IgVH Status Research Articles

The Amount of Spontaneous Apoptosis Is An Independent Strong Disease Progression Indicator in B-Cell Chronic Lymphocytic Leukemia (B-CLL)

Abstract 1252▪▪This icon denotes an abstract that is clinically relevant.Poster Board I-274Bcl-2 levels has emerged as the most important protein in predicting survival between 11 proteins in CLL cells that are implicated in the control of apoptosis, proliferation and differentiation (Faderl, 2002). In fact, malignant cells are arrested in the G0/early G1 phase of the cell cycle, and inhibition of spontaneous apoptosis with upregulation of the anti-apoptotic protein bcl-2 may define clinical prognosis. The today availability both of bcl-2 antisense oligonucleotides and of novel pro-apoptotic BH3 peptidomimetic prompted us to evaluate the real impact of apoptosis pathways on B-CLL prognosis. The primary aims of our study were: 1) to determine progression-free survival (PFS) and overall survival (OS) upon bax/bcl-2 ratio, 2) whether bax/bcl-2 ratio and ZAP-70 show additive prognostic impact and finally 3) whether bax/bcl-2 is an independent prognostic factor. Therefore we investigated 356 patients (pts), median age 65 years (range 37-89), 164 males and 192 females. With regard to modified Rai stages, 115 pts had a low stage, 229 an intermediate stage and 12 a high stage. Bax/bcl-2 ratio was determined by flow cytometry, dividing mean fluorescence intensity (MFI) of bax by MFI of bcl-2 on CD19+CD5+ B-CLL cells. We obtained the bax/bcl-2 ratio and the threshold was set at the median value >1.45 (range 0.27-13.6). ZAP-70 was quantified by multicolor flow cytometry and the cut-off was fixed at >20%. Two hundred-three pts were bax/bcl-2 ratio positive (203/356; 57%). Higher bax/bcl-2 ratio was significantly associated with low Rai stage (80/115; P=0.003), lymphocyte doubling time >12 months (183/299; P=0.0003), beta-2 microglobulin (B-2M) <2.2 mg/dl (129/200; P=0.001) and soluble CD23 (sCD23) <70 U/ml (141/228; P=0.005). Moreover, there were significant correlations between higher bax/bcl-2 ratio and IgVH gene mutated status (216 cases, 96/154; P=0.015) or low risk (normal or 13q-) FISH cytogenetics (255 cases, 109/187; P=0.011). Noteworthy, a very strict association was found between higher bax/bcl-2 ratio and lower ZAP-70 (147/220; P<0.00001), suggesting that low ZAP-70 expression is characterized by high apoptosis levels. With regard to clinical outcome, significant shorter PFS and OS were observed in pts with lower bax/bcl-2 ratio (10% vs 60% at 14 years; P<0.00001 and 51% vs 74% at 16 years; P=0.005, respectively) as well as in ZAP-70+ pts (5% vs 57% at 12 years; P<0.00001 and 30% vs 85% at 16 years, respectively). To further explore the prognostic impact of bax/bcl-2 ratio, we investigated its expression associated with ZAP-70 protein. As a matter of fact, higher bax/bcl-2 ratio plus ZAP-70 <20% identified the pts subset with the longest PFS (70% vs 2% at 12 years; P <0.00001, Figure) and OS (92% vs 33%; P<0.00001). The discordant pts presented an intermediate outcome (Figure). In multivariate analysis of PFS, in which cytogenetics, IgVH status, ZAP-70, CD38, bax/bcl-2, sCD23 entered, bax/bcl-2 (P=0.02), cytogenetics (P=0.02) and ZAP-70 (P=0.04), resulted to be independent prognostic factors. In conclusion, our apoptotic index (bax/bcl-2 ratio), performed by flow cytometry, was very useful to identify pts at different progression rate and since the ZAP-70 negative subset represents a large and heterogeneous B-CLL population with a variable progression, other biological factors, such as the amount of apoptosis, have to be added in order both to identify early and treat timely progressive pts. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Positive Direct Antiglobulin Test (DAT) in Chronic Lymphocytic Leukemia (CLL) Patients: Correlation with Prognostic Factors.

Abstract 2351Poster Board II-328 Background:although the clinical course of patients with B-cell chronic lymphocytic leukemia (CLL) is often complicated by autoimmune phenomena few data are available concerning the prevalence of autoimmune diseases in this lymphoprolipherative disorder as well as their clinical outcome. Autoimmune hemolytic anemia (AIHA) is the most common form, occurring in 10% to 25% of patients during CLL course. DAT may be positive at some time during the course of the disease in up to 35% of cases, but overt AIHA occurs less frequently. Although AIHA may occur in asymptomatic untreated CLL, it is more common in patients with advanced-stage disease. So far it is unknown if also the biological characteristics of CLL may have any influence on the risk of developing autoimmune phenomena. Recently, an exceedingly high prevalence of un-mutated IgVH, with an excess of VH1 and VH3 families has been described in patients with autoimmune thrombocytopenia and DAT status has been correlated with clinical and laboratory prognostic factors in CLL Chinese population. Aim:the current retrospective study was conducted to investigate the relation between incidence of positive DAT test and biological features of CLL patients. Patients and methods:in our institution 158 patients with CLL were studied with DAT at some time during the disease course. Characteristics of patients are summarized in the table. DAT resulted positive in 42 patients (26.6%), overt AIHA occurred in 28,6% of them. Univariate analysis by Fisher's exact test was performed to establish the association between DAT result and AIHA development with the following variables: age, sex, Binet stage, LDH and beta2 microglobulin (B2m) at diagnosis, IgVH status, VH families, ZAP70, CD38 and FISH analysis.Positive DATNegative DATN° of patients42116Median age67 y67yM/F %67/3362/38Binet stage%A5471B3323C136High LDH %2611High B2m %6943IgVH un-mutated%7648ZAP70+%7648CD38+%3242FISH %17p1335336q2118711q223523 Results:the incidence of positive DAT was significantly increased in presence of higher B2m (p 0.011) and highly correlated with IgVH un-mutated status (p 0.014). No prevalence or correlation for the IgVH families was recorded between DAT positive and negative patients. In the subgroup of patients with poor risk FISH chromosome aberration (deletion in 17p13, 11q22 or 6q21) we found a trend (p 0.064) for higher frequency of positive DAT compared to good risk (deletion in 13q14 or trisomy 12). A trend for increased positivity of DAT has been also found in patients with positive ZAP70 (p 0.083) and increased LDH value at diagnosis (p 0.051). Among DAT-positive patients none of biological and clinical characteristics listened above showed a significant correlation with the risk to develop AIHA. Conclusions:in our series DAT positivity correlated with poor risk biological features, however no difference was detected in terms of OS between patients with positive or negative test. Furthermore, we could speculate that patients with un-mutated IgVH status are characterized by a peculiar antibody reactivity, resulting in a major failure controlling the emergence of auto-antibodies from normal B cells.Although the results of this study warrant further confirmation from larger series, the documented association of poor biological features and DAT positivity in CLL patients, suggests that this laboratory test may improve prognostic evaluation at diagnosis. Disclosures:No relevant conflicts of interest to declare.

Family-Associated Monoclonal B Lymphocytosis Shows Differences From CLL That Suggest An Indolent Biology.

