E7 Oncogene Transcripts in Dysplastic and Nondysplastic Cervical Scrapes by Nested RT-PCR

GYNECOLOGIC ONCOLOGY ARTICLE NO.

69, 114 –121 (1998)

GO984994

Detection of Human Papillomavirus Type 16 E6/E7 Oncogene Transcripts in Dysplastic and Nondysplastic Cervical Scrapes by Nested RT-PCR Karl Sotlar,*,1 Hans-Christoph Selinka,* Michael Menton,† Reinhard Kandolf,* and Burkhard Bu¨ltmann* *Institute for Pathology and †Department of Obstetrics and Gynecology, University of Tu¨bingen, Tu¨bingen, Germany Received July 23, 1997

HPV-31 and -33) and ‘‘low risk’’ types (e.g., HPV-6 and -11) [5]. The oncogenic potential of high-risk HPV infections, as proven in transformation studies, is due to the viral transforming genes E6 and E7 [6]. The viral oncoprotein E6 initiates degradation of the cellular tumor suppressor protein p53 [7], whereas oncoprotein E7 leads to inactivation of another cellular tumor suppressor protein, the retinoblastoma gene product pRB [8]. These synergistic effects have been shown to be important steps in carcinogenesis, leading to a loss of cellcycle control [9, 10]. Transcriptional activity of both viral oncogenes, E6 and E7, is under control of a common promoter (p97), which is mainly suppressed by the viral E2 gene product [11]. In SCC, HPV persistence is achieved by integration of the viral DNA into the host genome. Usually, integration disrupts the viral DNA within the E1/E2 open reading frames (ORF) [12]. As a result of integration, control of the viral E6/E7 oncogene transcription is lost [13–15]. In contrast, oncogene transcription is absent or efficiently down-regulated in premalignant lesions and HPV-infected normal epithelium, in which papillomaviruses predominate as episomes [16, 17]. Transcription of the E6/E7 ORFs gives rise to three different splice products, due to alternative splicing, using a common splice donor site at nt 226 and two different splice acceptor sites at nt 409 and 526. The full-length transcript encodes the functional E6 protein, whereas the E7 protein is most likely encoded by the E6*I and E6*II splice products [18 –20]. Of these two, E6*I has been shown to be the major E6/E7 splice product [19]. Using reverse transcription-polymerase chain reaction (RT-PCR), Cornelissen et al. [20] demonstrated a uniform splicing pattern in SCC, SCC-derived cell lines, and CIN. Today, HPV screening programs are mainly based on PCR detection of HPV DNA, using consensus- and type-specific primers [21, 22]. However, the prevalence of HPV infections, especially in the young and sexually active population, is high compared to the number of women who will ever develop cervical cancer [12]. Thus, detection of E6/E7 oncogene transcripts of high-risk HPV types might serve as an additional risk evaluation factor for the development of CIN and for the

Infections with high-risk human papillomaviruses (e.g., HPV16) play an important role in the development of cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (IC). Continued expression of the viral E6 and E7 genes is believed to be a key factor for oncogenic transformation of infected cells. Two spliced transcripts of the E6/E7 oncogenes, termed E6*I and E6*II, can be detected by reverse transcriptase polymerase chain reaction (RT-PCR). To increase the sensitivity of detecting E6/E7 transcripts in cervical scrapes we took advantage of a nested RT-PCR (nRT-PCR) protocol. In a series of 30 HPV-16-positive patients with histologic diagnoses ranging from nondysplastic epithelium to IC, the application of nRT-PCR significantly improved the detection of E6/E7 transcripts compared to conventional RTPCR. The prevalence of E6/E7 spliced transcripts correlated with lesion severity and the nRT-PCR protocol allowed detection of these transcripts even in nondysplastic epithelium and CIN I lesions. Therefore, in epidemiologic follow-up studies, detection of E6/E7 transcripts by nRT-PCR should prove to be a useful diagnostic tool for risk evaluations regarding the development of CIN and its progression to cervical cancer, especially in high-risk HPVtype-infected patients with CIN 0 and CIN I. © 1998 Academic Press

