One of the most commonly presenting clinical complaint among postmenopausal women is  bleeding (PMB), which causes great concern because of its strong link with the endometrial carcinoma^[1]. The natural phenomenon of cessation of ovulation and decreased hormone production results in atrophy of the endometrium. Bleeding from the vagina occurring after 12 months from the beginning of menopause should be considered as abnormal and requires an urgent investigation^[2,3].

Causes of bleeding vary from non neoplastic conditions such as atrophic endometrium, polyps, and simple hyperplasia to frank malignancy^[4,5]. Even though about 80% of the cases are related to benign changes, PMB is the main symptom observed in 90% of patients with endometrial carcinoma^[6]. Endometrial polyps and hyperplasia can cause similar symptoms that also require early diagnosis^[7].

A biopsy test, preferably outpatient one, still remains the golden diagnostic method of choice regarding endometrial lesions^[8]. The development of the immuno-histochemistry (IHC) technique increased diagnostic and differential diagnostic ability drastically. Endometrioid carcinomas usually are stained positive for Estrogen receptors (ER), Progesterone Receptors (PR), and Vimentin; while more than 90% of serous carcinomas have All or None expression of p53 ^[9,10]. This research project aimed to study the histopathology of endometrium and IHC profile of diagnosed malignancies.

MATERIAL AND METHODS

The study was carried out by means of a descriptive study design with an overall study population of 300 patients. Non-probability consecutive sampling technique was employed, wherein all the endometrial samples available at our Department of Pathology, Government Medical College during the entire 18 months of study period, starting from April 2021 and ending October 2022, were included. The strict inclusion criterion for this study was the inclusion of only such endometrial samples as those which belong to postmenopausal women who presented with bleeding. To ensure adherence to the demographic scope of the study, endometrial samples of women in their premenopausal stage of life were not considered in the current study. The study protocol was reviewed and approved by our Institutional Ethics Committee. The research was conducted in full compliance with standard ethical guidelines for clinical and pathological studies, ensuring proper clearance before analyzing the patient samples collected during the study period from April 2021 to October 2022.

Endometrial samples were obtained via endometrial biopsy (53.7%), total abdominal hysterectomy with bilateral salphingo-oophorectomy (TAH+BSO) (37.3%), fractional curettage, and other surgical procedures. Tissue specimens were fixed in 10% neutral buffered formalin, routinely processed, and embedded in paraffin wax. Sections of 4-micrometer thickness were stained with Hematoxylin and Eosin (H&E) for morphological diagnosis. Stringent quality controls were applied in the process of tissue processing and interpretation. All the samples of the endometrial tissues collected were equally treated through fixation in 10% formalin, processing, and embedding in paraffin for sectioning and staining. In carrying out immunohistochemistry on the tissues, the process adopted the technique of antigen retrieval by heat treatment. Immunohistochemistry markers' interpretations adhered to a specified procedure; for example, the determination of estrogen receptor status (ER) was done using the Allred scoring technique that involved proportion and intensity.

Twenty-eight samples diagnosed with malignancy were subjected to IHC study using the Heat Induced Epitope Retrieval (HIER) method. The marker panel included ER, Vimentin, p16, AMACR, and p53. The ER status was interpreted using the Allred scoring system, which combines the percentage of positive cells (Proportion Score, 0-5) and the intensity of the reaction (Intensity Score, 0-3). Scores between 3 and 8 were considered positive. Vimentin positivity was defined by strong cytoplasmic and membrane staining, p16 by strong and diffuse block positivity, p53 by all or none pattern and AMACR by cytoplasmic granular positivity.

RESULTS

The age of the study participants ranged from 45 to 90 years, with a mean age of 53.87 ± 7.06 years. The majority of patients (44.3%) were in the 45-50 years age group. On histopathological examination, benign lesions were predominant, accounting for 245 cases (81.7%). Malignant lesions were found in 32 cases (10.7%), and 23 samples (7.7%) were inadequate for reporting.

