- Email: [email protected]
Detection of human papillomavirus types in cervical adenocarcinoma by the polymerase chain reaction
GYNECOLOG &OBSTETRIC International Journal of Gynecology & Obstetrics 63 (1998) 265-270
Article
Detection of human papillomavirus types in cervical adenocarcinoma by the polymerase chain reaction M.F. Leea, M.C. Changb, C.H. Wu”p* ‘Derrtment of Medical Research,Taichung VeteransGeneral Hospital, Taichung Taiwan Department of Pathology, Taichung VeteransGeneral Hospital, Taichung, Taiwan Received 4 June 1998; received in revised form 24 August 1998; accepted 1 September 1998
Abstract Objective: To determine the human papillomavirus (HPV) types in cervical adenocarcinomaof patients from Taiwan. Metho&: DNA wasextractedfrom fixed tissuesand polymerasechain reaction wasperformedin conjunction
with a unique probe, pRSA I, allowing simultaneous detection of HPV types 6, 11, 16, 18, 31 and 33 from amplified HPV DNAs after endonuclease,RsuI, digestion. Results: Of 69 tissues examined, 31.9% (22/69) were found to contain HPV DNA. Among 22 HPV-positive specimens,no HPV types6, 11,31 and 33 were detected.On the other hand, HPV 16 and HPV 18 were found in 11 (15.9%) and 10 (14.5%) of HPV-positive specimens, respectively. One specimen (1.5%) was found to contain both HPV 16 and 18 DNAs. Conclusions: Our findings support that HPV 18, along with HPV 16, may play a certain role in the adenocarcinoma pathogenesis of the uterine cervix. 0 1998 International Federation of Gynecology and Obstetrics. Keyworuk
Human papillomavirus; Cervical adenocarcinoma; Polymerase chain reaction
1. Introduction
Human papillomavirus types 6 and 11 are lowrisk types and identified mainly in genital condylomata. HPV 16, 18, 31, 35 and 39 are high-risk
*Corresponding author. Fax: +886 4 3592705; e-mail: [email protected]
types often present in invasive tumor. Unlike squamous cell carcinoma, adenocarcinoma has not been linked with sexual promiscuity which may facilitate transmission of HPV infection. However, it has been shown that 20-40% of adenocarcinomas coexist with cervical intraepithelial neoplasia [1,2]. The association of HPV with cervical adenocarcinoma has not been as extensively studied as with squamous cell carci-
0020-7292/98/$ - see front matter 0 1998 International Federation of Gynecology and Obstetrics PZZ SOO20-7292(98)00171-4
266
M.F. Lee et al. /International Journal of Gynecology& Ubstehics 63 (1998)Xi-270
noma. Previous studies revealed conflicting results that the prevalence of HPV 16 and 18 in cervical adenocarcinoma has ranged from 6 to 58% with in situ hybridization and/or Southern blot analysis [3-61 and from 15 to 85% by polymerase chain reaction (PCR) [4,6-181. The association of human HPV with cervical adenocarcinoma has not been studied extensively in Taiwan. The present study describes the detection of HPV types 6, 11, 16, 18, 31 and 33 in cervical adenocarcinoma of patients from Taiwan by PCR using the Ll consensus primers in conjunction with &a1 digestion and subsequent hybridization to a unique oligonucleotide probe, pRSA I, which dist&guishes six types of HPV DNA in one single step [19]. 2. Materials
and methods
2.1. Fixed tissue section
A total of 69 formalinfixed, paraffin-embedded tissue blocks of cervical adenocarcinoma and 14 normal cervical biopsies obtained from the Taichung Veterans General Hospital, Department of Pathology Tissue Archives of Taiwan, were used in this study. The diagnosis was based on the criteria for primary cervical adenocarcinoma proposed by Maier and Norris [2]. Each case was classified according to the most aberrant area within the specimen. 2.2. DNA extraction
Extraction of DNA from peon-embedded tissue was performed using a sonication method [20]. Briefly, 5-10 pm paraffin-sectioned samples were placed in a microcentrifuge tube, and 400 ~1 xylene was added and vortexed vigorously for 2 min to deparaffinize tissue samples. Xylene was discarded after ~ntr~gation at 100~ X g for 2 min, and the sample was dried on a 50°C heating block. About 2-5 ml of sample preparation buffer (SPB, 10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM MgCl,, 0.01% gelatin, 0.5% Tween 20 and 0.5 mg/ml proteinase K), pretreated glass beads (glycerol glass controlled pore 120-200 mesh, Sigma Chemicals, St. Louis, MO) and 100 ~1 of
