MyeChild & UKALL2011 trial custom FISH probe experience

Claire Schwab

Leukaemia Research Cytogenetics Group

Newcastle University

Leukaemia Research Cytogenetics Group

• Established in 1992 with funding from LLR/Bloodwise

• Collect cytogenetic data for UK treatment trials of acute leukaemia

• Discover and characterise new genetic abnormalities in acute leukaemia using cytogenetic and molecular approaches

• Investigate the epidemiology and prognostic relevance of new and existing genetic abnormalities

• Link between trial coordinators and regional cytogenetic laboratories.

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35000 Inter-B-NHL

LLR CellBankiAMP21study11q23WorkshopMyeChild01

AML16

AML15

AML14

AML12

AML11

AML10

RelapseTrialsUKALL60+

UKALL14

UKALLXII

UKALLXA

Accrual of cases to LRCG database

MyeChild01

• AML clinical trials use three prognostic cytogenetic groups: good, intermediate and poor risk

• International Trial in Children with AML, MyeChild01 opened in Summer 2016

• Review of existing and novel genetic risk factors for stratification of patients

MyeChild01 Risk Groups

~30%

~40%

~30%

Patient entered to trial

LRCG to inform cytogenetics lab by request form and email

Local lab to perform cytogenetics/FISH to identifyt(8;21), inv(16), MLL, 3q abn,

-5, -7, t(6;9), BCR-ABL1,abn 12p (ETV6)

If negative for all above test for

NUP98-NSD1 (Kreatech NUP98 BA probe)

If MLL+vedetermine

partner

Intermediate Riskt(9;11)(p21;q23)/MLL-MLLT3

t(11;19)(q23;p13.3)/MLL-MLLT1

Other MLL rearrangements

Poor Riskt(6;11)(q27;q23)/MLL-MLLT4t(4;11)(q21;q23)/MLL-AFF1

t(10;11)(p11-p14;q23)/ MLL-MLLT10

If negative test for MNX1-ETV6 (ETV6 BA probe)

If positive test withNUP98-NSD1 specific probe

If negative test with CBFA2T3-GLIS2 specific probe

If positive test withMNX1-ETV6

specific probe

If negative test for FLT3-ITD, NPM1 and CEBPA mutations

These tests can be

performed by LRCG

Cytogenetic testing in MyeChild01

All other positive cases – no further

testing required

Custom Probes – Cytocell MyProbes

MyeChild01 Results so far…

• 28 patients entered

• 3 with results pending….

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MyeChild01 Results so far…

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CBFB-MYH11

RUNX1-RUNX1T1

MyeChild01 Results so far…

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t(9;11)

t(11q23) MLL-ELL

Other

MyeChild01 Results so far…

NUP98-KDM5A

NUP98-NSD1

t(10;11)

CBFA2T3-GLIS2

t(6;11)

Monosomy 7

Monosomy 7/abn 3q

abn 12p/NUP98-KDM5A

FLT3-ITD

MyeChild01 Results so far…

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Case Study #1

• 1 year old girl referred to Glasgow Cytogenetics Laboratory

• 46,X,der(X)(Xpter->Xq24:),ins(5;11)(5pter->5q35::11p11.2->11p15::5q35->5qter)x2,der(11)(Xq24::11p11.2->11qter)

• NSD1-NUP98 fusion

WCPX/WCP11

NUP98/NSD1

Case Study #2

• 1 year old boy at Manchester Cytogenetics Laboratory

• 46,XY,der(18)t(1;18)(q22;p11.2)

• CBFA2T3-GLIS2 by FISH and RT-PCR

CBFA2T3/GLIS2

Case Study #3

• 1 year old boy referred to Cardiff cytogenetics

• 46,XY,der(7)del(7)(q11q36)t(7;12)(q36;p13),der(12)t(7;12)(q36;p13)[2]/49,sdl1,+13,+19,+22[7]/49,sdl2,+der(7)del(7)t(7;12),+10,+19,+21[5]

• MNX1-ETV6 fusion

MNX1/ETV6

Conclusions

• Successful opening of MyeChild01

• Fewer good risk patients than expected

• Full range of the rare cryptic abnormalities associated with a poor risk

• Cytogenetic and FISH essential for risk stratification and to identify markers for MRD monitoring

Cytogenetics of BCP-ALL

25%

30%4%

3%2%

1%1%

4%

30%

t(12;21)/ETV6-RUNX1

High Hyperdiploidy

11q23/MLL rearranged

t(9;22)/BCR-ABL1

iAMP21

t(17;19)/TCF3-HLF

Hypo/Haploidy

t(1;19)/TCF3-PBX1

B-other

Good risk Poor risk Intermediate

Chromosome abnormalities are the hallmark of childhood ALL and

are used in risk stratification

Roberts KG et al, NEJM, (2014) 371(11):1005-1015Moorman AV et al, Lancet Oncol. (2010) 11(5):429-438Schwab CJ et al, Haematologica. 2013;98(7):1081-1088Nebral K et al, Leukemia. 2009;23(1):134-143Russell LJ et al, JCO, (2014) 32(14):1453-1462Clappier E et al, Leukemia (2014) 28(1):70-7Yasuda T et al Nat. Genetics (2016) 48(5):569-74Lilljebjörn H et al Nat. Commun (2016). 7:11790*denotes IGH partner gene

IntermediateRisk

Good Risk

BCR-ABL1-like/Ph-like

Poor Risk

B-Other BCP-ALL

Gene expression profiling identifies Ph-like ALL/BCR-ABL1-like BCP-ALL

• Accounts for ~5% of BCP-ALL

• Mutually exclusive of other established cytogenetic subgroups

• Shares the same high-risk of relapse and poor outcome

• Show novel kinase activating fusion gene or activation of JAK-STAT signalling and CRLF2 overexpression

