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Introduction:

Despite impressive therapeutic progress represented by the advent of chemoimmunotherapy, chronic lymphocytic leukemia (CLL) remains incurable by conventional modalities. Refractory CLL defined by non-response to treatment or relapse/progression within 6 months is associated with multiple unfavourable prognostic factors such as p53 pathway disruption, deteriorating patient condition, increased risk of severe infections, and poor response to treatment, resulting in a very short overall survival. Therefore, refractory CLL represents a highly challenging situation for the hematologist as well as the patient. The amount and quality of data on refractory CLL are rather limited as clinical trials usually combine patients with relapsed and refractory disease. Therapeutic options for refractory CLL include alemtuzumab, ofatumumab, fludarabine- and bendamustine-based regimens, platinum-based aggressive protocols and high-dose corticosteroids combined with monoclonal antibodies. The recent introduction of ibrutinib and idelalisib, small molecules interfering with B-cell receptor pathways, revolutionized the treatment of refractory CLL by the novel concept of long-term, oral treatment leading to impressive progression-free and overall survival improvement. Allogeneic stem cell transplantation is still considered the only option with curative potential. This review focuses on the currently available therapeutic strategies for refractory CLL.

Clinical trials in chronic lymphocytic leukemia (CLL) have focused mainly on younger fit patients until recently. However, CLL is a disease of elderly and many patients have significant comorbid conditions which together with advanced age preclude the use of aggressive regimens like FCR (fludarabine, cyclophosphamide, rituximab). Therefore, parameters such as performance status, renal function and number/severity of comorbidities together with clinical judgment should be used to guide the decision-making process regarding intensity of treatment. Two large randomized trials recently demonstrated that addition of monoclonal anti-CD20 antibodies (obinutuzumab, rituximab, and ofatumumab) to chlorambucil in untreated comorbid patients lead to improvement in complete remission rate, progression-free survival and even overall survival (obinutuzumab-chlorambucil and rituximab-chlorambucil), with acceptable toxicity profile. Thus, chemoimmunotherapy combining chlorambucil with an anti-CD20 antibody is the new standard approach for elderly/comorbid CLL patients in the first line. Treatment of relapsed/refractory disease in this patient population is very challenging and data regarding this subpopulation are rather limited. Impressive efficacy of novel targeted small molecules interfering with B-cell receptor signaling, namely Bruton tyrosine kinase inhibitor ibrutinib and phosphatidylinositol-3 kinase delta inhibitor idelalisib, radically changed the treatment paradigms for relapsed/refractory CLL; relatively mild toxicity of these agents make them very good candidates for elderly/comorbid patients. Other options for relapsed/refractory disease include alemtuzumab, ofatumumab, high-dose glucocorticoids+rituximab and bendamustine+rituximab. This review summarizes the current knowledge on prognostication and therapy of elderly and comorbid patients with CLL.

Treatment of chronic lymphocytic leukemia (CLL) has recently undergone revolutionary changes. Two large randomized trials demonstrated superiority of chemoimmunotherapy combining fludarabine and cyclophosphamide with monoclonal anti-CD20 antibody rituximab (FCR) over fludarabine and cyclophosphamide (FC) alone in first line and relapse; this lead to establishment of FCR regimen as new gold standard in younger and physically fit patients. However, elderly and/or comorbid patients may not tolerate such aggressive approach due to high risk of unacceptable toxicity. To date, no randomized trials in this patient population have improved therapeutic results over chlorambucil; therefore, this agent remains the backbone of treatment against which the new protocols should be tested. Indeed, several currently running large trials investigate whether addition of an anti-CD20 monoclonal antibody (rituximab, obinutuzumab, ofatumumab) to chlorambucil yields better results. Performance status, biological age and number/severity of comorbid conditions should be incorporated into decision-making process with regard to intensity of treatment. Other emerging treatment alternatives for this patient population include fludarabine-based regimens in attenuated doses as well as protocols containing bendamustine or lenalidomide. Highdose steroids combined with rituximab might be a promising in relapsed/refractory CLL but infectious toxicity is serious. Finally, ofatumumab has been recently approved for the treatment of fludarabine and alemtuzumab-refractory patients. This article provides an overview of the current and future possibilities in the treatment of elderly and comorbid patients with CLL.

The use of novel small molecule inhibitors alone or in combination with anti-CD20 monoclonal antibodies for chronic lymphocytic leukemia (CLL) has raised a number of questions on efficacy, tolerability, long-term treatment adherence in patients with heterogeneous clinical features. To fill this gap, we designed a study focusing on treatment sequencing in patients with CLL in order to (i) compare the outcome of patients treated with chemoimmunotherapy (CIT) combinations in first-line versus those receiving Bruton's tyrosine kinase inhibitors (BTKi); (ii) characterize the efficacy and tolerability of venetoclax-based regimens; (ii) understand the impact of treatment sequencing when it comes to chemo-free options including venetoclax after BTKi and vice versa. Data from consecutive sets of patients diagnosed with CLL between 2000-2020 attended at 77 institutions affiliated with ERIC were collected and analyzed. Collected variables included: demographics, clinical stage at diagnosis, IGHV gene somatic hypermutation status; cytogenetic status for chromosomes 11q, 13q 17p and 12 determined by fluorescence in situhybridization; TP53gene mutation status; treatment; treatment response; discontinuation; reason for discontinuation; death. We included 9173 patients with a diagnosis of CLL who received at least one line of treatment. The median age at diagnosis was 67 years with a male:female ratio of 1.9. The median follow-up was 78 months (IQR, 48-120 months). Regarding novel targeted agents, 1860/9173 (20.2%) patients had received at least one line of treatment with BTKi (ibrutinib, n=1788; acalabrutinib, n=72) over the disease course; 631/9173 (6.9%) with venetoclax; and, 447/9173 (4.9%) with the PI3K inhibitor idelalisib. Seventy-nine patients were treated with both BTKi and venetoclax (59 BTKi followed by BCL2i, 20 vice versa). At last follow-up, 5870/9173 patients (64.0%) were alive, 3229/9173 (35.2%) died and 74/9173 (0.8%) were lost to follow-up. Patients treated with BTKi in first-line were enriched for TP53aberrations [del(17p) 27.6%, TP53mutation 26.3%] and unmutated IGHV genes (69%) and obtained an ORR of 87.7%. Of these, 136 (26.3%) discontinued treatment after a median of 1.2 years (0.07-5.98); main reasons of discontinuation were toxicity (40.5%) and failure (26.2%). Among 631 patients treated with venetoclax at any line, 100 (15.8%) received BCL2 +/- anti-CD20 as first-line; 170 (26.9%) as second line (125 previously treated with CIT, 27 with BTKi); and, 361 as third or subsequent line. ORR ranged between 71.5% (≥3 lines) with 30.5% CR/CRi to 90.3% (first-line) with 68.1% CR/CRi. Treatment discontinuation was due to toxicity in 28.6% of patients treated in the first-line, and 17.6% and 21.8% of patients treated in second and third-or-higher-line, respectively. Disease progression led to treatment discontinuation in 14.3%, 20.6% and 33.6% in first, second and third-or-higher line, respectively. CIT was used as front-line treatment in 5465 patients (59.6%). Of these, 2070 (37.9%) and 1018 (18.6%) patients received a second and third line of treatment, respectively. The great majority (865/1086 cases, 79.7%) of patients who received a second line before 2014 were retreated with CIT, most commonly Bendamustine-Rituximab (284/1086, 26.1%) and Fludarabine-Cyclophosphamide-Rituximab (252/1086, 23.2%); alemtuzumab monotherapy was used in 55/1086 (5%) of patients. After 2014, 415/984 patients (42.1%) were retreated with BTKi; 93 (9.5%) with venetoclax; 70 (7.2%) with idelalisib; 50 (5%) with Alemtuzumab monotherapy, and 315 (32%) with CIT. Similarly, in the third-or-higher line of treatment, most patients (86.3%) were retreated with CIT before 2014, while BTKi, BCL2i, and PI3Ki were mainly used after 2014 (in 43.1%, 15.7% and 14.7% of cases, respectively). Finally, our cohort included 1075 patients with TP53aberrations. The ORR of patients receiving BTKis (n=171) as first-line of treatment was 86.5% (22.2 CR+64.3 PR), while the ORR with venetoclax +/- anti-CD20 (n=15) was 91% (45.5% CR+45.5 PR). Patients treated with CIT (n=694) had an ORR of 68.7% (28.3% CR+40.4% PR). In conclusion, in a large international study we provide real world data regarding the selection and sequencing of treatment in CLL, charting a major shift in treatment patterns before and after the introduction of novel trargeted agents and confirming their efficacy even in high-risk CLL.

