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There is a growing demand for functional layers for the immobilization of (bio)molecules on different kinds of substrates in the field of biosensors, microarrays, and lab-on-a-chip development. These functional coatings should have the ability to specifically bind (bio)molecules with a high binding efficiency, while showing low unspecific binding during the following assay. In this paper we present rSbpA surface layer proteins (S-layer proteins) as a versatile immobilization layer for the development of DNA microarrays. S-layer proteins show the ability to reassemble into two-dimensional arrays on solid surfaces and their functional groups, such as carboxylic groups, are repeated with the periodicity of the lattice, allowing for immobilization of other (bio)molecules. Different fluorescently labeled amino functionalized DNA oligomers were covalently linked to the S-layer matrices to allow the characterization of DNA binding on S-layers. Hybridization and dissociation of DNA-oligomers were studied on S-layer coated slides, revealing low levels of unspecific adsorption of DNA on S-layer based immobilization matrices. In the following the principle was transferred to a DNA microarray design showing successful spotting and hybridization on whole microarray slides. Besides common laser scanning for fluorescence detection, S-layer based microarrays were evaluated with a compact, low cost platform for direct fluorescence imaging based on surface plasmon enhanced fluorescence excitation. It could be shown that S-layer protein layers are promising as immobilization matrices for the development of biosensors and microarrays.

Genetic fusion of two proteins frequently induces beneficial effects to the proteins, such as increased solubility, besides the combination of two protein functions. Here, we study the effects of the bacterial surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a on the folding of a C-terminally fused enhanced green fluorescent protein (EGFP) moiety. Although GFPs are generally unable to adopt a functional confirmation in the bacterial periplasm of Escherichia coli cells, we observed periplasmic fluorescence from a chimera of a 150-amino-acid N-terminal truncation of SgsE and EGFP. Based on this finding, unfolding and refolding kinetics of different S-layer-EGFP chimeras, a maltose binding protein-EGFP chimera, and sole EGFP were monitored using green fluorescence as indicator for the folded protein state. Calculated apparent rate constants for unfolding and refolding indicated different folding pathways for EGFP depending on the fusion partner used, and a clearly stabilizing effect was observed for the SgsE_C fusion moiety. Thermal stability, as determined by differential scanning calorimetry, and unfolding equilibria were found to be independent of the fused partner. We conclude that the stabilizing effect SgsE_C exerts on EGFP is due to a reduction of degrees of freedom for folding of EGFP in the fused state.

Eur J Clin Invest 2012; 42 (9): 953–960 Abstract Background G protein-coupled receptor 5D (GPRC5D) is a novel surface receptor. As this new subtype of G protein-coupled receptors was discovered, little is known about the role of this gene. Materials and methods In this retrospective study, we investigated GPRC5D mRNA expression by real-time polymerase chain reaction (RT-PCR) in bone marrow (BM) of 48 patients with multiple myeloma (MM). Results Highly variable levels of GPRC5D (median, 288; quartiles, 17–928) were detected in patients with MM, whereas only low expression was detected in normal tissues (median, 1; quartiles, 1–23). High mRNA expression of GPRC5D correlated positively with high plasma cell count in bone marrow (r = 0·64, P

The Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051(T) possesses a two-dimensional crystalline protein surface layer (S-layer) with oblique lattice symmetry composed of a single type of O-glycoprotein species. Herein, we describe a strategy for nanopatterned in vivo cell surface co-display of peptide and glycan epitopes based on this S-layer glycoprotein self-assembly system. The open reading frame of the corresponding structural gene spaA codes for a protein of 983 amino acids, including a signal peptide of 24 amino acids. The mature S-layer protein has a theoretical molecular mass of 105.95kDa and a calculated pI of 5.83. It contains three S-layer homology domains at the N-terminus that are involved in anchoring of the glycoprotein via a non-classical, pyruvylated secondary cell wall polymer to the peptidoglycan layer of the cell wall. For this polymer, several putative biosynthesis enzymes were identified upstream of the spaA gene. For in vivo cell surface display, the hexahistidine tag and the enhanced green fluorescent protein, respectively, were translationally fused to the C-terminus of SpaA. Immunoblot analysis, immunofluorescence staining, and fluorescence microscopy revealed that the fused epitopes were efficiently expressed and successfully displayed via the S-layer glycoprotein matrix on the surface of P. alvei CCM 2051(T) cells. In contrast, exclusively non-glycosylated chimeric SpaA proteins were displayed, when the S-layer of the glycosylation-deficient wsfP mutant was used as a display matrix.

