ALBERT

Beschreibung: It is well known that cytogenetic abnormalities, the IgVH mutational status, ZAP-70 and CD38 have a significant prognostic role in chronic lymphocytic leukemia (CLL). We therefore designed a 1st line treatment approach for young CLL patients stratified according to the biological features of the disease. Between November 2005 and July 2008, previously untreated CLL patients ≤60 years, with advanced or progressive disease, from 21 Italian centers, were included in this study. High risk (HR) patients were defined by the presence of an adverse biologic profile: a 17p deletion in ≥20% of analyzed cells, or a 11q deletion associated with at least one additional poor prognostic factor (IgVH germline, ZAP-70+ ≥10% or CD38+ ≥7%), or a germline IgVH or mutated VH3-21 status and at least 2 additional unfavorable prognostic factors (ZAP+ ≥10%, CD38+ ≥7%, 6q deletion or trisomy 12). Low risk (LR) patients were defined by the absence of the above mentioned characteristics. For HR patients, treatment consisted of 4 monthly courses of Fludarabine and Campath-1H (FluCam; Flu 30 mg/m2 iv; Campath-1H 30 mg iv, days 1–3). Patients who achieved a response with evidence of residual disease - by CT scan, flow cytometry and/or PCR - received a post-induction therapy including a reduced intensity PBSCs allogeneic transplant or, in the absence of a sibling donor, an autologous PBSC transplant or, in the absence of a sufficient harvest, Campath-1H sc (30 mg weekly for a maximum of 12 weeks). For LR patients, treatment included 6 monthly courses of Fludarabine and Cyclophosphamide (FluCy; Flu 30 mg/m2 iv and Cy 250 mg/m2, days 1–3). Patients with no response after 4 courses, were treated with Campath-1H sc (30 mg weekly for a maximum of 12 weeks). All patients received Darbepoietin alpha in case of anemia, G-CSF and Ciprofloxacin in case of severe granulocytopenia and PC prophylaxis with Bactrim. In addition, patients treated with FluCam underwent weekly CMV antigenemia monitoring and valacyclovir prophylaxis (2g/8h). So far, 74 young patients with advanced or progressive disease fulfilling the above criteria have been included in the study, 41 (55%) with a HR profile and 33 (45%) with a LR profile. Forty-five patients have completed the induction therapy, 24 HR patients and 21 LR patients. A response was observed in 17 HR patients: OR 71%, CR 30%, with 17% of patients obtaining an MRD- status; and in 20 LR patients: OR 95%, CR 57%, with a 19% MRD negativity. The 7 FluCam refractory patients were characterized by the presence of a 17p deletion in 3 cases and by multiple enlarged nodes in 5 (bulky nodes: 3 cases). Grade III–IV granulocytopenia was the most common toxicity after FluCam and after FluCy. However, long-lasting cytopenia was observed only in cases treated with FluCy. Asymptomatic CMV reactivation was detected in 3 cases treated with FluCam. Four patients, all treated with FluCy, have died. The causes of deaths were: febrile granulocytopenia in 2 cases, cerebral hemorrhage in 1 and multiple cerebral abscesses of unknown origin in 1. At present, 9 HR patients who achieved a response to FluCam have undergone a PBSC transplantation (allogeneic 3, autologous 6). In conclusion, the first analysis of this study, focused on young CLL patients with progressive disease stratified according to the biologic profile of the disease, has shown a high CR rate after FluCy given to patients with a LR profile and a considerable response rate with a low number of CMV reactivations after FluCam administered to patients with a HR profile. Factors predicting FluCy-related myelotoxicity warrant further investigation.

Beschreibung: BACKGROUD. The B-cell receptor inhibitor ibrutinib has significantly improved treatment and overall management of chronic lymphocytic leukemia (CLL). Although several data derived from clinical trials suggest that ibrutinib increases the risk of atrial fibrillation (AF), the incidence of AF in a real-life cohort of CLL patients is unknown. Furthermore, it would be clinically relevant to identify patients at high risk of AF during ibrutinib. The aim of this study is to report the prevalence and risk factors of AF in ibrutinib-naive CLL, in order to define a predictive model for the development of AF and to test it in a cohort of subjects receiving ibrutinib. METHODS. We retrospectively analyzed data from 860 ibrutinib-naive CLL patients, referred to the Padua University hospital. Comorbidities, clinical and biological prognostic markers were analyzed using the Mann-Whiney, Fisher exact or Chi-square tests, when appropriated. Time to AF (TTAF) and overall survival (OS) were evaluated with Kaplan-Meier method. Univariate and multivariate Cox models were run to identify independent factors associated with AF. Then, risk values were obtained based on the hazard ratios. The score for AF was calculated as the sum of each risk values. Subsequently, the model was evaluated in a cohort of 354 ibrutinib-treated patients referred from 8 Italian hematological centers. RESULTS. Among the 860 patients from Padua hospital, 60% were male, 49% were older than 65 years, 73% were Binet A stage at diagnosis and 41% underwent at least one line of treatment. A prior history of AF was present in 21 patients (2.4%) at CLL diagnosis, while, among the remaining 839 patients without a previous history of AF, 47 (5.6%) developed it after a median follow-up of 9.4 years, resulting in an estimated incidence of almost 0.8% cases/year. Moreover, the median OS for patients with AF was significantly shorter than that patients without AF (12 vs 22 years, p65 years (p=0.001, 1 point), male gender (p=0.003, 1 point), valvular hearth diseases (p=0.001, 2 points), cardiopathy (p

