Publications by authors named "Paul G Walfish"

61 Publications

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

Oncotarget 2018 Apr 13;9(28):19767-19782. Epub 2018 Apr 13.

Alex and Simona Shnaider Research Laboratory in Molecular Oncology, Sinai Health System, Toronto, ON, Canada.

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.
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http://dx.doi.org/10.18632/oncotarget.24833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5929424PMC
April 2018

Letter to the Editor referring to the retracted publication entitled "Straticyte demonstrates prognostic value over oral epithelial dysplasia grade for oral potentially malignant lesion assessment" by Hwang et al.

Oral Oncol 2018 05 23;80:103. Epub 2018 Mar 23.

Proteocyte Diagnostics Inc., MaRS Centre, South Tower, 101 College Street, Suite 200, Toronto, Ontario M5G 1L7, Canada; Laboratory Medicine and Pathobiology: Surgery, University of Toronto, 1 Kings College Circle, Toronto, Ontario M5S 1A8, Canada; Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.

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http://dx.doi.org/10.1016/j.oraloncology.2018.03.012DOI Listing
May 2018

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

Oral Oncol 2018 02;77:138

Proteocyte Diagnostics Inc., MaRS Centre, South Tower, 101 College Street, Suite 200, Toronto, OntarioM5G 1L7, Canada; Laboratory Medicine and Pathobiology: Surgery, University of Toronto, 1 Kings College Circle, Toronto, OntarioM5S 1A8, Canada; Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, OntarioM5G 1X5, Canada.

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http://dx.doi.org/10.1016/j.oraloncology.2017.12.019DOI Listing
February 2018

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

Oral Oncol 2017 09 29;72:1-6. Epub 2017 Jun 29.

Proteocyte Diagnostics Inc., MaRS Centre, South Tower, 101 College Street, Suite 200, Toronto, Ontario M5G 1L7, Canada; Laboratory Medicine and Pathobiology: Surgery, University of Toronto, 1 Kings College Circle, Toronto, Ontario M5S 1A8, Canada; Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.

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.
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http://dx.doi.org/10.1016/j.oraloncology.2017.06.024DOI Listing
September 2017

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

EBioMedicine 2017 Apr 24;18:50-55. Epub 2017 Mar 24.

Alex and Simona Shnaider Research Laboratory in Molecular Oncology, Mount Sinai Hospital, Toronto, ON, Canada; Joseph and Mildred Sonshine Family Centre for Head and Neck Diseases, Department of Otolaryngology-Head and Neck Surgery Program, Mount Sinai Hospital, Toronto, Ontario, Canada; Department of Otolaryngology-Head and Neck Surgery, Mount Sinai Hospital, Toronto, Ontario, Canada; Department of Otolaryngology-Head and Neck Surgery, University of Toronto, Toronto, Ontario, Canada; Department of Medicine, Endocrine Division, Mount Sinai Hospital, Toronto, Ontario, Canada; University of Toronto Medical School, Toronto, Ontario, Canada. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ebiom.2017.03.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5405181PMC
April 2017

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

Oral Surg Oral Med Oral Pathol Oral Radiol 2017 Mar 22;123(3):374-381. Epub 2016 Nov 22.

Proteocyte Diagnostics Inc., Toronto, Ontario, Canada; Oral Pathology/Oral Medicine, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada.

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.
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http://dx.doi.org/10.1016/j.oooo.2016.11.004DOI Listing
March 2017

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

Endocrine 2016 Nov 17;54(2):460-466. Epub 2016 May 17.

Department of Medicine, Endocrine Division & Department of Otolaryngology-Head & Neck Surgery Program, Joseph & Mildred Sonshine Family Centre for Head & Neck Diseases, Mount Sinai Hospital, University of Toronto Medical School, Toronto, ON, M5G 1X5, Canada.

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.
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http://dx.doi.org/10.1007/s12020-016-0989-3DOI Listing
November 2016

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

BMC Cancer 2016 07 16;16:486. Epub 2016 Jul 16.

