Nair Madhumathy G, Ramesh Rakesh S, Naidu Chandrakala M, Mavatkar Apoorva D, V P Snijesh, Ramamurthy Vishakha, Somashekaraiah Vidya M, C E Anupama, Raghunathan Kiruthiga, Panigrahi Anuradha, Das Manjula, Dhar Sujan K, Prabhu Jyothi S
Division of Molecular Medicine, St. John's Research Institute, St. John's Medical College, Bangalore 560034, India.
Department of Surgical Oncology, St. John's Medical College and Hospital, Bangalore 560034, India.
Cancers (Basel). 2023 Feb 7;15(4):1054. doi: 10.3390/cancers15041054.
Liquid biopsy is widely recognized as an efficient diagnostic method in oncology for disease detection and monitoring. Though the examination of circulating tumor cells (CTC) is mostly implemented for the assessment of genomic aberrations, the need of complex methodologies for their detection has impeded its acceptance in low-resource settings. We evaluated cell-free DNA (cfDNA) as a liquid biopsy tool and investigated its utility in breast cancer patients.
Total cell-free DNA was extracted from the plasma of breast cancer patients (n = 167) with a median follow-up of more than 5 years, at various stages of the disease. Quantitative PCR was performed to estimate the copy numbers of two fractions of ALU repetitive elements (ALU 115 and ALU 247), and DNA integrity (DI) was calculated as the ratio of ALU 247/115. Mutations in TP53 and PIK3CA in the cfDNA were estimated by next-gen sequencing (NGS) in a subset of samples. Associations of the levels of both the ALU fragments with various clinico-pathological factors and disease-free survival at various stages were examined. Nomogram models were constructed with clinical variables and ALU 247 levels to predict disease-free survival and the best performing model was evaluated by decision curve analysis.
DI and ALU 247 levels were significantly lower ( < 0.0001) in the post-operative plasma when compared to their pre-surgery levels. DI and ALU 247 were found to be significantly higher in patients with metastasis ( < 0.05). Patients with higher levels of ALU 247 in their post-operative plasma had significant poor disease-free survival ( = 0.005). Higher levels of ALU 247 in the circulation also correlated with low tumor-infiltrating lymphocytes (TIL) within their primary tumors in the ER-negative breast cancer subtype ( = 0.01). Cox proportional hazard analysis confirmed ALU 247 as an independent variable of disease-free survival both in univariate and multivariate analysis [HR 1.3 (95% CI 1.047 to 1.613, = 0.017)]. The nomogram model showed that the addition of ALU 247 with other variables significantly improved (C-index 0.823) the predictive ability of the model.
Our results confirm the utility of cfDNA as an evolving liquid biopsy tool for molecular analysis. Evaluation of larger fragments of cfDNA estimated through ALU 247 can provide vital information concurrent with the pathological process of disease evolution in breast cancer and warrants expansion to other cancer types.
液体活检作为肿瘤学中疾病检测和监测的一种有效诊断方法已得到广泛认可。尽管循环肿瘤细胞（CTC）检测主要用于评估基因组畸变，但其检测所需的复杂方法阻碍了其在资源匮乏地区的应用。我们评估了游离DNA（cfDNA）作为一种液体活检工具，并研究了其在乳腺癌患者中的效用。
从167例乳腺癌患者的血浆中提取总游离DNA，这些患者疾病处于不同阶段，中位随访时间超过5年。进行定量PCR以估计ALU重复元件的两个片段（ALU 115和ALU 247）的拷贝数，并将DNA完整性（DI）计算为ALU 247/115的比值。在一部分样本中通过下一代测序（NGS）估计cfDNA中TP53和PIK3CA的突变。检查了两个ALU片段水平与各种临床病理因素以及不同阶段无病生存期的相关性。构建了包含临床变量和ALU 247水平的列线图模型以预测无病生存期，并通过决策曲线分析评估了最佳表现模型。
与术前水平相比，术后血浆中的DI和ALU 247水平显著降低（<0.0001）。发现转移患者的DI和ALU 247显著更高（<0.05）。术后血浆中ALU 247水平较高的患者无病生存期明显较差（=0.005）。循环中较高水平的ALU 247也与雌激素受体阴性乳腺癌亚型原发肿瘤内低肿瘤浸润淋巴细胞（TIL）相关（=0.01）。Cox比例风险分析在单变量和多变量分析中均证实ALU 247是无病生存期的独立变量[风险比1.3（95%置信区间1.047至1.613，=0.017）]。列线图模型显示，将ALU 247与其他变量相加显著提高了模型的预测能力（C指数0.823）。
我们的结果证实了cfDNA作为一种不断发展的用于分子分析的液体活检工具的效用。通过ALU 247估计的cfDNA较大片段的评估可以提供与乳腺癌疾病演变的病理过程同时发生的重要信息，并值得扩展到其他癌症类型。
