Neck Cancer Specimens Research Articles

Augmented Reality-Guided Frozen Section Analysis: Bringing the Pathologist From the Laboratory to the Operating Room.

Due to the anatomic complexity of the head and neck and variable proximity between laboratory and operating room (OR), effective communication during frozen section analysis (FSA) between surgeons and pathologists is challenging. This proof-of-concept study investigates an augmented reality (AR) protocol that allows pathologists to virtually join the OR from the laboratory. Head and neck cancer specimens were scanned ex vivo using a 3-dimensional scanner and uploaded into an AR platform. Eight head and neck specimens were discussed by surgeons and pathologists in an AR environment. AR-guided intraoperative consultation was used for specimen orientation and discussion of FSA margin sampling sites. One patient had positive initial margins on FSA andwas re-resected to negative final margins. AR-guided FSA is possible and allows pathologists to join the operating from any location for intraoperative discussion.

Augmented-Reality Surgery to Guide Head and Neck Cancer Re-resection: A Feasibility and Accuracy Study.

Given the complex three-dimensional (3D) anatomy of head and neck cancer specimens, head and neck surgeons often have difficulty relocating the site of an initial positive margin to perform re-resection. This cadaveric study aimed to determine the feasibility and accuracy of augmented reality surgery to guide head and neck cancer re-resections. This study investigated three cadaveric specimens. The head and neck resection specimen was 3D scanned and exported to the HoloLens augmented reality environment. The surgeon manually aligned the 3D specimen hologram into the resection bed. Accuracy of manual alignment and time intervals throughout the protocol were recorded. The 20 head and neck cancer resections performed in this study included 13 cutaneous and 7 oral cavity resections. The mean relocation error was 4 mm (range, 1-15 mm) with a standard deviation of 3.9 mm. The mean overall protocol time, from the start of 3D scanning to alignment into the resection bed, was 25.3 ± 8.9 min (range, 13.2-43.2 min). Relocation error did not differ significantly when stratified by greatest dimension of the specimen. The mean relocation error of complex oral cavity composite specimens (maxillectomy and mandibulectomy) differed significantly from that of all the other specimen types (10.7 vs 2.8; p < 0.01). This cadaveric study demonstrated the feasibility and accuracy of augmented reality to guide re-resection of initial positive margins in head and neck cancer surgery.

Effect of formalin fixation on tumour size and margins in head and neck cancer specimens.

To quantify tumour size and margin shrinkage due to formalin fixation in head and neck cancer specimens and determine its effect on tumour staging and margin clearance. Tumour specimens were measured immediate post-resection and at 24 hours after fixation. Tumour was measured in 2 dimensions and one margin was measured. Shrinkage was categorised as < 10%, 10-20%, and > 20%. Effect of shrinkage on tumour stage and margin clearance were evaluated. A total of 50 specimens were analysed. The tumour AP (Anterior Posterior) and SI (Superior Inferior) measurements showed significant shrinkage with a mean difference of 22.93% and 21.69% respectively. > 20% shrinkage was noted in 78% of AP and 58% of SI measurements. Margins showed a mean difference of 25.61%. 84% of margins demonstrated > 20% shrinkage. In all, 46.7% of T3 and 23% of T2 tumours pre-fixation were downstaged to T2 and T1, respectively, post-fixation. Formalin fixation alone can be responsible for significant shrinkage of tumour and margin dimensions in head and neck specimens. It is suggested that decisions regarding the treatment plan should be made on clinical staging of primary tumour rather than pathological staging. In addition, post-excision pre-fixation margins should be considered for treatment planning.

β2-adrenergic receptor expression and the effects of norepinephrine and propranolol on various head and neck cancer subtypes.