Alex and Simona Shnaider Laboratory, Laboratory Medicine in Molecular Onocolgy, Mount Sinia Hospital, Room 6-318, 600 University Avenue, Toronto, ON, M5G 1X5, Canada.

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.
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http://dx.doi.org/10.1186/s12885-016-2507-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4947324PMC
July 2016

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

Oncotarget 2016 May;7(22):32318-28

Alex and Simona Shnaider Research Laboratory in Molecular Oncology, Mount Sinai Hospital, Toronto, Ontario, Canada.

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.
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http://dx.doi.org/10.18632/oncotarget.8698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5078015PMC
May 2016

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

Oncotarget 2016 Mar;7(13):17162-81

Department of Medicine, Alex and Simona Shnaider Research Laboratory in Molecular Oncology, Endocrine Division, Mount Sinai Hospital, Toronto, Canada.

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.
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http://dx.doi.org/10.18632/oncotarget.7751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941378PMC
March 2016

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

J Transl Med 2015 Aug 30;13:285. Epub 2015 Aug 30.

Alex and Simona Shnaider Laboratory of Molecular Oncology, Mount Sinai Hospital, 6-500, Toronto, ON, M5G 1X5, Canada.

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.
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http://dx.doi.org/10.1186/s12967-015-0637-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4553214PMC
August 2015

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

PLoS One 2015 24;10(7):e0133735. Epub 2015 Jul 24.

Alex and Simona Shnaider Research Laboratory in Molecular Oncology, Mount Sinai Hospital, Toronto, Canada; Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada; Joseph and Mildred Sonshine Family Centre for Head and Neck Diseases, Department of Otolaryngology-Head and Neck Surgery, Mount Sinai Hospital, Toronto, Canada; Department of Otolaryngology-Head and Neck Surgery, University of Toronto, Toronto, Canada.

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-β-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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133735PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4514673PMC
May 2016

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

Thyroid 2015 Nov 6;25(11):1224-34. Epub 2015 Aug 6.

1 Alex and Simona Shnaider Research Laboratory in Molecular Oncology, Mount Sinai Hospital , Toronto, Canada .

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.
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http://dx.doi.org/10.1089/thy.2015.0114DOI Listing
November 2015

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

Mol Oncol 2015 Oct 21;9(8):1720-35. Epub 2015 May 21.

Alex and Simona Shnaider Research Laboratory in Molecular Oncology, Mount Sinai Hospital, Toronto, Canada; Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada; Joseph and Mildred Sonshine Family Centre for Head and Neck Diseases, Department of Otolaryngology - Head and Neck Surgery, Mount Sinai Hospital, Toronto, Canada; Department of Otolaryngology - Head and Neck Surgery, University of Toronto, Toronto, Canada. Electronic address:

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/β-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.
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http://dx.doi.org/10.1016/j.molonc.2015.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5528795PMC
October 2015

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.

Endocrine 2015 Sep 20;50(1):130-7. Epub 2015 Mar 20.

Endocrine Division, Department of Medicine, Joseph and Mildred Sonshine Family Centre for Head & Neck Diseases, Mount Sinai Hospital, 413-7, 600 University Avenue, Toronto, ON, M5G 1X5, Canada,

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.
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http://dx.doi.org/10.1007/s12020-015-0575-0DOI Listing
September 2015

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

J Clin Endocrinol Metab 2015 May 9;100(5):1738-41. Epub 2015 Mar 9.

Department of Medicine (S.O., F.S., L.K., P.G.W.), Endocrine Division, and Otolaryngology-Head and Neck Surgery Program (P.G.W.), Mt. Sinai Hospital, Toronto, ON M5G 1X5, Canada and University of Toronto School of Medicine, Toronto, ON M5S 1A8, Canada.

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.
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http://dx.doi.org/10.1210/jc.2014-4560DOI Listing
May 2015

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

PLoS One 2015 19;10(2):e0107586. Epub 2015 Feb 19.

