Serum protein profiles to identify head and neck cancer.

PURPOSE: New and more consistent biomarkers of head and neck squamous cell carcinoma (HNSCC) are needed to improve early detection of disease and to monitor successful patient management. The purpose of this study was to determine whether a new proteomic technology could correctly identify protein expression profiles for cancer in patient serum samples. EXPERIMENTAL DESIGN: Surface-enhanced laser desorption/ionization-time of flight-mass spectrometry ProteinChip system was used to screen for differentially expressed proteins in serum from 99 patients with HNSCC and 102 normal controls. Protein peak clustering and classification analyses of the surface-enhanced laser desorption/ionization spectral data were performed using the Biomarker Wizard and Biomarker Patterns software (version 3.0), respectively (Ciphergen Biosystems, Fremont, CA). RESULTS: Several proteins, with masses ranging from 2778 to 20800 Da, were differentially expressed between HNSCC and the healthy controls. The serum protein expression profiles were used to develop and train a classification and regression tree algorithm, which reliably achieved a sensitivity of 83.3% and a specificity of 100% in discriminating HNSCC from normal controls. CONCLUSIONS: We propose that this technique has potential for the development of a screening test for the detection of HNSCC.     

Involvement of EphA2 in head and neck squamous cell carcinoma: mRNA expression, loss of heterozygosity and immunohistochemical studies

EphA2 is a 130-kDa transmembrane protein primarily found in adult human epithelial cells and is a member of one of the largest receptor tyrosine kinases. It is located on 1p36.1, a genetic hot spot in cancer. EphA2 overexpression has been observed in aggressive solid tumors and its potential role in tumorigenesis, which includes cell growth, survival, migration and angiogenesis have been reported. However, the role of EphA2 remains unknown in head and neck cancer. In this study, we investigated the genetic profile of EphA2 in primary head and neck squamous cell carcinoma (HNSCC) by determining mRNA level, status of loss of heterozygosity and protein expression. mRNA expression was also correlated with clinicopathological data. Infrequent loss of heterozygosity (20%) was observed, though a 10-fold increase of mRNA expression in tumors compared to normal tissues was noted. A significant number of samples with normal to high mRNA expression was observed among patients with regional metastasis, with T3-T4 tumor size and with moderate to poor differentiation. However, statistical studies did not show any correlation between mRNA expression and any of the clinicopathological parameters. Tumor cells expressed EphA2 protein, but only weakly. These results suggest that EphA2 might be involved in the early development of HNSCC although not directly responsible for its progression.     

SCCA1 expression in T-lymphocytes peripheral to cancer cells is associated with the elevation of serum SCC antigen in squamous cell carcinoma of the tongue

Squamous cell carcinoma (SCC) antigen has been used for the management of SCC arising in various cites including head and neck region. However, the true mechanism of the elevation of this protein in the serum of patients with SCC is still unknown. SCC antigen belongs to the superfamily of serine protease inhibitors. Recently, molecular studies show that serum SCC antigen is transcribed by two nearly identical genes (SCCA1 and SCCA2), and is mainly produced by SCCA1. The objective of this study is to clarify the mechanism of the elevation of SCC antigen in oral tongue SCC patients and to identify cells histologically, which are responsible for serum SCC antigen production. In this study, we examined SCCA1 expression in a series of four head and neck SCC (HNSCC) cell lines, and found that all expressed equal to low SCCA1 protein as compared with the normal human oral keratinocyte. Using the double immunohistochemical technique to examine the expression pattern of SCCA1 in 86 cases of oral tongue squamous cell carcinoma, SCCA1 immunostaining was observed in the cytoplasm of cancer cells and T-lymphocytes peripheral to cancer cells. We also compared the clinicopathological features including serum SCC antigen level of the oral tongue SCC cases with the immunohistochemical SCCA1 expression pattern, and found that elevated serum SCC antigen level was significantly correlated with SCCA1 expression not in cancer cells, but in T-lymphocytes peripheral to cancer cells. These results suggest that T-lymphocytes peripheral to cancer cells may be responsible for serum SCC antigen production in HNSCC patients.     

