Globally, colorectal cancer (CRC) constitutes a major public health issue with approximately 1.2 million new cases yearly [1]. There is a wide global variation in CRC mortality and incidence. In high-income developed countries the overall incidence of CRC is declining and CRC is a disease of the elderly, with most patients diagnosed after 60 years of age [2]. In contrast, developing low-income countries in Sub-Saharan Africa have shown an increase in the incidence of CRC in recent studies [3-5]. In Uganda and other Sub-Saharan African countries, CRC patients tend to present at an advanced stage and at a younger age [6-10]. Moreover, in developing low-income countries the stage for stage survival is significantly worse and the CRC death rates outnumber those of developed high-income countries [5,11].

Molecular markers such as CDX2 represent a bowel-specific tumour suppressor which inhibits the dissemination and progression of colorectal cancer [13]. A nuclear protein is produced by the specific tumour suppressor which is essential for the growth and proliferation of epithelial cells and expressed in bowel epithelium is CDX2. During tumorigenesis, CDX2 is frequently downregulated [14-16], whilst during tumour cell invasion and metastasis of colorectal adenocarcinoma, β-catenin which is an oncoprotein is expressed [17,18]. In colon cancer, CDX-2 has been found to inhibit the β-catenin/T-cell transcription factor transcriptional activity and cellular proliferation [19,20]. A lack of CDX-2 results in unregulated proliferation with loss of differentiation of the bowel epithelium [21-24].  However, the mechanism for loss of CDX-2 expression is not well understood and for early-stage CRC, this low expression has been found to be linked to a poor prognosis [25].

Due to the role of CDX-2 as a tumour suppressor, its loss is associated with advanced stage, high tumour grade and microsatellite instability in CRC [26-29]. A recent study by Dalerba et al., has shown that CDX-2 has been found to be absent in a subgroup of colon cancers characterised by high levels of ALCAM, a molecule which is expressed at high levels at the bottom of human colonic crypts [25,30,31]. The results of this study showed that CDX-2 negative tumours are associated with a poor prognosis and a lower rate of disease-free survival than CDX-2 positive tumours, independent of many known risk factors involving tumour grade [25]. However, although some studies have shown an unfavourable course associated with the lack of CDX-2 expression, other studies have not confirmed this finding [27-29]. Patients with stage II CDX2 negative colon cancer who are treated by surgery alone, may benefit from adjuvant chemotherapy [25].

The loss of CDX-2 tends to be more evident at the invasive front of the tumour where the metastatic process is initiated [26,32,33]. Expression of CDX-2 results in the formation of multicellular structures with demonstrable enterocyte and goblet cell features in an undifferentiated intestinal cell line, which supports a role for CDX-2 in bowel differentiation. This results in the upregulation of transcription from genes such as MUC2 and liver-intestine cadherin by CDX2 [34,35].

The relationship between intestinal differentiation and CDX-2 expression suggests that CDX-2 may serve as a marker of epithelial tumours in the gastrointestinal tract. This finding is supported by recent surveys which showed staining of colorectal adenocarcinomas using a commercially available monoclonal antibody to CDX2 [36-38]. Using the same anti-CDX2 monoclonal antibody, the objective of this study was to determine the relation between CDX-2 and clinicopathological features of colorectal adenocarcinoma in Ugandan patients.

Formalin-fixed and paraffin-embedded (FFPE) tissue blocks for the 1^st January 2008 to the 15^th September 2021 participants that had a histopathological diagnosis of colorectal adenocarcinoma were retrieved from the archives of the Department of Pathology, School of Biomedical Sciences, College of Health Sciences, Makerere University. From 16^th September 2019 to 16^th September 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 were 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 which represented more than 50% tumour, with not much mesenchymal tissue and with no necrosis was selected for the study.

Exclusion criteria included colorectal adenocarcinoma samples taken from patients after having had chemotherapy and radiotherapy treatment, poor quality tissue block samples and tissue samples with incomplete or unavailable data. There were 55 CDX-2 participants who met these selection criteria for CDX-2 immunohistochemistry (IHC) analysis.

Haematoxylin and eosin sections were prepared to establish the histopathological subtype, degree of differentiation of the tumour (grade), and lymphovascular invasion (LVI) status. The histopathological examination established the histopathological subtype (AC, MAC or SRCC), LVI status and the main features of the tumours and then the ABC method for immunohistochemistry was used for the CDX-2 marker.

