Marta Cuadros

Expression of the NF‐κB targets BCL2 and BIRC5/Survivin characterizes small B‐cell and aggressive B‐cell lymphomas, respectively

Nuclear factor kappa B (NF‐κB) activation has been proposed as a cardinal feature of tumourigenesis, although the precise mechanism, frequency, relevance, and extent of NF‐κB activation in lymphomas remain to be fully elucidated. In this study, expression profiling and tissue microarray studies of 209 and 323 non‐Hodgkins lymphomas (NHLs) respectively, including the most frequent sub‐types of NHL, were employed to generate a hypothesis concerning the most common NF‐κB targets in NHL. These analyses showed that NF‐κB activation is a common phenomenon in NHL, resulting in the expression of distinct sets of NF‐κB target genes, depending on the cell context. BCL2 and BIRC5/Survivin were identified as key NF‐κB targets and their expression distinguished small and aggressive B‐cell lymphomas, respectively. Interestingly, in the vast majority of B‐cell lymphomas, the expression of these markers was mutually exclusive. A set of genes was identified whose expression correlates either with BIRC5/Survivin or with BCL2. BIRC5/Survivin expression, in contrast to BCL2, was associated with a signature of cell proliferation (overexpression of cell cycle control, DNA repair, and polymerase genes), which may contribute to the aggressive phenotype and poor prognosis of these lymphomas. Strikingly, mantle cell lymphoma and chronic lymphocytic leukaemia expressed highly elevated levels of BCL2 protein and mRNA, higher than that observed in reactive mantle zone cells or even in follicular lymphomas, where BCL2 expression is deregulated through the t(14;18) translocation. In parallel with this observation, BIRC5/Survivin expression was higher in Burkitts lymphoma and diffuse large B‐cell lymphoma than in non‐tumoural germinal centre cells. In vitro studies confirmed that NF‐κB activation contributes to the expression of both markers. In cell lines representing aggressive lymphomas, NF‐κB inhibition resulted in a decrease in BIRC5/Survivin expression. Meanwhile, in chronic lymphocytic leukaemia (CLL)‐derived lymphocytes, NF‐κB inhibition resulted in a marked decrease in BCL2 expression. Copyright

Differential expression of NF-κB pathway genes among peripheral T-cell lymphomas

Nuclear factor kappa B (NF-κB) is one important pathway in T-cell proliferation and survival. In a previously reported microarrray study, we found NF-κB pathway genes differentially expressed between peripheral (PTCL) and lymphoblastic lymphomas. Here, we investigated the expression of NF-κB pathway genes using cDNA microarrays in a group of 62 PTCL and in reactive lymph nodes. We found two different subgroups of PTCL based on the expression of NF-κB pathway genes. One-third of PTCL showed clearly reduced expression of NF-κB genes, while the other group was characterized by high expression of these genes. This distinction was found among all T-cell lymphoma categories analyzed (PTCL unspecified, angioimmunoblastic, cutaneous and natural killer/T lymphomas) with the exception of anaplastic lymphomas (ALCL), which were characterized by reduced NF-κB expression in anaplastic cells. Quantitative RT-PCR and immunohistochemical analysis of NF-κB-p65 protein confirmed these differences among PTCL subgroups. Importantly, we found that differentiation between NF-κB-positive and -negative PTCL could be of clinical interest. The expression profile associated to reduced expression of NF-κB genes was significantly associated with shorter survival of patients and seems to be an independent prognostic factor in a multivariate analysis.

Identification of a Proliferation Signature Related to Survival in Nodal Peripheral T-Cell Lymphomas

