ALBERT

Description: BCR-ABL negative myeloproliferative neoplasms (MPNs), such as polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) are chronic myeloid malignancies characterized by overproduction of hematopoietic cells. JAK2 mutations are found in most patients with PV, and in only 50-60% of patients with ET and MF. JAK2 mutation testing has greatly simplified MPN diagnosis, but distinguishing JAK2-wildtype ET from reactive thrombocytosis remains a diagnostic challenge. Mutations in signalling pathways (MPL, LNK) and epigenetic regulators (TET2, DNMT3A, IDH1/2, EXH2, ASXL1) have been found in a minority of MPNs. However genome-wide data are lacking and the pathogenesis of MPNs that do not harbor JAK2 or MPLmutations remains obscure. Methods Exome sequencing was performed in 151 MPN patients on matched tumor and constitutional samples. CALR status was assessed in 3412 samples using Sanger sequencing and analysis of exome/genome sequencing data. Presence of CALR mutations in hematopoietic stem and progenitor cells was assessed by flow sorting and sequencing. Phylogenetic trees were established using hematopoietic colonies. Calreticulin cellular localisation was assessed in patient samples and cell lines expressing CALR variants by flow cytometry and immunofluorescence. Results Exome sequencing identified 1498 somatic mutations with a median of 6.5 mutations in PV and ET, and 13 in MF (MF vs ET, P=0.0002; MF vs PV, P=0.008). JAK2V617F was found in all cases of PV (n=48), 56% of ET (35/62), and 69% of MF (27/39), and MPL mutations in 7 ET and MF cases.  Mutations in epigenetic regulators TET2, DNMT3A, ASXL1, EZH2, and IDH1/2 were identified in 22, 12, 12, 4, 3 patients respectively, and components of the splicing machinery (U2AF1, SF3B1 or SRSF2) were mutated in 9 patients.  Mutations in rare genes reported to be mutated in MPNs were found in four patients (1 CBL; 2 NFE2; 1 SH2B3/LNK). We found novel somatic mutations in CHEK2 (1 PV, 1 ET and 1 MF) which have not been previously reported in MPNs.  The mutation spectrum showed a predominance of C〉T transitions. Pairwise associations between MPN genes demonstrated that ASXL1 and SRSF2 mutations were positively correlated with mutations in epigenetic modifiers. Novel somatic mutations in calreticulin (CALR) were identified by exome sequencing in the majority (26/31) of JAK2 or MPL unmutated patients. CALR and JAK2/MPL mutations were mutually exclusive, and 97% of patients harbored a mutation in 1 of these 3 genes. In an extended follow up screen of 1345 hematological malignancies, 1517 other cancers and 550 controls we found CALR mutations in 71% of ET (80/112), 56% of idiopathic MF (18/32), 86% of post ET-MF (12/14) and 8% of myelodysplasia (10/115), but not in other myeloid, lymphoid or solid cancers. Compared to JAK2-mutated MPNs, those with CALR mutations presented with higher platelet counts (Wilcoxon rank-sum, P=0.0003), lower hemoglobin levels (Student’s t test, P=0.02) and showed a higher incidence of transformation to MF (Fishers exact, P=0.03). All CALR mutations were insertions or deletions affecting exon 9, with 2 common variants L367fs*46 (52 bp deletion) and K385fs*47 (5 bp insertion). Loss of heterozygosity over CALR was seen in a minority of patients. Of 148 CALR mutations identified, there were 19 distinct variants. Remarkably, all generated a +1 basepair frameshift, which results in loss of most of the C-terminal acidic domain of the protein as well as the KDEL Golgi-to-endoplasmic reticulum (ER) retrieval signal, raising the possibility of compromised ER retention. Mutant proteins were readily detected in transfected cell lines and localised to the ER in the same manner as wildtype CALR, without Golgi or cell surface accumulation. These results are consistent with studies reporting KDEL-independent mechanisms of ER retention. Mutation of CALR was detected in highly purified hematopoietic stem/progenitor cells. Clonal analyses demonstrated CALR mutations in the earliest phylogenetic node in 5/5 patients, consistent with it being an initiating mutation in these individuals. Conclusions We describe the mutational landscape of BCR-ABL negative MPNs and demonstrate that somatic mutations in the endoplasmic reticulum chaperone CALR are found in the majority of patients with JAK2-unmutated MPNs. These results reveal a novel biological pathway as a target for tumorigenic mutations and will simplify diagnosis of MPN patients. Disclosures: Bowen: Celgene: Honoraria. Harrison:S Bio: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Shire: Speakers