Correlation of histology, human papillomavirus, and viral load in laryngeal papillomas of childhood.

The purpose of this study was to analyze 47 laryngeal papillomas in children for human papillomavirus (HPV) DNA by in situ hybridization and RT in situ PCR and to correlate these results with the histologic findings. HPV DNA was detected by in situ hybridization in 29 of 47 (62%) of the cases; all positive cases contained HPVs 6 or 11. HPV DNA detection was associated with a statistically significant increase in the presence of keratohyaline granules, nonuniform perinuclear halos, and marked papillomatosis (P<0.02). The viral load was low, defined by less than 20 HPV-positive cells per tissue with a correspondingly weak signal, in 19 of 29 (65%) of the positive cases. In comparison, a high viral load was evident in 19 of 21 (90%) of vulvar condylomas. The laryngeal lesions negative for HPV by in situ hybridization were tested for HPV by RT in situ PCR using primers specific for HPVs 6 and 11. The detection rate of HPV increased to 38 of 47 (81%) after PCR amplification. It is concluded that laryngeal papillomas in childhood are characterized, in general, by a relatively low HPV viral load and that the cases with productive viral infection, as demonstrated by in situ hybridization, are associated with nonuniform keratohyaline granules, nonuniform perinuclear halos, and marked papillomatosis.     

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.     

Buffered formalin is the superior fixative for the detection of HPV DNA by in situ hybridization analysis.

In situ hybridization is used commonly for detection of human papillomavirus (HPV) DNA. There is little information, however, on whether the detection of HPV DNA by in situ hybridization can be affected by the way in which the tissue is fixed. To address this question, the authors compared the hybridization signal using this technique under low stringency conditions for several genital condylomata containing HPV 6 or 11 that were randomly subdivided and fixed in various fixatives for 16 hours. In all cases, the largest proportion of cells with koilocytotic atypia that had detectable HPV DNA was in buffered formalin-fixed tissue (80%), followed by tissue fixed in unbuffered formalin (70%), Hartman's solution (40%), and Bouin's solution (10%). After a high stringency wash, the greatest decrease in the overall hybridization signal was with tissue fixed in Bouin's solution; a minimal decrease was noted with tissue fixed in buffered formalin. Fixation in Bouin's solution for 2 hours gave in situ hybridization results comparable with buffered formalin fixation but with poorer cytologic detail. It is concluded that, of the fixatives studied, buffered formalin is superior for the detection of HPV DNA by in situ hybridization analysis.     

Detection of human papillomavirus DNA on routine Papanicolaou's smears by in situ hybridization with the use of biotinylated probes.

Specific human papillomavirus (HPV) types have been implicated as playing a major role in the development of cervical neoplasias. HPV DNA types usually have been identified by nucleic acid hybridization assays with the use of radiolabeled probes. These techniques are sensitive and specific but are not suitable for large-scale clinical use. To detect specific HPV DNAs simply and rapidly, in situ hybridization (ISH) with the use of biotinylated HPV DNA 6/11 and 16/18 probes was done on destained Papanicolaou's (Pap) smears from 545 patients. All smears showed koilocytotic changes. HPV DNAs were demonstrated not only in cervical intraepithelial neoplasia (CIN) and atypia, but also in squamous cells with minimal nuclear changes (karyomegaly) and perinuclear halos. HPV DNA 16/18 was detected in 52% of the smears with CIN I, 44% of those with CIN II and III, 19% of those with atypia, and 4% of the minimal changes. HPV DNA 6/11 was detected in 27% of the smears with CIN I, 27% of those with atypia, and 6% of the minimal changes. HPV DNAs were also detected in smears without koilocytotic changes. Thus, ISH with the use of biotinylated probes can serve as an adjunct to Pap smears in detecting HPV infection.     

Comparison of Southern blot hybridization and polymerase chain reaction methods for the detection of human papillomavirus DNA.

