2008년 6월부터 2011년 9월까지 아주대학교 병원에서 CML로 처음 진단받은 환자 중 진단 후 지속적으로 아주대학교 병원에서 약물 치료를 받으며 추적 관 찰 중인 24명의 환자들을 대상으로 하였다. -75℃에 보관 되어 있던 환자들의 RNA 검체 중 진단 당시와 약물 치료 3개월에서 6개월 뒤 추적 검사 시의 RNA 검체를 사용하였으며 농도를 1㎍/㎕로 맞춘 후 사용하였다. 본 연구는 아주대학 교 병원 임상시험 심사위원회(IRB)의 승인을 받았다.
4
표 2. LC kit의 RQ-PCR 반응 시약.
Component Volume per reaction t(9;22) or G6PDH detection mix 6.2㎕
10X Reaction mix 2.0㎕
6
환자 검체와 같은 batch에서 standard plasmid (SP)를 사용하여 역전사와
8
IS-NCNsample = NCNsample ´ IS-Cal value/NCNcal MMR은 아래의 표에 따라 결정 되었다.
표 7. MMR 판정 기준.
IS-NCNsample ≤ 0.05 0.05 < IS-NCNsample <0.15 IS-NCNsample ≥0.15 Major Molecular Response Grey zone around the MMR
cut-off: inconclusive result
III. 결 과
A. 대상 환자군의 특징
환자들의 나이는 16세부터 77세까지 분포하였고 평균연령은 45.2세였으 며, 여자가 10명, 남자가 14명이었다. 환자 중 6명은 가속기에 속하였고 1명은 모 세포기, 그리고 17명은 만성기에 속하였다. 24명의 환자 모두 imatinib을 복용하고 있었으며 22명은 하루에 400mg을 1회 복용하였고 2명은 600mg을 복용하였다.
- 10 - 표 8. 대상 환자군의 특징.
환자번호 성별 연령 병기 LC kit 정량결과 IS-kit 정량결과 Initial Follow-up Initial Follow-up 1 F 30 모세포기 0.29 0.02 67.450992 6.8840097
B. MMR에 대한 검사법 사이의 일치율
진단 시와 추적 검사 시 LC kit로 검사한 BCR-ABL1 RQ-PCR 결과는 0.000343 ~ 0.35 (median 0.02)와 0 ~ 0.09 (median 0.000525) 이었으며, IS-kit로 검사한 결과는 각각 0.017905 ~ 100.153 (median 61.125)와 0 ~ 66.390 (median 0.805) 이었다.
MMR 상태를 결정함에 있어 IS-kit와 LC kit의 일치율은 92% (22/24)였으 며 P < 0.001로 두 검사법 사이에 유의한 차이가 있는 것으로 나타났다. 두 kit로 비교 시 서로 상이한 결과를 보인 환자들은 LC kit로 시행한 검사에서는 MMR 상태를 보였으나 IS-kit로 시행한 검사에서는 No MMR 상태로 나타났다.
표 9. 각각의 kit 에 의한 MMR 판정 결과.
IS-kit
LC kit
MMR No MMR
MMR 7 2
No MMR 0 15
- 12 -
A. B.
C. D.
E. F.
그림 3. LC kit와 IS-kit에 의한 BCR-ABL1 정량(예). LC kit를 사용하여 진단 시 BCR-ABL1 정량 (A와 B)을 시행 하였고 3개월 후 추적 검사 시 (C와 D) 정량 결 과는 3 log 이상 감소를 보여 MMR 상태를 보였으나 추적 검사 검체를 IS-kit로 검사 시 (E와 F) IS-NCN 수치는 No MMR 상태를 보였다.
