ALBERT

Description: Abstract 2341 Poster Board II-318 Background. The clinical heterogeneity of chronic lymphocytic leukemia (CLL) requires parameters to stratify patients into prognostic subgroups to adapt treatment ranging from ‘watch and wait’ to allogeneic stem cell transplantation. To this end, several parameters such as lymphocyte doubling time, β-2 microglobulin, CD38 and ZAP-70 expression, immunoglobulin variable heavy chain (IgVH) mutation status and genetic abnormalities, as assessed by fluorescence in situ hybridization (FISH), have been integrated in clinical practice. Aims. In the present study, we investigated by FISH the incidence of the known major cytogenetic alterations (+12 and 13q14, 17p13, 11q23 deletions) in a series of Binet A B-CLL patients included in the prospective O-CLL1 GISL study started in April 2007. Methods. Molecular markers characterization and FISH analyses were performed as previously reported (Cutrona et al. Haematologica, 2008; Fabris et al. GCC, 2008). A cut-off value of 2% was used to distinguish mutated and unmutated patients. CD38 and ZAP-70 were determined by flow-cytometry and a 30% cut-off was used to distinguish between positive or negative cases. Results. Up to date, 326 patients have been enrolled in the trial and FISH data concerning trisomy 12 and 13q14, 17p13, 11q23 deletions were available in 305 patients. At least one abnormality was found in 197 (64%) cases. The most frequent was del(13)(q14) (150/305, 49%), followed by +12 (40/303, 13%) (in one and three cases accompanied by 17p13 and 13q14 deletions, respectively), del(17)(p13) (7/305, 2%) and del(11)(q23) (17/305, 5%). 13q14 deletion was found as a sole abnormality in 134 patients; in the remaining cases, it was combined with +12 (3 pts) and 17p13 (3 pts) or 11q23 (10 pts) deletions. Among patients with 13q14 deletions, 99 were monoallelic, 12 biallelic and 39 showed a combination of the two patterns. Biomarkers data were available in all of the patients: 95/305 (31%) cases had unmutated IgVH genes; ZAP-70 and CD38 were positive in 117/305 (38%) and 72/305 (23%) cases, respectively. Concerning the distribution of cytogenetic aberrations, the unmutated IgVH group included 29/150 (19%) 13q14 deleted cases, 23/40 (57%) cases with trisomy 12 and 4/7 (57%) and 16/17 (94%) with 17p13 and 11q23 deletions, respectively. ZAP-70-positive groups included 43/150 (28%) 13q14 deleted cases, 26/40 (65%) cases showing trisomy 12 and 5/7 (71%) and 12/17 (70%) with 17p13 and 11q23 deletions, respectively. Finally, CD38-positive cases included 18/150 (12%) 13q14 deleted cases, 26/40 (65%) cases carrying trisomy 12 and 5/7 (71%) and 7/17 (41%) with 17p13 and 11q23 deletions, respectively. The percentages of IgVH mutations significantly correlated with cytogenetic alterations; namely, 5.8±0.3 for cases with del(13)(q14), 4.6±0.4 for normal karyotype, 2.6±0.5 in +12, 0.3±0.2 in del(11)(q23), and 1.7±0.9 in del(17)(p13) cases (p for trend

Description: Abstract 4613 Background: Biologic risk factors such as immunoglobulin variable heavy chain (IgVH) gene mutation status and CD38 and ZAP-70 expression levels, along with genomic aberrations, have been integrated in clinical prognostic evaluation of CLL. Additionaly, CLL subsets expressing a certain stereotyped B-cell receptors have also been indicated to share biological and clinical features. Aims: We investigated, by FISH, the incidence of the major cytogenetic alterations (+12 and 13q14, 17p13, 11q23 deletions), their clinical implication and their relationship with prognostic biomarkers in 344 out of 384 Binet A CLLs enrolled in the prospective multicenter O-CLL1 GISL trial. Stereotypy subsets identification have been performed in 324 patients. Methods: Molecular markers characterization and FISH protocols were previously reported (Cutrona et al. Haematologica, 2008; Fabris et al. Genes Chromosomes Cancer, 2008), while stereotyped subsets were defined according to Stamatopoulos et al (Blood, 2007) and Murray et al (Blood, 2008). Results: At least one abnormality was found in 225/344 (65.4%) cases. The most frequent abnormality was del(13q14), detected in 173 CLLs (50.3%) followed by +12 (44/344;12.8%) (one case harboring 17p13 deletion), del(17p13) (9/344, 2.6%) and del(11q23) (18/344, 5.2%). 