High‐risk HPV types and head and neck cancer

Although HPV16 has been strongly implicated in oropharyngeal carcinogenesis, the role of other high‐risk HPV types in the etiology of head and neck cancer remains unclear. To date, few data exist addressing the nature of the association between antibodies to oncogenic proteins of non‐HPV16 HPVs in relation to head and neck cancer. We examined the relationship between multiple HPV types (HPV6, 11, 16, 18, 31, 33, 45, 52, 58) and head and neck squamous cell carcinoma (HNSCC) in a large population‐based case–control study (1069 cases and 1107 controls). Serological measures for HPV types included antibodies to L1, E6 and/or E7. In a secondary analysis, we excluded HPV16 seropositive subjects to examine independent associations with other high‐risk HPVs. All analyses were adjusted for age, race, sex, education, smoking and alcohol consumption. Statistically significant associations were observed for HPV16, 18, 33 and 52 and risk of HNSCC after mutually adjusting for HPV types. Among HPV16 seronegative subjects, elevated risks of HNSCC were observed for HPV18 E6 (OR = 4.19, 95% CI = 1.26–14.0), HPV33 E6 (OR = 7.96, 95% CI = 1.56–40.5) and HPV52 E7 (OR = 3.40, 95% CI = 1.16–9.99). When examined by tumor type, associations with HPV18 and HPV33 remained statistically significant for oropharyngeal cancer, and HPV52 was associated with oral cancer. In addition, magnitude of associations for HNSCC increased markedly with increasing number of seropositive high‐risk HPV infections. High‐risk HPV types, other than HPV16, are likely to be involved in the etiology of HNSCC.     

Dysregulation of splicing proteins in head and neck squamous cell carcinoma

Signaling plays an important role in regulating all cellular pathways. Altered signaling is one of the hallmarks of cancers. Phosphoproteomics enables interrogation of kinase mediated signaling pathways in biological systems. In cancers, this approach can be utilized to identify aberrantly activated pathways that potentially drive proliferation and tumorigenesis. To identify signaling alterations in head and neck squamous cell carcinoma (HNSCC), we carried out proteomic and phosphoproteomic analysis of HNSCC cell lines using a combination of tandem mass tag (TMT) labeling approach and titanium dioxide-based enrichment. We identified 4,920 phosphosites corresponding to 2,212 proteins in six HNSCC cell lines compared to a normal oral cell line. Our data indicated significant enrichment of proteins associated with splicing. We observed hyperphosphorylation of SRSF protein kinase 2 (SRPK2) and its downstream substrates in HNSCC cell lines. SRPK2 is a splicing kinase, known to phosphorylate serine/arginine (SR) rich domain proteins and regulate splicing process in eukaryotes. Although genome-wide studies have reported the contribution of alternative splicing events of several genes in the progression of cancer, the involvement of splicing kinases in HNSCC is not known. In this study, we studied the role of SRPK2 in HNSCC. Inhibition of SRPK2 resulted in significant decrease in colony forming and invasive ability in a panel of HNSCC cell lines. Our results indicate that phosphorylation of SRPK2 plays a crucial role in the regulation of splicing process in HNSCC and that splicing kinases can be developed as a new class of therapeutic target in HNSCC.     

Serum levels of interleukin-6 in patients with primary head and neck squamous cell carcinoma

Interleukin (IL)-6 plays a central role as a differentiation and growth factor of tumor cells. IL-6 has been identified in a wide variety of malignancies, including head and neck squamous cell carcinomas (HNSCC). The aim of this study was to investigate the association between the serum levels of IL-6 in HNSCC patients and the biological characteristics of the tumor as well as the clinicopathological status of the patients. The circulating level of IL-6 in sera from patients with various HNSCC (n = 90) as well as from healthy normal controls (n = 39) was investigated. Serum IL-6 concentrations were determined as serum immunoreactivity using a quantitative sandwich enzyme immunoassay technique. For statistical analysis, the Kruskal-Wallis test was performed. The majority of the patients with HNSCC were found to have high serum IL-6 concentrations. The IL-6 levels in the sera of patients with cancer ranged from below the detection limit to 312.8 pg/ml (mean, 19.5 pg/ml). In contrast, the IL-6 serum levels in 39 healthy individuals ranged from below the detection limit to 52.2 pg/ml (mean, 6.0 pg/ml), with the concentration being significantly higher in HNSCC patients (p < 0.001). Furthermore, the correlation of the IL-6 serum concentration with tumor stage was significant (p = 0.04). Accordingly, there was a significant difference of IL-6 serum concentration of tumors with positive and negative lymph nodes (p = 0.045), with concentration being significantly higher in lymph node-positive tumors. Our data on elevated IL-6 serum levels in the majority of HNSCC cancer patients and its correlation with tumor stage and lymph node status suggest that serum IL-6 reflects the proliferative activity of the tumor in patients with head and neck cancer. IL-6 serum determinations might serve as a biological marker and help to identify advanced head and neck tumors.     

