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CCR Translation for this Article from Expression of Mutated IGHV3-23 Genes in Chronic Lymphocytic Leukemia Identifies a Disease Subset with Peculiar Clinical and Biological Features

Supplementary Data from Expression of Mutated IGHV3-23 Genes in Chronic Lymphocytic Leukemia Identifies a Disease Subset with Peculiar Clinical and Biological Features

Purpose: B-cell chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease whose outcome can be foreseen by investigating the mutational status of immunoglobulin heavy chain variable (IGHV) genes. Moreover, a different prognosis was reported for CLL expressing specific IGHV genes in the context or not of stereotyped B-cell receptors. Here we investigated novel associations between usage of specific IGHV genes and clinical features in CLL.Experimental Design: Among 1,426 CLL-specific IG-rearrangements, stereotyped B-cell receptor clusters never utilized the IGHV3-23 gene. Given this notion, this study was aimed at characterizing the IGHV3-23 gene in CLL, and identifying the properties of IGHV3-23–expressing CLL.Results: IGHV3-23 was the second most frequently used (134 of 1,426) and usually mutated (M; 109 of 134) IGHV gene in our CLL series. In the vast majority of M IGHV3-23 sequences, the configuration of the 13 amino acids involved in superantigen recognition was consistent with superantigen binding. Clinically, M IGHV3-23 CLL had shorter time-to-treatment than other M non–IGHV3-23 CLL, and multivariate analyses selected IGHV3-23 gene usage, Rai staging, and chromosomal abnormalities as independent prognosticators for M CLL. Compared with M non–IGHV3-23 CLL, the gene expression profile of M IGHV3-23 CLL was deprived in genes, including the growth/tumor suppressor genes PDCD4, TIA1, and RASSF5, whose downregulation is under control of miR-15a and miR-16-1. Accordingly, relatively higher levels of miR-15a and miR-16-1 were found in M IGHV3-23 compared with M non–IGHV3-23 CLL.Conclusions: Altogether, expression of the IGHV3-23 gene characterizes a CLL subset with distinct clinical and biological features. Clin Cancer Res; 16(2); 620–8

Background CD69 is expressed in several hemopoietic cells and is an early activation marker in chronic lymphocytic leukemia. Chronic lymphocytic leukemia is a clinically heterogeneous disease which needs novel prognostic parameters which can be easily and efficiently managed. Design and Methods We investigated CD69 by flow cytometry in a series of 417 patients affected by chronic lymphocytic leukemia and compared this to other biological and clinical prognosticators. Results CD69 was associated with Rai stages ( P =0.00002), β2-microglobulin ( P =0.0005) and soluble CD23 ( P

Nucleophosmin gene (NPM1) mutations characterize acute myeloid leukaemia (AML) with normal karyotype and frequently co-exist with FLT3 internal tandem duplications (ITD). We evaluated bcl-2, bax, NPM1 and FLT3-ITD in 222 AML patients. Bax/bcl-2 ratio >0.35 and NPM1 without FLT3-ITD were significantly associated (P = 0.0001). NPM1-mutated (mt)/FLT3-ITD negative patients showed a higher complete remission (CR) rate (90%, P = 0.0002) and a longer overall survival (OS, P = 0.00007). NPM1-mt/FLT3-ITD negative plus bax/bcl-2 > 0.35 subset showed a very high CR rate (96%), very long OS (P = 0.00005) and disease-free survival (P = 0.004). The favourable prognosis of NPM1-mt/FLT3-ITD negative patients might be explained by a higher bax/bcl-2 ratio.

