Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
Cancer Immunol Res ; 3(4): 326-32, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25527356

RESUMO

Programmed death 1 ligand 1 (PD-L1) is an immune regulatory molecule that limits antitumor immune activity. Targeting of PD-L1 and other immune checkpoint proteins has shown therapeutic activity in various tumor types. The expression of PD-L1 and its correlation with response to neoadjuvant chemotherapy in breast cancer has not been studied extensively. Our goal was to assess PD-L1 expression in a cohort of breast cancer patients treated with neoadjuvant chemotherapy. Pretreatment biopsies from 105 patients with breast cancer from Yale New Haven Hospital that subsequently received neoadjuvant chemotherapy were assessed for PD-L1 protein expression by automated quantitative analysis with a rabbit monoclonal antibody (E1L3N) to the cytoplasmic domain of PD-L1. In addition, tumor-infiltrating lymphocytes (TIL) were assessed on hematoxylin and eosin slides. PD-L1 expression was observed in 30% of patients, and it was positively associated with hormone-receptor-negative and triple-negative status and high levels of TILs. Both TILs and PD-L1 measured in the epithelium or stroma predicted pathologic complete response (pCR) to neoadjuvant chemotherapy in univariate and multivariate analyses. However, because they are strongly associated, TILs and PD-L1 cannot both be included in a significant multivariate model. PD-L1 expression is prevalent in breast cancer, particularly hormone-receptor-negative and triple-negative patients, indicating a subset of patients that may benefit from immune therapy. Furthermore, PD-L1 and TILs correlate with pCR, and high PD-L1 predicts pCR in multivariate analysis.


Assuntos
Antígeno B7-H1/biossíntese , Biomarcadores Tumorais/biossíntese , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Linfócitos do Interstício Tumoral/imunologia , Idoso , Neoplasias da Mama/patologia , Quimioterapia Adjuvante , Estudos de Coortes , Feminino , Humanos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Proteínas de Neoplasias/biossíntese , Prognóstico , Resultado do Tratamento
2.
Clin Cancer Res ; 18(16): 4449-57, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22661537

RESUMO

PURPOSE: To deepen our understanding of mutant ROS1 expression, localization, and frequency in non-small cell lung cancer (NSCLC), we developed a highly specific and sensitive immunohistochemistry (IHC)-based assay that is useful for the detection of wild-type and mutant ROS1. EXPERIMENTAL DESIGN: We analyzed 556 tumors with the ROS1 D4D6 rabbit monoclonal antibody IHC assay to assess ROS1 expression levels and localization. A subset of tumors was analyzed by FISH to determine the percentage of these tumors harboring ROS1 translocations. Using specific and sensitive IHC assays, we analyzed the expression of anaplastic lymphoma kinase (ALK), EGFR L858R, and EGFR E746-A750del mutations in a subset of lung tumors, including those expressing ROS1. RESULTS: In our NSCLC cohort of Chinese patients, we identified 9 (1.6%) tumors expressing ROS1 and 22 (4.0%) tumors expressing ALK. FISH identified tumors with ALK or ROS1 rearrangements, and IHC alone was capable of detecting all cases with ALK and ROS1 rearrangements. ROS1 fusion partners were determined by reverse transcriptase PCR identifying CD74-ROS1, SLC34A2-ROS1, and FIG-ROS1 fusions. Some of the ALK and ROS1 rearranged tumors may also harbor coexisting EGFR mutations. CONCLUSIONS: NSCLC tumors with ROS1 rearrangements are uncommon in the Chinese population and represent a distinct entity of carcinomas. The ROS1 IHC assay described here is a valuable tool for identifying patients expressing mutant ROS1 and could be routinely applied in clinical practice to detect lung cancers that may be responsive to targeted therapies.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas de Transporte/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Quinase do Linfoma Anaplásico , Animais , Sequência de Bases , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Transporte/genética , Linhagem Celular , Proliferação de Células , Expressão Gênica , Genes erbB-1 , Genótipo , Proteínas da Matriz do Complexo de Golgi , Humanos , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Camundongos , Mutação , Proteínas de Fusão Oncogênica/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transplante Heterólogo
3.
Cancer Res ; 72(13): 3312-23, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22570254