Beschreibung: Introduction Several studies have recently pointed out the adverse impact of KIT mutations (mutKIT) on relapse incidence (RI) and overall survival (OS) in AML pts with t(8;21). By contrast, the prognostic significance of mutKIT in pts with inv(16) remains unclear. Purpose of this study is to evaluate the prevalence and the prognostic impact of mutKIT in inv(16)(p13q22). Patients and Methods Fifty adults with inv(16) AML at diagnosis (median age 46.6 yrs, range: 17–88; M/F: 30 /20), were centrally analyzed for mutKIT in exon 2, 8, 10, 11 and 17. Mutations were detected using sequencing and other sensitive assays such as ARMS (amplification refractory mutation system) PCR for D816Y and D816H and enzymatic digestion with HINFI for D816V and with Tsp509I for N822K. Results Data showed a prevalence of KIT mutation of 34% (17/50 pts). Among the mutKIT cases, we detected mutations in exon 17 (n=12), exon 8 (n= 4) and exon 10 (n=1). There was no difference between the mutKIT vs the unmutated (KIT-) patients in the median of WBC count at presentation (WBC 13.9 x109/L, range 4.4 to 277.5 vs 19.4 x109/L, range 2.5 to 130; Mann-Whitney U test: p = 0.649). Of the 42 patients (age 〈 60 years) who received intensive chemotherapy, 13 resulted mutKIT upon mutational screening. Complete remission (CR) was achieved in 13/13 (100%) mutKIT vs 27/29 (93%) KIT- patients (Fisher’s exact test: p 〉 0.999). At a median follow-up of 26 months (range: 2–144), 9/13 (69%) mutKIT and 8/27 (29%)KIT- pts relapsed;the Kaplan-Meier plots revealed KIT mutations to be a significant factor adversely affecting RI (log-rank test: p = 0.017) but not OS (61% in mutKIT vs 75% in KIT-;log-rank test: p = 0.331). Conclusion KIT mutations are associated with a greater probability of relapse following CR, without affecting OS, in AML pts with inv(16) aged

Beschreibung: BACKGROUND: The WHO and iwCLL diagnostic criteria for CLL rely on morphology and immunophenotype based on the co-expression of CD19/CD5/CD23 on B-cells with weak CD20 and monoclonal sIg expression. These diagnostic criteria are likely to persist in the near future because there is no specific diagnostic molecular abnormality for CLL. The current criteria have some limitations affecting reproducibility, particularly flexibility in marker expression with many centres using a scoring system that permits absence of CD5 or CD23. Potentially informative new markers have been identified but there is no consensus yet on which should be routinely assessed. AIM: To identify reproducible criteria and to achieve a consensus on markers recommended for the diagnosis of CLL METHODS: ERIC/ESCCA members were invited to classify 35 flow-cytometry markers as being required or recommended for the diagnosis of CLL. Consensus was considered to be achieved if 〉75% of participants agreed on the marker classification. A diagnostic panel was identified by the steering committee and characteristics of component markers that could be reproducibly validated within an individual laboratory were identified. The proposed panel was assessed in 13 different centres. RESULTS: Responses were received from 154 members (100 laboratory staff, 14 clinicians and 36 from both laboratory and clinic) with a diagnostic workload 〉20 cases per week in 23/154 (15%), 5-20 in 82/154 (53%) and

