Current Techniques in Molecular Biology Friedel Nollet, Ph.D. Molecular Biology and Cytometry course May 16-17, 2013 Mol, SCK-CEN, Belgium
Sanger DNA sequencing
Kary Mullis received a Nobel Prize in chemistry in 1993, for his invention of the polymerase chain reaction (PCR). The process, which Kary Mullis conceptualized in 1983.
Reverse Transcriptase Real Time PCR reverse transcription of RNA to cdna (template for real time PCR) (m)rna is chemically stable but RNases are omnipresent! RNases cleanup RNAs which are not longer required RNases serve in the proces of maturation of all kind of RNAs intracellular RNAs are protected from RNase activity the human body uses RNases to defend against invading microorganisms retains substantially active after freeze-thaw or even autoclaving
@diagnosis for diagnosis and/or prognosis (RT-) PCR of fusion gene, a results of chromosome translocation (RT-) PCR of a DNA mutation PCR amplification of immunoglobulin and TCR gene rearrangements in B- and T-cell malignancies
(RT-) PCR in hematology Detection of translocations (eg. t(9;22)(q34.1;q11.2) in CML)
Major cytogenetic subgroups of AML (without APL) Döhner et al., Haematologica (2008) 93(7) 976-82
Hemavision kit (Biorad) 28 chromosomal rearrangements, (including more than 80 splice variants) in acute leukemia
Somatic mutations in AML Abnormality Frequency Prognosis NPM1 35% Good Flt3-ITD Flt3-TKD 25% 5-8% Adverse Not known, probably not the same adverse prognosis as Flt3-ITD CEBPα-sm CEBPα-dm 5(-10)% 2(-5)% Good IDH1 IDH2 5-10% 10% Neutral/adverse TET2 15-20% Unknown DNMT3A 20% Adverse ckit 2-10% Adverse for CBF-AML WT1 10% Adverse RUNX1 10-15% MLL-PTD 5% c-cbl, PTPN11, NRAS, KRAS, p53, JAK2, BCOR <5%
Mutation analysis in AML (genescan analysis) NPM1 & Flt3 NPM1 wild type NPM1 +4bp Flt3 wild type Flt3-ITD CEBPα bzip TAD2 TAD1 wild type TAD1 +5bp
JAK2 V617F mutation analyse Myeloproliferative disorders Malignant population (bone marrow stem cell) JAK2 V617F mutation Polycythemia Vera (Vaquez disease) red blood cell precursor 90-100% Essential Thrombocythemia Megakaryocytes 50% Ideopathic Myelofibrosis Megakaryocytes 50%
JAK2 V617F mutation analysis (2)
Immunoglobulin clonality analysis
Sensitivity of clonality analysis 100% 5% 10% 1%
@follow-up Ø a sensitive and quantitative technique Real time PCR
Minimal residual disease Disease Status Diagnosis/Relapse 1010 Clinical Remission Morphology/ karyo FISH 10-2 108 Cytogenetic Remission Flow cytometry 10-4 106 Molecular Remission PCR 10-6 Sensitivity Malignant cells 1012 Detection Technique
Förster Resonance Energy Transfer TAMRA ENERGY 6-FAM λ λ absorbed exciting light emitted fluorescence absorbed exciting energy light λ emitted fluorescence light Förster, T. (1948) Intermolecular Energy Migration and Fluorescence Ann. Phys 2:55-75.
TAQMAN Real Time PCR Mocellin et al. Trends Mol Med 2003 (www)
Analytical sensitivity? 5 molec. BCR-ABL p210 (plasmid): 12.8% negative (12/94 wells) when duplo analysis : 1.6% negative (0.128x0.128)
Molecular MRD detection Genetic targets Molecular techniques chromosome translocation real time QPCR (sensitivity 10-4 to 10-6) gene overexpression real time QPCR (sensitivity max. 10-4) eg. HOX11, HOX11L2, WT1 small indel mutations real time QPCR (sensitivity 10-4 to 10-6) eg. NPM1 mutations point mutations eg. JAK2 V617F, BRAF V600E, MPL(S505, W515), ckit (exon 8,17), p53, EZH2, TET2, ASXL1, clonal IgH/TCR rearrangements many different techniques (sensitivity max.10-2) competitive TAQMAN mutation detection assays (10-3) Patient specific QPCR assays (sensitivity 10-4 to 10-5) Genescan analysis (sensitivity 10-2)
Expression (FG/CG) at diagnosis fusion gene CN / control gene CN (Gabert et al., 2003 - EAC) AML AZ Sint-Jan Brugge t(15;17) PML-RARa 0.30 (BM) - 0.36 (PB) inv16 CBFB-MYH11 1.35 (type-a) 1.00 (type-b) 0.88 (type-d) t(8;21) AML1-ETO 2.1 (BM) - 4.2 (PB) t(12;21) TEL1-AML1 4.45 (BM) - 4.71 (PB) t(4;11) MLL-AF4 0.46 (BM) - 0.60 (PB) t(1;19) E2A-PBX1 44.6 (BM) - 42.7 (PB) T-ALL del(1p32) SIL-TAL1 0.12 (BM) - 0.09 (PB) CML t(9;22) p210 : 0.86 (BM) - 1.17 (PB) p190 : 0.80 (BM) - 0.68 (PB) 1.96 0.65 ALL BCR-ABL remark: >10% not quantifiable AML NPM1 type-a, B, D mutation / 15.7 B-ALL
Survival 1983-2008 (CML) Primary imatinib, 2002-2008 (CML IV) 5-year survival 93% Survival probability n = 2830 IFN or SCT, 1997-2008 (CML IIIA) 5-year survival 71% IFN or SCT, 1995-2008 (CML III) 5-year survival 63% (CML I, II) IFN, 1986-2003 5-year survival 53% Hydroxyurea, 1983-1994 Busulfan, 1983-1994 5-year survival 38% Courtesy of the German CML Study Group Year after diagnosis
Imatinib (Glivec Europe; Gleevec U.S.)
