KRAS amplification in colorectal cancer: correlations with clinicopathological features and prognosis in patients and prediction of response to targeted therapy

Introduction

Colorectal cancer (CRC) has a high incidence and high mortality rate, despite surgery and chemotherapeutic treatment options. EGFR-targeted therapies, used to treat RAS WT CRC patients, are still only 55-65% effective suggesting other mechanisms of EGFR therapy resistance. Mutated KRAS is one of the most common drivers of tumour initiation and progression in CRC and studies indicate that KRAS amplification may be a mediator of drug resistance. Increased KRAS copy number has been identified as an independent prognostic variable associated with poor survival rate. Recent studies have demonstrated a link between copy number variation (CNV) and immune infiltration into the tumour microenvironment. KRAS mutation is also thought to have a major role in the immunosuppressive tumour microenvironment of CRC and further studies demonstrated KRAS regulated CRC immune infiltrates using specific pathways involving CD8+ T-cells. The contribution of both KRAS amplification and KRAS mutation in CRC is poorly understood.

Main Points

• KRAS amplification, determined by qPCR, was present in 44/304 primary CRC patients that had surgery. Amplification was associated with age (>65), immunoscore, mutation status and tumour status but not gender, tumour location or nerve invasion.

• 6 patients with amplified KRAS also had mutant KRAS but patients with amplified KRAS were mostly KRAS wild type. Samples were divided into WT-KRAS/Non-Amplified, WT-KRAS/Amplified, Mut-KRAS/Non-Amplified and Mut-KRAS/Amplified.

• There was no significant difference in tumour stage, immunoscore or tumour location between the 4 groups. In the WT KRAS groups there was increased protein expression of KRAS in the amplified group compared to non-amplified, however, in the KRAS mutant groups, irrespective of mutation type, there was no significant difference in KRAS protein expression in amplified compared to non-amplified samples.

• RNA-seq analysis of amplified and non-amplified KRAS samples in the WT/KRAS group resulted in >4000 differentially expressed genes. Pathway analysis demonstrated many enriched pathways downstream of KRAS but also enrichment in immune-function pathways. KRAS pathways were confirmed by western blotting of p-AKT/AKT, p-ERK/ERK and p-MEK/MEK.

• Enrichment of immune regulated pathways prompted the authors to investigate CD8+ T cell density in 204 CRC tissue samples using immunohistochemistry. In the mutant KRAS group there was no difference in CD8+ T-cell density in the amplified KRAS samples compared to non-amplified. In the WT KRAS samples CD8+ T-cells were significantly less in the amplified group compared to non-amplified. This was supported by data from analysis of publicly available scRNA-seq CRC datasets.

• Xenografts from CRC patients were implanted into NOD/SCID mice. Tumour volumes and weights from WT KRAS amplified xenografts were consistently higher than WT KRAS non-amplified xenografts. This was also observed in isogenic HCT-116 cell xenografts with the WT KRAS amplified having significantly increased volume compared to non-amplified. EGFR-therapy efficiently inhibited the tumour growth in WT KRAS non-amplified xenografts, but only weak inhibition was observed in amplified xenografts.

• In a KRAS amplified xenografts a combination of CDK4/6 inhibitor and MEK inhibitor increased anti-tumour activity and slowed tumour growth compared to MEK inhibitor alone suggesting that combination therapies could be promising for KRAS amplified tumours.

Conclusion

The contribution of KRAS mutations and KRAS amplifications to initiation and progression of CRCs has not been studied in detail. This study demonstrated that KRAS amplification was more common in KRAS WT CRCs with hardly any CNV variations in CRCs harbouring KRAS mutations. In CRCs where KRAS amplification was detected prognosis was worse and efficacy of anti-EGFR therapies was significantly impaired. KRAS amplification in the absence of KRAS mutation was sufficient to activate downstream tumour promoting pathways in CRC and also resulted in reduced CD8+ T-cell infiltration to the tumour microenvironment. This study also demonstrated that resistance to EGFR therapies in KRAS amplified tumours could be overcome with a combination of MEK and CDK4/6 inhibitors.

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