Alex and Simona Shnaider Laboratory in Molecular Oncology, Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada; Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada; Joseph and Mildred Sonshine Family Centre for Head and Neck Diseases, Mount Sinai Hospital, Toronto, Ontario, Canada; Department of Medicine, Endocrine Division, Mount Sinai Hospital and University of Toronto, Toronto, Ontario, Canada; Department of Otolaryngology-Head and Neck Surgery, Mount Sinai Hospital, Toronto, Ontario, Canada.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0107586PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335027PMC
January 2016

Prognostic significance of cytoplasmic S100A2 overexpression in oral cancer patients.

J Transl Med 2015 Jan 16;13. Epub 2015 Jan 16.

Alex and Simona Shnaider Laboratory of Molecular Oncology, Mount Sinai Hospital, Toronto, Ontario, Canada.

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.
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http://dx.doi.org/10.1186/s12967-014-0369-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4324434PMC
January 2015

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

BMC Cancer 2014 Sep 29;14:726. Epub 2014 Sep 29.

Alex and Simona Shnaider Research Laboratory in Molecular Oncology, Mount Sinai Hospital, 600 University Avenue, Suite 6-318, Toronto M5G 1X5, Ontario, Canada.

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.
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http://dx.doi.org/10.1186/1471-2407-14-726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4190296PMC
September 2014

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.

BMC Cancer 2014 Jan 2;14. Epub 2014 Jan 2.

Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India.

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.
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http://dx.doi.org/10.1186/1471-2407-14-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890584PMC
January 2014

Slug is a predictor of poor prognosis in esophageal squamous cell carcinoma patients.

PLoS One 2013 18;8(12):e82846. Epub 2013 Dec 18.

Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India ; Department of Medicine, Endocrine Division, Mount Sinai Hospital and University of Toronto, Toronto, Ontario, Canada ; Alex and Simona Shnaider Research Laboratory in Molecular Oncology, Department of Pathology & Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada ; Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada ; Joseph and Mildred Sonshine Family Centre for Head and Neck Diseases, Department of Otolaryngology, Head and Neck Surgery, Mount Sinai Hospital, Toronto, Ontario, Canada.

Background: Slug, a regulator of epithelial mesenchymal transition, was identified to be differentially expressed in esophageal squamous cell carcinoma (ESCC) using cDNA microarrays by our laboratory. This study aimed to determine the clinical significance of Slug overexpression in ESCC and determine its correlation with clinicopathological parameters and disease prognosis for ESCC patients.

Methods: Immunohistochemical analysis of Slug expression was carried out in archived tissue sections from 91 ESCCs, 61 dysplastic and 47 histologically normal esophageal tissues. Slug immunopositivity in epithelial cells was correlated with clinicopathological parameters and disease prognosis over up to 7.5 years for ESCC patients.

Results: Increased expression of Slug was observed in esophageal dysplasia [cytoplasmic, 24/61 (39.3%) cases, p = 0.001, odd's ratio (OR) = 4.7; nuclear, 11/61 (18%) cases, p < 0.001, OR = 1.36] in comparison with normal esophageal tissues. The Slug expression was further increased in ESCCs [cytoplasmic, 64/91 (70.3%) p < 0.001, OR = 10.0; nuclear, 27/91 (29.7%) p < 0.001, OR = 1.42]. Kaplan Meier survival analysis showed significant association of nuclear Slug accumulation with reduced disease free survival of ESCC patients (median disease free survival (DFS) = 6 months, as compared to those that did not show overexpression, DFS = 18 months; p = 0.006). In multivariate Cox regression analysis nuclear Slug expression [p= 0.005, Hazard's ratio (HR) = 2.269, 95% CI = 1.289 - 3.996] emerged as the most significant independent predictor of poor prognosis for ESCC patients.

Conclusions: Alterations in Slug expression occur in early stages of development of ESCC and are sustained during disease progression. Slug may serve as a diagnostic biomarker and as a predictor of poor disease prognosis to identify ESCC patients that are likely to show recurrence of the disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0082846PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3867395PMC
October 2014

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

J Gen Virol 2014 Jan 17;95(Pt 1):142-152. Epub 2013 Oct 17.

