Circulating cell-free DNA (cfDNA) levels in BRCA1 and BRCA2 mutation carriers: A preliminary study

1. Introduction

Circulating cell-free DNA (cfDNA) levels originating from healthy or tumorous tissues [1, 2] are reportedly increased in several clinical scenarios: cancer, inflammation, autoimmune disease smoking, pregnancy and even intense exercise [3, 4]. Increased amounts of cfDNA collectively referred to as “liquid biopsy” are appealing as a possible additional diagnostic tool in cancer [5, 6, 7]. Indeed, several studies have examined the potential of liquid biopsy for early detection of breast cancer [8, 9], and in assessing recurrence risk [10]. Madhavan et al., reported the ability to apply a specific assay of cfDNA level determination for early detection of breast cancer in a cohort of 383 clinically distinct breast cancer cases [11]. Several other studies [12, 13, 14] and a meta-analysis [15] focused on the potential use of cfDNA as a biomarker in ovarian cancer diagnosis. Kamat et al. [13, 14] reported that cfDNA levels in advanced ovarian cancer samples were elevated compared with controls. Yet, No and coworkers [12] detected no significant difference between cfDNA levels of ovarian cancer patients and patients with benign disease. While most studies focused on the utility of liquid biopsy and cfDNA quantification in cancer affected individuals (reviewed by Bronkhorst et al. [16]), there is a paucity of data on cfDNA analysis in high risk BRCA germline mutation carriers. Dey and colleagues [17] reported that using a specifically designed assay to quantify telomere derived cfDNA, levels in unaffected BRCA1 BRCA2 mutation carriers ( n = 28) were lower than those of non-carriers. Given the paucity of data on cfDNA quantification in BRCA1 BRCA2 carriers the current study was carried out.

2. Materials and methods

3. Results

In this preliminary study, cfDNA levels measured using a robust assay, were significantly higher in cancer-free BRCA1/BRCA2 mutation carriers compared with cancer-free controls. The biological basis for this phenomenon may be associated with defective DNA repair mechanisms. Both BRCA genes encode large proteins that play an essential role in DNA damage response (DDR) and homologous repair (HR) of double stranded DNA damage [19]. In BRCA-associated cancers there are no active BRCA proteins somatically, a fact that may lead to accelerated apoptosis that could result in elevated levels of cfDNA. Indeed, Park et al. [20] reported that baseline cfDNA, measured by the same assay applied in the current study, is an effective predictor of relapse in triple negative breast cancer (TNBC) patients, a subtype of breast cancer much more prevalent amongst BRCA1 mutation carriers [21]. Dey and coworkers reported the presence of shorter telomere in 28 BRCA mutation carriers compared with controls [17]. In agreement with these studies, in non-BRCA carrier women with infertility problem we previously found an inverse relationship between cfDNA levels and telomere lengths [22]. Elevated cfDNA and high rate telomeres shortening are probably both caused by accelerated cell turnover.

Thus, it is plausible to assume that BRCA haploinsufficiency, a feature shared in all cells of a BRCA mutation carrier, may result in an increased apoptotic rate of non-cancer cells and cause cfDNA elevation. Noteworthy, this is presently a speculative explanation to account for the results of this study that should be viewed cautiously.

The assay employed herein is simple, robust, easy, and cheap. It has also been shown to be applicable in several clinical scenarios: cfDNA level had better discriminatory ability than the gold standard APACHE II score and procalcitonin (PCT) for 28-day mortality of patients with severe sepsis [23]. Post-dialysis cfDNA concentration was an independent predictor of all-cause mortality in patients undergoing hemodialysis [24]. Baseline pre-surgery cfDNA levels measured in patients with colorectal cancer were good predictors of disease-free survival at 5 years: all patients who died within 5 years had elevated cfDNA levels, while normal cfDNA levels ( 800 ng/ml) were highly predictive of disease-free survival [25]. Thus it seems that the assay employed herein is well accepted and validated.

There are inherent limitations to this study that should be enumerated. The numbers of cases and controls is small, the spectrum of BRCA1 and BRCA2 mutations is limited, there is no long-term follow up of mutation carriers, the mutational status of the controls was not determined. Given the rates of germline BRCA1 BRCA2 mutations in the Jewish Ashkenazi population (2.5%) [26], it seems that at the most we may have missed one possible mutation carrier in the controls. Lastly, we do not have all possible factors that may have affected the final cfDNA levels-smoking habits menstrual cycle, pregnancy status, to mention a few.

In conclusion, cfDNA levels are seemingly elevated in cancer-free BRCA mutation carriers. The results presented in this preliminary and admittedly limited scope study should be validated in a larger set of BRCA mutation carriers’ cases of diverse ethnicities. These studies should evaluate the sensitivity and specificity of sequential cfDNA measurements as a surrogate marker for early detection of breast or ovarian cancer in healthy BRCA1/2 carriers.