Molecular Analysis of Endometrial Tumorigenesis: Importance of Complex Hyperplasia Regardless of Atypia

Results

This retrospective study was conducted on serial specimens of endometrium with various histologic diagnoses, taken by aspiration biopsy or surgery during a 10-year surveillance of MMR gene mutation carriers (13), some of which developed (group I) and others did not develop (group II) endometrial carcinoma by the end of follow-up (see Materials and Methods and Table 1). Series of unrelated samples of normal endometrium and hyperplasia taken from consecutive sporadic cases undergoing endometrial biopsy or hysterectomy for nonmalignant reasons (group III) were used for comparison. Table 2 provides a summary of all molecular alterations observed in different groups.

DNA MMR status

Among MMR gene mutation carriers, results obtained from groups I and II were broadly comparable with each other (Table 2), which made it possible to combine these groups into a “Lynch syndrome group” for most subsequent analyses. In the Lynch syndrome group, decreased MMR protein expression corresponding to the germ-line mutation was observed in 2 of 29 (7%) in normal endometrium, 2 of 5 (40%) in simple hyperplasia, 3 of 3 (100%) in complex hyperplasia without atypia, 11 of 12 (92%) in complex hyperplasia with atypia, and 11 of 11 (100%) in endometrial carcinoma (Table 2). MSI frequencies in the same core series were 2 of 36 (6%), 1 of 6 (17%), 2 of 3 (67%), 5 of 13 (38%), and 7 of 11 (64%), respectively, suggesting that immunohistochemical detection of MMR protein expression was more sensitive than MSI analysis to identify MMR defects. Instability of mononucleotide repeats (BAT25 and BAT26) accounted for most cases with MSI, and among those cases with one unstable marker specifically, BAT26 was involved in 70% and D5S346 in 30%. By histology, results from immunohistochemistry and MSI analysis indicated that normal endometrium was MMR proficient (with the exception of two cases), whereas MMR defects were observed in most cases of complex hyperplasia, whether complex hyperplasia without atypia or complex hyperplasia with atypia, and endometrial carcinoma. Differences between normal endometrium versus complex hyperplasia without atypia + complex hyperplasia with atypia and normal endometrium versus endometrial carcinoma (but not normal endometrium versus simple hyperplasia) were statistically significant (P < 0.01) with respect to either MSI or abnormal MMR protein expression. The general trends for MSI between histologic categories persisted after the inclusion of additional cases (“extended” series; Table 2).

Temporal appearance of molecular genetic changes in endometrial tissues from MMR gene mutation carriers is depicted in Table 3 (only cases from which serial samples of different histologies were available are included). In group I, absent MMR protein was detectable in endometrial hyperplasia up to 3 years before the diagnosis of endometrial carcinoma (cases 2, 4, 7, and 8 in Table 3). Representative examples of immunohistochemistry and MSI results from simple hyperplasia, complex hyperplasia with atypia, and endometrial carcinoma specimens (originating from cases 8 and 24) are shown in Fig. 1.

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Fig. 1.

Immunohistochemical images (with MLH1 antibody) and corresponding MSI plots (with BAT25 marker) for patients 8 (A) and 24 (B) with germ-line mutation in MLH1. In patient 8, simple hyperplasia (SH; top left), complex hyperplasia with atypia (CAH; middle), and endometrial carcinoma (EC; top right) lacked MLH1 protein (red arrows), whereas stromal cells showed normal expression of MLH1 (black arrows) as indicated by brown staining. In the same patient, BAT25 mononucleotide repeat was stable in simple hyperplasia and unstable in complex hyperplasia with atypia (CH) and endometrial carcinoma. Short vertical lines, constitutional peaks of 121 bp; arrowheads, size shifts indicative of MSI. In patient 24, simple hyperplasia expressed and complex hyperplasia without atypia did not express MLH1 protein, and both specimens were microsatellite-stable.

In the reference group (group III), MSI was not observed at all in normal endometrium (Table 2). In regard to hyperplasia, MLH1 promoter methylation was used as a surrogate marker for MMR status because it is known that most sporadic MMR-deficient endometrial carcinomas arise through promoter methylation (21, 22), and only cases with MLH1 methylation (n = 4) were further examined for MMR protein expression and MSI. Two of 37 hyperplasia samples, one complex hyperplasia without atypia and one complex hyperplasia with atypia, had MLH1 protein loss and/or MSI (5%; data not shown).

