Nuclear and Cytoplasmic Accumulation of Ep-ICD Is Frequently Detected in Human Epithelial Cancers

BACKGROUND: We previously demonstrated that nuclear and cytoplasmic accumulation of the intracellular domain (Ep-ICD) of epithelial cell adhesion molecule (EpCAM) accompanied by a reciprocal reduction of its extracellular domain (EpEx), occurs in aggressive thyroid cancers. This study was designed to determine whether similar accumulation of Ep-ICD is a common event in other epithelial cancers. METHODOLOGY AND RESULTS: Ten epithelial cancers were immunohistochemically analyzed using Ep-ICD and EpEx domain-specific antibodies. The subcellular localization of EpEx and Ep-ICD in the human colon adenocarcinoma cell line CX-1 was observed using immunofluorescence. Nuclear and cytoplasmic Ep-ICD expression was increased in cancers of the breast (31 of 38 tissues, 82%), prostate (40 of 49 tissues, 82%), head and neck (37 of 57 tissues, 65%) and esophagus (17 of 46 tissues, 37%) compared to their corresponding normal tissues that showed membrane localization of the protein. Importantly, Ep-ICD was not detected in the nuclei of epithelial cells in most normal tissues. High nuclear and cytoplasmic Ep-ICD accumulation also occurred in the other six epithelial cancer types analyzed - lung, colon, liver, bladder, pancreatic, and ovarian. A concomitant reduction in membrane EpEx expression was observed in a subset of all cancer types. Receiver operating characteristic curve analysis revealed nuclear Ep-ICD distinguished breast cancers with 82% sensitivity and 100% specificity and prostate cancers with 82% sensitivity and 78% specificity. Similar findings were observed for cytoplasmic accumulation of Ep-ICD in these cancers. We provide clinical evidence of increased nuclear and cytoplasmic Ep-ICD accumulation and a reduction in membranous EpEx in these cancers. CONCLUSIONS: Increased nuclear and cytoplasmic Ep-ICD was observed in all epithelial cancers analyzed and distinguished them from normal tissues with high-sensitivity, specificity, and AUC. Development of a robust high throughput assay for Ep-ICD will facilitate the determination of its diagnostic, prognostic and therapeutic relevance in epithelial cancers

Proteomics of Thyroid Carcinoma: Detection of Potential Biomarkers of Aggressive and Non-aggressive Subtypes

In search of thyroid carcinoma biomarkers, proteins secreted by thyroid cancer cell lines, papillary-derived TPC-1 and anaplastic-derived CAL62, were analyzed using liquid chromatography-tandem mass spectrometry. Of forty six high-confidence identifications, six proteins were considered for verification in thyroid cancer patients’ tissues and blood. The localization of two proteins, nucleolin and prothymosin-alpha (PTMA), was confirmed in TPC-1 and CAL62 by confocal microscopy and immunohistochemically in xenografts of TPC-1 cells and human thyroid carcinomas. Increased nuclear and cytoplasmic expression of PTMA was observed in anaplastic carcinomas compared to normal thyroid tissues, papillary and poorly differentiated carcinomas. Importantly, six proteins were detected in thyroid cancer patients’ sera, warranting future analysis to confirm their potential as blood-based thyroid cancer markers. Herein we demonstrate the ability of secretome analysis of thyroid cancer cell lines to identify proteins that may be studied for application in management of thyroid carcinomas upon future validation.MAS

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

An Ep-ICD based index is a marker of aggressiveness and poor prognosis in thyroid carcinoma.

