Alteration of Markers after Neoadjuvant Chemotherapy 207 색검사는유방암치료과정에서기본적으로시행되고있다. 한편수술전항암화학요법은항암제의반응을확인할수있고유방보존술의시행가능성을증가시킨다는장점을바탕으로국소진행성유방암의치료방법중하나로

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1 Journal of Breast Cancer ISSN J Breast Cancer 2007; September 10 (3): ORIGINAL ARTICLE 수술전항암화학요법을전후한유방암종양수용체 (ER, PR, HER-2) 의발현변화 이종원ㆍ한원식ㆍ고은영ㆍ조지형ㆍ정소연ㆍ김은규ㆍ김범석 1 ㆍ임석아 1 ㆍ이호창 2 ㆍ박인애 2 ㆍ오승근ㆍ윤여규ㆍ김성원황기태ㆍ노동영서울대학교의과대학외과학교실, 1 내과학교실, 2 병리학교실 Alteration of Estrogen Receptor, Progesterone Receptor, and HER-2 Expression in Breast Cancer after Neoadjuvant Chemotherapy Jong Won Lee, Wonshik Han, Eunyoung Ko, Jihyoung Cho, So-Youn Jung, Eun-Kyu Kim, Bhumsuk Keam 1, Seock-Ah Im 1, Ho-chang Lee 2, In Ae Park 2, Seung Keun Oh, Yeo-Kyu Youn, Sung-Won Kim, Ki-Tae Hwang, Dong-Young Noh Departments of Surgery, 1 Internal Medicine, and 2 Pathology, Seoul National University College of Medicine, Seoul, Korea Purpose: We aimed to assess the concordance of the immunohistochemical profiles of core biopsy before administrating neoadjuvant chemotherapy with that of the surgical specimens after a definitive operation for breast cancer. Methods: We retrospectively reviewed the estrogen receptor (ER), progesterone receptor (PR), and HER-2 expressions in 130 consecutive patients who received neoadjuvant chemotherapy and were followed by surgery during the period between February 2002 and March The pathologic complete tumor response rate for this group was 4.6% (6/ 130). Both the pre- and post-operative immunohistochemical profiles were available in 32 of the 124 patients (25.8%). Immunohistochemical staining was done on the core biopsies before chemotherapy and on the surgical specimens after operation. Results: There were 12 markers from 11 patients that were altered out of the 96 total markers (ER, PR, or HER-2) from 32 patients: 2 ER (2/12, 16.7%), 4 PR (4/12, 33.3%), and 6 HER-2 (6/12, 50.0%). One patient simultaneously had changes in the expressions of PR and HER-2. Conversion of the hormone receptor status occurred in 3 patients (3/32, 9.4%): this was positive to negative in two, and vice versa in one. In addition, there were 6 conversions (6/32, 18.8%) of the HER-2 status from negative to positive. Conclusion: The hormone receptor status changed in 9.4% of the 32 patients and the HER-2 status changed in 18.8% of the 32 patients after neoadjuvant chemotherapy. We have concluded that conducting only a single immunohistochemical study about ER, PR, and HER-2 may not be enough to exactly estimate the tumor marker status in the neoadjuvant setting. Key Words : Breast cancer, Neoadjuvant chemotherapy, Tumor marker expression, Steroid hormone receptor, HER-2 중심단어 : 유방암, 수술전항암화학요법, 종양수용체발현, 스테로이드호르몬수용체, HER-2 책임저자 : 노동영 서울시종로구연건동 28, 서울대학교의과대학외과 Tel: , Fax: dynoh@plaza.snu.ac.kr 접수일 : 2007 년 5 월 2 일게재승인일 : 2007 년 6 월 21 일 * 본논문의요지는 2006 년춘계유방암학회에서구연되었음. 서론에스트로겐수용체 (Estrogen receptor, ER) 와프로게스테론수용체 (Progesterone receptor, PR) 는내분비요법에대한반응을예상할수있는예측인자로서이들에대한면역조직화학염 206

2 Alteration of Markers after Neoadjuvant Chemotherapy 207 색검사는유방암치료과정에서기본적으로시행되고있다. 한편수술전항암화학요법은항암제의반응을확인할수있고유방보존술의시행가능성을증가시킨다는장점을바탕으로국소진행성유방암의치료방법중하나로시행되고있다. 국소진행성유방암에대하여수술전항암화학요법을시행하는경우항암제를사용한전후로유방암종양수용체 (ER, PR, HER-2 등 ) 발현에변화가생길수있음이보고되어있다.(1, 2) 하지만이러한변화의임상적유의성과원인에대해서는논란이있다.(1-3) 이에저자들은수술전항암화학요법을시행한환자들을대상으로항암제사용전의침생검조직과항암제요법을시행한후수술적으로절제한조직에서의 ER, PR, HER-2에대한면역조직화학염색결과를비교해보았다. 이를통하여수술전항암화학요법을전후한종양수용체의발현변화양상을확인하고자본연구를수행하였다. 방법 1. 연구대상 2002년 2월부터 2006년 3월까지서울대학교병원에서유방암진단하에수술전항암화학요법을시행한뒤근치적수술을시행한 130 명의환자를대상으로하였다. 세사이클의 Docetaxel과 adriamycin 병합요법후 Pathologic Complete Response (pcr) 는 6명에서나타났으며 (6/130, 4.6%) 이들을제외한 124 명중항암제사용전침생검조직과근치적수술에의한조직에서면역조직화학검사가중복시행된 32 명의환자를대상으로 ER, PR, HER-2의면역조직화학검사상발현양상을비교하였다. 연구대상자 32명의평균연령은 42.8세였으며, 수술전항암 Table 1. Clinicopathologic characteristics 화학요법전의임상적병기는 lla 에서 lllc까지분포하였으며, 수술전항암화학요법후의근치적절제표본의병리학적병기는 l에서 lllc까지분포하였다. 30 예는조직학적으로침윤성관암이었으며 1예는점액성암, 1예는침윤성미세유두암이었다 (Table 1). Docetaxel과 adriamycin 병합요법에의한반응은 Partial Response (PR) 19예 (59%), Stable Disease (SD) 11예 (35%), Progressive Disease (PD) 2예 (6%) 였다. 근치적수술로는유방전절제술 18예 (56%) 와보존술 14예 (44%) 가시행되었다 (Table 2). 2. 방법면역조직화학염색을위하여파라핀포매된조직을 4 m 두께로절단하고 saline 또는 poly-l-lysin이처리된 slide에얹어통상의방법대로탈-파라핀하고증류수로세척한후, 3% 과산화수소로 5분간처치하였다. 이후 10 mm citrate buffer (ph 6.0) 에담근채 750 W microwave에 5분, trypsin에 5분간처치하고 phosphate buffered saline (PBS) 용액으로세척한후정상쥐혈청으로 20 분간반응시켰다. 이후각각에해당하는단클론항체와반응시켜관찰하였다. 반응후 PBS 용액으로세척한후표지항체 (labeled antibody; avidin-biotynylated peroxidase complex) 로 30 분간반응시켰다. Diaminobenzidine으로발색하고 hematoxylin으로대조염색하여 10배의고배율시야에서관찰하였다. 결과는한명의병리전문의에의해판독되었으며, ER 과 PR 은면역조직화학염색후, 고배율시야에서 10% 이상유방암세포의핵이명확히염색된경우발현양성으로판정하였고 HER-2는 NCL-CB-11 (Novocastra, New Castle, UK) 를이용하여염색한후세포막에염색된정도에따라 0에서 3+ 까지나누어 2+ 와 3+ 를발현양성으로판정하였다. Characteristics No. of patients % Mean age (yr) 42.8±9.4 Stage (pre-chemotherapy, clinical) llb 4 13 llla lllb 9 28 lllc 3 9 Stage (post-operative, pathologic) l 5 16 lla 1 3 llb llla 8 25 lllb 2 6 lllc 6 19 Histology Infiltrating ductal Mucinous 1 3 invasive micropapillary 1 3 Table 2. Treatment characteristics Characteristics No. of patients % Number of cycles (Docetaxel+Adriamycin) Chemotherapy response PR SD PD 2 6 Type of operation Mastectomy BCS PR=partial response; SD=stable disease; PD=progressive disease; BCS=breast conserving surgery.

