ALBERT

Description: Bendamustine has been demonstrated to be effective for the treatment of CLL, either alone compared with chlorambucil (Knauf et al, JCO 2009 and BJH 2012) or in combination with monoclonal antibodies such as rituximab both in second or more lines (Fischer et al, JCO 2011) and in first line treatment (Fischer et al, JCO 2012). However, the relationship between its activity with clinical and biological prognosticators has been addressed only in few studies. For this purpose, we evaluated the efficacy and safety of bendamustine, in a real-life contest, on 56 patients, median age 66 years (41-80), median number of previous regimens 1 (0-3, 32% previously untreated). Bendamustine was given for a median number of 6 cycles (70-90 mg/m2), in 82% of cases with rituximab at conventional doses. Overall (ORR) and complete response (CRR) rates were 73% and 44.6%, respectively. Obviously, CRR was higher (83.3%) for 18 patients treated in first line. A significant correlation was found between lower ORR and lymphocyte doubling time 30%) of alpha-4 integrin CD49d (OR 13.0; P=0.018), an important marker of bad prognosis in CLL (Bulian et al, JCO 2014). On the other hand, no significant correlations were found between ORR and CD38, ZAP-70 or IGHV mutational status. Similarly, no significant correlations were noted between ORR and FISH cytogenetics, excluding del(17)p, or NOTCH1 mutations, thus confirming the independence of response to bendamustine from some well-known important biologic prognostic factors. In fact, multivariate analysis confirmed a significant relationship only between ORR and TP53/del(17)p (OR 0.020; P=0.0015) and concomitant rituximab (OR 0.019; P=0.0074). The estimated 1-year OS and PFS were 57% and 86%, respectively. Side effects included grade 3-4 neutropenia, infections, thrombocytopenia and anemia which occurred in 21%, 12%, 12% and 5% of patients, respectively. Grade 3-4 non-hematologic toxicity, including infusion-related reactions, heart or kidney or liver failure were found almost exclusively in elderly patients treated with bendamustine after two or more lines of therapy (12.5%). In multivariate analisys of OS, calculated from the end of treatment with bendamustine, only response to bendamustine (P=0.008) was confirmed to be an independent prognostic factor, while both the number of previous therapies and the concomitant use of rituximab demonstrated no statistical significance. These our results confirm both the activity and safety of bendamustine, particularly in combination with rituximab, also in the setting of elderly patients, often affected by two or three comorbidities. Noteworthy, this effectiveness appears to be present also in patients with unfavorable clinical and biological features, excluding del(17)p or TP53 mutations, in which the employment either of modern oral BCR inhibitors or of BH3 mimetics anti-Bcl-2 will be definitely active, also in combination with the same bendamustine. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3906 Today the treatment target of CLL is the attainment of an optimal disease control combining chemotherapy with monoclonal antibodies (MoAbs). This approach produces more complete molecular remissions and longer response duration (RD), remaining often a minimal residual disease (MRD) detectable by flow cytometry. In addition, consolidation and maintenance therapy with MoAbs might provide a further RD and overall survival (OS) benefit in CLL, as it has been already clearly demonstrated in indolent non-Hodgkin lymphomas. We treated in first line 145 CLL symptomatic patients (pts), median age 63 years (37–80), with six monthly courses of intravenous (25 mg/m2) or oral fludarabine (30–40 mg/m2) and then, after a median time of 30 days, with four weekly doses (375 mg/m2) of rituximab (rtx). Before treatment, 15 pts had a modified low Rai stage, 127 an intermediate stage and only 3 a high stage. We defined as high risk pts having at least two of these markers: unmutated IgVH, CD38〉30%, ZAP-70〉20%, intermediate/poor cytogenetics (trisomy 12 or del11q or del17p). Sixty-three pts (43.5%) belonged to the high risk subset. For MRD flow cytometric study, the threshold was set at 〉1% CD19+CD5+CD79b+/− bone marrow (BM) CLL cells. Based on NCI criteria (Cheson, 1996), 111/145 (76%) pts achieved a complete remission (CR), 27/145 (19%) a partial remission (PR) and 7/145 (5%) no response or progression. Phenotypic CR (CD19+CD5+CD79b- BM cells

