There is an estimated annual 1.2 million new cases of colorectal cancer globally resulting in this non-communicable disease being a major public health issue [1,2]. The global mortality and incidence of CRC show wide variation between continents. CRC patients present with advanced disease and a younger age in Africa [3-5]. The advanced stage presentation of CRC may be due to a lack of screening in African patients. In resource deprived settings in Uganda and generally Sub-Saharan Africa, the most reliable diagnostic tool, namely colonoscopy is not easily accessible. However, in most parts of the world, carcinoembryonic antigen (CEA) is the most widely used tumour marker [5].

Circulating levels of carcinoembryonic antigen (CEA) is widely accepted after primary tumour resection to monitor tumour recurrence and this marker has proved to be useful in the management of colorectal cancer [6-11]. When used in conjunction with the TNM staging system, determining the presence of preoperative CEA may be of prognostic value. After curative resection of the primary tumour, the CEA molecular marker may be helpful in immunotherapy ad adjuvant chemotherapy trials by identifying patients at high risk of recurrence [12-14].

In some CRC patients, however, the clinical course did not correlate with the plasma CEA level. Previous studies have shown that in CRC patients with “poorly differentiated” tumours, the CEA levels did not monitor correctly the clinical course [15].

Some studies have reported strong staining intensity in well-differentiated adenocarcinoma. A low CEA content and weak staining intensity have been found in poorly differentiated adenocarcinoma [16,17]. In well-differentiated colorectal; adenocarcinoma the CEA molecular marker is primarily localized at the apical surface of cancer glands. Whilst in moderately differentiated adenocarcinoma, the CEA is localised to the intracytoplasmic and entire surface of the cancer cells (18).

CEA is localized to the luminal surface and has been found to be associated with lower serum CEA levels than CEA localized to the basolateral membrane or stromal tissue [18]. With these findings, one may conclude that CEA which flows into the ductal lumen contributes lower serum CEA than CEA flowing from interstitial tissue into the blood vessels. Vascular and lymphatic drainage of colon cancer contributes to a high serum CEA level [19].

CEA has been proposed for roles in both the diagnosis and prognosis of colorectal cancer. In terms of the prognostic value of CEA in patients with known colorectal cancer, the evidence has been conflicting. Several studies have shown that patients with high preoperative concentrations of CEA have a worse outcome than those with low levels [20]. Preoperative elevation of CEA, and the degree of elevation, is associated with an increased risk of recurrence and decreased long-term survival [21,22] with the highest level of evidence [23]. Also, when analysing subsets of patients (stage II/III), preoperative CEA is reported to be a significant prognostic factor [21]. Following potentially curative resection, CEA may rise if recurrence occurs, and the reported sensitivity and specificity are 64% and 91%, respectively [24]. Even when normal preoperatively, it will rise in at least 50% of patients with recurrent disease [25], making it useful in routine follow up programmes. Currently, CEA is most often measured preoperatively as a means of detecting disease recurrence or monitoring response to treatment [26].

Therefore, the objectives of this study were to correlate elevations in plasma CEA levels with the extent of tissue CEA and determine the association of clinicopathological features with tissue CEA in primary tumours in Uganda.

From 16^th September 2019 to 16^th September 2021, samples of colorectal biopsy specimens, resected colorectal carcinoma specimens and blood samples were obtained from patients in Masaka Regional Referral Hospital, Mulago National Referral Hospital, Uganda Martyrs’ Hospital Lubaga and Mengo Hospital. There were 119 consecutively recruited participants with corresponding formalin-fixed and paraffin-embedded (FFPE) tissue blocks and blood samples that were included for plasma and FFPE tissue CEA analysis. Using a standard pretested Data Extraction Form, data for all tissue samples was extracted from the clinical patients’ files in the respective hospitals. 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.