Pocket proteins suppress head and neck cancer

Head and neck squamous cell carcinomas (HNSCC) is a common cancer in humans long known to be caused by tobacco and alcohol use, but now an increasing percentage of HNSCC is recognized to be caused by the same human papillomaviruses (HPV) that cause cervical and other anogenital cancers. HPV-positive HNSCCs differ remarkably from HPV-negative HNSCCs in their clinical response and molecular properties. From studies in mice, we know that E7 is the dominant HPV oncoprotein in head and neck cancer. E7 is best known for its ability to inactivate pRb, the product of the retinoblastoma tumor susceptibility gene. However, loss of pRb function does not fully account for potency of E7 in causing head and neck cancer. In this study, we characterized the cancer susceptibility of mice deficient in the expression of pRb and either of two related "pocket" proteins, p107 and p130, that are also inactivated by E7. pRb/p107-deficient mice developed head and neck cancer as frequently as do HPV-16 E7 transgenic mice. The head and neck epithelia of the pRb/p107-deficient mice also displayed the same acute phenotypes and biomarker readouts as observed in the epithelia of E7 transgenic mice. Mice deficient for pRb and p130 in their head and neck epithelia showed intermediate acute and tumor phenotypes. We conclude that pRb and p107 act together to efficiently suppress head and neck cancer and are, therefore, highly relevant targets of HPV-16 E7 in its contribution to HPV-positive HNSCC.     

Mass Spectrometry-Based Salivary Proteomics for the Discovery of Head and Neck Squamous Cell Carcinoma

The 5-year survival rates for cases of head and neck squamous cell carcinoma (HNSCC) are only some 60%, mainly because 20%–40% of the patients develop a local relapse in the same or an adjacent anatomic region, even when the surgical margins are histologically tumour-free. Tumours are often discovered in an advanced stage because of the lack of specific symptoms and the diagnostic difficulties. The more advanced the stage of the tumour, the more invasive the diagnostic and treatment interventions needed. An early molecular diagnosis is therefore of vital importance in order to increase the survival rate. The aim of this study was to develop an efficient rapid and sensitive mass spectrometric method for the detection of differentially expressed proteins as tumour-specific biomarkers in saliva from HNSCC patients. Whole saliva samples were collected from patients with HNSCC and from healthy subjects. The proteins were profiled by using SDS PAGE, MALDI TOF/TOF mass spectrometry and the Mascot database search engine. Several potential tumour markers were identified, including annexin A1, beta- and gamma-actin, cytokeratin 4 and 13, zinc finger proteins and P53 pathway proteins. All of these proteins play a proven role in tumour genesis, and have not been detected previously in saliva. Salivary proteomics is a non-invasive specific method for cancer diagnosis and follow-up treatment. It provides facilities for the readily reproducible and reliable detection of tumours in early stages.     

Expression of SGLT-1 in preneoplastic and neoplastic lesions of the head and neck

Tumors show an increased glucose uptake that is mediated by glucose transport proteins. We have analyzed the expression of the sodium-dependent glucose co-transporters SGLT-1 and-2 in short-term cultures of squamous cell carcinomas of the head and neck (HNSCC) by RT-PCR. Distribution of the SGLT-1 protein in HNSCC tissues was investigated by immunohistochemistry. While we observed in 17/36 HNSCC short-term cultures the SGLT-1 mRNA, we found no SGLT-2 expression. SGLT-1 mRNA expression occurred preferentially in cultures originating from moderately and well differentiated HNSCC. In tumor tissues a heterogeneous SGLT-1 staining restricted to differentiated tumor cells was seen in 27/30 HNSCC analyzed. In normal mucosa SGLT-1 staining was also confined to differentiated compartments and lacked in dysplastic areas. Our data indicate a differentiation-dependent expression of SGLT-1 in HNSCC. This is important knowledge for the planning of glucose-targeting therapies and suggest SGLT-1 as a differentiation marker in head and neck tissues.     

