Auswahl der wissenschaftlichen Literatur zum Thema „HNSC CANCER TREATMENT“

Abstract Head and neck squamous cell carcinoma (HNSC) is a devastating disease accounting for 570,000 newly diagnosed cases and resulting in 344,000 deaths annually. HNSC arises from its histological precursor lesion, squamous dysplasia (HNSD); however, most patients are diagnosed with late-stage HNSC with no prior identification of pre-malignant disease. Earlier diagnosis and intervention could ameliorate the outsized burden of HNSC; therefore, there is an urgent unmet need to characterize the molecular changes underlying progression through HNSD to HNSC. To identify novel therapeutic targets or biomarkers that predict progression to HNSC, we have generated a three dimensional (3D) organoid library derived from dysplastic oral tissue harvested from mice treated with 4-Nitriquinoline N-oxide (4-NQO), a potent mimetic of tobacco smoke. Through RNA sequencing analysis of these organoids, we have identified two distinct gene expression states that accompany either early or late dysplasia. By performing gene set enrichment analysis of these two distinct gene signatures, we have identified c-Jun N-terminal Kinase (JNK) signaling as a potential biomarker that predicts or progression from HNSD to HNSC. Together, we have identified the salient transcriptional changes that accompany progression through the histological precursor lesion of HNSC. This study positions JNK signaling as a potential biomarker or therapeutic target for early intervention during the treatment of a deadly disease. Citation Format: Robert Agee, Samuel Flashner, Masataka Shimonosono, Andres Klein-Szanto, Hiroshi Nakagawa. Leveraging 3D organoids to identify molecular changes underlying HNSC initiation and development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3082.

Purpose: Identification of molecularly-defined cancer subgroups and targeting tumor-specific vulnerabilities have a strong potential to improve treatment response and patient outcomes but remain an unmet challenge of high clinical relevance, especially in head and neck squamous cell carcinoma (HNSC). Experimental design: We established a UCHL1-related gene set to identify and molecularly characterize a UCHL1-related subgroup within TCGA-HNSC by integrative analysis of multi-omics data. An extreme gradient boosting model was trained on TCGA-HNSC based on GSVA scores for gene sets of the MSigDB to robustly predict UCHL1-related cancers in other solid tumors and cancer cell lines derived thereof. Potential vulnerabilities of UCHL1-related cancer cells were elucidated by an in-silico drug screening approach. Results: We established a 497-gene set, which stratified the TCGA-HNSC cohort into distinct subgroups with a UCHL1-related or other phenotype. UCHL1-related HNSC were characterized by higher frequencies of genomic alterations, which was also evident for UCHL1-related cancers of other solid tumors predicted by the classification model. These data indicated an impaired maintenance of genomic integrity and vulnerability for DNA-damaging treatment, which was supported by a favorable prognosis of UCHL1-related tumors after radiotherapy, and a higher sensitivity of UCHL1-related cancer cells to irradiation or DNA-damaging compounds (e.g., Oxaliplatin). Conclusion: Our study established UCHL1-related cancers as a novel subgroup across most solid tumor entities with a unique molecular phenotype and DNA-damaging treatment as a specific vulnerability, which requires further proof-of-concept in pre-clinical models and future clinical trials.

Head and neck squamous carcinoma (HNSC) is the most prevalent malignancy of the head and neck regions. Long noncoding RNAs (lncRNAs) are vital in tumorigenesis regulation. However, the role of lncRNAs in HNSC requires further exploration. Herein, through bioinformatic assays using The Cancer Genome Atlas (TCGA) datasets, rapid amplification of cDNA ends (RACE) assays, and RNA-FISH, we revealed that a novel cytoplasmic transcript, HNSC-associated transcript 1 (HNSCAT1, previously recognized as linc01269), was downregulated in tumor samples and advanced tumor stages and was also associated with favorable outcomes in HNSC. Overexpression of HNSCAT1 triggered treatment efficacy in HNSCs both in vivo and in vitro. More importantly, through high-throughput transcriptome analysis (RNA-seq, in NODE database, OEZ007550), we identified KRT80, a tumor suppressor in HNSC, as the target of HNSCAT1. KRT80 expression was modulated by lncRNA HNSCAT1 and presented a positive correlation in tumor samples ( R = 0.52 , p < 0.001 ). Intriguingly, we identified that miR-1245 simultaneously interacts with KRT80 and HNSCAT1, which bridges the regulatory function between KRT80 and HNSCAT1. Conclusively, our study demonstrated that lncRNA HNSCAT1 functions as a necessary tumor inhibitor in HNSC, which provides a novel mechanism of lncRNA function and provides alternative targets for the diagnosis and treatment of HNSC.

Head and neck squamous carcinoma (HNSC) is a frequent and deadly malignancy that is challenging to manage. The existing treatment options have considerable efficacy limitations. Hence, the identification of new therapeutic targets and the development of efficacious treatments are urgent needs. Cuproptosis, a non-apoptotic programmed cell death caused by excess copper, has only very recently been discovered. The present study investigated the prognostic importance of genes involved in cuproptosis through the mRNA expression data and related clinical information of HNSC patients downloaded from public databases. Our results revealed that many cuproptosis-related genes were differentially expressed between normal and HNSC tissues in the TCGA cohort. Moreover, 39 differentially expressed genes were associated with the prognosis of HNSC patients. Then, a 24-gene signature was identified in the TCGA cohort utilizing the LASSO Cox regression model. HNSC expression data used for validation were obtained from the GEO database. Consequently, we divided patients into high- and low-risk groups based on the 24-gene signature. Furthermore, we demonstrated that the high-risk group had a worse prognosis when compared to the low-risk group. Additionally, significant differences were found between the two groups in metabolic pathways, immune microenvironment, etc. In conclusion, we found a cuproptosis-related gene signature that can be used effectively to predict OS in HNSC patients. Thus, targeting cuproptosis might be an alternative and promising strategy for HNSC patients.

Abstract It is right time to review the management of head and neck squamous cancer (HNSC) because of fundamental changes in both diagnostic and therapeutic modalities. Head and neck cancer affects area highly associated with the individual's. identity and can produce profound alteration in appearance, speech, and swallowing. Due to morbidity, disfigurement and problems of disease control clinicians used to have lot of reservations in treating complex HNSC cases. The field has taken a new vigor by incorporating important basic advances in understanding of cancer, new modalities of treatment and management of functional deficits. Although much progress has been made in understanding the molecular genetics of HNSC for novel diagnostic and therapeutic interventions, they are still far to go before becoming standard of care for head and neck cancer.

Aim: Cell invasion leading to metastasis is a major cause of treatment failure in head–neck cancers (HNCs). Identifying prognostic molecules associated with invasiveness is imperative for clinical applications. Materials & methods: A systemic approach was used to globally survey invasion-related genes, including transcriptomic profiling, pathway analysis, data mining and prognostic assessment using TCGA-HNSC dataset. Results: Six functional pathways and six hub molecules (LAMA3, LAMC2, THBS1, IGF1R, PDGFB and TGFβ1) were identified that significantly contributed to cell invasion, leading to poor survival in HNC patients. Combinations of multiple biomarkers substantially increased the probability of accurately predicting prognosis. Conclusion: Our six defined invasion-related molecules may be used as a panel signature in precision medicine for prognostic indicators or molecular therapeutic targets for HNC.