4µm thick sections from the 10% formalin-fixed paraffin-embedded tissue were taken to perform immunohistochemistry for CDX-2. The procedure used was first deparaffinization in xylene, rehydration in alcohol series, and brought to distilled water. Heat induced epitope retrieval in an alkaline novolink epitope retrieval buffer (pH9) was done. Then incubation with peroxide block, washing in Tris-buffer saline (TBS), incubation for 30 minutes with primary antibody which included 1:100 CDX-2 antibody, standard Novolink post-primary antibody, and Novolink polymer and washing in Tris-buffer saline (TBS) then development in DAB chromogen and counter-stained with Mayer’s haematoxylin. The CDX-2 antibody dilution was 1:100, source DAKO Agilent USA, clone DAK-CDX2 and reference IRO080.

Free peroxide splits the diaminobenzidine from the ABC complex and this localizes the antigen precisely in the nuclei and 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 on the paraffin-embedded tumoral samples for CDX-2 used a random scale for staining intensity which included: 0: (negative) none; weak (+1): CDX2 immunoreactivity in 1-10% of tumour cells; moderate (+2): CDX2 immunoreactivity in 10-50% of tumour cells; strong (+3): CDX-2 immunoreactivity in >50% tumour cells. This molecular marker was correlated with the grade and stage of the tumour.

Right-sided tumours included those in the caecum, ascending colon, hepatic flexure and transverse colon whilst left-sided tumours were those in the splenic flexure, descending colon, sigmoid colon, rectosigmoid and rectum.

Continuous numeric data were summarised by mean (standard deviation) and categorical data were summarized as frequencies and percentages. The distribution of the intensities of CDX-2 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. Pearson chi-square test was used to assess the association between CDX-2 expression and demographic and histopathological variables. Spearman’s rank correlation coefficients were used to determine and compare the correlations between the CDX-2 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 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 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.

Among 55 CDX2 participants, the mean age (SD) was 52.4 (15.8) years; 29 (52.7%) were male and 26 (47.3%) were female. There were 25 (49%) participants that had colon cancer; 26 (50.9%) had rectal cancer; 11 (21.6%) had right-sided colon cancer and 14 (27.4%) had left-sided colon cancer (Table 3). A significant proportion of participants, 32.7% (n=18) had stage III CRC, whereas 15(27.3%) had stage IV disease. Early-stage CRC comprised of 11 (20%) participants with stage I CRC and 9 (16.4%) participants with stage II disease. The stage was missing for 2 (3.6%) participants.

The histopathological features for the 55 CDX2 participants included, 6 (10.9%) mucinous adenocarcinoma, 6 (10.9%) SRCC and 43 (78.2%) had classical adenocarcinoma. Based on the grade of differentiation, grade II was most commonly seen in 34 (69.4%) participants, 8 (16.3%) had grade III, and 7 (14.3%) had grade I tumours. Positive lymphovascular invasion (LVI) was seen in 33 (60%) participants whilst 1 (1.8%) participant had no lymphovascular invasion (LVI). The LVI status was unknown in 21 (38.1%) participants.

There were 45 (81.8%) participants with CDX2 positive CRC tumours and 10 (18.2%) participants with CDX2 negative CRC tumours. There was no difference in the baseline characteristics of age, sex, grade, topography, histopathological subtype and LVI status between CDX2 positive and CDX2 negative tumours (Table 7). Representative images of CDX2 staining at different magnifications are shown in Figures 1-6.

The distribution of staining intensity of CDX2 regarding the tumour grade, tumour depth, stage, topography, histopathological subtype and LVI status is shown in Tables 1-6.

Table 1: CDX2 antibody distribution regarding CRC tumour grade.

Table 1 compares the CDX2 intensity of expression in CRC with tumour grade. G1 (well differentiated) tumours had 42.9% high intensity expression (+3) for CDX2 compared to G3 (poorly differentiated) tumours which were associated with 37.5% high intensity expression (+3) for CDX2. There were 28.6% G1 tumours that stained negatively for CDX2 and 25% G3 tumours that stained negatively for CDX2 (Table 1).

Table 2: CDX2 antibody distribution regarding CRC tumour depth (T) in bowel wall.

Table 2 compares the CDX2 intensity of expression in CRC with the tumour depth (T). Absent CDX2 was found in 25% T2 tumours, 20% T3 tumours and 19.1% T4 tumours compared to 0% T1 tumours. Maximal intensity of expression (+3) for CDX2 was found in 66.7% T1 tumours compared to 12.5% T2 tumours, 40% T3 tumours and 33.3% T4 tumours (Table 2).

Table 3: CDX2 antibody distribution regarding the CRC tumour stage.

