High CRLF2 Expression Research Articles

Genetic Abnormalities and Prognosis in Pediatric B–precursor Acute Lymphoblastic Leukemia Treated on Tokyo Children′s Cancer Study Group Protocol

Introduction; Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Most childhood cases of B-precursor ALL (BCP-ALL) may be subclassified by the presence of either gross or submicroscopic genetic alteration. Recent genomewide analyses have identified genetic or cytogenetic subtypes, such as Ph-like ALL, deletion of ERG, intrachromosomal amplification of chromosome 21 (iAMP21) in BCP-ALL. Deletion of ERG and iAMP21 were associated with frequent IKZF1 deletions, which were found in Ph-like ALL associated with poor prognosis. However, the frequency and prognostic implications of these genomic abnormalities have not been defined in Japan.Patients and Methods; We studied 208 children with de novo BCP-ALL enrolled on Tokyo Children Cancer Study Group (TCCSG) ALL L0416 and L0616 trials. All patients had a diagnosis of BCP-ALL without BCR-ABL1 phenotype confirmed by morphology, immunophenotyping and mRNA Real-time PCR. Alterations of ERG and iAMP21 were examined by Multiplex Ligation-dependent Probe Amplification (MLPA) kit P327-B1. To identify Ph-like ALL and BCR-ABL1 like ALL, We investigated gene expression of 235 BCP-ALL cases enrolled on TCCSG L0416 and L0616 trials by microarray GeneChip Human Genome U133 Plus 2.0 and analyzed using clustering analysis as described previously. To assess whether these genomic alterations overlap with known gene abnormalities, such as mutations of CRLF2,JAK1, JAK2 and IL7R, rearrangement of P2RY8-CRLF2, PAX5-JAK2 and PDGFRB-EBF1 genes and copy number alteration of IKZF1, CDKN2A/2B, PAX5, EBF1 and SH2B3 genes, mutational analyses of these genes were also performed using direct sequencing, RT-PCR or MLPA kit P335-A1 and P200.Results; We identified Ph-like signature in 9 (3.8%) patients and BCR-ABL1 like ALL signature in 11 (4.7%) patients. Five (55.6%) of 9 Ph-like ALL patients and 3 (27.3%) of 11 BCR-ABL1 like ALL patients have IKZF1 deletions. Five patients were classified into both Ph-like ALL and BCR-ABL1 like ALL. Among these 5 patients, one patient had CRLF2 high expression, JAK2 R683S/R683G mutation, CDKN2A/2B deletion and EBF1 deletion, and another patient have PAX5-JAK2 rearrangement. These two patients relapsed, and the latter patient died. Three of 9 Ph-like ALL patients relapsed and 2 died, while 7 of 11 BCR-ABL1 like ALL patients relapsed and 2 died. We identified iAMP21 in 5 (2.4%) patients and deletion of ERG in 8 (3.8%). One of 5 patients with iAMP21 and 3 of 8 patients with ERG deletion had IKZF1 deletion. One patient with iAMP21 had BCR-ABL1 like ALL signature, but not Ph-like ALL signature. CRLF2 high expression was identified in two of 5 patients with iAMP21, and P2RY8-CRLF2 rearrangement and SH2B3 deletion was identified in one of them. No mutations of CRLF2 and JAK2 were identified in patients with iAMP21 and ERG deletion. Among 5 iAMP21 patients, patient with BCR-ALB1 signature and IKZF1 deletion relapsed, and one patient died. No patients with ERG deletion relapsed or died.Conclusions; Using GeneChip microarray analysis, low frequency of Ph-like ALL signature and BCR-ABL1 like ALL signature was found. Using MLPA as a novel screenings technique, frequency of iAMP21 and ERG deletion in pediatric BCP-ALL was similar to that of previous studies. BCR-ABL1 like ALL and iAMP21 was associated poor prognosis, while ERG deletion was associated good prognosis in pediatric ALL. These data suggest that screening for these genetic abnormalities in pediatric patients with BCP-ALL is critical not only for outcome prediction but also for risk-adapted therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CRLF2 over-Expression Is a Poor Prognostic Marker in Children with High Risk T-Cell Acute Lymphoblastic Leukemia