Tumor suppressor promoter methylation

Among 24 tumor suppressor genes examined for promoter methylation by MS-MLPA, the average number of methylated loci increased progressively from normal endometrium to simple hyperplasia to complex hyperplasia (complex hyperplasia without atypia/complex hyperplasia with atypia) in both MMR gene mutation carriers (groups I and II combined) and reference series (group III) as evident from Table 2. When comparing the combined group of MMR gene mutation carriers with the reference group, the average number of methylated loci was significantly higher in the former versus the latter group for normal endometrium (1.1 versus 0.3; P = 0.0097) and simple hyperplasia (2.0 versus 0.6; P = 0.0016), whereas no difference was seen for either type of complex hyperplasia (between groups or relative to each other). The fact that a hypermethylated state is present already in histologically normal endometrium may predispose MMR gene mutation carriers to early endometrial carcinoma development compared with sporadic cases.

Aberrant DNA methylation stratified the different histologic categories precisely in the same way as MMR defects in the Lynch syndrome group. For simplicity, we combined groups I, II, and III, and based on values given in Table 2, the average number of methylated loci was similar for normal endometrium versus simple hyperplasia (0.8 versus 1.0; P = 0.5), whereas normal endometrium was significantly less methylated relative to either complex hyperplasia without atypia (0.8 versus 2.7; P < 0.0001) or complex hyperplasia with atypia (0.8 versus 2.5; P < 0.0001). Additionally, in group I, methylation in endometrial carcinoma was similar to that in complex hyperplasia with atypia (2.1 in both).

Figure 2 shows promoter methylation frequencies for individual loci by histology, separately for the Lynch syndrome (groups I and II combined) and reference series. Examples of MS-MLPA tracings for normal endometrium, complex hyperplasia without atypia, and complex hyperplasia with atypia, all chosen from the reference series, are depicted in Fig. 3. Three loci, CDH13, RASSF1(A), and GSTP1, were preferential targets for methylation in complex hyperplasia, whether originating from the Lynch syndrome group (75%, 63%, and 25%, respectively, for complex hyperplasia without atypia and 70%, 55%, and 30% for complex hyperplasia with atypia) or the reference group (85%, 69%, and 31% for complex hyperplasia without atypia and 62%, 54%, and 69% for complex hyperplasia with atypia). The same loci were frequently involved in endometrial carcinoma from group I (69%, 54%, and 15%). In contrast, lower methylation frequencies or no methylation at all were observed for these loci in simple hyperplasia and normal endometrium. Like MMR defects, aberrant tumor suppressor promoter methylation appeared before the diagnosis of endometrial carcinoma in group I (Table 3). For example, methylation at the CDH13 promoter in normal endometrium and/or complex hyperplasia with atypia was observed 3 and 12 years before endometrial carcinoma in cases 2 and 5, respectively. Regarding interpretation of data given in Table 3, complex hyperplasia with atypia cells were occasionally interspersed with normal cells, making it impossible to separate “pure” areas of complex hyperplasia with atypia and normal cells for DNA extraction. In such cases, results from DNA-based assays were considered to represent complex hyperplasia with atypia but not normal tissue (whereas immunohistochemical evaluation always allowed distinction between complex hyperplasia with atypia and normal cells).

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Fig. 2.

Comparison of MS-MLPA results for individual tumor suppressor loci in the Lynch syndrome series (groups I and II combined) and reference series (group III). Loci that showed promoter methylation in at least one sample are included. Results for normal endometrium, proliferatory and secretory combined (A), simple hyperplasia (B), complex hyperplasia without atypia (C), complex hyperplasia with atypia (D), and endometrial carcinoma, group I (E).

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Fig. 3.

Examples of MS-MLPA plots representing normal endometrium (top), complex hyperplasia without atypia (middle), and complex hyperplasia with atypia (bottom). Twenty-four different tumor suppressor loci were interrogated with specific probe pairs (two pairs of probes were included for RASSF1), combined with a set of control probes (C). The fragment size (increasing from left to right) identifies each locus. An amplification product (that is, visible peak) is obtained only if a given tumor suppressor locus is methylated (in contrast, control loci, which do not include HhaI sites, yield peaks invariably). Arrows, methylation of particular tumor suppressor gene loci in complex hyperplasia without atypia and complex hyperplasia with atypia, whereas no methylation is observed in normal endometrium.