Table 3 compares the CDX2 intensity of expression in CRC with the stage of CRC. Stage II and stage III CRC had a negative CDX2 expression in 22% of tumours, while stage IV tumours had CDX2 negative expression in 26.7%. There were 18.2% stage I tumours that had a negative expression for CDX2. Maximal CDX2 intensity of expression (+3) was found in 33.3% stage III and stage IV tumours compared to 36.4% stage I and 44.4% stage II tumours.

Table 4: CDX2 antibody distribution regarding the CRC tumour topography.

Table 4 shows that rectal tumours and left-sided colon tumours have a maximal CDX2 intensity of expression (+3) in 38.5% and 42.9% of tumours compared to 18.2% of right-sided colon tumours.

Table 5: CDX2 antibody distribution regarding histopathological subtype.

Table 5 shows that the absence of CDX2 expression was found in the AC histopathological subtype in 16.3% of tumours compared to 33.3% of MAC tumours and 16.7% of SRCC tumours. Maximal expression of CDX2 (+3) was found in 41.9% of AC tumours and 33.3% of SRCC tumours.

Table 6: CDX2 antibody distribution regarding lymphovascular invasion (LVI) status.

Table 6 shows that with a negative CDX2 expression, there were 0% that had no lymphovascular invasion compared to 12.1% that had lymphovascular invasion.

Relation between CDX2 Status Expression and Baseline Parameters

The relationship of CDX2 status expression with baseline demographic and histopathological parameters is shown in Table 7.The relationship of lack of CDX2 with baseline parameters with comparisons among the variables was compared using Pearson chi square tests in Table 8.

Table 7: Comparison of CDX2 status with baseline characteristics.

*Fischer’s exact test

Association of Lack of CDX2 Expression In Relation To Demographics and Histopathological Characteristics

Lack of CDX2 expression was significantly associated with the presence of lymphovascular invasion (p=0.005) (Table 34). Compared to 20% of CRC participants with stage I disease there were 40% of participants who presented with stage IV disease exhibited a lack of CDX2 expression, however, this did not reach statistical significance (p=0.329). There were 55.6% that presented with high-grade poorly differentiated adenocarcinoma compared to 22.2% presenting with low-grade well-differentiated adenocarcinoma in those that exhibited a lack of CDX2 expression, however, this was not statistically significant (p=0.126). There were 60% rectal participants and 40% colon participants in those exhibiting a lack of CDX2 expression however, this did not reach statistical significance (p=0.371).

There was a negative correlation between the CRC grading and CDX2 (r=-0.0235) which did not reach statistical significance (p=0.8729).

Table 8: Lack of CDX2 expression in relation to the demographics and some pathological characteristics.

In the present study, the loss of CDX-2 expression was 18.9% overall, with 40% being stage IV CRC. Studies conducted by Singh J and Baba Y [38] have reported a loss of CDX-2 expression in 33.6% and 29% of the patient population respectively [38]. Different studies have reported a variation in the loss of expression of CDX-2 which ranges from 4-30% [38,40,24].

Studies have reported that CDX-2 expression significantly varies with the stage of CRC with more CRC cases showing loss of CDX-2 expression with the advanced stage of CRC [38]. In the present study, 20% of stage I CRC lacked CDX-2 expression compared to 40% of stage IV cases, respectively. Female sex was associated with loss of CDX-2 expression in a study by Bae [26]. However, due to population heterogeneity, similar to the findings by Singh J the present study did not find any significant relation with gender or age.

In the present study, lymphovascular invasion (LVI) was associated with a lack of expression of CDX-2. These findings are in keeping with those reported in other studies [38,26,20,21,18,40,24,22,42,16,43,17,23,45]. The presence of LVI is a poor prognostic marker in CRC with a lack of expression of CDX2.

In the literature, the down regulation of CDX2 is correlated with poor differentiation, right-sided tumours, BRAF mutations and MMR deficiency [46]. Studies have shown no correlation between KRAS mutations, MMR deficiency and CDX2 expression. Compared to those without BRAF V600E mutation, there was a significantly lower CDX2 expression rate with BRAF V600E mutated CRC [46].

