Maie A. St. John

Salivary Transcriptome Diagnostics for Oral Cancer Detection

Purpose: Oral fluid (saliva) meets the demand for noninvasive, accessible, and highly efficient diagnostic medium. Recent discovery that a large panel of human RNA can be reliably detected in saliva gives rise to a novel clinical approach, salivary transcriptome diagnostics. The purpose of this study is to evaluate the diagnostic value of this new approach by using oral squamous cell carcinoma (OSCC) as the proof-of-principle disease. Experimental Design: Unstimulated saliva was collected from patients (n = 32) with primary T1/T2 OSCC and normal subjects (n = 32) with matched age, gender, and smoking history. RNA isolation was done from the saliva supernatant, followed by two-round linear amplification with T7 RNA polymerase. Human Genome U133A microarrays were applied for profiling human salivary transcriptome. The different gene expression patterns were analyzed by combining a t test comparison and a fold-change analysis on 10 matched cancer patients and controls. Quantitative polymerase chain reaction (qPCR) was used to validate the selected genes that showed significant difference (P < 0.01) by microarray. The predictive power of these salivary mRNA biomarkers was analyzed by receiver operating characteristic curve and classification models. Results: Microarray analysis showed there are 1,679 genes exhibited significantly different expression level in saliva between cancer patients and controls (P < 0.05). Seven cancer-related mRNA biomarkers that exhibited at least a 3.5-fold elevation in OSCC saliva (P < 0.01) were consistently validated by qPCR on saliva samples from OSCC patients (n = 32) and controls (n = 32). These potential salivary RNA biomarkers are transcripts of IL8, IL1B, DUSP1, HA3, OAZ1, S100P, and SAT. The combinations of these biomarkers yielded sensitivity (91%) and specificity (91%) in distinguishing OSCC from the controls. Conclusions: The utility of salivary transcriptome diagnostics is successfully demonstrated in this study for oral cancer detection. This novel clinical approach could be exploited to a robust, high-throughput, and reproducible tool for early cancer detection. Salivary transcriptome profiling can be applied to evaluate its usefulness for other major disease applications as well as for normal health surveillance.

Serum Circulating Human mRNA Profiling and Its Utility for Oral Cancer Detection

PURPOSE The purpose of this study is to explore the presence of informative RNA biomarkers from human serum transcriptome, and evaluate the serum transcriptome diagnostics for disease detection. Oral squamous cell carcinoma (OSCC) was selected as the proof-of-concept disease. PATIENTS AND METHODS Blood samples were collected from patients (n = 32) with primary T1/T2 OSCC and matched healthy patients (n = 35). Circulating RNA was isolated from serum and linearly amplified using T7 polymerase. Microarrays were applied for profiling transcriptome in serum from 10 cancer patients and controls. The differential gene expression was analyzed by combining the present calls, t tests, and fold-change statistics. Quantitative polymerase chain reaction (PCR) was used to validate the selected candidate RNA markers identified by microarray. Receiver operating characteristic curve and classification models were exploited to evaluate the diagnostic power of these markers for OSCC. RESULTS Human serum circulating mRNAs were presented by reverse transcriptase PCR. Microarray identified 2,623 +/- 868 probes assigned present calls in OSCC (n = 10) versus 1,792 +/- 165 in healthy patients (n = 10), indicating a higher complexity of serum transciptome in OSCC patients (P = .002, Wilcoxon test). Three hundred thirty-five serum RNAs exhibited significantly differential expression level between the two groups (P < .05, t test; fold > or = 2). Five cancer-related gene transcripts were consistently validated by quantitative PCR on serum from OSCC patients (n = 32) and controls (n = 35). The best combination of biomarkers yielded a receiver operating characteristic curve value of 88%, sensitivity (91%), and specificity (71%) in distinguishing OSCC. CONCLUSION The utility of serum transcriptome diagnostics is successfully demonstrated for OSCC detection. This novel concept could be developed as an adjunctive tool for disease diagnosis.

Surgery for Papillary Thyroid Carcinoma: Is Lobectomy Enough?

OBJECTIVE To further understanding of treatment of papillary thyroid carcinoma (PTC). DESIGN The Surveillance, Epidemiology, and End Results Program database was searched for patients who had undergone surgery for PTC. SETTING Areas covered by Surveillance, Epidemiology, and End Results population-based registries. PATIENTS Patients who had undergone PTC surgery between January 1, 1988, and December 31, 2001, were included in the study. MAIN OUTCOME MEASURES Disease-specific survival (DSS) and overall survival (OS). RESULTS Of the total 22,724 patients with PTC, 5964 patients underwent lobectomy. There were 2138 total and 471 disease-specific deaths. Controlling for tumor size, multivariate analysis revealed no survival difference between patients who had undergone total thyroidectomy and those who had undergone lobectomy. Increased tumor size, extrathyroidal extent, positive nodal status, and increased age displayed significantly worse DSS and OS (P < .001). Histologically, follicular PTC subtype did not affect DSS or OS. Patients who had received radioactive iodine had poorer DSS but improved OS. Patients undergoing external beam radiation therapy had poor DSS (hazard ratio, 4.48; 95% confidence interval, 3.30-6.06; P < .001) and OS (1.71; 1.42-2.07; P < .001). CONCLUSIONS The results of this study compel us to reinvestigate the current PTC surgical recommendations of total thyroidectomy based on tumor size because this may not affect survival across all populations. In addition, the current use of external beam radiation therapy for the treatment of PTC should be reexamined.