Applying the molecular biology and epigenetics of head and neck cancer in everyday clinical practice

During the past decade, there has been a significant increase in knowledge regarding the molecular biology and epigenetics of head and neck squamous cell carcinoma (HNSCC). This has been aided by the steady development of new technology and novel techniques aimed at elucidating additional aberrant molecular alterations characteristic of HNSCC, including the advent of high throughput assays and the development of more sophisticated bioinformatics tools. In addition, advancements in the field of cancer epigenetics and microRNA have increased the complexity of understanding HNSCC tumorigenesis. These advances have lead to an increasing number of translational studies in the diagnosis, prognosis, and treatment of head and neck cancer. The end result is that molecular biomarkers, gene detection panels and targeted therapeutics are becoming a reality for the care of patients with HNSCC. In this article, we will focus on the many implications of this research as it pertains to clinical practice and the treatment of HNSCC patients.     

Autoantibody approach for serum-based detection of head and neck cancer.

Currently, no effective tool exists for screening or early diagnosis of head and neck squamous cell carcinoma (HNSCC). Here, we describe an approach for cancer detection based on analysis of patterns of serum immunoreactivity against a panel of biomarkers selected using microarray-based serologic profiling and specialized bioinformatics. We biopanned phage display libraries derived from three different HNSCC tissues to generate 5,133 selectively cloned tumor antigens. Based on their differential immunoreactivity on protein microarrays against serum immunoglobulins from 39 cancer and 41 control patients, we reduced the number of clones to 1,021. The performance of a neural network model (Multilayer Perceptron) for cancer classification on a data set of 80 HNSCC and 78 control samples was assessed using 10-fold cross-validation repeated 100 times. A panel of 130 clones was found to be adequate for building a classifier with sufficient sensitivity and specificity. Using these 130 markers on a completely new and independent set of 80 samples, an accuracy of 84.9% with sensitivity of 79.8% and specificity of 90.1% was achieved. Similar performance was achieved by reshuffling of the data set and by using other classification models. The performance of this classification approach represents a significant improvement over current diagnostic accuracy (sensitivity of 37% to 46% and specificity of 24%) in the primary care setting. The results shown here are promising and show the potential use of this approach toward eventual development of diagnostic assay with sufficient sensitivity and specificity suitable for detection of early-stage HNSCC in high-risk populations.     

Evaluation of soluble CD44v6 as a potential serum marker for head and neck squamous cell carcinoma.

In recent years, the measurement of soluble CD44 levels in the circulation of patients with malignant diseases has been introduced as a new and simple diagnostic tool for the detection of human cancer. The high CD44v6 expression in head and neck squamous cell carcinoma (HNSCC) would enable the use of soluble CD44v6 proteins present in the circulation of HNSCC patients as a marker of disease. In the present study, we determined CD44v6 plasma levels using a domain-specific ELISA in healthy volunteers, non-cancer patients, and HNSCC patients before and after surgical removal of the tumor. A difference between the CD44v6 plasma levels of HNSCC patients and controls could not be observed. Moreover, surgical removal of the tumor did not result in a reduction of the CD44v6 plasma level in the HNSCC patients. In addition, the spectrum of soluble v6-containing CD44 proteins present in the plasma of HNSCC patients and controls was determined by immunoprecipitation experiments, but again, tumor-related isoforms could not be distinguished in patient samples. Additional experiments to unravel the biological source of these circulating proteins indicated surprisingly that the v6-containing proteins present in the circulation of healthy individuals are only released in part, if at all, by activated lymphocytes or other nucleated blood cells. Most circulating CD44v6 proteins seem to be derived from the normal epithelial cell compartments, including breast cells, colon cells, and squamous cells. Taken together, these data do not support the use of soluble CD44v6 as a tumor marker in HNSCC or any other tumor type that has developed from tissues producing soluble isoforms.     

Analysis of 724 cases of primary head and neck squamous cell carcinoma (HNSCC) with a focus on young patients and p53 immunolocalization

This study evaluated 724 primary head and neck squamous cell carcinoma (HNSCC) in young and old patients, with regard to clinical profile and immunohistochemical expression of p53 protein. Associations among age, epidemiological and clinicopathological parameters, and survival analysis were evaluated. HNSCC in young people occurred in 14.5% (median age 40.7 years; male-to-female ratio 5.9:1). A statistical association was demonstrated between age and family history of cancer, and between age and anatomical site. Among older patients, a higher presence of disease was noted in posterior sites. Expression of p53 was found in 71.7% of the samples and a higher expression was noted in lesions of young patients. Survival analysis showed that the age parameter is not a reliable prognostic factor for HNSCC. Among young patients, cervical metastasis was associated with worse survival. The presence of a family history of cancer in young patients could indicate genetic susceptibility and molecular disturbances in the p53 pathway in HNSCC of young and older patients seem to be distinct.     

