Subcellular differential expression of Ep-ICD in oral dysplasia and cancer is associated with disease progression and prognosis.

BACKGROUND: Identification of patients with oral dysplasia at high risk of cancer development and oral squamous cell carcinoma (OSCC) at increased risk of disease recurrence will enable rigorous personalized treatment. Regulated intramembranous proteolysis of Epithelial cell adhesion molecule (EpCAM) resulting in release of its intracellular domain Ep-ICD into cytoplasm and nucleus triggers oncogenic signaling. We analyzed the expression of Ep-ICD in oral dysplasia and cancer and determined its clinical significance in disease progression and prognosis. METHODS: In a retrospective study, immunohistochemical analysis of nuclear and cytoplasmic Ep-ICD and EpEx (extracellular domain of EpCAM), was carried out in 115 OSCC, 97 oral dysplasia and 105 normal oral tissues, correlated with clinicopathological parameters and disease outcome over 60 months for oral dysplasia and OSCC patients. Disease-free survival (DFS) was determined by Kaplan-Meier method and multivariate Cox regression analysis. RESULTS: In comparison with normal oral tissues, significant increase in nuclear Ep-ICD and membrane EpEx was observed in dysplasia, and OSCC (p = 0.013 and < 0.001 respectively). Oral dysplasia patients with increased overall Ep-ICD developed cancer in short time period (mean = 47 months; p = 0.044). OSCC patients with increased nuclear Ep-ICD and membrane EpEx had significantly reduced mean DFS of 33.7 months (p = 0.018). CONCLUSIONS: Our study provided clinical evidence for Ep-ICD as a predictor of cancer development in patients with oral dysplasia and recurrence in OSCC patients, suggesting its potential utility in enhanced management of those patients detected to have increased risk of progression to cancer and recurrence in OSCC patients.

Nuclear heterogeneous nuclear ribonucleoprotein D is associated with poor prognosis and interactome analysis reveals its novel binding partners in oral cancer.

BACKGROUND: Post-transcriptional regulation by heterogeneous ribonucleoproteins (hnRNPs) is an important regulatory paradigm in cancer development. Our proteomic analysis revealed hnRNPD overexpression in oral dysplasia as compared with normal mucosa; its role in oral carcinogenesis remains unknown. Here in we determined the hnRNPD associated protein networks and its clinical significance in oral squamous cell carcinoma (OSCC). METHODS: Immunoprecipitation (IP) followed by tandem mass spectrometry was used to identify the binding partners of hnRNPD in oral cancer cell lines. Ingenuity pathway analysis (IPA) was carried out to unravel the protein interaction networks associated with hnRNPD and key interactions were confirmed by co-IP-western blotting. hnRNPD expression was analyzed in 183 OSCCs, 44 oral dysplasia and 106 normal tissues using immunohistochemistry (IHC) and correlated with clinico-pathological parameters and follow up data over a period of 91 months. Kaplan-Meier survival and Cox-multivariate-regression analyses were used to evaluate the prognostic significance of hnRNPD in OSCC. RESULTS: We identified 345 binding partners of hnRNPD in oral cancer cells. IPA unraveled novel protein-protein interaction networks associated with hnRNPD and suggested its involvement in multiple cellular processes: DNA repair, replication, chromatin remodeling, cellular proliferation, RNA splicing and stability, thereby directing the fate of oral cancer cells. Protein-protein interactions of hnRNPD with 14-3-3ζ, hnRNPK and S100A9 were confirmed using co-IP-western blotting. IHC analysis showed significant overexpression of nuclear hnRNPD in oral dysplasia [p = 0.001, Odds ratio (OR) = 5.1, 95% CI = 2.1-11.1) and OSCCs (p = 0.001, OR = 8.1, 95% CI = 4.5-14.4) in comparison with normal mucosa. OSCC patients showing nuclear hnRNPD overexpression had significantly reduced recurrence free survival [p = 0.026, Hazard ratio = 1.95, 95% CI = 1.0-3.5] by Kaplan-Meier survival and Cox-multivariate-regression analyses and has potential to define a high-risk subgroup among OSCC patients with nodal negative disease. CONCLUSIONS: Our findings suggest novel functions of hnRNPD in cellular proliferation and survival, besides RNA splicing and stability in oral cancer. Association of nuclear hnRNPD with poor prognosis in OSCC patients taken together with its associated protein networks in oral cancer warrant future studies designed to explore its potential as a plausible novel target for molecular therapeutics.

