DOI: 10.4046/trd.2010.69.4.271 ISSN: 1738-3536(Print)/2005-6184(Online) Tuberc Respir Dis 2010;69:271-278 CopyrightC2010. The Korean Academy of Tuberculosis and Respiratory Diseases. All rights reserved. 비소세포폐암에서 PNA-Mediated PCR Clamping 을이용한 EGFR 돌연변이분석법 건국대학교의학전문대학원 1 내과학교실, 2 병리학교실이계영 1, 김희정 1, 김순종 1, 유광하 1, 김원동 1, 오서영 2, 김완섭 2 Original Article PNA-Mediated PCR Clamping for the Detection of EGFR Mutations in Non-Small Cell Lung Cancer Kye Young Lee, M.D., Ph.D. 1, Hee Joung Kim, M.D. 1, Sun Jong Kim, M.D. 1, Gwang Ha Yoo, M.D. 1, Won Dong Kim, M.D. 1, Seo Young Oh, M.S. 2, Wan Seop Kim, M.D. 2 Departments of 1 Internal Medicine, 2 Pathology, Konkuk University School of Medicine, Seoul, Korea Background: Recent studies have demonstrated that the epidermal growth factor receptor (EGFR) genotype is the most important predictive marker to EGFR-tyrosine kinase inhibitors (TKIs) and first-line gefitinib treatment will be approved in the near future for use in non-small cell lung cancer (NSCLC) patients with the EGFR mutation. Direct sequencing is known to be the standard for detecting EGFR mutations; however, it has limited sensitivity. Peptide nucleic acids (PNA)-mediated PCR clamping method is a newly introduced method for analyzing EGFR mutations with increased sensitivity and stability. Methods: A total of 71 NSCLC patients were analyzed for EGFR mutations using the PNA-mediated PCR clamping technique. Sixty-nine patients were analyzed for clinicopathologic correlation with EGFR genotype; 2 patients with indeterminate results were excluded. In order to determine EGFR-TKI drug response, 57 patients (42 gefitinib, 15 erlotinib) were included in the analysis. Results: The EGFR mutation rate was 47.8%. Being female, a non-smoker, and having adenocarcinoma were favorable clinicopathologic factors, as expected. However, more than a few smokers (33.3%), male (28.1%), and patients with non-adenocarcinoma (28.6%) had the EGFR mutation. Having a combination of favorable clinicopathologic factors did not increase the EGFR mutation rate significantly. Drug response to EGFR-TKIs showed significant differences depending on the EGFR genotype; ORR was 14.3% for wild type vs 69.0% for mutant type; DCR is 28.6% for wild type vs 96.6% for mutant type. The median EGFR-TKI treatment duration is 7.6 months for mutant type group and 1.4 months for wild type group. Conclusion: EGFR genotype determined using the PNA-mediated PCR clamping method is significantly correlated with the clinical EGFR-TKI responses and PNA-mediated PCR. Key Words: Peptide Nucleic Acids; Receptor, Epidermal Growth Factor; Carcinoma, Non-Small-Cell Lung 서 론 Address for correspondence: Kye Young Lee, M.D., Ph.D. Department of Internal Medicine, Konkuk University School of Medicine, 4-12, Hwayang-dong, Gwangjin-gu, Seoul 143-729, Korea Phone: 82-2-2030-7521, Fax: 82-2-2030-7748 E-mail: kyleemd@kuh.ac.kr Received: Sep. 3, 2010 Accepted: Oct. 15, 2010 Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) 가임상에도입된초기에는여성, 비흡연자, 선암, 동양인등과같은임상-병리적요소들이약제를선택하는기준이었지만 1-3, 최근에는 EGFR 돌연변이가 EGFR-TKI 에대한약제반응을결정하는가장중요한인자라는것이밝혀지면서 4,5 EGFR 돌연변이분석검사의 271
KY Lee et al: PNA-mediated PCR clamping for the detection of EGFR mutations 중요성이대두되고있다. 또한조만간 EGFR 돌연변이양성비소세포폐암환자는 1차항암약제로서세포독성항암제를지양하고 gefitinib 와같은 EGFR-TKI 가선택처방될것으로예상되는시점에서, EGFR 돌연변이분석검사는이제연구적가치를넘어서임상에서반드시필요한검사방법으로자리매김할것으로예상하고있으며, 정확하고예민한 EGFR 돌연변이분석검사방법의도입은필연적이라하겠다. EGFR 돌연변이를검사할수있는분석방법으로는 direct sequencing 이표준방법으로알려져있고현재대다수의문헌자료들은이방법에의한결과들이다 6. 그러나 direct sequencing 방법은민감도가떨어져서 mutant DNA 가최소 20% 이상의농도를유지해야검출되므로정상 DNA 에의한희석효과를감소시키기위하여조직에서종양세포의세포비율 (cellularity) 이 40% 이상은되어야하며정확한분석을위해서암세포조직에대한 laser capture microdissection이필요하다 7. 따라서예민도가높고임상적으로 EGFR-TKI 에대한약제반응과의일치도가높은검사방법이요구되고있는데, IPASS study 에서사용된 Scorpion 방법이널리알려져있기는하지만 8, 고비용과검사키트의물리적안정성등의문제점으로인해현재국내에널리보급되고있지않은상황이다. 최근 peptide nucleic acids (PNA) 를이용한방법, 즉 PNA-mediated PCR clamping 이란방법이개발되어 EGFR 돌연변이분석에이용하려는움직임이있어주목받고있으며 9, 국제적으로도이방법을이용한연구결과가이미보고되기시작하였다 10,11. 이에저자들은비소세포폐암환자에서 PNAmediated PCR clamping 방법을이용하여 EGFR 돌연변이검사를시행하고그결과와 EGFR-TKI 에대한약제반응과의일치도를분석조사하여비소세포폐암에서 PNAmediated PCR clamping을이용한 EGFR 돌연변이분석검사의임상적유용성을조사하여보고하는바이다. 대상및방법 1. 연구대상건국대학교병원에서비소세포폐암으로진단받은 71명의환자를대상으로하였고, 연령은 70.8±12.2세 (26 86세 ) 남녀비는 33:38 (46.5%:53.5%), 흡연력은비흡연자 36명 (50.7%), 과거흡연자 16명 (22.5%), 현재흡연자 19명 (26.8%) 이며, 선암이 64명 (90.1%) 으로대다수를차지하고있었으며기타평편상피암 3명 (4.2%), 대세 Table 1. Patient characteristics (n=71) Age, yr Average±SD 70.8±12.2 Median (range) 61 (26 86) Sex, No. (%) Male 33 (46.5) Female 38 (53.5) Smoking status, No. (%) Never smoker 36 (50.7) Ex-smoker 16 (22.5) Current smoker 19 (26.8) Pathology, No. (%) Adenocarcinoma 64 (90.1) Squamous cell carcinoma 3 (4.2) Large cell carcinoma 1 (1.4) Non-small cell carcinoma 3 (4.2) Biopsy vs. Cyotlogy, No. (%) Biopsy 45 (64.3) Cytology 26 (36.6) Stage, No. (%) IA 1 (1.4) IB 5 (7.0) IIA 1 (1.4) IIB 2 (2.8) IIIA 1 (1.4) IIIB 14 (19.7) IV 47 (66.2) 포암 1명 (1.4%), 비소세포폐암 3명 (4.2%) 이었으며, 조직검사시료가 45명 (64.3%) 이었으며세포진검사시료는 26명 (36.6%) 이었다 (Table 1). 