Head and neck squamous cell carcinoma (HNSC) exhibits genetic heterogeneity in etiologies, tumor sites, and biological processes, which significantly impact therapeutic strategies and prognosis. While the influence of human papillomavirus on clinical outcomes is established, the molecular subtypes determining additional treatment options for HNSC remain unclear and inconsistent. This study aims to identify distinct HNSC molecular subtypes to enhance diagnosis and prognosis accuracy. In this study, we collected three HNSC microarrays (n = 306) from the Gene Expression Omnibus (GEO), and HNSC RNA-Seq data (n = 566) from The Cancer Genome Atlas (TCGA) to identify differentially expressed genes (DEGs) and validate our results. Two scoring methods, representative score (RS) and perturbative score (PS), were developed for DEGs to summarize their possible activation functions and influence in tumorigenesis. Based on the RS and PS scoring, we selected candidate genes to cluster TCGA samples for the identification of molecular subtypes in HNSC. We have identified 289 up-regulated DEGs and selected 88 genes (called HNSC88) using the RS and PS scoring methods. Based on HNSC88 and TCGA samples, we determined three HNSC subtypes, including one HPV-associated subtype, and two HPV-negative subtypes. One of the HPV-negative subtypes showed a relationship to smoking behavior, while the other exhibited high expression in tumor immune response. The Kaplan–Meier method was used to compare overall survival among the three subtypes. The HPV-associated subtype showed a better prognosis compared to the other two HPV-negative subtypes (log rank, p = 0.0092 and 0.0001; hazard ratio, 1.36 and 1.39). Additionally, within the HPV-negative group, the smoking-related subgroup exhibited worse prognosis compared to the subgroup with high expression in immune response (log rank, p = 0.039; hazard ratio, 1.53). The HNSC88 not only enables the identification of HPV-associated subtypes, but also proposes two potential HPV-negative subtypes with distinct prognoses and molecular signatures. This study provides valuable strategies for summarizing the roles and influences of genes in tumorigenesis for identifying molecular signatures and subtypes of HNSC.

Objectives. Head and neck squamous cell carcinomas (HNSCs) are frequently diagnosed at the locoregional advanced stage (stage IVa), but controversy remains regarding whether stage IVa HSNCs should be treated with upfront surgery or definitive chemoradiation therapy (CRT). The purpose of this study was to compare overall survival (OS) and disease-free survival (DFS) in patients with stage IVa HNSC treated primarily by surgery with curative intent with/without (neo)adjuvant treatment (surgery group) versus those treated primarily with CRT (CRT group).Methods. We reviewed data of 1,033 patients with stage IVa HNSC treated with curative intent at 17 cancer centers between 2010 and 2016.Results. Among 1,033 patients, 765 (74.1%) received upfront surgery and 268 (25.9%) received CRT. The 5-year OS and DFS rates were 64.4% and 62.0% in the surgery group and 49.5% and 45.4% in the CRT group, respectively. In multivariate analyses, OS and DFS were better in the surgery group than in the CRT group (odds ratio [OR] for death, 0.762; 95% confidence interval [CI], 0.592–0.981; OR for recurrence, 0.628; 95% CI, 0.492–0.802). In subgroup analyses, the OS and DFS of patients with oropharyngeal cancer were better in the surgery group (OR for death, 0.548; 95% CI, 0.341–0.879; OR for recurrence, 0.598; 95% CI, 0.377–0.948). In the surgery group, patients with laryngeal cancer showed better OS (OR for death, 0.432; 95% CI, 0.211–0.882), while those with hypopharyngeal cancer DFS was improved (OR for recurrence, 0.506; 95% CI, 0.328–0.780).Conclusion. A survival benefit from surgery may be achieved even in patients with stage IVa HNSC, particularly those with oropharyngeal and laryngeal cancer. Surgery led to a reduction in the recurrence rate in patients with hypopharyngeal cancer.

Background As a complex group of malignancies, head and neck squamous cell carcinoma (HNSC) is one of the leading causes of cancer mortality. This study aims to establish a reliable clinical classification and gene signature for HNSC prognostic prediction and precision treatments. Methods A consensus clustering analysis was performed to group HNSC patients in The Cancer Genome Atlas (TCGA) database based on genes linked to programmed cell death (PCD). Differentially expressed genes (DEGs) between subtypes were identified using the “limma” R package. The TCGA prognostic signature and PCD-related prognostic genes were found using a least absolute shrinkage and selection operator (LASSO) regression analysis and univariate Cox regression analysis. The robustness of the LASSO analysis was validated using datasets GSE65858 and GSE41613. A cell counting kit-8 (CCK-8) test, Western blot, and real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) were used to evaluate the expression and viability of prognostic genes. Results Four molecular subtypes were identified in PCD-related genes. Subtype C4 had the best prognosis and the highest immune score, while subtype C1 exhibited the most unfavorable outcomes. Three hundred shared DEGs were identified among the four subtypes, and four prognostic genes (CTLA4, CAMK2N1, PLAU and CALML5) were used to construct a TCGA-HNSC prognostic model. High-risk patients manifested poorer prognosis, more inflammatory pathway enrichment, and lower immune cell infiltration. High-risk patients were more prone to immune escape and were more likely to be resistant to Cisplatin and 5-Fluorouracil. Prognosis prediction was validated in external datasets. The expression of CTLA4, CAMK2N1, PLAU and CALML5 was enhanced in CAL-27 and SCC-25 cell lines, and CALML5 inhibited CAL-27 and SCC-25 cell viability. Conclusion This study shares novel insights into HNSC classification and provides a reliable PCD-related prognostic signature for prognosis prediction and treatment for patients with HNSC.

This study examines efficacy and optimal treatment variables of photodynamic therapy (PDT) for human head and neck squamous cancer (HNSC) xenografts in athymic mice. Two and four days after injection of hematoporphyrin derivative (HPD), tumors were illuminated with red light from an argon-dye laser. Sixty-three tumors were treated. With HPD dose and light intensity constant at 7.5 mg/kg and 100 mW/cm2, respectively, the extent of tumor necrosis was strongly dependent on duration of light exposure. There was no substantial difference in results for 30- and 60-minute treatment durations between animals injected with HPD 2 and 4 days before treatment. After 30 minutes treatment time, responses were seen in 8 of 10 mice (2 days post-HPD) and 11 of 12 mice (4 days post-HPD). After 60 minutes treatment time, toxicity was high. We conclude that, in this model, PDT is effective in selective killing of HNSC. For future comparison studies in this model, if the indicated HPD dose and light intensity are used we recommend a 2-day delay after HPD injection and a light exposure duration of 30 minutes

Head and neck cancer is the sixth leading type of cancer with 90 percent of head and neck cancer arising from squamous cell lining on the epithelium of the oral and nasal cavity, pharynx, and salivary gland. Even with tremendous achievements on chemotherapeutic drugs and therapies, the long-term prognosis of patients with advanced head and neck squamous cell carcinoma (HNSCC) has shown little improvement over the last three decades. Cisplatin is one of widely used chemotherapeutic drugs for multiple cancers, including head and neck cancer, but the prolonged use of this drug is limited by its toxicity and by the development of resistance. To overcome these major roadblocks to improved prognosis requires mechanism-based therapeutic strategies to maximize the antitumor effect of drugs while limiting their toxicities. Cisplatin exerts anticancer effects via multiple mechanisms, yet its most prominent mode of action involves the generation of DNA lesions followed by the activation of the DNA damage response and the induction of BCL-2 family-dependent mitochondrial apoptosis. DNA damage activates a tumor suppressor p53 to induce apoptosis. One of its functions is to induce the expression of several pro-apoptotic proteins such as Noxa, which binds to an anti-apoptotic BCL-2 family protein, MCL-1 (myeloid leukemia cell-1) to inactivate its pro-survival function and induce apoptosis. We examined Noxa expression and apoptosis induced by cisplatin in p53-wild-type HN30 and HN31, p53-truncated and inactive HN4 and HN12, and p53-deleted HN22 and HN8 HNSCC cell lines. We found that Noxa was induced in HN30 and HN31 cells and down-regulation of Noxa by shRNA (short-hairpin RNA) decreased apoptosis, indicating Noxa contribution to cisplatin-induced apoptosis. Interestingly, cisplatin treatment induced Noxa and apoptosis even in p53-deleted HN22 and HN8 cells, suggesting the existence of the p53-independent pathways for the induction of Noxa. Based on these observations, we hypothesized that modulation of Noxa/MCL-1 axis could mimic cisplatin-induced cell death. We found that Noxa overexpression induced cell death in all cell lines tested regardless of p53 status. This finding could be applicable as a potential therapeutic strategy to treat head and neck cancer.

