ALBERT

Description: Diffuse large B-cell lymphomas (DLBCLs) can be divided into germinal-center B cell–like (GCB) and activated-B cell–like (ABC) subtypes by gene-expression profiling (GEP), with the latter showing a poorer outcome. Although this classification can be mimicked by different immunostaining algorithms, their reliability is the object of controversy. We constructed tissue microarrays with samples of 157 DLBCL patients homogeneously treated with immunochemotherapy to apply the following algorithms: Colomo (MUM1/IRF4, CD10, and BCL6 antigens), Hans (CD10, BCL6, and MUM1/IRF4), Muris (CD10 and MUM1/IRF4 plus BCL2), Choi (GCET1, MUM1/IRF4, CD10, FOXP1, and BCL6), and Tally (CD10, GCET1, MUM1/IRF4, FOXP1, and LMO2). GEP information was available in 62 cases. The proportion of misclassified cases by immunohistochemistry compared with GEP was higher when defining the GCB subset: 41%, 48%, 30%, 60%, and 40% for Colomo, Hans, Muris, Choi, and Tally, respectively. Whereas the GEP groups showed significantly different 5-year progression-free survival (76% vs 31% for GCB and activated DLBCL) and overall survival (80% vs 45%), none of the immunostaining algorithms was able to retain the prognostic impact of the groups (GCB vs non-GCB). In conclusion, stratification based on immunostaining algorithms should be used with caution in guiding therapy, even in clinical trials.

Description: The balance between actions of procoagulant and anticoagulant factors protects organisms from bleeding and thrombosis. Thus, antithrombin deficiency increases the risk of thrombosis, and complete quantitative deficiency results in intrauterine lethality. However, patients homozygous for L99F or R47C antithrombin mutations are viable. These mutations do not modify the folding or secretion of the protein, but abolish the glycosaminoglycan-induced activation of antithrombin by affecting the heparin-binding domain. We speculated that the natural β-glycoform of antithrombin might compensate for the effect of heparin-binding mutations. We purified α- and β-antithrombin glycoforms from plasma of 2 homozygous L99F patients. Heparin affinity chromatography and intrinsic fluorescence kinetic analyses demonstrated that the reduced heparin affinity of the α-L99F glycoform (KD, 107.9 ± 3nM) was restored in the β-L99F glycoform (KD, 53.9 ± 5nM) to values close to the activity of α-wild type (KD, 43.9 ± 0.4nM). Accordingly, the β-L99F glycoform was fully activated by heparin. Similar results were observed for recombinant R47C and P41L, other heparin-binding antithrombin mutants. In conclusion, we identified a new type of mosaicism associated with mutations causing heparin-binding defects in antithrombin. The presence of a fully functional β-glycoform together with the activity retained by these variants helps to explain the viability of homozygous and the milder thrombotic risk of heterozygous patients with these specific antithrombin mutations.

Description: Background FLT3, PIM1, PIM2 and CXCR4 are proteins implicated in the signal transduction of the regulation of proliferation, differentiation and survival of hematopoietic stem cells, at different levels. The impairing of these three processes, regulated by these molecules, constitutes the main hallmark of Acute Myeloid Leukemia (AML). The objectives of this work were to evaluate the expression level of the genes that codify such proteins in patients with AML; as well as correlate this level of expression with biological variables at diagnosis and survival. Methods peripheral blood or bone marrow samples from 31 healthy subject (HS) and 92 AML patients at diagnosis between 2004 and 2012 were studied. The median follow up was 14 months (69% of patients had died). We quantified the expression of FLT3, PIM1 and 2, and CXCR4 by real time PCR, employing primer pairs designed in our laboratory to amplify all known protein-coding transcripts; highlighting the presence of a 30.4% FLT3 ITD, 5.4% of other FLT3 mutations and 26% of NMP1 mutations. Results were then analyzed with the statistical package IBM“ SPSS” Statistics, v20 (IBM“, Nueva York). Results FLT3 was overexpressed in AML patients (FLT3 expression: controls 0.99 vs patients 36.97; p