BACKGROUND:Nuclear accumulation of the intracellular domain of epithelial cell adhesion molecule (Ep-ICD) in tumor cells was demonstrated to predict poor prognosis in thyroid carcinoma patients in our earlier study. Here, we investigated the clinical significance of Ep-ICD subcellular localization index (ESLI) in distinguishing aggressive papillary thyroid carcinoma (PTC) from non-aggressive cases. METHODS:Using domain specific antibodies against the intracellular (Ep-ICD) and extracellular (EpEx) domains of epithelial cell adhesion molecule, 200 archived tissues from a new cohort of patients with benign thyroid disease as well as malignant aggressive and non aggressive PTC were analyzed by immunohistochemistry (IHC). ESLI was defined as sum of the IHC scores for accumulation of nuclear and cytoplasmic Ep-ICD and loss of membranous EpEx; ESLI = [Ep-ICD(nuc) + Ep-ICD(cyt) + loss of membranous EpEx]. RESULTS:For the benign thyroid tissues, non-aggressive PTC and aggressive PTC, the mean ESLI scores were 4.5, 6.7 and 11 respectively. Immunofluorescence double staining confirmed increased nuclear Ep-ICD accumulation and decreased membrane EpEx expression in aggressive PTC. Receiver-operating characteristic (ROC) curve analysis showed an area under the curve (AUC) of 0.841, 70.2% sensitivity and 83.9% specificity for nuclear Ep-ICD for differentiating aggressive PTC from non-aggressive PTC. ESLI distinguished aggressive PTC from non-aggressive cases with improved AUC of 0.924, 88.4% sensitivity and 85.5% specificity. Our study confirms nuclear accumulation of Ep-ICD and loss of membranous EpEx occurs in aggressive PTC underscoring the potential of Ep-ICD and ESLI to serve as diagnostic markers for aggressive PTC. Kaplan Meier survival analysis revealed significantly reduced disease free survival (DFS) for ESLI positive (cutoff >10) PTC (p<0.05), mean DFS=133 months as compared to 210 months for patients who did not show positive ESLI. CONCLUSION:ESLI scoring improves the identification of aggressive PTC and thereby may serve as a useful index for defining aggressiveness and poor prognosis among PTC patients

Kaplan Meier survival analysis for ESLI.

<p>Kaplan Meier survival analysis showing significant association with reduced disease free survival (DFS) in ESLI positive PTC patients (p = 0.039) with a mean DFS = 133 months compared to ESLI negative patients with a mean DFS = 210 months.</p

Comparison of IHC scores for Ep-ICD, EpEx and ESLI in thyroid tissues.

<p>Mann-Whitney test <i>p</i> value for benign vs. PTC: cytoplasmic Ep-ICD <i>p</i> = 0.000; nuclear Ep-ICD <i>p</i> = 0.002 and ESLI <i>p</i> = 0.000. <i>p</i> value for Aggressive vs. Non-aggressive PTC: cytoplasmic Ep-ICD <i>p</i> = 0.000; nuclear Ep-ICD <i>p</i> = 0.000 and ESLI <i>p</i> = 0.000.</p

IHC analysis of Ep-ICD and EpEx subcellular localization in thyroid tissues.

<p>The IHC score cut-off values for positivity were defined as ≥2 for nuclear Ep-ICD positivity; ≥5 for cytoplasmic Ep-ICD positivity and ≤5 for loss of membranous EpEx expression. ESLI cut-off ≥6 was used to determine ESLI positivity for distinguishing PTC from benign cases; and a cut-off value of >10 was used to determine ESLI positivity for distinguishing aggressive PTCs from non-aggressive PTCs.</p

Immunohistochemical analysis of EpEx and Ep-ICD expression in papillary thyroid carcinomas and benign tissues.

<p>The representative photomicrographs show immunostaining of EpEx and Ep-ICD in paraffin-embedded thyroid benign nodule goiters, non-aggressive PTC and aggressive PTC tissues. Strong membranous EpEx immunostaining was observed in benign cases (A) and non-aggressive PTC tissues (C); reduced staining of membrane EpEx was observed in aggressive PTC cases (E, G). The benign thyroid nodules and non-aggressive PTC (D) showed predominant cytoplasm localization of Ep-ICD and no detectable nuclear Ep-ICD staining (B, D), while the aggressive PTC cases showed strong nuclear and cytoplasmic Ep-ICD accumulation (F, H). M, membrane staining; C, cytoplasmic staining; N, nuclear staining; Loss of M, loss of membrane expression. Original magnification × 400.</p

Clinicopathological characteristics of thyroid carcinoma patients.

<p>Clinicopathological characteristics of thyroid carcinoma patients.</p

Fluorescence immunostaining with anti-EpEx and anti-Ep-ICD antibodies in aggressive and non-aggressive papillary thyroid carcinomas.

<p>Secondary antibodies are FITC-anti-mouse (green) and TRITC-anti-rabbit (red). A-F images from a non-aggressive PTC; G-L Images from an aggressive PTC. A,G) EpEx; B,H) DAPI; C, I) Ep-ICD; D) EpEx and DAPI (A & C merged); E) Ep-ICD and DAPI (B & C merged); F) EpEx, Ep-ICD, and DAPI (A, B, C merged). J) EpEx and DAPI (G & I merged); K) Ep-ICD and DAPI (H & I merged); L) EpEx, Ep-ICD, and DAPI (G, H, I merged). M, Membranous staining; C, Cytoplasm staining; N, Nuclear staining. Original magnification × 400.</p