Head And Neck Squamous Cell Carcinoma Primary Tumors Research Articles

Post-radiochemotherapy PET radiomics in head and neck cancer – The influence of radiomics implementation on the reproducibility of local control tumor models

PurposeThis study investigated an association of post-radiochemotherapy (RCT) PET radiomics with local tumor control in head and neck squamous cell carcinoma (HNSCC) and evaluated the models against two radiomics software implementations. Materials and methods649 features, available in two radiomics implementations and based on the same definitions, were extracted from HNSCC primary tumor region in 18F-FDG PET scans 3 months post definitive RCT (training cohort n = 128, validation cohort n = 50) and compared using the intraclass correlation coefficient (ICC). Local recurrence models were trained, separately for both implementations, using principal component analysis (PCA) and the least absolute shrinkage and selection operator. The reproducibility of the concordance indexes (CI) in univariable Cox regression for features preselected in PCA and the final multivariable models was investigated using respective features from the other implementation. ResultsOnly 80 PET radiomic features yielded ICC > 0.8 in the comparison between the implementations. The change of implementation caused high variability of CI in the univariable analysis. However, both final multivariable models performed equally well in the training and validation cohorts (CI > 0.7) independent of radiomics implementation. ConclusionThe two post-RCT PET radiomic models, based on two different software implementations, were prognostic for local tumor control in HNSCC. However, 88% of the features was not reproducible between the implementations.

Abstract C144: LCL161, a SMAC mimetic, induces preferential radiosensitization in human papillomavirus negative head and neck squamous cell carcinoma through induction of apoptosis

Abstract Background and purpose: Evasion of apoptosis contributes to radioresistance of head and neck squamous cell carcinoma (HNSCC), calling for novel strategies to overcome apoptotic resistance. Second mitochondria-derived activator of caspase (SMAC) - mimetics are a new class of targeted drugs that specifically induce apoptotic cell death and block pro-survival signalling by antagonizing selected members of the inhibitor of apoptosis protein (IAP) family. The present study was designed to investigate the radiosensitizing effect of a SMAC mimetic, LCL161, in HNSCC and the underlying mechanisms for radiosensitization. Material and methods: We examined the correlation between mRNA expression of apoptotic pathway associated molecules and HPV status by using The Cancer Genome Atlas Database (TCGA) in which 279 HNSCC tumors are included. = Protein expression was investigated in 6 HNSCC cell lines (3 HPV[+], 3 HPV[-]), normal oral epithelial cells (NOE) by immunoblotting, and in primary HNSCC tumors by immunohistochemistry (IHC). Clonogenic survival and Alamar Blue assays were carried out in order to explore the potential of LCL161 as a radiosensitizer in HNSCC cell lines. Cell cycle analysis and Annexin-V assays were performed to investigate mechanisms of radiation-induced tumor cell death. Human tumor xenografts were generated to explore the radiosensitization effect of LCL161 on HPV[-] tumors in vivo. Results: TCGA database analysis indicated significantly higher mRNA expression of cIAP1 and RELA, lower expression of CASP3, CASP9, and BCL2 in HPV[-] compared with HPV[+] HNSCC tumors. Consistent with these findings, immunoblotting confirmed that that protein expression of cIAP1 and p65 encoded by RELA were elevated in HPV[-] HNSCC cells.LCL161 displayed minimal single-agent cytotoxicity with IC50 values between 32μM – 95μM in 6 HNSCC cells. LCL161 is a potent inhibitor of cIAPs, and caused downregulation of cIAP1 at nanomolar concentrations in less than 1 hour, while it showed no obvious effect on NFκB, Stat3, Erk, or Akt activation. Interestingly, we found that LCL161 could induce radiosensitization only in HPV[-] HNSCC cell lines, but not in HPV[+] HNSCC cells. LCL161 mediated radiosensitisation was associated with enhanced activation of caspases-3, -7, -8, -9, and PARP. Blockage of caspase activation via a pan-caspase inhibitor, z-VAD-fmk, largely abrogated LCL161 radiosensitization. Conclusion: These results suggest that LCL161, a SMAC-mimetic, significantly radiosensitizes a subset of HNSCC tumors, via a mechanism involving caspase activation and apoptosis induction. This agent holds promise for future clinical development as a novel radiosensitizer in the treatment HNSCC HPV[-] tumors. Citation Format: Linlin Yang, Bhavna Kumar, Mitchell Romito, Theodoros N. Teknosa, Amab Chakravarti, Terence M. Williams. LCL161, a SMAC mimetic, induces preferential radiosensitization in human papillomavirus negative head and neck squamous cell carcinoma through induction of apoptosis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C144.