The present study aimed to investigate expression of β2-adrenergic receptor (AR), the effect of the stress-related neurotransmitter norepinephrine (NE) on cell viability, proliferation and the therapeutic effect of propranolol, which is a typical β-blocker in various type of head and neck cancers for the first time. The β2-AR expression was investigated using immunohistochemistry and an immunoreactive scoring (IRS) system in 57 different head and neck cancer specimens, and reverse transcriptase-polymerase chain reaction and western blotting in four head and neck cancer cell lines (HNCCLs). Cell viability and proliferation assays were performed using 0, 1, 5 and 10 µM of NE and 1 µM of propranolol in four HNCCLs. The expression of β2-AR was positive in the majority of head and neck cancer tissues (55/57, 96.5%); however, it was significantly higher in oral cavity cancer than in pharyngeal cancer (median IRS: 9 vs. 3; P<0.001). All HNCCLs exhibited β2-AR expression, with a higher expression level detected in the oral cavity cancer cell line than in the others. NE stimulated viability (oral cavity, 206%; larynx, 156%; pharynx, 130%; nasal cavity, 137%; 10 µM NE) and proliferation (124, 176, 131 and 127%, respectively) in a dose-dependent manner in all HNCCLs. Conversely, propranolol attenuated such viability (55, 42, 18 and 22%, respectively) and proliferation (22, 40, 61 and 48%, respectively). In conclusion, the viability and proliferation of various head and neck cancers may be directly stimulated by stress and this may be attenuated by β-blockers.

Human Papillomavirus Testing in Head and Neck Squamous Cell Carcinoma: Impact of the 2018 College of American Pathologists Guideline Among Referral Cases at a Large Academic Institution.

Given the growing clinical significance of human papillomavirus status in oropharyngeal squamous cell carcinoma, the College of American Pathologists established a set of evidence-based recommendations for high-risk human papillomavirus testing for publication in a guideline. To evaluate the impact of the recommendations on human papillomavirus ancillary test ordering habits by comparing compliance before and after the guideline was published. We retrospectively reviewed head and neck squamous cell carcinoma biopsy or resection specimens from outside institutions during a 2.5-year period around guideline publication to determine whether human papillomavirus testing was performed in accordance with the guideline. Human papillomavirus testing deviated from the guideline in 45 of 107 cases (42.1%) before and 93 of 258 cases (36.0%) after its publication (P = .29). This included 6 of 26 cases of oropharyngeal squamous cell carcinoma (23.1%) before and 5 of 55 cases (9.1%) after (P = .16), with 5 of 5 (100.0%) after due to not performing p16 immunohistochemistry. This also included 30 of 68 cases of nonoropharyngeal carcinoma (44.1%) before and 69 of 163 (42.3%) after the guideline was published (P = .88), with 29 of 30 (96.7%) before and 67 of 69 (97.1%) after due to unnecessary use of p16 immunohistochemistry. Nodal metastasis testing deviated in 9 of 13 cases (69.2%) before and 19 of 40 cases (47.5%) after (P = .21) with marked variability in testing, including 3 of 9 (33.3%) before and 8 of 19 (42.1%) after, for not confirming certain p16 immunohistochemistry-positive tumors with human papillomavirus-specific testing. Pathologists continue to deviate from the testing guideline significantly in everyday practice. Further education and discussion about the appropriate handling of head and neck cancer specimens may be needed.

Hedgehog pathway proteins SMO and GLI expression as prognostic markers in head and neck squamous cell carcinoma.

