Prevalence of human papillomavirus in sinonasal papillomas: a study using polymerase chain reaction and in situ hybridization.

Inverted and fungiform papillomas of the sinonasal cavity share a common origin from the Schneiderian membrane, but they differ widely in their rates of recurrence and progression to carcinoma. To determine the role of human papillomavirus in the etiology of these lesions, 15 inverted papillomas, five fungiform papillomas, and two squamous cell carcinomas associated with inverted papilloma were examined for the presence of HPV by in situ hybridization (ISH) and polymerase chain reaction (PCR). ISH was carried out on formalin-fixed, paraffin-embedded material using HPV types 6/11, 16/18, and 31/33/35 DNA probes. Tissue DNA was amplified by PCR with HPV L1 consensus primers, and the product was detected by gel electrophoresis, Southern blotting, and hybridization with type specific probes (HPV types 6/11, 16, 18). Three of 15 inverted papillomas and two of five fungiform papillomas were positive for HPV 6/11 by ISH, whereas PCR detected HPV 6/11 sequences in two of 15 inverted and three of five fungiform papillomas. Biopsies from two patients who had serial resections contained HPV 6/11 in the original lesions and all recurrences. No HPV was detected in the carcinomas by ISH, whereas PCR detected HPV 16 in one carcinoma. These findings confirm the presence of HPV DNA sequences in both inverted and fungiform sinonasal papillomas as well as in an associated squamous carcinoma. This would suggest a role for HPV in the pathogenesis of Schneiderian membrane lesions. Furthermore, our data indicate that ISH and PCR are equally sensitive in detecting HPV in sinonasal papillomas.     

Detection of human papillomavirus on Papanicolaou-stained cervical smears using indirect in situ polymerase chain reaction hybridization

OBJECTIVE: Polymerase chain reaction (PCR) and indirect in situ hybridization were combined to detect human papillomavirus (HPV) DNA on Papanicolaou (PAP)-stained cervical smears. To our knowledge, this is the first report of an experiment using indirect in situ PCR (IS-PCR) on PAP-stained cervical smears. DESIGN: We collected native cell specimens from cervicovaginal lavage of 162 patients with squamous intraepithelial lesions. Solution-phase PCR (SP-PCR) was performed as the reference method in the detection of HPV DNA. Indirect IS-PCR was carried out for the same patients to detect the HPV DNA types 6/11 and 16/18 after the PAP-stained smears had been decolorized. Low-risk and high-risk HPV DNA types were also detected by both SP-PCR and indirect IS-PCR. RESULTS: In the evaluation by indirect IS-PCR, 48 of 81 PAP-stained cell smears of low-grade squamous intraepithelial lesions were positive for HPV DNA, as compared to 40 positive cell smears determined by indirect SP-PCR (sensitivity of indirect IS-PCR compared to SP-PCR, 98.1%). Forty-two of 42 high-grade squamous intraepithelial lesion samples were positive for HPV DNA, as determined by both methods (sensitivity of IS-PCR, 100%). Cell lines investigated in this study as positive or negative controls for HPV DNA were confirmed by indirect IS-PCR and SP-PCR. CONCLUSIONS: Our data show that in comparison to SP-PCR, indirect IS-PCR is a highly sensitive method to detect HPV DNA in cell smears from the uterine cervix. The advantages of indirect IS-PCR are (a) low numbers of cells needed, (b) the possibility of using PAP-stained specimens, and (c) cytologic details of smears can be preserved.     

Human papillomavirus infection and anal carcinoma. Retrospective analysis by in situ hybridization and the polymerase chain reaction.

To examine the association of human papillomavirus (HPV) infection with anal squamous cell carcinoma, the authors applied the highly sensitive polymerase chain reaction (PCR) and in situ hybridization (ISH) techniques to detect HPV DNA in formalin-fixed, paraffin-embedded tissues from 18 patients. The presence of HPV types 16/18 in 3 (16.7%) of 18 patients with anal carcinoma was found, using a colorimetric ISH technique for HPV types 6, 11, 16, 18, 31, 35, and 51. Results from one of these three patients were also positive for HPV 31, 35, 51 by ISH techniques. When the same series was analyzed using the PCR and consensus primers to the L1 open reading frame of the HPV genomes, the frequency of positive patients rose to 14 (77.8%) of 18. PCR analysis of the 14 lesions containing HPV DNA, using type-specific primers and probes for HPV 6, 11, 16, 18, and 33, showed that 1 contained HPV 6, 1 contained HPV 11, 4 contained HPV 16, 1 contained HPV 18, 1 contained HPV 33, 5 contained HPV of unclassified type(s), and 1 contained a mixture of three HPV types. There was concordance between typing of cases that were positive by ISH and PCR methods. These data agree with the concept that HPV, in particular type 16, is implicated in the pathogenesis of anal cancer.     

