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1 Ann Clin Microbiol Vol. 16, No. 2, June, ISSN Prevalence and Genotype of Human Papillomavirus Infection and Risk of Cervical Dysplasia among Asymptomatic Korean Women Soie Chung 1, Sue Shin 2,3, Jong Hyun Yoon 2,3, Eun Youn Roh 2, Sung Jun Seoung 2, Gyoung Pyoung Kim 2, Eui-Chong Kim 3 Department of Laboratory Medicine, 1 Seoul National University Hospital Healthcare System Gangnam Center, 2 Seoul National University Boramae Hospital, 3 Seoul National University College of Medicine, Seoul, Korea Background: The persistence of infection by high-risk human papillomavirus (HPV) may lead to cervical cancer. Recently, the American Society for Colposcopy and Cervical Pathology (ASCCP) announced that oncogenic HPV screening and the PAP smear are the main methods of screening for cervical cancer. The goal of this study was to investigate the prevalence and genotyping of HPV, as well as the risk of cervical dysplasia. Methods: HPV genotyping was conducted by a commercial chip assay. Cervical dysplasia was retrospectively reviewed using electronic medical records. The study participants were grouped together according to cervical dysplasia status: no dysplasia, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), and high-grade squamous intraepithelial lesion (HSIL). The HPV prevalence and genotyping were analyzed according to the cervical dysplasia group. Results: The overall prevalence of HPV was 17.6% (91 out of 518 patients). HPV-18 (2.3%), HPV-16 (2.1%), and HPV-58 (1.2%) were the three most frequent genotypes. The prevalence of HPV infection and the high-risk HPV positive rate was higher in the ASCUS, LSIL, and HSIL groups than in the no dysplasia group (P<0.05). Conclusion: In this study, basic data regarding the prevalence and distribution of HPV genotypes were obtained. Since HPV vaccination has been actively encouraged among Korean women, a change in the prevalence of HPV and cervical dysplasia is expected in the future. This study provided basic data describing the prevalence of HPV and its genotypes in the pre-hpv vaccination era. (Ann Clin Microbiol 2013;16:87-91) Key Words: Cervical dysplasia, HPV genotyping, Human papilloma virus INTRODUCTION 보건복지부의암등록통계에따르면 2009년자궁경부암은발생률이 7.5% 로위암, 폐암, 간암, 대장암, 유방암에이어 6번째로발생률이높은암이다 [1]. 기존의여러연구에서자궁경부에지속적인인유두종바이러스 (Human papillomavirus, HPV) 감염이자궁경부암을발생시킨다는것이밝혀졌고현재고위험 HPV 유전형의존재유무와더불어자궁경부질세포진검사가자궁경부암의선별에중요한요소로자리잡고있다 [2]. 또한고위험 HPV 유전형중에서 HPV-16과 HPV-18을표적으로하는백신이개발되어식품의약품안전청에서 2007년 6월에 Gardasil TM 을 2008년에 Cervarix TM 를사용허가하였다. 이에따라대한부 Received 9 November, 2012, Revised 5 December, 2012 Accepted 7 December, 2012 Correspondence: Sue Shin, Department of Laboratory Medicine, Seoul National University Boramae Hospital, 39 Boramae-gil, Dongjak-gu, Seoul , Korea. (Tel) , (Fax) , ( ) jeannie@snu.ac.kr 인종양ㆍ콜포스코피학회에서는백신접종을통한 HPV 감염및자궁경부암을예방하는것을권고하고있어 [3] 향후 HPV 유병률및유전형분포에변화가있을것으로판단된다. 기존의한국여성을대상으로한연구에서는부산지역대학생, 의료기관에방문한여성혹은성매매직업여성에대해 HPV 유병률에대한연구가이루어진바있으나 [4-6], 산부인과적증상이없는한국여성을대상으로한 HPV 유병률과 HPV 유전자형의분포에대한연구가제한적이다. 2002년에진행된국내대학병원건강증진센터에서 HPV 유병률과자궁경부세포진검사이상과의관련성을연구가있지만 [7] 이연구에서 HPV 유전형을분석하지는않았고 HPV가성을매개로감염된다는것을고려할때성생활변화에따른 HPV 유병률및유전형에대한연구가필요하다. 따라서이번연구에서는산부인과적증상이없는건강한한국여성을대상으로 HPV 유병률및 HPV 유전자형의분포와자궁이형성증과의연관성에대해알아보고향후자궁경부암백 87