Scarfo: Janssen: Honoraria, Other: Travel grants; Astra Zeneca: Honoraria; Abbvie: Honoraria. Iacoboni: BMS/Celgene, Gilead, Novartis, Janssen, Roche: Honoraria. Collado: Abbvie,: Other: pharmaceutical Company, Research Funding; Janssen: Other: Pharmaceutical Company, Research Funding. Galimberti: AbbVie, Janssen: Honoraria, Other: Travel grants; Incyte: Speakers Bureau. García-Serra: AbbVie: Other: Educational grands; Janssen: Other: Educational grants; Novartis: Other: Educational grants. Gozzetti: Janssen: Honoraria; AbbVie: Honoraria. Hatzimichael: Amgen, Roche, Genesis, Novartis, Bristol Mayer Squibb, Celgene, Pfizer: Consultancy; Abbvie, Amgen, Bristol Mayer Squibb, MSD, Gilead, Janssen Cilag, Genesis Pharma, Roche, Takeda: Honoraria. Herishanu: AbbVie: Honoraria, Research Funding; Janssen: Honoraria; Roche: Honoraria; Medison: Honoraria. Jaksic: Roche, Oktal-Pharma/Celtrion, Sandoz: Consultancy, Honoraria. Kater: Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; Abbvie: Honoraria, Other: Ad Board, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding; Genmab, LAVA: Other: Ad Board, Steering Committee. Kotsianidis: Astellas: Other: NONE, Research Funding, Speakers Bureau; Genesis: Consultancy, Other: NONE; Janssen Hellas: Consultancy, Other: NONE, Speakers Bureau; Bristol Hellas: Consultancy, Other: NONE, Research Funding, Speakers Bureau; Novartis Hellas: Consultancy, Other: NONE, Research Funding, Speakers Bureau; Abbvie: Consultancy, Other: NONE, Research Funding, Speakers Bureau. Kreitman: NIH: Patents & Royalties: Moxetumomab Pasudotox; Genentech: Research Funding; Teva: Research Funding; AstraZeneca/MedImmune: Research Funding; Innate: Research Funding; GSK/Novartis: Research Funding; Array BioPharma/Pfizer: Research Funding. Laribi: BeiGene: Other: Personal Fees; Jansen: Research Funding; Novartis: Other: Personal Fees, Research Funding; Astellas Phama, Inc.: Other: Personal Fees; AstraZeneca: Other: Personal Fees; Le Mans Hospital: Research Funding; Takeda: Other: Personal Fees, Research Funding; AbbVie: Other: Personal Fees, Research Funding; IQONE: Other: Personal Fees. Lopez-Garcia: Roche: Other: Speaker Honoraria, Travel and accommodation grants; Novonordisk: Other: Speaker Honoraria; Fresenius: Other: Speaker Honoraria; Celgene: Other: Speaker Honoraria; Abbvie: Other: Speaker Honoraria, Advisor, Travel and accommodation grants; Janssen: Other: Speaker Honoraria, Advisor, Travel and accommodation grants, Research Funding. Milosevic: Roche: Honoraria; Abbvie,: Honoraria; Janssen: Honoraria; Sandoz: Honoraria. Reda: Beigene: Consultancy; Astra Zeneca: Consultancy; Abbvie: Consultancy; Janssen: Consultancy. Ruchlemer: AbbVie: Consultancy, Honoraria, Research Funding. Šimkovič: Janssen, Gilead, Roche, AstraZeneca, and AbbVie: Other: consultancy fees, advisory board participation fees, travel grants, and honoraria; University Hospital Hradec Kralove: Current Employment; AbbVie: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Merck: Current equity holder in publicly-traded company; Eli Lilly: Current equity holder in publicly-traded company; J&J: Current equity holder in publicly-traded company; Gilead: Other: Travel, Accommodations, Expenses. Špaček: AbbVie, AstraZeneca, Gilead, Janssen, and Roche: Honoraria. Tadmor: Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding. Visentin: Italfarmaco and Gilead: Speakers Bureau. Vassilakopoulos: AstraZeneca: Honoraria; Amgen: Honoraria, Research Funding; Pfizer: Research Funding; Dr. Reddy's: Research Funding; Novartis: Consultancy, Honoraria; GlaxoSmithKline: Honoraria, Other: Travel; Merck: Honoraria, Research Funding; Integris: Honoraria; Roche: Consultancy, Honoraria, Other: Travel; Genesis Pharma: Consultancy, Honoraria, Other: Travel; Takeda: Consultancy, Honoraria, Other: Travel, Research Funding; AbbVie: Consultancy, Honoraria; Karyopharm: Research Funding. Vitale: Janssen: Honoraria. Yáñez: Gilead-Kite, Janssen, AbbVie, AstraZeneca, Beigene, Roche, Pfizer, Jazz, BMS, and Merck: Other: Advisory board participation fees ; Janssen, AbbVie, AstraZeneca, Gilead-Kite, Roche, Pfizer, and Merck: Speakers Bureau. Antic: AbbVie, Janssen, and Roche: Honoraria. Coscia: Janssen: Honoraria, Other, Research Funding; Gilead: Honoraria; AstraZeneca: Honoraria; AbbVie: Honoraria, Other. Cuneo: AstraZeneca: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau. Gaidano: Beigene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astrazeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Guièze: Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Astrazeneca: Consultancy, Honoraria. Laurenti: AstraZeneca: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria; Janssen: Consultancy, Honoraria; BeiGene: Honoraria. Murru: Abbvie: Consultancy, Honoraria, Other: travel and accommodation; Janssen: Consultancy, Honoraria. Sportoletti: AstraZeneca: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Tam: Beigene: Research Funding; Janssen: Research Funding; Abbvie: Research Funding; Loxo: Honoraria; Beigene: Honoraria; Janssen: Honoraria; Abbvie: Honoraria. Trněný: Amgen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Gilead Sciences: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; MorphoSys: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Celgene: Consultancy; 1st Faculty of Medicine, Charles University, General Hospital in Prague: Current Employment; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Portola: Honoraria, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses. Bosch Albareda: Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria; Abbvie: Consultancy; AstraZeneca: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Kite: Honoraria; Sanofi: Honoraria; Lilly: Honoraria. Doubek: Janssen-Cilag, AbbVie, AstraZeneca, Amgen, Gilead, Novartis: Honoraria, Research Funding. Chatzidimitriou: Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Ghia: AbbVie: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; ArQule/MSD: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Celgene/Juno/BMS: Consultancy, Honoraria; Gilead: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Sunesis: Research Funding. Stamatopoulos: AstraZeneca: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

Introduction: Chronic lymphocytic leukemia (CLL) is a disease with an extremely variable clinical course. Biological features of CLL with impact on prognosis have been well defined over the last decades, but there is little data on how comorbidities of CLL patients (pts) may influence overall survival. Importantly, most of the studies on this topic included selected patient populations from secondary or tertiary centers with significant bias caused by high proportion of referred patients with unfavourable disease course.

Introduction

No abstract.

No relevant conflicts of interest to declare.

