Immunosuppressive Glycodelin A is an independent marker for poor prognosis in endometrial cancer

Endometrial cancer is the fourth common carcinoma in women following cancer of breast, colon and lung and accounts for 5.6% of all malignancies [1]. The diagnosis of endometrial cancer is typically made at postmenopausal age [2] and its 5-year survival ranges between 75 and 83% [2].

Some risk factors for the development of endometrial cancer have been described [3–8], though the exact mechanisms in tumourigenesis are by far not explained. A fast tumour progression is most likely favoured by local immunosuppression, which decreases the body’s own anti-tumour immunoreactivity. Until today little is known about tumour induced, local immunosuppression in endometrial cancer.

Glycodelin (Gd), also known progestagen-associated endometrial protein, is a glycoprotein with immunosuppressive capacity, which is mainly produced in reproductive tissue [9, 10]. Four different isoforms have been described: GdS (in seminal vesicles and seminal plasma) [11], GdA (in endometrium/decidua, amniotic fluid, maternal serum) [12, 13], GdF (in follicular fluid und oviduct) [14] und GdC (in the cumulus oophorus) [15]. The isoforms share a common protein backbone but differ in glycosylation and biological activity [16, 17].

GdA holds several immunosuppressive abilities, which are best characterized in reproductive medicine [18]. These include the suppression of lymphocyte proliferation and inhibition of T- and B-cell activity [19–21]. Moreover, the induction of apoptosis via GdA has been investigated [22].

Recently, we found GdA to be of prognostic significance in ovarian cancer [23]. So far, there are very few results on endometrial cancer cells and Gd or GdA [24] and no clinical data on endometrial cancer. Therefore, the aim of this study was to assess the expression of Gd on mRNA and protein level. Further, we aimed to specify the proportion of the immunosuppressive glyko-modification GdA in tissue samples of a large cohort of endometrial cancer patients by using an extensively validated anti GdA antibody. Finally, we aimed to analyse the impact of Gd/GdA positivity on clinical and pathological features including patient outcome.

Endometrial cancer tissue of 292 patients (Table 1) was available. Data of 291 cases analysed for Gd, 289 cases stained for GdA and 254 cases analysed for Gd mRNA were included in the statistical analysis. Remaining cases (IHC: Gd: n = 1, GdA: n = 3 and ISH: Gd mRNA: n = 38) had to be excluded due to technical reasons. Mean patient age at primary diagnosis was 65.1 ± 0.6 years (range 35–88 years) and further patient characteristics are listed in Table 1.

Gd mRNA expression in endometrial cancer tissue

Intermediate (37.8%) or high (24.0%) expression of Gd mRNA (PAEP, progestagen-associated endometrial protein) was detected in the majority of cases investigated here (Figure 1). Though mRNA in situ hybridization revealed Gd transcripts to predominantly localize to the tumour epithelium, no significant difference in Gd mRNA expression was detected among histological tumour subtypes (Figure 1). In the current study positivity for Gd mRNA was neither statistically associated with histological tumour grade nor with the patients’ FIGO stage.

Gd and GdA in endometrial cancer tissue

Using a polyclonal antiserum we also proofed presence of Gd protein in endometrial tissue. Immunohistochemical staining showed endometrial cancer tissue to be intermediately or highly positive for Gd in 52.2% or 20.6% (Figure 2, Table 1), respectively. A significant proportion of endometrioid carcinoma cases were observed to produce an immunosuppressive Gd glyocmodification, termed GdA. The latter was present at intermediate or high levels in 62.6% or 9.0% of cases, respectively. Inspite the fact that immunoreactivity for the Gd protein was positively correlated with Gd mRNA expression (Correlation coefficient 0.155, p = 0.013), no such association was observed when Gd mRNA was correlated with the glyco-variant GdA. However, GdA immunoreactivity was closely correlated with Gd protein expression (Correlation coefficient 0.249, p < 0.001).

The highest median Gd expression was noted for the undifferentiated histological subtype (median IRS 8.0; mean IRS 7.80 ± 0.49, Additional file 3), followed by the endometrioid (median IRS 6.0; mean IRS 5.9 ± 0.23), the serous (median IRS 6.0; mean IRS 5.74 ± 0.76) and the mixed cell type (median IRS 6.0; mean IRS 4.97 ± 0.60), though differences among Gd expression and the histological subtypes did not reach statistical significance (p > 0.05) (Figures 2 and 3). Comparable results were observed for GdA expression and the histological subtype (Figure 2 and 3). The most common histological subtypes (endometrioid and serous) show median GdA expression of IRS 6.0. Also, no statistically significant differences in GdA expression were observed among the different histological subtypes (p > 0.05) (Figures 2, 3 and Additional file 3).

