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

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 (kappa = 0.661) and HPV18 (kappa = 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.     

A new nonisotopic detection of human papillomavirus DNA using polymerase chain reaction.

A novel technique using a two-step polymerase chain reaction (PCR) with specific primers detecting human papillomavirus (HPV) DNA of types 6/11, 16, and 18 and a final nonisotopic colorimetric detection has been developed. Sixty formalin-fixed and paraffin-embedded sections were treated with this methodology and the results compared with those obtained with in situ hybridization (ISH). Twenty cases displaying HPV DNA with ISH were positive with PCR. Seven (35%) of 20 cases negative for ISH but evocative of HPV infection with classic histology displayed HPV DNA with the two-step PCR. Only one case (5%) of 20 normal tissues and/or inflammatory lesions not evocative of HPV infection and negative upon ISH showed HPV DNA. This original technique allows rapid, highly sensitive, and specific detection of HPV DNA and is suitable for most laboratories.     

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

Human papillomaviruses (HPV) are the causative agents of most cervical carcinomas. A complete understanding of the HPV types that cause cervical carcinoma is needed as vaccines are designed. Fresh tissues are not always available for such studies. We therefore sought to determine the feasibility of HPV studies using formalin-fixed, paraffin-embedded sections of 56 cervical carcinomas, correlating typing information with the pathology and physical state of the HPV sequences within cells. Sections from each specimen were used to extract and purify DNA. Specific HPV types were identified using a PCR/reverse blot strip assay. Tyramide signal-amplified, fluorescent DNA in situ hybridization (FISH) was used to localize HPV within cells. Human beta-globin sequences were amplified in DNA from all specimens. HPV sequences from oncogenic types were identified in 52 of 56 (92.9%) by PCR/reverse blot strip assay, and in one additional case using an HPV 16 multiplex PCR assay. HPV 16 was the most commonly detected type, present in most cases as a solitary isolate. Thirty- five of 42 HPV 16 or HPV 18 PCR-positive specimens were also positive in the FISH assay, in most cases in a pattern consistent with viral integration. We conclude that HPV typing from formalin-fixed, paraffin-embedded sections of cervical carcinomas is possible, with a sensitivity that is similar to that found in studies using fresh tissue.     

Detection of human papillomavirus DNA in formalin-fixed tissues by in situ hybridization after amplification by polymerase chain reaction.

The authors describe the detection of human papillomavirus (HPV) 16 DNA in paraffin-embedded, formalin-fixed tissues of cervical squamous intraepithelial lesions (SILs) by in situ hybridization after amplification by the polymerase chain reaction (PCR). Using conventional in situ hybridization and a biotin-labeled probe, variable numbers of superficial cells and none of the basal cells in the SILs showed detectable HPV 16 DNA. When the in situ assay was done after amplification, increased numbers of superficial cells had detectable HPV DNA, and the hybridization signal was much more intense. HPV DNA was also detected in basal and parabasal cells at the site of the lesion whereas not detectable in directly adjacent, normal squamous epithelium. Amplified HPV DNA was demonstrated in formalin-fixed SiHa cells using a biotin-labeled probe, demonstrating the ability to detect one copy of HPV 16 DNA. This technique should allow for direct visualization in cells of other DNA sequences of low copy number from achival specimens otherwise undetectable by conventional in situ hybridization analysis.     

Detection of human papillomavirus in normal and dysplastic tissue by the polymerase chain reaction

Detection of human papilloma virus (HPV) types 16 and 18 in formalin-fixed, paraffin-embedded tissue by a new in vitro DNA amplification method, the polymerase chain reaction, was compared with detection with genomic DNA probes using in situ hybridization. The polymerase chain reaction replicates exponentially HPV DNA sequences present in a single 5- to 10-micron paraffin-embedded tissue section. The amplified sequences are detected with a DNA hybridization probe in a dot blot assay. The HPV polymerase chain reaction was able to detect on the average less than one HPV genome/cell as determined by tests of paraffin sections of cell pellets with known HPV genomic content. Cervical sections from 21 patients with HPV types 16, 18, or 31 as determined by in situ DNA hybridization were analyzed by the polymerase chain reaction. No disagreements between the two methods were detected. The sections comprising normal and dysplastic epithelium were further analyzed by the HPV polymerase chain reaction. The presence of virus correlated with the presence of dysplasia in the sections, though 3 of 10 normal sections contained HPV, and 1 of 21 sections with dysplasia lacked HPV 16 or 18. The polymerase chain reaction can specifically detect HPV 16 or 18 with high sensitivity from paraffin-embedded tissues.     

