RESUMEN
Three-dimensional structured illumination microscopy (3D-SIM) and fluorescence in situ hybridization on three-dimensional preserved cells (3D-FISH) have proven to be robust and efficient methodologies for analyzing nuclear architecture and profiling the genome's topological features. These methods have allowed the simultaneous visualization and evaluation of several target structures at super-resolution. In this chapter, we focus on the application of 3D-SIM for the visualization of 3D-FISH preparations of chromosomes in interphase, known as Chromosome Territories (CTs). We provide a workflow and detailed guidelines for sample preparation, image acquisition, and image analysis to obtain quantitative measurements for profiling chromosome topological features. In parallel, we address a practical example of these protocols in the profiling of CTs 9 and 22 involved in the translocation t(9;22) in Chronic Myeloid Leukemia (CML). The profiling of chromosome topological features described in this chapter allowed us to characterize a large-scale topological disruption of CTs 9 and 22 that correlates directly with patients' response to treatment and as a possible potential change in the inheritance systems. These findings open new insights into how the genome structure is associated with the response to cancer treatments, highlighting the importance of microscopy in analyzing the topological features of the genome.
Asunto(s)
Imagenología Tridimensional , Hibridación Fluorescente in Situ , Humanos , Hibridación Fluorescente in Situ/métodos , Imagenología Tridimensional/métodos , Translocación Genética , Cromosomas/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Interfase/genética , Cromosomas Humanos/genética , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
BACKGROUND: Few trials have evaluated the utility of liquid biopsies to detect epidermal growth factor receptor mutations (EGFRm) at the time of response evaluation and its association with the clinical characteristics and outcomes of non-small-cell lung cancer (NSCLC) patients. OBJECTIVE: This study aimed to evaluate, in a real-world clinical setting, the prevalence of plasma EGFRm and its association with the clinical characteristics, response and survival outcomes of NSCLC patients under treatment with EGFR-tyrosine kinase inhibitors (EGFR-TKIs). METHODS: This observational study enrolled advanced or metastatic NSCLC patients, with confirmed tumor EGFRm, receiving treatment with first- or second-generation EGFR-TKIs. Blood samples for the detection of plasma EGFRm were collected at the time of response evaluation and processed using the Target Selector™ assay. The main outcomes were the detection rate of plasma EGFRm, median Progression-Free Survival (PFS) and Overall Survival (OS) according to plasma EGFR mutational status. RESULTS: Of 84 patients, 50 (59.5%) had an EGFRm detected in plasma. After a median follow-up of 21.1 months, 63 patients (75%) had disease progression. The detection rate of plasma EGFRm was significantly higher in patients with disease progression than in patients with partial response or stable disease (68.3% versus 33.3%; P< 0.01). PFS and OS were significantly longer in patients without plasma EGFRm than among patients with plasma EGFRm (14.3 months [95% CI, 9.25-19.39] vs 11.0 months [95% CI, 8.61-13.46]; P= 0.034) and (67.8 months [95% CI, 39.80-95.94] vs 32.0 months [95% CI, 17.12-46.93]; P= 0.006), respectively. A positive finding in LB was associated with the presence of ⩾ 3 more metastatic sites (P= 0.028), elevated serum carcinoembryonic (CEA) at disease progression (P= 0.015), and an increase in CEA with respect to baseline levels (P= 0.038). CONCLUSIONS: In NSCLC patients receiving EGFR-TKIs, the detection of plasma EGFRm at the time of tumor response evaluation is associated with poor clinical outcomes.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Neoplasias Pulmonares/mortalidad , Inhibidores de Proteínas Quinasas/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Análisis Mutacional de ADN , Progresión de la Enfermedad , Receptores ErbB/sangre , Receptores ErbB/genética , Femenino , Humanos , Biopsia Líquida , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Masculino , Persona de Mediana Edad , Supervivencia sin Progresión , Inhibidores de Proteínas Quinasas/farmacología , Medición de Riesgo/métodos , Medición de Riesgo/estadística & datos numéricosRESUMEN
The introduction of the BCR-ABL kinase inhibitor, imatinib mesylate (Gleevec®, Novartis) led to significant changes in the treatment of chronic myeloid leukaemia (CML) patients. However, despite the impressive percentage of responding patients, some CML cases, particularly those in advanced phases of the disease, show primary resistance or relapse after the initial response. The second-generation BCR-ABL inhibitors nilotinib (Tasigna®, Novartis) and dasatinib (Sprycel®, Bristol-Myers Squibb) have shown significant activity in clinical trials in patients who failed imatinib therapy, but these agents are still incapable of inhibiting the T315I mutant of Bcr-Abl and present partial activity in advanced phases of CML. The acquired biological notions of the mechanisms of tyrosine kinase inhibitor (TKI) resistance has led to the development of new compounds, some of which have shown encouraging preliminary results in clinical trials, even against T315I mutants. In this paper we discuss the new emerging therapies which may overcome TKI resistance in CML patients.
A introdução do inibidor de tirosino quinase BCR-ABL mesilato de imatinibe (Glivec®, Novartis) levou a significantes mudanças no tratamento da LMC. Entretanto, a despeito de impressionante porcentagem de pacientes que respondem, alguns casos de LMC, particularmente em fases avançadas da doença mostram resistência primaria ou recidivas após terapêutica inicial. Inibidores de tirosino quinases de segunda geração como o nilotinibe (Tasigna®, Novartis) e o dasatinibe (Sprycel®, Bristol Myers Squibb) têm mostrado significante atividade nos estudos clínicos em paciente onde o imatinibe falhou. Porém, estes agentes não são capazes de inibir a mutação T315I do Bcr-Abl e apresentam atividade parcial em fases avançadas da LMC. As noções biológicas adquiridas sobre os mecanismos de resistência aos inibidores de TK levaram ao desenvolvimento de novos compostos alguns dos quais têm resultados preliminares encorajadores incluindo a mutação T315I. Neste trabalho nós discutimos os novos agentes emergentes e qual o potencial poderão atingir para ultrapassar a resistência aos inibidores de TK em pacientes com LMC.