Clinical researchers from ARUP Laboratories and the University of Utah have developed a modified approach to performing quantitative allele-specific PCR that obviates the need to generate standard curves, and have demonstrated its use to quantify BRAF V600E mutation frequencies in hairy cell leukemia as accurately as standard curve-based qPCR methods.
The technique, which the researchers have also used as part of an assay for the JAK2 V617F mutation, is platform-agnostic and can be used to detect mutations in blood and bone marrow samples and formalin-fixed, paraffin-embedded tissue.
As such, the technique's developers believe that it could prove useful to more quickly and efficiently quantitate any recurrent substitution mutation as part of a diagnostic test for cancers harboring such mutations or in basic research applications.
The researchers described their method in a paper published this month in the Journal of Molecular Diagnostics. As noted in their paper, allele-specific qPCR has emerged as the tool of choice for clinical labs to quantitate predominant recurrent base change mutations, which can be useful for understanding the kinetics of treatment response or prognosing or diagnosing certain cancers.
Allele-specific qPCR, however, usually requires the generation of standard curves that, in turn, rely on accurate DNA quantitation to calculate the absolute copy number of DNA molecules in a sample, the researchers wrote. This process is tedious and time-consuming, and is prone to errors resulting from variations in pipetting or miscalculation of the absolute copy numbers of standards.
According to Philippe Szankasi, first author on the paper and a research scientist at ARUP Laboratories, the underpinnings of the new technique were established a few years ago, when researchers at the lab developed an allele-specific test for the JAK2 V617F mutation — "a very common SNP that is a good diagnostic for various myeloproliferative disorders," Szankasi said. "This is a homebrew test … and when you do homebrew, it's hard to do standard curves."
In order to eliminate the need to generate standard curves, the group used a pair of calibrator plasmids, each containing fragments of the wild-type and mutant gene of interest, and added the plasmids at equal concentrations to the PCR reaction mixture.
"It was mixed in at supposedly equal amounts," Szankasi said. "But it turns out that from batch to batch … you'd get different results. [Someone] had the good idea, let's just make it one plasmid, a heterozygous plasmid. That's really the key to the whole story."
The group first applied its single, heterozygous plasmid calibrator to an allele-specific qPCR assay for BRAF V600E mutations. After realizing that it worked well, they adapted it to its original JAK2 V617F test.
The group's impetus for developing a BRAF V600E test came from a seminal paper published in 2011 in the New England Journal of Medicine demonstrating that the mutation was associated with nearly all cases of hairy cell leukemia and could serve as a specific diagnostic marker apart from traditional morphological and flow cytometric analysis.
"This is currently being offered as a clinical test at ARUP targeted towards hairy cell leukemia," said Todd Kelley, medical director of the Molecular Hematopathology Laboratory at ARUP, and corresponding author on the paper.
"Of course, BRAF mutations are found in lots of other circumstances, so there is potentially interest in the test from solid tumor folks," Kelley added. "Also, we designed and directed the test for use in HCL and blood and bone marrow, although it does work very well in FFPE tissue and solid tumors."
BRAF V600E mutations are also associated with response to Roche's melanoma drug Zelboraf (vemurafenib). The drug and a companion test — Roche's Cobas 4800 BRAF V600 Mutation Test — were simultaneously cleared by the US Food and Drug Administration in 2011.
In the JMD study, the group used previously described allele-specific primers for the BRAF V600E mutation and the wild-type mutation, and designed a dual-labeled hydrolysis probe to obtain quantitative data. Each PCR run, which was performed on a Roche LightCycler 480 instrument, included the calibrator plasmid that contained both a wild-type and V600E mutant fragment covering the region amplified by the PCR primers.
The researchers tested 18 HCL samples and found that the heterozygous plasmid method generated data comparable to those obtained via allele quantitation using conventional standard curves over a wide range of allelic ratios. The researchers also showed that their method had good correlation with pyrosequencing; and that it demonstrated a high degree of correlation with existing techniques when using FFPE solid tumor samples.
"The idea behind this is that we don’t have to have three, four, five different plasmid dilutions to make a standard curve," Kelley said. "We just have one plasmid … that has both the wild type and the mutant sequence cloned in, so it's a perfect 50-50 ratio, regardless of how much of that plasmid you're using."
"We thought it was a really simple but actually really advantageous and efficient method for doing quantitative PCR, and we've had a lot of success … using this general strategy in terms of maximizing efficiency in the clinical laboratory," he added.
Szankasi noted that the technique should prove particularly valuable for laboratory-developed tests. Even though the ARUP researchers used their own PCR assay along with a Roche qPCR platform, the technique could be used with any qPCR reagents or instrument platform.
"I think most people buy a kit because no one wants to generate their own standard curves, and that's a big service from, [for instance], companies that sell allele-specific PCR tests, and provide the standard curves and basically you don't have to worry about this whole [calibration]," Szankasi said. "But that also costs a lot of money. This technology is so cheap, and the focus of the paper is to really show that it's just as accurate."
Kelley noted that the test could also be useful for general research purposes, but said that it is not necessarily applicable to all qPCR assays.
"Historically, JAK2 V617F has been reported by most labs as a ratio of mutant to wild type," Kelley said. "In circumstances like that it makes a lot of sense to use this plasmid. However, in circumstances where, for instance, the qPCR results have classically been reported as a ratio to a reference gene — for example BCR-ABL is typically reported as a ratio of BCR-ABL to ABL — it doesn't make sense."
As such, the test is primarily applicable to substitution mutations commonly found in hematopathology, where clinicians desire a quantitative result, as opposed to solid tumors, where a qualitative test is usually — but not always — sufficient.
Kelley said he couldn't immediately think of other hematopathological diseases where the approach might be valuable, "but that's not to say that as these sorts of recurrent point mutations are discovered that this wouldn't be a good strategy to apply to those."
Lastly, Kelley noted that the method could be useful in the continuing push for laboratory-to-laboratory assay standardization.
"Currently everyone has their own method and their own way of quantifying and it's difficult to compare results from lab A to lab B, so, as a general statement, I think strategies like this could really propel standardization efforts," he said.
ARUP Laboratories has applied for a patent covering the method. Kelley said that there is "potential" for the approach to be commercialized outside ARUP's use of it in homebrewed molecular assays, but declined to comment further on the matter.