ALBERT

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: The prognosis of fludarabine-refractory CLL is poor with a median survival time of 10 months. Intravenous Campath-1H (Alemtuzumab) was approved for fludarabine refractory CLL based on a remission rate of 33% and a median survival time of 16 months (Keating et al., Blood 2002). While the standard route of Campath-1H administration is by 2 hour intravenous infusion, the subcutaneous route was shown to be safe and efficacious in first line treatment (Lundin et al., Blood, 2002). The CLL2H trial of the GCLLSG was initiated to evaluate the subcutaneous application of 3 x 30 mg Campath-1H weekly for a maximum of 12 weeks in fludarabine refractory CLL after intravenous dose escalation (3, 10, 30 mg). The current interim analysis is based on the first 50 consecutive patients enrolled until April 2004. Median age was 63 (range 35–79) years, 70% were male, and a median number of 4 (range 1–7) prior lines of therapy had been given. Dose escalation was given in 46, subcutaneous continuation of therapy in 44, and treatment has not been started in 4 patients, respectively. Intravenous dose escalation was accompanied by no or mild (grade I-II) rigors and fever after premedication with paracetamol and antihistamines in the majority of patients, while grade III/IV infusion reactions were rare (n=3). Subcutaneous treatment was continued on an outpatient basis in all cases but had to be temporarily interrupted in 28 patients due to neutropenia (n=18), anemia (n=1), thrombocytopenia (n=3), infections (n=6; CMV reactivations n=3), and was stopped early in 22 cases due to progression (n=9), CMV reactivation (n=3), hematotoxicity (n=7), hemolysis (n=1), side effects (n=1), and infection (n=1). There were 6 cases of CMV reactivation, 5 of which promptly responded to oral valgancyclovir. During subcutaneous treatment toxicity was mostly grade I/II apart from hematotoxicity (grade III/IV anemia: 14%, thrombocytopenia: 34%, neutropenia: 66%) and grade III/IV infections (24%). After a median follow up time of 9.3 months 18 deaths have occurred (progression n=12, sepsis n=3, not CLL related n=2, before treatment start n=1). The overall response rate was 36% (CR 2%, PR 34%), the median progression free survival time was 9.7 months, and median overall survival time was 13.1 months. Analyses of genetic risk factors showed an unmutated VH status in 62% and high-risk genomic aberrations in the majority of patients (17p-: 29%, 11q-: 29%, +12q: 18%, 13q- single: 16%). Responses (CR or PR) were observed in 14 of 27 VH unmutated, 5 of 13 11q-, and 7 of 13 17p- cases. In conclusion, Campath-1H given via the subcutaneous route appears to be feasible in an outpatient setting in a high risk population of fludarabine-refractory CLL and appears to be of similar efficacy as compared to intravenous administration. Most importantly, genetic high risk subgroups with unmutated VH, 11q- or 17p- appear to respond to Campath-1H. An amendment has been activated including prophylactic pegfilgrastim and allowing subcutaneous dose-escalation. An update of this trial with ongoing recruitment and extended follow-up will be presented.

Description: Hodgkin- and Reed-Sternberg (HRS) cells microdissected from 41 classical Hodgkin lymphomas (cHL) of 40 patients comprising 8 lymphocyte-rich (cHL-LR), 16 nodular sclerosis (cHL-NS), 15 mixed-cellularity (cHL-MC), and 2 lymphocyte-depletion (cHL-LD) subtypes were analyzed by comparative genomic hybridization for recurrently imbalanced chromosomal subregions. Chromosomal gains most frequently involved chromosome 2p (54%), 12q (37%), 17p (27%), 9p and 16p (24% each), and 17q and 20q (20% each), whereas losses primarily affected chromosome 13q (22%). Using fluorescence in situ hybridization, amplification of the REL oncogene was demonstrated within a distinct 2p15-p16 amplicon. The high frequency of 2p overrepresentations including REL, particularly in cHL-NS (88%), suggests that an alternative mechanism of constitutive activation of nuclear factor NF-κB is a hallmark of HRS cells. Hierarchical cluster analysis of chromosomal imbalances revealed a closer relationship among cHL-NS than other subtypes. Furthermore, there is a tendency for different subtypes of cHL-MC tumors characterized by different ages at the time of tumor onset and gain of chromosome 17p. The imbalance pattern of cHL subtypes suggests that different molecular pathways are activated, with REL or other genes on chromosomal band 2p15-p16 playing a fundamental role in the pathogenesis of classical Hodgkin lymphoma.

