Digital spatial profiling identifies phospho-JNK as a biomarker for early risk stratification of aggressive prostate cancer

Abstract

Background: Prostate cancer (PCa) is a highly heterogeneous disease, ranging from indolent to highly aggressive forms. Ongoing research focuses on identifying new biomarkers to improve early risk stratification in PCa, addressing current limitations to accurately evaluate disease progression. A promising new approach to aid PCa risk stratification is digital spatial profiling (DSP) of PCa tissue.

Methods: A total of 94 regions of interest from 38 PCa patients at first diagnosis were analyzed for the expression of 44 proteins, including components of the PI3K/AKT, MAPK, and cell death signaling pathways as well as immune cell markers. An additional validation cohort consisting of 154 PCa patients with long-term follow-up data was analyzed using immunohistochemistry (IHC) to assess the consistency of the identified biomarkers across a larger sample set.

Results: DSP identified the proliferation marker Ki-67 and phosphorylated c-Jun N-terminal protein kinase T183/Y185 (p-JNK), a member of the MAPK signaling pathway, as significantly upregulated proteins in aggressive PCa (Gleason grades 4 or 5) compared to indolent disease (Gleason grade 3; p<0.05). The upregulation of p-JNK was confirmed by IHC. High p-JNK expression was associated with a shorter time to biochemical recurrence (log-rank, p=0.1).

Conclusion: Our results indicate that p-JNK may contribute to PCa progression and serve as an early biomarker for aggressive PCa stratification. Identification of this biomarker through DSP could prove crucial in advancing disease management and addressing the critical unmet need for more targeted therapies in the treatment of PCa. Further studies are warranted to evaluate the role of p-JNK in PCa progression.

Keywords: biomarker discovery; digital spatial profiling (DSP); p-JNK; prostate cancer; risk stratification.

Figure 1

Representative GeoMx^® DSP scan of PCa specimen Gleason 3 and 4 TMA cores stained with morphology markers SYTO13, CD45 and PanCK along with H&E staining. Scale bar = 250 µm.

Figure 2

(A) Unsupervised clustering and heatmap of DSP protein expression data obtained from 94 Gleason grade ROIs from 38 PCa patients. Data normalized to house keeper GAPDH & S6. (B) Heatmap visualization of target protein expression across patient samples ordered by Gleason score.

Figure 3

(A) Volcano plot of 44 differentially expressed target proteins in 94 ROIs comparing Gleason 3 to Gleason 4 and 5. The vertical dotted lines represent the log₂(fold change) of 0.5 and − 0.5. The horizontal dotted line shows the adjusted p-value (Benjamini-Hochberg) < 0.1. Significantly upregulated proteins are p-JNK and Ki-67, highlighted in blue (p = 0.01 and 0.02, respectively). (B) A representative patient in the IHC cohort stained with p-JNK (Thr183/Tyr185). The staining intensity was scored on a scale of negative, weak, weak-moderate and moderate and used to calculate the IRS of p-JNK. Scale bar = 100 µm. (C) Histogram showing the distribution of p-JNK IRS in the validation cohort of 154 patients. (D) Kaplan–Meier curve for 154 patients of the validation cohort, stratified into patients with high (> 4.5; n = 24) or low (≤ 4.5; n = 130) p-JNK IRS.

Figure 4

Combined analysis of p-JNK status with pathology-derived parameters. (A) p-JNK status combined with Gleason score. (B) p-JNK status combined with T stage. (C) p-JNK status combined with both Gleason score and T stage. p-JNK expression was stratified into high and low according to the immunoreactive score (cutoff >4.5).