Interestingly, there is a significant reduction in Gd expression observed from FIGO III to FIGO IV (p = 0.044) (Figure 3). However, overall Gd/GdA immunoreactivities comparing cases of low vs. high FIGO stage were not significantly different (Additional file 4). There were no significant differences in Gd and GdA expression between different tumour grades (Figure 3). Immunoreactivity of Gd or GdA staining was not significantly different comparing cases being negative vs. positive for ERs, PRs or co-morbidities.

Prognostic value

Statistical analysis was also performed to test for a prognostic value of Gd or GdA expression. Univariate Kaplan Meier analysis revealed a good prognosis for intermediate and high Gd expression (p = 0.039) (Figure 4A). In contrast, highly positive GdA endometrial cancer patients had a poor outcome compared to intermediate and low GdA expression (p = 0.003) (Figure 4B). Gd mRNA expression was not significantly associated with patients’ outcome.

Besides tumour stage, grade and the concomitant diagnosis of hypertension (each p < 0.05), Cox-regression analysis (Table 3) showed GdA to be an independent prognostic marker for patient survival (p = 0.002, 95% CI 1.362-3.943).

			95% CI
Covariate	Coefficient (bi)	[HR Exp(bi)]	Lower	Upper	P-value
FIGO stage					<0.001
I	(0.000)	(1.00)
II	−3.064	.047	.011	.192	<0.001
III	−3.025	.049	.010	.233	<0.001
IV	−1.810	.164	.040	.669	.012
WHO grade					.023
1	(0.000)	(1.00)
2	−.820	.440	.246	.790	.006
3	−.561	.571	.325	1.002	.051
Histology					0.068
Endometrioid	(0.000)	(1.00)
Serous	−.297	.743	.233	2.372	.616
Clear cell	−.069	.934	.260	3.349	.916
Mucinous	−1.423	.241	.024	2.403	.225
Squamous cell	.074	1.077	.260	4.470	.918
Mixed cell	3.167	23.742	2.245	251.079	.008
Undifferentiated	−.163	.850	.255	2.828	.790
Lymph node metastasis	−.732	.481	.209	1.109	.086
Age (≤50 y vs. >50 y)	1.939	6.953	.909	53.204	.062
Diabetes	.451	1.570	.880	2.800	.127
Obesity	−.065	.937	.600	1.462	.774
Hypertension	.454	1.575	1.043	2.380	.031
Lymphangiosis	.216	1.241	.631	2.443	.532
Hemangiosis	.681	1.975	.476	8.187	.348
ER alpha	−.031	.970	.652	1.442	.880
PRA	−.156	.855	.572	1.279	.446
GdA	.840	2.317	1.362	3.943	.002
Gd	−.298	.743	.456	1.209	.232

Significant results are shown in bold.

Endometrial cancer can be subdivided into two histological subtypes, the estrogen-associated Type I and the estrogen-independent Type II carcinoma [36, 37]. The most common cause for endometrial Type I carcinoma is thought to be an excess of estrogens, which are inadequately antagonized by gestagens [38]. Therefore obesity, polycystic ovarian syndrome, menopausal hormone use are associated with a higher risk for endometrial cancer [3–5]. The Type II carcinoma, which comprises mostly the serous and clear cell histological subtypes, is known to metastasize more often and to have a worse survival. In contrast to endometrial Type I carcinomas estrogen dominance does not seem to be causally linked to this type of the disease, rather higher age and previous radiation therapy of the uterus [39].

The majority of cases are classified as Type I carcinoma and comprise the endometrioid adenocarcinomas. In literature it accounts for 75-85% of all adenocarcinomas [36, 37, 40], which is in accordance with our study population of 72.6% endometrioid tumours.

Interestingly, we found the concomitant diagnosis of hypertension to be a negative predictor in patients diagnosed with endometrial cancer. This finding is in accordance with newly published data by Nicholas et al., who reported diabetes and hypertension to adversely affect survival and demanded to give more attention to comorbidities, since they are gaining more influence on current health care and policy [41].

