Genetics of Adolescent/Young Genetics of Adolescent/Young Adult ALLAdult ALL

(Cytogenetics)(Cytogenetics)Christine J Harrison

Professor of Childhood Cancer Cytogenetics

Cytogenetics Group

Clinical Trial Service Unit

Clinical Trial Coordinators

UK Cancer Cytogenetics Group

Leukaemia Research Cytogenetics Group Leukaemia Research Cytogenetics Group

Cytogenetic subgroup by ageCytogenetic subgroup by age

0%

20%

40%

60%

80%

100%

0

1-4

5-9

10-14

15-19

20-24

25-29

30-34

35-39

40-44

45-49

50-54

55-59

Age (years)

Pro

porti

on o

f cas

es

t(4;11)/11q23 t(12;21) HeH t(9;22) t(1;19) 14q32 Other

<

Moorman 2007

AYA by Age at Diagnosis and Treatment Trial AYA by Age at Diagnosis and Treatment Trial 19901990--present (n=1,205)present (n=1,205)

0

40

80

120

160

200

13 14 15 16 17 18 19 20 21 22 23 24

Age at diagnosis

Num

ber o

f pat

ient

s

ALL2003 (N=384) ALL97 (N=221) UKALLXI (N=108) UKALLXII (N=492)

Age groups (n=1,205)Age groups (n=1,205)

15-19 yearsn=544, 45%

13-14 years n=432, 36%

20-24 years n=229, 19%

Sex Ratio (1.74M:1F)Sex Ratio (1.74M:1F)

Males, 765,63%

Females, 440, 37%

Immunophenotype (n=1,132)Immunophenotype (n=1,132)

Mature-B, N=4, 0%

BCP-ALL,N=894, 79%

T-ALL, N=234, 21%

Immunophenotype not known in 73 (6%) cases

AgeAge--specific incidence of Tspecific incidence of T--ALLALL

0%

20%

40%

60%

80%

100%

13 14 15 16 17 18 19 20 21 22 23 24

T-ALLBCP-ALL

Estimates of the incidence of Estimates of the incidence of TT--ALL specific abnormalitiesALL specific abnormalities

Abnormality Incidence

Children AYA Adults

22%

12%

2%

17%

2%

51%

4%

2%

SIL-TAL1/t(1;14) 16% 9%

t(11;14)(p13;q11)/LMO2 2% 0%

t(10;14)/TLX1 (HOX11) 4% 24%

t(5;14)/TLX3 (HOX11L2) 11% 6%

CALM-AF10 8% 0%

CDKN2A/B 46% 44%

MLL 5% 0%

NUP214-ABL1 3% 3%

Cytogenetics of BCPCytogenetics of BCP--ALL in 13ALL in 13--24 year olds 24 year olds (n=837)(n=837)

IGH@12%

IGH@-CRLF23%

Normal13%

Other27%

HeH16%

Hypo (<40)3%iAMP21

4%CRLF2

3%

t(17;19)0%t(4;11)

3%

11q231%

t(1;19)3%

t(9;22)8%

t(12;21)4%

Estimates of the incidence of BCPEstimates of the incidence of BCP--ALL ALL specific abnormalitiesspecific abnormalities

Abnormality No. Positive No. Tested Incidence <13 years >24 years

t(9;22) 68 781 9% 2% 20%t(1;19) 27 696 4% 3-5% 3-5%t(12;21) 25 531 5% 25% <1%t(17;19) 4 696 <1% <1% <1%t(4;11) 27 780 4% 2% 5-10%11q23 6 780 1% 2% 2%HeH 149 754 20% 35% 10%Hypo (<40) 23 754 3% 1% 5%iAMP21 26 531 5% <2% <2%IGH@ 31 216 14% 3% 15%IGH@-CRLF2 8 284 3% <1% ~5%CRLF2 5 115 4% ~5% ?Normal 102 696 15%Other 227 696 33%

4 cases had iAMP21 plus CRLF2 and 2 cases had iAMP21 plus an IGH translocation

““OthersOthers””

Total n=227

• Abnormal 9p ~50%• +21 ~4%• +8 ~4%• +5 ~4%

Duplication of chromosome 21 Duplication of chromosome 21 involving amplification of involving amplification of RUNX1RUNX1

Intrachromosomal amplification of chromosome 21 Intrachromosomal amplification of chromosome 21

iAMP21iAMP21

ETV6RUNX1

Interphase FISH Metaphase FISH Metaphase FISH

Demographic Profile of Demographic Profile of iAMP21 patientsiAMP21 patients

• Older children/Adolescents – Median age 10 years

• Common/Pre-B immunophenotype• Low WBC

EFS of 28 iAMP21 patients on MRC ALL97EFS of 28 iAMP21 patients on MRC ALL97

0.00

0.20

0.40

0.60

0.80

1.00

Eve

nt F

ree

Sur

viva

l

0 1 2 3 4 5 6 7 8 9Time from diagnosis (years)

t(12;21)

