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RATIONALE: Human epidermal growth factor receptor 2 (HER2) is a key driver of tumorigenesis, and over-expression as a result of HER2 gene amplification has been observed in a number of solid tumors. Recently HER2 has become an important biomarker for the monoclonal antibody treatment of HER2-positive metastatic breast and advanced gastric cancer. The HER2 targeting antibody trastuzumab treatment requires accurate measurement of HER2 levels for proper diagnosis. Droplet digital PCR (ddPCR) with highly direct, precise and absolute nucleic acid quantification could be used to detect HER2 amplification levels. OBJECTIVES: Our objective was to evaluate a robust, accurate and less subjective application of ddPCR for HER2 amplification levels and test the assay performance in clinical formalin-fixed paraffin-embedded (FFPE) breast and gastric carcinoma samples. METHODS: Genomic DNA from HER2 amplified cell line SK-BR-3 was used to set up the ddPCR assays. The copy number of HER2 was compared to the chromosome 17 centromere reference gene (CEP17), expressed as HER2:CEP17 ratio. Genomic DNAs of FFPE specimens from 145 Asian patients with breast and gastric carcinomas were assayed using both standard methods, immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH), and ddPCR. RESULTS: Based on 145 clinical breast and gastric carcinoma cases, our study demonstrated a high concordance of ddPCR results to FISH and IHC. In breast cancer specimens, the ddPCR results had high concordance with FISH and IHC defined HER2 status with a sensitivity of 90.9% (30/33) and a specificity of 100% (77/77). In gastric cancer specimens that were concordant in both FISH and IHC, our assay was 95.5% concordant with FISH and IHC (21/22). CONCLUSIONS: ddPCR has the advantage of automation and also allows levels of HER2 amplification to be easily evaluated in large numbers of samples, and presents a potential option to define HER2 status.
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Neoplasias de la Mama/genética , Reacción en Cadena de la Polimerasa/métodos , Receptor ErbB-2/genética , Neoplasias Gástricas/genética , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Fijadores/química , Formaldehído/química , Amplificación de Genes , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Persona de Mediana Edad , Parafina , Receptor ErbB-2/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/metabolismo , Adhesión del Tejido/métodos , Fijación del Tejido/métodosRESUMEN
The success of crizotinib in ALK-positive patients has elicited efforts to find new oncogenic fusions in lung cancer. These efforts have led to the discovery of novel oncogenic fusion genes such as ROS1 and RET. However, the molecular and clinicopathologic characteristics associated with RET or ROS1 fusion, compared with ALK fusion-positive lung cancer, remain unclear. We accordingly analyzed the clinicopathologic characteristics of RET- and ROS1-fusion-positive lung adenocarcinomas. We further performed immunohistochemistry and fluorescence in situ hybridization analysis (FISH) in 15 cases of RET and 9 cases of ROS1 fusion tumors by identified NanoString's nCounter screening. RET fusion-positive patients were younger in age, never-smokers, and in early T stage; ROS1 fusion-positive patients had a higher number of never-smokers compared with patients with quintuple-negative (EGFR-/KRAS-/ALK-/ROS1-/RET-) lung adenocarcinoma. Histologically, RET and ROS1 fusion tumors share the solid signet-ring cell and mucinous cribriform pattern, as previously mentioned in the histology of ALK fusion tumors. Therefore, it can be presumed that fusion gene-associated lung adenocarcinomas share similar histologic features. In immunohistochemistry, the majority of 15 RET and 9 ROS1 fusion-positive cases showed positivity of more than moderate intensity and cytoplasmic staining for RET and ROS1 proteins, respectively. In FISH, the majority of RET and ROS1 rearrangement showed two signal patterns such as one fusion signal and two separated green and orange signals (1F1G1O) and an isolated 3' green signal pattern (1F1G). Our study has provided not only characteristics of fusion gene-associated histologic features but also a proposal for a future screening strategy that will enable clinicians to select cases needed to be checked for ROS1 and RET rearrangements based on clinicohistologic features.
