Different gene expression in immunoglobulin-mutated and immunoglobulin-unmutated forms of chronic lymphocytic leukemia.

The mutation status of the immunoglobulin heavy chain variable regions (IgVH) has been found to be a good prognostic indicator for B-cell chronic lymphocytic leukemia (CLL) because unmutated VH genes are associated with rapid disease progression and shorter survival time. To study the differences in gene expression between the Ig-unmutated and Ig-mutated CLL subtypes, we performed gene expression profiling on 31 CLL cases and investigated the VH gene mutation status by sequencing. The array data showed that the greatest variances between the unmutated (20 cases) and the mutated (11 cases) group were in expressions of ZAP70, RAF1, PAX5, TCF1, CD44, SF1, S100A12, NUP214, DAF, GLVR1, MKK6, AF4, CX3CR1, NAFTC1, and HEX. ZAP70 was significantly more expressed in the Ig-unmutated CLL group, whereas the expression of all the other genes was higher in the Ig-mutated cases. These results corroborate a recent finding, according to which the expression of ZAP70 can predict the VH mutation status and suggest that RAF1, PAX5, and other differentially expressed genes may offer good markers for differentiating unmutated cases from mutated cases and thus serve as prognostic markers.

Molecular mechanisms of CD99-induced caspase-independent cell death and cell-cell adhesion in Ewing's sarcoma cells: actin and zyxin as key intracellular mediators.

CD99 is a unique 32-kDa cell surface molecule with broad cellular expression but still poorly understood biological functions. In cancer cells, CD99 is highly expressed in virtually all Ewing's sarcoma (ES). Engagement of CD99 induces fast homotypic aggregation of ES cells and caspase-independent apoptosis. In this study, we analysed signal transduction after CD99 engagement on ES cells. Findings obtained with selective inhibitors indicated that only actin cytoskeleton integrity was essential for cell-cell adhesion and apoptosis of ES cells. Indeed, CD99 stimulation induced actin repolymerization, further supporting the role of cytoskeleton in CD99 signaling. Gene expression profiling of ES cells after CD99 engagement showed modulation in the expression of 32 genes. Among the pool of upregulated genes reported to be involved in cell adhesion, we chose to analyse the role of zyxin, a cytoplasmic adherens junction protein found to play a role in the regulation of the actin cytoskeleton. Overexpression of zyxin after CD99 ligation was confirmed by real-time PCR and Western blot. Treatment of ES cells with zyxin antisense oligonucleotides inhibited CD99-induced cell aggregation and apoptosis, suggesting a functional role for this protein. Therefore, our findings indicate that CD99 functions occur through reorganization of cytoskeleton and identify actin and zyxin as the early signaling events driven by CD99 engagement.

Gene expression profile by blocking the SYT-SSX fusion gene in synovial sarcoma cells. Identification of XRCC4 as a putative SYT-SSX target gene.

Increasing evidence suggests that the SYT-SSX fusion gene plays an important role in synovial sarcoma development and progression. However, very little is known about the downstream targets of SYT-SSX. In this study, we used antisense oligonucleotides to block the expression of the SYT-SSX fusion gene in synovial sarcoma cells. By comparing SYT-SSX inhibited cells with noninhibited cells, the gene expression profile was analysed using cDNA microarray and established by real-time PCR. Herewith, using a filter containing 1176 cancer-relevant genes, we found that the DNA repair gene XRCC4 and the DNA mismatch repair gene MSH2 were downregulated, whereas the gene encoding for the serine/threonine protein kinase PRK (also known as CNK), and the macrophage inhibitory cytokine MICI (also known as PLAB) were upregulated after the inhibition of SYT-SSX. In comparison, expression of the XRCC4 gene was undergoing the strongest alteration. Consistently, the protein expression of XRCC4 was found to be decreased after SYT-SSX inhibition, whereas there were no detectable changes for the other gene products. Our study provides some clues to elucidate the signaling pathways of the SYT-SSX fusion gene, as well as it demonstrates a valuable model system for search for other SYT-SSX targets.

Lymphotoxin beta expression is high in chronic lymphocytic leukemia but low in small lymphocytic lymphoma: a quantitative real-time reverse transcriptase polymerase chain reaction analysis.