HeH

Other11q23/MLL

<40 chr’s

t(9;22)

iAMP21

Moorman et al (2007) Blood 109:2327

88%

83%

72%61%

50%

38%29%

Overall Survival - 5yr 69% (SE 8.6)

Event Free Survival - 5yr 26% (SE 8.3)

N=29

22 relapses (15 BM, 5 CNS, 2 BM&CNS)11 deaths (1 in 1st CCR)

October 2007 Update

0.00

0.25

0.50

0.75

1.00

Prop

ortio

n

0 1 2 3 4 5 6 7 8 9 10 11 12Years from Diagnosis

Leukaemia Research Cytogenetics Group

Outcome of iAMP21 patients on MRC ALL97

DecisionDecision

To treat iAMP21 patients as highTo treat iAMP21 patients as high--risk risk in the current childhood trial: ALL2003in the current childhood trial: ALL2003

iAMP21: outcome in ALL2003iAMP21: outcome in ALL20030.

000.

250.

500.

751.

00E

vent

Fre

e Su

rviv

al (%

)

0 1 2 3 4 5Years from diagnosis

Two deaths in remissionInfection following BMT One BM relapse

Post BMT9yr, Female

n=3933 treated as high risk14 received BMT

iAMP21iAMP21Duplication of chromosome 21 Duplication of chromosome 21

involving amplification of involving amplification of RUNX1RUNX1

Every abnormal chromosome 21 has a different Every abnormal chromosome 21 has a different morphologymorphology

7256

7619

8983

BAC BAC ProbesmBAND G-band

A

B

C

D

E

F

A B C D E F

mBANDINGmBANDINGG bandingG banding

Robinson et al (2007) Genes Chromosomes Cancer 46:318-26

A double-strand DNA break results in loss of atelomere and the formation of an unstable chromosome

Following replication the two sister chromatids fuse to form a dicentric chromosome

During anaphase this dicentric pulls apart resulting in breakage of the fusion bridge and production of an unstable chromosome with an inverted repeat

This process is repeated until the chromosome becomes stabilised by gaining a telomere. In this way it is possible to generate a chromosome with ladder like amplification.

Telomere

Centromere

RUNX1

KEY

The BreakageThe Breakage--FusionFusion--Bridge cycleBridge cycle

iAMP21iAMP21• iAMP21 defines a distinct patient subgroup of older

children/young adults with a poor prognosis• Chromosomal instability gives rise to complex

intrachromosomal rearrangements of chromosome 21• Genome wide they show the same abnormalities of B-

cell differentiation genes• No obvious differentially expressed genes• Studies are in progress to determine the initiating

mechanism • Currently FISH with probes

directed to RUNX1 is the only reliable diagnostic method

Normal 14

der(14)

der(19)

IGH@

Normal 19

BCL3

IGH@ IGH@ translocations in BCPtranslocations in BCP--ALLALL

RP11-270I13

Normal 19

der(19)

der(14)Normal 19

der(19)

der(14)

CEBPA

Within 6 nucleotides

IGH@-CEBP family

IGH Testing in ALL by Age (n=1,304)IGH Testing in ALL by Age (n=1,304)3% <10 yrs, 14% >10 years3% <10 yrs, 14% >10 years

NB Selected screeningNB Selected screening

2522

1 421

27

0

100

200

300

400

500

600

700

800

900

0-9 10-14 15-19 20-24 25-29 30

Age Group

Num

ber o

f pat

ient

s

Normal Translocation

t(14;19)(q32;q13)

der(19)

der(14)CEBPA

t(8;14)(q11;q32)CEBPD der(8)

der(14)

t(14;20)(q32;q13)CEBPB

der(20)

der(14)

t(14;14)(q11;q32)CEBPEder(14)

der(14)IGH@

translocations

IGH@IGH@--CEBP CEBP familyfamily

der(19)

der(14)

IGH@-CEBPG

IGH@translocations

t(8;14)(q11;q32)

t(14;19)(q32;q13)inv(14)(q11q32)

t(14;20)(q32;q13)

t(14;14)(q11;q32)

IGH@-CEBP family

0

1

2

3

4

5

6

7

8

9

t(8;14

)t(8

;14)

t(14;2

0)H

L60M

UTZ5

REH

SD1

TANO

UEPER36

5

ALL pati

ent (

C)

