Role of CD44 and CD24 Expression on 2-years Disease Free Survival in Patients with Advanced Epithelial Ovarian Carcinoma

Objective: Ovarian cancer is one of the most common cancers with a high mortality rate worldwide. Despite optimal surgical therapy and chemotherapy, recurrence is still common. Cancer stem cells expressing CD44 and CD24 are thought to be contributing factors in recurrence. Methods: A cohort retrospective study with survival analysis was carried out on advanced ovarian cancer patients who underwent optimal debulking surgery followed by 6 cycles of chemotherapy at Cipto Mangunkusumo General Hospital and Fatmawati General Hospital from January 2019 to March 2023. Immunohistochemical examination was performed on tumor tissue with CD44 and CD24 expression were assessed using the H-Score method then determined the cut off-point expression level using the ROC curve. Furthermore, the relationship between these expression levels with the disease-free survival was assessed using the survival curve. Results: There were 48 subjects who were included in the study. There were high expression levels of CD44 in 47.9% and CD24 in 50% of cases. High CD44 expression had mean and median survival of 13.2 ± 1.8 and 11 months (HR 5.05, 95% CI 1.84- 13.85). High CD24 expression had mean and median survival of 13.5 ± 2.4 and 7 months (HR 7.73, 95% CI 2.58 – 23.15). The combination of the two high expressions had mean and median survival of 10.44 ± 1.88 and 7 months. Conclusion: High expression of CD44 and CD24 will shorten the disease-free survival of patients with advanced ovarian cancer.


Introduction
Ovarian carcinoma is the third most common gynecological carcinoma worldwide after cervical and uterine carcinoma [1].In 2020, the World Health Organization (WHO) reported 313,959 cases of ovarian carcinoma in women globally through the Global Burden of Cancer (GLOBOCAN) data register, with a mortality rate of 207,252 [2].According to the data from the Indonesian Society of Gynecologic Oncology (INASGO) for the period 2019-2021, 1,600 cases of ovarian carcinoma were reported in Indonesia, with serous cystadenocarcinoma and mucinous cystadenocarcinoma dominating the cases [3].Given the poor diagnosis at the advanced stage and delay in diagnosis, ovarian carcinoma remains the second

Role of CD44 and CD24 Expression on 2-years Disease Free Survival in Patients with Advanced Epithelial Ovarian Carcinoma
leading cause of death in gynecological carcinoma.The five-year survival rate of ovarian carcinoma in women worldwide is approximately 30%-40% [4].
Despite cytoreductive surgery and chemotherapy, recurrence is common.Ovarian cancer recurrence rates range from 10% in stage I to 20% in stage II, with advanced stages (III and IV) accounting for up to 62%-85% of cases [5].One of the prognostic factors for recurrence that is currently being studied is the presence of cancer stem cell (CSC) factors.Recent studies have revealed the emerging role of CSC expression in contributing to poor clinical manifestations; therefore, the presence of CSC surface markers, such as CD24 and CD44, can be used to predict prognosis in ovarian carcinoma [6].High CD24 expression corresponds with grading, prognosis, and aggressiveness of ovarian cancer cells.In a study of epithelial ovarian cancer patients, CD24 expression was shown to be significantly correlated (p = 0.012) with overall survival after surgery as the primary treatment.Recurrences occur in approximately 60% of individuals with high CD24 and CD44 expression who have undergone primary surgery [7].Moreover, a meta-analysis study examining the significance of CD44 as a prognostic factor for ovarian carcinoma concluded that high CD44 expression was significantly associated with five-year survival rates for ovarian carcinoma [6].
To date, no studies have extensively investigated the association of disease-free survival in ovarian carcinoma and the number of cells expressing CSC surface markers, particularly CD24 and CD44.This study aimed to examine the relationship between the number of cells expressing CD24 and CD44 using H-score as a parameter with twoyear disease-free survival in epithelial ovarian carcinoma.

Study Design and Data Collection
This is a cohort retrospective study, and survival analysis was conducted using the survival curve.Data were obtained from medical records and the paraffin archive in the Dr. Cipto Mangunkusumo National Central General Hospital and the Fatmawati Central General Hospital in Jakarta.This research has received a certificate of passing the ethical reviews of Dr. Cipto Mangunkusumo National Central General Hospital (number KET-707/ UN2.F1/ETIK/PPM.00.02/2022) and Fatmawati General Hospital (number LB.02.2/VIII.2/I6295/2022).

