RESUMO
BACKGROUND: While NTRK fusion-positive cancers can be exquisitely sensitive to first-generation TRK inhibitors, resistance inevitably occurs, mediated in many cases by acquired NTRK mutations. Next-generation inhibitors (e.g., selitrectinib, repotrectinib) maintain activity against these TRK mutant tumors; however, there are no next-generation TRK inhibitors approved by the FDA and select trials have stopped treating patients. Thus, the identification of novel, potent and specific next-generation TRK inhibitors is a high priority. METHODS: In silico modeling and in vitro kinase assays were performed on TRK wild type (WT) and TRK mutant kinases. Cell viability and clonogenic assays as well as western blots were performed on human primary and murine engineered NTRK fusion-positive TRK WT and mutant cell models. Finally, zurletrectinib was tested in vivo in human xenografts and murine orthotopic glioma models harboring TRK-resistant mutations. RESULTS: In vitro kinase and in cell-based assays showed that zurletrectinib, while displaying similar potency against TRKA, TRKB, and TRKC WT kinases, was more active than other FDA approved or clinically tested 1st- (larotrectinib) and next-generation (selitrectinib and repotrectinib) TRK inhibitors against most TRK inhibitor resistance mutations (13 out of 18). Similarly, zurletrectinib inhibited tumor growth in vivo in sub-cute xenograft models derived from NTRK fusion-positive cells at a dose 30 times lower when compared to selitrectinib. Computational modeling suggests this stronger activity to be the consequence of augmented binding affinity of zurletrectinib for TRK kinases. When compared to selitrectinib and repotrectinib, zurletrectinib showed increased brain penetration in rats 0.5 and 2 h following a single oral administration. Consistently, zurletrectinib significantly improved the survival of mice harboring orthotopic NTRK fusion-positive, TRK-mutant gliomas (median survival = 41.5, 66.5, and 104 days for selitrectinib, repotrectinib, and zurletrectinib respectively; P < 0.05). CONCLUSION: Our data identifies zurletrectinib as a novel, highly potent next-generation TRK inhibitor with stronger in vivo brain penetration and intracranial activity than other next-generation agents.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Inibidores de Proteínas Quinases , Receptor trkA , Receptor trkB , Receptor trkC , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Receptor trkA/genética , Receptor trkA/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptor trkB/antagonistas & inibidores , Receptor trkB/genética , Receptor trkC/genética , Receptor trkC/antagonistas & inibidores , Linhagem Celular Tumoral , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Ratos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Pirazóis/farmacologia , Glioma/tratamento farmacológico , Glioma/genética , Glioma/patologia , Pirimidinas/farmacologia , Mutação , Feminino , Glicoproteínas de MembranaRESUMO
The prognosis of advanced/recurrent cervical cancer patients remains poor. We analyzed 54 fresh-frozen and 15 primary cervical cancer cell lines, along with matched-normal DNA, by whole-exome sequencing (WES), most of which harboring Human-Papillomavirus-type-16/18. We found recurrent somatic missense mutations in 22 genes (including PIK3CA, ERBB2, and GNAS) and a widespread APOBEC cytidine deaminase mutagenesis pattern (TCW motif) in both adenocarcinoma (ACC) and squamous cell carcinomas (SCCs). Somatic copy number variants (CNVs) identified 12 copy number gains and 40 losses, occurring more often than expected by chance, with the most frequent events in pathways similar to those found from analysis of single nucleotide variants (SNVs), including the ERBB2/PI3K/AKT/mTOR, apoptosis, chromatin remodeling, and cell cycle. To validate specific SNVs as targets, we took advantage of primary cervical tumor cell lines and xenografts to preclinically evaluate the activity of pan-HER (afatinib and neratinib) and PIK3CA (copanlisib) inhibitors, alone and in combination, against tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway (71%). Tumors harboring ERBB2 (5.8%) domain mutations were significantly more sensitive to single agents afatinib or neratinib when compared to wild-type tumors in preclinical in vitro and in vivo models (P = 0.001). In contrast, pan-HER and PIK3CA inhibitors demonstrated limited in vitro activity and were only transiently effective in controlling in vivo growth of PIK3CA-mutated cervical cancer xenografts. Importantly, combinations of copanlisib and neratinib were highly synergistic, inducing long-lasting regression of tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway. These findings define the genetic landscape of cervical cancer, suggesting that a large subset of cervical tumors might benefit from existing ERBB2/PIK3CA/AKT/mTOR-targeted drugs.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Sequenciamento do Exoma , Mutação , Receptor ErbB-2/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/terapia , Animais , Linhagem Celular Tumoral , Terapia Combinada , Variações do Número de Cópias de DNA , Feminino , Xenoenxertos , Humanos , Polimorfismo de Nucleotídeo Único , Neoplasias do Colo do Útero/patologiaRESUMO