Malignancies of the upper aero-digestive tract are a major public health problem, especially in South Asia. The major risk factors in South Asia remain smoked/smokeless tobacco, areca nut, alcohol abuse and poor diet, with limited evidence for human papillomavirus (HPV). Although HPV-associated head and neck squamous cell carcinoma (HNSCC) is well documented in the western world, studies on South Asian populations are few and inconsistent. However, the incidence of HPV-associated head and neck cancer (HNC) has increased in recent years. Certain high-risk types of HPV infection are regarded as well-established risk factors for cervical cancer and a subset of HNSCC; however, their true role and importance in the progression of HNSCC remain unclear. Although HPV-associated HNC patients generally have a better prognosis than those with HPV-negative disease, current chemo- and radio-therapies are largely non-specific and have considerable toxicities. RNA interference (RNAi), which has shown great promise as a highly specific therapy for other diseases, has potential for treating HPV-associated HNC, especially if disease progression is dependent on the continual expression of HPV oncogenes.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Dentistry and Oral Health
Griffith Health
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Cancer is a complex and multifaceted disease that continues to pose a significant challenge to global health. As the second leading cause of death worldwide. Early detection and noninvasive techniques of detecting cancer are necessary to improve treatment outcomes, save lives and improve the quality of life. Biopsies of tumors are often expensive and invasive and raise the risk of serious complications like infection, excessive bleeding, and puncture damage to nearby tissues and organs. Early detection biomarkers are often variably expressed in different patients and may even be below the detection level at an early stage. Hence PBMC that shows alteration in gene profile as a result of interaction with tumor antigens may serve as a better early detection biomarker. Also, such alterations in immune gene profile in PBMCs are more detectable in a wide variety of cancer patients despite their variability in different cancer mutants. Tumor cell biomarkers lack specificity, and tumor heterogeneity complicates accurate diagnosis and treatment. Changing biomarker expression affects treatment responses, and technical challenges impact utility. Synthetic drugs targeting tumor cells often trigger tumour cells to acquire resistance against them. Tumor progression is an outcome of tumor growth regulation in conjunction with tumor evasion by immune modulation. Therefore, understanding of immunological biomarkers is equally important. Hence, designing a prudent chemotherapeutic combination requires a detailed understanding of gene regulation altering cancer prognosis and its impact on immune regulation . Immunotherapy also has its side effects and does not provide an adequate response in all patients, and its inherent variability in patient response often makes them prohibitive. Hence, a concomitant targeting of tumour cells and modulation of immune cell function may be a particularly beneficial mechanism for cancer treatment. Machine learning tools are crucial for early cancer detection and immune modulation due to their ability to analyze complex data and identify patterns that may not be apparent through viii | P a g e traditional methods. Potential diagnostic biomarkers were predicted for breast cancer using eXplainable Artificial Intelligence (XAI) on XGBoost machine learning (ML) models trained on a binary classification dataset containing the expression data of PBMCs from 252 breast cancer patients and 194 healthy women. After effectively adding SHAP values further into the XGBoost model, ten important genes related to breast cancer development were discovered to be effective potential biomarkers. It was discovered that SVIP, BEND3, MDGA2, LEF1-AS1, PRM1, TEX14, MZB1, TMIGD2, KIT, and FKBP7 are key genes that impact model prediction. These genes may serve as early, non-invasive diagnostic and prognostic biomarkers for breast cancer patients. The impact of concomitant intervention cancer progression and immune regulation therefore necessitated identification of such biomarkers that have dual impact. Gene expression data of HNSC tumor samples and PBMCs of tumor patient datasets were analysed for the identification of differentially expressed genes. 110 DEGs were found to be common in both datasets. Further, it was identified that these 110 DEGs were involved in biological processes related to tumor regulation. Potential Immunological biomarkers were identified for HNSC cancer. The Genes that play a role in both tumour growth and immune suppression were identified by enrichment analysis followed by gene expression analysis. 10 such genes were shortlisted, Foxp3, CD274, IDO1, IL-10, SOCS1, PRKDC, AXL, CDK6, TGFB1, FADD. CD274 and IDO1 were found to have the highest degree of interaction based on their network of interactions. Synthetic drugs including many of FDA approved drugs might cause significant side effects, leading to adverse impacts on patients' quality of life. Additionally, some cancer cells may develop resistance to synthetic drugs over time, reducing treatment efficacy. Moreover, targeted therapies may only be effective in cancers with specific molecular characteristics, limiting their broad applicability. To address these limitations, ongoing research focuses on developing more targeted and personalized therapies, combining synthetic drugs with other ix | P a g e treatment modalities, and exploring alternative natural compounds with multi-target effects. Multi-target natural compounds offer the advantage of targeting multiple pathways involved in cancer progression without significant side effects. These compounds, derived from plants and other natural sources, hold promise in cancer treatment due to their diverse mechanisms of action and potential for reduced toxicity. Natural compounds that help in tumour suppression as well as functional immune modulation were identified for their dual roles. Np care and GEO databases were used for retravel of natural compounds. 102 potential anti-cancer natural compounds treatment gene expression data was analysed and key differentially regulated genes by them were identified. These 102 natural compounds were analysed for their ability to alter the expression of 110 commonly differentially expressed (identified in first objective). Salidroside was altering maximum number of 66 gene from them. Gallic acid and Shikonin were found to be the natural compounds that target CD274 and IDO1 respectively. Galic acid is extracted from leaves of bearberry, in pomegranate root bark, gallnuts, witch hazel, both in free-state and as part of the tannin molecule, whereas Shikonin is found in the extracts of dried root of the plant Lithospermum erythrorhizon. Studies have demonstrated that both Shikonin and Gallica acid exhibits anti-cancer properties. Single drug treatment can lead to the development of drug resistance, where cancer cells become less responsive to the treatment over time. Some cancers may be inherently resistant to certain drugs, restricting their effectiveness. Moreover, high doses of a single drug can cause severe side effects, impacting patients' quality of life. Additionally, single-drug therapy may not be effective due to the heterogeneity of cancer cells, allowing potential tumor recurrence. Combination therapy targets cancer cells through multiple pathways, reduces drug resistance, and enhances efficacy of treatment outcomes. Synergistic interactions can improve efficacy while minimizing side effects, advancing personalized cancer care for better patient outcomes. A combination of Salidroside, Ginsenoside Rd, Oridonin, Britanin, and Scutellarein was x | P a g e chosen such that they could alter the expression of 108 genes out of the selected 110 genes. The combination was further analyzed for regulating pathways and biological processes that were affected. Expression data analysis of HNSC cancer exhibited 1745 differentially expressed genes. Gallic acid treatment results in the downregulation of 120 genes and upregulation of 35 genes while Shikonin results in the downregulation of 660 genes and upregulation of 38 genes. Pathway analysis of these genes that were modulated by Gallic acid and Shikonin showed them to be crucially involved in pathways that were essential for cancer prognosis. Further Gallic acid and shikonin treatment impact on cancer cell line was analysed individually as well as in combination with the help of in vitro experiments. Gallic acid showed IC50 value of 46.87, 59.37, and 93.75 at 12h, 24h, and 48h treatment, respectively. Shikonin showed IC50 value of 13.86, 11.95, and 10.89 at 12h, 24h, and 48h treatment, respectively. Lowest percentage of cell viability was observed for combination of 80 µl Gallic acid and 16 µl of Shikonin. So, this combination of gallic acid and shikonin could be effective for the HNSC cancer treatment. Our studies showed a multifaceted, multi-dimensional tumor regression by altering autophagy, apoptosis, inhibiting cell proliferation, angiogenesis, metastasis and inflammatory cytokines production. Thus, the study has helped develop a unique combination of natural compounds that will markedly reduce the propensity of development of drug resistance in tumors and immune evasion by the tumors. This study is crucial to developing a combinatorial natural therapeutic cocktail with accentuated immunotherapeutic potential.

Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer, with a worldwide incidence of 275,000 new cases annually (Warnakulasuriya, 2009). Globally, the head and neck carcinoma represents a major cause of morbidity and mortality and is the sixth most commonly occurring cancer (Warnakulasuriya, 2009). A majority (>90%) of the head and neck cancers are squamous in origin and thus are linguistically referred to as head and neck squamous cell carcinoma (HNSCC) (Warnakulasuriya, 2009). HNSCC includes cancers of the oral cavity, larynx and pharynx; oral cancer being the most common (Warnakulasuriya, 2009). Although, HNSCC is the sixth most common cancer globally (Warnakulasuriya, 2009), the Indian scenario is graver. According to GLOBOCAN 2008 (http://globocan.iarc.fr), the worldwide age standardized incidence rate (ASR) for HNSCC (and thus OSCC) is 5.3 and 2.5 per 100,000 males and females respectively (Ferlay et al., 2010). In India, the ASR is 9.8 and 5.2 per 100,000 males and females respectively, clearly demonstrating a remarkably high incidence rate of OSCC (Ferlay et al., 2010; http://globocan.iarc.fr). OSCC is a peculiar cancer which is largely preventable and rarely presents as a familial disorder. The most common etiological factors associated with OSCC include tobacco and alcohol consumption (Johnson, 2001). Additionally, high risk human papillomaviruses (HPV strains 16 and 18) as well as genetic predispositions have been implicated. The treatment of OSCC mainly relies on surgical resection of the tumor. The site, size, depth of infiltration and proximity to the bone of the tumor determine whether a combination of surgery with radiation therapy or chemotherapy would be advised (Scully and Bagan, 2009). The concomitant chemo-radiation therapy is the most commonly used strategy in locally advanced cancer. Taxanes (e.g., paclitaxel and docetaxel) and platinum-based induction chemotherapy (e.g., cisplatin) are the options in the treatment of locally advanced cancer. Epidermal growth factor receptor (EGFR) targeted with cetuximab in combination with radiotherapy has been successfully tested in a large randomized trial and thus is currently a new option (Scully and Bagan, 2009). The success of cetuximab has paved the path for the development and implementation of molecules targeting various signaling pathways. Despite extensive research on oral squamous cell carcinoma (OSCC), the five-year survival rate has not changed in several decades with the exception of the targeted treatment strategies involving cetuximab as discussed above. The current chemotherapeutic approaches lack selectivity and are flagitious. Thus, effective treatment of OSCC requires the identification of molecular targets to design appropriate therapeutic strategies. To this end, the present study took three distinct approaches in order to validate the use of existing targets and to reveal novel prognostic biomarkers and therapeutic targets. 1) Targeting the PI3K-AKT-MTOR pathway in OSCC and identification of determinants of its sensitivity. 2) Gene expression analysis of ectopically overexpressed TSC2 to identify new therapeutic targets and prognostic biomarkers as well as to elucidate the genes regulated by it. 3) Expression profiling of CYP1B1 in order to validate the use of CYP1B1 based prodrug therapy in OSCC. Investigations pertaining to the changes in gene and protein expression profiles in malignant as well as pre-malignant lesions have documented the deregulation of the PI3K-AKT-MTOR (phosphoinositide 3-kinase-AKT-mechanistic target of rapamycin) and EGFR (epidermal growth factor receptor) pathways in OSCC which are being widely targeted in many therapeutic strategies (Molinolo et al., 2007; Chakraborty et al., 2008; Matta and Ralhan, 2009; Molinolo et al., 2009; Stransky et al., 2011). The PI3K-AKT-MTOR pathway is a central hub for controlling cellular proliferation and growth in response to various intracellular as well as extracellular stimuli. Crucial signaling cascades including WNT, RAS, HIF-1α and AMPK cross-talk with the PI3K-AKT-MTOR pathway at a variety of molecular junctions. Thus, making this pathway sensitive to perceiving various growth modulatory conditions, ranging from the presence of growth factors to hypoxia and nutrient deprivation (Sengupta et al., 2010; Yang and Guan, 2007). The aberrant expression of the PI3K-AKT-MTOR pathway in OSCC advocated the targeting of this coveted pathway (Chakraborty et al., 2008). In various cancers, the monotherapeutic treatments with inhibitors like LY294002 (PI3K inhibitor) and rapamycin (MTOR inhibitor) demonstrated reduced efficacies. Such reduced efficacies were attributed to the drug toxicity and non-specific action of LY294002 (Davies et al., 2000; Sun et al., 2005; Ikezoe et al., 2007; Wang et al., 2008; Liu et al., 2009), or the ablation of a feedback inhibition loop leading to the reactivation of the PI3K-AKT-MTOR pathway by rapamycin (O'Reilly et al., 2006; Carracedo et al., 2008). Thus, rapamycin or its analogues demonstrated mediocre efficacy due to cytostatic effects in clinical trials, primarily due to the paradoxical activation of major survival kinases namely MAPK and AKT (O'Reilly et al., 2006; Carracedo et al., 2008). The present study aimed at increasing the efficacy of these drugs by incorporating a combinatorial approach. The MTT assay demonstrated that prolonged monotherapeutic treatments with rapamycin led to a modest growth inhibition in three OSCC (KB, SCC131 and SCC084) and HeLa cell lines. Western blot analysis of the phosphorylation status of AKT and RPS6KB1 revealed that monotherapeutic treatments with rapamycin for 96 hr led to the reactivation of the PI3K-AKT-MTOR pathway. Thus, the modest growth inhibitory effect of rapamycin was attributed to the reactivation of the PI3K-AKT-MTOR pathway. A combinatorial treatment approach was hence believed to circumvent this problem in order to increase the efficacy of targeting the PI3K-AKT-MTOR pathway. The PI3K inhibitor LY294002 was used combinatorially with rapamycin. This prolonged dual combinatorial treatment regime was distinctly more efficacious than either of the drugs alone and led to a reduction in cellular viability accompanied by increased sub-G1 population, indicating marked cell death that was characterized as caspase-3 dependent apoptosis. The differential sensitivity of the cell lines towards this combinatorial treatment revealed a novel determinant of the sensitivity, the transactivation of EGFR. The cell lines (SCC131 and SCC084) that were capable of transactivating EGFR were relatively resistant to the dual targeting of PI3K and MTOR in comparison to cell lines that did not transactivate EGFR (HeLa and KB). Further, targeting PI3K, MTOR and EGFR simultaneously was more efficacious in the presence of EGFR transactivation than dually targeting PI3K and MTOR. The results conclusively proved that the combinatorial therapeutic approach dually targeting PI3K and MTOR is a promising treatment strategy as compared to a monotherapeutic treatment and a major factor determining the sensitivity towards this treatment is the status of autophosphorylation of EGFR (Tyr1173) which governs the potential for EGFR transactivation by the combinatorial treatment. Thus, this study demonstrated that the status of EGFR autophosphorylation (Tyr1173) can be used as a biomarker to predict the sensitivity towards the combinatorial targeting of PI3K and MTOR in OSCC. The PI3K-AKT-MTOR pathway is negatively regulated by TSC2 (tuberous sclerosis complex 2; tuberin) (Tee et al., 2002). The importance of the TSC2 gene in the regulation of cell growth and proliferation is irrefutable. TSC2 facilitates the crosstalk between a variety of cellular signals, making it a crucial hub where many cellular networks integrate like AKT, MAPK and AMPK (Clements et al., 2007; Rosner et al., 2007; Rosner et al., 2008). It is a tumor suppressor gene and is downregulated in many cancers including OSCC (Chakraborty et al., 2008). In order to identify the genes regulated by TSC2 in OSCC, we stably overexpressed TSC2 in KB cells and the changes in the gene expression profiles caused by this ectopic overexpression were observed using a whole genome expression microarray. The results showed differential regulation of 268 genes (107 genes were upregulated and 161 genes were downregulated, p<0.05, fold change ≥ 1.5). A majority of these genes were functionally associated with transcription, cell growth and proliferation, apoptosis, cell cycle and neurogenesis. Functional annotation and network analysis was performed by using the DAVID v6.7 and IPA version 8.7 softwares. The microarray data revealed a novel aspect in the crosstalk between WNT signaling and TSC2, namely the transcriptional regulation of WNT signaling by TSC2. Further, in the context of therapeutic applications, the microarray analysis revealed multiple genes that were functionally categorized to be involved in response to radiation, UV and drugs (e.g., SERPINB13 and IL1B). Future studies on the regulation of such genes that are involved in responses to drugs and radiation may give insights into the role of TSC2 in resistance or sensitivity towards chemotherapy and radiation therapy. Moreover, EREG, a member of the epidermal growth factor family, was found to be the most downregulated gene in the microarray analysis. Previous reports have documented elevated levels of EREG in tuberous sclerosis lesions and its association with poor clinical prognosis in OSCC patients (Li et al., 2008; Shigeishi et al., 2008), making its regulatory aspects intriguing. Additionally, published data on the transcriptional functions of TSC2 instigated us to analyze the role of TSC2 in the regulation of EREG. TSC2 has been shown to modulate the transcription mediated by members of the steroid receptor superfamily of genes (Henry et al., 1998) and was shown to bind specifically to ERα and inhibit estrogen induced proliferation (Finlay et al., 2004). Also, TSC2 has been shown to possess C-terminal transcriptional activation domains (Tsuchiya et al., 1996). We have therefore attempted to investigate the transcription related functional aspects of TSC2 by exploiting the observed transcriptional repression of EREG. The physiological roles of TSC1 and TSC2 that are independent of the PI3K-AKT-MTOR pathway have been termed as ‘non-canonical’ (Neuman and Henske, 2011). The repression of EREG by TSC2 was observed to be insensitive to rapamycin, suggesting that it was independent of MTORC1 and thus a non-canonical function of TSC2. To determine whether the repression in EREG was at the level of the promoter, we performed a dual luciferase reporter assay. The results showed that the EREG promoter was repressed by stable as well as transient overexpression of TSC2. In order to elucidate the mechanism of transcriptional regulation by TSC2, we performed the ChIP analysis to observe the in vivo binding of TSC2 to the EREG promoter. In the ChIP analysis with the anti-TSC2 antibody, we observed that TSC2 did not bind to the EREG promoter between the regions -857 bp to -302 bp or -325 bp to +165 bp. Further, in silico analysis revealed an interesting trend among the transcription factors that were differentially regulated by TSC2 and had putative binding sites on the EREG promoter. A majority of these transcription factors (17/21) were downregulated by the overexpression of TSC2. This observation suggested that the repression of EREG could be an indirect effect due to repression of transcription factors caused by overexpression of TSC2. On the whole, this study revealed novel functions of TSC2 in OSCC with implications in determining novel biomarkers and therapeutic targets. As discussed previously, OSCC has a very flagitious treatment regime. A prodrug approach is thought to aid in targeting chemotherapy (Rooseboom et al., 2004). CYP1B1, a member of the cytochrome P450 family, has been implicated in chemical carcinogenesis (Bandiera et al., 2005; Sliwinski et al., 2010). There exists a general accordance that this protein is overexpressed in a variety of cancers (e.g., colon, lung, renal, bladder, prostate, breast, endometrial and esophageal cancers), making it an ideal candidate for a prodrug therapy (McFadyen et al., 1999; Murray et al., 2001; McFadyen et al., 2004; Sissung et al., 2006; Wen and Walle, 2007; Sliwinski et al., 2010). The activation of the prodrug facilitated by CYP1B1 would enable the targeting of chemotherapy to tumor tissues in which CYP1B1 is specifically overexpressed as a result reducing the non-specific side effects that the current chemotherapy elicits (Rooseboom et al., 2004). This study was aimed at validating the use of CYP1B1 as a target for the prodrug therapy in OSCC. The expression profile of CYP1B1 was analysed in a panel of 51 OSCC tumors, their corresponding normal tissues, an epithelial dysplasia lesion and its matched normal tissue by qRT-PCR, Western blotting and Immunohistochemistry. Counterintuitively, CYP1B1 was found to be downregulated in 77.78% (28/36) tumor tissues in comparison to their corresponding normal tissues as well as in the epithelial dysplasia lesion compared to its matched normal tissue at the transcriptional level, and in 92.86% (26/28) of tumor tissues at the protein level. This clearly demonstrated the downregulation of CYP1B1 at the transcriptional and translational levels in tumor tissues in comparison to their corresponding normal tissues. These observations indicate that caution should be observed as this therapy may not be applicable universally to all cancers. Since CYP1B1 has been shown to be involved in the activation of pro-carcinogens (Murray et al., 2001; Bandiera et al., 2005; Sissung et al., 2006), its inhibition could facilitate the development of a prophylactic therapy for oral cancer. Overall, this study has identified the transactivation of EGFR as a determinant of sensitivity towards combinatorial targeting of PI3K and MTOR in OSCC and has demonstrated that the autophosphorylation of EGFR (Tyr1173) can be used as a marker to judge the sensitivity towards this treatment. In the clinical perspective, the identification of such markers would aid in predicting the efficacy of targeted therapies. Such investigations would enable the strategic treatment of OSCC patients, thus decreasing the time lost in trial and errors for determining the appropriate treatment. Additionally, this study elucidated a novel role of TSC2 in the transcriptional repression of EREG, a prognostic biomarker for OSCC. Further, the study revealed potential prognostic biomarkers as well as therapeutic targets that are regulated by TSC2 by using a whole genome expression microarray. Moreover, the counterintuitive downregulation of CYP1B1 in OSCC tumors suggested the possibility of a prophylactic therapy for oral cancer but also advised a precautionary note for the application of prodrug treatments based on CYP1B1 overexpression in OSCC.

Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer, with a worldwide incidence of 275,000 new cases annually (Warnakulasuriya, 2009). Globally, the head and neck carcinoma represents a major cause of morbidity and mortality and is the sixth most commonly occurring cancer (Warnakulasuriya, 2009). A majority (>90%) of the head and neck cancers are squamous in origin and thus are linguistically referred to as head and neck squamous cell carcinoma (HNSCC) (Warnakulasuriya, 2009). HNSCC includes cancers of the oral cavity, larynx and pharynx; oral cancer being the most common (Warnakulasuriya, 2009). Although, HNSCC is the sixth most common cancer globally (Warnakulasuriya, 2009), the Indian scenario is graver. According to GLOBOCAN 2008 (http://globocan.iarc.fr), the worldwide age standardized incidence rate (ASR) for HNSCC (and thus OSCC) is 5.3 and 2.5 per 100,000 males and females respectively (Ferlay et al., 2010). In India, the ASR is 9.8 and 5.2 per 100,000 males and females respectively, clearly demonstrating a remarkably high incidence rate of OSCC (Ferlay et al., 2010; http://globocan.iarc.fr). OSCC is a peculiar cancer which is largely preventable and rarely presents as a familial disorder. The most common etiological factors associated with OSCC include tobacco and alcohol consumption (Johnson, 2001). Additionally, high risk human papillomaviruses (HPV strains 16 and 18) as well as genetic predispositions have been implicated. The treatment of OSCC mainly relies on surgical resection of the tumor. The site, size, depth of infiltration and proximity to the bone of the tumor determine whether a combination of surgery with radiation therapy or chemotherapy would be advised (Scully and Bagan, 2009). The concomitant chemo-radiation therapy is the most commonly used strategy in locally advanced cancer. Taxanes (e.g., paclitaxel and docetaxel) and platinum-based induction chemotherapy (e.g., cisplatin) are the options in the treatment of locally advanced cancer. Epidermal growth factor receptor (EGFR) targeted with cetuximab in combination with radiotherapy has been successfully tested in a large randomized trial and thus is currently a new option (Scully and Bagan, 2009). The success of cetuximab has paved the path for the development and implementation of molecules targeting various signaling pathways. Despite extensive research on oral squamous cell carcinoma (OSCC), the five-year survival rate has not changed in several decades with the exception of the targeted treatment strategies involving cetuximab as discussed above. The current chemotherapeutic approaches lack selectivity and are flagitious. Thus, effective treatment of OSCC requires the identification of molecular targets to design appropriate therapeutic strategies. To this end, the present study took three distinct approaches in order to validate the use of existing targets and to reveal novel prognostic biomarkers and therapeutic targets. 1) Targeting the PI3K-AKT-MTOR pathway in OSCC and identification of determinants of its sensitivity. 2) Gene expression analysis of ectopically overexpressed TSC2 to identify new therapeutic targets and prognostic biomarkers as well as to elucidate the genes regulated by it. 3) Expression profiling of CYP1B1 in order to validate the use of CYP1B1 based prodrug therapy in OSCC. Investigations pertaining to the changes in gene and protein expression profiles in malignant as well as pre-malignant lesions have documented the deregulation of the PI3K-AKT-MTOR (phosphoinositide 3-kinase-AKT-mechanistic target of rapamycin) and EGFR (epidermal growth factor receptor) pathways in OSCC which are being widely targeted in many therapeutic strategies (Molinolo et al., 2007; Chakraborty et al., 2008; Matta and Ralhan, 2009; Molinolo et al., 2009; Stransky et al., 2011). The PI3K-AKT-MTOR pathway is a central hub for controlling cellular proliferation and growth in response to various intracellular as well as extracellular stimuli. Crucial signaling cascades including WNT, RAS, HIF-1α and AMPK cross-talk with the PI3K-AKT-MTOR pathway at a variety of molecular junctions. Thus, making this pathway sensitive to perceiving various growth modulatory conditions, ranging from the presence of growth factors to hypoxia and nutrient deprivation (Sengupta et al., 2010; Yang and Guan, 2007). The aberrant expression of the PI3K-AKT-MTOR pathway in OSCC advocated the targeting of this coveted pathway (Chakraborty et al., 2008). In various cancers, the monotherapeutic treatments with inhibitors like LY294002 (PI3K inhibitor) and rapamycin (MTOR inhibitor) demonstrated reduced efficacies. Such reduced efficacies were attributed to the drug toxicity and non-specific action of LY294002 (Davies et al., 2000; Sun et al., 2005; Ikezoe et al., 2007; Wang et al., 2008; Liu et al., 2009), or the ablation of a feedback inhibition loop leading to the reactivation of the PI3K-AKT-MTOR pathway by rapamycin (O'Reilly et al., 2006; Carracedo et al., 2008). Thus, rapamycin or its analogues demonstrated mediocre efficacy due to cytostatic effects in clinical trials, primarily due to the paradoxical activation of major survival kinases namely MAPK and AKT (O'Reilly et al., 2006; Carracedo et al., 2008). The present study aimed at increasing the efficacy of these drugs by incorporating a combinatorial approach. The MTT assay demonstrated that prolonged monotherapeutic treatments with rapamycin led to a modest growth inhibition in three OSCC (KB, SCC131 and SCC084) and HeLa cell lines. Western blot analysis of the phosphorylation status of AKT and RPS6KB1 revealed that monotherapeutic treatments with rapamycin for 96 hr led to the reactivation of the PI3K-AKT-MTOR pathway. Thus, the modest growth inhibitory effect of rapamycin was attributed to the reactivation of the PI3K-AKT-MTOR pathway. A combinatorial treatment approach was hence believed to circumvent this problem in order to increase the efficacy of targeting the PI3K-AKT-MTOR pathway. The PI3K inhibitor LY294002 was used combinatorially with rapamycin. This prolonged dual combinatorial treatment regime was distinctly more efficacious than either of the drugs alone and led to a reduction in cellular viability accompanied by increased sub-G1 population, indicating marked cell death that was characterized as caspase-3 dependent apoptosis. The differential sensitivity of the cell lines towards this combinatorial treatment revealed a novel determinant of the sensitivity, the transactivation of EGFR. The cell lines (SCC131 and SCC084) that were capable of transactivating EGFR were relatively resistant to the dual targeting of PI3K and MTOR in comparison to cell lines that did not transactivate EGFR (HeLa and KB). Further, targeting PI3K, MTOR and EGFR simultaneously was more efficacious in the presence of EGFR transactivation than dually targeting PI3K and MTOR. The results conclusively proved that the combinatorial therapeutic approach dually targeting PI3K and MTOR is a promising treatment strategy as compared to a monotherapeutic treatment and a major factor determining the sensitivity towards this treatment is the status of autophosphorylation of EGFR (Tyr1173) which governs the potential for EGFR transactivation by the combinatorial treatment. Thus, this study demonstrated that the status of EGFR autophosphorylation (Tyr1173) can be used as a biomarker to predict the sensitivity towards the combinatorial targeting of PI3K and MTOR in OSCC. The PI3K-AKT-MTOR pathway is