244 부합화는조직학적으로동정된암세포의핵내에서유전자의증폭여부를정확히구별할수있는장점을가지고있으며, 또한소량의조직에서도검색이가능하므로조기진단으로얻을수있는암조직의양이점점작아지는현실적인면에서도유리하다. 유방암환자에게서 HER-2/neu 유전자의증폭분석이면역조직화학염색보다예후를

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1 대한병리학회지 : 제 36 권제 4 호 2002 The Korean Journal of Pathology. 2002; 36: 유방암종의 HER-2/neu 검색을위한형광제자리부합화와면역조직화학염색법의비교 인제대학교상계백병원병리과 Comparing Fluorescence In Situ Hybridization and Immunohistochemistry to Determine the HER-2/neu Status in Breast Carcinoma Kyeongmee Park, Jungyoen Kim and Sungjig Lim Department of Pathology, Inje University Sanggye Paik Hospital, Seoul, Korea 접수 : 2002 년 5 월 8 일게재승인 : 2002 년 7 월 20 일 책임저자 : 박경미우 서울시노원구상계 7동 인제대학교상계백병원병리과전화 : Fax: ysdol@unitel.co.kr * 이논문은 2001년도인제대학교학술연구조성비보조에의한것임. Background : Identification of HER-2/neu status is important in predicting the response to specific chemotherapy in breast carcinoma patients and HER-2/neu status is associated with poor clinical outcome even with systemic chemotherapy. Introduction of fluorescence in situ hybridization (FISH) allows an accurate assessment of the level of gene amplification with information about distribution of gene copies in histologic sections. Methods : HER-2/neu status was performed on paraffin sections of 176 primary breast carcinomas by FISH, using PathVysion and by immunohistochemistry (IHC), using HercepTest. The results of HER- 2/neu amplification was compared with clinical and pathological prognostic factors. Results : HER-2/neu amplification and overexpression were detected in 51 tumors (29.0%) by FISH and 32 tumors (18.2%) by IHC. The results of each method agreed with each other in 157 tumors (concordance: 89.2%, kappa=0.783). HER-2/neu amplification was associated with poor nuclear grade, marked nuclear pleomorphism, and presence of the combined ductal carcinoma in situ in the invasive ductal carcinomas as well as Van Nuys grade of the ductal carcinoma in situ component (p<0.05). Conclusions : The comparison of FISH and IHC demonstrated an excellent correlation of HER-2/neu overexpression 2+ and 3+ with gene amplification. However, FISH may be a more accurate and reliable method for negative and 1+ cases. HER-2/neu amplification proves to be of prognostic relevance. Key Words : Mammary Neoplasms-In Situ Hybridization, Fluorescence-Genes, erbb-2, Immunohistochemistry HER-2/neu (c-erbb-2) 암유전자는 185 kda의 transmembrane growth factor receptor로서티로신키나아제작용을가지고있으며세포의성장을자극한다. 유방암종에서 HER-2/neu 발현율은 20-30% 로예후예측, 항암치료및호르몬치료에있어그중요성이증명되고있다. 1-3 HER-2/neu 증폭이나과발현을나타내는환자의경우 doxorubicin 치료에효과가있다는보고와 4-6 항암치료제의양을증가시키면효과가강화된다는보고들 5-7 은항암제선택에있어서 HER-2/neu의중요성을시사하였다. 최근 trastuzumab (HERCEPTIN ; Genentech, Inc., South San Francisco, CA, U.S.A.) 이라는 HER-2/neu에대한단클론항체치료제의개발과전이유방암종에대한높은치료효과는 HER-2/neu의검색이유방암종환자치료에필수적 임을말해준다. 8 유방암종환자에게서 HER-2/neu의발현이있는경우 cyclophosphamide, methotrexate, 5-fluorouracil (CMF) 치료에내성을보이는경우가많고 tamoxifen을포함한호르몬치료에내성을보인다는보고도계속되고있다 결국항암제치료를받는모든환자에게 HER-2/neu의검색은효과적인치료를위하여필수적인과정이되었다. HER-2/neu 의과발현을보기위한단백검색방법으로는면역조직화학염색이가장많이이용되지만, 이용되는항체의특이도와판정기준에따라결과가다르다. 12 HER-2/neu 유전자의경우단백의과발현보다는유전자의증폭이항암제에대한효과를더정확히예측할수있는지표이므로형광제자리부합화 (fluorescence in situ hybridization: FISH) 검색이필수적이다. 13,14 형광제자리 243

