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Although post-transplant cyclophosphamide (PT-Cy) is effective for graft-versus-host disease (GVHD) prophylaxis, it is associated with toxicities, which might be dose-dependent. We compared the outcomes with PT-Cy at 80 mg/kg to those with PT-Cy at 100 mg/kg in elderly patients undergoing haploidentical hematopoietic cell transplantation (HCT). Inclusion criteria included peripheral blood stem cells, hematological malignancy, and age65 years (or age60 years if cardiac event history). Thirty-eight patients received PT-Cy at 80 mg/kg and 55 100 mg/kg, divided in two doses. The cumulative incidences (CI) of acute grade II-IV, acute grade III-IV, and moderate/severe chronic GVHD were 32%, 16%, and 13% with PT-Cy at 80 mg/kg compared to 33%, 13%, and 16% with 100 mg/kg, respectively. In multivariable analysis, reducing PT-Cy dose had no significant impact on GVHD. Neutrophil and platelet engraftments were significantly improved, and CI of BK virus-associated hemorrhagic cystitis was reduced with 80 mg/kg of PT-Cy compared to 100 mg/kg. At 2 years, non-relapse mortality was 16% and 31%, progression-free survival 65% and 49%, overall survival 70% and 56%, and GVHD-free, relapse-free survival 52% and 36% with 80 mg/kg and 100 mg/kg, respectively. Reducing PT-Cy dose to 80 mg/kg is safe and associated with improved hematological recovery and lower CI of hemorrhagic cystitis in elderly patients undergoing haploidentical HCT.

PURPOSE Myelodysplastic syndromes (MDS) are heterogeneous myeloid neoplasms in which a risk-adapted treatment strategy is needed. Recently, a new clinical-molecular prognostic model, the Molecular International Prognostic Scoring System (IPSS-M) was proposed to improve the prediction of clinical outcome of the currently available tool (Revised International Prognostic Scoring System [IPSS-R]). We aimed to provide an extensive validation of IPSS-M. METHODS A total of 2,876 patients with primary MDS from the GenoMed4All consortium were retrospectively analyzed. RESULTS IPSS-M improved prognostic discrimination across all clinical end points with respect to IPSS-R (concordance was 0.81 v 0.74 for overall survival and 0.89 v 0.76 for leukemia-free survival, respectively). This was true even in those patients without detectable gene mutations. Compared with the IPSS-R based stratification, the IPSS-M risk group changed in 46% of patients (23.6% and 22.4% of subjects were upstaged and downstaged, respectively). In patients treated with hematopoietic stem cell transplantation (HSCT), IPSS-M significantly improved the prediction of the risk of disease relapse and the probability of post-transplantation survival versus IPSS-R (concordance was 0.76 v 0.60 for overall survival and 0.89 v 0.70 for probability of relapse, respectively). In high-risk patients treated with hypomethylating agents (HMA), IPSS-M failed to stratify individual probability of response; response duration and probability of survival were inversely related to IPSS-M risk. Finally, we tested the accuracy in predicting IPSS-M when molecular information was missed and we defined a minimum set of 15 relevant genes associated with high performance of the score. CONCLUSION IPSS-M improves MDS prognostication and might result in a more effective selection of candidates to HSCT. Additional factors other than gene mutations can be involved in determining HMA sensitivity. The definition of a minimum set of relevant genes may facilitate the clinical implementation of the score.

Human Herpesvirus-6 (HHV-6) can reactivate after allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT), and may lead to severe symptoms. HHV-6-specific immune responses after HSCT are largely unexplored. We conducted a prospective observational study on 208 consecutive adult patients who received allo-HSCT, to investigate HHV-6 reactivations and specific immune responses. IFN-γ-producing HHV-6-specific T cells were quantified by ELISpot assay. "HHV-6 reactivation" occurred in 63% of patients, at a median of 25 days (range 3-538) from allo-HSCT. Only 40% of these presented a "clinically relevant infection", defined according to center guidelines by the presence of classical HHV-6 End-Organ Diseases (EOD), based on European-Conference-on-Infections-in-Leukaemia (ECIL) guidelines, and other possible HHV6-related EODs. By multivariate analysis, we identified risk factors for HHV-6 reactivation: previous allo-HSCT (Hazard Ratio, HR=2.89; p-value4 days after the first viremia detection, predicted clinically relevant infections (p-value

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) with post-transplant cyclophosphamide is a curative treatment for many hematological malignancies, yet a majority of patients still suffers from recurrent infections. Post-transplant infusion of memory T cells could potentially enhance immunological protection without increasing the risk of eliciting acute graft-versus-host disease, which is mainly induced by naïve T cells. Here, we performed longitudinal analysis of the lymphocyte compartment in 19 haplo-HSCT patients previously enrolled in a phase II prospective clinical trial (ClinicalTrials.gov Identifier: NCT04687982), in which they received post-transplant CD45RA-depleted donor lymphocyte infusions (DLI). T cell receptor sequencing analysis showed that, surprisingly, CD45RA-depleted DLI do not increase T cell clonal diversity, but lead to prominent expansion of a selected number of infused memory T cell clones, suggestive of recruitment of these cells in the immune response. Pathogen-specific memory T cells, including cytomegalovirus (CMV)-specific cells, engrafted and were able to persist for at least one month. Deep immunophenotyping revealed strong polyfunctional effector CMV-specific T cell responses in the majority of patients, with their expansion correlating with the frequency of CMV-specific cells in the donor. These findings provide a rationale behind the suggested improved protection against viral infections for patients receiving CD45RA-depleted DLI.

