Molecular characterisation of Mozambican patients diagnosed with colorectal adenocarcinoma

Introduction

Colorectal cancer (CRC) is a significant healthcare burden worldwide, accounting for over two million diagnosed cases per year and approximately one million deaths in 2022 (1). Although this form of cancer ranked third in overall cancer incidence worldwide, it only appears as the fifth most frequent malignancy in Africa. According to GLOBOCAN report, over 70,000 new CRC cases have been diagnosed in 2022, and 46,087 disease-specific deaths have been recorded. More specifically, in Mozambique, CRC appears as the ninth most frequent cancer, with over 650 new cases per year (1).

Evidence on a global scale highlights the substantial increase of CRC in many low- and middle-income countries (LMICs), a phenomenon linked to socioeconomic transition. Between 2010 and 2019, the highest percentage increase in death counts due to CRC was observed in Djibouti (71%, 35–115%), Rwanda (67%, 38–100%), Cabo Verde (65%, 28–100%), Angola (63%, 31–105%), the Democratic Republic of the Congo (58%, 26–97%), Ethiopia (56%, 30–83%), and Mozambique (40%, 11–77%) (2). Although a systematic examination reported a significant increase of this malignancy in LMICs, limitations on the access to epidemiological data on CRC in sub-Saharan Africa (SSA) remain scarce (2). However, recent data showed that CRC manifests at a younger age and in more advanced stages in SSA, with over 60% of patients presenting with stage IV cancer and a higher prevalence of peritoneal metastases compared to the USA (3). Moreover, Gullickson et al. revealed that the average age at diagnosis in SSA is 58.1 years, ranging from 48.8 years in Maputo, Mozambique, to 62.2 years in Mauritius (4). Later-stage diagnosis correlates with poor survival, and patients aged 50 to 69 years demonstrate better survival rates than those in younger and older age groups (4).

In Mozambique, the limited available data from a hospital series study indicated a higher prevalence of female patients with a median age of 54 years (range, 20–99 years). On this study, poor prognosis was attributed to advanced stage at diagnosis (5).

Limited studies had revealed a higher rate of microsatellite instability (MSI) among Nigerian CRC patients and a more prevalent alteration in the RAS pathway in West Africa, emphasizing the need for more detailed molecular and genetic analysis (6-8). Interestingly, a genomics study of CRC in populations with African and European ancestry uncovered distinct features among those of African descent. CRC patients of African descent were younger, exhibited less frequently MSI tumours, and had a significantly higher frequency of alterations in KRAS, specifically on codons 12 and 13 (9). No relevant studies highlighting p53 protein status on CRC in this population were found.

Given the gaps in existing knowledge, we initiated a study on a series of CRC cases in Mozambique. Our aim was to assess the type and frequency of mutations in the KRAS, NRAS, BRAF, and TP53 genes, as well as the proportion of MSI-positive cases. The goal is to improve patient management and adapt treatment strategies to achieve more effective outcomes.

Methods

Patients from Maputo Central Hospital (MCH) diagnosed with CRC and treated in the institution between 2019 and 2022 were included (n=45). The study was based on the Cancer Registry of the MCH and was approved by the Joint Institutional Bioethics Committee of the Faculty of Medicine, Eduardo Mondlane University and MCH (No. CIBS FM&HCM/71/2017) and individual consent for this retrospective analysis was waived. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. FFPE tissue samples from all patients were retrieved from the biobank of the Pathology Department at MCH and were submitted to a series of procedures and analysis, as summarised in Figure 1.

Figure 1 Schematic workflow for molecular and data analysis using FFPE tissues from Mozambique CRC patients. Briefly, patients diagnosed with CRC underwent surgical resection of the tumour and histological procedures for diagnosis (microtomy and staining with haematoxylin and eosin). After patients’ selection, a molecular analysis was performed, including immunohistochemical analysis for p53 protein, mutation test analysis for KRAS, NRAS, and BRAF and MSI analysis using the Idylla^TM platform. Bioinformatic analysis using SPSS and Prism was implemented to integrate the clinicopathological and demographic data with molecular data. CRC, colorectal cancer; FFPE, formalin-fixed paraffin-embedded; MSI, microsatellite instability.

