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•We present real-world data on all ruxolitinib-treated myelofibrosis patients in a 10-million-resident region, with a follow-up of 2 years.•We found no evidence of an increased risk of developing lymphomas.

•Of 167 patients with ITP, 18.5% had somatic mutations typical of clonal hemopoiesis, 68% high-risk variants, and 8 multiple mutations.•Mutated patients were more frequently older males, with a shorter time from first to second-line therapy, and had a higher thrombotic risk.

Essential thrombocythemia (ET) and polycythemia vera (PV) are characterized by persistent platelet activation. The mechanisms involved in their clearance are poorly characterized. In the present study, we report that leukocytes were actively involved in platelet disposal in 51 patients with ET and 30 with PV, but not in 70 age- and sex-matched controls. The fraction of circulating neutrophils and monocytes that had phagocytosed platelets, as assessed by flow cytometry, was significantly higher in patients with PV or ET, independently of hydroxyurea treatment, than in controls. Platelet phagocytosis by circulating leukocytes was confirmed by confocal and electron microscopy. The lack of effect of hydroxyurea, which disrupts the P-selectin/P-selectin glycoprotein ligand 1 (PSGL-1) interaction, suggests a P-selectin–independent mechanism. This hypothesis was confirmed in an ad hoc animal model based on the in vivo injection of activated platelets from P-selectin+/+and P-selectin−/−mice. P-selectin expression was associated with an earlier and effective clearance of platelets by neutrophils. A second delayed, P-selectin–independent phase actively involved monocytes. Our results suggest that phagocytic clearance of platelets by leukocytes occurs in PV and ET, possibly involving P-selectin–dependent and -independent pathways, thus representing a novel mechanism to remove activated platelets from the circulation.

Essential thrombocythemia (ET) and polycythemia vera (PV) are characterized by persistent platelet activation. The mechanisms involved in their clearance are poorly characterized. In the present study, we report that leukocytes were actively involved in platelet disposal in 51 patients with ET and 30 with PV, but not in 70 age- and sex-matched controls. The fraction of circulating neutrophils and monocytes that had phagocytosed platelets, as assessed by flow cytometry, was significantly higher in patients with PV or ET, independently of hydroxyurea treatment, than in controls. Platelet phagocytosis by circulating leukocytes was confirmed by confocal and electron microscopy. The lack of effect of hydroxyurea, which disrupts the P-selectin/P-selectin glycoprotein ligand 1 (PSGL-1) interaction, suggests a P-selectin–independent mechanism. This hypothesis was confirmed in an ad hoc animal model based on the in vivo injection of activated platelets from P-selectin+/+ and P-selectin−/− mice. P-selectin expression was associated with an earlier and effective clearance of platelets by neutrophils. A second delayed, P-selectin–independent phase actively involved monocytes. Our results suggest that phagocytic clearance of platelets by leukocytes occurs in PV and ET, possibly involving P-selectin–dependent and -independent pathways, thus representing a novel mechanism to remove activated platelets from the circulation.

Rossi: Daiichi-Sankyo: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria. Elena:Novartis: Consultancy; Pfizer: Consultancy. Iurlo:Pfizer: Other: Speaker Honoraria; Incyte: Other: Speaker Honoraria; Novartis: Other: Speaker Honoraria.

In the PhilosoPhi34 study (EudraCT: 2012-005062-34) on 87 CP-CML patients, we analyzed the gene expression profiling (GEP) of bone marrow (BM) CD34+/lin- cells of 79 patients with chronic-phase chronic myeloid leukemia (CP-CML) patients at diagnosis and after 12 months of nilotinib treatment (Pungolino et al. AM J Hematol. 2018). FISH analyses identified CD34+/lin- Ph+ cells in all 79 CML-CP patients at diagnosis. 78/79 patients achieved at least a complete cytogenetic response after 12 months of nilotinib whereas 1/79 patients relapsed at 12 months. No Ph+ nuclei were detected in 78/79 patients at 12 months.

Rossi: Sandoz: Honoraria; Jazz: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Teva: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Honoraria; Novartis: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.

Rossi: Janssen: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Rossi: Teva: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees.

BackgroundChronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder which molecular base is represented by the bcr-abl fusion gene, encoding for the constitutionally activated BCR-ABL tirosine-kinase. Three Tirosin-Kinase Inhibitors (TKI) are approved for first line treatment: Imatinib (IM) and the second generation (2G) TKI Nilotinib (NIL) and Dasatinib. 2G TKI are known to provide faster and deeper molecular responses (MR) compared to Imatinib, but serious toxicities may hamper long term treatment with these molecules. Furthermore, 2G TKI were usually employed as second line after IM failure, while the inverse sequence from second to first generation TKI (like an induction-maintenancemodel) has not been explored yet.

