotyping, phosphoFlow, and intracellular cytokine detection. Human peripheral blood mononuclear cells (PBMCs) from healthy controls and patients were used for optimization, and CBD450 and CBD500, Pacific Orange succinimidyl ester, DyLight 350, and DyLight 800 were tested for barcoding 6 to 36 samples. Working concentrations ranging from 0 to 125 µg/mL were tested, and viability dye staining was also optimized to increase data robustness. We first measured fluorescence intensity (MFIs) of six serial dilutions (0, 0.75, 3.25, 12.5, 62.5 and 125 µg/mL) and the difference in intensity separating them. Second, a 3x3 matrix was prepared using three concentrations of two different dyes, and PMBCs were barcoded with nine different combinations of FCB dye concentrations. When gated with the dyes, nine lymphocyte populations were detected. A 4x(3x3) matrix was also designed using three different FCB dyes (CBD450, DyLight 800, and Pacific Orange) at various concentrations to barcode 36 samples simultaneously. Thus, each lymphocyte population was clearly identified. The separation of each population or purity of deconvolution is based on the distance between MFIs and CVs. When we calculated the purity of deconvolution in our experiments, human PBMCs displayed CVs of 10 - 25%. When MFIs were separated by 2-fold increase, barcoded populations could be identified with a good resolution, higher when MFIs were separated by 3- or more fold increase. The use of viability dyes as LIVE/DEAD Fixable Aqua viability dye together with the FCB showed increase of data robustness due to exclusion of dead cells from gating strategy. In combination with viability dye, we successfully performed a six-color phosphoFlow and a simple four-color stainings using the same FCB dye combinations and cytometer voltages, and also combining one healthy control and two different patient samples. Our methods using FCB dye alone and/or in combination with antibody staining should be useful to efficiently perform multiplex drug screening and lymphocyte characterization. FCB minimizes batches and technical variations, and also increases data robustness, thus improving the immune phenotyping of patients. Disclosures Young: GSK/Novartis: Research Funding.

Description: Eltrombopag (EPAG), a thrombopoietin receptor agonist, has been shown to improve hematopoiesis in patients with aplastic anemia (AA), but in MDS patients the effect of thrombopoietin mimetics in bone marrow function is still unclear. In this phase-2 dose escalation study, we investigated the safety and effectiveness of EPAG treatment in low to intermediate-2 risk MDS patients (NCT 00961064). Thirty patients were enrolled from March 2011 to July 2017. Preceding enrollment the majority of patients were either diagnosed with AA (n=13) or hypoplastic MDS (n=5). EPAG was started at 50 mg/day, up to a maximal dose of 150 mg/day, increasing the dose by 25mg every 2 weeks. The primary endpoint was hematologic response at 16 or 20 weeks, defined as either: (1) an increase in platelet counts ≥20.000/uL or transfusion independence for a minimum of 8 weeks; (2) hemoglobin (Hb) increase of ≥1.5g/dL from baseline, or a reduction in red blood cells (RBC) transfusion of at least 50%; or (3) an increase in absolute neutrophil counts (ANC) of ≥0.5x109/L or by at least 100% in patients with a baseline ANC 10g/dl, and thrombocytes 〉50.000/L, and ANC〉1000/L. However, peripheral blood cell counts significantly declined in 5/10 RR and EPAG was restarted per protocol. In 4 of these patients peripheral blood cell counts recovered. One patient did not achieve a second response. Based on International Prognostic Score System (IPSS), 4/30 (13%) patients progressed on study, including 3 non-responders and 1 responder, at a median follow-up of 4 months (3-35 months). The responding patient was diagnosed with increased bone marrow myeloblast 7 months after discontinuation of EPAG for robust response and 35 months after enrolling in the study. New cytogenetic abnormalities determined progression in non-responding patients (Figure). Novel dose limiting toxicities were not observed. Three patients developed CTCAE grade III hepatic toxicities. One of them discontinued EPAG at 3 months. Elevated transaminases returned to baseline after EPAG discontinuation in 2 patients. In both cases EPAG was resumed either at the same (150mg/day) or reduced dose (50mg/day) level. There were no treatment-related death cases. One patient died on study before the primary endpoint from acute respiratory distress syndrome. Sequential acquisition of genomic aberrations has been associated with malignant transformation. Targeting next-generation sequencing for somatic variants in genes previously associated with myeloid malignancies (Myeloid cancer genes, MCG) was performed in 29/30 patients with sufficient material (bone marrow