Bureau; Celgene: Honoraria; YM Bioscience: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Vannucchi:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Description: In Epstein-Barr virus (EBV) classical Hodgkin lymphoma (EBV+ cHL), Hodgkin-Reed Sternberg cell antigen presentation is intact, with viral expression restricted to sub-dominant latent-antigens including LMP1/2A. Large epidemiological studies have reported differential HLA-class I (HLA-I) susceptibility to EBV+ cHL. The functional basis for these observations is unknown. HLA-I molecules present viral peptides for recognition by CD8+ T-cells, and it may be that the relative risk of developing EBV+ cHL is due to HLA-I alleles influencing the magnitude of CD8+ T-cell immunity against relevant EBV-specific antigens. However this remains speculative, with immunological evidence lacking. Several non-HLA-I linked genetic susceptibility loci have been identified, and HLA-I associations may simply represent markers for genes of diverse functions that are in linkage disequilibrium to the HLA-I region. We undertook an Australasian Leukaemia and Lymphoma Group study to address this fundamental question, utilizing 4 distinct but complimentary experimental approaches. 1. 9 EBV+ cHL and 11 EBV-ve cHL pre-therapy PBMC samples were tested for ex-vivo IFNγ, TNFα and CD107a CD8+ T-cell immunity, using overlapping LMP1 and LMP2A peptide pools. The non-HRS expressed EBV-lytic protein BZLF1 was a control. Highly stringent FACS gating was used to maximize specificity. Results were interrogated using Profile and SPICE analysis. Interestingly IFNγ, TNFα and CD107 CD8+ T-cell responses in HLA-A*02 EBV+ cHL (but not EBV-ve cHL) patients were greater than non-HLA-A*02 (LMP1 p=0.002; LMP2A p=0.03; combined LMP1/LMP2A p=0.005), whereas BZLF1 was equivalent, indicating that HLA-I provides differential CD8+ T-cell immunity against relevant EBV-latent antigens in EBV+ cHL but not EBV-ve cHL. 2. However, up to 4 different HLA-A/B molecules can potentially present relevant EBV-derived epitopes in each individual, adding a confounding layer of complexity to single allele-based effects. To overcome this and enhance sensitivity, we used the mutant HLA-I 721.221 cell-line (pulsed with LMP2A), transfected with either HLA-A*01, HLA-A*02, HLA-A*03 or HLA-B*08 alleles, as antigen presenting cells to in-vitro expand LMP2A-specific CD8+ T-cells from HLA-A*02 heterozygotes. This found ∼90% of the HLA-I LMP2A response was restricted through HLA-A*02. 3. In contrast to EBV+ cHL, in EBV-post-transplant lymphoproliferative disorders (EBV+ PTLD) the immunogenic EBNA3A/3B/3C latent-antigens are expressed. We compared HLA-I associations in 110 cHL (35% EBV+ cHL) to 153 PTLD (63% EBV+ PTLD) patients. Using Bonferoni corrected statistics, we confirmed that HLA-A*02 and HLA-A*01 homozygotes had lower and higher susceptibility to EBV+ cHL respectively, and that HLA-B*37 was positively associated. Notably, no HLA-I associations with EBV+ PTLD were found. 4. To investigate the impact of HLA-I on the hierarchy of CD8+ T-cell immunity to sub-dominant (LMP1/2A) and immune-dominant (EBNA3A/3B/3C) EBV-latent proteins, we analysed the diversity of HLA-class I restricted T-cells in 30 healthy EBV+ participants. To supplement 30 ‘defined' (i.e. validated) HLA-I EBV-latent antigen epitopes and expand HLA-I coverage, we identified 31 ‘SYFPEITHI' bioinformatically ‘predicted' peptide epitopes for HLA-A*01, HLA-A*03 or HLA-B*37 restricted EBV-latent antigens. All SYFPEITHI scores were ≥21, and thermal stability circular dichroism analysis (HLA-A*01) or MHC stabilization assays on T2 cells (HLA-A*03) confirmed peptide binding to HLA-I. Ex-vivo CD107 CD8+ T-cell assays for the 61 peptides, found that sub-dominant LMP1/2A-specific peptide responses were largely confined to HLA-A*02 (Fig 1A), whilst immuno-dominant CD8+ T-cell responses were stimulated by peptides presented by numerous HLA-I alleles (Fig 1B). These data combined illustrate that differential HLA-I-associated susceptibility to EBV+ cHL reflects altered EBV latent antigen-specific CD8+ T-cell immune hierarchies. For lymphomas expressing a restricted set of poorly immunogenic proteins, even modest CD8+ T-cell responses against relevant tumor-associated proteins confer protection, with broad implications for EBV-vaccine design. Studies are required to determine if similar mechanisms are applicable to non-lymphoid EBV+ malignancies with restricted latency such as undifferentiated nasopharngeal carcinoma. Disclosures: No relevant conflicts of interest to declare.