Expression profiles of the selected genes in tumor and surgical margin in oral cavity cancer

In the past years, the number of anew diagnosed patients with head and neck squamous cell carcinoma (HNSCC) is still growing. Generally, HNSCC proliferates fast, is locally aggressive and has a high tendency to metastasize to cervical lymph nodes. Head and neck cancer is associated with gene-environment interaction. Several genetic alterations have been reported to contribute to the development of HNSCC, while cigarette smoking, alcohol drinking, chronic exposure to certain industrial agents and infection with specific subtypes of human papillomaviruses (HPV) are considered to be important risk factors, too. Primary HNSCC tumors and matching surgical margin samples were collected following surgical resection from 56 patients in the Clinic of Oncological and Reconstructive Surgery, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice. Their medical records provided necessary medical and demographic data including patient's age, gender, tumor grade and stage, regional lymph nodes, cigarette smoking and alcohol drinking. The Ethics Committee of Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology in Gliwice approved the study protocol (No KB/493-15/08 and KB/430-47/13). Average age of the patients was 56.05 (range: 29-77 years, median 58.5 years). The study included 37 males and 19 females. 80% of patients were smokers, 73% of them reported alcohol consumption, and 64% were both smoking and alcohol drinking. Obtained paired non-malignant tissues were situated over 10 mm from the tumor lesion margins and were histologically confirmed to be cancer-free. The aim of the study was to analyze the significance of p16, APC, MGMT, TIMP3, SFRP1, SFRP2, CDH1, RASSF1, RORA, DAPK1 genes expression in tumors and matching surgical margin samples. Total RNA was extracted with RNeasy Mini Kit (Qiagen, Germany) from frozen tissue samples. Isolated RNA; after being verified for integrity, quantity and purity; was used for cDNA synthesis (High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor; Applied Biosystems, USA). Gene expression levels for the above mentioned genes were analyzed by quantitative reverse transcription (qRT)-PCR in tumors and matching surgical margin samples. Reaction was performed using specific TaqMan Gene Expression Assays and TaqMan Gene Expression Master Mix in a 7300 Real Time PCR System (Applied Biosystems, USA). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control. Relative expression levels were calculated according to the 2-ΔΔCT formula. Two-sample parametrical Student's t-test for independent samples with equal/unequal variances showed statistically significant lower expression of SFRP1 in tumor compared to margin samples (0.30 vs 0.62, p < 0.01). One-way analysis of variance indicated a significant main effect of the grading stage (p < 0.05). NIR post-hoc test showed, that grade G3 patients have significantly higher values of DAPK gene expression than the G1 group (p < 0.05), as well as G2 (p < 0.01). No association was found between the genes expression and clinical parameters, except MGMT, which low expression was associated with smoking (0.87 vs 1.34, p = 0.065) and DAPK1, which low expression was associated with alcohol consumption (0.85 vs 1.97, p = 0.074). It has been known that the survivors of head and neck cancer presented increased morbidity and mortality and second primary tumors are the main reason of mortality in this group of patients. Second primary tumors or local recurrence are thought to be the consequences of cancerization and could be the results of exposure to carcinogens, frequently involving the development of multiple premalignant and malignant lesions. The initial step of field effect is associated with various molecular lesions, e.g. gene expression. In conclusion, an important implication is that field often remains after surgery and the remaining peripheral tissue (surgical margin) poses an increased risk of developing cancer. Diagnosis and treatment of cancers ought not to concentrate only on the tumor itself, but also on the cancer field effect.

Outlier Analysis Defines Zinc Finger Gene Family DNA Methylation in Tumors and Saliva of Head and Neck Cancer Patients.

Head and Neck Squamous Cell Carcinoma (HNSCC) is the fifth most common cancer, annually affecting over half a million people worldwide. Presently, there are no accepted biomarkers for clinical detection and surveillance of HNSCC. In this work, a comprehensive genome-wide analysis of epigenetic alterations in primary HNSCC tumors was employed in conjunction with cancer-specific outlier statistics to define novel biomarker genes which are differentially methylated in HNSCC. The 37 identified biomarker candidates were top-scoring outlier genes with prominent differential methylation in tumors, but with no signal in normal tissues. These putative candidates were validated in independent HNSCC cohorts from our institution and TCGA (The Cancer Genome Atlas). Using the top candidates, ZNF14, ZNF160, and ZNF420, an assay was developed for detection of HNSCC cancer in primary tissue and saliva samples with 100% specificity when compared to normal control samples. Given the high detection specificity, the analysis of ZNF DNA methylation in combination with other DNA methylation biomarkers may be useful in the clinical setting for HNSCC detection and surveillance, particularly in high-risk patients. Several additional candidates identified through this work can be further investigated toward future development of a multi-gene panel of biomarkers for the surveillance and detection of HNSCC.

MiR-124 Regulates the Epithelial-Restricted with Serine Box/Epidermal Growth Factor Receptor Signaling Axis in Head and Neck Squamous Cell Carcinoma.