병기별로는 IV병기가 47명 (66.2%) 으로가장많았고 IIIB가 14명 (19.7%) 으로뒤를이었다. 2. DNA 분리 병리의사가현미경으로관찰하여파라핀포매조직및세포슬라이드에서종양세포가최소 50% 이상포함되도록영역을표기하였다. 커버글라스를 xylene 으로제거한후 26 gauge 주사기바늘을이용하여암세포만을분리채취하였다. 10% Resin 이들어있는 DNA extraction buffer (50mMTris-cl ph 8.5, 1mMEDTA ph 8.0, 0.5% Tween 20) 50 100 μl 에채취된암세포만을분리하여넣고 200 μg/ml Proteinase K와혼합한후 56 o C에서최소 1시간이상처리한후 100 o C에서 10분동안가열하였다. 그리고 12,000 rpm에서 10 15 분동안원심분리하고 Resin이혼입되지않도록주의하면서상층액만을채취하여 PCR 반응에사용하였다 12,13. 272
Tuberculosis and Respiratory Diseases Vol. 69. No. 4, Oct. 2010 3. PNA-mediated PCR clamping 추출된 DNA 에서 EGFR 돌연변이분석검사는 PNA Table 2. The PCR protocol One cycle Pre-denaturation 94 o C 5 min 4-Step cycling Denaturation 94 o C 30 sec PNA clamping 70 o C 20 sec Annealing 63 o C 30 sec Extension 72 o C 30 sec No. of cycles: 40 cycles One cycle Final extension 72 o C 5 min Clamp TM EGFR mutation detection kit (PANAGENE, Daejeon, Korea) 를사용하여검출하였다. Real time PCR 기기는 CFX 384 (Bio-Rad, Hercules, CA, USA) 를사용하였으며 Table 2의반응조건으로실험하였다. PNAClamp TM EGFR mutation detection kit에서검출가능한돌연변이는 Table 3에나타내었다. 야생형유전자와혼성화되도록고안된 PNA 프로브가 EGFR 돌연변이코돈유전자부위에혼성화되어증폭을저해하게되면증폭이저해되어 Ct값이높게나타난다. 반면 EGFR 돌연변이코돈부위에돌연변이가발생한경우 PNA 프로브와혼성화되지못하고증폭되어 Ct값이낮게나타나게된다. Standard Ct값에서미지의시료로부터얻어진 Ct값을빼어얻어진 Ct의값을확인하여각코돈의돌연변이유무를확인하였다. Table 3. The PNA-mediated clamping method detects 29 mutations of the EGFR gene No. EGFR detection region Exon Amino acids change Base change 1 18 Point mutation p.gly719ala 2156 G>C 2 18 p.gly719ser 2155 G>A 3 18 p.gly719cys 2155 G>T 4 19 Deletion p.glu746_ala750del 2235_2249 del 15 5 19 p.glu746_thr751delinsile 2235_2252 AAT (complex) 6 19 p.glu746_ser752del 2236_2253 del 18 7 19 p.glu746_thr751delinsala 2237_2251 del 15 8 19 p.glu746_s752>ala 2237_2254 del 18 9 19 p.glu746_ser752delinsval 2237_2255>T (complex) 10 19 p.glu746_ala750del 2236_2250 del 15 11 19 p.glu746_ser752delinsasp 2238_2255 del 18 12 19 p.leu747_ala750>pro 2238_2248 >GC (complex) 13 19 p.leu747_thr751delinsgln 2238_2252 >GCA (complex) 14 19 p.leu747_glu749del 2239_2247 del 9 15 19 p.leu747_thr751del 2239_2253 del 15 16 19 p.leu747_ser752del 2239_2256 del 18 17 19 p.leu747_glu749del; p.ala750pro 2239_2248 TTAAGAGAAG>C 18 19 p.leu747_pro753delinsgln 2239_2258 >CA (complex) 19 19 p.leu747_thr751delinsser 2240_2251 del 12 20 19 p.leu747_pro753delinsser 2240_2257 del 18 21 19 p.leu747_thr751del 2240_2254 del 15 22 19 p.leu747_thr751deinspro 2239_2251>C (complex) 23 20 Point mutation p.thr790met 2369 C>T 24 20 p.ser768ile 2303 G>T 25 20 Insertion p.ala767_val769dupalaserval 2307_2308 ins9 26 20 p.his773duphis 2319_2320 inscac 27 20 p.asp770_asn771insgly 2310_2311 insggt 28 21 Point mutation p.leu858arg 2573 T>G 29 21 p.leu861gln 2582 T>A PNA: peptide nucleic acids; EGFR: epidermal growth factor receptor. 273
KY Lee et al: PNA-mediated PCR clamping for the detection of EGFR mutations 4. 통계분석 수집한자료는 SPSS version 17.0 (SPSS Inc., Chicago, IL, USA) 를이용하여통계처리하였다. 임상특성비교에있어서연령은 Mann-Whitney U test, 성별, 흡연력, 병기, Table 4. EGFR mutation testing results using PNA-mediated PCR clamping (n=71) Wild type 36 (50.7) Indeterminate 2 (2.8) Mutant type 33 (46.5) Exon19 del 18 (54.5) Exon21 L858R or L861Q 14 (42.4) Exon19 del+t790m 1 (3.0) Values are presented as number (%). EGFR: epidermal growth factor receptor; PNA: peptide nucleic acids. 조직형, 검체종류, 약제사용에대한반응은 chi-square test, 무진행생존율은 Kaplan Meier 법과 log rank test를이용하여분석하였다. p값이 0.05 미만인경우통계적으로유의한것으로판단하였다. 결 1. PNA-mediated PCR clamping 을이용한 EGFR 돌연변이분석 71명의비소세포폐암환자에서 PNA-mediated PCR clamping 방법을이용하여 EGFR genotyping 을시행한결과 36명 (50.7%) 에서는 wild-type 이 33명 (46.5%) 에서는 mutant-type 이결정되었고, 2명 (2.8%) 에서는 genotype 을결정할수없었다 (Table 4). 그이유는추출된 DNA 의질적인문제때문에 PCR 증폭이잘되지않은것으로생각 과 Table 5. Comparisons of clinicopathologic factors depending on EGFR genotype EGFR wild-type No. (%) (n=36 [52.2%]) EGFR mutant-type No. (%) (n=33 [47.8%]) Total No. (%) (n=69) p-value Sex, M:F 23:13 (63.9) 9:24 (27.3) 32:37 (46.4) 0.002 Male 23 (71.9) 9 (28.1) 32 (46.4) Female 13 (35.1) 24 (64.9) 37 (53.6) Age Median (range) 57 (36 86) 63 (26 81) 61 (26 86) Mean±SD 60.8±13.2 60.5±11.3 60.6±12.3 0.971 Smoking status 36 33 69 Non smoker 13 (36.1) 22 (66.7) 35 (50.7) 0.040 Current smoker 12 (33.3) 6 (18.2) 18 (26.1) Ex-smoker 11 (30.6) 5 (15.2) 16 (23.2) Never smoker 13 (36.1) 22 (66.7) 35 (50.7) 0.011 Ever smoker 23 (63.9) 11 (33.3) 34 (49.3) Pack-years 35.0±22.3 32.6±26.9 34.3±23.2 0.799 Stage IB 2 (5.6) 3 (9.1) 5 (7.2) 0.307 IIA 1 (2.8) 0 1 (1.4) IIB 2 (5.6) 0 2 (2.9) IIIA 1 (2.8) 0 1 (1.4) IIIB 8 (22.2) 6 (18.2) 14 (20.3) IV 22 (61.1) 24 (72.7) 46 (66.7) Pathology Adenocarcinoma 31 (86.1) 31 (93.9) 62 (89.9) 0.286 Squamous cell carcinoma 2 (5.6) 1 (3.0) 3 (4.3) Large cell carcinoma 1 (2.8) 0 1 (1.4) Non-small cell carcinoma 2 (5.6) 1 (3.0) 3 (4.3) Biopsy 22 (61.1) 22 (66.7) 44 (63.8) 0.632 Cytology 14 (38.9) 11 (33.3) 25 (36.2) EGFR: epidermal growth factor receptor. 274