Apoptosis plays a key role in the control of rapidly renewing tissues, such as the hematopoietic system and leukemia cells invariably have abnormalities in one or more apoptotic pathways, determining a survival advantage of these cells and the development of drug resistance. These defects are also frequently associated with a low rate of response to standard chemotherapy and with a poor survival in acute myeloid leukemia (AML). The major form of apoptosis proceeds through the mitochondrial pathway, with the mitochondrial outer membrane permeabilization, leading to the release of proteins normally found in the space between the inner and outer mitochondrial membranes (cytochrome C, AIF and others). Higher levels of anti-apoptosis proteins bcl-2, bcl-x(L), Mcl-1 block permeabilization of the membrane and are reported in AML patients presenting a poor outcome. On the contrary, activated pro-apoptotic bax or bad proteins allow this permeabilization and are correlated to a good prognosis in AML. Defects in the mitochondrial pathway induce multidrug-resistance and confer important prognostic information in AML. High ratios of bcl-2 to bax protein confer a poor prognosis with decreased rates of complete remission and overall survival. The prognostic information from the ratio of the proteins is greater than bcl-2 levels alone. Recently, we confirmed the impressive impact of the bax/bcl-2 ratio, determined by flow cytometry, on AML prognosis (complete remission and overall survival) in 255 AML patients. Bcl-2 down regulation might lower the apoptotic threshold of leukemic cells and, through this mechanism, favor response to chemotherapy. Phase II studies of oblimersen (antisense Bcl-2), cytarabine and daunorubicin or oblimersen plus gentuzumab, a cytotoxic antibody directed against CD33+ cells in relapsed AMLs, showed promising results. Defects in apoptosome proteins, such as APAF-1, are frequent in AML and treatment with 5-aza-2'-deoxycytidine, a specific inhibitor of DNA methylation, restored APAF-1 expression in leukemic cells. In conclusion, targeted therapies that are designed to induce apoptosis in leukemic cells, are the most promising anti-leukemia strategies. The elucidation of the apoptotic machinery and of its defects in AML lays the basis for developing new drugs able to trigger apoptosis and overcome therapy resistance.

The ability of bone to resist fracture depends on the intrinsic properties of the materials that comprise the bone matrix mineralization, the amount of bone (i.e. mass), and the spatial distribution of the bone mass (i.e. microarchitecture). Antiresorptive agents may prevent the decay of cancellous bone and cortical thinning, with no improvement of bone microstructure, leading to a partial correction of the principal bone quality defect in osteoporosis, the disruption of trabecular microarchitecture. Anabolic agents promote bone formation at both trabecular and endocortical surfaces, resulting in an increase of cancellous bone volume and cortical thickness. The improvement of cortical bone strength may be limited by an increase in cortical porosity. strontium ranelate improves trabecular network and cortical thickness that will contribute to anti-fracture efficacy at both vertebral and non-vertebral sites. The results of clinical and experimental studies are consistent with the mode of action of strontium involving dissociation between bone formation and resorption leading to a stimulation both trabecular and cortical bone formation without increasing cortical porosity.

Deletion at 13q14 is detected by fluorescence in situ hybridization (FISH) in about 50% of chronic lymphocytic leukemia (CLL). Although CLL with 13q deletion as the sole cytogenetic abnormality (del13q-only) usually have good prognosis, more aggressive clinical courses are documented for del13q-only CLL carrying higher percentages of 13q deleted nuclei. Moreover, deletion at 13q of different sizes have been described, whose prognostic significance is still unknown. In a multi-institutional cohort of 342 del13q-only cases and in a consecutive unselected cohort of 265 CLL, we investigated the prognostic significance of 13q deletion, using the 13q FISH probes locus-specific identifier (LSI)-D13S319 and LSI-RB1 that detect the DLEU2/MIR15A/MIR16-1 and RB1 loci, respectively. Results indicated that both percentage of deleted nuclei and presence of larger deletions involving the RB1 locus cooperated to refine the prognosis of del13q-only cases. In particular, CLL carrying

Abstract 3578 Introduction: Chronic lymphocytic leukemia (CLL) patients bearing 13q14 deletion are known to experience a more favorable clinical course. Recent studies, focusing on patients with loss of 13q as the sole cytogenetic aberration at diagnosis (del13q-only cases), showed that the number of malignant cells carrying this genetic lesion correlates with a more aggressive clinical behavior. However, whether the size of the 13q deletion may also influence the clinical outcome remains to be elucidated. Patients and Methods: Probes for chromosome 13q (LSI-RB1, LSI-D13S319), 11q (LSI-ATM), 17p (LSI-p53) and chromosome 12 (CEP12) were utilized on nuclei collected at diagnosis from: i) a multi-institutional CLL cohort (342 del13q-only cases) and ii) a consecutive unselected single-institution cohort of 265 cases. RB1 deleted cases (delRB1) were defined as having at least 5% of deleted nuclei. Time to treatment (TTT) intervals, as well as Rai staging, IGHV mutational status, CD38 and ZAP70 expression, B2-microglobulin levels, all evaluated at diagnosis, were also available for all cases that entered the study. Genome wide DNA profile was performed in a pilot series of 90 CLL samples using Affymetrix GeneChip Human SNP6 arrays. Results: According to genome wide DNA analysis, delRB1 occurred in a proportion of del13q-only cases (36/90; 40%), always comprising the deleted region detected with the LSI-D13S319 probe (that covers the miR-15a/16-1 cluster and the DLEU2 gene) and characterized by a larger chromosome loss (median size 2.07 Mb vs. a median size of 0.86 Mb for the canonical del13S319). Maximally selected log-rank statistics identified the 70% of nuclei bearing del13S319 as the most appropriate cut-off value capable of separating del13q-only cases into two subgroups with different TTT distributions. Consistently, del13q-only cases with at least 70% of nuclei bearing del13S319 showed a significantly shorter TTT than del13q-only cases with less than 70% deleted nuclei (p=0.0001). Del13q-only cases were then divided in four subsets according to the percentage of nuclei bearing del13S319 with or without a concomitant delRB1: del13S319 70% (group 3), 64 cases; del13S319 >70% + delRB1 (group 4), 39 cases. The median TTT of group 1 (not reached) was significantly longer than the median TTT of group 2 (92 months, p=0.012), group 3 (68 months, p70% of cells (with or without delRB1, group C, 25 cases) or a normal karyotype (group D, 75 cases) had shorter median TTT intervals (ranging from 105 to 129 months, p Conclusion: In the context of del13q-only cases, different clinical outcomes were associated to the percentage of 13q14 deleted cells, as well as to the size of the 13q14 deletion, as detected by the LSI-RB1 probe. Moreover, the presence of delRB1 emerged as a feature capable of refining the prognostic assessment in the context of CLL cases with Disclosures: No relevant conflicts of interest to declare.

