대한임상검사학회지 : 36 권제 2 호, 185-192, 2004 유방암의조직학적악성도에따른에스트로겐수용체 (ER) 와프로게스테론수용체 (PR), 그리고 C-erbB-2 종양단백질발현과의상관관계 연세대학교의과대학병리학교실 1 서울보건대학임상병리과 2 한경희 1 김태전 2 Correlation Between Expression of ER, PR and C-erbB-2 Protein as Histologic Grade of Breast Cancer Han, Kyung Hee 1., Kim, Tai Jeon 2 Department of Pathology, University Collage of Medicine, Seoul, Korea 1 Department of Clinical Laboratory Science, Seoul Health Collage, Sungnam, Korea 2 Overexpressions of the estrogen receptors(er), progesterone receptors(pr) and C-erbB-2 protein are important determiners of the response to chemotherapy in the breast cancer. For detecting ER, PR and C-erbB-2, immunohistochemistry are currently regarded as standard method. The purposes of this study compared to histologic grade and expression of the ER, PR and C-erbB-2 in breast cancer. We examined overexpression of ER, PR and C-erbB-2 protein in 84 breast carcinomas by using immunohistochemical stains. The following results were obtained. For histologic grade, 10 cases(11.9%) showed carcinoma in situ, 16 cases(19%) showed grade Ⅰ, 36 cases (42.9%) showed grade Ⅱ, and 22 cases(26.2%) showed grade Ⅲ among the 84 test samples. The average positive rate ER and PR was 63%, 46% showed carcinoma in situ, 80%, 60% showed grade Ⅰ, 64%, 41% showed grade Ⅱ, 34%, 23% showed grade Ⅲ, respectively. The induction of PR increased when induction of ER increased, thus showing significant relationship(p<0.05). The expression of C-erbB-2 protein was 9 cases(10.7%) in one positive(1+), 9 cases(10.7%) in two positive(2+), and 9 cases(10.7%) in three positive(3+). C-erbB-2 protein expression showed no statistical significance. In conclusion, ER and PR positive rates were inversely associated with histologic grades significantly(p<0.05). C-erbB-2 showed no significant difference with histologic grade. However ER, PR and C-erbB-2 showed significant relationship with each other(p<0.05). Therefore, these findings might be an important prognostic factor and might be arranged as a regular pathological examination in cases of breast cancer. Key Words : Histologic grade, Estrogen receptor, Progesterone receptor, C-erbB-2 1) 교신저자 : 한경희, ( 우 )120-752 서울시서대문구신촌동 134 번지, 연세대학교의과대학병리학교실 Tel : 02-361-5244, E-mail : hankh9121@hanafos.com 185
