Head And Neck Squamous Cell Carcinoma Lesions Research Articles

The expression of immune checkpoint proteins PD-L1 and TIM3 in mouse and human head and neck squamous cell carcinoma.

The aim of this study was to examine the expression of programmed death-ligand 1 (PD-L1) and of T cell immunoglobulin and mucin domain-containing protein (TIM3) in oral epithelial dysplasia and head and neck squamous cell carcinoma (HNSCC). Mouse HNSCC was induced with 4-nitroquinoline-1 oxide (4NQO). Oral epithelial dysplastic lesions, carcinoma in situ and HNSCC lesions were stained with anti-PD-L1 and TIM3 antibodies. The expression of PD-L1 and TIM3 in tumor cells and immune cells was semiquantitatively measured and compared. In parallel, human dysplasia and HNSCC were stained with anti-PD-L1 and anti-TIM3. The expression pattern of PD-L1+ and TIM3+ cells was further compared. In human and mouse samples both PD-L1 and TIM3 were found to be expressed in neoplastic and immune cells in HNSCC, but not in dysplasia. There was no significant difference in PD-L1 and TIM3 expression between metastatic and nonmetastatic HNSCC. We conclude that the 4NQO-induced mouse HNSCC model may be an excellent preclinical model for immune checkpoint therapy.

Analysis of HLA-G 14 bp Insertion/Deletion Polymorphism and HLA-G, ILT2 and ILT4 Expression in Head and Neck Squamous Cell Carcinoma Patients.

HLA-G is the checkpoint molecule involved in the suppression of the immune response. Increased expression of HLA-G and its ILTs receptors have been correlated with tumor progression in various cancer types. In head and neck squamous cell carcinoma (HNSCC) tumors, the effect of HLA-G, ILT2 and ILT4 expression on cancer development has to be explained. The 34 HNSCC patients and 98 controls were genotyped for the HLA-G 14 bp ins/del polymorphism. In HNSCC lesions, HLA-G, ILT2 and ILT4 mRNA expression was analysed using real-time PCR. The association between HLA-G, ILT2 and ILT4 mRNA expression and clinical variables (age at onset, TNM staging system and p16 positivity) was also evaluated. No genetic association between the HLA-G 14 bp ins/del and HNSCC risk was detected (p > 0.05). However, in the non-metastatic HNSCC group, a significantly higher HLA-G mRNA expression was noted in tumors in the T4 stage compared to those in the T1 and T2 stages (p = 0.0289). ILT2 mRNA expression was significantly increased in non-metastatic vs. metastatic tumors (p = 0.0269). Furthermore, a significantly higher ILT4 mRNA expression was noted in tumors in the T1+T2 stage compared to those in the T3 stage (p = 0.0495). Our results suggest that the HLA-G molecule creates an immunological microenvironment involved in HNSCC development.

Potential Roles of Activin in Head and Neck Squamous Cell Carcinoma Progression and Mortality.

Activin, a member of the TGF-β super family of cytokines, is involved in head and neck squamous cell carcinoma (HNSCC). This study examined the constituents of the activin axis in order to further elucidate the role of activin A in HNSCC progression. Immunohistochemistry (IHC), reverse transcription polymerase chain reaction (RT-PCR), MTT, and matrigel invasion assays, in addition to analysis of the tumor cancer genome atlas (TCGA), were employed. IHC in HNSCC and oral leukoplakia (OPL) lesions demonstrated increased expression of the inhibin subunit βA (INHBA) (p<0.0001), as well as activin receptor type IB (ACVR1B) (p<0.0032) compared to normal mucosa. TCGA analysis revealed increased INHBA expression was associated with lymph node positive tumors (p=0.024), decreased overall survival (p=0.0167), and decreased promoter methylation (p<0.0001). Concomitant up-regulated expression of gene pathways strongly correlated with INHBA expression demonstrated further deleterious effects on survival (p<0.0148). Activin may be an important component of early carcinogenesis in OPL and HNSCC with unfavorable effects on clinical end-points such as survival.