Identification of molecular targets for immunotherapy of patients with head and neck squamous cell carcinoma

To identify molecular targets for immunotherapy of head and neck squamous cell carcinoma (HNSCC) patients, we analyzed gene expression profile in matched tumor (HN) and normal fibroblast (FB) cell lines established from a HNSCC patient using microarray technique followed by real-time RT-PCR. Screening against a series of established normal and malignant cell lines followed by screening against a panel of normal human tissues led to the identification of 7 genes (AREG, CDH3, KLK10, NmU, SLPI, ANAX3 and MAL2), which were over-expressed at least 10-fold in tumors over any of the normal tissues. We determined the expression of mRNA encoding these genes against a panel of 15 HNSCC primary tumor samples. Relative expression of these genes was at least 20-fold. Expression of AREG, CDH3, KLK10, NmU and SLPI at the protein level was determined by immunohistochemistry in seven supraglottic laryngeal cancer specimens. All five proteins were expressed in these tumor samples with high intensity. We conclude that these molecules are potential targets for immunotherapy of HNSCC patients.     

Overexpression of cell cycle regulatory proteins correlates with advanced tumor stage in head and neck squamous cell carcinomas

Squamous cell carcinoma of the head and neck region (HNSCC) is the sixth most frequent cancer worldwide. In the USA, 30,000 new cases and 8,000 deaths are reported each year. Differences between normal epithelium and cancer cells from the upper aerodigestive tract arise from alterations in expression of specific genes controlling proliferation and immortalization. The protein products of these genes include growth factor receptors, cell cycle regulators, and tumor suppressors which affect a variety of intracellular signaling pathways. To determine how altered expression of these gene products contribute to HNSCC progression, we examined expression of epidermal growth factor receptor (EGFR), cyclins, p16INK4A, c-myc, proliferating cell nuclear antigen (PCNA), and telomerase in archival pathology specimens by immunohistochemistry. A substantial majority of HNSCC tumors showed loss of p16INK4A expression and dramatic overexpression of EGFR. Overexpression of this receptor correlated with increased cyclin A levels and high mitotic index. EGFR, cyclins A, -B1, -E, and c-myc overexpression was significantly increased in stage III and IV tumors compared to early stage cancers. hTERT was expressed in all tumors and primarily in the basal layer cells of dysplastic epithelial lesions. Suprabasal expression of hTERT was found in a significantly higher number of HNSCC cases than in dysplastic lesions. These results indicate that overexpression of cell cycle regulatory proteins correlates with advanced tumor stage in HNSCC.     

Expression of proinflammatory and proangiogenic cytokines in patients with head and neck cancer.

Altered immune, inflammatory, and angiogenesis responses are observed in patients with head and neck squamous cell carcinoma (HNSCC), and many of these responses have been linked with aggressive malignant behavior and a decrease in prognosis. In this study, we examined the hypothesis that HNSCC cells produce cytokines that regulate immune, inflammatory, and angiogenesis responses. We identified important regulatory cytokines in supernatants of well-defined and freshly cultured HNSCC cell lines by ELISA and determined whether these cytokines are detected in tumor cell lines and tissue specimens by immunohistochemistry. The serum concentration of the cytokines and cytokine-dependent acute phase inflammatory responses (i.e., fibrinogen, C-reactive protein, and erythrocyte sedimentation rate) from patients with HNSCC was determined, and the potential relationship of serum cytokine levels to tumor volume was analyzed. Cytokines interleukin (IL)-1alpha, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor were detected in similar concentration ranges in the supernatants of a panel of established University of Michigan squamous cell carcinoma (UM-SCC) cell lines and supernatants of freshly isolated primary HNSCC cultures. Evidence for the expression of IL-1alpha, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, and VEGF in HNSCC cells within tumor specimens in situ was obtained by immunohistochemistry. In a prospective comparison of the cytokine level and cytokine-inducible acute-phase proteins in serum, we report that cytokines IL-6, IL-8, and VEGF were detected at higher concentrations in the serum of patients with HNSCC compared with patients with laryngeal papilloma or age-matched control subjects (at P < 0.05). The serum concentrations of IL-8 and VEGF were found to be weakly correlated with large primary tumor volume (R2 = 0.2 and 0.4, respectively). Elevated IL-1- and IL-6-inducible acute-phase responses were also detected in cancer patients but not in patients with papilloma or control subjects (at P < 0.05). We therefore conclude that cytokines important in proinflammatory and proangiogenic responses are detectable in cell lines, tissue specimens, and serum from patients with HNSCC. These cytokines may increase the pathogenicity of HNSCC and prove useful as biomarkers or targets for therapy.     

