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.
Bibliographical noteFunding Information:
The authors thank Solange Paredes Moscoso and Claudio Villegas Llerena from the Centro de Genética y Biología Molecular (USMP), as well as all the participants for their valuable contributions to this study. This study was supported by the Programa Nacional de Innovación para la Competitividad y Productividad (Innóvate Perú), under the contract 138‐PNICP‐PIAP‐2015, Universidad de San Martín de Porres (Project E10012015038), and Oncosalud–AUNA.
- breast cancer
- cell-free DNA
- digital PCR
- liquid biopsy
- ultrasensitive detection method