The CDX2 expression rates in the different histologic subtypes of CRC have been evaluated in other studies [26]. The CDX2 expression rates have been found to be similar between mucinous carcinomas and classical adenocarcinomas, however, in our study the lack of CDX2 expression was more commonly found in classical adenocarcinoma. Other studies have shown that compared to other subtypes, the CDX2 expression rate of medullary carcinoma was significantly lower [26]. Previous studies have shown that CDX2 expression of medullary carcinoma was 0%, 10% and 25% respectively [38,48,49]

A specific marker of intestinal mucosa is CDX2 and its diagnostic role is suitable for metastatic CRCs. A study has found that the expression rates of metastatic CRCs were higher than those of primary CRCs [26]. Therefore compared to primary CRCs, the expression rates of metastatic CRCs were higher. For the differentiation of metastatic CRCs, CDX2 may be a useful marker. Previous studies have shown that a poor differentiation grade of CRCs is significantly correlated with CDX2 loss or downregulation [40,51,52]. Our study showed no significant correlation between CDX2 expression and tumour differentiation. Since there is no correlation between CDX2 expression and differentiation, between poorly differentiated carcinomas, the diagnostic impacts on the differential diagnosis are more important.

A study has shown that loss of CDX2 expression has been found to have a poor response to chemotherapy and is associated with less survival benefit [53]. However, the present study did not evaluate the role of chemotherapy in patients presenting with loss of CDX2 expression. Loss of CDX2 expression is related to a poor outcome since CDX2 has an important role in promoting cellular differentiation and proliferation of the bowel [54]. Since the loss of CDX2 expression is associated with a biologically aggressive tumour, it presents more commonly at an advanced stage [55].

The present study did not evaluate the effect of CDX2 loss on survival, however, other studies have shown diverse results on survival outcomes. Lugli A and Baba Y have shown in univariate analysis a poor survival for loss of CDX2 in CRC however the same results were not found in multivariate analysis [38,56]. Other studies have shown that in early-stage CRC a poor prognosis was associated with loss of expression of CDX2 [26,24,28].

The findings in the present study in Ugandan patients, in keeping with previous studies in the West found that loss of CDX2 expression was associated with poor prognostic markers such as lymphovascular invasion. These findings from colorectal cancer patients in Uganda are testimony to the role of loss of CDX2 as a poor prognostic factor in colorectal adenocarcinoma.

Loss of CDX2 expression is associated with poor prognostic markers such as lymphovascular invasion in Ugandan patients. The expression of CDX2 is a useful marker to differentiate between CRCs and its expression is useful as a predictor for the prognosis of CRC patients. Since loss of CDX2 expression is associated with a biologically aggressive tumour, it presents more commonly with an advanced stage. The response of CDX2-negative tumours to chemotherapy and its relation to survival need to be investigated by carrying out further studies in Uganda.

Figure 1: Immunohistochemical expression of CDX2 in colorectal carcinoma tissue, showing diffuse strong positive (3+) nuclear expression of CDX2 in all tumoral cells. Magnification: x4.

Figure 2: Immunohistochemical expression of CDX2 in colorectal carcinoma tissue, showing diffuse strong positive (3+) nuclear expression of CDX2 in all tumoral cells. Magnification: x20.

Figure 3: Immunohistochemical expression of CDX2 in colorectal carcinoma tissue, showing diffuse strong positive (3+) nuclear expression of CDX2 in all tumoral cells. Magnification: x20.

Figure 4: Immunohistochemical expression of CDX2 in colorectal carcinoma tissue, showing diffuse strong positive (3+) nuclear expression of CDX2 in all tumoral cells. Magnification: x10.

Figure 5: Immunohistochemical expression of CDX2 in colorectal carcinoma tissue, showing diffuse strong positive (3+) nuclear expression of CDX2 in all tumoral cells. Magnification: x4.

Figure 6: Immunohistochemical expression of CDX2 in colorectal carcinoma tissue, showing diffuse strong positive (3+) nuclear expression of CDX2 in all tumoral cells. Magnification: x40.

The number of rectal cancer patients excluded due to neoadjuvant chemoradiotherapy 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 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.

A weakness of the study was the duration of fixation of samples, particularly for those FFPE tissue blocks in the retrospective arm of the study. For the prospective arm of the study, the storage of the specimens was kept for a short period of time in 10% formalin for up to 24 hours with biopsy specimens and up to 72 hours for resection specimens however, there was no control over the quality of fixation.

Other limitations were inclusion of a heterogenous population of colon and rectal tumours. Instead of tissue microarray, the use of whole tissue sections were used which although labour 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, 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 colorectal cancer research project in the Department of Pathology, School of Biomedical Sciences, College of Health Sciences, Makerere University.

Abbreviations

CRC – colorectal cancer

LVI – lymphovascular invasion

AC – classical adenocarcinoma

CDX2 – caudal-type homeobox transcription factor 2

MAC – mucinous adenocarcinoma

SRCC – signet ring colorectal carcinoma

DAB chromogen – 3, 3’ – Diaminobenzidine chromogen

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