negatively regulated by TSC2 (tuberous sclerosis complex 2; tuberin) (Tee et al., 2002). The importance of the TSC2 gene in the regulation of cell growth and proliferation is irrefutable. TSC2 facilitates the crosstalk between a variety of cellular signals, making it a crucial hub where many cellular networks integrate like AKT, MAPK and AMPK (Clements et al., 2007; Rosner et al., 2007; Rosner et al., 2008). It is a tumor suppressor gene and is downregulated in many cancers including OSCC (Chakraborty et al., 2008). In order to identify the genes regulated by TSC2 in OSCC, we stably overexpressed TSC2 in KB cells and the changes in the gene expression profiles caused by this ectopic overexpression were observed using a whole genome expression microarray. The results showed differential regulation of 268 genes (107 genes were upregulated and 161 genes were downregulated, p<0.05, fold change ≥ 1.5). A majority of these genes were functionally associated with transcription, cell growth and proliferation, apoptosis, cell cycle and neurogenesis. Functional annotation and network analysis was performed by using the DAVID v6.7 and IPA version 8.7 softwares. The microarray data revealed a novel aspect in the crosstalk between WNT signaling and TSC2, namely the transcriptional regulation of WNT signaling by TSC2. Further, in the context of therapeutic applications, the microarray analysis revealed multiple genes that were functionally categorized to be involved in response to radiation, UV and drugs (e.g., SERPINB13 and IL1B). Future studies on the regulation of such genes that are involved in responses to drugs and radiation may give insights into the role of TSC2 in resistance or sensitivity towards chemotherapy and radiation therapy. Moreover, EREG, a member of the epidermal growth factor family, was found to be the most downregulated gene in the microarray analysis. Previous reports have documented elevated levels of EREG in tuberous sclerosis lesions and its association with poor clinical prognosis in OSCC patients (Li et al., 2008; Shigeishi et al., 2008), making its regulatory aspects intriguing. Additionally, published data on the transcriptional functions of TSC2 instigated us to analyze the role of TSC2 in the regulation of EREG. TSC2 has been shown to modulate the transcription mediated by members of the steroid receptor superfamily of genes (Henry et al., 1998) and was shown to bind specifically to ERα and inhibit estrogen induced proliferation (Finlay et al., 2004). Also, TSC2 has been shown to possess C-terminal transcriptional activation domains (Tsuchiya et al., 1996). We have therefore attempted to investigate the transcription related functional aspects of TSC2 by exploiting the observed transcriptional repression of EREG. The physiological roles of TSC1 and TSC2 that are independent of the PI3K-AKT-MTOR pathway have been termed as ‘non-canonical’ (Neuman and Henske, 2011). The repression of EREG by TSC2 was observed to be insensitive to rapamycin, suggesting that it was independent of MTORC1 and thus a non-canonical function of TSC2. To determine whether the repression in EREG was at the level of the promoter, we performed a dual luciferase reporter assay. The results showed that the EREG promoter was repressed by stable as well as transient overexpression of TSC2. In order to elucidate the mechanism of transcriptional regulation by TSC2, we performed the ChIP analysis to observe the in vivo binding of TSC2 to the EREG promoter. In the ChIP analysis with the anti-TSC2 antibody, we observed that TSC2 did not bind to the EREG promoter between the regions -857 bp to -302 bp or -325 bp to +165 bp. Further, in silico analysis revealed an interesting trend among the transcription factors that were differentially regulated by TSC2 and had putative binding sites on the EREG promoter. A majority of these transcription factors (17/21) were downregulated by the overexpression of TSC2. This observation suggested that the repression of EREG could be an indirect effect due to repression of transcription factors caused by overexpression of TSC2. On the whole, this study revealed novel functions of TSC2 in OSCC with implications in determining novel biomarkers and therapeutic targets. As discussed previously, OSCC has a very flagitious treatment regime. A prodrug approach is thought to aid in targeting chemotherapy (Rooseboom et al., 2004). CYP1B1, a member of the cytochrome P450 family, has been implicated in chemical carcinogenesis (Bandiera et al., 2005; Sliwinski et al., 2010). There exists a general accordance that this protein is overexpressed in a variety of cancers (e.g., colon, lung, renal, bladder, prostate, breast, endometrial and esophageal cancers), making it an ideal candidate for a prodrug therapy (McFadyen et al., 1999; Murray et al., 2001; McFadyen et al., 2004; Sissung et al., 2006; Wen and Walle, 2007; Sliwinski et al., 2010). The activation of the prodrug facilitated by CYP1B1 would enable the targeting of chemotherapy to tumor tissues in which CYP1B1 is specifically overexpressed as a result reducing the non-specific side effects that the current chemotherapy elicits (Rooseboom et al., 2004). This study was aimed at validating the use of CYP1B1 as a target for the prodrug therapy in OSCC. The expression profile of CYP1B1 was analysed in a panel of 51 OSCC tumors, their corresponding normal tissues, an epithelial dysplasia lesion and its matched normal tissue by qRT-PCR, Western blotting and Immunohistochemistry. Counterintuitively, CYP1B1 was found to be downregulated in 77.78% (28/36) tumor tissues in comparison to their corresponding normal tissues as well as in the epithelial dysplasia lesion compared to its matched normal tissue at the transcriptional level, and in 92.86% (26/28) of tumor tissues at the protein level. This clearly demonstrated the downregulation of CYP1B1 at the transcriptional and translational levels in tumor tissues in comparison to their corresponding normal tissues. These observations indicate that caution should be observed as this therapy may not be applicable universally to all cancers. Since CYP1B1 has been shown to be involved in the activation of pro-carcinogens (Murray et al., 2001; Bandiera et al., 2005; Sissung et al., 2006), its inhibition could facilitate the development of a prophylactic therapy for oral cancer. Overall, this study has identified the transactivation of EGFR as a determinant of sensitivity towards combinatorial targeting of PI3K and MTOR in OSCC and has demonstrated that the autophosphorylation of EGFR (Tyr1173) can be used as a marker to judge the sensitivity towards this treatment. In the clinical perspective, the identification of such markers would aid in predicting the efficacy of targeted therapies. Such investigations would enable the strategic treatment of OSCC patients, thus decreasing the time lost in trial and errors for determining the appropriate treatment. Additionally, this study elucidated a novel role of TSC2 in the transcriptional repression of EREG, a prognostic biomarker for OSCC. Further, the study revealed potential prognostic biomarkers as well as therapeutic targets that are regulated by TSC2 by using a whole genome expression microarray. Moreover, the counterintuitive downregulation of CYP1B1 in OSCC tumors suggested the possibility of a prophylactic therapy for oral cancer but also advised a precautionary note for the application of prodrug treatments based on CYP1B1 overexpression in OSCC.

