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Background Cell-mediated immunity (CMI) after anti-Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 vaccines has been poorly explored in recipients of allogeneic hematopoietic stem-cell transplantation (HSCT), especially with regard to the 3rd dose (booster). Aim of the study was to assess the specific T-cell responses before and after the 3rd dose of BNT162b2 mRNA vaccine in a cohort of allogeneic HSCT recipients, and compare it with healthy donors (HD). Methods Allogenic HSCT recipients and HD were enrolled before receiving the 3rd dose of BNT162b2 mRNA vaccine. Whole blood for T-cell specific responses was collected before (T1) and 8 weeks after (T2) the booster administration. T-cell responses were assessed with an Interferon (IFN)-γ release assay (IGRA), after overnight stimulation of heparin whole blood with pools of lyophilized peptides, covering the immunodominant sequence of the Spike (S) protein. IFN-γ production was assessed with an enzyme linked immunosorbent assay (ELISA). Statistical analysis was performed with GraphPadPrism. Results 14 HSCT recipients (8M, 6F) and 15 HD (7M, 8F) were enrolled (table 1). Median age was 47 [39-59] and 41 [31-48] years in the HSCT and HD groups, respectively. Time between the vaccine 2nd dose and T1 was significantly longer in HD than HSCT recipients (p< .001), while the time between T1 and T2 did not differ between the two groups. SARS-CoV-2 S specific T-cell responses at T1 were inferior in HSCT recipients compared to HD (median IFN-γ production: 463 vs 231 ng/ml, respectively), although the difference did not reach the statistical significance. No differences were observed at T2. In a before-after analysis, SARS-CoV-2 S specific T-cell responses were significantly increased in HSCT recipients at T2 compared to T1 (median IFN-γ production: 267 vs 881 ng/ml, p=0.02) (Figure 1). At T1, 3 HSCT recipients showed very low or no IFN-γ production, while at T2 only 1 patient still had undetectable IFN-γ production after S peptide stimulation. Table 1:Clinical characteristics of allo-HSCT recipients and HDHSCT: hematopoietic stem-cell transplantation; IQR: interquartile range; ALL: acute lymphoblastic leukemia; AML: acute myeloid leukemia; MDS: myelodysplastic syndrome; T-ALL: T-cell acute lymphoblastic leukemia; AA: aplastic anemia; PMF: primary myelofibrosis; MUD: matched unrelated donor; MMUD: mismatched unrelated donors; NA: not applicable; ns: not significant.Figure 1:IFN-γ Production after Spike peptide stimulation of whole blood from allogenic HSCT recipients and HD.Comparison of IFN-γ production after Spike peptide stimulation of whole blood in allogenic HSCT recipients and HD at T1 (1.A) and T2 (1.B) (before and after BNT162b2 mRNA vaccine 3rd dose, respectively). Before-after representation of IFN-γ production after Spike peptide stimulation in HD and allogenic HSCT recipients (1.C). Conclusion SARS-CoV-2 IGRA represents a useful tool to assess CMI, also in immunocompromised hosts. An additional 3rd BNT162b2 mRNA vaccine booster dose seems to enhance CMI in allogenic HSCT recipients. Further studies are needed to evaluate the duration of SARS-CoV-2 CMI in HSCT recipients compared to HD. Disclosures All Authors: No reported disclosures.

A matched-pair analysis of transplant-related outcomes was carried out in 116 of 255 consecutive patients who received transplants from an HLA identical sibling (n = 58) or haploidentical related donor (n = 58). The 2 patient series were matched with 9 variables: period of transplant, patient and donor age, sex, diagnosis, disease phase, conditioning regimen, donor-recipient sex, and cytomegalovirus (CMV) status combinations. As graft-versus-host disease (GVHD) prophylaxis, all patients received the standard cyclosporine and methotrexate association with the addition of anti-thymocyte globulins, mycophenolate mofetil, and basiliximab in haploidentical, unmanipulated bone marrow recipients. Anti-infectious management, transfusion policy, and supportive care were identical for all patients. By comparing the 2 patient series, no statistically significant difference was observed for the cumulative incidence of advanced acute and extensive chronic GVHD, transplant-related mortality, and relapse. With a median follow-up of 3.5 years, the 5-year disease-free survival was 37% ± 6% and 36% ± 6% for HLA identical sibling and haploidentical recipients, respectively. The results of transplant from HLA identical siblings and haploidentical donors are comparable. Regardless of the HLA matching, other factors known to affect the transplant outcomes, such as donor-recipient age, sex, and CMV status combinations, might drive the search for the best donor.

Simple Summary Upfront genetics/cytogenetics and minimal measurable disease (MRD) are becoming relevant biomarkers in the process of post-remission transplant allocation in AML. However, until recently the transplantation choice relied on the availability of a fully matched familiar donor, whereas individual patient- and disease-related characteristics played a secondary role in transplant allocation. In this paper we analyzed the evolution of the transplantation policy at our center in a 20-year time interval. At the beginning of our observation patients were submitted to allogeneic transplant, per protocol, mostly if a fully matched family donor was available or to autologous transplant if no fully matched family donor was identified (“donor vs. no donor” strategy) regardless of upfront genetics/cytogenetics or MRD status. Thereafter, persistence of MRD after consolidation cycle was included in the decision-making process for transplant selection. Patients with favorable and intermediate-risk cytogenetic risk were to receive allogeneic or autologous stem cell transplantation if MRD positive or negative, respectively, (“transplant vs. no transplant” strategy). In this cohort, patients with FLT3-ITD or adverse risk karyotype were submitted to allogeneic transplant as well. Abstract Measurable residual disease (MRD) is increasingly employed as a biomarker of quality of complete remission (CR) in intensively treated acute myeloid leukemia (AML) patients. We evaluated if a MRD-driven transplant policy improved outcome as compared to a policy solely relying on a familiar donor availability. High-risk patients (adverse karyotype, FLT3-ITD) received allogeneic hematopoietic cell transplant (alloHCT) whereas for intermediate and low risk ones (CBF-AML and NPM1-mutated), alloHCT or autologous SCT was delivered depending on the post-consolidation measurable residual disease (MRD) status, as assessed by flow cytometry. For comparison, we analyzed a matched historical cohort of patients in whom alloHCT was delivered based on the sole availability of a matched sibling donor. Ten-years overall and disease-free survival were longer in the MRD-driven cohort as compared to the historical cohort (47.7% vs. 28.7%, p = 0.012 and 42.0% vs. 19.5%, p = 0.0003). The favorable impact of this MRD-driven strategy was evident for the intermediate-risk category, particularly for MRD positive patients. In the low-risk category, the significantly lower CIR of the MRD-driven cohort did not translate into a survival advantage. In conclusion, a MRD-driven transplant allocation may play a better role than the one based on the simple donor availability. This approach determines a superior outcome of intermediate-risk patients whereat in low-risk ones a careful evaluation is needed for transplant allocation.

In acute lymphoblastic leukemia, flow cytometry detects more accurately leukemic cells in patients' cerebrospinal fluid compared to conventional cytology. However, the clinical significance of flow cytometry positivity with a negative cytology - occult central nervous system disease - is not clear. In the framework of the national Campus ALL program, we retrospectively evaluated the incidence of occult central nervous system disease and its impact on outcome in 240 adult patients with newly diagnosed acute lymphoblastic leukemia. All cerebrospinal fluid samples were investigated by conventional cytology and flow cytometry. The presence of ≥10 phenotypically abnormal events, forming a cluster, was considered as flow cytometry positivity. No central nervous system involvement was documented in 179 patients, while 18 were positive by conventional morphology and 43 were occult central nervous system disease positive. The relapse rate was significantly lower in central nervous system disease negative patients and the disease-free and overall survival were significantly longer in central nervous system disease negative patients than in those with manifest or occult central nervous system disease positive. In multivariate analysis, the status of manifest and occult central nervous system disease positivity was independently associated with a worse overall survival. In conclusion, we demonstrate that in adult acute lymphoblastic leukemia patients at diagnosis flow cytometry can detect occult central nervous system disease at high sensitivity and that the status of occult central nervous system disease positivity is associated with an adverse outcome. (Clinicaltrials.gov NCT03803670)