INTRODUCTION Cervical squamous cell carcinoma (SCC) develops on welldefined precursor lesions, termed cervical intraepithelial neoplasias (CIN). The etiologic role of human papillomavirus (HPV) infections in the development of both SCC and CIN is meanwhile generally accepted [1–3]. Today, more than 70 different HPV types have been characterized. About 25 HPV types preferentially infect the anogenital mucosa, causing a broad spectrum of lesions ranging from benign condyloma to SCC [4]. The association of certain HPV types with benign and malignant lesions has led to the definition of ‘‘high-risk’’ types (e.g., HPV-16 and -18), ‘‘intermediate-risk’’ types (e.g., 1

To whom correspondence and reprint requests should be addressed at Institut fu¨r Pathologie, Universita¨t Tu¨bingen, Liebermeisterstraße 8, D-72076 Tu¨bingen, Germany. Fax: (49) 7071 29 2258. E-mail: [email protected]. 0090-8258/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

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progression from CIN to cervical cancer, respectively. Former studies, dealing with the detection of E6/E7 oncogene transcripts in cervical scrapes, suffered from two major problems: either a lack in sensitivity has prevented the detection of HPV transcripts in nondysplastic or moderately dysplastic cervical epithelium [16], or laborious techniques with the use of radioactivity for primer labeling [23] have made them inconvenient for large scale epidemiologic follow-up studies. Therefore, we have established a nested RT-PCR (nRTPCR) assay which facilitates detection of HPV-16 E6/E7 oncogene transcripts in cervical scrapes from low-grade and nondysplastic epithelium. In clinical practice, these are the cases that may be followed up over quite some time without placing the patients at an increased risk of developing cervical cancer. MATERIALS AND METHODS Patients and Sample Preparation Between February and August 1996, 122 patients were referred to the colposcopy clinic at the Department of Gynecology, University of Tu¨bingen, Germany, because of atypical Papanicolaou smears. All patients underwent colposcopy and repeated cytologic examination. In all cases, two additional scrapes were taken for HPV DNA and mRNA detection, respectively. In cases where colposcopic findings were suspicious for the presence of CIN, colposcopically directed biopsies were taken for histopathologic examination (n 5 110). Swabs assigned for HPV DNA detection were transferred into a tube containing 400 ml proteinase K– buffer, or, if used for HPV-E6/E7 mRNA detection, transferred into a tube containing 400 ml Solution D (4 M guanidinium thiocyanate; 25 mM sodium citrate, pH 7; 0.5% N-lauryl sarcosyl; 0.1% 2-mercaptoethanol). All tubes were snap-frozen in liquid nitrogen and stored at 270°C until further processing. Human liver tissue was used as HPV-negative control. The human cervical carcinoma cell line CaSki (CRL 1550, American Tissue Culture Collection), carrying transcriptionally active HPV-16 genomes, served as positive control. PCR Primer Design For type-specific detection of HPV-16 genomic DNA, as well as for the detection of E6/E7 oncogene transcripts, PCR primers were designed to enclose splice sites within E6/E7, previously described by Cornelissen et al. [20]. The first PCR amplification of both DNA and cDNA was performed with primer pair S3/S4 (20 pmol each), specific for nt 142–161 and nt 666 – 647 of HPV-16 [24]. Oligonucleotides S1 (nt 192–211) and S2 (nt 586 –567) [25] were used as nested PCR primers. Figure 1A illustrates the primer positions relative to the E6 and E7 ORF and the expected sizes of amplification products of full-length transcripts and splice products.