13q14 deletion was found as a sole abnormality in 155 (45%) patients; in the remaining cases, it was combined with +12 (3 pts) and 17p13 (4 pts) or 11q23 deletions (11 pts). The 13q deletion was found as a monoallelic deletion in 139/173 (80.3%); the presence of a biallelic deletion (〉 20% of interphase nuclei) was found in the remaining 34 cases. No acquisition of new cytogenetic aberrations was evidenced among the 13 CLLs developing progressive disease (range, 6 to 32 months; median, 20 months); in only one case, the proportion of nuclei with 17p13 and 13q14 deletions increased from the time of diagnosis (from 33% to 92%). Biomarkers data were available in all of the patients. CD38 percentages (mean value ± sem) were 7.9±1.3, 15.1±1.9, 51.7±5.5, 22.0±7.8,40.8±13.2, 39.8±7.3 for del(13q14), normal karyotype, +12, del(11q23), del(17p13) and multiple alterations, respectively (p

Description: Abstract 2423 Chronic lymphocytic leukemia (CLL) is characterized by an extremely variable clinical course. Mutational status of the immunoglobulin heavy-chain variable (IGHV) region defines two disease subsets with different prognosis. A fraction of CLL cases carries highly homologous B-cell receptors (BCR), i.e. characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3). We performed sequence analysis to characterize IGHV regions in a panel of 1133 CLL patients investigated by a multicenter Italian study group. A total of 1148 rearrangements were identified; the analysis of stereotyped subsets was performed based on previously reported criteria (Messmer et al, J Exp Med 2004; Stamatopuolos et al, Blood 2007). Specifically, we compared all our sequences with those found in three different publicly available data sets (Stamatopoulos et al, Blood 2007; Murray et al, Blood 2008 and Rossi et al, 2009 Clin Cancer Res). In addition, a pairwise alignment within all sequences was performed in order to discover novel potential subsets (HCDR3 identity 〉 60%). Based on the 2% cut-off used to discriminate between Mutated (M) and Unmutated (UM) cases, 777 sequences (67.59%) were classified as M, while 371 sequences (32.3%) as UM. The most represented IGHV genes within mutated cases were IGHV4-34 (104/118) and IGHV3-23 (85/96), whereas IGHV1-69 (97/112) was the most frequently used in the UM group. Interestingly, the IGHV3-21 gene, reported to be frequently expressed in CLL patients from Northern Europe, was present in only a small fraction of cases (24; 2.07%), confirming a previous finding reported by Ghia et al (Blood 2005) in a smaller panel. In our series, stereotyped HCDR3 sequences were found in 407/1148 (35.45%) patients, 177 of whom were M and 230 were UM cases. Overall, we observed that stereotyped sequences were significantly associated with UM IGHV status (Fisher's exact test, P

Description: Abstract 1360 The arbitrary cut-off of 5000/μL chronic lymphocytic leukemia (CLL)-phenotype cells in peripheral blood is generally used to separate monoclonal B-cell lymphocytosis (MBL) from CLL. However, a major concern is the biological differentiation, if any, between MBL and CLL. We tried to address the issue therefore analyzing 261 Rai stage 0 patients enrolled in a Gruppo Italiano Studio Linfomi (GISL) prospective multicentre trial designed to validate biological parameters in early CLL as well as to assess the impact on clinical outcome of an early versus delayed policy of treatment with subcutaneous alemtuzumab in the high biological risk. In this cohort, biological characteristics of 105 (40.2%) patients who would be reclassified as MBL using the 2008 CLL diagnostic criteria were compared with those of the remaining 156 patients who had more than 5000/μL CLL-phenotype cells in peripheral blood and fulfilled diagnostic criteria of CLL. Male to female ratio was similar for MBL and CLL (54/53 vs. 92/66, P=0.21) as was median age (58.18 vs 58.18, P=0.98). Median absolute number of cells with CLL phenotype in peripheral blood was 3120/μL (range,400-4959) in MBL and 9925/μL (range, 5020–110000) in CLL (P

