Sabine Franke, PhDCHU Liège, Centre for Human Genetics

20/04/12 ULG

Acquired alterations of

IGH and TCR loci in lymphoproliferative

disorders

Interuniversity course - HUMAN GENETCIS

Overview

what are lymphoproliferative disorders?

what are IGH and TCR?

what are alterations and how to detect them?

Blood cell development

2 major types of lymphocytes: B and T cells

Lymphoproliferative disorders (LPDs)

LPDs refer to several conditions in which lymphocytes are produced in excessive quantities.

Chronic lymphocytic leukemia Acute lymphoblastic leukemia Hairy cell leukemia lymphomas Multiple myeloma Waldenstrom’s macroglobulinemia Wiskott-Aldrich syndrome Post-transplant lymphoproliferative disorder Autoimmune lymphoproliferative syndrome (ALPS) ‘Lymphoid interstitial pneumonia’

Lymphoid malignancy B lineage T lineage NK lineage

Acute lymphoblastic leukemia– children 82 – 86% 14 – 18% < 1%– adults 75 – 80% 20 – 25% < 1%

Chronic lymphocytic leukemias 95 – 97% 3 – 5% 1 – 2%

Non-Hodgkin lymphomas– nodal NHL 95 – 97% 3 – 5% < 2%– extranodal NHL 90 – 95% 5 – 10% < 2%– cutaneous NHL 30 – 40% 60 – 70% < 2%

Multiple myeloma 100% 0% 0%

B, T, and NK lineage of lymphoid malignancies

Blood cell development

Gene rearrangements

of the antigen receptor

genes occur during the

lymphoid proliferation

The ability to produce billions of different antibodies in humans results from the production of variable regions of light and heavy antibody genes by DNA rearrangement.

The five major classes of heavy chain are IgM, IgG, IgA, IgD, and IgE.

http://www.biology.arizona.edu

Schematic representation of an immunoglobulin

Identify lymphocyte populations derived from one single cell using the unique V-J gene rearrangements present within these antigen receptor loci.

These gene rearrangements generate products that are unique in length and sequence in each cell.

(junctional region)

stepwise rearrangement of V, D, and J gene segments

Genes encoding antigen receptors are unique:-high diversity -developing lymphocytes through V(D)J rearrangement.

The production of variable regions of light and heavy antibody genes by DNA rearrangement.

T-cells

T-cells have receptor gene rearrangement.

T-cell receptor gene rearrangement

Receptor for antigen on the majority of mature T-cells consists of two polypeptides alpha and beta that are linked by disulphide bonds and are associated with CD3

A small population of mature T-cells express a different TCR heterodimer in association with CD3. This is composed of two polypeptides designated gamma and delta

The variable domain of both the TCR α-chain and β-chain have three hypervariable or complementary determining regions (CDRs)

So how much variation is possible through recombining gene fragments?

Over 15,000,000 combinations of variable, diversity and joining

gene segments are possible.

Imprecise recombination and mutation increase the variability

into billions of possible combinations.

Estimated diversity of human Ig and TCR molecules

IgH Igα Igג TCR α β γ δ molecules molecules

Number gene segmentsV gene segments ~44 ~43 ~38 ~46 ~47 ~6 ~6D gene segments 27 2 3J gene segments 6 5 4 53 13 5 4

Combination diversity >2x10 6 2x10 6 <5000

Junctional diversity ++ + + + ++ ++ ++++

Total diversity >10 12 >10 12 >10 12

Clinically relevant testing

• Reactive versus malignant

• B-cell versus T-cell malignancy

• New lymphoma versus recurrence

• Assessment of remission and relapse

• Clinically relevant: bone marrow involvement (relation with prognosis)

• Evaluation of treatment effectiveness detection of minimal residual disease - treatment

PCRDiscrimination between monoclonal and polyclonal Ig/TCR gene PCR products

GeneScanning analysisFast, accurate, sensitivenon-quantitative monitoring of clonal proliferationsNeed sequence equipment

Heteroduplex analysisSensitivity ~5-10%

available to most laboratories

Design of novel primer sets for detection of Ig/TCR rearrangements

BIOMED-2 study (multiplex PCR)

Ig genes: IGH: VH-JH and DH-JH IGK: V-J and Kde rearrangements IGL: V-J

TCR genes: TCRB: V-J and D-J TCRG: V-J TCRD: V-J, D-D, D-J, and V-D

BIOMED-2 clonality strategy

Suspected B-cell lymphoma

Suspected lymphoma of unknown origin

SuspectedT-cell lymphoma

IGH V(D)J FR1, FR2, FR3IGH DJ(A)IGK-VJ and DE

TCRGVJ (A and B)TCRB V(D)J (A and B)