2 88 Ann Clin Microbiol 2013;16(2):87-91 신접종 10년후연구의기본자료를구성하고자하였다. MATERIALS AND METHODS 1. 연구대상 2011년 1월 3일부터 2011년 12월 30일까지서울특별시보라매병원건강증진센터에서 genotyping 검사가의뢰된환자들중, 동시에자궁경부질세포진검사를시행하고산부인과적증상이없는 518명의환자를대상으로하였다. 연령구성은 20-29세 4 명 (0.8%), 세 61명 (11.8%), 40-49세 130명 (25.1%), 세 182명 (35.1%), 60-69세 121명 (23.4%), 70-79세 19명 (3.7%), 80-89세 1명 (0.2%) 이었다. 본후향적의무기록연구는보라매병원의학연구윤리심의위원회의승인을받았다 ( ). 2. 연구방법 1) HPV genotyping: 환자의질분비물을 Digene Cervical brush and specimen transport medium (Digene, Silver Spring, MD, USA) 으로채취하여수송후, HPV genotyping chip (Goodgene, Seoul, Korea) 을사용하여제조사의지시에따라검사를시행하였다. 검사원리를간단히설명하자면다음과같다. HPV E6, E7, L1 유전자와사람베타글로빈유전자를증폭시킨후, 증폭된유전자산물을 DNA chip에얹어동소교잡반응을시키면 HPV 유전자형이형광으로나타난다. 이형광신호를스캐너로읽으면유전자형을알수있다 [8]. 이번연구에서진행한검사방법은다음과같다. L1, E6, E7 유전자와사람베타글로빈유전자를 Cy5-dUTP (NEN Life Science Products, Inc., Boston, USA) 로표지한후 10 μl의 HPV 증폭산물과 10 μl 의글로빈산물을 95 o C에서 2분간외가닥의 DNA로전환한후 3분간얼음위에식혔다. 검체를 50 μl의 GG 동소교잡버퍼 (Goodgene) 및후 0.2% SDS와혼합한후 DNA chip에얹었다. 동소교잡은 40 o C에서 2시간까지진행하였고, 3 SSPE에서 2분간세척후다시 1 SSPE로 2분간세척하여상온에서건조시켰다. 동소교잡신호는 Affymetrix 428 Array Scanner (Affymetrix, Inc., Santa Clara, CA, USA) 를이용하여시각화시켰다. 고위험 HPV 유전형 (HPV type 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68,69) 과저위험 HPV 유전형 (HPV type 6, 11, 34, 40, 42, 43, 44) 을검출하고자하였다 [9]. 2) Cerivical dysplasia 의구분 : 후향적으로환자의무기록검토를통하여자궁경부질세포진검사에서정상및염증소견을보인경우를 no dysplasia 군 으로분류하고, 미확정비정형편평상피세포 (atypical squamous cells of undetermined significance, ASCUS), 저등급편평상피내병변 (low-grade squamous intraepithelial lesion, LSIL), 고등급편평상피내병변 (high-grade squamous intraepithelial lesion, HSIL) 으로판독된경우는각각 ASCUS 군, LSIL 군, HSIL 군 으로분류하였다. 3) 통계분석 : 연령별 HPV 유병률의비교에 Chi-square 법을사용하였고자궁경부질세포진검사결과에따른 HPV 유병률에 Fisher's exact test를사용하였다. 통계계산은 SPSS (Chicago, IL, USA) 18 버전을사용하였다. 모든유의수준은 P<0.05를기준으로하였다. 1. 대상환자의특성 RESULTS 자궁경부질세포진검사결과에따른대상군의구성은다음과같았다. PAP smear 결과에서정상소견 (no dysplasia) 을보인예는 496예 (95.8%), ASCUS 11예 (2.1%), LSIL 7예 (1.4%), HSIL 4예 (0.8%) 였다. 2. HPV 유병률및유전형분포 전체 HPV 감염유병률은 17.6% (91/518) 였다. 각 HPV 유전형별유병률을분석하였을때 HPV-18 (2.3%), HPV-16 (2.1%), HPV-58 (1.2%) 순으로높았다 (Fig. 1). HPV 양성환자들중 12.1% (11/91) 에서는여러유전형의중복감염이있었다 (HPV-16, HPV-43; HPV-16, HPV-35, HPV-68; HPV-16, HPV-39, HPV-58, HPV-66, HPV-40, HPV-43; HPV-18, HPV-33; HPV-18, HPV-35; HPV-18, HPV-39; HPV-18, HPV-40; HPV-18, HPV-58; HPV-18, HPV-68; HPV-35, HPV-58; HPV-35, HPV-66). 고위험 (high risk) 유전형의유병률은 8.5% (44/518) 였으며, 저위험 (low risk) 유전형의유병률은 0.6% (3/518), 위험도가밝혀지지않은기타유전형 (Other type) 의유병률은 8.5% (44/518) 였다. 각연령군내에서 HPV 유병률은 20-39세 27.7% (18/65), 40-49세 16.9% (22/130), Fig. 1. Prevalence of human papilloma virus in each genotype. The number of HPV-infected patients in each genotype, including both single and multi-infection was divided by total number of patients (n=518). Abbreviation: HPV, human papilloma virus.

