B-other Cases Research Articles

Prognostic Value of Rare IKZF1 deletions in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia: An International Collaborative Study

BACKGROUNDDeletions in IKZF1 are found in approximately 15% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). There is strong evidence for the poor prognosis of the most common IKZF1 deletions affecting exons 4-7 (DEL 4-7) and exons 1-8 (DEL 1-8), but evidence for the remaining 33% of cases harboring other variants of IKZF1 deletions is lacking. In an international multi-centre study we analyzed the prognostic value of these rare variants.METHODSMultiplex ligation-dependent probe amplification (MLPA) assays were performed on genomic DNA from patients’ bone marrow aspirates at diagnosis by the national study groups. Each IKZF1-deleted case was matched to three wild-type controls based on cytogenetic subtype, treatment protocol, stratification arm, white blood cell count and age at diagnosis. Known high-risk factors age <1 year (infants), BCR-ABL1-positive, and MLL-rearranged cases were excluded. We compared the cumulative incidence of relapse with death as competing event (CIR) between cases and their controls using Gray’s test. Matched pair Cox regression was used for event-free survival (EFS) analysis, and the hazard ratio (HR) with 95% confidence interval (CI) was reported.RESULTSWe included 134 BCP-ALL cases with a rare IKZF1 deletion and 402 matched controls. Of these cases, 26 (19%) had a deletion in exon 2 to 3 (DEL 2-3), 32 (24%) in exon 2 to 7 (DEL 2-7), 15 (11%) in exon 2 to 8 (DEL 2-8), 27 (20%) in exon 4 to 8 (DEL 4-8), and 34 (25%) belonged to the remaining group (DEL-Other). All rare IKZF1 deletion variants together had a higher 5-year CIR compared with the matched wild-type controls (40% vs. 22%, p<0.001), and a lower matched pair EFS (HR 1.8, 95% CI: 1.4-2.3; p<0.001). Analysis of cases and matched controls within their own risk group (56 standard risk, 33 intermediate risk and 45 high risk cases), showed an unfavorable effect for rare IKZF1 deletions in all stratification groups.Rare IKZF1 deletions were found in all BCP-ALL subtypes. The frequency of ETV6-RUNX1-positive (12 cases, 9%), high-hyperdiploid (21 cases, 16%), and unclassified BCP-ALL (13 cases, 10%) was relatively low among rare IKZF1-deleted cases. Most cases were found in the B-other group (88 cases, 66%). These B-other cases had a higher 5-year CIR compared with wild-type controls (47% vs. 27%, p<0·001), which translated into a lower EFS (HR 1·8, 95% CI: 1·3-2·4, p=<0·001). CIR and EFS analysis of high-hyperdiploid cases revealed a weak trend for an adverse outcome associated with rare IKZF1 deletions (5-year CIR 29% vs. 18%, p=0·1 and HR 2·4, 95% CI: 0·8-6·7, p=0·1). No prognostic impact was seen for rare IKZF1 deletions in ETV6-RUNX1-positive BCP-ALLSeparate analyses per IKZF1 deletion type showed a higher 5-year CIR for DEL 2-7 (38% vs. 18%, p=0.05), for DEL 2-8 (60% vs. 31%, p=0.02), and for DEL-Other cases (45% vs. 24%, p=0.04). Matched pair analysis of EFS revealed a poor prognosis for DEL 2-7 (HR 2·0, p=0·03), DEL 2-8 (HR 2·2, p=0·002), and DEL-Other (HR 2·2, p<0·001). The CIR and EFS of DEL 2-3 cases displayed a trend for unfavorable outcome (5-year CIR 28% vs. 17%, p=0.06; HR 1.8, p=0.1) but not for DEL 4-8 (34% vs. 26%, p>0.1; HR 1.0, p>0.1). The prognosis of each rare variant, including DEL 2-3 and DEL 4-8, was equal or worse compared with the most frequently observed and unfavorable prognostic DEL 4-7 and DEL 1-8 variants.CONCLUSIONSAll types of rare IKZF1 deletions, with the possible exception of DEL 4-8 cases, had a significantly increased risk of relapse and poorer EFS compared with their matched wild-type controls. The prognosis of DEL 4-8 cases was as poor as those of the other rare variants and that of the known high-risk variants DEL 4-7 and DEL 1-8. We therefore conclude that all variants of IKZF1 deletions are equivalent in terms of their prognostic impact. DisclosuresNo relevant conflicts of interest to declare.

