ALBERT

Beschreibung: Objetives: Analyze the prognostic significance of CD38 expression compared to other prognostic factors. Patients and methods: We studied retrospectively and prospectively at our institution 160 patients (pts) with diagnostic of CLL who met the diagnostic criteria of National Cancer Institute-Working Group (NCI-WG), 97 men and 63 female with an age range of 33 – 90 years (median 62) diagnosed between January 1989 and December 2003 and evaluated up to June 2004. Sixty nine pts showed CD38 positive cells and 91 were CD38 negative. CD38 expression was determined by flow cytometry defining as a higher expression 〉 7% CD38 positive cells. (Krober A et al. Blood15: 1410–1416.2002). Event Free Survival (EFS) and Overall Survival (OS) were analized according to different characteristics at diagnosis. No. Patients %EFS 72 mo p= %OS 72 mo p= Binet Stage A /B-C 129/31 65/30 0.000 90/92 0.245 Rai Stage 0 /I – II – III – IV 92/68 72/37 0.000 94/94 0.339 TDLI 〈 / ≥ 12 mo 23/126 41/63 0.007 65/100 0.001 Light Chains kappa / lambda 94/66 68/50 0.473 90/95 0.702 CD38 Positive / Negative 69/91 32/82 0.000 81/100 0.000 Statistical significant difference was observed in Binet stage, Rai stage, TDLI and CD38 expression for EFS and in TDLI and CD38 expression for OS. Considering these variables with p value of

Beschreibung: Background. There are known prognostic factors in Acute myeloid leukemia (AML) patients, being the cytogenetic analysis the strongest as single predictor of disease relapse or poor therapy response. Recently, alterations in FLT3 gene (Internal tandem duplications-ITD and D835/836 mutations) are frequently detected by PCR in 30–35% of AML patients (pts) and would be associated with aggressive disease. This study reports the molecular characterization of 82 AML pts from Argentina and Uruguay, mostly of Spanish-Italian origin, studied between 1996 to 2005. Design and Methods. This study was based on 82 pts: 71 adults, median age 36 yrs, (range 25–80) and 11 children (median age: 11 yrs, range. 3–17 yrs). Cytogenetic risk was established in 77 pts by kariotyping, PCR and FISH: 49% (n=38) with low risk, 38% (n=29) with standard risk, and 13% (n=10)with high risk. The FAB distribution (n=75) was: M0=2,7% (n=2), M1= 6,7% (n=5); M2=17,3% (n=13); M3=46,7% (n=35); M4=16% (n=12); M5=6,7% (n=5); M6=4% (n=3). Clinical endpoints and follow up were available for 45 pts and 56 pts, respectively. A total of 42 pts achieved complete remission (CR), 12 pts had relapse of disease, 10 pts underwent early death without completing induction (ED pts), and 7 pts died after treatment. Prognostic factors considered were: Age 〉 55 years, WBC average, WBC 〉 100 x 106/L, % Blasts in bone marrow, Secondary etiology (therapy related/MDS). JM and TKD domain coding sequences were amplified by PCR for characterization of ITD and D835/836 mutations, respectively. Results and Interpretation. FLT3 mutations could be demonstrated in 23% (19/82 pts): ITD =16% (13/82), D835/836 =7% (6/82). The median follow-up time was 36 months (range 1 – 96 m). A total of 48% (n=27) of pts. were still alive without relapse at the end of this study. Higher incidence of Flt3 mutations [ITD+ and D835/836+] were found in: 41,7% (n=5) of pts with no achievement of CR (n=12) Vs 7,1% (n=3) pts with CR (n=42) (p=0.01), and in 38% (n=5) of the death patients group (n=13) Vs. 7,4% (n=2) pts still alive without relapse (n=27) (p=0,027).The WBC average was significantly higher in the ITD+ group (69,38x106/L) Vs ITD(−) group (9,27x106/L) (p=0.001). ITD mutation was more frequent in pts with WBC 〉100x106/L (83,3%) Vs WBC 100.106/L. Both type of FLT3 mutations (ITD and D835/836) were associated with early death in the cohort. This colaborative study showed that FLT3 mutational status had to be considered as important tool in prognosis of AML pts, however further follow up with larger number of pts is required to fully address its association with poor clinical outcome.

