RESUMO
BACKGROUND: High-grade serous ovarian carcinoma (HGSOC) is generally associated with a very dismal prognosis. Nevertheless, patients with similar clinicopathological characteristics can have markedly different clinical outcomes. Our aim was the identification of novel molecular determinants influencing survival. METHODS: Gene expression profiles of extreme HGSOC survivors (training set) were obtained by microarray. Differentially expressed genes (DEGs) and enriched signalling pathways were determined. A prognostic signature was generated and validated on curatedOvarianData database through a meta-analysis approach. The best prognostic biomarker from the signature was confirmed by RT-qPCR and by immunohistochemistry on an independent validation set. Cox regression model was chosen for survival analysis. RESULTS: Eighty DEGs and the extracellular matrix-receptor (ECM-receptor) interaction pathway were associated to extreme survival. A 10-gene prognostic signature able to correctly classify patients with 98% of accuracy was identified. By an 'in-silico' meta-analysis, overexpression of FXYD domain-containing ion transport regulator 5 (FXYD5), also known as dysadherin, was confirmed in HGSOC short-term survivors compared to long-term ones. Its prognostic and predictive power was then successfully validated, both at mRNA and protein level, first on training than on validation sample set. CONCLUSION: We demonstrated the possible involvement of FXYD5 and ECM-receptor interaction signal pathway in HCSOC survival and prognosis.
Assuntos
Cistadenocarcinoma Seroso/metabolismo , Resistencia a Medicamentos Antineoplásicos , Canais Iônicos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/metabolismo , Receptores de Superfície Celular/metabolismo , Idoso , Análise de Variância , Antineoplásicos/uso terapêutico , Carboplatina/uso terapêutico , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/mortalidade , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Canais Iônicos/genética , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/mortalidade , Prognóstico , Intervalo Livre de Progressão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sobrevida , Transcriptoma , Regulação para CimaRESUMO
Carcinosarcomas (CSs) of the uterus and ovary are highly aggressive neoplasms containing both carcinomatous and sarcomatous elements. We analyzed the mutational landscape of 68 uterine and ovarian CSs by whole-exome sequencing. We also performed multiregion whole-exome sequencing comprising two carcinoma and sarcoma samples from six tumors to resolve their evolutionary histories. The results demonstrated that carcinomatous and sarcomatous elements derive from a common precursor having mutations typical of carcinomas. In addition to mutations in cancer genes previously identified in uterine and ovarian carcinomas such as TP53, PIK3CA, PPP2R1A, KRAS, PTEN, CHD4, and BCOR, we found an excess of mutations in genes encoding histone H2A and H2B, as well as significant amplification of the segment of chromosome 6p harboring the histone gene cluster containing these genes. We also found frequent deletions of the genes TP53 and MBD3 (a member with CHD4 of the nucleosome remodeling deacetylase complex) and frequent amplification of chromosome segments containing the genes PIK3CA, TERT, and MYC Stable transgenic expression of H2A and H2B in a uterine serous carcinoma cell line demonstrated that mutant, but not wild-type, histones increased expression of markers of epithelial-mesenchymal transition (EMT) as well as tumor migratory and invasive properties, suggesting a role in sarcomatous transformation. Comparison of the phylogenetic relationships of carcinomatous and sarcomatous elements of the same tumors demonstrated separate lineages leading to these two components. These findings define the genetic landscape of CSs and suggest therapeutic targets for these highly aggressive neoplasms.
Assuntos
Histonas/genética , Neoplasias Ovarianas/genética , Proteína Supressora de Tumor p53/genética , Neoplasias Uterinas/genética , Idoso , Idoso de 80 Anos ou mais , Carcinossarcoma/genética , Carcinossarcoma/patologia , Classe I de Fosfatidilinositol 3-Quinases/genética , Proteínas de Ligação a DNA/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Mutação , Neoplasias Ovarianas/patologia , PTEN Fosfo-Hidrolase/genética , Telomerase/genética , Neoplasias Uterinas/patologiaRESUMO
BACKGROUND: The existence of cancer stem cells (CSCs) within a tumor bulk has been demonstrated for many solid tumors including epithelial ovarian carcinoma (EOC). CSCs have been associated to tumor invasion, metastasis and development of chemoresistant recurrences. In this context, we aim to characterize EOC CSCs from the molecular point of view in order to identify potential biomarkers associated with chemoresistance. METHODS: We isolated a population of cells with stem-like characteristics (OVA-BS4 spheroids) from a primary human EOC cell line under selective conditions. OVA-BS4 spheroids were characterized for drug response by cytotoxicity assays and their molecular profile was investigated by microarray and RT-qPCR. Finally, we performed a gene expression study in a cohort of 74 high-grade serous EOC (HGSOC) patients by RT-qPCR. RESULTS: Spheroids exhibited