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 Jung Department of Pathology, Jeollabukdo Gunsan Medical Center, Gunsan 573-73, Korea The genotypes of Human Papilloma Virus (HPV) are important in the carcinogenesis of uterine cervical cancer. Diagnosis of uterine cervical cancer screening has been executed using Papanicolau method (Pap) and HPV DNA Chip method. We researched the interrelation of HPV DNA genotypes in single and multiple infections and analyzed the results of Pap and HPV DNA Chip tests at Gunsan Medical Center (GMC). The correlation analysis was surveyed on collected results from 599 patients who have been tested with both Pap and HPV DNA chip tests from November 200 to May 200 at GMC. The inconsistency between Pap and HPV DNA Chip tests was.%. The HPV DNA Chip genotype related with high risk cases were type 6 (3.5%), type 52 (0.5%), type 58 (0.%), and type 8 (3.%). Those related with low risk cases were type 70 (8.9%), type 6 (.7%), type 0 (.2%), type (.3%), and other types (.3%). Among the 95 cases of HPV positive status, 6 cases were associated with single infection; 08 (67.%) cases were related with high risk genotype; 9 (.8%) cases were low risk genotype; 3 (2.%) cases were related with other types. 29 cases were associated with double infections; 23 (79.3%) cases were high risks; 5 (7.2%) cases were mixed high and low risks; (3.5%) case was low risk. Key Words : Human papillomavirus (HPV), Papanicolau smear test (Pap). HPV DNA Chip. 서론 우리나라에서자궁경부암은유방암, 위암과대장암에이 어네번째로많이발생하며 ( 강과이, 2008), 자궁경부상피 내암을포함할경우여성암중발병빈도가높은질환이다. 자궁경부암과 Human papilloma virus (HPV) 감염과의많 은연구가이루어지면서, 자궁경부암의발생은전세계에서 HPV 감염과연관되어있다고보고되고있다 (Walboomers 등, 999; 김등, 2009). HPV 는약 8,000 개의염기쌍으로 구성된이중나선 DNA 유전체를가진바이러스로, 현재까 Corresponding author : Lee, Young-Ju. Department of Pathology, Jeollabukdo Gunsan Medical Center, Gunsan 573-73, Korea TEL : 063-72-5373, Fax: 063-72-537 E-Mail: lyj9929@naver.com Received: 23 February 20 Return for modification : 3 March 20 Accepted : 6 March 20 지알려진 HPV는 00가지이상이며, 지금까지연구를통해 0여가지가생식기감염과연관되어있음이밝혀졌고, 이중 20여가지아형이자궁경부암환자에게서분리되었다 (Walboomers 등, 999; 정등, 200). HPV genotype 은발암성의발생기전과관련하여고위험군과저위험군으로나누며고위험군에속하는아형으로는 HPV 6, 8, 3, 33, 35, 39, 5, 5, 52, 56, 58,59, 68, 73, 82이고저위험군은 6,, 0, 2, 3,, 5, 6, 70, 72, 8 등이다 (Munoz 등, 2003). 자궁경부암과전암성병변의선별검사로자궁경부세포진검사는편리성저비용등의장점으로많이사용되고있으며, HPV 감염여부를진단하는방법으로는 polymerase chain reaction (PCR), in situ hybridization, hybrid-capture assay, HPV DNA chip 검사가소개되고있다 ( 강과이, 2008). HPV 감염여부를알기위한여러가지방법들중에서 PCR 방법을이용한 HPV DNA chip은 20여종이상의다양한아형의 HPV를파악할수있을뿐만아니라다중의감염 26