Beschreibung: Abstract 2655 Poster Board II-631 Introduction: The predictive power of measuring the effect of anticancer treatments on whole living tumor cells freshly removed from cancer patients, called Individualized Tumor Response Testing (ITRT), has been recently further validated in a clinical trial, the UK's LRF CLL4 trial (Bosanquet ASH 2007). It predicts resistance better than sensitivity. We present a novel approach to ITRT based on measuring drug induced apoptosis of tumor cells in whole blood ex vivo (in vitro using freshly extracted samples). It uses a novel automated flow cytometry platform (ExviTech) capable of evaluating hundreds of drugs and drug combinations used in current treatment protocols, and can address the significant scaling of potential future protocols induced by a number of new drug approvals in each indication. Patients and Methods: We evaluated 47 samples of peripheral blood or bone marrow from patients diagnosed with hematological malignancies: 20 chronic Lymphocytic Leukemia (CLL), 14 Acute Lymphoblastic Leukemia (ALL), 7 Multiple Myeloma (MM), and 6 Acute Myeloblastic Leukemia (AML). After informed consent, samples, collected into heparin, were processed the same or the next day. Whole blood was diluted and incubated with drugs for 24 and 48 hours. Whole blood was used to retain erythrocytes and serum proteins enabling more clinically relevant physiological conditions. Three types of drugs were tested: 1) Approved drugs for each indication, including all possible pair wise combinations, and combinations administered within current and experimental protocols as advised by the PETHEMA groups in Spain. 2) Concomitant medicines (Con-Meds), including alternative drugs within the same class of antacids, antiemetics, etc… to test whether they may also induce apoptosis 3) Drugs in clinical trials, preferentially Phase III drugs, alone and in combination with approved drugs, which may form the basis of future treatment protocols. Drugs were plated at a final concentration equivalent to their reported plasma Cmax concentration. Synergistic drug combinations were identified as one drug potentiating the effect of the other. Results: The efficacy of each drug and combination tested was categorized as highly resistant, intermediate or highly sensitive. Highly resistant drug results were contraindicated. Among the highly sensitive treatments ex vivo, often those that effectively killed all malignant cells, we selected those whose drugs were significantly less toxic as treatment guidelines, highlighting those treatment protocols that act faster ex vivo (24 vs 48 hours) and/or show synergistic combinations. The final result was a set of multiple reasonable ex vivo options for hematologists. The efficacy of individual drugs varied notably from patient to patient, , as reported earlier by other methods. Drug-drug combinations show surprising results. Some combinations, effective at high doses, kill 80% of malignant cells when combined in low concentrations at which the individual drugs kill only 10%20% of these cells. On the contrary, many drug combinations were antagonistic, effectively turning them into cytoprotectors and the patient into potential resistance. Specific combinations that show consistent efficacy across samples are indicative of potential new protocols. Surprisingly, for a proportion of patients, some of the Con-Meds were highly efficient in killing malignant cells selectively. For example, in a particular CLL patient an antacid and an antiviral drug had similar efficacies as the best approved cytotoxic drugs. In other patients, drugs still in clinical trials showed high sensitivity and highly selective apoptosis – suggesting that those patients could be referred for inclusion into these trials, which could represent new alternatives especially for refractory patients with few therapeutic options available. Conclusions: We have developed a Personalized Medicine Multi-Drug ex vivo test, evaluating the efficacy of hundreds of drugs and drug combinations in whole blood. This scale could address the predictable expansion of multi-drug potential treatments as the existing extensive drug pipeline delivers new drug approvals, exploring hundreds of new protocols ex vivo. Promising results obtained ex vivo (in vitro using freshly extracted samples) need to be verified in clinical trials. Disclosures: Bennett: Vivia Biotech: Employment. Sapia:Vivia Biotech SL: Employment. Primo:Vivia Biotech SL: Employment. Suarez:Vivia Biotech SL: Employment. Lago:Vivia Biotech SL: Employment. Matoses:Vivia Biotech SL: Employment. Espinosa:Vivia Biotech SL: Employment. Tudela:Vivia Biotech SL: Employment. Arroyo:Vivia Biotech SL: Employment. Gorrochategui:Vivia Biotech SL: Employment. Jackson:Vivia Biotech SL: Employment. Okun:Vivia Biotech SL: Research Funding. Lopez:Vivia Biotech SL: Employment. Gornemann:Vivia Biotech SL: Employment. Diez:Vivia Biotech SL: Employment. Gonzáalez:Vivia Biotech SL: Consultancy. Bosanquet:Vivia Biotech SL: Consultancy. Orfao:Vivia Biotech SL: Research Funding. Ballesteros:Vivia Biotech SL: Equity Ownership.