PURPOSE Nodal peripheral T-cell lymphomas (PTCLs) constitute a heterogeneous group of neoplasms, suggesting the existence of molecular differences contributing to their histologic and clinical variability. Initial expression profiling studies of T-cell lymphomas have been inconclusive in yielding clinically relevant insights. We applied DNA microarrays to gain insight into the molecular signatures associated with prognosis. MATERIALS AND METHODS We analyzed the expression profiles of 35 nodal PTCLs (23 PTCLs unspecified and 12 angioimmunoblastic) using two different microarray platforms, the cDNA microarray developed at the Spanish National Cancer Centre and an oligonucleotide microarray. RESULTS We identified five clusters of genes, the expression of which varied significantly among the samples. Genes in these clusters seemed to be functionally related to different cellular processes such as proliferation, inflammatory response, and T-cell or B-cell lineages. Regardless of the microarray platform used, overexpression of genes in the proliferation signature was associated significantly with shorter survival of patients. This proliferation signature included genes commonly associated with the cell cycle, such as CCNA, CCNB, TOP2A, and PCNA. Moreover the PTCL proliferation signature showed a statistically significant inverse correlation with clusters of the inflammatory response (P < .0001), as well as with the percentage of CD68(+) cells. CONCLUSION Our findings indicate that proliferation could be an important factor in evaluating nodal PTCL outcome and may help to define a more aggressive phenotype.

Expression Profiling of T-Cell Lymphomas Differentiates Peripheral and Lymphoblastic Lymphomas and Defines Survival Related Genes

Purpose: T-Cell lymphomas constitute heterogeneous and aggressive tumors in which pathogenic alterations remain largely unknown. Expression profiling has demonstrated to be a useful tool for molecular classification of tumors. Experimental Design: Using DNA microarrays (CNIO-OncoChip) containing 6386 cancer-related genes, we established the expression profiling of T-cell lymphomas and compared them to normal lymphocytes and lymph nodes. Results: We found significant differences between the peripheral and lymphoblastic T-cell lymphomas, which include a deregulation of nuclear factor-κB signaling pathway. We also identify differentially expressed genes between peripheral T-cell lymphoma tumors and normal T lymphocytes or reactive lymph nodes, which could represent candidate tumor markers of these lymphomas. Additionally, a close relationship between genes associated to survival and those that differentiate among the stages of disease and responses to therapy was found. Conclusions: Our results reflect the value of gene expression profiling to gain insight about the molecular alterations involved in the pathogenesis of T-cell lymphomas.

Frequent inactivation of the p73 gene by abnormal methylation or LOH in non‐Hodgkin's lymphomas

p73 is a candidate tumor suppressor and imprinted gene that shares significant homology with the p53 gene. It is located on 1p36, a region frequently deleted in neuroblastoma and other tumors. To investigate the pattern of inactivation of this gene in human lymphomas, we studied 59 tumors to identify abnormal methylation in exon 1 and loss of heterozygosity (LOH) at this locus. p73 was methylated in 13/50 (26%) B cell lymphomas. There was no evidence of p73 methylation in the 9 T cell lymphomas analyzed. Burkitts lymphomas showed the highest proportion of methylated cases (36%), although this alteration also affected other aggressive lymphomas such as diffuse large cell and some marginal zone lymphomas. LOH at the p73 locus was detected in 4/34 (11%) B and 1/9 (11%) T cell lymphomas. The p73 expression analysis showed absence or low level of p73 product in methylated lymphomas, whereas p73 was always detected in unmethylated tumors. We found monoallelic expression in normal peripheral blood samples, consistent with imprinting. None of the tumors showed LOH and methylation of the remaining allele simultaneously, suggesting that alteration of the expressed allele could lead to the total inactivation of the gene. Our results show that deletion or methylation of the p73 gene could be important mechanisms in suppressing p73 expression in B cell non‐Hodgkins lymphomas.

Estrogen and progesterone receptor gene polymorphisms and sporadic breast cancer risk: a Spanish case-control study.

Estrogens, and to a lesser extent progesterones, influence the proliferation, differentiation and physiology of breast tissue as well as the development and progression of breast cancer. Genetic variants in the steroid hormone receptor genes ESR1 and PGR (belonging to the nuclear receptor superfamily) could therefore modify sporadic breast cancer susceptibility. Two studies have shown a protective effect associated with variants in ESR1 in 2 distinct populations. We studied 4 single nucleotide polymorphisms (SNPs) in ESR1 and 4 in PGR in 550 consecutive and unrelated sporadic Spanish breast cancer patients and 564 healthy Spanish controls. We observed a dominant protective effect for the S10S variant in ESR1, with an estimated odds ratio (OR) of 0.75 (95% CI = 0.58–0.97; p = 0.03) although functional studies did not show changes in the RNA stability. A small subset of individuals carried a haplotype combination that corroborates this protection. No other SNP considered in either gene was found to be associated with sporadic breast cancer. Our results obtained in a European population confirm the protective role of the S10S variant in ESR1, previously reported in an Asian and a European–American population.

