Cytokine Receptor-like Factor 2 Overexpression Research Articles

CRLF2 Expression in Mexicans with Acute Lymphoblastic Leukemia across the Age Spectrum

Introduction Acute lymphoblastic leukemia (ALL) is a hematological malignancy that primarily affects children also occurs in adults. Certain subtypes of ALL, such as Philadelphia chromosome-like ALL (Ph-like ALL), present unique challenges and are associated with poor outcomes. Alterations in the CRLF2 (Cytokine Receptor-like Factor 2) gene have emerged as critical contributors to the pathogenesis of Ph-like ALL. CRLF2 gene rearrangements result in the overexpression of CRLF2 protein, leading to dysregulated signaling pathways and enhanced leukemogenesis. The prevalence and clinical implications of CRLF2 overexpression in Mexicans with ALL remain underexplored. Hence, this study aimed to investigate the prevalence of CRLF2 overexpression in Mexican patients with ALL across different age groups. Patients and Methods: We conducted a prospective study on a cohort of ALL patients from a single institution in Mexico that cares for patients across the age spectrum. Consecutive patients with a diagnosis of ALL were enrolled in the study and were assessed for CRLF2 overexpression using flow cytometry and fluorescent in situ hybridization (FISH). Patients were treated with a local BFM-based chemotherapy regimen according to age and risk stratification. In adolescents and young adults ≥16, a pediatric-inspired regimen was used and in patients with positive measurable residual disease (MRD) allogeneic hematopoietic cell transplantation was sought. To determine the impact of CRLF2 overexpression on clinical characteristics and treatment response, we analyzed various parameters, including response rates, MRD, and survival. Statistical analyses were performed using SPSS 25, encompassing descriptive analysis, chi-squared tests, and survival analysis with the Kaplan-Meier method. Results: A total of 79 patients were enrolled with a median age of 17 years (2-70 years), 34 were female and 45 males with a median white blood cell count of 10.1x103/L (.63-649.57). Our findings revealed that 21 out of 79 patients (26.6%) exhibited CRLF2 overexpression. The proportion of CRLF positive ALL increased with age; it was 20.5% in patients aged 0-16 years, 34.6% in 17-39 and 40% in >60 years. There were no statistically significant differences observed in white blood cell counts, central nervous system involvement, response to treatment, or minimal residual disease levels in CRLF2 positive or negative patients (Table 1). In the Kaplan-Meier survival analysis, it was observed that positive CRLF2 expression was significantly associated with lower overall survival (OS) with a median of 24.9 months (0.2-65.4 months); with 85.8% OS at 2 years in CRLF2 negative vs 41.2% in CRLF2 positive patients (Figure 1). Event-free survival rate at 2 years was also lower with 70.8% vs 35.3% at 2 years in CLRF2 positive patients. Moreover, this association was found to be consistent across all age subgroups, including patients aged 0-16, 17-39, and over 40 years (P < 0.05). Conclusion: Our study highlights the significance of CRLF2 overexpression as a cost-effective biomarker indicative of an unfavorable prognosis in ALL across the age spectrum. Early detection of this alteration through flow cytometry plays a vital role in risk stratification and should prompt the implementation of tailored therapeutic interventions to improve clinical outcomes. Additionally, our study underscores the importance of conducting population-specific research to gain comprehensive insights into the genetic landscape and disease characteristics of ALL in different ethnic groups.

Poor outcome of pediatric B-cell acute lymphoblastic leukemia associated with high level of CRLF2 gene expression in distinct molecular subtypes.

Overexpression of the cytokine receptor-like factor 2 (CRLF2) gene is the most common feature in the Philadelphia chromosome (Ph)-like subtype of B-cell acute lymphoblastic leukemia (B-ALL). However, the predictive value of CRLF2 overexpression for the prognosis of pediatric B-ALL patients remain controversial. The molecular mechanisms that upregulate CRLF2 expression level in patients has not been fully elucidated. In this study, the prognostic impact of CRLF2 expression level on molecular types of B-ALL in pediatric patients from Zhujiang Hospital (n = 111) was retrospectively analyzed. Youden index analysis was used to categorize CRLF2 expression into 3 groups, and these categories more precisely described the differences in the prognosis of patients with varying expression levels of CRLF2 in both the Zhujiang Hospital cohort and the TARGET cohort. We used the Zhujiang Hospital cohort as a discovery cohort to determine the cutoff value of CRLF2 expression. CRLF2-high patients accounted for approximately 6%. In addition, the percentage of bone marrow blast cells and initial white blood cell count in CRLF2-high patients were higher than those in CRLF2-low patients, and MRD turned negative slower. The results were validated in the TARGET cohort and indicated that CRLF2 overexpression could be subdivided by CRLF2 expression levels into 2 categories: CRLF2-high with a poor survival and CRLF2-medium with a good OS and EFS. Such heterogeneity was attributed to the different molecular mechanisms leading to CLRF2 upregulation, where the CRLF2 overexpression level was high in Ph-like B-ALL and medium in high hyperdiploid B-ALL. This study highlights the importance of the molecular mechanisms of the upregulation of CRLF2 expression in predicting the prognosis of pediatric B-ALL patients.

