Cytokine Receptor-like Factor 2 Overexpression Research Articles

JAK2 mutations and CRLF2 rearrangements in Down Syndrome-Associated Acute Lymphoblastic Leukemia in Japan

Abstract 1451Children with Down syndrome (DS) have a 10- to 20-fold increased risk of developing acute lymphoblastic leukemia (ALL). In DS-associated ALL (DS-ALL), the chromosome aberrations which are generally common in childhood ALL, such as hyperdiploidy and t(12;21), are less frequent. Recent studies have shown that activating JAK2 mutations and overexpression of cytokine receptor-like factor 2 (CRLF2) gene are identified in approximately 20% and 50–60% of DS-ALL in Western countries, respectively. Most of the patients with CRLF2 overexpression have been reported to be associated with interstitial deletions of the pseudoautosomal region 1 (PAR1) of the sex chromosomes and the P2RY8-CRLF2 fusion gene. In addition, one report showed that the activating CRLF2 F232C mutation was identified in about 10% of DS-ALL. However, there have been no studies to determine the incidence of these genetic aberrations in Asian patients with DS-ALL. In this study, 23 patients with DS-ALL in Japan were screened for mutations in the pseudokinase domain of the JAK2 gene, the P2RY8-CRLF2 fusion gene, and the CRLF2 F232C mutation by PCR/RT-PCR and direct sequencing. Fourteen patients, whose bone marrow RNAs were available, were also screened for CRLF2 overexpression by real-time quantitative RT-PCR. We identified the JAK2 R683G mutation in 2 patients (9%) and the P2RY8-CRLF2 fusion gene in 4 patients (17%). The CRLF2 F232C mutation was not detected in any patient. CRLF2 overexpression was observed in 2 of 14 patients examined (14%). Although bone marrow RNA was available in only 1 of 4 patients positive for P2RY8-CRLF2, high-level expression of CRLF2 was confirmed in this patient. The other patient with CRLF2 overexpression was negative for P2RY8-CRLF2, indicating the involvement of the other type of CRLF2 rearrangement, IGH@-CRLF2 in this patient. We also performed a preliminary study on JAK1, JAK3, and Interleukin-7 receptor-α (IL7R) mutations, and 14, 11, and 12 patients were screened for mutations in the pseudokinase domain of JAK1, JAK3, and exon 5 and 6 of IL7R, respectively. However, no mutations were identified in any patient. Our results show the lower incidence of CRLF2 rearrangements in DS-ALL patients in Japan than that in Western countries. Gene alterations other than CRLF2 rearrangements may contribute to leukemogenesis in Japanese patients with DS-ALL. To clarify if the incidences of the mutations in JAK1-3, CRLF2, and IL7R are also lower in DS-ALL patients in Japan than those in Western counties, more patients need to be studied. Disclosures:No relevant conflicts of interest to declare.

IKZF1 Deletion Is Strongly Associated with Risk of Relapse in Intermediate Risk Group in JACLS ALL02 Cohort

