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1.
Cancer Res ; 79(22): 5884-5896, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31585941

RESUMO

Gastric cancer is the world's third leading cause of cancer mortality. In spite of significant therapeutic improvements, the clinical outcome for patients with advanced gastric cancer is poor; thus, the identification and validation of novel targets is extremely important from a clinical point of view. We generated a wide, multilevel platform of gastric cancer models, comprising 100 patient-derived xenografts (PDX), primary cell lines, and organoids. Samples were classified according to their histology, microsatellite stability, Epstein-Barr virus status, and molecular profile. This PDX platform is the widest in an academic institution, and it includes all the gastric cancer histologic and molecular types identified by The Cancer Genome Atlas. PDX histopathologic features were consistent with those of patients' primary tumors and were maintained throughout passages in mice. Factors modulating grafting rate were histology, TNM stage, copy number gain of tyrosine kinases/KRAS genes, and microsatellite stability status. PDX and PDX-derived cells/organoids demonstrated potential usefulness to study targeted therapy response. Finally, PDX transcriptomic analysis identified a cancer cell-intrinsic microsatellite instability (MSI) signature, which was efficiently exported to gastric cancer, allowing the identification, among microsatellite stable (MSS) patients, of a subset of MSI-like tumors with common molecular aspects and significant better prognosis. In conclusion, we generated a wide gastric cancer PDX platform, whose exploitation will help identify and validate novel "druggable" targets and optimize therapeutic strategies. Moreover, transcriptomic analysis of gastric cancer PDXs allowed the identification of a cancer cell-intrinsic MSI signature, recognizing a subset of MSS patients with MSI transcriptional traits, endowed with better prognosis. SIGNIFICANCE: This study reports a multilevel platform of gastric cancer PDXs and identifies a MSI gastric signature that could contribute to the advancement of precision medicine in gastric cancer.


Assuntos
Neoplasias Gástricas/genética , Transcrição Genética/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/genética , Feminino , Perfilação da Expressão Gênica/métodos , Genes ras/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Estadiamento de Neoplasias/métodos , Fenótipo , Prognóstico , Neoplasias Gástricas/patologia
3.
Neoplasia ; 20(5): 443-455, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29574251

RESUMO

Patient-Derived Xenografts (PDXs), entailing implantation of cancer specimens in immunocompromised mice, are emerging as a valuable translational model that could help validate biologically relevant targets and assist the clinical development of novel therapeutic strategies for gastric cancer. More than 30% of PDXs generated from gastric carcinoma samples developed human B-cell lymphomas instead of gastric cancer. These lymphomas were monoclonal, Epstein Barr Virus (EBV) positive, originated tumorigenic cell cultures and displayed a mutational burden and an expression profile distinct from gastric adenocarcinomas. The ability of grafted samples to develop lymphomas did not correlate with patient outcome, nor with the histotype, the lymphocyte infiltration level, or the EBV status of the original gastric tumor, impeding from foreseeing lymphoma onset. Interestingly, lymphoma development was significantly more frequent when primary rather than metastatic samples were grafted. Notably, the development of such lympho-proliferative disease could be prevented by a short rituximab treatment upon mice implant, without negatively affecting gastric carcinoma engraftment. Due to the high frequency of human lymphoma onset, our data show that a careful histologic analysis is mandatory when generating gastric cancer PDXs. Such care would avoid misleading results that could occur if testing of putative gastric cancer therapies is performed in lymphoma PDXs. We propose rituximab treatment of mice to prevent lymphoma development in PDX models, averting the loss of human-derived samples.


Assuntos
Xenoenxertos/efeitos dos fármacos , Linfoma de Células B/tratamento farmacológico , Rituximab/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Animais , Linfócitos B/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias/patologia , Transplante Heterólogo/métodos
4.
Oncotarget ; 6(7): 5182-94, 2015 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-25691052

RESUMO

The ROS1 tyrosine kinase is activated in lung cancer as a consequence of chromosomal rearrangement. Although high response rates and disease control have been observed in lung cancer patients bearing rearranged ROS1 tumors (ROS1+) treated with the kinase inhibitor crizotinib, many of these patients eventually relapse.To identify mechanisms of resistance to ROS1 inhibitors we generated resistant cells from HCC78 lung cancer cells bearing the SLC34A2-ROS1 rearrangement. We found that activation of the RAS pathway in the HCC78 cell model, due to either KRAS/NRAS mutations or to KRAS amplification, rendered the cells resistant to ROS1 inhibition. These cells were cross-resistant to different ROS1 inhibitors, but sensitive to inhibitors of the RAS signaling pathway. Interestingly, we identified focal KRAS amplification in a biopsy of a tumor from a patient that had become resistant to crizotinib treatment.Altogether our data suggest that the activation of members of the RAS family can confer resistance to ROS1 inhibitors. This has important clinical implications as: (i) RAS genetic alterations in ROS1+ primary tumors are likely negative predictors of efficacy for targeted drugs and (ii) this kind of resistance is unlikely to be overcome by the use of more specific or more potent ROS1 targeting drugs.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Proteínas ras/genética , Apoptose , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células , Crizotinibe , Rearranjo Gênico , Humanos , Técnicas Imunoenzimáticas , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mutação/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras) , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/metabolismo , Células Tumorais Cultivadas , Proteínas ras/metabolismo
5.
Oncotarget ; 6(4): 2315-30, 2015 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-25537513

