Cancer-associated mutations in normal human endometrium: Surprise or expected?

The monoclonal composition of endometrial glands reminds us of the presence of stem/progenitor cells, from which descendant cells compose a whole gland with clonal expansion; otherwise, the proliferative potential would become exhausted after several dozen menstrual cycles. Endometrial epithelial stem cells are supposed to localize at the stratum basalis of the gland because they appear to be protected from shedding during menstruation. Extensive efforts have been made to identify endometrial epithelial stem cells. The side population (SP) phenotype is a universal marker of adult stem cells that is characterized as a low fluorescent phenotype by flow cytometry with Hoechst 33342 staining. 13 SP cells have been identified in cultured endometrial cells, 14 and endometrial SP cells sorted from short‐term cultures have been maintained in three‐dimensional (3D) culture for 3 mo, whereas non‐SP cells became senescent in shorter periods, which concurs with the longevity associated with adult stem cells. Endometrial SP cells preferentially express several endothelial cell markers 15 and can differentiate into various phenotypes, including glandular epithelial, stromal, and endothelial cells in vitro. Furthermore, endometrial SP, but not non‐SP, cells can successfully reconstitute endometrium‐like tissues containing glandular components when transplanted into severely immunodeficient mice, but are mainly composed of endothelial tissues (46%), stromal tissues, (13%), and rarely, epithelial tissues (0.02‐8%), indicating their heterogeneity. 15 Several other candidate markers for endometrial epithelial stem cells have been identified. The embryonic stem cell‐surface marker, stage‐specific embryonic antigen 1 (SSEA‐1), marks human endometrial basal glandular cells. 16 Cultured SSEA‐1 + cells have increased telomerase activity and longer telomeres and generate larger gland‐like spheroids in 3D culture than do SSEA‐1 – cells and N‐cadherin has been identified as a potential marker for endometrial epithelial progenitors. 17 Serial cloning assay has revealed that N‐cadherin + epithelial cells exhibit greater self‐renewal capacity than do N‐cadherin – endometrial epithelial cells. N‐cadherin + epithelial cells can differentiate into glandular structures lined with cytokeratin + luminal cells in organotypic culture. Some other potential markers for endometrial epithelial cells have also been proposed and reviewed 18 , 19 , 20 , 21 , 22 , 23 , 24 (Table  1 ). Despite these extensive studies, no definitive marker specific to endometrial epithelial stem cells has been identified. One problem may lie in the controversy regarding the specificity of the antibodies used, as the reliability of some antibodies was not verified. Another concern is that a greater number of cells with candidate markers are present in the normal endometrium compared with the expected number for an adult stem cell population. Furthermore, the difficulty in establishing a reconstitution model in which the selected cell population, hopefully a single cell, with specific markers can reconstitute a whole endometrial gland, with or without other components, in 3D culture or a mouse model remains an urgent issue. Overcoming these issues could be expected to purify further glandular stem/progenitor cells, which is important for understanding the mechanisms of clonal expansion as well as whether they are mutation targets. Proposed markers for endometrial epithelial stem cells Abbreviations: LGR5, leucine‐rich repeat‐containing G‐protein‐coupled receptor 5; LRC, label‐retaining cell; SOX9, sex‐determining region Y (SRY)‐box 9 protein; SP, side population; SSEA‐1, stage‐specific monoclonal antigen‐1

Mouse studies have provided useful information on the effect of driver mutations in endometrium. As PTEN homozygous null mice are embryonic lethal, PTEN heterozygous mice are widely used for cancer studies in earlier periods. 32 PTEN heterozygous females have been reported to show AEH. 33 The development of the Cre ‐loxP system has enabled conditional knockout in endometrium, and some studies with PTEN homozygous null mice have demonstrated the development of carcinoma, 34 whereas others have reported the development of precursor lesions, such as atypical hyperplasia. 35 , 36 The additional introduction of PIK3CA mutation to PTEN ‐null mice efficiently created cancer in endometrium. 35 Cheng et al developed a mouse model of endometriosis in which the endometrium of transgenic mice with conditional knock‐in of KRAS G12V/+ was subcutaneously transplanted into immunocompetent recipient mice. 37 The implanted endometrium developed endometriosis‐like lesions with intact glandular structures, while that of the control mice with the wild‐type allele formed smaller lesions with many dying cells. This model suggests that oncogenic KRAS mutation in eutopic endometrium plays an essential role in the development of endometriotic lesions via implantation. A recent knockout/knock‐in mouse study indicated the role of ARID1A and PIK3CA mutations in normal endometrium. 38 Neither conditional knockout of ARID1A nor knock‐in of PIK3CA mutant allele in normal endometrial epithelia had any gross phenotypes, however the combination of both conferred phenotypic change as atypical hyperplasia. Thereafter, in a mouse model, the above authors challenged the mice to mimic the spread of endometrium into the peritoneal cavity. 39 Conditional knockout mice displaying a loss of ARID1A expression concomitant with oncogenic PIK3CA H1047R expression in endometrium were prepared and subjected to a surgical procedure to facilitate endometrial spread into the peritoneal cavity, in which salpingectomy was performed to remove the barrier of the uterotubal junction, facilitating mutant endometrial cells to invade outside the uterus. Following salpingectomy, endometriotic lesions were formed in the mice on various sites in the pelvic space, whereas no specific lesions were formed in control mice with the wild‐type allele. These findings support the hypothesis that mutated phenotypes of eutopic endometrium are essential for the development of ectopic sites. Overall, these mouse studies demonstrated that a single hit of driver mutations in endometrial epithelial cells is insufficient for the acquisition of complete cancer phenotypes; additional hits are required. They also demonstrated that KRAS mutations in eutopic endometrium may play pivotal roles in the development of endometriosis.

