원저 Lab Med Online Vol. 5, No. 1: 1-5, January 진단혈액학 골수증식종양환자에서 MPL W515 돌연변이빈도와임상혈액학적특징 Frequency and Cl

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김재룡 1 함지연 2 서장수 2 김유경 3 Sung Gyun Park, M.D. 1, Kyoung Bo Kim, M.D. 1, Wonmok Lee, M.D. 1, Jung Sook Ha, M.D. 1, Nam Hee Ryoo, M.D. 1, Dong Seok Jeon, M.D. 1, Jae Ryong Kim, M.D. 1, Ji Yeon Ham, M.D. 2, Jang Soo Suh, M.

Pathology 2, Kyungpook National University School of Medicine, Daegu; Department of Laboratory Medicine 3, Yeungnam University School of Medicine, Daegu, Korea Background: Recently,

1 원저 Lab Med Online Vol. 5, No. 1: 1-5, January 2015 진단혈액학 골수증식종양환자에서 돌연변이빈도와임상혈액학적특징 Frequency and Clinicohematologic Characteristics of Mutations in Patients with Myeloproliferative Neoplasms 박성균 1 김경보 1 이원목 1 하정숙 1 류남희 1 전동석 1 김재룡 1 함지연 2 서장수 2 김유경 3 Sung Gyun Park, M.D. 1, Kyoung Bo Kim, M.D. 1, Wonmok Lee, M.D. 1, Jung Sook Ha, M.D. 1, Nam Hee Ryoo, M.D. 1, Dong Seok Jeon, M.D. 1, Jae Ryong Kim, M.D. 1, Ji Yeon Ham, M.D. 2, Jang Soo Suh, M.D. 3, Yu Kyung Kim, M.D. 4 계명대학교의과대학진단검사의학교실 1, 경북대학교의학전문대학원임상병리학교실 2, 영남대학교의과대학진단검사의학교실 3 Department of Laboratory Medicine 1, Keimyung University School of Medicine, Daegu; Department of Clinical Pathology 2, Kyungpook National University School of Medicine, Daegu; Department of Laboratory Medicine 3, Yeungnam University School of Medicine, Daegu, Korea Background: Recently, myeloproliferative leukemia (MPL) W515 mutations have been reported to be molecular markers for myeloproliferative neoplasms (MPNs). We studied the association between mutations and the clinico-hematological features of patients with MPNs. Methods: Our study included 154 consecutive patients diagnosed with MPNs (31 had polycythemia vera [ PV]; 106, essential thrombocythemia [ ET]; and 17, primary myelofibrosis [ PMF]). mutations were detected by real-time PCR and direct sequencing methods. Results: The L mutation was found in 4 patients and the A mutation was detected in 1 patient. These 5 patients were diagnosed with JAK2 V617F-negative ET, and they accounted for 12.5% of patients with JAK2 V617F-negative ET. The patients with -positive ET showed significantly lower hemoglobin levels and WBC counts than did patients