3D Structural Revelations of Neoadjuvant Hormonal Therapy Treatment in High Risk Localized Prostate Cancer by Light-Sheet Imaging.

Abstract

e17124 Background: Neoadjuvant hormonal therapy for high-risk (Gleason score ≥ 8), high volume localized prostate cancer produces heterogeneous architectural changes that are difficult to assess on 2D histology. We assessed whether three-dimensional (3D) light-sheet imaging may add additional qualitative information for assessing Minimal Residual Disease (MRD) after neoadjuvant treatment. Methods: Ten patients of the neoadjuvant GUNS clinical trial (NCT04812366) receiving 24 weeks of doublet hormonal therapy (LHRHa + APA) contributed paired pre-treatment biopsies (n = 10) and post-treatment radical prostatectomy (RP) tissue (n = 10). FFPE tissues were cleared using a modified iDISCO protocol and stained with an H&E fluorescent analogue (To-Pro-3 for nuclei, eosin for stroma). Stained and cleared tissues were imaged on the Aurora 3Di light-sheet microscope and reviewed in 3D and 2D by a genitourinary pathologist. Qualitative features recorded included tumor volume, cellular and acinar connectivity, stromal remodeling, and degenerative changes. Results: High-volume carcinoma in biopsies frequently corresponded to highly connected residual carcinoma in RP specimens (cases 007, 021, 030, 046, 047). Several cases demonstrated reduced tumor volume with 3D structure still being highly connected (056, 070), while others showed predominantly isolated single cells with minimal connectivity (033, 034) only detectable in 3D. Similar to cellular connectivity, structural connectivity of tumor acini was visible as an interconnected angulated tubular system in 3D while appearing as discreet cavities in 2D imaging . One RP specimen (061) showed no residual carcinoma in 3D or 2D. 3D analysis highlighted infiltrative projections, acinar connectivity and reconstruction, stromal expansion, hyalinization, and heterogeneous degeneration after neoadjuvant treatment that were underestimated or not visible in 2D histology. Correlation with 2D confirmed residual tumor and added additional details on nuclear morphology. Conclusions: 3D light-sheet microscopy revealed treatment-related structural patterns as well as residual tumor architecture and volume that were not captured by 2D histology alone. Persistent lesion connectivity, architectural fragmentation, and stromal changes were readily identified in 3D. These findings suggest the use of 3D pathology could inform post-neoadjuvant response classification in prostate cancer once future work validate quantitative metrics and correlate structural features with clinical outcomes.