Description: IGHV3-21–using chronic lymphocytic leukemia (CLL) is a distinct entity with restricted immunoglobulin gene features and poor prognosis and is more frequently encountered in Northern than Southern Europe. To further investigate this subset and its geographic distribution in the context of a country (Italy) with both continental and Mediterranean areas, 37 IGHV3-21 CLLs were collected out of 1076 cases enrolled by different institutions from Northern or Central Southern Italy. Of the 37 cases, 18 were identified as homologous (hom)HCDR3–IGHV3-21 CLLs and were found almost exclusively (16 of 18) in Northern Italy; in contrast, 19 nonhomHCDR3–IGHV3-21 cases were evenly distributed throughout Italy. Clinically, poor survivals were documented for IGHV3-21 CLLs as well as for subgroups of mutated and homHCDR3–IGHV3-21 CLLs. Negative prognosticators CD38, ZAP-70, CD49d, and CD79b were expressed at higher levels in homHCDR3 than nonhomHCDR3–IGHV3-21 cases. Differential gene expression profiling (GEP) of 13 IGHV3-21 versus 52 non–IGHV3-21 CLLs identified, among 122 best-correlated genes, TGFB2 and VIPR1 as down- and up-regulated in IGHV3-21 CLL cases, respectively. Moreover, GEP of 7 homHCDR3 versus 6 nonhomHCDR3–IGHV3-21 CLLs yielded 20 differentially expressed genes, with WNT-16 being that expressed at the highest levels in homHCDR3–IGHV3-21 CLLs. Altogether, IGHV3-21 CLLs, including those with homHCDR3, had a peculiar global phenotype in part explaining their worse clinical outcome.

Description: B-cell chronic lymphocytic leukemia (B-CLL) follows heterogeneous clinical courses, and several biological parameters need to be added to the current clinical staging systems to predict which patients will experience an indolent or an aggressive outcome. This study analyzed CD38 expression by flow cytometry and soluble APO1/Fas (sAPO1/Fas), Bcl-2 (sBcl-2), and CD23 (sCD23) proteins by immunoenzymatic methods to evaluate their effect on the clinical course of 168 unselected B-CLL patients. Intermediate/high risk modified Rai stages were characterized by a higher CD38+ B-cell number (P = .0002) and higher sCD23 levels (P 

Description: B-cell chronic lymphocytic leukemia (CLL) is a heterogeneous disease with highly variable clinical courses which can be at least in part foreseen by investigating the expression of known prognosticators, including the IGHV gene mutational status or CD38/ZAP-70 expression. By analysing the coordinated expression of several surface antigens in a series of CLL with known clinical courses, we identified the simultaneous over-expression of CD38 and CD49d as part of the signature characterizing the subgroup with the worst outcome. The aim of the present study was to investigate the relationship between CD49d and other well-established biologic prognosticators (CD38 and ZAP-70 expression, IGHV gene mutational status), or markers of tumor burden (Rai clinical stage, beta2-M, sCD23), and to define the independent prognostic impact of CD49d in predicting overall survival (OS) and disease progression (evaluated as time-to-treatment, TTT) in CLL patients. The study includes samples from 303 patients affected by CLL according to the current diagnostic criteria (median age: 63.5 years, range 32–97). The entire cohort of patients was utilized to investigate the impact of CD49d and other prognosticators (CD38, ZAP-70, IGHV gene mutational status) on OS. TTT information and additional laboratory parameters (beta2-M, sCD23) were available for 232/303 patients, whose therapies were established according to NCI-WG criteria. CD49d expression was determined by three-color flow cytometry combining anti-CD49d, anti-CD19 and anti-CD5 mAbs; its expression was demonstrated to be stable over-time and the 30% of positive CD5+CD19+CLL cells was chosen as cut-off to discriminate CD49dlow from CD49dhigh cases. CD49d, whose expression was strongly associated with that of CD38 (p=2.2×10exp-16) and ZAP-70 (p=2.6×10exp-5), or with IGHV gene status (p=1.1×10exp-6), was independent predictor for OS (HR=4.39; p=0.0081) along with IGHV status (HR=5.54; p=0.0005) or, if this parameter was omitted, with ZAP-70 (HR=2.90; p=0.0092). CD49d also effectively predicted TTT and refined the prognostic relevance of all the investigated prognosticators. Notably, a CD49dhigh phenotype, while not changing the outcome of good prognosis (ZAP-70low, mutated-IGHV) CLL, was necessary to correctly predict the bad clinical courses of ZAP-70high (HR=3.12; p=0.023) or unmutated-IGHV (HR=2.95; p=0.002) cases. These findings support the introduction of CD49d detection in routine prognostic assessment of CLL patients, and suggest both pathogenetic and therapeutic implications for CD49d expression in CLL, e.g. envisioning the use of a humanized anti-CD49d monoclonal antibody, currently employed in multiple sclerosis (Natalizumab), for selcted CLL patients.

