BCR ABL1 like ALL: molekuliniai mechanizmai ir klinikinė reikšmė. IKAROS delecija: molekulinė biologija, prognostinė reikšmė. ASH 2015 naujienos

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1 BCR ABL1 like ALL: molekuliniai mechanizmai ir klinikinė reikšmė. IKAROS delecija: molekulinė biologija, prognostinė reikšmė. ASH 2015 naujienos

2 Ph like ALL BCR ABL1 like acute lymphoblastic leukemia (ALL) is a recently identified B cell ALL (B ALL) subtype with poor outcome that exhibits a gene expression profile similar to BCR ABL1 positive ALL but lacks the BCR ABL1 fusion protein.

3 Istorija Gene expression profiling of ALL Eng Juh Yeoh et al. Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling Gene expression profile similar to BCR ABL January. Mullighan, C.G. et al. Deletion of IKZF1 and prognosis in acute lymphoblastic leukemia. First Ph like February. Den Boer, M.L. et al. A subtype of childhood acute lymphoblastic leukaemia with poor treatment outcome: a genome wide classification study.

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6 One of the most surprising observations from this study was the identification remarkable difference of the individual leukemia subtypes. HG_U95Av2 oligonucleotide array (Affymetrix) transkriptų

7 Istorija Gene expression profiling of ALL Eng Juh Yeoh et al. Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling Gene expression profile similar to BCR ABL January. Mullighan, C.G. et al. Deletion of IKZF1 and prognosis in acute lymphoblastic leukemia. First Ph like February. Den Boer, M.L. et al. A subtype of childhood acute lymphoblastic leukaemia with poor treatment outcome: a genome wide classification study.

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9 Despite best current therapy, up to 20% of pediatric patients with acute lymphoblastic leukemia (ALL) have a relapse. Recent genome wide analyses have identified a high frequency of DNA copy number abnormalities in ALL, but the prognostic implications of these abnormalities have not been defined. Genetic alteration of IKZF1 is associated with a very poor outcome in B cell progenitor ALL....deletion of IKZF1 is frequent in BCR ABL1 ALL, suggests that IKAROS abnormalities are important in the pathogenesis of both BCR ABL1 positive B cell progenitor ALL and BCR ABL1 negative ALL that is associated with a poor outcome. The gene expression signatures of patients with poor outcome (IKZF1 deleted) ALL in the original and validation cohorts were very similar to each other and to the signature of BCR ABL1 positive ALL, a subtype of ALL in which IKZF1 deletion is very common. Since BCR ABL1 ALL has a poor prognosis, these findings suggest that the deletion of IKZF1 is a key determinant of a poor outcome both in patients with BCR ABL1 positive and patients with BCR ABL1 negative disease transkriptų (Affymetrix)

10 25% of precursor B ALL cases are genetically unclassified and have intermediate prognosis. We constructed a classifier based on gene expression in 190 children with newly diagnosed ALL. Classifying gene probe sets revealed a new ALL subtype, the underlying genetic abnormalities of which were characterised by comparative genomic hybridisation arrays and molecular cytogenetics. The prognosis of BCR ABL1 like disease was similar to that of BCR ABL1 positive ALL. 36 (82%) of the patients with BCR ABL1 like disease had deletions in genes involved in B cell development, including IKZF1, TCF3, EBF1, PAX5, and VPREB transkriptų (Affymetrix)

11 Istorija Gene expression profiling of ALL Eng Juh Yeoh et al. Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling Gene expression profile similar to BCR ABL January. Mullighan, C.G. et al. Deletion of IKZF1 and prognosis in acute lymphoblastic leukemia. First Ph like February. Den Boer, M.L. et al. A subtype of childhood acute lymphoblastic leukaemia with poor treatment outcome: a genome wide classification study. Transcriptome and whole genome sequencing on 15 cases of Ph like ALL Roberts KG. et al. Genetic alterations activating kinase and cytokine receptor signaling in high risk acute lymphoblastic leukemia. A total of 154 patients with Ph like ALL underwent detailed genomic analysis Roberts KG, et al. Targetable Kinase Activating Lesions in Ph like Acute Lymphoblastic Leukemia

12 We performed transcriptome and whole genome sequencing from 15 patients with Phlike ALL. Strikingly, we identified alteration of genes encoding cytokine receptors and regulators of tyrosine kinase signaling in all 15 cases studied.