Background. Hodgkin lymphoma (HL) that develops in the course of chronic lymphocytic leukemia (CLL) is a rare event. Optimal management of these patients (pts) is not defined. We analyzed outcome of 10 pts treated for Hodgkin´s variant (HV) of Richter syndrome.

van Oers: Roche: Consultancy. Off Label Use: Ofatumumab is a human anti-CD20 monoclonal antibody that has proven efficacy as monotherapy in refractory CLL. The purpose of its use in this clinical trial is to investigate its use in a maintenance setting for CLL. . Smolej:GlaxoSmithKline: Consultancy, Honoraria, Travel funds Other; Roche: Consultancy, Honoraria, Travel funds, Travel funds Other; Sanofi: Consultancy, Honoraria, Travel funds, Travel funds Other; Janssen: Consultancy, Honoraria, Travel funds, Travel funds Other. Gupta:GlaxoSmithKline: Employment. Phillips:GlaxoSmithKline: Employment. Williams:GlaxoSmithKline: Employment. Lisby:Genmab: Employment. Geisler:Roche: Consultancy; GlaxoSmithKline: Consultancy; Celgene: Consultancy; Janssen: Consultancy.

Greil: Roche: Honoraria; Pfizer: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria; Astra-Zeneca: Honoraria; Novartis: Honoraria; Genentech: Honoraria, Research Funding; Janssen-Cilag: Honoraria; Merck: Honoraria; Mundipharma: Honoraria, Research Funding; Eisai: Honoraria; Amgen: Honoraria, Research Funding; Celgene: Consultancy, Research Funding; Cephalon: Consultancy, Honoraria, Research Funding; Bristol-Myers-Squibb: Consultancy, Honoraria; Sanofi Aventis: Honoraria; GSK: Research Funding; Ratiopharm: Research Funding. Off Label Use: Rituximab in Maintenace Treatment of CLL. Kozak:Roche: Honoraria, Travel grants Other. Girschikofsky:Pfizer: Honoraria, Research Funding; Mundipharm: Consultancy, Honoraria. Petzer:Celgene: Honoraria, unrestricted grant Other. Egle:Roche: Honoraria, Research Funding, Travel Grants Other.

Smolej: Janssen: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Travel grants, Travel grants Other; GlaxoSmithKline: Consultancy, Honoraria, Travel grants, Travel grants Other; Roche: Consultancy, Honoraria, Research Funding, Travel grants Other. Brychtova:Roche: Travel grants Other. Doubek:Roche: Consultancy; GlaxoSmithKline: Research Funding; Janssen: Consultancy. Spacek:Roche: Consultancy, Travel grants Other. Belada:Celgene: Research Funding; Roche: Consultancy, Research Funding, Travel grants, Travel grants Other; GlaxoSmithKline: Research Funding. Motyckova:Roche: Travel grants Other. Prochazka:Roche: Honoraria, Travel grants Other; Takeda: Speakers Bureau. Kozak:Roche: Honoraria, Travel grants Other.

Background: Combination of fludarabine, cyclophosphamide and rituximab (FCR) is the current gold standard for physically fit patients (pts) with chronic lymphocytic leukemia (CLL). Nevertheless, many CLL patients cannot tolerate this intensive regimen owing to advanced age and/or serious comorbidities. Combination protocols based on dose-reduced fludarabine demonstrated promising results in pilot studies. Therefore, the Czech CLL Study Group initiated Project Q-lite, an observational study to assess efficacy and safety of low-dose FCR regimen used in elderly/comorbid patients with CLL/SLL. Updated results including progression-free survival (PFS), overall survival (OS) and multivariate analysis are presented. Patients and Methods: Between March 2009 and July 2012, a total of 207 pts with active disease (CLL, n=196, SLL, n=11) were treated by low-dose FCR at 16 centers cooperating within Czech CLL Study Group. Dose reductions of chemotherapy compared to full-dose FCR were: 50% of fludarabine (12 mg/m2i.v. or 20 mg/m2orally on days 1-3) and 60% of cyclophosphamide (150 mg/m2i.v./p.o. on days 1-3). Rituximab was administered in standard schedule (375mg/m2i.v. day 1 in 1st cycle, 500mg/m2i.v. day 1 from 2nd cycle). Treatment was repeated every 4 weeks; antimicrobial prophylaxis with sulfamethoxazol/trimethoprim and aciclovir or equivalents was recommended. The basic characteristics are summarized in Table 1. Results: Based on intention-to-treat principle, the overall response rate / complete responses including clinical CR (without bone marrow biopsy) and CRi were 81/37% in 1st line and 63/30% in relapsed/refractory (R/R) disease. Serious (CTCAE grade III/IV) neutropenia was frequent (56 and 50%) but grade III/IV infections were only 15 and 18%. The most common causes of death were CLL progression and infections. At the median follow-up of 25 months, median progression-free survival (PFS) for previously untreated and R/R patients was 28 and 15 months; median overall survival (OS) has not been reached in previously untreated pts (75 % at 30 months) and was 30 months in R/R pts. Multivariate analysis identified del 11q, del 17p, bulky lymphadenopathy (1st line) and del 17p (R/R) as independent predictors of shorter PFS; absence of therapeutic response was the only factor associated with shorter OS (both in 1st line and R/R pts). Conclusions: Low-dose FCR appears to be an effective treatment for elderly/comorbid patients with CLL/SLL in first-line as well as R/R setting. Toxicity was acceptable and manageable. In historical comparison, efficacy of low-dose FCR compares favourably with chlorambucil monotherapy and is similar to obinutuzumab-chlorambucil combination from German CLL11 study. Interestingly, neither CIRS score nor creatinine clearance were predictive of PFS/OS. The study is registered at www.clinicaltrials.gov (NCT02156726).

Ofatumumab was given a conditional approval in the EU on April 2010 for the treatment of CLL refractory to fludarabine (F-ref) and alemtuzumab (A-ref), encouraging the retrieval of further data in patients treated in a “daily life” setting and to investigate treatment safety.The main objective of this study was to obtain information on the safety profile of ofatumumab given outside clinical trials in patients with previously treated CLL. The secondary endpoints were efficacy, progression-free-survival (PFS), and overall survival (OS).This was an observational, retrospective study. Patients were eligible regardless of prior treatments or disease status and provided they had not been included in ofatumumab clinical trials. Data on patients’ characteristics at diagnosis, prior treatment, adverse events response rate, PFS and OS were recorded.One-hundred and twenty patients were screened of which 103 from 25 centers in 10 European countries were eventually eligible for the study. There were 71 males; median age at initiation of ofatumumab was 64 years (range, 38-84); 66% patients were in advanced clinical stage (Rai III-IV/Binet C) and 33 patients presented bulky lymphadenopathy. Number of prior lines of therapy was 4 (range, 1-13). 94% had received prior F-based therapy, 54% received A-based therapy and 51% received both. Eighty-one percent had been previously exposed to rituximab-combination regimens. Fifty-four percent were F-ref, 70% A-ref and 41% were both F- and A-refractory. Cytogenetics within 3 months prior therapy was available in 52 patients of which 34 presented abnormalities (11 patients: 17p-; 9 patients: 11q-; 2 patients: 13q-; 1 patient: trisomy 12; 11 patients: two or more abnormalities including 17p- or 11q-). Forty-two of 50 patients showed unmutated IGHV genes. The median number of cycles of ofatumumab given was 9 (range, 0-16) and the median percentage of given/planned cycles was 83.3% (range, 0-133). In most patients the treatment dose and schedule were as follows: 300 mg 1st infusion followed by 2000 mg for subsequent infusions (8 weekly followed by 4 doses monthly). One hundred and sixty-one adverse events were reported in 68 patients, with 28 (17%) of them being considered as ofatumumab-related. Infusion related-reactions occurred in 19 (28%) patients (III-IV: 6%). Neutropenia was reported in 26% (III-IV: 19%), thrombocytopenia in 15% (III-IV: 12%) and anemia in 15% (III-IV: 7%). The non-hematological adverse events, included infection 44% (III-IV: 36%), fatigue 10% (III-IV: 4%), fever 10% (III-IV: 6%), rash 10% (III-IV: 3%), cough 7% (III-IV: 1%), diarrhea 6% (grade III-IV: 0%) and nausea 1% (III-IV: 0%). Hematologic toxicity correlated with the number of prior lines of therapy. Autoimmune hemolytic anemia and Richter syndrome were reported in one patient each. Two heavily pre-treated patients (5 and 6 prior lines of therapy, respectively) developed progressive multifocal leukoencephalopathy. The overall response rate (ORR) was 23% and the median PFS and OS were 5 and 12 months, respectively. The main causes of death were disease progression (61%) and infection (28%).The safety profile of ofatumumab given outside clinical trials to patients with poor-prognosis and heavily pre-treated CLL was consistent with that observed in clinical trials. Although not unexpectedly the ORR was lower in this study, PFS and OS were in line with those reported in phase II trials.Montillo: Roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; GSK: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Mundipharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Bloor:GSK: Consultancy, Honoraria, Paid speaker Other. Schuh:GSK: Honoraria; Celgene: Honoraria; Mundipharma: Honoraria. Geisler:Roche: Consultancy; GSK: Consultancy. Hillmen:GlaxoSmithKline: Honoraria, Research Funding. Stilgenbauer:GSK: Honoraria, support Other. Smolej:GSK: Consultancy, Honoraria, travel grants Other. Jaeger:GSK: Honoraria, Research Funding. Leblond:Roche : Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Mundipharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Kimby:Roche: Consultancy, Honoraria, Research Funding; celgene: Consultancy, Honoraria, Research Funding; Teva: Consultancy, Honoraria, Research Funding; Emergent BioSolutions: Consultancy, Honoraria, Research Funding; Gilead Sciences: Consultancy, Honoraria, Research Funding; Jansen: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding.

