ALBERT

Description: A new mutation is described in the X-linked geneGATA1, resulting in macrothrombocytopenia and mild dyserythropoietic features but no marked anemia in a 4-generation family. The molecular basis for the observed phenotype is a substitution of glycine for aspartate in the strictly conserved codon 218 (D218G) of the amino-terminal zinc finger loop of the transcription factor GATA1. Zinc finger interaction studies demonstrated that this mutation results in a weak loss of affinity of GATA1 for its essential cofactor FOG1, whereas direct D218G-GATA1 binding to DNA was normal. The phenotypic effects of this mutation in the patients' platelets have been studied. Semiquantitative RNA analysis, normalized for β-actin messenger RNA, showed extremely low transcription of the GATA1 target genes GPIbβ and GPIXbut also a significantly lower expression of the nondirectly GATA1-regulated Gsα gene, suggestive of incomplete megakaryocyte maturation. In contrast, GPIIIa expression was close to normal in agreement with its early appearance during megakaryocyte differentiation. Flow cytometric analysis of patient platelets confirmed the existence of a platelet population with abnormal size distribution and reduced GPIb complex levels but with normal GPIIIa expression. It also showed the presence of very immature platelets lacking almost all membrane glycoproteins studied (GPIbα, GPIbβ, GPIIIa, GPIX, and GPV). Patients' platelets showed weak ristocetin-induced agglutination, compatible with the disturbed GPIb complex. Accordingly, electron microscopy of the patients' platelets revealed giant platelets with cytoplasmic clusters consisting of smooth endoplasmic reticulum and abnormal membrane complexes. In conclusion,GATA1 mutations can lead to isolated X-linked macrothrombocytopenia without anemia.

Description: Abstract 905 Introduction: Though dose-intensive strategies using chemoimmunotherapy have significantly improved mantle cell lymphoma (MCL) outcomes with prolonged progression-free survival (PFS), most patients still relapse over time. In the relapsed setting, MCL patients often develop chemoresistance and have a poor overall prognosis. The immunomodulatory agent lenalidomide has demonstrated tumoricidal and antiproliferative effects in MCL and clinical activity and safety in multiple phase II studies in aggressive non-Hodgkin's lymphoma. The objective of the MCL-001 “EMERGE” study was to examine the safety and efficacy of single-agent lenalidomide in subjects with MCL who relapsed or were refractory to bortezomib. Methods: This phase II, multicenter, single-arm, open-label study examined single-agent lenalidomide administered at 25 mg/d PO on days 1–21 of a 28-day cycle until disease progression, unacceptable toxicity, or voluntary withdrawal. The subjects were required to have had prior treatment with rituximab, cyclophosphamide and anthracycline (or mitoxantrone), and to have relapsed or progressed (

