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      Correlation of HPV16 Gene Status and Gene Expression With Antibody Seropositivity and TIL Status in OPSCC

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          Abstract

          Introduction

          Human papillomavirus 16 (HPV16) is the main cause of oropharyngeal squamous cell carcinoma (OPSCC). To date, the links between HPV16 gene expression and adaptive immune responses have not been investigated. We evaluated the correlation of HPV16 DNA, RNA transcripts and features of adaptive immune response by evaluating antibody isotypes against E2, E7 antigens and density of tumor-infiltrating lymphocytes (TIL).

          Material and Methods

          FFPE-tissue from 27/77 p16-positive OPSCC patients was available. DNA and RNA were extracted and quantified using qPCR for all HPV16 genes. The TIL status was assessed. Immune responses against E2 and E7 were quantified by ELISA (IgG, IgA, and IgM; 77 serum samples pre-treatment, 36 matched post-treatment).

          Results

          Amounts of HPV16 genes were highly correlated at DNA and RNA levels. RNA co-expression of all genes was detected in 37% (7/19). E7 qPCR results were correlated with higher anti-E7 antibody (IgG, IgA) level in the blood. Patients with high anti-E2 IgG antibody (>median) had better overall survival (p=0.0311); anti-E2 and anti-E7 IgA levels had no detectable effect. During the first 6 months after treatment, IgA but not IgG increased significantly, and >6 months both antibody classes declined over time. Patients with immune cell-rich tumors had higher levels of circulating antibodies against HPV antigens.

          Conclusion

          We describe an HPV16 qPCR assay to quantify genomic and transcriptomic expression and correlate this with serum antibody levels against HPV16 oncoproteins. Understanding DNA/RNA expression, relationship to the antibody response in patients regarding treatment and outcome offers an attractive tool to improve patient care.

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          Most cited references52

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          Global Cancer Statistics 2018: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries

          This article provides a status report on the global burden of cancer worldwide using the GLOBOCAN 2018 estimates of cancer incidence and mortality produced by the International Agency for Research on Cancer, with a focus on geographic variability across 20 world regions. There will be an estimated 18.1 million new cancer cases (17.0 million excluding nonmelanoma skin cancer) and 9.6 million cancer deaths (9.5 million excluding nonmelanoma skin cancer) in 2018. In both sexes combined, lung cancer is the most commonly diagnosed cancer (11.6% of the total cases) and the leading cause of cancer death (18.4% of the total cancer deaths), closely followed by female breast cancer (11.6%), prostate cancer (7.1%), and colorectal cancer (6.1%) for incidence and colorectal cancer (9.2%), stomach cancer (8.2%), and liver cancer (8.2%) for mortality. Lung cancer is the most frequent cancer and the leading cause of cancer death among males, followed by prostate and colorectal cancer (for incidence) and liver and stomach cancer (for mortality). Among females, breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death, followed by colorectal and lung cancer (for incidence), and vice versa (for mortality); cervical cancer ranks fourth for both incidence and mortality. The most frequently diagnosed cancer and the leading cause of cancer death, however, substantially vary across countries and within each country depending on the degree of economic development and associated social and life style factors. It is noteworthy that high-quality cancer registry data, the basis for planning and implementing evidence-based cancer control programs, are not available in most low- and middle-income countries. The Global Initiative for Cancer Registry Development is an international partnership that supports better estimation, as well as the collection and use of local data, to prioritize and evaluate national cancer control efforts. CA: A Cancer Journal for Clinicians 2018;0:1-31. © 2018 American Cancer Society.
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            Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

            The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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              Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction

              Background Choosing appropriate primers is probably the single most important factor affecting the polymerase chain reaction (PCR). Specific amplification of the intended target requires that primers do not have matches to other targets in certain orientations and within certain distances that allow undesired amplification. The process of designing specific primers typically involves two stages. First, the primers flanking regions of interest are generated either manually or using software tools; then they are searched against an appropriate nucleotide sequence database using tools such as BLAST to examine the potential targets. However, the latter is not an easy process as one needs to examine many details between primers and targets, such as the number and the positions of matched bases, the primer orientations and distance between forward and reverse primers. The complexity of such analysis usually makes this a time-consuming and very difficult task for users, especially when the primers have a large number of hits. Furthermore, although the BLAST program has been widely used for primer target detection, it is in fact not an ideal tool for this purpose as BLAST is a local alignment algorithm and does not necessarily return complete match information over the entire primer range. Results We present a new software tool called Primer-BLAST to alleviate the difficulty in designing target-specific primers. This tool combines BLAST with a global alignment algorithm to ensure a full primer-target alignment and is sensitive enough to detect targets that have a significant number of mismatches to primers. Primer-BLAST allows users to design new target-specific primers in one step as well as to check the specificity of pre-existing primers. Primer-BLAST also supports placing primers based on exon/intron locations and excluding single nucleotide polymorphism (SNP) sites in primers. Conclusions We describe a robust and fully implemented general purpose primer design tool that designs target-specific PCR primers. Primer-BLAST offers flexible options to adjust the specificity threshold and other primer properties. This tool is publicly available at http://www.ncbi.nlm.nih.gov/tools/primer-blast.
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                Author and article information

                Contributors
                Journal
                Front Oncol
                Front Oncol
                Front. Oncol.
                Frontiers in Oncology
                Frontiers Media S.A.
                2234-943X
                26 January 2021
                2020
                : 10
                : 591063
                Affiliations
                [1] 1 CRUK and NIHR Experimental Cancer Medicine Center & School of Cancer Sciences, Faculty of Medicine, University of Southampton , Southampton, United Kingdom
                [2] 2 Department of Otorhinolaryngology, Head & Neck Surgery, University of Ulm , Ulm, Germany
                [3] 3 Southampton University Hospitals NHS Foundation Trust , Southampton, United Kingdom
                [4] 4 Department Affaires Médicinales, Research, Project, Transgene SA , Illkirch, France
                [5] 5 Department of Otorhinolaryngology, Head & Neck Surgery, Poole Hospital , Poole, United Kingdom
                [6] 6 Liverpool Head and Neck Centre, Institute of Translational Medicine, Department of Molecular & Clinical Cancer Medicine, University of Liverpool , Liverpool, United Kingdom
                Author notes

                Edited by: Francois Mouawad, Centre Hospitalier Regional et Universitaire de Lille, France

                Reviewed by: Stuart Charles Winter, University of Oxford, United Kingdom; Stefania Staibano, University of Naples Federico II, Italy

                *Correspondence: Christian H. Ottensmeier, c.ottensmeier@ 123456liverpool.ac.uk

                This article was submitted to Head and Neck Cancer, a section of the journal Frontiers in Oncology

                Article
                10.3389/fonc.2020.591063
                7871909
                33575210
                055beb3f-e9cb-4085-9575-21e0bb95acae
                Copyright © 2021 von Witzleben, Currall, Wood, Chudley, Akinyegun, Thomas, Bendjama, Thomas, Friedmann, King, Laban and Ottensmeier

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 03 August 2020
                : 02 November 2020
                Page count
                Tables: 2, Equations: 1, References: 52, Pages: 13, Words: 6601
                Funding
                Funded by: Deutsche Forschungsgemeinschaft 10.13039/501100001659
                Award ID: # WI 5255/1-1:1, GRK-2254
                Categories
                Oncology
                Original Research

                Oncology & Radiotherapy
                oropharyngeal squamous cell carcinoma,human papillomavirus 16,antibody isotype,gene expression,immune response

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