ALL pati

ent (

M)

BM

Samples

Fol

d C

han

ge

CEBPB

CEBPD

qRTPCR

IGH@IGH@--CEBP CEBP familyfamily

Translocation M:Fratio

Age range(median)

WBC rangex109/L

(median)

Current statuswhere available

t(14;19)(q32;q13) 2:7 10-44(19)

1-71(5)

1 dead4 CR

t(14;19)(q32;q13) 0:1 32 94 NA

t(14;20)(q32;q13) 1:2 13-35(15)

3-103(75)

2 CR

t(8;14)(q11;q32) 5:5 3-49(14)

2-375(7)

1 dead2 CR

t(14;14)(q11;q32)inv(14)(q11q32)

4:0 15-45(20)

1-24(13)

3 CR

IGH@IGH@--CEBP CEBP familyfamily

Four IGH@ translocations

Involve five partner genes from the same gene family – CCAAT enhancer binding-proteins

One subtype of haematological disease, B-cell precursor ALL in older children and young adults

Basic leucine zipper transcription factors implicated in proliferation and differentiation

Expressed in haematopoietic system – control of myeloid differentiation

Tumour suppressor and oncogenic effects in leukaemogenesis

Summary – IGH@-CEBP family

IGH@IGH@--ID4ID4

der(6)Normal 6

der(14)ID4

Centromere Telomere

A B

bHLH family of transcription factors – inhibitory proteins which regulate growth, differentiation, senescence and apoptosis

• qRTPCR

020406080

100120140160

6120 16503 Normal Thyroid BCP-ALLpatient without

t(6;14)

BCP-ALLpatient without

t(6;14)

Normal bonemarrow

Samples

Fold

cha

nge

ID4

IGH@-ID4

IGH@IGH@--ID4ID4

Patients• 13 BCP-ALL patients – recurrent

translocation• Low WBC (median 3x109/l,

range 1-11x109/l)• Age higher than expected for BCP-ALL

(median 16 yrs, range 6-48 years)

IGH@IGH@--EPOREPOR

EPORC10orf39

G248P81743H6G248P85762A4 G248P81603G11

G248P82252A3

RGL3LPPR2Telomere Centromere

G248P89814C12

der(14) der(19)

(C)

5kb

IGH@-EPOR

0

50

100

150

200

250

300

350

1067

2 diag

nosis

1067

2 rem

ission

(1)

1067

2 rela

pse

1067

2 rem

ission

(2)

All with

out tr

anslo

catio

nIG

H posit

ive ALL

High H

yperd

iploid

Saliva

ry BMFo

ld C

hang

e

EPOR

• qRTPCR

~900Kb~250Kb

IGH@CRLF2

50Kb

wcpX wcpY

IGH@IGH@--CRLF2CRLF2TSLP TSLP ((thymicthymic stromalstromal derived derived lymphopoietinlymphopoietin))

IGH@-CRLF2

• 33 patients

• BCP-ALL • CD34+ and CD33+

• Median age 16yrs (range 3-76yrs)

266 4 t(X;14)(p22;q32)

t(Y;14)(p11;q32)

Unknown

IGH@-CRLF2

11 patients

27 BCP-ALL cell lines – 2 with t(Y;14)

i

L1477/04 BCP-ALL cell line

7243

L326/05

L1200/028765

7116

7108

12882

L759/02

CRLF2 CSF2RA10kb

PAR1PAR2

L889/01

11687

BCP-ALL cell line

P2RY8

152kbYp11.32

• LDI-PCRXp22.33

IGH@-CRLF2

HIGH

low

• Expression

Patien

ts with

tran

sPati

ent w

ithout tr

ans

Cell lin

es w

ith tr

ans

Cell lin

es w

ithout tr

ans

Normal

bone marr

ow

0123456789

101112131415161718192021

p<0.0001 p<0.0001

Del

ta C

t val

ues

IGH@IGH@--CRLF2CRLF2Children (n=19)10 events: 8 relapses (7 died);