A 10mls blood sample was obtained from the 119 prospectively recruited participants, preoperatively to obtain the plasma CEA level and these blood samples were transported using a cold chain system at 4-80C to the Department of Immunology & Molecular Biology, School of Biomedical Sciences, College of Health Sciences, Makerere University. The inclusion criteria for the preoperative plasma CEA levels included patients with index histologically diagnosed colorectal adenocarcinoma and patients had to be willing to provide their blood samples for plasma CEA preoperatively. Blood samples with incomplete or unavailable data were excluded.

The FFPE tissue block samples came from patients with histologically confirmed colorectal adenocarcinoma and who fulfilled the following selection criteria: Inclusion criteria included index histologically diagnosed 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 (LVI) 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 CEA marker. 4µm thick sections from 10% formalin-fixed paraffin-embedded tissue were taken to perform immunohistochemistry for CEA. The procedure used was first deparaffinization in xylene, rehydration in alcohol series, and brought to distilled water. Heat-induced epitope retrieval in alkaline novolink 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 1:100 CEA 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 CEA antibody dilution was 1:100, the source was DAKO Agilent USA, clone I17 and reference IR622.

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 on the paraffin-embedded tumoral samples for CEA used a scale for staining intensity, adopted by Vrabie et al., 2008 which included: negative (0) (none); weak (+1) CEA immunoreactivity in 1-10% of tumour cells; moderate (+2): CEA immunoreactivity in 10-50% of tumour cells; strong (+3): CEA immunoreactivity in >50% tumour cells. The CEA molecular marker was correlated with the grade and stage of the tumour. The histological grade and LVI status was determined by two experienced consultant pathologists who reviewed all the histological slides as soon as they were received and prepared by the laboratory technicians. Internal quality control of the immunohistochemical experiments was carried out and screened by two independent pathologists to ensure the reliability of the experimental study.

For the CEA antibody, the laboratory control tissue had a proven positive slide. At every run of the day, a section of negative and positive controls were used. A standard laboratory research protocol was devised for the study to analyze CEA. The quality control sample selection criteria included those with primary tumours that were ≥1mm thickness, with an estimated ≥50% tumour content and with an acceptable morphology. The two pathologists were blinded to any clinical information that may link them to the colorectal cancer cells in the internal clinical databases. All plasma samples for CEA levels were stored at 4-8⁰C.

Left-sided tumours were splenic flexure tumours or tumours located distal to this site including the rectal tumours [27,28]. Right-sided tumours included tumours in the caecum, ascending colon, hepatic flexure, and transverse colon. [27,28].

Continuous numeric data were summarized by mean (standard deviation) and categorical data were summarized as frequencies and percentages. The distribution of the intensities of CEA 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 CEA expression and demographic and histopathological variables. Spearman’s rank correlation coefficients were used to determine and compare correlations between the CEA biomarker and grade. A p-value of ≤0.05 was considered statistically significant. Average plasma CEA levels (ng/ml) were compared to tissue CEA intensities using the analysis of variance (ANOVA) technique. A p-value of ≤0.05 was considered statistically significant.

Baseline Characteristics for CEA Participants

In this study, there were 119 CEA participants with CRC including 67 male participants and 52 female participants. The median age (SD) of all participants was 59.9(15.3) years. Among 119 participants, 56(50.9%) had colon cancer; 54(49.1%) had rectal cancer; 26(23.6%) had right-sided colon cancer and 30(27.3%) had left-sided colon cancer (Table 1).

A significant proportion of participants, 29.4% (n=35) had stage III CRC, whereas 33(27.7%) had stage IV disease. Early-stage CRC consisted of 17(14.3%) participants with stage I CRC and 30(25.2%) participants with stage II disease. The stage was missing for 4(3.4%) participants.

The histological subtypes for all CEA participants included 99(85.3%) participants with classical adenocarcinoma, 8(6.9%) participants with MAC and 9(7.8%) participants with SRCC.

