Prognosis Of Head And Neck Squamous Cell Carcinoma Research Articles

OP050: Expression of fascin in squamous cell carcinoma of the oral cavity: Clinicopathological, prognostic significance and cell line study

Head and neck squamous cell carcinoma (HNSCC) is one of the most common head and neck cancers. The prognosis of HNSCC is usually poor because of extensive local invasion at initial diagnosis. Fascin, an actin-bundling protein, has been reported responsible for cell motility and overexpression of Fascin contributes to an unfavorable clinical course in several malignancies. Nevertheless, the roles of Fascin protein playing in aggressiveness of HNSCC and their potential mechanisms need to be elucidated. Tca8113(cell lines of OSCC) via the vector-based small interfering RNA (siRNA) to suppress the expression of the Fascin gene were used. Subsequent analyses and observation regarding the expression of Fascin protein and cyto-morphological alterations were detected by Western blot and Boyden chamber invasion assay, cell migration assay and adhesion assay were also applied to investigate the functional changes of Tca8113. Paraffin sections using tissue microarrays of 84 patients with OSCC were investigated immunohistochemically, to determine the level of expression of Fascin labelling index in tumor specimens. The role of Fascin in OSCC was evaluated by correlation with clinical-pathological parameters. There were statistically significant differences ( P < 0.05) of Fascin expression before and after silencing. Down-regulation of Fascin protein directly resulted in suppression of migration, invasion and increase of adhesion in cell line ( P < 0.05). Overexpression of Fascin was significantly associated with regional lymph node metastases ( P = 0.044),advanced T stage (T1 vs T2-4, P = 0.046) advanced tumor stage (I/II vs III/IV, P = 0.049), histologic grade (well vs moderately/poorly, P = 0.018). In 54 patients with tongue carcinoma, overexpression of Fascin was significantly associated with muscle invasion ( P = 0.002). Our results suggest that expression of Fascin protein may play an essential role in regulation of progression of OSCC. Overexpression of Fascin contributes to a more aggressive clinical course and suggests that Fascin has the potential of being a new molecular target for therapeutic intervention.

Lysophosphatidic acid receptor 4 signaling potentially modulates malignant behavior in human head and neck squamous cell carcinoma cells

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common non-skin cancer worldwide. Despite improvement in therapeutic strategies, the prognosis of advanced HNSCC remains poor. The extacellular lipid mediators known as lysophosphatidic acids (LPAs) have been implicated in tumorigenesis of HNSCC. LPAs activate G-protein-coupled receptors not only in the endothelial differentiation gene (Edg) family (LPA1, LPA2, LPA3) but also in the phylogenetically distant non-Edg family (LPA4, LPA5, LPA6). The distinct roles of these receptor isoforms in HNSCC tumorigenesis have not been clarified. In the present study, we investigated the effect of ectopic expression of LPA4 in SQ-20B, an HNSCC cell line, expressing a trivial level of endogenous LPA4. LPA (18:1) stimulated proliferation of SQ-20B cells, but did not affect proliferation of HEp-2, an SCC cell line expressing higher levels of LPA4, comparable to those of with LPA1. LPA-stimulated proliferation of SQ-20B cells was attenuated by Ki16425 and Rac1 inhibitor, but not by Y-27632. Infection with doxycycline-regulatable adenovirus vector expressing green fluorescent protein-tagged LPA4 (AdvLPA4G) abolished LPA-stimulated proliferation in SQ-20B cells with the accumulation of G2/M-phasic cells. Ectopic LPA4 induction further downregulated proliferation of Ki16425-treated SQ-20B cells, of which downregulation was partially recovered by LPA. Ectopic LPA4 induction also downregulated proliferation of Rac1 inhibitor-treated SQ-20B cells, however, LPA no longer recovered it. Finally, LPA-induced cell motility was suppressed by ectopic LPA4 expression as well as by Ki16425, Rac1 inhibitor or Y-27632. Our data suggest that LPA4 signaling potentially modulates malignant behavior of SQ-20B cells. LPA signaling, which is mediated by both Edg and non-Edg receptors, may be a determinant of malignant behavior of HNSCC and could therefore be a promising therapeutic target.