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Adenocarcinoma/genética , Reordenamiento Génico , Neoplasias Pulmonares/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Proto-Oncogénicas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Adulto , Factores de Edad , Anciano , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ret/metabolismo , Adulto JovenRESUMEN
RATIONALE: Constitutive activation of the epidermal growth factor receptor (EGFR) is prevalent in epithelial cancers, particularly in non-small cell lung carcinoma (NSCLC). Mutations identified in EGFR predict the sensitivity to EGFR-targeted therapy. Detection of these mutations is mainly based on tissue biopsy, which is invasive, expensive, and time consuming. OBJECTIVES: Noninvasive, real-time, inexpensive detection and monitoring of EGFR mutations in patients with NSCLC is highly desirable. METHODS: We developed a novel core technology, electric field-induced release and measurement (EFIRM), which relies on a multiplexible electrochemical sensor that can detect EGFR mutations directly in bodily fluids. MEASUREMENTS AND MAIN RESULTS: We established EFIRM for the detection of the EGFR mutations in vitro and correlated the results with tumor size from xenografted mice. In clinical application, we demonstrated that EFIRM could detect EGFR mutations in the saliva and plasma of 22 patients with NSCLC. Finally, a blinded test was performed on saliva samples from 40 patients with NSCLC. The receiver operating characteristic analysis indicated that EFIRM detected the exon 19 deletion with an area under the curve of 0.94 and the L858R mutation with an area under the curve of 0.96. CONCLUSIONS: Our data indicate that EFIRM is effective, accurate, rapid, user-friendly, and cost effective for the detection of EGFR mutations in the saliva of patients with NSCLC. We termed this saliva-based EGFR mutation detection (SABER).
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Carcinoma de Pulmón de Células no Pequeñas/genética , Técnicas Electroquímicas , Genes erbB-1/genética , Neoplasias Pulmonares/genética , Mutación/genética , Saliva , Anciano , Animales , Técnicas Biosensibles , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Modelos Animales de Enfermedad , Femenino , Humanos , Neoplasias Pulmonares/diagnóstico , Masculino , Ratones , Persona de Mediana Edad , Sensibilidad y Especificidad , Método Simple CiegoRESUMEN
Cholesteryl ester transfer protein (CETP) shuttles various lipids between lipoproteins, resulting in the net transfer of cholesteryl esters from atheroprotective, high-density lipoproteins (HDL) to atherogenic, lower-density species. Inhibition of CETP raises HDL cholesterol and may potentially be used to treat cardiovascular disease. Here we describe the structure of CETP at 2.2-A resolution, revealing a 60-A-long tunnel filled with two hydrophobic cholesteryl esters and plugged by an amphiphilic phosphatidylcholine at each end. The two tunnel openings are large enough to allow lipid access, which is aided by a flexible helix and possibly also by a mobile flap. The curvature of the concave surface of CETP matches the radius of curvature of HDL particles, and potential conformational changes may occur to accommodate larger lipoprotein particles. Point mutations blocking the middle of the tunnel abolish lipid-transfer activities, suggesting that neutral lipids pass through this continuous tunnel.
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Proteínas de Transferencia de Ésteres de Colesterol/química , Ésteres del Colesterol/química , Modelos Moleculares , Fosfatidilcolinas/química , Triglicéridos/química , Animales , Sitios de Unión , Células CHO , Proteínas de Transferencia de Ésteres de Colesterol/genética , Cricetinae , Cricetulus , Cristalografía por Rayos X , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Mutación Puntual , Unión Proteica , Conformación ProteicaRESUMEN
ALK, ROS1 and RET gene fusions are important predictive biomarkers for tyrosine kinase inhibitors in lung cancer. Currently, the gold standard method for gene fusion detection is Fluorescence In Situ Hybridization (FISH) and while highly sensitive and specific, it is also labour intensive, subjective in analysis, and unable to screen a large numbers of gene fusions. Recent developments in high-throughput transcriptome-based methods may provide a suitable alternative to FISH as they are compatible with multiplexing and diagnostic workflows. However, the concordance between these different methods compared with FISH has not been evaluated. In this study we compared the results from three transcriptome-based platforms (Nanostring Elements, Agena LungFusion panel and ThermoFisher NGS fusion panel) to those obtained from ALK, ROS1 and RET FISH on 51 clinical specimens. Overall agreement of results ranged from 86-96% depending on the platform used. While all platforms were highly sensitive, both the Agena panel and Thermo Fisher NGS fusion panel reported minor fusions that were not detectable by FISH. Our proof-of-principle study illustrates that transcriptome-based analyses are sensitive and robust methods for detecting actionable gene fusions in lung cancer and could provide a robust alternative to FISH testing in the diagnostic setting.
