Marker In Chronic Lymphocytic Leukemia Research Articles

The Relative Significance of ZAP-70 Promoter Methylation As a Prognostic Factor in Previously Untreated Chronic Lymphocytic Leukemia: Validation of Results Using a Second Large CLL Research Consortium (CRC) Patient Data Set

AbstractAbstract 3865ZAP-70 expression is a prognostic marker differentiating chronic lymphocytic leukemia (CLL) patients with indolent disease from those rapidly progressing after diagnosis and requiring treatment. We previously demonstrated in four independent clinical cohorts that DNA methylation at a single CpG dinucleotide in the ZAP-70 promoter region impacts on ZAP-70 transcriptional regulation and is prognostic in CLL (Claus et al. J Clin Oncol. 2012;30(20):2483–91). Here, we confirm and extend our previous findings by investigating the significance of ZAP-70 methylation relative to established prognostic biomarkers in CLL: IGHV mutation status or expression of ZAP-70 protein or CD38.DNA methylation analysis of the ZAP-70 core promoter region was conducted in mononuclear cells of 295 untreated CLL patients, previously reported by the CRC (Rassenti et al. Blood 2008;112:1923–30) using MALDI-TOF mass spectrometry (MassARRAY, Sequenom). The 295 patients had a median age of 55 years (range 26–82), 69% were male, and 46% were Rai stage 0, 47% stage I/II, and 7% stage III/IV at diagnosis. ZAP-70 protein and CD38 expression were measured by four-color flow cytometry and IGHV mutational status was assessed, as previously described.By hierarchical clustering, the previously identified single CpG dinucleotide (“CpG unit 1”, position +223 relative to the transcription start) separated patients with high methylation levels across the entire region from those with lower methylation levels at this specific site. With a median follow-up of 3.9 years in 50 patients who had not started treatment and 5.6 years among those still alive (n=196), patients with lower methylation levels at CpG unit 1 had shortened time to first treatment (TTT) and overall survival (OS) (both p<0.0001), confirming the prognostic relevance of this site. In our previous study, an optimal cut-off for CpG unit 1 methylation across four data sets was between 10% and 20%. In this cohort, the optimal cut-off across the two endpoints was between 15% and 25%. A natural gap in CpG unit 1 methylation levels occurred between 20% and 25%, adding potential biological significance, as lower values may represent patient cell samples in which both alleles are unmethylated. Applying a 20% cut-off, TTT and OS were significantly different for those with lower and higher methylation levels (both p<0.0001). The 5-year TTT estimates were 22% (95 % CI: 0.16–0.28) and 64% (95% CI: 0.52–0.74), respectively, for patients with low versus high methylation levels; 5-year OS estimates were 80% (95% CI: 0.73–0.85) and 90% (95% CI: 0.81–0.95). Low ZAP-70 methylation was significantly associated with CLL-cell expression of ZAP-70, CD38, or unmutated IGHV status (each p<0.0001). In a model of TTT, expression of ZAP-70 modified the strong effect of ZAP-70 methylation. In patients with CLL cells lacking ZAP-70 expression, high methylation was protective (hazard ratio [HR]=0.37, 95% CI: 0.23–0.58). In contrast, methylation had no significant effect in ZAP-70+ CLL (HR=1.47, 95% CI: 0.81–2.68); TTT was short regardless of methylation levels (Figure 1). IGHV mutation status and expression of CD38 did not provide significant additional prognostic information. The effect modification described for TTT was not observed for OS (p=0.57). Here, ZAP-70 methylation was the strongest risk factor (hazard ratio=0.32, 95% CI: 0.19–0.54; p<0.0001), and expression of ZAP-70, CD38 or unmutated IGHV did not significantly improve the ability to explain differences in OS.Hence, the prognostic value of ZAP-70 CpG unit 1 methylation was confirmed in a large patient cohort with extensive follow-up. We defined a clinically applicable threshold for ZAP-70 methylation assessment and determined its relative value to current prognostic markers in CLL. ZAP-70 methylation predicted longer TTT among patients with CLL cells lacking ZAP-70 expression, but not among those with ZAP-70+ CLL cells, although this could reflect sampling bias due to the small number of patients (n=16) in our cohort with both high methylation and expression of ZAP-70. Lastly, methylation was the strongest risk factor for OS, and neither IGHV mutation status nor expression of ZAP-70 or CD38 significantly improved the ability to predict prognosis. In conclusion, ZAP-70 DNA methylation represents a clinically valuable biomarker for predicting TTT and OS in previously untreated patients with CLL. [Display omitted] Disclosures:Gribben:Gilead: Honoraria; Mundipharma: Honoraria; GSK: Honoraria; Pharmacyclics: Honoraria; Roche: Honoraria; Celgene: Honoraria. Kay:Celgene: Research Funding.