Epithelial-restricted with serine box (ESX), a member of the ETS transcription factor family, is elevated and regulates EGFR in head and neck squamous cell carcinoma (HNSCC). However, the molecular mechanisms that contribute to ESX dysregulation remain to be elucidated. In this study, in silico analysis of the 3'-untranslated region (UTR) of ESX predicted two miR-124-binding sites. Delivery of miR-124 inhibited the 3'UTR ESX-driven reporter activity by 50% (P < 0.05) confirming ESX as a direct target of miR-124. Loss of miR-124 was found to be a frequent event in HNSCC. miR-124 expression was significantly depleted in the primary tumor compared with matched normal tissue in 100% (12/12) of HNSCC patients; relative mean miR-124 expression of 0.01197 and 0.00118 (P < 0.001, n = 12) in matched normal adjacent tissue and primary HNSCC tumor, respectively. Overexpression of miR-124 decreased ESX and EGFR levels in miR-124(low)/ESX(high)/EGFR(high) SCC15 HNSCC cells and reduced cell invasion, migration, proliferation, and colony formation. SCC15 cells with miR-124 restoration were less tumorigenic in vivo than miR-control SCC15 cells (70% inhibition, P < 0.01). Restoration of miR-124 in SCC15 cells enhanced the antiproliferative efficacy of the EGFR/Her2 tyrosine kinase inhibitors. Furthermore, recapitulation of EGFR in miR-124-overexpressing SCC15 cells was sufficient to completely block the antiproliferative effects of lapatinib and afatinib. Taken together, our work provides intriguing evidence that miR-124 is a novel therapeutic approach to reduce ESX/EGFR, and may be a tractable strategy to enhance the response rate of HNSCC patients to current anti-EGFR/Her2 therapies.

TU-F-CAMPUS-I-01: Head and Neck Squamous Cell Carcinoma: Short-Term Repeatability of Apparent Diffusion Coefficient and Intravoxel Incoherent Motion Parameters at 3.0T

Purpose: Many published studies have recently demonstrated the potential value of intravoxel incoherent motion (IVIM) analysis for disease evaluation. However, few have questioned its measurement repeatability/reproducibility when applied. The purpose of this study was to determine the short-term measurement repeatability of apparent diffusion coefficient ADC, true diffusion coefficient D, pseudodiffusion coefficient D* and perfusion fraction f, in head and neck squamous cell carcinoma (HNSCC) primary tumors and metastatic nodes. Methods: Ten patients with known HNSCC were examined twice using echo-planar DW-MRI with 12 b values (0 to 800 s/mm2) 1hour to 24 hours apart before radiation treatment. All patients were scanned with the customized radiation treatment immobilization devices to reduce motion artifacts and to improve image registration in repeat scans. Regions of interests were drawn in primary tumor and metastases node in each patient (Fig. 1). ADC and IVIM parameters D, D* and f were calculated by least squares data fitting. Short-term test–retest repeatability of ADC and IVIM parameters were assessed by measuring Bland–Altman limits of agreements (BA-LA). Results: Sixteen HNSCC lesions were assessed in 10 patients. Repeatability of perfusion-sensitive parameters, D* and f, in HNSCC lesions was poor (BA-LA: -144% to 88% and −57% to 96% for D* and f, respectively); a lesser extent was observed for the diffusion-sensitive parameters of ADC and D (BA-LA: −34% to 39% and −37% to 40%, for ADC and D, respectively) (Fig. 2). Conclusion: Poor repeatability of D*/f and good repeatability for ADC/D were observed in HNSCC primary tumors and metastatic nodes. Efforts should be made to improve the measurement repeatability of perfusion-sensitive IVIM parameters.

Abstract 5306: Shotgun proteomic analysis of human head and neck squamous cell carcinoma cell line SQ20B with diminished AHSG expression

Abstract The Alpha-Heremans-Schmid Glycoprotein (AHSG) has tumor promoting properties in animal models of breast cancer and lung cancer (1, 2). These cancer cells do not synthesize AHSG, instead utilizing the liver-generated glycoprotein that is abundant in serum. We have reported that head and neck squamous cell carcinoma (HNSCC) cell lines synthesize AHSG (in press) and have also detected abundant AHSG in primary HNSCC tumors (unpublished). Growth in serum-free medium and in vitro tumorigenic properties, including proliferation, adhesion and migration, are diminished in the HNSCC SQ20B cell line modified with AHSG-specific shRNA to express only twenty percent of the wild-type SQ20B cell line (SQ20B-AH20) compared to SQ20B modified with empty vector alone and expressing the wild-type amount of AHSG (SQ20B-EV) (in press). Here we have used shotgun proteomic analysis to identify additional proteins whose expression may also affect these in vitro properties of tumorigenesis associated with AHSG. The two cell lines were cultured in serum-free medium to avoid the contribution of exogenous AHSG in serum. Cell lysates were obtained, and the proteins were separated in polyacrylamide gels in duplicate. Each full protein lane was cut into 10 pieces, in-gel digested with trypsin and analyzed with tandem liquid chromatography mass spectrometry (LC-MS). MS data were analyzed against a recent human protein database. Proteins differentially expressed were quantified using a spectral counting approach. A total of 1386 distinct protein groups were identified in the two samples. Forty-eight proteins were differentially expressed in these samples (z score &amp;gt; 3, which corresponds to p ≤ 0.001, and Benjamini-Hochberg [statistical] FDR &amp;lt; 0.05). Proteins associated with the cytoskeleton, adhesion and apoptosis were over-represented in the group of differentially expressed proteins. Here we have shown that the human HNSCC cell line SQ20B exhibits changes in the proteome when AHSG expression is diminished. These proteins are critical for the tumorigenic properties of adhesion and migration. These data will help to identify mechanisms of tumorigenesis associated with AHSG in HNSCC.

