Diagnosis of cervical intraepithelial neoplasia and human papillomavirus infection: punch biopsy versus cervical smear

Summary In 102 patients referred to our colposcopy clinic because of one to three Papanicolaou smears indicating cervical intraepithelial neoplasia (CIN) and/or abnormal colposcopy, routine smears and colposcopically directed punch biopsies were taken simultaneously. For detection and typing of human papillomavirus (HPV)-DNA in situ hybridization was performed in all biopsies and in 46 of the cervical smears. In cases of dysplastic lesions the number of HPV 16/18 (40.5%) and 31/33 (42.9%) was markedly higher than HPV 6/11 (16.6%) infection rate. In cases where simultaneous in situ hybridization in biopsy specimen and cervical smears was performed 21.7% showed a HPV negative smear and a positive biopsy, in 6.5% the results were the other way round. In 34.9% of cases with CIN I and 9.5% of cases with CIN II verified by punch biopsy the cytological smear did not indicate dysplasia. Our data show that mild and moderate CIN lesions of the cervix as well as HPV infection are detected more frequently by a combination of cervical smear and colposcopically directed punch biopsy than by cervical smear alone.     

Detection of human papillomavirus types 16 and 18 DNA sequences in the screening of the cells related with carcinoma in situ of the uterine cervix--application of in situ hybridization for cytologic diagnosis

In order to investigate the relationship between the presence of human papillomavirus (HPV) DNA and cervical carcinoma, we examined the cervical screening cells as well as the biopsy specimens obtained from 3 cases of severe dysplasia, 13 cases of carcinoma in situ (CIS) and 2 cases of microinvasive carcinoma for the presence of HPV types 6, 11, 16 and 18 DNA by DNA-DNA in situ hybridization using the biotinylated HPV DNA probes. The results of in situ hybridization analysis revealed that HPV 16 DNA sequences were detected in the nuclei of koilocytosis of severe dysplasia and CIS cases. The nuclei of atypical cells obtained from cervical screening cells were positive for HPV 16 or 18 DNA sequences. Two CIS cases were positive for the presence of HPV 16 and 18 DNA sequences. None of them contained HPV 6/11 DNA sequences. Eighteen cervical screening cases were examined and 10 contained HPV 16 DNA sequences and 6 contained HPV 18 DNA sequences. We suggest that the identification of HPV DNA types in cervical screening cells by in situ hybridization might be of diagnostic and prognostic value in early cervical neoplasia.     

Superficial nuclear enlargement without koilocytosis as an expression of human papillomavirus infection of the uterine cervix: an in situ hybridization study.

Nuclear enlargement of the superficial cervical epithelial cells in the absence of koilocytosis is frequently observed. The purpose of this study was to investigate whether this change represents human papillomavirus (HPV) infection. We reviewed 257 cervical biopsies with the diagnosis of "suggestive of condyloma," mild or moderate dysplasia, or both. Of the 257 consecutive biopsies, 23 fulfilled the two criteria: the superficial cells had a nuclear diameter of at least twice that of the basal nuclei, and complete absence of koilocytosis was seen. Parallel sections from each paraffin block were hybridized with biotinylated probes for HPV 6/11, 16/18, and 31/35/51 under high-stringency conditions. The cases that were negative at high-stringency conditions were then hybridized under low-stringency conditions with a mixture of the three HPV probes. Twelve of the 23 cases (52.17%) were positive for HPV, including one positive for HPV 16/18 in one area and for HPV 31/35/51 in another area, four positive for HPV 31/35/51, two positive for HPV 16/18, two positive for HPV 6/11, and three positive for HPV probe mixture at low-stringency conditions. The positive in situ hybridization was located predominantly in the enlarged nuclei in the superficial layers. In conclusion, HPV infection can be expressed as nuclear enlargement in the superficial layers of the cervical epithelium in the absence of koilocytosis.     