Philadelphia-Like Signature In Childhood Acute Lymphoblastic Leukemia: The AIEOP Experience

▪ BackgroundDespite the current risk-based stratification and therapies, up to 20% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) experience relapse. A major research effort is dedicated to identifying poor prognostic subgroups in the larger subset of patients with intermediate-risk disease, where most relapses occur. Recently, two groups in the US and the Netherlands independently identified a novel BCP-ALL subtype negative for the BCR/ABL fusion gene but with a gene-expression profile similar to Philadelphia chromosome-positive (Ph+) ALL. This ‘Ph-like' (or ‘BCR/ABL-like') subtype encompasses 10-15% of BCP-ALL patients, predicts high incidence of relapses and defines a candidate subgroup for targeted treatment with tyrosine kinase inhibitors and alternative drugs. AimAim of this project was to identify BCP-ALL Ph-like cases and their prognosis in patients treated in Study Protocols of the Italian Association of Pediatric Hematology and Oncology (AIEOP) MethodsIn the context of the Microarray Innovations in LEukemia (MILE) study, gene expression profiling was successfully performed on 400 Italian childhood BCP-ALL cases enrolled in AIEOP-BFM ALL2000/R2006 protocols, from whom material was available. The MILE classifier recognized the most common genetic subgroups, namely t(4;11), t(12;21), t(1;19) or t(9;22) positive, or high hyperdiploid (DNA index≥1.16). Out of 400 BCP-ALL cases, 143 negative for common fusion transcripts, non-high hyperdiploid (DNA index<1.16) and non-Down Syndrome, were defined as ‘B-others'. ResultsUsing signatures of known ALL subgroups, the likelihood to be close to one of the ‘known' classes was defined for each of the B-others samples. Specifically, 43 B-other cases (43/143=30.1%, about 10% of total BCP-ALL cohort) presented as a cluster with a gene expression signature close to the BCR/ABL signature, therefore referred to as ‘Ph-like'. Among B-others, Ph-like cases had a significantly increased proportion of males, age>10 years and WBC>20x109/L. The 5y event-free survival (EFS) of Ph-like patients was 54.8% (SE 8.2) vs 83.1% (3.9) in the remaining B-others patients (p<0.001), mostly due to an increased cumulative incidence of relapse (CIR: 33.9% (7.4) vs 14.9% (3.7); p=0.009). Notably, Ph-like patients did not differ for prednisone (PDN) and minimal residual disease (MRD) response or final risk stratification. Indeed, out of 36/43 stratified by MRD, only 7 patients had high-risk MRD, while 20 were MRD intermediate and 9 even MRD standard risk. Only 3 non-HR cases by MRD were then classified as HR based on PDN response. Of relevance, the significant difference in 5y EFS was confirmed in the 33 Ph-like patients with no HR features (59.8% (9.2) vs 88.5% (3.9) in the remaining B-others patients with similar features (p<0.001), with a significantly increased CIR (32.1% (8.4) vs 10.2% (3.7); p=0.005). Among 43 Ph-like cases, 1 was resistant and 1 died during the induction phase. Out of the 14 relapses, 10 were isolated medullary, and 4 combined with an extramedullary site (no CNS relapses). Two patients died in remission and no secondary malignancies occurred during the observation period. Overall, 25/43 (58.1%) Ph-like patients are alive in complete remission versus 84/100 (84.0%) in B-others, non Ph-like patients. Conclusions and perspectivesWe identified a ‘Ph-like' subgroup in the Italian cohort of children with BCP-ALL, associated to a poor outcome. Ph-like patients, independently of the other known risk features, could be considered eligible for alternative treatments. The genetic characterization of this subgroup is ongoing within the AIEOP-BFM research trial cooperative group, which is aimed at further defining the pathogenetic mechanisms and identifying the therapeutic translation. Disclosures:No relevant conflicts of interest to declare.

BCR-ABL1-Like Cases In Pediatric Acute Lymphoblastic Leukemia: A Comparison Between COG/St. Jude and Dutch DCOG Signatures