C. 검사법 사이의 상관관계
- 14 - (FISH), Southern blot, Western blot, 그리고 reverse transcription polymerase chain reaction (RT-PCR) 등이 있는데, 이러한 방법들은 민감도가 낮을 뿐만 아니라 검 사 절차가 복잡하며 정량이 불가능하고 골수 검체를 사용하여야 한다는 단점이 있다. 이에 반해 RQ-PCR은 100,000개 이상의 정상 세포에서 한 개의 종양 세포
를 검출할 수 있을 정도로 민감도가 높으며 골수 검체와 말초 혈액의 RQ-PCR
- 16 -
MMR 상태를 결정함에 있어 IS-kit는 기존의 LC kit의 결과와 92% (22/24)
- 18 -
RQ-PCR의 표준화와 IS가 제안 되었으며 IS-kit는 2005년 CML 전문가들이 제안 한 표준화된 프로토콜과 IS에 기반하여 제작되었다.
본 연구는 단일 기관에서 24명의 환자를 대상으로 IS-kit와 표준화 이전 에 개발된 LC kit를 비교하였으며, 새롭게 개발된 IS-kit는 MMR 상태 평가에 있 어 기존의 검사 kit와 높은 일치율을 보일 뿐만 아니라 보다 더 정확하게 MMR 상태 도달을 판단할 수 있을 것으로 보여 기존 kit를 대체 할 수 있을 것으로 보 인다.
V. 결 론
본 연구는 IS 에 기반을 두고 최근 개발된 BCR-ABL Mbcr IS-MMR Dx kit 와 기존의 LightCycler t(9;22) quantification kit 을 비교하였으며 BCR-ABL Mbcr IS-MMR Dx kit 는 LightCycler t(9;22) quantification kit 와 비교 시 MMR 상태 평가에 있어 높은 일치율을 보일 뿐만 아니라 보다 더 정확하게 MMR 상태 도달을 판단할 수 있을 것으로 보인다.
- 20 -
참고문헌
1. Baccarani M, Cortes J, Pane F, Niederwieser D, Saglio G, Apperley J, Cervantes F, Deininger M, Gratwohl A, Guilhot F, Hochhaus A, Horowitz M, Hughes T, Kantarjian H, Larson R, Radich J, Simonsson B, Silver RT, Goldman J, Hehlmann R:
Chronic myeloid leukemia: An update of concepts and management recommendations of European Leukemia Net. J Clin Oncol 27: 6041–6051, 2009 2. Baccarani M, Saglio G, Goldman J, Hochhaus A, Simonsson B, Appelbaum F,
Apperley J, Cervantes F, Cortes J, Deininger M, Gratwohl A, Guilhot F, Horowitz M, Hughes T, Kantarjian H, Larson R, Niederwieser D, Silver R, Hehlmann R: Evolving concepts in the management of chronic myeloid leukemia: Recommendations from an expert panel on behalf of the European LeukemiaNet. Blood 108: 1809–1820, 2006
3. Cross NCP: Standardisation of molecular monitoring for chronic myeloid leukaemia.
Best Pract Res Clin Haematol 22(3): 355-365, 2009
4. Druker BJ, Guilhot F, O’Brien SG, Gathmann I, Kantarjian H, Gattermann N, Deininger MW, Silver RT, Goldman JM, Stone RM, Cervantes F, Hochhaus A, Powell BL, Gabrilove JL, Rousselot P, Reiffers J, Cornelissen JJ, Hughes T, Agis H, Fischer T, Verhoef G, Shepherd J, Saglio G, Gratwohl A, Nielsen JL, Radich JP, Simonsson B, Taylor K, Baccarani M, So C, Letvak L, Larson RA: Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med 355:
2408–2417, 2006
5. Gabert J, Beillard E, van der Velden V, Bi W, Grimwade D, Pallisgaard N, Barbany G, Cazzaniga G, Cayuela JM, Cavé H, Pane F, Aerts JL, De Micheli D, Thirion X, Pradel V, González M, Viehmann S, Malec M, Saglio G, van Dongen JJ:
Standardization and quality control studies of ‘realtime’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program. Leukemia 17: 2318-2357, 2003
6. Hughes T, Deininger M, Hochhaus A, Branford S, Radich J, Kaeda J, Baccarani M, Cortes J, Cross NC, Druker BJ, Gabert J, Grimwade D, Hehlmann R, Kamel-Reid S, Lipton JH, Longtine J, Martinelli G, Saglio G, Soverini S, Stock W, Goldman JM:
Monitoring CML patients responding to treatment with tyrosine kinase inhibitors:
Review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood 108: 28–37, 2006
7. Hughes TP, Hochhaus A, Branford S, Müller MC, Kaeda JS, Foroni L, Druker BJ, Guilhot F, Larson RA, O'Brien SG, Rudoltz MS, Mone M, Wehrle E, Modur V, Goldman JM, Radich JP: Long-term prognostic significance of early molecular response to imatinib in newly diagnosed chronic myeloid leukemia: an analysis from the International Randomized Study of Interferon and STI571 (IRIS). Blood 116(19):