Prognostic significance of cytoplasmic S100A2 overexpression in oral cancer patients.

BACKGROUND: Oral squamous cell carcinoma (OSCC) patients are at high risk of loco-regional recurrence and 5-year survival rates are about 50%. Identification of patients at high risk of recurrence will enable rigorous personalized post-treatment management. Most novel biomarkers have failed translation for clinical use because of their limited successful validation in external patient cohorts. The aim of this study was to determine the prognostic significance of alterations in sub-cellular expression of S100A2, a pro-tumorigenic calcium binding protein, identified as a candidate biomarker in our proteomic analysis in OSCC and validation of its clinical utility in an external cohort. METHODS: In a retrospective study, immunohistochemical analysis of S100A2 was carried out in 235 Indian OSCC (Test set) and 129 normal oral tissues, correlated with clinicopathological parameters and disease outcome over 122 months for OSCC patients following the REMARK criteria. The findings were validated in an external cohort (Validation set 115 Canadian OSCC and 51 normal tissues) and data analyzed using the R package. RESULTS: Significant increase in cytoplasmic and decrease in nuclear S100A2 expression was observed in OSCC in comparison with normal tissues. Cox multivariable regression analysis internally and externally validated cytoplasmic S100A2 association with tumor recurrence. Kaplan Meier analysis of patients stratified to high and low risk groups showed significantly different recurrence free survival (Test set- log rank test, p = 0.005, median survival 16 and 69 months respectively and Validation set - p < 0.00001, median survival 9.4 and 59.9 months respectively); 86% and 81% of patients who had recurrence were correctly stratified into the high risk group. Seventy percent and 81% patients stratified into low risk group did not show cancer recurrence within 1 year in Test and Validation sets. CONCLUSIONS: Our study provided clinical evidence for the potential of cytoplasmic S100A2 overexpression as a predictor of recurrence risk in OSCC patients. A unique translational aspect of our study is validation of S100A2 as prognostic marker in two independent cohorts (Canadian and Indian) suggesting this protein is likely to find widespread utility in clinical practice for identifying oral cancer patients at high risk of disease recurrence.

S100A7 overexpression is a predictive marker for high risk of malignant transformation in oral dysplasia.

Early detection of oral lesions (OLs) at high risk of cancer development is of utmost importance for intervention. There is an urgent unmet clinical need for biomarkers that allow identification of high-risk OLs. Recently, we identified and verified a panel of five candidate protein biomarkers namely S100A7, prothymosin alpha, 14-3-3ζ, 14-3-3σ and heterogeneous nuclear ribonucleoprotein K using proteomics to distinguish OLs with dysplasia and oral cancers from normal oral tissues. The objective of our study was to evaluate the potential of these candidate protein biomarkers for identification of oral dysplastic lesions at high risk of cancer development. Using immunohistochemistry, we analyzed expressions of these five candidate protein biomarkers in 110 patients with biopsy-proven oral dysplasia and known clinical outcome and determined their correlations with p16 expression and HPV 16/18 status. Kaplan-Meier survival analysis showed reduced oral cancer-free survival (OCFS) of 68.6 months (p = 0.007) in patients showing cytoplasmic S100A7 overexpression when compared to patients with weak or no S100A7 immunostaining in cytoplasm (mean OCFS = 122.8 months). Multivariate Cox regression analysis revealed cytoplasmic S100A7 overexpression as the most significant candidate marker associated with cancer development in dysplastic lesions (p = 0.041, hazard ratio = 2.36). In conclusion, our study suggested the potential of S100A7 overexpression in identifying OLs with dysplasia at high risk of cancer development.

The adenovirus 55 residue E1A protein is a transcriptional activator and binds the unliganded thyroid hormone receptor.