Beschreibung: Abstract 4414 Introduction Discovery of novel non cytotoxic drugs for cancer focuses on targets selectively expressed in malignant cells, only testing at the end if they are toxic to patients. We have developed a novel approach to discover these drugs starting at the end; we screen 2.000 approved drugs with proven safety, directly on freshly extracted (ex vivo) blood samples of patients with Chronic Lymphocytic Leukemia (CLL). These screens are enabled by a novel technology platform based on automated flow cytometry we call ExviTech for ex vivo technology. Patients and Methods All screening studies were performed directly on either peripheral blood or bone marrow samples from 44 patients diagnosed with various subtypes of B-cell malignancies, after informed consent. Patient samples were diluted and plated with each of the 2.000 drugs individually, retaining the erythrocyte population and serum proteins to enable clinically relevant concentrations. The experimental assay was setup the same or a day after sample extraction. Each sample was diluted to achieve a leukemic cell concentration of approximately 3,000 cells/μl; then 45μl of the suspension is added to each well of 96-well plates that contain the pharmacological agents (final concentration of 30μM). The compound plates were then sequentially incubated for 24 hours at 37°C with 5% CO2 for screening (sterile conditions). After incubation, the erythrocytes were lysed and the leucocytes incubated with Annexin V-FITC, anti-CD45-APC and anti-CD19-PE added to each well. The plates were then transferred to an automated flow cytometry system where the contents of each well were aspirated and analyzed by a CyAn flow cytometer. Candidates from the primary screens were validated in additional samples with dose-responses, combinations with approved drugs, multiple incubation times, etc… Results Analyzing primary screens from 24 CLL patients, three related compounds (Vivia007, Vivia008 and Vivia009) were found to consistently induce apoptosis of nearly all leukemic B-cells from most of the patient samples diagnosed with B-cell chronic lymphocytic leukemia at levels equal to or greater than known CLL active cytotoxic agents. Notably, these candidates are equally effective against samples of p53 mutated patients. These 3 drugs are pharmacologically me-too drugs sharing the same target and mechanism of action, and are non cytotoxic drugs with a known and good safety profile, administered to millions of patients over many years. Validation experiments were done on 20 additional CLL patients and Vivia009 emerged as the most effective agent with an average EC50 of 18.2μM. The mechanism of action is different than the known mechanism of Vivia009 and its class members for their approved indications. Consistent with this observation, only 3 of 15 members of the same pharmacological drug class were efficacious against CLL malignant cells. All 3 Vivia′s candidates were equally efficacious against other B-Cell Malignancies such as B-ALL (pediatric and adult), and Multiple Myeloma. These drugs are not effective in their current oral formulation and require a novel intravenous formulation. Interestingly, kinetics of induction of apoptosis were faster for Vivia009 than for fludarabine, cyclophosphamide and mitoxantrone. Vivia009 requires only 1 hour of incubation with fresh cells to induce maximal apoptosis. This timeline is less than the 3 hours in which Vivia009 was found present at high concentrations in bone marrow of rats using a single intravenous bolus. Thus, Vivia009 seems to fulfill the pharmacokinetic criteria to eliminate all leukemic cells with a single intravenous bolus, which would be a major advantage over current treatments (5-days fludarabine or 3 days FCR). Animal models are ongoing to confirm the non cytotoxic nature of the candidates in the novel IV formulation and the fewer days needed to reach remission, both compared with fludarabine monotherapy. Conclusions In summary, our results demonstrate the potential of the ExviTech technology platform as a successful model for the systematic search of new uses for already existing approved drugs directly on patient samples of hematological malignancies. A new drug candidate with excellent safety profile has been identified with similar efficacy ex vivo as the best approved cytotoxic drugs, which is a non-cytotoxic drug with fast kinetics that might enable significantly safer and shorter treatments. Disclosures: Bennett: Vivia Biotech: Employment. Sapia:Vivia Biotech SL: Employment. Primo:Vivia Biotech SL: Employment. Suarez:Vivia Biotech SL: Employment. Lago:Vivia Biotech SL: Employment. Matoses:Vivia Biotech: Employment. Espinosa:Vivia Biotech: Ana Espinosa, Employment. Tudela:Vivia Biotech SL: Employment. Arroyo:Vivia Biotech SL: Employment. Jackson:Vivia Biotech SL: Employment. Okun:Vivia Biotech SL: Research Funding. Lopez:Vivia Biotech SL: Employment. Gornemann:Vivia Biotech SL: Employment. Diez:Vivia Biotech SL: Employment. González:Vivia Biotech SL: Consultancy. Dominguez-Gil:Vivia Biotech SL: Consultancy. Troconiz:Vivia Biotech SL: Consultancy. Rodriguez de Fonseca:Vivia Biotech SL: Consultancy. Saunders:Vivia Biotech: Consultancy. Montejo:Vivia Biotech SL: Consultancy. Caveda:Vivia Biotech SL: Employment. Orfao:Vivia Biotech SL: Research Funding. Ballesteros:Vivia Biotech SL: Equity Ownership.