The Akt inhibitor KP372-1 inhibits proliferation and induces apoptosis and anoikis in squamous cell carcinoma of the head and neck

Therapies that target signaling pathways critical to the pathogenesis and progression of squamous cell carcinoma of the head and neck (HNSCC) are needed. One such target, phosphatidylinositol 3-kinase, and its downstream target serine/threonine kinase, Akt, are up-regulated in HNSCC. Targeted therapy could consist of inhibitors of these kinases or, alternatively, of inhibitors of the pathways that they regulate. To explore the effect of Akt inhibition on the growth and survival of HNSCC tumors, we evaluated the effect of a novel Akt inhibitor, KP372-1, on the growth, survival, and sensitivity to anoikis of HNSCC cell lines in culture. Using Western blotting of head and neck cancer cell lines and squamous mucosa and carcinoma specimens, we found that Akt was highly phosphorylated in head and neck cancer cell lines and human head and neck squamous carcinoma specimens. Treatment of HNSCC cell lines with KP372-1 blocked the activation of Akt, inhibited head and neck cancer cell proliferation, and induced apoptosis and anoikis in several HNSCC cell lines. Furthermore, KP372-1 decreased the phosphorylation of the S6 ribosomal (Ser240/244) protein, which is a downstream target of Akt. Taken together, these findings indicate that KP372-1 may be a useful therapeutic agent for HNSCC and should be further evaluated in preclinical models of HNSCC.     

Cyclin A as a predictive factor for chemotherapy response in advanced head and neck cancer.

PURPOSE: Overall survival of head and neck squamous cell cancer (HNSCC) patients has not improved despite advances in our understanding of the biology and molecular features of this disease. In particular, patients with advanced HNSCC have the poorest prognosis. To understand more about the contribution of cell cycle alterations to HNSCC development and their possible value in predicting prognosis and response to chemotherapy, we evaluated the levels of proteins involved in cell cycle control in patients diagnosed with advanced HNSCC. EXPERIMENTAL DESIGN: A tissue microarray was made with 122 HNSCC specimens obtained from biopsy material. Protein expression was evaluated by immunohistochemistry and correlated with clinical and pathological characteristics. RESULTS: Multiple alterations at various checkpoints of cell cycle progression were observed. Loss of P16 protein was less common in oropharyngeal tumors than at other HNSCC locations (P = 0.02). Evaluation of the simultaneous expression of different proteins highlighted direct correlations (P < 0.05) such as that of the cyclin-dependent kinases with their cyclin-partners, and the Ki-67 protein with cyclin-dependent kinases 1, cyclin A (CA) and cyclin B1. Median overall survival and time-to-progression were longer in patients with CA-expressing tumors (not reached versus 34.4 months, P = 0.02) and (47.3 versus 14.6 months, P = 0.006), respectively. Moreover, expression of CA in tumors predicted a better response to chemotherapy. Positive expression of cyclin E in tumors was also associated with an increased median time-to-progression (14.6 versus 25.8 months, P = 0.04). Finally, patients with cyclin D1-expressing tumors had shorter median overall survival (29.6 months versus not reached, P = 0.05) and shorter median time-to-progression (21.5 months versus not reached, P = 0.06). However, in a multivariate analysis a CA-negative-expressing tumor was the only independent poor prognostic factor in the entire cohort of HNSCC patients [odds ratio, 2.3; 95% confidence interval (CI) = 1.2-4.5; P = 0.01]. CONCLUSIONS: Our results provide detailed information on the molecular profile of cell cycle components in HNSCCs and identify CA-negative-expressing tumors as an independent marker of tumor progression and poor response to chemotherapy in patients diagnosed with advanced HNSCC.