Description: Abstract 1369 Introduction: Single-agent cladribine (2CdA) given intravenously or subcutaneously (sc) is highly effective in Hairy cell leukemia (HCL). However, HCL patients are projected to progress in their life-span or, in a minority, are refractory and may benefit from other treatment strategies. Parameters predicting efficacy of (sc)2CdA in HCL are scarcely known. Aims: In a national multicentre clinical trial (EudraCT code: ICGHCL2004) we prospectively sought parameters able to predict efficacy of sc2CdA in classic HCL requiring first treatment (1987 Consensus Criteria). Methods: Clinical data and samples were collected centrally for diagnostic revision according to the WHO criteria and for molecular analyses prior to treatment. Tumor IGH and TP53 analyses or Genome-wide DNA profiling was performed in the cases with 〉10% or 〉50% hairy cells (HC) in the test sample, respectively (Forconi, Blood 2009). Patients entering the study received 0.5–0.7 mg/kg sc2CdA as a single course. Efficacy endpoints were response to sc2CdA, treatment free interval (TFI), relapse free survival (RFS), and overall survival (OS). Responses were assessed on month 2 and 6 after treatment and defined according to the 1987 Consensus criteria. Complete Remissions (CR) and Partial Remissions (PR) were rated as beneficial responses (CR/PR), while minor Responses (mR) and No Responses (NR) were rated as failures (mR/NR). TFI was measured in all patients from sc2CdA initiation to time of new treatment requirement. RFS was measured in patients with CR/PR from sc2CdA initiation to relapse. OS was measured from sc2CdA initiation to death. Results: Of 148 patients, 116 were males (78,4%). Median values were: age 52 years (range 30–83), splenomegaly below costal margin 2 cm (0-16), hemoglobin 11,8 (5-17 g/dL), PLT 77×10e9/L (14-255), WBC 2,7×10e9/L (0,7-48), HC 0,2×10e9/L (0-38). Serum B2microglobulin was twice above upper limit (B2M〉2X) in 9/122 patients (7,4%), IGH unmutated (UM) in 7/62 (11,3%) and TP53 mutated (mutTP53) in 2/62 (3,2%). Onehundred-forty patients (94,6%) had a CR/PR (101 CR, 68,2%; 39 PR, 26,4%) and 8 (5,4%) a mR/NR (5 mR, 3,4%; 3 NR, 2%). Risk factors of sc2CdA failure were splenomegaly (p=.001, best-cutoff〉 3 cm bcm by ROC and Youden's Test), WBC (p10×10e9/L), HC counts (p5×10e9/L), and B2M〉2X (p=.013). In 62 patients investigated for molecular features, UM-IGH and TP53 were confirmed as risk factors for mR/NR (p10×10e9/L (p5×10e9/L (p3cm, UM-IGH and mutTP53), UM-IGH (HR=8.1, 95%CI 1.7–38.1, p=.008, median TFI=8.5 months), high HC (HR=6.9, 95%CI 1.5–31.6, p=.012, TFI=27,4 months) and splenomegaly (HR=3.7, 95%CI 1.1–12.8, p=.039, TFI=49,3 months) scored as independent risk factors. Quality of response also predicted risk of short TFI (NR=mR〉PR〉CR, p5×10e9 (p=.016) and PR (p=.001)as risk factors of short RFS. Cox multivariate analysis revealed PR as the sole independent risk factor of relapse after a median of 46 months (HR=4.5, 95%CI 1,7-12,3, p=.003). 2CdA dose schedule was among other factors that did not associate with TFI or RFS. Conclusion: This data confirm that sc2CdA has potent activity in HCL. Tumor UM-IGH status, splenomegaly, high HC count (or leukocytosis) are confirmed as independent risk factors of treatment failure and rapid progression. RFS analysis identify PR as the sole independent factor of relapse risk in the patients initially responsive to sc2CdA. This analysis may have important implications for the selection of HCL patients that will require treatments alternative to single-agent 2CdA. Disclosures: No relevant conflicts of interest to declare.