Departments of Oncology, Microbiology & Immunology, University of Western Ontario and London Regional Cancer Centre, London, Ontario N6A 4L6, Canada.

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.
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http://dx.doi.org/10.1099/vir.0.056838-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3917064PMC
January 2014

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

Int J Cancer 2014 Mar 8;134(6):1379-88. Epub 2013 Oct 8.

Alex and Simona Shnaider Laboratory in Molecular Oncology, Mount Sinai Hospital, Joseph & Wolf Lebovic Health Complex, Toronto, ON, Canada.

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.
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http://dx.doi.org/10.1002/ijc.28473DOI Listing
March 2014

Transglutaminase 2 overexpression in tumor stroma identifies invasive ductal carcinomas of breast at high risk of recurrence.

PLoS One 2013 13;8(9):e74437. Epub 2013 Sep 13.

Alex and Simona Shnaider Laboratory in Molecular Oncology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Joseph and Wolf Lebovic Health Complex, Toronto, Ontario, Canada.

Introduction: 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.

Methods: 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.

Results: 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.

Conclusions: 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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0074437PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772876PMC
June 2014

Clinical significance of altered expression of β-catenin and E-cadherin in oral dysplasia and cancer: potential link with ALCAM expression.

PLoS One 2013 28;8(6):e67361. Epub 2013 Jun 28.

Alex and Simona Shnaider Laboratory in Molecular Oncology, Department of Pathology and Laboratory Medicine, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.

Background: Perturbations in cell adhesion molecules are linked to alterations in cadherin-catenin complexes and likely play major roles in invasion and metastasis; their impact on early precancerous stages remains yet unknown. We showed ALCAM overexpression in early oral lesions and its cytoplasmic accumulation in oral squamous cell carcinoma (OSCC) to be a predictor of disease progression and poor prognosis. This study tested the hypothesis that alterations in E-cadherin and β -catenin expressions are early events in oral tumorigenesis, associated with disease prognosis, and correlate with perturbations in ALCAM expression.

Methods: Expressions of E-cadherin and β-catenin were analyzed in the same cohort of 105 OSCCs, 76 oral lesions and 30 normal oral tissues by immunohistochemistry and correlated with clinicopathological parameters and prognosis. The effect of siRNA mediated ALCAM knockdown on E-cadherin and β -catenin was determined using western blot, confocal microscopy and RT-PCR analysis in oral cancer cells.

Results: Significant loss of membranous E-cadherin and β-catenin expression was observed from normal, hyperplasia, dysplasia to OSCCs (p(trend) <0.001); and correlated with cytoplasmic ALCAM accumulation in OSCCs (p  = 0.006). Multivariate analysis revealed β-catenin membrane loss and ALCAM/β-catenin(nuclear/cytoplasmic) accumulation to be significant predictors for late clinical stage (p<0.001, OR = 8.7; p = 0.006, OR = 9.9, respectively) and nodal metastasis (p = 0.003, OR = 3.8; p = 0.025, OR = 3.4 respectively). Cox's regression showed E-cadherin membrane loss/ALCAM cytoplasmic expression [p<0.001; HR = 4.8] to be independent adverse prognosticators in OSCCs. siRNA mediated silencing of ALCAM resulted in concurrent increase in E-cadherin and β-catenin both at the transcript and protein levels.

Conclusions: Losses of E-cadherin and β-catenin expressions are early events in oral tumorigenesis; their associations with aggressive tumor behavior and disease recurrence underscore their potential as prognostic markers. Correlation of loss of E-cadherin and β-catenin with cytoplasmic ALCAM accumulation both in vitro and in in vivo suggests that these dynamic changes in cell adhesion system may play pivotal role in oral cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0067361PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696121PMC
January 2014

Secretome proteins as candidate biomarkers for aggressive thyroid carcinomas.

