Ziwei Yu

Molecular Classification Identifies a Subset of Human Papillomavirus–Associated Oropharyngeal Cancers With Favorable Prognosis

PURPOSE We sought to determine the prevalence of biologically relevant human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OSCC). Retinoblastoma (Rb) downregulation by HPV E7 results in p16 upregulation. We hypothesized that p16 overexpression in OSCC defines HPV-induced tumors with favorable prognosis. METHODS Using real-time polymerase chain reaction for HPV16, we determined HPV16 viral load in a cohort of 79 OSCCs annotated with long-term patient follow-up. A tissue microarray including these cases was also analyzed for p53, p16, and Rb utilizing in situ quantitative protein expression analysis. Seventy-seven tumors were classified into a three-class model on the basis of p16 expression and HPV-DNA presence: class I, HPV-, p16 low; class II, HPV+, p16 low; and class III, HPV+, p16 high. RESULTS Sixty-one percent of OSCCs were HPV16+; HPV status alone was of no prognostic value for local recurrence and was barely significant for survival times. Overall survival was improved in class III (79%) compared with the other two classes (20% and 18%; P = .0095). Disease-free survival for the same class was 75% versus 15% and 13% (P = .0025). The 5-year local recurrence was 14% in class III versus 45% and 74% (P = .03). Only patients in class III had significantly lower p53 and Rb expression (P = .017 and .001, respectively). Multivariable survival analysis confirmed the prognostic value of the three-class model. CONCLUSION Using this system for classification, we define the molecular profile of HPV+ OSCC with favorable prognosis, namely HPV+/p16 high (class III). This study defines a novel classification scheme that may have value for patient stratification for clinical trials testing HPV-targeted therapies.

Quantitative determination of nuclear and cytoplasmic epidermal growth factor receptor expression in oropharyngeal squamous cell cancer by using automated quantitative analysis

Background: Several lines of evidence support the epidermal growth factor receptor (EGFR) as a molecular target for therapy in head and neck squamous cell carcinomas (HNSCC). Determination of tumor EGFR levels by conventional immunohistochemistry has not always predicted antitumor efficacy. Quantitative assays may provide more accurate assessment of the level of EGFR receptor in the tumor, which may thus provide more reliable prognostic and predictive information. We studied the prognostic value of quantitative assessment of EGFR in oropharyngeal squamous cell cancers treated with radiotherapy. Experimental Design: We studied EGFR protein expression on a tissue microarray composed of 95 oropharyngeal cancer cases using an in situ molecular-based method of quantitative assessment of protein expression (AQUA) and correlated those with clinical and pathologic data. Automated, quantitative analysis uses cytokeratin to define pixels as cancer (tumor mask) within the array spot and measures intensity of EGFR expression using a Cy5-conjugated antibody within the mask. A continuous index score is generated, which is directly proportional to the number of molecules per unit area, and cases were defined as high expressing if they were above the median expression level. Results: The mean follow-up time for survivors was 44.9 months, and for the entire cohort was 34.8 months. Patients with high tumor EGFR expression levels had a local recurrence rate of 58% compared with 17% for patients with low EGFR tumor expression (P < 0.01). Similarly, patients with high nuclear EGFR expression had a local recurrence rate of 54% compared with 21% for patients with low EGFR nuclear expression (P < 0.05). Additionally, patients with high tumor and nuclear EGFR levels had inferior disease-free survival compared with low expressors (19% versus 43% and 19% versus 45%, respectively. P < 0.05 for each). In multivariate analysis adjusting for well-characterized prognostic variables, high tumor and nuclear EGFR expression levels retained their prognostic significance. Conclusion: The AQUA system provides a continuous measurement of EGFR on paraffin-embedded tissue and was able to reveal the association between EGFR expression and outcome expected from the biological role of EGFR. In the future, EGFR AQUA score may be useful in predicting response to EGFR-targeted therapies.

