Immunohistochemical expression of p53 in Type I and II epithelial ovarian cancer among Sudanese women: a cross- sectional study [version 1; peer review: 1 approved with reservations]

Background: Epithelial ovarian cancer (EOC) represents the leading cause of death from gynecologic malignancies worldwide. In Sudan, ovarian cancer represents the fourth most frequent tumors among females. TP53 somatic mutations is a defining feature of ovarian highgrade serous carcinoma. However, p53 sequencing is not feasible in most lowand middle-income countries, like Sudan, and its frequency varies greatly. The study aimed to determine the frequency of p53 overexpression and its relationship with tumor types I and II and tumor grade among Sudanese women with EOC. Methods: In this cross-sectional, hospital-based study a total of 114 paraffin-embedded tissue blocks previously diagnosed as epithelial ovarian cancer were collected from six governmental hospitals in Khartoum state, Sudan, in the period 2013-2016. Immunohistochemistry was performed on tissue microarray slides to measure the protein expression of p53 in the EOC. Results: Overexpression of p53 was detected in 35.1% (n=40/114) of EOC samples, with a higher frequency in women with Type II 53.7% (n= 29/54)  than type I 18.5% (n= 10/54)  (P= 0.000). Also, a high frequency of p53 overexpression was evident in 49.2% (n= 30/61) of high-grade carcinoma compared with 16.7% (n= 1/6) of non-graded borderline tumors, and in 19.1% (n= 9/47) of low-grade tumors (P= 0.003). A high-grade serous carcinoma harbor p53 overexpression in 53.7% (n= 29/54) and none of low-grade serous carcinoma harbor p53 overexpression. Our result showed a significant association between Open Peer Review


Introduction
Ovarian cancer is a fatal disease, the mortality rate ranks the highest of all gynecological malignancies 1 . It is considered the third most common cancer in the female reproductive system (following uterine cervix and corpus) and the leading cause of death from gynecologic malignancies in the United States as estimated by the American Cancer Society for the year 2019 1,2 . Ovarian cancer refers to a group of morphologically and genetically heterogeneous neoplasms 3,4 . In Sudan, ovarian cancer represents the fourth most frequent tumor type in women 5 .
Based on clinicopathological and molecular studies, epithelial ovarian cancer (EOC) is classified as type I or type II. Type I tumors are genetically quite stable, typically present at a low stage, and reveal distinct, morphologic differences than type II tumors 6 . These include different histotypes: low-grade serous, endometrioid, clear-cell, and mucinous ovarian carcinoma. Type I tumors are characterized by distinct molecular genetics profiles, such as mutations in KRAS, BRAF, PIK3CA, PTEN and ERBB2, but not TP53 6 . Type II tumors are generally high-grade serous (about 90% of all EOCs). They are highly aggressive, develop rapidly, present in an advanced stage in most cases, genetically unstable and express a mutated TP53 7-12 . TP53 mutations have an important role in the prognosis and treatment of ovarian cancer 13 . Mutations in TP53 are found in high grade and rarely in low grade serous ovarian cancers. TP53 encodes the 53 kDa nuclear protein, their mutations leading to gain or loss of function of its protein product. TP53 mutation leads either to overexpression of p53 protein or complete lack of expression, while wild-type p53 is associated with focal expression 14,15 .
Immunohistochemical staining for p53 was considered as an essential biomarker for clinical trials targeting mutant p53 and used in the diagnostic workup of carcinomas of multiple sites, including ovarian cancers 16 . It is used as a substitute for TP53 mutational analysis, these mutations were global in high-grade serous ovarian cancer (HGSOC) (over 96% were mutated), so were used to discriminates between high-and lowgrade serous carcinomas 17-20 . Access to TP53 sequencing is not feasible in many low-and middle-income countries; pathologists there used p53 immunohistochemistry, which is quick, easy to perform, inexpensive and can approach 100% specificity for the presence of TP53 mutation. Its high negative predictive value is clinically useful as it can exclude the possibility of a low-grade serous tumor 18-20 . To our knowledge, there are no published reports about the frequency of p53 immunostaining in type I and II EOC in Sudan. The study aimed to determine the frequency of p53 overexpression and its relationship with tumor types I and II and tumor grades among Sudanese women with EOC.

