Feasibility study of a human papillomavirus E6 oncoprotein test for diagnosis of cervical precancer and cancer

In a feasibility study using a prototype, lateral-flow test for human papillomavirus type 16, 18, and/or 45 (HPV16/18/45) E6 oncoproteins, 51 of 75 (68%; 95% confidence interval [95% CI] of 56 to 78%) of HPV16/18/45 DNA-positive specimens from women with a diagnosis of CIN3+ (cervical intraepithelial neoplasia grade 3+ or cervical cancer) tested positive for HPV16/18/45 E6 oncoprotein. None of 16 (95% CI of 0 to 37%) HPV16/18/45 DNA-positive cervical specimens from women with a negative or CIN1 diagnosis tested positive for HPV16/18/45 E6 oncoprotein.     

Detection of human papillomavirus DNA by the nucleic acid sandwich hybridization method from cervical scraping

Cervical scrapes collected from 100 consecutive patients participating in a prospective follow-up study for cervical human papillomavirus (HPV) infections were tested for the presence of HPV 11 DNA by the nucleic acid sandwich hybridization method, which allows testing the specimens in a crude form. Part of each specimen was processed through phenol extraction and DNA purification to a dot blot hybridization assay. The dot blots were serially hybridized with HPV 6, 11, 16, and 18 probes as well as with an Alu-repeat probe to estimate the number of cells in the specimen. In PAP smears, HPV-infection was suspected in 63 patients whereas in 37 patients the smear was negative. In the first group, the dot blot assay revealed three cases of HPV 11, two of HPV 16, and one of HPV 18 infection. In the second group with normal PAP smear, one additional HPV 18 infection was found. The sandwich hybridization assay detected 5 HPV 11 infections, including the three mentioned above. All HPV DNA-positive samples contained at least 1.6 X 10(6) cells. Since we considered this a prerequisite for successful diagnosis, only 25 specimens in the first group and 15 in the second were adequate specimens. Thus the HPV-DNA detection rate was 32% (8/25) in the first group and 1/15 in the second. This study demonstrates that sandwich hybridization, detecting 1-3 X 10(5) HPV 11 molecules is a reliable diagnostic method. Cervical scrape is a valuable alternative to punch biopsy, but the number of cells collected is critical for the outcome of the assay.     

An ELISA capture assay for the E7 transforming proteins of HPV16 and HPV18.

ELISA capture assays were established for the E7 transforming proteins of HPV16 and HPV18, based on a range of previously characterised polyclonal and monoclonal antibodies. No cross-reactivity was observed in the ELISAs between HPV18 E7 and HPV16 E7. Immunoreactive E7 protein (iE7) was measured in a series of HPV-transformed cell lines, and ranged from 0.6 to 17.7 ng iE7/mg cell protein. iE7 was labile at 22 degrees C (t1/2 = 37 min) but relatively more stable at 4 degrees C (t1/2 = 210 min). HPV16 E7 protein at concentrations from 0.10 to 0.69 ng iE7/mg cell protein was detected in 5 of 13 smears from women with abnormal cervical cytology. Assay of E7 protein may play a role in the detection of HPV-induced cervical lesions with malignant potential.     

Antibodies against human papillomavirus type 16 and 18 E6 and E7 proteins in cervicovaginal washings and serum of patients with cervical neoplasia.

Serum antibodies against the E6 and E7 proteins of human papillomavirus (HPV) 16 and 18 are associated with cervical cancer. The aim of this study was to investigate the presence of local antibodies against HPV in cervicovaginal washings (CWs). In this study antibodies against the native HPV16 and HPV18 E6/E7 proteins were detectable in CWs (48%) and sera (29%) from patients with cervical cancer (n = 21) utilizing a sandwich protein enzyme-linked immunosorbent assay (ELISA). In paired CWs and sera from patients with cervical intraepithelial neoplasia (n = 38) and from healthy women (n = 22) no antibodies against these proteins were found. In 10 of 11 patients, the antibody response corresponded with the HPV type in the cervical smear and/or tumor tissue, which indicates the HPV type specificity of the assay. In 7 of 11 patients with antibody reactivity against HPV16 or HPV18 E6 and/or E7 proteins a higher level of antibody reactivity in the CWs than in the paired serum samples was found at similar inputs of total IgG. This suggests that the antibodies in the CWs against the investigated HPV proteins in these patients were locally produced.     

