Evaluation of a combined triple method to detect causative HPV in oral and oropharyngeal squamous cell carcinomas: P16 Immunohistochemistry, Consensus PCR HPV-DNA, and in Situ Hybridization

Giuseppe Pannone, Vito Rodolico, Angela Santoro, Lorenzo Lo Muzio, Renato Franco, Gerardo Botti, Gabriella Aquino, Maria Pedicillo, Simona Cagiano, Giuseppina Campisi, Corrado Rubini, Silvana Papagerakis, Gaetano De Rosa, Maria Lina Tornesello, Franco M. Buonaguro, Stefania Staibano, Pantaleo Bufo

Research output: Contribution to journalArticle

Abstract

Background. Recent emerging evidences identify Human Papillomavirus (HPV) related Head and Neck squamous cell carcinomas (HN-SCCs) as a separate subgroup among Head and Neck Cancers with different epidemiology, histopathological characteristics, therapeutic response to chemo-radiation treatment and clinical outcome. However, there is not a worldwide consensus on the methods to be used in clinical practice. The endpoint of this study was to demonstrate the reliability of a triple method which combines evaluation of: 1. p16 protein expression by immunohistochemistry (p16-IHC); 2. HPV-DNA genotyping by consensus HPV-DNA PCR methods (Consensus PCR); and 3 viral integration into the host by in situ hybridization method (ISH). This triple method has been applied to HN-SCC originated from oral cavity (OSCC) and oropharynx (OPSCC), the two anatomical sites in which high risk (HR) HPVs have been clearly implicated as etiologic factors. Methylation-Specific PCR (MSP) was performed to study inactivation of p16-CDKN2a locus by epigenetic events. Reliability of multiple methods was measured by Kappa statistics. Results. All the HN-SCCs confirmed HPV positive by PCR and/or ISH were also p16 positive by IHC, with the latter showing a very high level of sensitivity as single test (100% in both OSCC and OPSCC) but lower specificity level (74% in OSCC and 93% in OPSCC). Concordance analysis between ISH and Consensus PCR showed a faint agreement in OPSCC ( = 0.38) and a moderate agreement in OSCC ( = 0.44). Furthermore, the addition of double positive score (ISHpositive and Consensus PCR positive) increased significantly the specificity of HR-HPV detection on formalin-fixed paraffin embedded (FFPE) samples (100% in OSCC and 78.5% in OPSCC), but reduced the sensitivity (33% in OSCC and 60% in OPSCC). The significant reduction of sensitivity by the double method was compensated by a very high sensitivity of p16-IHC detection in the triple approach. Conclusions. Although HR-HPVs detection is of utmost importance in clinical settings for the Head and Neck Cancer patients, there is no consensus on which to consider the 'golden standard' among the numerous detection methods available either as single test or combinations. Until recently, quantitative E6 RNA PCR has been considered the 'golden standard' since it was demonstrated to have very high accuracy level and very high statistical significance associated with prognostic parameters. In contrast, quantitative E6 DNA PCR has proven to have very high level of accuracy but lesser prognostic association with clinical outcome than the HPV E6 oncoprotein RNA PCR. However, although it is theoretically possible to perform quantitative PCR detection methods also on FFPE samples, they reach the maximum of accuracy on fresh frozen tissue. Furthermore, worldwide diagnostic laboratories have not all the same ability to analyze simultaneously both FFPE and fresh tissues with these quantitative molecular detection methods. Therefore, in the current clinical practice a p16-IHC test is considered as sufficient for HPV diagnostic in accordance with the recently published Head and Neck Cancer international guidelines. Although p16-IHC may serve as a good prognostic indicator, our study clearly demonstrated that it is not satisfactory when used exclusively as the only HPV detecting method. Adding ISH, although known as less sensitive than PCR-based detection methods, has the advantage to preserve the morphological context of HPV-DNA signals in FFPE samples and, thus increase the overall specificity of p16/Consensus PCR combination tests.