In our retrospective study, 16 patients affected by advanced cutaneous T cell lymphoma (CTCL) underwent allogeneic hematopoietic stem cell transplantation (HSCT). Two patients (12.5%) were in complete remission (CR), nine (56.3%) in partial remission (PR), and five (31.2%) with active disease. The patients were transplanted from an HLA-identical (n = 7) from a mismatched (n = 1) or haploidentical (n = 1) sibling, from matched unrelated donor (n = 5), or from a single cord blood unit (n = 2). Conditioning regimen was standard myeloablative in 6 patients and at reduced intensity in 10. Seven patients died from non relapse mortality (NRM) and four patients relapsed or progressed, three of them achieved a second CR after donor lymphocyte infusion (DLI) or chemotherapy plus DLI. To date, with a median follow-up of 76 months (range 6-130), nine patients are alive, eight in CR, and one with active disease. Overall survival (OS) and disease-free survival (DFS) at 1 and 10 years are 61% (95% CI 40-91%) and 54% (95% CI 33-86%), 40% (95% CI 22-74%), and 34% (95% CI 16-68%), respectively. The time from diagnosis to transplant seems to influence negatively both OS (log-rank p

BACKGROUND From 2011 to 2014, a total of 71% of the 3834 patients with hematologic malignancies successfully identified a matched unrelated donor (MUD) through the Italian Bone Marrow Donor Registry (IBMDR), corresponding to a transplant efficiency of 62%. STUDY DESIGN AND METHODS From 2006, the Rome Transplant Network (RTN) followed a hierarchical selection strategy for the alternative donor search: first MUD, second cord blood, and third haploidentical donor. Using a low-resolution HLA, a preliminary query (PQ) was performed in all cases with assignment of good or poor score if more or less than 10 MUDs were identified in Bone Marrow Donors Worldwide. Herein we assessed the utility of PQ and of high-resolution (HR) HLA from the start of the search. Moreover, we compared the donor identification and the transplant efficiency between IBMDR and RTN. RESULTS At RTN 79% of 417 patients met a good PQ with a 50% MUD identification versus 12.5% with poor PQ. Our policy led to 78 and 74% of alternative donor identification and transplant efficiency, respectively, higher than IBMDR data equal to 71% (p = 0.007) and 62% (p

High-dose melphalan (HDMel) is the most common conditioning chemotherapy regimen for autologous stem cell transplantation (SCT) in patients affected by multiple myeloma (MM). No consensus exists for the emetogenicity or prophylaxis of chemotherapy-induced nausea and vomiting (CINV) in this regimen. Data on the incidence and efficacy/safety of CINV prophylaxis among patients affected by MM undergoing autologous SCT with the HDMel regimen was extracted from electronic databases and analyzed. Eleven studies involving multiple CINV prophylaxis regimens were identified and included. No consensus on HDMel emetogenicity was reached, but most studies summarized the emetogenicity as moderate-high risk. An aprepitant-based three-drug regimen (aprepitant + serotonin receptor antagonist (5HT3RA) + dexamethasone) showed better efficacy than a two-drug regimen (5HT3RA + dexamethasone) for CINV prevention without increasing the frequency in adverse events. The aprepitant-based three-drug regimen should be the regimen of choice for CINV prophylaxis for MM patients undergoing autologous SCT with HDMel conditioning.

Incidence and effect on outcome of central nervous system (CNS) involvement in adult patients with acute myeloid leukemia (AML) is not clearly defined. To address this issue, 103 consecutive adult patients with newly diagnosed AML, regardless of neurologic symptoms, were submitted to a routine explorative lumbar puncture. Cerebrospinal fluid (CSF) samples were collected from 65 males and 38 females. All 103 CSF samples were examined by conventional cytology (CC) whereas 95 (92%) also by flow cytometry (FCM). At diagnosis, 70 patients (68%) were CNS negative (CNS-), whereas 33 (32%) were CNS positive (CNS+). In 11 of 33 (33%), CNS infiltration was documented either by CC or FCM , in 21 (67%) only by FCM. CNS positivity was significantly associated with a M4-M5 phenotype of the underlying AML (P = .0003) and with high levels of lactate dehydrogenase (P = .006). Overall, 80 of 103 (78%) achieved complete remission with no significant differences between CNS+ and CNS- patients. Five-year disease-free survival and overall survival were found to be shorter in CNS+ patients than in those CNS- (18% vs 50%, P = .006 and 19% vs 46%, P = .02, respectively). In multivariate analysis, CNS status and age were found to affect independently overall survival. In conclusion, the incidence of CNS involvement in adult patients with newly diagnosed AML is higher than expected. Regardless of neurologic symptoms, it should always be searched at diagnosis; CSF samples should routinely be investigated by FCM since a certain proportion of CNS involvements might remain undetected if examination is exclusively CC based.

We assessed by flow cytometry minimal residual disease (MRD) in patients with acute myeloid leukemia (AML) given standard-dose (SDAC) and high-dose ARA-C (HDAC) regimens. Of 163 patients enrolled, 130 (median age, 45 years; range, 18-59 years) qualified for analysis, all achieving complete remission after treatment with SDAC (n = 78) or HDAC (n = 52) plus etoposide and daunorubicin. Consolidation consisted of intermediate-dose ARA-C and daunorubicin. MRD negativity was significantly more frequent in the SDAC vs. HDAC arm after both induction (37% vs. 15%, P = 0.007) and consolidation (44% vs. 18%, P = 0.002). Respective median residual leukemic cell counts with SDAC and HDAC use were 1.5 × 10(-3) and 4 × 10(-3) (P = 0.033) after induction and 5.7 × 10(-4) and 2.9 × 10(-3) (P = 0.008) after consolidation. Based on ARA-C schedule and post-consolidation MRD status, the four patient groups (SDAC-MRD(-) , HDAC-MRD(-) , SDAC-MRD(+) , and HDAC-MRD(+) ) displayed 5-year overall survival rates of 60%, 33%, 24%, and 42% (P = 0.007), respectively, with 24%, 35%, 74%, and 48% (P

Background: Mutations of the gene encoding Fms Related Tyrosine Kinase 3 (FLT3), at the juxta-membrane level (ITD), represent the most common lesions found in Acute Myeloid Leukemia (AML), identifying a subgroup of patients (pts) with unfavorable prognosis. FLT3-ITD mutations are considered an unreliable tool for measurable residual disease (MRD) monitoring, due to their intraclonal heterogeneity and instability during the course of disease. Instead, multiparametric flow cytometry (MFC) may represent an alternative to monitor MRD in this molecular subset. In fact, through the recognition and monitoring of leukemia associated immunophenotypes, MFC is applicable to > 90% of AML patients with a sensitivity of 10-4. Aims: The aim of our study was to investigate the reliability of MFC in MRD assessment of 72 FLT3-ITD positive pts whose treatment allocation was prospectively decided according to the genetic/cytogenetic profile at diagnosis and post consolidation MRD. FLT3-ITD pts were to receive, after induction and consolidation, allogeneic stem cell transplant (ASCT), whatever the source of stem cells. In this subgroup analysis, we investigated if FLT3-ITD mutated pts have a different propensity to achieve high quality (e.g. MRD negative) complete remission as compared to FLT3 wildtype ones. Furthermore, we seek for a correlation between different levels of MRD and overall (OS) and disease-free survival (DFS). Methods: We included in the analysis 72 pts with de novo AML carrying FLT3-ITD mutations whose MRD assessment at the post-consolidation timepoint was available. Pts were defined as MRD-negative, when obtaining a residual leukemic cells count below the threshold of 3.5x10-4 (0.035%). MRD positive pts (with MRD ≥ 3.5x10-4 RLC) were stratified into 3 classes according to the levels of MRD (0.035%-0.1%; >0.1%-1%; >1%). We compared the MRD status and clinical outcome with a matched group of FLT3 wildtype AML (n = 203) treated in the same protocol. Results: Overall median age was 49 (range 18-60.9). The 2 cohorts were balanced in terms of age and sex distribution. In the FLT3-ITD group, 80/126 (64%) cases carried a concomitant NPM1 mutation vs 107/374 (28.6%) of FLT3 wildtype ones (p 0.1% and > 1%, respectively as compared to 65/203 (33.0%) and 15/203 (7.4%) of FLT3 wildtypeones, respectively (p=0.017). When considering the different MRD stratification levels of FLT3-ITD pts, OS probability at 24 months was 57.2% (27 pts), 71.4% (7 pts), 53.6% (28 pts) and 20% (10 pts), for the MRD categories 0.1%-1%, >1%, respectively (p=0.028). DFS probability at 24 months was 53.8% (27 pts), 71.4% (7 pts), 34.9% (27 pts) and 20% (10 pts), for the MRD categories 0.1%-1%, >1%, respectively (p=0.038). Summary/Conclusion: We demonstrated that MRD determination by MFC is a reliable tool to assess remission quality and prognosis in FLT-ITD positive patients. This subpopulation shows a lower propensity to obtain a MRD negative CR, with the majority of pts maintaining an amount of MRD > 0.1% after standard treatment. Even though most of these pts were addressed to ASCT, post-consolidation MRD maintained its negative impact on OS and DFS, particularly for those pts with MRD >1%. In the attempt to improve the quality of response, prevent leukemia recurrence and pursue a durable remission, delivery of FLT3 inhibitors as a maintenance after transplant may represent a promising option. Disclosures Venditti: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees. Buccisano:Janssen: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Background: Chronic inflammation leads to gray matter damage in progressive multiple sclerosis (MS), but the mechanism linking inflammation and neurodegeneration is unclear. Objective: The objective of this paper is to investigate the synaptic mechanism of inflammatory neurodegeneration in progressive forms of MS. Methods: Cytokine and neurofilament-light were determined in cerebrospinal fluid (CSF) of MS patients. In vitro electrophysiology and cell swelling experiments were performed to measure the effects of inflammatory cytokines in the CSF of MS patients on synaptic transmission and neuronal integrity. Results: Tumor necrosis factor-α (TNF) was higher in CSF of progressive MS subjects, and caused excitotoxic neuronal death in vitro. In murine brain slices incubated in the presence of CSF from progressive MS, in fact, we observed increased spontaneous excitatory postsynaptic currents (sEPSCs) and glutamate-mediated neuronal swelling through a mechanism dependent on enhanced TNF signaling. We also suggested a pathogenic role of B cells in TNF CSF increase, exacerbation of glutamatergic transmission and neuronal damage, since CNS depletion of B cells with intrathecal rituximab caused a dramatic reduction of TNF levels, of TNF-induced sEPSC alterations, and of neurofilament CSF concentrations in a patient with progressive MS. Conclusion: Our results point to TNF as a primary neurotoxic molecule in progressive forms of MS.