Antibodies against Early Proteins of Human Papillomaviruses as Diagnostic Markers for Invasive Cervical Cancer

Cervical cancer is the most prevalent tumor in developing countries and the second most frequent cancer among females worldwide. Specific human papillomaviruses (HPVs) and, most notably, HPV types 16 and 18 are recognized as being causally associated with this malignancy. Antibodies against early HPV proteins E6 and E7 have been found more often in patients with tumors than in controls. Existing peptide enzyme-linked immunosorbent assays (ELISAs) for the detection of anti-E6 and anti-E7 antibodies in human sera have low levels of sensitivity and specificity and thus are not suitable for use as diagnostic tools. Based on highly purified recombinant native proteins, we developed four sandwich ELISAs for the detection of antibodies against HPV type 16 and 18 E6 and E7 proteins. We demonstrate their sensitivities and high degrees of specificity for cervical cancer. Among a total of 501 serum specimens from unselected patients with invasive cervical cancer, 52.9% reacted positively in at least one of the four assays. In contrast, among 244 serum specimens from control subjects without cervical cancer, only 2 reactive serum specimens (0.8%) were found. For 19 of 19 antibody-positive patients, the HPV type indicated by seroreactivity was identical to the HPV DNA type found in the tumor, which also indicates a high degree of specificity for antibody detection with respect to HPV type. In a direct comparison of 72 serum specimens from patients with cervical cancer, 56% of the specimens reacted in at least one of the four protein ELISAs, whereas 40% reacted in at least one of seven peptide ELISAs covering the four antigens. These assays could be of value for the detection of invasive cervical cancer in settings in which cytology-based early tumor screening is not available, for the clinical management of patients diagnosed with cervical cancer, and for the immunological monitoring of E6 and E7 vaccination trials.     

Human papillomavirus is a necessary cause of invasive cervical cancer worldwide

A recent report that 93 per cent of invasive cervical cancers worldwide contain human papillomavirus (HPV) may be an underestimate, due to sample inadequacy or integration events affecting the HPV L1 gene, which is the target of the polymerase chain reaction (PCR)-based test which was used. The formerly HPV-negative cases from this study have therefore been reanalysed for HPV serum antibodies and HPV DNA. Serology for HPV 16 VLPs, E6, and E7 antibodies was performed on 49 of the 66 cases which were HPV-negative and a sample of 48 of the 866 cases which were HPV-positive in the original study. Moreover, 55 of the 66 formerly HPV-negative biopsies were also reanalysed by a sandwich procedure in which the outer sections in a series of sections are used for histological review, while the inner sections are assayed by three different HPV PCR assays targeting different open reading frames (ORFs). No significant difference was found in serology for HPV 16 proteins between the cases that were originally HPV PCR-negative and -positive. Type-specific E7 PCR for 14 high-risk HPV types detected HPV DNA in 38 (69 per cent) of the 55 originally HPV-negative and amplifiable specimens. The HPV types detected were 16, 18, 31, 33, 39, 45, 52, and 58. Two (4 per cent) additional cases were only HPV DNA-positive by E1 and/or L1 consensus PCR. Histological analysis of the 55 specimens revealed that 21 were qualitatively inadequate. Only two of the 34 adequate samples were HPV-negative on all PCR tests, as against 13 of the 21 that were inadequate ( p< 0·001). Combining the data from this and the previous study and excluding inadequate specimens, the worldwide HPV prevalence in cervical carcinomas is 99·7 per cent. The presence of HPV in virtually all cervical cancers implies the highest worldwide attributable fraction so far reported for a specific cause of any major human cancer. The extreme rarity of HPV-negative cancers reinforces the rationale for HPV testing in addition to, or even instead of, cervical cytology in routine cervical screening. Copyright © 1999 John Wiley & Sons, Ltd.     

