ALBERT

Description: Despite recent advancements, approximately 50% of patients with acute myeloid leukemia (AML) do not respond to induction therapy (primary induction failure, PIF) or relapse after

Description: BACKGROUND Multiple Myeloma (MM) is an incurable malignancy heralded by an immunosuppressive tumor microenvironment (TME). Therapeutic efforts to reprogram the TME and release innate and adaptive immunity may synergize with current anti-MM drugs, deepen responses and ultimately prolong long-term survival. We have developed an ex vivo gene therapy approach that locally delivers interferon-alpha (IFNa) into the TME through the Tie2+ myeloid progeny of transplanted gene-modified hematopoietic stem and progenitor cells (HSPCs). Preclinical studies in the immunocompetent Vk*Myc murine model as well as in the hematochimeric xenotransplantation setting demonstrate that IFNa gene therapy has anti-MM effect and may subvert the immunosuppressive MM TME, acting both on myeloma cells as well as on the immune infiltrate. Additional toxicology data, demonstrates that a mixture of genetically modified and unmodified CD34+ cells can be transplanted safely. We have therefore progressed into the clinic at Ospedale San Raffaele in Milan, Italy, with our gene therapy approach (Temferon). METHODS The TEM-MM-101 study recruits patients with MM in early relapse (within 18 months) after intensive front line therapy defined as triplet induction (at least 3 cycles including a proteasome inhibitor and/or immunomodulatory drug [IMID]) followed by single or double autologous stem cell transplantation, or, triplet or quadruplet induction including at least a proteasome inhibitor and an IMID, followed by consolidation/maintenance treatment. Three cohorts of 3 patients will be included in the study. Patients will be dosed sequentially in each cohort with data review by an independent data monitoring committee before dose escalation to the next cohort. Key eligibility criteria include achieving at least a VGPR after second line salvage therapy, age 18-70 years, ECOG 70%, adequate cardiac, renal, hepatic and pulmonary function, and willingness to use appropriate contraception if relevant. Important exclusion criteria include the presence of active autoimmune disease or ineligibility for autologous bone marrow transplant. After confirmation of eligibility, patients undergo autologous peripheral blood HSPC mobilization with lenograstim and plerixafor, before undergoing apheresis. After purification, CD34+ cells undergo transduction with a third-generation, vesicular stomatitis virus-G pseudo-typed lentiviral vector driving myeloid-specific IFNa2 expression. Patients will be offered maintenance treatment from successful HSPC collection until Temferon release. A reduced intensity conditioning regimen is administered at Day -2 followed by ASCT and administration of Temferon at Day 0. Following hematological recovery, patients will be discharged from the Transplant Unit with regular outpatient follow up. After Day +100 from Temferon infusion, patients will resume anti-myeloma consolidation/maintenance treatment according to best clinical practice, unless there is Temferon engraftment and absence of disease on Day +100 evaluations, documented by stringent complete response (sCR) according to the IMWG response criteria, imaging (PET, MRI) negativity, and bone marrow minimal residual disease 〈 10-4 by multi-parameter flow cytometry, to be confirmed by next generation sequencing. If these response criteria are met on Day +100, patients may stay off maintenance treatment, as long as MRD≤10-5 documented on bone marrow aspirates performed every 3 months, starting from the Day +180 visit. The primary endpoints for this study are: Engraftment of Temferon between Day +30 and Day +100The proportion of patients achieving hematologic recovery by Day +30 from ASCTSafety of Temferon (short-term tolerability of Temferon; stable blood counts and absence of cytopenias 〉 grade 2, unless related to myeloma progression or concomitant medications; absence of systemic IFNa toxicity; absence of Replication Competent Lentivirus; absence of hematologic malignancy that is distinct from progression of the primary neoplasm) The study is actively recruiting patients in Italy. In parallel, a study using Temferon in patients with glioblastoma multiforme with a similar study design and approach (TEM-GBM-001) is also in progress with the first cohort fully recruited. Disclosures Gentner: Genenta Science: Consultancy, Equity Ownership, Research Funding. Mazzoleni:Genenta Science: Employment, Equity Ownership. Russo:Genenta Science: Consultancy, Equity Ownership. Naldini:Genenta Science: Consultancy, Equity Ownership; Magenta Therapeutics: Equity Ownership; San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was then licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial..