BCR-ABL1-likeDen Boer (2009) Lancet Oncology 10:125-134

Ph-like ALLHarvey RC (2010) Blood 116:4874-4884

Novel kinase activating fusion genes

EBF1-PDGFRB +ve patients treated with imatinib

Roberts et al 2012 Cancer Cell 22: 153-66; Lengline et al 2013 Haematologica 98: 146-8;

Weston et al 2013 J Clinical Oncology 31: 413-6Schwab et al 2016 Blood 127(18):2214-8

Homegrown Probes and/or RT-PCR for rare partners

FISH screening for fusionsCommercial Probes for PDGFRB, CRLF2 and JAK2

Cytocell MyProbes for ABL1 and ABL2Most common partner genes EBF1, SSBP2, PAX5, NUP214, MEF2D

PDGFRB FISH also detects CSF1R rearrangements

1R0G2F

PDGFRB FISHEBF1 FISH

0R0G2F

EBF1 FISH

dup(5)(q14q32)

SSBP2-CSF1R fusion associated with duplication of 5q

SSBP2-CSF1Rfusion

SSBP2 5q14

CSF1R 5q33CSF1R 5q33

SSBP25q14

SSBP2 CSF1R

2R2G2F

CSF1R break-apart probe

Case Study

• 13 year old girl

• 46,XX,t(1;5)(q22;q32)[14]/

52-53,idem,+der(5)t(1;5),

+6,+21,+21,inc[cp3]

• PDGFRB +ve by FISH

1R1G1F 1R2G1F

CSF1R FISH MEF2D FISH

1R1G1F 1R2G1F

MEF2D-CSF1R fusion

FISH screening of UKALL2003 B-other

B-other FISH

Frequency in B-other

ALL

Fusions

ABL1

5/486

1%

ETV6-ABL1 (n=2)

Unknown (n=3)

PDGFRB/CSF1R

17/491

3.4%

EBF1-PDGFRB(n=10)

ATF7IP-PDGFRB (n=1)

SSBP2-CSF1R(n=1)

MEF2D-CSF1R(n=1)

Unknown (n=4)

JAK2

2/481

<1%

PAX5-JAK2 (n=2)

ABL2

1/454

<1%

ZC3HAV1-ABL2 (n=1)

CRLF2*

65/503

13%

P2RY8-CRLF2(n=43)

IGH-CRLF2

(n=22)

*FISH and/or MLPA

Outcome

• The 5-year event free survival (EFS) of the entire B-other cohort was 82% with a relapse rate (RR) of 12%

• Patients with rearrangements of PDGFRB/CSF1R (n=17) and showed an inferior outcome, with EFS rates of 31% RR of 64% re (p<0.005)

• PDGFRB rearrangements associated with high MRD and induction failure

ABL-class fusions – D28 MRD levels and outcome

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B-other ABL-class

ETV6-ABL1

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B-other (n=738)

ABL-class (n=17)

5 yr RR: 63% v 12%Hazard ratio = 7.1, p<0.001Still significant even after adjusting for MRD and restricting to regimen C

Genetics of ALL2003 patients with refractory disease (M2/M3 marrow or >5% MRD post induction)

T-ALL32%

ETV6-RUNX1

2%

HeH13%

MLL7%

iAMP211%

Hap/HoTr1%

Failed/ND8%

PDGFRB/CSF1R9%

ABL1/ABL2 2%

CRLF2-d 3%

Unknown22%

B-other36%

What action is required by the local genetics lab?• FISH with PDGFRB dual colour break apart probe• Review BCR-ABL1 FISH results for ABL1 involvement• Review cytogenetic result for 1q25 (ABL2) involvement

Criteria for testingPoor responding patients who have: • Induction failure or M2/M3 marrow at the end of induction• MRD >1% at day 28 or >0.5% at week 14AND• B-other ALL or T-ALL

What diagnostic testing is performed by the LRCG?• If 1q25 is abnormal by cytogenetics, FISH with ABL2 probe• If BCR-ABL1 FISH shows extra ABL1 signal, FISH with ABL1 break apart

probe • Identify partner gene in positive cases

What happens if the case has an ABL-class fusion? • Discuss result with UKALL2011 trial coordinator and consider

treatment with a tyrosine kinase inhibitor

UKALL2011 screening algorithm for ABL-class fusions

1 2 3

ZC3HAV1 ex12ABL2 ex2

RT-PCR

ABL2 FISH

• 17yr old male

• 46,XY,t(1;7)(q25;q3?5),add(3)(p1?3)

• >1% MRD day 28

• Imatinib 400mg with first salvage FlA-Ida

• Marrow at end of this shows 2% blasts by flow – 0% by morphology MRD awaited

• Switched to Dasatinib due to low level disease

• Allograft planned with sibling donor following this course

ZC3HAV1-ABL2 fusion associated with t(1;7)(q25;q34)

Other B-other FISH

Targeted screening to

patients <2yrsor visible 15q

abn(n=91)

Abnormals

BRD9-NUTM1(n=1)

Unknown (n=1)

UKALL2003 trialB-other n=810, with FC n=526, with DNA n=308

NUTM1

n=94

dic(9;12) n=7PAX5-JAK2

n=2Unknown n=5

PAX5

n=155

n=12?TCF3

?TAF15?EWSR1?ARID1B

ZNF384

n=122

n=3?CSF1R?BCL9

MEF2D

ETV6 (n=425)

ETV6 rea (n=6)IKZF1 rea

(n=1)

ETV6-RUNX1-like

IKZF1 (n=54)

Acknowledgements

Leukaemia Research Cytogenetics GroupChristine HarrisonAnthony MoormanAll members of LRCG past and present

UKCCG member laboratories

Cytocell MyProbes Team