bhs training course 2013-2015: seminar 1 laboratory …friedel nollet, ph.d. molecular biologist...
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Friedel Nollet, Ph.D. Molecular Biologist
Laboratory Medicine AZ Sint-Jan Brugge-Oostende AV
Molecular Hematology
BHS TRAINING COURSE 2013-2015: SEMINAR 1
Laboratory techniques
The Human Genome
Printed:
3300 books of 1000 pages
1000 base pairs per page.
Computer data storage:
3.3 billion base pairs recorded at 2 bits per pair
= 786 megabytes
or = one CD
Wellcome Collection, London
Genome Browser
CTCTCTCTCACTTTGATCTCCATATTCCAGGCTTACACAGGGGTTTCCTCAGAACGTTGATGGCAGTTGCAGGTCCATATAAAGGGACCAAAGCACATTGTATCCTCATCTATAGTCATG
CTGAAAGTAGGAGAAAGTGCATCTTTATTATGGCAGAGAGAATTTTCTGAACTATTTATGGACAACAGTCAAACAACAATTCTTTGTACTTTTTTTTTTCCTTAGTCTTTCTTTGAAGCA
GCAAGTATGATGAGCAAGCTTTCTCACAAGCATTTGGTTTTAAATTATGGAGTATGTGTCTGTGGAGACGAGAGTAAGTAAAACTACAGGCTTTCTAATGCCTTTCTCAGAGCATCTGTT
TTTGTTTATATAGAAAATTCAGTTTCAGGATCACAGCTAGGTGTCAGTGTAAACTATAATTTAACAGGAGTTAAGTATTTTTGAAACTGAAAACACTGTAGGACTATTCAGTTATATCTT
GTGAAAAAGGAAAGCAATGAAGTTAAAAGTAGAAGGTTACAATGCCCAAACAATAGAGTATTATAGTAAACAAATGTCTATAAAACATTTTGTGTTCATGATAGCAAAAGAGATTATGGC
AGGTTCAACATAACATTGGAATAACTGGCCTTTTCAGTACAAACTTATCTGGAATTATGAAGACAAAGCATATAAATGATACACTTAATTTTTAATGGAACTGACAGAAATGATTATGTT
GATATGATACTAGATATATTTTTTGGCTAAATTTAGGTGTTCACAGAAACTACTAAAAGTATAAATCGTACCCCATGCTTTAATACTATACAGGCATGCCTCATTTTATTGCACCTTGCT
TTATTGTGCTTCTTAGATATTGTATTTTTTACATATTGAAGGTTTACGGCAACCCAGTGTCTAGCAACTCTGTCAGCAACATTTTCCCAACAGCATGTGCTCATTTCATGTCTCTGTGTC
ATATTTTGGTAATTCTAGCAACATTTCAAACTTTAAAAAAATCATATGGTGATCTGTGATCAGTAATCTTTAATGCTAGTATTGTAATTATTCTGGGGTGTCCCAAACAGAGAGAATATA
CTCTCTCTCACTTTGATCTCCATATTCCAGGCTTACACAGGGGTTTCCTCAGAACGTTGATGGCAGTTGCAGGTCCATATAAAGGGACCAAAGCACATTGTATCCTCATCTATAGTCATG
CTGAAAGTAGGAGAAAGTGCATCTTTATTATGGCAGAGAGAATTTTCTGAACTATTTATGGACAACAGTCAAACAACAATTCTTTGTACTTTTTTTTTTCCTTAGTCTTTCTTTGAAGCA
GCAAGTATGATGAGCAAGCTTTCTCACAAGCATTTGGTTTTAAATTATGGAGTATGTTTCTGTGGAGACGAGAGTAAGTAAAACTACAGGCTTTCTAATGCCTTTCTCAGAGCATCTGTT
TTTGTTTATATAGAAAATTCAGTTTCAGGATCACAGCTAGGTGTCAGTGTAAACTATAATTTAACAGGAGTTAAGTATTTTTGAAACTGAAAACACTGTAGGACTATTCAGTTATATCTT
GTGAAAAAGGAAAGCAATGAAGTTAAAAGTAGAAGGTTACAATGCCCAAACAATAGAGTATTATAGTAAACAAATGTCTATAAAACATTTTGTGTTCATGATAGCAAAAGAGATTATGGC
