ALBERT

Description: Bcl-2 plays a key role in the regulation of apoptosis. We investigated the role of a novel regulatory single-nucleotide polymorphism (−938C〉A) in the inhibitory P2 BCL2 promoter in B-cell chronic lymphocytic leukemia (B-CLL). The −938C allele displayed significantly increased BCL2 promoter activity and binding of nuclear proteins compared with the A allele. Concomitantly, Bcl-2 protein expression in B cells from CLL patients carrying the −938 AA genotype was significantly increased compared with CC genotypes. Genotype distribution between 123 CLL patients (42 AA, 55 AC, 26 CC) and 120 genotyped healthy controls (36 AA, 63 AC, 21 CC) was not significantly different, suggesting that genotypes of this polymorphism do not increase the susceptibility for B-CLL. However, median time from first diagnosis to initiation of chemotherapy and median overall survival were significantly shorter in patients with −938AA genotype (38 and 199 months, respectively) compared with AC/CC genotypes (120 and 321 months, respectively; P = .008 and P = .003, respectively). Multivariable Cox regression identified the BCL2−938AA genotype as an independent prognostic factor for the time to first treatment (hazard ratio [HR] 1.9; P = .034) together with disease stage at diagnosis (HR 2.5; P = .004) and ZAP-70 status (HR 3.0; P = .001). The BCL2−938AA genotype is associated with increased Bcl-2 expression and a novel unfavorable genetic marker in patients with B-CLL.

Description: Bcl-2 plays a key role in the regulation of apoptosis. We investigated the role of a novel regulatory single nucleotide polymorphism in the BCL2 gene (−938C〉A) in B-CLL. Bcl-2 protein expression in B-cells from CLL patients carrying the −938 AA genotype was significantly increased compared to AC and CC genotypes. Moreover, the −938C- and A-alleles differed with regard to the binding of nuclear proteins. Genotype distributions between 123 CLL patients (42 AA, 55 AC, 26 CC) and in 109 age-matched healthy controls (38 AA, 55 AC, 16 CC) were not significantly different suggesting that this polymorphism does not increase the susceptibility for B-CLL. However, time from first diagnosis to initiation of chemotherapy was significantly shorter in patients with −938AA genotype (38 months) compared to AC/CC genotypes (120 months; p=0.0077), especially in CD38 and ZAP-70 negative CLL patients. Multivariate Cox regression identified the BCL2 −938AA genotype as an independent prognostic factor for progression-free survival (hazard ratio, HR, 1.9; p=0.034) together with disease stage at diagnosis (HR 1.6; p=0.018) and ZAP-70 status (HR 2.9; p=0.001). Furthermore, the BCL2 −938C〉A polymorphism in combination with the two common prognostic factors CD38 and ZAP70 status provides additional prognostic information beyond that obtained from single or double marker analysis. To the best of our knowledge this is the first report demonstrating the influence of a novel regulatory polymorphism in the BCL2 gene upon the progression of B-CLL, which, of course, requires confirmation from independent studies. On the other hand, the results presented here appear plausible with regard to differences on the protein level depending on genotype and the known impact of Bcl-2 on disease progression. Thus, our report exceeds other genetic association studies in that a mechanistic link between genotype and observed phenotype can be proposed. In conclusion, carriage of the BCL2 −938AA genotype is a novel unfavorable genetic marker in patients with B-CLL associated with increased Bcl-2 expression.

Description: B-cell chronic lymphocytic leukemia (B-CLL) is a heterogenous disease with a highly variable clinical course and analysis of ZAP-70 and CD38 expression on B-CLL cells allowed for identification of patients with good (ZAP-70−CD38−) and poor (ZAP-70+CD38+) prognosis. DNA microarray technology was employed to compare eight ZAP-70+CD38+ with eight ZAP-70−CD38− B-CLL cases. The expression of 358 genes differed significantly between the two subgroups including genes involved in B cell receptor signaling, angiogenesis and lymphomagenesis. Three of these genes, i.e. IRTA4/FcRH2, angiopoietin 2 and Pim2 were selected for further validating studies in a cohort of 94 B-CLL patients. IRTA4/FcRH2 expression as detected by flow cytometry was significantly lower in the poor prognosis subgroup as compared to ZAP-70−CD38− B-CLL cells. In healthy individuals IRTA4/FcRH2 protein expression was associated with a CD19+CD27+ memory cell phenotype. Angiopoietin 2 plasma concentrations were two-fold higher in the poor prognosis subgroup (p