A methodologic study was performed to compare the polymerase chain reaction (PCR) and Southern blot hybridization, two commonly used testing strategies for the detection of human papillomavirus (HPV) infection. Three laboratories tested masked aliquots of exfoliated cervical cell specimens obtained from 120 women by cervicovaginal lavage. The study population included 32 women with condylomatous atypia or cervical intraepithelial neoplasia and 88 control women with no known history of cervical neoplasia. Two laboratories used PCR with different sets of consensus primers for HPV detection. The third laboratory used low-stringency Southern blot hybridization to identify all HPV types, followed by high-stringency Southern and/or dot blot hybridization to confirm specific HPV types. One of the PCR primer sets detected HPV types with a differential efficiency that was not predicted by analysis of DNA sequences or direct testing of HPV-containing plasmids. In contrast, the second PCR primer set was shown to be a much broader consensus system, detecting the same HPV types as Southern blotting, though requiring much less clinical specimen. Over 80% of women with cervical intraepithelial neoplasia or condylomatous atypia were found to be HPV infected both by Southern blotting and by the second PCR primer set. Among the control women, 11% were HPV positive by Southern blotting, while 31% were positive with the second set of primers. Most of the HPV infections found only by PCR were not due to HPV type 6, 11, 16, 18, 31, 33, or 45. These known HPV types were uncommon among normal women in the study population, even as determined by the PCR method.     

Oligonucleotide primers for DNA amplification of the early regions 1, 6, and 7 from human papillomavirus types 6, 11, 16, 18, 31, and 33

Summary Human papillomavirus (HPV) type-specific sequences required for polymerase chain reaction (PCR) mediated amplification of HPV DNA sequences are presented. One primer pair within the E1 open reading frame (ORF) was shared by HPV 6, HPV 11, HPV 16, and HPV 31, whereas the other primer pair within the E1 ORF was specific for HPV 16. Eight primer pairs from the E6 and E7 ORFs specifically detected HPV 6, HPV 16, HPV 18, and HPV 33 sequences. This system has been used for detection of HPV DNA in biopsies, cytological smears and sections of formalin-fixed tissues.     

Nested polymerase chain reaction and in situ hybridization for detection of nucleopolyhedrosis

A nested polymerase chain reaction (PCR) and in situ hybridization were developed for detection of baculoviruses in insects or other arthropods with nucleopolyhedrosis. The nested PCR was based on the sequences of polyhedrin genes from baculoviruses. Two sets of primers were designed, primers set, 35/36, was for the first step of amplification and yielded a product of around 680 bp, the second primer, 35-1/36-1, was designed to yield a product of around 335bp from the fragment amplified by the first primer set. The sensitivity of this two-step amplification was 100 to 1000 times higher than that of the one-step amplification by primer set (35/36). Samples which contained baculovirus DNA yielded an amplification product showing the expected DNA fragment mobility, whereas nucleic acid extracted from tissue samples of clinically healthy insects or uninfected cells showed no such DNA fragment, thereby confirming the specificity of the primers. Using the 35/36 amplicon as a probe, the PenuNPV-infected cells show positive reaction by in situ hybridization. Two-step DNA amplification and in situ hybridization with the DNA probe developed in the present paper provide effective detection and diagnostic tools for screening insects or other arthropods, especially crustacean species, crabs and shrimps, for baculovirus infections, and may be important in preventing (and/or controlling/enhancing) the infection of baculoviruses.     

A general primer pair for amplification and detection of genital human papillomavirus types.