BackgroundTargeted therapy with Tyrosine-Kinase-Inhibitors (TKIs) totally modified the course of treatment of Chronic Myeloid Leukemia (CML). The objectives and the needs of treatment have been modified during the last years and the discontinuation of therapy is now a feasible aim. However, a lot of biological data acquired in the last twenty years, showed that degree and mechanisms of Leukemic Stem Cells (LSCs) clearance during TKI treatment are not clearly established as well as the predictive criteria for a stable and prolonged Treatment Free Remission (TFR). The multicentre, prospective, single-arm PhilosoPhi34 study (EudraCT: 2012-005062-34) was designed by the Rete Ematologica Lombarda (REL), to verify the in-vivo activity and time-course of first-line Nilotinib (NIL) therapy on Bone Marrow (BM) CD34+/lin-Ph+ cells clearance. An exploratory Gene Expression Profiling (GEP) study of CD34+/lin- cells at diagnosis and at 12 months (mos) of treatment, for the first 30 evaluable pts, was included. Preliminary GEP data suggested a correlation between different NFKBIAexpression at diagnosis and at 12 mos and the achievement of a deeper Molecular Response (MR) (Pungolino et al, AJH 2018).

Introduction

BackgroundChronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder, hallmarked by the BCR-ABL fusion gene which encodes for a constitutively activated BCR/ABL tyrosine-kinase, effectively treated with Tyrosine-Kinase Inhibitors (TKIs). The first line TKI Imatinib (IM) can obtain a high cumulative CCyR; compared to IM, deeper and faster molecular responses can be obtained in newly diagnosed CML patients with the second generation TKIs, Nilotinib (NIL) and Dasatinib. Despite the three TKIs have been evaluable as first line therapy for a long time, our experience largely concern the second generation TKIs after IM but not evidence are reported regarding IM after second generation TKIs.

Introduction:Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder with heterogeneous biological and clinical features. The biomolecular mechanisms of CML response to tyrosine-kinase inhibitors (TKI) are not fully defined. Despite the TKI nilotinib being a very effective drug for the treatment of CML, drug resistance can emerge. In the contest of the REL-PhilosoPhi34 study (EudraCT: 2012-005062-34) on behalf of the Rete Ematologica Lombarda, we undertook gene expression profiling (GEP) of selected bone marrow (BM) CD34+/lin- cells of 30 patients with CP-CML at diagnosis vs. the same patients after 12 months of nilotinib treatment to investigate molecular signatures characterizing both conditions.

Orlandi: Ariad: Honoraria; BMS: Honoraria; Novartis: Honoraria.

BackgroundCML is a clonal disorder characterized by the presence of the Philadelphia (Ph) chromosome which encodes for the bcr-abl tyrosine-kinase (TK). Target therapy with the TK inhibitors (TKIs)) has greatly improved its outcome.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Background.Chronic Myeloid Leukaemia (CML) can be effectively treated with the first generation Tyrosine Kinase Inhibitor (TKI) Imatinib, and more effectively with the second generation TKI, like Nilotinib. However, despite the deeper and faster responses induced by nilotinib in a large proportion of patients, the possible eradication of the pathological stem cells is not yet clearly elucidated. In fact, in vitro data suggest that quiescent stem cells are not sensitive to Bcr/Abl inhibition (Corbin AS, et al 2011; Hamilton A, et al 2012). A preliminary in-vivo study (Defina M, et al 2012) shows that in patients in CCyR even after short-term of nilotinib therapy, residual leukemic progenitors are rarely detected.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

This study aimed to determine the gene expression profiling (GEP) of bone marrow (BM) CD34+/lin- cells of CML patients at diagnosis and after nilotinib treatment.Microarray was performed on 30 CML patients at diagnosis as well as 8 patients after 12 months of nilotinib treatment using the latest generation Affymetrix GeneChip HTA 2.0 to further investigate genomic complexity.

Chronic myeloid leukemia (CML) is a disease of stemming from genetic damage to a hematopoietic stem cell. Despite nilotinib being a very effective drug for the treatment of CML, drug resistance can emerge.