Abstract IA020: Targeting mTOR signaling in oral premalignant lesions: From bench to clinic and back

Abstract Despite encouraging recent results from novel treatment options, such as immunotherapy, for head and neck squamous cell carcinoma (HNSCC), limited progress has been made in improving outcomes for most patients. Prevention and early detection are key to improving the prognosis of HNSCC. Our team has focused on decoding the oncogenic signaling circuitries driving HNSCC initiation and progression, aimed at identifying novel druggable targets to treat and prevent this aggressive malignancy. These efforts led to the early discovery that persistent activation of PI3K/mTOR signaling circuitry is the most frequent dysregulated signaling mechanism in HNSCC, and that in turn, the overreliance on PI3K/mTOR for HNSCC initiation and progression can be exploited for therapeutic purposes. Evidence will presented that mTOR inhibition exerts a potent antitumor activity in HNSCC patients in a recently reported window of opportunity clinical trial (NCT01195922). Thus, mTOR inhibitors can be considered for the prevention of HNSCC development and for the treatment of existing HNSCC lesions. However, their safety profile and tolerability may hamper their potential long-term use for HNSCC prevention. In this regard, we have shown that the repurposed drug metformin, which is safely used by millions of type 2 diabetes patients, decreases mTOR signaling in HNSCC and displays potent chemopreventive activity in experimental oral premalignancy models. Based on these findings, we have conducted a Phase IIa Clinical Trial using metformin for HNSCC prevention (NCT02581137) in patients with oral premalignant lesions (OPL), which was recently completed. Metformin administration resulted in mTOR inhibition, and improvement in the histological severity of 60% of the OPLs, including a subset (17%) of patients that exhibited complete responses. Ongoing experimental and planned clinical studies will be presented, which may provide a mechanistic framework for the use of metformin as a precision preventive agent for HNSCC. Citation Format: J. Silvio Gutkind. Targeting mTOR signaling in oral premalignant lesions: From bench to clinic and back [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA020.

COL3A1: Potential prognostic predictor for head and neck cancer based on immune-microenvironment alternative splicing.

We aimed to identify a novel prognostic biomarker for head and neck squamous cell carcinoma (HNSCC) based on tumor immunology-related alternative splicing (AS). Data for 502 HNSCC and 44 normal samples were obtained from the TCGA database and used to establish an AS-related risk model through univariate, least absolute shrinkage, and selection operator Cox regression analyses. Fresh HNSCC and normal oral tissues were surgically obtained from 44 HNSCC patients. Western blotting and quantitative reverse transcription-PCR were used to assess gene expression levels. Kaplan-Meier was performed to evaluate patients' overall survival (OS) rate. The CIBERSORT algorithm, single-sample gene set enrichment analysis, and immune checkpoint analyses were performed to compare immune activities between subgroups. The risk model was established using 10 pivotal AS events first. Collagen Type III Alpha 1 Chain (COL3A1) were screened based on |log2FC| ≥ 1 and FDR < 0.05 criteria. COL3A1 expression levels in HNSCC tissues were elevated relative to normal tissues (p < 0.001). Moreover, COL3A1 was a reliable biomarker for HNSCC patients' prognostic prediction in both cohorts (p < 0.001, p=0.0085, respectively). COL3A1 protein (p=0.0054) and mRNA (p < 0.0001) levels were correlated with HNSCC differentiation. Furthermore, the T stage was correlated with COL3A1 expression (p=0.043), and COL3A1 expression was an independent prognostic predictor for HNSCC patients (p=0.006). Compared with the risk model, COL3A1 was better at evaluating immune cell infiltrations, immune activities, and immune checkpoint gene expressions of HNSCC lesions.

Identification of NUTF2 as a Candidate Diagnostic and Prognostic Biomarker Associated with Immune Infiltration in Head and Neck Squamous Cell Carcinoma.

BackgroundHead and neck squamous cell carcinoma (HNSC) is one of the most common tumors worldwide. Nuclear transport factor 2 (NUTF2) plays a key role in cell death and immune processes. However, few reports have studied correlations between NUTF2 gene expression and the occurrence and development of HNSC.MethodsThe expression of NUTF2 was analyzed using publicly available databases, including the Cancer Genome Atlas and Human Protein Atlas and Gene Expression Omnibus (GEO) database, which was validated by RT-PCR. We evaluated the functions of NUTF2 with Kaplan–Meier curve, logistic regression were used to study the relationship between clinicopathological features and the expression of NUTF2. Cox regression analyses were used to identify the effects of NUTF2 expression on survival. Gene Ontology and Gene Set Enrichment Analysis were used to explore relevant biological pathways. The relationship between NUTF2 and tumor-infiltrating immune cells was investigated with on-line bioinformatic tools.ResultsNUTF2 was significantly upregulated in HNSC lesions and is associated with tumor size (P < 0.01). Increased expression of NUTF2 was linked to shorter overall and progress-free survival in HNSC. Cox regression analyses revealed that NUTF2 is an independent prognostic factor in HNSC. GSEA analysis demonstrated that NUTF2 negatively regulates several immune pathways. NUTF2 was correlated with the infiltrating levels of B cells and CD8+ T cells and was negatively correlated with diverse immune marker sets in HNSC.ConclusionNUTF2 is highly expressed in HNSC and correlates with poor prognosis. Correlation with immune functions suggests that NUTF2 may serve as a biomarker and therapeutic target for HNSC.

