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Ph-like (BCR/ABL1-like): un apporoccio baato sul target molecolare
Sabina Chiaretti, MD PhD
LE SFIDE DELLA MEDICINA DI PRECISIONE IN EMATOLOGIABologna 28 Giugno, 2018
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Topics: BCR/ABL1-like and other subgroups
• Molecular background
• Incidence
• Diagnosis
• Outcome and MRD
• Treatment
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• Molecular background
• Incidence
• Diagnosis
• Outcome
• Treatment
Topics: BCR/ABL1-like
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Haferlach et al, Blood 2005Chiaretti et al, CCR 2005
First report in adult ALLs
2005: first identification, by GEP, of a subset of adult B-lineage ALL clustering together with BCR/ABL1+ ALL cases
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Characterization of BCR-ABL1-like in children
GEP: Identification, within children (n=297), of a subset with a transcriptional profile resembling that of BCR/ABL1+cases (≈15-20%)
Clinical features: Hyperleukocytosis, poor response to VCR, ASP and DNM, poor prognosis (reduced DFS at 5 years andincreased resistance to induction)
Array-CGH: IKZF1, PAX5, TCF3 and VPREB1 deletions , CRLF2 deregulationDen Boer et al, Lancet 2009; Mullighan et al, NEJM 2009
Deletions are visualised in red, whereas amplifications are shown in blue.
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Impact of CRLF2 expression on B-ALL survival
CRLF2-high
p=0.004
Surv
ival
pro
bab
ility
Chiaretti et al, Leuk Res 2016
CRLF2-low
del(X)(p22.33p22.33)del(Y)(p11.32;p11.32)
t(X;14)(p22;q32)t(Y;14)(p11;q32)
Point mutations(F232C)
• 5-7% of pediatric BCR/ABL1-negative cases • >50% of cases associated with Down's syndrome• 5-15% of adult BCR/ABL1-negative cases • Associated with mutant JAK and IKZF1
d-CRLF2
P2RY8/CRLF2
IGH@/CRLF2
CRLF2-low CRLF2-highCRLF2 deregulationDCt>8 DCt<8
100
75
50
25
00 10 20 30 40 50 60 70
Months
Associated with the BCR/ABL1-like profile, but not useful as a diagnostic marker
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In high risk ALL, RNA-seq has identified novel mutations that involve TKs in the majority of cases. They appear to havetransforming capability and to respond to TKIs.
Roberts KG. Cancer Cell 2012; 22:153-66 Roberts KG, et al. N Engl J Med 2014;371:1005–1015
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BCR/ABL1-like ALL in adults. Genetics
Roberts KG et al, JCO 2016
Higher frequency of other kinase involvement.Some cases do no have any lesions.
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Kinase fusions identified in Ph-like ALLKinase gene
Fusion partners, n
Patients, n 5’ genes
ABL1 6 14 ETV6, NUP214, RCSD1, RANBP2, SNX2, ZMIZ1
ABL2 3 7 PAG1,* RCSD1,* ZC3HAV1*
CSF1R 1 4 SSBP2*
PDGFRB 4 11 EBF1, SSBP2,* TNIP1,* ZEB2*
CRLF2 2 30 IGH, P2RY8
JAK2 10 19 ATF7IP,* BCR, EBF1,* ETV6, PAX5, PPFIBP1,* SSBP2, STRN3, TERF2,* TPR*
EPOR 2 9 IGH, IGK*
DGKH 1 1 ZFAND3*
IL2RB 1 1 MYH9*
NTRK3 1 1 ETV6†
PTK2B 2 1 KDM6A,* STAG2*
TSLP 1 1 IQGAP2*
TYK2 1 1 MYB*
Roberts KG, et al. N Engl J Med 2014;371:1005–1015
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• Molecular background
• Incidence
• Diagnosis
• Outcome
• Treatment
Topics: BCR/ABL1-like
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BCR-ABL1-like. Incidence
Incidence is higher in AYA (10% in children; 27% in AYA). NEVER detected in cases positive for known fusion transcripts (BCR/ABL1, KMT2A-r, TCF3/PBX1)
However:- It highly depends on the denominator (all B-lineage ALL or B-neg ALL) and
- on the assay used for BCR/ABL1-like identification
- More adult cases are being evaluated →incidence in adults is almost equal to AYA ≥ 25%
Roberts KG, NEJM 2014 371:1005-15; Heroldt T, NEJM 2014 371:2235; Chiaretti S et al, in press
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• Molecular background
• Incidence
• Diagnosis
• Outcome
• Treatment
Topics: BCR/ABL1-like
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BCR/ABL1-like ALL diagnosis: a difficult issue
• Well identified subgroup by GEP
• Poor prognosis documented: therapy?
