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Background: Follicular lymphoma (FL) remains an incurable disease for most patients (pts), characterized by a relapsing and remitting pattern. For pts with relapsed/refractory (r/r) FL, treatment outcomes typically worsen with each subsequent line of therapy, highlighting an unmet need. Tisagenlecleucel (tisa-cel) has demonstrated clinical benefits and manageable safety in pediatric and young adult pts with r/r B-cell acute lymphoblastic leukemia and adult pts with r/r diffuse large B-cell lymphoma. In a prior phase (Ph) 2a study of 14 pts with r/r FL, 71% achieved durable complete remission with tisa-cel (Chong et al. ICML 2019). Here we present a planned interim analysis of ELARA, a Ph 2, international trial of tisa-cel in adults with r/r FL. Methods: Adults with histologically confirmed FL (grades [Gr] 1-3A) being r/r within 6 mo after second-/later-line therapy (including an anti-CD20 monoclonal antibody with an alkylating agent) or relapsed after autologous hematopoietic stem cell transplant (autoHSCT), and with ECOG performance score of 0-1 were eligible. Pts with histologic transformation, prior allogeneic HSCT, or active CNS involvement were excluded. All pts received lymphodepleting chemotherapy followed by tisa-cel infusion of 0.6-6×108 CAR-T cells (bridging therapy prior to infusion was permitted). Disease was reassessed prior to infusion for all pts who received bridging therapy to establish a new baseline. After infusion, disease assessments were performed every 3 mo. The primary endpoint was complete response rate (CRR) based on best response by central review (Lugano 2014 criteria). Pts evaluable for efficacy had measurable disease at infusion and ≥6 mo follow-up from infusion or discontinued early. Secondary endpoints included overall response rate (ORR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. Results: As of May 26, 2020, 122 pts were screened, 98 were enrolled, 97 received tisa-cel (median follow-up, 6.5 mo), and 52 were evaluable for efficacy (median follow-up, 9.9 mo). At study entry, median age among treated pts was 57 y (range, 29-73), 66% of pts were male, 84% had stage III-IV disease, and 60% had a FLIPI score ≥3. The median number of prior lines of therapy was 4 (range, 2-13), including prior autoHSCT in 36%; 77% were refractory to the last prior treatment (75% ≥2 prior regimens), and 60% had disease progression within 2 y of initial anti-CD20-containing treatment. Overall, 43% of pts received bridging therapy; 18% were treated as outpatient. Of the first 52 pts evaluable for efficacy, CRR was 65.4% (34/52; 99.5% CI, 45.1-82.4) in the intent-to-treat (ITT) population and 71.1% (33/46; 99.5% CI, 56.5-84.0) in the per-protocol (PP) population; ORR was 82.7% (43/52; 95% CI, 69.7-91.8) in the ITT population and 84.8% (39/46; 95% CI, 71.1-93.7) in the PP population. In pts with best response of CR, probability of response lasting ≥6 mo was 89.7% (84.4% for all responding pts [CR+PR]). CRR and ORR were consistent across key prognostic subgroups and per investigator assessment. Median DOR, PFS, OS, and time to next antilymphoma treatment were not reached. Of 97 pts evaluable for safety, 69% experienced Gr ≥3 adverse events, most commonly neutropenia (Gr 3, 13%; Gr 4, 15%); 48% of pts had cytokine release syndrome related to tisa-cel (CRS; Gr 1, 29%; Gr 2, 20%; Gr ≥3, 0%; per Lee scale). To treat CRS, 15% of pts required tocilizumab and 3% required steroids. Any grade serious neurologic events (NEs; per CTCAE v4.03) occurred in 10% of pts; 2% had Gr ≥3 and all recovered (1 pt developed Gr 4 immune-effector cell neurotoxicity syndrome related to tisa-cel concomitant with possible HHV6 encephalitis; 1 pt developed Gr 3 delirium unrelated to tisa-cel after progression of disease and start of salvage therapy). Median time to CRS and serious NEs onset were 4 and 8.5 d, respectively, with respective median time to resolution of 4 and 2 d. Three pts died from progressive disease; no deaths were treatment-related. Conclusions: Preliminary data from ELARA suggest that tisa-cel is effective in extensively treated r/r FL, resulting in high CRRs and ORRs, including in high-risk pts. The overall safety profile is favorable, with no severe CRS and very low NE reported, requiring limited anticytokine therapy. Updated safety and efficacy results on 97 pts (including dose and cellular kinetics data) will be presented at the meeting. Disclosures Fowler: TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Research Funding. Dickinson:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Merck Sharp & Dohme: Consultancy; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau. Dreyling:Astra Zeneca: Consultancy; Abbvie: Research Funding; Roche: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Beigene: Consultancy; Gilead: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy. Martinez-Lopez:Janssen: Consultancy, Honoraria; Novartis: Consultancy; Janssen-cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Incyte: Consultancy, Research Funding. Kolstad:Merck: Research Funding; Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees, Research Funding. Popplewell:Pfizer: Research Funding; Novartis: Research Funding; Roche: Research Funding. Chavez:Genentech: Speakers Bureau; Epizyme: Speakers Bureau; Gilead: Consultancy; Verastem: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; BeiGene: Speakers Bureau; Novartis: Consultancy; Kite, a Gilead Company: Consultancy, Speakers Bureau; Pfizer: Consultancy; AstraZeneca: Speakers Bureau; Morphosys: Consultancy, Speakers Bureau; Merck: Research Funding; Bayer: Consultancy; Celgene: Consultancy. Bachy:Beigene: Membership on an entity's Board of Directors or advisory committees; Roche, Celgene, Amgen, Janssen, Gilead, Novartis, Sanofi: Honoraria; Amgen: Research Funding; Roche, Gilead: Consultancy. Kato:AbbVie, AstraZeneca, Celgene, Chugai, Eisai, Janssen, and Novartis: Consultancy; hugai, Takeda, Kyowa-Kirin, Abbvie, Novartis, Eisai, Janssen, Celgene, Ono: Research Funding; Chugai, Takeda, MSD, Kyowa-Kirin, Janssen, Celgene, Ono, Mundi, Dainippon-Sumitomo, Novartis: Honoraria. Harigae:Novartis, Chugai, BMS: Honoraria. Kersten:BMS: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Celgene: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Amgen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Janssen/Cilag: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Miltenyi Biotech: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Takeda: Research Funding; Roche: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Kite/Gilead: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); MSD: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Novartis: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company). Andreadis:Novartis: Research Funding; Gilead/Kite: Consultancy; Merck: Research Funding; Incyte: Consultancy; Karyopharm: Honoraria; Jazz Pharmaceuticals: Honoraria; BMS/Celgene/Juno: Honoraria, Research Funding; Genentech: Consultancy, Current equity holder in publicly-traded company. Riedell:Bayer: Honoraria; Karyopharm Therapeutics: Honoraria; Morphosys: Research Funding; Celgene/Bristol-Myers Squibb Company: Honoraria, Research Funding; Verastem Oncology: Honoraria; Kite Pharmaceuticals/Gilead: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Nastoupil:Karus Therapeutics: Research Funding; Gamida Cell: Honoraria; Gilead/KITE: Honoraria; Novartis: Honoraria, Research Funding; Merck: Research Funding; Celgene: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Bayer: Honoraria; Janssen: Honoraria, Research Funding; LAM Therapeutics: Research Funding; TG Therapeutics: Honoraria, Research Funding; Genentech, Inc.: Honoraria, Research Funding. von Tresckow:Takeda: Honoraria, Other: Travel support, Research Funding; Amgen: Honoraria; Pfizer: Honoraria; Kite/Gilead: Honoraria; Roche: Honoraria; MSD Sharp & Dohme: Honoraria, Research Funding; Takeda: Honoraria, Other: Travel support, Research Funding; Novartis: Other: Travel support, Research Funding. Ferreri:Morphosys: Research Funding; Hutchinson: Research Funding; BMS: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding. Teshima:Sharp & Dohme Corp: Consultancy, Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer Japan Inc.: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; TEIJIN PHARMA LIMITED: Honoraria; The Center of Innovation Program from Japan Science and Technology Agency: Other; Fuji Pharma Co., Ltd.: Honoraria; NIPPON SHINYAKU CO., LTD.: Honoraria; Janssen Pharmaceutical K.K.: Other; Japan Society for the Promotion of Science KAKENHI (17H04206): Other; Novartis Pharma K.K.: Consultancy, Other: Manuscript preparation, Research Funding; Takeda Pharmaceutical Company Limited: Consultancy, Honoraria; Chugai Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Merck: Consultancy, Honoraria; Sanofi K.K.: Research Funding. Patten:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Honoraria; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria. McGuirk:Novartis: Research Funding; Fresenius Biotech: Research Funding; Astellas: Research Funding; Juno Therapeutics: Consultancy, Honoraria, Research Funding; Allo Vir: Consultancy, Honoraria, Research Funding; Kite Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pluristem Ltd: Research Funding; Gamida Cell: Research Funding; Bellicum Pharmaceutical: Research Funding. Petzer:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: 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; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Viardot:Amgen: Honoraria, Other: advisory board; Novartis: Honoraria, Other: advisory board; Kite/Gilead: Honoraria, Other: advisory board; Roche: Honoraria, Other: advisory board. Zinzani:ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immune Design: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSA Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immune Design: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Portola: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyowa Kirin: Consultancy, Speakers Bureau; Eusapharma: Consultancy, Speakers Bureau; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kirin Kyowa: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Therapeutics, Inc.: Honoraria, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Malladi:Novartis: Consultancy, Honoraria. Bubuteishvili Pacaud:Novartis: Current Employment. Forcina:Novartis: Current Employment. Zia:Novartis: Current Employment. Schuster:Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding; AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria. Thieblemont:Cellectis: Speakers Bureau; Roche, Amgen, Kyte Gilead, Celgene, Abbvie, Novartis, Cellectis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Roche, Hospita: Research Funding.

Background: The PI3K/AKT/mTOR pathway is an established therapeutic target in indolent B-cell non-Hodgkin lymphoma (B-NHL). Several PI3K inhibitors are approved to treat relapsed/refractory (R/R) follicular lymphoma (FL) and, more recently, marginal zone lymphoma (MZL). Nonetheless, potential class-specific and PI3K-isoform-related toxicities may limit their clinical utility. Capivasertib is an oral, potent, selective inhibitor of the 3 AKT isoforms that demonstrated a manageable safety profile (n>1500) and survival benefit in combination with other agents in patients (pts) with solid tumors. Unlike PI3K inhibitors, colitis or pneumonitis have not been a concern with monotherapy. Preclinically, capivasertib was active both in vitro in a large panel of B-NHL cell lines and in vivo in PTEN-null GCB-DLBCL PDX models, where it downregulated the oncogenic factor MYC. In the JVM2 mantle cell lymphoma (MCL) cell line, capivasertib showed superior activity vs PI3Ka/d (AZD8835) and PI3Kb/d (AZD8186) inhibitors. Of note, AKT activation following dysregulation of PTEN has been frequently described in FL (where >85% of tFL or FL/DLBCL exhibited a GCB phenotype). Therefore, capivasertib may represent an effective treatment option for pts with R/R B-NHL. Trial Design: This modular phase II study will enroll pts in 3 cohorts according to B-NHL type. Core inclusion criteria include: age ≥18 years and ECOG PS ≤2. Module-specific criteria include: cohort 1A: R/R FL after ≥2 prior systemic lines (max 5), including an anti-CD20 monoclonal antibody (mAb) and an alkylating agent; cohort 1B: R/R MZL of splenic, nodal, and extranodal subtypes after ≥2 prior systemic lines (max 5) including ≥1 anti-CD20 mAb; cohort 1C: R/R MCL after ≥2 prior systemic lines (max 4), previously exposed to anti-CD20 mAb and a BTK inhibitor. Pts with FL grade 3B, transformed disease, blastoid or TP53 mutation MCL, active CNS disease, diabetes requiring insulin, or being Funding: AstraZeneca Clinical Trial Registration: NCT050080552 Citation Format: Michael Wang, Ho-Jin Shin, Alex F. Herrera, Moshe Levy, Wong Seog Kim, Tae Min Kim, Jin Seok Kim, Dok Hyun Yoon, Vincent Ribrag, Miguel Canales, John Radford, Dima El-Sharkawi, Brandy Hemmer, Richa Manwani, Veerendra Munugalavadla, Robert Chen, Andrew Mortlock, Alessandra Forcina, Franck Morschhauser. Trial in progress: A phase II, open-label, multicenter study of capivasertib, a potent, oral pan-AKT inhibitor in patients with relapsed or refractory B-cell non-hodgkin lymphoma (CAPITAL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT127.

