Yolanda Campos-Martín

Nodal marginal zone lymphoma: gene expression and miRNA profiling identify diagnostic markers and potential therapeutic targets

Nodal marginal zone lymphoma (NMZL) is a small B-cell neoplasm whose molecular pathogenesis is still essentially unknown and whose differentiation from other small B-cell lymphomas is hampered by the lack of specific markers. We have analyzed gene expression, miRNA profile, and copy number data from 15 NMZL cases. For comparison, 16 follicular lymphomas (FLs), 9 extranodal marginal zone lymphomas, and 8 reactive lymph nodes and B-cell subtypes were included. The results were validated by quantitative RT-PCR in an independent series, including 61 paraffin-embedded NMZLs. NMZL signature showed an enriched expression of gene sets identifying interleukins, integrins, CD40, PI3K, NF-κB, and TGF-β, and included genes expressed by normal marginal zone cells and memory B cells. The most highly overexpressed genes were SYK, TACI, CD74, CD82, and CDC42EP5. Genes linked to G(2)/M and germinal center were down-regulated. Comparison of the gene expression profiles of NMZL and FL showed enriched expression of CHIT1, TGFB1, and TACI in NMZL, and BCL6, LMO2, and CD10 in FL. NMZL displayed increased expression of miR-221, miR-223, and let-7f, whereas FL strongly expressed miR-494. Our study identifies new candidate diagnostic molecules for NMZL and reveals survival pathways activated in NMZL.

Worse outcome in primary glioblastoma multiforme with concurrent epidermal growth factor receptor and p53 alteration.

Primary glioblastoma multiforme (GBM), in contrast with secondary GBM, has been associated with the presence of EGFR amplification and absence of p53 mutation. In this study, we analyzed relevant molecular and clinical variables in 194 primary GBMs and tested them for survival analysis. Although most of the tumors showed a mutually exclusive pattern, concurrent alterations of EGFR and p53 were detected. Survival analysis of CDK4 amplification revealed a highly significant association with a worse clinical outcome (P = .01), whereas MDM2, CDK6, PTEN, and p21 were not associated with patient survival. Multivariate analysis including the significant clinical and molecular variables revealed CDK4 amplification, age, and radiotherapy to be markers with independent prognostic value. In addition, the primary GBM tumors showing simultaneous EGFR and p53 alterations were significantly associated with worse survival (P < .01). These results highlight the prognostic value of CDK4 amplification and of simultaneous EGFR-p53 alterations in the clinical outcome of patients with primary GBM.

Stimulation of the midkine/ALK axis renders glioma cells resistant to cannabinoid antitumoral action

Identifying the molecular mechanisms responsible for the resistance of gliomas to anticancer treatments is an issue of great therapeutic interest. Δ9-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids inhibit tumor growth in animal models of cancer, including glioma, an effect that relies, at least in part, on the stimulation of autophagy-mediated apoptosis in tumor cells. Here, by analyzing the gene expression profile of a large series of human glioma cells with different sensitivity to cannabinoid action, we have identified a subset of genes specifically associated to THC resistance. One of these genes, namely that encoding the growth factor midkine (Mdk), is directly involved in the resistance of glioma cells to cannabinoid treatment. We also show that Mdk mediates its protective effect via the anaplastic lymphoma kinase (ALK) receptor and that Mdk signaling through ALK interferes with cannabinoid-induced autophagic cell death. Furthermore, in vivo Mdk silencing or ALK pharmacological inhibition sensitizes cannabinod-resistant tumors to THC antitumoral action. Altogether, our findings identify Mdk as a pivotal factor involved in the resistance of glioma cells to THC pro-autophagic and antitumoral action, and suggest that selective targeting of the Mdk/ALK axis could help to improve the efficacy of antitumoral therapies for gliomas.

Genetic alterations associated with progression and recurrence in meningiomas.

