RESUMEN

BACKGROUND: Cell-free DNA (cfDNA) analysis using peripheral blood represents an exciting, minimally invasive technology for cancer diagnosis and monitoring. The reliability of testing is dependent on the accuracy and sensitivity of specific molecular analyses to detect tumor-associated genomic variants and on the quantity and quality of cfDNA available for testing. Specific guidelines for standardization and design of appropriate quality programs focused specifically on cfDNA isolation are lacking, as are standardized quality control reagents. CONTENT: This report describes and illustrates quality control and quality assurance processes, supported by generation of in-house quality control material, to ensure the reliability of the preanalytical phase of cfDNA analysis. SUMMARY: We have developed a robust quality program to support high-volume automated cfDNA extraction from peripheral blood by implementing processes and procedures designed to monitor the adequacy of specimen collection, specimen stability, efficiency of cfDNA extraction, and cfDNA quality.

Asunto(s)

Recolección de Muestras de Sangre/normas , ADN Tumoral Circulante/aislamiento & purificación , Servicios de Laboratorio Clínico/normas , Guías como Asunto , Neoplasias/diagnóstico , ADN Tumoral Circulante/genética , Servicios de Laboratorio Clínico/organización & administración , Análisis Mutacional de ADN , Humanos , Mutación , Neoplasias/sangre , Neoplasias/genética , Control de Calidad , Mejoramiento de la Calidad , Reproducibilidad de los Resultados

RESUMEN

Anti-tumor immunity is driven by self versus non-self discrimination. Many immunotherapeutic approaches to cancer have taken advantage of tumor neoantigens derived from somatic mutations. Here, we demonstrate that gene fusions are a source of immunogenic neoantigens that can mediate responses to immunotherapy. We identified an exceptional responder with metastatic head and neck cancer who experienced a complete response to immune checkpoint inhibitor therapy, despite a low mutational load and minimal pre-treatment immune infiltration in the tumor. Using whole-genome sequencing and RNA sequencing, we identified a novel gene fusion and demonstrated that it produces a neoantigen that can specifically elicit a host cytotoxic T cell response. In a cohort of head and neck tumors with low mutation burden, minimal immune infiltration and prevalent gene fusions, we also identified gene fusion-derived neoantigens that generate cytotoxic T cell responses. Finally, analyzing additional datasets of fusion-positive cancers, including checkpoint-inhibitor-treated tumors, we found evidence of immune surveillance resulting in negative selective pressure against gene fusion-derived neoantigens. These findings highlight an important class of tumor-specific antigens and have implications for targeting gene fusion events in cancers that would otherwise be less poised for response to immunotherapy, including cancers with low mutational load and minimal immune infiltration.

Asunto(s)

Antígenos de Neoplasias/genética , Inmunoterapia/métodos , Neoplasias/inmunología , Neoplasias/terapia , Linfocitos T Citotóxicos/inmunología , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/inmunología , Fusión Génica , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/terapia , Humanos , Factores de Transcripción NFI/genética , Factores de Transcripción NFI/inmunología , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/inmunología , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Secuenciación Completa del Genoma

RESUMEN

Double-stranded DNA (dsDNA) in the cytoplasm triggers the production of interleukin 1ß (IL-1ß) as an antiviral host response, and deregulation of the pathways involved can promote inflammatory disease. Here we report a direct cytosolic interaction between the DNA-damage sensor Rad50 and the innate immune system adaptor CARD9. Transfection of dendritic cells with dsDNA or infection of dendritic cells with a DNA virus induced the formation of dsDNA-Rad50-CARD9 signaling complexes for activation of the transcription factor NF-κB and the generation of pro-IL-1ß. Primary cells conditionally deficient in Rad50 or lacking CARD9 consequently exhibited defective DNA-induced production of IL-1ß, and Card9(-/-) mice had impaired inflammatory responses after infection with a DNA virus in vivo. Our results define a cytosolic DNA-recognition pathway for inflammation and a physical and functional connection between a conserved DNA-damage sensor and the innate immune response to pathogens.

Asunto(s)

Proteínas Adaptadoras de Señalización CARD/inmunología , Enzimas Reparadoras del ADN/inmunología , ADN Viral/inmunología , Proteínas de Unión al ADN/inmunología , Interleucina-1beta/biosíntesis , Virus Vaccinia/inmunología , Ácido Anhídrido Hidrolasas , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Proteína 10 de la LLC-Linfoma de Células B , Proteínas Adaptadoras de Señalización CARD/genética , Línea Celular , Citosol/inmunología , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Células Dendríticas/inmunología , Activación Enzimática , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Noqueados , FN-kappa B/inmunología , Transducción de Señal , Receptor Toll-Like 4/biosíntesis , Receptor Toll-Like 9/biosíntesis , Virus Vaccinia/genética

RESUMEN

The DNA damage response (DDR) is activated by oncogenic stress, but the mechanisms by which this occurs, and the particular DDR functions that constitute barriers to tumorigenesis, remain unclear. We established a mouse model of sporadic oncogene-driven breast tumorigenesis in a series of mutant mouse strains with specific DDR deficiencies to reveal a role for the Mre11 complex in the response to oncogene activation. We demonstrate that an Mre11-mediated DDR restrains mammary hyperplasia by effecting an oncogene-induced G2 arrest. Impairment of Mre11 complex functions promotes the progression of mammary hyperplasias into invasive and metastatic breast cancers, which are often associated with secondary inactivation of the Ink4a-Arf (CDKN2a) locus. These findings provide insight into the mechanism of DDR engagement by activated oncogenes and highlight genetic interactions between the DDR and Ink4a-Arf pathways in suppression of oncogene-driven tumorigenesis and metastasis.

Asunto(s)

Neoplasias de la Mama/genética , Carcinogénesis , Proteínas de Unión al ADN/metabolismo , Oncogenes , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Daño del ADN/genética , Proteínas de Unión al ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Hiperplasia/genética , Proteína Homóloga de MRE11 , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Ratones , Metástasis de la Neoplasia/genética