DOI: 10.4046/trd.2011.70.2.132 ISSN: 1738-3536(Print)/2005-6184(Online) Tuberc Respir Dis 2011;70:132-138 CopyrightC2011. The Korean Academy of Tuberculosis and Respiratory Diseases. All rights reserved. 비소세포폐암에서 Maspin 의발현과임상적의의 Original Article 부산대학교의학전문대학원 1 내과학교실, 2 병리학교실윤성훈 1, 김원진 1, 신경화 1, 김미현 1, 조우현 1, 김기욱 1, 박혜경 1, 전두수 1, 김윤성 1, 이창훈 2, 이민기 1, 박순규 1 Maspin Expression and Its Clinical Significance in Non-Small Cell Lung Cancer Seong Hoon Yoon, M.D. 1, Won Jin Kim, M.D. 1, Kyung Hwa Shin, M.D. 1, Mi Hyun Kim, M.D. 1, Woo Hyun Cho, M.D. 1, Ki Uk Kim, M.D. 1, Hye-Kyung Park, M.D. 1, Doo Soo Jeon, M.D. 1, Yun Seong Kim, M.D. 1, Chang Hun Lee, M.D. 2, Min Ki Lee, M.D. 1, Soon Kew Park, M.D. 1 Departments of 1 Internal Medicine, 2 Pathology, Pusan National University School of Medicine, Busan, Korea Background: Maspin (mammary serine protease inhibitor) is a member of the serpin superfamily. A few studies have examined the role of maspin in tumor suppression of non-small cell lung cancer (NSCLC); however, its role in the development and progression of NSCLC still remains controversial. We evaluated the immunohistochemical expression of maspin in order to elucidate its clinical significance in NSCLC. Methods: We analyzed 145 patients with pathologically confirmed NSCLC, including 66 cases of squamous cell carcinomas (SCCs) and 79 cases of adenocarcinomas (ADCs). We performed a immuno-histochemical stain with maspin and PCNA (proliferating cell nuclear antigen) using tissue microarray blocks. Results: There were 108 men and 37 women in the study population. The mean age of patients in the study was 63.7 years (range, 40.0 82.0; median, 65.0). The proportion of maspin expression was significantly higher in SCCs (52/66, 78.8%; p<0.01) than in ADCs (17/79, 21.5%; p<0.01). Maspin expression was not associated with PCNA (p=0.828), lymph node involvement (p=0.483), or tumor stage (p=0.216), but showed correlation with well-to-moderate tumor differentiation (p=0.012). There was no observed correlation between maspin expression and survival with NSCLC (p=0.218). Conclusion: The present study suggests that maspin expression was significantly higher in SCCs than in ADCs and was associated with low histological grade. However, maspin expression was not an independent factor to predict a prognosis in NSCLC. Key Words: Maspin; Non-Small Cell Lung Cancer; Carcinoma, Squamous Cell; Pathology; Prognosis 서 Mammary serine protease inhibitor (maspin) 단백은 serine protease inhibitor (serpin) 의일종으로, 1994년정 Address for correspondence: Soon Kew Park, M.D. Department of Internal Medicine, Pusan National University School of Medicine, 1-10, Ami-dong, Seo-gu, Busan 602-739, Korea Phone: 82-51-240-7222, Fax: 82-51-254-3127 E-mail: snkpark@pusan.ac.kr Received: Feb. 24, 2010 Accepted: Feb. 5, 2011 론 상유선조직에서처음분리되었다 1. 