Tuberculosis and Respiratory Diseases Vol. 69. No. 4, Oct. 2010 된다. EGFR 돌연변이는 exon 19 del이 18명 (54.5%) 으로가장빈도가높았고뒤이어 exon 21 L858R이 14명 (42.4%), 그리고 1명의환자에서는 exon 19 del과획득내성돌연변이인 T790M이동시에발견되었다. 2. EGFR genotype과임상-병리인자들과의비교 EGFR genotype이결정된 69명환자들을야생 (wildtype) 군과변이 (mutant) 군으로구분하여주요임상- 병리인자들에대하여비교분석하여보았다 (Table 5). EGFR 돌연변이는예상대로여성 (64.9%:28.1%), 비흡연자 (62.9%: 37.1%) 에서유의하게높은빈도를보였지만상대적으로적지않은숫자의남성, 흡연자에서도 EGFR 돌연변이가발견되었다. 연령, 흡연량, 병기에대해서는유의한차이를발견할수없었고병리학적으로는선암에서는 62명중 31명, 즉 50% 의양성률을보이고비선암에서는 7명중 2 명 28.6% 의양성률을보였으나통계적유의성을발견할수없었던것은비선암의환자수가절대적으로적었기때 문이라고생각된다. 조직검사로진단된경우와세포진검사로진단된경우사이에서 EGFR 돌연변이검출률에는유의한차이를관찰할수없었다. EGFR-TKI 에대한약제반응이좋다고알려진임상-병리인자인여성, 비흡연자, 선암등의인자와돌연변이의빈도와의관련성을비교해볼때우호적인자들이두가지조합, 심지어세가지조건모두를가지고있는경우에도 EGFR 돌연변이율이크게증가하지않는다는사실을확인할수있어 (Table 6), 임상-병리인자들만가지고 EGFR 돌연변이를예측해서는안되고반드시 EGFR 돌연변이검사를시행해야한다는것을시사하는자료라고생각한다. 한편 EGFR 돌연변이가확인된 33명의환자들을분석해보면우호적인자가하나도없는경우는 1명 (3.0%) 에서만돌연변이양성이었고, 두가지인자를가지고있는경우는 Table 6. EGFR mutation positive rate depending on favorable clinicopathologic factors Female 24/37 (64.9%) Non-smoker 22/35 (62.9%) Adenocarcinoma 31/62 (50.0%) Female and non-smoker 19/35 (54.3%) Female and adenocarcinoma 23/35 (65.7%) Non-smoker and adenocarcinoma 21/33 (63.6%) Female and non-smoker and 18/28 (64.3%) adenocarcinoma EGFR: epidermal growth factor receptor. Figure 1. The proportions of favorable clinicopathologic factors and each combination in EGFR mutation positive group (n=33). Table 7. The concordance rate between EGFR genotypes and TKI responses EGFR wild type No. (%) (n=28 [49.1%]) EGFR mutant type No. (%) (n=29 [50.9%]) Total No. (%) (n=57) p-value Gefitinib 17 (60.7) 24 (82.8) 41 (71.9) 0.064 Erlotinib 11 (39.3) 5 (17.2) 16 (28.1) Response evaluation CR 0 0 0 <0.001 PR 4 (14.3) 20 (69.0) 24 (42.1) SD 4 (14.3) 8 (27.6) 12 (21.1) PD 20 (71.4) 1 (3.4) 21 (36.8) Overall RR (ORR) (CR+PR) 4/28 (14.3) 20/29 (69.0) 24/57 (42.1) <0.001 Disease control rate (CR+PR+SD) 8/28 (28.6) 28/29 (96.6) 36/57 (63.2) <0.001 Duration of TKI, Median (range), mo 1.7 (0.6 15.7) 8.3 (1.8 29.5) 4.7 (0.6 29.5) <0.001 EGFR: epidermal growth factor receptor; TKI: tyrosine kinase inhibitors. 275