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Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Reordenamiento Génico , Neoplasias Pulmonares/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Quinasa de Linfoma Anaplásico , Línea Celular Tumoral , Humanos , Hibridación Fluorescente in Situ , Proteínas de Fusión Oncogénica/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ret/metabolismoRESUMEN
Variation in CETP has been shown to play an important role in HDL-C levels and cardiovascular disease. To better characterize this variation, the promoter and exonic DNA for CETP was resequenced in 189 individuals with extreme HDL-C or age. Two novel amino acid variants were found in humans (V-12D and Y361C) and an additional variant (R137W) not previously studied in vitro were expressed. D-12 was not secreted and had no detectable activity in cells. C361 and W137 retained near normal amounts of cholesteryl ester transfer activity when purified but were less well secreted than wild type. Torcetrapib, a CETP inhibitor in clinical development with atorvastatin, was found to have a uniform effect on inhibition of wild type CETP versus W137 or C361. In addition, the level of variation in other species was assessed by resequencing DNA from nine cynomolgus monkeys. Numerous intronic and silent SNPs were found as well as two variable amino acids. The amino acid altering SNPs were genotyped in 29 monkeys and not found to be significantly associated with HDL-C levels. Three SNPs found in monkeys were identical to three found in humans with these SNPs all occurring at CpG sites.
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Proteínas Portadoras/genética , Variación Genética , Glicoproteínas/genética , Macaca fascicularis/sangre , Polimorfismo de Nucleótido Simple , Negro o Afroamericano/genética , Anciano de 80 o más Años , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas de Transferencia de Ésteres de Colesterol , HDL-Colesterol/sangre , Ensayos Clínicos como Asunto , Glicoproteínas/antagonistas & inhibidores , Humanos , Datos de Secuencia Molecular , Pan troglodytes , Fenotipo , Quinolinas/farmacología , Alineación de Secuencia , Población Blanca/genéticaRESUMEN
Anaplastic lymphoma kinase (ALK) fusion is a common mechanism underlying pathogenesis of non-small cell lung carcinoma (NSCLC) where these rearrangements represent important diagnostic and therapeutic targets. In this study, we found a new ALK fusion gene, SEC31A-ALK, in lung carcinoma from a 53-year-old Korean man. The conjoined region in the fusion transcript was generated by the fusion of SEC31A exon 21 and ALK exon 20 by genomic rearrangement, which contributed to generation of an intact, in-frame open reading frame. SEC31A-ALK encodes a predicted fusion protein of 1,438 amino acids comprising the WD40 domain of SEC31A at the N-terminus and ALK kinase domain at the C-terminus. Fluorescence in situ hybridization studies suggested that SEC31A-ALK was generated by an unbalanced genomic rearrangement associated with loss of the 3'-end of SEC31A. This is the first report of SEC31A-ALK fusion transcript in clinical NSCLC, which could be a novel diagnostic and therapeutic target for patients with NSCLC.
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Adenocarcinoma/genética , Neoplasias Pulmonares/genética , Fusión de Oncogenes , Proteínas de Fusión Oncogénica/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas de Transporte Vesicular/genética , Adenocarcinoma/tratamiento farmacológico , Quinasa de Linfoma Anaplásico , Exones/genética , Humanos , Hibridación Fluorescente in Situ , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Proteínas de Fusión Oncogénica/química , Proteínas Tirosina Quinasas Receptoras/química , Proteínas de Transporte Vesicular/químicaRESUMEN
REarranged during Transfection (RET) fusion genes are detected in approximately 1% of lung adenocarcinomas and known primarily as oncogenic driver factors. Here, we found a novel RET fusion gene, KIAA1217-RET, and examined the functional differences of RET51 and RET9 protein, fused with KIAA1217 in cancer progression and drug response. KIAA1217-RET, resulting from the rearrangement of chromosome 10, was generated by the fusion of KIAA1217 exon 11 and RET exon 11 from a non-small cell lung cancer patient. Expression of this gene led to increased cell growth and invasive properties through activations of the PI3K/AKT and ERK signaling pathways and subsequently enabled oncogenic transformation of lung cells. We observed that cells expressing KIAA1217-RET9 fusion protein were more sensitive to vandetanib than those expressing KIAA1217-RET51 and both isoforms attenuated cellular growth via cell cycle arrest. These results demonstrated that KIAA1217-RET fusion represents a novel oncogenic driver gene, the products of which are sensitive to vandetanib treatment, and suggested that the KIAA1217-RET-fusion gene is a promising target for lung cancer treatment.