Abstract 1241Poster Board I-263 Background and SignificanceMonoclonal B lymphocytosis (MBL) is a hematologic syndrome characterized by accumulations of clonal B lymphocytes in the peripheral blood. Most MBL have a CLL-like immunophenotype, though other, less common phenotypes are observed. Although some MBL, particularly those with MBL cell counts >1.9 × 109 / L, progress to CLL, “low count” MBL (<0.2 × 109 MBL / μL) have little potential to progress to MBL and may have different biologic characteristics from MBL with lymphocytosis or Rai Stage 0 CLL. Our hypothesis was that detailed characterization of family-associated MBL may also provide biologic insights into the pathogenesis of CLL. MethodsIndividuals with MBL were identified by flow cytometry screening of fresh and cyropreserved peripheral blood from unaffected members of CLL kindreds ascertained by Genetic Epidemiology of CLL Consortium (GEC) sites. We defined MBL as populations of CD5+, CD19+, CD20lo, CD23+ B cells that comprised at least 0.02% of the PBMC and did not exceed 5.0 × 109 MBL cells / L. Flow cytometry was used to determine the surface immunophenotype including prognostic parameters of CD38, intracellular ZAP-70, immunoglobulin isotype, CD49d, and the ratio of CD69:71. mRNA and genomic DNA from single MBL cells isolated by flow cytometric sorting were analyzed using PCR to determine immunoglobulin heavy and light chain sequences (IGVH status). MBL cells were sorted in bulk for FISH determination of genetic loci associated with clinical CLL. ResultsFifty-four unaffected family members were found to have MBL, of these 48 (89%) showed a typical CLL immunophenotype (CLL-like MBL). We observed significant variability in the size of the MBL clone as a percentage of the CD19+ B cell compartment (mean 30%, median 16%; range 2% - 97%). CD38 positive (defined as CD38 surface expression in ≥ 30% of MBL cells) was observed in 6 of 38 (16%) subjects tested. ZAP-70 positive (defined as intracellular expression in ≥ 20% of MBL cells) was expressed in 5 of 28 (18%) participants. CD49d positive (defined as surface expression in ≥ 45% of MBL cells) was observed in 4 of 17 (24%) subjects. The ratio of surface expression of CD69:CD71, a measure of cellular activation that also correlates with an unmutated IGVH, was ≥2.0 in 3 of 34 (9%) subjects tested. Among 36 subjects tested, 9 (25%) MBL expressed both surface IgD and IgM (defined as surface expression ≥ 40% for both IgM and IgD), 12 (33%) expressed IgD only, 2 (6%) expressed IgM only, and 13 did not express IgD or IgM (36%). Analysis of IGVH status has been completed in 10 individuals. Both immunoglobulin heavy chain variable (IGVH) region mutated (n = 14) and unmutated (n = 5) sequences were observed. Six of 10 individuals had 2 or more unrelated MBL clones (range 2 - 4), including two individuals with both unmutated and mutated clones. Among the 19 MBL clones identified in these 8 subjects, VH3 or VH4 rearrangements were observed in all MBL clones. The most commonly rearranged IGVH genes were 3-07 (3 MBL clones), 3-15 (3), and 4-34 (3). No VH1 family gene rearrangements were observed. MBL cells were bulk sorted for FISH from 14 subjects. Mono or biallelic deletion of 13q14.3 was observed in 9 subjects, 4 were normal, and one showed trisomy 12. ConclusionsOur data affirms that CLL-like MBL are commonly observed among the unaffected family members from CLL kindreds. We found that family associated MBL clones (most of which were small clones) express ZAP-70, CD38, and CD49d at an apparently lower frequency than observed in CLL. Unlike in CLL, the surface immunoglobulin isotype showed co-expression of IgM and IgD in only one third of cases. Moreover, small MBL clones are commonly oligoclonal and predominantly express mutated IGVH genes with an IGVH usage that also appears different from CLL. Taken together, these findings suggest that small MBL clones, though phenotypically similar to CLL, have important biologic differences from CLL that may explain the limited potential of these clones to progress to CLL. The clinical outcome of these MBL clones in relation to our baseline prognostic characterization will be of interest. In addition the further investigation of family associated MBL is being conducted and may clarify the genetics and immunobiology of familial CLL. DisclosuresNo relevant conflicts of interest to declare.

ZAP-70 Expression Assessed by Immunohistochemistry Correlates with Time to First Treatment in Patients with Chronic Lymphocytic Leukemia.

Abstract 4686 BackgroundZAP-70 expression is a prognostic indicator in chronic lymphocytic leukemia (CLL). Initial studies measured intracellular ZAP-70 expression by flow cytometry and defined a cut-off of 20% as predictive of time from diagnosis to initial therapy and overall survival. However, due to technical challenges, standardization of methods for evaluation of ZAP-70 expression has been difficult. This has resulted in a limitation of applicability for a potentially useful clinical test. Immunohistochemistry (IHC) is easy to perform and interpret, and therefore may be a more reliable and accurate means for assessing ZAP-70 expression. We investigated the potential prognostic implications of ZAP-70 expression assessed by IHC. MethodsWe searched our CLL database for all patients seen at Weill Cornell Medical College between 2007 and 2009 with an identifiable date of diagnosis, date of first therapy, IgVH mutation status, and ZAP-70 status. All patients gave informed consent to participate in the research database. Paraffin-embedded cell blocks were prepared from formalin-fixed peripheral blood mononuclear cells and evaluated by IHC for PAX5, CD3, and ZAP-70. The entire cell clot was reviewed. If more than 20% of cells faintly expressed ZAP-70, the ZAP-70 status was considered to be positive. Patients had IgVH mutations status determined by a commercial laboratory with <98% sequence homology used as a cut-off. Time from diagnosis to first treatment (TT) was estimated by the Kaplan-Meier method; comparisons were performed by log-rank test. Pearson's pairwise correlation coefficient was used to evaluate the relationship between ZAP-70 and IgVH. ResultsOne-hundred thirty-seven patients with date of diagnosis and date of first treatment were identified in the database. Sixty-six patients have been treated to date. ZAP-70 status was available for 106 patients, 62 were positive, 44 were negative. In a subset of 20 patients, two blinded pathologists reviewed IHC and found perfect agreement on ZAP-70 status. IgVH mutation status was available for 86 patients, 34 were unmutated, and 52 were mutated. Eighty-two patients had both ZAP-70 status and IgVH mutation status available. There was good correlation between ZAP-70 and IgVH status (r=0.36, p=0.0008). Seventy-five percent of those with unmutated IgVH were ZAP-70 positive. Sixty-one percent of patients with mutated IgVH were ZAP-70 negative. However, only 57% of ZAP-70 positive patients were IgVH unmutated. The predicted median TT was 54 months for the whole cohort (range 0-376+ months, 95% CI 45-96 months). The median TT was 27 months (range 0-112 months) for ZAP-70-positive patients compared to 96 months (range 0-376+ months) for ZAP-70-negative patients (p=0.002). The median TT was 32 months (range 0-112 months) for IgVH unmutated patients compared to 88 months (range 0-376+ months) for IgVH mutated patients (p=0.01). ConclusionsZAP-70 as measured by IHC was feasible in a large cohort of patients and ZAP-70 status correlated with time to first treatment. These data suggest that ZAP-70 assessment by IHC may be a reliable prognostic tool in CLL and should be evaluated in larger, multicenter clinical studies. Disclosures:No relevant conflicts of interest to declare.

Detail Characterization of 22q11 Deletion and Mir-650 Role in CLL Patients.

