INTRODUCTION
Endometrial hyperplasia (EH) is the abnormal proliferation of endometrial glands that occur as a result of a relative progesterone deficiency as well as chronic exposure to estrogen.[1] Irregular proliferation of the endometrium is characterized by abnormal gland-stroma ratio, histopathological complexity, and atypical features. However, it should not be forgotten that endometrial cancer (EC) may develop in untreated cases of EH.[1-3]
EHs were divided into two categories according to the presence of cytological atypia by WHO in 2014. Here, cases without atypia were classified as EH, while cases with atypia were classified as endometrial intraepithelial neoplasia.[4]
Although hyperplasia without atypia increases the risk of EC approximately four times, most cases resolve with curettage and hormonal therapy.[5] Considering that the incidence of EH is about three times the number of EC cases and that EH is the precursor lesion of EC, early diagnosis can prevent progression to cancer.[6]
Levonorgestrel-releasing intrauterine system [LNG-IUS]) and oral progestogens can be used in the treatment of EH without atypia. However, LNG-IUS is more preferred because the incidence of side effects is less and the rate of regression of the disease is higher.[7] Surgical treatment is required in case of atypical EH development during treatment, non-recovery of the disease despite 12 months of treatment, relapse of EH, non-stopping of abnormal bleeding, and an incompatible patient who refuses to follow-up during and after treatment. Bilateral salpingo-oophorectomy and total hysterectomy may be recommended for a postmenopausal patient requiring surgical operation.[2]
Hyaluronan (HA) is a simple glycosaminoglycan that is abundant in the extracellular matrix. HA plays an important role in the regulation of processes such as adhesion, proliferation, differentiation, migration, and invasion in cells.[8] CD44 is responsible for cell proliferation, differentiation, angiogenesis and uptake of hyaluronic acid (HA) into the cell by endocytosis.[9] HAS2 plays an important role in the synthesis of HA.[10] The presence of HAS2, which is associated with cell migration and invasion in some cancers, has also been demonstrated immunohistochemically in endometrial tissues.[11,12] In addition, it has been shown that HA accumulation increases as a result of decreased hyaluronidase levels in EC.[13]
The discovery of changes in membrane or intracellular components in cancer cells has paved the way for the development of new methods in cancer treatments.[14] In our study, we investigated HAS2 and CD44 immunoreactivity in predicting resistance to progesterone therapy in patients with EH without atypia, which is the leading lesion of EC.
MATERIALS AND METHODS
This retrospective case-control study was conducted after obtaining ethics committee approval. The study was conducted following the principles of the Declaration of Helsinki.
Selection of cases
Endometrial biopsy results obtained by carmen cannula and dilation and curettage between 2010 and 2020 in Firat University, Gynecology and Obstetrics Clinic and evaluated in Firat University Pathology Laboratory were scanned. During this period, all pathology reports diagnosed as EH without atypia were obtained. The previous pathology results of the case with each pathology result obtained were searched, and the old pathology results were reached. Cases who received uninterrupted progesterone treatment for 6 months were determined from the file information. After reaching the biopsy results of these cases 6 months ago, 4–6 μm thick sections from paraffin blocks were taken on polylysine slides for immunohistochemical staining. Since the number of patients who had complete pathology records for 10 years and who received continuous progesterone therapy for 6 months and did not improve was 20, the other groups were also formed using 20 patients. No distinction was made between the cases who used LNG-IUS for 6 months as progesterone treatment and who used medroxy progesterone acetate and norethisterone.
Group 1 (G1) (n = 20) = Normal PE group [Figure 1a]
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Figure 1: Normal proliferative endometrium (H and E), endometrial hyperplasia without atypia (H and E), and HAS2 and CD44 immunohistochemical images of all groups a = Normal proliferative endometrium. b = Endometrial hyperplasia without atypia. c = Endometrial hyperplasia without atypia persisted with treatment. d = Endometrial hyperplasia without atypia improved with treatment. e = HAS2 immune-positive cells are rarely seen in G1 (black arrow). f = There is a significant increase in HAS2 positive cells in G2 compared to G1, (black arrow). g = There is a marked increase in HAS2 immune-positive cells in G3 compared to G1, (black arrow). h = Compared to G3, there is a marked decrease in HAS2 immune-positive cells in G4 (black arrow). i = CD44 immune-positive cells are rarely seen in G1, (black arrow). j = An increase in CD44 immune-positive cells is observed in G2 compared to G1, (black arrow). k = An increase in CD44 immune-positive cells is observed in G3 compared to G1, (black arrow). l = An increase in CD44 immune-positive cells is observed in G4 compared to G1, (black arrow)
Group 2 (n = 20) = EH group without atypia [Figure 1b]
Group 3 (n = 20) = Group whose hyperplasia without atypia persisted with treatment [Figure 1c]
Group 4 (n = 20) = Group in which hyperplasia without atypia improved with treatment [Figure 1d].