Shotgun proteomic analysis of human head and neck squamous cell carcinoma cell line SQ20B with diminished AHSG expression

Background The Alpha-Heremans-Schmid Glycoprotein (AHSG) has tumor promoting properties in animal models of breast cancer and lung cancer [1,2]. These cancer cells do not synthesize AHSG, instead utilizing the liver-generated glycoprotein that is abundant in serum. We have reported that head and neck squamous cell carcinoma (HNSCC) cell lines synthesize AHSG (in press) and have also detected abundant AHSG in primary HNSCC tumors (unpublished). Growth in serum-free medium and in vitro tumorigenic properties, including proliferation, adhesion and migration, are diminished in the HNSCC SQ20B cell line modified with AHSG-specific shRNA to express only twenty percent of the wild-type SQ20B cell line (SQ20B-AH20) compared to SQ20B modified with empty vector alone and expressing the wild-type amount of AHSG (SQ20B-EV) [3]. Here we have used shotgun proteomic analysis to identify additional proteins whose expression may also affect these in vitro properties of tumorigenesis associated with AHSG.

MDA-9/Syntenin regulates differentiation and angiogenesis programs in head and neck squamous cell carcinoma.

Little is known about the molecular pathways regulating poor differentiation and invasion of head and neck squamous cell carcinoma (HNSCC). In the present study, we aimed to determine the role of MDA-9/Syntenin, a metastasis associated molecule in HNSCC tumorigenesis. Elevated MDA-9/Syntenin expression was evident in 67% (54/81) primary HNSCC tumors (p=0.001-0.002) and 69% (9/13) pre-neoplastic tissues (p=0.02-0.03). MDA-9/Syntenin overexpression was associated with the stage (p=0.001), grade (p=0.001) and lymph node metastasis (p=0.0001). Silencing of MDA-9/Syntenin in 3 poorly differentiated HNSCC cell lines induced squamous epithelial cell differentiation, disrupted angiogenesis and reduced tumor growth in vitro and in vivo. We confirmed SPRR1B and VEGFR1 as the key molecular targets of MDA-9/Syntenin on influencing HNSCC differentiation and angiogenesis respectively. MDA-9/Syntenin disrupted SPRR1B expression interacting through its PDZ1 domain and altered VEGFR1 expression in vitro and in vivo. VEGFR1 co-localized with MDA-9/Syntenin in HNSCC cell lines and primary tumor. Downregulation of growth regulatory molecules CyclinD1, CDK4, STAT3, PI3K and CTNNB1 was also evident in the MDA-9/Syntenin depleted cells, which was reversed following over-expression of MDA-9/Syntenin in immortalized oral epithelial cells. Our results suggest that early induction of MDA-9/Syntenin expression influences HNSCC progression and should be further evaluated for potential biomarker development.

Caspase-8 mutations in head and neck cancer confer resistance to death receptor-mediated apoptosis and enhance migration, invasion, and tumor growth

Little is known regarding molecular markers in head and neck squamous cell carcinoma (HNSCC) that predict responsiveness to different therapeutic regimens or predict HNSCC progression. Mutations in procaspase-8 occur in 9% of HNSCC primary tumors, but the functional consequences of these mutations are poorly understood. In this study, we examined the impact of four, representative, HNSCC-associated procaspase-8 mutations on activation of the extrinsic apoptosis pathway, as well as cellular migration and invasion, and in vivo tumor growth. All four mutant proteins acted to potently inhibit activation of apoptosis following treatment with TRAIL or agonistic anti-Fas. In contrast to wild-type procaspase-8, the mutant proteins were not recruited to FADD following treatment with TRAIL or anti-Fas, but may be constitutively bound by FADD. Three of the four procaspase-8 mutants promoted enhanced cellular migration and invasion through matrigel, relative to that seen with the wild-type procaspase-8 protein. Procaspase-8 mutation also stimulated the growth of HNSCC xenograft tumors. These findings indicate that HNSCC-associated procaspase-8 mutations inhibit activation of the extrinsic apoptosis pathway and are likely to represent markers for resistance to therapeutic regimens incorporating death receptor activators. Moreover, procaspase-8 mutations may serve as markers of HNSCC tumor progression, as exemplified by enhanced migration, invasion, and tumor growth.

PD-L1 is preferentially expressed on CD44+ tumor-initiating cells in head and neck squamous cell carcinoma

