B-cell chronic lymphocytic leukemia (B-CLL) is a heterogenous disease with a highly variable clinical course. Recent studies have shown that expression of the protein tyrosine kinase ZAP-70 may serve as a prognostic marker in B-CLL. Employing a semiquantitative RT-PCR assay, we examined purified leukemia B cells of 39 CLL patients for the expression of ZAP-70 mRNA transcripts. Significant ZAP-70 mRNA levels exceeding those found in control samples with 5% T cells were detected in 36% of the CLL cases. Patients in the ZAP-70 positive cohort were characterized by an unfavorable clinical course with a significantly shorter progression-free survival as compared to the ZAP-70-negative patients (64%). These results were confirmed by flow-cytometric analysis of the ZAP-70 protein, and expanded to a larger patient cohort (n ¼ 67). A combined statistical analysis of 79 patients showed that the two patient subgroups also differed with regard to overall survival and a panel of known clinical prognostic factors including LDH, thymidine kinase serum levels and expression of the CD38 surface antigen by the leukemic cell clone. The level of ZAP-70 expression did not change over time in the majority of patients where sequential samples were available for analysis.

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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

Purpose Although CD49d is an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL), definitive validation evidence is lacking. A worldwide multicenter analysis was performed using published and unpublished CLL series to evaluate the impact of CD49d as an overall (OS) and treatment-free survival (TFS) predictor. Patients and Methods A training/validation strategy was chosen to find the optimal CD49d cutoff. The hazard ratio (HR) for death and treatment imposed by CD49d was estimated by pooled analysis of 2,972 CLLs; Cox analysis stratified by center and stage was used to adjust for confounding variables. The importance of CD49d over other flow cytometry–based prognosticators (eg, CD38, ZAP-70) was ranked by recursive partitioning. Results Patients with ≥ 30% of neoplastic cells expressing CD49d were considered CD49d+. Decrease in OS at 5 and 10 years among CD49d+ patients was 7% and 23% (decrease in TFS, 26% and 25%, respectively). Pooled HR of CD49d for OS was 2.5 (2.3 for TFS) in univariate analysis. This HR remained significant and of similar magnitude (HR, 2.0) in a Cox model adjusted for clinical and biologic prognosticators. Hierarchic trees including all patients or restricted to those with early-stage disease or those age ≤ 65 years always selected CD49d as the most important flow cytometry–based biomarker, with negligible additional prognostic information added by CD38 or ZAP-70. Consistently, by bivariate analysis, CD49d reliably identified patient subsets with poorer outcome independent of CD38 and ZAP-70. Conclusion In this analysis of approximately 3,000 patients, CD49d emerged as the strongest flow cytometry–based predictor of OS and TFS in CLL.

Soluble or membrane-anchored ligands of NKG2D and their receptor have a critical role in the elimination of tumor cells and disease progression. Plasma samples of 98 patients with B-cell chronic lymphocytic leukemia (CLL) were analyzed with specific ELISA systems for soluble major histocompatibility complex class I-related chains (sMICA and sMICB) and UL-16-binding proteins (ULBP1, 2, and 3). The flow cytometric analysis of MICA on CLL cells and natural killer group 2 member D (NKG2D) receptors on NK cells was performed after thawing of frozen peripheral blood lymphocytes of CLL patients (N ¼ 51). Levels of sMICA, sMICB, and sULBP2 were significantly increased (Po0.001) compared with 48 controls, whereas sULBP1 3 were not detectable in patients and controls. Levels of sMICA4990 pg/ml (P ¼ 0.014), sMICB4200 pg/ml (P ¼ 0.0001), and sULBP24105 pg/ml (Po0.0001) were associated with poor treatment-free survival (TFS). Neither MICA nor NKG2D expression could be related to clinical parameters. In multivariate analysis Binet stage (P ¼ 0.002), sULBP2 (P ¼ 0.002) and ZAP-70 (P ¼ 0.002) were independent predictive factors for TFS. In patients with Binet stage A, sULBP2 levels4105 pg/ml were strongly associated (P ¼ 0.0025) with poor TFS. Our data show that soluble but not membrane-anchored NKG2D ligands or receptors are of prognostic significance in CLL. Moreover, sULBP2 seems to be useful to identify early-stage patients with risk of disease progression.