Description: Hairy cell leukemia (HCL) is a rare B-cell neoplasm generally responsive to Cladribine. Cladribine is generally administered intravenously either as a continuous weekly infusion or as a 2-hour daily or weekly infusion for 7 days. Subcutaneous Cladribine is an alternative route with 100% bioavailability and with efficacy similar to intravenous 2CdA at the dose of 0.7 mg/kg/cycle. In indolent non-Hodgkin lymphomas other than HCL, reduction to 0.5 mg/kg/cycle determined equivalent efficacy and lower toxicity. In a national multicentre clinical trial (protocol EudraCT code: ICGHCL 2004), we have evaluated efficacy and toxicity of subcutaneous Cladribine given 0.1mg/kg/die for 5 (total dose 0.5 mg/kg, arm A) or for 7 days (total dose 0.7 mg/kg, arm B) as a single course in newly diagnosed HCL requiring treatment. Responses to treatment were assessed on day 60 and day 180 after treatment and defined according to the 1987 Consensus criteria. Complete Remissions (CR) and Partial Remissions (PR) were considered as beneficial responses, while minor Responses (mR) and No Responses (NR) were rated as treatment failures. Toxicity was assessed from day 0 to day 60 after treatment, according to the 2003 NCI/CTCAE v3 criteria. In this interim analysis, 92 of the 132 patients currently recruited (45 patients in arm A and 47 in arm B) were evaluated for toxicity and response to treatment. 2CdA was administered at the proposed regimen with no modifications in all patients. Eighty-five of 92 patients (92%) had a beneficial response to treatment (57/92 CR, 62%; 28/92 PR, 30%). The 7/92 treatment failures scored as 3/92 mR (3%) and 4/92 (5%) NR. Responses were equivalent in the two arms (p=0.7), with 41/45 (91%) beneficial responses (27/45 CR, 60%; 14/45 PR, 31%; 2/45 mR, 4% and 2/45 NR, 4%) in arm A versus 44/47 (94%) beneficial responses in arm B (30/47 CR, 64%; 14/47 PR, 30%; 1/47 mR, 2%; 2/47 NR, 4%). Overall grade 3–4 toxicity was recorded in 16/92 (17%) patients (8/92 FUO, 9%; 4/92 documented infections, 6%, 3/92 skin rashes, 3%; 1/92 hepatic toxicity, 1%) and appeared less frequent in arm A (4/45, 9%) than in arm B (12/47, 25%) (p=0.05). Analysis of distribution of toxicities in the two arms (arm A: 2/45 FUO, 4%; 0/45 documented infections; 1/45 skin rash, 1%; 1/45 hepatic toxicity, 1%; arm B: 6/47 FUO, 13%; 4/47 documented infections, 9%; 2/47 skin rashes, 4%) revealed a significantly lower frequency of FUO and infections in arm A (2/45, 4%) than in arm B (10/47, 22%) (p=0.02), to suggest a higher risk of infection in the 7 day regimen. The present data indicate that overall activity of subcutaneous 2CdA is similar to the intravenous formulation (Cheson, 1998). Furthermore, the current interim analysis suggests that subcutaneous 2CdA given at 25% reduced doses (0.5 mg/kg) has equivalent activity and lower toxicity than subcutaneous 2CdA at standard doses (0.7 mg/kg) and is easy to give in an outpatient setting.