Purification and characterisation of the E7 oncoproteins of the high-risk human papillomavirus types 16 and 18

E7 proteins are major oncoproteins of human papillomaviruses (HPVs) which play a key role in virus-associated cervical carcinogenesis. The E7 oncoprotein of HPV-16 has been shown to interact with a variety of cellular target proteins and these interactions are considered essential for the transforming properties of this oncoprotein. Several additional HPV types associated etiologically to cervical cancer have been described, the second most common being HPV-18. Less is known about the biochemical functions and interactions of HPV-18 E7. As a first step to determine biochemical properties common to the E7 proteins of the high-risk HPV types 16 and 18 these E7 proteins were expressed in bacteria and purified to homogeneity. Purified E7 proteins were used to investigate the in vitro interaction with the pocket protein p107 and insulin-like growth factor-binding protein-3 (IGFBP-3) that are known to interact with the amino-terminal and the carboxyl-terminal part of IGFBP-3, respectively. Both purified E7 proteins interacted strongly with p107 and, as demonstrated here for the first time, HPV-18 E7 was capable of binding to IGFBP-3, albeit to a lesser extent than HPV-16 E7. These findings suggest that the purified recombinant E7 proteins retain, at least in part, their biochemical activities.     

Antibody response for L1, E6 and E7 HPV 16 and HPV 18 antigens in Tunisian women with cervical cancer and controls

Results obtained in the present work indicated that the Luminex assay is more sensitive than ELISA. The reactivity to the early antigens E6 and E7 was 37% versus 42% for HPV 16 and 21% versus 20% for HPV 18 among cervical cancer cases using ELISA. However, these ratios were 44% and 61%, respectively, for E6 and E7 HPV 16 versus 28% and 21% for E6 and E7 HPV 18 when using the Luminex technique. Data also indicated that HPV 16 and HPV 18 showed distinct profiles for the different antigens tested. Finally, the differences in antibody responses between cervical cancer cases and benign cases toward the different antigens were significant.     

Detection of human papillomavirus in matched cervical smears and biopsy specimens by non-isotopic in situ hybridisation.

AIMS: To determine the relative diagnostic sensitivity of non-isotopic in situ hybridisation (NISH) for the diagnosis of human papillomavirus (HPV) on matched smears and biopsy specimens; to compare the NISH signal type in the two samples; and to correlate the NISH data with the morphological diagnosis. METHODS: HPV samples were assayed individually by NISH with digoxigenin labelled probes (HPV6, 11, 16, 18, and 33) on routinely collected paraffin wax embedded cervical biopsy specimens and for high risk HPVs with a cocktail of similarly labelled probes (HPV16, 18, 33) on matched smears. These were taken at the same colposcopic examination from 32 patients investigated for an abnormal cervical Papanicolaou (PAP) stained smear. RESULTS: An HPV signal was present in 18 (56%) biopsy specimens and in 14 (44%) smears. There was higher concordance of sets of data in the presence of cytopathic wart virus changes. The superiority of biopsy over smear in detecting HPV was mainly the result of examining the entire cervical biopsy specimen rather than cells scraped from the cervical surface. The NISH signal type in both biopsy specimen and smear was similar; it has been shown that NISH type 1 signal correlates with episomal viral replication and type 2 and 3 signals with viral integration. CONCLUSIONS: These data show that NISH on cervical smears is a worthwhile primary screen for HPV infection. The NISH signal types in cervical smears are similar to those previously described in cervical biopsy specimens.     

Independent association of antibodies against human papillomavirus type 16 and E7 proteins with cervical cancer

The E4 open reading frame (ORF) of human papillomaviruses (HPVs) is transcribed in abundant mRNAs encoding an fusion gene during the productive infection, and the HPV 16 E7 ORF encodes an oncoprotein detectable in the cell lines derived from cervical carcinoma. We examined 421 human sera, which included 108 samples from the patients with cervical carcinoma, for the presence of IgG antibodies against the HPV 16 E4 and E7 proteins by enzyme-linked immunosorbent assay. Bacterially expressed fusion protein lac- and nonfusion protein E7 were purified and used as antigens. All of the 22 serum samples positive for anti-E7 antibody and the 11 out of 15 samples positive for anti- antibody were from the patients with cervical carcinoma, but only one sample was found to contain both anti- and anti-E7 antibodies. These findings show specific and independent association of these antibodies with cervical carcinoma.     