properties of self-renewal and a pronounced expression of well-known stem cell genes. Moreover, they demonstrated greater resistance towards several anticancer drugs compared to parent cell line, consistent with their higher ABCG2 gene expression. From microarray studies MAL (T-cell differentiation protein) emerged as the most up-regulated gene in spheroids, compared to parent cell line. In HGSOC patients, MAL was significantly overexpressed in platinum-resistant compared to platinum-sensitive patients and resulted as an independent prognostic marker of survival. CONCLUSIONS: This investigation provides an important contribution to the identification of molecular markers of ovarian CSCs and chemoresistance. Successful translation of molecular findings would lead to a better comprehension of the mechanisms triggering chemoresistant recurrences, to the individuation of novel therapeutic targets and to the personalization of treatment regimens.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Neoplasias Císticas, Mucinosas e Serosas/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Progressão da Doença , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Neoplasias Císticas, Mucinosas e Serosas/metabolismo , Neoplasias Císticas, Mucinosas e Serosas/mortalidade , Neoplasias Císticas, Mucinosas e Serosas/cirurgia , Células-Tronco Neoplásicas , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/cirurgia , Prognóstico , Regulação para CimaRESUMO
MicroRNAs (miRNAs) belong to a family of small non-coding RNAs (sncRNAs) playing important roles in human carcinogenesis. Multiple investigations reported miRNAs aberrantly expressed in several cancers, including high-grade serous ovarian carcinoma (HGS-OvCa). Quantitative PCR is widely used in studies investigating miRNA expression and the identification of reliable endogenous controls is crucial for proper data normalization. In this study, we aimed to experimentally identify the most stable reference sncRNAs for normalization of miRNA qPCR expression data in HGS-OvCa. Eleven putative reference sncRNAs for normalization (U6, SNORD48, miR-92a-3p, let-7a-5p, SNORD61, SNORD72, SNORD68, miR-103a-3p, miR-423-3p, miR-191-5p, miR-16-5p) were analysed on a total of 75 HGS-OvCa and 30 normal tissues, using a highly specific qPCR. Both the normal tissues considered to initiate HGS-OvCa malignant transformation, namely ovary and fallopian tube epithelia, were included in our study. Stability of candidate endogenous controls was evaluated using an equivalence test and validated by geNorm and NormFinder algorithms. Combining results from the three different statistical approaches, SNORD48 emerged as stably and equivalently expressed between malignant and normal tissues. Among malignant samples, considering groups based on residual tumour, miR-191-5p was identified as the most equivalent sncRNA. On the basis of our results, we support the use of SNORD48 as best reference sncRNA for relative quantification in miRNA expression studies between HGS-OvCa and normal controls, including the first time both the normal tissues supposed to be HGS-OvCa progenitors. In addition, we recommend miR-191-5p as best reference sncRNA in miRNA expression studies with prognostic intent on HGS-OvCa tissues.
Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias Císticas, Mucinosas e Serosas/genética , Neoplasias Ovarianas/genética , Pequeno RNA não Traduzido/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Simulação por Computador , Feminino , Perfilação da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Pequeno RNA não Traduzido/metabolismo , Padrões de Referência , Reprodutibilidade dos Testes , Análise de Sequência de RNA , SoftwareRESUMO
Uterine serous carcinoma (USC) is a biologically aggressive subtype of endometrial cancer. We analyzed the mutational landscape of USC by whole-exome sequencing of 57 cancers, most of which were matched to normal DNA from the same patients. The distribution of the number of protein-altering somatic mutations revealed that 52 USC tumors had fewer than 100 (median 36), whereas 5 had more than 3,000 somatic mutations. The mutations in these latter tumors showed hallmarks of defects in DNA mismatch repair. Among the remainder, we found a significantly increased burden of mutation in 14 genes. In addition to well-known cancer genes (i.e., TP53, PIK3CA, PPP2R1A, KRAS, FBXW7), there were frequent mutations in CHD4/Mi2b, a member of the NuRD-chromatin-remodeling complex, and TAF1, an element of the core TFIID transcriptional machinery. Additionally, somatic copy-number variation was found to play an important role in USC, with 13 copy-number gains and 12 copy-number losses that occurred more often than expected by chance. In addition to loss of TP53, we found frequent deletion of a small segment of chromosome 19 containing MBD3, also a member of the NuRD-chromatin-modification complex, and frequent amplification of chromosome segments containing PIK3CA, ERBB2 (an upstream activator of PIK3CA), and CCNE1 (a target of FBXW7-mediated ubiquitination). These findings identify frequent mutation of DNA damage, chromatin remodeling, cell cycle, and cell proliferation pathways in USC and suggest potential targets for treatment of this lethal variant of endometrial cancer.