Introduction: Although risk-adapted therapy improved the prognosis of pediatric T-ALL in the last decades, patients with T-ALL still have a worse outcome compared to B Cell Precursor (BCP)-ALL, and therefore they would benefit from the identification of new prognostic markers for a better treatment stratification and novel strategies to complement current chemotherapy regimens.Among newly reported genomic abnormalities, a subset of BCP-ALL patients is characterized by the over-expression of the Cytokine Receptor-like Factor 2 (CRLF2) gene, due to either an intra-chromosomal deletion causing the P2RY8-CRLF2 fusion or the IGH@-CRLF2 translocation. These two CRLF2 rearrangements were shown to correlate with poor outcome in BCP-ALL patients. In T-ALL, alterations of CRLF2 were not reported yet, but recently, mutations in its partner IL7Rα have been identified in about 10% of T-ALL patients.Aim: To estimate the incidence of CRLF2 aberrations and their prognostic value in pediatric T-ALL.Methods: We analyzed CRLF2 gene expression in 120 T-ALL patients, consecutively enrolled in the AIEOP-BFM ALL2000 study in Italian centers (AIEOP) from September 2000 to July 2005, and, as a validation cohort, in 92 consecutive patients treated with the same protocol in German centers (BFM-G), from July 1999 to December 2004. CRLF2 transcript levels were analyzed by RQ-PCR. Relative gene expression of CRLF2 was quantified by the 2-DDCt method. The DDCt for AIEOP and BFM-G samples was referred to the median DCt of their respective cohort.Results: An heterogeneous expression of CRLF2 was observed among AIEOP T-ALL patients (range: 0.06 to 82 fold change). Seventeen patients (14.2%) presented an expression 5 times higher than the median ('CRLF2-high’). Interestingly, none of the CRLF2-high cases resulted to be positive for P2RY8-CRLF2 fusion, 1/5 was positive for the IGH@ translocation and 1/7 showed a supernumerary X chromosome. JAK2 and CRLF2 mutations were absent in all 120 cases, while IL7R mutations were detected in 5/107 patients (4.7%), but unexpectedly they were not associated to CRLF2 over-expression.CRLF2-high patients had a significantly inferior 5-y EFS (41.2%±11.9 vs. 68.9%±4.6, p=0.006) and an increased, although not statistically significant, cumulative incidence of relapse (CIR) compared to CRLF2-low patients (41.2%±11.9 vs. 26.3%±4.3, p=0.17).The prognostic value of CRLF2 over-expression was confirmed in the BFM-G cohort (5-y EFS in 12 CRLF2-high patients was 50.0%±14.4 vs 83.8%±4.1, p-=0.008; CIR: 33.3%±13.6 vs. 11.3%±3.5, p= 0.06).Interestingly, CRLF2-high patients were more frequently allocated to the high risk (HR) T-ALL subgroup (20.9% in HR vs.8.3% in no-HR in the two cohorts analyzed together). In this subgroup, CRLF2 over-expression was significantly associated to a poor prognosis (5-y EFS: 31.6%±10.7 vs. 62.5%±5.7, p-value=0.008; CIR: 47.4%±11.5 vs. 29.2%±5.4, p=0.14).When analyzed according to prednisone (PDN) response, 17/28 (61%) were prednisone poor responders (PPR), and in the 'PPR-only’ subgroup (non-HR by other features) 4/9 (44%) CRLF2-high patients relapsed versus 4/36 (11%) CRLF2-low.Cox model analysis adjusted by risk group showed that CRLF2-high expression had a relevant prognostic impact with a 2-fold increased risk of relapse (Hazard ratio 2.12; 95% CI 1.07-4.21; p=0.03).The mechanisms responsible for CRLF2 over-expression in T-ALL and its contribution to the pathogenesis of the disease is still being investigated. Notably, gene set enrichment analysis (GSEA) showed an inverse correlation between the expression of CRLF2 and positive cell cycle regulators, thus suggesting that CRLF2-high blasts have a low proliferating activity and may therefore be less sensitive to conventional chemotherapy.Conclusions: CRLF2 over-expression is a poor prognostic marker not only in BCP-ALL patients, but also in T-ALL, identifying a subset of HR T-ALL patients with extremely severe outcome. Specifically, this marker would allow identifying T-ALL patients that could benefit from alternative therapy, potentially targetting the CRLF2 pathway. DisclosuresNo relevant conflicts of interest to declare.

BCR-ABL1-like Acute Lymphoblastic Leukemia Is Associated with IKZF1 and JAK2 Alterations and inferior Outcome in Adults

