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We evaluated the impact of genetic analysis combining cytogenetics and broad molecular screening on leukemia diagnosis according to World Health Organization (WHO) and on genetic risk assignment. A two-step nested multiplex RT-PCR assay was used that allowed the detection of 29 fusion transcripts. A total of 186 patients (104 males (56%), 174 adults (94%), 12 children (6%), 155 AML (83%), 31 ALL (17%)) characterized by morphology and immunophenotyping were included. Of these 186 patients, 120 (65%) had a genetic abnormality. Molecular typing revealed a fusion transcript in 49 (26%) patients and cytogenetic analysis revealed an abnormal karyotype in 119 (64%). A total of 27 (14%) cases were genetically classified as favorable, 107 (58%) intermediate and 52 (28%) unfavorable. For 38 (20%) patients, there was a discrepancy in the genetic risk assignments obtained from broad molecular screening and cytogenetics. Cryptic fusion transcripts in nine (5%) patients changed the genetic risk assignment in four and the WHO classification in four patients. In 34 patients (18%), cytogenetics defined the risk assignment by revealing structural and numerical chromosomal abnormalities not detected by molecular screening. Broad molecular screening and cytogenetics are complementary in the diagnosis and genetic risk assignment of acute leukemia.

Contains fulltext : 50765.pdf (Publisher’s version ) (Closed access) Keratin 8 (KRT8) is one of the major intermediate filament proteins expressed in single-layered epithelia of the gastrointestinal tract. Transgenic mice over-expressing human KRT8 display pancreatic mononuclear infiltration, interstitial fibrosis and dysplasia of acinar cells resulting in exocrine pancreatic insufficiency. These experimental data are in accordance with a recent report describing an association between KRT8 variations and chronic pancreatitis. This prompted us to investigate KRT8 polymorphisms in patients with pancreatic disorders. The KRT8 Y54H and G62C polymorphisms were assessed in a cohort of patients with acute and chronic pancreatitis of various aetiologies or pancreatic cancer originating from Austria (n=16), the Czech Republic (n=90), Germany (n=1698), Great Britain (n=36), India (n=60), Italy (n=143), the Netherlands (n=128), Romania (n=3), Spain (n=133), and Switzerland (n=129). We also studied 4,234 control subjects from these countries and 1,492 control subjects originating from Benin, Cameroon, Ethiopia, Ecuador, and Turkey. Polymorphisms were analysed by melting curve analysis with fluorescence resonance energy transfer probes. The frequency of G62C did not differ between patients with acute or chronic pancreatitis, pancreatic adenocarcinoma and control individuals. The frequency of G62C varied in European populations from 0.4 to 3.8%, showing a northwest to southeast decline. The Y54H alteration was not detected in any of the 2,436 patients. Only 3/4,580 (0.07%) European, Turkish and Indian control subjects were heterozygous for Y54H in contrast to 34/951 (3.6%) control subjects of African descent. Our data suggest that the KRT8 alterations, Y54H and G62C, do not predispose patients to the development of pancreatitis or pancreatic cancer.

Background: Previous trials from the Swiss Group for Clinical Cancer Research (SAKK) and the Nordic Lymphoma Group (NLG) showed that therapy with single-agent rituximab can produce long-term remissions in a sizeable subset of follicular lymphoma (FL) patients, with overall survival not inferior to chemo-immunotherapy, providing the rationale for the development of chemotherapy-free treatment strategies. Promising results have also been reported with the combination of rituximab and lenalidomide. The SAKK 35/10 phase-2 study was developed and conducted by the SAKK in cooperation with the NLG to compare the activity of rituximab plus lenalidomide versus single-agent rituximab in the first-line FL therapy. Methods: Patients with histologically confirmed untreated FL, grade 1, 2, 3a and in need of systemic therapy, were randomized either to rituximab monotherapy (R) (8 infusions of 375mg/m2 at day 1 of weeks 1, 2, 3, 4, and repeated at day 1 of weeks 12, 13, 14 and 15) or to rituximab (given at the same schedule) in combination with lenalidomide (RL) (lenalidomide given orally, 15 mg daily, starting 14 days before the first rituximab administration and continuously until 14 days after the last). The primary endpoint was the complete response (CR/CRu) assessed at week 23, defined according to the NCI standardized criteria (Cheson et al 1999). The study sample size was calculated to allow the detection of a 20% increase of the CR/CRu rate with RL over R, with 90% power and a type I error of 0.10. The 2 arms were compared using a one-sided Z-test with unpooled variance for proportions. Trial treatment was discontinued in patients who at week 10 did not achieve at least a minimal response, defined as reduction of more than 25% in the sum of product of tumor diameters (SPD), and rescue chemotherapy was given at the discretion of the treating physician. Results: In total, 154 patients were randomized; 77 (40 women and 37 men; median age 63yrs, range 29-85; 52% with stage IV and 47% with poor-risk FLIPI score) were allocated to arm R and 77 (42 women and 35 men; median age 61yrs, range 26-80; 48% with stage IV and 47% with poor-risk FLIPI score) to arm RL. Treatment was discontinued by 21 (28%) patients in arm R, in 16 due to lack of response at week 10 and in 1 due to toxicity, and by 19 (25%) patients in arm RL, in 3 due to lack of response at week 10 and in 13 due to toxicity. Adverse events of any grade were reported in 91% of patients in arm R and 100% in arm RL and adverse events of grade ≥3 were more common in arm RL than in arm R (51% vs 18% of patients). Grade 3-4 neutropenia was observed in 5% of patients in arm R and 19% in arm RL. The primary endpoint analysis (using the response assessment from the local investigators, reviewed by the study chairs) showed a significantly higher CR/CRu rate in patients treated with RL in comparison with those receiving R. This difference was observed both in the intent-to-treat (CR/CRu rate, 36% vs. 25%, respectively; p=0.056) and the per-protocol population (CR/CRu rate, 42% vs. 28%, respectively; p=0.049). Conclusions: The addition of lenalidomide to rituximab results in a significantly better CR/CRu at the cost of an expected increased toxicity. Further follow-up is needed to ascertain whether the response improvement will translate into prolonged time to next treatment and superior progression-free and overall survival rates. TableInvestigators’ assessment of the response at week 23 in the intent-to-treat (ITT) population Rituximab (N=77) Rituximab+Lenalidomide (N=77)Response category n (%)[95% C.I.]n (%)[95% C.I.]CR/CRu19 (25) [16-36%]28 (36)[26-48%] PR28 (36) [26-48%]35 (45)[34-57%]SD6 (8) [3-16%]4 (5)[1.4-13%]PD/relapse2 (3) [0.3-9%]3 (4)[0.8-11%]Not evaluable*22 (29) [19-40%]7 (9)[4-18%]*Patients with no assessment at week 23, including patients not achieving at least a minimal response at week 10. Disclosures Kimby: Roche: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: Lenalidomide, not approved for follicular lymphoma. Mey:Celgene: Membership on an entity's Board of Directors or advisory committees. Ferreri:Celgene: Research Funding. Bargetzi:Celgene: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Krasniqi:Roche, Takeda: Membership on an entity's Board of Directors or advisory committees. Zucca:Roche, Mundivarma, Novartis: Consultancy, Honoraria, Research Funding.