Based on the grade of differentiation, grade II was most commonly seen in 77(72.6%) participants, 16(15.1%) had grade III, and 13(12.3%) had grade I tumours. There were 79(66.4%) participants that had positive lymphovascular invasion (LVI), whilst 1(0.8%) participant had no lymphovascular invasion (LVI). The LVI status was unknown in 39(32.8%) participants.

Relation of CEA Expression with Baseline Parameters

The relationship of CEA expression with baseline demographic and histopathological parameters is shown in Table 1. The expression of CEA increased with the increasing stage of CRC. In stage I, there were 12(15.1%) participants whose tumours stained positively for CEA, compared to 23(26.7%) for stage II and stage III and this increased to 27(31.4%) for stage IV. However, compared to those that lacked CEA staining this did not reach statistical significance (p=0.480). There were 58(73.4%) colorectal tumours that were moderately differentiated and 9(11.4%) that were poorly differentiated with CEA-positive tissue tumours (Table 1). Representative images of CEA staining at different magnifications are shown in Figures 1-5.

Table 1: Relationship of CEA with baseline and pathological parameters.

^*Fischers exact test          

There were 58(98.3%) participants that had a positive tissue CEA, however were associated with no lymphovascular invasion. All participants whose tissues lacked CEA, 21(100%) were associated with no lymphovascular invasion. There was no difference between the presence and absence of CEA in the colorectal tissues for lymphovascular invasion (p=0.737).

CEA Positive Colorectal Tumours

Among the positive CEA participants, the proportion that stained positively was 26.7% for stage III versus 15.1% for stage I and this was borderline statistically significant (p=0.057). Compared to stage I, there were 31.4% CRC tissues that stained positively for CEA in stage IV and this reached statistical significance (p=0.0101). (Table 2). Compared to 15.2% of CRC tissues with grade I CRC that stained positively for CEA there were 73.4% with grade II CRC which stained positively for CEA and this reached statistical significance (p=0.0000). There were more participants with AC (85%) compared to MAC (5.8%) and SRCC (9.2%) that stained positively for CEA and this reached statistical significance (p=0.0000) (Table 2).

Table 2: Relation of the presence of CEA in CRC tissue with baseline parameters.

A positive correlation between the grade of colorectal cancer and CRC tissue CEA was found (r=+0.2204) and this was statistically significant (p=0.0232) using the Spearman correlation.

The distribution of CEA antibody staining intensity with tumour grade, tumour depth, stage, topography, histopathological subtype and lymphovascular invasion is shown in tables 3-8.

Table 3: CEA antibody distribution regarding CRC tumour grade.

Table 3 compares the CEA intensity of expression in CRC with the tumour grade. It shows that the highest CEA expression (+3) was more commonly associated with poorly differentiated (G3) tumours in 31.3% of CRC compared to 23.1% of well-differentiated (G1) CRC.

Table 4: CEA antibody distribution regarding CRC tumour depth (T).

Table 4 compares the CEA intensity of expression in CRC with the tumour depth (T). T1 and T2 tumours were associated with a 16.7% CEA intensity (+3). However, T4 tumours were associated with a 31.7% CEA intensity (+3) and T3 tumours were associated with a 14% CEA intensity (+3).

Table 5: CEA antibody distribution regarding the CRC stage.

Table 5 compares the CEA intensity of expression in CRC with the stage of CRC. Stage II CRC was associated with a 16.7% CEA intensity (+3) compared to stage I CRC which was associated with a 5.9% CEA intensity (+3). Stage IV CRC was associated with a 33.3% CEA intensity (+3) compared to 20% CEA intensity (+3) for stage III disease.

Table 6: CEA antibody distribution regarding CRC tumour topography.

Table 6 compares the CEA intensity of expression in CRC with the location of the tumour. Right-sided colon tumours were associated with a 23.1% CEA intensity (+3) and left-sided colon tumours were associated with a comparable 20% CEA intensity (+3). Rectal tumours were also associated with a comparable 18.5% CEA intensity (+3).