I. 서론 유방암은우리나라여성에서가장높은발생빈도를차지하고있으며, 최근에는자가검진과유방촬영술의발달로발견율이더욱높아지고있다. 유방암의발생에는에스트로겐호르몬이관련되어있다고알려져있으며, 유방암환자의호르몬수용체유무는예후와치료에있어서중요한역할을한다고알려져있다 (Taylor 등, 1981 ; Scottish 등, 1993). 이에스트로겐수용체 (estrogen receptor, ER) 가음성인유방암의항호르몬치료에대한반응은 3~10% 인반면, ER에양성인경우에는 50~60% 에서좋은반응을보인다 (Osborne 등, 1980; Wittliff, 1984). 특히 ER과프로게스테론수용체 (progesterone receptor, PR) 모두에서양성인유방암에서는 75~80% 의높은호전율을보인다 (Wittliff, 1984). C-erbB-2 단백질은유방, 타액선, 장상피, 신세뇨관등의정상선상피에존재하며, C-erbB-2 단백질의발현은 C-erbB-2(HER-2/neu) 유전자의증폭을반영하여악성종양에서증폭되어나타난다 (Park 등, 2000). 그래서면역조직화학염색에의한 C-erbB-2 종양단백질의발현을이종양유전자의증폭지표로사용할수있다고알려져있다 (De Potter 등, 1989). 유방암의약 20% 에서 C-erbB-2 단백질이과발현되고이는나쁜예후와관련되어있다고한다 (Ali 등, 1989; Berger 등, 1988 ; Wright 등, 1989; Slamon 등, 1987; Riou 등, 2001). 또한유방암의관내암종과침윤성관암종에서 C-erbB-2 단백질이높은발현율을보인다고보고되었다 (Gusterson 등, 1988 ; Barnes 등, 1992). 따라서본연구에서는유방관암종의조직학적악성도를평가하고, 면역조직화학염색을통하여예후인자인 ER 및 PR의양성률과 C-erbB-2 종양단백질의발현율을평가하여, 조직학적악성도와의상관관계를알아보고, 예후인자들상호간의관련성을비교해보고자하였다. II. 재료및방법 1. 재료본실험에사용된재료는 2003년 1월부터 2003년 12월사이에신촌세브란스병원에서유방암으로진단을받고유방절제수술을시행한환자의종양조직을사용하였으며, 유방관암종 (breast ductal carcinoma) 으로병리조직학 적진단을받은 84예로하였다. 연령분포는 28~76세로평균 47.8세였다. 2. 방법 1) 유방암의조직학적악성도검사외과적수술로절제된유방암조직을일반적인조직표본제작과정인 10% 포르말린용액에고정한후, 파라핀침투과정을거쳐포매하여파라핀블록을만들었다. 파라핀블록은박절기 (microtome, Microm) 를이용하여 3μm두께로박절하여유리슬라이드에부착시키고, xylene용액으로탈파라핀과정을거친후단계적알코올로함수과정을실행한다음, hematoxylin & eosin 염색액으로핵과세포질을염색하였고, 알코올로탈수과정을거쳐, xylene 으로투명과정을실시한후, histomount액으로봉입하였다. 유방암의조직학적악성도판정은 Elston과 Ellis (1991) 의반정량적방법을사용하여병리의사에의해광학현미경으로검경하여판정하였다. 2) ER, PR과 C-erbB-2의면역조직화학적검사조직표본파라핀블록을박절기를이용하여 3μm두께로박절한다음, 3-aminopropyltriethoxy silane(sigma Chemical, Germany) 으로처리된코팅슬라이드에박절된리본을부착시켰다. 탈파라핀과함수과정을거친후, 3% H 2 O 2 에 10분간담그고수세한다음, 10mM citrate buffer(ph6.0) 를 microwave에넣고 10분간예열한후, 슬라이드를담그고다시 15분끓인다. 실온에서 30분간방치한후, PBS(phosphate buffer solution, ph 7.5) 에 10분간담그고, 1차항체로 ER, PR과 C-erbB-2를 Table 1에있는배율로희석하여슬라이드에떨어뜨리고, 냉장고에 18시간넣어둔다. 그다음 PBS(pH 7.5) 에 10분간담그고, 2차항체 (link antibody, anti-mous and anti-rabbit Ig) 에 20분간작용시키고, PBS(pH 7.5) 에 10분담근다. streptavidin peroxidase에 20분간작용시키고, PBS(pH 7.5) 에 10분간담근다음, DAB(diaminobenzidine) 발색제에 5분발색시키고, PBS(pH 7.5) 에 3분간담근후수세한다. Mayer's hematoxylin에 5분대조염색하고, 수세한다음, 단계적알코올에탈수시키고, xylene에투명하여봉입한다. 3) 면역조직화학염색의양성판정및평가 ER과 PR은면역조직화학적으로염색하여핵에염색된것을양성으로판정하여양성 (+) 과양성백분율 (%) 로표 186