이후전립선, 흉선, 고환, 소장, 대장등의정상상피조직에서도발견되었다 2. Maspin 과종양과의관계에서일부연구들은 maspin 이유방암에서세포운동성, 침습및전이의억제를통해종양억제기능을가진다고했지만 3,4, 다른연구들에서는유방암, 췌장암및난소암에서 maspin 의발현이나쁜예후및종양의진행과연관이있다고하였다 5,6. 이처럼아직까지종양에서 maspin 의정확한기능은정립되어있지않은실정이다. 원발성폐암은전세계적으로암사망률의가장흔한원인이며, 우리나라에서도 2007년기준, 암으로인한사망 132
Tuberculosis and Respiratory Diseases Vol. 70. No. 2, Feb. 2011 원인중 1위이다 7,8. 비소세포폐암은원발성폐암의 80% 를차지하며 9, 비소세포폐암의임상적인예후지표에대한필요성이제기되었으나그간의연구에서는특정한지표를정립하지못하였다. 최근여러연구에서비소세포폐암에서 maspin 의발현을보고하고있지만, 아직이단백의정확한기능및역할에대해서는보고마다차이가있고, maspin 의세포내발현위치에따라서도기능이다르다고알려져있다. Smith 등 10 은, 세포핵내에서발현되는 maspin은좋은예후와관련된다고보고했지만, Hirai 등 11 은세포질에서발현되는 maspin 은종양의진행및나쁜예후와연관이있다고보고하였다. 이상에서보듯이비소세포폐암에서아직 maspin의정확한기능은잘정립되어있지않다. 따라서본연구에서는비소세포폐암의주요조직학적유형에따른 maspin 의발현에대해분석하였고, 비소세포폐암의예후에대한 maspin 의역할에대해알아보고자하였다. 또한병기, 조직학적분화도, 종양의증식능과 maspin 발현과의연관성에대해서도평가를하였으며, 기존의예후인자로알려진 나이, 종양의증식, TNM 병기, 림프절병기및조직학적분화도와예후와의연관성에대한분석도시행하였다. 대상및방법 1. 대상환자 1998년 4월 1일부터 2008년 4월 30일까지부산대학교병원에서조직학적으로비소세포폐암으로확진된 145명의환자를대상으로하였고, 이들의조직학적분류, 병기, 증식정도및임상적소견을후향적으로분석하였다. 전체환자중편평상피세포암은 66명이었고, 선암은 79명이었다. 평균나이는 63.7세였고평균추적관찰기간은 44 개월이었으며, 145명중남자가 109명, 여자가 36명이었다. 모든환자에서의무기록및영상학적사진 ( 엑스레이, 흉부전산화단층촬영등 ) 을검토하였고 maspin 의발현과종양의증식정도는면역조직화학염색을이용하여분석하였으며, 병리학적병기는기존의 TNM 병기분류법 12 을따랐다. Figure 1. Immunohistochemical staining for maspin using the tissue microarray ( 200). (A) ADC without maspin expression. (B) ADC with maspin expression. (C) SCC without maspin expression. (D) SCC with maspin expression. 133
SH Yoon et al: Maspin expression in non-small cell lung cancer 2. 면역조직화학염색 Maspin 의종양세포내발현정도를보기위해조직미세배열블록 (tissue microarray block) 을사용하여 maspin 에대한면역조직화학염색을하였다 (Figure 1). 각증례에서종양세포핵의 5% 이상에서암갈색의염색반응을보일경우양성으로하였고세포질에서의발현은무시하였다. 종양의증식정도를평가하기위해서는 proliferating cell nuclear antigen (PCNA) 에대한면역조직화학염색을실시하였다 (Table 1). PCNA 의양성소견은종양세포의핵에서암갈색의염색소견을보이는경우로하였고, 각증례에서 1,000개의종양세포중양성소견을보이는종양세포를계수하여백분율 (PCNA index) 로표시하였다. 3. 통계학적분석 Maspin의발현과여러임상병리학적특징들 ( 나이, 성별, 조직학적유형, 종양의증식정도, TNM 병기등 ) 과의연관성은 Chi-square 검사를이용하였고, 생존분석은 Kaplan-Meier법및 Cox's proportional hazards model 을이용하였다. 모든통계학적분석은 SPSS version 15.0 (SPSS Inc., Chicago, IL, USA) 를이용하였고 p값이 0.05 미만인경우유의한것으로판정하였다. 결과 1. 대상환자의특성및 maspin의발현율전체환자 145명중, 69명 (47.6%) 에서 maspin 단백은양성발현을보였다. 환자군의중간연령인 65세를기준으로하였을때, 65세이상인환자에서 maspin 의발현율이 51.9% (40/77) 였고 65세이하에서는 42.6% (29/68) 를보였으나의미있는차이를보이지는않았다 (p=0.263). 전체환자중남성에서는 maspin의발현율이 58.3% (63/ 108) 였고, 여성에서는 16.2% (6/37) 로남성에서 maspin 의발현율이유의하게더높았다 (p<0.01). 조직학적유형에따른 maspin 의발현율은편평상피세포암은 78.8% (52/66) 였고, 선암은 21.5% (17/79) 로편평상피세포암에서 maspin의발현율이더높았다 (p<0.01, Table 2). 2. 임상병리학적특성과의관련성 Maspin의발현과종양의조직학적분화도 (differentiation) 와의관련성을살펴보면, 조직의분화도등급이낮을때 (well or moderate differentiation), maspin의발현 Table 1. Data sheet for maspin and PCNA Antigen Antibody Clone Source Dilution Detection Antigen retrieval (min) Maspin Gpab C-20 Santa Cruz, USA 1:400 Envision Kit, DAKO Room temperature (60) PCNA Mmab PC10 SIGMA, USA 1:2,000 Envision Kit, DAKO Overnight Gpab: polyclonal gout antibody; PCNA: proliferating cell nuclear antigen; Mmab: monoclonal mouse antibody. Table 2. Characteristics of patients and maspin expression No. of patients Negative (<5) Maspin expression Positive ( 5) p-value All patients 145 (100) 76 (52.4) 69 (47.6) Age, yr 63.7±9.5 62.22±8.93 65.33±8.2 0.043 <65 68 (46.9) 39 (57.4) 29 (42.6) 0.263 65 77 (53.1) 37 (48.1) 40 (51.9) Sex Male 108 (74.5) 45 (41.7) 63 (58.3) <0.01 Female 37 (25.5) 31 (83.8) 6 (16.2) Histological type Squamous cell carcinoma 66 (45.5) 14 (21.2) 52 (78.8) <0.01 Adenocarcinoma 79 (54.5) 62 (78.5) 17 (21.5) Values are presented as number (%) unless otherwise indicated. 134
Table 3. Relationship between maspin expression and clinicopathologic features Tuberculosis and Respiratory Diseases Vol. 70. No. 2, Feb. 2011 Patients Negative (<5) Maspin expression Positive ( 5) p-value All patients 145 (100) 76 (52.4) 69 (47.6) Differentiation Well, moderate 117 (82.9) 55 (47) 62 (53) 0.012* Poor 24 (17.1) 18 (75) 6 (25) T classification T1 48 (33.1) 26 (54.1) 22 (45.9) 0.216 T2 4 97 (66.9) 47 (48.5) 50 (51.5) Lymph node involvement N0 101 (69.7) 51 (50.5) 50 (49.5) 0.483 N1 3 44 (30.3) 25 (56.8) 19 (43.2) Pathologic stage I 93 (64.1) 50 (53.8) 43 (46.2) 0.284 II 19 (13.1) 7 (36.8) 12 (63.2) IIIA 33 (22.8) 19 (57.6) 14 (42.4) Values are presented as number (%). *Regardless of histologic type, maspin expression was significantly associated with low histological grade (p-value: SCC, <0.01; ADC, 0.04); I, II vs. IIIA. Table 4. Relationship between maspin expression and PCNA Tumors Maspin expression No. of patients PCNA (%) p-value NSCLC Positive 68 52.8±2.5 0.828 Negative 76 53.2±2.8 SCC Positive 52 52.7±2.8 0.21 Negative 14 60.7±7.2 ADC Positive 16 53.4±6.4 0.77 Negative 62 51.5±3.0 NSCLC: non-small cell lung cancer; SCC: squamous cell carcinoma; ADC: adenocarcinoma; PCNA: pro-liferating cell nuclear antigen. 