Several studies in mouse model of invasive aspergillosis (IA) and in healthy donors have shown that different Aspergillus antigens may stimulate different adaptive immune responses. However, the occurrence of Aspergillus-specific T cells have not yet been reported in patients with the disease. In patients with IA, we have investigated during the infection: a) whether and how specific T-cell responses to different Aspergillus antigens occur and develop; b) which antigens elicit the highest frequencies of protective immune responses and, c) whether such protective T cells could be expanded ex-vivo. Forty hematologic patients have been studied, including 22 patients with IA and 18 controls. Specific T cells producing IL-10, IFN-γ, IL-4 and IL-17A have been characterized through enzyme linked immunospot and cytokine secretion assays on 88 peripheral blood (PB) samples, by using the following recombinant antigens: GEL1p, CRF1p, PEP1p, SOD1p, α1–3glucan, β1–3glucan, galactomannan. Specific T cells were expanded through short term culture. Aspergillus-specific T cells producing non-protective interleukin-10 (IL-10) and protective interferon-gamma (IFN-γ) have been detected to all the antigens only in IA patients. Lower numbers of specific T cells producing IL-4 and IL-17A have also been shown. Protective T cells targeted predominantly Aspergillus cell wall antigens, tended to increase during the IA course and to be associated with a better clinical outcome. Aspergillus-specific T cells could be successfully generated from the PB of 8 out of 8 patients with IA and included cytotoxic subsets able to lyse Aspergillus hyphae. Aspergillus specific T-cell responses contribute to the clearance of the pathogen in immunosuppressed patients with IA and Aspergillus cell wall antigens are those mainly targeted by protective immune responses. Cytotoxic specific T cells can be expanded from immunosuppressed patients even during the infection by using the above mentioned antigens. These findings may be exploited for immunotherapeutic purposes in patients with IA.

3483 Defibrotide (DF) has been shown to be effective in the treatment and prophylaxis of the veno-occlusive disease (VOD) of the liver, a relatively common complication after allogeneic stem cell transplant (SCT). We report our experience in 46 consecutive adult patients (pts). The pts (mean age 41 yrs, range 16–66) were affected by leukemia (22), lymphoma (15), multiple myeloma (4), myelodisplasia (2), solid tumor (2), Fanconi anemia (1). Risk factors for VOD were: resistant disease 9, previous transplant 8, B virus hepatitis 6, liver metastases 1, lymphoma of the liver 1, increased transaminases before conditioning 7. All were transplanted from their HLA-identical siblings; the conditioning regimen was reduced in 17, while it was conventional in 29. All the pts received a combination of drugs as conditioning regimen, 23 of them received Busulfan and 8 of them oral Busulfan. 15 received bone marrow and 31 peripheral blood as source of stem cells. None was T-cell depleted; the GVHD prophylaxis regimen was: CsA alone or combined with MTX 45, FK506+MTX 1. For VOD prophylaxis no heparin was administered, while DF was given at the dose of 10 mg/Kg in continuous iv infusion starting on day +1 until day +21 after the SCT. DF was very well tolerated and no hemorrhagic complications were seen. Blood coagulation significant alterations were: prolonged PT (4/46), prolonged aPTT (3/46), elevated fibrinogen (24/46), (never over 800 mg/dl), low level (less than 50%) of ATIII and/or protein C and or protein S (2/46). By using the VOD Baltimore criteria, only 1 case of VOD was observed on day +29 in a patient who died at day +36 with VOD, aspergillosis and CMV pneumonia. The bilirubin was more than 2 times the normal value in 26/46; US-scan of the liver and Doppler, performed in 16 pts with a possible sign of VOD, was positive only in the patient who died for VOD. We documented 32 infectious complications (16 FUO, 9 gram positive bacteremias, 3 pneumonia and 4 invasive aspergillosis). We documented acute GVHD in 14 pts (12 grade I-II and 2 grade III). Five pts died between +36 and +100 but none for VOD (3 for progression of their disease and 2 for aspergillosis). Our laboratory data demonstrated modest alterations of the coagulative parameters, low consumption of coagulative factors and reduced fibrinolysis. Since in this at risk transplanted population only 1 VOD has been observed, DF low-dose continuous infusion, without heparin, seems able to play a relevant role in the VOD prophylaxis. On considering the favourable results by us and others, the absence of toxicity and the low cost of the drug, we intend to continue this experience with DF as VOD prophylaxis, even if we foretell a large randomized study to find better indications. Disclosures: No relevant conflicts of interest to declare.

