Nuclear Ep-ICD accumulation predicts aggressive clinical course in early stage breast cancer patients

Abstract Background Regulated intramembrane proteolysis of Epithelial cell adhesion molecule (EpCAM) results in release of its intracellular domain (Ep-ICD) which triggers oncogenic signalling. The clinical significance of Ep-ICD in breast cancer remains to be determined. Herein, we examined the expression of nuclear and cytoplasmic Ep-ICD, and membranous extracellular domain of EpCAM (EpEx) in breast cancer patients, to determine its potential utility in predicting aggressive clinical course of the disease. Methods In this retrospective study, 266 breast cancers and 45 normal breast tissues were immunohistochemically analyzed to determine the expression patterns of nuclear and cytoplasmic Ep-ICD and membranous EpEx and correlated with clinicopathological parameters and follow up. Disease-free survival was determined by Kaplan-Meier method and multivariate Cox regression analysis. Results Nuclear Ep-ICD was more frequently expressed in breast cancers compared to normal tissues. Significant association was observed between increased nuclear Ep-ICD expression and reduced disease-free survival in patients with ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) (p < 0.001). Nuclear Ep-ICD was positive in all the 13 DCIS and 25 IDC patients who had reduced disease-free survival, while none of the nuclear Ep-ICD negative DCIS or IDC patients had recurrence during the follow up period. Notably, majority of IDC patients who had recurrence had early stage tumors. Multivariate Cox regression analysis identified nuclear Ep-ICD as the most significant predictive factor for reduced disease-free survival in IDC patients (p = 0.011, Hazard ratio = 80.18). Conclusion Patients with nuclear Ep-ICD positive breast cancers had poor prognosis. The high recurrence of disease in nuclear Ep-ICD positive patients, especially those with early tumor stage suggests that nuclear Ep-ICD accumulation holds the promise of identifying early stage patients with aggressive disease who are likely to be in need of more rigorous post-operative surveillance and/or treatment

Biotinidase is a novel marker for papillary thyroid cancer aggressiveness.

Biotinidase was identified in secretome analysis of thyroid cancer cell lines using proteomics. The goal of the current study was to analyze the expression of biotinidase in thyroid cancer tissues and fine needle aspiration (FNA) samples to evaluate its diagnostic and prognostic potential in thyroid cancer. Immunohistochemical analysis of biotinidase was carried out in 129 papillary thyroid cancer (PTC, 34 benign thyroid tissues and 43 FNA samples and correlated with patients' prognosis. Overall biotinidase expression was decreased in PTC compared to benign nodules (p = 0.001). Comparison of aggressive and non-aggressive PTC showed decrease in overall biotinidase expression in the former (p = 0.001). Loss of overall biotinidase expression was associated with poor disease free survival (p = 0.019, Hazards ratio (HR) = 3.1). We examined the effect of subcellular compartmentalization of nuclear and cytoplasmic biotinidase on patient survival. Decreased nuclear expression of biotinidase was observed in PTC as compared to benign tissues (p<0.001). Upon stratification within PTC, nuclear expression was reduced in aggressive as compared to non-aggressive tumors (p<0.001). Kaplan-Meier survival analysis showed significant association of loss of nuclear biotinidase expression with reduced disease free survival (p = 0.014, HR = 5.4). Cytoplasmic biotinidase expression was reduced in aggressive thyroid cancers in comparison with non-aggressive tumors (p = 0.002, Odds ratio (OR) = 0.29) which was evident by its significant association with advanced T stage (p = 0.003, OR = 0.28), nodal metastasis (p<0.001, OR = 0.16), advanced TNM stage (p<0.001, OR = 0.21) and extrathyroidal extension (p = 0.001, OR = 0.23). However, in multivariate analysis extrathyroidal extension emerged as the most significant prognostic marker for aggressive thyroid carcinomas (p = 0.015, HR = 12.8). In conclusion, loss of overall biotinidase expression is a novel marker for thyroid cancer aggressiveness

Nuclear Ep-ICD Expression Is a Predictor of Poor Prognosis in “Low Risk” Prostate Adenocarcinomas

<div><p>Introduction</p><p>Molecular markers for predicting prostate cancer (PCa) that would have poor prognosis are urgently needed for a more personalized treatment for patients. Regulated intramembrane proteolysis of Epithelial cell adhesion molecule results in shedding of the extracellular domain (EpEx) and release of its intracellular domain (Ep-ICD) which triggers oncogenic signaling and might correlate to tumor aggressiveness. This study aimed to explore the potential of Ep-ICD and EpEx to identify PCa that have poor prognosis.</p><p>Methods</p><p>Immunohistochemical analysis of Ep-ICD and EpEx was carried out in normal prostate tissues (n = 100), benign prostate hyperplasia (BPH, n = 83), and prostate cancer (n = 249) using domain specific antibodies. The expression of Ep-ICD and EpEx was correlated with clinico- pathological parameters and disease free survival (DFS).</p><p>Results</p><p>Reduced expression of nuclear Ep-ICD and membrane EpEx was observed in PCa in comparison with BPH and normal prostate tissues (p = 0.006, p < 0.001 respectively). For patients who had PCa with Gleason Score less than 7, preserved nuclear Ep-ICD emerged as the most significant marker in multivariate analysis for prolonged DFS, where these patients did not have recurrence during follow up of up to 12 years (p = 0.001).</p><p>Conclusion</p><p>Reduced expression of nuclear Ep-ICD was associated with shorter disease free survival in patients with a Gleason Score less than 7 and may be useful in identifying patients likely to have aggressive tumors with poor prognosis. Furthermore, nuclear Ep-ICD can differentiate between normal and prostate cancer tissues for ambiguous cases.</p></div

