Differential expression of genes mapping to recurrently abnormal chromosomal regions characterize neuroblastic tumours with distinct ploidy status

<p>Abstract</p> <p>Background</p> <p>Neuroblastic tumours (NBTs) represent a heterogeneous spectrum of neoplastic diseases associated with multiple genetic alterations. Structural and numerical chromosomal changes are frequent and are predictive parameters of NBTs outcome. We performed a comparative analysis of the biological entities constituted by NBTs with different ploidy status.</p> <p>Methods</p> <p>Gene expression profiling of 49 diagnostic primary NBTs with ploidy data was performed using oligonucleotide microarray. Further analyses using Quantitative Real-Time Polymerase Chain Reaction (Q-PCR); array-Comparative Genomic Hybridization (aCGH); and Fluorescent <it>in situ </it>Hybridization (FISH) were performed to investigate the correlation between aneuploidy, chromosomal changes and gene expression profiles.</p> <p>Results</p> <p>Gene expression profiling of 49 primary near-triploid and near-diploid/tetraploid NBTs revealed distinct expression profiles associated with each NBT subgroup. A statistically significant portion of genes mapped to 1p36 (<it>P </it>= 0.01) and 17p13-q21 (<it>P </it>< 0.0001), described as recurrently altered in NBTs. Over 90% of these genes showed higher expression in near-triploid NBTs and the majority are involved in cell differentiation pathways. Specific chromosomal abnormalities observed in NBTs, 1p loss, 17q and whole chromosome 17 gains, were reflected in the gene expression profiles. Comparison between gene copy number and expression levels suggests that differential expression might be only partly dependent on gene copy number. Intratumoural clonal heterogeneity was observed in all NBTs, with marked interclonal variability in near-diploid/tetraploid tumours.</p> <p>Conclusion</p> <p>NBTs with different cellular DNA content display distinct transcriptional profiles with a significant portion of differentially expressed genes mapping to specific chromosomal regions known to be associated with outcome. Furthermore, our results demonstrate that these specific genetic abnormalities are highly heterogeneous in all NBTs, and suggest that NBTs with different ploidy status may result from different mechanisms of aneuploidy driving tumourigenesis.</p

Differential expression of genes mapping to recurrently abnormal chromosomal regions characterize neuroblastic tumours with distinct ploidy status

<p>Abstract</p> <p>Background</p> <p>Neuroblastic tumours (NBTs) represent a heterogeneous spectrum of neoplastic diseases associated with multiple genetic alterations. Structural and numerical chromosomal changes are frequent and are predictive parameters of NBTs outcome. We performed a comparative analysis of the biological entities constituted by NBTs with different ploidy status.</p> <p>Methods</p> <p>Gene expression profiling of 49 diagnostic primary NBTs with ploidy data was performed using oligonucleotide microarray. Further analyses using Quantitative Real-Time Polymerase Chain Reaction (Q-PCR); array-Comparative Genomic Hybridization (aCGH); and Fluorescent <it>in situ </it>Hybridization (FISH) were performed to investigate the correlation between aneuploidy, chromosomal changes and gene expression profiles.</p> <p>Results</p> <p>Gene expression profiling of 49 primary near-triploid and near-diploid/tetraploid NBTs revealed distinct expression profiles associated with each NBT subgroup. A statistically significant portion of genes mapped to 1p36 (<it>P </it>= 0.01) and 17p13-q21 (<it>P </it>< 0.0001), described as recurrently altered in NBTs. Over 90% of these genes showed higher expression in near-triploid NBTs and the majority are involved in cell differentiation pathways. Specific chromosomal abnormalities observed in NBTs, 1p loss, 17q and whole chromosome 17 gains, were reflected in the gene expression profiles. Comparison between gene copy number and expression levels suggests that differential expression might be only partly dependent on gene copy number. Intratumoural clonal heterogeneity was observed in all NBTs, with marked interclonal variability in near-diploid/tetraploid tumours.</p> <p>Conclusion</p> <p>NBTs with different cellular DNA content display distinct transcriptional profiles with a significant portion of differentially expressed genes mapping to specific chromosomal regions known to be associated with outcome. Furthermore, our results demonstrate that these specific genetic abnormalities are highly heterogeneous in all NBTs, and suggest that NBTs with different ploidy status may result from different mechanisms of aneuploidy driving tumourigenesis.</p

Structure versus function: correlation between outer retinal and choroidal thicknesses measured by swept-source OCT with multifocal electroretinography and visual acuity

