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Updated: Dec 9, 2025
Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens
Published on: January 21, 2019
Multiplex Immunofluorescence in Formalin-Fixed Paraffin-Embedded Tumor Tissue to Identify Single-Cell-Level PI3K
Konrad H Stopsack1,2, Ying Huang3, Svitlana Tyekucheva4,5
1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.
Purpose:
Identifying cancers with high PI3K pathway activity is critical for treatment selection and eligibility into clinical trials of PI3K inhibitors. Assessments of tumor signaling pathway activity need to consider intratumoral heterogeneity and multiple regulatory nodes.
Experimental Design:
We established a novel, mechanistically informed approach to assessing tumor signaling pathways by quantifying single-cell-level multiplex immunofluorescence using custom algorithms. In a proof-of-concept study, we stained archival formalin-fixed, paraffin-embedded (FFPE) tissue from patients with primary prostate cancer in two prospective cohort studies, the Health Professionals Follow-up Study and the Physicians' Health Study. PTEN, stathmin, and phospho-S6 were quantified on 14 tissue microarrays as indicators of PI3K activation to derive cell-level PI3K scores.
Results:
In 1,001 men, 988,254 tumor cells were assessed (median, 743 per tumor; interquartile range, 290-1,377). PI3K scores were higher in tumors with PTEN loss scored by a pathologist, higher Gleason grade, and a new, validated bulk PI3K transcriptional signature. Unsupervised machine-learning approaches resulted in similar clustering. Within-tumor heterogeneity in cell-level PI3K scores was high. During long-term follow-up (median, 15.3 years), rates of progression to metastases and death from prostate cancer were twice as high in the highest quartile of PI3K activation compared with the lowest quartile (hazard ratio, 2.04; 95% confidence interval, 1.13-3.68).
Conclusions:
Our novel pathway-focused approach to quantifying single-cell-level immunofluorescence in FFPE tissue identifies prostate tumors with PI3K pathway activation that are more aggressive and may respond to pathway inhibitors.