Human tumors consist of heterogeneous populations with different phenotypic characteristics, and these subpopulations can often be distinguished by different surface marker expression. In head and neck squamous cell carcinoma (HNSCC), CD44 has been identified to be a marker of a subpopulation, containing the tumor-initiating cells (or cancer stem cells), which are capable of self-renewal and differentiation into cells lacking this capacity. Significant evidence indicates that these tumor-initiating cells have an immune suppressive phenotype, allowing them to evade the antitumor host immune response, leading some to hypothesize that these cells may contribute to long-term tumor dormancy. In this study, we profiled 21 primary human HNSCC tumors and/or patient-derived xenografts for CD44 expression and PD-L1 (B7-H1) expression by flow cytometry and quantitative RT-PCR. The tumors were categorized into PD-L1-negative and PD-L1-positive tumors. In the PD-L1-positive tumors, we found elevated expression of PD-L1 on CD44+ cells, both at the protein level by flow cytometry and at the transcript level by qRT-PCR. Interestingly, treatment of the tumor cells with recombinant IFN-γ resulted in preferential up-regulation of PD-L1 on CD44+ cells (as compared to the CD44- population) in both PD-L1-negative and PD-L1-positive tumors. To assess the functional significance of the preferential PD-L1 expression on the CD44+ tumor cells, autologous tumor-infiltrating lymphocytes (TILs) were cultured from the primary tumors. By ELISA, autologous co-cultures of CD8+ TILs with sorted subsets of tumor cells showed higher secretion of IFN-γ when incubated with CD44- cells compared to CD44+ cells, suggesting that the CD44+ cells are more immunosuppressive. Blocking the TILs with anti-PD-L1 and anti-PD-1 antibodies partially restored IFN-γ secretion by the TILs incubated with the CD44+ cells, indicating that part of the immunosuppressive properties of CD44+ cells is mediated by the PD-L1-PD-1 signaling pathway. Our results indicate that the preferential PD-L1 expression on tumor-initiating cells may function as one of multiple mechanisms by which long-term tumor-initiating progenitor cells evade the immune response and provide rationale for targeting the PD-1 axis in the adjuvant therapy setting.

Human Papillomavirus-Associated Head and Neck Squamous Cell Carcinoma Survival: A Comparison by Tumor Site and Initial Treatment

Recent evidence suggests that human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) patients have better survival than HPV-negative patients. However, it is unclear if similar patterns for survival exist across different tumor sites, and whether HPV-associated prognosis is modified by type of treatment. We prospectively tested 222 histologically confirmed HNSCC primary tumors for HPV DNA by PCR and HPV E6/E7 RNA by RT-PCR prior to treatment at a large urban health center. Cox proportional hazard ratio models were constructed to assess HPV-associated differences in overall and disease-specific survival adjusting for clinical and demographic covariates. HPV detection varied significantly by primary HNSCC tumor site, from 35 % for oropharynx, to 25 % for hypopharynx, 5 % for larynx, and 3 % for oral cavity (p < 0.0001), with HPV16 accounting for the majority (95 %) of HPV-positive tumors. The hazard-risk of overall and disease-specific death comparing HPV16-positive versus negative oropharyngeal HNSCC was reduced by 74 and 89 %, respectively (p values < 0.05), and was independent of other prognostic indicators; no statistically significant changes in outcomes were observed for non-oropharyngeal HNSCC sites. Prediction of overall survival was better with combined DNA and RNA HPV16 positive PCR detection. There was no difference in HPV16-associated survival whether patients received either surgery or (chemo)radiotherapy as their initial treatment modality. Improved HPV-associated HNSCC survival is limited to patients with oropharyngeal primaries. No selective treatment advantage is observed for HPV-positive tumors, although clinical trials are needed to evaluate which treatment modalities provide the most benefit for HPV-positive HNSCC.

Genetic and Chemical Targeting of Epithelial-Restricted with Serine Box Reduces EGF Receptor and Potentiates the Efficacy of Afatinib

EGF receptor (EGFR) is elevated in more than 90% of head and neck squamous cell carcinoma (HNSCC). However, a majority of patients with HNSCC do not respond to anti-EGFR therapeutics. Insensitivity to EGFR inhibitors may be due to kinase-independent actions of EGFR and/or activation of Her2. Strategies to reduce EGFR and Her2 protein levels in concert may be an optimal approach to enhance the efficacy of current anti-EGFR molecules. In this study, knockdown of epithelial-restricted with serine box (ESX) decreased EGFR and Her2 promoter activity, expression, and levels. ESX was elevated in primary HNSCC tumors and associated with increased EGFR and Her2. Genetic ablation of ESX decreased EGFR and Her2 levels and enhanced the antiproliferative effects of EGFR/Her2 tyrosine kinase inhibitors (TKI), lapatinib and afatinib. Biphenyl isoxazolidine, a novel small-molecule ESX inhibitor, reduced EGFR and Her2 levels and potentiated the antiproliferative efficacy of afatinib. Single-agent biphenyl isoxazolidine retarded the in vivo tumorigenicity of CAL27 cells. Importantly, the combination of biphenyl isoxazolidine and afatinib was significantly superior in vivo and resulted in a 100% response rate with a 94% reduction in tumor volume. Targeting EGFR/Her2 levels with an ESX inhibitor and EGFR/Her2 kinase activity with a TKI simultaneously is a highly active therapeutic approach to manage HNSCC. Our work provides evidence to support the further development of ESX inhibitors as an adjuvant to enhance the response rate of patients with HNSCC to current anti-EGFR/Her2 therapeutics.

Abstract 4528: Folate receptor targeted iron oxide nanoparticles loaded with cisplatin for imaging and therapy in head and neck squamous cell carcinoma (HNSCC).