Febrile neutropenia and sepsis are common and life-threatening complications in hematological diseases. This study was performed retrospectively in 514 patients treated for febrile neutropenia at our institute, to investigate the clinical usefulness of a molecular tool, LightCycler® SeptiFast test (SF), to promptly recognize pathogens causing sepsis in hematological patients. We collected 1837 blood samples of 514 consecutive hematological patients. The time of processing is short. Overall, 757 microorganisms in 663 episodes were detected by molecular test and standard blood cultures (BC): 73.6% Gram-positive bacteria, 23.9% Gram-negative bacteria, and 2.5% fungal species. This large analysis demonstrated a significant episode-to episode agreement (71.9%) between the two methods, higher in negative samples (89.14%), and a specificity of 75.89%. Clinical variables that gave a statistically significant contribution to their concordance were absolute neutrophil count, ongoing antimicrobial therapy, timing of test execution, and organ localization of infection. The large analysis highlights the potential of molecular-based assays directly performed on blood samples, especially if implementing the detection of antibiotic resistance genes, which was lacking in the used study.

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.

Introduction For patients (pts) with relapsed or refractory follicular lymphoma (r/r FL) beyond front-line therapy, there is no well-defined standard of care (SOC) treatment, especially in the third-line or later (3L+) setting. Treatment decisions for symptomatic patients depend on comorbidities, extent of disease, lines of prior therapy and duration of response to initial anti-CD20 containing treatment. Treatment options for 3L+ may include similar options to the ones in earlier lines, with a preference for non-cross-resistant schemes. Recently, a plethora of new compounds are being studied in clinical trials. A systematic literature review (SLR) was conducted to identify relevant evidence on clinical outcomes in pts with r/r FL, including conventional treatments and emerging compounds, within the 3L+ setting. Methods We performed a SLR on March 17, 2020. Clinical trials and observational studies were searched through Embase, PubMed, and Cochrane Central Register of Controlled Clinical Trials from 1998 to 2020, followed by relevant conference proceedings and regulatory documents. Evidence assessing any intervention as 3L+ FL and published in English language was included. If a study included broader indolent non-Hodgkin's lymphoma (iNHL) pts, only FL data was reported; if a study included pts with fewer than 3L+, 3L+ data was included, with mixed line results excluded. Conventional treatments, defined as approved or clinical guideline recommended treatments, included anti-CD20 monoclonal antibody (mAb)-containing regimen, mAb alone, chemotherapy alone, phosphatidylinositol 3-kinase (PI3K) inhibitors (idelalisib, copanlisib, duvelisib), lenalidomide + rituximab (R2), radio-immunotherapy (RIT) with yttrium-90 (90Y) ibritumomab tiuxetan, tazemetostat, autologous and allogeneic stem cell transplantation (auto- and allo-SCT). Emerging compounds included new treatments that are not approved but were tested in the context of clinical trials (bruton tyrosine kinase inhibitors, bortezomib, polatuzumab vedotin, daratumumab, inotuzumab, bi-specific T-cell engaging CD19 mAb, anti-CD19 chimeric antigen receptor T-cell [CAR-T] therapy). Results Of the 3747 publications identified, 74 studies assessing 26 treatment regimens, including conventional ones like rituximab (R)-containing immunochemotherapy to emerging compounds such as CAR-T therapies, were selected. Across the conventional regimens, 7 studies reported clinical trial data with relatively large 3L+ FL populations (Table 1). With R monotherapy, PI3K inhibitors and tazemetostat, the reported complete response (CR) rates and overall response rates (ORR) ranged from 1-20% and 34-77%, while median duration of response (mDOR) ranged between 7.9-16.3 months. Three clinical trials with PI3K inhibitors reported proportions of pts achieving a response of at least 6 months in duration ranging from 18-30%; median progression-free survival (mPFS) ranging from 8.3-11.2 months. For tazemetostat, mPFS was 11.0 months in EZH2 mutant vs 5.7 months in wild-type FL. With allo-SCT, 2-yr PFS rate was 88% and 57% for pts with CR and PR, respectively. Overall survival (OS) data varied: median overall survival (mOS) was 28-38 months for PI3K inhibitors while mOS was up to 85 months after allo-SCT, despite high non-relapse mortality (NRM) rates with most common causes of death being graft-versus-host disease (GvHD) and infections. Safety profiles were also different across treatments, with most common side effects being hepatotoxicity, diarrhea and infections (PI3K inhibitors) to GvHD (allo-SCT). Three trials presented patient-reported outcomes (PRO) data, all using the Functional Assessment of Cancer Therapy (FACT) questionnaire. Two studies with PI3K inhibitors demonstrated favorable or clinically significant improvement on pts quality of life (QoL), but 1 allo-SCT trial did not show a significant difference between baseline and 2 years post-transplant scores. Conclusion To our knowledge, this is the first SLR focusing on 3L+ treatments of FL. Heterogeneity in study design, patient population, safety profile and reported outcomes make it challenging to identify an optimal treatment regimen for FL in the 3L+ setting. More PRO data are needed considering the important role pt QoL plays in treatment selection. Disclosures Ma: Novartis:Current Employment.Kaur:Novartis:Current Employment.Zhao:Novartis:Current Employment.Zhang:Novartis:Current Employment.Ramos:Novartis:Current Employment.Kumar:Novartis:Current Employment.John:Novartis:Current Employment.Bubuteishvili Pacaud:Novartis:Current Employment.Forcina:Novartis:Current Employment.

Introduction: Pembrolizumab (pembro) has shown clinical activity in r/r DLBCL after failing tisagenlecleucel (tisa), an autologous anti-CD19 chimeric antigen receptor (CAR)-T therapy (Chong et al. Blood. 2017). Methods: PORTIA is a phase 1b, multicenter, open-label trial investigating the safety and efficacy of tisa plus pembro in adult patients (pts) with r/r DLBCL. Pts receive a single tisa IV infusion (target: 0.6-6.0×108 cells) on Day (D)1 and pembro at 200mg every 21 days, for up to 6 doses. Pembro was started on D15 post tisa in Cohort 1, with the option of moving to D8 (Cohort 2) or D22 based on safety profile and dose-limiting toxicities (DLTs). Primary endpoints: proportion of pts receiving pembro per schedule, incidence of DLTs (dose-timing selection phase), overall response rate (dose-expansion phase). Results: As of 5 March 2019, 5 patients were screened for Cohort 1; 4 pts were enrolled. Median age was 54 (range, 35-79). Median follow-up after tisa infusion was 46 days (range, 36-85). Pts received 1.7-3.0×108 CAR-positive T cells and 1, 2, 2, and 4 pembro doses, respectively, with no delays. All 4 pts experienced ≥1 adverse event (AE), with no exacerbation or recurrence of tisa-related AEs post pembro infusion. No pembro-related AEs, DLTs or grade 3/4 treatment-related AEs (TRAEs) were observed (Table 1). All pts experienced initial expansion between D6-15 post-tisa infusion; overall exposure is consistent with that observed in the JULIET trial. Two pts discontinued pembro treatment (after 1 and 2 doses, respectively) due to disease progression. With limited follow-up, 1 partial response has been observed. Table 1.TRAEs and Grade 3/4 AEs Observed in Day 15 Cohort Interim ReadAdverse EventPatients With Grade 3/4 AE, n (%)Patients With Grade 3/4 AE, n (%)Patients With Grade 3/4 TRAE, n (%)Anemia1 (25)a00Cytokine release syndrome01 (25)a0Pancreatitis1 (25)a00Tachycardia01 (25)a0AE, adverse event; TRAE, treatment-related adverse event.aAnemia developed on D14 after tisa infusion but preceding pembro initiation, and again on D36 while the pt was on pembro (during the time from first pembro administration to 30 days after the last pembro administration date). Pancreatitis developed on D28 in the same pt. Both are likely related to lymphoma progression.bOne pt developed grade 2 cytokine release syndrome according to the Lee scale (D7-11) and concomitant sinus tachycardia, both attributed to tisa and resolved prior to pembro infusion; no neurological events occurred. Conclusions: PD-1 blockade with pembro was feasible and showed a manageable safety profile in the first 4 pts. No DLTs or clinically significant exacerbation of AEs were observed. Efficacy and safety data for Cohorts 1 and 2 (D15 and D8) with longer follow-up will be presented at the congress. Clinical trial information: NCT03630159 Citation Format: Ulrich Jaeger, Nina Worel, Joseph P. McGuirk, Peter A. Riedell, Isabelle Fleury, Peter Borchmann, Simon Newsome, Ahmed M. Abdelhady, Alessandra Forcina, Lida Bubuteishvili Pacaud, Edmund K. Waller. The phase 1b PORTIA study: Safety and efficacy of tisagenlecleucel plus pembrolizumab in patients with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT162.

Multidrug-resistant Gram-negative bacteria (MDR-GNB) are an emerging cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Three-hundred forty-eight consecutive patients transplanted at our hospital from July 2012 to January 2016 were screened for a pretransplant MDR-GNB colonization and evaluated for clinical outcomes. A pretransplant MDR-GNB colonization was found in 16.9% of allo-HSCT and in 9.6% of auto-HSCT recipients. Both in auto- and in allo-HSCT, carriers of a MDR-GNB showed no significant differences in overall survival (OS), transplant-related mortality (TRM), or infection-related mortality (IRM) compared with noncarriers. OS at 2 years for carriers compared with noncarriers was 85% versus 81% (P =.262) in auto-HSCT and 50% versus 43% (P =.091) in allo-HSCT. TRM at 2 years was 14% versus 5% (P =.405) in auto-HSCT and 31% versus 25% (P =.301) in allo-HSCT. IRM at 2 years was 14% versus 2% (P =.142) in auto-HSCT and 23% versus 14% (P =.304) in allo-HSCT. In multivariate analysis, only grade III to IV acute graft-versus-host disease was an independent factor for reduced OS (P

Background Infections and graft-vs-host disease (GvHD) still represent major, not easily predictable complications in allogeneic hematopoietic stem cell transplant (allo-HSCT). Both conditions have been correlated to altered enteric microbiome profiles during the peritransplant period. The main objective of this study was to identify possible early microbiome-based markers useful in pretransplant risk stratification. Methods Stool samples were collected from 96 consecutive patients at the beginning of the pretransplant conditioning regimen (T0) and at 10 (T1) and 30 (T2) days following transplant. When significant in univariate analysis, the identified microbiome markers were used in multivariate regression analyses, together with other significant clinical variables for allo-HSCT-related risk stratification. Four main outcomes were addressed: (1) septic complications, (2) GvHD, (3) relapse of the underlying disease, and (4) mortality. Results The presence of >5% proinflammatory Enterobacteriaceae at T0 was the only significant marker for the risk of microbiologically confirmed sepsis. Moreover, ≤10% Lachnospiraceae at T0 was the only significant factor for increased risk of overall mortality, including death from both infectious and noninfectious causes. Finally, a low bacterial alpha-diversity (Shannon index ≤ 1.3) at T1 was the only variable significantly correlating with an increased risk of GvHD within 30 days. Conclusions Microbiome markers can be useful in the very early identification of patients at risk for major transplant-related complications, offering new tools for individualized preemptive or therapeutic strategies to improve allo-HSCT outcomes.

Infection-related mortality (IRM) is a substantial component of nonrelapse mortality (NRM) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). No scores have been developed to predict IRM before transplantation. Pretransplantation clinical and biochemical data were collected from a study cohort of 607 adult patients undergoing allo-HSCT between January 2009 and February 2017. In a training set of 273 patients, multivariate analysis revealed that age >60 years (P = .003), cytomegalovirus host/donor serostatus different from negative/negative (P

CD1c self-reactive T cells recognize a novel class of self-lipids that are accumulated on leukemia cells., T cells that recognize self-lipids presented by CD1c are frequent in the peripheral blood of healthy individuals and kill transformed hematopoietic cells, but little is known about their antigen specificity and potential antileukemia effects. We report that CD1c self-reactive T cells recognize a novel class of self-lipids, identified as methyl-lysophosphatidic acids (mLPAs), which are accumulated in leukemia cells. Primary acute myeloid and B cell acute leukemia blasts express CD1 molecules. mLPA-specific T cells efficiently kill CD1c+ acute leukemia cells, poorly recognize nontransformed CD1c-expressing cells, and protect immunodeficient mice against CD1c+ human leukemia cells. The identification of immunogenic self-lipid antigens accumulated in leukemia cells and the observed leukemia control by lipid-specific T cells in vivo provide a new conceptual framework for leukemia immune surveillance and possible immunotherapy.

