A comparison of methods for the detection of human papillomavirus DNA by in situ hybridization with biotinylated probes on human carcinoma cell lines. Application to wart sections.

We compared nine different techniques for the detection of biotinylated DNA-DNA HPV hybrids on HeLa cells with 10-50 copies of HPV 18 DNA per cell. CaSki cells with 600 copies of HPV 16 DNA per cell and tissue sections from frozen or paraffin-embedded biopsy specimens. The cell samples were either cell deposits or cytocentrifuged or cultured slides. In most cases, the samples (cell deposits and tissue sections) were denatured with hybridization mixture prepared under stringent conditions (Tm = -17 degrees C) containing biotinylated DNA probes (cloned HPV types 1, 2, 6, 11, 16 and 18), at 90 degrees C for 10 min. In other cases (cytocentrifuged or cultured cells), the denaturation was performed by HCl hydrolysis and mild heating at 50 degrees C; the probes were denatured separately by heating. All the samples were further incubated overnight at 37 degrees C. For HPV DNA detection, three amplification levels were used on cell deposits. Only the techniques involving a three-step reaction (a rabbit anti-biotin antibody - a biotinylated goat anti-rabbit antibody - a complex of streptavidin-alkaline phosphatase or streptavidin-gold or streptavidin-fluorescein) gave satisfactory results, on both cell lines. With the one step reaction (an avidin-horseradish peroxidase, or streptavidin-alkaline phosphatase or streptavidin-fluorescein complex), no labeling of HeLa cells was observed with any of the HPV probes, including HPV 18. The techniques involving four steps (avidin or streptavidin - anti-avidin goat antibody or anti-streptavidin rabbit antibody - a biotinylated anti-goat (or anti-rabbit) antibody - a complex of avidin-biotin-peroxidase or streptavidin-biotin-alkaline phosphatase or streptavidin-biotin-horseradish peroxidase) resulted in high background on both cell lines. For the reproducible detection of low copy number of HPV DNA (less than 50 copies) such as occur in HeLa cells our data suggested that the three-step technique with the streptavidin-alkaline phosphatase complex was the method of choice. The most intense labeling was always obtained with cell deposits and the technique was successfully applied to frozen and paraffin-embedded tissue sections from typical warts.

Langerhans cells and epithelial cell modifications in cervical intraepithelial neoplasia: correlation with human papillomavirus infection.

The study of a series of 18 cervical intraepithelial neoplasia (CIN) grade II and III was aimed at determining the distribution and phenotype of immunocompetent cells (Langerhans cells, T and NK cells) and the alteration in the expression of EGF receptors and beta 2-microglobulin in correlation with human papillomavirus (HPV) infection (viral antigen and DNA typing with biotinylated probes). These lesions were characterized by a reduced number of Langerhans cells and a dense infiltrate. HPV infection did not induce HLA-DR expression in the infected epithelial cells. We observed an enhanced expression of epidermal growth factor (EGF) receptors by epithelial cells and a reduced beta 2-microglobulin reactivity by both epithelial and immunocompetent cells. Most of CIN showed foci of infected cells. No significant differences were observed in immunological markers of CIN harboring benign HPV 6/11 DNA or oncogenic HPV 16/18 DNA. Viral antigen was not detected in these lesions. These changes in the epithelial cells of CIN and their microenvironment associated to the lack of HLA-DR expression in the infected cells hamper the squamous epithelial cells to function as antigen presenting cells. This may facilitate a decrease in the immunological surveillance and may contribute to the severity of such lesions.

Human papillomavirus infection and filaggrin expression in paraffin-embedded biopsy specimens of extragenital Bowen's disease and genital bowenoid papulosis.

