ALBERT

Description: Background: The trombopoietin receptor agonists (TRAs) romiplostim and eltrombopag are effective and safe in the treatment of chronic immune thrombocytopenia (ITP). However, when no response is achieved or when adverse events occur with one TRA the value of the sequential use of romiplostim and eltrombopag has not been clearly established. Here we have evaluated the efficacy and tolerance of using eltrombopag after romiplostim in ITP. Methods: Fifty-one primary ITP patients (aged 18 years or more) who had been sequentially treated first with romiplostim and then with eltrombopag in the Spanish Eltrombopag Registry were retrospectively evaluated. In accordance with the usual standards, complete response was defined as a platelet count of 100x109/L and a response as a platelet count of 30x109/L or a count of at least twice the initial (pre-treatment) value. This study was performed in accordance with the standards of the Helsinki declaration and approved by the Hospital Universitario de Burgos Ethics Committee. Results: The median age of our cohort was 49 [range, 18–83] years. There were 32 women and 19 men. According to the standard definition, patients were allocated to newly diagnosed (n=2), persistent (n=5) and chronic (n=44) ITP groups. The median number of therapies prior to administration of eltrombopag was 4 [range, 2–9], including splenectomy (39%), rituximab (33%) and romiplostim (100%). The median duration of romiplostim use before switching to eltrombopag was 12 (IQR 5–21) months. The reasons for switching from the romiplostim to eltrombopag were: lack of efficacy of romiplostim (n=25), patient's preference (n=16), platelet-count fluctuation (n=6), and side-effects (n=4). The initial response rate to eltrombopag was 41/51 (80.5%), including 67% (n=34) of cases with complete remission. After a median follow-up of 13 months with eltrombopag, 39 patients maintained their response. When eltrombopag was used for patients who were refractory to the maximum romiplostim dose the initial response rate of eltrombopag was 25%. However, 83% of patients who relapsed after their initial response to romiplostim responded to eltrombopag. Sixteen romiplostim responders requested their physicians to switch them to eltrombopag because they preferred an oral drug. The efficacy was maintained after switching in all 16 patients. In the platelet-count fluctuation group, the initial response rate was also 100%. All 4 patients who were switched to eltrombopag because they experienced side-effects of romiplostim achieved complete remission with eltrombopag and their adverse events were resolved. 16 / 51 (33%) patients experienced one or more adverse event during treatment with eltrombopag. The frequency of grade 3–4 adverse events during treatment with eltrombopag was 9.8%. Conclusion: The use of eltrombopag after romiplostim for treating ITP is effective and safe. The reason for discontinuing romiplostim was associated with the response to eltrombopag. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Melphalan, in combination with bortezomib, should be maintained as one of the standards of care for the treatment of elderly MM patients. Complete response and particularly flow complete response should be an important goal in the treatment of elderly myeloma patients.