mononuclear cells) available from baseline, primary endpoint, and at time of progression. At baseline, 22/29 (76%) patients were found with at least one mutation:TET2 (14.5%), ASXL1 (12.5%), SF3B1 (8.3%), SETBP1 (8.3%), ATM (8.3%), and ZRSR2 (8.3%). After EPAG, additional somatic variants in different genes were detected in 4/14 responders and 7/16 non-responders. Variants present at baseline were no longer detected in post EPAG samples from 4 responding and 6 non-responding patients. The VAF of variants detected at both time points were similar, indicating no selective expansion of clones with EPAG in neither responder, non-responder nor patients with progression based on IPSS. In conclusion, our results suggest that EPAG is well-tolerated and effective in restoring hematopoiesis in patients with low to intermediate-2 risk MDS, particular with a prior history of hypoplastic bone marrow failure syndromes. EPAG was discontinued for robust response in the majority of responders but declining blood cell counts were observed in about 50% of them. Variants in MCG were more common at study entry compared to patients with aplastic anemia (Yoshizato, NEJM, 2015). However, EPAG appears not to selectively promote expansion of clones harboring MCGs in this patient population. Disclosures Townsley: National Institute of Health: Research Funding. Scheinberg:Pfizer: Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Janssen: Honoraria, Research Funding. Dunbar:National Institute of Health: Research Funding. Young:GlaxoSmithKline: Research Funding; CRADA with Novartis: Research Funding; National Institute of Health: Research Funding. Winkler:National Institute of Health: Research Funding.

Description: About 30% of patients with newly diagnosed multiple myeloma (NDMM) are older than 75 years. Immunomodulatory drugs (IMIDs) have improved response rates and outcomes of NDMM, except for patients older than 75 years more vulnerable to side effects of IMIDs because of their frailty and comorbidities. We evaluated efficacy, toxicity and health-related quality of life (HRQOL) associated with continuous alternate-day low dose lenalidomide (LD-R, 10 mg on alternate days) and low dose prednisone (15 mg/day) (LD-RP) in 7 octogenarian NDMM patients (5 males and 2 females) with a median age of 82 years (range 80-87). All octogenarian patients had IgG MM, except 1 oligosecretory lambda chain MM; all were in Durie-Salmon stage III, except 1 in stage II, and had poor WHO performance status (median: 2, range 1-3). Patients were evaluated at baseline and every 6 months for HRQOL according to MM-specific questionnaire QLQ-MY20 of European Organisation for Research and Treatment of Cancer (EORTC). All patients received aspirin thromboprophylaxis, 57% of them requiring from diagnosis erythropoietin and zoledronic acid treatment. In these 7 octogenarian NDMM patients completing at least three months of therapy, the overall response rate (ORR) was 86%, including 1 complete remission (CR), 2 very good partial remission (VgPR) and 3 PR. After a median follow-up of 12 months (range 3-24), the quality of response improved with continuous LD-RP treatment with a cumulative median reduction in monoclonal protein levels of 85% (range 20-100%); none of the patients required discontinuation of treatment secondary to specific hematologic and/or extra-hematologic toxicity. In addition, QLQ MY-20 questionnaires revealed that 70% of patients treated with continuous LD-RP reported improvements of QOL scores. Two out of 7 octogenarian patients died (1 for progression after 12 months and 1 for sepsis no treatment-related), and 2-year overall survival and progression-free survival estimates were 41% and 75%, respectively. Noteworthy, all patients treated with continuous alternate-day LD-RP showed a progressive increase in the percentage of CD3+ CD56+ NK cells during the first 6 months of LD-RP therapy reaching a plateau maintained until +12 months after initiation of therapy: the median percentage of NK cells was 4% before LD-RP treatment versus 10%, 13%, 30%, 31%, and 27% at +1, +3, +6, +9 and +12 months, respectively. Mean fold increase of NK cells during LD-RP therapy was 1.5, 2.5, and 6.5 at +1, +3 and +6 months, respectively. Progressive increase of NK cells was concomitantly associated with reduction in tumor-linked monoclonal immunoglobulin in all patients and increased circulating NK cells further support that this drug may mediate its anti-MM effect, at least in part by modulating NK-cell number and function. Our data provide evidence that continuous alternate-day low dose lenalidomide is a manageable and effective frontline treatment for octogenarian NDMM patients and increases circulating NK cells. These preliminary results require further validation in prospective larger studies. Disclosures: No relevant conflicts of interest to declare.