with -negative ET or JAK2 V617F-positive ET. Conclusions: mutation is a useful diagnostic marker for JAK2 V617F-negative MPNs and it is associated with specific hematologic characteristics such as lower hemoglobin levels and WBC counts. Kew Words: Myeloproliferative neoplasm, Essential thrombocythemia,, JAK2 protein tyrosine kinase 서론 골수증식종양 (myeloproliferative neoplasm, MPN) 이란, 하나이 상의혈구계열에서성숙세포가과도하게생성되는클론성조혈모 세포질환이며, 2008 WHO 분류에따라 BCR-ABL1 양성만성골수 성백혈병 (chronic myelogenous leukemia, CML), 만성호중구백혈 Corresponding author: Jung-Sook Ha Department of Laboratory Medicine, Keimyung University School of Medicine, 56 Dalsung-ro, Joong-gu, Daegu , Korea Tel: , Fax: , ksksmom@dsmc.or.kr Received: March 4, 2014 Revision received: May 29, 2014 Accepted: July 1, 2014 This article is available from , Laboratory Medicine Online This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 병 (chronic neutrophilic leukemia), 진성적혈구증가증 (polycythaemia vera, PV), 일차골수섬유증 (primary myelofibrosis, PMF), 진성고혈소판증가증 (essential thrombocythaemia, ET), 만성호산구백혈병 (chronic eosinophilic leukemia, NOS), 비만세포증 (mastocytosis) 및미분류골수증식종양 (myeloproliferative neoplasm, unclassifiable) 등이이에속한다 [1]. 그중 classic MPN으로분류되는진성적혈구증가증, 진성고혈소판증가증, 일차골수섬유증은최근까지뚜렷한분자유전학적표지자가보고되지않았으나, 2005년 JAK2 티로신인산화효소의 V617F 돌연변이 ( JAK2 V617F) 가보고되면서진단과연구의새로운전환점을맞이하게되었다 [2-6]. 그러나, PV에서는 95% 이상에서양성인반면, ET와 PMF에서는 30-40% 이상이 JAK2 V617F가관찰되지않아, JAK2 V617F 이외새로운분자유전학적표지자에대한필요성이지속적으로제기되어왔다 [3-6]. 최근트롬보포이에틴수용체 (thrombopoietin receptor, TPO-R; myeloproliferative leukemia, MPL) 10번엑손에위치한트립토판 eissn

2 (tryptophan) 이다른아미노산으로치환되는 돌연변이가 MPN에서보고되었다. 이돌연변이는 JAK2 V617F 음성 PMF의약 7-25%, ET의 1-24% 에서관찰되고 [7-15], 돌연변이양성군은음성군에비해낮은혈색소와총백혈구수, 높은혈소판수를보인다 [13-16]. 한국에서 돌연변이에관한보고는, 2010년 Kim 등이 59명의 ET 환자와 4명의진성혈소판증가증후골수섬유증 (post- ET myelofibrosis) 환자 63명을대상으로 2명 (3%) 의 돌연변이 (L 1명, K 1명 ) 를보고하였을뿐, 현재까지 ET 이외의다른 MPN에서돌연변이의빈도분석이나연관된임상양상및검사소견에대한보고는없다 [9]. 본연구에서는 classic MPN 154명에대한 돌연변이의빈도및그에따른임상양상및검사소견을분석하였다. 재료및방법 1. 대상 2007년 3월부터 2013년 7월까지 MPN으로진단받은환자중, DNA분석이가능했던 154명의골수검체를이용하여검사를시행하였다. 대상환자는남자 80명, 여자 74명으로중간연령은 67 (22-86) 세였다. MPN의진단은 2008년 WHO 진단기준에따라재검토하였고 [17], ET 106명, PMF 17명, PV 31명으로구성되었다. 본연구는본원연구윤리심의위원회의승인을받은후진행되었다. 2. 