3 Soie Chung, et al. : HPV and Asymptomatic Korean Women 89 Table 1. Prevalence of human papilloma virus infection according to genotype and patients age group Age group HPV any High-risk* HPV HPV genotype group Low-risk HPV PAP smear Other HPV Negative Normal ASCUS LSIL HSIL (n=65) (n=130) (n=182) 60 (n=141) 18 (27.7) 22 (16.9) 29 (15.9) 22 (15.6) 8 (12.3) 13 (10.0) 9 (4.9) 14 (9.9) 1 (0.8) 1 (0.5) 1 (0.7) 10 (15.4) 8 (6.2) 19 (10.4) 7 (5.0) 47 (72.3) 108 (83.1) 153 (84.1) 119 (84.4) 63 (96.9) 122 (93.8) 176 (96.7) 135 (95.7) 2 (3.1) 3 (2.3) 3 (1.6) 3 (2.1) 3 (2.3) 2 (1.1) 2 (1.4) 2 (1.6) 1 (0.5) 1 (0.8) The values are presented as number and percentage (%) in parentheses. *High-risk HPV genotype includes both single high-risk genotype infection and multiple genotype infection including any high-risk genotype. Abbreviations: HPV, human papilloma virus; ASCUS, atypical squamous cells of undetermined significance; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; PAP smear, papanicolaou smear. Table 2. The genotype-specific human papilloma virus prevalence stratified by PAP smear findings Normal (n=496) ASCUS (n=11) LSIL (n=7) HSIL (n=4) Fig. 2. Percent human papilloma virus positive cases according to PAP smear findings. The high-risk HPV genotypes are more prevalent among HSIL (4/4, 100.0%), LSIL (3/7, 42.9%), and ASCUS (3/11, 27.3%) patients than no dysplasia patients (34/496, 6.9%) (no dysplasia vs. ASCUS, P=0.008; no dysplasia vs. LSIL, P=0.003; no dysplasia vs. HSIL, P<0.001). Abbreviation: HPV, human papilloma virus 세 15.9% (29/182), 60세이상 15.6% (22/141) 였다 (Table 1). 각연령군내에서고위험 HPV 유병률은 20-39세 12.3% (8/65), 40-49세 10.0% (13/130), 50-59세 4.9% (9/182), 60세이상 9.9% (14/141) 였다. 39세이하의환자의경우 40세이상의환자에비해 HPV 유병률이유의하게높았다 (27.7% vs. 16.1%, P=0.022). 고위험 HPV 유병률또한 39세이하의환자의경우 40세이상의환자에비해유의하게높았다 (12.3% vs. 7.8%, P=0.037). 3. 자궁경부질세포진검사와 HPV 자궁경부질세포진검사결과에따른 HPV genotyping 검사결과 HPV 감염유병률은 no dysplasia 군 (15.7%) 에비해 ASCUS 군 (45.5%), LSIL 군 (57.1%), HSIL 군 (100.0%) 에서유의하게높았다 (no dysplasia vs. ASCUS, P=0.008; no dysplasia vs. HPV any HPV-16 HPV-18 HPV-31 HPV-33 HPV-35 HPV-39 HPV-45 HPV-52 HPV-56 HPV-58 HPV-59 HPV-66 HPV-68 HPV-6 HPV-34 HPV-40 HPV-43 Other 78 (15.7) 10 (2.0)* 9 (1.8)* 4 (0.8)* 3 (0.6) 2 (0.4)* 2 (0.4)* 41 (8.3) 5 (45.5) 2 (18.2) 4 (57.1) 4 (100.0) 1 (25.0) 1 (25.0) The values are presented as number and percentage (%) in parentheses. *Total of 11 cases were known as multiple genotype infections (HPV-16, HPV-43 coinfection; HPV-16, HPV-35, and HPV-68 coinfection; HPV-16, HPV-39, HPV-58, HPV-66, HPV-40, and HPV-43 coinfection; HPV-18, HPV-33 coinfection; HPV-18, HPV-35 coinfection; HPV-18, HPV-39 coinfection; HPV-18, HPV-40 coinfection; HPV-18, HPV-58 coinfection; HPV-18, HPV-68 coinfection; HPV-35, HPV-58 coinfection; HPV-35, HPV-66 coinfection). LSIL, P=0.003; no dysplasia vs. HSIL, P<0.001). 또한고위험유전형의유병률이 no dysplasia 군 (6.9%) 에비해 ASCUS 군 (27.3%), LSIL 군 (42.9%), HSIL 군 (100.0%) 에서유의하게높았다 (no dysplasia vs. ASCUS, P=0.008; no dysplasia vs. LSIL, P=0.003; no dysplasia vs. HSIL, P<0.001) (Fig. 2). 자궁경부질세포진검사결과의유전자형특이분포는 Table 2에서보는바와같았다.