The early region 1A (E1A) of human adenovirus types 2 and 5 is differentially spliced to yield five distinct mRNAs that encode different proteins. The smallest E1A RNA transcript encodes a 55 residue (55R) protein that shares only 28 amino acid residues with the other E1A proteins. Even though it is the most abundant E1A transcript at late times post-infection, little is known about the functions of this E1A isoform. In this study, we show that the E1A 55R protein interacts with, and modulates the activity of the unliganded thyroid hormone receptor (TR). We demonstrate that E1A 55R contains a signature motif known as the CoRNR box that confers interaction with the unliganded TR; this motif was originally identified in cellular corepressors. Using a system reconstituted in the yeast Saccharomyces cerevisiae, which lack endogenous TR and TR coregulators, we show that E1A 55R nonetheless differs from cellular corepressors as it functions as a strong co-activator of TR-dependent transcription and that it possesses an intrinsic transcriptional activation domain. These data indicate that the E1A 55R protein functions as a transcriptional regulator.

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

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.

Mitogen activated protein kinase kinase kinase 3 (MAP3K3/MEKK3) overexpression is an early event in esophageal tumorigenesis and is a predictor of poor disease prognosis.

BACKGROUND: Mitogen-activated protein kinase kinase kinase3 (MAP3K3/MEKK3) was identified to be differentially expressed in esophageal squamous cell carcinoma (ESCC) using cDNA microarrays by our laboratory. Here in we determined the clinical significance of MEKK3 in ESCC. METHODS: Immunohistochemical analysis of MEKK3 expression was carried out in archived tissue sections from 93 ESCCs, 47 histologically normal and 61 dysplastic esophageal tissues and correlated with clinicopathological parameters and disease prognosis over up to 7.5 years for ESCC patients. RESULTS: MEKK3 expression was significantly increased in esophageal dysplasia and ESCC in comparison with normal mucosa (ptrend < 0.001). Kaplan Meier survival analysis showed significantly reduced median disease free survival median DFS = 10 months in patients with MEKK3 positive ESCCs compared to patients with no immunopositivity (median DFS = 19 months, p = 0.04). ESCC patients with MEKK3 positive and lymph node positive tumors had median DFS = 9 months, as compared to median DFS = 21 months in patients who did not show the alterations (p = 0.01). In multivariate Cox regression analysis, combination of MEKK3 overexpression and node positivity [p = 0.015, hazard ratio (HR) = 2.082, 95% CI = 1.154 - 3.756] emerged as important predictor of reduced disease free survival and poor prognosticator for ESCC patients. CONCLUSIONS: Alterations in MEKK3 expression occur in early stages of development of ESCC and are sustained during disease progression; MEKK3 in combination with lymph node positivity has the potential to serve as adverse prognosticator in ESCC.

Secretome proteins as candidate biomarkers for aggressive thyroid carcinomas.

Using proteomics in tandem with bioinformatics, the secretomes of nonaggressive and aggressive thyroid carcinoma (TC) cell lines were analyzed to detect potential biomarkers for tumor aggressiveness. A panel of nine proteins, activated leukocyte cell adhesion molecule (ALCAM/CD166), tyrosine-protein kinase receptor (AXL), amyloid beta A4 protein, amyloid-like protein 2, heterogeneous nuclear ribonucleoprotein K, phosphoglycerate kinase 1, pyruvate kinase isozyme M2, phosphatase 2A inhibitor (SET), and protein kinase C inhibitor protein 1 (14-3-3 zeta) was chosen to confirm their expression in TC patients' sera and tissues. Increased presurgical circulating levels of ALCAM were associated with aggressive tumors (p = 0.04) and presence of lymph node metastasis (p = 0.018). Increased serum AXL levels were associated with extrathyroidal extension (p = 0.027). Furthermore, differential expression of amyloid beta A4 protein, AXL, heterogeneous nuclear ribonucleoprotein K, phosphoglycerate kinase 1, pyruvate kinase muscle isozyme M2, and SET was observed in TC tissues compared to benign nodules. Decreased nuclear expression of AXL can detect malignancy with 90% specificity and 100% sensitivity (AUC = 0.995, p < 0.001). In conclusion, some of these proteins show potential for future development as serum and/or tissue-based biomarkers for TC and warrant further investigation in a large cohort of patients.

Immunohistochemical analysis based Ep-ICD subcellular localization index (ESLI) is a novel marker for metastatic papillary thyroid microcarcinoma.