Abstract Background Cisplatin (DDP) is a widely used anticancer drug but its application is limited by side effects. We have previously shown that selective delivery of DDP to tumors using novel nanoparticles could significantly enhance antitumor effects with reduced toxicity. Since our group has shown that 45% of 105 HNSCC primary tumors are positive for folate receptor (FR) in Head Neck 31:475-81, 2009, we further developed DDP-loaded folate-targeted iron oxide nanoparticles (FA-IO-DDP) for HNSCC imaging and therapy. Methods HNSCC cell lines were screened for FR expression by Western blot analysis and its surface expression was confirmed by FACS. Intracellular platinum (Pt) and Pt-DNA adducts were quantified by ICP-mass spectrometry. MRI was used to detect T2 signal changes induced by iron oxide. Near infrared dye (Cy-5.5) conjugated nanoparticles were used to assess accumulation of the nanoparticles in the tumor tissues and cells. Efficacy and toxicity were assessed using nude mice bearing FR expressing SQCCY1 xenograft using 1mg/kg of free DDP and IO-DDP and FA-IO-DDP equivalent to 0.3mg/kg of free DDP. Results Five HNSCC cell lines expressing surface FR were identified among 16 HNSCC cell lines screened. Intracellular Pt and Pt-DNA adducts were increased in FR-positive PCI15B cells treated with FA-IO-DDP by 1.6-fold and 3.8-fold, respectively, compared to cells treated with non-targeted IO-DDP and free DDP (p&amp;lt;0.05). A similar degree of enhanced uptake was observed in FR-expressing SQCCY1 cells but not in FR-negative Tu212 cells. In a subcutaneous SQCCY1 xenograft model in nude mice, tumor targeted FA-IO-DDP showed significantly enhanced efficacy compared to free DDP (p&amp;lt;0.05). Nanoparticles had better efficacy compared to free drug even at lower concentration. The average tumor volume in the FA-IO-DDP treated group was 243.0±412.4 mm3 compared to 541.4±744.5 mm3 and 3324.6±501.3 mm3 in the IO-DDP and free DDP groups, respectively. A notable change in T2 signal in MRI and infrared signal in optical imaging was detected in nude mice bearing SQCCY1 cells 24 hours after treatment with FA-IO-DDP. Conclusion Our study showed that novel FR-IO-DDP nanoparticles remarkably enhanced the antitumor effects of DDP in vivo in nude mice bearing FR-positive HNSCC cells and can accumulate in FR-expressing tumors in vivo as detected by MRI and near infrared imaging. Results from the in vitro studies suggest that the effect is mediated by increased intracellular Pt concentration and Pt-DNA adducts. Further analyses in tissues from xenograft mice are ongoing. This work was supported by Chester Rochfort Endowment Fund/Winship Cancer Institute of Emory University and NIH U01CA151802-02, 1R01CA154846-01 and 1U01CA151810-02. Citation Format: Hyunseok Kang, Jing Huang, Xianghong Peng, Zhuo (Georgia) Chen, Hui Mao, Dong M. Shin. Folate receptor targeted iron oxide nanoparticles loaded with cisplatin for imaging and therapy in head and neck squamous cell carcinoma (HNSCC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4528. doi:10.1158/1538-7445.AM2013-4528

YM155 Reverses Cisplatin Resistance in Head and Neck Cancer by Decreasing Cytoplasmic Survivin Levels

Cisplatin is one of the commonly used chemotherapeutic drugs for the treatment of head and neck squamous cell carcinoma (HNSCC). However, acquisition of cisplatin resistance is common in patients with HNSCC, and it often leads to local and distant failure. In this study, we showed that survivin expression is significantly upregulated in HNSCC primary tumors and cell lines. In addition, survivin levels were significantly higher in human papilloma virus-negative patients that normally respond poorly to cisplatin treatment. Survivin expression was further increased in cisplatin-resistant cells (CAL27-CisR) as compared with its parent cells (CAL27). Therefore, we hypothesized that targeting of survivin in HNSCC could reverse the resistant phenotype in tumor cells, thereby enhancing the therapeutic efficacy of cisplatin. We used both in vitro and in vivo models to test the efficacy of YM155, a small molecule survivin inhibitor, either as a single agent or in combination with cisplatin. YM155 significantly decreased survivin levels and cell proliferation in a dose-dependent manner. In addition, YM155 pretreatment significantly reversed cisplatin resistance in cancer cells. Interestingly, YM155 treatment altered the dynamic localization of survivin in cells by inducing a rapid reduction in cytoplasmic survivin, which plays a critical role in its antiapoptotic function. In a severe combined immunodeficient mouse xenograft model, YM155 significantly enhanced the antitumor and antiangiogenic effects of cisplatin, with no added systemic toxicity. Taken together, our results suggest a potentially novel strategy to use YM155 to overcome the resistance in tumor cells, thereby enhancing the effectiveness of the chemotherapy in HNSCC.