The Tumor Microenvironment Contribution to Development, Growth, Invasion and Metastasis of Head and Neck Squamous Cell Carcinomas

Head and neck squamous cell carcinoma (HNSCC) is a complex tissue that contains tumor cells and the surrounding stroma, which is populated by different types of mesenchymal cells and the extracellular matrix (ECM). Collectively, they are referred to as the tumor microenvironment (TME). Recent studies have shown that TME has a more profound influence on the growth and metastasis of HNSCC than was previously appreciated. Because carcinoma-associated fibroblasts (CAFs) are frequently observed in the stroma of the tumor, this review focuses on the potential role of tumor-CAFs interactions in progression of HNSCC. Tumor-CAFs crosstalk enhances the production of growth factors, cytokines, chemokines, matrix metalloproteinases (MMPs), and inflammatory mediators, which eventually facilitates tumor growth. In fact, factors and cells that do not support tumor growth are usually down regulated or mitigated in TME. Therefore TME may determine the fate of the tumors at the site of invasion and metastasis. For tumor cells that survive at these sites, stromal activation may serve to establish a supportive tumor stroma, fostering the outgrowth of the metastatic cells. The concept of tumor-stromal interactions and microenvironmental niche has profound consequences in tumor growth and metastasis and therefore, it's understanding will open up new strategies for the diagnosis, prognosis and therapy of HNSCC.

Prognostic value of human papillomavirus and squamous cell carcinoma antigen in head and neck squamous cell carcinoma

To clarify the synergistic influence of human papillomavirus (HPV) status and squamous cell carcinoma antigen (SCCA) mRNA expression on head and neck squamous cell carcinoma (HNSCC) prognosis, HPV DNA presence and SCCA1 and SCCA2 mRNA expression were determined by PCR and quantitative real-time RT-PCR, respectively, in 121 patients with primary HNSCC who were receiving curative treatment. HPV DNA was detected in 28.1% (34/121) of HNSCC cases, and only high-risk types (HPV-16, HPV-33, HPV-35 and HPV-58) were observed. Positive HPV status showed a significantly better prognosis than negative HPV status (P = 0.022). An elevated SCCA2/SCCA1 mRNA ratio was an independent predictor of disease recurrence (P = 0.004). In addition, HPV-negative patients with a high SCCA2/SCCA1 ratio (>0.27) had a significantly lower recurrence-free survival rate than HPV-negative patients with a low SCCA2/SCCA1 ratio (P < 0.011). Our findings revealed that both HPV status and the SCCA2/SCCA1 mRNA ratio are independently associated with prognosis in HNSCC. Patients with both a HPV-negative status and a high SCCA2/SCCA1 ratio might need intensified treatment and rigorous follow up after treatment because of the high risk of recurrence.

PRAME expression in head and neck cancer correlates with markers of poor prognosis and might help in selecting candidates for retinoid chemoprevention in pre-malignant lesions

PRAME (Preferentially Expressed Antigen in Melanoma) is a tumor-associated antigen recognized by immunocytes, and it induces cytotoxic T cell-mediated responses in melanoma. PRAME expression in tumors interferes with retinoic acid receptor (RAR) signaling thus promoting tumor progression. Here, we study PRAME expression in head and neck squamous cell carcinoma (HNSCC) to determine its potential clinical significance. PRAME expression in HNSCC was evaluated by immunohistochemistry in tissue microarrays of primary tumors (n=53), metastatic lymph nodes (n=8) and normal oral mucosa (n=11). Biopsies of dysplastic oral lesions (n=12) were also examined. PRAME expression levels in tissues were correlated with markers of poor prognosis in HNSCC. PRAME mRNA in HNSCC cell lines and in normal immortalized human keratinocytes (HaCaT cell line) was measured by qRT-PCR, and the protein expression by flow cytometry and western blots. PRAME was expressed in HNSCC cell lines and HNSCC lesions. PRAME expression in dysplastic mucosa was variable. No or only weak expression was found in normal cells or tissues. PRAME expression levels significantly correlated with the tumor grade, size, nodal involvement and the clinical status of HNSCC patients. Elevated PRAME expression associates with clinicopathologic markers of poor outcome in HNSCC and might identify potential candidates with pre-cancerous lesions for chemoprevention with retinoids.