2 non-remitter/early death9 patients on ALL2003 – all in 1st CCR

0.00

0.25

0.50

0.75

1.00

Pro

porti

on S

urvi

ving

Eve

nt F

ree

0 2 4 6Years from diagnosis

IGH@ Partners

3p14 - FOXP1

2p13 - BCL11A

4p16 - FGFR34p16 - WHSC14p13 - RHOH

BCP-ALL

Previously reported

Published by LRCG and collaborators

On-going

Mature leukaemia/lymphoma

1q21- MUC11q24 - LHX4

1p34 – LAPTM5 1p22 - BCL10

1q21- FCGR2B1q21 - BCL9

3q27 - BCL6

6p25 - IRF46p22 - ID4*6p21 - CCND3

7q21 - CDK67q21 - ERVWE1

8q11 - CEBPD8q24 – MYC 9p13 - PAX5

10q24 - NFKB211q13 - CCND1

11q23 - DDX611q23 - PCSK7

11q23 - PAFHA1B212q23 - CHST11

12p13 – BCL114q11 - CEBPE14q11 - TCRA

15q12 - BCL816q23 - MAF

18q21 - BCL218q21 - MALT1

19q13 - CEBPA19q13 - CEBPG

19q13 - BCL3

20q11 - MAFB20q13 - CEBPB

22q11 - IGL

5q31 - IL3

1q21 – ITRA119p13 - EPOR

17q21 – IGF2BP1

Xp22/Yp11 – CRLF2

7p14 – TRG@

IGH@Translocations

IGH@ Partnersin BCP-ALL

BCP-ALL

Previously reported

Published by LRCG and collaborators

On-going

1q24 - LHX4

1q21 - BCL9

8q11 - CEBPD

14q11 - CEBPE

20q13 - CEBPB

5q31 - IL3

6p22 - ID4*

12p13 – BCL1

19q13 - CEBPA19q13 - CEBPG

19p13 - EPOR

Xp22/Yp11 – CRLF2

7p14 – TRG@

IGH@Translocations

IGH@ IGH@ translocationstranslocations

• IGH@ is a promiscuous locus: common link to the genes involved and their interrelated pathways

• Majority of patients are older children or adolescents

• Cytogenetics still identifies new translocations and subgroups

Conclusions to genetics of AYAConclusions to genetics of AYA

• They show abnormalities in common with childhood ALL, although the incidences are different

• There are some novel abnormalities emerging which are common in this age group

• Detailed analysis may highlight some as these as specific targets for therapy

AcknowledgementsAcknowledgements

Newcastle, UKNewcastle, UK•• Anthony MoormanAnthony Moorman•• Leukaemia Research Leukaemia Research

Cytogenetics GroupCytogenetics Group

Leicester, UKLeicester, UK•• Martin DyerMartin Dyer

Kiel, GermanyKiel, Germany•• Reiner SiebertReiner Siebert

Paris, FranceParis, France•• Olivier BernardOlivier Bernard

To findTo find IGH@IGH@ positive positive casescases

Normal 14 der(14)

der(19)

Screen by FISH with IGH@ breakapart probe

Not: • ETV6-RUNX1 positive• High hyperdiploidy• BCR-ABL1• t(1;19)

IGH@-CRLF2

13 14 15 16 17 18 19 20 21 22

1 2 3 4 5 6 7 8 9 10 11 12

PAX5

CDKN2A/B

IKZF1

• aCGH

X Y

IGH@-CRLF2

0

0.2

0.4

0.6

0.8

1

1.2

siCRLF2 Negative control siRNA

Fold

cha

nge

siMENIN + CRLF2 AbsiCRLF2 + CRLF2 Ab

siCRLF2 + IgG2 Ab

siMENIN + CRLF2 AbsiCRLF2 + CRLF2 Ab

siCRLF2 + IgG2 Ab

• Knockdown

Untreated unstained

Untreated unstained

IGH@-CRLF2

Gated

0

10

20

30

40

50

60

70

80

90

UL UR LL LR

Quadrant

% o

f cel

ls

siCRLF2siMENINMOCK

siMENIN + pSTAT5 AbsiCRLF2 + pSTAT5 Ab

Isotype control + pSTAT5 Ab

No difference in;

Apoptosis

pSTAT5 Why?

Cell cycle

• Biological consequences

siCRLF2 siMENIN

IGH@-CRLF2• JAK2 mutation?

WT - TTCTGCTTATCAGAGAAGAA

GGA - R683G

TTC - I682F

IGH@-CRLF2

hCRLF2 – number and diameter

Data from Dr Melania Capasso

Mouse fetal liver cells

• Retroviral transfection

Data from Dr Melania Capasso

•CRLF2 expressing cells are less differentiated compared to EV cells

•Low CRLF2 expressing cells are more differentiated than high CRLF2 expressing cells

CD43+/CD19+

IGH@-CRLF2

Numbers of AYA by Trial and Year of diagnosis (N=1,179)

0

20

40

60

80

100

1990

1991

1992

1993

1994

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

Year of diagnosis

Num

ber o

f pat

ient

s

ALL2003 (N=335) ALL97 (N=243) UKALLXI (N=107) UKALLXII (N=493)