Endothelial Activation and Stress Index (EASIX) is a prognostic score reflecting endothelial damage. It can identify cohorts of patients at higher risk of non-relapse mortality (NRM) after allogeneic stem cell transplantation (SCT) from a matched-related or -unrelated donor. No data are available in the setting of haploidentical-SCT with post-transplant cyclophosphamide (PT-Cy). We retrospectively analyzed the role of EASIX score in a cohort of 266 patients receiving Haplo-SCT with PT-Cy at our center. By a decision-tree model, 1-year NRM was 16% vs. 29% and overall survival was 59% vs. 32%, respectively, for patients with a pre-transplant EASIX (EASIX-PRE)0.8 vs. ≥0.8 (p 0.001). By multivariable analysis, EASIX-PRE was an independent predictor of NRM (hazard ratio [HR] 2.43, p 0.001) and overall survival (HR: 1.64, p = 0.011). EASIX-PRE did not predict patients at higher risk of developing acute graft-versus-host disease (GVHD) but was an independent predictor of 1-year NRM (3.2 cutoff, HR 6.61, p = 0.002;3.2 vs. ≥3.2: 10% vs. 56%, p 0.001) in patients developing acute GVHD. EASIX score can also represent an important tool to predict mortality in the setting of Haplo-SCT with PT-Cy. It may help to make therapeutic decisions both before the transplant and at the onset of acute GVHD.

Risk factors for cytomegalovirus (CMV) reactivation and the impact of CMV reactivation on patient outcomes have been extensively investigated after matched related or unrelated donor transplantation, but little is known in the setting of haploidentical stem cell transplantation (Haplo-SCT) with post-transplantation cyclophosphamide (PT-Cy), in which recipients are considered more severely immunocompromised. We retrospectively analyzed a cohort of 554 consecutive patients undergoing Haplo-SCT with PT-Cy at 3 different centers. Early CMV reactivation (occurring within the first 120 days post-transplantation) occurred in 242 patients, for an estimated cumulative incidence of 44%. Among those patients, 74 (30%) had recurrent CMV and 20 (8%) had CMV disease. On multivariable analysis, positive recipient CMV serostatus (hazard ratio [HR]2.5; P.001), disease histology (lymphoid versus myeloid: HR, 0.66; P = .003) and increasing recipient age (HR, 1.01; P = .015) were independent predictors of CMV reactivation. At a 4-month landmark analysis, CMV reactivation was associated with higher 1-year and 5-year cumulative incidence of nonrelapse mortality (NRM) relative to patients without reactivation: 13% versus 5% and 22% versus 9%, respectively (P.001). On multivariable analysis, CMV reactivation was an independent negative predictor of NRM (HR, 2.69; P.001) and was close to statistically significant for overall survival (HR, 1.38; P = .062). Our results suggest that CMV reactivation plays an important role at determining NRM. Because patient CMV serostatus is the main predictor of CMV reactivation, it should be considered when evaluating strategies for preventing CMV reactivation. 2022 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

INTRODUCTION Primary and post-ET/PV myelofibrosis are myeloproliferative neoplasms harboring in most cases driving mutations in JAK2, CALR or MPL, and a variable number of additional mutations in other genes. Molecular analysis represents a powerful tool to guide prognosis and clinical management. Only about 10% of patients with myelofibrosis harbor alterations in MPL gene. No data are available about the transplantation outcome in the specific MPL-mutated group. PATIENTS We collected the data of 18 myelofibrosis patients(primary: 14; post-ET: 4) transplanted in 4 EBMT centers (Hamburg, Paris, Essen, and Hannover) between 2005 and 2016. RESULTS Before the transplant, we explored the molecular profile by NGS and reported the frequency of mutations occurring in a panel of genes including JAK2, MPL, CALR, U2AF1, SRSF2, SF3B1, ASXL1, IDH1, IDH2, CBL, DNMT3A, TET2, EZH2, TP53, IKZF1, NRAS, KRAS, FLT3, SH2B3, and RUNX1. The 1-year transplant-related mortality was 16.5%, 5-years overall survival and 5-y relapse-free survival 83.5%. The only relapse occurred in a patient who harbored mutations in both ASXL1 and EZH2 genes. CONCLUSION These retrospective data suggest that MPL-mutated myelofibrosis patients have a favorable outcome after allogeneic transplantation with very low rate of disease relapse (5.5%) in comparison with the available historical controls regarding myelofibrosis in all.

Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is a curative treatment for many hematological malignant and non-malignant diseases. A major complication of the procedure is the donor T-cell-mediated graft-versus-host disease (GvHD). GvHD accounts for about 10% of early mortality after transplantation. GVHD is also the major cause of morbidity and disability in the late follow-up phase of transplanted patients, mainly because of the low response to first-line steroids, and the lack of efficient second-line standard treatments. The increasing knowledge regarding GVHD pathogenesis provides new pharmacological targets, potentially exploitable in clinical practice, in order to prevent and treat this complication. This review provides a description of GVHD pathogenesis, with a focus on the central role of the Janus kinase-related mechanisms. The first inflammatory innate-immunity response is triggered by a JAK/STAT dependent pathway, and JAK inhibition impairs antigen-presenting cell differentiation and activation and downregulates the expression of signals for T-cell triggering. The chronic evolution of alloreactivity, characterized by the long-term maintenance of inflammation and fibrosis, is also dependent on JAK/STAT activation. Based on preclinical data, we reviewed the rationale behind the clinical use of JAK-inhibitors in GVHD, presenting available results of clinical trials and reports, and looked at future implementation of this new promising treatment approach.

Allogeneic stem cell transplantation (alloSCT) is a curative procedure for myelofibrosis. Elderly people are mainly affected, limiting the feasibility of myeloablative regimens. The introduction of reduced-intensity conditioning (RIC) made alloSCT feasible for older patients. Nevertheless, the incidence of myelofibrosis is not negligible in young patients, who are theoretically able to tolerate high-intensity therapy. Very few data are available about the efficacy of RIC-alloSCT in younger myelofibrosis patients. This study included 56 transplanted patients aged

OBJECTIVES Major complications affecting the central nervous system (CNS) present a challenge after allogeneic stem cell transplantation (allo-SCT). METHODS Incidence, risk factors, and outcome were retrospectively analyzed in 888 patients in a monocentric study. RESULTS Cumulative incidence (CI) of major CNS complications at 1 year was 14.8% (95%CI 12.3%-17.2%). Median follow-up is 11 months. CNS complications were documented in 132 patients: in 36 cases, classified metabolic; 26, drug-related neurotoxicity (14 attributed to cyclosporine A, 4 to antilymphocyte globulin); 11, cerebrovascular (ischemic n = 8, bleeding n = 3); 9, infections; 9, psychiatric; and 9, malignant. The cause of CNS symptoms remained unclear for 37 patients (28%). Multivariate analysis demonstrated an association of CNS complication with patient age (P

Busulfan (Bu) is an alkylating agent routinely used for conditioning regimens before allogeneic stem cell transplantation (allo-SCT). Bu shows wide pharmacokinetic (PK) variability among patients. Patients can have a higher systemic exposure (expressed as area under the curve [AUC]) with an increased risk of toxicity or a lower AUC with a higher probability of graft rejection and/or disease relapse. After i.v. administration, an optimal Bu therapeutic window (AUC target of 16,000 to 24,000 μM·minute) has been identified. The use of PK-guided Bu dosing leads to improved overall survival (OS) and progression-free survival (PFS) compared with fixed-dose administration in a variety of hematologic diseases. The aim of this study was to evaluate the outcomes and feasibility of a reduced-toxicity conditioning (RTC) regimen comprising thiotepa, Bu, and fludarabine (TBF) with therapeutic drug monitoring of Bu in patients with hematologic disorders. We report on 41 adult patients with myeloid or lymphoid malignancies who underwent an allo-SCT with a PK-guided Bu-based RTC regimen between January 2019 and October 2020. Patients received a total Bu dose to achieve a target AUC of 16,000 μM·minute in combination with Flu and thiotepa. The median time to absolute neutrophil count recovery and transfusion-independent platelet count recovery was 23 days (range, 15 to 42 days) and 29 days (range, 14 to 97 days), respectively. The cumulative incidence (CI) of nonrelapse mortality was 7% at 100 days and 13% at 1 year. Grade 3 liver toxicity was observed in 6 patients. One patient developed sinusoidal obstruction syndrome at day +27. Grade 3 mucositis occurred in 18 patients. Looking at grade ≥3 infections, the CI was 29% at 30 days, 34% at 60 days, 44% at 100 days, and 56% at 1 year. The 180-day CI of grade II-IV acute graft-versus-host disease (GVHD) was 15%, and the 1-year CI of overall chronic GVHD was 20%. With a median follow-up of alive patients of 14.4 months (range, 3.2 to 24 months), the CI of relapse at 1 year was 6%. The 1-year PFS was 81%, and 1-year OS was 84%. In conclusion, these data support the efficacy of PK-guided Bu dose in the context of a TBF conditioning regimen and the feasibility of therapeutic dosage monitoring of i.v. Bu for patients with hematologic diseases. © 2021 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Low-dose total body irradiation (TBI) has long been used in nonmyeloablative conditioning (NMAC) regimens before allogeneic stem cell transplantation from haploidentical donors (haplo-SCT). More recently, the use of total marrow lymphoid irradiation (TMLI) instead of TBI in conditioning is increasing. This study aimed to evaluate outcomes in a cohort of patients treated with low-dose TMLI in terms of engraftment, full donor chimerism status, graft-versus-host disease (GVHD), and extrahematologic toxicities, and to compare these outcomes with those in a cohort of patients receiving conventional TBI-containing conditioning. This retrospective single-center study included 100 patients with advanced hematologic malignancies who underwent haplo-SCT. Between 2009 and 2011, the NMAC regimen consisted of cyclophosphamide, fludarabine, and low-dose TBI (2 Gy), and after 2011, TBI was replaced with TMLI (2 Gy). Patients received post-transplantation cyclophosphamide, calcineurin inhibitor, and mycophenolate mofetil as GVHD prophylaxis. For all patients, the median time to absolute neutrophil count (ANC) recovery to >0.5 × 109/L was 21 days (range, 15 to 49 days), the 30-day incidence of ANC recovery was 97% (95% confidence interval [CI], 89% to 99%), the median time to achieve an unsupported platelet count >20 × 109/L was 26 days (range, 12 to 67 days), and the 60-day rate of platelet engraftment was 99% (95% CI, 89% to 100%). Cumulative incidence of full donor chimerism by day 100 was 88% (95% CI 79-90). Grade II-IV acute GVHD occurred in 35% of the patients (95% CI, 26% to 45%) at a median of 40 days (range, 23 to 166 days). The incidence of moderate to severe chronic GVHD was 5% (95% CI, 2% to 10%). No differences between the TBI and TMLI cohorts were seen in terms of engraftment, full donor chimerism, and GVHD. No organ toxicity was observed in the first months after transplantation in either cohort. The overall 2-year OS and PFS rates were 63%, and 54%, respectively, and were comparable in the 2 groups (P = .548). The strongest finding was that TBI can be safely replaced by TMLI in terms of engraftment, achievement of full donor chimerism status, GVHD incidence, and extrahematologic toxicities.