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Variações do Número de Cópias de DNA , Mutação , Neoplasias Uterinas/genética , Sequência de Aminoácidos , Animais , Pareamento Incorreto de Bases , Feminino , Humanos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: The aim of the present study was to investigate within ovarian carcinoma and normal ovarian biopsies the gene expression of multiple secretoglobin family members relative to mammaglobin B, which we previously reported as a promising novel ovarian carcinoma prognostic marker. METHODS: Using quantitative real-time Reverse Transcription PCR we tested 53 ovarian carcinoma and 30 normal ovaries for the expression of 8 genes belonging to the secretoglobin family: mammaglobin A, lipophilin A, lipophilin B, uteroglobin, HIN-1, UGRP-1, RYD5 and IIS. Next, we decided to expand the LipB gene expression analysis to a further 48 ovarian carcinoma samples, for a total of 101 tumor tissues of various histologies and to study its protein expression by immunohistochemistry in formalin-fixed paraffin-embedded tumors and normal ovaries. Finally, we correlated lipophilin B gene and protein expression to conventional patient clinico-pathological features and outcome. RESULTS: We found significant mammaglobin A, lipophilin A, lipophilin B and RYD5 gene overexpression in ovarian carcinomas compared to normal ovaries. Lipophilin B mRNA showed a higher presence in tumors (75.4%) compared to normal ovaries (16.6%) and the most significant correlation with mammaglobin B mRNA (rs =0.77, p < 0.001). By immunohistochemical analysis, we showed higher lipophilin B expression in the cytoplasm of tumor cells compared to normal ovaries (p < 0.001). Moreover, lipophilin B gene overexpression was significantly associated with serous histology (serous vs clear cell p = 0.027; serous vs undifferentiated p = 0.007) and lower tumor grade (p = 0.02). Lower LipB mRNA levels (low versus high tertiles) were associated to a shorter progression-free (p = 0.03, HR = 2.2) and disease-free survival (p = 0.02, HR = 2.5) by univariate survival analysis and, importantly, they remain an independent prognostic marker for decreased disease-free (p = 0.001, HR = 3.9) and progression-free survival (p = 0.004, HR = 2.8) in multivariate Cox regression analysis. CONCLUSIONS: The present study represents the first quantitative evaluation of secretoglobin gene expression in normal and neoplastic ovarian tissues. Our results demonstrate lipophilin B gene and protein upregulation in ovarian carcinoma compared to normal ovary. Moreover, lipophilin B gene overexpression correlates with a less aggressive tumor phenotype and represents a novel ovarian carcinoma prognostic factor.
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Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/metabolismo , Secretoglobinas/metabolismo , Idoso , Biomarcadores Tumorais/metabolismo , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Neoplasias Ovarianas/diagnóstico , Ovário/metabolismo , Fenótipo , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Resultado do Tratamento , Regulação para CimaRESUMO
BACKGROUND: Human epididymis protein 4 (HE4), kallikrein 6 (KLK6), osteopontin (OPN) and soluble mesothelin-related peptide (SMRP) are new promising biomarkers that could integrate CA125 in epithelial ovarian cancer (EOC) diagnosis. The autoantibody response to tumor antigens is a potential tool for improving the diagnostic performances of biomarkers. The aim of this study was to assess the diagnostic potential of these biomarkers in the form of free markers and immunocomplexed with immunoglobulin M (IgM). Moreover, we analyzed the association between these markers and clinico-pathological characteristics of EOC patients. METHODS: Serum and plasma samples of 60 healthy controls, 60 ovarian benign cysts, 60 endometriosis and 60 EOCs, collected before any treatment, were tested for CICs and free antigens by immunoassays. RESULTS: Immunocomplexes were characterized by poor sensitivity and specificity, since they allowed the detection only of a small number of EOC patients and were increased in patients with benign gynecological pathologies. However, the markers in the form of free antigens showed good diagnostic performances. Of note, CA125 and HE4 showed high sensitivity in the detection of the malignancy and HE4 emerged as a useful biomarker in differential diagnosis between EOC and endometriosis. Finally, elevated KLK6 and OPN, were associated with advanced FIGO stage, high grade disease, suboptimally debulked tumor and ascites. CONCLUSIONS: This study confirms the diagnostic role of CA125, HE4, KLK6, OPN and SMRP, and for the first time showed that CA125, HE4, KLK6, OPN and SMRP immunocomplexed with IgM are not a potential tool for EOC diagnosis.
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Biomarcadores Tumorais/sangue , Antígeno Ca-125/sangue , Imunoconjugados/sangue , Imunoglobulina M/sangue , Neoplasias Epiteliais e Glandulares/sangue , Neoplasias Ovarianas/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/química , Biomarcadores Tumorais/imunologia , Carcinoma Epitelial do Ovário , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas Ligadas por GPI/sangue , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/imunologia , Humanos , Imunoensaio , Imunoconjugados/química , Imunoconjugados/imunologia , Imunoglobulina M/química , Imunoglobulina M/imunologia , Calicreínas/sangue , Calicreínas/química , Calicreínas/imunologia , Masculino , Mesotelina , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/diagnóstico , Osteopontina/sangue , Osteopontina/química , Osteopontina/imunologia , Neoplasias Ovarianas/diagnóstico , Proteínas/análise , Proteínas/química , Proteínas/imunologia , Proteína 2 do Domínio Central WAP de Quatro DissulfetosRESUMO
BACKGROUND: The purpose of this study was to assess the diagnostic and prognostic impact of preoperative serum determination of human epididymis protein 4 (sHE4), and to investigate its potential correlation with clinicopathological features and survival endpoints in endometrial cancer patients. METHODS: Preoperative serum samples from 193 endometrial cancer patients and 125 women with normal endometrium were measured for sHE4 and serum CA125 (sCA125) concentrations by quantitative chemiluminescent microparticle immunoassays on the automated Architect instrument. RESULTS: sHE4 concentrations were significantly higher in endometrial cancer patients regardless of tumour stage and grade compared with normal controls. Setting the specificity at 95 % , the sensitivities in detecting endometrial cancer patients were 66 % for HE4, 33 % for CA125 and 64 % for the combination of the two markers. High concentrations of both HE4 and CA125 significantly correlated with all clinicopathological features characterising a more aggressive tumour phenotype.In multivariate analysis, only high preoperative sHE4 concentrations, but not sCA125, were independent prognostic factors for shorter Overall Survival, Disease-Free Survival and Progression-Free Survival. CONCLUSIONS: HE4 is more sensitive and specifi c than CA125in distinguishing endometrial cancer patients from women with normal endometrium, regardless of tumour stage and grade. sHE4 appears to be associated with a more aggressive tumour variant and it could be clinically useful, in identifying high-risk endometrial cancer patients, for a tailored surgical and postoperative therapy. HE4 significant correlation with decreased Overall Survival, Disease Free Survival and Progression Free Survival suggests its potential role as a novel prognostic marker for endometrial cancer.
