Holger Nückel

ZAP-70 expression is a prognostic factor in chronic lymphocytic leukemia.

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=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.

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.

CD49d Is the Strongest Flow Cytometry–Based Predictor of Overall Survival in Chronic Lymphocytic Leukemia

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.

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.

Anti-CD20 monoclonal antibody therapy in relapsed MALT lymphoma of the conjunctiva

Abstract:  Low‐grade non‐Hodgkins lymphomas of the conjunctiva may be cured by radiotherapy, but complications are frequent and relapses may occur. Other treatment modalities including resection, cryotherapy, injection of interferon‐α or systemic chemotherapy have been used with varying success. We treated two patients with relapsed extranodal marginal zone lymphoma (ENMZL) of mucosa‐associated lymphoid tissue (MALT) of the conjunctiva with the anti‐CD20 monoclonal antibody rituximab (375 mg/m2 intravenously once weekly for 4 wk) which has previously been shown to be effective in a variety of other B‐cell non‐Hodgkins lymphomas. Treatment was well tolerated and resulted in one partial and one complete remission. With a follow‐up of 32 or 30 months, respectively, further recurrences have not been observed. Rituximab is a highly effective and well‐tolerated treatment of conjunctival MALT lymphoma, which may not only be of value in relapse, but also in cases of contraindication to radiotherapy.

Granulocyte colony–stimulating factor–induced blood stem cell mobilisation in patients with chronic heart failure

Bone marrow–derived stem cells may contribute to the regeneration of non–haematopoietic organs. In order to test whether an increase in circulating stem cell numbers improves impaired myocardial function we treated 16 male patients with chronic heart failure due to dilated (DCM; n = 7) or ischaemic cardiomyopathy (ICM; n = 9) with the stem cell mobilising cytokine granulocyte colony–stimulating factor (G–CSF; four 10–day treatment periods interrupted by treatment–free intervals of equal length). Safety and efficacy analyses were performed at regular intervals.Peak CD34+ cell counts remained constant from cycle to cycle. Cardiac side effects in ICM patients included occasional episodes of dyspnea or angina and one episode of fatal ventricular fibrillation. Nine (4 DCM, 5 ICM) of 12 patients receiving four full G–CSF cycles experienced an improvement by one New York Heart Association (NYHA) class and a statistically significant increase in six–minute walking distance. By contrast, none of 8 ICM historical controls had a change in NYHA class during a similar time period. Statistically significant changes in echocardiographic parameters were not recorded.Sequential administration of G–CSF is feasible and possibly effective in improving physical performance in patients with chronic heart failure. Patients with ICM may be at risk of increased angina and arrhythmias.

The GNAS1 T393C Polymorphism Is Associated with Disease Progression and Survival in Chronic Lymphocytic Leukemia

Purpose: B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of monoclonal mature B cells. The G protein Gαs subunit has been linked to proapoptotic processes in cancer cell lines. The TT genotype of the GNAS1 T393C polymorphism is associated with increased Gαs transcript levels and a more favorable clinical course in different solid cancers. Experimental Design: We retrospectively genotyped 144 patients with B-CLL to examine a potential association between T393C genotypes with progression-free survival (time from diagnosis to initiation of chemotherapy) and overall survival. Results: The C-allele frequency in the patient group was 0.57 and not significantly different from that of healthy blood donors. Median progression-free survival was significantly different between genotypes (TT 130 months; TC 100 months; CC 31 months; P = 0.0066). Multivariable analysis showed that besides of ZAP-70 (P = 0.005) and Binet stage (P < 0.001), the T393C polymorphism was an independent prognostic factor for progression-free survival [hazard ratio (HR) CC versus TT 2.7; P = 0.010]. In Binet A stages, ZAP-70–positive patients with CC genotypes had a HR of 4.4 to receive first therapy compared with ZAP-70–negative patients with T-alleles (P = 0.0001). Regarding overall survival, CC genotypes (median overall survival, 197 months) were at highest risk for death compared with T-alleles (median overall survival, 310 months) in both univariate (HR, 4.8; P < 0.0001) and multivariable analysis (HR, 5.6; P = 0.002). Conclusions: Here, we show that the GNAS1 T393C status is a novel independent prognostic marker in patients with B-CLL. These results could help to define patients who could benefit from an early individualized therapy.

Trisomy 19 is associated with trisomy 12 and mutated IGHV genes in B‐chronic lymphocytic leukaemia

The occurrence of trisomy 19 was investigated in 705 cases of B‐chronic lymphocytic leukaemia (CLL) by metaphase cytogenetic and/or fluorescence in situ hybridisation analyses. Trisomy 19 was detected in 11 cases (1·6%), all of which also carried a trisomy 12; nine of 10 had mutated IGHV genes. In contrast, B‐CLL cases with trisomy 12 lacking trisomy 19 mostly had unmutated IGHV genes. Karyotypes of the present study and the literature identified a strong correlation to trisomy 18 in addition to trisomy 12. Trisomy 19 seems to be a secondary event in B‐CLL with trisomy 12, mostly originating from mutated B cells.

Aberrant hypomethylation of the cancer–testis antigen PRAME correlates with PRAME expression in acute myeloid leukemia

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.

1513A/C polymorphism in the P2X7 receptor gene in chronic lymphocytic leukemia: absence of correlation with clinical outcome

Purinergic P2X7 receptors are ligand‐gated cation channels expressed on the cells of the immune and hemopoietic system which have been shown to mediate the ATP‐induced apoptotic death of monocytes, macrophages and lymphocytes. A common single nucleotide polymorphism within the P2X7 gene has been described in exon 13 (1513A/C), the gene products encoding fully active and non‐functional proteins. We genotyped the P2X7 1513A/C polymorphism using DNA from 111 patients with chronic lymphocytic leukemia (CLL) and 97 healthy controls using polymerase chain reaction (PCR) amplification followed by Hha1 restriction analysis. We found no significant difference in allele frequency between CLL patients and controls. Time periods from diagnosis to initiation of chemotherapy, a surrogate marker for disease progression, were not different in patients displaying the combined 1513A/C and C/C or the 1513A/A genotype (P = 0.97). Similar results were observed in a subgroup analysis of prognostically more favorable CD38‐negative and ZAP‐70‐negative CLL patients. In conclusion, our data do not support a role of the P2X7 genotype as a prognostic marker in B‐cell CLL.