Allogeneic stem cell transplantation from haploidentical donor using post-transplantation cyclophosphamide has been used to cure hematological diseases. Because of slow immunological reconstitution, there is an increased incidence of viral infection. The aim of our study was to prospectively evaluate the efficacy and the feasibility of a CD45RA+ depleted donor lymphocytes infusion (DLI) in terms of reduction of viral infection early after haploidentical transplantation. This a prospective single-center study. We enrolled 23 patients, of whom 19 were evaluable. Graft-versus-host disease (GVHD) prophylaxis was the same for all patients. The primary endpoint was 100-day cumulative incidence of viral infections. The primary endpoint was met, because the 100-day cumulative incidence of viral infection was 32%. The median time from transplantation to first CD45RA+ depleted DLI was 55 days (range, 46-63). 28% of patients had cytomegalovirus reactivation, no patients reactivated human herpesvirus-6; 1 patient developed BK virus related hemorrhagic cystitis. Most of the patients received the planned 3 infusions. Only 1 patient had development of grade 2 acute GVHD, and 2 patients had moderate chronic GVHD. All evaluable patients were off immunosuppressive therapy at last follow-up. The median follow-up was 12 months (range, 3-23), the 1-year overall survival and progression-free survival were 79% and 75%, respectively; the 100-day and 1-year non-relapse mortality were 5% and 12%, respectively. CD45RA+ depleted DLI are feasible in patients treated with haploidentical transplantation. The toxic profile is good with a low risk for development of both acute and chronic GVHD.

Infections are a major cause of morbidity and mortality in hematological patients. We prospectively tested a new molecular assay (Verigene®) in 79 consecutive hematological patients, with sepsis by gram-negative bacteria. A total of 82 gram-negative microorganisms were isolated by blood cultures, of which 76 cases were mono-microbial. Considering the bacteria detectable by the system, the concordance with standard blood cultures was 100%. Resistance genes were detected in 20 of the isolates and 100% were concordant with the phenotypic antibiotic resistance. Overall, this new assay correctly identified 66/82 of all the gram-negative pathogens, yielding a general sensitivity of 80.5%, and providing information on genetic antibiotic resistance in a few hours. This new molecular assay could ameliorate patient management, resulting in a more rational use of antibiotics.

Despite being the second least represented granulocyte subpopulation in the circulating blood, eosinophils are receiving a growing interest from the scientific community, due to their complex pathophysiological role in a broad range of local and systemic inflammatory diseases as well as in cancer and thrombosis. Eosinophils are crucial for the control of parasitic infections, but increasing evidence suggests that they are also involved in vital defensive tasks against bacterial and viral pathogens including HIV. On the other side of the coin, eosinophil potential to provide a strong defensive response against invading microbes through the release of a large array of compounds can prove toxic to the host tissues and dysregulate haemostasis. Increasing knowledge of eosinophil biological behaviour is leading to major changes in established paradigms for the classification and diagnosis of several allergic and autoimmune diseases and has paved the way to a “golden age” of eosinophil-targeted agents. In this review, we provide a comprehensive update on the pathophysiological role of eosinophils in host defence, inflammation, and cancer and discuss potential clinical implications in light of recent therapeutic advances.