A comparison of slot blot, southern blot, and in situ hybridization analyses for human papillomavirus DNA in genital tract lesions

Genital tract lesions were analyzed for human papillomavirus (HPV) DNA by in situ hybridization using probes of HPVs 6/11, 16/18, and 31/33/35. All of the HPVs detected in vulvar and perianal condylomata by in situ hybridization were HPV 6/11-related, whereas the majority of HPVs detected in cervical intraepithelial grade I lesions were types 16/18- and 31/33/35-related. None of the lesions with histologic features equivocal for HPV infection had detectable HPV DNA by in situ hybridization, though some did contain HPV DNA sequences as ascertained by filter hybridization analysis. The sensitivity of in situ hybridization was compared with that of filter hybridization (slot blot and/or Southern blot). The correlation was high (28 of 30) for cases that contained HPVs 6/11 or 16, as deduced by filter hybridization, and was much less (ten of 29) for cases that contained HPVs distinct from types in the filter hybridization probe cocktail (HPVs 6/11, 16, 18, 31, 35 and 51). There was a high concordance between the results of Southern blot hybridization and slot blot hybridization analyses, especially with cases that contained HPVs 6/11 and 16. In situ hybridization, slot blot, and Southern blot hybridization analyses are all very effective in detecting the common HPV types (HPVs 6/11 or 16). In situ hybridization is useful in differentiating cervical lesions that contain HPV 6/11 from those that contain HPV 16 or other types with oncogenic potential. However, filter hybridization is superior to in situ hybridization when analyzing cases with histologic findings equivocal for HPV infection or cases that contain HPV types related to, but distinct from, the types included in the probe.     

Prevalence of different types of human papillomavirus in cervical infection of north Italian women.

We investigated the prevalences of human papillomavirus (HPV) of type 6, 11, 16, 18, 31, 33, 35 and 42 in 276 cervical tissue samples biopsied from a group of North-Italian women in which a HPV infection of the cervix had been suspected. The specimens were evaluated by conventional histological examination and by HPV typing, using Southern blot hybridization on the extracted DNA. We related different degrees of squamous cervical dysplasia, showing or not viral cytopathic effects, with the HPV types harboured in the lesions. Significant rising of HPV 16 prevalences was observed proceeding from mild to moderate and to severe dysplasias both in presence (r = 0.9623, P less than 0.01) and in absence (r = 0.879, P less than 0.05) of koilocytosis, while an opposite trend was detected for HPV 6 and 11. In our sampling we did not find HPV 35 DNA sequences; in all occasions HPV 31 was associated with mild dysplasias. The absolute prevalences of types 18, 33 and 42 were generally low (from 2 to 3%); they did not show any association with specific histological damage of the cervix or with significant patterns related with the progression of cervical dysplasia.     

Human papilloma virus in uterine cervix: a comparison of detection by morphology and by dot-blot hybridization.

Colposcopically directed cervical biopsies, smears, and swabs obtained from 210 women with a previous abnormal cervical cytology were evaluated for the presence of human papilloma virus (HPV) using morphology and dot-blot hybridization. The diagnosis of HPV infection in biopsies and smears examined morphologically was rendered using established criteria for condyloma/cervical intraepithelial neoplasia (CIN). In hybridization studies, DNA was isolated from cells obtained from cervical swabs and annealed with probes that detected HPV types 6/11, 16/18, and 31/33/35 using a dot-blot procedure. Ninety-five cases demonstrated morphologic evidence of condyloma/CIN; 51 of these (54%) were positive for HPV DNA (five cases 6/11, 21 cases 6/18, 20 cases 31/33/35, and five cases two different probes). HPV DNA was also detected in 6 of the 115 cases (5.2%) that were morphologically negative (three cases 16/18, three cases 31/33/35). The results demonstrated that morphology was more sensitive than dot-blot hybridization for detection of HPV-related lesions. The dot-blot hybridization did detect HPV DNA in a small percentage of the cases that showed no morphologic abnormality and was useful for typing of the HPV. At this juncture, however, the clinical significance of the latter findings is unclear.     