Description: Background Targeted immunotherapy with T cells expressing a chimeric antigen receptor (CAR) can produce dramatic anti-tumor responses. We previously demonstrated complete remissions (CRs), prolonged persistence, and sustained responses in children and adults with relapsed/refractory (r/r) acute lymphoblastic leukemia (ALL) treated with CD19-specific CAR-modified T cells (CTL019). However, a subset (~25%) of patients has limited persistence of CTL019, which can increase the risk of relapse. We hypothesized that repeat infusion may prolong persistence in some patients but would be ineffective in patients with immune-mediated rejection. As most CAR single chain variable fragment (scFv) domains, including that of CTL019, are of murine origin, anti-mouse reactivity is one potential cause of immunogenicity that may be overcome by fully human or humanized CAR design. We now report on retreatment with murine or humanized CD19-directed CAR T cells. Aims We sought to 1) prolong persistence by reinfusion of murine CTL019 in children with r/r CD19+ ALL and evidence of poor persistence and 2) determine the safety and efficacy of CD19-directed CAR T cells with a humanized scFv domain in children with B cell recovery or CD19+ relapse after prior CAR T cell therapy. Method T cells collected from patients were transduced with a lentiviral vector encoding a CAR composed of murine or humanized anti-CD19 scFv, CD3z, and 4-1BB domains, activated/expanded ex vivo with anti-CD3/anti-CD28 beads, cryopreserved, and then infused. The humanized scFv was developed by grafting the complementary determining regions of both the heavy and light chains onto human germline acceptor frameworks. Results Of 50 patients in CR at 1 month after CTL019 infusion (50/53 CR), 14 patients received a repeat infusion of murine CTL019 at 3 and/or 6 months after initial infusion and 11 are evaluable for response. Indications were B cell recovery (n=4), CD19+ minimal residual disease (MRD, n=2), or undetectable CTL019 in peripheral blood by flow cytometry (≤0.1% CTL019+ of CD3+, n=5). Minimal toxicities were observed and included fever and fatigue. CTL019 reinfusion induced B cell aplasia for a second time in 1 of 4 children treated for B cell recovery. Two of these patients later relapsed, 1 with CD19+ and 1 with CD19- disease. Of 2 patients with CD19+ MRD, 1 had no response and subsequently experienced a CD19+ relapse and the second became MRD-negative but had B cell recovery. All 5 children reinfused for poor CTL019 persistence demonstrated continued B cell aplasia 3-15 months after repeat infusion with detectable CTL019 in peripheral blood by flow cytometry (0.2-2.8% CTL019+ of CD3+) in 4 patients. Of this group, 4 patients remain in remission 12-21 months after initial infusion, and 1 experienced a CD19- relapse. To overcome the potential for anti-murine immune-mediated rejection, patients previously treated with CAR-modified T cells were eligible for a phase 1 study of humanized CD19-directed CAR T cells (CTL119). Five children were infused for B cell recovery (n=3) or CD19+ relapse (n=2). Cytokine release syndrome (CRS), seen in 2 patients, was limited to fever, headache, and nausea and did not require vasopressor or respiratory support. Three patients showed no response and progressed (n=1) or relapsed with CD19+ (n=1) or CD19- (n=1) ALL. Responses were demonstrated in 2 patients: 1) MRD-negative CR in a patient treated for CD19+ relapse and 2) reduction in MRD and return of B cell aplasia in a patient treated for CD19+ MRD and B cell recovery, with subsequent progression to CD19+ relapse. Both responding patients were previously resistant to murine CTL019 reinfusion, suggesting a possible immune-mediated rejection mechanism. Ongoing studies are investigating anti-CAR responses. Conclusion Repeat infusion of murine CTL019 may prolong B cell aplasia in patients with early evidence of poor persistence and in a fraction of patients with B cell recovery. In the first demonstration of humanized