Background High quality response [very good partial remission (VGPR), complete remission (CR)] is associated with improved long-term outcome in multiple myeloma (MM) therapy. Lenalidomide (Len)/ dexamethasone (Dex) (Rd) is considered a standard 2nd-line regimen. However, after 1st-line MM therapy containing a “novel agent” like thalidomide, only a relatively small fraction (21.3%) of patients (pts) achieved VGPR or better when RD was used in relapsed/refractory MM (RRMM)1. Bendamustine (Ben) is an alkylating agent with superior activity compared to melphalan/prednisone. The combination of Ben, Len and Dex (BRd) in pts with advanced RRMM resulted in dose-limiting hematotoxicity, which restricted its efficacy in extensively pretreated pts2. However, recent phase I experience demonstrated the feasibility of BRd as 2nd- and 3rd-line myeloma therapy3. We therefore evaluated the efficacy and toxicity of the BRd-regimen as 2nd line therapy for RRMM. Patients and methods This multicenter Phase II study was designed to enroll 50 pts with RRMM undergoing 2nd-line MM therapy [including also pts with prior autologous stem cell transplantation (ASCT)]. Pts had to have measurable disease, ECOG performance status 0-2, adequate hematological values and a creatinine clearance > 50 ml/min. Study treatment consisted of Ben 75 mg/m2 i.v. day (d) 1 & 2 and Len 25 mg p.o. d 1-21 for a total of six 28-day induction cycles. Dex (40 and 20 mg p.o. for pts 75 years of age, respectively) was given on d 1, 8, 15, and 22. Pegfilgrastim 6 mg s.c. was administered on d 3 in case of severe neutropenia, treatment delay due to neutropenia or febrile neutropenia according to predefined application rules. Induction treatment was followed by 12 cycles (28 d) of maintenance therapy with Rd at the same dose. The primary study endpoint was the CR/VGPR rate after induction therapy, based on standard IMWG criteria. A Simon's two stage design was used to differentiate between 20% (considered uninteresting) vs 40 % (considered promising, power of 80%, p=5%) of high quality responses. At least 13 pts with VGPR or better after induction therapy were required to meet the statistical threshold predefined for promising activity of the BRd regimen in this study. Results 50 pts were enrolled between 04/2012 and 07/2014 (median age 68 years [46-84y], 73% men). 49% pts had ISS stage II/III, 42.5% had undergone prior SCTs, and 13% had known high risk cytogenetic aberrations. 91% of pts had received novel agents (thalidomide, bortezomib) during 1st-line therapy. At the time of abstract submission, data from 38 pts having completed induction treatment were available. Final analysis of the primary study endpoint including all patients will be updated and presented at the meeting. Of the currently 38 evaluable pts, 20 (52.6%, Table 1) achieved a CR/VGPR after a median of 3.3 induction cycles. Only 1 patient experienced disease progression (PD) during induction phase. 77% of pts received pegfilgrastim. Dose reduction during induction therapy was required in 7.7% of pts. 42.5% received < 6 scheduled induction cycles (50% due to toxicities, 50% for other reasons). 49% had treatment-related SAEs. Grade 3/ 4 neutropenia and thrombocytopenia occurred in 51%/25.5% and 23.4%/8.5% of pts, respectively. Only 1 patient developed CTC grade 3 febrile neutropenia. Most common grade 3/4 non-hematologic toxicities were infections (14%), rash (9.5%), and diarrhea (9.5%). Anaphylactic reaction grade 4 related to Ben and pulmonary embolism grade 3 occurred in 1 patient each. A cerebral insult grade 4 occurred in a patient non-compliant with the anti-thrombotic prophylaxis required per protocol. One death due to respiratory failure (considered to be unlikely related to study treatment) was reported for an 83 year old male. Conclusion BRd is a safe and efficacious regimen for 2nd line treatment of RRMM patients whose 1st-line therapy included thalidomide or bortezomib. High quality responses (>= VGPR) can be achieved in a considerable proportion (52.6%) of these pts. Our study suggests that the fraction of patients achieving VGPR or better after BRd treatment may be substantially higher than attainable with Rd alone. References 1 Wang et al. Blood, 2008, 112: 4445-51 2 Lentzsch et al. Blood, 2012, 119: 4608-13 3 Poenisch et al., Br J Haematol, 2013, 162, 202–9 Table 1 Best response after induction CR VGPR PR MR SD total (n=38) 3 17 15 1 2 prior ASCT (n=16) 2 6 7 1 0 no prior ASCT (n=22) 1 11 8 0 2 Disclosures Mey: Mundipharma: Honoraria, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: Use of Bendamustine in relapsed/refractory Multiple Myekoma. To investigate the efficacy and safety of Bendamustine in combination with the standard backbone of Lenalidomide/ Dexamethasone in patients with replaced/refractory MM.. Bargetzi:Celgene: Membership on an entity's Board of Directors or advisory committees. Taverna:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Schmid:Celgene: Honoraria. Knauf:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. von Moos:Amgen: Consultancy, Honoraria, Research Funding. Hiendlmeyer:Celgene: Research Funding; Mundipharma: Research Funding; Amgen: Research Funding. Hitz:Celgene: Research Funding. Driessen:Mundipharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees.