OBJECTIVE: Aberrant expression of HER2/neu and PIK3CA gene products secondary to amplification/mutations are common in high-grade-serous-endometrial (USC) and ovarian-cancers (HGSOC). Because scant information is currently available in the literature on the potential negative effect of PIK3CA mutations on the activity of afatinib, in this study we evaluate for the first time the role of oncogenic PIK3CA mutations as a potential mechanism of resistance to afatinib in HGSOC and USC overexpressing HER2/neu. METHODS: We used six whole-exome-sequenced primary HGSOC/USC cell-lines and three xenografts overexpressing HER2/neu and harboring mutated or wild-type PIK3CA/PIK3R1 genes to evaluate the role of PI3K-mutations as potential mechanism of resistance to afatinib, an FDA-approved pan-c-erb-inhibitor in clinical trials in USC. Primary-USC harboring wild-type-PIK3CA gene was transfected with plasmids encoding oncogenic PIK3CA-mutations (H1047R/E545K). The effect of afatinib on HER2/PI3K/AKT/mTOR pathway was evaluated by immunoblotting. RESULTS: We found PI3K wild-type cell-lines to be significantly more sensitive (lower IC50) than PI3K-mutated cell-lines pâ¯=â¯0.004). In vivo, xenografts of primary cell-line USC-ARK2, transfected with the PIK3CA-H1047R or E545K hotspot-mutations, exhibited significantly more rapid tumor growth when treated with afatinib, compared to mice harboring ARK2-tumors transfected with wild-type-PIK3CA (pâ¯=â¯0.041 and 0.001, respectively). By western-blot, afatinib effectively reduced total and phospho-HER2 proteins in all cell-lines. However, H1047R/E545K-PIK3CA-transfected-ARK2-cells demonstrated a greater compensatory increase in phosphorylated-AKT proteins after afatinib exposure when compared to controls ARK2. CONCLUSIONS: Oncogenic PI3K mutations may represent a major mechanism of resistance to afatinib. Combinations of c-erb with PIK3CA, AKT or mTOR inhibitors may be necessary to more efficiently block the PIK3CA/AKT/mTOR pathway.
Assuntos
Afatinib/farmacologia , Classe I de Fosfatidilinositol 3-Quinases/genética , Neoplasias dos Genitais Femininos/tratamento farmacológico , Fosfatidilinositol 3-Quinases/genética , Adulto , Idoso , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/biossíntese , Classe Ia de Fosfatidilinositol 3-Quinase , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Neoplasias dos Genitais Femininos/enzimologia , Neoplasias dos Genitais Femininos/genética , Humanos , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Mutação , Fosfatidilinositol 3-Quinases/biossíntese , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The i.p. administration of chemotherapy in ovarian and uterine serous carcinoma patients by biodegradable nanoparticles may represent a highly effective way to suppress peritoneal carcinomatosis. However, the efficacy of nanoparticles loaded with chemotherapeutic agents is currently hampered by their fast clearance by lymphatic drainage. Here, we show that a unique formulation of bioadhesive nanoparticles (BNPs) can interact with mesothelial cells in the abdominal cavity and significantly extend the retention of the nanoparticles in the peritoneal space. BNPs loaded with a potent chemotherapeutic agent [epothilone B (EB)] showed significantly lower systemic toxicity and higher therapeutic efficacy against i.p. chemotherapy-resistant uterine serous carcinoma-derived xenografts compared with free EB and non-BNPs loaded with EB.
Assuntos
Adesivos/administração & dosagem , Sistemas de Liberação de Medicamentos , Nanopartículas/administração & dosagem , Animais , Proliferação de Células/efeitos dos fármacos , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/patologia , Epotilonas/administração & dosagem , Epotilonas/farmacologia , Epotilonas/uso terapêutico , Feminino , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Injeções Intraperitoneais , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Nanopartículas/química , Nanopartículas/ultraestrutura , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/patologiaRESUMO
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
OBJECTIVE: Up to 12% of all endometrial-carcinomas (EC) harbor DNA-polymerase-ε-(POLE) mutations. It is currently unknown whether the favorable prognosis of POLE-mutated EC is derived from their low metastatic capability, extraordinary number of somatic mutations thus imparting immunogenicity, or a high sensitivity to chemotherapy. METHODS: Polymerase-chain-reaction-amplification and Sanger-sequencing were used to test for POLE exonuclease-domain-mutations (exons 9-14) 131 EC. Infiltration of CD4+ and CD8+ T-lymphocytes (TIL) and PD-1-expression in POLE-mutated vs POLE wild-type EC was studied by immunohistochemistry (IHC) and the correlations between survival and molecular features were investigated. Finally, primary POLE-mutated and POLE-wild-type EC cell lines were established and compared in-vitro for their sensitivity to chemotherapy. RESULTS: Eleven POLE-mutated EC (8.5%) were identified. POLE-mutated tumors were associated with improved progression-free-survival (P<0.05) and displayed increased numbers of CD4+ (44.5 vs 21.8; P=0.001) and CD8+ (32.8 vs 13.5; P<0.001) TILs when compared to wild-type POLE EC. PD-1 receptor was overexpressed in TILs from POLE-mutated vs wild-type-tumors (81% vs 28%; P<0.001). Primary POLE tumor cell lines were significantly more resistant to platinum-chemotherapy in-vitro when compared to POLE-wild-type tumors (P<0.004). CONCLUSIONS: POLE ultra-mutated EC are heavily infiltrated with CD4+/CD8+ TIL, overexpress PD-1 immune-check-point (i.e., features consistent with chronic antigen-exposure), and have a better prognosis when compared to other molecular subtypes of EC patients. POLE-mutated tumor-cell lines are resistant to platinum-chemotherapy in-vitro suggesting that the better prognosis of POLE-patients is not secondary to a higher sensitivity to chemotherapy but likely linked to enhanced immunogenicity.