AimsBecause the hedgehog signalling pathway plays a major role in many types of cancer and can nowadays be targeted by specific compounds, we aimed to investigate the role of this pathway in squamous cell carcinoma of the head and neck.Methods and resultsNinety‐eight treatment‐naive head and neck cancer specimens were immunohistologically stained for SMO, GLI‐1, p53 and p16 expression and correlated with clinicopathological factors. Immunoreactivity for SMO and GLI‐1 was found in 20 (20.4%) and 52 (53.1%) cases of tumours, respectively. SMO expression correlated with GLI‐1 expression (ρ = 0.258, P = 0.010) in univariate and multivariate analysis (P = 0.007, t = 2.81). In univariate analysis, high SMO expression was associated with shorter overall survival (HR = 0.56; 95% CI = 0.32–0.98; P = 0.044) and disease‐free survival (HR = 0.53; 95% CI = 0.30–0.95; P = 0.034). In multivariate cox regression analysis SMO expression showed a trend towards an independent predictor for shorter overall survival (HR = 0.57; 95% CI = 0.30–1.05; P = 0.072) and disease‐free survival (HR = 0.53; 95% CI = 0.28–1.02; P = 0.056). In head and neck cancer patients with low tumour p16 expression, SMO expression was an independent factor for overall survival (HR = 0.49; 95% CI = 0.24–0.98; P = 0.043) and disease‐free survival (HR = 0.45; 95% CI = 0.22‐0.96; P = 0.037).ConclusionAlthough it needs to be confirmed in larger cohorts, our results suggest that targeting SMO might be a potentially therapeutic option in patients with head and neck cancer. In line, molecular pathological analyses including mutation analysis in the hedgehog pathway might point to additional therapeutic leads.

Abstract 1109: S100A10 contributes to cancer stemness, invasion and metastasis of head and neck cancer cells

Abstract Head and neck cancer is one of the most common cancers worldwide, and an innovating therapy is awaited. We attempted to identify proteins associated with cancer stem cell phenotypes, and found an EF-hand protein S100A10 is highly expressed in the stem cell population. Together with its binding partner, Annexin-A2, S100A10 regulates a spectrum of dynamic membrane-related events, such as actin cytoskeleton control, vesicle formation and intercellular interaction. Immunohistochemical analysis using clinical head and neck cancer specimens showed that elevated S100A10 expression is typically present in the invasion front of tumors, and S100A10-high cases were associated with poor prognosis. S100A10 knockdown and knockout resulted in significant reduction of cell migration and invasion in vitro. Cell movement was slowed down by S100A10 knockdown. Accordingly, in vivo tumor formation was impaired by S100A10 depletion. Cell cycle progression was delayed by the depletion, suggesting its potential role in proliferation controls. These results suggest that S100A10 has a significant role in head and neck cancer cell proliferation and invasion. Citation Format: Nobuyukit Tanaka, Taketo Nishikawaji, Naoko Ogama. S100A10 contributes to cancer stemness, invasion and metastasis of head and neck cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1109.

Specimen mapping in head and neck cancer using fluorescence imaging.

OverviewAlthough the agreed‐upon standard is circumferential pathology analysis of the interface between the resected specimen and the patient, there is currently no consensus on the optimal methodology to achieve this in head and neck cancer specimens. This is most commonly conducted by either sampling the wound bed after resection or obtaining samples from the specimen. Regardless of the technique, only a fraction of the area of interest can be sampled due to the labor‐intensive nature of frozen sections.ObjectiveThis review will cover and define the possible role for optical mapping of the surgical specimen using fluorescence imaging in head and neck cancer.Level of EvidenceNA

Human papillomavirus oncogenic E6 protein regulates human β-defensin 3 (hBD3) expression via the tumor suppressor protein p53.

Human β-defensin-3 (hBD3) is an epithelial cell-derived innate immune regulatory molecule overexpressed in oral dysplastic lesions and fosters a tumor-promoting microenvironment. Expression of hBD3 is induced by the epidermal growth factor receptor signaling pathway. Here we describe a novel pathway through which the high-risk human papillomavirus type-16 (HPV-16) oncoprotein E6 induces hBD3 expression in mucosal keratinocytes. Ablation of E6 by siRNA induces the tumor suppressor p53 and diminishes hBD3 in HPV-16 positive CaSki cervical cancer cells and UM-SCC-104 head and neck cancer cells. Malignant cells in HPV-16-associated oropharyngeal cancer overexpress hBD3. HPV-16 E6 induces hBD3 mRNA expression, peptide production and gene promoter activity in mucosal keratinocytes. Reduction of cellular levels of p53 stimulates hBD3 expression, while activation of p53 by doxorubicin inhibits its expression in primary oral keratinocytes and CaSki cells, suggesting that p53 represses hBD3 expression. A p53 binding site in the hBD3 gene promoter has been identified by using electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP). In addition, the p63 protein isoform ΔNp63α, but not TAp63, stimulated transactivation of the hBD3 gene and was co-expressed with hBD3 in head and neck cancer specimens. Therefore, high-risk HPV E6 oncoproteins may stimulate hBD3 expression in tumor cells to facilitate tumorigenesis of HPV-associated head and neck cancer.