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

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.     

Sinonasal papillomas and human papillomavirus: human papillomavirus 11 detected in fungiform Schneiderian papillomas by in situ hybridization and the polymerase chain reaction.

A series of 19 paraffin-embedded sinonasal papillomas (four squamous papillomas, three fungiform papillomas, nine inverted papillomas, and three cylindrical cell papillomas) were investigated for evidence of human papillomavirus (HPV) infection using immunohistochemistry (polyclonal antibody to HPV capsid antigen), in situ hybridization (DNA probes for HPV 6/11, 16/18, and 31/33/35), and the polymerase chain reaction (primers and probes for HPV 6, 11, 16, 18, and 33). All three fungiform papillomas were positive by all three techniques: immunohistochemistry, in situ hybridization for HPV 6/11, and the polymerase chain reaction for HPV 11. None of the other lesions contained detectable HPV using the specific probes included in this study. These results support the continued classification of fungiform papilloma as a distinctive variant of schneiderian papilloma characterized by a predominantly exophytic growth pattern and an association with HPV 11.     

Detection of human papillomavirus infection in tissue blocks by in situ hybridization as compared with a polymerase chain reaction procedure.

Tissue blocks from 25 cases of condyloma and/or dysplasia were used for human papillomavirus typing with DNA in situ hybridization, compared with the very sensitive polymerase chain reaction. Only one of these cases was negative with both methods: a case of vaginal "koilocytosis." Polymerase chain reaction, as expected, was the more sensitive method, positive in 24 cases, with seven double infections. In situ hybridization was positive in 18 cases, with only two detected double infections. There was excellent agreement between the two methods in typing results. In all cases in situ hybridization showed a positive reaction in areas of koilocytosis and/or dysplasia.     

The use of polymerase chain reaction in generation of biotinylated human papillomavirus DNA probes for in situ hybridization.

The polymerase chain reaction (PCR) was used to produce biotin-labelled human papillomavirus (HPV) 16- and 18-specific DNA probes for in situ hybridization (ISH). PCR was performed by using Amplitaq DNA amplification reagent kit according to the manufacturer's instructions, except that dTTP was substituted by different concentrations of biotinylated dUTP (bio-11-UTP). As template DNA, DNA extracted either from CaSki or HeLa cells was used. The reaction mixture was taken through up to 40 cycles of amplification in a Perkin-Elmer Cetus Thermal Cycler. The highest yield was achieved when the concentrations of dTTP and biotinylated dUTP were 150 and 50 microM, respectively. ISH results compatible with those obtained with biotinylated whole genomic HPV DNA probes were demonstrated when primers from E7 and E6 ORF of the HPV-18 genome were used to produce the biotinylated probe by PCR. With HPV-16, several areas of the genome had to be amplified to generate a PCR probe with equal sensitivity as the whole genomic probe. The background staining was always stronger with the PCR probes than with the whole genomic probes. The sensitivity of the PCR probes does not seem to bear a clear-cut correlation with the size or nucleotide content of the probe, but it might rather depend on the three dimensional structure of the probe and the availability of biotin for the detection system by ISH.     

用聚合酶链反应检测食管癌组织中人乳头瘤病毒DNA

应用聚合酶链反应（ＰＣＲ）技术对汕头市区６８例食管癌的石蜡包埋标本进行人乳头瘤病毒（ＨＰＶ）ＤＮＡ序列检测，结果显示，ＨＰＶＤＮＡ总阳性率为６６．１８％（４５／６８），检出型别主要为ＨＰＶ６、１１、１６，检出率分别为２７．９４％、３６．７６％和２７．９４％，经统计学处理三型间无显著性差异；ＨＰＶ－１８及未定型别各占８．８２％。值得注意的是ＨＰＶ感染中多重感染占阳性病例的５３．３３％（２４／４５）。初步结果表明，汕头市食管癌高发区有较高的ＨＰＶ感染率，此与食管癌的发生，可能有密切关系。     