KY Lee et al: PNA-mediated PCR clamping for the detection of EGFR mutations Figure 2. Progression free survival (duration of EGFR-TKI treatment) according to EGFR genotype. 9명 (27.3%), 그리고세가지인자모두를가지고있는경우는 18명 (69.7%) 으로서우호적인자가많을수록돌연변이를가지고있을확률은증가한다는사실을확인할수있었다 (Figure 1). 3. EGFR-TKI 약물반응과 EGFR genotype EGFR genotype이결정된 69명의환자중에서 EGFR- TKI 를사용한 57명의환자를대상으로 EGFR 돌연변이유무에따른 EGFR-TKI 에대한약제반응을분석하여보았다 (Table 7). 분석대상 57명의환자중 28명 (49.1%) 이야생형이었고 29명 (50.9%) 이변이형이었으며 42명은 gefitinib를 15명은 erlotinib 를처방받았다. 변이형에서는 gefitinib 처방이상대적으로월등히많았음을확인할수있었는데이는 gefitinib에대한국내처방기준과연관이있을것으로생각된다. 양군에서완전관해환자는 1명도없었으며, 변이형군에서부분관해 69.0%, 안정질환 27.6% 로서질병조절률이 96.6% 로서야생형의각각 14.3%, 14.3%, 28.6% 의결과보다현저한차이를보이고있음을확인할수있었다 (p<0.001). 또한 EGFR-TKI 약제사용기간 ( 무진행생존기간 ; progression free survival) 도중앙값이변이형이 7.6개월인반면야생형은 1.4개월로뚜렷하고도유의한차이를나타내었다 (Figure 2). 고 찰 PNA 는인공 DNA 의일종으로 DNA 보다유전자를인식하는능력이뛰어나고 nuclease 와같은생물학적효소에대하여매우안정적인특징을가지고있어서, PNA-mediated PCR clamping 방법은 EGFR 돌연변이를 PCR 반응만으로신속하고정확하게찾아낼수있는방법으로알려져있다 10,14,15. 일본에서는 PNA-LNA PCR clamp를이용한 EGFR mutation kit가상용화되어있고이미이를이용한임상시험결과들이보고된바있으며, 임상연구수준이아니라일반적인폐암임상에정규검사로시행되고있다고한다 16. 이러한배경에서 PNA에대한대량생산에대한기술적원천특허권을국내기업인 ( 주 )PANAGENE이가지고있다는사실을생각해볼때, 국내에서 PNA를이용한 EGFR mutation kit가이제개발되어그임상자료가이제발표된다는사실은뒤늦은감이없지않다고생각된다. 더욱이 EGFR 돌연변이의임상적중요성과그빈도가우리나라와같은동양인에많다는사실에착안하여이에대한관심을배가시킬필요가있다고생각한다. 본연구에서 71명의환자시료를대상으로검사를시행한결과두명의환자에서는명확한 EGFR 유전형을결정하지못하였는데, 이두예는모두 DNA 추출에문제가있었던것으로생각된다. 이는돌연변이를분석및검출하는방법도중요하지만이에앞서양질의 DNA 를분리하는것이선행되어야함을확인할수있는사실이라고생각된다. 본연구에서현재표준방법으로알려져있는 direct sequencing 방법과의직접적인비교연구는시행하지못하였지만, EGFR 유전자형에따른임상-병리인자들과의상관성과 EGFR-TKI 에대한약물반응과의일치도결과에서볼때 PNA-mediated PCR clamping 방법은비소세포폐암에서 EGFR 돌연변이를검출하는데매우유용한검사방법이될것으로예상된다. 본연구결과에서보듯이 EGFR 돌연변이는선암, 여성, 비흡연자에서월등히그빈도가높다는사실을확인할수있었지만, 적지않은숫자의흡연자, 남성그리고때로는비선암환자에서도 EGFR 돌연변이가관찰되고이러한 EGFR 돌연변이는 EGFR-TKI 에대한약제반응과명백한관련성이있다는사실에서 EGFR 돌연변이검사는이제폐암의진료에있어서일상적으로시행되는검사방법이되어야할것으로생각한다. 또한본연구의시료분포를보면 61.1% 가조직검사시료인반면 38.9% 가세포진검사시료를이용하여조직검사시료에비하여그검사결과의예민도가떨어지지않았다는점에서 PNA-mediated PCR clamping 방법의예민도를증명할수있었다고생각한다. 이방법은 1% 의돌연변이 DNA 만있어도검출해낼수있는예민한방법이라고알려져있다 10. 반면 direct sequencing 방법은정상 DNA 에의한희석효과때문에돌 276