BackgroundPediatric precursor B-cell acute lymphoblastic leukemia (BCP-ALL) with the BCR-ABL1 translocation forms a small group with a poor prognosis. Approximately 15% of BCP-ALL cases are characterized by a gene expression signature similar to BCR-ABL1-positive disease. This so-called BCR-ABL1-like group shares the poor prognosis of the BCR-ABL1-positive subtype but without presence of a BCR-ABL1 translocation. Using different cohorts and methods, two different BCR-ABL1-like signatures have been identified (Den Boer et al., Lancet Oncol 2009; Mullighan et al., New Engl J Med 2009). Both signatures identify groups of patients with a poor prognosis and with deletions in B-cell development genes (Den Boer et al., Lancet Oncol 2009; Roberts et al., Cancer Cell 2012). The aim of this study was to compare these two signatures and the association to molecular aberrations and clinical outcome in diagnostic samples of the Dutch Childhood Oncology Group (DCOG) and the German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia (COALL). Patients and methodsThe BCP-ALL study cohort consisted of 452 patients, including 146 patients with B-precursor ALL without known recurrent aberrations (BCR-ABL1 translocation, ETV6-RUNX1 translocation, MLL rearrangements, TCF3 rearrangements, hyperdiploidy). BCR-ABL1-like cases were identified in these 146 B-precursor cases using Prediction Analysis of Microarrays (PAM) with 257 probe sets (Roberts et al., Cancer Cell 2012) and separately by Hierarchical Clustering (HC) with 110 probe sets (Den Boer et al., Lancet Oncol 2009), using Affymetrix U133Plus2 expression data. Frequencies of molecular aberrations detected by multiplex ligation-dependent probe amplification, array-comparative genomic hybridization and Sanger sequencing, including IKZF1 deletions, JAK2 mutations and CRLF2 aberrations, were compared between groups of patient samples classified by the two prediction algorithms using Fisher's Exact test. We analyzed the cumulative incidence of relapse (CIR) with death as a competing event. ResultsIn the DCOG/COALL cohort, which included patients from all ALL risk groups, we identified 33 BCR-ABL1-like cases with the PAM signature, of which 24 (73%) were high-risk according to the NCI-Rome criteria (WBC ≥ 50 cells/nl and/or age ≥ 10 years), and 79 cases with the HC signature, of which 57 (72%) were high-risk. Compared with 59 B-other cases not identified by either of the two BCR-ABL1-like signatures, the most enriched molecular aberrations in the PAM-identified group were IKZF1 deletions (52% vs 19%, p=0.002), high CRLF2 expression (39% vs 8%, p<0.001), intrachromosomal amplification of chromosome 21 (iAMP21; 32% vs 0%, p<0.0001), and RB1 deletions (23% vs 6%, p=0.04). Enriched aberrations in the HC-identified group included IKZF1 deletions (38% vs 19%, p=0.02), dic(9;20) (28% vs 4%, p<0.001), and iAMP21 (15% vs 0%, p=0.002), while BTG1 deletions occurred less frequently (6% vs 17%, p=0.04). Directly comparing PAM and HC-identified BCR-ABL-like cases indicated significantly higher proportions of CRLF2-high (p=0.004) and JAK2 mutations (p=0.02) in the PAM group, and higher dic(9;20) in the HC group (p=0.03). Next, we compared the overlap between cases identified by the two signatures. PAM identified 32% (25/79) of the BCR-ABL1-like cases identified by HC, and vice versa HC identified 76% (25/33) of the cases identified by PAM. The cumulative incidence of relapse at 5-years in the BCR-ABL1-like groups identified by PAM (20%, p=0.04), HC (27%, p<0.0001), or both methods (18%, p=0.15), was worse than in the non-BCR-ABL1-like/BCR-ABL1-negative/MLL wild-type BCP-ALL reference group (8.1%). Cases identified only by HC (31%, p<0.0001) or only by PAM (25%, p=0.05) also had higher 5-year cumulative relapse incidences than the reference group. ConclusionsBoth BCR-ABL1-like expression signatures defined by HC or PAM identified a group of patients with an unfavorable prognosis in the DCOG/COALL study. The two groups largely overlapped, but each signature also identified unique patients not identified by the other signature. Importantly, each of the signatures was associated with poor outcome, while patients identified by both signatures (PAM/HC-double positive) did not have a worse outcome than those identified only by PAM or HC. Since the two signatures are partially complementary, we suggest that both should be used. Disclosures:Evans:St. Jude: In accordance with institutional policy, St. Jude allocates a portion of the income it receives from licensing inventions and tangible research materials to those researchers responsible for creating this intellectual property., In accordance with institutional policy, St. Jude allocates a portion of the income it receives from licensing inventions and tangible research materials to those researchers responsible for creating this intellectual property. Patents & Royalties, Under this policy, I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics. Other.

Leukemia Cells with a BCR-ABL1-Like signature and/or IKZF1 deletions, but Not High CRLF2 Expression, Are Predictive of an Unfavorable Prognosis in Childhood B Cell Precursor Acute Lymphoblastic Leukemia

