NEW YORK – A team of researchers at New York University has systematically whittled down the "tissue-to-result" time of a neuro-oncology digital PCR workflow to perform molecular testing in under 20 minutes. The method is being developed for use in the operating room to assist neurosurgeons in determining boundaries of brain tumors during resection surgeries, but it may also have other surgical oncology and rapid disease testing applications.
During a neurosurgical procedure to remove a tumor, surgeons typically rely on histology and prior experience to determine when to stop removing tissue. Postoperatively, an MRI will show if any tumor tissue was left behind.
While molecular testing of margin tissue can pick up cancer mutations and help map the noncancerous brain cells, the process has been too time-consuming to perform during an operation. Digital PCR, for example, has the advantages of being resistant to inhibitors and providing absolute quantification of nucleic acids in a sample, but the standard workflow takes between two and five hours.
Yet, "we've always been fascinated by this almost science fiction idea of a surgeon having molecular insight into which tissue to cut out during the surgery," said Gilad Evrony, coauthor of a study describing the method published Tuesday in Med.
"Ideally, we would like a surgeon to be able to repeatedly assay tissue that's removed," he added.
Called ultra-rapid droplet digital PCR, or UR-ddPCR, the new method the team developed speeds up the workflow using elements of the Bio-Rad Laboratories' QX200 Droplet Digital PCR system, such that it can go from a tissue biopsy to an actionable result in the range of 15 minutes.
However, the sophistication of rapid molecular testing during brain surgery was only made possible by an old-fashioned methods development slog.
Along with coauthor and NYU neurosurgeon Daniel Orringer, Evrony and his lab first surveyed all available technologies to find any that could potentially be sped up.
The team chose to use Bio-Rad's droplet generator and reader but needed to forge novel sample preparation and thermal cycling protocols to achieve its rapid test times.
Emulsion-based PCR is like a vinaigrette of oil and water, with PCR reaction dispersed in each tiny droplet, so rapid detergent-based nucleic acid extraction would be incompatible. The team tried many homebrew and commercial methods, settling on a non-detergent extraction called SwiftX from Xpedite Diagnostics.
For the PCR steps, they built upon an approach dubbed extreme PCR that involves upping primer, probe, and enzyme concentrations. The enzyme typically used for ddPCR requires a long heat activation step, Evrony said. However, "every minute matters for us," he said, so the team set about troubleshooting alternative enzymes, as well, and ultimately found the fastest time by spiking in a temperature-sensitive, aptamer-inhibited Taq polymerase custom ordered from New England Biolabs.
Finally, to achieve ultra-rapid thermal cycling, the team went "back to really old school PCR methods" for inspiration, Evrony said, and substituted stainless steel tubes for the glass capillary tubes of foundational techniques.
These tubes, readily available from different suppliers, could increase the surface area to volume ratio and increase the thermal conductance, Evrony said.
The final workflow reduced extraction time to approximately five minutes, droplet generation to three minutes, thermal cycling to less than five minutes, and droplet reading to approximately three minutes.
For the Med validation study, the team performed UR-ddPCR in a room adjoining an operating room while a surgeon was performing resection at the margins for brain tumor patients.
Specifically, the team ran two ddPCR assays — IDH1 R132H and BRAF V600E — to detect clonal mutations in gliomas and melanomas, respectively.
They also combined UR-ddPCR to calculate tumor cell percentages. When coupled with a rapid stimulated Raman histology technique pioneered by Orringer, the team was able to intraoperatively estimate tumor cell densities ranging from greater than 1,300 tumor cells per square millimeter within a tumor core to fewer than 5 tumor cells/mm2 at the tumor margins.
Overall, the team tested 78 samples during a total of 22 surgeries.
The "tissue-to-results" time averaged 15 minutes and 20 seconds when profiling one sample at a time, and 27 minutes and 25 seconds when testing four samples in parallel, according to the study.
For 70 individually processed IDH1 R132H assays, the average time was 14 minutes and 39 seconds, while for eight individually processed BRAF V600E assays, the average time was 17 minutes and 7 seconds.
Meanwhile, the additional cost compared to standard ddPCR was minimal, Evrony noted, in the range of a few dollars per sample for the extra reagents and stainless steel capillary tubes.
Evrony said his team focused on droplet-based technologies for this work, and Bio-Rad provided reagents and an instrument but otherwise did not play any role in the research.
Although rapid intraoperative targeted sequencing approaches have also been developed, for the iterative testing needed in the resection of tumor borders, they would still be too slow at roughly 30 minutes. And, the library prep in particular is difficult to speed up, Evrony said. Intraoperative methods using qPCR, CRISPR assays, and methylation profiling are likewise slower and less precise than the team's UR-ddPCR approach.
In addition to resections using known hot-spot mutations, Evrony also noted that V600E can also be used to discover cases called papillary craniopharyngiomas that might be treated with BRAF and MEK inhibitors. It isn't known whether a tumor has these mutations, but with UR-ddPCR, "the surgeon can do a small biopsy, see if it is, and if it is then they can basically stop the surgery," he said.
The team has filed a provisional patent on the method and hopes to commercialize it in the future after further streamlining and automating the process, potentially using microfluidic chips. They also expect to begin a clinical trial soon to test whether giving the UR-ddPCR results to surgeons during surgeries can better guide the operations.