Folate-targeted iron oxide nanoparticles loaded with cisplatin for imaging and therapy in head and neck squamous cell carcinoma (HNSCC).

e13110 Background: Cisplatin (DDP) is a widely used anticancer drug but its application has been limited by side effects and drug resistance. We have previously showed that selective delivery of DDP to tumors using novel nanoparticles could significantly enhance antitumor effects with reduced toxicity. As our group has reported that 45% of 105 HNSCC primary tumors were positive for folate receptor (FR) in Head Neck 31:475-81, 2009, we further developed DDP-loaded folate-targeted iron oxide nanoparticle (FA-IO-DDP) for HNSCC imaging and therapy. Methods: HNSCC cell lines were screened for FR expression by Western blot and surface expression was confirmed by FACS. Intracellular platinum (Pt) and Pt-DNA adducts were quantified by ICP-mass spectrometry. MTT assay was used for cytotoxicity test. T2 weighted MRI was used to detect signal changes induced by FA-IO-DDP. Biodistribution of the nanoparticles was studied using nude mice bearing high FR expressing KB3-1 xenograft. Results: Five cell lines expressing surface FR were identified among sixteen cell lines screened. Three cell lines, PCI15B, SQCCY1 and UMSCC11B with high, intermediate, and negative FR expression respectively, were selected for further investigation. FA-IO-DDP showed significantly enhanced cytotoxicity. Intracellular Pt and Pt-DNA adducts were increased in FR-positive PCI15B cells treated with FA-IO-DDP by 4 fold and 3.2 fold, respectively, compared to cells treated with non-targeted IO-DDP. Similar degree of enhanced uptake was observed in SQCCY1 but not in UMSCC11B. A significant change in T2 signal induced by increased internalization of IO in FA-IO-DDP treated PCI15B cells was detected by MRI. In KB3-1 xenograft model, FA-IO-DDP showed 5.4 fold higher accumulation in tumor compared to free DDP. Conclusions: Our study showed that novel FA-IO-DDP targeting FR remarkably enhanced antitumor effects of DDP in FR-positive HNSCC cells by increasing intracellular Pt concentration and Pt-DNA adducts. MRI may be used to monitor DDP delivery by IO nanoparticles in vivo. Ongoing studies are focused on antitumor effect and toxicities of the FA-IO-DDP using nude mice bearing PCI15B, SQCCY1 and UMSCC11B cells.

Abstract 4019: Galanin has a tumor suppressor activity and is frequently inactivated by aberrant promoter methylation in head and neck cancer

Abstract Purpose: Chromosome 11q13 alterations have repeatedly been reported to be associated with bad prognosis in head and neck squamous cell carcinoma (HNSCC). Our recent findings support a growth regulatory function for GALR1 since antibody blockade of this receptor enhances proliferation of on HNSCC cells and Galanin and GALR1 induce a marked and prolonged ERK1/2 activation, up-regulation of p27Kip1 and p57Kip2, down-regulation of cyclin D1, and consequent inhibition of cell proliferation. Furthermore, expression-negative squamous cell cancers and cell lines exhibit hypermethylation of CpG islands in the GALR1 promoter region. Based on the hypothesis that CpG hypermethylation might silence the Galanin gene, promoter methylation status and gene expression were assessed in a large panel of HNSCC cell lines and primary tumors. Experimental Design: Promoter methylation of Galanin in 62 UM-SCC and 10 UT-SCC cell lines and 100 primary tumor samples using methylation-specific PCR. Galanin expression and methylation status of 12 UM-SCC cell lines were analyzed further by Quantitative real-time PCR and bisulfite sequencing analysis. To establish that methylation was responsible for silencing Galanin gene expression, we treated three cell lines (UM-SCC-2, -10B and -54). These cell lines that showed absence of Galanin expression and hypermethylation were cultured with 5-Azacytidine alone, TSA alone, or 5-Azacytidine plus TSA. UM-SCC-54 cell line was used in a relative colony-forming assay. Wild-type preprogalanin in the pCMV-SPORT6 construct or empty pCMV-SPORT6 construct were transfected into UM-SCC-54 cell line exhibiting Galanin hypermethylation and loss of Galanin expression. Results: MSP analysis revealed that the Galanin promoter was fully or partially methylated in 24 of 72 cell lines (33.3%) but was unmethylated in the majority 19/20 (95.0%) of non-malignant lines. Methylation correlated with decreased Galanin expression. Galanin methylation found in 24/100 HNSCC tumor specimens significantly correlated with GALR1 methylation status (P&amp;lt;0.001). The presence of Galanin promoter hypermethylation statistically correlated with a decrease in disease-free survival (DFS) (log-rank test, P &amp;lt;0.01).). In multivariate logistic-regression analysis, methylation of Galanin and methylation of the gene pair Galanin and GALR1 were associated with an odds ratio for recurrence of 8.95 (95% CI, 2.29 to 35.03; P=0.002) and 23.84 (95% CI, 2.74 to 207.17; P=0.004), respectively. In UM-SCC-54 cells that have hypermethylated Galanin and GALR1-proficient cell lines, exogenous expression of Galanin and stimulation suppressed cell proliferation. Conclusions: Frequent promoter hypermethylation, gene silencing, association with prognosis, and growth suppression after reexpression support the hypothesis that Galanin may act as a tumor suppressor activity in 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 4019. doi:1538-7445.AM2012-4019