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Biomarcadores Tumorais/sangue , Neoplasias do Endométrio/diagnóstico , Proteínas/metabolismo , Idoso , Antígeno Ca-125/sangue , Neoplasias do Endométrio/sangue , Neoplasias do Endométrio/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Curva ROC , Sensibilidade e Especificidade , Resultado do Tratamento , Proteína 2 do Domínio Central WAP de Quatro DissulfetosRESUMO
BACKGROUND: Endometrial cancer is the most common gynecologic malignancy in developed countries. Trop-2 is a glycoprotein involved in cellular signal transduction and is differentially overexpressed relative to normal tissue in a variety of human adenocarcinomas, including endometrioid endometrial carcinomas (EEC). Trop-2 overexpression has been proposed as a marker for biologically aggressive tumor phenotypes. METHODS: Trop-2 protein expression was quantified using tissue microarrays consisting of formalin-fixed paraffin-embedded specimens from 118 patients who underwent surgical staging from 2001-9 by laparotomy for EEC. Clinicopathologic characteristics including age, stage, grade, lymphovascular space invasion, and medical comorbidities were correlated with immunostaining score. Univariate and multivariate analyses were performed for overall survival, disease-free survival, and progression-free survival in relation to clinical parameters and Trop-2 protein expression. RESULTS: Clinical outcome data were available for 103 patients. Strong Trop-2 immunostaining was significantly associated with higher tumor grade (p=0.02) and cervical involvement (p<0.01). Univariate analyses showed a significant association with reduced disease-free survival (DFS) (p=0.01), and a trend towards significance for overall and progression-free survival (p=0.06 and p=0.05, respectively). Multivariate analyses revealed Trop-2 overexpression and advanced FIGO stage to be independent prognostic factors for poor DFS (p=0.04 and p <0.001, respectively). CONCLUSIONS: Trop-2 protein overexpression is significantly associated with higher tumor grade and serves as an independent prognostic factor for DFS in endometrioid endometrial cancer.
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BACKGROUND: Traditional prognostic factors in epithelial ovarian cancer (EOC) are inadequate in predicting recurrence and long-term prognosis, but genome-wide cancer research has recently provided multiple potentially useful biomarkers. The gene codifying for Mammaglobin B (MGB-2) has been selected from our previous microarray analysis performed on 19 serous papillary epithelial ovarian cancers and its expression has been further investigated on multiple histological subtypes, both at mRNA and protein level. Since, to date, there is no information available on the prognostic significance of MGB-2 expression in cancer, the aim of this study was to determine its prognostic potential on survival in a large cohort of well-characterized EOC patients. METHODS: MGB-2 expression was evaluated by quantitative real time-PCR in fresh-frozen tissue biopsies and was validated by immunohistochemistry in matched formalin fixed-paraffin embedded tissue samples derived from a total of 106 EOC patients and 27 controls. MGB-2 expression was then associated with the clinicopathologic features of the tumors and was correlated with clinical outcome. RESULTS: MGB-2 expression was found significantly elevated in EOC compared to normal ovarian controls, both at mRNA and protein level. A good correlation was detected between MGB-2 expression data obtained by the two different techniques. MGB-2 expressing tumors were significantly associated with several clinicopathologic characteristics defining a less aggressive tumor behavior. Univariate survival analysis revealed a decreased risk for cancer-related death, recurrence and disease progression in MGB-2-expressing patients (p < 0.05). Moreover, multivariate analysis indicated that high expression levels of MGB-2 transcript (HR = 0.25, 95%, 0.08-0.75, p = 0.014) as well as positive immunostaining for the protein (HR = 0.41, 95%CI, 0.17-0.99, p = 0.048) had an independent prognostic value for disease-free survival. CONCLUSION: This is the first report documenting that MGB-2 expression characterizes less aggressive forms of EOC and is correlated with a favorable outcome. These findings suggest that the determination of MGB-2, especially at molecular level, in EOC tissue obtained after primary surgery can provide additional prognostic information about the risk of recurrence.