Human papillomavirus infection in non-neoplastic uterine cervical disease in Hong Kong.

The polymerase chain reaction (PCR) was used to detect and identify human papillomavirus (HPV) in 108 cases of formalin-fixed, paraffin-embedded, non-neoplastic uterine cervical biopsy tissue retrieved from the surgical pathology archives of the Department of Pathology, Caritas Medical Centre, Hong Kong. After DNA extraction, HPV L1 gene primers were used to detect the presence of HPV, and type-specific primers (to HPV types 6, 11, 16, 18, 31 and 33) were used to identify the specific HPV type on HPV L1-positive cases. PCR amplification of the beta-globin gene was used to ensure the quality of amplifiable DNA extracted. Of 94 cases that yielded sufficient good-quality DNA for PCR analysis, three (one endocervical polyp, one chronic inflammation with erosion, and a normal biopsy) had detectable HPV infection. Two of these had high-risk HPV type 16; the other had an uncommon HPV type. In view of the low incidence of HPV found in these patients, large-scale population screening of clinical samples using PCR to detect the presence of HPV and identify high-risk asymptomatic patients would not be cost-effective.     

In situ hybridization for cytomegalovirus DNA in AIDS patients.

Infection by cytomegalovirus (CMV) is a frequent cause of morbidity and mortality in patients with acquired immune deficiency syndrome (AIDS). The authors studied the distribution of CMV in 4 patients with AIDS using a commercially available, biotin-labeled CMV DNA probe for in situ hybridization and immunohistochemical staining for the detection of CMV antigen in formalin-fixed paraffin-embedded tissues. The sensitivity and specificity of the hybridization procedure was demonstrated by appropriate controls. The immunohistochemical test for the detection of CMV antigen in routine histologic sections was less sensitive than the in situ hybridization method. CMV DNA was detected not only in cytomegalic inclusion cells, but also in nuclei and cytoplasm of histologically normal-appearing cells such as endothelial cells, pneumocytes, hepatocytes, biliary epithelium, gastrointestinal epithelium, Langerhans islet cells, acinar and duct epithelium of pancreas, adrenal cortical and medullary cells, and prostate epithelium. In addition, CMV DNA, but not CMV antigen, was found in polymorphonuclear leukocytes. These cells may serve as intermediate host or reservoir of CMV and may transmit posttransfusion CMV infection. In situ hybridization on routine histologic sections with a biotinylated CMV DNA probe is a rapid, sensitive, and specific method for diagnostic and experimental pathology.     

Rapid detection of xenotransplanted human tissues using in situ hybridization.

A rapid and sensitive method for the identification of human tissues xenotransplanted in nude mice was developed. An in situ hybridization technique made it possible to distinguish between cells of human origin and cells of murine origin in formalin-fixed paraffin sections. High-molecular-weight DNAs extracted from human or mouse tissues were sonicated, nick-translated with 32P-dCTP, and used as hybridization probes. Dot blot hybridization of 32P-labeled probes revealed clear species-specific signals. Formalin-fixed paraffin-embedded tissue samples from repopulated tracheal transplants, containing either human tracheal epithelial cells or human renal tubular cells, were used. Cells of human and murine origin were distinguishable by in situ hybridization with sonicated DNA probes. This method has several advantages; simple preparation of probes, high sensitivity, and applicability to formalin-fixed paraffin-embedded tissue sections. In situ hybridization with sonicated DNA probes should provide a powerful tool for verifying the human origin of xenotransplanted tissues in nude mice.     

The identification of cells containing JC papovavirus DNA in progressive multifocal leukoencephalopathy by combined in situ hybridization and immunocytochemistry

A double-labelling technique combining in situ hybridization and immunocytochemistry is described which was used to characterize cells in the central nervous system containing JC virus DNA in formalin-fixed, paraffin-embedded tissues from four cases of progressive multifocal leukoencephalopathy. All four cases showed positive nuclear labelling for JC virus in both oligodendrocytes and astrocytes. The latter gave a strongly positive cytoplasmic staining reaction using antibodies to glial fibrillary acidic protein and vimentin. No nuclear labelling of neurones or endothelial cells was noted. The results confirm previous suggestions that glia are the main cells infected by JC virus in this disorder and show that the distribution of viral DNA in the brain is more extensive than suggested by routine microscopy alone. In situ hybridization for JC virus may be useful in confirming the diagnosis of progressive multifocal leukoencephalopathy in both surgical biopsies and post-mortem brain tissue.     