CRLF2 overexpression results in reduced B-cell differentiation and upregulated E2F signaling in the Dp16 mouse model of Down syndrome

Children with Down syndrome (DS) are 10-fold more likely to develop B-cell acute lymphoblastic leukemia (B-ALL), with a higher frequency of rearrangements resulting in overexpression of cytokine receptor-like factor 2 (CRLF2). Here, we investigated the impact of CRLF2 overexpression on B-cell progenitor proliferation, immunophenotype, and gene expression profile in the Dp(16)1Yey (Dp16) mouse model of DS compared with wild-type (WT) mice. CRLF2 overexpression enhanced immature B-lymphoid colony development and increased the proportion of less differentiated pre-pro-B cells, with a greater effect in Dp16 versus WT. In CRLF2-rearranged (CRLF2-R) B-ALL patient samples, cells with higher CRLF2 expression exhibited a less differentiated B-cell immunophenotype. CRLF2 overexpression resulted in a gene expression signature associated with E2F signaling both in Dp16 B-progenitors and in DS-ALL patient samples, and PI3K/mTOR and pan-CDK inhibitors, which reduce E2F-mediated signaling, exhibited cytotoxicity in CRLF2-R B-ALL cell lines and patient samples. CRLF2 overexpression alone in Dp16 stem and progenitor cells did not result in leukemic transformation in recipient mice. Thus, CRLF2 overexpression results in reduced B-cell differentiation and enhanced E2F signaling in Dp16 B-progenitor cells and DS-ALL patient samples. These findings suggest a functional basis for the high frequency of CRLF2-R in DS-ALL as well as a potential therapeutically targetable pathway.

Prognostic significance of CRLF2 overexpression and JAK2 mutation in Egyptian pediatric patients with B-precursor acute lymphoblastic leukemia

BackgroundThe prognostic significance of cytokine receptor-like factor 2 (CRLF2) overexpression in pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) is still controversial. We aimed to investigate the role of CRLF2 overexpression and JAK2 mutation in the diagnosis and prognosis of newly diagnosed pediatric B-ALL patients. MethodsCRLF2 expression was assessed by real-time quantitative polymerase chain reaction (PCR) in 115 pediatric patients newly diagnosed with precursor B-ALL patients compared with 24 age- and sex-matched controls. JAK2 R683G mutation status was performed by the qBiomarker Somatic Mutation PCR Assay. ResultsCRLF2 overexpression was identified in 21 patients (18.3%), while the JAK2 R683G mutant type was found in only in 7 patients (6.1%). There was a significant CRLF2 overexpression in patients with high initial TLC, high blast count in blood, and organomegaly (P .04, 0.005 & 0.05 respectively). No patients with CRLF2 overexpression expressed any recurrent cytogenetic translocations. 4 patients with CRLF2 overexpression showed JAK2 R683G mutation. CRLF2 levels and JAK2 R683G mutation status did not have a significant impact on either overall survival or disease-free survival. ConclusionCRLF2 expression was significantly higher in Egyptian precursor B-ALL pediatric patients. CRLF2 overexpression was associated with a number of unfavorable prognostic factors with high tumor load, but was not an adverse independent parameter in pediatric BCP-ALL patients. Some patients with CRLF2 overexpression display JAK2 mutation, which may benefit from targeted therapy by kinase inhibitors.

IKZF1 deletions associate with CRLF2 overexpression leading to a poor prognosis in B-cell precursor acute lymphoblastic leukaemia

Cytokine Receptor-Like Factor 2 (CRLF2) overexpression occurs in 5-15% of B-cell precursor acute lymphoblastic leukaemia (B-ALL). In ∼50% of these cases, the mechanisms underlying this dysregulation are unknown. IKAROS Family Zinc Finger 1 (IKZF1) is a possible candidate to play a role in this dysregulation since it binds to the CRLF2 promoter region and suppresses its expression. We hypothesised that IKZF1 loss of function, caused by deletions or its short isoforms expression, could be associated with CRLF2 overexpression in B-ALL. A total of 131 paediatric and adult patients and 7 B-ALL cell lines were analysed to investigate the presence of IKZF1 deletions and its splicing isoforms expression levels, the presence of CRLF2 rearrangements or mutations, CRLF2 expression and JAK2 mutations. Overall survival analyses were performed according to the CRLF2 and IKZF1 subgroups. Our analyses showed that 25.2% of patients exhibited CRLF2 overexpression (CRLF2-high). CRLF2-high was associated with the presence of IKZF1 deletions (IKZF1del, p = 0.001), particularly with those resulting in dominant-negative isoforms (p = 0.006). Moreover, CRLF2 expression was higher in paediatric samples with high loads of the short isoform IK4 (p = 0.011). It was also associated with the occurrence of the IKZF1 plus subgroup (p = 0.004). Furthermore, patients with CRLF2-high/IKZF1del had a poorer prognosis in the RELLA05 protocol (p = 0.067, 36.1 months, 95%CI 0.0-85.9) and adult cohort (p = 0.094, 29.7 months, 95%CI 11.8–47.5). In this study, we show that IKZF1 status is associated with CRLF2-high and dismal outcomes in B-ALL patients regardless of age.

