Journal of Breast Cancer ISSN 1738-6756 J Breast Cancer 2007; September 10 (3): 193-8 ORIGINAL ARTICLE 유방암환자에서 3 차원조직배양항암제감수성검사의유용성 정용식ㆍ조영업 1 ㆍ서영진 2 ㆍ김정수 2 ㆍ오세정 2 ㆍ임철완 3 ㆍ김문보 4 ㆍ박흥규 5 아주대학교의과대학외과학교실, 1 인하대학교의과대학외과학교실, 2 가톨릭대학교의과대학외과학교실, 3 순천향대학교의과대학외과학교실, 4 ( 주 ) 메타바이오, 5 가천의과학대학교의과대학외과학교실 Can the Histoculture Drug Response Assay Predict the Clinical Results of Chemotherapy in Breast Cancer? Yong Sik Jung, Young Up Cho 1, Young Jin Suh 2, Jeong Soo Kim 2, Se-Jeong Oh 2, Cheol Wan Lim 3, Moon Bo Kim 4, Heung Kyu Park 5 Department of Surgery, Ajou University, School of Medicine, Suwon; 1 Department of Surgery, Inha University, College of Medicine, Incheon; 2 Department of Surgery, the Catholic University of Korea, College of Medicine, Seoul; 3 Department of Surgery, Soonchunhyang University, College of Medicine, Seoul; 4 MetaBio Incorporated, Seoul; 5 Department of Surgery, Gachon University of Medical and Science, Incheon, Korea Purpose: The behavior of invasive carcinomas in human can be very varied with different individual responses to chemotherapy. Individualization is crucial to the optimization of chemotherapy. Therefore, the prediction of a tumor s sensitivity to anticancer agents has been the subject of intensive investigation. In order to investigate the pathobiology of breast cancer, it is necessary to maintain or recreate the characteristics of the three-dimensional architecture of the tissues in culture. In this study, we have evaluated the relationship between the Histoculture Drug Response Assay (HDRA) assessment and chemotherapy responses in breast cancer patients. Methods: Tumor specimens from 30 patients with breast cancer were evaluated using the HDRA. Tumor tissues were cultured on gelfoam sponge gel in 24-well plates, followed by treatment with a variety of chemotherapeutic agents. All treatments were conducted in triplicate. The sensitivity of a chemotherapy regimen was defined as a tumor inhibition rate (IR) in excess of 30%. Neoadjuvant or palliative chemotherapy for patients, using anthracycline or taxane, was conducted on the basis of the established protocols. The responses to treatments were compared with the results of the HDRA. Results: The mean IR for the combinations of doxorubicin and docetaxel and for FAC and AC were 48, 45, and 36%, respectively. The above partial rate of response to chemotherapy was 81.1%. The sensitivity and specificity of the HD- RA assessment, with a 