4 90 Ann Clin Microbiol 2013;16(2):87-91 DISCUSSION 이번연구에서 HPV 유병률은 17.6% 로나타났다. 이는성매매직업여성을대상으로한연구 [5] 에서제시한 40.7% 의유병률과 18개의연구를기반으로메타분석을한기존논문 [4] 의유병률 (23.9%) 과비교하였을때낮은수치이나이는연구대상의특성에따른차이로보인다. 그근거로다수의남성과성관계를가진여성에서 HPV 유병률이그렇지않은여성에비해최고 6.9배의교차비를보였다는보고가있다 [6]. 또한기존연구 [4] 의메타분석에들어간상당수의연구대상이조직학적검사상다수의 HSIL 혹은자궁경부암환자를포함하고있기때문에산부인과적증상이없이선별검사의목적으로건강건강증진센터에방문한여성들을대상으로한이번연구에서보다유병률이높았던것으로판단된다. 2002년에건강증진센터방문여성을대상으로진행된연구 [7] 에따르면유병률은 15.5% 로이번연구와유사하다고볼수있다. 이번연구의자궁경부질세포진검사결과 no dysplasia 로분류된환자에서 HPV-16 (2.0%), HPV-18 (1.8%), HPV-58 (0.8%) 의세유전형이기존의메타분석연구 [4] 와동일하게가장유병률이높은것으로나타났다. 또한 2,308명의성매매직업여성을대상으로유전형분석을한연구에서도 HPV-16 (23.0%), HPV-58 (9.8%), HPV-18 (8.7%) 의순으로세가지유전형이유병률이높다는결과가보고되었다 [5]. HPV-58 은외국에비해우리나라에서자궁경부암이나 HSIL 환자에서유병률이높은것으로알려져있다 [2,4,10]. 따라서상대적으로 HPV-58의유병률의높은우리나라에서향후 HPV-16, HPV-18을표적으로하는백신접종후발암기전에 HPV-58이어떤작용을하는지밝히는것이중요할것이며이를토대로지역에따라서는 HPV-58을표적으로하는백신을개발할필요도있을것이다. 이번연구에서는산부인과적증상이없는건강한한국여성을대상으로 HPV 유병률및 HPV 유전자형의분포와자궁이형성증과의연관성에대한자료를구축하였다. 최근자궁경부 암백신접종이적극적으로권장되고있어향후한국여성에서 HPV와자궁이형성증유병률의변화가있을것으로생각되며저자들은이번연구가기본자료로역할을할것이라판단된다. REFERENCES 1. Statistics Korea. Incidence and mortality of cancer in Korea. jsp?idx_cd=2770&bbs=indx_001 [Online] (last visited on 24 October 2012). 2. Clifford GM, Smith JS, Aguado T, Franceschi S. Comparison of HPV type distribution in high-grade cervical lesions and cervical cancer: a meta-analysis. Br J Cancer 2003;89: Kim YT. Current status of cervical cancer and HPV infection in Korea. J Gynecol Oncol 2009;20: Bae JH, Lee SJ, Kim CJ, Hur SY, Park YG, Lee WC, et al. Human papillomavirus (HPV) type distribution in Korean women: a meta-analysis. J Microbiol Biotechnol 2008;18: Rhee JE, Shin MY, Kim CM, Kee HY, Chung JK, Min SK, et al. Prevalence of human papillomavirus infection and genotype distribution among high-risk Korean women for prospecting the strategy of vaccine development. Virol J 2010;7: Shin HR, Franceschi S, Vaccarella S, Roh JW, Ju YH, Oh JK, et al. Prevalence and determinants of genital infection with papillomavirus, in female and male university students in Busan, South Korea. J Infect Dis 2004;190: Joo WD, Kim SH, Kim DY, Suh DS, Kim JH, Kim YM, et al. Prevalence of human papillomavirus infection in Korean women: risks of abnormal pap smear and cervical neoplasia. Korean J Gynecol Oncol Colposc 2004;15: Kim KH, Yoon MS, Na YJ, Park CS, Oh MR, Moon WC. Development and evaluation of a highly sensitive human papillomavirus genotyping DNA chip. Gynecol Oncol 2006;100: Muñoz N, Bosch FX, de Sanjosé S, Herrero R, Castellsagué 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;348: Clifford GM, Smith JS, Plummer M, Muñoz N, Franceschi S. Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis. Br J Cancer 2003;88:63-73.