Cytokine Receptor-like Factor 2 (CRLF2) Overexpression in Myeloid Malignancies Secondary to Multiple Isodicentric Y Chromosomes: A Novel Cytogenetic Entity?

Background:Cytokine receptor-like factor 2 (CRLF2) overexpression is well-defined in B-cell and Ph-like ALL (~50%) with the CRLF2/IGH (76%) and P2RY8/CRLF2 (17%) rearrangements being the most common. CRLF2 is located on the pseudoautosomal region or PAR1 of both the short arm of X (Xp22.3) and Y (Yp11.3) chromosomes and its over-expression has not been previously described in myeloid neoplasms. Multiple isodicentric Y chromosomes, which have two centromeres and two copies of the short-arm of the Y chromosome, have the potential to produce amplification of CRLF2, leading to overexpression. We describe three cases of myeloid neoplasia, two with a diagnosis of myelodysplastic syndrome (MDS) and one with a diagnosis of acute myeloid leukemia (AML), in which cytogenetic studies demonstrated multiple copies of an isodicentric Y chromosome. CRLF2 overexpression was anticipated and confirmed via RNA sequencing in one patient. The clinical significance of CRLF2 overexpression is profound as it is known to activate the JAK-STAT pathway and is potentially targetable using tyrosine kinase inhibitors (TKIs).Case series:A similar genetic pattern was identified in three older males with a known diagnosis of myeloid malignancy, two of whom had MDS and one AML. The first patient is a 57-year-old man with a diagnosis of NPM-1/FLT3 ITD wt AML. Diagnostic cytogenetic abnormalities included 46-53, X, idic(Y)(q11.2)x1-8[cp12]/46,XY[8]. The second case is that of an 88-year-old man with monoclonal gammopathy of undetermined significance (MGUS), which progressed to multiple myeloma (MM) with trisomy 11. Two years after his diagnosis of MM, he was diagnosed with MDS with ringed sideroblasts (MDS-RS) with less than 5 per cent blasts, and the diagnostic karyotype result was 50-52,X,idic(Y)(q11.22)x3-4,+12. He had been treated with lenalidomide, dexamethasone and bortezomib. The third case is that of a 71-year-old man with transfusion-dependent anemia diagnosed as MDS-EB-1, with cytogenetic abnormalities at diagnosis being 50-54,XY,+idic(Y)(q11.2)x2-4,+8,+21[cp10]/46,XY[10]. Next generation sequencing identified pathogenic mutations in ASXL1 (c.1900_1922del; p.Glu635Argfs*15, 14%), IDH2 (c.419G>A; p.Arg140Gln, 37%) and SRSF2 (c.284_307del; p.Pro95_Arg102del, 36%). A bone marrow sample from this patient was subjected to RNA sequencing, and demonstrated overexpression of CRLF2, but not of SRY; confirming our hypothesis of multiple dicentric Y chromosomes resulting in CRLF2 overexpression (Figure 1).Discussion and Conclusions:Activation of JAK-STAT signaling is seen in Philadelphia chromosome positive (Ph) B- lymphoblastic lymphoma/leukemia (B-ALL) or Ph- like B- ALL and is therapeutically targetable with TKIs. Further, rearrangement of CRLF2 is not only associated with mutation of JAK kinases but also alteration of IKZF1, hispanic/latino ethnicity and a poor outcomes in pediatric B- ALL. Although the IGH-CRLF2 and P2RY8-CRLF2 rearrangements have been seen in up to 76 and 17 per cent of cases of adult Ph-like B-ALL cases respectively, there exist no reports of CRLF2 expression abnormalities in myeloid neoplasms (Jain N et al. Blood 2017).With the help of our series we document that: 1. Multiple isodicentric Y chromosomes are a unique cytogenetic abnormality that merits recognition and further clinical characterization in myeloid neoplasia. 2. The presence of up to eight isodicentric Y chromosomes, with up to 16 copies of the Y short arm, suggests the possibility of CRLF2 overexpression. 3. RNA sequencing can help confirm these results; thus providing relapsed/refractory patients with potential targeted salvage therapies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CRLF2 and IKZF1 abnormalities in Mexican children with acute lymphoblastic leukemia and recurrent gene fusions: exploring surrogate markers of signaling pathways.

