Colorectal cancer (CRC) is the second-leading cause of cancer deaths worldwide accounting for 9.1% of cancer deaths each year and the third most commonly diagnosed cancer [1]. Over the coming decades, the mortality and incidence of CRC are expected to increase, with the largest relative increases in middle- and developing low-income countries [2]. Recent studies in Sub-Saharan Africa and data from the Kampala Cancer Registry in Uganda have shown an increasing incidence of CRC cases in this region with patients presenting at an advanced stage and at a younger age compared to developed high-income countries [3-5].

This increase may be due to improved cancer reporting, management modalities and screening, or an increased prevalence of modifiable risk factors with Western diets and lifestyles [5-8]. A known risk factor for CRC in Uganda is urbanization which is associated with high consumption of processed meat and a Westernized diet [9,10]. Urbanization is associated with smoking, alcohol consumption, obesity and a sedentary lifestyle which are associated with CRC [11].

Invasion and metastasis are the most life-threatening aspects of the colorectal neoplastic process. Many studies have shown that invasion, metastasis and solid tumour growth is dependent on new blood vessel formation from established vasculature [12-15]. Several angiostatic and angiogenetic factors control angiogenesis.

The most effective angiogenetic protein known is the vascular endothelial growth factor, also known as the vascular permeability factor [15-18]. An active mitogen of vascular endothelial cells, providing the opportunity for the organization, migration and neovascularization of micrometastasis is vascular endothelial growth factor which is a 34-42 kDa dimeric, heparin-binding glycoprotein. It is derived by alternative mRNA splicing and is expressed in four isoforms which include VEGF206, VEGF189, VEGF165 and VEGF121 (Davies MM, 2000). The smaller forms, VEGF121 and VEGF165 are soluble proteins which are detected with immunoassay in the serum and the larger forms, VEGF189 and VEGF206 are bound to the cell surface [19].

Various known cancers and cultured tumour cells have been repeated to secrete and synthesise VEGF [20-23]. Studies have reported a relationship between tumour growth, distant metastasis and a poor prognosis with an increased expression of VEGF [24-26]. The aim of this study was to analyse the correlation between VEGF with clinicopathological characteristics of Ugandan colorectal cancer patients.

There were 52 formalin-fixed and paraffin-embedded (FFPE) blocks, which were obtained from the 1^st January 2008 to the 15^th September 2021 from participants that had a histopathological diagnosis of colorectal adenocarcinoma. From the 1^st January 2008 to the 15^th September 2019 FFPE CRC tissue blocks were obtained from the archives of the Department of Pathology, School of Biomedical Sciences, College of Health Sciences, Makerere University. Whilst from the 16^th September 2019 to the 16^th September 2021, samples of resected colorectal carcinoma specimens and colorectal biopsy specimens were obtained from Masaka Regional Referral Hospital, Mulago National Referral Hospital, Uganda Martyrs’ Hospital Lubaga and Mengo Hospital. The biopsy specimens were obtained during colonoscopy and the resected colorectal specimens at operation. 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 demographics (age, sex), stage and topography of the tumour. Left-sided tumours were splenic flexure tumours, descending colon tumours, sigmoid tumours, rectosigmoid tumours and rectal tumours [27,28]. Right-sided tumours included tumours located in the caecum, ascending colon, hepatic flexure and transverse colon [27,28].

Haematoxylin and eosin staining was used to determine the grade, LVI status and the histopathological subtype of colorectal adenocarcinoma (AC, MAC and SRCC). 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.

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. Exclusion criteria included those that had previous neo-adjuvant chemotherapy or preoperative radiotherapy, poor quality tissue block samples and tissue samples with incomplete or unavailable data. The quality control samples selection criteria included those with primary tumours that were ≥1mm thickness, with an estimated ≥50% tumour content and with an acceptable morphology.

Fifty-two FFPE tissue blocks were processed for VEGF immunohistochemistry examination. 4µm thick sections from 10% formalin-fixed paraffin-embedded tissue were taken to perform immunohistochemistry for VEGF. The procedure used was first deparaffinization in xylene, rehydration in alcohol series, and brought to distilled water. Heat-induced epitope retrieval in an alkaline novalink 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 1:25 VEGF-1 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 VEGF antibody dilution was 1:25, the source was DAKO Agilent USA, clone VG1 and reference M7273.

Free peroxidase splits 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 Nixon eclipse microscope. The immunohistochemical technique applied on the paraffin-embedded tumoral samples for VEGF used a scale for staining intensity which included 0: none and (+1) present. The VEGF molecular marker was correlated with the grade and stage of the tumour.

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 VEGF 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.

Statistical Analysis

Continuous numeric data were summarized by mean (standard deviation) and categorical data were summarized as frequencies and percentages. The distribution of the intensities of VEGF 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 VEGF expression and demographic and histopathological variables. Spearman’s rank correlation coefficients were used to determine and compare correlations between the VEGF biomarker and grade. A p-value of ≤0.05 was considered statistically significant.

Out of the 52 VEGF-1 participants, there were 16 (30.8%) tumours which stained positively for VEGF-1 and there were 36 (69.2%) tumours which stained negatively for VEGF-1. The distribution of staining intensity of VEGF-1 regarding tumour grade, tumour depth, stage, topography, histopathological subtype and LVI status is shown in Tables 1-6.

Table 1: VEGF-1 antibody distribution regarding CRC tumour grade.

Table 1 compared the VEGF-1 intensity of expression in CRC with tumour grade. It shows that the VEGF-1 expression (+1) was more commonly associated with G3 (poorly differentiated) tumour in 42.8% of tumours compared to 33.3% of G1 (well differentiated) tumours.

Table 2: VEGF-1 antibody distribution regarding CRC tumour depth (T) in bowel wall.

Table 2 compares the VEGF-1 intensity of expression in CRC with the tumour depth (T). There were 35.3% T3 and 33.3% T4 tumours that were positive for VEGF-1 compared to 16.7% T1 and 28.6% T2 tumours that were positive for VEGF-1. Lack of VEGF-1 expression was found in 66.7% T4 tumours compared to 83.3% T1 tumours.

Table 3: VEGF-1 antibody distribution regarding CRC tumour stage.

Table 3 compares VEGF-1 expression in CRC with the stage of CRC. VEGF-1 expression was more commonly found in 46.7% stage IV tumours and 33.3% stage III tumours compared to 20% stage I tumours and 22.2% stage II tumours. Lack of VEGF-1 expression was more common with stage I tumours (80%) compared to stage IV tumours (53.3%).

Table 4: VEGF-1 antibody distribution regarding the CRC tumour topography.

Table 4 shows that VEGF-1 was expressed in 36.4% of rectal tumours, 33.3% of right-sided colon tumours and 28.6% left-sided colon tumours.

Table 5: VEGF-1 antibody distribution regarding histopathological subtype.

Table 5 shows that VEGF-1 was more commonly expressed with the signet ring colorectal carcinoma histopathological subtype. 40% of SRCC tumours had VEGF-1 expression compared to 14.3% MAC tumours and 32.5% AC tumours.

Table 6: VEGF-1 antibody distribution regarding lymphovascular invasion (LVI) status.

Table 6 shows that in the presence of lymphovascular invasion, VEGF-1 was expressed in 27.3% of tumours. There were 72.7% tumours with lymphovascular invasion that did not express VEGF-1. One tumour with no lymphovascular invasion did not express VEGF-1.

Representative images of VEGF staining at different magnifications are shown in Figures 1-4. Among 52 VEGF-1 participants, the mean age (SD) was 52.9 (16.3) years; 25 (48.1%) were male and 27 (51.9%) were female. There were 26 (54.2%) participants that had colon cancer; 22 (45.8%) had rectal cancer; 12 (46.2%) had right-sided colon cancer and 14 (53.8%) had left-sided colon cancer (Table 7). A significant proportion of participants, 15 (30.6%) had stage III CRC, whereas 15 (30.6%) had stage IV disease. Early-stage CRC consisted of 10 (20.4%) participants with stage I CRC and 9 (18.4%) with stage II disease.

The histopathological features for the VEGF-1 participants included, 40 (76.9%) classical adenocarcinoma, 7 (13.5%) mucinous adenocarcinoma and 5 (9.6%) had SRCC. Based on the grade of differentiation, grade II was most commonly seen in 33 (71.7%) participants, 7 (15.2%) had grade III, and 6 (13.0%) had grade I tumours. Positive lymphovascular invasion (LVI) was seen in 31 (59.6%) participants, whilst 21 (40.4%) participants had no lymphovascular invasion (LVI).

The relationship of VEGF-1 status expression with baseline demographic and histopathological parameters is shown in Table 7. The relationship of the presence of VEGF-1 expression with baseline parameters and comparisons among the variables was carried out using Pearson chi-square tests (Table 8).

Table 7: Comparison of VEGF-1 status with baseline characteristics.

*Fischer’s exact test

Association of the presence of VEGF-1 expression in relation to demographics and histopathological characteristics

The presence of VEGF-1 expression was significantly associated with stage IV CRC, grade II CRC and the classical adenocarcinoma histological subtype. All VEGF-1 participants, 9(100%), had the presence of LVI (Table 8).