Immunohistochemical staining
4–6 μm thick sections taken from paraffin blocks were placed on polylysine slides. Deparaffinized tissues were passed through a graded alcohol series and boiled in citrate buffer solution at pH: 6 in a microwave oven (750W) for 10 minutes for antigen retrieval. Deparaffinized tissues were passed through a graded alcohol series and boiled in citrate buffer solution at pH: 6 in a microwave oven (750W) for 12 minutes for antigen retrieval, and then the tissues were left to cool at room temperature for approximately 20 minutes. After the tissues had cooled, they were washed 3 × 5 minutes with PBS (Phosphate Buffered Saline, P4417, Sigma-Aldrich, USA). After cooling, the tissues were washed with PBS (Phosphate Buffered Saline, P4417, Sigma-Aldrich, USA) for 3 × 5 minutes and incubated in hydrogen peroxide block solution for 5 minutes to block endogenous peroxidase activity (Hydrogen Peroxide Block, TA). -125-HP, Lab Vision Corporation, USA). After washing with PBS three times for 5 min each time, the sections were treated with Ultra V Block solution (TA-125-UB, Lab Vision Corp.) to prevent background staining. Sections were incubated with primary antibodies (Mouse HAS 2 antibody: sc-365263, Dallas, Texas, USA and Rabbit CD44 antibody, K007092P, Beijing, China) diluted 1:100 for 60 min in a humid chamber at room temperature. Primary antibodies were then applied to the tissues and washed with PBS for 3 × 5 minutes. After washing, the tissues were incubated for 30 min at room temperature with secondary antibodies in moist environment (biotinylated goat anti-polyvalent (anti-mouse/rabbitIgG), TP-125-BN, Lab Vision Corporation, USA and donkey anti-goat, sc-2042, Santa Cruz Biotechnology, USA). The tissues were treated with streptavidin peroxidase (TS-125-HR, Lab Vision Corporation, USA) for 30 minutes in a humid condition at room temperature before being rinsed with PBS for 3 × 5 minutes. The tissues were dripped with a solution of 3-amino-9-ethylcarbazole (AEC) Substrate + AEC Chromogen (AEC Substrate, TA-015 and HAS, AEC Chromogen, TA-002-HAC, Lab Vision Corporation, USA), and the image signal was captured using a light microscope before being rinsed with PBS. IgG from rabbit served as a negative control. After counterstaining the tissues with Mayer’s hematoxylin, they were rinsed with PBS and distilled water and covered with occlusion solution (Large Volume Vision Mount, TA-125-UG, Lab Vision Corporation, USA). The prepared slides were examined, evaluated, and photographed under the Leica DM500 microscope (Leica DFC295).
The extent of immunoreactivity in staining (0. 1: <25%, 0.4: 26–50%, 0.6: 51–75%, 0.9: 76–100%) and severity (0: no, +0.5: very little, +1: little, +2: moderate, +3: severe) histoscore was established. Histoscore = prevalence x severity.[15]
Statistical analysis
Mean ± standard deviation was used to determine the data obtained. IBM SPSS Statistics 22.0 (IBM Corporation, Armonk, NY, USA) for data entry, and analysis package program was used. Intergroup evaluation was done with One-way ANOVA and posthoc tukey test. P < 0.05 values were considered statistically significant.
RESULTS
HAS2 Immunoreactivity
HAS2 immunoreactivity was higher in G2 [Figure 1f] than in G1 [Figure 1e], (P = 0.003). When G1 and G3 [Figure 1g] were compared, a significant increase was observed in G3 (P < 0.001). When G1 and G4 [Figure 1h] were compared, HAS2 immunoreactivity was similar (P = 0.536).