Description: Background Hodgkin lymphoma (HL) is a clonal B-cell hematologic malignancy usually affecting only the lymphoid tissue, as extra-nodal (EN) involvement (e.g., bone, lung or liver) is seen only in ~15% of cases, much less than what is observed in non-Hodgkin lymphomas. To date, a small number of studies worldwide have investigated the epidemiology and clinical characteristics associated with HL with EN manifestations. Aim To describe risk factors and prognosis of EN involvement in HL patients. Methods Patients diagnosed with HL at King Abdullah University hospital (KAUH) between January 2004 and April 2018 were retrospectively reviewed for EN involvement, confirmed by histopathology of involved tissues. Risk factors assessed in our cohort of EN HL patients were: age, gender, presenting symptoms, B Symptoms, LDH levels, histological subtypes, CBC with differential, CT scans and PET/CT scans. International prognostic score (IPS) was also calculated. Results Among 140 HL patients included in our study, 16 of them (11.4%) had EN involvement. The mean age at diagnosis was 30 years old, ranging from 14-59. As in classic HL, men with EN involvement were slightly more affected than female (M/F, 10/6; 62.5% vs. 37.5%). Most of patients (N=8; 50%) had HL with mixed cellularity (MC); 6 patients (37.5%) had nodular sclerosing (NS); and 2 patients (12.5%) were classified as lymphocyte depleted (LD) subtype. Bone was the most compromised extra-nodal tissue as shown by the observation that 8 patients (50%) had bone involvement. Lungs pleura were affected in 7 patients (43.8%) and liver in 4 (25%) patients. The remaining subjects (N=4; 25%) showed involvement in other sites (skin, CNS and muscles). Out of our 16 EN HL patients, 6 of them (37.5%) had two EN tissues involved. EN HL patients were younger than patients without EN involvement (mean age, 30 vs. 37 years old respectively, P=.036 by unpaired t-test).EN HL patients with bone involvement were younger than those with liver or lung localization (mean age, 23.1 vs. 30.8 vs. 30.6 years old, respectively; P = 0.018 by unpaired t-test). We also observed a significant association between liver involvement and MC subtype. (P =0.032 by chi-square test). There was significant association between positive B symptoms and EN involvement. (P=.006 by chi-square test). There was no significant association between gender, high LDH levels, anemia, leukocytosis, lymphocytosis, monocytopenia, low albumin levels and EN involvement. There was significant association between IPS and EN involvement (56.25% of patients with EN involvement have high risk IPS, P=.00002 by chi-square test). All patients received ABVD as a first-line treatment. Seven patients (43.75%) experienced disease relapse during the follow-up period. There was a significant relationship between disease relapse and the type of HL, as 6 patients who experienced relapse had NS (P=0.0004 by chi-square), while only one MC. Overall survival rate was 75%, and 5-years survival rate was 45%. Four out of 7 patients who had disease relapse (57.1%) died, suggesting that disease relapse in EN HL might be a poor prognostic factor (P =0.045 by Kaplan Meier). There was no significant association between EN involvement and overall survival rate (P=.35 by Kaplan Meier). Conclusion Prevalence of EN involvement in patient with HL in Jordan is 11.4%, higher than that in Western countries and United States. In our cohort, EN involvement frequently presented as MC histological subtype; however, patients with NS showed the highest relapse rate. According to previous observations, bone and lungs were the most involved sites in our cohort, and patients with bone involvement were younger than those with other extra-nodal sites. Of note, our study might suggest some differences in the primary disease site in patients with HL possibly due to some geographic and ethnic variations. We also showed that risk and prognostic factors of classic HL are well applicable to EN HL, highlighting possible roles of histological subtypes in prognostication of EN HL. However, our preliminary findings need further validation in larger prospective studies. Disclosures No relevant conflicts of interest to declare.