A comparative study of sensitivities of immunocytochemistry, koilocytosis and transmission electron microscopy in detecting HPV in the cervical condyloma and intraepithelial neoplasia as compared to DNA hybridization

The sensitivity in detection of human papillomavirus (HPV) by immunocytochemistry, histological observation of koilocytosis and electron microscopy with reference to the results of Southern blot DNA hybridization were reviewed in 41 lesions (37 patients) of cervical and vaginal condylomata acuminata and intraepithelial neoplasia. HPV DNA was demonstrated in all but one lesion of moderate dysplasia (98%). HPV capsid antigens were demonstrated by immunocytochemistry in approximately 60% of the lesions of condyloma and mild dysplasia. Koilocytosis was present in approximately 90% of the lesions of condyloma and mild dysplasia as well. But the rate of HPV detection by immunocytochemistry and by observation of koilocytosis declined markedly in severe lesions; immunocytochemistry was positive in 22% and koilocytosis was present in 60% in the lesions of moderate dysplasia: positive in 17% and 33% respectively in the lesions of severe dysplasia: 0% and 20% respectively in the lesions of CIS. Intranuclear virus-like particles were observed in all of 8 lesions subjected to electron microscopy. The negative findings of immunocytochemistry and koilocytosis in advanced cervical intraepithelial neoplasia (CIN) have very little significance in relation to the actual presence of HPV in these lesions, although they may be useful in detecting HPV in condyloma acuminatum and mild dysplasia (over 50%).     

The presence of HPV DNA in squamous metaplasia, dysplasia and carcinoma in situ of the uterine cervix

HPV DNA was detected in 11% (4/36) of cervical squamous metaplasia, 43% (6/14) of mild dysplasia, 39% (7/18) of moderate dysplasia, 50% (7/14) of severe dysplasia and 50% (7/14) of CIS. The incidence of HPV16 DNA increased with the severity of dysplasia through CIS. It was distributed almost evenly in the nuclei of the total epithelial layers in severe dysplasia and CIS. Especially in CIS, HPV DNA was found significantly in the nuclei of both basal and parabasal cells, suggesting the possibility of involvement in the carcinogenesis of cervical cancer. The presence of HPV DNA in squamous metaplasia (36 cases) was investigated in three different pathological situations. Group 1 was squamous metaplasia in squamo-columnar junction; Group 2 in the polyp; and Group 3 accompanied by dysplasia or CIS. HPV DNA was positive only in squamous metaplasias with polyps. In these HPV DNA positive cases, HPV DNA was distributed not only in the metaplastic lesion, but also in the adjacent stromal lesion.     

In situ hybridization analysis of HPV DNA in cervical precancer and cervical cancers from China

Summary A series of 103 cervical biopsies derived from 103 women during July 1958 to September 1963 from Beijing, China were investigated with in situ hybridization for the presence of HPV6, 11, 16, 18, 31 and 33 DNA. The mean age of the patients was 46.1 + 10.6 years with a range of 24–74 years. Morphological features of HPV infection were found in 80 (77.7%) biopsies. Invasive cervical cancer was diagnosed in 43 biopsies and cervical intraepithelial neoplasia CIN I, CIN II and CIN III in 9, 9, and 27 cases, respectively. A total of 63.1% (65/103) of the lesions had morphological features of HPV infections associated with CIN or invasive carcinomas. Altogether, 31.1% (32/103) of the biopsies were shown to contain HPV DNA. Of the cases showing HPV morphology, 43.1% were HPV DNA positive. HPV16 (30/32) was the most frequent type, followed by HPV11 and 18, whereas no lesions with HPV6, 31 or 33 were found. A total of 19/43 (44.2%) of the invasive carcinomas contained HPV DNA. HPV DNA positivity and the grade of CIN showed a statistically significant correlation (P=0.0011). Our study demonstrated the presence of HPV in cervical lesions among Chinese women in the late 1950's and early 1960's when a single sexual partner was the rule and also supports the concept that HPV has as an important etiological role in cervical cancer, the highest risk being associated with HPV type 16. The applicability of in situ hybridization in retrospective assessment is emphasized.     

