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Histopathological patterns of primary malignant ovarian neoplasms in different age groups in Almadinah Almunawwarah region, KSA

Objectives In the literature, the epidemiological pattern of ovarian neoplasms in different age groups in the Almadinah Almunawwarah region in KSA has not been completely elucidated. Moreover, an unusually frequent diagnosis of adult granulosa cell tumour (AGCT) has been observed in patients in Almadinah Almunawwarah, KSA. This study aimed to describe the pattern of ovarian neoplasms in different age groups in the Almadinah Almunawwarah region with particular emphasis on AGCT. Methods Histopathological records of all ovarian specimens diagnosed from 2011 January to 2016 December were collected from the Maternity and Children Hospital in Almaadinah Almunawwarah, KSA. Hematoxylin and eosin (HE)-stained microscopic slides of serous and mucinous epithelial borderline neoplasms and of malignant epithelial, sex cord-stromal and germ line neoplasms were identified and examined. The tissue sections from the AGCT were stained immunohistochemically with BRCA-1 antibody. Results A total of 301 ovarian specimens were obtained. Of the specimens, 217 (72%) were neoplastic and 84 (28%) were non-neoplastic. In total, 135 (63%) of the neoplastic specimens were benign, 16 (7%) were borderline tumours, and 66 (30%) were malignant tumours. Moreover, 41 (62%) of the malignant tumours were surface epithelial carcinomas, 17 (26%) were sex cord-stromal tumours, and 8 (12%) were germ cell tumours. The incidence of AGCT was unusually high, which accounts for 26% (16/66) of all malignant ovarian neoplasms. Altered BRCA-1 expression was observed in only two specimens. Conclusion In this study, malignant ovarian neoplasms accounted for 30% of all neoplastic ovarian specimens, and the incidence of AGCT was remarkable. Such tumours did not show a significantly altered expression of BRCA-1. Further studies must be conducted to explore the underlying molecular causes of this condition.

Objectives: In the literature, the epidemiological pattern of ovarian neoplasms in different age groups in the Almadinah Almunawwarah region in KSA has not been completely elucidated. Moreover, an unusually frequent diagnosis of adult granulosa cell tumour (AGCT) has been observed in patients in Almadinah Almunawwarah, KSA. This study aimed to describe the pattern of ovarian neoplasms in different age groups in the Almadinah Almunawwarah region with particular emphasis on AGCT.
Methods: Histopathological records of all ovarian specimens diagnosed from 2011 January to 2016 December were collected from the Maternity and Children Hospital in Almaadinah Almunawwarah, KSA. Hematoxylin and eosin (HE)-stained microscopic slides of serous and mucinous epithelial borderline neoplasms and of malignant epithelial, sex cord-stromal and germ line neoplasms were identified and examined. The tissue sections from the AGCT were stained immunohistochemically with BRCA-1 antibody.

Introduction
Ovarian neoplasm is the seventh most common malignant tumour in women, and it is the most fatal gynecologic malignancy. After surgery and chemotherapy, the 5-year survival rate of patients is 30%, which is extremely poor. 1 The most important factor in improving the outcome of ovarian malignancies is early diagnosis with a full understanding of the molecular mechanisms underlying the development and progression of the disease. 2 A positive family history of the disease, particularly among first-degree relatives, is one of the most significant risk factors of ovarian carcinoma. 3 .
Several molecular abnormalities, such as alteration in the expression of tumour suppressor genes (BRCA-1, BRCA-2) 4 as well as dysfunction in the P53 and Kras genes, 1 play an important role in the development and prognosis of ovarian cancer.
To the best of our knowledge, the prevalence of ovarian neoplasms and distribution in different age groups in Almadinah Almunawwarah region, KSA, have not been fully elucidated. Surface epithelial tumours are the most common type according to different regional studies. 5e8 During our practice in the histopathology laboratory of Maternity and Children Hospital (MCH) in Almadinah Almunawwarah, we observed an unusually frequent diagnosis of adult granulosa cell tumours (AGCT), which is a type of ovarian cancer that is considered rare worldwide. 9 A recent report has shown that the incidence rate of AGCT in KSA is 2.3%. 10 GCT belongs to the group of ovarian sex cordstromal tumours, which differ from the common ovarian epithelial malignant tumours presenting in younger individuals and the indolent course of the disease.
The molecular basis underlying the development of GCT is less understood compared with that of the common surface epithelial tumours. 11 BRCA genes are tumour suppressor genes that normally maintain the integrity of the genome. BRCA mutation plays a significant role in the development of some ovarian tumours, such as ovarian serous carcinoma. However, their role in the development of GCT is not known. 12 Thus, the present study aimed to 1. assess the frequency of the different histopathological types of primary malignant ovarian tumours in Almadinah Almunawwarah region. 2. describe the age distribution of each tumour category compared with the reported figures in other regions of KSA and other countries worldwide. 3. assess the frequency of AGCT compared with that of other ovarian neoplasms 4. analyse the pattern of BRCA-1 expression in individuals with AGCT to identify if its expression is altered in an attempt to obtain a possible explanation for the high incidence of AGCT.

