The prognostic significance of soluble NKG2D ligands in B-cell chronic lymphocytic leukemia.

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=51). Levels of sMICA, sMICB, and sULBP2 were significantly increased (P<0.001) compared with 48 controls, whereas sULBP1 3 were not detectable in patients and controls. Levels of sMICA>990 pg/ml (P=0.014), sMICB>200 pg/ml (P=0.0001), and sULBP2>105 pg/ml (P<0.0001) 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=0.002), sULBP2 (P=0.002) and ZAP-70 (P=0.002) were independent predictive factors for TFS. In patients with Binet stage A, sULBP2 levels>105 pg/ml were strongly associated (P=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.

Percentage of smudge cells determined on routine blood smears is a novel prognostic factor in chronic lymphocytic leukemia.

Recently developed molecular prognostic tests in patients with early Binet stage chronic lymphocytic leukemia (B-CLL) are costly and often require a high level of technologic expertise. Recent data give evidence for the prognostic relevance of the percentage of smudge cells in B-CLL. In our study we analysed the prognostic potential of this novel marker in a cohort of 100 CLL patients. The percentage of smudge cells ranged from 0% to 70% (median 21%). Patients with 20% smudge cells. Multivariate Cox regression analysis identified percentages of smudge cells, stage according to Binet and CD38 expression as independent prognostic markers. The percentage of smudge cells was significantly lower in CD38+, ZAP-70+ and unmutated IgVH patients. Combined analysis of smudge cell percentages with CD38 expression provided complementary prognostic information identifying three patient subgroups with good, intermediate and poor prognosis. Comparing gene expression profiles in a subset of 12 patients we identified eight differentially expressed genes in groups with high vs. low percentage of smudge cells suggesting a role of these differentially expressed genes, especially for Tribbles homolog 2 (Trib2), in the disease progression of high risk CLL patients. In conclusion, our data confirm previous studies showing that the simple and inexpensive microscopic detection of smudge cells on blood smears prepared for routine diagnostic purposes is a novel independent factor predicting overall survival in CLL.

Bone marrow fibroblasts induce expression of PI3K/NF-kappaB pathway genes and a pro-angiogenic phenotype in CLL cells.

Microarray-based gene expression profiling (GEP) was used to study how stroma modulates the survival of CLL cells in an in vitro coculture model employing the murine fibroblast cell line M2-10B4. CLL cells cultured in direct contact with the stromal layer (STR) showed a significantly better survival than cells cultured in transwell (TW) inserts above the M2-10B4 cells. STR as compared to TW conditions induced a significant up-regulation of PI3K/NF-kappaB pro-survival pathway genes and mediated a pro-angiogenetic switch in the CLL cells by up-regulation of vascular endothelial growth factor (VEGF) and osteopontin (OPN) and down-regulation of the anti-angiogenetic molecule thrombospondin-1 (TSP-1).

Gene expression signatures separate B-cell chronic lymphocytic leukaemia prognostic subgroups defined by ZAP-70 and CD38 expression status.

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-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, 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-CD38- B-CLL cells. In healthy individuals, IRTA4/FcRH2 protein expression was associated with a CD19+CD27+ memory cell phenotype. ANGPT2 plasma concentrations were twofold higher in the poor prognosis subgroup (P<0.05). Pim2 was significantly overexpressed in poor prognosis cases and Binet stage C. Disease progression may be related to proangiogenic processes and strong Pim2 expression.

Combined analysis of ZAP-70 and CD38 expression as a predictor of disease progression in B-cell chronic lymphocytic leukemia.