Description: Abstract 880 On behalf of the German Low-Grade Lymphoma Study Group (GLSG) and the European MCL Network Background: In younger patients with mantle cell lymphoma (MCL), autologous stem cell transplantation (ASCT) significantly prolonged response duration with a trend towards improved overall survival in a randomized trial of the European MCL Network (Dreyling et al., ASH 2008). A recently updated clinical trial of the German Low-Grade Lymphoma Study Group (GLSG) also reported a significantly prolonged response duration by the addition of Rituximab to first-line CHOP (Hoster et al., ASH 2008). By pooled analysis of these trials we investigated whether Rituximab and ASCT independently prolong response duration. Methods: The analysis included all advanced stage MCL patients of the trials “CHOP vs. MCP” (Nickenig et al., Cancer 2006), “CHOP vs. R-CHOP” (Lenz et al., JCO 2005) and European MCL trial 1 (Dreyling et al., Blood 2005) with complete or partial remission to either CHOP or R-CHOP first-line induction and randomization between ASCT and Interferon-α maintenance. Response duration was defined as time from the end of successful induction chemotherapy to relapse or death from any cause, overall survival from the end of successful induction chemotherapy to death from any cause. Stratified Kaplan-Meier curves for response duration and overall survival were calculated for the four treatment groups (CHOP without ASCT, CHOP with ASCT, R-CHOP without ASCT and R-CHOP with ASCT). By multiple Cox regression we tested whether the impact of Rituximab (R) and ASCT was independent and additive. Results: One-hundred and eighty patients with MCL were evaluable, 80 treated with R-CHOP, 78 with ASCT (CHOP without ASCT: 56, CHOP with ASCT: 46, R-CHOP without ASCT: 44 and R-CHOP with ASCT: 34). Median age was 55 years (range 34-65), the MIPI classified 71% as low risk, 22% as intermediate risk, and 6% as high risk, and baseline characteristics were comparable between treatment groups. With a median follow-up of 63 months, median response duration was 16 months after CHOP without ASCT, 26 months after R-CHOP without ASCT, 39 months after CHOP with ASCT, and 41 months after R-CHOP with ASCT. In multiple Cox regression including R and ASCT, the hazard ratios of R (0.60, 95% CI 0.42-0.86, p = 0.0056) and ASCT (0.50, 95% CI 0.35-0.70, p = 0.0001) were independently significant. There was no interaction between R and ASCT (p=0.43). Median overall survival was 54 months after CHOP without ASCT, 66 months after R-CHOP without ASCT, 90 months after CHOP with ASCT, and not reached after R-CHOP with ASCT. The hazard ratios for OS were 0.70 (95% CI 0.44-1.12, p = 0.14) for R and 0.63 (95% CI 0.41-0.97, p = 0.0379) for ASCT. Conclusions: Our results indicate an additive effect of ASCT and Rituximab in combination with CHOP on response duration in advanced stage MCL patients and support strategies of several study groups to combine Rituximab-containing induction therapies with ASCT in younger MCL patients. However, even after combined treatment approaches, delayed relapses have been observed, supporting the use of maintenance therapy and the introduction of molecular targeted therapeutical strategies. Disclosures: Hoster: Roche: Travel Support. Off Label Use: Rituximab in Mantle Cell Lymphoma. Pfreundschuh:Roche: Membership on an entity's Board of Directors or advisory committees. Dührsen:Roche: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Gisselbrecht:Roche: Research Funding, Speakers Bureau. Unterhalt:Roche: Travel Support. Dreyling:Roche: Honoraria, Research Funding.

Description: Initial findings from the Phase 3 trial of Zevalin consolidation of first remission in patients with advanced follicular lymphoma showed a significant improvement in PFS following Zevalin consolidation as compared to no further treatment, ie, 37.5 vs. 13 months (Hagenbeek et al, ASH 2007). Patients had been randomised to either no further therapy or Zevalin consolidation (250 mg/m2 rituximab on day -7 and on day 0, followed on day 0 by Zevalin 0.4 mCi/kg; maximal dose 32 mCi). Herein we report on an additional 1-year follow-up of the FIT study, at a median of 42 months (range: 2 – 77), which includes 409 patients who achieved a complete or partial response (CR or PR), after induction therapy. Patient demographics remain similar in the Zevalin (n=202) and control groups (n= 207): males 48%/50%; median age 55/53 yrs; at diagnosis, stage III 35.2%/31%, stage IV 64%/66%. Response status prior to randomization was also similar between study arms: 107 patients in CR/CRu and 100 pts in PR in the Zevalin arm and 108 pts in CR and 88 pts in PR in the control arm. The 48-month PFS is 52% in the Zevalin arm compared with 31% in the control arm. As shown in the figure 1A, patients with a CR after induction therapy achieved a median PFS of 〉67 months for the Zevalin arm compared with 30.8 months for patients who received no further treatment (HR 0.61 [95% CI .41–.91]; P= 0.015). In figure 1B, among patients with a PR after induction, the median PFS was 29.6 months for those on the Zevalin arm vs 6.7 months for those on the control arm (HR 0.36 [95% CI .25–.51]; P

Description: In recent reports, granulocyte PRV-1 mRNA has been shown to be over-expressed in the majority of polycythemia vera (PV) patients and with a variable frequency in essential thrombocythemia (ET) patients. Here we evaluate PRV-1 mRNA quantification in the diagnosis of PV and ET. Following patients were included in the study: 23 PV patients; 5 secondary erythrocytosis (SE) cases, 4 by lung disease and 1 by a hemoglobinopathy (Hb San Diego); 4 relative erythrocytosis (RE) cases; and 13 ET patients. PV and ET diagnosis was made according to the WHO criteria. At time of sampling 43% of PV patients and 54% of ET patients were receiving myelosuppressive therapy. To establish a cut-off value 28 healthy persons (15 female, 13 male) were also analysed. RNA was extracted from whole-blood leucocytes and quantified by real-time TaqMan PCR. ABL was used to control the mRNA quality (Ct ABL 〈 27.0 – mean 24.7) and to normalise the PRV-1 values. PRV-1 primer and probe sequences originated from Brohée D et al, Blood, 2002, 100:3146a and ABL primer and probe sequences from Beillard E et al, Leukemia, 2003, 17:2474. The results were expressed as a ratio PRV-1/ABL which could be calculated using the ΔΔCt method as the efficiencies of both PCR reactions were almost equal (for PRV-1 93% and ABL 94%). Within-run and between-run reproducibility of a normal sample had a CV of 10.1 % and 28.6% and of a highly expressing sample a CV of 9.5% and 21.6%. The values of the healthy controls had a normal distribution and a mean of 0.28 with a SD of 0.35. A cut-off level was determined as the upper 95% percentile and was 0.98. Results of the different patient groups are summarised in the table. The differences between PV vs healthy controls, PV vs SE/RE and ET vs healthy controls were significant with a p-value of respectively

