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방광암의진단에서요세포검사, Nuclear Matrix Protein 22 (NMP22), Fluorescence in Situ Hybridization () 의효용성비교 Comparison of the Efficacy of Urine Cytology, Nuclear Matrix Protein 22 (NMP22), and Fluorescence in Situ Hybridization () for the Diagnosis of Bladder Cancer Won Tae Kim, Kyeongmee Park 2, Nam Hoon Cho 1, Won Sik Ham, Jin Sun Lee, Hee Jeong Ju, Yong Uk Kwon 3, Young Deuk Choi From the Department of Urology, Urological Science Institute, 1 Department of Pathology, Yonsei University College of Medicine, 2 Department of Pathology, Inje University College of Medicine, Seoul, 3 Department of Urology, College of Medicine, Kwandong University, Goyang, Korea Purpose: We compared the efficacy of urine cytology, nuclear matrix protein 22 (NMP22), and fluorescence in situ hybridization () for the detection of bladder cancer. Materials and Methods: Washing urine samples from 156 patients were evaluated for the detection of bladder cancer. Patients were divided into 3 groups. Group 1 was 106 patients with bladder cancer, group 2 was 30 patients with benign prostatic hyperplasia who underwent transurethral resection of the prostate without bladder cancer, and group 3 had gross hematuria without bladder cancer. The sensitivity and specificity of cytology, NMP22, and were compared. NMP22 positivity was defined as 10U/ml. was done with the UroVysion R system and positivity was defined as 2 abnormal urothelial cells with an abnormal signal from any out of 4 probes. Results: The overall sensitivity of urine cytology, NMP22, and was 60.4%, 75.5%, and 84.9%, respectively (p<0.001). The overall specificity of cytology, NMP22, and was 96.7%, 83.3%, and 93.3%, respectively (p=0.168). In group 3, the false-positive rates of cytology, NMP22, and were 20.0%, 55.0%, and 10.0%, respectively. In these patients with gross hematuria, the false-positive rate with NMP22 was significantly higher than with cytology or (p=0.004). The sensitivity of cytology, NMP22, and in low-grade bladder cancer patients was 25.9%, 51.9%, and 77.8%, respectively, and that in pta-1 bladder cancer patients was 40.6%, 65.6%, and 78.1%, respectively. In