High-dose methylprednisolone (HDMP) in combination with rituximab is active in the treatment of relapsed/refractory chronic lymphocytic leukemia (CLL) but serious infections are frequent. Recently published data suggest that high-dose dexamethasone might be equally effective to HDMP despite lower cumulative dose.To assess efficacy and safety of high-dose dexamethasone combined with rituximab (R-dex) in relapsed/refractory CLL.A total of 60 pts (pts) with relapsed/refractory CLL treated at a single tertiary center between September 2008 and October 2012 were included in this retrospective analysis. Basic characteristics are summarized in Table 1. The schedule of R-dex consisted of rituximab 500 mg/m2 i.v. day 1 (375 mg/m2 in cycle 1) and dexamethasone 40 mg orally on days 1-4 and 10-13. Treatment was repeated every 3 weeks for a maximum of 8 cycles. All pts received antimicrobial prophylaxis with sulfamethoxazole/trimethoprim and aciclovir.Median number of administered R-dex cycles was 6 (range, 1-8). The overall response (ORR)/complete remissions (CR) were achieved in 75/3%. At the median follow-up of 9 months, median progression-free survival was 8 months and median overall survival 24 months. Significant predictors of short PFS in univariate analysis were bulky lymphadenopathy (p=0.023) and refractoriness to fludarabine (p=0.02). Interestingly, activity of R-dex in bulky fludarabine-refractory CLL was similar to ofatumumab (ORR 62 %, median PFS, 4 months, median OS, 12 months) (Wierda et al., 2010). R-dex was successfully used as a debulking regimen before allogeneic stem cell transplantation in 8 patients. Serious (CTCAE grade III/IV) infections occurred in 29% of patients; 19% pts developed steroid diabetes requiring temporary short-acting insulin.Our data show that R-Dex is an active and feasible treatment for patients with relapsed/refractory CLL; however, major infections remain relatively frequent despite combined antimicrobial prophylaxis. In addition, durable responses are infrequent. Therefore, further optimization of this therapeutic approach is warranted. Updated results will be presented.No relevant conflicts of interest to declare.

The O-dex regimen shows relatively high ORR and CR, with promising findings for PFS and OS (including pts. harboring p53 defects), as compared to published data on rituximab plus dexamethasone regimen or ofatumumab in monotherapy. The infectious toxicity in 1/4 of pts. represents the most frequent side effect for this regimen.The study was registered at www.clinicaltrials.gov (NCT01310101).Doubek: GlaxoSmithKline: Research Funding. Mayer:Roche: Consultancy, Research Funding; Glaxo: Consultancy, Research Funding.

Ofatumumab was given a conditional approval in the EU on April 2010 for the treatment of CLL refractory to fludarabine (F-ref) and alemtuzumab (A-ref), encouraging the retrieval of further data in patients treated in a “daily life” setting and to investigate treatment safety.

The treatment of relapsed/refractory chronic lymphocytic leukemia (CLL) remains a challenging clinical issue despite remarkable improvements in prognostication and therapy. One emerging treatment option for relapsed/refractory CLL is the use of high-dose corticosteroids. High-dose methylprednisolone or dexamethasone combined with rituximab are active in relapsed/refractory CLL, but serious infections are frequent and progression-free survival (PFS) is short. The purpose of this clinical trial was to determine the efficacy and toxicity of ofatumumab-dexamethason (O-dex) combination in relapsed or refractory CLL population.

The clinical course of patients with chronic lymphocytic leukemia (CLL) is remarkably heterogeneous. All CLL patients who require therapy are destined to relapse. The prognosis and management of these relapsed patients differs based on the nature of the first-line therapy and the quality and duration of remission to that therapy, as well as biological prognostic factors. Currently, the first treatment option for younger and fit CLL patients is FCR (fludarabine, cyclophosphamide, rituximab) chemoimmunotherapy. Although it regards a very potent treatment option, patients inevitably relapse after the treatment and even approximately 7% of the patients, during initial FCR regimen treatment would fit the standard definition of refractory CLL. So far, little is known about the efficacy of subsequent therapy of patients who relapsed after FCR or were refractory to FCR. Therefore, the goal of this study was to analyze the fate of these patients in the context of routine hematological practice.We retrospectively analyzed the data of 119 patients (85 males; 34 females) consecutively entered into the databases of three large tertiary Czech hematological centers. All patients were indicated to receive FCR regimen in standard doses (F: 25mg/m2i.v. day 1–3; C: 250mg/m2i.v. day 1–3; R: 375mg/m2i.v.day 0 cycle 1, and 500mg/m2day 1 of all subsequent cycles).The median age was 57 years (range, 31–75) at the time of the CLL diagnosis, and 60 years (range, 32–78) at the start of FCR therapy. The median follow-up was 42 months (range, 3–114). Our cohort of patients consisted of two groups of patients which received the FCR regimen as first-line therapy (Group 1; n=63) and patients receiving FCR in second or subsequent line of treatment (Group 2; n=56). With respect to the basic parameters (age, clinical stage, FISH cytogenetics, molecular genetics), the statistics of these groups did not differ significantly. In Group 1, the overall response rate (ORR) was 84%, in Group 2, 78% (p=ns) after FCR. Complete remission (CR) rate was 53% in Group 1, and 38% in Group 2 (p=0.04). The median progression-free survival (PFS) was 18.6 months for Group 1 and 14.7 months for Group 2 (p=ns). With subsequent therapy (repeated FCR, alemtuzumab, R-CHOP, rituximab plus high-dose corticosteroids) for relapsed disease after FCR, ORR was 59% in Group 1 (33% CR) and 44% in Group 2 (21% CR) (p=ns). PFS after subsequent therapy after FCR was 13.3 months (Group 1) and 5.9 months (Group 2) (p=0.01). The median OS was insignificantly shorter in Group 2 (44.5 months in Group 2 vs. not reached in Group 1; p=ns). After FCR therapy, there was no statistically significant change in cytogenetic findings, however, new occurrence of 17p deletion was observed in 5 patients. Prior to FCR therapy and during relapse after FCR, the p53 function was analyzed in 37 patients. After FCR, the new mutation of p53 was newly found in 6 patients (16%). These patients received a higher cumulative dose of FCR than the patients who did not develop the mutation (F 591 mg vs. 334 mg; C 5072 mg vs. 3375mg; R 3700mg vs. 2000 mg) (p=ns).Subsequent treatment for patients who relapse after FCR or are FCR refractory is very heterogeneous. Regardless of the type of therapy selected, the prognosis of these patients is poor. In addition, new p53 mutations/deletions occur after FCR.No relevant conflicts of interest to declare.