Study background
A cross-sectional, hospital-based study was implemented. All 114 available formalin-fixed paraffin-embedded tissue blocks (convenience sampling) previously diagnosed as epithelial ovarian cancer were collected during the period 2013-2016, in six governmental hospitals in Khartoum state, Sudan (The National Public Health Laboratory, Maternity Hospital, Military Omdurman Hospital, Alribat, Bahri, and Omdurman Teaching Hospital). Well-preserved tissue blocks with adequate tissue left for tissue microarray (TMA) procedure were included. Inadequate tissue blocks, and cases with missing tissue blocks were excluded. Slides from the original paraffin blocks were stained with hematoxylin and eosin (H&E), were reviewed according to 2014 WHO classification of ovarian tumors 21 , and were graded and typed according to the Kurman model 8 .

Construction of a microarray
Available paraffin-embedded blocks from tumors were used for the construction of a tissue microarray (TMA). Representative areas of the tumor were identified and TMA blocks were constructed using two cores from each case. Sections were obtained from each TMA and were placed on negatively charged slides for immunohistochemistry.

Immunohistochemistry
Immunohistochemistry was performed to measure the protein expression of p53 monoclonal antibodies in ovarian carcinoma cases, as follow: Sections were cut into widths of 3-4 µm and placed on clean, electrostatically charged glass slides. Sections were dried by placing on a hot plate at 60°C for 15 minutes. Sections were dewaxed in two changes of xylene for two minutes. Sections were then hydrated through an ethanol series (100%, 90%, 70%, 50%) and water two minutes for each. Slides were retrieved using the water bath heat-retrieval technique 22 and then treated with 3% hydrogen peroxide for 10 minutes. After that, sections were washed in phosphate buffer saline (PBS) (pH 7.4) for five minutes and treated with a 10% casein solution for 10 minutes. Sections were treated with ready-to-use primary antibody of mouse monoclonal antibody to p53 protein (clone DO7 IgG2b; catalog no AM239-5M; BioGenex, CA) for 30 minutes at room temperature in a humidity chamber, then rinsed in PBS before being treated with Super Sensitive polymer -HRP IHC Detection System (catalog no QD420-YIKE; BioGenex, CA) by incubated with enhancer reagents 15 min at room temperature, followed by a polymer-HRP reagent conjugated to anti-mouse and anti-rabbit secondary antibody for 15 minutes at room temperature, and rinsed in PBS. The entire antibody-enzyme complex is then made visible by incubation with a chromogenic substrate 3,3 diaminobenzidine for 7 minutes then washed in PBS for five minutes. For the staining step, sections were counter-stained in Mayer's hematoxylin for one minute washed and blued in running tap water before they were dehydrated through ascending concentrations of ethanol (50%, 70%, 90%, 100%). Sections were finally cleared in xylene and mounted using DPX. A known p53-positive breast cancer tumor was used as positive control. As the negative control, tumor specimens were immunostained under the same conditions without the primary antibody. Both the quantity of nuclear positivity and the staining intensity were measured in the immune slides examined under a light microscope (Olympus BX41, Japan). The intensity of staining was reported as negative, weak, moderate, or strong (0, 1+,2+,3+) in comparison with the positive controls (internal or external) and it indicated the average staining intensity of the tumor nuclei on the entire slide. An IHC score of p53 staining intensity was categorized as 0 for none, (no brownish color seen using x40 magnification), +1 for weak (brownish color seen using x20 and x40), +2 for moderate (brownish color seen using x10 magnification) and +3 for strong staining (brown color visible using x4 magnification).
The percentage of positive tumor cells was quantified by counting cells manually in at least 100 cells in 10 high power fields, averaged and categorized as ≥75% of cells considered as high overexpression, ≤75-50% considered as moderate expression and less than 50% considered as focal expression. Only 75-100% positive tumor cells with moderate and strong staining intensity considered as positive results.

Statistical analysis
The data were analyzed using the statistical package for social sciences (SPSS version 24) to describe the variables. Pearson's Chi-square test was used to determine a statistically significant association between p53 expression and clinicopathological variables.

Ethical consideration
The study was approved by the ethical committees of Alzaiem Alazhari University and the Ministry of Health, Sudan. Informed consent from patients was waived by the committees, since patients' identity was anonymized, and only laboratory numbers were used.