When G2 and G3 were compared, HAS2 immunoreactivity was significantly increased in G3 (P < 0.001). When G2 and G4 were compared, score values were similar in both groups (P = 0.187). In addition, HAS2 immunoreactivity was significantly decreased in Group 4 (P < 0.001) compared to G3 [Table 1].
Table 1: HAS2 Immunoreactivity histoscore for each group. Values are given as mean±standard deviation
HAS2 immunoreactivity scores in all groups are shown in Table 1.
CD44 immunoreactivity
When CD44 immunoreactivity (black arrow) was examined using immunohistochemical staining under light microscopy in comparison with G1 [Figure 1i], CD44 immunoreactivity was statistically significantly higher in G2 [Figure 1j], G3 [Figure 1k], and G4 [Figure 1l], (P = 0.001, P = 0.001, and P = 0.008, respectively). However, when compared to G2, no significant difference was observed in G3 and G4 (P = 0.728, P = 0.407, respectively). There was no significant difference in CD44 immunoreactivity (P = 0.918) in G4 compared to G3, (P = 0.918), [Table 2].
Table 2: CD44 immunoreactivity histoscore for each group. Values are given as mean±standard deviation
CD44 immunoreactivity scores in all groups are shown in Table 1.
DISCUSSION
In this study, we showed that HAS2 immunoreactivity was significantly increased in all of our EH cases when compared with the regular proliferative endometrium (PE) group, while it decreased in the proliferation group in the EH cases responding to treatment. CD44 immunoreactivity was found to be significantly higher in hyperplasia groups that responded and did not respond to treatment compared to our proliferation group. These results show that HAS2 immunoreactivity can be evaluated as an immunohistochemical marker in predicting resistant EH cases. However, we showed that CD44 immunoreactivity is not effective in showing resistance to therapy.
Progesterone therapy should be continued for at least 6 months to ensure regression of hyperplasia. It is recommended that the cases be followed up with endometrial sampling every 6 months.[2] In our study, patients were given progesterone therapy for 6 months for the treatment of EH without atypia. After 6 months of treatment, control biopsies were performed in all cases.
The effectiveness of some immunohistochemical markers in predicting the probability of transition from EH to EC has been investigated in the literature.[16] Progesterone receptor-B expression,[17] COX-2 expression,[18] p53 expression,[19] lamin receptor-1 expression,[20] TRPM2, and TRPM7[21] ion channel activities can be given as examples of these biomarkers investigated. We also considered it appropriate to study HAS2 and CD44 immunoreactivity as an immunohistochemical biomarker to investigate the potential of detecting EH cases resistant to progesterone therapy.
HA, also known as HA, plays a role in the emergence of cancer and some chronic/acute diseases as well as normal physiological processes. Experimental studies have shown that HYAL-1 hyaluronidase, HAS, and HA receptors induce angiogenesis as well as tumor growth and metastasis. Due to its close association with carcinogenesis, the HA family of molecules has been seen as a potential diagnostic and prognostic marker for some carcinomas such as bladder, breast, ovary, endometrium, and prostate. Each HAS is expressed alone or together. Expression of HAS contributes to the processes of angiogenesis, proliferation, tumor growth, invasion, and metastasis in xenografts. On the other hand, it has been shown that blocking these genes can inhibit the carcinogenesis process.[22] Therefore, in our study, we considered it appropriate to evaluate HAS2 immunoreactivity as a marker for detecting treatment-resistant EH cases, due to its proliferative, angiogenic, adhesion, migration, and metastatic properties.
CD44 is a family of transmembrane glycoproteins expressed at different levels in many cell types such as embryonic stem cells, connective tissues, and bone marrow. CD44 is used as a cancer stem cell marker in some cancers. Overexpression of CD44 induces cell regeneration and transition from epithelial to mesenchymal. The CD44 gene has standard (CD44s) and variant (CD44v) isoforms. The interaction of such isoforms, particularly with HA, osteopontin, and matrix metalloproteinases, induces carcinogenesis. CD44 plays a role in the development of resistance to oxidative stress. This contributes to the survival and growth of cancer cells. CD44 contributes to the prevention of cell damage by maintaining low levels of reactive oxygen species.[23] The binding of HA to the CD44 ligand-binding domain induces a variety of cellular changes and activates pathways associated with proliferation, adhesion, migration, invasion, and wound healing.[24,25]
In our study, we preferred to study HAS2 and its receptor CD44 activity due to proliferation and their strong relationship with each other. We investigated HAS2 immunoreactivity in reversible and resistant hyperplasia, with the thought that HAS2 blockade in hyperplasias may provide benefit in treatment by stopping proliferation.