Abstract 4603

Both radiological (chest X-rays and High Resolution Computed Tomography) and PCR methods are essential for viral pneumonia diagnostic procedures. Bronchoalveolar lavage (BAL) samples obtained from patients with signs of lower respiratory tract infection were tested and analysed for viral agents.Forty seven BAL samples were obtained from 32 recipients (20 males and 12 females) of allogeneic haematological stem cell transplantation. Of those, 30 patients (93 %) were transplanted for haematological malignancy (52 % with acute myeloid leukemia, 12 % with chronic lymphatic leukemia) and 2 for aplastic anemia. BAL sampling was performed in locations of maximum signs of pathological process according to X-ray or HRCT. For testing, 4× 50 ml of F1/1 was installed in alveoli and 3rd and 4th portions were used. Viral infection diagnosis was based on real-time PCR detection of nucleic acid and/or immunochromatography detection of viral antigens.The following examinations were commonly performed on BAL samples: microscopic and cytologic examination (including staining for Pneumocystis jiroveci and branched fungi), cultivation (bacteriological, mycological and mycobacterial), real-time PCR (adenovirus, cytomegalovirus (CMV), herpes simplex virus (HSV) type 1,2, influenza virus A/B/H1N1, parainfluenza virus 1,2,3, respiratory syncytial virus (RSV), varicella zoster virus, rhinovirus, Epstein Barr virus, human metapneumovirus, Mycobacterium sp. et TBC, Streptococcus pneumoniae, Listeria monocytogenes, Chlamydia pneumoniae, Legionella pneumophila, Aspergillus fumigatus, panfungal DNA, Pneumocystis jiroveci (PCP)) and immunochromatography/immunofluorescence detection of antigens (Adenovirus, influenza virus A/B, respiratory syncytial virus, Chlamydia pneumoniae) and Aspergillus galactomannan.Viral agents were detected in 20 (out of 47) samples (in some of those, several tests yielded more than one positive or grey zone result). No viral infection was detected in 27 samples. Dual viral infection was diagnosed in 4 cases. Overall, 20 tests proved significant viral load (104cp/ml) (see Tab. 1).Tab. 1 Viruses examined and detected by PCR and immunochromatography (therapy: G – ganciclovir, F – foscarnet, A - aciclovir, T oseltamivir, R – ribavirin).Note: Influenza is mostly detected from nasal swab – this type of sample was not included in our study.The examination of BAL is very helpful for early diagnosis of viral pneumonia.The analysis must be performed from the 3rd and 4th portion of lavage fluid that comes in close contact with the terminal part of lungs - alveoli. As we expected CMV, HSV and RSV were found to be the most frequent causes of viral pneumonia. PCP pneumonia was not detected because of the sulfamethoxazolum/trimethoprimum prophylaction. In case of low viral load all other clinical, radiological and other laboratory findings must be evaluated to confirm viral etiology. High viral load is always alarming and it is necessary to start administration of causative antiviral therapy as soon as possible if it is available.Smolej: GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants; Roche: Honoraria, Travel Grants; Genzyme: Honoraria, Travel Grants.

Results: Seven pts (4 male) with a medianLittle is known about the pharmacokinetics (PK) and pharmacodynamics of CD20 monoclonal antibody (mAb) with chemotherapy in patients (pts) with CLL. Ofatumumab (O) is a human mAb targeting a membrane-proximal small-loop epitope on CD20 and mediates efficient complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity. Safety and efficacy of O at 2 dose levels in combination with fludarabine and cyclophosphamide (FC) were evaluated in previously untreated pts with CLL. Relationship between O PK, baseline characteristics, and clinical outcomes were studied.Pts with active CLL were randomized to O 500 mg (n=31) or 1000 mg (n=30) on Day 1 with F 25 mg/m2 and C 250 mg/m2 on Days 2–4 (Course 1) or Days 1–3 (Courses 2–6) every 4 weeks for 6 courses. O dose at Course 1 was 300 mg for both groups. Response (1996 NCI-WG criteria) was assessed by an Independent Review Committee up to 3 months after last course. Serial blood samples were collected at Courses 1 and 6 for noncompartmental PK analyses; pre- and post-infusion samples were collected at other courses. Relationship between PK parameters and baseline pt characteristics and disease factors was evaluated by univariable and multivariable analyses. Associations between PK and complete response (CR), overall response (OR), or progression-free survival (PFS) were explored using univariable regression analyses.22/31 (71%; 500 mg) and 19/30 (63%; 1000 mg) of pts received all 6 O doses; 2 pts at 1000 mg did not receive FC for Course 6. CR (primary endpoint) rates of 32% and 50% and OR rates of 77% and 73% were observed in the 500 mg and 1000 mg groups, respectively. O PK parameters are summarized (Table). O PK at Dose 6 appeared proportional to dose. Factors associated with PK in multivariable analyses are shown (Table). The factor most associated with PK at Dose 1 was sex, with higher Cmax/AUC and lower CL and Vss/longer t½ in women vs. men. PK at Dose 6 was not consistently associated with any factor tested.Median Cmax and Cmin values were similar at first dose between pts who had CR, partial response (PR)/nPR, and stable disease (SD)/progressive disease (PD; Figure); at later doses, median Cmax and Cmin values appeared different between CR and the other groups, although number of subjects decreased over time, especially in the SD/PD group. Based on univariable analyses, higher Cmax and Cmin at Dose 3 and higher Cmax and AUC at Dose 6 were associated with increased likelihood of CR (P<.05); higher Cmin before Dose 6 was associated with increased likelihood of OR and longer PFS.PK of O in combination with FC appeared proportional to dose after repeated dosing. Higher concentrations at Doses 3 and 6 were associated with CR. O PK was similar at first dose and different at later doses between patients who had CR, PR/nPR, and SD/PD, suggesting that response to O-FC treatment leads to clearance differences due to decreased B-cell mass and thus concentration differences with continued dosing. Further analyses of associations between disease-related factors, PK, and treatment response will be performed at study completion.Wierda: GlaxoSmithKline: Research Funding; Genentech: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Merck: Consultancy; Abbott: Research Funding. Off Label Use: Ofatumumab is an anti-CD20 monoclonal antibody approved for the treatment of fludarabine- and alemtuzumab-refractory chronic lymphocytic leukemia, and is currently under development for the treatment of B-cell malignancies (chronic lymphocytic leukemia, diffuse large B-cell lymphoma, Waldenstroms macroglobulinemia and follicular lymphoma), as well as autoimmune diseases (rheumatoid arthritis and multiple sclerosis). Jewell:GlaxoSmithKline: Employment. Kipps:Gilead Sciences: Consultancy, Research Funding; GSK: Research Funding; Genentech: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Abbot Industries: Research Funding; Celgene: Consultancy, Research Funding; Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding. Dürig:Santaris Pharma: Consultancy, Research Funding; GSK: Speakers Bureau; Roche: Speakers Bureau; Celgene: Research Funding. Stilgenbauer:Genmab: Research Funding; GSK: Consultancy, Honoraria, Research Funding. Smolej:Roche: Honoraria, Travel Grants; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants; Genzyme: Honoraria, Travel Grants. Hernandez-Ilizaliturri:Genmab: Research Funding; Celgene: Honoraria; Amgen: Research Funding. Fang:PharStat: Employment; GSK: Consultancy; Gilead Sciences: Consultancy; Pharmasset Inc: Consultancy. Gorczyca:GlaxoSmithKline: Employment. Chan:GlaxoSmithKline: Employment. Gupta:GlaxoSmithKline: Employment. Lisby:Genmab A/S: Employment.

Smolej: GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants; Roche: Honoraria, Travel Grants; Genzyme: Honoraria, Travel Grants.

Abstract 4577

Smolej: GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants; Roche: Honoraria, Travel Grants; Genzyme: Honoraria, Travel Grants.