SPB without proteinase K were added to sample tubes. Sonication was achieved using the Branson Model 2200 sonicating water bath (Branson Ultrasonics, Danbury, CT) at 45°C for 5-10 min, followed by boiling for 10 min and spun for 20 s in a ~cro~ntri~ge. 2.3. Primers and probes An Ll consensus primer pair, S’GCMCAGGGWCATAAYAATGG3’ (MYll) and S’CGTCC~R~AWA~GATC3’ (MYO9) [21] that are capable of detecting genital HPV types 6, 11, 16, 18, 30, 31, 33, 35, 39, 40,42,45, 51, 52, 53, 54, 55, 57, 58, 59, and at least another 20 yet undetermined types [22] were selected for PCR amplification of HPV DNA. A unique probe pRSA I, 5’~~G~GA~GG~GG3’ [191 was used for hybridization (R = A + G, Y = C + T, M=A+C, W=A+T and H=A+C+T). Complementary oligonucleotides were made using Applied Biosystems DNA synthesizer (Foster City, CA). The PCR was performed on all samples ~ntain~g human ~globin-spec~c primers, GH20 and PC04 [lo], used as an internal control. 2.4. PCR ~plification was performed in a 100~~1reaction mixture containing 10 mM Tris-HCl (pH 9.0), 50 mM KCI, 2.5 mM MgCl,, 0.01% gelatin, 0.1% Triton X-100, 50 pmol of each primer, 0.2 mM each of the four dNTPs, 0.5 units of Thermoprime PlusDNA polymerase (Advanced Biotechnologies, Leatherhead, Surrey, UK& and appropriate amounts of specimen DNA and controls. DNA was denatured at 95°C for 1 min, annealed at 55°C for 1 min, and then extended for 2 rain at 72°C. The amplification was carried out for 40 cycles using DNA Thermal Cycler 480 (Perkin Elmer, Norwalk, CT>. PCR products were analyzed on an agarose gel. 2.5. Digestion of amplification product with RsaI
Digestion was performed in a 30-~1 reaction mixture contain~g 10 mM Tris-HCl, pH 7.5, 10 mM MgCI,, 1.0 mM DTT, 15 ~1 of PCR products,
M.F. Lee et al. /International
Journal of Gynecology & Obstetrics 63 (1998) 265-270
and 0.1 U/p1 of the restriction endonuclease Z?.suI(Boehringer Mannheim, Mannheim, FGR). The mixture was overlaid with mineral oil and incubated at 37°C overnight. Aliquots of digestion products were separated on a 2% agarose. 2.6. Southern blot hybridization
The gel containing digested products was submerged in denaturation solution (1.5 M NaCl containing 0.5 M NaOH) at room temperature for 1 h. After neutralizing with 0.5 M Tris-HCl, pH 7.0 containing 1.5 M NaCl at room temperature for 1 h, DNAs were transferred from gel to nylon membrane by diffusion blotting. The membrane was washed with 2 x SSC, and cross-linked using UV light. Membrane was prehybridized at 50°C overnight in a solution containing 6 X SSC, 0.5% SDS, 5 x Denhardt’s solution and 100 pg/rnl salmon sperm DNA. Digoxigenin (DIG) was bound to uridine-nucleotides and incorporated enzymatically into pRSA I probe by 3’ end labeling. Hybridization was performed in the same solution at 50°C for 6 h with labeled probes. The filter was washed twice with 2 x SSC containing 0.1% SDS for 3 min at room temperature and then twice with 2 x SSC containing 0.1% SDS at 50°C for 30 min. After hybridization and blocking, DIG-labeled probes were detected by an alkaline phosphatase labeled anti-DIG antibody (Boehringer Mannheim Biochemica, Mannheim, Germany). Luminescence was performed by 2-min exposure to Amersham Hyperfilm-MP (Amersham International plc, Amersham, UK). 3. Results A total of 69 paraffin-embedded, formalin-hxed tissues of invasive cervical adenocarcinoma and 14 normal cervical biopsies were examined by PCR using the MY09 and MY11 primers to amplify target sequence. PCR amplified products containing a distinct band at 450 base pairs is defined as HPV-positive [21]. To classify the various types of HPV strain, a unique oligonucleotide, pRSAI, was used as the probe to distinguish HPV types 6,11, 16,18,31 and 33 from the amplified HPV DNAs after endonuclease RsuI
Fig. 1. Agarose gel electrophoresis (A) and Southern blot analysis (B) of amplified DNAs and controls digested with enzyme RsaI. Lanes a and j, 4174 DNA/HaeIII marker; lane b, negative control; lane c, HPV 16 and 18 positive controls and lanes d-i, fixed tissue specimens.