- 22 - 3758-3765, 2010
8. Hughes TP, Kaeda J, Branford S, Rudzki Z, Hochhaus A, Hensley ML, Gathmann I, Bolton AE, van Hoomissen IC, Goldman JM, Radich JP: Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N Engl J Med 349: 1423–1432, 2003
9. Iacobucci I, Saglio G, Rosti G, Testoni N, Pane F, Amabile M, Poerio A, Soverini S, Bassi S, Cilloni D, Bassan R, Breccia M, Lauria F, Izzo B, Merante S, Frassoni F, Paolini S, Montefusco E, Baccarani M, Martinelli G: Achieving a major molecular response at the time of a complete cytogenetic response (CCgR) predicts a better duration of CCgR in imatinib-treated chronic myeloid leukemia patients. Clin Cancer Res 12: 3037–3042, 2006
Significance of increasing levels of minimal residual disease in patients with Philadelphia chromosome-positive chronic myelogenous leukemia in complete cytogenetic response. J Clin Oncol 27: 3659-3663, 2006
12. Kantarjian H, Talpaz M, O’Brien S, Jones D, Giles F, Garcia-Manero G, Faderl S,
Ravandi F, Rios MB, Shan J, Cortes J: Survival benefit with imatinib mesylate versus interferon-based regimens in newly diagnosed chronic-phase chronic myelogenous leukemia. Blood 108: 1835-1840, 2006
13. Müller MC, Saglio G, Lin F, Pfeifer H, Press RD, Tubbs RR, Paschka P, Gottardi E, O'Brien SG, Ottmann OG, Stockinger H, Wieczorek L, Merx K, König H, Schwindel U, Hehlmann R, Hochhaus A : An international study to standardize the detection and quantitation of BCR-ABL transcripts from stabilized peripheral blood preparations by quantitative RT-PCR. Haematologica 92(7): 970-973, 2007
14. National Comprehensive Cancer Network. NCCN: Clinical Practice Guidelines in Oncology. Chronic Myelogenous Leukemia, Version 2, 2010. Available at http://www.nccn.org/professionals/physician_gls/f_guidelines.asp.
15. O’Brien SG, Guilhot F, Larson RA, Gathmann I, Baccarani M, Cervantes F, Cornelissen JJ, Fischer T, Hochhaus A, Hughes T, Lechner K, Nielsen JL, Rousselot P, Reiffers J, Saglio G, Shepherd J, Simonsson B, Gratwohl A, Goldman JM, Kantarjian H, Taylor K, Verhoef G, Bolton AE, Capdeville R, Druker BJ: Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 348: 994–1004, 2003
16. Press RD, Galderisi C, Yang R, Rempfer C, Willis SG, Mauro MJ, Druker BJ, Deininger MW: A half-log increase in BCR-ABL RNA predicts a higher risk of relapse in patients with chronic myeloid leukemia with an imatinib-induced complete
- 24 -
cytogenetic response. Clin Cancer Res 13: 6136–6143, 2007
17. Press RD, Love Z, Tronnes AA, Yang R, Tran T, Mongoue-Tchokote S, Mori M, Mauro MJ, Deininger MW, Druker BJ: BCR-ABL mRNA levels at and after the time of a complete cytogenetic response (CCR) predict the duration of CCR in imatinib mesylate-treated patients with CML. Blood 107: 4250–4256, 2006
18. Radich JP: How I monitor residual disease in chronic myeloid leukemia. Blood 114:
3376–3381, 2006
19. Ren R: Mechanisms of BCR-ABL in the pathogenesis of chronic myelogenous leukaemia. Nat Rev Cancer 5: 172–183, 2005
20. Shtivelman E, Lifshitz B, Gale RP, Canaani E: Fused transcript of abl and bcr genes in chronic myelogenous leukaemia. Nature 315: 550–554 , 1985