RESUMO

Ovarian cancer is the leading cause of death from gynecologic cancer. Improvement in the clinical outcome of patients is likely to be achieved by the identification of molecular events that underlie the oncogenesis of ovarian cancer. Here we show that the anaplastic lymphoma kinase (ALK) is aberrantly activated in ovarian cancer. Using an unbiased and global phosphoproteomic approach, we profiled 69 Chinese primary ovarian tumor tissues and found ALK to be aberrantly expressed and phosphorylated in 4 tumors. Genetic characterization of these ALK-positive tumors indicated that full-length ALK expression in two serous carcinoma patients is consistent with ALK gene copy number gain, whereas a stromal sarcoma patient carries a novel transmembrane ALK fusion gene: FN1-ALK. Biochemical and functional analysis showed that both full-length ALK and FN1-ALK are oncogenic, and tumors expressing ALK or FN1-ALK are sensitive to ALK kinase inhibitors. Furthermore, immunohistochemical analysis of ovarian tumor tissue microarray detected aberrant ALK expression in 2% to 4% serous carcinoma patients. Our findings provide new insights into the pathogenesis of ovarian cancer and identify ALK as a potential therapeutic target in a subset of serous ovarian carcinoma and stromal sarcoma patients.


Assuntos
Neoplasias Ovarianas/tratamento farmacológico , Receptores Proteína Tirosina Quinases/metabolismo , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Quinase do Linfoma Anaplásico , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida , Primers do DNA , Feminino , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fosforilação , Receptores Proteína Tirosina Quinases/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem , Análise Serial de Tecidos
4.
PLoS One ; 6(1): e15640, 2011 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-21253578

RESUMO

Cholangiocarcinoma, also known as bile duct cancer, is the second most common primary hepatic carcinoma with a median survival of less than 2 years. The molecular mechanisms underlying the development of this disease are not clear. To survey activated tyrosine kinases signaling in cholangiocarcinoma, we employed immunoaffinity profiling coupled to mass spectrometry and identified DDR1, EPHA2, EGFR, and ROS tyrosine kinases, along with over 1,000 tyrosine phosphorylation sites from about 750 different proteins in primary cholangiocarcinoma patients. Furthermore, we confirmed the presence of ROS kinase fusions in 8.7% (2 out of 23) of cholangiocarcinoma patients. Expression of the ROS fusions in 3T3 cells confers transforming ability both in vitro and in vivo, and is responsive to its kinase inhibitor. Our data demonstrate that ROS kinase is a promising candidate for a therapeutic target and for a diagnostic molecular marker in cholangiocarcinoma. The identification of ROS tyrosine kinase fusions in cholangiocarcinoma, along with the presence of other ROS kinase fusions in lung cancer and glioblastoma, suggests that a more broadly based screen for activated ROS kinase in cancer is warranted.


Assuntos
Neoplasias dos Ductos Biliares/enzimologia , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/enzimologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Humanos , Imunoensaio , Camundongos , Camundongos Nus , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fosforilação , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/metabolismo
5.
Am J Surg Pathol ; 33(7): 984-91, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19363441

RESUMO

NUT midline carcinoma (NMC) is a uniformly lethal malignancy that is defined by rearrangement of the nuclear protein in testis (NUT) gene on chromosome 15q14. NMCs are morphologically indistinguishable from other poorly differentiated carcinomas, and the diagnosis is usually made currently by fluorescence in situ hybridization (FISH). As normal NUT expression is confined to testis and ovary, we reasoned that an immunohistochemical (IHC) stain for NUT would be useful in diagnosing NMC. To this end, we raised a highly specific rabbit monoclonal antibody, C52, against a recombinant NUT polypeptide, and developed an IHC staining protocol. The sensitivity and specificity of C52 staining was evaluated in a panel of 1068 tissues, predominantly diverse types of carcinomas (n=906), including 30 NMCs. Split-apart FISH for NUT rearrangement was used as a "gold standard" diagnostic test for NMC. C52 immunoreactivity among carcinomas was confined to NMCs. IHC staining had a sensitivity of 87%, a specificity of 100%, a negative predictive value of 99%, and a positive predictive value of 100%. Two new cases of NMC containing BRD4-NUT fusions were detected by C52 IHC, but missed by conventional FISH. In both instances, these tumors contained cryptic BRD4-NUT rearrangements, as confirmed by FISH using a refined set of probes. Some germ cell tumors, including 64% of dysgerminomas, showed weak NUT immunoreactivity, consistent with the expression of NUT in normal germ cells. We conclude that IHC staining with the C52 monoclonal antibody is a highly sensitive and specific test that reliably distinguishes NMC from other forms of carcinoma. The NUT antibody is being prepared for commercial release and will be available in the near future.


