# Quantification of the Human Satellite 2 (HSAT2) Repeat in the Plasma Cell-Free DNA of Patients with Colon Cancer

## TL;DR

This study explores how a specific HSAT2 DNA fragment is more common in the blood of colon cancer patients compared to controls.

## Contribution

The first study to use a hybridization capture assay for targeted enrichment of HSAT2 cell-free DNA in plasma.

## Key findings

- 95 bp-HSAT2 was more abundant in colon cancer patients than in controls.
- Hybridization capture assay provided HSAT2 levels comparable to DNA extraction methods.
- Lower 114 bp-HSAT2 to 95 bp-HSAT2 ratios were observed in cancer patients.

## Abstract

Background/Objectives: Liquid profiling of molecular and epigenetic markers in bodily fluids is an expanding field of cancer biomarker research. Recent research activity also reveals the human satellite 2 (HSAT2) repetitive element cell-free DNA (cfDNA) as a potential cancer biomarker. Based on our recent results from targeted sequencing of HSAT2 cfDNA, we tested whether a specific HSAT2 sequence (e.g., 95 bp-HSAT2) shows greater cancer enrichment than 114 bp-SAT2, from which it derives, in patients with colon cancer. Methods: By comparing the ratio of 114 bp-HSAT2 to 95 bp-HSAT2, we investigated the increased cancer enrichment of 95 bp-HSAT2 in cfDNA samples obtained from plasma DNA extraction and a hybridization capture assay, in which HSAT2 sequences were captured from plasma using a biotin-labeled probe, in samples from colon cancer patients (n = 60) and polyp-controls (n = 60), and polyp-free controls (n = 60). Results: A correlation analysis between Ct values from DNA extraction and the hybridization capture assay for both 95 bp- and 114 bp-HSAT2 showed a positive correlation in patients with colon cancer and control subjects, indicating that the hybridization capture assay provides HSAT2 levels comparable to those obtained by DNA extraction. With both approaches, we found a lower 114 bp-HSAT2 to 95 bp-HSAT2 ratio in patients with colon cancer than in the control groups. The median ratio of extracted DNA was 62, 78, and 79 in patients with colon cancer, polyp-controls (p = 0.23), and polyp-free controls (p = 0.067), respectively. Capture assay values were 49, 87, and 64 in patients with colon cancer, polyp controls (p = 0.016), and polyp-free controls (p = 0.19), respectively. Even though statistical significance was not achieved in some comparisons, these results suggest that 95 bp-HSAT2 is more abundant in the blood of patients with colon cancer than 114 bp-HSAT2 in non-malignant patients. Conclusions: To our knowledge, this is the first study to conduct a hybridization capture assay using a biotinylated probe as a feasible approach for targeted enrichment of cfDNA from plasma. Our results confirm the outcomes of our recent article based on targeted sequencing and reveal that some specific HSAT2 sequences may exhibit increased cancer abundance.