인유두종바이러스 DNA 검사정도평가소개 Introduction of Human Papillomavirus (HPV) DNA Proficiency Testing Programs Abstract Cervical cancer is the second most common type of cancer among women worldwide and Human Papilloma Virus (HPV) infection is linked to more than 99 % of cervical cancer incidence. HPV 16 and HPV 18, the most prevalent high-risk types of HPV, account for about 70 % of all invasive cervical cancers worldwide. Accurate and internationally comparable HPV DNA detection and genotyping assays play essential roles in HPV epidemiological studies, HPV surveillance, vaccination impact monitoring, and prevention of cervical cancer by HPV infection. Currently, several HPV DNA detection and genotyping methodologies are used for diagnosis and monitoring of HPV-related diseases in clinical and research settings. However, there are some problems with reagents and laboratory performance, such as cross-reactions, false negatives, and contamination. To evaluate the accuracy and limitations of HPV detection, established HPV-infected cell lines have been used or plasmids containing the HPV genome have been constructed for proficiency testing. In this report, we introduced the developed HPV DNA proficiency testing programs. 질병관리본부국립보건연구원면역병리센터에이즈 종양바이러스과 이지은, 기미경, 송경주, 강춘 1) 들어가는말 인유두종바이러스 (Human Papillomavirus, HPV) 감염이지속되면자궁경부암 (cervical cancer) 또는생식기사마귀 (genital wart) 가유발되는주요원인으로알려져있다 [1]. 자궁경부암발생과관련하여 HPV 유전형중 HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -68은고위험군 (high-risk types) 으로, HPV-6 형과 11 형은저위험군 (low-risk types) 으로분류된다 [2, 3]. 고위험군 HPV 유전형중특히중요한 -16, -18형은자궁경부암환자의약 70% 에서 세포및 HPV 동시검사에관한관심이증가하고있다 [7]. HPV DNA 검사는염기서열분석, 중합효소연쇄반응, 유전형분석법등을기반으로다양한검사제품들이개발되어있다. 국내에서는주로 HPV 유전형검사에관한제품이다수개발되어식품의약품안전처의인증을받아임상검사에사용되고있으나, HPV 검사제품, 검사실, 검사자차이에따른정확도와민감도등평가를위한정도평가시스템은없는실정이다. 이에본글에서는세계보건기구 (The World Health Organization, WHO) 와외국의관련기관에서수행되고있는 HPV DNA 검사정도평가프로그램을소개하고자한다. 발견되고 [4], 생식기사마귀병변에서는저위험군인 HPV-6, 11형의감염이주로관찰된다 [5]. HPV DNA 유전형검사는질환을유발하는원인 HPV 유전형분석뿐만아니라, HPV 백신효과를평가하는데널리사용되고있다 [6]. 자궁경부암선별검사인세포검사 (Pap smear) 와동시에 HPV DNA 검사를하였을때자궁경부암에대한예측도가높게나타남으로인하여 몸말 WHO 는효과적인 HPV 감염률조사와 HPV 백신접종 효과를모니터링하기위하여 HPV 검사실서비스를질적으로 1) 교신저자 (ckang@nih.go.kr/ 043-719-8410) 790 www.cdc.go.kr
