DNA methylation as a biomarker for the detection of hidden carcinoma in endometrial atypical hyperplasia
Introduction
Endometrial carcinoma (EC) is one of the most common cancers of the female genital tract, although the incidence varies between countries [1]. Prolonged exposure to estrogen promotes the development of endometrial hyperplasia (EH), which leads to atypical hyperplasia (AH); 25–40% of patients with AH subsequently progress to EC. Although AH is the least common type of hyperplasia, it is the type most likely to progress to type 1 EC, which accounts for more than 80% of uterine cancers [2]. EC is usually confined to the inner lining of the uterus, which can be removed by hysterectomy. Unfortunately, even in stage I EC, there is a significant risk of tumor recurrence, distant metastasis, and death [3].
EH is classified into two categories by the World Health Organization: 1) EH, including simple and complex hyperplasia without atypia, and 2) endometrial AH, including simple and complex hyperplasia with atypia [4]. Several studies have shown that cytological atypia, which is the major criterion for the diagnosis of AH and the most reliable indicator of progression from EH to EC, has poor reproducibility [2], [5], [6]. In 12.7–42.6% of cases, EC coexists in patients with a diagnosis of AH [7]. The high rate of unrecognized cancer among women diagnosed preoperatively with AH reflects the fact that the histologic criteria for differentiating AH from some types of EC on dilation and curettage (D&C) are controversial and subject to different interpretations [8], [9], [10].
Because of overlap in the histologic picture of AH and low-grade EC in the limited tissue sample evaluated before major surgery, differentiation on pathologic grounds alone can be very difficult or impossible [5], [10], [11]. Although D&C before hysterectomy is the gold standard method for the diagnosis of endometrial lesions, detection of AH cannot rule out a more severe lesion [12], [13]. EC found at the time of hysterectomy for AH may be associated with deep myometrial (10%) or cervical stroma (5%) invasion [14].
Hysterectomy is the main therapeutic modality for AH. Conservative approaches such as high-dose progestin may be acceptable treatment options in certain situations (e.g., to maintain fertility), but the risks of progression to malignancy and of concurrent EC remain high [14]. At present, there is no established biomarker to differentiate endometrial AH and EC. Such a marker could be hysterectomy sparing for AH patients without EC. Even when EC arising from endometrial premalignant lesions is clearly defined, the possibilities for EC screening are very limited. Reliable determination of the presence or absence of EC would allow for better surgical decisions about hysterectomy and staging. The reassurance of patients given fertility-sparing management for AH may alleviate unnecessary anxiety. There is, therefore, a need to develop new, molecular-based, complementary tools that could improve the pathological diagnosis.
Epigenetic studies have demonstrated that silencing of genes, such as tumor-suppressor genes (TSGs), can act as a mechanism of carcinogenesis [15], [16]. The addition of a methyl group to the cytosine–guanine (CpG) island results in gene silencing. Because epigenetic silencing of TSGs by promoter hypermethylation is observed commonly in human cancers, it is possible that DNA methylation could be used for the early diagnosis of cancer. This concept, and its application in gynecologic cancers, has been gaining acceptance during the past few years, especially in diagnosing and treating cervical cancer (screening and triage) and ovarian cancer (prognosis) [17], [18], [19], [20]. However, similar studies of EC are relatively limited. It is known that the progression of EC involves a multistep process, and both genetic and epigenetic events have been shown to play important roles. Although gene promoter CpG islands epigenetically marked by de novo DNA methylation may serve as biomarkers in EC, they have been rarely studied in AH [21], [22], [23], [24]. Such epigenetic biomarkers could be useful for identifying EC in AH.
Our previous research on the epigenomics of cervical cancer using methylomic approaches identified several candidate genes that are methylated in cervical cancer tissues. Several candidate genes were significantly hypermethylated in CIN3 + lesions [19], [25]. Because the uterine cervix and endometrium both originate from the Müllerian duct system, this close embryologic relationship between the uterine cervix and endometrium may be reflected in adulthood in the form of malignant lesions. We hypothesized that some of the genes hypermethylated in cervical cancer may also be hypermethylated in EC. We initially tested 28 development-related genes. To test further the feasibility of using these new biomarkers in identifying endometrial lesions, we converted the methylation analysis to a quantitative methylation-specific polymerase chain reaction (QMSP) approach and tested its application value. We found that the following several genes were potentially implicated in endometrial carcinogenesis: adherens junction-associated protein 1 (AJAP1), heparan sulfate d-glucosamyl 3-O-sulfotransferase-2 (HS3ST2), sex-determining region Y, box 1 (SOX1), prostanoid receptor gene, prostaglandin D2 receptor (PTGDR), and LIM-homeobox gene 1A (LMX1A). These candidate genes, which could be used for the triage of AH, were validated in The Cancer Genome Atlas (TCGA) EC database. The aim of the present study was to analyze the DNA methylation status of AJAP1, HS3ST2, SOX1, PTGDR, and LMX1A genes as biomarkers for EC diagnosis in patients with endometrial AH. The discovery of reliable epigenetic biomarkers for diagnosis may open a new avenue for the management of AH patients with and without EC.
Section snippets
Patients and clinical samples
Samples of endometrium from patients with endometrioid-type EC (n = 20; 8 G1 cases, 8 G2 cases, 4 G3 cases) and with dysfunctional uterine bleeding (n = 20) were included as cancer and normal controls, respectively. Specimens were obtained from tissue blocks for methylation analysis of the candidate genes. Endometrial biopsy tissues of patients with AH (n = 61) were collected for methylation analysis. All patients underwent hysterectomy within 3 months after endometrial sampling. The clinicopathologic
DNA methylation of AJAP1, PTGDR, HS3ST2, SOX1, and LMX1A in EC
We first examined the methylation status of the five genes AJAP1, PTGDR, HS3ST2, SOX1, and LMX1A from EC patients and compared it with that in samples from normal endometrium. We randomly chose 20 samples of EC and 20 normal controls for analysis. The methylation status of AJAP1, PTGDR, HS3ST2, and SOX1 in the EC and normal controls is shown in Fig. 1. The EC samples showed a higher methylation index for AJAP1, PTGDR, HS3ST2, and SOX1 compared with normal controls (p < 0.0001, p = 0.0002, p = 0.0004,
Discussion
Endometrial carcinogenesis is a multistep process involving a precursor lesion with underlying genetic and epigenetic events. Therefore, molecular diagnostic methods have been proposed as new ancillary tools for the detection of undetected cancers and differential diagnosis of premalignant and malignant lesions. Abnormal patterns of DNA methylation have been recognized in various cancers. An increase in DNA methylation in gene promoter regions often precedes apparent malignant changes, which
Conflict of interest statement
The authors declare that no conflict of interest exists.
Acknowledgments
This study was supported by grants from the Tri-Service General Hospital (TSGH-C101-073, TSGH-C102-074, TSGH-C103-076, TSGH-C103-006-008-S01, TSGH-C103-006-008-S02, and TSGH-C103-006-008S03), NSC-102-2628-B-038-010-MY3, NSC-102-2314-B-016-038 and NSC-102-2314-B-016-043 from the National Science Council, Taiwan, Republic of China, TMUTOP103005-1 and 103TMU-SHH-11 from Taipei Medical University, MOHW103-TDU-B-212-113001 and MOHW103-TDU-PB-211-122025 from Ministry of Health and Welfare.
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HC Lai and YC Wang contributed equally to this work.