We observed the occurrence of celiac disease following allogeneic bone marrow transplantation in a patient transplanted for acute leukemia. The marrow donor was his HLA-identical sister, who had suffered from celiac disease since birth. The post-transplant period was characterized by recurrent episodes of diarrhea. Detailed workup showed atrophic intestinal mucosa on histology and anti-gliadin and anti-endomysium antibodies in the serum, features that were not present before transplantation. GVHD was absent at that time. The patient remains free of symptoms on gluten-free diet and slight immunosuppression. This case suggests transmission of celiac disease by bone marrow transplantation and supports the T cell concept in celiac disease.

The potential benefits of extended rituximab treatment have been investigated in a randomized trial comparing the standard schedule with prolonged treatment in 202 patients with newly diagnosed or refractory/relapsed follicular lymphoma (FL). All patients received standard treatment (rituximab 375 mg/m(2) weekly x 4). In 185 evaluable patients, the overall response rate was 67% in chemotherapy-naive patients and 46% in pretreated cases (P

Contains fulltext : 57434.pdf (Publisher’s version ) (Closed access) To assess the contribution of interphase fluorescence in situ hybridization (I-FISH) toward the detection of recurring unbalanced chromosomal anomalies at diagnosis, a systematic screening of -5/del(5)(q31), -7, and chromosome 8 aneuploidy was performed on 110 patients with acute myelocytic leukemia or high-risk myelodysplastic syndrome. We searched for monosomy 5/del(5q) by one-color I-FISH with a probe specific for the 5q31 region and for -7/8 by dual-color I-FISH with centromeric probes for chromosomes 7 and 8. Discrepancies between conventional cytogenetics (CC) and I-FISH were observed in 8 of the 110 patients (7.3%). For -5/del(5)(q31), a discordance was observed in two patients with complex abnormalities involving chromosome 5. Whereas no discordance was observed for -7, I-FISH detected a trisomy 7 unnoticed by CC in two cases. In six patients, I-FISH revealed a chromosome 8 aneuploidy not detected by CC. Our results illustrate that, when using this specific set of probes, I-FISH is of special interest for the detection of minor clones with chromosome 8 aneuploidy, breakpoint assessment, and sequence identification (markers). Also, to avoid misinterpretations, I-FISH should not be used alone at disease presentation, particularly in cases complex changes that have clearly established prognostic significance.

Cyclophosphamide with granulocyte colony stimulating factor (G-CSF) is commonly used to mobilize stem cells in multiple myeloma. Timing of collection is variable and incidence and severity of side effects is substantial. To optimize timing of collection, to reduce side effects and to limit costs of the procedure, we evaluated vinorelbine, a drug shown to have activity in multiple myeloma, in combination with G-CSF as mobilizing regimen. A total of 19 consecutive patients with advanced stage multiple myeloma received one dose of vinorelbine 35 mg/m(2) intravenously on day 1 in an outpatient setting and G-CSF 10 microg/kg/day from day 4 divided in two daily doses. Median CD34+ cell blood counts measured on day 8 of mobilization were 142 x 10(6)/l (range 57-467). One 15-l apheresis on day 8 resulted in sufficient stem cells (median 11.1 x 10(6) CD34+ cells/kg, range 6.2-36.0 prior and median 7.5 x 10(6) CD34+ cells/kg, range 4.0-20.2 post-positive CD34+ cell selection) for transplantation. Hematopoietic recovery was swift with ANC >0.5 x 10(9)/l on day 11 median (range 10-15) and platelets >20 x 10(9)/l on day 12 median (range 10-15) after reinfusion of the stem cells on day 0. No episodes of febrile neutropenia were observed during mobilization. In our institutions cost reduction for the procedure was about 1700 euros compared to the mobilization with cyclophosphamide and G-CSF. Vinorelbine and G-CSF allow precise timing and harvesting of sufficient stem cells, and might be an alternative to cyclophosphamide in the mobilization of stem cells for autologous transplantation in multiple myeloma.

The objective of this study was to evaluate a regimen of full doses of docetaxel and cisplatin, using an alternating schedule, as first-line therapy for patients with inoperable non-small cell lung cancer (NSCLC). The standard concomitant schedule does not allow full doses of both drugs to be administered. We wanted to see if there was an advantage to be gained by administering full doses of both docetaxel and cisplatin, using a different schedule. Docetaxel 100 mg/m2 was given once every 6 weeks from week 1 and cisplatin (120 mg/m2 for two doses and 100 mg/m2 thereafter) once every 6 weeks from week 4, for six cycles (three docetaxel and three cisplatin). Thirty-six of the 44 patients enrolled were evaluable for efficacy. Forty-eight percent of the patients had good (KPS 90-100%) performance status. A median of five cycles was administered, for which no dose reductions were necessary. There were 13 of 36 partial responses (36%; 95% CI 21-54%) and 15 of 36 patients achieved stable disease (42%). The median duration of response was 10.5 months, the median time to progression was 4.5 months and the median survival was 9 months. The 1 and 2 year survival rates were 39 and 16%, respectively. The most frequent grade 3-4 toxicities were nausea (23% of patients), vomiting (18%) and neutropenia (77%). Infections were also common, but not severe. The alternating schedule produced response, toxicity and survival figures that compared favorable with those using the concomitant schedule. This study could serve as a model for future studies of non-cisplatin-containing regimens, in which full doses of docetaxel could alternate with full doses of other new agents active against NSCLC.