Description: Fludarabine-refractory CLL has a poor prognosis with a median overall survival time of less than 12 months despite salvage chemotherapy and intravenous alemtuzumab (Campath-1H) is the approved treatment based on a remission rate of 33% and a median survival time of 16 months (Keating et al., Blood 2002). The CLL2H trial of the GCLLSG was initiated to evaluate the subcutaneous application of 3 × 30 mg alemtuzumab weekly in fludarabine refractory CLL. The current analysis is based on 109 consecutive patients enrolled until completion of the trial in April 2006. Median age was 63 (36–81) years, 71% were male. A median number of 3 (1–9) prior lines of therapy had been given. Subcutaneous treatment was performed on an outpatient basis in all cases and had to be temporarily interrupted in 68 patients due to neutropenia (43%), anemia (6%), thrombocytopenia (3%), infections (40%, CMV reactivations 30%), and was stopped early in 63 cases due to insufficient response (44%), hematotoxicity (16%), infection (17%), and CMV reactivation (13%). The median alemtuzumab dose given was 722 (3–2203) mg. Toxicity was mostly grade I/II apart from hematotoxicity (grade III/IV anemia: 42%, thrombocytopenia: 52%, neutropenia: 54%) and grade III/IV infections (25%). After a median follow up time of 21.4 months, 56 deaths have occurred (due to progression 52%, infections 39%, not CLL related 9%). The overall response rate was 33% (CR 4%, PR 27%), the median progression free survival time was 7.7 months, and median overall survival time was 19.1 months. Genetic high-risk factors were present in the vast majority of cases (unmutated VH 66%, 17p–29%, 11q–19%, TP53 mutation 39%). Responses (CR or PR) were observed in 22% of VH unmutated, 24% of 11q-, 39% of 17p-, and 33% of TP53 mutated cases. Progression free survival and overall survival were not significantly different when comparing the genetic subgroups, particularly TP53 mutated, 11q-, and 17p- (see figure). In conclusion, subcutaneous alemtuzumab is feasible in an outpatient setting in a high-risk population of fludarabine-refractory CLL and appears to be of similar efficacy as by intravenous administration. Most importantly, genetic high risk subgroups with unmutated VH, 11q- or 17p- appear to respond to alemtuzumab. Figure Figure

Description: Abstract 2937 Introduction: Chromosomal aberrations in myelodysplastic syndromes (MDS) play a major role in diagnostics, pathogenesis, prognosis, and, more recently, in treatment allocations. The acquisition of clonal abnormalities in pts with initially normal karyotype, the expansion of an aberrant cell clone with a given anomaly or the occurrence of new abnormalities are called karyotype evolution (KE). A model of stepwise cytogenetic changes is proposed, but only a few systematic studies had focused on this phenomenon as yet. Chromosomal anomalies can be detected by conventional chromosome banding analyses of bone marrow (bm) metaphases and most of them are provable by Fluorescence in Situ Hybridization (FISH) of circulating CD34+ cells from peripheral blood (pb). Aims: To closely follow chromosomal aberrations from pb, to assess karyotype evolution and rare anomalies and to correlate the molecular-cytogenetic results with pb blood counts, bm morphology and treatment modalities. For this purpose, we initiated a German multicenter prospective, non-interventional diagnostic study in October 2008. Methods: Sequential FISH analyses were performed on immunomagnetically enriched circulating CD34+ cells from pb as follows: a “super-panel” (D7/CEP7, EGR1, CEP8, CEP XY, D20, p53, IGH/BCL2, TEL/AML1, RB1, MLL, 1p36/1q25, CSF1R) was used for initial screening, every 12 months during follow-up and in every case of suspected progression. A “standard-panel” (EGR1, D7/CEP7, CEP8, p53, D20, CEP X/Y, TEL/AML1) was performed for analyses at short intervals every 2 months in the 1st year and every 3 months during the 2nd