Colposcopy, punch biopsy, in situ DNA hybridization, and the polymerase chain reaction in searching for genital human papillomavirus (HPV) infections in women with normal PAP smears

To assess the prevalence of HPV infection in the genital tracts of women with normal PAP smears, a random series of 109 women was reexamined using colposcopy, a further PAP smear, and punch biopsies taken from the cervix (in 33 cases), vagina (212 cases), and anus (20 cases). The biopsy material was examined using routine histological investigations, in situ hybridization (ISH) with a 35S-labelled DNA probe cocktail (HPV 6, 11, 16, 18), and the polymerase chain reaction (PCR) to detect HPV DNA. Changes consistent with HPV infection were seen in 6.9% (18/262) of the biopsy specimens. Seven biopsy specimens (2.7%) from seven different women were found to contain HPV DNA using ISH. All of these ISH-positive lesions were diagnosed as morphologically characteristic HPV lesions: six flat condylomas and one papillary condyloma. Using PCR, the HPV DNA detection rate was highest in the cervical biopsy specimens (50%) and lowest (28.6%) in the anal biopsy specimens. A total of 35.5% of the 93 biopsy specimens studied using PCR contained HPV DNA. The commonest type was HPV 11 (54.5%), followed by HPV 18 (33.3%). Four of the nine biopsy specimens (44.4%) from colposcopically normal areas proved HPV DNA-positive using PCR. Of 17 biopsy specimens in which the histology was normal, seven were examined using PCR and three were DNA-positive. The discovery of HPV DNA using PCR in 32/92 of the biopsy specimens (34.8%) which had been found to be HPV DNA-negative when routine ISH was used is noteworthy. The results suggest that the light microscopy criteria currently used in diagnosing HPV infections are of no value in predicting latent HPV infections and that acetowhite staining is unable to distinguish between subclinical and latent infections on the one hand and changes unrelated to HPV on the other.     

Detection of HPV in Norwegian cervical biopsy specimens with type-specific PCR and reverse line blot assays.

BACKGROUND: Although the association of HPV16 and HPV18 DNA with cervical cancers has been well studied, the prevalence of these types in high-grade cervical intraepithelial neoplasias (CIN2/3) may differ from the prevalence found in cervical cancer specimens. OBJECTIVE: To determine the prevalence of specific HPV types found in high-grade CIN2/3 biopsy samples. STUDY DESIGN: One thousand eight hundred and forty-eight cervical biopsy specimens were obtained from Norwegian women. HPV16 and HPV18 type and gene-specific PCR assays were performed amplifying a portion of the E6, E7 and L1 genes. In addition, the reverse line blot assay was performed on a subset of specimens, in which a portion of L1 was amplified and hybridized to strips coated with complimentary HPV sequences. RESULTS: The prevalences of HPV16 and HPV18 in the 1848 biopsy cohort were 32.3% and 6.0%, respectively. HPV16 was detected in 47.5% and HPV18 in 5.9% of diagnosed CIN2/3 specimens. Approximately 12% of the CIN2/3 specimens contained two HPV types and 2.5% contained three HPV types. CONCLUSIONS: The prevalence of HPV16 increases from grades CIN1 to CIN2 to CIN3. The prevalence of HPV18 did not change significantly with increasing CIN grade. The majority of infections diagnosed as CIN2/3 contained a single HPV type. The type-specific PCR assay had increased detection sensitivity over the reverse line blot assay.     

Independent association of antibodies against human papillomavirus type 16 E1/E4 and E7 proteins with cervical cancer.

The E4 open reading frame (ORF) of human papillomaviruses (HPVs) is transcribed in abundant mRNAs encoding an E1/E4 fusion gene during the productive infection, and the HPV 16 E7 ORF encodes an oncoprotein detectable in the cell lines derived from cervical carcinoma. We examined 421 human sera, which included 108 samples from the patients with cervical carcinoma, for the presence of IgG antibodies against the HPV 16 E4 and E7 proteins by enzyme-linked immunosorbent assay. Bacterially expressed fusion protein lac-E1/E4 and nonfusion protein E7 were purified and used as antigens. All of the 22 serum samples positive for anti-E7 antibody and the 11 out of 15 samples positive for anti-E1/E4 antibody were from the patients with cervical carcinoma, but only one sample was found to contain both anti-E1/E4 and anti-E7 antibodies. These findings show specific and independent association of these antibodies with cervical carcinoma.     

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

AIM--To assess the value of detecting human papillomavirus (HPV) DNA in false negative archival cervical smears in population based screening programmes for cervical cancer. METHODS--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. RESULTS--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. CONCLUSIONS--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.     