Description: Background:DNA methyltransferase 3A(DNMT3A) is a member of the DNA methyltransferase family primarily involved in de novo gene methylation. Mutations in DNMT3A are associated with a wide range of hematological malignancies, most frequently acute myeloid leukemia (AML). DNMT3A mutations are thought to produce a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. Mutations in DNMT3A often coexist with secondary lesions in leukemia-related genes such as NPM1 and FLT3 (Ley T et al., N Engl J Med, 2010). Furthermore, healthy individuals harboring DNMT3A-driven clonal hematopoiesis are at increased risk of future hematologic malignancies and all-cause mortality (Jaiswal S et al., N Engl J Med, 2014). Despite these important clinical associations, the mechanisms by which DNMT3A mutations contribute to malignant transformation have not been well-defined. Dnmt3a-knockout (KO)mouse hematopoietic stem cells (HSCs) preferentially self-renew rather than undergo differentiation, leading to their accumulation in the bone marrow (Challen GA et al., Nat Genet, 2011). DNMT3A loss has also been shown to drive hypomethylation and subsequent activation of leukemia-related genes (Lu R et al., Cancer Cell, 2016; Yang L et al., Cancer Cell, 2016). However, these findings have not been recapitulated using human tissue. The goals of this study were thus to determine the transcriptional and biological effects of DNMT3A mutations which contribute towards malignant transformation in human cells. Methods:To elucidate the effects of DNMT3A mutation, we introduced DNMT3A frameshift mutations into K562 cells using the CRISPR/Cas9 gene-editing system. We then performed various functional and genomic assays to better elucidate effects of DNMT3A loss. Results and Discussion:We successfully created 4 DNMT3A-KO K562 clones and 1 clone containing a mutation that produces an altered DNMT3A protein with an intact catalytic domain (DNMT3A-alt). We first assessed effects of DNMT3A loss on cell growth and apoptosis. DNMT3A-KO clones exhibited impaired growth compared to wild-type (WT) cells. DNMT3A-KO clones also displayed significantly increased apoptotic activity after exposure to 5-fluorouracil (5-FU). The DNMT3A-alt clone had similar growth and apoptotic activity to WT cells. We examined how DNMT3A loss impacted differentiation using phorbal 12-myristate 13-acetate (PMA), known to induce megakaryocytic differentiation of K562 cells. After overnight exposure to PMA, DNMT3A-KO clones exhibited less CD61 expression, a marker of megakaryocytic differentiation, than did WT cells. Again, the differentiation of the DNMT3A-alt clonewas comparable to WT. Finally, we performed karyotype analysis to elucidate a potential role of DNMT3A in maintaining genomic integrity. Surprisingly, DNMT3A-KO clones exhibited profound cytogenetic variability and genomic instability compared to WT, with most DNMT3A-KO clones containing dicentric chromosomes and ring forms in multiple spreads (Figure 1). The DNMT3A-alt clone had a karyotype identical to WT. CRISPR/Cas9-edited K562 clones without DNMT3A mutation (transfected WT or tWT) also had identical karyotypes to WT K562. TA cloning and mRNA sequencing were employed to elucidate whether loss of DNMT3A would lead to transcriptome instability. DNMT3A-KOand DNMT3A-altclones exhibited distorted splicing patterns, while tWT cell lines were comparable to WT. To further assess the effect of DNMT3A ablation on genomic integrity, we examined DNA-damage responses by measuring DNA double-stranded breaks (DSBs) after treatment with 5-FU. DNMT3A-KO clones were significantly more susceptible to DNA damage than were WT cells, while the DNMT3A-alt clone exhibited more DNA DSBs compared to WT only at high concentrations of 5-FU. Conclusion:CRISPR/Cas9-mediated DNMT3A-KO K562 cells may be used to model effects of DNMT3A mutations in human cells. Consistent with previous reports, our data suggest that DNMT3A is involved in the differentiation of multipotent progenitors. Novel to this approach, our findings implicate induction of genomic instability as a mechanism by which DNMT3A mutations might predispose to malignancy. Disclosures Hosokawa: Aplastic Anemia and MDS International Foundation: Research Funding. Townsley:Novartis: Research Funding. Young:GSK/Novartis: Research Funding.