AGGTTCAACATAACATTGGAATAACTGGCCTTTTCAGTACAAACTTATCTGGAATTATGAAGACAAAGCATATAAATGATACACTTAATTTTTAATGGAACTGACAGAAATGATTATGTT
GATATGATACTAGATATATTTTTTGGCTAAATTTAGGTGTTCACAGAAACTACTAAAAGTATAAATCGTACCCCATGCTTTAATACTATACAGGCATGCCTCATTTTATTGCACCTTGCT
TTATTGTGCTTCTTAGATATTGTATTTTTTACATATTGAAGGTTTACGGCAACCCAGTGTCTAGCAACTCTGTCAGCAACATTTTCCCAACAGCATGTGCTCATTTCATGTCTCTGTGTC
ATATTTTGGTAATTCTAGCAACATTTCAAACTTTAAAAAAATCATATGGTGATCTGTGATCAGTAATCTTTAATGCTAGTATTGTAATTATTCTGGGGTGTCCCAAACAGAGAGAATATA
Kary Mullis PCR-process conceptualized in 1983 Nobel Prize in Chemistry in 1993
PCR : polymerase chain reaction
Mg2+
dCTP
dGTP
dUTP
dATP
Taq DNA Polymerase
Synthetic forwards and reverse primers
Isolated DNA
PCR : reaction components
1 cycle = 2 Amplicon
2 cycle = 4 Amplicon
3 cycle = 8 Amplicon
4 cycle = 16 Amplicon
5 cycle = 32 Amplicon
6 cycle = 64 Amplicon
7 cycle = 128 Amplicon
No. Of No. Amplicon Cycles Copies of Target
1 2
2 4
3 8
4 16
5 32
6 64
20 1,048,576
30 1,073,741,824
PCR : amplification
Reverse-Transcriptase PCR
RNA
Omnipresence of RNases
• all organisms have different classes of RNases (endo-RNases & exo-RNases)
• biological role of RNases :
– degradation of RNA’s no longer in use
– processing of RNA’s
– defence against RNA virusses
• characteristics of RNAses:
– omnipresent
– extremely stable : withstand high t (>100 C), extreme pH, …
• in cellular granules!
Molecular diagnostics of hematological malignancies
@diagnosis
– Detection of chromosome translocations
– Detection of mutations
– Detection of clonality
@follow-up
– minimal residual disease detection
stem cell transplantation
- HLA alloantigen typing
- DNA fingerprinting (chimerism testing)
Molecular diagnostics of hematological malignancies
@diagnosis
– Detection of chromosome translocations
– Detection of mutations
– Detection of clonality
@follow-up
– minimal residual disease detection
stem cell transplantation
- HLA alloantigen typing
- DNA fingerprinting (chimerism testing)
t(9;22) in CML
1960 : Philadelphia chromosome 1984 : BCR-ABL1 gene rearrangement
Detection of BCR/ABL1 by RT-PCR
pat
ien
t-1
pat
ien
t-2
pat
ien
t-3
pat
ien
t-4
pat
ien
t-5
pat
ien
t-6
pat
ien
t-7
pat
ien
t-8
pat
ien
t-9
pat
ien
t-1
0
BCR/ABL P210 b3a2 (e14a2)
BCR/ABL P190 e1a2
normal donor
water control
100 bp ladder
multiplex RT-PCR method based on Cross et al. (1994) Leukemia 8(1):186-189