Abstract 4097: Integrative analysis of epigenetic and transcriptional alterations in head and neck squamous cell carcinomas

Abstract Objective: Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer worldwide with a 50% patient survival rate 5 years post-diagnosis despite current therapeutic strategies. A direct comprehensive genome wide integrated analysis of epigenetic and transcriptional alteration in primary HNSCC has not been performed before. Such data would greatly enrich our knowledge about HNSCC biology and would be beneficial for the development of the individual targeted therapy. Experimental Design: 44 primary HNSCC and 25 normal mucosal samples were used in the Affymetrix GeneChip Human Exon 1.0 array and for the Illumina Infinium Human Methylation 27 array. To analyze the data we employed a novel screening approach based on Cancer Outlier Profile Analysis (COPA). With use of this approach we determined 81 significant differentially expressed and 37 significant differentially methylated genes with both oncogenic and tumor suppressing properties. Results: Out of total 118 candidate genes we picked 36 candidate tumor suppressing genes with strong correlation between hypermethylation and decrease of expression in tumor samples, such as BANK1, ADFP, MAPK4, ZNF71 and others. 20 genes out of total 36 were validated by bisulfite sequencing in a separate cohort of 32 primary HNSCC tumor and 16 normal mucosal tissues. Out of the total 20 genes, 13 demonstrated at least 40% difference in methylation, most notably for BANK1 81% of tumors were more methylated as compared to normals. 7 of these newly discovered genes out of the 36 belong to Zinc Finger Proteins (ZNF) group and they have never been reported as tumor suppressors in HNSCC before. Bisulfite sequencing and quantitative real time PCR (qRT-PCR) analysis of ZNFs revealed that they are strongly hypermethylated and underexpressed in HNSCC tumor samples. Conclusions: Our new screening approach allowed identification of 36 candidate tumor suppressor genes aberrantly methylated and transcriptionally suppressed in HNSCC. Validation of gene expression and methylation allowed the detection of ZNF genes whose downregulation may play a significant role in HNSCC tumor development. 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 4097. doi:1538-7445.AM2012-4097

Low-Level Expression of miR-375 Correlates with Poor Outcome and Metastasis While Altering the Invasive Properties of Head and Neck Squamous Cell Carcinomas

Small, noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. We used comparisons of global miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) samples and adjacent normal tissue to rank those miRNAs that were most significantly altered in our patient population. Rank Consistency Score analysis revealed miR-375 to have the most significantly lowered miRNA levels in tumors relative to matched adjacent nonmalignant tissue from the same patient among 736 miRNAs that were evaluated. This result has been previously observed by other groups; however, we extend this finding with the unique observation that low miR-375 expression levels correlate significantly with cancer survival and distant metastasis. In a study of 123 primary HNSCC patients using multivariable Cox proportional hazard ratios (HR) and 95% confidence intervals (CI), both death from disease (HR: 12.8, 95% CI: 3 to 49) and incidence of distant metastasis (HR: 8.7, 95% CI: 2 to 31) correlated with lower expression levels of miR-375 regardless of the site or stage of the tumor. In addition, we found that oral cavity tumor cell lines (eg, UMSCC1 and UMSCC47) overexpressing miR-375 were significantly less invasive in vitro than their matched empty vector controls. We conclude that miR-375 represents a potential prognostic marker of poor outcome and metastasis in HNSCC and that it may function by suppressing the tumor's invasive properties.

MicroRNA-107 functions as a candidate tumor-suppressor gene in head and neck squamous cell carcinoma by downregulation of protein kinase Cɛ

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide with about 600,000 new cases diagnosed each year. Understanding the molecular pathways that lead to HNSCC is crucial to identify new targets for anti-cancer drug development. Protein kinase Cε (PKCε) is elevated in HNSCC and regulates the activation of Akt, Stat3, and Rho GTPases. To date, the molecular mechanism of PKCε dysregulation in HNSCC remains to be elucidated. In silico analysis identified three putative microRNA-107 (miR-107) binding sites in the 3'-untranslated region (UTR) of PKCε. An inverse relationship was revealed between miR-107 and PKCε in HNSCC cell lines. Delivery of miR-107 reduced PKCε levels in SCC15, SCC25, and CAL27, three HNSCC cell lines with high PKCε and low miR-107. The activity of a luciferase reporter construct containing the 3'-UTR of PKCε was down-regulated by miR-107 and mutations in the three cognate miR-107 binding sites completely ablated the regulation by miR-107. Treatment with miR-107 significantly blocked cell proliferation, DNA replication, colony formation, and invasion in SCC25 and CAL27 cells. Ectopic expression of miR-resistant PKCε was sufficient to partially rescue the loss-of-function phenotype in miR-107-overexpressing SCC25 cells. Tumor growth in nude mice was retarded by 93 ± 7% in CAL27/miR-107 cells compared to CAL27/miR-control cells. Lastly, human primary HNSCC tumors with elevated PKCε had reduced miR-107 expression. Our results demonstrate that PKCε is directly regulated by miR-107 and moreover, suggest that miR-107 may be a potential anti-cancer therapeutic for HNSCC.