Nonlinear microscopy for detection of prostate cancer: analysis of sensitivity and specificity in radical prostatectomies

Intraoperative evaluation of specimens during radical prostatectomy using frozen sections can be time and labor intensive. Nonlinear microscopy (NLM) is a fluorescence microscopy technique that can rapidly generate images that closely resemble H&E histology in freshly excised tissue, without requiring freezing or microtome sectioning. Specimens are stained with nuclear and cytoplasmic/stromal fluorophores, and nonlinear microscopy evaluation can begin within 3 minutes of grossing. Fluorescence signals can be displayed using an H&E color scale, facilitating pathologist interpretation. This study evaluates the accuracy of prostate cancer detection in blinded reading of nonlinear microscopy images compared to the gold-standard of formalin fixed, paraffin embedded H&E histology. A total of 122 freshly excised prostate specimens were obtained from 40 patients undergoing radical prostatectomy. The prostates were grossed, dissected into specimens of ~10×10 mm with 1–4 mm thickness, stained for 2 minutes for nuclear and cytoplasmic/stromal contrast, and then rinsed with saline for 30 seconds. Nonlinear microscopy images were acquired and multiple images were stitched together to generate large field of view, centimeter-scale digital images suitable for reading. Specimens were then processed for standard paraffin H&E. The study protocol consisted of training, pre-testing, and blinded reading phases. After a washout period, pathologists read corresponding paraffin H&E slides. Three pathologists achieved a 95% or greater sensitivity with 100% specificity for detecting cancer on nonlinear microscopy compared to paraffin H&E. Pooled sensitivity and specificity was 97.3% (93.7%−99.1%; 95% confidence interval) and 100.0% (97%−100%), respectively. Interobserver agreement for nonlinear microscopy reading had a Fleiss κ=0.95. The high cancer detection accuracy and rapid specimen preparation suggest that nonlinear microscopy may be useful for intraoperative evaluation in radical prostatectomy.