Mark A. Merkley

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.

Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes.

BackgroundInfection with high-risk type human papilloma viruses (HPVs) is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features.ResultsSix replicate measurements were performed for each group using two-dimensional difference gel electrophoresis (2D-DIGE). The median within-group coefficient of variation was 19–21%. Significance of between-group differences was tested based on Significance Analysis of Microarray and fold change. Expression of 170 (23%) of the protein features changed significantly in immortalized cells compared to primary keratinocytes. Most of these changes were qualitatively similar in cells immortalized by E6, E7, or E6/7 expression, indicating convergence on a common phenotype, but fifteen proteins (~2%) were outliers in this regulatory pattern. Ten demonstrated opposite regulation in E6- and E7-expressing cells, including the cell cycle regulator p16INK4a; the carbohydrate binding protein Galectin-7; two differentially migrating forms of the intermediate filament protein Cytokeratin-7; HSPA1A (Hsp70-1); and five unidentified proteins. Five others had a pattern of expression that suggested cooperativity between the co-expressed oncoproteins. Two of these were identified as forms of the small heat shock protein HSPB1 (Hsp27).ConclusionThis large-scale analysis provides a framework for understanding the cooperation between E6 and E7 oncoproteins in HPV-driven carcinogenesis.

Use of Combination Proteomic Analysis to Demonstrate Molecular Similarity of Head and Neck Squamous Cell Carcinoma Arising From Different Subsites

OBJECTIVE To evaluate head and neck squamous cell carcinomas (HNSCCs) for differences in protein expression between oral cavity, oropharynx, larynx, and hypopharynx subsites. DESIGN Retrospective proteomic analysis using tissue microarray (TMA) and 2-dimensional difference gel electrophoresis (2D-DIGE). For the TMA, automated quantitative protein expression analysis was used to interrogate levels of 4 cell-cycle regulatory proteins chosen for their known roles in cancer (cyclin D1, p53, Rb, and p14). For the 2D-DIGE, lesional and normal adjacent tissues were enriched by laser capture microdissection. Total protein was extracted, analyzed by 2D-DIGE with saturation dye labeling, and evaluated for relative abundance levels of individual protein spots. SETTING Two tertiary-care academic medical centers. PATIENTS Seventy-one patients with HNSCC for TMA, and 14 patients with HNSCC with frozen tumor and normal tissue for 2D-DIGE. RESULTS The automated quantitative analysis of protein expression analysis revealed no difference between subsite for cyclin D1, p53, Rb, or p14 expression. The 2D-DIGE study was based on 28 gels (14 cancer gels and 14 adjacent normal gels), and 732 spots were identified as matching across more than 90% of gels. Significance was evaluated based on false discovery rate (FDR) estimated from permuted data sets. There were no significant differences in protein expression between subsites (FDR greater than or equal to 30% in all instances). CONCLUSIONS Observed differences in outcomes between HNSCCs from different subsites may not reflect differences in tumor biologic characteristics between subsites. Rather, it is possible that observed clinical heterogeneity among HNSCCs may be based on other factors, such as viral vs chemical carcinogenesis.