Discussion

Early detection is crucial for the outcome of malignant disease, and endometrial carcinoma constitutes no exception to the general rule (2). In our previous study, tumors from MMR gene mutation carriers who participated in surveillance were of an earlier stage than those diagnosed based on symptoms, and no deaths from endometrial carcinoma occurred in the surveillance group (13). Endometrial hyperplasia, which may precede endometrial carcinoma, is common: it may explain 15% of postmenopausal uterine bleeding in the general female population (23) and was present in 8% of MMR gene mutation carriers screened by Renkonen-Sinisalo et al. (13). It is therefore important to diagnose developing endometrial malignancies and their precursors as early as possible to guide management decisions, including the possible need of prophylactic hysterectomy.

To our knowledge, no systematic molecular genetic study of serial follow-up samples of endometrium from MMR gene mutation carriers has been conducted before. Some investigations do exist that report the presence of molecular alterations in hyperplasia from individuals with Lynch syndrome. Berends et al. (24) studied 15 women from Lynch syndrome families, who underwent endometrial sampling for nonmalignant reasons, and found MLH1 or MSH2 protein loss in 3 of 4 patients with endometrial hyperplasia (2 with complex hyperplasia with atypia and 1 with complex hyperplasia without atypia). de Leeuw et al. (25) analyzed five cases of hyperplasia from patients who also had endometrial carcinoma and observed absent MMR protein corresponding to the germ-line mutation in all cases of hyperplasia (three complex hyperplasia with atypia, one complex hyperplasia without atypia, and one simple atypical hyperplasia). Ichikawa et al. (26) describe a MSH2 mutation carrier showing the loss of MSH2 protein in complex hyperplasia without atypia 7 months before endometrial carcinoma diagnosis. In a sporadic setting, Faquin et al. (27) conducted a retrospective study on patients who subsequently developed MSI(+) endometrial carcinoma, and in 1 of 13 patients, MSI was detectable in an endometrial sample with histologic diagnosis of “inactive” endometrium 7 years before the onset of endometrial carcinoma. Our study corroborates and extends these previous findings and shows that aberrant MMR protein expression, MSI, or tumor suppressor promoter methylation may be present in endometrial hyperplasia of various histologies, and even in normal endometrium, from MMR gene mutation carriers up to 12 years before endometrial carcinoma diagnosis (Table 3).

In colorectal carcinoma, it is debated if MSI analysis or immunostaining should be the primary method of prescreening for Lynch syndrome (28), whereas information available from endometrial carcinoma is limited. An important observation from our study was a higher sensitivity of immunostaining compared with MSI analysis in the detection of MMR defects in endometrial hyperplasia and endometrial carcinoma (Tables 2 and 3). Our data (with all hyperplasias originating from MLH1 mutation carriers, except for one) are in agreement with de Leeuw et al. (25) who showed that all three cases of endometrial hyperplasia from MLH1 mutation carriers were microsatellite-stable despite immunohistochemical loss of MLH1 expression. There may be a better concordance between immunohistochemical and MSI findings for MSH2-associated cases [ref. 25; a single complex hyperplasia without atypia from a MSH2 mutation carrier in our study, displaying MSH2 protein loss and MSI with 3 of 3 markers (data not shown)]. Of note, even in the present Lynch syndrome series, one third of endometrial carcinoma tumors were stable with all three microsatellite markers (Table 2), and the proportion of stable tumors may be still higher for certain other types of tumors (brain and adenocarcinomas of the kidney) arising in MMR gene mutation carriers (20). There are several potential explanations for the absence of MSI despite immunohistochemical loss of MMR protein, one of them being intratumoral heterogeneity (20, 29).

Previous studies have implicated PTEN as an early marker of endometrial tumorigenesis, either through mutations or reduced expression, which may occur at premalignant stages (30, 31). Even more striking, PTEN-null glands may be present in histologically normal endometrium in 40% of premenopausal women, and because only a small minority of such individuals will ever develop endometrial carcinoma, it was suggested that the initial PTEN alterations are not rate-limiting (32). Although we did not study gene expression, presumable inactivation of PTEN by promoter methylation occurred in <15% of our endometrial samples (Fig. 2). By comparing PTEN expression in endometrial hyperplasia from individuals, who subsequently developed versus did not develop (sporadic) endometrial carcinoma, a recent investigation found that loss of PTEN expression was neither associated with nor a sensitive and specific marker of progression to endometrial carcinoma (33).