The gene fusions BCR‐ABL1, TCF3‐PBX1, and ETV6‐RUNX1 are recurrent in B‐cell acute lymphoblastic leukemia (B‐ALL) and are found with low frequency in coexistence with CRLF2 (cytokine receptor‐like factor 2) rearrangements and overexpression. There is limited information regarding the CRLF2 abnormalities and dominant‐negative IKZF1 isoforms associated with surrogate markers of Jak2, ABL, and Ras signaling pathways. To assess this, we evaluated 24 Mexican children with B‐ALL positive for recurrent gene fusions at diagnosis. We found CRLF2 rearrangements and/or overexpression, dominant‐negative IKZF1 isoforms, and surrogate phosphorylated markers of signaling pathways coexisting with recurrent gene fusions. All the BCR‐ABL1 patients expressed CRLF2 and were positive for pCrkl (ABL); most of them were also positive for pStat5 (Jak2/Stat5) and negative for pErk (Ras). TCF3‐PBX1 patients with CRLF2 abnormalities were positive for pStat5, most of them were also positive for pCrkl, and two patients were also positive for pErk. One patient with ETV6‐RUNX1 and intracellular CRLF2 protein expressed pCrkl. In some cases, the activated signaling pathways were reverted in vitro by specific inhibitors. We further analyzed a TCF3‐PBX1 patient at relapse, identifying a clone with the recurrent gene fusion, P2RY8‐CRLF2, rearrangement, and phosphorylation of the three surrogate markers that we studied. These results agree with the previous reports regarding resistance to treatment observed in patients with recurrent gene fusions and coexisting CRLF2 gene abnormalities. A marker phosphorylation signature was identified in BCR‐ABL1 and TCF3‐PBX1 patients. To obtain useful information for the assessment of treatment in B‐ALL patients with recurrent gene fusions, we suggest that they should be evaluated at diagnosis for CRLF2 gene abnormalities and dominant‐negative IKZF1 isoforms, in addition to the analyses of activation and inhibition of signaling pathways.

Characterization of CRLF2 Expression in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is a heterogeneous disease with several underlying genetic ab-normalities. Several studies have tried to elucidate the prognostic significance of cytokine receptor-like factor 2 (CRLF2) overexpression in pediatric B-cell precursor (BCP)-ALL; however, it is still controversial. CRLF2 expression was assessed by flow cytometry in 87 newly diagnosed BCP-ALL pediatric patients, and 80 age and gender-matched control group. Janus Kinase2 (JAK2) (R683) mutation analysis was also performed in those identified to have CRLF2 overexpression with adequate DNA samples by direct sequencing. CRLF2 overexpression was identified in 26/87 (29.9%) of our patients with cutoff set at mean fluorescence intensity (MFI = 3.8) using the Receiver Operating Characteristic (ROC) curve. There were no significant differences in the clinical and laboratory features between patients with high and low-CRLF2 expression, apart from thrombocytopenia which showed statistically significant association with the low-expression group (p = 0.041). Sequence analysis of samples with high CRLF2 expression (n = 23) revealed that 2/23 (8.7%) cases harbored the mutation JAK2 (R683). CRLF2 levels did not have a significant impact on either overall survival (OS) or disease free survival (DFS) (p = 0.601; p = 0.212, respectively). CRLF2 overexpression was not an adverse parameter in pediatric BCP-ALL patients. However, patients with CRLF2 overexpression may harbor the JAK2 mutation presenting a group that can benefit from targeted therapy by kinase inhibitors. The usage of CRLF2 expression to monitor minimal residual disease of BCP-ALL would be an area of interest for further evaluation.

Prognostic significance of Cytokine Receptor-Like Factor 2 (CRLF2) gene expressions in pediatric acute lymphoblastic leukemia.

Background: The Cytokine Receptor-Like Factor 2 (CRLF2) genes play an important role in early Bcell development. Aberrations in CRLF2 activate the JAK-STAT signaling pathway that contributes to B-cell Acute Lymphoblastic Leukemia (B-ALL). The prognostic significance of CRLF2 overexpression in various B-ALL risk subgroups has not been well established. Methods: Thirty patients with newly diagnosed childhood B-ALL were enrolled from the Egyptian population. All the patients in this study were assessed by detailed history taking, a complete clinical examination required radiological and routine laboratory investigations, and flow cytometry to detect the subtype of ALL. Peripheral Blood and bone marrow samples were taken from patients, and the CRLF2 expression was measured using RT-PCR. Results: The prevalence of CRLF2 overexpression status was analyzed, and the prognostic impact of CRLF2 overexpression on B-ALL was evaluated by assessing their influence on overall survival and event-free survival. CRLF2 overexpression was found in 56% of the whole cohort. The results showed that CRLF2 overexpression was associated with a poor outcome in unselected B-ALL. Conclusion: From the present study's findings, we can conclude that CRLF2 gene expression at diagnosis has a significant prognostic impact on the clinical outcome in childhood ALL.