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DNA Extraction Tubes containing cervical swabs in 400 ml of proteinase K– buffer were vortexed and swabs were removed from the tubes. Total DNA was extracted from exfoliated cells by proteinase K digestion and phenol/chloroform/isoamyl alcohol (25:24:1 v/v/v) extraction. The extracted DNA was resuspended in 30 ml of distilled water and concentrations were determined in a spectrophotometer at 280 nm. RNA Extraction and Reverse Transcription RNA was extracted from cell lines and tissue samples according to the protocol described by Chomczynski and Sacchi [26], with minor modifications. The tubes containing cervical swabs and solution D were thawed on ice. After vortexing for 1 min, swabs were removed. Sequentially, 40 ml of 2 M NaAc, 400 ml of water-saturated ultrapure phenol (pH 4.5), and 80 ml of chloroform/isoamyl alcohol (24:1 v/v) were added. The mixture was vortexed and cooled on ice for 15 min. After 20 min centrifugation (10,000g) at 4°C, the aqueous phase was transferred into a tube containing 800 ml of cold isopropanol (4°C). The remaining phenol phase was washed with 200 ml RNase-free water, aqueous phases were combined, and the samples were stored at 220°C for at least 2 h to precipitate the RNA. After a 30-min centrifugation (10,000g) at 4°C the pellet was washed in 75% ethanol, vacuum dried, and resuspended in 30 ml of RNase-free water. Total RNA of CaSki cells was extracted by the same procedure. RNA concentrations were determined in a spectrophotometer at 260 nm. Reverse transcription of about 2 mg of total RNA was carried out in a final volume of 20 ml using avian myeloblastosis virus reverse transcriptase (AGS, Heidelberg, FRG) and the HPV-16-specific primer S4 (nts 666 – 647). The mixture was incubated for 5 min at 65°C, 1 h at 42°C, and 5 min at 95°C and immediately cooled on ice. Polymerase Chain Reaction For PCR amplification 200 ng of extracted total DNA or cDNA was used as template. All PCRs were performed in a final volume of 50 ml under identical conditions on a Perkin– Elmer GeneAmp PCR System 9600. Briefly, the first PCR amplification was performed with primer pair S3/S4 (20 pmol each), using 1 U of AmpliTaq DNA polymerase (Perkin– Elmer). The PCR mixture was subjected to 30 cycles of amplification consisting of denaturation at 94°C for 1 min, annealing at 54°C for 45 s, and elongation at 72°C for 1 min. The first cycle was preceded by 3 min of denaturation at 94°C and the last cycle was followed by a 10-min incubation at 72°C. For nested PCR as well as nested RT-PCR, 2 ml of the first PCR reaction mixture was used. Oligonucleotides S1 and S2 (20 pmol each) [25] were used as nested PCR primers. The integrity of extracted mRNA was tested by amplification of a 248-bp splice product of the human glyceraldehyde-3-phos-

