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1 Journal of Breast Cancer ISSN J Breast Cancer 2008; March 11 (1): 25-9 ORIGINAL ARTICLE 한국인젊은여성유방암환자에서 CHEK2 1100delC 돌연변이의발생빈도 최두호 조대연 1 이민혁 2 박희숙 3 Bruce G. Haffty 4 순천향대학교의과대학방사선종양학교실, 1 ㄜ랩지노믹스, 2 순천향대학교의과대학외과학교실. 3 내과학교실, 4 예일대학교의과대학치료방사선과 Frequency of the CHEK2 1100delC Mutation in Korean Women with Early Onset Breast Cancer Doo Ho Choi, Dae Yeon Cho 1, Min Hyuk Lee 2, Hee Sook Park 3, Bruce G. Haffty 4 Departments of Radiation Oncology, 2 Surgery, 3 Internal Medicine, Soonchunhyang University College of Medicine, Seoul; 1 LabGenomics Clinical Research Institute, LabGenomics, Seoul, Korea; 4 Department of Therapeutic Radiology, Yale University School of Medicine, New Haven CT, USA Purpose: Sequence variants in the cell cycle checkpoint kinase 2 (CHEK2 1100delC) are associated with an increased risk for breast cancer in women carrying this mutation. It is a lowpenetrance breast cancer susceptibility allele, frequently observed in patients with a family history of breast cancer and/or young age, with the frequency varying according to race or ethnicity. In this study, we evaluated the significance of CHEK2 1100delC in predisposition to breast cancer by assessing its frequency in material from 101 Korean women patients with early-onset breast cancer. Methods: One hundred and one Korean patients with earlyonset breast cancer (40 yr old or younger) were selected for this study. All the patients had been screened for BRCA1 and BRCA2 mutations and 14 patients had deleterious mutations. Of the 101 patients entered for this study, 14 had family history of breast cancer and 7 had bilateral breast cancers. Mutation detection of CHEK2 1100delC was based on analysis of primer extension products generated from previously-amplified genomic DNA using a chip based MALDI-TOP mass spectrometry platform (Sequenom, Inc., San Diego, CA, USA). Results: None of the 101 Korean patients with a family history of breast cancer and early-onset breast cancer who were candidates for the BRCA1 and BRCA2 test carried the 1100 delc mutation, which is observed in Caucasians with limited frequency. Conclusion: We previously observed higher or comparable prevalence of BRCA1 and BRCA2 mutations in Korean patients with breast cancer compared to Caucasians. However, the CHEK2 1100delC mutation is absent or infrequent in Korean patients with breast cancer who have a high risk of BRCA1 and BRCA2 mutations, making its screening irrelevant. Key Words : Breast cancer, CHEK2, Mutation 중심단어 : 유방암, CHEK2, 돌연변이 책임저자 : 최두호 서울시용산구한남동 657, 순천향대학교병원방사선종양학과 Tel: , Fax: dohochoi@hanmail.net 접수일 : 2007년 12 월 10일게재승인일 : 2008년 1월 21 일 * 본연구는한국유방건강재단 2005년학술연구비로수행되었음. * 본논문의요지는 Global Breast Cancer Conference 2007에포스터발표되었음. 서론유방암은서구여성에서가장빈발하는암이며우리나라에서도그빈도가급격히증가하는추세로 2001년이래로여성암중에서발생률 1위가되었다.(1) 유방암은가족력이있는경우가많으며현재까지연구된결과에의하면 BRCA1과 BRCA2 돌연변이가유전성유방암의많은부분을차지하고있다. 이러한유전자돌연 25