We describe the case of a patient with a Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) treated with dasatinib plus steroids as the first-line therapy who achieved a molecular complete remission and then underwent a matched, unrelated donor allogeneic transplant. Five months after the transplant, he experienced a disease relapse with an T315I mutation, which was resistant to salvage chemotherapy. Once the details of the T315I mutation were acquired, we initiated ponatinib treatment at a standard dosage and observed a rapid decrease of minimal residual disease (MRD) at molecular assessment. The bone marrow evaluation after 2, 3, 6, 10 and 13 months was negative for MRD. After starting ponatinib, the patient experienced a skin graft-versus-host disease (GVHD), whereas no occurrence of GVHD was observed after transplant, suggesting that the efficacy of ponatinib could be related not only to the direct antileukemic effect, but also to its ability to promote an indirect graft-versus-leukemia effect. Ponatinib was well tolerated but a thyroid dysfunction mimicking a cardiovascular toxicity was observed and solved with hormonal substitutive treatment.

Purpose of review In the last few years, mobilized peripheral blood has overcome bone marrow as a graft source, but, despite the evidence of a more rapid engraftment, the incidence of chronic graft-versus-host disease is significantly higher with, consequently, more transplant-related mortality on the long follow-up. Overall, the posttransplant outcome of mobilized peripheral blood recipients is similar to that of patients who are bone marrow grafted. More recently, the use of bone marrow after granulocyte colony-stimulating factor (G-CSF) donor priming has been introduced in the transplant practice. Herein, we review biological acquisitions and clinical results on the use of G-CSF-primed bone marrow as a source of hematopoietic stem cells (HSC) for allogeneic stem cell transplantation. Recent findings G-CSF the increases the HSC compartment and exerts an intense immunoregulatory effect on marrow T-cells resulting in the shift from Th1 to Th2 phenotype with higher production of anti-inflammatory cytokines. The potential advantages of these biological effects have been translated in the clinical practice by using G-CSF primed unmanipulated bone marrow in the setting of transplant from human leukocyte antigen (HLA)-haploidentical donor with highly encouraging results. Summary For patients lacking an HLA-identical sibling, the transplant of G-CSF primed unmanipulated bone marrow from a haploidentical donor combined with an intense in-vivo immunosuppression is a valid alternative achieving results that are well comparable with those reported for umbilical cord blood, HLA-matched unrelated peripheral blood/bone marrow or T-cell-depleted haploidentical transplant.

Introduction. In a modern context of improved management of acute lymphoblastic leukemia (ALL), central nervous system (CNS) involvement at diagnosis remains an obstacle towards long-term cure. We have previously reported that flow cytometry (FCM) is better than conventional cytology (CC) in demonstrating the presence of leukemic cells in patients' (pts) cerebrospinal fluid (CSF), especially in samples with low cell counts. In the framework of the national Campus ALL program aimed at improving the management of adult ALL patients enrolled in the GIMEMA protocols, we retrospectively evaluated the incidence of occult CNS positivity and its impact on outcome in 221 adult pts with newly diagnosed ALL from 11 centers. Methods. Ninety-four patients (42%) were females and 127 (58%) males, with a median age of 44 years (range 17-80), a median white blood cell (WBC) count of 10.6x109/L (range 0.1-457). One hundred and seventy pts (77%) had B-lineage ALL. Cytogenetic/genetic data were available in 167 (75%) pts: 58 (35%) had a BCR/ABL rearrangement, 14 (8%) a complex karyotype and 11 (6%) a MLL rearrangement. Pts were treated according to the GIMEMA/NILG ALL protocols or with the Hyper-CVAD program. Ninety-eight pts underwent an allogeneic stem cell transplant (ASCT). Median follow up was 26.8 months (range 1-136.6). All CSF samples were evaluated both by CC and FCM. The presence of ≥10 clonally restricted or phenotypically abnormal events was regarded as a FCM positivity. Based on the results of CSF examination, three different categories were recognized: manifest CNS+ (CC+FCM+), occult CNS+ (CC-FCM+) and CNS- (CC-FCM-). Results. Overall, 16 (7%) pts had manifest CNS+, 39 (17%) occult CNS+ and 166 (75%) were CNS-Median age, WBC count, B/T lineage, cytogenetic/genetic features did not differ significantly between the three categories. A complete remission (CR) was achieved in 178 (80%) pts, 9 (4%) died early in induction and 104/178 (58%) experienced a relapse. The frequency of CR rate did not vary significantly across the three identified categories. In univariate analysis, the CNS status correlated significantly with the incidence of relapse (p=.004) and with censor (45 years (RR:1.45, 95% CI 1.47-2.43, p=0.04)were independently associated with a lower OS. Conclusions. Our large, multicenter CAMPUS ALL study showed that i) in ALL adult pts, FCM allows to detect occult CNS disease, even in conditions of low spinal fluid leukemic count; ii) the presence of occult CNS disease is associated with an unfavorable outcome. Further prospective studies on larger series are needed to confirm these data. Figure. Figure. Disclosures Del Principe: Gilead: Membership on an entity's Board of Directors or advisory committees. Fracchiolla:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees. Foà:NOVARTIS: Speakers Bureau; CELGENE: Other: ADVISORY BOARD, Speakers Bureau; AMGEN: Other: ADVISORY BOARD; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; CELTRION: Other: ADVISORY BOARD; GILEAD: Speakers Bureau; ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; INCYTE: Other: ADVISORY BOARD; ROCHE: Other: ADVISORY BOARD, Speakers Bureau.

BACKGROUND: Platelet (PLT) gel has been successfully used in tissue regeneration of diabetic/surgical wounds through the releasing of growth factors such as basic fibroblast growth factor and PLT-derived growth factor. Therefore, the PLT gel could represent a therapeutic tool in treating the deep and painful wounds sometimes occurring during graft-versus-host disease (GVHD). STUDY DESIGN AND METHODS: The aim of this study was to verify the efficacy and safety of PLT gel for treating GVHD ulcers. Allogeneic hemocomponents were used to obtain PLT gel with an automated system for the on-site preparation and application of patient (autologous) or healthy blood donor (allogeneic)-derived fibrin sealant or PLT-rich fibrin (Vivostat system, Vivostat A/S). Six patients with multiple lesions involving dermis (Grade I, n = 2), subcutaneous (Grade II, n = 4), or oral mucosa and related to GVHD underwent PLT gel as local therapy. RESULTS: After the second PLT gel application, the pain disappeared in all cases and the granulation tissue was observed in the four patients with Grade II lesions. After a median of eight PLT gel applications (range, 4-10), five of six patients showed a complete response, while one patient with a partial response died early from multiorgan failure. No side effects were documented. CONCLUSION: These preliminary data show that the PLT gel may be used as a safe and effective tool in the management of mucosal skin lesions related to the GVHD.