Description: Abstract 1679 Recent data suggest that second-generation tyrosine-kinase inhibitor (TKI) dasatinib and nilotinib can be responsible for increased non-hematologic adverse events in comparison to imatinib. In particular, there are a few reports of severe peripheral arterial occlusive disease (PAOD) and other vascular occlusive events (infarction) in patients receiving nilotinib. With this in mind we retrospectively evaluated incidence of PAOD or other vascular occlusive events in our cohort including 82 consecutive chronic myeloid leukemia (CML) patients treated at our institution with imatinib alone (n=55) or nilotinib as first-line (n=17) or second-line treatment after imatinib failure (n=10). After a median time of exposition to nilotinib of 24 months (range, 7–34 months) 4 (14.8%) out of 27 patients developed an episode of severe and previously unrecognized PAOD or other vascular occlusive events (2 PAOD, 1 myocardial infarction, 1 ictus). All 4 patients were more than 60 years old and 3 out of 4 were male while obesity was never observed. A history of nicotine abuse could be found in 2 out of 4 patients. The same applied when we looking for the presence of arterial hypertension (2/4) or dyslipidemia (2/4). When 55 patients treated with imatinib were analyzed for PAOD or other vascular occlusive event incidence we detected only one patient who experienced myocardial infarction after 135 months of therapy. We then evaluated the likelihood of developing PAOD in the subset of patients treated only with imatinib and in those who received nilotinib, respectively. The projected 10-year actuarial probability of remaining PAOD-free was 100% in the imatinib group and 67% in the nilotinib group (HR, 14.6; P=0.0008). Interestingly, the two patient cohorts were alike with respect to age (P=0.76), gender (P=0.80), number cardiovascular risk factors (P=0.62) and body mass index (P=0.59). The only difference we observed was a significantly longer exposition to drug among patients treated only with imatinib in comparison to those who received nilotinib (P

Description: Abstract 701 Introduction: Hairy cell leukemia (HCL) is generally responsive to intravenous Cladribine (iv2CdA). Subcutaneous Cladribine (sc2CdA) is an alternative route with 100% bioavailability. In indolent non-Hodgkin lymphomas other than HCL, at the dose of 0.7 mg/kg/cycle, efficacy of sc2CdA is similar to iv2CdA and reduction to 0.5 mg/kg/cycle determines equivalent efficacy and lower toxicity. Aims: In a national multicentre clinical trial (protocol EudraCT code: ICGHCL 2004), we prospectively evaluated toxicity and efficacy of sc2CdA given 0.1mg/kg/die for 5 (total dose 0.5 mg/kg, arm A) or for 7 consecutive days (total dose 0.7 mg/kg, arm B) as a single course in classic HCL requiring first treatment. Method: Clinical data and diagnostic samples were collected from all patients for central revision of classical HCL diagnosis (WHO criteria) and for molecular analyses at the University of Siena, prior to study entry. Early grade 3–4 toxicity was assessed on days 7 to 30 after treatment, according to the 2003 NCI/CTCAE v3 criteria and occurrence of second tumors was assessed at every subsequent follow-up. Enpoints of sc2CdA efficacy were response to treatment, treatment free interval (TFI), relapse free survival (RFS), time to second tumor (TTST) and overall survival (OS). Responses to treatment were assessed on days 60 and 180 after treatment and defined according to the 1987 Consensus criteria. Complete Remissions (CR) and Partial Remissions (PR) were rated as beneficial responses (CR/PR), while minor Responses (mR) and No Responses (NR) were rated as treatment failures (mR/NR). TFI was measured as the time elapsed from sc2CdA initiation to second treatment because of new progression or failure to sc2CdA. RFS was measured in patients with a CR/PR from treatment inititation to relapse. OS was measured from sc2CdA initiation to death for any cause. Result: of 156 patients screened centrally, 148 scored