Cervical Biopsy Specimens Research Articles

Human papillomavirus detection by hybrid capture in paired cervicovaginal lavage and cervical biopsy specimens.

Infection of the uterine cervix with human papillomavirus (HPV) is associated with dysplastic lesions that may progress to malignancy. Certain HPV types are associated with higher risk of cervical cancer than other genital HPVs. The goal of this study was to determine if cells obtained by cervicovaginal lavage contain similar HPV types as paired cervical biopsy in women referred because of abnormal cervical cytology. Thirty-four paired lavage and biopsy samples were analyzed for HPV DNA by hybrid capture, using "low risk" (HPV types 6. 11, and related types and "high risk" group (HPV types 16, 18, and related types) HPV. HPV was detected in 24 lavage samples and 18 biopsies. High risk types were predominant. In 14 of 18 HPV-positive biopsies, the paired lavage was also positive for the same HPV group. Four biopsies were HPV-positive at low levels, and the paired lavage was HPV-negative. The mean viral copy numbers of the biopsies from patients with positive and negative lavage samples were 2.7 and 0.1, respectively (P = .02). Ten low level HPV infections were detected by lavage that were not detected by biopsy. HPV detection by hybrid capture in cells obtained by cervicovaginal lavage reflects the results of HPV testing in cervical biopsies.

Detection and typing of human papillomavirus in biopsy and cytological specimens by polymerase chain reaction and restriction enzyme analysis: a method suitable for semiautomation.

Screening for high-risk human papillomavirus (HPV) types allows the detection of women at a high risk of cervical squamous carcinomas, thereby defining a subset of patients targeted for more intensive screening and follow-up. Thirty-four cervical biopsy specimens and isolated cells from cervical smears of normal women or women diagnosed with high-grade intraepithelial lesion (HGSIL) were screened for the presence of HPV by in situ hybridization (ISH) and/or by polymerase chain reaction (PCR). The exact HPV type was determined using a novel restriction typing method. The detection of HPV was facilitated greatly by the use of a PCR-enzyme-linked immunosorbent assay (ELISA)-based method. HPV was detected by PCR in 32% of the biopsy specimens, whereas only 23% had a positive staining by ISH. In one case, a double infection was detected by ISH as well as by PCR. In two cases, the presence of HPV was detected by both methods but the exact type was different. Analyzing cells isolated from cervical smears by the PCR-ELISA technique or by PCR followed by agarose gel electrophoresis, HPV was detected only in patients with HGSIL and not in the control group. The PCR system is more sensitive than conventional ISH, and the PCR-ELISA system presented in this study is efficient in screening large series of cytological samples. Furthermore, this system allows exact HPV typing on the microtiter plate. These innovations may allow the application of HPV detection and typing as a routine screening method to identify patients with a high risk of developing cervical neoplasia.

Vascular Permeability Factor (Vascular Endothelial Growth Factor) Expression and Angiogenesis in Cervical Neoplasia

Angiogenesis is a critical factor in the progression of solid tumors, including cervical cancers. The mechanisms responsible for angiogenesis in cervical neoplasia, however, are not well defined. Our goal was to determine the relationship between angiogenesis and the expression of the angiogenic cytokine vascular permeability factor (VPF), also known as vascular endothelial growth factor, and its receptors in cervical neoplasia. Sixty-six cervical biopsy specimens were evaluated; among these, 16 samples were designated as benign, 17 as low-grade squamous intraepithelial lesions, 18 as high-grade squamous intraepithelial lesions, and 15 as invasive squamous cell carcinomas. Histologic sections immunostained for factor VIII-related antigen were evaluated quantitatively for microvessel density and for the presence of epithelial-stromal vascular cuffing. Sections were also evaluated for VPF messenger RNA (mRNA) expression by in situ hybridization. VPF mRNA expression, epithelial-stromal vascular cuffing, and microvessel density counts were significantly increased in invasive carcinoma and in high-grade intraepithelial lesions as compared with low-grade intraepithelial lesions and benign squamous epithelium. Vascular cuffing and increased microvessel density counts were also significantly associated with increased VPF mRNA expression. These observations suggest that VPF is an important angiogenic factor in cervical neoplasia.