Hemorrhagic cystitis (HC) occurring after allogeneic transplantation significantly affects quality of life and, in some cases, becomes intractable, increasing the risk of death. To date, its therapy is not established. We used the hemostatic agent fibrin glue (FG) to treat 35 patients with refractory post-transplantation HC. Of 322 adult patients undergoing an allogeneic transplantation for hematological malignancy, 35 developed grade ≥ 2 HC refractory to conventional therapy and were treated with FG, diffusely sprayed on bleeding mucosa by an endoscopic applicator. The cumulative incidence of pain discontinuation and complete remission, defined as regression of all symptoms and absence of hematuria, was 100% at 7 days and 83% ± 7%, respectively, at 50 days from FG application. The 6-month probability of overall survival for all 35 patients and for the 29 in complete remission was 49% ± 8% and 59% ± 9%, respectively. In the matched-pair analysis, the 5-year probability of overall survival for the 35 patients with HC and treated with FG was not statistically different from that of the comparative cohort of 35 patients who did not develop HC (32% ± 9% versus 37% ± 11%, P = not significant). FG therapy is a feasible, effective, repeatable, and affordable procedure for treating grade ≥2 HC after allogeneic transplantation.

We assessed by flow cytometry minimal residual disease (MRD) in patients with acute myeloid leukemia (AML) given standard-dose (SDAC) and high-dose ARA-C (HDAC) regimens. Of 163 patients enrolled, 130 (median age, 45 years; range, 18-59 years) qualified for analysis, all achieving complete remission after treatment with SDAC (n = 78) or HDAC (n = 52) plus etoposide and daunorubicin. Consolidation consisted of intermediate-dose ARA-C and daunorubicin. MRD negativity was significantly more frequent in the SDAC vs. HDAC arm after both induction (37% vs. 15%, P = 0.007) and consolidation (44% vs. 18%, P = 0.002). Respective median residual leukemic cell counts with SDAC and HDAC use were 1.5 × 10(-3) and 4 × 10(-3) (P = 0.033) after induction and 5.7 × 10(-4) and 2.9 × 10(-3) (P = 0.008) after consolidation. Based on ARA-C schedule and post-consolidation MRD status, the four patient groups (SDAC-MRD(-) , HDAC-MRD(-) , SDAC-MRD(+) , and HDAC-MRD(+) ) displayed 5-year overall survival rates of 60%, 33%, 24%, and 42% (P = 0.007), respectively, with 24%, 35%, 74%, and 48% (P 0.0001) respective cumulative incidence of relapse estimates. MRD may serve as a biomarker for optimal biologic dosing of ARA-C, and SDAC regimen appears to yield more frequent MRD negativity.

Conventional cytology (CC) of cerebrospinal fluid (CSF) fails to demonstrate malignant cells in up to 45 % of patients with acute lymphoblastic leukemia or lymphoblastic lymphoma (ALL/LL) in whom occult leptomeningeal disease is present. Flow cytometry (FCM) is considered more sensitive than CC, but clinical implications of CC negativity/CC positivity are not yet established. CSF samples from 38 adult patients with newly diagnosed ALL/LL were examined. Five (13 %) and nine (24 %) specimens were CC positive-FC positive (FCM(pos)/CC(pos)) and CC negative-FC positive (CC(neg)/FCM(pos)), respectively. The remaining 24 (63 %) samples were double negative (CC(neg)/FCM(neg)) (p = 0.001). CC(neg)/FCM(pos) patients showed a significantly shorter overall survival (OS) compared to CC(neg)/FCM(neg) ones. In multivariate analysis, the status of single FCM positivity was demonstrated to affect independently duration of OS (p = 0.005). In conclusion, FCM significantly improves detection of leptomeningeal occult localization in ALL/LL and appears to anticipate an adverse outcome. Further prospective studies on larger series are needed to confirm this preliminary observation.

Background Routine diagnostic lumbar puncture is not recommended in adult patients with acute myeloid leukemia (AML) without Central Nervous System (CNS) symptoms and little is known about the incidence of CNS involvement and its impact on survival in these patients. Furthermore, several studies have demonstrated that flow cytometry (FCM) is superior to conventional cytology (CC) for detection of CNS involvement in lymphoproliferative disorders but the role of this approach for the investigation of cerebrospinal fluid (CSF) in AML is unknown. Design and Methods The aims of our study were 1) to determine the incidence of occult/manifest CNS disease in a homogenous series of AML patients; 2) to correlate CNS disease with clinico-biologic parameters; 3) to examine the impact of CNS involvement on outcome. CSF samples were collected from 98 newly diagnosed AML patients, 62 males and 36 females, median age 53 years (range 18-75). Sixty-five and 33 patients aged 60 years, respectively.Seventy-one patients received standard and 22 high-dose-ARA-C-based regimens, 5 supportive care. All of 98 CSF samples were examined by CC whereas 90 (91%) also by FCM. CC positivity was defined as unequivocal morphologic evidence of leukemic blast in CSF and/or white blood cells count (WBCc) ≥ 5/µl with less than 10 erythrocytes/µl. A cluster of at least 10 phenotypically abnormal events was regarded as a proof of FCM positivity. Results Sixty-seven patients were CNS negative (CNS-) while thirty-one (31%) were CNS positive (CNS+). Among the last, 10 (10%) were positive on both CC and FCM (manifest CNS+) and 21 (21%) only on FCM (occult CNS+). There was an equal male/female distribution among CNS- and CNS+ patients, as well as median age (52 years, range, 20-71, vs 56 years, range, 18-75, p=NS) and WBCc(27.5 x109/L, range, 1,20-223 x109/L, vs. 11,6 x109/L, range, 0,70-315 x109/L, p= NS) were similar in both groups. Instead, higher levels of lactate dehydrogenase (LDH) were observed among CNS+ than CNS- patients (p=. 01). Forty-seven patients (48%) had monoblastic/monocytic or myelomonocytic AML and belonging to one of these categories was significantly associated with a condition of CNS positivity (55% vs 45%, P = 002). Cytogenetic/genetic data were available in 82/98 (84%). Twenty-for patients (29%), 33 (39%), 12 (14%) and 12 (14%), belonged to the category of favorable, intermediate-I, intermediate-II, and adverse karyotype, respectively. Cytogenetic/genetic characteristics did not differed significantly between CNS+ and CNS- patients. Overall, response rate was 70%, with complete remission rate being not statistically different between CNS+ and CNS- patients (69% vs 81% p= NS). Five-year DFS and OS were found to be significantly shorter in occult or manifest CNS+ patients than in those CNS- (23% vs 50% p= .03 and 19% vs 46%, p=.02, respectively)(Figure 1A and 1B). The prognostic variables achieving a statistical significance in univariate analysis (CNS status, age , WBCc, favorable vs adverse karyotype) were challenged in a multivariate model to determine to what extent they affected treatment outcome. In multivariate analysis, CNS positivity was found to be independently and significantly associated with a shorter duration of DFS.(p=.03 HR= 0.46). Age >50 years was found to be the only independent prognostic factor affecting OS (p=.01 HR= 2.26). Conclusion Our data suggest that incidence of CNS involvement in newly diagnosed AML pts is higher than expected. Regardless of neurologic symptoms, manifest and occult CNS positivity should always be sought at diagnosis since it may affect outcome and influence therapeutic decision. Further prospective studies on larger series are warranted to confirm this data. Figure 1 DFS and OS based on CNS status Figure 1. DFS and OS based on CNS status Disclosures Lo Coco: Teva: Consultancy, Honoraria, Speakers Bureau; Lundbeck: Honoraria, Speakers Bureau; Novartis: Consultancy; Baxalta: Consultancy; Pfizer: Consultancy.

