While the list of cancer driver genes capable of conferring growth advantages in tumor cells has advanced considerably, there has been a slower understanding regarding the timing, necessity, and sufficiency of the mutations in normal cells required to initiate cancer growth. The acquisition of somatic mutations is a part of the aging process and is itself thought to account for increased cancer risk as one ages [[1], [2], [3], [4]]. Concurrent with this understanding, DNA sequencing of tissues aims to become a routine part of clinical care. Importantly, this has been extended into liquid biopsies for screening and diagnosis of a growing number of cancers [[5], [6], [7]]. These next-generation deep sequencing approaches have begun revealing unexpected findings, namely, the existence of somatic mutations present within histologically normal, non-cancerous tissues [[8], [9], [10]]. Moreover, cancer gene driver mutations are even detectable from individuals without cancer [[10], [11], [12]]. While a majority of mutations in normal cells will not ultimately be associated with malignant transformation, certain somatic mutations may nonetheless increase that cell's ability to proliferate as evidenced through clonal expansion [13,14]. Studies defining which genes are mutated, their rates of mutation, as well as the causes and consequences of their being mutated within different tissues and cell types is unknown but critically needed.

Endometrial cancer (EndoCA) is the most prevalent cancers of the female genital tract and is one of the few cancers in which incidence and death rates continue to rise [15]. One of the major drivers of its increasing incidence is obesity and increased BMI [16,17]. By 2030, EndoCA is projected to surpass colorectal cancer to become the third most prevalent cancer among US women [18]. The 5-year survival rate for EndoCA is >90% for localized disease but drops to nearly 20% if detected in its late stages [19]. Despite the clear survival advantages associated with early-stage disease, there are no effective screening methods and diagnosis generally requires invasive biopsy.

As part of an overall effort to develop effective molecular screening tools for diagnosing EndoCA we began the molecular analysis of uterine lavage fluid collected in women undergoing hysteroscopy [11]. Using targeted next-generation sequencing (NGS) gene panels, we identified tumor-specific mutations in genomic DNA from cells and circulating free DNA within lavage fluid from women with EndoCA. While demonstrating high sensitivity to detect tumor-specific mutations in even microscopic-sized cancers, we simultaneously, and unexpectedly, also detected known EndoCA hot spot cancer driver mutations in approximately half of the women without cancer [11]. The origin of these mutations in women without cancer was unknown, but their existence has since been confirmed by others [[20], [21], [22], [23], [24], [25], [26], [27]].

To gain an understanding of not only the origin but also prevalence and possible associations of these mutations with known EndoCA risk factors, specifically including age and BMI, we now specifically investigated the genomic landscape of uterine tissue from women without cancer. We obtained multiple samplings of uterine tissue from geographically distinct anatomic sites and tissue layers from women undergoing total hysterectomy for non-cancer conditions. This allowed us to interrogate both possible mutation rate differences between: (1) regions of the uterus, given that EndoCA almost always arises in the uterine body and fundus but only rarely in the lower uterine segment, and (2) uterine endometrium and myometrium, given that in general, different tissue types throughout the body display different mutation rates [13,14]. We used a comprehensive targeted NGS panel focusing on 125 cancer-relevant genes and sequenced all samples at an ultra-deep level (>2000×). Through these findings, we demonstrate important caveats for future development of liquid biopsy-based tests to detect EndoCA and a possible link to a previously unrecognized EndoCA protective mechanism.