RESUMO
The worldwide approval of the combination maintenance therapy of olaparib and bevacizumab in advanced high-grade serous ovarian cancer requires complex molecular diagnostic assays that are sufficiently robust for the routine detection of driver mutations in homologous recombination repair (HRR) genes and genomic instability (GI), employing formalin-fixed (FFPE) paraffin-embedded tumor samples without matched normal tissue. We therefore established a DNA-based hybrid capture NGS assay and an associated bioinformatic pipeline that fulfils our institution's specific needs. The assay´s target regions cover the full exonic territory of relevant cancer-related genes and HRR genes and more than 20,000 evenly distributed single nucleotide polymorphism (SNP) loci to allow for the detection of genome-wide allele specific copy number alterations (CNA). To determine GI status, we implemented an %CNA score that is robust across a broad range of tumor cell content (25-85%) often found in routine FFPE samples. The assay was established using high-grade serous ovarian cancer samples for which BRCA1 and BRCA2 mutation status as well as Myriad MyChoice homologous repair deficiency (HRD) status was known. The NOGGO (Northeastern German Society for Gynecologic Oncology) GIS (GI-Score) v1 assay was clinically validated on more than 400 samples of the ENGOT PAOLA-1 clinical trial as part of the European Network for Gynaecological Oncological Trial groups (ENGOT) HRD European Initiative. The "NOGGO GIS v1 assay" performed using highly robust hazard ratios for progression-free survival (PFS) and overall survival (OS), as well a significantly lower dropout rate than the Myriad MyChoice clinical trial assay supporting the clinical utility of the assay. We also provide proof of a modular and scalable routine diagnostic method, that can be flexibly adapted and adjusted to meet future clinical needs, emerging biomarkers, and further tumor entities.
RESUMO
During hypoxia, a cellular adaptive response activates hypoxia-inducible factors (HIFs; HIF-1 and HIF-2) that respond to low tissue-oxygen levels and induce the expression of a number of genes that promote angiogenesis, energy metabolism, and cell survival. HIF-1 and HIF-2 regulate endothelial cell (EC) adaptation by activating gene-signaling cascades that promote endothelial migration, growth, and differentiation. An HIF-1 to HIF-2 transition or switch governs this process from acute to prolonged hypoxia. In the present study, we evaluated the mechanisms governing the HIF switch in 10 different primary human ECs from different vascular beds during the early stages of hypoxia. The studies demonstrate that the switch from HIF-1 to HIF-2 constitutes a universal mechanism of cellular adaptation to hypoxic stress and that HIF1A and HIF2A mRNA stability differences contribute to HIF switch. Furthermore, using 4 genome-wide mRNA expression arrays of HUVECs during normoxia and after 2, 8, and 16 h of hypoxia, we show using bioinformatics analyses that, although a number of genes appeared to be regulated exclusively by HIF-1 or HIF-2, the largest number of genes appeared to be regulated by both.-Bartoszewski, R., Moszynska, A., Serocki, M., Cabaj, A., Polten, A., Ochocka, R., Dell'Italia, L., Bartoszewska, S., Króliczewski, J., Dabrowski, M., Collawn, J. F. Primary endothelial cell-specific regulation of hypoxia-inducible factor (HIF)-1 and HIF-2 and their target gene expression profiles during hypoxia.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Adaptação Fisiológica/genética , Aorta/citologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Meia-Vida , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Artéria Ilíaca/citologia , Especificidade de Órgãos , Cultura Primária de Células , Artéria Pulmonar/citologia , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Pele/irrigação sanguínea , Útero/irrigação sanguíneaRESUMO
Basaloid squamous cell carcinoma (BSCC) and carcinosarcoma of the esophagus are rare entities, making up fewer than 2% of esophageal malignancies. Comparative genomic hybridization (CGH) in 1 case of BSCC and 2 cases of carcinosarcoma and subsequent array CGH in 1 case each of BSCC and carcinosarcoma revealed common chromosomal gains at 2p25.3-2p12, 7q21.3-7q22.3, and 11q13.2-11q13.4. Chromosomal losses at 13q31qter were observed in both carcinosarcomas. In addition, progression of genomic instability from in situ to invasive carcinosarcoma could be demonstrated by using array CGH. Our observations suggest a common genetic origin of BSCC and carcinosarcoma.