Original languageEnglish
Article number4
JournalInfectious Agents and Cancer
Volume7
Issue number1
DOIs
Publication statusPublished - 2012

Fingerprint

In Situ Hybridization
Squamous Cell Carcinoma
Immunohistochemistry
Polymerase Chain Reaction
DNA
Paraffin
Formaldehyde
Head and Neck Neoplasms
RNA
Virus Integration
Oropharynx
Oncogene Proteins
Epigenomics
Methylation
Mouth
Epidemiology
Guidelines
Radiation

Keywords

  • DNA consensus PCR
  • Epigenetic
  • Head and neck squamous cell carcinoma
  • HN-SCC
  • HPV
  • Human papillomavirus
  • IHC
  • Immunohistochemistry
  • Methylation-Specific PCR
  • OPSCC
  • OSCC
  • p16-IHC

ASJC Scopus subject areas

  • Infectious Diseases
  • Oncology
  • Epidemiology
  • Cancer Research

Cite this

Evaluation of a combined triple method to detect causative HPV in oral and oropharyngeal squamous cell carcinomas : P16 Immunohistochemistry, Consensus PCR HPV-DNA, and in Situ Hybridization. / Pannone, Giuseppe; Rodolico, Vito; Santoro, Angela; Lo Muzio, Lorenzo; Franco, Renato; Botti, Gerardo; Aquino, Gabriella; Pedicillo, Maria; Cagiano, Simona; Campisi, Giuseppina; Rubini, Corrado; Papagerakis, Silvana; De Rosa, Gaetano; Tornesello, Maria Lina; Buonaguro, Franco M.; Staibano, Stefania; Bufo, Pantaleo.

In: Infectious Agents and Cancer, Vol. 7, No. 1, 4, 2012.

Research output: Contribution to journalArticle

Pannone, Giuseppe ; Rodolico, Vito ; Santoro, Angela ; Lo Muzio, Lorenzo ; Franco, Renato ; Botti, Gerardo ; Aquino, Gabriella ; Pedicillo, Maria ; Cagiano, Simona ; Campisi, Giuseppina ; Rubini, Corrado ; Papagerakis, Silvana ; De Rosa, Gaetano ; Tornesello, Maria Lina ; Buonaguro, Franco M. ; Staibano, Stefania ; Bufo, Pantaleo. / Evaluation of a combined triple method to detect causative HPV in oral and oropharyngeal squamous cell carcinomas : P16 Immunohistochemistry, Consensus PCR HPV-DNA, and in Situ Hybridization. In: Infectious Agents and Cancer. 2012 ; Vol. 7, No. 1.
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abstract = "Background. Recent emerging evidences identify Human Papillomavirus (HPV) related Head and Neck squamous cell carcinomas (HN-SCCs) as a separate subgroup among Head and Neck Cancers with different epidemiology, histopathological characteristics, therapeutic response to chemo-radiation treatment and clinical outcome. However, there is not a worldwide consensus on the methods to be used in clinical practice. The endpoint of this study was to demonstrate the reliability of a triple method which combines evaluation of: 1. p16 protein expression by immunohistochemistry (p16-IHC); 2. HPV-DNA genotyping by consensus HPV-DNA PCR methods (Consensus PCR); and 3 viral integration into the host by in situ hybridization method (ISH). This triple method has been applied to HN-SCC originated from oral cavity (OSCC) and oropharynx (OPSCC), the two anatomical sites in which high risk (HR) HPVs have been clearly implicated as etiologic factors. Methylation-Specific PCR (MSP) was performed to study inactivation of p16-CDKN2a locus by epigenetic events. Reliability of multiple methods was measured by Kappa statistics. Results. All the HN-SCCs confirmed HPV positive by PCR and/or ISH were also p16 positive by IHC, with the latter showing a very high level of sensitivity as single test (100{\%} in both OSCC and OPSCC) but lower specificity level (74{\%} in OSCC and 93{\%} in OPSCC). Concordance analysis between ISH and Consensus PCR showed a faint agreement in OPSCC ( = 0.38) and a moderate agreement in OSCC ( = 0.44). Furthermore, the addition of double positive score (ISHpositive and Consensus PCR positive) increased significantly the specificity of HR-HPV detection on formalin-fixed paraffin embedded (FFPE) samples (100{\%} in OSCC and 78.5{\%} in OPSCC), but reduced the sensitivity (33{\%} in OSCC and 60{\%} in OPSCC). The significant reduction of sensitivity by the double method was compensated by a very high sensitivity of p16-IHC detection in the triple approach. Conclusions. Although HR-HPVs detection is of utmost importance in clinical settings for the Head and Neck Cancer patients, there is no consensus on which to consider the 'golden standard' among the numerous detection methods available either as single test or combinations. Until recently, quantitative E6 RNA PCR has been considered the 'golden standard' since it was demonstrated to have very high accuracy level and very high statistical significance associated with prognostic parameters. In contrast, quantitative E6 DNA PCR has proven to have very high level of accuracy but lesser prognostic association with clinical outcome than the HPV E6 oncoprotein RNA PCR. However, although it is theoretically possible to perform quantitative PCR detection methods also on FFPE samples, they reach the maximum of accuracy on fresh frozen tissue. Furthermore, worldwide diagnostic laboratories have not all the same ability to analyze simultaneously both FFPE and fresh tissues with these quantitative molecular detection methods. Therefore, in the current clinical practice a p16-IHC test is considered as sufficient for HPV diagnostic in accordance with the recently published Head and Neck Cancer international guidelines. Although p16-IHC may serve as a good prognostic indicator, our study clearly demonstrated that it is not satisfactory when used exclusively as the only HPV detecting method. Adding ISH, although known as less sensitive than PCR-based detection methods, has the advantage to preserve the morphological context of HPV-DNA signals in FFPE samples and, thus increase the overall specificity of p16/Consensus PCR combination tests.",
keywords = "DNA consensus PCR, Epigenetic, Head and neck squamous cell carcinoma, HN-SCC, HPV, Human papillomavirus, IHC, Immunohistochemistry, Methylation-Specific PCR, OPSCC, OSCC, p16-IHC",
author = "Giuseppe Pannone and Vito Rodolico and Angela Santoro and {Lo Muzio}, Lorenzo and Renato Franco and Gerardo Botti and Gabriella Aquino and Maria Pedicillo and Simona Cagiano and Giuseppina Campisi and Corrado Rubini and Silvana Papagerakis and {De Rosa}, Gaetano and Tornesello, {Maria Lina} and Buonaguro, {Franco M.} and Stefania Staibano and Pantaleo Bufo",
year = "2012",
doi = "10.1186/1750-9378-7-422376902",
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volume = "7",
journal = "Infectious Agents and Cancer",
issn = "1750-9378",
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TY - JOUR

