The global cancer burden increased by 9.6 million new cases in 2018, according to global data from the World Health Organization [1]. Colorectal cancer (CRC) accounts for 12.6% (242,000 deaths) of cancer-related deaths in Europe and is one of the most common cancers in women and men [2]. The lowest CRC incidence rates for both sexes are found in all regions of Africa, as well as in Southern Asia [3,4]. In Uganda, the CRC incidence is low; however, the Kampala Cancer Registry has reported a steady increase in the past 20 years. In males, there has been a rise from 7.8 to 11 per 100,000 people in the period 2006 to 2015. While in female CRC, there has been an increase from 8.0 to 9.2 per 100,000 population during the same time period [5]. For the years 2008–2012, the prevalence of colon cancer was 4.0 per 100,000 in men and 4.1 per 100,000 in women [6]. For the same time period, the global age-standardized incidence rate for rectal cancer was 3.8 per 100,000 males and 3.5 per 100,000 females [6].

The topography of colon tumour is also different, with right-sided colon tumours more commonly observed in developed, high-income countries, while many sub-Saharan African countries report a high proportion of left-sided colon tumours especially rectal tumours [7]. These differences are mainly due to the interaction of several genetic mutations and environmental factors. Occurring in about fifty percent of cases of colorectal carcinoma, the p53 gene mutation is a frequent phenomenon [8,9]. Cell cycle regulation is controlled by the 53kd protein (wild type), which is a protein product of the p53 gene. An abnormal protein with a long half-life is produced from the mutation of the p53 gene, resulting in early detection of the protein by immunohistochemistry. The wild-type p53 normally acts as a recessive tumour suppressor gene, while the dominant oncogene is the mutant form of the p53 gene [10]. The conversion of late adenomas to carcinoma in the adenoma-carcinoma sequence is due to the p53 mutation in colorectal carcinoma [11].

In many tumours, a poor prognosis and reduced survival have been associated with the detection of p53 protein in malignant cells [12,13]. The objective of this study was to determine the proportion of p53 expression in Ugandan CRC patients compared to the developed Western world and to determine the association of p53 expression with the grade, stage, LVI status, histopathological subtype, and topography of a colorectal tumour.

There were 404 formalin-fixed and paraffin-embedded (FFPE) blocks for the 1^st January 2008 to the 15^th September 2021 participants that had a histopathological diagnosis of colorectal adenocarcinoma. From September 16^th, 2019 to September 16^th, 2021, samples of colorectal biopsy specimens and resected colorectal carcinoma specimens were obtained from patients in Masaka Regional Referral Hospital, Mulago National Referral Hospital, Uganda Martyrs’ Hospital Lubaga, and Mengo Hospital. Using a standard pretested Data Extraction Form, data for all tissue samples was extracted from the clinical patients’ files in the respective hospitals and the Kampala Cancer Registry. The data included age, sex, stage, grade of colorectal cancer, and topography. For those patients that had more than one FFPE tissue block following a colorectal resection, all the blocks were examined, and the one that represented more than 50% tumour, with not much mesenchymal tissue and no necrosis, was selected for the study.

There were 353 CRC FFPE tissue block samples, which were excluded due to poor quality tissue block samples and from participants after having had chemotherapy and/or radiotherapy treatment. Therefore, out of the 404 participants, 51 were selected for p53 IHC analysis. The FFPE tissue block samples came from patients with histologically confirmed colorectal adenocarcinoma who fulfilled the following selection criteria: The inclusion criteria included index histologically diagnosed colorectal adenocarcinoma samples. Exclusion criteria included colorectal adenocarcinoma samples taken from patients after having had chemotherapy or radiotherapy treatment, poor-quality tissue block samples, and tissue samples with incomplete or unavailable data.

