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. 2023 Sep 16;14(9):1808.
doi: 10.3390/genes14091808.

Correlation between KRAS Mutation and CTLA-4 mRNA Expression in Circulating Tumour Cells: Clinical Implications in Colorectal Cancer

Sharmin Aktar  1   2   3 , Farhadul Islam  4 , Tracie Cheng  1   2 , Sujani Madhurika Kodagoda Gamage  1   2   5 , Indra Neil Choudhury  2 , Md Sajedul Islam  1   2   6 , Cu Tai Lu  7 , Faysal Bin Hamid  1   2 , Hirotaka Ishida  1 , Ichiro Abe  1 , Nan Xie  1 , Vinod Gopalan  1   2 , Alfred K Lam  1   2   8
Affiliations

Correlation between KRAS Mutation and CTLA-4 mRNA Expression in Circulating Tumour Cells: Clinical Implications in Colorectal Cancer

Sharmin Aktar et al. Genes (Basel). .

Abstract

Combination strategies of KRAS inhibition with immunotherapy in treating advanced or recurrent colorectal carcinoma (CRC) may need to be assessed in circulating tumour cells (CTCs) to achieve better clinical outcomes. This study aimed to investigate the genomic variations of KRAS in CTCs and matched CRC tissues and compared mRNA expression of KRAS and CTLA-4 between wild-type and KRAS-mutated CTCs and CRC tissues. Clinicopathological correlations were also compared. Six known mutations of KRAS were identified at both codon 12 and codon 13 (c.35G>T/G12V, c.35G>A7/G12D, c.35G>C/G12A, c.34G>A/G12S, c.38G>C/G13A, and c.38G>A/G13D). Three CTC samples harboured the identified mutations (16.7%; 3/18), while fifteen matched primary tumour tissues (65.2%, 15/23) showed the mutations. CTCs harbouring the KRAS variant were different from matched CRC tissue. All the mutations were heterozygous. Though insignificant, CTLA-4 mRNA expression was higher in patients carrying KRAS mutations. Patients harbouring KRAS mutations in CTCs were more likely to have poorly differentiated tumours (p = 0.039) and with lymph node metastasis (p = 0.027) and perineural invasion (p = 0.014). KRAS mutations in CTCs were also significantly correlated with overall pathological stages (p = 0.027). These findings imply the genetic basis of KRAS with immunotherapeutic target molecules based on a real-time platform. This study also suggests the highly heterogeneous nature of cancer cells, which may facilitate the assessment of clonal dynamics across a single patient's disease.

Keywords: CTLA-4; KRAS; circulating tumour cells; colorectal carcinoma; immune checkpoint molecules.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Enumeration of circulating tumour cells (CTCs) in colorectal carcinoma patients. (A) A representative image of CTCs detected from patients with colorectal carcinoma (Scale bar: 50 µm), (B) a comparison of the number of different subpopulations of CTCs detected in patients with colorectal carcinoma.
Figure 2
Figure 2
Exonic mutations were detected in CTCs and matched CRC tissues. Representative wild-type and mutant sequences of KRAS as seen via high-resolution melt (HRM) versus Sanger sequencing analysis in exon 2 and exon 3 are represented. Black arrow in HRM curve represents the heterozygous variant deviation against wild type. Different colured lines in chromatogram indicates the presence of six known heterozygous mutations, (A) c.35G>T/G12V, (B) c.35G>A7/G12D, (C) c.35G>C/G12A, (D) c.34G>A/G12S, (E) c.38G>C/G13A, and (F) c.38G>A/G13D against the respective wild type.
Figure 2
Figure 2
Exonic mutations were detected in CTCs and matched CRC tissues. Representative wild-type and mutant sequences of KRAS as seen via high-resolution melt (HRM) versus Sanger sequencing analysis in exon 2 and exon 3 are represented. Black arrow in HRM curve represents the heterozygous variant deviation against wild type. Different colured lines in chromatogram indicates the presence of six known heterozygous mutations, (A) c.35G>T/G12V, (B) c.35G>A7/G12D, (C) c.35G>C/G12A, (D) c.34G>A/G12S, (E) c.38G>C/G13A, and (F) c.38G>A/G13D against the respective wild type.
Figure 2
Figure 2
Exonic mutations were detected in CTCs and matched CRC tissues. Representative wild-type and mutant sequences of KRAS as seen via high-resolution melt (HRM) versus Sanger sequencing analysis in exon 2 and exon 3 are represented. Black arrow in HRM curve represents the heterozygous variant deviation against wild type. Different colured lines in chromatogram indicates the presence of six known heterozygous mutations, (A) c.35G>T/G12V, (B) c.35G>A7/G12D, (C) c.35G>C/G12A, (D) c.34G>A/G12S, (E) c.38G>C/G13A, and (F) c.38G>A/G13D against the respective wild type.
Figure 3
Figure 3
KRAS and CTLA-4 mRNA expression level in CTCs and CRC tissues and their comparison with KRAS mutation status in patients with CRC. (A,B) Fold change (log2) expression of KRAS and CTLA-4 in CTCs calculated relative to healthy donor blood samples and in CRC tissues calculated relative to the expression in adjacent non-neoplastic mucosa tissue and normalised by β-actin as an internal control. (C,D) Relative fold change expression level of KRAS and CTLA-4 in KRAS wild type (KRAS WT) and KRAS mutant (KRAS MT) cases detected in both CTC fractions and CRC tissues. The dashed line indicates the normal fold change value. ns; not significant.
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