Human herpesvirus 6 (HHV-6) is increasingly recognized as an opportunistic and potentially life-threatening pathogen in recipients of allogeneic Hematopoietic Stem Cell Transplantation (HSCT). Reported clinical manifestations of HHV-6 infection in transplanted patients are skin rash, interstitial pneumonia, bone marrow suppression and encephalitis. Moreover, an increasing number of clinical reports suggest that HHV-6 can facilitate the occurrence of other severe clinical complications of allogeneic HSCT, including Graft-versus-Host Disease (GvHD), ultimately increasining transplant-related mortality. Still, the actual incidence of HHV-6 infection in recipients of HSCT and the causative link between infection and clinical complications remain elusive, mostly due to the small and heterogeneous patient cohorts analyzed to date.From January 2009 to July 2011, we retrospectively evaluated 43 consecutive adult patients (median age 51 years) who developed positivity to HHV-6 after allogeneic HSCT for high-risk hematological malignancies. Stem cell donor was for 30 patients family haploidentical, for 5 an HLA identical sibling, and for 8 an unrelated volunteer (1 of which cord blood). The viral load was determined by quantitative PCR in cell-free body fluids such as plasma, bronchoalveolar lavage, cerebrospinal fluid, bone marrow aspirates or in gastrointestinal biopsies. At the time of positivity all patients were receiving acyclovir as viral prophylaxis except 5. Sixteen patients had clinical acute GvHD at time of HHV-6 positivity (grade III-IV in 14), and 33 were profoundly immunosuppressed with variable association of 2–4 immunosuppressive drugs (steroids included). Moreover concomitant CMV positivity was detected in 11 patients, while a severe neutropenia in 12.Median time from allogeneic HSCT to HHV-6 reactivation was 36 days (range: 7–625). In 19 patients HHV-6 was detected in plasma, with a median viral load of 19,454 cp/mL (34-4,524,600); 15 had concomitant fever, 5 skin rash of new onset, 4 impaired liver function, and 5 developed cytopenia subsequently to the infection. In 7 patients HHV-6 was detected in the bone marrow: the median viral load was 163'800 cp/mL (568-1'552'982). In 8 patients, all febrile, HHV-6 was observed in bronchoalveolar lavage samples with a median of 4'149 cp/mL (85–39250). In 16 patients, 10 with documented gut aGvHD, 11 with diarrhoea, HHV-6 was detected in gastrointestinal biopsies with a median of 7'510 cp/mL (120-4'524'600). HHV-6 was found in cerebrospinal fluid in 4 cases (all within 30 days after HSCT); the median viral load was 29'352 cp/mL (4'508-1'552'982); all these patients experienced encephalitis with confusion and anxiety, 2 suffered seizures and 3 showed abnormal findings on brain MRI. Amongst patients with organ localizations of HHV-6 only 28% had concomitant plasma positivity. HHV-6 positivity led to antiviral pharmacological treatment only when associated with clinical manifestations (n=21), using as first choice therapy foscarnet. Amongst the total 43 patients with documented HHV-6 positivity 11 completely solved the clinical event, whereas 19 (44%) died.HHV-6 infection/reactivation is associated with high morbidity and mortality in patients who undergo allogeneic HSCT. HHV-6 infection typically occurred close to the time of neutrophil engraftment. HSCT from an HLA-mismatched donor and steroid administration were associated with increased risk of active HHV-6 infection. Development of encephalitis was associated with high HHV-6 viral load. The regular monitoring of HHV-6 DNA in allogeneic HSCT recipients, 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.No relevant conflicts of interest to declare.

Abstract 1960

consolidation of complete remission (CR) with high-dose chemotherapy and autologous (auto) stem cell transplantation (SCT) can be an alternative option for AML and MDS patients (pts) when allogeneic SCT is not feasible. Elderly pts. are not usually offered a SCT for the high risk of letal toxicities; moreover, their long term survival is less then 10–15% also when a CR after induction chemotherapy has been obtained. New treatment strategies are warranted to improve the outcome of these poor prognosis pts. Treosulfan can induce a myelotoxicity comparable to busulfan, without CNS and mucosal toxicities, known to occur with busulfan. The fludarabine-cytarabine (FLA) association is effective as induction treatment of acute myeloid leukaemia (AML) and myelodisplastic syndrome (MDS). We have combined high-dose treosulfan with FLA in a new conditioning regimen prior to autoSCT for AML and MDS elderly pts.evaluation of preliminary data on toxicity and efficacy of a treosulfan-based conditioning regimen prior to autoSCT in MDS and AML pts.period 7/2006 to 2/2010, 10 pts, median age 68 (60-76). Diagnosis and risk factors: 5 AML with MDS-related changes (1 complex and 4 normal karytotype), 1 AML with minimal differentiation, 1 AMML with hyperleucocytosis (>100.000/microl), 1 AMML with normal karyotype and 1 MDS (RAEB2) with del5q. Chemo cycles before autoSCT: median 2 (range 2–3), at least 1 containing HDara-C (8 gr/sqm total dose). All pts were in CR at autoSCT, 57 days (median, range 44–132) after first CR documentation. Conditioning regimen (FLAT): treosulfan 10 gr/sqm for 3 days, fludarabine 30 mg/sqm for 5 days, cytarabine 2 gr/sqm for 5 days, PEG filgrastim 1 s.c. vial after autoSCT (8 pts). Transplant: PBSC, median 6.2×10e6 CD34+/kg BW (range 3.8–9).the overall median toxicity grade (CTC) was 1 (0-3), with hepatic (3 cases) and cardiac (2 cases of reversible atrial fibrillation) involvement. No deaths due to treatment occurred. Grade 4 neutropenia and thrombocytopenia median duration was 12 (range 9–37) and 27 (range 11–71) days, respectively. Grade 4 neutropenia in pts who received PEG filgrastim lasted 10.5 days (range 9–25). Four out of 10 pts did not experience either fever or sepsis. After autoSCT, 5 pts relapsed (4 haematologic, 1 only cytogenetic) and 4 of them died. Six pts are currently alive, 5 in first CR and 1 in second CR (after allogeneic SCT for cytogenetic relapse) 1025 days (median, range 60–1448) after autoSCT. Overall, EFS from autoSCT was 368 days (median, range 60–1448) with a 45% probability of survival at 3 years.the FLAT regimen is myeloablative, as demonstrated by the prolonged cytopenia in all pts. It proved to be feasibile anyway, remarkably in these elderly pts. Furthermore, prolonged survival free from disease was achieved. This new regimen is promising for AML/MDS pts as it could be an effective option to intensify the consolidation of their initial CR, with limited extra-hematologic toxicites. A phase II study in pts older than 65 is ongoing at our Center to confirm these data.No relevant conflicts of interest to declare.

