title: Human papillomavirus multiplex ligation-dependent probe amplification assay for the assessment of viral load, integration, and gain of telomerase-related genes in cervical malignancies.
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.
authors: Department of Molecular Cell Biology, GROW - School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht 6200MD, The Netherlands.
authors: Wendy
authors: W
authors: Theelen
authors: Rogier J N T M
authors: RJ
authors: Litjens
authors: Svetlana
authors: S
authors: Vinokurova
authors: Annick
authors: A
authors: Haesevoets
authors: Martin
authors: M
authors: Reijans
authors: Guus
authors: G
authors: Simons
authors: Frank
authors: F
authors: Smedts
authors: C Simon
authors: CS
authors: Herrington
authors: Frans C S
authors: FC
authors: Ramaekers
authors: Magnus
authors: M
authors: von Knebel Doeberitz
authors: Ernst-Jan M
authors: EJ
authors: Speel
authors: Anton H N
authors: AH
authors: Hopman
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000473:pathology / Q000821:virology*; D002584:Cervix Uteri / Q000473:pathology / Q000821:virology*; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D018628:Gene Dosage; D052162:Human papillomavirus 16 / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D052161:Human papillomavirus 18 / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D030361:Papillomavirus Infections / Q000473:pathology / Q000821:virology*; D012313:RNA / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D015203:Reproducibility of Results; D019098:Telomerase / Q000235:genetics; D002583:Uterine Cervical Neoplasms / Q000473:pathology / Q000821:virology*; D019562:Viral Load; D016662:Virus Integration; D002578:Uterine Cervical Dysplasia / Q000473:pathology / Q000821:virology*
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase
keywords: APOT; Cervical carcinoma; DIPS; HPV FISH; HPV integration