Beatriz Martínez-Delgado

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.

Genetic anticipation is associated with telomere shortening in hereditary breast cancer.

There is increasing evidence suggesting that short telomeres and subsequent genomic instability contribute to malignant transformation. Telomere shortening has been described as a mechanism to explain genetic anticipation in dyskeratosis congenita and Li-Fraumeni syndrome. Since genetic anticipation has been observed in familial breast cancer, we aimed to study telomere length in familial breast cancer patients and hypothesized that genetic defects causing this disease would affect telomere maintenance resulting in shortened telomeres. Here, we first investigated age anticipation in mother-daughter pairs with breast cancer in 623 breast cancer families, classified as BRCA1, BRCA2, and BRCAX. Moreover, we analyzed telomere length in DNA from peripheral blood leukocytes by quantitative PCR in a set of 198 hereditary breast cancer patients, and compared them with 267 control samples and 71 sporadic breast cancer patients. Changes in telomere length in mother-daughter pairs from breast cancer families and controls were also evaluated to address differences through generations. We demonstrated that short telomeres characterize hereditary but not sporadic breast cancer. We have defined a group of BRCAX families with short telomeres, suggesting that telomere maintenance genes might be susceptibility genes for breast cancer. Significantly, we described that progressive telomere shortening is associated with earlier onset of breast cancer in successive generations of affected families. Our results provide evidence that telomere shortening is associated with earlier age of cancer onset in successive generations, suggesting that it might be a mechanism of genetic anticipation in hereditary breast cancer.

Gross SDHB deletions in patients with paraganglioma detected by multiplex PCR: A possible hot spot?

Pheochromocytoma and paraganglioma are rare neuroendocrine tumors that arise in the adrenal medulla and the extra‐adrenal paraganglia, respectively. Inheritance of these tumors is mainly a result of mutations affecting the VHL, RET, NF1, and SDH genes. Germ‐line mutations of the SDH genes have been found to account for nearly 10% of apparently sporadic cases. Nevertheless, alterations other than point mutations have not yet been well characterized. In this study, we investigated the frequency of gross SDH deletions in 24 patients who tested negative for point mutations and had at least one of the recommended features for genetic testing. For this purpose, we used a technique that is easy to implement in the lab to specifically detect gross deletions affecting SDHB, SDHC, and SDHD. We identified 3 heterozygous SDHB deletions (3/24) in 3 independent cases with paraganglioma: 1 whole SDHB deletion and 2 deletions exclusively affecting exon 1. These latter mutations match the unique gross deletion previously reported, indicating this region could be a hot spot for gross SDHB deletions. It seems likely that these alterations can account for a considerable number of both familial and apparently sporadic paraganglioma cases. Although this is the first report describing the presence of gross deletions in patients with apparently sporadic paragangliomas, the extra‐adrenal location of the tumor seems to constitute a determining factor for whether to include these patients in genetic testing for gross deletions in the SDHB gene.

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.

Tumor microRNA expression profiling identifies circulating microRNAs for early breast cancer detection.

BACKGROUND The identification of novel biomarkers for early breast cancer detection would be a great advance. Because of their role in tumorigenesis and stability in body fluids, microRNAs (miRNAs) are emerging as a promising diagnostic tool. Our aim was to identify miRNAs deregulated in breast tumors and evaluate the potential of circulating miRNAs in breast cancer detection. METHODS We conducted miRNA expression profiling of 1919 human miRNAs in paraffin-embedded tissue from 122 breast tumors and 11 healthy breast tissue samples. Differential expression analysis was performed, and a microarray classifier was generated. The most relevant miRNAs were analyzed in plasma from 26 healthy individuals and 83 patients with breast cancer (36 before and 47 after treatment) and validated in 116 healthy individuals and 114 patients before treatment. RESULTS We identified a large number of miRNAs deregulated in breast cancer and generated a 25-miRNA microarray classifier that discriminated breast tumors with high diagnostic sensitivity and specificity. Ten miRNAs were selected for further investigation, of which 4 (miR-505-5p, miR-125b-5p, miR-21-5p, and miR-96-5p) were significantly overexpressed in pretreated patients with breast cancer compared with healthy individuals in 2 different series of plasma. MiR-505-5p and miR-96-5p were the most valuable biomarkers (area under the curve 0.72). Moreover, the expression levels of miR-3656, miR-505-5p, and miR-21-5p were decreased in a group of treated patients. CONCLUSIONS Circulating miRNAs reflect the presence of breast tumors. The identification of deregulated miRNAs in plasma of patients with breast cancer supports the use of circulating miRNAs as a method for early breast cancer detection.