Description: Introduction Graft versus leukemia (GvL) effect after hemotopoietic stem cell transplantation (HSCT) is mediated by donor immune cells recovery. Natural Killer (NK) cell alloreactivity is controlled by the interaction of activatory receptors and inhibitory killer-immunoglobulin-like receptors (KIRs) with major histocompatibility locus class I antigens on the leukemia cells. Haploidentical setting is a plattform for NK cell alloreactivity however HLA identical setting remains unclear. Methods and Patients We performed KIR-genotyping of HLA-identical sibling donors in 35 pediatric CD34 selection peripheral blood stem cell transplantations to identify genetic factors affecting leukemia relapse and overall survival. Univariate analysis of leukemia relapse and KIR genotyping was performed in order to identify independent variables predictive of outocome for pediatric acute lymphoblastic leukemia (ALL), (n=20) and acute myeloblastic leukemia (AML), (n=15). Results Donor B haplotype was observed in 21 cases (60%). Statistical analysis shown that donor B haplotype was associated with significantly more relapse in leukemia pediatric patients (38% vs. 0%) and worse overall survival (40% vs. 7%). Further analysis revealed that 2DL5a, 2DS3 and 2DS5 were associated with an increased rate of leukemia relapse (47% vs. 6%, 54% vs. 10% and 58% vs. 13%, respectively) and 2DL5a, 2DS1 and 3DS1 were associated with a worse overall survival (48% vs. 6%, 64% vs. 10% and 58% vs. 13%, respectively). No difference was observed in patients KIR haplotype or donor-recipient KIR haplotype mismatch. Conclusion In our study, which included only reduced intensity conditioning without antithymocyte globulin followed by related peripheral blood stem cell transplantation, we found a significant worse impact of donor KIR B haplotype in stem cell transplantation outcome. Our results suggest that donor KIR B haplotype (2DL5a, 2DS3 and 2DS5) increases leukemia relapse and also donor KIR B haplotype (2DL5a, 2DS1 and 3DS1) confer significant survival damage to HLA-identical sibling HSCT. Figure 1. Donor KIR genotyping (2DL5A, 2DS1 and 3DS1) impacts in overall survival. Figure 1. Donor KIR genotyping (2DL5A, 2DS1 and 3DS1) impacts in overall survival. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Acute myeloid leukemia (AML) is characterized by dysregulated gene expression and aberrant DNA methylation status. One alternative treatment for AML is hypomethylating agents such as 5-Azacytidine (AZA) and Decitabine (DAC), but just around 30% of the patients obtain a clinical response. These agents are slow acting requiring 3-4 cycles to demonstrate some efficacy, for both reasons looking for biomarkers to identify sensitive patients before treatment could be very beneficial to the patients. Objective: Design an ex-vivo model using the automated flow cytometry ExviTech® platform to identify the antiproliferative effect of hypomethylating agents such as 5-Azacytidine and Decitabine in leukemic cells from AML whole bone marrow patient samples. This assay may capture changes of hypomethylating agents effect that may potentially predict ex vivo the patient clinical outcome. Methods: Bone-marrow samples from 10 patients diagnosed of AML were sent to Vivia from 24 hospitals across Spain within 24h. Whole sample were incubated for 72h in in well plates containing 8 concentrations of AZA and DAC. To induce blast proliferation, AML cells were resuspended in RPMI medium and human cytokines including GM-DSF, IL3, SCF, G-CSF, EPO and transferrin. The number of proliferative and no proliferative live leukemic cells was determined using the CFDA dye. Blast population and viability was determined labeling with monoclonal antibodies and Annexin V. Dose-response curves of AZA and DEC were measured in these 10 patient samples. Results: To corroborate the hypothesis that AZA or DAC induce cell cycle arrest and this effect is different from cytotoxicity, we evaluated the effect of DAC in an AML cell line (KG-1) that grow spontaneously with medium and serum. As shown in figure 1, a decrease in the number of cells is observed when the AML cell line (KG-1) is incubated with DAC (blue line). Interestingly, an exactly proportional increase in the Mean Fluorescent Intensity (MFI) of CFDA is observed (red line), the symmetry between these curves indicate they measure the same effect; the drug-concentration dependent decrease in proliferating cells. Green line represents the percentage of apoptosis, as a measurement of the cytotoxic activity, clearly different that the other 2 measurements and only relevant at much higher doses, indicating that DAC has an essentially antiproliferative effect in the cell line. Once tested the hypothesis, we translate the idea to fresh AML samples inducing the blast proliferation with a very complete cytokine medium. In all patients included in this study, an average population of 47.2%±16.9% proliferating cells was identified. A clear difference in the sensitivity of no proliferative vs proliferative cell subset was observed, while DAC had no activity on the no proliferating cells (flat dose response in Figure 2), it was potently (1 uM) inhibiting almost completely proliferation in the proliferating cell subset. Conclusion: Although the mechanism of action of hypomethylating agents is not still entirely clear, we hypothesize that is associated with antiproliferative effects. In this study, using our Exvitech platform we have developed an ex-vivo model to measure the antiproliferative effect of hypomethylating agents, using whole stimulated bone marrow samples from AML patients. If this new assay would correlate with patient treatment response, it could become a valuable tool to personalize hypomethylating treatments for AML patients. Figure 1 Figure 1. Figure 2 Figure 2. Figure 1 Figure 2 Disclosures Hernandez: Vivia Biotech: Employment. Gomez:Vivia Biotech: Employment. Robles:Vivia Biotech: Employment. Gorrochategui:Vivia Biotech: Employment. Sanchez:Vivia Biotech: Employment. Ballesteros:Vivia Biotech: Employment. Martinez:Vivia Biotech: Membership on an entity's Board of Directors or advisory committees.

