Determination of Breast Cancer Dormancy: Analysis of Circulating Free DNA Using SNP 6.0 Arrays

Abstract

New biomarkers are needed in breast cancer to monitor minimal residual disease. Specific point mutations, promoter methylation and loss of heterozygosity have been demonstrated previously in paired tumor and circulating cell-free DNA (cfDNA) isolated from plasma. Moreover, acquired alterations unique to cfDNA have also been found, suggesting disease progression. This prompted us to characterize the “circulating” breast cancer genome, thus testing the hypothesis that cfDNA acts as a surrogate liquid biopsy of breast cancer. This was achieved using Affymetrix SNP 6.0 technology and bioinformatics to map single nucleotide polymorphism (SNP) and copy number variation (CNV), comparing cfDNA with matched normal leucocyte and primary tumor DNA in breast cancer patients and paired normal leucocytes and cfDNA in healthy female controls. Our results show that concordance of SNP genotype calls in paired leucocytes and cfDNA can distinguish between primary breast cancer patients and healthy controls (p < 0.0001), and between pre-surgical breast cancer patients and patients on follow-up after surgery and treatment (p = 0.0016). In 50 patients on follow-up, a significant difference (p = 0.0006) was seen between cfDNA samples taken an average of 3 years apart, suggesting disease progression. Considering CNVs, amplification was observed in matched tumor and cfDNA at multiple loci on different chromosome arms but was quiet or absent in normal DNA. Many of these tumor-specific CNVs contributed significantly to disease through binary logistic regression analysis. Furthermore these CNVs remained detectable in cfDNA up to 12 years after diagnosis and treatment despite no other evidence of disease. Taken together these cfDNA results suggest breast cancer dormancy in the majority of the patients on follow-up.