Detection of HPV genotypes in cervical lesions by the HPV DNA Chip and sequencing

Transcription

1 Gynecologic Oncology 98 (2005) Detection of HPV genotypes in cervical lesions by the HPV DNA Chip and sequencing Yoo-Duk Choi a, Woon-Won Jung c, Jong-Hee Nam a, Ho-Sun Choi b, Chang-Soo Park a, * a Department of Pathology, Chonnam National University Medical School, 5, Hak 1-Dong, Dong-Gu, Gwangju , Republic of Korea b Department of Obstetrics and Gynecology, Chonnam National University Medical School, Gwangju, Republic of Korea c MyGene Bioscience Institute, MyGene Company, Seoul, Republic of Korea Received 23 February 2005 Available online 15 July 2005 Abstract Objective. A newly introduced HPV detection technique in cervical lesion, the HPV DNA Chip test, contains 24 HPV probes and has the advantage of being able to detect 24 HPV types at once. We performed HPV DNA sequencing and compared the results with that of the HPV DNA Chip for evaluation of the accuracy of the DNA Chip test. Methods. The HPV DNA sequencing was performed in samples of 282 patients, where specific HPV type had been shown in HPV DNA Chip test. The sixteen cases where multiple HPV types had been found in HPV DNA Chip test were included in 282 cases. The sequencing was also performed in HPV-other type samples of 95 patients, where positive in HPV-PCR, but specific HPV type had not been found. Results. In 257 cases (91.1%) of 282 cases, the HPV types of the HPV DNA sequencing test were in agreement with types of the HPV DNA Chip. In 16 cases (5.7%), the sequencing types were different from the types of HPV DNA Chip. But, in 9 of 16 cases, types in HPV DNA sequencing were absent types in HPV DNA Chip test. The interpretation of HPV DNA sequencing was impossible in nine cases (3.2%). The HPV DNA sequencing test of 95 cases of HPV-other type showed that the sequencing types from 94 cases (98.9%) were absent types in HPV DNA Chip test. In sequencing test of HPV-other type, HPV-81 (20.0%), HPV-62 (14.7%), HPV-84 (13.7%), and HPV-61 (13.7%) were frequently detected. Conclusion. HPV DNA Chip is an accurate method for detecting the 24 HPV genotypes. D 2005 Elsevier Inc. All rights reserved. Keywords: Cervix; HPV DNA Chip; HPV DNA sequencing; HPV-other type Introduction Cervical cancer is the second most common cancer in terms of both incidence and mortality worldwide [1]. To date, there seems to be a limitation to cytology-based screening programs in terms of reducing cervical carcinoma incidence and mortality rates [2,3]. Recently, there is some agreement that the availability of high sensitive human papillomavirus (HPV) tests offers the potential for replacement of conventional cytologic screening programs [4]. In addition, the importance of HPV testing has been increasing because genetic typing is required to predict clinical progression [5]. * Corresponding author. Fax: address: daniel5438@hanmail.net (C.-S. Park). The more than 35 distinctive HPV types have been associated with cervical epithelial neoplasia (CIN) and cervical cancer [6,7]. But, the traditional methods for HPV detection, such as morphological and immunological methods, showed low sensitivity and specificity and did not detect the specific HPV type [8]. Digene hybrid-capture (HC-2) technology was widely used for it is highly sensitive, and if appropriately used, highly specific as well. A new HPV detection technique using a DNA microarray (DNA Chip) has been recently developed. This technique is based on the polymerase chain reaction (PCR) method with high sensitivity and has an ability to detect single and multiple infection at once [9,10]. By identifying which HPV types are highly associated with cervical precancerous and cancerous lesions, the biologic course of the HPV type /$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi: /j.ygyno