Description: Introduction . T-cell large granular lymphocytosis (T-LGL) is a low grade lymphoproliferative disorder, often clinically manifest as bone marrow failure. Treatment with immunosuppressive therapies is effective, but the dominant clone may persist even in responding patients. The pathogenesis of T-LGL has not been fully elucidated. In this study, we performed single cell RNA sequencing (sc-RNA seq) and V(D)J profiling to discern clonotypes and gene expression patterns of T lymphocytes from T-LGL patients who were sampled before and after treatment. Methods. Blood was obtained from patients participating in a phase 2 protocol of alemtuzumab as second line therapy (NCT00345345; Dumitriu B et al, Lancet Haematol 2016). Leukapheresis was performed in 13 patients (M/F 7/6; median age 51 years, range 26-85) before and after 3-6 months alemtuzumab administration and in 7 age-matched healthy donors. Cryopreserved blood was enriched for T cells with the EasySep Human T cell Isolation Kit (Stem cell). sc-RNA seq was performed on the 10XGenomics Chromium Single Cell V(D)J + 5' Gene Expression platform, and sequencing obtained on the HiSeq3000 Platform. Barcode assignment, alignment, unique molecular index counting and T cell receptor sequence assembly were performed using Cell Ranger 2.1.1. Results. Four hundred fifty thousand cells from 13 patients and 107,000 cells from 7 healthy donors were profiled. We measured productive TCR chains (which fully span the V and J regions, with a recognizable start codon in the V region and lacking a stop codon in the V-J region, thus potentially generating a protein). We detected at least one productive TCR α-chain in 50%, one productive TCR β-chain in 69% and paired productive αβ-chains in 47% of all cells. There was loss of TCR repertoire diversity in patients which was quantified by Simpson's diversity index; most patients showed oligoclonal or, less frequently, monoclonal expansion of the TCR repertoire (Fig. A). Regardless of clinical response, alemtuzumab treatment did not correct the low TCR repertoire diversity. TCR repertoires can be classified as "public", when they express identical TCR sequences across multiple individuals, or "private", when each individual displays distinct TCR clonotypes. No TCRA or TCRB CDR3 homology among patients was observed: most TCR clonotypes appeared to be private. Our data suggests that T-LGL is etiologically heterogenous disease, consistent with T cell expansion in response to a variety antigens, in diverse HLA contexts, or randomly. Despite differences of TCR among patients and healthy donors, and the presence of large clones in patients, distribution of TCR diversity followed the power law distribution in healthy donors and patients (Fig. B, showing the negative linear relationship between logarithmic expression of clone frequency and clone size). The observed distribution is consistent with a somatic evolution model, in which cell fitness depends on cellular receptor response to specific antigens and stimulation of cells by cytokine and other signals from the environment; fitted clones have higher birth-death ratios and thus expand (Desponds J et al, PNAS 2016). CD4 and CD8 T cells can be virtually separated by imputation from their transcriptomes (Fig. C). Comparison of gene expression between patients and healthy donors showed dysregulation of genes involved in pathways related to the immune response and cell apoptosis, consistent with a pathophysiology of T cell clonal expansion. We used diffusion mapping, which localizes datapoints to their eigen components in low-dimesional space, to characterize sources contributing to the gene expression phenotype: the first component was mainly from T cell activation and the second was associated with TCR expression. In LGL the T cell transcriptome appeared to be shaped by both lineage development and TCR rearrangement. Conclusion. We describe at the single cell level T clonal expansion profiles in T-LGL, pre- and post-treatment. Single cell analysis allows accurate recovery of paired α and β chains in the same cell and demonstrates a continuum of cell lineage differentiation. We found a range of differences in transcriptome and TCR repertoires across patients. Transcriptome data, coupled with detailed TCR-based lineage information, provides a rich resource for understanding of the pathology of T-LGL and has implications for prognosis, treatment, and monitoring in the clinic. Figure. Figure. Disclosures Young: GlaxoSmithKline: Research Funding; CRADA with Novartis: Research Funding; National Institute of Health: Research Funding.