Human papillomavirus in false negative archival cervical smears: implications for screening for cervical cancer.

To assess the value of detecting human papillomavirus (HPV) DNA in false negative archival cervical smears in population based screening programmes for cervical cancer. Cytomorphologically classified false negative archival Pap smears (n = 27) taken from 18 women up to six years before cervical cancer was diagnosed were blindly mixed with 89 smears from hospital patients with a variety of gynaecological complaints and tested for HPV by the polymerase chain reaction (PCR). Corresponding cervical cancer biopsy specimens were also available for HPV analysis. Neither the examining cytopathologist nor the molecular biologist was aware of the study design. HPV DNA was detected in the smears of 16 patients with cervical cancer missed previously by cytology. HPV 16 and 18 were found predominantly in those smears taken up to six years before the diagnosis of cervical cancer. The smears of the two remaining patients were reclassified as inadequate for cytology or contained no suitable DNA for PCR. In 15 patients the same HPV type could be found in the smears and the cervical cancer biopsy specimens. The results indicate that high risk HPV types can be detected in archival smears classified as false negative on cytology and that cytological screening errors may be reduced if combined with PCR testing for HPV.

Identification of genital human papillomaviruses in cervical biopsy specimens: Segregation of specific virus types in specific clinicopathologic lesions

We have established a critical identification method for the full spectrum of genital human papillomaviruses (HPVs) in clinical specimens. It was based on the recognition of PstI, BanI and MspI cleavage patterns of HPV DNA detected by blot hybridization with HPV 58 DNA probe at Tm -40 degrees C. By this method, we identified 24 different types of genital HPV including 5 novel types (HPV 59, 61, 62, 64 and 67) in the specimens collected at one hospital and found almost all the HPVs with the authentic cleavage patterns of their respective prototypes. In 235 cervical biopsy specimens, HPV 6 or 11 was found in exophytic condyloma acuminatum (15/15) but not in any cervical intraepithelial neoplasia (CIN) specimens. In contrast, HPV 18, 30, 43, 54, 56, 59, 62, 66 and 67 were identified in CIN I (28/71) or II (4/56) but not in CIN III, while HPV 16, 31, 33, 35, 39, 51, 52 and 58 were identified in CIN III (83/93) as well as in CIN I (34/71) and II (47/56). The result indicates that heterogeneous genital HPVs prevail all over the world. In addition, HPV 6 and 11 are etiologic agents only of exophytic condyloma, whereas the other HPVs are etiologic agents of CIN with the segregation of specific HPVs in CIN III. We propose a new clinicopathologic grouping of genital HPVs founded on nucleotide homology of the HPV genome.

Diagnosis of genital infection caused by human papillomavirus using in situ hybridisation: the importance of the size of the biopsy specimen.

To determine the size of a cervical biopsy specimen with human papillomavirus (HPV) infection required to enable in situ hybridisation to be carried out with a guarantee of a reliable result. In situ hybridisation was carried out in 142 cervical uterine biopsy specimens classified histologically as low grade and high grade squamous intraepithelial lesions. Epithelial length at the level of the basal membrane was measured by image analysis. The specimens were divided into 10 groups based on epithelial length. Of the biopsy specimens, 61.2% were HPV positive. In specimens with an epithelial length below 5 mm 31.9% were HPV positive; in those between 5 and 9 mm in length 67.5% were HPV positive; and in those greater than 9 mm in length 81.8% were positive for HPV. For low grade squamous intraepithelial lesions (n = 90), 68.4% of specimens with an epithelial length greater than 5 mm were HPV positive. For high grade squamous intraepithelial lesions (n = 52), 86.8% of specimens with an epithelial length greater than 5 mm were HPV positive. For a diagnosis of HPV infection using in situ hybridisation, the minimum length of epithelium in a cervical biopsy specimen should be 5 mm. For high grade squamous intraepithelial lesions, specimens over 5 mm in length are suitable. For low grade squamous intraepithelial lesions, to minimise the number of false negative results, the ideal minimum length is 10 mm.