In the United States, there are 48,000 new cases of head and neck cancer (HNC) annually. Although HNC used to be associated mainly with smoking and drinking, it is now found in many nonsmokers and nondrinkers in their 50s due to the spread of HPV. Pain is typically present at the time of diagnosis. Treatment usually includes radiation, chemotherapy, and/or surgery, which address the mass effect and pain. Yet, patients continue to experience pain during and after treatment, because the treatment modalities can cause significant inflammation and neuropathy and can lead to central sensitization. Painful mucositis is a complication of chemotherapy and radiation treatment; it can become severe, impacting patients’ ability to speak and eat, and sometimes limiting treatment. Pain treatment for HNC is multimodal, and includes preemptive approaches to prevent neuropathy and central sensitization with antiepileptics, such as gabapentin and pregabalin. Mucositis pain is treated using a stepwise protocol.

Head and neck cancer (HNC) refers to tumours arising from the epithelial lining of the upper aerodigestive track, including the oral cavity, larynx, pharynx, paranasal sinuses, and salivary glands. There are 50,000 cases of HNC diagnosed annually within the United States. The majority of tumours (> 90%) are squamous cell carcinomas. Risk factors include tobacco, alcohol, and areca nuts; human papilloma virus (HPV) or Epstein-Barr virus; and mucosal irritation. Previously considered to be a disease of older adults, the epidemic of HPV-associated oropharyngeal cancers has led to a striking increase in HNC among middle-aged adults. Symptoms are usually present at the time of diagnosis and remain problematic through the terminal phase. For those patients who are cured, long-term biopsychosocial sequelae may persist for years. Thus, assessment and treatment of palliative issues is an intrinsic and vital component of care for the HNC patient.

AbstractHigh-risk human papillomavirus (HPV)-related and most cases of HPV-negative locoregionally advanced head and neck squamous cell carcinoma (LA-HNSCC) have substantial risks of relapse despite definitive therapy, and thus represent conditions of unmet clinical need. The ability now exists to detect molecular residual disease (MRD) in these patients post-definitive treatment such as surgery or (chemo)radiotherapy using novel and highly sensitive and specific technologies to measure cancer-derived circulating biomarkers. The positive and negative predictive values of these assays to forecast cancer recurrence, as well as the lead time of circulating tumor DNA (ctDNA) detection before clinical relapse, are relevant as these parameters rationalize the design of clinical trials for cancer interception in the MRD setting. Currently, there is evidence that interception in the MRD setting yields benefit in clinical outcome in some cancers, but such data do not yet exist in LA-HNSCC and will require prospective testing via clinical trials.

AbstractBody fluids of cancer patients have attracted increasing attention in biomedical research within the last 15 years since—as so-called liquid biopsies—they represent a non-invasive source of clinically exploitable biomarkers, including circulating tumor cells (CTCs) and cell-free tumor DNA (ctDNA). Assessment of CTCs in peripheral blood from solid cancer patients has proven useful for detection of subclinical disease which otherwise remains invisible for current staging techniques. Based on results from large cohort studies in breast and colon cancer, diagnostic tests for enumeration of CTCs have been developed which can be used for tumor staging, prognosis, and post-treatment surveillance. Circulating plasma DNA derived from Epstein–Barr or human papilloma viruses has been established as a sensitive and highly specific biomarker for early cancer detection and disease monitoring. More recently, first studies have been initiated for studying the diagnostic value of mutant variants in plasma-derived ctDNA for treatment selection, response assessment and early detection of treatment failure.Advanced Head and Neck Squamous Cell Carcinoma (HNSCC) represents a malignancy associated with locoregionally advanced stage at presentation, dismal prognosis and little improvement in treatment outcome over the past decade, especially for patients with metastatic disease. HNSCC patients might therefore benefit from incorporation of liquid biopsy-based assays in clinical management. In the following chapters, I will summarize current evidence of the diagnostic value of liquid biopsies in HNSCC and give examples of potential clinical applications.

AbstractTreatment of localized cancer with protons therapy (PT) seems an appealing alternative to photons. PT has been available for decades and has unique dose distribution properties with the so-called Bragg-peak enabling protons to stop after their maximum depth is reached within millimeters. PT allows sparing of normal tissues and organs to a much greater extent than photons, even when modern photon techniques like intensity-modulated radiotherapy (IMRT) and volumetric modulated arc therapy (VMAT) are involved. Whereas IMRT and VMAT techniques have demonstrated their superiority to older 3D-conformal irradiation techniques, there is still a large gap between the theoretical advantages of PT due to its superior dose distribution and high-level clinical evidence, particularly in head and neck cancer (HNC). So far, mostly non-randomized clinical studies exist with clinical results in oropharyngeal, nasopharyngeal, sinonasal, periorbital, and salivary gland cancer. The limited broad availability of this treatment method and its unknown cost-effectiveness need to be evaluated. In this chapter, we discuss the currently available evidence of PT for HNCs and viable options to generate further evidence like the model-based approach.

AbstractOverall Survival (OS) and late quality of life of patients with locally advanced, HPV-negative Head and Neck Squamous Cell Cancer (HNSCC) are not satisfactory. Nutritional status at the beginning of both surgical and non-surgical treatment with curative intent has been linked to OS and quality of life. Weight loss, body-mass index, functional parameters, and biochemical examinations have been associated with the risk of treatment-related adverse events, mortality, quality of life and outcome. Moreover, there is a strong need for effective preventive approaches that could be implemented after completion of curative treatment to reduce recurrences, second tumors and improve quality of life. Aerobic exercise training, which is known to stimulate the immune system, and nutritional interventions represent easy, acceptable and reproducible methods to increase immune and nutritional competence, impacting thus on the aforementioned objectives. Participating in exercise training programs has been well accepted by patients with HNSCC, underlining the feasibility of such an intervention. There are also immunological arguments to promote an adequate physical activity in HNSCC patients. Baseline and after treatment immune competence of HNSCC should be weighted, as it may impact on cancer recurrence and OS. Patients with HNSCC have significantly lower absolute numbers of CD3+, CD4+ and CD8 + T cells than normal controls, and lymphocyte counts, neutrophil/lymphocyte ratio and the prognostic nutritional index (PNI) are associated with prognosis. Aerobic exercise training prevents immune senescence, and therefore may prevent cancer development. The immune system activity is accompanied by an increased rate of metabolism, requiring energy sources, substrates for biosynthesis and regulatory molecules, which are ultimately derived from the diet. Hence, an adequate supply of a wide range of nutrients is essential to support the immune system to function optimally. Therefore, a regular nutritional counselling combined with personalized exercise training is a simple and very cheap way that could improve patient’s nutritional condition and immunological function, and ultimately impact on survival and quality of life. There is a strong need for well conducted clinical trials aimed at evaluating, in homogeneous groups of HNSCC patients, the impact of nutritional interventions (also comprising immunonutrition) and physical exercise. The evaluation of surrogate endpoints like circulating immune cells should also be explored to identify feasible and effective interventions. Integrating these interventions within immunotherapy approaches represents another area deserving further studies.