Beschreibung: C-KIT gene mutations are not a rare event in Core Binding Factor Leukemia (CBFL). To investigate their prognostic impact in this setting , we attempted at correlating the KIT mutational status with the WBC count, the level of the WBC-index, the presence of extramedullary disease (EMD) and the outcome, in t(8;21)(n=30) and inv(16) (n=20) AML patients. On the basis of genomic DNA analysis of c-KIT gene exons 2, 8, 10, 11, 17 for mutation detection, we categorized 21 patients (42%) in the “KIT mutated group” (KIT+) and 29 patients (58%) in the “KIT unmutated group” (KIT−). The median age at diagnosis was 46.4 and 45.6 years for the KIT(+) and KIT(−) groups, respectively (P=0.872). Among the KIT(+) patients, we found mutations in exon 8 (n = 4), exon 10 (n =1), exon 11 (n = 1) and exon 17 (n = 15). We recorded mean WBC counts of 28.6 x 109/L vs 34.5 x 109/L (P=0.051), and a mean WBC-index, expressed as WBC x (% Bone Marrow blasts/100), of 37.2 vs 15.5 (P=0.0395), for KIT(+) and KIT(−), respectively. Seven out of 50 patients experienced an EMD: in 6 cases (28.57%) this event occurred in the KIT(+) group. 40 patients (age 〈 60 years) were evaluable for clinical response; 18 out of them were KIT mutated. At a median follow-up of 24 months (range 10–107), we recorded for KIT(+) and KIT(−), respectively: CR incidence of 88.8 % (16/18) vs 100% (22/22) (P=0.38); Relapse Incidence 81.3% (13/16) vs 31.81% (7/22), P=0.0072; OS 27.7% (5/18) vs 77.3% (17/22), P=0.0049; DFS 16.7% (3/18) vs 77.3% (17/22), P=0.0005. Using a log-it linear model for univariate and multivariate regression analysis, we found a significant contribution to death (P=0.048) and relapse (P= 0.045) exerted by mutation in the whole group of patients; this effect was more evident when the analysis was restricted to patients

Beschreibung: Introduction: Optimized therapeutic targeting of CD20 is one among several currently explored strategies to improve the overall prognosis of patients with chronic lymphocytic leukemia (CLL). Obinutuzumab (GA101) is an optimized (i.e. glycoengineered and type 2) CD20-targeting antibody that has been investigated in the CLL11 study comparing obinutuzumab plus chlorambucil (G-Clb) with rituximab plus chlorambucil (R-Clb) and with chlorambucil alone (Clb) in patients with previously untreated CLL and comorbidities. The study had demonstrated clinically meaningful and statistically significant superiority of G-Clb over Clb (with regard to PFS and OS) and over R-Clb (with regard to PFS, but not for OS) at previous pre-planned analyses. Here, we report updated results on survival and time to next treatment (TTNT) from a pre-planned analysis with data cut-off in May 2015. Methods: A total of 781 treatment-naïve patients with CLL in need of therapy and cumulative illness rating scale (CIRS) total score 〉 6 and/or estimated creatinine clearance (CrCl) 〈 70 mL/min were randomized in a 1:2:2 fashion to receive Clb alone (0.5 mg/kg po d1 and d15 q28 days, 6 cycles), R-Clb (rituximab: 375 mg/m2 iv d1 cycle 1, 500 mg/m2 d1 cycles 2-6), or G-Clb (obinutuzumab: 100 mg iv d1, 900 mg d2, 1000 mg d8 and d15 of cycle 1, 1000 mg d1 cycles 2-6). The study was powered for PFS as the primary endpoint, but not for OS and TTNT as secondary endpoints. P values are taken from a log rank test, hazard ratios (HR) from stratified Cox regression. Results: The study population was characterized by a median age of 73 years and median CIRS total score of 8. No new safety signals have been identified in this updated analysis. Updated results of the G-Clb (n=238) vs. Clb (n=118) comparison: The median observation time was 42.4 months. Confirming previously reported results, treatment with G-Clb compared with Clb alone was associated with substantially improved outcome (median PFS 31.1 vs. 11.1 months - HR 0.20, 95%CI 0.15-0.26, p

