Ultra-rapid droplet digital PCR enables intraoperative tumor quantification.

Abstract

Background: The diagnosis and treatment of tumors often depend on molecular-genetic data. However, rapid and iterative access to molecular data is not currently feasible during surgery, complicating intraoperative diagnosis and precluding measurement of tumor cell burdens at surgical margins to guide resections.

Methods: Here, we introduce Ultra-Rapid droplet digital PCR (UR-ddPCR), a technology that achieves the fastest measurement, to date, of mutation burdens in tissue samples, from tissue to result in 15 min. Our workflow substantially reduces the time from tissue biopsy to molecular diagnosis and provides a highly accurate means of quantifying residual tumor infiltration at surgical margins.

Findings: We demonstrate UR-ddPCR assays for the IDH1 R132H and BRAF V600E clonal mutations that are present in many low-grade gliomas and melanomas, respectively, and whose intraoperative detection would shape surgical decision-making. We illustrate the clinical feasibility of UR-ddPCR by performing it intraoperatively for 22 brain tumor cases, and we further combine UR-ddPCR tumor cell percentage measurements with UR-stimulated Raman histology intraoperatively to estimate tumor cell densities ranging from >1,300 tumor cells/mm² within a tumor core to <5 tumor cells/mm² at tumor margins. UR-ddPCR measurements were virtually identical to standard ddPCR measurements performed on the same samples (R² = 0.995).

Conclusions: The technology and workflow developed here enable intraoperative molecular-genetic assays with unprecedented speed and sensitivity. We anticipate that our method will facilitate novel point-of-care diagnostics and molecularly guided surgeries that improve clinical outcomes.

Funding: This study was funded by the National Institutes of Health and NYU Grossman School of Medicine institutional funds. Reagents and instruments were provided in kind by Bio-Rad.