Transitional cell carcinoma of the bladder: low incidence of human papillomavirus DNA detected by the polymerase chain reaction and in situ hybridization

Viral studies on mammalian urothelium have shown an association between the bovine papillomavirus and cancer of the bladder in cattle. However, the evidence for human papillomavirus (HPV) involvement in urinary bladder in man is less clear. The aim of this study was to investigate the association between HPV DNA and transitional cell carcinoma of the bladder, using the highly sensitive polymerase chain reaction (PCR) and non-isotopic DNA in situ hybridization on formalin-fixed paraffin-embedded tissues from 76 patients. An HPV type specific set of primers was localized on the E6-gene for HPV 16/18 DNA. The second and third set of primers were specific for HPV 6/11 DNA. A biotinylated DNA probe which recognizes HPV 6/11, 16/18, and 31/33/35 was used for in situ hybridization. Of the 76 cases investigated, PCR analysis showed positive signals in seven (9.2%) of cases–six for HPV 16 DNA, and one for HPV 16 DNA and HPV 6 DNA. Four (5.2%) were also reactive for HPV 16/18 DNA using in situ hybridization. Most transitional cell carcinomas (71.4%) associated with HPV DNA were of high pathological grade/stage. One case had koilocytosis. Our results suggest that HPV DNA in transitional cell carcinoma is probably a rare occurrence, although the finding of the high risk HPV 16 DNA may indicate a role for it in this tumour's aetiology.     

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.     

Human papillomavirus DNA in respiratory papillomatosis detected by in situ hybridization and the polymerase chain reaction.

The authors have demonstrated the presence of human papillomavirus (HPV) types 6 and 11 in 10 of 13 (77%) juvenile laryngeal papillomatosis by in situ DNA hybridization using as probes the radiolabeled DNAs of HPVs 6, 11, 16, and 18. Of six specimens from adult laryngeal papillomatosis assayed by the same technique, only 33% were positive. Immunohistochemistry to detect HPV capsid antigens performed on serial sections gave positive signals in 44% (8 of 18) of the specimens, all from juvenile lesions. These results were in agreement with in situ hybridization, except in two cases. When both series (juvenile and adult) were analysed by amplification of a 450-bp fragment corresponding to the L1 ORF of the HPV genomes directed by the polymerase chain reaction, the frequency of positive specimens rose to 100%. Our data agree with the concept that HPV is implicated in the etiology of laryngeal papillomatosis.     

Occurrence of multiple types of human papillomavirus in genital tract lesions. Analysis by in situ hybridization and the polymerase chain reaction.

More than 22 types of human papillomavirus (HPV) have been detected in genital tract squamous cell intraepithelial lesions. Seven of two hundred eighty-six (2.4%) genital tract tissues in which HPV DNA was detected by in situ hybridization contained two or more different HPV types. When analyzed by site, 5 of 204 (2.4%) of cervical intraepithelial lesions were infected by more than one type, compared with 2 of 82 (2.4%) of vulvar lesions. The rate for low-grade lesions was similar (5/218; 2.3%) to that for high-grade lesions (2/68; 2.9%). In contrast, two different HPV types were detected in 6/33 (18%) of tissues by the polymerase chain reaction (PCR) using type-specific primers for eight HPV types. It is concluded that infection by one HPV type is rarely associated with concurrent 'active' infection by a second HPV type, even though DNA of a different viral type can be detected by PCR in about one fifth of such cases. Further study is required to determine if an existing HPV infection can inhibit replication by a different HPV type.     

Human papillomavirus localization in cervical adenocarcinoma and adenosquamous carcinoma using in situ polymerase chain reaction: review of the literature of human papillomavirus detection in these carcinomas