Though Gd has been identified in a range of different tissue types, not all of them do indeed synthesize the protein, which is made evident by the presence and absence of Gd mRNA [14, 15, 42, 43]. Our immunhistochemistry results were confirmed by in situ hybridization showing not only the presence of Gd in endometrial cancer but also its synthesis and thus underline its role in carcinogenesis. To our best knowledge this is the first study reporting Gd to be present on both mRNA and protein level in endometrial cancer. Moreover, existence of Gd mRNA and its close correlation to Gd protein immunoreactivity suggests that endometrial cancer cells themselves possess the ability to synthesize the Gd protein. Interestingly, no significant association of Gd mRNA and the immunosuppressive Gd glyo-epitope GdA was observed, implying that GdA positivity marks a subfraction of endometrioid cancer that cannot be predicted by sole presence of Gd mRNA. Unfortunately, due to the very limited amount of tissue available protein extraction and western blot analysis, which would allow direct quantification of Gd/GdA of the same sample, was not possible.

In hormone-dependent tumours, Gd has been described to have various effects through reduced expression of oncogens and raised expression of tumour suppressor genes. Among these Gd induced chances are reduced tumour growth, decreased metastatic properties or decreased chemoresistance [24, 44]. Hautala et al. showed glycodelin to reduce breast cancer tumour growth in vivo [44]. Koistinen et al. transfected endometrial adenocarcinoma HEC-1B cells with Gd cDNA in both antisense and sense orientations [24]. They observed sense-transfected, Gd-producing carcinoma cells to have a reduced proliferation, morphologic changes, and altered expression of cancer-related genes in comparison to native and antisense-transfected carcinoma cells [24]. These results illustrate some aspects of Gd’s potential in gynecolocical cancers. In some hormone-depending tumours, Gd expression has been shown to go along with a favourable outcome, like in breast and ovarian cancer [45, 46]. Results on ductal carcinoma in situ and invasive breast revealed that Gd positivity is inversely correlated with the occurrence of metastasis [45]. These data are in line with our findings, though Gd expression reached only univariate prognostice significance in Kaplan Meier analysis.

Recently histone deacetylase inhibitors (HDACIs) have been highlighted as promising new anti-cancer agents. In 2006 the HDACI suberoylanilide hydroxamic acid (SAHA, Vorinostat (rINN), Zolinza®) has been approved by the FDA for the treatment of cutaneous T-cell lymphoma and has further been evaluated in patients suffering from e.g. glioblastoma multiforme [47], non-small-cell lung [48] cancer or myelodysplastic syndroms [49]. Uchida et al. [50–52] demonstrated that SAHA is capable of up-regulating Gd in endometrial cancer and choriocarcinoma cell lines and further that SAHA induced Gd in fact influences cell differentiation and migration in the model system employed. Since we found that Gd is significantly associated with prolonged overall survival in endometrial cancer, it remains challenging to investigate whether endometrial cancer patients might also benefit from the application of SAHA. Of course randomized and properly powered clinical trials are indispensable in order to validate this hypothesis on a clinical basis.

Depending on Gd glycosylation status, it can induce apoptosis in T cells and monocytes. These in vivo results on Gd and GdA may explain the partially contradictory results in clinical studies. In contrast to Gd, we observed a poor outcome in patients expressing the immunosuppressive isoform GdA. This result was made not only on the basis of univariate but also multivariate survival analysis and is in concordance with a recently published study on ovarian cancer and GdA, where we report GdA to be a prognostic marker for poor outcome in advanced stage ovarian cancer [23]. Nevertheless, there are controversial results on Gd expression and patient survival [23, 45, 46, 53]. These may be attributable to various mono- and polyclonal antibodies being either peptide-specific or glycosylation specific. Bearing in mind that differently glycosylated Gd isoforms may exert opposing actions may at least partially explain the conflicting research results published on this issue [54]. Functional analysis e.g. employing an endometrial cancer animal model is thus needed to further clarify the immunomodulatory actions of Gd/GdA.

In conclusion, Gd and GdA are commonly expressed in endometrial cancer tissue and seem to be of relevance in tumourigenesis. They differ not only in glycosylation but also in their biological activity, since Gd is associated with a better survival, whereas GdA holds prognostic significance for a poor outcome in endometrial cancer patients. Therefore, Gd and especially GdA might help to select patients for a more individualized tumour therapy.

As stated above the current study has been approved by the ethics committee of the Ludwig-Maximilians University Munich (approval number: 063–13) and has been carried out in compliance with the guidelines of the Helsinki Declaration of 1975. All specimens included in this study were left over samples collected during routine clinical diagnostics. Patient data were fully anonymised and the current study has been approved by the ethics committee of the LMU Munich.