low-grade or in pta-1 patients, the sensitivity of the three diagnostic tools was significantly different (low grade; p<0.001, pta-1; p<0.001). Conclusions: is more sensitive in low-grade bladder cancer than is urine cytology and can be used as a diagnostic tool for the detection of primary and recurrent bladder cancer. NMP22 was affected by gross hematuria and thus has limitations for screening of bladder cancer. However, it can be used to follow-up bladder cancer. (Korean J Urol 2009;50:6-11) Key Words: Transitional cell carcinoma; In situ hybridization, fluorescence; Nuclear matrix protein 22 대한비뇨기과학회지제 50 권제 1 호 2009 연세대학교의과대학비뇨기과학교실, 비뇨의과학연구소, 1 병리학교실, 2 인제대학교의과대학병리학교실, 3 관동대학교의과대학비뇨기과학교실 김원태ㆍ박경미 2 ㆍ조남훈 1 함원식ㆍ이진선ㆍ주희정권용욱 3 ㆍ최영득 접수일자 :2008년 6월 25일채택일자 :2008년 11월 5일 교신저자 : 최영득연세대학교의과대학비뇨기과학교실서울시서대문구성산로 250 120-752 TEL: 02-2228-2317 FAX: 02-312-2538 E-mail: youngd74@yuhs.ac 6

김원태외 : 방광암진단검사법의효용성비교 7 서론방광암은처음진단시약 70-75% 가표재성방광암 (nonmuscle invasive; non-mibc, stage Ta, T1, or Tis) 이며, 이중에서 70% 이상이재발하고, 재발의 20-30% 가진행한다. 따라서표재성방광암은조기발견에따른적절한처치후세밀한추적관찰이필요하다. 1-5 요세포검사는방광경의보조적진단법으로일차성방광암의진단이나상피내암과같은잘보이지않는병소의진단및방광암의내시경처치후방광암재발을발견하기위하여주로사용되어왔다. 그러나요세포검사는높은특이도를보이는반면, 저등급분화도암에서민감도가낮다. 이에방광암진단에있어서민감도가높은진단기법이필요한경우가많다. 6,7 요세포검사의낮은민감도를보완하기위해, bladder tumor antigen (BTA), telomerase, urinary bladder cancer antigen (UBC), nuclear matrix protein 22 (NMP22) 와 fluorescence in situ hybridization assay () 등다양한검사법이개발, 시도되고있다. 는소변내비정상적인요상피세포를발견하는보조적인방법으로요상피세포의유전자이상을발견하여직접형광접합법을통해유전자이상을확인하는방법으로방광암진단에있어민감도와특이도가높은검사법이다. 현재는 3번, 7번, 17번, 그리고 9p21번염색체위치에 4가지색깔의직접형광접합을시행하는 multitarget (M-), UroVysion R (Abbott/Vysis, Downers Grove, USA) 이개발되어사용되고있다. 는요세포검사를보완하여병행시행하면방광암의진단과재발, 추적관찰에효과적이라는보고가많다. 6,8 한편방광암의종양지표로현재많이사용되고있는 NMP22는핵내부구조골격의부분으로 DNA 합성및복제조절, RNA 전사, 그리고유사분열에관여한다. 방광암환자의요중에는 NMP22 값이상승하는것으로알려져있어방광암의진단및재발등에이용되고있다. 9 본연구에서는방광암의진단에있어방광경검사와병용되는기존의요세포검사와비교하여, NMP22 및 의사용에따른각각의유용성에대해서알아보고자하였다. 대상및방법 1. 환자선택및검체채취소변검체로요세포검사및 NMP22, 를동시에시행한 156명의환자를대상으로하였다. 방광암으로경요도방 광종양절제술을시행받고방광이행상피암으로진단받은 106명을대상군으로하였고 (1군), 특이도를분석하기위한대조군으로과거방광암의병력이없고, 현재방광암이없는전립선비대증환자에서경요도전립선절제술을시행받는 30명의환자를 2군으로하였다. 방광암대상군의경우신장결석, 신장염, 신장암등의신장질환의기왕력이있거나, 방광수술기왕력, 방사선치료, 항암치료, 육안적혈뇨, 요로감염이있는경우는본연구에서제외하였다. 