Assuntos
Epitélio/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas da Mielina/biossíntese , Proteínas da Mielina/fisiologia , Neoplasias Ovarianas/metabolismo , Proteolipídeos/biossíntese , Proteolipídeos/fisiologia , Uteroglobina/biossíntese , Uteroglobina/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Estudos de Coortes , Feminino , Humanos , Imuno-Histoquímica/métodos , Mamoglobina B , Pessoa de Meia-Idade , Neoplasias Ovarianas/diagnóstico , Prognóstico , Recidiva , Risco , SecretoglobinasRESUMO
OBJECTIVE: The purpose of this study was to develop and characterize a human antibody in a single-chain antibody fragment format (scFv) that is directed specifically against claudin-3 (CLDN3). STUDY DESIGN: The synthetic ETH-2 Gold human antibody phage display library was used to select scFv specific against CLDN3. scFv binding properties were analyzed by surface plasmon resonance; specificity was confirmed with enzyme-linked immunosorbent assay, immunofluorescence, and flow cytometry on a panel of ovarian and uterine serous carcinoma cell lines. RESULTS: Surface plasmon resonance studies indicated scFv H6 to be the clone with the highest affinity against CLDN3 (K(D) of 23.60 nmol/L). scFv H6 efficiently stained CLDN3-expressing cells and recognized its epitope in enzyme-linked immunosorbent assay that was performed with uterine serous papillary carcinoma native protein extract, which suggested that a conformational epitope is recognized by this antibody. Cell surface immunofluorescence with laser scanning confocal microscopy confirmed the specific binding to the native membrane CLDN3. CONCLUSION: scFv H6 may represent a novel antitumor agent against chemotherapy-resistant ovarian and serous papillary carcinomas and other human malignancies that overexpress CLDN3.
Assuntos
Carcinoma Papilar/imunologia , Fragmentos de Imunoglobulinas/imunologia , Proteínas de Membrana/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias do Colo do Útero/imunologia , Carcinoma Papilar/metabolismo , Linhagem Celular Tumoral , Separação Celular , Claudina-3 , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Biblioteca Gênica , Humanos , Fragmentos de Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Confocal , Neoplasias Ovarianas/metabolismo , Replicação de Sequência Autossustentável , Sensibilidade e Especificidade , Ressonância de Plasmônio de Superfície , Neoplasias do Colo do Útero/metabolismoRESUMO
INTRODUCTION: Kallikrein-related peptidases are secreted serine proteases that exert stimulatory or inhibitory effects on tumor progression. A recent study demonstrated that kallikrein-related peptidase 5 (KLK5) concentration is elevated in serum of patients with ovarian carcinoma. At the moment, the presence of KLK5 in other ovarian pathological lesions is not clearly determined. Moreover, the possibility of a spontaneous humoral immune response to KLK5 has not been studied yet. METHODS: In this study, we examined KLK5 levels and antibody (IgG and IgM) response to KLK5 in the serum of 50 healthy women, 50 patients with benign pelvic masses, 17 patients with ovarian borderline tumors, and 50 patients with ovarian carcinomas, using 3 enzyme-linked immunosorbent assay tests available in-house. RESULTS: At 95% specificity on healthy controls, 52% of patients with ovarian carcinoma showed high serum KLK5 (sKLK5) levels, whereas patients with benign pathological lesions or borderline tumors showed almost undetectable sKLK5 levels. Moreover, sKLK5 levels were positively associated to International Federation of Gynaecologists and Obstetricians stage suggesting a possible role of sKLK5 in ovarian cancer progression. Our results about humoral response showed elevated levels of KLK5-specific antibodies in 20% of patients with benign masses, 26% of patients with borderline tumors, and 36% of patients with ovarian carcinomas when compared with healthy controls. Interestingly, KLK5 antibodies were also found in patients with undetectable sKLK5 levels. CONCLUSIONS: In conclusion, our results showed that KLK5 is a potential new biomarker to be used in combination with other biomarkers for ovarian cancer detection. Moreover, the existence of KLK5 antibodies suggests that KLK5 might represent a possible target for immune-based therapies.
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Carcinoma/imunologia , Imunidade Humoral , Calicreínas/sangue , Calicreínas/imunologia , Neoplasias Ovarianas/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antineoplásicos/análise , Anticorpos Antineoplásicos/sangue , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/imunologia , Carcinoma/sangue , Carcinoma/diagnóstico , Carcinoma/patologia , Estudos de Casos e Controles , Feminino , Humanos , Imunidade Humoral/fisiologia , Imunoensaio/métodos , Pessoa de Meia-Idade , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/patologia , Curva ROC , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto JovemRESUMO
OBJECTIVE: The purpose of this study was to identify genes that are highly differentially expressed in metastatic serous papillary ovarian tumors (MET) when compared with primary ovarian serous carcinomas (OSPC). STUDY DESIGN: An oligonucleotide microarray with probe sets complementary to >14,500 human genes was used to determine whether patterns of gene expression may differentiate OSPC from MET in 31 snap-frozen serous papillary ovarian carcinomas (ie, 14 primary OSPC and 17 omental metastasis [MET]). RESULTS: Hierarchic cluster analysis of gene expression in OSPC and MET identified 156 genes that exhibited > 2-fold differences (P < .05) and that distinguished OSPC from MET. A number of invasion and metastasis predictive genes (including plasminogen activator; matrix metalloproteinase; matrix structural constituent genes encoding products with collagen, heparin, and hyaluronic acid binding activity; genes encoding receptors for insulin-like growth factors; vascular endothelial growth factor; endothelin type A; fibroblast growth factor; thrombospondin 1 and 2; type A and B integrins, and chemokines [stromal cell-derived factor 1 (CXCL12)]) were found among the 120 genes that were highly differentially overexpressed in MET, when compared with OSPC. Down-regulated genes in MET compared with OSPC included hepsin and testisin. Overexpression of CXCL12, matrix metalloproteinase, plasminogen activator, type A and B integrins, and hepsin genes was validated by quantitative real-time polymerase chain reaction in all samples. Finally, overexpression of CXCL12 in MET, when compared with OSPC, was validated at the protein level by immunohistochemistry. CONCLUSION: Gene expression profiling may differentiate metastatic ovarian cancer from primary OSPC. The identification of metastasis-associated genes may provide a foundation for the development of new type-specific diagnostic strategies and treatment for metastatic ovarian cancer.
