Introduction

Colorectal cancer (CRC) is one of the world's most prevalent malignant neoplasms It accounts for 1.9 million new cases and 935,000 deaths, as estimated in the year 2020. It represents about one in 10 cancer cases and deaths worldwide, ranking third in terms of incidence and second in mortality. CRC is responsible for a significant burden of disease in the Indian population. Colon carcinoma is reported to develop in 1 in 298 individuals and rectal carcinoma in 1 in 295 individuals.^[1]

Many hypotheses have been postulated for its development. Among all, the cancer stem cell hypothesis has garnered a lot of attention in recent years and has been the subject of much ongoing research. Cancer stem cells are a small population of cells in cancer that are immortal.^[2] They have selectively acquired tumor-related features like uncontrolled growth and the ability to metastasize while maintaining their property of self-renewal and so are responsible for tumor initiation, growth, and recurrence.^[3-5] They were discovered as early as the mid-19th century by Rudolf Virchow ^{[3, 6]} but were first isolated from Acute myeloid leukemia by Bonnet and Dick in the late 20th century.^[7] The colorectal stem cells were first identified in the year 2007 by Brien et al. ^{[6, 8]}

Several stem cells, such as CD44, CD133, EpCAM, and ALDH1, have been studied in colorectal carcinomas. CD44, also known as Hermes antigen,^[9] is a membrane receptor for hyaluronic acid, found to be more concentrated in the malignant and premalignant cells.^[10] After getting activated by binding to hyaluronic acid, it regulates stem cell homing and attracts tumor-associated macrophages in the niches. These macrophages produce growth factors like platelet-derived growth factor, which helps to maintain tumor cells in a continuous proliferative phase. The important functions of CD44 are angiogenesis, cell adhesion, cell migration, cell-to-cell interaction,^{[11, 12]} epithelial-mesenchymal transition, tumor invasion, and metastasis.^[11] In normal colonic mucosa and hyperplastic colonic epithelium, the CD44 cells are expressed at the base of crypts,^[13] their over-expression is
 

reported in inflammation, autoimmune diseases, and in neoplastic conditions.^[10] A Pubmed search of English literature revealed human and animal studies of CD44 staining and expression in colorectal carcinoma and adenoma. However, to the best of the authors' knowledge, there are only a few human studies that studied its expression in adenoma with varying grades of dysplasia and compared the degree of expression with carcinoma, and there was no published study on CD44 expression in colorectal adenoma from Indian population. The role of CD44 has also been studied in many other neoplasms like carcinoma of the breast, pancreas, stomach, and lungs.^[14-17]

Materials and Methods

Participants and procedures

The present study was an ambispective study conducted from September 2019 to August 2021. Our study included histo-pathologically proven CRC cases and adenomas diagnosed during the study period and archived blocks of cases from January 2018 to August 2019. A total of 30 cases of CRC and 20 cases of colorectal adenoma were included in the study. All cases of CRC with a history of previous treatment or those with only incisional biopsy were excluded. Informed consent was taken from all the patients, and the study adhered to the Declarations of Helsinki. The Institutional Ethics Committee approved the study.

Histopathological evaluation

Histological examinations of tumor type and grade were performed according to the criteria outlined in the World Health Organization Classification of tumors, 2019. The parameters evaluated were tumor depth, lymphovascular invasion (LVI), perineural invasion (PNI), and lymph node status. The pathological stage was assessed according to AJCC 8th edition. The presence of mitoses and necrosis was also recorded. The demographic and clinical data included age, gender, and location of the tumor. Adenomas included in the study were classified as adenomas with low-grade dysplasia and high-grade dysplasia.

Immunohistochemical evaluation

The immunohistochemical marker CD44 was evaluated by secondary labeling method on formalin-fixed paraffin-embedded tissue sections (4-5micron thick) mounted on poly L-lysine coated slides. The monoclonal antibody used was CD44 (clone- BSB-12). Tonsillar tissue was taken as a positive control. Negative control was performed by skipping the step of adding a primary antibody.

The assessment of the staining of CD44 was done by two observers independently. When there was discordance between the observers, the slides were reviewed again by the two observers to reach a consensus opinion.

Membranous expression of CD44 was recorded in adenoma, carcinoma, and adjacent uninvolved epithelium. As tabulated in Table 1, the intensity of staining was graded into four groups: 0- none, 1- mild, 2- moderate, and 3- strong staining. The percentage of staining was also graded into four groups: 1: expression in <5%; 2: 5-20%; 3: 21-50%; and 4: in >50% of cells.

For evaluation of staining, the cases were divided into two groups based on the addition of intensity and percentage score, a cumulative score of 0-2: negative staining and 3-7: positive staining. The expression was evaluated based on multiplying the intensity and percentage of cells stained positive. A score of <100 was assigned as a low expression and >100 as a high expression (Figures 1a-1c).

Statistical analysis

Analysis was conducted with SPSS 20 software. To check the association between two categorical variables, chi-square and Fisher exact tests were used. For all statistical tests, a p-value less than 0.05 was considered significant.

Results and Discussion

Clinicopathological parameters

Our study comprised 30 cases of CRC. The average age of patients was 62.7±12.5 years. The study included 17 males and 13 females, with an M: F ratio of 1.3:1; most cases had a tumor size of ≤5 cm. The most common site of carcinoma was the left colon (53%). The maximum number of cases were well differentiated (70%), and only one case of poorly differentiated carcinoma was observed. Most of the cases belonged to Stage III (60%), with none in Stage IV. The presence and absence of lymphovascular invasion (LVI) were almost similar, with 53% of cases that showed LVI. However, most cases showed the absence of perineural invasion (70%). Lymph node metastasis was observed in 60% of the cases. All the clinicopathological observations are tabulated in Table 2.

Abbreviations: SD- Standard deviation; AJCC- American Joint Committee on Cancer; LVI- Lymphovascular Invasion; PNI- Perineural Invasion; LN- Lymph node; HPF- High power field.

Staining of adjacent uninvolved epithelium

Out of the 30 carcinoma cases, adjacent uninvolved mucosa was included in 18 cases. Out of these 18 tissue samples, 77.8% showed positive staining of the uninvolved epithelium. The staining was noted in the crypts and was moderate in intensity in 28.5% and strong in 71.5% of cases.

Correlation of CD44 with clinicopathological parameters

The correlation of CD44 staining and expression with clinicopathological parameters is tabulated in Table 3. Amongst the cases of CRC, 66.7% cases showed positive staining, and 43.3% showed high expression with CD44.

*Fisher's exact test

Abbreviations: N- Number of cases; AJCC- American Joint Committee on Cancer; LVI- Lymphovascular Invasion; PNI- Perineural Invasion; LN- Lymph node; HPF- High power field.