The pivotal role of integrin β1 in metastasis of head and neck squamous cell carcinoma.

This study aimed to understand the prognostic value of integrin β1 expression in head and neck squamous cell carcinoma (HNSCC) and the mechanism underlying its association with metastatic HNSCC. Archival HNSCC tissues including 99 nonmetastatic primary tumors and 101 metastatic primary tumors were examined for the association of integrin β1 expression with metastasis and disease prognosis by appropriate statistical methods. Fluorescence-activated cell sorting was used to separate the integrin β1(high/+) cell population from the integrin β1(low/-) population in HNSCC cell lines. These two populations and integrin β1 shRNA knockdown HNSCC cells were examined for the effect of integrin β1 on invasion in vitro and on lymph node and lung metastases in a xenograft mouse model. Expression and activation of matrix metalloproteinases (MMP) were examined by zymography. Statistical analysis showed that integrin β1 expression was significantly higher in the metastatic primary tumors than in the nonmetastatic tumors (42.6% vs. 24.8%, P < 0.0001 and P < 0.0001 by univariate and multivariate analyses, respectively). In patients with lymph node metastasis, integrin β1 expression was inversely correlated with overall survival (P = 0.035). The integrin β1 knockdown or integrin β1(low/-) HNSCC cells showed a significant reduction in lymph node and lung metastases in vivo (P < 0.001 and P < 0.05, respectively). Significantly reduced Matrigel invasion capability was also found in integrin β1 knockdown or integrin β1(low/-) HNSCC cells (P < 0.01). Finally, zymography results showed integrin β1-affected HNSCC invasion by regulating MMP-2 activation. These findings indicate that integrin β1 has a major impact on HNSCC prognosis through its regulation of metastasis.

Abstract 727: Homozygosity for Arg of p53 codon 72 correlates with poor prognosis in Puerto Rican patients with head and neck squamous cell carcinoma

Abstract Objective: Genetic variations at codon 72 of p53 gene results in altered activities for p53 and are suggested as a potential cancer risk. However, epidemiologic findings have been inconsistent. The p53 gene has a polymorphic site at exon 4 codon 72, which, in an occurrence of a base substitution, can produce Proline or Arginine (Arg72Pro). This polymorphism is differentially dispersed among different ethnic populations. The objective of this study is to evaluate the association between p53 polymorphism and head and neck squamous cell carcinoma (HNSCC) in the Puerto Rican population. Methods: Genotyping of the p53 polymorphism at exon 4 codon 72 was performed using PCR-based restriction fragment polymorphism (PCR-RFLP) in a study with 72 HNSCC patients. Correlations between clinicopathological parameters and p53 polymorphism were analyzed by Fisher's exact test and chi-square when suitable. Survival analysis of HNSCC patients, with each type of p53 genotype, was performed using Mantel-Cox test. Results: The frequencies for homozygote Arg allele were 34.7% in HNSCC cases and 31.1% in controls. The Pro allele carriers (Arg/Pro or Pro/Pro) had a frequency of 65.3% in HNSCC cases and 68.9% in healthy controls. The OR of HNSCC cancer-associated with the Arg-Arg genotypes of codon 72 was 1.18 [95% confidence interval (CI): 0.53-2.61]. Survival analysis showed that HNSCC patients with the Arg/Arg allele had a poorer survival rate compared to patients with the Pro allele (Hazard Ratio = 2.40, 95% CI: 1.27-4.56, P=0.0074). Also, there were differences in survival based on patient's genotype and response to treatment. However, no statistically significant association was found between p53 polymorphic variants and gender, age, risk factors and staging. Conclusion: Our results suggest that homozygosity for Arg of p53 Arg72Pro may have a role in the progression of HNSCC in the Puerto Rican population. Detection of the p53 polymorphic variants may be used as an indicator of prognosis in HNSCC patients. Accurate prognosis in HNSCC will help clinicians to establish proper treatment and increase the survival of HNSCC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 727. doi:1538-7445.AM2012-727

Abstract 726: Copy number amplification of the PIK3CA gene is associated with poor prognosis in early-stage head and neck squamous cell carcinoma