Prognostic predictions in B-cell chronic lymphocytic leukemia (B-CLL) at early clinical stage are based on biological disease parameters, such as ZAP-70 and CD38 protein levels, genomic aberrations as well as immunoglobulin variable heavy chain gene (IgV(H)) mutation status. In the current study, ZAP-70 and CD38 expressions were examined by flow cytometry in 252 patients with B-CLL. Cytoplasmic ZAP-70 expression in more than 20% (ZAP-70(+)) and surface CD38 expression on more than 30% (CD38(+)) of B-CLL cells were associated with an unfavorable clinical course. The levels of ZAP-70 and CD38 did not change over time in the majority of patients where sequential samples were available for analysis. Combined analysis of ZAP-70 and CD38 yielded discordant results in 73 patients (29.0%), whereas 120 patients (47.6%) were concordantly negative and 59 patients (23.4%) were concordantly positive for ZAP-70 and CD38 expression. Median treatment-free survival times in patients whose leukemic cells were ZAP-70(+)CD38(+) was 30 months as compared to 130 months in patients with a ZAP-70(-)CD38(-) status. In patients with discordant ZAP-70/CD38 results, the median treatment-free survival time was 43 months. Thus, ZAP-70 and CD38 expression analyses provided complementary prognostic information identifying three patient subgroups with good, intermediate and poor prognosis. Over-representation of high-risk genomic aberrations such as 17p deletion or 11q deletion and distribution of the IgV(H) mutation status in B-CLL discordant for ZAP-70/CD38 pointed toward a distinct biologic background of the observed disease subgroups. This finding was also supported by microarray-based gene expression profiling in a subset of 35 patients. The expression of 37 genes differed significantly between the three groups defined by their expression of ZAP-70 and CD38, including genes that are involved in regulation of cell survival and chemotherapy resistance.

[Prognostic significance of expression of p53, bcl-2 and bax in squamous epithelial carcinoma of the larynx--a multivariate analysis]. / Prognostische Bedeutung der Expression von p53, bcl-2 und bax im Plattenepithelkarzinom des Larynx--Eine multivariate Analyse.

BACKGROUND AND OBJECTIVE: Conventional clinicopathologic parameters do not accurately reflect the clinical outcome of patients with head and neck carcinoma. The establishment of additional prognostic factors that may give insight into the biologic features of a tumor is therefore an essential goal. The present study analyses the expression patterns of p53, bcl-2, and bax with regard to their relationships with conventional tumor parameters and to their prognostic significance in patients with laryngeal squamous cell carcinoma. PATIENTS/METHODS: Paraffin-embedded tissue sections of 88 primary laryngeal squamous cell carcinomas diagnosed and treated between 1986 and 1996 were investigated for p53, bcl-2, and bax protein expression by immunohistochemistry. The mean follow-up time was 45.9 months. RESULTS: Bcl-2 immunoreactivity was positively correlated with an advanced clinical stage, a high T category, regional lymph node metastasis, and a high histological grading. Significant relationship between clinicopathologic parameters and p53 or bax expression were not detectable. The age of the patients, advanced disease, positive bcl-2 expression, and a high level of p53 expression were significantly associated with shortened disease-specific survival in univariate analysis. In multivariate analysis, age, clinical stage, and p53 expression had independent prognostic value. CONCLUSIONS: Although expression of p53 and bcl-2 was found to be clinically relevant in univariate analysis, only p53 but not bcl-2 was an independent predictor of patient outcome. This superiority of p53 in multivariate analysis points to its central role within cell cycle and death regulation, with which it influences two important parameters of tumor progression.

Prognostic significance of p53/bcl-2 co-expression in patients with laryngeal squamous cell carcinoma.

OBJECTIVE: The p53, bcl-2, and bax genes are known to be involved in control of cell cycle progression and regulation of apoptotic cell death. Although they are frequently altered in laryngeal squamous cell carcinoma, their clinical relevance is not yet fully understood. In the present study, individual and combined expressions of these genes were related with patient survival as well as with proliferative and apoptotic activity. DESIGN: Retrospective study. METHODS: Paraffin-embedded tissue sections of 88 laryngeal squamous cell carcinomas that were diagnosed and treated between 1986 and 1996 were investigated for p53, bcl-2, and bax protein expression by immunohistochemistry. Apoptotic cells were visualized using the nick end labeling method. To assess proliferative activity of tumors, mitotic indices were determined. RESULTS: Age of patients, advanced disease (stages HI and IV), high mitotic activity, positive bcl-2 expression, high level of p53 expression, and p53/bcl-2 co-expression were significantly associated with shortened overall survival in univariate analysis. In multivariate analysis, only age and p53/bcl-2 co-expression had independent prognostic value. Other combinations of genes, i.e., bcl-2-to-bax and p53-to-bax ratios, were not associated with patient outcome. A significant positive correlation was found between apoptotic and mitotic activity. However, protein levels of p53, bcl-2, and bax were unrelated to proliferation and apoptosis of tumor cells. CONCLUSIONS: The co-expression of p53/bcl-2 was an independent predictor of patient outcome and had a prognostic value superior to both parameters considered separately. The rate of apoptosis mainly counterbalanced proliferative activity but appeared not to be significantly influenced by p53, bcl-2, and bax.