Immunohistochemical Expression of p53 in Epithelial Ovarian Carcinoma and Its Correlation with Clinicopathological Parameters.

This observational cross-sectional study was conducted at the Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh, from July 2022 to June 2023. A total of 50 women with diagnosed EOCs admitted for definitive surgery were selected purposively. In addition, convenience sampling was applied to recruit participants based on their availability. Women aged ≥ 18 years with suspected EOC planned for primary debulking surgery were included. Exclusions were women with suspected non-EOC or benign conditions, pregnant and lactating women with ovarian tumors, secondary metastatic ovarian cancer, those undergoing interval or secondary debulking surgery, women with recurrent EOC, those who received chemotherapy, and those receiving p53 inhibitors. A semiquantitative histochemical score was used to record results of p53 nuclear staining. More than 1,000 tumor cells, in multiple high-power fields, were counted for assessing the percentage. Also, the average staining intensity was considered. The slides were checked more than once to exclude subjectivity. Positive and negative control slides were included in each run of staining. Positive control slides were prepared from a case known to be positive for p53. While negative control slides were prepared from the same tissue block incubated with Tris-buffered saline instead of the primary antibody. Categorical variables were analyzed using the chi-square test or Fisher's exact test, as appropriate. Continuous variables that were not normally distributed were assessed with the Mann–Whitney test. Correlations between various parameters were determined using the Spearman's correlation coefficient.

A total of 50 patients with EOC were enrolled in the study. Sociodemographic and clinicopathological characteristics of the study participants are shown in Table 1 . Majority (38%) of the women were 51 years and older, followed by 41 to 50 years (30%). Above four-fifths (82%) were married while 10.0% were widowed. Half (50%) had education up to primary level, while 34% had secondary education and above. Most (90.0%) of the women were housewives and the majority (52%) of the women were from the lower middle-class group. The mean age of women with p53 nonmutated EOC was slightly higher (47.5 ± 15.05 years) than those with p53-mutant EOC (47.0 ± 10.66 years). According to the Asian Development Bank Institute: Razafimandimby AR. (2017). Middle-class composition and growth in middle-income countries . ADBI Working Paper No. 753. Tokyo: Asian Development Bank Institute. Available at https://www.adb.org/publications/middle-class-composition-and-growth-middle-income-countries . Among study participants, 31 (62%) had a p53 mutation, whereas in 19 (38%) women with EOC TP53 mutation was absent. Among women with a positive family history, 14 (100%) exhibited p53 mutation, while none in this category were nonmutant. In contrast, among those with a negative family history, 17 (47.2%) have p53 mutation and 19 (52.8%) were nonmutant. Moreover, p53 expression was found to be significantly associated with a family history of ovarian cancer (chi-square test, p  = 0.001). Among total 50 cases of different histological subtypes, 39 (78%) cases were serous, 9 (18%) cases were mucinous, 1 (2%) case was seromucinous, and 1 (2%) case was of endometrioid variety ( Table 1 ). A significant association was found between p53 mutation status and histological subtype (Fisher's exact test, p  = 0.046). In the present study, the median preoperative CA-125 level in patients with early-stage serous ovarian carcinoma was significantly lower than that in patients with advanced-stage serous ovarian carcinoma (133.5 vs. 641 U/mL; Mann–Whitney test, p  = 0.016). Our study revealed that the median preoperative CA-125 level in patients with LGSOC was significantly lower than that in patients with HGSOC (100 vs. 371 U/mL; Mann–Whitney test, p  = 0.001). The present study showed that the median preoperative CA-125 level in patients with p53 nonmutant serous ovarian carcinoma was lower than that in patients with p53 mutant serous ovarian carcinoma (110 vs. 321 U/mL; Mann–Whitney test, p  = 0.155). Table 2 shows the relationship between p53 expression and clinicopathological parameters in patients with serous EOC. Among 39 cases of serous carcinoma, 24 (61.5%) were in early stage (Federation of Gynecology and Obstetrics [FIGO] stage I and II) and 15 (38.5%) were in advanced stage (FIGO stage III and IV). Out of 24 early-stage serous carcinoma, 16 (66.67%) cases were p53 mutant and out of 15 advanced serous carcinoma, 12 (80%) cases were p53 mutant. Fisher's exact test revealed no significant association between the stage of carcinoma and p53 mutation status ( p  = 0.477). Additionally, Spearman's correlation coefficient ( ρ  = 0.144) indicated a weak positive correlation between the stage and p53 mutation status. Among 39 cases of serous carcinoma, 33 (84.6%) were HGSOC and 6 (15.4%) were LGSOC. Number of p53 positive cases in HGSOC was 26/33 (78.8%) and in LGSOC it was 2/6 (33.33%). The grade of the carcinoma indicated a significant association with p53 mutation status (Fisher's exact test, p  = 0.042), and Spearman's correlation coefficient ( ρ  = 0.364) demonstrated a moderate positive correlation between the grade and p53 mutation status. These findings suggest that higher-grade carcinomas are significantly associated with the presence of p53 mutations.