2 26 Doo Ho Choi, et al. 변이가있으면전생애에걸쳐유방암이생길가능성은 80% 에이르며난소암은 BRCA1의경우 40%, BRCA2의경우 20% 가발생하며현재약 10% 정도의유방암은이두유전자의돌연변이에의해발생되는것으로추정되고있다.(2, 3) BRCA1과 BRCA2 돌연변이뿐만아니라다른유전성돌연변이도유방암의발생위험성을높이는데영향을미치며 Li-Fraumeni 증후군을가진 TP53 돌연변이가족이나 Cowen 증후군을가진 PTEN 돌연변이, 그리고세포주기조절자인 CHEK2의돌연변이등도빈도는흔하지않으나젊은여성에게서종종발생한다.(4, 5) 유전성유방암은젊은나이때부터정기검진을통해조기발견하고, 매우민감한유전자검사를통해유전성을밝힐수있으며예방적수술을통해유방암의발생빈도를줄이고생존율향상을기대할수있다.(6) 그러나유전성유방암의경우조기발견을위한정기검진도자주시행하여야하고 MRI 등고가의장비를이용한검사를해야효과적이며예방적치료를위한유방절제술이나난소절제술등은매우침습적인방법이므로젊은여성에서생기는유방암이유전성인지아닌지를판별하는것이중요하다. 우리나라의여성유방암은구미선진국등과달라발생연령이매우낮아유방암진단의중앙나이가 45 세정도이며 50 세이하가전체암의 60% 정도를차지하고있다.(1) 한국인에서 BRCA1과 BRCA2 돌연변이는가족력이있는경우또는젊은여성에서많은빈도로발견되고있으며,(7-9) 고위험군환자는의료보험적용까지받으며검사를시행할수있다. 그러나가족력을가졌거나젊은여성유방암중에서 BRCA1과 BRCA2 돌연변이를제외한비교적흔하지않은유전성돌연변이에대한연구는전무한실정이다. 이에저자들은 40세이하의젊은여성유방암환자중 BRCA1과 BRCA2 돌연변이를조사하기위해검사를시행하였던환자를대상으로 CHEK2 돌연변이에대한빈도조사를계획하였다. CHEK2는세포주기체크포인트키나제2로써 DNA 의손상반응경로에서중요한역할을하는매개자로몇개의변이가유방암과의관계가있음이연구되었으나그중에서도 CHEK2*1100delC 돌연변이가주로 BRCA1과 BRCA2 돌연변이가없는가족성유방암이나젊은여성유방암에서빈번한빈도로발견되었다. 이연구의목적은한국의젊은여성유방암환자에서도 CHEK2*1100delC 돌연변이가존재하는지, 빈도가얼마인지알아보기위해검사를행하였다. 방법 1. 대상환자 1995년 1월부터 2000년 12 월까지순천향대학병원에서침윤성유방암으로수술을시행받은 40 세이하의여성유방암환자 60 명 을무작위로선정하였으며그환자들의혈액 10 ml 를채취하여 BRCA1/2 full sequencing에대해전매특허를가지고있는미국 Salt Lake City에보냈다. 그리고같은 DNA 중남은것을 PCR 증폭을해서연구협력기관이었던미국예일대학교부속병원을통해확보하였다. 그리고백인, 흑인, 한국인젊은여성유방암환자중에서유방암유전자돌연변이의인종간의특성을연구하기위해 Susan G. Komen 연구비의지원으로추가로시행했던 41 명의같은대상환자의혈액을 ( 주 ) LabGenomic의연구실에서 DNA 를추출하였다. 모든환자 101 명은유방암유전자검사를위해 IRB 를통과하고동의서를받았으며 CHEK2*110delC 돌연변이는 LabGenomics Clinical Research Institute에서검사를시행하였다. 환자들의연령은 22-40세이었으며 ( 중앙연령 35 세 ), 종양의크기는 2 cm 이하가 55명, 2-5 cm이 44 명이었고림프절전이는 33 명이 1-3 개이었고 8명은 4개이상이었다. 3대에걸친유방암의가족력은모두 13 명이었고난소암의가족력은 1명이확인되었으며 17 명이국소재발이나원격전이를보였다 (Table 1). 2. 검사방법 제조사의프로토콜에따라 G-DEX TM II Genomic DNA Extraction Kit (Intron, Seoul, Korea) 를이용하여 genomic DNA 를추출하였으며, chip-based MALDI-TOF mass spectrometry platform (Sequenom, Inc., San Diego, CA, USA) 를이용하여증폭된 DNA로부터얻어진 primer extension products Table 1. Clinicopathologic characteristics of patients (n=101) Characteristics Numbers (%) Median age (range) 35 (22-40 yr) Tumor size (cm) (54.5) (43.5) (2.0) Lymph node metastasis No 61 (60.4) (31.7) 4+ or more 8 (7.9) BRCA status Negative 74 Positive 14 Unknown variant 22* Family history of breast and/or ovary No 87 Yes 14 Relapse during follow-up No 84 Yes 17 *9 patients had both deleterious mutations and variants of unknown significance.