BACKGROUND: Thyroid cancer is among the fastest growing malignancies; almost fifty-percent of these rapidly increasing incidence tumors are less than or equal to 1cm in size, termed papillary thyroid microcarcinoma (PTMC). The management of PTMC remains a controversy due to differing natural history of these patients. Epithelial cell adhesion molecule (EpCAM) is comprised of an extracellular domain (EpEx), a single transmembrane domain and an intracellular domain (Ep-ICD). Our group reported nuclear Ep-ICD correlated with poor prognosis in thyroid cancer (Ralhan et al., BMC Cancer 2010,10:331). Here in, we hypothesized nuclear and cytoplasmic accumulation of Ep-ICD and loss of membranous EpEx may aid in distinguishing metastatic from non-metastatic PTMC, which is an important current clinical challenge. To test our hypothesis, Ep-ICD and EpEx expression levels were analyzed in PTMC and the staining was correlated with metastatic potential of these carcinomas. METHODS: Thirty-six PTMC patients (tumor size 0.5 - 1cm; metastatic 8 cases and non-metastatic 28 cases) who underwent total thyroidectomy were selected. The metastatic group consisted of patients who developed lymph node or distant metastasis at diagnosis or during follow up. The patients' tissues were stained for Ep-ICD and EpEx using domain specific antibodies by immunohistochemistry and evaluated. RESULTS: PTMC patients with metastasis had higher scores for nuclear and cytoplasmic Ep-ICD immunostaining than the patients without metastasis (1.96 ± 0.86 vs. 1.22 ± 0.45; p = 0.007 and 5.37 ± 0.33 vs. 4.72 ± 1.07; p = 0.016, respectively). Concomitantly, the former had lower scores for membrane EpEx than the non-metastatic group (4.64 ± 1.08 vs. 5.64 ± 1.51; p = 0.026). An index of aggressiveness, Ep-ICD subcellular localization index (ESLI), was defined as sum of the IHC scores for accumulation of nuclear and cytoplasmic Ep-ICD and loss of membranous EpEx; ESLI = [Ep - ICDnuc + Ep - ICDcyt + loss of membranous EpEx]. Notably, ESLI correlated significantly with lymph node metastasis in PTMC (p = 0.008). CONCLUSION: Nuclear and cytoplasmic Ep-ICD expression and loss of membranous EpEx were found to correlate positively with metastasis in PTMC patients. In addition, ESLI had the potential to identify metastatic behavior in PTMC which could serve as a valuable tool for solving a current dilemma in clinical practice.

Identification of CCR4 and other essential thyroid hormone receptor co-activators by modified yeast synthetic genetic array analysis.

Identification of thyroid hormone receptor (TR) co-regulators has enhanced our understanding of thyroid hormone (TH) action. However, it is likely that many other co-regulators remained unidentified, and unbiased methods are required to discover these proteins. We have previously demonstrated that the yeast Saccharomyces cerevisiae is an excellent system in which to study TR action, and that defined TR signaling complexes in a eukaryotic background devoid of complicating influences of mammalian cell co-regulators can be constructed and analyzed for endogenous yeast genes, many of which are conserved in mammals. Here, a modified synthetic genetic array analysis was performed by crossing a yeast strain that expressed TRbeta1 and the co-activator GRIP1/SRC2 with 384 yeast strains bearing deletions of known genes. Eight genes essential for TH action were isolated, of which 4 are conserved in mammals. Examination of one, the yeast CCR4 and its human homolog CCR4/NOT6 (hCCR4), confirmed that (i) transfected CCR4 potentiates a TH response in cultured cells more efficiently than established TR co-activators and (ii) knockdown of CCR4 expression strongly inhibited a TH response (>80%). TH treatment promoted rapid and sustained hCCR4 recruitment to the TH-responsive deiodinase 1 promoter and TR co-localizes with hCCR4 in the nucleus and interacts with hCCR4 in 2-hybrid and pull-down assays. These findings indicate that a modified yeast synthetic genetic array strategy is a feasible method for unbiased identification of conserved genes essential for TR and other nuclear receptor hormone functions in mammals.

Prediction of recurrence free survival for esophageal cancer patients using a protein signature based risk model.