Description: Abstract 2924 Background: Clonal composition and clone dynamic changes of neoplasms are a controversial issue, whose investigation is now facilitated by the development of massive parallel sequencing. Here we have analyzed the changes in the mutational spectrum associated with progression, treatment response and relapse in a multiple myeloma patient. We sequenced exomes for the primary quiescent-tumor, the progression and the relapsed samples. M&M: Patient and samples description Samples from asymptomatic, progression and relapse phases were compared by FISH and Whole exome massive parallel sequencing in a multiple myeloma patient carrying the t(4;14)(p16.3;q32) alteration. At relapse the cytogenetic study identified the presence of two major clones, 13q14 deletion and t(4;14)(p16.3;q32) in the 60% of the cells, and 17p13 deletion in the 12% of the cells. Whole exome sequencing was performed at CNAG (Barcelona, Spain) following standard protocols for high-throughput paired-end 76pb sequencing on the Illumina HiSeq2000 instruments (Illumina Inc., San Diego, CA). The variant calling was performed using an in house written software calling potential mutations showing a minimum independent multi-aligner evidence. Results: We performed whole exome sequencing on 3 tumor samples from the same patient: the first one at the time of diagnosis correspond to bone marrow infiltrated by 7% of plasma cells. The two additional samples, at progression and relapse, were done in CD138+ bone marrow cells, at this moment the percentage of infiltration was of 84% and 64% respectively. The germinal DNA from the same patient was used as reference. The mean coverage obtained for the four samples were 93x, with around 85% of bases with at least 20X coverage. After filtering, a total of 104 single nucleotide variations (SNV) were identified, some of them in more than one sample. The variations were classified into silent (25), missense (71), nonsense (6), and essential splice (2), according to their potential functional effect. In addition to t(4;14) and del13q14, progression and relapse samples shared 36 common SNVs. There were also some variants gained and/or loss in the different time points, suggesting the presence of at least five different clones, independent but related in their evolution. The two main clones were present in progression and relapse samples, but the ratio of the mutant alleles decreased in parallel to the decrement in the percentage of cells carrying on the described cytogenetic alterations Conclusions: There is a coupling between the cytogenetic and tumor sequence changes indicating that tumor at progression was composed by a dominant clone, together with multiple minor clones. Relapse after treatment was associated with multiple changes in the clone dynamics, progressive reduction of the main clone, emerging of new subclones and lost of minor clones. Dynamic changes along progression could be facilitated/induced by the therapy received. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2647 We have reported in B-NHL cell lines that the p38 MAPK was constitutively activated and was involved in the regulation of tumor cell resistance to cytotoxic drugs. Further, inhibition of this pathway reversed drug resistance. Based in these findings we hypothesized that the activation of the p38 MAPK pathway in patients with B-NHL may be associated with unresponsiveness to cytotoxic drug therapy. This study was designed to test this hypothesis. Eighty patients with Diffused Large B Cell Lymphoma (DLBCL) were used for analysis. Freshly derived tumor tissues from these patients were obtained from biopsies prior to any treatment. Tissue microarrays were prepared and examined by immunohistochemistry for the expression of both p38 MAPK and phosphorylated p38 MAPK (active). The antibodies were tested for specificity. The frequency of stained cells

