ALBERT

Description: Introduction. Chronic lymphocytic leukemia (CLL) with 11q deletion has been associated to a poor prognosis, but the clinical course of patients carrying this lesion is variable. This aberration, most often monoallelic, is present in 10-17% of newly diagnosed CLL and in 20-30% of patients with progressive or chemorefractory disease. The minimal deleted region (MDR) (2-3 Mbp) is located on the 11q22.3-q23.1 region and includes ATM. Moreover, 30-40% of 11q- CLL have also an inactivating ATM mutation on the other allele. The deleted region on 11q can also include BIRC3, a gene that is often deleted or mutated in advanced/chemorefractory stages of the disease. Although BIRC3 disruption has been associated to a poor prognosis, its prognostic implications in addition to ATM deletion are not well defined. The aim of this study was to perform a copy number aberration (CNA) and gene sequencing analyses on a cohort of CLL patients with 11q- in order to identify subgroups with potential prognostic relevance based on: i) the inclusion of BIRC3 in the deleted region; ii) the presence of BIRC3 mutation; iii) the presence of other CNAs. Methods. The study has included 55 untreated CLL patients followed at our Institution or enrolled in GIMEMA clinical trials (2003-2013). Genomic DNA was extracted from peripheral blood samples. CNA analysis was performed by genomic hybridization on the CytoScan HD array (Affymetrix), which contains more than 2.6 x 106 markers for copy number analysis and 750.000 SNPs. Data were analyzed using both Partek Genomics Suite and ChAS (Chromosome Analysis Suite, Affymetrix) software. The resulting CNAs were verified by visual examination of the plotted copy number profiles. BIRC3 mutations (exons 6-9) were evaluated by Sanger sequencing. Time to first treatment (TFT) was calculated from the date of diagnosis to the date of first therapy or last follow-up; progression-free survival (PFS) from the date of first therapy to the date of progression, death or last follow-up. Results. Baseline characteristics of the 55 cases were as follows: median age at diagnosis 59 years (range 39-84), male gender in 81.8% of patients, progressive disease in 62%. All patients showed 11q- by FISH (median 80%, range 25-99% of nuclei); germline IGHV were present in 96.4% of cases; TP53 deletion in 1 case and TP53 mutation in none; NOTCH1 mutation in 4/40 cases; SF3B1 mutation in 5/40 (all mutually exclusive with only 1 case having both SF3B1 and BIRC3 mutations). By CytoScan HD array, the size of 11q- was very variable, ranging from 0.36 Mbp to 65.14 Mbp; the MDR was located on 11q22.3 region, encompassing 4 genes (ACAT1, ATM, CUL5, NPAT). BIRC3 was included in the deleted region in 45/55 cases (81.8%) and was mutated in 4/54 (7.5%), being always deleted on the other allele. Beside 11q-, 51 cases (92.7%) showed several additional CNAs (average 4.9, range 1-14 per patient), with 5 recurrent lesions: 2p gain in 11 cases, del4(p15.2) in 6, del19(p13.3) in 6, 8q gain in 5 and del4(q22.1) in 4. BIRC3 deletion was not associated to the number of additional CNAs nor to specific CNA. After a follow-up of 59.6 months (range 7.4-229.7), 40 of 47 evaluable patients have received treatment (median TFT 15.8 months, range 0-167). BIRC3 deleted cases (n=37) showed a TFT not significantly different from WT cases (n=10). Conversely, BIRC3 mutation was associated to a shorter TFT (p 3 CNAs larger than 5 Mb (n=14) or 〉10 CNAs (n=5), or the presence of 2p gain, del4(p15.2), del19(p13.3) or 8q gain. So far, 22 patients have been evaluated for PFS after first-line therapy (median 28.7 months). BIRC3 deleted cases (n=17) were not associated to a shorter PFS compared to WT cases (n=5), in line with the results from Rose-Zerilli et al (Haematologica 2014). Conclusions. Among CLL with 11q-: 1) BIRC3 deletion involves more than 80% of cases, whilst the mutation is rare (7.5%); 2) BIRC3 deletion is not associated to a higher genomic complexity; 3) BIRC3 deletion does not seem to influence TFT or PFS of 11q- CLL; 4) BIRC3 mutation is strongly associated to a short TFT; 5) BIRC3 biallelic lesions can be associated to a marked hyperleucocytosis at diagnosis and immediate need of treatment. Disclosures No relevant conflicts of interest to declare.