Abstract Meningiomas are the most common primary brain tumors; they arise from the coverings of the brain. Although meningiomas are generally benign, some are more clinically aggressive, as reflected by their histopathological features or by their unexpected recurrence. We hypothesized that recurrent histologically benign meningiomas might have genetic features in common with those showing a more aggressive histology. By comparing gene expression profiles associated with meningioma progression and recurrence in 128 tumor samples (i.e. 83 benign World Health Organization [WHO] Grade I, 37atypical WHO Grade II, and 8 anaplastic WHO Grade III) from121patients, we identified a 49-gene signature of meningioma aggressivity. This signature classified the tumors into 2 groups showing different clinical and pathological behaviors. The signature was composed of genes involved in the cell cycle (TMEM30B, CKS2, and UCHL1) and other pathways previously described as being altered in meningiomas, that is, WNT (SFRP1 and SFRP4) and transforming growth factor-&bgr; pathways (LTBP2 and LMO4). Overall, gene downregulation was observed in advanced and recurrentsamples versus benign and original ones. We propose that this gene repression may be caused by gene promoter hypermethylation, as in the case of UCHL1 and SFRP1, suggesting that this epigenetic event, together with loss of specific chromosomal regions, may play an important role in meningioma progression and recurrence.

Differential expression profiling analyses identifies downregulation of 1p, 6q, and 14q genes and overexpression of 6p histone cluster 1 genes as markers of recurrence in meningiomas

The majority of meningiomas are probably benign but a number of tumors display considerable histological and/or clinical aggressivity, sometimes with unexpectedly high recurrence rates after radical removal. Understanding the potential behavior of these tumors in individual patients is critical for rational therapeutic decision-making. This study aimed to identify gene expression profiles and candidate markers associated with original and recurrent meningiomas. Unsupervised hierarchical clustering of the samples confirmed 2 main groups of meningiomas with distinct clinical behaviors. The gene expression profiling study identified genes and pathways potentially associated with meningioma recurrence, revealing an overall lower level of gene expression. The differential gene expression profiling analyses of original and recurrent meningiomas identified 425 known genes and expressed sequence tags related to meningioma recurrence, with SFRP1 (8p12), TMEM30B (14q23), and CTGF (6q23) showing the most disparate expression. Most of the differentially expressed genes were located at 1p, 6q, and 14q and were underexpressed in recurrences. Loss of such chromosomal regions has previously been associated with a higher risk of meningioma recurrence or malignant progression. Thus, at these locations, we propose the existence of novel candidate genes that could be involved in meningioma recurrence. In addition, the overexpression of genes of histone cluster 1 (6p) in recurrent meningiomas is reported here for the first time. Finally, the altered genes related to meningioma recurrence are involved in pathways such as Notch, TGFβ, and Wnt, as described previously, and in other pathways such as cell cycle, oxidative phosphorylation, PPAR, and PDGF, not related before to meningioma recurrence.

Gene expression changes associated with erlotinib response in glioma cell lines

Erlotinib (ERL), a tyrosine kinase inhibitor that acts on the epidermal growth factor receptor (EGFR), is used as a second line treatment for glioma therapy, with controversial findings regarding its response. Here, we analysed the gene expression profiles of a series of human glioma cell lines with differing sensitivities to ERL to identify the gene expression changes associated with ERL response. The varying responses to ERL were associated with different expression levels of specific genes (HRAS, CTFG, ERCC5 and HDAC3) and genes associated with specific pathways (apoptosis and cell death). PI3K pathway genes were primarily affected by ERL, as we found that PIK3R3 was repressed by ERL treatment in sensitive glioma cell lines. The cell cycle and ubiquitin pathways were also affected by EGFR inhibition, as GAS5, PLK1 and BIRC5 were the most significantly affected genes. In this study we have identified several genes such as PIK3R3 and GAS5, that can be targeted in order to enhance the response to ERL therapy.