Assuntos
Carcinoma/genética , Carcinoma/imunologia , DNA Polimerase II/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Carboplatina/farmacologia , Carcinoma/química , Sobrevivência Celular/efeitos dos fármacos , DNA Polimerase II/análise , Intervalo Livre de Doença , Neoplasias do Endométrio/química , Feminino , Humanos , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Mutação , Proteínas de Ligação a Poli-ADP-Ribose , Células Tumorais CultivadasRESUMO
BACKGROUND: Clinical options for patients harbouring advanced/recurrent uterine serous carcinoma (USC), an aggressive variant of endometrial tumour, are very limited. Next-generation sequencing (NGS) data recently demonstrated that cyclin E1 (CCNE1) gene amplification and pik3ca driver mutations are common in USC and may therefore represent ideal therapeutic targets. METHODS: Cyclin E1 expression was evaluated by immunohistochemistry (IHC) on 95 USCs. The efficacy of the cyclin-dependent kinase 2/9 inhibitor CYC065 was assessed on multiple primary USC cell lines with or without CCNE1 amplification. Cell-cycle analyses and knockdown experiments were performed to assess CYC065 targeting specificity. Finally, the in vitro and in vivo activity of CYC065, Taselisib (a PIK3CA inhibitor) and their combinations was tested on USC xenografts derived from CCNE1-amplified/pik3ca-mutated USCs. RESULTS: We found that 89.5% of the USCs expressed CCNE1. CYC065 blocked cells in the G1 phase of the cell cycle and inhibited cell growth specifically in CCNE1-overexpressing USCs. Cyclin E1 knockdown conferred increased resistance to CYC065, whereas CYC065 treatment of xenografts derived from CCNE1-amplified USCs significantly reduced tumour growth. The combination of CYC065 and Taselisib demonstrated synergistic effect in vitro and was significantly more effective than single-agent treatment in decreasing tumour growth in xenografts of CCNE1-amplified/pik3ca-mutated USCs. CONCLUSIONS: Dual CCNE1/PIK3CA blockade may represent a novel therapeutic option for USC patients harbouring recurrent CCNE1-amplified/pi3kca-mutated tumours.
Assuntos
Ciclina E/genética , Mutação , Proteínas Oncogênicas/genética , Fosfatidilinositol 3-Quinases/genética , Neoplasias Uterinas/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Ciclina E/metabolismo , Variações do Número de Cópias de DNA , Feminino , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Hibridização in Situ Fluorescente , Técnicas In Vitro , Camundongos , RNA Mensageiro/genética , Análise Serial de Tecidos , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismoRESUMO
BACKGROUND: Uterine serous carcinoma is an aggressive form of endometrial cancer that carries an extremely poor prognosis. Solitomab is a novel bispecific single-chain antibody construct that targets epithelial cell adhesion molecule on tumor cells and also contains a CD3 binding region. We evaluated the expression levels of epithelial cell adhesion molecule and the in vitro activity of solitomab against primary uterine serous carcinoma cell lines in vitro and ex-vivo in the ascites of patients with uterine serous carcinoma. OBJECTIVE: The purpose of this study was to determine the frequency of expression of epithelial cell adhesion molecule on uterine serous carcinoma cell lines and the ability of solitomab to modulate immune responses (T-cell proliferation, activation, cytokine production, and tumor killing) to tumor cells when it is combined with lymphocytes and epithelial cell adhesion molecule-positive cell lines or epithelial cell adhesion molecule-positive ascitic fluid in vitro. STUDY DESIGN: Epithelial cell adhesion molecule expression was evaluated by flow cytometry in a total of 14 primary uterine serous carcinoma cell lines. Sensitivity to solitomab-dependent cellular-cytotoxicity was tested against a panel of primary uterine serous carcinoma cell lines that express different levels of epithelial cell adhesion molecule in standard 4-hour chromium release assays. The proliferative activity, activation, cytokine secretion (ie, type I vs type II), and cytotoxicity of solitomab in autologous tumor-associated T cells in the ascitic fluid of patients with uterine serous carcinoma was also evaluated by carboxyfluorescein succinimidyl ester and flow-cytometry assays. Differences in epithelial cell adhesion molecule expression, solitomab-dependent cellular-cytotoxicity levels were analyzed with the use of an unpaired t test. T-cell activation marker increase and cytokine release were analyzed by a paired t