Detection of human papillomavirus DNA in cell scrapes and formalin-fixed, paraffin-embedded tissue of the uterine cervix by filter in situ hybridisation

Filter in situ hybridisation (FISH) was used to detect the presence of DNA of human papillomavirus (HPV) types 6/11 or 16/18 in cell scrapes (CYTOFISH) and formalin-fixed, paraffin-embedded biopsies (HISTOFISH) taken from the uterine cervices of 19 women. Paraffin tissue sections collected for HISTOFISH were either digested with pepsin or lysed with alkali/Triton X-100. The digest or lysate of the tissue sections and cell scrapes were applied to nylon or nitrocellulose membranes for nucleic acid hybridisation using 32P-labeled HPV-DNA probes. CYTOFISH and HISTOFISH were compared directly by taking samples for each method from the cervices of the same women. Of 19 women examined by colposcopy, cytology, and histology, eight were assessed as normal and 11 had evidence of a cervical disorder and/or the presence of HPV infection. Whereas no HPV-DNA was detected in the normal cases, the presence of HPV-DNAs was detected by both CYTOFISH and HISTOFISH in 11 cases with histological evidence of HPV infection and/or dysplasia. In these HPV positive cases, eight contained HPV 16/18, two HPV 6/11, and one a mixed infection of HPV 6/11/16/18. The high correlation between the results of CYTOFISH and HISTOFISH shows that formalin-fixed, paraffin-embedded cervical biopsies are suitable specimens for the detection and typing of HPV-DNA by FISH. Both CYTOFISH and HISTOFISH should facilitate studies on the prevalence and distribution of HPVs and their association with neoplasia.     

Automated procedure for dewaxing and rehydration of paraffin-embedded tissue sections for DNA flow cytometric analysis of breast tumors.

Flow cytometric DNA analysis of paraffin-embedded tumors is an important diagnostic and prognostic tool in clinical pathology. The technique is limited, however, by the time-consuming multistep procedure for dewaxing and rehydrating tissue. The authors developed an automated procedure to complete the dewaxing and rehydration of tissue using a routine histologic tissue processor with a 24-hour timer. This technique provided excellent tissue recovery and reproducible DNA histograms comparable to those obtained by manual methods. Subsequently, the authors analyzed the DNA content of 93 paraffin-embedded breast cancer tissues. The automation of a significant portion of the routine processing required for paraffin-embedded tissue makes cytometric DNA analysis a more practical procedure in the laboratory.     

Ultrastructural visualization of human papillomavirus DNA in verrucous and precancerous squamous lesions.

Human papillomavirus (HPV) DNA was ultrastructurally localized by the non-isotopic in situ hybridization technique in formalin-fixed, paraffin-embedded specimens of verruca vulgaris of the skin, condyloma acuminatum of the penis and severe dysplasia of the uterine cervix. Biotinylated DNA probe cocktails were employed for the visualization of HPV-DNA, types 6 and 11 (HPV 6/11) and types 16 and 18 (HPV 16/18). The papillomavirus genus-specific antigen was also visualized by pre-embedding immunoelectron microscopy using rabbit antiserum. In verruca vulgaris, HPV antigen-positive 50-60 nm-particles of mature viral size were observed in the nuclei of the granular cells and parakeratotic cells with perinuclear haloes, whereas HPV 6/11 and HPV 16/18 DNA were negative. In condyloma acuminatum, the nuclei were positive for the HPV antigen and HPV 6/11 DNA, but were negative for HPV 16/18 DNA. More cells were labeled for the viral DNA than for the viral antigen. The ultrastructural observation indicated the presence of the naked (plasmid) viral DNA as fine particles sized 15-20 nm. In the dysplastic cervical mucosa, dot-like positivity of HPV 16/18 DNA was recognized. The HPV antigen and HPV 6/11 DNA were undetectable. HPV 16/18 DNA was localized in part of the nuclear chromatin. This pattern of localization may suggest integration of the viral DNA into the host cell DNA.     