anti-CD19 CAR efficacy, retreatment with CTL119 expressing a humanized scFv induced remission of ALL refractory to prior CD19-directed murine CAR T cell therapy, suggesting that immune-mediated rejection may be an important mechanism of resistance in the subset of patients with rapid CAR cell loss and loss of B cell aplasia. Further investigation into anti-CAR responses will be vital to improve durable remission rates in this highly refractory population. Disclosures Maude: Novartis: Consultancy, Research Funding. Off Label Use: CTL019 and CTL119 for relapsed/refractory ALL. Rheingold:Endo: Other: Husband's employer, has equity interest; Novartis: Consultancy. Aplenc:Sigma Tau: Consultancy. Teachey:Novartis: Research Funding. Shaw:Novartis: Research Funding. Brogdon:Novartis: Employment. Loew:Novartis: Employment. Zheng:Novartis: Patents & Royalties. Levine:Novartis: Patents & Royalties, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. June:Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. Grupp:Novartis: Consultancy, Research Funding.

Description: Alternative pre-mRNA splicing (AS) is a normal epigenetic phenomenon, a key regulator of gene expression, yields multiple transcripts and thus a variety of proteins from a single gene. Mutations in the spliceosome components resulting in aberrant splicing isoforms are common in AML, and other myeloid neoplasms, and may generate leukemia-specific neoantigens targetable with an antibody-drug conjugates (ADCs) or blocking antibodies. Our previous studies revealed that the FLT3 cell surface receptor is one of the most commonly misspliced genes in AML (54-63% of ~400 AML patients). We conducted cloning and sequencing analyses in AML cells and identified multiple aberrant splice-variants of FLT3 that resulted from either skipping of one or more exons or activation of cryptic splicing sites. Transfection of cDNA with three of these variants in TF-1 (AML cell line) cells resulted in expression of Flt3 variant proteins on the cell surface. We successfully generated rabbit polyclonal antiserum against a unique peptide sequence present in the most commonly expressed abnormal splice variant, which we termed Flt3Va. Immunoblots performed with the polyclonal antibody identified a ~160 kDa protein expressed by TF-1 cells transfected with FLT3Va, and the antibody did not react with untransfected TF-1 cell lysate. Using standard techniques, we generated rabbit hybridomas and evaluated the clones by flow cytometry and western blotting experiments. Based on these data, we selected one antibody clone (15-7) for further experiments. The 15-7 anti-Flt3Va rabbit monoclonal antibody identified Flt3Va protein expressed on the cell surface and within the cytoplasm of transfected TF-1 cells by flow cytometry and western blotting. However, no Flt3Va protein was detected in untransfected TF-1 cells or normal CD34+ bone marrow cells. The 15-7 antibody bound to 26 of 52 primary AML samples and 5 of 10 primagraft samples (PDX models) of human AML. Immunoblotting analyses of PDX models and patient samples confirmed binding to a protein of the expected size (130-160 kDa). Additionally, multi-parameter flow cytometry in 10 PDX models and 52 primary demonstrated that putative AML stem cells (as defined by the CD45dim, CD34, CD38, CD33, c-Kit cell surface expression) co-expressed Flt3Va antigen in 50% samples evaluated. An analysis of Flt3Va protein localization by live cell imaging showed a punctate distribution of Flt3Va on the cell surface. Furthermore, we observed that overexpression of Flt3Va in TF-1 cells led to GM-CSF growth factor independence. Analysis of TF-1 cells in the absence of GM-CSF and Flt3 ligand demonstrated constitutive activation of STAT5, an important mediator of Flt3 signaling, in Flt3Va overexpressing cells. In addition, Erk1/2 phosphorylation was also increased in Flt3Va overexpressing cells, another downstream effector of Flt3. In an effort to determine if Flt3Va+ cells had tumor repopulating ability, we sorted 