Evaluation of the Expression Intensity of Glucose Transporter-1 Marker and its Diagnostic Value in Differentiating Between Borderline and Malignant Ovarian Epithelial Tumors

Background : Ovarian cancer ranks second among gynecological cancers worldwide. This study aimed to compare the glucose transporter-1 (GLUT-1) expression in benign, borderline, and malignant ovarian epithelial tumors and evaluate GLUT-1 expression as a diagnostic tool for distinguishing tumors in the ovary. Materials and Methods: This descriptive-analytical cross-sectional study analyzed 69 pathological samples of patients diagnosed with ovarian epithelial tumors who underwent oophorectomy. Immunohistochemical staining was performed using GLUT-1 antibody. The intensity of cell membrane staining and the proportion of positive neoplastic cells were graded to score immunostaining. Chi-square/Fisher’s exact test was used to analyze the data. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), and staining accuracy for GLUT-1 in distinguishing borderline from invasive tumors were calculated by standard methods ( P < 0.05). Results: In all benign tumors, GLUT-1 staining was negative. In addition, weak staining intensity was observed in 38.5% of borderline tumors, and 96% of invasive tumors had strong staining intensity ( P < 0.001). Strong GLUT-1 staining was found in 94.7% of Papillary Serous Carcinoma, 9/1% of Borderline Serous tumors, 100% of Brenner tumors, and clear cell carcinoma. The results demonstrated a high diagnostic value of GLUT-1 expression intensity in differentiating between borderline and malignant ovarian epithelial tumors (Accuracy: 97.10, Sensitivity: 96%, Specificity: 97.73). Conclusion: Overall, GLUT-1 expression could help distinguish benign from borderline, especially borderline from malignant ovarian epithelial tumors. Thus, it seems that it provides useful prognostic information, particularly for the borderline category.

fertility and have a good prognosis even with conservative treatment (8).
The diagnosis of an ovarian lesion is difficult; nonetheless, it is critical in the preoperative environment to plan appropriate treatment operations and influence patient management. The growth of cancer cells is an energy-related process that is aided by enhanced glucose metabolism (9), indicating that a similar increase in glucose transporter protein uptake is necessary.
Glucose transporter-1 (GLUT-1), a facilitative cell surface glucose transport protein, is a member of the GLUT family (10). It is physiologically expressed and immunohistochemically detectable in erythrocytes, endothelial cells, placenta, and blood-tissue barriers (11). Glut-1 is a useful marker in pathology, and its expression is utilized to distinguish self-limiting infantile hemangiomas from other vascular diseases (12). GLUT-1 is mainly undetectable by immunohistochemistry on normal epithelial tissues and benign epithelial tumors but is expressed in a variety of malignancies (1). Thus, the expression of GLUT-1 appears to be a potential marker of malignant transformation. Further, its expression in thymic epithelial malignancies was previously measured by the percentage of positive cells, intensity of immunostaining, or a score integrating both of these variables (13).
Many studies have been conducted to differentiate benign tumors from malignant or invasive tumors (14)(15)(16). However, no study has so far evaluated GLUT1 expression in differentiating the ovarian borderlines (low malignant) from invasive tissues. Accordingly, this study attempted to compare the expression of GLUT-1 in benign, borderline, and malignant ovarian epithelial tumors. We evaluated the use of GLUT-1 as a diagnostic tool in distinguishing between morphologically dubious borderline and malignant changes in the ovary.