internal amplification
control
Translocations in acute leukemia
Karyotype Gene(s) Adults (freq.,%)
Children (freq.,%)
t(9;22) BCR-ABL1 15-25 2-3
t(1;14) TAL-1 10-15 5-10
t(10;14) HOX11 5-10 <5
t(8;14), t(8;22), t(2;8)
C-MYC 5 2-5
t(14q32) IgH 5 <5
t(1;19), t(17;19) TCF3-PBX1,E2A-HLF
<5 3-5
t(5;15) HOX11L2 1 2-3
Karyotype Gene(s) Adults (freq.,%)
Children (freq.,%)
t(8;21) AML1-ETO 5-12 7-16
t(15;17) PML-RAR 10 2-10
inv(16) CBF-MYH11 5 3-11
der(11q23) MLL 4 14-22
t(9;22) BCR-ABL1 2 0-1
t(6;9) DEK-CAN <1 1-2
t(1;22) OTT-MAL <1 0-3
AML ALL
Hemavision kit (Biorad)
28 chromosomal rearrangements, (including more than 80 splice variants) in acute leukemia
Hemavision kit (full version)
M1A t(X;11)(q13;q23) MLL(11q23)-FOXO4(Xq13.1) M5A t(4;11)(q21;q23) MLL(11q23)-AFF1(4q21.3)
M1B t(6;11)(q27;q23) MLL(11q23)-MLLT4(6q27) M5B t(10;11)(p12;q23) MLL(11q23)-MLLT10(10p12)
M1C t(11;19)(q23;p13.1) MLL(11q23)-ELL(19p13.1) M5C t(11;19)(q23;p13.3) MLL(11q23)-MLLT1(19p13.3)
M1D t(10;11)(p12;q23) MLL(11q23)-MLLT10(10p12) M5D t(9;11)(p22;q23) MLL(11q23)-MLLT3(9p22)
M5E t(1;11)(q21;q23) MLL(11q23)-MLLT11(1q21)
M2A t(1;11)(p32;q23) MLL(11q23)-EPS15(1p32) M6A inv(16)(p13;q22) CBFB(16q22.1)-MYH11(16p13.11)
M2B t(11;17)(q23;q12-21) MLL(11q23)-MLLT6(17q21) M6B t(9;22)(q34;q11) BCR(22q11)-ABL1(9q34.1)
M2C t(11;19)(q23;p13.3) MLL(11q23)-MLLT1(19p13.3) M6C t(9;12)(q34;p13) ETV6(12p13)-ABL1(9q34.1)
M2D t(10;11)(p12;q23) MLL(11q23)-MLLT10(10p12) M6D t(5;12)(q33;p13) ETV6(12p13)-PDGFRB(5q33)
M2E t(9;11)(p22;q23) MLL(11q23)-MLLT3(9p22) M6E t(12;22)(p13;q11-12) ETV6(12p13)-MN1(22q12.1)
M3A t(1;19)(q23;p13) TCF3(19p13.3)-PBX1(PRL)(1q23.3) M7A t(6;9)(p23;q34) DEK(6p23)-NUP214(9q34)
M3B t(17;19)(q22;p13) TCF3(19p13.3)-HLF(17q22) M7B t(9;9)(q34;q34) SET(9q34)-NUP214(9q34)
M3C t(12;21)(p13;q22) ETV6(12p13)-RUNX1(21q22.3) M7C inv(16)(p13;q22) CBFB(16q22.1)-MYH11(16p13.11)
M3D TAL1(40_kb_deletion) STIL(1p32)-TAL1(1p32) M7D t(3;21)(q26;q22) RUNX1(21q22.3)-EAP(3q26)
M4A t(8;21)(q22;q22) RUNX1(21q22.3)-RUNX1T1(8q22) M8A t(11;17)(q23;q12-21) ZBTB16(11q23)-RARA(17q12)
M4B t(3;21)(q26;q22) RUNX1(21q22.3)-MDS1(3q26) M8B t(3;21)(q26;q22) RUNX1(21q22.3)-EVI1(3q26)
M4C t(16;21)(p11;q22) FUS(16p11.2)-ERG(21q22.3) M8C t(15;17)(q22;q12) PML(15q22)-RARA(17q12)