Description: Background: Several studies have suggested that genetic variability related with single nucleotide polymorphisms (SNPs) within the genes involving Ara C pathway could influence in treatment outcome of AML patients treated with this cytotoxic drug. However, the association of these polymorphisms with toxicity of combinations of Ara C and anthracyclines in AML patients remains undetermined. Methods: The SNPs of Ara C metabolism genes previously described in literature (DCK: rs2306744, rs11544786, rs4694362; CDA: rs2072671, rs3215400, rs532545, rs602950; NT5C2: rs11598702; RRM1: rs9937; NME1: rs2302254) were evaluated in 109 adult patients of a single center at initial diagnosis of AML, using a Sequenom (iPLEX) mass spectrometry–based multiplex genotyping assay (Sequenom, San Diego, CA). All patients were treated with intensive induction chemotherapy consisting of idarubicin plus Ara C (PETHEMA-AML 99, 2007 and 2010 trials). Genotypes were grouped as dichotomous variables (dominant and recessive model). Efficacy of first induction cycle was evaluated comparing complete remission (CR) vs. partial remission or resistance. Patients dying during induction were considered as no evaluable for efficacy. Based on WHO grading scale, toxicities were grouped as binary variables (grade 0-1 vs. grade 2-4). The grade of toxicity assigned to an organ group was the maximum grade of all the specific toxicities within that group. Overall toxicity was grouped with grade 3-4 assessment. Hematologic toxicity was measured with the time to neutropenia and thrombocytopenia recovery since first day of chemotherapy. Categorical and continuous variables were assessed using χ2 test with Yates correction if needed and Mann–Whitney U test, respectively. Multivariate analyses were performed using the Cox method. Results: The median age of patients was 53 years (17-78 years). Among the baseline characteristics analyzed (age, gender, leukocyte count, hemoglobin level, platelet count and percentage of peripheral or BM blasts) there was statistically significant difference in the genotype distributions of CDA polymorphisms regarding age (patients with variant alleles of rs2072671, rs532545, rs602950, and wild type of rs3215400 were older, P= 0.006, 0.025, 0.025 and 0.012, respectively) and gender (men had higher proportion of variant alleles than women for rs2072671, rs3215400 and rs602950, P= 0.04, 0.046 and 0.039). The variant homozygous of DCK SNP rs11544786, enzyme that catalyzes the limiting first phosphorylation in activation of Ara C, showed a trend towards a prolonged neutropenia duration (67.5 vs. 34.2 days, P=0.058). Concerning the CDA polymorphisms, the main inactivating enzyme in the Ara C, wild genotype of rs2072671 was associated with higher frequency of grade 2-4 liver toxicity (83.3% vs. 51.0%, P= 0.034) and time to thrombocytopenia recovery (36.1 vs. 20.6 days, P= 0.026) compared to variant alleles, which is consistent with the CDA activity reduction attributed to this SNP. Multivariable regression models including age and gender as covariates were performed in CDA SNPs analysis with similar results for the association between rs2072671 and liver toxicity (OR=0.209, 95%IC=0.043-1.002, P=0.026), but not for time to thrombocytopenia recovery. We did not found any association between any of the investigated cytarabine pathway gene polymorphisms and the CR rates. Conclusions: This study reveals associations between polymorphisms of metabolic genes of Ara C and the toxicity during induction therapy in adult AML patients. Further studies with larger population are needed to validate these associations, especially in SNPs with low variant allele frequency, such as in DCK. A better knowledge of the patient’s risk of toxicity related to genetic variability could improve the treatment outcomes in AML. Disclosures No relevant conflicts of interest to declare.