Background: BCR-ABL1-like (Ph-like) B-precursor acute lymphoblastic leukemia (ALL) displays a gene expression profile closely related to B-precursor ALL with t(9;22)(q34;q11). In addition, Ph-like ALL patients (pts) are characterized by distinct genetic alterations and inferior prognosis in pediatric trials. The purpose of this study was the genetic and clinical characterization of Ph-like ALL in adults.Methods: Affymetrix gene expression profiles (GEP) generated from diagnostic bone marrow samples of 306 adult B-precursor and T-ALL pts (median age 41 years, range 16-84 years) were classified as Ph-like ALL according to published algorithms (Roberts et al., Cancer Cell 2012) and separated from BCR-ABL1-positive and B-other ALL pts (BCR-ABL1-negative; non Ph-like). The incidence and genetic characteristics of the Ph-like subset were analyzed in the overall cohort, whereas clinical and outcome analysis were restricted to B-precursor ALL pts treated within GMALL trials 06/99 and 07/03 (n=107). The median age of this population was 30 (16-64) years. The routine diagnostic work-up included immunophenotyping, fluorescent in situ hybridization (FISH) for BCR-ABL1 and KMT2A (MLL) rearrangements, cytogenetics and molecular analyses of BCR-ABL1 translocations and MLL rearrangements. A subgroup of pts with B-precursor ALL was analyzed for CRLF2 alterations by FISH (n=88) and by RT-PCR for the P2RY8-CRLF2 translocation (n=117). Multiplex ligation-dependent probe amplification (MLPA) for common copy number variations (SALSA MLPA probemix P335-B1) and targeted amplicon sequencing of 131 genes recurrently mutated in ALL were performed in BCR-ABL1 positive (n=30), Ph-like (n=16) and B-other ALL pts (n=23).Results: Of the 306 pts, we classified 26 pts (9%) as Ph-like ALL based on their GEP and the absence of the BCR-ABL1 translocation, corresponding to an incidence of 13% (26/207) in B-precursor ALL and 24% (26/110) among BCR-ABL1-negative B-precursor ALL.Nineteen of 107 B-precursor ALL pts treated within the GMALL trials displayed a Ph-like phenotype. There were no significant differences in baseline characteristics like age, sex, white-cell count, hemoglobin or platelet count and risk group in comparison to the B-other subgroup (n=51). All 19 Ph-like pts showed no MLL rearrangement and 58% belonged to the standard risk group. The complete remission rate after induction was similar for Ph-like and B-other pts (96% vs 100%; p>0.05). At 5 years, the Ph-like ALL subgroup had a lower probability of continuous complete remission (RD: 24% vs 63%; p=0.004) and overall survival (OS: 22% vs 56%; p=0.05) compared to B-other ALL pts. After exclusion of pts with MLL rearrangement from the B-other group (n=11), these differences remained significant (RD: 24% vs 62%; p<0.001; OS: 22% vs 64%; p=0.006).MLPA and amplicon sequencing revealed specific genetic alterations associated with the Ph-like ALL subgroup (Figure 1). All pts with IGH-CRLF2 (n=6) were identified in the Ph-like subgroup, whereas all pts with P2RY8-CRLF2 (n=2) were found in the B-other group (p<0.001 and p>0.05, respectively). Additionally, most pts with high CRLF2 expression clustered in the Ph-like ALL subgroup (13/26, 50% vs 8/79, 10% of B-other; p<0.001). Deletions of IKZF1 were significantly more common in Ph-like ALL (13/16, 81%; p=0.003) and in BCR-ABL1 positive ALL (21/30, 70%; p=0.005) in contrast to B-other ALL (7/21, 30%). Mutations in JAK2 were exclusively found in the Ph-like subgroup (0/53, 0% vs 7/16, 44%; p<0.001). In our data set, FISH for IGH-CRLF2 and sequencing of JAK2 was sufficient to identify Ph-like cases with a sensitivity and specificity of 59% and 100%, respectively.Conclusion: Ph-like ALL in adults is associated with inferior survival in a homogenously treated group of pts. Additionally, molecular analysis revealed distinct genetic alterations identifying this specific ALL subtype. Since gene expression analysis could be difficult to be implemented in routine diagnostics our data suggest, that testing for JAK2 mutations and the IGH-CRLF2 translocation could be options for the diagnosis of the Ph-like subtype. Future treatment strategies should be explored to improve the dismal prognosis for these high risk pts. [Display omitted] DisclosuresNo 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.

Independent prognostic value of BCR-ABL1-like signature and IKZF1 deletion, but not high CRLF2 expression, in children with B-cell precursor ALL

AbstractMost relapses in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are not predicted using current prognostic features. Here, we determined the co-occurrence and independent prognostic relevance of 3 recently identified prognostic features: BCR-ABL1-like gene signature, deletions in IKZF1, and high CRLF2 messenger RNA expression (CRLF2-high). These features were determined in 4 trials representing 1128 children with ALL: DCOG ALL-8, ALL9, ALL10, and Cooperative ALL (COALL)-97/03. BCR-ABL1-like, IKZF1-deleted, and CRLF2-high cases constitute 33.7% of BCR-ABL1–negative, MLL wild-type BCP-ALL cases, of which BCR-ABL1-like and IKZF1 deletion (co)occurred most frequently. Higher cumulative incidence of relapse was found for BCR-ABL1-like and IKZF1-deleted, but not CRLF2-high, cases relative to remaining BCP-ALL cases, reflecting the observations in each of the cohorts analyzed separately. No relapses occurred among cases with CRLF2-high as single feature, whereas 62.9% of all relapses in BCR-ABL1–negative, MLL wild-type BCP-ALL occurred in cases with BCR-ABL1-like signature and/or IKZF1 deletion. Both the BCR-ABL1-like signature and IKZF1 deletions were prognostic features independent of conventional prognostic markers in a multivariate model, and both remained prognostic among cases with intermediate minimal residual disease. The BCR-ABL1-like signature and an IKZF1 deletion, but not CRLF2-high, are prognostic factors and are clinically of importance to identify high-risk patients who require more intensive and/or alternative therapies.