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Adenocarcinoma/genética , Neoplasias Pulmonares/genética , Proteínas de Fusión Oncogénica/genética , Proteínas/genética , Proteínas Proto-Oncogénicas c-ret/genética , Adenocarcinoma/metabolismo , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Células 3T3 NIH , Proteínas de Fusión Oncogénica/metabolismo , Piperidinas/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-ret/metabolismo , Quinazolinas/farmacología , Trasplante HeterólogoRESUMEN
Genetic variation in the human cholesteryl ester transfer protein (CETP) promoter is associated with HDL cholesterol levels and cardiovascular disease with much of the genetic variation in CETP attributed to the promoter region. In this region, there are several single nucleotide polymorphisms as well as a variable length tandem repeat located 1946 base pairs upstream of the CETP transcription start that is highly polymorphic with respect to both length and sequence. There are more than 10 different long alleles and these vary in their repeat structure. We find that the short allele of this repeat is associated with high HDL cholesterol levels in vivo (P<0.0001). In males, this association is independent of the nearby -629 polymorphism. In addition, the variable length GAAA repeat can stimulate an adjacent GGGGA repeat to form a structure that hinders DNA amplification and sequencing. This structure also has an effect in vivo as shown by orientation effects and cloning efficiency in Escherichia coli.
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Proteínas Portadoras/genética , ADN/química , Glicoproteínas/genética , Repeticiones de Minisatélite , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Proteínas Portadoras/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol , HDL-Colesterol/sangre , HDL-Colesterol/química , ADN/metabolismo , Femenino , Glicoproteínas/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Polimorfismo Genético , Alineación de SecuenciaRESUMEN
To better understand the role of cholesteryl ester transfer protein (CETP) in cardiovascular disease, nine polymorphisms spanning the gene from the upstream promoter region to beyond the 3'UTR were genotyped in 2553 individuals from multiple ethnic groups and with different cardiovascular disease profiles. The frequency of four of these SNPs varied by 40-300% between Caucasians and African Americans. SNPs in each ethnic group fell into two haploblocks with significant linkage disequilibrium within each block. SNPs in the 5' haploblock were significantly associated with HDL cholesterol while SNPs in the 3' haploblock were, at best, only weakly associated with HDL-C. One SNP in the 3' haploblock (rs1800774 in intron 12) was highly associated with history of myocardial infarction even though it was not associated with HDL-C. This association was driven by the effect in Caucasian women where 11.9% of the women with no history of MI are homozygous for the less common allele while 23.7% of those with a history of MI share this genotype. In addition, this SNP was highly associated with BMI among Caucasians (p < 0.0001). The association of HDL-C with CETP genotype was found to be independent of smoking or alcohol consumption. These results replicate some earlier findings and also help to explain some of the apparent contradictions in the literature surrounding the role of CETP in modulating HDL-C and cardiovascular disease.