Beschreibung: Purpose: To evaluate the efficacy of FCR in improving complete remission (CR), disease-free survival (DFS) and overall survival (OS) rates in patients previously treated with chlorambucil-prednisolone and untreated CLL patients. Patients and methods: A total of 45 CLL patients started FCR. Forty-one patients completed treatment: 16 following previous relapse and 25 previously untreated with progressive disease. Four patients are still receiving treatment. Median patient age was 63 years (range 34–88 years). Binet’s stage were: A: 8%, B: 34% and C: 58%. CD38 expression was positive (〉 7% of cells) in 56% of patients and negative in 44%. FCR consisted of: fludarabine (25 mg/m2/day × 3); cyclophosphamide (250 mg/m2/day × 3) and rituximab (375 mg/m2/day × 1), all given intravenously, every 4 weeks for 4–6 cycles. CR was defined by CLL/NCI-WG criteria. Minimal residual disease (MRD) negativity was 〈 1% CD19, CD5 positive cells in peripheral blood and bone marrow. Results: The results of this study are detailed in Table 1. To summarise: CR: 69%; nodular partial remission (PR): 15%; PR: 7% and stable disease: 2%. Grade 3–4 neutropenia occurred in 33% of patients and 34% required hospitalization due to infections. Table 1: Patient details following FCR treatment Patients % CR % MRD negative % DFS 36 mo P % OS 36 mo P Untreated 25 87 96 90 95 Treated 16 53 87 75 0.10 89 0.90 Total 41 74 92 83 93 CD38 Positive 23 71 90 71 0.02 87 0.27 CD38 Negative 18 76 94 100 100 Conclusions: FCR induces a high CR (74%) and DFS (83%) rate and increases MRD negativity (92%). Significantly higher DFS rates at 36 months were observed in patients who were CD38 negative vs. CD38 positive (100 % vs. 71% P=0.02).

Beschreibung: Introduction: During the last decade, strong prognostic factors and new compounds emerged for the treatment of CLL. The initial experience with the combination of FCR at MDACC as salvage therapy and later in untreated patients (pts) promote us to use these regimen since March 2001. Purpose: To evaluate the efficacy of FCR in improving the complete remission (CR), disease free survival (DFS) and overall survival (OS) rates in pts previously treated with chlorambucil-prednisone as a single therapy and untreated CLL pts. Patients and methods: A total of 32 CLL pts, 16 after relapse and 16 untreated with progressive disease completed treatment and evaluation, 3 pts are still on treatment. FCR consisted of Fludarabine 25 mg/m2 iv on days 1 to 3, Cyclophosphamide 250 mg/m2 iv on day 1 to 3 and Rituximab 375 mg/m2 iv on day 1 given at 4 weeks interval up to 4–6 cycles. Response was assessed 4–8 weeks after treatment. CR was defined by CLL/NCI-WG. Minimal residual disease (MRD) negative was defined as 7% of cells) in 53% and negative in 47% of the cases. Results: The CR was obtained in 66% of the pts, nodular partial remission (NPR) in 19%, PR in 9% and stable disease in 6%. Characteristics Pts. # % CR % MRD negative % DFS 24 mo P= # OS 24 mo P= Untreated Pts 16 81 100 81 0.64 90 0.81 Treated Pts 16 50 81 76 87 CD38 Positive 17 65 82 65 0.01 80 0.28 CD38 Negative 15 67 87 100 100 Total 32 66 84 78 89 Neutropenia grade 3–4 was observed in 31% of the courses, 5% of the pts required hospitalisation due to infeccious episodes. Conclusion: In patients with CLL, FCR induce a high CR, MRD negative and DFS rate. Patients with CD38 negative expression correlated with a better DFS.