INTRODUCTION Allogeneic stem cell transplantation (alloSCT) is the only curative procedure for primary and secondary myelofibrosis (PMF, SMF). Elderly people are mainly affected, limiting the feasibility of intensive myeloablative chemotherapy regimens. The introduction of reduced-intensity conditioning (RIC) made alloSCT feasible and effective for old patients. Nevertheless, the incidence of PMF and SMF is not negligible in young patients, theoretically able to tolerate also high-intensity therapy. Very few data are available about the efficacy of RIC-alloSCT in the particular setting of young-aged MF patients. PATIENTS AND METHODS This study includes 56consecutive Myelofibrosis young patients (age RESULTS Engraftment rate was 98%, with a median neutrophil engraftment time of 15 days. Platelet engraftment was reached by 51 patients (91%) in a median time of 19 days. Four patients (7%) developed poor graft function, successfully treated with CD34+ selected PBSC boost. After a median follow up of 8.6 y estimated 5y PFS and OS was 68% and 82% respectively. DIPSS risk and donor HLA-matching resulted the only significant impacting factors on OS. Neither cytogenetic nor molecular abnormalities (included ASXL1) were significantly related to OS. Twenty-five patients (44%) experienced acute GVHD grade >1. Chronic GVHD was observed in 34 patients (65%), mostly (82%) beginning in the first 300 days after transplantation. Cumulative incidence of TRM was 7% at 1 year, with a plateau after the first year (5y TRM = 12%). TRM was observed only in patients with maximal grade (3) of marrow fibrosis (p=0.00). Furthermore, TRM never occurred in previously splenectomized patients (p=0.00), but no significant impact from splenectomy on OS was observed (p=0.32). After transplant, 11 patients (20%) relapsed: 1 died without any treatment because of infection, 9 received DLI with durable recovery of complete remission in 3 cases, 7 patients (6 after DLI) underwent a second alloSCT, with long-term survival in 5 cases. CONCLUSION Reduced intensity conditioning followed by allogeneic SCT is a curative treatment approach for younger patients with myelofibrosis with a low NRM. The most important outcome-determining factor is donor HLA-matching. Interestingly, marrow high grade fibrosis showed to significantly impact TRM. Biological markers such as ASXL1 mutation and cytogenetics, largely known as highly predictive for poor prognosis in the disease natural course, did not show any impact on survival, suggesting that patients harboring these abnormalities could get the greatest benefit from alloSCT. According to these data, RIC-alloSCT ensures optimal engraftment and low relapse rate, being able to lead also younger MF patients to cure. Further data collection, and a prospective randomized trial are needed to confirm our conclusion. Disclosures No relevant conflicts of interest to declare.