Longitudinal assessment of PD-L1 expression and gene expression profiles in patients with head and neck cancer reveals temporal heterogeneity

BackgroundProgrammed death-ligand 1 (PD-L1) is the most validated predictive biomarker used for the treatment of head and neck squamous cell carcinoma (HNSCC) with immune checkpoint inhibitors (ICI). Several gene expression-based signatures surrogate of the activation of IFN-gamma pathway and of the presence of tertiary lymphoid structures (TLS) have also been proposed as potential biomarkers. While they may have a potential therapeutic implication, the longitudinal changes of either PD-L1 or gene expression profiles between the initial and recurrent HNSCC lesions is unknown. MethodsPD-L1 immunohistochemistry (IHC) and targeted RNA-sequencing of 2,549 transcripts were analyzed on paired specimens from the initial diagnosis and recurrent HNSCC. PD-L1 status was defined using the combined positive score (CPS). PD-L1 mRNA levels were compared with protein expression levels by IHC. Enrichment scores of surrogate signatures for TLS and IFN-gamma (IFN-γ) pathway activation were computed using the single sample gene set enrichment analysis (ssGSEA). ResultsPD-L1 status was 64% (21/33) concordant between the initial and recurrent lesions using a CPS 1 threshold and 67% (22/33) concordant using a CPS 20 threshold. CPS score was associated with PD-L1 gene expression levels. There was a 43% (15/35) and 66% (23/35) concordance for the IFN-γ and TLS signature scores, respectively. ConclusionOur study reveals temporal heterogeneity of PD-L1 status and TLS/IFN-γ gene expression surrogates in HNSCC that need to be considered when interpreting biomarker studies.

Human Papillomavirus Infection in Head and Neck Squamous Cell Carcinomas: Transcriptional Triggers and Changed Disease Patterns.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of cancers. Collectively, HNSCC ranks sixth in incidence rate worldwide. Apart from classical risk factors like tobacco and alcohol, infection of human papillomavirus (HPV) is emerging as a discrete risk factor for HNSCC. HPV-positive HNSCC represent a distinct group of diseases that differ in their clinical presentation. These lesions are well-differentiated, occur at an early age, and have better prognosis. Epidemiological studies have demonstrated a specific increase in the proportions of the HPV-positive HNSCC. HPV-positive and HPV-negative HNSCC lesions display different disease progression and clinical response. For tumorigenic-transformation, HPV essentially requires a permissive cellular environment and host cell factors for induction of viral transcription. As the spectrum of host factors is independent of HPV infection at the time of viral entry, presumably entry of HPV only selects host cells that are permissive to establishment of HPV infection. Growing evidence suggest that HPV plays a more active role in a subset of HNSCC, where they are transcriptionally-active. A variety of factors provide a favorable environment for HPV to become transcriptionally-active. The most notable are the set of transcription factors that have direct binding sites on the viral genome. As HPV does not have its own transcription machinery, it is fully dependent on host transcription factors to complete the life cycle. Here, we review and evaluate the current evidence on level of a subset of host transcription factors that influence viral genome, directly or indirectly, in HNSCC. Since many of these transcription factors can independently promote carcinogenesis, the composition of HPV permissive transcription factors in a tumor can serve as a surrogate marker of a separate molecularly-distinct class of HNSCC lesions including those cases, where HPV could not get a chance to infect but may manifest better prognosis.

Immune Escape Mechanisms and Their Clinical Relevance in Head and Neck Squamous Cell Carcinoma.