IKZF1andCRLF2gene alterations correlate with poor prognosis in JapaneseBCR‐ABL1‐negative high‐risk B‐cell precursor acute lymphoblastic leukemia

Genome-wide analysis studies have demonstrated that IKZF1, CRLF2, and JAK2 gene alterations correlate with poor prognosis in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the prognostic significance for these gene alterations has not been clarified in Japanese patients. A total of 194 patients with BCP-ALL enrolled in the Japanese Children's Cancer & Leukemia Study Group ALL 2004 clinical trial were assessed for the presence of three different gene alterations: IKZF1 deletions, CRLF2 expression and JAK2 mutation. IKZF1 deletions and CRLF2-high expression were identified in 22 of 177 (12%) patients and in 15 of 141 (11%) patients, respectively. However, JAK2 R683 mutation was detected only one of 177 patients. The 4-year event-free survival (4y-EFS) was different when comparing patients with or without IKZF1 deletions (68.2% vs. 85.2%; P = 0.04) and was also different when comparing patients with different CRLF2 expression levels (high, 66.7% vs. low, 88.1%; P = 0.03). The differences in 4y-EFS were statistically significant in patients with ALL in the National Cancer Institute (NCI)-high risk group (HR-ALL) (IKZF1 deletions: yes, 58.3% vs. no, 87.0%, P = 0.02; CRLF2 expression: high, 55.6% vs. low, 85.3%, P = 0.04) but not in patients with ALL in the NCI-standard risk group (SR-ALL; IKZF1 deletions: yes, 80.0% vs. no, 84.4%, P = 0.75; CRLF2 expression: high, 83.3% vs. low, 89.2%, P = 0.77). Coexistence of IKZF1 deletions and CRLF2-high expression associated with poor outcomes. IKZF1 deletions and CRLF2-high expression predicted poor outcomes in patients with HR-ALL but not in patients with SR-ALL in our Japanese cohort.

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 CRLF2 Genomic Lesions in Patients with Pediatric B-Precursor Acute Lymphoblastic Leukemia (BCP ALL) by Flow Cytometry or Quantitative RT-PCR: A Children's Oncology Group (COG) Stud.

AbstractAbstract 2529▪▪This icon denotes a clinically relevant abstractGenomic alterations involving the CRLF2 gene lead to over-expression of intact CRLF2 and have significant prognostic value in pediatric BCP-ALL. Not only do patients with these lesions have inferior outcomes, they also have a very high frequency of JAK1 and JAK2 mutations and may be candidates for targeted therapies. The two major CRLF2 lesions include cryptic translocations that produce IgH@-CRLF2 and interstitial deletions of the pseudoautosomal region of X/Y causing P2RY8-CRLF2 fusion. Both lesions can be detected by fluorescence in situ hybridization (FISH), and genomic PCR or RT-PCR can identify P2RY8-CRLF2. To develop rapid and inexpensive assays for detection/screening of these events, we developed a flow cytometry based method to measure CRLF2 expression and compared this assay to quantitative RT-PCR (qPCR) measurement of CRLF2 expression by evaluating their performance in an unselected cohort of 279 newly diagnosed pediatric BCP-ALL patients consecutively enrolled on the COG AALL03B1 biology/classification study between 10/30/09-5/1/10. Flow cytometry was performed first in real time on diagnostic specimens shipped to a central COG reference laboratory and then residual diagnostic material was shipped to a separate laboratory for RNA isolation and qPCR analysis. Of the 279 cases analyzed by flow, 257 (92%) yielded sufficient RNA quality and quantity for qPCR analysis. In our previous studies with qPCR and CRLF2 it was shown that CRLF2 lesions occurred only among those cases with the highest expression (ΔCt < 8). In order to assure that we identified all cases with CRLF2 lesions, we performed FISH and P2RY8-CRLF2 PCR on all cases with qPCR expression ΔCt < 10 (n = 109) and an additional 14 cases with a flow blast/lymph CRLF2 mean fluorescence intensity (MFI) ratio >1.15. Of these 123 cases, 11 were determined by FISH to have the IGH@-CRLF2 translocation and 15 were shown to have P2RY8-CRLF2 fusions by PCR. Figure 1 shows the locations of these genomic lesion-positive cases among the qPCR (panel A) and flow cytometry (panel B) CRLF2 expression data. The overall frequency of CRLF2 lesions among these patients is 10.1% (assuming all lesions were identified among the highest expressing cases) and, surprisingly, the frequencies of IgH@-CRLF2 and P2RY8-CRLF2 were very similar (4.3% and 5.8%, respectively). With both methods, the 11 IgH@-CRLF2 cases were found to be the highest expressing (among the top 12 cases by qPCR and 16 cases by flow). Receiver operating curve analysis of each method identified cutoffs with excellent performance: qPCR cutoff CRLF2 ΔCt = 5.47 with 96.9% specificity and 88.5% sensitivity; flow cutoff MFI CRLF2 ratio of 2.04 with 95.9% specificity and 92.3% sensitivity. The broader dynamic range of the qPCR assay may be necessary for the identification of poor risk cases with high CRLF2 expression that lack genomic lesions, however both methods are rapid, highly effective and very comparable for finding ALL cases that harbor CRLF2 genomic lesions, and suitable for incorporation in large scale clinical trials. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Outcome modeling with CRLF2, IKZF1, JAK, and minimal residual disease in pediatric acute lymphoblastic leukemia: a Children's Oncology Group Study

As controversy exists regarding the prognostic significance of genomic rearrangements of CRLF2 in pediatric B-precursor acute lymphoblastic leukemia (ALL) classified as standard/intermediate-risk (SR) or high-risk (HR), we assessed the prognostic significance of CRLF2 mRNA expression, CRLF2 genomic lesions (IGH@-CRLF2, P2RY8-CRLF2, CRLF2 F232C), deletion/mutation in genes frequently associated with high CRLF2 expression (IKZF1, JAK, IL7R), and minimal residual disease (MRD) in 1061 pediatric ALL patients (499 HR and 562 SR) on COG Trials P9905/P9906. Whereas very high CRLF2 expression was found in 17.5% of cases, only 51.4% of high CRLF2 expressors had CRLF2 genomic lesions. The mechanism underlying elevated CRLF2 expression in cases lacking known genomic lesions remains to be determined. All CRLF2 genomic lesions and virtually all JAK mutations were found in high CRLF2 expressors, whereas IKZF1 deletions/mutations were distributed across the full cohort. In multivariate analyses, NCI risk group, MRD, high CRLF2 expression, and IKZF1 lesions were associated with relapse-free survival. Within HR ALL, only MRD and CRLF2 expression predicted a poorer relapse-free survival; no difference was seen between cases with or without CRLF2 genomic lesions. Thus, high CRLF2 expression is associated with a very poor outcome in high-risk, but not standard-risk, ALL. This study is registered at www.clinicaltrials.gov as NCT00005596 and NCT00005603.

Unfavorable Prognostic Value of IKAROS but Not CRLF2 in Children with BCRABL1-positive Acute Lymphoblastic Leukemia,

Abstract 3528Despite the relative high success-rate to treat children (<18 yrs) with precursor B-ALL (5-year event-free survival >80%), the prognosis of the subtype of BCRABL1- positive ALL remains poor (5-year event-free survival <50%). Patients carrying this translocation are treated according to intensified high-risk protocols which are, however, associated with therapy-related side-effects and mortality. A different treatment for those cases at high risk of treatment failure and less treatment for those at low risk of treatment failure is preferred to improve overall outcome results in children with BCRABL1 -positive ALL. Deletions in the gene encoding Ikaros (IKZF1) and an aberrant high expression level of the cytokine receptor-like factor 2 gene (CRLF2) have both recently been associated with a poor prognosis of children with newly diagnosed precursor B-ALL. We here studied whether these genomic abnormalities can also serve as prognostic factor within the subset of BCRABL1 -positive pediatric ALL. Methods;Leukemic cells of BCRABL1- positive patients enrolled in two consecutive treatment protocols of the Dutch Childhood Oncology Group (DCOG) were analyzed for IKZF1 and CRLF2 status. IKZF1 deletions were determined by Multiplex Ligation Probe-based Amplification assays (MLPA) and comparative genomic hybridization arrays (aCGH; Agilent, Sureprint G3 Human CG 180K). The p335 MPLA assay (MRC Holland) was used for discovery of IKZF1 deletions and data were validated by aCGH and a second MLPA assay (p202; MRC Holland) with higher resolution for affected regions. CRLF2 expression levels were determined by Affymetrix U133 plus 2.0 gene expression arrays. A large cohort of 459 newly diagnosed ALL cases served as reference. A high level of CRLF2 expression was assigned to 10% of all cases ranked according to CRLF2 expression level. Results;Deletions in IKZF1 were found in 65% of the cases (n=11); 10 cases had a partial deletion between exon 2 and 7 of IKZF1 resulting in a dominant negative variant and 1 case had a deletion of the complete IKZF1 gene. Analysis of patient characteristics revealed that cases with deletions in IKZF1 have a median 4.5-fold higher white blood cell count compared to unaffected cases (p=0.009). Analysis of the cumulative incidence of relapse (pCIR) with death as a competing risk indicated that children with IKZF1 -deleted BCRABL1 -positive ALL had a significantly increased risk of developing a relapse compared with those with an unaffected IKZF1 gene (73% versus 17%, p=0.02). As a consequence, the 5-year event-free survival was also lower for the IKZF1- deleted group compared to the unaffected group (18% versus 67% p=0.04, Log-Rank). The expression level of CRLF2 in BCRABL1- positive cases was median 91.4 arbitrary units (p25-p75: 84.2–97.2) compared to 91.3 arbitrary units (p25-p75: 82.6–107.3) for BCRABL1 -negative cases (p>0.05). Expression of none of the BCRABL1 -positive ALL cases was ranked within the highest 10% percentile of the reference group. Hence, high expression of CRLF2 cannot explain a poor prognosis of BCRABL1- positive cases. In conclusion,our data show that a deletion of IKZF1 but not a high expression of CRLF2 is a strong discriminative prognostic factor in BCRABL1- positive pediatric precursor B-ALL. We therefore advocate to implement the IKZF1 status as a new prognostic factor in BCRABL1- positive pediatric ALL. Disclosures:No relevant conflicts of interest to declare.

IKZF1 and CRLF2 Gene Alterations Correlate with Poor Prognosis in BCR-ABL1-Negative High Risk B-Cell Precursor Acute Lymphoblastic Leukemia

Abstract 566▪FN2▪This icon denotes a clinically relevant abstract Introduction:Despite improved outcome of current therapy, up to 20% of pediatric patients with B-cell precursor Acute Lymphoblastic Luekemia (BCP-ALL) still relapse. Recent genome-wide analysis showed that IKZF1, JAK2 and CRLF2 gene alterations correlate with poor prognosis in these patients. We analyzed theses gene alterations in Japanese patient cohort. Materials and Methods:Two hundred patients with BCR-ABL1-negative BCP-ALL between 1 and 18 years old, who were enrolled in Japanese Children’s Cancer & Leukemia Study Group (JCCLSG)-2004 study from 2004 to 2008, were analyzed in this study. DNA in 179 patients and RNA in 147 patients, respectively, were extracted from the initial diagnostic bone marrow or peripheral blood samples. Using DNA samples, JAK2R683 point mutation was analyzed by direct sequencing, and the copy number alterations of IKZF1 by multiplex ligation-dependent probe amplification (MLPA). CRLF2 expression in RNA samples were measured by real-time quantitative polymerase chain reaction (RQ-PCR). At the same time, we detected IKZF1 transcript of Ik6 isoform (del. exon 4 to 7) and P2RY8-CRLF2 fusion gene expression by reverse transcribed (RT) - PCR. Event-free survival (EFS) and relapse-free interval (RFI) was calculated by Kaplan-Meier method. Event was defined with induction failure, relapse, death of any cause in EFS, and with relapse in RFI. Result:Twenty-two (12%) of 179 patients had some exon deletions in IKZF1. Seven (32%) of 22 patients had only one copy of all exon (exon 1 to 8) and 6 of 22 (27%) patients had deletion from exon 4 to 7, which expressed Ik6 isoform. The older the age was, the higher the frequency of IKZF1 deletion was. The frequency of IKZF1 deletions (12/59) in the patients of NCI-HR group was significantly higher than that (10/120) of the patients of NCI-SR group (p=0.029). In outcome, EFS for the patients with IKZF1 deletions was significantly worse than that for without IKZF1 deletions (EFS; 66.4%±12.1% vs. 89.2%±2.7% respectively, p=0.007). And RFI for the patients with IKZF1 deletions was also worse than that for without it, (RFI; 73.1%±12.4% vs. 90.9%±2.6%, respectively, p=0.047). IKZF1 deletions seemed to be poor prognostic factor in NCI-HR patients, while they are not in NCI-SR, as shown in Table.High CRLF2 expression (10 times higher than median value, 14.4 copies) was detected in 15 (10%) of 147 patients. Only 2 of 15 CRLF2-high patients had P2RY8-CRLF2 fusion gene by RT-PCR. We also detected 3 of 135 CRLF2-low patients had this fusion. EFS for CRLF2-high expression patients had also significantly worse than that for CRLF2-low patients (EFS; 62.2%±13.7% vs. 90.6%±2.7%, p=0.003). And RFI for CRLF2-high patients was 73.1%±12.4%, while RFI for CRLF2-low patients was 90.9%±2.6% (p=0.069). EFS for CRLF2-high showed significantly worse than that for CRLF2-low in NCI-HR patients (53.3%±17.3% vs. 87.0%±6.1%, p=0.028), but not significantly in NCI-SR (p=0.445). Regardless of patients with or without CRLF2-high expression, they showed similar RFI in NCI-HR and SR. Five patients had IKZF1 deletions and CRLF2-high expression, simultaneously, and showed extremely poor prognosis (4 of 5 showed induction failure or relapse). None of 179 patients had JAK2R683 mutation in this study. Discussion:While IKZF1 and CRLF2 alterations were detected in similar frequency to previous reports, P2RY8-CRLF2 fusion was found only 13% of CRLF2-high patients. Moreover, JAK2R683 mutation was not detected in any patients in this study. These findings suggest that there might be racial difference in genetic mechanism of leukemogenesis. IKZF1 and CRLF2 alterations might be a poor prognostic factor in NCI-HR patients, but not in NCI-SR. These independency would be disclosed in next large-scale national study. Disclosures:No relevant conflicts of interest to declare.

Identification of novel cluster groups in pediatric high-risk B-precursor acute lymphoblastic leukemia with gene expression profiling: correlation with genome-wide DNA copy number alterations, clinical characteristics, and outcome

AbstractTo resolve the genetic heterogeneity within pediatric high-risk B-precursor acute lymphoblastic leukemia (ALL), a clinically defined poor-risk group with few known recurring cytogenetic abnormalities, we performed gene expression profiling in a cohort of 207 uniformly treated children with high-risk ALL. Expression profiles were correlated with genome-wide DNA copy number abnormalities and clinical and outcome features. Unsupervised clustering of gene expression profiling data revealed 8 unique cluster groups within these high-risk ALL patients, 2 of which were associated with known chromosomal translocations (t(1;19)(TCF3-PBX1) or MLL), and 6 of which lacked any previously known cytogenetic lesion. One unique cluster was characterized by high expression of distinct outlier genes AGAP1, CCNJ, CHST2/7, CLEC12A/B, and PTPRM; ERG DNA deletions; and 4-year relapse-free survival of 94.7% ± 5.1%, compared with 63.5% ± 3.7% for the cohort (P = .01). A second cluster, characterized by high expression of BMPR1B, CRLF2, GPR110, and MUC4; frequent deletion of EBF1, IKZF1, RAG1-2, and IL3RA-CSF2RA; JAK mutations and CRLF2 rearrangements (P < .0001); and Hispanic ethnicity (P < .001) had a very poor 4-year relapse-free survival (21.0% ± 9.5%; P < .001). These studies reveal striking clinical and genetic heterogeneity in high-risk ALL and point to novel genes that may serve as new targets for diagnosis, risk classification, and therapy.

Presence of the P2RY8-CRLF2 rearrangement is associated with a poor prognosis in non–high-risk precursor B-cell acute lymphoblastic leukemia in children treated according to the ALL-BFM 2000 protocol

AbstractHigh-level expression of the cytokine receptor-like factor 2 gene, CRLF2, in precursor B-cell acute lymphoblastic leukemia (pB-ALL) was shown to be caused by a translocation involving the IGH@ locus or a deletion juxtaposing CRLF2 with the P2RY8 promoter. To assess its possible prognostic value, CRLF2 expression was analyzed in 555 childhood pB-ALL patients treated according to the Acute Lymphoblastic Leukemia Berlin-Frankfurt-Münster 2000 (ALL-BFM 2000) protocol. Besides CRLF2 rearrangements, high-level CRLF2 expression was seen in cases with supernumerary copies of the CRLF2 locus. On the basis of the detection of CRLF2 rearrangements, a CRLF2 high-expression group (n = 49) was defined. This group had a 6-year relapse incidence of 31% plus or minus 8% compared with 11% plus or minus 1% in the CRLF2 low-expression group (P = .006). This difference was mainly attributable to an extremely high incidence of relapse (71% ± 19%) in non–high-risk patients with P2RY8-CRLF2 rearrangement. The assessment of CRLF2 aberrations may therefore serve as new stratification tool in Berlin-Frankfurt-Münster–based protocols by identifying additional high-risk patients who may benefit from an intensified and/or targeted treatment.

DOWN'S Syndrome Acute Lymphoblastic LEUKEMIA: A HIGHLY Heterogeneous DISEASE DRIVEN by an Aberrant CRLF2/JAK2 Cooperation – A REPORT FROM the Ibfm-STUDY GROUP.

