Project description:Background: The incidence of human papillomavirus (HPV) associated oropharyngeal carcinomas has increased rapidly over the last thirty years. These tumours behave as a distinct biological entity when compared to classical smoking- and alcohol-related disease. To gain more information regarding the pathway through pre-malignancy in HPV positive/HPV negative oropharyngeal carcinoma (OSCC), we investigated genomewide expression profiles in histopathologically confirmed tumour samples with site-matched normal epithelial controls. Methods: Twenty-four patients with primary oropharyngeal squamous cell carcinoma (4 with stage III and 20 with stage IV disease) were included in this prospective clinical trial (UKCRN11945). The tumour tissues were each assessed by a histopathologist with HPV status and typing by p16INK4A, consensus PGMY PCR, type-specific HPV16 DNA & RNA PCR and DNA sequencing. Fresh tissue samples were subjected to whole transcriptome analysis using the Illumina Bead Array (~47,000 transcripts) and the results validated with quantitative Real Time-PCR (qRT-PCR). In a separate cohort of twelve OSCC patients, laser capture micro-dissection of formalin fixed paraffin embedded (FFPE) tissue allowed RNA extraction from regions of in situ malignant change (with matched normal and invasive SCC samples). Results: The majority of OSCC patients (28/36) displayed evidence of high risk HPV positivity. Predictable fold changes of RNA expression in HPV-associated disease included multiple transcripts within the p53 oncogenic pathway (e.g. CDKN2A/CCND1). Other candidate transcripts found to have altered levels of expression in this study have not previously been established in HPV16 associated oropharyngeal cancer. These were involved in cell differentiation, proliferation and invasion and demonstrated considerable overlap with expression analysis data in other oncogenic pathways (SFRP1/CRCT1/DLG2/SYCP2/CRNN). SYCP2 showed the highest consistent fold change from baseline in both fresh frozen and FFPE tissue (qRT-PCR fresh frozen tissue [P<0.04]; FFPE tissue pre-invasive [P<0.01]; FFPE tissue invasive [P<0.01]), and aberrant expression of this meiosis-specific protein may contribute to genetic instability during HPV-associated cancer development. Conclusion: Investigation of differentially expressed genes in HPV-positive tumours may reveal unique pathways that can explain their different natural history and biological properties. The data from this study reveal SYCP2 (amongst others) as a potential biomarker in HPV positive oropharyngeal carcinoma, and if corroborated on a larger scale, may facilitate the development of a non-invasive screening tool. Twenty four samples (12 matched invasive oropharyngeal carcinoma and normal epithelial controls).

Project description:The global rise of HPV(+) oropharyngeal squamous cell carcinoma (OPSCC) has generated considerable interest underlying its etiology and management. Despite an overall decline in head and neck malignancies, the incidence in OPSCC has by contrast sharply risen, with HPV(+) subtypes now comprising 80% of all OPSCC.1,2 Relative to HPV(-) OPSCC, HPV(+) patients are more responsive to chemoradiation and harbor a 52% risk-of-death reduction.3 Despite this distinct outcome, treatment regimens remain the same for all OPSCC subtypes (smoking-driven and virus-driven), rather than an adaptive approach to what most consider distinct diseases. The short-term effects (e.g., mucositis, odynophagia) and long-term toxicities (e.g., xerostomia, dysphagia, ototoxicity) from treatment substantially affect quality of life, and rival the impact of the cancer itself. Recently published as well as ongoing trials are actively examining deintensification approaches2,4-9, with the goal of diminishing treatment sequelae for HPV(+) subtypes. While deintensification may decrease chemoradiation-related toxicities, it nonetheless may also undertreat a meaningful percentage of HPV(+) patients who may then recur. In examining national trials, 19% of HPV(+) patients had disease progression after therapy, with a two-year survival rate of 60%.10 Current methods to ascertain HPV status involve p16 staining. However, on comparison with gold standard E6/E7 expression by qPCR, p16 harbored a 15% false positivity rate11, suggesting limited utility as a sole biomarker for deintensification. Improved molecular stratification would greatly enhance the clinician’s ability to precisely tailor treatment while minimizing the risk of jeopardizing outcomes. One approach encompasses in-depth proteomic profiling of HPV(+) OPSCC to reveal distinct protein expression profiles and delineate clinically relevant upstream pathways. In turn, these proteomic differences may distinguish higher-risk disease (cases predisposed to recurrence that may benefit from treatment intensification) from lower-risk phenotypes (cases whose treatment response is sufficiently robust to warrant deintensification). Here, two HPV(+) OPSCC cohorts stratified by treatment response are compared via a hybrid data dependent acquisition/data independent acquisition (DDA/DIA) approach via mass spectrometry. We focused on detection of low-abundance proteins to highlight proteomic signatures that can be potentially exploited for treatment stratification.