Immunotherapy has been recently approved for the treatment of relapsed and metastatic human papilloma virus (HPV) positive and negative head and neck squamous cell carcinoma (HNSCC). However, the response of patients is limited and the overall survival remains short with a low rate of long-term survivors. There exists growing evidence that complex and partially redundant immune escape mechanisms play an important role for the low efficacy of immunotherapies in this disease. These are caused by diverse complex processes characterized by (i) changes in the expression of immune modulatory molecules in tumor cells, (ii) alterations in the frequency, composition and clonal expansion of immune cell subpopulations in the tumor microenvironment and peripheral blood leading to reduced innate and adaptive immune responses, (iii) impaired homing of immune cells to the tumor site as well as (iv) the presence of immune suppressive soluble and physical factors in the tumor microenvironment. We here summarize the major immune escape strategies of HNSCC lesions, highlight pathways, and molecular targets that help to attenuate HNSCC-induced immune tolerance, affect the selection and success of immunotherapeutic approaches to overcome resistance to immunotherapy by targeting immune escape mechanisms and thus improve the HNSCC patients’ outcome.

Pathway-Specific Genome Editing of PI3K/mTOR Tumor Suppressor Genes Reveals that PTEN Loss Contributes to Cetuximab Resistance in Head and Neck Cancer.

Cetuximab, an mAb targeting EGFR, is a standard of care for the treatment for locally advanced or metastatic head and neck squamous cell carcinoma (HNSCC). However, despite overexpression of EGFR in more than 90% of HNSCC lesions, most patients with HNSCC fail to respond to cetuximab treatment. In addition, there are no available biomarkers to predict sensitivity or resistance to cetuximab in the clinic. Here, we sought to advance precision medicine approaches for HNSCC by identifying PI3K/mTOR signaling network-specific cetuximab resistance mechanisms. We first analyzed the frequency of genomic alterations in genes involved in the PI3K/mTOR signaling circuitry in the HNSCC TCGA dataset. Experimentally, we took advantage of CRISPR/Cas9 genome editing approaches to systematically explore the contribution of genomic alterations in each tumor suppressor gene (TSG) controlling the PI3K-mTOR pathway to cetuximab resistance in HNSCC cases that do not exhibit PIK3CA mutations. Remarkably, we found that many HNSCC cases exhibit pathway-specific gene copy number loss of multiple TSGs that normally restrain PI3K/mTOR signaling. Among them, we found that both engineered and endogenous PTEN gene deletions can mediate resistance to cetuximab. Our findings suggest that PTEN gene copy number loss, which is highly prevalent in HNSCC, may result in sustained PI3K/mTOR signaling independent of EGFR, thereby representing a promising mechanistic biomarker predictive of cetuximab resistance in this cancer type. Further prospective studies are needed to investigate the impact of PTEN loss on cetuximab efficacy in the clinic.

Syngeneic animal models of tobacco-associated oral cancer reveal the activity of in situ anti-CTLA-4

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Tobacco use is the main risk factor for HNSCC, and tobacco-associated HNSCCs have poor prognosis and response to available treatments. Recently approved anti-PD-1 immune checkpoint inhibitors showed limited activity (≤20%) in HNSCC, highlighting the need to identify new therapeutic options. For this, mouse models that accurately mimic the complexity of the HNSCC mutational landscape and tumor immune environment are urgently needed. Here, we report a mouse HNSCC model system that recapitulates the human tobacco-related HNSCC mutanome, in which tumors grow when implanted in the tongue of immunocompetent mice. These HNSCC lesions have similar immune infiltration and response rates to anti-PD-1 (≤20%) immunotherapy as human HNSCCs. Remarkably, we find that >70% of HNSCC lesions respond to intratumoral anti-CTLA-4. This syngeneic HNSCC mouse model provides a platform to accelerate the development of immunotherapeutic options for HNSCC.

Targeting CD44v6 for fluorescence-guided surgery in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is an often highly invasive tumor, infiltrating functionally important tissue areas. Achieving complete tumor resection and preserving functionally relevant tissue structures depends on precise identification of tumor-free resection margins during surgery. Fluorescence-guided surgery (FGS), by intraoperative detection of tumor cells using a fluorescent tracer, may guide surgical excision and identify tumor-positive resection margins. Using a literature survey on potential surface molecules followed by immunohistochemical validation, we identified CD44 variant 6 (CD44v6) as a constitutively expressed antigen in the invasion zone of HNSCC lesions. The monoclonal anti-CD44v6 antibody BIWA was labeled with both a near-infrared fluorescent dye (IRDye800CW) and a radioactive label (Indium-111) and dual-modality imaging was applied in a locally invasive tumor mouse model. BIWA accurately detected human HNSCC xenografts in mice with a tumor uptake of 54 ± 11% ID/g and invasion regions with an accuracy of 94%. When dissected under clinical-like conditions, tumor remnants approximately 0.7 mm in diameter consisting of a few thousand cells were identified by fluorescence imaging, resulting in reliable dissection of invasive microregions. These data indicate that CD44v6 is a suitable target for reliable near-infrared detection and FGS of invasive HNSCC lesions in vivo.

