Insights on PRAME and osteosarcoma by means of gene expression profiling.

BACKGROUND: Osteosarcoma (OS) is the most frequent bone tumor in children and adolescents. Tumor antigens are encoded by genes that are expressed in many types of solid tumors but are silent in normal tissues, with the exception of placenta and male germ-line cells. It has been proposed that antigen tumors are potential tumor markers. OBJECTIVES: The premise of this study is that the identification of novel OS-associated transcripts will lead to a better understanding of the events involved in OS pathogenesis and biology. METHODS: We analyzed the expression of a panel of seven tumor antigens in OS samples to identify possible tumor markers. After selecting the tumor antigen expressed in most samples of the panel, gene expression profiling was used to identify osteosarcoma-associated molecular alterations. A microarray was employed because of its ability to accurately produce comprehensive expression profiles. RESULTS: PRAME was identified as the tumor antigen expressed in most OS samples; it was detected in 68% of the cases. Microarray results showed differences in expression for genes functioning in cell signaling and adhesion as well as extracellular matrix-related genes, implying that such tumors could indeed differ in regard to distinct patterns of tumorigenesis. CONCLUSIONS: The hypothesis inferred in this study was gathered mostly from available data concerning other kinds of tumors. There is circumstantial evidence that PRAME expression might be related to distinct patterns of tumorigenesis. Further investigation is needed to validate the differential expression of genes belonging to tumorigenesis-related pathways in PRAME-positive and PRAME-negative tumors.

SAGE analysis demonstrates increased expression of TOSO contributing to Fas-mediated resistance in CLL.

To identify novel genes involved in the molecular pathogenesis of chronic lymphocytic leukemia (CLL) we performed a serial analysis of gene expression (SAGE) in CLL cells, and compared this with healthy B cells (nCD19(+)). We found a high level of similarity among CLL subtypes, but a comparison of CLL versus nCD19(+) libraries revealed 55 genes that were over-represented and 49 genes that were down-regulated in CLL. A gene ontology analysis revealed that TOSO, which plays a functional role upstream of Fas extrinsic apoptosis pathway, was over-expressed in CLL cells. This finding was confirmed by real-time reverse transcription-polymerase chain reaction in 78 CLL and 12 nCD19(+) cases (P < .001). We validated expression using flow cytometry and tissue microarray and demonstrated a 5.6-fold increase of TOSO protein in circulating CLL cells (P = .013) and lymph nodes (P = .006). Our SAGE results have demonstrated that TOSO is a novel over-expressed antiapoptotic gene in CLL.

Higher expression of transcription targets and components of the nuclear factor-kappaB pathway is a distinctive feature of umbilical cord blood CD34+ precursors.

Delayed engraftment, better reconstitution of progenitors, higher thymic function, and a lower incidence of the graft-versus-host disease are characteristics associated with umbilical cord blood (UCB) transplants, compared with bone marrow (BM). To understand the molecular mechanisms causing these intrinsic differences, we analyzed the differentially expressed genes between BM and UCB hematopoietic stem and progenitor cells (HSPCs). The expressions of approximately 10,000 genes were compared by serial analysis of gene expression of magnetically sorted CD34(+) cells from BM and UCB. Differential expression of selected genes was evaluated by real-time polymerase chain reaction on additional CD34(+) samples from BM (n = 22), UCB (n = 9), and granulocyte colony stimulating factor-mobilized peripheral blood (n = 6). The overrepresentation of nuclear factor-kappaB (NF-kappaB) pathway components and targets was found to be a major characteristic of UCB HSPCs. Additional promoter analysis of 41 UCB-overrepresented genes revealed a significantly higher number of NF-kappaB cis-regulatory elements (present in 22 genes) than would be expected by chance. Our results point to an important role of the NF-kappaB pathway on the molecular and functional differences observed between BM and UCB HSPCs. Our study forms the basis for future studies and potentially for new strategies to stem cell graft manipulation, by specific NF-kappaB pathway modulation on stem cells, prior to transplant.

PRAME is a membrane and cytoplasmic protein aberrantly expressed in chronic lymphocytic leukemia and mantle cell lymphoma.

The preferentially expressed antigen in melanoma (PRAME) gene is aberrantly expressed in chronic lymphoproliferative disorders (CLD). We produced and characterized an anti-PRAME monoclonal antibody (MoAb), which was then applied in a quantitative flow cytometric (QFC) method to evaluate PRAME expression in leukemic cells from the peripheral blood (PB) of 47 patients with chronic lymphocytic leukemia and seven with mantle cell lymphoma as well as in the PB mononuclear cells (PBMCs) and B lymphocytes from 15 healthy subjects. Approximately 90% of CLD, but none of the normal samples, presented more than 20% of PRAME+ lymphocytes. Moreover, the intensity of PRAME expression was significantly higher in CLD cells compared to normal B lymphocytes and PBMCs. By immunofluorescence microscopy and by permeabilized flow cytometry we demonstrated that PRAME is a membrane antigen and a cytoplasmic protein aberrantly expressed in malignant CLD. Our results suggest that the analysis of PRAME protein may contribute for the distinction between normal and leukemic cells in CLD, and that PRAME may be a potential target for therapy.

Expression of cancer testis antigens in head and neck squamous cell carcinomas.