A general primer pair localized in the E7 and E1 regions was identified and used for the detection of genital human papillomaviruses (HPVs). The genital HPV types 6b, 11, 16, 18, 31 and 33 were amplified and detected by the polymerase chain reaction (PCR) performed at a high stringency annealing temperature (60 degrees C). HPV-2, -3, -7, -13 and -30 were amplified only at lower temperatures. Twelve biopsies from women with invasive cancer in the cervix were analysed with the general primer pair. The amplification product specific for the general primer pair was detected in 11 of the 12 biopsies. The eleven HPV DNA positive specimens were shown to contain HPV-6b, HPV-16 and/or HPV-18 by Southern blot hybridization of the PCR products. The general primers were also used for analysis of 57 cervical scrapes from women with normal cytology, condyloma or CIN. By ethidium bromide staining after agarose gel electrophoresis we could detect 21 positives. Slot-blot analysis of the amplification products from all 57 scrapes confirmed the specificity of the 21 positives and revealed 5 additional positives. Among the 57 scrapes, 15/21 CIN scrapes, 10/21 condyloma scrapes and 1/15 normal scrapes contained HPV DNA. Eight different HPV types were detected. The general primer pair from the E7/E1 region is thus a powerful tool for the detection of HPV in clinical samples. The amplimer obtained offers a possibility for further typing by slot-blot hybridization using HPV-type specific probes.     

Useful cytological confirmation of HPV 13 in lesional mucosa enhances diagnosis of focal epithelial hyperplasia

An 11-year-old female presented with multiple oral lesions for several months. Histopathological findings suggested focal epithelial hyperplasia (FEH), also known as Heck disease. FEH is strongly associated with Human papillomavirus (HPV), especially genotypes 13 and 32. An oral swab of a mucosal lesion was subsequently obtained for cytology, immunohistochemistry and in situ hybridization. In addition, in situ hybridization and immunohistochemistry were also performed retrospectively on the biopsy specimen for correlation. The cytology specimen showed squamous cells with enlarged, slightly atypical nuclei and rare perinuclear halos. The histology findings included papillomatosis with acanthosis, mild nuclear atypia and focal perinuclear halos. The immunohistochemistry for the consensus HPV L1 capsid protein was found in both the cytology and biopsy specimens indicating that the lesion was HPV-related. High viral copy numbers of HPV 13 were detected by in situ hybridization in both the cytology and histology specimens. Although histologic features of FEH have been well characterized in the literature, to our knowledge, this is the first case to describe in FEH with adjunct immunohistochemistry and in situ hybridization results. Furthermore, these findings assisted in our diagnosis since the patient's clinical presentation was a diagnostic challenge with smooth dome-shaped papules instead of the typically described flat-topped verrucous lesions seen in FEH. In summary, our case reveals that there is a high concordance between the HPV 13 detection in the cytology and histology of FEH, and that performing cytology in addition to histology can be used to optimize diagnostic evaluation towards appropriate patient care.     

An improved technique for the in situ detection of DNA after polymerase chain reaction amplification.

In situ detection of polymerase chain reaction (PCR)-amplified DNA in cell and tissue preparations previously required 5 to 7 primer pairs designed to generate a long (greater than 1,000 base pair) product. The authors describe a nonisotopic PCR in situ technique, employing a single primer pair and target sequences as short as 115 base pairs, that can detect one target molecule per cell. The essential procedural change is to withhold the DNA polymerase or primers until the reaction temperature approaches 80 degrees C. The Hot-Start method greatly increased amplification specificity which, more than product size, appears to determine success of in situ PCR. The marked improvement in specificity may permit target detection by direct incorporation of labeled nucleotides.     

肺鳞癌人乳头状瘤病毒感染的原位杂交检测和观察

经多重多聚酶链反应，检测４９例肺鳞癌中发现７例人乳头瘤病毒阳性的肿瘤组织，采用生物素标记ＨＰＶＤＮＡ探针，进行原位杂交检测，结果发现在５例肿瘤组织中显示ＨＰＶＤＮＡ阳性信号，其中ＨＰＶ１１型阳性３例；ＨＰＶ１６例阳性１例；１例为ＨＰＶ１１例和１６型均阳性。原杂交ＨＰＶＤＮＡ阳性信号，大多位于凹空样肿瘤细胞或低分化鳞癌细胞的核内，分子生物学研究表明ＨＰＶ感染可能与部分鳞有关。     