Description: Background: Eltrombopag is effective and safe for treating chronic immune thrombocytopenia (ITP) patients who have not responded to previous therapy. Interestingly, some patients in whom hemostatic platelet counts are achieved with eltrombopag may sustain the platelet response when eltrombopag ceases to be administered. However, the frequency of sustained responses after discontinuing eltrombopag without additional therapy for ITP is largely unknown. Methods: A total of 260 adult patients (aged 18 years or more) with primary ITP treated with eltrombopag included in the Spanish Eltrombopag Registry were retrospectively evaluated. The study was performed in accordance with the standards of the Helsinki declaration and approved by the Hospital Universitario de Burgos Ethics Committee. Results: The median age was 62 [range, 18–93] years. There were 165 women and 95 men. According to the standard definition, patients were allocated to newly diagnosed (n=29), persistent (n=36) and chronic (n=195) ITP groups. The median time from diagnosis to eltrombopag initiation was 24 [range, 1–480] months. The median number of previous therapies was 3 [range, 0–10], including splenectomy (22%), rituximab (23%) and romiplostim (19%). The initial response rate to eltrombopag was 231/260 (89%), including 77% (n=201) cases of complete remission (platelet count ≥100 x 109/L). The median duration of eltrombopag treatment was 6 [range, 1–54] months. Eltrombopag was discontinued in 80 out of 201 (39.8%) patients who achieved CR. Reasons for eltrombopag discontinuation were: persistent response despite a reduction in dose over time (n=33), platelet count 〉400x109/L (n=29), patient’s request (n=5), aspartate aminotransferase elevation (n=3), diarrhea (n=3), thrombosis (n=3) and other reasons (n=4). For analysis of discontinuation, patients with follow-up 〈 6 months (n=15), newly diagnosed ITP (n=11) or patients who received concomitant or previous (6 months before) treatments at the start of eltrombopag use (n=5) were excluded. Of the 49 evaluable patients, 22 (45%) had an immediate relapse after stopping eltrombopag. One patient with sustained response after stopping treatment relapsed at 10 months. A total of 26 patients (53%) showed sustained response after discontinuing eltrombopag without additional ITP therapy, with a median follow-up of 9 [range, 6–25] months. These patients were characterized by a median time since ITP diagnosis of 46.5±114.1 months, with 4/26 having ITP

Description: Introduction Epstein-Barr virus (EBV) viral load and serum free light chains (sFLC) are easily measurable and are possible biomarkers for screening and prognosis in HIV-related lymphomas. EBV load and sFLC levels have been shown to be increased at the diagnosis of HIV-related lymphomas but there is a lack of data on their levels at complete response (CR). We aimed to determine the clinical relevance and the potential use of combining EBV load and sFLC measurements as tumor markers in patients with HIV-related lymphomas. Methods Retrospective study of HIV-infected patients diagnosed with non-Hodgkin (NHL) and Hodgkin’s lymphoma (HL) treated in a single institution from 1998 to 2012. EBV loads were determined in plasma by means of a commercial real-time PCR technique (EBV PCR kit, Qiagen GmbH, Hilden, Germany). Levels of κ and λ sFLC were measured in serum using a standardized assay (FREELITE, The Binding Site, Birmingham, UK). sFLC were considered elevated when κ, λ, or the sum of κ and λ were above the upper normal limit. Clinical and biological data were collected from the clinical records. Results Sixty-eight patients were studied (53 NHL and 15 HL). At diagnosis (N=47), levels of κ, λ, and sum of κ and λ sFLC were elevated in 87%, 70%, and 81% of patients, respectively. At CR (N=29), the percentage of patients with elevated κ, λ, and κ+λ was 79%, 45%, and 62%, respectively. EBV load was detectable in 60% of the patients at diagnosis (N=50) and in 7.4% at CR (N=27). Median levels of κ, λ, and κ+λ sFLC at diagnosis were higher than at CR (6.06 mg/dL vs 3.19 mg/dL, P=0.006; 3.94 mg/dL vs 2.21 mg/dL, P=0.021; 11.38 mg/dL vs 5.43 mg/dL, P=0.004; respectively). Median levels of EBV loads at diagnosis were also higher than at CR (121.5 copies/mL vs. 0 copies/mL, P

Description: Introduction: MCL is a mature B-cell neoplasm characterized by t(11;14) (q13;q32) and cyclin D1 (CCND1) overexpression. Molecular studies have revealed other alterations in cell-cycle regulation, DNA damage response and cell survival pathways, with a landscape of somatic mutations being recently identified. CNS involvement is a well known complication, occurring in 4-26% of MCL at five years, with an ominous significance. Although different clinical variables have been identified as risk factors for CNS infiltration, the biological parameters related to this complication have not been extensively studied. The aim of the study was to explore the biological parameters associated with CNS involvement in a multicentre and retrospective series of MCL patients. Patients and Methods: 285 patients (M:74%; 64 yr) diagnosed of MCL between 1990-2014 (median survival of 4 years) were analysed. In addition to standard clinico-biological variables, IGHV mutational analysis, chromosomal alteration studies and Sanger sequencing of NOTCH1, NOTCH2, TP53, BIRC3, WHSC1, MEF2B, MLL2, TLR2 and PRDM1 were performed. Results: CNS involvement was observed in 15/285 MCL patients (5.2%), with a 5-yr risk of 9.1% (95%CI: 4.6-13.6), one patient at diagnosis, and at first or second/ulterior progressions in 7 cases each. The clinical, pathological and molecular risk factors identified are detailed in the Table. In addition to what has been already described, CNS involvement was usually observed in MCL cases with a clinical nodal presentation (p=0.05). In fact, no indolent MCL with a non-nodal presentation developed this complication during the follow-up period. No differences were observed in the risk of CNS involvement between patients treated in first-line with conventional or high-dose intense treatment (5-yr risk: 6.1%+/-6% vs. 10.7%+/-10.6%, p=ns). Regarding the biological features, no differences in terms of the IGHV mutational status were observed in cases developing CNS involvement compared to the others (75% vs. 68.7%, using 97% identity cut-off). Similarly, the IGHV gene usage of CNS involved cases corresponded to the more frequent IGHV genes observed in MCL (usually IGHV1-18, IGHV3-23, IGHV4-34, IGHV4-59). Although not significant, a predominance of high number copy number alterations (CNA) (〉4) could be observed in the genetic study of MCL cases with CNS involvement as could be expected for the enrichment in blastoid variants (up to 50% of these cases). In fact, we did not observe any case with CNS involvement among those cases with 3 or less CNA. CNS involvement was not related to common poor prognosis genetic alterations such as 9p, 11p and 17p losses, but the presence of 8q gains was associated with a higher risk of CNS involvement (p=0.05). We did not find any significant association between CNS involvement and the large number of oncogenic mutations studied. Conclusions: CNS involvement in MCL is associated with initial aggressive clinico-biological characteristics. Non-nodal MCL cases with a low number of genetic alterations did not present CNS involvement. Finally, the presence of 8q gains was associated with a higher