2 370 Y.-D. Choi et al. / Gynecologic Oncology 98 (2005) specific cervical lesions may be predicted, thus providing appropriate patient care according to HPV types [11]. And, several studies demonstrated that persistent HPV infection with a high-risk type is strongly associated with cervical dysplasia and carries a greater risk of subsequent progression to cervical cancer [12,13]. In this study, we evaluated the accuracy of HPV DNA Chip test for detection and typing of HPV in cervical lesions by comparing with result of HPV DNA sequencing of same samples. In addition, we desired to find out the types which were not detected in HPV DNA Chip test by HPV DNA sequencing of HPV-other types. Materials and methods Study subjects HPV DNA sequencing was performed in 377 cervical samples obtained from 377 patients, in which positive reaction had been shown by HPV DNA Chip test. These samples were composed of 282 samples, in which specific HPV genotypes had been detected in HPV DNA Chip test and 95 samples, in which had shown amplified HPV- PCR product, but specific HPV genotypes had not been detected (HPV-other type samples). The 282 study samples included 266 samples of single HPV genotype (single infection) and 16 of multiple genotypes (multiple infection) in HPV DNA Chip test. The types of HPV DNA sequencing were compared with the types of HPV DNA Chip test. In cases of multiple infection, when the type of HPV DNA sequencing was one of the types of HPV DNA Chip test, we considered that sequencing type agrees with the type of HPV DNA Chip. In the HPV DNA sequencing test of 95 HPV-other type samples, we checked whether the sequencing types of HPV-other type samples are present or not in HPV DNA Chip test. HPV genotyping in HPV DNA Chip test We used the HPV DNA Chip, PCR-based DNA microarray system, as an HPV genotyping method (provided by MyGene Company, Seoul, South Korea) for HPV typing. The HPV DNA Chip contains 24 type specific probes; 15 types from high-risk types (HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-53, HPV-56, HPV-58, HPV-59, HPV-66, and HPV- 68) and 9 types of low-risk types (HPV-6, HPV-11, HPV-34, HPV-40, HPV-42, HPV-43, HPV-44, HPV-54, and HPV-70). Twenty-four type-specific 30-mer oligonucleotide probes containing an amine group at the 5V terminus were immobilized onto a chip slide glass. A slide has eight chambers, and each chamber is used for a test. Therefore, a slide tests eight samples at one time. Briefly, DNA was isolated from swab samples using a DNA isolation kit (MyGene. Co, Seoul, Korea), and target L1 regions of HPV DNA were amplified and labeled by a single dye, indocarbocyanine-dutp, (MEN Life Science Products, Inc.,Boston, MA), using consensus GPd5+/GP6d+ primers. h-globin was amplified using PCR as internal controls. The PCR products of all samples were detected by electrophoresis through a 2.5% agarose gel, and the product size of HPV DNA was 150 base pairs (bp). Ten microliter of the HPV-amplified product was denaturated for 5 min at 95-C. The samples were mixed with a hybridization solution (MyGene. Co., Seoul, Korea) then applied onto the DNA Chip. Hybridization was performed at 43-C for 90 min and was followed by washing with 3 SSPE for 5 min and 1 SSPE for 5 min and drying at room temperature. Hybridized HPV DNA was visualized using a DNA Chip scanner (Scanarray lite; GSI Lumonics\, Ottawa, Ontario, Canada). HPV amplicons can be hybridized with corresponding type-specific oligonucleotide probe and visualized on HPV DNA Chip slides as double positive spots (Fig. 1) when HPV DNA is present in amplified PCR product. The samples that showed a positive band of 150 bp on the gel electrophoresis but were negative on the HPV DNA Chip slide were deginated as HPV-other. None of the negative controls (without DNA) revealed HPV positivity. HPV genotyping in HPV DNA sequencing The primed PCR product was added to the sequencing reaction mixture. Sequencing was performed bidirectionally Fig. 1. Examples of the HPV DNA Chip. This HPV DNA Chip is available for detection of 24 HPV genotypes, all of which were amplified form HPV-PCR reaction and subsequently hybridized to oligonucleotide probes that are specific to each genotypes. The positive signal was represented as double spots. (A) A single infection of HPV-16. (B) A single infection of HPV-53. (C) A single infection of HPV-11. (D) A double infection of HPV-35 and 53.

