CD44 as a prognostic marker in early colorectal cancer: single center experience “South Egypt cancer institute”, Egypt

ABSTRACT Background Colorectal cancer (CRC) is a common and lethal disease; it remains the third most frequently occurring cancer among both genders. In Egypt, colon cancer represents the ninth most common cancer while rectal cancer ranks the 18th most common cancer by incidence. The application of cancer stem cells (CSC) as prognostic markers in CRC is now studied, among which is CD44, which is implicated in cellular growth, differentiation, survival as well as the metastatic behavior of cancer cells. The prognostic role of CD44 in CRC is still controversial. The study aims at inspecting CD44 immunoreactivity in the epithelium of colorectal cancer specimens and to detect its association with the patients’ clinicopathological features as well as its prognostic significance. Methods This retrospective cross-sectional study included 85 CRC specimens and 16 adenoma specimens collected from the Pathology Department, South Egypt Cancer Institute, during the period from 2018 to 2020. All specimens were stained by anti-CD44 antibody. Results CD44 epithelial expression in colon cancer tissue specimens was substantially higher than in normal mucosa. It was observed that 95% (n = 81) of the studied colorectal carcinoma cases showed positive CD44 epithelial expression compared to distant normal mucosa as controls as well as adenoma cases where only 43.8% (n = 7) of adenoma cases showed positivity with highly significant p-value P = 0.000 and it correlates with cancer progression and aggressiveness. Low H score expression H ≤ 150 had better 3 years disease-free survival DFS estimated by 83.3% compared to 79.6% with high H score expression with significant P-value (P = 0.041). Discussion Our current study showed a statistically significant association between CD44 expression and tumor size (T), lymph node metastasis (N) as well as disease stage with P-values (P = 0.009), (p = 0.000) and (p = 0.000), respectively. CD44 epithelial immunostaining showed to be of an adverse prognostic significance in early CRC associated with more aggressive behavior and worse survival.


Introduction
Colorectal cancer (CRC) is a common and lethal disease; it remains the third most frequently occurring cancer among males and females worldwide representing 8% of all estimated newly occurring cancers in 2022.CRC showed male predominance with 80,690 estimated new male cases and 70,340 new female cases [1].
Worldwide, the median age for diagnosis of CRC is 66 years in males and 69 years in females.However, the age at diagnosis is less for rectal cancer (age 62 and 63, respectively) than that for colon cancer (age 67 and 71, respectively) [2].
The overall mortality rate of CRC is 60%, which represents the second leading cause of cancer-related death in western societies.Over the past few decades, the mortality rates of CRC patients have been reduced and attributed to early diagnosis and treatment [3].The relative five-year survival rate is 90% for colorectal cancers diagnosed at an early stage compared with only 13% for those diagnosed at advanced stages [4].
The variation in CRC patients' prognosis prompts an urgent need for new molecular biomarkers to boost the accuracy for predicting the CRC patients' prognosis as a complement to the traditional Tumor-Node-Metastasis (TNM) staging system for clinical practice [3].
Cancer stem cells (CSCs) or tumor initiating cells are defined as a group of undifferentiated cells within a tumor that have the ability of self-renewal and initiation of carcinogenesis [9].A variety of CSC have been proposed for CRC [10].Among which is CD44, which is a cell hyaluronate transmembrane glycoprotein implicated in cell growth, differentiation, survival as well as the metastatic behavior of some cancer cells [11].
Various studies indicated that lymphoma, breast, and endometrial cancer have raised levels of CD44 mRNA.Besides, CD44 is extensively overexpressed in other cancer types including gallbladder, prostate, ovarian, oral squamous cell carcinoma, and gastric cancer, correlating with aggressive biological behavior, worse prognosis as well as incidence of tumor relapse [12,13].
The aim of this research is to study CD44 in the epithelial cells of colorectal cancer specimens and to detect its association with the patients' clinicopathological features and its prognostic significance.

Study cohort
In this cross-sectional study, we have analyzed 16 adenoma specimens (including tubular, villous and Tubulovillous), 85 early colorectal adenocarcinoma specimens at diagnosis, and 10 distant normal mucosal specimens attended to South Egypt Cancer Institute (SECI) from January 2018 to December 2020.The formalin-fixed paraffin-embedded tissues were collected from the oncological pathology department, South Egypt Cancer Institute, Assiut University from patients after primary surgery for colorectal cancer.All pathological data and grading were revised by an oncologic pathologist according to WHO 2019.Cases presented with primary synchronous metastasis, recurrent adenocarcinoma, and cases with any other malignancies were excluded from the study.
The clinicopathological as well as survival data were collected from the patients' records.Ethical approval was obtained from Institutional Board Review (IBR) (SECI-IRB) IORG0006563 approval No.509 Date September 8 2020.