Proteomics 2013 Mar;13(5):771-87

Alex and Simona Shnaider Laboratory in Molecular Oncology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.

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.
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http://dx.doi.org/10.1002/pmic.201200356DOI Listing
March 2013

Iodine nutrition during pregnancy in Toronto, Canada.

Endocr Pract 2013 Mar-Apr;19(2):206-11

Department of Medicine, University of Manitoba, Winnipeg, MB, Canada.

Objective: To evaluate the status of iodine nutrition among pregnant women presenting for routine antenatal care in Toronto, Canada, as determined by the median urine iodine concentration (UIC) of this population.

Methods: A cross-sectional, observational study was conducted involving 142 pregnant women recruited from four low-risk antenatal outpatient clinics in Toronto, Canada. Subjects completed a questionnaire and provided a spot urine sample for the measurement of iodine concentration.

Results: Mean maternal age was 33.8 ± 4.3 years. Mean gestational age was 29.3 ± 7.8 weeks. The median UIC was 221 μg/L (interquartile range, 142 to 397 μg/L). Six women (4.2%) had urine iodine levels <50 μg/L, and 36 women (25.4%) had levels between 50 and 150 μg/L.

Conclusion: This cohort of primarily Caucasian, well-educated, and relatively affluent pregnant women in Toronto, Canada, are iodine sufficient, perhaps due to universal salt iodization and/or other dietary and lifestyle factors.
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http://dx.doi.org/10.4158/EP12193.ORDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3968857PMC
November 2013

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

BMC Cancer 2012 Nov 15;12:523. Epub 2012 Nov 15.

Alex and Simona Shnaider Laboratory in Molecular Oncology, Department of Pathology & Laboratory Medicine, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 60 Murray Street, Suite L6-304, Toronto, ON, M5T 3L9, Canada.

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.
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http://dx.doi.org/10.1186/1471-2407-12-523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3518100PMC
November 2012

Activated leukocyte cell adhesion molecule is a marker for thyroid carcinoma aggressiveness and disease-free survival.

Thyroid 2013 Feb;23(2):201-8

Alex and Simona Shnaider Research Laboratory in Molecular Oncology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada.

Background: Currently, there are no protein biomarkers for aggressive subtypes of thyroid carcinomas (TC) in clinical use that would allow for early detection and patient management. We hypothesized that activated leukocyte cell adhesion molecule (ALCAM or CD166) expression in thyroid tissues will reveal ALCAM to be a potential diagnostic and/or prognostic marker for TC aggressiveness.

Methods: Forty-five benign and 158 malignant thyroid tissues were analyzed for ALCAM expression using immunohistochemistry. ALCAM expression was correlated with different subtypes and clinicopathological features of TC, as well as patient disease-free survival.

Results: Combined membranous and cytoplasmic (total) expression of ALCAM was significantly reduced in patients with poorly/undifferentiated (aggressive) TC as compared to well-differentiated (nonaggressive) tumors (p<0.001; area-under-curve=0.865, sensitivity=82%, specificity=74%). The decreased ALCAM expression in TC correlated significantly with extrathyroidal extension, distant metastasis, and TC histotype. Notably, Kaplan-Meier survival analysis for follow-up data of 134 patients revealed significantly reduced disease-free survival for patients with TC with decreased ALCAM membranous, cytoplasmic, and total expression. Median survival of patients with decreased cytoplasmic ALCAM expression was 6 years, as compared to 13.7 years for patients with higher ALCAM expression (p<0.001).

Conclusion: ALCAM has the potential to serve as a diagnostic and prognostic biomarker for aggressive TC. This protein can be taken forward for analysis in sera of patients with TC to determine its applicability as a minimally invasive serum biomarker for TC aggressiveness and patient disease-free survival.
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http://dx.doi.org/10.1089/thy.2012.0405DOI Listing
February 2013

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

PLoS One 2012 25;7(9):e42893. Epub 2012 Sep 25.

Alex and Simona Shnaider Laboratory in Molecular Oncology, Department of Pathology & Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042893PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458098PMC
April 2013