Detection of KRAS mutation in colorectal cancer (CRC) is important in the prediction of response to target therapy. The study aims to develop a novel mutation detection platform called the "PNA-LNA molecular switch" for the detection of KRAS mutation in CRC. We employed the enhanced binding specificity of peptide nucleic acid (PNA) and locked nucleic acid (LNA) in conjunction with a loop-mediated isothermal amplification (LAMP) approach to identify the mutation status of KRAS oncogene codon 12 (c.35G>T/G12V and c.35G>A/G12D) using synthetic oligonucleotides and colon cancer cell lines (Caco-2 and SW480). This method specifically blocked the amplification of the wild-type sequences while substantially amplifying the mutated ones, which was visualized by both colorimetric and fluorescence assays. We then checked the mutation profile of KRAS codon 12 in the DNA derived from tumor tissue samples (number of samples, n = 30) and circulating tumor cells (n = 24) from CRC patients. Finally, we validated the results by comparing them with the data obtained from DNA sequencing of colon tumors (n = 21) of the same CRC patients. This method showed excellent sensitivity (1 DNA copy/μl), reproducibility [relative standard deviation (%RSD) < 5%, for n = 3], and linear dynamic range (1 ag/μl-10 pg/μl, R² = 0.94). This platform is significantly faster, relatively cheaper, has superior sensitivity and specificity, and does not require any high-end equipment. To conclude, this method has the potential to be translated into clinical settings for the detection of mutations in diverse diseases and conditions.

Information regarding genetic alterations of driver cancer genes in circulating tumour cells (CTCs) and their surrounding immune microenvironment nowadays can be employed as a real-time monitoring platform for translational applications such as patient response to therapeutic targets, including immunotherapy. This study aimed to investigate the expression profiling of these genes along with immunotherapeutic target molecules in CTCs and peripheral blood mononuclear cells (PBMCs) in patients with colorectal carcinoma (CRC). Expression of p53, APC, KRAS, c-Myc, and immunotherapeutic target molecules PD-L1, CTLA-4, and CD47 in CTCs and PBMCs were analysed by qPCR. Their expression in high versus low CTC-positive patients with CRC was compared and clinicopathological correlations between these patient groups were analysed. CTCs were detected in 61% (38 of 62) of patients with CRC. The presence of higher numbers of CTCs was significantly correlated with advanced cancer stages (p = 0.045) and the subtypes of adenocarcinoma (conventional vs. mucinous, p = 0.019), while being weakly correlated with tumour size (p = 0.051). Patients with lower numbers of CTCs had higher expression of KRAS. Higher KRAS expression in CTCs was negatively correlated with tumour perforation (p = 0.029), lymph node status (p = 0.037), distant metastasis (p = 0.046) and overall staging (p = 0.004). CTLA-4 was highly expressed in both CTCs and PBMCs. In addition, CTLA-4 expression was positively correlated with KRAS (r = 0.6878, p = 0.002) in the enriched CTC fraction. Dysregulation of KRAS in CTCs might evade the immune system by altering the expression of CTLA-4, providing new insights into the selection of therapeutic targets at the onset of the disease. Monitoring CTCs counts, as well as gene expression profiling of PBMCs, can be helpful in predicting tumour progression, patient outcome and treatment.

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.

Faysal Bin Hamid was not included as an author in the original publication