Purpose: B-cell chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease whose outcome can be foreseen by investigating the mutational status of immunoglobulin heavy chain variable (IGHV) genes. Moreover, a different prognosis was reported for CLL expressing specific IGHV genes in the context or not of stereotyped B-cell receptors. Here we investigated novel associations between usage of specific IGHV genes and clinical features in CLL. Experimental Design: Among 1,426 CLL-specific IG-rearrangements, stereotyped B-cell receptor clusters never utilized the IGHV3-23 gene. Given this notion, this study was aimed at characterizing the IGHV3-23 gene in CLL, and identifying the properties of IGHV3-23–expressing CLL. Results: IGHV3-23 was the second most frequently used (134 of 1,426) and usually mutated (M; 109 of 134) IGHV gene in our CLL series. In the vast majority of M IGHV3-23 sequences, the configuration of the 13 amino acids involved in superantigen recognition was consistent with superantigen binding. Clinically, M IGHV3-23 CLL had shorter time-to-treatment than other M non–IGHV3-23 CLL, and multivariate analyses selected IGHV3-23 gene usage, Rai staging, and chromosomal abnormalities as independent prognosticators for M CLL. Compared with M non–IGHV3-23 CLL, the gene expression profile of M IGHV3-23 CLL was deprived in genes, including the growth/tumor suppressor genes PDCD4, TIA1, and RASSF5, whose downregulation is under control of miR-15a and miR-16-1. Accordingly, relatively higher levels of miR-15a and miR-16-1 were found in M IGHV3-23 compared with M non–IGHV3-23 CLL. Conclusions: Altogether, expression of the IGHV3-23 gene characterizes a CLL subset with distinct clinical and biological features. Clin Cancer Res; 16(2); 620–8

Background ZAP-70 is an independent negative prognostic marker in chronic lymphocytic leukemia (CLL). Usually, its expression is investigated by flow cytometric protocols in which the percentage of ZAP-70 positive CLL cells is determined in respect to isotypic control (ISO-method) or residual ZAP-70 positive T cells (T-method). These methods, however, beside suffering of an inherent subjectivity in their application, may give discordant results in some cases. The aim of this study was to assess the prognostic significance of these methods in comparison with another in which ZAP-70 expression was evaluated as a Mean-Fluorescence-Intensity Ratio between gated T and CLL cells (T/B Ratio-method). Methods Cytometric files relative to ZAP-70 determination according to the three readouts were retrospectively reviewed on a cohort of 173 patients (test set), all with complete clinical and biological prognostic assessment and time-to-treatment (TTT) available. Findings were then validated in an independent cohort of 341 cases from a different institution (validation set). Results The optimal prognostic cut-offs for ZAP-70 expression were selected at 11% (ISO-method) or 20% of positive cells (T-method), as well as at 3.0 (T/B Ratio-method) in the test set; these cut-offs yielded 66, 60 and 73 ZAP-70+ cases, respectively. Univariate analyses resulted in a better separation of ZAP-70+ vs. ZAP-70- CLL patients utilizing the T/B Ratio, compared to T- or ISO-methods. In multivariate analyses which included the major clinical and biological prognostic markers for CLL, the prognostic impact of ZAP-70 appeared stronger when the T/B-Ratio method was applied. These findings were confirmed in the validation set, in which ZAP-70 expression, evaluated by the T- (cut-off = 20%) or T/B Ratio- (cut-off = 3.0) methods, yielded 180 or 127 ZAP-70+ cases, respectively. ZAP-70+ patients according to the T/B Ratio-method had shorter TTT, both if compared to ZAP-70- CLL, and to cases classified ZAP-70+ by the T-method only. Conclusions We suggest to evaluate ZAP-70 expression in routine settings using the T/B Ratio-method, given the operator and laboratory independent feature of this approach. We propose the 3.0 T/B Ratio value as optimal cut-off to discriminate ZAP-70+ (T/B Ratio less than 3.0) from ZAP-70- (T/B Ratio more/equal than 3.0) cases.