Description: BACKGROUND: the combination of rituximab (RTX) and lenalidomide (LEN) has been shown to be synergistic in pre-clinical models and in patients (pts) with previously untreated and relapsed follicular lymphoma (FL). Furthermore, LEN demonstrated single agent efficacy in pts with mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL). We hypothesized that the combination of obinutuzumab (GA), a glycoengineered type II anti-CD20 antibody, with LEN (GALEN) might be even more efficient while retaining a similarly manageable safety profile. In 2012, we started a phase Ib/II study to assess the safety and efficacy of this combination (ClinicalTrials.gov: NCT01582776) in pts with relapsed/refractory lymphoma. Phase IB defined the recommended dose (RD) of LEN as being 20mg in combination with GA. Here, we report the results of the Phase II part assessing efficacy and safety of GALEN therapy during a 6 months induction followed, for responders, by a reduced LEN maintenance GALEN regimen (in a cohort of relapsed/refractory aggressive NHL (DLBCL or MCL) pts. METHODS: pts with a histologically confirmed DLBCL (de novo or transformed low-grade NHL) or MCL were eligible if they had a ECOG PS of ²2, a life expectancy 〉3 months (mo) and had previously received ³1 prior therapy including at least one RTX-containing regimen. Induction treatment consisted of LEN 20 mg given orally once daily on days 1-21 of a 28-day cycle for the first cycle and on days 2-22 of a 28-day cycle from cycles 2 to 6. Intravenous infusions of GA were given at a flat dose of 1000mg on days 8, 15, and 22 of cycle 1 and at D1 of cycles 2 to 6 (total of 8 infusions). Responding pts then received 12 cycles of LEN at 10 mg daily on days 2-22 every 28 days for a total of 18 cycles and GA 1000mg every 8 weeks for 12 additional cycles until progression or unacceptable toxicity. All pts were required to take daily aspirin (100 mg) for deep vein thrombosis (DVT) prophylaxis during study period. Pts who were unable to tolerate aspirin and had a prior history of DVT or were at high risk for a DVT received low molecular weight heparin therapy or warfarin (coumadin) treatment. The primary study endpoint was overall response rate (ORR) by investigator assessment at the end of induction according to 1999 IWG criteria. Secondary endpoints included ORR and complete response (CR) according to IWG 2007, best ORR, duration of response, progression-free survival, overall survival (OS) and safety. RESULTS: ninety-one pts (DLBCL [n=77 including 18 transformed], MCL [n=13], and other [n=1]) were enrolled between June 2014 and March 2015 at 22 LYSA centers in France and Belgium. At baseline, median age was 70 (range, 48-84) years with 64.7% men, ECOG PS of 1 (47.1%) or 2 (18.8%), 83.5% Ann Arbor stage III-IV and 66.3% with elevated LDH. Median number of prior systemic therapies was 2 (range, 1-9) including haematopoietic stem cell transplantation in 17pts (20%); 61% were refractory to a RTX-containing regimen and 53% refractory to last prior therapy. Three pts were withdrawn before receiving any treatment due to major protocol violation, concurrent illness or ulceration of the tumor in the stomach and 3 pts received one of both study drugs, leaving 85 pts assessable for efficacy. Cut-off date was March 25th 2016. With amedianfollow-up of 14.5 mo,39pts (45.8%) completed induction (32 DLBCL and 7 MCL) and 20 (23.5%) are still ongoing in maintenance (16 DLBCL, 4 MCL). At the time of the cut-off, 44pts (51.8%) had died mainly due to lymphoma (88.6%). Response at the end of induction, best ORR during induction and OS are summarized in the table. Table Analysis of other secondary endpoints and correlation with cell of origin is ongoing. The most common treatment emergent AEs during induction (all grades 〉10%pts/ % grade 3/4) were neutropenia (51.1/31.8), infections (45.5/12.5), constipation (30.7/0.0), cough (21.6/1.1), diarrhea (17.0/3.4), nausea (17.0/0) anemia (15.9/10.2), thrombocytopenia (15.9/10.2), muscle spasms (13.6/0.0), fatigue (12.5/1.1) and dyspnea (11.4/4.5). Febrile neutropenia occurred in 4.5% pts. There was onlyone secondprimary malignancy (basal carcinoma). CONCLUSION: oral LEN plus GA infusion is effective in relapsed or refractory DLBCL and MCL pts with no unexpected toxicity. Longer follow-up and additional translational studies are needed to identify the subset of pts most benefiting of this regimen. Correlative studies will be presented at the meeting. Table Table. Disclosures Morschhauser: celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; servier: Consultancy, Honoraria; gilead: Consultancy, Honoraria; janssen: Honoraria. Cartron:Roche: Consultancy, Honoraria; Celgene: Honoraria; Gilead: Honoraria; Jansen: Honoraria. Salles:Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Gilead: Honoraria, Research Funding; Mundipharma: Honoraria; Novartis: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding. Maerevoet:ARGN-X: Membership on an entity's Board of Directors or advisory committees; roche: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Bonnet:ROCHE: Membership on an entity's Board of Directors or advisory committees; SERVIER: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; JANSSEN: Membership on an entity's Board of Directors or advisory committees.