Abstract 4905

Abstract 2526

Abstract 1793

Patients with refractory CLL have poor outcome despite currently used salvage treatment. Regimens based on high-dose corticosteroids seem to offer a promising treatment option in this scenario. High-dose methylprenisolone combined with rituximab (R-HDMP) demonstrated significant activity in relapsed/refractory CLL but serious infectious complications occurred in a substantial proportion of patients. Pilot data have shown that combination of dexamethasone and rituximab (R-Dex) may provide comparable results with less toxicity.We performed a retrospective analysis of the efficacy and safety of R-Dex in patients (pts) with CLL treated at two tertiary centers between April 2006 and February 2010. Patients received two versions of R-Dex regimen: the dose of rituximab was either 500 mg/m2 on day 1, 8, 15, 22 (375 mg/m2 in 1st cycle), repeated every 4 weeks (n=25) or 500 mg/m2 on day 1 (375 mg/m2 in 1st cycle) repeated every 3 weeks (n=16). The dose of dexamethasone was identical in both regimens: 320 mg per cycle (40 mg on day 1–4 and 10–13 or 15–18).R-Dex was administered to 41 patients (19 males) with median age of 68 years (range, 44–81) indicated for treatment according to NCI-WG criteria. Autoimmune hemolytic anemia or thrombocytopenia was the only indication for the treatment in 7 patients. Rai stage III/IV was present in 37/41 pts. IgVH genes were unmutated in 24/29 pts with available results. Cytogenetic aberrations detected by FISH (n=33) revealed del 17p in 7 patients; del 11q in 11 patients; del 13q in 15 patients and trisomy 12 in 5 patients. Median number of previous therapies was 2 (0-8); 29/41 pts were previously treated with fludarabine-based regimens. The effect of R-Dex in evaluable patients without hemolysis (n=32) was: overall response rate (ORR), n= 21 (62%), complete remission (CR), n=6 (18%), partial remission (PR), n=15 (44%), stable disease (SD), n=4 (12%) and progressive disease (PD), n=5 (15%). All patients treated with R-Dex for autoimmune cytopenia achieved complete resolution of hemolysis. Grade III or IV toxicity included infections in 13 patients (32%), steroid diabetes in 6 patients (15%) and rituximab infusion-related side effects in 3 patients (7%). At the time of analysis (February 2010), median progression free survival (PFS) was 9 months; median overall survival has not been reached. There was no difference in ORR, PFS or OS between the two versions of R-Dex regimen.This pilot study shows that R-Dex is a feasible and effective treatment for relapsed/refractory CLL. In particular, R-Dex appears to be highly effective in CLL with autoimmune cytopenias. However, infectious toxicity remains a serious issue. In addition, long-term disease control can be expected in minority of patients only. Interestingly, higher dose of rituximab per cycle did not result in improved efficacy.Supported by research project MZO 00179906 from Ministry of Health, Czech Republic, by research grant MSM 0021620808 and by the Czech Leukemia Study Group for Life.Smolej: Roche: Honoraria; Bayer-Schering: Honoraria; Genzyme: Honoraria.

Combination of fludarabine, cyclophosphamide and rituximab (FCR) is currently considered the treatment of choice in physically fit patients (pts) with chronic lymphocytic leukemia (CLL). However, many patients cannot tolerate this aggresive treatment because of advanced age and/or serious comorbid conditions. For these patients, chlorambucil has remained so far the standard of treatment. Low-dose fludarabine-based regimens have recently demonstrated promising results in small studies.to assess efficacy and safety of low-dose FC or FCR regimen used in elderly/comorbid patients with CLL.Between March 2009 and June 2010, we treated 74 pts with active disease (CLL, n=70, SLL, n=4, 57% males, median age, 70 years [range, 58–83], median Cumulative Illness Rating Score 4 [range, 0–10]) by low-dose FC/FCR at fourteen centers cooperating within Czech CLL Study Group. Dose reduction of chemotherapy was as follows: fludarabine to 50% (12 mg/m2 i.v. or 20 mg/m2 orally on Days 1–3), cyclophosphamide to 60% (150mg/m2 i.v./p.o. D1-3). The dose of rituximab was standard (375mg/m2 in 1st cycle, 500mg/m2 from 2nd cycle). The choice of regimen (FC vs FCR) was at the discretion of the attending physician. Treatment was repeated every 4 weeks. Antimicrobial prophylaxis with cotrimoxazole and aciclovir or equivalents was recommended. Fifty per cent of pts were treated in first line, the remaining half had relapsed/refractory CLL. Rai stage III/IV was present in 57% pts; 39% had bulky disease. IgVH genes were unmutated in 74%; according to hierarchical model, del 11q was present in 32% and del 17p in 8%.FCR was used in 72 pts, FC in 2. Based on intention-to-treat principle, the overall response/complete response rate (including clinical CR and CR with incomplete blood count recovery) was 70/35%; 34 pts are still on treatment. No data on PFS/OS are available yet. Serious (CTC grade III/IV) neutropenia occurred in 51%, thrombocytopenia in 13% and anemia in 10% of pts. Serious infections were diagnosed in 13% of pts. Three pts (4%) died, all of them after failure of treatment (pneumonia, n=2, pulmonary embolism, n=1).Treatment of elderly/comorbid CLL patients with low-dose FC/FCR demonstrated very promising results. Toxicity was acceptable and manageable. Longer follow-up is needed for the assessment of PFS and OS. Supported by research project MZO 00179906 from Ministry of Health, Czech Republic.Smolej: Roche: Honoraria; Bayer-Schering: Honoraria; Genzyme: Honoraria. Off Label Use: Low-dose FCR in elderly/comorbid patients with CLL. Belada: Roche: Consultancy, Honoraria. Motyckova: Roche: Honoraria.

Abstract 2466

Abstract 4629

Chemoimmunotherapy regimens have become the treatment standard for patients with CLL. Ofatumumab is a human monoclonal antibody that targets a unique small-loop epitope on CD20 and elicits rapid and efficient in vitro complement-dependent cytotoxicity, as well as antibody-dependent cellular cytotoxicity. Recent studies demonstrated single-agent ofatumumab activity, with high overall response rates (ORR) in patients with refractory CLL. We conducted an international, randomized, parallel group, Phase II trial with two doses of ofatumumab combined with fludarabine and cyclophosphamide (FC) in previously untreated patients with CLL to evaluate the efficacy and tolerability of this chemoimmunotherapy regimen.Previously untreated patients (N=61) with active CLL (by NCI-WG guidelines) were randomized to receive ofatumumab 500 mg (Group A) or 1000 mg (Group B) on day 1, combined with fludarabine (25 mg/m2 IV daily; days 1–3) and cyclophosphamide (250 mg/m2 IV daily; days 1–3) every 4 weeks for a total of 6 courses. In both Groups, the first dose of ofatumumab was 300 mg. Dose reduction of FC, but not ofatumumab, was allowed. Premedication for ofatumumab was paracetamol and antihistamine prior to each infusion, and glucocorticoid prior to infusions 1 and 2. Neutrophil growth factor and anti-infective prophylaxis were not mandated. The primary endpoint was complete response (CR) rate (1996 NCI-WG criteria) assessed by an Independent Review Committee (IRC), measured from the start of treatment until 3 months after the last infusion. Safety evaluations included investigator-reported adverse events (AEs) and deaths. Follow-up continues for collection of time-to-event endpoints.Data from all 61 patients were available for response assessment (primary endpoint). Pretreatment characteristics are shown in the Table. 71% and 57% of patients in Groups A and B, respectively, completed all 6 courses of O-FC treatment. The CR rate (95% CI) by IRC evaluation was 32% (17, 51%) for Group A and 50% (31, 69%) for Group B; the ORR (95% CI) was 77% (59, 90%) and 73% (54, 88%), respectively (Table). The median progression-free survival has not been reached with the short median follow up of 8 months. No CTC grade 3–4 infusion-related reactions on the day of ofatumumab infusion were reported. During treatment and up to 30 days following the last dose, the most common (>10% of patients) grade 3–4 AEs reported by investigators were infections in 11 patients (Group A, n=4; Group B, n=7) including febrile neutropenia in 3 patients in each Group, and hematologic AEs including neutropenia in 29 patients (Group A, n=11; Group B, n=18), anemia in 8 patients (Group A, n=2; Group B, n=6) and thrombocytopenia in 9 patients (Group A, n=2; Group B, n=7); grade 3–4 hemolytic anemia occurred in 2 patients in Group A and 1 in Group B; one patient in Group B died (19 days from last dose) with dyspnea (etiology unknown). Beyond the AE reporting period mentioned above, one patient in Group A died (50 days from last dose) due to febrile neutropenia during the follow up period. Results from additional analysis of data will be presented at the meeting.The O-FC regimen is highly active in previously untreated patients with CLL at both ofatumumab doses investigated and may offer a new chemoimmunotherapy treatment option in these patients. AEs with the O-FC regimen were manageable with no unexpected toxicities. The 1000 mg dose of ofatumumab is currently being evaluated in combination with chemotherapy in other studies for patients with CLL.Wierda: Genmab, GlaxoSmithKline: Honoraria, Research Funding. Off Label Use: Ofatumumab is an investigational anti-CD20 monoclonal antibody, currently under development for the treatment of B-cell malignancies (chronic lymphocytic leukemia, diffuse large B-cell lymphoma, Waldenstroms macroglobulinemia and follicular lymphoma) as well as autoimmune diseases (rheumatoid arthritis and multiple sclerosis). Kipps:Physicians' Education Resource, Educational Concepts: Speakers Bureau; Genmab, Abbott Industries, Celgene, Biogen Idec, Cephalon, sanofi-aventis, Medimmune, Memgem, Genentech: Research Funding. Dürig:GlaxoSmithKline: Honoraria. Griskevicius:GlaxoSmithKline, Genmab: Research Funding. Stilgenbauer:GlaxoSmithKline, Genmab: Consultancy, Honoraria, Research Funding. Mayer:GlaxoSmtihKline: Consultancy. Smolej:Bayer Schering: Honoraria. Padmanabhan:GlaxoSmithKline: Consultancy, Honoraria; Celgene, Genentech: Consultancy. Gorczyca:GlaxoSmithKline: Employment. Chan:GlaxoSmithKline: Employment. Gupta:GlaxoSmithKline: Employment. Andersen:H. Lundbeck A/S: Shares ownership; Novo Nordisk A/S, H. Lundbeck A/S and Genmab A/S: Consultancy. Strange:Genmab: Employment. Nielsen:Genmab: Employment. Russell:Genmab: Employment, Equity Ownership.