Description: In CLL patients, ZAP-70 expression is associated with an unmutated status of the variable immunoglobulin heavy chain gene (IgVH) region. Both features bear an adverse prognostic value. However, in various published series there is a proportion of cases with discordance in the expression of these two markers (i.e. ZAP-70-/IgVH unmutated or ZAP-70+/IgVH mutated) ranging from 5 to 30%. In order to clarify the outcome of this subgroup of patients, information on the clinico-biological features of the discordant cases is becoming increasingly more relevant. From November 2002 to April 2006, we evaluated ZAP-70 and CD38 expression, IgVH mutation status and cytogenetic aberrations by FISH in 125 young and untreated patients: 69 males, 56 females, with a median age of 51 years (range 29–64). According to Binet’s staging system, 81% were stage A, 15% stage B and 4% stage C. Eighty % of patients presented stable disease and 20% progressive disease. After a median follow-up of 40 months (range 3–191) from diagnosis, 32% of cases have required therapy. ZAP-70, evaluated by immunocytochemistry and flow-cytometry, was positive (≥7%) in 36% of cases, IgVH genes were unmutated (≥98% homology) in 25% and CD38 was positive (≥7%) in 18%. The correlations between ZAP-70/IgVH, CD38/IgVH and CD38/ZAP-70 were highly significant (p≤0.001 each) with a proportion of discordant cases of 21%, 16% and 24%, respectively. Focusing on 114 cases with available data for both ZAP-70 and IgVH mutation status, three groups were identified: 23 ZAP-70+/IgVH unmutated, 67 ZAP-70-/IgVH mutated, 24 discordant cases. Significant differences were found in terms of distribution of cytogenetic aberrations, CD38 expression and atypical lymphocyte morphology (Table I). Only 2 cases showed V3-21 usage: both were IgVH mutated, 1 was ZAP-70+ and the other ZAP-70-. Of the 24 discordant cases, 18 (75%) were ZAP-70+/IgVH mutated and 6 (25%) ZAP-70-/IgVH unmutated. No significant difference was shown between the two groups regarding the presence of poor risk genetic abnormalities del(17p), del(11q) and +12 (p=0.17), CD38 expression (p=0.2), atypical lymphocyte morphology (p=0.12). Although the follow-up is still short, ZAP-70 and IgVH status significantly predicted treatment-free interval (TFI), individually (p

Description: This study was conducted to determine the feasibility, safety and efficacy of the FAND plus Campath-1H combination (FAND-Cam) given to 19 previously treated CLL patients with adverse prognostic features. The median age was 55 years (range 43–65), the median duration of CLL was 68 months; 89 % of patients were unmutated, 74% CD38+ and 75% ZAP-70+. A 17p− deletion was observed in 5/11 evaluable cases. The median number of prior treatments was 2 (range 1–6). Seventeen patients (89%) were refractory to prior therapy, which included fludarabine in 14 (fludarabine, n = 3; fludarabine-cyclophosphamide, n = 10; fludarabine-cyclophosphamide-rituximab, n = 1). The first 6 patients received 2 courses of FAND, for tumor debulking, followed by 2 FAND-Cam courses. However, a high infection rate was observed. In an attempt to reduce the risk of infection, the subsequent 13 patients received 2 courses of FAND-Cam without prior debulking with FAND. The treatment regimen of FAND was as follows: fludarabine, 25 mg/m2 intravenously (IV) administered daily on days 1–3 (0, 24 and 48 hours); Ara-C, 700 mg/m2 IV on days 1–3 (4, 28 and 52 hours); mitoxantrone, 10 mg/m2 IV on day 1 at 6 hours; dexamethasone, 20 mg IV on days 1–3. Before the first FAND-Cam course, Campath-1H was dose escalated from 3 mg 10 mg and then to 30 mg IV. After the FAND regimen, patients received Campath-1H, 30 mg IV on 3 consecutive days. G-CSF was used in the event of severe neutropenia. Infection prophylaxis consisted of trimethoprim-sulfamethoxazole, fluconazole, acyclovir 200 mg 3 times a day, for the first 9 patients, valacyclovir 2g 3 times a day for the last 10 patients. CMV viremia was performed weekly. To date, the response is evaluable in 18 patients. All 6 patients who received 2 courses of FAND for tumor debulking, followed by 2 courses of FAND-Cam, responded to treatment (complete response [CR], n = 1; cytometric CR, n = 4; partial response [PR], n = 1). However, infections were recorded in all cases (Nocardia pneumonia + CMV reactivation, n = 