5 Soie Chung, et al. : HPV and Asymptomatic Korean Women 91 = 국문초록 = 건강증진센터를방문하는여성의인유두종바이러스의유병률및유전형의분포와자궁이형성증과의관계 1 서울대학교병원강남센터, 2 서울대학교보라매병원진단검사의학과, 3 서울대학교의과대학검사의학교실정소이 1, 신수 2,3, 윤종현 2,3, 노은연 2, 성승준 2, 김경평 2, 김의종 3 배경 : 지속적인인유두종바이러스 (Human papillomavirus, HPV) 의감염이자궁경부암을발생시킨다는것이밝혀졌고현재고위험 HPV 유전형의존재유무와자궁경부질세포진검사가자궁경부암선별에중요한요소로자리잡고있다. 이에고위험 HPV를표적으로하는백신이개발되어실제임상에서쓰이고있다. 따라서산부인과적증상이없는여성에서인유두종바이러스의유병률및유전형의분포, 자궁이형성증과의관계를알아보고자하였다. 방법 : 환자의질분비물을 HPV genotyping chip을이용하여유전형의분포를분석하였다. 자궁이형성증은후향적으로환자의의무기록검토를통하여 no dysplasia 군, 미확정비정형편평상피세포 (atypical squamous cells of undetermined significance, ASCUS) 군, 저등급편평상피내병변 (low-grade squamous intraepithelial lesion, LSIL) 군, 고등급편평상피내병변 (high-grade squamous intraepithelial lesion, HSIL) 군 으로분류하여 HPV 유병률및유전형의분포, 자궁이형성증과 HPV 감염과의관계를분석하였다. 결과 : 전체 HPV 감염유병률은 17.6% (91/518) 였다. HPV-18 (2.3%), HPV-16 (2.1%), HPV-58 (1.2%) 순으로높은유전자형의분포를보였다. 자궁경부질세포진검사결과에따른 HPV genotyping 검사결과 HPV 감염유병률및고위험 HPV 유전형의유병률은 no dysplasia 군 에비해 ASCUS 군, LSIL 군, HSIL 군 에서유의하게높았다 (P<0.05). 결론 : 이번연구에서는산부인과적증상이없는건강한한국여성을대상으로 HPV 유병률및 HPV 유전자형의분포와자궁이형성증과의연관성에대한자료를구축하였다. 최근자궁경부암백신접종이적극적으로권장되고있어향후한국여성에서 HPV와자궁이형성증유병률의변화가있을것으로생각되며저자들은이연구가기본자료로역할을할것이라판단된다. [Ann Clin Microbiol 2013;16:87-91] 교신저자 : 신수, , 서울시동작구보라매길 39 서울특별시보라매병원진단검사의학과 Tel: , Fax: jeannie@snu.ac.kr

Korean J Clin Lab Sci. 20, 3() : 을발견하기도용이하며, PCR 방법을사용하였기때문에낮은개체수의 HPV도검출할수있어민감도가높아그유용성이기대되고있다 ( 이등, 2005). 이에군산의료원산부인과에서의뢰되는 Papanicolau smear

26 Korean J Clin Lab Sci. 20, 3() : 26-32 ISSN 738-35 The Relationship between Papanicolaou Smear Test and Human Papilloma Virus DNA Chip Test in the Uterine Cervix Young-Ju Lee, Ji-Hun Jung, and Da-Young