Imatinib mesylate (Gleevec, Glivec, STI-571) 2-phenylaminopyrimidine
Standardised International Scale (IS) Hughes et al. Blood 2006 anchored to International Randomized Study of Interferon vs STI571 (IRIS) trial (100% IS) with a major molecular response (MMR) corresponding to 0.1% IS specific conversion factor (IS % ratio = local % ratio x conversion factor). How to standardize? By a hierarchical and serial strategy of sample exchange a conversion factor (CF) is calculated for each participating laboratory (EUTOS in Europe) 2010 : WHO primary reference material for, secondary reference materials now available. http://www.whereareyouontheis.com/
MRD detection in CML Early Molecular Response : 10% IS BCR-ABL Major Molecular response (MMR) : 0.1% IS BCR-ABL Complete Molecular Response (CMR): not detectable <0.01% IS BCRABL (MR4.0) MR3.5, MR4.0, MR4.5,
MolecularDiagnostics.be test directory 15-03-2013 JAK2 V617F muta3on analysis BCR/ABL p210 quan3ta3ve analysis BCR/ABL p190 quan3ta3ve analysis IgH monoclonality analysis TCRgamma monoclonality analysis BCR/ABL qualita3ve analysis bcl2/igh qualita3ve analysis bcl1/igh qualita3ve analysis IgH hypermuta3on analysis IgK monoclonality analysis Flt3- ITD muta3on analysis PML/RARalpha quan3ta3ve analysis chimerism analysis WT1 overexpression analysis Hemavision kit (screening for acute leukemia) NPM1 muta3on analysis KRAS muta3on analysis CBFB/MYH11 quan3ta3ve analysis PML/RARalpha qualita3ve analysis AML1/ETO quan3ta3ve analysis MLL/AF4 quan3ta3ve analysis TEL/AML1 quan3ta3ve analysis MLL transloca3on t(11q23) detec3on CBFB/MYH11 qualita3ve analysis E2A/PBX1 qualita3ve analysis SIL/TAL1 qualita3ve analysis AML1/ETO qualita3ve analysis FIP1L1/PDGFRA qualita3ve analysis Abelson muta3on analysis ckit D816V muta3on analysis #labs 15 15 14 13 12 11 10 9 9 9 8 8 8 8 7 6 6 6 6 6 5 5 5 5 5 4 4 4 4 4
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The PCR Song http://www.youtube.com/watch?v=7uafuvnkuzg
pre-/post conversion values from UKNEQAS EQC reports
Real-time PCR : quantification methods standard curve method deltact method 2-(C gene-c housekeeping gene) t t condition : equal efficiencies of both reaction of ~100%
Nucleophosmin-1 mutations in AML * Falini, B. et al. NEJM 2005; 352:254-266
Prognostic significance of MRD-negativity (NPM1mut) After induction End of treatment Krönke J et al. JCO 2011;29:2709-2716
Conclusions Molecular Techniques : QPCR (5 molecules, 10-4 10-6) > Genescan analysis (1-10%) > Sanger DNA-sequencing (20%) How to increasing sensitivity? Increase quality of samples cdna synthesis? More sample input per reaction 2, 3, 4,.. fold reactions NGS CML : IS? reference material, peer group comparison B-cell malignancies : currently only Genescan clonality analysis feasible, (<->leukemia specific QPCR), hopeful for NGS AML : sensitive molecule marker (QPCR target: recurrent translocations, NPM1 type-a, -B, -D mutations) for 30-40% of AML patients. Interventional study protocols?