Eighty patients with high-risk hematologic malignancies underwent unmanipulated, G-CSF–primed BM transplantation from an haploidentical family donor. Patients were transplanted in first or second complete remission (CR, standard-risk: n =45) or insecond CR or active disease (high-risk: n =35). The same regimen for GVHD prophylaxis was used in all cases. The cumulative incidence (CI) of neutrophil engraftment was 93% 0.1%. The 100-day CIs for II-IV and III-IV grade of acute GVHD were 24% 0.2% and 5% 0.6%, respectively. The 2-year CI of extensive chronic GVHD was 6% 0.1%. The 1-year CI of treatment-related mortality was 36% 0.3%. After a median follow-up of 18 months, 36 of 80 (45%) patients are alive in CR. The 3-year probability of overall and disease-free survival for standard-risk and high-risk patients was 54% 8% and 33% 9% and 44% 8% and 30% 9%, respectively. In multivariate analysis, disease-free survival was significantly better for patients who had standard-risk disease and received transplantations after 2007. We conclude that unmanipulated, G-CSF–primed BM transplantation from haploidentical family donor provides very encouraging results in terms of engraftment rate, incidence of GVHD and survival and represents a feasible, valid alternative for patients with high-risk malignant hematologic diseases, lacking an HLA identical sibling and in need to be urgently transplanted.Haploidentical, unmanipulated, G-CSF-primed bone marrow transplantation. Haploidentical hematopoietic stem cell transplantation for hematologic malignancies.

Objective Aim of our prospective study was to investigate accuracy of bowel ultrasonography in detecting gastrointestinal acute graft versus host disease (GVHD), when using clinical assessment as gold standard. In a subgroup of patients, bowel ultrasonography was compared with colonoscopy and histology in diagnosing of gastrointestinal acute GVHD. Methods Fifty-two patients underwent allogeneic hematopoietic stem cell transplantation and developed gastrointestinal symptoms. Results Clinical assessment lead to a diagnosis of gastrointestinal acute GVHD in 17/52 patients, no gastrointestinal acute GVHD was detected in 20/52 patients, while 15 patients were not able to complete the study. Bowel ultrasonography detected either bowel wall thickness of the ileum and the colon or dilation in 16/17 patients and showed 94% sensitivity (95% CI 0.69–0.99), 95% specificity (95% CI 0.73–0.99), and 94.5% accuracy. Colonoscopy was performed in 13/52 patients, showing gastrointestinal acute GVHD in 11/13. In these 11 patients, histology confirmed the diagnosis of gastrointestinal acute GVHD, and bowel ultrasonography detected findings compatible with gastrointestinal acute GVHD in all 11 patients, and was negative in the 2 patients with no gastrointestinal acute GVHD. Conclusion Bowel ultrasonography can be considered a valuable tool to add to clinical assessment for patients with suspected gastrointestinal acute GVHD for addressing a prompt and appropriate treatment.

Background. During the last years, hematopoietic stem cell transplant from haploidentical donor is increasing by using the unmanipulated graft. We report the results of allogeneic bone marrow transplantation (BMT) from haploidentical donor primed with G-CSF in patients (pts) with high-risk acute myeloid leukemia (AML), who received an uniform TBF (Thiotepa, Busulfan, Fludarabine) conditioning regimen including antitymocyte globulin and an identical GVHD prophylaxis combining MTX+CSA, MMF and an anti-CD25 monoclonal antibody (Basiliximab). Patients and Methods. Since August 2005 to December 2014, 165 pts with high-risk hematological malignancy underwent unmanipulated BMT from fully haploidentical donor primed with 4µg/kg/day of G-CSF over 7 days. Of these 165 pts, 71 (median age 49 yrs, (range5-66) were affected by high-risk AML and were in early (CR1 and CR2: n=58) or advanced (>CR2 or active disease: n=13) phase at time of transplant. All pts received the same myeloablative (MAC: n=47) or reduced intensity (RIC: n=24) TBF conditioning regimen including antithymocyte globuline for 4 days (days-4 through -1) and an identical graft-versus-host disease (GVHD) prophylaxis consisting of the classical association of MTX+CSA combined with MMF from day +7 to day +100 and the administration of 20 mg of Basiliximab at days 0 and +4. The donor were represented by: son (n=25); brother (n=18), mother (n=11), daughter (n=8), sister (n=6) and father (n=3). Anti-infective management, supportive care and transfusion policy were identical for all pts. The experience was conducted at 2 Institutions: Stem Cell Transplant Unit at University Tor Vergata of Rome (n=45) and at Civil Hospital of Pescara (n=26), Italy. Results. All pts engraftedwith full donor chimerism: median time 20 days (range, 11-35) and CI 94±3%. With a median follow-up (FU) of 3.9 yrs (range, 0.7-7.4), the 7-yrs probability of overall survival (OS) and disease free survival (DFS) for all pts was 45±6% and 43±6%, respectively. OS calculated by disease status at transplant was 53±7% at 7 yrs for 58 pts transplanted in early phase and 0.0% at 4 yrs for 13 transplanted in advanced phase (p=0.0027), DFS was 49±7% and 15±10% at 7 and 2 yrs (p=0.007), respectively. TBF-MAC Regimen: 47 pts (median age 43 yrs, range 5-63), in early (n=38) or advanced (n=9) phase at transplant received a TBF-MAC regimen. For all these pts the CI of engraftment was 94±4% at a median of 20 days (range, 12-35). The 100 day CI of acute GVHD (AGVHD) grade II-IV and III-IV was 32±7% and 6±4%, respectively. The CI of extensive chronic-GVHD (CGVHD) was 19±7%. In the patients transplanted in early phase the CI of transplant related mortality (TRM) at 6 months and 7 yrs was 21±7 and 27±7%, respectively. The CI of relapse at 1 and 7 yrs was 16±6% and 24±7%, respectively. With a median FU of 3.9 yrs (range, 1.3-7.4), the 7-yr probability of OS and DFS was respectively 60±8% and 52±8% with a plateau of the curve from 2 yrs after transplant. None of the 15 variables considered in multivariate analysis (Cox and Fine and Gray models) influenced significantly the outcomes. TBF-RIC Regimen: 24 pts (median age 61.5 yrs, range 41-66) were conditioned with TBF-RIC. All pts engrafted with full donor chimerism (CI 96±6%). The 100 day CI of AGVHD grade II-IV and III-IV was 33±10% and 13±7%, respectively. The CI of extensive CGVHD was 8±8%.Four pts were in advanced and 20 in early phase at transplant. For these last pts, the CI of TRM at 6 months and 6 yrs was 15±8% and 25±10%, respectively. The CI of relapse at 1 and 6 yrs was 15±8% and 27±11%, respectively. With a median FU of 4.1 yrs (range, 0.8-6), the 6-yr probability of OS and DFS was respectively 42±12% and 41±12% with a plateau of the curve from 2 yrs after transplant. In multivariate analysis, no significant factor was identified. Conclusions. Although the number of pts does not allow an extensive analysis of the results, the homogeneity of our series in terms of diagnosis, patient selection, conditioning regimen and GVHD prophylaxis supports the conclusion that haploidentical, G-CSF primed, unmanipulated BMT is a valid alternative for high-risk AML pts lacking an HLA identical sibling. The regimen of GVHD prophylaxis is highly effective and can be proposed in alternative to the use of post-transplant cyclophosphamide, proposed by the Baltimore group. Finally, TBF-RIC regimen can be confidently offered to unfit or older AML pts candidate to an haploidentical transplant. Disclosures No relevant conflicts of interest to declare.

Despite advances in treatment and supportive therapies, outcome of acute myeloid leukemia (AML) remains dismal with approximately 40% of younger and less than 20% of elderly patients becoming long-term survivors. Stem cell transplantation (SCT) is a well-established post-remission therapeutic option, and in both its variants, autologous (AuSCT) and allogeneic (ASCT), it is often incorporated in modern treatment programs for it may reliably improve long-term prognosis. Ten years ago we demonstrated that the levels of minimal residual disease (MRD) before autologous stem cell transplant (AuSCT), assessed at the post-consolidation time point by multiparametric flow cytometry (MFC), affected outcome (1). Moreover, we have preliminarily observed that in MRD positive (MRDpos) patients, allogeneic stem cell transplant (ASCT) attenuates the negative prognostic impact of pre-transplant MRD positivity by conferring a significant survival advantage in terms of either overall (OS) or disease free survival (DFS) (2). At variance with this, others have shown that even in the setting of ASCT, pre-transplant MRD positivity is associated with a poor prognosis regardless of the graft-versus-leukemia (GVL) effect (3). The aim of the present analysis was to evaluate, in 81 MRDpos patients submitted to ASCT (45) or AuSCT (36), the impact on clinical outcome of different MRD levels. As previously reported, counting 3.5x10-4 (0.035%) residual leukemic cells (RLCs) or more in the bone marrow (BM) upon full hematological recovery after consolidation cycle, was regarded as a condition of MRD positivity. Patients with or above 3.5x10-4 RLC were arbitrarily divided into 3 different cohorts: 1) ≥0.035%≤0.1% (13 patients, 6 ASCT and 7 AuSCT); 2) >0.1%≤1% (52 patients, 31 ASCT and 21 AuSCT); 3) >1% (16 patients, 8 ASCT and 8 AuSCT). In the category no. 2, ASCT gave a significant 5-years OS (64,9% vs 17,9%, p1%) MRD positive patients, ASCT does not guarantee a long-term control of leukemia and further reduction of relapse risk is expected to be facilitated by novel strategies. Disclosures No relevant conflicts of interest to declare.