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phate dehydrogenase gene (GAPDH) using primers specific to nt 3932–3949 and nt 4355– 4372 of the GAPDH DNA [27]. Amplification products were analyzed by electrophoresis on 2% agarose gels and stained with ethidium bromide. The specificity of PCR products from CaSki cells and several patient samples was verified by dye deoxy terminator cycle sequencing on a 373 DNA Sequencer (ABI, Weiterstadt, FRG) according to the manufacturer’s instructions. Southern Blot Analysis For Southern blot hybridization, agarose gels were denatured for 30 min in 1.5 M NaCl/0.5 N NaOH, washed in distilled water, and neutralized for 15 min by incubation in 1 M Tris/1.5 M NaCl, pH 7.4. DNA was transferred from agarose gels to a nylon filter (Hybond-N1, Amersham) by overnight diffusion blotting in 103 SSC– buffer. After DNA transfer, filters were washed in 53 SSC for 5 min and DNA was cross-linked to the filters using a UV-Stratalinker 1800 (Stratagene) at 1200 mJ/cm2. Subsequently, filters were prehybridized at 60°C for 1 h in 20 ml hybridization buffer consisting of 0.4% SDS, 63 SSC, 100 mg/ml hering-sperm-DNA, RNasefree water, and 53 Denhardt’s solution (503 Denhardt’s solution: 5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin in 500 ml water). Hybridization was performed overnight at 60°C using an oligonucleotide corresponding to nt 531–565 of the HPV-16 E6/E7 oncogene sequence [24]. The oligonucleotide was labeled with 32P-dCTP to a specific activity of 4 3 108 2 1 3 109 cpm/mg. Posthybridization, filters were washed twice in 23 SSC/0.5% SDS at 42°C and twice in 13 SSC/1% SDS at 52°C for 30 min. Autoradiography was performed at 270°C for 3 and 25 days, respectively. RESULTS PCR Amplification of HPV-16 E6/E7 Oncogene DNA and mRNA in CaSki Cells To monitor the presence of HPV-16 on DNA and mRNA levels, PCR primers were used for amplification of the E6/E7 viral oncogenes (Fig. 1A). Qualitative evaluation of these primers for their application in PCR and nPCR assays was performed with CaSki DNA and cDNA, respectively (Fig. 1B, left). As PCR template, 100 ng of CaSki DNA or cDNA, respectively, was used. PCR with CaSki DNA as template resulted in the amplification of products of 525 bp (first PCR) and 395 bp (nPCR). Amplification of CaSki cDNA yielded PCR products of 525 bp (E6/E7 full-length), 343 bp (E6*I), and 225 bp (E6*II) in the first PCR, and products of 395 bp (E6/E7 full-length), 213 bp (E6*I), and 95 bp (E6*II) for the nested PCR assay. These results demonstrate the feasibility of the S3/S4 and S1/S2 primer combinations to detect PCR products corresponding to full-length and spliced transcripts (E6*I and E6*II) of HPV-16 E6/E7 oncogenes in CaSki cells. Subsequent to PCR amplification, Southern blot hybridization,

FIG. 1. (A) Schematic overview of the HPV-16 E6 and E7 oncogenes (open bars) and their transcripts (solid bars). Positions and 59 to 39 orientations of PCR primers (S1-S4) are indicated by arrows. (B) Amplification of HPV-16 E6/E7 DNA and cDNA from CaSki cells by PCR and nested PCR (left). Southern blot hybridization using an internal oligonucleotide probe (nt 531– 565 of HPV-16) verified the specificity of amplified PCR products (right). Size markers of HaeIII-digested phage FX174 DNA are indicated by M.

using a HPV-16 E6/E7-specific, 32P-labeled internal probe, was performed to verify the specificity of amplified PCR products (Fig. 1B, right). Improved Sensitivity of HPV-16 DNA Detection in Cervical Scrapes by Nested PCR Conventional PCR with primer pair S3/S4 yielded products of 525 bp in cervical scrapes from 23 of the 122 patients (19%) tested in this study, 17 of which are shown in Fig. 2A (lanes 1–17). Subsequent application of the nPCR protocol with primer pair S1/S2 resulted in amplification of the characteristic 395-bp product in 30 cases. Twenty-three of these cases were already positive in the conventional PCR assay and 7 additional cases were positive by the nPCR assay. Thus, nPCR confirmed the specificity of the PCR results and led to a 30% (23/122 vs 30/122) increase of sensitivity in the detection of HPV-16 DNA (Fig. 2B). Histopathologic Diagnoses Of 110 cases in which histologic diagnoses were available, 52% (n 5 57) had no evident cervical dysplasia (CIN 0), 19% (n 5 21) each had CIN I and CIN II, respectively, 6% (n 5 7)

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fication product of the E6*I major transcript is present in lanes 10, 12, and 16. Subsequent Southern blot hybridization of the amplified RT-PCR products with an exposure time of 3 days did not increase the sensitivity of HPV-16 E6/E7 mRNA detection (Fig. 3B). A marked increase in sensitivity, however, was achieved after 25 days of exposure (Fig. 3C). Of the 17 cases shown, the E6*I major splice product could be detected in 3 additional cases (lanes 2, 4, and 14), with a total of 8 cases (Table 1, cases 2, 4, 10, 12, 14, 16, 24, and 29). The E6*II minor splice product, undetectable by gel electrophoresis and Southern blot hybridization with short time exposure, was detectable after an exposure time of 25 days in 4 cases (lanes 4, 10, 12, and 16). By the use of nRT-PCR, amplification products of the E6*I and E6*II spliced transcripts were detectable by simple gel electrophoresis in 22 (73%) of the 30 HPV-16 DNA-positive cases, as demonstrated on 17 patients in Fig. 4A (lanes 1–17). Subsequent Southern blot hybridization of the nRT-PCR prod-