Detection of human papillomavirus DNA in cervical lesions by in situ hybridization using biotinylated DNA probes

In situ hybridization (ISH) for human papillomavirus (HPV)-6, -11, -16, -18, and -31 DNA was performed on 615 formalin-fixed paraffin-embedded cervical biopsies using biotinylated DNA probes. Results were obtained from 584 samples with 266 (45.5%) containing HPV-DNA sequences. Ninety percent of condyloma acuminatum specimens were positive for HPV-DNA with 18 of 19 positive cases containing HPV-6 or -11 DNA. The detection rate of HPV in cervical intraepithelial neoplasia (CIN) lesions was 50.6% (239 of 472), while only 8 of 91 (8.9%) cervical biopsies considered to be histologically normal or with minimal dysplasia contained HPV-DNA as demonstrated by ISH. The prevalence of HPV-16, -18, and/or -31 DNA increased with the severity of the lesions, with 20 of 20 (100%) positive CIN-III lesions containing these viral types compared with 102 of 157 (65.0%) positive CIN-I lesions. ISH with biotinylated DNA probes appears helpful in identifying lesions containing higher risk viral strains.     

DNA-ploidy and HPV infection in epithelial lesions of the lower female genital tract

DNA content was related to the type of human papillomaviruses (HPV) in a series of 87 lesions of the lower female genital tract. Nineteen condylomas, 32 biopsies with slight dysplasia, 19 with moderate dysplasia and 17 with severe dysplasia-carcinoma in situ were studied. HPV status was assessed by in situ hybrization (ISH) with biotinylated probes (HPV 6/11, 16/18, 31/35/51) and the polymerase chain reaction (PCR) (HPV 16,18). DNA ploidy was measured by Feulgen DNA cytophotometry. Positivity for HPV by ISH and PCR was obtained in 48% and 59% of the biopsies, respectively. Seventy-eight per cent of the lesions were diploid or tetraploid and the remaining 22% were aneuploid. The percentage of aneuploid DNA increased with the severity of the lesions. By comparing DNA-ploidy and HPV types by ISH, diploid DNA was more frequently found in HPV 6/11 positive lesions (93%) than in HPV 16/18 positive (81%) or HPV 31/35/51 positive (57%). PCR was more sensitive for detecting HPV in aneuploid dysplastic lesions than ISH, probably indicating the HPV copies are scarce in such lesions. In summary, the results indicate some relationship between aneuploidy and HPV types 16/18 and 31/35/51, which supports an oncogenic potential of these subtypes of HPV.     

Progression of squamous cell carcinoma of the uterine cervix from cervical intraepithelial neoplasia infected with human papillomavirus: a retrospective follow-up study by in situ hybridization and polymerase chain reaction.

A retrospective study was conducted to reveal the natural history of cervical carcinoma infected with human papillomavirus (HPV) and to document latent persistence of HPV infection. Thirty-eight formalin-fixed, paraffin-embedded hysterectomy specimens of cervical carcinoma and cervical intraepithelial neoplasia (CIN) III were examined for the presence of HPV 6, 11, 16, 18, 31, 33, and 35 DNA by in situ hybridization (ISH) with biotinylated DNA probes. The HPV 6 and 11 were not detected, but HPV 16, 18, 31, 33, and 35 were detected in 63% (24 of 38) of CIN III and cervical carcinoma. The HPV-negative specimens by ISH were subjected to polymerase chain reaction (PCR) for detection of HPV 16 and 52b with high sensitivity. In 11 of 14 specimens, specific amplification of HPV 16 was detected and HPV DNA was demonstrated in 92% (35 of 38). A retrospective examination for the presence of HPV DNA by ISH and PCR was performed on sequential biopsy specimens of eight cases whose hysterectomy specimens were judged as HPV 16/18 positive. In the eight cases, HPV 16 was constantly demonstrated in all biopsy specimens throughout the course, from early dysplastic change to carcinoma. This finding indicates long (up to 10 years) persistence of HPV infection in the natural course of progression to carcinoma of the uterine cervix.     

HPV 11, 16 and 18 DNA sequences in cervical swabs from women with cervical dysplasia: prevalence and associated risk of progression.

Cervical swabs obtained from 164 women with histologically proven preinvasive and invasive cervical neoplasia were analysed for HPV type 11, 16 and 18 DNA by filter in situ hybridisation. HPV 16 or 18 was detected in 8 of 24 swabs from patients with invasive squamous cell carcinoma (33%), in 59 of 100 patients with carcinoma in situ or severe dysplasia (59%) and in 16 of 40 patients with mild or moderate dysplasia (40%). HPV 6 or 11 was found in only 2% of all swabs. Thirty-eight of the patients participated in a prospective follow-up study and were monitored non-invasively by cytology and colposcopy for 8 to 36 months. 25 patients had persisting or progressive lesions, 13 of which harboured HPV 16 or 18. Of 13 patients who had complete resolution of the dysplasia, only 2 were HPV-positive. The study indicates a significantly higher risk of malignant progression when the cervical dysplasia is associated with HPV 16 or 18 infection.     