Assuntos
Anticorpos Monoclonais , Carcinoma/diagnóstico , Animais , Especificidade de Anticorpos , Carcinoma/genética , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Proteínas de Neoplasias , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/imunologia , Proteínas de Fusão Oncogênica , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade
6.
Clin Cancer Res ; 15(9): 3023-8, 2009 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19366827

RESUMO

PURPOSE: Activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are found in approximately 10% to 20% of non-small-cell lung cancer (NSCLC) patients and are associated with response to EGFR inhibitors. The most common NSCLC-associated EGFR mutations are deletions in exon 19 and L858R mutation in exon 21, together accounting for 90% of EGFR mutations. To develop a simple, sensitive, and reliable clinical assay for the identification of EGFR mutations in NSCLC patients, we generated mutation-specific rabbit monoclonal antibodies against each of these two most common EGFR mutations and aimed to evaluate the detection of EGFR mutations in NSCLC patients by immunohistochemistry. EXPERIMENTAL DESIGN: We tested mutation-specific antibodies by Western blot, immunofluorescence, and immunohistochemistry. In addition, we stained 40 EGFR genotyped NSCLC tumor samples by immunohistochemistry with these antibodies. Finally, with a panel of four antibodies, we screened a large set of NSCLC patient samples with unknown genotype and confirmed the immunohistochemistry results by DNA sequencing. RESULTS: These two antibodies specifically detect the corresponding mutant form of EGFR by Western blotting, immunofluorescence, and immunohistochemistry. Screening a panel of 340 paraffin-embedded NSCLC tumor samples with these antibodies showed that the sensitivity of the immunohistochemistry assay is 92%, with a specificity of 99% as compared with direct and mass spectrometry-based DNA sequencing. CONCLUSIONS: This simple assay for detection of EGFR mutations in diagnostic human tissues provides a rapid, sensitive, specific, and cost-effective method to identify lung cancer patients responsive to EGFR-based therapies.


Assuntos
Anticorpos Monoclonais , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutação/imunologia , Animais , Bioensaio , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/secundário , Análise Mutacional de DNA , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Imunoglobulina G/imunologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Coelhos , Sensibilidade e Especificidade , Deleção de Sequência , Transplante Heterólogo , Células Tumorais Cultivadas
7.
Cell ; 131(6): 1190-203, 2007 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-18083107

RESUMO

Despite the success of tyrosine kinase-based cancer therapeutics, for most solid tumors the tyrosine kinases that drive disease remain unknown, limiting our ability to identify drug targets and predict response. Here we present the first large-scale survey of tyrosine kinase activity in lung cancer. Using a phosphoproteomic approach, we characterize tyrosine kinase signaling across 41 non-small cell lung cancer (NSCLC) cell lines and over 150 NSCLC tumors. Profiles of phosphotyrosine signaling are generated and analyzed to identify known oncogenic kinases such as EGFR and c-Met as well as novel ALK and ROS fusion proteins. Other activated tyrosine kinases such as PDGFRalpha and DDR1 not previously implicated in the genesis of NSCLC are also identified. By focusing on activated cell circuitry, the approach outlined here provides insight into cancer biology not available at the chromosomal and transcriptional levels and can be applied broadly across all human cancers.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/genética , Quinase do Linfoma Anaplásico , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Ativação Enzimática , Fusão Gênica , Humanos , Neoplasias Pulmonares/genética , Modelos Biológicos , Dados de Sequência Molecular , Fosforilação , Fosfotirosina/genética , Proteínas Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo
8.
Proc Natl Acad Sci U S A ; 104(31): 12784-9, 2007 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-17640904

RESUMO

Protein 4.1B is a 4.1/ezrin/radixin/moesin domain-containing protein whose expression is frequently lost in a variety of human tumors, including meningiomas, non-small-cell lung cancers, and breast carcinomas. However, its potential tumor-suppressive function under in vivo conditions remains to be validated. In a screen for genes involved with prostate cancer metastasis, we found that 4.1B expression is reduced in highly metastatic tumors. Down-regulation of 4.1B increased the metastatic propensity of poorly metastatic cells in an orthotopic model of prostate cancer. Furthermore, 4.1B-deficient mice displayed increased susceptibility for developing aggressive, spontaneous prostate carcinomas. In both cases, enhanced tumor malignancy was associated with reduced apoptosis. Because expression of Protein 4.1B is frequently down-regulated in human clinical prostate cancer, as well as in a spectrum of other tumor types, these results suggest a more general role for Protein 4.1B as a negative regulator of cancer progression to metastatic disease.


