De moleculaire testen voor maligne lymfoom
Patricia Groenen
UMC Nijmegen
For the BIOMED-2 group
Moleculaire Diagnostiek in de Pathologie 15.02.08
Moleculaire diagnostiek voor maligne lymfoom
•
•
•
Diagnose: maligne lymfoom/ reactief proces
Moleculaire clonaliteits analyse
Welk type lymfoom: Classificatie
Detectie van chromosomale translocaties
Prognose ?
IGH mutatiestatus bepaling bij CLL
Diagnostische verrichtingen
160
140
120
100
2002
2003
2004
80
2005
2006
2007
60
40
CLL
t(11;18) RT-PCR
Clonaliteit
0
t(14;18) PCR
20
• Weefsel nr (multiple weefsels/patiënt)
• Ca. 65 % B-cel clonaliteit, ca. 35% T-cel clonaliteit
• Ca.60% consultancy cases
Moleculaire test (1)
•
Diagnosticeren: maligne lymfoom/ reactief proces
•
Genetische identiteit van verdacht maligne cellen
⇒ In principe zijn B-/T-cel maligniteiten clonale ziekten
•
Moleculaire clonaliteits analyse:
Detectie Ig en TCR genherschikkingen
Antigen receptors
V
IgH
D
IgH D
J
V
J
C
V
C
J
J
C
C
C
B-lymphocyte cell membrane
IgL
TCRα
V
C
C
TCRβ
C
C
CD79a
C
CD79b
IgL
V
V
J
D
J
C
C
CD3
ε CD3
δ
CD3
γ
T-lymphocyte membrane
CD3
ξ
CD3
ξ
Gene Rearrangements IGH
IGH 14q32
VH
1
2
3
4
7
n
DH
JH
s
1 2 3 4
1 2 3 4 5 6
C
D to J joining
1
2
3
4
7
n
1 23 4 5 6
V to DJ joining
1
2
3
4
7
23 4 5 6
Heavy chain
IGK gene rearrangement
Ig expressed
IGL gene rearrangement
Rearrangeable gene segments in Ig and TCR genes
Gene segments
(non-polymorphic)
IGH
IGK
IGL
TCRA
TCRB
TCRG
TCRD
44 (7)
66 (7)
43 (7)
76 (7)
38 (10)
56 (11)
46 (32)
54 (32)
47 (23)
67 (30)
6 (4)
9 (4)
8
8
27 (7)
–
–
–
2
–
3
6
6
5
5
4
5
53
61
13
13
5
5
4
4
V segments
Functional (family)
Rearrangeable (family)
D segments
Rearrangeable (family)
J segments
Functional
Rearrangeable
Detectie genherschikkingen mbv PCR approach
BIOMED-2 report, LEUKEMIA 2003;17:2257-2317
BIOMED-2 Concerted Action BMH4-CT98-3936:
PCR-based clonality studies in lymphoproliferations
Coordination
NL
Rotterdam
J.J.M. van Dongen
A
B
C
D
E
NL/BE
ES
PT
GB (south)
GB (north)
F
DE
G
FR
Pathology Review Panel
(+ national pathologists)
Rotterdam
Salamanca
Lisboa
Southampton
Leeds
Kiel
Paris
A.W. Langerak
J.F. San Miguel
A. Parreira
J.L. Smith
G.J. Morgan
M. Kneba
E.A. Macintyre
J.H.J.M. van Krieken
Rotterdam
Salamanca
Lisboa
Southampton
Leeds
Kiel
Paris
NL
GB (north)
A.W. Langerak
R. García Sanz
M. Gonzalez Diaz
D. Gonzalez
J. Diamond
P. Gameiro
R. Fragoso
L.Lavender
E. Hodges
H. White
P.A. Evans
J. Droese
M. Brüggemann
C. Pott
E. Delabesse
K. Beldford
V. Asnafi
J.H.J.M. van Krieken
Ph.M. Kluin
A.H. Mulder
S.T. Pals
A. Jack
Groningen
Ph.M.Kluin
E. Schuuring
Madrid
Porto
London
C. Sambade
L. Foroni
Nijmegen
R. Villuendas
B. Martinez-Delgado
P.J.T.A. Groenen
London
T.C. Diss
Amsterdam
Cardiff
M. Spaargaren
K. Mills
Roeselare
E. Moreau
E. Boone
Cambridge
D. Pearson
Nottingham
I. Carter
Norwich
B. Jennings
Aberdeen
B.J. Milner
M. Vickers
Münster
Rouen
C. Kersting
C. Bastard
ES
Berlin
Paris
M. Hummel
F. Davi
J.F. García
T. Flores Corral
M.A. Piris
Heidelberg
Créteil
C.R. Bartram
T. Flohr
M.H. Delfau-Larue
Göttingen
L. Trümper
W. Jung
Rotterdam
J.W.W. Coebergh
Würzburg
(epidemiologist)
M. Ott
P. Starostik
Toulouse
T. Al Saati
Paris
T. Molina
Pierre-Benite
G. Salles
L. Bassegio
PT
J.Cabeçadas
C. Sambade
GB (south)
B. Jasani
P. Isaacson
B.S. Wilkins
DE
H. Herbst
H. Stein
M. Ott
R. Parwaresch
M. Tiemann
M.L. Hansmann
S. Oeschger
FR
T.J. Molina
G. Delsol
F. Berger
D. Canioni
P. Gaulard
C. Copie
F. Charlotte
S. Laberge
BIOMED-2 Concerted Action BMH4-CT98-3936: PCR-based clonality studies
Objectives of BIOMED-2 Concerted Action
1. Design and standardization of PCR primer sets and PCR
protocols for detection of Ig/TCR gene rearrangements and
well-defined chromosome aberrations as targets for clonality
assessment.