Sensitivity of in situ hybridization techniques using biotin- and 35S-labeled human papillomavirus (HPV) DNA probes

In order to evaluate the sensitivity of our modified in situ DNA hybridization technique using biotinylated probes, formalin fixed, paraffin embedded biopsies from 20 cervical lesions known to contain human papillomavirus (HPV) DNA were re-examined by the technique using both 35S-labeled- and biotinylated HPV DNA probes. The probe concentrations as well as the detection limits of biotin probing were screened by spotting known amounts of HPV 16 DNA on nylon filter, and allowed to hybridize with biotinylated HPV 16 DNA probe. By this method, 4 pg of HPV 16 DNA could be detected using a probe concentration of 0.2 micrograms/ml. HPV DNA could be demonstrated in all 20 biopsies with both hybridization techniques. However, signals in subrabasal cells were detected more frequently with biotin- than with 35S-labeled probes. Additional experiments were performed using three cervical cancer cell lines (with known copy numbers of HPV DNA), to assess the detection limits of HPV infections by the in situ hybridization techniques. The CaSki cells (500-600 HPV 16 copies/cell) were unequivocally positive with both labelling systems. HeLa cells (10-50 HPV 18 copies/cell) were positive with the biotin probing in 10/10 smears, as compared to 7/10 smears when 35S-labeled probes were used. Radioactive probing was inferior to biotinylated probing in detecting the signals in SiHa cells (1-2 HPV 16 copies/cell). This is because even weak background signals could mask true positive signals when 35S-labeled probes are used. In contrast, no background is generated with the biotinylated probes, detected with streptavidin-biotinylated alkaline phosphatase complex. In situ hybridization with biotinylated DNA probes is as sensitive as techniques using 35S-labeled probes for detecting HPV infections in routine cervical biopsies or smears.     

An improved in situ DNA hybridization protocol for detection of human papillomavirus (HPV) DNA sequences in paraffin-embedded biopsies

In situ DNA hybridization is becoming rapidly an important technique for detection and typing of human papillomaviruses (HPV) in epithelial lesions, some of which (those due to HPV 16, 18 and 31) seem to possess an increased risk for progression into an invasive squamous cell carcinoma. An improved in situ DNA hybridization technique (Technique II) was described, and the results obtained in a series of cervical and penile HPV lesions were compared with those given by the in situ hybridization technique (Technique I) previously used in our laboratory. Special emphasis was made to increase the sensitivity with three basic alterations of the hybridization protocol; omission of the 0.2 N HCl wash, use of increased proteinase K concentration (from 50 micrograms/ml to 1 mg/ml), and elevated denaturation temperature (obtained by a heating block instead of an incubator). Poly-D-lysine as a slide-coating medium was replaced by Kodak Photo-Flo 200 to improve the attachment of the tissue sections on the slides. Identical HPV DNA types were discovered by the two hybridization techniques. The attachment of the tissue sections was equal on the slides coated with either poly-D-lysine or Kodak Photo-Flo 200, and the latter did not interfere with the sensitivity of in situ hybridization. The hybridization signals for HPV DNA were weak or moderate in 15/16 lesions with Technique I, but intense in 10/16 lesions with Technique II (P less than 0.001). Furthermore, the resolution of Technique II seemed to be superior to that of Technique I, while being capable of disclosing HPV DNA in the intermediate cell layers (P less than 0.001) and in basal/parabasal cell layers (P less than 0.025) of both the cervical and penile lesions. The discovery of HPV DNA in the parabasal cells provides important clues to the understanding of the biology of HPV infection in the squamous epithelium, and makes this improved in situ DNA hybridization technique invaluable in assessing the lesions, where low copy numbers of HPV are to be expected.     

Assessment of a fully automated high-throughput DNA extraction method from formalin-fixed,paraffin-embedded tissue for KRAS, and BRAF somatic mutation analysis

Preoperative biopsies or imbedded cytological cells will become more and more a primary source of tissue for molecular diagnostic analyses as a result of novel neo-adjuvant treatment regimens for several cancer types. Furthermore there is a growing need to examine metastatic cancer tissue. Hence, nucleic acids need to be reliably isolated and analyzed from small amounts of formalin-fixed and paraffin-embedded (FFPE) tissue. The limited numbers of (tumor) cells in these samples make high quality and sensitive DNA isolation challenging. Also demands for faster turnaround times are growing. Therefore, we evaluated a fully automated DNA/RNA isolation system and compared this with a manual, classical routine molecular pathology method. We compared the quality of the isolates from both tissue cores and micro-dissection for detection of hotspot mutations in KRAS, BRAF applying hydrolysis probe assays. In addition we determined whether the automated method decreases the hands-on-time and turnaround times in routine molecular pathology workflow.     