Materials and Methods
This descriptive-analytical cross-sectional study was performed at the Department of Pathology of Motahari Hospital, a public teaching medical center in Iran, from 2020 to 2021. Sixty-nine pathological samples of patients diagnosed with ovarian epithelial tumors who underwent oophorectomy were included in the study. The information about these patients was obtained by searching the computer system. The patients who had undergone chemotherapy or radiotherapy before the surgery were excluded from the study. Patients' age, histological type of tumor, and degree of malignancy were recorded, and GLUT-1 transporters were detected by immunohistochemistry using the labeled streptavidinbiotin procedure. The prepared glass slides were removed from the archive, and diagnosis and re-grading were performed after re-examining. If the slides were not of good quality or were broken, new sections were made from the existing paraffin blocks, and hematoxylin and eosin were stained.
Then, the appropriate block for each case was selected for immunohistochemical staining. Fourmicron sections were selected from selected blocks, and immunohistochemical staining was performed, in which paraffin was removed first. The tissue with paraffin was transferred to the slides, and after sticking to the slide, we had to remove paraffin from the slide, which required washing the tissue. The slides were washed twice for five minutes with xylene, then soaked in 50%, 75%, 95%, and 100% alcohol for five minutes, and finally, they were washed with a buffer. An appropriate amount of diluted GLUT 1 antibody was added dropwise to all areas fixed on the slide.
Afterwise, the tissue was incubated with the antibody for about 10-20 minutes. Then, the slides were cooled down with TBS and washed for five minutes. In addition, the coloring pattern and color intensity of each case were examined, and the slides were divided into grades 0-4 based on staining intensity. The grading method represented 0, 1, 2, and 3 for no, low, moderate, and severe staining, respectively. The percentage of positively stained cells was classified as 0: < 10%, 1: 10-50%, and 2: > 50%. The final intensity score was calculated by multiplying the staining intensity score by the staining percentage score. All cases were subsequently classified into four expression groups according to the final scores (0, 1, 2, and 3 for negative (-), weak ( + ), moderate ( + + ), and strong ( + + + ), respectively). For determining the diagnostic value of the GLUT-1 biomarker in distinguishing between morphologically dubious borderline and malignant changes of the ovary, the intensities of 0, 1, and 2 were considered negative, while 3 was considered positive.
The mean, standard deviation (SD), and frequency (percentage) were reported for continuous variables The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), and staining accuracy for GLUT-1 in distinguishing benign and borderline from invasive tumors were calculated by standard methods. Chi-square/Fisher's exact test was employed to test the association between the histologic types and grades of ovarian epithelial tumors with GLUT-1 immunoreactivity. Statistical analysis was performed using the SPSS statistical package; in all statistical analyses, P < 0.05 was considered significant. One-way ANOVA was used to compare the age of patients between the three ovarian tumor categories.
Sixty-seven of 69 patients were correctly categorized (Accuracy = 97.10%) with sensitivity and specificity of 96% and 97.73 %, respectively. Positive GLUT-1 expression (score = 3) was observed in 1 and 24 patients with benign and borderline and invasive ovarian epithelial tumors, respectively. Based on the results, 96% of patients with positive GLUT-1 expression truly had invasive ovarian epithelial tumors (PPV = 96%). In addition, 97.73% of patients with a negative GLUT-1 expression truly had benign and borderline ovarian epithelial tumors (NPV = 97.73%). Based on the clinical findings, the association between GLUT-1 marker results and grades of ovarian epithelial tumors in histological findings was statistically significant (Pearson chi-square = 60.61, P < 0.001).