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Enfermedades Cardiovasculares/genética , Proteínas Portadoras/genética , HDL-Colesterol/sangre , Glicoproteínas/genética , Polimorfismo de Nucleótido Simple , Negro o Afroamericano/genética , Índice de Masa Corporal , Proteínas de Transferencia de Ésteres de Colesterol , Femenino , Frecuencia de los Genes , Genotipo , Homocigoto , Humanos , Desequilibrio de Ligamiento , Masculino , Registros Médicos , Persona de Mediana Edad , Infarto del Miocardio/genética , Factores Sexuales , Población Blanca/genéticaRESUMEN
The aim of this study was to determine the distribution of known oncogenic driver mutations in female never-smoker Asian patients with lung adenocarcinoma. We analyzed 214 mutations across 26 lung cancer-associated genes and three fusion genes using the MassARRAY LungCarta Panel and the ALK, ROS1, and RET fusion assays in 198 consecutively resected lung adenocarcinomas from never-smoker females at a single institution. EGFR mutation, which was the most frequent driver gene mutation, was detected in 124 (63%) cases. Mutation of ALK, KRAS, PIK3CA, ERBB2, BRAF, ROS1, and RET genesoccurred in 7%, 4%, 2.5%, 1.5%, 1%, 1%, and 1% of cases, respectively. Thus, 79% of lung adenocarcinomas from never-smoker females harbored well-known oncogenic mutations. Mucinous adenocarcinomas tended to have a lower frequency of known driver gene mutations than other histologic subtypes. EGFR mutation was associated with older age and a predominantly acinar pattern, while ALK rearrangement was associated with younger age and a predominantly solid pattern. Lung cancer in never-smoker Asian females is a distinct entity, with the majority of these cancers developing from oncogenic mutations.
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Adenocarcinoma/genética , Pueblo Asiatico/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutación/genética , Oncogenes/genética , Fumar/genética , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , PronósticoRESUMEN
The cholesteryl ester transfer protein (CETP) gene has been implicated in the variation of HDL levels but most studies have focused on only one or a few genetic variations. In order to properly understand the role of CETP in determining phenotype, it is necessary to examine the entire gene and all its common polymorphisms. The coding regions, adjacent introns, and proximal 5' and 3' regions were resequenced from an ethnically diverse population. Novel and previously known polymorphisms were then characterized and associations with HDL and CETP mass levels determined. The polymorphism most highly associated with CETP was 629 bp upstream of the transcription start site while the polymorphism most highly associated with HDL was a VNTR 1946 bp upstream of the transcription start site. Genetic variation in the CETP gene is associated with protective HDL levels. The ethnic diversity of some SNPs and complex interplay among them dictate careful analysis of the whole gene prior to conclusions about the role of individual polymorphisms.
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Pueblo Asiatico/genética , Proteínas Portadoras/genética , HDL-Colesterol/metabolismo , Predisposición Genética a la Enfermedad , Glicoproteínas , Hipercolesterolemia/etnología , Hipercolesterolemia/genética , Polimorfismo Genético , Población Blanca/genética , Análisis de Varianza , Secuencia de Bases , Proteínas de Transferencia de Ésteres de Colesterol , HDL-Colesterol/sangre , Estudios de Cohortes , Femenino , Frecuencia de los Genes , Marcadores Genéticos/genética , Humanos , Masculino , Repeticiones de Minisatélite , Datos de Secuencia Molecular , Fenotipo , Reacción en Cadena de la Polimerasa/métodos , Probabilidad , Valores de Referencia , Sensibilidad y EspecificidadRESUMEN
INTRODUCTION: The most common mechanism underlying overexpression and activation of anaplastic lymphoma kinase (ALK) in non-small-cell lung carcinoma could be attributed to the formation of a fusion protein. To date, five fusion partners of ALK have been reported, namely, echinoderm microtubule associated protein like 4, tropomyosin-related kinase-fused gene, kinesin family member 5B, kinesin light chain 1, and protein tyrosine phosphatase, nonreceptor type 3. METHODS: In this article, we report a novel fusion gene huntingtin interacting protein 1 (HIP1)-ALK, which is conjoined between the huntingtin-interacting protein 1 gene HIP1 and ALK. Reverse-transcriptase polymerase chain reaction and immunohistochemical analysis were used to detect this fusion gene's transcript and protein expression, respectively. We had amplified the full-length cDNA sequence of this novel fusion gene by using 5'-rapid amplification of cDNA ends. The causative genomic translocation t(2;7)(p23;q11.23) for generating this novel fusion gene was verified by using genomic sequencing. RESULTS: The examined adenocarcinoma showed predominant acinar pattern, and ALK immunostaining was localized to the cytoplasm, with intense staining in the submembrane region. In break-apart, fluorescence in situ hybridization analysis for ALK, split of the 5' and 3' probe signals, and isolated 3' signals were observed. Reverse-transcriptase polymerase chain reaction revealed that the tumor harbored a novel fusion transcript in which exon 21 of HIP1 was fused to exon 20 of ALK in-frame. CONCLUSION: The novel fusion gene and its protein HIP1-ALK harboring epsin N-terminal homology, coiled-coil, juxtamembrane, and kinase domains, which could play a role in carcinogenesis, could become diagnostic and therapeutic target of the lung adenocarcinoma and deserve a further study in the future.