Difficult to identify these cases by techniques otherthan GEP
Furthermore, non univocal gene signature
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Still a difficult issue…
LDA
Q-RT-PCR
Quantification of expression of 15 transcripts(Kang BW et al, ASH 2013)
Quantification of expression of 10 transcripts(Chiaretti S et al, BJH 2018)
LDA, FISH, RT-PCR, NGS (RNA-seq, WGS, WES) (Roberts KG et al, NEJM 2014)
FISH, RT-PCR, Q-RT-PCR, NGS (Fasan A et al, ASH 2015)
Known fusion transcripts, JAK2 mutations , CRLF2-r(Herold T et al, Haematologica 2016)
CRLF2 expression (FC), JAK2 mutations, FISH for TK-rearrangements and CRLF2-r (Jain N et al, ASH 2017)
As far as possible, diagnostic assays should be available in most centers (or in centralized laboratories)
Integrated algorithms
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BCR/ABL1-like ALL diagnosis: a difficult issue
• Well identified subgroup by GEP
• Poor prognosis documented: therapy?
Difficult to identify these cases by techniques other than GEP
Furthermore, non univocal gene signature
To set up a diagnostic assay for a straightforward identification of BCR/ABL1-like ALL cases
To analyze the clinico-biologic and molecular features of BCR/ABL1-like adult ALL cases
To verify BCR/ABL1-like cases response to TKIs (ponatinib)
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BCR/ABL1-like ALL cases: methods (I)
342 Probsets
337 Probsets
Harvey R.C. et al.
9
CRLF2
• By meta-analysis of published and in house GEP data of B-NEG ALL, selection of 9 BCR/ABL1-like specifictranscripts (FC 1.5, p<0.001) + CRLF2
• Confirm the differential expression of the 10 transcripts by a Q-PCR approach
• Build a “BCR/ABL1-like predictor” on the basis of Q-PCR results (easy, fast and economic!)
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BCR/ABL1-like ALL cases: methods (II)
26 negative B-ALL
52 adult ALL samples previously evaluated by GEP
• Q-RT expression values shrinked into 3 principal components.
• A logistic regression model was used to examine the association among the 3 principalcomponents and BCR/ABL1-like cases.
• Generation of a score on principal components, used to classify the remaining samples(screening panel).
26 BCR/ABL1-like ALL cases
Discovery panel
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Development of the BCR/ABL1-like predictor and screening
Selection of BCR/ABL1-like specificgenes and validation by Q-PCR
Development ofBCR/ABL1-like predictor*
Screening
Factor loadings
PC1 PC2 PC3Gene1 2^(-ΔCt) 0.87921 -0.08451 0.19315
Gene2 2^(-ΔCt) 0.87162 0.41262 0.05974
Gene3 2^(-ΔCt) 0.80540 0.30143 0.22085
Gene4 2^(-ΔCt) 0.66775 0.40903 0.41369
Gene5 2^(-ΔCt) 0.57903 0.48726 0.48781
Gene6 2^(-ΔCt) 0.17357 0.90884 0.08792
Gene7 2^(-ΔCt) 0.61788 0.68881 0.14372
Gene8 2^(-ΔCt) 0.13521 0.65104 0.55717
Gene9 2^(-ΔCt) 0.17012 0.01622 0.90066
Gene10 2^(-ΔCt) 0.22407 0.26363 0.89565
54/194 newly identified BCR/ABL1-like patients (28%)- 9.5% children, 29% AYA, 30% adults -
1. Q-PCR of predictor genes in 129 B-NEG ALL
2. BCR/ABL1-like predictor
1. Selection of 10 predictor genes
2. Validation by Q-PCR in 52 B-NEG ALL
1. Identification of principal components (PCs)
2. Definition of a score
Chiaretti S et al, BJH in press*https://redcap.gimema.it/redcap
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BCR/ABL1-like ALL cases: clinical features
BCR/ABL1-like (n=54)
non-BCR/ABL1-like (n=140)
P-value
Gender (M/F) 36/18 78/62 p=ns
Age 32 (6-72) 28 (0-78) p=p=ns
Wbc x 109/l 22.6 (1.89-239) 12.4 (0.6-425) p=0.023
Plts x 109/l 47 (0.15-283) 47 (1-308) p=ns
Hb g/dl 9.7 (4.1-15.3) 8.9 (3.7-15.8) p=ns
CR rate 77.8% 89.2% p=0.06
No significant differences in age and gender: significantly higher WBC and lower remission rate