Assuntos
Carcinoma Basocelular/genética , Carcinoma Basoescamoso/genética , Carcinoma de Células Escamosas/genética , Carcinossarcoma/genética , Hibridização Genômica Comparativa/métodos , Neoplasias Esofágicas/genética , Idoso , Carcinoma in Situ/genética , Carcinoma in Situ/patologia , Carcinoma in Situ/cirurgia , Carcinoma Basocelular/patologia , Carcinoma Basocelular/cirurgia , Carcinoma Basoescamoso/patologia , Carcinoma Basoescamoso/cirurgia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/cirurgia , Carcinossarcoma/patologia , Carcinossarcoma/cirurgia , Aberrações Cromossômicas , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 2/genética , Cromossomos Humanos Par 7/genética , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/cirurgia , Evolução Fatal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Tomografia Computadorizada por Raios XRESUMO
We describe two newly established malignant mesothelioma (MM) cell lines derived from a pleural effusion of a male. One cell line, designated as MM-Z03E, reveals an epithelioid cobblestone morphology, while the second one, designated as MM-Z03S and subcloned after in vivo selection, exhibits a sarcomatoid storiform growth pattern. Both cell lines showed the immunologic profile characteristic for MM (i.e., expression of cytokeratin, CK18, calretinin, and vimentin in both phenotypes). Cytogenetics, multicolor fluorescence in situ hybridization, comparative genomic hybridization, and oligonucleotide array CGH were performed on both cell lines. Aberrations shared by both cell lines included chromosomal losses of 1q34 approximately qter, 4, 9p, 10p, 13, 14, 16q, 18, and 22, as well as a complex structural aberration involving chromosome 17. Aberrations exclusive to MM-Z03E included gains of 3q11q27 and 5p, while gain of 9q and losses of 3q27qter, 11q, and 18 in MM-Z03S were exclusive to MM-Z03E. Both cell lines were able to develop solid transplant tumors in nude mice within 16 weeks, and immunophenotyping of tumor xenografts revealed an overall retained expression profile of the markers used. Remarkably, one xenograft from MM-Z03E revealed overexpression of p53 and widely invasive growth. In conclusion, both cell lines are useful in vivo and in vitro model systems to study the underlying genetic mechanisms of biphasic differentiation in MM, which can be of certain value considering the increasing relevance of assessing MM tumor biology for the clinical management of this disease.
Assuntos
Linhagem Celular Tumoral , Aberrações Cromossômicas , Células Clonais , Mesotelioma/genética , Animais , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Derrame Pleural Maligno , Transplante HeterólogoRESUMO
Psychotic disorders affecting up to 1% of the human population represent pathological changes to the metabolic homeostasis of the brain. Increasing evidence in the literature suggests complex biochemical and/or transcriptional alterations accompanying schizophrenia-like phenomena. Sub-chronic treatment with sub-anaesthetic doses of ketamine induces schizophrenia-related psychotic alterations that can be used as an animal model in the study of this disorder. Ampullosporin A belongs to a specific group of pore-forming fungal peptides, peptaibols. We focused on the analysis of molecular events occurring in the brain of ketamine-pre-treated rats after administration of Ampullosporin A with neuroleptic-like activity. The complex experimental approach allowed us to correlate the use of low molecular weight substances with a transcriptome fingerprint in the prefrontal cortex. We found 63 genes to be up-regulated and 22 genes suppressed, with transthyretin, syndecan-1 and NeuroD1 showing the highest degree of up-regulation. Our results suggest the possibility that Ampullosporin A belongs to the group of neuroleptic-like compounds, inducing massive changes in neurotransmitter receptor composition, calcium signalling cascades and second messenger systems, and leading to the plastic reorganization of brain tissue, metabolic pathways and synapses.