Transglutaminase 2 Overexpression in Tumor Stroma Identifies Invasive Ductal Carcinomas of Breast at High Risk of Recurrence

<div><p>Introduction</p><p>Molecular markers for predicting breast cancer patients at high risk of recurrence are urgently needed for more effective disease management. The impact of alterations in extracellular matrix components on tumor aggressiveness is under intense investigation. Overexpression of Transglutaminase 2 (TG2), a multifunctional enzyme, in cancer cells impacts epithelial mesenchymal transition, growth, invasion and interactions with tumor microenvironment. The objective of our study is to determine the clinical relevance of stromal TG2 overexpression and explore its potential to identify breast cancers at high risk of recurrence.</p><p>Methods</p><p>This retrospective study is based on immunohistochemical analysis of TG2 expression in normal breast tissues (n = 40) and breast cancers (n = 253) with clinical, pathological and follow-up data available for up to 12 years. TG2 expression was correlated with clinical and pathological parameters as well as disease free survival (DFS) of breast cancer patients.</p><p>Results</p><p>Stromal TG2 overexpression was observed in 114/253 (45.0%) breast cancer tissues as compared to breast normal tissues. Among invasive ductal carcinomas (IDC) of the breast, 97/168 (57.7%) showed strong TG2 expression in tumor stroma. Importantly, IDC patients showing stromal TG2 accumulation had significantly reduced DFS (mean DFS = 110 months) in comparison with patients showing low expression (mean DFS = 130 months) in Kaplan-Meier survival analysis (p<0.001). In Cox multivariate regression analysis, stromal TG2 accumulation was an independent risk factor for recurrence (p = 0.006, Hazard’s ratio, H.R. = 3.79). Notably, these breast cancer patients also showed immunostaining of N-epsilon gamma-glutamyl lysine amino residues in tumor stroma demonstrating the transamidating activity of TG2.</p><p>Conclusions</p><p>Accumulation of TG2 in tumor stroma is an independent risk factor for identifying breast cancer patients at high risk of recurrence. TG2 overexpression in tumor stroma may serve as a predictor of poor prognosis for IDC of the breast.</p></div

Immunohistochemical analysis of Ep-ICD and EpEx in prostate tissues.

<p>Panel shows (a) Nuclear/Cytoplasmic Ep-ICD immunostaining in (I) Normal, (II) BPH, and Prostate Adenocarcinoma: (III) Gleason Score < 7, (IV) Gleason Score = 7; and (V) Gleason Score > 7. Panel (b) Membrane EpEx immunostaining in (I) Normal (II) BPH, and Prostate Adenocarcinoma (III) Gleason Score < 7, (IV) Gleason Score = 7; and (V) Gleason Score > 7. Arrows show nuclear (N) Ep-ICD staining and Membrane (M) EpEx staining (Original Magnification x 400).</p

Kaplan Meier survival analysis of Ep-ICD and EpEx in prostate cancer patients.

<p>Kaplan-Maier analysis was performed dividing the patients as biomarker (Ep-ICD nuclear, Membrane EpEx) positive (green) and negative (blue). These were then further stratified by Gleason score. All the differences were significant and p-values are reported in figure as well as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107586#pone.0107586.t002" target="_blank">Table 2</a>.</p

Analysis of Ep-ICD and EpEx expression in BPH and prostate cancer and correlation with clinic-pathological parameters.

<p>Analysis of Ep-ICD and EpEx expression in BPH and prostate cancer and correlation with clinic-pathological parameters.</p

Kaplan Meier Survival Analysis.

<p><b>Panel</b> (<b>a</b>) Kaplan Meier survival analysis in breast cancer patients. Panel shows breast cancer patients with TG2 overexpression in tumor stroma have significantly reduced DFS (mean DFS = 112 months, p = 0.002) as compared to patients with low or no detectable expression of stromal TG2 (mean DFS = 127 months). <b>Panel</b> (<b>b</b>) shows patients with IDCs of the breast demonstrating stromal TG2 overexpression have reduced DFS (mean DFS = 110 months, p<0.001) in comparison with patients showing lower TG2 expression in tumor stroma (mean DFS = 130 months).</p

Study scheme of patient cohort.

<p>Patient cohort details are listed in a hierarchy manner.</p