Tuberculosis and Respiratory Diseases Vol. 69. No. 4, Oct. 2010 연변이 DNA가 20% 이상은되어야검출해낼수있다고알려져있다. 하지만 PNA-mediated PCR clamping 방법은 direct sequencing 과는달리새로운돌연변이를검출해낼수는없는단점이있다. 즉, 기존의잘알려진돌연변이형에대한 kit를개발한검사법이고본연구에사용한 PANAGENE 사의 PNAClamp TM EGFR mutation detection kit는지금까지알려진돌연변이형중임상적으로중요하고빈도가흔한 29가지종류를망라하고있어서임상적으로중요한돌연변이를놓칠가능성은높지않다고생각한다. 실제로 EGFR 돌연변이는 EGFR 유전자의 TK domain (exon 18 21) 에예외없이존재하는데현재까지 250여가지이상의돌연변이가보고되고있지만이중에서임상적중요성이알려진돌연변이는 10개를넘지않고있다고알려져있다 17-19. 다만아직임상시료에대한분석경험이많지않아이에대한충분한임상적용이필요한상태이다. 현재저자를중심으로 200여명의비소세포폐암환자에서파라핀포매병리조직을이용하여 direct sequencing 과 PNA-mediated PCR clamping 방법간의비교연구가진행중인데이연구가끝나면보다정확한비교임상자료를얻을수있을것으로생각된다. EGFR 돌연변이는민감성 (sensitizing) 돌연변이와내성 (resistant) 돌연변이로대별되는데 exon 19의 deletion 과 exon 21의 L858R point mutation이가장중요한민감성돌연변이로서약 85 90% 를차지하고있고 exon 19 del 돌연변이가 TKI에대한민감성이더좋은것으로알려져있다 20. 반면 exon 20의 T790M 점돌연변이는가장중요한내성돌연변이로서획득내성환자의 50% 이상에서발견되는것으로알려져있다 21. 따라서 T790M 점돌연변이의검출문제는 EGFR-TKI 의획득내성의문제가부각되고이에대한대안으로개발되고있는 BIBW2992 와같은제2세대 EGFR-TKI 의임상도입에있어서매우중요한이슈로부각될수있는문제이기에예민도가높은 PNAmediated PCR clamping 과같은방법을혈장 DNA 에적용하는시도가필요할것으로예상된다 22,23. 원칙적으로 T790M 검출에대해서는재조직검사 (rebiopsy) 가필요하지만현실적으로재조직검사에대한어려움이너무크므로혈장 DNA에서돌연변이를검색할수있는방법들에대한많은연구가진행중이어서조만간이에대해서도그임상적유용성에대한결론이내려질것으로생각된다 24. 결론적으로본연구를통하여비소세포폐암환자에서 EGFR 돌연변이를검출하는데있어서 PNA-mediated PCR clamping 방법은신속하고예민한방법임을확인할수있었으며 EGFR-TKI 에대한약물반응과의일치도가비교적매우정확하다는점에서그임상적유용성이높다고판단되며, 국내비소세포폐암환자에서 EGFR 돌연변이에관한임상자료를얻는데향후기여도가매우높은검사방법으로자리매김할수있도록다양한임상연구가진행되어야할것으로생각한다. 감사의글 This study was supported by a grant from Korean Association for the Study of Lung Cancer (KASLC-1002). 참고문헌 1. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129-39. 2. Paez JG, Jänne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304: 1497-500. 3. Pao W, Miller VA. Epidermal growth factor receptor mutations, small-molecule kinase inhibitors, and nonsmall-cell lung cancer: current knowledge and future directions. J Clin Oncol 2005;23:2556-68. 4. Cappuzzo F, Hirsch FR, Rossi E, Bartolini S, Ceresoli GL, Bemis L, et al. Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-smallcell lung cancer. J Natl Cancer Inst 2005;97:643-55. 5. Zhu CQ, da Cunha Santos G, Ding K, Sakurada A, Cutz JC, Liu N, et al. Role of KRAS and EGFR as biomarkers of response to erlotinib in National Cancer Institute of Canada Clinical Trials Group Study BR.21. J Clin Oncol 2008;26:4268-75. 6. Dacic S. EGFR assays in lung cancer. Adv Anat Pathol 2008;15:241-7. 7. Pao W, Ladanyi M. Epidermal growth factor receptor mutation testing in lung cancer: searching for the ideal method. Clin Cancer Res 2007;13:4954-5. 8. Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 2009;361:947-57. 9. Lee KY. Molecular diagnosis in lung cancer. J Lung 277
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