test. RESULTS: Surface expression of epithelial cell adhesion molecule was found in 85.7% (12 of 14) of the uterine serous carcinoma cell lines that were tested by flow cytometry. Epithelial cell adhesion molecule-positive cell lines were found resistant to natural killer cells or T-cell-mediated killing after exposure to peripheral blood lymphocytes in 4-hour chromium-release assays (mean killing ± standard of the mean, 2.7% ± 3.1% after incubation of epithelial cell adhesion molecule-positive cell lines with control bispecific antibody construct). In contrast, after incubation with solitomab, epithelial cell adhesion molecule-positive uterine serous carcinoma cells became highly sensitive to T-cell cytotoxicity (mean killing, 25.7% ± 4.5%; P < .0001) by peripheral blood lymphocytes. Ex vivo incubation of autologous tumor-associated lymphocytes with epithelial cell adhesion molecule that expressed malignant cells in ascites with solitomab resulted in a significant increase in T-cell proliferation in both CD4+ and CD8+ T cells, increase in T-cell activation markers (ie, CD25 and HLA-DR), and a reduction in number of viable uterine serous carcinoma cells in ascites (P < .001). CONCLUSION: Solitomab induces robust immunologic responses in vitro that result in increased T-cell activation, proliferation, production of cytokines, and direct killing of tumor cells. These findings suggest that solitomab may represent a novel, potentially effective agent for the treatment of recurrent/metastatic and/or chemo-resistant uterine serous carcinoma-overexpressing epithelial cell adhesion molecule.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antígenos de Neoplasias/efeitos dos fármacos , Antígenos de Neoplasias/imunologia , Antineoplásicos/farmacologia , Complexo CD3/imunologia , Carcinoma Papilar/tratamento farmacológico , Moléculas de Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/imunologia , Neoplasias Císticas, Mucinosas e Serosas/tratamento farmacológico , Neoplasias Uterinas/tratamento farmacológico , Antígenos de Neoplasias/análise , Líquido Ascítico/patologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Carcinoma Papilar/química , Carcinoma Papilar/imunologia , Moléculas de Adesão Celular/análise , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Molécula de Adesão da Célula Epitelial , Feminino , Citometria de Fluxo , Humanos , Ativação Linfocitária/efeitos dos fármacos , Neoplasias Císticas, Mucinosas e Serosas/química , Neoplasias Císticas, Mucinosas e Serosas/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Neoplasias Uterinas/química , Neoplasias Uterinas/imunologiaRESUMO
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.
Assuntos
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
Identification of micrometastatic disease at the time of surgery remains extremely challenging in ovarian cancer patients. We used fluorescence microscopy, an in vivo imaging system and a fluorescence stereo microscope to evaluate fluorescence distribution in Claudin-3- and -4-overexpressing ovarian tumors, floating tumor clumps isolated from ascites and healthy organs. To do so, mice harboring chemotherapy-naïve and chemotherapy-resistant human ovarian cancer xenografts or patient-derived xenografts (PDXs) were treated with the carboxyl-terminal binding domain of the Clostridium perfringens enterotoxin (c-CPE) conjugated to FITC (FITC-c-CPE) or the near-infrared (NIR) fluorescent tag IRDye CW800 (CW800-c-CPE) either intraperitoneally (IP) or intravenously (IV). We found tumor fluorescence to plateau at 30 min after IP injection of both the FITC-c-CPE and the CW800-c-CPE peptides and to be significantly higher than in healthy organs (p < 0.01). After IV injection of CW800-c-CPE, tumor fluorescence plateaued at 6 hr while the most favorable tumor-to-background fluorescence ratio (TBR) was found at 48 hr in both mouse models. Importantly, fluorescent c-CPE was highly sensitive for the in vivo visualization of peritoneal micrometastatic tumor implants and the identification of ovarian tumor spheroids floating in malignant ascites that were otherwise not detectable by conventional visual observation. The use of the fluorescent c-CPE peptide may represent a novel and effective optical approach at the time of primary debulking surgery for the real-time detection of micrometastatic ovarian disease overexpressing the Claudin-3 and -4 receptors or the identification of residual disease at the time of interval debulking surgery after neoadjuvant chemotherapy treatment.