3 Y.-D. Choi et al. / Gynecologic Oncology 98 (2005) Fig. 2. Examples of HPV DNA sequencing. (A) HPV-18. (B) HPV-31.

4 372 Y.-D. Choi et al. / Gynecologic Oncology 98 (2005) with the BigDye3 terminator cycle sequencing kit (PE Applied Biosystems) using ABI PRISM 310 Genomic Analyser (PE Applied Biosystems) at a dispensing pressure of 600 mbar with 8-ms open times and 65-s cycle times. The sequencing procedure was carried out by stepwise elongation of the primer strand upon cyclic dispensation of the different deoxynucleoside triphosphates (Amersham Pharmacia Biotech). A CCD camera detected the light output resulting from nucleotide incorporation. The data were obtained in graphic format. Results In 257 (91.1%) of 282 samples, the types of sequencing were in agreement with the types of HPV DNA Chip test (Fig. 2). Among 16 samples of multiple HPV genotypes, the agreement was described in nine samples. One type was designated in all nine samples, and the type in HPV DNA sequencing was one of the types of HPV DNA Chip test. In the remaining seven samples of multiple HPV genotypes, genotyping was impossible due to showing the mixed peak in sequencing data. Furthermore, genotyping was impossible in two samples of single HPV genotype due to same cause. The types of HPV DNA sequencing were different from the types of HPV DNA Chip test in 16 samples (5.7%). In 9 of 16 samples, the types of HPV DNA sequencing were the absent types used at the HPV DNA Chip instrument. In Table 2 The types of HPV DNA sequencing in 95 HPV-other samples Type Number (%) The present type at used HPV DNA Chip test (n =1) 11 1 (1.1%) The absent types used at HPV DNA Chip test (n = 94) 32 5 (5.3%) (13.7%) (14.7%) 64 1 (1.1%) 69 1 (1.1%) 71 7 (7.4%) 72 8 (8.4%) 73 1 (1.1%) (20.0%) 82 7 (7.4%) 83 5 (5.3%) (13.7%) Total 95 seven samples, the types of sequencing were the present types (Table 1). The result of HPV DNA sequencing in about 95 HPVother samples was designated in Table 2. The types of HPV DNA sequencing were the absent types used at the HPV DNA Chip instrument except one sample (98.9%). HPV-81 (20.0%) was frequently detected in sequencing of HPVother samples. The next types were HPV-62, HPV-84, and HPV-61 (Fig. 3). Discussion Table 1 The comparison of type of HPV DNA sequencing and type of HPV DNA Chip test in studied 282 samples, in which specific HPV genotype was detected in HPV DNA Chip test Agreement samples 257 (91.1%) Multiple HPV genotypes samples 9 Disagreement samples 16 (5.7%) The samples in which the type in sequencing is the present type in HPV DNA Chip (n =7) The samples in which the type in sequencing is the absent type in HPV DNA Chip (n =9) The type in HPV DNA Chip The samples in which genotyping is 9 (3.2%) impossible Multiple HPV genotypes samples The type in HPV sequencing HPV DNA Chip test is an accurate method for detection and genotyping of HPV. All studied samples showed positive reaction for HPV detection. The type of HPV DNA Chip test was in agreement with type of HPV DNA sequencing in 91.1% of HPV-positive cervical samples. The types of HPV DNA sequencing in 94 HPV-other samples (98.9%) were the absent types in the HPV DNA Chip test. It was the additional feature to certify the accuracy of HPV DNA Chip test for HPV genotyping. Genotyping in HPV DNA sequencing was impossible in nine cases. The nine cases consist of seven samples of multiple genotypes and two samples of a single genotype in HPV DNA Chip test. In HPV DNA sequencing of specimen with multiple genotypes, the detection of specific type depends on the proportional dominance and number of genotypes present in the amplicon. At present, sequencing might not be particularly useful for identifying infection genotypes with more than one HPV genotype because multiple infections give sequence signals from all of the available types in the specimen [14]. But, in this study, genotyping was impossible in two samples of single HPV genotype. We considered the possibility that the genotypes which are absent in HPV DNA Chip might be combined with type detected in HPV DNA Chip. Occasionally, the genotyping in HPV DNA sequencing may be possible even

5 Y.-D. Choi et al. / Gynecologic Oncology 98 (2005) Fig. 3. Examples of HPV DNA sequencing in HPV-other types. (A) HPV-81. (B) HPV-84. (C) HPV-61. (D) HPV-62.