2. Assessment of Ig/TCR gene rearrangement patterns and
chromosome aberrations in WHO-defined disease categories,
using the standardized PCR primers and PCR protocols
Management structure
More than 47 European institutes (including 30 PCR laboratories) in
seven national networks (NL, ES, PT, GB-south, GB-north, DE, FR)
Gene Rearrangements IGH
IGH 14q32
VH
1
2
3
DH
4
7
n
JH
1 2 3 4
s
C
1 2 3 4 5 6
D to J joining
1
2
3
4
7
n
1 23 4 5 6
V to DJ joining
1
2
3
4
7
23 4 5 6
Genescanning
PCR GeneScan analysis of IGH tube A: VH-JH rearrangements
VH
DH
6 VH-FR1 primers
(VH family primers)
JH
JH primer
CTGTGCAAGAGCGGGCTATGGTTCAGGGAGTTATGGCTACTACGGTATGGACGTCTGG
CTGTGCAAGAGGACGAAACAGTAACTGCCTACTACTACTACGGTATGGACGTCTGG
CTGTGCAAGAGAGATAGTATAGCAGCTCGTACAACTGGTTCGACTCCTGG
CTGTGCAAGAGATCCGGGCAGCTCGTTTTGCTTTTGATATCTGG
CTGTGCAAGAGCCTCTCTCCACTGGGATGGGGGGCTACTGG
CTGTGCAAGAGCAGCAGCTCGGCCCCCTTTGACTACTGG
CTGTGCAAGAGGACTTTGGATGCTTTTGATATCTGG
CTGTGCAAGAGGGTGGGAGCTACTAGACTACTGG
CTGTGCAAGGGTAGCTAAACCTTTGACTACTGG
CTGTGCAATATCTACTTTGACTACTGG
IGH VJ
Heterogeniteit in grootte
FR3
FR2
FR1
*
Heteroduplex PCR analysis of Ig / TCR genes
monoclonal cells
monoclonal cells in
polyclonal background
polyclonal cells
denaturation / renaturation
1
heteroduplexes
homoduplex
1
2
3
2
3
BIOMED-2 primers for Ig/TCR gene rearrangements
Basic principles
– recognition of (virtually) all functional gene segments
– design of primers for multiplexing
– position of multiplexed primers retain triplet spacing in case of
complete in-frame V-(D-)J rearrangements
Complementarity of BIOMED-2 primers
– recognition of as many gene segments as possible
e.g. multiple primers at different FR positions (IGH)
– inclusion of multiple types of rearrangements per locus
e.g. V-D-J, D-J, V-D
e.g. unmuted Ig targets: DH-JH and Kde
– inclusion of multiple Ig and TCR loci
clonal results in more than one tube
Gene Rearrangements IGH
IGH 14q32
VH
1
2
3
DH
4
7
n
JH
1 2 3 4
s
C
1 2 3 4 5 6
D to J joining
1
2
3
4
7
n
1 23 4 5 6
V to DJ joining
1
2
IGH V(D)JComplete genherschikking
3
4
7
23 4 5 6
Gene Rearrangements IGH
IGH 14q32
VH
1
2
3
DH
4
7
n
JH
1 2 3 4
s
C
1 2 3 4 5 6
D to J joining
1
2
3
4
7
n
1
23 4 5 6
V to DJ joining
IGH DJIn-complete genherschikking
Pro-B-cel
BIOMED-2 Concerted Action BMH4-CT98-3936: PCR-based clonality studies
Multiplex Multi target PCRs
PCR targets for diagnostic clonality studies
Ig genes:
IGH:
IGK:
IGL:
VH-JH
Vκ-Jκ
Vλ-Jλ
TCR genes
TCRB:
TCRG:
TCRD:
Vβ-Jβ
and Dβ-Jβ
Vγ-Jγ
Vδ-Jδ, Dδ-Dδ, Dδ-Jδ, and Vδ-Dδ
⇒ Complementariteit:
Multiple targets voor B- en T-NHL
Multiple PCRs per target
Inclusie ‘ongemuteerde’targets: IGH-DJ en IGK-DE
⇒ Hoge detectie graad
and
and
DH-JH
ΚDE
Resultaat van B-/T-cel clonalititeits analyse
Polyclonaal
Oligoclonaal
Monoclonaal
Complementarity: multiple PCR targets
Reactive dd T-cell malignancy (AILD) ?