Many studies have suggested that human papillomavirus (HPV) infection plays an important role in the carcinogenesis of the cervical adenocarcinoma. However, the prevalence of HPV infection in cervical adenocarcinoma and adenosquamous carcinoma varies among the studies. Cervical adenocarcinoma (24 cases) and adenosquamous carcinoma (16 cases), including the underlying non-neoplastic epithelium were examined for HPV-DNA using in situ polymerase chain reaction (PCR), which enabled visualization of the localization on a glass slide. In adenocarcinoma, HPV-DNA was found in 13 cases (54%) and in eight cases in underlying non-neoplastic epithelium, resulting in a total of 21 positive cases (88%). In adenosquamous carcinoma, HPV-DNA was detected in 12 cases (75%) and and the HPV-DNA localization of each component was pure adenocarcinoma, 28.6%; mixed, 54.5%; and pure squamous cell carcinoma, 83.3%. In the underlying non-neoplastic epithelium, HPV-DNA was found more frequently in the squamous epithelium (73.3%) than the cervical glands (6.3%). In conclusion, HPV-DNA was detected in 54% of adenocarcinoma, and the rate was elevated by HPV localization in the underlying non-neoplastic epithelium. HPV infection in the underlying squamous epithelium might be related to the carcinogenesis, even in cervical adenocarcinoma. HPV-DNA localization was different in each component of adenosquamous carcinoma.     

Unsuccessful effort to detect human papillomavirus DNA in urinary bladder cancers by the polymerase chain reaction and in situ hybridization

The association of human papillomavirus (HPV) with urinary bladder carcinogenesis is now a controversial issue. In order to certify the presence of HPV DNA in urinary bladder cancers, the polymerase chain reaction (PCR) using five primer sets for detecting various HPV types was used in this study as weli as in sltu hybridizetion (ISH) for HPV 16 and 18 detection. In the PCR study of 93 DNA samples extracted from formalin-fixed and paraffin-ernbedded urinary bladder cancem, no HPV DNA was detected in these tumor samples. The ISH study was also performed on the same tumor samples, but failed to demonstrate any HPV 16-or 18-positive signals in ail except one of the tumor samples. However, the FCR failed to demonstrate HPV 16 DNA even in the bladder cancer positive for HPV 16 DNA by the ISH. This ISH technique was able to demonstrate HPV 16 and 18 DNA in eight of 13 paraffinembedded cervical cancers, in which HPV 16 or 18 DNA had already been detected by the PCR. Our HPV study using PCR and ISH revealed that the HPV status of urinary bladder carcinomas was far different from that of cervical cancers.     

HPV in situ hybridization with catalyzed signal amplification and polymerase chain reaction in establishing cerebellar metastasis of a cervical carcinoma.

We report an unusual case of cerebellar metastasis from a cervical adenosquamous carcinoma in which molecular techniques assisted in establishing the correct diagnosis. The patient was a 43-year-old woman with surgically unresectable cervical carcinoma diagnosed 2 years before presenting with neurological symptoms. A magnetic resonance imaging scan showed a large, enhancing cerebellar lesion with significant brain stem compression. The excised cerebellar tumor resembled a small cell carcinoma and was initially not thought to be a metastasis from the cervical adenosquamous carcinoma. In situ hybridization with catalyzed signal amplification and polymerase chain reactions with primers specific for human papilloma virus (HPV) types 16 and 18 were used to determine the relationship between the cervical and the cerebellar neoplasms. A positive signal was present in the nuclei of both neoplasms by in situ hybridization using HPV16/18 DNA probes. Polymerase chain reaction revealed the presence of HPV-18 DNA sequences in the cervical and cerebellar neoplasms confirming that the cerebellar neoplasm was a metastasis from the cervical primary.     

Human papillomavirus (HPV) detection using in situ hybridization in histologic samples: correlations with cytologic changes and polymerase chain reaction HPV detection

Although in situ hybridization (ISH) and polymerase chain reaction (PCR) have extensively been used on cytology specimens, there have been limited reports of the usefulness of these techniques in relation to confirmed histologic findings. In this study, we used PCR and ISH to detect human papillomavirus (HPV) in cytologic and histologic specimens, respectively. By using positive and negative likelihood ratios, we attempted to identify any predictive role of ISH testing alone or in combination with PCR for the development of high-grade histologic lesions (cervical intraepithelial neoplasia [CIN] 2+). In our study, ISH was a useful method for detection of HPV, even in a large fraction of samples with normal cytologic or biopsy findings. We suggest that when used together and evaluated in conjunction with histologic sections, ISH is a useful tool for ancillary molecular testing of HPV infection in cervical lesions, especially in CIN 2+ histological lesions where its analytic sensitivities and specificities were as good as those of PCR testing.     

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.     