T1 - Evaluation of a combined triple method to detect causative HPV in oral and oropharyngeal squamous cell carcinomas

T2 - P16 Immunohistochemistry, Consensus PCR HPV-DNA, and in Situ Hybridization

AU - Pannone, Giuseppe

AU - Rodolico, Vito

AU - Santoro, Angela

AU - Lo Muzio, Lorenzo

AU - Franco, Renato

AU - Botti, Gerardo

AU - Aquino, Gabriella

AU - Pedicillo, Maria

AU - Cagiano, Simona

AU - Campisi, Giuseppina

AU - Rubini, Corrado

AU - Papagerakis, Silvana

AU - De Rosa, Gaetano

AU - Tornesello, Maria Lina

AU - Buonaguro, Franco M.

AU - Staibano, Stefania

AU - Bufo, Pantaleo

PY - 2012

Y1 - 2012

N2 - Background. Recent emerging evidences identify Human Papillomavirus (HPV) related Head and Neck squamous cell carcinomas (HN-SCCs) as a separate subgroup among Head and Neck Cancers with different epidemiology, histopathological characteristics, therapeutic response to chemo-radiation treatment and clinical outcome. However, there is not a worldwide consensus on the methods to be used in clinical practice. The endpoint of this study was to demonstrate the reliability of a triple method which combines evaluation of: 1. p16 protein expression by immunohistochemistry (p16-IHC); 2. HPV-DNA genotyping by consensus HPV-DNA PCR methods (Consensus PCR); and 3 viral integration into the host by in situ hybridization method (ISH). This triple method has been applied to HN-SCC originated from oral cavity (OSCC) and oropharynx (OPSCC), the two anatomical sites in which high risk (HR) HPVs have been clearly implicated as etiologic factors. Methylation-Specific PCR (MSP) was performed to study inactivation of p16-CDKN2a locus by epigenetic events. Reliability of multiple methods was measured by Kappa statistics. Results. All the HN-SCCs confirmed HPV positive by PCR and/or ISH were also p16 positive by IHC, with the latter showing a very high level of sensitivity as single test (100% in both OSCC and OPSCC) but lower specificity level (74% in OSCC and 93% in OPSCC). Concordance analysis between ISH and Consensus PCR showed a faint agreement in OPSCC ( = 0.38) and a moderate agreement in OSCC ( = 0.44). Furthermore, the addition of double positive score (ISHpositive and Consensus PCR positive) increased significantly the specificity of HR-HPV detection on formalin-fixed paraffin embedded (FFPE) samples (100% in OSCC and 78.5% in OPSCC), but reduced the sensitivity (33% in OSCC and 60% in OPSCC). The significant reduction of sensitivity by the double method was compensated by a very high sensitivity of p16-IHC detection in the triple approach. Conclusions. Although HR-HPVs detection is of utmost importance in clinical settings for the Head and Neck Cancer patients, there is no consensus on which to consider the 'golden standard' among the numerous detection methods available either as single test or combinations. Until recently, quantitative E6 RNA PCR has been considered the 'golden standard' since it was demonstrated to have very high accuracy level and very high statistical significance associated with prognostic parameters. In contrast, quantitative E6 DNA PCR has proven to have very high level of accuracy but lesser prognostic association with clinical outcome than the HPV E6 oncoprotein RNA PCR. However, although it is theoretically possible to perform quantitative PCR detection methods also on FFPE samples, they reach the maximum of accuracy on fresh frozen tissue. Furthermore, worldwide diagnostic laboratories have not all the same ability to analyze simultaneously both FFPE and fresh tissues with these quantitative molecular detection methods. Therefore, in the current clinical practice a p16-IHC test is considered as sufficient for HPV diagnostic in accordance with the recently published Head and Neck Cancer international guidelines. Although p16-IHC may serve as a good prognostic indicator, our study clearly demonstrated that it is not satisfactory when used exclusively as the only HPV detecting method. Adding ISH, although known as less sensitive than PCR-based detection methods, has the advantage to preserve the morphological context of HPV-DNA signals in FFPE samples and, thus increase the overall specificity of p16/Consensus PCR combination tests.