Assuntos
Proteínas de Membrana/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Supressoras de Tumor/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Progressão da Doença , Regulação para Baixo , Humanos , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Neoplasias da Próstata/genética , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Cancer Res ; 65(21): 9789-98, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16267000

RESUMO

Dissemination to draining lymph nodes is a frequent first step in prostate cancer metastasis. Although tumors metastasize to lymph nodes via the lymphatics, the importance of lymphangiogenesis in mediating the process remains controversial. Here, we inhibit intratumoral lymphangiogenesis in s.c. and surgical orthotopic implantation mouse models of human prostate cancer using several strategies. Stable expression of small interfering RNAs (siRNA) targeted against human vascular endothelial growth factor-C (VEGF-C) in PC-3 cells reduced intratumoral lymphatics by 99% in s.c. tumors, indicating that tumor-secreted VEGF-C is necessary for lymphangiogenesis. Expression of siRNAs against human VEGF-A somewhat reduced tumor lymphangiogenesis. Secretion of a soluble VEGF receptor-3/Flt4 fusion protein by PC-3 cells reduced intratumoral lymphatics by 100% in s.c. tumors. Combination of soluble Flt4 and VEGF-C siRNA yielded >92% reduction of intratumoral lymphatics in orthotopic prostate tumors. However, metastasis to lymph nodes was not significantly affected regardless of intratumoral lymphatic vessel density. The abundance of marginal lymphatics at the tumor-stromal interface was unchanged in orthotopic tumors whose intratumoral lymphatics were inhibited, suggesting that these marginal vessels could be sufficient for lymph node metastasis. Hematogenous metastasis (blood tumor burden, lung metastasis) correlated with degree of lymph node invasion. We also analyzed the lymphatics in spontaneous transgenic adenocarcinomas of the mouse prostate which metastasize to lymph nodes. Progression from well-differentiated prostate intraepithelial neoplasia to metastatic, undifferentiated adenocarcinoma was accompanied by loss of lymphatics. These results suggest that tumor-secreted VEGF-C and, to a lesser extent, VEGF-A, are important for inducing prostate cancer intratumoral lymphangiogenesis but are unnecessary for lymph node metastasis.


Assuntos
Adenocarcinoma/patologia , Linfonodos/patologia , Neoplasias da Próstata/patologia , Fator C de Crescimento do Endotélio Vascular/fisiologia , Adenocarcinoma/metabolismo , Animais , Humanos , Linfangiogênese/fisiologia , Metástase Linfática , Masculino , Camundongos , Transplante de Neoplasias , Neoplasias da Próstata/metabolismo , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator C de Crescimento do Endotélio Vascular/biossíntese , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo
10.
Mol Cancer Ther ; 4(8): 1186-97, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16093434

RESUMO

OSI-930, a potent thiophene inhibitor of the Kit, KDR, and platelet-derived growth factor receptor tyrosine kinases, was used to selectively inhibit tyrosine phosphorylation downstream of juxtamembrane mutant Kit in the mast cell leukemia line HMC-1. Inhibition of Kit kinase activity resulted in a rapid dephosphorylation of Kit and inhibition of the downstream signaling pathways. Attenuation of Ras-Raf-Erk (phospho-Erk, phospho-p38), phosphatidyl inositol-3' kinase (phospho-p85, phospho-Akt, phospho-S6), and signal transducers and activators of transcription signaling pathways (phospho-STAT3/5/6) were measured by affinity liquid chromatography tandem mass spectrometry, by immunoblot, and by tissue microarrays of fixed cell pellets. To more globally define additional components of Kit signaling temporally altered by kinase inhibition, a novel multiplex quantitative isobaric peptide labeling approach was used. This approach allowed clustering of proteins by temporal expression patterns. Kit kinase, which dephosphorylates rapidly upon kinase inhibition, was shown to regulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2. Interactions with SH2 domain adapters [growth factor receptor binding protein 2 (Grb2), Cbl, Slp-76] and SH3 domain adapters (HS1, cortactin, CD2BP3) were attenuated by inhibition of Kit kinase activity. Functional crosstalk between Kit and the non-receptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk was observed. Inhibition of Kit modulated phosphorylation-dependent interactions with pathways controlling focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, Wasp, Fhl-3, G25K, Ack-1, Nap1, SH3P12/ponsin) and septin-actin complexes (NEDD5, cdc11, actin). The combined use of isobaric protein quantitation and expression clustering, immunoblot, and tissue microarray strategies allowed temporal measurement signaling pathways modulated by mutant Kit inhibition in a model of mast cell leukemia.


Assuntos
Antineoplásicos/farmacologia , Leucemia de Mastócitos/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/efeitos dos fármacos , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tiofenos/farmacologia , Humanos , Mutação , Proteínas de Neoplasias/metabolismo , Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/fisiologia , Análise Serial de Tecidos , Células Tumorais Cultivadas
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...