Oncogenesis of CRLF2 Overexpression and Effect of JAK2 Inhibitor in CRLF2 Overexpressed B-Cell Acute Lymphoblastic Leukemia

Backgroud: Cytokine receptor-like factor 2 (CRLF2) plays an important role in the development of normal B lymphocytes, which can mediate the proliferation of early B cells. However, the diect oncogenic effect of CRLF2 overexpression in acute lymhpoblastic leukemia (ALL) is far yet to be clarified. Here, we explored the effect of CRLF2 overexpression on cell proliferation and the effect of the novel JAK2 inhibitor on B-ALL cells with CRLF2 overexpression. Methods: The 83 patients with newly-diagnosed ALL (56 B-cell and 27 T-cell ALL; range from 14 to 77 years old) between June 2008 and June 2016 were studied at Zhongda Hospital Southeast University. The 21 normal bone marrow subjects were enrolled as controls. The qPCR method is developed for detection CRLF2 expression and the CRLF2 overexpression was determined with a cutoff value more than the highest sample of normal bone marrow control. Median differences between the cohorts were evaluated using a Mann-Whitney U-test. Frequency differences were analyzed using uni- and multivariate Cox model. Event-free survival (EFS) and overall survival (OS) were estimated by the Kaplan-Meier method and compared by log-rank test. CRLF2 F232C gain-of-function mutant which we previously reported or CRLF2 were expressed in Nalm6 and 697 B-ALL cells with lentiviral transduction. WST-1 cell proliferation assay and in vitro clonogenic assay were performed upon JAK2 inhibitor (BBT594) treatment. Nalm6-CRLF2-luc, Nalm6-F232C-luc, and Nalm6-vector-luc cells were injected via tail vein into the NSG mice. The leukemia engraftment was monitored once a week by living imaging. Results: The expression of CRLF2 in patietns with ALL was significantly higher than the normal control (P<0.0001). Patients with CRLF2 overexpression had a significantly higher WBC count (53*10^9/L vs. 29.5*10^9/L, P=0.041). Survival analysis showed that the patients with CRLF2 overexpression had a worse EFS and OS, the differences were statistically significant (11 months vs. 26 months, P=0.043 and 15 months vs. 32 months, P=0.015). Also, the CRLF2 expression is determined with flow cytometry after staining with FITC-CRLF2 antibody in 28 samples. The correlation analysis was performed on the CRLF2 expression detected by qPCR and flow cytometry, respectively. A significant positive correlation of the two methods was observed(r=0.957, P<0.0001). These data not only indicate that CRLF2 overexpression is a marker of poor outcome, but also reveal the qPCR might be a simple and quick method for screening CRLF2 overexpression in the clinic compared to flow cytometry which is commonly used. We further found that expression of CRLF2 or CRLF2 F232C mutant into Nalm6 and 697 B-ALL cells dramatically increase the CRLF2 mRNA level, which is 69 times than vector-only control. Moreover, CRLF2 or CRLF2 F232C significantly promotes the cell proliferation of Nalm6 and 697 cells compared to vector only (P<0.001). In addition, JAK2 inhibitor (BBT594) treatment showed the significant dose-dependent cell proliferation arrest and clonogenic inhibition in CRLF2 or CRLF2 F232C overexpressed Nalm6 and 697 cells compared to vector-only control. Furthermore, in vivo we observed the 5-fold higher signal intensity of leukemia engraftment in the mice injected with Nalm6-CRLF2-luc or Nalm6-F232C-luc compared to that of Nalm6-vector-luc control 1-3 weeks after the injection(P<0.001). The Nalm6-CRLF2-luc and Nalm6-F232C-luc infiltrations were observed in bone marrow, central nervous system, liver and spleen of the mice. Conclusion: We showed that CRLF2 overexpression could enhance the proliferation and infiltration of human B-ALL cells, and for the first time indicated that JAK2 inhibitor could suppress the cell proliferation and clonogenesis of the CRLF2 overexpressed B-ALL cells. Our data provide direct evidence of the oncogenic role of CRLF2 overexpression and the new therapeutic potential for targeting CRLF2 overexpressed B-ALL with JAK2 inhibitor. Disclosures No relevant conflicts of interest to declare.

CRLF2 rearrangement in Ph-like acute lymphoblastic leukemia predicts relative glucocorticoid resistance that is overcome with MEK or Akt inhibition.

Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is a genetically heterogeneous subtype of B-cell ALL characterized by chromosomal rearrangements and mutations that result in aberrant cytokine receptor and kinase signaling. In particular, chromosomal rearrangements resulting in the overexpression of cytokine receptor-like factor 2 (CRLF2) occur in 50% of Ph-like ALL cases. CRLF2 overexpression is associated with particularly poor clinical outcomes, though the molecular basis for this is currently unknown. Glucocorticoids (GCs) are integral to the treatment of ALL and GC resistance at diagnosis is an important negative prognostic factor. Given the importance of GCs in ALL therapy and the poor outcomes for patients with CRLF2 overexpression, we hypothesized that the aberrant signal transduction associated with CRLF2 overexpression might mediate intrinsic GC insensitivity. To test this hypothesis, we exposed Ph-like ALL cells from patient-derived xenografts to GCs and found that CRLF2 rearranged (CRLF2R) leukemias uniformly demonstrated reduced GC sensitivity in vitro. Furthermore, targeted inhibition of signal transduction with the MEK inhibitor trametinib and the Akt inhibitor MK2206, but not the JAK inhibitor ruxolitinib, was sufficient to augment GC sensitivity. These data suggest that suboptimal GC responses may in part underlie the poor clinical outcomes for patients with CRLF2 overexpression and provide rationale for combination therapy involving GCs and signal transduction inhibitors as a means of enhancing GC efficacy.

Potential efficacy and prognosis of silencing the CRLF2‑mediated AKT/mTOR pathway in pediatric acute B‑cell lymphoblastic leukemia.

Acute B-cell lymphoblastic leukemia (B-ALL) is a common type of blood cancer, which is associated with aberrant gene expression. Cytokine receptor-like factor 2 (CRLF2) serves a crucial role in the growth and allergic and inflammatory responses of dendritic cells and T cells. The purpose of the present study was to investigate the potential therapeutic and prognostic effect of silencing the CRLF2-mediated RAC-α serine/threonine-protein kinase (AKT)/serine/threonine-protein kinase mTOR (mTOR) pathway in B-ALL. In our study, bone marrow specimens were collected from 128 children with B-ALL and 26 healthy children. The expression of CRLF2 in bone marrow tissue was detected using immunohistochemistry. The survival rates were compared among the children with high and low CRLF2 expression levels. BaF3 leukemia cells were treated with CRLF2 short hairpin RNA knockdown and/or the AKT/mTOR pathway specific inhibitor LY294002. mRNA and protein expression associated with CRLF2 and the AKT/mTOR pathway in each group was detected by reverse transcription-quantitative polymerase chain reaction analysis and western blotting. The viability of BaF3 cells in all the groups was assessed by Cell Counting Kit-8 assay; the migration and invasion of BaF3 cells were determined by wound healing and Transwell invasion assays; and the sensitivity of BaF3 cells to the chemotherapeutic drug imatinib was detected using flow cytometry. The results demonstrated that CRLF2 overexpression is associated with a poor prognosis in B-ALL, and the CRLF2/AKT/mTOR pathway is involved in the migration, invasion and chemotherapeutic agent-induced apoptosis of BaF3 cells.

CRLF2-Positive B-Cell Acute Lymphoblastic Leukemia in Adult Patients: A Single-Institution Experience.

Cytokine receptor-like factor 2 ( CRLF2 ) rearrangement is found in approximately 50% of pediatric Ph-like B-cell acute lymphoblastic leukemia (B-ALL), and around 50% of CRLF2 + cases harbor JAK mutations. We analyzed CRLF2 expression and studied its correlation with CRLF2 rearrangement in adult patients with B-ALL. Multiparameter flow cytometry (MFC) was performed consecutively in 126 patients. CRLF2 overexpression was detected in 30 (27%) patients, 28 (41%) of 69 patients with B-ALL not otherwise specified, 14 (21%) of 67 untreated patients, and 16 (27%) of 59 patients with relapsed B-ALL, with the highest among Hispanic patients (25/55, 45%). Of CRLF2+ cases, 21 (100%) of 21 cases showed CRLF2 rearrangement by fluorescence in situ hybridization, preferentially involving IGH@CRLF2 (15/15). The entire coding region of JAK2 was sequenced in 14 patients with CRLF2+ B-ALL, and nine (64%) were positive for JAK2 mutations. MFC allows a rapid, inexpensive, and reliable detection of B-ALL with CRLF2 rearrangement that would further facilitate testing for JAK2 mutations for targetable therapy.

Prognostic significance of P2RY8-CRLF2 and CRLF2 overexpression may vary across risk subgroups of childhood B-cell acute lymphoblastic leukemia.

The cytokine receptor-like factor 2 (CRLF2) gene plays an important role in early B-cell development. Aberrations in CRLF2 activate the JAK-STAT signaling pathway that contributes to B-cell acute lymphoblastic leukemia (B-ALL). The prognostic significance of CRLF2 overexpression and P2RY8-CRLF2 fusion in various B-ALL risk subgroups has not been well established. Two hundred seventy-one patients with newly diagnosed childhood B-ALL were enrolled from a Chinese population. The prevalence of CRLF2 overexpression, CRLF2-P2RY8 fusion, CRLF2 F232C mutation, and JAK2 and IL7R mutational status were analyzed, and the prognostic impact of CRLF2 overexpression and P2RY8-CRLF2 on B-ALL was evaluated by assessing their influence on overall survival and event-free survival. CRLF2 overexpression and P2RY8-CRLF2 were found in 19% and 10%, respectively, in the whole cohort. No correlation between CRLF2 overexpression and P2RY8-CRLF2 was observed. CRLF2 F322C and IL7R mutations were not detected in B-ALL cases overexpressing CRLF2, and no JAK2 mutations were found in the whole cohort either. The results showed that CRLF2 overexpression and P2RY8-CRLF2 were associated with a poor outcome in unselected B-ALL. Moreover, in an intermediate risk B-ALL subgroup P2RY8-CRLF2 was correlated with worse survival, whereas in high- and low-risk subgroups, CRLF2 overexpression predicted a poor outcome. Our findings suggest that P2RY8-CRLF2 is an independent prognostic indicator in intermediate risk B-ALL, while CRLF2 overexpression is correlated with an inferior outcome in high- or low-risk B-ALL. Our study demonstrates that the impact of P2RY8-CRLF2 and CRLF2 overexpression on B-ALL survival may differ across risk subgroups. © 2016 Wiley Periodicals, Inc.

