A novel xenonucleic acid-mediated molecular clamping technology for early colorectal cancer screening

Abstract

Background: Colorectal cancer (CRC) is one of the leading causes of cancer-related death. Early detection is critical to reduce CRC morbidity and mortality. In order to meet this need, we developed a molecular clamping assay called the ColoScape TM assay for early colorectal cancer diagnostics.

Methods: Nineteen mutations in four genes (APC, KRAS, BRAF and CTNNB1) associated with early events in CRC pathogenesis are targeted in the ColoScapeTM assay. Xenonucleic Acid (XNA)-mediated qPCR clamping technology was applied to minimize the wild-type background amplification in order to improve assay sensitivity of CRC mutation detection. The assay analytical performance was verified and validated, cfDNA and FFPE CRC patient samples were evaluated, and an ROC curve was applied to evaluate its performance.

Results: The data showed that the assay analytical sensitivity was 0.5% Variant Allele Frequency, corresponding to ~7-8 copies of mutant DNA with 5 ng total DNA input per test. This assay is highly reproducible with intra-assay CV of <3% and inter-assay CV of <5%. We have investigated 380 clinical samples including plasma cfDNA and FFPE samples from patients with precancerous and different stages of CRC. The preliminary assay clinical specificity and sensitivity for CRC cfDNA were: 100% (95% CI, 80.3-97.5%) and 92.2% (95% CI, 94.7-100%), respectively, with AUC of 0.96; 96% specificity (95% CI, 77.6-99.7%) and 92% sensitivity (95% CI, 86.1-95.6%) with AUC of 0.94 for CRC FFPE; 95% specificity (95% CI, 82.5%-99.1%) and 62.5% sensitivity (95% CI, 35.8%-83.7%) with AUC of 0.79 for precancerous lesions cfDNA.

Conclusions: The XNA-mediated molecular clamping assay is a rapid, precise, and sensitive assay for the detection of precancerous lesions cfDNA and CRC cfDNA or FFPE samples.

Fig 1. XNA structure and its function in the assay.

(A). XNA structure and hybridization with DNA. (B). Principle of the Coloscape^TM assay mutation detection in targeted genes. XNAs hybridize tightly to complementary DNA target sequences only if the sequence is a complete match. When there is a mutation in the target site, and therefore is a mismatch, the XNA: DNA duplex is unstable, allowing strand elongation by DNA polymerase. Addition of an XNA whose sequence is a complete match to the wild-type DNA to a PCR reaction blocks amplification of wild-type DNA allowing selective amplification of mutant DNA. This enrichment of the mutation amplicons enables mutation detection by qPCR.

Fig 2. Comparison of XNA-based qPCR and qPCR without XNA.

(A). CTNNB1 T41 amplification curves with XNA. a: 5% VAF of CTNNB1 T41 mutant, CT = 29.1 (ACTB, CT = 30.7, not shown here due to the use of Cy5 channel), b: CTNNB1 T41 wildtype, CT = 38.3 (ACTB, CT = 30). This indicates that XNA-based qPCR has a Δ Ct of ~9 for mutant to wildtype. (B). CTNNB1 T41 amplification curves without XNA. a, 5% VAF of CTNNB1 T41 mutant, CT = 26.9 (ACTB, CT = 30.4). b, CTNNB1 T41 wildtype, CT = 27.2 (ACTB, CT = 30). This indicates that qPCR without XNA has a Δ Ct of ~0.3 for mutant to wildtype. (C). Sanger sequencing for amplicons from CTNNB1 T41 assay with XNA, confirming that there is only mutant sequence GCC at CTNNB1 T41 (red arrow). (D). Sanger sequencing for amplicon from CTNNB1 T41 assay without XNA showing that there is a mix of wildtype and mutant A/GCC of CTNNB1 T41(red arrow). (E). Amplification profile of the ColoScape^TM multiplex qPCR assay with various concentrations of reference gDNA. a, BRAFV600E with 1%, 0.5%, 0.1% and 0% VAF (Fam as the probe-labeling dye). b. KRAS G13 with 1%, 0.5%, 0.1% and 0% VAF (Hex as the probe-labeling dye); c. CTNNB1 S45 with 1%, 0.5%, 0.1% and 0% VAF (CFR610 as the probe-labeling dye); and d. Beta-Actin (internal control, QS670 as the probe-labeling dye).

Fig 3. Receiver Operating Characteristic (ROC) curve and area under the curve (AUC) to evaluate the performance of the assay.

a, precancerous lesions cfDNA samples analysis. b, CRC FFPE samples analysis; c, CRC cfDNA sample analysis.