CHEK2 Mutation in Korean Women with Breast Cancer 27 Sample Blood Genomic DNA PCR Amplify MALDI-TOF analysis I base amplification reaction (SBE: Single Baes Extension) result Peak analysis

결과총 101 명의환자중에서돌연변이는 14 명 16 개 ( 두명은 BRCA1 과 BRCA2에각각한개씩의돌연변이가있었음 ) 이었고 22 명에서의미가불분명한돌연변이가있었으며이중 9명은돌연변이와의미가불분명한변이를동시에가지고있었다 (Table 1).

3 CHEK2 Mutation in Korean Women with Breast Cancer 27 Sample Blood Genomic DNA PCR Amplify MALDI-TOF analysis I base amplification reaction (SBE: Single Baes Extension) result Peak analysis Confirmation of the results Spotting on Chip Matrix Biochip MALDI-TOF Mass Spectrometer analysis MALDI-TOF MS CHEK2 Mutation analysis Flow chart Fig 2. Example of MALDI-TOP CHEK2 mutation (wild type). 결과총 101 명의환자중에서돌연변이는 14 명 16 개 ( 두명은 BRCA1 과 BRCA2에각각한개씩의돌연변이가있었음 ) 이었고 22 명에서의미가불분명한돌연변이가있었으며이중 9명은돌연변이와의미가불분명한변이를동시에가지고있었다 (Table 1). BRCA1 과 BRCA2 양성인 14명의환자뿐만아니라 87 명의 non-brca1/ 2 mutation 환자에서도 CHEK2 1100delC 돌연변이는발견되지않았다. Fig 2는실제환자의결과 1100delC 돌연변이가없는 wild type의예를보여주고있다. Fig 1. MALDI-TOP MS CHEK2 mutation analysis flow chart. 고 찰 를분석하여 SNP 를탐지하였다. PCR 을한이후에 unincorporated dntps는 0.3 U of Shrimp alkaline phosphatase를첨가하고섭씨 37 에서 20 분간배양후효소비활성화를위해섭씨 85 에서 5분간반응하여제거하였다. CHEK2 1100delC의 extension primer는 5 -CCT TTT GTA CTG AAT TTT AGA TTA 이었으며각반응의총량은 hme enzyme (Thermosequenase; Amersham Pharmacia Biotech, London, UK), ACG termination mix 와 5 m of extension primer를포함하여 9 L 이었다. Primer extension protocol은섭씨 94 에서 2 분간시행후94 에서 5초, 52 에서 5초, 72 에서 5초를시행하는것을 55 번시행하였고반응부산물을 SpectroCLEAN (Sequenom, Inc.) 로탈염화시킨후 SpectroJET (Sequenom, Inc.) 를사용하여 SpectroCHIP (Sequenom, Inc.) 에분배하였다. SpectroCHIP 은 MALDI-TOF MassARRAY system (Bruker-Sequenom, CA, USA) 를이용하여자동화모드로분석하였으며불량한피크가있는경우수동으로다시체크하였다. Fig 1은 MALDI-TOP MS CHEK2 돌연변이 flow chart를보여주고있다. 젊은여성에서유방암이발생하는경우 BRCA1이나 BRCA2가없는환자중에서투과도 (penetrance) 가비교적낮은유전자의돌연변이가상당수있다.(10) 그중에서도 CHEK2의특이한돌연변이인 CHEK2 1100delC는북미나유럽의여러나라들에서유방암의발생에일정한역할을한다고알려져있다. CHEK2는세포주기를조절하는키나제로 DNA 이중나선구조가손상되면 DNA 의손상복구과정에중요한역할을하는데 CHEK2 1100delC가있으면키나제의도메인과 CHEK2의활동이떨어지게되며남아있는정상적인대립유전자도소실되면 CHEK2의기능이완전이상실하게되고세포는 S와 G2 주기에주로모이게되고유전자의불안전성을보이게되고유방암뿐만아니라전립선암, 대장-직장암등다른암의발생가능성도높아지며암의치료에도돌연변이가있으면다른반응을보일수있다. CHEK2 1100delC 돌연변이와유방암의위험도와의관계는처음에 CHEK2 Breast Cancer Consortium (11) 에의해처음보고되었다. 당시의연구결과에의하면가족성유방암이있으며 BRCA1이나 BRCA2 돌연변이가없는환자중에서 CHEK delc 돌연변이빈도가높았다. 추가적으로 Vahteristo 등 (12) 의