FIG. 2. PCR amplification of HPV-16 DNA in clinical samples. (A) Use of primer pair S3/S4 resulted in a 525-bp product in cervical scrapes of patients 2– 6, 9 –13, and 17. (B) Nested DNA PCR with use of primer pairs S3/S4 (outer primers) and S1/S2 (inner primers) markedly increased the sensitivity of HPV-16 E6/E7 DNA detection, represented by a 395-bp amplification product in all 17 patients. DNA of human liver tissue and water served as negative controls and CaSki DNA as HPV-16 positive control in both PCR assays. M indicates size markers of HaeIII-digested phage FX174 DNA.

TABLE 1 Comparison of Histologic Diagnoses and Nested RT-PCR Results in HPV-16 DNA-Positive Patients Case No.

Histologya

E6/E7b

E6*Ib

E6*IIb

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

CIN 0 CIN II CIN I CIN II CIN I CIN I CIN I CIN 0 CIN II CIN III IC (adeno) CIN II CIN II CIN III CIN II IC (squa) CIN II CIN II CIN II CIN I CIN 0 CIN II CIN 0 IC (squa) CIN I CIN 0 CIN I CIN II CIN III CIN 0

2 1 2 2 1 1 1 2 2 1 2 1 2 2 1 2 1 1 2 2 2 2 2 2 1 2 1 1 2 2

1 1 2 1 1 1 2 2 1 1 1 1 2 1 2 1 1 1 1 2 2 1 2 1 1 1 1 1 1 1

1 2 2 1 1 2 2 2 2 1 1 1 2 1 2 1 2 2 1 2 2 1 2 1 1 2 1 2 1 1

were diagnosed as CIN III, and 4% (n 5 4) displayed invasive cancer (IC). Of these 110 patients, 80% (n 5 88) were found to be HPV-DNA-positive, using a nested PCR assay with L1 consensus primers, suitable for the detection of HPV-6, -11, -16, -18, -31, -33, -35, -42, -43, -44, -45, -52, -56, -58, and others [22, 28, data not shown]. Correlation of overall HPV detection with histology revealed the presence of HPV infections in 100% (4/4) of IC, in 100% (7/7) of CIN III, in 95% (20/21) of CIN II, in 90% of CIN I (19/21), and in 67% (38/57) of CIN 0. Results of the histopathologic examinations of 30 HPV-16 DNA-positive cases are shown in Table 1. These results included the diagnoses of IC in 3 cases, 2 of which were of squamous and 1 of adenopapillary differentiation. Moreover, 3 cases were classified as CIN III and in 11 cases the diagnosis of CIN II was established. Seven biopsies were classified as CIN I and 6 cases displayed nondysplastic epithelium (CIN 0). Detection of HPV-16 E6/E7 Oncogene Transcripts Using RT-PCR, the major E6/E7 transcript, E6*I, could be detected in 5 of the 30 (17%) cases of this study, which were HPV-16 DNA-positive by nPCR. In Fig. 3A, 17 of these 30 cases (lanes 1–17) are shown as examples. The 343-bp ampli-

a

CIN, cervical intraepithelial neoplasia; CIN 0, no dysplasia; IC, invasive carcinoma; adeno, adenopapillary, squa, squamous. b E6/E7, full-length transcript or genomic HPV DNA; E6*I, E6*II, spliced transcripts.

