Breast cancer (BC) is the most prevalent cancer worldwide, and mortality remains high in low-income countries due to few options for early detection. For individuals with dense breast tissue, the current method for early BC detection involves mammography plus ultrasound-assisted core needle biopsy for BC diagnosi s and staging. However, mammography has low sensitivity with a high number of false negatives for dense breast tissue and needle biopsies may miss small lesions. A noninvasive information-rich method that could more accurately diagnose cancer and optimize therapy could avoid these drawbacks. Prior studies of circulating tumor DNA (ctDNA), small nucleic acid fragments of 134–144bp, have shown high concordance between the ctDNA molecular profile and traditional tumor tissue testing methods. Next-generation sequencing (NGS) has simplified and improved the speed for identifying and testing ctDNA genomic alterations, which could improve patient outcomes.

This study investigated what molecular markers might be associated with breast cancer in 32 patients that were sorted into four groups: mammography positive/needle biopsy negative diagnosis (pos-neg), positive for both mammography and needle biopsy (pos-pos) plus segmenting the positive breast cancer diagnosis into Class I (pos-pos I) and Class II (pos-pos II). Plasma samples from individuals were taken after the initial mammography and sorted into various groups after the needle biopsy results. Tumor-derived DNA levels was estimated before the microbiopsy but not found to be statically significant among the four groups after biopsy results, but there was a larger mean tumor DNA was present in the pos-pos stage II individuals.

Next-generation sequencing and data analysis of ctDNA were used to determine which copy number alterations (CNA) single nucleotide polymorphisms (SNPs) and insertions/deletions (Indels) were present and potentially associated with the diagnostic groupings. The results were further analyzed for cancer genes, coding and noncoding variants. The pos-neg individuals had deletion and gain CNAs that overlapped across 11 known oncogenes identified from other cancers plus three deletion CNAs of associated with three breast cancer oncogenes. For pos-pos individuals, five gain CNAs were found in five different breast-cancer associated oncogenes.

The pos-pos group also had 10 SNP and indel variants that were associated with cancer, including one that is a known breast cancer risk factor located in SERAC1, which plays a role in splicing. Another variant occurred in F-box protein FBXW4. Decreased expression of FBXW4 correlated with poor prognosis for patients with other cancer types. Another pos-pos variant was identified in telomere function, one identified with a role in inflammation while the remaining variants were associated with known breast cancer risk genes. For the pos-neg group, there were six oncogene SNP and Indel variants identified that are associated with breast cancer, including a calcium-dependent protein kinase. While this study had limitations including a small sample size, no normal tissue testing from each subject and no healthy control individuals, researchers identified some CNAs, SNPs and Indels associated exclusively with either the pos-neg or pos-pos groups that look promising as a potential noninvasive early test for breast cancer.

Keywords: cell-free DNA, breast cancer, copy number alterations, circulating tumor DNA, tumor fraction, minor allele frequency, multiple nucleotide polymorphisms

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