Description: Introduction: The CellaVision Advanced Red Blood Cell (RBC) Software Application is a new software for advanced morphological analysis of RBC which automatically performs a preliminary characterization and grouping of RBC into 21 morphological categories, including schistocytes. Upon automated classification, the software offers the possibility of reclassification of RBC by the operator. The aim of this study was to evaluate the schistocyte analysis by the CellaVision Advanced RBC Application. Methods: Schistocyte counts were evaluated comparing the automated count on a CellaVisionTM DM96, both before and after reclassification, with the reference manual microscopic method. 76 samples of both hospitalized patients and healthy donors were analyzed. Results: Within-run, between-run and between-observer coefficients of variation were lower when counted with the CellaVision compared to the manual microscopic count. The very high sensitivity but rather poor specificity implicates the need for reclassification by the operator, following automated analysis. After reclassification, method comparison studies revealed good agreement with the manual microscopic method, with however slightly higher values of schistocytes for the automated analysis. Conclusion: The CellaVision Advanced RBC Software Application provides a sensitive, accurate and reproducible measurement of schistocytes in peripheral blood. Furthermore, it is an easy-to-use software and an excellent teaching tool that might contribute to standardization in the investigation of schistocyte-related conditions. Disclosures No relevant conflicts of interest to declare.

Description: Few studies have reported on the efficacy of donor lymphocyte infusions (DLI) for myelodysplastic syndromes (MDS) relapsing after T-cell depleted allogeneic stem cell transplantation. Since 1996 we have treated 11 patients with DLI as a single therapeutic modality (without chemotherapy) for a relapse of MDS after allogeneic stem cell transplantation. Median age of the patients was 56 years (range 23 to 73 years). Initial diagnosis was RAEB (4 patients), RAEBt (3 patients), secondary AML(3 patients) and MDS with fibrosis (1 patient). Conditioning was myeloablative in 10 patients ( TBI -Cyclophosphamide, Busulfan-Cyclophosphamide or Busulfan-Melphalan) and non-myeloablative in 1 patient (Busulfan-Fludarabine-ATG). Source of stem cells was peripheral blood in all patients. Donors were HLA-identical siblings in 10 patients and HLA-matched unrelated in 1 patient. Allografts were T-cell depleted with Campath 1H in the bag in 8 of 11 patients. GVHD prophylaxis was cyclosporin in all patients combined with methotrexate in the 3 patients receiving unmodified stem cells. Relapse of MDS occurred at a median of 123 days (54 to 373 days) after allograft. At the time of DLI the percentage of blast cells in the bone marrow ranged from 3 % to 50 % (median 6 %); donor chimerism ranged from 14 % to 91 % (median 72%). The total dose of CD3 lymphocytes ranged from 0.1 to 30 x 10 7 /kg (median 0.6 x 10 7/kg) and was administered in 1 to 4 infusions (median 1). Time interval between first and second DLI varied between 7 and 180 days.Three of the 11 patients achieved a durable complete remission with 100 % donor chimerism after DLI. These 3 patients are currently disease free at 20, 30 and 40 months from the first DLI. All durable responses were associated with limited chronic GVHD requiring temporary immunosuppressive therapy. Time interval between the first DLI and the achievement of complete donor chimerism varied between 6 weeks and 9 months. One of the 11 patients achieved a short state of complete chimerism for 4 weeks and died from relapse 146 days after DLI. Five of the 11 patients progressed after DLI and 4 of them died from disease progression at 29 to 112 days after first DLI. Two patients died at 26 and 97 days after the first DLI from acute GVHD grade IV after having received only 1 x 10 6 CD3 lymphocytes /kg from HLA-identical sibling donors. None of the 6 patients with a complex karyotype responded to DLI while 3 of 3 patients with a single chromosomal abnormality ( t(1,14), t(1,17), t(1,3)) had a durable response (p-value = 0.01). We conclude that DLI is a powerful therapy for about 30 % of MDS patients relapsing after T-cell depleted allogeneic stem cell transplantation. DLI may result in durable remissions particularly in patients with non-complex cytogenetic abnormalities. However, even low doses of donor T lymphocytes may cause mortality from severe GVHD.