TCRB DJ

Van Dongen et al Leucemia 2003

polyclonallym phocytes

m onoclonalleukem ia

IG H tube C : V -FR 3H J H

IG H tube C : V -FR 3H J H

BIO M ED -2 C oncerted Action BM H 4-C T98-3936: PC R-based clonality stud ies

PCR GeneScan analysis

region

region

IG H tube B V -FR 2–JH H

Mw

M

PB

-MN

C

ton

s il

ES

-6G

BN

-10

FR

-5

200 -

300 -

400 -

500 -600 -

700 -

800 -

bp 50100

0

150

100 400300200

100200

0

300

100 400300200

12502500

0

3750

100 400300200

15003000

0

4500

100 400300200

15003000

0

4500

100 400300200

250-295 nt

D H JH

J prim erHV -FR 2 prim ersH

V H

BIO M ED -2 report: Leukem ia 2003; 17: 2257-2317

tonsil

ES-6

G BN -10

FR -5

PB-M N C

Example BIOMED-2 multiplex IGHVH-FR2–JH

Use of controls

From the patient to the analysis

Selection of material protocol Check DNA quality Clonality analysis

Examples of cases

Lymphoma? Relapse?

Case 1

Female 63 years Lymphoma in dec. 2004 FR1 polyclonal, FR3 monoclonal Feb. 2009 biopsy Relapse?

GeneScan analysis of controls

H2O

Case 1: Relapse?

FR3

12/2004

2/2009BiopsieFR3: 152 bp

12/2004BiopsieFR3: 152 bp

2000

600

Results

Controls okB-cell targets: IGH(VDJ) FR3 clonal

Molecular conclusion:Clonal rearrangement of the IGH gene was detected in this specimen.This gene rearrangement profile is identical to the one detected in the biopsie of 12/2004 and confirms the relapse of the disease.

Case 2

Female 81 years Biopsie Lymphoma?

Case 2

3/2009biopsieFR1327,11 bp

control

2700

100

Results

Controls okB-cell targets: IGH(VDJ) FR1 monoclonal

FR2 monoclonal

Molecular conclusion:Clonal rearrangements of the IGH gene were detected in this specimen.This gene rearrangement profile fits to the presense of a monoclonal B-cell population/B-NHL.

J 1.1J 1.2J 1.3J 1.4J 1.5J 1.6J 2.2J 2.6J 2.7

G TG G TC TAAG TG TC AAC ATC C ATTCC TG G TC C AATTG G C AAC ATC C ATTCTTC AAC C G AG TG AC AAC ATC C ATTCC TTG G G TC G AG AG AC AG AAC C C ATACC TG AG C TG AG AG G TAG G ATC C ATTCG TC C G AG TG AC AC TG TC C ATACTC C G AC TG G C ATG AC C C ATTCTC C G AC TG G C AC G AC C C G C TCG TC C G AG TG C C AATG TC C ATTC

(+53)(+53)(+55)(+56)(+55)(+58)(+56)(+58)(+52)

3 ' 5 '

D J

V fam ily prim ers J prim ers

TC R B tubes A and BV 2V 4V 5/1V 6a/11V 6b/25V 6cV 7V 8aV 9V 10V 11V 12a/3/13a/15V 13bV 13c/12b/14V 16V 17V 18V 19V 20V 21V 22V 23/8bV 24

(-204)(-201)(-197)(-201)(-201)(-201)(-198)(-201)(-198)(-201)(-198)(-198)(-198)(-198)(-201)(-198)(-201)(-201)(-193, )(-201)(-201)(-201)(-197)

inv

AAC TATG TTTTG G TATC G TC A C AC G ATG TTC TG G TAC C G TC AG C A C AG TG TG TC C TG G TAC C AAC AG AAC C C TTTATTG G TAC C G A C A ATC C C TTTTTTG G TAC C AAC AG AAC C C TTTATTG G TATC AA C AG C G C TATG TATTG G TAC AAG C AC TC C C G TTTTC TG G TAC AG AC AG AC C G C TATG TATTG G TATAAAC AG TTATG TTTAC TG G TATC G TAAG AAG C C AAAATG TAC TG G TATC AAC AA ATAC ATG TAC TG G TATC G AC AAG AC G G C C ATG TAC TG G TATAG AC AAG G TATATG TC C TG G TATC G AC AAG A TAAC C TTTATTG G TATC G AC G TG T G G C C ATG TAC TG G TAC C G AC A TC ATG TTTAC TG G TATC G G C AG TTATG TTTATTG G TATC AAC AG AATC A C AAC C TATAC TG G TAC C G AC A TAC C C TTTAC TG G TAC C G G C AG ATAC TTC TATTG G TAC AG AC AAATC T C AC G G TC TAC TG G TAC C AG C A C G TC ATG TAC TG G TAC C AG C A

5' 3 ’

V

TC R B tubes A and C : J A prim ers

J 2.1J 2.3J 2.4J 2.5

AG TG G C AC G ATC C ATTC TTC CAC TG TC AC G AG C C ATTC G C C CAG AG TC AC G AC C C ATTC G AC CC AC G AG C C AC AC G C G C

(+59)(+58)(+59)(+57)

3 ' 5 '