Abstract Background: Deregulation of the EGFR signaling pathway is one of the most frequently observed genetic abnormalities that drive cancer development. Mutations in downstream components of the EGFR signaling pathway, including KRAS, BRAF and PIK3CA, have been reported in numerous cancers. However, extensive mutation analysis of these genes in clinical samples has not been performed for head and neck squamous cell carcinoma (HNSCC). Patient and methods: We examined mutations and copy number alterations of KRAS, BRAF and PIK3CA in 115 clinical specimens of HNSCC obtained from surgically treated patients. Results: We identified three mutations in K-RAS and two mutations in PIK3CA. Copy number amplification was found in 42 cases (36.4%) for PIK3CA, 12 cases (10.4%) for K-RAS and nine cases (7.8%) for BRAF. Tumor recurrence and death occurred in 49 (42.6%) and in 22 (19.3 %) patients during the median follow-up of 723 days, respectively. Kaplan-Meier survival analysis revealed that in patients with early-stage carcinoma (Stages I-III), copy-number amplification of PIK3CA was drastically associated with cancer relapse. Conclusion: Copy number amplification of the PIK3CA is associated with poor prognosis in early-stage HNSCC. The PIK3CA copy number status will serve as a marker of poor prognosis in patients with HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 726. doi:1538-7445.AM2012-726

Metformin suppresses growth of human head and neck squamous cell carcinoma via global inhibition of protein translation

Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer in the world; the main risk factors are alcohol and tobacco use. Advancements in therapies have yet to improve the prognosis of HNSCC. The connection between diabetes and cancer is being recognized, and metformin has been shown to decrease cancer incidence in diabetic patients. Accordingly, here, for the first time, we investigated metformin’s efficacy on the growth and viability of human HNSCC FaDU and Detroit cells. Our results show that metformin treatment (5–20 mM) dose-dependently inhibits the growth of both cell lines. In FaDU cells, metformin caused 18–57% and 35–81% growth inhibition after 48 and 72 h treatments, respectively. Similarly, in Detroit 562 cells, 48 and 72 h metformin treatment resulted in 20–57% and 33–82% inhibition, respectively. Mechanistically, metformin caused G1 arrest, which coincided with a decrease in the protein levels of CDKs (2, 4 and 6), cyclins (D1 and E) and CDK inhibitors (p15, p16, p18 and p27), but no change in p19 and p21. Metformin also decreased the levels of oncogenic proteins Skp2 and β-Trcp. In other studies, metformin decreased the phosphorylation of 4E-BP1 at Ser65, Thr37/46 and Thr70 sites, but drastically increased the phosphorylation of EF2 at Thr56 and AMPK at Thr172, which results in global translational inhibition. In summary, the observed wide spectrum of mechanistic effects of metformin on HNSCC cells provides support for the anticancer capability of the drug and its potential use in future therapies.

Prognostic Significance of Plasma Osteopontin in Patients with Locoregionally Advanced Head and Neck Squamous Cell Carcinoma Treated on TROG 02.02 Phase III Trial

High plasma osteopontin (OPN) levels have been reported to be an adverse prognostic factor in head and neck squamous cell carcinomas (HNSCC), correlate with tumor hypoxia, and be predictive of benefit from hypoxia-targeted therapy. We sought to confirm the prognostic and predictive significance of OPN in patients treated on a large international trial. Patients with stage III/IV HNSCC were randomized to receive definitive radiotherapy concurrently with cisplatin or cisplatin plus the hypoxic cell cytotoxin, tirapazamine (TPZ). Eligibility criteria for this prospective substudy included plasma sample availability for OPN assay by ELISA and absence of major radiation therapy deviations (N = 578). OPN concentrations were analyzed for overall survival (OS) and time to locoregional failure (TTLRF), adjusting for known prognostic factors. Additional analysis was carried out in patients with available tumor p16(INK4A) staining status. The median OPN level was 544 ng/mL (range: 7-2,640). High OPN levels were not associated with worse OS (relative HR, 1.03 for highest tertile) or TTLRF (relative HR 0.91 for highest tertile). There was no interaction between OPN and treatment arm for OS or TTLRF (P = 0.93 for OS; P = 0.87 for TTLRF). For the highest tertile the 2-year OS was 66% on control arm and 67% on TPZ arm (HR = 1.11, P = 0.67). Similarly for p16(INK4A) negative patients in the highest tertile, the 2-year OS was 61% on control arm and 63% on TPZ arm (HR = 1.05, P = 0.86). We found no evidence that high plasma OPN levels were associated with an adverse prognosis in HNSCC, or were predictive of benefit with hypoxia targeting therapy.