Vol. 8 No. 34 PUBLIC HEALTH WEEKLY REPORT, KCDC 향상시키고표준화하고자 2005 년 WHO HPV 검사실네트워크 Table 1. HPV DNA proficiency panel composition (HPV LabNet) 을구성하였다. HPV LabNet 의주요활동은 HPV or cell types (s) HPV (IU/genome equivalents per 5μl) HPV 백신에대한연구와백신접종효과를규명할수있는 HPV 검사에대한정도평가방법, 국제적평가기준과정도평가용표준물질등을개발하는것이다. HPV 유전형각각을독립적으로포함된생물학적자원확보가쉽지않기때문에, WHO는표준물질로특정유전형의 HPV DNA를포함하는재조합플라스미드형태로 14종의고위험유전형 (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68b) 과 2종의저위험유전형 (HPV-6, 11) 을포함하였다. WHO는 2008년첫정도평가를시작한이후전세계에서수행되고있는 HPV 검사와유전형분석현황을파악하기위하여 1-2 년간격으로정도평가를하였고, 2011년정도평가결과를최근에보고하였다 [8]. 2011년정도평가에서는고위험유전형인 HPV-68a 가추가되어총 17 종의유전형으로구성된 43개샘플과 DNA 추출과정에대한정도평가를위하여자궁경부암세포주 (HPV 감염혹은비감염 ) 를포함한 3개샘플이더해져총 46개의샘플로패널을구성하였다 (Table 1). HPV DNA 검사정도평가프로그램에참여의사를밝힌 100 여개의전세계검사실을대상으로정도평가를하여총 134 건의결과를보고받았고, 그결과에대하여검사실간비교등다양한방식으로분석하였다. 2008년, 2010 년 HPV DNA 검사정도평가와 -30 이는지속적인정도평가프로그램을수행함으로인하여 HPV 검사능력이향상되었음을보여주고있었다 (Table 2). 미국 CAP (College of American Pathologists) 에서는 Clinical Laboratory Improvement Amendments of 1988 (CLIA 1988) 를근간으로 HPV 감염유-무를검출하는정도평가를실시하고있으며, 인증제도운영으로종합적평가가최소 80% 이상인경우에한하여 HPV 검사에관한숙련도를인정하고, 80% 미만의경우는 0점을부여하고있다. 최근한연구에서는질병통제예방센터 (Centers for Disease Control and Prevention, CDC) 의지원을받아 pap smear와 HPV 검사를동시에수행하고있는검사실을대상으로 2008년, 2009 년, 2010 년에수행한정도평가결과를논문에게재하였다 [9]. 16 50 16 5 18 50 18 5 6 500 6 50 11 500 11 50 31 500 31 50 33 500 33 50 35 500 35 50 39 500 39 50 45 500 45 50 51 500 51 50 52 500 52 50 56 500 56 50 58 500 58 50 59 500 59 50 66 500 66 50 68a 500 68a 50 68b 500 68b 50 6, 56, 58, 68a 500 6, 56, 58, 68a 50 11, 18, 31, 51 500 11, 18, 31, 51 50 16, 33, 45, 51 500 16, 33, 45, 51 50 35, 39, 59, 66, 68b 500 35, 39, 59, 66, 68b 50 None 0 HPV16-positive 25 HPV-negative 0 HPV16-positive 2,500 Abbreviation: HPV= Human Papillomavirus www.cdc.go.kr 791
Table 2. Proficiency of detecting HPV types by laboratories that participated in 2011 proficiency panel, with data from 2008 and 2010 in comparison with all data sets submitted in 2011 Proficiency (%) Identical assays used in: No. of proficient assays / total no. of assays (%) All tests run by laboratories that participated in: 2008 2010 2011 2008 2010 2011 No. of proficient data sets / total no. of data sets from 2011 (%) 100 8/25 (32) 17/47 (36) 20/47 (42) 8/32 (25) 18/61 (29) 28/66 (42) 54/134 (40) 90-99 2/25 (8) 4/47 (8.5) 5/47 (11) 2/32 (6.2) 5/61 (8.2) 10/66 (15) 15/134 (11) 80-89 2/25 (8) 6/47 (13) 6/47 (13) 4/32 (12) 6/61 (9.8) 10/66 (15) 20/134 (15) < 80 4/25 (16) 5/47 (11) 6/47 (13) 5/32 (16) 9/61 (15) 6/66 (9.1) 12/134 (8.9) Not proficient 9/25 (36) 15/47 (32) 10/47 (21) 13/32 (41) 23/61 (38) 12/66 (18) 33/134 (25) Table 3. Responses for challenges by survey year, intended