The Role of the BCR Class Expressed by Eμ-TCL1tg Mice and Human CLL

AbstractAbstract 182The B-cell antigen receptor (BCR) plays a critical role in the development and progression of B-cell lymphomas. In chronic lymphocytic leukemia (CLL), the existence of stereotyped heavy-chain complementarity determining regions (HCDR3) suggested that binding of external antigens might play a role in CLL pathogenesis. In contrast, we recently reported that BCRs derived from both human CLL patients and from Eμ-TCL1tg mice have the unique function to induce antigen-independent signaling. This capacity is mediated by the HCDR3 through binding to a BCR internal motif in adjacent BCRs on the same cell (Dühren-von Minden et al., Nature 2012). Mature B cells as well as CLL B cells co-express IgM and IgD with the same HCDR3. In this study, we address the respective roles of these expressed BCR classes in lymphoma pathogenesis in Eμ-TCL1tg mice and in human CLL.By mating Eμ-TCL1tg-mice with IgM−/− mice, which lacks the μ constant heavy domain (μCH) and instead expresses IgD in all developmental stages, we demonstrate a significantly lower frequency of CD19+CD5+IgM+IgDlow lymphoma cells in heterozygous IgM+/−TCL1 mice compared to conventional IgM+/+TCL1 mice (p=0.007). Furthermore, IgM+/−TCL1 mice show a delayed or slowed disease progression compared to TCL1tg mice carrying both IgM alleles. In both TCL1tg mice strains, lymphoma development was exclusively linked to expression of surface IgM, since no IgD single positive lymphoma was detected in IgM+/−TCL1 mice (n=12). TCL1tg mice that lack both μCH alleles showed an accumulation of CD19+CD5+ cells in the spleen at the age of 6 months. According to the genotype of these mice, this population was indeed IgD single positive. However, no further progression could be observed during follow-up to an age of 8 months, indicating a benign form of lymphoproliferation. In contrast to BCRs derived from Eμ-TCL1 mice, analysis of the signaling properties of BCRs derived from IgM−/−TCL1 mice failed to show any autonomous signaling capacity, even when they were expressed as IgM. To address whether IgD in general is able to mediate autonomous signaling reported for TCL1tg- and CLL-derived BCRs, we tested these receptors for autonomous signaling capacity when expressed as IgD. However, expression as IgD led to a complete loss of autonomous signaling capacity in all cases (n=10).In conclusion, whereas autonomous signaling is a characteristic feature of TCL1tg- and CLL-derived BCRs, the pathogenesis of CLL is dependent on the expression of their BCR as the IgM isotype. Expression of IgD-BCRs leads to loss of autonomous signaling capacity, and mice that lack μCH fail to develop malignant lymphoproliferation.To address the question if differential expression of IgD and IgM also had an impact on the clinical behavior of human CLL, we measured the relative expression levels of surface IgD and IgM on circulating lymphoma cells from 67 CLL patients by flow cytometry with simultaneous staining. According to previous reports (Mockridge et al., Blood 2007), unmutated (UM-CLL) cases (n=22) had a higher level of total surface Ig compared to mutated CLL (M-CLL) cases (n=45). Based on our results that IgM is more potent to drive lymphoproliferation, we calculated the ratio of mean fluorescence intensities for IgD over IgM, further called DvM-Score, for every case. A significant difference in the expression pattern as represented by the DvM-Score was observed for UM-CLL and M-CLL (p=0.003) as well as for ZAP70+ and ZAP70- cases (p=0.0002). Both UM-CLL cases as well as ZAP70+ cases show a higher amount of IgM compared to IgD represented by a DvM-Score of <1, whereas the majority of M-CLL and ZAP70− cases express less IgM than IgD and show a DvM-Score of >1. Based on receiver operating characteristics, an DvM cut-off value of 1.15 was identified to optimally discriminate mutational status (AUC: 0.72) and ZAP70 expression (AUC: 0.78). Preliminary data show that CLL samples with a DvM-Score <1.15 had a more aggressive disease course as indicated by a median time to first treatment (TTFT) of 39 months, whereas cases with a DvM-Score of >1.15 show a median TTFT of 154 months (log rank test, p=0.0014).In summary, our results demonstrate an important role of the BCR class, especially with respect to the pathogenetic role of autonomously active IgM BCRs expressed by CLL B cells, for the outcome of the disease. In addition, the DvM score may represent a convenient and novel prognostic marker for CLL. Disclosures:No relevant conflicts of interest to declare.

Matriptase Is Highly up-Regulated in Chronic Lymphocytic Leukemia and Promotes Cancer Cell Invasion

AbstractAbstract 4612Proteases are known to play a critical role in cancer development and progression. Matriptase is an epithelial transmembrane serine protease essential for skin development and barrier function. Matriptase overexpression has been reported in many human solid tumors including prostate, breast, ovarian, kidney and lung cancers, indicating that matriptase may play an important role in cancer. In mice, dysregulated matriptase activity promotes malignant transformation and carcinogenesis.In human blood, low levels of matriptase mRNA expression were detected in monocytes and B-lymphocytes but the biological significance is unclear. In this study, we examined matriptase mRNA and protein expression in 15 hematological cancer cell lines and 223 bone marrow samples from patients with leukemia by reverse transcription PCR (RT-PCR), quantitative real-time PCR (qRT-PCR) and Western blotting. Flow cytometry and immunostaining were used to determine the cell membrane expression of matriptase in B cell-derived Namalwa cells and bone marrow cells from leukemia patients. Small hairpin RNA (shRNA) interference and a recombinant matriptase inhibitor, hepatocyte growth factor activator inhibitor (HAI)-1, were used to inhibit matriptase expression and activity in Namalwa, Raji and chronic lymphocytic leukemia (CLL) cells to test the effect of matriptase inhibition on cancer cell invasion in matrigel-based assays.Our results showed that matriptase mRNA was highly expressed in Namalwa and Raji cells and in bone marrow cells from patients with CLL. Such an expression profile was not found in bone marrow cells from patients with acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). By Western blotting, matriptase protein expression was confirmed in bone marrow samples from the majority (79/81, 97.5%) of CLL patients. By immunostaining and flow cytometry, matriptase protein was localized on the surface of Namalwa and CLL cells. In these cells, the matriptase inhibitors, HAI-1 and HAI-2, were not detected, indicating that the upregulated matriptase activity may not be inhibited in CLL cells. In functional studies, silencing matriptase gene expression by specific shRNAs markedly reduced Namalwa and Raji cells and bone marrow cells from CLL patients in invading matrigels. Similarly, a recombinant HAI-1 dose-dependently inhibited matrigel invasion of these cancer cells.In conclusion, our results showed that matriptase was highly expressed on the surface of CLL cells. Inhibition of matriptase by gene silencing or specific inhibitor prevented CLL cells in invading matrigels, indicating that matriptase contributes to the invasiveness of the cancer cells. Our results suggest that matriptase may represent a new cell surface marker for CLL and that blocking matriptase expression or activity may be used as a therapeutic strategy to inhibit CLL progression. Disclosures:No relevant conflicts of interest to declare.

Regulatory T-cells in B-cell chronic lymphocytic leukemia: their role in disease progression and autoimmune cytopenias

Regulatory T-cells (Tregs) have been shown to be important for the balance of autoimmunity and oncogenesis. Tregs have a protective role in autoimmune diseases and conversely promote oncogenesis. Chronic lymphocytic leukemia (CLL) is unique in being at the cross-roads of oncogenesis and autoimmunity. We studied Tregs, defined as CD4+CD25highCD127lowFOXP3+, in 32 treatment-naive patients with CLL. Our study shows that patients with CLL had a higher absolute Treg count than the control group (p < 0.001). A progressive increase of Tregs was noted in advanced stages of the disease (p < 0.001). The increase in absolute Treg count is more significant than the increase in percentage Tregs. The absolute Treg count appears to be more important in disease pathogenesis. The absolute Treg count was significantly higher in those patients having autoimmune cytopenias. There was an inverse correlation between lymphocyte doubling time and absolute Treg count (p = 0.03). The absolute Treg count may be used as a prognostic marker in CLL.

Cross‐talk between DNA methylation and active histone modifications regulates aberrant expression of ZAP70 in CLL

Zeta-associated protein of 70 kD (ZAP70) is a recognized adverse prognostic marker in chronic lymphocytic leukaemia (CLL) associated with enhanced B-cell receptor signalling, significantly more aggressive disease course and poor overall survival. Zeta-associated protein of 70 kD is ordinarily expressed in T cells where it has a crucial role in T-cell receptor signalling, whereas its aberrant expression in CLL leads to enhanced B-cell receptor signalling and significantly more aggressive disease course. Although much is known about the activation of ZAP70 following engagement of T-cell receptor, there are little data on the regulation of ZAP70 gene expression in normal T cells or CLL. To understand the molecular events underpinning epigenetic regulation of ZAP70 gene in CLL, we have defined ZAP70 promoter region and outlined the regions crucial in regulating the gene activity. Following a direct comparison of ZAP70+ and ZAP70− primary CLLs, we show ZAP70 promoter in expressing CLLs to be associated with a spectrum of active histone modifications, some of which are tightly linked to aberrant DNA methylation in CLL. Cross-talk between histone modifications and reduced DNA methylation culminates in transcriptional de-repression of ZAP70. Moreover, treatment with histone deacetylase (HDAC) and DNA methylation inhibitors results in recovery of ZAP70 expression, which provides a possible explanation for the failure of HDAC inhibitors in CLL treatment and might serve as a cautionary warning for their future use in treatment of this leukaemia.

Copper levels in patients with hematological malignancies

BackgroundCopper levels are elevated in cancer patients compared to normal subjects. However, few studies have investigated the relationship between copper and hematological malignancies. Methods84 patients with hematological diseases were studied, along with 50 healthy individuals. Copper was measured by flame atomic absorption spectrometry. The patients were classified to 2 homogeneous groups, acute and chronic hematological neoplasms, respectively. For the patients with acute hematological malignancies, relapse and remission were investigated in relation to serum copper levels. For chronic hematological neoplasms, serum copper was connected either with stable or progressive disease. Zeta-chain-associated protein kinase 70 (ZAP70) and CD38 expression, along with the unmutated VH immunoglobulin genes (IgVH) status were also determined for the 22 chronic lymphocytic leukemia (CLL) patients. Results54 patients with relapse or progressive disease had elevated copper levels (mean value 1.8mg/l), whereas 30 patients either in remission or in stable disease had normal copper levels (mean value 1.01mg/l) (normal range 0.8–1.3mg/l). ConclusionHence, our study indicates that serum elevated copper levels are associated with hematological malignancies either in relapse or in disease progression, whereas normal copper levels are linked with hematological neoplasms in remission or in stable disease. Furthermore, we report for the first time an association between high serum copper levels and several adverse prognostic markers in CLL, such as increased expression of ZAP70 and CD38, along with elevated percentage of unmutated IgVH.