p53 overexpression, indicative of TP53 mutations, is common in EOC, especially in high-grade serous carcinomas. A higher prevalence of p53 mutations in those with a positive family history suggests inherited genetic factors influence these mutations. Increased p53 expression in high-grade carcinomas highlights its critical role in the pathogenesis and treatment of ovarian cancer.

Number of p53 mutant cases was more in women who were under 50 years of age compared with women who were over 50 years of age (61.3% vs. 38.7%). The average age of women with p53 nonmutated EOC was slightly higher than those with p53-mutant EOC, though the difference was not statistically significant. Darcy et al (2008) found that p53 overexpression was not related to age at enrollment or race/ethnicity, but it was linked to worse progression-free survival. 16 Chang et al reported the mean age at EOC diagnosis as 52.8 years old. 17 A majority of these women were married, had up to primary education, were housewives, and belonged to lower middle-class families. In this study, all the women with a positive family history had p53 mutations, while none were p53 nonmutant. This suggests that p53 mutations in EOC may be influenced by inherited genetic factors related to ovarian cancer risk within families. The present study exhibited preoperative CA-125 levels were higher in women with high-grade EOCs than in women with low-grade cancer. In contrast, Li et al in their study revealed that low-grade EOC patients had significantly low level (125.34 ± 115.42 U/mL) of CA-125 than the high-grade EOCs (2224.43 ± 4225.11 U/mL), with p -value < 0.001. 18 Nayak et al found the mean CA-125 value significantly higher in HGSOC (2059 ± 1460.55) compared with LGSOC (553.37 ± 278.52) ( p  < 0.01). The difference may result from the small sample size and individual biological variability of CA-125 levels. 14 Compared with patients with advanced-stage serous ovarian carcinoma, individuals with early-stage serous ovarian cancer had a substantially lower median preoperative CA-125 level. Charkhchi et al in their study revealed that serum CA-125 levels were elevated in 50% of early-stage compared with 92% of advanced-stage epithelial ovarian tumors. 15 The majority of high-grade serous cases had p53 mutations, while most low-grade serous and mucinous carcinoma cases did not. In the seromucinous carcinoma and endometrioid subtypes, there were no p53 mutations at all. Nayak et al conferred that only 50% cases of LGSOC were positive for p53 immunostaining, it was 100% in case of HGSOC. 14 Kaur and Singh also exhibited that 86.6% of the malignancies were p53 positive and among which maximum number was that of serous carcinomas (50%), followed by mucinous carcinomas (10%), two cases (6.6%) each of dysgerminomas and adult granulosa cell tumor, followed by one case each (3.3%) of malignant Brenner tumor, malignant mixed germ cell tumor, immature teratoma, and squamous cell carcinoma of the ovary. 19 These correlated with the present study findings. The current study demonstrated that the median preoperative CA-125 level in patients with p53 nonmutant serous ovarian carcinoma was lower than that of those with p53 mutant serous ovarian carcinoma. Tiwari et al exhibited that the mean preoperative CA-125 level was strongly and positively correlated with p53 expression (Spearman's rank correlation, ρ  = 0.639). 20 In this study, a little over half of early-stage ovarian cancers had p53 mutations, while slightly less than half did not. Among advanced-stage ovarian cancers, a larger proportion had p53 mutations compared with those without. Although not statistically significant, this data hints at a potential trend. In low-grade cancers, the number of p53 mutations was lower than p53 nonmutations. In high-grade cancers, a majority had p53 mutations, while a significant number did not. This notable difference in distribution hints at a correlation between p53 mutations and histological grade. In the research conducted by Sallum et al ( p  < 0.0001) 21 and Naik et al ( p  < 0.05), 22 a statistically significant difference was observed in p53 expression concerning serous carcinoma grade, aligning with our study ( p  = 0.018).