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Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/secundário , Perfilação da Expressão Gênica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica/genéticaRESUMO
BACKGROUND: Epithelial ovarian cancer (EOC) is a spectrum of different diseases, which makes their treatment a challenge. Forkhead box M1 (FOXM1) is an oncogene aberrantly expressed in many solid cancers including serous EOC, but its role in non-serous EOCs remains undefined. We examined FOXM1 expression and its correlation to prognosis across the three major EOC subtypes, and its role in tumorigenesis and chemo-resistance in vitro. METHODS: Gene signatures were generated by microarray for 14 clear-cell and 26 endometrioid EOCs, and 15 normal endometrium snap-frozen biopsies. Validation of FOXM1 expression was performed by RT-qPCR and immunohistochemistry in the same samples and additionally in 50 high-grade serous EOCs and in their most adequate normal controls (10 luminal fallopian tube and 20 ovarian surface epithelial brushings). Correlations of FOXM1 expression to clinic-pathological parameters and patients' prognosis were evaluated by Kaplan-Meier and Cox proportional-hazards analyses. OVCAR-3 and two novel deeply characterized EOC cell lines (EOC-CC1 and OSPC2, with clear-cell and serous subtype, respectively) were employed for in vitro studies. Effects of FOXM1 inhibition by transient siRNA transfection were evaluated on cell-proliferation, cell-cycle, colony formation, invasion, and response to conventional first- and second-line anticancer agents, and to the PARP-inhibitor olaparib. Gene signatures of FOXM1-silenced cell lines were generated by microarray and confirmed by RT-qPCR. RESULTS: A significant FOXM1 mRNA up-regulation was found in EOCs compared to normal controls. FOXM1 protein overexpression significantly correlated to serous histology (p = 0.001) and advanced FIGO stage (p = 0.004). Multivariate analyses confirmed FOXM1 protein overexpression as an independent indicator of worse disease specific survival in non-serous EOCs, and of shorter time to progression in platinum-resistant cases. FOXM1 downregulation in EOC cell lines inhibited cell growth and clonogenicity, and promoted the cytotoxic effects of platinum compounds, doxorubicin hydrochloride and olaparib. Upon FOXM1 knock-down in EOC-CC1 and OSPC2 cells, microarray and RT-qPCR analyses revealed the deregulation of several common and other unique subtype-specific FOXM1 putative targets involved in cell cycle, metastasis, DNA repair and drug response. CONCLUSIONS: FOXM1 is up-regulated in all three major EOCs subtypes, and is a prognostic biomarker and a potential combinatorial therapeutic target in platinum resistant disease, irrespective of tumor histology.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Proteína Forkhead Box M1/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/mortalidade , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Cistadenocarcinoma Seroso/genética , Reparo do DNA , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Prognóstico , Isoformas de Proteínas , RNA Interferente Pequeno/genéticaRESUMO
Innovative therapies in cervical cancer (CC) remain a priority. Recent data indicate that human immunodeficiency virus (HIV)-protease inhibitors used in highly active antiretroviral therapy can exert direct antitumor activities also in HIV-free preclinical and clinical models. The aim of the present study was to evaluate the antineoplastic effects of various HIV-protease inhibitors (indinavir, ritonavir and saquinavir) on primary and established CC cell lines. Two CC cell lines established in our laboratory and four commercially available CC cell lines were treated with indinavir, ritonavir and saquinavir at different concentrations and for different times. Proliferation, clonogenicity and radiosensitivity were evaluated by crystal violet staining. Proteasomal activities were assessed using a cell-based assay and immunoblotting. Cell cycle was analyzed by propidium iodide staining and flow cytometric analysis. Invasion was tested with Matrigel chambers. A t-test for paired samples was used for statistical analysis. In all cell lines, saquinavir was more effective than ritonavir in reducing cell proliferation and inhibiting proteasomal activities (P≤0.05). Conversely, indinavir exerted a negligible effect. The saquinavir concentrations required to modulate the proteasome activities were higher than those observed to be effective in inhibiting cell proliferation. In HeLa cells, saquinavir was strongly effective in inhibiting cell invasion and clonogenicity (P≤0.05) at concentrations much lower than those required to perturb proteasomal activities. Saquinavir did not contribute to increase the sensitivity of HeLa cells to X-rays. In conclusion, the present results demonstrate that saquinavir is able to significantly reduce cell proliferation, cell invasion and clonogenicity in a proteasome-independent manner in in vitro models of CC, and suggest that saquinavir could be a promising CC therapeutic agent.