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Adenocarcinoma/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Proteínas de Unión al ADN/genética , Neoplasias Pulmonares/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Tirosina Quinasas Receptoras/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/tratamiento farmacológico , Adulto , Quinasa de Linfoma Anaplásico , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Femenino , Reordenamiento Génico , Humanos , Técnicas para Inmunoenzimas , Hibridación Fluorescente in Situ , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamiento farmacológico , Pronóstico , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Translocación GenéticaRESUMEN
Approximately 7% of non-small cell lung carcinomas (NSCLCs) harbor oncogenic fusions involving ALK, ROS1, and RET. Although tumors harboring ALK fusions are highly sensitive to crizotinib, emerging preclinical and clinical data demonstrate that patients with ROS1 or RET fusions may also benefit from inhibitors targeting these kinases. Using a transcript-based method, we designed a combination of 3' overexpression and fusion-specific detection strategies to detect ALK, ROS1 and RET fusion transcripts in NSCLC tumors. We validated the assay in 295 NSCLC specimens and showed that the assay is highly sensitive and specific. ALK results were 100% concordant with fluorescence in situ hybridization (FISH) (n = 52) and 97.8% concordant with IHC (n = 179) [sensitivity, 96.8% (95% CI 91.0%-98.9%); specificity, 98.8% (95% CI 93.6%-99.8%)]. For ROS1 and RET, we also observed 100% concordance with FISH (n = 46 and n = 15, respectively). We identified seven ROS1 and 14 RET fusion-positive tumors and confirmed the fusion status by RT-PCR and FISH. One RET fusion involved a novel partner, cutlike homeobox 1 gene (CUX1), yielding an in-frame CUX1-RET fusion. ROS1 and RET fusions were significantly enriched in tumors without KRAS/EGFR/ALK alterations. ALK/ROS1/RET/EGFR/KRAS alterations were mutually exclusive. As a single-tube assay, this test shows promise as a more practical and cost-effective screening modality for detecting rare but targetable fusions in NSCLC.
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Pruebas Genéticas/métodos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Quinasa de Linfoma Anaplásico , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/genética , Femenino , Orden Génico , Humanos , Hibridación Fluorescente in Situ , Masculino , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Transcripción Genética , Translocación GenéticaRESUMEN
INTRODUCTION: Anaplastic lymphoma kinase (ALK) fusion is the most common mechanism for overexpression and activation in non-small-cell lung carcinoma. Several fusion partners of ALK have been reported, including echinoderm microtubule-associated protein-like 4, TRK-fused gene, kinesin family member 5B, kinesin light chain 1 (KLC1), protein tyrosine phosphatase and nonreceptor type 3, and huntingtin interacting protein 1 (HIP1). METHODS AND RESULTS: A 60-year-old Korean man had a lung mass which was a poorly differentiated adenocarcinoma with ALK overexpression. By using an Anchored Multiplex polymerase chain reaction assay and sequencing, we found that tumor had a novel translocated promoter region (TPR)-ALK fusion. The fusion transcript was generated from an intact, in-frame fusion of TPR exon 15 and ALK exon 20 (t(1;2)(q31.1;p23)). The TPR-ALK fusion encodes a predicted protein of 1192 amino acids with a coiled-coil domain encoded by the 5'-2 of the TPR and juxtamembrane and kinase domains encoded by the 3'-end of the ALK. CONCLUSIONS: The novel fusion gene and its protein TRP-ALK, harboring coiled-coil and kinase domains, could possess transforming potential and responses to treatment with ALK inhibitors. This case is the first report of TPR-ALK fusion transcript in clinical tumor samples and could provide a novel diagnostic and therapeutic candidate target for patients with cancer, including non-small-cell lung carcinoma.