Background: Tisagenlecleucel is an autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy, approved for the treatment of relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) after ≥ 2 prior lines of therapy. T-cell exhaustion due to an immunosuppressive environment has been a hypothesized mechanism for CAR-T cell therapy failure. Subgroup analyses of the JULIET trial suggested an association between programmed cell death 1 (PD-1) and programmed death ligand 1 (PD-L1) interaction in baseline biopsies and lack of response (Agoulnik et al. EHA. 2018). Moreover, the anti-PD-1 monoclonal antibody pembrolizumab has shown clinical activity in r/r DLBCL after failing tisagenlecleucel therapy (Chong et al. Blood. 2017). PORTIA is a phase 1b, multicenter, open-label trial investigating the safety and efficacy of tisagenlecleucel plus pembrolizumab in patients with r/r DLBCL. We report data from a completed cohort of the ongoing study. Methods: Eligible patients must be ≥ 18 years old with a confirmed diagnosis of DLBCL that has relapsed after or is refractory to ≥ 2 prior lines of therapy and Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Patients treated with prior allogeneic stem cell transplantation, anti-CD19 therapies, or checkpoint inhibitors are excluded. Lymphodepleting chemotherapy consists of fludarabine-cyclophosphamide. Patients receive a single tisagenlecleucel intravenous infusion (target dose: 0.6-6.0x108 cells) on Day 1. Pembrolizumab is given at 200 mg every 21 days, for up to 6 doses. Pembrolizumab was started on Day 15 post-tisagenlecleucel in Cohort 1, with the option of moving to Day 8 or 22 in subsequent cohorts, based on observed data and guided by a Bayesian Logistic Regression Model with Escalation with Overdose Control principle, evaluating the distribution of dose-limiting toxicities (DLTs) occurring in the 21 days following the first pembrolizumab dose. As per study protocol, a total of 20 patients will be treated at the optimal dose timing. Primary endpoints are the proportion of patients receiving pembrolizumab per protocol schedule, the incidence of DLTs in the dose-timing selection phase, and the overall response rate in the dose-expansion phase. Secondary outcomes include duration of response, progression-free survival, overall survival, safety, cellular kinetics, and immunogenicity. Results: As of 5 March 2019, 5 patients were screened for Cohort 1. Four patients were enrolled and received tisagenlecleucel and pembrolizumab. Median age was 54 (range, 35-79). Median follow-up from time from tisagenlecleucel infusion to data cut-off was 46 days (range, 36-85). Patients received 1.7-3.0x108 CAR-positive T cells, and 1, 2, 2 and 4 pembrolizumab doses, respectively, with no delays. All 4 patients experienced at least 1 adverse event (AE), with no exacerbation or recurrence of tisagenlecleucel-related AEs following pembrolizumab infusion. No pembrolizumab-related AEs were observed. No DLTs or grade 3-4 treatment-related adverse events (TRAEs) were observed. TRAEs and AEs are summarized in Table 1. Two patients discontinued pembrolizumab treatment (after 1 and 2 doses, respectively) due to disease progression. All 4 patients experienced initial expansion between Days 6 and 15 post-tisagenlecleucel infusion, with peak transgene levels ranging from 1,980 to 77,200 copies/µg DNA (Figure 1). No secondary expansion was observed after pembrolizumab administration. The overall exposure is consistent with the observed exposure in r/r DLBCL patients in the JULIET trial. With very limited follow-up, 1 partial response has been observed. Cohort 2 (pembrolizumab starting Day 8) was ongoing at the time of submission. Conclusions: Overall, PD-1 blockade with pembrolizumab on Day 15 after tisagenlecleucel infusion was feasible and showed a manageable safety profile in the first 4 patients. No DLTs and no clinically significant exacerbation of AEs were observed, supporting the initiation of Cohort 2. Efficacy and safety data with an updated cutoff for Cohort 1 and new data from Cohort 2 will be presented at the congress. Clinical trial information: NCT03630159. Disclosures Jaeger: Novartis, Roche, Sandoz: Consultancy; AbbVie, Celgene, Gilead, Novartis, Roche, Takeda Millennium: Research Funding; Amgen, AbbVie, Celgene, Eisai, Gilead, Janssen, Novartis, Roche, Takeda Millennium, MSD, BMS, Sanofi: Honoraria; Celgene, Roche, Janssen, Gilead, Novartis, MSD, AbbVie, Sanofi: Membership on an entity's Board of Directors or advisory committees. Worel:Sanofi Genzyme, Malinckrodt Therakos: Research Funding; Jazz, Sanofi, Celgene, Novartis, Malinckrodt Therakos: Honoraria; Sanofi Genzyme, Malinckrodt Therakos: Speakers Bureau. McGuirk:Juno Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bellicum Pharmaceuticals: Research Funding; Astellas: Research Funding; Fresenius Biotech: Research Funding; Novartis: Research Funding; ArticulateScience LLC: Other: Assistance with manuscript preparation; Pluristem Ltd: Research Funding; Gamida Cell: Research Funding; Kite Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Riedell:Kite/Gilead: Honoraria, Research Funding, Speakers Bureau; Novartis: Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Speakers Bureau. Fleury:AstraZeneca: Consultancy; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Chu:Novartis: Employment. Abdelhady:Novartis: Employment. Forcina:Novartis: Employment. Bubuteishvili Pacaud:Novartis: Employment. Waller:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Other: Travel expenses, Research Funding; Cerus Corporation: Other: Stock, Patents & Royalties; Chimerix: Other: Stock; Cambium Oncology: Patents & Royalties: Patents, royalties or other intellectual property ; Amgen: Consultancy; Kalytera: Consultancy.

NA A major hurdle to the field of allogeneic hematopoietic stem cell transplantation (HSCT) is the lack of validated biomarkers of protective immunity against opportunistic infections, including CMV reactivation syndromes. This issue is particularly relevant to alternative-donor settings, for example, HLA-haploidentical HSCT (haplo-HSCT) and cord blood transplantation, where aggressive GvHD prophylaxis results in a profound and long-lasting state of immune incompetence. Despite the introduction of routine post-transplant viral monitoring and preemptive antiviral therapy, CMV reactivation syndromes remain a major cause of transplant related mortality. Accordingly, serological evidence of prior CMV infection is still one of the main negative prognostic factors in HSCT. Different studies have demonstrated that a faster recovery of CMV-specific T-cell responses associates with a reduced CMV reactivation incidence in HSCT from HLA-identical sibling and matched unrelated donors. Given its clinical and biological peculiarities, it is currently unknown whether these observations are directly translatable to the haplo-HSCT setting. In this study, while aiming to follow the recovery of CMV-specific T-cell responses after haplo-HSCT, we unexpectedly found that protective levels of CMV immunity were associated with a reduced incidence of severe infections overall.

Despite the considerable progress in understanding the molecular bases of acute myeloid leukemia (AML), new tools to link disease biology to the unpredictable patient clinical course are still needed. Herein, high-throughput metabolomics, combined with the other “-omics” disciplines, holds promise in identifying disease-specific and clinically relevant features. In this study, we took advantage of nuclear magnetic resonance (NMR) to trace AML-associated metabolic trajectory employing two complementary strategies. On the one hand, we performed a prospective observational clinical trial to identify metabolic changes associated with blast clearance during the first two cycles of intensive chemotherapy in nine adult patients. On the other hand, to reduce the intrinsic variability associated with human samples and AML genetic heterogeneity, we analyzed the metabolic changes in the plasma of immunocompromised mice upon engraftment of primary human AML blasts. Combining the two longitudinal approaches, we narrowed our screen to seven common metabolites, for which we observed a mirror-like trajectory in mice and humans, tracing AML progression and remission, respectively. We interpreted this set of metabolites as a dynamic fingerprint of AML evolution. Overall, these NMR-based metabolomic data, to be consolidated in larger cohorts and integrated in more comprehensive system biology approaches, hold promise for providing valuable and non-redundant information on the systemic effects of leukemia. Electronic supplementary material The online version of this article (doi:10.1186/s13045-016-0346-2) contains supplementary material, which is available to authorized users.

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.

Carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) infections are an emerging cause of death after hematopoietic stem cell transplantation (HSCT). In allogeneic transplants, mortality rate may rise up to 60%. We retrospectively evaluated 540 patients receiving a transplant from an auto- or an allogeneic source between January 2011 and October 2015. After an Institutional increase in the prevalence of KPC-Kp bloodstream infections (BSI) in June 2012, from July 2012, 366 consecutive patients received the following preventive measures: (i) weekly rectal swabs for surveillance; (ii) contact precautions in carriers (iii) early-targeted therapy in neutropenic febrile carriers. Molecular typing identified KPC-Kp clone ST512 as the main clone responsible for colonization, BSI and outbreaks. After the introduction of these preventive measures, the cumulative incidence of KPC-Kp BSI (P=0.01) and septic shocks (P=0.01) at 1 year after HSCT was significantly reduced. KPC-Kp infection-mortality dropped from 62.5% (pre-intervention) to 16.6% (post-intervention). Day 100 transplant-related mortality and KPC-Kp infection-related mortality after allogeneic HSCT were reduced from 22% to 10% (P=0.001) and from 4% to 1% (P=0.04), respectively. None of the pre-HSCT carriers was excluded from transplant. These results suggest that active surveillance, contact precautions and early-targeted therapies, may efficiently control KPC-Kp spread and related mortality even after allogeneic HSCT.

Coadministration of posaconazole and sirolimus in allogeneic hematopoietic stem cell transplant recipients

We retrospectively studied 181 patients with polycythaemia vera (n=67), essential thrombocythaemia (n=67) or primary myelofibrosis (n=47), who presented a first episode of splanchnic vein thrombosis (SVT). Budd-Chiari syndrome (BCS) and portal vein thrombosis were diagnosed in 31 (17.1%) and 109 (60.3%) patients, respectively; isolated thrombosis of the mesenteric or splenic veins was detected in 18 and 23 cases, respectively. After this index event, the patients were followed for 735 patient years (pt-years) and experienced 31 recurrences corresponding to an incidence rate of 4.2 per 100 pt-years. Factors associated with a significantly higher risk of recurrence were BCS (hazard ratio (HR): 3.03), history of previous thrombosis (HR: 3.62), splenomegaly (HR: 2.66) and leukocytosis (HR: 2.8). Vitamin K-antagonists (VKA) were prescribed in 85% of patients and the recurrence rate was 3.9 per 100 pt-years, whereas in the small fraction (15%) not receiving VKA more recurrences (7.2 per 100 pt-years) were reported. Intracranial and extracranial major bleeding was recorded mainly in patients on VKA and the corresponding rate was 2.0 per 100 pt-years. In conclusion, despite anticoagulation treatment, the recurrence rate after SVT in myeloproliferative neoplasms is high and suggests the exploration of new avenues of secondary prophylaxis with new antithrombotic drugs and JAK-2 inhibitors. TB and AMV were supported by a grant from Associazione Italiana per la Ricerca sul Cancro (AIRC, Milano) ‘Special Program Molecular Clinical Oncology 5 × 1000’ to AGIMM (AIRC-Gruppo Italiano Malattie Mieloproliferative). VDS was supported by an unrestricted grant from the Bruno Farmaceutici Foundation.

The optimal duration of treatment with vitamin K antagonists (VKA) after venous thromboembolism (VTE) in patients with Philadelphia-negative myeloproliferative neoplasms (MPNs) is uncertain. To tackle this issue, we retrospectively studied 206 patients with MPN-related VTE (deep venous thrombosis of the legs and/or pulmonary embolism). After this index event, we recorded over 695 pt-years 45 recurrences, venous in 36 cases, with an incidence rate (IR) of 6.5 per 100 pt-years (95% confidence interval (CI): 4.9-8.6). One hundred fifty-five patients received VKA; the IR of recurrent thrombosis per 100 pt-years was 4.7 (95% CI: 2.8-7.3) on VKA and 8.9 (95% CI: 5.7-13.2) off VKA (P=0.03). In patients receiving VKA, the IR of recurrent thrombosis per 100 pt-years was 5.3 (95% CI: 3.2-8.4) among 108 patients on long-term VKA and 12.8 (95% CI: 7.3-20.7) after discontinuation among the 47 who ceased treatment (P=0.008), with a doubled risk of recurrence after stopping VKA (hazard ratio: 2.21, 95% CI: 1.19-5.30). The IR of major bleeding per 100 pt-years was 2.4 (95%: CI: 1.1-4.5) on VKA and 0.7 (95% CI: 0.08-2.5) off VKA (P=0.08). In conclusion, in MPN patients with VTE recurrent thrombosis is significantly reduced by VKA and caution should be adopted in discontinuation; however, the incidence of recurrence on treatment remains high, calling for clinical trials aimed to improve prophylaxis in this setting.