Description: Abstract 3951 Aim: The aims of this study were on one hand, to evaluate the significance of achieving molecular response (MR) by fluorescent-PCR (F-PCR) of Ig genes in a prospective way, particularly in patients with singular cytogenetics' features. On the other hand, to compared MR with immunophenotypic response (IR) and complete remission by immunofixation (CR). Patients: 130 new MM patients who had achieved CR or VGPR were analyzed by PCR, multiparametric flow cytometry (MFC) and immunofixation at diagnosis, and after induction therapy or transplantation: 48 patients received conventional chemotherapy as induction therapy followed with ASCT and were included in the GEM2000 trial and the other 82 patients were enrolled in the GEM2005 clinical trials in which the induction and maintenance therapy were based on Bortezomib and/or Thalidomide. Methods: The molecular analysis of Immunoglobulin (Ig) gene rearrangements at diagnosis was carried out by fluorescent PCR in DNA from bone marrow samples according to the Biomed-2 protocols. Briefly three different multiplex PCRs: DHJ; Ig Kappa light chain (IGK) rearrangements K-VJ and Kappa deleting element (KDE). DHJ and light chain rearrangements detected at the diagnosis were used in the follow-up. The sensitivity of PCR was between 1/103 and 1/104 as Martínez P. et al previously published in BJH, 2008. Regarding MFC the following monoclonal antibody combinations (FITC/PE/PerCP-Cy5.5/APC) were used at the diagnosis to identify different aberrant plasma cells phenotypes which were used as patient-specific probes for Minimal Residual Disease. Results: 64 patients had negative F-PCR and 66 patients had positive F-PCR after induction therapy. The OS was 77.32% and 59.18%, respectively and the medians OS were 116 and 67 months for each group (P= 0.03). Regarding the PFS, 28 of the 64 F-PCR negative patients relapsed while there were 44 relapses in the group of positive F-PCR. The 5y PFS was 56.44% and 27.43% respectively; the medians PFS were 60 and 36 months respectively (P= 0.0005). The multivariate analysis showed that achieving molecular response by F-PCR had a independent prognostic value in PFS (P=0.003, HR 2.3). Interestingly amongst patients who achieved CR, F-PCR identified a population of patient with a better PFS, figure A (P= 0.04, HR 2.179).The applicability of the F-PCR was almost equivalent to that observed by MFC (91.5 vs. 92%). Nevertheless we found discordant results between these two response criteria. Approximately the 20% of patients who had achieved a MR no obtained IR. The opposite pattern also was observed (IR/noMR) in 10% of the patients. In order to see if the PCR showed differences in terms of survival in patients with high or low risk cytogenetics features at the diagnosis, we made two independent analysis: in one hand we analyzed 14 patients with high-risk cytogenetics [t(4;14), t(14;16) and/or del(17p)] and on the other hand 72 patients with standard-risk cytogenetics. In the former analysis, 4 of the 8 who had negative F-PCR relapsed after the induction therapy while the totality of the other six with positive F-PCR did. The medians PFS were 32 and 17 months respectively (P= 0.0002). In connection with the 72 patients with standard-risk cytogenetics, 13 of the 34 who had negative F-PCR relapsed while 24 of the 38 with positive F-PCR did. The medians PFS times were 75 and 38 months respectively, figure B (P= 0.01). Conclusion: F-PCR of IgH rearrangements is a simple, cheap and feasible method for evaluating response in MM patients after induction therapy or transplantation. Additionally achieving MR provides a similar prognostic value to IR in patients differentially treated, and particularly in patients with singular cytogenetics' features as well as in patients with CR by immunofixation. Disclosures: Paiva: Jansen-Cillag: Honoraria; Celgene: Honoraria. Rosiñol:Janssen: Honoraria; Celgene: Honoraria. Alegre:Janssen: Honoraria; Celgene: Honoraria. Blade:Centocor Ortho Biotech Research & Development: Research Funding. San Miguel:Janssen-Cilag: Honoraria; Celgene: Honoraria. Lahuerta:Celgene: Honoraria; Janssen: Honoraria.

Description: Key Points FOXP1 is downregulated in germinal centers, inversely to BCL6, whereby it regulates a network of genes, half of which are also BCL6 targets. In transgenic mice, constitutive FOXP1 expression impairs GC formation and function, which might contribute to B-cell lymphomagenesis.

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: Background Bortezomib-based combinations, including alkylating agents (VMP or CyBorD) or immunomodulatory drugs (VTD or RVD) have been established as regimens widely used in newly diagnosed MM patients. Bendamustine is a bifunctional alkylating agent effective in relapsed and/or refractory MM patients, and approved in Europe in combination with prednisone for elderly newly diagnosed MM. Since bendamustine may be more efficient than other alkylators, an attractive possibility would be to explore it in combination with bortezomib and prednisone (BVP) in newly diagnosed MM patients both transplant and non transplant candidates. Patients and Methods 60 newly diagnosed MM patients were included in the trial. The first cycle consisted on bendamustine at 90 mg/m2 given IV on days 1 and 4, in combination with bortezomib at 1,3 mg/m2 given IV on days 1, 4, 8, 11, 22, 25, 29 and 32 and prednisone at 60 mg/m2 given PO on days 1 to 4. In the following cycles, bendamustine was given on days 1 and 8, and bortezomib on days 1, 8, 15 and 22 (weekly schedule), and prednisone as it was previously described. Patients younger than 65 years proceeded to peripheral blood stem cell collection (PBSC) using growth factors alone after 4 cycles; HDT-ASCT was performed after 6 cycles. Patients older than 65 years received up to nine 28-day cycles. Results Between May 2011 and July 2012 enrollment was completed (60 pts). Median age was 61 years (range 38-82; 18 pts ≥65), 67% had ISS stage II/III, and 67% had unfavorable cytogenetics: t(4;14), t(14;16), del 17p or 1q gains by FISH. After a median of 6 cycles (2-9), 75% of patients achieved at least PR, including 16% of sCR, 9% CR and 28% of VGPR. Although the differences were not statistically significant, there was a trend to higher CR rate in the group of patients