In Philadelphia-positive (Ph+) Acute Lymphoblastic Leukemia (ALL) patients (pts), resistance to tyrosine kinase inhibitors (TKIs) is frequently associated with the selection of one or more mutations in the BCR-ABL1 kinase domain (KD). The swift emergence of mutant clones as early as during induction therapy supports the hypothesis that, at least in some cases, mutations may already be present at diagnosis. Next Generaton Sequencing (NGS) has been proposed as an alternative to Sanger sequencing (seq) for BCR-ABL1 KD mutation screening because of its greater sensitivity and accuracy, but no studies have so far evaluated its prospective use in Ph+ ALL. Between 2015 and 2018, we have used NGS in parallel to Sanger seq to analyze a consecutive series of 126 Ph+ ALL pts who were newly diagnosed (n=39) or who had relapsed/refractory disease (n=87) on TKI therapy. In 22 cases, both bone marrow and peripheral blood were analyzed and compared. NGS of ≈400bp amplicons generated by nested RT-PCR was performed on a Roche GS Junior (until April 2017) or on an Illumina MiSeq (from May 2017 on). Read alignment and variant calling (with a lower limit set to 3%) were done with the AmpSuite software (SmartSeq srl). When multiple mutations mapped within the same sequence reads, assessment of cis vs trans configuration was done correcting for the probability of PCR recombination. Three out of 39 (7.7%) de novo Ph+ ALL pts had low burden point mutations detectable by NGS: one had a V289A (variant frequency, 3.4%); one had a D276G (4.0%) and a F359V (3.5%); one had an E255K mutation (3.3%). The first pt was enrolled in the GIMEMA LAL1811 study of frontline ponatinib; the second and the third pts were enrolled in the GIMEMA D-ALBA study of frontline sequential treatment with dasatinib and blinatumomab. All pts achieved molecular remission, consistently with the mutations being sensitive to the TKIs received. The 35INS insertion/truncation mutant was detected in 27 (69%) pts, who all have so far achieved molecular remission. This is in line with the report by O'Hare et al (Blood 2011) suggesting that the 35INS variant is kinase-inactive and does not contribute to TKI resistance. For this reason, the 35INS was excluded from subsequent analyses. Relapsed/refractory pts positive for mutations by Sanger seq were 57 (65%); those positive for mutations by NGS were 69 (79%). Fifty-six out of 87 (49%) pts had >1 mutation (up to 13) detected by NGS. NGS identified low burden mutations (i.e., mutations present in a proportion of transcripts between 3 and 20%) in 12 pts who were negative for mutations by Sanger seq. Most importantly, NGS provided a more accurate picture of BCR-ABL1 mutations status in 40 (46%) pts who turned out to have one or more low burden mutations in addition to the dominant mutation(s) detectable by Sanger seq. In all cases, each low burden mutation detected by NGS could be recognized as poorly sensitive either to the TKI the pt was receiving at the time of testing, or to the previous TKI. The clonal nature of NGS-based analysis further proved its utility i) in 4 pts where Sanger seq had shown 2 base substitutions in the same codon so that the actual amino-acid change(s) were impossible to infer (a ponatinib-resistant pt with a T315M mutation, 2 dasatinib-resistant pts with various combinations of F317I, F317C and/or F1317L, a dasatinib-resistant pt with 2 different nucleotide substitutions both leading to the V299L), and ii) in 48/56 pts who had ≥2 mutations whose clonal configuration could not be resolved. Twenty-eight out of these 48 pts were found to carry one or more (up to 3) compound mutants. Compound mutants were more common in pts who had failed ≥2 lines of therapy, whereas polyclonality was more common in pts who had failed first line therapy. The most frequent compound mutants were T315I+E255K and T315I+E255V. Interestingly, the latter was associated with poor or no response to ponatinib. Our results in a relatively large series of Ph+ ALL pts suggest that an NGS-based approach provides a more accurate characterization of the complexity of BCR-ABL1 KD mutation status, including compound mutants some of whom may be poorly sensitive even to ponatinib. Mutations may already be detected at the time of diagnosis. It remains to be assessed whether more sensitive techniques like digital PCR may identify a greater number of pts with pre-therapy mutations and whether the detection of pre-therapy mutations may be used to guide 1st-line treatment selection. Disclosures Soverini: Incyte Biosciences: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Pagano:Gilead: Speakers Bureau; Basilea: Speakers Bureau; Merck: Speakers Bureau; Janssen: Speakers Bureau; Pfizer: Speakers Bureau. Abruzzese:Ariad: Consultancy; BMS: Consultancy; Novartis: Consultancy; Pfizer: Consultancy. Martinelli:Roche: Consultancy; Celgene: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Abbvie: Consultancy; Janssen: Consultancy; Ariad/Incyte: Consultancy; Amgen: Consultancy. Cavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Human herpesvirus 6 (HHV-6) is increasingly recognized as a potentially life-threatening pathogen in allogeneic hematopoietic stem cell transplantation (alloSCT). We retrospectively evaluated 54 adult patients who developed positivity to HHV-6 after alloSCT. The median time from alloSCT to HHV-6 reactivation was 34 days. HHV-6 was present in plasma samples from 31 patients, in bone marrow (BM) of 9 patients, in bronchoalveolar lavage fluid and liver or gut biopsy specimens from 33 patients, and in cerebrospinal fluid of 7 patients. Twenty-nine patients developed acute graft-versus-host disease (GVHD), mainly grade III-IV, and 15 had concomitant cytomegalovirus reactivation. The median absolute CD3+ lymphocyte count was 207 cells/µL. We reported the following clinical manifestations: fever in 43 patients, skin rash in 22, hepatitis in 19, diarrhea in 24, encephalitis in 10, BM suppression in 18, and delayed engraftment in 11. Antiviral pharmacologic treatment was administered to 37 patients; nonetheless, the mortality rate was relatively high in this population (overall survival [OS] at 1 year, 38% ± 7%). A better OS was significantly associated with a CD3+ cell count ≥200/µL at the time of HHV-6 reactivation (P = .0002). OS was also positively affected by the absence of acute GVHD grade III-IV (P = .03) and by complete disease remission (P = .03), but was not significantly influenced by steroid administration, time after alloSCT, type of antiviral prophylaxis, plasma viral load, or organ involvement. Although HHV-6 detection typically occurred early after alloSCT, better T cell immune reconstitution seems to have the potential to improve clinical outcomes. Our findings provide new insight into the interplay between HHV-6 and the transplanted immune system.

To the editor: Graft-versus-host disease (GVHD), both acute and chronic, is still a leading cause of nonrelapse mortality after transplantation.[1][1] High-dose, posttransplantation cyclophosphamide (PTCy) is an attractive approach for in vivo allodepletion across the HLA barrier in allogeneic