AB - Background. Recent emerging evidences identify Human Papillomavirus (HPV) related Head and Neck squamous cell carcinomas (HN-SCCs) as a separate subgroup among Head and Neck Cancers with different epidemiology, histopathological characteristics, therapeutic response to chemo-radiation treatment and clinical outcome. However, there is not a worldwide consensus on the methods to be used in clinical practice. The endpoint of this study was to demonstrate the reliability of a triple method which combines evaluation of: 1. p16 protein expression by immunohistochemistry (p16-IHC); 2. HPV-DNA genotyping by consensus HPV-DNA PCR methods (Consensus PCR); and 3 viral integration into the host by in situ hybridization method (ISH). This triple method has been applied to HN-SCC originated from oral cavity (OSCC) and oropharynx (OPSCC), the two anatomical sites in which high risk (HR) HPVs have been clearly implicated as etiologic factors. Methylation-Specific PCR (MSP) was performed to study inactivation of p16-CDKN2a locus by epigenetic events. Reliability of multiple methods was measured by Kappa statistics. Results. All the HN-SCCs confirmed HPV positive by PCR and/or ISH were also p16 positive by IHC, with the latter showing a very high level of sensitivity as single test (100% in both OSCC and OPSCC) but lower specificity level (74% in OSCC and 93% in OPSCC). Concordance analysis between ISH and Consensus PCR showed a faint agreement in OPSCC ( = 0.38) and a moderate agreement in OSCC ( = 0.44). Furthermore, the addition of double positive score (ISHpositive and Consensus PCR positive) increased significantly the specificity of HR-HPV detection on formalin-fixed paraffin embedded (FFPE) samples (100% in OSCC and 78.5% in OPSCC), but reduced the sensitivity (33% in OSCC and 60% in OPSCC). The significant reduction of sensitivity by the double method was compensated by a very high sensitivity of p16-IHC detection in the triple approach. Conclusions. Although HR-HPVs detection is of utmost importance in clinical settings for the Head and Neck Cancer patients, there is no consensus on which to consider the 'golden standard' among the numerous detection methods available either as single test or combinations. Until recently, quantitative E6 RNA PCR has been considered the 'golden standard' since it was demonstrated to have very high accuracy level and very high statistical significance associated with prognostic parameters. In contrast, quantitative E6 DNA PCR has proven to have very high level of accuracy but lesser prognostic association with clinical outcome than the HPV E6 oncoprotein RNA PCR. However, although it is theoretically possible to perform quantitative PCR detection methods also on FFPE samples, they reach the maximum of accuracy on fresh frozen tissue. Furthermore, worldwide diagnostic laboratories have not all the same ability to analyze simultaneously both FFPE and fresh tissues with these quantitative molecular detection methods. Therefore, in the current clinical practice a p16-IHC test is considered as sufficient for HPV diagnostic in accordance with the recently published Head and Neck Cancer international guidelines. Although p16-IHC may serve as a good prognostic indicator, our study clearly demonstrated that it is not satisfactory when used exclusively as the only HPV detecting method. Adding ISH, although known as less sensitive than PCR-based detection methods, has the advantage to preserve the morphological context of HPV-DNA signals in FFPE samples and, thus increase the overall specificity of p16/Consensus PCR combination tests.

KW - DNA consensus PCR

KW - Epigenetic

KW - Head and neck squamous cell carcinoma

KW - HN-SCC

KW - HPV

KW - Human papillomavirus

KW - IHC

KW - Immunohistochemistry

KW - Methylation-Specific PCR

KW - OPSCC

KW - OSCC

KW - p16-IHC

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