CD49d/α4-integrin is variably expressed in chronic lymphocytic leukemia (CLL). We evaluated its relevance as independent prognosticator for overall survival and time to treatment (TTT) in a series of 303 (232 for TTT) CLLs, in comparison with other biologic or clinical prognosticators (CD38, ZAP-70, immunoglobulin variable heavy chain (IGHV) gene status, cytogenetic abnormalities, soluble CD23, β2-microglobulin, Rai staging). Flow cytometric detection of CD49d was stable and reproducible, and the chosen cut-off (30% CLL cells) easily discriminated CD49dlow from CD49dhigh cases. CD49d, whose expression was strongly associated with that of CD38 (P < .001) and ZAP-70 (P < .001), or with IGHV mutations (P < .001), was independent prognosticator for overall survival along with IGHV mutational status (CD49d hazard ratio, HRCD49d = 3.52, P = .02; HRIGHV = 6.53, P < .001) or, if this parameter was omitted, with ZAP-70 (HRCD49d = 3.72, P = .002; HRZAP-70 = 3.32, P = .009). CD49d was also a prognosticator for TTT (HR = 1.74, P = .007) and refined the impact of all the other factors. Notably, a CD49dhigh phenotype, although not changing the outcome of good prognosis (ZAP-70low, mutated IGHV) CLL, was necessary to correctly prognosticate the shorter TTT of ZAP-70high (HR = 3.12; P = .023) or unmutated IGHV (HR = 2.95; P = .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.

BACKGROUND. Rituximab in sequential combination with fludarabine (Flu) allowed patients with B-cell chronic lymphocytic leukemia (B-CLL) to achieve higher remission rates and longer response duration. Based on their recent experience in indolent non-Hodgkin lymphomas, in this study, the authors attempted to demonstrate whether consolidation/maintenance therapy with rituximab could prolong the response duration in this patient population. METHODS. This Phase II study was based on a consolidation/maintenance therapy with rituximab for patients in complete remission (CR) or partial remission (PR) who were positive for minimal residual disease (MRD), as determined by flow cytometry. Seventy-five symptomatic, untreated patients with B-CLL received 6 monthly cycles of Flu (25 mg/m2 for 5 days) followed by 4 weekly doses of rituximab (375 mg/m2). Then, 28 patients who were positive for MRD were consolidated with 4 monthly cycles of rituximab (375 mg/m2) followed by 12 monthly low doses of rituximab (150 mg/m2). RESULTS. Based on National Cancer Institute criteria, 61 of 75 patients (81%) achieved a CR, 10 of 75 patients (13%) had a PR, and 4 of 75 patients (5%) had either no response or disease progression. MRD-positive patients in CR or PR who received consolidation therapy (n = 28 patients) had a significantly longer response duration (87% vs 32% at 5 years; P = .001) compared with a subset of patients who did not receive consolidation therapy (n = 18 patients). All patients experienced a long progression-free survival from the end of induction treatment (73% at 5 years). It was noteworthy that, within the subset of ZAP-70-positive patients, MRD-positive, consolidated patients (n = 12 patients) had a significantly longer response duration (69% vs 0% at 2.6 years; P = .007) compared with MRD-positive, unconsolidated patients (n = 11 patients). CONCLUSIONS. The addition of a consolidation and maintenance therapy withrituximab prolonged response duration significantly in patients with MRD-positive B-CLL. Cancer 2008. © 2007 American Cancer Society.

Abstract 3887 Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic monoclonal expansion of Disclosures: No relevant conflicts of interest to declare.