Assuntos
Enterotoxinas/administração & dosagem , Corantes Fluorescentes/administração & dosagem , Micrometástase de Neoplasia/patologia , Neoplasias Ovarianas/patologia , Animais , Claudina-3/metabolismo , Claudina-4/metabolismo , Feminino , Humanos , Camundongos , Camundongos SCID , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
BACKGROUND: Solitomab is a novel, bispecific, single-chain antibody that targets epithelial cell adhesion molecule (EpCAM) on tumor cells and also contains a cluster of differentiation 3 (CD3) (T-cell coreceptor) binding region. The authors evaluated the in vitro activity of solitomab against primary chemotherapy-resistant epithelial ovarian carcinoma cell lines as well as malignant cells in ascites. METHODS: EpCAM expression was evaluated by flow cytometry in 5 primary ovarian cancer cell lines and in 42 fresh ovarian tumor cell cultures in ascites from patients with mainly advanced or recurrent, chemotherapy-resistant disease. The potential activity of solitomab against EpCAM-positive tumor cells was evaluated by flow cytometry, proliferation, and 4-hour chromium-release, cell-mediated cytotoxicity assays. RESULTS: EpCAM expression was detected by flow cytometry in approximately 80% of the fresh ovarian tumors and primary ovarian tumor cell lines tested. EpCAM-positive, chemotherapy-resistant cell lines were identified as resistant to natural killer cell-mediated or T-cell-mediated killing after exposure to peripheral blood lymphocytes in 4-hour chromium-release assays (mean±standard error of the mean, 3.6%±0.7% of cells killed after incubation of EpCAM-positive cell lines with control bispecific antibody). In contrast, after incubation with solitomab, EpCAM-positive, chemotherapy-resistant cells became highly sensitive to T-cell cytotoxicity (mean±standard error of the mean, 28.2%±2.05% of cells killed; P<.0001) after exposure to peripheral blood lymphocytes. Ex vivo incubation of autologous tumor-associated lymphocytes with EpCAM-expressing malignant cells in ascites with solitomab resulted in a significant increase in T-cell activation markers and a reduction in the number of viable ovarian tumor cells in ascites (P<.001). CONCLUSIONS: Solitomab may represent a novel, potentially effective agent for the treatment of chemotherapy-resistant ovarian cancers that overexpress EpCAM.
Assuntos
Anticorpos Biespecíficos/farmacologia , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Biespecíficos/imunologia , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/imunologia , Complexo CD3/imunologia , Carcinoma Epitelial do Ovário , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Molécula de Adesão da Célula Epitelial , Feminino , Citometria de Fluxo , Humanos , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/imunologia , Neoplasias Ovarianas/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
OBJECTIVES: We evaluated the role of PIK3CA-mutations as mechanism of resistance to trastuzumab in primary HER2/neu-amplified uterine-serous-carcinoma (USC) cell lines. METHODS: Fifteen whole-exome-sequenced USC cell lines were tested for HER2/neu-amplification and PIK3CA-mutations. Four HER2/neu-amplified USC (2-harbouring wild-type-PIK3CA-genes and 2-harbouring oncogenic-PIK3CA-mutations) were evaluated in in vitro dose-titration-proliferation-assays, cell-viability and HER2 and S6-protein-phosphorylation after exposure to trastuzumab. USC harbouring wild-type-PIK3CA were transfected with plasmids encoding oncogenic PIK3CA-mutations (i.e., H1047R/R93Q) and exposed to trastuzumab. Finally, trastuzumab efficacy was tested by using two USC xenograft mouse models. RESULTS: Seven out of fifteen (46%) of the USC cell lines were HER2/neu-amplified by fluorescence in situ hybridisation. Within these tumours four out of seven (57%) were found to harbour oncogenic PIK3CA-mutations vs two out of eight (25%) of the HER2/neu not amplified cell lines (P=0.01). HER2/neu-amplified/PIK3CA-mutated USC were highly resistant to trastuzumab when compared with HER2/neu-amplified/wild-type-PIK3CA cell lines (P=0.02). HER2/neu-amplified/PIK3CA wild-type cell lines transfected with oncogenic PIK3CA-mutations increased their resistance to trastuzumab (P<0.0001). Trastuzumab was effective in reducing tumour growth (P=0.001) and improved survival (P=0.0001) in mouse xenografts harbouring HER2-amplified/PIK3CA wild-type USC but not in HER2-amplified/PIK3CA-mutated tumours. CONCLUSIONS: Oncogenic PIK3CA mutations are common in HER2/neu-amplified USC and may constitute a major mechanism of resistance to trastuzumab treatment.