The mesophilic organism Lysinibacillus sphaericus CCM 2177 produces the surface (S)-layer protein SbpA, which after secretion completely covers the cell surface with a crystalline array exhibiting square lattice symmetry. Because of its excellent in vitro recrystallization properties on solid supports, SbpA represents a suitable candidate for genetically engineering to create a versatile self-assembly system for the development of a molecular construction kit for nanobiotechnological applications. The first goal of this study was to investigate the surface location of 3 different C-terminal amino acid positions within the S-layer lattice formed by SbpA. Therefore, three derivatives of SbpA were constructed, in which 90, 173, or 200 C-terminal amino acids were deleted, and the sequence encoding the short affinity tag Strep-tag II as well as a single cysteine residue were fused to their C-terminal end. Recrystallization studies of the rSbpA/STII/Cys fusion proteins indicated that C-terminal truncation and functionalization of the S-layer protein did not interfere with the self-assembly capability. Fluorescent labeling demonstrated that the orientation of the crystalline rSbpA(31-1178)/STII/Cys lattice on solid supports was the same, like the orientation of wild-type S-layer protein SbpA on the bacterial cell. In soluble and recrystallized rSbpA/STII/Cys fusion proteins, Strep-tag II was used for prescreening of the surface accessibility, whereas the thiol group of the end-standing cysteine residue was exploited for site-directed chemical linkage of differently sized preactivated macromolecules via heterobifunctional cross-linkers. Finally, functionalized two-dimensional S-layer lattices formed by rSbpA(31-1178)/STII/Cys exhibiting highly accessible cysteine residues in a well-defined arrangement on the surface were utilized for the template-assisted patterning of gold nanoparticles.

Vascular endothelial growth factor (VEGF) is produced in neoplastic cells in various myeloid neoplasms and may act as an autocrine growth-regulator. We have examined the expression of five VEGF receptors (VEGR1/Flt-1, VEGFR2/KDR, Flt-4, neuropilin-1 = NRP-1, NRP-2) in leukemic cells obtained from patients with acute myeloid leukemia (n = 28), chronic myeloid leukemia (n = 14), chronic eosinophilic leukemia (n = 3), chronic myelomonocytic leukemia (n = 9), or mast cell leukemia/systemic mastocytosis (n = 3) as well as in respective cell lines. Expression of VEGFR mRNA was analyzed by RT-PCR, and expression of VEGFR protein by immunocytochemistry. In most patients, leukemic cells expressed NRP-1 mRNA and NRP-2 mRNA independent of the type of disease. By contrast, transcripts for Flt-1, KDR, and Flt-4 were expressed variably without a clear correlation to the type of leukemia. Expression of VEGF receptors was also demonstrable at the protein level in all cases tested. In conclusion, neoplastic cells in myeloid leukemias frequently express VEGFR including NRP-1 and NRP-2.

Lipoprotein lipase (LPL) is a prognostic marker in B-cell chronic lymphocytic leukemia (B-CLL) related to immunoglobulin V(H) gene (IgV(H))mutational status. We determined gene expression profiles using Affymetrix U133A GeneChips in two groups of B-CLLs selected for either high ('LPL+', n=10) or low ('LPL-', n=10) LPL mRNA expression. Selected genes were verified by real-time PCR in an extended patient cohort (n=42). A total of 111 genes discriminated LPL+ from LPL- B-CLLs. Of these, the top three genes associated with time to first treatment were Septin10, DMD and Gravin (P

