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.

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.

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.

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.

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.

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.

Serial post-surgical stimulated and unstimulated highly sensitive thyroglobulin measurements in low- and intermediate-risk papillary thyroid carcinoma patients not receiving radioactive iodine.

The purpose of this study was to determine the natural temporal trends of serial thyroglobulin (Tg) among low/intermediate-risk PTC patients not receiving radioactive iodine (RAI) using TSH-stimulated Tg (Stim-Tg) and unstimulated highly sensitive Tg (u-hsTg). We prospectively analyzed serial Stim-Tg measurements after total thyroidectomy ± therapeutic central neck dissection among 121 consecutive low/intermediate-risk PTC patients who did not receive RAI, of whom 104 also had serial u-hsTg measurements available. Median follow-up was 6.5 years with Stim-Tg measurements commencing 3 months after surgery and u-hsTg commencing 1.8 years after surgery (when the assay became available). TSH stimulation was performed with 9-day T3 withdrawal, 22-day T4 withdrawal, or using recombinant human TSH (rhTSH). To account for within-patient correlations of repeated Tg measurements, temporal trends in Stim-Tg and u-hsTg were assessed using Generalized Estimating Equations. Stim-Tg models were adjusted for the method of TSH stimulation, whereas the u-hsTg models were adjusted for concurrent TSH level. Linear regression modeling was used to assess the trend in serial Stim-Tg and u-hsTg measurements as a function time from time of surgery throughout the duration of follow-up. The main outcome measured was the change in u-hsTg and Stim-Tg measurements over time. A total of 337 Stim-Tg (2.8/patient) and 602 u-hsTg (5.8/patient) measurements were analyzed. Among the 337 Stim-Tg measurements, Stim-Tg was assessed using rhTSH in 202 (60 %), T4 withdrawal in 41 (12 %), and T3 withdrawal in 94 (28 %) measurements. The overall mean ± 1SD for Stim-Tg and u-hsTg measured was 1.0 ± 1.2 and 0.2 ± 0.1 µg/L, respectively. When adjusted for method of TSH stimulation, serial Stim-Tg measurements did not significantly change over time (all p = NS). The estimated changes in Stim-Tg per year for rhTSH, T4 withdrawal, and T3 withdrawal were 0.01, -0.08, and 0.04 µg/L, respectively. Upon exclusion of 73 patients with an initial undetectable Stim-Tg (n = 48), serial Stim-Tg measurements did not change significantly over time (all p = NS). For these patients, the estimated changes in Stim-Tg per year for rhTSH, T4 withdrawal, and T3 withdrawal were -0.09, -0.10, and 0.01 µg/L, respectively. Serial u-hsTg measurements did not significantly change over time after adjusting for TSH level (p = NS). The estimated change in u-hsTg per year was -0.003 µg/L. No patients had any clinical or imaging evidence of a recurrence during the duration of their follow-up. Among low/intermediate-risk PTC patients not treated with RAI, serial post-surgical Stim-Tg and u-hsTg measurements do not change significantly over a median follow-up of 6.5 years.

Programmed death-ligand 1 overexpression is a prognostic marker for aggressive papillary thyroid cancer and its variants.

Programmed death-ligand 1(PD-L1) expression on tumor cells is emerging as a potential predictive biomarker in anti-PD-L1 directed cancer immunotherapy. We analyzed PD-L1 expression in papillary thyroid carcinoma (PTC) and its variants and determined its prognostic potential to predict clinical outcome in these patients. This study was conducted at an academic oncology hospital which is a prime referral centre for thyroid diseases. Immunohistochemical subcellular localization (IHC) analyses of PD-L1 protein was retrospectively performed on 251 archived formalin fixed and paraffin embedded (FFPE) surgical tissues (66 benign thyroid nodules and 185 PTCs) using a rabbit monoclonal anti-PD-L1 antibody (E1L3N, Cell Signaling Technology) and detected using VECTASTAIN rapid protocol with diaminobenzidine (DAB) as the chromogen. The clinical-pathological factors and disease outcome over 190 months were assessed; immunohistochemical subcellular localization of PD-L1 was correlated with disease free survival (DFS) using Kaplan Meier survival and Cox multivariate regression analysis. Increased PD-L1 immunostaining was predominantly localized in cytoplasm and occasionally in plasma membrane of tumor cells. Among all combined stages of PTC, patients with increased PD-L1 membrane or cytoplasmic positivity had significantly shorter median DFS (36 months and 49 months respectively) as compared to those with PD-L1 negative tumors (DFS, both 186 months with p < 0.001 and p < 0.01 respectively). Comparison of PD-L1+ and PD-L1- patients with matched staging showed increased cytoplasmic positivity in all four stages of PTC that correlated with a greater risk of recurrence and a poor prognosis, but increased membrane positivity significantly correlated with a greater risk of metastasis or death only in Stage IV patients. In conclusion, PD-L1 positive expression in PTC correlates with a greater risk of recurrence and shortened disease free survival supporting its potential application as a prognostic marker for PTC.

ER maleate is a novel anticancer agent in oral cancer: implications for cancer therapy.