Assuntos
Antineoplásicos/administração & dosagem , Cistadenocarcinoma Seroso/tratamento farmacológico , Fosfatidilinositol 3-Quinases/genética , Receptor ErbB-2/genética , Trastuzumab/administração & dosagem , Neoplasias Uterinas/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Sobrevivência Celular/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases , Cistadenocarcinoma Seroso/genética , Feminino , Amplificação de Genes , Humanos , Camundongos , Mutação , Trastuzumab/uso terapêutico , Neoplasias Uterinas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Uterine serous carcinoma (USC) is an aggressive subtype of endometrial cancer that carries an extremely poor prognosis. Up to 35 % of USC may overexpress the epidermal growth factor receptor-2 (HER2/neu) at strong (i.e., 3+) level by immunohistochemistry (IHC) or harbor HER2/neu gene amplification by fluorescence in situ hybridization (FISH). In this study, we assessed the sensitivity of a panel of USC cell lines with and without HER2/neu gene amplification to dacomitinib (PF-00299804), an irreversible pan-human epidermal growth factor receptor tyrosine kinase inhibitor. Eight primary cell lines (i.e., four harboring HER2/neu gene amplification by FISH and four FISH- cell lines), all demonstrating similar in vitro growth rates, were evaluated in viability/proliferation assays. The effect of dacomitinib on cell growth, cell cycle distribution, and signaling was determined using flow cytometry-based assays. Dacomitinib caused a significantly stronger growth inhibition in HER2/neu FISH+ USC cell lines when compared to FISH- USC (dacomitinib half maximal inhibitory concentration (IC50) mean ± SEM = 0.02803 ± 0.003355 µM in FISH+ versus 1.498 ± 0.2209 µM in FISH- tumors, P < 0.0001). Dacomitinib growth inhibition was associated with a significant and dose-dependent decline in phosphorylated HER2/neu and S6 transcription factor and a dose-dependent and time-dependent cell cycle arrest in G0/G1 in FISH+ USC. Dacomitinib is remarkably effective against chemotherapy-resistant HER2/neu gene-amplified USC. Clinical studies with dacomitinib in HER2/neu FISH+ USC patients resistant to standard salvage chemotherapy are warranted.
Assuntos
Cistadenocarcinoma Seroso/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Quinazolinonas/administração & dosagem , Receptor ErbB-2/genética , Neoplasias Uterinas/tratamento farmacológico , Anticorpos Monoclonais Humanizados/administração & dosagem , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Feminino , Amplificação de Genes , Humanos , Hibridização in Situ Fluorescente , Receptor ErbB-2/antagonistas & inibidores , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologiaRESUMO
OBJECTIVE: Around 7-10% of endometrial carcinomas are characterized by polymerase-ε-(POLE) exonuclease-domain-mutations, an ultra-mutated-phenotype and a favorable prognosis. It is currently unknown whether POLE ultra-mutated-tumors are more immunogenic when compared to the other groups of endometrial cancers. METHODS: We used autologous-dendritic-cells (DC) pulsed with whole-tumor-extracts to assess the level of CD8+ and CD4+ T-cell-activation induced by POLE-ultramutated (+) and POLE wild-type (-) endometrial cancer cells in vitro. T-lymphocyte-proliferations were evaluated using CFSE and/or ([3H])thymidine-incorporation-assays while the ability to specifically kill autologous-tumor-cells by cytotoxic-T-lymphocyte (CTL) was tested in standard 4-h-(51)Cr-cytotoxicity-assays. In order to correlate cytotoxic activity and proliferation by CD4+ and CD8+ T-lymphocytes, respectively, with a particular lymphoid subset, two-color-flow-cytometric analysis of intracellular-cytokine-expression (IFN-γ vs IL-4) at the single cell level was also performed. RESULTS: DC-pulsed with tumor extracts were able to induce CTL-responses against autologous-tumor-cells in both POLE (+) and POLE (-) cancer patients (P=0.305). These CD8+ T-cell-populations were cytotoxic against tumor-cells but they did not lyse PHA-stimulated-autologous-lymphocytes or autologous-EBV-transformed-lymphoblastoid-control-cell-lines. In contrast, only POLE (+) tumor-lysate-pulsed-DC were able to induce significant proliferation and high IFN-γ expression (i.e., Th1-cytokine-bias) in autologous in vitro DC-stimulated CD4+ T-cells as well as naïve CD4+ and CD8+ T-cells from patients-peripheral-blood (P<0.05). CONCLUSIONS: POLE ultra-mutated-tumors are significantly more immunogenic when compared to POLE (-) tumors, in particular to the helper arm of the immune system. These data lend support to the hypothesis that the better prognosis of patients with POLE (+) tumors may at least in part be linked to their enhanced immunogenicity.
