January 1, 2014 (Vol. 34, No. 1)

Somatic Mutation PCR Testing Suppresses Amplification of Wild-Type Template DNA, Selectively Amplifies Mutant Templates

In the context of personalized cancer treatment, the “gold standard” for tumor mutational analysis is DNA sequencing. This approach, however, is not entirely satisfactory. Recent studies indicate that it is one of the least sensitive methods for characterizing mutation. In fact, with DNA sequencing, a mutation must be present in 10–20% of the sample to be readily detected.

Below this threshold, tumorigenic mutations may lurk unseen. Such may be the case with colon tumors, most if not all of which are polyclonal and heterogeneous. In one study of colon tumors, investigators affiliated with the FDA concluded that tumorigenic mutations may be undetectable using standard DNA sequencing methods.

To improve sensitivity, several alternative approaches have been developed. These include developments in real-time PCR-based detection methods, such as allele-specific PCR (ASPCR) and hydrolysis-based probes. Although these techniques show better sensitivity, lowering the detection threshold to at least 5%, they still fall short of a crucial goal—detecting a mutation that is present in less than 1% of tissue samples.

These techniques are inadequate because they cannot eliminate large amounts of wild-type genomic DNA in the backgrounds of samples. In addition, hydrolysis-based probes lack specificity, failing to detect EGFR T790M and JAK2 Exon 14 mutant detections.

Recently, droplet digital PCR (ddPCR) has been developed to improve the sensitivity of PCR so that it can reach below a detection limit of 0.1% mutated DNA. The technology makes millions of droplets to separate the mutated DNA from wild-type genomic DNA. However, some droplets generated by the machine contain both wild-type and mutated DNA.

The presence of such droplets poses a problem when one is working with clinical samples. Wild-type DNA continues to form a large background, so sensitivity does not quite reach below 0.1% sensitivity. It is much more challenging to detect and quantitate below 1% mutant abundance in either mixed tissue biopsies or in cfDNA in plasma or serum.

Another Alternative

To reduce the wild-type background and improve sensitivity, a molecular clamp has been designed to hybridize selectively to wild-type template DNA and block its amplification. This molecular clamp consists of a synthetic, sequence-specific Xeno-nucleic acid (XNA) probe. It is called QClamp™.

In the presence of a mutation such as a single nucleotide polymorphism (SNP) gene deletion, insertion, or rearrangement in the region of the XNA probe sequence, the XNA probe molecule melts off the mutant template DNA during the PCR cycling process, and only mutant templates are amplified efficiently (Figure 1).

QClamp has been shown to be a sensitive and precise quantitative PCR (qPCR) technology. It is able to block the amplification of wide-type DNA from samples. In addition, it can detect genetic mutations in less than 0.01% DNA sample. This level of sensitivity enables detection of gene mutations in the oncology therapeutic clinical setting utilizing patient biopsy, surgical tissue, or formalin-fixed, paraffin-embedded (FFPE) tissue.

The QClamp technology is particularly suited to “liquid biopsy” applications for the detection of tumor-derived, cell-free DNA in plasma or urine, which can achieve sensitivities equivalent or even better than droplet digital PCR (ddPCR). Utilizing only standard qPCR real-time instrumentation, QClamp technology detects below 0.01% mutated DNA.

Figure 1. The modified DNA oligo probe binds—or clamps—to wild-type DNA and blocks further wild-type DNA amplification. This probe, or XNA clamp, does not bind to mutated DNA, allowing it to be amplified and detected.

Evaluation of QClamp Technology

QClamp technology has been intensively evaluated at several large hospitals, service labs, and biopharmaceutical companies. Recently, a premier diagnostic services company in United States has been proactively evaluating platforms for cancer diagnostics to detect EGFR T790M mutations in exon 20 from tissue and FFPE samples. QClamp successfully detected all the clinically relevant EGFR mutations, including T790M, directly from the FFPE samples without DNA extraction, which was further demonstrated by high-resolution melting curves (Figure 2).

Moreover, during the evaluation, leukocyte-depleted whole blood was spiked with JAK2 wild-type and MUT 617 DNA over a range of ratios (50%, 20%, 10%, 5%, 1%, 0.5%, and 0%). Spiked samples were extracted with PCR-ready lysis buffer QZol™ and tested using the QClamp JAK2 mutation assay. The results demonstrated that QClamp technology can detect below 0.1% mutated DNA directly from whole blood without DNA purification. It strongly suggested that QClamp can be a tool for liquid biopsy applications.

One investigator, Dr. Sumanta Goswami, an associate professor at Albert Einstein College of Medicine in New York, is using the QClamp KRAS assay to detect low-frequency drug-resistance “driver” mutations. The tissue samples are fine-needle aspirates taken from lung cancer patients as part of an outpatient procedure. This application shows that besides offering sensitivity and precision, QClamp provides a level of convenience that could prove useful in medical oncology and personalized diagnostics.

Figure 2. High-resolution melting (HRM) profiles of clinically relevant EGFR mutations generated by the QClamp EGFR test.

Speed and Flexibility

QClamp technology includes a rapid direct sample treatment protocol that utilizes a proprietary sample lysis buffer (QZol) and does not require sample DNA purification. An aliquot of the lysed sample solution can be used directly in the PCR reaction. QZol sample treatment gives the same results as purified DNA in less time, taking only 20 minutes.

The QClamp assays can also be formatted as endpoint colorimetric or chemiluminescence “Amplicon Capture Assays” and mutations detected by a standard plate-based ELISA reader, a luminometer, or simple lateral flow-strip assay formats. QClamp currently detects a broad range of mutations including EGFR, KRAS, NRAS, BRAF, JAK2, ALK, PIK3CA, and cKIT. Products for each of these mutations are currently marketed for Research Use Only (RUO) and are in the process of obtaining CE-IVD approval, which may be granted in early 2014.

Michael Powell, Ph.D. ([email protected]), is CSO and Aiguo Zhang, Ph.D., is founder and CEO of DiaCarta.

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