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PURPOSE: Liquid biopsy specimen genomic profiling is integrated in non-small-cell lung cancer (NSCLC) guidelines; however, data on the clinical relevance for ALK /ROS1 alterations are scarce. We evaluated the clinical utility of a targeted amplicon-based assay in a large prospective cohort of patients with ALK/ROS1-positive NSCLC and its impact on outcomes. PATIENTS AND METHODS: Patients with advanced ALK/ROS1-positive NSCLC were prospectively enrolled in the study by researchers at eight French institutions. Plasma samples were analyzed using InVisionFirst-Lung and correlated with clinical outcomes. RESULTS: Of the 128 patients included in the study, 101 were positive for ALK and 27 for ROS1 alterations. Blood samples (N = 405) were collected from 29 patients naïve for treatment with tyrosine kinase inhibitors (TKI) or from 375 patients under treatment, including 105 samples collected at disease progression (PD). Sensitivity was 67% (n = 18 of 27) for ALK/ROS1 fusion detection. Higher detection was observed for ALK fusions at TKI failure (n = 33 of 74; 46%) versus in patients with therapeutic response (n = 12 of 109; 11%). ALK-resistance mutations were detected in 22% patients (n = 16 of 74) overall; 43% of the total ALK-resistance mutations identified occurred after next-generation TKI therapy. ALK G1202R was the most common mutation detected (n = 7 of 16). Heterogeneity of resistance was observed. ROS1 G2032R resistance was detected in 30% (n = 3 of 10). The absence of circulating tumor DNA mutations at TKI failure was associated with prolonged median overall survival (105.7 months). Complex ALK-resistance mutations correlated with poor overall survival (median, 26.9 months v NR for single mutation; P = .003) and progression-free survival to subsequent therapy (median 1.7 v 6.3 months; P = .003). CONCLUSION: Next-generation, targeted, amplicon-based sequencing for liquid biopsy specimen profiling provides clinically relevant detection of ALK/ROS1 fusions in TKI-naïve patients and allows for the identification of resistance mutations in patients treated with TKIs. Liquid biopsy specimens from patients treated with TKIs may affect clinical outcomes and capture heterogeneity of TKI resistance, supporting their role in selecting sequential therapy.
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BACKGROUND: Members of the ZFP36 family of RNA-binding proteins regulate gene expression post-transcriptionally by binding to AU-rich elements in the 3'UTR of mRNA and stimulating mRNA degradation. The proteins within this family target different transcripts in different tissues. In particular, ZFP36 targets myogenic transcripts and may have a role in adult muscle stem cell quiescence. Our study examined the requirement of ZFP36L1 and ZFP36L2 in adult muscle cell fate regulation. METHODS: We generated single and double conditional knockout mice in which Zfp36l1 and/or Zfp36l2 were deleted in Pax7-expressing cells. Immunostained muscle sections were used to analyse resting skeletal muscle, and a cardiotoxin-induced injury model was used to determine the regenerative capacity of muscle. RESULTS: We show that ZFP36L1 and ZFP36L2 proteins are expressed in satellite cells. Mice lacking the two proteins in Pax7-expressing cells have reduced body weight and have reduced skeletal muscle mass. Furthermore, the number of satellite cells is reduced in adult skeletal muscle and the capacity of this muscle to regenerate following muscle injury is diminished. CONCLUSION: ZFP36L1 and ZFP36L2 act redundantly in myogenesis. These findings add further intricacy to the regulation of the cell fate of Pax7-expressing cells in skeletal muscle by RNA-binding proteins.
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Desarrollo de Músculos , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Células Satélite del Músculo Esquelético/metabolismo , Tristetraprolina/metabolismo , Animales , Factor 1 de Respuesta al Butirato , Diferenciación Celular , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/citología , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Proteínas Nucleares/genética , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Células Satélite del Músculo Esquelético/citología , Tristetraprolina/genéticaRESUMEN
Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications.
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Biopsia Líquida/métodos , Análisis de Secuencia de ADN/métodos , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/genética , ADN Tumoral Circulante/sangre , Estudios de Cohortes , Humanos , Mutación , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
INTRODUCTION: Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes. MATERIALS AND METHODS: We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material. RESULTS: The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA. CONCLUSIONS: These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine.
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Biomarcadores de Tumor/análisis , Ácidos Nucleicos Libres de Células/análisis , Análisis Mutacional de ADN/métodos , Mutación , Neoplasias/diagnóstico , Células Neoplásicas Circulantes/patología , Adulto , Alelos , Biomarcadores de Tumor/genética , ADN Tumoral Circulante/análisis , ADN de Neoplasias/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Biopsia Líquida/métodos , Masculino , Células Neoplásicas Circulantes/química , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Individual human epidermal cells differ in their self-renewal ability. To uncover the molecular basis for this heterogeneity, we performed genome-wide pooled RNA interference screens and identified genes conferring a clonal growth advantage on normal and neoplastic (cutaneous squamous cell carcinoma, cSCC) human epidermal cells. The Hippo effector YAP was amongst the top positive growth regulators in both screens. By integrating the Hippo network interactome with our data sets, we identify WW-binding protein 2 (WBP2) as an important co-factor of YAP that enhances YAP/TEAD-mediated gene transcription. YAP and WPB2 are upregulated in actively proliferating cells of mouse and human epidermis and cSCC, and downregulated during terminal differentiation. WBP2 deletion in mouse skin results in reduced proliferation in neonatal and wounded adult epidermis. In reconstituted epidermis YAP/WBP2 activity is controlled by intercellular adhesion rather than canonical Hippo signalling. We propose that defective intercellular adhesion contributes to uncontrolled cSCC growth by preventing inhibition of YAP/WBP2.