Immunohistochemistry

Briefly, 3 µm CRC tumour sections were manually deparaffinized and rehydrated in a series of alcohols and water. After hydration with running water, the slides were submitted to an antigen unmasking using a microwave for twenty minutes with boiling ethylenediaminetetraacetic acid (EDTA) 1 mM pH 9 (E177, VWR, Radnor, PA, USA). Subsequently, endogenous peroxidases were blocked with 3% hydrogen peroxide (DS9800, Leica, Wetzlar, Germany) for 5 minutes and unspecific background was blocked posteriorly with bovine serum albumin (BSA) for 1 hour at room temperature (5% BSA). Primary antibody (p53 protein; DO-7 clone, M7001, Leica) was added subsequently to the sections for 1 hour at room temperature at a dilution of 1:250. Primary antibody was detected through a two-step immunohistochemistry method following manufacturer’s instructions (BOND Polymer Refine Detection, DS9800, Leica). Nuclei were counterstained with haematoxylin (3801560E, Leica) for 5 minutes. Positive and negative control sections were tested in parallel to assess procedure’s reliability. Immunohistochemical evaluation was double-blindly assessed by two independent pathologists. In case of disagreement, the slides were reviewed, and consensus was reached. Immunohistochemical signal was evaluated in terms of extension (percentage of positive tumour cells) and intensity of staining. Image data was acquired using a BA310 Trinocular microscope with an integrated MOTICAM X5 Plus and a Motic ImagePlus 3.0 software (Motic, Hong Kong, China).

IdyllaTM mutation test analysis

The tumour areas were marked by pathologists, all specimens (10 µm of thickness) were macrodissected and were subjected to the Idylla™ KRAS and NRAS-BRAF mutation tests and the MSI assay. The Idylla™ KRAS mutation test (A0020/6, Biocartis, Mechelen, Belgium) is a single-use cartridge-based test designed for the qualitative detection of 21 different mutations, including those in KRAS codons 12, 13, 59, 61, 117, and 146. The Idylla™ NRAS-BRAF mutation test (A0030/6, Biocartis) detects 18 mutations in codons 12, 13, 59, 61, 117, and 146 of the NRAS oncogene and five mutations in codon 600 of the BRAF oncogene. The Idylla™ MSI Assay (A0100/6, Biocartis) tests a set of seven short homopolymers in the ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, and SULF2 genes (10). The evaluation of the Idylla™ KRAS and NRAS-BRAF mutation tests and the MSI Assay was performed in the Department of Laboratory Genetics, Portuguese Institute of Oncology, Porto, Portugal.

Statistical analysis and illustration

Statistical Package for Social Sciences (SPSS) for MacOS (version 27; IBM, Armonk, NY, USA) and GraphPad Prism software (version 9; Dotmatics, Woburn, MA, USA) were used to perform statistical analysis. Categorical variables were compared using Chi-squared analysis and unpaired t-test without Welch’s correction was used for continuous variables. Comparison of estimates was done using log-rank tests. Clustering analysis using a tree diagram (dendrogram) was used to organize data according with their similarity in terms of molecular profiling and clinicopathological data. Scientific illustration was performed using BioRender online platform.

Results

Patient sample set selection and characterization

A series of 45 patients from MCH diagnosed with CRC were selected to enter the study. Inclusion of patients in the analytical study was based on the histological type and the tumour representation on the histological sample, as demonstrated in Figure 2. Five cases were excluded from the study since they were classified as non-adenocarcinoma (four squamous cell carcinoma and one neuroendocrine tumour). Finally, 10 cases were excluded in accordance with the tumour representation on the histological sample, as they present less than 30% of tumour representation on the haematoxylin and eosin staining. A total of 30 cases were included in the analytical study.