Nuclear localization of MUC1 extracellular domain in breast, head and neck, and colon cancer.

The glycoprotein MUC1 is overexpressed and underglycosylated in cancer cells. MUC1 is translated as a single polypeptide that undergoes autocleavage into 2 subunits (the extracellular domain and the cytoplasmic tail), and forms a stable heterodimer at the apical membrane of normal epithelial cells. The MUC1 cytoplasmic tail localizes to the cytoplasm of transformed cells and is targeted to the nucleus. To study the expression of the MUC1 extracellular subunit in cell nuclei of neoplastic breast, head and neck, and colon samples. 330 primary tumor samples were analyzed: 166 invasive breast carcinomas, 127 head and neck tumors, and 47 colon tumors; 10 benign breast disease (BBD) and 40 normal specimens were also included. A standard immunohistochemical method with antigen retrieval was performed. Nuclear fractions from tissue homogenates and breast cancer cell lines (ZR-75, MDA-MB-231, MCF7, and T47D) were obtained and analyzed by Western blotting (WB). The anti-MUC1 extracellular subunit monoclonal antibody HMFG1 was used for immunohistochemistry. 37/166 breast cancer specimens, 5/127 head and neck cancer specimens, 2/47 colon cancer samples, and 3/10 BBD samples showed immunohistochemical staining at the nuclear level. No nuclear reaction was detected in normal samples. By WB, breast and colon cancer purified nuclear fractions showed reactivity at 200 kDa in 3/30 breast and 3/20 colon cancer samples as well as purified nuclear fractions obtained from breast cancer cell lines. This study shows that the MUC1 extracellular domain might be translocated to the cell nucleus in breast, head and neck, and colon cancer as well as BBD.

RAS/PI3K Crosstalk and Cetuximab Resistance in Head and Neck Squamous Cell Carcinoma

Cetuximab, an antibody directed against the EGF receptor, is an effective clinical therapy for patients with head and neck squamous cell cancer (HNSCC). Despite great clinical promise, intrinsic or acquired cetuximab resistance hinders successful treatment outcomes but little is known about the underlying mechanism. To study the role of oncogenic HRAS in cetuximab resistance in HNSCC, the frequency of oncogenic HRAS mutations was determined in a cohort of 180 genomic DNAs from head and neck cancer specimens. We also used a combination of cetuximab-resistant cell lines and a transgenic mouse model of RAS-driven oral cancer to identify an oncogenic RAS-specific gene expression signature that promotes cetuximab resistance. Here, we show that activation of RAS signaling leads to persistent extracellular signal-regulated kinase 1/2 signaling and consequently to cetuximab resistance. HRAS depletion in cells containing oncogenic HRAS or PIK3CA restored cetuximab sensitivity. In our study, the gene expression signature of c-MYC, BCL-2, BCL-XL, and cyclin D1 upon activation of MAPK signaling was not altered by cetuximab treatment, suggesting that this signature may have a pivotal role in cetuximab resistance of RAS-activated HNSCC. Finally, a subset of patients with head and neck cancer with oncogenic HRAS mutations was found to exhibit de novo resistance to cetuximab-based therapy. Collectively, these findings identify a distinct cetuximab resistance mechanism. Oncogenic HRAS in HNSCC promotes activation of ERK signaling, which in turn mediates cetuximab resistance through a specific gene expression signature. Clin Cancer Res; 20(11); 2933-46. ©2014 AACR.