Table 7: CEA antibody distribution regarding histopathological subtype.

Table 7 compares CEA intensity with the histopathological subtype. There were more SRCC tumours with a higher 55.6% CEA intensity (+3) compared to 25% CEA intensity (+3) for MAC and 19.2% CEA intensity (+3) for AC.

Table 8: CEA antibody distribution regarding lymphovascular invasion (LVI) status.

Table 8 demonstrates the CEA antibody distribution with the presence or absence of lymphovascular invasion. The presence of LVI in CRC was more commonly associated with the presence of CEA at (+2) and (+3) CEA intensities. The presence of LVI was associated with a 32.9% CEA intensity (+2) compared to lack of LVI which was associated with a 0% CEA intensity (+2). The presence of LVI was associated with a 21.5% CEA intensity (+3) compared to lack of LVI which was associated with a 0% CEA intensity (+3).

Plasma CEA Level versus CRC Tissue Level

The mean plasma CEA level was 23.6 ng/ml. The average plasma CEA levels (ng/ml) did not differ by CEA intensity in CRC tissue (p=0.725). An ANOVA analysis was used to assess this association (Table 9). Therefore there was no relation between preoperative plasma CEA level and CEA in CRC tissue.

Table 9: Relation between preoperative plasma CEA (ng/ml) and CRC tissue CEA.

The present study evaluated the CRC tissue CEA and the correlation of the results with preoperative plasma CEA levels, stage, grade, topography and histopathological subtype of CRC.

In this study, no significant correlation between CEA tissue expression and serum CEA was found in the colon cancer cohort and rectal cancer cohort. There was no significant correlation found between preoperative serum CEA and CEA tumour intensity. Previous studies have shown that other factors can affect serum CEA levels including smoking, benign disease, location of the CEA receptor on the cell membrane and tumour necrosis [28,29]. There was no correlation found between the pT stage, stage, location of CRC and histopathological subtype of CRC when compared to CEA tumour intensity.

However, there was a positive correlation between the intensity of CEA in the tissue and the grade of colorectal cancer. This is in keeping with findings from a study by Vrabie CD, which also demonstrated a positive correlation between the intensity of tissue CEA with the grade of CRC which was statistically significant [30]. In the study by Vrabie, it was demonstrated that irrespective of tumour stage there was the same CEA tissue intensity in most CRC cases, similar to the findings in this study [30].

Park et al., showed that CEA-negative tumours have significantly lower serum CEA concentrations than CEA-positive CRC tumours [31]. This association has not been reproduced in other studies [32,33]. Compared to colon adenocarcinoma cases, more cases of rectal adenocarcinoma in this study showed CEA expression. This CEA tissue intensity was independent of the preoperative serum CEA level. Therefore CEA-targeted agents might benefit all patients with rectal cancer independent of their serum CEA level. However, regarding the role of CEA in tumour response to chemotherapy no consensus has been reached [34-36].

Preoperative tumour extent, recurrence and outcomes have been shown to be associated with CEA levels in CRC tissue [37]. Studies have shown that more serum CEA is produced from well-differentiated CRCs than from poorly differentiated CRCs [38,39]. This is probably due to the fact that it is not clearly defined whether it is cellular CEA, free CEA or both that is responsible for cell differentiation [40].

A study by Tong G, showed that tumour topography, size, and TNM stage were not significantly associated with CRC tissue CEA expression [41] similar to the findings in this study. Tong G et al., also showed a significant correlation between a higher CEA CRC tissue expression and grade of CRC similar to this study. Poorly differentiated CRC has a higher incidence of lymph node metastasis and a higher CRC tissue CEA expression. Poor CRC differentiation (high-grade CRC) and increasing numbers of positive lymph nodes are associated with a high tissue CEA expression [41].