Abstract 2692 Cytogenetic aberrations are considered major prognostic indicators for predicting the survival of chronic lymphocytic leukemia (CLL) patients (pts) [Dohner et al, 2000]. Given the difficulties in obtaining abnormal metaphases in CLL, fluorescent in situ hybridization (FISH) with specific probes is generally used to detect the most frequent abnormalities. Interphase FISH (I-FISH) is a rapid and sensitive technique for analysis of chromosome aberrations in CLL, but the limited number of DNA probes available to screen B-CLL results in a high number of cases presenting normal cytogenetics. Moreover, deletion 13q14 on FISH analysis which is the most common cytogenetic abnormality in CLL is a favourable prognostic biomarker when detected as a sole abnormality, but a higher percentage of 13q- nuclei was found to be associated with significantly shorter time to treatment (P Disclosures: No relevant conflicts of interest to declare.

Abstract 3574 Chronic lymphocytic leukemia (B-CLL) is a very heterogeneous disease with some patients experiencing rapid disease progression and others living for years without requiring treatment and therefore it is mandatory to find new prognostic markers. CD69 overexpression which resembles B cells at an earlier and greater state of activation (Damle, 2002 and 2007) and induces increased proliferation and survival of leukemic B-lymphocytes, may reflect an aggressive and progressive clinical outcome. The primary endpoints of our research were: 1) to determine progression free survival (PFS) and overall survival (OS) upon CD69 in univariate analysis; 2) to correlate CD69 with other clinical or biological prognostic factors such as age, Rai stages, lymphocyte doubling time, beta-2 microglobulin, CD38, CD49d, ZAP-70, cytogenetics by FISH and IgVH status and finally, 3) to confirm CD69 as an independent prognostic factor. We investigated 417 patients (pts), median age 66 years (range 33–89), 239 males and 178 females. With regard to modified Rai stages, 127 pts had a low stage, 272 an intermediate stage and 18 a high stage. CD69 was determined by multicolor flow cytometry, fixing the cut-off value at 30%. CD69+ pts were 111/417 (26.6%). CD69 12 months (260/337; P=0.0006), beta-2 microglobulin or Disclosures: No relevant conflicts of interest to declare.

The unbalance between anti-apoptotic (bcl-2) and pro-apoptotic mitochondrial proteins (bax) represents a critical mechanism explaining the high rate of resistance and treatment failure in acute myeloid leukemia (AML) (Del Poeta, 2003). Moreover, the availability of novel and effective pro-apoptotic compounds such as bortezomib (Attar, 2008) and gemtuzumab (Del Poeta, 2008) moved us to widely investigate the impact of mitochondrial proteins such as bcl-2, bax and 7A6 on AML prognosis. 7A6 antigen has been found to be exposed on the mitochondrial membrane during the early stages of apoptosis and is recognized by monoclonal antibody APO2.7. For this purpose, a large series of 420 non M3 AML patients (pts), median age 63 years, treated with intensive chemotherapy regimens, were tested. The aims of our research were: to correlate bax/bcl-2 and APO2.7/bcl-2 ratios, as measures of mitochondrial apoptosis, with other well-known prognostic factors such as age, cytogenetics, FLT3-ITD, P-glycoprotein; to evaluate whether mitochondrial apoptosis was able to dissect normal karyotype AML with regard to prognosis, and finally to confirm that mitochondrial apoptosis is an independent prognostic factor. Bcl-2, bax and 7A6 proteins were assessed by multicolor flow cytometry and bax/bcl-2 or APO2.7/bcl-2 ratios were obtained by dividing mean fluorescence intensity (MFI) of bax/MFI bcl-2 or MFI of APO2.7/MFI bcl-2. The thresholds of positivity were set at the median values >0.35 and >0.60, respectively. Fifty-nine percent of pts were bax/bcl-2 positive and 50% were APO2.7/bcl-2 positive. There were strict correlations between higher bax/bcl-2 or higher APO2.7/bcl-2 and FAB M2 or M4/M5 AML subgroups (p0.35 or APO2.7/bcl-2>0.60 and CD34 negativity (p