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Proteínas Adaptadoras Transductoras de Señales/genética , Proliferación Celular/genética , Proteínas Nucleares/genética , Células Madre/metabolismo , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular , Línea Celular Tumoral , Células Cultivadas , Células Epidérmicas , Femenino , Regulación de la Expresión Génica , Humanos , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Proteínas Nucleares/metabolismo , Células Madre/citología , Transactivadores , Factores de Transcripción/metabolismoRESUMEN
Metastatic colonization of distant organs underpins the majority of human-cancer-related deaths, including deaths from head and neck squamous cell carcinoma (HNSCC). We report that miR-203, a miRNA that triggers differentiation in multilayered epithelia, inhibits multiple postextravasation events during HNSCC lung metastasis. Inducible reactivation of miR-203 in already established lung metastases reduces the overall metastatic burden. Using an integrated approach, we reveal that miR-203 inhibits metastasis independently of its effects on differentiation. In vivo genetic reconstitution experiments show that miR-203 inhibits lung metastasis by suppressing the prometastatic activities of three factors involved in cytoskeletal dynamics (LASP1), extracellular matrix remodeling (SPARC), and cell metabolism (NUAK1). Expression of miR-203 and its downstream effectors correlates with HNSCC overall survival outcomes, indicating the therapeutic potential of targeting this signaling axis.
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Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , MicroARNs/genética , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Animales , Carcinoma de Células Escamosas/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proteínas del Citoesqueleto/antagonistas & inhibidores , Neoplasias de Cabeza y Cuello/metabolismo , Xenoinjertos , Humanos , Proteínas con Dominio LIM/antagonistas & inhibidores , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Metástasis de la Neoplasia , Osteonectina/antagonistas & inhibidores , Pronóstico , Proteínas Quinasas , Proteínas Represoras/antagonistas & inhibidores , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
Stem cell function is essential for the maintenance of adult tissue homeostasis. Controlling the balance between self-renewal and differentiation is crucial to maintain a receptive satellite cell pool capable of responding to growth and regeneration cues. The mitogen-activated protein kinase p38α has been implicated in the regulation of these processes but its influence in adult muscle remains unknown. Using conditional satellite cell p38α knockout mice we have demonstrated that p38α restricts excess proliferation in the postnatal growth phase while promoting timely myoblast differentiation. Differentiation was still able to occur in the p38α-null satellite cells, however, but was delayed. An absence of p38α resulted in a postnatal growth defect along with the persistence of an increased reservoir of satellite cells into adulthood. This population was still capable of responding to cardiotoxin-induced injury, resulting in complete, albeit delayed, regeneration, with further enhancement of the satellite cell population. Increased p38γ phosphorylation accompanied the absence of p38α, and inhibition of p38γ ex vivo substantially decreased the myogenic defect. We have used genome-wide transcriptome analysis to characterize the changes in expression that occur between resting and regenerating muscle, and the influence p38α has on these expression profiles. This study provides novel evidence for the fundamental role of p38α in adult muscle homeostasis in vivo.
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Células Madre Adultas/química , Células Madre Adultas/enzimología , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/enzimología , Animales , Animales Recién Nacidos , Procesos de Crecimiento Celular/fisiología , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Músculos/lesiones , Músculos/fisiología , Fosforilación , Regeneración/fisiologíaRESUMEN
Tissue generation and repair requires a stepwise process of cell fate restriction to ensure that adult stem cells differentiate in a timely and appropriate manner. A crucial role has been implicated for Polycomb-group (PcG) proteins and the H3K27me3 repressive histone mark in coordinating the transcriptional programmes necessary for this process, but the targets and developmental timing for this repression remain unclear. To address these questions, we generated novel genome-wide maps of H3K27me3 and H3K4me3 in freshly isolated muscle stem cells. These data, together with the analysis of two conditional Ezh2-null mouse strains, identified a critical proliferation phase in which Ezh2 activity is essential. Mice lacking Ezh2 in satellite cells exhibited decreased muscle growth, severely impaired regeneration and reduced stem cell number, due to a profound failure of the proliferative progenitor population to expand. Surprisingly, deletion of Ezh2 after the onset of terminal differentiation did not impede muscle repair or homeostasis. Using these knockout models and the RNA-Seq and ChIP-Seq datasets, we show that Ezh2 does not regulate the muscle differentiation process in vivo. These results emphasise the lineage and cell-type-specific functions of Ezh2 and Polycomb repressive complex 2.