Description: Introduction: Treatment of mantle cell lymphoma (MCL) in younger patients (pts) is still a challenge, with questions about best induction regimen before autologous stem cell transplantation (ASCT) and impact of Rituximab. We report here the final results with extended follow-up of a prospective phase II trial. Methods: Patients under 66 years with histologically proven, stage III-IV, MCL were included. Treatment consisted of three courses of CHOP with Rituximab at the third one and three courses of RDHAP. Peripheral blood stem cells harvest was performed and responding pts were eligible for an ASCT after high dose radio-chemotherapy with TAM6 (TBI 10 Gy, Aracytine 6 g/m², Melphalan 140 mg/m²) or BEAM if TBI could not be performed. Results: From May 2000 to September 2003, 60 pts were included. Median age was 57 years. Characteristics of patients were as follow : bone marrow involvement 85%, leukemic disease 48%, gastrointestinal involvment 52%, PS〉1 6%, LDH 〉 1N 38%. Overall response rate was high with 93% after (R)CHOP and 95% after RDHAP. Interestingly, CR was uncommon after (R)CHOP (12%), whereas high proportion of patients (61%) were in CR after RDHAP, suggesting higher efficacy of high dose AraC. Forty-nine pts were autografted (41 with TAM6) : all patients but two (96%) were in CR. With a median follow-up of 67 months, median EFS was 83 months and median OS was not reached. Five years OS was 75%. Neither toxic death nor unexpected toxicities were observed. The comparison with our previous French oligocentric study using the same regimen but without Rituximab (Lefrere, Hematologica 2007) suggests a better outcome when Rituximab is added (median EFS : 51 months). Conclusion: This study confirms that regimens containing Aracytine and Rituximab are safe and prolong survival and may even induce cure in MCL patients. Thus, they should be used in induction treatment before ASCT. This regimen is currently compared with the classical RCHOP induction in a multicentric European protocol within the EMCL network.

Description: There is no generally established prognostic index for patients with mantle cell lymphoma (MCL), because the International Prognostic Index (IPI) and Follicular Lymphoma International Prognostic Index (FLIPI) have been developed for diffuse large cell and follicular lymphoma patients, respectively. Using data of 455 advanced stage MCL patients treated within 3 clinical trials, we examined the prognostic relevance of IPI and FLIPI and derived a new prognostic index (MCL international prognostic index, MIPI) of overall survival (OS). Statistical methods included Kaplan-Meier estimates and the log-rank test for evaluating IPI and FLIPI and multiple Cox regression for developing the MIPI. IPI and FLIPI showed poor separation of survival curves. According to the MIPI, patients were classified into low risk (44% of patients, median OS not reached), intermediate risk (35%, 51 months), and high risk groups (21%, 29 months), based on the 4 independent prognostic factors: age, performance status, lactate dehydrogenase (LDH), and leukocyte count. Cell proliferation (Ki-67) was exploratively analyzed as an important biologic marker and showed strong additional prognostic relevance. The MIPI is the first prognostic index particularly suited for MCL patients and may serve as an important tool to facilitate risk-adapted treatment decisions in patients with advanced stage MCL.