Hematopoietic stem cell transplantation (HSCT) from human leukocyte antigen (HLA) haploidentical family donors is a promising therapeutic option for high-risk hematologic malignancies. Here we explored in 121 patients, mostly with advanced stage diseases, a sirolimus-based, calcineurin-inhibitor-free prophylaxis of graft-versus-host disease (GvHD) to allow the infusion of unmanipulated peripheral blood stem cell (PBSC) grafts from partially HLA-matched family donors (TrRaMM study, Eudract 2007-5477-54). Conditioning regimen was based on treosulfan and fludarabine, and GvHD prophylaxis on antithymocyte globulin Fresenius (ATG-F), rituximab and oral administration of sirolimus and mycophenolate. Neutrophil and platelet engraftment occurred in median at 17 and 19 days after HSCT, respectively, and full donor chimerism was documented in patients' bone marrow since the first post-transplant evaluation. T-cell immune reconstitution was rapid, and high frequencies of circulating functional T-regulatory cells (Treg) were documented during sirolimus prophylaxis. Incidence of acute GvHD grade II-IV was 35%, and occurrence and severity correlated negatively with Treg frequency. Chronic GvHD incidence was 47%. At 3 years after HSCT, transpant-related mortality was 31%, relapse incidence 48% and overall survival 25%. In conclusion, GvHD prophylaxis with sirolimus-mycophenolate-ATG-F-rituximab promotes a rapid immune reconstitution skewed toward Tregs, allowing the infusion of unmanipulated haploidentical PBSC grafts. Hematopoietic stem cell transplantation (HSCT) from human leukocyte antigen (HLA) haploidentical family donors is a promising therapeutic option for high-risk hematologic malignancies. Here we explored in 121 patients, mostly with advanced stage diseases, a sirolimus-based, calcineurin-inhibitor-free prophylaxis of graft-versus-host disease (GvHD) to allow the infusion of unmanipulated peripheral blood stem cell (PBSC) grafts from partially HLA-matched family donors (TrRaMM study, Eudract 2007-5477-54). Conditioning regimen was based on treosulfan and fludarabine, and GvHD prophylaxis on antithymocyte globulin Fresenius (ATG-F), rituximab and oral administration of sirolimus and mycophenolate. Neutrophil and platelet engraftment occurred in median at 17 and 19 days after HSCT, respectively, and full donor chimerism was documented in patients’ bone marrow since the first post-transplant evaluation. T-cell immune reconstitution was rapid, and high frequencies of circulating functional T-regulatory cells (Treg) were documented during sirolimus prophylaxis. Incidence of acute GvHD grade II–IV was 35%, and occurrence and severity correlated negatively with Treg frequency. Chronic GvHD incidence was 47%. At 3 years after HSCT, transplant-related mortality was 31%, relapse incidence 48% and overall survival 25%. In conclusion, GvHD prophylaxis with sirolimus– mycophenolate–ATG-F–rituximab promotes a rapid immune reconstitution skewed toward Tregs, allowing the infusion of unmanipulated haploidentical PBSC grafts.

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.

Infection-related mortality (IRM) still represents a major determinant of non-relapse mortality (NRM) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Despite the curative potential of allo-HSCT, the profound and prolonged status of immune incompetence following transplantation poses patients at risk for lethal opportunistic infections. This is particularly important in transplants from HLA-mismatched donors. Although numerous studies have investigated the role of pre-transplant clinical variables for the prediction of IRM, very few have focused on biological culprits. This study was aimed at the development of a composite clinico-biological prognostic scoring system for the prediction of early and late IRM after allo-HSCT. A total of 492 consecutive adult patients receiving allo-HSCT for hematological disorders were studied from January 2009 to May 2015. Second transplants were excluded, as well as patients for which pre-transplant biological data were missing. The Receiver Operating Characteristics (ROC) curve analysis defined the optimal cut-offs of pre-transplant biological variables predicting 100-days IRM. All variables were then challenged in multivariate analysis. Based on the value of the coefficients selected in the final multivariate model, a weighted score predicting IRM was elaborated in the training cohort of patients (n=273, from January 2012 to May 2015), and then tested in an internal validation cohort of patients (n=219, from January 2009 to December 2011). The median follow-up was of 31 months (range, 1-85). Acute leukemia was the main indication to transplant, accounting for the 61% (n=309) of patients. Fifty-one percent of patients received an HLA-haploidentical donor graft, 27% a fully HLA-matched (10/10) or a single mismatch (9/10) unrelated donor (MUD) graft, 19% a HLA-identical sibling donor and 3% a cord blood unit. Noticeably, 49% (n=248) patients underwent transplant for advanced diseases. To assess uniformity between the training and validation cohorts, we calculated the incidence of NRM as well as overall survival (OS) and progression-free survival (PFS) in each group. OS at 2 yrs was 53% (95% CI: 47% to 60%) in the training cohort and 46% (95% CI: 40% to 53%) in the validation cohort (P=0.050). PFS at 2 yrs was 23% (95% CI: 16% to 23%) in the training cohort and 17% (95% CI: 12% to 27%) in the validation cohort (P=0.440). The 2-yr incidences of NRM and IRM were 28% (95% CI, 23% to 24%) and 22% (95% CI, 18% to 28%), respectively, in the training cohort and 34% (95% CI: 28% to 40%) and 23% (95% CI: 18% to 29%), respectively, in the validation cohort (P=0.371 and P=0.702). Only four variables were found independent predictors of IRM: age >60 yrs (P=0.003), CMV host/donor serostatus different from negative/negative (P 11.11 points). Considering the entire cohort, the 2-yr IRM was 12% (95% CI: 8% to 17%) for low-risk patients (n=196), 23% (95% CI: 17% to 30%) for intermediate-risk patients (n=175) and 37% (95% CI: 28% to 46%) for high-risk patients (n=108) (P Figure 1 shows IRM according to the 3-tiered prognostic model in the training group (A) and in the validation group (B). Additionally, the 2-yrs OS was significantly different among the 3 groups, being 59% (95% CI: 52% to 67%), 50% (95% CI: 43% to 59%) and 37% (95% CI: 29% to 48%), for low, intermediate and high-risk patients, respectively (P=0.0001). The proposed clinico-biological scoring system is able to predict IRM after allo-HSCT and may foster active surveillance and early pre-emptive antimicrobial strategies in patients at higher risk for infectious complications. Moreover, this score pave the way for the prospective investigation of prophylactic infusions of IgM/IgA-enriched immunoglobulins to reduce IRM after allo-HSCT and to improve its overall outcome. Figure 1 Figure 1. Disclosures Ciceri: MolMed SpA: Consultancy.

BACKGROUND: Allogeneic HSCT (allo-HSCT) is the only cure for several patients with hematological malignancies. Recent advances in therapies have significantly reduced transplant-related mortality (TRM); nevertheless infections and graft-versus-host disease (GvHD) still represent major complications. Recent studies indicate that patients undergo dramatic alterations of intestinal microbiota during allo-HSCT, potentially affecting the outcome. An alarming emergence of gram-negative multi-drug-resistant (GN-MDR) pathogens has been increasingly reported from various cancer centers, and the primary site of colonization is mainly the gut. Moreover, GvHD was associated with major shifts in the composition of intestinal microbiota, able to modulate the severity of intestinal inflammation. METHODS: Between October 2014 and March 2016, we have conducted a prospective observational study to examine the intestinal microbiota by NGS techniques in 100 consecutive adult patients, who received allo-HSCT for high-risk hematological malignancies (63.5% acute leukemia). Stem cell donors were family haploidentical (n=45), HLA identical sibling (n=15), unrelated volunteer (n=35), cord blood (n=5). Stem cell source was mainly T-cell replete PBSCs. Fecal specimens have been collected before conditioning (T0), during aplasia (T10) and after engraftment (T30). The fecal microbiome have been analyzed using the 454 GS Junior System, and QIIME software. We defined a threshold of normality for the main phyla on the basis of the control. RESULTS: We observed an important difference in relative percentages of phyla populating the gut between our pts and control. Our patients showed a progressive reduction of the intestinal microbial diversity (alpha diversity) during the transplant process, with a specific decrease of anaerobic species belonging to both Bacteroidetes and Firmicutes phyla, and a relative increase of facultative anaerobic gram-positive families such as Enterococcaceae and Staphylococcaceae. A great variation was observed, particularly between T0 and T30, where we found a significant decrease in alpha diversity (p < 0.001). A significantly different distribution in the baseline microbiome was reported in patients who will experience different clinical outcomes. Patients (n=23) who developed a microbiologically-confirmed sepsis show an increase of Proteobacteria phylum (cut-off 5%; p We identified by multivariate analysis some associations between the relative increase of a specific phylum and clinical variables at baseline. Previous allo-HSCT and prior infections represented strong risk factors for developing colonization by Proteobacteria (exceeding 5%). Sepsis by GN-MDR bacteria was associated to increase in Enterobacteriaceae(exceeding 5%; p 0.011). Interestingly, significant microbioma changes were observed at 10 days after transplant, in patients who developed a subsequent acute GvHD, with a predominant role played by gram-positive bacteria belonging to Firmicutes. More in details, the presence of Lachnospiraceae was associated to a decreased risk of developing acute GvHD (p=0.04 and RR= 4.35), whereas dominance of Enterococcaceae (p CONCLUSIONS: Longitudinal study of microbioma profile allows early identification of patients at risk for major transplant-related complications such as sepsis by GN-MDR or acute GvHD, offering a new tool for individualized pre-emptive or therapeutical strategies to improve the outcome of allo-HSCT. Disclosures Ciceri: MolMed SpA: Consultancy.

Introduction Pre-transplant risk assessment is a crucial issue to improve the HCT decision-making process. Several transplant-related models have been designed to optimize decision-making about suitable candidates for allogeneic HCT. The refined Disease Risk Index (DRI) was developed to stratify disease risk acrosshistologiesand HCT regimens. However, few recipients of haploidentical HCT were originally included in the DRI study cohorts. In 2015 a first large cohort of non-myeloablativehaploidentical HCT with post-transplant cyclophosphamide (PTCy) confirmed the validity of DRI also in this setting. Beside this, in the past few years the novel composite end point of GVHD-free, relapse-free survival (GRFS) after HCT spreads out. GRFS acknowledge that both survival and rates of grade 3-4 acute GVHD, chronic GVHD requiring systemic treatment, relapse, or death are clinically meaningful. GRFS therefore represents ideal recovery from HCT and a measure of cure without ongoing morbidity. Methods We analyzed risk-stratified GRFS according to the refined DRI in haploidentical HCT at our Center, where it was exploited - since 2006 - asirolimus-based,calcineurininhibitor-free prophylaxis of GvHD to allow the safe infusion of unmanipulated haploidentical HCT. We analyze data collected between 2006 and 2014 including 207 adult pts. Data were prospectively collected in Institutional database. A written consent was given by pts allowing the use of medical records for research in accordance with the Declaration of Helsinki. All consecutive pts receiving a haplo HCT as 1st allogeneic transplantation were included - pts receiving haplo HCT as 2nd or 3rd HCT were excluded from the present analysis. Results Baseline characteristics of the 207 pts are outlined in table 1. With 4-year median follow-up, 4-year probabilities of transplant related mortality (TRM),relapse, progression-free survival (PFS), and overall survival (OS) were 25,8%, 42,5%, 31,7%, and 34,4%, respectively. Day-100 cumulative incidence of grade II-IV and III-IV acute graft-versus-host-disease (GvHD) were 30,1% and 15,5% respectively. The 4-year cumulative incidence of chronic GvHD was 33,5% (moderate-severe chronic GvHD 26,2%). Considering the composite end point of GRFS, for the entire population the 4-year GRFS was 17,8%. By refined DRI group, low-intermediate (n 69), high (n 105), and very high (n 33) risk groups had 4-year GRFS estimates of 31,1%, 13,7%, and 3,0% (p < .0001), with corresponding 4-year OS estimates of 56,7%, 28,9%, and 6,1% (p < .0001). On a multivariable Cox model we considered as covariates age, host/donor sex mismatch, host/donor CMV status, stem cell source, conditioning intensity, GvHD prophylaxis ATG-based versusPTCy-based, DRI stratification, HCT Comorbidity Index Score (HCT-CI). On multivariable analyses, the DRI was statistically significantly associated with GRFS, OS, PFS, relapse, TRM and grade II to IV acute GvHD (ref table 2). HCT-CI was statistically significantly associated with GRFS, OS, PFS and TRM. Conditioning intensity was associated with PFS and relapse, while GvHD prophylaxis (PTCyvs ATG) was only associated with OS. Interestingly no risk factors were clearly emerging for chronic GvHD. Conclusions The combination of a refined DRI and GRFS provide a valid tool to improve the HCT decision-making process and will help optimize patient outcomes. Disclosures 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.