digestion. Southern blot analysis showing bands of 149,216,307, 125,379, and 236 base pairs were considered positive for the presence of HPV types 6, 11, 16, 18, 31 and 33, respectively [19]. Stringent precautions, as recommended by Kwok and Higuchi 1231have been taken to avoid false positives with PCR by exogenous contamination. All of 69 specimens and 14 normal cervical biopsies tested contained amplifiable DNA as confirmed by the positive reaction for @globin. Negative controls were performed on reaction mixtures containing Chlamydia truchomatis DNA and water, and in each assay have produced the predicted results. Of 69 specimens, 31.9% (22/69) were found to contain HPV DNAs by PCR, while HPV DNA was detected in the cervical biopsies of one of 14 (7.1%) normal individuals. The only
268
h4.F.Lee et al. /International Journal of Gynecology& Obstem’cs63 (1998)265-270
HPV DNA-positive sample from normal cervical biopsies was found to be type 11. The presence of the six most common genital I-WV types was subsequently determined in HPV-positive specimens by Southern blot analyses using pRSAI probe after endonuclease RFaI digestion. Of 22 HPV-positive specimens, no HPV types 6, 11, 31 and 33 were detected. HPV 16 and 18 were found in 11 (15.9%) and 10 (14.5%) of the HPV-positive specimens, respectively, and one specimen (1.5%) was found to contain both HPV 16 and HPV 18 DNAs, and some of these results are shown in Fig. 1. Though one amplified product was barely visible in the ethidium bromide-stained agarose gel, it was clearly,demonstrated in the transferred DNA hybridization with DIG-labeled probe (Fig. 1, lane d). 4. Discussion Cervical carcinoma is the leading cancer among women in Taiwan with an annual incidence rate of 31.0 per 100000 (Cancer Registry Annual Report in Taiwan Area, 1995). It has been shown that HPV is associated with a continuum of genital tract disease from dyplasia to invasive squa-
mous cell carcinoma. The high prevalence of HPV DNAs, predominantly types 16, l&31,33, and 35, have been detected in Taiwanese women with cervical carcinoma [ 11,24-261. The association of HPV in cervical adenocarcinoma have been reported [2-181, and some of these show conflicting results (Table 1). The prevalence of HPV types 16 and 18 (31.9%) in cervical adenocarcinoma found in this study appears to be lower in comparison with some studies (Table 1) using hybridization (44% to 58.3%) and PCR (42% to 85%). However, we believe that our results are significant since the prevalence of HPV DNAs found in normal cervical biopsies is less than 10% in Taiwan [24,26], and no HPV 16 and 18 was detected in normal controls in the present study. Similar results, 18% by the hybridization and 15% to 34% by the PCR in cervical adenocarcinoma, were also reported by other groups (Table 1). The sampling sizes, the different detecting techniques, and the geographic differences in the distribution of specific HPV types probably reflect the remarkable variability of frequency among reports. HPV 18 has been found to be the predominant type in cervical adenocarcinoma by most of the
Table 1 Frequency of HPV 16 and/or 18 DNA in cervical adenocarcinoma by hybridization and PCR Author
Country
Patient no.
Techniques
HPV (o/o)
HPV16 (a)
HPV 18 (%)
HP 16 and 18 (%)
Leminen Duggan Das Griffin Bjersing Johnson Hording Lee Chen Yamakawa Tenti Iwasawa Parker Uchiyama Ferguson Tenti Lee (current study)
Finland Canada India England Sweden US, Michigan Denmark US, Vermont Taiwan Japan Italy Finland US, Washington DC Japan US, Michigan Italy Taiwan
106 77 12 16 26 22 50 20 42 43 138 108 32 32 27 74 69
In situ hybridization (16, 18) Dot blot hybridization (16,181 In situ hybridization (16,181 PCR (16,18 and Southern) PCR (E6/E7 and Southern) PCR (E6/E7, and Southern) PCR (16,18 and dot blot) PCR (16,18 and Southern) PCR (Ll, E6/E7, MY09, MY11) PCR (16,18 and Southern) PCR (16,18 and Southern) PCR (16,18, MY09, MY111 PCR (16,181 PCR (Ll, 16,18 and MselI and RsaI) PCR (Ll and M&I and RFaI) PCR (16,18 and Southern) PCR (MYO9, MY11 and Southern and RsaI)
18 44 58.3 31.3 42.0 82 70 15 67 56 85 75 50 34 59 76 32
2 18 41.6 25 15 23 18 10 19 21 28 17 22 19 26 20 16
14 23 16.6 6.3 27 59 52 5 48 33 30 56 28 12 26 33 14.5
2 Not mentioned 0 0 0 0 0 0 0 2 27 3 0 3 Not mentioned 23 1.5
h4.F.Lee et al. /International Journal of Gynecology& Obstetrics63 (1998)265-270
reports [3,5,8,11,12,20,211.Only a few studies revealed that the rates of HPV 16 DNAs, 80% (4/5) [4], 71.4% (5/7) [61, 66.7% (2/3) [lo] and 55% (6/U) [16] were higher than HPV 18 in cervical adenocarcinoma. No such prevalence was found in our study. Of 22 HPV-positive specimens, 50% of HPV 16, 45.5% of HPV 18 and 4.5% of HPV 16 and 18 were detected. Nevertheless, HPV 16 and HPV 18 DNAs were detected with significant frequency in the tissue sections obtained from cervical adenocarcinoma patients. Taken together, our findings support that HPV 18, along with HPV 16, may play a possible role in the pathogenesis of adenocarcinoma of the uterine cervix. More than 20 HPV types have been found in the genital tract and the possibility of tumor specimens containing HPV types other than those screened for here cannot be excluded. Acknowledgements
This study was supported by the Taichung Veternans General Hospital Grant TVGH 847321, Taiwan, Republic of China. References
111 Paavonen J, Koutsky LA, Kiviat N. Cervical neoplasia and other STD-related genital and anal neoplasia. In: Holmes KK, Mardh PA, Sparling PF, Wiesner PJ, editors. Sexually transmitted diseases. New York: McGraw-Hill, 1989:561-592. Bl Maier RC, Norris HJ. Coexistence of cervical intraepithelial neoplasia with primary adenocarcinoma of the endocervix. Obstet Gynecol 1980;56:361-364. [31 Leminen A, Paavonen J, Vesterinen E, Wahlstrom T, Rantala I, Lehtinen M. Human papillomavirus types 16 and 18 in adenocarcinoma of the uterine cervix. Am J Clin Path01 1991;95:647-652. [41 Griffin NR, Dockey D, Lewis FA, Wells M. Demonstration of low frequency of human papillomavirus DNA in cervical adenocarcinoma and adenocarcinoma in situ by the polymerase chain reaction and in situ hybridization. Int J Gynecol Path01 1991;10:36-43. 151 Duggan MA, Benoit JL, McGregor SE, Nation JG, Inoue M, Stuart GC. The human papillomavirus status of 114 endocervical adenocarcinoma cases by dot blot hybridization. Hum Path01 1993;24:121-125. [61 Das BC, Gopalkrishna V, Das DK, Sharma JK, Singh V, Luthra UK. Human papillomavirus DNA sequences in adenocarcinoma of the uterine cervix in Indian women. Cancer 1993;72:147-153.