BACKGROUND: Biomarkers to predict the risk of disease recurrence in Esophageal squamous cell carcinoma (ESCC) patients are urgently needed to improve treatment. We developed proteins expression-based risk model to predict recurrence free survival for ESCC patients. METHODS: Alterations in Wnt pathway components expression and subcellular localization were analyzed by immunohistochemistry in 80 ESCCs, 61 esophageal dysplastic and 47 normal tissues; correlated with clinicopathological parameters and clinical outcome over 86 months by survival analysis. Significant prognostic factors were identified by multivariable Cox regression analysis. RESULTS: Biomarker signature score based on cytoplasmic ß-catenin, nuclear c-Myc, nuclear DVL and membrane α-catenin was associated with recurrence free survival [Hazard ratio = 1.11 (95% CI = 1.05, 1.17), p < 0.001, C-index = 0.68] and added significant prognostic value over clinical parameters (p < 0.001). The inclusion of Slug further improved prognostic utility (p < 0.001, C-index = 0.71). Biomarker Signature Scoreslug improved risk classification abilities for clinical outcomes at 3 years, accurately predicting recurrence in 79% patients in 1 year and 97% in 3 years in high risk group; 73% patients within low risk group did not have recurrence in 1 year, with AUC of 0.76. CONCLUSIONS: Our comprehensive risk model predictive for recurrence allowed us to determine the robustness of our biomarker panel in stratification of ESCC patients at high or low risk of disease recurrence; high risk patients are stratified for more rigorous personalized treatment while the low risk patients may be spared from harmful side effects of toxic therapy.

Secretome-based identification and characterization of potential biomarkers in thyroid cancer.

In search of thyroid cancer 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 46 high-confidence identifications, 6 proteins were considered for verification in thyroid cancer patients' tissue and blood. The localization of two proteins, nucleolin and prothymosin-α (PTMA), was confirmed in TPC-1 and CAL62 cells by confocal microscopy and immunohistochemically in xenografts of TPC-1 cells in NOD/SCID/γ mice and human thyroid cancers (48 tissues). Increased nuclear and cytoplasmic expression of PTMA was observed in anaplastic compared to papillary and poorly differentiated carcinomas. Nuclear expression of nucleolin was observed in all subtypes of thyroid carcinomas, along with faint cytoplasmic expression in anaplastic cancers. Importantly, PTMA, nucleolin, clusterin, cysteine-rich angiogenic inducer 61, enolase 1, and biotinidase were detected in thyroid cancer patients' sera, warranting future analysis to confirm their potential as blood-based thyroid cancer markers. In conclusion, we demonstrated the potential of secretome analysis of thyroid cancer cell lines to identify novel proteins that can be independently verified in cell lines, xenografts, tumor tissues, and blood samples of thyroid cancer patients. These observations support their potential utility as minimally invasive biomarkers for thyroid carcinomas and their application in management of these diseases upon future validation.

EpCAM nuclear localization identifies aggressive thyroid cancer and is a marker for poor prognosis.

BACKGROUND: Proteolytic cleavage of the extracellular domain (EpEx) of Epithelial cell adhesion molecule (EpCAM) and nuclear signaling by its intracellular oncogenic domain Ep-ICD has recently been implicated in increased proliferation of cancer cells. The clinical significance of Ep-ICD in human tumors remains an enigma. METHODS: EpEx, Ep-ICD and beta-catenin immunohistochemistry using specific antibodies was conducted on 58 archived thyroid cancer (TC) tissue blocks from 34 patients and correlated with survival analysis of these patients for up to 17 years. RESULTS: The anaplastic (ATC) and aggressive thyroid cancers showed loss of EpEx and increased nuclear and cytoplasmic accumulation of Ep-ICD. In contrast, the low grade papillary thyroid cancers (PTC) showed membranous EpEx and no detectable nuclear Ep-ICD. The ATC also showed concomitant nuclear expression of Ep-ICD and beta-catenin. Kaplan-Meier Survival analysis revealed reduced overall survival (OS) for TC patients showing nuclear Ep-ICD expression or loss of membranous EpEx (p < 0.0004), median OS = 5 months as compared to 198 months for patients who did not show nuclear Ep-ICD or demonstrated only membranous EpE. CONCLUSION: We report reciprocal loss of membrane EpEx but increased nuclear and cytoplasmic accumulation of Ep-ICD in aggressive TC; nuclear Ep-ICD correlated with poor OS of TC patients. Thus nuclear Ep-ICD localization may serve as a useful biomarker for aggressive TC and may represent a novel diagnostic, prognostic and therapeutic target for aggressive TC.

