2019 NCCN CML 지침 및 droplet digital PCR을 이용한 BCR-ABL1 fusion transcript 검사

건국대학교병원 진단검사의학과 정희정

2019 NCCN 지침에 따른 CML의 MRD 검사법과 ddPCR을 이용한 detection capability 를 소개합니다.

Tyrosine kinase inhibitor (TKI) 치료제의 발전으로 만성골수성백혈병(chronic myeloid leukaemia: CML) 환자의 수명은 정상인의 기대 여명과 차이가 없어졌다. 최근에는 CML 치료의 목표를 질환의 완치에 삶의 질 향상까지 확장하기 위한 TKI 치료중단(discontinuation) 이 화두이다[1]. TKI 중단 대상 및 시점 선택 및 이를 위한 MRD monitoring을 위해서는 충분히 낮은 농도에서 BCR-ABL1 검출능이 중요해졌다[2].

1. BCR-ABL1 fusion transcript 정량 검사의 표준화 및 보고단위

CML 환자 관리를 위한 European LeukemiaNet 및 National Comprehensive Cancer Network(NCCN)의 최근 실무 지침에서는 BCR-ABL1 fusion transcript 검출을 위해 real-time quantitative PCR (RQ-PCR)과 같은 민감한 PCR 분석법의 사용을 요구한다[3,4]. NCCN CML 최신지침에서는 molecular response에 도달하였는지 판단을 위해 3 개월마다 BCR-ABL1 검사를 하고 표준화 된 International Scale (% IS) 단위로 결과를 보고하도록 한다[5]. 충분히 낮은 농도에서 BCR-ABL1결과를 정확하게 측정하지 못하면, 임상적 결정에 영향을 미쳐 환자의 치료에 부정적 결과를 초래할 수 있으므로[6,7], MR4 (0.01% IS) 또는 MR4.5 (0.0032% IS)까지 MRD 검출을 위해서는 적절한 측정 감도가 보장되어야 한다[8]. 따라서 현재 가장 널리 사용되고 있는, RQ-PCR 분석법이나 droplet digital PCR과 같은 분석법의 검출 한계(limit of detection: LOD) 및 정량 한계(limit of quantification: LOQ) 검증은 매우 중요하다[6].

Table 1.% IS values, associated molecular log reduction (MR) level, and clinical response.

% IS MR Response (2019 NCCN guideline 참고)
10 1.0 Early molecular response (EMR)
1 2.0
0.32 2.5
0.1 3.0 Major Molecular Response (MMR)
0.032 3.5
0.01 4.0
0.032 4.5 Complete molecular response (CMR)
0.002 4.7
0.001 5.0

BCR-ABL1 fusion transcript 정량 보고는 WHO primary reference materials 에 traceability 가 있는 IS calibrator 도입으로 표준화가 이루어졌고, 검사실 간, 검사법 결과값의 호환이 가능하게 되었다[9]. % IS와 molecular log reduction (MR) level 의 관계는 다음과 같다.

2. Discontinuation of TKI therapy in patients with Chronic Myeloid Leukemia : NCCN Guidelines Version 1.2019

Table 2. Criteria for hematologic, cytogenetic, and molecular response and relapse by NCCN CML guideline v2019.01

Complete hematologic response [10]
  • Complete normalization of peripheral blood counts with leukocyte count <10 x 109/L
  • Platelet count <450 x 109/L
  • No immature cells, such as myelocytes, promyelocytes, or blasts in peripheral blood
  • No signs and symptoms of disease with disappearance of palpable splenomegaly
Cytogenetic response [11]
  • Complete cytogenetic response (CCyR) - No Ph-positive metaphases (CCyR typically correlates with BCR-ABL1 (IS) ≤1% (>0.1%–1%))
  • Major cytogenetic response (MCyR) - 0%–35% Ph-positive metaphases
  • Partial cytogenetic response (PCyR) - 1%–35% Ph-positive metaphases
  • Minor cytogenetic response - >35%–65% Ph-positive metaphases
Molecular response [12]
  • Early molecular response (EMR) - BCR-ABL1 (IS) ≤10% at 3 and 6 months
  • Major molecular response (MMR) - BCR-ABL1 (IS) ≤0.1% or ≥3-log reduction in BCR-ABL1 mRNA from
    • the standardized baseline, if qPCR (IS) is not available
  • Complete molecular response (CMR) is variably described, and is best defined by the assay’s level of sensitivity (eg, MR4.5)
Relapse
  • Any sign of loss of response (defined as hematologic or cytogenetic relapse)
  • 1-log increase in BCR-ABL1 transcript levels with loss of MMR should prompt bone marrow evaluation for loss of CCyR but is not itself defined as relapse (eg, hematologic or cytogenetic relapse)

2019년 National Comprehensive Cancer Network (NCCN) CML 지침에서 명시하는 CML 치료 반응 criteria는 표 2와 같다. Complete molecular response를 정의하려면 측정법의 분석 민감도가 보장되어야 하며, MR4.5 가 가장 바람직하다. 현재 널리 사용되고 있는 RT-QPCR의 LOD는 4.0MR (0.01% IS), LOQ는 제조사가 제시하지 않고 있다. BCR-ABL1 감시는 최신 지견을 따르려면 검사법상 기술적인 보완 및 더 측정 민감도가 좋은 방법의 도입이 필요하다.