M4D t(15;17)(q22;q12) PML(15q22)-RARA(17q12) M8D t(5;17)(q35;q12) NPM1(5q35)-RARA(17q12)
M8E t(3;5)(q25.1;q35) NPM1(5q35)-MLF1(3q25.1)
M8F t(9;22)(q34;q11) BCR(22q11)-ABL1(9q34.1)
Molecular diagnostics of hematological malignancies
@diagnosis
– Detection of chromosome translocations
– Detection of mutations
– Detection of clonality
@follow-up
– minimal residual disease detection
stem cell transplantation
- HLA alloantigen typing
- DNA fingerprinting (chimerism testing)
Detection versus screening • Mutation detection :
– Minisequencing – Pyrosequencing – RFLP (restriction fragment length polymorphism) – Sequence Specific Oligonucleotides (SSO) (InnoLipa, Innogenetics) – Oligo ligation assay – Sequence specific primer PCR (SSP-PCR) / allele specific PCR (AS-PCR) – ARMS-PCR (amplification refractory mutation system) – Mutation/SNP detection by MALDI-TOF mass spectrometry (MassCleave™) – TAQMAN allelic discrimination assay – MLPA (multiplex ligation-dependent probe amplification) – DNA microarray technology (oligo arrays : SNP-arrays, resequencing arrays) – …
• Mutation screening – Direct sequencing – Protein Truncation Test (PTT) – Enzymatic cleavage methods – Single Strand Conformational Polymorphism (SSCP) / Conformation sensitive gel
electrophoresis (CSGE) – Denaturing Gradient Gel Electrophoresis (DGGE) – Heteroduplex analysis – PAGE Gel electrophoresis – dHPLC – Mutation screening by MALDI-TOF mass spectrometry (MassCleave™) – Hi-Res melting (Idaho) – DNA microarray technology (oligo arrays : SNP-arrays, resequencing arrays) – -…
SSP-PCR/AS-PCR
example : Factor-V Leiden mutation analysis
S1 WT MUT
S2 WT MUT
S3 WT MUT
WILD TYPE CTRL WT MUT
HETERO- ZYGOTE CTRL WT MUT
WATER CTRL WT MUT
internal amplification
control
JAK2 V617F mutation analysis
Myeloproliferative
neoplasms
Malignant population
(bone marrow stem cells)
JAK2 V617F
Polycythaemia Vera Red blood cell recursor 90-100%
Essential thrombocythaemia Megakaryocytes 50%
Primary myelofibrosis Megakaryocytes 50%
JAK2 exon 14 : TCTTTCTTTGAAGCAGCAAGTATGATGAGCAAGCTTTCTCACAAGCATTTGGTTTTAAATTATGGAGTATGTGTCTGTGGAGACGAGA JAK2 V817F exon 14 sequence: TCTTTCTTTGAAGCAGCAAGTATGATGAGCAAGCTTTCTCACAAGCATTTGGTTTTAAATTATGGAGTATGTTTCTGTGGAGACGAGA
LNA based QPCR
Denys et al., J Mol Diagn. 2010 July; 12(4): 512–519.
