XNA Molecular Clamp Technology

Optimal Choice for Early Cancer Detection

XNA, xenonucleic acids, are innovative new nucleic acid molecular oligomers that hybridize by Watson-Crick base pairing to target DNA sequences, and yet have a modified chemical backbone. XNA oligomers are highly effective at hybridizing to targeted normal DNA sequences and can be employed as molecular clamps in quantitative real-time polymerase chain reactions (qPCR) or as highly specific molecular probes for detection of nucleic acid target sequences. The XNA tightly bind to the wild-type sequence that is 100% complementary and block DNA polymerase from DNA elongation; only the mutant target sequence gets amplified because the XNA:mutant DNA duplex is not stable due to mismatch and falls off from the template in PCR reactions.

Introducing XNA Molecular Clamp Technology

XNA molecular clamps assays are highly sensitive for liquid biopsy and FFPE samples. The limit of detection (LoD) can reach as low as 0.1% (7 or 8 copies of mutant DNA) using 5 ng of ctDNA, equivalent to 2 ml of blood from a patient. Since the presence of circulating tumor cells (CTCs) has been found to be associated with poor survival in multiple cancers, and lowering the CTC threshold from unfavorable (≥5 CTCs/7.5 ml of blood) to favorable levels (<5 CTCs/7.5 ml of blood) improves survival, the number of CTCs in blood can be used as a predictive factor for cancer treatment response. Considering its sensitivity, the XNA technology may be used to find cancer mutations and to glean meaningful information for cancer treatment. In the context of precision medicine for cancer, finding the cancer driver mutations such as those in growth pathway and in tumor suppressor genes is critical, as it is generally recognized that these mutations contribute to early development of different types of cancers. Detection of these mutations is not only essential for early cancer detection, but for providing strategies for cancer therapy as a companion diagnostic tool.

Early Cancer detection potential by highly sensitive XNA technology

XNA molecular clamps assays are highly sensitive for use with liquid biopsy and FFPE samples.

One of the examples for cancer gene mutation detection using the XNA technology is ColoScape™ colorectal cancer mutation detection kit we developed for FFPE and liquid biopsy samples. We obtained a sensitivity of about 60% in advanced adenoma tissue samples (stage 0) compared to 42% pre-cancer detection achieved by the ColoGuard kit. For stage I to IV colon cancer, the sensitivity was 95.5%. The beauty of using XNA technology is achieving high sensitivity using regular qPCR TaqMan assays that can be performed in common pathology and genetic biomarker discovery labs without investing in other expensive instrumentation.

 

Analytical sensitivity in plasma (QClamp® KRAS Mutation Detection Test) powered by XNA technology

Analytical sensitivity in FFPE (QClamp® KRAS Mutation Detection Test) powered by XNA technology

Precision Medicine and Early Cancer Detection

Precision Medicine Initiative defines precision medicine as “an emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle for each person.” Using the precision medicine approach doctors make treatment decisions based on an individual’s genetic makeup rather than providing a generic “one-size-fits-all” solution.

In the context of precision medicine for cancer, finding the cancer driver mutations, such as those involved in growth pathway and tumor suppressor genes is critical, as it is generally recognized that these mutations contribute to early development of different types of cancers. Detection of these mutations is essential not only for early cancer detection, but also for providing strategies for using cancer therapy as a companion diagnostic tool.

The Invasive Way with FFPE Samples

Challenges for Early Cancer Detection – Invasive Method

Traditionally, tumor gene mutations have been detected using DNA isolated from formalin-fixed paraffin embedded (FFPE) tumor biopsy tissue samples. But tissue biopsy poses a few challenges:

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Surgical tumor biopsies are invasive, sometimes risky, and are not always easily accessible

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Mutations in a distant organ are not necessarily detected in the biopsy samples and doctors miss the opportunity to provide the appropriate treatment regimen

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The dynamic mutational changes that occur with the progression of cancer are not identified by tissue biopsy, thus altering therapeutic strategies over the course of the disease becomes difficult

