BHS TRAINING COURSE 2013-2015: SEMINAR 1 Laboratory …Friedel Nollet, Ph.D. Molecular Biologist...

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







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)

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)


@diagnosis




@follow-up





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


@diagnosis




@follow-up





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


B- and T-cell clonality analysis

polyclonal

monoclonal

heteroduplex analysis Capillary electroforese

polyclonal

monoclonal


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)


@diagnosis




@follow-up





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)


@diagnosis




@follow-up





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

BHS TRAINING COURSE 2013-2015: SEMINAR 1 Laboratory …Friedel Nollet, Ph.D. Molecular Biologist...

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Transcript of BHS TRAINING COURSE 2013-2015: SEMINAR 1 Laboratory …Friedel Nollet, Ph.D. Molecular Biologist...