269
[71 Bjersing L, Rogo K, Evander M, Gerdes U, Stendahl U,
Wadell G. HPV 18 and cervical adenocarcinomas. Anticancer Res 1991;11:123-127. is1 Johnson TL, Kim W, Plieth DA, Sarkar FH. Detection of HPV 16/18 DNA in cervical adenocarcinoma using polymerase chain reaction (PCR) methodology. Mod Path01 1992;5:35-40. [91 Hording U, Teglbjaerg CS, Visfeldt J, Bock JE. Human papillomavirus types 16 and 18 in adenocarcinoma of the uterine cervix. Gynecol Oncol 1992;46:313-316. [lOI Lee KR, Howard P, Heintz NH, Collins CC. Low prevalence of human papillomavirus types 16 and 18 in cervical adenocarcinoma in situ, invasive adenocarcinoma, and glandular dysplasia by polymerase chain reaction. Mod Path01 1993;6:433-437. 1111Chen TM, Chen CA, Wu CC, Huang SC, Chang CF, Hsieh CY. The genotypes and prognostic significance of human papillomavirus in cervical cancer. Int J Cancer 1994;57:181-184. WI Yamakawa Y, Forslund 0, Teshima H, Hasumi K, Kitagawa T, Hansson BG. Human papillomavirus DNA in adenocarcinoma and adenosquamous carcinoma of the uterine cervix detected by polymerase chain reaction (PCR). Gynecol Path01 1994;53:190-195. D31 Tenti P, Romagnoli S, Silini E, Zappatore R, Spinillo A, Giunta P, Cappellini A, Vesentini N, Zara C, Camevali L. Human papillomavirus types 16 and 18 infection in infiltrating adenocarcinoma of the cervix: PCR analysis of 138 cases and correlation with histologic type and grade. Am J Clin Path01 1996;106:52-56. [141 Iwasawa A, Nieminen P, Lehtimen M, Paavonen J. Human papillomavirus DNA in uterine cervix squamous cell carcinoma and adenocarcinoma detected by polymerase chain reaction. Cancer 1996;77:2275-2279. D51 Parker MF, Arroyo GF, Geradts J, Sabichi AL, Park RC, Taylor RR, Birrer MJ. Molecular characterization of adenocarcinoma of cervix. Gynecol Oncol 199764: 242-251.
[161 Uchiyama M, Iwasaka T, Matsuo N, Hachisuga T, Mori M, Sugimori H. Correlation between human papillomavirus positivity and ~53 gene overexpression in adenocarcinoma of uterine cervix. Gynecol Oncol 1997;65:23-29. D71 Ferguson AW, Svoboda-Newman SM, Frank TS. Analysis of human papillomavirus infection and molecular alterations in adenocarcinoma of cervix. Mod Path01 1998;11:11-18. Us1 Tenti P, Pavane110S, Padovan L, Spinillo A, Vesentini N, Zappatore R, Migliora P, Zara C, Ranzani GN, Camevali L. Analysis and clinical implications of ~53 gene mutations and human papillomavirus type 16 and 18 infection in primary adenocarcinoma of the uterine cervix. Am J Path01 1998;152:1057-1063. [I91 Chen S, Tabrizi SN, Fairley CK, Borg AJ, Garland SM. Simultaneous detection and typing strategy for human papillomaviruses based on PCR and restriction endonuclease mapping. BioTechniques 1994;17:138-142.
M.F. Lee et al. /International
270
Journal of Gynecology & Obstehics 63 (1998) 265-270
DO1 Heller MJ, Burgart LI, TenEyck CT, Anderson ME,
D31 Kwok S, Higuchi R. Avoiding false positive with PCR.
Greiner TC, Robinson RA. An efficient method for the extraction of DNA from formalinfixed, paraffin-embedded tissue by son&cation. BioTechniques 1991;ll:
[241 Wu CH, Lee MP, Chang MC, Ho SC. Detection of
372-377.
ml
Ting Y, Manos MM. Detection and typing of genital human papihomaviruses. In: Imris MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. San Diego: Academic Press,
ml
Bauer HM, Ting Y, Greer CE, Chambers JC, Tashiro CJ, Chimera J, Reingold A, Manos MM. Genital human papillomavirus infection in female university students as determined by a PCR-based method. J Am Med Assoc 1991:265:472-477.
1990~356-367.
Nature 1989;338:237-238. human papillomavirus types in cervical lesions of patients from Taiwan by the polymerase chain reaction. Sex Transm Dis 1994;21:309-314. Pao CC, Kao SM, Tang GC, Lee K, Si J, Ruan S. t251 Prevalence of human papillomavirus DNA sequences in an area with very high incidence of cervical carcinoma. Br J Cancer 1994;70:694-696. D61 Liaw KL, Hsing AW, Chen CT, Schiffman MH, Zhang TY, Hsieh CY. Human papihomavirus and cervical neoplasia: a case-control study in Taiwan. Int Cancer 1995;62:565-571.