Description: Heterogeneous clinical behavior of B-CLL makes difficult for physicians to identify which pts experience a slowly progressive clinical course and which ones may benefit from an early and/or more aggressive treatment. The development of interphase FISH techniques allowed to detect selected chromosome abnormalities in non-dividing cells. In 325 CLL pts, multivariate analysis identified 17p- and 11q- abnormalities as variables associated with shorter overall survival (OS) (Dohner, 2000). Moreover, the lack of IgVH gene mutation has been shown to predict a rapid disease progression (DP) and an inferior OS (Damle, Hamblin, 1999). B-CLL cells that use non-mutated IgVH genes express ZAP-70 protein, associated with an enhanced B cell receptor signaling and with an early DP risk. The aims of our study were: 1) to determine progression-free survival (PFS) and OS upon cytogenetic groups and ZAP-70 expression; 2) whether ZAP-70 could predict varied outcome within interphase cytogenetic groups; and 3) whether ZAP-70 and interphase cytogenetic groups were independent prognostic factors. We investigated 216 pts, median age 64 years, 69 pts belonging to low Rai stage, 140 to intermediate stage and 7 to high stage. To date, we have completed analysis of interphase cytogenetics in 137 pts, and ZAP-70 was quantified in 216 pts by a multicolor flow cytometric method using a cut-off value of 20%. With regard to cytogenetic groups, 73 (53.3%) pts had a normal karyotype and 35 (25.5%) pts had 13q-. Twenty-nine (21.2%) pts with trisomy 12, 17p- and 11q- were pooled together and defined as “poor-risk” cytogenetic subset. ZAP-70+ pts were 81/216 (37.5%) and there was a significant correlation between high or low ZAP-70 expression and Ig V gene mutational status (P