Figure 2 Patient selection criteria. Patients admitted at Mozambique hospital and diagnosed with CRC were initially included in the study (n=45). After inclusion criteria application, thirty patients enrolled the final analysis. All cases were staged based on the criteria from the UICC Classification of Colorectal, Appendiceal, and Anal Carcinoma and histologically confirmed as malignant by the pathologists (M.I. and C.C.). CRC, colorectal cancer; UICC, Union for International Cancer Control.

Briefly, the analytical study included 11 males (37%) and 19 females (63%), with a median age of 55 years (ranging from 34 to 75 years), diagnosed with adenocarcinoma (Table 1). Most cases were diagnosed as adenocarcinoma of the rectum (83%), in all clinical stages of the disease. Tumour staging evaluation revealed 17% of cases in stage I, 23% in stage II, 20% in stage III, and 33% in stage IV. Remaining characterization of the selected patient sample set is summarized in Table 1.

Mutational profiling of colorectal adenocarcinoma in Mozambique

Mutational profiling was performed through immunohistochemistry for p53 protein and with the Idylla^TM platform for KRAS, NRAS, BRAF, and MSI. Immunohistochemical analysis of the p53 protein unveiled overexpression in over 70% of cases, presumably resulting from the dominant negative effect of TP53 missense mutations, whereas the other 30% of the cases showed normal p53 expression (Figure 3A). KRAS mutations were detected in over 63% of cases, with higher incidence in codon 12 of the gene (43% of total cases; Figure 3B). Additional mutations on codons 13, 59, and 117 of KRAS were found in lower frequencies (3–10%, Figure 3B). MSI was only detected in two patients (7%; Figure 3C) and a NRAS mutation was detected in one case (Figure 3D). No mutation in the BRAF gene was observed in our study (Figure 3D).

Figure 3 Mozambique CRC patients present frequent alterations on p53 protein status, as well as recurrent mutations on codon 12 of the KRAS gene. (A) Patients diagnosed with colorectal adenocarcinoma in Mozambique present frequent overexpression of p53 protein (70% of cases), as demonstrated by immunohistochemistry, counterstained with haematoxylin, 100× (left panels) and 400× (right panels). (B) Approximately 63% of CRC patients present mutations on codons 12, 13, 59, and 117 of the KRAS gene. The most frequent mutations were observed on codon 12 (G12S, G12D, G12R, G12V). A representative illustration of Idylla^TM curve report for WT and mutated KRAS (KRAS G12D) is presented. (C) MSI was only found in 7% of CRC patients (n=2). (D) Only one patient presented a NRAS mutation (NRAS G12A/V) and no BRAF mutation was observed. A representative illustration of Idylla^TM amplification curve report for WT and mutated NRAS and BRAF is presented. CRC, colorectal cancer; MSI, microsatellite instability; WT, wild type.

Mutational profiling of CRC and clinical relevance

Mutational status of KRAS/NRAS/BRAF oncogenes (RAS/RAF), p53 tumour suppressor gene or the MSI phenotype did not correlate with age of the patient, even when multiple molecular alterations were present (Figure 4).

Figure 4 Age distribution in different molecular sub-groups [mutations in RAS/RAF (A), p53 protein status (B), MSI (C), or combinations of different molecular fingerprints (D-F)]. No differentiation was observed among molecular groups according with the age of the patients [(A) P=0.45; (B) P=0.62; (C) P=0.27; (D) P=0.52; (E) P=0.18, and (F) P=0.37]. All comparisons were performed using an unpaired t-test, after normality evaluation (Shapiro-Wilk). Statistical significance was considered when P<0.05. RAS/RAF refers to KRAS/NRAS/BRAF mutations. MSI, microsatellite instability; MSS, microsatellite stability.