Immunohistochemical staining
The formalin-fixed, paraffin-embedded tissues were cut into sections 5-microns thick and placed on positively charged slides.Tissues were de-paraffinized then rehydrated in graded alcohols to distilled water.Slides were incubated in (Tris EDTA) in a heated water bath at a temperature of 97°C for 20 min for antigen retrieval.Primary mouse monoclonal anti-Human CD44, antibody (Catalog #BSB 6238, Bio SB, USA) at 1/200 concentration (optimum dilution) was used for tissue sections and incubated for 1 h at room temperature in a humid chamber.Then, washing of the slides was done.Thereafter, immunostaining was performed using a universal staining kit "Ultra Vision Detection System Ant Polyvalent, HRP/DAB (Ready-To-Use)" (LAB VISION corporation, catalog # TP-015-HD, Fremont, California 94,539-6406, USA) following the manufacturer's instructions.Lastly, (DAB solution) was added to the slides for 5-10 min.The counterstain used was Mayer's hematoxylin.Sections from reactive lymph node were used as positive control.However, for negative control, we used sections of tissue-specific positive control but without the primary antibody.

Interpretation of Immunohistochemical staining
CD 44 expression was determined by membranous staining technique.The histochemical score (H-score) was calculated as the product of multiplying the intensity (I) of staining scored as (0) Absent, (1) weak, (2) Moderate, (3) Strong by the percentage of positive tumor cells (P) detected.The H-score ranges from 0 to 300 with 150 as a cut-off value.
According to previous literatures where H score < 150 is considered low and H score ≥ 150 is considered high [7].

Survival analysis
Survival data of patients meeting our inclusion criteria were obtained by reviewing the files of colorectal cancer patients attended to SECI from January 2018 to December 2020.

Statistical analysis
The data were collected, tabulated, and statistically analyzed using SPSS (Statistical package for the social science; SPSS Inc., Chicago, IL, USA) version 26.Quantitative data were presented as mean (± SD) and median or range if not normally distributed.Qualitative data were presented in terms of frequencies (number) and relative frequencies (percentages).Comparison of quantitative variables was done using student t-test for normally distributed data and Mann-Whitney U-test for non-normally distributed data.For comparing categorical data, Chi-square (χ2) test was performed.Fisher Exact test was used instead when the expected frequency is less than 5. P-value is always two-tailed set significant at 0.05 level.

Results
We evaluated 16 adenoma specimens and 85 colorectal cancer specimens.The clinicopathological data including age, gender, type of adenoma, and degree of dysplasia are shown in Table 1 while that of pathologically diagnosed colorectal cancer are described in Table 2.
It was observed that 95% (n = 81) of the studied colorectal carcinoma cases show positive CD44 epithelial expression compared to distant normal mucosa obtained from specimens of the same patients away at least 10 cm from the tumor.Similarly, the studied colorectal carcinoma cases showed more positive CD44 epithelial expression compared to adenoma cases where only 43.8% (n = 7) of adenoma cases showed positivity with highly significant p value P = 0.000 as shown in Table 3.
The current study revealed that 67% (n=57) of the studied CRC cases showed high CD44 epithelial membranous expression (H score ≥ 150 as shown in Figure 1).A highly statistically significant association was observed between either high epithelial expression and the presence of lymph node invasion, LVI, PNI (P=0.000) as well as advanced stage (p=0.000)as shown in Table 4.
The cumulative survival in relation to H score is demonstrated in Figure 2, where low H score expression (H < 150) represented by the blue line had longer cumulative survival where after 3 years of follow up, the percentage of patients who survived were 94% versus 67.5% for those with high H score expression with significant value (P = 0.009).On the right-side, Kaplan-Meier survival curve shows the relation between disease-free survival DFS and H score expression where low H score had better 2 years DFS estimated by 83.3% compared to 79.6% with high H score expression with significant P value (P = 0.041).