The way in which human endometrial glands regenerate after shedding during menstruation is not well known. While the stratum functionalis sheds during menstruation, the stratum basalis remains, providing proliferating glandular cells for the next cycle of regeneration. The clonality of human endometrial glands is crucial for understanding the mechanism of cyclic regeneration. Therefore, we investigated the clonality of normal human endometrial glands using X chromosome inactivation patterns as informative markers of tissue clonality. 9 During embryogenesis, DNA methylation randomly inactivates either the paternal or maternal X chromosome, and methylated alleles become resistant to DNA restriction enzymes. 10 Following treatment with restriction enzymes, polymerase chain reaction (PCR) can amplify intact alleles on the methylated, but not the unmethylated X chromosome, which generates 2 distinct PCR bands in electrophoresis, derived from the paternal and maternal alleles, respectively, because of allelic polymorphisms in polyclonal normal tissues. However, if the PCR generates the single band, the tissue is considered to comprise monoclonal cell population. DNA isolated from individual glands was treated with restriction enzymes, and the alleles of the androgen receptor gene on the X chromosome were amplified by PCR. The DNA recovered from almost all the glands of each patient produced a single band upon Hha I digestion (Figure  1A ), suggesting that the glands were composed of a monoclonal cell population of either paternal or maternal origin. We further examined gland clonality in a mouse harboring a green fluorescent protein gene on either the maternal or paternal X chromosome. 11 The cells of this mouse harboring the transgene on the unmethylated active X chromosome exhibited fluorescence, whereas those with the transgene on the inactive X chromosome did not. 12 Therefore, this mouse genus, known as “green mice,” can be used to analyze the clonality of tissues of interest. Under fluorescence microscopy, histological sections of the uteri of this mouse revealed that the individual glands were composed of either fluorescent or nonfluorescent cells at similar ratios (Figure  1B ), suggesting that the glands are composed of a monoclonal cell population. Clonality analysis of human endometrial glands. A, The principle of the clonality assay for androgen receptor (AR) gene is illustrated. Endometrial glands were individually isolated with microscopic manipulation as shown in the photograph. DNA from each gland was digested with Hha I, followed by PCR to amplify the CAG repeats in the AR gene. After digestion with Hha I, the DNA yielded single PCR bands (shown as red circles) derived from either paternal or maternal alleles, suggesting that glandular cells are monoclonal. DNA from each patient's white blood cells (WBC) were used as controls for the polyclonal population. B, Uterine sections of a female hemizygous transgenic mouse carrying an X‐linked green fluorescent protein transgene. Left: Histological view of endometrium. Right: Fluorescence microscopy shows endometrial glands composed of either fluorescent (A) or nonfluorescent (B) cells. (Adapted with alterations and permission from reference 9 )