Abstract 1231Poster Board I-253 BackgroundChromosomal abnormalities are present in the majority of CLL cases (>80%) and recently, a novel deletion of 22q11 was described in CLL patients. This locus encodes lambda immunoglobulin light chain (IgL) subgenes and several protein-coding genes, which distinguishes IgL from kappa Ig or heavy Ig locus. Gunn et al. (2008) previously characterized the minimally deleted region of 22q11 in CLL as 0,34Mb (containing PRAME, ZNF280A, ZNF280B, GGTLC2) and described the PRAME as a candidate gene with prognostic impact in CLL. Interestingly, the IgL variable segment (V2-8) located at 22q11 includes an annotated miR gene miR-650. According to the performed BLAST search also other subgenes of V2 family include homologues for miR-650, which overlay the leader exon of the V2 subgenes. This could be of particular importance because the expression of miRNAs was previously shown to be associated with known prognostic markers in CLL: 13q deletion, IgVH status (Calin, 2002, 2005) and p53 deletion/mutation (Mraz, 2009). Moreover, miRNA pathways are involved in the regulation of immune system functions including IgG production and V(D)J recombination (Vigorito, 2007; Koralov, 2008). AimWe analyzed genomes of 40 CLL patients using array-CGH (CGH Microarray 4×44 K, Agilent) and FISH (del13q14, del17p13, del11q23, +12), and in particular focused on detailed characterization of 22q11 deletion. An expression analyses (RT-qPCR, TaqMan miRNA Assay, ABI) was performed for protein-coding genes and miR-650 located at 22q11. ResultsUsing array-CGH additional aberrations to FISH were found in the majority of samples and among them, the deletion of 22q11 locus was observed most often (ranging in size up to ∼ 0,77 Mb). Altogether, 40% of patients (16/40) had detectable deletion in this locus and according to our finding, all observed deletions were a consequence of a VJ rearrangement event, which lead to an elimination of specific genes located between VJ elements. These rearrangements were characterized in detail by PCR detection (BIOMED-2 protocol) and sequencing. In our cohort, deletions of 22q11 containing PRAME gene constituted only ∼44% (n=7) and 56% patients (n=9) harbored smaller deletions not involving PRAME. To analyze the gene expression, we performed a Real-Time PCR analyses for previously mentioned protein-coding genes and miR-650. The Real-Time PCR assay for miR-650 reported higher expression of miR-650 in CLL cells utilizing V2-8, V2-5, V2-14, V2-23 subgenes for IgL (n=5) compared to samples utilizing different V lambda family or expressing kappa Ig (n=36). It remains to be elucidated how and whether all the V2 subgenes give rise to a mature and functional miRNA. The overlap between miR-650 and the leader exon of IgL gene could provide a unique mechanism for coordinated IgL and miRNA expression. To assess the function of miR-650 we performed a microarray (Human OneArray, Phalanx) expression profiling 24/48 hours post transfection of CLL cells with a short RNA mimicing miR-650 (miRIDIAN Mimic, Dharmacon). The transfection led to a significant down-regulation of dozens of mRNAs that could be considered as potential targets for miR-650. Interestingly, the predicted targets for miR-650 include early B cell factor (EBF3; target with highest total score – TargetScan) and VPREB1 (pre B lymphocyte gene 1) whose expression is important for immature phenotype of pre-B lymphocytes prior to light chain recombination. The coupled activation of miR-650 and lambda Ig expression following productive rearrangement of IgL locus could potentially affect the expression of VPREB1/EBF3. ConclusionDeletion of 22q11 is frequently detected in CLL patients and we suggest that it is caused by physiological process of lambda immunoglobulin variable locus rearrangement. This leads to the elimination of protein-coding genes located between VJ elements. Interestingly, the V2 family of variable segments for lambda light chain at 22q11 includes an annotated miR gene miR-650 and its homologues. The productive lambda locus rearrangement utilizing V2 family could be also coupled with activation of miR-650 expression with consequent relevance for B cell biology. This work is in progress and in further analyses we expect complex characterization of miR-650 functions in B cells and CLL. Supported by IGA MZ CR NR9293-3/2007 and IGA MZ CR NS10439-3/2009. DisclosuresMayer:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy; Fresenius: Consultancy; Roche: Research Funding; Pfizer: Research Funding.

Distribution of Genetic and Molecular Prognostic Markers in Young Patients with Chronic Lymphocytic Leukemia (CLL) in Different Phases of the Disease.

Abstract 4373In chronic lymphocytic leukemia (CLL), the distribution and prognostic impact of genetic and molecular markers have been mostly investigated in retrospective studies including patients in different phases of the disease. From November 2002 to May 2009, we prospectively assessed the clinical and biologic features of 219 young CLL patients ('65 years), distributed in three different cohorts: 124 cases at diagnosis, of which 99 in Binet stage A (group 1), 68 at first progression (group 2), 27 at progression after one or more therapies (group 3). Median age was 52 years for group 1, 57 years for group 2 and 61 years for group 3. Advanced disease defined as Rai stage III/IV was present in 6%, 19% and 48% patients and as Binet stages B/C in 19%, 68% and 78% patients in the 3 cohorts, respectively. The proportion of unmutated IgVH cases progressively and significantly increased, being 36% in group 1, 48.5% in group 2 and 75% in group 3 (p 0.001). The same trend was found for CD38 (p 0.01). The incidence of ZAP-70 expression and p53 mutations showed a trend towards a progressive increase among the three groups (41%, 55% and 56.5% for ZAP-70 and 4%, 7% and 12.5% for p53 mutations), without however reaching significance. The incidence of del(17p) ≥20% raised progressively from group 1, to groups 2 and 3 (2%, 4.5% and 18.5%, respectively; p 0.001). The incidence of del(11q) increased from group 1 to 2 (10% and 16%; p 0.18), though it was not higher in group 3 (7.5%). Focusing the comparison to groups 1 and 2 only, i.e. patients with CLL at diagnosis in all stages and patients at first progression of the disease requiring therapy, the distribution of prognostic markers did not differ significantly, except for a lower proportion of cases expressing CD38, although the proportion of del(17p) >20% and >10% and of +12 doubled. When the analysis was restricted to stage A CLL at diagnosis, there was a highly significant difference in the lower proportion of unmutated IgVH (0.003), CD38 expression (p 0.009), ZAP-70 expression (p 0.004), del(17p) (p 0.034) and in the higher proportion of del(13q) (p 0.028) in comparison with CLL at first progression. No patient with stage A CLL at diagnosis showed del(17p) ≥20%, whilst 2% showed del(17p) >10% and 3% harbored p53 mutations. Of the 40 patients evaluated by FISH at multiple time points, 35% showed clonal evolution. Only previous treatment was significantly associated with the development of clonal evolution, whilst no correlation with the IgVH mutational status, ZAP-70 or CD38 expression was found. Patients of group 1 required treatment after a median time of 49.6 months from diagnosis. Treatment-free interval (TFI) was significantly shorter in cases with IgVH unmutated vs. mutated (at 48 months, 16.5% and 68.3%, respectively p<0.0001), CD38 pos vs. neg (at 48 months 12.5% vs. 61.3%, p<0.0001) and ZAP-70 pos vs neg (39.1% vs. 58.9%, p=0.0049). Cases with del(17p) or del(11q) had a TFI at 48 months of 0% versus 25% for cases with +12 and 61.7% for cases with del(13q) or no abnormalities (p<0.0001). IgVH status and white blood cell count were the only independent predictors of TFI in multivariate analysis (p=0.0005 and p=0.012, respectively). The same prognostic impact was shown in the stage A subgroup. The variable incidence of the biologic factors with prognostic impact has to be considered when patients in different phases of the disease are investigated, both in terms of the timing of the assessment and of the design of clinical trials. Reassessment of genetic abnormalities during the course of CLL is warranted, especially after treatment. Disclosures:No relevant conflicts of interest to declare.

High Resolution Array-CGH Provides New Insights Into the Prognosis of Chronic Lymphocytic Leukemia (CLL): Is 8p Loss Worse Than 17p Loss?.