Novel Genomic Alterations and Mechanisms Associated With Tumor Progression in Oligodendroglioma and Mixed Oligoastrocytoma

Combined 1p/19q deletions are very prevalent in oligodendrogliomas (OGs) and, to a lesser extent, in oligoastrocytomas (OAs). These losses are associated with responsiveness to therapy. Using array-based comparative genomic hybridization, we screened for recurrent genomic alterations in OG and oligoastrocytoma subtypes on chromosome 19. Concomitant 1p/19q loss was detected in most of the tumors with allelic loss, but array-based comparative genomic hybridization revealed some tumors to have unrelated 1p/19q arm losses, suggesting alternative mechanisms of loss to that related to the reported t(1;19) translocation. Analyses of 1p/19q loss by fluorescence in situ hybridization and loss of heterozygosity assays and correlations of genomic data with the Ki-67 proliferation marker were also performed. Four 1q (or 19p) and 2 1p (or 19q) fluorescence in situ hybridization probe signals together with homozygosity of the 1p/19q microsatellites suggested a hypothetical mechanism of genome duplication consecutive to the loss of the derivative chromosome der(1p;19q) from the t(1;19)(1q;19p) translocation. This genome duplication was frequent in high-grade OGs and was strongly correlated with Ki-67 expression; thus, it could be related to tumor progression. Finally, in addition to the frequent 1p/19q loss, we report a novel 17q amplified region in OGs with BIRC5 as one of the possible candidate target genes of the amplicon.

Glomerular C3d as a novel prognostic marker for renal vasculitis.

Pauci-immune necrotizing crescentic glomerulonephritis is the histologic substrate of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. Several studies in animal models have demonstrated the crucial role of complement activation in the pathogenesis of ANCA-associated vasculitis, but only small series have analyzed the prognostic implications of complement glomerular deposits. This study aimed to assess the clinical and prognostic implications of C3d- and C4d-positive glomerular staining in renal vasculitis. Eighty-five patients with a diagnosis of pauci-immune necrotizing crescentic glomerulonephritis were included in the study. C3d and C4d were analyzed by immunohistochemical staining using a polyclonal antibody. The primary predictors were glomerular C3d- and C4d-positive staining. The primary end point was the cumulative percentage of patients who developed end-stage renal disease. Glomerular staining for C3d and C4d was observed in 42 (49.4%) of 85 biopsies and 38 (44.7%) of 85 biopsies, respectively. C3d-positive staining was associated with the severity of renal impairment and with a lower response rate to treatment (P=.003 and P=.04, respectively). Renal survival at 2 and 5 years was 60.9% and 51.8% in C3d-positive patients compared with 87.7% and 78.9% in C3d-negative patients (P=.04). C4d-positive staining did not show any impact in renal outcome. When adjusted by renal function and other histologic parameters, C3d staining remained as an independent predictor for renal survival (hazard ratio, 2.5; 95% confidence interval, 1.1-5.7; P=.03). Therefore, this study demonstrates that C3d-positive glomerular staining is an independent risk factor for the development of end-stage renal disease in ANCA-associated renal vasculitis.

Copy Number Alterations in Glioma Cell Lines

Established tumor-derived cell lines are widely and routinely used as in vitro cancer models for various kinds of biomedical research. The easy management of these cell cultures, in contrast to the inherent difficulty in establishing and mantaining primary tumoral cultures, has contributed to the wide use of these inmortalized cell lines in order to characterize the biological significance of specific genomic aberrations identified in primary tumors. Therefore, it has been assumed that the genomic and expression aberrations of long-term established cell lines resemble, and are representative, of the primary tumor from which the cell line was derived. Indeed, the cell line-based research has been performed, not only for the definition of the molecular biology of several cancer models, but also for the investigation of new targeted therapeutic agents in a prior step to clinical practice. The use of tumor-derived cell lines has been highly relevant for the testing and development of new therapeutical agents, with several cancer cell-line panels having been developed for drug sensitivity screening and new agents’ discovery (Sharma et al, 2010). Controversial concerning the ability of tumor-derived cell lines to accurately reflect the phenotype and genotype of the parental histology has been documented. A previous report of Greshock and coworkers using array-based Comparative Genomic Hybridization (aCGH) data of seven diagnosis-specific matched tumors and cell lines showed that, on average, cell lines preserve in vitro the genetic aberrations that are unique to the parent histology from which they were derived, while acquiring additional locus-specific alterations in long-term cultures (Greshock et al, 2007). In contrast, a study on breast cancer cell lines and primary tumors highlight that cell lines do not always represent the genotypes of parental tumor tissues (Tsuji et al, 2010). Furthermore, a parallel genomic and expression study on glioma cell lines and primary tumors states that in this specific cancer type, cell lines are poor representative of the primary tumors (Li et al, 2008). Given the importance of the use of cell lines as models for the study of the biology and development of tumors, and for the testing of the mode of action of new therapeutical agents, the knowledge of which genomic alterations are tumor-specific or which are necessary for the maintenance of the cell line in culture, becomes essential.

