원저 Lab Med Online Vol. 7, No. 3: , July 진단혈액학 HLA-DR 과 CD34 음성인급성골수성백혈병의빈도및특징 : 전형적급성골수성백혈병과급성전골수

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1 원저 Lab Med Online Vol. 7, No. 3: , July 2017 진단혈액학 HLA-DR 과 CD34 음성인급성골수성백혈병의빈도및특징 : 전형적급성골수성백혈병과급성전골수구백혈병사이의중간적특질 Frequency and Distinct Characteristics of Acute Myeloid Leukemia Lacking HLA-DR and CD34 Expression: Features Intermediate between Typical Acute Myeloid Leukemia and Acute Promyelocytic Leukemia 이혜영 조영욱 유은경 장성수 서을주 박찬정 Hye-Young Lee, M.D., Young-Uk Cho, M.D., Eunkyoung You, M.D., Seongsoo Jang, M.D., Eul-Ju Seo, M.D., Chan-Jeoung Park, M.D. 울산의대서울아산병원진단검사의학과 Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea Background: The objective of this study was to investigate the frequency and characteristics of HLA-DR - /CD34 - acute myeloid leukemia (AML) also known as acute promyelocytic leukemia (APL)-like AML. Methods: This study included 683 newly diagnosed patients with AML. After exclusion of 211 patients with recurrent genetic abnormalities, one with acute panmyelosis with myelofibrosis, two with myeloid leukemia associated with Down syndrome, and two devoid of metaphase cells, we classified the remaining 467 patients as follows: group 1, HLA-DR + /CD34 + (typical AML); group 2, HLA-DR + /CD34 - or HLA-DR - /CD34 + ; group 3, APL-like AML. Results: Group 1 comprised 294 patients, group 2 comprised 133, and group 3 comprised 40. Therefore, the frequency of APL-like AML among 683 unselected patients with AML was 5.9%. Group 3 patients had significantly higher leukocyte counts and bone marrow (BM) blast percentages, higher frequencies of normal karyotypes and NPM1 mutation, higher fractions of CD33-positive cells, higher concentrations of fibrin degradation products and D-dimers, lower frequencies of complex karyotypes, monosomal karyotypes and poor cytogenetic risk, lower fractions of CD13-positive cells, and lower fibrinogen concentrations, compared with group 1 patients. The values of the BM blast percentage, number of CD33-positive cells, and DIC score of the patients with APL-like AML were intermediate between those of the patients with typical AML and APL. Conclusions: This study demonstrates that APL-like AML is not uncommon, and it has characteristics distinguishable from those of typical AML. APL-like AML may have some pathophysiological relationships with APL, which need further investigation. Key Words: Acute promyelocytic leukemia, Acute myeloid leukemia, HLA-DR, CD34, Immunophenotype, DIC, NPM1 mutation 서론 Corresponding author: Young-Uk Cho Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea Tel: , Fax: , yucho@amc.seoul.kr Received: August 22, 2016 Revision received: December 24, 2016 Accepted: January 4, 2017 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. 