Gene expression profiling identifies ARSD as a new marker of disease progression and the sphingolipid metabolism as a potential novel metabolism in chronic lymphocytic leukemia

Several studies demonstrated IGVH mutational status and ZAP70 expression as the most relevant prognostic markers in Chronic Lymphocytic Leukemia (CLL), suggesting the separation of two patient subgroups: with good mutated ZAP70 negative (MTZAP70(-) and poor unmutated ZAP70 positive (UMZAP70(+)) prognosis. We determined the gene expression of B cells in 112 CLL patients divided into three classes: class 1 with MTZAP70(-), class 2 with UMZAP70(+), and class 3 included both UMZAP70(-) and MTZAP70(+). We found LPL, AGPAT2, MBOAT1, CHPT1, AGPAT4, PLD1 genes encoding enzymes involved in lipid metabolism overexpressed in UMZAP70(+). In addition, this study identified ARSD, a gene belonging to the sphingolipid metabolism, as a new gene significantly overexpressed in UMZAP70(+) compared to MTZAP70(-). Western blots confirmed that ARSD protein levels were significantly different between the 3 classes of patients and normal controls. Statistical analysis identified a significant correlation between ARSD and IGVH; however, both ARSD protein level and IGVH were independently associated with the need for therapy of CLL patients. ARSD is a novel prognostic factor as the time to start therapy is shorter in patients with high levels of ARSD protein and sphingolipid metabolism could represent a new biological mechanism in CLL.

Cell surface expression of CD25 antigen (surface IL-2 receptor alpha-chain) is not a prognostic marker in chronic lymphocytic leukemia: results of a retrospective study of 281 patients

Using the database of the Israeli CLL Study Group, we investigated the incidence and prognostic significance of CD25 expression on the surface of lymphocytic leukemia cells. Strong CD25 expression was found in 46 (16.4 %) of 281 tested cases and was correlated with the presence of splenomegaly (p = 0.04), expression of CD38 antigen (p = 0.001), and FMC-7 (p = 0.04). Age, gender, Binet stage, circulating lymphocyte count, presence of anemia or thrombocytopenia, atypical cell morphology, serum beta 2-microglobulin level, and ZAP-70 expression did not differ in patients with or without cell surface CD25. There was no correlation between CD25 expression and time to first treatment or overall survival. CD25 expression does not appear to be a prognostic factor in CLL.

Quantitative detection of ZAP-70 expression in chronic lymphocytic leukemia.

6581 Background: Intracellular ZAP-70 protein was recently recognized as prognostic marker in chronic lymphocytic leukemia (CLL). The specific objective of this study was to develop a simple and sensitive assay, for the quantitative detection of ZAP-70 protein in leukemic cells. Methods: Components of the assay system include sample preparation, immunomagnetic fluorescence assay, Signalyte-II spectrofluorometer. The leukemic cells were isolated from CLL patient blood samples and lysed to release the intracellular ZAP-70 protein. ZAP-70 protein was captured by magnetic beads coated with anti-ZAP70 capture antibody, and recognized by a fluorescent detector antibody, forming an immuno-sandwich complex. This complex was dissociated for measurement of the fluorescence signal, which was proportional to ZAP-70 concentration, using the spectrofluorometer. Results: The assay conditions were extensively optimized by selecting an optimal pair of capture/detector antibodies, conjugation of fluorescence dye, cell lysis condition and excitation/emission wavelengths. The protocol was further validated with two positive controls, Jurkat cell lysate and recombinant ZAP70 protein (rZAP70). The limit of detection was determined to be lower than 125 Jurkat cells and 39 pg of rZAP70 protein. The signal response was linear over a wide range of concentration, from 625 to 40,000 Jurkat cells per test (R2=0.9987) and from 0 to 40,000 pg rZAP70 protein per test (R2=0.9928). The results from 20 CLL patients correlated strongly with flow cytometry analysis. The concordance between the two methods for positive and negative results was 100% (7/7) and 92% (12/13), respectively, while the overall concordance between the two methods was 95%. Conclusions: The assay is a simple, reliable, and reproducible method for quantitative detection of ZAP-70 in patient leukemic cells, without the need for cell fixation or permeabilization. The entire assay could be completed in 5.5 hours. The ZAP-70 signal was linear over a wide dynamic range, which we believe enables quantitative assessment of small changes in ZAP-70 expression over the course of the disease and in response to therapeutic intervention.

Mutational Status and Gene Repertoire of IGHV-IGHD-IGHJ Rearrangements in Serbian Patients With Chronic Lymphocytic Leukemia

The mutational status and configuration of immunoglobulin heavy variable (IGHV) gene rearrangements was analyzed in 85 Serbian patients with chronic lymphocytic leukemia (CLL). We found that 55.3% of cases belonged to mutated and 44.7% to unmutated CLL, progressive disease predominating in the unmutated subset. IGHV gene use resembled that obtained for Mediterranean countries, except for underrepresentation of the IGHV4 subgroup in our cohort. Chronic lymphocytic leukemia (CLL) results from the clonal expansion of mature B lymphocytes and is characterized by extreme clinical heterogeneity. One of the most reliable prognostic markers in chronic lymphocytic leukemia (CLL) is the mutational status of immunoglobulin heavy variable (IGHV) genes, which defines 2 subsets, mutated CLL (M-CLL) and unmutated CLL (U-CLL), with different clinical courses. Biased IGHV gene use between M-CLL and U-CLL clones, as well as population differences in the IGHV gene repertoire have been reported. In this study, mutational status and configuration of IGHV-IGHD-IGHJ rearrangements in 85 Serbian patients were analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing methodology. We found that 55.3% of cases belonged to M-CLL and 44.7% belonged to U-CLL, with progressive disease predominating in the unmutated subset. Most frequently expressed was the IGHV3 subgroup (55.7%), followed by IGHV1 (27.3%), IGHV4 (12.5%), IGHV5 (2.3%), IGHV2 (1.1%), and IGHV6 (1.1%). The distribution of IGHD subgroups was as follows: IGHD3, 39.1%; IGHD2, 21.8%; IGHD6, 12.6%; IGHD1, 10.3%; IGHD4, 8%; IGHD5, 6.9%; and IGHD7, 1.1%. The most frequent IGHJ gene was IGHJ4 (48.9%), followed by IGHJ6 (28.4%), IGHJ3 (11.4%), and IGHJ5 (11.4%). In 15.3% of cases, heavy complementarity-determining region 3 (VH CDR3) amino acid sequences could be assigned to previously defined stereotyped clusters. Our study showed a strong correlation between IGHV gene mutational status and clinical course of CLL. IGHV gene use was comparable to that obtained for Mediterranean countries, with the exception of the IGHV4 subgroup, which was underrepresented in our cohort.