Graft Failure (GF) occurs in 5–27% of patients (pts) after allogeneic hamatopoietic stem cell transplant (HSCT) and is associated with high morbidity and mortality related to infections and hemorrhagic events. Graft function may be poor as result of graft rejection, primary disease relapse or Poor Graft Function (PGF). The incomplete recovery of blood counts is defined primary PGF and the decreasing blood counts after successful engraftment secondary PGF. Several factors may determine GF: disease risk and status, conditioning regimen, HSC source, HLA compatibility, T cell content, immunosuppression, GvHD, viral infections, drugs. GCSF and Rhu-EPO are readily available and effective in PGF but with no effects on platelets. Second transplantation from the same donor, with or without conditioning therapy, can boost the haematopoietic recovery in pts with GF. Unfortunately, both a second peripheral CD34+ mononuclear cells (MNC) mobilization and a marrow harvest in the operating room may be contraindicated early after the first donation as not safe for donors. Intrabone SCT can overcome the risk of graft failure even with a low number of CD34+ MNC, as it has been demonstrated in cord blood transplant. Here we investigate in three adult pts with GF a bone-to-bone boost (BBB) with a small marrow harvest from respective donors, unfit for a second conventional donation.to evaluate the feasibility of the BBB technique in 3 pts with graft failure.pts were 2 males (57, 53 y) with PGF with a diagnosis of AML and CMML, respectively, and a female (44 y) with graft rejection and AML relapse. In the first two patients prolonged pancytopenia and hypoplastic marrow were documented, with diagnosis of primary PGF and secondary PGF, respectively, donor chimerism ranging from 80–100% (STR and HLA), without evidence of leukemia. In the third patient, after prolonged pancytopenia an AML relapse was documented with 89% blasts on bone marrow aspirate. In PGF patients no conditioning regimen was administered before the boost at day 30 and 72 after SCT, respectively. In the patient with AML relapse Melphalan 200 mg/mq was given 48 h before the infusion, at day 35 after SCT. The 3 donors were related, haploidentical. For the BBB procedure small quantities of bone marrow (< 200ml) were collected from the posterior iliac crest bilaterally of the donors, at the bedside, during deep sedation and analgesya. Shortly after the unmanipulated marrow harvested was infused in superior-posterior iliac crest mono- or bilaterally, depending on the volume, during deep sedation and analgesya. In pt 1 Mononucleated cell (MNC) dose was 0.9 × 10^8/Kg for a volume of 166 ml. In pt 2 MNC dose was 0.4 × 10^8/Kg for a volume of 88 ml. In pt 3 MNC dose was 0.3 × 10^8/Kg for a volume of 140 ml.In this cases the BBB technique proved feasible and safe for both the donor and the patient. Patient 1 received a second PBSC boost, without conditioning, 3 months after the BBB, and he's now alive, in CR, 13 months after the first transplant. Patient 2 died 3 months after the first transplant for pneumonia and sepsis. Patient 3 is alive, in CR, 4 months after the first HSCT. This practice can give the chance of HSC boost to patients with GF without the need of a GCSF mobilization for donors, with a minimal invasive operation. This can give the option to overcome and resolve infectious and hemorrhagic complications, bridging patients to further therapies and procedures. The intra-bone SCT may be a facilitating tool for microenvironment reconstitution, seeding and subsequent differentiation and may as well have a tolerogenic effect, through the mesenchymal stromal cells infused with the harvest. Further studies are necessary to assess the efficacy of this procedure.No relevant conflicts of interest to declare.