Description: Background Over the last decade, the advent of next-generation sequencing (NGS) has dramatically improved our understanding of the genomic landscape that characterize acute myeloid leukemia at time of diagnosis, leading to the identification of new prognostic subsets and therapeutic targets. Still, few studies have addressed how this landscape changes upon treatment, and in particular after allogeneic hematopoietic cell transplantation (allo-HCT). Moreover, the links between the mutational profile of AML and mechanisms of post-transplantation immune evasion remain to date largely unknown. Methods Taking advantage of the HaloPlex High Sensitivity (HS) technology, which allows a more precise definition of mutations and clonal architecture through the use of unique molecular identifiers, we designed and tested a NGS panel covering genes and miRNAs known to be involved in the pathogenesis of myeloid malignancies (n=112), in the DNA damage response (n=50) or in immune-related processes (n=30). By using an Illumina HiSeq2500n instrument,we sequenced a total of 200 AML samples collected from 131 patients, including 84 diagnoses, 45 relapses after chemotherapy (CT) and 71 relapses after allo-HCT, 31 of which characterized by genomic loss of HLA (Vago et al, N Engl J Med, 2009). Variant calling was performed using a pipeline based on the FreeBayes algorithm. Results Sequencing coverage was uniform across all target amplicons, with a 855x average depth-of-sequencing, and a mean of 122 unique barcodes for each region. Among the 84 diagnosis samples we identified 253 oncogenic mutations, with a median of 3 mutations per patient (range 0-8). Both the frequency and the prognostic value of mutations detected at diagnosis were in line with the largest published dataset on AML genomics (Papaemmanuil et al, N Engl J Med, 2016; R2= 0.88, p

Description: Haploidentical stem cell transplantation (SCT) can be used in relapsed haematological malignancies for patients lacking a matched sibling or unrelated donor. Major barriers of this strategy are the poor immune reconstitution and the high risk of relapse. Here, we report results of a phase I–II trial evaluating early add-backs of CD8-depleted donor lymphocytes (DLIs) (from 1x10^4 up to 1x10^5 cells/kg starting at day+45 up to day +105 at monthly intervals) after a reduced intensity conditioning (RIC) regimen [thiotepa (10 mg/kg), fludarabine (120 mg/sqm), cyclophosphamide (60 mg/kg) and TBI (2 Gy)]. Ex-vivo and in-vivo TCD were carried out by CD34+ cell selection using the CliniMACS device and alemtuzumab (15mg/m2, day-2), respectively. Twenty-one patients [n= 10 NHL (n=5 CLL, n=5 high-grade NHL), n=7 HL, n=1 MM, n=1 ALL, n=2 AML] were transplanted with advanced disease: 16 (76%) failed a previous autograft and 13 (62%) had refractory disease. A median of 10.4x10^6/Kg CD34+, 1x10^4/kg CD3+, 10x10^4/kg CD19+, 0.9 x10^4/kg NK+ were infused. All patients engrafted with full donor chimerism from day +90. At a median follow-up of 12 months (range, 4–41 months), 12 of 21 pts are alive (7 CR, 2 PR and 3 PD) and 9 died [n=3 infection with GVHD (+610, +187, +253), n=6 disease]. The estimated 2-year overall survival was 49%: pts transplanted in remission had better outcome (83% versus 31%, p=0.13). The estimated 2-year cumulative incidence of TRM and relapse were 27% and 58%, respectively. CMV reactivation and hospital readmissions for opportunistic infections occurred in 76% and 57% of patients, respectively. For CD8 depletion of donor leukaphereses, a new depletion protocol using clinical grade CD8 microbeads (Miltenyi) was applied. This procedure is efficient to reduce the content of CD8 T cells by 3 logs while the median cell recovery of CD3+, CD4+, CD56+/CD3+, CD 20+ was 60%, 86%, 54%, 72%, respectively. Before DLIs, only 2 of 21 patients (10%) developed acute GVHD (no grade III–IV). A total of 36 CD8-depleted DLIs were administered to 17 pts without any acute toxicity. Following DLIs, 6 pts (35%) developed acute GVHD (grade II) and 5 (30%) chronic GVHD (n=2 limited, n=3 extensive). Overall, the incidence of acute GVHD is higher (50% vs 22%, p=0.33) in pts receving larger numbers of donor cells (10–15x10^4/kg versus 3–5x10^4/kg CD8-depleted DLIs). The median values of CD4+/uL, CD8+/uL and NK+ were 100, 280 and 680 at 4 months and 220, 200, 500 at 6 months after SCT in patients receiving CD8-depleted DLIs. Measurable TREC/ucg DNA (mean value 316; mean value donors 3740) and polyclonal T cell repertoire, evaluated by spectratyping, were observed at 9 months in patients younger than 40 years and/or without GVHD. Our results suggest that: haploidentical SCT with RIC regimen provides high engraftment rate T-cell addback allows the achievement of more than 100/uL CD4+ at 4 months after SCT in the majority of patients Survival rate is promising in patients who had transplantation in remission suggesting that this strategy should be evaluated earlier in high risk haematological diseases.