ER maleate [10-(3-Aminopropyl)-3, 4-dimethyl-9(10H)-acridinone maleate] identified in a kinome screen was investigated as a novel anticancer agent for oral squamous cell carcinoma (OSCC). Our aim was to demonstrate its anticancer effects, identify putative molecular targets and determine their clinical relevance and investigate its chemosensitization potential for platinum drugs to aid in OSCC management. Biologic effects of ER maleate were determined using oral cancer cell lines in vitro and oral tumor xenografts in vivo. mRNA profiling, real time PCR and western blot revealed ER maleate modulated the expression of polo-like kinase 1 (PLK1) and spleen tyrosine kinase (Syk). Their clinical significance was determined in oral SCC patients by immunohistochemistry and correlated with prognosis by Kaplan-Meier survival and multivariate Cox regression analyses. ER maleate induced cell apoptosis, inhibited proliferation, colony formation, migration and invasion in oral cancer cells. Imagestream analysis revealed cell cycle arrest in G2/M phase and increased polyploidy, unravelling deregulation of cell division and cell death. Mechanistically, ER maleate decreased expression of PLK1 and Syk, induced cleavage of PARP, caspase9 and caspase3, and increased chemosensitivity to carboplatin; significantly suppressed tumor growth and increased antitumor activity of carboplatin in tumor xenografts. ER maleate treated tumor xenografts showed reduced PLK1 and Syk expression. Clinical investigations revealed overexpression of PLK1 and Syk in oral SCC patients that correlated with disease prognosis. Our in vitro and in vivo findings provide a strong rationale for pre-clinical efficacy of ER maleate as a novel anticancer agent and chemosensitizer of platinum drugs for OSCC.

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.

Anticancer Activity of Apaziquone in Oral Cancer Cells and Xenograft Model: Implications for Oral Cancer Therapy.

Oral squamous cell carcinoma (OSCC) patients diagnosed in late stages have limited chemotherapeutic options underscoring the great need for development of new anticancer agents for more effective disease management. We aimed to investigate the anticancer potential of Apaziquone, [EOquin, USAN, E09, 3-hydroxy-5- aziridinyl-1-methyl-2(1H-indole-4,7-dione)-prop-ß-en-α-ol], a pro-drug belonging to a class of anti-cancer agents called bioreductive alkylating agents, for OSCC. Apaziquone treatment inhibited cell proliferation and induced apoptosis in OSCC cells in vitro. Apaziquone treated OSCC cells showed increased activation of Caspase 9 and Caspase 3, and Poly (ADP ribose) polymerase (PARP) cleavage suggesting induction of apoptosis by apaziquone in oral cancer cells. Importantly, apaziquone treatment significantly reduced oral tumor xenograft volume in immunocompromised NOD/SCID/Crl mice without causing apparent toxicity to normal tissues. In conclusion, our in vitro and in vivo studies identified and demonstrated the pre-clinical efficacy of Apaziquone, as a potential novel anti-cancer therapeutic candidate for oral cancer management.

Immunohistochemical Subcellular Localization of Protein Biomarkers Distinguishes Benign from Malignant Thyroid Nodules: Potential for Fine-Needle Aspiration Biopsy Clinical Application.

BACKGROUND: It is of critical clinical importance to select accurately for surgery thyroid nodules at risk for malignancy and avoid surgery on those that are benign. Using alterations in subcellular localization for seven putative biomarker proteins (identified by proteomics), this study aimed to define a specific combination of proteins in surgical tissues that could distinguish benign from malignant nodules to assist in future surgical selection by fine-needle aspiration biopsy (FNAB). METHODS: Immunohistochemical subcellular localization (IHC) analyses of seven proteins were retrospectively performed on surgical tissues (115 benign nodules and 114 papillary-based thyroid carcinomas [TC]), and a risk model biomarker panel was developed and validated. The biomarker panel efficacy was verified in 50 FNAB formalin-fixed and paraffin-embedded cell blocks, and 26 cytosmears were prepared from fresh surgically resected thyroid nodules. RESULTS: Selection modeling using these proteins resulted in nuclear phosphoglycerate kinase 1 (PGK1) loss and nuclear Galectin-3 overexpression as the best combination for distinguishing TC from benign nodules (area under the curve [AUC] 0.96 and 0.95 in test and validation sets, respectively). A computed malignancy score also accurately identified TC in benign and indeterminate nodules (test and validation sets: AUC 0.94, 0.90; specificity 98%, 99%). Its efficacy was confirmed in surgical FNAB cell blocks and cytosmears. CONCLUSION: Using surgical tissues, it was observed that a combination of PGK1 and Galectin-3 had high efficiency for distinguishing benign from malignant thyroid nodules and could improve surgical selection for TC among indeterminate nodules. Further validation in prospective preoperative FNAB will be required to confirm such a clinical application.

Anticancer activity of pyrithione zinc in oral cancer cells identified in small molecule screens and xenograft model: Implications for oral cancer therapy.