8560 Background: Standard conditioning regimens for ASCT are often not tolerated by elderly patients. Single agent high-dose melphalan has been shown to be safe and active in elderly patients with multiple myeloma. Y90-IT is a well-tolerated lymphoma treatment and feasible in the transplantation setting. We therefore investigated this combination of high-dose melphalan and Y90-IT as a conditioning regimen for elderly patients. Methods: Patients ≥65 years with relapsed or chemotherapy resistant CD20-positive lymphoma in PR or CR after salvage chemotherapy could be enrolled prior to stem cell mobilization. Myeloablation regimen consisted of standard dose Y90-IT followed by melphalan at escalating doses (100, 140, 170 and 200mg/m2, with a 3+3 phase I design) and by ASCT. The primary objective was to identify the MTD of melphalan in combination with standard dose Y90-IT (as defined < 1 DLT in 3 patients); secondary endpoints were toxicity and CR rate 100 days after transplantation. Results: Between 2006-2012 twenty patients were included. Median age was 72 years (range 66-77). One patient was considered retrospectively ineligible and was evaluable for toxicity but not for DLT. Thirteen patients received the treatment and were transplanted. Eleven patients were evaluable for DLT. No DLT occurred. Non-hematological grade 3 or higher treatment related adverse events were: infection (n=6, including 2 cases of febrile neutropenia), diarrhea (n=3), mucositis, anorexia, viral hepatitis, hypokalemia, dehydration and multi-organ failure (n=1). Seven patients did not start treatment because of mobilization failure (n=3), progressive disease (n=2), worsening of cardiac failure (n=1) and grade 3 dyspnea (n=1). Seven patients achieved a CR/CRu and 2 patients were stable 100 days after transplantation. Conclusions: The combination ofstandard dose Y90-IT and high dose melphalan (200mg/m2) is a safe and feasible conditioning regimen before ASCT for patients ≥65 years. The results show promising activity. Clinical trial information: NCT00392691.

Optimum treatment of severe neutropenia, a major factor for morbidity and mortality in T-large granular lymphocyte (LGL) leukemia, is undefined. We observed a rapid improvement of the neutrophil count in a patient with T-LGL leukemia and severe neutropenia after the combined administration of antilymphocyte-globulin (ALG), cyclosporin A, prednisone, and granulocyte colony-stimulating factor (G-CSF). Although G-CSF treatment was terminated after 7 days, the neutrophil count has persisted above 1.0 x 10(9)/1 for up to 6 months now. Oral methotrexate is given continuously as treatment for T-LGL leukemia. The response to this immunosuppressive regimen suggests a T-cell-mediated mechanism as the underlying cause for neutropenia in T-LGL leukemia.

The role of splenectomy in aplastic anaemia (AA) is controversial. The hazards of operating on a severely pancytopenic patient, the fear of compromising the patient’s immune function, and the improvement of non-surgical treatment have made splenectomy unpopular in this disease. We have evaluated positive and adverse effects of splenectomy in 80 patients with severe aplastic anaemia (SAA) treated with antilymphocyte globulin (ALG) (group A), using 52 nonsplenectomized ALG patients as controls (group B). All patients survived the operation. Nonfatal complications of surgery occurred in 10 (12.5%). Splenectomy induced a significant increase of peripheral blood neutrophils, reticulocytes and platelets within 2 weeks, followed by a continuous increase of all values over the following weeks. 28/132 patients (21%) developed a late clonal disorder of haemopoiesis, paroxysmal nocturnal haemoglobinuria (PNH) or myelodysplastic syndrome (MDS), or both. Their incidence was identical in groups A and B. 13/28 (59%) died, 10/17 (59%) in group A and 3/11 (27%) in group B (not significant (n.s.)). Overall probability of survival at 18 years after ALG was 51 ± 6% for group A and 61 ± 7% for group B (n.s.). We conclude that splenectomy in AA is safe. It induces an immediate increase of peripheral blood counts and, thereafter, a continuous improvement of haemopoiesis. It does not increase the incidence of late clonal complications but has a borderline effect on mortality from these disorders. Splenectomy should be reconsidered in selective nontransplanted patients who have prolonged transfusion requirements despite otherwise optimal treatment.