Snail Promotes CXCR2 Ligand–Dependent Tumor Progression in Non–Small Cell Lung Carcinoma

Purpose: As a transcriptional repressor of E-cadherin, Snail has predominantly been associated with epithelial-mesenchymal transition, invasion, and metastasis. However, other important Snail-dependent malignant phenotypes have not been fully explored. Here, we investigate the contributions of Snail to the progression of nonsmall cell lung cancer (NSCLC). Experimental Design: Immunohistochemistry was done to quantify and localize Snail in human lung cancer tissues, and tissue microarray analysis was used to correlate these findings with survival. NSCLC cell lines gene-modified to stably overexpress Snail were evaluated in vivo in two severe combined immunodeficiency murine tumor models. Differential gene expression between Snail-overexpressing and control cell lines was evaluated using gene expression microarray analysis. Results: Snail is upregulated in human NSCLC tissue, and high levels of Snail expression correlate with decreased survival (P < 0.026). In a heterotopic model, mice bearing Snail-overexpressing tumors developed increased primary tumor burden (P = 0.008). In an orthotopic model, mice bearing Snail-overexpressing tumors also showed a trend toward increased metastases. In addition, Snail overexpression led to increased angiogenesis in primary tumors as measured by MECA-32 (P < 0.05) positivity and CXCL8 (P = 0.002) and CXCL5 (P = 0.0003) concentrations in tumor homogenates. Demonstrating the importance of these proangiogenic chemokines, the Snail-mediated increase in tumor burden was abrogated with CXCR2 blockade. Gene expression analysis also revealed Snail-associated differential gene expression with the potential to affect angiogenesis and diverse aspects of lung cancer progression. Conclusion: Snail upregulation plays a role in human NSCLC by promoting tumor progression mediated by CXCR2 ligands. (Clin Cancer Res 2009;15(22):68209)

Proinflammatory Mediators Upregulate Snail in Head and Neck Squamous Cell Carcinoma

Purpose: Inflammatory cytokines have been implicated in the progression of head and neck squamous cell carcinoma (HNSCC). Herein we investigate the mechanisms by which interleukin-1β (IL-1β) might contribute to Epithelial-Mesenchymal Transition (EMT) in HNSCC. Experimental Design: We evaluated the effect of IL-1β on the molecular events of EMT in surgical specimens and HNSCC cell lines. We examined the correlation with tumor histologic features, and a SCID xenograft model was used to assess the effects of Snail overexpression. Results: Cyclooxygenase-2 (COX-2)-dependent pathways contribute to the modulation of E-cadherin expression in HNSCC. An inverse relationship between COX-2 and E-cadherin was shown in situ by double immunohistochemical staining of human HNSCC tissue sections. Treatment of HNSCC cells with IL-1β caused the downregulation of E-cadherin expression and upregulation of COX-2 expression. This effect was blocked in the presence of COX-2 small hairpin RNA. IL-1β–treated HNSCC cell lines showed a significant decrease in E-cadherin mRNA and an increase in the mRNA expression of the transcriptional repressor Snail. IL-1β exposure led to enhanced Snail binding at the chromatin level. Small hairpin RNA–mediated knockdown of Snail interrupted the capacity of IL-1β to downregulate E-cadherin. In a SCID xenograft model, HNSCC Snail-overexpressing cells showed significantly increased primary and metastatic tumor burdens. Conclusions: IL-1β modulates Snail and thereby regulates COX-2–dependent E-cadherin expression in HNSCC. This is the first report indicating the role of Snail in the inflammation-induced promotion of EMT in HNSCC. This newly defined pathway for transcriptional regulation of E-cadherin in HNSCC has important implications for targeted chemoprevention and therapy. (Clin Cancer Res 2009;15(19):6018–27)

Histopathologic findings of HPV and p16 positive HNSCC

Human papilloma virus (HPV) and p16INKa (p16) positivity in head and neck squamous cell carcinomas (HNSCCs) is currently thought to be an encouraging prognostic indicator. However, the histopathologic changes responsible for this behavior are poorly understood. It is our objective to elucidate these histopathologic characteristics to help define the clinical utility of these markers.

Inflammation, epithelial to mesenchymal transition, and epidermal growth factor receptor tyrosine kinase inhibitor resistance.