2D-DIGE proteomic characterization of head and neck squamous cell carcinoma

Objective: Identify proteins that are differentially expressed between head and neck squamous cell cancer (HNSCC) and patient-matched normal adjacent tissue, and validate findings in a separate patient cohort. Study Design: Cross-sectional study of surgical specimens. Setting: Tertiary care academic medical center. Subjects and Methods: Laser capture microdissection and two-dimensional difference gel electrophoresis were used previously to establish proteomic profiles for tumor and normal adjacent tissue from 14 patients. Here, significance analysis of microarray was used to rank candidate biomarkers. Spots meeting statistical and biological criteria of significance were analyzed by liquid chromatography and tandem mass spectrometry to obtain protein identifications. The expression pattern of the highest-ranked candidate biomarker (cornulin) was validated in a larger, independent patient cohort (n = 68) by immunohistochemical staining of a tissue microarray. Results: Of 732 spots, 117 (15.9%) met criteria for significance. Identities were obtained for 39 spots, representing 17 different proteins. Four proteins were novel in the context of HNSCC: glutathione synthetase, which was upregulated; and cornulin (squamous epithelial heat shock protein 53), guanylate binding protein 6, and heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa), which were downregulated. Cornulin functions in the stress response in normal squamous epithelium, and reduced expression has been proposed as a marker of susceptibility to laryngopharyngeal reflux and other stressors. Loss of cornulin expression was confirmed in an independent HNSCC patient cohort (P < 0.001). Conclusions: Downregulation of cornulin is a prominent feature of the molecular signature of HNSCC identified by comparative proteomics. Cornulin may represent a link between HNSCC and other pathologies arising in stratified squamous epithelium.

Significance of HSPB1 expression in Head and Neck Squamous Cell Carcinoma: A Meta‐Analysis of published literatures

OBJECTIVE HSPB1 functions to prevent stress-induced cellular damage and has is elevated in multiple cancer types. The significance of HSPB1 in HNSCC remains controversial. We sought to perform a meta-analysis of HSPB1 expression to clarify previous findings. STUDY DESIGN Meta-analysis of all published studies of HSPB1 in HNSCC patients using IHC techniques. METHODS A literature review was performed on PubMed and Google Scholar search engines using terms HSP27, HSPB1, Heat Shock Proteins, Cancer, Head and Neck Squamous Cell Carcinoma. Additional studies were added by review of manuscript bibliographies. Means and standard deviations for continuous data were obtained for overall HSPB1 expression (in cancer, normal and dysplasia), nodal status and TNM stage. Chi-square and Cochrans Q test were used to test statistical significance. RESULTS There were 77 studies identified in the context of HSPB1 and cancer in general. Of these, 7 studies (total patients n=347) met inclusion criteria and reported findings in HNSCC using IHC scoring techniques. For the mean difference in HSPB1 expression; cancer vs. normal, cancer vs. dysplasia, and dysplasia vs. normal all showed significance (p<0.0001) however the difference was not homogeneous across studies for cancer vs. dysplasia and normal. The difference was homogeneous for dysplasia vs. normal. There was no significant difference for HSPB1 expression by nodal status or stage. CONCLUSION HSPB1 is elevated in HNSCC and may be a useful biomarker for this disease.

Multi-spectral imaging analysis of HSPB1 expression in Head and Neck Squamous Cell Carcinoma

The objective of this study was to obtain more accurate quantitation of HSPB1 expression in HNSCC using a novel quantitative protein expression analysis system based on multispectral imaging. The study was a retrospective laboratory study of HNSCC patients treated at tertiary care academic medical center. Archival tissue samples from forty seven patients with HNSCC were subjected to immunohistochemistry using primary antibody to HSPB1. Seven of the patients had early stage cancers (TNM stage I/II) and forty patients had advanced stage cancers (TNM stage III/IV). HSPB1 expression was increased in advanced stage versus early stage cancers. Further investigation of HSPB1 as a potential biomarker for HNSCC is warranted.

Survival with Spindle Cell Carcinoma Compared to Conventional Squamous Cell Carcinoma

Objectives: (1) Compare survival of patients with spindle cell carcinoma variant (SpCC) to survival of patients with conventional squamous cell carcinoma (SCC). (2) Describe the impact of patient demographics and tumor characteristics on survival with SpCC. Methods: A retrospective cohort study was conducted with 18,416 cases entered into the Surveillance, Epidemiology, and End Results Program database between 2004 and 2009. Variables including age, sex, race, tumor grade, size, stage group, TNM stage, and treatment modality of 18,298 SCC patients and 118 SpCC patients were extracted and combined into oral cavity, oropharynx, and larynx sites. Characteristics and survival of SpCC and SCC patients were compared in total and at each site. The effect of variables on survival with SpCC was assessed. Results: In total, SpCC patients experienced worse survival than conventional SCC patients (P < .05). Overall, SpCC patient mortality was worse than SCC patients within the first 3 years (P < .05) but was not different at 5 or 10 years. Of the 3 sites, survival was only significantly worse for SpCC patients within the oropharynx site (P < .05). Survival of SpCC patients was worse with higher stage group and TNM stage (both P < .05), but did not differ by sex, race, or tumor grade. Conclusions: SpCC carries a worse prognosis than conventional SCC when located in the oropharynx. Survival with SpCC was associated with stage group and TNM stage, but was unaffected by sex, race, or grade.