Plectin promotes migration and invasion of cancer cells and is a novel prognostic marker for head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is usually found at a late stage and distant metastasis occurs at high frequency; therefore, novel prognostic markers are needed. This study was aimed to identify novel tumor markers in HNSCC. We identified 65 proteins which were significantly increased or decreased in the tumors by 2D-DIGE using 12 HNSCC and adjacent non-cancer tissues. Western blotting and immunohistochemical analysis confirmed that the expression of plectin was significantly increased in most cancer tissues as compared with non-cancer tissues. Strikingly, the suppression of endogenous plectin using siRNA inhibited the proliferation, migration and invasion of HNSCC cells and down-regulated Erk 1/2 kinase. Furthermore, immunohistochemistry using paraffin-embedded tissues from 62 patients showed not only that the frequency of recurrence was correlated with the plectin expression but that the prognosis of patients with a high plectin was extremely poor. Moreover, the survival rate of patients with a high plectin was significantly lower than that of patients with low E-cadherin levels, which is known to correlate with the poor prognosis of HNSCC. Our findings suggest that plectin promotes the migration and invasion of HNSCC cells through activation of Erk 1/2 kinase and is a potential prognostic biomarker of HNSCC.

Expression of Y-box-binding protein YB-1 allows stratification into long- and short-term survivors of head and neck cancer patients

Background:Histology-based classifications and clinical parameters of head and neck squamous cell carcinoma (HNSCC) are limited in their clinical capacity to provide information on prognosis and treatment choice of HNSCC. The primary aim of this study was to analyse Y-box-binding protein-1 (YB-1) protein expression in different grading groups of HNSCC patients, and to correlate these findings with the disease-specific survival (DSS).Methods:We investigated the expression and cellular localisation of the oncogenic transcription/translation factor YB-1 by immunohistochemistry on tissue micro arrays in a total of 365 HNSCC specimens and correlated expression data with clinico-pathological parameters including DSS.Results:Compared with control tissue from healthy individuals, a significantly (P<0.01) increased YB-1 protein expression was observed in high-grade HNSCC patients. By univariate survival data analysis, HNSCC patients with elevated YB-1 protein expression had a significantly (P<0.01) decreased DSS. By multivariate Cox regression analysis, high YB-1 expression and nuclear localisation retained its significance as a statistically independent (P<0.002) prognostic marker for DSS. Within grade 2 group of HNSCC patients, a subgroup defined by high nuclear and cytoplasmic YB-1 levels (co-expression pattern) in the cells of the tumour invasion front had a significantly poorer 5-year DSS rate of only 38% compared with overall 55% for grade 2 patients. Vice versa, the DSS rate was markedly increased to 74% for grade 2 cancer patients with low YB-1 protein expression at the same localisation.Conclusion:Our findings point to the fact that YB-1 expression in combination with histological classification in a double stratification strategy is superior to classical grading in the prediction of tumour progression in HNSCC.

Abstract B40: micro-RNA-128 regulated gene expression in head and neck squamous cell carcinoma