JAK2 forward primer 5′-AGCAGCAAGTATGATGAGCAAG-3′
JAK2 reverse primer 5′-GAGAAAGGCATTAGAAAGCCTGTAG-3′
Mutation probe 5′-TGGAGTATGTTTCTGTGGA-3′
LNA oligonucleotide 5′-TATGTGTCTGT−3′
Control probe 5′-ACAAGCATTTGGTTTTAAATTATGGAG-3′
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
CEBP sm CEBP -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%
Risk categorisation in AML (excl. APL - European LeukemiaNet)
Genetic group Subsets -------------------------------------------------------------------------------- Favorable t(8;21)(q22;q22); RUNX1-RUNX1T1 inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11 Mutated NPM1 without FLT3-ITD (normal karyotype) Mutated CEBPA (normal karyotype) Intermediate-I* Mutated NPM1 and FLT3-ITD (normal karyotype) Wild-type NPM1 and FLT3-ITD (normal karyotype) Wild-type NPM1 without FLT3-ITD (normal karyotype) Intermediate-II t(9;11)(p22;q23); MLLT3-MLL Cytogenetic abnormalities not classified as favorable or adverse Adverse inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1 t(6;9)(p23;q34); DEK-NUP214 t(v;11)(v;q23); MLL rearranged –5 or del(5q); –7; abnl(17p); complex karyotype -------------------------------------------------------------------------------- Frequencies, response rates, and outcome measures should be reported by genetic group, and, if sufficient numbers are available, by specific subsets
indicated; excluding cases of acute promyelocytic leukemia. * Includes all AMLs with normal karyotype except for those included in the favorable subgroup; most of these cases are associated with poor prognosis, but they should be reported separately because of the potential different response to treatment. For most abnormalities, adequate numbers have not been studied to draw firm conclusions regarding their prognostic significance. Three or more chromosome abnormalities in the absence of one of the WHO designated recurring translocations or inversions, that is, t(15;17), t(8;21), inv(16) or t(16;16), t(9;11), t(v;11)(v;q23), t(6;9), inv(3) or t(3;3); indicate how many complex karyotype cases have involvement of chromosome arms 5q, 7q, and 17p.
Döhner et al., Blood 2010 115(3) 453-74
Multiplex GeneScan
NPM1 wild type
NPM1 +4bp
bZIP
TAD2 TAD1 wild type
TAD1 +5bp
Flt3 wild type
Flt3-ITD
CEBP
NPM1 & Flt3
AMLSG Genotype specific treatment trials
TKI resistance in CML
Soverini et al. Blood (2011) 118:1208-1215 O’Hare et al. Blood. 2007; 110(7); 2242-9
mutation
BCR/ABL kinase domain mutation screening
Molecular diagnostics of hematological malignancies
@diagnosis
– Detection of chromosome translocations
– Detection of mutations
– Detection of clonality
@follow-up
– minimal residual disease detection
stem cell transplantation
- HLA alloantigen typing
- DNA fingerprinting (chimerism testing)
ALL MM CLL Lymphomas
Hematopoietic stem cell
Neutrophils
Eosinophils
Basophils
Monocytes
Platelets
Red cells
Myeloid progenitor
Myeloproliferative neoplasms AML
Lymphoid progenitor T-lymphocytes
Plasma cells
B-lymphocytes
naïve
B- and T-cell receptors
IgH
IgL IgL
V V
C C
J J
IgHV V
D DJ J
C C
C
C
C
C
C
C
CD
79a
CD
79b
CD3CD3 CD3
CD3 CD3
V
J
C
V
D
J
C
TCR TCR
B-lymphocyte T-lymphocyte
CD3CD3 CD3
CD3 CD3
V
J
C
V
D
J
C
TCR TCR
T-lymphocyte
Ton Langerak, Erasmus MC, Rotterdam
IgH gene rearrangements
1 2 3 4 5 6 66 1 2 3 4 1 2 3 4 5 6
VH DH JH Cs
D J joiningH H
V D -J joiningH H H
precursor mRNAIGH
mature mRNAIGH
transcription
RNA splicingV DJ C
27
IgH
IgL IgL
V V
C C
J J
IgHV V
D DJ J
C C
C
C
C
C
C
C
CD
79a
CD
79b