1; pneumonia, n = 1; pneumonia + CMV reactivation, n = 1; sinus infection, n = 1; CMV reactivation, n = 2). In the second group of patients who, without prior debulking, received only 2 courses of FAND-Cam, a response was observed in 10/12 (83%) evaluable cases (CR, n = 3; cytometric CR, n = 3; PR, n = 4; no response [NR], n = 2). An infection was recorded in 8/12 cases (Pseudomonas sepsis, n = 1; Pseudomonas sepsis + CMV reactivation, n = 1; Aspergillus, n = 1; Pneumonia, n = 3; CMV reactivation, n = 2). Prophylactic treatment with valacyclovir reduced the rate of CMV reactivation from 67% (6/9 pts) to 11% (1/9pts). During treatment there were no deaths related to infection. Six patients who achieved a response after Fand-Cam underwent allogeneic peripheral stem cell transplantation. CLL refractory patients with adverse prognostic factors respond poorly to therapy and show a high infection rate due to the severe immunodeficiency related to both refractory disease and treatment. In this study, FAND-Cam combination proved to be a feasible and effective salvage therapy in most patients. Infections were common. Careful surveillance and extended prophylaxis for infection are therefore required.

Description: 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.

Description: Background. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of relatively mature B cells and by a very variable clinical course. This clinical heterogeneity is sustained by different biologic parameters, such as the mutational status of the immunoglobulin variable genes (IgVH), CD38 and ZAP-70 expression. In order to investigate the potential role of protein kinase (PK) inhibitors in CLL, we evaluated the gene expression profile of 1324 probesets annotated as PK using the HGU133 Plus2.0 Affymetrix arrays. Methods. We evaluated 44 CLL and 137 acute lymphocytic leukemia (ALL) patients. Two additional sets of CLL (49 cases) were utilized to validate the results obtained. Probesets identified as PK genes were used for all the analyses, namely unsupervised clustering, Analysis of Variance (ANOVA) and t-test analysis. ANOVA was performed using a p-value of ≤0.001: further selection was performed retaining only those probesets whose mean expression level was ≥300 in at least one group and showed a fold change difference of ≥1.5 across all groups. Finally, to specifically identify genes differentially expressed between different subclasses of CLL, a t-test was applied: probesets were required to have a p-value ≤0.05 and a fold change〉1.5. Results. Unsupervised analysis, performed on CLL samples and different ALL subgroups, highlighted in CLL a unique and very homogeneous pattern characterized by the overexpression of a large set of PK; these results were further confirmed by ANOVA. Moreover, we identified 16 PK genes that were highly expressed in all 3 CLL sets analyzed. These genes codify for proteins with tyrosine kinase activity (SYK, LYN, BLK, LCK, JAK1, CSK and FGR), serin-threonin kinase activity (PIM2, PFTK1, TLK1, MAP4K1, PDPK1, PRKCB1 and STK10) or both (GRK6 and WEE1). Some of the selected genes are members of important protein kinase families, involved in cellular signaling, such as Src kinases (SFK), MAPK and JAK kinase family. PK expression was also analyzed in different CLL subclasses, subdivided according to different prognostic factors; in particular, we compared IgVH mutated vs unmutated patients, CD38+ vs CD38- cases and, finally, ZAP-70+ vs ZAP-70- patients in the 3 experimental CLL sets. Comparison between IgVH mutated vs unmutated cases highlighted a differential expression of ZAP-70 in all the 3 sets analyzed. Contrariwise, no PK was associated with the other prognostic parameters. Thus, these analyses did not show a specific signature associated with the abovementioned biologic features, suggesting that PK overexpression is specific of the disease itself rather than of CLL subclasses. Conclusions. Our results show that CLL is characterized by a very peculiar PK signature and identify some potential molecular targets. Moreover, our findings indicate that a common mechanism of PK-mediated deregulation is operational in CLL cells, independently of other prognostic factors. Based on these pre-clinical data, we propose that second generation PK inhibitors may have a role in the management of all CLL patients.