Abstract 3098 Background. Upfront treatment for advanced Hodgkin lymphoma (HL) is a matter of debate. The wide difference in progression-free survival (PFS) or freedom from treatment failure observed between the intensified regimen escalated (esc) BEACOPP and other standard intensity schedules - such us ABVD, ABVD hybrids and BEACOPP baseline - is in contrast with a small difference in overall survival (OS) [NEJM 2011;365 :203 and JCO 2009;27 :4548]. This fact suggests that high-dose therapy with autologous stem-cell transplantation (HDT/ASCT) can salvage more patients treated with standard intensity schedules than escBEACOPP treated patients. Methods. In order to better understand this subject we analyzed all Swiss cases of refractory/relapsed HL that received HDT/ASCT after failure of at least 4 cycles of upfront escBEACOPP (4 to 8 cycles), given alone or combined with other regimens (cases, n=22) and compared outcomes with a control group of 33 patients transplanted after failure of upfront ABVD (N=18), ABDV-based combinations (hybrids, N=7), BEACOPP baseline (N=5) or up to 3 cycles of escBEACOPP plus radiotherapy (N=3). The high-dose schedule used before transplant was BEAM for 51/55 patients and CBV in 1 case and 3 controls. Results. Controls and cases were statistically similar in terms of timing of transplant (median year 2006 vs 2007), age at transplant (median 33 vs 28 years), sex (males 64% vs 68%), HL subtype, Ann Arbor stage, international prognostic score, ESR (38 vs 59 mm/1h) and bulky presentation (35% vs 60%), but there were significantly more B-symptoms among escBEACOPP patients (77% vs 23%, p=0.007). Furthermore, chemosensitivity (94% vs 82%), timing of relapse after upfront treatment (0–3 months: 30% vs 22%, 3–12 months: 18% vs 23% and >12 months: 52% vs 55%), median number of CD34+ cells reinfused (4.11 vs 2.99 ×10*6/kg), and need for a second salvage (12% vs 27%) were not significantly different in the two groups. After a median post transplant follow up of 42.5 months, median PFS was 15.4 months for escBEACOPP, and was not reached for control cases (p=0.145). Two-years OS was 84% for controls (95% CI 65%-93%) and 72% for escBEACOPP (95% CI 45%-87%), while 5-years OS was 68% (95% CI 44%-84%) and 65% (95% CI 37%-83%) for controls and cases, respectively. Four of 33 controls (12%) and 3 of 22 (14%) upfront escBEACOPP cases received an allograft for relapsed HL after HDT/ASCT (p=NS). The incidence of severe (grade 3–4) anemia, thrombocytopenia, neutropenia, diarrhea, stomatitis and neutropenic fever was similar in the two groups. Regarding uncommon/unexpected toxicities of HDT/ASCT, one patient in the escBEACOPP group developed grade 4 encephalopathy, whilst in the control group we observed two cases of acute renal failure, one ARDS and one fatal stroke. One patient in the control group was diagnosed with a T-cell lymphoma after transplant (p=NS). Three t-MDS plus one t-AML were recorded among patients in the escBEACOPP group, with no cases of t-AML/t-MDS occurring among those treated with the less intensive approaches (p=0.023). Conclusion. In our series, overall survival of chemosensitive HL patients autografted after failure of either upfront escBEACOPP or standard intensity regimens is encouraging. HDT/ASCT consolidation of chemosensitive patients failing upfront escBEACOPP seems justified. Disclosures: No relevant conflicts of interest to declare.

Abstract 1555 An increase in micro vessel density has been shown in Acute Myeloid Leukemia (AML) bone marrow biopsies at diagnosis, correlating with increased expression of Vascular Endothelial Growth Factor (VEGFA) (de Bont et al, 2001, 2002; Padro et al, 2000; Aguayo et al, 1999). Previously we reported heterogeneity in AML bone marrow vessel patterns, and three subgroups can be distinguished: (a) low vessel count', (b) angiogenic sprouting' (biopsies exhibiting high vessel count with mainly a network of small vessels with thin walls, narrow lumen and branching) and (c) ‘vessel hyperplasia’ (biopsies displaying high number of vessels with predominantly a large lumen and thin walls) (Weidenaar et al, abstract ASH 2010; 2011). In this study we set out to confirm the previously defined morphology groups in a larger group of patients, and investigated the relationship between vascular morphology and clinical outcome in the HOVON81 study which is a multicenter phase II trial evaluating the additional value of Bevacizumab (Roche, Basel, Switzerland) (5 mg/kg to a maximum of 10 mg/kg) at day 1 and 15 of cycle I and II to standard intensive chemotherapy in elderly AML patients (>60 years). AML bone marrow biopsies at diagnosis (n=93) were immunohistochemical stained for FVIII (endothelial cell marker) and SMA (pericytes marker). The three previously reported AML vessel morphology patterns could be confirmed in this cohort (Fig 1A-B). The median percentage of vessels covered by pericytes was 50.1% (range 2.1–100.0, n=66), and was not significantly (P=.27) different between the three groups. In addition it was shown that patients with ‘angiogenic sprouting’ and ‘low vessel count’ (EFS 2 yr: 10%) have a decreased EFS as compared to patients with 'vessel hyperplasia' (EFS 2 yr 30%) (P=.017). For OS, a trend for unfavorable outcome was observed for the ‘angiogenic sprouting’ subgroup (P=.055). Interestingly, treatment with Bevacizumab significantly increased EFS and tended to be associated with a beneficial OS for patients displaying ‘low vessel count’ profile (P=.023 and P=.099 respectively). EFS and OS were not increased in patients with ‘angiogenic sprouting’ or ‘vessel hyperplasia’ upon Bevacizumab treatment. Multivariate analysis established vessel morphology (HR: 2.2 (95% CI 1.1–4.0)) as a prognostic indicator independent of other statistical significant risk factors (for EFS (P=.015)). In conclusion, our results demonstrate that different vasculature patterns in AML bone marrow biopsies are related to treatment outcome in AML patients. Patients displaying an ‘angiogenic sprouting’ profile seem to constitute an unfavorable subset of patients. In addition, AML patients with ‘low vessel count’ might be good candidates for Bevacizumab treatment. Our results show that AML vascular morphology provides prognostic information; therefore, it might be useful to study vessel patterns at diagnosis. Fig. 1. AML biopsies prior to treatment divided into three morphology groups. (A) Scatterplot representing the AML biopsies at diagnosis. Biopsies with a ‘low vessel count’ are displayed below the Y-axis reference line (13 microvessels/hpf, group I). The X-axis reference line divides AML biopsies at diagnosis with a ‘high vessel count’ into two subgroups according to the median Chalkley count of 5.4 in AML at diagnosis. Cut-off points are based on previously described results. Samples with a Chalkley count ≤5.4 were defined as ‘angiogenic sprouting’ (group II) and samples with a Chalkley count >5.4 as ‘vessel hyperplasia’ (group III). (B) A representative picture of the three groups is shown. Fig. 1. AML biopsies prior to treatment divided into three morphology groups. (A) Scatterplot representing the AML biopsies at diagnosis. Biopsies with a ‘low vessel count’ are displayed below the Y-axis reference line (13 microvessels/hpf, group I). The X-axis reference line divides AML biopsies at diagnosis with a ‘high vessel count’ into two subgroups according to the median Chalkley count of 5.4 in AML at diagnosis. Cut-off points are based on previously described results. Samples with a Chalkley count ≤5.4 were defined as ‘angiogenic sprouting’ (group II) and samples with a Chalkley count >5.4 as ‘vessel hyperplasia’ (group III). (B) A representative picture of the three groups is shown. Disclosures: No relevant conflicts of interest to declare.