4 28 Doo Ho Choi, et al. 연구와 Oldenburg 등 (13) 의연구에서도 BRCA1/2 음성인가족성유방암환자에서 CHEK2 1100delC 가중요한역할을하고있음을밝히고있다. 그러나일부의연구보고서는대조군에서뿐만아니라유방암환자중에서도 CHEK2 1100delC 보유자가낮은빈도를보이고있으며차이가없다고하고 (14) 스페인의유방암환자에서는 CHEK2 1100delC 돌연변이가없다고하였다. 많은환자와대조군을통해시행한최근의연구들에따르면 5개국에서만명이상의유방암환자와만명가까운대조군을통한연구분석에서 CHEK2 1100delC 보유자는유방암위험도가 2배이상증가한다고하였으며유방암가족력이없는젊은환자에서도발생비율이높다고보고하였으며 (15) 덴마크에서도만명가까운환자를통한연구에서는 3배정도유방암의위험도가증가한다고하였다.(16) 그리고 CHEK2 100delC의비율이높은경우는 BRCA1 과 BRCA2 돌연변이가없는가족성유방암환자에서약 5% 까지이돌연변이가있음을보고하고있다.(17) 유럽과미국의연구결과를제외하면타인종에대한 CHEK2 1100delC 돌연변이에대한연구결과는거의없는실정이다. 동양인의경우인도인을대상으로시행한연구에의하면 22 명의유방암가족력이있는환자에서 BRCA1과 BRCA2가발견되었으나 CHEK2 1100delC는발견되지않았다는것이유일한자료이다.(18) 본연구는유방암가족력이있고 BRCA1 또는 BRCA2 돌연변이가없는환자뿐만아니라가족력과관계없는젊은유방암환자에서도 CHEK2 1100delC 를찾을수없었다. 최근에 CHEK2 와임상-병리적관계에관한연구에의하면 CHEK2 양성환자는에스트로겐수용체양성비율이 CHEK2 음성환자와비슷하고 CHEK2 양성환자만의다른특징도찾을수없지만 (19, 20) 2차암 ( 반대측유방암 ) 이발생할가능성이 CHEK2 1100delC 음성인유방암환자에비해 2배높고무병생존율이낮다고하며예후가나쁜것은 2차유방암이발생하는것만으로는설명할수없다고한다.(20) 그리고 Meyer 등 (21) 에의하면초기유방암에서유방보존수술과방사선치료를시행한환자에서 CHEK2 1100delC 양성환자는무전이생존율 (metastasis free survival rate) 에서 T 병기다음으로나쁜예후인자로작용한다고하였다. 남성유방암은 BRCA1 또는 BRCA2 돌연변이와관련성이많으며한국남성유방암환자에서도보고되었는데 (9) Cowen 증후군이있는 PTEN 돌연변이에도남성유방암의빈도가높으므로 CHEK2 와남성유방암의관계도연구가되어왔으며초기의자료들은그가능성을시사하였으나후속적인결과들은남성유방암에서 CH- EK2 1100delC 양성비율은대조군과비슷하다고한다.(22-24) CHEK2 1100delC 돌연변이가있으면 60 세이상의환자가신체질량지수 25 이상인경우 10 년내에유방암이발생할가능성이 24% 가된다고하며 (16) 방사선에피폭시유방암이증가할가능성 이있으므로 (25) CHEK2 1100delC 돌연변이빈도가높은환자에대한 screening이필요하다. BRCA1과 BRCA2 유전자는각각 1,800개이상과 3,600개이상의아미노산을결정하는거대한염기서열을지니고있으며어떤장소에서도돌연변이가생길수있으므로모든부위를다 full sequencing해야하므로복잡하고비용도많이들지만 CHEK2 1100delC 돌연변이는단일한 genotyping test이므로비용이별로들지않으며보유자를 100% 찾아낼수있다. 이번연구를통해한국인젊은여성유방암환자에서가족력을가진 BRCA1/2 음성환자뿐만아니라가족력이없는환자도이돌연변이가발견되지않았다. 그러나 1100delC는주로백인들을통한연구에서도각민족에따른빈도가크게다르며다른인종에대한연구는거의시행되지않았으므로한국인도이돌연변이의빈도가얼마나되는지또는유방암발생에얼마나기여하는지연구할필요가있다. 이연구는모두 100 명정도의환자를대상으로시행했으므로실제로한국인은이돌연변이의빈도가낮거나거의존재하지않는지결론을내리기에는대상환자숫자가비교적적다. 따라서이돌연변이가한국인에서유방암발생에기여하는정도를보다정확하게연구하기위해서는유방암가족력이있는환자, 젊은여성유방암환자, 양측성유방암환자및남성유방암환자를대상으로보다많은대상의추가적인연구가필요하다. 그리고일정비율의돌연변이가발견될경우정상대조군과의비교연구도필요할것으로생각된다. 이에따라본연구자들은추가적인연구를진행하고있다. 결론유방암유전자 BRCA1/2 감사를시행한젊은여성유방암환자를대상으로세포주기조절기능을가진 CHEK2의 1100delC 돌연변이를조사한결과한국인에서 CHEK2 1100delC는유방암발생과관련이없는것으로나타났다. 보다정확한빈도와유방암의발생과의관련성을연구하기위해유방암가족력이있거나젊은여성유방암등 CHEK2 1100C 돌연변이가잘생기는인자를가진많은환자를대상으로추가적인연구가필요하다. 참고문헌 1. Korean Central Cancer Registry Annual report of the Korean Central Cancer Registry. Gwacheon: Ministry of Health and Walfare; King MC, Marks JH, Mandell JB. Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2. Science 2003;302:

5 CHEK2 Mutation in Korean Women with Breast Cancer Malone KE, Daling JR, Neal C, Suter NM, O brien C, Cushing-Haugen K, et al. Frequency of BRCA1/BRCA2 mutations in a population-based sample of young breast carcinoma cases. Cancer 2000;88: Lynch ED, Ostermeyer EA, Lee MK, Arena JF, Ji H, Dann J, et al. Inherited mutations in PTEN are associated with breast cancer, Cowen disease, and juvenile polyposis. Am J Hum Genet 1997;61: Rapakko K, Allinen M, Syrjakoski K, Vahteristo P, Huusko P, vahakangas K, et al. Gernline TP53 alterations in Finnish breast cancer families are rare and occur at conserved mutation-prone sites. Br J Cancer 2001;84: Meijers-Heijboer H, van Geel B, van Putten WL, Henzen-Logmans SC, Seynaeve C, Menke-Pluymers MB, et al. Breast cancer after prophylactic bilateral mastectomy in women with a BRCA1 or BRCA2 mutation. N Eng J Med 2001;345: Choi DH, Lee MH, Bale AE, Carter D, Haffty BG. Incidence of BRCA1 and BRCA2 mutations in young Korean breast patients. J Clin Oncol 2004;22: Kang HC, Kim IJ, Park JH, Kwon HJ, Won YJ, Heo SC, et al. Germline mutations of BRCA1 and BRCA2 in Korean breast and/or ovarian cancer families. Hum Mutat 2002;20: Ahn SH, Hwang UK, Kwak BS, Yoon HS, Ku BK, Kang HJ, et al. Prevalence of BRCA1 and BRCA2 mutations in Korean breast cancer patients. J Korean Med Sci 2004;19: Antoniou AC, Pharoah PD, McMullan G, Day NE, Ponder BA, Easton D. Evidence for further breast cancer susceptibility genes in addition to BRCA1and BRCA2 in a population-based study. Genet Epidemiol 2001;21: Meijers-Heijbor H, van den Ouweland A, Klijn J, Wasielewski M, de Snoo A, Oldenburg R, et al. Low-penetrance susceptibility to breast cancer due to CHEK2(*)1100delC in noncarriers of BRCA1 and BRCA2 mutations. Nat Genet 2002;31: Vahteristo P, Bartkova J, Eerola H, Syrjakoski K, Ojala S, Kilpivaara O, et al. A CHEK2 genetic variant contributing to a substantial fraction of familial breast cancer. Am J Hum Genet 2002;71: Oldenburg RA, Kroeze-Jansema K, Kraan J, Morreau H, Klijn JG, Hoogerbrugge N, et al. The CHEK2*1100delC variant acts as a breast cancer risk modifier in non-brca1/brca2 