Article
Detection of human papillomavirus types in cervical adenocarcinoma by the polymerase chain reaction M.F. Leea, M.C. Changb, C.H. Wu”p* ‘Derrtment of Medical Research,Taichung VeteransGeneral Hospital, Taichung Taiwan Department of Pathology, Taichung VeteransGeneral Hospital, Taichung, Taiwan Received 4 June 1998; received in revised form 24 August 1998; accepted 1 September 1998
Abstract Objective: To determine the human papillomavirus (HPV) types in cervical adenocarcinomaof patients from Taiwan. Metho&: DNA wasextractedfrom fixed tissuesand polymerasechain reaction wasperformedin conjunction
with a unique probe, pRSA I, allowing simultaneous detection of HPV types 6, 11, 16, 18, 31 and 33 from amplified HPV DNAs after endonuclease,RsuI, digestion. Results: Of 69 tissues examined, 31.9% (22/69) were found to contain HPV DNA. Among 22 HPV-positive specimens,no HPV types6, 11,31 and 33 were detected.On the other hand, HPV 16 and HPV 18 were found in 11 (15.9%) and 10 (14.5%) of HPV-positive specimens, respectively. One specimen (1.5%) was found to contain both HPV 16 and 18 DNAs. Conclusions: Our findings support that HPV 18, along with HPV 16, may play a certain role in the adenocarcinoma pathogenesis of the uterine cervix. 0 1998 International Federation of Gynecology and Obstetrics. Keyworuk
Human papillomavirus; Cervical adenocarcinoma; Polymerase chain reaction
1. Introduction
Human papillomavirus types 6 and 11 are lowrisk types and identified mainly in genital condylomata. HPV 16, 18, 31, 35 and 39 are high-risk
*Corresponding author. Fax: +886 4 3592705; e-mail: [email protected]
types often present in invasive tumor. Unlike squamous cell carcinoma, adenocarcinoma has not been linked with sexual promiscuity which may facilitate transmission of HPV infection. However, it has been shown that 20-40% of adenocarcinomas coexist with cervical intraepithelial neoplasia [1,2]. The association of HPV with cervical adenocarcinoma has not been as extensively studied as with squamous cell carci-
0020-7292/98/$ - see front matter 0 1998 International Federation of Gynecology and Obstetrics PZZ SOO20-7292(98)00171-4
266
M.F. Lee et al. /International Journal of Gynecology& Ubstehics 63 (1998)Xi-270
noma. Previous studies revealed conflicting results that the prevalence of HPV 16 and 18 in cervical adenocarcinoma has ranged from 6 to 58% with in situ hybridization and/or Southern blot analysis [3-61 and from 15 to 85% by polymerase chain reaction (PCR) [4,6-181. The association of human HPV with cervical adenocarcinoma has not been studied extensively in Taiwan. The present study describes the detection of HPV types 6, 11, 16, 18, 31 and 33 in cervical adenocarcinoma of patients from Taiwan by PCR using the Ll consensus primers in conjunction with &a1 digestion and subsequent hybridization to a unique oligonucleotide probe, pRSA I, which dist&guishes six types of HPV DNA in one single step [19]. 2. Materials
and methods
2.1. Fixed tissue section
A total of 69 formalinfixed, paraffin-embedded tissue blocks of cervical adenocarcinoma and 14 normal cervical biopsies obtained from the Taichung Veterans General Hospital, Department of Pathology Tissue Archives of Taiwan, were used in this study. The diagnosis was based on the criteria for primary cervical adenocarcinoma proposed by Maier and Norris [2]. Each case was classified according to the most aberrant area within the specimen. 2.2. DNA extraction
Extraction of DNA from peon-embedded tissue was performed using a sonication method [20]. Briefly, 5-10 pm paraffin-sectioned samples were placed in a microcentrifuge tube, and 400 ~1 xylene was added and vortexed vigorously for 2 min to deparaffinize tissue samples. Xylene was discarded after ~ntr~gation at 100~ X g for 2 min, and the sample was dried on a 50°C heating block. About 2-5 ml of sample preparation buffer (SPB, 10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM MgCl,, 0.01% gelatin, 0.5% Tween 20 and 0.5 mg/ml proteinase K), pretreated glass beads (glycerol glass controlled pore 120-200 mesh, Sigma Chemicals, St. Louis, MO) and 100 ~1 of
SPB without proteinase K were added to sample tubes. Sonication was achieved using the Branson Model 2200 sonicating water bath (Branson Ultrasonics, Danbury, CT) at 45°C for 5-10 min, followed by boiling for 10 min and spun for 20 s in a ~cro~ntri~ge. 2.3. Primers and probes An Ll consensus primer pair, S’GCMCAGGGWCATAAYAATGG3’ (MYll) and S’CGTCC~R~AWA~GATC3’ (MYO9) [21] that are capable of detecting genital HPV types 6, 11, 16, 18, 30, 31, 33, 35, 39, 40,42,45, 51, 52, 53, 54, 55, 57, 58, 59, and at least another 20 yet undetermined types [22] were selected for PCR amplification of HPV DNA. A unique probe pRSA I, 5’~~G~GA~GG~GG3’ [191 was used for hybridization (R = A + G, Y = C + T, M=A+C, W=A+T and H=A+C+T). Complementary oligonucleotides were made using Applied Biosystems DNA synthesizer (Foster City, CA). The PCR was performed on all samples ~ntain~g human ~globin-spec~c primers, GH20 and PC04 [lo], used as an internal control. 2.4. PCR ~plification was performed in a 100~~1reaction mixture containing 10 mM Tris-HCl (pH 9.0), 50 mM KCI, 2.5 mM MgCl,, 0.01% gelatin, 0.1% Triton X-100, 50 pmol of each primer, 0.2 mM each of the four dNTPs, 0.5 units of Thermoprime PlusDNA polymerase (Advanced Biotechnologies, Leatherhead, Surrey, UK& and appropriate amounts of specimen DNA and controls. DNA was denatured at 95°C for 1 min, annealed at 55°C for 1 min, and then extended for 2 rain at 72°C. The amplification was carried out for 40 cycles using DNA Thermal Cycler 480 (Perkin Elmer, Norwalk, CT>. PCR products were analyzed on an agarose gel. 2.5. Digestion of amplification product with RsaI