돌연변이와 JAK2 V617F 의분석 돌연변이검출은실시간중합효소연쇄반응법으로, Real-Q L/K screening Kit (Biosewoom, Seoul, Korea) 을이용하여제조사의지침에따라진행하였고, MPL 10번엑손의직접염기서열분석법으로다시한번확인하였다. JAK2 V617F은지침 (Biosewoom) 에따라, 대립형질특이중합효소연쇄반응법으로검출하였다. 유화정도 ) 는 Mann-Whitney test를사용하여검정하였다. 모든분석은 SPSS v20.0 (SPSS Inc., Chicago, IL, USA) 를사용하여시행하였고, 양측검정상 P값이 0.05 미만일때통계학적으로유의하다고판단하였다. 결과 1. 돌연변이의빈도분석및 JAK2 V617F와의관계 실시간중합효소연쇄반응으로분석한결과, 총 4명의환자에서 L 변이가검출되었고, 직접염기서열분석으로확인한결과모두같은 L가관찰되었다 (Fig. 1). 실시간중합효소연쇄반응으로 돌연변이가확인되지않은나머지 150개의검체를직접염기서열분석으로다시확인한결과, 1명의 MPL W515A 환자가추가로발견되었다 (Fig. 1). 이 5명의환자는모두 ET 환자였고, JAK2 V617F 음성이었다. 따라서본연구결과, 전체 MPN 환자에서의 돌연변이의빈도는약 3.2% (5/154), ET환자에서는약 4.7% (5/106) 로나타났으며, JAK2 V617F 음성 ET환자에서는 12.5% (5/40) 로나타났다. PMF와 PV환자에서 MPL W515 돌연변이는관찰되지않았고, JAK2 V617F와 돌연변이가동반된경우는없었다. 2. ET 환자에서 돌연변이와임상양상및검사소견과의연관성 106명의 ET 환자를 양성군 (MPL + ) 과 음성 Normal 3. 임상양상및검사소견의분석진단당시의임상양상 ( 성별, 나이, 장기비대증의유무 ) 과검사소견 ( 혈색소, 백혈구수, 혈소판수, 핵형분석결과, 골수섬유화정도 ) 은의무기록을통하여조사하였다. 진단이후추적관찰기간동안발생한혈전색전질환의유무를의무기록을통하여조사하였다. 4. 통계분석 돌연변이와범주변수 ( 핵형분석결과, 장기비대증의유무, 혈전색전질환의유무, 성별 ) 는 Fisher s exact test를통해비교하였고, 연속변수 ( 나이, 총백혈구수, 혈색소, 혈소판수, 골수섬 c. 1544G>T (p.w515l) c.1543_1544tg>gc (p.w515a) Fig. 1. Electropherograms of normal control (upper), L mutation (middle), and A mutation (lower). 2

3 Table 1. Comparison of laboratory and clinical features between patientswith positive ET and negative ET positive JAK2 V617F positive negative JAK2 V617F negative Number (%) 5 (4.7) 66 (62.3) 35 (33.0) 101 (95.3) Total positive vs. MPL W515 negative P value positive vs. JAK2 V617F positive positive vs. JAK2 V617F negative Men:Women 4:1 33:33 15:20 48: Median age, yr (range) 69 (62-72) 68 (22-86) 63 (27-84) 67 (22-86) Median WBC count, 10 9 /L (range) 8.1 ( ) 13.3 ( ) 9.9 ( ) 11.7 ( ) Median Hb, g/dl (range) 11.1 ( ) 13.9 ( ) 12.9 ( ) 13.5 ( ) Median platelet, 10 9 /L (range) 1,187.0 ( ) 1,038.0 ( ) 1,086.0 ( ) 1,071.0 ( ) Organomegaly (%) 2/2 (100) 21/30 (70) 6/11 (54.5) 27/41 (65.9) Thromboembolic event (%) 2/5 (40) 18/58 (31) 6/32 (18.8) 24/90 (26.7) Median fibrosis grade (range) 0 (0-3) 1 (0-3) 1 (0-3) 1 (0-3) Abnormal karyotype (%) 0/5 (0) 2/52 (3.8) 4/30 (13.3) 6/82 (7.3) Abrreviations: MPL, myeloproliferative leukemia; JAK2, Janus kinase 2; WBC, white blood cell; Hb, hemoglobin. 