RESUMO
Membrane protein claudin3 has been recently suggested as a marker for biologically aggressive tumors and a possible target for the therapeutic delivery of active anti-cancer compounds. Claudin3-binding molecules such as the Clostridium perfringens enterotoxin (CPE), CPE-related molecules, and murine and chimeric antibodies have shown promising antitumor efficacy in preclinical oncological settings. We first engineered a fully human anti-claudin3 IgG1 antibody (IgGH6) by fusing the human IgG1 Fc-domain to the anti-claudin3 scFvH6 previously isolated from a pre-immune phage display library. The construct was expressed in mammalian cells and specifically targeted claudin3 endogenously expressed on the surface of different human ovarian cancer cell lines. No detectable cross-reactivity with other homologous claudins was observed. The epitope recognized by IgGH6 is located within the minor extracellular domain of claudin3 and becomes accessible only in tumor cells characterized by incomplete junction formation. Confocal microscopy experiments demonstrated that IgGH6 was actively internalized in tumor cells after binding to native claudin3 and co-localized, likely within intracellular vesicles, with the C-CPE peptide. Preliminary results indicate that IgGH6 accumulated in vivo in free claudin3 ovarian carcinoma xenografts. For its selective uptake in tumor cells and its human nature, IgGH6 represents a valuable candidate for antibody-drug conjugate therapeutic applications in ovarian cancer patients.
Assuntos
Anticorpos Antineoplásicos/farmacologia , Claudina-3/imunologia , Sistemas de Liberação de Medicamentos/métodos , Imunoglobulina G/farmacologia , Neoplasias Ovarianas/imunologia , Animais , Afinidade de Anticorpos , Antineoplásicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Portadores de Fármacos/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Camundongos , Camundongos SCID , Microscopia Confocal , Microscopia de Fluorescência , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Ressonância de Plasmônio de Superfície , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Accurate normalization is a primary component of a reliable gene expression analysis based on qRT-PCR technique. While the use of one or more reference genes as internal controls is commonly accepted as the most appropriate normalization strategy, many qPCR-based published studies still contain data poorly normalized and reference genes arbitrarily chosen irrespective of the particular tissue and the specific experimental design. To date, no validated reference genes have been identified for endometrial cancer tissues. In this study, 10 normalization genes (GAPDH, B2M, ACTB, POLR2A, UBC, PPIA, HPRT1, GUSB, TBP, H3F3A) belonging to different functional and abundance classes in various tissues and used in different studies, were analyzed to determine their applicability. In total, 100 endometrioid endometrial cancer samples, which were carefully balanced according to their tumor grade, and 29 normal endometrial tissues were examined using SYBR Green Real-Time RT-PCR. The expression stability of candidate reference genes was determined and compared by means of geNorm and NormFinder softwares. Both algorithms were in agreement in identifying GAPDH, H3F3A, PPIA, and HPRT1 as the most stably expressed genes, only differing in their ranking order. Analysis performed on the expression levels of all candidate genes confirm HPRT1 and PPIA as the most stably expressed in the study groups regardless of sample type, to be used alone or better in combination. As the stable expression of HPRT1 and PPIA between normal and tumor endometrial samples fulfill the basic requirement of a reference gene to be used for normalization purposes, HPRT1 expression showed significant differences between samples from low-grade and high-grade tumors. In conclusion, our results recommend the use of PPIA as a single reference gene to be considered for improved reliability of normalization in gene expression studies involving endometrial tumor samples at different tumor degrees.
Assuntos
Carcinoma Endometrioide/genética , Neoplasias Ovarianas/genética , Actinas/genética , Actinas/normas , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Endometrioide/metabolismo , Carcinoma Endometrioide/patologia , Estudos de Coortes , Endométrio/metabolismo , Endométrio/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/normas , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , RNA/isolamento & purificação , RNA/metabolismo , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , Valores de Referência , SoftwareRESUMO
BACKGROUND: Two commercial immunoassays for HE4 have been compared and the diagnostic accuracy of HE4, CA 125 and the combinatory ROMA algorithm for epithelial ovarian cancer (EOC) has been evaluated. METHODS: HE4 and CA125 were measured on sera obtained from 259 women (73 healthy, 90 with benign ovarian or adnexal diseases, 96 with EOC). The ARCHITECT CMIA HE4 assay was compared with the Fujirebio EIA HE4, and the risk for EOC by the combinatory ROMA algorithm (HE4+CA 125) was assessed with both HE4 assays. RESULTS: The CMIA HE4 assay showed a good linearity (r>0.9998) and precision (interassay and total CVs <4%). The correlation with EIA HE4 was linear (r=0.994), with an average bias of 0.4%. By ROC curve analysis, the sensitivity for EOC at a fixed specificity of 90%, 95% and 99% was 89.6%, 84.4% and 79.2% by CMIA HE4, 84.4%, 83.3% and 79.2% by EIA HE4, 86.5%, 76.0% and 59.4% by CMIA CA125. The accuracy of the ROMA algorithm determined by CMIA or EIA HE4 was very similar (AUC 87.1% vs. 87.6%; p=n.s.) and greater in menopause. CONCLUSIONS: The two HE4 assays showed a good correlation and similar clinical value, with a greater precision for CMIA. HE4 was more specific and accurate than CA125, supporting its use in addition to clinical and imaging criteria for the discrimination of benign from malignant ovarian lesions. The ROMA algorithm showed a good accuracy for discriminating women at high risk for EOC.