Abstract 2339Poster Board II-316 BACKGROUND:CLL, the most common adult-onset leukemia in the Western world, has a heterogeneous clinical course. Many advances have led to a better understanding of its pathogenesis and to improvements in treatment strategies, but striking solutions are still missing. We conducted a study to evaluate the impact of genomic aberrations on the clinical course. METHODS:From January 1980 to May 2008, 395 frozen samples of CLL patients, were prospectively collected in four centers. Extracted DNA was analyzed with Affymetrix Human Mapping 6.0 arrays. Normal matched DNA was analyzed for one fourth of the cases. Correlations between minimal common regions (MCR) and clinical parameters were evaluated with the Fisherôs-exact test and their impact on OS with the log-rank test. A p-value after Bonferroni multiple test correction (MTC) (p-adj.) <0.05 was considered as statistically significant. Up to now 266 samples have been analyzed. RESULTS:Analysis of the clinical parameters (CPs) and known risk factors (Rai/Binet, age, doubling time, LDH, beta2, IGVH status, p53 mutations, telomere length, CD38, 11q, 17p) was consistent to previous published series. ZAP70 did not affect the clinical course, likely due inter-laboratories variability. After a median follow up of 53 months, 143/239 (60%) of the patients have started therapy and 63/261 (24%) died. 5-yr OS was 82%. Fisher test between the MCRs and CPs revealed an inverse relation between the presence of trisomy 12 by FISH and del13q14.3, an association between del17p and losses of 8p regions and between CD38 and 12q gain. Before MTC, 46 MCRs had a significant impact on OS and 67. After MTC, 3 regions maintained their role: 8p22 loss (38/248, 15%, p-adj.=0.002, median OS: 26 months vs. 48), 17p13.3-11.2 loss (20/248, 8%, p-adj.=0.001; median OS: 10 months vs. 48). In univariate analysis, the log-rank test among pts with 8p-/17p- (8/248, 3%), 8p- (30/248, 12%), 17p- (12/248, 5%), wild type (198/248, 80%) was statistically significant (p<0.001; see figure). Importantly, none of the analyzed clinical and biological parameters was associated with this aberration. [Display omitted] CONCLUSIONS:Loss of 8p22 designated a CLL subgroup with a worse outcome among all patients and in the subset with 17p loss. Our data suggested that this aberration might constitute an independent prognostic factor to be evaluated in independent studies. Results, including a Cox regression model, will be presented on all 395 cases. Disclosures:No relevant conflicts of interest to declare.

Clinical Relevance of Circulating Poly-Ubiquitin as a Prognostic Indicator in Chronic Lymphocytic Leukemia.

Abstract 1240Poster Board I-262The ubiquitin-proteasome pathway is implicated in the pathogenesis of many hematological malignancies. We have reported that proteasome activities in plasma samples can be detected in patients with chronic lymphocytic leukaemia (CLL). Here we examined the correlation of plasma ubiquitin protein levels with clinical behavior in CLL patients. Using the Meso Scale Discovery (MSD) platform, we quantified poly-ubiquitin levels in plasma samples from 138 patients with CLL and 101 healthy control subjects, and examined the associations of ubiquitin levels with various clinical and laboratory variables and overall survival. Patients with CLL had significantly higher levels of poly-ubiquitin proteins (median=158.2; range=35.0-281.2) than did normal control subjects (median=57.3; range=22.0-160.2) (P<0.001). Poly-ubiquitin levels showed no correlation with Rai stage, performance status, beta-2 microglobulin (B2M), or the mutation status of IgVH. However, when considered as a continuous variable, poly-ubiquitin levels correlated negatively with survival. Higher levels of poly-ubiquitin were associated with longer survival (P=0.04). In a multivariate model incorporating poly-ubiquitin, B2M, and IgVH mutation status only B2M (P=0.00004) and poly-ubiquitin P=0.04) levels, but not IgVH mutation status (P=0.12), were independent predictors of survival. When a cut- point corresponding to the upper quartile was used, patients with poly-ubiquitin levels >193 ng/mL had significantly better survival than did those with lower levels. Using B2M and poly-ubiquitin in combination allowed us to stratify patients into 3 different groups : survival was greatest in patients with high poly-ubiquitin and low B2M levels, lowest in those with low poly-ubiquitin and high B2M levels, and intermediate in those with high levels of B2M and ubiquitin or low levels of both (P<0.00001). In conclusion, these findings confirm the importance of the proteasome-ubiquitin system in the pathophysiology of CLL. Moreover, our data also suggest that plasma levels of poly-ubiquitin, along with B2M, should be explored as a prognostic factor in CLL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bendamustine Combined with Rituximab (BR) in First-Line Therapy of Advanced CLL: A Multicenter Phase II Trial of the German CLL Study Group (GCLLSG).

Abstract 205 Introduction:Bendamustine has shown considerable activity in monotherapy for lymphoid malignancies including chronic lymphocytic leukemia (CLL). In vitro studies have demonstrated synergistic pro-apoptotic effects of bendamustine and the CD20 antibody rituximab (BR) in primary CLL cells. Encouraging results have also been obtained using the BR combination treatment in previously treated CLL. This multicenter phase II trial (CLL2M) is the first study assessing the efficacy and toxicity of bendamustine in combination with rituximab in previously untreated CLL patients (pts). Patients and Methods:Between March 2007 and September 2008 117 pts with untreated CLL requiring therapy were enrolled into the protocol. Bendamustine was given at a dose of 90 mg/m2 on day 1 and 2, combined with 375 mg/m2 rituximab for the first cycle and 500 mg/m2 for subsequent cycles. BR treatment was administered every 28 days for up to 6 courses. Blood samples were taken for analysis by fluorescence in situ hybridization (FISH), the IgVH mutational status and expression of ZAP70/CD38. Minimal residual disease (MRD) was evaluated in peripheral blood and bone marrow by 4-color flow cytometry. Results:Data on the entire study population of 117 pts (median age 64 years) with a total of 583 treatment cycles are available. As of June 2009 the median observation time was 15.4 months (mo). 11.1% of the pts presented with stage Binet A, 41.0% with Binet B and 47.9% with Binet C disease. A mean number of 5.0 courses were delivered. 114 pts were evaluable for toxicity, 110 for response and 113 for progression free survival (PFS). The most frequent adverse events based on 583 treatment cycles were myelosuppression and infections: grade 3/4 anemia occurred in 4.9%, grade 3/4 leukopenia in 14.6%, grade 3/4 neutropenia and thrombocytopenia in 6.5% and 6.1% of all given courses, respectively. 29 episodes of CTC grade >3 infections were documented (5.1% of all courses). Treatment related mortality occurred in 2.6% of the pts: one liver failure after attempt of suicide with antihistamines, one fatal pneumonia and one sepsis in neutropenia. The overall response rate was 90.9% with 32.7% (36 pts) clinical complete remissions (CR). A nodular partial remission (nPR) was achieved in 2.7% (3 pts) and a partial response (PR) in 55.5% of the pts (61 pts), respectively. 9.1% of the pts (10 pts) had stable disease (SD) whereas none of the pts was progressive (PD). After 18 mo 75.8% of the pts were still in remission, median PFS has not been reached. An MRD level below 10E-4 was observed after completion of therapy in 29 of 50 evaluable pts in peripheral blood, while 7 of 25 pts achieved MRD negativity in bone marrow. Differences in response were observed among the genetic subgroups: 19 of 21 pts with 11q- achieved a remission with 10 PR and 9 CR (ORR: 90.5%). Accordingly, 17 of 19 patients with +12 responded (14 PR, 3 CR, ORR: 89.5%). In the high-risk group with 17p-, 3 of 7 pts showed a partial response (ORR: 42.9%). 56 of 63 pts (ORR: 88.9%) with unmutated IgVH status responded to BR. Conclusion:Bendamustine plus rituximab (BR) is effective and safe in the first-line treatment of CLL. Major side effects (myelosuppression and infections) were infrequent. Based on these encouraging phase II data the German CLL Study group is presently investigating the efficacy of BR in comparison to fludarabine-based immunochemotherapy (FCR) in the first-line treatment of CLL within a randomized phase III trial (CLL10 protocol). Disclosures:Fischer:Roche: travel expenses. Cramer:Roche: travel grants. Stilgenbauer:Bayer: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding. Fink:Roche: . Boettcher:Roche: Research Funding. Ritgen:Roche: Research Funding. Kneba:Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Döhner:Roche: Research Funding. Eichhorst:Roche: Honoraria, Research Funding; Mundipharma: Research Funding; Hospira: Honoraria. Hallek:Roche: Consultancy, Honoraria, Research Funding. Wendtner:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer Schering: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Immunochemotherapy with Fludarabine (F), Cyclophosphamide (C), and Rituximab (R) (FCR) Versus Fludarabine (F), Cyclophosphamide (C) and MabCampath (Cam) (FCCam) in Previously Untreated Patients (pts) with Advanced B-Chronic Lymphocytic Leukemia (B-CLL) : Experience On Safety and Efficacy within a Randomised Multicenter Phase III Trial of the french Cooperative Group On CLL and WM (FCGCLL/MW) and the “Groupe Ouest-Est d'Etudes Des Leucémies