0.3X10^6 Flt3Va+ and Flt3Va- cells from a PDX sample and injected the sorted populations or unsorted bulk tumor cells into NSG mice. The human cell engraftment in the mice was detected by the expression of human CD45, CD33, CD34, CD38, and c-kit antigens in the peripheral blood. In two experiments, mice injected with Flt3Va+ cells had detectable circulating leukemic cells by ~18 days after injection, while those injected with Flt3Va- cells had detectable circulating leukemic cells after the 4th week. These results suggest both Flt3Va+ and Flt3Va- cell populations are able to reconstitute leukemia after transplantation in NSG mice. However, Flt3Va+ may be expressed by an aggressive AML clone that facilitate early tumor engraftment. Overall, these studies suggest that Flt3Va is a leukemia-specific neoantigen and is an attractive potential immunotherapeutic target in AML. Proteins such as Flt3Va generated by alternative splicing are common in AML and may be targets for of novel blocking antibodies or ADCs, minimizing effects on normal tissues. Disclosures Adamia: Janssen: Research Funding. Nemeth:Janssen: Employment. Attar:Janssen: Employment. Letai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding. Steensma:Millenium/Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Ariad: Equity Ownership; Genoptix: Consultancy. Weinstock:Novartis: Consultancy, Research Funding. DeAngelo:Novartis: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; Baxter: Consultancy; Celgene: Consultancy; Incyte: Consultancy; Amgen: Consultancy. Stone:Agios: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celator: Consultancy; Juno Therapeutics: Consultancy; Roche: Consultancy; Jansen: Consultancy; Pfizer: Consultancy; ONO: Consultancy; Sunesis Pharmaceuticals: Consultancy; Merck: Consultancy; Xenetic Biosciences: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Amgen: Consultancy; Karyopharm: Consultancy; Seattle Genetics: Consultancy. Griffin:Janssen: Research Funding; Novartis: Consultancy, Research Funding.

Description: Key Points An international panel established the first ever diagnostic criteria for iMCD based on review of 244 clinical cases and 88 tissue samples. The criteria require multicentric lymphadenopathy with defined histopathology, ≥2 clinical/laboratory changes, and exclusion of iMCD mimics.

Description: Anti-CD20 monoclonal antibodies are widely used for the treatment of hematological malignancies or autoimmune disease but may be responsible for a secondary humoral deficiency. In the context of COVID-19 infection, this may prevent the elicitation of a specific SARS-CoV-2-antibody response. We report a series of 17 consecutive patients with profound B-cell lymphopenia and prolonged COVID-19 symptoms, negative IgG-IgM SARS-CoV-2 serology and a positive RNAemia measured by digital PCR who were treated with four units of COVID-19 convalescent plasma. Within 48 hours following transfusion, all patients except one experienced an amelioration of their clinical symptoms. The inflammatory syndrome abated within a week. Only one patient who needed mechanical ventilation for severe COVID-19 disease died of bacterial pneumonia. SARS-CoV-2 RNAemia decreased to below the sensitivity threshold in 9 out of 9 evaluated patients. Analysis of virus-specific T-cell responses using T-cell enzyme linked immunoSpot (ELISPOT) assay was analyzed before convalescent plasma transfusion in 3 patients. All showed a conserved SARS-CoV-2 T-cell response and poor cross-response to other coronaviruses. No adverse event was reported. In COVID-19 patients unable to mount a specific humoral response to SARS-CoV-2, convalescent plasma with anti-SARS-CoV-2 antibodies appears to be a very promising approach in the context of protracted COVID-19 symptoms.

Description: Key Points CIP4 affects the remodeling of both plasma membrane and cortical cytoskeleton in megakaryocytes. CIP4 in platelet biogenesis involves cortical tension, as does WASP, and WASP-independent plasma membrane reorganization.

