Korean Journal of HBP Surgery Vol. 13, No 2, June 원저 간세포암에서 Vimentin 과 MMP2 발현의임상적의의 Clinical Importance of Vimentin and MMP 2 Expression in Hepatocellular Carcinoma Background / Purpose: Hepatocellular carcinoma (HCC) is a common cancer with a poor prognosis. A better understanding of the molecular mechanisms underlying the genesis of HCC, as well as the progression of HCC, allow for improved prediction of the prognosis of patients with HCC and more effective treatment. In this study, we determined the expression of vimentin and matrix metalloproteinase 2 (MMP 2) and evaluated the clinical significance in (HCC). Methods: Immunohistochemical staining was performed for vimentin and MMP 2 in 98 surgically resected HCC specimens using the tissue microarray method. The clinicopathologic data and the outcomes were reviewed, and the levels of expression of vimentin and MMP 2 were compared. Results: Positive expression of vimentin and MMP 2 was observed in 7.1% and 41.8% of specimens, respectively. The overexpression of vimentin and MMP 2 had a positive correlation with tumor cell proliferative activity, as measured by the Ki-67 labeling index (p<0.001 and p=0.043, respectively), but was not correlated with the TUNEL labeling index. Other clinicopathological factors, such as platelet count, serosal invasion, Edomondson grade, capsule infiltration, TNM stage(uicc, 6 th edition) and extrahepatic metastases in patients with recurrences had a significant correlation with vimentin. The presence of portal vein thrombosis approached statistical significance with MMP 2 expression. In the survival analysis, overexpression of vimentin and MMP 2 was correlated with a poor overall survival rate based on univariate analysis (p=0.002 and, p=0.047, respectively), but not based on multivariate analysis. Conclusion: In HCC, vimentin and MMP 2 may have a role in cancer progression with more aggressive potential, thus suggesting their use as a prognostic markers in HCC. Key Words : Hepatocellular carcinoma, Vimentin, MMP 2, Immunohistochemistry, Survival analysis 중심단어 : 간세포암, vimentin, MMP 2, 면역조직화학염색, 생존분석 이은신, 홍성우, 강윤경, 1 이우용, 장여구, 백인욱, 이혁상 인제대학교서울백병원외과학교실 Eun Sin Lee, M.D., Seong Woo Hong, M.D., Yun Kyung Kang, M.D. 1, Woo Yong Lee, M.D., Yeo Goo Chang, M.D., In Wook Paik, M.D., and Hyucksang Lee, M.D. Department of Surgery and 1 Pathology, Seoul Paik Hospital, Inje University, Seoul, Korea 책임저자 이혁상인제대학교서울백병원외과학교실서울시중구저동 2 가 85 번지, 우 100-032 Tel: 02-2270-0016 Fax: 02-2270-0373 E-mail: hyuckslee@gmail.com Received: 2009. 4. 8. Accepted: 2009. 6. 8. 서론 Vimentin은세포골격단백질을이루는중간섬유의한종류로서세포질내에서각세포소기관을연결하고지하여세포의유연성을유지해주는역할을한다. Ben Ze와 Raz A 1 는 1985년흑색종에서 Vimentin의과발현이암의전이와관련이있음을보고하였고, 이후 Domagala 등 2 이유방암에서발현을보고하였으며이후다른상피세포암 ( 자궁경부암, 전립선암, 신장암, 두경부암등 ) 에서도같은결과가보고 되었다. 3-6 Matrix metalloproteinase2 (MMP2) 는세포외벽 (extracellular matrix) 을분해하는단백분해효소의한종류로서간질콜라겐 (stroma collagen) 을분해함으로서종양세포의침습과전이의첫째방어벽인세포외벽을해체하여세포의이동 (migration) 과침습을용이하게하는역할을한다. 7,8 간세포암 (hepatocellular carcinoma, HCC) 은전세계적으로는물론국내에서도높은발병율을보이는흔한암으로 9 불량한예후를보이는데수술이가능한경우에는 5년생존률이평균 11%~75% 까지보고되고있으나전이가있는수술불가능한경우에는 5% 미만으로매우낮게보고되고있다. 10-12 최근이러한불량한예후를나타내는암의침습과전이, 진행 89
한국간담췌외과학회지 : 제 13 권제 2 호 2009 과정을세포화학적으로분석하여각세포단계에작용하는유전자의변화와악성도의관련성을설명하는보고가많이있다. 13-14 Vimentin을포함한특정세포골격단백질의과발현이세포-세포간의연결 (cell-cell adhesion) 의손실을조장하고, MMP2와같은유전자의과발현은세포방어벽 (cell barrier) 의파괴를용이하게하여암세포의전이와진행에중요한역할을한다. 15,16 간세포암에서 vimentin과 MMP2의발현을임상연구와병행하여예후인자로서의의미를연구한보고는저자들의논문검색에서는찾을수없었다. 저자들은간세포암에서면역조직화학염색을이용하여 Table 1. Ki-67 labeling index (LI) in correlation with vimentin and MMP2 expression Number Ki-67 LI * p-value Vimentin Positive 6 30.0 ± 13.0 <0.001 Negative 88 12.8 ± 9.9 MMP2 Positive 38 16.7 ± 12.7 0.043 Negative 56 12.0 ± 9.1 * : Values are means ± standard deviation Table 2. TUNEL labeling index (LI) in correlation with vimentin and MMP2 expression Number TUNNEL LI * p-value Vimentin Positive 6 4.2 ± 3.1 0.352 Negative 86 3.3 ± 2.1 MMP2 Positive 38 3.5 ± 2.2 0.632 Negative 54 3.3 ± 2.1 * : Values are means ± standard deviation vimentin과 MMP2의발현을조사하고, 세포증식지수와세포자멸사지수 (apoptosis index) 와의관련성을알아보며, 환자의임상병리학적자료와생존자료를분석하여, 간세포암에서 vimentin과 MMP2 발현이예후를나타내는지표로서임상적의의가있는지알아보고자본연구를계획하였다. 대상및방법 1. 대상인제대학교서울백병원에서 1991년부터 2002년까지간세포암으로간절제술을시행받은환자중에서파라핀포매조직이사용가능한 98예에대해 SuperBioChips 사 ( 서울 ) 에의뢰하여조직배열 (tissue array) 을제작하였다. 전향적으로수집된환자의자료와의무기록을참고하여환자의임상병리학적자료를조사하였고, 추적조사를통해환자의술후성적을조사하였다. 2. 면역조직화학염색방법조직배열블록을 4μm두께로박절한후면역조직화학염색을시도하였다. 파라핀을제거하고내인성과산화효소를억제하기위하여 0.3% 과산화수소수로처리하였다. Vimentin 일차항체 (clone V9, 1:100 dilution; Neomarkers, Fremont, CA) 와 MMP2일차항체 (clone A-Gel VC2, 1:400 dilution; Neomarkers) 를실온에서 1시간반응시켰으며, phophatebuffered saline으로세척후 biotynylated 이차항체를반응시켰고, avidin-biotin streptavidin-peroxidase complex Fig. 1. Immunohistochemical expression of cytoplasmic vimentin in hepatocellular carcinoma tissue array (A:ⅹ40, B: x200). 90
이은신외 : 간세포암에서 Vimentin 과 MMP2 발현의임상적의의 Fig. 2. Immunohistochemical expression of cytoplasmic MMP2 in hepatocellular carcinoma tissue array (A:ⅹ40, B: x200). (Vectastatin Elite ABC kit; Vector Laboratories, Burlingame, CA) 로배양하였다. 발색제로는 3,3 -diaminobenzidine tetrahydrochloride를사용하였고, Mayer s hemotoxylin으로대조염색하였다. 음성대조군은일차항체없이같은과정으로염색하였다. 면역조직화학염색의판독은공저자인병리과전문의에의해음성과양성발현으로판독하였다. 양성의기준은종양세포의 10% 이상에서 세포질에발현하는경우로하였다. 세포증식지수는 Ki-67에대한다클론항체 (Dako, Glostrup, Denmark) 를이용한면역화학염색으로조사하였다. 1000개의종양세포를조사하여세포핵에염색된세포의백분율을세포증식지수 (Ki-67 labeling index, LI) 로구하였다. 세포의자멸사지수는 ApopTag In Situ Apoptosis Detection Kit (Intergen, Purchase, NY) 를이용하여 1000개의종양세포중에양성인세포의수로 terminal deoxynuclotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) labelling index (LI) 를구하였다. Fig. 3. Overall survival in patients with hepatocellular carcinoma according to vimentin expression (p=0.002). 3. 통계방법통계는평균의비교는독립표본 T-test를범주형자료의비교는 chi-square test를이용하였다. 생존율은 Kaplan-Meier 방법을이용하여구했으며, 그비교는 Log-Rank 방법을사용하였고 p<0.05를유의하다고하였다. 다변량분석은 Cox proportion hazard model을사용하였다. 91
한국간담췌외과학회지 : 제 13 권제 2 호 2009 결과 1. 간세포암조직에서 vimentin과 MMP2의발현간세포암에서 vimentin은 98예중양성이 7예 (7.1%), 음성이 91예 (92.8%) 였다 (Fig. 1). MMP2의발현은 98예중양성이 41예 (41.8%), 음성이 57예 (58.2%) 에서관찰되었다 (Fig. 2). 2. Vimentin과 MMP2 의발현과세포증식지수및세포자멸지수와의관계세포증식지수 (Ki-67 labelling index) 는 vimentin 양성군에서 30.0 ± 13.0이며음성인군에서 12.8 ± 9.9로유의한차이를보였고 (p<0.001), MMP2 양성인군에서 16.7 ± 12.7이며음성인군에서 12.0 ± 9.1로차이를나타냈다.(p=0.043)(Table 1). 세포자멸지수 (TUNEL labeling index) 는 vimentin, MMP2 모두양성과음성인군에서유의한차이를나타내지았다 (p=0.352, p= 0.632)(Table 2). 3. Vimentin, MMP2 의발현과임상병리학적인자들과의관계환자의 B형간염표면항원의유무, C형간염항체의유무, 만성간염또는간경화의유무, Child-Pugh 분류, ICG R15, Fig. 4. Overall survival in patients with hepatocellular carcinoma according to MMP2 expression (p=0.047). 혈소판의수, 종양의크기, 알파태아단백, 종양의수, 문맥의종양혈전, 종양의장막 (serosa) 침습유무, Edomondson 분류, 종양캡슐의암세포침습 (capsule infiltration) 유무, TNM 병기 (UICC, 6thedition), 간외장기재발유무에따른 vimentin, MMP2의발현을비교하였다. Vimentin은혈소판수치가 270,000/uL이상인경우 (p=0.036), 종양의장막침습이있는경우 (p=0.013), Edomondson grade가높은경우 (p<0.001), 종양캡슐에암세포침습이있는경우 (p=0.021), TNM 병기 (UICC,6 th edition) 가 3기이상인경우 (p=0.013) 와재발시간전이와함께간외장기에전이가있는경우 (p=0.015) 에발현율이높았고, MMP2의과발현이있는경우에는문맥의종양혈전이있는경우에통계학적인유의성에접근하였다 (p=0.057)(table 3). 4. 수술후예후와 vimentin 및 MMP2 의발현수술후생존율분석결과수술후 vimentin 양성인환자의 1, 3, 5년생존율은각각 71.4%, 0%, 0% 와중앙생존기간이 15개월로음성인군의 73.6%, 37.4%, 26.4% 와중앙생존기간 23개월에비해좋지았다 (p=0.002)(fig. 3). MMP2의발현에따른생존율은양성인환자는 1, 3, 5년생존율이각각 77.5%, 27.5%, 15% 와중앙생존기간이 21개월로나타났고음성인군은 70.7%, 39.7%, 31% 와중앙생존기간 27개월로나타나통계학적으로유의하게양성인군이음성인군에비해낮았다 (p=0.047)(fig. 4). 단변량분석에서생존에유의한차이를보이는임상병리학적인자인간질환 (chronic hepatitis or cirrhosis) 의유무 (p= 0.003), 높은 ICG 수치 (p= 0.014), 높은혈소판수치 (p= 0.032), B 이상의 Child-Pugh class(p=0.018), 다발성암 (p= 0.005), 문맥의종양혈전이있는경우 (p= 0.006), 수술시완전절제의여부 (p< 0.001), Edmondson grade(p= 0.018), 높은 TNM 병기 (UICC, 6 th edition)(p < 0.001) 를다변량분석하였고, ICG R15와 3기이상의 TNM 병기 (UICC, 6 th edition) 가독립적인예후인자로의미가있었다. vimentin과 MMP2는통계학적유의성을얻지못하였다 (Table 5). 92
이은신외 : 간세포암에서 Vimentin 과 MMP2 발현의임상적의의 Table 3. Clinicopathological factors and vimentin and MMP2 expression Vimentin MMP2 Parameters Negative Positive p-value Negative Positive p-value HBsAg Negative 25 1 0.446 13 13 0.266 Positive 66 6 45 27 Anti-HCV Negative 84 7 0.446 56 35 0.098 Positive 7 0 2 5 Liver disease Normal 5 0 0.422 3 2 0.929 CH * 43 2 26 19 Cirrhosis 42 5 29 18 Child-Pugh stage A 80 6 0.931 52 34 0.448 B 10 1 6 5 C 1 0 0 1 ICGR15 10% 44 2 0.312 28 18 0.749 >10% 47 5 30 22 Platelet count < 270 74 3 0.036 47 30 0.538 (1000/uL) 270 14 3 9 8 Tumor size 5cm 47 2 0.239 30 19 0.681 > 5cm 44 5 28 21 α FP (ng/ml) 20 28 4 0.152 21 11 0.366 >20 63 3 37 29 Tumor mutiplicity Solitary 58 3 0.272 37 24 0.703 Multiple 33 4 21 16 Portal tumor thrombi Absence 37 1 0.168 27 11 0.057 Presence 54 6 31 29 Serosa invasion Absence 54 1 0.013 32 23 0.688 Presence (M) 21 3 16 8 Presence(G) 8 3 5 6 Penetrate 8 0 5 3 Edmondson grade II 27 0 <0.001 20 7 0.153 III 54 2 7 27 IV 10 5 9 6 Capsule infiltration Absence 7 2 0.021 5 4 0.470 Presence 60 2 42 20 TNM stage(uicc,6 th I 54 1 0.013 32 23 0.688 edition) II 21 3 16 8 III 8 3 5 6 IV 8 0 5 3 Recurrent site Liver 39 0 0.015 25 14 0.113 Other organ 18 3 9 12 * :chronic hepatitis :Indocyanine green 15minute retention rate :alpha-fetoprotein :Microscopic presence :Grossly presence :Liver with extrahepatic organ(lung or Bone) was involved. 93
한국간담췌외과학회지 : 제 13 권제 2 호 2009 Table 4. Survival rate and vimentin, MMP2 expression Vimentin MMP2 Positive Negative p-vlaue Positive Negative p-vlaue Overall 1 yr. 71.4% 73.6% 0.002 77.5% 70.7% 0.047 (%) 3yr. 0% 37.4% 27.5% 39.7% 5yr. 0% 26.4% 15% 31.0% Median(month) 15 23 21 27 고찰 HCC는불량한예후를보이는흔한암중의하나로암세포의진행과정및원인에대해많은보고가있다. 9-12 최근에는암의침습과전이과정을세포화학적으로설명하고각단계에서작용하는유전자에대해연구함으로써암진행의원인과과정을밝히고예후를예측하며나아가치료방법을모색하는연구가많이진행되고있다. 13,14 암세포의진행은여러과정을거치게된다. 먼저특정유전자의변화로인해세포-세포간의연결메커니즘 (cell-cell adhesion mechanism) 이파괴되고세포외벽 (extracellular matrix) 의손상이용이해짐에따라세포가운동성을획득하게되고, 운동성을가진암세포가 원발부위를떠나주변조직으로침습하고혈관내벽을침입하여전신순환 (systemic circulation) 으로들어가게된다. 이렇게혈류를타고다른장기로이동하여새로운환경 (new host environment) 에서자라게된다. 15 이러한암진행의과정에서 vimentin 같은특정세포골격단백질과 MMP2를포함한특정단백질파괴유전자의과발현이세포벽파괴를용이하게해줌으로서암세포의전이와진행에중요한역할을하는것으로알려져있다. 16 이러한암세포의진행과정에관여하는여러유전자가보고되고있는데 13,14 Vimentin과 MMP2는여러고형암에서과발현이보고되었으나 1-8 암의예후와직접적인관련성에대해서는아직정확한통계학적보고가없는상황이다. 저자들은간세포암에서면역조직화학염색을통해 Table 5. Univariate and multivariate survival analysis in hepatocellular carcinoma patients Prognostic factors Univariate Multivariate Relative risk 95% CI p-value Vimentin 0.002 1.561 0.597-4.083 0.364 MMP2 0.047 1.240 0.755-2.038 0.396 Liver disease * 0.003 1.603 0.932-2.756 0.088 Child-Pugh stage (A vs B or C) 0.032 1.715 0.823-3.576 0.150 ICGR15 >10% 0.014 1.846 1.098-3.105 0.021 Platelet( 270,000) 0.032 1.715 0.823-3.576 0.150 Multiple tumor 0.005 1.349 0.808-2.250 0.252 PVT 0.006 1.553 0.775-3.114 0.215 Radicality <0.001 1.624 0.933-2.827 0.087 Edmondson grade 0.018 0.93 0.447-1.939 0.848 (II vs III or IV) TNM stage <0.001 2.360 1.017-5.475 0.046 (I or II vs III or IV) * :Chronic hepatitis or Cirrhosis :Indocyanine green 15minute retention rate :Portal vein tumor thrombi 94
이은신외 : 간세포암에서 Vimentin 과 MMP2 발현의임상적의의 vimentin과 MMP2의발현을조사하고이를임상병리학적인자들과연관하여생존율과의관련성에대해분석하였다. 본연구에서 vimentin이암세포에서발현한예는 7.1% 로나타났고 MMP2의발현은 41.8% 였고두인자사이의연관성은없었다 (p=0.087). Vimentin, MMP2의발현과세포의증식또는자멸사의상관관계를알아보기위해세포증식지수 (Ki-67 labelling index) 와세포자멸사지수 (TUNEL labeling index) 를구하였다. 세포증식지수는 vimentin과 MMP2의발현이있는예에서높게측정되는경향을보였는데 (p<0.001, p=0.043) Ki-67을이용한세포증식지수는간세포암의악성도와예후에유의한인자로인정되고있어 17,18 vimentin과 MMP2의발현이간세포암에서암세포의활발한증식과관련이있으며, 악성도를나타내는인자로의미가있음을보여준다고할수있다. 세포의자멸사과정 (apoptotic signaling cascade) 에서유전자 (DNA fragment) 분석을통한세포자멸사지수는암의진행에불충분한자멸사 (insufficient apotosis) 가관여한다는관점에서예후인자로서의역할이보고되고 있는데 19,20 본연구에서 vimetin 과 MMP2 의발현과유의한 관련성을나타내지는았다. 간세포암에서예후에영향을미치는것으로알려진다른주요한임상병리학적인자와비교해보았을때 vimentin은혈소판수치가높은경우, 종양의장막침습이있는경우, Edmondson grade가나쁜경우, 종양캡슐에암세포침습이있는경우, TNM 병기가높은경우, 재발시간전이와함께간외장기에전이가있는경우에통계학적으로유의하게상관관계가있으며, MMP2의발현은문맥의종양혈전이있는경우에통계학적인유의성에접근하였다 (Table 4). 이와같이조직병리학적으로악성도가높음을나타내는여러인자들과 vimentin과 MMP2의발현은상관관계가있음을보였고수술후재발율의차이는없었으나재발이있는환자의경우를분석했을때간재발과함께간외장기재발 ( 폐전이, 골전이 ) 가있는모든경우에 vimentin의발현이관찰되어불량한예후를시사하였다. Atsushi Shiga 등 21 이세포분화도가나쁜경우에 vimentin의과발현을보고하였고, Liang Hu. 등 22 은원격전이가없는간세포암과비교했을때원격전이가있는환자에서 vimentin의과발현이관찰을보고하였으며, Chun-Feng Lee등 23 은임파선전이가없는경우와비교할때전이가있는간세포암에서 MMP2의발현이 3배이상높은것으로보고하여본연구와같이 vimentin과 MMP2가종양의악성도와관련이있음을보여주었다. 수술후생존율과평균생존기간을분석한결과 vimentin과 MMP2의발현이있는환자에서통계적으로유의하게생존율및생존기간이낮은것으로나타나예후인자로서의미가 있음을보여주었다. 그러나본연구에서 vimentin의발현이총 7예에서만관찰되어임상적인의의는더많은예에대한분석이요할것으로사료된다. 단변량분석에서생존에유의한차이를보이는여러임상병리학적인자들을 vimentin, MMP2와함께다변량분석했을때 ICG R15와 3기이상의 TNM 병기 (UICC, 6 th edition) 만이독립적인예후인자로의미가있었고 vimentin과 MMP2는의미있는결과를얻지못하였다. 결 론 간세포암에서 vimentin은조직병리학적으로분화도가좋지은종양에서관찰되어종양의재분화과정에관여하는것으로보이며, 재발한환자에서간외장기전이가있는모든경우에서 vimentin이발현되어불량한예후를시사하였고 MMP2는종양의문맥침범이있는경우에발현이증가되어종양의악성도와관련이있었다. 다변량분석에서유의한결과를얻지는못했으나 Ki-67로대변되는세포의증식도, 수술후생존율및생존기간과상관관계를보여 vimentin과 MMP2가환자의생존에불량한예후인자로사용될수있는가능성을시사하였다. 참고문헌 1. Ben Ze ev and Raz A. Relationship between the organization and synthesis of vimentin and the metastatic capability of B16 melanoma cells. Cancer Res 1985;45:2632-2641. 2. Domagala W, Striker G, Szadowska A, et al. P53 protein and vimentin in invasive ductal NOS breast carcinoma-relationship with survival and sites of metastases. Eur J Cancer 1994;30:1527-1534. 3. Gilles C, Polette M, Piette J, et al. High level of MT-MMP expression is associated with invasiveness of cervical cancer cells. Int J Cancer 1996;65:209-213. 4. Moch H, Schraml P, Bubendorf L, et al. Identification of prognostic parameters for renal cell carcinoma by cdna arrays and cell chips. Verh Dtsch Ges Pathol 1999;83:225-232. 5. Lang SH, Hyde C, Reid IN, et al. Enhanced expression of vimentin in motile prostate cell lines and in poorly differentiated and metastatic prostate carcinoma. Prostate. 2002;52:253-263. 6. Mandal M, Myers JN, Lippman SM, et al. Epithelial to mesenchymal transition in head and neck squamous carcinoma: association of Src activation with E-cadherin down-regulation, vimentin 95
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