Detection of human papillomavirus in cervical intra-epithelial neoplasia, using in situ hybridization and various polymerase chain reaction techniques

Abstract

One hundred and forty-eight randomly chosen neutral-buffered formaldehyde-fixed cervical biopsies in which cervical intra-epithelial neoplasia (CIN) I-III had been diagnosed were tested for HPV (human papilloma virus) DNA by in situ hybridization (ISH) and polymerase chain reaction (PCR). For ISH, we utilized a biotinylated panprobe and type-specific, genomic probe sets. For PCR, we used the general primers GP5/GP6 and their recently described, elongated version GP5+/GP6+, and included the modification of hot-start PCR. Amplified DNA was detected by gel electrophoresis and slot blot hybridization. The positivity rate of ISH was 59% for all biopsies and 69%, 62% and 46% for CIN I, II and III, respectively. The sensitivity of GP5/GP6 was 74% with cold-start PCR and 78% with hot-start PCR. When GP5+/GP6+ was used, the sensitivity increased to 89% with cold-start PCR and to 95% with hot-start PCR. Based on the most sensitive PCR technique, HPV detection was 93%, 95% and 96% in CIN I, II and III, respectively. The number of HPV types decreased with the severity of the lesion, and HPV 16 was the predominant type. Multiple HPVs were rare and almost all HPV-positive cases could be typed. ISH and slot blot hybridization correlated well regarding HPV typing specificity. Our results confirm that distinct HPV types are present in a high proportion of cases of CIN. The sensitivity of ISH is lower than that of PCR. Furthermore, the modified general primers GP5+/GP6+ give a higher yield than GP5/GP6, while hot-start PCR increases sensitivity even further.

Tumourigenesis Driven by the Human Papillomavirus Type 16 Asian-American E6 Variant in a Three-Dimensional Keratinocyte Model

“…During an in vivo infection in a human host, HPV genomes are not found in every keratinocyte of the stratified squamous epithelium. Instead, HPV infection can only be evidenced focally [30], [31]. In creating our keratinocyte model, we utilized a transfection method we had developed earlier [29] (described in detail in Materials and Methods), which met these requirements.…”

Comparison of prevalence, viral load, physical status and expression of human papillomavirus-16, -18 and -58 in esophageal and cervical cancer: a case-control study

“…This low figure was also found in a Chinese study of EC tissues (<1 to 157 copies/cell) [13,26] and a Finnish study of head and neck SCCs (4.6 to 49 copies/cell) [27]. Extremely low levels of HPV DNA load (even 0.01 copies/cells) in ESCC tissues may exceed the detection ability of some techniques such as regular PCR and conventional in situ hybridization (ISH), which limits detection to <10-50 copies/cell [28]. Our positive results for <10 copies of HPV DNA would be out of the detection range with use of general PCR and ISH technology.…”

IFN-κ, a novel type I IFN, is undetectable in HPV-positive human cervical keratinocytes

“…The probes are sensitive and specific for each type without cross-hybridization. 24,25 In addition, PCR analysis 26 and p16-based testing using the CINtec p16 immunocytochemistry staining kit (CINtec p16INK4a Cytology 29 Fibroblast containing collagen rafts without NIKS served as negative controls. 29 After 14 days in the liquid/air interface, cultures were harvested.…”