Abstract 11Children with Down's Syndrome (DS) have an increased risk for acute lymphoblastic leukemia (ALL). We have previously identified somatic mutations in R683 of JAK2 in 20% of DS-ALLs (Bercovich et al, Lancet 2008;372:1484). However the cooperating cytokine receptor and other molecular features have been obscured. Here we report that gene expression analysis reveals that DS-ALL is a highly heterogeneous disease not definable as unique ALL subtype. However, 62% (33 of 53) of the DS-ALL samples analyzed were characterized by aberrant expression of the type I cytokine receptor CRLF2 caused by two types of genomic aberrations. CRLF2 encodes one chain of the heterodimeric receptor of the TSLP cytokine involved in inflammation and lymphoid development and has been recently reported to be involved in translocations to the IgH locus or micro-deletions in 3% of ALLs in children without DS. We have confirmed its cell surface expression on primary DS-ALL cells by Flow cytometry analysis. Consisting with a possible role of CRLF2 as a cytokine receptor, all specimens with the R683 mutated JAK2 had high CRLF2 expression. We further show that CRLF2 and R683 mutated JAK2 cooperate in conferring cytokine independent growth of pro-B cells. Furthermore in 3 of 23 DS-ALL patients with wt. JAK2 we identified a novel activating somatic mutation in CRLF2 replacing the juxta-membrane F232 with Cystein. Mutated CRLF2 but not wild type CRLF2 conferred cytokine independent growth of BaF3 cells associated with phosphorylation of STAT5. Aberrant expression of CRLF2 was also associated with a younger age at diagnosis and worse prognosis. Intriguingly, bioinformatic analysis revealed that gene expression of DS-ALLs is enriched with DNA damage and BCL6 responsive genes suggesting the possibility that DS is associated with B-cell lymphocytic genomic instability leading to the genomic aberrations activating CRLF2. Thus DS confers increased risk for genetically highly diverse ALLs with frequent somatic anomaly in CRLF2, whose higher expression further selects for the activating mutations in itself or in JAK2 (figure). Our data also suggests that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathway. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

CRLF2/JAK Signaling Confers Susceptibility to JAK Inhibitors and Small Molecule Inhibitors of Protein Kinase C.

Abstract 3767Poster Board III-703 IntroductionWe and others recently identified the type I-like cytokine receptor CRLF2 as an oncoprotein in adult and high-risk pediatric precursor B-cell acute lymphoblastic leukemia (B-ALL). Wild-type CRLF2 heterodimerizes with IL7R and signals in response to thymic stromal lymphopoietin (TSLP) through unclear mechanisms that include STAT5 activation. In cases of B-ALL that overexpress CRLF2, the CRLF2 locus is rearranged through either a translocation with IGH or an intrachromosomal deletion. CRLF2 rearrangements are confined to patients who lack translocations involving MLL, E2A, TEL, and BCR/ABL. A subset of cases with CRLF2 overexpression also harbors a CRLF2 Phe232Cys gain-of-function mutation, while a separate subset has mutations in Janus Kinases (JAK). Cells transformed by CRLF2 Phe232Cys or the combination of wild-type CRLF2 with JAK2 exon 16 mutants have constitutive activation of STAT5 and ERK, and upregulation of STAT target genes. However, only the cells that express JAK2 mutants have constitutive phosphorylation of JAK2, suggesting that CRLF2 Phe232Cys signals through an alternate kinase. We used transcriptional profiling to identify candidate pathways and then screened available kinase inhibitors for selective toxicity in cells transformed by CRLF2 and JAK2 mutant alleles. MethodsWe reviewed transcriptional profiles obtained from 22 adult patients with B-ALL enrolled on Gruppo Malattie Ematologiche dell'Adulto (GIMEMA) protocols LAL2000 and LAL2004. All samples were confirmed to have high (n=8) or low (n=14) CRLF2 expression by quantitative RT-PCR. Samples were assayed on the Affymetrix HG-U133A platform, which contains a single probe set (208303_s_at) that targets the CRLF2 transcript (both complete and partial coding sequence, as well as expression sequence tags). Supervised analysis was performed using a t-test: probe sets were 1.5, and expression≥0.005 and fold change ≤required to have a p-value >100 in at least one group. For the kinase inhibitor screen, BaF3 cells that stably express CRLF2, IL7R and/or JAK2 alleles were incubated with compound for 48 hours in the absence of IL3. Viability was measured by ATP content with luminescence using CellTiterGlo and read on an Envision plate reader. ResultsSupervised analysis defined a 105 gene (130 probe set) signature associated with CRLF2 overexpression. This signature included upregulation of CD10, CD99, the STAT5 induced regulator SOCS2 and Protein Kinase C (PKC) iota. Downregulated genes included class I and class II major histocompatibility antigens. Based on the involvement of JAKs in CRLF2 signaling and the overexpression of PKC iota, we carried out a kinase inhibitor screen. Compared with cells that express wild-type CRLF2/IL7R grown in the presence of TSLP, cells transformed by CRLF2 Phe232Cys or wild-type CRLF2/mutant JAK2 had 10-100-fold lower EC50 for small-molecule inhibitors of JAK signaling and agents with activity against PKC family kinases (Go6976, UCN-01, k252a). Assays to confirm these findings in primary B-ALL specimens are underway. ConclusionCRLF2/JAK signaling confers a unique pattern of kinase inhibitor sensitivity that can be elucidated through transcriptional profiling and targeted with available agents. Disclosures:No relevant conflicts of interest to declare.