AbstractImmune checkpoint inhibitors have changed the standard of care for recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC). However, only a minority of patients respond, hence the search for predictive biomarkers. Potential predictive biomarkers for immune checkpoint inhibitors discussed in this chapter include (1) Immune checkpoint ligand expression e.g., PD-L1, (2) biomarkers of a T-cell inflamed tumour microenvironment (TME) such as gene expression profiles of activated T cells, (3) biomarkers of tumour neoepitope burden such as tumour mutation burden (TMB) and (4) multidimensional quantitative techniques. At present only PD-L1 expression has been shown to have clinical utility in head and neck cancer. It enriches for populations more likely to respond, but the false positive predictive value remains high. In the pivotal Keynote−048 trial that established a role for pembrolizumab (anti-PD1) monotherapy and pembrolizumab + chemotherapy as treatment options in first-line R/M HNSCC, primary endpoints included overall survival in defined subgroups based on PD-L1 expression. In this trial the combined positive score (CPS) was used which takes into account PD-L1 expression in tumour and immune cells. Based on this trial regulatory approvals for first-line pembrolizumab in R/M HNSCC require assessment of PD-L1 expression using the CPS. Finally we discuss emerging evidence that locoregionally advanced HPV-associated oropharyngeal cancers that have high expression of CD103 positive CD8 T cells have an excellent prognosis and features that suggest increased probability of responding to anti-PD1/PD-L1, raising the possibility of incorporating these immune therapies as part of a de-escalation trial strategy.

AbstractSince the initial reports of activity of pembrolizumab in recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC), investigation of the role of immune therapies has been the major focus of clinical trials in R/M HNSCC. Randomised trials initially with nivolumab and later with pembrolizumab established overall survival benefit in patients with R/M HNSCC previously treated with platinum compared to physicians choice of 2nd line therapy, and have led to regulatory approval around the world. More recently the Keynote-048 trial has compared both pembrolizumab monotherapy and pembrolizumab + platinum/5FU to the Extreme regimen of platinum/5FU/cetuximab in the first-line R/M setting. The key findings from this trial are that pembrolizumab monotherapy compared to Extreme improved overall survival in patients with PD-L1 combined positive score (CPS) ≥ 20 and ≥ 1, and that pembro/chemotherapy improved OS in CPS ≥ 20, CPS ≥ 1 and the total population. Relative to Extreme there was less toxicity in the monotherapy arm and comparable toxicity in the pembro/chemo arm. Based on this trial use of pembrolizumab as part of first-line treatment for R/M HNSCC is appropriate for the majority of patients, and represents a new standard of care. The focus has now moved to identifying combinations that may be superior to pembrolizumab monotherapy or to chemotherapy + pembrolizumab. Some of the more promising approaches under investigation in HNSCC are discussed in this chapter. In summary, immune therapies are now the cornerstone of management of R/M HNSCC with the approval of pembrolizumab in the first-line R/M setting.

AbstractAnti-PD-1 immune checkpoint inhibitors have recently revolutionized the treatment of recurrent and/or metastatic head and neck squamous cell carcinoma (HNSCC) patients, both in the first and second recurrent and metastatic settings. However, not all patients respond to PD-1 blockade, nor derive prolonged benefit from these immunotherapies, requiring further development of immune-oncology strategies beyond PD-1/PD-L1 inhibitors. There has been an important therapeutic development with the evaluation of many new immune checkpoints molecules and other type of immunomodulatory molecules, along with combinations of these new agents with PD-1/PD-L1 inhibitors, but very few of these strategies have shown significant anti-tumor activity as single agent in HNSCC patients, and further results are awaited from ongoing trials. All randomized trials assessing novel immune-oncology drugs in combination with an anti-PD1/PD-L1 agents have failed so far in HNSCC patients. Many other immune-oncology drugs are still in early clinical development and will hopefully improve HNSCC patient outcomes.

AbstractDerived from intracranial stereotactic radiosurgery, stereotactic body radiation therapy (SBRT) was introduced to clinical practice in 1991. Based on delivering precisely targeted high doses of radiation in one or several fractions, the concept of SBRT has been rapidly adopted by many institutions. This chapter aims to summarize the available literature regarding the use of SBRT in the reirradiation setting of recurrent and second primary head and neck cancer (HNC) as well as in oligometastatic HNC. Several studies have been published showing safety and efficacy of SBRT-based reirradiation in recurrent disease or a second malignancy. According to a meta-analysis of 10 papers published between 2006 and 2016, SBRT-based reirradiation seems to be a promising modality with acceptable safety and short overall treatment times in patients with inoperable recurrent or second primary HNC. However, overall survival following SBRT-based reirradiation remains moderate, which might be due to insufficient doses. There is thus a need for well-designed trials of SBRT-based reirradiation in terms of dose escalation and combination with systemic therapy. In HNC, the evidence for treatment of oligometastatic disease using SBRT is less clear when compared to other tumor types. We need to understand which patients benefit the most from local ablation, when the right moment is to intervene, how SBRT compares with surgery in operable patients and with other modalities (e.g. radiofrequency ablation), and what the impact of combination strategies is (e.g. with immune checkpoint inhibitors). All these unmet needs underline the importance of conducting dedicated studies in oligometastatic squamous HNC patients.

AbstractDespite the fact that elderly people are the main incidental and continuously growing patient group with head and neck cancer, prospective trials focusing on special issues regarding head and neck surgery in elderlies are missing. To avoid complications during and after surgery in that patient category, comprehensive evaluation of functional status, comorbidities, performance status, social support and mental condition is mandatory. Regarding functional parameters, cardiac and respiratory conditions play a major role for any primary surgical procedure. Nevertheless, other comorbidities, medication and patients view on self-determination have carefully to be taken into consideration. It has repeatedly been shown that fit elderly individuals may benefit from intensive therapies like reconstructive surgery with microvascular free tissue transfer, concurrent chemoradiotherapy in the locoregionally advanced disease setting, and even from the standard first- and second-line palliative systemic therapies. Since it is well known that tolerance of systemic nonsurgical treatments in elderly people is less and therefore death from noncancer-related causes in that population is higher, moderate surgical procedures can be even more effective regarding quality of life in situations facing higher comorbidities, or functional constraints with limited life expectancy compared to nonsurgical standard approaches. Older people usually are at increased risk of postoperative complications. In particular, organ failure progresses much faster in multiple organ failure. The preoperative clarification of comorbidity for the avoidance of surgical complications is therefore of major importance. Close coordination with anesthesia and rapid postoperative mobilization are essential for this. Decision-making and treatment based on specific assessment in an experienced multidisciplinary team is key.

Squamous cell carcinoma (SCC) is the most commonly diagnosed oral cancer. It is a type of head and neck squamous cell carcinoma (HNSCC) oral cancer affects more than 300,000 people in a year. Oral cancer is the sixth most common malignant cancer. The traditional methods of treatment were used through surgery, followed by chemotherapy, but these methods are not effective enough for the treatment, so treatment was focused on using magnetic nanoparticles. Magnetic nanoparticles demolish only the cancer cells directly without affecting healthy cells. They can also be used to increase the effectiveness of the other treatment methods. Iron oxide nanoparticles, maghemite (Fe2O3) and magnetite (Fe3O4) are widely used in the diagnosis and treatment of cancerous diseases. Iron oxides NPs have distinctive properties as they have good biodegradability, very low toxicity, modifiability, and ease of preparation. the method of hyperthermia is one of the effective methods in the treatment of cancer. Because cancer cells show greater sensitivity to high temperature compared to normal cells.