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Molecular features of the cases identified by the BCR/ABL1-like predictor
BCR/ABL1-like (N=28) non-BCR/ABL1-like (N=26)
• 96% of cases had a lesion typical of the BCR/ABL1-like subset• JAK/STAT mutations often concurrent with CRLF2 overexpression• CRLF2 overexpression detected in 69% of BCR/ABL1-like cases, though not exclusively• ABL-class rearrangements detected in 18% of BCR/ABL1-like ALL• TK-r cases often do not express high levels of CRLF2 Chiaretti S et al, BJH 2018
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• Molecular background
• Incidence
• Diagnosis
• Outcome and MRD
• Treatment
Topics: BCR/ABL1-like
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Outcome in childrenAssociated with male gender, hyperleucocytosis and increased MRD levels, also when patients are considered as standard-risk
Roberts KG et al, JCO 2014
Intensive and MRD-driven treatment seems to abolish the negative prognosticimpact of the BCR/ABL1-like signature in childhood ALL
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Response to induction therapy in adults
• Contrasting results on the CR rate
- Lower than in other B-neg ALL cases (den Boer J et al, Haematologica 2015; Chiaretti S et al, BJH in press)
-No differences with other ALL cases (Herold T et al, Haematologica 2016; Jain N et al, Blood 2017)
-MRD persistence more frequent in BCR/ABL1-like cases (Roberts KG et al, JCO 2016; Herold T et al Haematologica 2017; Jain N etal Blood 2017; personal data). Not yet clear if in adults MRD negativity overcomes BCR/ABL1-like signature
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• Significantly inferior survival (EFS, DFS, OS) in all reported studies
Survival in adults
Roberts KG et al, JCO 2016
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• Molecular background
• Incidence
• Diagnosis
• Outcome and MRD
• Treatment: two options
Topics: BCR/ABL1-like
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Treating the target
Pui CH et al, Clin Lymp Myel Leuk, 2017
Requires a deep knowledge of the genomic background of each case. Time and cost consuming. Feasible only in a few centers.
9 R/R pts have been treated. Median age 24 yrs (range 18-62).8 pts treated on the ruxolitinib arm (7 pts CRLF2-high, 1 with a JAK2 fusion (HMBOX1-JAK2).1 pt on the dasatinib arm (NUP214/ABL1).
No DLT, but no reponse on ruxo or dasa.
Jain N et al, ASH 2017
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In vitro use of ponatinib on primary cells: effect on
proliferation and apoptoticresponse similar in
BCR/ABL1+ and BCR/ABL1-like cases (2 EBF1/PDGFRB-
positive, 1 JAK2-mutated and P2RY8/CRLF2-positive, 1
RCSD1/ABL1, 3 WT for JAK/STAT and RAS
mutations)
Wide-spectrum appraoch. Ponatinib
Chiaretti S et al, BJH 2018
p=0.0007
p=0.023
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Conclusions
BCR-ABL1-like ALL represents a novel ALL subtype.
- Detected only in B-neg ALL cases and more frequently from adolescence onwards.
- The genomic background is highly heterogeneous and variable from case to case. It can be summarized in lesions involving ABL class genes, JAK/STAT genes and other kinases. Some cases are devoid of lesions.
- Diagnosis is still not standardized and represents an unmeet need.
- Therapy should include a TKI, possibly in combination with steroids and chemotherapy.
Upfront? At MRD persistence (different strategies in children and adults). In combination with immunotherapy?
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Monica Messina Alessia LaurettiAlfonso PiciocchiNadia PeragineGiorgio InghiramiOliver Elemento Antonella VitaleAnna GuariniRobin Foà
Acknowledgments