Programmed death-ligand 1 expression by digital image analysis advances thyroid cancer diagnosis among encapsulated follicular lesions.

Recognition of noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) that distinguishes them from invasive malignant encapsulated follicular variant of papillary thyroid carcinoma (EFVPTC) can prevent overtreatment of NIFTP patients. We and others have previously reported that programmed death-ligand 1 (PD-L1) is a useful biomarker in thyroid tumors; however, all reports to date have relied on manual scoring that is time consuming as well as subject to individual bias. Consequently, we developed a digital image analysis (DIA) protocol for cytoplasmic and membranous stain quantitation (ThyApp) and evaluated three tumor sampling methods [Systemic Uniform Random Sampling, hotspot nucleus, and hotspot nucleus/3,3'-Diaminobenzidine (DAB)]. A patient cohort of 153 cases consisting of 48 NIFTP, 44 EFVPTC, 26 benign nodules and 35 encapsulated follicular lesions/neoplasms with lymphocytic thyroiditis (LT) was studied. ThyApp quantitation of PD-L1 expression revealed a significant difference between invasive EFVPTC and NIFTP; but none between NIFTP and benign nodules. ThyApp integrated with hotspot nucleus tumor sampling method demonstrated to be most clinically relevant, consumed least processing time, and eliminated interobserver variance. In conclusion, the fully automatic DIA algorithm developed using a histomorphological approach objectively quantitated PD-L1 expression in encapsulated thyroid neoplasms and outperformed manual scoring in reproducibility and higher efficiency.

Transient neonatal hypothyroidism is associated with elevated serum anti-thyroglobulin antibody levels in newborns and their mothers.

Transient congenital hypothyroidism (TCH) was detected in 6 of 35,067 newborns (1:5845 births) screened in Iran. Antithyroglobulin antibodies positivity was present in 4 of 6 (66.7%) of those with TCH and in 6 of 106 (5.7%) of those with "transient hyperthyrotropinemia and normal" diagnoses (P = .0005), but positivity was similar in newborns with transient hyperthyrotropinemia versus normal neonates (P = .397).

Extrathyroidal extension in well-differentiated thyroid cancer: macroscopic vs microscopic as a predictor of outcome.

OBJECTIVE: To examine the prognostic difference in well-differentiated thyroid cancer between macroscopic extrathyroidal extension (ETE), which is appreciated in the operating room, vs microscopic ETE, which is only appreciated under the microscope by the pathologist. DESIGN: Retrospective medical record review. SETTING: Tertiary care academic hospital. PATIENTS: Among 582 patients, those who were surgically treated for stage III well-differentiated thyroid cancer with a minimum 5-year follow-up were included. Fifty-five patients (10%) (17 males and 38 females [mean age, 53.1 years]) met the selection criteria. MAIN OUTCOME MEASURES: Disease-specific survival and overall survival. RESULTS: Thirty-two patients (58%) had macroscopic ETE, while 23 patients (42%) had microscopic ETE. Twenty-year disease-specific survival in the macroscopic group was 47% (8 of 17) and 45% (5 of 11) in the microscopic group (P=.45). Twenty-year overall survival in the macroscopic group was 27% (3 of 11) and 24% (4 of 17) in the microscopic group (P=.59). The only confounding factor was external beam radiation therapy (EBRT). More patients with macroscopic ETE were treated with EBRT (P=.007). When survival was stratified according to EBRT, patients with macroscopic ETE who did not receive EBRT had diminished disease-specific survival (P=.07) and overall survival (P=.12). On multivariate analysis, EBRT was the only predictor of improved disease-specific survival (P=.02) and overall survival (P=.06). CONCLUSIONS: In selected patients with macroscopic ETE, we recommend postoperative EBRT. Further investigation is required to determine whether macroscopic ETE vs microscopic ETE is an independent predictor of outcome.

Programmed Death - Ligand 1 Expression Distinguishes Invasive Encapsulated Follicular Variant of Papillary Thyroid Carcinoma from Noninvasive Follicular Thyroid Neoplasm with Papillary-like Nuclear Features.