Description: Abstract 1784 Introduction: The introduction of whole exome sequencing has allowed to unravel novel molecular lesions in CLL. NOTCH1, SF3B1 and BIRC3 mutations are detected, according to the phases of disease, in 4–12%, 5–17% and 4–24% of patients, respectively. In retrospective studies, their presence has been shown to correlate with overall survival (OS) and treatment-free interval shortening. Aims: To define the incidence, correlation with known prognostic factors and clinical impact of NOTCH1, SF3B1 and BIRC3 mutations in CLL patients undergoing first-line treatment. Methods: We evaluated 162 CLL patients enrolled in the GIMEMA LLC0405 protocol (n=80) for patients aged 65 yrs or 60–65 if not eligible for fludarabine-based programs). In the GIMEMA LLC0405 protocol, patients were stratified into low and high-risk: patients with del17p or with del11q plus an unmutated IGHV status and/or CD38 positivity and/or ZAP70 positivity were considered as high-risk (HR) and underwent Fludarabine plus Campath, followed by stem cell transplantation procedures, whereas low-risk patients received Fludarabine and Cyclophosphamide. The MLL21445 protocol consisted of 8 cycles of Chlorambucil and 6 of Rituximab induction treatment. NOTCH1 (exon 34), SF3B1 (exons 14 and 15) and BIRC3 (exons 2–9, including splicing sites) were screened by Sanger sequencing on either genomic DNA (gDNA) or whole genome amplified DNA (WGA) collected at the time of treatment. These studies were not part of the clinical protocols. Results: NOTCH1 mutations were detected at the time of treatment in 18 cases (22%) enrolled in the LLC0405 study. There was a significant association with high-risk stratification (p=0.036), namely with an IGHV unmutated status (p=0.0035), CD38 (p=0.03), +12 (p=0.034) and, partly, ZAP-70 expression (p=0.059). While the overall response rate (ORR) did not differ between NOTCH1 mutated vs wild-type (WT) cases (82% vs 77%, respectively), the complete response (CR) rate was significantly lower in NOTCH1 mutated patients (43% for WT vs 17% for NOTCH1 mutated cases; p=0.05). So far, no significant difference between mutated and WT patients has emerged in terms of OS and progression-free survival (PFS); this may be contributed by the fact that most NOTCH1 mutated cases were HR and were therefore treated more aggressively. SF3B1 mutations were recorded in 9 cases (11%); no significant associations were found with known biological parameters and, so far, with the ORR and CR rate. A single case harbored a BIRC3 mutation; this patient had an IGHV unmutated status, no FISH abnormalities and a concomitant SF3B1 mutation. In the ML21445 cohort, NOTCH1 mutations were found in 12 cases (15%), were associated with an unmutated IGHV status (p=0.047) and ZAP-70 expression (p=0.007), and did not impact on the ORR and CR rate. SF3B1 mutations were found in 11 cases (13%); no significant associations were found with known biological parameters and the ORR rate. Of interest, only 1/11 SF3B1 mutated patients achieved a CR. BIRC3 mutations were recorded in 3 patients (3.6%); of these, 2 were IGHV mutated, 1 had no cytogenetic abnormalities and 1 carried a del11q, while the third patient was IGHV unmutated status and had no cytogenetic abnormalities. No NOTCH1 and/or SF3B1 mutations were detected. Overall, NOTCH1, SF3B1 and BIRC3 mutations were largely mutually exclusive among each other and with TP53 lesions in the whole cohort. Conclusions: This study confirms the association of NOTCH1 mutations with unfavorable biologic markers and +12, while the presence of SF3B1 mutations was not coupled to poor prognostic markers in CLL patients requiring first-line treatment. Furthermore, it suggests that NOTCH1 mutations impact on the CR rate of young patients receiving Fluda-based regimens, while SF3B1 appears to impact on the CR rate of elderly patients treated with Chlorambucil and Rituximab. Given the small numbers of patients harboring BIRC3, it is at present difficult to draw any conclusion on the clinical impact of this mutation in the cohort of patients hereby analyzed. Disclosures: No relevant conflicts of interest to declare.