year. Results were also correlated with those of conventional banding and FISH analyses performed on bm samples, which typically were collected every 6 months. Results: As yet, 205 pts have been included in the study. Concerning median age, gender distribution, and MDS subtypes (according to WHO and IPSS) the study cohort was representative for the disease. Up to date, chromosomal aberrations were detectable in 126 pts. (62%) by FISH from pb. Most of them had 1 or 2 anomalies, but there were even pts with 4 or more aberrations. The most common aberrations- as known from other studies based on bm analyses- were del(5q) (64%), aberrations of chromosome 7 (del(7q)/-7) (25%), allelic loss of p53 (12%), +8 (11%), del(20q) (10%) and aberrations of chromosome 21 (del(21q)/-21/+21) (7%). Interestingly, a 12p-deletion was detectable as often as a trisomy 8 (11%), and in most cases as part of complex aberrations. A loss of p53 was significantly associated with aberrations of chromosome 7: 67% of pts with aberrations of chromosome 7 showed an allelic loss of p53 at the same time. In 4 cases, a coexistence of del(7q) and -7 in the same clone was observed. Within a median observation time of 7 months (2-21), a KE was detectable from pb by FISH in 17 pts (13%) out of 135 with at least 2 sequential analyses. In most cases del(7q)/-7 developed as a new anomaly, and was followed in frequency by aberrations of chromosome 21. Conclusions: FISH analyses of enriched circulating CD34+ cells are a feasible, less-invasive method to detect chromosomal aberrations in MDS pts and to follow the clone size from peripheral blood. By that method we are able to observe karyotype evolution as a step wise process in detail and to follow even rare abnormalities to learn more about their clinical and prognostic impact. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1632 Background: OFA is a fully human monoclonal antibody that binds to both the large and small extracellular loops of CD20. OFA is currently approved for patients (pts) with refractory chronic lymphocytic leukemia and has demonstrated activity in non-Hodgkin's lymphomas, including follicular lymphoma (FL). We previously reported results of a phase II study of OFA in combination with CHOP (cyclophosphamide 750 mg/m2, doxorubicin 50 mg/m2, vincristine 1.4 mg/m2, prednisone 100 mg daily for 5 days) chemotherapy (O-CHOP) in pts with previously untreated FL (Czuczman et al. Br J Haematol. 2012;157:438). We now report updated efficacy, safety and pharmacokinetic (PK) follow-up data for this study. This trial is registered at www.clinicaltrials.gov (NCT00494780). Methods: Fifty-nine pts with previously untreated FL were randomized to OFA 500 mg (n = 29) or 1000 mg (n = 30) on day 1, with CHOP on day 3, every 3 weeks for 6 cycles. The primary end point was overall response rate (ORR), as assessed by an independent end points review committee. Secondary end points included complete response (CR), progression-free survival (PFS), overall survival, adverse events (AEs) and PK. Follow-up assessments after therapy were done every 3 months (mo) until mo 12 and then every 6 mo until alternative FL therapy or mo 60. Positron emission tomography (PET) was done at baseline and 3 mo after last therapy. Blood samples for PK analyses were collected to determine OFA serum concentrations, and noncompartmental methods were used to estimate PK parameter values. Results: Fifty-eight pts received therapy; 1 pt in the 1000-mg group withdrew before initiation of therapy. The ORR was 90% for the 500-mg group (n=29) and 100% for the 1000-mg group (n=29); 55% of pts achieved CR or unconfirmed CR (CRu), including 67% of pts with a Follicular Lymphoma International Prognostic Index (FLIPI) score of 3–5. At baseline, 57 pts were PET positive, and 49 pts underwent repeat PET scans after therapy. Forty of 49 pts (82%) became PET negative, including 27 of 29 (93%) pts who achieved CR/CRu