급성전골수구백혈병 (acute promyelocytic leukemia, APL) 의전형적인면역표현형은 HLA-DR과 CD34의소실, 균질하면서도강한 CD33의발현, 그리고 CD33에비해상대적으로약하고불균질한 CD13의발현으로요약된다 [1]. APL은파종혈관내응고 (disseminated intravascular coagulation, DIC) 를잘동반하므로, 신속한진단을통해치명적인출혈합병증을예방하여초기사망률을낮추는것이중요하다 [2]. 다변량유세포분석을이용한면역표현형검사는검사소요시간이짧고, 폭넓게보급되어있으며, APL의특징적인소견이이미확립되어있어 APL의예비진단에충분히활용될수있다. 그러나 APL의대표적면역표현형인 HLA-DR과 CD34의 eissn

2 소실은 PML-RARA 유전자재배열을보이지않는 AML에서도관찰될수있음이알려져있다 [1-7]. 대부분의이전연구는 HLA-DR 음성인 AML을대상으로하였다. 지금까지알려진 HLA-DR 음성이면서 APL이아닌 AML의특징으로는높은 CXCR-4 발현율, 컵모양의핵, CD34 및 CD2의소실, 증가된백혈구와혈소판수, 그리고 FLT3-ITD와 NPM1 돌연변이와의연관성등이있다 [3-7]. 그러나 HLA-DR - /CD34 - AML 환자군만을분석한보고는하나밖에없었다 [8]. 이연구에따르면 HLA- DR - /CD34 - 이면서 NPM1 돌연변이양성인 AML이 APL과구분되는특징적인면역표현형을보였다고하였다. 그러나대상환자수가적어전체 AML 환자군에서 APL이아니면서도 HLA-DR - /CD34 - 인 AML의정확한빈도를파악하기어렵고, 면역표현형외다른특징에대한기술이없었다는제한점이있다. 본연구에서는 HLA-DR과 CD34를모두발현하지않으면서 PML- RARA 재배열또는 t(15;17)(q22;q12) 를보이지않는 AML (APL-유사 AML) 의빈도와임상적그리고병리학적특성을분석하고자하였다. 더나아가 APL과 HLA-DR 및 CD34 모두양성인전형적 AML 환자군과의특성을비교함으로써 APL-유사 AML이두아형사이의연속선상에있는중간적특질을띠는지여부도조사하여병태생리학적연관성을유추해보고자하였다. 대상및방법 1. 대상 2011년 1월부터 2015년 12월까지서울아산병원에서새로진단받은 683명의 AML 환자를대상으로하였다. 대상환자중남자는 387명 (56.7%), 여자는 296명 (43.3%) 이었고, 연령의중앙값은 54세였다. AML의진단은 2008년 WHO 진단기준에의거하였다. 전체 AML 환자군을대상으로 APL-유사 AML 환자군의빈도를계산하였다. 그러나 HLA-DR과 CD34의발현양상에따른환자군분류와각군간의특성비교에는반복유전자이상 AML (N =211), 골수섬유증동반급성범골수증 (N =1), 다운증후군관련골수성백혈병 (N =2) 환자는제외하였다. 왜냐하면각아형마다그자체만으로고유한특성을가지므로 APL-유사 AML 환자군의특징을도출하는데바이어스로작용할수있기때문이다. 또한중기분열세포를얻지못해핵형분석을실시하지못한 2명의환자도비교분석에서제외하였다. 결과적으로총 467명의환자를 HLA-DR과 CD34의발현양상에따라다음과같이세군으로분류하였다 : 제1군, HLA- DR + /CD34 + ( 전형적 AML); 제2군, HLA-DR + /CD34 - 또는 HLA-DR - / CD34 + ; 제3군, HLA-DR - /CD34 - (APL-유사 AML). 환자군의인구학적특성, 임상적및검사실적소견, 면역표현형, 세포유전학적소견그리고돌연변이결과는의무기록을통해후향적으로조사하였다. 본검사실의 AML에대한진단적접근은다음과같다. 면역표현형검사, 다중역전사효소 PCR, 핵형검사, FLT3-ITD는모든환자에서기본적으로시행하였다. 그러나 NPM1, CEBPA, 그리고 MLL-PTD 와같은돌연변이검사는다중역전사효소 PCR에서반복유전자이상 AML에해당하는융합유전자가검출되지않는경우에만시행하였다. 본검사실에서 CEBPA 돌연변이검사는 2012년 5월부터, MLL-PTD는 2013년 9월부터시작하였다. 본연구는본원임상연구심의위원회의승인 ( ) 을받은후진행되었다. 2. 면역표현형검사면역표현형분석은주로진단당시적혈구를용해시킨골수검체를이용하였다. 세포의수는각시험관마다 개로조정하였다. 백혈병세포의항원발현은다변량유세포분석기인 FACSCanto II (Becton Dickinson Biosciences, San Diego, CA, USA) 를이용하여분석하였다. 사용된형광색소는 flurorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin-chlorophyll protein (PerCP), allophycocyanin (APC) 이었다. 형광색소에접합된단클론항체들의조합 (FITC/PE/PerCP/APC) 은다음과같다 : CD14/CD33/CD41/ CD45, CD19/CD13/CD7/CD45, CD56/CD117/CD34/CD45, CD65/ CD10/CD3/CD45, CD15/CD2/-/CD45, HLA-DR/-/-/CD45, -/MPO/-/ CD45, TdT/cyCD22/cyCD3/CD45. 각시험관마다최소한 50,000개의형광발현 (event) 을측정하였으며, CD45와 side scatter cytogram 패턴에따라백혈병세포군을지정 (gating) 하여분석하였다. 양성기준은 20% 로설정하였으며, TdT의경우 10% 이상을양성으로판정하였다. 3. 