Minimal Residual Disease Quantification Is an Independent Predictor of Progression-Free and Overall Survival in Chronic Lymphocytic Leukemia: A Multivariate Analysis From the Randomized GCLLSG CLL8 Trial

To determine the clinical significance of flow cytometric minimal residual disease (MRD) quantification in chronic lymphocytic leukemia (CLL) in addition to pretherapeutic risk factors and to compare the prognostic impact of MRD between the arms of the German CLL Study Group CLL8 trial. MRD levels were prospectively quantified in 1,775 blood and bone marrow samples from 493 patients randomly assigned to receive fludarabine and cyclophosphamide (FC) or FC plus rituximab (FCR). Patients were categorized by MRD into low- (< 10(-4)), intermediate- (≥ 10(-4) to <10(-2)), and high-level (≥ 10(-2)) groups. Low MRD levels during and after therapy were associated with longer progression-free survival (PFS) and overall survival (OS; P < .0001). Median PFS is estimated at 68.7, 40.5, and 15.4 months for low, intermediate, and high MRD levels, respectively, when assessed 2 months after therapy. Compared with patients with low MRD, greater risks of disease progression were associated with intermediate and high MRD levels (hazard ratios, 2.49 and 14.7, respectively; both P < .0001). Median OS was 48.4 months in patients with high MRD and was not reached for lower MRD levels. MRD remained predictive for OS and PFS in multivariate analyses that included the most important pretherapeutic risk markers in CLL. PFS and OS did not differ between treatment arms within each MRD category. However, FCR induced low MRD levels more frequently than FC. MRD levels independently predict OS and PFS in CLL. Therefore, MRD quantification might serve as a surrogate marker to assess treatment efficacy in randomized trials before clinical end points can be evaluated.

RNA-based markers as prognostic factors in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is most often indolent at diagnosis but has a highly variable clinical course, and many patients will eventually progress and require treatment. Currently, there are a number of clinical and molecular markers known to be predictive of prognosis in CLL that can be applied to discriminate patients that are more likely to develop a progressive disease. Gene-expression profiling studies have identified genes with differential expression between prognostic subgroups in CLL, and research on these RNA-based prognostic markers has expanded during recent years. For example, high lipoprotein lipase and CLLU1 mRNA expression have recently been shown to be strong markers of poor clinical outcome. In this review we will provide a summary of the most significant prognostic markers in CLL, focusing on the recent category of RNA-based markers in particular.

The CD49d/CD29 complex is physically and functionally associated with CD38 in B-cell chronic lymphocytic leukemia cells

CD49d and CD38 are independent negative prognostic markers in chronic lymphocytic leukemia (CLL). Their associated expression marks a disease subset with a highly aggressive clinical course. Here, we demonstrate a constitutive physical association between the CD49d/CD29 integrin complex and CD38 in primary CLL cells and B-cell lines by (i) cocapping, (ii) coimmunoprecipitation and (iii) cell adhesion experiments using CD49d-specific substrates (vascular-cell adhesion molecule-1 or CS-1/H89 fibronectin fragments). The role of CD38 in CD49d-mediated cell adhesion was studied in CD49d(+)CD38(+) and CD49d(+)CD38(-) primary CLL cells, and confirmed using CD38 transfectants of the originally CD49d(+)CD38(-) CLL-derived cell line Mec-1. Results indicate that CD49d(+)CD38(+) cells adhered more efficiently onto CD49d-specific substrates than CD49d(+)CD38(-) cells (P < 0.001). Upon adhesion, CD49d(+)CD38(+) cells underwent distinctive changes in cell shape and morphology, with higher levels of phosphorylated Vav-1 than CD49d(+)CD38(-) cells (P = 0.0006) and a more complex distribution of F-actin to the adhesion sites. Lastly, adherent CD49d(+)CD38(+) cells were more resistant to serum-deprivation-induced (P < 0.001) and spontaneous (P = 0.03) apoptosis than the CD49d(+)CD38(-) counterpart. Altogether, our results point to a direct role for CD38 in enhancing CD49d-mediated adhesion processes in CLL, thus providing an explanation for the negative clinical impact exerted by these molecules when coexpressed in neoplastic cells.