Korean J Clin Lab Sci. 20, 3() : 26-32 을발견하기도용이하며, PCR 방법을사용하였기때문에낮은개체수의 HPV도검출할수있어민감도가높아그유용성이기대되고있다 ( 이등, 2005). 이에군산의료원산부인과에서의뢰되는 Papanicolau smear test (Pap 검사 ) 와 HPV DNA chip법의병행을통하여자궁경부암진단의선별검사를시행하게된이후두검사간의결과를분석하고 HPV DNA genotype, 단일및중복감염등에대하여알아보고자한다. 재료및방법. 재료 200년 월부터 200년 5월까지군산의료원산부인과에내원한환자중에 Pap 검사와 HPV DNA chip을병행한환자 599명을대상으로하였다 2. 방법 ) Papanicolau smear 검사 Pap 검사는 cytobrush 을이용한통상적인세포진검사방법으로시행되었으며검사결과분류는 Bethesda system (TBS) 을사용하였다. 2) HPV DNA chip HPV DNA chip 검사는환자의질에멸균된 speculum 을삽입하고검체채취용 sampler 에포함된 brush로자궁경부세포를채취하고 cytobrush 자체를전용용기에담아오면삼광의료재단에검사를의뢰하였다. HPV DNA chip (My Gene Company, Seoul, Korea) 은채취된자궁경부세포에서 HPV DNA를추출하여 PCR 법을통해증폭시킨후각각의저위험군과, 고위험군을포함하여 HPV 특이염기서열이점적된 oligonucleotide chip에반응시켜유전자형을검사하는방법으로삼광의뢰재단에의뢰하여얻어진결과를가지고시행하였다. HPV Genotype 6, 8, 3, 33, 35, 39, 5, 5, 52, 53, 5, 56, 58, 59, 66, 68을고위험군으로 6,, 3, 0, 2, 3,, 70을저위험군으로하여각각의 HPV 감염여부를판정하였다. 3. 분석 Pap 과 HPV DNA chip 검사결과를바탕으로두검사결 과의일치 / 불일치, HPV DNA genotype, 단일및중복감염 등에대하여분석하였다. 결과. 대상환자군의연령분포 본원에서시행한 Pap 및 HPV DNA Chip 을시행한총 599 명의환자들의평균연령은 2.2 세 (25-82 세 ) 였으며연 령별구성은 5-29 세 (2.2%), 30-39 세 (7.%), 0-9 세 (27%), 50-59 세 (27%), 60-69 세 (3.5%), 70 세이상 (5.3%) 이었다 (Table ). Table. Age distribution of patients Age (yrs) Number of patients (%) 5-29 30-39 0-9 50-59 60-69 >70 3 (2.2) 0 (7.) 62 (27) 62 (27) 8 (3.5) 32 (5.3) Total 599 2. Papanicolau smear 검사결과 자궁경부세포진검사의결과는음성은 26 예 (3.6%), atypical squamous cells of undetermined significance (ASCUS) 는 92 예 (32.%), atypical glandular cells of undetermined significance (AGUS) 는 예 (0.7%), atypical squamous cells suspicious of high grade squamous intraepithelial lesion (ASC-H) 은 9 예 (.5%), low grade squamous intraepithelial lesion (LSIL) 은 예 (9%), high grade squamous intraepithelial lesion (HSIL) 은 0 예 (.7%), carcinoma (CA) 는 6 예 (%), carcinoma in situ (CIS) 는 2 예 (0.3%), (Table 2). unsatisfactory 는 예 (0.%) 순으로관찰되었다 The Relationship between Papanicolaou Smear and HPV DNA Chip test 27