Prognostic Significance of p16 Protein Levels in Oropharyngeal Squamous Cell Cancer

Purpose: Functional inactivation of p16 is an early and frequent event in head and neck squamous cell cancers. In this study, we sought to determine whether p16 expression is of prognostic importance in oropharyngeal squamous cell carcinoma. Experimental Design: p16 protein expression was evaluated by immunohistochemistry in a tissue microarray composed of 123 oropharyngeal squamous cell cancers with a mean patient follow-up time of 33 months. Results: p16 overexpression was associated with more advanced Tumor-Node-Metastasis stage and higher histologic grade. Despite this association with unfavorable features, p16 overexpression was associated with decreased 5-year local recurrence rates (11 versus 53%) and increased 5-year disease-free survival (62 versus 19%) and overall survival (60 versus 21%). In multivariate analysis, p16 expression status remained an independent prognostic factor for local recurrence, disease-free survival, and overall survival. Conclusions: In patients with oropharyngeal squamous cell carcinoma, overexpression of p16 as determined by immunohistochemistry is associated with significantly improved prognosis and lower local recurrence rates.

Effect of Epidermal Growth Factor Receptor Expression Level on Survival in Patients with Epithelial Ovarian Cancer

Background: Several lines of laboratory evidence support the epidermal growth factor receptor (EGFR) as an adverse prognostic indicator in ovarian cancers. However, different methods of immunohistochemical assessment have yielded conflicting results. Here, we sought to determine the prognostic value of EGFR in ovarian cancer using a novel method of compartmentalized in situ protein analysis. Methods: A tissue array composed of 150 advanced-stage ovarian cancers uniformly treated, with surgical debulking followed by platinum-paclitaxel combination chemotherapy, was constructed. For evaluation of EGFR protein expression, we used an immunofluorescence-based method of automated in situ quantitative measurement of protein analysis (AQUA). Results: Mean follow-up time for the entire cohort was 34.4 months. Eighty-one of 150 cases had sufficient tissue for AQUA analysis. High tumor EGFR expression was associated with poor outcome for overall survival (P = 0.0001) and disease-free survival (P = 0.0005) at 3 years. In multivariable analysis, adjusting for well-characterized prognostic variables, EGFR expression status was the most significant prognostic factor for disease-free and overall survival. Conclusion: The conflicting results in the literature regarding the prognostic value of EGFR may be due to the technical difficulties inherent in assessing EGFR with immunocytochemistry. In the present study, we show that measurement of EGFR protein levels in ovarian cancer using AQUA is feasible and can give important prognostic information.

Phosphorylation of Akt (Ser473) Predicts Poor Clinical Outcome in Oropharyngeal Squamous Cell Cancer

Background: Several lines of laboratory evidence support a role of persistent activation of Akt pathway in oropharyngeal squamous cell carcinoma (OSCC) progression. Loss of phosphatase PTEN is one of the proposed mechanisms of Akt activation. We sought to determine the prognostic significance of Akt activation in a cohort of patients with OSCC as well as the association between phosphorylated (activated) Akt and PTEN levels. Methods: Using a novel system of in situ quantitative protein expression analysis (AQUA), we studied the protein expression levels of phosphorylated Akt (p-Akt) and PTEN on a tissue microarray. The array included 79 OSCCs with a mean follow-up of 36 months. Results: Patients with tumors expressing low tumor p-Akt levels had lower 5-year local recurrence rates (5% versus 38%). Additionally, these patients had improved 5-year overall survival rates (45% versus 27%). This survival effect was likely due to disease recurrence, as there was no difference in death without recurrence between low- and high-expressing groups. In adjusted analysis, tumor p-Akt expression was a strong predictor of local recurrence. A significant inverse relationship was found between nuclear p-Akt and nuclear PTEN: Tumors with high nuclear p-Akt had low nuclear PTEN and vice versa. Conclusions: Akt activation in OSCC is associated with adverse patient outcome, indicating that Akt is a promising molecular target in OSCC. PTEN loss may be one of the mechanisms of Akt activation in OSCC. (Cancer Epidemiol Biomarkers Prev 2007;16(3):553–8)

Defining molecular phenotypes of human papillomavirus–associated oropharyngeal squamous cell carcinoma: Validation of three-class hypothesis

