Facultad de Medicina Humana, Centro de Investigación de Genética y Biología Molecular, Instituto de Investigación, Universidad de San Martín de Porres, Lima, Perú.
Facultad de Farmacia y Bioquímica, Universidad Nacional Mayor de San Marcos, Lima, Perú.
J Clin Lab Anal. 2021 Apr;35(4):e23720. doi: 10.1002/jcla.23720. Epub 2021 Feb 1.
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.
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.
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.
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.
无细胞 DNA（cfDNA）用于临床研究以鉴定癌症的诊断和随访生物标志物。在这里，我们提出了一种快速创新的方法，使用传统管家基因作为 cfDNA 靶标进行拷贝数分析。我们专注于使用高灵敏度技术，如数字 PCR（dPCR），通过定量血浆样本中的 PUM1 和 RPPH1（RNase P）区域来区分乳腺癌（BC）患者和对照。
我们进行了一项病例对照研究，纳入了 82 名 BC 患者和 82 名健康女性。使用磁性珠从血浆中分离 cfDNA，并通过分光光度法定量以估计总 cfDNA。然后，通过 dPCR 特异性定量 both PUM1 和 RPPH1 基因。使用不同浓度的参考基因组 DNA 校准数据分析。
我们发现患者组中 RNase P 和 PUM1 值相关（组内相关系数 [ICC] = 0.842），但在健康女性中没有相关性（ICC = 0.519）。在 dPCR 定量中，PUM1 具有区分早期患者和对照的良好特异性（98.67%）和敏感性（100%）的能力。相反，RNase P 在三阴性 BC 患者中的 cfDNA 水平较低，与腔面亚型相比（两者均 p < 0.025），证实了它们在患者分类中的应用。
我们提出 PUM1 基因作为 BC 早期诊断的 cfDNA 标志物，RNase P 作为与激素状态和 BC 亚型分类相关的 cfDNA 标志物。需要进一步进行更大样本量的研究。