It has been shown that increased serum HA levels in EC cases are associated with the depth of myometrial invasion, histological grade, and lympho-vascular space involvement.[26] In this study, we showed that the immunoreactivity score of HAS2 immunoreactivity increased in patients with treatment-resistant EH. In our study, we could not obtain information about lymphovasular and myometrial invasion, since we excluded cases of EH with atypia and early stage EC from the evaluation. This is because our study objective was only to investigate the resistance to treatment in EHs without atypia.
In a study by McLaughlin et al.[27] by inhibiting the synthesis of HA with 4-methylumbeliferon (4-MU), endometrial epithelial and stroma cells were reduced to peritoneal mesothelial cells (PMCs) compared to control. PMCs showed reduced attachment, migration and invasion. It has been suggested that this effect is mediated by a reduction in HAS2, HAS3, and CD44. Wang et al.[28] showed that HAS2 upregulation-induced endometrial malignancy, while knockdown of HAS2 reversed oncogenic effects. Saegusa et al.[29] suggested that although CD44v upregulation was evident in endometrial carcinomas, it was not an effective marker for cancer progression. In our study, although we found an increase in CD44 immunoreactivity in all hyperplasia groups compared to the normal PE group; we did not observe a significant difference in CD44 immunoreactivity between treatment-resistant and responder groups. Zhan et al.[30] found no significant difference in CD44 immunoreactivity with HA receptor between ectopic and eutopic endometrium in women with or without endometriosis. They reported that other mechanisms may play a role in HA-associated endometriosis, rather than the expression change of CD44. Increased CD44v-mRNA levels were detected in menstrual endometrial cells obtained from women with endometriosis. In addition, higher levels of CD44 forms have been detected in the peritoneal fluids of women with endometriosis. It has been reported that this phenomenon may indicate the presence of a compensatory defense mechanism against endometriosis. This is because it has been shown that the soluble form of CD44, a competitive HA inhibitor, can reduce the ability of endometriotic cells to attach to the peritoneal cavity.[30] Based on this information, the high level of CD44 immunoreactivity in all hyperplasia groups may be a defense mechanism of the body, despite the decrease in HAS2 immunoreactivity in our group who recovered with treatment. This is because the absence of atypia in our patients with persistent hyperplasia despite treatment may suggest that a similar defense mechanism may exist in endometriosis.
The limitations of our study are that the number of cases was limited, it was a retrospective study, and the amount of HA in the tissue could not be measured biochemically. In addition, the fact that HAS2 and CD44 gene expressions could not be studied is another limitation of our study. However, since our aim was to search for an easily accessible immunohistochemical marker, we only conducted our study with immunohistochemical staining.
The strengths of our study are that, unlike the studies above, our subjects were previously diagnosed with EH and the control biopsy results were evaluated after progesterone treatment. In addition, it is the first investigation of HAS2 and CD44 immunoreactivity immunohistochemically to determine treatment resistance in hyperplasia without atypia.
CONCLUSION
HAS2 can be used as an immunohistochemical marker in the detection of treatment-resistant EH cases, perhaps in predicting the progression to malignancy. Larger studies can be conducted where molecules that inhibit HAS2 biosynthesis can be used as therapeutic agents in EH and EC. In addition, CD44 has increased immunoreactivity compared to normal endometrial tissue in cases of EH and does not seem to have an effect on determining the prognosis.
Ethical approval
This retrospective case-control study was approved scientifically and ethically by the Ethics Committee of Firat University with the decision dated 21.07.2020 and numbered 2020/11-04.
Financial support and sponsorship
This prospective case-control study was financed by Firat University, Scientific Research Grant (Project number: TF.21.36).
This clinical study is derived from the master thesis of Dr. Emine Boybay.
Conflicts of interest
There are no conflicts of interest.
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