Discussion
CD44 is an important membrane receptor for hyaluronic acid (HA).It is accepted as a CSC marker in many solid tumors [14].In several studies, it has been proven that CD44 is associated with several biological behaviors of the neoplasms, including proliferation, metastasis, recurrence, and resistance to therapy [15].However, there are still controversies about its prognostic role [16].In our current study, we have investigated 16 adenoma cases specimens (divided as tubular, villous, and Tubulovillous) and 85 colorectal adenocarcinoma specimens as regard clinicopathological features and CD44 epithelial expression in each group.There was a highly statistically significant relation between positive epithelial expression of CD44 in colorectal carcinoma cases compared to distant normal mucosa with P value (P = 0.000), this result is similar to that of Holah et al [7] and Liu et al [17] who found that CD44 positive cells were not present in the normal colonic mucosa adjacent to colorectal tumors and this might be attributed to the fact that  normal colonic tissue does not express variant isoforms of CD44, whereas tumor cells seemed to express a wide variety of CD44 isoforms [18].Our current study showed that only 7 (43.8%)adenoma cases showed positive membranous expression of CD44, and this agrees with the findings of Holah et al [7] who also found that colorectal adenomatous polyps showed membranous expression of CD44 and this is explained by the fact that variant forms of CD44−mRNA might be expressed in the early stage of colorectal carcinogenesis.
Positive CD44 epithelial expression was observed in 95.3% of CRC cases (n = 81) compared with 57.1% reported by Holah et al [7], 51% reported by Lugli et al [19], and 70% reported by Chun et al [20].This might be attributed to either the fact that CD44 is implicated in the carcinogenesis of CRC as it is involved in the activation of the tyrosine kinase receptor c-Met, which is responsible for the regulation of proliferation, motility, invasion, and metastasis [21] or due to the expression of a wide variety of CD44 isoforms, which might have remarkable homology in their antigenic repertoire [18].
Our current study showed a statistically significant association between CD44 expression and tumor size (T), lymph node metastasis (N) as well as disease stage with P values (P = 0.009), (p = 0.000) and (p = 0.000), respectively, in agreement with Zhao et al [22] who found a significant association between CD44 expression and stage II and III.This relationship was is in contrary with Chen et al [21] where CD44 expression showed no significant relationship with disease stage.
High epithelial expression of CD44 was statistically significant associated with the presence of LVI, PNI as well as lymph node metastasis (P = 0.000).This is in agreement with Li et al [23] and Zhao et al (Zhao, Lin et al. [22]) who found a significant association between CD44 expression and lymph node metastasis.
Survival analysis was performed for CD44 expression and there was a statistically significant association  The current study revealed that 67% (n=57) of the studied CRC cases showed high CD44 epithelial membranous expression (H score ≥ 150 as shown in Figure 1.A highly statistically significant association was observed between either high epithelial expression and the presence of lymph node invasion, LVI, PNI (P=0.000) as well as advanced stage (p=0.000)as shown in Table 4.
between low epithelial expression (H score) and 3-year OS (p = 0.009) as well as a statistically significant association between low epithelial expression and 3-year DFS (p = 0.041).Our results agreed with Huh and Kim et al [24] who similarly found that CD44 overexpression was associated with lower cancer-related survival.Also, a study done by Yan et al [25] concluded that overexpression of CD44 is associated with short  disease-free survival.However, our results disagreed with Galizia et al [26] and Holah et al [7] who found no significant association between CD44 and overall survival.This difference could be attributed to the difference in the number of the studied cases, and different techniques for detection of CD44 in different studies.
Our study has a limitation of small number of cases due to the limited availability of CD44 Kits and evaluation of CD44 by other methods as measurement of its m RNA by real-time PCR [27].

Conclusions
Our study concluded the presence of a significant statistical correlation between high CD44 epithelial expression (expressed by high H score), lymph node metastasis, and advanced stage considered a poor prognostic factor associated with shorter overall survival and early recurrence.We recommend further studies on expression of CD44 using a larger sample size.

Figure 1 .
Figure 1.Intensity of CD44 immunostaining either negative, weak, moderate or strong in epithelial cells captured by Olympus BX 45 Microscope.

Figure 2 .
Figure 2. Survival function in relation to H score.

Table 2 .
Clinicopathological data of the studied colorectal carcinoma CRC cases (n = 85).

Table 3 .
Comparison between immunohistochemical expression of CD44 in studied CRC specimens compared with distant normal mucosa and adenoma specimens.

Table 4 .
The relationship between epithelial expression of CD44 and clinicopathological details of studied colorectal carcinoma (n = 85).