Description: RIC followed by allo-SCT is an effective salvage treatment for some relapsed hematologic malignancies due to the postulated graft versus tumour (GVT) effect. In order to evaluate the quality of the clinical response, we have investigated the molecular status of patients receiving allo-SCT for relapsed disease. Forty-four patients (19 chronic lymphocytic leukemias (CLL), 21 follicular lymphomas (FCL) and 4 small lymphocytic lymphomas (SLL)) were enrolled in a prospective phase II study. The median age was 54 years (range: 32–69 years). The median number of previous chemotherapy regimens was 2 (range: 1–5) and 23% of patients had already failed an auto-SCT. Before transplant 34% of patients were chemorefractory and 34% of the chemosensitive patients were in complete remission (CR). The conditioning regimen consisted of thiotepa 10mg/kg, fludarabine 60mg/ms and cyclophosphamide 60mg/kg; short course of methotrexate and cyclosporin were used as GVHD prophylaxis. Minimal residual disease (MRD) was monitored by nested PCR for IgH or Bcl-2 genes; in PCR-positive patients a TaqMan based quantitative monitoring was also employed. All patients engrafted. On day +30 after transplant 39% of patients achieved CR. Acute GVHD (aGVHD) was observed in 57% of patients and 52% of 42 evaluable patients developed chronic GVHD; no difference in the incidence of GVHD between FCL and CLL/SLL was observed. In 30 of 44 patients (68%) a PCR marker for MRD monitoring was found. Twenty-five patients (10 CLL, 2 SLL, 13 FCL) of 37 patients in CR after allo-SCT were monitored by nested PCR and 4 PCR-positive patients were monitored by TaqMan PCR. At a median molecular follow up of 15 months (range: 3–62) 15 of 25 patients (60%) were alive and in molecular remission; one CLL patient died of TRM in molecular remission (MR); five of these patients were chemorefractory. Nine patients (3 FCL, 5 CLL, 1 SLL) never achieved PCR negativity and 3 of them relapsed (2 CLL; 1 SLL) after a median time of 270 days. In one of these patients the TaqMan PCR system could detect a continuous increase of tumour genomes in the marrow prior to the clinical relapse. The SLL patient achieved MR after chemotherapy and DLI, developing limited cGVHD; the other two patients never developed GVHD, even after DLI. Eighty percent of PCR-negative patients developed GVHD and it preceded or was concomitant with the achievement of MR. The better molecular outcome of FCL seems to be due to a longer follow up (19 months vs 12 months) if compared to CLL/SLL, in which a slow clearance of MRD has been observed. In conclusion, MR can be achieved in relapsed and chemorefractory patients affected by indolent lymphoproliferative disorders; quantitative PCR monitoring can be used to modulate post-transplant immunotherapy; a longer follow up is warranted to evaluate if the GVT effect can sustain MR in the long-term.