In resectable CRC, elevated preoperative plasma CEA levels have been found to be associated with a poorer prognosis [42]. An association between tumour stage and levels of plasma CEA have been found [43]. After tumour resection postoperative CEA levels will decrease. In developed high-income countries, CEA has been found to be useful in monitoring disease progression and is the antigen of choice to predict prognosis after diagnosis of CRC [44]. Colorectal tissue CEA has also been found to be associated with a poor prognosis.

The findings from this study show that increasing CEA intensity in CRC is associated with a poorer grade in Ugandan patients. This finding suggests a poorer prognosis for patients expressing high levels of CEA in their colorectal cancer tissue in Uganda.

The present study showed that colorectal cancer tissue CEA expression has a higher prognostic value than that of preoperative plasma CEA. There was no correlation found between preoperative plasma CEA and tissue CEA. Therefore, the CEA tissue intensity was independent of the preoperative serum CEA level. This study showed a significant correlation between a higher CEA CRC tissue expression and the grade of CRC. Poorly differentiated CRC have a higher incidence of lymph node metastasis and a higher CRC tissue CEA expression. This finding suggests a poorer prognosis for patients expressing high levels of CEA in their colorectal cancer tissue in Uganda.

Limitations

A limitation for analyzing the preoperative plasma CEA levels in this study was that different time intervals elapsed between plasma CEA measurement concentrations and colorectal resection. Underrating or overrating the plasma CEA level may have influenced the outcome of the study due to the timing of the measurement of plasma CEA.

For this 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. High standards of laboratory testing were also followed. High standard of laboratory testing means using procedures based on sound quality control and quality assurance. Quality control referred to measures that were taken to ensure that the results and interpretation meets a specified standard of quality. Quality assurance refers to measures that were taken to monitor, verify, and document performance by the laboratory.

Other limitations were inclusion of a heterogenous population of colon and rectal tumours. The number of rectal cancer patients excluded due to neoadjuvant chemoradiotherapy was small and therefore it is unlikely that selection bias was introduced. However, the effect of neoadjuvant chemoradiotherapy on tissue CEA expression should be explored in future studies. Instead of tissue microarray, the use of whole tissue sections were used which although labour intensive, avoided false-negative results.

Figure 1: Carcinoembryonic antigen (CEA) distribution in colorectal cancer. Diffuse-cytoplasmic pattern of CEA with low intensity. Magnification x10.

Figure 2: Carcinoembryonic antigen (CEA) distribution in colorectal cancer. Diffuse-cytoplasmic pattern of CEA with low intensity. Magnification x4.

Figure 3: Carcinoembryonic antigen (CEA) distribution in colorectal cancer. Diffuse-cytoplasmic pattern of CEA with low intensity. Magnification x20.

Figure 4: Carcinoembryonic antigen (CEA) distribution in colorectal cancer. Diffuse-cytoplasmic pattern of CEA with low intensity. Magnification x20.

Figure 5: Carcinoembryonic antigen (CEA) distribution in colorectal cancer. Diffuse-cytoplasmic pattern of CEA with low intensity. Magnification x4.

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). Written informed consent was obtained from prospective participants included in the study before completing the Data Extraction Form. 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.

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. Michael Odida and Henry Wabinga, 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. We are grateful to Ms Dorothy Nabbale for the laboratory technical work which involved immunohistochemistry on the colorectal cancer tissues 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. Lastly, we are also grateful to Mr. Francis Wasswa, for the laboratory technical work which involved extracting plasma from the blood samples to determine the plasma CEA levels in the Department of Immunology and Molecular Biology, School of Biomedical Sciences, College of Health Sciences, Makerere University.

Abbreviations

CRC – Colorectal cancer

LVI – Lymphovascular invasion

AC – Classical adenocarcinoma

CEA – Carcinoembryonic antigen

MAC – Mucinous adenocarcinoma

SRCC – Signet ring colorectal carcinoma

DAB Chromogen – 3, 3’– Diaminobenzidine chromogen

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