as classical HCL and entered the study. Gender (male total 116/148, 78,4%; arm A: 62/77, 80,5% vs arm B: 54/71, 76,1%), age (total: median age 52 years, range 30–83; arm A: median 56, range 30–83; arm B: median 51, range 33–82), clinical and laboratory parameters prior to treatment (including spleen, hemoglobin, platelet, leucocyte and hairy cell counts) were equally balanced in the two arms. Dose reduction was required in no patients from arm A and in 2 patients from arm B due to concurrent infection. Overall hematological toxicity was no different among the two treatment arms. Requirement of platelet transfusions (total 7/148, 4,7%; arm A: 2/77, 2,6% vs arm B: 5/71, 7%), red blood cell transfusion (total 32/148; 21,6%, arm A: 15/77, 19,5% vs arm B: 17/71, 23,9%), or G-CSF (total 102/148; 68,9%, arm A: 55/77, 71,4% vs arm B: 47/71, 66,2%) was no different among the two arms. However, a significantly higher non hematological toxicity (total 28/148; 18,9%, arm A: 9/77, 11,7% vs arm B: 19/71, 26,8%) was observed in arm B (p=.019). Non haematological toxicity was mainly represented by infections or FUO (total 25/148; 16,9%) that were significantly more frequent in arm B (arm A: 8/77, 10,4% vs arm B: 17/71, 23,9%, p=.028). Higher prevalence of toxicity resulted in a higher hospitalization rate in arm B than in arm A (total 29/148; 19,6%, arm A: 9/77, 11,7% vs arm B: 20/71, 28,2%, p=.012) and one early death was experienced because of lung aspergillosis in arm B. Onehundred-forty patients (94,6%) had a beneficial response (101 CR, 68,2%; 39 PR, 26,4%) and 8 (5,4%) failed treatment (5 mR, 3,4%; 3 NR, 2%). Responses were equivalent in the two arms, with 72/77 (93,5%) beneficial responses (49/77 CR, 63,6%; 23/77 PR, 29,9%; 4/77 mR, 5,2% and 1/77 NR, 1,3%) in arm A versus 68/71 (95,8%) beneficial responses in arm B (52/71 CR, 73,2%; 16/71 PR, 22,5%; 1/71 mR, 1,4%; 2/71 NR, 2,8%). After a median follow-up of 36 months (range 12–66), 5 year TFI, RFS, TTST and OS were 67%, 71% 87% and 94%, respectively. Causes of late death were 2 cardiac events and 3 second tumors. No differences of TFI, RFS, TTST and OS were observed in the 2 arms of treatment. Conclusion: The present data indicate that overall activity of sc2CdA is similar to iv2CdA (Cheson, 1998). Furthermore, this study indicates that sc2CdA given at 25% reduced doses (0.5 mg/kg) has equivalent activity and significantly lower toxicity than sc2CdA at standard doses (0.7 mg/kg). The reduced infection rates and hospitalization rates of sc2CdA have important pharmaco-economic implications. Disclosures: No relevant conflicts of interest to declare.

Description: Hairy cell leukemia (HCL) is generally responsive to single-agent cladribine, and only a minority of patients are refractory and with poor prognosis. HCLs generally express mutated (M) and, in a minority, unmutated (UM) IGHV. In a multicenter clinical trial in newly diagnosed HCL, we prospectively investigated clinical and molecular parameters predicting response and event-free survival after single-agent cladribine. Of 58 HCLs, 6 expressed UM-IGHV (UM-HCL) and 52 M-IGHV (M-HCL). Beneficial responses were obtained in 53 of 58 patients (91%), whereas treatment failures were observed in 5 of 58 patients (9%). Failures were associated significantly with UM-IGHV (5 of 5 failures vs 1 of 53 beneficial responses had UM-IGHV, P 〈 .001), leukocytosis (3 of 5 vs 3 of 53, P = .006), and bulky spleen (4 of 5 vs 4 of 53, P 〈 .001). The UM-HCL not benefiting from cladribine characteristically had bulky spleen (4 of 5, 80%), leukocytosis (3 of 5, 60%), and TP53 defects (2 of 5, 40%), and progressed rapidly after first treatment (median event-free survival, 7.5 months). Our data suggest that UM-HCLs identify the minor subgroup failing cladribine treatment and with more aggressive disease. High incidence of TP53 dysfunction indicates a potential mechanism of resistance to cladribine in the UM-HCL group. Overall, our data provide new molecular elements relevant for treatment concerns in HCL.