육안적혈뇨가검사결과에미치는영향을알아보기위해육안적혈뇨가있고, 전산화단층촬영및방광경에서방광암이없는환자 20명을 3군으로하여위양성률을분석하였다. 소변검체는전산화단층촬영이나초음파, 방광경에서방광암이의심되어경요도방광종양절제술을시행받는환자에서경요도방광종양절제술을시행하기전방광을세척하여소변검체를얻었고, 검체는요세포검사, NMP22, 의검사를위해 3종류의샘플로나누어각각의검사실로보냈다. 이후경요도방광종양절제술을실시하여, 조직검사결과를확인하였다. 방광암의형태및분화도분석은 1998년 World Health Organization/International Society of Urological Pathology (WHO/ISUP) 의분류를따랐고, 10 방광암의병기는 American Joint Committee on Cancer (AJCC) 의 TNM classification을따랐다. 11 대조군 (2군) 의소변검체는경요도전립선절제술을시행하기전에방광세척을통해검체를얻었고, 위양성률확인을위한 3군환자는방광경시에방광세척을통해검체를얻었다. 2. 요세포검사, NMP22, 의검사방법및분석요세포검사는통상적인방법으로관찰하였으며, 암세포가의심이될경우부터양성으로간주하였다. 를위한검체는 10분동안원심분리하고침전물을슬라이드에도말건조시켰다. 슬라이드를 73 o C에서 2분동안 2x standard saline citrate (SSC) 용액에담근후, 10분동안 37 o C, protease 용액 (Vysis, Dowmers Grove, USA) 에서처리시켰다. 이후실온에서 PBS로 5분동안세척하고 5분동안 1% 포름알데히드용액에서고정한후다시실온에서 PBS 용액으로세척했다. Carnoy 고정액으로고정하고, 70, 80, 100% acetic acid로각각 5분간탈수해고정과탈수를 3회반복하였다. 이후슬라이드를 73 o C 2xSSC/70% 포름알데히드용액에 5분간담가변성시키고 70%, 85%, 100% 에탄올에차례로 1분씩탈수시켰다. 건조시킨슬라이드에 5μl의 Vysis probe (Vysis, Downers Grove, USA) 를떨어뜨리고하룻밤을 hybridization시켜, NP-40 용액으로헹굼, 건조시켰다. DAPI용액 (4,6-diamidine, 2-phenylindole dihydrochloride) 으로대조염색을하였다. 각각의염색체는 Red (3번), Green

8 대한비뇨기과학회지 : 제 50 권제 1 호 2009 (7번), Aqua (17번), Yellow (9p21번) 로염색되었다. 염색체 3, 7, 17에서각각의형광표식인자가 3개이상으로증가하거나, 염색체 9p21에서형광표식인자가 2개이상증가하거나감소하는경우를비정상세포즉방광암세포로판정, 양성으로하였다. NMP22 검사는 NMP22 R test kit (Matritech Inc., Newton, USA) 를이용하여세척을통해얻은소변을원심분리하여상층액으로 EIA (enzyme immunoassay) 방법을이용하여요중 NMP22 값을구하였고, NMP22의값이 10 이상일경우 NMP22 양성으로하였다. 3. 자료분석 요세포검사, NMP22, 의민감도와특이도의분석을위한통계분석은 SPSS 12K를이용하였고, chi-square test로분석하였으며, p값이 0.05 미만인경우를통계적으로의의있는것으로판정하였다. 1. 환자분석 Table 1. Overall results of cytology, NMP22, and of each group Cytology NMP22 결 환자들의평균나이는 62.9±13.4세였고, 경요도방광종양절제술후방광암의분화도는저등급분화도가 54명, 고등급분화도가 52명이었다. 방광암의병기는 pta-1이 64명, pt2-4가 42명이었다. 2. 요세포검사, NMP22, 의민감도및특이도분석 방광암환자 106명에서요세포검사의민감도는 60.4% (64명), NMP22 75.5% (80명), 84.9% (90명) 로통계적으로유의한차이를보였다 (p<0.001). 2군에서각검사의특 Positive Negative Positive Negative Positive Negative Group 1 (n=106) 64 42 80 26 90 16 Group 2 (n=30) 1 29 5 25 2 28 Group 3 (n=20) 4 16 11 9 2 18 NMP: nuclear matrix protein, : fluorescence in situ hybridization, Group 1: bladder tumor patients group, Group 2: benign prostatic hyperplasia group, Group 3: hematuria or infection group 과 이도는요세포검사 96.7%, NMP22 83.3%, 93.3% 로 NMP22가상대적으로낮았지만, 통계적으로유의한차이는없었다 (p=0.168) (Table 1, 2). 3. 요세포검사, NMP22, 의위양성률분석 2군에서의위양성률은요세포검사, NMP22, 가각각 3.3%, 16.7%, 6.6% 로각군간에통계적으로유의한차이가없었다 (p=0.168). 3군에서위양성률은요세포검사, NMP22, 가각각 20.0%, 55.0%, 10.0% 로각군간에통계적으로유의한차이가있었다 (p=0.004). 요세포검사, NMP22, 결과에육안적혈뇨가미치는영향을보기위한 2군과 3군을비교한결과육안적혈뇨가있는경우특히 NMP22에서위양성률이상대적으로높게증가하였다 (Table 3). 4. 방광암의분화도와병기에따른요세포검사, NMP22, 의민감도 저등급분화도 54명의환자에서민감도는요세포검사 25.9%, NMP22 51.9%, 77.8% 로통계적으로유의한차이가있어 (p<0.001), 저등급분화도방광암의경우 의민감도가의의있게높았다. 고등급분화도 52명의환자에서민감도는요세포검사 96.2%, NMP22 100%, 92.3% 로고등급분화도방광암의경우검사간에통계적으로유의한차이가없었다 (p=0.125). 그러나전체적으로고등급 Table 2. Overall sensitivity, specificity, positive predictive value, and negative predictive value of urine cytology, NMP22, and for the detection of bladder tumors Cytology NMP22 p-value Sensitivity (%) 60.4 75.5 84.9 <0.001 Specificity (%) 96.7 83.3 93.3 0.168 PPV (%) 98.5 94.1 97.8 0.250 NPV (%) 40.8 49.0 63.6 0.059 NMP: nuclear matrix protein, : fluorescence in situ hybridization, PPV: positive predictive value, NPV: negative predictive value Table 3. False-positive rates of group 2 and group 3 Cytology NMP22 p-value False positive rate of group 2(%) 3.3 16.7 6.6 0.168 False positive rate of group 3(%) 20.0 55.0 10.0 0.004 NMP: nuclear matrix protein, : fluorescence in situ hybridization

김원태외 : 방광암진단검사법의효용성비교 9 Table 4. Sensitivity of cytology, NMP22, and for the detection of bladder tumors according to grade and stage Cytology NMP22 p-value Grade Low (n=54) (%) 25.9 51.9 77.8 <0.001 High (n=52) (%) 96.2 100 92.3 0.125 Stage pta-1 (n=64) (%) 40.6 65.6 78.1 <0.001 pt2-4 (n=42) (%) 90.5 90.5 95.2 0.648 NMP: nuclear matrix protein, : fluorescence in situ hybridization Table 5. The overall sensitivity, specificity, positive predictive value, and negative predictive value of combination of cytology, NMP22, and for the detection of bladder tumors NMP22 NMP22+ NMP22+ p-value Sensitivity (%) 56.6 54.7 69.8 52.8 0.051 Specificity (%) 96.7 96.7 100 100 0.565 PPV (%) 98.4 98.3 100 100 0.535 NPV (%) 38.7 37.7 48.4 37.5 0.522 Cyto: cytology, NMP: nuclear matrix protein, : fluorescence in situ hybridization, PPV: positive predictive value, NPV: negative predictive value 분화도를보이는경우민감도는증가하였다 (Table 4). 방광암의병기에따른민감도의분석결과 64명의 pta-1 의경우요세포검사가 40.6%, NMP22 65.6%, 78.1% 를보여각검사간에통계적으로유의한차이가있었다 (p< 0.001). 42명의 pt2-4 방광암환자에서민감도는요세포검사 90.5%, NMP22 90.5%, 95.2% 로각검사간에통계적으로유의한차이가없었다 (p=0.648). 병기역시전체적으로병기가증가함에따라각검사의민감도가증가하였다. 5. 두가지이상검사를병행했을경우의결과 방광암진단에있어요세포검사, NMP22, 중두가지검사를병행한경우에서민감도는요세포검사와 NMP22 를병행시에 56.6%, NMP22와 를병행시에 54.7%, 요세포검사와 를병행시에 69.8%, 그리고 3가지검사를병행시에 52.8% 로통계적으로유의한차이가없었다 (p=0.051). 특이도역시 96.7%, 96.7%, 100%, 100% 로통계적으로유의한차이가없었다 (p=0.565). 양성예측도 (positive predictive value; PPV) 는 98.4%, 98.3%, 100%, 100% 로나타났고 (p=0.535), 음성예측도 (negative predictive value; NPV) 는 38.7%, 37.7%, 48.4%, 37.5% 로각각통계적으로유의한 Table 6. Sensitivity of combination of cytology, NMP22, and for the detection of bladder tumors according to grade and stage NMP22 NMP22+ NMP22+ p-value Grade Low (n=54) (%) 18.5 22.2 48.1 18.5 0.001 High (n=52) (%) 96.2 88.5 92.3 88.5 0.444 Stage pta-1 (n=64) (%) 34.4 34.4 59.4 31.3 0.003 pt2-4 (n=42) (%) 90.5 85.7 85.7 85.7 0.890 Cyto: cytology, NMP: nuclear matrix protein, : fluorescence in situ hybridization 차이가없었다 (p=0.522). 두가지이상의검사를병행할때분화도와병기에따른민감도를보면요세포검사와 NMP22를병행시, 요세포검사와 를병행시, NMP22와 를병행시, 그리고 3 가지검사를병행시에저등급분화도방광암의경우각각 18.5%, 22.2%, 48.1%, 18.5% 로 NMP22와 를병행시에민감도가통계적으로유의하게높았다 (p=0.001). 고등급분화도방광암의경우는각각 96.2%, 88.5%, 92.3%, 88.5% 로각검사간통계적으로유의한차이는없었다 (p=0.444). 또한 pta-1인경우각각 34.4%, 34.4%, 59.4%, 31.3% 로역시 NMP22와 를병행시에민감도가통계적으로유의하게높았다 (p=0.003). pt2-4인경우는각각 90.5%, 85.7%, 85.7%, 85.7% 로높은병기에서는두가지이상의검사를병행하는경우통계적으로유의한차이가없었다 (p=0.890) (Table 5, 6). 고 NMP22는핵유사분열장치 (nuclear mitotic apparatus; NuMA) 의구성요소로서, 정상요로상피세포에비해방광암세포에서약 25배의세포내농도가증가한다. 12 또한이미방광암세포의 NMP22의측정값이방광암의분화도와연관이있어분화도가나쁜방광암에서증가한다. 13 그러나, NMP22의경우육안적혈뇨시에위양성률이높아, 다른연구들에서이런환자들을배제하면특이도와양성예측값이증가한다. 14,15 본연구에서도육안적혈뇨의유무에따라위양성률이 16.7% 에서 55.0% 로증가를보였다. 그러나, 요세포검사 찰

10 대한비뇨기과학회지 : 제 50 권제 1 호 2009 나 는의미있는증가를보이지않아상대적으로육안적혈뇨에영향을적게받음을알수있었다. 1980년대부터방광암을포함한이행세포암의 9번장완과단완의염색체변화에대한연구가시작되었고, 최근기술의발전으로홑염색체 (monosomy) 9번에의해시작, 세염색체 (trisomy) 7번에의해좀더침습성을지니는 2개의세포유전경로를파악하였다. 16,17 다양한세포유전학적인데이터를얻을수있게되면서다인자탐색법의개발이시작되었고, 이것은방광암환자에서진단, 추적관찰의보조적인방법으로사용되게되었다. 또한 Meloni 등 18 은최초로방광암환자의소변이나세척뇨에서나온세포를이용하여 와방광암의조직을동시에비교하였다. 는높은민감도와특이도를보여방광암환자의소변이나세척뇨를통해비침습적으로방광암을발견할수있다. 1,6,19-21 최근에는방광경검사에서음성을보이는방광암환자의재발위험을예측하는데도움이된다는연구들이나오고있다. 1,22,23 본연구에서도저등급분화도방광암환자에서요세포검사의낮은민감도에비해, 가비교적높은민감도를보여방광경검사에서육안적으로관찰하기어려운병변등에서 가방광암의조기진단에유용하리라생각한다. 본연구에서는방광암진단시요세포검사및 NMP22, 를동시에시행하여이들의결과를비교분석하였다. 이미국내에서도방광암에서의요세포검사및 NMP22, 등의결과가보고된바있으나이는 NMP22 및 에대한각각결과들만이보고되었다. 국내에서보고된 의유용성에대한연구는 Choi 등 20 이 의민감도와특이도를각각 88.9% 와 100% 로보고하였고, Kim 등 21 은 의민감도와특이도를각각 50% 와 97.5% 로보고하였다. 본연구에서 의민감도와특이도는각각 84.9% 와 93.3% 를보여, Choi 등 20 과비슷한결과를보였고, Kim 등 21 에비해다소높은민감도를보였다. 이러한차이는아마도배뇨소변과방광경시세척소변에대한검체의차이와고등급분화도및고병기환자의분포차이에의한것으로여겨진다. 발표연구에약간의차이는있으나방광암에서특이도는요세포검사, NMP22, 모두높아통계적차이는없으나민감도는요세포검사및 NMP22에비해 가통계적으로의의있게민감도가높다. 따라서 의경우높은민감도와특이도로방광암의조기진단에이용할수있을것으로생각한다. NMP22 에대한국내연구는 Kim 등 24 이기준값을 7.70U/ml 로할때 80% 의민감도를보고했고, 다른연구들에서기준값을 10U/ml로할때 Kim 등 25 은 73.1% 와 67.7% 의민감도와특이도를, Park 등 26 은 96.7% 와 65.5% 의민감도와특이도를, Kwon과 Hong 27 은 61.8% 와 95.8% 의민감도와특이도를각각보고했다. 본연구에서는 75.5% 와 83.3% 의민감도와특이도를보여비슷한값을보였으나, 위양성률이 50% 로높게나타나진단을위한 screening에는제한이있을것으로생각한다. 본연구에서 2개이상의검사를병행하는경우양성의진단기준을두가지검사에서모두다양성으로나오는것으로삼아상대적으로민감도와특이도가증가하지못했다. 요세포검사의민감도가저등급방광암에서상대적으로낮아서요세포검사와다른검사를병행할경우결과가더낮게나왔으나, 저등급방광암을제외하면통계적인유의성은없었다. 따라서각각의검사의특성을파악하여 NMP22의경우높은위양성률로방광암의진단 screening에적용하기보다는방광암치료후재발추적관찰에이용하는것이도움이되리라생각한다. 각검사에따른경제적관점을고려할경우, 현재요세포검사의검사비는 6,930원 ( 보험가 ), NMP22의검사비는 17,630 원 ( 보험가 ) 이다. 의경우는아직까지고가 ( 약 20만원 ) 의경제적문제로연구용으로주로시행되고있으며, 다른검사에비해상대적으로많은시약과장비및노력이요구되어방광암진단에손쉽게사용되기에는현실적으로어려움이있다. 많은연구들로좀더간편하고저렴하게시행할수있는 검사법이등장한다면추후에방광경검사를대체할수는없지만, 방광경검사의횟수를줄여주고, 요세포검사를대체할수있는 screening법및방광암의재발을추적관찰할수있는검사법이될수있을것으로생각한다. 결 저등급방광암의경우요세포검사에비해 의민감도가높아 는방광경과병용하여일차성방광암의진단이나방광암의재발에유용한방법으로생각한다. 그러나 의경우는경제적측면을고려할때좀더많은개발이요구된다. NMP22의경우는육안적혈뇨에대한위양성률이높아방광암의진단을위한 screening에는제한이있으며방광암의재발추적관찰에유용하리라생각한다. 론 REFERENCES 1. Bubendorf L, Grilli B, Sauter G, Mihatsch MJ, Gasser TC, Dalquen P. Multiprobe for enhanced detection of bladder cancer in voided urine specimens and bladder washings. Am J Clin Pathol 2001;116:79-86 2. Dalbagni G, Herr HW. Current use and questions concerning intravesical bladder cancer group for superficial bladder cancer. Urol Clin North Am 2000;27:137-46

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