Due to the lack of cellular atypia in endometrial cells, cytological screening of endometrial cancer is not easy compared with that of cervical cancer. Several studies have proposed the utility of genetic analysis for cancer screening using cytological samples or washing fluid in the endometrial cavity. The use of Sanger sequencing targeting representative cancer‐associated genes has frequently revealed somatic mutations in 80%‐90% of endometrial cancer specimens, but rarely in normal endometrial samples. 40 , 41 A targeted NGS panel for 8 genes ( ARID1A , CTNNB1 , KRAS , MTOR , PIK3CA , POLE , PTEN and TP53 ) detected somatic mutations in over 90% of endometrial cancer specimens, while only 6% of women without cancer exhibited PIK3CA mutations. 42 Collectively, these studies demonstrated the potential utility of genetic analysis for the early detection or screening of endometrial cancer, based on the assumption that normal endometrium has an extremely low frequency of cancer‐associated mutations. However, Nair et al reported the results of NGS for 12 genes using uterine lavage fluids. 43 Of 95 women without cancer, 51 were identified as having somatic mutations; the most frequent driver mutations were KRAS G12C (8 patients), KRAS G12S (10 patients), and PIK3CA H1047R (8 patients) with relatively high VAFs (1%‐30.4%). These findings suggest that NGS is likely to detect unexpectedly frequent somatic mutations in normal endometrial samples, and that additional or alternative strategies are needed to distinguish cancerous from noncancerous mutations for screening or early detection of endometrial cancer.

Recent NGS technologies have detected unexpectedly greater numbers of somatic mutations in endometrial epithelial cells compared with in stromal cells in an age‐dependent manner, with substantial rates of glands having cancer‐associated mutations. The biological significance of such mutations remains unclear, but may confer a growth advantage to glands, thereby contributing to efficient regeneration in a menstrual cycle; this should be confirmed in functional studies. Somatic mutations in endometriosis show a similar set of driver mutations, but with higher MAFs. Endometrial epithelial cells with driver mutations, especially KRAS mutation, may therefore evolve clonally to develop ectopic sites, which supports retrograde menstruation theory (Figure  3 ). It is, however, unclear when and where additional genetic hits occur within eutopic or ectopic sites during the development of endometriosis. Proposed consequence of somatic mutations in normal endometrial glands. Stem/progenitor cells in normal endometrial glands, possibly located in the stratum basalis, may clonally expand with or without somatic mutations in cancer‐ or noncancer‐associated genes. Both populations, probably the former in favor, may have a growth advantage, forming clonal colonization. Additional genetic hits, including POLE mutation, mismatch repair (MMR) deficiency, and/or copy number alterations (CNAs), facilitate malignant conversion. Similarly, endometriosis may arise via clonal evolution, possibly in the population with KRAS mutation. AEH, Atypical endometrial hyperplasia; CA, cancer‐associated mutation; EIN, Endometrial intraepithelial lesion; MT, mutation Endometrial cancer also has a similar set of drive mutations, but with a much higher frequency. The very high mutation loads attributed to DNA mismatch repair deficiency and POLE mutations, as well as structural variants and copy number changes, were specific to endometrial cancer. Malignant conversion of normal epithelial cells requires these additional hits accumulated in an age‐dependent manner, and may therefore be a rare lifetime event (Figure  3 ). Knockout/knock‐in mouse studies also support multiple hits of key driver mutations required for the development of cancer. How somatic mutations are consistently retained after complete shedding of the functionalis during menstruation remains unclear. This is a characteristic feature of the endometrium, unlike other regenerative tissues, such as the colon. “Stem cell hit theory” may explain this consistency clearly; descendants will take them over with clonal expansion during each menstrual cycle. The rate and spectra of mutation status in endometrial stem cells are crucial to gaining a better understanding of the biological significance of somatic mutations in normal endometrial glands and the pathogenesis of both endometriosis and endometrial cancer. Although specific markers for endometrial epithelial stem cells have not been clearly identified, organoid cultures derived from single cells under selected culture conditions may be available to resolve such issues. Finally, caution should be urged when detecting cancer‐associated mutations for cancer screening and diagnosis. Molecular‐based screening or diagnostic strategies require further improvements to distinguish cancerous from noncancerous mutations.

While somatic mutations in epithelial cells have been extensively analyzed, little information has been available for those in stromal cells. Suda et al performed targeted‐gene sequencing of laser‐microdissected stromal samples from endometriotic lesions and normal endometria. 30 In total, 18 and 16 mutations were detected in endometriotic stromal and normal endometrial stromal samples, respectively, but without any mutations shared with paired epithelial samples. Furthermore, the MAFs of mutations in stromal samples were extremely low compared with those of the paired epithelial samples. The completely different mutation profiles between paired samples suggested their independent origin and biology. In relation, Noë et al 31 analyzed 19 somatic passenger mutations in 24 patients with DIE lesions and found that these mutations were predominantly detected in the epithelial compartment, whereas very few mutations were observed in the stromal ones.