and 13 of 20 pts (65%) who achieved partial response (PR). With a median follow-up of 33.8 mo, the median PFS for the 500-mg group was 27.6 mo and the median PFS for the 1000-mg group was not reached (P=0.46). Median PFS for pts with FLIPI scores of 0–1 (n=17), 2 (n=20) and 3–5 (n=21) was not reached, 27.6 mo and 27.6 mo, respectively (P=0.68). Median PFS for pts (n=32) who achieved CR/CRu was also not reached and was 28.3 mo for pts (n=23) achieving PR. Median PFS for PR pts who were PET positive and PET negative after therapy was not reached and 28.3 mo, respectively. No deaths have been reported. No hematologic serious AEs (SAEs) were experienced during the follow-up period. During the follow-up period, non-hematologic SAEs were reported in 1 pt in the 500-mg group (pneumonia) and 5 pts in the 1000-mg group (abdominal hernia, erysipelas, intervertebral disc protrusion, meniscus lesion and vulval cancer); none were ofatumumab-related. After repeated dosing, OFA clearance values were 6.3 and 5.9 mL/h, and half-life values were 27.2 and 26.8 days in the 500-mg and 1000-mg groups, respectively. Conclusions: O-CHOP achieved durable remissions in previously untreated pts with FL. There were no observed PK or PFS differences between the 500-mg and 1000-mg arms, but the study was not powered to detect such differences. O-CHOP was effective in pts with high-risk FLIPI scores, and CR/CRu and PFS rates were not affected by FLIPI score. PET status after therapy did not predict PFS in responding pts, although the study was too small to make such a determination. These results indicate that O-CHOP should be studied as a therapy for FL pts with high-risk FLIPI scores. Disclosures: Czuczman: GlaxoSmithKline: Advisory board Other, Honoraria. Off Label Use: Ofatumumab in follicular lymphoma. Belada:GlaxoSmithKline: Research Funding. Mayer:Roche: Consultancy, Research Funding; GlaxoSmithKline: Consultancy, Research Funding. Gupta:GlaxoSmithKline: Employment. Lin:GlaxoSmithKline: Employment, Equity Ownership. Winter:GlaxoSmithKline: Employment, Equity Ownership. Goldstein:GlaxoSmithKline: Employment, Equity Ownership. Jewell:GlaxoSmithKline: Employment, Equity Ownership. Lisby:Genmab: Employment, Equity Ownership.

Description: The unique clinicopathologic features of Hodgkin lymphoma (HL) are due to the multiple cytokines produced by its neoplastic cells, the Hodgkin and Reed-Sternberg (HRS) cells. Cytokine signaling is mediated through the signal transducer and activator of transcription (STAT) family of transcription factors. Immunoblotting and immunohistochemistry were used to examine cell lines and tissue sections derived from patients with HL and non-Hodgkin lymphoma (NHL) for expression of activated STAT proteins. Constitutive phosphorylation of STAT6 and STAT3 was common in HL. STAT6 was constitutively phosphorylated in 5 of 5 HL cell lines and in HRS cells from 25 of 32 (78%) classical HL cases. STAT3 was constitutively phosphorylated in 4 of 5 HL cell lines and in HRS cells from 27 of 31 (87%) classical HL cases. Only 4 of 24 NHL cases demonstrated constitutive STAT6 activation, whereas STAT3 activation was observed in 6 of 13 (46%) cases of B-cell NHL and 8 of 11 (73%) cases of T-cell NHL. Constitutive STAT5 phosphorylation was not a common feature of HL or NHL. STAT6 mediates signaling by interleukin 13 (IL-13), a cytokine frequently expressed by HRS cells. Antibody-mediated neutralization of IL-13 resulted in significant decreases in both cellular proliferation and levels of phosphorylated STAT6 of HL cell lines. In conclusion, constitutive STAT6 phosphorylation is a common and distinctive feature of HRS cells in classical HL, whereas STAT3 activation was regularly present in both HL and NHL. These results suggest that IL-13 signaling is largely responsible for the constitutive STAT6 activation observed in HRS cells and further implicate IL-13 as an important growth factor in classical HL.