BACKGROUND: There is considerable interest in the expression of cancer testis (CT) antigens in human cancers, because they may serve as the basis for diagnostic tests or an immunologic approach to therapy, or as prognostic markers. METHODS: On this basis, we evaluated by semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) the expression of genes that code for tumor antigens (melanoma antigen-1 [MAGE-1], MAGE-4, MAGE-10, MAGE-12, B melanoma antigen, CTL-recognized antigen melanoma antigen (CT antigen 2) [LAGE], New York esophageal squamous cell carcinoma antigen (CT antigen 1) [NYESO-1], and preferentially expressed antigen of melanoma [PRAME]) in surgical samples of the tumors, margins, and lymph nodes (when present) from patients with a diagnosis of head and neck carcinoma. The study was conducted on 33 patients (31 men and two women), aged 31 to 94 years (mean, 56 years), with squamous cell carcinomas located in the mouth (15 cases), larynx (14 cases), and pharynx (four cases). RESULTS: The findings were compared with the clinical course and laboratory data. Expression of at least one antigen was observed in 66.6% of cases, with different rates of expression according to tumor staging (100% of T4, 57% of T3, 50% of T1 and T2) and smoking habit. There was a significantly higher expression of multiple genes (two or more) in tumors in advanced stages. CONCLUSIONS: We conclude that the tumor-specific antigen genes are expressed in variable frequencies and intensities in the primary lesions of head and neck squamous cell carcinomas and in their metastases, with expression of the PRAME gene being always present in the metastastatic lymph nodes. In primary lesions, gene expression correlated with smoking habit and with advanced tumors with a higher malignant potential, with the frequent expression of two or more of these genes.

Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT, WNT and TGFbeta signalling pathways.

Microarray studies have revealed the differential expression of several genes in mantle cell lymphoma (MCL), but it is unknown which of these differences are dependent on the transformed MCL cell itself or on the tumour microenvironment. To investigate which genes and signalling pathways are aberrantly expressed in MCL cells we used oligonucleotide microarrays to perform gene expression profiling of both purified leukaemic MCL cells and their normal counterparts, the naive B cells. A total of 106 genes were differentially expressed at least threefold in MCL cells compared with naive B cells; 63 upregulated and 43 downregulated. To validate the microarray results in a larger set of samples, we selected 10 differentially expressed genes and quantified their expression by real-time polymerase chain reaction in peripheral blood of MCL patients (n=21), purified MCL cells (n=6) and naive B cells (n=4), obtaining fully concordant results. A computer-assisted approach was used to procure specific molecular signalling pathways that were aberrantly expressed in MCL cells. Several genes related to apoptosis and to the PI3K/AKT, WNT and tumour growth factor beta signalling pathways were altered in MCL cells when compared with naive B cells. These pathways may play a significant role in the pathogenesis of MCL and deserve further investigation as candidates for new therapeutic targets.

Mesenchymal stem cells can be obtained from the human saphena vein.

Mesenchymal stem cells (MSC) can be isolated from many sites adults and the fetus. Cells with osteoblastic, chondrogenic, leiomiogenic and stromogenic potentials have been obtained from the bovine artery wall, and we now show that MSC can be isolated also from the adult human vein wall. Cells detached from internal surface of the saphenous vein are cultured in vitro for 2-3 weeks and replated weekly. The culture forms a semi-confluent layer of spindle-shaped cells that are CD13(+), CD29(+), CD44(+), CD34(-), CD45(-), CD14(-), CD133(-), CD31(-), CD33(-), CD54(+), CD106(-), CD90(+), KDR(-), cadherin-5-, HLA class I(+) and HLA-DR- and differentiate in vitro into osteoblasts, chondrocytes and adipocytes. Gene expression, when compared with seven other normal tissues, shows strong similarity with MSC obtained from other sources. Three genes more expressed in saphenous MSC than in the other two MSC are related to angiogenesis, and the expression of two of them is shared by endothelial cells. These results demonstrate that the human vein wall contains mesenchymal cells with morphologic features, immunophenotypic markers, gene expression profile and differentiation potential that are similar to MSC obtained from the bone marrow and from the umbilical vein.

The profile of gene expression of human marrow mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent precursors present in adult bone marrow, that differentiate into osteoblasts, adipocytes and myoblasts, and play important roles in hematopoiesis. We examined gene expression of these cells by serial analysis of gene expression, and found that collagen I, secreted protein acidic and rich in cysteine (osteonectin), transforming growth factor beta- (TGF-beta) induced, cofilin, galectin-1, laminin-receptor 1, cyclophilin A, and matrix metalloproteinase-2 are among the most abundantly expressed genes. Comparison with a library of CD34(+) cells revealed that MSCs had a larger number of expressed genes in the categories of cell adhesion molecule, extracellular and development. The two types of cells share abundant transcripts of many genes, some of which are highly expressed in myeloid progenitors (thymosin-beta 4 and beta 10, fos and jun). Interleukin-11 (IL-11), IL-15, IL-27 and IL-10R, IL-13R and IL-17R were the most expressed genes among the cytokines and their receptors in MSCs, and various interactions can be predicted with the CD34(+) cells. MSCs express several transcripts for various growth factors and genes suggested to be enriched in stem cells. This study reports the profile of gene expression in MSCs and identifies the important contribution of extracellular protein products, adhesion molecules, cell motility, TGF-beta signaling, growth factor receptors, DNA repair, protein folding, and ubiquination as part of their transcriptome.

The expression of PRAME in chronic lymphoproliferative disorders.

The PRAME gene encodes an antigen recognized by autologous T lymphocytes and is expressed in trophoblasts, testis and frequently in human solid cancers and acute leukemias, making it a candidate for immunotherapy and for detecting MRD. We demonstrate expression of PRAME by RT-PCR in the peripheral blood or bone marrow of 26% of 58 patients with CLD (38 cases of CLL, 4 cases of PLL and 16 cases of NHL). Seven out 16 cases of MCL, 2 out 4 of PLL and 6 cases of CLL demonstrated some degree of gene expression. Thus, CLD are among the hematopoietic malignancies for which PRAME may be the target of immunological therapy or used to evaluate MRD. The stronger and more frequent expression of PRAME in MCL is apparently an additional distinguishing feature on this group of lymphoproliferative disorders.