A team at the University of Toledo Health Sciences Campus in Ohio has developed a multiplexed method for two-color fluorometric, real-time, reverse transcriptase qPCR from formalin-fixed, paraffin-embedded fine needle aspirate biopsies.
The assay, published in PLoS One in February, uses a patented internal standards mixture, as well as external standards and a pre-amplification step, to enable measurement of transcription levels of three lung cancer-associated genes and a control gene. These genes are part of a previously reported lung cancer diagnostic test. The group is also developing the method to be useful in both PCR and next-generation sequencing.
Key elements of the group's approach – multiplex PCR with an internal standards mixture, and the use of two rounds of PCR – received a US patent in 2009. This method, including the ISM, is licensed to Wilmington, NC-based AccuGenomics. Started in 2010 by CEO Nick Lazaridis, this company is a new incarnation of Gene Express, which closed in 2008. Tom Morrison, former senior director at BioTrove (a company acquired by Life Technologies in 2009, now part of Thermo Fisher Scientific), serves as CSO of AccuGenomics.
James Willey, a professor of medicine and pathology at University of Toledo, was the corresponding author on the paper describing the RT-qPCR method. In an interview with PCR Insider he said that getting reliable quantification out of this particular type of sample is vital for diagnostics. "Increasingly, cancers are diagnosed through fine needle aspiratates," he said, "So having adequate quality control as well as amplification ability for analysis of FFPE samples is important."
Willey said the new method builds on similar two-color fluorometric technology for HIV testing, such as that used by COBAS, but includes internal controls not just for genes of interest but also for loading control genes. "The Covance HIV test is a single analyte measured against a single internal standard, using two different fluors to sort them out during the PCR process," Willey said. His method uses "a mixture of internal standards for multiple targets, both loading control and target genes ... In each case, we have sequence-specific probes for the native template and the internal standard for each target gene measured."
The ISM is "a constant reference material that can be used by different experimenters in different laboratories, and when you add that mixture, which has the internal standards at the same constant stoichiometric ratio, to each cDNA sample, then you have a reliable measurement across laboratories," Willey said.
Another innovative aspect of the method was the pre-amplification step. For this, the group essentially ran two rounds of PCR. The first round was a multiplex PCR that included four pairs of primers for each of the four genes they were measuring. The second round contained primers for each individual target and probes for the native template as well as for the ISM.
The ISM controlled for the pre-amplification step, and was important to be able to quantify RNA levels in the FFPE samples, according to Willey. "You cannot do this type of two rounds of PCR and get quantitative measurements unless you have internal standards present throughout the whole process, because the PCR becomes non-linear and unpredictably exponential," he said. The group used the ABI 7500 platform for real-time PCR.
Importantly, the pre-amplification step was able to affect signal-to-noise ratio in FFPE samples. "Unless you do that pre-amplification, a lot of the amplification curves you're going to get are going to be very close to the background noise. By doing the pre-amplification, it moves your quantification cycle threshold to more like six or seven cycles away from the background," Willey said.
The group also developed an external standards mixture to control for inter-experimental variation. This step compensated for changes in cycle threshold between experiments. The ESM also had the advantage of controlling for any possible variations in fluormetric intensity of the probes, which may sometimes occur with freezing and thawing, Willey said.
The group used the Qiagen RNeasy FFPE kit to isolate RNA from the fine needle aspirates. Willey said they ran a comparison with another brand, but got better results from the Qiagen kit. To overcome FFPE-induced obstacles, they chose short PCR products, and gene-specific priming as opposed to random hexamers. For this type of sample, "Nobody uses oligo-dT because these things are pretty degraded," Willey said, "but a lot of people use random hexamers. We report in this manuscript that you get much greater signal if you use gene-specific priming in FFPE."
In their study, Willey and colleagues demonstrated that their test, when performed on surgical FFPE samples, had a diagnostic accuracy of 93 percent — similar to that previously reported for analysis of fresh samples.
In parallel to tackling PCR quality control, Willey's group is also working on sequencing. The group published in PLoS One in November on studies "to develop targeted PCR amplicons for libraries for sequencing," Willey said.
Wiley called this a "very powerful approach" to doing highly quality controlled gene expression measurement of hundreds or even thousands of genes, using multiplex PCR followed by sequencing on an Illumina HiSeq platform.
"Because we are measuring each gene relative to a known number of internal standard molecules at the beginning of the PCR, even though we are doing multiple rounds of multiplex PCR, which ordinarily would cause starting amounts to become unpredictable ... it is possible to get reliable data on the sequencer," he said.
The group is planning to publish results soon that show this sequencing technology can also be used to assess FFPE. Willey said a portion of the products quantified in the four-gene, two-color fluorometric multiplex real-time RT-qPCR were also sequenced, and, "because they share those characteristics of internal standards and multiplexing, the results were very highly correlated," he said. "I'm very excited about the ability to develop one set of reagents that allows you to assess both by PCR and by sequencing."
AccuGenomics' Lazaridis told PCR Insider that the ISM is available for sublicensing for research and development use, and may also be obtained for limited other uses. The company is currently in discussions with pharmaceutical and diagnostic companies to utilize the standardization technology for PCR and next-generation sequencing, Lazaridis said. In addition, AccuGenomics has two lung cancer-related tests — one for high risk and one for diagnosis — also developed in the Willey lab. The company is currently looking for partners with CLIA lab certification to run pivotal studies with these tests. Willey will be presenting the latest results on the risk and diagnostic work at the American Association for Cancer Research meeting this weekend in San Diego.