Multidrug resistant Gram-negative (MDR-GN) bacteria are responsible for severe infections in immune-compromised hosts. Clinical outcome is often poor, especially in patients undergoing hematopoietic stem cell transplantation (HSCT) with mortality rates exceeding 50%. In patients with a documented pre-transplant MDR-GN bacterial infection, HSCT is usually regarded as a potentially harmful procedure, despite its curative potential. Recently, active surveillance strategies based on rectal swab evaluation have been recommended with the aim of early identification of MDR-GN carriers, although they are not routinely performed in every transplant center. After an Institutional outbreak of K. pneumoniae carbapenemase -producing (KPC-Kp) infections in our Centre in June 2012, we decided to start an Institutional programme of active surveillance. No patient was excluded from HSCT, despite a documented colonization. In this study, we retrospectively analysed the results of the active surveillance programme and the impact of a pre-transplant colonization on HSCT outcomes. From July 2012 to January 2016 (43 months), 362 consecutive patients (254 undergoing allogeneic HSCT (allo-HSCT) and 108 autologous HSCT (auto-HSCT) were examined. All patients had a pre-transplant rectal swab evaluable. Blood cultures were performed at each febrile episode. Neutropenic patients developing fever were treated according to internal antimicrobial guidelines, usually with multiple targeted antimicrobial therapy in case of a previously known MDR-GN bacterial colonization. Median follow-up was of 602 days. Pre-transplant carriers were 8% and 17% in auto-HSCT and allo-HSCT recipients, respectively. Both in auto-HSCT and allo-HSCT, 30% of carriers were colonized by KPC-Kp. Among auto-HSCT, the overall survival (OS) did not significantly differ in carriers versus non-carriers (86% vs 84% at 2 years; P=0.231). Moreover, neither transplant-related mortality (TRM) nor infection-related mortality (IRM) were significantly different among the two groups, being of 0% vs 4% at 100 days (P=0.32) and of 0% vs 2% at 100 days (P= 0.099), respectively. In allo-HSCT, the OS was not significantly influenced by the carrier status nor univariate (43% vs 50% at 2 years; P=0.091) or in multivariate analysis (HR 1.41; P=0.129). Furthermore, TRM and IRM were comparable in MDR-GN bacteria carriers versus non-carriers, being 31% vs 25% at 2 years (P=0.301) and 23% vs 14% at 2 years (P=0.304). A MDR-GN bacterial colonization was not an independent risk factor for TRM and IRM, with an hazard ratio (HRs) of 1.428 (P=0.268) and of 1.69 (P=0.215). Furthermore, we did not observe any significant variation in the incidence of severe acute GvHD when comparing carriers and non-carriers (HR 1.86; P=0.087). In multivariate analysis severe acute GvHD was the only independent factor for a worse OS, TRM and IRM ( HRs of 3.177 (P< 0.001), 7,933 (P< 0.001) and 18,690 (P < 0.001), respectively. We registered a total of 168 Gram-negative bloodstream infections (BSIs) and 41/168 (24%) were due to a MDR bacteria, of which 6/41 in auto-HSCT patients and 35/41 in allo-HSCT recipients. Extended spectrum β-lactamases (ESBL+) E. coli (39%) and KPC-Kp (24%) were most frequently isolated from blood cultures. Thanks to the early initiation of a combined antimicrobial therapy guided by the antimicrobial susceptibly test deriving from rectal swabs in patients developing neutropenic fever, we witnessed a significant reduction in 30-day MDR-GN bacteria-related mortality (0% for auto-HSCT and 17% for allo-HSCT patients). Noticeably, in our Center 30-day KPC-Kp-related mortality was only 30%. Our retrospective single-centre study was able to demonstrate that, both in auto-HSCT and allo-HSCT, a pre-transplant colonization status is not significantly associated with a reduced OS, nor with a reduced TRM and IRM. Moreover, the prompt initiation of an adequate empirical therapy based on information coming from rectal swab is able to reduce MDR-GN bacteria-related mortality and to improve the overall HSCT outcome. Disclosures Ciceri: MolMed SpA: Consultancy.

The clinical outcome of allogeneic hematopoietic stem cell transplantation (HSCT) is strongly influenced from the potential complications arising during the delicate phase of post-transplant immune restoration. The quantitative aspects of immune-cell repopulation after HSCT and the qualitative features their functional restitution have been extensively reported. Nevertheless, measurable immune biomarkers predicting the clinical outcome of HSCT await formal validation. The aim of this review is an appraisal of most studies published so far on the predictive value of different T and NK-cell biomarkers after HSCT with emphasis on defined thresholds endorsed by multivariate analysis.

Infections are the major cause of treatment-related morbidity and mortality in hematological patients, mostly after allogeneic hematopoietic stem cell transplantation (HSCT). Although the proportion of infection-related deaths has decreased in the past two decades, the alarming emergence of multidrug resistant (MDR) gram-negative pathogens, increasingly reported from various cancer treatment centers, represents a significant challenge. Dramatic infection-related mortality (64.4%) have been observed in allogeneic HSCT, due to carbapenem-resistant Klebsiella pneumoniae (CRKp). Timely microbiological diagnosis, active surveillance and early therapeutic strategies represent critical aspects for the management of MDR infections after allogeneic HSCT. In this study, we investigated whether a new molecular tool could be clinically relevant in the management of high-risk hematological patients, particularly in allogeneic HSCT. At San Raffaele Hematology and BMT Unit, all blood cultures positive for gram-negative bacteria in the last year, were prospectively collected and in parallel tested with Verigene® Gram-negative blood culture (BC-GN) test (Nanosphere, Northbrook, IL, USA), a microarray-based system allowing a rapid identification of genus, species, and genetic resistance determinants for a broad panel of gram-negative bacteria directly from positive blood cultures. Initially, we evaluated the reliability of the BC-GN test on 50 consecutive patients undergoing chemotherapy (n=16), autologous (n=4) or allogeneic (n=30) HSCT. The concordance with the standard blood culture was 100% considering the bacteria detectable by the system. Resistance genes (CTX-M or carbapenemases, as KPC and VIM) were detected in 15% of the isolates, and 100% were associated to the same phenotypic antibiotic resistance. We observed only 8% of phenotypic resistances not detected by the test, belonging to other kinds of resistance mechanisms not related to the genes included in the panel. Overall, Verigene BC-GN assay correctly identified 40/50 of all the gram-negative pathogens (3 Klebsiella pneumonia, 25 Escherichia coli, 5 Pseudomonas aeruginosa, 4 Acinetobacter spp, 1 Enterobacter spp, 2 Citrobacter spp), yielding a general sensitivity of 80%, which increased to 100% if only the genera and species included in the panel were considered. Then, we examined the potential clinical impact of this molecular approach in allogeneic HSCT recipients (n=30), either in an inpatient (n=24) or outpatient (n=6) management. As far as transplanted patients' care is concerned, the BC-GN test strongly influenced clinical practice. In the majority of cases we were able to early start targeted antibiotic therapy (78%), sparing or interrupting non-specific antimicrobial therapy (56%), thus reducing useless and/or potentially more toxic antibiotics and their potential impact in favouring antimicrobial resistance. Moreover, in 5/8 cases the 'triple therapy', recommended at fever onset for all CRKp-colonized patients, was spared thanks to BC-GN test results, showing the absence of resistance markers. In 95% of patients immunosuppressive prophylaxis was not reduced, thanks to a rapid control of sepsis, avoiding the risk of undesired GVHD. Early contact isolation was possible in 32% of patients, preventing the spread of infections from a patient to the others and allowing infection control. Infection-related mortality was reported only in one patient. An outpatient management was continued in 3/6 haemodynamically stable patients, avoiding unnecessary hospitalization. While conventional microbiological methods required 2-4 days, BC-GN test provided detailed results within 2 hours from blood culture positivity. The mean gain in time comparing BC-GN test to fast blood cultures was reported to be about 20 hours. In our experience, this new microarray test has provided highly accurate identification results and earlier potentially important information on antibiotic susceptibility, both confirming and excluding the presence of an MDR phenotype. The contribution to a rapid diagnosis and an earlier targeted antimicrobial therapy of Verigene BC-GN test is of high clinical relevance for the infections by MDR bacteria, a significant public health challenge worldwide, particularly in allogeneic HSCT recipients. Disclosures No relevant conflicts of interest to declare.

Invasive fungal infections (IFI) have emerged as a leading cause of morbidity and infection-related mortality among allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients over the past two decades. Several new drugs have recently been introduced as antifungal prophylaxis, supported by results of prospective randomized trials. In agreement with the international guidelines, a mold-active antifungal prophylaxis is strongly recommended in high-risk patients receiving allo-HSCT, considering efficacy, different pharmacokinetic, drug-drug interactions, and toxicity profile of the various antifungal drugs. Posaconazole, a triazole with broad-spectrum antifungal activity and a favorable toxicity profile, is the drug of choice for patients with graft versus host disease (GVHD). Consequently, posaconazole prophylaxis in allo-HSCT recipients is administered in combination with immunosuppressive drugs for GVHD prophylaxis and/or treatment, most commonly cyclosporine (CsA) or tacrolimus. Sirolimus is an inhibitor of mammalian target of rapamycin (mTOR), largely used for GVHD prevention and treatment, but extensively metabolized by CYP3A4. As posaconazole strongly inhibits CYP3A4 its coadministration with sirolimus is contraindicated by the manufacturer of posaconazole, and up to now poorly described in literature. In the San Raffaele Haematology and Bone Marrow Transplantation Unit, from 2010 to 2015, we retrospectively identified sixty-six patients that received posaconazole oral suspension as primary (n=43) or secondary (n=23) antifungal prophylaxis after allo-HSCT. The majority of patients were affected by acute leukaemia (67%), not in remission at time of allo-HSCT (55%), and with a previous allo-HSCT reported for 50% of them, carrying an high risk for developing a post-transplant IFI. Median follow up was 357 days (range 43-1884) after HSCT. Posaconazole administration was given for a median of 221 days (range 13-966). Thirty-two patients (49%) received posaconazole during engraftment phase, while 28 (42%) and 19 (29%) patients for IFI prophylaxis in the setting of acute or chronic GvHD respectively. A concomitant administration of sirolimus was performed in 49 patients (74%). Sirolimus concentrations were monitored two times a week, while posaconazole was controlled in patients' serum on a weekly basis, during the first two months of treatment. We observed a 55-70% steady-state dose reduction of sirolimus in 19 patients, after posaconazole introduction. Alternatively, patients with ongoing posaconazole prophylaxis received an initial empiric sirolimus dose reduction at 1 mg/day. The co-administration of posaconazole and sirolimus resulted safe. Discontinuation was reported mainly in patients with documented IFI, who required a change of the antifungal drug. No adverse events potentially associated with sirolimus overexposure (i.e. sinusoidal obstructive syndrome, sirolimus-related thrombotic microangiopathy) were reported, although one-third of the patients experienced a transient and moderate elevation of sirolimus serum levels in the first week of coadministration. In this analysis post-transplant IFI occurred in 14 cases. The risk of developing IFI was influenced by type of prophylaxis, resulting in 12% and 39% of IFI during primary and secondary prophylaxis respectively (p value 0.013; OR 4.89, CI 1.39-17.11). However the most significant and strong association was reported in concomitant with insufficient posaconazole serum levels ( In patients with adequate posaconazole serum levels (>0.5 ml/L) the incidence of IFI was 5%, supporting the utility of therapeutic drug monitoring (TDM) in such conditions and generating interest for the use of the upcoming posaconazole tablets with improved bioavailability in allo-HSCT recipients. In our experience concurrent sirolimus and posaconazole use was well tolerated, with a 55% to 70% sirolimus dose reduction at posaconazole initiation and close monitoring of serum sirolimus and posaconazole trough levels in the first months of co-administration. Disclosures No relevant conflicts of interest to declare.

Abstract 666 Background: Results of haploidentical stem cell transplantation (SCT) after standard extensive T-cell depletion for advanced leukemias are poor (Ciceri et al, 2008). In contrast, significant leukemia-free-survivals are produced after T-cell replete SCT from matched related, unrelated and cord-blood donors, even in advanced phases of disease (Kolb HJ, 2009). New protocols based on T-cell repletion are warrented in patients receiving haplo-SCT, in order to offer to all candidates patients with advanced leukemia the potential of cure of allogeneic SCT.Rapamycin is an immunosuppressive drug that arrests cell cycle in G1 phase through the inhibition of DNA transcription, RNA translation and protein synthesis. Morover, in contrast to calcineurin inhibitors, it promotes the generation and expansion of T regulatory cells (Tregs). Aim: We investigated the safety of infusion of T-cell replete unmanipulated peripheral blood stem cells (PBSC) from family haploidentical donor with a combination Rapamycin, Mycophenolate and ATG as GvHD prophylaxis, to preserve early Treg function (TrRaMM study, Eudract 2007-5477-54). Patients and Methods: Since 2007, forty-six patients underwent allogeneic transplantation for AML (25 pts), ALL (7 pts), sAML (6 pts), MDS (3 pts), CML-BC (2 pts), NHL (2 pts) or HD (1 pt). The median age was 50 years (range 14-69). At time of transplantation 5 pts were in early phase, and 41 were in advanced phase. Median time from diagnosis to transplantation was 351 days (range 81-1387); 8 patients were enrolled for relapse after allogeneic SCT from MRD or MUD. Median comorbodity index score was 1 (0-5). The conditioning regimen included Treosulfan (14 g/m2 for 3 days), Fludarabine (30 mg/m2 for 5 days) and an in vivo T and B-cell depletion, by ATG-Fresenius (10 mg/kg for 3 times) and Rituximab (a single 500 mg dose). All pts received allogeneic peripheral blood cells from an HLA-haploidentical related donor without any in vitro positive selection of CD34+ cells. GvHD prophylaxis consisted of Rapamycin (target level 8-15 ng/ml, till day +60) and MMF (15 mg/kg tid till day +30). Results: All patients engrafted, and all but eight were in disease remission at first marrow evaluation on day +30. Cumulative incidence of grade 2-4 aGvHD was 33% (95% CI: 18-48); cumulative incidence of grade 3-4 aGvHD was 12% (95% CI: 2-22). Interestingly, half of patients with GvHD developed it at immunosuppressive prophylaxis withdrawal for disease relapse. Only six patients developed cGvHD. Cumulative incidence of TRM and relapse incidence were 26% (95% CI: 11-41) and 53% (95% CI: 35-71) respectively. None developed EBV reactivation. Patients experienced an early and sustained immunoreconstitution with a median 221 circulating CD3+cells/μL (range 43-1690) from day 30. The immune-reconstitution was polarized towards central memory cells (CD45RA-CD62L+ cells 32.7% ± 4.8) that produced IL-2 (IL-2+ cells 26.2% ± 5.3). Of interest, at day +90 from transplant, Tregs were significantly expanded (CD4+CD25+CD127-Foxp3+ cells 15.6% ± 4.8 on total CD3+ cells, P Conclusions: Rapamycin-Mycophenolate-ATG are effective to prevent GvHD in T-cell replete unmanipulated haploidentical peripheral stem cell transplantation for advanced haematological malignancies. This associates with an early T-cell immunoreconstitution characterized by the in vivo expansion of early-differentiated T cells and Tregs, and translates in promising leukemia-free survival in patients with advanced resistant leukemia. Further studies are warranted to gain insight on the role of rapamycin as platform for exploitation of Tregs in allogeneic HSCT from mismatched donor. Disclosures: No relevant conflicts of interest to declare.