Inflammation is an important contributor to lung tumor development and progression. In addition, inflammatory signaling may promote epithelial to mesenchymal transition, development of aggressive metastatic tumor phenotypes, and play a role in resistance to targeted therapies. New insights in inflammatory signaling have led to the evaluation of combination therapies that target these specific pathways. In addition to developing the optimal combination of targeted agents, biomarker-based selection of patients who will likely benefit will be critical to the success of this strategy. Here we focus on the potential contribution of inflammatory mediator-induced resistance to epidermal growth factor receptor tyrosine kinase inhibitors.

Cancer of unknown primary: Does treatment modality make a difference?

We systematically reviewed the published experience on the treatment outcomes of patients with head and neck cancer of unknown primary (CUP) to determine if treatment modality affects survival outcomes.

The role of ZEB1 in the inflammation-induced promotion of EMT in HNSCC:

Objectives: To determine the role of ZEB1 in the inflammation-induced promotion of the epithelial-mesenchymal transition (EMT) in head and neck squamous cell carcinoma (HNSCC). Study Design: A molecular biology study. Real-time quantitative reverse-transcriptase polymerase chain reaction (RT-PCR), Western blot analysis, and immunohistochemical staining of human HNSCC tissue sections were used to determine how inflammation affects the transcriptional repressor, ZEB1. Setting: An academic hospital laboratory. Subjects and Methods: Relative ZEB1 RNA levels were determined by RT-PCR, and protein expression was evaluated in situ by immunohistochemical staining of human HNSCC tissue sections. Results: IL-1β-treated HNSCC cell lines demonstrated a significant decrease in E-cadherin mRNA and an increase in the mRNA expression of the transcriptional repressor ZEB1. IL-1β exposure led to enhanced ZEB1 binding at the chromatin level, as determined by chromatin immunoprecipitation assays (ChIP). An inverse relationship between ZEB1 and E-cadherin was demonstrated in situ by immunohistochemical staining of human HNSCC tissue sections. Conclusions: Our recent investigations indicate that inflammatory mediators are potent regulators of EMT in HNSCC. This is the first report indicating the role of ZEB1 in the inflammation-induced promotion of EMT in HNSCC. This newly defined pathway for transcriptional regulation of E-cadherin in HNSCC has important implications for targeted chemoprevention and therapy.

Epidemiology and Treatment of Lacrimal Gland Tumors: A Population-Based Cohort Analysis

IMPORTANCE Primary tumors of the lacrimal gland are rare and are associated with substantial morbidity and mortality. The literature regarding these tumors is limited to case series and case reports. OBJECTIVE To examine the incidence, treatment, and overall survival (OS) and disease-specific survival (DSS) of patients with cancer of the lacrimal gland. DESIGN, SETTING, AND PARTICIPANTS Population-based cohort analysis using the Surveillance, Epidemiology, and End Results (SEER) database to identify patients with primary tumors of the lacrimal gland from 1973 to 2010. MAIN OUTCOMES AND MEASURES Overall survival and DSS. RESULTS A total of 321 patients with nonlymphoid tumors of the lacrimal gland were identified. The most common histological subtypes were adenoid cystic carcinoma (ACC) (32.1%) and squamous cell carcinoma (SCC) (29.9%). Survival analysis revealed a 5-year OS and DSS for all lacrimal gland tumors of 60% and 75%, respectively. On univariate analysis, low tumor grade (P = .04) and surgical treatment (P < .001) were associated with significantly better OS. For ACC tumors, surgery (P = .009), but not radiotherapy (P = .44), was found to significantly improve OS. For SCC tumors, surgical treatment significantly improved both OS (P < .001) and DSS (P = .004); radiation therapy also significantly improved OS (P = .03). Using a multivariable analysis model, age (hazard ratio [HR], 1.03 [95% CI, 1.01-1.04]; P < .001), surgery (HR, 0.43 [95% CI, 0.25-0.75]; P = .003), and T stage at presentation (HR, 1.18 [95% CI, 1.01-1.37]; P = .03) were found to be independent predictors of OS. For ACC alone, age (HR, 1.04 [95% CI, 1.02-1.06]; P < .001) and surgery (HR, 0.35 [95% CI, 0.13-0.91]; P = .03) were independent predictors of OS. For SCC, age (HR, 1.05 [95% CI, 1.02-1.09]; P = .005), surgical resection (HR, 0.31 [95% CI, 0.12-0.83]; P = .02), and radiation therapy (HR, 0.33 [95% CI, 0.14-0.80]; P = .01) were independent predictors of OS. CONCLUSIONS AND RELEVANCE Our study demonstrates that ACC is the most common malignant epithelial neoplasm of the lacrimal gland. Determinants of survival for tumors of the lacrimal gland include age at diagnosis and surgical therapy. Radiation therapy is associated with improved DSS in SCC but not in ACC.