Correlation of serum lactate dehydrogenase and alkaline phosphatase in different histological grades of head and neck squamous cell carcinoma and premalignant lesions.

Head neck cancer (HNCA) in North-East India accounts for 54.48% cancers at all sites, one of the highest in the country. Increased serum lactate dehydrogenase (LDH) activity is considered as a marker of cellular necrosis while serum alkaline phosphatase (ALP) is recognized as an important marker of induction of tumor cell differentiation. Considering the importance and need of biomarker in HNCA and there being no previous study on tumor markers from this highly prevalent region, we intended to examine the role of serum LDH and ALP in head and neck squamous cell carcinoma (HNSCC) and epithelial precursor lesions (EPLs) and also to find their correlation with the different histological grades of tumor. This is a prospective observational study on patients with HNSCC and precancerous lesions attending Department of ENT of a Teaching Hospital. Serum LDH and ALP was measured in HNSCC, EPL and control groups and the results were statistically analyzed and compared using analysis of variance, Pearson correlation coefficient, and sensitivity-specificity analysis. Serum LDH was found to be a significant marker of HNSCC. Total serum LDH level was high in both premalignant and HNSCC cases. There was a significant correlation between serum LDH and grades of HNSCC showing highest levels of expression in moderately differentiated SCC. Elevated serum LDH was also seen in erythroleukoplakia, leukoplakia, and verrucous lesion, but serum ALP levels were not significant. This is the first study from this highly prevalent region of HNCA showing that serum LDH could be regarded as a biomarker for malignant and premalignant conditions of the head and neck.

The Association and Clinical Significance of CDKN2A Promoter Methylation in Head and Neck Squamous Cell Carcinoma: a Meta-Analysis

Background/Aims: The association between cyclin-dependent kinase inhibitor 2A (CDKN2A) hypermethylation and head and neck squamous cell carcinoma (HNSCC) risk has been investigated by a number of studies. However, these studies have not demonstrated consistent results. Moreover, the role of CDKN2A methylation in HNSCC carcinogenesis and its clinical significance remain unclear. Methods: We performed a systematic meta-analysis based on 72 articles (including 3399 HNSCCs, 668 premalignant lesions, and 2393 normal controls) from the PubMed, Google Scholar, Web of Science, Embase, China National Knowledge Infrastructure and Wanfang databases. Results: Our study showed a significant increase in the frequency of CDKN2A methylation during HNSCC carcinogenesis (HNSCC vs. normal controls, odds ratio (OR) = 6.72, P < 0.01; HNSCC vs. precancerous lesions, OR = 1.89, P < 0.05; precancerous lesions vs. normal controls, OR = 14.70, P < 0.01). Moreover, CDKN2A methylation was significantly associated with gender (OR = 1.34; P < 0.05) and lymph node metastasis (OR = 2.32; P < 0.01). The area under summary receiver operating characteristic curve (AUC) for diagnosis of HNSCC based on all samples and saliva sample subgroup were 0.77 and 0.96, respectively. Additionally, CDKN2A hypermethylation was significantly associated with shorter overall survival (OS) (hazard ratio (HR) = 1.01, P < 0.05) and recurrence-free survival (RFS) (HR = 1.77, P < 0.05). Conclusion: Our findings indicate CDKN2A methylation is involved in the carcinogenesis, progression, and metastasis of HNSCC. Furthermore, methylated CDKN2A could be a potential diagnostic and prognostic biomarker for HNSCC.