Background: To correlate retina-choroidal anatomy as assessed via swept-source OCT (SS-OCT) with retinal function as determined by best-corrected visual acuity (BCVA) and multifocal electroretinogram (mfERG). Methods: Thirty-three eyes from 33 patients including 16 with neovascular AMD (nvAMD) and 17 controls were included. Patients were included in the present study after a complete ophthalmologic examination, including BCVA, slit-lamp study, intraocular pressure measurement, dilated fundus examination after tropicamide instillation, SD-OCT, SS-OCT, fundus photographs and mfERG. Age, sex, BCVA, number of anti-VEGF intravitreal injections in the nvAMD group, were recollected. Outer retinal and choroidal thickness were determined at the fovea and 500 μm temporal, superior, nasal and inferior. First-order response from mfERG was collected. P1 amplitude was recorded in R1, R2 and the average of R1 + R2. The measurements recollected from the SS-OCT, mfERG and BCVA were compared. Results: Better BCVA was found with thicker outer retina foveal thickness (r = 0.349; P = 0.047), with thicker subfoveal choroidal thickness (r = 0.443; P = 0.010), and with higher amplitude in P1 at R1 (r = 0.346; P = 0.037). Outer retina foveal thickness did not correlate with P1 amplitude at R1 (r = 0.072; P = 0.692), R2 (r = 0.265; P = 0.137) either with the average P1 amplitude at R1 + R2 (r = 0.253; P = 0.156). A thicker subfoveal choroidal thickness was related with higher amplitude in P1 at R1 (r = 0.383; P = 0.028), R2 (r = 0.409; P = 0.018) and the average of R1 + R2 (r = 0.419; P = 0.015). Conclusions: Choroidal thickness demonstrated a positive correlation with retinal function in the sample studied, so a thicker choroid is related to a better retinal function measured with mfERG and BCVA

DNA Methylomes Reveal Biological Networks Involved in Human Eye Development, Functions and Associated Disorders

This work provides a comprehensive CpG methylation landscape of the different layers of the human eye that unveils the gene networks associated with their biological functions and how these are disrupted in common visual disorders. Herein, we firstly determined the role of CpG methylation in the regulation of ocular tissue-specification and described hypermethylation of retinal transcription factors (i.e., PAX6, RAX, SIX6) in a tissue-dependent manner. Second, we have characterized the DNA methylome of visual disorders linked to internal and external environmental factors. Main conclusions allow certifying that crucial pathways related to Wnt-MAPK signaling pathways or neuroinflammation are epigenetically controlled in the fibrotic disorders involved in retinal detachment, but results also reinforced the contribution of neurovascularization (ETS1, HES5, PRDM16) in diabetic retinopathy. Finally, we had studied the methylome in the most frequent intraocular tumors in adults and children (uveal melanoma and retinoblastoma, respectively). We observed that hypermethylation of tumor suppressor genes is a frequent event in ocular tumors, but also unmethylation is associated with tumorogenesis. Interestingly, unmethylation of the proto-oncogen RAB31 was a predictor of metastasis risk in uveal melanoma. Loss of methylation of the oncogenic mir-17-92 cluster was detected in primary tissues but also in blood from patients.The research leading to these results was supported by European Research Council Advanced Grant EPINORC, RecerCaixa Foundation, Federación Española de Enfermedades Raras (FEDER), Federación Española de Enfermedades Neuromusculares (ASEM), Fundación Isabel Gemio, COST CM1406, Instituto de Salud Carlos III (PI/00816) and Health and Sciences Departments of the Catalan Government (Generalitat de Catalunya). M.E. is an Institució Catalana de Recerca i Estudis Avançats (ICREA) Research Professor. We thank the staff of the Biobank Facility at the Bellvitge Biomedical Research Institute (IDIBELL), Spanish National Cancer Research Center (CNIO), Institute of Rare Diseases Research (BioNER-ISCIII), Vall d’Hebron Research Institute (VHIR) and Banc de Sang i Teixits (BST) of the Catalan Ministry of Health. We also thank Dr. Mercedes Hurtado (Department of Ophthalmology, University and Polytechnic Hospital La Fe) and Dr. Dolores Pinazo (Department of Ophthalmology, Dr. Peset University Hospital) for obtaining samples from glaucomatous patients. We thank the patients and their families.S

Evaluation of AQP4 functional variants and its association with fragile X-associated tremor/ataxia syndrome