OBJECTIVE: The purpose of this study was to determine if oropharyngeal squamous cell carcinoma (OSCC) classified into three groups based on human papillomavirus (HPV) 16 DNA presence and p16 expression display different protein expression patterns. STUDY DESIGN: Cross-sectional study. SETTING: A laboratory-based study of patients with OSCC treated at a tertiary care academic medical center. SUBJECTS AND METHODS: Paraffin-embedded OSCC specimens from 77 patients classified into the three-class model (HPV negative, HPV inactive [HPV16+/p16–], and HPV active [HPV16+/p16 +]) were queried for the expression of 14 tumor progression proteins using AQUA (HistoRx, New Haven CT). Protein expression between groups was assessed by analysis of variance. Global expression patterns were determined by unsupervised hierarchical clustering. RESULTS: There were significant differences in expression of β-catenin (P = 0.009), epidermal growth factor receptor (P = 0.009), and vascular endothelial growth factor (P = 0.028) between groups. HPV-active tumors had overexpression of β-catenin. Hierarchical clustering showed HPV-negative and HPV-inactive tumors displayed association patterns distinct from HPV-active tumors. CONCLUSIONS: Tumors classified by HPV DNA presence and p16 expression have different molecular phenotypes. This is the first demonstration of overexpression of β-catenin (also found in HPV-caused cervical cancer) in HPV-active OSCC. HPV-active OSCC may share a similar ontogeny to HPV-caused cervical cancer.

Correlates and Determinants of Nuclear Epidermal Growth Factor Receptor Content in an Oropharyngeal Cancer Tissue Microarray

Background: We have previously reported nuclear localization of epidermal growth factor receptor (EGFR) protein in oropharyngeal cancer tissue. Nuclear EGFR levels were inversely correlated with survival and response to radiotherapy. Here, we sought to identify the determinants and correlates of nuclear EGFR content. Methods: We analyzed an oropharyngeal cancer tissue microarray for the expression of the key molecules of the EGFR signaling cascade using an automated image analysis technique (AQUA) scored on a scale of 0 to 255, which permits protein quantitation and subcellular localization. Patients with oropharyngeal squamous cell cancer treated with radiotherapy or surgery and radiotherapy were eligible. Data were analyzed using Spearman correlations and multiple linear regression with robust SEs. Results: Of the 95 tumors included in this study, 72 (75%) had sufficient tissue for analysis of nuclear EGFR. Nuclear EGFR levels were associated with membranous/cytoplasmic EGFR levels (ρ = 0.82, P < 0.001), nuclear extracellular signal-regulated kinase-2 (ρ = 0.30, P = 0.01), and nuclear proliferating cell nuclear antigen (PCNA; ρ = 0.36, P = 0.003). Nuclear phosphorylated-Akt, cyclin D1, phosphatase and tensin homolog (mutated in multiple cancers 1) (PTEN), p53, and proliferation marker Ki-67 levels did not correlate with nuclear EGFR level. In multivariable analysis, only PCNA retained its significant association (P = 0.01). Conclusions: These results are consistent with preclinical data showing that EGFR may function as a tyrosine kinase in the nucleus, phosphorylating and stabilizing PCNA. The nuclear activity of EGFR may constitute a novel therapeutic target. (Cancer Epidemiol Biomarkers Prev 2008;17(6):1486–92)

β-Catenin Functions Mainly as an Adhesion Molecule in Patients with Squamous Cell Cancer of the Head and Neck