Description: Background: CLL is characterized by an heterogeneous clinical course, with some patients living many years without therapy and other patients presenting an aggressive disease. Different biologic properties of the leukemic cells have important prognostic implications. In particular, the IgVH gene mutational status, chromosomal abnormalities, expression of CD38 and ZAP-70 have been correlated with the clinical course of the disease. There is also growing evidence that angiogenesis may be linked to the pathogenesis and outcome of CLL. In particular, vascular endothelial growth factor A (VEGF-A) has been detected in the serum and bone marrow biopsy cells analyzed by immunostaining: both events have been correlated with a poor prognostic likelihood. Aims: The goal of this study was to obtain a more complete characterization of the angiogenic potential of CLL cells, to correlate the angiogenic capacity with other prognostic parameters and to investigate the possibility of overcoming this property by specific VEGF-signaling inhibitors. Methods: Eighteen patients with a diagnosis of CLL, based on morphologic and immunophenotypic criteria, have been evaluated for the expression of CD38 and ZAP-70, and for the IgVH gene mutational status. An Angiokit test (TCS Cell Works) was utilized to evaluate the capacity of purified CD19+ CLL cells to stimulate the growth and tubule formation of human endothelial cells and the relative roles of an anti-VEGF specific antibody and of PTK787/ZK 222584 (PTK/ZK, Schering, Novartis), a novel, oral angiogenesis and lymphangiogenesis inhibitor that blocks tyrosine kinase signaling from all known VEGFRs. The quantification of endothelial tubule formation was carried out using a specific software. After stimulation with a biotinylated goat anti-human IgM F(ab)2, leukemia cells were evaluated for gene expression profiling using the Affymetrix HGU133 Plus 2.0 gene chip. Results: In 9 patients, the leukemic cells were CD38+, ZAP-70+ and showed an unmutated IgVH status; in the remaining 9, the leukemic cells were CD38−, ZAP-70− and with a mutated IgVH status. In CLL patients with poor prognostic features - CD38+, ZAP-70+ and unmutated IgVH - purified leukemic cells showed a significantly greater capacity to stimulate the growth and tubule formation (3407 ± 968 tubules) of human endothelial cells compared to leukemic cells obtained from CLL patients with good prognostic factors, i.e. CD38−, ZAP-70− and mutated IgVH, (1550 ± 624 tubules). This property was amplified following sIgM cross-linking, that resulted in the upregulation of angiogenetic genes, including: EGR-1, SDF2L1, TNFAIP3, PTGER4, FIBP, NR4A3. A reduction of VEGF-A-induced tubule number was observed when leukemic cells were cultured with the anti-VEGF antibody (43–82% of inhibition) and, to a further extent, with the PTK/ZK inhibitor (90–96% of inhibition) compared to purified CLL cells alone. Conclusions: In CLL patients with poor prognostic factors, leukemic cells display an angiogenic capability significantly higher than that of cells from patients with favorable prognostic factors. The current findings provide the rationale for investigating the potential clinical role of anti-angiogenic agents, such as PTK/ZK, in the management of CLL patients.