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plified from cDNA of the E6/E7 full-length transcript or from E6/E7 genomic DNA. Amplification of extracted total RNA, without preceding RT reaction, again yielded the 395-bp product in all 12 cases, whereas spliced transcripts (E6*I, E6*II) were not detected. Thus, in our study the 395-bp product, as obtained by nRT-PCR, mainly seemed to have originated from HPV-16 genomic DNA than from the E6/E7 full-length transcript. In the remaining 18 of the 30 HPV-16 DNA-positive cases of this study, no 395-bp product was detected (Table 1), presumably due to the specificity of the Chomczynski protocol [26] for the extraction of cellular RNA but not genomic DNA. Correlation of Histologic Findings and PCR Results Histologic findings and PCR results of the 30 HPV-16 DNA-positive patients of this study are shown in Tables 1 and 2. By conventional PCR, 23 patients were found to harbor HPV-16 infections. Seven additional patients were found to be HPV-16-infected by nPCR. In this group, 3 patients were diagnosed as CIN 0, 2 patients had CIN I lesions, and 1 patient each was diagnosed as CIN III and IC. Of the 7 patients who

FIG. 3. Detection of HPV-16 E6/E7 transcripts in cervical scrapes by RT-PCR. (A) RT-PCR with primer pair S3/S4. In cases 10, 12, and 16 weak amplification products of 343 bp, corresponding to the major splice product E6*I, could be detected. In CaSki cells (lane 20) all three transcripts of the viral oncogenes could be amplified: the full-length E6/E7 transcript (525 bp), as well as E6*I (343 bp) and E6*II (225 bp) splice products. (B) Southern blot hybridization. A 72-h exposure confirmed the specificity of the PCR amplification products. (C) Southern blot hybridization. After prolonged exposure time (25 days), E6/E7 spliced transcripts were detected in six cases (lanes 2, 4, 10, 12, 14, and 16). Human liver DNA (lane 18) and water (lane 19) served as negative controls. M corresponds to the HaeIII-digested phage FX174 DNA size markers.

ucts followed by a 3-day exposure did not increase the sensitivity of HPV mRNA detection (Fig. 4B). In contrast to the results obtained by RT-PCR and Southern blot hybridization, a prolonged exposure time (25 days) did not lead to a further increase in sensitivity of detecting E6/E7 spliced transcripts, demonstrating the high sensitivity of the nRT-PCR assay. While the E6*I major splice product was detectable in all 22 cases, the E6*II minor splice product could be detected in 15 of these cases only. All E6*II transcripts were detected in cases in which E6*I was also present. By nRT-PCR, a 395-bp product could be amplified in 12 of the 30 HPV-16 DNA-positive cases of this study. In 10 of these 12 cases, spliced transcripts (E6*I and/or E6*II) of the E6/E7 mRNAs were also detected by nRT-PCR (Table 1). However, it cannot be distinguished whether the 395-bp product is am-

FIG. 4. Detection of HPV-16 oncogene transcripts in cervical scrapes by nested RT-PCR. (A) Transcriptionally active HPV-16 was detected by amplification of products of 213- or 95-bp length, corresponding to the E6*I and E6*II splice products (cases 1, 2, 4 – 6, 9 –12, 14, 16, and 17). The 395-bp amplification product may represent both E6/E7 DNA and E6/E7 full-length mRNA. (B) Southern blot hybridization of nested RT-PCR products with a 32 P-labeled internal probe and 72-h exposure of the autoradiographs confirmed the specificity of amplification products. Human liver DNA (lane 18) and water (lane 19) served as negative controls. Amplification of CaSki cDNA by nRT-PCR revealed the expected products of 395 bp (E6/E7), 213 bp (E6*I), and 95 bp (E6*II) length (lane 20). Size markers of HaeIII-digested phage FX174 DNA are indicated by M.