244 부합화는조직학적으로동정된암세포의핵내에서유전자의증폭여부를정확히구별할수있는장점을가지고있으며, 또한소량의조직에서도검색이가능하므로조기진단으로얻을수있는암조직의양이점점작아지는현실적인면에서도유리하다. 유방암환자에게서 HER-2/neu 유전자의증폭분석이면역조직화학염색보다예후를더정확히예측할수있다는연구결과도형광제자리부합화의장점을보여주는것이다.

2 244 부합화는조직학적으로동정된암세포의핵내에서유전자의증폭여부를정확히구별할수있는장점을가지고있으며, 또한소량의조직에서도검색이가능하므로조기진단으로얻을수있는암조직의양이점점작아지는현실적인면에서도유리하다. 유방암환자에게서 HER-2/neu 유전자의증폭분석이면역조직화학염색보다예후를더정확히예측할수있다는연구결과도형광제자리부합화의장점을보여주는것이다. 1 그러나여러장점을가지고있음에도불구하고실제적으로형광제자리부합화를이용한 HER-2/neu 유전자증폭분석은보편화되지못하고있다. 형광제자리부합화를시행하기위해서는형광물질을분석할수있는특수현미경및필터가필요하며검사에사용되는시약가격의저렴화도요구된다. 본연구는유방암종조직에서형광제자리부합화와면역조직화학염색을이용하여 HER-2/neu 유전자증폭과단백질과발현의결과를비교분석하고자시행되었다. 재료와방법 1989년 9월부터 2000년 8월까지인제대학교상계백병원에서유방암종절제술을받은환자중임상정보를갖춘침윤관암종 176예를대상으로하였다. 침윤관암종은 Nottingham 분류법 15 에의한조직학적등급을사용하였고동반된관내암종은 Van Nuys 분류법 16 에의하여등급을나누었다. 를선택하여 CEP 녹색신호와 HER-2/neu 오렌지신호의수를관찰하여비율을구하였다. HER-2/neu 오렌지신호가 CEP 녹색신호의 2배이상인경우를유전자증폭으로간주하였다 (Fig. 1). 면역조직화학염색두께 3.5 m의슬라이드를탈파라핀하여 의전처리용액 (HercepTest kit; DAKO, Carpinteria, CA, U.S.A.) 에 40분처리한후실온에서 20분간식혔다. 세척완충액으로세척한후과산화효소차단제 (HercepTest kit; DAKO) 를 5분간적용하였다. 200 L의일차항체를조직위에 30분간적용한후세척하였다. 200 L의 Visualization Reagent (HercepTest kit; DAKO) 를 30분간적용한후세척하였다. 200 L의 diaminobenzidine tetrahydrochloride (DAB) 를 10분간적용한후세척하고 Mayer s hematoxylin으로대조염색을시행하였다. 암세포의세포막에갈색으로염색이되는정도에따라다음과같이점수를주었다. 음성반응을나타내거나 10% 미만의암세포에서약하게염색되는경우를 0, 암세포의 10% 이상에서염색은되었으나세포막의일부가약하게염색된경우는 1+, 암세포의 10% 이상에서세포막의전체가중등도로염색된경우를 2+, 암세포의 10% 이상에서세포막의전체가강하게염색된경우를 3+ 로판독하였으며, 그중염색점수가 2+ 와3+ 인경우를 HER-2/neu 단백질의과발현으로간주하였다 (Fig. 2). 형광제자리부합화 두께 3.5 m의슬라이드를 56 로 24시간두었다가탈파라핀한후 100% 알코올로탈수시켜 상태를유지하였다. 0.2 N HCl에 20분담근후증류수에 3분간넣고세척완충액 (Vysis Inc., Downers Grove, IL, U.S.A.) 에 3분간두었다. 80 의전처리용액 (Vysis) 에 30분간처리하고증류수에 1분간담근후세척완충액에 5분동안 2회세척하였다. 37 의단백분해효소용액 (Vysis) 에 10분동안처리한후세척완충액에 5분씩 2회세척하고 상태를유지하면서탈수하였다. 포르말린에 10분간고정시킨후세척완충액으로 5분씩 2회세척하고 상태를유지하면서탈수하였다. 72 의변성용액 (Vysis) 에 5분간적용한후 70%, 85%, 100% 알코올에순서대로 1분씩담그고 상태로탈수하였다. 10 L의 LSI HER-2/CEP probe (Vysis) 를적용한후커버슬립으로덮고 37 로 14-18시간동안유지하였다. 실내온도의후부합화세척완충액 (Vysis) 에넣어커버슬립을제거한후 72 의후부합화세척완충액에 2분간적용한다음슬라이드를어두운곳에세운상태로탈수하였다. 10 L의 4,6-diamidino-2-phenylindole (DAPI, Vysis) 로염색하여관찰하였다. 현미경의 1,000배시야에서암세포가한층으로배열된부위 Fig. 1. Fluorescence in situ hybridization shows increased HER- 2/neu gene copy number in breast cancer tissue (HER-2/neu gene probe, orange signals; chromosome 17 centromere control, green signals).