Project description:Background: The incidence of human papillomavirus (HPV) associated oropharyngeal carcinomas has increased rapidly over the last thirty years. These tumours behave as a distinct biological entity when compared to classical smoking- and alcohol-related disease. To gain more information regarding the pathway through pre-malignancy in HPV positive/HPV negative oropharyngeal carcinoma (OSCC), we investigated genomewide expression profiles in histopathologically confirmed tumour samples with site-matched normal epithelial controls. Methods: Twenty-four patients with primary oropharyngeal squamous cell carcinoma (4 with stage III and 20 with stage IV disease) were included in this prospective clinical trial (UKCRN11945). The tumour tissues were each assessed by a histopathologist with HPV status and typing by p16INK4A, consensus PGMY PCR, type-specific HPV16 DNA & RNA PCR and DNA sequencing. Fresh tissue samples were subjected to whole transcriptome analysis using the Illumina Bead Array (~47,000 transcripts) and the results validated with quantitative Real Time-PCR (qRT-PCR). In a separate cohort of twelve OSCC patients, laser capture micro-dissection of formalin fixed paraffin embedded (FFPE) tissue allowed RNA extraction from regions of in situ malignant change (with matched normal and invasive SCC samples). Results: The majority of OSCC patients (28/36) displayed evidence of high risk HPV positivity. Predictable fold changes of RNA expression in HPV-associated disease included multiple transcripts within the p53 oncogenic pathway (e.g. CDKN2A/CCND1). Other candidate transcripts found to have altered levels of expression in this study have not previously been established in HPV16 associated oropharyngeal cancer. These were involved in cell differentiation, proliferation and invasion and demonstrated considerable overlap with expression analysis data in other oncogenic pathways (SFRP1/CRCT1/DLG2/SYCP2/CRNN). SYCP2 showed the highest consistent fold change from baseline in both fresh frozen and FFPE tissue (qRT-PCR fresh frozen tissue [P<0.04]; FFPE tissue pre-invasive [P<0.01]; FFPE tissue invasive [P<0.01]), and aberrant expression of this meiosis-specific protein may contribute to genetic instability during HPV-associated cancer development. Conclusion: Investigation of differentially expressed genes in HPV-positive tumours may reveal unique pathways that can explain their different natural history and biological properties. The data from this study reveal SYCP2 (amongst others) as a potential biomarker in HPV positive oropharyngeal carcinoma, and if corroborated on a larger scale, may facilitate the development of a non-invasive screening tool.

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	01dez_Marisol_200min_cid_16.raw	Raw
	01dez_Marisol_200min_cid_5.raw	Raw
	01dez_Marisol_200min_cid_6.raw	Raw
	13jan_Marisol_16.raw	Raw

Dataset Information

New Insights into the impact of Human Papillomavirus on Oral Cancer in Young Patients: Proteomic Approach Reveals a Novel Role for S100A8

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New Insights into the Impact of Human Papillomavirus on Oral Cancer in Young Patients: Proteomic Approach Reveals a Novel Role for S100A8.

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