Abstract 1455▪▪This icon denotes a clinically relevant abstract Introduction:Despite the progress of the current therapy, approximately 20 % of pediatric patients with B-cell-presursor (BCP)-ALL experience relapse who had no conventional adverse prognostic factor. Recently, alteration of the IKZF1 gene has been reported to be associated with a poor outcome in pediatric BCP-ALL without BCR-ABL. In addition, it is also reported that a significant proportion of these cases with the alteration of IKZF1 shared the point mutation of JAK2 and over-expression of cytokine receptor-like factor 2 (CRLF2). Herein, in order to assess the prognostic value of these genetic abnormalities in Japanese cohort, we conducted genetic analysis of IKZF1, JAK2 and CRLF2 in pediatric BCP-ALL. Materials and Methods:Diagnostic bone marrow or peripheral blood samples of 215 pediatric BCP-ALL patients treated according to Japan Association of Childhood Leukemia Study (JACLS) ALL02 protocol from April, 2002 to May, 2008 were examined in this study. All patients were categorized into high risk (HR) group defined as follows;(1) initial white blood cell counts more than 10,000 /μl, (2) age at diagnosis is > 10 years and (3) good response to initial predonisolone (PSL) treatment ( blast counts in peripheral blood is less than 1,000 /μl after one week PSL treatment). Ph+ALL and infantile ALL patients were excluded in this protocol. The patients with Down syndrome were also excluded in this genetic analysis. The deletion of IKZF1 was determined using multiplex ligation-dependent probe amplification (MLPA) in 212 patients whose diagnostic DNA samples were available. The expression of isoform of IKZF1 was determined by RT-PCR in 113 patients whose diagnostic RNA samples were available. The expression level of CRLF2 was also determined by real time RT-PCR in 112 patients and over-expression was defined as over ten times more than median expression value. The presence of P2RY8-CRLF2 fusion was examined by RT-PCR or MLPA in the patients with CRLF2 over-expression or IKZF1 deletion. JAK2 mutations were also determined by direct sequencing of the exon 16, 20 and 21 in the patients with IKZF1 deletion. Results:The deletion of IKZF1 gene was present in 19 of 212 (9.0 %) patients. In detail, the mono-allelic deletion of entire IKZF1 gene was present in 10 of 19 patients. On the other hand, the expression of dominant-negative IK6 isoform was present in 9 of 112 (8.0%) patients including 12 patients with IKZF1 deletion. The expression of IK6 was present in 4 of 12 (33.3%) IKZF1 deleted patients. Interestingly, 5 of 9 patients with IK6 expression had no alteration of IKZF1 gene. In terms of CRLF2, over-expression was detected in 16 of 112 patients (14.3 %). However, P2RY8-CRLF2 fusion was not detected in these 16 patients with altered CRLF2 expression. Strikingly, none of the patients with IKZF1 deletion (n=19) had either P2RY8-CRLF2 fusion or JAK2 exon 16, 20 and 21 mutation. Patients with IKZF1 gene deletion had significantly worse relapse rate than those without IKZF1 deletion (7/19 vs 22/193, p<0.01). On the contrary, none of the patients with IK6 expression experienced relapse. Discussions:This study confirmed that the presence of IKZF1 deletion was strongly correlated with risk of relapse in intermediate risk group in JACLS ALL02 cohort. Thus, we expect that IKZF1 deletion is an independent predictor of treatment outcome and represents a candidate of prognostic marker to be integrated in future algorithms for early risk stratification in pediatric BCP-ALL. Strikingly, point mutation of JAK2 exon 16, 20 and 21 or P2RY8 -CRLF2 fusion was rarely present even in BCP-ALL patients with IKZF1 deletion. There might be unrevealed class I mutation cooperating with IKZF1 alteration in Japanese BCP-ALL cohort. Disclosures:No relevant conflicts of interest to declare.

Relationship of CRLF2 expression and outcome in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL): A report from the Children’s Oncology Group.

9505 Background: We previously reported a highly significant association between over-expression of CRLF2 (cytokine receptor-like factor 2) and poor clinical outcomes in 207 uniformly-treated children with high-risk B-ALL (Blood 115:5312, 2010). To validate and extend these studies to standard risk cases we studied 896 B-ALL patients from COG P9905. Eligible patients included 344 with NCI high-risk (HR) features and 552 with standard-risk (SR) features. Methods: Quantitative RT-PCR assays were used to determine CRLF2 expression in the diagnostic leukemic cells. Averages of duplicate assays were used to establish ΔCt values relative to a control gene (EEF2). Using the ΔCt threshold determined from our prior studies, 117/896 B-ALL samples had elevated CRLF2 expression. These 117 samples and other 35 samples below the threshold were tested for genomic rearrangements of CRLF2 to detect the P2RY8-CRLF2 fusion or CRLF2 F232C mutations. Studies to detect the IGH@-CRLF2 translocation, IKZF1 (IKAROS) deletions, and JAK mutations are ongoing. Outcomes were evaluated using relapse-free survival (RFS) and other known risk factors. Results: CRLF2 overexpression was associated with a poorer outcome in HR, but not in SR, B-ALL. Among the 246 HR cases lacking detectable end-induction minimal residual disease, CRLF2 expression levels could further distinguish those B-ALL patients who had a very good (>80%) vs. poor (<40%) RFS (Hazard Ratio = 2.59; P=0.002). While P2RY8/CRLF2 fusions were seen only in high CRLF2-expressors, 6/17 CRLF2 F232C mutations were among low CRLF2 expressors. Down syndrome ALL cases had a relatively good outcome regardless of CRLF2 expression levels or NIH risk status. Conclusions: Elevated CRLF2 expression predicts a significantly poorer outcome in HR, but not SR, B-ALL and can be used with other prognostic factors to improve ALL risk classification. Standard-risk RFS High-risk RFS N Hazard ratio P N Hazard ratio P All cases (n=896) 552 – – 344 – – High CRLF2 81 1.20 .510 36 1.84 .019 Down syndrome (DS) 35 1.25 .571 5 0.94 .952 Genomic abnormalities 99 53 P2RY8 fusion 37 1.00 .988 14 1.64 .034 F232C mutation 15 – – 2 – –