Detection of herpes simplex virus DNA by in situ hybridization technique and polymerase chain reaction in Papanicolaou-stained cervicovaginal smears

Papanicolaou-stained cervicovaginal smears from six patients with herpes simplex virus (HSV) infection were destained and reprocessed by means of in situ hybridization (ISH) technique to demonstrate the presence of HSV DNA utilizing biotinylated probe. Positive results were obtained in all six cases with intense staining signal for the HSV DNA in the nuclei of cells having a ground-glass nuclear appearance as well as in multinucleated giant cells. Furthermore, a hybridization signal was also noted in smears that had been prepared as much as 3 yr previously. HSV type 2-specific antigen was confirmed in six destained smears by means of immunoperoxidase staining. Moreover, polymerase chain reaction (PCR) was also performed for four patients from Pap-destained cervicovaginal smears. Amplified HSV DNA was detected in all four cases as 106 basepair PCR products by polyacrylamide gel electrophoresis. The combined use of cytology and the ISH technique and PCR amplification was of great value for the rapid cytodiagnosis of genital infection of HSV.     

Detection of human papilloma virus (HPV) genomes by the primed in situ (PRINS) labelling technique

Primed in situ Labelling, a technique based on primer mediated DNA synthesis, has become a useful tool in cytogenetics, especially for chromosome mapping, banding and the investigation of sequence organization in fresh metaphase preparations. Its application in the routine surgical pathology laboratory has been hampered by the fact that the technique did not work on paraffin-embedded, formalin-fixed tissue. We investigated cervical biopsies (n = 20) with morphological signs of HPV infection and found that the PRINS method is at least as sensitive as a classical in situ hybridization assay for detecting HPV DNA in paraffin-embedded, formalin-fixed tissue. In all investigated cases (n = 20), HPV DNA was found by both methods. The PRINS method was able to demonstrate HPV DNA not only in superficial koilocytotic squamous cells but also in non-koilocytotic cells in the deeper spinous cell layers, and even in some basal cells. We describe an economical protocol using conventional consensus HPV oligonucleotide DNA primers. The described method is rapid (approximately 3 hours) and easy to perform for screening and subtyping HPV infection in the routine surgical pathology laboratory.     

In situ hybridization analysis of HPV 16 DNA sequences in early cervical neoplasia

The authors examined 18 cervical intraepithelial neoplasms (CIN) for the presence of human papillomavirus (HPV) DNA sequences by Southern blot hybridization and DNA-DNA in situ hybridizations for HPV DNA sequences and compared the epithelial distribution of HPV 16 DNA sequences with HPV 6/11 sequences in selected condylomas. Fifteen of the 18 CIN lesions contained HPV 16 DNA as determined by Southern blot hybridization. With the use of biotinylated HPV 16 DNA probes, 10 of the 18 were positive by in situ hybridization, 9 of which were also positive by Southern blot hybridization. In situ hybridization to HPV 16 probes was found primarily in areas of CIN which contained either maturation or koilocytotic atypia, although in two cases hybridizing sequences were detected in superficial cells from epithelium with no discernible maturation. Staining in both condylomas and CIN lesions varied in distribution and intensity. However, in some CIN lesions staining from cell to cell varied considerably. This greater variability in staining appeared to correlate with greater morphologic variations which characterize CIN, and which may influence greater variation in HPV DNA replication. Thus, some differences in patterns of hybridization for HPV DNA between CIN and condylomas may be explained by morphologic differences in the two classes of lesions. Differences in viral gene expression between condylomas and CIN and their relationship to morphologic findings remain to be clarified.     