Allogeneic haemopoietic stem cell transplantation (HSCT) is the best therapeutic option for high-risk hematological disease (HRHD), particularly acute myeloid and lymphoblastic leukemia (AML/ALL). A widely application of HSCT is limited by lack of availability of a suitable donor for every candidate patients (pts). In order to offer a donor to every candidate pts, several centers had developed in the last decade alternative strategy of HSCT, such as umbilical cord blood (UCB) or family haploidentical HSCT (haplo-HSCT). With the aim to treat high-risk leukemia in the ideal appropriate time by allogeneic HSCT, we adopted a policy relying upon HLA typing at entry of every pts diagnosed with HRHD, followed, in absence of a MRD, by the prompt activation of the MUD search. Patients lacking a MRD or a MUD at the appropriate timing are proposed for haplo-HSCT.Here we report an intention-to-treat analysis of alternative donor HSCT at our Institution for pts diagnosed with high risk AML or ALL. Data were obtained from local institutional database.Between Jan-2004 and July-2010 241 pts (median age 48y, r 15–72) with diagnosis of ALL (60 pts; median age 33y, r 15–64; male 39) or AML (181 pts, median age 51y, r 19–72; male 83) were defined as “high risk status” and received an indication to HSCT according to EBMT (European Group for Blood and Marrow Transplantation) and Northern Italy Leukemia Group (NILG, www.nilg.it) recommendations.In the ALL group 3 pts died before proceed to HSCT, 5 pts are waiting for the identification of a donor. Overall, 52/60 (86%) pts underwent HSCT, the status of disease at transplant was of complete remission (CR) for 27 pts, persistence of disease (PD) for 25.In the AML group 6 pts died before proceed to HSCT, 15 pts are waiting for the identification of a donor. Overall, 160/181 (88%) pts underwent HSCT, the status of disease at transplant was of CR for 97 pts, PD 63.Overall 92 pts activated the research of a MUD, 42 proceed to transplant, 7 received a UCB, 26 received a haplo-HSCT due to absence of a suitable donor in the appropriate time frame or failure to met the criteria to engage a MUD donor. The median time from diagnosis to registry activation was 69 days (r 5–876), the median time from activation to transplant 84 days (r 28–348).In the group of pts in CR at transplant, 37 underwent HSCT from a MRD and 36 from a MUD, 4 pts received a UCB and 47 a haplo-HSCT. Seventy-two pts are alive and in CR at last follow-up, 3/72 after a second transplant from a different donor (haplo-HSCT) and 3/72 after chemotherapy and adoptive immunotherapy to treat hematological relapse. Fifty-two pts died and the causes of death were: relapse (27), infection-related (19), graft-versus-host-disease (GvHD; 5), acute myocardial infarction (1).In the group of pts in PD at transplant, 16 underwent HSCT from a MRD and 6 from a MUD, 3 pts received UCB and 63 a haplo-HSCT. Fifteen pts are alive, 14 in CR, 1 pts under adoptive immunotherapy; 1/14 pts received a second transplant from a different donor (haplo-HSCT) to treat hematological relapse. Seventy-three pts died and the causes of death were: relapse (43), infection (23), GvHD (7).Overall, the median survival is 382 days and the median follow-up for pts alive is 548 days. The 1-year overall survival (OS)/transplant related mortality (TRM)/relapse incidence (RI) are 50,76% 26,96% and 40% respectively.For pts transplanted in CR the 1y OS/TRM/RI are 77,2%, 20,01% and 25% respectively. The outcome analysis per donor source (MRD vs MUD vs haplo-HSCT) is comparable (p=ns).For pts transplanted in PD the 1y OS/TRM/RI are 19,7%, 41% and 67% respectively. The outcome analysis per donor source (MRD vs MUD vs haplo-HSCT) shows a trend of lower RI and TRM in the haplo-SCT vs MRD.The policy adopted provided an allogeneic HSCT in > 80% of candidate high-risk acute leukemia patients. No significant differences were registered in outcome for patients transplanted from matched-related, unrelated or family haploidentical donors. Further evaluation and a long-term follow-up will add important evaluation in term of long-term disease control and long-term toxicities.Bonini: MolMed: Consultancy. Bordignon: Molmed: Employment.