Description: Clinical trials of monoclonal antibodies in combination with chemotherapy have reported previously unattained response rates in CLL because this approach reduces disease burden to levels detectable only by flow cytometry or molecular methods. The latest studies from CALGB have demonstrated that rituximab added concurrently or sequentially to fludarabine (Flu) for symptomatic, untreated CLL allows to achieve higher remission rates, longer progression free and overall survival. Nevertheless, the analysis of different biologic parameters could better explain the discordant outcome independent of treatment observed in these studies. Recent literature data indicate that unmutated VH genes, CD38 and/or ZAP-70 protein tyrosine kinase overexpression may predict both a lower response and a shorter survival. We performed at our Institution a phase II study that added rituximab sequentially to Flu as initial therapy for symptomatic, untreated CLL in order to evaluate either the toxicity or the clinical response or outcome. Complete remission (CR) was also assessed by a multiparametric flow cytometric method. ZAP-70 protein and CD38 antigen were determined before chemotherapy on mononuclear cells by flow cytometry using an anti-ZAP-70 and an anti-CD38 antibody, respectively. Forty-nine B-CLL patients, median age 59 years (range 37–74) received six monthly courses of Flu (25 mg/m2 for 5 days) and four weekly doses of rituximab (375 mg/m2) starting on an average of thirty days (range 5–180) after completion of the Flu therapy. According to modified Rai stages, 4 pts had a low stage, 42 an intermediate stage and 3 a high stage. Three out of 49 pts experienced fever, chills and rigors, during the first infusion of rituximab and only 1 patient presented grade 3 infective lung toxicity according to WHO. Hematologic toxicity included neutropenia (grade 1 and/or 2 in 12 pts, grade 3 and/or 4 in 22 pts), thrombocytopenia (grade 1 and/or 2 in 4, grade 3 and/or 4 in 3 pts) and anemia (grade 2 in 3 pts). Based on the NCI criteria, 45/49 (91.8%) pts achieved a CR, 3/49 (6.1%) a partial remission (PR) and 1/49 (2%) no response (NR). The median follow-up duration was 29 months. Median duration of CR and PR has not been reached. Noteworthy, our B-CLL pts treated sequentially with Flu and rituximab experienced a very long progression-free survival (PFS) from treatment (72% at 3 years). ZAP-70 and CD38 were positive (〉20%) in 19/48 (39.5%) and in 13/48 (27%) pts, respectively. Minimal residual disease (MRD) performed on bone marrow by flow cytometry was positive (〉5% CD19+CD5+CD79b- CLL cells) in 7/37 (19%) analysed pts. A significant shorter PFS was observed in ZAP-70+ pts (38% vs 100% at 3 years; P=0.003), in CD38+ pts (39% vs 92% at 3 years; P=0.007) and also in pts with higher MRD after treatment (50% vs 81% at 2 years; P=0.04). Therefore, the addition of monoclonal antibodies, such as rituximab, to chemotherapy allowed a better outcome in CLL, exerting a key role to eradicate MRD. Moreover, the stratification of pts within different risk classes using novel biologic predictive factors, such as ZAP-70 and CD38, might allow us to offer more tailored treatment strategies, reserving experimental approaches and/or transplantation procedures only to CLL subsets with proved adverse biologic and clinical features.

Description: Multidrug resistance and recurrent disease are key problems in the variable response of AML pts to treatment. Tumor cells in a high proliferative state have a high density of transferrin receptors, as demonstrated in breast cancer (Yang DC, 2001) and in adult T-cell leukemia/lymphoma (Moura IC, 2004). On the other hand, defects in apoptotic pathways such as higher levels of bcl-2 and Mcl-1 were reported in “poor risk” AML pts. Current availability of antisense oligonucleotides targeted both to the transferrin receptor genes and bcl-2 incited us to evaluate the impact of proliferative and/or apoptotic pathways on AML prognosis. Therefore, a large series of 325 pts, affected by de novo AML, except FAB M3, median age 55 years, treated with intensive chemotherapy regimens, were studied. The aims of our research were: 1) to correlate bax/bcl-2 ratio with the proliferation levels, determined by the transferrin receptor (CD71) and 2) to demonstrate that the clinical significance of spontaneous apoptosis is independent from proliferation. CD71, bcl-2 and bax proteins were determined by multicolor flow cytometry. CD71 was evaluated as mean fluorescence intensity (MFI) and bax/bcl-2 ratio was obtained by dividing MFI bax/MFI bcl-2. One hundred-seventy five pts (53.8%) were bax/bcl-2 ratio positive and 204/324 (63%) were CD71 positive, respectively. There was a close correlation between higher CD71 expression and Ki-67 positive staining by flow cytometry (r=0.86), confirming that transferrin receptor overexpression is really linked to increased cellular proliferation in AML. No significant correlation was found between a higher bax/bcl-2 ratio and a lower CD71 MFI (p=0.16), confirming that an apoptosis resistant protein profile may have variable proliferation levels. A significant lower complete remission (CR) rate was found in pts with lower bax/bcl-2 ratio (43% vs 72%, p

