Despite the advance in modern oncology, there was limited improvement over the survival outcome of head and neck cancers. Until the discovery of the etiological association between Human Papillomavirus (HPV) and oropharyngeal cancer, an accelerated evolution in the field of head and neck oncology began. This viral-related tumor has ignited tremendous effort in American and European countries to explore the optimal treatment approaches. In contrast, the paucity of comprehensive and robust studies commonly exists in many Chinese and Asian countries. Little is known about the current situation of HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) in Hong Kong. This retrospective local multi-institutional study attempted to explore the HPV-associated OPSCC in Hong Kong from various aspects, including demographics, risk factors, clinical and histological features, molecular profile, as well as clinical outcomes. Finally, attempts were made to determine any predictive factors to stratify high-risk patients in this distinct disease entity and explore the most appropriate detection algorithm of the biologically active HPV infection in our locality. With the support from the Hong Kong Cancer Registry and nine public hospitals, 141 (43.3%) of 326 newly diagnosed OPSCC in the whole population of Hong Kong between 2005 and 2009 were recruited. Inclusion criteria were histologically proven OPSCC with tumor specimens available for prospective laboratory tests. Those with non-SCC, non-oropharyngeal in origin and incomplete clinical records were excluded. Prospective HPV PCR and genotyping, and immunohistochemical staining with p16, p53, cyclin D1 and HER-2 were performed. Univariate and multivariate analyses were performed to assess the correlations between various parameters and HPV-associated OPSCC. Epidemiologically, based on the combined positivity in HPV PCR and p16, the prevalence of HPV-associated OPSCC was 22%. Thirty (96.8%) of thirty one were HPV-16, the remaining one was HPV-18. In the univariate analysis, this cancer directly correlated with female gender (p=0.014), younger age (p=0.012), non-smoker (p=0.02), non-drinker (p=0.06) and early primary tumor (p=0.001). Histologically, basaloid differentiation (p<0.001), non-keratinization (p=0.007) and high tumor infiltrating lymphocyte (TIL) level (p<0.001) showed significant correlations. A distinct molecular profile was identified, with p16 positivity noted in all cases (p<0.001), few of p53 (p<0.001) and cyclin D1 positivity (p<0.001) and absence of HER2 over-expression. Significantly superior prognosis was demonstrated in HPV-associated OPSCC. The 5-year overall and disease specific survivals were 67.0% and 88.6% compared with 27.8% (p<0.001) and 41.3% (p<0.001) in the non-viral counterpart respectively. Other good prognostic factors identified for OS and DSS included early primary disease (T1/T2) (p=0.02; p=0.001), absence of distant metastasis (both p<0.001), high TIL level (both p<0.001), p16-positivity (p=0.002; p=0.003), non-smoker (p=0.021; p=0.014). In the multivariate analysis, the HPV-associated tumor (HR: 0.28; 95% CI: 0.08 – 0.93; p=0.038), early primary tumor (HR: 0.52; 95% CI 0.30 – 0.89; p=0.017) and absence of distant metastasis (HR=0.15 95% CI: 0.07 – 0.3; p<0.001) were associated with lower risk of death from any causes after controlling other confounding factors. Most importantly, high TIL level in HPV-associated OPSCC patients was associated with 89% (HR: 0.11; 95% CI: 0.02 – 0.61; p=0.012) lower risk of death from any causes than those with low TIL level, but not present in non-HPV counterpart. In addition, either HPV PCR complemented with typical high-risk genotyping results or p16 IHC positivity complemented with HPC PCR positivity using specific designed primers were two reasonably sensitive and specific detection algorithms based on the local genotypic distribution of this disease. In summary, this is the first most comprehensive and robust local study indicating the importance of HPV-associated OPSCC in Hong Kong. It successfully illustrated various distinctive characteristics of this viral-related cancer. Additionally, it has suggested a potential predictor to identify the minority with more aggressive diseases and the most effective laboratory detection algorithms in this locality. It definitely facilitates the next step in exploring this disease via larger and prospective trials in coming future. / published_or_final_version / Surgery / Master / Master of Medical Sciences

Source: Masters Abstracts International, Volume: 45-04, page: 1935. Adviser: Erich M. Sturgis. Includes bibliographical references.

