Contents Introduction Real-time quantitative PCR Clinical applications Molecular monitoring of CML Molecular monitoring of fusion genes in acute leuke

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1 대한진단혈액학회 2015 심포지엄 Real-time quantitative PCR in diagnostic hematology : Reporting & interpretation Young-Uk Cho Department of Laboratory Medicine, University of Ulsan, College of Medicine and Asan Medical Center 1

2 Contents Introduction Real-time quantitative PCR Clinical applications Molecular monitoring of CML Molecular monitoring of fusion genes in acute leukemia Reporting of the result Summary & Conclusion 2

3 Introduction VII. 분자진단 분자진단의이론과술기 필수학습목표 Real-time PCR 반응의원리를이해하고분석법을알고있어야한다. 보험급여또는비급여로등재된분자진단검사항목을파악하고, 각검사를시행하는방법을설명할수있어야한다. 필수술기 PCR 기반의검사및 real-time PCR 기반의검사를직접시행하고판독한다 ( 각 10 건 ) 종양의분자진단 필수학습목표 혈액암의대표적인유전자재배열 (BCR/ABL1, RUNX1/RUNX1T1, PML/RARA 등 ) 의정성및정량검출법을이해하고검사의의의및해석법을이해한다. 혈액암에서미세잔존질환의진단을위해사용될수있는검사법과의의및해석법을이해한다. 필수술기 대표적인유전자재배열에대해분자진단검사결과를판독하고보고서를작성한다 (5 건 ). 대표적인미세잔존질환검사법에대해검사결과를판독하고보고서를작성한다 (5 건 ). 3 전공의수련목표집제 3 판 (2014)

4 Introduction 분류번호코드분류질환 노 -596 유전자돌연변이검사 [ 기타검사 ] CZ807 CZ808 CZ809 CZ810 CZ821 CZ823 CZ824 PML/RARa 유전자재배열, 정량 [ 실시간역전사중합효소연쇄반응 ] AML1/ETO 유전자재배열, 정량 [ 실시간역전사중합효소연쇄반응 ] BCR/ABL 유전자재배열, 정량 [ 실시간역전사중합효소연쇄반응 ] TH (tyrosine hydroxylase) 유전자, 정량 [ 실시간역전사중합효소연쇄반응 ] CBFB/MYH11 유전자재배열, 정량 [ 실시간역전사중합효소연쇄반응 ] NPM1 유전자, mrna 정량 [ 실시간역전사중합효소연쇄반응 ] BAALC 유전자, mrna 정량 [ 실시간역전사중합효소연쇄반응 ] AML (APL) AML CML, ALL Neuroblastoma AML AML AML CZ825 WT1 유전자, mrna 정량 [ 실시간역전사중합효소연쇄반응 ] AML, ALL, CML CZ826 TEL/AML1 유전자재배열, 정량 [ 실시간역전사중합효소연쇄반응 ] ALL 4 건강보험요양급여비용

5 Real-time quantitative PCR RQ-PCR 형광신호 (fluorescence signal) 의검출및정량 표적증폭과신호검출이동일한튜브에서동시에시행됨 (homogeneous) 각 PCR 주기마다반복적으로측정 (kinetic) 형광역치 (threshold) 역치주기 (threshold cycle, C T ) 형광방출량이고정된역치를초과하는첫번째 PCR 주기의수 표적주형 (target template) 의양과상관성을가짐» 표적핵산의 starting copy number 의양이많을수록형광의유의한증가가더빨리검출됨 (inverse linear relationship) 5

6 RQ-PCR 신호검출방법 DNA 결합염료 SYBR Green High-resolution dye (Roche, LC480 HRM dye)» Melting curve analysis 염기순서특이탐색자 (probe) Linear probes» TaqMan probes» Hybridization probes Structured probes» Molecular beacons» Scorpions 6

7 Double dye oligonucleotide TaqMan = Taq polymerase + PacMan Taq DNA polymerase 의 5-3 exonuclease 활성 TaqMan probe chemistry mechanism

8 Normalization 변이 (variation) 를제어하는방법 Extraction yield 검체의양또는핵산의회수율 RNA 변성 검체또는핵산의질 Reverse-transcription yield cdna 합성의효율 검체 loading 및피펫에러 Efficiency of amplification PCR 억제제및기타영향인자 8