translation
junctional regionTon Langerak, Erasmus MC, Rotterdam
Hypervariable regions
random deletion and insertion of nucleotides
D-exon
J-exon V-exon junctional
Region
(CDR3)
CDR1 CDR2 FR1 FR2 FR3
Ton Langerak, Erasmus MC, Rotterdam
B- and T-cell clonality analysis
polyclonal
monoclonal
heteroduplex analysis Capillary electroforese
polyclonal
monoclonal
Ton Langerak, Erasmus MC, Rotterdam
B-cell clonality analysis
BIOMED-2 B-cell malignancy report : PAS Evans et al., leukemia 2007
T-cell clonality analysis
BIOMED-2 T-cell malignancy report : PAS M/ Brüggemann et al., leukemia 2007
* 20 to 25% of anaplastic large cell lymphomas do not have TCR gene rearrangements (null-ALCL)
Molecular diagnostics of hematological malignancies
@diagnosis
– Detection of chromosome translocations
– Detection of mutations
– Detection of clonality
@follow-up
– minimal residual disease detection
stem cell transplantation
- HLA alloantigen typing
- DNA fingerprinting (chimerism testing)
TAQMAN probe
TAQ polymerase
5’-3’ exonuclease activiteit
Föster Ann. Phys., 1948
(Reporter (R); Quencher (Q))
FRET (Förster Resonance Energy Transfer)
Real-time PCR
Real-time PCR
cycle threshold value (Ct)
Minimal residual disease detection N
um
be
r o
f le
uke
mic
cel
ls
BC
R-A
BL / A
BL ratio
100%
10%
1%
0.1%
0.01%
0.001%
0.0001%
1012
1011
1010
109
108
107
106
Diagnosis, pretreatment or hematological relapse
Complete hematological response
Complete cytogenetic response
Major Molecular Response
Undetectable transcript (complete molecular response)
Morphology FISH
Immuno- phenotyping
PCR
Krönke J et al. JCO 2011;29:2709-2716
After induction
End of treatment
Prognostic significance of MRD-negativity (NPM1mut)
Examples relapsed AML patients (RQ-PCR NPM1 type-A, B and D mutation)
Molecular diagnostics of hematological malignancies
@diagnosis
– Detection of chromosome translocations
– Detection of mutations
– Detection of clonality
@follow-up
– minimal residual disease detection
stem cell transplantation
- HLA alloantigen typing
- DNA fingerprinting (chimerism testing)
HLA- class I
loci : HLA-A,-B,-C
interacts with CD8 lymphocytes
HLA- class II
loci : HLA-DR,-DP,-DQ
interacts with CD4 lymphocytes
Hematopoietic stem cell transplantation Human leucocyte antigen complex
HLA typering
SSO SSP SBT
DNA fingerprinting : microsatellites
Chimerisme test
patient
donor
sample (29.1% donor)
moleculardiagnostics.be test directory (9.10.2013)
TEST #LABS PERFORMING TEST
BCR/ABL p210 quantitative analysis 15
JAK2 V617F mutation analysis 15
BCR/ABL p190 quantitative analysis 14
IgH monoclonality analysis 13
TCRgamma monoclonality analysis 12
BCR/ABL qualitative analysis 11
bcl2/IgH qualitative analysis 10
bcl1/IgH qualitative analysis 9
IgH hypermutation analysis 9
IgK monoclonality analysis 9
chimerism analysis 8
Flt3-ITD mutation analysis 8
PML/RARalpha quantitative analysis 8
WT1 overexpression analysis 8
Hemavision kit (screening for acute leukemia) 7
AML1/ETO quantitative analysis 6
CBFB/MYH11 quantitative analysis 6
NPM1 mutation analysis 6
PML/RARalpha qualitative analysis 6
CBFB/MYH11 qualitative analysis 5
E2A/PBX1 qualitative analysis 5
MLL/AF4 quantitative analysis 5
TEL/AML1 quantitative analysis 5
Abelson mutation analysis 4
cKIT D816V mutation analysis 4
FIP1L1/PDGFRA qualitative analysis 4
SIL/TAL1 qualitative analysis 4
SIL/TAL1 quantitative analysis 4
TCRbeta monoclonality analysis 4
TEL/AML1 qualitative analysis 4
THE END
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