21. Vigil CE, Griffiths EA, Wang ES, Wetzler M: Interpretation of cytogenetic and molecular results in patients treated for CML. Blood Rev 25: 139–146, 2010
22. Westbrook CA, Hooberman AL, Spino C, Dodge RK, Larson RA, Davey F, Wurster-Hill DH, Sobol RE, Schiffer C, Bloomfield CD: Clinical significance of the BCR-ABL fusion gene in adult acute lymphoblastic leukemia: a Cancer and Leukemia Group B Study (8762). Blood 80: 2983–2990, 1992
23. White HE, Matejtschuk P, Rigsby P, Gabert J, Lin F, Lynn Wang Y, Branford S, Müller MC, Beaufils N, Beillard E, Colomer D, Dvorakova D, Ehrencrona H, Goh HG, El Housni H, Jones D, Kairisto V, Kamel-Reid S, Kim DW, Langabeer S, Ma
ES, Press RD, Romeo G, Wang L, Zoi K, Hughes T, Saglio G, Hochhaus A, Goldman JM, Metcalfe P, Cross NC: Establishment of the first World Health Organization international genetic reference panel for quantitation of BCR-ABL mRNA. Blood 116: 111-117, 2010
24. Zhang T, Grenier S, Nwachukwu B, Wei C, Lipton JH, Kamel-Reid S. Inter-laboratory comparison of chronic myeloid leukemia minimal residual disease monitoring: summary and recommendations. J Mol Diagn. 9(4): 421-430, 2007
- 26 -
-
ABSTRACT-Quantification of Major BCR-ABL1 Fusion Gene Transcripts for Monitoring Therapeutic Response to Tyrosine Kinase Inhibitors in Chronic Myelogenous Leukemia; Comparison of LightCycler t(9;22)
Quantification Kit and BCR-ABL1 Mbcr IS-MMR Dx Kit.
Sunhyun Ahn
Department of Laboratory Medicine The Graduate School, Ajou University
(Supervised by Associate Professor Sung Ran Cho)
Background : CML is in >90% of cases characterized by the presence of the Philadelphia chromosome (Ph) and the corresponding fusion gene, BCR-ABL1. For disease monitoring, standardize BCR-ABL1 quantification and International Scale (IS) has been suggested, and this study is to compare IS-based IS-kit with non-IS-based LightCycler-t(9;22) Quantification kit (LC kit; Roche, Germany) for determination of MMR status.
Methods: We used stored RNA samples from 24 patients who had been newly diagnosed with CML in Ajou university hospital from Jun 2008 to Sep 2011. We reviewed the patients’
medical records including age at diagnosis, gender, phase of the disease, medication, and BCR-ABL1 quantification results done by LC kit.
Results: The mean age at diagnosis was 45 (16~77) and M:F ratio was 1.4:1. BCR-ABL1 transcript levels by the LC kit at diagnosis and follow-up were 0.000343~0.35 (median 0.02) and 0~0.09 (median 0.000525), respectively. IS-NCN by the IS-kit at diagnosis and follow-up were 0.017905~100.156 (median 61.125) and 0~66.390 (median 0.805). The IS-kit and the LC kit showed a concordance rate of 92% (22/24) for determination of MMR status.
Two patients with discrepancies were determined to achieve MMR by LC kit, but not by IS-kit. When reviewing further follow-up samples of the 2 patients, BCR-ABL1 transcript levels increased thereafter, meaning they might not truly achieve MMR.
Conclusions: Despite of a good concordance between the IS-kit and non-IS-based kit, our data suggest that the standardized IS-based kit would be more promising for accurate determination of MMR in CML.
Keyword : CML, BCR-ABL1 mRNA, RQ-PCR, MMR, IS