Real-time PCR-based intraoperative molecular boundary diagnosis of corticotroph pituitary neuroendocrine tumors

Corticotroph pituitary neuroendocrine tumors (PitNETs) causing Cushing's disease require complete surgical resection to achieve endocrinological remission. Although endoscopic transsphenoidal surgery is the standard first-line treatment, accurate intraoperative delineation of tumor boundaries remains challenging because the pseudocapsule is often thin or ill-defined and tumor cells may infiltrate adjacent tissues. Given that intraoperative histopathology and conventional molecular methods are limited by time constraints, rapid molecular detection of tumor-specific mutations may serve as an intraoperative surrogate marker for tumor cell presence.

We employed a microfluidic real-time PCR platform (GeneSoC®) capable of detecting mutations within a short timeframe for intraoperative molecular boundary diagnosis of corticotroph PitNETs.

Sanger sequencing of archival frozen samples identified the USP8 P720R mutation, which was used to validate the real-time PCR assay. Using a fluorescence intensity cutoff value of 20, real-time PCR reliably discriminated mutation-positive from mutation-negative samples and detected mutations at variant allele frequencies of ≥ 2%. Intraoperatively, four samples obtained from different anatomical sites were analyzed, and real-time PCR detected the USP8 P720R mutation in all samples.

Real-time PCR rapidly provided diagnostic accuracy comparable to that of immunohistochemistry, Sanger sequencing, and droplet digital PCR. In one sample, the real-time PCR result was discordant with the hematoxylin and eosin staining findings.

Real-time PCR may serve as a rapid and objective method for intraoperative molecular boundary assessment of corticotroph PitNETs, although further validation is required. This method could potentially be extended to other tumors harboring detectable genetic mutations.