유방암종의 HER-2/neu 검색 245 A B C D Fig. 2. Scores for HER-2/neu overexpression range from 0 to 3+ (A) 0: Negative; No staining.

(C) 2+: Week positive; A weak to moderate complete membrane staining is observed in >10% of tumor cells.

05 미만인것을의미있는것으로하였다. 측정인자들간의상관성검증에는 Chi-square 법을사용하였으며 Spearman s correlation coefficient (kappa) 에의하여형광제자리부합화와면역조직화학염색결과의일치성에대한상관관계를구하였다. kappa 계수가 1에근접할수록완전한일치에가깝고 0.4 이하는불량한일치로보았다.

9%) 가관내암종을동반하였고, 그중광범위관내암종인경우는 41예 (31.5%) 였다. 동반된관내암종의 Van Nuys에의한등급은 I이 11예 (8.5%), II가 47예 (36.2%), III이 72예 (55.4%) 였다.

3 유방암종의 HER-2/neu 검색 245 A B C D Fig. 2. Scores for HER-2/neu overexpression range from 0 to 3+ (A) 0: Negative; No staining. (B) 1+: Negative; A faint membrane staining is detected in >10% of tumor cells. The cells are only stained in part of their membrane. (C) 2+: Week positive; A weak to moderate complete membrane staining is observed in >10% of tumor cells. (D) 3+: Strong positive; A strong complete membrane staining is observed in >10% of tumor cells. 통계학적분석통계분석은 Statistical Package for the Social Science (SPSS) 8.0 (Systat, Chicago, U.S.A.) 통계프로그램을이용하였고 p 값이 0.05 미만인것을의미있는것으로하였다. 측정인자들간의상관성검증에는 Chi-square 법을사용하였으며 Spearman s correlation coefficient (kappa) 에의하여형광제자리부합화와면역조직화학염색결과의일치성에대한상관관계를구하였다. kappa 계수가 1에근접할수록완전한일치에가깝고 0.4 이하는불량한일치로보았다. 결과침윤관암종 176예중 Nottingham 분류에의한조직학적등급은 I이 22예 (12.5%), II가 83예 (47.2%), III이 71예 (40.3%) 였고, Black의분류에의한핵등급은 I이 75예 (42.6%), II가 84예 (47.7%), III이 17예 (9.7%) 였다. 침윤관암종의 130예 (73.9%) 가관내암종을동반하였고, 그중광범위관내암종인경우는 41예 (31.5%) 였다. 동반된관내암종의 Van Nuys에의한등급은 I이 11예 (8.5%), II가 47예 (36.2%), III이 72예 (55.4%) 였다. 총 176예에대하여 HER-2/neu 검색을위한형광제자리부합화와면역조직화학염색을시행한결과, 형광제자리부합화와면역조직화학검사에서 HER-2/neu 유전자증폭과단백질과발 Table 1. Correlation between results of FISH and immunohistochemistry for the detection of HER-2/neu amplification and overexpression in 176 breast carcinomas FISH 0 IHC Total No amplification Amplification Total Concordance: 89.2%. FISH: fluorescence in situ hybridization, IHC: immunohistochemistry. 현을나타낸예는각각 51예 (29.0%) 와 32예 (18.2%) 였다. 형광제자리부합화와면역조직화학결과에서일치를보인경우는 157 예 (89.2%) 였으며 kappa 계수는 0.783였다. 검사결과의불일치를보인 19예모두형광제자리부합화에서는 HER-2/neu 유전자증폭이관찰되었으나면역조직화학염색에서는 HER-2/neu 단백의과발현이관찰되지않았다. 그중에서단백발현의점수가0과1+ 인경우가각각 11예, 8예였다 (Table 1). 병리학적소견과비교해보았을때침윤관암종의핵등급이나쁠수록 HER-2/neu 유전자증폭이증가하였고동반된관내암종의 Van Nuys 등급이높을수록 HER-2/neu 유전자의증폭이증가되었다 (p<0.05). HER-2/neu 유전자증폭은관내암종이동반된침윤관암종에서동반되지않은경우보다자주관찰되었다 (p<0.05). 그러나조직학적등급이나동반된광범위관내암종의유무및 Ki-67 표지지수와는상관성이없었다. 침윤관암종의조직학적등급을위하여사용하는 Nottingham분류법의 3가지

4 246 Table 2. Relationship between HER-2/neu amplification and pathological parameters Table 3. Relationship between HER-2/neu amplification and clinical parameters HER-2/neu (%) No Amplification Amplification p-value HER-2/neu (%) No Amplification Amplification p-value Histologic grade I 20 (90.9) 2 (9.1) II 63 (75.9) 20 (24.1) III 53 (74.6) 18 (25.4) Tubule formation (81.8) 2 (18.2) 2 25 (78.1) 7 (21.9) (76.7) 31 (23.3) Nuclear pleomorphism (100.0) 0 (0.0) 2 31 (88.6) 4 (11.4) (73.5) 36 (26.5) Mitotic index (81.8) 16 (18.2) 2 28 (75.7) 9 (24.3) 3 36 (70.6) 15 (29.4) Nuclear grade I 54 (72.0) 21 (28.0) II 66 (78.6) 18 (21.4) III 16 (94.1) 1 (5.9) DCIS Absent 42 (91.3) 4 (8.7) Present 94 (72.3) 36 (27.7) EIC Absent 105 (77.8) 30 (22.2) Present 31 (76.2) 10 (23.8) Van Nuys grade of associated DCIS (10.0) 0 (0.0) 2 36 (80.0) 9 (20.0) 3 43 (61.4) 27 (38.6) Ki-67 labeling index (%) <10 71 (78.0) 20 (22.0) (75.6) 20 (23.5) DCIS: ductal carcinoma in situ, EIC: extensive intraductal carcinoma. 요소를 HER-2/neu 유전자증폭과비교해보았을때핵의다형성이심할수록 HER-2/neu 유전자증폭이증가하였지만 (p<0.05), 관구조형성의정도나유사분열의정도와는상관성을나타내지않았다 (Table 2). 침윤관암종에서 HER-2/neu 유전자증폭과환자의연령, 종양의크기, 림프절전이, 호르몬수용체, 병기, 재발및생존율등과유의한상관성은없었다 (Table 3). 고찰 유방암종환자에게서 HER-2/neu 유전자검색의주목적은가장효과적인치료약제의선택에있다. 특히전이성유방암종치료제인 trastuzumab (Herceptin ) 이미국 Food and Drug Administration (FDA) 에서승인되면서 HER-2/neu의중요성 Age (yr) <50 90 (77.6) 26 (22.4) (76.7) 14 (23.3) Size (cm) <2 36 (75.0) 12 (25.0) (80.0) 22 (20.0) >5 12 (66.7) 6 (33.3) Node metastasis (73.0) 24 (27.0) (81.4) 8 (18.6) 4 36 (81.8) 8 (18.2) ER Negative 72 (76.6) 22 (23.4) Positive 49 (78.0) 18 (22.0) PR Negative 55 (77.5) 16 (22.5) Positive 81 (77.1) 24 (22.9) Stage (76.3) 9 (23.7) 2 67 (76.1) 21 (23.9) 3 34 (81.0) 8 (19.0) 4 6 (75.0) 2 (25.0) ER: estrogen receptor, PR: progesterone receptor. 이더욱강조되고있다. 이와함께 HER-2/neu 검사의면역조직화학염색인 HercepTest 역시 FDA의승인을받게되었다. 현재까지 HER-2/neu 유전자검색으로는면역조직화학염색이보편적으로사용되어왔으나, 최근형광제자리부합화와면역조직화학검사간의불일치가계속보고되고있으므로 HER- 2/neu 유전자단백의과발현보다는유전자의증폭을증명할수있는형광제자리부합화검사가요구되고있다. 12,17-19 본연구에서는총 176예의침윤관암종을대상으로형광제자리부합화와 HercepTest 면역조직화학염색결과를비교하여일치율 89.2% 와 kappa 값 0.783의결과를얻었는데, 이는다른보고에서와마찬가지로좋은성적이었다 특히면역조직화학결과가 2+, 3+ 인경우형광제자리부합화에서도 HER-2/neu 유전자증폭을확인할수있었다. 그러나면역조직화학염색점수가 0이거나1+ 인경우의소수에서 HER-2/neu 유전자증폭을나타냈다. 이러한결과는다른연구자들이보고하였듯이저등도의유전자증폭을나타냈거나면역조직화학염색의기술적인문제로생각된다. 21,22 그러므로면역조직화학검사의위음성을극복하려면형광제자리부합화가시행되어야함을시사한다. 많은연구자들이보고하였듯이 21,22 HER-2/neu 유전자증폭은겨드랑림프절전이, 1,4,23 호르몬수용체의결핍, 1,4,24-26 종양의크기 1,4,23 등과같은불량한예후와상관성이있는것으로알려져있다. 본연구에서는이러한임상적예후인자들과의미있는상관성을나타내지않았지만향후항암치료제에따른환자의재분류를시

5 유방암종의 HER-2/neu 검색 247 도하여 HER-2/neu 유전자증폭과치료방법에따른예후와의상관성을확인할필요성이있을것이다. 본연구결과처럼 HER-2/neu 유전자와호르몬수용체발현간의의미있는상관성을발견하지못한보고도있다. 27 조직학적등급과 Ki-67 표지지수도 HER-2/neu 유전자와상관성을나타내지않았고다른문헌에서도그결과가다양하다. 24,27 광범위관내암종은유방암종의재발과관련이있다고알려져있지만 28,29 HER-2/neu의증폭과유의한상관성은없었다. 이는광범위관내암종이유방암종의분포범위와상관은있으나암세포의생물학적특성과는직접적관련이없기때문이라고생각한다. 그러나침윤관암종의핵등급과동반된관내암종의 Van Nuys 등급은 HER- 2/neu의증폭과의미있는상관관계를나타냈다. 16,24 이러한결과는 HER-2/neu 유전자의증폭이불량한예후와연관이있음을시사한다. Nottingham 분류에의한조직학적등급에사용하는 3가지인자중핵의다형성만 HER-2/neu 유전자의증폭과상관성을나타냈다는것은유방암종의조직학적분화보다는핵의분화가절대적으로예후와상관성이크다는것을암시하는데, 다른문헌에서도 HER-2/neu 유전자의증폭이핵의등급과밀접한관계가있다고보고하였다. 30 침윤관암종이관내암종을동반하는경우에 HER-2/neu 유전자의증폭이의미있게관찰되었다. 결론적으로 HER-2/neu 암유전자검색방법에있어면역조직화학염색에서 2+, 3+ 를나타낸경우에는형광제자리부합화와높은일치율을나타냈으나 0 또는 1+ 경우에는제한점이있으므로형광제자리부합화가면역조직화학염색의단점을보완할수있는좋은방법이될것으로생각한다. 참고문헌 1. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987; 235: Paik S, Hazan R, Fisher ER, et al. Pathologic finding from the national surgical adjuvant breast and bowel project: prognostic significance of erbb-2 protein overexpression in primary breast cancer. J Clin Oncol 1990; 8: Toikkanen S, Helin H, Isola J, Joensuu H. Prognostic significance of HER-2 oncoprotein expression in breast cancer: a 30-yr follow-up. J Clin Oncol 1992; 10: Muss HB, Thor AD, Berry DA, et al. c-erbb-2 expression and response to adjuvant therapy in women with node-positive early breast cancer. N Engl J Med 1994; 330: Paik S, Bryant J, Park C, et al. erbb-2 and response to doxorubicin in patients with axillary lymph node-positive, hormone receptor-negative breast cancer. J Natl Cancer Inst 1998; 90: Thor AD, Berry DA, Budman DR, et al. erbb-2, p53, and efficacy of adjuvant therapy in lymph node-positive breast cancer. J Natl Cancer Inst 1998; 90: Clark GM. Should selection of adjuvant chemotherapy for patients with breast cancer be based on erbb-2 status? J Natl Cancer Inst 1998; 90: Pegram M, Hsu S, Lewis G, et al. Inhibitory effects of combinations of HER-2/neu antibody and chemotherapeutic agents used for treatment of human breast cancers. Oncogene 1999; 18; Allred DC, Clark GM, Tandon AK, et al. HER-2/neu in node-negative breast cancer: prognostic significance of overexpression influenced by the presence of in situ carcinoma. J Clin Oncol 1992; 10: Tetu B, Brisson J, Plante V, Bernard P. p53 and erbb-2 as markers of resistance to adjuvant chemotherapy in breast cancer. Mod Pathol 1998; 11: Bitran JD, Samuels B, Trujillo Y, Klein L, Schroeder L, Martinec J. Her2/neu overexpression is associated with treatment failure in women with high-risk stage II and stage IIIA breast cancer (>10 involved lymph nodes) treated with high-dose chemotherapy and autologous hematopoietic progenitor cell support following standard-dose adjuvant chemotherapy. Clin Cancer Res 1996; 2: Press MF, Hung G, Godolphin W, Slamon DJ. Sensitivity of HER- 2/neu antibodies in archival tissue samples: potential source of error in immunohistochemical studies of oncogenic expression. Cancer Res 1994; 54: Kallioniemi OP, Kallioniemi A, Kurisu W, et al. ERBB2 amplification in breast cancer analyzed by fluorescence in situ hybridization. Proc Natl Acad Sci USA 1992; 89: Andrulis IL, Bull SB, Blackstein ME, et al. neu/erbb-2 amplification identifies a poor prognosis group of women with node-negative breast cancer. Toronto breast cancer study group. J Clin Oncol 1998; 16: Elston CW, Ellis IO. Pathological prognostic factors in breast cancer: I. The value of histologic grade in breast cancer-experience from a large study with long-term follow-up. Histopathology 1991; 19: Silverstein MJ, Poller DN, Waisman JR, et al. Prognostic classification of breast ductal carcinoma-in-situ. Lancet 1995; 345: Jacobs TW, Gown AM, Yaziji H, Barnes MJ, Schnitt SJ. Specificity of HercepTest in determining Her-2/neu status of breast cancer using the United States Food and Drug Administration-approved scoring system. J Clin Oncol 1999; 17: Roche PC, Ingle JN. Increased Her-2 with US Food and Drug Administration approved antibody. J Clin Oncol 1999; 17: Tubbs RR, Pettay JD, Roche PC, Stoler MH, Jenkins RB, Grogan

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Jkbcs016(92-97).hwp

Jkbcs016(92-97).hwp Expression of bcl-2 and Apoptosis and Its Relationship to Clinicopathological Prognostic Factors in Breast Cancer - A Study with Long Term Follow-up correlated with the survival rate.(journal of Korean