multiple case families. Cancer Res 2003;63: Offit K, Pierce H, Kirchhoff T, Kolachana P, Rapaport B, Gregersen P, et al. Frequence of CHEK2*1100delC in New York breast cancer cases and controls. BMC Med Genet 2003;4: The CHEK2 Breast Cancer Case-Control Consortium: CHEK2* 1100delC and susceptibility to breast cancer: a collaborative analysis involving 10,860 breast cancer cases and 9,065 controls from 10 studies. Am J Hum Genet 2004;74: Weischer M, Bojesen SE, Tybjoerg-Hansen A. Axelsson CK, Nordestgaard BG. Increased risk of breast cancer associated with CHEK2* 1100delC. J Clin Oncol 2007;25: Walsh T, Casadei S, Coats KH, Swisher E, Stray SM, Higgins J, et al. Spectrum of mutations in BRCA1, BRCA2, CHEK2, and TP53 in families at high risk of breast cancer. JAMA 2006;295: Rajkumar T, oumittra N, Nancy NK, Swaminathan R, Sridevi V, Shanta V. BRCA1, BRCA2 and CHEK2 (1100 delc) germline mutations in hereditary breast and ovarian cancer families in South India. Asian Pac J Cancer Prev 2003;4: Cybulski C, Gorski B, Huzarski T, Byrski T, Gronwald J, Debniak T, et al. CHEK2-positive breast cancers in young Polish women. Clin Cancer Res 2006;12: Schmidt MK, Tollenaar RA, de Kemp SR, Broeks A, Cornelisse CJ, Smit VT, et al. Breast cancer survival and tumor characteristics in premenopausal women carrying the CHEK2*1100delC Germline mutation. J Clin Oncol 2007;25: Meyer A, Dork T, Sohn C, Karstens JH, Bremer M. Breast cancer in patients carrying a germ-line CHEK2 mutation: outcome after breast conserving surgery and adjuvant radiotherapy. Radiother Oncol 2007; 82: Syrjakoski K, Kuukasjarvi T, Auvinen A, Kallioniemi OP. CHEK2 1100delC is not a risk factor for male breast cancer population. Int J Cancer 2004;108: Neuhausen S, Dunning A, Steele L, Yakumo K, Hoffman M, Szabo C, et al. Role of CHEK2*1100delC in unselected series of Non-BRCA1/ 2 male breast cancers. Int J Cancer 204;108: Ohayon T, Gal I, Baruch RG, Szabo C, Friedman E. CHEK2*1100delC and male breast cancer risk in Israel. Int J Cancer 2004;108: Bernstein JL, Teraoka SN, John EM, Andrulis IL, Knight JA, Lapinski R, et al. The CHEK2*1100delC allelic variant and risk of breast cancer: screening results from the Breast Cancer Family Registry. Cancer Epidemiol Biomarkers Prev 2006;15:

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