DETECTION OF HPV-16 E6/E7 mRNAs BY NESTED RT-PCR

TABLE 2 Detection of HPV-16 E6/E7 DNA and mRNA in Correlation to PCR Protocol HPV-16 E6/E7 DNA Histologya CIN CIN CIN CIN IC

0 I II III

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DISCUSSION

b

HPV-16 E6/E7 mRNA

PCR

nPCR

RT-PCR

nRT-PCR

3/6 5/7 11/11 2/3 2/3

6/6 7/7 11/11 3/3 3/3

0 0 3/11 3/3 2/3

3/6 4/7 9/11 3/3 3/3

n 5 23

n 5 30

n58

n 5 22

a

CIN, cervical intraepithelial neoplasia; CIN 0, no dysplasia; IC, invasive carcinoma. b E6*I and/or E6*II spliced transcripts.

were positive for HPV-16 by nPCR, E6/E7 oncogene transcripts could be detected in 4 patients by nested RT-PCR. The histologic findings in these 4 patients were CIN 0, CIN I, CIN III, and IC, respectively. Thus, in the total group of 30 HPV-16 DNA-positive patients, there was no correlation between lesion severity and the method— conventional PCR or nested PCR— by which HPV-16 DNA was detected (Table 2). After RT-PCR amplification of cDNA present in cases 1 to 30 and subsequent Southern blot hybridization of the RT-PCR products with 25 days exposure of cases 1 to 17, E6/E7 spliced transcripts were detected in 8 of 30 HPV-16 DNA-positive cases. Correlation of these results with histopathologic findings revealed the presence of spliced E6/E7 oncogene transcripts in 3 of 11 patients with CIN II lesions, in 3 of 3 patients with CIN III lesions, and in 2 of 3 patients with IC (Tables 1 and 2). Thus, E6/E7-specific transcripts could only be detected in cervical scrapes from cases with the histopathologic diagnosis of CIN II or more severe lesions. After nRT-PCR amplification, the E6*I major splice product could be detected in 22 of 30 (73%) HPV-16 DNA-positive cases (Tables 1 and 2). This group of patients comprised 3 of 6 (50%) cases without cervical dysplasia (CIN 0), 4 of 7 (58%) cases classified as CIN I, and 9 of 11 (81%) patients with CIN II lesions. The remaining 6 cases included all patients with the diagnoses of CIN III and IC, respectively. Thus, in contrast to the RT-PCR results, by nRT-PCR, E6/E7-specific transcripts could be detected even in HPV-16-infected nondysplastic cervical epithelium, as well as in all grades of CIN and cervical cancer, respectively. Moreover, the prevalence of the E6*I transcript was found to be increased, corresponding to lesion severity. The E6*II minor splice product was detected in 15 of the 30 HPV-16 DNA-positive cases (50%) of this study, comprising 2 of 6 (33%) CIN 0, 3 of 7 (43%) CIN I, 4 of 11 (36%) CIN II, and all 6 CIN III and IC lesions. Thus, in contrast to E6*I, the E6*II transcript was less prevalent in CIN 0, CIN I, and CIN II lesions (37%), however ubiquitous in more severe lesions.