Introduction. Genomic loss of an HLA haplotype encoding incompatible alleles ("HLA loss") has been described in previous single-center studies as a mechanism by which leukemic cells evade the graft-versus-leukemia effect mediated by alloreactive donor T cells and outgrow into a clinically evident relapse. HLA loss accounts for up to 30% of relapses after HLA-haploidentical transplants (Crucitti, Leukemia 2015), but the actual frequency and clinical relevance of this phenomenon in unrelated donor HSCTs, including cord blood transplants, are largely unknown. Here we present the first global collaborative study to investigate the incidence of HLA loss across different transplant settings. Methods. Twenty transplant centers from Europe (n=16), North America (n=3) and Asia (n=1) joined to form the HLALOSS consortium. To date, we collected a total of 619 cases of hematologic relapse from adult patients with acute myeloid leukemia (78.5%), acute lymphoblastic leukemia (13.9%), myelodysplastic syndromes (4%) or myeloproliferative neoplasms (1.1%) after allogeneic HSCT from HLA-haploidentical relatives (31.7%), HLA-mismatched unrelated donors (MMUD, 21.3%), 10/10-matched unrelated donors (MUD, 37.2%), or unrelated cord blood units (UCB, 9.8%). Where available, the donor and patient germlines and the patient pre-transplant disease were collected in parallel. Until today, 476 cases were analyzed using conventional HLA typing of sorted leukemic blasts, the recently developed HLA-KMR assay (Ahci and Toffalori, Blood, 2017) or a novel Next-Generation Sequencing (NGS) method. The latter was developed adapting the HLA typing strategy in use at the DKMS (Lange, BMC Genomics 2013) to the study of chimeric samples, and allowing to cover all possible HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles and to analyze at least 48 different cases in a single run. Results. Out of the 476 relapses analyzed to date, 396 (83.2%) were informative for the study of HLA loss. Of these, 155 occurred after haploidentical HSCT, 101 after MMUD HSCT, 93 after 10/10-matched, HLA-DPB1 mismatched MUD, and 47 after UCB HSCTs. Three-hundred-two (76.2%) of cases were analyzed using the NGS platform. This method resulted particularly robust, reliable and sensitive in analyzing large sample series: the mean coverage across the 6 sequenced loci was over 8500x, up to 0.5% of the HLA allele of interest could be detected in artificial chimerism curves, and relapse samples tested in parallel via the sequencing platform and HLA-KMR (n=10) showed remarkable concordance between the two methods (R2=0.86, p<0.0001). In total, we detected 51 HLA loss post-transplantation relapses out of the 396 cases analyzed (12.8%). Of these, 35 occurred after haploidentical HSCT (22.6% of relapses in this setting), 12 after MMUD HSCT (11.9%), 4 after 10/10 MUD HSCT (4.3%) and, notably, none after UCB HSCT. Conclusions. The present data, obtained from the largest collaborative study on the immunobiology of relapse to date, confirm the clinical relevance of HLA loss as a major mechanism of immune evasion and post-transplantation relapse after allogeneic HSCT, with an incidence which is proportional to the number of donor-recipient HLA mismatches. The only exception is represented by UCB HSCT which, despite being often performed across multiple major HLA incompatibilities, does not appear to be associated with this relapse modality. This finding might reflect the fact that in UCB HSCT, multiple HLA mismatches are often not encoded in cis on the same chromosome, thereby reducing the selective advantage for leukemic cells that undergo an HLA haplotype loss. This phenomenon might in turn contribute to the lower incidence of relapse reported for UCB HSCT compared to other stem cell sources. Disclosures Vago: Moderna TX: Research Funding; GENDX: Research Funding. Stoelzel:Neovii: Speakers Bureau. Gojo:Novartis: Membership on an entity's Board of Directors or advisory committees; Merck inc: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. Busca:Novartis: Speakers Bureau; Jazz Pharmaceuticals: Honoraria; Pfizer Pharmaceuticals: Honoraria, Speakers Bureau; Merk: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Luznik:WIndMIL Therapeutics: Equity Ownership, Patents & Royalties. Kobbe:Amgen: Honoraria, Research Funding; Celgene: Honoraria, Other: Travel Support, Research Funding; Roche: Honoraria, Research Funding. Kroeger:Novartis: Honoraria, Research Funding; Sanofi: Honoraria; Riemser: Honoraria, Research Funding; Neovii: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; JAZZ: Honoraria. Finke:Neovii: Consultancy, Honoraria, Other: travel grants, Research Funding; Medac: Consultancy, Honoraria, Other: travel grants, Research Funding; Riemser: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Other: travel grants, Research Funding. Mohty:Takeda: Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria; Servier: Consultancy; MaaT Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; Molmed: Consultancy; Jazz Pharmaceuticals: Honoraria, Research Funding, Speakers Bureau; Bristol Myers: Consultancy, Research Funding; Janssen: Honoraria, Research Funding, Speakers Bureau. Beelen:Medac: Consultancy, Other: Travel Support. Fleischhauer:GENDX: Research Funding.

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BCR-ABL1 kinase domain mutation testing in tyrosine kinase inhibitor (TKI)-resistant Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukaemia (ALL) patients is routinely performed by Sanger sequencing (SS). Recently, next-generation sequencing (NGS)-based approaches have been developed that afford greater sensitivity and straightforward discrimination between compound and polyclonal mutations. We performed a study to compare the results of SS and NGS in a consecutive cohort of 171 Ph+ ALL patients. At diagnosis, 0/44 and 3/44 patients were positive for mutations by SS and NGS respectively. Out of 47 patients with haematologic resistance, 45 had mutations according to both methods, but in 25 patients NGS revealed additional mutations undetectable by SS. Out of 80 patients in complete haematologic response but with BCR-ABL1 ≥0Á1%, 28 (35%) and 52 (65%) were positive by SS and NGS respectively. Moreover, in 12 patients positive by SS, NGS detected additional mutations. NGS resolved clonal complexity in 34 patients with multiple mutations at the same or different codons and identified 35 compound mutations. Our study demonstrates that, in Ph+ ALL on TKI therapy, NGS enables more accurate assessment of mutation status both in patients who fail therapy and in patients with minimal residual disease above 0Á1%.