TP53 mutation status and consensus molecular subtypes of colorectal cancer in patients from Rwanda

Abstract

Background: Mutations in the TP53 tumor suppressor gene are well-established drivers of colorectal cancer (CRC) development. However, data on the prevalence of TP53 variants and their association with consensus molecular subtype (CMS) classification in patients with CRC from Rwanda are currently lacking. This study addressed this knowledge gap by investigating TP53 mutation status concerning CMS classification in a CRC cohort from Rwanda.

Methods: Formalin-fixed paraffin-embedded (FFPE) tissue blocks were obtained from 51 patients with CRC at the University Teaching Hospital of Kigali, Rwanda. Exons 4 to 11 and their flanking intron-exon boundaries in the TP53 gene were sequenced using Sanger sequencing to identify potential variants. The recently established immunohistochemistry-based classifier was employed to determine the CMS of each tumor.

Results: Sequencing analysis of cancerous tissue DNA revealed TP53 pathogenic variants in 23 of 51 (45.1%) patients from Rwanda. These variants were predominantly missense types (18/23, 78.3%). The most frequent were c.455dup (p.P153Afs*28), c.524G > A (p.R175H), and c.733G > A (p.G245S), each identified in three tumors. Trinucleotide sequence context analysis of the 23 mutations (20 of which were single-base substitutions) revealed a predominance of the [C > N] pattern among single-base substitutions (SBSs) (18/20; 90.0%), with C[C > T]G being the most frequent mutation (5/18, 27.8%). Furthermore, pyrimidine bases (C and T) were preferentially found at the 5' flanking position of the mutated cytosine (13/18; 72.2%). Analysis of CMS subtypes revealed the following distribution: CMS1 (microsatellite instability-immune) (6/51, 11.8%), CMS2 (canonical) (28/51, 54.9%), CMS3 (metabolic) (9/51, 17.6%), and CMS4 (mesenchymal) (8/51, 15.7%). Interestingly, the majority of TP53 variants were in the CMS2 subgroup (14/23; 60.1%).

Conclusion: Our findings indicate a high frequency of TP53 variants in CRC patients from Rwanda. Importantly, these variants are enriched in the CMS2 subtype. This study, representing the second investigation into molecular alterations in patients with CRC from Rwanda and the first to explore TP53 mutations and CMS classification, provides valuable insights into the molecular landscape of CRC in this understudied population.

Keywords: TP53; Africa; Colorectal cancer; Consensus molecular subtypes; Mismatch repair; Mutation pattern; Mutation spectrum; Rwanda.

Fig. 1

Location and type of TP53 mutations in colorectal cancer (CRC) of patients from Rwanda. A: Types and locations of exonic TP53 mutations (n = 23) on the protein structure using the cBioPortal mutation mapper. Lollipop plots represent mutation frequencies, with the height proportional to frequency. The most frequent codon with mutations is 245 (3× p.G245S, 1× p.G245D). Colors denote mutation type: green - missense, black - truncating, pink - others. TAD: TP53 transactivation motif (residues 6–30), TAD2: TP53 transactivation motif 2 (residues 35–59), DBD: DNA binding domain (residues 100–288), Tetramerization motif (residues 319–357). B: Distribution of TP53 mutation types (n = 23) detected in patients with CRC from Rwanda. The pie chart shows the proportion of missense (blue), frameshift (orange), and nonsense (gray) variants. Missense variants are the most frequent type of mutations in Rwanda at 78.3%

Fig. 2

Immunohistochemical staining of mismatch repair (MMR) protein markers for colorectal cancer in patients from Rwanda. A-D: Representative images of retained expression (A: MLH1, B: PMS2, C: MSH2, D: MSH6). E, F: Representative images of loss of expression (E: PMS2, F: MSH6). Scale bar = 100 μm

Fig. 3

Representative positive and negative immunohistochemistry staining results for the consensus molecular subtype (CMS) protein markers of CK (AE1/AE3, CDX2, FRMD6, HTR2B, ZEB1, and β-catenin). The photomicrograph on the positive side indicates representative images of CMS protein expression in our colorectal cancer (CRC) cohort. The panels on the negative side indicate low or a lack of CMS protein expression in our CRC cohort. Scale bar = 100 μm

Fig. 4

Immunohistochemical staining of consensus molecular subtype (CMS) protein markers for colorectal cancer in patients from Rwanda. Representative images of protein markers [CK AE1/AE3, CDX2, FRMD6, HTR2B, ZEB1, and β-catenin] used for CMS classification (CMS2, CMS3, and CMS4) are shown. Strong cytoplasmic CK AE1/AE3 and nuclear CDX2 staining in CMS2 and CMS3 highlight their epithelial origin. β-catenin staining serves as a tie-breaker: strong nuclear, cytoplasmatic, and membrane staining is observed in CMS2, while CMS3 is negative. CMS4 (mesenchymal) exhibits higher keratin content with CK AE1/AE3 but weaker nuclear CDX2 expression. Additionally, CMS4 shows strong cytoplasmic staining for HTR2B, weak to moderate staining for FRMD6, and moderate nuclear staining for ZEB1. These features are consistent with mesenchymal-like subtypes (p > 0.6), as confirmed by the online CMS classifier (https://crcclassifier.shinyapps.io/appTesting/). Scale bar = 100 μm