RESUMO

Basal-like breast cancer is an aggressive tumor subtype with a poor response to conventional therapies. Tumor formation and relapse are sustained by a cell subset of Breast Cancer Stem Cells (BrCSCs). Here we show that miR-100 inhibits maintenance and expansion of BrCSCs in basal-like cancer through Polo-like kinase1 (Plk1) down-regulation. Moreover, miR-100 favors BrCSC differentiation, converting a basal like phenotype into luminal. It induces the expression of a functional estrogen receptor (ER) and renders basal-like BrCSCs responsive to hormonal therapy. The key role played by miR-100 in breast cancer free-survival is confirmed by the analysis of a cohort of patients' tumors, which shows that low expression of miR-100 is a negative prognostic factor and is associated with gene signatures of high grade undifferentiated tumors. Our findings indicate a new possible therapeutic strategy, which could make aggressive breast cancers responsive to standard treatments.


Assuntos
Neoplasias da Mama/genética , Diferenciação Celular/genética , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Células MCF-7 , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Prognóstico , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tamoxifeno/farmacologia , Transplante Heterólogo
6.
Cancer ; 116(4): 843-51, 2010 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-20041483

RESUMO

BACKGROUND: The authors investigated the expression of serum amyloid A (SAA) in endometrial endometrioid carcinoma and evaluated its potential as a serum biomarker. METHODS: SAA gene and protein expression levels were evaluated in endometrial endometrioid carcinoma and normal endometrial tissues, by real-time polymerase chain reaction (PCR), immunohistochemistry (IHC), and flow cytometry. SAA concentration in 194 serum samples from 50 healthy women, 42 women with benign diseases, and 102 patients including 49 grade 1, 38 grade 2, and 15 grade 3 endometrial endometrioid carcinoma was also studied by a sensitive bead-based immunoassay. RESULTS: SAA gene expression levels were significantly higher in endometrial endometrioid carcinoma when compared with normal endometrial tissues (mean copy number by real-time PCR = 182 vs 1.9; P = .001). IHC revealed diffuse cytoplasmic SAA protein staining in poorly differentiated endometrial endometrioid carcinoma tissues. High intracellular levels of SAA were identified in primary endometrial endometrioid carcinoma cell lines evaluated by flow cytometry, and SAA was found to be actively secreted in vitro. SAA concentrations (microg/mL) had medians of 6.0 in normal healthy women and 6.0 in patients with benign disease (P = .92). In contrast, SAA values in the serum of endometrial endometrioid carcinoma patients had a median of 23.7, significantly higher than those of the healthy group (P = .001) and benign group (P = .001). Patients harboring G3 endometrial endometrioid carcinoma were found to have SAA concentrations significantly higher than those of G1/G2 patients. CONCLUSIONS: SAA is not only a liver-secreted protein, but is also an endometrial endometrioid carcinoma cell product. SAA is expressed and actively secreted by G3 endometrial endometrioid carcinoma, and it is present in high concentration in the serum of endometrial endometrioid carcinoma patients. SAA may represent a novel biomarker for endometrial endometrioid carcinoma to monitor disease recurrence and response to therapy.


Assuntos
Carcinoma Endometrioide/metabolismo , Neoplasias do Endométrio/metabolismo , Proteína Amiloide A Sérica/metabolismo , Idoso , Biomarcadores Tumorais/metabolismo , Carcinoma Endometrioide/sangue , Linhagem Celular Tumoral , Células Cultivadas , Neoplasias do Endométrio/sangue , Endométrio/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo
7.
Int J Gynecol Cancer ; 19(5): 860-6, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19574774

RESUMO

To evaluate the potential of epithelial cell adhesion molecule (Ep-CAM/TROP-1)-specific immunotherapy against epithelial ovarian carcinomas (EOCs), we have analyzed the expression of Ep-CAM at RNA and protein level in patients harboring primary, metastatic, and chemotherapy-resistant/recurrent EOC. Epithelial cell adhesion molecule expression was evaluated by real-time polymerase chain reaction and immunohistochemistry in 168 fresh-frozen biopsies and paraffin-embedded tissues. In addition, Ep-CAM surface expression was evaluated by flow cytometry in several freshly established ovarian carcinoma cell lines derived from patients harboring tumors resistant to chemotherapy in vivo as well as in vitro. Epithelial cell adhesion molecule transcript was found significantly overexpressed in primary, metastatic, and recurrent EOC when compared with normal human ovarian surface epithelium cell lines and fresh-frozen normal ovarian tissue (P < 0.001). Similarly, by immunohistochemistry, Ep-CAM protein expression was found significantly higher in primary, metastatic, and recurrent EOC when compared with normal ovarian tissues. Of interest, metastatic/recurrent tumors were found to express significantly higher levels of Ep-CAM protein when compared with primary ovarian carcinomas (P < 0.001). Finally, a high surface expression of Ep-CAM was found in 100% (5/5) of the chemotherapy-resistant ovarian carcinoma cell lines studied by flow cytometry. These results demonstrate high Ep-CAM overexpression in ovarian carcinoma, especially in metastatic and recurrent/chemotherapy-resistant ovarian disease. The lack of Ep-CAM expression on the chelomic epithelium in the peritoneal cavity, combined with the recent development of fully human monoclonal antibodies against this surface molecule, suggest Ep-CAM as a promising target for antibody-mediated therapies in ovarian carcinoma patients harboring tumors refractory to standard treatment modalities.