Description: INTRODUCTION Cytomegalovirus (CMV) infection is common after allogeneic haemopoietic stem cell transplant (HSCT), especially in the first 12 months when the recovering immune system is immature. We are conducting an early phase clinical trial of 3rd party donor-derived, partially HLA matched, virus-specific T cells (VST) for initial reactivation of CMV after HSCT. RESULTS At data cutoff for this analysis (July 15 2018), we had administered 22 infusions of CMV VST to 15 HSCT recipients (age 1-70 years, median 57; male n=9, female n=6) with reactivation of CMV within 7 days of first commencement of antiviral pharmacotherapy. Up to 3 VST infusions were given (1 infusion n=9, 2 n=5 and 3 n=1). Diagnoses include AML/MDS (n=5), ALL (n=2), NHL (n=3), CML, CMML, MDS/MPN, mucopolysaccharidosis type 1 and ARPC1B deficiency (n=1 each). Patients received reduced intensity conditioning (n=10) and myeloablative conditioning (n=5) and were transplanted from matched unrelated donors (n=9), mismatched unrelated donors (n=3), a matched sibling (n=1) and haploidentical donors (n=2). Haploidentical graft recipients received graft versus host disease (GVHD) prophylaxis with post-transplant cyclophosphamide, cyclosporine and mycophenolate mofetil. All other patients with the exception of the sibling graft recipient and two pediatric graft recipients received thymoglobulin. Two pediatric graft recipients received alemtuzumab in vivo and CD34 ex vivo selected stem cells respectively. Eleven patients were CMV D+/R+, 4 patients were D-/R+. Median day of first VST infusion was day 49 post-transplant (range 34-83). There were no significant infusion related reactions. Of the fifteen patients who received treatment for CMV, median maximum CMV copy number prior to first VST infusion was 6088 copies/ml (range 2342 to 53140). 12 patients (80%) achieved a complete virological response (defined as at least one negative PCR test after infusion). The remaining 3 patients achieved a virological PR (at least 50% reduction in CMV copy number); in 2 of these 3 cases CMV fell below the level of assay quantitation but remained detectable. Median time to CR was 21 days (range 9-53) and to PR was 20.5 days (range 12-48). Twelve of the 22 infusions (55%) were followed by complete virological response. Median number of days in hospital after VST infusion was 0 (0-83). Median number of days of CMV antiviral administration was 21 (range 7-72). All but 2 patients had additional non-bacterial pathogens isolated post-transplant (1 n=4, 2 n=5, ≥3 n=4). 3 patients developed acute graft versus host disease, 2 post VST infusion (grades I and II skin), 1 both before (grade IV gut GVHD) and after VST infusion (grade IV gut GVHD) on steroid weaning. One patient has developed mild chronic GVHD affecting mouth and liver. CMV pneumonitis was observed in 1 patient after VST infusion as part of complex lung pathology comprising pulmonary veno-occlusive disease, pulmonary fibrosis and positive lung PCR testing for CMV, EBV and HHV6. 5 patients were censored at post-transplant days 111 (relapse CMML), 122 (secondary graft rejection after CD34 selected transplant), 155 (unmanipulated DLI for falling myeloid chimerism), 160 (relapse ALL) and 275 (ATG for second line therapy of grade IV gut GVHD). At a median follow up of 165 days (42-279) 14 of 15 patients (93%) remain alive (1 death due to relapse). At date of last follow up, 12 patients had ECOG status 0, 1 had ECOG 2 and 2 had ECOG 4. Post VST infusion CMV specific immune reconstitution was observed with a rise in pp65 specific cellular immune response measured by IFN-g elispot (median 152 spot forming unitsx104/L before infusion to median peak of 2710 after infusion; p=0.006; Fig 1). HLA mismatched 3rd party cells were detectable in the peripheral blood in 4 of 9 patients at low levels (mean 0.012%) by a high sensitivity digital PCR chimerism assay up to day 97 post infusion. CONCLUSION Early administration of 3rd party partially HLA matched CMV-specific T-cells in association with standard antiviral treatment is non-toxic and does not induce a high rate of acute GVHD. These preliminary data suggest that this approach is associated with good viral control and performance status in allogeneic transplant recipients. Recruitment of additional patients, longer follow up and ultimately a randomized study will be required to determine whether addition of early cell therapy of this kind adds value to standard antiviral treatment. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: IPI-504 is a novel, water-soluble Heat Shock Protein 90 (Hsp90) inhibitor. It is the chemical reduction product of 17-AAG, a well-characterized Hsp90 inhibitor. 17-AAG is currently in clinical trials, but suffers from very poor aqueous solubility and must be administered to patients in either suboptimal DMSO formulations, or newer cremophor or emulsion formulations. In contrast, IPI-504 exists as a hydrochloride salt which is soluble in water in excess of 200 mg/mL. Therefore, IPI-504 is 4000-fold more soluble than 17-AAG. It has previously been shown that IPI-504 inter-converts with 17-AAG and exists in a pH and enzyme-mediated dynamic redox equilibrium which has been observed in human clinical trials as well. In preclinical animal studies, IPI-504 has demonstrated in vitro and in vivo anti-tumor effects in a variety of cancers including xenograft and orthotopic models of multiple myeloma (MM). Methods: An open-label phase I dose-escalation trial of IPI-504 infused on days 1, 4, 8 and 11 of a 21 day cycle is being conducted in patients (pts) with relapsed/refractory MM. IPI-504 is administered to pts in 250cc of normal saline over thirty minutes. The primary objectives of the study are to determine the maximum tolerated dose (MTD) and characterize the pharmacokinetic (PK) and pharmacodynamic (PD) marker effects of IPI-504. Levels of Hsp70 protein were measured as a marker of IPI-504’s biologic activity in peripheral blood leukocytes. Toxicity is evaluated by NCI CTCAE (v3.0) and response by EBMT. An accelerated dose titration design was employed with dose escalation of 100% for the first three dose levels with 1 pt / cohort. The first three pts received 22.5, 45 and 90 mg/m2 respectively. Per protocol, once the 150 mg/m2 level was achieved, dose escalation converted to a modified Fibonacci schema with 3 pts per cohort. Subsequently dose escalation occurred in increments of 66%, 50%, 33% and 25%. Results: Currently, 15 pts (11 female / 4 male), average age 61.3 years, have been treated. IPI-504 has been well-tolerated at doses ranging from 90 to 300 mg/m2. Patients continue to be enrolled at the 400 mg/m2 dose level. Dose limiting toxicities (DLTs), defined as grade 4 or greater hematologic toxicity and/or grade 3 or greater non-hematologic toxicity within the first 21 days of treatment, have not been observed to date. Dose escalation will continue until a recommended phase 2 dose is identified. Analysis of PK, safety, tolerability, and biologic response to IPI-504 is ongoing. The above support the planning of further clinical exploration of IPI-504 including combination with agents such as bortezomib that have proven synergistic in animal models of MM.

Description: To obtain a comprehensive genomic profile of presenting multiple myeloma cases we performed high-resolution single nucleotide polymorphism mapping array analysis in 114 samples alongside 258 samples analyzed by U133 Plus 2.0 expression array (Affymetrix). We examined DNA copy number alterations and loss of heterozygosity (LOH) to define the spectrum of minimally deleted regions in which relevant genes of interest can be found. The most frequent deletions are located at 1p (30%), 6q (33%), 8p (25%), 12p (15%), 13q (59%), 14q (39%), 16q (35%), 17p (7%), 20 (12%), and 22 (18%). In addition, copy number-neutral LOH, or uniparental disomy, was also prevalent on 1q (8%), 16q (9%), and X (20%), and was associated with regions of gain and loss. Based on fluorescence in situ hybridization and expression quartile analysis, genes of prognostic importance were found to be located at 1p (FAF1, CDKN2C), 1q (ANP32E), and 17p (TP53). In addition, we identified common homozygously deleted genes that have functions relevant to myeloma biology. Taken together, these analyses indicate that the crucial pathways in myeloma pathogenesis include the nuclear factor-κB pathway, apoptosis, cell-cycle regulation, Wnt signaling, and histone modifications. This study was registered at http://isrctn.org as ISRCTN68454111.