Purpose Although CD49d is an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL), definitive validation evidence is lacking. A worldwide multi-center analysis was performed using published and unpublished CLL series to evaluate the impact of CD49d as overall survival (OS) and treatment free survival (TFS) predictor. Patients and Methods A training/validation strategy was chosen to find the optimal CD49d cut-off. The hazard ratio (HR) for death and treatment imposed by CD49d was estimated by pooled analysis of 2,972 CLL, and Cox analysis stratified by center and stage was used to adjust for confounding variables. The importance of CD49d over other flow cytometry-based prognosticators (CD38, ZAP-70) was ranked by recursive partitioning. Results Patients with ≥30% of neoplastic cells expressing CD49d were considered CD49d+. The decrease in OS at 5 and 10-years among CD49d+ cases was 7% and 23% (decrease in TFS 26% and 25% respectively). The pooled HR of CD49d for OS was 2.5 (2.3 for TFS) in univariate analysis. This HR remained significant and of similar magnitude (HR=2.0) in a Cox model adjusted for clinical and biological prognosticators. Hierarchical trees including all cases, or restricted to early stage or patients ≤65 years, always selected CD49d as the most important flow-cytometry-based biomarker, with negligible additional prognostic information added by CD38 or ZAP-70. Consistently, by bivariate analysis, CD49d reliably identified patients' subsets with poorer outcome independent of CD38 and ZAP-70. Conclusions In this analysis of ∼3000 patients, CD49d emerged as the strongest flow cytometry-based predictor of OS and TFS in CLL. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Burger:Pharmacyclics: Research Funding.

B-cell chronic lymphocytic leukaemia (B-CLL) is a heterogenous disease with a highly variable clinical course and analysis of zeta-associated protein 70 (ZAP-70) and CD38 expression on B-CLL cells allowed for identification of patients with good (ZAP-70 À CD38 À ) and poor (ZAP-70 þ CD38 þ ) prognosis. DNA microarray technology was employed to compare eight ZAP-70 þ CD38 þ with eight ZAP-70 À CD38 À 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, that is, immune receptor translocation-associated protein 4 (IRTA4)/Fc receptor homologue 2 (FcRH2), angiopoietin 2 (ANGPT2) 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 À CD38 À B-CLL cells. In healthy individuals, IRTA4/FcRH2 protein expression was associated with a CD19 þ CD27 þ memory cell phenotype. ANGPT2 plasma concentrations were twofold higher in the poor prognosis subgroup (Po0.05). Pim2 was significantly overexpressed in poor prognosis cases and Binet stage C. Disease progression may be related to proangiogenic processes and strong Pim2 expression.

PRAME is a tumor-associated antigen, which belongs to the family of cancer-testis antigens (CTA). The expression of CTA is mainly restricted to the testis and various tumors. In contrast to other CTA, PRAME expression is also frequently detected in acute and chronic leukemias. Due to this expression pattern, PRAME has attracted great interest as a prognostic tumor marker that can be used for the detection of minimal residual disease and as a potential target for immunotherapy. In acute myeloid leukemia (AML), PRAME expression has been observed in 30-64% of cases. To evaluate whether epigenetic mechanisms contribute to PRAME activation in AML, we studied DNA methylation of 15 CpG dinucleotides within a CpG-rich region located in the intron 1 of the PRAME gene. DNA methylation was determined by sequence analysis of cloned PCR products generated from bisulfite-treated genomic DNA. Methylation patterns were correlated with PRAME mRNA levels as determined by microarray analysis and real-time PCR. We found almost complete methylation in mononuclear blood cells from two healthy donors and in bone marrow cells of four PRAME-negative AML patients. In contrast, the degree of PRAME methylation was clearly reduced in four PRAME-positive AML bone marrow samples. In particular, these samples were characterized by the presence of clones, which were completely devoid of methylation. The significant inverse correlation between the degree of methylation and PRAME expression suggests a causal role of DNA methylation in PRAME regulation. Such a role is further supported by the observation that treatment of PRAME-negative cell lines U-937 and THP-1 with the demethylating agent 5'-Aza-2'dC resulted in a dose-related upregulation of PRAME expression.