Description: Aptamers or chemical antibodies are single-stranded DNA or RNA oligonucleotides that bind proteins and small molecules with high affinity and specificity by recognizing tertiary or quaternary structures as antibodies. Aptamers can be easily produced in vitro through a process known as systemic evolution of ligands by exponential enrichment (SELEX) or a cell-based SELEX procedure. Aptamers and modified aptamers, such as slow, off-rate, modified aptamers (SOMAmers), can bind to target molecules with less polar and more hydrophobic interactions showing slower dissociation rates, higher stability, and resistance to nuclease degradation. Aptamers and SOMAmers are largely employed for multiplex high-throughput proteomics analysis with high reproducibility and reliability, for tumor cell detection by flow cytometry or microscopy for research and clinical purposes. In addition, aptamers are increasingly used for novel drug delivery systems specifically targeting tumor cells, and as new anticancer molecules. In this review, we summarize current preclinical and clinical applications of aptamers in malignant and non-malignant hematological diseases.

Description: The identification of new molecular markers in Chronic Lymphocytic Leukemia (CLL) allowed to better define prognosis and clinical outcome. The actual staging systems could estimate the prognosis, but not the rapidity of disease evolution. Neither the identification of new molecular markers did allow to foresee the evolution and clinical response, because discordant findings were mostly reported. The aim of the present study was (1) to confirm the independent prognostic role of CD49d as a single marker in CLL patients, (2) to investigate the relationship between CD49d and other well-established CLL-membrane predictor markers (CD5, CD11c, CD20 and CD38) or clinical staging systems and (3) to evaluate the role of an immunophenotypic score based on the flow-cytometric detection of CD5, CD11c, CD20, CD38 and CD49d in the work up of CLL staging. Heparinized whole blood was collected from 68 CLL patients for immunophenotyping using the following antibodies: anti kappa, anti-lambda, CD5, CD11c, CD19, CD20, CD23, CD38, CD45, CD49d. A scoring system was elaborated combining 5 membrane markers: CD5, CD11c, CD20, CD38 and CD49d. Antigens were divided in two groups, favorable (CD5 and CD20) and unfavorable (CD11c, CD38 and CD49d) prognostic markers, and the cut-off of positivity was chosen according to the literature (30% for CD5, CD11c, CD20 and CD38, and 45% for CD49d). A value of "0" or "1" ("2" only for CD49d positivity) was assigned according to antigen expression. Finally, we defined a favorable phenotype when the sum of all the cytometric features was equal or less than 2, conversely the unfavorable phenotype was defined for a sum equal or greater than 3 (between 3 and 6). Flow cytometric analysis showed high CD49d expression in CD19+ cells in 47% of patients (n=32), and high CD38 expression in 44% of subjects (n=30), simultaneously expressed in 28% of patients (n=19). The 19% (n=13) of all CLL patients were CD5-, and interestingly the 85% of them showed higher expression of CD49d. Linear correlation was found between CD49d and CD38 (r2=0.08772, p=0.0142), and between CD49d and CD20 expression (r2=0.2490, p

Description: Cytomegalovirus (CMV) reactivation is one of the most frequent complication after hematopoietic stem cell transplantation (HSCT). Pre-transplant CMV-positive recipient serostatus is the most significant independent variable for viral reactivation. Oral valgancyclovir (VGCV) is a prodrug of intravenous gancyclovir (GCV) and is an effective and safety alternative for the management of CMV reactivation prophylaxis and preemptive therapy. However, VGCV at standard dose (900 mg twice a day) increases risk of myelosuppression in HSCT recipients. The efficacy of low dose (LD, 450 mg daily) oral VGCV was retrospectively evaluated in 30 allogeneic HLA-matched related patients and 2 unrelated, with a median age of 40 years (range, 18-59) and a median follow-up of 30 months (range, 3-56). Primary diseases were acute myeloid leukemia (AML, n=19), acute lymphoblastic leukemia (n=4), non-Hodgkin’s lymphoma (n=3), multiple myeloma (n=3) and myelodysplastic syndrome (n=3). Seventeen of twenty-three acute leukemia (AL) patients were transplanted in first complete remission (CR), while the remaining (n=6) were transplanted in 2nd CR. Five patients suffered from AML secondary to long-lasting MDS (n=3) or Hodgkin disease and breast cancer (n=2). Based upon CMV serostatus (D/R, donor/recipient), thirty (94%) of HSCT recipients were classified as high risk (D-/R+ = 3 and D+/R+ = 27) for CMV reactivation and only 6% as low risk (D-/R- = 2); none of the patients was in the intermediate risk group (D+/R-). Fifteen and 17 patients received a myeloablative and RIC regimens, respectively. Twenty-one patients received GvHD prophylaxis with cyclosporin A (CsA, 1 mg/kg intravenously from day -1 to +21, then 8 mg/kg orally for at least 6 months) and short-course methotrexate (MTX, 10 mg/kg on days +1, +3, +6 and +11). The others (n=11) received CsA with MTX and antithymocyte globulin (ATG, as a part of the conditioning regimen at 10 mg/kg at days -3, -2 and -1). According to the Glucksberg scoring system, thirteen patients experienced grade I-II and two grade III-IV acute GvHD, while 7 patients developed limited (n=6, 18%) and extensive (n=3, 10%) chronic GvHD. Starting from time of engraftment, LD oral VGCV was given prophylactically for at least 6 months. CMV infection was monitored weekly using polymerase chain reaction (PCR) in high risk seropositive recipients and we started preemptive therapy when the peak viral load exceeding 1000 copies/mL in two consecutive plasma samples. Six patients (4 early and 2 late) developed a positive PCR after a mean of 59 days post-HSCT successfully treated with 900 mg of VGCV twice a day for at least when PCR negative (in a median of 12 days). Only one patient developed late fatal gastrointestinal CMV disease. Indeed, asymptomatic early and late CMV-DNA PCR reactivation occurred only in 17% (n=5) of high risk seropositive HSCT recipients, in contrast to 37% and 18% of early and late CMV reactivation observed in matched gender, disease phase, graft source and CMV serostatus cohort of 32 HSCT recipients treated prophylactically with oral acyclovir (ACV, 15 mg/kg daily) and high dose intravenous immunoglobulins (IVIG, 0.4 gr/kg weekly for at least 6 months) . Seven patients presented hematological toxicity do not requiring drug discontinuation. The rate of non CMV-related infections was 25% and was similar in both groups with and without CMV reactivation. At the end of the follow-up, 18 of 32 (56%) patients were alive with a median follow-up of 31 months (range, 2-56). Relapsed-related mortality was 20%, transplant-related mortality was 9% and did not differ between group with and without CMV reactivation. Our data provide evidence that LD-VGCV is safe and effective as CMV reactivation prophylaxis in allogeneic HSCT recipients. These results require further validation in randomized studies. Disclosures: No relevant conflicts of interest to declare.

Description: Hodgkin and Reed-Sternberg (HRS) cells in classical Hodgkin Lymphoma (HL) are surrounded by a rich inflammatory infiltrate which aids in their survival and escape from cytotoxic CD8+ T cells (CTLs) and Natural Killer cells (NKs). Within HL environment, T regulatory cells (Tregs) directly suppress the activity of CTLs and NKs, enhancing the tolerance against HRS cells. B regulatory cells (Bregs) have been shown to support the differentiation of Tregs through IL-10 production; thus, we hypothesized that they could have a role in the pathophysiology of HL. We evaluated 30 classic HL patients (M/F: 18/12; median age, 31 years, range 15-62) and 5 healthy controls (HC) for circulating peripheral blood (PB) Bregs, Tregs, CTLs, NKs, and NKTs. Twenty-four of them were new-diagnosed patients (NwHL) and 6 received a previous diagnosis of HL but were in complete remission (CR) for more than 12 months (PvHL). NwHL patients were divided according to the International Prognostic Score (IPS) and the Ann-Arbor Staging System. All subjects were treated following the ABVD protocol (doxorubicin 25 mg/m2, bleomycin 10 U/m2, vinblastine 6 mg/m2, and dacarbazine 375 mg/m2). Flow cytometry was performed on heparinized PB samples with a 5-color Beckman Coulter Cytomics FC500 flow cytometer. Breg (CD19+CD24+), Treg (CD3+CD4+CD25+), CTL (CD3+CD8+), NK (CD3-CD56+), and NKT (CD3+CD56+) levels were measured simultaneously with the PET/CT evaluations, ie at diagnosis, at the end of the second ABVD administration, at the end of treatment, and at 6 and/or 12 months off-therapy. Moreover, Breg levels were compared to IPS and Ann-Arbor staging groups, and also were correlated to the erythrocyte sedimentation rate (ESR) and to the absolute lymphocyte count (ALC). We found decreased circulating Bregs in NwHL and PvHL patients compared to controls (0.39% vs 0.875% vs 1.813%, respectively, p