Abstract 351: mTOR-ERK co-targeting strategies for head and neck cancer therapy

Abstract Despite therapeutic advances in the past decade, head and neck squamous cell carcinoma (HNSCC) continues to be a major public health concern. Although recent studies of human cancer genomes identified remarkable complexity of genomic alterations in HNSCC, surprisingly, most aberrations have fallen within key driver signaling pathways. Among them, PI3K/AKT/mTOR pathway is most frequently activated in over 80% of the HNSCC patients. We showed that the majority of HNSCC lesions exhibit high levels of phosphorylated ribosomal S6 protein (pS6), key downstream target of the AKT-mTOR pathway, and that inhibition of mTOR by the use of rapamycin causes a rapid decrease in the level of pS6 and the apoptotic death of HNSCC tumor xenografts, thereby causing tumor regression. Our initial studies identified the AKT-mTOR pathway as a potential therapeutic target for HNSCC and provided strong rational to initiate multiple clinical trials. Indeed, inhibitors of PI3K/AKT/mTOR block mTOR activity and exert a beneficial response in HNSCC patients. However, therapeutic resistance was evidenced in several trials, which might be due to the activation of adaptive survival signaling in tumor cells. To explore the therapeutic option that can overcome the resistance, we performed a synthetic lethality screen in HNSCC cell lines using shRNA libraries. We found that multiple molecules involved in ERK pathway were highly represented. Furthermore, treating HNSCC cell lines harboring activated mTOR with small molecule inhibitors that target PI3K/AKT/mTOR pathway can sequentially induce ERK activation, which is consistent with the findings observed in numerous clinical trials. We demonstrated that co-targeting mTOR and ERK using trametinib, a MEK1/2 inhibitor or cetuximab, an EGFR monoclonal antibody, exhibited the synergistic effect by sensitizing HNSCC to priming with the mTOR inhibitor. Specifically, mTOR-ERK co-targeting prevented the growth of HNSCC tumor xenografts by decreasing cell proliferation, reducing lymphangiogenesis and activating apoptosis. In addition, we have recently used a phosphoproteomics approach to identify the molecular mechanisms of ERK feedback activation caused by PI3K/AKT/mTOR inhibition. Overall, our promising findings from these preclinical studies suggest that the use of mTOR-ERK co-targeting strategies may provide the novel alternative approach to achieve durable responses (cure) in HNSCC cancer patients. Citation Format: Zhiyong Wang, Esteban Delgado, Kosuke Yamaguchi, Ramiro Iglesias-Bartolomé, Panomwat Amornphimoltham, Alfredo Molinolo, Qianming Chen, J.Silvio Gutkind. mTOR-ERK co-targeting strategies for head and neck cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 351. doi:10.1158/1538-7445.AM2017-351

Hypoxia Mediates Differential Response to Anti-EGFR Therapy in HNSCC Cells

Despite advances in the head and neck squamous cell carcinoma (HNSCC) treatment modalities, drug resistance and cancer recurrence are often reported. Hypoxia signaling through hypoxia-inducible factor 1 (HIF-1) promotes angiogenesis and metastasis by inducing epithelial-mesenchymal-transition (EMT). The aim of this study was to evaluate the impact of hypoxia on response to therapy as well as EMT and expression of stem cell markers in HNSCC cells. Five HNSCC cell lines (UT-SCC-2, UT-SCC-14, LK0412, LK0827, and LK0923) were selected for this study. The treatment sensitivity for radiation, cisplatin, cetuximab, and dasatinib was assessed by crystal violet assay. Gene expression of EMT and cancer stem cell (CSC) markers as well as protein level of EGFR signaling molecules were analyzed by qPCR and western blotting, respectively. Unlike UT-SCC-14 and LK0827, the LK0412 cell line became significantly more sensitive to cetuximab in hypoxic conditions. This cetuximab sensitivity was efficiently reversed after suppression of HIF-1α with siRNA. Additionally, hypoxia-induced EMT and expression of stem cell markers in HNSCC cells was partially revoked by treatment with cetuximab or knockdown of HIF-1α. In summary, our study shows that hypoxia might have a positive influence on the anti-EGFR therapy effectiveness in HNSCC. However, due to heterogeneity of HNSCC lesions, targeting HIF-1α may not be sufficient to mediate such a response. Further studies identifying a trait of hypoxia-specific response to cetuximab in HNSCC are advisable.