Overexpression of p53
Positive p53 immunostaining was seen in 35.1% (40/114) of ovarian epithelial carcinoma. From the 40 positive cases, staining intensity was as follows: 25 cases exhibited strong staining (+3), and 15 cases were moderate staining (+2). While the remaining 74 cases were considered as negative results (complete absence of p53 expression seen in 45 cases and +1 staining in 29 cases) (Figure 1-Figure 4).

Discussion
Immunohistochemical staining for p53 was used as a surrogate for TP53 mutational analysis to discriminate between high (over 96% was mutated) and low-grade serous carcinomas 17-20 .  The present study showed that p53 marker was overexpressed in (53.7%) of type II, and (18.5%) of type I. This result agreed with Carter et al., 2018, who reported that p53 was highly expressed in type II EOC (68.8%) than type I (33.3%) 4 . HGSOC is the most frequent type of ovarian cancer and has been associated with a poor clinical outcome 34 . According to The Cancer Genome Atlas report, mutations in TP53 are the most common events in EOC, especially in HGSCs 35 .
The results of some of these studies may be conflicting primarily because of the indiscriminate grouping of TP53 mutations, which can result in either loss of function or gain of function 36 . GOF mutations can convert p53 protein from a tumor suppressor to an oncogene, leading to expression of a mutant p53 protein at a high level, while LOF mutants leading to loss of p53 protein expression 37 . TP53 mutations are classified according to their function as oncomorphic, loss of function and unclassified. Around 21% of all ovarian cancer patients harbor oncomorphic TP53 mutations, which had the highest p53 protein levels and contribute to chemoresistance and cancer progression, and the tumors with unclassified TP53 mutations express the mutated p53 protein at a fairly high level 33 . The differences found between the studies in the frequency of p53 expression may be due to the differences in scoring of p53 expression and interpretation of results: some studies scored this as overexpression (OE), complete absence (CA), cytoplasmic (CY) or normal/wild type (WT) 18,20 . Some authors consider complete absence of p53 expression as a mutant also because not all TP53 mutations alter the expression of the protein 15,17,25 . Complete absence of p53 expression does not indicate TP53 mutation, as a lack of immunoexpression may be found in normal cells 16 . So, we believe that true overexpression (more than 75% of the cells stained positive) was the most important type of mutation to consider due to their importance in clinical practice, as they are chemoresistant mutations 33 and could also be interpreted easily in the immune-slide without any confusion.

Study limitations
The limitations of our study were related to the relatively small sample size. Many cases were found in the lab records but lacking tissue blocks, and some blocks contain less amount of tissue for TMA.

Conclusion
Our study showed that the overexpression of p53 tumor marker is associated with EOC, histological subtype and tumor grade, and found that high-grade serous tumors had a higher percentage of p53 expression in more than 50% of cases, while low grade serous was negative in 100% of the cases. We recommended the use of p53 immunohistochemical staining in the pathologic workup of ovarian carcinomas. Careful attention to laboratory protocols and practical works, including adequate controls, and training in interpretation is needed to make this a reliable test informing diagnosis and subsequent management of ovarian carcinoma.

Daniel R Barnes
Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, University of Cambridge, Cambridge, UK

GENERAL COMMENTS/OVERVIEW
In this study, the authors present their findings from analysing p53 expression in a sample of 114 tissue microarrays obtained from Sudanese women diagnosed with ovarian cancer. They report statistically significant associations of differences in p53 expression and ovarian cancer histotype, grade and type.
The paper is generally well written, with all experiments and analyses well described. Table 1: The chi-square test used to test the association between ovarian cancer subtype and negative/positive p53 expression is likely to be inappropriate, stemming from the small table cell counts (and even some zeroes). Here I feel that, although it is unlikely to make any difference to the reported association P-value and conclusions, a Fisher exact test is the correct statistical test. Table 2: Similar to the comment on the statistical analysis for data presented in Table 1, a Fisher exact test would be more appropriate to analyse these data. The P-value presented for the association between cancer type and p53 status was "0.000". Could the authors present the Pvalue in scientific notation if it is P<0.001? Table 2: What was the magnitude of these associations? Could the authors fit logistic regression models to these data to estimate odds ratios of a p53 negative/positive expression by cancer type and cancer grade?

MAJOR CONCERNS
Discussion, first and second paragraphs: The authors discuss the findings of many other studies in lengthy detail, showing numbers and percentages. It is somewhat difficult to read. A more general overview with fewer details and providing references should suffice.