Estimation of ALU Repetitive Elements in Plasma as a Cost-Effective Liquid Biopsy Tool for Disease Prognosis in Breast Cancer

Abstract

Background: 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. Methods: 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. Results: DI and ALU 247 levels were significantly lower (p < 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 (p < 0.05). Patients with higher levels of ALU 247 in their post-operative plasma had significant poor disease-free survival (p = 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 (p = 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, p = 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. Conclusion: 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.

A Case–Control Study by ddPCR of ALU 260/111 and LINE-1 266/97 Copy Number Ratio in Circulating Cell-Free DNA in Plasma Revealed LINE-1 266/97 as a Potential Biomarker for Early Breast Cancer Detection

“…Of note, all studies with ALU 260/111 or LINE-1 266/97 observed a decrease in cfDI in cancer patients versus healthy controls [7,10,19], as we previously noted [6]. On the contrary, other targets such as ALU 245/115 [15,18], ALU 247/60 [20], and LINE-1 259/97 [17] showed an increase in the cfDI of cancer patients versus healthy controls. Thus, the difference seems to be related to the target, although the explanation deserves further investigation.…”

“…In 50% of human cancers, an increase in LINE-1 transposable activity is observed, and their insertion in tumor suppression genes or their methylation status has been related to malignancy [8,12]. In this respect, many studies targeted ALU and LINE-1 repeats in cfDNA of BC patients to determine cfDI by quantitative PCR [7,[13][14][15][16][17][18]. In BC patients, the majority of the studies targeted LINE-1 266 bp and 97 bp [7,14,16]; only Miao et al targeted LINE-1 259 bp and 97 bp [17].…”

“…In BC patients, the majority of the studies targeted LINE-1 266 bp and 97 bp [7,14,16]; only Miao et al targeted LINE-1 259 bp and 97 bp [17]. The targeting of ALU sequences in cfDNA of BC patients was ALU 245 bp and 115 bp [15,18] or ALU 260 bp and 111 bp [7,13,14,16]. In the plasma cfDNA of patients with early BC diagnosis or healthy control women study groups, we targeted ALU 260 bp and 111 bp and LINE-1 266 bp and 97 bp copy number by digital droplet PCR.…”

Circulating Tumor DNA in Human Papillomavirus–Mediated Oropharynx Cancer

“…De Sarkar et al 77 demonstrated that specific prostate cancer ctDNA profiles had nucleosome patterns associated with transcriptional activity in key regions. Studies in breast cancer have shown that PIK3CA mutations, high DNA integrity, and ALU 247 repeat elements were associated with lower levels of tumor-infiltrating lymphocytes, as well as poor outcomes 78,79 . Furthermore, variable posttreatment PIK3CA levels may be explained by intratumoral heterogeneity in advanced breast cancer 80 .…”

“…Studies in breast cancer have shown that PIK3CA mutations, high DNA integrity, and ALU 247 repeat elements were associated with lower levels of tumor-infiltrating lymphocytes, as well as poor outcomes. 78,79 Furthermore, variable posttreatment PIK3CA levels may be explained by intratumoral heterogeneity in advanced breast cancer. 80 In summary, these data suggest promise for using ctDNA assays to reveal information regarding tumor phenotypes and microenvironment; however, further studies are needed in HPV-mediated oropharyngeal cancer.…”