Clinical and diagnostic relevance of NOTCH2-and KLF2-mutations in splenic marginal zone lymphoma.

Splenic marginal zone lymphoma (SMZL) is a small Bcell lymphoma distinguished by frequent 7q loss and the biased use of IGHV01-02. Massive sequencing techniques have demonstrated NOTCH2 and KLF2 gene mutations to be the most frequent mutations in SMZL, with NOTCH2 mutations occurring in 10-25% of SMZL cases, and transcription factor KLF2 mutations present in 12-44% of SMZLs. The clinical impact of NOTCH2 alterations is controversial, with different studies reaching very distinct conclusions. Herein, we have analysed NOTCH2 and KLF2 mutations in a large series of SMZLs and examined the associations of these alterations with clinicopathological features. Our results confirm the association of the NOTCH2 mutation with shorter median treatment-free survival and suggest the possible usefulness of the identification of these changes for the diagnosis of SMZL. The study panel comprised a series of 84 SMZL patients, and, for comparison purposes, 76 non-SMZL B-cell lymphomas, including 12 splenic diffuse red pulp B-cell lymphoma (SDRPL), 13 chronic lymphocytic leukemia (CLL), 14 mantle cell lymphoma (MCL), 14 follicular lymphoma (FL), 1 hairy cell leukemia (HCL) and 18 lymphoplasmacytic lymphoma (LPL). Four cases of monoclonal B-cell lymphocytosis (MBL) with non-CLL phenotype were also included. The diagnosis of all the lymphoma types included in the series was performed according to WHO criteria and Matutes et al. MBL cases had a B-cell monoclonal population without lymphadenopathy or splenomegaly. The NOTCH2 C-terminal coding exon 34 and the KLF2 exons 1-3 were sequenced by Sanger sequencing for all tumor samples in the study herein (Online Supplementary Table S1). We identified 17 NOTCH2 mutations in 14 of the 84 (16.6%) cases of SMZL, noting double mutations in 3 cases. Their somatic nature was confirmed in 5 cases for which non-tumor DNA was available. Of the 17 NOTCH2 mutations identified in SMZL, 9 were truncating (frameshift or nonsense) mutations. These deleterious mutations were localized in the nuclear localization signal (NLS), transactivation domain (TAD) and PEST domains (Figure 1), where they are predicted to alter the correct transport of the notch intracellular domain (NICD) to the nucleus or to eliminate degradation signals in the PEST domain, increasing the stability of the NICD. The remaining 8 mutations seen in SMZL were missense variants localized in the TAD and PEST domains. Two of them were predicted to be deleterious by the PolyPhen-2 program (Online Supplementary Table S2). No NOTCH2 abnormality was seen in the other B-cell lymphomas examined: in SDRPL (0/9), CLL (0/13), MCL (0/14), FL (0/14), HCL (0/1), LPL (0/15) and MBL (0/4). Consistent with our findings, this alteration has been reported only occasionally in other B-cell lymphomas, such as extranodal marginal zone lymphoma (1.5%), in diffuse large Bcell lymphoma (3.7-4.3%), MCL (5.2%) and FL (1.94%). It is of note that NOTCH-mutated FLs showed significantly more frequent splenic involvement than wildtype cases. The analysis of the relationship of NOTCH2 mutations with other alterations involved in SMZL pathogenesis has revealed an association with the deletion of 7q31-32,3 IgHV1-2 overrepresentation and higher promoter-methylation status.