Beschreibung: Loss of chromosome 7 (−7) or deletion of the long arm (7q−) are recurring chromosome abnormalities in myeloid leukemias. The association of −7/7q− with myeloid leukemia suggests that these regions contain novel tumor suppressor gene(s), whose loss of function contribute to leukemic transformation or tumor progression. Based on chromosome banding analysis, two critical regions have been identified, one in band q22 and another in bands q32-q35. Presently there are no data available on the molecular delineation of the distal critical region. In this study we analyzed bone marrow and blood samples from 13 patients with myeloid leukemia (de novo myelodysplastic syndrome [MDS] , n = 3; de novo acute myeloid leukemia [AML], n = 9; therapy-related (t-) AML, n = 1) which, on chromosome banding analysis, exhibited deletions (n = 12) or in one case a balanced translocation involving bands 7q31-qter using fluorescence in situ hybridization (FISH). As probes we used representative clones from a contig map of yeast artificial chromosome (YAC) clones that spans chromosome bands 7q31.1-qter. In the 12 cases with loss of 7q material, we identified a commonly deleted region of approximately 4 to 5 megabasepairs in size encompassing the distal part of 7q35 and the proximal part of 7q36. Furthermore, the breakpoint of the reciprocal translocation from the patient with t-AML was localized to a 1,300-kb sized YAC clone that maps to the proximal boundary of the commonly deleted region. Interestingly, in this case both homologs of chromosome 7 were affected: one was lost (−7) and the second exhibited the t(7q35). The identification and delineation of translocation and deletion breakpoints provides the first step toward the identification of the gene(s) involved in the pathogenesis of 7q35-q36 aberrations in myeloid disorders.

Beschreibung: Introduction. The fludarabine, cyclophosphamide, rituximab (FCR) regimen is associated with high complete response (CR) rates and a negative residual disease status in a significant proportion of cases and is considered the optimal front-line treatment for fit patients with chronic lymphocytic leukemia (CLL). In addition, long-term follow-up of patients treated with FCR at the MD Anderson Cancer Center, in the multicenter German CLL8 study and at Italian institutions indicate that a sizable fraction of patients characterized by a favorable biologic profile remains free from progression in excess of 10 years. FC combined with ofatumumab (FC-O), a human monoclonal antibody which targets an epitope of the CD20 molecule, has also been associated with a high CR rate. The aim of this study was to evaluate whether a double dose of ofatumumab (O2) combined with FC could improve the CR rate in young (≤65 yrs) and fit patients with CLL. Methods. Sixty-one fit CLL patients from 15 Italian institutions were enrolled in this front-line study and treated with the FC-O2 regimen based on the FC schedule (F 25 mg/sqm i.v. d1-3, C 250 mg/sqm i.v. d1-3) combined with 13 doses of O (300 mg i.v. d14; 1000 mg d21 at the first cycle; 1000 mg d1 and d15 at cycles 2-6 and d28 at cycle 6). As infection prophylaxis, patients received bactrim and peg-filgrastim in order to prevent granulocytopenia. CLL diagnosis, treatment requirement and response were assessed according to the 2008 iwCLL guidelines. Minimal residual disease (MRD) was evaluated by flow cytometry in the peripheral blood (PB) and bone marrow (BM), and also by RQ-PCR in flow negative cases. CT scan evaluation was included in the response assessment. Adverse events (AEs) were graded according to the NCI-CTCAE. Results. The median age of patients was 60 years (range 36-65), Binet stages B and C were recorded in 86% of cases, B-symptoms in 21%, increased β2M values in 74% and bulky nodes (≥5 cm) in 10%. An IGVH unmutated status was recorded in 60% of cases, deletion 13q in 37%, no aberrations in 33%, deletion 11q in 14%, trisomy 12 in 12%, 17p deletion and/or TP53 mutation were found in 10% of cases. At present, the median follow-up of patients is 7 months (range 1-20). Response to treatment has been assessed in 29 patients after a median number of 6 courses of treatment (range 2-6). The overall response rate is 90%, with a CR rate of 69% (20 patients). No evidence of MRD was observed by flow cytometry in both PB and BM in 15/20 CR patients (75%). To date, 11 patients with cytometric MRD negative CR have been evaluated by RQ-PCR and no residual disease was detected in 3. Grade 3-4 granulocytopenia was recorded in 4 patients (7%), a severe infection in 4 (7%) and 5 patients (8%) experienced a severe infusion-related reaction during ofatumumab administration. Treatment was discontinued in 8 patients as a result of toxicity (infection, 2 cases; FUO, 1; infusion-related toxicity, 1; autoimmune hemolytic anemia, 1; recurrent granulocytopenia, 1; tachyarrhythmia, 1; non-specified toxicity,1). A non-treatment-related death (traumatic aortic transaction due to a dislocated aortic endoprostheses) has been recorded in a patient after 2 months from treatment discontinuation and 1 showed a disease progression after 4 courses of FC-O2. Conclusions. Taken together, the first analysis of this ongoing front-line study suggests that the combination of FC with an increased dose of ofatumumab is well tolerated with acceptable and no unexpected toxicity. Our preliminary results show that the FC-O2 treatment is associated with a high rate of cytometric MRD-negative CR in young and fit patients with previously untreated CLL. Disclosures Carella: Seattle Genetics Inc.: Research Funding. Foà:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Beschreibung: Extensive submicroscopic deletions adjacent to the breakpoint on derivative chromosome 9 [der(9)] have been reported in a subset of Chronic Myeloyd Leukemia (CML) patients and have been associated with an adverse outcome with conventional drugs and α-interferon (α-IFN). Huntly et al (Blood.2003; 102.2205–12) reported 275 CML pts who were treated with imatinib in CP, suggesting that der(9) deletions were associated with lower response rates and a shorter time to progression. Different data were reported by Quintas-Cardama et al (Blood.2005; 105:2281–6), who did not find any difference related with der(9) deletions in other 320 patients treated with imatinib. In these 2 studies, some patients began imatinib in early CP (51 and 152, respectively) while many patients (224 and 168, respectively) were treated in late CP. To establish the relationship of der(9) deletions with the response to imatinib in early CP patients, we planned a prospective study involving 3 consecutive multicentric national studies of the GIMEMA (Gruppo Italiano Malattie Ematologiche dell’Adulto) CML Working Party. 421 CML patients in early chronic phase were enrolled between January, 2004 and January, 2006; Fluorescence in situ hybridization (FISH) analysis of bone marrow cells was performed using BCR/ABL extra-signal, D-FISH or dual-color dual-fusion probes. At diagnosis, 52 (12%) of them had der(9) deletion and 369 (88%) had not. The 2 groups, with/without deletions, were comparable (no difference in age, Sokal risk, imatinib dose). Median observation time is 12 months. At 3 months, the CHR rates in with/without deletions patients were 87%/92%. At 6 months, the complete cytogenetic response (0% Ph-pos; CCgR) rates were 80%/80%, with major molecular response (MMolR, defined as a Bcr-Abl/Abl x 100 ratio 〈 0.1%) rates of 52%/51%, respectively. At 12 months, CCgR rates were 89%/86% and MMolR 52%/61%. No difference is significant. We conclude that the presence of der(9) deletions at diagnosis do not constitute a negative factor for response to imatinib: the haematological, cytogenetic and molecular response rates resulted equal between the 2 groups of patients with and without der(9) deletions. This finding is relevant to the long term effect of imatinib treatment, since both the CCgR and the MMolR are important and established indicator of long term survival.