Challenges for Early Cancer Detection – Non-invasive Method

Recently, minimally invasive procedures, commonly called liquid biopsies, such as collecting blood samples and monitoring mutations in the circulating cell-free tumor DNA (ctDNA) in the blood, have gained traction in early cancer detection. This method has a few limitations:

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Success depends on the sensitivity of the detection technologies, which becomes a limiting factor. That can also be the major reason why most of the current cancer studies and detections have been done from malignant cancers

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Premaligancy studies or early cancer detection using genomic-wide analysis has been very limited due to the difficulty in detection of cancer gene mutations in a small sub-population of cells before cancer is developed

The Non-invasive Way with Liquid Biopsy

Limitations of Current Technologies for Early Cancer Detection

Finding tumor mutations in ctDNA is like finding a needle in a haystack because the proportion of cancer cell DNA present in a population of healthy cells in circulation is miniscule compared to wild-type DNA. Detecting such minute amount of tumor cell DNA (barely 1% and as little as 0.01% of total ctDNA during early stages) requires much more sensitive techniques compared to techniques used in advanced cancers, where the proportion of tumor DNA is high and may contribute to the majority of circulating DNA in the blood.

 

Source:
Genomics, Circuits, and Pathways in Clinical Neuropsychiatry, 2016
Analysis of DNA Methylation by Pyrosequencing, 2016
What is next-generation sequencing? 2013
Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data, 2017
Understanding PCR, 2017

Sanger Sequencing

Sanger sequencing is the process of selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.

Advantage: accurate result and is, therefore, the gold standard
Disadvantage:  low sensitivity (20% to 25% VAF)

NGS

Next-generation sequencing (NGS) or massively parallel or deep sequencing describes a DNA sequencing technology where millions of DNA fragments are sequenced in parallel. NGS has revolutionized genomic research making genomics easily accessible.

Advantage:  high-throughput and good sensitivity (1% to 5% VAF, or even better)
Disadvantage: costly and time-consuming (7 to 10 days)

qPCR Analysis

Quantitative polymerase chain reaction (qPCR) is a primer-probe-based method by which the amount of the PCR product can be determined in real-time and is very useful for investigating gene expression

Advantage: Sensitivity can reach 1% VAF for some targets. Rapid with minimal hands-on work.
Disadvantage: Multiple methods available for qPCR and a lot of variations in sensitivity. Some of them are only 10% VAF

Pyrosequencing Assays

Pyrosequencing is a technique that uses a sequencing-by-synthesis system, designed to quantify single-nucleotide polymorphisms (SNPs).

Advantage: better sensitivity and throughput than Sanger sequencing; the early form of NGS assays
Disadvantage:
 low sensitivity (5% to 8% VAF)

ddPCR

Droplet Digital PCR (ddPCR) technology is a method of performing PCR in a sample that is fractionated into thousands of water-oil emulsion droplets. It utilizes Taq polymerase in a standard PCR reaction to amplify a target DNA fragment in each individual droplet using pre-validated primer or primer/probe assays.

Advantage: high sensitivity (claimed to be 0.001% VAF)
Disadvantage:  much less sensitivity observed in testing than claimed and suffers from false-positive results

XNA Technology

Sensitivity reaches 0.1% to 0.5% VAF; much more sensitive than regular qPCR and other qPCR-derived techniques.
Achieves ultra-sensitivity by amplifying only mutant DNA and blocking wild-type sequences.

 

Features and Benefits of XNA Molecular Clamps Assays

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Regular qPCR reactions on most of common instruments

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Synthetic oligomers (15 to 25 nt long) resistant to any known nucleases

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Much higher binding affinity for target sequence independent of salt concentration

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Large melting temperature difference (ΔTm = 15-20ºC) in single-nucleotide (SNP’s) and insertion/deletions (indels) (5-7ºC for natural DNA)

XNA Technology Novel Applications

Single Gene Mutation Detection

Colorectal Cancer Mutation Detection

NGS Targeted Sequencing

CRISPR/Cas9 Gene Editing Test

Adapter Dimer Removal for NGS

Resources

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Email us: information@diacarta.com

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