Deregulated miRNAs in hereditary breast cancer revealed a role for miR-30c in regulating KRAS oncogene.

Aberrant miRNA expression has been previously established in breast cancer and has clinical relevance. However, no studies so far have defined miRNAs deregulated in hereditary breast tumors. In this study we investigated the role of miRNAs in hereditary breast tumors comparing with normal breast tissue. Global miRNA expression profiling using Exiqon microarrays was performed on 22 hereditary breast tumors and 15 non-tumoral breast tissues. We identified 19 miRNAs differentially expressed, most of them down-regulated in tumors. An important proportion of deregulated miRNAs in hereditary tumors were previously identified commonly deregulated in sporadic breast tumors. Under-expression of these miRNAs was validated by qRT-PCR in additional 18 sporadic breast tumors and their normal breast tissue counterparts. Pathway enrichment analysis revealed that deregulated miRNAs collectively targeted a number of genes belonging to signaling pathways such as MAPK, ErbB, mTOR, and those regulating cell motility or adhesion. In silico prediction detected KRAS oncogene as target of several deregulated miRNAs. In particular, we experimentally validated KRAS as a miR-30c target. Luciferase assays confirmed that miR-30c binds the 3′UTR of KRAS transcripts and expression of pre-miR-30c down-regulated KRAS mRNA and protein. Furthermore, miR-30c overexpression inhibited proliferation of breast cancer cells. Our results identify miRNAs associated to hereditary breast cancer, as well as miRNAs commonly miss-expressed in hereditary and sporadic tumors, suggesting common underlying mechanisms of tumor progression. In addition, we provide evidence that KRAS is a target of miR-30c, and that this miRNA suppresses breast cancer cell growth potentially through inhibition of KRAS signaling.

Characterization of the A673 cell line (Ewing tumor) by molecular cytogenetic techniques

The A673 cell line was established from a patient with a primary rhabdomyosarcoma (RMS), which is referred to in the literature either as a Ewing tumor (ET) or as RMS. Although the two tumoral types are associated with specific and well-characterized translocations, no cytogenetic report on this cell line has been published. We characterized the A673 cell line using a combination of spectral karyotyping (SKY), fluorescence in situ hybridization (FISH), and reverse transcriptase polymerase chain reaction (RT-PCR), which revealed the presence of a complex karyotype and a translocation involving chromosomes 11 and 22 and the fusion of EWS and FLI1 genes, both events being specific to ET. Neither cytogenetics nor molecular alterations specific to RMS were found.

Shorter telomere length is associated with increased ovarian cancer risk in both familial and sporadic cases

Background Alterations in telomere maintenance mechanisms leading to short telomeres underlie different genetic disorders of ageing and cancer predisposition syndromes. It is known that short telomeres and subsequent genomic instability contribute to malignant transformation, and it is therefore likely that people with shorter telomeres are at higher risk for different types of cancer. Recently, the authors demonstrated that the genes BRCA1 and BRCA2 are modifiers of telomere length (TL) in familial breast cancer. The present study analysed TL in peripheral blood leucocytes of hereditary and sporadic ovarian cancer cases, as well as in female controls, to evaluate whether TL contributes to ovarian cancer risk. Methods TL was measured by quantitative PCR in 178 sporadic and 168 hereditary ovarian cases (46 BRCA1, 12 BRCA2, and 110 BRCAX) and compared to TL in 267 controls. Results Both sporadic and hereditary cases showed significantly shorter age adjusted TLs than controls. Unconditional logistic regression analysis revealed an association between TL and ovarian cancer risk with a significant interaction with age (p<0.001). Risk was higher in younger women and progressively decreased with age, with the highest OR observed in women under 30 years of age (OR 1.56, 95% CI 1.34 to 1.81; p=1.0×10−18). Conclusion These findings indicate that TL could be a risk factor for early onset ovarian cancer.