Expression of myelomonocytic antigens in Egyptian B-cell chronic lymphocytic leukemia ( impact on prognosis)

Background B-cell chronic lymphocytic leukemia (CLL) is a clonal malignancy of mature B cells that displays immense clinical heterogeneity. Therefore, there has been considerable interest in identifying prognostic markers that can be used to distinguish those patients who may have an aggressive form of CLL. Clinical staging systems lack the ability to predict disease progression. Numerous cellular and molecular markers with potential prognostic and therapeutic significance have been identified. Among these markers are the myelomonocyte-associated markers whose expression on neoplastic lymphocytes in CLL has been reported to correlate with an unfavorable prognosis. Aim of the study We aimed to study the expression of the myelomonocytic markers CD13 and CD11b in Egyptian CLL patients in comparison with a healthy control group and assess their relation to other prognostic factors and patient outcomes. This study also aimed to reveal the association of CD38 and FMC7 with prognosis and disease progression. Participants and methods Expressions of CD13 and CD11b were detected by flow cytometry in 30 newly diagnosed CLL cases and 10 healthy individuals. Percentages of CD38-positive and FMC7-positive cells were also measured. Results CD13 was expressed in three cases (10%), whereas CD11b was expressed in four cases (13.3%) of CLL. CD38 was expressed in 13 cases (43.3%) and FMC7 in 11 cases (36.6%). None of the individuals in the healthy control group expressed CD13 or CD11b. The expression of CD13 and CD38 was significantly correlated with adverse results of complete blood count ( P P =0.04). Expression of all studied markers (CD13, CD11b, CD38, and FMC7) was significantly correlated with the presence of 11q deletion (11qdel) ( P =0.004, P P =0.001, and P P =0.005, 0.01, P P P =0.002, 0.01, P P P =0.02 and P P =0.01). Conclusion CD13 and CD11b expressions were found to be correlated with the standard adverse prognostic factors in CLL and with shorter survival, which questions their future possible role as prognostic markers in CLL. FMC7 and CD38 expression was also associated with unfavorable prognosis and poor outcome.

Lipoprotein lipase: a new prognostic factor in chronic lymphocytic leukaemia.

The clinical course of patients with chronic lymphocytic leukemia (CLL) is highly heterogeneous. Gene expression analyses have revealed that leukemic cells with unmutated immunoglobulin heavy chain genes (IgV H) differ from CLL cells with mutated IgV H in the expression level of some genes, i.e. encoding kinase ZAP-70 and antigen CD38. Recently, additional markers in CLL, including the expression level of apoptosis-regulating genes/proteins (Bcl-2, Mcl-1) and microRNAs, have been suggested. In this review, we attempt to provide data concerning the properties of lipoprotein lipase (LPL), as well as to present its prognostic value in CLL. LPL mRNA expression level was able to predict mutational status in a high percentage of CLL cases and high LPL expression was associated with shorter treatment-free survival. Importantly, since LPL activity is low (or absent) in other blood cell types, its expression can be determined by PCR technique in peripheral blood mononuclear cells or in lysed blood samples.

Front-Line Therapy for Chronic Lymphocytic Leukemia

Historically, alkylator-based therapy has been used to treat patients with chronic lymphocytic leukemia (CLL). More effective therapies, such as the use of monoclonal antibodies in combination with chemotherapy, have been shown to prolong both progression-free survival and overall survival. Improvements in the identification of prognostic markers for CLL, as well as novel combinations for chemoimmunotherapy regimens, have improved the outcome for patients with CLL. We examine the diagnosis of CLL, the role of prognostic factors in determining treatment goals, and current data on front-line management of CLL. The benefits of single-agent and combination therapies are associated with prolonged progression-free and overall survival. While more aggressive management may therefore be warranted, each patient's comorbidities and performance status must be weighed against the benefits, availability, cost, treatment goals, and incidence of adverse effects associated with each therapy. New single agents and novel treatment combinations have shown promising results in phase I/II studies. The ultimate therapeutic goals of prolonged survival and improved quality of life will be validated only by ongoing clinical and laboratory research and by continuous enrollment of patients in clinical trials.

ZAP-70 Expression Measured by Quantitative PCR (QPCR) As a Prognostic Marker in Chronic Lymphocytic Leukaemia (CLL)

Abstract 4602 AimZAP-70 expression provides important prognostic information in CLL, however, routine flow cytometric assessment has been limited by difficulties in method standardisation. We aimed to test whether ZAP-70 expression measured by quantitative PCR was superior to flow cytometric assessment. MethodsWe determined ZAP-70 expression by QPCR on CD19 selected peripheral blood B-cells in 95 patients with CLL and compared this to ZAP-70 measured by flow cytometry. Utility as a predictor of IgVH mutation status and clinical outcome was compared along with other standard prognostic markers. Results95 patients were analysed: median age 62years, 62% male, Binet stage at diagnosis (A 84%, B 11%, C 5%). 46% progressed to require treatment at a median time to first treatment of 7.6 years. Median overall survival has not been reached at a median follow-up of 5 years. 72% were ZAP-70 positive (defined as >20%) by flow cytometry, 32% were CD38 positive and 41% had unmutated IgVH status. Median ZAP-70 by QPCR was 3,437 per 10,000 copies of ABL (range, 163 to 30,452). ZAP-70 by flow cytometry and QPCR were significantly correlated (Spearman's correlation coefficient 0.52, p=0.002). Positive ZAP-70 by flow cytometry had a sensitivity of 100% and specificity of 56% for an unmutated IgVH status. Using a cut-off of 5000, ZAP-70 by QPCR had a sensitivity of 91% and specificity of 88% for an unmutated IgVH status.Correcting for Binet stage, a shorter time to first treatment was predicted by higher ZAP-70 by QPCR (p=0.001), CD38 positivity (p=0.004) and unmutated IgVH status (p=0.003), but not ZAP-70 positivity by flow cytometry (p=0.09). ConclusionZAP-70 expression determined by QPCR provides better prediction of IgVH mutation status and time to first treatment in patients with CLL than does ZAP-70 expression as measured by flow cytometry.This research was supported by a grant from the Leukaemia Foundation of Australia. Disclosures:Banh:Leukaemia Foundation of Australia: Research Funding.

