Patients and clinicians are in need of more accurate biomarkers to predict the prognosis of prostate cancer, especially for intermediate grade tumors . Few markers have been reported that reliably predict treatment failure (e.g. PSA recurrence after surgery). Altered nuclear shape and size are a hallmark of cancer and reflect changes in chromatin structure. Image analyses of heterochromatin content and nuclear shape have been reported to improve the prediction of PCa prognoses . HP1γ is a member of the HPI family of proteins involved in chromatin packaging and gene regulation. The current study represents the first in depth analysis of HP1γ expression for any cancer. We observe an increase in HP1γ expression in the majority of prostate cancers. Furthermore, by analyzing staining in distinct cellular compartments in a cohort of patients with extended follow-up, we find a role for the expression of this gene in predicting treatment failure.
A unique strength of this study lies in our use of the newly validated VECTRA™ imaging technology. The platform merges automated slide scanning, multi-spectral imaging technology, and pattern recognition software into a system for biomarker analysis. VECTRA™ has many advantages over other imaging systems for biomarker quantitation . VECTRA™ allows researchers to objectively analyze expression within separate epithelial and stromal tissue compartments. Nuclear and cytoplasmic staining within these compartments is concurrently assessed and multiple markers may be addressed simultaneously. The system is highly automated and uses pattern recognition to measure expression on a per-cell, core, or compartment basis on TMAs. The algorithm designed to segment tissue compartments by our genitourinary pathologist [WH] had a 97% rate of acceptable tissue segmentation. In the current analysis, VECTRA™ permitted a reproducible assessment of HP1γ, and its association with Ki-67, in multiple cell compartments for a large cohort of patients.
HP1γ and the other HP1 family members have been primarily studied during embryogenesis and development, including that of the prostate . Expression of HP1γ was noted in the fetal prostate at 14 and 24 weeks of gestation. This is in contrast to HP1α which is not expressed at those developmental stages . Decreased expression of HP1γ is required for normal cellular differentiation [5, 6]. Our study demonstrates that expression of HP1γ occurs in 98% of metastatic and 76% of localized PCa tumors compared to 26% of benign tissues. This cut-off was determined by ROC analysis to highlight the marked differences between patients with benign versus cancer. Adjusting this cut point for Hp1-gamma overexpression can increase the sensitivity or specificity depending on its required use. Total expression was associated with higher Gleason score (p = 0.04).
One notable finding was that HP1γ was not only an independent predictor of PSA-recurrence for patients who underwent radical prostatectomy, but was superior to pathological Gleason score using both a multivariate Cox model and a Kaplan-Meier analysis (Table 3 and Figure 4). Few biomarkers to date are able to predict PSA recurrence more robustly than Gleason score. Recently, Cuzick et al. reported a panel of 31 RNA markers had a more significant P value compared to Gleason score for predicting PSA recurrence . One explanation for the robustness of HP1γ is that its prognostic value is not collinearly associated with other clinicopathologic variables (Table 1). Given the role HP1γ plays in gene repression [3, 21] and development , we postulate that aberrant expression of this protein may be central to the pathophysiology of PCa.
Increased expression of HP1γ in the nucleus compared to the cytoplasm is consistent with data demonstrating this protein is primarily localized to centromeric and telomeric heterochromatin [3, 8] and euchromatin [22, 23]. It was of interest to find expression of the protein in the cytoplasm and note that this carried prognostic significance. Its presence in the cytoplasm may represent altered protein processing, or may have some as of yet unknown functional significance. Ki-67 is a nuclear protein that correlates with cell proliferation and is a known marker of PCa progression . The C-terminal domain has been found to bind to all three members of the HP1 family . We found a significant association between HP1γ and Ki-67 on a per core basis (Figure 2). Given the more uniform expression of HP1γ across prostate cancer cells when compared to Ki-67, it clearly has other functions independent from Ki-67 in the regulation of heterochromatin.