Prognostic significance of cytokine receptor-like factor 2 alterations in acute lymphoblastic leukemia: a meta-analysis.

Cytokine receptor-like factor 2 (CRLF2) has been shown to play a role in the pathogenesis of acute lymphoblastic leukemia (ALL). Studies have examined the relationship between CRLF2 alterations such as over-expression or deregulation and clinical outcome in childhood ALL, but the results are conflicting. This meta-analysis aimed to explore the association between CRLF2 alterations and survival of pediatric patients with ALL. Electronic databases updated to March 2014 were searched for relevant studies. A meta-analysis was made of twelve studies including 5945 patients to evaluate the prognostic significance of CRLF2 alterations on survival in childhood ALL. Hazards ratios (HRs) with 95% confidence intervals (CIs) were pooled across the studies using a fixed-effects model. CRLF2 over-expression in childhood ALL was associated with poor prognosis in terms of relapse-free survival (RFS; HR=1.70, 95% CI=1.28-2.24, P=0.000), event-free survival (EFS; HR=1.78, 95% CI=1.05-3.01, P=0.032), and overall survival (OS; HR=2.28, 95% CI=1.42-3.65, P=0.001). The combined data also suggested that CRLF2 deregulation in childhood ALL was correlated with poor EFS (HR=1.95, 95% CI=1.46-2.61, P=0.000), RFS (HR=2.20, 95% CI=1.53-3.18, P=0.000), and OS (HR=1.89, 95% CI=1.24-2.87, P=0.003). Subgroup analysis on multivariate HRs showed that CRLF2 deregulation independently predicted a poor prognosis for childhood ALL. The present meta-analysis reveals that both CRLF2 over-expression and deregulation are associated with poor prognosis in pediatric patients with ALL.

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.

Integrated Analysis Of CRLF2 Signaling In Acute Lymphoblastic Leukemia Identifies Polo-Like Kinase 1 As a Therapeutic Target

IntroductionAcute lymphoblastic leukemia is the most common cancer in children, and the five-year survival rate among patients older than 20 years old is still poor. Recently, Cytokine Receptor-Like Factor 2 (CRLF2) was identified as a prognostic factor in 15% of precursor B cell-acute lymphoblastic leukemia (B-ALL) that lack typical gene rearrangements (Yoda et al., Proc Natl Acad Sci U S A. 2010). In addition, several independent studies also demonstrated that CRLF2 acts as an oncoprotein (Mullighan et al., Nat Genet. 2009; Russell et al., Blood. 2009; Chapiro et al., Leukemia. 2010). The Children's Oncology Group also found that high expression of CRLF2 confers an adverse prognosis in high-risk B-ALL patients (Chen et al., Blood. 2012). The mechanism of CRLF2-associated leukemogenesis, however, is still not clear. In CRLF2-rearranged B-ALL cases, CRLF2 is overexpressed because of genetic rearrangements. Interestingly, one-half of cases co-harbor an activating mutation in exon 16 of JAK2, most commonly involving R683 or I682. In a murine progenitor B cell line, BaF3, it has been shown that the combination of CRLF2 overexpression and JAK2 R683G confers IL-3-independent growth. This illustrates that CRLF2 overexpression could synergize with JAK2 R683G to trigger leukemic proliferation. In our previous phosphoproteomics analysis of BaF3 cells expressing CRLF and JAK2 R683G, the combination upregulated phosphorylation of Protein phosphatase 1, regulatory subunit 12A (Ppp1r12a) (van Bodegom et al., Blood. 2012), which is a negative regulator of Polo-like kinase 1 (Plk1) (Yamashiro et al., Dev Cell. 2008). In this study, we aimed to focus on novel approaches to target CRLF2 signaling in B-ALL. MethodsTo identify effective kinase inhibitors against leukemic CRLF2 signaling, we subjected BaF3 cells with CRLF2 overexpression and JAK2 R683G (BJC cells) to a kinase inhibitor screening system that consisted of 73 kinase inhibitors and other small molecules. An in vitro cell proliferation assay was coupled to observe growth inhibition at 37°C for 3 days (Tyner et al., Cancer Res. 2012). To validate ex vivo efficacy of the Plk1 inhibitor (BI-2536), which scored in the screen, we injected BJC cells with/without pre-treatment for 24 hours at 100 nM into the NOD-scid IL2Rγnull (NSG) mice and compared spleen/liver sizes 15 days after injection. To compare in vitro efficacy of Plk1 inhibitor in CRLF2-rearranged B-ALL xenografts, we used human hTERT-transformed bone marrow stromal cells as a feeder layer and green fluorescent dye (488 nm) viability staining for flow cytometry analysis 48 hours after treatment. To investigate the downstream effect of dysregulated Plk1 in BJC cells, morphology of centrosomes was examined using anti-g-tubulin immunofluorescence staining. ResultsThe kinase inhibitor screening showed that the Plk1 inhibitor BI-2536 is particularly potent against BJC cells with an IC50 of 11 nM. To validate this screening result and test the efficacy of BI-2536 ex vivo, NSG mouse transplantation experiments showed that BJC cells that were pretreated with BI-2536 had significantly smaller spleen (0.47% vs. 1.45% of total body weight, p<0.01) and liver (5.53% vs. 7.62% of total body weight, p<0.01) sizes compared with BJC cells without treatment. The efficacy of BI-2536 against human CRLF2-rearranged B-ALL xenografts was evaluated in vitro. In comparison with vehicle control, the viability of CRLF2-rearranged B-ALL cells treated with BI-2536 was lower (48.8% of vehicle) than a wild-type CRLF2 B-ALL with BI-2536 (105.3% of vehicle). Because Plk1 is a major regulator of centrosome, we carried out immunofluorescence staining of γ-tubulin, a specific marker for centrosomes. While morphology of centrosomes showed normal patterns in BaF3 cells, in cells treated with BI-2536, replicated centrosomes failed to part from each other, surrounded by M phase chromosomes. ConclusionsBy integrating results from prior phosphoproteomics analysis, kinase inhibitor screening, ex vivo therapy in mouse transplantation experiments and in vitro viability assay using human xenografts, Plk1 inhibitor BI-2536 was shown to have potent activity against CRLF2-rearranged B-ALL cells. Our result suggests future therapy for this subset of B-ALL could be directed against Plk1 and its associated signaling pathways. Disclosures:Tyner:Incyte Corporation: Research Funding.

IKZF1 deletion is associated with a poor outcome in pediatric B‐cell precursor acute lymphoblastic leukemia in Japan

Genetic alterations of Ikaros family zinc finger protein 1 (IKZF1), point mutations in Janus kinase 2 (JAK2), and overexpression of cytokine receptor-like factor 2 (CRLF2) were recently reported to be associated with poor outcomes in pediatric B-cell precursor (BCP)-ALL. Herein, we conducted genetic analyses of IKZF1 deletion, point mutation of JAK2 exon 16, 17, and 21, CRLF2 expression, the presence of P2RY8-CRLF2 fusion and F232C mutation in CRLF2 in 202 pediatric BCP-ALL patients newly diagnosed and registered in Japan Childhood Leukemia Study ALL02 protocol to find out if alterations in these genes are determinants of poor outcome. All patients showed good response to initial prednisolone (PSL) treatment. Ph+, infantile, and Down syndrome–associated ALL were excluded. Deletion of IKZF1 occurred in 19/202 patients (9.4%) and CRLF2 overexpression occurred in 16/107 (15.0%), which are similar to previous reports. Patients with IKZF1 deletion had reduced event-free survival (EFS) and overall survival (OS) compared to those in patients without IKZF1 deletion (5-year EFS, 62.7% vs. 88.8%, 5-year OS, 71.8% vs. 90.2%). Our data also showed significantly inferior 5-year EFS (48.6% vs. 84.7%, log rank P = 0.0003) and 5-year OS (62.3% vs. 85.4%, log rank P = 0.009) in NCI-HR patients (n = 97). JAK2 mutations and P2RY8-CRLF2 fusion were rarely detected. IKZF1 deletion was identified as adverse prognostic factor even in pediatric BCP-ALL in NCI-HR showing good response to PSL.

Poor prognosis for P2RY8-CRLF2 fusion but not for CRLF2 over-expression in children with intermediate risk B-cell precursor acute lymphoblastic leukemia

Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has achieved an 80% cure rate as a result of a risk-adapted therapy largely based on minimal residual disease (MRD) monitoring. However, relapse is still the most frequent adverse event, occurring mainly in the patients with intermediate MRD levels (intermediate risk, IR), emphasizing the need for new prognostic markers. We analyzed the prognostic impact of cytokine receptor-like factor 2 (CRLF2) over-expression and P2RY8-CRLF2 fusion in 464 BCP-ALL patients (not affected by Down syndrome and BCR-ABL negative) enrolled in the AIEOP-BFM ALL2000 study in Italy. In 22/464 (4.7%) samples, RQ-PCR showed CRLF2 over-expression (≥20 times higher than the overall median). P2RY8-CRLF2 fusion was detected in 22/365 (6%) cases, with 10/22 cases also showing CRLF2 over-expression. P2RY8-CRLF2 fusion was the most relevant prognostic factor independent of CRLF2 over-expression with a threefold increase in risk of relapse. Significantly, the cumulative incidence of relapse of the P2RY8-CRLF2 + patients in the IR group was high (61.1% ± 12.9 vs 17.6% ± 2.6, P<0.0001), similar to high-risk patients in AIEOP-BFM ALL2000 study. These results were confirmed in a cohort of patients treated in Germany. In conclusion, P2RY8-CRLF2 identifies a subset of BCP-ALL patients currently stratified as IR that could be considered for treatment intensification.

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.