Cutaneous Bowen's disease (BD) and genital bowenoid papulosis (BP) are considered as precancerous or cancerous lesions that are sometimes infected with human papillomavirus (HPV). We studied retrospectively paraffin-embedded sections of 11 samples of cutaneous BD and 6 samples of genital BP from the general population for HPV infection and filaggrin expression. Using in situ hybridization with biotinylated probes of HPV types 1, 2, 5, 6, 11, 16, and 18, under stringent and/or non-stringent conditions and a streptavidin-alkaline-phosphatase complex for hybrid detection, HPV DNA was detected in 6/17 cases (5 BD and 1 BP). Positive nuclei were located in intermediate or upper epithelial cell layers. HPV 16 was found in 2 cases of BD but associated either with HPV 2 or 18. Three additional lesions reacted only under non-stringent conditions; HPV could not be typed with the probes used. The positive case of BP reacted with the four probe types 1, 2, 16, 18 and was negative with HPV 6 or 11. Viral antigen was not detected by indirect immunofluorescence with a rabbit antiserum directed to group-specific viral capsid antigen. Differentiation disorders were observed in the intermediate and upper cell layers of these specimens, as shown by a reduced expression of filaggrin/profilaggrin, a marker of terminal differentiation, in extragenital BD (7/11 cases), and an increased expression in genital BP (4/5 cases) although viral DNA was not always detectable. This study shows that in situ hybridization is a valuable technique for HPV DNA detection and its typing in BD and BP lesions on deparaffinized sections. The positive nuclei were located in the cell layers that exhibited abnormal expression of differentiation. There is no relation between the HPV infecting type and the filaggrin expression.

Filaggrin expression in cutaneous and mucosal human papillomavirus induced lesions.

A series of 32 human papillomavirus induced lesions derived from epidermis and mucosa was studied for the modulation of filaggrin-profilaggrin (F-PF) expression according to the degree of virus infection as compared to normal skin and mucosa biopsies. This investigation was carried out on frozen sections using indirect immunofluorescence for filaggrin detection and group specific viral antigen and by in situ hybridization with biotinylated probes for viral DNA detection and typing. The 9 cutaneous warts showed an increase of F-PF expression in upper layer cells as compared to normal epidermis, which could be related to the high production of virus (viral antigen and HPV types 1 or 2). The 5 condyloma acuminata displayed also an enhanced expression of these components which was located in several upper layers but virus infection was confirmed in 2 of them with HPV types 6, 11 or 16. The 6 laryngeal papillomas exhibited a granular reactivity pattern for F-PF in suprabasal cell layers with an increase in the upper layers; viral antigen was found in 4 cases and HPV DNA types 6, 11 or 16 were detected in 4 specimens. Conversely among 12 cervical intraepithelial neoplasia, F-PF was expressed only in very superficial layers in few cases, without any correlation with the DNA detection (6, 11 or 16, 18). Taken together these data are suggestive of an intense expression of F-PF in benign lesions which can replicate the virus and a discrete or an absent expression of these components in premalignant or malignant lesions.

Influence of fixation on human papillomavirus DNA detection in frozen and embedded paraffin lesions by in situ hybridization with biotinylated probes.

Series of frozen or paraffin-embedded tissues from various body sites, taken from non-immunosuppressed or immunosuppressed patients with persistent papilloma lesions were examined for the presence of group specific antigen from human papillomavirus (HPV) by indirect immunofluorescence or HPV DNA by in situ hybridization with biotinylated probes. We have shown that it is possible to detect HPV DNA after fixation of tissues in neutral formalin, Bouin's or Baker's solution. However, the sensitivity was reduced as compared to frozen tissues. The HPV DNA was detected in nuclei of heavily infected epithelial cells such as plantar or hand warts or in dispersed cells containing high copy numbers of HPV DNA from lesions such as squamous cell carcinomas or keratoacanthomas. In premalignant or malignant lesions of both immunosuppressed or non-immunosuppressed patients, HPV DNA was rarely detected after fixation. HPV types commonly described for skin and genital samples were identified in non-immunosuppressed patients, whereas in transplant recipients oncogenic HPV type 16 was identified in benign warts as well as in premalignant or malignant lesions. Positive reactions with several HPV types were more frequent in lesions from grafted patients than from the normal population. Virus antigen was detectable more frequently in frozen sections than in fixed tissues. Our findings indicate that in situ hybridization is an appropriate and rapid technique to study the presence of HPV infection. However, numerous controls are needed to avoid misinterpretations.

[Human papillomavirus typing by molecular hybridization in situ with biotinylated probes. Optimization of sample preparation and hybridization time]. / Typage de papillomavirus humains par hybridation moléculaire in situ avec des sondes biotinylées. Optimisation de la préparation des échantillons et du temps d'hybridation.

