Abstract
Abstract Background: The PI3K pathway is activated in a large proportion of solid cancers, including prostate cancer. Loss of its inhibitor PTEN is associated with higher risk of lethal prostate cancer. However, clinical trials of PI3K and Akt inhibitors have had mixed results in prostate cancer, likely because patients were not selected for PI3K pathway activation. Here, we established a novel assessment of PI3K pathway activity on a single-cell level. Methods: Formalin-fixed, paraffin-embedded tumor tissue from patients diagnosed with primary prostate cancer during prospective follow-up of the Health Professionals Follow-up Study (HPFS) and the Physicians' Health Study (PHS) underwent centralized re-review by genitourinary pathologists. Tissue microarrays (TMAs) were sequentially stained for PTEN, stathmin, and phospho-S6 (pS6) as indicators of PI3K activation, and for AMACR. Slides were scanned on Vectra 3 (PerkinElmer), followed by multispectral imaging analysis. Tumor cells were identified using histology-based machine learning and AMACR staining intensity. Software for visualization of histomorphology and marker colocalization was developed. Cell-level PI3K activation scores were defined using a predefined analytical approach and compared to a data-driven approach. An independent, genomically validated core-level immunohistochemistry for PTEN loss and an mRNA signature of 105 PI3K/Akt/mTOR target genes served for validation. Patients were followed prospectively for lethal disease (metastases and cancer-specific death). Odds ratios (OR) with 95% confidence intervals (CIs) were estimated using logistic regression. Results: In 1,103 patients (719 from HPFS, 384 from PHS), 1.05 million tumor cells were assessed on 14 TMAs (median, 669 tumor cells per patient; interquartile range, 208 to 1362). Visualizations revealed intratumoral heterogeneity in PI3K activation in some tumors. Predefined and data-driven approaches to quantifying PI3K activity had similar results, with cell-level PI3K scores being driven by higher stathmin and pS6 and lower cell-level PTEN intensity. PI3K scores were higher in tumors with higher Gleason grade (ptrend < 0.001), core-level PTEN loss (p < 0.001; n = 866), and a higher transcriptional signature (ptrend = 0.025; n = 295). Over a median follow-up of 12.5 years with 95 lethal events, tumors in the highest quartile of PI3K activation were more than twice as likely to become lethal (OR, 2.56; 95% CI, 1.40–4.66; ptrend < 0.001) than those in the lowest quartile, even after adjusting for core-level PTEN status and Gleason grade (OR, 2.27; 95% CI, 1.01–5.08). Conclusion: Single-cell multiplex assessment of immunofluorescence PI3K markers can validly identify tumors with high activity of the PI3K pathway and may harbor more information than PTEN status alone. It provides a scalable measure that can be applied to routine specimens, including archival tissue from observational studies, and that can be used for patient selection into clinical trials of PI3K inhibitors. Citation Format: Konrad H. Stopsack, Ying Huang, Svitlana Tyekucheva, Travis A. Gerke, Clyde Bango, Habiba Elfandy, Xueliang Gao, Zhenying Cai, Thomas M. Roberts, Philip W. Kantoff, Lorelei A. Mucci, Massimo Loda. Single-cell multiplex immunofluorescence of formalin-fixed, paraffin-embedded prostate cancer tissue identifies PI3K pathway activation in two prospective cohort studies [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B31.
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