Inhibition of the epidermal growth factor (EGF)-receptor (EGFR) has become a promising anticancer treatment strategy. In addition, application of retinoids yields encouraging results for cancer prevention and therapy. Many tumors express no or low amounts of retinoic acid receptor-beta2 (RAR-beta2) due to epigenetic silencing via DNA hypermethylation. RAR-beta2 is the main mediator of the antiproliferative effect of retinoids. RAR-beta2 re-expression causes reversal of transformation, cell cycle arrest, and restoration of retinoid sensitivity. RAR-beta2 is thus a tumor suppressor. Western blotting, colorimetric in vitro cell proliferation assays, and reverse transcription-polymerase chain reaction demonstrated that the EGFR inhibitor PD153035 not only blocked activation of EGFR and inhibited cell growth, but also stimulated RAR-beta expression in MDA-MB-468 breast and OVCAR-3 ovarian carcinoma cells. Upregulation of RAR-beta by PD153035 was confirmed by real-time reverse transcription-polymerase chain reaction. In contrast, expression of other retinoid receptors and of estrogen receptor-alpha was not affected. PD153035-mediated re-induction of RAR-beta was associated with demethylation of the RAR-beta2 gene promoter P2 as demonstrated by methylation-specific polymerase chain reaction. These novel results on the ErbB/retinoid receptor cross-talk may be useful for designing future anticancer combination regimens.

Internal tandem duplication of the FLT3 gene (FLT3/ITD) has been linked to poor outcome in acute myeloid leukemia (AML). However, the prognostic value of FLT3/ITD in various cytogenetic risk groups is still a matter of debate. The aim of this study was to evaluate the prognostic significance in patients with de novo AML and a normal karyotype or a t(15;17), t(8;21) or inv(16) (good risk group).Diagnostic samples of 100 patients were investigated by single-step PCR of exons 11 and 12 of the FLT3 gene in a single center retrospective analysis. Subgroups included 53 patients with normal karyotype, 21 patients with t(15;17), 9 patients with t(8;21) and 17 patients with inv(16).FLT3/ITD was found in 26 out of 100 patients: 30% of patients with a normal karyotype and 38% of t(15;17) patients tested positive. The complete remission (CR) rates for the ITD(+) or ITD(-) groups were 50 vs 76% in normal karyotypes, and 100 vs 53% in t(15;17) patients, while the relapse rates were 75 vs 25% in normal karyotypes and 50 vs 42% in t(15;17) patients. Overall survival (OS) and disease free survival (DFS) at 5 years were significantly different in patients with normal cytogenetics: ITD(+) vs ITD(-): OS 6 vs 28% (P0.003); DFS 13 vs 41% (P0.02) Interestingly, FLT3/ITD had no significant effect on the outcome of t(15;17) patients: ITD(+) vs ITD(-): OS 85 vs 53% (P=0.056), DFS 45 vs 60% (P=0.6) at 50 months.These data suggest a high prognostic value of FLT3/ITD in patients with normal cytogenetics. However, we find no evidence that FLT3/ITD is a predictive marker for patients with t(15;17).

Colloids, polymers, gels, and biological materials are widely used for numerous technological applications. The design, fabrication, and understanding of the physico-chemical properties of such (bio)materials, however, represent a challenge for scientists and technologists. This review is a concise update of the latest achievements in surface and bulk analytical techniques applied to biomaterials and soft matter systems. Specific emphasis is devoted to their structural, mechanical, surface, and chemical properties. The review introduces the basics of atomic force microscopy (AFM) and discusses its combination with optical microscopies and spectroscopic techniques. In particular, we report the AFM combination with fluorescence, confocal microscopy, stimulated emission depletion, Raman, and infrared spectroscopy. The review highlights the impact and the potential applications of the combination of such experimental techniques on current problems and questions related to material and polymer science, cell biology, biochemical engineering, and protein chemistry. This review is an accessible introduction to AFM for the newcomer and a source of new experimental information for the experienced researcher.