Subjects
The subjects were all patients diagnosed with epithelial ovarian carcinoma between January 2019 and March 2023 and who met the following inclusion criteria: advanced epithelial carcinoma (stages IIB-IVA) with or without recurrence within two years after complete treatment (surgery and six cycles of chemotherapy) and optimal debulking (residual tumor <1 cm after surgery).The exclusion criteria included patients with non-epithelial ovarian carcinoma or epithelial type in stages IA-IIA as well as patients with progressive disease while undergoing chemotherapy (platinum refractory), double primary tumors, and incomplete medical records or inconclusive paraffin block samples.Recurrence criteria were obtained from clinical and supporting examinations (imaging and tumor markers), while mortality data were obtained from medical records or family follow-up.

Slide Preparations and Cell Counts
Immunohistochemical staining is a gold standard in observing the expression of CD24 and CD44.In this study, reagent optimization was agreed upon at an optimal dilution ratio of 1:400.CD44 primary antibody GTX102111 (GeneTex, Inc.) and CD24 primary antibody GTX37755 (GeneTex, Inc.) were utilized.Reagent staining techniques were performed according to the standardized immunohistochemical staining protocol.Further observation was performed using a light microscope at 100x magnification at five fields of view; cells were then counted up to 100 cells per field of view for a total of 500 cells, using ImageJ software.CD24positive staining was observed on the cytoplasm, and CD44-positive staining was observed on the membrane (Figures 1 and 2).A semiquantitative scoring method was used to calculate the CD44 and CD24 intensities of expressions: values of 0 (none), 1 (low), 2 (moderate), and 3 (high).The total score of cells in each field of view and the number of cells with each intensity of stain were calculated using the Histochemical-score (H-score) formula given below: H-score = (% of cells stained in the 0 category ˟ 0) + (% of cells stained in the 1 category ˟ 1) + (% of cells stained in the 2 category ˟ 2) + (% of cells stained in the 3 category ˟ 3).

Statistical Analysis
The data from this study are displayed in the form of text, tables, and/or images.Data were analyzed using SPSS software version 24.The sample demographic data variables are provided in tabular form, including frequency and standard deviation.The parameters used in receiver operating characteristic curve (ROC) are the H-scores of both CD24 and CD44 expressions.With the sensitivity and specificity value obtained from the curve, a cut-off point was determined.Then, H-score cut-off points were classified as "high expression (≥ cut-off point)" and "low expression (< cut-off point)" based on these cut-off values.The survival curves indicate the relationship between the combination of CD44 and CD24 expression and two-year disease-free survival.

Results
Fifty-four patients were eligible for the study and 48 samples met the inclusion criteria, without applying the exclusion criteria.The mean age of the sample was 53.02 ± 3.78 years; the most frequent histopathological type was high-grade serous (50%), with most FIGO stage being IIIB-IIIC-stage macroscopic abdominal spread (58.3%) (Table 1).
The H-score for the CD44 expression was 86.63 ± 62.87 (4-242), with a median of 78; the H-score for the CD24 expression was 88.17 ± 34.85 (24-174), with a median of 95 (Table 2).The AUC value of the CD44 expression was 0.859 (p = 0.000), while that for the CD24 expression was 0.795 (p = 0.000) (Figure 3 and Supplementary Table 1).Based on both sensitivity and specificity curves of the CD44 H-score, the cut-off point was at 87, with a sensitivity value of 77% and specificity of 76.9% (Supplementary Figure 1).The cut-off point of the CD24 H-score was 97, with a sensitivity value of 77% and specificity of 76.9% (Supplementary figure 2).Data were then grouped into "high expression (≥ 87 for CD44 and ≥ 97 for CD24)" and "low expression (<87 for CD44 and <97 for CD24)" based on these cut-off values.
In high CD44 expression, the mean disease-free survival was 13.2 ± 1.8 months.This value was significantly shorter compared to that in low CD44 expression, which    2).Furthermore, in high CD24 expression, the mean disease-free survival was 3.5 ± 2.4 months, while in low CD24 expression, a longer period was also significantly observed with a mean of 37.4 ± 2.5 months (p = 0.000, HR = 7.73; 95% CI, 2.58-23.15)(Figure 5 and Supplementary Table 3).Further, a high expression on both CD44 and CD24 markers (CD44high /CD24low) was found in 15 cases (31.3%).A positive expression only on the CD44 marker (CD44high /CD24low) was found in 10 cases (20.8%), and a positive expression only on the CD24 marker (CD44low/CD24 high) was found in 7 cases (14.6%) (Supplementary Table 4).Most of the recurrence cases (13 cases, 86.7%) were a combination of CD44 high and CD24 high.A high expression on either CD44 or CD24 revealed similar percentages-50% on CD44high/ CD24low and 57.1% on CD44low/CD24high.In the group of CD44low/CD24low, no recurrence occurred after two years of complete treatment (Supplementary Table 5).The combination of high CD44 and CD24 had the highest two-year recurrence trend compared to the expression of only one CD (mean rank 34.45 vs. 26.21;p = 0.000) (Supplementary Table 6).