3 208 Jong Won Lee, et al. 결 대상환자 32명에대해각기 ER, PR, HER-2 3가지종류의종양수용체, 총 96 개종양수용체의발현여부를항암제사용전후로나누어비교하였다. 이중 12 개 (12/96, 12.5%) 의수용체가발현양상에변화가있었으며, ER 이 2예 (2/12, 16.7%), PR이 4예 (4/12, 33.3%), HER-2가 6예 (6/12, 50.0%) 였다. 항암제사용전 Table 3. Alteration patterns of ER and PR expression on IHC after neoadjuvant chemotherapy (n=32) Specimen type & IHC results 과 Surgical specimen + - ER Conversion rate=6.25% (2/32) PR Conversion rate=12.5% (4/32) Hormone receptor status +* Conversion rate=9.4% (3/32) ER=estrogen receptor; PR, progesterone receptor; IHC=immunohistochemistry. *Hormone receptor (+)=ER (+) and/or PR (+); Hormone receptor (-)= ER (-) and PR (-). 침생검조직과비교하여항암제사용후수술적절제조직에서의결과를보았을때, ER 의경우 1예는발현음성에서양성으로 1예는발현양성에서음성으로변하였다. PR 의경우 1예는발현음성에서양성으로 3예는발현양성에서음성으로변하였다. HER-2 의경우 6예모두발현음성에서양성으로변하였다. 1명의환자에서 PR 의발현음성변화와 HER-2의발현양성변화가동시에발생하였기때문에수용체자체의변화는 12예 (12/96, 12.5%) 였으나수용체변화가있는환자는 11예 (11/32, 34.4%) 였다 (Table 3, 4). 보조적호르몬요법의임상적관점에서볼때, ER 과 PR 각각의수용체변화로인해스테로이드호르몬수용체의전체적인발현상태가변화한환자는 3예 ( 환자 1, 4, 5) 로 9.4% (3/32) 였다. 환자 1의경우스테로이드호르몬수용체양성 (ER 과 PR 중최소하나이상이발현양성인경우 ) 으로변화하였으며, 환자 4와 5는스테로이드호르몬수용체음성 (ER 과 PR 모두음성 ) 으로변화하였다 (Table 3, 5). Table 4. Alteration patterns of HER-2 on IHC after neoadjuvant chemotherapy Specimen type & IHC results Conversion* rate=18.8% (6/32) IHC=immunohistochemistry. *Conversion=(- or 1+) to (2+ or 3+) or vice versa. Surgical specimen Table 5. Clinicopathologic characteristics of 11 female patients who experienced receptor expression changes after neoadjuvant chemotherapy Stage Pt Age Dx NG/HG Clinical Pathlogic CTx response Op Marker status (ER/PR/HER-2) Initial After 1 31 T2N1 T1N0 IDC 3/lll PR BCS -/-/2+ +/-/ T4N1 T4N1 IDC 2/lll SD MRM +/+/- -/+/- 3* 45 T3N2 T3N2 IDC 3/lll SD MRM +/+/+ +/-/ T4N1 T2N1 IDC 3/NA PR BCS -/+/- -/-/ T3N2 T3N0 IDC 3/lll SD MRM -/+/- -/-/ T2N2 T2N3 MC 2/NA PR BCS +/-/- +/+/ T2N2 T2N2 IDC 3/lll SD MRM +/+/+ +/+/ T4N1 T2N2 IDC 2/ll PR BCS +/+/- +/+/ T3N1 T3N1 IDC 3/lll SD MRM -/-/+ -/-/ T4N1 T4N2 IDC 2/ll PD MRM +/-/- +/-/ T2N2 T1N3 IMC NA PR BCS -/-/- -/-/3+ Pt=patient s number; Dx=histologic diagnosis; NG=nuclear grade; HG=histologic grade; CTx=chemotherapy; Op=operation type; ER=estrogen receptor; PR, progesterone receptor; IDC=infiltrating ductal carcinoma; MC=medullary carcinoma; IMC=invasive micropapillary carcinoma; PR= partial response; SD=stable disease; PD=progressive disease; BCS=breast conserving surgery; MRM=modified radical mastectomy; NA=not available. *One patient (pt 3) simultaneously experienced PR down-expression and HER-2 up-expression.