A new method for direct analysis of polymerase chain reaction-amplified human papillomavirus using DNA enzyme immunoassay.

We describe use of a polymerase chain reaction in conjunction with a DNA enzyme immunoassay for the simultaneous detection of multiple types of human papillomavirus in a single microtiter plate. The method is specific, sensitive, rapid, and capable of detecting human papillomavirus-6, -11, -16, -18, and -33 in cervical biopsy specimens.

Global DNA hypomethylation increases progressively in cervical dysplasia and carcinoma.

Global DNA hypomethylation has been observed in some human neoplasms and has been implicated as an important factor in carcinogenesis. The current study was designed to assess whether DNA hypomethylation occurs in cervical dysplasia and cancer, and to determine the relationship between the degree of DNA hypomethylation and the grade of neoplasia. Cervical biopsy specimens were obtained from colposcopically identifiable lesions in 41 patients with abnormal Pap smear results. The extent of global DNA methylation was assessed by incubating the extracted DNA with [3H]-S-adenosylmethionine and Sss1 methyltransferase, an enzyme that specifically catalyzes the transfer of methyl groups to cytosine residues in the cytosine-guanine doublet. The degree of exogenous 3H-methyl group incorporation into the DNA therefore is related reciprocally to the extent of endogenous DNA methylation. These data were compared with the histopathologic classification of the lesions. The extent of 3H-methyl group incorporation was increased threefold and sevenfold in the DNA from cervical dysplasia and cancer, respectively, compared with the DNA from normal cervical tissue (P = 0.006, analysis of variance). Significant incremental increases in DNA hypomethylation were observed in the progression from normal and low grade squamous intraepithelial lesions (SIL) to high grade SIL and to cancer (P < 0.0001, trend). These data show that global DNA hypomethylation is a significant epigenetic event in cervical carcinogenesis and that the degree of DNA hypomethylation increases with the grade of cervical neoplasia. These data suggest that global DNA methylation may serve as a biochemical marker of cervical neoplasia.

Interobserver variation in the diagnosis and grading of dyskaryosis in cervical smears: specialist cytopathologists compared with non-specialists.

To compare the assessment of dyskaryosis in cervical smears made by specialist consultant cytopathologists and consultant general histopathologists. One hundred and ten cervical smears were circulated to 10 observers from five district general hospital histopathology departments and five major departments of cytopathology. Their responses were analysed by five consultant general histopathologists and five consultant specialist cytopathologists. In 54 of the 110 cases, the histology of a corresponding cervical biopsy specimen was compared with the smear assessments. Specialist cytopathologists were more consistent than non-specialists when diagnosing and grading dyskaryosis. They chose the higher grades of dyskaryosis more frequently than the non-specialists. The cytopathologists recommended referral for colposcopy more frequently, but if they asked for a repeat smear, they wanted it done within three months more frequently than the histopathologists. The specialists were more frequently in agreement with the biopsy grade of intra-epithelial neoplasia than the non-specialists, whose smear diagnoses tended to underestimate the severity of the histopathological abnormality. This study has shown major differences between specialist and non-specialist cytopathologists in the diagnosis and grading of cervical smears and in the recommended management of patients with abnormal smears. These differences may result in uneven clinical management of women with smear abnormalities. It is therefore important to explore possible strategies for standardising the reporting of cervical smears, such as centralisation of screening services, accreditation in cytopathology for non-specialist consultants, and the value of participation in external quality assessment schemes.

Cytokeratin expression and acetowhite change in cervical epithelium.