Fragile X-associated tremor/ataxia syndrome (FXTAS, OMIM# 300623) is a late-onset neurodegenerative disorder with reduced penetrance that appears in adult FMR1 premutation carriers (55-200 CGGs). Clinical symptoms in FXTAS patients usually begin with an action tremor. After that, different findings including ataxia, and more variably, loss of sensation in the distal lower extremities and autonomic dysfunction, may occur, and gradually progress. Cognitive deficits are also observed, and include memory problems and executive function deficits, with a gradual progression to dementia in some individuals. Aquaporin 4 (AQP4) is a commonly distributed water channel in astrocytes of the central nervous system. Changes in AQP4 activity and expression have been implicated in several central nervous system disorders. Previous studies have suggested the associations of AQP4 single nucleotide polymorphisms (SNPs) with brain-water homeostasis, and neurodegeneration disease. To date, this association has not been studied in FXTAS. To investigate the association of AQP4 SNPs with the risk of presenting FXTAS, a total of seven common AQP4 SNPs were selected and genotyped in 95 FMR1 premutation carriers with FXTAS and in 65 FMR1 premutation carriers without FXTAS. The frequency of AQP4 -haplotype was compared between groups, denoting 26 heterozygous individuals and 5 homozygotes as carriers of the minor allele in the FXTAS group and 25 heterozygous and 2 homozygotes in the no-FXTAS group. Statistical analyses showed no significant associations between AQP4 SNPs/haplotypes and development of FXTAS. Although AQP4 has been implicated in a wide range of brain disorders, its involvement in FXTAS remains unclear. The identification of novel genetic markers predisposing to FXTAS or modulating disease progression is critical for future research involving predictors and treatments

Specific gene expression profiles and chromosomal abnormalities are associated with infant disseminated neuroblastoma

Background: Neuroblastoma (NB) tumours have the highest incidence of spontaneous remission, especially among the stage 4s NB subgroup affecting infants. Clinical distinction of stage 4s from lethal stage 4 can be difficult, but critical for therapeutic decisions. The aim of this study was to investigate chromosomal alterations and differential gene expression amongst infant disseminated NB subgroups. Methods: Thirty-five NB tumours from patients diagnosed at < 18 months (25 stage 4 and 10 stage 4s), were evaluated by allelic and gene expression analyses. Results: All stage 4s patients underwent spontaneous remission, only 48% stage 4 patients survived despite combined modality therapy. Stage 4 tumours were 90% near-diploid/tetraploid, 44% MYCN amplified, 77% had 1p LOH (50% 1p36), 23% 11q and/or 14q LOH (27%) and 47% had 17q gain. Stage 4s were 90% near-triploid, none MYCN amplified and LOH was restricted to 11q. Initial comparison analyses between stage 4s and 4 < 12 months tumours revealed distinct gene expression profiles. A significant portion of genes mapped to chromosome 1 (P < 0.0001), 90% with higher expression in stage 4s, and chromosome 11 (P = 0.0054), 91% with higher expression in stage 4. Less definite expression profiles were observed between stage 4s and 4 < 18m, yet, association with chromosomes 1 (P < 0.0001) and 11 (P = 0.005) was maintained. Distinct gene expression profiles but no significant association with specific chromosomal region localization was observed between stage 4s and stage 4 < 18 months without MYCN amplification. Conclusion: Specific chromosomal aberrations are associated with distinct gene expression profiles which characterize spontaneously regressing or aggressive infant NB, providing the biological basis for the distinct clinical behaviour

EWS/FLI Confers Tumor Cell Synthetic Lethality to CDK12 Inhibition in Ewing Sarcoma

Many cancer types are driven by oncogenic transcription factors that have been difficult to drug. Transcriptional inhibitors, however, may offer inroads into targeting these cancers. Through chemical genomics screening, we identified that Ewing sarcoma is a disease with preferential sensitivity to THZ1, a covalent small-molecule CDK7/12/13 inhibitor. The selective CDK12/13 inhibitor, THZ531, impairs DNA damage repair in an EWS/FLI-dependent manner, supporting a synthetic lethal relationship between response to THZ1/THZ531 and EWS/FLI expression. The combination of these molecules with PARP inhibitors showed striking synergy in cell viability and DNA damage assays in vitro and in multiple models of Ewing sarcoma, including a PDX, in vivo without hematopoietic toxicity. Iniguez et al. find that inhibition of CDK12 is synthetic lethal with EWS/FLI expression. CDK12/13 inhibitors impair DNA damage repair in cells expressing EWS/FLI, and the combination of CDK12/13 and PARP inhibitors synergistically reduces tumor growth and extends survival in Ewing sarcoma mouse models