Fig. 1. Immunohistochemical staining for ER and PR in breast ductal carcinoma(abc, 400). A ; Showing ER positive in nuclei of cancer cells(arrow). B ; Showing PR positive in nuclei cancer cells(arrow). Table 1. Antibodies, dilution, and sources Antibody Source Clone Dilution Microwave Estrogen Receptor LAB-VISION SP1 1:100 ~ 1:400 Yes Progesterone Receptor DAKO PgR636 1:50 Yes C-erbB-2 oncoprotein DAKO Polyclonal 1:100 ~ 1:200 Yes 시하였고, C-erbB-2 단백질은세포막을따라염색된것을양성으로하여종양조직에서양성세포의백분율이 0% 인경우음성 (-), 1-30% 인경우 1+, 31-60% 인경우 2+, 그리고 61% 이상인경우 3+ 로하였다. 4) 통계분석통계분석은 SAS프로그램을이용하여조직학적악성도와예후인자들인 ER, PR과 C-erbB-2 종양단백질사이에상관관계를 Chi-square검정과 Fisher의정확검정을사용하여검정하였고, 통계학적유의성은 p<0.05인경우로하였다. Table 2. Histologic grades of breast ductal carcinoma Histologic grade No. of case(%) in situ 10(11.9%) Ⅰ 16(19.0%) Ⅱ 36(42.9%) Ⅲ 22(26.2%) (19%), grade Ⅱ가 36예 (42.9%), grade Ⅲ가 22예 (26.2%) 로나타났다 (Table 2). III. 결과 1. 유방암의조직학적악성도유방암의조직학적악성도는 H&E로염색한슬라이드를광학현미경으로관찰하여핵의다형성및분열수, 세관의형성등을종합하여판정하였으며, 총 84예중관내암종 (in situ ca) 이 10예 (11.9) 였고, grade Ⅰ이 I6예 2. ER 및 PR의발현 ER 및 PR의발현율을검사하기위해면역조직화학염색을하여관찰한결과, 유방암의종양세포핵에적갈색으로염색된것을양성으로하였다 (Fig. 1A, B). 유방관암종에서검체의 ER, PR 양성률 (%) 의평균은관내암종에서 63%, 46% 였고, grade Ⅰ에서 80%, 60% 였으며, grade Ⅱ에서 64%, 41% 였고, grade Ⅲ에서 34%, 23% 로나타났다 (Fig. 2). 또한 ER, PR의양성검체수는관내암 187
Table 3. Histologic grade vs. ER, PR, and in infiltrating duct carcinoma of the breast Histiologic graded (n) ER - + - + PR in situ 10(11.9%) 1(1.19%) 9(10.71%) 2(2.38%) 8(9.52%) Ⅰ 16(19.0%) 0(0%) 16(19.05%) 2(2.38%) 14(16.67%) Ⅱ 36(42.9%) 6(7.14%) 30(35.71%) 12(14.29%) 24(28.57%) Ⅲ 22(26.2%) 12(14.29%) 10(11.9%) 12(14.29%) 10(11.9%) Total 84(100%) 19(22.62%) 65(77.38%) 28(33.33%) 56(66.67%) p-value 0.0003 0.0388 90% 80% 80% 70% 60% 63% 60% 64% 50% 40% 46% 41% 34% ER PR 30% 23% 20% 10% 0% IDC in situ 1 2 3 histologic grade Fig. 2. ER and PR average positive rates as histologic grades. 종에서 9예 (10.7%), 8예 (9.5%) 였고, grade Ⅰ에서 16예 (19%), 14예 (16.7%) 였으며, grade Ⅱ에서 30예 (35.7%), 24 예 (28.6%) 였고, grade Ⅲ에서 10예 (11.9%), 10예 (11.9%) 였다 (Table 3). 조직학적악성도와 ER, PR사이의상관관계는카이제곱검정한결과 p<0.05를나타내어통계학적으로유의성이있는것으로나타났다. 3. C-erbB-2 종양단백질의발현유방암의종양조직을면역조직화학염색을통하여 C-erbB-2 종양단백질을발현시켜광학현미경으로관찰한결과종양세포의세포막을따라적갈색으로염색되었으며핵은 hematoxylin에의해청색으로염색되었다 (Fig. 3). C-erbB-2 종양단백질은총 84예중음성 (-) 이 57예 (67.9%) 였으며, 1+~3+ 모두에서각각 9예 (10.7%) 를나타냈다. 조직학적악성도에따라 C-erbB-2 종양단백질의발현율은 Table 4와같다. Fig. 3. Immunohistochemical staining for C-erbB-2 in breast ductal carcinoma(abc, 400). C-erbB-2 protein shows the strong membranous cancer cells. 4. ER, PR 과 C-erbB-2 의상관관계 유방암에서병리조직학적예후인자들인 ER, PR 과 C-erbB-2 사이의상관관계를비교하여보았다 (Table 5). ER, PR과 C-erbB-2사이의상관성을카이제곱검정한결과 p값이 ER과 PR 모두에서 p<0.05로나타나통계학적으로유의성이있었다. IV. 고 찰 유방암은현재여성암중에서발생률이가장높으며수술에의해종양을제거하고수술후치료는예후인자인액와림프절의전이유무, ER, 및 PR의존재여부, 종양의 188