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14 We next performed recurrence screening of extended cohorts of high risk B ALL to determine the frequency of these genetic alterations. 231 cases, 40 Ph like (14%) Twenty five cases (8.8%) had high CRLF2 expression, 19 of which were Ph like and 6 non Ph like. JAK mutations were present in ten cases with high CRLF2 expression, all of which were Ph like. The EBF1 PDGFRB fusion was detected in three Ph like patients (8% of Ph like ALL). Two cases with NUP214 ABL1 rearrangement. No ABL1, JAK2, or PDGFRB rearrangements were identified in the MPN, CMML, and AML cohorts and have not been detected in other childhood B ALL subtypes studied by WGS and mrna seq, indicating these genetic lesions are highly enriched in the Ph like subtype. IL7R mutations in 5/40 Ph like patients (12,5%). One Ph like case with SH2B3 mutation. Four Ph like cases with FLT3 mutations.

15 Istorija Gene expression profiling of ALL Eng Juh Yeoh et al. Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling Gene expression profile similar to BCR ABL January. Mullighan, C.G. et al. Deletion of IKZF1 and prognosis in acute lymphoblastic leukemia. First Ph like February. Den Boer, M.L. et al. A subtype of childhood acute lymphoblastic leukaemia with poor treatment outcome: a genome wide classification study. Transcriptome and whole genome sequencing on 15 cases of Ph like ALL Roberts KG. et al. Genetic alterations activating kinase and cytokine receptor signaling in high risk acute lymphoblastic leukemia. A total of 154 patients with Ph like ALL underwent detailed genomic analysis Roberts KG, et al. Targetable Kinase Activating Lesions in Ph like Acute Lymphoblastic Leukemia

16 We performed genomic profiling of 1725 patients with precursor B cell ALL and detailed genomic analysis of 154 patients with Ph like ALL. Transcriptome sequencing, whole genome sequencing, and whole exome sequencing.

17 Genomic alterations activating kinase signaling were identified in 91% of patients with Ph like. Subgroups: ABL1 fusions (12.6 %), rearrangements of EPOR or JAK2 (3.9%), rearrangements of CRLF2 (49.7%); Other JAK STAT (IL7R, FLT3, SH2B3, JAK1, JAK3, TYK2, IL2RB) (12.6%) Ras pathway mutations (4.3%) uncommon fusions (e.g., involving NTRK3 or DGKH) (0.9%) no kinase alterations (4.8%) material for analysis was not available (3.9%). Notably, ABL class rearrangements were more common among children, and JAK2 rearrangements were more frequent among young adults

18 123 of 264 patients with Ph like ALL had high CRLF2 expression, with the frequency ranging from 24% among children with standard risk ALL to 60% among adolescents with ALL (among patients with high CRLF2 expression, P2RY8 CRLF2 in 45 patients and IGH CRLF2 in 61 patients). Sixty eight patients (55%) with CRLF2 rearrangement had Janus kinase mutations, most commonly in JAK2. We found additional alterations activating JAK STAT in 5 of 11 patients who had CRLF2 rearrangement but no JAK mutations; these alterations included IL7R mutations (4 patients), an FLT3 mutation (1 patient), and a deletion of SH2B3. Sequence mutations and focal deletions activating JAK STAT signaling, including in IL7R, FLT3, SH2B3, JAK1, and JAK3, were identified in 31 patients without CRLF2 rearrangement or other kinase fusions. Fifteen patients had alterations in the Ras pathway only, including NRAS, KRAS, PTPN11, NF1, and BRAF.

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20 Acute lymphoblastic leukemia (ALL), a cancer of white blood cells, is the most common malignancy in children and remains the most frequent cause of cancer death in the US among persons younger than 20 years old. The prevalence of Ph like ALL increases with age and that the older age groups, adolescents and young adults, have inferior outcomes (Table 1). Among all age groups studied, Ph like ALL was an independent adverse prognostic factor (NHI data).

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22 ABL1 fusions (12.6 %), rearrangements of EPOR or JAK2 (3.9%), rearrangements of CRLF2 (49.7%); Other JAK STAT (IL7R, FLT3, SH2B3, JAK1, JAK3, TYK2, IL2RB) (12.6%) Ras pathway mutations (4.3%) uncommon fusions (e.g., involving NTRK3 or DGKH) (0.9%) no kinase alterations (4.8%) material for analysis was not available (3.9%).