Nucleophosmin gene mutations (NPM1-Mt) are the hallmark of a large adult acute myeloid leukemia (AML) subgroup with normal karyotype and interact with p53 and its regulatory molecules (Arf, Hdm2/Mdm2), thus lowering cell proliferation and increasing apoptosis (Falini, 2007). Moreover, AML pts show co-existing NPM1-Mt and internal tandem duplications of FLT3 (FLT3-ITD) which increase potential for cell proliferation. Furthermore, genes and proteins involved in apoptosis, such as bcl-2 and bax, have been demonstrated to be relevant in response to treatment and outcome (Del Poeta, 2003). Therefore, we analysed NPM1-Mt, FLT3-ITD and apoptosis proteins (bcl-2 and bax) in 222 pts, affected by de novo non-M3 AML, median age 60 years, treated with intensive chemotherapy regimens according to GIMEMA-EORTC protocols. The aims of our study were: to correlate NPM1-Mt or FLT3-ITD with bax/bcl-2 ratio levels, as a measure of spontaneous apoptosis; to assess the independent prognostic significance of NPM1-Mt and FLT3-ITD. Bcl-2 and bax proteins were determined by multicolor flow cytometry and bax/bcl-2 ratio was obtained by dividing mean fluorescence intensity (MFI) of bax/MFI bcl-2. The threshold of positivity was set at the median value >0.35 (range 0.01–9.1). NPM1 mutations and FLT3-ITD were detected by multiplex PCR and capillary gel electrophoresis. One hundred-twenty-one/222 (54.5%) pts were bax/bcl-2 ratio positive, 54/222 (24.3%) were NPM1-Mt and 52/222 (23.4%) presented FLT3-ITD; 17/222 (7.6%) pts carried both FLT3-ITD and NPM1-Mt. There was a strong correlation between higher WBC counts (>100x109/L) and FLT3-ITD (P0.35) and NPM1-Mt without FLT3-ITD were significantly associated (30/37; P=0.0001), demonstrating that NPM1-Mt alone express high amount of spontaneous apoptosis. Moreover, NPM1-Mt cases were significantly related to FAB M4 or M5 AML (P=0.03). A normal karyotype was found in 37/45 (82%) NPM1-Mt pts (P=0.00001) and almost all NPM1-Mt cases were CD34 negative (47/54; P

CD69 membrane protein is expressed early in the activation of lymphoid cells and is inducible by B-cells through cross-linking of surface immunoglobulins. Moreover, B-CLL cells exhibit features of activated and of antigen-experienced B lymphocytes overexpressing activation markers such as CD23, CD25, CD69 and CD71 (Damle, 2002). Furthermore, we have demonstrated that ZAP-70+ CLL subgroup shows a rapid disease progression and an inferior overall survival (Del Principe, 2006). The generation of antimurine CD69 monoclonal antibodies able to induce down-modulation or partial depletion of CD69+ cells (Sancho, 2006), prompted us to evaluate the real impact of CD69 expression on B-CLL prognosis. The primary aims of our study were: to determine progression-free survival (PFS) and overall survival (OS) upon CD69 expression; whether CD69 could predict varied outcome within ZAP-70+ and ZAP-70 negative subgroups; and finally whether CD69 was an independent prognostic factor. Therefore, we investigated 247 pts, median age 65 years, 131 males and 116 females. With regard to modified Rai stages, 69 pts had a low stage, 167 an intermediate stage and 11 a high stage. CD69 was determined by multicolor flow cytometry fixing a cut-off value of 30%. CD69+ B-CLL pts were 73/247 (30%). CD69 >30% was significantly associated with an intermediate/high Rai stage (p=0.001), lymphocyte doubling time (LDT) 2.2 mg/dl (p=0.002). Lower CD69 expression and IgVH mutated status (>2%) were significantly correlated (73/92; p=0.0003). Furthermore, we found significant associations between lower CD69 and lower CD38 (137/184; p=0.02) or lower ZAP-70 (110/144; p=0.01). Lower levels of soluble CD23 (sCD23) were strongly associated with lower CD69 (127/158; p Figure Figure