율은전체 117명중 62명으로 53% 를나타냈고등급이높을때는 (poor differentiation) 발현율이전체 24명중 6명으로 25% 를나타내었다. 분화도의등급이낮을때, maspin의발현율이의미있게더높았으며 (p=0.012), 이는조직학적유형별분화도에따른비교에서도똑같은결과를보였다. 반면, maspin 의발현은다른임상병리학적특징들 (T병기, 림프절의침범, TNM 병기 ) 에따른유의한차이를보이지않았다 (Table 3). Figure 2. Comparison of cumulative survival according to maspin expression in non-small cell lung cancer. 3. Maspin 의발현과종양의증식정도 종양의증식을나타내는 PCNA 의경우, maspin 이양성인군에서 52.8% 였고음성인군에서 53.2% 를보였으나의미있는차이는보이지않았다 (p=0.828). 조직학적유형을나누어살펴보면, 편평상피세포암의경우 maspin 이양성인군에서 PCNA 가 52.7% 였고 maspin 이음성인군에서는 60.7% 를나타내었다. 선암의경우는각각 53.4% 와 51.5% 였다. 조직학적유형에따라살펴보았지만 maspin 135
SH Yoon et al: Maspin expression in non-small cell lung cancer Table 5. Multivariate analysis of the prognostic factors in non-small cell lung cancer Factors Beta p-value Hazard ratio (95% CI) PCNA, % ( 50 vs. <50) 1.178 0.01 0.31 (0.18 0.49) Age, yr ( 65 vs. <65) 1.438 <0.001 0.24 (0.15 0.31) N-stage (+ vs. ) 0.815 0.033 0.44 (0.17 0.89) PCNA: proliferating cell nuclear antigen; CI: confidence interval. 의발현과종양의증식은유의한연관성이없었다 (Table 4). 4. Maspin의발현과비소세포폐암의예후 Maspin 이양성인군에서는비소세포폐암의 5년생존율이 52% 인반면, maspin 이음성인군에서는 5년생존율이 65% 를나타냈다. 하지만, 전체추적기간중에는 maspin 의발현과생존율은유의한연관성이없었다 (Figure 2). 조직학적유형에따라서도 maspin 의발현과생존율과는유의성이없었다 (ADC, p=0.677; SCC, p=0.563). 5. 임상병리인자들의예후적의의종양의증식능, TNM 병기, 나이, 림프절병기, 조직학적분화도와생존과의연관성을살펴보면, PCNA, 나이및림프절병기가생존율과유의한연관성을보였고 (p=0.029 for PCNA; p=0.009 for age; p=0.029 for N stage), 이러한인자를다변량분석을통해위험비를산출하면종양의증식이심할수록, 나이가많을수록, 림프절병기가진행될수록나쁜예후를나타내는것을알수있었다 (Table 5). 고찰 Maspin 은 18번염색체에위치하는유전자에의해발현되며, 9개의 α-나선 (helices), 3개의 β-시트 (sheets) 및 reative site loop로구성된다 13. Maspin 은 1994년 subtractive hybridization 방법에의해유선상피조직에서처음분리가되었고, 이후전립선, 흉선, 고환등의상피세포뿐만아니라정상폐조직에서도발현되었다 14. 기존의몇몇연구들은정상폐조직에서세포의종류및위치에따라서 maspin의발현율이차이가난다고보고하였는데, Yatabe 등 15 에의하면, 기관지상피의기저세포와샘꽈리 (glandular acinus) 에는 maspin 이잘발현되지만말초폐세포에는전반적으로발현율이떨어진다고하였다. 이러한결과는여러연구에서다른조직학적유형보다말초 폐에서주로기인하는선암의 maspin 발현율이떨어지는것으로설명할수있다. 본연구에서도 maspin 의발현율이선암에서편평상피세포암보다유의하게더낮음을알수있었다. 종양조직에서 maspin 의발현은주로유방암, 전립선암, 췌장암, 난소암등에서기능및임상적중요성에대한연구가많이진행되었는데, Hendrix 16 는유방암및전립선암에서 maspin 은종양세포의운동, 침범및전이등을억제하여종양억제기능을한다고하였다. 또한 Zou 등 17 은 maspin 이 p53을통하여종양의진행을억제하고좋은예후와연관된다고하였다. 이와는대조적으로다른몇몇연구에서는유방암및췌장암에서 maspin 의발현이종양의진행및나쁜예후와연관된다고보고하였다 18,19. 따라서아직종양의진행및예후에있어서 maspin 의정확한역할은정립되어있지않다. 비소세포폐암에서도 maspin 의기능및임상적중요성에대한여러보고가있었으나아직정확한역할은잘모르는실정이다. 특히생존과관련된예후측면에서여러보고가있었는데, Nakagawa 등 20 은편평상피세포암에서 maspin 의발현은좋은예후를나타내는지표가된다고하였고, 다른한연구에서는 maspin의발현이기존에잘알려진 p53이아닌다른전사인자 (transcription factor) 인 p63에의해나타나고폐암의침범을억제하는역할을한다고하였다 21. 이와대조적으로 Hirai 등 11 은비소세포폐암에서 maspin의발현은종양의진행및나쁜예후와관련됨을보고하였다. 이렇게예후인자로서 maspin 의역할이다른결과를보이는이유중일부는면역조직화학염색시 maspin 발현에대한정확한정량화가어렵기때문으로생각된다 22. 다른연구에서는세포내 maspin 의발현위치 ( 핵 / 세포질 ) 에따라다른예후를보고하기도하였는데, 핵내의 maspin 발현은좋은예후와관련되고세포질내발현은나쁜예후와연관된다고보고하였다 10,11. 본연구에서는비소세포폐암의핵내의 maspin 발현만을양성으로판정해서연구하였고예후와는의미있는결과를보이지못하였다. 136
Tuberculosis and Respiratory Diseases Vol. 70. No. 2, Feb. 2011 기존의한연구에서 maspin 의발현은림프절의침범이나높은 T병기와연관된다고하였는데 21, 본연구에서는 maspin의발현과조직학적분화도, T병기, N병기및 TNM병기와의관련성에대해평가하였으나조직학적분화도만이유의한연관성을가졌으며분화도의등급이낮을때 maspin 의발현율이더높았다 (p=0.012). 또한종양의증식능과의상관성을보기위해 PCNA 를이용하여면역조직화학적염색방법을통해정량적으로분석을하였으나 maspin 과종양의증식능과는유의한연관성이없었다 (p=0.828). 조직학적유형을나누어서평가를하였지만모두의미가없었다 (SCC, p=0.21; ADC, p=0.77). 한편, 지금까지널리알려진비소세포폐암의예후인자와생존과의상관성을살펴보기위해다변량분석을하였을때, 나이가많을수록, 종양의증식이심할수록, 림프절침범이있을수록나쁜예후를나타냈다. 결론적으로 maspin 은선암보다는편평상피세포암에서더많이발현되고, 남자에서더흔히발현됨을알수있었다. 또한, 여러임상병리학적특징중조직학적분화도와연관이되며, 비소세포폐암의예후와는유의한연관성이없음을알수있었다. 본연구는대상인원의수가적고후향적으로연구되었기때문에환자의선택및생존분석에있어제한점이있었다. 따라서, 비소세포폐암에서 maspin 의발현과예후를정확하게평가하기위해서는더많은환자를대상으로객관적인방법으로수치를정량화할수있는전향적인연구가필요할것으로생각된다. 감사의글 This work was supported for two years by Pusan National University Research Grant. 참고문헌 1. Zou Z, Anisowicz A, Hendrix MJ, Thor A, Neveu M, Sheng S, et al. Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells. Science 1994;263:526-9. 2. Pemberton PA, Tipton AR, Pavloff N, Smith J, Erickson JR, Mouchabeck ZM, et al. Maspin is an intracellular serpin that partitions into secretory vesicles and is present at the cell surface. J Histochem Cytochem 1997; 45:1697-706. 3. Sheng S, Carey J, Seftor EA, Dias L, Hendrix MJ, Sager R. Maspin acts at the cell membrane to inhibit invasion and motility of mammary and prostatic cancer cells. Proc Natl Acad Sci USA 1996;93:11669-74. 4. Sheng S, Truong B, Fredrickson D, Wu R, Pardee AB, Sager R. Tissue-type plasminogen activator is a target of the tumor suppressor gene maspin. Proc Natl Acad Sci USA 1998;95:499-504. 5. Umekita Y, Ohi Y, Sagara Y, Yoshida H. Expression of maspin predicts poor prognosis in breast-cancer patients. Int J Cancer 2002;100:452-5. 6. Bièche I, Girault I, Sabourin JC, Tozlu S, Driouch K, Vidaud M, et al. Prognostic value of maspin mrna expression in ER alpha-positive postmenopausal breast carcinomas. Br J Cancer 2003;88:863-70. 7. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin 2007;57:43-66. 8. Korea National Statistical Office. Deaths and death rate by cause. Daejeon: Korea National Statistical Office; 2007. 9. Buccheri G, Ferrigno D. Prognostic value of stage grouping and TNM descriptors in lung cancer. Chest 2000;117:1247-55. 