Kidney spontaneous rupture is not a recognized complication neither for amyloidosis nor of autologous stem cell transplantation (ASCT). A 46-year-old white woman, affected by nephrotic syndrome, was diagnosed as AL amyloidosis by renal biopsy. We report the singular case of a bilateral spontaneous kidney rupture during ASCT for AL with renal rescue.

Patients with neutropenia are exposed to a high risk for infections in which fever is often the unique symptom [1]. Systemic infections remain the main cause of mortality in these patients therefore, the policy for infection management is to promptly administer empirical antibiotic therapy in order to avoid the increased risk of mortality related to the treatment delay [2]. However, microbiological diagnostic tests are not sufficiently rapid, sensitive or specific to indentify the microbial causes of fever, and a considerable number of patients suffer febrile episodes over a prolonged period without a definite microbiological etiology.

Multiparametric flow cytometry (MPFC) detection of minimal residual disease (MRD) represents a robust surrogate for the quality of complete remission (CR) and reliably predicts clinical outcome. In our experience, MRD detection provides prognostically relevant information when assessed at the post-consolidation time point. Ten years ago we demonstrated that the amount of MRD before autologous stem cell transplant (AuSCT) affected outcome. More recently, other authors have extended this observation to allogeneic stem cell transplant (ASCT) showing that pre-transplant MRD is a major determinant of prognosis regardless of graft-versus-leukemia (GVL) effect. The aim of our study was to evaluate, in an extended series of patients submitted to AuSCT or ASCT, the impact of a pre-transplantation MRD positive (MRDpos) or negative (MRDneg) status on overall survival (OS) and disease free survival (DFS). We analyzed 173 MRDpos and 53 MRDneg patients of whom 67 were submitted to AuSCT and 51 to ASCT. Eighty-two patients received no transplant because of age, poor performance status or insufficient stem cell harvest whereas 26, all in the MRDpos group, relapsed before transplant delivery. In the AuSCT group, before transplant, 32/67 (48%) were MRDneg and 35/67 (52%) MRDpos, with MRDneg group showing a superior OS (55% vs 20%, p=0.007). In the ASCT group, before transplant, 45/51 (88%) were MRDpos and 6/51 (12%) MRDneg. For 21 out of 51 (41%) sources of stem cells were matched unrelated donors (12) or haploidentical donors (9). In this subgroup, MRDpos and MRDneg patients shared a comparable 5-years OS (60% vs 56%, p=NS), with a 36% survival gain for those MRDpos who received ASCT as compared to AuSCT. Among MRDneg patients, no survival differences were demonstrated between those submitted to AuSCT or ASCT (55% vs 60%, p=NS). Such a lack of difference is likely due to the higher treatment related mortality (ASCT 3/6, 50% vs AuSCT 2/32, 6%, p=0.003) which counterbalanced the lower relapse rate in the ASCT group (ASCT 0/6, 0% vs AuSCT 11/32, 34%, p=NS). In conclusion, ASCT confers a significant survival advantage to MRDpos patients, attenuating the negative prognostic impact of pre-transplant MRD positivity. ASCT may expose MRDneg patients to an excess of toxicity suggesting that in these patients the allogeneic option should be postponed after a second remission. In MRDpos patients, AuSCT does not represent a valid therapeutic choice and ASCT, which should be timely delivered, also considering alternative sources of stem cells. Disclosures No relevant conflicts of interest to declare.

Patients and Methods. At the Rome Transplant Network, a JACIE accredited metropolitan transplant program, a matched-pair analysis has been conducted on 116 of 255 patients transplanted between January 2008 and December 2012. The patients transplanted from Id Sib (n=58) or Haplo related donors (n=58) were completely matched for the following features: patient age, gender, diagnosis (7 subgroups), disease phase (early: CR1+CR2; advanced: >CR2+active disease), myeloablative or reduced intensity conditioning regimen consisting of Thiotepa, i.v. Busulphan and Fludarabine (TBF) association, donor age and donor/recipient sex, AB0 and CMV combinations. As GVHD prophylaxis, all patients received the standard CSA and MTX combination with the addition of ATG, MMF and Basiliximab in Haplo bone marrow recipients. The transfusion policy, supportive care and antinfectious prophylaxis were identical for all patients. Results. By comparingId Sib to Haplo recipients, the cumulative incidence (CI) of grade II-IV and III-IV acute-GVHD was 18±5% vs 42±7% (p=0.002) and 7±3% vs 14±5% (p=ns), respectively. The CI of extensive chronic GVHD was 23±6% for both patient series. The 5-year CI of TRM was 30±6% vs 36±6% (p=ns), respectively. The main causes of TRM were infections and the 6-month CI of Infection Related Mortality (IRM) was 26±6% in Haplo transplant and 10±4% in Id Sib (p=0.04). Although not statistically significant, the 5-year CI of relapse was higher in Id Sib (40±7%) than in Haplo recipients (28±6%). With a median follow-up of 3.5 years (range, 1 - 6), the 1- and 5-year disease free survival (DFS) was respectively 50±7% and 37±6% for Id Sibs and 45±7% and 36±6% for Haplo (Figure 1). Since 2 years after transplant, DFS curves of Id Sib and Haplo patients remained at plateau and were overlapping. Conclusions. This analysis considered a high number of factors for matching patients, who were grafted according to an identical transplant program. We can conclude that as consequence of a more intensive GVHD prophylaxis and therapy ensuing from higher incidence of >II grade acute GVHD, Haplo recipients are mainly exposed to a risk of early infection mortality. On the other hand, Id Sib patients seem to express less graft-versus-tumor activity with increasing risk of relapse after transplant. The identical long-term DFS justifies to consider the unmanipulated bone marrow transplant from haploidentical donor a valid alternative for patients lacking an HLA identical sib. Finally, it can appear at present provocative, but certainly realistic in perspective, the hypothesis that the donor choice in a familiar setting will mainly take into account other favourable donor/recipient combined characteristics rather than HLA compatibility. Figure 1 - Disease Free Survival Figure 1. - Disease Free Survival Disclosures No relevant conflicts of interest to declare.

B-cell chronic lymphocytic leukemia (B-CLL) is an indolent lymphoproliferative disorder with a progressive accumulation of small, morphologically mature B lymphocytes in the bone marrow, blood and lymphoid tissues. Alemtuzumab (MabCampath) is a IgG1 monoclonal antibody (mAb) that target the CD52 antigen, a glycosylated peptide highly expressed on B-CLL cells. This mAb is active in patients with B-CLL refractory to alkylating agents and purine nucleoside analogues. The primary adverse event so far reported is the induction of a profound and prolonged depletion of CD4 and CD8 subpopulations leading to an immunodeficient status and the development of opportunistic infections [1,2]. Recently, Epstein-Barr virus (EBV) related lymphoma have been reported in patients with T and B cell lymphoproliferative disorder after treatment with alemtuzumab [2–5]. Here, we report a case of an EBV related lymphoproliferative disorder secondary to alemtuzumab therapy for B-CLL. A 53 years old man diagnosed in January 1999 as having stage Rai II classical B-CLL, was admitted to our department in June 2007 as a consequence of disease progression. Analysis of IgVH mutational status was not performed. However, peripheral blood flow cytometry analysis revealed that tumor cells expressed ZAP-70, but were negative for CD38 expression. He was previously treated with two cycles of fludarabine 25 mg/m with no response. In March 2001, he received six doses of rituximab 375 mg/m obtaining a partial remission. In December 2005, because of disease progression associated with Coombs positive autoimmune hemolytic anemia (AHA) he was treated with rituximabþfludarabineþ cyclophosphamide (FCR) for six cycles obtaining a partial remission with resolution of the (AHA). From June 2007 to July 2007, he received 12 doses of alemtuzumab 30 mg/m, obtaining the clearance of bone marrow neoplastic cells with persistence of lymph nodes involvement. CD4 and CD8 levels prior alemtuzumab therapy were 517/mL and 1380/mL, respectively. After 4 weeks of anti CD52 therapy, treatment was interrupted because of CMV reactivation which was successfully treated with ganciclovir. In October 2007, the patient presented with fever AHA recrudescence and hepatomegaly. Laboratory analysis showed high LDH level (1299 UI/L), hypoalbuminemia (2.5 g/dL), elevated liver enzyme (ALT: 92 UI/L; AST: 87 UI/L) and anemia (Hb 8.4 g/dL). Lymphocyte count on peripheral blood was less than 40/mL. A CT scan revealed the presence of multiple enlarged lymph nodes at both sides of the diaphragm and liver enlargement. An abdominal ecography revealed multiple hypoechogenic liver lesions which were biopsied and showed large B cells CD20þ. In situ hybridisation for EBV-RNA was strongly positive and serum EBV DNA viral load showed more than 6500 copies/mL. Bone marrow evaluation didn’t show any infiltration by neoplastic cells. The diagnosis of stage IV CD20þ