AbstractAbstract 880 BackgroundThe prognosis of childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL) has improved enormously due to risk-adapted therapeutic stratification of patients. Still, most relapses occur in the group that is supposed to have an intermediate relapse risk. To overcome this problem, new prognostic markers are needed. Recently, new high-risk molecular features were identified in BCP-ALL: the BCR-ABL1-like signature, IKZF1 deletions and high CRLF2 expression (CRLF2-high). These features can occur together; however, whether they confer independent prognostic value is currently unknown. In this study we determined the frequency, co-occurrence and prognostic relevance of these new molecular features in four independent cohorts of children with ALL. MethodsBCR-ABL1-like gene signature (110-gene probe classifier), IKZF1 deletions (Multiplex Ligation-dependent Probe Amplification) and CRLF2 expression levels (Affymetrix U133 plus 2.0) were determined in leukemic cells (>90% blasts) from 1128 children with newly diagnosed ALL enrolled in three consecutive Dutch Childhood Oncology Group trials (DCOG ALL-8/-9/-10) and in the German COALL-97/03 trial. The cumulative incidence of relapse (CIR) was calculated using death as a competing event. ResultsBCR-ABL1-like, IKZF1-deleted and CRLF2-high cases were found in 16%, 17% and 10% of the BCP-ALL cases, respectively. Collectively, these three features constitute 34% of BCP-ALL cases for which 4% of those had all three, 29% had any two and 67% had only one these features. The BCR-ABL1-like signature and IKZF1-deletions co-occurred the most frequent of these three features (22%). The BCR-ABL1-like signature was only found in B-other cases negative for the genetic abnormalities ETV6-RUNX1, BCR-ABL1, hyperdiploid, TCF3- and MLL-rearrangement, whereas IKZF1-deleted and CRLF2-high cases were also found in other subtypes of BCP-ALL. The frequency of IKZF1- deletions was highest in BCR-ABL1- positive (70%) and BCR-ABL1-like (40%) cases. The BCR-ABL1-like signature, IKZF1 deletions and CLRF2-high were associated with prognosis in four, three and one out of 4 studied treatment protocols, respectively. Protocol-stratified analysis revealed that five-year CIR was higher in BCR-ABL1-like (32%, p<0.001) and IKZF1- deleted (34%, p<0.001) compared to other BCP-ALL cases (11% and 13%, respectively), whereas no unfavorable CIR was associated with the CRLF2-high feature. IKZF1- deleted cases also were at higher risk of relapse in the hyperdipoid (5-year CIR 34%, p=0.04) and non-BCR-ABL1- like B-other group (5-year CIR 40%, p=0.02) compared to 9% for non-deleted BCP-ALL cases whereas IKZF1-deletions had no (additive) prognostic value in ETV6-RUNX1- positive or BCR-ABL1-like cases. Cases with only the BCR-ABL1-like signature (HR 5.3) or only an IKZF1 deletion (HR 4.4) as well as those cases positive for both features (HR 3.7) were at higher risk to develop an adverse event than those cases negative for both features (p<0.001). Together these two features accounted for 62.9% of all relapses in BCP-ALL whereas no relapse occurred in the cases with high CRLF2 expression as single feature. The adverse prognosis associated with BCR-ABL1-like signature and/or IKZF1-deletions was independent of other risk factors including age and white blood cell count. Moreover, the BCR-ABL1-like signature (HR=3.7 p=0.026) and IKZF1- deletions (HR 2.7, p=0.043) were predictive of an unfavorable prognosis in the group of BCP-ALL cases with intermediate minimal residual disease levels at day 79 of treatment. ConclusionsHigh CRLF2 expression is the least predictive, whereas the BCR-ABL1-like signature and IKZF1- deletions –both individually and combined- are strong independent prognostic factors and have additional prognostic value on top of intermediate minimal residual disease levels in children with BCP-ALL. These findings demonstrate that these two molecular features are important clinically for identifying high-risk patients who may require more intensive therapy. Disclosures:No relevant conflicts of interest to declare.

Identification of new microRNA genes and aberrant microRNA profiles in childhood acute lymphoblastic leukemia

MicroRNAs (miRNAs) control the expression of protein-coding genes in normal hematopoietic cells and, consequently, aberrant expression may contribute to leukemogenesis. To identify miRNAs relevant to pediatric acute lymphoblastic leukemia (ALL), we cloned 105 known and 8 new miRNA genes expressed in patients' leukemia cells. Instead of known miRNA genes, new miRNA genes were not evolutionarily conserved. Quantification of 19 selected miRNA genes revealed an aberrant expression in ALL as compared with normal CD34+ cells (P <or= 0.02); both upregulated (14/19) and downregulated (5/19) expressions were observed. Eight miRNAs were differentially expressed between MLL and non-MLL precursor B-ALL cases (P<0.05). Most remarkably, miR-708 was 250- up to 6500-fold higher expressed in 57 TEL-AML1, BCR-ABL, E2A-PBX1, hyperdiploid and B-other cases than in 20 MLL-rearranged and 15 T-ALL cases (0.0001<P<0.01), whereas the expression of miR-196b was 500-fold higher in MLL-rearranged and 800-fold higher in 5 of 15 T-ALL cases as compared with the expression level in the remaining precursor B-ALL cases (P<0.001). The expression did not correlate with the maturation status of leukemia cells based on immunoglobulin and T-cell receptor rearrangements, immunophenotype or MLL-fusion partner. In conclusion, we identified new miRNA genes and showed that miRNA expression profiles are ALL subtype-specific rather than linked to the differentiation stadium associated with these subtypes.