분자유전및세포유전검사 RUNX1-RUNX1T1, CBFB-MYH11, PML-RARA, MLLT3-MLL, 그리고 BCR-ABL1과같은반복유전자재배열은다중역전사효소 PCR (HemaVision multiplex RT-PCR kit, DNA Diagnostic, Risskov, Denmark) 을통해선별하였다. FLT3-ITD, NPM1 돌연변이, CEBPA 돌연변이, MLL-PTD는기존방법을사용하여검출하였다 [9-12]. 세포유전검사는표준 G-분열법을사용하였고, 염색체이상은분열중기세포에서관찰하였다. 세포유전학적이상에따른위험군은 National Comprehensive Cancer Network (NCCN) 체계에따라분류하였다 [13]. 이에따라복잡핵형 (complex karyotype) 은클론성의염색체이상이 3개이상관찰되는경우로정의하였다. 단일염색체핵형 (monosomal karyotype) 은상염색체의단일염색체 (monosomy) 가 2개이상관찰되거나 1개의단일염색체가최소한 1개이상의구조적이상과동반될때로정의하였다 [14]

3 4. 통계분석각환자군간연속변수는 Wilcoxon rank-sum test로, 범주변수는 χ 2 test를이용하여비교하였다. 환자군비교는각각 1군 vs. 2군, 1군 vs. 3군, 2군 vs. 3군, 1군 vs. APL군, 2군 vs. APL군, 3군 vs. APL 군으로나누어시행하였다. 특정변수의각환자군간빈도의경향성은 χ 2 test for trends를이용하여검정하였다. 통계프로그램은 MedCalc program version (MedCalc Software, Acacialaan, Belgium) 을이용하였다. P <0.05를통계적으로의미가있다고해석하였다. 결과 1. APL-유사 AML의빈도및특성 HLA-DR과 CD34의발현패턴에따라제1군은 294명, 제2군은 133명, 그리고제3군은 40명으로분류되었다. 따라서 APL-유사 AML 환자군 ( 제3군 ) 의빈도는 683명의선별되지않은전체환자군에서는 5.9% (40/683), 주로반복유전자이상 AML을제외한 467명에서는 8.6% (40/467) 이었다. 반복유전자이상 AML 환자들중 APL 은 55명이었다. Table 1. Comparison of demographics, and clinicopathological features among the patient groups Group 1* (N=294) Group 2 (N=133) Group 3 (N=40) APL (N=55) P value 1 v 2 1 v 3 2 v 3 1 v APL 2 v APL 3 v APL Age, yr (range) 56.5 (3-83) 57 (0.1 90) 59 (1 80) 46 (7 74) < Pediatric age (%) 16 (5.4) 11 (8.3) 3 (7.5) 2 (3.6) Male sex (%) 184 (62.6) 72 (54.1) 23 (57.5) 22 (40.0) WBC count, 10 9 /L (range) 4.45 ( ) 13.2 ( ) 17.8 ( ) 5.1 ( ) Hemoglobin level, g/dl (range) 8.8 ( ) 8.5 ( ) 8.75 ( ) 8.5 ( ) Platelet count, 10 9 /L (range) 55.5 (3 1019) 75 (4 796) 58.5 (11 312) 40 (7 135) BM blast, % (range) 49.6 ( ) 53.2 ( ) 73.1 ( ) 83.6 ( ) < < FAB subtype (%) < M0 19 (6.5) 3 (2.3) 2 (5.0) M1 89 (30.3) 29 (21.8) 20 (50.0) M2 146 (49.7) 43 (32.3) 12 (30.0) M4 17 (5.8) 32 (24.1) 3 (7.5) M5 3 (1.0) 14 (10.5) 1 (2.5) M6 13 (4.4) 7 (5.3) 2 (5.0) M7 7 (2.4) 5 (3.8) 0 WHO subtype (%) < AML, NOS 145 (49.3) 48 (36.1) 30 (75.0) AML with MRC 127 (43.2) 83 (62.4) 8 (20.0) taml 22 (7.5) 2 (1.5) 2 (5.0) Cytogenetics (%) NK 102/289 (35.3) 63/130 (48.5) 26/37 (70.3) Complex 71/289 (24.6) 17/130 (13.1) 4/37 (10.8) MK 63/289 (21.8) 14/130 (10.8) 3/37 (8.1) Cytogenetic risk Poor 98/289 (33.9) 22/130 (16.9) 4/37 (10.8) Intermediate 191/289 (66.1) 108/130 (83.1) 33/37 (89.2) Mutations (%) FLT3-ITD 44/291 (15.1) 29/132 (22.0) 9/39 (23.1) 12 (21.8) NPM1 13/274 (4.7) 42/126 (33.3) 28/37 (75.7) < < < CEBPA ll 17/179 (9.5) 0/80 0/ NA MLL-PTD 8/124 (6.5) 6/66 (9.1) 0/ All continuous variables are expressed as a median (with range). *Group 1 consisted of patients with HLA-DR + /CD34 + (typical AML); group 2 consisted of those with either HLA-DR + /CD34 - or HLA-DR - /CD34 + ; group 3 consisted of those with HLA-DR - /CD34 - (APL-like AML); We excluded eleven cases with suboptimal metaphase (<20 metaphase with no cytogenetic abnormalities) from the analysis; ll Only biallelic CEBPA mutations were calculated. Abbreviations: APL, acute promyelocytic leukemia; v, versus; WBC, white blood cell; BM, bone marrow; FAB, French-American-British; NOS, not otherwise specified; MRC, myelodysplasia-related changes; taml, therapy-related acute myeloid leukemia; NK, normal karyotype; MK, monosomal karyotype; ITD, internal tandem duplication; NA, not available; PTD, partial tandem duplication

4 환자의나이와성별은 3개의환자군간유의한차이가없었다. 그러나 APL 환자군이다른군보다낮은연령을보였다 (1군 vs. APL 군, P<0.0001). 말초혈액의백혈구수와골수의모세포수는제3군에서다른환자군보다유의하게높았다 (1군 vs. 3군, 각각 P = 과 P = ). FAB 분류상제3군에서상대적으로 M1의비율이높았고 (2군 vs. 3군, P = 0.008), 제2군의경우 M4와 M5의비율이높았다 (1군 vs. 2군, P <0.001). WHO 분류상제3군에서는상세불명 AML의비율이 (2군 vs. 3군, P <0.0001), 제2군에서는골수형성이상관련 AML의비율이유의하게높았다 (1군 vs. 2군, P = ). 세포유전학적이상측면에서는제3군에서정상핵형의비율이유의하게높았고, 복잡핵형과단일염색체핵형의비율이유의하게낮았다 (1군 vs. 3군, P = ). 이와같은소견이반영되어세포유전학적예후불량군의비율은제3군에서유의하게낮았다 (1군 vs. 3군, P = 0.008). NPM1 돌연변이는제3군에서유의하게높은빈도로검출되었다 (1군 vs. 3군, P <0.0001). 이와는반대로 CEBPA 돌연변이는제1군에서만검출되었고, 제2군과 3군에서는검출되지않았다 (1군 vs. 2군, P = 0.01). 각환자군간 FLT3-ITD와 MLL-PTD의분포는유의한차이를보이지않았다 (Table 1). 2. APL-유사 AML의면역표현형적특성및섬유소용해표지자와의상관관계 CD117 (1군 vs. 3군, P<0.0001; 2군 vs. APL군, P<0.0001) 과 CD13 (1군 vs. 2군, P = 0.014; 2군 vs. APL군, P = 0.037) 은제1군과 APL군에서의양성률이유의하게높았다. CD33의양성률은환자군간 유의한차이가없었으나, CD33 양성세포의비율은제3군과 APL군에서유의하게높았다 (1군 vs. 3군, P<0.0001; 1군 vs. APL군, P<0.0001). 반면 CD13 양성세포의비율은제1군과 APL군에서유의하게높았다 (1군 vs. 3군, P <0.0001; 2군 vs. APL군, P <0.0001). 이러한소견이반영되어 CD13 양성세포의비율에대한 CD33 양성세포의비율의비 (ratio) 는제1군에서가장낮았고, 제3군에서가장높았다 (1 군 vs. 2군, P <0.0001; 1군 vs. 3군, P <0.0001). CD65의양성률은제2군과 APL군에서유의하게높았고 (1군 vs. 2군, P <0.0001; 1군 vs. APL군, P <0.0001), CD15 의양성률은제2군에서유의하게높았다 (1군 vs. 2군, P = ). 세포표면항원의계열교차성발현 (cross-lineage antigen expression) 양상은 CD7이제1군에서유의하게높은빈도를보였다 (1군 vs. 2군, P=0.0003; 1군 vs. 3군, P=0.003). CD2의경우 APL군에서다른환자군에비해유의하게높은빈도를보였다 (1 군 vs. APL군, P<0.0001; 3군 vs. APL군, P =0.0001). CD56 과 TdT는환자군에따른유의한차이를보이지않았다 (Table 2). 섬유소원의농도는제3군 (1군 vs. 3군, P = 0.009; 2군 vs. 3군, P = 0.012) 과 APL군 (1군 vs. APL군, P<0.0001; 2군 vs. APL군, P<0.0001) 에서유의하게낮았다. 반면섬유소 ( 원 ) 분해산물 (fibrin/fibrinogen degradation products, FDP) 의농도는제3군 (1군 vs. 3군, P<0.0001; 2군 vs. 3군, P <0.0001) 과 APL군 (1군 vs. APL군, P <0.0001; 2군 vs. APL군, P <0.0001) 에서유의하게상승하였다. 이러한유의성을 D- dimer에서도동일하게관찰되었다. 이와같은소견이반영되어세계혈전지혈학회 (International Society on Thrombosis and Hemostasis, ISTH) 의 DIC 점수 [15] 가 5점이상의빈도는제1군 (1군 vs. 2 Table 2. Comparison of immunophenotypic characteristics among the patient groups Group 1* (N=294) Group 2 (N=133) Group 3 (N=40) APL (N=55) P value 1 v 2 1 v 3 2 v 3 1 v APL 2 v APL 3 v APL CD117, N (%) 283 (96.3) 79 (59.4) 31 (77.5) 53 (96.4) < < CD33, N (%) 277 (94.2) 130 (97.7) 39 (97.5) 55 (100.0) CD13, N (%) 284 (96.6) 120 (90.2) 37 (92.5) 55 (100.0) CD33, % (range) ( ) 91.8 ( ) ( ) 98.6 ( ) < < < < CD13, % (range) 81.7 ( ) 69.7 ( ) 56.8 ( ) 93.5 ( ) < < < < < CD33%/CD13%, ratio (range) 1.0 (0-50.7) 1.2 ( ) 1.7 ( ) 1.0 ( ) < < < < CD65, N (%) 118 (40.1) 85 (63.9) 14 (35.0) 42 (76.4) < < CD15, N (%) 158 (53.7) 98 (73.7) 18 (45.0) 27 (49.1) CLE ll CD56, N (%) 40 (13.6) 23 (17.3) 11 (27.5) 8 (14.5) CD2, N (%) 20 (6.8) 5 (3.8) 1 (2.5) 22 (40.0) < < CD7, N (%) 73 (24.8) 12 (9.0) 1 (2.5) 4 (7.3) TdT, N (%) 15 (5.1) 1 (0.8) NA Others, N (%) 8 (2.7) 6 (4.5) 1 (2.5) All continuous variables are expressed as a median (with range). *Group 1 consisted of patients with HLA-DR + /CD34 + (typical AML); group 2 consisted of those with either HLA-DR + /CD34 - or HLA-DR - /CD34 + ; group 3 consisted of those with HLA-DR - /CD34 - (APL-like AML); The percentage (%) indicates the proportion of CD33 or CD13-positive cells among the total number of cells tested; ll The distributions of cross-lineage antigen expression may overlap with each other. For example, the patient expressing both CD56 and CD7 was categorized as CD56 and CD7. Abbreviations: APL, acute promyelocytic leukemia; v, versus; N, number of a positive case; CLE, cross-lineage expression; NA, not available

5 Table 3. Comparison of concentration of markers for fibrin formation and fibrinolysis among the patient groups Group 1* (N=294) Group 2 (N=133) Group 3 (N=40) APL (N=55) P value 1 v 2 1 v 3 2 v 3 1 v APL 2 v APL 3 v APL Fibrinogen (mg/dl) 350 (54-845) 367 (28 907) 308 (52 771) (5 384) < < < FDP (μg/ml) ll 5.1 (0 87.6) 6.3 ( ) ( ) ( ) < < < < D-dimer (μg/ml FEU) 1.14 ( ) ( ) ( ) ( ) < < < < DIC score 5, N (%)** 23 (12.3) 21 (23.6) 17 (51.5) 38 (70.4) < < < All continuous variables are expressed as a median (with range). *Group 1 consisted of patients with HLA-DR + /CD34 + (typical AML); group 2 consisted of those with either HLA-DR + /CD34 - or HLA-DR - /CD34 + ; group 3 consisted of those with HLA-DR - /CD34 - (APL-like AML); Test results were available for 190 patients of group 1, 89 of group 2, 34 of group 3, and 54 of APL; ll Test results were available for 167 patients of group 1, 83 of group 2, 30 of group 3, and 52 of APL; Test results were available for 197 patients of group 1, 98 of group 2, 33 of group 3, and 54 of APL; **Scoring results were available for 187 patients of group 1, 89 of group 2, 33 of group 3, and 54 of APL. Abbreviations: APL, acute promyelocytic leukemia; v, versus; FDP, fibrin/fibrinogen degradation products; FEU, fibrinogen-equivalent units; DIC, disseminated intravascular coagulation. (%) BM blast 76.0% CD33-positive cell 91.4% DIC score 5 군과 APL 사이의중간적특질을나타내었다. 