Description: CLL is a heterogeneous disease with patients (pts) experiencing rapid disease progression and others living for years without requiring treatment. Recently, next generation sequencing has revealed new molecular alterations, targeting the NOTCH1 and BIRC3 genes which occur in about 10% CLL at diagnosis and correlate with poor outcome. Given the possibility of targeting NOTCH1 and BIRC3 with drugs currently under development, the primary endpoints of our research were: 1) to determine overall survival (OS) upon IGHV, NOTCH1, TP53 and BIRC3 in univariate analysis; 2) to correlate these genomic aberrations with other biological or clinical prognostic factors, and finally 3) to confirm NOTCH1, BIRC3 and TP53 as independent prognostic factors. We investigated 475 pts with a median age of 65 years (range 33-89), whose 160 had low Rai stage, 301 intermediate stage and 14 high stage. NOTCH1 mutations (mut) were studied by ARMS PCR for c.7544-7545delCT and by Sanger sequencing of NOTCH1 exon 34. Mutations of TP53 were analysed by DNA direct sequencing, while BIRC3 disruption (disr) was studied by Sanger sequencing for mutations and by interphase FISH for deletions. All these alterations were studied at diagnosis or before any chemotherapeutic approach. NOTCH1mut and TP53mut pts were 52 (10.9%) and 36/475 (7.6%), respectively. Thirty four patients were BIRC3mut (7.2%) and 26 BIRC3 deleted (5.5%) for a total of 46 cases (9.7%) BIRC3disr. NOTCH1, TP53 and BIRC3 alterations were mutually exclusive. There were significant correlations between NOTCH1 (P

Description: Abstract 2870 Chronic lymphocytic leukemia (CLL) is a very heterogeneous disease ranging from rapid disease progression leading to death to a nearly normal life expectancy and therefore it is mandatory to find valid prognostic markers. Recent studies showed that activating mutations of NOTCH1 proto-oncogene occur in about 10% CLL at diagnosis and are associated with an unfavorable clinical outcome (Rossi et al, 2012). About 85% of NOTCH1 mutated CLL cases displayed a DCT7544–7545 frameshift deletion (hereafter NOTCH1 mutation), that has been demonstrated to predict NOTCH1 degradation impairment through the truncation of the C-terminal PEST domain. Given the possibility of targeting NOTCH1 with drugs currently under development, the primary endpoints of our research were: 1) to correlate NOTCH1 mutation with other clinical and biological prognostic factors; 2) to determine time to first treatment (TTFT) and overall survival (OS) upon NOTCH1 mutation in univariate analysis; 3) to validate NOTCH1 mutation as an independent prognostic factor. We investigated 463 pts, median age 65 years (range 33–89), 256 males and 207 females. With regard to modified Rai stages at diagnosis, 159 had a low stage, 290 an intermediate stage and 14 a high stage. NOTCH1 mutation was investigated by amplification refractory mutation system (ARMS) PCR at diagnosis or before any chemotherapeutic approach. The ARMS PCR approach was set up in order to identify NOTCH1 mutation when present in at least 10% of the alleles. Using this approach, NOTCH1 mutated pts were 45/463 (9.7%). Considering the association with markers of tumor burden and proliferation, NOTCH1 mutation correlated with intermediate/high Rai stages (37/45; P=0.002), multiple thoracic/abdominal lymphadenopathies and/or splenomegaly (26/45, P=0.003), beta-2 microglobulin 〉2.2 mg/ml (27/45; P=0.02), lymphocyte doubling time 70 U/ml (26/39; P=0.00001). Significant associations were also found with the main biologic prognostic markers in CLL. In this regard, NOTCH1 mutation was associated with an unmutated IGHV status (available for 446 total cases, 30/43; P30% (26/45, P20% (33/45; P30% (22/34; P=0.009). Finally, considering associations with specific chromosomal aberrations defined by FISH cytogenetics (available in 417 cases), significant correlations (P=0.003) were found between NOTCH1 mutation and trisomy 12 (14/41; 25%), and del11q (7/41;16% ), whereas only 2/43 NOTCH1 mutated cases presented 17p deletion. With regard to clinical outcome, 30/45 (67%) NOTCH1 mutated pts received chemotherapy vs 193/418 (46%) among NOTCH1 germ line CLL (P=0.01), with 15/45 (33%) vs 48/418 (11%) cases, belonging to the same subgroups, undergoing at least two lines of treatment (P=0.001). Moreover, both significant shorter TTFT and OS were observed in NOTCH1 mutated pts (7% vs 35% at 12 years, P=0.0006 and 34% vs 78% at 14 years, P20%, 188 pts) and unmutated IGHV (