Introduction Haematopoietic stem cell transplantation (HSCT) is the only curative option for patients (pts) affected by high-risk haematological diseases (HRHD). However, the availability of a match donor is still an unmet-medical need. Recently alternative donor transplantations have been broadly exploited, reaching results similar to transplants from a well match donor. Anyway, whoever the donor is, the most important complication remains Graft-versus-Host Disease (GvHD). Aim of the study We evaluated incidence, characterization, stratification, treatment, treatment response and outcome at medium and long-term follow-up for both acute (a-) and chronic (c-) GvHD in haploidentical setting. We also evaluated the impact of the NIH consensus criteria on GvHD in a routine clinical application to confirm their feasibility in daily practice and not only in clinical trials use. Methods A population of 257 pts was selected from our Institutional database on the basis of their having an HRHD with indication to allogeneic HSCT and having received a HSCT from an haploidentical family donor without exclusion. HSCT were performed between January 2004 and December 2013 at our Center. No distinction between first HSCT or subsequent one was made, all consecutive haploidentical HSCT were captured. Results Time of median follow-up was 10 months; 1-year overall-survival (OS) was 46%, with better outcome for pts transplanted in complete remission (p 0.0001) confirming disease status as the leading factor for overall outcome. Transplant related mortality was estimated to be 30% at 1 year and the leading cause of death was infections. The 6-months a-GvHD incidence was 45% (119/257) - median day of presentation 21 post HSCT (range 8-89). Late-onset a-GvHD was documented in 15 pts. Grade I GvHD was documented in 33 pts (27.7%), grade II in 44 (37%), grade III-IV in 36 (30.3%) – 6 not evaluable. Skin was the most frequently involved organ (77.3%), both as single manifestation or combined. One-hundred and five pts received a first line therapy based on high-dose prednisone (2mg/Kg) and 37/105 completely abrogated the a-GvHD. At a 3-months evaluation 46% of affected pts showed complete resolution of a-GvHD, while the mortality rate was 29%. C-GvHD affected 69/257 pts and 1-year risk of onset was 25% - median day of presentation was 139 post HSCT (range 40-809) and 36/69 pts were off immunosuppressive therapy (52.3%) at presentation. According to onset presentation 53.7% were de novo, 24.6% progressive and 18.8% quiescent GvHD. Forty-one pts (59.5%) presented at declaration with overlap features. The most frequent involved organ was skin (grade I to III), as reported in 53 pts (76.8%). Skin lesions were usually accompanied with mouth lesions (35 pts – 50.7%), liver (23 pts – 33.3%) or eyes (27 pts – 39.1%) dysfunctions. The median number of involved organs was 3 (range 1-7). Mild c-GvHD was diagnosed in 10 pts (14.5%), who received topical therapy. Fifty-nine pts – diagnosed with moderate (32, 46.4%) or severe (27, 39.1%) - received a systemic treatment for c-GvHD: prednisone 1 mg/Kg alone or 0,5 mg/kg plus mTOR- or calcineurin-inhibitor for pts that were not likely to tolerate high dose steroid due to comorbidity (namely active infections, diabetes, cardiovascular diseases). At a 12-months evaluation the overall response was 48% (complete resolution 25%, partial resolution 23%), while the mortality rate was 36%. The Landmark analysis of OS at 3 months after HSCT shows that a-GvHD affected pts had a worse outcome (p= 0,068), on the contrary the Landmark analysis of OS at 18 months shows that c-GvHD did not associate with a worse outcome (p ns) but the follow up is still short. Confirming previous reports, overlap c-GvHD was related to worse survival in comparison with classic c-GvHD (p 0.0098). Conclusion Haploidentical HSCT is a valid and feasible option for pts in need of a transplant. GvHD is manageable after haploidentical HSCT, as in full matched setting. Better knowledge and insight in a/c-GvHD are providing advance in improving pts outcome. The NIH-consensus criteria are manageable in daily clinical practice and able to translate in a tailored approach to GvHD with benefit on general outcome. Further advance in the development of specific biomarkers for GvHD will provide additional crucial information for management, diagnosis and prognostication in GvHD. Figure 1 Figure 1. Disclosures Bonini: MolMed S.p.A.: Consultancy. Bordignon:MolMed: Employment.

BACKGROUND: Human herpesvirus type 6 (HHV-6) is increasingly recognized as an opportunistic and potentially life-threatening pathogen in recipients of allogeneic hematopoietic stem cell transplantation (AlloSCT). HHV-6 is a member of the beta herpesvirus subfamily (genus Roseolovirus). HHV-6 infection is recognized as the cause of a febrile disease and exanthem subitum in early childhood. Approximately 60% of solid organ transplant and 40% of patients after alloSCT experienced HHV-6 reactivation. Reported clinical manifestations of HHV-6 infection in transplanted patients are skin rash, interstitial pneumonia, bone marrow suppression and encephalitis. Moreover, some clinical reports suggest that HHV-6 can facilitate the occurrence of severe clinical complications of alloSCT, increasing transplant-related mortality. METHODS: From January 2009 to February 2013, we retrospectively evaluated 54 consecutive adult patients (median age 50 years) who developed positivity to HHV-6 after alloSCT for high-risk hematological malignancies. Stem cell donors were family haploidentical (37), HLA identical sibling (8), unrelated volunteer (6), cord blood (3). The viral load was determined by quantitative PCR (Nanogen Advanced Diagnostic S.r.L) in cell-free body fluids such as plasma, bronchoalveolar lavage (BAL), cerebrospinal fluid (CSF), bone marrow (BM) aspirates or in gastrointestinal biopsies. RESULTS: Median time from alloSCT to HHV-6 reactivation was 34 days (range: 0-705). Thirty-one patients presented HHV-6 positive in plasma, 9/54 in BM, 33/54 in gut biopsies or BAL, 7/54 in CSF. At the time of viral positivity all pts were receiving acyclovir as viral prophylaxis except five. Twenty-nine patients had acute graft versus host disease (GvHD). Twenty-two out of these twenty-nine patients experienced a grade III-IV acute GvHD, requiring high dose steroids in twenty-six cases. A concomitant CMV positivity was detected in 15/54 patients. The median absolute count of CD3+ lymphocytes was 207 cells/mcl. In 52/54 cases we reported HHV-6 clinical manifestations: fever (43), skin rash (22), hepatitis (19), diarrhoea (24), encephalitis (10), BM suppression (18), delayed engraftment (11). HHV-6 positivity led to antiviral pharmacological treatment in 37/54 cases, using as first choice therapy foscarnet. Amongst the total fifty-four patients with documented HHV-6 positivity thirty-one solved the clinical event. However the mortality rate was relatively high in this population (overall survival (OS) ±SE at 1 year after HHV-6 reactivation was 38% ± 7%), mainly related to severe infections or GvHD. A better OS is significantly associated with CD3+ cells ≥200/mcl at the time of HHV-6 reactivation (fig 1) (OS at 1 year 63% compared to 11% for patients with CD3 CONCLUSIONS: This retrospective study confirms a correlation of HHV-6 with high morbidity and mortality rates after alloSCT, thus suggesting a regular HHV-6 monitoring in alloSCT recipients. The regular monitoring of HHV-6 DNA, using a real-time PCR assay, may be useful for identifying active HHV-6 infection and for the introduction of a pre-emptive treatment, possibly reducing the incidence of the most severe clinical complications. Despite HHV-6 detection typically occurred early after alloSCT, a better immune reconstitution has the potential to improve clinical outcome. Figure 1: Overall survival after alloSCT in HHV-6 positive patients: green line showed patients with more than 200/mcl CD3+ cells, blue line the ones with less than 200/mcl CD3+ cells at HHV-6 reactivation. P value is provided by Log Rank test. Figure 1:. Overall survival after alloSCT in HHV-6 positive patients: green line showed patients with more than 200/mcl CD3+ cells, blue line the ones with less than 200/mcl CD3+ cells at HHV-6 reactivation. P value is provided by Log Rank test. Disclosures Bonini: MolMed S.p.A.: Consultancy.

BACKGROUND: Febrile neutropenia and sepsis are frequent and life-threatening complications in patients with haematological malignancies. Although the proportion of infectious deaths in haematological patients has decreased over the last two decades, much remains to be done to further reduce these events. More effective preventive and prophylactic strategies for high-risk patients are necessary. Blood cultures (BC) identify a pathogen in only 20 to 30% of febrile episodes, the culturing and pathogen identification process is lengthy, postponing the start of a pathogen-targeted treatment. Thereby, a sensitive tool to promptly recognize pathogens causing sepsis is of high clinical relevance. METHODS: We assessed the diagnostic usefulness of the LightCycler SeptiFast test (SF; Roche Molecular Systems), a PCR-based multiplex assay performed on peripheral blood and capable of detecting 25 among the most common species isolated in sepsis. The assay uses dual fluorescent resonance energy transfer (FRET) probes against the species-specific internal transcribed spacer (ITS) regions, a non-coding sequence interspaced among highly conserved bacterial and fungal RNA. Time from processing to result is remarkably short (less than 6 hours). In this study, blood samples from febrile haematological patients were concomitantly tested by traditional blood culture (BacT/Alert 3D; bioMerieux). RESULTS: A total of 1941 blood samples were collected from 516 consecutive haematological patients treated for febrile neutropenia at the San Raffaele Haematology and Bone Marrow Transplantation Unit, from 2009 to 2013. Out of the total 1941 episodes examined, positive results were detected in 537 samples by SF (28%), and in 263 by BC (14%). Together, the two methods identified a total of 586 microorganisms in 509 (26%) episodes: Gram-positive bacterial species (80%), Gram-negative bacterial species (17%), and fungal species (3%). Concordance between the two methods was 77%, with most of the discordant samples that tested negative by culture but positive using the molecular approach (50% of the total positive samples). The cases positive by SF alone were mostly samples from patients already receiving antimicrobial therapy, or, importantly, sample positive for fungal pathogens such as Aspergillus fumigatus, which is hard to detect by the traditional approaches. CONCLUSION: This analysis demonstrates a significant correlation between the molecular test and the standard BC in haematological patients with febrile neutropenia. A molecular test such as SF, in combination with traditional assays, can play an important role in the diagnosis of sepsis, particularly in persistent fever despite antibacterial therapy, when a non-responding bacterial infection or an invasive fungal infection is suspected, therefore leading to a rapid diagnosis and an earlier targeted antimicrobial therapy. Disclosures No relevant conflicts of interest to declare.