response, media, and method Factor No. of Laboratory responses Total correct, % p value Survey year 0.19 2008 3588 97.9 2009 4759 98.0 2010 6564 98.1 Challenge type (Intented response) 0.07 Positive 8086 97.9 Negative 6825 98.2 Media 0.002 ThinPrep 6684 98.0 Mixed media 4217 98.6 SurePath 2196 96.2 Digene STM 1814 99.2 Method <0.001 Digene Hybrid Capture 10323 99.1 Cervista 2465 96.3 Third Wave Invader 576 93.2 Use-developed method 530 98.5 Other commercial kit 465 90.5 Other (noncommercial) method 192 92.7 Roche AMPLOCOR 115 99.1 Access Genetics 85 98.8 Roche Linear Array 50 100.0 792 www.cdc.go.kr
Vol. 8 No. 34 PUBLIC HEALTH WEEKLY REPORT, KCDC 논문에따르면정도평가물질은 HPV 음성및 HPV 양성세포를배양하여이용하였고, HPV 검사에서흔히사용되고있는 4가지종류의 pap smear 수송용기 (media) 를이용하였다. pap smear 수송용기는각기관이선택한용기에정도평가물질을담아서검사실에배송하였다. 3년간총 476개의실험실이 14,911 건의검사결과를보고하였고, 정확도는평균 98% 였다. 연도별양성과음성의결과정확도는유의한차이가없었으나, 정도평가용물질제작에사용된 pap smear 수송용기 (media), HPV 검사방법간에는다소차이를보였다. 따라서논문에서는 HPV 검사의정확도를위하여 pap smear 수송용기 (media) 와 HPV 검사방법을선택하는데유용한정보를제공할것이라고보고하였다 (Table 3). 또한, 중국의국립임상검사소 (National center for clinical laboratories) 에서는 2012년 HPV 유전형분석을하고있는검사실에대한정도평가결과를발표하였다 [10]. 최근중국에서는자궁경부암선별검사법의하나로다양한종류의 HPV 유전형을구별할수있는검사법을사용하고있다. 각검사실에서사용하고있는바이러스검사법에대한표준화와분석능력을향상시키기위하여중국내에서감염률이높은 9개의 HPV 유전형 (HPV-6, 11, 16, 18, 31, 33, 39, 51, 52) 에대한정도평가를하였다. 국립임상검사소에서는우선 9개유전형의바이러스 DNA를포함하는재조합플라스미드를제조하였고, 제조한플라스미드를이용하여 23개의바이러스양성, 1개음성으로구성된총 24개의샘플로패널을제조하여 76개검사실에배포하였다. 이들검사결과를통하여중국에서사용하고있는다양한 HPV 유전형검사법의민감도와단일또는혼합형의바이러스유전형을 Table 4. HPV DNA proficiency panel composition and the performance of participants HPV types Number of HPV IU or GE/ml % participants correct (no./total) a The 2 false-positive types were from the same dataset False-positive results 16 10 6 98.7 (74/75) 39 16 10 5 93.3 (70/75) 18;18 16 10 4 88.0 (66/75) - 18 10 6 96.0 (72/75) 16 18 10 5 94.7 (71/75) - 18 10 4 86.7 (65/75) 16 6 10 6 94.4 (68/72) 42;53;52 6 10 5 94.4 (68/72) - 11 10 6 55.6 (40/72) 16 11 10 5 45.8 (33/72) - 31 10 6 96.0 (72/75) - 31 10 5 84.0 (63/75) 18 33 10 6 92.0 (69/75) 35, 58 a 33 10 5 92.0 (69/75) 31 39 10 6 21.3 (16/75) 16 39 10 5 18.7 (14/75) 52;35 51 10 6 42.7 (32/75) 58 51 10 5 36.0 (27/75) 66 52 10 6 88.0 (66/75) 33;16;51 52 10 5 80.0 (60/75) 51;11;51 18, 31 10 6 89.3 (67/75) 6, 16 a 33, 39, 52 10 6 17.3 (13/75) - 6, 16, 51 10 6 33.3 (24/72) - Negative - 94.7 (71/75) 18, 31 a ;31;16;11 구별해내는검사실의검사능력을평가하였다 (Table 4). 본정도평가를 시작으로매년정기적인정도평가를실시하고있으며, 검사실에서 수행하고있는 HPV 검사정확도를분석함으로 HPV 검사실의 질향상을목적으로하고있다. 맺음말 고위험 HPV 지속감염으로발생한자궁경부암은백신접종및 정기적인검진, 적절한치료를통해충분히예방이가능한 질환으로서, 2014 년미국식품의약품안전처에서는 pap smear 와 동시에실시한 HPV DNA 검사를자궁경부암선별검사방법으로 www.cdc.go.kr 793