TC R B tubes B and C : J B prim ers

D J

D 1 prim er J prim ersD 2 prim er

(-252)

TC R B tube CD 1 G C C AAAC AG C C TTAC AAAG AC

5' 3 'D 2 (-137) TTTC C AAG C C C C AC AC AG TC

5' 3 '

BIO M ED -2 report: Leukem ia 2003; 17: 2257-2317

Analysis of TCRB gene rearrangements

D J

V fam ily prim ers J B prim ers

V

BIO M ED -2 report: Leukem ia 2003; 17: 2257-2317

Mw

M

PB

-MN

C

thym

us

Cel

l lin

e P

EE

R

ES

-9

ES

-6

TC R B tube B V -J

200 -

400 -

bp

800 -

250500

200 400300

0

750

450900

200 400300

0

1350

400800

200 400300

0

120016002000

7001400

200 400300

0

2100

ho

240-285 n t

he

ss

PB-M N C

cell line PEER

ES-6

ES-9

BIOMED-2 multiplex TCRB tube B

Use of clonality analysis

1. Making the diagnosisNormal reactive malignant

2. Involvement (staging)

3. Assessment of remission and relapseNormal reactive malignant

4. Evaluation of treatment effectiveness- detection of minimal residual disease (MRD)

MRD-based risk-group stratification (treatment reduction or treatment intensification)

Detection of abnormality at molecular level

PCR

Southern blot

FISH

Southern blot

Large region Large quantity of high molecular weight DNA Less sensitive Labor intensive

FISH – Fluorescence in situ hybridization

Relative large region Translocation partner has not te be known On metaphases and nuclei

breakpointreg ion

G ene A

no translocation translocation

Advantages:- detection of aberrations is independent of partner genes- m inim isation of fa lse positiv ity- identification of partner gene (or chrom osom e region), if m etaphases are present

Split-signal FISH

IG H gene com plex (#14q32.3)

1

V 3H V 66H D H JHV 2HV 1H

2 3 4 1 32 456

s s s sC C C 3 C 1 C 1

s s C 2 C 4 C 2s C s

IG K gene com plex (#2p11.2)V 1 V 2 V 3 V 76

1 32 4 5

J C Kde

IG L gene com plex (#22q11.2)JV 56V 3V 2V 1 J J C 3C 2C 1 J J J 654 J C 7

27

iE

3’E

iE 3’Eintr

on

RS

S

E

IG H -U(612 kb)

IG H -D(460 kb)

IG K -U IG K -D

IG L -U(309 kb)

IG L -D(343 kb)

IG L -U(520 kb)

Split-signal FISH for human Ig genes

TC R A TC R D and gene com plex (#14q11.2)

TC R B gene com plex (#7q34)

TC R G gene com plex (#7p14)

V 1 V 2 V 3 V 4 V 5 V n V 1 Rec

V 2 D J C

J J C

V 1 V 2 V 3 V 4 V 5 V n D 1 J 1 C 1 D 2 J 2 C 2

1 2 3 1 24 3

1 2 3 4 5 6 71 2 3 4 5 6

V 3

V 2 V 3 V 4 V 5 V 7 V 10 V 11V 8 V 9 J 1 C 1 J 2 C 2

1 2 3 31

functional V : 46 functional J : 53

functional V : 47

functional V : 6

TCR A/D -U TCR A/D -D

TCR B -U TCR B -D~20 kb

TCR G -U TCR G -D

Split-signal FISH for human TCR genes

Chromosome Genes Effect Occurrence Prognosis aberration involvedt(1;19)(q23;p13) E2A-PBX1 fusion 30% pre-B-ALL intermediate

t(4;11)(q21;q23) MLL-AF4 fusion 40% infant ALL poor

t(9;22)(q34;q11) BCR-ABL fusion 35% adult ALL poor

t(12;21)(p13;q22) TEL-AML1 fusion 25% childhood ALL good

del(1p32) SIL-TAL1 TAL1 15% childhood T-ALL intermediate

t(8;14)(q24;q32) IGH-MYC MYC 90% Burkitt’s lymphoma good

t(11;14)(q13;q32) BCL1-IGH Cyclin D1 >90% MCL poor

t(14;18)(q32;q21) BCL2-IGH BCL2 >90% FCL intermediate

t(2;5)(p23;q35) NPM-ALK fusion 50% ALCL intermediate

Prognostic value of chromosomal aberrations

FISH

• Excellent diagnostic tool for detection of well-defined chromosome aberrations

• Can also be used on tissue sections

• Requires limited handlingsusage of directly labeled probes

• Split-signal FISH has several major advantages– detection of aberrations, independent of partner gene– minimization of false-positive results – identification of partner gene or affected chromosome region, if metaphases are available

Summary

Gene rearrangements of the antigen receptor

genes occur

during the lymphoid proliferation These gene rearrangements generate products

that are

unique in length and sequence in each cell. Unique length allows by PCR discrimination of

monoclonality and polyclonality Standardized protocol (BIOMED-2)