9 Normalization 내부대조 : reference gene (housekeeping gene) GAPDH, G6PDH, ABL, B2M, GUSB Europe Against Cancer (EAC) 에서추천한유전자 표적유전자와내부대조와의비율 (quotient) Target gene/reference gene Gene Genomic structure/pseudogene Regulation GAPDH Multigene family; genes >200 in mouse mostly pseudogenes : lung, pancreatic, colon cancer : insulin, EGF β2-microglobulin No pseudogenes : NHL G6PDH No pseudogenes growth factors : kidney, stomach tumor : hormones, oxidant stress PBGD β-actin ABL No pseudogenes Multigene family; >20 genes 1 active locus 20 pseudogenes No pseudogenes : hormones of thyroid gland : stomach tumor GUSB No pseudogenes 9

10 PBGD (porphobilinogen deaminase) revealed the highest variation of expression ABL Constant expression in the PB of healthy individuals Expression level in the range of the fusion genes at diagnosis Good correlation with other housekeeping genes Weisser M, et al. The use of housekeeping genes for real-time PCR-based quantification of fusion 10 gene transcripts in acute myeloid leukemia. Leukemia 2004;18:

11 이상적인 PCR 곡선 - Steep 증폭곡선 - High 신호강도 - Low C p Threshold: Background fluorescent signal 의 3-10 SD Standard curve method 11

12 2 배희석시 1 cycle 차이 10 배희석시 3.3 cycle 차이 è 10 배희석으로작성한표준곡선의기울기 = -3.3 To double or not to double, that s the question! 허용범위 - Slope : -3.2 ~ Efficiency : Error :

Molecular monitoring of CML Technically demanding à intrinsic variables No method clearly best No means to assess the analytical validity varying from 1.

13 Molecular monitoring of CML Technically demanding à intrinsic variables No method clearly best No means to assess the analytical validity varying from 1.6 to 3 log at the same dilution Common procedures improved comparability Zhang T, et al. Inter-laboratory comparison of chronic myeloid leukemia minimal residual 13 disease monitoring: summary and recommendations. J Mol Diagn 2007;9:

14 Differences in Amount of RNA extracted RNA integrity Efficacy of reverse transcription Relate the number of copies to those of a housekeeping control gene (internal control for both the quantity and quality of cdna for each sample) Schematic outline of BCR-ABL RQ-PCR analysis Cross NC. Standardisation of molecular monitoring for chronic myeloid leukemia. Best Pract Res 14 Clin Haematol 2009;22:

15 Ideal control gene Expressed uniformly in different cell types regardless of their proliferative status Unaffected by therapeutic regimens Invariant between individuals Expressed at a level similar to BCR-ABL EAC group ABL Beta-2-microglobulin (b2m) Beta-glucuronidase (GUSB) 15

16 High level distortion The primers for quantification of ABL also amplify BCR-ABL Actual unit : BCR-ABL/(ABL+BCR-ABL) non-linear in the high BCR-ABL level ABL remains the most commonly used Much of the published evidence The clear value of accurate RQ-PCR analysis è sequential quantification of relatively low levels of disease 16

17 RQ-PCR analysis in the IRIS trial Centralized in three centers Adelaide, London, Seattle Reproducible differences in median BCR-ABL values at specific time-points Standardized baseline 30 pretreatment samples Major molecular response (MMR) 3-log reduction from the standardized baseline è Improved comparability Hughes TP, et al. Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine 17 in newly diagnosed chronic myeloid leukemia. N Engl J Med 2003;349:

18 The international scale for BCR-ABL International Scale (IS) Detectable disease as a percentage Ph-positive metaphases by standard cytogenetics Hughes T, et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts 18 and kinase domain mutations and for expressing results Blood 2006;108:28-37.

19 Conversion factors (CF) Initiated by the Adelaide laboratory CF calculation Exchange of a series of samples (20-30) Span at least 3 logs of detectable disease Not exceed an IS value of roughly 10% Over a period of time CF validation Exchange of a further set of samples in a similar manner Bias of within ±1.2-fold compared to the reference laboratory è Suitable for conversion of the test laboratory results to the IS Pragmatic compromise The concept of regional or national reference laboratories Branford S, et al. Desirable performance characteristics for BCR-ABL measurement on an international reporting scale to allow consistent interpretation of individual patient response and comparison 19 of response rates between clinical trials. Blood 2008;112:

20 Development of internationally accredited reference reagents for BCR-ABL K562/HL60 mixtures 10%, 1%, 0.1%, and 0.01% on the IS Proposed to the WHO The 1 st International Genetic Reference Panel for the quantitation of BCR-ABL mrna White HE, et al. Establishment of the first World Health Organization International Genetic Reference 20 Panel for quantitation of BCR-ABL mrna. Blood 2010;116:e111-7.

CF: 0.7218 CF: 0.46 Balasubramanian P, et al.

21 CF: CF: 0.46 Balasubramanian P, et al. International reporting scale of BCR-ABL1 fusion transcript in chronic myeloid 21 leukemia: first report from India. Acta Haematol 2012;127:

22 Commercial kit BCR-ABL Mbcr IS-MMR kit (Ipsogen, Marseille, France) NCN (normalized copy number) = (Mbcr CN /ABL CN ) 100 IS-NCN sample = NCN sample IS-Cal value/ncn cal Each lot of the IS-MMR-calibrator has an assigned value (IS-Cal value) derived from a calibration against the NIBSC WHO certified primary reference material. 22

23 Commercial kit Xpert BCR-ABL Monitor Assay On Cepheid GeneXpert Instrument System 1 wash reagent 2 rinse reagent 3 elution reagent S sample 23

24 E ΔCt : the efficiency of the PCR calculated from the calibration curve slope (10 (1/slope) ) 24

25 r=0.881 r=0.878 r=0.881 Lopez-Jorge CE, et al. Comparative study of BCR-ABL1 quantification: Xpert assay, a feasible solution 25 to standardization concerns. Ann Hematol 2012;91:

26 Response criteria of CML 26 Baccarani M, et al. Definition and treatment of resistance to tyrosine kinase inhibitors in chronic myeloid leukemia. Expert Rev Hematol 2014;7:

Treatment-free remission (TFR) Complete molecular response (CMR) Molecularly

27 Levels of molecular response in CML. MCyR, major cytogenetic response; MR, molecular response. Treatment-free remission (TFR) Complete molecular response (CMR) Molecularly undetectable leukemia Mahon FX et al. Deep molecular response in chronic myeloid leukemia: the new goal of therapy. Clin Cancer Res 2014;20:

28 Molecular monitoring of fusion genes in acute leukemia AML PML-RARA RUNX1-RUNX1T1 CBFB-MYH11 NPM1 mutations ALL minor BCR-ABL1 TEL-AML1 28

29 21 consecutive AML patients with t(8;21) RQ-PCR TaqMan technology TBP as the housekeeping gene Results: ΔC t according to standard curve Leroy H, et al. Prognostic value of real-time quantitative PCR (RQ-PCR) in AML with t(8;21). Leukemia ;19:

30 Outcomes of log reduction in BM at remission in t(8;21) patients. < 1 log reduction BM log reduction > 3 log reduction 278 CBF AML patients 163 with t(8;21) 115 with inv(16)/t(16;16) RQ-PCR The ABI 7900HD platform Results: absolute copy numbers of fusion gene transcripts normalized to ABL (per 10 5 copies of ABL) Yin JA, et al. Minimal residual disease monitoring by quantitative RT-PCR in core binding factor AML allows risk stratification and predicts relapse: results of the United Kingdom MRC AML-15 trial. Blood 2012;120:

31 Outcomes by CBFB-MYH11 copy numbers in PB at remission in inv(16) patients. > 500 copies PB copy numbers < 10 copies Yin JA, et al. Minimal residual disease monitoring by quantitative RT-PCR in core binding factor AML allows risk stratification and predicts relapse: results of the United Kingdom MRC AML-15 trial. Blood 2012;120:

32 Sequential MRD monitoring during follow-up in t(8;21) patients. > 4 weeks after completion of consolidation course 4 BM > 500 copies PB > 100 copies Yin JA, et al. Minimal residual disease monitoring by quantitative RT-PCR in core binding factor AML allows risk stratification and predicts relapse: results of the United Kingdom MRC AML-15 trial. Blood 2012;120:

33 Sequential MRD monitoring during follow-up in inv(16) patients. BM > 50 copies PB > 10 copies Yin JA, et al. Minimal residual disease monitoring by quantitative RT-PCR in core binding factor AML allows risk stratification and predicts relapse: results of the United Kingdom MRC AML-15 trial. Blood 2012;120:

Minimal residual disease monitoring by quantitative RT-PCR in core binding factor AML

34 Quantitative RT-PCR analysis in paired BM and PB samples after induction, during consolidation, and at follow-up. t(8;21) inv(16) Yin JA, et al. Minimal residual disease monitoring by quantitative RT-PCR in core binding factor AML allows risk stratification and predicts relapse: results of the United Kingdom MRC AML-15 trial. Blood 2012;120:

Reduction in fusion transcript ratio by CBF subset and treatment arm.

BM samples Results (transcript ratio) [fusion gene/abl1] 100 Time points MRD 1 before

Before 3rd consolidation Jourdan E, et al.