Abstract 538▪▪This icon denotes an abstract that is clinically relevant. Introduction:Recent data suggest that FCR immunochemotherapy improves response rates and Progression-Free Survival (PFS) of previously untreated CLL pts. The monoclonal antibody alemtuzumab, a humanized anti-CD52 antibody (Campath), has shown activity alone and in combination in CLL pts. In order to validate the place of Campath in combination with the synergic association FC, the FCGCLL/MW and the GOELAMS conducted a multicenter French and Belgian phase III trial, CLL2007FMP, to evaluate the efficacy and toxicity of FCCam versus FCR in previously untreated patients with advanced CLL. PFS was the primary-end-point of this trial. Methods and Patients:A cohort of 178 fit medically pts (cumulative illness rating scale (CIRS) score of up to 6), less than 65 years old, without 17p deletion, were enrolled between November 2007 and January 2009. Pts were randomly assigned to receive 6 oral courses of FC (F 40mg/m2 d1-3 and C 250 mg/m2 d1–3; q 28 days) in combination with either R (n=83; 375 mg/m2 i.v. d 0 at first cycle and 500 mg/m2 d1 all subsequent cycles; q 28 days) or Cam (n=82; 30 mg s/cut d1-3; q 28 days). Patients were stratified according to IgVH mutational status and 11q deletion. Anti-infective prophylaxis included trimethoprim-sulfamethoxazole and valaciclovir during immunochemotherapy and until the CD4-positive lymphocyte count reached 0,2 109/l. In the FCCAm arm, CMV monitoring was monthly performed by either PCR or antigenemia. The use of GSCF was recommended. The trial's recruitment was stopped in January 2009 after 165 pts had been randomized (83 and 82 respectively in the FCR and FCCam arms) because of an excess of mortality in the FCCam arm, while 13 patients were enrolled but not randomized because of this decision. Results:We reported here a preliminary analysis including baseline characteristics and response rates in the first 100 included pts but safety analysis of the whole cohort ; among the first 100 pts, 81% were Binet B, 19 % Binet C ; median age was 56.8 years (range 52.8 to 60.6). Percentages of pts with 11q deletion, unmutated IgVH status, and elevated β2m, were 18.5%, 48.4%, and 77.6%, respectively. A number of 76.5% (FCR arm) and 71.4% (FCCam arm) of pts received 6 cycles. Reasons for discontinuation were mainly related to persistent grade 3-4 neutropenia. Safety analysis data were availabel for 165 pts. Number of patients reported with Common Toxicity Criteria (CTC) grade 3-4 adverse events were observed in 87.8% (FCCam arm) versus 90.2% (FCR arm) (p = 0.76). Grade 3-4 neutropenia was the most frequently reported adverse event (79.6% with FCCam and 74.6% with FCR arm). Interestingly, percentage of observed grade 4 neutropenia was stable during FCR treatment (17.6% for cycle 1 and 17.9% for cycle 6) but increased during FCCam treatment (28.4% for cycle 1 and 45.5% for cycle 6). A total of 63 Serious Adverse events (SAE) were declared (19 with 18 pts in FCR arm, and 44 with 35 pts in FCCam arm) consisting mostly of the cases in febrile neutropenia (13 with FCR arm, 27 with FCCam arm); 7 patients died, all in the FCCam arm : 3 of B diffuse large B-cell lymphoma (one of them EBV positive), 1 of mucormycosis, 1 of septic shock due to P.aeruginosae and 2 of heart failure during neutropenia. The Overall Response Rate ( ORR) in the first 100 patients was 96% in the FCR arm compared to 85% in the FCCam arm (p=0.086). The Complete Response rate was 78% (FCR arm) versus 58% (FCCam arm) (p=0.072). PFS and OS are not yet evaluable. Conclusion:the FCCam regimen for the treatment of advanced CLL appeared to be associated with an unfavourable safety profile representing a significant limitation of its use in this indication. Other combinations with Alemtuzumab may be studied. Disclosures:No relevant conflicts of interest to declare.

Dic(17;18)(p11.2;p11.2) Is a Recurring Abnormality in Chronic Lymphocytic Leukemia Associated with Aggressive Disease.

Abstract 1233Poster Board I-255 IntroductionCytogenetic analysis has become a routine part of the evaluation of patients with chronic lymphocytic leukemia (CLL) because specific chromosomal aberrations have been shown to assist in predicting disease progression, treatment efficacy, and overall survival. One such abnormality is del(17p13.1), which has been found to be associated with a need for early therapy, poor response to conventional treatment, and shortened survival. Identification of recurring abnormalities in this region represents great interest to identify genes relevant to CLL progression and poor prognosis. Patients and MethodsWe performed an extensive review of 1213 patients undergoing metaphase cytogenetics study at our institution and identified 16 (1.3%) with a recurrent unbalanced translocation between the p arms of chromosomes 17 and 18 that results in a dicentric chromosome with loss of much of 17p and 18p. The clinical features of these patients were characterized. ResultsA total of 16 patients were identified to have dic(17;18)(p11.2;p11.2) representing a 1.3% occurrence rate. The median age at diagnosis of these patients was 57 years (range 37-68). The dic(17;18)(p11.2;p11.2) was associated with a complex (3 or more unrelated cytogenetic abnormalities) karyotype in 12 patients (75%) at the time that the abnormality was first identified, and eventually associated with a complex karyotype in 94% of patients. This abnormality was associated with trisomy 12 in 7 patients (44%) and with del(13q) in 5 patients (31%) with no overlap between these two abnormalities. IgVH mutational analysis was un-mutated in 88% of cases. Except for one patient who was diagnosed with CLL incidentally during a workup for metastatic tonsillar cancer, all patients identified with dic(17;18)(p11.2;p11.2) met criteria for disease treatment, with a median time from diagnosis to first treatment of 15 months. Of the 12 patients who received fludarabine-based therapy, 7 (58%) were refractory. Three patients have received stem cell transplant for recurrent/refractory disease, and 4 are currently undergoing chemotherapy. With a mean follow-up of 54 months, 4 patients have died, 21, 42, 49, and 92 months after diagnosis. ConclusionsOur study combined with small series reported by others demonstrate that dic(17;18)(p11.2;p11.2) is a novel recurrent cytogenetic abnormality in CLL. Here we demonstrate the clinical significance of this recurring abnormality that is associated with young age at diagnosis, accelerated disease progression, decreased response to fludarabine, and shortened overall survival. The dic(17;18)(p11.2;p11.2) is frequently accompanied by other negative prognostic markers including a complex karyotype and unmutated IgVH status. Additional studies to further characterize the prevalence of this abnormality and genes that are disrupted by the translocation are warranted. DisclosuresNo relevant conflicts of interest to declare.

Expanded Proliferation Centers (PCs) Identify a Histological Subtype of Chronic Lymphocytic Leukemia (“Accelerated” CLL) with Adverse Clinical Outcome.