G protein-coupled receptor 5D (GPRC5D) is a novel surface receptor. As this new subtype of G protein-coupled receptors was discovered, little is known about the role of this gene.In this retrospective study, we investigated GPRC5D mRNA expression by real-time polymerase chain reaction (RT-PCR) in bone marrow (BM) of 48 patients with multiple myeloma (MM).Highly variable levels of GPRC5D (median, 288; quartiles, 17-928) were detected in patients with MM, whereas only low expression was detected in normal tissues (median, 1; quartiles, 1-23). High mRNA expression of GPRC5D correlated positively with high plasma cell count in bone marrow (r = 0·64, P0·001), high β(2) -microglobulin (r = 0·42, P = 0·003) and poor-risk cytogenetics: deletion 13q14 (rb-1), P = 0·003; and 14q32 translocation t(4;14)(p16;q32), P = 0·029. GPRC5D mRNA expression showed a significant correlation with overall survival (P = 0·031). The estimated overall survival of patients expressing GPRC5D above or below the median of 288 was 43·9% vs. 70·2% at 48 months. Here, we report, for the first time, the association of GPRC5D expression and cancer.Overexpression in poor-risk myeloma, low expression in normal tissues and cell surface expression identify GPRC5D as a potential novel cancer antigen. Our data demonstrate that GPRC5D is a prognostic factor in MM correlating with other major risk factors.

This work reports for the first time on the fabrication of a bi-functional S-layer tandem fusion protein which is able to self-assemble on solid supports without losing its functionality. Two variants of the green fluorescent protein (GFP) were genetically combined with a self-assembly system having the remarkable opportunity to interact with each other and act as functional nanopatterning biocoating. The S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a was fused with the cyan ECFP donor protein at the SgsE N-terminus and with the yellow YFP acceptor protein at the C-terminus. The fluorescence energy transfer was studied with spectrofluorimetry, confocal microscopy and flow cytometry, whilst protein self-assembly (on silicon dioxide particles) and structural investigations were carried out with atomic force microscopy (AFM). The fluorescence resonance energy transfer efficiency of reassembled SgsE tandem protein was 20.0 ± 6.1% which is almost the same transfer efficiency shown in solution (19.6 ± 0.1%). This work shows that bi-fluorescent S-layer fusion proteins self-assemble on silica particles retaining their fluorescent properties.

S-layer fusion protein technology was used to design four different fluorescent fusion proteins with three different GFP mutants and the red fluorescent protein mRFP1. Their absorption spectra, steady-state fluorescence, and fluorescence lifetime were investigated as a function of pH. It was found that fluorescence intensities and lifetime of the GFP mutant S-layer fusion proteins decreased about 50% between pH 6 and pH 5. The spectral properties of the red S-layer fusion protein were minimally affected by pH variations. These results were compared with His-tagged reference fluorescent proteins, demonstrating that the S-layer protein did not change the general spectral properties of the whole fusion protein. In addition, the pK(a) values of the fluorescent S-layer fusion proteins were calculated. Finally, it was shown that the S-layer fusion proteins were able to self-assemble forming 2D nanostructures of oblique p2 symmetry with lattice parameters of about a = 11 nm, b = 14 nm, and gamma = 80 degrees . The fluorescence tag did not hinder the natural self-assembly process of the S-layer protein. The combination of the fluorescence properties and the self-assembly ability of the engineered fusion proteins make them a promising tool to generate biomimetic surfaces for future applications in nanobiotechnology at a wide range of pH.

This paper describes the development of planar and fiber optic oxygen sensors utilizing surface layer (S-layer) proteins as immobilization matrix for oxygen sensitive dyes. S-layer proteins have the intrinsic capability to reassemble into two-dimensional arrays in suspension and at interfaces. Due to their crystalline character the distribution of functional groups, such as carboxylic groups, is repeated with the periodicity of the lattice and thus allows the reproducible and geometrically distinct binding of functional molecules. For the development of oxygen sensors an oxygen sensitive Pt(II) porphyrin dye was covalently bound to the S-layer matrix. Measurement of the oxygen concentration was performed by phase modulation fluorimetry. Setups comprising low cost optoelectronic components like LEDs and silicon photodiodes were constructed. For both sensor setups (planar and fiber optic) variations in the oxygen concentrations resulted in distinct and reproducible changes in luminescence lifetime and intensity. The luminescence quenching efficiency of these sensors was found to be 1.5-1.9 (expressed as the ratio of signal under nitrogen and air) which compares well to other sensor systems using luminophores embedded in polymer matrices. These results demonstrated the application potential of S-layers as immobilization matrices in the development of (bio-)sensors.