Cancer metabolism, stemness and tumor recurrence

Here, we interrogated head and neck cancer (HNSCC) specimens (n = 12) to examine if different metabolic compartments (oxidative vs. glycolytic) co-exist in human tumors. A large panel of well-established biomarkers was employed to determine the metabolic state of proliferative cancer cells. Interestingly, cell proliferation in cancer cells, as marked by Ki-67 immunostaining, was strictly correlated with oxidative mitochondrial metabolism (OXPHOS) and the uptake of mitochondrial fuels, as detected via MCT1 expression (p < 0.001). More specifically, three metabolic tumor compartments were delineated: (1) proliferative and mitochondrial-rich cancer cells (Ki-67+/TOMM20+/COX+/MCT1+); (2) non-proliferative and mitochondrial-poor cancer cells (Ki-67−/TOMM20−/COX−/MCT1−); and (3) non-proliferative and mitochondrial-poor stromal cells (Ki-67−/TOMM20−/COX−/MCT1−). In addition, high oxidative stress (MCT4+) was very specific for cancer tissues. Thus, we next evaluated the prognostic value of MCT4 in a second independent patient cohort (n = 40). Most importantly, oxidative stress (MCT4+) in non-proliferating epithelial cancer cells predicted poor clinical outcome (tumor recurrence; p < 0.0001; log-rank test), and was functionally associated with FDG-PET avidity (p < 0.04). Similarly, oxidative stress (MCT4+) in tumor stromal cells was specifically associated with higher tumor stage (p < 0.03), and was a highly specific marker for cancer-associated fibroblasts (p < 0.001). We propose that oxidative stress is a key hallmark of tumor tissues that drives high-energy metabolism in adjacent proliferating mitochondrial-rich cancer cells, via the paracrine transfer of mitochondrial fuels (such as L-lactate and ketone bodies). New antioxidants and MCT4 inhibitors should be developed to metabolically target “three-compartment tumor metabolism” in head and neck cancers. It is remarkable that two “non-proliferating” populations of cells (Ki-67−/MCT4+) within the tumor can actually determine clinical outcome, likely by providing high-energy mitochondrial “fuels” for proliferative cancer cells to burn. Finally, we also show that in normal mucosal tissue, the basal epithelial “stem cell” layer is hyper-proliferative (Ki-67+), mitochondrial-rich (TOMM20+/COX+) and is metabolically programmed to use mitochondrial fuels (MCT1+), such as ketone bodies and L-lactate. Thus, oxidative mitochondrial metabolism (OXPHOS) is a common feature of both (1) normal stem cells and (2) proliferating cancer cells. As such, we should consider metabolically treating cancer patients with mitochondrial inhibitors (such as Metformin), and/or with a combination of MCT1 and MCT4 inhibitors, to target “metabolic symbiosis.”

Knockdown of β-catenin controls both apoptotic and autophagic cell death through LKB1/AMPK signaling in head and neck squamous cell carcinoma cell lines

The Wnt/β-catenin pathway regulates the viability and radiosensitivity of head and neck squamous cancer cells (HNSCC). Increased β-catenin predisposes HNSCC patients to poor prognosis and survival. This study was conducted to determine the mechanism by which β-catenin regulates the viability of HNSCC. AMC-HN-3, -HN-8, UM-SCC-38, and -SCC-47 cells, which were established from human head and neck cancer specimens, and underwent cell death following β-catenin silencing. β-Catenin silencing significantly induced G1 arrest and increased the expression of Bax and active caspase-3, which demonstrates the sequential activation of apoptotic cascades following treatment of HNSCC with targeted siRNA. Intriguingly, β-catenin silencing also induced autophagy. Here, we confirm that the number of autophagic vacuoles and the expression of type II light chain 3 were increased in cells that were treated with β-catenin siRNA. These cell death modes are most likely due to the activation of LKB1-dependent AMPK following β-catenin silencing. The activated LKB1/AMPK pathway in AMC-HN-3 cells caused G1 arrest by phosphorylating p53 and suppressing mTOR signaling. In addition, treating AMC-HN-3 cells with LKB1 siRNA preserved cell viability against β-catenin silencing-induced cytotoxicity. Taken together, these results imply that following β-catenin silencing, HNSCC undergo both apoptotic and autophagic cell death that are under the control of LKB1/AMPK. To the best of our knowledge, these results suggest for the first time that novel crosstalk between β-catenin and the LKB1/AMPK pathway regulates the viability of HNSCC. This study thus presents new insights into our understanding of the cellular and molecular mechanisms involved in β-catenin silencing-induced cell death.