Settings
The histopathological reports of primary ovarian specimens obtained from January 2012 to December 2016 were collected from Maternity and Children Hospital (MCH), which is a tertiary hospital that provides services to patients from Almadinah Almunawwarah and other surrounding areas. It is the referral center for ovarian surgical specimens. H&E-stained tissue slides and formalin-fixed paraffinembedded blocks of ovarian malignant tumours were identified and collected.

Specimens
The inclusion criteria included ovarian specimens obtained in the MCH histopathology and cytology department or received from other nearby hospitals on the condition that their data are found in the database of the department of pathology. For descriptive purposes, malignant tumours were classified into three groups according to the age of the patients (<20 years, 21e50 years, and >50 years). The incidence and percentage of each type in each age group were calculated. Tumours were classified into these age groups because ovarian tumours are heterogeneous in their age at presentation, and some tumours are most common during postmenopausal age. Moreover, tumours, such as juvenile granulosa cell tumour and yolk sac tumours, are most common at a young age. Meanwhile, others, such as adult sex cord-stromal tumours, are common during middle age. This stratification was used in similar previous studies for similar purposes. 8 The representative sections from each specimen were collected for a detailed histopathological examination, and tumours were classified into surface epithelial, germ cell, and sex cord-stromal tumours according to the recent WHO classification. 2 Each type was further classified into subtypes, which primarily depends on the following histological features: surface epithelial tumours classified into serous, mucinous, endometrioid, transitional, clear cell adenocarcinoma, and borderline. Sex cord-stromal tumours were subtyped into granulosa-stromal cell tumours and Sertoli-stromal cell tumours, and the former was further subtyped into the granulosa cell tumour group (adult and juvenile) and thecoma-fibroma group. The germ cell tumours were subdivided into dysgerminoma, choriocarcinoma, yolk sac tumour, embryonal carcinoma, and immature teratoma according to the recent WHO classification. 2 An unusually high frequency of AGCT compared to the known average international frequency was observed. The diagnosis was made via H&E staining and was confirmed via inhibin alpha immunohistochemical staining. 13 Because of the known role in the genetic predisposition of some ovarian tumours and the high incidence of certain genetic disorders, such as inherited hemolytic anemias, in certain regions of KSA, we assessed the level of BRCA-1 protein expression in these tumours to identify if the cause of the unusual increase in the incidence is the aberrant expression of BRCA-1.

Immunohistochemical staining
Sixteen formalin-fixed paraffin-embedded blocks of AGCT were identified. In total, 15 specimens with normal ovarian surface epithelium and 15 specimens with normal tubal epithelium were used as controls. Tissue sections from each specimen were stained using the Rabbit anti-BRCA1 polyclonal antibody. Staining was performed using an immunoperoxidase technique with the autostainer Bench-Mark ULTRA instrument. In summary, paraffin sections were deparaffinized, rehydrated, treated in 3% H 2 O 2 for 5 min, and washed. The sections were then incubated with the primary Ab (BRCA-1, Origene; TA310042, polyclonal; 1:100) for 45 min at room temperature. Antigeneantibody reaction was visualized using diaminobenzidine, counterstained with hematoxylin, dehydrated, and mounted.
Assessment of BRCA-1 expression was conducted using a semiquantitation approach. The intensity of nuclear staining was scored in a scale of 0e3: 0: negative; 1: weak; 2: moderate; and 3: strong. The same approach was used in several studies. 14

Statistical analysis
Statistical analysis was performed using the Statistical Package for the Social Sciences software version 21 for Windows (SPSS Inc., Chicago, IL, the USA). Descriptive analysis using frequencies and percentages was carried out.