Haematoxylin and eosin sections were prepared to establish the histopathological subtype, degree of differentiation of the tumour (grade), and Lymphovascular invasion status. The histopathological examination established the histopathological subtype of colorectal adenocarcinoma (AC, MAC, or SRCC), LVI status, and the main features of the tumours and then the ABC method for immunohistochemistry was used for the p53 marker. Fifty-one FFPE tissue block cases were processed for p53 immunohistochemistry examination. 4µm-thick sections from 10% formalin-fixed paraffin-embedded tissue were taken to perform immunohistochemistry for p53. The procedure used was first deparaffinization in xylene, rehydration in the alcohol series, and then distilled water. Heat-induced epitope retrieval in alkaline novo link epitope retrieval buffer (pH9) was done. Then there was incubation with peroxide block, washing with Tris-buffer saline (RBS), incubation for 30 minutes with primary antibody, which included a 1:100 p53 antibody standard, Novolink post-primary antibody, and Novo link polymer, and washing in Tris-buffer saline (TBS), then development in DAB chromogen and counter-stained with Mayer’s haematoxylin. The p53 antibody dilution was 1:100; the source was DAKO Agilent USA, clone DO-7, and reference IR6 16.

Free peroxide splits the diaminobenzidine from the ABC complex, and this localizes the antigen precisely in the nuclei of the cytoplasm by providing a brown precipitate. The nuclei, which were counterstained with Mayer’s haematoxylin followed by dehydration, were then mounted in an organic medium. The specimen slides were mounted and then photographed on a Nikon eclipse microscope. The immunohistochemical technique applied to the paraffin-embedded tumoral samples for p53 used a scale for staining intensity that included 0: none and (+1) present. The p53 molecular marker was correlated with the grade and stage of the tumour.

Left-sided tumours were splenic flexure tumours or tumours located distal to this site, including the rectal tumours [14,15].

Continuous numeric data were summarized by mean (standard deviation), and categorical data were summarized as frequencies and percentages. The distribution of the intensities of p53 was determined and compared by age (≤median age and above median age), sex (male and female), grade (I-III), stage, LVI status, histopathological subtypes, and topography. A Pearson chi-square test was used to assess the association between p53 expression and demographic and histopathological variables. Spearman’s rank correlation coefficients were used to determine and compare correlations between the p53 biomarker and grade. A p-value of ≤0.05 was considered statistically significant.

This study was approved by the Higher Degrees Research and Ethics Committee, School of Biomedical Sciences, College of Health Sciences, Makerere University (reference number: SBS-HDREC-630), and the Uganda National Council for Science and Technology (reference number: HS-2574). Written informed consent was obtained from prospective participants included in the study before completing the questionnaire form. A waiver of consent was obtained from the Higher Degrees Research and Ethics Committee, School of Biomedical Sciences, College of Health Sciences, Makerere University, for the colorectal adenocarcinoma FFPE tissue blocks obtained retrospectively from the archives of the Department of Pathology, School of Biomedical Sciences, College of Health Sciences, Makerere University, and to access and abstract the corresponding data from the Kampala Cancer Registry and from the case files in the respective hospitals. All the data and specimens pertaining to the research were kept confidential. The ethical standards that apply to research were applied according to the Helsinki Declaration.

Representative images of p53 staining at different magnifications are shown in Figures 1, 2, and 3. Among 51 IHC p53 participants, the mean age (SD) was 52.5 (17.1) years; 24 (47.1%) participants were male and 27 (52.9%) participants were female. There were 25 (49.0%) participants who had colon cancer; 26 (50.9%) had rectal cancer; 15 (29.4%) had right-sided colon cancer; and 36 (70.6%) had left-sided colon cancer (Table 1). Early-stage CRC consisted of 10 (19.6%) participants with stage I CRC and 12 (23.5%) participants with stage II disease. Late-stage CRC consisted of 16 (31.4%) participants with stage III CRC and 12 (23.5%) participants with stage IV disease.