Abstract 51: Stromal AKT2 controls epithelial invasions

Abstract The tumor microenvironment is gaining importance in the regulation of tumorigenesis, invasion and metastasis. Loss of PTEN, a negative regulator of AKT, in stromal fibroblasts results in an increased tumor occurrence in ErbB2 expressing murine mammary tumors. Furthermore, we have shown that activation of AKT in stromal fibroblasts, following loss of the Retinoblastoma Protein (Rb), induces epithelial invasions in organotypic raft cultures, via an increase in Keratinocyte Growth Factor (KGF) expression. In addition, we have shown that loss of the PTEN tumor suppressor in stromal fibroblasts and subsequent activation of AKT, increases invasive potential of Human Papilloma Virus Type 16 E6 and E7 expressing keratinocytes. This is mediated by an increase in KGF expression. There are three isoforms of AKT expressed in human fibroblasts, AKT1, AKT2 and AKT3. By depleting each individual AKT isoform in stromal fibroblasts we have been able to establish that AKT2 is essential in regulating the cross talk between epithelium and stroma, and depletion of stromal AKT2 inhibits invasions. However, loss of stromal AKT2 does not function exclusively through regulation of KGF levels as it activates a second, protective pathway, which is able to counteract the effects of KGF. This protective pathway is mediated by increased Interleukin1 beta levels produced by the fibroblasts. Preliminary data from head and neck cancer specimens show that AKT is activated in stroma of cells associated with tumor but not normal tissue. Future work will determine the AKT dependent pathways that lead to increased KGF and Interleukin1 beta expression as well as how these factors mediate invasions. 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 51. doi:1538-7445.AM2012-51

Shrinkage of head and neck cancer specimens after formalin fixation

BackgroundWe conducted this study to investigate whether formalin fixation is associated with the shrinkage of head and neck cancer specimens. MethodsPatients scheduled to undergo operation were eligible for enrollment. Fresh specimens were measured immediately in the operating room, and the measurements were repeated after formalin fixation. ResultsA total of 100 specimens were collected. Nearly half of them were obtained from the oral cavity (n=49), and a large majority were squamous cell carcinoma (n=69). The average decreases in length, width, and depth after formalin fixation were 1.50mm (4.40%), 1.52mm (6.18%), and 0.67mm (4.10%), respectively. There was no significant difference in the shrinkage percentage associated with gender, age, tumor site, tumor size, or histology. ConclusionWe found that head and neck cancer specimens shrink after formalin fixation. Therefore, we recommend that the specimen be measured immediately in order to avoid the underestimation of tumor size.

A comparative immunofluorescence analysis of three clinical-stage antibodies in head and neck cancer

BackgroundThe antibody-based targeted delivery of bioactive molecules to tumour vasculature is an attractive avenue to concentrate therapeutic agents at cancer sites, while sparing normal organs. L19, F8 and F16 are three fully human monoclonal antibodies, specific to splice isoforms of fibronectin and tenascin-C, which bind to sites of active tissue remodeling and which are currently in Phase I and II clinical trials as radio-immunoconjugates and immunocytokines in patients with cancer and arthritis.In this article, we report the first comparative analysis of expression patterns for the extra domains EDB and EDA of fibronectin and A1 of tenascin-C in both primary and metastatic head and neck cancer lesions.MethodsWe performed a comparative immunofluorescence analysis with the L19, F8 and F16 antibodies in 40 freshly frozen human head and neck cancer specimens.ResultsOn average, F8 and F16 exhibited similar staining intensities, which were typically stronger than L19. Interestingly, some specimens exhibited striking differences in staining by the three antibodies.ConclusionsThese results suggests that an individualized treatment procedure (e.g., choice of L19, F8 or F16 based on immuno-PET or immunofluorescence procedure) may represent the most logical avenue for offering the best possible antibody to any given patient.