Table 1: Histopathological Diagnosis of Endometrial Samples

Age distribution correlated with the type of lesion: benign lesions were most common in the younger age group (40-50 years), while the incidence of malignant lesions peaked in the 51-60 years age group (68.8% of all malignancies).

Immunohistochemical Profile

Twenty-eight malignant samples were analyzed using IHC (Table 2). Of the 26 endometrioid adenocarcinoma (EEA) cases, 20 were Grade I and 6 were Grade II. All EEA cases demonstrated 100% strong positivity for ER and Vimentin, and complete negativity for p53.

The single case of clear cell carcinoma showed intense, diffuse granular cytoplasmic positivity for AMACR, while testing negative for ER, Vimentin, and p53. The serous carcinoma case exhibited all or None staining pattern for p53, consistent with mutated Tp53, and lacked expression of the other markers.

Table 2: Immunohistochemical Marker Profile of Analyzed Malignancies (n=28)

Figure 1 Histopathology and Immunohistochemistry of various samples

DISCUSSION

Postmenopausal bleeding is one of the important symptom of an underlying malignancy, hence, the need for a high index of suspicion in diagnosis. In our study, the mean age at diagnosis was 53.87 years, which is almost equal to the age of 53.22 years recorded by Vijayakumar et al^[4], while the age of Mallick et al.^[5] is higher at 57.12 years.

Non neoplastic lesions were identified in 81.7% of our patients. The atrophic endometrium was found in 31.8% of those cases; this finding corresponds well with postmenopausal physiology since deprivation of estrogen results in atrophy of glands. Atrophic endometrium has a similar prevalence of 32% according to Srivatsa et al.,^[13] but it is much less compared to the prevalence of 40.7% documented by Mallick et al.^[5]. The endometrial polyp, accounting for 28.6% of the benign conditions, was much more prevalent than the only 4.6% recorded by Mallick et al.^[5]

Endometrial malignancy was seen in 10.7% of the cases. This incidence is similar to Srivatsa et al.'s study (10.4%), whereas Mallick et al. document a prevalence of 52.8%.^[5,13] Most endometrial malignancies diagnosed in our study had endometrioid morphology (81.3%).

It is necessary to distinguish between type I and type II endometrial cancers based on the different molecular mechanisms involved in their pathogenesis. Immunohistochemical staining showed that type I endometrial adenocarcinoma was negative for p53 but positive for ER and vimentin, a result that agrees with current literature^[14]. Type II serous endometrial carcinoma stained positively for p53, indicating a mutated TP53 protein, thus, distinguishing this carcinoma from the others. Clear cell endometrial carcinoma showed a strong positivity for AMACR protein^[15].

Strengths of the Study

The main strength of this research is a large sample size consisting of 300 women who are postmenopausal, making it highly representative with respect to the range of histopathological changes associated with endometrial lesions in this population group. In addition, the methodological design of the study transcends mere identification through a combination of an extensive array of immunochemistry (ER, Vimentin, p53, p16, AMACR) in all 28 types of malignant cases. This feature of IHC adds a lot to the diagnostic power of this research, providing a clear distinction between various endometrial carcinomas.

Limitations of the Study

Though the study is robust, there are a few limitations that the study is subject to. First, being a descriptive study done in a government medical college, the results may only be applicable to a particular demographic within the community. Secondly, out of the 300 samples used in the study, 23 of them (7.7%) were deemed unsuitable for histopathology report. Thirdly, though there were many samples in total, the number of each type of high-grade malignancy, which included clear cell carcinoma, serous carcinoma, and carcinosarcoma, was very low (each with one sample).

CONCLUSION

Most common cause of postmenopausal bleeding is non neoplastic conditions, which mainly  include atrophic endometrium. Nevertheless, due to the fact that malignancy is associated with the aging process, it becomes imperative to conduct histopathologic evaluation for all such cases. Endometrioid adenocarcinoma is the type of malignancy frequently observed. Immunohistochemistry, which includes ER, vimentin, p53, and AMACR, plays a key role in subtyping such lesions.

REFERENCES

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