Abstract Purpose: Each year in the United States there are more than 65,000 Americans diagnosed with head and neck cancer and 25,000 die from this disease. Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease with complex molecular abnormalities. African Americans have an incidence approximately twice higher than Caucasian men. The overexpression of BMI1 polycomb ring finger oncogene (BMI-1) and epidermal growth factor receptor (EGFR) in HNSCC cases has been shown to be an indicator of poor prognosis and survival in HNSCC. MicroRNAs are a class of posttranslational regulators of gene expression and have been shown to regulate a number of biological processes. In this study, we investigate that mechanism of microRNA-128 (miR-128) involved the regulation of BMI-1 and EGFR gene expressions in HNSCC. Methods: We generated stably expressing exogenous miRNA-128 in HNSCC of the Larynx (JHU-22) using lentivirus infection, performed colony formation assays to determine cell growth and proliferation, QRT-PCR to analyze gene expression, and western blot analysis to evaluate protein expression. Results: The transfected JHU-22miR-128 cell line stably expressed exogenous miR-128 in vitro and in vivo tumor xenografts. Compared with the control vector cells, the cell growth and proliferation decreased significantly in JHU-22miR-128 cells and JHU-22miR-128 tumor xenografts. However, the level of BMI-1proteins was decreased more than EGFR in transfected JHU-22miR-128 cells. Conclusion: miR-128 is able to significantly inhibit BMI-1protein expression, partially affects EGFR protein expression. miR-128 has a putative target site within the mRNA 3′UTR of Bmi-1 and EGFR. Therefore, miR-128 regulates the proliferative pathway in HNSCC. MiR −128 have potential to serve as a therapeutic approach to specifically target EGFR in HNSCC. This work was supported in part by grants P20 CA118770 from National Cancer Institute. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B40.

Association of FANCC and PTCH1 with the Development of Early Dysplastic Lesions of the Head and Neck

Alteration of chromosome 9q22.3 region is an early and frequent event in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to understand the association of candidate tumor suppressor genes PHF2, FANCC, PTCH1, and XPA located in this region in the development of HNSCC. The alterations (deletion, promoter methylation, mutation, expression) of these genes were analyzed in 65 dysplastic head and neck lesions and 84 primary HNSCC samples. Clinicopathologic correlations were made with alterations of the genes. Overall alterations (deletion, promoter methylation) of FANCC and PTCH1 were high in mild dysplasia and comparable in subsequent stages of tumor progression. However, PHF2 alteration was low in mild dysplasia, but increased in moderate and severe dysplasias. Alterations (deletion, promoter methylation) of FANCC and PTCH1 showed association with each other. Two novel mutations in GLI binding sites of PTCH1 promoter and a novel microsatellite marker hmPTCH1 with four alleles at immediate upstream of the gene were identified. In a case-control study, the (CGG)7 allele of hmPTCH1 was found to be susceptible for HNSCC development. Concordance was seen in the expression (RNA, protein) of these genes with their molecular alterations. Alterations of FANCC and PTCH1 could be used as molecular marker for early diagnosis and prognosis of HNSCC.

Education, tobacco smoking, alcohol consumption, and IL-2 and IL-6 gene polymorphisms in the survival of head and neck cancer

The association of education, tobacco smoking, alcohol consumption, and interleukin-2 (IL-2 +114 and -384) and -6 (IL-6 -174) DNA polymorphisms with head and neck squamous cell carcinoma (HNSCC) was investigated in a cohort study of 445 subjects. IL-2 and IL-6 genotypes were determined by real-time PCR. Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (95%CI) of disease-specific survival according to anatomical sites of the head and neck. Mean age was 56 years and most patients were males (87.6%). Subjects with 5 or more years of schooling had better survival in larynx cancer. Smoking had no effect on HNSCC survival, but alcohol consumption had a statistically significant effect on larynx cancer. IL-2 gene +114 G/T (HR = 0.52; 95%CI = 0.15-1.81) and T/T (HR = 0.22; 95%CI = 0.02-3.19) genotypes were associated with better survival in hypopharynx cancer. IL-2 +114 G/T was a predictor of poor survival in oral cavity/oropharynx cancer and larynx cancer (HR = 1.32; 95%CI = 0.61-2.85). IL-2 -384 G/T was associated with better survival in oral cavity/oropharynx cancer (HR = 0.80; 95%CI = 0.45-1.42) and hypopharynx cancer (HR = 0.68; 95%CI = 0.21-2.20), but an inverse relationship was observed for larynx cancer. IL-6 -174 G/C was associated with better survival in hypopharynx cancer (HR = 0.68; 95%CI = 0.26-1.78) and larynx cancer (HR = 0.93; 95%CI = 0.42-2.07), and C/C reduced mortality in larynx cancer. In general, our results are similar to previous reports on the value of education, smoking, alcohol consumption, and IL-2 and IL-6 genetic polymorphisms for the prognosis of HNSCC, but the risks due to these variables are small and estimates imprecise.