Identifying human papillomavirus subtypes in cervical biopsies with in situ DNA hybridization with biotinylated probes.

To test the utility of biotinylated DNA probes against various subtypes of human papillomavirus (HPV), we performed in situ DNA hybridization on routinely processed archival material from 30 patients with serial cervical biopsies including conization (group I) and a prospective group of 35 patients whose cervical biopsies showed various degrees of koilocytotic atypia and/or dysplasia (group II). Commercially available biotinylated probe cocktails against HPV types 6 and 11, 16 and 18, and 31, 35 and 51 were detected via the avidin-biotin horseradish peroxidase technique. Virus was found in 87% (26/30) of group I and 57% (20/35) of group II. Almost exclusively, viral types 16, 18, 31, 35 and 51 were detected in group I; 54% (19/35) of group II stained for types 16, 18 or 31, 35 and 51; 2.9% (1/35) stained for types 6 and 11. Nine percent of group II (3/35) showed coinfection with types 16, 18 and 31, 35 and 51. Three of six vulvar condylomata (50%) stained for types 6 and 11. In general, weaker staining was associated with greater dysplasia. In situ hybridization using biotinylated DNA probes is useful in identifying patients infected with dysplasia/carcinoma-associated HPV subtypes and can be performed easily on routine surgical specimens.     

Study on the localization of human papillomavirus DNA in the vulvar disease by means of in situ hybridization

We investigated human papillomavirus (HPV) DNA of vulvar diseases obtained from 18 cases of condyloma acuminatum, 3 cases of hyperplastic dystrophy without atypia, 4 cases of lichen sclerosus, 4 cases of Bowenoid papulosis, one case of Bowen disease and 8 cases of squamous cell cancer by in situ hybridization with biotinylated HPV 6, 11, 16 and 18 DNA probes. The results of in situ hybridization analysis showed that HPV 6/11 was positive in 94.4% (17/18) of condyloma acuminatum. In 17 cases of HPV 6/11 positive condyloma acuminatum, 3 cases were positive for HPV 16 and one for HPV 18, respectively. HPV 16 was positive in 75.0% (3/4) of Bowenoid papulosis and one case of Bowen disease was positive for HPV 18. In two cases of Bowenoid papulosis which were positive for HPV 16, cervical carcinoma in situ containing HPV 16 DNA sequences was also found during the follow up period. In 8 cases of squamous cell cancer, 2 cases were positive for HPV 18, one for HPV 16 and one for HPV 6/11. All cases of hyperplastic dystrophy without atypia and lichen sclerosus were negative for HPV DNA. Our results suggested HPV is closely associated with vulvar neoplasia.     

新疆维吾尔族妇女子宫颈癌活检组织中人类乳头状瘤病毒DNA的检测

目的：研究我国宫颈癌高发区新疆维吾尔族妇女宫颈癌与人类乳头状瘤病毒（ＨＰＶ）感染的关系。方法：对６５例新疆维吾尔族妇女的宫颈癌活检组织标本，应用原位杂交法检测ＨＰＶ６／１１、１６／１８和３１／３３／３５ＤＮＡ；Ｌ１共有序列引物聚合酶链反应（ＰＣＲ）及Ｅ６特异型引物ＰＣＲ检测其中的５８例ＨＰＶ６、１６和１８ＤＮＡ。结果：６５例宫颈癌患者中，原位杂交法检测ＨＰＶＤＮＡ有２８例阳性（４３．１％）；Ｌ１ＰＣＲ和Ｅ６ＰＣＲ检测ＨＰＶＤＮＡ阳性分别为１３例（２２．４％）和４５例（７７．６％）。结论：对宫颈癌标本中的ＨＰＶＤＮＡ检测，Ｅ６特异性引物ＰＣＲ敏感性最高，Ｌ１ＰＣＲ及原位杂交相对较低。新疆维吾尔族妇女宫颈癌发病与ＨＰＶ感染有密切关系。     