Epstein-Barr virus and Hodgkin''s disease. A correlative in situ hybridization and polymerase chain reaction study.

The authors studied typical Hodgkin's disease along with the nodular, lymphocyte predominance subtype by both the polymerase chain reaction (PCR) and in situ hybridization for evidence of the Epstein-Barr virus (EBV). By PCR, EBV DNA was detected in 12/23 cases of typical Hodgkin's disease and 2/13 cases of the nodular, lymphocyte predominance subtype. EBV RNA was detected by in situ hybridization studies within Reed-Sternberg cells and variants in 11/23 cases of typical Hodgkin's disease and 0/13 cases of nodular, lymphocyte predominance Hodgkin's disease. Other cells positive for EBV, identified as both B and T cells in double-labeling immunohistochemical/in situ hybridization studies, were found in 20/23 cases of typical Hodgkin's disease, 9/13 cases of nodular, lymphocyte predominance Hodgkin's disease, 4/6 cases of progressive transformation of germinal centers, and 7/10 normal lymphoid tissues. It is concluded that EBV is significantly associated with Reed-Sternberg cells in approximately one-half cases of typical Hodgkin's disease but not in the nodular, lymphocyte predominance subtype. EBV-infected B and T cells are also present in a majority of cases of Hodgkin's disease as well as in reactive conditions.     

Localization of hepatitis B virus DNA in hepatocellular carcinoma by polymerase chain reaction in situ hybridization.

The polymerase chain reaction in situ hybridization (PCR-ISH) is a new technique that combines the sensitivity of PCR with the localizing ability of ISH. To investigate the expression pattern of hepatitis B virus (HBV) in the tissue of hepatocellular carcinoma (HCC), we detected HBV-DNA with PCR-ISH in paraffin-embedded tumor and corresponding non-tumor tissues from 11 HCC patients. HBV-DNA was detected in 4 of 11 tumor tissues and in 7 of 10 non-tumor tissues. In tumor tissues, positive signals were scattered in the tissue with occasional clustering, and were found mainly in the cytoplasm of HCC cells rather than in the nucleus. In non-tumor tissues, the number of positive signals was higher than in tumor tissues and they were found in regenerating nodules with differing patterns and intensities. When we compared the detection rate of PCR-ISH with nested PCR among 10 tissue samples, HBV-DNA was detected in 5 tissue samples by PCR-ISH, but the S gene was detected in 10, precore gene in 9 and X gene in 8 by nested PCR. The findings suggest that PCR-ISH is a sensitive technique for localizing HBV in tissue sections and that the low level of HBV replication persists in HCC cells.     

CMV and transplant-related coronary atherosclerosis: an immunohistochemical, in situ hybridization, and polymerase chain reaction in situ study.

Accelerated graft coronary atherosclerosis is the main obstacle to long-term survival in patients who have had a heart transplant. A possible involvement of the human cytomegalovirus (HCMV) in this type of coronary atherosclerosis has been postulated by many authors but has not been definitively demonstrated. In an attempt to clarify the role of HCMV infection in the pathogenesis of this complication, we looked for in situ antigens or DNA of HCMV in 30 coronary artery segments obtained at necropsy from patients who had undergone orthotopic cardiac transplantation at the S?o Paulo Heart Institute. We tried to correlate these HCMV markers with the presence of inflammation and/or atherosclerosis in histologic sections. The patients were grouped as follows: GI, less than 170 days of graft survival and absent/mild atherosclerosis; GII, more than 170 days of graft survival and absent/mild atherosclerosis; GIII, more than 170 days of graft survival and severe/moderate atherosclerosis (170 days was the shortest graft survival time associated with atherosclerosis). The search for HCMV genome and antigens in the coronary artery sections was performed using immunohistochemistry, in situ hybridization, and polymerase chain reaction in situ techniques. Immunohistochemistry and in situ hybridization revealed no evidence of HCMV in all 30 cases. Polymerase chain reaction in situ revealed scarce HCMV-positive lymphocytes in two cases (one each from GI and GIII) located in the adventitial layer. These findings preclude a direct role for the HCMV in the pathogenesis of accelerated graft coronary atherosclerosis. However, the possibility of an indirect effect of the virus, such as an immune-mediated inflammatory response by the host that increases the expression of histocompatibility antigens, leading to tissue injury, cannot be excluded.