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No. of bitstreams: 2 Dissertação - Cesar Augusto Sam Tiago Vilanova Costa - 2006.pdf: 1500361 bytes, checksum: 3401843db66da8f77a90a41b7a08f826 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2006-09-29 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Hypo pharynx is the under region of pharynx and serves both respiratory and digestive tract, acting as a passageway to air and food. It’s composed by squamous nonkeratinized epithelial tissue. Statistical studies indicates that 85 to 90% of neoplasies rush pyriform sinus and are characterized by fast dissemination and metastization. Epidemiological trends of hypo pharynx cancer in Goiania on period 1998 to 2003 analyzing data derived from Cancer Registry of Association for Cancer Combat of Goias, shows that the relative survival rate for men diagnosed with hypo pharynx cancer was 38% past 60 months while women presented a survival rate of 100%. Surgery was the treatments that present fewer rates (34%) and chemotherapy was associated to the most survival rate (54%). Patients that use tobacco presented a survival rate of 43% while non-tabagists present a rate of 60% of surviving. Relative survival rate for patients diagnosed with hypo pharynx tumor was 38% and estimated censure for 5 years was just 5%. Survival rate was extremely decreased due to 45% of cases were diagnosed in advanced stages of disease. It is important to point out that preventive campaign is important to evade the increasing of these pathologies that are related to behavior of tabagism and etilism. Present study also evaluates cytogenetically individuals diagnosed with hypo pharynx cancer. Cytogenetic analysis of exfoliated cells is largely used to evaluate cytotoxic and genotoxic alterations on people exposed to potentially mutagens. A number of 36 individuals were analyzed, representing case group, and 36 healthy individuals composed control group. Data was collected and analysis was performed to estimate main nuclear alterations. Standard protocols were adopted to collect and counting cells. Micronuclei test was used to determinate the frequency of cytotoxic and genotoxic alterations. Besides than micronuclei others alterations like broken-eggs, binucleated cells and piknotic nuclei are good markers of cell alterations because they are resulted from cytotoxic events frequently related to occupational exposure. Behaviors like tabagism, etilism and exposition time were also investigated to correlate cells alterations. The correlation between micronuclei frequencies of case and control group test doesn’t show statistic difference. Broken egg and binucleated cells were the most observed anomalies on case group and showed up significant statistic difference (p=0.03). Results indicates a high grade of cytotoxic anomalies on individuals with hypo pharynx cancer, specially in tabagists and chronic etilists individuals, behaviors that are correlated to induct and promote tumors in susceptible people due their capabilities to affect cell cycle. Studies about relationship between Human Papillomavirus (HPV) and cancer development are performed since 80’s. Although the correlation of HPV and cervical cancer are well established, the possible role of HPV and head and neck cancer are still unclear. Among controversial factors of HPV and carcinogenesis of squamous epithelial cells, there is a viral incidence that oscillates from 0 to almost 100% on studies that includes a myriad of viral genome detect methods, such Blotting and PCR. Importance of HPV infection and epithelial carcinogenesis is related to capabilities of High risk HPV 16 and 18 to promote cell alterations, by tumor suppressor proteins inactivation, tumor suppressor genes blockage and insertions of HPV oncogenes. On present study, HPV detection and typing was performed on tumors derived from hypo pharynx of 24 patients diagnosed with cancer (CID-O: C12,0 to C13,9). All samples was submitted to PCR with generic primers MY09/11 and GP05/06, as specific primers to HPV 6, 11, 16, 18, 33, 35, 45 and 58. Results shows that on 9 of 24 samples, HPV were detected by generic primers (a rate of 37,5%) and HPV types 16, 18 and 45 were identified, with a sample presenting coinfection of HPV 16 and 18. / A hipofaringe, região inferior da faringe, também chamada de laringofaringe ou faringolaríngea, serve aos sistemas respiratório e digestivo, funcionando como passagem para o ar e os alimentos. É composta de tecido epitelial escamoso estratificado não-queratinizado, com grande quantidade de glândulas seromucosas. As estatísticas mostram que entre 85 e 90% dos cânceres que acometem a hipofaringe são carcinomas espino-celulares. A lesão inicial no seio piriforme apresenta-se macroscopicamente sob o aspecto de um nódulo ou úlcera, tendo como característica principal, a rápida disseminação pela submucosa. Atualmente, com a associação de cirurgias radicais e radioterapia pós-peratória aumentou-se o controle locorregional destes tumores, contudo, a sobrevida global não sofreu alterações em decorrência das metástases