Heat Shock Protein 90 (Hsp90) Activity Status Is An Independent Prognostic Marker in Chronic Lymphocytic Leukemia (CLL) and Correlates with IgVH Mutational Status, ZAP-70 Expression and Time to First Treatment

Abstract 2544The clinical presentation of chronic lymphocytic leukemia (CLL) is variable and includes at least two distinct clinical subtypes: indolent or progressive disease. Several prognostic markers such as the mutational status of the IgVH genes or ZAP-70 expression identify patients that will have more aggressive clinical courses. We have previously reported that ZAP-70 expression in CLL requires the support and stability provided by the Hsp90 activated complex (Hsp90-AC) and that Hsp90 inhibitors induce apoptosis in CLL cells preferentially in patients with high–risk/rapid progressive disease.Hsp90 requires the conformation of a multimeric complex with other co-chaperones (Hop, p23) to become active and function as a chaperone for mutated, over-expressed, misfolded or constitutively activated proteins. However, to measure Hsp90-AC activity is challenging and requires protein assays that are semi-quantitative and not always can provide a functional read out. To address this problem we designed a test to measure Hsp90-AC by taking advantage of the higher avidity of this complex to bind Hsp90 inhibitors such as 17-AAG. We hypothesize that Hsp90-AC activity is an independent prognostic marker of disease progression in CLL and to test this we measured the ability of 17-AAG to inhibit Hsp90-AC and promote apoptosis in vitro in a cohort of previously untreated CLL patients. This evaluation was performed in samples collected at the time of diagnosis and the results were correlated with the time from diagnosis to first treatment (t-Tx1) as well as the presence of other known prognostic markers.Ninety-five previously untreated CLL patients were included in this cohort. In vitro sensitivity/apoptosis to Hsp90-AC inhibition using 17-AAG (1μg/ml) was measured by flow cytometry using DiOC6 and PI after 48 hours of incubation. Only samples with more than 60% viability were included and we selected a cut-point of ≥ 53% induction of apoptosis to determine if a sample was sensitive or not to Hsp90-AC inhibition (This cut-point was selected using a ROC analysis for optimal diagnostic performance).We found that 37 patients (39%) were sensitive to 17-AAG. These patient had a median time from diagnosis to first treatment (t-Tx1) of 7 years compared to 4.2 years in patients that were resistant to 17-AAG (p=0.04). In addition, 37 patients (39%) had unmutated IgVH genes with a median t-Tx1 of 8.7 years compared to 4.2 years in patients with mutated IgVH genes (p=0.02) and 41 patients (43%) had high levels of ZAP-70 expression (>20% by flow cytometry) and this group of patients had a median t-Tx1 of 7 years vs. 4.2 years in ZAP-70 negative patients (p=0.03). We evaluated the association between the degree of Hsp90-AC inhibition (17-AGG sensitivity) and IgVH mutational status and found a strong correlation between these two variables with shorter t-Tx1 in patients that were IgVH unmutated and sensitive to Hsp90 inhibitor (p=0.0003). We found a similar strong correlation between ZAP-70 expression and sensitivity to Hsp90-AC inhibitor (p=0.003). In our patient cohort, we saw that IgVH, ZAP-70 and Hsp90 inhibitor sensitivity are independent prognostic markers in CLL (Cox regression, p=0.002). We found that the sensibility and specificity of ZAP-70 test for high-risk CLL (sensitivity 81.1% [95% IC 67–95%] and specificity 77.6%[95% IC 65–90%]) was increased when the Hsp90-AC inhibitor sensitivity test was added (sensitivity 87% [95% IC 71–100%] and specificity 91%[95% IC 79–100%]).In conclusion, in vitro inhibition of Hsp90-AC in CLL using 17-AAG is an independent prognostic maker and strong predictor of the need for treatment. Hsp90-AC inhibition showed a high correlation with IgVH mutational status and ZAP-70 expression. Additionally, patients sensitive to 17-AAG whose leukemic cells also highly express ZAP-70 appear to have the worst prognosis with the shortest t-Tx1. Our data suggest that in vitro sensitivity to 17-AAG at the time of diagnosis serves as a quantitative surrogate marker for Hsp90-AC activity and correlates with disease progression in CLL. This is the first time that such correlation has been established in a cohort of cancer patients and shows the relevance of the Hsp90 chaperone system in cancer biology and disease progression. Disclosures:No relevant conflicts of interest to declare.

Sex Chromosome Loss May Represent a Disease-Associated Clonal Population in Chronic Lymphocytic Leukemia (CLL),