In situ hybridization with non radioactive probes is more and more used to detect viral infections, especially human papillomavirus (HPV) infections. The quality of the reaction depends on several factors, such as sample preparation (including fixation and pretreatments). Their importance was evaluated on a model with cell lines including CaSki cells (harboring about 600 copies of HPV 16 DNA per cell) and Hela cells (containing 10-50 copies of HPV 18 DNA per cell). These cell lines were chosen in order to evaluate cytological and histological difficulties. Several parameters were studied: preparation of samples, fixation and hybridization duration. DNAs of biotinylated probes of HPV types 16 and 18 and cells were simultaneously denatured 10 min at 95 degrees C. Hybridization was carried out at 37 degrees C for various periods of time; it was followed by a 3-step reaction for detection of biotinylated DNA-DNA hybrids with immunoenzymatic staining using streptavidin-alkaline phosphatase complex. Typical intranuclear granulations were seen either in cell deposits fixed with acetone, methanol-acetic acid, paraformaldehyde, formaline, Bouin's, Baker's or Carnoy's fixatives; or in cytocentrifuged cells fixed with formaline, Bouin's or Baker's fixatives. The detergent pretreatment was unnecessary. On the contrary, the protease pretreatment was required with formaline, Bouin's or Baker's fixatives. In order to detect constantly HPV 16 in CaSki cells and HPV 18 in HeLa cells, hybridization should be performed for more than 4 h. The sensitivity of the technique could therefore be evaluated to few copies of HPV DNA per cell. This technique is reliable, sensitive and rapid; et can be applied to biopsy specimens fixed with Bouin's or Baker's fixatives and paraffin-embedded; it allows routine detection of HPV infections.

Human papillomavirus detection in cervical cells by in situ hybridization with biotinylated probes.

We have investigated the applicability of human papillomavirus (HPV) DNA detection by in situ hybridization with biotinylated probes in epithelial cells obtained from the cervix using a cotton tip swab. We describe a simple procedure for obtaining homogeneous cell samples and good preservation of cellular structure. This is achieved by pretreatment of cells with L-cysteine before hybridization. Separate denaturation of cellular DNA and probe DNA is also necessary for satisfactory results. Both benign HPV DNA 6/11 and potentially oncogenic HPV DNA 16/18 could be identified in our series. In situ hybridization on cervical scrapes is a rapid, simple and very specific method for detecting patients infected with oncogenic HPV types.

In situ detection of human papillomavirus types 13 and 32 in focal epithelial hyperplasia of the oral mucosa.

17 cases of focal epithelial hyperplasia of the oral mucosa (FEH, Heck's disease) were investigated for the presence of human papillomavirus (HPV) nucleic acid sequences by means of in situ DNA hybridization using biotinylated DNA probes of HPV types 1, 6, 11, 13, 16, 18, and 32. Ten of 17 cases were positive for HPV 13 DNA in contrast to 6 of 17 positive cases obtained after application of the HPV 32 probe, with a double infection in one case. The results of our study suggest, that HPV 13 and HPV 32 are very specifically found in lesions of FEH and can be detected in a high percentage of cases using in situ hybridization.

Study of stringency conditions for human papillomavirus DNA detection on cell lines, frozen and paraffin-embedded tissue sections by in situ hybridization with biotinylated probes.

In situ hybridization was mainly used for typing human papillomavirus (HPV) in paraffin-embedded or frozen sections under stringent conditions (SC). We tested 5 different conditions of stringency with biotinylated HPV 1, 2, 16 and 18 probes on 3 cell lines (Sihà and CaSki with HPV16, HeLa with HPV18) by varying the concentration of formamide in the hybridization mixture and washings in order to determine the stringency conditions to be used to assess the presence of HPV and its typing: A-low stringency, hybridization at 35 degrees C below the melting temperature of DNA (Tm-35 degrees C) and washings without formamide; B-low stringency, hybridization and washings at Tm-35 degrees C; C-medium stringency, hybridization at Tm-35 degrees C and washings at Tm-12 degrees C; D-high stringency, hybridization at Tm-12 degrees C and washing without formamide; E-very high stringency, hybridization and washings at -12 degrees C. This study showed that HPV typing required a high stringency. On the contrary, under non stringent conditions (NSC), each cell line was positive with the heterologous probes. When 3 to 5 stringency conditions were assayed on 4 frozen samples, similar results were obtained. Typing required high stringency conditions whereas NSC allowed HPV detection. Furthermore, this study demonstrated the specificity of the reaction in lesions positive with more than one type. Stringent (Tm-12 degrees C) and non stringent (Tm-35 degrees C) conditions of hybridization were further applied to 57 biopsy sections (17 frozen and 40 paraffin-embedded specimens) from typical wart lesions and lesions suspected of HPV.(ABSTRACT TRUNCATED AT 250 WORDS)