Allogeneic transplantation of haemopoietic stem cells (allo-SCT) from an HLA- matched related (MRD) or unrelated donor (MUD) is a curative option for patients (pts) with high-risk haematological disease (HRHD). In the absence of a MRD or MUD, pts have been offered investigational transplant strategies such as umbilical cord blood (UCB) or family haploidentical SCT (haplo-SCT). In our Institution, all patients with HRHD are typed at entry; if a suitable MRD donor is missing a MUD search is promptly activated through the Bone Marrow Donors Worldwide Registry (BMDW). Our policy is to offer an haplo-SCT to adult pts lacking an MRD or MUD in order to adequately treat HRHD in the ideal appropriate time according to clinical indications for allo-SCT. This policy is based on our ongoing protocols for primary disease.Here we report the intention-to-treat (ITT) analysis of alternative donor transplantation at our Institution. Data were obtained from Institutional database; all HRHD patients were included in analysis.Between Jan-2004 and July-2010, 410 pts (100% of the following ITT analysis; median age 48y, range 15–76, 91 pts over-60y; male 264) received indication to allo-SCT according to EBMT recommendations. The majority of pts presented with a diagnosis of acute leukaemia (15% lymphoblastic, 45% myeloid), while 12% presented with non Hodgkin lymphoma, 8% myelodysplastic syndrome, 6% Hodgkin disease, 5% multiple myeloma, 2% chronic myeloid leukaemia, 2% chronic lymphocytic leukaemia, 2% severe aplastic anaemia, 1% primary myelofibrosis, 2% others.Eighty-nine pts (22%) received a transplant from a MRD; 190 pts (46%) activated an MUD search in the BMDW registry; 84 pts (20% of total pts, 44% of MUD searching) received a MUD transplant; 42 pts (10%-21%) received an haplo-SCT due to lacking of a suitable MUD in the appropriate timing according to disease status, or absence of suitable criteria to engage a MUD donor; 11 pts (3%-6%) received a UCB if lacking a suitable haplo donor. Overall, 149 pts received an haplo-SCT (36%): 42 after BMDW research, 107 up-front. Twenty-one pts died before receiving a transplant (5%), 37 (9%) are still searching for a suitable donor and an eligible donor is under final evaluation for 19 pts (5%).One-hundred-fifty pts underwent transplant in complete remission (CR), 12 in early phase, 9 in very-good-partial remission (VGPR), 7 in partial response (PR), while 155 were in progression of disease (PD). The median time from diagnosis to transplant was 165 days, the median time from MUD search to transplant 103 days (range 28–824 days).The overall survival (OS) analysis in intention to treat is 51% at 1 year, 39% at 3y; the 1y and the 3y OS for pts transplanted in CR/early phase/VGPR are 74% and 57% respectively, for the pts transplanted in PR/PD 29% and 21% (p<0.0001). Interestingly, the OS according to donor source (MRD vs MUD vs haplo-SCT) is comparable (p = ns) in pts transplanted in CR/early phase/VGPR.At last evaluation 149 pts are alive, with a median follow-up of 802,5 days (range 2–2315); 93/149 received transplant in CR, 10 in early phase, 6 in VGPR, 3 in PR, 37 in PD. Twenty out of 149 experienced disease relapse and were rescue with salvage treatment and adoptive immunotherapy in 15 cases, while 5 pts received a second transplant from a different donor. Up to 155 pts transplanted in PD, 37 (24%) are alive with a median follow-up of 499 days, of these 22 pts received a haplo-SCT, 8 MUD, 6 MRD, 1 UCB. The median follow-up for pts dead is of 118 days (range 2–2315), the cause of death were disease relapse (53,26%), graft-versus-host-disease (8,7%), infection (31,5%), multi organ failure (3,8%), cardiovascular disease (1,09%), haemorrhage (1,63%).In ITT analysis, 81% of overall pts candidate received an allo-SCT (59% from an alternative donor, 22% from a MRD) and the outcome achieved for pts transplanted in CR is comparable within the three major donor source (MRD – MUD – haplo-SCT).This result confirm the need to offer to every candidate pts a transplant option in the appropriate time, but also support the use of alternative donors, particularly haplo-SCT, in absence of an available MRD or MUD. The highly committed policy performed in the alternative-SCT setting and the implementation of an alternative donor option are essential prerequisite to obtain these results, offering to every candidate pts a concrete opportunity of treatment.Bordignon: Molmed: Employment.