30% inhibition rate, were 81.5 and 66.7%, respectively. The positive and negative response prediction values were 91.7 and 44.4%, respectively. The responses to treatments and the results of the HDRA assessment were not correlated with the expressions of the hormonal receptor or c-erbb2. Conclusion: In cases in which the inhibition rate is in excess of 30%, the HDRA assessment yielded a high positive response prediction value. The sensitivity to chemotherapy, as determined by the HDRA, appears to be a good guide for selection in breast cancer patients. Thus the results presented herein should be integrated into future research on the subject. Key Words : Breast cancer, Chemotherapy, Histoculture Drug Response Assay (HDRA) 중심단어 : 유방암, 항암화학요법, 3차원조직배양항암제감수성검사 책임저자 : 박흥규 405-760 인천광역시남동구구월동 1198, 가천의과학대학외과, 길병원유방센터 Tel: 032-460-3268, Fax: 032-460-3247 E-mail : hgjh@gilhospital.com 접수일 : 2007 년 5 월 21 일게재승인일 : 2007 년 7 월 24 일 * 본논문의요지는 2006 년추계외과학회구연발표되었음. 서론현재한국여성의유방암발생은해마다빠르게증가하고있으며이중많은여성들이수술후보조적인항암화학요법을시행 193
194 Yong Sik Jung, et al. 받고있다. 계속해서새로운항암제가개발되고있으며수술과약물요법, 그리고방사선요법을병용하여치료의효과를극대화시키고있으나재발과전이는계속해서발생하고있다. 또한같은약제에대해서도개개인에따라다양한치료반응과결과를보이므로가장효과적인항암제를선택하기위한많은연구가진행되어왔다.(1, 2) 항암제감수성검사는세포배양을이용한 2차원모델과조직배양을이용한 3차원모델로크게나눌수있으며이중 2차원모델은비교적쉽게할수있다는장점이있어널리이용되고있으나 stroma 조직이결핍되어있어인체내의조직에서와같은미세환경과는차이가있을것으로생각된다.(3) 암의발생과진행형태는매우다양한모습을보인다. 이것은조직의미세환경내에서세포와세포간의지속적인상호작용에의한것으로생각되며많은호르몬과성장인자, adhesion molecule, cytokine 등에의한신호전달체계가관여하고있다. 따라서인체내의미세한경과와유사한조건을만들어약제의감수성을검사하는것이좀더정확한결과를얻을수있을것이고이러한목표하에많은조직배양방법이등장하였다. Histoculture Drug Response Assay (HDRA) 는미국 Anticancer사의기술을기반으로개발되었으며, collagen sponge 위에암조직을 3차원적으로배양한뒤항암제의감수성을평가하는방법으로최근많은연구가진행되고있으며, 2차원적인세포배양방법에비해정확한결과를얻을수있을것으로기대되는방법이다.(4) 본연구에서는수술전항암요법을받을유방암환자혹은전이성유방암환자를대상으로 HDRA 검사를시행하여단독혹은병용투여된항암제의감수성을평가하고실제환자의항암화학요법후의결과와비교하여 3차원조직배양항암제감수성검사의유효성을평가하고자하였다. 방법 1. 대상 2005년 5월부터 2006년 4월까지 7개병원을방문한국소진행성유방암환자및전이성유방암환자 30 명을대상으로전향적연구를하였으며정확하고신속한결과를얻기위하여대상환자군을전이성유방암혹은수술전신보강항암요법을시행할예정인환자로국한하였다. 2. 조직배양및항암제감수성검사치료시작전총조직검사를시행하여채취한조직을 transport media (Hank s Balanced Salt Solution [include 10% FBS, 200 mg/l Gentamycine Sulfate, 1X Non-Essential Amino Acids]) 에넣어 4 를유지하여운반한후, cryostat을이용하여유방암조직을확인하였으며, 운반된조직을약 1 mm 크기로 chopping 한후조직을 culture media (Minimum Essentials Media [include Sodium Bicarbonate, 100 ml/ L FBS, 2 ml/l Gentamycin Sulfate, 1X Non-Essential Amino Acids]) 에옮긴후하룻밤동안배양하였다. 조직을 4-6 mg 씩동일하게측정하여배양액이담긴 collagen sponge 위에올려놓고각 well에 control로사용되는 PBS와 1% dimethylsulfoxide (DMSO, Sigma, USA) 및시험항암제를처리하였으며, 감수성검사에사용된항암제와농도는 Table 1과같다. 