Update on Intraoperative Ultra-Rapid PCR for BRAF Mutation Detection in Craniopharyngioma

Recent molecular studies have shown that approximately 90% of papillary craniopharyngioma contain BRAF V600E mutations. A phase II trial of BRAF V600E+ papillary craniopharyngioma treated with BRAF/MEK inhibitors showed 94% response rate and 91% tumor volume reduction. During resection of craniopharyngioma, disruption of the pituitary/hypothalamic axis is often inevitable, requiring hormone replacement. Determination of tumor genotype prior to surgical resection can alter the available treatments. Despite interest in developing “liquid-biopsies” for noninvasive preoperative detection and diagnosis of CNS tumors, results thus far have been unsuccessful. There is considerable interest in bringing molecular grade diagnostics to the operating room for intraoperative use. However, traditional molecular testing is time-intensive and unable to produce results quickly enough to make decisions in real-time. The development of ultra-rapid droplet digital PCR testing can provide accurate information on a tumor’s mutational status within a span of 15-20 minutes. Methods: Bead homogenization in the presence of a detergent-free DNA extraction buffer was used to lyse the cells from the tissue sample. DNA was isolated from associated proteins and cellular debris. An ultra-rapid ddPCR based assay determined the mutational status intraoperatively. Histopathological analysis (H&E and immunohistochemistry) was performed on permanent pathology specimen. We present four cases of patients with craniopharyngiomas that underwent standard endoscopic endonasal approaches. Upon visualizing the tumor, biopsy was performed using cup forceps and sent for permanent pathology and an ultra-rapid BRAF V600E PCR analysis. Results: Four craniopharyngioma cases underwent endoscopic endonasal approaches for biopsy or resection. The average time to result for the droplet digital PCR testing was 17 minutes and 12 seconds. PCR findings were subsequently corroborated by standard immunohistochemical analysis with 4/4 (100%) concordance. Conclusion: Through use of an intraoperative ultra-rapid PCR, we can rapidly and accurately determine BRAF V600E mutational status, which can directly impact our clinical decision in real-time. This presents an innovative approach for intraoperative diagnosis and characterization of craniopharyngioma, with significant treatment implications. Publication History Article published online: 27 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany