132 Mi Hee Sung, et al. p16 INK4a /Ki-67 Dual Immunostaining in Liquid-based Cytology ORIGINAL ARTICLE Korean J Clin Lab Sci. 2015, 47(3):132-139 http://dx.doi.org/10.15324/kjcls.2015.47.3.132 pissn 1738-3544 eissn 2288-1662 Evaluation of p16 INK4a /Ki-67 Dual Immunostaining in Liquid-based Cytology for Diagnosis of Uterine Cervical Dysplasia and Cancer Mi Hee Sung 1, Hoon Taek Lee 3, Min Shik Shin 1, Seo Young Oh 1, and Wook Youn Kim 1,2 1 Department of Pathology, Konkuk University Medical Center, Seoul 05030, Korea 2 Department of Pathology, Konkuk University School of Medicine, Seoul 05029, Korea 3 Department of Animal Biotechnology, Konkuk University, Seoul 05029, Korea 자궁경부이형성증과암의진단을위한액상세포검체에서 p16 INK4a /Ki-67 이중면역염색의평가 성미희 1, 이훈택 3, 신민식 1, 오서영 1, 김욱연 1,2 1 건국대학교병원병리과, 2 건국대학교의학전문대학원병리학교실, 3 건국대학교동물생명공학과 Recently, p16 INK4a /Ki-67 dual immunostaining has been introduced as a new biomarker protocol for early detection of uterine cervical dysplasia and cancer in liquid-based cytology (LBC). We performed the p16 INK4a /Ki-67 dual immunostaining using a CINtec R PLUS kit in a total of 109 LBC cases of cervicovaginal smear and compared its results with those from LBC, HPV hybrid capture II (HC II) test and histological diagnosis. Expression of p16 INK4a and Ki-67 was significantly associated with cases of LSIL or higher in cytological diagnosis and cases of cervical intraepithelial neoplasia (CIN) 1 or higher in histological diagnosis ( and, respectively). Among forty-six cases of atypical squamous cells of undetermined significance (ASCUS) in LBC, p16 INK4a and Ki-67 was expressed in 31 (67.4%), which were positively associated with cases of CIN I lesion or higher in histology. The sensitivity of p16 INK4a /Ki-67 dual immunostaining for finding lesions of CIN 1 or higher was 89.0%, which was higher than LBC. The specificity was 73.5%, which was higher than that of the HC II test. Based on these results, the p16 INK4a /Ki-67 dual immunostaining method can be a useful diagnostic marker for improving the sensitivity of LBC and the specificity of HC II test. Keywords: Liquid-based cytology, p16 INK4a /Ki-67 dual immunostaining, CINtec plus kit, HPV, Cervical cancer, Cervical dysplasia Corresponding author: Wook Youn Kim Department of Pathology, Konkuk University School of Medicine, Seoul 05029, Korea Tel: 82-2-2030-5646 E-mail: 20100182@kuh.ac.kr This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright 2015 The Korean Society for Clinical Laboratory Science. All rights reserved. Received: June 9, 2015 Revised 1 st : June 15, 2015 Revised 2 nd : July 17, 2015 Accepted: July 20, 2015 서론자궁경부암은국내여성에서발생하는생식기암중가장빈도가높은암으로 (National Cancer Center, 2011), 전암단계를거치기때문에조기에진단될경우치료효과가매우양호하다 (Lim 등, 2010). 자궁경부암의조기진단에대한방법으로는고식적도말검사와액상세포검사법이보편적으로사용되고있고, 자궁경부암의원인인인유두종바이러스 (human papilloma virus, HPV) 를검출하는분자진단법 (HPV DNA test) 이보조적으로사용되고있다. 그러나세포검사법들은관찰자간에주관적인이견으로재현성이낮
Korean J Clin Lab Sci. Vol. 47, No. 3, Sep. 2015 133 다고하였고 (Stoler 등, 2001), 특히위축성상피세포와미성숙화생상피세포는고등급편평상피내병변세포와감별이어려울수있다고보고하였다 (Nucci 등, 2003). 이처럼세포학적으로감별이어려운경우에는세포주기조절과세포증식의표지지수와관계되는 Ki-67 및세포주기조절에관련된 p16 유전자면역조직화학염색법이정확한진단에도움이된다고하였다 (Keating 등, 2001; Kim 등, 2003). 그리고 HPV DNA test는일시적인감염과종양형감염사이를구별할수없다는제한점이있어 (Waldstrom 등, 2013), 자궁경부암조기진단의정확성, 민감도의증가를위해세포검체에 p16과 Ki-67이라는새로운표지자에대한면역세포염색법연구가진행되었다 (Keating 등, 2001). p16 유전자는 CDK (cyclin-dependent kinase) 를불활성화시켜세포주기를조절한다고밝혀져있다 (Sano 등, 1998). 또한자궁경부암주요원인인 HPV의 E7 종양단백질이종양억제단백질 prb (phosphorylated retinoblastoma) 를불활성화시켜세포주기가활성화되는데 (Zappacosta 등, 2013), 이것은 E7 단백질에의해 prb가불활성화됨으로써음성되먹임기전으로조절되던 p16 단백질의발현이상대적으로증가한다고하였다 (Klaes 등, 2001). Ki-67은비히스톤핵단백질로세포주기에서발현되는세포의증식능을반영하는대표적인단백질로알려져있으며 (Dona 등, 2012), 자궁경부상피내종양의이형성정도와연관성이있어상피내종양의등급결정및진행에대한예측인자로밝혀졌다 (Kruse 등, 2004). 이에본연구에서는자궁경부액상세포와조직검체를대상으로 p16 INK4a /Ki-67 (INK4a: Inhibitor of CDK 4, a type) 이중면역염색을시행하여자궁경부액상세포검체를이용한 p16 INK4a /Ki-67 이중면역염색법이자궁경부암을조기진단하는선별검사로서유용한검사법이라는것을확인하고자실시하였다. 재료및방법 1. 연구재료본연구는건국대학교병원임상연구심의위원회 (Institutional Review Board, IRB) 에서승인을얻은액상세포와조직검체를이용하여시행하였다 ( 승인번호 KUH1210026). 검체는 2013년 8월부터 2014년 5월까지건국대학교병원에서자궁경부액상세포검사를받고, 세포검사후시행한조직학적검사로확진을받은환자의액상세포, 조직검체각각 109례를대상으로실험하였다. 2. 자궁경부액상세포 p16 INK4a /Ki-67 이중면역염색및평가자궁경부액상세포는 Surepath (TriPath imaging, USA) 의잔 류검체로새로운슬라이드를제작하였고, p16 INK4a /Ki-67 이중면역염색은 CINtec R PLUS kit (Roche mtm laboratories, Germany) 를사용하였으며 polymer법으로시행하였다. 항원을복구하기위해서구연산완충액에슬라이드를넣고, water bath 내에서 97 o C (95 99 o C) 의온도로 15분간유지하였다. 다음단계로는실온에서 20분간방치한후, 비특이적반응을차단하기위해 peroxidase blocking solution을 5분간처리하였다. 1차항체로 RTU (ready to use) 인 p16 INK4a (clone E6H4, mouse monoclonal antibody), Ki-67 (clone 274-11 AC3, rabbit monoclonal antibody) 을 30 분동안반응을시킨후, 1차항체의반응을가시화하기위해서 horseradish peroxidase 와 alkaline phosphatase 를각각 15분동안염색하였다. 발색은 3.3'-diamonibenzidine substrate 를 10 분동안그리고 fast red substrate에서 15분간씩 2회반응시켰다. Mayer hematoxylin (DAKO, Denmark) 으로대조염색하고봉입하여광학현미경 (Olympus BX51, Japan) 으로관찰하였다. p16 INK4a /Ki-67 이중면역염색의판정은동일세포내에서세포질은 p16에의해갈색으로염색되고핵은 Ki-67에의해적색으로염색된세포가최소하나또는그이상일때를양성으로판정하였고, 슬라이드에이중염색된세포가하나도없거나세포질과핵어느한곳에만염색되면음성으로판정하였다 (Wentzensen 등, 2012; Edgerton 등, 2013). 3. 자궁경부조직 p16 INK4a /Ki-67 이중면역염색자궁경부조직검체대상은대표파라핀블록을선정하여 3 m 로절편하였고, p16 INK4a /Ki-67 이중면역염색에사용하였다. 표본슬라이드는탈파라핀과함수과정을거쳐구연산완충액 (epitope retrieval solution) 에담가 water bath 내에서 97 o C (95 99 o C) 의온도로 15분간처리하였다. 1차항체로반응후 3.3'-diamonibenzidine substrate를 10분그리고 fast red substrate에서 15분동안발색하였다. Mayer hematoxylin (DAKO, Denmark) 으로대조염색하고광학현미경 (Olympus) 에서검경하였다. 4. Hybrid capture II high-risk HPV DNA test Hybrid capture II (HC II) 검사는고위험군 (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 및 68) 의탐침자를사용하여실시하였다. 검체의채취는자궁경부액상세포와동일한부위에서동시에 cervical sampler (QUIAGENE, USA) 를이용하여세포를채취하였다. 채취된세포는 denaturation buffer를넣고 66 o C에서 1시간반응시킨후, 고위험군 HPV RNA 탐침자혼합액과 66 o C에서 1시간교잡반응을시행하였다. 검체는 RNA/DNA 교잡에대한특이항체로도포된 microplate에옮겨담아 1시간동안반응시킨후,
134 Mi Hee Sung, et al. p16 INK4a /Ki-67 Dual Immunostaining in Liquid-based Cytology detection reagent 1을이용하여 alkaline phosphatase-conjugated antibody 와 30분반응하였다. 세척후, detection reagent 2를이용하여형광반응 (CDP-Star with Emerald II) 시켜, 검체에존재하는 HPV DAN의양을 luminometer를사용하여측정하였다 (DML2000, Digene, UK). 양성대조군 (HPV 16 DNA, 1 pg/ml) 은키트에있는 3개의평균값을 cut off 값으로정하고, 검체측정값을 cut off 값으로나눈값이 1.0미만이면음성, 1.0이상이면양성으로판독하였다. 5. 통계통계학적분석은 SPSS Windows version 18.0 프로그램을이용하였고, χ 2 검정법으로분석하였다. p 값이 0.05 이하인경우통계학적으로유의성이있다고평가하였다. 결과 1. 자궁경부액상세포검체에서 p16 INK4a /Ki-67 이중면역염색의발현액상세포검체에서 p16 INK4a /Ki-67 이중면역염색의발현은 Fig. 1에서보는바와같이 NILM (Negative for intraepithelial lesion or malignancy) 은일부세포에서 p16이세포질에갈색으로염색되었지만, 동일세포에 p16과 Ki-67이동시에염색된세포는관찰되지않았으며 (Fig. 1A), LSIL (Low grade squamous intraepithelial lesion), HSIL (High grade squamous intraepithelial lesion), squamous cell carcinoma 로갈수록강한양성발현을보였다 (Fig. 1B, C, D). Table 1에서제시한대로 NILM은세포면역염색결과 10례 (100%) 모두에서음성이었고, 반면에, ASCUS (Atypical squamous cells of undetermined significance) 는 46 례중 31례 (67.4%), LSIL은 33 례중 25 례 (75.8%), HSIL과 carcinoma 에서는 16례, 4례모두 (100%) 에서양성이었다. 또한조직검사결과에따른세포면역염색양성율은 NILM 34례중 9례 (26.5%), CIN I (Cervical intraepithelial neoplasia I) 37례중 31례 (83.8%), CIN II/CIN III 29례중 27례 (93.1%), carcinoma 7례중 7례 (100%) 에서양성소견을보였다. 액상세포잔유검체를이용한 p16 INK4a /Ki-67 이중면역염색검사는세포학적및조직학적이상소견의중증도에따라양성율이증가하였고, 로유의한차이를확인하였다. 2. 자궁경부조직검체에서 p16 INK4a /Ki-67 이중면역염색의발현자궁경부조직검체에서 p16 INK4a /Ki-67 이중면역염색을적용한결과, Fig. 1을보면 NILM은동일세포에 p16과 Ki-67이동시에발현되지않았고 (Fig. 1E), CIN I, CIN III, squamous cell carcinoma 에서는강한양성반응을확인하였다 (Fig. 1F, G, H). Table 2에서제시한대로 NILM 34례중 1례 (2.9%), atypical cells 2례중 1례 (50%), CIN I 37례중 27례 (73%), CIN II/CIN III 29례중 29례 (100%) 그리고 carcinoma 7례중 7례 (100%) 에서양성소견을보였다. 또한자궁경부조직검체에서 p16 INK4a /Ki-67 이중면 Fig. 1. Results of p16 INK4a /Ki-67 dual immunostaining for liquid-based cytology (A, B, C, D) and tissue (E, F, G, H). (A) Metaplastic cells cytoplasm staining for p16 interpreted as negative ( 400). (B) LSIL showing positivity for p16 INK4a /Ki-67 dual immunostaining ( 1000). (C) HSIL, p16 INK4a /Ki-67 dual immunostaining in positive cells showed strongly staining ( 1000). (D) Squamous cell carcinoma, p16 INK4a /Ki-67 dual immunostaining in positive cells showed strongly staining ( 1000). (E) Negative, nuclear of basal layer in Ki-67 staining interpreted as negative ( 200). (F) CIN I in p16 INK4a /Ki-67 dual immunostaining showed positive in focal lesion ( 200). (G) CIN III in p16 INK4a /Ki-67 dual immunostaining showed positive ( 200). (H) Squamous cell carcinoma in p16 INK4a /Ki-67 dual immunostaining showed positive ( 200).
Korean J Clin Lab Sci. Vol. 47, No. 3, Sep. 2015 135 Table 1. Correlation between cytologic diagnosis, histologic diagnosis and p16 INK4a /Ki-67 dual immunocyochemistry p16 INK4a /Ki-67 dual immunocyochemistry No. (%) Negative Positive Total Cytologic Dx NILM 10 (100.0) 0 (0.0) 10 (100.0) ASCUS 15 (32.6) 31 (67.4) 46 (100.0) LSIL 8 (24.2) 25 (75.8) 33 (100.0) HSIL 0 (0.0) 16 (100.0) 16 (100.0) Carcinoma 0 (0.0) 4 (100.0) 4 (100.0) Total 33 (30.3) 76 (69.7) 109 (100.0) Histologic Dx Negative 25 (73.5) 9 (26.5) 34 (100.0) Atypical cells 0 (0.0) 2 (100.0) 2 (100.0) CIN I 6 (16.2) 31 (83.8) 37 (100.0) CIN II/CIN III 2 (6.9) 27 (93.1) 29 (100.0) Carcinoma 0 (0.0) 7 (100.0) 7 (100.0) Total 33 (30.3) 76 (69.7) 109 (100.0) p-value Dx, diagnosis; NILM, Negative for intraepithelial lesion or malignancy; ASCUS, Atypical squamous cells of undetermined significance; LSIL, Low grade squamous intraepithelial lesion; HSIL, High grade squamous intraepithelial lesion. Table 2. Results of p16 INK4a /Ki-67 dual immunohistochemistry according to histologic diagnosis p16 INK4a /Ki-67 dual immunohistochemistry No. (%) Negative Positive Total Histologic Dx Negative 33 (97.1) 1 (2.9) 34 (100.0) Atypical cells 1 (50.0) 1 (50.0) 2 (100.0) CIN I 10 (27.0) 27 (73.0) 37 (100.0) CIN II/CIN III 0 (0.0) 29 (100.0) 29 (100.0) Carcinoma 0 (0.0) 7 (100.0) 7 (100.0) p16 INK4a /Ki-67 immunocytochemistry Negative 29 (87.9) 4 (12.1) 33 (100.0) Positive 15 (19.7) 61 (80.3) 76 (100.0) p-value Dx, diagnosis; CIN I, Cervical intraepithelial neoplasia I; CIN II, Cervical intraepithelial neoplasia II; CIN III, Cervical intraepithelial neoplasia III. 역염색과액상세포검체의 p16 INK4a /Ki-67 이중면역염색간의일치율은총 109례중 90례 (82.3%) 이었다. 따라서조직학적이상소견의정도에따라 p16 INK4a /Ki-67 이중면역조직염색의양성율이증가하였으며조직과세포검체에서 p16 INK4a /Ki-67 이중면역염색의일치율은 로통계학적으로유의성이높은것으로나타났다. 3. 자궁경부액상세포 p16 INK4a /Ki-67 이중면역염색과 Hybrid capture II 결과비교자궁경부액상세포검체 p16 INK4a /Ki-67 이중면역염색의양성은총 76례로 HC II의양성이 69례 (90.8%) 이었고, HC II viral load 는 RLU (relative light unit) 1 50에서 16례 (55.2%), RLU 51-100 에서 5례 (83.3%), RLU>100 에서 48례 (82.8%) 로나타내었다. 따라서 p16 INK4a /Ki-67 이중면역세포염색은 HC II 검사결과가양성 이고, HC II viral load 값이클때양성율이증가하였으며, 통계학적으로유의한결과를보였다 (p<0.05). 4. 자궁경부액상세포 p16 INK4a /Ki-67 이중면역염색의임상적유용성자궁경부액상세포검체에서 p16 INK4a /Ki-67 이중면역염색의임상적유용성은민감도, 특이도, 예측도로확인하였다 (Table 5). 그결과, CIN I 이상의병변에서 p16 INK4a /Ki-67 이중면역세포염색은 89.0% 의민감도, 73.5% 의특이도를보여액상세포검사에서 57.5% 의민감도와 HC II 검사에서 29.4% 의특이도보다높았으며통계적으로유의한상관관계를나타내었다 (). 5. 경계성진단에서 p16 INK4a /Ki-67 이중면역세포염색의발현경계성진단인 ASCUS로진단된자궁경부병변은생검으로조직
136 Mi Hee Sung, et al. p16 INK4a /Ki-67 Dual Immunostaining in Liquid-based Cytology Table 3. Correlation between p16 INK4a /Ki-67 dual immunocytohemistry and Hybrid capture II test p16 INK4a /Ki-67 dual immunocyochemistry No. (%) Negative Positive Total p-value HC II Negative 9 (56.3) 7 (43.8) 16 (100.0) p=0.012* Positive 24 (25.8) 69 (74.1) 93 (100.0) HC II viral load (RLU) 1 50 13 (44.8) 16 (55.2) 29 (100.0) p=0.019* 51 100 1 (16.7) 5 (83.3) 6 (100.0) >100 10 (17.2) 48 (82.8) 58 (100.0) HC II, Hybrid capture II High-Risk HPV DNA test; RLU, Relative light unit. Table 4. Sensitivity, specificity and predictive values of p16 INK4a /Ki-67 dual immunocytochemistry in CIN I+, CIN II+ Sensitivity (%) Specificity (%) PPV (%) NPV (%) p-value CIN I+ LBC 57.5 67.7 79.3 42.6 p16 INK4a /Ki-67 89.0 73.5 87.8 75.8 HC II 93.2 29.4 73.9 66.7 CIN II+ LBC 55.6 100.0 100.0 68.0 p16 INK4a /Ki-67 94.4 73.4 79.1 92.6 HC II 91.7 29.4 57.9 76.9 LBC, Liquid-based cytology; PPV, Positive predictive value; NPV, Negative predictive value; CIN I+, Cervical intraepithelial neoplasia of grade I or higher; CIN II+, Cervical intraepithelial neoplasia of grade II or higher; HC II, see table 3. Table 5. Detection rate of p16 INK4a /Ki-67 dual immunocytochemistry for ASCUS in liquid-based cytology p16/ki-67 dual immunocyochemistry Total No. Negative % Positive % Negative 14 2 14.3 Atypical 2 2 100.0 CIN I 18 13 72.2 CIN II/CIN III 11 11 100.0 Carcinoma 1 1 100.0 p-value See Table 2. 을절제하여조직학적으로확진하였다 (Table 5). 자궁경부액상세포검사에서 ASCUS 로분류된 46례는조직학적검사결과 NILM 14 례, atypical 2례, CIN I 18례, CIN II/CIN III 11례, carcinoma 1례로분류되었다. 그리고 ASCUS 로진단된세포검체에 p16 INK4a /Ki-67 이중면역염색을적용해본결과, CIN I에서는 18례중 13례에서양성반응을보여 72.2% 의양성율이확인되었고, CIN II/CIN III 11 례, carcinoma 1례가모두 100% 의양성율을보여유의한결과를확인하였다 (). 고찰본연구에서는자궁경부액상세포검체에 p16 INK4a /Ki-67 이중면역염색을시행하여자궁경부암의조기발견과세포검사의불확실한진단을보완하기위한검사법으로유용성을확인하였다. p16 INK4a /Ki-67 이중면역염색검사에대한선행연구결과를보면 Yoshida 등 (2011), Dona 등 (2012) 그리고 Byun 등 (2013) 은세포학적및조직학적이상소견의중증도에따라양성율이증가하여자궁경부병변을평가하는검사방법으로유용하다고보고하였다. Lee 등 (2006) 은 HPV 고위험군이자궁경부암의원인으로 99% 이상을차지한다고하였고, Klaes 등 (2001) 과 Keating 등 (2001) 은
Korean J Clin Lab Sci. Vol. 47, No. 3, Sep. 2015 137 HPV에감염된자궁경부병변에서 p16이과발현된다고하였으며, Stanley 등 (2002) 은 p16의과발현은 HPV가단순히감염된상태를넘어활동적인상태에있다는것을의미한다고하였다. 또한 Choi 등 (2004) 은 Ki-67이이형성증의정도가진행될수록발현율이증가한다고하였다. 본연구에서도액상세포검체와세포검사후에조직생검, 자궁경부원추형절제술및자궁적출술등을통해얻어진조직검체에 p16 INK4a /Ki-67 이중면역염색을동시에적용해본결과, 양성율은 NILM 0%, HSIL과 carcinoma 100% 로분석되어세포와조직에서병변의중증도에따라증가하였고, LSIL 또는 CIN I 이상의병변과관련이있었다 (Table 1, 2). 또한 HC II가양성일때 p16 INK4a /Ki-67 이중면역염색의양성율이높아자궁경부암의주요원인인 HPV 감염과관련이있는검사법으로확인되었고, 선행연구와유사한결과가관찰되었다 (Table 3). 따라서 p16 INK4a /Ki-67 이중면역염색은자궁경부병변을검출할수있는검사방법이며, 자궁경부암을조기에발견하기위해액상세포검체에적용하는것이유용하다고사료된다. 경계성진단인 ASCUS는 Yeo 등 (2005) 의연구에서세포학적진단기준의모호성으로임상적치료에있어논란이된다고하였다. Kwon 등 (2005) 은반복적인세포진검사로인한비용의과다가발생할수있으며과도한조직검사로인한불안을야기한다는문제점을제시하였다. 이러한문제점을개선하기위해 ASCUS로진단된세포검체로분자검사법과면역화학적검사법에대한연구가선행되어졌고, 액상세포검체에 p16 INK4a /Ki-67 이중면역염색을적용한결과, Wentzensen 등 (2012) 은 40.2%, Dona 등 (2012) 은 43.7% 의양성율을보고하였다. 또한 Schmidt 등 (2011) 은 ASCUS 병변중조직검사결과, CIN II 이상으로확진된경우에 p16 INK4a /Ki-67 이중면역염색을적용하면민감도와특이도가높다고보고하였다. 본연구의결과에서는 ASCUS 의증례에서 67.4% 의양성율을보여 (Table 2) 선행연구보다높은양성율을관찰하였다. 그이유는대상군의차이, 관찰자간의이견, 액상세포검체의차이라고본다. 또한총 46례중에서 31례가 CIN I 이상의병변으로확진되었으며, CIN II 이상의병변에서는 100% 의양성율을보여 (Table 5) 선행된연구결과와유사하였다. 따라서세포학적으로판단이어려운 ASCUS 의증례에서는액상세포검사와 p16 INK4a /Ki-67 이중면역염색을병행해서검사하는것이미확정비정형세포로인한세포진단의문제점을보완하고, 불필요한조직검사와재검을줄이기위하여필요하다고생각된다. 조직학적진단인 CIN II 이상의병변을발견하기위해서는세포검사만시행했을때보다 HPV 분자검사를같이시행했을때민감도가높다고하였다 (Reuschenbach 등, 2010; Petry 등, 2011; Overstad 등, 2011; Szarewski 등, 2012). 그러나 HPV 분자검사 법은낮은특이도가한계로지적되고있어 (Ikenberg 등, 2013) p16 INK4a /Ki-67 이중면역염색을적용한결과, HPV 분자검사보다특이도가높다고보고하였다 (Waldstrom 등, 2013). 본연구에서도 CIN I 이상의병변에서 p16 INK4a /Ki-67 이중면역염색은 89.0% 의민감도, 73.5% 의특이도를보였다 (Table 4). 액상세포검사만진행했을경우는 p16 INK4a /Ki-67 이중면역염색을시행했을때보다민감도와특이도가낮게나타나재현성이낮은검사로생각되었고, HC II 검사는 p16 INK4a /Ki-67 이중면역염색보다민감도가높지만특이도가낮아위양성의가능성이높기때문에정확한진단을내리기위해서는 p16 INK4a /Ki-67 이중면역염색을시행하는것이유용하다고사료된다. 결론적으로본연구에서는 p16 INK4a /Ki-67 이중면역염색의양성율은세포학적및조직학적이상소견의중증도에따라의미있게증가하였고, 경계성진단, 상피내병변및자궁경부암에적용한결과유용성이높은것으로나타났다 (p<0.05). 그리고자궁경부암의선별검사에서 p16 INK4a /Ki-67 이중면역염색법이세포검사의낮은민감도와 HC II의낮은특이도를보완하여세포검사의정확도를높이는검사방법으로확인되었다. 따라서자궁경부암을조기에발견하여치료하기위해서는액상세포검사와민감도가높은 HC II 검사뿐만아니라특이도가높은 p16 INK4a /Ki-67 이중면역염색법을병행하는것이정확한결과를판정하는데유용하다고생각한다. 향후연구과제로더정확한진단을내리기위해서는많은증례를대상으로세포진단의정확성을향상시키는연구가필요하다고사료된다. 요약최근 p16 INK4a /Ki-67 이중면역염색은액상세포검사에서자궁경부이형성증과암을조기에발견하기위한새로운생체표지자로대두되고있다. 저자들은자궁경부질도말의액상세포검체총 109 례에서 CINtec R PLUS kit를사용하여 p16 INK4a /Ki-67 이중면역염색을시행하였고, 그결과를액상세포검사, HPV hybrid capture II (HC II) 검사그리고조직학적진단과서로비교하였다. p16 INK4a / Ki-67 양성발현은세포학적진단에서저등급편평상피내병변이상의증례그리고조직학적진단에서자궁경부상피내종양 1등급이상의증례에서유의미하게높았다. 액상세포검사상비정형편평상피세포소견을보이는 46례중, 31례 (67.4%) 가 p16 INK4a 과 Ki-67 양성소견을보였고, 이러한양성증례들은조직검사에서도대부분자궁경부상피내종양 1등급이상의병변에해당하였다. 자궁경부상피내종양 1등급이상의병변을발견하기위한 p16 INK4a /Ki-67 이중면역염색의민감도는액상세포검사보다높은 89.0% 였고,
138 Mi Hee Sung, et al. p16 INK4a /Ki-67 Dual Immunostaining in Liquid-based Cytology 특이도는 73.5% 로 HC II 검사보다높게분석되었다. 따라서, p16 INK4a /Ki-67 이중면역염색방법은액상세포검사법의민감도와 HC II 검사법의특이도를보완하기위한진단적검사로유용하다고할수있다. Acknowledgements: None Funding: None Conflict of interest: None References 1. Byun SW, Lee A, Kim S, Choi YJ, Lee YS, Park JS. Immunostaining of p16 INK4a /Ki-67 and L1 capsid protein on liquid-based cytology specimens obtained from ASC-H and LSIL-H cases. Int J Med Sci. 2013, 10:1602-1607. 2. Choi SK, Kim TJ, Hong SB, Lee HT. Expression of p53 and Ki-67 in cervical dysplasia with human papilloma virus infection or non-infection. Korean J Clin Lab Sci. 2004, 36(2):178-184. 3. Dona MG, Vocaturo A, Giuliani M, Ronchetti L, Rollo F, Pescarmona E, et al. p16/ Ki-67 dual staining in cervico-vaginal cytology: correlation with histology, Human Papillomavirus detection and genotyping in women undergoing colposcopy. Gynecol Onocol. 2012, 126:198-202. 4. Edgerton N, Cohen C, Siddiqui MT. 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