Abstract 2350 In this study we investigated the feasibility and clinical value of non T-cell depleted bone marrow transplantation (BMT) from HLA haploidentical related donor in patients with high risk hematological malignancies. Materials and Methods. Between August 2005 and May 2010, 71 patients were transplanted for acute myeloid leukemia (AML) (n=42), acute lymphoblastic leukemia (n=13), chronic myeloid leukemia (n=5), Hodgkin lymphoma (n=5), plasmacell leukemia (n=3), myelofibrosis (n=2) and myelodisplastic syndrome (n=1). Their median age was 35 years (5-71). At time of BMT, all patients were at very high risk: 39 were in early (complete remission 1 or 2) and 31 in more advanced stage of disease. Seventeen of them were given a previous transplant either autologous (n=14) or allogenic (n=3). All donors were HLA identical at 1 haplotype and mismatched for 2 (n=24) or 3 (n=47) loci on the unshared haplotype. As pretransplant regimen, 10 patients received a reduced intensity conditioning consisting of Fludarabine (Flu) alone (n=1), Flu + Thiotepa (Thio) + Melphalan (n=2) or Flu + Thio + i.v.Busulphan (Bu) (n=7), and 61 patients received a myeloablative therapy consisting of Aracytin + Cyclophosphamide combined with TBI (n=7) or Treosulphan (n=11) or Bu (n=11), whereas the last 32 consecutive patients underwent transplant after conditioning with the association of Thio + Flu + i.v. Bu. All patients received an identical graft-versus-host disease (GvHD) prophylaxis consisting of Fresenius Antithymocyte Globulin (5 mg/Kg/d from day -4 to -1) combined with Cyclosporine (1,5-3 mg/Kg/day i.v. from day -7 to +28 and orally 5 mg/Kg until day +365), Methotrexate (15 mg/sqm on day +1 and 10 mg/sqm on day +3, +6 and +11), Mycophenolate Mofetil (1 g/d from day +7 to +100) and the anti-CD 25 monoclonal antibody Basiliximab (20 mg i.v. on day 0 and +4). Bone marrow cells were harvested from all donors after priming with Filgrastim at 3–4 microg/Kg/d from day -7 to -1. Bone marrow cells were infused fresh and unmanipulated on day 0. Results. The median dose of total nucleated, CD34+ and CD3+ cells infused was 7.8 (1-28) ×108/kg, 2.1 (0.8-11) ×106/Kg and 28 (10-98) ×106/Kg, respectively. One patient had a primary graft failure and 5 patients died too early to be evaluated for engraftment. Results in terms of cumulative incidence (CI) of PMN engraftment, acute and chronic GvHD, relapse, transplant related mortality (TRM) and overall survival (OS) estimated with the Kaplan Meyer method are given in the Table. After a median follow-up of 16 (3-56) months, the 3 years probability of OS and disease-free survival (DFS) was 43% and 37% respectively for all patients (see Figure). The 3 years OS for the 42 patients with AML was 48% (61% for 30 patients transplanted in early stage and 18% for 12 patients transplanted in advanced stage, P=0.01). The 1 year OS for the 32 patients transplanted with the conditioning therapy employed in the last 2 years (Thio + Flu + i.v. Bu) was 72% in 18 patients transplanted in early stage and 52% in 14 patients transplanted in advanced stage (P=0.47). Early stage Advanced stage All patients P value N. 39 32 71 CI of PMN engraftment at day 30 87±0.3 81±0.5 88+0.7 NS CI of acute GvHD grade II - IV 34±0.7 16±0.6 26±0.3 NS CI of acute GvHD grade III - IV 9±0.4 3±0.1 6±0.1 NS CI of chronic GvHD 8±0.2 14±0.8 11±0.1 NS CI of relapse at 2 years 30±0.7 37±1 33±0.4 NS CI of TRM at 1 year 21±0.4 41±0.5 30±0.3 NS CI of TRM at 2 years 24±0.5 48±1.3 36±0.6 NS OS at 3 years 54±9.2 26±9.9 43±7 0.03 Download : Download high-res image (91KB) Download : Download full-size image Conclusions. BMT from haploidentical donor using G-CSF primed, unmanipulated bone marrow cells and an intensive regimen for GVHD prophylaxis is correlated with high engraftment rate, low incidence of acute and chronic GVHD, acceptable TRM and favourable patient outcome. The results seem particularly encouraging for AML patients grafted at an early disease stage. In alternative to transplant from matched unrelated donor or cord blood, this approach can be offered to high risk patients with hematological malignancies particularly for those who are on urgency for a transplant. Results are given as % ± standard error Disclosures: No relevant conflicts of interest to declare.

Abstract 1195 Poster Board I-217 The policy of the Rome Transplant Network (RTN), a metropolitan network of transplant Centers, for patients candidates to an allogeneic hematopoietic stem cell transplant (HSCT) and lacking an HLA identical sibling is the contemporary search for one the HSC alternative sources such as Matched Unrelated Donor (MUD),Cord Blood Unit (CBU) or Haploidentical Related Donor (HRD). The main aim of the RTN policy is the identification of a suitable donor in order to perform transplant in adequate timing. The selection criteria for MUD consist of a 8/8 HLA loci matching tested at low resolution for class I HLA and at high resolution (HR) for class II; one difference in C Ag is considered acceptable in case of both I and II class HR identity. CBU's selection criteria are instead based on cell doses (TNC≥2.5×107/kg and CD34+≥1× 105/kg) and on a HLA-compatibility ≥to 4/6 HLA Ag. From April 2006, the haplodentical option was also simultaneously considered, so all closer family members have been tested for the HLA. Here, we report the results of the intention to treat (ITT) analysis on the potential therapeutic impact of our transplant policy. Data were obtained from RTN database. From April 2006 to date, 196 pts have been candidated to receive an allogeneic HSCT for hematological disease. Sixty-six out of 196 (34%) underwent HSCT from HLA identical sibling, while a search process for an alternative donor was activated for 130 pts. Of 130 pts, 9 (7%) lost the eligibility to transplant early during the search process and 19 (15%) died of early disease progression in most cases: a suitable MUD or CBU had been identified for 13 of 19 within 3 months from the start of the search and only 6 pts (5%) died without an alternative donor had been found. To date, 73/102 evaluable pts (72%) lacking an HLA identical sibling have been transplanted (n=66: 23 MUD; 24 CBU; 19 HRD) or are willing to proceed towards the transplant (n=7: 3 MUD;2 CBU; 2 HRD). In summary, for all 196 candidates to an allogeneic transplant the eligibility was confirmed for 187 (95%), a suitable donor could be identified for 181 (92%) of all pts or 97% of the eligible ones and an allogeneic transplant could be performed for 168 (86%) of all candidates or 93% of those eligible. From this ITT analysis, we can conclude that, by adopting the RTN policy of widespread donor search and multiple transplant options, the allogeneic transplant can be offered as potential therapeutic procedure to a large majority of pts. Disclosures: No relevant conflicts of interest to declare.