Description: Background VMP and Rd are two of the most efficient and widely accepted regimens in the treatment of elderly newly diagnosed MM patients. In order to further improve the outcome of elderly patients, one possibility would be to use regimens including all these drugs simultaneously, but this may result into high toxicity. Alternatively, the use of these regimens (VMP and Rd) in a sequential or alternating scheme could improve the treatment of elderly patients. We hypothesized the alternating scheme would minimize the emergence of resistant clones, and would reduce the cumulative toxicity. In order to test this hypothesis we decided to compare VMP and RD in a sequential vs an alternating scheme. Patients and methods 241 patients were randomized to receive a sequential scheme consisting on 9 cycles of VMP followed by 9 cycles of Rd or the same regimens in an alternating approach (one cycle of VMP alternating with one Rd (half of the patients started by VMP and half by Rd) up to 18 cycles). VMP included the iv administration of bortezomib 1.3 mg/m2 twice weekly for 1 six-weeks cycle, followed by once weekly for 8 four-weeks cycles in combination with oral melphalan 9 mg/m2 and prednisone 60 mg/m2 once daily on days 1–4 of each cycle. Rd treatment consisted on lenalidomide 25 mg daily on days 1-21 plus dexamethasone 40 mg weekly. Results 121 patients were allocated to receive the sequential scheme and 120 the alternating regimen. Both arms were well balanced according to the baseline characteristics. 52% patients in the sequential arm and 55% in the alternating and had high risk cytogenetic abnormalities (t(4;14), t(14;16), del17p or 1q gains). After 9 cycles of treatment, in the sequential arm, 35 out of 66 (54%) achieved at least VGPR vs 51 out of 65 patients (78%) in the alternating arm (p=0.002), including sCR/CR rate of 28% vs 38% in the sequential and alternating arms, respectively (p=NS). Seven patients in each arm achieved immunophenotypic CR. Moreover, while four patients progressed in the sequential arm under treatment with VMP, no patients in the alternating scheme developed disease progression during the first 9 cycles, After a median follow up of 12 months, there was a trend for shorter TTP in the sequential as compared with the alternating scheme (18 m-TTP of 83% vs 89% (p=NS)). In terms of OS, 83% of patients in the sequential arm were alive at 18 m versus 93% in the alternating (p=NS). Patients who achieved sCR/CR had a significantly longer 18 m-TTP as compared with patients who didn’t achieve it in both sequential (100% vs 71%; p=0.006) and alternating arms (100% vs 79%; p=0.006) and this translated into a significant benefit in OS. No differences were observed in overall response rates and CR rates in standard and high risk patients. The 18 m-TTP was similar in standard and high risk groups in both sequential (86% vs 81%) and alternating arms (84% vs 94%), noting that 94% of patients receiving the alternating scheme were progression-free at 18 months. Regarding hematologic toxicity, the frequency of G3-4 neutropenia was slightly lower in the sequential than in the alternating arm (16% and 23%) and the same trend was observed for G3-4 thrombocytopenia (16% vs 20%). Concerning non-hematologic toxicity, 5% and 4% of the patients in the sequential and alternating arms developed G3-4 infections, respectively; the rate of G3-4 skin rash was 4% in the sequential and 3% in the alternating arm; 4% of patients in the sequential arm developed G3-4 peripheral neuropathy and 3% in the sequential arm. The rate of grade 3-4 thrombotic events was 2% in both arms. Nevertheless, the detailed evaluation of the toxicity will be done at the completion of the trial when all patients will have received the same amount of drugs in either a sequential or an alternating scheme (at the present time, 42 patients in the sequential arm were not yet at risk for the development of lenalidomide-related side effects). Conclusions The administration of melphalan, bortezomib, lenalidomide and steroids in elderly MM patients in a sequential or alternating scheme is feasible. Although longer follow-up is necessary, the alternating scheme may be superior in terms of response rate and outcome, as result of the early exposure of the plasma cell to different agents. Toxicity profile is acceptable. Aparently both schemes of therapy seems to overcome the poor prognosis of high risk cytogenetic. Disclosures: Mateos: Janssen, Celgene: Honoraria. Off Label Use: Lenalidomide plus dexamethasone is not approved for newly diagnosed MM patients. Ocio:Onyx: Consultancy, Research Funding; Novartis: Consultancy; Array Biopharma: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Celgene: Consultancy, Research Funding. San Miguel:Janssen, Celgene: Membership on an entity’s Board of Directors or advisory committees.