Ethical approval for this study was obtained from the Health and Research Ethics committee of Aminu Kano Teaching Hospital, Kano (ethical review reference number: AKTH/MAC/SUB/12A/P-3/V1/2904). Additional consent of individuals whose samples were used in this study was waived, as is the institutional ethical committee’s policy for retrospective studies involving non-human subjects. Patients’ identities were concealed at all times, and records were kept under lock.

This retrospective, descriptive, hospital-based study was carried out in the Histopathology Laboratory of the Department of Histopathology, Aminu Kano Teaching Hospital, Kano (north-western Nigeria). Aminu Kano Teaching Hospital is a tertiary hospital with over 700-bed capacity. The histopathology department of Aminu Kano Teaching Hospital receives an average of 5500 histological samples per annum, and renders services including cytology, histology, autopsy, and immunohistochemistry.

Adenoma cases included in this study were all cases that were histologically diagnosed with colorectal adenoma in the study centre from 01 January 2015 to 31 December 2019. Cases with insufficient clinical information, particularly biodata, missing or damaged blocks, and tissue blocks with insufficient tissue were excluded (in this regard, excluded case is zero).

Data, such as age, site of lesion, histologic diagnosis, and grade of the disease was obtained from Kano cancer registry, pathology request forms, patients’ case notes, duplicate copies of histopathological reports, and slide reviews of cases. The patient’s identity was concealed at all times. The cases were classified and graded according to the World Health Organization classification of tumours of the colon and rectum.¹⁹ The two-tiered system of grading was used based on prognosis: low grade and high grade.¹⁹

Immunohistochemical staining was performed using anti-COX-2 rabbit polyclonal antibody (Elabscience, Houston, Texas, United States; catalogue No. E-AB-70031), used at a 1:500 dilution according to standard immunohistochemical staining protocols. The antibody was stored at −20 °C (using −25 °C freezer 110 L, Biobase Scientific, Jinan, Shandong, China) and it was centrifuged (using LC-4K-2 Centrifuge, Biobase Scientific, Jinan, Shandong, China) before opening to ensure complete recovery of contents. A kidney sample (an autopsy sample) with intact renal tubules was used as a positive control, while a negative control was obtained by replacing the primary antibody with distilled water.

Statistical analysis was performed using Statistical Package for Social Sciences version 25 (IBM^® Corp, Armonk, New York, United States). Chi-square was used with statistical significance set at p < 0.05 to test the association between the COX-2 expression and clinicopathological parameters such as age, sex, histological subtype, and grade. Proportions and central tendencies of nominal and continuous variables were obtained by descriptive statistics of frequency, mean and median.

Twenty-nine adenoma cases were analysed, and 26 cases were endoscopic biopsies. The age distribution of colorectal adenoma cases ranged from 12 years to 80 years with a mean age of 53.4 (standard deviation = 16.0) years. The largest proportion of cases (23 of 29 cases; 79.3%) clustered within the category of patients aged 40 years to 79 years and peaked in the 6th decade. Eleven cases of adenomas (37.9%) occurred before age 50 years while only six cases (20.7%) occurred at age 70 years or more (Table 2).

There were 20 male cases and nine female cases, giving a male-to-female ratio of 2.2:1. Male cases occurred in individuals aged 12 years to 75 years, with a mean age of 51.7 years. The nine cases that were observed among the female patients occurred from 39 years to 80 years, with a mean age of 57.2 years (Table 2).

Eighteen (62.1%) cases of colorectal adenoma were tubular adenomas; of these, eight cases were high-grade lesions. Eight (27.6%) cases were tubulovillous adenomas (five of which were high-grade), and only three (10.3%) cases were villous adenomas (including one high-grade lesion, Figure 1). There were no significant associations between histological type and the age group (χ² = 15.8538 degrees of freedom [df] = 14, p = 0.3224) or sex (χ² = 0.2529, df = 2, p = 0.8812).

Fifteen cases (51.7%) of adenoma were high-grade adenomas (Table 2). This comprised nine cases of tubular, five cases of tubulovillous, and one case of villous adenoma. Fourteen cases (48.3%) were low-grade. Among the female patients, there were only five cases (33.3%) of high-grade adenoma, and four cases (28.6%) in the low-grade tumours category. There were no statistically significant associations between the grade of tumour and age (χ² = 8.8808, df = 7, p = 0.2613) or sex (χ² = 0.0762, df = 1, p = 0.7818).