Abstract 2335Poster Board II-312The proliferation compartment in lymphoid tissues involved by CLL is essentially represented by large-cells that form proliferation centers (PCs) whose biological and clinical significance is a matter of investigation. For this, tissue biopsies consecutively obtained from 100 patients diagnosed with CLL were analyzed. Histological review was independently performed by three investigators (EC, AM and DM) in a BX51 Olympus microscope. Each biopsy was analyzed for the presence and size of proliferation centers (PCs), delineated by p27 negative immunostains, and for the proliferation rate inside the PCs assessed by means of mitosis count and Ki-67 immunostain. Histological characteristics were correlated with the main clinical characteristics and survival from the time of biopsy. To define the best cut-offs for histological variables in predicting survival, the Maximally Selected Rank Statistics was applied. CLL/SLL diagnosis was established in 78 cases, whereas DLBCL transformation was observed in 22 cases. Upon variables selection by the Maximally Selected Rank Statistics, CLL cases with enlarged and confluent PCs (broader than 20x field) (p=0.001), >2.4 mitosis per PCs (p=0.019), or >30% Ki-67 per PCs (p=0.024) were considered as having “accelerated” CLL (n= 23). Positive p53 immunostaining was observed in 24% “accelerated” CLLs compared to 5% CLLs without adverse features in the biopsy (p=0.02). The presence of p53 positivity was also frequently observed in DLBCL transformed cases (67%). “Accelerated” CLL cases were detected early in the course of the disease (median time from diagnosis: 16 months; 39% of cases at diagnosis), as opposite to DLBCL transformed cases (median time form diagnosis: 45 months; 14% at diagnosis). Tissue biopsy was performed in 73% of cases to rule out a transformation into an aggressive lymphoma. “Accelerated” CLL/SLL displayed high beta-2-microglobulin levels, elevated ZAP-70 expression and unmutated IgVH status more frequently than CLL/SLL without adverse features in the biopsy (p< 0.05). Median survival of “accelerated” CLLs from the time of biopsy was 34 months, whereas it was of 75 months (HR 2.2, 95%CI HR 1.21-3.86; p=0.008) for cases without adverse features in the tissue biopsy. Patients diagnosed with DLBCL transformation (n=22) had a median survival of 4 months. Notably, only one patient with an “accelerated” CLL pattern transformed into DLBCL after a median follow-up of 5 years. In conclusion, in lymph node biopsies from CLL/SLL patients, enlarged and confluent PCs and/or an increased proliferation activity in the PCs without DLBCL criteria identifies a new histological subtype of CLL/SLL (“accelerated CLL) with adverse biological features and inferior survival. Whether these patients require specific therapeutic approaches warrants investigation. Disclosures:No relevant conflicts of interest to declare.

Phase I Study of RO5072759 (GA101) in Relapsed/Refractory Chronic Lymphocytic Leukemia.

Abstract 884 Background:RO5072759 (GA101) is the first humanized and glycoengineered type II monoclonal anti-CD20 antibody to enter clinical trials. In vitro, GA101 exhibits increased antibody-dependent cytotoxicity (ADCC) and strongly enhanced direct cell death compared to rituximab. Methods:A Phase I study was initiated to determine the safety, tolerability, dose-limiting toxicity (DLT), and pharmacokinetics of GA101 given as a single agent to patients with CD20+ B-CLL for whom no therapy of higher priority was available. A flat, intravenous dose ranging from 400 mg to 2000 mg was given in a safety-driven, dose escalating, 3 × 3 design on days 1, 8 and 22 and subsequently every 3 weeks for a total of 9 infusions. Here, we present the phase I data on 13 B-CLL patients, 33% [9/13 evaluable] with high risk cytogenetics (17p or 11q) and 70% [7/10 evaluable] displaying unmutated IgVH status, who received a median of 3 [1-8] prior regimens, including fludarabine (13/13) and rituximab-containing therapy, (8/13). Baseline median hematology parameters included hemoglobin 12.6 g/dL [9.4 -14.9], WBC 51.8 × 109/L [10-124], platelets 191 × 109/L [48-404], lymphocytes 47.4 × 109/L [7.0-119.3]. Results:GA101 was well tolerated with no DLTs and no dose reductions. The most common adverse events were Grade 1 or 2 infusion related reactions essentially limited to the first infusion. GA101-related Grade 3/4 hematological toxicities included transient neutropenia (n=9), febrile neutropenia (n=1) and transient thrombocytopenia (n=1). Neutropenia recovered spontaneously or with G-CSF. SAEs were reported in 3 patients (febrile neutropenia, thrombocytopenia, bronchitis, gingivitis, neutropenia and tumor lysis syndrome). Ten patients had infections (17 episodes with only three being Grade 3). No significant changes to baseline immunoglobulin levels were observed. Measurement of plasma cytokines during and immediately after the 1st infusion showed a transient increase in IL6 and IL8 with smaller increases in IL10, IFN-γ and TNFa. Activation of complement was not observed (C3a, C4a, C5a). Concurrent to cytokine increase was a decrease in T-cell subsets and NK cell counts (peripheral blood) after the first infusion. At the end of treatment, CD3 and CD8 counts had recovered while median CD4 and CD16/56 counts remained just below the normal range, with no clinical sequelae observed. Immunologic monitoring is ongoing.B-cell (CD19+) depletion was almost complete for all 13 patients and sustained following the first infusion. Best overall response rate according to IWCLL criteria was 62% (8/13) with 1 CRi, 7 PR and 5 SD observed across all FcγIIIRA [158F/V polymorphism] genotypes with no clear dose relationship established. Responses are ongoing with durations ranging from 3.5+ to 8+ months. End of treatment minimal residual disease (MRD) from 7/11 evaluable patients was detectable for 6 patients (median reduction of 2 log, range 2-4) and negative for one (despite a stable disease, as assessed by CT-scan). GA101 pharmacokinetics were characterized by one linear and one time-dependent saturable clearance components, consistent with target-mediated disposition, which is also observed with rituximab. Whilst the plasma concentrations demonstrate a dose-dependent increase, there was significant inter and intra-patient variability. Time-dependent clearance is consistent with a reduction in target-mediated antibody clearance with increasing duration of treatment. With the same doses of GA101, clearance is faster in B-CLL patients than NHL patients (Salles et al ASH 2008, 2009). Conclusion:These phase I results indicate that GA101 has promising activity when given as single agent to heavily pre-treated B-CLL patients and has a similar safety profile to that observed in NHL patients (Salles et al, ASH 2008, 2009) with an increased incidence of transient neutropenia in B-CLL. GA101 is currently being explored as a single agent in phase II in relapsed/refractory B-CLL and indolent/aggressive NHL and in combination with chemotherapy in a phase Ib study. Disclosures:Morschhauser:Roche Products Limited : Honoraria. Cartron:Roche Products Limited : Consultancy, Honoraria. Milpied:Roche Products Limited : Honoraria. Weisser:Roche Diagnostics : Employment. Birkett:Roche Products Limited : Employment. Salles:Roche Products Limited : Honoraria.

14q Deletions Are Recurrent Aberrations in CLL and Other Mature B-Cell Neoplasms and Show a High Coincidence with An Unmutated IgVH Status and Trisomy 12, and Are Associated with An Intermediate Overall Survival and a Shorter Time to Treatment.