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Abstract 2385

Abstract 4596

Abstract 4438

when allogeneic transplantation is not feasible, ASCT is an alternative option as post-remission therapy for patients (pts) with AML, as it can prolong their disease-free survival (DFS). Relapse is the main cause of AML treatment failure after initial complete response; AML relapse after ASCT is partly due to contamination with leukemic blasts of the PBSC products, although neither morphological nor genetic evidence of disease are detected in the bone marrow before leukapheresis. Thus, identification and quantification of a reliable minimal residual disease (MRD) marker in the collected PBSC could be relevant in determining the relapse risk after ASCT. The WT1 gene is overexpressed in leukemic blasts of most AML cases; several studies have shown that quantification with RT-PCR of WT1 in the bone marrow and peripheral blood of AML pts in complete remission has prognostic value. We have determined the WT1 transcript levels in autologous PBSC of AML pts autografted for complete remission (CR) consolidation; preliminary results and data interpretation are here presented.to evaluate quantitative WT1 transcript levels in autologous PBSC collections and to compare results with outcome in AML patients who received an ASCT as consolidation of CR, at our Institute.9 pts, period 06/2006-03/2009, median age 68 years (range 41-76). At diagnosis cytogenetic prognostic risk was intermediate for all pts. All pts were in morphological and genetic CR at the time of PBSC collection and before ASCT. Eight patients were in first and 1 in second CR. PBSC collection by leukapheresis (COBE Spectra cell separator): median count of CD34+ 9.75 ×106/kg (3.79-32). Conditioning regimen: Treosulfan 30 mg/sqm, Fludarabine 150 mg/sqm and Cytarabine 5 g/sqm. Median count of CD34+ cells infused: 5×106/kg (range 3.3-8.5×106/kg). RT-PCR quantification of WT1 transcript was performed using TaqMan technology starting from 1 μg of RNA extracted from mononucleated cells of fresh (4) or cryopreserved (5) PBSC samples. The housekeeping gene ABL was used as the control gene for these quantifications with WT1 level being normalised to 104 copies of ABL per sample. We used the Mann-Whitney-U-test to determine if median WT1 levels in the PBSC products of relapsed and not-relapsed pts was statistically different. Than we used the log-rank test to compare RFS and median WT1 value in the PBSC products.at last follow-up 4 pts relapsed and 5 were still in CR. The WT1 levels in the autologous PBSC of the 4 relapsed pts were 89.96, 193.87, 779.43 and 839.63, of the 5 CR pts were 8.40, 16.96, 36.45, 74.89 and 82.49. Overall median WT1 in the PBSC products was 82,49 copies. The median WT1 levels in the PBSC products of relapsed and not-relapsed pts were 486.65 (89.96–839.63) and 36.35 (8.40–82.49) copies, respectively; this difference was statistically significant (p<0.05). Overall, median relapse free survival (RFS) from ASCT was 534 (93-1096) days. Median RFS was 360 days for pts with a WT1 level > 82,49 copies (n=4), and has not been reached for pts with a WT1 level ' 82,49 (n=5) (p=ns).these results suggest that RT-PCR quantification of the WT1 transcript in autologous PBSC could predict AML relapse in pts who receive ASCT in CR. Higher WT1 levels should reflect higher PBSC product contamination with leukemic blasts, indicating an increased risk of relapse after ASCT. These last pts could probably benefit of other strategies than ASCT. We conclude that, if our preliminary data will be confirmed in a larger number of pts, RT-PCR quantification of WT1 transcripts should be used for MRD detection and quantification in autologous PBSC, before proceeding to ASCT; according to our preliminary data the cut-off level could be about 80 WT1 copies to discriminate which pts should receive the ASCT and which should not.No relevant conflicts of interest to declare.

Definition of leukemia remission requires the lack of disease markers at sub-microscopic level. This is highly important after a potentially curative approach as allogeneic hematopoietic stem cell transplant (HSCT), where early detection of relapse at molecular level may lead to modulation of immunosuppressive therapy or donor lymphocyte infusion (DLI). In AML various approaches has been used to define MRD, but still the majority of AML cases do not have a useful and sensitive MRD marker. Over-expression of Wilms' tumor gene 1 (WT1) in leukemic blasts has been reported in >80% of AML and >40% of MDS. Physiologic hematopoietic stem cell compartments also express WT1, however, a ‘malignant’ WT1 expression can be clearly distinguished based on quantitative detection methods such as semiquantitative RT-PCR. Quantification of WT1 expression levels can detect frequencies of leukemic cells in bone marrow (BM) and peripheral blood (PB) as low as 10-3 and 10-5, respectively. Therefore WT1 expression levels provide a new marker for leukemic blast cells regardless of the type of leukemia especially in the relevant percentage of patients that lacks a specific molecular marker of their disease and thus may be a useful marker MRD and may predict the relapse after allogeneic HSCT.We measured quantitative expression of WT1 at diagnosis, before and after allogeneic transplant monthly for the first six months and then every three months. The quantitative assessment of the WT1 transcript amount was performed by real-time quantitative polymerase chain reaction (RQ-PCR).Our study included 19 AML and 6 MDS pts who underwent allogeneic HSCT in our Institute between 12/2007 and 6/2009. Median age at diagnosis was 49 years (range 22-68). Bone marrow samples at diagnosis showed a WT1 median expression level of 5851.66 copies (range 77.98-31203.57). Fifteen pts (60%) had a specific cytogenetic marker that could allow MRD monitoring. At HSCT 17 pts (68%) were in CR, 5 (20%) had a refractory or relapsed disease, while 3 (12%) were transplanted upfront. Six pts (24%) received grafts from a matched sibling donors, 6 (24%) were transplanted from a matched unrelated donor (MUD), 10 (40%) from a familiar haploidentical donor and 3 (12%) received a cord blood unit. Myeloablative conditioning regimen consisted of Treosulfan 42 g/sqm, Fludarabine 150 mg/sqm, ALG 30 mg/kg and Rituximab 500 mg (last two drugs only for alternative donors). After HSCT a rapid decline of WT1 expression levels was observed in all pts that obtained or maintained a condition of CR. Two pts (8%) relapsed and both had an increase in WT1 expression before relapse. In the first relapsed pt, analysis of WT1 showed a dramatically increase between pre transplant level and day +28, while STR showed 100% donor chimerism. Relapse occurred on day +43, still on IST, with 69% of blast at AM evaluation and 40% donor chimerism. This pt was successfully reinduced with chemotherapy followed by allogeneic PBSC infusion without GvHD profilaxys obtaining a rapid reduction of WT1 (43.66). The pt developed GvHD that required a new IST, confirming a strong immune-surveillance of HSCT. The second pt was still on immunosuppressive treatment (IST) at the time of relapse (+136). WT1 in BM on day +30 was 30 cp and then increased gradually to 167 and 190 cp on day +67 and +102, respectively. Cytogenetic analysis and STR chimerism still showed a cCR with 100% donor chimerism. At relapse AM showed 10% blasts, WT1 expression level was 7447 cp with >95% donor chimerism. IST was discontinued and one month later WT1 expression level decreased to 1640 with >95% donor chimerism. In this situation we reasoned that DLI was the best available treatment. The total dose of donor T cell infused was 5 ×105 CD3/Kg. This procedure allowed an immune-mediated leukemia control with reduction of WT1 (1.58), cCR and 100% donor chimerism.These data show that WT1 may be useful as a non-specific leukemia marker for monitoring MRD in AML and MDS after allogeneic HSCT and should enable the detection of early relapse allowing intervention at a more favourable stage than at overt relapse. We observed a complete concordance between WT1 expression levels and status of AML before and after allogeneic HSCT. Based on these results cases with increase of WT1 levels after HSCT and without GVHD may be candidate to immune intervention such as discontinuation of immunosuppression and/or DLI.Bonini: MolMed S.p.A.: Consultancy.