Description: Background: Flotetuzumab (FLZ; MGD006/S80880) is a novel CD123 x CD3 bispecific DART® protein being tested in a Phase 1/2 study (NCT02152956) in patients with relapsed/refractory acute myeloid leukemia (AML). As with all T-cell redirecting therapies, cytokine secretion, inherent in T-cell activation, with ensuing potential for cytokine release syndrome (CRS), remains an important side effect. We have previously reported that multi-step dosing mitigates CRS severity (1). CRS diagnosis and treatment is guided by the occurrence of non-specific clinical signs, such as fever, chills, hypotension and tachycardia. Therefore, identification of predictors of CRS will be useful for optimal pt. management. Here we report on potential biomarkers of CRS severity that may help guide CRS management. Methods: Data from pts treated with FLZ at RP2D (lead-in dose of 30ng/kg/d for 3d, 100ng/kg/d for 4d for week 1 followed by 500ng/kg/d CIV week 2-4 of cycle 1, and a 4d-on/3d-off schedule for Cycle 2 and beyond in 28-day cycles) was collected and analyzed. Incidence and severity of CRS were analyzed for correlation with cytokine levels and changes in BM blasts. Relation between immune cells (T-cell subsets, monocytes) with tumor burden, percent CD123+ AML blasts, and CD123 expression, were interrogated as potential determinants of CRS. Administration, dose and frequency of tocilizumab (TCZ), an IL-6 receptor antagonist, were evaluated for their relationship with CRS severity, frequency, CRP and cytokine levels. Results: 30 pts were dosed at RP2D. Most pts experienced mild to moderate CRS (G1 26.7%, G2 60%) of short duration (median 1 day(d), range 1-26 d) and were conservatively managed to full resolution. Grade ≥ 3 events occurred in 4/30 pts (13%), with vasopressors use in 2 pts, and a median duration of 2.5 d (range 2-13 d). CRS frequency decreased with time on treatment: 42% occurred within the first week (LID), 39.6% occurring in week 2 (step up to 500ng/kg/day) and 18% occurring during week 3 and 4. IL-6 levels showed the best relationship with CRS severity, as previously shown (1). IL-6 levels, however, did not correlate with response. Twenty pts (67%) received at least one dose of TCZ (median 2 doses/pt; range 1-12 doses). As anticipated, mean IL-6 levels increased after administration of TCZ. CRS severity showed a relationship with the baseline frequency of circulating CD4+ cells (median 47% in G1 vs 73% in G ≥2, p = 0.0082), while CD8+ cell frequency did not correlate with CRS. Disease burden (absolute AML blasts, % CD123 AML blasts), CD123 expression on AML blasts, monocytes levels or effector-to-target ratio in the peripheral blood did not show a relationship with CRS severity. Importantly, CRS severity was not correlated with FLZ anti-leukemic activity. Conclusion: The frequency of CD4+ cells at baseline may be a potential biomarker for identifying pts at risk of more severe CRS. Early use of TCZ can effectively modify the activity of IL-6, a significant contributor to CRS, and blunt CRS severity. Since severity of CRS and IL-6 levels did not correlate with FLZ anti-leukemic activity, blunting its severity should be aggressively pursued. Early identification of pts at greater CRS risk together with multistep dosing (1) and early use of TCZ can effectively manage CRS with no impact on FLZ anti-leukemic activity.Jacobs et al. ASH 2017, abstract #3856 Disclosures Jacobs: MacroGenics: Employment. Viero:Servier: Employment. Baughman:MacroGenics: Employment. Sun:MacroGenics: Employment. Ying:Macrogenics: Employment. Muth:MacroGenics: Employment. Hong:MacroGenics: Employment. Sweet:BMS: Honoraria; Agios: Consultancy; Phizer: Consultancy; Astellas: Consultancy; Jazz: Speakers Bureau; Jazz: Speakers Bureau; BMS: Honoraria; Novartis: Consultancy, Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Agios: Consultancy; Phizer: Consultancy; Celgene: Honoraria, Speakers Bureau; Astellas: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau. Uy:Curis: Consultancy; GlycoMimetics: Consultancy. Ravandi:Xencor: Research Funding; Macrogenix: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Abbvie: Research Funding; Jazz: Honoraria; Amgen: Honoraria, Research Funding, Speakers Bureau; Orsenix: Honoraria; Abbvie: Research Funding; Orsenix: Honoraria; Seattle Genetics: Research Funding; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Sunesis: Honoraria; Sunesis: Honoraria; Xencor: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Amgen: Honoraria, Research Funding, Speakers Bureau; Macrogenix: Honoraria, Research Funding; Jazz: Honoraria. Foster:Celgene: Research Funding; Macrogenics: Research Funding; Pfizer: Research Funding; Shire: Honoraria. Rizzieri:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Arog: Consultancy, Membership on an entity's Board of Directors or advisory committees; Teva: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees. Arellano:Cephalon: Research Funding. Rettig:Amphivena Therapeutics: Research Funding; Novimmune: Research Funding. Topp:F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees, Research Funding; Regeneron Pharmaceuticals, Inc.: Honoraria, Research Funding; Boehringer Ingelheim: Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding. Lelièvre:Servier: Employment. Lowenberg:Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; international Scientific Advisory Board, Institute Gustave Roussy, Paris: Membership on an entity's Board of Directors or advisory committees; "Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherlands: Membership on an entity's Board of Directors or advisory committees; Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands): Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Editorial Board "International Journal of Hematology": Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; Editorial Board "The Netherlands Journal of Medicine": Membership on an entity's Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees. Wigginton:MacroGenics: Employment. Davidson-Moncada:MacroGenics: Employment.