Our study emphasizes early and widespread tumor suppressor promoter methylation changes in endometrial tumorigenesis. In Lynch syndrome (groups I and II), increased DNA methylation, especially CDH13 promoter methylation, was prevalent already in normal endometrium, being present in 42% of specimens (13 of 31) compared with 0% in the reference series (Fig. 2). Among 24 tumor suppressor genes studied, CDH13, RASSF1, and GSTP1 were the most frequently methylated genes in endometrial hyperplasia and endometrial carcinoma (Fig. 2). The overall methylation profile in the present endometrial carcinomas diagnosed in association with a surveillance project was comparable with that in an independent series of 38 Lynch syndrome–related endometrial carcinomas examined previously (18). Furthermore, our data are broadly concordant with reports on sporadic endometrial carcinoma of the endometrioid type, whereas endometrial hyperplasias have not previously been characterized relative to DNA methylation changes to any significant extent. CDH13, which encodes H-cadherin, a calcium-dependent cell adhesion protein, has been reported to be methylated in 35% to 71% of endometrial carcinoma samples (34, 35). RASSF1 codes for a Ras association domain family 1 protein (isoform A), a tumor suppressor, and is methylated in 33% to 85% of endometrial carcinoma (35–37) possibly correlating with advanced stage (38). GSTP1 encodes glutathione S-transferase π, which is involved in carcinogen metabolism, and promoter methylation (present in 31%) may be associated with myometrial invasion of endometrial carcinoma (39). Additionally, MSI phenotype that occurs in 20% to 30% of sporadic endometrial carcinoma of the endometrioid type is mostly explained by biallelic MLH1 promoter methylation (21, 22), whereas, not surprisingly, MLH1 methylation was rare in the present series of endometrial carcinoma from Lynch syndrome (Fig. 2).

The literature on endometrioid carcinoma precursors is usually based on retrospective sample materials taken from symptomatic women. Heterogeneity of endometrial tissue, incomplete representativeness of the sampled material, poor reproducibility of histopathologic diagnostics, and limited possibility to monitor the clinical behavior, particularly of complex hyperplasia without atypia and complex hyperplasia with atypia, have all hampered studies on the evolution of the endometrioid neoplastic process. Also, it is uncertain to which extent the diagnostic procedure and nonsurgical therapeutic intervention may affect the natural history of the neoplastic process. Largely based on the seminal study by Kurman et al. (4), nuclear atypia has been considered as the most important risk factor for progression to malignancy. In agreement, in sporadic cases investigated by Esteller et al. (40), MLH1 promoter methylation was primarily restricted to complex hyperplasia with atypia, the difference in methylation frequencies between complex hyperplasia without atypia and complex hyperplasia with atypia being statistically significant [1 of 33 (3%) versus 7 of 21 (33%); P = 0.0039]. In the present study on Lynch syndrome and sporadic cases, aberrant tumor suppressor promoter methylation, MMR protein expression, and MSI were equally common in complex hyperplasia without atypia versus complex hyperplasia with atypia, and the alteration frequencies in these complex hyperplasias were comparable with endometrial carcinoma but different from simple hyperplasia and normal endometrium. Our Lynch syndrome series mainly consisted of MLH1 mutation carriers and it remains to be seen if tumorigenesis is similar or different in carriers of mutations in other MMR genes, such as MSH6, which is particularly associated with endometrial cancer predisposition (41). Although confirmation in larger series is warranted, our findings suggest that complex hyperplasia without atypia and complex hyperplasia with atypia are equally important as precursor lesions of malignancy. The present results are supported by Pijnenborg et al. (37) who observed RASSF1A promoter methylation in 4 of 8 (50%) among endometrial hyperplasia specimens, and although the exact histology was not specified, the authors wrote that “atypia was present in only one case with endometrial hyperplasia, indicating that RASSF1A methylation was not restricted to those cases with cellular atypia.”

In conclusion, our results provide important new insights into the timing and molecular nature of the critical events involved in endometrial tumorigenesis and may turn out useful in the design of improved surveillance strategies for endometrial carcinoma in the future. Molecular alterations in biopsy specimens could serve as an indicator for prophylactic hysterectomy, for example, in MMR mutation carriers who would not primarily opt for prophylactic surgery (42).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.