à distância, o que não representa uma falha no tratamento realizado e sim uma evolução natural devido à característica invasiva desse tipo de neoplasia. Os tumores malignos de hipofaringe não são freqüentes, representando entre 5 a 10% das neoplasias das vias aerodigestivas superiores e apenas 0,5 % de todos os cânceres. Porém, do conjunto das neoplasias malignas da região da cabeça e pescoço, tumores de hipofaringe apresentam o pior prognóstico. A avaliação das tendências epidemiológicas apresentadas pelo câncer de hipofaringe em Goiânia no período de 1998 a 2003, analisando os dados disponíveis pelo Registro de Câncer de Base Populacional da Associação de Combate ao Câncer em Goiás, mostrou uma sobrevida relativa dos pacientes do sexo masculino diagnosticados com câncer de hipofaringe foi de 38% após 60 meses de segmento, enquanto que para o feminino apresentou-se uma sobrevida máxima de 100%, com todas as pacientes curadas. Dentre os tratamentos realizados, a cirurgia apresentou a menor taxa de sobrevida (34%) ao passo que para a quimioterapia foi associada uma sobrevida de 54%. A sobrevida dos pacientes tabagistas (43%) se mostrou muito inferior à dos indivíduos que não eram tabagistas (60%). Em fim, ao longo de cinco anos de segmento deste estudo, a taxa de sobrevida relativa para portadores de tumores malignos da hipofaringe foi de aproximadamente 38%, estimada para cinco anos com uma censura de apenas 5%. A taxa de sobrevida relativa é diminuída uma vez que cerca de 45% dos casos, o diagnóstico é feito tardiamente. O presente estudo também avaliou as alterações citogenéticas nucleares de pacientes portadores de câncer na região da hipofaringe. Foram analisados 36 indivíduos do grupo de casos, constituído de pacientes diagnosticados com tumores de hipofaringe, e 36 indivíduos saudáveis do grupo controle. Para obtenção dos dados foram considerados hábitos de consumo como tabagismo e etilismo e principalmente o tempo de exposição. Foram contadas 5000 células por indivíduo e as freqüências de micronúcleo, binucleadas, broken eggs e demais aberrações nucleares, foram calculadas para ambos os grupos. A freqüência de micronúcleo mostrada entre o grupo controle e o de casos não apresentaram diferença estatística. Porém, foi observado, por parte do grupo de casos, um grande número de células com broken eggs e binucleadas. Tais resultados demonstraram diferença estatística (p = 0,03). A binucleação e a formação de dois núcleos em forma de broken egg sugere que agentes como o tabaco e o alcoolismo crônico podem estar relacionados com a indução e progressão de tumores em pessoas susceptíveis, devido à capacidade desses mutágenos afetarem o ciclo celular. Dentre os fatores que geram controvérsias, podemos destacar o achado freqüente de variados tipos de HPV em mucosa oral normal, numa incidência que varia de 0% a quase 100% em diversos estudos, com numerosos métodos de identificação, incluindo entre eles um método altamente sensível, a PCR. A importância da infecção pelo HPV na carcinogênese de células epiteliais é suportada pela capacidade dos HPVs de alto risco, principalmente os tipos 16 e 18, de imortalizar os queratinócitos orais, quando em análise in vitro. O processo de imortalização pode envolver a desativação de proteínas supressoras de tumores pré-formadas pelas oncoproteínas virais, o bloqueio da transcrição de genes supressores de tumores como resultado da inserção do oncogene do HPV, ou a estimulação da transcrição do oncogene celular pela inserção de seqüências ativadoras de transcrição derivadas do próprio genoma do HPV. No presente estudo, o grupo amostral foi constituído de 24 pacientes diagnosticados com carcinoma de hipofaringe. Os casos foram obtidos do Registro de Câncer de Base Populacional de Goiânia/GO da Associação de Combate ao Câncer em Goiás. Todos os pacientes considerados foram diagnosticados como carcinomas de hipofaringe (CID-O: C12.0 a C13.9). Todas as amostras foram submetidas a uma PCR com os primers genéricos MY09/11 e GP05/06 para detecção em amplo espectro do genoma de HPV. Posteriormente, utilizou-se a mesma abordagem, com os primers tipo-específico para HPVs 6, 11, 16, 18, 33, 35, 45 e 58. Em 9 das 24 amostras utilizadas, foi observada amplificação com os primers genéricos, identificando-se o genoma de HPV em 37,5% (6/24) dos casos. Em 67% (6/9) das amostras, o genoma viral foi detectado por MY09/11 e em 33% (3/9) pelos primers GP05/06. As amostras positivas foram então submetidas a uma segunda PCR para a genotipagem dos HPVs, tendo sido observados os tipos 16, 18 e 45. Os tipos virais mais associados ao câncer da hipofaringe, no presente estudo, foram os HPV 16 e 18, presente em 6 amostras e o HPV45, encontrado em 1 amostra. Em um paciente foi observada a co-infecção de tipos virais de HPVs 16 e 18. Contudo, mesmo diante de tantas pesquisas realizadas, há muito para ser explorado, apesar de uma hipótese possível para a carcinogênese ser a interação sinérgica de carcinógenos químicos, virais, oncogenes e genes supressores de tumores.

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.