Table 4. Histologic grade vs. C-erbB-2 in infiltrating ductal carcinoma of the breast Histiologic C-erbB-2 grade (n) - 1+ 2+ 3+ in situ 10 7(8.33%) 2(2.38%) 0(0%) 1(1.19%) Ⅰ 16 12(14.29%) 2(2.38%) 1(1.19%) 1(1.19%) Ⅱ 36 22(26.19%) 5(5.95%) 4(4.76%) 5(5.95%) Ⅲ 22 16(19.05%) 0(0%) 4(4.76%) 2(2.38%) Total 84 57(67.86%) 9(10.71%) 9(10.71%) 9(10.71%) Table 5. ER, PR vs. C-erbB-2 expression in breast ductal carcinoma C-erbB-2-1+ 2+ 3+ ER - 19(22.6%) 0 0 0 + 38(45.2%) 9(10.7%) 9(10.7%) 9(10.7%) p-value 0.0088 PR - 28(33.3%) 0 0 0 + 29(34.5%) 9(10.7%) 9(10.7%) 9(10.7%) p-value 0.0002 크기및조직학적진단에의해이루어진다 (Tandon 등, 1989). 유방암환자에있어서호르몬수용체는예후나치료를결정하는중요한인자중의하나로서반드시검사되어야한다 (Taylor 등, 1981 ; Osborn 등, 1980 ; Mirecki 등, 1985). 원발성유방암의 55~66% 가호르몬수용체를가지고있으며전이성유방암에서호르몬수용체는 45~55% 이다. 특히갱년기이후에발생한유방암인경우에호르몬수용체는대체적으로발현율이높다고한다 (Wittliff 등, 1984). 호르몬수용체가양성인유방암에서는수술후추가치료를안한경우도종양크기가작은음성보다생존율이높고, 항호르몬치료에양호한반응을보인다 (Pertschuk 등, 1990; Hahnel 등, 1979). 호르몬수용체양성인종양은약 55% 에서항호르몬치료에좋은반응을보이고, 음성인경우의반응은 3~10% 미만이며또한항암요법에대한세포독성이높다 (Osborn 등, 1980 ; Wittliff, 1984). 유방관암종의면역조직화학염색에서 ER과 PR의양성률이높으면항호르몬치료에대한반응률도높다 (Osborn 등, 1980 ; Bloom 등, 1957 ; Chang 등, 1990). 즉호르몬수용체양성인경우에는보다생존율이높고재발율이낮다고알려져있다 (Mireck 등, 1985 ; Hahnel 등, 1979). 면역조직화학검사결과의호르몬수용 체 (ER, PR) 양성빈도는 50~80% 로다양하게보고되어졌으며 (Tesch 등, 1993 ; Shintaku 등, 1987 ; Vielh 등, 1992 ; Lozowski 등, 1990), 본연구에서도 ER과 PR의평균양성률은 77.4% 와 66.7% 로유사하게나타났다 (Table 3). Osborne 등 (1980) 은 ER과 PR의양성률은서로상관관계가있으므로두가지중하나만시행해도된다고주장하였고 Vollenweider-Zerargui 등 (1986) 은 ER이 PR보다더욱민감한표지자역할을하며 ER+/PR+ 인경우와 ER+/PR-인경우가서로대등한관계라고하였다. 본연구에서도유방관암종의조직학적악성도가높아짐에따라 ER과 PR의양성률이낮아지는것으로나타나통계학적으로유의성을나타냈으며 (p<0.05), ER의발현율이상승함에따라서 PR의발현율도상승하여유사한경향을나타내통계학적으로유의성을보였으며, 이러한경향으로보아조직학적악성도와 ER, PR의사이에상관관계가있다고보여졌다. Khan 등 (1994) 과 Karayiannakis 등 (1996) 은 ER 및 PR은양성종양보다악성종양에서발현율이높고 ER과 PR의발현율은서로연관성이있다고보고한것과본연구결과와차이가없었다. 그리고 ER 및 PR의발현율과 C-erbB-2 종양단백질발현율과의사이에도통계학적유의성이있었으나 (Table 5), 조직학적악 189
성도와 C-erbB-2 사이에는유의한차이가없었다 (Table 4). 유선이나자궁내막의성장에있어서스테로이드호르몬의영향을받는다는것은이미알려진사실이다. 그러나이러한호르몬이표적세포에작용하기위해서는세포의핵막에존재하는수용체와결합해야하며, 스테로이드호르몬의영향을받으므로유방암의치료에있어서방사선치료나항암화학요법보다는호르몬치료요법이효과적이라는것을시사한다. 세포성장에관여하는 C-erbB-2 종양단백질은염색체 17q21에위치하는 tyrosine 활성을가지는 185kd의막당단백질 (transmembrane glycoprotein) 이며표피성장인자수용체 (epidermal growth factor receptor) 와유사한구조를가지고있다 (Schechter 등, 1985 ; Fukushige등, 1986). C-erbB-2 단백질은정상선상피에서존재하며면역조직화학염색에서세포막을따라염색되고악성종양에서증폭되어나타난다. 특히유방암에서약 20% 가과발현되는것으로보고되었다 (Ali 등, 1988; Berger 등, 1988; Wright 등, 1989). 본연구에서는유방관암종에서약 32% 가발현되어약간높게나타났으며, 조직학적악성도와는유의한상관성이없었다 (Table 4). Wright 등 (1989) 은 C-erbB-2 종양단백질발현과림프절전이와는상관관계가없다고하였으나, Tandon 등 (1989) 은상관관계가있다고하였다. Berger 등 (1988) 은 C-erbB-2 종양단백질과조직학적악성도와의상관관계는유의하다고하였고, Tsuda 등 (1992) 은상관관계가없는독립적인예후인자라고하였다. 본연구에서는조직학적악성도와 C-erbB-2 종양단백질사이에는통계학적유의성은없었으나, 조직학적악성도의모든등급에서 C-erbB-2가총 32.1% 발현되어유방암의종양단백질이증폭하여존재하는것으로보여지므로 C-erbB-2 종양단백질이양성인경우에는 C-erbB-2(HER-2/neu) 유전자의검출을시행해야한다고생각된다. 또한유방관암종에서스테로이드호르몬수용체와 C-erbB-2 종양단백질사이에는유의한상관관계가있었다. 그러므로유방암에서 ER과 PR은양호한예후인자라고판단되어지며, 이들예후인자들은유방암환자의예후및치료에필요한보다정확한정보를제공할수있다고생각된다. V. 결론 유방암의종양조직총 84예를 H&E염색으로조직학적악성도검사를하였으며, 면역조직화학염색을하여 ER 및 PR과 C-erbB-2 종양단백질을평가하여, 조직학적악성도와 ER, PR과 C-erbB-2 종양단백질사이에상관관계를비교분석하여다음과같은결론을얻었다. 1. 조직학적악성도는총 84예중관내암종이 10예 (11.9%) 였고, grade Ⅰ이 16예 (19%) 였으며, grade Ⅱ가 36예 (42.9%) 였고, grade Ⅲ가 22예 (26.2%) 로나타났다. 2. ER과 PR은조직학적악성도가높아짐에따라반대로양성률이저하되는것으로나타났으며, ER의발현이증가하면 PR의발현도증가하였고, 통계학적으로유의한차이 (p<0.05) 를나타내었다. 3. C-erbB-2 종양단백질발현은총 84예중음성 (-) 이 57예 (67.86%), 1+ 가 9예 (10.7%), 2+ 가 9예 (10.7%), 3+ 가 9예 (10.7%) 로나타났으며, 조직학적악성도와통계학적유의성은없었다. 4. ER 및 PR과 C-erbB-2 종양단백질과의상관관계는통계학적유의성을나타내어 (p<0.05), 예후인자들간에상호연관성이있는것으로나타났다. 이상을종합하면, ER과 PR의발현은유방암의양호한예후인자로볼수있으며, C-erbB-2 종양단백질은조직학적악성도와상관관계가적으나모든등급에서발현되므로 C-erbB-2(HER-2/neu) 유전자검출방법인 FISH (fluorescent in situ hybridization) 법등을이용하여유전자검출을실행하여야한다고생각되며, 이러한연구결과는유방암환자의예후및치료에대한많은정보를제공할수있어병리조직검사에있어서반드시필요하다고생각된다. 참고문헌 1. Ali IU, Campbell G, Lidereau R, Callahen R. Amplification of c-erbb-2 and aggressive human breast tumors. Science 240:1795-1796, 1988 2. Barnes DM, Barkova J, Camplejohn RS, Gullick WJ, Smith PJ, Millis RR. Overex pression of the c-erbb-2 oncoprotein: Why does this occur more frequently in ductal carcinoma in situ than in invasive mammary carcinoma and is this progno stic significance?. Eur J Cancer 28:644-648, 1992 3. Berger MS, Locher GW, Saurer S, Gallick WJ. Correlation of c-erbb-2 gene amplification and protein 190
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