Abstract 207

Introduction: Angiogenesis is an essential process in the biology of solid tumors as well as hematological malignancies. Several studies have recently reported increased markers of angiogenesis in chronic myeloid leukemia (CML). However, much less is known on the possible impact of tyrosine kinase inhibitor imatinib mesylate (IM), the current cornerstone of CML treatment, on angiogenic signaling.

Several studies have demonstrated potential prognostic importance of angiogenesis in chronic lymphocytic leukemia (CLL). Elevated expression of angiogenic activator angiopoietin-2 (Ang-2) was recently reported in CLL but data regarding prognostic significance of Ang-2 in CLL is limited. Therefore, we evaluated potential relationship between Ang-2 expression and other prognostic factors in patients with CLL. Ang-2 mRNA was quantified by real-time quantitative PCR in purified mononuclear cells of 33 untreated CLL patients. Ang-2 transcript levels were further analyzed with regard to clinical stage, disease course, IgVH mutation status, CD38 and ZAP-70 expression. Elevated Ang-2 mRNA was detected in 12 cases; twenty-one patients had very low or undetectable levels of Ang-2 transcript. High Ang-2 mRNA levels were significantly associated with unmutated IgVH genes (p=0.010) and high CD38 expression (p=0.011) but not with ZAP-70 expression (p=0.784), Rai stage (p=0.305) or stable vs. progressive clinical course (p=0.443). Interestingly, there was a trend towards shorter progression-free survival in patients with high Ang-2 expression, but it did not reach statistical significance (median 15 months vs. not reached; p=0.090). In conclusion, our data show that Ang-2 mRNA is differentially expressed in patients with CLL and its increased expression appears to be associated with poor prognostic features.

Angiogenesis has been studied in chronic lymphocytic leukemia (CLL) since mid 1990s. Given the extraordinary heterogeneity of clinical course of CLL patients, angiogenesis assessment may potentially improve prognostic stratification. There are numerous reports on increased markers of angiogenesis in CLL; however, data on possible association of angiogenic markers with modern prognostic factors are very scarce. Therefore, we evaluated prognostic significance of angiogenic cytokines, namely basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), endostatin, and angiopoietin-2 (Ang-2) in patients with untreated CLL. Plasma levels of angiogenic factors were quantified using enzyme-linked immunosorbent assay (bFGF, VEGF, n=106; endostatin, n=83, Ang-2, n=55). Eighty healthy donors served as control group. Relationship of angiogenic markers to modern prognostic factors (IgVH mutation status, cytogenetic aberrations detected by fluorescent in situ hybridization [FISH]) was also analyzed. Results: bFGF, VEGF, and endostatin were significantly elevated in CLL patients when compared to controls (bFGF, VEGF: p<0.0001, endostatin: p=0.039); Ang-2 levels were not different from the control (p=0.192). There were no differences in angiogenic factors with regard to gender, clinical course or modified Rai stage. Similarly, no association of angiogenic factors with IgVH or FISH was identified. Interestingly, in a pilot subgroup of 16 patients with serial measurements before and after successful fludarabine-based combination treatment, levels of bFGF as well as VEGF decreased significantly (p<0.0001 and p=0.0027, respectively). In conclusion, our study confirms that angiogenic signaling is intensive in CLL. Fludarabine-based treatment may have intrinsic antiangiogenic properties. Further research is needed for better understanding of angiogenic processes, especially with respect to possible refinement of individual patient's prognosis in this disease.

The p53 gene, metaphorically named “the Guardian of the Genome“ by David Lane in 1992, is one of the most important decision makers inside the cell. By interacting with an array of downstream genes, p53 can regulate cell fate, either by precipitating events leading to cell death by apoptosis, or by acting as a regulatory factor during G1/S phase allowing the cell to repair minor damage to DNA and proceed to the next stage of cell division. Playing such a pivotal role within the cell, p53 itself is subjected to a tight and orchestrated control, creating a network of positive and negative regulations via a number of interacting proteins (MDM-2, Wip-1, Cyclin G, p14ARF). Yet another level of p53 regulation is represented by post-translational modifications, modulating its transcriptional/transactivational ability. These modifications include phosphorylations on serines 15, 18 and 20 or acetylations at C-terminal lysines of p53. Recently, it has been found that the regulation of p53 is also substantially controlled at the transcriptional level. They identified nine different splicing variants of p53 with distinct biological characteristics, resulting from combinations of an alternative splicing of intron 2, 9 and/or aberrant transcription, starting at so-far unrecognized cryptic promoter in intron 4. Herein we present evidence of a novel splicing variant of p53 gene, termed delta ex6 that is differentially expressed in patients with chronic lymphocytic leukemia (CLL) as compared to healthy donors. The delta ex6 variant was identified in 109 out of 127 (86%) CLL patients, while in healthy individuals it was not detected. Delta ex6 variant is devoid of transactivational activity as determined in vitro by FASAY (Functional Analysis of Separated Alleles in Yeast). To test the biological properties of the delta ex6 variant we have cloned its whole coding sequence and transfected the p53-double-knock-out model cell line H1299 to produce stable integrants. Stable H1299 cell lines expressing the delta ex6 variant were distinguished by a remarkable loss of intercellular contacts and semi-suspension growth properties, in contrast to the strictly adherent growth of the parental cells and mock-transfected cells. Four stable delta ex6 producing H1299 cell lines, as well as control cell lines in doublets (parental H1299 harboring the cloning vector, and H1299 stably transfected with wild type p53) were subjected to the Affymetrix GeneChip Human Exon 1.0 ST array expression analysis. The microarray data corroborated the accented and proliferative phenotype as observed in vitro in the tissue culture: overexpression of a number of cyclins (A1, G1, G2, F, I, B2, A2, T2), matrix metalloproteinases, hyaluronidases and caspase inhibitors; and downregulation of adhesion molecules and molecules of the intercellular matrix. Our data on the presence of the delta ex6 p53 variant in CLL patients supports the recent evidence on dysregulation of p53 splicing pattern in malignancies. Moreover, as assessed in vitro, overexpression of the delta ex6 variant leads to an accented and proliferative phenotype, a finding further supporting the biological role of the novel delta ex6 p53 variant in vivo.

