Human papillomavirus multiplex ligation-dependent probe amplification assay for the assessment of viral load, integration, and gain of telomerase-related genes in cervical malignancies

Wendy Theelen, Rogier J. N. T. M. Litjens (Lead / Corresponding author), Svetlana Vinokurova, Annick Haesevoets, Martin Reijans, Guus Simons, Frank Smedts, C. Simon Herrington, Frans C. S. Ramaekers, Magnus von Knebel Doeberitz, Ernst-Jan M. Speel, Anton H. N. Hopman

    Research output: Contribution to journalArticle

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    Abstract

    We evaluated the reliability of a novel multiplex ligation-dependent probe amplification (MLPA) assay in detecting integration of human papillomavirus (HPV) based on the viral E2/E6 copy number ratio in formalin-fixed and paraffin-embedded cervical lesions. The MLPA results were compared with those of amplification of papillomavirus oncogene transcripts for RNA, detection of integrated papillomavirus sequences for DNA, and HPV fluorescence in situ hybridization (FISH). DNA was isolated from 41 formalin-fixed and paraffin-embedded HPV-positive cervical lesions (cervical intraepithelial neoplasia grade 3 lesions, squamous cell carcinomas, and adenocarcinomas) for MLPA analysis. From 13 matching frozen samples, DNA and RNA were isolated for the detection of integrated papillomavirus sequences and/or the amplification of papillomavirus oncogene transcripts, respectively. Integrated HPV16, HPV18, or both were identified. The MLPA assay detected viral integration in 12 of these 13 cases, and episomal copies also were detected in 7 cases. In 20 of the 24 cases with exclusive viral integration or episomal viral copies as detected by FISH, MLPA confirmed the physical status of the virus. In the cases classified as mixed by FISH, the presence of excess episomal copies complicated the recognition of viral integration by MLPA. Furthermore, the feasibility of detecting gain of the telomerase genes with the HPV MLPA assay was evaluated. The MLPA confirmed the FISH data in 12 of 13 cases in which the status of copy number gain for telomerase RNA component was known. In conclusion, the HPV MLPA assay can be performed on routinely processed cervical lesions for the detection of viral load and HPV integration.

    Original languageEnglish
    Pages (from-to)2410-2418
    JournalHuman Pathology
    Volume44
    Issue number11
    Early online date19 Aug 2013
    DOIs
    Publication statusPublished - Nov 2013

    Fingerprint

    Virus Integration
    Telomerase
    Multiplex Polymerase Chain Reaction
    Viral Load
    Genes
    Fluorescence In Situ Hybridization
    Neoplasms
    Oncogenes
    Paraffin
    Formaldehyde
    RNA
    Cervical Intraepithelial Neoplasia
    DNA
    Squamous Cell Carcinoma
    Adenocarcinoma
    Viruses