10. Smith SL, Watson SG, Ratschiller D, Gugger M, Betticher DC, Heighway J. Maspin - the most commonly-expressed gene of the 18q21.3 serpin cluster in lung cancer - is strongly expressed in preneoplastic bronchial lesions. Oncogene 2003;22:8677-87. 11. Hirai K, Koizumi K, Haraguchi S, Hirata T, Mikami I, Fukushima M, et al. Prognostic significance of the tumor suppressor gene maspin in non-small cell lung cancer. Ann Thorac Surg 2005;79:248-53. 12. Mountain CF. Revisions in the international system for staging lung cancer. Chest 1997;111:1710-7. 13. Ngamkitidechakul C, Warejcka DJ, Burke JM, O'Brien WJ, Twining SS. Sufficiency of the reactive site loop of maspin for induction of cell-matrix adhesion and inhibition of cell invasion. Conversion of ovalbumin to a maspin-like molecule. J Biol Chem 2003;278:31796-806. 14. Futscher BW, Oshiro MM, Wozniak RJ, Holtan N, Hanigan CL, Duan H, et al. Role for DNA methylation in the control of cell type specific maspin expression. Nat Genet 2002;31:175-9. 15. Yatabe Y, Mitsudomi T, Takahashi T. Maspin expression in normal lung and non-small-cell lung cancers: cellular property-associated expression under the control of promoter DNA methylation. Oncogene 2004;23:4041-9. 16. Hendrix MJ. De-mystifying the mechanism(s) of mas- 137
SH Yoon et al: Maspin expression in non-small cell lung cancer pin. Nat Med 2000;6:374-6. 17. Zou Z, Gao C, Nagaich AK, Connell T, Saito S, Moul JW, et al. p53 regulates the expression of the tumor suppressor gene maspin. J Biol Chem 2000;275:6051-4. 18. Maass N, Hojo T, Ueding M, Lüttges J, Klöppel G, Jonat W, et al. Expression of the tumor suppressor gene Maspin in human pancreatic cancers. Clin Cancer Res 2001;7:812-7. 19. Maass N, Teffner M, Rösel F, Pawaresch R, Jonat W, Nagasaki K, et al. Decline in the expression of the serine proteinase inhibitor maspin is associated with tumour progression in ductal carcinomas of the breast. J Pathol 2001;195:321-6. 20. Nakagawa M, Katakura H, Adachi M, Takenaka K, Yanagihara K, Otake Y, et al. Maspin expression and its clinical significance in non-small cell lung cancer. Ann Surg Oncol 2006;13:1517-23. 21. Kim S, Han J, Kim J, Park C. Maspin expression is transactivated by p63 and is critical for the modulation of lung cancer progression. Cancer Res 2004;64:6900-5. 22. Katakura H, Takenaka K, Nakagawa M, Sonobe M, Adachi M, Ito S, et al. Maspin gene expression is a significant prognostic factor in resected non-small cell lung cancer (NSCLC). Maspin in NSCLC. Lung Cancer 2006;51:323-8. 138