Background Hematopoietic stem cell transplantation (HSCT) is so far the only potentially curative option for patients with aggressive hematological malignancies. Since many patients cannot find a suitable HLA-identical (related or unrelated) donor, it is essential to analyze the safety of alternative graft sources, such as haploidentical donors. The use of reduced-intensity conditioning regimens in patients with an advanced disease did not improve the outcome because of a higher incidence of relapse. Here we present an interim analysis of this phase II prospective clinical trial that investigates a new conditioning regimen for allogeneic HSCT based on a well established one (Fludarabine and Treosulfan) intensified adding 4 Gy TBI with the aim of maintaining an acceptable toxicity profile while reducing the incidence of relapse in high-risk patients (TrRaMM4Gy study, Eudract 2011-001534-42). Patients and Methods Ninety-six patients underwent allogeneic HSCT for AML (n=46), ALL (n=13), acute biphenotypic leukaemia (n=2), HD (n=8), NHL (n=7), MDS (n=6), Myelofibrosis (n=5), MM (n=4), CML (n=2), CMML (n=2) or CLL (n=1). The median age was 45 years (range 17-67). At the time of the transplantation most patients (n=76) were in advanced disease phase, while the remaining 20 patients were in early phase. Twenty-eight patients were enrolled for relapse after a previous allogeneic HSCT. Median time from diagnosis to transplantation was 443 days (range 56-5249). Median comorbodity index score (according to Sorror criteria) was 2 (range: 0-8). Sixty-two patients received the graft from haploidentical donors, 17 from MUD, 14 from HLA-identical sibling and 3 patients from a single cord blood unit. Median number of CD34+ and CD3+ cells/kg were 6.46 millions and 241 millions respectively. The conditioning regimen included Treosulfan (14 g/m2 for 3 days), Fludarabine (30 mg/m2 for 5 days) and 4 Gy TBI split in 2 fractions. Patients receiving HSCT from haploidentical donor (arm A) were also treated with ATG-Fresenius (10 mg/kg for 3 days) and Rituximab (200 mg/m2 in single dose), patients receiving HSCT from HLA-identical sibling, MUD or single cord blood unit and in early disease status (arm B) were also treated with ATG-Fresenius (5 mg/kg for 3 days) and Rituximab (200 mg/m2 in single dose), while patients receiving HSCT from HLA-identical sibling, MUD or single cord blood unit and in advanced disease status (arm C) did not receive any ATG or Rituximab. GvHD prophylaxis consisted of Sirolimus (target concentration 8-15 ng/ml, till day +60) and Mycophenolate Mofetil (10 mg/kg tid till day +30). Results Neutrophils engraftment was reported at a median time of 17 days (range: 9-46) in all 91 evaluable patients; five patients died of transplant-related causes (n=4) or disease progression (n=1) before day +15. Platelets engraftment occurred after a median time of 17 days (range: 4-153) in 75 patients (82%). At the first marrow evaluation on day +30 all the evaluable patients showed full donor chimerism, 77 patients (90%) were in complete remission and 9 were in stable disease or progression. After a median follow-up of 425 days, cumulative incidence of grade 2-4 and 3-4 aGvHD were 40% and 26% respectively. Cumulative incidence of moderate or severe cGvHD (according to NIH criteria) was 57%. Cumulative incidence of NRM at day +100 and +365 were 23% and 36% respectively; cumulative incidence of relapse at day +365 was 33%. Overall survival and progression-free survival 1 year after HSCT were 51% and 43% respectively. EBV reactivation occurred in 7 patients, but no PTLD was observed. Conclusions The new conditioning regimen we investigated proved to be feasible in this high-risk population. We did not observe any major difference in terms of NRM and aGvHD compared to the data from our previous trial (TrRaMM, Eudract 2007-5477-54) with similar inclusion criteria and patient characteristics, but a reduced-intensity conditioning regimen based on Treosulfan and Fludarabine. Interestingly, we observed a trend of lower relapse incidence, resulting in a better OS and PFS. The low toxicity profile of Treosulfan and Fludarabine should be considered as a myeloablative platform for further intensification; more studies are warranted in order to find the association for a conditioning regimen endowed with a low toxicity profile but still a strong anti-tumor activity. Disclosures: Bonini: MolMed SpA: Consultancy.

Background Antithymocyte globulins Fresenius (ATG-F) are purified, concentrated preparations of polyclonal immunoglobulin G from hyperimmune serum of rabbits immunized with human thymus activated lymphocytes. These immunoglobulins induce immunosuppression through T-cell depletion and immune modulation. The polyclonal nature of ATG-F is responsible for its effects on the immune system: T-cell depletion in blood and peripheral lymphoid tissues through complement-dependent lysis and T-cell activation and apoptosis; modulation of molecules involved in leukocyte-endothelium interactions; induction of apoptosis in B-cell lineages; interference with dendritic cells. ATG-F administered before allogeneic hematopoietic stem cell transplantation (allo-HSCT) reduces the risks of graft rejection and graft-versus-host disease (GvHD), but the slow clearance of the xenoserum might delay immune reconstitution, increase the risk of disease relapse and impair the activity of a donor lymphocyte infusion (DLI) performed early after allo-HSCT. Methods We studied 24 patients with hematologic malignancies, who underwent allo-HSCT from familiar or unrelated donors, after a conditioning regimen based on myeloablative treosulfan and fludarabine. As graft rejection and GvHD prophylaxis, 17 patients received ATG Fresenius at the dose of 10 mg/kg over 16 hours at day -4,-3 and -2 before HSCT; 5 patients received in vivo T-cell depletion at the dose of 20 mg/kg at day -4,-3 and -2 before HSCT; 2 more patients received ATG Fresenius at the dose of 10 mg/kg much earlier before allo-HSCT (day -14, -13 and -12) since the treatment protocol included a donor DLI at day +3 after transplantation. We collected serum samples at different timepoints, from the first ATG dose to at least 3 weeks after HSCT. We used a flow cytometry-based assay to detect the concentration of free T-cell specific rabbit IgG (SRIgG) which corresponds to the serum biological activity against human T-lymphocytes, as opposed to the levels of unspecific rabbit IgG (RIgG) by ELISA, which lack anti-T-cell function. Results In our cohorts of patients we observed a concentration peak at 64 hours after the first ATG administration, corresponding to the end of the last immunoglobulin dose administration. Interestingly, patients who received the 20 mg/kg dose of serum reached four times higher levels of SRIgG, suggesting a non-linear correlation between the administered dose and the measured plasmatic peak concentrations. Moreover, the terminal elimination half life of SRIgG is significantly shorter than the one of RIgG: 14 vs 67 days (data not shown), indicating that, for the dose of 10 mg/kg, 10 days after HSCT, SRIgG titre has already reached sub-therapeutic levels. Conclusions Previous results indicated that in vivo specific activity of rabbit ATG Fresenius disappears from circulation around day +7 after their last administration (at the dose of 10 mg/kg, day -4, -3, 2). Based on our current data, we can suggest that residual specific anti-lymphocyte activity is dependent upon i. timing and ii. dosage of ATG administration, although this correlation might not be linear. According to these results, current policies of a fixed schedule for DLI infusion should be revisited and possibly adapted to each patient, based on specific conditioning protocols. Disclosures: Seitz: Fresenius Biotech GmbH: Employment. Martinius:Fresenius Biotech GmbH: Employment.

Background Human herpesvirus type 6 (HHV-6) is a member of the beta herpesvirus subfamily (genus Roseolovirus) and two distinct variants have been described: types A and B. HHV-6 infection is recognized as the cause of a febrile disease and exanthem subitum in early childhood. The infection rarely causes serious events in healthy individuals, but viral reactivation in immunocompromised patients is frequently associated with severe clinical manifestations. Above all HHV-6 is increasingly recognized as an opportunistic and potentially life-threatening pathogen in recipients of allogeneic hematopoietic stem cell transplantation (AlloSCT). Approximately 60% of solid organ transplant and 40% of patients undergoing alloSCT experience HHV-6 reactivation, mainly of variant B. Reported clinical manifestations of HHV-6 infection in transplanted patients are skin rash, interstitial pneumonia, bone marrow suppression and encephalitis. Moreover, some clinical reports suggest that HHV-6 can facilitate the occurrence of severe clinical complications of alloSCT, increasing transplant-related mortality. Methods From January 2009 to February 2013, we retrospectively evaluated 54 consecutive adult patients (median age 50 years) who developed positivity to HHV-6 after alloSCT for high-risk hematological malignancies. Stem cell donors were family haploidentical (37), HLA identical sibling (8), unrelated volunteer (6), cord blood (3). The viral load was determined by quantitative PCR (Nanogen Advanced Diagnostic S.r.L) in cell-free body fluids such as plasma, bronchoalveolar lavage (BAL), cerebrospinal fluid (CSF), bone marrow (BM) aspirates or in gastrointestinal biopsies. Results Median time from alloSCT to HHV-6 reactivation was 34 days (range: 0-705). Thirty-one patients presented HHV-6 positive in plasma, 9/54 in BM, 33/54 in gut biopsies or BAL, 7/54 in CSF. At the time of viral positivity all pts were receiving acyclovir as viral prophylaxis except five. Twenty-nine patients had acute graft versus host disease (GvHD). Twenty-two out of these twenty-nine patients experienced a grade III-IV acute GvHD, requiring high dose steroids in twenty-six cases. A concomitant CMV positivity was detected in 15/54 patients. The median absolute count of CD3+ lymphocytes was 262 cells/mcl. In 52/54 cases we reported HHV-6 clinical manifestations: fever (43), skin rash (22), hepatitis (19), diarrhoea (24), encephalitis (10), BM suppression (18), delayed engraftment (11). HHV-6 positivity led to antiviral pharmacological treatment in 37/54 cases, using as first choice therapy foscarnet. Amongst the total fifty-four patients with documented HHV-6 positivity thirty-one solved the clinical event. However the mortality rate was relatively high in this population (only 30% of patients were alive), mainly related to severe infections or GvHD. A better overall survival is significantly associated with CD3+ cells higher than 200/mcl (p-value 0.011) and time after alloSCT more than 2 months (p-value 0.035). In this analysis the overall survival was not significantly influenced by steroids administration, presence of acute GvHD, plasma viral load and organ involvement. Conclusions This retrospective study further demonstrates the correlation between HHV-6 reactivation and high morbidity and mortality rates in patients after alloSCT. Despite HHV-6 detection typically occurred in the first month after AlloSCT, a better immune reconstitution has the potential to improve the outcome. The regular monitoring of HHV-6 DNA, using a real-time PCR assay, may be useful for identifying active HHV-6 infection and for the introduction of a pre-emptive treatment, possibly reducing the incidence of the most severe clinical complications. Disclosures: Bonini: MolMed SpA: Consultancy.

Introduction Achieving a cure for relapsed leukemia remains a challenge. Hematopoietic stem cell transplantation (HSCT) from an HLA-haploidentical relative provides a potentially curative treatment for such patients in urgent need. Based on previous studies on the feasibility and low toxicity profile of haploidentical HSCT with high-dose post-transplant Cyclophosphamide (PT-Cy), we planned to intensify this protocol for patients with active disease by the use of: 1) myeloablative conditioning, 2) peripheral blood stem cells (PBSCs) graft instead of bone marrow (BM), and 3) sirolimus instead of tacrolimus, for its direct anti-leukemic effect. Patients and Methods Here we report the results on 18 patients treated between November 2012 and July 2013. The median age was 59 years (range, 30–78). Diagnoses included AML (10 refractory, 3 CR1, and 2 CR>1), ALL (2 refractory), and 1 CML in lymphoid blast crisis. The majority (78%) of patients were not in remission at the time of HSCT. Consistently, 13 (72%) patients scored high/very high accordingly to the CIBMTR disease score (Todisco et al., Leukemia 2013). Fourteen (78%) patients had a Sorror comorbidity score ≥ 3. Conditioning consisted of treosulfan (14 g/m2/day) on days –6 to –4, fludarabine (30 mg/m2/day) on days –6 to –2, and melphalan (70 mg/m2/day) on days –2 and –1, followed by T-repleted peripheral blood stem cells (PBSCs). Postgrafting immunosuppression consisted of PT-Cy (50 mg/kg/day) on days 3 and 4, followed by mycophenolate mofetil for 30 days, and sirolimus for 3 months. Results PBSC grafts contained an infused median TNC/kg of 10.5 e8 (range 5.5–20.7 e8), CD3/kg 18.4 e7 (range 10.5–40.9 e7) and a CD34/kg of 7.01 e6 (range 4.3–8.02 e6). All patients but 1 engrafted (94%), with a median time to neutrophil and platelet recovery of 18 days (range 14–26) and 19 days (range 9–51) respectively. Notably, the patient with primary graft failure had a HLA-DP mismatch in HvG direction. No patient experienced secondary graft failure. Post-HSCT recovery of lymphocyte subsets was broad and fast: the median CD3 count at day 30 after HSCT was 642/µl and exceeded 1000/µl by day 60, with a median value of CD4 T cell counts of 329/µl at day 30; NK cells reached a median value of 192/µl at day 30 and remained stable thereafter, while B cells steadily increased from a median value of 2/µl at day 30 to 48/µl at day 180. Circulating T cells comprised both naïve and memory subsets, with a recovery of CD31+ recent thymic emigrants (RTEs) starting from day 30. All patients had a significantly higher proportion of circulating RTEs at day 30, 90 and 180 compared to their pre-HSCT levels, suggesting an improvement in their thymic function after HSCT. Peri-engraftment syndrome (PES) occurred in 4 patients with a median time to onset of 15 days and was promptly resolved with short-course steroids. Interestingly, the percentage of circulating Ki-67+, dividing, effector memory CD8 T cells at day 8 post HSCT was able to accurately predict the occurrence of PES. Grade I/II skin acute GvHD occurred in 6 patients, while 4 patients experienced grade III/IV acute GvHD (22%). Acute GvHD were accompanied by a rise in circulating Ki-67+ CD8 T cells, while response to first line therapy resulted in a drop in Ki-67 expression. Chronic GVHD occurred in 2 of the 7 patients at risk, but only one patient required systemic therapy. Of the 16 patients at high risk, 7 had CMV reactivations. No patient had EBV-related PTLD. With a median follow-up of 100 days (range 15–260), 14 (78%) out of the 18 patients are alive. Only two patients died of non-relapse mortality (NRM, 11%). All patients in CR at time of transplant are alive and disease-free, while 8 (57%) out of 14 patients with active disease at transplant are alive and leukemia-free. In 4 patients tumor-specific T cells against PRAME or WT1 were detectable both in the peripheral blood and BM up to 180 days after HSCT, suggesting that PT-Cy did not hamper the GvL effect. Conclusions Myeloablative haploidentical HSCT with PBSCs, PT-Cy and sirolimus is feasible and might be a valid option for patients with refractory/active leukemia. If confirmed in a larger cohort of patients and with a longer follow-up, these results suggest that the acceptable rates of GvHD and NRM as well as the favorable immune reconstitution profile open the way for combining it with novel immunomodulatory or cellular therapies to improve leukemia-free survival. Disclosures: Bonini: MolMed SpA: Consultancy.