Discussion
Histological differentiation of borderline from malignant ovarian epithelial tumors can be challenging because they often exhibit similar behaviors to invasive carcinomas (17). Researchers have emphasized diagnostic biomarkers to differentiate between tumors (18). To our knowledge, this study is the first of its kind that compared GLUT-1 expression in benign, borderline, and malignant ovarian epithelial tumors and distinguished borderline from invasive tumors.
The findings of this study demonstrated that there was no significant difference in the mean age of the patients with benign, borderline, and malignant tumors. In addition, Abdul Hamid et al reported no significant difference between the median age for the study groups with phyllodes tumors (19). In line with other studies (1,20), our results represented a strong relationship between GLUT-1 expression and ovarian epithelial tumors. On the other hand, Xiong et al (21) reported that GLUT-1 expression can be utilized to distinguish between benign endometrial lesions and endometrial cancer; however, it has little predictive significance in women who have this cancer, which is contrary to the findings of this study.
Furthermore, the results showed that the majority (94.7%) of the malignant epithelial tumors, including papillary serous carcinoma (18/19), showed strong GLUT-1 staining, indicating the usefulness of this marker in assisting diagnosis. Additionally, only one case of borderline serous tumors had strong GLUT-1 staining (1/11), and GLUT-1 staining was negative among 11 benign serous cysts. This finding is almost the same with those of Ullah et al and Khabaz et al (1,22); the only difference is that both of these studies indicated that benign tumors had positive GLUT-1 staining, but the majority of benign tumors were negative in our study. Accordingly, patients with positive GLUT-1 expression tend to have a poorer prognosis than those with negative GLUT-1 expression, suggesting the biomarker's predictive relevance. In agreement with this statement, Szablewski reported that the overexpression of GLUT-1 was strongly related to poor survival in patients with various malignancies (23).
In this study, moderate staining intensity for GLUT-1 was found in most borderline mucinous (5/9) and serous (9/11) tumors, and strong staining was in malignant papillary serous carcinoma (18/19). The difference in architecture and proliferative activity between both tumors is due to the stratified papillary structure of its tumor cells, which is accompanied by fewer vascular channels. None of the borderline and invasive cases showed negative (0) staining. These findings align with a study conducted by Nagib et al (24) in which weak to moderate GLUT-1 expression was reported in most borderline cases. Furthermore, Cantuaria et al (25) demonstrated weak and moderate positivity in most borderline cases. On the contrary, in a study by Ruby et al, moderate to strong GLUT-1 expression (score = 2 or 3) was only observed in malignant tumors (20).
Moreover, twenty-four (95%) malignant epithelial tumors stained positively with anti-GLUT-1 among 25 cases in the present study. In positive cases, staining had strong intensity and was more extensive than in borderline tumors, and immunoexpression was observed in the majority of cell membranes. These findings conform to the results of Yan et al, representing moderate to strong GLUT-1 staining intensity in 96% of invasive cases (26).
Likewise, Cai et al (9) concluded that staining was absolutely negative in normal ovarian tissue, whereas GLUT-1 and P63 expression were greater in borderline tumors and adenocarcinoma cysts. These results are consistent with the findings of our study both in terms of the high sensitivity (95%) of GLUT-1 marker expression in distinguishing malignant tumors from borderline or benign tumors and the negative staining of benign ovarian tumors with GLUT-1 marker expression. Conversely, Ruby et al reported that GLUT-1 is not overly sensitive in determining whether a tumor is borderline or invasive (20).
According to the obtained results of the present investigation, GLUT-1 expression progresses slowly through all stages of ovarian tumors (benign, borderline, and malignant). Elbasateeny et al confirmed this result and showed that GLUT-1 expression in ovarian cancers is progressively stained (27). This finding suggests that the degree of GLUT-1 expression is closely linked to the histopathological grade of the malignant transformation of ovarian epithelial tumors, implying that they have an increased need for glucose metabolism. The weak points of this study are the relatively small sample size and the semi-quantitative interpretation of immunostaining. However, studies with large sample sizes are undoubtedly of great value for estimating the diagnostic and prognostic

Conclusion
In general, it was revealed that GLUT-1 is involved in glucose uptake by ovarian epithelial tumor cells, leading to increased growth and biological aggressiveness. Our findings support GLUT-1 as a diagnostic tool to distinguish borderline from malignant ovarian tumors and suggest its association with different grades of ovarian epithelial tumors. Moreover, its relatively strong expression in serous tumors as compared to mucinous represents its association with the histological characteristics of the tumors. As a result, the predictive significance of GLUT-1 overexpression could be used to identify patients with a poor prognosis who would benefit from the future therapeutic targeting of overexpressed markers.