4 Alteration of Markers after Neoadjuvant Chemotherapy 209 고찰이연구는수술전항암화학요법에의한 ER, PR, HER-2의면역조직화학검사상의결과변화와그변화의임상적의의와보조적치료에미치는영향을확인하고자하였다. 본연구에서는총 32 예의환자중 3예의환자에서수술전항암화학요법을전후하여면역조직화학검사에서스테로이드호르몬수용체상태가변화하였다. 구체적으로환자 1의경우에 ER/PR (-/-) 에서 ER/PR (+/ -) 로스테로이드호르몬수용체양성으로변화하여보조호르몬요법의대상자가되었다. 환자 4와 5의경우에는 ER/PR (-/+) 에서 ER/PR (-/-) 로스테로이드호르몬수용체음성으로변화하여보조호르몬요법의대상에서빠지는결과가되었다 (Table 3, 5). Piper 등 (2) 은수술전항암화학요법을받은 35 명의대상환자들을 34 명의항암화학요법을받지않은대조군과비교하여 ER, PR, HER-2의발현변화율이수술전항암화학요법을받은군에서유의하게높다고보고하였다. Taucher 등 (3) 은수술전항암화학요법을전후한 ER 과 PR 의발현변화양상이특히발현양성에서발현음성으로유의하게변함을보고하였다. 하지만이러한종양수용체음성발현은조직고정과정의불완전성에의해서도발생할수있는데 Mann 등 (4) 은침생검조직이수술적절제조직보다유의하게진하게염색되었다고보고하였다. 또한수술적절제조직보다침조직생검에의한조직이고정이더완전하게되기때문에면역조직화학검사에서보다신뢰도가높은것으로간주하였다. 이들의연구는항암제에의한종양세포의단백질발현양상의변화보다는조직고정과정의차이에의한염색상의문제에초점을맞춘것이었다. 이에반하여유방암종양수용체의면역조직화학염색상의발현변화가항암요법과는무관하다는주장도있는데, Arens 등 (5) 은수술전항암화학요법을받은군과받지않은군에서 ER, PR, Ki67, p53, HER-2의발현변화율에서통계적인유의성을찾을수없었다. 또한 Fluorescent in situ hybridization (FISH) 에의한 HER-2 gene copy number에서도두군간에유의한차이를발견할수없었다. Lee 등 (1) 도 56 명의수술전항암화학요법을받은환자군을동수의항암화학요법을받지않은대조군과비교하였는데양군모두동일하게 3명 (5%) 에서스테로이드호르몬수용체상태의변화가있었으며, 그원인으로는조직채취과정에서의차이에기인한것으로결론지었다. 그러나이러한변화는향후보조적치료에영향을끼칠수있기때문에수술적으로절제한조직에서수용체발현에대한재검을권장하였다. 한편종양수용체중 HER-2가 ER 이나 PR 보다항암제에의한영향에안정적이라는주장이있다. Varga 등 (6) 은특히 ER과 PR 의면역조직화학검사에서의발현변화율은 30% 인데비해 HER- 2 gene의증폭변화율은 FISH 검사에서 13% 로상대적으로적었다. 앞서기술한 Taucher 등 (3) 의연구에서 ER 과 PR 의발현이수술전항암화학요법을전후하여발현양성에서발현음성으로유의하게변화하였으나비슷한시기의그의연구에서 Epirubicin 과 Docetaxel 병용요법에의한수술전항암화학요법이 HER-2 발현변화에미치는영향은유의하지않았다. 본연구와달리이들은 FISH를이용하여 HER-2의발현변화를비교하였다. 이미면역조직화학염색은경제성면에서는장점이있으나판독시발생할수있는병리전문의에의한주관성의문제로인해결과의일관성이결여되며특히약양성의경우에는 FISH와비교할때면역조직화학염색법이 HER-2의발현을정확히예측하지못한다.(8-10) 본연구에서는 HER-2의발현변화가전체 12예중 6예로가장많았다. 비록 1명의병리전문의가판독을하여주관성의문제를극복하려하였으나, HER-2 발현의변화가있었던 6예중 5예에서그변화의양상이 0 혹은 1+ 에서 2+ 로변한경우임을고려해야하겠다 (Table 4, 5). 본연구는대조군과의비교가없어 Docetaxel과 Adriamycin 병용요법에의한수술전항암화학요법이 ER, PR, HER-2의면역조직화학적발현에의미있는영향을미치는지는알수없다. 그러나본연구결과를통해서수술전항암화학요법을전후하여스테로이드호르몬수용체에대한면역조직화학검사결과가 9.3% (3/32) 에서달라질수있음을알수있었다. 만일이환자들이수술전항암화학요법을전후한면역조직화학검사상의스테로이드호르몬수용체결과중공교롭게도음성의결과를근거로보조적호르몬요법여부를결정하게된다면이를통해얻을수있는치료효과를잃는결과가될것이다. 따라서수술전항암화학요법의사용이늘어나는현실에서수술전항암화학요법이 ER 과 PR 의발현에미치는영향은아직명확히알수없으나임상의는면역조직화학검사를수술전항암화학요법전후로중복시행하여둘중어느한경우에서라도호르몬수용체양성인경우에는보조적호르몬요법을시행하고, anti-her-2 therapy가필요한경우 - 특히면역조직화학염색이약양성 (2+) 일때에는 FISH 검사법으로확진하는차선책을고려해야할것이다. 향후면역조직화학염색법의단점을극복하고,(11) 항암제가유방암종양수용체발현에미치는영향에대한기전을파악하기위해서는보다세밀한연구가필요할것이다. 결론유방암의예후및예측인자로서중요한 ER, PR, HER-2의면역조직화학검사에의한발현양상은수술전항암화학요법을전후하여변할수있다. 따라서항암제사용을전후하여면역조직화