Human papillomavirus DNA in glandular dysplasia and microglandular hyperplasia: presumed precursors of adenocarcinoma of the uterine cervix

Presumed precursors of adenocarcinoma of the uterine cervix were investigated with specific techniques to identify human papillomavirus (HPV) DNA. The presence of HPV DNA in 36 lesions of glandular dysplasia and 16 lesions of microglandular hyperplasia of the uterine cervix was studied by in situ hybridization using 3H-labeled HPV 16 and HPV 18 DNA probes. Only two of 36 lesions (6%) of glandular dysplasia contained HPV 18 DNA, although 64% of coexisting adenocarcinoma in situ, microinvasive adenocarcinoma, and cervical squamous intraepithelial neoplasia III lesions contained HPV 18 and/or HPV 16 DNA. Two lesions of HPV 18 DNA-positive glandular dysplasia coexisted with adenocarcinoma in situ that contained the same type of HPV DNA. None of the microglandular hyperplasia lesions contained HPV 16 DNA or HPV 18 DNA. These results suggest that, if HPV infection is an initial step toward carcinogenesis, it is unlikely that glandular dysplasia and microglandular hyperplasia are precursor lesions of adenocarcinoma of the uterine cervix. A large proportion of glandular dysplasia may represent reactive lesions of endocervical columnar epithelium. Two lesions of HPV 18 DNA-positive glandular dysplasia may represent well-differentiated components of adenocarcinoma in situ of the uterine cervix.     

Detection of HPV DNA in the uterine cervical lesions by polymerase chain reaction and in situ hybridization]

Human papillomavirus (HPV) is found in close association with carcinogenesis of the uterine cervix. We applied a new in vitro gene amplification technology, the polymerase chain reaction (PCR) to detect HPV 16 and 18 in cervical exfoliated cells. HPV infections were detected in 5 (16%) of 31 women with no pathological lesions of the uterine cervix (normal), 16 (24%) of 67 with cervical intraepithelial neoplasia (CIN) and 6 (38%) of 16 with invasive cervical cancer. Moreover, 10% formalin-fixed and paraffin-embedded tissue sections were prepared from the uterine cervix of these 27 women with PCR-proven HPV infection and were examined for the histological localization of HPV-DNA by in situ hybridization with biotin-labeled DNA probes of HPV types 6/11, 16/18 and 31/33/35. HPV-DNA type 16/18 was detected in 3 of 5 normal women, 2 of 4 CINs I, 2 of 3 CINs II, 6 of 9 CINs III and 6 of 6 invasive cervical cancers. HPV-DNA type 6/11 was detected in 6 of 6 condylomas. Viral DNA sequence was detected in the superficial cells of CIN I and II, and it was distributed through entire thickness layer of undifferentiated cells derived from CIN III and squamous cell carcinoma. In addition, the staining intensity became weak as the lesion progressed. These differences between lesions might be due to the difference in the viral form in the nuclei, ie whether an episomal or integrated form. Thus, an in situ hybridization technique with a biotin-labeled DNA probe as well as the PCR method is useful for the detection of HPV in clinical samples.     

Human papillomavirus DNA in situ hybridization may be used for the quality control of genital tract biopsies

Biopsies of human papillomavirus (HPV)-related lesions of the lower female genital tract were studied using in situ hybridization for HPV DNA. The probes included HPV types 6, 11, 16, 18, 31, 33, 35, 41, 43, 44, 45, 51, 52, and 56. In cervical intraepithelial neoplasia (CIN) 1 lesions, 64 of 70 (91%) of formalin-fixed tissues were HPV DNA-positive; in vulvar condylomata, 34 of 36 (94%) were positive. Only two of 52 (4%) of the lesions diagnosed as equivocal for CIN 1 or condyloma were positive. Higher-grade CIN and vulvar intraepithelial neoplasia lesions had a lower rate of HPV DNA positivity. It is suggested that in situ hybridization may be used as a quality control procedure for the histologic diagnosis of HPV-related lesions.