We report high expression of the maternally imprinted gene PEG10 in high-risk B-CLL defined by high LPL mRNA expression. Differential expression was initially identified by microarray analysis and confirmed by real time PCR in 42 B-CLL patients. mRNA expression ranged from 0.3- to 375.4-fold compared to normal peripheral blood mononuclear cells (PBMNC). Expression levels in CD19+ B-CLL cells were 100-fold higher than in B-cells from healthy donors. PEG10 expression levels in B-CLL patient samples remained stable over time even after chemotherapy. High PEG10 expression correlated with high LPL expression (p=0.001) and a positive Coombs' test (p=0.04). Interestingly, similar expression patterns were observed for the neighbouring imprinted gene sarcoglycan-epsilon (SGCE). Monoallelic expression and maintained imprinting of PEG10 were found by allele- or methylation-specific PCR. The intensity of intracellular staining of PEG10 protein corresponded to mRNA levels as confirmed by immunofluorescence staining. Short term knock-down of PEG10 in B-CLL cells and HepG2 cells was not associated with changes in cell survival but resulted in a significant change in the expression of 80 genes. However, long term inhibition of PEG10 led to induction of apoptosis in B-CLL cells. Our data indicate (i) a prognostic value of PEG10 in B-CLL patients; (ii) specific deregulation of the imprinted locus at 7q21 in high-risk B-CLL; (iii) a potential functional and biological role of PEG10 protein expression. Altogether, PEG10 represents a novel marker in B-CLL.

Inhibiting epidermal growth factor-receptor (ErbB-1) represents a powerful anticancer strategy. Activation of retinoid pathways is also in development for cancer treatment. Retinoic acid receptor-β—the tumor suppressor and main retinoid mediator—-is silenced in many tumors. The ErbB-1 inhibitor PD153035 cooperates with retinoic acid during growth inhibition and induces retinoic acid receptor-β suggesting that ErbB-1 controls retinoic acid receptor-β. However, here we demonstrate that ErbB pathways are not involved in PD153035-mediated retinoic acid receptor-β-upregulation. PD153035 inhibits ErbB-1-phosphorylation, whereas its derivative EBE-A22 is inactive. Yet both inhibit cell growth and upregulate retinoic acid receptor-β in ErbB-1-overexpressing (MDA-MB-468), moderately expressing (OVCAR-3), ErbB-1-negative (MDA-MB-453) or ErbB-negative cells (CEM, Jurkat). Both bind DNA, whereas the closely related ErbB-1 inhibitors AG1478 and ZD1839, which are inactive on retinoic acid receptor-β, do not significantly bind DNA. None of the other ErbB-1/ErbB-2 inhibitors tested (RG-14620, LFM-A12, AG879, AG825) affect retinoic acid receptor-β. PD153035 decreases methylation of the retinoic acid receptor-β2 promoter. In OVCAR-3, it stimulates dislodgement of histone deacetylase 1 from the promoter and acetylation of histones H3 and H4. Consequently, PD153035 facilitates recruitment of RNA polymerase II to the promoter and stimulates transcriptional activity. Moreover, PD153035 increases the retinoic acid receptor-β mRNA half-life. No other retinoid receptor, nor estrogen receptor-α, nor RASSF1A is upregulated by PD153035. Thus PD153035 induces retinoic acid receptor-β by ErbB-independent transcriptional and post-transcriptional mechanisms. This report highlights a triple action for an ErbB-1 inhibitor (ErbB-1 inhibition, DNA intercalation, retinoic acid receptor-β-induction). Such multitargeting drugs bear great potential for cancer treatment. J. Cell. Physiol. 211: 803–815, 2007. © 2007 Wiley-Liss, Inc.