Abstract 2291 Poster Board II-268 Haploidentical bone marrow transplantation (BMT) is an alternative treatment to patients with high-risk hematologic malignancy lacking a HLA-matched donor and those urgently need transplantation. We used a haploidentical-BMT protocol without ex vivo T cell depleted based on the knowledge that marrow grafts have 10 times fewer lymphocytes compared to peripheral blood stem cell grafts and granulocyte colony-stimulating factor (G-CSF) donor priming reduce the incidence of acute GvHD. Materials and Methods: 40 patients (median age of 32, 12-63) with advanced disease or leukemia with poor prognostic features underwent unmanipulated haplo-BMT: 22 with AML, 9 with ALL, 3 with CML, 3 with Hodgkin lymphoma and 3 with plasmacell leukemia. Status at disease: 22 early (first or second comple! te remission), 18 advanced (progressive or refractory disease). All pairs of donors and recipients were identical for one HLA haplotype and incompatible at 2 or 3 loci.The myeloablative conditioning regimens used were different; antithymocyte globuline, cyclosporine, metotrexate, mycophenolate mofetil and basiliximab were used for GvHD prophylaxis. Donors were primed with filgrastim at 4 micrograms/Kg/d for 7 consecutive days. Bone marrow cells were harvest on the 8 day and were infused unmanipulated. Results: the median dose of total nucleated, CD34+ and CD3+cells was 7×10e8/Kg (1.01-28.7), 2.3×10e6/Kg (1.17-6.0) and 23.3×10e6/Kg (9.7-66.6) respectively. 1 patients had a primary graft failure and 5 patients died early prior to engraftment. In the remaining 34 patients, engraftment was seen with median time to granulocyte and platelet recovery of 22 and 27 days respectively; acute GvHD was grade 0 in 17 patients (50%), grade I in 9 (26%), grade II in 7 (20%) and grade IV in 1 (3%). In 29 evaluable patients, chronic GvHD was limited in 3 (10%) and extensive in 1 (3%). Transplant-related mortality at 6 months for early and advantage stage was 22% and 35% respectively. After a median follow up of 18 (3-42) months, 8 patients relapsed; 11 patients (50%) in the early stage and 4 (22%) in advanced phase are now living in haematological remission. The 1-year Kaplan-Meyer probability of disease-free survival is 45% for all patients. Conclusion: the high engraftment rate, low incidence of grade II-IV acute GvHD and an acceptable TRM suggest that G-CSF-primed marrow grafting along with sequential immunosuppression could provide an excellent alternative for patients who lack matched donors. Disclosures: No relevant conflicts of interest to declare.

Implementation of transplant procedures and the availability of alternative sources of stem cells for patients without HLA identical siblings has made allogeneic stem cell transplant (ASCT) a suitable therapy for many patient affected by acute myeloid leukemia (AML); consequently, in AML patients transplant strategies must be carefully designed according to stringent risk/benefit analysis. To date, risk assessment relies on identification of baseline prognostic parameters such as karyotype, which has become critical in order to choose post-remissional therapy. Furthermore, there are strong evidences that the presence of specific gene abnormalities, such as mutations of FLT3, NPM and c-kit, allow to identify, within homogeneous cytogenetic groups, subsets of patients with distinct clinical outcome. We hypothesized that, in order to guide the post-remission decisional process, more robust informations may derive from the combined evaluation of conventional baseline and delayed prognosticators. Minimal residual disease (MRD) detection is potentially the most efficient tool to investigate the quality of remission and might represent the ideal parameter to be associated with baseline biological features for prognostic purposes. Therefore, we aimed to determine whether combined analysis of karyotype and MRD, using multiparametric flow-cytometry (MPFC), could help to refine risk assessment in adult patients with AML, allowing the most appropriate post-remissional strategy to be selected. We analyzed 132 patients with AML who had intensive chemotherapy with EORTC/GIMEMA protocols. According to MRC classification, 22 (17%), 104 (78%) and 6 (5%), respectively, had good, intermediate and poor risk karyotype. Thirteen of 107 (12%) carried a FLT3-ITD. Within good and intermediate risk categories, MRD positivity (≥ 3.5×10−4 residual leukemic cells at the post-consolidation time-point) was associated with a worse prognosis in terms relapse free (RFS) and overall survival (OS). In fact, we observed that: MRD negative good and intermediate risk categories shared the same favorable prognoses with RFS and OS of 80% vs. 69% and 84% vs 54%, respectively; MRD positive good and intermediate risk categories fared as worse as those with poorrisk karyotype or FLT3-ITD in terms of RFS (28% vs. 17% vs. 0%) and OS (42% vs. 21% vs. 17%). Using this approach the conventional cytogenetic classification that uses three categories is simplified into 2 prognostically defined groups: favorable, including MRD negative good and intermediate risk karyotype; unfavorable, including MRD positive good and intermediate risk karyotype, poor risk karyotype and FLT3-ITD positive cases (Fig. 1). Based on these observations, we believe that ASCT is recommended, not only for poor-risk karyotype or FLT3 positive AML, but also for good/intermediate risk categories not gaining MRD negativity, being this option able to provide a superior chance of prolonged RFS (Maurillo et al, JCO 2008). On the other hand, patients belonging to MRD negative good/intermediate risk categories, who can experience a 5-years survival higher than 60%, may have their life expectancy hampered by the choice of a therapeutic strategy with a disadvantageous risk/benefit ratio: for this category a standard intensification procedure (chemo and/or autologous transplant) is indicated. In conclusion, the combined assessment of baseline prognosticators (cytogenetics) and parameters inherent the quality of response (MRD), is useful to define discrete categories of risk within the respective karyotypic groups, allowing tailored therapeutic approaches to be applied according to the actual clinical risk and avoiding under or over treatments. Figure 1 Figure 1.

Background. Similar probabilities of survival have been reported for patients transplanted from Matched Unrelated Donor (MUD), Umbilical Cord Blood (UCB) or Haploidentical (Haplo) donors as alternative hematopoietic stem cell sources. However, few studies have compared these results with those obtained in patients transplanted from HLA Id-siblings (Id-sib). Moreover, all reported studies are retrospective and the criteria of donor selection were not predefined. We report the intention to treat (ITT) analysis results on 238 patients with high-risk acute myeloid leukemia (AML) prospectively transplanted according to the policy of the Rome Transplant Network (RTN), a metropolitan transplant program established in Rome in 2006. Patients and Methods. For AML patients eligible to an allogeneic transplant, the RTN policy consists of an algorithm of donor choice based on a hierarchy according to the following criteria: 1) HLA identical sibling; 2) MUD ≥8/10 HLA 3) UCB as single unit selected on the base of cell dose and number of HLA disparities (0-1/6 HLA: TNC ≥2.5x107/kg and CD34 ≥1x105/kg; 2/6 HLA: TNC ≥3.5x107/kg and CD34 ≥2x105/kg); 4) G-CSF primed, unmanipulated bone marrow Haplo donor. Myeloablative (MAC) or reduced intensity (RIC) TBF (Tiothepa, Busulfan, Fludarabine) conditioning regimen was identical for all patients, GVHD prophylaxis was uniform for each categories. of transplant Results. From January 2006 to December 2014, 238(89%) out of 303 adult patients candidates to an allogeneic transplant for high-risk AML were considered eligible. Overall, a donor was available for 205 (86%) of 238 eligible patients. At time of the analysis, 17 of these 205 patients (8%) had lost the transplant eligibility and 4 (2%) were still scheduled for transplant, therefore 184/205 (90%) patients with an available donor were finally transplanted from Id-sib (n=76), MUD (n=38), UCB (n=17) or Haplo (n=53) donors. The 8-yrs overall survival (OS) of the 238 eligible patients from time of HLA typing and of the 184 transplanted patients from time of the graft was 40±4%and 43±4%, respectively. By excluding the low number of UCB recipients (n=17), the OS was particularly dismal for the 34 patients transplanted in advanced disease phase (7±4% at 4 yrs), whereas for the 132 patients transplanted in early (CR1+ CR2) phase the 8-yr OS was 56±5%: 58±7% for 61 HLA Id-sib, 50±8% for 40 Haplo and 63±10% for 27 MUD recipients (P=NS). The OS of patients transplanted in early phase was 63±5% for 97 patients receiving MAC and 33±9% for RIC recipients. For these 97 patients, the survival by type of donor was 62±10% either for 47 Id-sib or 28 Haplo and 70±10% for 22 MUD recipients (P=NS). The results were analyzed by various donor/recipient (D/R) combinations such as age, sex and CMV status. The median donor age was 39 years (range, 18-70) and the median patient age was 43 years (range, 16-59): the 8-yr OS of patients (n=30) with younger D/R combination (D Conclusions. RTN policy allowed donor identification in 86% of all eligible AML patients and allowed an allogeneic transplant to be carried out in 90% of them with no substantial differences in terms of long-term survival between initially eligible (ITT analysis) and definitively transplanted patients. Only the definition of a specific transplant policy and the ITT analysis allow to evaluate the real impact of a transplant program. As the probability of survival is not substantially different comparing different donor stem cell sources, the final conclusion which can be drawn from our study is that for patients with AML undergoing an allogeneic transplant, the HLA matching is unlikely to remain the first criterion for donor identification. Other factors such as D/R age, sex and CMV status should drive the search for the best donor. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures No relevant conflicts of interest to declare.