Korean J Clin Lab Sci. 20, 3() : 26-32 Table 2. Cytologic diagnosis of patients by Papanicolau smear Diagnosis* Number of patients (%) Negative ASCUS AGUS ASC-H LSIL HSIL CA CIS Unsatisfactory 26 (3.6) 92 (32.) (0.7) 9 (.5) (9) 0 (.7) 6 () 2 (0.3) (0.) Total 599 * ASCUS, atypical squamous cells of undetermined significance; AGUS, atypical glandular cells of undetermined significance; ASC-H, atypical squamous cells suspicious of high grade squamous intraepithelial lesion; LSIL, low grade squamous intraepithelial lesion; HSIL, high grade squamous intraepithelial lesion; CIS, carcinoma in situ; CA, carcinoma 3. HPV DNA chip 결과및아형분포 HPV DNA chip 검사결과는음성이 67.%, 양성이 32.6% 이었다. 고위험군 (67.7%), 저위험군 (0.3%), 고 - 저위험 군 (.6%), 기타 (7.%) 의순으로나타났으며, 단일감염은 82.6%, 이중감염은.9% 이었으며, 삼중감염과사중감염 도각각.0% 와.5% 로나타났다. HPV DNA genotype 은고위험군 6 형 (3.5%), 52 형 (0.6%), 58 형 (0.%), 53 형 (7.6%), 66 형, 33 형은 (.6%), 3 형 (3.85%), 8 형 (3.%), 39 형 (3.%), 68 형 (3.0%), 35 형 (2.%), 56 형 (2.%), 5 형 (.3%), 5 형 (0.8%), 5 형 (0.8%), 59 형 (0.%) 의순으로나타났다. 저위험군은 70 형 (8.9%), 6 형 (.7%), 0 형 (.3%), 형 (0.9%), 3 형 (0.%), 형은 (0.%) 의순으로나타났고, 그이외의유전형을보인예가 모두.3% 를나타났다 (Fig. ).. Pap 결과와 HPV DNA chip 결과비교 Fig.. Numbers of cases infected with individual HPV genotype Pap 검사음성의 HPV DNA Chip 결과는음성 (80.%), 고위험군 (3.%), 저위험군군 (.5%), (3.8%) 순으로관찰되었다 (Table 3). 혼합형 (.2%), 기타 ASCUS 의 HPV DNA Chip 결과는음성 (63.5%), 저위험 군 (5.7%), 고위험군 (2.%), 고 - 저위험군 (.6%), 기타 (7.8%) 로관찰되었고, ASC-H 의 HPV DNA Chip 결과는 음성 (55.6%), 고위험군 (33.3%), 기타 (.%), LSIL 의 HPV DNA Chip 결과는음성 (56.%), 저위험군 (%), 고위험군 (30.7%), 고 - 저위험군 (2.6%), 기타 (6.2%) 순으로관찰되었 다 (Table 3). HSIL 의 HPV DNA Chip 결과는음성 (0%), 고위험군 (90%) 로관찰되었으며, CIS 의 HPV DNA Chip 결과는고위 험군 (00%) 를 CA 는 HPV 음성 (33.3%), 고위험군 (66.7%) 로 관찰되었고 AGUS 는 HPV DNA Chip 결과고위험군 (75%), Table 3. HPV DNA Groups according to cytologic diagnosis HPV DNA Group* Cases of Cytologic diagnosis (%) Negative ASCUS ASC-H LSIL HSIL CIS CA AGUS Unsatisfactory High risks 35 (3.) (2.) 3 (33.3) 35 (30.7) 9 (90) 2 (66.7) 3 (75) 32 Low risks (.5) (5.7) 5 () 20 High-low risks 3 (.2) 3 (.6) 3 (2.6) 9 Others 0 (3.8) 5 (7.8) (.) 7 (6.2) (25) 3 Negative 209 (80.) 22 (63.5) 5 (55.6) 6 (56.) (0) 2 (33.3) 0 Total 26 92 9 0 2 6 599 * High risks of HPV are type 6, 8, 3, 33, 35, 39, 5, 5, 52, 53, 5, 56, 58, 59, 66, and 68. Low risks are type 6,, 3, 0, 2, 3,, and 70. Abbreviations are the same of Table 2. Total 28 The Relationship between Papanicolaou Smear and HPV DNA Chip test