Abstract 2181 Background: Acute Myeloid Leukemia (AML) in the elderly is notoriously difficult to treat and has a low remission rate with very few long term survivors when using standard treatment approaches. Azacytidine, a hypomethylating agent, has been shown to induce remission and prolong survival in patients with myelodysplastic syndromes; studying this approach to patients with AML is therefore warranted. We present results of an ongoing phase II trial treating elderly or frail AML patients with Azacytidine. Methods: AML elderly or frail patients, and therefore unfit for an intensive chemotherapy regimens, with a WHO performance status ≤ 3 were considered for this trial. Trial therapy consisted of 100mg/m2 of Azacytidine injected subcutaneously on 5 consecutive days every 28 days up to 6 cycles, stopping at 6 months if no hematological improvement achieved, or earlier in the case of progression or complications. Treatment was continued beyond 6 months in responding patients. Trial therapy was considered uninteresting if the response rate (CR + PR) within 6 months of therapy initiation was 15% or less and promising if 34% or more. Using the exact single-stage phase II design by A’Hern with a 5% significance level and 90% power, 43 patients were required: If 10 or fewer achieved a response within 6 months the trial therapy should not be considered for further investigation in its current format for this indication and patient population. Results: Between September 2008 and January 2010, 45 evaluable patients across 10 Swiss centers were accrued with a median follow-up of 7 months (range: 0 – 13). 27 (60%) were male, median age was 74 (range: 55 – 86) years and 35 (78.8%) had performance status 0–1. Patients had been excluded from more intensive chemotherapy regimens because of age (n = 37) or due to comorbidities or patient refusal (n=8). Five patients had therapy related AML. Patients received a median of 3 (range: 1 – 10) cycles. Treatment was stopped for not achieving a response by the 6th cycle in 2 patients and earlier in 26 patients (for disease progression in 5, toxicity in 3, patient refusal in 2, recurrent infections in 1, and death in 8). Seventeen patients remain on therapy. The median time spent in the hospital was 12 days (1 - 30) in 24/38 patients hospitalized during the first treatment cycle and 13 days (2 - 28) in 15/31 patients hospitalized during subsequent cycles. Adverse events of grade III or higher most frequently reported were constitutional or hematologic, i.e. fatigue in 5, febrile neutropenia in 8, infections in 6, dyspnea in 6, anemia in 3, neutropenia in 12 and thrombocytopenia in 10, hemorrhage in 2 and retinal detachment in 5. Based on available data on 38 patients, CR/CRi or hematologic improvement or stable disease within 6 months of trial registration was observed in a proportion of patients. Final and mature data, determining whether the predefined proportion of responding patients has been reached or not, will be presented at the conference. Up to now there were a total of 26 deaths. Median overall survival time was 5.7 months (95% CI: 3.1, 8.7). Conclusions: The current results of this slightly modified Azacytidine schedule demonstrate a feasible new therapy option for elderly or frail AML patients in an outpatient setting with moderate, mainly hematologic toxicity. Disclosures: No relevant conflicts of interest to declare.

Besides its established role in the treatment of patients with multiple myeloma, the proteasome inhibitor bortezomib is active in patients with a variety of indolent non-Hodgkin’s lymphomas, notably mantle cell lymphoma and follicular lymphoma. Bendamustine was originally designed as a bifunctional anticancer compound combining an alkylating and an antimetabolite function. It has strong efficacy in non-Hodgkin’s lymphoma and multiple myeloma, and apparently low cross-resistance with other alkylating agents. This open label, single-center phase 1/2 study evaluated a weekly combination of bortezomib and bendamustine in patients with relapsed or refractory indolent non-Hodgkin’s lymphoma. The primary endpoint was to define the maximal tolerated dose (MTD). Secondary endpoints were tolerability and response. On days 1, 8, 15, and 22 of a 35-day cycle, patients received intravenous bolus bortezomib 1.6 mg/m2 for a maximum of 3 cycles. Bendamustine was administered as 30-min. intravenous infusion on days 1, 8, and 15. Dose escalation was started at a dose of 60 mg/m2 bendamustine. Response was assessed at the end of study treatment. Four patients entering the first dose level showed no dose-limiting toxicity (DLT). Thereupon, bendamustine dosage was increased to 80 mg/m2. In 3 out of 5 patients, DLT was observed. Dose-limiting adverse events were grade 3 diarrhea with dehydration, fatigue, and grade 4 thrombocytopenia, respectively. Adverse events with an overall incidence of ≥20% were diarrhea, nausea, vomiting, thrombocytopenia, and fatigue. There were no infectious or dose-limiting neurological adverse events. The 9 patients (7 females) in the phase 1 part of this trial, 5 with relapsed, 4 with refractory stage III (n=2) or stage IV (n=7) disease, received a median of 2 treatment cycles (range 2–3). Median age was 71 yrs (range 55–85). Detailed histological diagnoses were mantle cell lymphoma (n=4), follicular lymphoma (n=4), and Waldenstroem’s macroglobulinemia (n=1). All patients were pretreated (median 3 lines of treatment, range 2–8). Prior treatments comprised rituximab (n=7), anthracyclines (n=4), ibritumomab tiuxetan (n=2), bortezomib (n=2), and autologous stem cell transplantation (n=1). The reasons for not completing the planned 3 treatment cycles were DLT (n=2), and disease progression (n=3). As best response, partial remission was achieved in 6 patients, while disease progressed in 3 patients. Among the different types of lymphoma, partial remissions were observed in all 4 mantle cell lymphoma patients, 1 out of 4 follicular lymphoma patients, and in the Waldenstroem’s macroglobulinemia patient. The trial’s phase 2 part is currently ongoing. In conclusion, weekly bortezomib and bendamustine (1.6 mg/m2 d1, 8, 15, & 22 and 60 mg/m2 d1, 8, & 15 q5w, respectively) was found to have acceptable toxicity. Moreover, this study demonstrates initial evidence of efficacy of the combination in heavily pretreated patients with indolent non-Hodgkin’s lymphoma, particularly mantle cell lymphoma.

