用于乳腺癌早期检测的ctDNA来源DNA回文序列全基因组分析

Background and Objective: Circulating tumor DNA (ctDNA) in the blood provides valuable information about all aspects of patient care, including real-time tumor burden and therapeutic targets. While ctDNA tests currently focus on detecting small mutations and abnormal DNA methylations in targetedgenomic regions, structural variants (SVs) are also common in tumors and could serve as important cancer biomarkers. Despite the growing use of liquid biopsy in cancer detection and management, an effective agnostic SV detection method remains a missing piece. To address this, we target DNA palindromes, a chromosomal structural abnormality also known as fold-back inversions and inverted repeats. We explored the potential of an approach called GAPF-Seq (Genome-wide Analysis of Palindrome Formation: GAPF with NGS) for ctDNA detection. GAPF-seq enriches palindromic DNA from very small amounts of genomic DNAthrough intramolecular annealing (Tanaka et al., Nat Genet 2005). Next-generation sequencing (NGS) analysis of the enriched DNA would enable us to identify SVs across the genome. Methods: Breast tumor DNA was processed by the successive denaturation and renaturation. DNA from palindromes would form double-stranded DNA by intramolecular annealing, while normal and nonpalindromic DNA would remain single-stranded, which would be eliminated by S1 nuclease. Tumor-derived DNA palindromes were amplified by PCR, sequenced by NGS, and subjected to bioinformatic analysis, including ROC validation and chromosomal distribution profiling. Results: (1) ROC analysis demonstrated high diagnostic accuracy. Using the top 1000 high coverage bins(HCBs) among the 1 million 1-kb bins in the genome, we found that the AUC value for tumor DNA calling was 0.9885, with sensitivity 92.3% and specificity 97.4%, enabling robust distinction between cancer and normal samples, including Stage I tumors. Comparable performance was achieved between 30 ng and 100 ng of DNA.(2) As GAPF-Seq is based on Structural variant detection genome-wide, it allows comprehensive genome-wide screening beyond single-gene mutation or a subset of CpG methylation analyses. Chromosomal mapping revealed uniform distribution of top 1000 HCBs in normal samples, while tumor samples exhibited chromosome-specific enrichment of HCBs. Subtype-specific analysis showed accumulation around the CCND1 gene on chromosome 11 in Luminal type, and enrichment near the ERBB2 gene on chromosome 17 in HER2 type. Conclusion: GAPF-Seq has the potential to enable accurate detection of early breast cancer even from minimal DNA input. Its agnostic, genome-wide profiling capability provides an additional benefit. Potential clinical applications include cancer screening and monitoring minimal residual disease.