The detection of high-risk HPV E6/E7 transcripts might serve as an additional risk evaluation factor for the development of cervical disease. In former studies, RT-PCR with 32 P-labeled primers [23] or conventional RT-PCR with subsequent Southern blot hybridization was used to detect E6/E7 transcripts in cervical scrapes [16, 25]. However, the applied techniques were either too laborious for use in large-scale studies [23] or not sensitive enough for the detection of E6/E7 oncogene transcripts in CIN 0 or CIN I lesions [16, 25]. In the present study, application of the nPCR method generally resulted in a significant increase in sensitivity for detection of HPV-16 nucleic acids. Notably, on the DNA level, 7 of 30 HPV-16 DNA-positive scrapes would have been missed by conventional PCR, including 2 patients with diagnoses of CIN III and invasive carcinoma, respectively, as well as 4 patients with transcriptionally active E6/E7 oncogenes (Table 2). According to Cornelissen et al. [20], only the detection of the E6*I and E6*II splice products is unequivocal proof of HPV-16 E6/E7 oncogene transcription. This is in correspondence with our results, which also show that RT-PCR products of the size of the E6/E7 full-length transcript mainly seem to have originated from the viral genomic DNA. Thus, to monitor the transcriptional activity of the HPV-16 oncogenes, only cases with detectable E6/E7 spliced transcripts were taken into account. On the RNA level, the application of nRT-PCR and simple gel electrophoresis allowed the detection of HPV-16 E6/E7 spliced transcripts in 73% of HPV-16 DNA-positive cases of this study (Fig. 4A). The sensitivity of nRT-PCR was underlined by the fact that no further increase in sensitivity could be achieved by subsequent Southern blot hybridization, even with exposure times of 25 days. In this regard, Southern blot hybridization only verified the specificity of nRT-PCR results (Fig. 4B). Importantly, by nRT-PCR, E6/E7 spliced transcripts could be detected in about 50% of the HPV-16-infected normal and mild dysplastic epithelia. Further correlation of nRT-PCR and histomorphologic results revealed increasing prevalence in detection of E6/E7 oncogene transcripts with lesion severity. These results are supported by recent findings of Fujii et al. [29], who used a similar PCR assay. In their study, however, which was also performed on cervical scrapes, prevalence of HPV-16 E6/E7 transcripts in all grades of cervical disease was generally lower. In contrast to the results of Hsu et al. [23], who found three different transcription profiles (E6*I only, E6*II only, and both E6*I and E6*II) equally distributed and unrelated to histopathologic findings, we detected the E6*II minor splice product in a smaller subset of cases in which the E6*I major splice product was present. In addition, in our study, the presence of E6*I correlated with lesion severity, while the presence of E6*II did not. In former studies, the HPV-16 E6/E7 splicing

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pattern has been reported to have no prognostic significance in cervical carcinomas [30]. However, it is unknown whether the same is also true for CIN lesions or nondysplastic epithelium. Thus, the detection of the HPV-16 E6/E7 splicing pattern and the follow-up of affected cases might help to clarify the biologic functions of the E6*I and E6*II derived proteins, especially in the development of CIN and its progression to cervical cancer. Persistence and progression of cervical lesions have been reported to correlate with lesion severity and HPV-type infection: High-grade lesions (e.g., CIN II and CIN III) and highrisk HPV-type-infected lesions, respectively, are much more likely to persist or progress [31–33]. In our study, the increasing prevalence of HPV-16 E6/E7 transcripts, corresponding to lesion severity, might reflect these findings and thus might help to identify patients in whom cervical lesions will persist or progress to invasive cervical cancer. Recently, Jeon and Lambert [34] reported an increased stability of HPV-16 E6/E7 mRNA transcribed from integrated HPV sequences. Thus integration, typically found in cervical cancers, but only infrequently found in CIN lesions [35], appears to be an important event in cervical carcinogenesis. It will be interesting to learn whether a correlation between the detection of integration and transcriptional activity can be demonstrated. To address this question, HPV integration will be studied on the material described in this paper. For better understanding of HPV-associated cervical carcinogenesis, viral risk factors must be characterized. Regarding the detection of HPV-16 E6/E7 oncogene transcripts in cervical scrapes, this study has demonstrated the feasibility of nRT-PCR as a highly sensitive and specific diagnostic tool even in nondysplastic cervical epithelium or low-grade cervical lesions. At present, however, detection of transcriptionally active HPV infections should not result in prophylactic treatment, but in intensified surveillance. To substantiate the prognostic relevance of the detection of HPV E6/E7 oncogene transcripts and to define a population of patients with low or increased risk of developing cervical cancer, follow-up studies on a larger scale of patients are currently being pursued.

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ACKNOWLEDGMENTS

18. Smotkin D, Wettstein FO: Transcription of human papillomavirus type 16 early genes in a cervical cancer and cancer derived cell line and identification of the E7 protein. Proc Natl Acad Sci 83:4680 – 4684, 1986

This work was supported in part by a grant from the Federal Ministry of Education and Research and the Interdisciplinary Clinical Research Center Tu¨bingen (IKFZ; 01KS9602).

19. Smotkin D, Prokoph H, Wettstein FO: Oncogenic and nononcogenic human genital papillomaviruses generate the E7 mRNA by different mechanisms. J Virol 63:1441–1447, 1989

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