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).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).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).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<0,05 vs donor controls). After a median follow-up of 6 months, overall survival is 64% (95% CI: 50-78), and projected OS at 1 year is 46% (95% CI: 31-61).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.No relevant conflicts of interest to declare.

the majority of AML and poor risk MDS cases in the elderly (>60 years) are resistant to induction chemotherapy or relapse after initial response; thereafter, most of these patients (pts) receive only supportive treatments, with an expected survival of few weeks. Allogeneic (allo) stem cell transplantation (SCT) could be the only potentially salvage strategy; actually, for the high probability of letal toxicites due to the conditioning regimen, acute graft-versus-host disease (aGvHD) and infections, these pts are rarely offered this procedure.Since 2005, we have transplanted 12 pts, age>60, with refractory or relapsed AML/MDS, from an allogeneic donor. Data on feasibility and outcome are here reported.to retrospectively evaluate data on alloSCT in elderly pts with refractory or relapsed AML and MDS, transplanted at our Institute.period 9/2005 to 4/2009, 12 pts, median age 65 (61-72). Performance status (Karnofsky): median 90% (80-100), HCT-CI: median score 1 (0-4). Diagnosis (WHO): AML 3, AMLMD 4, RAEB2 2, MDS/MPD 2, therapy-related AML 1. Median number of chemo cycles before SCT: 2 (1-7), all pts received at least 1 cycle with intermediate or high-dose cytarabine, 2 pts also received an autologous SCT. Donors: matched sibling 2, matched unrelated 2, related haploidentical 7, cord blood 1. Disease status at SCT: primary refractory 7, relapsed 5. Conditioning regimen: treosulfan (TREO) 14 g/sqm for 3 days, fludarabine (FLU) 30 mg/sqm for 5 days, ATG 10 mg/kg for 3 days for SCT from alternative donors. GvHD prophylaxis: T-cell depletion in 2 cases, cyclosporine (CSA)/methotrexate in 7, rapamycin/mycophenolate mofetil (MMF) in 3.11/12 (92%) pts engrafted and were in CR at day +30, with 95-100% donor chimerism (VNTR). Deaths within day 30 and day 100 were 0 and 3 (25%), respectively. Acute GvHD: 6 pts (55%), grade II (3) or III (3), only of skin in 3 cases, skin+liver in 1, only intestinal in 2. Relapses were 4 (36%). Overall TRM was 25% (3 pts), with all deaths due to infections during immunosuppression for aGVHD; 5 more pts died because of disease progression. Median OS from SCT of all 12 pts was 180 (56-1150) days, DFS (11 pts) was 128 (24-1114) days. At last follow-up (FU) 4 pts (33%) are alive, all in CR with a median FU of 406 (128-1150) days.alloSCT proved to be feasible in our elderly pts with AML/MDS refractory to induction or relapsed after initial complete remission. Early letal toxicities, due to the conditioning treatment, were absent; aGVHD, infections and relapses were the principal causes of mortality. Up to now, prolonged survival (>1 year) free from disease has been obtained in 25% of pts; of the 4 pts alive in CR, only 1 is receiving immunosuppression (CSA), for chronic GVHD. In conclusion, the TREO-FLU association showed a reduced-toxicity profile in these frail pts and a substantial anti-leukemic effect. Better prevention of aGVHD should be obtained, expecially after SCT from alternative donors, possibly with the rapamycin/MMF combination, which we are currently investigating at our Institute. AlloSCT after TREO-FLU conditioning can be considered an effective option for AML/MDS elderly pts with active disease after failure of previous intensive treatments.bonini: MolMed S.p.A.: Consultancy.

Abstract 4488

Abstract 4690

Abstract 666

Abstract 4318