In stage III CRC there were 5 (31.3%) participants compared to 2 (12.5%) in stage I CRC, however, this did not reach statistical significance (p=0.1985). There were 7 (43.7%) participants with stage IV disease compared to 2 (12.5%) with stage I disease and this reached statistical significance (p=0.0479). Moderately differentiated grade II constituted 11 (68.8%) participants compared to 2 (12.5%) participants with grade I CRC and this was statistically significant (p=0.0012). There were 13 (81.3%) participants with AC compared to 1 (6.3%) with MAC and 2 (12.5%) with SRCC that had positive VEGF-1 expression and this reached statistical significance (p=0.0000).

There was a negative correlation between CRC grading and VEGF-1 expression (r= -0.0565) which did not reach statistical significance (p=0.7091).

Table 8: Presence of VEGF-1 expression in relation to demographics and some pathological characteristics.

Vascular endothelial growth factor-1 has been found to be important for angiogenesis which is important for the progression, development and metastasis of colorectal tumours [29]. VEGF-1 has an important role in colorectal cancer in determining the vascular phenotype and the modulation of tumour angiogenesis. Globally, several studies investigating the relationship between clinicopathologic parameters and VEGF-1 expression have been conducted regarding colorectal adenocarcinoma. The relationship between the CRC stage and topography of the tumour with the expression of VEGF1 has been studied by Bendardaf R [30]. A strong relationship was observed between the stage of CRC and the expression intensity of VEGF-1. The same study observed that compared to the right colon, the VEGF intensity of expression was higher in the rectum and left colon (45% vs 61%) [30]. In the present study, more CRC tissues were stained for VEGF at an advanced stage (III and IV) compared to early stage (I and II) disease and this reached statistical significance for stage IV disease.

There was a similar distribution of VEGF-1 intensity between left-sided, right-sided colon and rectal cancers. VEGF-1 is expressed in colorectal cancer more commonly than in normal colorectal tissue. Hashim AF found that 18.2% of normal controls, in contrast to 51.9% of colorectal cancer patients exhibited VEGF-1 expression [31]. In this study there was no significant difference observed between the three stages (I, II, III) of colorectal adenocarcinoma with the intensity of VEGF-1 expression however, the grade was significantly associated with the intensity of VEGF-1 expression [31].

The present study did find a significant relationship between the grade II and grade I of colorectal adenocarcinoma and the intensity of VEGF-1 expression as the number of cases that positively stained for VEGF-1 increased with grade II moderate differentiation of CRC. This study also confirmed that although the number of cases of positive VEGF-1 increased with increasing stage of CRC. Regarding histopathological subtypes, more cases of AC had a tendency to present with more VEGF-1 expression than MAC and SRCC. This is due to AC being more frequent and presenting at an advanced stage even though the biological behaviours of SRCC and MAC subtypes are more aggressive and tend to present at an advanced stage, grade and LVI compared to AC. This finding is in agreement with the findings of another study [32].

Immunohistochemical analysis of colorectal adenocarcinoma was also used to evaluate the prognostic significance of VEGF-1 in a study by Zafirellis K [33]. Compared to tumours without lymph node metastasis, the intensity of staining for tumours with lymph node metastasis was higher. The same study showed that compared to stage I and II, the intensity of VEGF-1 staining with stage III was higher (p<0.05) [33]. The present study also showed a tendency for the intensity of VEGF-1 expression to be more commonly associated with lymph node metastasis and hence stage III disease, however, this did not reach statistical significance. In the present study, stage IV CRC was associated with more cases of staining for VEGF1 and these cases with metastasis were significantly associated with positive VEGF-1.

In a study by Kekec Y, it was shown that when the histologic grade is significantly lower there were more patients who did not show any VEGF-1 expression in the colorectal adenocarcinoma [34]. In the present study in Uganda, histological grades 2 showed more patients that did confirm positive VEGF-1 expression.

Cano D, investigated the VEGF-1 and HIF-1α expression in colorectal adenocarcinoma and its correlation with prognostic and clinical implications [35]. The expression of VEGF-1 and HIF-1α were found to be 56.34% and 54.93% respectively, and the two molecular markers were significantly associated with lymph node involvement, metastasis and also tumour stage (p<0.05) [35]. However, the present study and other studies have shown a significant correlation between distant metastasis and the expression of VEGF-1 and more patients expressed VEGF-1 in stage IV disease [36].

The prognostic and clinicopathologic value of HER-2/neu and VEGF-1 marker expression has also been evaluated in colon adenocarcinoma in a study by Qingguoli [37]. The expression of HER2/neu and VEGF-1 in the colorectal adenocarcinoma tumour cells was 15.5% and 55.5% respectively. There was a significant correlation found between tumour size, lymph node metastasis, distant metastasis, stage and the VEGF-1 expression [37]. The results of the present study, however, showed that out of these four variables, only distant metastasis was significantly associated with the expression of VEGF-1.