Introduction: This trial aimed to determine the efficacy and toxicity of an induction immunochemotherapy consisting of rituximab and cladribine (2-chlorodeoxyadenosine, 2-CDA) in patients (pts) suffering from chronic lymphocytic leukemia (CLL). Methods: Inclusion criteria were CLL at first diagnosis or after one treatment with alkylating agents. The regimen consisted of four remission induction cycles. In cycle 1, 2-CDA (0.1 mg/kg/day) was administered for 5 days. In cycles 2-4, Rituximab (375 mg/m^2) was given on day 1 followed by 2-CDA (0.1 mg/kg body weight), in intervals of 28 days. Responding pts (complete remission (CR), very good partial remission (VGPR) or nodular partial remission (NPR)) underwent stem cell mobilization chemotherapy with Cyclophosphamide (4g/m^2 on day 2) G-CSF (10 microgram/kg s.c. daily, from day 4 on), and Rituximab (375 mg/m^2) on day 1 and 8 as in vivo purging. If no CR, VGPR or NPR was achieved, up to 4 cycles CHOP were administered. Primary endpoint was CR, secondary endpoints were VGPR, NPR and toxicity after induction and feasibility of stem cell mobilization. For response evaluation, staging procedures included clinical examination as well as bone marrow biopsies and CT-scans. A total of 41 pts was planned using Simon’s two-stage design with 5% significance and 80% power for the null hypothesis of CR rate < 25% and the alternative hypothesis of CR rate > 45%. Results: 42 pts were included, median age 53.8 y (range 38 – 65), WHO performance status 0 in 33 pts and 1 in 9 pts, stage Binet B in 20 pts, Binet C in 8 pts and progressive A in 14 pts. 2 pts were not evaluable for response. 9 pts reached CR (22.5%, 95% CI: 11–38%). Overall response rate including 15 VGPR and 2 NPR was 65% (CI: 48–79%). Of the 14 non-responders, 8 underwent CHOP treatment of which 2 achieved VGPR. 20 patients underwent mobilization and 8 pts refused further protocol treatment. 14 pts had leucapheresis. Stem cell harvest was feasible in 7 pts, all with ≥ 2×10^6 cells/kg. Fever and infection were reported respectively in 13 and 9 pts. Infusion related adverse events of Rituximab were moderate and occurred mainly after the first infusion. 42 of 158 cycles (27%) were associated with grade 3 or 4 neutropenia and 6 of 158 cycles (4%) with grade 3 thrombocytopenia. Conclusions: Although the expected CR rate was not achieved, a combination of Rituximab and 2-CDA is an effective and well tolerated treatment.

EVI1 is a transcription factor that is targeted for de-regulation in 10% of patients with acute myeloid leukemia (AML). Overexpression of EVI1 is associated with a particularly poor prognosis. Various karyotypic abnormalities involving chromosomal region 3q26 have been shown to inappropriately activate expression of EVI1, the most frequent alterations being inv(3)(q21q26)and t(3;3)(q21;q26). Rearrangements of 3q are frequently associated with trilineage myelodysplasia, especially dysmegakaryocytosis. So far, translocation t(3;8)(q26;q24) has been reported in one patient with MDS and two cases of AML without further molecular analysis. No other recurrent chromosome aberration with a breakpoint in 8q24 has been shown in AML. We report a case of a 58 year old male patient with a multilineage AML featuring a t(3;8)(q26;q24) as the sole chromosomal aberration in 80% of bone marrow cells analysed by conventional cytogenetics. The normocellular bone marrow aspirate showed 25–50% atypical blasts, dyserythropoiesis, dysplastic myelopoiesis and hyperactivated megakaryopoiesis with many micromegakaryocytes. Immunophenotyping of the blast population by flow cytometry confirmed the myeloid nature of the cells with CD13+, CD33+, CD64+, CD117+, HLA−DR+ phenotype and variable expression of CD14 and CD34. Persisting pancytopenia occurred after two cycles of chemotherapy and a relapse was evident 4 months later. By real time quantitative RT-PCR (RQ-PCR) we showed that EVI1 is highly overexpressed (>100:1) in this patients bone marrow cells as compared with an AML patient without 3q26 involvement. Fluorescence in situ hybridisation (FISH) is currently being performed to characterize the 8q24 and 3q26 breakpoints in more detail. The chromosomal translocation t(3;8)(q26;q24) leads to overexpression of EVI1 and, as in other aberrations involving 3q, shows a poor prognosis in AML.