To investigate the distribution of cytokeratins 10, 13, 14 and 19 in biopsy specimens taken from acetowhite and non-acetowhite areas of the cervix. Cervical biopsy specimens were taken from both acetowhite and non-acetowhite areas from 44 patients who presented with abnormal cervical cytology. The specimens were snap frozen in liquid nitrogen and multiple sections taken from each specimen. Staining was performed for cytokeratins 10, 13, 14, 19 and NADPH diaphorase enzyme. The areas of each section positive for the various markers were measured. Cytokeratin 10 positive cells were greatly increased in number in acetowhite biopsy specimens compared with non-acetowhite samples (45.1% v 2.8%; p < 0.0001). Cytokeratin 19 was also increased, but to a lesser extent (17.8% v 5.5%; p < 0.0001). In contrast, the almost universal expression of cytokeratin 13 was reduced in acetowhite biopsy specimens (86.2% v 96.9%; p < 0.0001). Cytokeratin 14 was found diffusely in the basal region of the stratified squamous epithelium and was marginally more apparent in the acetowhite biopsy specimens (p = 0.04). It is suggested that the presence of cytokeratin 10 may be an essential requirement for the formation of acetowhite change in association with the cellular swelling caused by acetic acid.

Detection of human papillomavirus in squamous intraepithelial lesions by consensus and type-specific polymerase chain reaction

Polymerase chain reaction (PCR) was used to identify human papillomavirus (HPV) in 216 cervical biopsy specimens from women referred to the gynecological out-patient unit for colposcopy because of an abnormal smear. HPV DNA was screened using type-specific primers for HPV6, 11, 16, 18, 31 and 33 (TS-PCR) as well as a consensus primer located in the El region of the HPV genome (C-PCR). TS-PCR specificity was validated by Southern blot analysis. Low-grade (SIL 1) and high-grade (SIL 2) squamous intraepithelial lesions were found in 165 biopsies. HPV 16 detection was better with PCR than Southern blot, particularly for SIL 1 and SIL 2. The fact that 10% of HPV 16 (all SIL 2) were not detected by C-PCR indicates that both PCR techniques should be performed. C-PCR also detects uncharacterized HPV types (8.6% prevalence in our results), mainly in SIL 1 and SIL 2. HPV 16, the most frequently isolated type (prevalence 21%), was associated with SIL 2 in 83% of cases. A low HPV prevalence was found in specimens without dysplastic cells. These results suggest that PCR may be an important tool for identifying women at risk for developing dysplasia or cervical cancer.

Proton MR and human cervical neoplasia: ex vivo spectroscopy allows distinction of invasive carcinoma of the cervix from carcinoma in situ and other preinvasive lesions.

The concept that high-resolution (8.5-T) hydrogen-1 magnetic resonance (MR) spectroscopy can be used as an adjunct to conventional histologic diagnosis of cervical neoplasia was investigated. Cervical biopsy specimens (n = 159) were examined with H-1 MR spectroscopy and the results compared with results of histopathologic analysis. A high-resolution lipid MR spectrum was observed in 39 of 40 invasive carcinomas, whereas the 119 preinvasive samples showed little or no lipid spectrum but were characterized by a strong unresolved resonance between 3.8 and 4.2 ppm. Peak ratios of the methylene/methyl and the unresolved/methylene resonances allowed accurate distinction between invasive and preinvasive epithelial malignancy (P < .0001). Since MR spectroscopic examination does not destroy the specimen, the specimens remained intact for further testing and histopathologic analysis. The authors conclude that H-1 MR spectroscopy can independently allow distinction between invasive and preinvasive lesions of the cervix and has the potential to assist in clinical management of cervical cancer.

Detection of human papillomavirus DNA by in situ hybridization and polymerase chain reaction in human papillomavirus equivocal and dysplastic cervical biopsies