In this paper the use of a special class of self assembling protein materials for optical sensor technology is discussed. These S-layer proteins were reassembled into well defined nanostructures on solid sensor surfaces. The covalent immobilization of fluorescent dyes onto these protein structures was investigated. Following this approach a new oxygen sensitive system based on S-layers functionalized with Pt(II) metalloporphyrin is described. A luminescence lifetime based measurement system for dissolved oxygen sensing was successfully developed.

Karyotype is an important prognostic factor in patients with newly diagnosed acute myeloblastic leukaemia (AML). The prognostic value of cytogenetics on the outcome of patients with AML in relapse has not yet been well defined. We analysed the clinical outcome of 152 patients with de novo, chemotherapy-treated AML in first relapse according to the cytogenetic classification of the United Kingdom Medical Research Council. The rate of second complete remission (CR) (88, 64 and 36%) and the probability of survival at 3 years (43, 18 and 0%) were significantly different between the favourable, intermediate and adverse cytogenetic risk groups, respectively. Compared to the favourable group, the relative risk (RR) of death (multivariate analyses) was 2.6 (confidence interval (CI): 1.5-4.4, P

We analyzed in vitro growth characteristics of bone marrow mononuclear cells (BMMCs) from 322 patients with acute myeloid leukemia (AML) in relation to cytogenetic abnormalities. Median colony growth was low in each of the cytogenetic changes associated with a favorable outcome. Most karyotypic abnormalities in the intermediate prognosis group were associated with low growth potential, but 11q23 abnormalities exhibited 8 times higher in vitro growth. Cytogenetic changes that included abn(3q) seemed to display the highest colony growth in the unfavorable prognosis group, whereas isolated -7 may have been associated with limited growth potential. In vitro growth behavior was predictive of neither rate of complete remission (CR) nor survival of AML patients within the 3 cytogenetic risk groups. In contrast, colony growth differed significantly in the subgroup of patients with a normal karyotype who achieved remission with induction treatment and those who had no remission (10 versus 81.5/105 BMMCs;P = .015). Significantly more patients with normal cytogenetics and colony growth below the 50th percentile went into CR than did patients with colony growth above the 50th percentile (82.8% versus 71.2%). Only 4 (6.8%) of the patients in the low growth group had no remission, compared with 12 (23.1%) of the patients with higher in vitro growth (P = .031, chi-square test). In conclusion, colony growth may prove useful as a prognostic factor for early treatment failure in AML patients with a normal karyotype.Int J Hematol. 2003;78:241-247.

Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins.

Four different coloured pH dependent biocolloids have been designed with fluorescent S-layer fusion proteins. The intrinsic self assembling capability of the S-layer protein SgsE on silicon dioxide particles lead to crystalline p2 lattice symmetry which was combined with the fluorescent properties of fused cyan ECFP, green EGFP, yellow YFP and red mRFP1. Confocal microscopy was used to monitor the pH dependence of the fluorescent S-layer coating. Electrophoretic measurements were carried out to understand the colloidal behaviour of the S-layer protein coated particles. It was found with flow cytometry that the stable SgsE-ECFP, SgsE-EGFP, SgsE-YFP and SgsE-mRFP bioparticle suspensions lost 50% of their fluorescence emission at the pKa of the chromophores. These novel fluorescent S-layer biocolloids can be used as pH sensors and might have an important role in surface pH determination.