In a study by Hedaya MS, it was observed that VEGF-1 and COX-2 were intensely expressed in the advanced grade and stage of colorectal adenocarcinoma from CRC resections [38].

In the present study survival rates of VEGF stained cases were not compared to those that did not stain for VEGF-1 in Ugandan patients. In the absence of expression of VEGF-1, a study by Li W et al has shown improved survival rates [37]. The same study showed that histological grade, stage and lymph node metastasis were related to VEGF-1 expression [39].

The present study showed that in parallel with an increase in tumour grade, there was a gradual increase in the frequency of expression of VEGF-1 (in grade 1 there was 33.3% VEGF-1 expression, in grade II 33.3% and grade III 42.8%). However, among the three degrees of differentiation, grade II showed a significant difference with more VEGF-1 staining noted in Ugandan patients. These findings are supported by other studies that reached similar conclusions [40,41]. However, there was no correlation between VEGF-1 and the grade of CRC in Ugandan patients.

There was also a tendency in the present study for the frequency of VEGF-1 expression to increase with increasing stage of CRC and this reached statistical significance for stage IV patients and all patients that were positive for VEGF-1 had lymphovascular invasion. These findings show that there is a tendency for VEGF-1 to be associated with a poor prognosis in Ugandan patients. Studies have shown a decrease in tumour size and a decrease in angiogenesis with anti-VEGF which includes bevacizumab (anti-VEGF treatment).

Anti-VEGF antibodies administered to mice have been shown experimentally to inhibit metastasis and also decrease tumour CRC growth [42,43]. The combination of bevacizumab and doxorubicin has been found to be more significant in decreasing tumour size [44-46]. The mechanism responsible for increasing the results of chemotherapy with anti-VEGF therapy is not entirely understood. However, tumour apoptosis may be increased with inhibition of angiogenesis.

In Uganda, there is a need for more efficient and well-tolerated medical treatments using new combinations of anti-VEGF and chemotherapy treatments such as capecitabine and oxaliplatin (XELOX) or oxaliplatin and 5-FU/LV (FOLFOX-4) for treating patients with colorectal cancer [47].

Among those patients that expressed VEGF-1, there was a tendency to increase expression with increasing stage of CRC. Stage IV and hence the presence of metastasis was significantly associated with increasing expression of VEGF-1. There were more patients with positive VEGF-1 expression associated with an increasing grade of CRC. These findings showed that there is a tendency for VEGF-1 expression to be associated with a poor prognosis in Ugandan patients. In Uganda, there is a need for more efficient and well-tolerated medical treatments using new combinations of anti-VEGF1 and chemotherapy treatments such as capecitabine and oxaliplatin (XELOX) or oxaliplatin and 5-FU/LV (FOLFOX-4) for treating patients with colorectal cancer.

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 VEGF. High standards of laboratory testing were also followed. The storage of the specimens was kept for a short period of time. The number of rectal cancer patients excluded due to neoadjuvant chemoradiotherapy was small and therefore it is unlikely that selection bias was introduced.

Other limitations were the inclusion of a heterogeneous population of colon and rectal tumours. Instead of tissue microarray, the use of whole tissue sections was used which although labour intensive, avoided false-negative results. 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.Figure 1: Immunohistochemical expression of VEGF-1 in colorectal carcinoma tissue, showing positive cytoplasmic (+1) immunohistochemical expression of VEGF-1. Magnification x200.

Figure 2: Immunohistochemical expression of VEGF-1 in colorectal carcinoma tissue, showing positive cytoplasmic (+1) immunohistochemical expression of VEGF-1. Magnification: x4.

Figure 3: Immunohistochemical expression of VEGF-1 in colorectal carcinoma tissue, showing positive cytoplasmic (+1) immunohistochemical expression of VEGF-1. Magnification: x10.

Figure 4: Immunohistochemical expression of VEGF-1 in colorectal carcinoma tissue, showing positive cytoplasmic (+1) immunohistochemical expression of VEGF-1. Magnification: x20.

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 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.

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 immunohistochemical slides. Michael Odida, Henry Wabinga, Josephat Jombwe, and Emmanuel Elobu performed critical reviews of the manuscript for intellectual content. All authors approved the final manuscript for publication.

Acknowledgments

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

VEGF-1 – vascular endothelial growth factor-1

AC – classical adenocarcinoma

MAC – mucinous adenocarcinoma

SRCC – signet ring colorectal carcinoma

DAB chromogen – 3, 3’ – Diaminobenzidine chromogen

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