Clinical studies of monoclonal antibodies combined with chemotherapy have attained more complete responses and longer duration of responses in CLL because this approach reduces disease burden to levels detectable only by immunological or molecular methods. Recent literature data indicate that VH genes, CD38, ZAP-70 protein, and cytogenetic status have a major impact on CLL prognosis. We performed a phase II study that added rituximab sequentially to fludarabine (Flu) as initial therapy for symptomatic, untreated CLL in order to evaluate both the clinical response and outcome. Remission status was also assessed by a multiparametric flow cytometric method based on the detection of CD19+CD5+CD79b− residual B-CLL lymphocytes. VH mutational status, CD38 and cytogenetics were obtained in all pts before treatment. ZAP-70 protein was performed by flow cytometry using an anti-ZAP-70 Alexa Fluor 488 conjugated antibody. Seventy-five CLL pts, median age 60 years, 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 after completion of Flu therapy. According to modified Rai stages, 6 pts had a low stage, 66 an intermediate stage and 3 a high stage. Based on NCI criteria, 61/75 (81%) pts achieved a complete remission (CR), 10/75 (13%) a partial remission (PR) and 4/75 (5%) no response. Three pts presented grade 3 (WHO) infective lung toxicity and 1 patient acute fatal B hepatitis. Hematologic toxicity included neutropenia (grade 3 and/or 4 in 38 pts) and thrombocytopenia (grade 3 and/or 4 in 4 pts). Twenty eight pts, either with CD5+CD19+CD79b− (MRD) bone marrow (BM) cells >1% (n=17 pts) or with CD19+CD5+CD79b− (MRD) peripheral blood lymphocytes (PBL) >1000/microl (n=11 pts) within six months after completion of the induction treatment, underwent consolidation/maintenance therapy with four monthly cycles of rituximab at 375 mg/m2 followed by twelve monthly doses of rituximab at 150 mg/m2. The median follow-up duration was 37 months. Noteworthy, all pts experienced a long progression-free survival (PFS) from treatment (67% at 5 years). Nevertheless, CLL pts that underwent consolidation therapy (n=28) showed a significant longer duration of response (75% vs 27% at 5 years, P=0.002) in comparison with the subset of not consolidated and BM or PBL MRD positive (n=18) CLL pts. Interestingly, BM and PBL MRD negative pts (n=26) showed a duration of response similar to that of the consolidated pts. Moreover, a significant shorter PFS was observed within CD38+ pts (29% vs 79% at 5 years, P=0.005), unmutated pts (0% vs 92% at 2 years, P=0.001), ZAP-70+ pts (29% vs 92% at 5 years; P=0.00003) and within the “poor risk” [trisomy 12 or del 11q or del 17p] cytogenetic subset (0% vs 79% at 2 years, P=0.03). Interestingly, within the ZAP-70+ subset (n=35), the consolidated pts (n=12) showed a longer duration of response (68% vs 0% at 2.6 years, P=0.007) in comparison with the unconsolidated pts (n=11). Therefore, the addition of a consolidation/maintenance therapy with rituximab prolongs significantly the duration of response allowing a better outcome. Moreover, rituximab improves significantly PFS of pts notoriously at worse prognosis, such as ZAP-70+ B-CLL.

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.

Abstract 2364 Poster Board II-341 The treatment goal of B-cell chronic lymphocytic leukemia (B-CLL) has already shifted from symptom palliation to the attainment of maximal disease control combining purine analogs with monoclonal antibodies. This immunochemotherapeutic approach resulted both in more complete responses (CR) and longer response duration, often remaining only a minimal residual disease (MRD) detectable by flow cytometry. We treated in first line 120 B-CLL symptomatic patients (pts), median age 62 years, with six monthly courses of intravenous or oral fludarabine at conventional doses and then, after a median time of 31 days, with four weekly doses (375 mg/sqm) of rituximab (rtx). Fourteen pts had a low Rai stage, 103 an intermediate stage and 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 unfavorable cytogenetics (trisomy 12 or del11q or del17p). Based on NCI criteria, 92/120 (77%) pts achieved a CR, 24/120 (20%) a partial remission (PR) and 4/120 (3%) no response or progression. Ten pts underwent grade 3 (WHO) infective lung toxicity, 1 patient acute fatal B hepatitis and 2 pts progressed towards Richter's syndrome. Hematologic toxicity included mainly neutropenia (grade 3 and/or 4 in 56 pts) and thrombocytopenia (grade 3 and/or 4 in 8 pts). Fifty-four pts either in CR with B-CLL bone marrow cells >1% (MRD+, n=16 pts) or in CR MRD negative, but with B-CLL peripheral cells going up >1000/microl within 1 year after induction (n=22 pts) or in PR (n=16 pts), underwent consolidation and maintenance therapy with four monthly cycles of rtx at 375 mg/sqm followed by twelve monthly low doses of rtx (150 mg/sqm). The median follow-up duration was 50 months. All treated pts experienced a long progression-free survival from the end of induction treatment (40% at 9 years). On the other hand, global overall survival (OS) was 54% at 10 years. Nevertheless, CLL pts undergoing consolidation and maintenance therapy (n=54) showed a longer response duration vs MRD+ not consolidated pts (n=16; 75% vs 9% at 4 years; P1 year) pts (n=43) showed a very long response duration (79% at 6 years, Figure). Moreover, OS was shorter in MRD+ not consolidated pts (0% vs 79% at 15 years; P=0.0007). Noteworthy, within the high risk subset (n=48), consolidated pts (n=17) showed a longer response duration (56% vs 0% at 2.5 years, P=0.003) vs MRD+ not consolidated pts (n=11). Therefore, rituximab consolidation and maintenance immunotherapy improve response duration in B-CLL, thus potentially increasing OS, also within the high risk subset. Disclosures: No relevant conflicts of interest to declare.