Assuntos
Linfócitos T CD4-Positivos/imunologia , DNA Polimerase II/genética , Neoplasias do Endométrio/enzimologia , Neoplasias do Endométrio/imunologia , Mutação , Adulto , Idoso , Linhagem Celular Tumoral , DNA Polimerase II/imunologia , Células Dendríticas/imunologia , Neoplasias do Endométrio/genética , Feminino , Humanos , Ativação Linfocitária , Pessoa de Meia-Idade , Proteínas de Ligação a Poli-ADP-Ribose , Linfócitos T Citotóxicos/imunologiaRESUMO
OBJECTIVE: Carcinosarcoma is a deadly gynecologic malignancy with few effective treatment options. The study of new therapies is difficult because of its rarity. The objective of this study was to determine the efficacy of neratinib in the treatment of HER2 amplified carcinosarcoma. METHODS: The efficacy of neratinib in the treatment of HER2 amplified carcinosarcoma was determined in vitro using seven primary carcinosarcoma cell lines with differential expression of HER2/neu. Data regarding IC50, cell cycle distribution, and cell signaling changes were assessed by flow cytometry. The efficacy of neratinib was determined in treating mice harboring HER2 amplified carcinosarcoma xenografts. RESULTS: Two of seven (28.5%) carcinosarcoma cell lines were HER2/neu amplified. HER2/neu amplified cell lines SARARK6 and SARARK9 were significantly more sensitive to neratinib than the five non-HER2/neu amplified carcinosarcoma cell lines (mean±SEM IC50:0.014µM±0.004vs.0.164µM±0.019 p=0.0003). Neratinib treatment caused a significant build up in G0/G1 phase of the cell cycle, arrest auto phosphorylation of HER2/neu and activation of S6. Neratinib inhibited tumor growth (p=0.012) and prolonged survival in mice harboring HER2 amplified carcinosarcoma xenografts (p=0.0039). CONCLUSIONS: Neratinib inhibits HER2 amplified carcinosarcoma proliferation, signaling, cell cycle progression and tumor growth in vitro. Neratinib inhibits HER2/neu amplified xenograft growth and improves overall survival. Clinical trials are warranted.
Assuntos
Carcinossarcoma/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Quinolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Animais , Carcinossarcoma/enzimologia , Carcinossarcoma/genética , Carcinossarcoma/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Amplificação de Genes , Humanos , Camundongos , Camundongos SCID , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: To evaluate the efficacy of taselisib, a selective inhibitor of PIK3CA, against primary uterine serous carcinomas (USC) harboring PIK3CA mutations and HER2/neu gene amplification. METHODS: Sensitivity to taselisib was evaluated by flow-cytometry viability assays in vitro against nine primary USC cell lines. Cell cycle distribution and downstream signaling were assessed by measuring the DNA content of cells and by phosphorylation of the S6 protein by flow-cytometry. Preclinical efficacy of taselisib was also evaluated in vivo in a mouse model. RESULTS: Four USC cell lines harbored HER2/neu gene amplification by FISH and two of them harbored oncogenic PIK3CA mutations. Taselisib caused a strong differential growth inhibition in both HER2/neu FISH positive and HER2/neu FISH positive/PIK3CA mutated USC cell lines when compared to lines that were FISH negative and PIK3CA wild type (taselisib IC50 mean±SEM=0.042±0.006µM in FISH+ versus 0.38±0.06µM in FISH-tumors, P<0.0001). Taselisib growth-inhibition was associated with a significant and dose-dependent increase in the percentage of cells in the G0/G1 phase of the cell cycle and dose-dependent decline in the phosphorylation of S6. Taselisib was highly active at reducing tumor growth in vivo in USC mouse xenografts harboring PIK3CA mutation and overexpressing HER2/neu (P=0.007). Mice treated with taselisib had significantly longer survival when compared to control mice (P<0.0001). CONCLUSIONS: Taselisib represents a novel therapeutic option in patients harboring PIK3CA mutations and/or HER2/neu gene amplification.
Assuntos
Antineoplásicos/farmacologia , Carcinoma/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Imidazóis/farmacologia , Neoplasias Císticas, Mucinosas e Serosas/genética , Oxazepinas/farmacologia , Fosfatidilinositol 3-Quinases/genética , Receptor ErbB-2/genética , Neoplasias Uterinas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Amplificação de Genes , Humanos , Hibridização in Situ Fluorescente , Técnicas In Vitro , Camundongos , Pessoa de Meia-Idade , Inibidores de Fosfoinositídeo-3 Quinase , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVES: Uterine serous carcinoma (USC) represents an aggressive variant of endometrial cancer and accounts for a large proportion of deaths annually. HER2/neu amplification is associated with USC in approximately 30-35% of cases. The objective of this study was to determine the sensitivity of a panel of primary USC cell lines to the small tyrosine kinase inhibitor neratinib, an ErbB1 and HER2 inhibitor, both in vitro and in vivo. METHODS: HER2/neu amplification was determined by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in 24 USC cell lines. Flow cytometry was used to determine the effects of neratinib on cell viability, cell cycle distribution and signaling in vitro. Mice harboring HER2/neu amplified xenografts were treated with neratinib to assess the efficacy of the drug in vivo. RESULTS: HER2/neu amplification was noted in 8/24 primary cell lines. Data regarding the efficacy of neratinib was determined using 4 HER2 amplified cell lines and 4 non-amplified cell lines with similar growth rates. Data revealed that cell lines with HER2/neu amplification were exquisitely more sensitive to neratinib compared to non-amplified cell lines (mean ± SEM IC50: 0.011µM ± 0.0008 vs. 0.312µM ± 0.0456 p<0.0001). Neratinib caused arrest in the G0/G1 phase of the cell cycle and resulted in decreased autophosphorylation of HER2 and activation of S6. Neratinib treated mice harboring xenografts of HER2/neu amplified USC showed delayed tumor growth and improved overall survival compared to vehicle (p=0.0019). CONCLUSIONS: Neratinib may be a potential treatment option for patients harboring HER2/neu amplified USC. Clinical trials for this subset of endometrial cancer patients are warranted.