Description: Abstract 2418 Introduction: The human leukocyte antigen G (HLA-G) molecule exhibits limited tissue distribution and exerts multiple immunoregulatory functions. Since the net result of these effects is immunosuppression, HLA-G expression in tumor cells may favor their escape from antitumor immune responses, thus allowing tumor progression. The HLA-G protein polymorphism is extremely restricted in comparison to the classical HLA antigens. However, specific HLA-G polymorphisms in exons 3, 4, 5, 7 and parts of the 3`UTR encode for different haplotypes. Demonstrating the extensive and proven prognostic role of HLA-G in various diseases and especially in B-CLL we investigated the role of the HLA-G haplotypes in B-CLL. Methods: Genotyping was performed on the basis of examining exons 3, 4, 5, 7 and parts of the 3`UTR by pyrosequencing in 190 patients with B-CLL. In total, we found 17 different haplotypes in the cohort. Results: The haplotype distributions between 190 B-CLL patients and 190 healthy controls were not different, arguing that the haplotypes of HLA-G do not increase the susceptibility for B-CLL. However, combining four different haplotypes (*01:06, *01:12; *01:12 variant and *01:13N) we evaluated a risk score (0=no risk allele; 1=one risk allele; 2=two risk alleles) for TFS. 77 patients showed no risk allele, 90 patients one risk allele and 23 patients two risk alleles. The TFS for those patients with two risk alleles were significantly shorter (median TFS 33 months) than for those with one risk allele (median TFS 49 months) and those with no risk alleles (median TFS 88 months)(log rank test: p=0.03). In multivariate analysis we could show that the stage according to Binet (HR 1.7, 95% CI 1.2–2.6, p=0.005), CD38 status (HR 1.8, 95% CI 1.0–3.2, p=0.04) and the haplotype risk model (HR 1.7, 95% CI 1.1–2.6, p=0.011) were independent predictors for first therapy. Conclusion: Our study is the first study demonstrating that the combination of different alleles of the HLA-G gene is associated with the risk of first therapy in B-CLL patients. This fact emphasizes the extensive role of this gene by the tumor escape mechanism and could be responsible for progress in other cancers. Disclosures: No relevant conflicts of interest to declare.

Description: BACKGROUND: In contrast to other B-cell neoplasias, chronic lymphocytic leukemia (CLL) is not only characterized by a clonal expansion of specific B-cells, but also by an increase in non-leukemic T-cells, most likely involved in sustaining the growth of the leukemic B-cell clone. Based on ZAP-70, CD38 and the IgVH mutation status, two prognostic groups of CLL patients can be identified. Our aim was to characterize the replicative histories of the B- and T-cells in the two groups of CLL patients compared to healthy individuals. PATIENTS and METHODS: Blood samples from 73 patients with CLL (ZAP-70−/CD38−: n = 29, ZAP-70+/CD38+: n = 30, ZAP-70/CD38 discordant: n = 14) were analyzed. The quantity and characteristics of the lymphocyte subsets was assessed by a cell counter and by immunophenotypic analysis. The replicative histories of naive and memory T-cells as well as B-cells was determined by measurements of telomere length in peripheral blood leukocytes of CLL patients and healthy individuals by automated multicolor flow-FISH. RESULTS: As expected, the average telomere length of the clonal B-cells was short. The telomere length was, however, significantly shorter for the ZAP-70+/CD38+ patient samples (2.46 ± 1.08 kb) than for the ZAP-70−/CD38− patient samples (5.06 ± 1.76 kb, p 〈 6.7 x 10−9). Interestingly, also the naive and memory T-cells from ZAP-70+/CD38+ CLL patients exhibited significantly shorter average telomere lengths (mean ± std: 4.85 ± 1.58 kb; 4.39 ± 1.09 kb) than T-cells from ZAP-70−/CD38− CLL patients (6.64 ± 1.72 kb, p 〈 2.2 x 10−4; 6.22 ± 1.5 kb, p 〈 7.4 x 10−6). These results are in line with the observed higher absolute T-cell numbers in the ZAP-70+/CD38+ CLL patients compared to ZAP-70−/CD38− CLL patients. Moreover, the average telomere loss in relation to time from primary diagnosis to sample date was higher for naive T-cells than memory T-cells in ZAP-70+/CD38+ patients (7.8 vs. 5.8 bp/month). When we compared the telomere length to age-related percentiles calculated from over 400 healthy individuals aged 0–102 years practically all telomere length values of the naive and memory T-cells from the ZAP-70+/CD38+ CLL patients fell below the 50th percentile, whereas the values of naive and memory T-cells from the ZAP-70−/CD38− CLL patients were within the normal distribution. CONCLUSIONS: We can confirm significantly shorter telomere length values for the B-cells of the ZAP-70+/CD38+ CLL patients. In addition, we can also demonstrate significantly shorter telomeres in T-cells of ZAP-70+/CD38+ CLL patients, which are below the 50th percentile compared to controls, and a higher telomere loss over time for naive T-cells of ZAP-70+/CD38+ CLL patients. As telomere length shortens approximately 50 to 100 bp per cell division the observed decrease in telomere length of the T-cells in ZAP-70+/CD38+ CLL patients equals to approximately 18 to 36 population doublings. This is by far more than expected by the slightly higher T-cell numbers in the peripheral blood. Our observations imply an extensive expansion of the T-cell compartment in ZAP-70+/CD38+ CLL patients and suggest an important role of T-cells in this subgroup of CLL patients.