Digestion was performed in a 30-~1 reaction mixture contain~g 10 mM Tris-HCl, pH 7.5, 10 mM MgCI,, 1.0 mM DTT, 15 ~1 of PCR products,
M.F. Lee et al. /International
Journal of Gynecology & Obstetrics 63 (1998) 265-270
and 0.1 U/p1 of the restriction endonuclease Z?.suI(Boehringer Mannheim, Mannheim, FGR). The mixture was overlaid with mineral oil and incubated at 37°C overnight. Aliquots of digestion products were separated on a 2% agarose. 2.6. Southern blot hybridization
The gel containing digested products was submerged in denaturation solution (1.5 M NaCl containing 0.5 M NaOH) at room temperature for 1 h. After neutralizing with 0.5 M Tris-HCl, pH 7.0 containing 1.5 M NaCl at room temperature for 1 h, DNAs were transferred from gel to nylon membrane by diffusion blotting. The membrane was washed with 2 x SSC, and cross-linked using UV light. Membrane was prehybridized at 50°C overnight in a solution containing 6 X SSC, 0.5% SDS, 5 x Denhardt’s solution and 100 pg/rnl salmon sperm DNA. Digoxigenin (DIG) was bound to uridine-nucleotides and incorporated enzymatically into pRSA I probe by 3’ end labeling. Hybridization was performed in the same solution at 50°C for 6 h with labeled probes. The filter was washed twice with 2 x SSC containing 0.1% SDS for 3 min at room temperature and then twice with 2 x SSC containing 0.1% SDS at 50°C for 30 min. After hybridization and blocking, DIG-labeled probes were detected by an alkaline phosphatase labeled anti-DIG antibody (Boehringer Mannheim Biochemica, Mannheim, Germany). Luminescence was performed by 2-min exposure to Amersham Hyperfilm-MP (Amersham International plc, Amersham, UK). 3. Results A total of 69 paraffin-embedded, formalin-hxed tissues of invasive cervical adenocarcinoma and 14 normal cervical biopsies were examined by PCR using the MY09 and MY11 primers to amplify target sequence. PCR amplified products containing a distinct band at 450 base pairs is defined as HPV-positive [21]. To classify the various types of HPV strain, a unique oligonucleotide, pRSAI, was used as the probe to distinguish HPV types 6,11, 16,18,31 and 33 from the amplified HPV DNAs after endonuclease RsuI
Fig. 1. Agarose gel electrophoresis (A) and Southern blot analysis (B) of amplified DNAs and controls digested with enzyme RsaI. Lanes a and j, 4174 DNA/HaeIII marker; lane b, negative control; lane c, HPV 16 and 18 positive controls and lanes d-i, fixed tissue specimens.
digestion. Southern blot analysis showing bands of 149,216,307, 125,379, and 236 base pairs were considered positive for the presence of HPV types 6, 11, 16, 18, 31 and 33, respectively [19]. Stringent precautions, as recommended by Kwok and Higuchi 1231have been taken to avoid false positives with PCR by exogenous contamination. All of 69 specimens and 14 normal cervical biopsies tested contained amplifiable DNA as confirmed by the positive reaction for @globin. Negative controls were performed on reaction mixtures containing Chlamydia truchomatis DNA and water, and in each assay have produced the predicted results. Of 69 specimens, 31.9% (22/69) were found to contain HPV DNAs by PCR, while HPV DNA was detected in the cervical biopsies of one of 14 (7.1%) normal individuals. The only
268
h4.F.Lee et al. /International Journal of Gynecology& Obstem’cs63 (1998)265-270
HPV DNA-positive sample from normal cervical biopsies was found to be type 11. The presence of the six most common genital I-WV types was subsequently determined in HPV-positive specimens by Southern blot analyses using pRSAI probe after endonuclease RFaI digestion. Of 22 HPV-positive specimens, no HPV types 6, 11, 31 and 33 were detected. HPV 16 and 18 were found in 11 (15.9%) and 10 (14.5%) of the HPV-positive specimens, respectively, and one specimen (1.5%) was found to contain both HPV 16 and HPV 18 DNAs, and some of these results are shown in Fig. 1. Though one amplified product was barely visible in the ethidium bromide-stained agarose gel, it was clearly,demonstrated in the transferred DNA hybridization with DIG-labeled probe (Fig. 1, lane d). 4. Discussion Cervical carcinoma is the leading cancer among women in Taiwan with an annual incidence rate of 31.0 per 100000 (Cancer Registry Annual Report in Taiwan Area, 1995). It has been shown that HPV is associated with a continuum of genital tract disease from dyplasia to invasive squa-
mous cell carcinoma. The high prevalence of HPV DNAs, predominantly types 16, l&31,33, and 35, have been detected in Taiwanese women with cervical carcinoma [ 11,24-261. The association of HPV in cervical adenocarcinoma have been reported [2-181, and some of these show conflicting results (Table 1). The prevalence of HPV types 16 and 18 (31.9%) in cervical adenocarcinoma found in this study appears to be lower in comparison with some studies (Table 1) using hybridization (44% to 58.3%) and PCR (42% to 85%). However, we believe that our results are significant since the prevalence of HPV DNAs found in normal cervical biopsies is less than 10% in Taiwan [24,26], and no HPV 16 and 18 was detected in normal controls in the present study. Similar results, 18% by the hybridization and 15% to 34% by the PCR in cervical adenocarcinoma, were also reported by other groups (Table 1). The sampling sizes, the different detecting techniques, and the geographic differences in the distribution of specific HPV types probably reflect the remarkable variability of frequency among reports. HPV 18 has been found to be the predominant type in cervical adenocarcinoma by most of the
Table 1 Frequency of HPV 16 and/or 18 DNA in cervical adenocarcinoma by hybridization and PCR Author
Country
Patient no.