35 Reduction in fusion transcript ratio by CBF subset and treatment arm. 198 CBF AML patients 96 with t(8;21) 102 with inv(16)/t(16;16) RQ-PCR Ipsogen plasmids BM samples Results (transcript ratio) [fusion gene/abl1] 100 Time points MRD 1 before initiation of the first consolidation course MRD 2 Before 2nd consolidation MRD 3 Before 3rd consolidation Jourdan E, et al. Prospective evaluation of gene mutations and minimal residual disease in patients with core binding factor acute myeloid leukemia. Blood 2013;121:

36 Outcome by MRD2 response. Reduction less than 3-log Reduction 3-log or more Jourdan E, et al. Prospective evaluation of gene mutations and minimal residual disease in patients with core binding factor acute myeloid leukemia. Blood 2013;121:

Prospective evaluation of gene mutations and minimal residual

37 Similar results when replacing MRD2 reduction by absolute MRD2 level 0.1% Jourdan E, et al. Prospective evaluation of gene mutations and minimal residual disease in patients 37 with core binding factor acute myeloid leukemia. Blood 2013;121:

38 38 Ommen HB, et al. Strikingly different molecular relapse kinetics in NPM1c, PML-RARA, RUNX1- RUNX1T1, and CBFB-MYH11 acute myeloid leukemia. Blood 2010;115:

39 Relation between molecular and flow cytometric detection of residual disease during and after treatment in childhood acute myeloid leukemia. AML1-ETO CBFB-MYH11 MLL Inaba H, et al. Comparative analysis of different approaches to measure treatment response in39acute myeloid leukemia. J Clin Oncol 2012;30:

40 Event-free survival (EFS) for patients with acute myeloid leukemia included in this study according to minimal residual disease (MRD) by flow cytometry. After induction 1 After induction 2 After induction 1 After induction 2 Inaba H, et al. Comparative analysis of different approaches to measure treatment response in40acute myeloid leukemia. J Clin Oncol 2012;30:

41 A poor correlation among the methods overall MRD levels by flow cytometry strongly correlated with clinical outcome AML1-ETO and CBFB-MYH11 transcripts persist in many patients who remained in long-term remission Low levels of MRD by PCR (undetectable by flow cytometry) could be controlled by subsequent chemotherapy or immune reconstitution The presence of fusion transcripts could result from the persistence of preleukemic cells or from partially differentiated leukemic cells that have lost not only their aberrant immunophenotypic features but also their clonogenic potential Inaba H, et al. Comparative analysis of different approaches to measure treatment response in41acute myeloid leukemia. J Clin Oncol 2012;30:

42 Reporting of the result Molecular pathology report 의일반원칙 Communicates results of a molecular test and their significance in a concise manner to help guide patient management Should be written such that a nonspecialist physician can understand and act on its content Should provide an interpretation of results in the context of the setting and indications for testing in that particular patient 42

43 Specimen Result Interpretation Method Sign 43

44 Specimen PB or BM Time points Result Post-induction 1 or more Post-consolidation 1 or more Post-HSCT (OOO day or O month) Major BCR-ABL1 IS-NCN OOOO Others Fusion transcript CN ABL1 CN Normalized CN OOOOO OOOO OOO ABL1 CN가일정 cut-off 이하 à RNA의질이떨어지거나양이부족한상태이므로정량결과의 precision을확보할수없음 44

45 Interpretation 정량누적결과 년 11 월 28 일 (Bone Marrow) : 년 12 월 11 일 (Bone Marrow) : 년 1 월 13 일 (Bone Marrow) : 0.89 : 현재 post-induction 상태로진단시와비교하여 3-log 이상감소하였습니다. 그래프? Method Real-time quantitative PCR using ipsogen FusionQuant Kit of CBFB-MYH11 A (QIAGEN) on the LC-480 instrument. It has not been approved by the US FDA. However, such approval is not required for clinical implementation, and test results have been shown to be clinically useful. This laboratory is CAP accredited. Sign 45

46 Patient demographics Specimen : bone marrow (post-induction 1) Results : Fusion transcript CN ABL1 CN Normalized CN 675 7, Interpretation : 진단시와비교하여 3-log 이상감소하였습니다. Log 10 NCN CBF-MYH11 정량보고서예 Method : Sign 0-1 Diagnosis Induction D14 Post-induction 1 46

47 Summary & Conclusion RQ-PCR 질환특이유전자변이정량에사용됨 CML 치료반응모니터링의표준으로인정받고있음 WHO 표준물질개발을통한 international scale 로보고함 급성백혈병등기타질환 미세잔존질환측정의유용한도구로널리사용되고있음 일반적으로 normalized CN 형태로보고함 CML 과달리검사법및보고체계에관해아직표준화되어있지않으므로 RQ-PCR 을시행하거나의뢰하는검사실마다보고및해석에관한원칙수립이필요함 47

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