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Adenocarcinoma/genética , Reordenamiento Génico , Neoplasias Pulmonares/genética , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Translocación Genética/genética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Quinasa de Linfoma Anaplásico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Exones/genética , Humanos , Cinesinas , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , PronósticoRESUMEN
Notch signaling mediates breast cancer cell survival and chemoresistance. In this report, we aimed to evaluate the antitumor efficacy of PF-03084014 in combination with docetaxel in triple-negative breast cancer models. The mechanism of action was investigated. PF-03084014 significantly enhanced the antitumor activity of docetaxel in multiple xenograft models including HCC1599, MDA-MB-231Luc, and AA1077. Docetaxel activated the Notch pathway by increasing the cleaved Notch1 intracellular domain and suppressing the endogenous Notch inhibitor NUMB. PF-03084014 used in combination with docetaxel reversed these effects and demonstrated early-stage synergistic apoptosis. Docetaxel elicited chemoresistance by elevating cytokine release and expression of survivin and induced an endothelial mesenchymal transition (EMT) phenotype by increasing the expressions of Snail, Slug, and N-cadherin. When reimplanted, the docetaxel-residual cells not only became much more tumorigenic, as evidenced by a higher fraction of tumor-initiating cells (TICs), but also showed higher metastatic potential compared with nontreated cells, leading to significantly shortened survival. In contrast, PF-03084014 was able to suppress expression of survivin and MCL1, reduce ABCB1 and ABCC2, upregulate BIM, reverse the EMT phenotype, and diminish the TICs. Additionally, the changes to the ALDH(+) and CD133(+)/CD44(+) subpopulations following therapy corresponded with the TIC self-renewal assay outcome. In summary, PF-03084014 demonstrated synergistic effects with docetaxel through multiple mechanisms. This work provides a strong preclinical rationale for the clinical utility of PF-03084014 to improve taxane therapy.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Antígeno AC133 , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Antígenos CD/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Docetaxel , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Glicoproteínas/metabolismo , Humanos , Receptores de Hialuranos/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones SCID , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas del Tejido Nervioso/metabolismo , Péptidos/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal/efectos de los fármacos , Taxoides/administración & dosificación , Tetrahidronaftalenos/administración & dosificación , Factores de Tiempo , Valina/administración & dosificación , Valina/análogos & derivados , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Evaluating the effects of novel drugs on appropriate tumor models has become crucial for developing more effective therapies that target highly tumorigenic and drug-resistant cancer stem cell (CSC) populations. In this study, we demonstrate that a subset of cancer cells with CSC properties may be enriched into tumor spheroids under stem cell conditions from a non-small cell lung cancer cell line. Treating these CSC-like cells with gemcitabine alone and a combination of gemcitabine and the novel CHK1 inhibitor PF-00477736 revealed that PF-00477736 enhances the anti-proliferative effect of gemcitabine against both the parental and the CSC-like cell populations. However, the CSC-like cells exhibited resistance to gemcitabine-induced apoptosis. Collectively, the spheroid-forming CSC-like cells may serve as a model system for understanding the mechanism underlying the drug resistance of CSCs and for guiding the development of better therapies that can inhibit tumor growth and eradicate CSCs.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos , Células Madre Neoplásicas/efectos de los fármacos , Esferoides Celulares/efectos de los fármacos , Animales , Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Benzodiazepinonas/administración & dosificación , Biomarcadores de Tumor/metabolismo , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Sinergismo Farmacológico , Femenino , Humanos , Ratones , Ratones Desnudos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Pirazoles/administración & dosificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Células Tumorales Cultivadas , GemcitabinaRESUMEN
Figitumumab (CP-751,871), a potent and fully human monoclonal anti-insulin-like growth factor 1 receptor (IGF1R) antibody, has been investigated in clinical trials of several solid tumors. To identify biomarkers of sensitivity and resistance to figitumumab, its in vitro antiproliferative activity was analyzed in a panel of 93 cancer cell lines by combining in vitro screens with extensive molecular profiling of genomic aberrations. Overall response was bimodal and the majority of cell lines were resistant to figitumumab. Nine of 15 sensitive cell lines were derived from colon cancers. Correlations between genomic characteristics of cancer cell lines with figitumumab antiproliferative activity revealed that components of the IGF pathway, including IRS2 (insulin receptor substrate 2) and IGFBP5 (IGF-binding protein 5), played a pivotal role in determining the sensitivity of tumors to single-agent figitumumab. Tissue-specific differences among the top predictive genes highlight the need for tumor-specific patient selection strategies. For the first time, we report that alteration or expression of the MYB oncogene is associated with sensitivity to IGF1R inhibitors. MYB is dysregulated in hematologic and epithelial tumors, and IGF1R inhibition may represent a novel therapeutic opportunity. Although growth inhibitory activity with single-agent figitumumab was relatively rare, nine combinations comprising figitumumab plus chemotherapeutic agents or other targeted agents exhibited properties of synergy. Inhibitors of the ERBB family were frequently synergistic and potential biomarkers of drug synergy were identified. Several biomarkers of antiproliferative activity of figitumumab both alone and in combination with other therapies may inform the design of clinical trials evaluating IGF1R inhibitors.