Background: Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for many haematological diseases. Despite advances in supportive therapies, acute Graft-versus-Host Disease (aGvHD) remains the leading cause of early morbidity and mortality. There are shortcomings in the prediction of aGVHD, indicating the urgent need for non-invasive and reliable laboratory tests to allow a precision-medicine tailored prophylactic approach. Aims: We conducted a prospective observational study to ascertain the potential usefulness of the levels of eleven biomarkers, measured pre- and at +7 days post-alloHSCT, in predicting the development and severity of aGvHD in allo-HSCT patients. These time-points were chosen as early risk stratification may provide a window for additional prophylactic measures. Methods: We collected data from 120 consecutive patients (41 female and 79 male; median age 52, range 19-77 years) who underwent allo-HSCT at our institute between 2014 and 2015. Most patients were affected by myeloid malignancies (AML=52%, MDS=11%, MPN=5%) while 32% were of lymphoid origin (ALL, MM and lymphomas). Revised disease-risk index (DRI, Armand et al.) was low or intermediate for 41% of pts, high for 44% and very high for 15% of them. Most patients (n=101) received peripheral blood stem cells (PBSC). Stem cell donors were unrelated (n=39, HLA matching 9/10 in 14 and 10/10 in 25), family haploidentical (n=56), HLA-identical sibling (n=21), or cord blood (n=4). Post-transplant GvHD prophylaxis was PT-Cy-bases in 65 patients, ATG-bases in 28 patients, both agents in 18 cases whilst 9 patients received neither. Additionally, sirolimus and MMF were used as additional GvHD prophylaxis according to institutional guidelines. The following biomarkers were measured 7 days pre- and post-transplant: interleukin-6 (IL6), Ceruloplasmin(CER), Cholinesterase (CHE), Albumin, Immunoglobulin A, Gammaglutamyl-transferase (GGT), White Blood Cells, Neutrophils, Haemoglobin,Platelets and Glycaemia. ROC curves were used to define optimal cut-offs of the biochemical variables to predict 100-day severe aGvHD and 100-days TRM by logistic regression. Then cumulative incidences curves (CI) for aGvHD and TRM and overall-survival (OS) curves were computed comparing patients under and over the identified cut offs for the variables with the best reported sensitivity and specificity. Multivariate Cox proportional hazard regression was finally applied. Results: The 100-days CI of grade III-IV aGvHD was 13.5%, with a 100-days CI of TRM of 12%. The 1-year OS was 64%. Death was reported in 49 of the total 120 patients: 5/49 deaths were attributable to aGvHD. At baseline, IL6 threshold value of 2.5 pg/ml was significantly associated with the prediction of 100-days severe aGVHD (sens 65%, spec 61%) and TRM (sens 75%, spec 61%). Patients with IL-6 concentration equal or superior to 2.5 pg/ml had higher 100-days CI of severe aGvHD (16% Vs 6%, p 0,03) and TRM (12% Vs 3%, p 0,06). Interestingly, higher IL6 concentrations were associated to worse 1-year OS (42% Vs 82%, p= 129 U/l levels had higher 100-days CI of severe aGVHD (15% vs 7%, p 0,16) and TRM (13% vs 4%, p 0,01), in addition to worse OS (53% vs 71% at 1y, p 0,03). By multivariate analysis (adjusting for age, DRI, Sorror comorbidity index, type of donor, source of stem cells, and backbone of GvHD prophylaxis) pre-transplant IL6 concentrations were significantly associated to severe aGvHD (HR 3, p 0.04), TRM (HR 3, p 0.06), and OS (HR 2.6, p 0,004). Conclusion: In our series, baseline IL6 levels are predictors of aGvHD, especially with regards to grade III/IV aGvHD and translate in a higher risk of TRM and worse OS. This biomarker could be incorporated in a treatment algorithm to increase primary GvHD prophylaxis in patients at risk of severe aGVHD, potentially improving the final outcome of allo-HSCT. Disclosures Ciceri: MolMed SpA: Consultancy.

Introduction Tyrosine kinase inhibitor (TKi) has become the standard of care in patients (pts) with chronic myeloid leukemia (CML) and an unavoidable tool in the combined therapy for pts with Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). Nevertheless, because of resistance to TKI and side-effects, allogeneic stem cell transplantation (HSCT) remains the standard therapy of ALL Ph+ and of CML pts failing 1st line therapy with TKi, with failure or insufficient response or intolerance or mutations resistant to 2nd generation TKI, or in the advanced phase at diagnosis (accelerated phase and blast crisis). Unfortunately, despite greater remission with the use of TKi pre-transplant, HSCT transplant outcome have not improved largely due to high incidence of relapse after transplant. In the past decade several multi-institutional studies confirmed the feasibility and safety of post-HSCT imatinib administration as prophylactic or therapeutic strategy. Second and 3rd generation TKi administration after HSCT - targeting mutational status and according to pre-HSCT activity - is today under investigation. Methods Here we are reporting our experience in post-HSCT treatment with the 3rd generation TKi ponatinib in 5 pts (4 CML, 1 ALL Ph+) treated between 2011 and 2016 at our Institution. Pts data and information were collected from Institutional database and chapters revision. A written consent was given by pts allowing the use of medical records for research in accordance with the Declaration of Helsinki. Results Pts and diseases features are reported in table 1. Stem cell source was peripheral blood in all cases, 3 pts were transplanted from a family mismatched donor (haplo), 1 from a family matched donor, 1 from a matched unrelated donor (MUD). The 3 haplo-transplanted pts previously underwent a MUD HSCT. All pts received a treosulfan based conditioning regimen and GvHD prophylaxis consisted on co-administration of MMF and rapamycin. Pre-transplant treatment for the ALL Ph+ consisted of chemotherapy combined with dasatinib, followed by a first MUD HSCT and dasatinib in maintenance. The patient relapsed 1 year after HSCT with documentation of mutation V299L. Ponatinib was introduced as salvage treatment to bridge second haplo HSCT. Pre-transplant treatment for the CML patients consisted of TKi therapy with combination of chemotherapy in case of uncontrolled progression of disease. Two pts received a first MUD HSCT but relapsed respectively 5 months and 4 years later. Four pts received ponatinib 45 mg daily before the last HSCT: one patient achieved sustained major molecular response, 3 pts obtained transient response. All pts were presenting 2nd generation TKi resistant mutation (ref table 1). Ponatinib was started at a median time of 157 days after HSCT (range, 117-583): in 3 cases as salvage treatment in overt relapse, while in one case as prophylaxis and one case as preemptive therapy. Acute GvHD was diagnosed in 4 pts before ponatinib administration, 2 of them also experienced chronic GvHD. No new cases of GvHD were observed after initiation of ponatinib. Immunosuppressive treatment and azoles treatment were discontinued before ponatinib in all but one patient who was under combined treatment for chronic GvHD: therapeutic drug monitoring was closely performed without evidence drug-drug interaction. Pts were regularly evaluated for toxicities. No serious adverse events were reported in our experience: we administered ponatinib at a median maximum dosage of 30 mg daily (range, 15-45 mg), for a median of 24 weeks (range, 4 - 116 weeks). Two pts required anti hypertension drugs. One patient was closely monitored for multifactorial liver cholestasis never requiring ponatinib discontinuation. At last evaluation one patient maintained the status of molecularly undetectable leukemia (follow-up post HSCT 30 months) and two pts obtained molecular response (follow-up post HSCT 25 months and 5 months). Two patients who received therapeutic ponatinib in overt relapse didn't respond and died for progressive disease. Conclusions Ponatinib is safe and well tolerated as bridge to HSCT and to maintain the disease control after transplant. Prophylaxis targeted therapy and pre-emptive therapy with ponatinib may lead the reduction of disease relapse for high-risk Ph+ leukemia. Disclosures Bonini: Molmed SpA: Consultancy; TxCell: Membership on an entity's Board of Directors or advisory committees. Ciceri:MolMed SpA: Consultancy.