Ovarian cancer is the most lethal gynecologic malignancy involving diverse tumors. 1 Its late presentation is due to the ovary's anatomical location and complex histology, complicating management. 2 Epithelial tumors are categorized by epithelial proliferation, invasion, and histotype. 3 Cancerous epithelial tumors are called carcinomas, comprising 85 to 90% of malignant ovarian cancers. A woman's lifetime risk is 1 in 78, with a 1 in 108 chance of dying. 4 Globally, there were over 295,000 cases, almost 185,000 deaths, and over 750,000 women living within 5 years of diagnosis. 5 In India (2004–2005), ovarian cancer varied from 1.7 to 8.7% of all female cancers. In Bangladesh (2020), it ranked 13th in age-standardized death rates, with 3,122 new cases and a 5-year prevalence of 7,044. 6 Epithelial ovarian carcinoma (EOC) has five main groups: high-grade serous (HGSOC), low-grade serous (LGSOC), clear cell, endometrioid, and mucinous carcinoma. Low-grade epithelial ovarian cancers are diagnosed at a younger age and have an indolent clinical course. Unlike HGSOCs, which develop de novo, LGSOCs follow a continuum model from benign tumor to carcinoma in situ to LGSOC. Less common malignant epithelial neoplasms include malignant Brenner tumors and seromucinous carcinoma. 7 Type I EOC tumors can arise from the ovarian surface epithelium and Mullerian inclusions that are considered low grade and have an excellent prognosis when confined to the ovary. In contrast, type II EOC tumors present at an advanced stage in greater than 75% of cases and are characterized by p53 mutations and a poor prognosis. This type has a phenotype that resembles the fallopian tube mucosa. Type I tumors include low-grade serous, endometrioid, clear cell, and mucinous carcinomas, while type II cancers are predominantly HGSCs, but also include carcinosarcomas and undifferentiated carcinomas. 8 Risk factors for EOC include the number of lifetime ovulations (nulliparity, early menarche, late menopause), family history, smoking, benign gynecological conditions (e.g., endometriosis, polycystic ovary syndrome, pelvic inflammatory disease), and possibly talcum powder use. 9 Higher body mass index (> 30) increases risk. 10 BRCA1 and BRCA2 genes cause 65 to 75% of hereditary EOC, mainly high-grade serous EOC. Lynch syndrome, associated with endometrioid or clear-cell tumors, accounts for 10 to 15% of hereditary EOC. 11 Some of the theories for the pathogenesis of EOC include: (1) repeated ovulation with trauma, (2) increased estrogen concentrations because of excess gonadotropin secretion, (3) high androgen concentrations, and (4) stromal hyperactivity. 12 The p53 tumor suppressor gene, located on chromosome 17p13.1, is the “guardian of the genome.” Its protein, made of 393 amino acids, functions in the G1 phase to repair deoxyribonucleic acid damage and prevent cell entry into S phase or lead to apoptosis in damaged cells. Mutation in p53 hinders its ability to trigger cell death, causing uncontrolled cell growth and tumorigenesis. Normally, undetectable immunohistochemically mutated p53 accumulates in the nucleus and is detectable with monoclonal antibodies. Tumors with normal p53 respond better to irradiation and chemotherapy than those with mutated alleles. 13 Mutations of the p53 gene as determined by mutation analysis and/or positive immunohistochemical (IHC) staining for p53 are common in ovarian cancer and have been associated with poor clinical outcomes. Studies have mentioned that the p53 gene is mutated in approximately 50 to 80% of ovarian carcinoma. 14 LGSOC lacks p53 gene mutations and is considered to arise from borderline tumors. In contrast, HGSOC arises as de novo and it has been suggested that 100% of HGSOC are in fact p53 mutated. However, the findings of several studies on the prognostic value of p53 expression in ovarian cancer have been inconclusive. 15 This study was aimed to determine the IHC expression of p53 in EOC and its correlation with clinicopathological parameters.