Korean J Clin Lab Sci. 20, 3() : 26-32 Table. Effects of quality improvement process in histological fixation process of modified radical mastectomy tissue Cytologic diagnosis Negative 29 (26.9) ASCUS 35 (32.) AGUS 3 (2.8) ASC-H 3 (2.8) LSIL 27 (25) HSIL 6 (5.5) CA 5 (.6) Total 03 Single infection (n=6) HPV DNA multiple infection Dual infection (n=29) Triple infection (n=2) Quadruple infections (n=3) High Low Others High Low H-Low High H-Low H-Low (2.05) (57.9) (2.05) 9 0 (29.) 5 (.2) (2.9) (2.9) 7 (20.6) 3 6 (26.) 6 (26.) 7 (30.) 3 (3.) (.3) 22 (20) 2 (0) 2 (0) 5 (33.3) (33.3) (33.3) 3 Total (%) 52 70 50 9 6 89 기타 (25%), unsatisfactory 는 HPV DNA Chip결과음성 (00%) 로관찰되었다 (Table 3). 이들결과의진단일치율은 58.9% 이었다. 5. 중복감염 HPV DNA Chip 결과양성으로중단일감염의고위험군 (67.%), 저위험군 (/.8%), 기타 (2.%) 로관찰되었으며, 이중감염은고위험군 (79.3%), 고-저위험군 (7.2%), 저위험군 (3.5%) 를삼중감염은고위험군 (50%), 고-저위험군 (50%) 를사중감염은고-저위험군 (00%) 로관찰되었다 (Table ). 6. Multiple HPV infection 환자의 pap 결과비교단일감염의고위험군를나타내는환자의 Pap 결과는 negative (26.9%), ASCUS (32.%), AGUS (2.8%), ASC-H (2.8%), LSIL (25%), HSIL (5.5%), CA (.6%), 저위험군의환자는 negative (2.05%), ASCUS (57.9%), LSIL (2.05%), 기타형은 negative (29.%), ASCUS (.2%), AGUS (2.9%), ASC-H (2.9%), LSIL (20.6%) 로관찰되었으며이중감염의고위험군환자의 Pap 결과는 negative (26.%), ASCUS (26.%), LSIL (30.%), HSIL (3.%), CA (.3%) 로저위험군환자는 LSIL (00%) 로고-저위험군환자는 negative (20%), ASCUS (0%), LSIL (0%) 로관찰되었고삼중감염의고위험군환자는 LSIL (00%), 고-저위험군환자는 negative (00%) 로관찰, 사중감염은고-저위험군으로 negative (33.3%), ASCUS (33.3%), LSIL (33.3%) 로나타났다 (Table ). 고찰 현재까지자궁경부암의원인에대해명백히밝혀진바는없으나역학적연구결과들에의하면성적접촉 ( 다수의성적접촉, 특히젊은나이에시작된성적접촉 ), 흡연, 감염등이연관성이있는것으로알려져왔고, 특히 HPV의지속적인생식기감염이자궁경부암및전암병변의발생과밀접하 The Relationship between Papanicolaou Smear and HPV DNA Chip test 29
Korean J Clin Lab Sci. 20, 3() : 26-32 게연관된것으로알려지면서 HPV의감염을진단할수있는선별검사법에대한연구가활발히진행되고있다 (Ho 등, 995; Drain 등, 2002; 박등, 2003). 자궁경부암의조기진단선별검사인 Pap 검사가보편화되면서자궁경부암의발생률과사망률을크게감소시키고있으나위음성률이높아완벽한검사를할수는없으며, 자궁경부암의조기발견이가능한질환이나 Pap 검사의진단의정확도전체적으로 5% 에불과하다고하였다 (Lee 등, 997). 본원에서 Pap 검사를보완고자 HPV DNA Chip검사를추가로시행후결과를비교하였을때결과는두검사간의진단일치율은 58.9%, 불일치율은.% 였다. 