The histopathological subtypes of the participants included 40 (78.4%) classical adenocarcinomas (AC), 7 (13.7%) mucinous adenocarcinomas (MAC), and 4 (7.8%) signet ring colorectal carcinoma (SRCC). Grade II was most commonly seen in 38 (74.5%) participants; 7 (13.7%) had grade III, and 6 (11.7%) had grade I tumours. Positive Lymphovascular invasion (LVI) was seen in 36 (70.6%) participants, while 15 (29.4%) participants had no Lymphovascular invasion (LVI).

The relationship of p53 status expression with baseline demographic and histopathological parameters is shown in Table 1. The relationship between the presence of p53 expression and baseline parameters and comparisons among the variables were compared using Pearson chi-square tests in Table 2.

Table 1: Comparison of P53 Status with Baseline Characteristics.

^*Fischer’s exact test

The presence of p53 expression was significantly associated with left-sided colon tumours, grade II+III tumours, the presence of LVI, and the classical adenocarcinoma histological subtype (Table 2).

There were 20 (74.1%) left-sided colon tumours that presented with p53 expression compared to 7 (25.9%) right-sided colon tumours that presented with p53 expression, and this reached statistical significance (p = 0.0004). Moderately and poorly differentiated tumours constituted 23 (85.2%) of all tumours that had p53 expression, compared to only 4 (14.8%) well differentiated tumours that presented with p53 expression, and this was statistically significant (p = 0.0000).

Compared to 10 (37% of tumours) that had p53 expression but no Lymphovascular invasion, there were 17 (63% of tumours) that had p53 expression and were associated with Lymphovascular invasion. This finding reached borderline statistical significance (p = 0.0561). There were 22 (81.5%) participants with AC compared to 5 (18.5%) with MAC and SRCC that had positive p53 expression, and this reached statistical significance (p = 0.0000).

There was a negative correlation between the CRC grading and p53 expression (r = -0.1189), which did not reach statistical significance (p = 0.4059). A negative correlation was also found between CRC stage and p53 expression (r = -0.1702), and this was not statistically significant (p = 0.2324).

Table 2: Presence of P53 Expression in Relation To Demographics and Some Pathological Characteristics.

Globally, the prevalence of p53 protein expression has been reported to range from 52.5% to 61.4% [14-20]. In the present study, p53 protein expression has been found in 52% of colorectal carcinomas, similar to global findings. Other studies have found p53 expression varying between 43% and 48% [21-23]. Studies have shown that the p53 mutation is positively correlated with p53 expression [23-25]. P53 gene mutations are important in the pathogenesis of colorectal cancer, and the overexpression of p53 in the present study, together with previous studies, supports this theory.

The p53 gene mutation is responsible in both high- and low-risk populations for a subset of sporadic colorectal adenocarcinomas. Studies from the Middle East have shown that the presence of p53 in low- and high-risk populations is similar, possibly due to a common pathway involving the p53 mutation in colorectal carcinogenesis [26]. In low- and high-risk populations, smoking and alcohol consumption have been found to be responsible for mutations in p53 in colorectal adenocarcinoma [25-27]. The present study found that past alcohol drinkers in Ugandan patients increased the likelihood of CRC and therefore were partly responsible for mutations in p53 in our population.

It has been shown that the pathogenesis of right- and left-sided colon tumours is different, with differences in prognosis. These pathological and clinical differences between left- and right-sided colorectal tumours have been reported in many studies [16,19,28]. In the present study in Uganda, a higher proportion of p53-positive tumours have been found with left-sided colon cancers compared to right-sided colon cancers. This finding may imply that the pathogenesis of right- and left-sided colon cancers is different, and hence their prognosis is different. In sporadic left-sided colon cancer, p53 mutations are more common than in right-sided colon cancer [21,29]. Therefore, in Uganda, this finding may suggest that many colorectal cancers are sporadic. Right-sided colon tumours tend to be associated with hereditary nonpolyposis colorectal cancer (HNPCC) [30,31]. Since adenomatous polyposis syndromes have rarely been reported in Uganda, dietary factors tend to play a key role in the pathogenesis of left-sided colon cancer [29,30]. A change in diet, particularly with increased consumption of red meat, has been found to partly explain the increase in the incidence of CRC in Uganda [32-34].