BACKGROUND: The noninvasive Encapsulated follicular variant of papillary thyroid cancer (EFVPTC) has been reclassified as Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) without a significant risk for malignant behavior. However the evaluation remains a challenge for clinicians. We sought to determine whether programmed death-ligand 1 (PD-L1) expression may serve as a biomarker to predict invasiveness of EFVPTC and assist to distinguish these neoplasms from NIFTP. METHODS: Immunohistochemical staining of PD-L1 expression was performed in sections of 174 Formalin-fixed paraffin-embedded (FFPE) tissue blocks from surgery removed thyroid nodules. RESULTS: Cytoplasmic PD-L1 expression was significantly increased in invasive EFVPTC (4.76±1.49) as compared to NIFTP (3.06±2.16, p<0.001). Increased cytoplasmic PD-L1 expression was associated with invasiveness in EFVPTC (p<0.001); PD-L1 positive EFVPTC cases were at 3.16 folds higher risk in developing invasion than the PD-L1 negative cases. No significant difference in cytoplasmic PD-L1 expression was observed between NIFTP and benign nodules. CONCLUSION: PD-L1 expression may serve as a useful biomarker in predicting invasiveness of EFVPTC and distinguishing NIFTP from invasive EFVPTC. To our knowledge this is the first report suggesting the application of a protein biomarker to confirm NIFTP as benign indolent neoplasms.

RETRACTED: Straticyte demonstrates prognostic value over oral epithelial dysplasia grade for oral potentially malignant lesion assessment.

OBJECTIVES: Straticyte™ was previously shown to be a more effective prognostic assessment than the current standard of care, histopathological dysplasia grading, to assess progression risk of oral epithelial dysplasia to invasive cancer [Hwang JT, Gu YR, Shen M, Ralhan R, Walfish PG, Pritzker KP, et al. Individualized five-year risk assessment for oral premalignant lesion progression to cancer. Oral Surg Oral Med Oral Pathol Oral Radiol. 2017;123:374-81]. In this follow-up study, our aim is to confirm the prognostic value of Straticyte using an independent cohort of oral biopsy cases previously assessed as epithelial dysplasia of various grades. MATERIALS AND METHODS: Using Visiopharm image analysis system, we analyzed an independent retrospective cohort of 51 oral biopsy samples with known outcomes and a follow-up history of up to 12years, to verify Straticyte, an individualized 5-year risk assessment for progression of oral potentially malignant lesions to invasive squamous cell carcinoma. RESULTS: Straticyte classified the lesions more accurately than histopathological oral epithelial dysplasia grading for risk for progression to cancer over five years. The sensitivity of low-risk vs. non-low-risk Straticyte groups was 100% compared to 68% for mild vs. non-mild dysplasia. The sensitivity of high-risk vs. non-high-risk Straticyte was 71% compared to 3% for severe vs. non-severe dysplasia. Furthermore, the Negative Predictive Value (NPV) for Straticyte was 100% for low-risk vs. non-low-risk, whereas the NPV for mild vs. non-mild dysplasia was 38%. CONCLUSION: In this cohort, Straticyte ascertains as a more useful assessment for risk of cancer progression in oral potentially malignant lesions than oral epithelial dysplasia grade.

Individualized five-year risk assessment for oral premalignant lesion progression to cancer.

OBJECTIVE: The standard of care for premalignant lesion risk assessment is dysplasia grading by histopathology. With significant overlap between dysplasia grades and high inter- and intraobserver variations, histopathology dysplasia grading alone is not a useful prognostic tool. Our aim is to investigate whether a method for quantitatively assessing S100A7, a prognostic biomarker, using image analysis can better predict clinical outcome in cases with oral dysplasia. STUDY DESIGN: Using the Visiopharm image analysis system, we analyzed a cohort of 150 oral biopsy samples to build and test Straticyte, a model for individualized assessment of the 5-year risk of progression of oral precancerous lesions to invasive squamous cell carcinomas. RESULTS: Straticyte classified lesions more accurately than histopathological dysplasia grading for risk to progression to cancer over the following 5 years. The sensitivity of low-risk versus intermediate- and high-risk Straticyte groups was 95% compared to 75% for mild versus moderate and severe dysplasia. Furthermore, the negative predictive value for low-risk versus intermediate- and high-risk Straticyte groups was 78% compared to 59% for mild versus moderate and severe dysplasia. CONCLUSION: By quantitatively assessing S100A7, Straticyte better defines the risk for developing oral squamous cell carcinoma than histopathological dysplasia grading alone.