Techniques
HPV (o/o)
HPV16 (a)
HPV 18 (%)
HP 16 and 18 (%)
Leminen Duggan Das Griffin Bjersing Johnson Hording Lee Chen Yamakawa Tenti Iwasawa Parker Uchiyama Ferguson Tenti Lee (current study)
Finland Canada India England Sweden US, Michigan Denmark US, Vermont Taiwan Japan Italy Finland US, Washington DC Japan US, Michigan Italy Taiwan
106 77 12 16 26 22 50 20 42 43 138 108 32 32 27 74 69
In situ hybridization (16, 18) Dot blot hybridization (16,181 In situ hybridization (16,181 PCR (16,18 and Southern) PCR (E6/E7 and Southern) PCR (E6/E7, and Southern) PCR (16,18 and dot blot) PCR (16,18 and Southern) PCR (Ll, E6/E7, MY09, MY11) PCR (16,18 and Southern) PCR (16,18 and Southern) PCR (16,18, MY09, MY111 PCR (16,181 PCR (Ll, 16,18 and MselI and RsaI) PCR (Ll and M&I and RFaI) PCR (16,18 and Southern) PCR (MYO9, MY11 and Southern and RsaI)
18 44 58.3 31.3 42.0 82 70 15 67 56 85 75 50 34 59 76 32
2 18 41.6 25 15 23 18 10 19 21 28 17 22 19 26 20 16
14 23 16.6 6.3 27 59 52 5 48 33 30 56 28 12 26 33 14.5
2 Not mentioned 0 0 0 0 0 0 0 2 27 3 0 3 Not mentioned 23 1.5
h4.F.Lee et al. /International Journal of Gynecology& Obstetrics63 (1998)265-270
reports [3,5,8,11,12,20,211.Only a few studies revealed that the rates of HPV 16 DNAs, 80% (4/5) [4], 71.4% (5/7) [61, 66.7% (2/3) [lo] and 55% (6/U) [16] were higher than HPV 18 in cervical adenocarcinoma. No such prevalence was found in our study. Of 22 HPV-positive specimens, 50% of HPV 16, 45.5% of HPV 18 and 4.5% of HPV 16 and 18 were detected. Nevertheless, HPV 16 and HPV 18 DNAs were detected with significant frequency in the tissue sections obtained from cervical adenocarcinoma patients. Taken together, our findings support that HPV 18, along with HPV 16, may play a possible role in the pathogenesis of adenocarcinoma of the uterine cervix. More than 20 HPV types have been found in the genital tract and the possibility of tumor specimens containing HPV types other than those screened for here cannot be excluded. Acknowledgements
This study was supported by the Taichung Veternans General Hospital Grant TVGH 847321, Taiwan, Republic of China. References
111 Paavonen J, Koutsky LA, Kiviat N. Cervical neoplasia and other STD-related genital and anal neoplasia. In: Holmes KK, Mardh PA, Sparling PF, Wiesner PJ, editors. Sexually transmitted diseases. New York: McGraw-Hill, 1989:561-592. Bl Maier RC, Norris HJ. Coexistence of cervical intraepithelial neoplasia with primary adenocarcinoma of the endocervix. Obstet Gynecol 1980;56:361-364. [31 Leminen A, Paavonen J, Vesterinen E, Wahlstrom T, Rantala I, Lehtinen M. Human papillomavirus types 16 and 18 in adenocarcinoma of the uterine cervix. Am J Clin Path01 1991;95:647-652. [41 Griffin NR, Dockey D, Lewis FA, Wells M. Demonstration of low frequency of human papillomavirus DNA in cervical adenocarcinoma and adenocarcinoma in situ by the polymerase chain reaction and in situ hybridization. Int J Gynecol Path01 1991;10:36-43. 151 Duggan MA, Benoit JL, McGregor SE, Nation JG, Inoue M, Stuart GC. The human papillomavirus status of 114 endocervical adenocarcinoma cases by dot blot hybridization. Hum Path01 1993;24:121-125. [61 Das BC, Gopalkrishna V, Das DK, Sharma JK, Singh V, Luthra UK. Human papillomavirus DNA sequences in adenocarcinoma of the uterine cervix in Indian women. Cancer 1993;72:147-153.