Nucleic acid sandwich hybridization in detection of HPV 16 DNA: technique and its clinical application

Nucleic acid sandwich hybridization technique was used for detection of HPV 16 specific DNA in cervical scrapes. Alternating HPV 16-PstI fragments were cloned into plasmid pBR322 and phage M13mp10. pBR322-clones were used as 32P-labelled probe reagents and the M13mp10 clones as catching reagents in the assay. The detection limit of the test proved to be 1-5 X 10(5) HPV 16 molecules per test. A weak cross reaction was seen with HPV 31 DNA but not with the other types tested, e.g. HPV 6, 11, 18 and 33. Cervical scrapings obtained from 163 consecutive patients included in a prospective follow up study were analyzed for HPV 16 DNA with the sandwich hybridization method, dot-blot hybridization being used as a reference method. Sandwich hybridization assay detected 25 positive cases out of 163 specimens (15.3%). Of these 6 and 3 additional specimens were positive in dot-blot hybridization assay. HPV 16 DNA was related to higher PAP grades, and HPV 16 appeared more frequently in HPV CIN than HPV NCIN lesions. None of the infections caused by HPV 16 regressed, and 24% progressed during the follow up.     

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. © 1996 Wiley-Liss, Inc.     

HPV 16 E7 antibody levels in cervical cancer patients: Before and after treatment

Antibody response to HPV16 E7 oncoprotein may represent a marker of cervical cancer. A HPV16 GST-E7 fusion protein was used in a Western Blot assay to analyse the HPV16 E7 antibody response in 30 patients before and after treatment for cervical carcinoma (stage IIB or IIIB). Patients were treated with three courses of cisplatin/bleomycin therapy followed by surgery, or with surgery alone. Thirteen out of 30 patients had serum antibodies to HPV16 E7 antigen. Three months after chemotherapy little or no change in antibody titre was detected. In contrast, after surgery, a significant decrease in antibody titre was observed in 9/10 patients. In two cases the titre declined to zero 3 and 9 months after treatment, respectively. These results confirm the usefulness of studying anti-HPV16 E7 antibody profile in cervical cancer patients and suggest that the serum response correlates with tumour burden. J. Med. Virol. 54:192–195, 1998. © 1998 Wiley-Liss, Inc.     

Detection of HPV DNA in cervical carcinomas by PCR and hybrid capture assay

HPV DNA was detected in exfoliated cervical cells of 73% (85/116) cervical cancer patients by PCR using HPV consensus primers and by hybrid capture assay (HC II) (Digene Corp., USA) in 77 of the 85 cases found HPV positive by PCR. Presence of HPV 16/18 DNA were investigated in the 79 cases by PCR using type specific primers. HPV 16 was detected in 31 (39%) patients, HPV 18 in 7 (8.8%), both HPV 16 and 18 in 19 (24%) and HPVs other than 16/18 in 22 (27.8%) cases. Age and clinical stages had no significant effect on HPV prevalence. Double infection of HPV 16 and 18 was significantly (p<0.05) high in the older patients (56 years or more) compared to younger group. Results indicated that cervical cancers in India are strongly associated with high-risk type HPV infection. HC II assays and PCR results for detection of HPV in cervical smears were comparable.     

Subcellular localization of the human papillomavirus 16 E7 oncoprotein in CaSki cells and its detection in cervical adenocarcinoma and adenocarcinoma in situ

E7 is the major oncoprotein of high-risk human papillomaviruses (HPV) which causes cervical cancer. To date E7 oncoproteins have not been investigated in cervical adenocarcinoma. In this study we generated a rabbit monoclonal anti-HPV-16 E7 antibody, RabMab42-3, which recognizes a conformational epitope in the E7 carboxy-terminal zinc-finger resulting in a strong increase in the sensitivity for the detection of cell-associated HPV-16 E7 protein relative to conventional polyclonal anti-HPV-16 E7 antibodies. Using RabMab42-3, we show that the subcellular localization of endogenous HPV-16 E7 oncoprotein varies during the cell cycle in cervical cancer cells. Moreover, we demonstrate for the first time that the HPV-16 E7 oncoprotein is abundantly expressed in cervical adenocarcinoma in situ and adenocarcinoma, suggesting an important role of HPV-16 E7 for the development of these tumors. Our findings suggest that the HPV-16 E7 oncoprotein could be a useful marker for the detection of cervical adenocarcinoma and their precursors.