Background: β-catenin, depending on subcellular localization, plays a dual role in carcinogenesis: as a signaling factor (in the nucleus) and as an adhesion molecule (in cell membrane). In this study, we sought to determine the role of β-catenin in head and neck carcinogenesis. Methods: First, we studied the incidence of mutations of β-catenin in a cohort of 60 head and neck squamous cell cancers (HNSCC). We subsequently evaluated the protein expression levels of β-catenin in a cohort of oropharyngeal squamous cell cancer tissue microarray using a novel in situ method of quantitative protein analysis and correlated those with cyclin D1 levels and clinical and pathologic data. Results: The mean follow-up time for survivors was 45 months and for all patients was 35 months. We found no mutations in the cohort of 60 HNSCC. β-catenin displayed primarily membranous expression pattern. Patients with high tumor-node-metastasis stage were more likely to have high expression of β-catenin (P = 0.040). Patients with low β-catenin expression had a local recurrence rate of 79% compared with 29% for patients with high β-catenin tumors (P = 0.0021). Univariate Cox regression revealed a hazard ratio for low β-catenin tumors of 3.6 (P = 0.004). Kaplan-Meier analysis showed that patients with low β-catenin expressing tumors trended toward worse 5-year disease-free survival (P = 0.06). In multivariate analysis, only β-catenin expression status was an independent prognostic factor (P = 0.044) for local recurrence. Tumors with high β-catenin had low cyclin D1 and vice versa (P = 0.007). Conclusions: The absence of activating β-catenin mutations combined with the inverse correlation between β-catenin levels with cyclin D1 levels and outcome suggest that β-catenin mainly functions as an adhesion and not signaling molecule in HNSCC.

Subcellular Localization and Protein Levels of Cyclin-Dependent Kinase Inhibitor p27 Independently Predict for Survival in Epithelial Ovarian Cancer

Purpose: p27 protein is regarded as a valuable prognostic biomarker in cancer with a potential use as a molecular target. However, different methods of immunohistochemical assessment have yielded conflicting results. Here, we sought to determine the prognostic value of p27 in ovarian cancer using a novel method of compartmentalized in situ protein analysis. Experimental Design: A tissue array composed of 150 advanced stage ovarian cancers uniformly treated, with surgical debulking followed by platinum-paclitaxel combination chemotherapy, was constructed. For evaluation of p27 protein expression, we used an immunofluorescence-based method of automated in situ quantitative measurement of protein analysis [automated quantitative analysis (AQUA)]. Results: The mean follow-up time of the patients was 34.3 months. Patients with low Fédération Internationale des Gynaecologistes et Obstetristes stage were more likely to have low nuclear p27 expression (P = 0.008). Low nuclear p27 expression was associated with improved 3-year overall survival (66% versus 20%, P = 0.0047) and disease-free survival (27% versus 12%, P = 0.022). In multivariable analysis, adjusting for well-characterized prognostic variables, low nuclear p27 expression level was the most significant prognostic factor for both disease-free and overall survival. Conclusions: Our results indicate that quantitative assessment of nuclear p27 expression level by automated in situ quantitative analysis is a strong predictor for outcome in ovarian cancer.

Effects of Altered Consciousness on the Protective Glottic Closure Reflex

Objectives: The sphincteric function of the larynx, essential to lower airway protection, is most efficiently achieved through strong reflex adduction by both vocal folds. We hypothesize that central facilitation is an essential component of a bilateral brain stem-mediated adductor reflex and that its disturbance by altered consciousness or physiologic sleep could result in weakened sphincteric closure. Methods: In 10 adult pigs the glottic closure response was evaluated under light and deep isoflurane anesthesia. The internal branch of the left superior laryngeal nerve was stimulated through bipolar platinum-iridium electrodes, and recording electrodes were positioned in the ipsilateral and contralateral thyroarytenoid muscles. The force of evoked glottic closure was measured with a pressure transducer positioned between the vocal folds. Results: Consistent threshold responses (>90%) were obtained ipsilaterally from 0.5 to 2.0 minimal alveolar concentration (MAC) anesthesia. However, the contralateral reflex responses declined to 6.4% in successive trials as anesthetic levels approached 1.5 to 2.0 MAC. Furthermore, glottic closing force closely reflected these electromyographic changes, declining from 383 mm Hg at 0.5 to 1.0 MAC to 114 mm Hg at 1.5 to 2.0 MAC. Conclusions: Alteration of central facilitation by progressively deeper loss of consciousness abolishes a lower brain stem-mediated crossed adductor reflex, predisposing the subject to a weakened glottic closure response.