One hundred twenty-one paraffin-embedded cervical biopsy specimens were tested for the presence of human papillomavirus (HPV) DNA by in situ hybridization and polymerase chain reaction. By in situ hybridization using probes for HPV types 6 11 , 16 18 , 31/33/35, 42/43/44, 51 52 , and 45 56 , HPV DNA was found in none of 20 normal/squamous metaplasia biopsy specimens, in one of 76 HPV equivocal biopsy specimens, in seven of 12 condyloma/mild dysplasia biopsy specimens, and in 12 of 13 moderate/severe dysplasia biopsy specimens. Polymerase chain reaction using HPV L1 consensus sequence primers followed by filter hybridization of the amplification products was positive for HPV DNA in two of 20 normal/squamous metaplasia biopsy specimens, in 23 of 76 HPV equivocal biopsy specimens, in eight of 12 condyloma/mild dysplasia biopsy specimens, and in 12 of 13 moderate/severe dysplasia biopsy specimens. Among biopsies that tested positive by polymerase chain reaction but that were negative by in situ hybridization, the most commonly identified HPV was type 16. We conclude that although HPV equivocal biopsy specimens contain HPV DNA more frequently than histologically normal tissue, the majority of biopsy specimens in this category test negative for HPV DNA. The clinical significance of a positive test for HPV, in the absence of unequivocal histologic changes, remains to be determined.

Cytomegalovirus infection of the cervix detected by cytology and histology: a report of five cases.

We present four cases of cytomegalovirus (CMV) infection detected by cervical cytology of asymptomatic young women. One patient who had undergone bone marrow transplantation was immunosuppressed, but no factors predisposing to CMV infection were identified in the other three patients. A cervical biopsy specimen from a fifth patient, who was also asymptomatic, demonstrated the locus of CMV infection to be in endocervical gland cells. Immunocytochemical and polymerase chain reaction studies on this biopsy specimen confirmed that the histological changes were caused by CMV. The finding of CMV-infected cells in cervical cytological or biopsy specimens is a rarity. Our observations in asymptomatic, non-immunocompromised women suggest their presence is an incidental finding, unlikely to have any clinical significance.

Detection of human papillomavirus in cervical swab samples by ViraPap and in cervical biopsy specimens by in situ hybridization.

Human papillomavirus (HPV) detection in cervical swab specimens by ViraPap and in cervical biopsies by the ViraType in situ hybridization assay was correlated with concurrent cytologic and histologic findings in 149 patients. Seven percent of patients with normal samples were positive for HPV DNA, compared with 29% and 69% of patients with low- and high-grade squamous intraepithelial lesions (SILs), respectively. The performance of both assays increased the rate of HPV detection in low-grade lesions but not in high-grade cases. ViraPap was more sensitive than the ViraType in situ hybridization assay in high-grade lesions. The present investigation suggests that HPV DNA detection may be useful in separating confirmed lesions from histologically questionable biopsies.

Origin of cervical collagenase during parturition

Cervical biopsy specimens were obtained under standard conditions from the posterior lip of the uterine cervix in 105 patients. A significant increase of collagenase activity was observed during parturition as determined with an assay with iodine 125-labeled native triple-helical collagen type I as the substrate. The collagenase was not likely to originate from cervical fibroblasts because in situ hybridization failed to detect synthesis of the specific procollagenase messenger ribonucleic acid. However, migration of polymorphonuclear leukocytes into the cervical stroma occurred on onset of labor, and an antibody specific for human leukocyte collagenase that did not cross react with fibroblast collagenase revealed the presence of the enzyme in the granules of polymorphonuclear leukocytes and subsequently in the extracellular matrix of the cervix. Therefore it is likely that the cells critically involved in collagen degradation during cervical dilatation are not resident fibroblasts but rather polymorphonuclear leukocytes emigrating from blood vessels.

High prevalence of human papillomavirus transcripts in all grades of cervical intraepithelial glandular neoplasia.

Cervical biopsy specimens containing cervical intraepithelial glandular neoplasia (CIGN) were examined for the presence of human papillomavirus (HPV) RNA transcripts by in situ hybridization with iodine 125-labeled riboprobes. HPV RNA was detectable in 95.2% of biopsy specimens. HPV 16 RNA was present in 12, HPV 18 in 27, and both in 1 of the 42 cases examined. Among HPV-positive cases, HPV RNA was detectable in all grades of CIGN and, in three cases, in glands displaying only minimal nuclear abnormality insufficient for a diagnosis of CIGN. Patients with HPV RNA-positive CIGN were younger than those with negative findings for HPV, and patients with less severe grades of CIGN showed a trend toward a younger age of presentation than patients with severe glandular lesions. Increasing grades of CIGN may reflect progressive stages in the development of cervical adenocarcinoma, and this progression may closely involve HPV gene expression from its earliest stages.