Combination of CD44 and CD24 Expression in Relation to Disease-free Survival
In this study, a combination of high expression in both markers (CD44high/CD24high) had the lowest mean survival, with a period of 10.44 ± 1.88 months and a median of seven months.On the other hand, in the CD44high/CD24low combination group, the mean survival was 16.94 ± 2.71 months, with a median of 17 months.Moreover, CD44low/CD24high expression had a mean survival of 16.81 ± 5.49 months, with a median of 9 months (Table 3).The combination of high CD44 and CD24 had a mean disease-free periode of 10.44 ± 1.88 months, with a median of 7 months, while a longer disease-free period was observed in the group of either one of the high expression markers (18.94 ± 3.18 and a median of 12 months) (Table 3 and Figure 6).

Discussion
The mean age of the sample was approximately 53.02 ± 3.78 years.Half of the sample in this study included high-grade serous ovarian carcinoma cases (50%).The second most frequent histopathological type was clear cells (27.1%), followed by endometrioid (10.4%) and mucinous (12.5%).According to this study, stages IIIB and IIIC were the most common stages (58.3% of the cases).CA-125 levels were found the most in the range of ≥ 200 U/l (64.6%).This can be thought that the most common histopathological type of the sample was serous carcinoma, and therefore, CA-125 was secreted the most [8].
The H-score calculation for the CD44 expression was 86.63 ± 62.87 (4-242), with a median of 78, while that for  the CD24 expression was 88.17 ± 34.85 (24-174), with a median of 95.To the best of our knowledge, no previous studies have ever examined the H-score values for CD44 and CD24 expression.The mean survival of patients with a high CD44 expression was 13.2 ± 1.8 months.This range was shorter compared to patients with a low CD44 expression, which could reach 35.4 ± 3 months (p = 0.000, HR = 5.05; 95% CI, 1.84-13.85).Several studies have stated that CD24 and CD44 expressions contribute to poor clinical manifestations in ovarian cancer [6,9].A meta-analysis study assessing the role of CD44 as a prognostic factor for ovarian cancer concluded that high CD44 expression was significantly associated with fiveyear survival (RR = 1.42, 95% CI 1.01-2.00,P = 0.05) [6].This result is also in line with the study by Gao et al. [10], which stated that a strong CD44 expression reduced the disease-free survival period compared to the weak expression group.Another study conducted in Surabaya concluded that the higher the expression of CD44, the faster the recurrence of ovarian carcinoma.Patients with CD44 expression ≥12.50 are 5.667 times more likely to have a relapse in less than six months compared to patients with CD44 expression < 12.50 [11].Overexpression of the CD44 marker implied a one-year disease free survival rate of 60% (p = 0.036) [10].
In this study, the group with a high CD24 expression had an average survival of only 13.5 ± 2.4 months, while the group with a low CD24 expression had a longer survival period of 37.4 ± 2.5 months (p = 0.000, HR = 7.73; 95% CI, 2.58-23.15).A study also concluded that the expression level of CD24 was found to be significantly associated with progression-free survival (p < 0.01) and overall survival (p < 0.05).Further, the percentage of recurrence-free survival was 57.5% (follow-up range of 29 months) compared to CD24-negative ovarian cancer cells (p < 0.01) [12].
Research on epithelial ovarian carcinoma has found that CD24 expression is significantly correlated (p = 0.012) to the overall survival of patients who received surgery as primary therapy.Furthermore, CD24 expression was evaluated at surgery after neoadjuvant chemotherapy and a high CD24 expression was found (p = 0.0025).Nagare et al. [7] showed that approximately 60% of patients with high CD24 expression after primary surgery experienced recurrences.No recurrences were found within a two-year range in the group with low combined expression on both markers (CD44low/ CD24low); this contrasted with high expression on either or both CD44 and CD24, which showed a variety of recurrence events in each combination.Sihombing et al. [13] suggested that the CD44+/CD24-combination had a poor prognostic role and chemoresistance in ovarian carcinoma.This difference in results could be due to the different examination methods compared to the study by Sihombing et al. [13], which used the dual-stain CD44 and CD24 reagent staining method, while the current study used separate primary antibody reagents for CD44 and CD24, thereby resulting in different expressions for each cell and field of view [13].
In conclusions, high expressions of CD44 and CD24 correlate with a shorter mean of disease-free survival.This

Table 2 .
H-Score Calculation of CD44 and CD24 Expressions