Abstract 1262Poster Board I-28414q deletions are rare aberrations in malignant neoplasia but recurrent in hematologic malignancies, where they occur most frequently in CLL and other mature B-cell neoplasms. The breakpoints and the size of the 14q deletion have not been exactly characterized in a larger cohort yet. Based on chromosome banding analysis we identified 47 cases with 14q deletion out of 3054 cases (1.5%) with mature B-cell neoplasms (CLL: 1863, CLL/PL: 92, other: 1099). 32 cases with 14q deletion were classified as CLL, 5 as CLL/PL and 10 as other mature B-cell neoplasms based on immunophenotyping or cytomorphology. Therefore, the incidence of 14q deletions was 1.7% in CLL, 4.7% in CLL/PL and 0.9% in other mature B-cell neoplasms. These cases were further analyzed by a panel of FISH probes localized in 14q22.1 (BAC RP11-218E20), 14q24.1 (BACs PR11-35D12, RP11-226F19), 14q32.33 (BAC RP11-521B24) (BlueGnome, Oxford, UK) and the IgH-locus (14q32.3). The study revealed that 14q deletions are heterogeneous in size. A breakpoint cluster on the centromeric side was identified in 14q24.1 between BAC PR11-35D12 and BAC RP11-226F19 (62% of cases), whereas the most frequent breakpoint on the telomeric side was within the IgH-locus (14q32.3) between the IgH3'-flanking probe and IgHV probe (45% of cases). In 16 cases (34%) breakpoints occurred within 14q24.1 and within 14q32.3. In 19% of patients the 14q deletion was the sole abnormality whereas 30% showed one, 15% two and 36% three or more additional aberrations. Trisomy 12 (45%) was the most frequent aberration accompanying 14q deletion. In comparison to a cohort of 1579 cases with mature B-cell neoplasms without 14q deletion which were analyzed for IgVH mutation status in the same time period, patients with 14q deletions showed more often an unmutated IgVH status (39% vs. 61%, p=0.001). In the subgroup with breakpoint in 14q24.1 and 14q32.3 even 82% showed an unmutated IgVH-status. This subgroup was also associated with a CLL immunophenotype (81%). Clinical follow up data was available in 28 patients. CLL and CLL/PL with 14q deletion were compared to 390 CLL and CLL/PL cases without 14q deletion. While overall survival (OS) did not differ between both groups (OS at 3 yrs 86.4% vs. 88.7%, p=0.195), time to treatment (TTT) was significantly shorter in cases with 14q deletion (21.0 months vs. 80.1 months, p=0.015). Next, all 418 cases were assigned into 6 subgroups with known prognostic relevance: 1) 14q deletion, 2) 17p deletion, 3) 11q deletion, 4) trisomy 12, 5) 13q deletion as the sole abnormality, and 6) none of the mentioned abnormalities. OS at 3 yrs was 86.4%, 31.4%, 77.5%, 96.9%, 93.4% and 93.9%, respectively. For TTT the respective data were 21.0 months, 21.6 months, 20.9 months, not reached, 79.6 months and 147.0 months. In conclusion, 14q deletions are recurrent abnormalities in CLL and other mature B-cell neoplasms. Comparable to 13q deletions the size of 14q deletions varies. However, recurrent breakpoints in 14q24.1 and 14q32.3 were identified. Our data suggest to add FISH probes for 14q24.1 and for the IGH locus to the standard FISH panel in CLL and other mature B-cell neoplasms to pick up cases with 14q deletion, who would otherwise be assigned to “normal” FISH subgroup. In conclusion, 14q deletions are frequently accompanied by trisomy 12, an unmutated IgVH status and are associated with an intermediate overall survival and a shorter time to treatment comparable to other high risk cytogenetic aberrations (17p deletion, 11q deletion). DisclosuresReindl:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Monoclonal B-Cell Lymphocytosis (MBL) Is Closely Related to Chronic Lymphocytic Leukemia (CLL) and May Be Better Classified as Early-Stage CLL.

Abstract 2349Poster Board II-326Monoclonal B-cell lymphocytosis (MBL) is separated from chronic lymphocytic leukemia (CLL) mainly by the somewhat arbitrary cut-off of 5000/μl CLL-phenotype cells in peripheral blood. While MBL in general shows an indolent clinical course this is also true for early-stage CLL. This may call into question the adequateness of separating MBL from CLL. Therefore, we prospectively analyzed a series of 298 cases with MBL by immunophenotyping, fluorescence in situ hybridization (FISH; probes for detection of del(6q21), del(11q22.3) (ATM), +12, del(13q14) (D13S25, D13S319), del(17p13) (TP53), and t(11;14)(q13;q32) (IGH-CCND1)), chromosome banding analysis (CBA) and molecular genetics (analysis of IgVH mutation status) for parameters which are established as prognostically relevant in CLL. Data was compared to a previously published series of 356 cases with CLL (Cytometry B Clin Cytom 2009;Epub.). Male:female ratio was similar for MBL and CLL (2.1:1 vs. 1.8:1, n.s.) as was mean±SD age (66.5±10.5 vs. 65.7±10.2 years, n.s.). Mean±SD cells with CLL phenotype in peripheral blood amounted to 2,417±1,497/μl in MBL and to 27,771±39,607/μl in CLL (p<0.001). ZAP-70 expression (mean±SD MFI ratio T-cells:B-cells 4.3±3.0 vs. 5.1±3.3, p=0.011) and CD38 expression (mean±SD % positive cells 33.5±28.9 vs. 26.5±31.5, p=0.004) were stronger in MBL. FISH analysis revealed similar frequencies of del(11q22.3) (8.1% vs. 11.7%, n.s.). In contrast, +12 (22.8% vs. 13.7%, p=0.003) and t(11;14) (2.1% vs. 0.0%, p=0.008) were observed more frequently in MBL while del(6q21) (1.8% vs. 6.1%, p=0.008), del(13q14) (45.1% vs. 64.3%, p<0.001), del(13q14) as sole abnormality (35.0% vs. 47.7%, p=0.002), and del(17q13) (1.4% vs. 8.0%, p<0.001) were more frequent in CLL. CBA demonstrated a normal karyotype (31.5% vs. 21.6%, p=0.004) and trisomies (10.7% vs. 6.2%, p=0.045) more often in MBL while deletions were observed less often (30.5% vs. 39.3%, p=0.021). Analysis of IgVH revealed a mutated status more frequently in MBL (76.3% vs. 60.6%, p<0.001). Thus, while some good risk parameters have been encountered more frequently in MBL compared to CLL there was no clear predominance of all good risk parameters but rather a mixed distribution between MBL and CLL. We next analyzed the prognostic impact of the above parameters in cases with MBL. Time to therapy (TTT) was negatively affected by a higher CD38 expression (p=0.007), del(11q22.3) (p=0.01), the presence of independent clones as identified by CBA, and an unmutated IgVH status (p=0.001). Multivariate analysis revealed a higher CD38 expression (p=0.026) as the only independent parameter affecting TTT. Overall survival (OS) was negatively affect by del(6q21) (p=0.001) and by the presence of independent clones as identified by CBA (p=0.056) while a normal karyotype by CBA was associated with a better OS (p=0.031). When analyzing both MBL and CLL cohorts together, +12 (p=0.011) was found to be related to shorter TTT and del(13q14) as sole abnormality (p=0.038) and a higher ZAP-70 ratio T-cells:B-cells (p=0.007) were related to longer TTT. Neither the amount of cells with CLL phenotype in peripheral blood nor the presence of MBL were significantly related to TTT. The only parameters independently related to TTT were del(11q22.3) (p=0.007) and +12 (p=0.037). Parameters negatively affecting OS were MBL (p=0.006), the presence of independent clones as identified by CBA (p=0.038) and a female gender (p=0.047). Multivariate analysis demonstrated MBL (p=0.021) and the presence of independent clones as identified by CBA (p=0.046) as independently related to OS. The present data indicates that biologic characteristics of CLL are found in MBL and that there is no general predominance of good risk parameters in MBL as compared to CLL. Thus, MBL may not be considered a distinct disease but rather an early stage of CLL. This is further supported by the lack of impact of MBL as compared to CLL on the TTT. Furthermore, it is suggested that cases classified as MBL should undergo assessment of prognostic parameters including CBA as one prognostic parameter as shown for CLL cases. Disclosures:Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Relationship Between Expression of TLRs and Disease Activity in CLL.

Abstract 4379 BackgroundToll-like receptors (TLRs) are major agents of innate immunity and initiators of adaptive immune response by acting as costimulatory signals for B cells and inducing maturation, proliferation and antibody production after pathogen recognition. They are also involved in the self-antigen recognition and could play a role in autoimmune phenomena. It is well known that chronic lymphocytic leukemia (CLL) is characterized by an increased incidence of autoimmune phenomena and immunodeficiency, which can greatly influence the disease outcome leading to a variable clinical course. AimsTo evaluate the correlation between the gene expression of TLRs in 41 patients with CLL (mean age±SD 66±18 years, range 42-90, 15 female and 26 male), the clinical course of the disease and the expression of prognostic factors (mutational status of IgVH region, CD38 and ZAP70 expression, and cytogenetic alterations). MethodsThe gene expression of TLR4, TLR9, and TLR10 was studied. Total RNA was extracted from B cells of patients and controls (pool of 10 healthy donors), cDNA was synthesized, and real-time PCR was performed. For each reaction 50 ng of cDNA were mixed with 1.25μl of TaqMan primer/probe set and 10.25μl of TaqMan Universal Master Mix. The TLR4, TLR9, and TLR10 expression was normalized according to GAPDH as an internal control gene and it was expressed as percentage of control. ResultsTLR4 gene expression was significantly lower in CLL patients compared to controls (18±3%, mean±SE) while TLR9, and TLR10 gene expression was higher (3457±500% and 2897±440%, respectively). Moreover, considering “progressive” CLL patients (n=20), TLR4 gene expression was significantly lower compared with “indolent” ones (n=21) (10.2±2.2% vs 24.9±4.9%, p=0.01). Patients with unmutated IgVH expression and unfavorable cytogenetic alterations (mostly 11q-) showed even more reduced TLR4 gene expression, but the small numbers did not allow statistical evaluation. TLR4 gene expression was lower in patients with infective diathesis, although not significantly (11.5±2.9% vs 20.0±3.9).The 6 “progressive” CLL patients with autoimmune diseases (hemolytic anemia, Evan's syndrome and phemphigus) showed significantly higher TLR4 gene expression compared with “progressive” patients without autoimmune phenomena (17.7±4.4% vs 6.9±2.1%, p=0.02), possible expression of activated immune system. ConclusionThe reduced TLR4 gene expression in CLL patients with “progressive” disease, unmutated IgVH status, and unfavourable cytogenetic alterations is consistent with a reduced ability to a proper immune response to Gram-negative bacterial lipopolysaccarides and consequent increased susceptibility to infections. Disclosures:No relevant conflicts of interest to declare.