Description: Abstract 3816 Background Conventional chromosome banding (CCB) analyses of bone marrow (bm) metaphases represent the gold standard of cytogenetic diagnostics in myelodysplastic syndromes (MDS), but they are not suitable for frequent follow-up analyses. Most aberrations can also be detected by fluorescence in situ hybridisation (FISH), and they are provable in CD34+ cells from peripheral blood (pb). In our prospective multicenter German diagnostic study “Screening and genetic monitoring of patients with MDS under different treatment modalities by cytogenetic analyses of circulating CD34+cells” (ClinicalTrails.gov NCT01355913) we followed MDS pts by sequential FISH analyses. Methods CD34+ pb cells were enriched by immunomagnetic cell sorting (MACS®) and analysed by FISH using a “Superpanel” (D7/CEP7, EGR1, CEP8, CEP XY, D20, TP53, IGH/BCL2, TEL/AML1, RB1, MLL, 1p36/1q25, CSF1R, all Abbott® Products) at initial screening, every 12 months during follow-up and in case of suspected disease progression and a “Standardpanel” (EGR1, D7/CEP7, CEP8, TP53, D20, TEL/AML1, CEP XY, plus -if necessary- another informative probe) every 2 months in the 1st and every 3 months in the 2nd and 3rd year. If bm aspirate was available, additional CCB and FISH analysis of CD34+ and native bm cells were performed. Cut-off values for each FISH probe were evaluated in our lab. Cytogenetics, bm morphology, clinical course and therapies were documented in a database. All pts gave their written informed consent. The study was approved by all local ethic committees. Results After 3 years of study time, 361 patients (25 AZALE (University of Dresden), 110 LEMON5 (University of Duesseldorf), 226 CD34+FISH) have been included in the study, resulting in a total number of 19,516 FISH analyses: Median age, gender distribution and MDS subtypes were typical for the disease, median follow-up at the time of analysis was 8.2 (1–36) months. Chromosomal aberrations could be detected by FISH of CD34+ pb cells in 71.5% of pts (55% of CD34+FISH-cohort, 99% of LEMON5-trial pts, 100% of AZALE-trial pts). FISH and CCB were highly correlated: p

Description: Abstract 404 Comparison of conventional chemotherapy with high-dose therapy followed by autologous stem cell transplantation (HDT/ASCT) administered to young, high-risk patients with aggressive B-cell lymphoma as part of first-line therapy gave conflicting results; none of the randomized studies used rituximab (R) in combination with conventional or HDT. In March 2003 we started a randomized phase III study for young (18-60 years), high-risk (age-adjusted IPI 2 or 3) patients with aggressive lymphoma. For patients with B-cell lymphomas this study compared 8 cycles of CHOEP-14 (CHOP + etoposide 300 mg/m2 given every 2 weeks) with MegaCHOEP. The MegaCHOEP program used cyclophoshamide (1500 mg/m2 in cycle 1; 4500 mg/m2 in cycles 2 and 3; 6000 mg/m2 in cycle 4), doxorubicin (70 mg/m2 in all cycles), vincristine (2 mg, all cycles), etoposide (600 mg/m2 , cycle 1; 960 mg/m2 , cycles 2 and 3; 1480 mg/m2, cycle 4), and prednisone (500 mg, all cycles) to be administered every 21 days. Hematopoietic stem cells were harvested after cycles 1 and 2 and reinfused after HDT cycles 2, 3, and 4. Feasibility, safety, and efficacy of MegaCHOEP + / - R have been described (Glass et al. Blood 2006 and BMT 2006). The phase III study originally had four arms (8 × CHOEP – 14, 8 × CHOEP – 14 and 6 × R, MegaCHOEP, and MegaCHOEP and 6 × R). Treatment arms without R were closed in June 2004 because other studies (e.g the MInT study) had shown major improvement in outcome parameters when R was added to chemotherapy. The study continued comparing 8 × CHOEP – 14 and 6 × R (375 mg/m2) with MegaCHOEP and 6 × R (375 mg/m2). At the time of this analysis 346 patients (pts) had been recruited; 216 pts. (median age 48 years, LDH 〉 N 97 %, stage III or IV 96%, ECOG 〉 1 35%) had been randomized until 07 / 07 and were availabel for this planned interim analysis ( 8 × CHOEP – 14 + 6 × R, n = 91; MegaCHOEP + 6 × R, n = 94; 8 × CHOEP – 14, n = 15; MegaCHOEP, n = 16). Major toxicities included mucositis, diarrhea, and infections all of which were significantly more frequent in the MegaCHOEP arm of the study. Treatment – related deaths occurred in 5 / 94 pts. ( 5.3%) in the MegaCHOEP arm and in 1 / 91 pts. (1.1 %) in the R – CHOEP arm (p = 0.211). Surprisingly, the 3 – year event – free survival ( EFS : time from randomization to either disease