    Keywords

    • HPV integration
    • APOT
    • DIPS
    • HPV FISH
    • CERVICAL CARCINOMA

    Cite this

    Theelen, Wendy ; Litjens, Rogier J. N. T. M. ; Vinokurova, Svetlana ; Haesevoets, Annick ; Reijans, Martin ; Simons, Guus ; Smedts, Frank ; Herrington, C. Simon ; Ramaekers, Frans C. S. ; von Knebel Doeberitz, Magnus ; Speel, Ernst-Jan M. ; Hopman, Anton H. N. / Human papillomavirus multiplex ligation-dependent probe amplification assay for the assessment of viral load, integration, and gain of telomerase-related genes in cervical malignancies. In: Human Pathology. 2013 ; Vol. 44, No. 11. pp. 2410-2418.
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    abstract = "We evaluated the reliability of a novel multiplex ligation-dependent probe amplification (MLPA) assay in detecting integration of human papillomavirus (HPV) based on the viral E2/E6 copy number ratio in formalin-fixed and paraffin-embedded cervical lesions. The MLPA results were compared with those of amplification of papillomavirus oncogene transcripts for RNA, detection of integrated papillomavirus sequences for DNA, and HPV fluorescence in situ hybridization (FISH). DNA was isolated from 41 formalin-fixed and paraffin-embedded HPV-positive cervical lesions (cervical intraepithelial neoplasia grade 3 lesions, squamous cell carcinomas, and adenocarcinomas) for MLPA analysis. From 13 matching frozen samples, DNA and RNA were isolated for the detection of integrated papillomavirus sequences and/or the amplification of papillomavirus oncogene transcripts, respectively. Integrated HPV16, HPV18, or both were identified. The MLPA assay detected viral integration in 12 of these 13 cases, and episomal copies also were detected in 7 cases. In 20 of the 24 cases with exclusive viral integration or episomal viral copies as detected by FISH, MLPA confirmed the physical status of the virus. In the cases classified as mixed by FISH, the presence of excess episomal copies complicated the recognition of viral integration by MLPA. Furthermore, the feasibility of detecting gain of the telomerase genes with the HPV MLPA assay was evaluated. The MLPA confirmed the FISH data in 12 of 13 cases in which the status of copy number gain for telomerase RNA component was known. In conclusion, the HPV MLPA assay can be performed on routinely processed cervical lesions for the detection of viral load and HPV integration.",
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    Theelen, W, Litjens, RJNTM, Vinokurova, S, Haesevoets, A, Reijans, M, Simons, G, Smedts, F, Herrington, CS, Ramaekers, FCS, von Knebel Doeberitz, M, Speel, E-JM & Hopman, AHN 2013, 'Human papillomavirus multiplex ligation-dependent probe amplification assay for the assessment of viral load, integration, and gain of telomerase-related genes in cervical malignancies', Human Pathology, vol. 44, no. 11, pp. 2410-2418. https://doi.org/10.1016/j.humpath.2013.04.026

    Human papillomavirus multiplex ligation-dependent probe amplification assay for the assessment of viral load, integration, and gain of telomerase-related genes in cervical malignancies. / Theelen, Wendy; Litjens, Rogier J. N. T. M. (Lead / Corresponding author); Vinokurova, Svetlana; Haesevoets, Annick; Reijans, Martin; Simons, Guus; Smedts, Frank; Herrington, C. Simon; Ramaekers, Frans C. S.; von Knebel Doeberitz, Magnus; Speel, Ernst-Jan M.; Hopman, Anton H. N.

    In: Human Pathology, Vol. 44, No. 11, 11.2013, p. 2410-2418.

    Research output: Contribution to journalArticle

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    AU - Vinokurova, Svetlana

    AU - Haesevoets, Annick

    AU - Reijans, Martin

    AU - Simons, Guus

    AU - Smedts, Frank

    AU - Herrington, C. Simon

    AU - Ramaekers, Frans C. S.

    AU - von Knebel Doeberitz, Magnus

    AU - Speel, Ernst-Jan M.

    AU - Hopman, Anton H. N.

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    N2 - We evaluated the reliability of a novel multiplex ligation-dependent probe amplification (MLPA) assay in detecting integration of human papillomavirus (HPV) based on the viral E2/E6 copy number ratio in formalin-fixed and paraffin-embedded cervical lesions. The MLPA results were compared with those of amplification of papillomavirus oncogene transcripts for RNA, detection of integrated papillomavirus sequences for DNA, and HPV fluorescence in situ hybridization (FISH). DNA was isolated from 41 formalin-fixed and paraffin-embedded HPV-positive cervical lesions (cervical intraepithelial neoplasia grade 3 lesions, squamous cell carcinomas, and adenocarcinomas) for MLPA analysis. From 13 matching frozen samples, DNA and RNA were isolated for the detection of integrated papillomavirus sequences and/or the amplification of papillomavirus oncogene transcripts, respectively. Integrated HPV16, HPV18, or both were identified. The MLPA assay detected viral integration in 12 of these 13 cases, and episomal copies also were detected in 7 cases. In 20 of the 24 cases with exclusive viral integration or episomal viral copies as detected by FISH, MLPA confirmed the physical status of the virus. In the cases classified as mixed by FISH, the presence of excess episomal copies complicated the recognition of viral integration by MLPA. Furthermore, the feasibility of detecting gain of the telomerase genes with the HPV MLPA assay was evaluated. The MLPA confirmed the FISH data in 12 of 13 cases in which the status of copy number gain for telomerase RNA component was known. In conclusion, the HPV MLPA assay can be performed on routinely processed cervical lesions for the detection of viral load and HPV integration.

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    KW - APOT

    KW - DIPS

    KW - HPV FISH

    KW - CERVICAL CARCINOMA

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    DO - 10.1016/j.humpath.2013.04.026

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