Assuntos
Algoritmos , Proteínas Secretadas pelo Epidídimo/análise , Imunoensaio/métodos , Neoplasias Epiteliais e Glandulares/sangue , Neoplasias Ovarianas/sangue , Antígeno Ca-125/sangue , Carcinoma Epitelial do Ovário , Técnicas de Laboratório Clínico , Feminino , Humanos , Proteínas de Membrana/sangue , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade , beta-DefensinasRESUMO
We evaluated the expression of tissue factor (TF) in ovarian cancer (EOC) and the potential of hI-con1, an antibody-like molecule targeting TF, as a novel form of therapy against chemotherapy-resistant ovarian disease. We studied the expression of TF in 88 EOC by immunohistochemistry (IHC) and real-time-PCR (qRT-PCR) and the levels of membrane-bound-complement-regulatory-proteins CD46, CD55 and CD59 in primary EOC cell lines by flow-cytometry. Sensitivity to hI-con1-dependent-cell-mediated-cytotoxicity (IDCC), complement-dependent-cell-cytotoxicity and inhibition of IDCC by γ-immunoglobulin were evaluated in 5-h (51)chromium-release-assays. Cytoplasmic and/or membrane TF expression was observed in 24 out of 25 (96%) of the EOC samples tested by IHC, but not in normal ovarian-tissue. EOC with clear cell histology significantly overexpress TF when compared to serous, endometrioid, or undifferentiated tumors by qRT-PCR. With a single exception, all primary EOC that overexpressed TF demonstrated high levels of CD46, CD55 and CD59 and regardless of their histology or resistance to chemotherapy, were highly sensitive to IDCC. The effect of complement and physiologic doses of γ-immunoglobulin on IDCC in ovarian cancer cell lines overexpressing TF was tumor specific and related to the overexpression of CD59 on tumor cells. Small-interfering-RNA-mediated knockdown of CD59 expression in ovarian tumors significantly increased hI-con1-mediated cytotoxic activity in vitro. Finally, low doses of interleukin-2 further increased the cytotoxic effect induced by hI-con1 (P < 0.01). hI-con1 molecule induces strong cytotoxicity against primary chemotherapy-resistant ovarian cancer cell lines overexpressing TF and may represent a novel therapeutic agent for the treatment of ovarian tumors refractory to standard treatment modalities.
Assuntos
Regulação Neoplásica da Expressão Gênica , Imunoconjugados/uso terapêutico , Imunoterapia/métodos , Neoplasias Ovarianas/tratamento farmacológico , Tromboplastina/uso terapêutico , Linhagem Celular Tumoral , Fator VII/imunologia , Fator VII/metabolismo , Fator VII/uso terapêutico , Feminino , Humanos , Imunoconjugados/imunologia , Imunoconjugados/metabolismo , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/metabolismo , Tromboplastina/genética , Tromboplastina/imunologia , Tromboplastina/metabolismoRESUMO
BACKGROUND: The aim of this work was to analyze the diagnostic and prognostic value of serum human epididymis protein 4 (HE4) and Risk for Ovarian Malignancy Algorithm (ROMA) in epithelial ovarian cancer (EOC). METHODS: Preoperative serum samples of 419 women (140 healthy controls, 131 ovarian benign cysts, 34 endometriosis, and 114 EOC) were tested for CA125 and HE4 using fully automated methods (Abbott ARCHITECT) and validated cutoff values. RESULTS: For the discrimination of benign masses from EOC, in premenopausal women, the sensitivity and specificity were 92.3% and 59.4% for CA125, 84.6% and 94.2% for HE4, and 84.6% and 81.2% for ROMA, whereas in postmenopausal women, the sensitivity and specificity were 94.3% and 82.3% for CA125, 78.2% and 99.0% for HE4, and 93.1% and 84.4% for ROMA. In patients with EOC, elevated CA125, HE4, and ROMA levels were associated with advanced Federation of Gynaecologists and Obstetricians (FIGO) stage, suboptimally debulking, ascites, positive cytology, lymph node involvement, and advanced age (all P ≤ 0.05). Elevated HE4 and ROMA (both P ≤ 0.01), but not CA125 (P = 0.0579), were associated with undifferentiated tumors. In multivariable analysis, elevated HE4 and ROMA (all P ≤ 0.05) were independent prognostic factors for shorter overall, disease-free, and progression-free survival. CONCLUSIONS AND IMPACT: This study underlines the high specificity of HE4 in discriminating endometriosis and ovarian benign cysts from EOC and the high sensitivity of CA125 in detecting EOC. We showed HE4 and ROMA as independent prognostic factors. Multicenter studies are needed to draw firm conclusions about the applicability of HE4 and ROMA in clinical practice.