Gene expression analysis of chromosomal regions with gain or loss of genetic material detected by comparative genomic hybridization

Comparative genomic hybridization (CGH) has been widely used to detect copy number alterations in cancer and to identify regions containing candidate tumor‐responsible genes; however, gene expression changes have been described only in highly amplified regions (amplicons). To study the overall impact of slight copy number changes on gene expression, we analyzed 16 T‐cell lymphomas by using CGH and a custom‐designed cDNA microarray containing 7,657 genes and expressed sequence tags related to tumorigenesis. We evaluated mean gene expression and variability within CGH‐altered regions and explored the relationship between the effects of the gene and its position within these regions. Minimally overlapping CGH candidate areas (6q25, 13q21–q22, and 19q13.1) revealed a weak relationship between altered genomic content and gene expression. However, some candidate genes showed modified expression within these regions in the majority of tumors; these candidate genes were evaluated and confirmed in another independent series of 23 T‐cell lymphomas by use of the same cDNA microarray and by FISH on a tissue microarray. When all the CGH regions detected for each tumor were considered, we found a significant increase or decrease in the mean expression of the genes contained in gained or lost regions, respectively. In addition, we found that the expression of a gene was dependent not only on its position within an altered region but also on its own mechanism of regulation: genes in the same altered region responded very differently to the gain or loss of genetic material. Supplementary material for this article can be found on the Genes, Chromosomes, and Cancer website at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat/index.html.

Coincidental LOH regions in mouse and humans: evidence for novel tumor suppressor loci at 9q22–q34 in non-Hodgkin’s lymphomas

Using an animal model for the study of murine thymic lymphomas, frequent losses of heterozygosity (LOHs) on six chromosomal regions were reported. To determine the existence of LOH loci in human lymphomas, we screened 43 non-Hodgkins lymphomas by using polymorphisms mapped in each of the orthologous human region. All LOH regions (1p32-p36, 9p21, 9p22-p23, 9q22-q34 and 10q23-q24) were observed in B-cell lymphomas at different frequencies. In addition, analysis of the tumor suppressor genes, p16(INK4a), p73 and PTEN located in 9p21, 1p36 and 10q23, respectively, revealed the participation of p16(INK4a) and p73 but not of PTEN. Importantly, two subregions of LOH were observed in 9q22-q34, one of them not previously described. Our results support the usefulness of murine models to study human lymphoid neoplasias and reveal novel regions on 9q22-q34 candidate for containing tumor suppressor genes involved in human lymphomas.

Identification of amplified and highly expressed genes in amplicons of the T-cell line huT78 detected by cDNA microarray CGH

BackgroundConventional Comparative Genomic Hybridization (CGH) has been widely used for detecting copy number alterations in cancer and for identifying regions containing candidate tumor responsible genes. Recently, several studies have shown the utility of cDNA microarray CGH for studing gene copy changes in various types of tumors. However, no such studies on T-cell lymphomas have been performed. To date T-cell lymphomas analyzed by the use of chromosome CGH have revealed only slight copy number alterations and not gene amplifications.ResultsIn the present study, we describe the characterization of three amplicons of the T-cell line huT78 located at 2q34-q37, 8q23-q24 and 20p, where new amplified and overexpressed genes are found. The use of a cDNA microarray containing 7.657 transcripts allowed the identification of certain genes, such as BCLX, PCNA, FKBP1A, IGFBP2 and cMYC, that are amplified, highly expressed, and also contained in the amplicons on 20p and 2q. The expresion of these genes was analyzed in 39 T-cell lymphomas and 3 other T-cell lines.ConclusionBy the use of conventional CGH and CGH and expression cDNA microarrays we defined three amplicons in the T-cell line huT78 and identified several novel gene amplifications (BCLX, PCNA, FKBP1A, IGFBP2 and cMYC). We showed that overexpression of the amplified genes could be attributable to gene dosage. We speculate that deregulation of those genes could be important in the development of T-cell lymphomas and/or in the maintenance of T-cell lines.