Assuntos
Quinolinas/uso terapêutico , Receptor ErbB-2/biossíntese , Neoplasias Uterinas/tratamento farmacológico , Neoplasias Uterinas/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Técnicas In Vitro , Camundongos , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias Uterinas/patologiaRESUMO
Since their discovery in 2002, BRAF mutations have been identified as clear drivers of oncogenesis in several cancer types. Currently, their incidence rate is nearly 7% of all solid tumors with BRAF V600E constituting approximately 90% of these diagnoses. In melanoma, thyroid cancer, and histiocytic neoplasms, BRAF hotspot mutations are found at a rate of about 50%, while in lung and colorectal cancers they range from 3% to 10% of reported cases. Though present in other malignancies such as breast and ovarian cancers, they constitute a small portion of diagnoses (<1%). Given their frequency along with advancements in screening technologies, various methods are used for the detection of BRAF-mutant cancers. Among these are targeted next-generation sequencing (NGS) on tumor tissue or circulating tumor DNA (ctDNA) and immunohistochemistry (IHC)-based assays. With advancements in detection technologies, several approaches to the treatment of BRAF-mutant cancers have been taken. In this review, we retrace the milestones that led to the clinical development of targeted therapies currently available for these tumors.
RESUMO
Intraperitoneal co-delivery of chemotherapeutic drugs (CDs) and immune checkpoint inhibitors (ICIs) brings hope to improve treatment outcomes in patients with peritoneal metastasis from ovarian cancer (OC). However, current intraperitoneal drug delivery systems face issues such as rapid drug clearance from lymphatic drainage, heterogeneous drug distribution, and uncontrolled release of therapeutic agents into the peritoneal cavity. Herein, we developed an injectable nanohydrogel by combining carboxymethyl chitosan (CMCS) with bioadhesive nanoparticles (BNPs) based on polylactic acid-hyperbranched polyglycerol. This system enables the codelivery of CD and ICI into the intraperitoneal space to extend drug retention. The nanohydrogel is formed by cross-linking of aldehyde groups on BNPs with amine groups on CMCS via reversible Schiff base bonds, with CD and ICI loaded separately into BNPs and CMCS network. BNP/CMCS nanohydrogel maintained the activity of the biomolecules and released drugs in a sustained manner over a 7 day period. The adhesive property, through the formation of Schiff bases with peritoneal tissues, confers BNPs with an extended residence time in the peritoneal cavity after being released from the nanohydrogel. In a mouse model, BNP/CMCS nanohydrogel loaded with paclitaxel (PTX) and anti-PD-1 antibodies (αPD-1) significantly suppressed peritoneal metastasis of OC compared to all other tested groups. In addition, no systemic toxicity of nanohydrogel-loaded PTX and αPD-1 was observed during the treatment, which supports potential translational applications of this delivery system.
Assuntos
Quitosana , Sistemas de Liberação de Medicamentos , Hidrogéis , Inibidores de Checkpoint Imunológico , Nanocompostos , Neoplasias Ovarianas , Neoplasias Peritoneais , Animais , Hidrogéis/química , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/secundário , Neoplasias Peritoneais/patologia , Camundongos , Quitosana/química , Quitosana/análogos & derivados , Feminino , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/química , Inibidores de Checkpoint Imunológico/administração & dosagem , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Nanocompostos/química , Humanos , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/administração & dosagem , Camundongos Endogâmicos BALB C , Glicerol/química , Glicerol/análogos & derivados , Linhagem Celular Tumoral , Polímeros/química , PoliésteresRESUMO
Activating mutations in PIK3CA are frequently found in estrogen-receptor-positive (ER+) breast cancer, and the combination of the phosphatidylinositol 3-kinase (PI3K) inhibitor alpelisib with anti-ER inhibitors is approved for therapy. We have previously demonstrated that the PI3K pathway regulates ER activity through phosphorylation of the chromatin modifier KMT2D. Here, we discovered a methylation site on KMT2D, at K1330 directly adjacent to S1331, catalyzed by the lysine methyltransferase SMYD2. SMYD2 loss attenuates alpelisib-induced KMT2D chromatin binding and alpelisib-mediated changes in gene expression, including ER-dependent transcription. Knockdown or pharmacological inhibition of SMYD2 sensitizes breast cancer cells, patient-derived organoids, and tumors to PI3K/AKT inhibition and endocrine therapy in part through KMT2D K1330 methylation. Together, our findings uncover a regulatory crosstalk between post-translational modifications that fine-tunes KMT2D function at the chromatin. This provides a rationale for the use of SMYD2 inhibitors in combination with PI3Kα/AKT inhibitors in the treatment of ER+/PIK3CA mutant breast cancer.