Studies have shown that the overexpression of p53 in left-sided colon tumors is associated with short survival and a poor prognosis [14,22,25,19,29,35,36]. The present study showed that p53 expression was not correlated with the grade of CRC in Ugandan patients. The relationship between p53 expression and the grade of colorectal carcinoma has been shown to differ between studies [29,35,36]. Zgurova N found no correlation between p53 in colorectal adenocarcinoma and the grade of differentiation of the tumour [36]. There was also no correlation between p53 expressions and the TNM stage, topography, size, gender, or age [36]. In contrast to these findings, Kapiteijin E found that p53 was overexpressed in 70% of tumours that were poorly differentiated. However, less than 30% of moderately and well-differentiated tumours had overexpression of p53 [29]. These findings were in contrast to those from a study by Rambeau PE which showed that, compared to poorly differentiated tumours, p53 was overexpressed in well-differentiated tumours [35]. The same study showed a trend that with a worsening grade of CRC, there was a decreased p53 expression. The findings are in contrast to those in the present study, which showed that in Ugandan patients, p53 is not influenced by the degree of CRC differentiation. However, among those tumours that had p53 expression, there were more patients that were moderately and poorly differentiated compared to those that were well differentiated. Similar to findings by Caneiro FP, the present study found no relationship between the histological differentiation (grade) of the tumour and p53 expression [37,38]. However, this finding is in contrast to findings from other previous studies [29,35].

Differences in the expression of p53 among populations may explain these divergent results. A p53 mutation cannot be ruled out due to a lack of p53 nuclear staining. Mutations that produce a deletion will not produce any protein and hence will need to be detected by genetic sequencing and will not be detected by immunohistochemistry [18,21,24]. In missense mutations where there is an increase in protein stability, detection of p53 is easily carried out on immunohistochemistry [21]. Bazen V found that patients with advanced tumour CRC stage and grade had markers due to deletions [21]. In the present study, genetic sequencing for p53 was not carried out on all fifty-one p53 samples, and therefore the type of p53 mutation could not be ascertained. However, in the present study, the marker used for mutation of the p53 gene was p53 protein expression, and immunohistochemistry has been found to detect between 75% and 94% of CRC tumours having the actual gene mutation [29,39]. The advantage of using immunohistochemistry for the detection of p53 expression is that it is cheaper than molecular analysis and more feasible.

In the literature, there are many discrepancies regarding the correlation between protein immunopositivity and the type of gene mutation [18,40]. Generally, those studies reporting no association between prognosis and overexpression of p53 are equal to those that report poor survival in positive p53 cases [35,36,40]. The present study also showed more Lymphovascular invasion among those tumours that positively expressed p53, and this reached borderline statistical significance. This is in keeping with findings from Yousse Rahman, OAE, and Powell E et al., which showed that p53 expression is associated with Lymphovascular invasion [41,42]. Therefore, p53-positive tumours have a higher tendency to metastasize, and Powell et al. showed that mutant p53 has a role in tumorigenesis, invasion, and metastasis [41].

The evaluation of p53 expression in rectal tumours and the sensitivity of the tumour to radiotherapy is still unclear [43]. Rodel C et al. and Saw RP et al. demonstrated that neither p53 nor bcl-2 status has a correlation with response to radiotherapy [44,45]. However, Rebischung et al. showed that the sensitivity to radiotherapy was correlated with the presence of p53 mutations [46]. In these studies, the response to radiotherapy was evaluated by using histologic tumour regression grade and pathologic down staging following radiotherapy treatment [47-49]. The present study did not evaluate the role of radiotherapy in the response to rectal tumours that have positive p53 expression; however, future studies may evaluate this role in Ugandan patients.