Description: Abstract 3592 Introduction: Increasing experimental evidence supports the idea of aberrant microRNA (miRNA) expression in cancer pathogenesis, especially in chronic lymphocytic leukemia (CLL). Recently, aberrant expression of miR-29c and miR-223 has been associated with CLL outcome. Moreover, low expression of miR-34a in CLL is associated with p53 inactivation and also chemotherapy-refractory disease. Therefore, we investigated miR-29c, miR-223 and miR-34a expression in a large representative cohort of 110 CLL patients in order to assess the role of these miRNAs in risk prediction in B-CLL. Methods: Mononuclear cells of B-CLL were isolated from whole blood by centrifugation on a Ficoll/Hypaque gradient and cryopreserved in liquid nitrogen. MicroRNA was extracted from native liquid-nitrogen frozen cells using the QIAGEN miRNeasy® mini kit (Qiagen, Hilden, Germany). The relative expression of mature miRNAs was quantified using the TaqMan MicroRNA RT kit and TaqMan® MicroRNA Assays (Applied Biosystems, Foster City, California) on the ABI 7500 Real Time PCR System (Applied Biosystems, Foster City, California). RNU6B was used as internal control. The relative expression of each microRNA of interest to RNU6B was calculated using the formula miRNA of interest/RNU6B=2-deltaCt(miRNA-RNU6B). Result: The impact of the three miRNA expressions on treatment-free survival (TFS) and overall survival (OS) was assessed by performing “Receiver Operating Characteristics” (ROC) curve analysis. Patients with low miR-29c, miR-223 or miR-34a expression had a shorter TFS and OS than those patients with high expression: miR-29c: TFS 49 vs 91 months (p=0.081); OS 164 months vs not reached (p=0.093) miR-223: TFS 27 vs 84 months (p=0.035); OS 132 months vs not reached (p=0.001) miR-34a: TFS 46 vs 83 months (p=0.084); OS 132 months vs not reached (p=0.001) Furthermore, we investigated whether combining information on the expression of miRNAs could help refine the prognostic information provided by either of the three risk factors (RF) alone. Adding the number of risk factors this approach allowed for highly significant separation of our patient cohort into four subgroups, which differed significantly with regard to their clinical outcome. TFS: 0 RF 99 months; 1 RF 75 months; 2 RF 26 months; 3 RF 22 months (p=0.008) OS: 0 RF not reached; 1 RF not reached; 2 RF 164 months; 3 RF 132 months (p