Beschreibung: Cyclin-dependent kinase inhibitors (CKI) regulate cell division resulting aberrantly expressed in many types of cancer. Alterations of CKI have been reported in acute leukemia, as the result of gene promoter methylation. Despite the common frequency of these alterations, little has been reported on the role of CKI aberrant protein expression and results are less clear, especially in acute lymphoblastic leukemia (ALL). The aim of this study was to analyze p21, p15 and p16 protein expression and their gene methylation status in primary cells from adult ALL cases enrolled in the LAL2000 GIMEMA protocol. Normal peripheral blood lymphocytes (PBL) and 91 primary samples from untreated ALL patients were evaluated in this study. The p21, p15 and p16 protein expression was analyzed by Western blot using the specifically MoAbs. The CKI gene methylation status was investigated using a widely accepted method based on bisulfite modification of DNA, followed by the use of the methylation-specific PCR assay (MSP). This assay was further validated in vitro by SSI methylase. Normal PBL from 10 healthy donors, as described, did not expressed all CKIs and resulted unmethylated. The p21 expression was found in 28/91 cases (30.8%); in contrast, samples were found constantly unmethylated. The p15 expression was found in 44/85 cases (51.8%) and its gene methylated in 41.7%; a significant correlation was found between absence of protein expression and gene methylation (P=0.040). The p16 resulted never expressed in adult ALL, while its promoter was found methylated in 8/42 cases (19.1%). A significant association (P=0.037) was observed between p21 expression and immunophenotype; in fact, 3/24 (12.5%) T-ALL and 24/65 (36.9%) B-lineage ALL expressed this protein. The p16 methylation was associated with T-ALL (P=0.082). Achievement of CR was not influenced by single protein expression, nor by gene methylation status. However, the co-expression of p15 and p21 was associated with failure to induction treatment; in fact, only 6/67 (9%) of patients co-expressing p15 and p21 achieved CR (P=0.021). In summary, in adult ALL p21 is not methylated and p16 is never found expressed, and CR achievement is adversely affected by the co-expression of p21 and p15. In conclusion, we report that in addition to CKI methylation, aberrant expression of CKI, namely p21 and p15, is associated with poor outcome in adult ALL, suggesting that chemotherapy resistance may be promoted in these cases by cell cycle arrest and/or abnormal survival.