Results
Between 2012 January and 2016 December, data about 301 primary ovarian specimens were retrieved from the computerized database of the histopathology laboratory of MCH in Almadinah Almunawwarah region. Nonneoplastic lesions were identified in 84 (30%) of 301 specimens. A total of 217 specimens were diagnosed as ovarian neoplasms. Of the 217 neoplasms, 135 (63%), 16 (7%), and 66 (30%) were benign, borderline neoplasms, and malignant neoplasms, respectively.
Majority of sex cord-stromal tumours were granulosa cell tumours of adult type (16/66), followed by malignant Sertoli cell tumour. 2 All AGCT were unilateral, and of the 16 AGCT, 9 (56%) were found in the left ovary. Variable histological patterns were observed in individuals with AGCT. The tumours were composed of proliferating granulosa cells forming diffuse sheets, cords, trabeculae, characteristic Call-Exner bodies, and a combination of all previous patterns. Granulosa cells showed few cytoplasms and ovoid nucleus with longitudinal groove and prominent nucleoli, and mitosis varied from 1 to 5 HPF. Moreover, 1 (6%) of 16 tumours presented with pleomorphism and brisk mitoses. The diagnosis of all AGCT was confirmed via inhibin alpha staining.
The mean age of the participants was 41 years, and majority of cases (n ¼ 48) were in the 20e49 age group, except  for germ cell tumours in which more than half of the cases involved patients younger than 20 years. Women older than 50 years primarily presented with surface epithelial tumours (n ¼ 12; 63%), followed by sex cord-stromal tumours (31.5%). The mean age of the patients with AGCT was 44.5 (age range: 30e75) years.

BRCA-1 expression in granulosa cell tumours
All normal ovarian surface and fallopian tube epithelium showed a uniform strong positive expression of BRCA-1. All granulose cell tumours were positive for BRCA-1 expression, and of the 16 specimens, 14 presented with a strong positive expression with heterogenous intensity. A weak expression was detected in two specimens (Figure 1).