Description: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only cure for several patients with high-risk hematological malignancies. Recent advances in supportive therapies have significantly reduced the treatment-related mortality over the last decades. Nevertheless graft-versus-host disease (GvHD) still represents a major complication, and research is needed to achieve further progress in this area of transplantation. Moreover, although initial studies suggested relative equivalence, recent and large prospective studies showed higher rates of acute and chronic GvHD, and worse survival in allo-HSCT from unrelated donors (URD) compared with HLA-identical sibling donors. Following our encouraging results in haplodentical setting with the more recently post-transplantation cyclophosphamide (PTCy) and sirolimus as GvHD prophylaxis (Cieri et al, 2015), in this study we investigated whether the incorporation of low dose anti-thymocyte globulin (ATG) to PTCy and sirolimus GvHD prophylactic approach, may alleviate some of the increased alloreactivity associated with URD transplants. We retrospectively evaluated a cohort of 21 haematological patients receiving URD allo-HSCT in the San Raffaele Haematology and BMT Unit, from 2013 to 2015. All subjects had high-risk hematologic malignancies, with the most common diagnosis being myeloid diseases (52%). A total of 11 patients had active disease at HSCT, while 9 were in hematologic remission. Nine patients scored high according to disease risk index defined by Armand et al, 10 scored intermediate, while only 2 had a low disease score. Myeloablative conditioning consisted of treosulfan (14 g/m2/day) on days -6 to -4, fludarabine (30 mg/m2/day) on days -6 to -2, and melphalan (70 mg/m2/day) on days -2 and -1, followed by T-replete G-CSF-mobilized PBSCs (n=19) or BM grafts (n=2). Whether 12 patients received stem cells from HLA- matched URD (fully 10/10 HLA matched), the remaining 9 had one-locus HLA-mismatched from their donors. During conditioning patients received ATG-Fresenius 5 mg/kg per day IV on day -4 to -2. In vivo B-cell depletion was obtained by rituximab (i.v. 375mg/m2 given as a single dose on day -1). Postgrafting immunosuppression consisted of PTCy (50 mg/kg/day) on days 3 and 4. Sirolimus was given orally from day 5, and monitored to maintain a target therapeutic plasma level of 5-14 ng/ml. According to chimeric status and evidence of GVHD, the dosage of sirolimus was tapered during the second month post-transplantation, ending in complete withdrawal within the sixth month after HSCT. Mycophenolate mofetil (MMF) was initiated orally at 10 mg/kg t.i.d. on day 5 after HSCT, and withdrawn on day 30. Median follow up was 247 days (range 29-486) from HSCT. All patients were engrafted in a median time of 17 days (range, 14-51 days), with full donor chimerism achieved by day 30. Poor graft function was observed in 11 patients. Post-HSCT recovery of lymphocyte subsets was lengthy as compared to other unmanipulated HSCT, with a median time to CD3〉100/ml of 41 days (range, 22-389 days) by flow-cytometry, while 5 patients never reached immune reconstitution. All except one developed febrile neutropenia during aplasia. We observed a high rate of serious infections: severe pneumonia in 7 cases (5 required NIV, 4 were admitted to ICU), CMV reactivation in 13 (3 CMV disease), HHV6 reactivation in 10, BK virus haemorrhagic cystitis in 2 patients. Although the overall mortality associated with these complications was limited, as evidenced by a favourable NRM, it caused significant morbidity in patients and often required aggressive therapies, potentially contributing to poor graft function. The incidences of acute and chronic GVHD were low. A grade II-IV acute GvHD was reported in 10% patients, while five patients developed chronic GvHD (mild in 4 cases, only one moderate). Non-relapse mortality (NRM) at 100 days and 1 year was 4.8% and 20.7%, respectively. The estimated 1-year overall survival (OS) and relapse incidence were 51% and 22.5% respectively. Although limited by a small number of patients with relatively short follow-up, our study suggests that URD allo-HSCT with ATG, PTCy and sirolimus-based GvHD prophylaxis, can be very effective in preventing GvHD, but at the expense of delayed immune reconstitution and high rate of infectious complications, while warranting acceptable NRM and relapse incidence. Disclosures Bonini: MolMed S.p.A: Consultancy.