군 (MPL - ) 으로나누고,MPL- 환자군은다시 JAK2 V617F 양성군 (MPL - JAK2 + ) 과 JAK2 V617F 음성군 (MPL - JAK2 - ) 으로나누어분석하였다. MPL+ ET 환자는 MPL- 환자에비해총백혈구수 {8.1 ( ) 10 9 /L vs ( ) 10 9 /L, P = 0.015)}, 혈색소 {11.1 ( ) g/dl vs ( ) g/dl, P = 0.019} 가유의하게낮았고, MPL - JAK2 + 환자군에비해서도총백혈구수 {8.1( ) 10 9 /L vs. 9.9 ( ) 10 9 /L, P = 0.002)} 와혈색소 {11.1( ) g/dl vs ( ) g/dl, P = 0.010} 가유의하게낮은결과를보였다 (Table 1). 그러나 MPL + 환자군과 MPL - JAK2 - 환자군사이에는통계학적으로유의한차이를보이지않았다. 성별, 나이, 혈소판, 장기비대증의유무, 혈전색전질환의유무, 골수섬유화정도, 비정상핵형분석의빈도에서는환자군간유의한차이를보이지않았다 (Table 1). 고찰 MPL은트롬보포이에틴신호전달에관여하는세포막수용체로거대핵세포분화와혈소판증식의주요조절인자및조혈모세포의재생, 증식에관여한다. MPL 막통과-세포질접합부 ( 엑손 10) 에위치한 5개의양친매성아미노산모티브 (KWQFP) 는 MPL의자발적활성화를억제하는데중요한역할을한다. 돌연변이는이아미노산모티브내위치한 515번째아미노산트립토판이다른아미노산으로치환되는기능획득돌연변이이다 [8, 11-13]. 변이는주로 MPN 중 PMF와 ET에서보고되어있다. JAK2 V617F 음성 PMF의약 7-25%, ET의 1-24% 에서검출되며 [7-10, 12-15], PV에서는현재까지단 2개의증례만이보고된상태이다 [18]. 본연구에서도 JAK2 V617F 음성 ET의약 12.5% 에서 MPL W515 돌연변이가관찰되어앞선보고와유사한결과를보였으나, PMF에서는변이가관찰되지않았다. JAK2 V617F와 돌연변이가동시에존재하는경우는전체 양성환자의최고 30% 까지다양하게보고되고있으나, 본연구에서는 JAK2 V617F와 가동시에존재하는경우또한발견되지않아앞선보고와차이를보였다 [7, 12-16]. 이러한차이는낮은빈도로관찰되는 돌연변이로인한적은연구대상의수에서기인함으로볼수있다. MPN에서 돌연변이의아형은지금까지총 5가지가알려져있고, 트립토판이류신으로치환되는 W515L [8], 라이신 (lysine) 으로치환되는 W515K [8], 아스파라긴 (asparagines) 으로치환되는 W515R [10], 알라닌 (alanine) 으로치환되는 W515A [11], 세린 (serine) 으로치환되는 W515S [15] 이다. 그중 W515L과 W515K가가장흔하며, ET에서각각 55%, 21%, PMF에서각각 93%, 7% 로 W515L이더흔히관찰된다 [15, 19-21]. 본연구에서는총 101명의 ET환자에서 L 4명이관찰되고 K는관찰되지않아 L이가장흔하다는앞선보고들과유사한결과를나타내었다. 본연구에서는 A 1예가관찰되었는데, 이돌연변이는 2008년 Chaligne 등에의해최초로보고되었고, PMF 와 ET 모두에서 1% 미만에서매우드물게나타난다고알려져있다 [10-12, 15]. 본연구에서확인된 A 환자는 62세남자환자로, 골수생검결과정상세포충실성을보이며거대핵세포의개수가증가한전형적인 ET 소견을보였으며, JAK2 V617F는동반되지않았다. 돌연변이와임상양상의연관성에대한몇몇보고에따르면, ET의경우, 돌연변이를가질경우 JAK2 V617F 양성에비해혈색소와총백혈구수가낮고혈소판수가높았다 [

4 15]. 골수소견상으로는세포충실도가비교적낮고적혈구계열의혈구수가적었지만, 거대핵세포수는비교적많았다 [13-15]. 본연구에서도 돌연변이양성 ET 환자군은음성환자군에비해혈색소가낮고, 총백혈구수도낮아, 이전보고들과유사한결과를나타내었다 [15]. 그러나, 혈소판수에는유의한차이가없었고, 나이, 성별, 장기비대증, 골수섬유화의정도, 혈전색전증의유무, 비정상핵형의유무등은 돌연변이의유무와큰연관성이없음을알수있었다. 이러한결과는 돌연변이가있을경우, 적혈구계열이나백혈구계열의혈구수보다는거대핵세포의증식에조금더특이적이라는사실을뒷받침한다 [13, 14, 22]. 본연구에서는 L과 W515K 검출에특이적인실시간중합효소연쇄반응키트를사용하여분석하여총 4명의환자에서검출하였고, 직접염기서열분석법으로확인한결과일치하는소견을보였다. 일반적으로실시간중합효소연쇄반응법이더높은민감도를가져 L/K 검출에는더유리하지만, W515L/K 이외의소수의아형들을확인하기위해서는추가적인직접염기서열분석법이필요할것으로생각된다. 본연구는몇가지한계점을가진다. 