Assuntos
Antígenos de Neoplasias/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Moléculas de Adesão Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Recidiva Local de Neoplasia/metabolismo , Neoplasias Ovarianas/metabolismo , Adenocarcinoma de Células Claras/tratamento farmacológico , Adenocarcinoma de Células Claras/metabolismo , Adenocarcinoma de Células Claras/secundário , Adenocarcinoma Mucinoso/tratamento farmacológico , Adenocarcinoma Mucinoso/metabolismo , Adenocarcinoma Mucinoso/secundário , Adulto , Antígenos de Neoplasias/genética , Western Blotting , Carcinoma Papilar/tratamento farmacológico , Carcinoma Papilar/metabolismo , Carcinoma Papilar/secundário , Moléculas de Adesão Celular/genética , Quimioterapia Adjuvante , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/secundário , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/secundário , Molécula de Adesão da Célula Epitelial , Feminino , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/tratamento farmacológico , Compostos Organoplatínicos/administração & dosagem , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Ovário/metabolismo , Ovário/patologia , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Resultado do Tratamento , Células Tumorais Cultivadas
8.
J Virol ; 83(13): 6779-89, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19386711

RESUMO

Papillomavirus-like particles (VLPs) based on L1 capsid protein represent a promising prophylactic vaccine against human papillomavirus (HPV) infections. However, cell-mediated immune responses against this antigen are believed to be of limited therapeutic value in established HPV-infected cervical lesions and, for this reason, have not been intensively investigated in cervical cancer patients. In this study we analyzed and quantified by real-time PCR (RT-PCR) the RNA expression levels of E6, E7, and L1 genes in flash-frozen HPV-16 cervical carcinomas. In addition, the kinetics of expression of E6, E7, and L1 in HPV-16-infected primary cell lines established as long-term cultures in vitro was also evaluated at RNA and protein levels. Finally, in order to evaluate the therapeutic potential of L1-specific CD4(+) and CD8(+) T lymphocytes responses in cervical cancer patients, L1 VLP-loaded dendritic cells (DCs) were used to stimulate peripheral blood lymphocytes from cervical cancer patients and such responses were compared to those elicited by the E7 oncoprotein. We show that 22 of 22 (100%) flash-frozen cervical biopsy samples collected from HPV-16-positive cervical cancer patients harbor L1, in addition to E6 and E7 RNA, as detected by RT-PCR. E7 RNA copy number (mean, 176.2) was significantly higher in HPV-16-positive cervical cancers compared to the E6 RNA copy number (mean, 47.3) and the L1 copy number (mean, 58.3) (P < 0.0001 and P < 0.001, respectively). However, no significant differences in expression levels between E6 and L1 were found. Kinetic studies of E6, E7, and L1 RNA and protein expression levels in primary tumors showed a sharp reduction in L1 expression after multiple in vitro passages compared to E6 and E7. Autologous DCs pulsed with HPV-16 VLPs or recombinant full-length E7 elicited strong type 1 L1- and E7-specific responses in CD4(+) and CD8(+) T cells from cervical cancer patients. Importantly, L1 VLP-specific CD8(+) T lymphocytes expressed strong cytolytic activity against autologous tumor cells and were as effective as E7-specific cytotoxic T lymphocytes in lysing naturally HPV-16-infected autologous tumor cells. Taken together, these data demonstrate a consistent expression of L1 in primary cervical tumors and the possibility of inducing effective L1/tumor-specific CD4(+) and CD8(+) T-lymphocyte responses in patients harboring HPV-infected cervical cancer. These results may have important implications for the treatment of patients harboring established HPV-infected lesions with L1 VLPs or combined E7/L1 DC-based vaccinations.


Assuntos
Papillomavirus Humano 16/imunologia , Infecções por Papillomavirus/imunologia , Linfócitos T Citotóxicos/imunologia , Neoplasias do Colo do Útero/imunologia , Adulto , Idoso , Vacinas Anticâncer/imunologia , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Feminino , Perfilação da Expressão Gênica , Papillomavirus Humano 16/genética , Humanos , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/imunologia , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus , RNA Viral/metabolismo , Proteínas Repressoras/metabolismo , Linfócitos T Citotóxicos/virologia , Neoplasias do Colo do Útero/virologia , Adulto Jovem
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