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Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Receptor IGF Tipo 1/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Análisis por Conglomerados , Variaciones en el Número de Copia de ADN , Sinergismo Farmacológico , Perfilación de la Expresión Génica , Humanos , Concentración 50 Inhibidora , Mutación , Sitios de Carácter Cuantitativo , Receptor IGF Tipo 1/metabolismo , Transducción de SeñalRESUMEN
Anaplastic lymphoma kinase gene (ALK) fusions have been identified in approximately 5% of non-small-cell lung carcinomas (NSCLCs) and define a distinct subpopulation of patients with lung cancer who are highly responsive to ALK kinase inhibitors, such as crizotinib. Because of this profound therapeutic implication, the latest National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology recommend upfront ALK screening for all patients with NSCLC. The Food and Drug Administration-approved companion diagnostic test (ie, fluorescence in situ hybridization) for identification of ALK-positive patients, however, is complex and has considerable limitations in terms of cost and throughput, making it difficult to screen many patients. To explore alternative screening modalities for detecting ALK fusions, we designed a combination of two transcript-based assays to detect for presence or absence of ALK fusions using NanoString's nCounter technology. By using this combined gene expression and ALK fusion detection strategy, we developed a multiplexed assay with a quantitative scoring modality that is highly sensitive, reproducible, and capable of detecting low-abundant ALK fusion transcripts, even in samples with a low tumor cell content. In 66 archival NSCLC samples, our results were highly concordant to prior results obtained by fluorescence in situ hybridization and IHC. Our assay offers a cost-effective, easy-to-perform, high-throughput, and FFPE-compatible screening alternative for detection of ALK fusions.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Expresión Génica , Neoplasias Pulmonares/genética , Fusión de Oncogenes , Proteínas Tirosina Quinasas Receptoras/genética , Quinasa de Linfoma Anaplásico , Secuencia de Bases , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Neoplasias Pulmonares/diagnóstico , Datos de Secuencia Molecular , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Análisis de Secuencia de ADNRESUMEN
Targeting cancers with amplified or abnormally activated c-Met (hepatocyte growth factor receptor) may have therapeutic benefit based on nonclinical and emerging clinical findings. However, the eventual emergence of drug resistant tumors motivates the pre-emptive identification of potential mechanisms of clinical resistance. We rendered a MET amplified gastric cancer cell line, GTL16, resistant to c-Met inhibition with prolonged exposure to a c-Met inhibitor, PF-04217903 (METi). Characterization of surviving cells identified an amplified chromosomal rearrangement between 7q32 and 7q34 which overexpresses a constitutively active SND1-BRAF fusion protein. In the resistant clones, hyperactivation of the downstream MAPK pathway via SND1-BRAF conferred resistance to c-Met receptor tyrosine kinase inhibition. Combination treatment with METi and a RAF inhibitor, PF-04880594 (RAFi) inhibited ERK activation and circumvented resistance to either single agent. Alternatively, treatment with a MEK inhibitor, PD-0325901 (MEKi) alone effectively blocked ERK phosphorylation and inhibited cell growth. Our results suggest that combination of a c-Met tyrosine kinase inhibitor with a BRAF or a MEK inhibitor may be effective in treating resistant tumors that use activated BRAF to escape suppression of c-Met signaling.