RESUMO
Advanced lung cancer has poor survival with few therapies. EGFR tyrosine kinase inhibitors (TKIs) have high response rates in patients with activating EGFR mutations, but acquired resistance is inevitable. Acquisition of the EGFR T790M mutation causes over 50% of resistance; MET amplification is also common. Preclinical data suggest synergy between MET and EGFR inhibitors. We hypothesized that EGFR-MET dimerization determines response to MET inhibition, depending on EGFR mutation status, independently of MET copy number. We tested this hypothesis by generating isogenic cell lines from NCI-H1975 cells, which co-express L858R and T790M EGFR mutations, namely H1975L858R/T790M (EGFR TKI resistant); H1975L858R (sensitized) and H1975WT (wild-type). We assessed cell proliferation in vitro and tumor growth/stroma formation in derived xenograft models in response to a MET TKI (SGX523) and correlated with EGFR-MET dimerization assessed by Förster Resonance Energy Transfer (FRET). SGX523 significantly reduced H1975L858R/T790M cell proliferation, xenograft tumor growth and decreased ERK phosphorylation. The same was not seen in H1975L858R or H1975WT cells. SGX523 only reduced stroma formation in H1975L858R. SGX523 reduced EGFR-MET dimerization in H1975L858R/T790M but induced dimer formation in H1975L858R with no effect in H1975WT. Our data suggests that MET inhibition by SGX523 and EGFR-MET heterodimerisation are determined by EGFR genotype. As tumor behaviour is modulated by this interaction, this could determine treatment efficacy.
Assuntos
Adenocarcinoma/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Células HEK293 , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Reprodutibilidade dos TestesAssuntos
Biomarcadores Tumorais , Linfoma de Burkitt , Imunofenotipagem , Linfonodos , Linfoma Folicular , Linfoma Difuso de Grandes Células B , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Linfoma de Burkitt/genética , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/patologia , Regulação Neoplásica da Expressão Gênica , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Linfonodos/imunologia , Linfonodos/patologia , Linfoma Folicular/genética , Linfoma Folicular/imunologia , Linfoma Folicular/patologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
Fluorescence in situ hybridisation (FISH) analysis is now widely employed in the diagnosis and risk stratification of a wide range of malignant diseases. While this technique is used successfully with formalin-fixed paraffin-embedded (FFPE) sections from numerous tissue types, FISH analysis of FFPE tissue sections from trephine biopsy specimens has been less widely reported, possibly due to technical limitations relating to the decalcification protocols employed. During the last 4 years FISH analysis has been carried out successfully in 42 out of 55 (76%) consecutive trephine biopsy specimens received as part of the standard diagnostic service at our institution. Samples decalcified using EDTA-based protocols were analysed successfully in 31/31 cases (100%), whereas only 11/24 samples (46%) decalcified using formic acid-based protocols were successful. In our experience, FISH analysis of trephine biopsy specimens is a highly reproducible technique and a very useful adjunctive tool in the diagnostic armoury; however, its use in a standard diagnostic setting relies on the use of EDTA-based decalcification protocols.
Assuntos
Doenças da Medula Óssea/diagnóstico , Exame de Medula Óssea/métodos , Medula Óssea/patologia , Aberrações Cromossômicas , Hibridização in Situ Fluorescente , Transtornos Linfoproliferativos/diagnóstico , Doença Aguda , Idoso , Biópsia/métodos , Doenças da Medula Óssea/genética , Doença Crônica , Técnica de Descalcificação , Feminino , Formaldeído , Humanos , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/genética , Linfoma de Células B/diagnóstico , Linfoma de Células B/genética , Transtornos Linfoproliferativos/genética , Pessoa de Meia-Idade , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/genética , Inclusão em Parafina , Fixação de Tecidos/métodosRESUMO
AIMS: In recent years the genetic aberrations associated with diffuse large B-cell lymphoma and the new subtype described in the 2008 revision of the WHO classification, 'B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma' have been increasingly well defined. Recurrent genetic abnormalities include rearrangements involving MYC (8q24), BCL2 (18q21) and BCL6 (3q27); as the prognostic and therapeutic implications associated with these abnormalities are clarified their accurate identification at diagnosis is becoming increasingly critical. We describe our experience of using a panel of fluorescence in situ hybridisation (FISH) probes on formalin-fixed paraffin-embedded tissue sections in the diagnostic work-up of 162 patients with non-Burkitt high grade B-cell non-Hodgkin's lymphomas (HG-BNHL). METHODS: BCL6, IGH-BCL2 and MYC status were determined prospectively in sequential patients presenting with HG-BNHL, with respect to the presence of rearrangements and copy number changes. Small numbers of samples were analysed retrospectively or were studied at relapse in previously untested patients. RESULTS: FISH analysis was successful in 160/162 (99%) cases, with abnormalities detected in 118/160 (74%). CONCLUSIONS: FISH analysis of formalin-fixed paraffin-embedded tissue sections is a highly reproducible technique with an excellent success rate for the detection of genetic abnormalities which will play an increasingly important role in improving risk stratification of patients with HG-BNHL.