RESUMEN
BACKGROUND: Cell-free DNA (cfDNA) is used in clinical research to identify biomarkers for diagnosis of and follow-up on cancer. Here, we propose a fast and innovative approach using traditional housekeeping genes as cfDNA targets in a copy number analysis. We focus on the application of highly sensitive technology such as digital PCR (dPCR) to differentiate breast cancer (BC) patients and controls by quantifying regions of PUM1 and RPPH1 (RNase P) in plasma samples. METHODS: We conducted a case-control study with 82 BC patients and 82 healthy women. cfDNA was isolated from plasma using magnetic beads and quantified by spectrophotometry to estimate total cfDNA. Then, both PUM1 and RPPH1 genes were specifically quantified by dPCR. Data analysis was calibrated using a reference genomic DNA in different concentrations. RESULTS: We found RNase P and PUM1 values were correlated in the patient group (intraclass correlation coefficient [ICC] = 0.842), but they did not have any correlation in healthy women (ICC = 0.519). In dPCR quantification, PUM1 showed the capacity to distinguish early-stage patients and controls with good specificity (98.67%) and sensitivity (100%). Conversely, RNase P had lower cfDNA levels in triple-negative BC patients than luminal subtypes (p < 0.025 for both), confirming their utility for patient classification. CONCLUSION: We propose the PUM1 gene as a cfDNA marker for early diagnosis of BC and RNase P as a cfDNA marker related to hormonal status and subtype classification in BC. Further studies with larger sample sizes are warranted.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/genética , Proteínas de Unión al ARN/genética , Ribonucleasa P/genética , Biomarcadores de Tumor/sangre , Neoplasias de la Mama/sangre , Estudios de Casos y Controles , Estrógenos/metabolismo , Femenino , Fluorescencia , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Proteínas de Unión al ARN/metabolismo , Curva ROC , Ribonucleasa P/metabolismo , Sensibilidad y Especificidad , Neoplasias de la Mama Triple Negativas/sangre , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
BACKGROUND: The detection of circulating DNA is considered a promising strategy in cancer patients. Digital PCR has emerged as a sensitive method able to quantify both circulating free and tumour DNA. AIM: The aim of this study was to prospectively evaluate the clinical value of a chip-based digital PCR for the detection of circulating DNA. METHODS: Digital PCR was used in 34 metastatic colorectal cancer patients to detect and quantify circulating free and tumour DNA based on K-ras mutational status. Clinical outcomes were analyzed according to circulating DNA measurements. RESULTS: Digital PCR yielded a detection rate of 69% for circulating tumour DNA. The median concentrations of circulating free and tumour DNA were 20 and 6.8 ng/mL, respectively, with significant correlation between both biomarkers (p<0.001). Median overall survival was 4.8 months in patients with high circulating free DNA (>75% quartile) versus not reached in patients with a low level (<25% quartile) (p=0.029). Moreover, median overall survival was significantly decreased in patients with detectable circulating tumour DNA compared to those without (respectively 11.8 months versus not reached, p=0.04). CONCLUSIONS: Chip-based digital PCR is a simple and non-invasive method allowing the efficient detection of circulating DNA. Our results highlight that levels of these circulating markers may have a potential prognostic value.