5 210 Jong Won Lee, et al. 학검사의재검이필요하며이결과를유방암환자의보조적치료방침을결정하는데고려할필요가있다. 참고문헌 1. Lee SH, Chung MA, Quddus MR, Steinhoff MM, Cady B. The effect of neo- adjuvant chemotherapy on estrogen and progesterone receptor expression and hormone receptor status in breast cancer. Am J Surg 2003;186: Piper GL, Patel NA, Patel JA, Malay MB, Julian TB. Neoadjuvant chemotherapy for locally advanced breast cancer results in alterations in preoperative tu- mor marker status. Am Surg 2004;70: Taucher S, Rudas M, Gnant M, Thomanek K, Dubsky P, Roka S, et al. Sequential steroid hormone receptor measurements in primary breast cancer with and without intervening primary chemotherapy. Endocr Relat Cancer 2003;10: Mann GB, Fahey VD, Feleppa F, Buchanan MR. Reliance on hormone receptor assays of surgical specimens may compromise outcome in patients with breast cancer. J Clin Oncol 2005;23: Arens N, Bleyl U, Hildenbrand R. HER2/neu, p53, Ki67, and hormone receptors do not change during neoadjuvant chemotherapy in breast cancer. Virchows Arch 2005;446: Varga Z, Caduff R, Pestalozzi B. Stability of the HER2 gene after primary chemotherapy in advanced breast cancer. Virchows Arch 2005;446: Taucher S, Rudas M, Mader RM, Gnant M, Sporn E, Dubsky P, et al. Influence of neoadjuvant therapy with epirubicin and docetaxel on the expression of HER2/neu in patients with breast cancer. Breast Cancer Res Treat 2003;82: Lebeau A, Deimling D, Kaltz C, Sendelhofert A, Iff A, Luthardt B, et al. HER-2 analysis in archival tissue samples of human breast cancer: comparison of immunohistochemistry and fluorescence in situ hybridization. J Clin Oncol 2001;19: Perez EA, Roche PC, Jenkins RB, Reynolds CA, Halling KC, Ingle JN, et al. HER2 testing in patients with breast cancer: poor correlation between weak positivity by immunohistochemistry and gene amplification by fluorescence in situ hybridization. Mayo Clin Proc 2002;77: Hammock L, Lewis M, Phillips C, Cohen C. Strong HER-2 protein overexpression by immunohistochemistry often does not predict oncogene amplification by fluorescence in situ hybridization. Hum Pathol 2003;34: Tawfik OW, Kimler BF, Davis M, Donahue JK, Persons DL, Fan F, et al. Comparison of immunohistochemistry by automated cellular imaging system (ACIS) versus fluorescence in-situ hybridization in the evaluation of HER-2 expression in primary breast carcinoma. Histopathology 2006;48:

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