Description: Abstract 1237 Poster Board I-259 Introduction Free light chains (FLC) have prognostic significance in monoclonal gammopathy of undetermined significance, solitary plasmocytoma of bone, smouldering myeloma, multiple myeloma, Waldenstroms macroglobulinaemia and AL amyloidosis. Although monoclonal protein secretion is a typical feature of plasma cell dyscrasias, it can also be detected in other B cell malignancies including chronic lymphocytic leukemia (CLL). Recent data suggest a significant correlation between abnormal ratio of FLC and outcome. Therefore, we investigated FLC in a large cohort of 120 patients in order to assess the role of FLC in CLL. Methods and Results Plasma samples which had been previously cryopreserved and collected at the time before the initiation of therapy or six months after finishing therapy were used. The levels of FLC were assessed using nephelometric immunoassays (The Binding Site) and quantified nephelometrically with the BNII analyser. A normal FLC range (κlγ) was defined as 0.26-1.65. Moreover, in all cases we evaluated the M band on immunofixation (IF). Abnormal FLC ratios were found in 71 patients (59%) whereas the IF was positive in only 32 cases (27%). In 48 cases the FLC ratio was positive while IF was negative and in only 9 cases the IF was positive while the FLC ratio was normal. In total, 23 patients had both a positive IF and an abnormal FLC ratio. Patients with an abnormal FLC ratio for γ had a significantly shorter treatment-free survival (TFS) than patients with an abnormal ratio for κ or with a normal FLC ratio (median TFS: 34 versus 78 versus 109 months, p=0.042). Evaluation of several disease characteristics in association with FLC of the patients' B-CLL cells showed no significant differences for FLC in the different risk groups (ZAP-70 status, CD38 status, cytogenetics and Binet stage) suggesting no correlation of the FLC with these already established adverse prognostic factors. Conclusion FLC can be detected in a substantial fraction of patients with CLL and the FLC technique improves detection of M-proteins. Moreover, an abnormal FLC ratio is associated with worse outcome, particularly those with a low abnormal FLC ratio. Evaluation of the prognostic significance of abnormal FLC in a larger cohort is currently under way. This data will be presented at the meeting. Future studies are warranted to elucidate the role of FLC as biomarkers of disease and as a prognostic factor for response. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 913 Altered numbers and functions of T-cells have previously been demonstrated in chronic lymphocytic leukemia (CLL) patients. However, dynamics and specific T-cell subset alterations have not been studied in great detail. Therefore, we determined numbers of blood lymphocyte subsets of CLL patients in a longitudinal manner. We found that dynamic expansions of the peripheral blood CD4+ and CD8+ T-cell numbers were consistently associated with a progressively increasing CLL leukemic compartment. Additionally, we performed gene expression profiling (GEP) of blood CD3+ T-cells of CLL patients and normal donors. We identified a list of 135 genes that had significantly increased expression and 11 genes that had significantly decreased expression in CLL T-cells. The up-regulated genes included killer cell lectin-like receptor familiy members KLRA1, KLRC2, KLRD1 (CD94), KLRK1 and KLRF1 as well as CD244 (NK-cell receptor 2B4), CD160 (NK cell receptor BY55), PRF1 (perforin 1) and CRTAM (class-I MHC-restricted T-cell associated molecule). These up-regulated genes are known to be preferentially expressed by CD8+ T-cells with an effector memory phenotype. We used Gene Set Enrichment Analysis (GSEA) to investigate whether CLL T-cell genes correlate with a previously published gene expression signature of effector memory CD8+ T-cells (Willinger et al., Journal of Immunolgy 175[9], 2005). This analysis revealed a highly significant enrichment of CLL T-cell genes within the effector memory CD8+ T-cell signature (p