Moreover, no clear pattern between molecular and clinicopathological data (clinical stage, gender, and tumour location) was evident in the clustering analysis (Figure 5A), although a significant association between mutations in the RAS/RAF pathway and overexpression of p53 protein was unveiled in male patients (Figure 5A,5B). Indeed, all male patients that present mutations in KRAS/NRAS oncogenes, concomitantly overexpress p53 protein (P=0.02; Figure 5B, Table S1). Moreover, a statistically significant association between overexpression of p53 protein with the patient’ gender was disclosed (P=0.006; Table 2), as all male patients overexpressed the p53 protein when compared with 53% in female patients (Table 2). No further association between clinicopathological/demographic data with different molecular fingerprints was observed (Table 2, Tables S1-S3).

Figure 5 Male patients presenting mutations in KRAS or NRAS, concomitantly present alterations on p53 protein expression. (A) Clustering analysis based on p53 protein status, mutations in RAS/RAF, MSI, and clinicopathological data (gender, tumour location, and clinical stage). No clear pattern or association was evident between molecular data and the clinicopathological information. (B) All male patients diagnosed with CRC that present mutations in KRAS, NRAS, or BRAF concomitantly overexpress p53 protein (P=0.02). All comparisons were performed using a Chi-squared test. Statistical significance was considered when P<0.05. RAS/RAF refers to KRAS/NRAS/BRAF mutations. CRC, colorectal cancer; MSI, microsatellite instability.

Focusing the mutational profile of KRAS solely, it did not correlate statistically with the clinicopathological data (data not shown), however on the clustering analysis it is possible to observe an enrichment of mutated KRAS cases on early stages of the disease (clinical stage I and II) with no statistical significance (Figure 6A). Furthermore, mutated KRAS was the most recurrent feature in both tumour locations, 60% in colon adenocarcinomas and 64% in rectal adenocarcinomas (Figure 6A,6B). In colon, mutations were found only in codons 12 or 13 of KRAS. Rectal adenocarcinomas presented mutations in codons 12 and 13 (over 50% of cases), but also mutations in codons 59 and 117 (12%; Figure 6B). KRAS G12D was the most represented mutation on the subset of rectal adenocarcinomas (28%; Figure 6B). No predominance of specific mutation was observed on the subset of colon carcinomas (G12S: 20%; G12V: 20%; G13D: 20%; Figure 6B).

Figure 6 No clear pattern or association was perceptible between KRAS mutational data and the clinicopathological information. (A) Clustering analysis based on mutations in KRAS and clinicopathological data (gender, tumour location and clinical stage). Mutational status of KRAS did not correlate statistically with the clinicopathological data. An enrichment of mutated KRAS cases on early stages of the disease (clinical stage I and II) could be observed. (B) Mutated KRAS was the most frequent signature both in colon and rectum. In both locations, approximately 60% of cases presented mutations in KRAS gene. In colon adenocarcinoma, mutations were found on codons 12 and 13 of KRAS. Mutations on codons 12, 13, 59, and 117 of KRAS were found in the adenocarcinomas that rise for rectum epithelium. The most frequent mutation found in rectum adenocarcinomas was KRAS G12D (28%). WT, wild type.

