Paul M. Weinberger

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.

E6 and E7 Gene Silencing and Transformed Phenotype of Human Papillomavirus 16-Positive Oropharyngeal Cancer Cells

BACKGROUND The E6 and E7 genes of human papillomavirus type 16 (HPV16) encode oncoproteins that bind and degrade p53 and retinoblastoma (pRb) tumor suppressors, respectively. We examined the effects of repressing E6 and E7 oncogene expression on the transformed phenotype of HPV16-positive oropharyngeal cancer cell lines. METHODS Human oropharyngeal squamous cell cancer 147T and 090 (harboring integrated HPV16 DNA) and 040T (HPV DNA-negative) cells were infected with retroviruses that expressed a short hairpin RNA (shRNA) targeting the HPV16 E6 and E7 genes or a scrambled-sequence control shRNA. Flow cytometry, terminal deoxynucleotidyltransferase-mediated UTP end-labeling assay, and immunoblotting for annexin V were used to assess apoptosis in shRNA-infected cell lines. Biochemical analysis involved quantitative real-time polymerase chain reaction analysis of p53- and pRb-target gene expression and immunoblotting for p53 and pRb protein expression. RESULTS In 147T and 090 cells, shRNA-mediated inhibition of HPV16 E6 and E7 expression reduced the E6 and E7 mRNA levels by more than 85% compared with control cells that expressed a scrambled-sequence shRNA. E6 and E7 repression resulted in restoration of p53 and pRB protein expression, increased expression of p53-target genes (p21 and FAS), decreased expression of genes whose expression is increased in the absence of functional pRb (DEK and B-MYB), and induced substantial apoptosis in 147T and 090 cells compared with the control shRNA-infected cells (from 13.4% in uninfected to 84.3% in infected 147T cells and from 3.3% in uninfected to 71.2% in infected 090 cells). CONCLUSION Repression of E6 and E7 oncogenes results in restoration of p53 and pRb suppressor pathways and induced apoptosis in HPV16-positive oropharyngeal squamous cell cancer cell lines.

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.

Cisplatin, Fluorouracil, and Leucovorin Induction Chemotherapy Followed by Concurrent Cisplatin Chemoradiotherapy for Organ Preservation and Cure in Patients With Advanced Head and Neck Cancer: Long-Term Follow-Up

PURPOSE The poor functional outcome in patients with advanced head and neck squamous cell carcinoma (HNSCC) with surgery and radiation has led to alternative approaches to advanced disease. We conducted a phase II study of induction chemotherapy followed by concurrent chemoradiotherapy for organ preservation in patients with advanced resectable and unresectable (nasopharyngeal) tumors. PATIENTS AND METHODS Forty-two patients with stage III to IV resectable HNSCC and nasopharyngeal tumors received induction chemotherapy with two courses of cisplatin (20 mg/m2/d continuous infusion [CI]), fluorouracil (800 mg/m2/d CI), and leucovorin (500 mg/m2/d CI; PFL) for 4 days followed by concurrent therapy with cisplatin (100 mg/m2/d on days 1 and 22) and approximately 70 Gy of external-beam radiotherapy. RESULTS Response to induction chemotherapy included partial response rate of 52% and complete response rate of 24%. The most common grade 3 or 4 toxicity was neutropenia (59%). After cisplatin chemoradiotherapy the complete response rate was 67%. Toxicities of cisplatin chemoradiotherapy consisted of grade 3 or 4 mucositis (79%) and neutropenia (51%). At a median follow-up of 71.5 months, 43% of the patients are still alive and disease-free. The 5-year progression-free survival (PFS) rate was 60%, and the 2- and 5-year overall survival (OS) rates were 67% and 52%, respectively. Three patients died of second primaries. Late complications of treatment included xerostomia and hoarseness. One patient had persistent dysphagia and required laser epiglotectomy 108 months after treatment. CONCLUSION Induction chemotherapy with PFL followed by concurrent cisplatin chemoradiotherapy is well tolerated and results in a good likelihood of organ preservation and excellent PFS and OS.

Activation of Wnt Signaling Pathway by Human Papillomavirus E6 and E7 Oncogenes in HPV16-Positive Oropharyngeal Squamous Carcinoma Cells

We sought to determine the role of human papillomavirus (HPV) E6 and E7 oncogenes in nuclear β-catenin accumulation, a hallmark of activated canonical Wnt signaling pathway. We used HPV16-positive oropharyngeal cancer cell lines 147T and 090, HPV-negative cell line 040T, and cervical cell lines SiHa (bearing integrated HPV16) and HeLa (bearing integrated HPV18) to measure the cytoplasmic and nuclear β-catenin levels and the β-catenin/Tcf transcriptional activity before and after E6/E7 gene silencing. Repression of HPV E6 and E7 genes induced a substantial reduction in nuclear β-catenin levels. Luciferase assay showed that transcriptional activation of Tcf promoter by β-catenin was lower after silencing. The protein levels of β-catenin are tightly regulated by the ubiquitin/proteasome system. We therefore performed expression analysis of regulators of β-catenin degradation and nuclear transport and showed that seven in absentia homologue (Siah-1) mRNA and protein levels were substantially upregulated after E6/E7 repression. Siah-1 protein promotes the degradation of β-catenin through the ubiquitin/proteasome system. To determine whether Siah-1 is important for the proteasomal degradation of β-catenin in HPV16-positive oropharyngeal cancer cells, we introduced a Siah-1 expression vector into 147T and 090 cells and found substantial reduction of endogenous β-catenin in these cells. Thus, E6 and E7 are involved in β-catenin nuclear accumulation and activation of Wnt signaling in HPV-induced cancers. In addition, we show the significance of the endogenous Siah-1–dependent ubiquitin/proteasome pathway for β-catenin degradation and its regulation by E6/E7 viral oncoproteins in HPV16-positive oropharyngeal cancer cells. Mol Cancer Res; 8(3); 433–43

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)

Human papillomavirus-active head and neck cancer and ethnic health disparities

Mortality for black males with head and neck squamous cell carcinoma (HNSCC) is twice that of white males or females. Human papillomavirus (HPV)‐active HNSCC, defined by the concurrent presence of high‐risk type HPV DNA and host cell p16INK4a expression, is associated with decreased mortality. We hypothesized that prevalence of this HPV‐active disease class would be lower in black HNSCC patients compared to white patients.

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.