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 LB-243: Targeting the PI3K-mTOR signaling with metformin for the prevention of HPV-associated malignancies in HIV- and HIV+ individuals

Abstract The majority of head and neck squamous cell carcinoma (HNSCC) lesions exhibit persistent activation of the PI3K-mTOR pathway, including cases associated with traditional risk factors or human papillomavirus (HPV) infection. This overreliance on the PI3K-mTOR signaling circuitry for tumor growth can potentially be exploited for therapeutic purposes. Indeed, we have shown that mTOR inhibitors are highly effective in decreasing tumor burden and metastasis in multiple in vitro and in vivo models. However, currently available PI3K and mTOR inhibitors may raise safety concerns regarding their long term use in patients diagnosed with potential premalignant oral lesions. We have recently shown that the first-line, oral antidiabetic drug metformin prevents HNSCC development in chemically-induced experimental oral premalignant lesions by reducing mTOR activity through stimulation of the AMP-activated protein kinase (AMPK) signaling pathway. Noteworthy, metformin is safely used by more than two million Americans on a daily basis. We have optimized the oral delivery of metformin to achieve clinically relevant blood levels, and explored its preclinical activity in representative HNSCC tumor xenograft models. HNSCC cells harboring PIK3CA mutations or HPV oncogenes were very sensitive to the growth inhibitory activity of metformin in vitro, concomitant with AMPK activation and mTOR inhibition. These effects were dependent on the expression of organic cation transporter 3 (OCT3), a metformin uptake transporter that is highly expressed in dysplastic oral squamous epithelium and HNSCC. Furthermore, metformin administration in vivo caused a dramatic, OCT3-dependent reduction in tumor growth of PIK3CA and HPV+ HNSCC cells transplanted into immunocompromised mice. In this regard, the incidence of HPV-related malignancies, including HNSCC, is increasing among HIV-infected individuals, irrespective of the restoration of their immune function by the use of highly active antiretroviral therapy. This prompted us to examine mTOR activation and OCT3 expression as surrogate biomarkers for metformin sensitivity, in a large panel of HNSCC lesions from HIV- and HIV-infected (HIV+) individuals. We found that in HIV- patients (n=490), 21% of the HNSCC lesions were HPV+, whereas 35% of the HNSCC in HIV+ patients were HPV+ (n=164, p&amp;lt;0.001). The majority of these lesions, as well as perianal and cervical SCCs arising in HIV+ patients, exhibit increased mTOR activity and high OCT3 expression. We can conclude that activation of the PI3K-mTOR pathway is a widespread event in SCCs, including HPV- and HPV+ lesions arising in HIV+ patients, all of which express OCT3. These observations may provide a rationale for the clinical evaluation of metformin to halt SCC development from precancerous lesions, including in HIV-infected individuals that are at high risk of developing HPV-associated cancers. Citation Format: Dmitriy Madera, Lynn Vitale-Cross, Daniel Martin, Abraham Schneider, Alfredo Molinolo, J. Silvio Gutkind. Targeting the PI3K-mTOR signaling with metformin for the prevention of HPV-associated malignancies in HIV- and HIV+ individuals. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-243. doi:10.1158/1538-7445.AM2014-LB-243

DSG3 as a biomarker for the ultrasensitive detection of occult lymph node metastasis in oral cancer using nanostructured immunoarrays

The diagnosis of cervical lymph node metastasis in head and neck squamous cell carcinoma (HNSCC) patients constitutes an essential requirement for clinical staging and treatment selection. However, clinical assessment by physical examination and different imaging modalities, as well as by histological examination of routine lymph node cryosections can miss micrometastases, while false positives may lead to unnecessary elective lymph node neck resections. Here, we explored the feasibility of developing a sensitive assay system for desmoglein 3 (DSG3) as a predictive biomarker for lymph node metastasis in HNSCC. DSG3 expression was determined in multiple general cancer- and HNSCC-tissue microarrays (TMAs), in negative and positive HNSCC metastatic cervical lymph nodes, and in a variety of HNSCC and control cell lines. A nanostructured immunoarray system was developed for the ultrasensitive detection of DSG3 in lymph node tissue lysates. We demonstrate that DSG3 is highly expressed in all HNSCC lesions and their metastatic cervical lymph nodes, but absent in non-invaded lymph nodes. We show that DSG3 can be rapidly detected with high sensitivity using a simple microfluidic immunoarray platform, even in human tissue sections including very few HNSCC invading cells, hence distinguishing between positive and negative lymph nodes. We provide a proof of principle supporting that ultrasensitive nanostructured assay systems for DSG3 can be exploited to detect micrometastatic HNSCC lesions in lymph nodes, which can improve the diagnosis and guide in the selection of appropriate therapeutic intervention modalities for HNSCC patients.

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.