Application of the Hybrid Capture 2 assay to squamous cell carcinomas of the head and neck

A growing proportion of head and neck squamous cell carcinoma (HNSCC) is caused by the human papillomavirus (HPV). In light of the unique natural history and prognosis of HPV-related HNSCCs, routine HPV testing is being incorporated into diagnostic protocols. Accordingly, there is an escalating demand for an optimal detection strategy that is sensitive and specific, transferrable to the diagnostic laboratory, standardized across laboratories, cost-effective, and amenable to broad application across specimen types including cytologic preparations. Cytologic preparations (fine-needle aspirates [FNAs] and brushes) were obtained from surgically resected HNSCCs and evaluated for the presence of high-risk HPV using the Hybrid Capture 2 assay. HPV analysis was also performed on the corresponding tissue sections using HPV in situ hybridization and p16 immunohistochemistry. In cases in which the immunohistochemical and in situ hybridization results were discordant, HPV status was determined by real-time polymerase chain reaction detection of E7 expression. HPV status in the tissues and corresponding cytologic samples was compared. Based on benchmark HPV testing of the tissue sections, 14 HNSCCs were classified as HPV positive and 10 as HPV negative. All corresponding cytologic preparations were correctly classified using the Hybrid Capture 2 assay. The Hybrid Capture 2 strategy, already widely used for the detection of high-risk HPV in cervical brushes, is readily transferrable to HNSCCs. Consistent accuracy in cytologic preparation suggests its potential application in FNAs from patients who present with lymph node metastases, and may eliminate the need to obtain tissue solely for the purpose of HPV testing.

Relationship between Epidermal Growth Factor Receptor Status, p16INK4A, and Outcome in Head and Neck Squamous Cell Carcinoma

Human papilloma virus (HPV) infection is a powerful prognostic biomarker in head and neck squamous cell carcinoma (HNSCC). Increased epidermal growth factor receptor (EGFR) gene copy number and protein expression have been reported to be negative predictors of outcome. This study examined the relationship between HPV status, EGFR gene copy number, EGFR protein expression, and clinical outcome in HNSCC patients treated with chemoradiation. HPV status was determined using p16(INK4A) immunohistochemistry (IHC), EGFR gene copy number was evaluated with FISH, and EGFR protein expression by IHC in 212 subjects. EGFR FISH was positive in 41 of 204 (20%) patients and was negatively correlated with failure-free survival (FFS; HR = 1.84, P = 0.027) and overall survival (OS; HR = 1.78, P = 0.082). For p16(INK4A), 85 of 200 (42.5%) patients were found to be p16 positive, including 75 of 131 (57%) with oropharyngeal cancer. Patients with p16-positive oropharyngeal cancer had significantly improved FFS (HR = 0.28, P < 0.001) and OS (HR = 0.31, P = 0.002). Only 2 of 126 (1.6%) oropharyngeal cancer patients were found to be p16+/EGFR FISH+. EGFR IHC was positive in 81 of 93 (87%) of patients and was associated with poorer FFS (HR = 1.98, P = 0.35) and OS (HR = 2.52, P = 0.22). Increased EGFR gene copy number is largely restricted to p16(INK4A)-negative oropharyngeal cancer. Although p16(INK4A) and EGFR FISH are both predictive of outcome in univariate analyses, only p16(INK4A) remains independently predictive. Knowledge of HPV and EGFR status can have implications for treatment options and prognosis in HNSCC.

Prognostic significance of plasma osteopontin in patients with locally advanced head and neck squamous cell carcinoma (HNSCC) treated with chemoradiation on TROG 02.02 phase III trial.