Similar to other parts of the world, in Ugandan patients, p53 expression is more commonly present in left-sided colon tumours. Therefore, this may support the theory in the West that left-sided colon tumours have a different pathogenesis than right-sided colon tumours and hence have a different prognosis.

The intensity of p53 expression is not influenced by the degree of differentiation (grade) or stage of CRC. P53 expression is more commonly present in left-sided tumours which in Ugandan patients is similar to other parts of the world. This finding may support the theory in the West that left-sided colon tumours have a different pathogenesis than right-sided colon tumours and hence have a different prognosis. Further studies should be carried out to determine the types of genetic mutations or epigenetic factors responsible for the difference in prognosis between left-sided and right-sided CRC in Ugandan patients.

Figure 1: Immunohistochemical Expression of p53 in Colorectal Carcinoma Tissue, Showing Diffuse Strong Positive (3+) Nuclear Expression of p53. Magnification x100.

Figure 2: Immunohistochemical Expression of p53 in Colorectal Carcinoma Tissue, Showing Diffuse Strong Positive (3+) Nuclear Expression of p53. Magnification x200.

Figure 3: Immunohistochemical Expression of p53 in Colorectal Carcinoma Tissue, Showing Diffuse Strong Positive (3+) Nuclear Expression of p53. Magnification x40.

Limitations

The number of rectal cancer patients excluded due to neoadjuvant chemo radiotherapy was small, and therefore it is unlikely that selection bias was introduced. The FFPE tissue blocks in the retrospective arm of the study may have been influenced to some extent by the antigen degradation of archival materials. To mitigate this influence, FFPE tissue blocks were randomly selected mainly from the prospective arm of the study and assessed for tissue quality prior to carrying out IHC for p53. High standards of laboratory testing were also followed. The storage of the specimens was kept for a short period of time.

Other limitations included the inclusion of a heterogeneous population of colon and rectal tumours. Instead of tissue microarrays, whole tissue sections were used, which, although labor-intensive, avoided false-negative results.

Ethical Approval

This study was part of the PhD work, which was approved by the Doctoral Committee and Higher Degrees Research and Ethics Committee of the School of Biomedical Sciences, College of Health Sciences, and Makerere University for the corresponding author (SBS-HDREC-630). Final approval of this research study was obtained from the Uganda National Council for Science and Technology (HS-2574).

Consent for Publication

Consent was obtained from all the participants enrolled in this study.

Competing Interests

The authors declare that they have no competing interests.

Funding

The authors declare that they received no specific funding for this work. However, the corresponding author personally funded this part of his PhD research study. No payment was received by the authors to write and publish this part of the study.

Authors’ Contributions

Richard Wismayer conceived the concept and proposal, collected data, performed data analysis and wrote the first draft. Julius Kiwanuka performed data analysis and provided statistical support. Michael Odida and Henry Wabinga interpreted all the immunhistochemical slides and performed critical reviews of the manuscript for intellectual content. All authors approved the final manuscript for publication.

Acknowledgements

The authors wish to thank the clinical staff and research assistants, particularly Dr. Sulaiman Ishaq Mahmud and Dr. Justus Atuhaire who recruited the participants from the Department of Surgery of Masaka Regional Referral Hospital, Mulago National Referral Hospital, Uganda Martyrs’ Hospital Lubaga and Mengo Hospital for their support in this research project. Lastly, we are also grateful to Ms Dorothy Nabbale for the laboratory technical work carried out for this part of the research project in the Department of Pathology, School of Biomedical Sciences, College of Health Sciences, Makerere University.

Abbreviations

CRC – colorectal cancer

LVI – lymphovascular invasion

P53 protein – tumour protein p53

AC – classical adenocarcinoma

MAC – mucinous adenocarcinoma

SRCC – signet ring colorectal carcinoma

DAB chromogen – 3, 3’ – Diaminobenzidine chromogen

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