Discussion

In our single-centre retrospective study, we present a landscape of the main KRAS, NRAS, and BRAF somatic mutations, in combination with p53 protein expression and MSI phenotype, in a patient sample set of 30 Mozambican CRC patients of African progeny. To our knowledge, this is the first comprehensive assessment of hotspot mutations in this population, opening an opportunity for future clinical interventions based on mutational profiling of these tumours. About 50% of cases were diagnosed at locally advanced or metastatic stages of the disease, essentially as rectal adenocarcinomas, similarly to the Nigerian population (6). Moreover, and in line with reports from Alatise et al., our data also suggest an earlier onset of the disease in the African population, with median age at diagnosis of 55 years (ranging from 34 to 75) (6,11). Recent population studies have unveiled that the average patient’s age at diagnosis is decreasing in the African population (3,9,12). Reports suggest that patients diagnosed at younger ages tend to have worst prognosis, making it imperative to address the clinical management of these patients in a state-of-the-art manner, already implemented at the developed countries (3). Additionally, a predominance of female patients on our patient sample set could be observed, in accordance with observations made in a Senegal-based study (11). Nevertheless, the molecular mechanisms for the development of cancer remain poorly understood in these populations. CRC is a multifactorial disease, however lifestyle changes (obesity and diabetes), as well as increase of the human immunodeficiency virus (HIV) infection, appears as the main risk factors for development of CRC in the Mozambican population (12). Nevertheless, somatic mutations is one of the promotors for the colorectal carcinogenesis cascade in this population, similarly to the European and American populations.

According with our study, Mozambican CRC patients present frequent alterations in KRAS and p53, with uncommon alterations of the NRAS and BRAF genes, as well as of MSI. Several studies developed in African cohorts highlighted major differences on the mutation frequency rates among different populations, however only a few studies reported the same tendencies as observed in our study for KRAS and BRAF genes and MSI (8,9,11,13,14). Regarding KRAS, our study identified mutations in more than 60% of the cases, which is significantly higher than the frequency obtained previously in Senegal (45%), Gana (34%), or Nigeria (21%) (8,9,11,13,14). The opposite was found for the BRAF gene, with no mutations detected on our patient sample set contrasting to reports in Senegal (55% of incidence) or Nigeria (4.5%) (11,14). Only Ghanaian CRC patients present same tendency for BRAF gene (0% of incidence) (8). However, Aldera et al., similarly to us, have reported that BRAF mutations, as well as MSI, are uncommon molecular signatures in Africa (13). In our patient sample, this phenotype was only identified in 2 patients (7%), contrasting with the results obtained in the Nigerian population (41%) (8). Interestingly, recent report sustain that African patients present double the prevalence of MSI when compared with European and North American individuals (14). Indeed, in a Nigerian study, the MSI phenotype in the African descents was significantly higher compared to Nigerian patients with European ancestry, suggesting that ancestry could determine the prevalence of MSI phenotype in certain populations (7). Furthermore, in 2022, an American comparative study between European and African ancestry unveiled distinct molecular signatures among the two ancestries, with different patient outcome (9). Patients of African ancestry exhibited higher frequency of mutations in the KRAS gene (codons 12 and 13) and low frequency of MSI tumours, associated with worst prognosis (9). These results are in line with the data of our retrospective study but not with the Nigerian report, emphasizing the need for more-in-depth knowledge of population-based determinants of CRC. Discrepancies between studies could be related to small sample size patient sets, associated with poorly developed national oncology registries in these countries, warranting future studies with larger sample set conglomerating different African countries with different ancestries. Moreover, differences in the proportion of cases of colon and rectum (adeno)carcinomas among studies could justify the dissimilarities between them, as exploited by Ciepiela et al. (15).