Bronchiolitis Obliterans (BO) initially affects terminal and respiratory bronchioles, a region of the lung largely unexplored by spirometry, which is only altered in advanced disease. In contrast, the Impulse Oscillation System (IOS) and the nitrogen multiple breath washout (N2-MBW) are techniques characterized by a high sensitivity to peripheral airway changes and potentially more suited to early detection of small airways disease. In a cross sectional study, a total of 161 patients, divided into 4 groups: healthy controls (n=41), bone marrow transplant candidates (n=47), haematopoietic stem cell transplantation (HSCT) recipients (n=65) and patients with chronic obstructive pulmonary disease (COPD; n=8), were assessed by IOS, N2-MBW, spirometry, body plethysmography and diffusing capacity for carbon monoxide (DLCO) in order to describe respiratory function changes in post-transplant patients without pulmonary graft versus host disease (GVHD) and in order to characterize the pattern of peripheral airway changes in BO. All subjects were able to perform IOS and N2-MBW without difficulty. Significant variables are illustrated in the table.ControlNo BOSpBOSpMSDMSDMSDRV/TLC %9714109240.04414422 Stem cell transplantation, even without respiratory complications, does not affect spirometry but appears to cause an increase in air trapping, a reduction in DLCO and enhanced ventilation inhomogeneity both in conductive (Scond*VT) and acinar (Sacin*VT) airways. Patients with BO (n=8) were characterized by further DLCO reduction, increase in oscillometric indices sensible to peripheral airways involvement (Z5Hz, DR5-20Hz, X5Hz, Resonant frequency and Rperipheral) and a further three-fold increase in Sacin* VT. Compared to patients with BO, COPD patients with the same degree of spirometric obstruction (FEV1/FVC< 0.7, FEV1 50% predicted) showed only half the increase in predicted Sacin*VT (p= 0.03). At a cut off of 321% of predicted value, Sacin* VT could distinguish the subjects with BO from recipients, with good accuracy (87%), sensibility (87.5%) and specificity (89.5%). We conclude that IOS and N2-MBW are simple tests able to detect changes following HSCT as well as those specific to Bronchiolitis Obliterans. Disclosures: No relevant conflicts of interest to declare.

Abstract 3139 Conditioning in allogeneic stem cell transplantation has been widely explored in the last decades, since the introduction of the Reduced Intensity Conditioning (RIC) allowed the extension of the feasibility of allotransplants in previously ineligible patients. Outcome of both treatments remains comparable because of an higher incidence of relapse although reduction of Non Relapse Mortality (NRM) in RIC. Treosulfan is a bifunctional alkylating agent without major extra-haematological toxicities. Here we show the results of a Treosulfan based regimen in use at our Center in patients enrolled in “AlloTreo” trial (Eudract 2005–005182–11). We analyzed 123 pts transplanted between 2004 and 2011 consecutively enrolled at our center. Patient's median age was 50 years (21–69) and median Sorror comorbidity index was 1 (0–7). Diagnosis: acute myeloid leukemia (41 pts), acute lymphoblastic leukemia (14 pts), Myelodysplastic syndrome (17 pts), Non Hodgkin Lymphoma (23 pts), Hodgkin Lymphoma (5 pts), Myeloproliferative neoplasms (11 pts), and others (12 pts). Fourty-three pts were in first CR, 18 pts were transplanted upfront, 46 pts in persistence of disease; 16 pts in CR2 or CR3. Donor was unrelated (UD) in 57% of pts; source of stem cells were BM, PBSC and CB respectively in 5, 106 and 12 pts. Conditioning was: Treosulfan (14 g/msq for 3 days) and Fludarabine (30 mg/msq for 5 days). In vivo T and B-cell depletion was performed by ATG-Fresenius (10 mg/kg for 3 days) and Rituximab (single 500mg dose) only in pts receiving an UD. GvHD prophylaxis was: Cyclosporine A and short course of Methotrexate, excepting CB. Median CD34+ infused for BM-PBSC was 6.6×106/kg (1–17). Median follow-up was 48 months (1–83). At day 60 cumulative incidence (CI) of neutrophil engraftment was 93±2%, with a median time 17 days. In evaluable pts molecular chimerism at day 30 was full-donor in 89% thus confirming myeloablative properties of full dose treosulfan. Regimen was very well tolerated, median grade of regimen-related toxicity (CTC score) was 1 (0–4), mostly a transient rise of bilirubin (74%). Grade 4 toxicity was observed in 2 pts, with significant increase of bilirubin without signs of VOD. Grades II-IV aGvHD occurred in 42 pts (34%), grade III-IV in 21 (17%). Among evaluable pts after day 100, 46% developed chronic GvHD, half of which was extensive. Thirty days, 100 days and 4 years CI of NRM were respectively 2%, 11% and 21%; in multivariate analysis (MVA) an advanced stage of disease (HR=6.4, p=0.01) was associated with increased TRM. 4 years PFS was 44%, in MVA factor associated with a decreased PFS was advanced stage disease at transplant (HR=2.31, p=0.004). Thirty days, 100 days and 4 years CI of relapse were respectively 3%, 15% and 35%; no risk factors found. OS at 4 years was 51%, in MVA once again advanced stage of disease was a risk factor (HR=2.62, p=0.003). Importantly, even if a trend of increased NRM was found in UD transplants, it does not meet the significance criteria; in MVA, indeed, UD were not associated with increased risk of NRM, PFS, RI or worst OS. 57 pts died after transplant, 26 for relapse and 31 for transplant related causes (GvHD 32%, infection 45%). Treosulfan is a safe and effective myeloablative drug with low toxicities. Use of ATG as in-vivo T-cell depletion guarantees similar results in unrelated donor settings without enhancing NRM or worsening OS. Anti-leukemic effect, rapid engraftment as well as relatively low toxicity and NRM configure this regimen as a possible paradigm of the so called Reduced Toxicity Conditioning. However strategies to ameliorate outcome in advanced diseases are warranted. Disclosures: No relevant conflicts of interest to declare.

Abstract 4191 Introduction: HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a readily available therapeutic option for patients suffering from high-risk hematological malignancies who lack an HLA-compatible donor or for whom there is no time to find one. To avoid GVHD, haplo-HSCT has been classically performed under conditions of strict T-cell depletion, in the form of CD34-selected (“naked”) grafts. Unfortunately, naked haplo-HSCT is followed by a profound state of immune incompetence, which associates with high non-relapse mortality (NRM) rates, mainly due to opportunistic infections. Over the last decade, different strategies have been developed in order to speed-up immune reconstitution after haplo-HSCT, including the delayed infusion of donor T cells (DLI), the selective depletion of T-cell subsets from the graft or the use of an unmanipulated graft followed by novel strategies of immune suppression. Aim: To analyze in depth the early phenomena of immune reconstitution following haplo-HSCT in order to find early surrogate biomarkers of NRM. Results: From 2004 to 2010, we prospectively studied multiple parameters of T-cell immune reconstitution in 89 pts treated at our Center with haplo-HSCT. Time-points of analysis were pre-transplant, at day 30, day 90 and day 180 post-transplant. Underlying diseases included: high-risk AML 53 pts (60%), ALL 11 pts (12%), CLL 2 pts (2%), tyrosine kinase inhibitors-resistant CML 4 pts (5%), Hodgkin lymphoma 6 pts (7%), NHL 2 pts (2%), MDS with high International Prognostic Scoring System 7 pts (7%), other 4 pts (4%). Eighteen patients (20%) were given prophylactic suicide gene-modified DLI starting at day 30 post-transplant, while 64 patients (80%) received an unmanipulated graft followed by rapamycin until day 100. Overall, the incidence of grade III-IV acute GvHD was as low as 12%. Chronic extensive GvHD developed in 28% of pts. Compared with historical naked haplo-HSCT, the recovery of T-cell counts was accelerated: at day 90, median CD3+ cells were 378 per μL (0–2817), CD4+ 127 (0–804), CD8+ 173 (0–1922). Higher T-cell counts, however, did not clearly associate with lower NRM rates. After the initial expansion of effector memory cells (CD45RA−/CD62L−) in patients given DLI (P95%) pts that did not reactivate the virus at later time points. Strikingly, while in pts with 1000 spots/mL, this was 0% (P Conclusions: These results clearly indicate that the early reconstitution of T-cell immunity to CMV after haplo-HSCT does not only protect from subsequent viral reactivation, but also surrogates for lower NRM rates. Moreover, they warrant the investigation of a CMV-specific IFN-γ ELISPOT cut-off value of 1000 spots/mL as a predictive biomarker in larger, multicenter series. Disclosures: No relevant conflicts of interest to declare.

Abstract 4198 Background and Methods. Chronic Graft-versus-Host-Disease (GvHD) remains a serious complication after allogeneic stem cell transplantation. In 2005 the National Institutes of Health (NIH) established new criteria for chronic GvHD based on retrospective data and expert recommendations. Here we are reporting the prospectively evaluation of the incidence of NIH-defined chronic GvHD and its prognostic impact in 113 consecutive patients who underwent unmanipulated peripheral blood graft from an HLA-haploidentical relative after a treosulfan based conditioning at our Institution. The peculiarity of this setting relies on the exploitation of a rapamycin-based, calcineurin-inhibitor-free regimen for prophylaxis of GvHD to induce in-vivo expansion of T-regulatory cells (Tregs). Results. Chronic GvHD was evaluable in 81 patients: 41 patients were diagnosed with chronic GvHD according to the revised Seattle criteria, 28 were classified as having extensive chronic GvHD and 13 were classified as having limited. The 1-year cumulative incidence was 43 +/− 9%. According to the NIH Consensus Criteria, 1 patient was reclassified as having late-acute GvHD, 20 patients as having overlap syndrome and 20 patients as having classic chronic GvHD. The late-acute consisted of late onset subtype. The 40 patients with classic chronic GvHD and overlap syndrome were graded as moderate (26 patients) and severe (14 patients) by NIH consensus criteria. The onset was classified as progressive in 4 cases, de novo in 27 cases, quiescent in 9 cases. The median number of organs involved was 3 (range 1–7), the majority of patients presented with skin and oral involvement, but also eyes, liver, joint, genitalia and gastro-intestinal damages were present – maximum severity of chronic GvHD according to organ manifestation is presented in figure 1. Twenty-seven out of 40 patients started a specific immunosuppressive treatment for chronic GvHD, noteworthy 21 patients restarted rapamycin (in combination with prednisone, or alone) with clinical benefit. In univariable analysis patients with overlap GvHD tended to present a lower overall survival (OS) compared to those with classic chronic GvHD (36% versus 63%, p 0.07). Patients with severe chronic GvHD at onset had a significantly lower OS compared to those with moderate chronic GvHD (19% versus 69%, p0.017). OS was significantly worse for patients with platelets counts below 100 g/l at onset of chronic GvHD (29% vs 67%, p0.003)– figure 2. Conclusion. This analysis confirms in haploidentical setting thrombocytopenia, overlap category and severe presentation as prognostic items for overall survival after allogeneic stem cell transplantation. Further investigations on implementation of immunosuppressive treatment strategy for high-risk GvHD patients (namely patients with overlap GvHD diagnosis and concomitant thrombocytopenia and/or progressive onset) are actually ongoing to enhance response and improve outcome. Disclosures: Bordignon: MolMed SpA: Employment. Bonini:MolMed S.p.A.: Consultancy.

Abstract 4471 Allogeneic stem cell transplantation (HSCT) is an emerging risk factor for cardiovascular disease. Long-term survivors seem to be at higher risk for premature arterial vascular disease compared with sex- and age-matched population. In HSCT, pre-transplant treatments and the conditioning regimen play a key role in endothelial and organ damage. The use of immunosuppressant drugs to prevent graft-versus-host disease (GvHD) influences as well the development of cardiac disease. To better understand the impact of treosulfan-based conditioning regimens in leading to the emergence of cardiovascular disease, we retrospectively evaluated 94 consecutive patients who had survived longer than 1 year, transplanted at Our Institution between 2002 and 2010 (62 males – median age 46, range 14–69). For 48 patients an HLA-identical sibling was available, 6 patients found an unrelated donor while 40 patients underwent haploidentical HSCT from family donors. Forty-six patients received in vivo T-cell depletion and 72 B-cell depletion. GvHD prophylaxis was based upon cyclosporine + methotrexate for the HLA-identical transplants (48 – median EBMT risk score 3) whereas rapamycin + MMF were used for patients undergoing transplantation from HLA-haploidentical donors (35 – median EBMT risk score 4); 9 did not receive any immunosuppression because of application of suicide gene strategy as tool to prevent GvHD (median EBMT risk score 3). We compared clinical variables (i.e. GvHD, OS, TRM, major cardiac events and comorbidities) with biochemical and functional tests (i.e. cardiac function, lipid profile and ferritin levels) both before and 1 year after transplantation. After a median follow-up of 29 months, only 3 patients experienced a major cardiovascular event; no one experienced a late congestive heart failure. In all series, we found that ferritin levels significantly decrease after 1 year of follow-up, compared to pre-transplant values (P Disclosures: No relevant conflicts of interest to declare.