Abstract 3512: Regulation of hypoxia inducible factor alpha in clear cell renal cell carcinoma

Abstract Clear cell renal cell carcinoma (CCRC) is the most common kidney cancer with high incidence of hypoxia-inducible factor 1 and 2 alpha (HIF-α) expression as a consequence of the nonfunctional Von Hippel-Lindau (VHL). The purpose of the study is: a) to investigate the expression levels and relationship between HIF-α and prolyl hydroxylases (PHDs), the negative regulators of HIF-α in tissue microarray (TMA) of human surgical specimens. b) to demonstrate whether methylselenenic acid (MSA) would degrade HIF-α, independent of VHL. c) to determine whether inhibition of HIF-α would enhance cytotoxicity of docetaxel.The results of immunohistochemical evaluation revealed that 92% (81 out of 88) combined incidence of both HIF-1α and HIF-2α with low levels of PHD2 (35%, 31 out of 88) and completely deficient with PHD3, the enzymes responsible for the hydroxylation of HIF-α and subsequent degradation in proteasome. In contrast, we found high incidence of PHD2 positive cases with low HIF-α in both head and neck (86% PHD2 with 38% HIF-α, n=210) and colorectal (90% PHD2 with 26% HIF-α, n=64) indicate high incidence of PHD2 is associated with low incidence of HIF-α. The expression of PHD3 was found in both head &amp; neck (21%) and colorectal (50%) cancer specimens which have low HIF-α incidence compared to CCRC. These data indicate that there is an inverse relationship between HIF-α and PHD2 in CCRC clinical samples in addition to VHL deficiency. In agreement with the surgical specimens, CCRC cells RC2 and 786- O express PHD2 protein but not PHD3 with constitutive expression of HIF-1α in RC2 and HIF-2α in 786 O. We have demonstrated the degradation of both HIF-1α and HIF-2α by MSA that leads to the significant reduction of HIF-α transcriptionally regulated gene VEGF in RC2 and 786-O. MSA exhibits unique property of degrading both HIF-1α and HIF-2α that has not been reported earlier by any other agent. Proteasome inhibitor MG132 reversed the HIF-1α degradation by MSA indicates HIF-1α degradation is proteasome dependent and VHL independent since VHL is nonfunctional in CCRC. Based on these findings we hypothesize that the degradation of HIF-α by MSA is VHL independent. We found RC2 cells with constitutive HIF-1α are 5 fold resistant to docetaxel compared to RC2-VHL cells with no detectable HIF-1α due to overexpression of VHL. As we expected, the combination of MSA enhanced the docetaxel efficacy only in RC2 cells, but not in RC2-VHL shows that the effect of MSA is through HIF-1α. These findings demonstrated the therapeutic potential of selenium for the treatment of CCRC. Thus, the unique molecular properties of CCRC being VHL deficient, express high constitutive HIF-1α and HIF-2α protein with low PHD2 and deficient in PHD3 could provide the rational for the development and evaluation of selenium in combination with the clinically approved drugs for kidney cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3512. doi:10.1158/1538-7445.AM2011-3512