23 132 adult ALL patients (age years, median 54 years) (lacking of BCR ABL1, MLL AF4 and E2A PBX1) GEP (Roberts et al., 2014) FISH targeting ABL1, ABL2, CSF1R, PDGFRB and JAK2 Fusion specific RT PCR for the identification of the respective ABLandJAK fusion partner Quantitave RT PCR for CRLF2 overexpression 13/132 Ph like pts 11/132 Ph like pts 4/132 with rearrangements ABL1 (SNX2 ABL1; n=1), and PDGFRB (EBF1 PDGFRB; n=3) 15 Ph like (11 overexp. CRLF2 + 4 ABL, PDGFRB fusions) combination of quantitative RT PCR, FISH and fusion specific RT PCR identified 15 cases (12%) compared to 13 cases (10%) identified by GEP with Ph like ALL. 7 pts IGH CRFL rearranged (FISH) 2 pts NRAS+ 8 pts JAK2+ 13 pts were assigned to the Ph like subset by both approaches, 2 patients by alternative approuches. while no patient was identified by GEP only. IKZF1 deletions in 12/15 patients. 1) Analysis of CRLF2 expression. 2) FISH targeting ABL and JAK pathway activating fusions involving the genes ABL1, ABL2, CSF1R, PDGFRB and JAK2. 3) Fusion specific RT PCR for the identification of the respective ABL and JAK fusion partner and sensitive MRD monitoring.

24 1) Analysis of CRLF2 expression. 2) FISH targeting ABL and JAK pathway activating fusions involving the genes ABL1, ABL2, CSF1R, PDGFRB and JAK2. 3) Fusion specific RT PCR for the identification of the respective ABL and JAK fusion partner and sensitive MRD monitoring.

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26 Here we report the development and validation of a robust gene expression classifier that can prospectively identify Ph like ALL patients for therapeutic intervention. Affymetrix U133_Plus_2.0 The 15 gene LDA classifier was able to predict Ph or Ph like ALL in the test set with a high degree of sensitivity (93.0%) and specificity (89.7%). 15 of the 38 genes (IGJ, SPATS2L, MUC4, CRLF2, CA6, NRXN3, BMPR1B, GPR110, CHN2, SEMA6A, PON2, SLC2A5, S100Z, TP53INP1, IFITM1). We have developed and validated a highly robust gene expression classifier for the prospective identification of Ph like ALL. Rapidly screened patients will then undergo targeted sequencing to confirm the presence of specific genomic lesions.

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28 There are significant differences in 5 year eventfree survival, with JAK2/EPOR rearranged cases (26.1±8.5) and CRLF2 rearranged JAK2 mutant cases (38.8±7.0) having a poor prognosis. Whilst cases harboring Other JAK STAT (68.3±9.9) and Ras pathway only alterations (85.7±11.5) have a more favorable outcome.

29 IKZF1 deletion Genetic alterations of IKZF1 influence the transcriptome of both Ph+ ALL and Ph ALL with a poor outcome. Mullighan et al The association between IKZF1 status and outcome was independent of age, leukocyte count at presentation, cytogenetic subtype; this indicates that detection of IKZF1 alterations at diagnosis might be useful in identifying patients with a high risk of treatment failure. Since BCR ABL1 ALL has a poor prognosis, these findings suggest that the deletions of IKZF1 is a key determinant of a poor outcome both in patients with BCRABL1 positive and patients with BCR ABL1 negative disease.

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31 IKZF1 alterations were more common in patients with Ph like ALL who had kinase fusions (140 of 180 [78%]) than in those with a sequence mutation (14 of 43 [33%], P<0.001). Furthermore, patients with Ph like ALL who had an IKZF1 alteration had inferior median (±SD) 5 year event free survival, as compared with patients who had Ph like ALL without an IKZF1 alteration;

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33 We performed a comprehensive analysis of the impact of IKZF1 del in a large cohort of children (n=1223) with BCR ABL1 negative BCP ALL treated in the EORTC CLG trial Patients with IKZF1 del had a lower 8 year event free survival (EFS, 67.7% versus 86.5%; hazard ratio (HR)=2.41; 95%confidence interval (CI)= ; P<0.001). Thus, IKZF1 del retains independent prognostic significance in the context of current risk adapted protocols.

34 IKZF1 positive IKZF1 positive IKZF1 positive IKZF1 positive V.Jodzevičiūtė E.Rožėnas K.Kosonia A.Malūkas BCR ABL1 positive BCR ABL1 positive BCR ABL1 negative BCR ABL1 negative, E2A positive Ph like?