5506 Background: High plasma osteopontin (OPN) levels have been reported to be an adverse prognostic factor in HNSCC, to correlate with tumor hypoxia and to be predictive of benefit from hypoxia targeted therapy. We sought to confirm the prognostic and predictive significance of OPN in patients treated on a large international trial. Methods: Patients with previously untreated Stage III or IV HNSCC were randomized to receive definitive radiotherapy concurrently with cisplatin or cisplatin plus the hypoxic cell cytotoxin, tirapazamine (TPZ). As previously reported there were no significant differences in any outcome parameter by randomization arm. Eligibility criteria for this prospective substudy included availability of plasma samples and absence of major radiation therapy deviations. Plasma OPN was measured by ELISA. OPN concentrations according to tertiles and median were analysed for overall survival (OS), and time to locoregional failure (TTLRF), adjusting for prognostic factors (site, T category, N category, hemoglobin, performance status). An additional analysis was performed in patients with available p16 immunohistochemistry results. Results: 578/853 patients were eligible and had a median OPN level of 544 µg/L (range: 7 - 2640). High OPN levels were not associated with worse overall survival (HR 1.03 for highest tertile and 0.98 for middle tertile, p=0.95) or time to locoregional failure (HR 0.91 for highest and 0.93 for middle, p=0.91). There was no interaction between OPN and treatment arm for OS or TTLRF (p=0.51 for both). For the highest tertile the 2-year OS was 70% on control arm and 71% on TPZ arm (HR=1.11, p=0.67). Similarly for p16 negative patients in the highest tertile, the 2 year OS was 69% on control arm and 73% on TPZ arm (HR=1.13, p=0.82). Conclusions: In contrast to previous reports we found no evidence that high plasma OPN levels were associated with an adverse prognosis in HNSCC, or were predictive of benefit with hypoxia targeting therapy. Supported by 1 R01 CA118582 and NHMRC project grant.

Persistent Krüppel‐like factor 4 expression predicts progression and poor prognosis of head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is one prevalent human cancer worldwide. No molecular markers are presently used for predicting prognosis in HNSCC. Krüppel-like factor 4 (KLF4) is a transcription factor with diverse physiological functions, and possesses opposing roles in different human cancers. The expression and roles of KLF4 in HNSCC remain to be elucidated. In this study, immunohistochemical (IHC) analysis of KLF4 in 62 HNSCC was firstly performed. IHC results demonstrated that 42 (67.7%) had decreased KLF4 expression compared with surrounding normal epithelium, while persistent KLF4 expression was demonstrated in 20 (32.3%). The IHC results were further verified by Western blot and real-time PCR analyses to confirm the robustness of staining and interpretation. Interestingly, persistent KLF4 expression independently correlated with a worse disease-specific survival (P = 0.005), especially in patients with advanced disease. In consistent with clinical observation, all five HNSCC cell lines tested revealed a low level of baseline KLF4 expression. Moreover, enforced KLF4 expression in cell line SAS significantly increased in vitro migration/invasion abilities, multi-drug resistance, and in vivo tumorigenicity. These results clearly illustrate that persistent KLF4 expression predicts poor prognosis and confers aggressiveness in HNSCC. Our data therefore provides valuable information that HNSCC with persistent KLF4 expression might require intensified combination treatment in future practice.

Inhibition of EGFR-STAT3 Signaling with Erlotinib Prevents Carcinogenesis in a Chemically-Induced Mouse Model of Oral Squamous Cell Carcinoma

Chemoprevention of head and neck squamous cell carcinoma (HNSCC), a disease associated with high mortality rates and frequent occurrence of second primary tumor (SPT), is an important clinical goal. The epidermal growth factor receptor (EGFR)-signal transducer and activator of transcription (STAT)-3 signaling pathway is known to play a key role in HNSCC growth, survival, and prognosis, thereby serving as a potential therapeutic target in the treatment of HNSCC. In the current study, the 4-nitroquinoline-1-oxide (4-NQO)-induced murine model of oral carcinogenesis was utilized to investigate the chemopreventive activities of compounds that target the EGFR-STAT3 signaling pathway. This model mimics the process of oral carcinogenesis in humans. The drugs under investigation included erlotinib, a small molecule inhibitor of the EGFR, and guggulipid, the extract of an Ayurvedic medicinal plant, which contains guggulsterone, a compound known to inhibit STAT3. Dietary administration of guggulipid failed to confer protection against oral carcinogenesis. On the other hand, the mice placed on erlotinib-supplemented diet exhibited a 69% decrease (P < 0.001) in incidence of preneoplastic and neoplastic lesions compared with mice on the control diet. Immunostaining of dysplastic lesions demonstrated modest decreases in STAT3 levels, with both drug treatments, that were not statistically significant. The results of the present study provide the basis for exploring the efficacy of erlotinib for prevention of HNSCC in a clinical setting.