progression, no CR / CRu at the end of treatment, initiation of salvage therapy, relapse or death from any cause) was better after conventional than after HDT / ASCT: 71.0% after 8 × CHOEP-14 + 6 × R vs. 56.7 % after MegaCHOEP + 6 × R (p = 0.050). After a median observation time of 29 months the estimated 3-year overall survival was 83.8 % after 8 × CHOEP – 14 + 6 × R and 75.3 % after MegaCHOEP + 6 × R (p = 0.142). Progression – free survival was 76.0 % after 8 × CHOEP – 14 + 6 × R and 64.6 % after MegaCHOEP + 6 × R (p = 0.119). A comparison of the rituximab-containing treatment arms (8 × CHOEP 14 + 6 × R and Mega CHOEP + 6 × R) with the chemotherapy – only arms (8 × CHOEP -14 and MegaCHOEP) revealed a 27.1 % difference in the 3-year EFS-rate ( p = 0.003 ) pointing to the unexpectedly high efficacy of R particularly in untreated, young, high-risk patients with aggressive B-NHL. These data were presented to the members of the study group and the data safety and monitoring committee who decided to stop the MegaCHOEP arm of the study. In conclusion, 8 × CHOEP -14 + 6 × R gave excellent results in young, high-risk patients with untreated aggressive B cell lymphoma. The 3-year EFS and OS are the best ever reported for this group of patients. MegaCHOEP + 6 × R was no better than aggressive conventional chemotherapy regarding any of the study endpoints; EFS (primary endpoint of the study) was significantly worse. Because of higher toxicity and inferior survival the MegaCHOEP arm was discontinued. HDT / ASCT has no role to play as part of first-line therapy for patients with high-risk aggressive B cell lymphoma if rituximab is combined with aggressive conventional chemotherapy. Disclosures: Schmitz: Roche: Honoraria, Research Funding. Nickelsen:Roche: Honoraria. Trümper:Roche: Honoraria, Research Funding. Pfreundschuh:Roche: Consultancy, Honoraria, Research Funding. Glass:Roche: Honoraria, Research Funding.

Description: The phosphoinositide 3-kinase (PI3K)-AKT pathway is a relevant signal transduction axis which regulates survival, growth and proliferation of hematopoietic cells through a variety of downstream targets. Constitutive activation of the PI3K-AKT pathway is observed in up to 70% of acute myelogenous leukemia (AML), primarily due to activating mutations in receptor tyrosine kinases (e.g. c-KIT) or RAS and is also present in T-cell-non-Hodgkins-lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, Hodgkins Lymphoma and myelodysplastic syndrome (MDS). We analyzed the effect of activating point mutations in the catalytic subunit p110alpha of class IA PI3K (PIK3CA) in hematopoietic cells. We transfected early hematopoietic, IL-3 dependent cells (Ba/F3 cell line) with point mutations in the helical (exon 9, E542K, E545A) and kinase domain (exon 20, H1047R) of the p110alpha catalytic subunit and determined the effect of PI3K-AKT pathway activation on cellular proliferation, survival and leukemogenic expansion in vitro and in vivo. We demonstrate that the p110alpha point mutations constitutively activated the PI3K-AKT pathway and resulted in factor-independent growth of Ba/F3 cells. Proliferation and survival of the cells were inhibited in a time- and dose-dependent manner using inhibitors of either PI3K or AKT. The mammalian target of rapamycin (mTOR) was demonstrated to be important for mitogenic, but not anti-apoptotic signaling of mutant PIK3CA. In a syngenic mouse model, hematopoietic cells expressing mutant p110alpha induced a leukemia-like disease. FACS analysis demonstrated a median chimerism of 68% in the bone marrow and 35% in peripheral blood of diseased mice, which were characterized by anemia, leukemic infiltration of hematopoietic organs, and 90% mortality within 5 weeks. No substantial differences were observed between E542K, E545A and H1047R. Mice, which were transplanted with an activating c-KIT point mutation (D814V), showed a significantly reduced survival and 100% died within 9 days. In conclusion our data show, that PIK3CA point mutations, by activating the PI3K-AKT pathway, confer factor-independence to hematopoietic cells in vitro and induce leukemogenic activity in vivo. As mutant c-KIT (D814V) showed enhanced leukemogenic potential, signaling through c-KIT may involve additional downstream pathways which cooperate with activated PI3K in leukemia progression. Our model is useful for the differential investigation of the leukemogenic relevance of the PI3K-AKT pathway and pharmacological inhibitor studies.