검사간의진단이일치하지않은경우는 ) 세포도말표본의오류 ( 건조, 두껍게도말, 염증등 ), 2) 표본처리과정의오류, 3) 채취오류, ) 판독자의판독오류등의원인을추정해볼수있다. 임상의사들간의검체채취의처리과정이다르고두검사를동시에시행하는데있어 Pap 검사의판독의객관성의결여를생각할수있다. Pap 검사와 HPV 진단의일치율을높이기위해서는임상의사교육과병리의사의판독능력을높이기위한지속적인교육과훈련이필요하고병리의사와임상의사간의정보교환을통해반복되는오류의원인에대한해결책을찾아개선해야한다고생각한다. 세계적으로가장많은감염을보이는아형은 HPV 6, 8, 5, 33 이지만지역마다차이를보이며, HPV 6, 8은자궁경부암과가장밀접한관련이있는걸로알려져있지만 HPV 3, 33, 52, 58도자궁경부암과관련이있다고한다 ( 이등, 2005). 한국보고에의하면가장높은감염률을보이는아형이 HPV 6이며그다음은 58, 52로보고하였다 (An 등, 2003; Hwang 등, 200). 본병원에서 HPV DNA Chip검사아형의발생빈도는고위험군에서는 6형, 52형, 58형, 52형, 53형, 33형, 3형 39 형, 8형, 66형, 68형, 35형의순이며저위험군에서는 70형, 6형이관찰되었다. 정등 (200) 의보고에의하면 HPV 70 은저위험군에속하여생식기부위사마귀를나타낸다고알려져있으나고위험군의세포변화를보이며특히 0대이상에서 HPV 70감염이나타나면정기적인추적관찰을통한관리를해야할것으로생각된다 ( 정등, 200). 다중감염은상피내종양과연관성이높은것으로보고 되고있지만임상적인의미에대해서는연구자들간의차이 를보이며결론을내리기는어려운실정이다 (Fife 등, 200; Sasagawa 등, 200; Beerens 등, 2005). 다중감염과자궁경 부암과의연관성에대한보고도있는데, Lee 등은다중감염 이있는경우, 정상인에비해자궁경부암의상대위험성이 3.8 배인데반하여, 단일감염의경우는 9.9 배로서다중감 염시자궁경부암의발생빈도가더높다고보고하고있다 (Lee 등, 2003). HPV 가감염되었더라하더라도일시적이며환자의면역 체계에의해제거되며자궁경부암은서서히세포변화가진 행되는질환이므로세포검사나 HPV DNA Chip 검사, 조직 검사를병행하면서계속적으로추적관찰이필요할것으로 생각된다. 참고문헌. 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, 97:672-680. 2. Beerens E, Van Renterghem L, Praet M, Sturtewagen Y, Weyers S, Temmerman M, et al. Human papillomavirus DNA detection in women with primary abnormal cytology of the cervix: prevalence and distribution of HPV genotypes. Cytopathology. 2005, 6:99-205. 3. Drain PK, Holmes KK, Hughes JP, Koutsky LA. Determinants of cervical cancer rates in developing countries. Int J Cancer. 2002, 00:99-205.. Fife KH, Cramer HM, Schroeder JM, Brown DR. Detection of multiple human papillomavirus types in the lower genital tract correlates with cervical dysplasia. J Med Virol. 200, 6:550-559. 5. Ho GY, Burk RD, Klein S, Kadish AS, Chang CJ, Palan P, et al. Persistent genital human papillomavirus infection as a risk factor for persistent cervical dysplasia. J Natl Cancer Inst. 995, 87:365-37. 6. Hwang HS, Park M, Lee SY, Kwon KH, Pang MG. Distribution and prevalence of human papillomavirus genotypes in routine pap smear of 2,70 korean women determined by DNA chip. Cancer Epidemiol Biomarkers Prev. 200, 3:253-256. 30 The Relationship between Papanicolaou Smear and HPV DNA Chip test
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