승인하였다. 이와같은자궁경부암선별검사와 HPV 백신효과를모니터링하기위한필수적인변수로 HPV DNA 검출과유전형분석에대한관심이높아지면서 HPV 검사에대한질관리와정도평가의중요성이증가되었다. 소개한바와같이 WHO를비롯한몇몇외국의보건기관에서는임상검체, HPV 감염세포주, 혹은자체제작된정도평가물질을활용하여 HPV 검사실을대상으로정도평가를하고있다. 국내에서도대한진단검사의학회 (HPV-16, -18 감염및음성세포주이용 ) 와대한병리학회 (WHO 로부터구매한 HPV-16, -18 표준물질활용 ) 를중심으로 HPV 정도평가를수행하고있다. 그러나 HPV 검사를하고있는국내검사실을대상으로동일한기준에서 HPV 검출능력을재현성있게비교할정도평가시스템이없으므로그필요성이더욱증대되고있다. 이에국내에적합한 HPV 검사법에대한정도평가프로그램을개발하여국내의 HPV 검사현황을파악하고각검사실에서사용되고있는검사방법에대한표준화및질향상을유도함으로써국민보건향상에기여할수있을것으로기대된다. 5. Ault K.A. 2006. Epidemiology and natural history of human papillomavirus infections in the female genital tract. Infect. Dis. Obstet. Gynecol. Suppl: 40470. 6. Sepehr N., Brotherton J.M.L., et al. 2012. Fall in human papillomavirus prevalence following a national vaccination program. J. Infect. Dis. 206:1645-1651. 7. Katki H.A., Kinney W.K., et al. 2011. Cervical cancer risk for women undergoing concurrent testing for human papillomavirus and cervical cytology: a population-based study in routine clinical practice. Lancet. Oncol. 12:663-672. 8. Eklund C., Forslund O., et al. 2014. Global improvement in genotyping of Human papillomavirus DNA: the 2011 HPV labnet international proficiency study. J. Clin. Microbiol. 52:449-459. 9. Moriarty A.T., Bentz J.S., et al. 2013. The college of american pathologists first 3 years experience with high-risk human papillomavirus proficiency testing for cytology and other laboratories. Arch. Pathol. Lab. Med. 137:606-609. 10. Zhang R., Huang J., et al. 2013. Proficiency test for human papillomavirus genotyping in China. Intervirology 56:295-301. 참고문헌 1. Bulkmans N.W., Berkhof J., et al. 2007. Human papillomavirus DNA testing for the detection of cervical intraepithelial neoplasia grade 3 and cancer: 5-year follow-up of a randomised controlled implementation trial. Lancet. 370:1764-1772. 2. 2. Muῇoz N., Bosch F.X., et al. 2003. 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. 348:518-527. 3. Smith J.S., Lindsay L., et al. 2007. Human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: a meta-analysis update. Int. J. Cancer. 121:621-632. 4. Bosch F.X., Burchell A. N., et al. 2008. Epidemiology and natural history of human papillomavirus infections and typespecific implications in cervical neoplasia. Vaccine. 26:K1-K16. 794 www.cdc.go.kr