Thirteen cases (44.8%) of adenoma were colonic in origin, while the remaining cases were rectal tumours (Table 2). There were no statistically significant associations between the site of tumour and age (χ² = 5.2217, df = 7, p = 0.6329).

The only case that showed COX-2 positivity (strong positive staining) was a low-grade tubular adenoma in a 60-year-old man (Figure 1). There was no statistical association between COX-2 over-expression and age, sex, or histological type of the tumour.

In the present study, COX-2 expression in the adenomas was 3.4% (1 out of 29 cases). The available data demonstrate relatively lower COX-2 expression in adenoma than in the CRC. The possible reason why COX-2 expression is higher in CRCs than in adenomas may be that although COX-2 expression is involved in tumour initiation, it is also a marker for advanced lesions (from dysplasia in adenomas to frank malignancy and invasiveness in carcinomas).^30,31 For example, it has been shown that COX-2 expression has a positive correlation with adenoma size, dysplasia, and grade but not with the tumour location or configuration, and the expression is higher in the CRC.^{30,32,33,34,35} This may also explain why the expression of COX-2 in colorectal adenomas demonstrates a wide variation similar to that of the CRC, from 38% to 100%, because of adenoma size, presence of dysplasia, histological subtype, and key genetic mutation.^35,36 Early adenomas are usually small and the progression of most small adenomas is slow, over several years, even though not all adenomas progress. Some may remain stable, regress or new ones may form. The progression in the spectrum of the adenoma-carcinoma sequence may involve more genetic mutations and molecular interplay, including over-expression of COX-2. To illustrate this, immunohistochemical studies have shown that 37.9% of 39 cases of adenoma were COX-2 positive; 90.9% of these positive cases had a Kirsten rat sarcoma mutation that correlates with tumour size (larger tumour), and the positivity is more frequent in tubulovillous (63.3%) than tubular adenoma (36.4%).³⁶ Contrary to this assertion that the COX-2 expression is a marker of progression, a COX-2 immunohistochemical study of nine advanced CRC and 12 adenoma cases in Japan by Bamba et al. showed a higher expression in adenomas than in CRCs.³⁷ Bamba et al.’s contrary observation is difficult to decode, but it may stem from the low sample size in their study.³⁷

The COX-2 expression (3.4%, 1 out of 29 cases) in this study confirms the assertion that COX-2 is a marker of progression in the adenoma-carcinoma spectrum, but it is relatively lower than other studies. It is difficult to determine the reasons, but the possibilities may include racial factors, as most studies involve non-African subjects, genetic make-up – particularly COX-2 gene polymorphism, and the fact that most adenomas analysed in this current study were colonoscopic biopsies, which may not represent the entire tumour heterogeneity, even though the adenomas from three colectomy cases did not express COX-2 as well. More studies are needed for Africa to explain the possible racial disparity in COX-2 expression in adenomas, because most available COX-2 studies on colorectal adenomas and CRCs are from America and Asian countries. Carriers of the COX-2 A-1195G AG polymorphism have increased risk for CRC and adenoma, and mutation in the adenomatous polyposis coli (APC) gene has been shown to correlate with COX-2 expression in both adenomas and CRCs.^10,38,39 More studies are needed to establish the role of race in COX-2 expression in adenoma.

Colorectal adenomas are known precursor lesions of CRC with similar risk factors.^6,7,8 Thus efforts are required to interrupt the possibility of the progression of adenomas to colorectal cancer.⁶ The expression of COX-2 provides vital information for the uses of nonsteroidal anti-inflammatory drugs in the prevention and treatment of adenoma and hurting adenoma progression into carcinoma. Thus, in terms of the prognosis and COX-2 targeted therapy in the near future, this study provides a strong rationale for additional studies in Nigeria and Africa at large.

The limitation of this study is the inability to include rare histological sub-types of adenoma and relatively low cases of adenomas in this study. To improve this, a multicentre study across Africa will give a better reflection of COX-2 expression in colorectal adenoma in Africa.

Adenomas occurred twice as commonly in men as in women. Tubular adenomas constituted 62.1% of all adenomas, and 51.7% of adenomas are high-grade adenomas. The rate of over-expression of COX-2 in adenomas is 3.4%, and this may imply its early involvement in colorectal tumourigenesis.