Background: The course of chronic lymphocytic leukemia (CLL) is highly variable. Therefore, there is a need for prognostic factors that are readily performed and have a high predictive power. Methods: The occurrence of translocations, a recently identified prognostic factor in CLL (Blood2006;107:742–751), was studied in 148 previously untreated, mostly early-stage patients and compared with respect to treatment-free survival (TFS) to several prognostic factors (Binet stage, mutational status of immunoglobulin genes, CD38, thymidine kinase serum concentration and cytogenetic aberrations detected by interphase FISH). To investigate chromosomal translocations, we applied a new method, CpG oligodeoxynucleotide stimulation that allows efficient preparation of metaphase spreads from CLL cells. Results: The occurrence of translocations classified the majority of patients with poor prognosis. If translocations were investigated in addition to the currently used prognostic factors they identified those patients who were classified to be in a low-risk group based on traditionally used criteria, who had in fact a high risk for progression. Vice versa, patients in the high-risk groups for progression who did not have translocations had a long TFS. There was a substantial overlap of patients who had translocations and additional risk factors. But when we omitted patients who had translocations in addition to a given risk factor, we found that the respective risk factor lost its prognostic significance for the remaining patients. The two factors that retained their prognostic power in these patients were translocations and the Binet stage. This could suggest that the prognostic significance of the currently used factors derives from their frequent co-occurrence with translocations. Finally, multivariate analyses demonstrated that Binet stage (p=0.02) and translocations (p=0.0005) are the factors with the highest impact on TFS in our study cohort. Conclusion: We present a method for efficient preparation of metaphase spreads in CLL cells in order to investigate chromosomal translocations. The occurrence of translocations is an independent prognostic marker in CLL. Finally, translocations occur not as a late event in the course of the disease and may define a new biological subgroup in this disease entity.

Introduction: To establish surrogate markers for IgVH mutation status in B-CLL, we performed microarray analysis with mutated and unmutated patient samples. Among the most differentially expressed genes was PEG10, a maternally imprinted gene located on chromosome 7q21. PEG10 is known to be expressed during placental development and in hepatocellular carcinoma. Here we have investigated the association of PEG10 with B-CLL subtypes and risk factors as well as its transcriptional regulation. In addition, we studied its expression in other hematologic malignancies. Methods and Results: In our microarray experiments, PEG10 was highly expressed (2.1-fold) in unmutated samples. We confirmed these results by real time PCR in 42 B-CLL patients and found a significant correlation between PEG10 expression and unmutated IgVH status (P=0.007). High PEG10 expression was associated with chromosomal del(11q) (P=0.008), a shorter time to treatment (P=0.009) as well as a positive Coombs test (P=0.04). High expression was found in B-CLL and sorted CD19+ B-CLL cells, plasmoblastic DLBCL as well as in the Burkitt’s lymphoma cell line Ramos. In normal tissues, relatively high levels were seen in brain, liver, lung and kidney, but these expression levels were lower than in CD19+ B-CLL cells. Expression in sorted B cells from healthy donors was lower than two orders of magnitude. Intracellular expression of the PEG10 protein in B-CLL cells was confirmed by immunofluorescence studies using antibodies that recognize specifically PEG10-RF1. To address the question whether PEG10 overexpression results from loss of imprinting, we performed allele-specific as well as methylation-specific PCR. Imprinting was maintained in 6 patients with B-CLL. Analysis of a mother and son, both having B-CLL, confirmed that at least in this familial case the maternal PEG10 allele was silenced as expected. Interestingly, similar expression patterns were observed for the neighbouring gene sarcoglycan epsilon indicating a common mechanism of deregulation of the 7q21 locus in high risk B-CLL. Conclusions: We show that PEG10 is differentially expressed in B-CLL and is associated with del(11q), a shorter time to treatment and unmutated IgVH mutation status, suggesting its role as a marker for high risk B-CLL. PEG10 overexpression is unlikely to result from loss of imprinting. Further studies of the gene locus at 7q21 are in progress.

We investigated the pattern of lipoprotein lipase (LPL) expression in B-cell chronic lymphocytic leukemia (B-CLL) and assessed its prognostic and potential functional relevance. LPL mRNA levels in peripheral blood mononuclear cells (PBMNC) from 55 CLL patients differed by 2 orders of magnitude between cases with mutated (N=28) or unmutated (N=27) IGVH (median: 1.62 vs. 100.43 compared to normal PBMNC). LPL expression correlated strongly with IGVH mutational status (R=0.78; P *J Exp Med 2001