예를들면골수에서의모세포백분율, CD33 양성세포비율, 섬유소원, FDP, D-이합체의농도등이제1군에서 APL로갈수록일정한방향성을유지하며유의하게증가하거나감소하는양상을보였다 (Tables 1-3). 이를도식화한 Fig. 1에서는골수에서의모세포백분율, CD33 양성세포비율, 그리고 ISTH 체계의 DIC 점수모두전형적 AML인제1군에서가장낮았고, APL에서가장높았으며, APL-유사 AML은그두군사이의중간값을가지는양상을확인할수있다. 이러한양상은통계적으로유의하였다 (3개의변수모두 χ 2 test for trends에의한 P <0.001). Fig. 1. The distribution and frequency of cases with high bone marrow blasts, CD33-positive cells, and DIC score. Patients with APL-like AML (group 3) showed an intermediate feature between HLA-DR + /CD34 + (group 1) and true APL cases. The cut-off values for bone marrow blasts (76.0%) and CD33-positive cells (91.4%) were derived from values corresponding to the 75 percentile of patients with group 1. The cut-off value for DIC score (5) was based on the ISTH consensus proposal. The frequencies of BM blast 76.0% were 25.2% in group 1, 22.6% in group 2, 45.0% in group 3, and 69.1% in APL. The frequencies of CD33-positive cell 91.4% were 25.2% in group 1, 52.6% in group 2, 70.0% in group 3, and 96.3% in APL. The frequencies of ISTH DIC score 5 were 12.3% in group 1, 23.6% in group 2, 51.5% in group 3, and 70.4% in APL. All of these three variables (BM blast, CD33-positive cell, DIC score) were significant (each, P<0.001) by Chi-square test for trends. 군, P = 0.026; 1 군 vs. 3 군, P <0.0001) 에서가장낮았고, APL 군에서 가장높았다 (1 군 vs. APL 군, P <0.0001; 2 군 vs. APL 군, P <0.0001; Table 3) Group 1 Group 2 Group 3 APL 3. APL- 유사 AML 의중간적특질 APL- 유사 AML 의일부지표들은 HLA-DR + /CD34 + ( 제 1 군 ) 환자 고찰 HLA-DR과 CD34는조혈전구세포 (hematopoietic progenitor) 에서발현되는대표적인세포표면항원이다. 특히골수모구 (myeloblast) 는 CD34와 HLA-DR을비롯하여 CD117, CD38, CD13, 그리고 CD33을발현하며, CD34는초기골수모세포에서부터발현된다. 호중구로의성숙이진행될수록일부항원은소실되고, 일부항원은획득된다. 전골수구단계에서 HLA-DR과 CD34가소실되고, 골수구단계에서 CD117이소실된다. 반면 CD11b 와 CD11c 는골수구단계에서획득된다 [15]. 그러므로 HLA-DR과 CD34의소실은 APL 을강하게시사하는면역표현형소견으로간주된다. 하지만다수의이전연구들을통해 HLA-DR - /CD34 - AML이반드시 APL을의미하는것이아님이알려졌고, 그빈도는대상환자군에따라매우다양하였다 [3-7]. 그러나대부분의이전보고들은 HLA-DR 음성 AML을대상으로하여 HLA-DR - /CD34 - AML만의빈도와특성을도출하기어려웠다. 단, 최근이루어진한연구에따르면 HLA-DR - / CD34 - NPM1 돌연변이양성 AML은 APL에비해 CD2 발현이없고, CD33의발현강도가유의하게낮고, CD110 발현율이유의하게높았다 [8]. 하지만이연구는분석대상자의수가적었고 (40명의 APL 과 12명의 NPM1 돌연변이양성환자 ), 두군간의면역표현형만을 107

6 비교하였기때문에변수의종류가매우제한적이었을뿐아니라 NPM1 돌연변이양성환자에국한하였기때문에 HLA-DR - /CD34 - 인 APL-유사 AML의특성이전반적으로반영되었다고는볼수없다. 본연구는선별되지않은연속환자 683명을일차적인분석대상으로삼았을뿐아니라기본적인임상적, 병리학적소견에서부터섬유소용해지표까지보다다양한변수들을비교하여 APL-유사 AML의특성을포괄적으로제공하였다는장점이있다. 먼저 APL-유사 AML의빈도는선별하지않은전체 AML 환자군에서 5.9% 이었고, 주로반복유전자이상 AML을제외한환자군에서는 8.6% 에이르렀다. 거의대부분의반복유전자이상 AML은 HLA- DR과 CD34 중하나이상에서양성을보이므로 [1], 반복유전자이상 AML에서 HLA-DR - /CD34 - 의빈도는매우미미할것으로생각된다. 그러므로전체 AML의약 6% 에서 APL-유사 AML 양상을보인다고해도무리가없다고할수있다. 선별하지않은성인일차성 AML 환자 350명을대상으로한초창기연구에서도본연구에서의 APL-유사 AML과매우유사한면역표현형 (HLA-DR - /CD34 ± /CD33 + / CD13 low /56 + /CD11a + /CD15 low /CD16 - ) 패턴을보이는 AML이 6% 에서관찰되었다고하였다 [17]. 이러한상황은혈액병리전문의로하여금중요한임상적판단을요구한다. 왜냐하면 APL과 APL-유사 AML의구분은치료방침결정에핵심적인역할을하기때문이다. HLA-DR과 CD34 모두양성인전형적인 AML과대비되는 APL- 유사 AML의특성은다음과같다. 첫째, APL-유사 AML의절반은 FAB M1 아형에속하였다. 이로인해 APL-유사 AML 환자는제1군에비해유의하게높은골수모구의백분율을보였다. 둘째, APL-유사 AML의약 76% 에서예후양호인자인 NPM1 돌연변이가양성이었고, 이는제1군에비하면압도적으로높은빈도이다. APL-유사 AML 환자군의세포유전학적특성 ( 높은정상핵형빈도, 낮은복잡핵형및단일염색체핵형의빈도, 낮은세포유전학적예후불량군의비율 ) 은모두 NPM1 돌연변이양성 AML의특징과연관된다. HLA-DR 음성환자군을대상으로한이전연구에서유의하게높은 NPM1 돌연변이의빈도는반복적으로확인된결과이다 [6-8]. NPM1 돌연변이양성 AML의가장현저한유전자발현패턴은 HOX 와같은전사인자유전자의활성화와 CD34와 CD133과같은조혈모세포- 연관유전자의억제이다 [18, 19]. 몇몇 HOX 유전자들의발현정도는조혈모세포에서높고, 세포가분화할수록감소한다. 그러므로 NPM1 돌연변이양성세포에서의 HOX 유전자활성화는줄기세포표현형을유지하는데관여한다고할수있다 [18]. 셋째, APL-유사 AML의가장현저한면역표현형적특징은 HLA-DR - / CD34 - 외에도 CD33 양성세포의분율이높은반면 CD13 양성세포의분율은매우낮다는점이다. 이는 CD33의발현이 CD13에비해상대적으로강하고균질한상태를의미한다. 이러한패턴은 APL 의주요한면역표현형적특징이므로 APL-유사 AML을면역표현형 분석을통해실제 APL과구분하는것이결코용이하지않음을시사한다. 그러므로 APL에비해 APL-유사 AML에서 CD2 발현이거의없다는사실이두질환을구분하는데도움이될것으로생각한다 [8]. 본연구결과가장주목할만한소견은 APL-유사 AML의중간적또는과도기적 (transitional) 특질이다. 특히섬유소분해지표 ( 섬유소원, FDP, D-이합체 ) 와이에근거한 DIC 점수의중간적특성은임상적으로도중요한의미를지닌다. 왜냐하면 DIC는기저질환과상관없이그자체만으로치명적일수있으므로임상의의감별진단에포함되어예방적조치를취하는것이중요하기때문이다. 이와같은측면에서본다면 DIC는일반적으로 APL과연관되는임상양상으로인식되어있으므로 APL이아닌일반적인 AML에서는저평가되어있을가능성이높다. 실제로 NCCN 지침에따르면임상적으로그리고병리학적으로 APL이의심되는환자에서는, 비록 APL 의분자유전학적확진결과가없더라도, ATRA를조기에투여하여치명적인출혈부작용을예방할것을권고하고있다 [13]. 따라서면역표현형적으로 APL-유사 AML 패턴이관찰되면검사실과임상의의활발한의사소통을통해 PML-RARA 재배열여부와관계없이반드시 DIC 진단을위한검사를시행하고, ATRA의조기투여를적극적으로검토해야할것으로생각한다. 일부연구자들은 NPM1 돌연변이양성환자에서 ATRA와 arsenic oxide (ASO) 가표준항암치료의보완요법으로유효함을보고하였다 [8-10]. APL-유사 AML 환자의많은수에서 NPM1 돌연변이가양성이고, 면역표현형을비롯한여러지표에서 APL과유사점을보인다는점을고려하면 NPM1 돌연변이양성 AML 환자가 APL 치료에사용되는약제에반응한다는사실이흥미롭다. APL 환자치료에서 ATRA의주요역할은 PML-RARA에의해억제된전사과정을재활성화시켜세포의분화를유도하는것이다. 또한 ATRA와 ASO 는모두 PML-RARA 융합단백자체를분해함으로써결국 PML- RARA에매개되는전사억제기능을무력화시킨다 [22]. NPM1 돌연변이양성 AML에서의 ATRA와 ASO의항백혈병효과의기전도약제에의한 NPM1 종양단백의분해로설명된다. NPM1 돌연변이단백과 PML-RARA 융합단백의시스테인 (cysteine) 에 ATO가결합하면산화스트레스에민감해지고, 산화된단백은 proteasome 기제의최종목표가되므로결국단백의분해로이어지게된다 [21]. 최근에는단순히종양단백의분해로인한효과뿐아니라 NPM1 돌연변이양성 AML에서의 PML 단백의변화로 ATRA와 ATO의항백혈병기전을설명하려는시도가제시되었다 [21]. 그러나 NPM1 돌연변이단백과 PML 단백과의상호작용에대해서는향후보다심도있는연구가필요할것으로생각된다. 본연구는국내에서는처음으로대규모환자군을대상으로 HLA- DR과 CD34가모두음성인 APL-유사 AML의빈도와특성을보고 108

7 하였다. APL-유사 AML은전체 AML의약 6% 를차지하여적지않은빈도임을알수있었다. APL-유사 AML은전형적 AML과구별되는특성을가지고있었고, 일부지표는전형적 AML과 APL 사이의중간적특질을보였다. 특히, DIC와의높은연관성은검사실과임상의의적극적인의견교환이필요함을시사한다. 본연구결과와관련참고문헌리뷰를통해 APL-유사 AML과 APL 사이에병태생리적공유점이있을것으로추측되지만, 향후심화연구가필요한상황이다. 요약 배경 : 본연구의목적은 HLA-DR - /CD34 - AML (APL-유사 AML) 의빈도와특성을분석하고자하였다. 방법 : 새로진단받은 683명의 AML 환자를대상으로하였다. 211명의반복유전자이상 AML, 1명의골수섬유증동반급성범골수증, 2 명의다운증후군관련골수성백혈병, 그리고중기분열세포를획득하지못한 2명의환자를제외한나머지 467명을다음과같이분류하였다 : 제1군, HLA-DR + /CD34 + ( 전형적 AML); 제2군, HLA-DR + / CD34 - 또는 HLA-DR - /CD34 + ; 제3군, APL-유사 AML. 결과 : 제1군은 294명, 제2군은 133명, 제3군은 40명으로분류되었다. 