External Validation On Biological Basis of New Prognostic Index in Early Asymptomatic Chronic Lymphocytic Leukemia (CLL) Patients: The Gruppo Italiano Studio Linfomi (GISL) Experience.

Abstract 2375Poster Board II-352A prognostic index based on widely available clinical and laboratory features was recently proposed to predict survival in patients with previously untreated patients with chronic lymphocytic leukemia (CLL) by MD Anderson investigators. However, whether proposed clinical risk categories may surrogate new biological variables of prognostic relevance (i.e., mutational status of the IgVH gene regions, ZAP-70 or CD-38 expression, cytogenetic abnormalities) is unclear thus far.In a series of 160 asymptomatic Binet stage A patients enrolled in a Gruppo Italiano Studio Linfomi (GISL) multicentre trial designed to validate prospectively biological parameters in early CLL as well as to assess the impact on clinical outcome of an early versus delayed policy of treatment with subcutaneous alemtuzumab in the high biological risk, we evaluated whether clinical categories derived from newly proposed prognostic index reflected biological risk.Since the original prognostic index was derived from a database including cases with more advanced disease we used an optimal cutoff search to determine how to best split Binet stage A patients in different prognostic groups. To this purpose an independent patient cohort consisting of 310 Binet stage A patients included in a GIMEMA (Gruppo Italiano Malattie EMatologiche Maligne dell'Adulto) database was used. According to recursive partitioning (RPART) model, a classification tree was built that identified two subsets of patients who scored respectively: 0-3 (low risk) and 4-7 (high risk). Therefore, by prognostic index, 48.7% and 51.2% of 160 asymptomatic stage A patients, respectively, met criteria of low risk and high risk disease.In our prospective series high- risk score was more frequently associated with both unmutated IgVH status (P=0.009) and higher CD38-expression (P=0.002); in contrast only a trend towards an increased ZAP-70 expression could be found (P=0.06). As far as cytogenetic abnormalities are concerned, we observed that 11q deletion occurred more frequently among patients belonging to high-risk score (P=0.005), while cases with 13q deletion or trisomy 12 were homogeneously distributed among low- and high-risk patient category(P=0.151 and P=0.452, respectively). We did not consider suitable for correlation analysis 17p deletion since observed only in 2 out of 160 Binet stage A patients.In conclusion, our results demonstrate in a prospective cohort of patients with early CLL that clinical categories of a revised score index may surrogate biological parameters of prognostic relevance. The observation reinforces the revised IWCLL guidelines recommendations to assess the risk of CLL patients on clinical basis and to deserve biological studies to patients eligible for clinical trials. Disclosures:No relevant conflicts of interest to declare.

Correlation of flow cytometrically determined expression of ZAP‐70 using the SBZAP antibody with IgVH mutation status and cytogenetics in 1,229 patients with chronic lymphocytic leukemia

ZAP-70 provides an important prognostic information in chronic lymphocytic leukemia (CLL); however, the most appropriate antibody clone and way of analysis have not yet been defined. We determined ZAP-70 expression in 1,229 patients with CLL using the SBZAP clone by applying three different ways of analysis (% positive B-cells, mean fluorescence intensity (MFI) in B-cells, MFI ratio T-cells:B-cells). ZAP-70 expression was related to somatic hypermutation status of IgVH genes for all three ways of analysis (P < 0.0001 each). The strongest correlation was found for MFI ratio (r = -0.475, P < 0.0001); cases with mutated and unmutated IgVH status significantly differed in their mean MFI ratio (6.33 vs. 3.34, P < 0.0001). ZAP-70 expression was weaker in sole del(13q) (5.44 vs. 4.44, P = 0.001) and stronger in del(11q) (3.31 vs. 5.29, P < 0.0001) and del(6q) (3.29 vs. 5.18, P = 0.005). MFI ratio was significantly related to time to therapy (P = 0.004, RR = 0.79) in 221 evaluable cases. Multivariate analysis proved MFI ratio (P = 0.043, RR = 0.64) and CD38 (P = 0.047, RR = 1.20 per 10%) as only parameters independently related to time to therapy. Determination of ZAP-70 expression using SBZAP and applying MFI ratio T-cells:B-cells results in significant relations to IgVH mutation status, cytogenetics, and outcome and should be further analyzed and considered for routine application in CLL.

Relevance of the immunoglobulin VH somatic mutation status in patients with chronic lymphocytic leukemia treated with fludarabine, cyclophosphamide, and rituximab (FCR) or related chemoimmunotherapy regimens

AbstractAlthough immunoglobulin VH mutation status (IgVH MS) is prognostic in patients with chronic lymphocytic leukemia (CLL) who are treated with alkylating agents or single-agent fludarabine, its significance in the era of chemoimmunotherapy is not known. We determined the IgVH somatic mutation status (MS) in 177 patients enrolled in a phase 2 study of fludarabine, cyclophosphamide, and rituximab (FCR) and in 127 patients treated with subsequent chemoimmunotherapy protocols. IgVH MS did not impact significantly on the complete remission (CR) rate of patients receiving FCR or related regimens. However, CR duration was significantly shorter in patients with CLL that used unmutated IgVH than those whose CLL used mutated IgVH (TTP 47% vs 82% at 6 years, P < .001). In a multivariate model considering all baseline characteristics, IgVH MS emerged as the only determinant of remission duration (hazard ratio 3.8, P < .001). Our results suggest that postremission interventions should be targeted toward patients with unmutated IgVH status.

CD49d, Zap‐70, and CD38 Expression in Chronic Lymphocytic Leukemia

Zeta‐chain (TCR) associated protein kinase 70kDa (Zap‐70), and CD38 expression are predictors of IgVH mutation and prognosis in chronic lymphocytic leukemia (CLL). Previous studies indicate that Zap‐70 and CD38 are usually positive in cases of CLL with non‐mutated immunoglobulin variable region genes (IgVH), and may be used to predict IgVH mutation status and prognosis. Usually, cases of CLL positive for Zap‐70 or CD38 indicate a worse prognosis. In the present investigation, 62 cases of CLL were evaluated for CD38 expression, and 32 cases were evaluated for both Zap‐70 and CD38 expression. Of the 62 cases, 40% positively expressed CD38. Of the 32 cases evaluated for Zap‐70, 65% were positive, while only 35% were positive for CD38 expression; the remaining cases were negative for CD38. More recently CD49d, an integrin molecule, has been added as a predictor of prognosis in CLL. The results of this study show that Zap‐70 expression may be a better indicator of the mutational status of IgVH and prognosis of CLL than CD38 expression, and that CD49d represents an improved prognostic capability in CLL cases. In addition, CD38 negativity does not necessarily indicate that IgVH mutation has occurred. These data point to the need for a broader study to evaluate the significance of Zap‐70 and CD38 expression as indicators of IgVH mutation status, and of CD49d in the prognosis of CLL patients.