Expression of angiogenic mediators in head and neck cancer

e17032 Background: In cancer, systematic analysis of mRNA expression levels can contribute to define a molecular network of lung carcinogenesis and establish predictive and prognostic molecular markers. Altered mRNA expression in certain angiogenic and anti-angiogenic genes such as vascular endothelial growth factor family of ligands and their receptors together with other molecules implicated in angiogenesis could predict disease outcome and add prognostic information at diagnosis. Methods: We performed RT-qPCR in frozen head and neck cancer specimens from untreated patients who had undergone surgical resection (n = 23). Samples were processed for mRNA extraction following standard procedures and quantification of gene expression was expressed as relative concentration normalized by an endogenous gene. The following angiogenic genes were quantified: PDGF-A, PlGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, COX-2, bFGF, HGF, IL-8; and anti-angiogenic genes: Angiomotin and endostatin. Results: Median age was 56.5 years, range (31–79), 17 patients presented squamous carcinoma of the larynx; 8 patients were stage III and the other 15 stage IV. None of the patients received pre-surgery chemo or radiotherapy and 10 patients reveived post-surgery treatment. Our results show that tumor samples had significant higher expression of PDGF, PlGF, COX-2, and IL-8 and a deep down-regulation of angiomotin. However, those data show no correlation between the levels of expression and stage of the disease. Conclusions: Our results reveal that there is an increase in the expression of the pro-angiogenic mediators PDGF, PlGF, COX-2, and IL-8 and a downregulation of angiomotin that may mediate the inhibitory effect of angiostatin on tube formation and the migration of endothelial cells toward growth factors during the formation of new blood vessels in the tumoral area. This data suggest that more research are needed in other angiogenic mediators that VEGF, that PlGF and angiomotin have some role in cancer progression and could be a promising new biomarker in head and neck cancer. No significant financial relationships to disclose.

P226 Lack of Exon 19 and 21 EGFR Mutations in Squamous Cell Head and Neck Cancer Specimens and Cell Lines

termined the global gene expression profiles of primary OSCCs and identified a subset of deregulated genes that correlated with more advanced-stage tumors. A subset of these genes are involved in signal transduction genetic networks. Our objective is thus to examine the expression of these genes in OSCCs. Design: Tissue samples were obtained at the time of surgery from the Toronto General Hospital and snap frozen in liquid nitrogen for RNA extraction. Quantitative real-time reverse-transcriptase polymerase chain reaction using the ABI PRISM 7700 Sequence Detection System and the Sequence Detection System software (PE Applied Biosystems, Foster City, Calif) was used for relative quantification of gene expression. Data analysis was performed using the Delta Delta Ct method. Patients: Sixty-eight patients with OSCC who had surgery as primary treatment were included in this study. Results: Deregulated expression of -catenin, a gene involved in maintaining cell-to-cell adhesion in normal cells mediating the Wnt signal transduction pathway was detected. WISP1, WISP2, TCF4, and LEF1 genes are also associated with the Wnt pathway and were thus included in this study to further investigate whether the Wnt/b-catenin pathway is deregulated in head and neck cancer. Conclusion: Deregulated expression of genes involved the Wnt/beta-catenin pathway in OSCC.

The effect of nimesulide, a selective cyclooxygenase‐2 inhibitor, on Ets‐1 and Ets‐2 expression in head and neck cancer cell lines

The protooncogenes Ets-1 and Ets-2 are involved in carcinogenesis of different tumors. Nimesulide, a selective cyclooxygenase-2 (COX-2) inhibitor, has antiproliferative effects on tumor cells. The question arises whether nimesulide influences Ets-1 and Ets-2 synthesis in head and neck tumors. Expression of Ets-1 and Ets-2 was analyzed in tumor tissues by immunohistochemistry. The influence of nimesulide and an extracellular signal-regulated kinase (ERK) inhibitor on cell proliferation of two head and neck cancer cell lines and Ets-1 and Ets-2 expression was determined by automated cell counting and Western blotting, respectively. Immunohistochemistry showed a high expression of Ets-1 and Ets-2 in tumor tissues. In both cell lines, Ets-1 and Ets-2 expression were reduced after 24 and 48 hours by nimesulide. Both Ets-1 and Ets-2 are overexpressed in head and neck cancer specimens. Inhibition of Ets-1 and Ets-2 expression in head and neck cancer cell lines by nimesulide might explain the proapoptotic property of this COX-2 inhibitor.