따라서선별되지않은 683명의 AML 환자군에서의 APL-유사 AML의빈도는 5.9% 이었다. APL-유사 AML은제1군에비해백혈구수와골수의모세포분율, 정상핵형과 NPM1 돌연변이의비율, CD33 양성세포의분율, 섬유소 ( 원 ) 분해산물및 D-이합체의농도가유의하게높았으며, 복잡핵형, 단일염색체핵형, 세포유전학적예후불량군의빈도, CD13 양성세포의분율, 그리고섬유소원의농도가유의하게낮았다. 골수모세포분율, CD33 양성세포분율, 파종혈관내응고점수의경우 APL-유사 AML에서전형적 AML과 APL 사이의중간값을보였다. 결론 : 본연구는 APL-유사 AML이드물지않고, 전형적 AML과구별되는특성을보임을제시하였다. APL-유사 AML과 APL 사이에는병태생리적공유점이있을것으로추측되지만심화연구가필요할것으로생각한다. 이해관계 저자들은본연구와관련하여어떠한이해관계도없음을명시함. REFERENCES 1. Arber DA, Vardiman JW, Brunning RD, Porwit A, Le Beau MM, Thiele J, et al. Acute myeloid leukaemia with recurrent genetic abnormalites. In: Swerdlow SH, Campo E, et al. eds. WHO classification of tumours of haematopoietic and lymphoid tissues. 4th ed. Lyon: IARC, 2008: Park JH, Qiao B, Panageas KS, Schymura MJ, Jurcic JG, Rosenblat TL, et al. Early death rate in acute promyelocytic leukemia remains high despite all-trans retinoic acid. Blood 2011;118: Wetzler M, McElwain BK, Stewart CC, Blumenson L, Mortazavi A, Ford LA, et al. HLA-DR antigen-negative acute myeloid leukemia. Leukemia 2003;17: Moon H, Lee S, Huh J, Chung WS. Characteristics of acute myeloid leukemia without HLA-DR expression. Korean J Lab Med 2007;27: Syampurnawati M, Tatsumi E, Furuta K, Takenokuchi M, Nakamachi Y, Kawano S, et al. HLA-DR-negative AML (M1 and M2): FLT3 mutations (ITD and D835) and cell-surface antigen expression. Leuk Res 2007;31: Syampurnawati M, Tatsumi E, Ardianto B, Takenokuchi M, Nakamachi Y, Kawano S, et al. DR negativity is a distinctive feature of M1/M2 AML cases with NPM1 mutation. Leuk Res 2008;32: Oelschlaegel U, Mohr B, Schaich M, Schakel U, Kroschinsky F, Illmer T, et al. HLA-DRneg patients without acute promyelocytic leukemia show distinct immunophenotypic, genetic, molecular, and cytomorphologic characteristics compared to acute promyelocytic leukemia. Cytometry B Clin Cytom 2009;76: Ferrari A, Bussaglia E, Ubeda J, Facchini L, Aventin A, Sierra J, et al. Immunophenotype distinction between acute promyelocytic leukaemia and CD15- CD34- HLA-DR- acute myeloid leukaemia with nucleophosmin mutations. Hematol Oncol 2012;30: Kottaridis PD, Gale RE, Frew ME, Harrison G, Langabeer SE, Belton AA, et al. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood 2011;98: Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L, et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med 2005;352: Wouters BJ, Löwenberg B, Erpelinck-Verschueren CA, van Putten WL, Valk PJ, Delwel R. Double CEBPA mutations, but not single CEBPA mutations, define a subgroup of acute myeloid leukemia with a distinctive gene expression profile that is uniquely associated with a favorable outcome. Blood 2009;113: Schnittger S, Kinkelin U, Schoch C, Heinecke A, Haase D, Haferlach T, 109

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