Description: Abstract 2462 Background: Two of the largest trials ever conducted in patients with chronic lymphocytic leukemia (CLL) have shown that the addition of rituximab to fludarabine plus cyclophosphamide (R-FC) significantly improves outcome. However, myelotoxicity and immunosuppression limit the use of this regimen in patients with impaired performance status and pre-existing co-morbidities, predominantly in the elderly. Chlorambucil (CLB) remains a first-line treatment option for such patients. The use of CLB in combination with R is thus an attractive therapeutic option in view of the potentially increased activity compared to CLB alone and the likely good tolerability. This study was designed to determine whether the R-CLB combination is feasible and beneficial as first-line treatment for elderly patients with CLL and to define the role of maintenance R. Patients and Methods: Between October 2008 and January 2010, 97 elderly patients with untreated CD20+ CLL requiring therapy according to the IWCLL criteria were enrolled into the protocol. CLB treatment was administered every 28 days for up to 8 courses at a dose of 8 mg/m2/day p.o. on days 1–7 combined with 375 mg/m2 R for cycle 3 and 500 mg/m2 for cycles 4–8. Responsive patients were randomized to R maintenance (375 mg/m2 every 2 months for 2 years) versus observation. At baseline, blood samples were taken for FISH analysis, IgVH mutational status and expression of Zap-70 and CD38. Minimal residual disease (MRD) was planned to be evaluated on peripheral blood (PB) and bone marrow (BM) cells by four-color flow cytometry and, when required, by PCR. The primary endpoint was the overall response rate at the end of the induction phase defined according to the IWCLL 2008 on the intention-to-treat (ITT) population (all enrolled patients who received at least 1 dose of R). Secondary endpoints included the adverse event (AE) profile, progression-free and overall survival. Results: These are the data of the planned interim analysis based on the first 54 evaluable patients from 19 Italian centers, including tumor response at the end of the induction phase and safety. The median age of patients was 70.5 years (range 61–84): 14.8% were between 61 and 64, 31.5% between 65 and 69, 31.5% between 70 and 74, 16.7% between 75 and 79, and 5.6% were ≥80 years; thus, 53.8% of patients were over the age of 70; 70.4% were males; 25.9% were Binet stage A, 57.4% stage B and 16.7% stage C. The overall incidences of trisomy 12 and abnormalities of 13q, 11q23 and 17p13 were 24.5%, 52.8%, 20.8% and 5.7%, respectively; 7.5% of patients had p53 mutations. Of the 51/54 patients analyzed for the IgVH mutational status, 64.7% were unmutated; of the 53/54 patients studied, 39.6% were CD38+ and 71.7% were Zap-70+. The overall response rate on an ITT analysis was 81.4% (44/54 patients); a CR assessed by CT scan and trephine immunohistochemistry was found in 16.7% of cases (9 patients: 4 in Binet stage A, 3 in stage B and 2 in stage C), a CRi in 3.7% (2 patients), a nPR in 1.9% (1 patient) and a PR in 59.3% (32 patients). Eight of the 9 CR cases were investigated for MRD by flow cytometry and all proved positive: 6/8 had MRD levels

Description: The genetic lesions identified to date do not fully recapitulate the molecular pathogenesis of chronic lymphocytic leukemia (CLL) and do not entirely explain the development of severe complications such as chemorefractoriness. In the present study, BIRC3, a negative regulator of noncanonical NF-κB signaling, was investigated in different CLL clinical phases. BIRC3 lesions were absent in monoclonal B-cell lymphocytosis (0 of 63) and were rare in CLL at diagnosis (13 of 306, 4%). Conversely, BIRC3 disruption selectively affected 12 of 49 (24%) fludarabine-refractory CLL cases by inactivating mutations and/or gene deletions that distributed in a mutually exclusive fashion with TP53 abnormalities. In contrast to fludarabine-refractory CLL, progressive but fludarabine-sensitive patients were consistently devoid of BIRC3 abnormalities, suggesting that BIRC3 genetic lesions associate specifically with a chemorefractory phenotype. By actuarial analysis in newly diagnosed CLL (n = 306), BIRC3 disruption identified patients with a poor outcome similar to that associated with TP53 abnormalities and exerted a prognostic role that was independent of widely accepted clinical and genetic risk factors. Consistent with the role of BIRC3 as a negative regulator of NF-κB, biochemical studies revealed the presence of constitutive noncanonical NF-κB activation in fludarabine-refractory CLL patients harboring molecular lesions of BIRC3. These data identify BIRC3 disruption as a recurrent genetic lesion of high-risk CLL devoid of TP53 abnormalities.