첫번째로대상이되는환자군, 특히 PMF 질환군의숫자가적었다. 따라서 PMF 질환군에서앞선보고와는다르게 돌연변이가발견되지않았다. 둘째, 돌연변이의유전자변이량에대한연구가이루어지지않았고, 추적관찰이비교적짧아, 다양한인자에대한예후관련분석이이루어지지못하였다. 이러한한계점에도불구하고, 본연구는한국인에서 돌연변이가 JAK2 V617F 음성 ET의진단에중요한표지자가됨을보여주고있으며, 앞서다른보고들과같이, 돌연변이를가진경우낮은혈색소와총백혈구수를보인다는사실을확인할수있었다. 더많은수의 PMF, PV 환자군에서 돌연변이빈도분석과임상양상및검사소견과의연관성에대한추가적인연구가이루어져야하겠고, 장기적추적관찰을통해, 급성백혈병이나이차골수섬유화로의진행등의예후관련분석이이루어져야하겠다. 요약 배경 : 최근 myeloproliferative leukemia (MPL) W515 돌연변이가골수증식종양 (MPN) 에서발견되었고, 새로운분자유전학적표지자로제시되었다. 본연구에서는 돌연변이빈도및임상혈액학적지표들과의상관성을알아보고자하였다. 방법 : 진성적혈구증가증 (PV) 31명, 일차골수섬유증 (PMF) 17명, 진성고혈소판증 (ET) 106명을포함한총 154명의 MPN 환자를대상으로 돌연변이를실시간중합효소연쇄반응으로검출하 였고, 직접염기서열분석을통해재확인하였다. 결과 : 전체 154명의환자중 5명 (3.2%) 의환자에서 돌연변이가확인되었다. 이들은모두 ET환자였고, JAK2 V617F 음성으로, JAK2 V617F 음성 ET환자의 12.5% 를차지하였다. 돌연변이양성 ET 환자들은 음성이나 JAK2 V617F 양성 ET 환자에비해총백혈구수와혈색소가유의하게낮았다. 결론 : 돌연변이는 JAK2 V617F 음성 MPN 환자의진단에중요한표지자로이용할수있으며, 심한빈혈양상이나낮은백혈구수등특징적인임상혈액학적소견을가져 JAK2 V617F 음성 ET 및다른 MPN 병태생리의이해에도움이될것이다. REFERENCES 1. Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 2009;114: Jones AV, Kreil S, Zoi K, Waghorn K, Curtis C, Zhang L, et al. Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood 2005;106: Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005;365: Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005;352: James C, Ugo V, Le Couédic JP, Staerk J, Delhommeau F, Lacout C, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005;434: Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005;7: Pardanani AD, Levine RL, Lasho T, Pikman Y, Mesa RA, Wadleigh M, et al. MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients. Blood 2006;108: Pikman Y, Lee BH, Mercher T, McDowell E, Ebert BL, Gozo M, et al. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Med 2006;3:e Kim HJ, Jang JH, Yoo EH, Kim HJ, Ki CS, Kim JW, et al. JAK2 V617F and L/K mutations in Korean patients with essential thrombocythemia. Korean J Lab Med 2010;30:

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<4D F736F F F696E74202D203920B9E6BCF6B9CC28B0F8B0B3292E BC8A3C8AF20B8F0B5E55D> MPD MPN in WHO 2008 Chronic myelogenous leukemia, BCR-ABL1 positive Chronic neutrophilic leukemia Polycythemia vera Primary myelofibrosis Essential thrombocythemia Chronic eosinophilic leukemia, not otherwise