35 ASH 2015 naujienos: adult ALL

36 The prevalence of Ph like ALL increases with age and accounts for over 25% of patients with B progenitor ALL between the ages of years. However, the frequency, outcome and genetic basis of Ph like ALL in adults over the age of 39 is unknown. 692 adults with B ALL. Ph like ALL comprised 26% of patients between years of age and 20% of patients aged Similar to previous reports, 99 of 186 (53%) of patients with Ph like ALL had high expression of CRLF2. Of 75 cases tested, 56 harbored IGH CRLF2 and 19 P2RY8 CRLF2. Of the 87 Ph like ALL patients with low CRLF2 expression, we identified rearrangements involving tyrosine kinase or cytokine receptor genes in 45 patients: ABL1 (n=5 patients), ABL2 (n=7), CSF1R (n=1), EPOR (n=8), JAK2 (n=18),pdgfra(n=1), PDGFRB (n=2), PTK2B (n=1) and TYK2 (n=2). Nine of these 45 fusions have not previously been identified in Ph like ALL. Exome sequencing is being performed on cases that do not harbor a kinase fusion by RNA seq analysis.

37 The WES analysis of the 41 Ph negative cases identified 735 point mutations and 25 mutations that occur in splicing sites in 651 genes. The average number of somatic coding mutations was 17 per case (range 1 47). Many studies found that deletion of CDKN2 was associated with poor prognosis in ALL,and CDKN2 deletion also as a frequent cytogenetic aberration in Ph+ ALL patients. Here we study about the prognostic significance of the CDKN2 in Ph+ ALL in TKIs era. 135 Ph+ patients (98 denovo and 37 relapsed cases). The prevalence of CDKN2 deletions in all study population was 27.4%(37/135). Deletions of CDKN2 were significantly associated with poor outcomes including decreased overall survival (OS) (P<0.001), lower disease free survival (DFS) (P<0.001), and increased cumulative incidence of relapse (P=0.003). Deletion of CDKN2 by I FISH is a frequent event in Ph positive ALL, and frequently acquired during leukemia progression and are a poor prognostic marker of long term outcomes even though treated by adding dasatinib. We also found CDKN2 deletion patients had higher CD20 expression, this group patients may be benefit from rituximab.

38 We analyzed 70 adult B ALL patients (median age 45 years, range 18 83) Deletions:CDKN2A (48.6%), IKZF1 (40%), PAX5 (24.2%), BTG1 (17.1%), ETV6(15.7%), BTLA (14.3%), RB1 (5.7%), EBF1 (5. 7%), LEF1 (2.9%), and TCF3 (2.9%). Gains: ERG (30%), ETS2 (21.4%), MYB (20%), UBASH3B (20%), PTEN (20%), PRKCH (18.6%), CDK6 (17.1%), andetv6 (12.9%). 27 samples obtained at the time of relapse were screened for the recurrent CNAs identified in the diagnostic samples. Incidence of CNAs at these genes was lower in the relapse cohort than the initial diagnostic cohort, although there was a higher frequency of CNAs at PAX5, BTLA, ETS2, RB1, LEF1, PTEN, and TCF3 in the relapse samples. The average number of CNAs at diagnosis and relapse was the same. The combination of both CDK2NA and IKZF1 deletions (26%) correlated with a significantly worse OS than having only one or neither of these deletions. Age was the only other covariate significant in univariate analyses for OS, yet IKZF1/CDKN2Aco deletion remained significant in multivariate analysis adjusting for age. When identified in diagnostic samples, co deletion of CDKN2A/IKZF1 is a negative prognostic marker in adult B ALL.

39 Seventy patients had a single relapse, 15 cases had 2 relapses, and 8 cases developed a second tumor of different lineage (B cell lymphoma, chronic myeloid leukemia (n=1 each) and acute myeloid leukemia (n=6)). Diagnosis and relapse samples were studied using Affymetrix SNP 6.0 microarrays and whole genome or whole exome sequencing. The number of CNAs did not differ significantly between diagnosis (with a median of 9) and relapse (compared to a median of 10). The median number of mutations was 12 (range 0 70) at diagnosis (n=91), 21 (range 0 858) at relapse. The most frequently mutated genes were NOTCH1 (n=33), NRAS (n=24), CREBBP (n=20) and KRAS (n=16). Clonal evolution analyses showed multiple patterns of evolution. Comprehensive germline analyses are underway.