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Liquid biopsies are revolutionizing the detection and management of malignant diseases. While repetitive DNA sequences, such as LINE-1 and ALU are established in cell-free DNA (cfDNA) research, their clinical applications remain limited. In this study, we explore human satellite 2 (HSATII), a prevalent repeat DNA sequence in plasma that exhibits increased levels in cancer patients, thereby positioning it as a potential pan-cancer biomarker. We employed targeted sequencing and copy number variation (CNV) analysis using two primer pairs to assess the differential abundance of HSATII sequences in the plasma of breast cancer patients compared to healthy individuals. PCR amplicons of HSATII from 10 patients and 10 control subjects were sequenced, generating 151 bp paired-end reads. By constructing a pooled reference dataset, HSATII copy ratios were estimated in the patients. Our analysis revealed several significant CNVs in HSATII, with certain sequences displaying notable gains and losses across all breast cancer patients, suggesting their potential as biomarkers. However, we observed pronounced fragmentation of cfDNA in cancer, leading to the loss of longer PCR amplicons (>180 bp). While not all observed losses can be attributed to fragmentation artifacts, this phenomenon does introduce complexity in interpreting CNV data. Notably, this research marks the first instance of targeted HSATII sequencing in a liquid biopsy context. Our findings lay the groundwork for developing sequencing-based assays to detect differentially represented HSATII sequences, potentially advancing the field of minimally-invasive cancer screening. [ABSTRACT FROM AUTHOR]

Tumor marker determinations are valuable tools for the guidance of breast cancer patients during the course of disease. They are assessed on diverse analytical platforms that may be associated with differences according to the methods applied and the clinical performance. To investigate the method dependency and clinical significance of breast cancer protein tumor markers, CEA, CA 15-3, CA 125, CA 19-9 and AFP were measured in a total of 154 biobanked samples from 77 patients with breast cancer, 10 with DCIS, 31 with benign breast diseases and 36 healthy controls using a Millipore multiplex biomarker panel (MP) and an automized version of the routinely used Vista LOCI technology. The markers were compared between methods and investigated for diagnostic performance. CEA, CA 15-3 and AFP showed good correlations between both platforms with correlation coefficients of R = 0.85, 0.85 and 0.92, respectively, in all samples, but similarly also in the various subgroups. CA 125 and CA 19-9 showed only moderate correlations (R = 0.71 and 0.56, respectively). Absolute values were significantly higher for CEA, CA 15-3, CA 125 and AFP in the Vista LOCI as compared with the MP method and vice versa for CA 19-9. The diagnostic performance for discrimination of breast cancer from healthy controls was similar for both methods with AUCs in ROC curves for CEA (MP 0.81, 95% CI 0.72–0.91; LOCI 0.81; 95% CI 0.72–0.91) and CA-15-3 (MP 0.75, 95% CI 0.65–0.86; LOCI 0.67, 95% CI 0.54–0.79). Similar results were obtained for the comparison of breast cancer with benign breast diseases regarding CEA (AUC MP 0.62, 95% CI 0.51–0.73; LOCI 0.64, 95% CI 0.53–0.74) and CA-15-3 (MP 0.70, 95% CI 0.6–0.81; LOCI 0.66, 95% CI 0.54–0.77). Both platforms show moderate to good method comparability for tumor markers with similar clinical performance. However, absolute levels in individual patients should be interpreted with care. [ABSTRACT FROM AUTHOR]

Breast cancer is the most common cancer affecting women worldwide. It is a malignant and heterogeneous disease with distinct molecular subtypes, which has prognostic and predictive implications. Circulating tumor DNA (ctDNA), cell-free fragmented tumor-derived DNA in blood plasma, is an invaluable source of specific cancer-associated mutations and holds great promise for the development of minimally invasive diagnostic tests. Furthermore, serial monitoring of ctDNA over the course of systemic and targeted therapies not only allows unparalleled efficacy assessments but also enables the identification of patients who are at risk of progression or recurrence. Droplet digital PCR (ddPCR) is a powerful technique for the detection and monitoring of ctDNA. Due to its relatively high accuracy, sensitivity, reproducibility, and capacity for absolute quantification, it is increasingly used as a tool for managing cancer patients through liquid biopsies. In this review paper, we gauge the clinical utility of ddPCR as a technique for mutational profiling in breast cancer patients and focus on HER2, PIK3CA, ESR1, and TP53, which represent the most frequently mutated genes in breast cancers. [ABSTRACT FROM AUTHOR]