Overall, our preliminary study of this population unveiled molecular signatures, essentially associated with KRAS gene mutations, p53 protein overexpression and low frequency of MSI phenotype, creating novel knowledge that could be translated to the clinic. According to our clinical data, patients in Mozambique are majorly unimodally treated with surgery (60%), with only 13% of cases being treated palliatively with conventional chemotherapy. Our study opened a window of opportunity to introduce in an informed manner the targeted treatment regimens applied on developed countries in the different stages of the disease. For example, tumours with MSI benefit from the application of novel immunotherapies, such as immunocheckpoint inhibitors, however such therapy could not represent a major impact in Mozambican CRC patients (16,17). Moreover, frequent mutations in KRAS gene could predict treatment outcome, making it an important marker for clinical decision on CRC. Tumours that present mutations in KRAS do not respond to targeted therapies against epidermal growth factor receptor (EGFR), such as cetuximab and panitumumab (18). Accordingly, approximately 70% of Mozambican CRC patients would not be eligible to anti-EGFR therapies, highlighting the need for the development of novel targeted therapies to block the activation of this oncogenic pathway, the same way as sotorasib (inhibitor of KRAS G12C in lung cancer) (19). On the other hand, several studies have reported TP53 gene status as an independent predictive marker for the effect of adjuvant 5-fluorouracil (5-FU) in stage III colon cancer (20). Also, Oden-Gangloff et al. reported that mutations in TP53 are predictive of cetuximab sensitivity, mainly in patients without KRAS mutations (21). Moreover, in a case report, a patient with concomitant mutations in TP53 and KRAS presented increased response when submitted to FOLFIRI (folinic acid, fluorouracil, and irinotecan) and bevacizumab combination chemotherapy (22). However, limited information on p53 protein status on Mozambican population until this moment had limited the putative association of TP53 mutations and patient response to treatment in these patients. These reports reinforce the importance of this pilot study of the mutational profiling of Mozambican CRC patients, regarding KRAS, NRAS, BRAF, TP53, and MSI. Further investigations should be performed to validate our results in an independent larger cohort.

By identifying the mutational pattern of Mozambican CRC patients, this study can help clinicians better determine which targeted therapies will most likely benefit their patients, thus optimizing treatment planning and outcomes. Understanding the mutational landscape of CRC in Africa is also important for designing clinical trials that evaluate novel therapies and treatment strategies. This knowledge can help ensure the inclusion of African populations in clinical research, making the benefits of medical advancements accessible to patients in this region. Further endeavours should be made to create a molecular biology laboratory in Mozambique to support future oncology clinical trials in this population.

Conclusions

Our study sheds light, for the first time, on the mutation landscape of CRC in Mozambican patients, making possible to create the basilar knowledge to better understand the molecular mechanisms underlying the carcinogenesis and the clinical outcome, towards the improvement of patient management. The high frequency of RAS mutations (63.3%), particularly in codon 12 of KRAS, coupled with the absence of BRAF mutations, p53 protein overexpression and low frequency of MSI tumours, underscores a unique genetic profile of CRC in this population. Our findings align with previous studies indicate variations in mutation rates across not only ancestries, but also populations within same ancestry. This highlights the importance of population studies instead of generalisation or extrapolation. Future research with larger cohorts is warranted to enhance our understanding of the frequency and impact of KRAS, NRAS, BRAF, p53, and MSI genotypes in Mozambican CRC patients. Such endeavours will contribute to improve the clinical management tailored to the unique genetic landscape of CRC in this region.

Acknowledgments

The authors would like to acknowledge all individuals recruited at Hospital Central de Maputo who kindly accepted to participate in this study.

Footnote

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-24-677/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-24-677/prf

Funding: This study received financial support from PT national funds [Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES)] through CI-IPOP project UIDP/00776/2020. This work was cofounded by the project “The Porto Comprehensive Cancer Center Raquel Seruca” with the reference NORTE-01-0145-FEDER-0726678 – Consorcio PORTO.CCC – Porto. Comprehensive Cancer Center Raquel Seruca, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), Portugal. FCT is cofinanced by European Social Fund (ESF) under Human Potential Operation Programme (POPH) from National Strategic Reference Framework (NSRF). The authors also thank the financial support of the Portuguese Oncology Institute of Porto – Research Centre (CI-IPOP-29-2017-2024) and Calouste Gulbenkian Foundation. The authors thank the Maputo Central Hospital for the provision of biological samples for the development of this work. The authors also wish to acknowledge funding from FCT for researcher position 2020.09384.BD (DF; DOI 10.54499/2020.09384.BD).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-24-677/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Joint Institutional Bioethics Committee of the Faculty of Medicine, Eduardo Mondlane University and MCH (No. CIBS FM&HCM/71/2017), and individual consent for this retrospective analysis was waived.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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