6 374 Y.-D. Choi et al. / Gynecologic Oncology 98 (2005) in multiple infection, provided one type is solidly dominant, with low background signal from other existing genotype(s) [14]. In this study, this circumstance is applicable to nine cases where one type was detected in HPV DNA sequencing, even if multiple genotypes had been found in HPV DNA Chip test. The type of HPV DNA sequencing was different from the type of HPV DNA Chip test in 16 samples (5.7%). This disagreement was illustrated in three ways. First, HPV DNA Chip test does not accurately detect the HPV types of cervical samples. Second, the cervical samples were the status with multiple HPV genotypes. When (1) the multiple HPV genotypes might be made up of the present type and the absent type(s) in HPV DNA Chip and (2) the absent type was more dominant than the present type in performing HPV DNA sequencing, we think that the difference of type between two tests could occur. This probability was applicable to nine cases where the types of sequencing were the absent types at the HPV DNA Chip instrument. Third, each of the types detected in HPV DNA Chip and HPV DNA sequencing have homologous base sequence, and misleading occurs in HPV DNA sequencing. But, this information alone may be insufficient. Thus, additional further evaluation is necessary to explain this problem. At present, other HPV detection tests have been widely used. The Southern blot hybridization test, highly sensitive test for HPV DNA, is considered a gold standard for HPV detection. This test, however, is unsuitable for clinical use because it is labor-intensive and requires fresh samples [15]. The hybrid capture test is a proprietary nucleic acid hybridization signal amplification system and a sensitive reliable test for detecting 13 cancer-associated viral types of HPV in cervical specimens [16]. This is a simple procedure that uses a single test to detect any of the 13 high oncogenic risk HPV types, but it fails to distinguish between HPV types. [4,6]. The polymerase chain reaction using consensus primers designed from E6 and E7 open reading frames (ORFs) followed by restriction fragment length polymorphism (RFLP) is also a highly sensitive test for the detection of HPV DNA but also has some limitations in its sensitivity and genotyping capability [17,18]. HPV-81, 61, 62, 84, 72, 71, and 82 were detected at considerable rate in the sequencing of HPV-other types. These types are not included in HPV DNA Chip test. No article did not describe the characteristic features of this types. In one article, HPV-61 and 81 were classified as lowrisk types [19]. Mitsuishi et al. suggested that HPV-61 and 62 are often identified in Bowen s disease [20]. On the phylogenetic HPV, HPVs that were usually detected are categorized in the following manner: A9 (HPV-16 group; 31, 33, 35, 52, 58, and 67), A7 (HPV-18 group; 39, 45, 59, and 68), A5 (51 and 69), A6 (HPV-53 group; 56 and 66), A10 (6, 11, 44, and 55), and A8 (40, 43, and 54) [9,21]. In recent article, a newly detected HPV-61, 62, 72, 81, 83, and 84 were categorized in A3 phylogenetic subgroup, and this A3 group was by far the most frequently observed viral types in immunosuppressive condition such as AIDS [22]. But, further study will be needed to demonstrate the association between A3 group and immunosuppression. Furthermore, this A3 group should be added in HPV DNA Chip test. A recent study found that multiple infection is an important factor in persistent HPV infection [23]. The multiple infection could include low-risk types as well as high-risk types. Persistent infection was also important for development of cervical dysplasia. But, HC-2 cannot detect multiple infection. The detection of HPV genotype needs two steps in HC-2. However, in HPV DNA Chip test, that is more rapidly accomplished. The cost of HPV DNA Chip test is lower than those of HC-2 [24]. In conclusion, HPV DNA Chip test have been used as a diagnostic tools since microarray discriminates many HPV genotypes easily and also identifies multiple infections. Furthermore, in this study, the accuracy of HPV DNA Chip test for HPV genotyping could be certified by comparison with sequencing data. The HPV DNA Chip test may have clinical value in decision-making as to which patient with equivocal smear needs colposcopy and biopsy versus regular follow-up. Extensive, prospective, controlled, clinical trials are needed to evaluate the true diagnostic yield of this emerging technology. References [1] Parkin DM, Muir CS. Cancer incidence in five continents. Comparability and quality of data. IARC Sci Publ 1992;120: [2] Van der Graaf Y, Vooijs GP, Gaillard HL, Go DM. Screening errors in cervical cytologic screening. Acta Cytol 1987 (Jul Aug);31(4): [3] Koss LG. The Papanicolaou test for cervical cancer detection. A triumph and a tragedy. JAMA 1989 (Feb);261(5): [4] Hwang TS, Jeong JK, Park M, Han HS, Choi HK, Park TS. Detection and typing of HPV genotypes in various cervical lesions by HPV oligonucleotide microarray. Gynecol Oncol 2003 (Jul);90(1):51 6. [5] Choi BS, Kim O, Park MS, Kim KS, Jeong JK, Lee JS. Genital human papillo-mavirus genotyping by HPV oligonucleotide microarray in Korean commercial sex workers. J Med Virol 2003 (Nov); 71(3): [6] Kim CJ, Jeong JK, Park M, Park TS, Park TC, Namkoong SE, et al. HPV oligonucleotide microarray-based detection of HPV genotypes in cervical neoplastic lesions. Gynecol Oncol 2003 (May);89(2): [7] Bosch FX, Manos MM, Munoz N, Sherman M, Jansen AM, Peto J, et al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. J Natl Cancer Inst 1995 (Jun);87(11): [8] Liu CH, Ma WL, Shi R, Ou YQ, Zhang B, Zheng WL. Possibility of using DNA chip technology for diagnosis of human papillomavirus. J Biochem Mol Biol 2003 (Jul);36(4): [9] Cho NH, An HJ, Jeong JK, Kang S, Kim JW, Kim YT, et al. Genotyping of 22 human papillomavirus types by DNA chip in Korean women: comparison with cytologic diagnosis. Am J Obstet Gynecol 2003 (Jan);188(1): [10] Oh TJ, Kim CJ, Woo SK, Kim TS, Jeong DJ, Kim MS, et al. Development and clinical evaluation of a highly sensitive DNA microarray for detection and genotyping of human papillomaviruses. J Clin Microbiol 2004 (Jul);42(7):

7 Y.-D. Choi et al. / Gynecologic Oncology 98 (2005) [11] An HJ, Cho NH, Lee SY, Kim IH, Lee C, Kim SJ, et al. Correlation of cervical carcinoma and precancerous lesions with human papillomavirus (HPV) genotypes detected with the HPV DNA chip microarray method. Cancer 2003 (Apr);97(7): [12] Ho GYF, Burk RD, Klein S, Kadish AS, Chang CJ, Palan P, et al. Persistent genital HPV infection as a risk factor for persistent cervical dysplasia. J Natl Cancer Inst 1995 (Sep 20);87(18): [13] Chua K-L, Hjerpe A. Persistence of HPV infections preceding cervical carcinoma. Cancer 1996 (Jan 1);77(1): [14] Gharizadeh B, Kalantari M, Garcia CA, Johansson B, Nyren P. Typing of human papillomavirus by pyrosequencing. Lab Invest 2001 (May); 81(5): [15] Ferenczy A. Viral testing for genital human papillomavirus infections: recent progress and clinical potentials. Int J Gynecol Cancer 1995 (Sep);5(5): [16] Lorincz AT. Hybrid capture method for detection of human papillomavirus DNA in clinical specimens: a tool for clinical management of equivocal Pap smears and for population screening. J Obstet Gynaecol Res 1996 (Dec);22(6): [17] Pizzighella S, Pisoni G, Bevilacqua F, Vaona A, Palu G. Simultaneous polymerase chain reaction detection and restriction typing for the diagnosis of human genital papillomavirus infection. J Virol Methods 1995 (Oct);55(2): [18] Kay P, Meehan K, Williamson AL. The use of nested polymerase chain reaction and restriction fragment length polymorphism for the detection and typing of mucosal human papillomaviruses in samples containing low copy numbers of viral DNA. J Virol Methods 2002 (Aug);105(1): [19] Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, et al. International agency for research on cancer multicenter cervical cancer study group. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003 (Feb);348(6): [20] Mitsuishi T, Sata T, Matsukura T, Iwasaki T, Kawashima M. The presence of mucosal human papillomavirus in Bowen s disease of the hands. Cancer 1997 (May 15);79(10): [21] Chan SY, Delius H, Halpern AL, Bernard HU. Analysis of genomic sequences of 95 papillomavirus types: uniting typing, phylogeny, and taxonomy. J Virol 1995 (May);69(5): [22] Broker TR, Jin G, Croom-Rivers A, Bragg SM, Richardson M, Chow LT, et al. Viral latency The papillomavirus model. Dev Biol (Basel) 2001;106: [discussion 452 3, ]. [23] Sasagawa T, Basha W, Yamazaki H, Inoue M. High-risk and multiple human papillomavirus infections associated with cervical abnormalities in Japanese women. Cancer Epidemiol, Biomarkers Prev 2001 (Jan);10(1): [24] Lee GY, Kim SM, Rim SY, Choi HS, Park CS, Nam JH. Human papillomavirus (HPV) genotyping by HPV DNA chip in cervical cancer and precancerous lesions. Int J Gynecol Cancer 2005 (Jan Feb); 15(1):81 7.