약제처리 3일후배양액을제거하고각각의 well 에 0.1 mg/ dl collagenase (type I) 를포함하는 HBSS와 5 mg/dl의 PBS 에녹여진 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, USA) 를각각 100 L씩넣어주고, 37 에서 4시간동안배양한다. 이후에배양액을제거하고 0.5 ml의 DMSO (Sigma, USA) 를넣고 shaker 위에올려놓고 1시간이상두어 formazan crystal을추출하였다. 540 nm에서분광흡광계 (SPECTRA max 340PC, Molecular Devices) 로흡광도를측정하였으며, 측정된흡광도를이용하여 inhibition rate (IR) 를계산하였으며공식은아래와같다. Inhibition rate (%)=(1-T/C ) 100 T: Drug을처리한 well의조직 g 당흡광도 C: Control well의조직 g 당흡광도 Inhibition rate가 30% 이상이면각항암제의 cutoff 농도에대한종양의감수성이양성인것으로판정하였다. 실험의정도관리를위하여 Glucose consumption test (Quantichron TM Glucose Assay kit [DIGL-200]) 를이용한 Table 1. Concentrations and referencces of anti-cancer drugs in Histoculture Drug Response Assay Anti-cancer drugs Concentrations ( L/mL) References Adriamycin 6 J. Surgical Oncol. 1991;47:253-60 Cyclophosphamide 20 J. Pharmacol. Exp. Ther. 1999;288(3):928-37 5-Fluorouracil 40 Anticancer Res. 1992;12:1055-62 Docetaxel 50 Oncology 2002;63:205-212 FAC 40:6:20 J. Natl Cancer Inst. 1998;90:1346-60 AC 6:20 Curr Oncol Rep. 2003;5(1):66-71 AD 6:50 Cancer Letters. 2004;215:53-9 FAC=combination of 5FU, adriamycin, and cyclophosphamide; AC= combination of adriamycin and cyclophosphamide; AD=combination of adriamycin and docetaxel.
The Value of Histoculture Drug Response Assay in Breast Cancer Treatment 195 배지내포도당소모량을측정하였으며, TUNEL assay로약제에의한 apoptosis 확인도함께실시하였다 (Fig 1). 대한평가는 Response Evaluation Criteria in Solid Tumor (RECIST) 에따라시행하였다 (Table 3). 3. 항암화학요법및치료반응판정 항암제감수성검사의결과와상관없이현재가장많이쓰이는 anthracycline 중심혹은 anthracycline과 taxane의병용요법을선택하여항암화학요법을진행하였으며항암제의용량은일반적으로투여되는제품표시기재사항의 용법및용량 에설명된범위내에서투여하였다. 27 명의환자에서는처음선택한요법으로치료후평가하였으며, 3명은 1차선택치료후 stable disease 혹은 progressive disease를나타내어약제를바꾸어다시치료하여총 33 예의치료에대한결과를얻었다. 26예는 anthracycline과 taxane의병용요법이었으며, 7 예는 anthracycline 중심의화학요법으로치료하였고, 연구에사용된항암제와용량, 주기는 Table 2와같다. 항암화학요법시행전초음파혹은컴퓨터단층촬영을시행하여종양의장축을측정하여치료후같은방법의검사를통해치료전과후의종양의크기변화를측정하였으며, 치료반응에 Table 2. Regimens and mean dosage of chemotherapy were treated to patients Regimens (mean dosage) Number Anthracycline 7 AC (50, 500/m 2 ) 3 FAC (500, 50, 500/m 2 ) 4 Anthracycline+Taxane 26 Adriamycin+Docetaxel (60, 80/m 2 ) 26 4. 통계실제항암제치료의결과를반응그룹 (complete response, partial response) 과불응그룹 (stable disease, progressive disease) 으로분류하고사용된약제의 HDRA 검사의결과를감수성양성그룹 (chemosensitive) 과음성그룹 (chemoresistant) 으로분류하여 Pearson chi square test를이용하여교차분석을시행하였으며 HDRA의결과와치료반응여부를평가하고, ER, PR, c-erbb2 발현과의관계도교차분석을시행하였다. 통계프로그램은 SPSS 11.0을이용하였으며 p 값이 0.05 이하일때통계적으로유의성이있는것으로판단하였다. 결과 1. 환자의임상적특성환자들의평균연령은 48.6세였으며대부분 ECOG 1 이상이었다. 30 명중 7명은진단당시타장기에전이가있는 4기유방암이었으며나머지 23 명의평균종양크기는 5.8 cm이었다. 대부분침윤성관상피암이었고 2예의침윤성소엽세포암과 1예의 apocrine carcinoma가포함되어있었다. 항암화학요법의평균투여횟수는 3.2회 (cycle) 였다 (Table 4). 항암화학요법에대한 Table 3. Response evaluation criteria in solid tumors (RECIST) AC=combination of adriamycin, cyclophosphamide; FAC=combination of 5FU, adriamycin, cyclophosphamide. Complete response (CR) Partial response (PR) Stable disease (SD) Progressive disease (PD) Disappearance of disease 30% or more decrease Neither PR nor PD criteria met 25% or more increase No CR, PR, SD documented before increased disease Specimen in transport media Tumor is minced Glucose Consumption 측정 MTT stain, Quantified in a plate reader Inhibition rate 측정 Fig 1. Schematic process of 3 dimensional histoculture drug response assay. Table 4. Clinico-pathologic characteristics of patients Age at diagnosis (yr) 48.6 (range: 29-77) Physical status ECOG 0 : 24 ECOG 1 : 5 ECOG 2 : 1 Tumor diameter 5.8 cm (1.7-11 cm) Histologic type Infiltrating ducta carcinoma: 27 Infiltrating lobular carcinoma: 2 Apocrine carcinoma: 1 Estrogen receptor Positive: 10 Negative: 19 Progesterone receptor Positive: 7 Negative: 22 c-erbb2 Positive: 14 Negative: 15
196 Yong Sik Jung, et al. % 50 45 40 35 30 25 20 15 10 50 37 36 Average inhibition rete Fig 2. Average inhibiton rate in 3-dimensional histoculture drug response assay. ADR=adriamycin; CTX=cyclophosphamide; FAC=combination of 5FU, adriamycin, cyclophosphamide; AC=combination of adriamycin, cyclophosphamide; AD=combination of adriamycin, docetaxel. 반응은 partial response 이상이 27예로 81.8%, stable disease 를보인경우가 6예로 18.2% 였다. 2. Histoculture Drug Response Assay 단독항암제와병용요법을포함하여 8개의약제에대해항암제감수성검사를시행한결과전체적인 inhibition rate는 36% 였으며 5-FU, adriamycin, cyclophosphamide의병용요법과 adriamycin, docetaxel의병용요법이평균 inhibition rate 가 43% 로가장높게나와서실제로현재가장많이쓰이는요법과일치하는결과를나타내었다 (Fig 2). 3. HDRA 결과와치료반응과의상관관계 28 ADR 5FU CTX Taxol Docetaxel FAC AC AD 항암화학요법후실제로 partial response 이상의반응을보인 27예의 HDRA 결과중 IR 이 30% 미만이었던 resistant group 은 5예였으며 IR 30% 이상의 sensitive group은 22 예였으며, 항암화학요법에반응이없었던 6예중 4예는 HDRA의결과가 IR 30% 미만, 2예는, IR 30% 이상이었다 (Table 5). HDRA 검사의예민도 (sensitivity) 는 81.5%, 특이도 (specificity) 는 66.7 %, 양성반응예측률 (positive response prediction value) 은 91.7%, 음성반응예측률 (negative response prediction value) 은 44.4% 였다. 전체적인검사의정확도는 78.2% 였으며전이성암의경우 89.9% 의정확도를나타내었고, 수술전항암화학요법의경우 75% 의정확도를나타내었다. 항암화학요법에반응이있었던경우의평균 IR은 44.5% 였으며반응이없었던경우의평균 IR은 19.5% 였다. HDRA 검사결과와항암화학요법에대한반응모두호르몬수용체의발현이나 c-erbb2의과다발현과는관계가없는것으로나타났다. 고 37 국소진행성혹은전이성유방암의치료에있어서항암화학요 찰 28 43 36 43 Table 5. Correlation between result of HDRA and response to adriamycin with or without docetaxel combination Response SD, PD PR, CR Total p- value Resistant group 4 5 9 (30% >IR) Sensitive group 2 22 24 <0.05 (30% IR) Total 6 27 HDRA=histoculture drug response assay; SD=stable disease; PD= progressive disease; PR=partial response; CR=complete response; IR=inhibition rate. 법은매우큰비중을차지하고있으며다양한항암약제들이임상에서사용되고있다. 그러나같은형태학적특징을나타내는종양이라할지라도항암화학요법에대한반응은환자개개인에따라다양하게나타날수있으며이러한다양성은종양세포의성장에따라획득되는유전체상의특성에기인하는것으로생각이되며 DNA의메틸화, 전사후변형등의기전으로인해더욱복잡한양상을띄게된다. 따라서이렇게다양한비균질성을극복하고항암화학요법의반응을높이는것이종양전문의들의주된관심으로대두되고있다. 최근에각광을받는방법은종양의특수한표지자를목적으로하는 targeted therapy와개별종양에대한약제감수성을평가하는 individualized therapy가있으며후자인항암제감수성검사의경우세포의배양방법이나세포측정방법에따라여러가지다양한방법들이개발되었고현재많은방법들이연구목적혹은임상치료에서사용이되고있다. 그러나이러한항암제감수성검사의경우그동안많은제한점들로인해널리이용되지못한것이사실이며, 특히 2차원적인배양의경우자발적으로성장할수있는세포들을대상으로하므로세포배양의환경이생체내에서일어나는다양한요소들, 즉미세혈관의생성, 저산소증에따른여러인자들, 주위기질세포들이나종양세포들간의세포 - 세포간의상호작용이나호르몬혹은성장단백질등의미세한작용들을정확히반영할수없다는점이가장큰약점으로지적되어왔다. 또한과거의 2차원적인배양방법의경우실험의성공률과재현성이낮다는점과세포성장시발생할수있는가소성등으로인하여측정오류가생기는문제점도임상적적용을유보하게만든원인중하나였다.(1) HDRA는미국 Anticancer사의기술을기반으로개발되었으며, collagen sponge위에암조직을 3차원적으로배양한뒤항암제의감수성을평가하는방법으로 collagen sponge gel을이용해생체내의간질조직과유사한구조를형성하고원래의종양과주위조직을유지시켜 3차원적으로배양하는방법으로생체
The Value of Histoculture Drug Response Assay in Breast Cancer Treatment 197 내미세환경과유사한조건을제공하고종양세포의성장또한실제종양과유사한구조로성장하게함으로써 2차원적인배양방법에비해이론적인장점이있는것으로평가되고있다.(5-8) 실제로기존의연구에서 HDRA가실험의성공률과재현성이높게나타나며몇몇종양에대한연구에서도실제생존율이 HDRA에의한항암제감수성이있는그룹에서높은것으로보고되어임상적유용성에대한기대를높이고있다.(4, 9, 10) HDRA의임상적인유용성에대해서는유방암을포함한다양한종양에대해연구가진행되고있으며두경부종양, 폐의비소세포암, 구강의편평상피암, 방광암및난소암등의연구에서실제치료반응과의높은일치도혹은생존율의향상을보고하고있으나연구의대부분이소규모로진행되어추가적인연구또한필요한상황이다.(11-15) 본연구의결과 HDRA 검사의전체양성반응예측률은 91.7% 였으며 adriamycin과 docetaxel의병용요법을사용한 27예의양성반응예측률은 94.4% 로상당히높게나타났으며이는기존의연구에서보고되는경험적신보강항암화학요법의반응률인 80-85% 를훨씬웃도는결과로환자의개개인에따른항암제선택에도움이될것으로기대가되나음성반응예측률은전체 44.4 %, adrimycin과 docetaxel의병용요법을사용한경우 50% 로낮은예측률을보여 HDRA 검사에서낮은 inhibition rate를보였을때항암제의선택에는신중한결정이필요할것으로사료된다. 이러한낮은음성반응예측률의원인에대해서는연구가더필요할것으로생각되며, HDRA 검사와치료반응이일치하지않은환자의대부분의 inhibition rate가 20% 에서 30% 사이에분포하고있었으며 inhibition rate의 cut-off 값을 20% 로낮추었을때의양성반응예측률은 95.2%, 음성반응예측률은 80% 로상승하는것으로계산되어추후유방암에대한 inhibition rate에대한검증또한필요할것으로생각된다. 호르몬수용체나 c-erb B2의발현등은 HDRA 검사결과나치료반응과관련이없는것으로관찰되었으나연구에참여한환자의수가적어현재로써는정확한상관관계를언급하기힘들며연구에참여한환자의수가제한적이고항암제용량의표준화가정확히이루어지지않은것등은아쉬운점이나유방암에서 HD- RA 의유용성에대한국내의임상적연구가거의보고된바없는상태이므로앞으로의지속적인연구가필요할것으로생각된다. 결론유방암환자에서 HDRA를이용한항암제감수성검사결과실제항암화학요법에대해높은양성반응예측률을보여향후국소진행성유방암이나전이성유방암치료영역에서치료약제의 선택에도움을줄수있을것으로기대가되며이러한선택적치료가생존율의향상이나부작용의감소에효과가있는지에대해서도추가적인연구가필요할것이다. 참고문헌 1. Kern DH. Wiesenthal LM. Highly specific prediction of antineoplastic drug resistance with an in vitro assay using suprapharmacologic drug exposure. J Natl Cancer Inst 1990;82:582-8. 2. Hoffman RM. In vitro assays for chemotherapy sensitivity. Crit Rev Oncol Hematol 1993;15:99-111. 3. Kim JB, Stein R, O Hare MJ. Three-dimensional in vitro tissue culture models of breast cancer-a review. Breast Cancer Res Treat 2004; 85:281-91. 4. Furukawa T, Kubota T, Hoffman RM. Clinical applications of the histoculture drug response assay. Clin Cancer Res 1995;1:305-11. 5. Furukawa T, Kubota T, Watanabe M, Takahara T, Yamaguchi H, Takeuchi T, et al. High in vitro-in vivo correlation of drug response using sponge-gel-supported three-dimensional histoculture and the MTT end point. Int J Cancer 1992;51:489-98. 6. Hoffman RM. To do tissue culture in two or three dimensions? that is the question. Stem Cells 1993;11:105-11. 7. Sourla A, Doillon C, Koutsilieris M. Three-dimensional type I collagen gel system containing MG-63 osteoblasts-like cells as a model for studying local bone reaction caused by metastatic cancer cells. Anticancer Res 1996;16:2773-80. 8. Jacquot J, Spilmont C, Burlet H, Fuchey C, Buisson AC, Tournier JM, et al. Glandular-like morphogenesis and secretory activity of human tracheal gland cells in a three-dimensional collagen gel matrix. J Cell Physiol 1994;161:407-18. 9. Kang HJ, Ko CD, Yoon HS, Kim MB, Ahn SH. The reliability of histoculture drug response assay (HDRA) in chemosensitivity tests for breast cancer. Cancer Res Treat 2001;33:392-8. 10. Vescio RA, Connors KM, Kubota T, Hoffman RM. Correlation of histology and drug response of human tumors grown in native-state three-dimensional histoculture and in nude mice. Proc Natl Acad Sci USA 1991;88:5163-6. 11. Singh B, Li R, Xu L, Poluri A, Pastel S, Shaha AR, et al. Prediction of survival in patients with head and neck cancer using the histoculture drug response assay. Head Neck 2002;24:437-42. 12. Yoshimasu T, Oura S, Maebeya S, Tanino H, Bessho T, Arimoto J, et al. Histoculture drug response assay on non-small cell lung cancer.
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