BACKGROUND AND OBJECTIVES: The lymphotoxin beta (LTB) gene, localized to the major histocompatibility complex region on chromosome 6p21.3, has an important role in the formation of germinal center reactions and regulation of immune response and apoptosis. Our aim was to determine LTB gene expression in different hematologic neoplasias. DESIGN AND METHODS: We determined the expression of LTB using quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) on a series of RNA samples from CD3(+) T cells and CD19(+) B cells isolated from peripheral blood (n=7); CD19(+) B cells isolated from lymph nodes (n=11) and from patients with acute lymphoblastic leukemia (ALL; n=16), acute myeloid leukemia (AML; n=43), chronic myeloid leukemia (CML; n=12), mantle cell lymphoma (MCL; n=19), chronic lymphocytic leukemia (CLL; n=32) and small lymphocytic lymphoma (SLL; n=22). RESULTS: The expression level of LTB in CD3(+) T cells and CD19(+) B cells isolated from blood was ten to forty times lower than that in normal CD19(+) B cells isolated from lymph nodes. In malignant myeloid cells the expression levels were very low, whereas in malignant lymphoid cells the expression was higher than in myeloid cells, being highest in MCL and CLL (20.2+/-14.0 ng/microL and 81.0+/-116.3 ng/microL) and low in SLL (4.5+/-3.6 ng/microL; p=0.001). We did not find correlations between LTB expression and hematoclinical parameters (risk groups, immunophenotypes and overall survival). INTERPRETATION AND CONCLUSIONS: A distinct difference in expression of LTB in CLL and SLL indicates that these morphologically similar B-cell malignancies are molecularly different. Further studies are needed to investigate the prognostic value of LTB and any role that LTB may have in determining whether the malignant B cells manifest a leukemia or lymphoma.

Genome-wide allelotyping of 104 Finnish colorectal cancers reveals an excess of allelic imbalance in chromosome 20q in familial cases.

We have allelotyped a series of 104 Finnish colorectal cancers (CRCs) using 372 polymorphic markers spaced, on average, at 10 cM intervals, and have made a comparison of the differences in the frequency of allelic imbalance (AI) between familial and sporadic cases. Differences in the frequency of allelic imbalance (loss of heterozygosity or amplification) at a number of loci were detected and these were evaluated through analysis of additional series of cancers using specific markers. The most consistent difference was observed at chromosome 20q13.1-13.3 characterized by a two fold difference between familial and nonfamilial disease in a total of 99 familial and 186 sporadic Finnish cases. This difference was not observed in a UK set of 67 familial and 96 sporadic CRCs. The genome-wide effort resulted in a large data set giving clues to the location of putative CRC predisposition genes in the genome. The approach provides an alternative strategy for detecting cancer predisposition genes solely reliant on the molecular analysis of single cases obviating the requirement to collect multiple samples from families.

Abnormal expression of apoptosis-related genes in haematological malignancies: overexpression of MYC is poor prognostic sign in mantle cell lymphoma.

The expression of apoptosis-related genes BCL2, BAX, BCL2L1, BCL2A1, MCL1, DAPK1 and MYC was studied by quantitative real-time polymerase chain reaction on total RNA samples from patients with acute lymphoblastic leukaemia (ALL, n = 16), acute myeloid leukaemia (AML, n = 27), chronic myeloid leukaemia (CML, n = 12), mantle cell lymphoma (MCL, n = 19) and chronic lymphoid leukaemia (CLL, n = 32). BCL2, BAX, BCL2A1, MCL1, DAPK1 and MYC were overexpressed in all patient groups. BCL2L1 was underexpressed in CLL and CML, but not in AML, ALL and MCL. MCL1 levels were significantly higher in CD13 and CD33-positive ALL, and in CD56-positive AML samples. BCL2, BCL2L1, BCL2A1 and MCL1 were overexpressed and DAPK1 was underexpressed in CLL samples with a 11q23 deletion. MYC overexpression was significantly associated with shorter overall survival in MCL (P < 0.01). AML patients with a normal karyotype showed a higher frequency of BCL2A1 overexpression (P < 0.001) than those with an abnormal karyotype.

Investigatory and analytical approaches to differential gene expression profiling in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a non-Hodgkin's lymphoma of B-cell lineage. The blastoid variant of MCL, characterized by high mitotic rate, is clinically more aggressive than common MCL. We used the cDNA array technology to examine the gene expression profiles of both blastoid variant and common MCL. The data was analysed by regression analysis, principal component analysis and the naive Bayes' classifier. Eight genes were identified as differentially deregulated between the two groups. Oncogenes CMYC, BCL2 and PIM1 were upregulated more frequently in the blastoid variant than in common MCL. This implied that the gp130-mediated signal transducer and activator of transcription 3 (STAT3) signalling pathway was involved in the blastoid variant transformation of MCL. Other differentially deregulated genes were TOP1, CD23, CD45, CD70 and NFATC. By using the eight differentially deregulated genes, we created a classifier to distinguish the blastoid variant from common MCL with high accuracy. We also identified 18 genes that were deregulated in both groups. Among them, BCL1, CALLA/CD10 and GRN were suggested to be oncogenes. The products of RGS1, RGS2, ANX2 and CD44H were suggested to promote tumour metastasis. CD66D was suggested to be a tumour suppressor gene.

A cluster of familial malignant mesothelioma with del(9p) as the sole chromosomal anomaly.