Relationship of Human Papillomavirus Type to Grade of Cervical Intraepithelial Neoplasia

OBJECTIVE--To determine the relationship of human papillomavirus (HPV) type to grade of cervical intraepithelial neoplasia (CIN) in a large series of cases. DESIGN--A survey of HPV types in CIN lesions detected using a new, highly accurate method for typing HPV that is based on restriction fragment length polymorphism analysis of amplimers produced during polymerase chain amplification of the conserved L1 region of HPV using consensus primers. SETTING--Private gynecologists' offices and inner-city colposcopy clinics. PATIENTS--A convenience sample of 276 HPV DNA-positive cervical biopsy specimens or samples from patients undergoing colposcopy for abnormal Papanicolaou smears. INTERVENTION--None. MAIN OUTCOME MEASURE(S)--Human papillomavirus type(s). RESULTS--Cervical intraepithelial neoplasia 1 lesions were relatively heterogeneous with regard to associated HPV types. Nineteen percent of CIN 1 lesions were associated with HPV types 6 or 11; 29% contained HPV types 16, 18, or 33; and 19% were associated with "novel types" of HPV. It was also found that 22% of CIN 1 lesions were associated with more than one HPV type. In contrast to CIN 1, both CIN 2 and CIN 3 were relatively homogeneous with regard to associated HPV types. Eighty-eight percent of CIN 2 and 3 lesions contained HPV types 16, 18, or 33. Unlike CIN 1 lesions, which often contained multiple types of HPV, only 7% of CIN 2 and 3 lesions were associated with multiple HPV types. CONCLUSIONS--Cervical intraepithelial neoplasia should be classified into two separate categories--low-grade and high-grade CIN. Since only 29% of low-grade lesions are associated with HPV types 16, 18, or 33, HPV type could potentially play a role in determining the most appropriate clinical management of patients with low-grade CIN. However, prospective follow-up studies of lesional behavior based on HPV type are required before clinical recommendations can be made.

Origin of cervical collagenase during parturition

Cervical biopsy specimens were obtained under standard conditions from the posterior lip of the uterine cervix in 105 patients. A significant increase of collagenase activity was observed during parturition as determined with an assay with iodine 125-labeled native triple-helical collagen type I as the substrate. The collagenase was not likely to originate from cervical fibroblasts because in situ hybridization failed to detect synthesis of the specific procollagenase messenger ribonucleic acid. However, migration of polymorphonuclear leukocytes into the cervical stroma occurred on onset of labor, and an antibody specific for human leukocyte collagenase that did not cross react with fibroblast collagenase revealed the presence of the enzyme in the granules of polymorphonuclear leukocytes and subsequently in the extracellular matrix of the cervix. Therefore it is likely that the cells critically involved in collagen degradation during cervical dilatation are not resident fibroblasts but rather polymorphonuclear leukocytes emigrating from blood vessels.

Amplification of human papillomavirus type 16 transforming genes from cervical cancer biopsies and lymph nodes of Hungarian patients.

We have used a polymerase chain reaction (PCR) to examine cervical cancer biopsy specimens and pelvic lymph nodes for the presence of human papillomavirus type 16 (HPV 16) DNA. Of the 75 cervical specimens tested, 36 (48%) were positive for HPV 16 in the PCR. A total of 65 pelvic lymph nodes removed during radical surgery on 35 women were also analyzed. Lymph nodes originating from 19 patients whose cervical biopsy specimens were negative for HPV 16 seemed to lack HPV 16 sequences. For 16 women with positive PCR results for cervical biopsy specimens, 9 of 10 lymph node metastases were positive in the PCR, while 11 of their 36 histologically negative lymph nodes were also shown to contain HPV 16 DNA.