Oral squamous cell carcinoma (OSCC) patients diagnosed in late stages have limited chemotherapeutic options, underscoring the great need for development of new anticancer agents for more effective disease management. We aimed to identify novel anticancer agents for OSCC using quantitative high throughput assays for screening six chemical libraries consisting of 5170 small molecule inhibitors. In depth characterization resulted in identification of pyrithione zinc (PYZ) as the most effective cytotoxic agent inhibiting cell proliferation and inducing apoptosis in OSCC cells in vitro. Further, treatment with PYZ reduced colony forming, migration and invasion potential of oral cancer cells in a dose-dependent manner. PYZ treatment also led to altered expression of several key components of the major signaling pathways including PI3K/AKT/mTOR and WNT/ß-catenin in OSCC cells. In addition, treatment with PYZ also reduced expression of 14-3-3ζ, 14-3-3σ, cyclin D1, c-Myc and pyruvate kinase M2 (PKM2), proteins identified in our earlier studies to be involved in development and progression of OSCCs. Importantly, PYZ treatment significantly reduced tumor xenograft volume in immunocompromised NOD/SCID/Crl mice without causing apparent toxicity to normal tissues. Taken together, we demonstrate in vitro and in vivo efficacy of PYZ in OSCC. In conclusion, we identified PYZ in HTS assays and demonstrated in vitro and in vivo pre-clinical efficacy of PYZ as a novel anticancer therapeutic candidate in OSCC.

Post-operative stimulated thyroglobulin and neck ultrasound as personalized criteria for risk stratification and radioactive iodine selection in low- and intermediate-risk papillary thyroid cancer.

The purpose of this study was to demonstrate the utility of a personalized risk stratification and radioactive iodine (RAI) selection protocol (PRSP) using post-operative stimulated thyroglobulin (Stim-Tg) and neck ultrasound in low- and intermediate-risk papillary thyroid carcinoma (PTC) patients. Patients with PTC tumors ≥1 cm were prospectively followed after total thyroidectomy and selective therapeutic central compartment neck dissection. Low/intermediate risk was defined as PTC confined to the thyroid or central (level VI) lymph nodes. Stim-Tg and neck ultrasound were performed approximately 3 months after surgery and used to guide RAI selection. Patients with Stim-Tg < 1 µg/L did not receive RAI, while those with Stim-Tg >5 µg/L routinely did. Those with Stim-Tg 1-5 µg/L received RAI on the basis of several clinical risk factors. Patients were followed for >6 years with serial neck ultrasound and basal/stimulated thyroglobulin. Among the 129 patients, 84 (65 %) had undetectable Stim-Tg after initial surgery, 40 (31 %) had Stim-Tg of 1-5 µg/L, and 5 (4 %) had Stim-Tg >5 µg/L. RAI was administered to 8 (20 %) patients with Stim-Tg 1-5 µg/L and 5 (100 %) with Stim-Tg >5 µg/L. Using this approach, RAI therapy was avoided in 17/20 (85 %) patients with tumors >4 cm, in 72/81 (89 %) patients older than 45 years, and in 6/9 (67 %) patients with central lymph node involvement. To date, 116 (90 %) patients in this cohort have not received RAI therapy with no evidence of residual/recurrent disease, whereas among the 13 patients who received RAI, 1 (8 %) had pathologic residual/recurrence disease. Using the proposed PRSP, RAI can be avoided in the majority of low/intermediate-risk PTC patients. Moreover, traditional risk factors considered to favor RAI treatment were not always concordant with the PRSP and may lead to overtreatment.

Induction of painless thyroiditis in patients receiving programmed death 1 receptor immunotherapy for metastatic malignancies.

CONTEXT: Immunotherapies against immune checkpoints that inhibit T cell activation [cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed cell death 1 (PD-1)] are emerging and promising treatments for several metastatic malignancies. However, the precise adverse effects of these therapies on thyroid gland function have not been well described. CASE DESCRIPTION: We report on 10 cases of painless thyroiditis syndrome (PTS) from a novel etiology, following immunotherapy with anti-PD-1 monoclonal antibodies (mAb) during treatment for metastatic malignancies. Six patients presented with transient thyrotoxicosis in which thyrotropin binding inhibitory immunoglobulins (TBII) were absent for all, whereas four patients had evidence of positive antithyroid antibodies. All thyrotoxic patients required temporary beta-blocker therapy and had spontaneous resolution of thyrotoxicosis with subsequent hypothyroidism. Four patients presented with hypothyroidism without a detected preceding thyrotoxic phase, occurring 6-8 weeks after initial drug exposure. All of these patients had positive antithyroid antibodies and required thyroid hormone replacement therapy for a minimum of 6 months. CONCLUSIONS: Patients receiving anti-PD-1 mAb therapy should be monitored for signs and symptoms of PTS which may require supportive treatment with beta-blockers or thyroid hormone replacement. The anti-PD-1 mAb is a novel exogenous cause of PTS and provides new insight into the possible perturbations of the immune network that may modulate the development of endogenous PTS, including cases of sporadic and postpartum thyroiditis.

Nuclear Ep-ICD expression is a predictor of poor prognosis in "low risk" prostate adenocarcinomas.

INTRODUCTION: 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. METHODS: 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). RESULTS: 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). CONCLUSION: 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.

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.

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.