269
[71 Bjersing L, Rogo K, Evander M, Gerdes U, Stendahl U,
Wadell G. HPV 18 and cervical adenocarcinomas. Anticancer Res 1991;11:123-127. is1 Johnson TL, Kim W, Plieth DA, Sarkar FH. Detection of HPV 16/18 DNA in cervical adenocarcinoma using polymerase chain reaction (PCR) methodology. Mod Path01 1992;5:35-40. [91 Hording U, Teglbjaerg CS, Visfeldt J, Bock JE. Human papillomavirus types 16 and 18 in adenocarcinoma of the uterine cervix. Gynecol Oncol 1992;46:313-316. [lOI Lee KR, Howard P, Heintz NH, Collins CC. Low prevalence of human papillomavirus types 16 and 18 in cervical adenocarcinoma in situ, invasive adenocarcinoma, and glandular dysplasia by polymerase chain reaction. Mod Path01 1993;6:433-437. 1111Chen TM, Chen CA, Wu CC, Huang SC, Chang CF, Hsieh CY. The genotypes and prognostic significance of human papillomavirus in cervical cancer. Int J Cancer 1994;57:181-184. WI Yamakawa Y, Forslund 0, Teshima H, Hasumi K, Kitagawa T, Hansson BG. Human papillomavirus DNA in adenocarcinoma and adenosquamous carcinoma of the uterine cervix detected by polymerase chain reaction (PCR). Gynecol Path01 1994;53:190-195. D31 Tenti P, Romagnoli S, Silini E, Zappatore R, Spinillo A, Giunta P, Cappellini A, Vesentini N, Zara C, Camevali L. Human papillomavirus types 16 and 18 infection in infiltrating adenocarcinoma of the cervix: PCR analysis of 138 cases and correlation with histologic type and grade. Am J Clin Path01 1996;106:52-56. [141 Iwasawa A, Nieminen P, Lehtimen M, Paavonen J. Human papillomavirus DNA in uterine cervix squamous cell carcinoma and adenocarcinoma detected by polymerase chain reaction. Cancer 1996;77:2275-2279. D51 Parker MF, Arroyo GF, Geradts J, Sabichi AL, Park RC, Taylor RR, Birrer MJ. Molecular characterization of adenocarcinoma of cervix. Gynecol Oncol 199764: 242-251.
[161 Uchiyama M, Iwasaka T, Matsuo N, Hachisuga T, Mori M, Sugimori H. Correlation between human papillomavirus positivity and ~53 gene overexpression in adenocarcinoma of uterine cervix. Gynecol Oncol 1997;65:23-29. D71 Ferguson AW, Svoboda-Newman SM, Frank TS. Analysis of human papillomavirus infection and molecular alterations in adenocarcinoma of cervix. Mod Path01 1998;11:11-18. Us1 Tenti P, Pavane110S, Padovan L, Spinillo A, Vesentini N, Zappatore R, Migliora P, Zara C, Ranzani GN, Camevali L. Analysis and clinical implications of ~53 gene mutations and human papillomavirus type 16 and 18 infection in primary adenocarcinoma of the uterine cervix. Am J Path01 1998;152:1057-1063. [I91 Chen S, Tabrizi SN, Fairley CK, Borg AJ, Garland SM. Simultaneous detection and typing strategy for human papillomaviruses based on PCR and restriction endonuclease mapping. BioTechniques 1994;17:138-142.
M.F. Lee et al. /International
270
Journal of Gynecology & Obstehics 63 (1998) 265-270
DO1 Heller MJ, Burgart LI, TenEyck CT, Anderson ME,
D31 Kwok S, Higuchi R. Avoiding false positive with PCR.
Greiner TC, Robinson RA. An efficient method for the extraction of DNA from formalinfixed, paraffin-embedded tissue by son&cation. BioTechniques 1991;ll:
[241 Wu CH, Lee MP, Chang MC, Ho SC. Detection of
372-377.
ml
Ting Y, Manos MM. Detection and typing of genital human papihomaviruses. In: Imris MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. San Diego: Academic Press,
ml
Bauer HM, Ting Y, Greer CE, Chambers JC, Tashiro CJ, Chimera J, Reingold A, Manos MM. Genital human papillomavirus infection in female university students as determined by a PCR-based method. J Am Med Assoc 1991:265:472-477.
1990~356-367.
Nature 1989;338:237-238. human papillomavirus types in cervical lesions of patients from Taiwan by the polymerase chain reaction. Sex Transm Dis 1994;21:309-314. Pao CC, Kao SM, Tang GC, Lee K, Si J, Ruan S. t251 Prevalence of human papillomavirus DNA sequences in an area with very high incidence of cervical carcinoma. Br J Cancer 1994;70:694-696. D61 Liaw KL, Hsing AW, Chen CT, Schiffman MH, Zhang TY, Hsieh CY. Human papihomavirus and cervical neoplasia: a case-control study in Taiwan. Int Cancer 1995;62:565-571.