Selective inhibition of HDAC6 regulates expression of the oncogenic driver EWSR1-FLI1 through the EWSR1 promoter in Ewing sarcoma

Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor of children and young adults in which the principal driver is a fusion gene, EWSR1-FLI1. Although the essential role of EWSR1-FLI1 protein in the regulation of oncogenesis, survival, and tumor progression processes has been described in-depth, little is known about the regulation of chimeric fusion-gene expression. Here, we demonstrate that the active nuclear HDAC6 in EWS modulates the acetylation status of specificity protein 1 (SP1), consequently regulating the SP1/P300 activator complex binding to EWSR1 and EWSR1-FLI1 promoters. Selective inhibition of HDAC6 impairs binding of the activator complex SP1/P300, thereby inducing EWSR1-FLI1 downregulation and significantly reducing its oncogenic functions. In addition, sensitivity of EWS cell lines to HDAC6 inhibition is higher than other tumor or non-tumor cell lines. High expression of HDAC6 in primary EWS tumor samples from patients correlates with a poor prognosis in two independent series accounting 279 patients. Notably, a combination treatment of a selective HDAC6 and doxorubicin (a DNA damage agent used as a standard therapy of EWS patients) dramatically inhibits tumor growth in two EWS murine xenograft models. These results could lead to suitable and promising therapeutic alternatives for patients with EWS.Research in the E.D.A. lab is supported by Asociación Española Contra el Cáncer (AECC), the Ministry of Science of Spain-FEDER (CIBERONC, PI1700464, PI2000003, RD06/0020/0059)S. D.G.D. and L.H.P. are supported by CIBERONC (CB16/12/00361). D.G.D., M.J.R. and L.H.P. are PhD researchers funded by the Consejería de Salud, Junta de Andalucía (PI-0197-2016, ECAI F2-0012-2018 and PI-0013-2018, respectively).Peer reviewe

DNA methylomes reveal biological networks involved in human eye development, functions and associated disorders

This work provides a comprehensive CpG methylation landscape of the different layers of the human eye that unveils the gene networks associated with their biological functions and how these are disrupted in common visual disorders. Herein, we firstly determined the role of CpG methylation in the regulation of ocular tissue-specification and described hypermethylation of retinal transcription factors (i.e., PAX6, RAX, SIX6) in a tissue-dependent manner. Second, we have characterized the DNA methylome of visual disorders linked to internal and external environmental factors. Main conclusions allow certifying that crucial pathways related to Wnt-MAPK signaling pathways or neuroinflammation are epigenetically controlled in the fibrotic disorders involved in retinal detachment, but results also reinforced the contribution of neurovascularization (ETS1, HES5, PRDM16) in diabetic retinopathy. Finally, we had studied the methylome in the most frequent intraocular tumors in adults and children (uveal melanoma and retinoblastoma, respectively). We observed that hypermethylation of tumor suppressor genes is a frequent event in ocular tumors, but also unmethylation is associated with tumorogenesis. Interestingly, unmethylation of the proto-oncogen RAB31 was a predictor of metastasis risk in uveal melanoma. Loss of methylation of the oncogenic mir-17-92 cluster was detected in primary tissues but also in blood from patients

Mutational heterogeneity in cancer and the search for new cancer genes

Major international projects are now underway aimed at creating a comprehensive catalog of all genes responsible for the initiation and progression of cancer. These studies involve sequencing of matched tumor–normal samples followed by mathematical analysis to identify those genes in which mutations occur more frequently than expected by random chance. Here, we describe a fundamental problem with cancer genome studies: as the sample size increases, the list of putatively significant genes produced by current analytical methods burgeons into the hundreds. The list includes many implausible genes (such as those encoding olfactory receptors and the muscle protein titin), suggesting extensive false positive findings that overshadow true driver events. Here, we show that this problem stems largely from mutational heterogeneity and provide a novel analytical methodology, MutSigCV, for resolving the problem. We apply MutSigCV to exome sequences from 3,083 tumor-normal pairs and discover extraordinary variation in (i) mutation frequency and spectrum within cancer types, which shed light on mutational processes and disease etiology, and (ii) mutation frequency across the genome, which is strongly correlated with DNA replication timing and also with transcriptional activity. By incorporating mutational heterogeneity into the analyses, MutSigCV is able to eliminate most of the apparent artefactual findings and allow true cancer genes to rise to attention