Abstract 1256 Poster Board I-278 Introduction In the last years, the B cell receptor (BCR) has become a key molecule in chronic lymphocytic leukemia (CLL), given the correlation between mutational status of immunoglobulin heavy chain variable (IGHV) genes and disease prognosis. Recently, a fraction of CLL has been shown to preferentially express specific IGHV genes, often in a non-random combination with homologous heavy chain complementarity-determining region-3 (HCDR3) and peculiar light chains. Some of these stereotyped BCR mark CLL subsets with peculiar clinical behavior regardless of IGHV mutations. These data suggest a role for BCR in defining the clinical and biological features of CLL, also beyond the mutational status of IGHV genes. Patients and Methods A HCDR3-driven clustering of 1,426 IG sequences (1,398 patients) was performed using ClustalX(1.83). Time to treatment (TTT) intervals, Rai staging, IGHV mutational status, CD38, ZAP-70, and karyotype abnormalities evaluated by FISH were available for 617 patients. Gene expression profiling (GEP) and quantitative real-time PCR experiments (QRT-PCR) were performed on purified CLL cells. Results IGHV3-23 was totally absent in 71 identified stereotyped clusters despite being the second most frequently used IGHV gene, such distribution was significantly skewed (p Conclusion Expression of IGHV3-23 marks a subset of M CLL with a worse prognosis; such a peculiar clinical behavior may be related to superantigen stimulation combined with down-regulation of specific growth/tumor suppressor genes and up-regulation of miR-15a and miR-16-1. Disclosures No relevant conflicts of interest to declare.

Abstract 1257 Poster Board I-279 Introduction Expression of the T-cells constitutive ZAP-70 protein by CLL cells has been the focus of many studies in the last few years, due to its ability to stratify indolent and more aggressive disease subsets. Although the strength of ZAP-70 as independent negative prognosticator was demonstrated by several studies, concerns about its use derive from a lack of multicentric standardization of flow-cytometric protocols. Analyses in clinical settings are usually performed according to two methods, respectively evaluating the percentage of CLL cells expressing ZAP-70 compared to isotypic control (ISO-method), or to autologous T-cells (T-method). Of note, while the two methods yield concordant results in most patients, a fraction of cases may be discordant as for evaluation of ZAP-70 positivity. Moreover, either method suffers of an operator dependent variability, mainly related to subjectivity in cursor placement to determine the percentage of ZAP-70+ cells. The aim of this study was to compare the ISO- and T-methods with the expression of ZAP-70 evaluated as Mean-Fluorescence-Intensity (MFI) Ratio between gated T and CLL cells (T/B-Ratio-method), and to assess the prognostic significance of the three approaches. Methods Cytometric files relative to ZAP-70 determination according to the three readouts were retrospectively reviewed with BD-DiVa software on a cohort of 173 patients (test set), all with complete clinical and biological prognostic assessment and time-to-treatment (TTT) available. Findings were then validated in an independent cohort of 341 cases from a different institution (validation set). Notably, in the two cohorts, ZAP-70 assessment was accomplished using two different antibody combinations and instrumentations for data acquisition and analysis. Results ZAP-70 expression was reviewed in the test set by applying the ISO- and T-methods. Utilizing respectively 11% for ISO-method and 20% of ZAP-70+ cells for T-method, both selected as optimal cut-offs with prognostic relevance by ROC-analysis and maximally selected log-rank statistics, 66 (ISO-method) and 60 (T-method) ZAP-70+ cases were defined. By applying the T/B-Ratio-method, a value of 3.0 was identified as the optimal prognostic cut-point. According to this value, 73 ZAP-70+ cases (i.e. with T/B-Ratio Conclusions We suggest to evaluate ZAP-70 expression in routine settings using the T/B-Ratio-method, given the operator and laboratory independent feature of this approach. We propose the 3.0 T/B-Ratio value as optimal cut-off to discriminate ZAP-70+ (T/B-Ratio less than 3.0) from ZAP-70− (T/B-Ratio more/equal than 3.0) cases Disclosures No relevant conflicts of interest to declare.