Abstract 3534Sex chromosome loss is observed in the healthy population and increases with advancing age. It is also observed in hematological malignancies with variable clinical value. Its significance as a disease marker in CLL is not clearly defined. We undertook this study to evaluate whether Y or X loss is an age- and/or a CLL-associated abnormality, since it has not yet been reported.Out of 198 patients with stage B/C CLL analyzed by karyotype (K), 6 (3%) exhibited a loss of sex chromosome (Sutton et al, blood 2011). We also identified 14/745 (2%) additional patients in our CLL-database (2002–11). All patients had a Matutes score ≥ 4. K was performed on peripheral blood using TPA until 2005, and then CPG + IL2. FISH analyses were carried out to detect trisomy 12 and 11q22 (ATM), 13q14, 17p13 (TP53), X/Y deletions. IGVH gene mutational status was analyzed by PCR with a sequence homology cutoff of 98%. B- and T-cells were sorted by flow cytometry from peripheral blood of 5 patients (purity ≥ 94%) using CD19+ and CD3+ respectively. Polynuclear and mononuclear cells were enriched by Ficoll (purity ≥ 99%) for 4 patients.There were 13 males and 7 females (n =20). At the time of the K, there were 11 Binet stage A and 9 stage B, and median age was 64 (36–78). Regarding IGVH status, 9/12 (75%) cases were mutated. We observed 13 -Y and 7 -X, chromosome loss being the sole abnormality in 10 (50%) cases. The percentage of cells with -X or -Y ranged between 3 to 20/20 mitoses. K was complex (≥ 3 abnormalities) in 4 cases. When associated with another chromosomal abnormality, -X or -Y was the primary change in 2 cases, in the same clone in 7 cases, and as a sub-clone change in one case. FISH identified 16/20 (80%) del13q, 4/20 (20%) tri12, 3/20 (15%) delATM, 2/20 (10%) delTP53. Among del13q, there were 8 monoallelic, 7 concomitant mono- and biallelic, and 1 biallelic deletion. Sex chromosome loss was confirmed by interphase FISH in 14 patients, with percentage of loss between 5 to 84% (median: 68%). Co-hybridization with two probes including the X/Y probe was performed for 13 patients. The two analyzed abnormalities were present in independent clones in 4 cases, in the same cells in 2 cases; -X or -Y appeared as the primary change in 4 cases, and as a sub-clone change in 3 cases. The analyses of polynuclear and mononuclear populations in 4 patients showed a maximum of 2% of polynuclear cells with X or Y loss, whereas the mononuclear cells exhibited a significant higher loss frequency (range 6–87%, p =.03). The analyses of B- and T-cells in 5 samples showed a significant increase in losses frequency in B-cells (range 88–96%) compared to T-cells (2%, 2%, 3%, 4%, 6%) (p =.008).The incidence of -X in peripheral blood lymphocytes is about 3% of the cells in healthy women aged 16–50, rising to 5% in women aged over 85 years. The incidence of Y loss in men is distinctly lower, with a frequency less than 1% of the cells before 85 years (Wodja et al, J Appl Genet, 2003). The median of cells with sex chromosome loss in our series is 68%, with only 2 patients with low percentage: a 66-year old female (5%) and a 68-year old male (6%). However these percentages are higher than those observed in healthy people. Moreover, we never observed the loss of a sex chromosome in more than 2% in polynuclear cells of any of the patients, indicating that sex chromosome loss in CLL is associated with the tumor cells. It is not clear whether the low proportion of loss of sex chromosome in CD3+ cells that exceed in one case only slightly our laboratory threshold of 5% is due to contamination. Sex chromosome loss may occur during K evolution, as well as the acquisition of additional abnormalities may occur in cells with primary sex chromosome loss. Even if loss appears through an age-related effect in elderly cells, our data support an oncogenic property of this abnormality. One patient with –Y as the sole aberration in CLL, has developed a myelodysplastic syndrome upon treatment, with complex karyotype without -Y, suggesting that Y loss had been associated to CLL. In addition, del13q, especially when biallelic, appears to be closely associated to the loss of sex chromosome, as compared to our database (del13q: 16/20 (80%) vs 141/279 (50%), p =.01; biallelic del13q: 8/16 (50%) vs 24/141(17%), p =1×10−5).In conclusion, sex chromosome loss has to be considered as a clonal abnormality in CLL, is significantly associated with biallelic 13q deletion, and may participate to oncogenic transformation. Disclosures:No relevant conflicts of interest to declare.

Combined normal donor and CLL: Single tube ZAP‐70 analysis

Zeta-chain-associated protein kinase 70 (ZAP-70) has been identified as an independent prognostic marker in chronic lymphocytic leukemia (CLL). Based on our previous studies, we have developed a combined one-tube technology with multiple internal controls to optimize ZAP-70 assessment. Forty-eight untreated CLL cases were examined for ZAP-70 expression using a modified 7-color one-tube assay. Normal donor (ND) whole blood is stained with CD3 APC-Cy7 and CD19 APC. In a second tube, patient whole blood is stained with CD5 PE-Cy7, CD19 PerCP-Cy5.5, and CD20 eFluor450. After surface staining and fixation, these two tubes are combined. After saponin permeabilization, the cells were stained with two anti-ZAP-70 clones (1E7.2/AF488 and SBZAP/PE). The results obtained from this modified tube were compared with those obtained concurrently using the non-mixed single sample tubes. Five different methods of ZAP-70 expression analysis were evaluated: percentage positive cells using ND T-cells as a reference; the internal patient T-cell/clone ratio; ND T-cell/clone ratio; clone/ND B-cell ratio; and modified Z-index. Overall, the combined patient and ND mix tube performed better than the non-mixed single sample tube. The strongest correlations between ZAP-70 expression and immunoglobulin heavy chain variable (IGHV) mutational status were seen with percentage positive ND T-cell, ND T-cell/clone ratio, and clone/ND B-cell ratio for both 1E7.2 and SBZAP clone (P < 0.0001). The modified one tube method combining the ND and patient sample provides highly reliable results that correlate with the IGHV mutational status. This method should be considered as part of the next step in standardization of the ZAP-70 assay in CLL.