Abstract 3286: Overexpression of cornulin in histologically normal adjacent tissue predicts occult nodal metastases in head and neck cancer patients

As with many epithelial cancers, presence of nodal metastases in patients with Head and Neck Squamous Cell Carcinoma (HNSCC) predicts early recurrence and poor survival. Determination of occult metastases in the clinically N-zero (cN0) patient remains problematic and therefore prophylactic neck dissections are routinely performed for high risk cN0 patients. A cohort of 7 cN0 HNSCC patients who subsequently underwent planned neck dissection as part of their treatment was selected. Snap frozen tissue was enriched for tumor and adjacent normal tissue by laser capture microdissection. Total protein was extracted and separated by saturation-labeling 2D difference in-gel electrophoresis (2D-DIGE). Significance analysis of Microarray (SAM) method was used to evaluate for differential protein expression between patients with (n=3) and without (n=4) occult nodal metastases on neck dissection pathologic evaluation. Spot maps were generated for 14 gels (7 HNSCC, 7 normal) and relative abundance values calculated for each of 662 protein spots common to >90% of gels. A 5% false discovery rate and 2-fold change were used as criteria for significance in the defintion of candidate biomarkers. Protein spots meeting these criteria were then extracted, digested and analyzed by liquid chromatography and tandem mass spectrometry to obtain protein identifications. For tumor tissue, there was no significant difference between groups. There were considerable differences in protein expression for normal adjacent tissue between patients with and without occult metastases, with 60 spots meeting criteria for significance (FDR Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3286.

Molecular Similarity of HNSCC from Different Subsites

Problem Squamous cell carcinomas arising from various subsites within the head and neck (HNSCC), while histologically identical, have substantial differences in survival and recurrence rates. Controversy exists as to whether this reflects physical differences or fundamental molecular heterogeneity between subsites. In this study, we used two proteomics approaches to evaluate HNSCCs for differences in protein expression between subsites including oral cavity, oropharynx, larynx and hypopharynx. Methods Tumor tissue from a total of 85 patients with HNSCC was queried by two complementary proteomics techniques. Frozen tissue from 14 patients with histologically confirmed HNSCC was enriched for tumor and normal tissue by laser capture microdissection. Total protein was extracted, separated by 2D-difference gel electrophoresis (2D-DIGE), and evaluated for differential protein expression between subsites. A tissue microarray (TMA) was constructed consisting of 71 patients with HNSCC. This TMA was queried for expression of 4 cell-cycle and regulatory proteins chosen a priori for their known roles in cancer, using Automated Quantitative Analysis of protein expression (AQUA). Results From the first cohort, proteins from tumor and matched normal tissue were successfully isolated and 732 spots identified as matching across >90% of gels. After supervised statistical analysis, we identified no significant differences in protein expression between subsites. AQUA analysis of the second, larger cohort likewise revealed no difference between subsite for cyclin D1, p53, Rb, or p14 expression. Conclusion Using two validated, complementary proteomics methods we identified no significant differences between HNSCCs from different head and neck subsites. Significance It is possible that heterogeneity among HNSCCs may be based not on anatomic subsite, but rather on fundamentally different carcinogenic pathways – i.e. virally induced versus carcinogenic insult (tobacco, alcohol). Support NIHR33-CA-5941(WSD); AAO/HNSF Resident Research Grant 26357 (PMW).