We describe a family of three sisters affected by malignant mesothelioma (MM) (2 pleural and 1 peritoneal) and one brother affected by pleural plaques. All members of the family had been subjected to previous asbestos exposure of environmental-residential type. For 13 years, from 1951 to 1964, their housing was provided by the father's employer, an asbestos cement factory, and the factory warehouse was on the ground floor of the building they lived in. DNA extracted from paraffin-embedded MM samples was used to search for chromosomal alterations by a comparative genomic hybridization (CGH) method. In two cases, a loss at 9p was found to be the only change. The loss at 9p is a frequent event in malignant mesothelioma and the fact that this anomaly was diagnosed in two sisters as the only alteration suggests that this region may be the site of one or more oncosuppressor genes that could play an important role in the development of MM and in inducing greater genetic susceptibility to the carcinogenic effects of asbestos.

Genetic profile, PTEN mutation and therapeutic role of PTEN in glioblastomas.

New therapeutic strategies are needed to improve survival in glioblastoma (GBM) the most malignant astrocytic tumor. We evaluated: a) the genetic status of 22 GBMs by comparative genomic hybridization (CGH); b) the specific role of mutation and/or homozygous deletion of PTEN in the genesis of GBM; and c) the possible therapeutic role of PTEN against GBM, in vitro. CGH demonstrated that the most frequent region of gain was at chromosome 7p, whereas the most frequent losses occurred at chromosomes 10q and 13q. Losses at chromosome 10 were found in 36% of patients, and PTEN was mutated in 27% of the 22 GBMs, including 4 point mutations and 2 homozygous deletions. The possible therapeutic role of PTEN in GBM was also studied in a system based on retroviral infection of the GBM cell line A172, homozygously deleted at the PTEN locus. A172 growth and proliferation rate were reduced by 50% after PTEN transduction. Moreover, we showed that inhibition of cell growth occurred through the PI3K/Akt/p27 pathway. Our findings suggest that PTEN participates in the genesis of GBM, and might be further studied as a candidate therapeutic agent in other testing systems.

Gene expression analysis of 1,25(OH)2D3-dependent differentiation of HL-60 cells: a cDNA array study.

The alterations in gene expression associated with 1,25(OH)2D3-induced differentiation of HL-60 cells were studied in order to identify potential targets for further investigation of the genetic basis of acute myeloid leukaemia. Atlas human haematology filters, including 406 genes (Clontech), were used to study gene expression in response to 1,25(OH)2D3 (concentration, 5 x 10-8 mol/l) for 24 and 72 h. Compared with untreated cells, expression differences were found in 43 genes. Downregulated genes at both time-points were: IL2RA, CMYC, NPM, DEK, AF4, FLI1, htlf, MNDA, BCR, IKAROS, BPI and NFAT4. Upregulated genes at both time-points were IL1B, CD14 and MCL1. CD55, CD58, IRF2, CREB1, ATF4, RAC1, TIAR, KIAA0053, BAT2, BTK, RCK, EV12B and EDN were downregulated at 24 h, while SPI1, MKK3, BTG1 and IL8 were upregulated. At 72 h the upregulated genes were IL1RA, IL2RG, CXCR4, SCYA1, SCYA3, SCYA4, SCYA5, SCYA22, ANX2, CD83 and UPAR. cDNA array results were confirmed on randomly selected genes using quantitative real-time polymerase chain reaction for three upregulated (CXCR4, IL1B and CD14) and three downregulated (DEK, AF4 and FLI1) genes. Gene expression analysis after differentiation induction may provide a tool to study the roles of DEK, AF4 and FLI1 in cell proliferation and differentiation. To demonstrate the genes that initiate differentiation, sequential gene expression analysis has to be performed during the first 24 h of the differentiation process.

Recurrent DNA sequence copy losses on chromosomal arm 6q in capillary hemangioblastoma.

Capillary hemangioblastomas (CHB) of the central nervous system, the most common tumor in von Hippel-Lindau (VHL) disease, usually show mutations in the VHL tumor suppressor gene on chromosome 3p25-p26. Because little is known concerning the cytogenetic changes in these tumors, we studied 22 cases through comparative genomic hybridization to screen for DNA copy number changes in both sporadic and VHL-associated CHB. Our analysis revealed that 6 of 22 samples (27%) contained DNA copy number losses, whereas no gains were observed. The most recurrent finding was loss of chromosomal arm 6q, seen in five cases. In two of these cases also loss of chromosome 3 was noted. The third aberration observed was loss of chromosome 8, seen in one case. No differences were noted between VHL-associated and sporadic tumors, nor did the cytogenetic aberrations correlate with the clinical outcome. The loss of 6q, seen in this study and previously in other VHL-associated tumors (renal cell carcinomas and pheochromocytomas) and other tumors, suggest that this chromosome area may contain tumor suppressor genes involved in the early steps of tumorigenesis.