A new nitrocellulose-based ELISA technique, the line immunobinding assay (LIBA), permits the quantitative detection of multiple antigen-antibody reactions in one single assay. The antigen solutions are sprayed in thin lines onto a nitrocellulose sheet which is then cut into several strips before incubation in a multi-channel tray with serially diluted serum. The reacting antibodies are visualized by a peroxidase system. Comparison with other techniques routinely used for the detection of antibodies against measles, mumps and rubella, suggest that the LIBA offers specificity with sensitivity and may have a broad range of applications.

Administration of interleukin-1 beta (IL-1 beta) to humans initiates a cascade of metabolic, hematologic, and cardiovascular events. To investigate the role of the sympathetic nervous system in the early cardiovascular response to IL-1 beta in humans, we recorded the heart rate, blood pressure, and changes in hand vein compliance in five patients with malignant melanoma treated with a 30-min infusion of 10,000-20,000 U/kg of human recombinant IL-1 beta. All patients developed fever, chills, and marked hemodynamic changes. During or shortly after the infusion, a dramatic decrease in hand vein compliance occurred (vasoconstriction). At the time of maximum venoconstriction (35 min after the start of the IL-1 beta infusion), the mean heart rate and systolic blood pressure were significantly increased by 30 mm Hg and 31 beats/min, respectively. Venoconstriction always preceded the onset of chills by several minutes (mean of 7 min), was closely correlated with the heart rate, and could be reversed by local administration of the alpha-antagonist phentolamine, indicating involvement of catecholamines. Our study shows that cardiovascular responses that occur early after IL-1 beta administration in humans are most likely the result of adrenergic stimulation possibly through its effect on the central nervous system.

Flecainide acetate, a class Ic antiarrhythmic agent, is eliminated to a larger extent by renal excretion and to a minor extent by the liver. In patients with impaired renal function or with elevated urinary pH, however, its elimination is dominated by hepatic metabolism. Recent evidence suggests that the in vivo metabolism of flecainide is controlled by the genetic polymorphism of the debrisoquin/sparteine type; i.e., it is a substrate of cytochrome P450IID6. We investigated the inhibitory effect of flecainide on bufuralol 1'-hydroxylation in human liver microsomes in vitro and on the metabolic dextromethorphan urinary ratio in eight healthy male volunteers. Both bufuralol and dextromethorphan are well-known substrates of cytochrome P450IID6. Microsomal bufuralol 1-hydroxylation was competitively inhibited by flecainide with an apparent Ki of 0.954 mumol/L. Moreover, a statistically significant increase in the urinary metabolic ratio (MR) of dextromethorphan/dextrorphan after 1 week of administration of oral flecainide was observed (p = 0.013) in all subjects. One individual increased the urinary MR to a value consistent with the poor metabolizer phenotype. We conclude that flecainide is a potent inhibitor of cytochrome P450IID6 in vitro and in vivo and that careful drug monitoring is required with respect to renal function, debrisoquine phenotype, and concomitant drug administration.

Abstract 2803 Introduction: Mantle cell lymphoma (MCL) accounts for 6% of all B-cell lymphomas and remains incurable for most patients. Those who relapse after first line therapy or hematopoietic stem cell transplantation have a dismal prognosis with short response duration after salvage therapy. On a molecular level, MCL is characterised by the translocation t[11;14] leading to Cyclin D1 overexpression. Cyclin D1 is downstream of the mammalian target of rapamycin (mTOR) kinase and can be effectively blocked by mTOR inhibitors such as temsirolimus. We set out to define the single agent activity of the orally available mTOR inhibitor everolimus (RAD001) in a prospective, multi-centre trial in patients with relapsed or refractory MCL (NCT00516412). The study was performed in collaboration with the EU-MCL network. Methods: Eligible patients with histologically/cytologically confirmed relapsed (not more than 3 prior lines of systemic treatment) or refractory MCL received everolimus 10 mg orally daily on day 1 – 28 of each cycle (4 weeks) for 6 cycles or until disease progression. The primary endpoint was the best objective response with adverse reactions, time to progression (TTP), time to treatment failure, response duration and molecular response as secondary endpoints. A response rate of ≤ 10% was considered uninteresting and, conversely, promising if ≥ 30%. The required sample size was 35 pts using the Simon's optimal two-stage design with 90% power and 5% significance. Results: A total of 36 patients with 35 evaluable patients from 19 centers were enrolled between August 2007 and January 2010. The median age was 69.4 years (range 40.1 to 84.9 years), with 22 males and 13 females. Thirty patients presented with relapsed and 5 with refractory MCL with a median of two prior therapies. Treatment was generally well tolerated with anemia (11%), thrombocytopenia (11%), neutropenia (8%), diarrhea (3%) and fatigue (3%) being the most frequent complications of CTC grade III or higher. Eighteen patients received 6 or more cycles of everolimus treatment. The objective response rate was 20% (95% CI: 8–37%) with 2 CR, 5 PR, 17 SD, and 11 PD. At a median follow-up of 6 months, TTP was 5.45 months (95% CI: 2.8–8.2 months) for the entire population and 10.6 months for the 18 patients receiving 6 or more cycles of treatment. Conclusion: This study demonstrates that single agent everolimus 10 mg once daily orally is well tolerated. The null hypothesis of inactivity could be rejected indicating a moderate anti-lymphoma activity in relapsed/refractory MCL. Further studies of either everolimus in combination with chemotherapy or as single agent for maintenance treatment are warranted in MCL. Disclosures: Off Label Use: everolimus for the treatment of mantle cell lymphoma.