BACKGROUND: Although Human herpesvirus 6 (HHV6) reactivation in healthy individuals usually occurs without significant morbidity, in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is associated with severe clinical manifestations and increased transplant-related mortality (TRM). The role of HHV6 in transplant-related complications remains in question, considering that both latent and active viral infection can occur. Moreover, only limited experiences are reported on HHV6-specific immune responses after HSCT, and their correlation with clinical outcome is largely unexplored. METHODS: From February 2013 to October 2015, we conducted a prospective observational study to investigate HHV6 reactivation in 213 consecutive adult patients (median age 52 years) who received allo-HSCT for high-risk hematological malignancies (57% acute leukemia) in our institute. Stem cell donors were family haploidentical (104), HLA identical sibling (39), unrelated (63), cord blood (7). Stem cell source was mainly T-cell replete PBSCs (87%). Viral load was weekly monitored by quantitative PCR in plasma within the first month after HSCT. Numbers of IFNγ-producing HHV6-T-cells were determined by enzyme-linked immunospot assay (ELISPOT). We challenged patients PBMC against a library of overlapping peptides covering the entire sequence of the immunodominant virus protein U54, expressed during the lytic cycle of virus replication. Patients were evaluated at a median of 34 days after HSCT (HHV6-; 57 patients) for controls or by the 4th day after the first HHV6 DNAemia (median 32 days) for reactivating patients (HHV-6+; 54 patients). RESULTS: HHV6-reactivation occurred in 56% of patients at 100 days, with a median time of 28 days after HSCT. HHV6 was detected in plasma for 86% of patients, while 33% resulted positive in other materials: 9 BM aspirates, 39 gut biopsies, 3 BAL, 5 CSF. All patients received acyclovir as prophylaxis. Only 41% of reactivating patients presented a clinically relevant HHV6 infection (HHV6 positivity in presence of HHV6-related clinical symptoms and/or HHV6-disease). Clinical manifestations were: fever (25), skin rash (37), hepatitis (19), diarrhoea (28), encephalitis (5), BM suppression (30). According to center guidelines, antiviral treatment was given in 23% of reactivating patients, for uncontrolled clinically relevant HHV6 infection. Overall survival (OS) was not different in HHV6 reactivating patients compared to controls (p=0,2). Relapse incidence and TRM were not affected by HHV6. All HSCT recipients showed a better OS with CD3+ cells≥200/mcl at 30 days (p The number of IFNγ-producing HHV6-specific T-cells was significantly higher in HHV6 reactivating patients (p= 0.0149; mean number of specific T-cells 43.48 per 10^5 PBMC) than in non-reactivating patients (specific T-cells 12.57 per 10^5 PBMC), especially in the presence of active and clinically relevant HHV6 infection (p CONCLUSIONS: In this study, we observed that active disease status before HSCT, haploidentical donors, especially using PT-Cy, CMV reactivation, GvHD and lower CD3+ counts at 30 days, are strong predictors of HHV6 reactivation. HHV6-specific T-cells, detectable by ELISPOT assay despite extremely low T-cell numbers and immunosuppressive therapy, are significantly associated with active and clinically relevant HHV6 infections, representing a new and promising tool to unravel the role of HHV6 positivity in allo-HSCT recipients. Disclosures Ciceri: MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.