risk of CNS infiltration. Table Initial Clinical Features Category N 5 yr-CNS involvement (%, 95%CI) HR p Performance status (ECOG) 〉 1 8/51 41.5 (+/-28) 4.2 .003 ≤ 1 7/128 9.4 (+/-5.5) Nodal disease Yes 14/185 13.3 (+/-7.6) 6.1 .05 No 1/77 1.4 (+/-2.7) Hemoglobin (g/L) 〈 105 12/93 24.7 (+/-14.7) 3.2 .05 ≥ 105 3/78 5.3 (+/-6.3) LDH 〉 ULN 4/89 27.1(+/-19.4) 6.7 ULN 11/114 21.6 (+/-14.9) 3.5 .04 〈 ULN 3/66 8.7(+/-10) Molecular & Pathological data Histological variant Blastoid 6/58 17.3 (+/-13.7) 3.5 .02 Others 8/156 1.3 (+/-7.2) Ki-67 〉 30% 5/44 17.5 (+/-14.9) 3.6 .06 ≤ 30% 3/61 6.7 (+/-9.4) SOX11 Positive 8/153 2.9 (+/- 5.7) 2.6 ns Negative 1/42 2.1 (+/-2.4) IGHV ≥97% 6/109 9.5 (+/-9.6) 1.9 ns 4 2/87 3.9 (+/-5.7) 1.1 ns ≤ 4 1/44 2.3 (+/-4.3) Chromotripsis Yes 1/17 12.5 (+/-22) 3.2 ns No 2/106 1.9 (+/-2.7) 8q gain Yes 2/30 13.1(+/-19) 7.5 .05 No 2/97 1 (+/-1.96) CNA: copy number alteration; IGHV: immunoglobulin heavy chain; LDH: Lactate dehydrogenase Disclosures No relevant conflicts of interest to declare.

Description: The genetic heterogeneity of multiple myeloma (MM) makes it unlikely that established or novel chemotherapy could be equally effective in all genetic subgroups. Therefore, genetics alone is insufficient to fully capture different disease outcomes, and there is growing body of evidence showing that detection of minimal residual disease (MRD), using immunophenotypic or molecular-based approaches, also provides powerful independent prognostic information particularly among transplant-eligible patients. However, it is perhaps in elderly MM, the major patient subgroup and in which optimal balance between efficacy and toxicity is critical, that sensitive response assessment could help to tailor patients’ treatment. Here, we used for the first time sensitive 8-color multidimensional flow cytometry (cut-off of 10-5) to monitor MRD among elderly MM patients included in the PETHEMA/GEM2010MAS65 trial (sequential chemotherapy with 9 cycles of bortezomib-melphalan-prednisone (VMP) followed by 9 cycles of lenalidomide-low dose dexamethasone (Rd), or alternating cycles of VMP and Rd up to 18 cycles). A single 8-color antibody combination (CD45-PacB/CD138-OC515/CD38-FITC/CD56-PE/CD27-PerCPCy5.5/CD19-PECy7/CD117-APC/CD81-APCH7) was used to detect phenotypically aberrant clonal plasma cells (PCs), and MRD-negativity was defined when

Description: INTRODUCTION AND AIMS Almost seventy percent of Acute Myeloid Leukemia (AML) patients will relapse. Fludarabine, Ara-C, Idarrubicine, G-CSF (FLAG-Ida) and FLAG-Ida and Gentuzumab-Ozogamicin (FLAGO-Ida) is frequently used before allogenic stem cell transplant (ASCT). There are several studies analyzing the risk factors for survival in relapsed/refractory patients, but none of them were performed exclusively in patients treated with FLAG-Ida. We analyzed the results of in the Spanish trials conducted by PETHEMA. We also present a predictive score system of survival prognosis based on the data of the patients enrolled METHODS This is a retrospective, multicentre study of the PETHEMA AML epidemiologic Registry. In PETHEMA protocols 98,99,2007,2010 and 2011, FLAG-Ida was included as salvage treatment in patients relapsed or resistant after a 2ndinduction based in 3+7. In PETHEMA trial 2007, FLAG-Ida (or FLAGO-Ida) was administered if complete remission (CR) was not attained in the first induction cycle. The use of FLGO in PETHEMA 2007 trial depended on the availability of GO for each centre. We use overall survival (OS) as final end point of the analysis. Univariate survival analysis was performed by Long-Rank test. Stepwise backward variable selection and Cox proportional hazard multivariable analysis was performed (covariables selected if p