TCRB- DJ
T04-15567 (1288)
50 ng
200 ng
Polyclonal
-80% T-cells of which 25% suspect malignant
-DNA quality sufficient (par: 300 bp)
-NB: Detection of polyclonal VDJ-rearanged TCRB genes
TCRG
Clonaliteits analyse
weefsel en cytologische preparaten
T02-02025 DNA 1351, Case 1 workshop
C07-03546 Giemsa staining
Mantle zone
Germinal center
Large and small cells
“bont beeld”, probably reactive
Clonaliteits analyse
cytologisch preparaat (C07-03546)
IGH-DJ
IGH-VJ
C07-03546
monoclonal
polyclonal
FR1
FR3
IGK-VJ
IGH-VJ
C07-03546
monoclonal
polyclonal
FR2
B-/T-cel Clonaliteits analyse
Kracht en Mogelijkheden
•
Complementariteit van multi-target PCR benadering, inclusief de analyse
van”unmutated B-cell targets” (IGH-DJ en IGK-DE)
⇒ Hoge mate van detectie clonale herschikkingen in lymfomen
97% van B-NHL , 95-99% T-NHL
•
•
Clonaliteits onderzoek in de context van pathologie, klinische bevindingen
Afstemming patholoog – moleculair bioloog
Moleculaire test (2)
•
Welk type lymfoom: Classificatie
•
Detectie van chromosomale translocaties
PCR -benadering
Fluorescent In Situ Hybridisatie
PCR analysis of BCL2 –IGH gene rearrangements
18q21 (BCL2)
60 - 70%
10 - 20%
MBR
mcr
BCL2 gene
Exon I
Exon II
Exon III
5’
3’
mcr1
MBR2
MBR1
Far 3´ -MBR
10 %
5´ -mcr
3%
Gebruik van JH consensus primer
% literatuur
mcr2
t(14;18)(q32:q21); Follicular lymphoma
MBR
1576
1577
Far 3’ MBR
mcr / 5’ mcr
Pos / neg
controles
1576
1577
Pos / neg
controles
1576
1577
Pos / neg
controles
MBR / IGH junction
MBR
I
II
JH
III
E
s
C
5’
MBR2 MBR1
JH
⇒ Patient met recidief ? vergelijk lymfklieren 1985 en 2005
⇒ Translocatie; exact hetzelfde breukpunt bevestigd (sequentie analyse)
t(14;18)(q32:q21); Follicular lymphoma
Fluorescence in situ hybridisatie
Bcl2- Split signal-probe
18q21 (BCL2)
MBR
Bcl 2; 18q21
I
Telomere
II
mcr
III
Centromere
Bcl2 (18q21) split-signal probe, folliculair lymfoom, weefselcoupe
⇒ Bcl2- breukpunt, fusiepartner ?
Moleculaire test (3)
IGH-mutatie status bepaling CLL
Tobin et al., 2003
Kaplan-Meier curve of the CLL patient material investigated in the study showing mutated (n = 79),
unmutated(n = 134), and VH3-21 cases (n = 31) with a median survival of 137, 64, and 83 months, respectively
IGH mutation status
• QC DNA of CLL blood / BM, % tumorcells >90%
• PCR and sequence analysis based on IGH-clonality assessment (Biomed-2)
Hypermutation test 1
330 bp
Hypermutation test 2
530 bp
Sequence analysis
(2060)
Blast in database IMGT
Comparison sequence sample with
the most homologous sequence in
the database
Samenvattend:
•
•
Diagnostiseren / classificeren van maligne lymfoom:
Moleculaire clonaliteits analyse
Detectie van chromosomale translocatie
Integratie uitslag moleculaire diagnostiek binnen PALGA
Risk–stratificatie, Prognose ?
IGH mutatiestatus bepaling bij CLL (Pathologie)
Chromosomale afwijkingen
(Antropogenetica, sectie cytogenetica)
(17p del en/of 11 q del en/of trisomy 12; high risk)
Uitslag naar de heamatoloog
?
BIOMED-2/Euroclonality Workshop
‘Clonality Assessment in Pathology’
Nijmegen
Feb 2006, 2007, 2008
Barcelona
03.11.2006
Istanbul
14.09.2007
Prof. dr. Jacques van Dongen, Dr. Patricia Groenen, Dr. Ton Langerak,
Prof. dr. Han van Krieken for the BIOMED-2 group for clonality testing