Description: Introduction Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the best curative option for many patients with Acute Myeloid Leukemia and related malignancies, mostly due to the antileukemic activity of immune cells contained in the graft. Although in most of the cases HSCT can accomplish apparent disease eradication, frequently residual leukemic cells are able to evade elimination, eventually outgrowing and resulting in clinical relapse. Apparently indistinguishable from disease at diagnosis, relapses commonly display a more aggressive behavior, and most of the available therapeutic options are ineffective. Next-generation sequencing provides the opportunity to track specific alterations and disease subclones during the clinical history of patients, and from this information to identify mechanisms by which leukemic cells evade elimination. Methods Serial disease samples collected longitudinally during the complex clinical history of a patient with high-risk myelodysplastic syndrome were analyzed by genomic HLA typing and by high-depth exome sequencing (minimum depth of coverage 70x). Patient fibroblasts and mononucleated peripheral blood cells harvested at remission served as reference for germline HLA typing and exome sequence. Whenever possible, leukemic blast were FACS-purified. Sequencing data were aligned to the reference genome using the BWA aligner and analyzed using the tools developed from the Cancer Genome Analysis group at the Broad institute to identify newly acquired mutations, clonal segregation of novel and pre-existing mutations and quantitative clonal dynamics. Results A 54 year old patient with high-risk refractory anemia with excess blasts (pancytopenia, blasts 19%, adverse cytogenetics) underwent unmanipulated T cell-repleted HLA-haploidentical HSCT in the presence of active disease. After one year of remission a first relapse occurred. HLA typing of the leukemic blasts harvested at relapse demonstrated the selective genomic loss of the mismatched HLA haplotype targeted by donor T cells, a frequent mechanism of leukemia immune evasion after haploidentical HSCT, first described by our group (Vago et al, N Engl J Med, 2009). The patient was re-transplanted from a different haploidentical donor mismatched for the HLA alleles retained by the mutated variants; accordingly, T cells from this donor were expectedly alloreactive against the relapsed leukemia. The patient re-obtained complete remission, which was maintained for five years, before the occurrence of a second relapse. Unexpectedly, at this relapse leukemic cells had lost the HLA molecules mismatched with the second donor, and expressed those that had gone amiss at first relapse. Since at both relapses HLA haplotype loss was due to a stable genomic alteration, chromosome 6p acquired uniparental disomy (aUPD), any linear relation between the first and second relapse could be ruled out (Figure 1). Leukemic samples at diagnosis, first relapse and second relapse were further characterized by high-depth exome sequencing, revealing that most of the mutations present in each of the three samples were not found in the other two: only a minute fraction of the mutations could be identified in all three disease presentations, possibly comprising the “driver mutations” harbored by the original leukemic stem cell clone. Amongst these shared mutations we are currently validating the functional and epidemiological relevance of missense alterations in the TP53 tumor suppressor gene, in the NHEJ1 gene (related to chromosomal instability), and in the BTNL8 gene, encoding the costimulatory receptor B7-H5, which may putatively play a role in leukemia recognition by T cells. Conclusions Our results demonstrate that antileukemic immunity has a profound impact on leukemia clonal evolution, driving the selection of immuno-privileged subclones. Importantly, the genetic alterations shared between the leukemic variants that emerged at years of distance during the clinical history of our patient suggest the long-term persistence of a reservoir of leukemic stem cells which are resistant to, or in stable equilibrium with, the immune pressure that sculpted their progeny. Finally, the longitudinal approach adopted in this study holds promise for the identification of leukemia “driver” mutations, to provide new insights into leukemogenesis and new rationales for targeted eradicating therapies. Disclosures: Bordignon: MolMed SpA: Employment. Bonini:MolMed SpA: Consultancy.

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