1 Balanced Translocation detected by FISH. 2 Red- Chrom. 5 probe Green- Chrom. 8 probe.
MyeChild & UKALL2011 trial custom FISH probe … & UKALL2011 trial custom FISH probe experience ......
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Transcript of MyeChild & UKALL2011 trial custom FISH probe … & UKALL2011 trial custom FISH probe experience ......
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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5000
10000
15000
20000
25000
30000
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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6
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14
Good Risk Intermediate Risk Poor Risk
Nu
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Pat
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MyeChild01 Results so far…
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4
6
8
10
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14
Good Risk Intermediate Risk Poor Risk
Nu
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of
Pat
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ts
CBFB-MYH11
RUNX1-RUNX1T1
MyeChild01 Results so far…
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4
6
8
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14
Good Risk Intermediate Risk Poor Risk
Nu
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Pat
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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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6
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Good Risk Intermediate Risk Poor Risk
Nu
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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
0
20
40
60
80
100
120
140
160
180
B-other ABL-class
ETV6-ABL1
0.0
00.2
50.5
00.7
51.0
0R
R
0 2 4 6 8 10Years
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