Description: Abstract 2640 Poster Board II-616 Introduction: Deletion of 13q14 (del13q14) is the most common abnormality of B-chronic lymphocytic leukemia (CLL). However, for a long time investigations of this region did not detect the relevant pathogenetic mechanism. Micro-RNA genes MIRN15a and MIRN16-1 located on 13q14.3 were postulated to close this gap. Down-regulated expression of these miRNAs was shown to increase the anti apoptotic B cell lymphoma 2 (Bcl2) proteins. However, relevance and frequency of deregulated micro-RNA genes was differentially described. To understand the influence of deletion of chromosomal region 13q14 on gene expression, chromosomal abnormalities detected by single nucleotide polymorphism (SNP) chips were compared with gene expression analyzed by gene expression profiling in CLL. Furthermore, impact of deletion 13q14 on MIRN15A and MIRN16-1 expression and correlation to BCL2 protein expression were investigated. Methods: 15 B-CLL cases harboring del 13q14 were investigated for the extend of deletion with the 50k Xbal SNP array. Gene expression of the genes located in the aberrant region evaluated by Affymetrix U133A gene chip of 13 of these cases was compared between cases with and without this aberration. FISH analysis was done to validate SNP-data. Expression of MIRN15a and MIRN16-1 was evaluated by real-time PCR from further 20 B-CLL with TaqMan MicroRNA assays. From the 25 cases Western blot analysis for Bcl2 was performed. Results: SNP-chip and FISH analysis with probes for regions RB1, D13S25 and D13S319 gave consistent results. The minimal deleted region of del13q14 reaches from physical position 49543165 to 50272626 and mostly includes the location of MIRN15a and 16-1. 10 gene probes were located in the aberrant chromosomal 13q14 region and entered statistical analysis. In 4 of 5 genes with monoallelic deletions gene expression was significantly reduced. In regions harbouring mono- and biallelic deletions in 4 of 5 genes the monoallelic deleted cases showed decreased expression compared to the normal cases. Evaluation of MIRN15a and MIRN16-1 revealed strong down-regulation of expression in CLL harbouring biallelic del13q14 compared to other CLL and healthy B-cells (p=0.002; p=0.002), whereas there were no statistically significant differences between CLL with or without monoallelic deletion or healthy B-cells (p=0.534; p=0.665). Bcl2 Western blot analysis revealed stronger Bcl2 expression in CLL harbouring del13q14 in contrast to CLL without this abnormality (p=0.002). However, no difference between cases with mono- or biallelic del13q14 was detected (p=0.400). Conclusion: SNP chip analysis is a helpful tool to detect chromosomal abnormalities on a high resolution. Although detailed genetic analyses failed to demonstrate the consistent involvement of any of the genes located in the deleted region of B-CLL harboring del13q14, reduced expression occurred in most of these genes indicating gene dosage effects. MIRN15a and MIRN16-1 expression are frequently reduced in B-CLL with biallelic but not monoallelic deletion of 13q14 without an influence on Bcl2 protein levels. Disclosures: Off Label Use: intrapleural and intraperitoneal use of rituximab.

Description: BACKGROUND: In chronic lymphocytic leukemia (CLL) short telomeres were shown to be associated with mutational status, progression free survival (PFS) and overall survival (Damle et al., 2004). Chromosomal instability increases with shortening of telomeres. Recently, a relationship between telomere length and number of chromosomal aberrations has been shown if telomere length was investigated by quantitative real-time polymerase chain reaction (Tel-PCR) (Roos et al., 2008). The aim of the present study was to correlate average telomere length of individual cells measured by multicolor flow-FISH to established prognostic factors and genomic aberrations. PATIENTS and METHODS: Blood samples from 64 patients with CLL were analyzed. Flow cytometry was performed for quantification of ZAP-70 and CD 38 expression with a cut off at 20%. Immunoglobulin variable heavy chain (IGVH) genes were sequenced. An IGVH gene sequence with less than 98% homology with the corresponding germ-line sequence was considered to be mutated. Chromosomal alterations were investigated by fluorescence in situ hybridization (FISH) with the following gene probes: LSI 13q14, LSI 13q34, CEP 12, LSI 17p13, LSI 11q22–23. Copy number changes were also detected in 18 samples by SNP-chip analysis. The average length of telomere repeats at chromosome ends was measured by multicolor flow-FISH. Values of telomere length from CLL cells were correlated to values of telomere lengths of B lymphocytes from healthy age matched individuals (delta telomere length=Δtel). RESULTS: The average telomere length of the clonal B-cells was short. Patient samples from advanced Binet stages (B/C) had significantly shorter telomeres (Δtel −4.8 ± 1.0 kb) than patients samples from Binet A (Δtel −3.4 ± 1.2 kb, p=0.03). The average telomere length was significantly shorter for ZAP-70+ (Δtel −5.0 ± 0.5 kb) and CD38+ (Δtel −4.9 ± 0.7 kb) patient samples than for ZAP-70− (Δtel −2.4 ± 0.8 kb) and CD38− (Δtel −3.0 ± 1.0 kb) patient samples, respectively (p