표3은 NCCN에서 제시한 TKI 중단 기준으로 10가지를 모두 만족하여야 한다. 이 중 검사실에서 BCR-ABL1 농도 감시와 관련하여 5, 6, 7번이 중요하다. 특히6번의 최소한 MR4.5 (BCR-ABL1 ≤0.0032% IS)에서 신뢰할 만한 측정 민감도를 보장하여야 한다는 내용에 주목할 필요가 있다. EURO-SKI 연구 결과에 따르면 3 년 이상 MR4.0 (BCR-ABL1 ≤0.01% IS)이하의 농도를 나타내는 것이 이마티닙 치료 중단 성패를 결정하는 가장 중요한 예측 변수로 나타났다[13].

Table 3. Criteria for TKI Discontinuation by NCCN CML guideline v2019.01

TKI discontinuation should be considered only if ALL of the criteria included in the list below are met
  1. Age ≥18 years.
  2. Chronic phase CML. No prior history of accelerated or blast phase CML.
  3. On approved TKI therapy for at least 3 years.
  4. Prior evidence of quantifiable BCR-ABL1 transcript.
  5. Stable molecular response (MR4; BCR-ABL1 ≤0.01% IS) for ≥2 years, as documented on at least 4 tests, performed at least 3 months apart [13].
  6. Access to a reliable qPCR test with a sensitivity of detection of at least MR4.5 (BCR-ABL1 ≤0.0032% IS) and that provides results within 2 weeks.
  7. Monthly molecular monitoring for one year, then every 6 weeks for the second year, and every 12 weeks thereafter (indefinitely) is recommended for patients who remain in MMR (MR3; BCR-ABL1 ≤0.1% IS) after discontinuation of TKI therapy.
  8. Prompt resumption of TKI within 4 weeks of a loss of MMR with molecular monitoring every 4 weeks until MMR is re-established, then every 12 weeks thereafter is recommended indefinitely for patients who have reinitiated TKI therapy after a loss of MMR. For those who fail to achieve MMR after 3 months of TKI resumption, BCR-ABL1 kinase domain mutation testing should be performed, and monthly molecular monitoring should be continued for another 6 months.
  9. Consultation with a CML Specialty Center to review the appropriateness for TKI discontinuation and potential risks and benefits of treatment discontinuation, including TKI withdrawal syndrome.
  10. Reporting of the following to an NCCN CML Panel Member is strongly encouraged:
    A. Any significant adverse event believed to be related to treatment discontinuation.
    B. Progression to accelerated or blast phase CML at any time.
    C. Failure to regain MMR after 3 months following treatment reinitiation.
Data from the EURO-SKI study suggest that MR4.0 (BCR-ABL1 ≤0.01% IS) for 3 years or more was the most significant predictor for successful discontinuation of imatinib. Total duration of imatinib therapy for at least 6 years was also predictive of successful discontinuation [13].

2. Validation of detection capability of droplet digital PCR

Droplet digital PCR (ddPCR) 분석법은 RQ-PCR의 기본원리에 각 샘플을 20,000개의 나노 리터 크기의 droplet으로 분리하는 단계를 추가한 검사법이다. 개별 droplet 개수만큼 PCR 반응이 증폭되어 분석의 정확성과 재현성이 향상되므로 낮은 농도 target을 검출하거나 정량할 때 유용한 검사법이다[14-16]. 이론적으로 절대 정량이 가능하고 RT-QPCR등 상대 정량법에 비해 표준 곡선이 불필요하다는 장점이 있다. 최근 ddPCR 기반 체외진단(IVD) 제품이 최초로 출시되었는데, CML-MRD를 위한 QXDx BCR-ABL% IS (Bio-Rad, Hercules, CA, USA)로 미국 FDA 허가와 CE 마크를 득하였다. 저자는 임상 검체 및 certified reference material을 이용하여 automated Droplet Generator (Bio-Rad), CFX96 thermal cycler (Bio-Rad), QX200 Droplet Reader (Bio-Rad) 검사 시스템에서 CLSI EP17-A2 [17] 에 따라 제조사의 LOD/LOQ claims (0.002% IS = MR4.7, for both)을 검증하였다[18]. LOD검증의 경우 0.0033% IS (MR4.5), 0.0023% IS (MR4.6)로 조제된 물질을 각각 12반복 측정한 24 개의 측정치 중 23 개 측정치(95.8%)가 제조사가 제시한 LOD (>0.002% IS) 이상으로 측정되었고, 한 개의 측정치만 제조사가 제시한 LOD (<0.002% IS) 미만으로 측정되었다. LOQ 검증의 경우 85% 이상인 87.5% (24 개의 측정치 중 21 개)의 측정치가 허용 가능한 범위(accuracy goal of ± 15% total error) 이내로 관찰되어 제조사 claim을 검증할 수 있었다[data in press]. QXDx BCR-ABL% IS ddPCR assay의 detection capability는 NCCN가이드라인에 따른 CML환자의MRD감시에 충분한 것으로 관찰되었으나, 국내 임상 검사실에 도입되기 위해서는 신의료기술 및 가격 등이 해결되어야 할 것이다.

참고문헌

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