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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
Topics: BCR/ABL1-like and other subgroups
• Molecular background
• Incidence
• Diagnosis
• Outcome and MRD
• Treatment
• Molecular background
• Incidence
• Diagnosis
• Outcome
• Treatment
Topics: BCR/ABL1-like
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
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.
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
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
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.
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
• Molecular background
• Incidence
• Diagnosis
• Outcome
• Treatment
Topics: BCR/ABL1-like
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
• Molecular background
• Incidence
• Diagnosis
• Outcome
• Treatment
Topics: BCR/ABL1-like
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
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
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)
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!)
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
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
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
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
• Molecular background
• Incidence
• Diagnosis
• Outcome and MRD
• Treatment
Topics: BCR/ABL1-like
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
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
• Significantly inferior survival (EFS, DFS, OS) in all reported studies
Survival in adults
Roberts KG et al, JCO 2016
• Molecular background
• Incidence
• Diagnosis
• Outcome and MRD
• Treatment: two options
Topics: BCR/ABL1-like
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
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
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?
Monica Messina Alessia LaurettiAlfonso PiciocchiNadia PeragineGiorgio InghiramiOliver Elemento Antonella VitaleAnna GuariniRobin Foà
Acknowledgments