The endometrium is a unique component of the uterus characterized by cyclic regeneration during menstrual cycles. After shedding during menstruation, the endometrium vigorously regenerates during a 14‐d proliferative phase, followed by a secretory change persisting until the next menstruation. Endometriosis is a disease experienced by many women of reproductive age in which the uterine endometrium grows outside the uterus. It is accompanied by a variety of complications, including dysmenorrhea, pelvic pain, and infertility. Sampson's retrograde menstruation theory, which states that normal endometrial cells flow back into the peritoneal cavity during menstruation, leading to the development of ectopic endometriotic lesions, is widely accepted. 1 Endometrial cancer also arises from epithelial cells in the human endometrium through atypical hyperplasia as precursors or de novo from those of the atrophic endometrium in postmenopausal women. Therefore, the vast majority of studies have used normal human endometrium as experimental controls for both diseases. While the somatic mutational landscape of cancer in humans has been comprehensively characterized, 2 knowledge of somatic mutations in normal cells remains limited because of difficulties in accessing tissue, the small amounts of samples collected, the likely low frequency of mutations, and insufficient technical resolution. Recent advances in next‐generation sequencing (NGS) technology have helped to overcome these issues and uncover the mutational landscape of a variety of normal tissues, including the colon, 3 skin, 4 esophagus, 5 and endometrium. 6 , 7 , 8 This review introduces the clonal growth properties of endometrial glands, possibly via stem/progenitor cells, and summarizes recent findings regarding their mutational status, focusing on the roles of somatic gene mutations in the biology of endometrium and the pathogenesis of endometriosis and endometrial cancer.

None of the authors has any conflicts of interest to disclose.

In 2014, for the first time, Li et al 25 performed whole‐exome sequencing of endometriosis samples from 16 patients. This was the first study to detect unexpectedly numerous somatic mutations in endometriotic lesions and to find that the patients harbored various somatic mutations, including genes encoding histone methyltransferase, which is involved in histone H3 lysine 4 modification. In 2017, Anglesio et al reported somatic mutations in deeply infiltrating endometriotic (DIE) lesions. 26 Whole‐exome sequencing identified 80 nonsynonymous somatic mutations in 19 (79%) of 24 patients, with a mean of 3.3 mutations per lesion. Five patients (21%) had a known cancer driver mutation in either ARID1A , PIK3CA , KRAS , or PPP2R1A . Targeted sequencing and a droplet PCR assay identified additional KRAS mutations, and 10 (26%) of 39 DIE lesions had driver mutations, all of which were confined to the epithelial compartment of endometriotic lesions; these were not observed in stromal components, which raised a hypothesis regarding the differing origins of epithelial and stromal components. Eutopic endometria in 3 patients with DIE lesions harboring KRAS mutations were available, but were confirmed to have no detectable mutations. Overall, the frequency of cancer‐associated mutations detected in noncancerous DIE lesions surprised researchers, and was thought to explain the aggressive phenotype of this disease, which raises the question of whether superficial endometriosis has similar spectra of mutations. The same research group further examined mutation status in incisional endometriosis (IE), a type of iatrogenic endometriosis resulting from surgical procedures such as a cesarean section. 27 Endometriosis tissue samples from patients with IE or DIE lesions were examined by target‐gene sequencing for 33 genes. Four (10%) of 40 patients with IE harbored somatic mutations in either KRAS , PIK3CA , or ERBB2 . No somatic mutations were detected in any of the 4 corresponding eutopic endometrial specimens available. Eight patients (22.2%) with DIE lesions had somatic mutations in either KRAS or CTNNB1 . Simultaneous immunohistochemical analyses for the loss of PTEN or ARID1A expression added potentially mutated cases, and the overall rates of somatic driver mutations in IE and DIE lesions were 27.5% and 36.1%, respectively. These findings demonstrated that both lesions had similar rates of somatic driver mutations. As IE arises from surgical implantation/endometrial seeding, whether these mutations are attributable to the eutopic endometrium prior to implantation/seeding remains a great concern, although no mutations were detected in eutopic endometria in the above study. In 2018, Suda et al 6 performed whole‐exome sequencing of 13 ovarian endometriotic epithelial samples using laser microdissection techniques. Several cancer‐associated mutations, including KRAS , PIK3CA , FBXWJ , PPP2R1A , and PIK3R1 , were recurrently detected, with KRAS and PIK3CA being the most frequent. These mutations were located within the hotspot, leading to gain‐of‐function mutations, which activated downstream signaling pathways that may lead to a growth advantage. Representative cancer‐associated mutations had high mutant allele frequencies (MAFs), being ranked in the top 5%, reaching nearly clonal status (MAF of 0.5). The authors examined the patterns of mutation sharing in multiple endometriotic regions collected from the same individual; KRAS and PIK3CA mutations were frequently shared across multiple regions of an individual subject, indicating the growth advantage of clonal populations harboring such mutations.