The sensitivity of in situ hybridization for detection of human papillomavirus

We studied a sensitivity of HPV DNA detection by in situ hybridization method using 3H labeled HPV DNA. The materials were CaSki cells and SiHa cells which were derived from as a negative control. The total cellular DNAs extracted from these cell lines were estimated copy numbers of HPV 16 DNA using Southern blot hybridization. In our result, CaSki cell has 400 copies/cell, SiHa cell were appeared to have 1-5 copies/cell. Simultaneously these cells were fixed by periodate-buffered lysine-paraformaldehyde-glutaraldehyde (PLPG) and were detected HPV 16 DNA using in situ hybridization. We detected HPV 16 DNA in CaSki cells and SiHa cells by in situ hybridization also. We concluded that the sensitivity of our in situ hybridization technique is 1-5 copies/cell.     

A sensitive and specific detection of Human herpesvirus 8 by polymerase chain reaction and dot blot hybridization

Polymerase chain reaction (PCR) is a powerful technique of detecting Human herpesvirus 8 (HHV-8), but has a limited sensitivity and specificity. A new assay of HHV-8 based on combination of PCR with dot blot hybridization (DBH) was developed and evaluated for its sensitivity and specificity. An HHV-8-specific primer pair, ORF26out was used for amplification of target DNA. When the PCR product was detected visually the limit of detection was 0.1 ng DNA isolated from HHV-8-infected BC-3 cells. For DBH, the DNA amplified with the primer pair ORF26in specific for HHV-8 was labeled with digoxigenin (DIG). This DIG-labeled probe was capable of detecting 1.0 ng of DNA isolated from HHV-8-infected BC-3 cells. On the other hand, PCR combined with DBH (PCR/DBH) was more sensitive than PCR or DBH alone and also very specific. The sensitivity of PCR/DBH was higher than that of PCR and DBH alone. The PCR/DBH assay can be applied efficiently to confirm the presence of HHV-8 in clinical samples and to differentiate specifically HHV-8 infection from other viral infections.     

Thermus aquaticus DNA polymerase-catalysed chain reaction for the detection of human papillomaviruses

To delineate the conditions for the polymerase chain reaction (PCR) using primers specific for human papillomavirus (HPV) types 6b, 16 and 18, a number of important technical features were analysed. Buffer, concentrations of magnesium, Taq polymerase, primers and DNA templates, annealing temperature, and extension time were studied by a combination of gel electrophoresis, Southern and slot-blot hybridization. Amplification of E6 gene fragments of HPV-16 and HPV-18 generated bands of 110 bp and 154 bp respectively, as predicted. However, amplification of a segment within the long control region of HPV 6b yielded an unexpected size of 340 bp. Different conditions were found for each HPV type-specific primer pair. These results, and the applications of PCR in HPV research and in an increasingly wide range of fields in medical virology are discussed.     

Oncogenic human papillomaviruses and ploidy in cervical lesions.

AIM: To compare ploidy measurements obtained on tissue sections of selected low and high grade squamous intraepithelial lesions containing oncogenic HPV (types 16, 18 or 33) detected by in situ hybridisation (ISH) or PCR. METHODS: DNA ploidy was assessed by image cytometry after Feulgen staining of contiguous serial sections of eight lesions exhibiting atypical squamous cells or squamous atypia and 53 low and 63 high grade squamous intraepithelial lesions in which HPV had been detected by ISH or PCR. RESULTS: Aneuploidy was strongly associated with the presence of oncogenic HPV, being detected in 50% of lesions with squamous atypia and 75.5% of the low and 95.2% of the high grade squamous intraepithelial lesions. The multiploid profile was highly associated with high grade lesions and with the pattern of HPV DNA integration. CONCLUSIONS: The presence of aneuploidy is strongly suggestive of the presence of oncogenic HPV types. Combining the detection of HPV by ISH and PCR with DNA image cytometry may provide the pathologist and the physician with important prognostic information about low grade lesions, especially when these lesions have a multiploid DNA profile and contain oncogenic HPV.