Description: Abstract 1377 Targeting the CLL microenvironment with immunomodulatory agents like lenalidomide results in antileukemic activity. Addition of lenalidomide to an effective regimen such as fludarabine and cyclophosphamide (FC) could further increase therapeutic activity. On this basis, we designed a study aimed at evaluating the activity and safety profile of FC combined with lenalidomide (FCL) in previously treated CLL patients. Treatment consists of 6 monthly courses of FC (F, fudarabine: 30 mg/m2 iv and C, cyclophosphamide: 250 mg/m2, days 1–3) combined with 14 consecutive days of lenalidomide administration (days1-14). The first phase of this study was focused on defining the maximum tolerated dose (MTD) of lenalidomide given in combination with FC. In all patients, lenalidomide was given at the starting dose of 2.5 mg daily during the first course. For the subsequent courses the dose of lenalidomide was progressively escalated to reach a maximum daily dose of 5 mg, 10 mg and 15 mg, respectively, in each 3 cohorts of 3 patients, unless a dose limiting toxicity (DLT) was experienced. DLT was defined as persistent and severe hematologic toxicity, grade ≥2 tumor lysis syndrome (TLS), grade ≥3 tumor flare reaction (TFR) or other grade ≥3 toxicities. Supportive treatment included primary prophylaxis of granulocytopenia (G-CSF, days 6–11), PC and HVZ prophylaxis, thromboembolic prophylaxis with low molecular weight heparin (enoxaparin, 4000 units/sc/day) and TLS prophylaxis (oral hydration, allopurinol 300 mg/day, days -3 - 90). Strict contraceptive measures were required for all males and females with childbearing potential. At present, 9 relapsed CLL patients with advanced disease have been included in the first phase of the study focused on identifying the MTD for lenalidomide given in combination with FC. The median age was 63 years (range: 50–68); the median number of prior treatments was 2 (range: 1–2). All patients but 1 had been previously treated with the FC/FCR regimens; the majority showed a considerably high white blood count (range: 33–216 × 109/L) and 2 had a bulky nodal disease. Adverse cytogenetic abnormalities were detected by FISH in 5 cases (17p-, 2 cases; 6q-, 3 cases). While no DLT was observed in 3 patients who reached the 5 mg dose of lenalidomide given in combination with FC, a DLT was recorded in 2 of 3 patients who received 10 mg of lenalidomide (pneumonia; grade 3 hepatic toxicity). The 5 mg dose of lenalidomide was tested in 3 further patients with no DLT. Thus, 5 mg of lenalidomide was defined as the MTD of lenalidomide given in combination with FC. A marked reduction of white blood count was observed within the first 2 weeks of treatment in all cases but 1. A response was observed in 6 patients (67%; CR, 3; PR, 3), while a treatment-failure was recorded in 3 cases (SD, 2; Richter's syndrome,1). Despite G-CSF prophylaxis, the majority of patients experienced transient grade 3–4 neutropenia. Grade 1 fatigue and constipation were frequently observed during the first course of treatment. A transient grade 1 TFR was documented, while no episodes of TLS and thrombosis were recorded. Our findings indicate that 5 mg daily is the MTD of lenalidomide given in combination with FC. Initial responses suggest that FCL is a promising investigational regimen for patients with CLL who have failed prior treatment with fludarabine-based combinations. In the ongoing phase 2 of this study 32 patients are planned to receive 5 mg of lenalidomide in combination with FC. Disclosures: Foà: Celgene: Membership on an entity's Board of Directors or advisory committees.