Comparison of Real-Time PCR Signal-Amplified In Situ Hybridization and Conventional PCR for Detection and Quantification of Human Papillomavirus in Archival Cervical Cancer Tissue

Abstract

Archival paraffin-embedded tumor specimens offer a wealth of information for both cancer research and for routine clinical applications. However, the use of formalin-fixed, paraffin-embedded specimens for quantitative real-time PCR is not yet a standard diagnostic method in many laboratories, in particular for the quantification of human papillomavirus (HPV). Particularly high-risk HPV types are involved in almost 100% of the carcinogenesis of cervical cancer. We compared the diagnostic applicability and sensitivity of real-time PCR to that of chromogenic tyramide-signal-amplified in situ hybridization and conventional PCR for the detection of HPV from archival tissue in 164 cases of carcinoma in situ and cervical cancer. Furthermore, we examined whether the viral load of HPV is of prognostic relevance. Our findings indicate that patients in tumor stage I with a lower viral load of HPV type 16 (HPV16; up to 1,000 copies/ng of DNA) had a significantly better survival than HPV 16-negative patients (P ‫؍‬ 0.037). We observed a greater sensitivity of both real-time PCR and conventional PCR for the detection of HPV16 and -18 compared to signal amplified in situ hybridization. We found a considerable concordance between HPV16 ( ‫؍‬ 0.661) and HPV18 ( ‫؍‬ 0.781) status as measured by real-time PCR and conventional PCR, indicating similar sensitivities. We recognized an inhibitory effect of formalin fixation and paraffin embedding on the evaluation of real-time PCR quantification.Formalin-fixed, paraffin-embedded (FFPE) tissues retained in pathology archives are an important resource for molecular pathology studies and for diagnostic applications. The use of in situ hybridization (ISH) and PCR assays have both been reported for DNA analysis in formalin-fixed archival tissues (6,7,25,40,42,48). Both assays have the advantage that only small amounts of tissue are required, and both tolerate the degradation of target nucleic acids due to the formalin fixation and paraffin-embedding procedures. Each assay per se has its own advantages and disadvantages.For a pathologist, the retention of morphology and the histological context of a tumor is very important. Especially in the case of cervical carcinoma, the phenomenon of koilocytosis indicates an infection with human papillomavirus (HPV) (4) that can only be detected by microscopic analysis of tissue sections. Moreover, episomal and integrated viral DNA can only be differentiated by ISH due to the staining pattern (8). With the use of chromogenic tyramide-signal-amplified ISH (CISH) it is even possible to detect and localize single or very few HPV copies within infected nuclei (15,36,49). However, this technique is rather cost-intensive and quite involved for routine applications.Conventional PCR is held to be the most sensitive detection method available (9,16,20,27,37,41). Although this is probably true, it has some limitations, as does ISH, in that it cannot quantify viral load in a prognostic-diagnostic significant manner. This restriction can be circumvented by the use o...

Detection of specific human papillomavirus types in paraffin‐embedded sections of cervical carcinomas

“…The authors found two distinct signal patterns: punctate and diffuse nuclear, thought by the authors to correspond to integrated or episomal HPV. Biedermann et al [2004] analyzed paraffin-embedded tissue sections of 164 CIS or invasive cancers using tyramide-amplified in situ hybridization, real time PCR, or conventional PCR using MY09/11 L1 primers. HPV typing was performed only for HPV 16 or HPV 18.…”

Evaluation of a Commercialized In Situ Hybridization Assay for Detecting Human Papillomavirus DNA in Tissue Specimens from Patients with Cervical Intraepithelial Neoplasia and Cervical Carcinoma

“…Each has its strengths and drawbacks. Although the PCR assay is highly sensitive for detecting HPV DNA, there are disadvantages to using PCR for HPV DNA detection in FFPE tissue specimens (5,34). In particular, PCR inhibitors in DNA extracts from FFPE cervical tissue can interfere with the ability of PCR to detect HPV (10).…”

Urothelial carcinoma with prominent squamous differentiation in the setting of neurogenic bladder: role of human papillomavirus infection

“…In situ hybridization, although not as sensitive as PCR, has been widely applied 42 with the important advantage of direct visualization of the virus in the cell and evaluation of the episomal versus integrated state of the viral DNA. 43,44 The optimal detection method, however, still remains unclear and is likely a combination of both molecular detection and direct visualization.…”