Several studies have demonstrated potential prognostic importance of angiogenesis in chronic lymphocytic leukemia (CLL). Elevated expression of angiogenic activator angiopoietin-2 (Ang-2) was recently reported in CLL but data regarding prognostic significance of Ang-2 in CLL is limited. Therefore, we evaluated potential relationship between Ang-2 expression and other prognostic factors in patients with CLL. Ang-2 mRNA was quantified by real-time quantitative PCR in purified mononuclear cells of 33 untreated CLL patients. Ang-2 transcript levels were further analyzed with regard to clinical stage, disease course, IgVH mutation status, CD38 and ZAP-70 expression. Elevated Ang-2 mRNA was detected in 12 cases; twenty-one patients had very low or undetectable levels of Ang-2 transcript. High Ang-2 mRNA levels were significantly associated with unmutated IgVH genes (p=0.010) and high CD38 expression (p=0.011) but not with ZAP-70 expression (p=0.784), Rai stage (p=0.305) or stable vs. progressive clinical course (p=0.443). Interestingly, there was a trend towards shorter progression-free survival in patients with high Ang-2 expression, but it did not reach statistical significance (median 15 months vs. not reached; p=0.090). In conclusion, our data show that Ang-2 mRNA is differentially expressed in patients with CLL and its increased expression appears to be associated with poor prognostic features.

Angiogenesis has been studied in chronic lymphocytic leukemia (CLL) since mid 1990s. Given the extraordinary heterogeneity of clinical course of CLL patients, angiogenesis assessment may potentially improve prognostic stratification. There are numerous reports on increased markers of angiogenesis in CLL; however, data on possible association of angiogenic markers with modern prognostic factors are very scarce. Therefore, we evaluated prognostic significance of angiogenic cytokines, namely basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), endostatin, and angiopoietin-2 (Ang-2) in patients with untreated CLL. Plasma levels of angiogenic factors were quantified using enzyme-linked immunosorbent assay (bFGF, VEGF, n=106; endostatin, n=83, Ang-2, n=55). Eighty healthy donors served as control group. Relationship of angiogenic markers to modern prognostic factors (IgVH mutation status, cytogenetic aberrations detected by fluorescent in situ hybridization [FISH]) was also analyzed. Results: bFGF, VEGF, and endostatin were significantly elevated in CLL patients when compared to controls (bFGF, VEGF: p<0.0001, endostatin: p=0.039); Ang-2 levels were not different from the control (p=0.192). There were no differences in angiogenic factors with regard to gender, clinical course or modified Rai stage. Similarly, no association of angiogenic factors with IgVH or FISH was identified. Interestingly, in a pilot subgroup of 16 patients with serial measurements before and after successful fludarabine-based combination treatment, levels of bFGF as well as VEGF decreased significantly (p<0.0001 and p=0.0027, respectively). In conclusion, our study confirms that angiogenic signaling is intensive in CLL. Fludarabine-based treatment may have intrinsic antiangiogenic properties. Further research is needed for better understanding of angiogenic processes, especially with respect to possible refinement of individual patient’s prognosis in this disease.

Introduction: Angiogenesis is an essential process in the biology of solid tumors as well as hematological malignancies. Several studies have recently reported increased markers of angiogenesis in chronic myeloid leukemia (CML). However, much less is known on the possible impact of tyrosine kinase inhibitor imatinib mesylate (IM), the current cornerstone of CML treatment, on angiogenic signaling. Aims: To analyze peripheral blood levels of angiogenic cytokines in patients with newly diagnosed CML and during imatinib treatment. Methods: Peripheral blood plasma concentrations of key angiogenic activators, namely vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), soluble endoglin (sCD105), and angiopoietin-2 (Ang-2) were quantified using commercially available sandwich enzyme-linked immunosorbent assay (ELISA) in 27 patients with chronic-phase CML and 80 healthy blood donors; furthemore, repeated samples during the therapy with IM were analyzed. Results: We detected significantly elevated levels of VEGF (p<0.0001), bFGF (p=0.026), and sCD105 (n=25, p<0.0001) in comparison to the control group. Elevation of Ang-2 was of borderline significance (n=14, p=0.051). Levels of VEGF and sCD105 significantly decreased in patients who achieved complete cytogenetic response (p<0.0001 and p=0.01) whereas bFGF or Ang-2 did not change significantly (p=0.978 and p=0.625, respectively). VEGF and sCD105 remained low in repeated samples taken during IM treatment. Interestingly, there was a trend towards higher VEGF in patients who did not achieve complete cytogenetic response (p=0.058). Conclusions: Peripheral blood levels of angiogenic activators VEGF, bFGF and sCD105 (and borderline Ang-2) were elevated in CML patients. Successful treatment with IM resulted in significant and durable suppression of circulating VEGF and sCD105 but not bFGF and Ang-2. Our results point to the potential significance of angiogenic signaling (especially VEGF pathway) in CML biology. Further research in this field is therefore highly desirable.

The p53 gene, metaphorically named “the Guardian of the Genome“ by David Lane in 1992, is one of the most important decision makers inside the cell. By interacting with an array of downstream genes, p53 can regulate cell fate, either by precipitating events leading to cell death by apoptosis, or by acting as a regulatory factor during G1/S phase allowing the cell to repair minor damage to DNA and proceed to the next stage of cell division. Playing such a pivotal role within the cell, p53 itself is subjected to a tight and orchestrated control, creating a network of positive and negative regulations via a number of interacting proteins (MDM-2, Wip-1, Cyclin G, p14ARF). Yet another level of p53 regulation is represented by post-translational modifications, modulating its transcriptional/transactivational ability. These modifications include phosphorylations on serines 15, 18 and 20 or acetylations at C-terminal lysines of p53. Recently, it has been found that the regulation of p53 is also substantially controlled at the transcriptional level. They identified nine different splicing variants of p53 with distinct biological characteristics, resulting from combinations of an alternative splicing of intron 2, 9 and/or aberrant transcription, starting at so-far unrecognized cryptic promoter in intron 4. Herein we present evidence of a novel splicing variant of p53 gene, termed delta ex6 that is differentially expressed in patients with chronic lymphocytic leukemia (CLL) as compared to healthy donors. The delta ex6 variant was identified in 109 out of 127 (86%) CLL patients, while in healthy individuals it was not detected. Delta ex6 variant is devoid of transactivational activity as determined in vitroby FASAY (Functional Analysis of Separated Alleles in Yeast). To test the biological properties of the delta ex6 variant we have cloned its whole coding sequence and transfected the p53-double-knock-out model cell line H1299 to produce stable integrants. Stable H1299 cell lines expressing the delta ex6 variant were distinguished by a remarkable loss of intercellular contacts and semi-suspension growth properties, in contrast to the strictly adherent growth of the parental cells and mock-transfected cells. Four stable delta ex6 producing H1299 cell lines, as well as control cell lines in doublets (parental H1299 harboring the cloning vector, and H1299 stably transfected with wild type p53) were subjected to the Affymetrix GeneChip Human Exon 1.0 ST array expression analysis. The microarray data corroborated the accented and proliferative phenotype as observed in vitro in the tissue culture: overexpression of a number of cyclins (A1, G1, G2, F, I, B2, A2, T2), matrix metalloproteinases, hyaluronidases and caspase inhibitors; and downregulation of adhesion molecules and molecules of the intercellular matrix. Our data on the presence of the delta ex6 p53 variant in CLL patients supports the recent evidence on dysregulation of p53 splicing pattern in malignancies. Moreover, as assessed in vitro, overexpression of the delta ex6 variant leads to an accented and proliferative phenotype, a finding further supporting the biological role of the novel delta ex6 p53 variant in vivo.