DNA extraction by sonication: a comparison of fresh, frozen, and paraffin-embedded tissues extracted for use in polymerase chain reaction assays.

DNA extraction from fixed tissues can be the most laborious and complex step in amplifying DNA by the polymerase chain reaction (PCR). We have previously reported a rapid and efficient method for extracting DNA by the use of sonication and glass beads. We have extended our experiences with this technique using fresh, frozen, and formalin-fixed paraffin-embedded tissues with and without the use of glass beads and report their results. Multiple tissue types were obtained at autopsy or as part of a surgical specimen. DNA was extracted from identical tissue when the sample was fresh, frozen, or formalin-fixed paraffin-embedded. Our results indicate that in most instances the sonication technique, which takes only 30 min from start to finish, can rapidly extract fresh, frozen, or formalin-fixed paraffin-embedded tissue and is superior to other rapid extraction techniques in terms of quality and quantity of DNA. It is much more rapid than those techniques that use long digestion periods. This technique will be of great value to those investigators extracting DNA for polymerase chain reaction assays.     

Ultrastructural Visualization of Human Papillomavirus DNA in Verrucous and Precancerous Squamous Lesions

Human papillomavirus (HPV) DNA was ultrastructurally localized by the non-isotopic in situ hybridization technique in formalin-fixed, paraffin-embedded specimens of verruca vulgaris of the skin, condyloma acuminatum of the penis and severe dysplasia of the uterine cervix. Biotinylated DNA probe cocktails were employed for the visualization of HPV-DNA, types 6 and 11 (HPV 6/11) and types 16 and 18 (HPV 16/18). The papillomavirus genus-specific antigen was also visualized by pre-embedding immunoelectron microscopy using rabbit antiserum. In verruca vulgaris, HPV antigen-positive 50-60 nm-particles of mature viral size were observed in the nuclei of the granular cells and parakeratotic cells with perinuclear haloes, whereas HPV 6/11 and HPV 16/18 DNA were negative. In condyloma acuminatum, the nuclei were positive for the HPV antigen and HPV 6/11 DNA, but were negative for HPV 16/18 DNA. More cells were labeled for the viral DNA than for the viral antigen. The ultrastructural observation indicated the presence of the naked (plasmid) viral DNA as fine particles sized 15-20 nm. In the dysplastic cervical mucosa, dotlike positivity of HPV 16/18 DNA was recognized. The HPV antigen and HPV 6/11 DNA were undetectable. HPV 16/18 DNA was localized in part of the nuclear chromatin. This pattern of localization may suggest integration of the viral DNA into the host cell DNA. Acta Pathol Jpn 41: 757-762, 1991.     

Detection and localization of human papillomavirus DNA in human genital condylomas by in situ hybridization with biotinylated probes

We have examined the distribution of human papillomavirus (HPV) DNA in paraffin sections of humans warts by in situ hybridization with biotin-labeled DNA probes. Recombinant plasmid DNAs (HPV-1, -6, -11, -16) were labeled by nick translation with biotinylated deoxyuridine triphosphate. Paraffin sections were hybridized with the probes for 18 h in stringent or non-stringent conditions, and DNA-DNA hybrids were detected by immunocytochemistry. Paraffin sections of warts were also examined for the presence of HPV capsid antigen with the avidin-biotin peroxidase complex method for immunocytochemistry. HPV DNA was detected and localized in paraffin sections from a plantar wart, a laryngeal papilloma, and seven anogenital condylomas. The specific HPV type present in each lesion was determined by hybridization under stringent conditions with the homologous DNA probe. The papillomas were found to contain many more cells with replicating virus DNA, as demonstrated by in situ hybridization, than was apparent from the number of cells containing detectable virus antigen. In situ hybridization with biotin-labeled probes is an effective technique for the identification of HPV infection in routinely collected and processed tissue specimens.