Analysis of CRLF2/JAK Expression and Mutation Status In Adult ALL Patients

AbstractAbstract 758Adult acute lymphoblastic leukemia (ALL) is a heterogeneous disease with low overall survival. Compared to pediatric ALL, outcomes for adults have not improved despite recent advances in therapy and treatment support. Novel targets and biomarkers will aid in the development of new therapeutic strategies. Recent studies described mutations in the cytokine receptor-like factor 2 (CRLF2), and Janus kinase 1 (JAK1) and JAK2 in ALL patients (pts). These activating mutations have been associated with poor outcome in adult and pediatric series. Interestingly, the gene expression profile (GEP) of pts with these abnormalities overlaps with Philadelphia chromosome (Ph) positive ALL GEP. In order to further assess the prognostic value of these genetic abnormalities in adult ALL we analyzed RNA from available pretreatment bone marrow (BM) and peripheral blood (PB) samples of 138 pts enrolled in SWOG studies S9400, and S0333.The expression levels of CRLF2 were examined in 85 available specimens with more than 50% blasts. B-glucoronidase, GUSB, was used to correct for RNA integrity. Expression fold change was calculated relative to the pool of RNA from the PB of 7 healthy donors using the comparative Ct analyses (2-DDCt). CRLF2 levels varied significantly in the examined population, ranging from no detectable expression to 8735-fold greater than the calibrator. High-expressers were defined by expression fold change 10-times above the median (n=21, 25%). Deletion of pseudoautosomal region 1 of chromosomes × and Y, with juxtaposition of the P2RY8 promoter to the CRLF2 coding region, was tested in 126 pts, and 20 pts were positive for P2RY8-CRLF2 (16%). Sixty-seven percent of CRLF2 high-expressers displayed this mutation (14 out of 21). CRLF2 point mutation F232C was identified in 6% of the samples (7 out of 126). Less than 2% of samples (1.6%, 2 out of 120) displayed JAK1 mutations (V658F and V658I). Seven percent of tested pts (9 out of 129) had JAK2 mutations: D869V, L884P, N622T, R683G, and R683S (1, 2, 1, 4, and 1 pts, respectively).CRLF2 over-expression, the presence of P2RY8-CRLF2, or mutations in CRLF2, JAK1, or JAK2 did not significantly correlate with, gender, age or baseline WBC. No significant correlation with outcome variables (CR, RD, RFS, or OS) was found when the cohort was analyzed as a whole, or when pts with known T-cell lineage ALL are excluded (n=115). Though not a statistically significant result, the two pts with JAK1 were reported living at 9 and 12 years of follow-up. Subgroup analysis of Ph negative non-T-ALL pts (n=86) showed a modest correlation of CRLF2 high expression with a lower CR rate (50% vs. 76% CR rate chi-square test p=0.046). However, this trend in CR rate is not seen in the analysis of continuous CRLF2 expression levels and overall survival is not significantly correlated with CRLF2 expression. Conclusion:Our results do not confirm previous reports of associations between clinical outcomes and expression of CRLF2 and mutations in CRLF2, JAK1 and JAK2 in adult and pediatric ALL. The discrepancy between previous and current studies may be attributed to differences in age (adult vs. children) or treatment protocol. CRLF2 is a potential therapeutic target and biomarker for response for pediatric and adult ALL. Disclosures:No relevant conflicts of interest to declare.