Discussion
This study described the pattern of ovarian neoplasms in different age groups in Almadinah Almunawwarah region and compared with the known pattern of these tumours worldwide.
In our cohort, 70% of the surgically resected ovarian specimens collected at MCH were ovarian neoplasms, and 30% of which were malignant tumours. Our finding is within the range reported in studies on different ethnic populations worldwide. The reported rates of malignant ovarian tumours in previous studies were as follows: 15% from Africa, 15 40% from Asia, 16 20e25% from the western population, 17 and 22% from other regions in KSA. 5 The most common type of malignant neoplasms was the surface epithelial tumours with the serous cystadenocarcinoma, which is the most frequent type. They comprise 62% of malignant neoplasms in our cohort, which is similar to the data reported in KSA 5 and other countries worldwide.
Sex cord-stromal tumours were diagnosed in 26% of our specimens. Approximately 94% of such tumours were AGCT. Our rate (26%) was significantly higher than that reported from other areas in KSA (7.6%), 5 in studies of women from Asia (2e3%), 18,19 and in the western population where sex cord-stromal tumours account for 5e 10% of all ovarian malignancies. 17 This category of tumours originates from the stroma of the ovary, which originate from the sex cord cells of the embryonic gonad. The sex cord of the embryonic gonads is differentiated during development into granulosa and theca cells in women and to Sertoli and Leydig cells in men. Therefore, tumours arising from the ovarian stroma can be any of these four cell types; granulose cell tumours that are usually the mixture of granulosa and theca cells, Sertoli cell tumours, or Leydig cell tumours. 17 The molecular carcinogenic mechanisms underlying sex cord-stromal tumours are not well understood. By reviewing the literature, we observed a variation in the incidence of AGCT among the ethnic populations. In a study in the USA, a higher incidence of sex cord-stromal tumour was observed in black women than in white woman, whereas a significantly higher frequency of surface epithelial tumours was observed in white woman than in black woman. 20 These AGCTs were almost unilateral and have varying histological patterns with little pleomorphism and low mitotic activity. Our histopathological findings are in accordance with the known features of these tumours. 18 The histopathological morphological pattern of GCT is sometimes challenging for pathological diagnosis as it may mimic other carcinomas, particularly endometrioid carcinoma. We stained the GCT with inhibin for the confirmation of diagnosis. Inhibin is a protein secreted with granulosa cells and is a specific marker for the diagnosis and follow-up of GCT. 11,13 We examined the reason behind the high incidence rate of GCT in our patients. Moreover, it may be a familial cause due to certain genetic abnormalities that can be inherited from one generation to another in Almadinah Almunawwarah region. Some diseases, such as hemolytic anemias, have significantly high rates in regions in KSA than in other regions worldwide. 21 In individuals with ovarian carcinomas, the familial genetic abnormality in the BRCA-1 gene is well documented in surface epithelial tumours. BRCA-1 is a normal gene located in chromosome 17q21. The encoded protein functions as a tumour suppressor gene by repairing DNA damage. 22 The reported rate of BRCA-1 mutation among patients with ovarian cancer varied among studies from 5% in some studies 23 to 60% in other studies. 24 We examined GCT for the expression pattern of BRCA-1 to validate if the aberrant expression of this protein has a role in the development of GCT. All our specimens showed a positive expression of the protein except in two cases in which the expression was low. Such finding indicates that BRCA-1 expression does not play a significant role in the carcinogenesis of these tumours.
Advances in molecular-based studies showed that genetic mutation through changes in the gene sequence itself is not the only mechanism associated with aberrant genetic function. Abnormal gene function may result from epigenetic changes in which the abnormal pattern of gene methylation has a direct impact on gene activity. In a study examining epigenetic changes in GCT, researchers have found that the hypermethylation of the promoters of certain genes, such as CDH13, DKK3, and forkheadbox L2 (FOXL2), are significantly higher in GCT specimens compared to normal tissues. 25 Other studies have suggested that the main molecular event underlying GCT is an abnormal high proliferation of the granulose cells due to mutation in the FOXL2 gene known as c.402C>G mutation. This mutation was found in majority of GCT both the primary and metastatic tissues. Due to its high prevalence in GCT, authors have recommended its use in the diagnosis of challenging GCT cases. 11 All these aforementioned changes in the activation of growth factors or methylation have been caused by exposure to environmental factors, such as pollutants and dietary components. 26 There may be certain undiscovered environmental factors in the Almadinah Almunawwarah region. These genetic changes have not been examined in the current study; however, it would be interesting to examine epigenetic changes in our tumour samples.
Germ cell tumours in our study were defined in 13% of cases, which is within the reported range in other studies 7 and is similar to other regions in KSA. 27 The mean age of the patients in this study was 41 years. This age is younger than that described in other regional studies with a comparable range from 7 to 91 years in both studies, and most cases were observed in the 20e50 age group, which was obviously younger than that of other findings in different regional studies. 5,27 These findings indicated the need of investigating the genetic predisposition of different ovarian malignant neoplasms in the Almadinah Almunawwarah region.
In conclusion, malignant ovarian neoplasms in individuals in Almadinah Almunawwarah region accounted for 30% of neoplastic ovarian specimens. Despite the overall low frequency of malignant ovarian tumours in Almadinah Almunawwarah region, a remarkably frequent AGCT was observed. These tumours did not show a significantly altered expression of BRCA-1, which increases the risk of surface ovarian tumours. However, this conclusion should be taken with caution due to the limited number of samples that were assessed in the study. Further studies must be conducted to explore the underlying molecular causes behind the observed high incidence of GCT. data and participated in writing the draft of the manuscript. AB: Provided logistic support. All authors have critically reviewed and approved the final draft and are responsible for the content and similarity index of the manuscript.