CRLF2 Expression Research Articles

The application of targeted RNA sequencing for the analysis of fusion genes, gene mutations, IKZF1 intragenic deletion, and CRLF2 overexpression in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is characterized by highly genetic heterogeneity, owing to recurrent fusion genes, gene mutations, intragenic deletion, and gene overexpression, which poses significant challenges in clinical detection. RNA sequencing (RNA-seq) is a powerful tool for detecting multiple genetic abnormalities, especially cryptic gene rearrangements, in a single test. Sixty samples (B-ALL, n = 49; T-ALL, n = 9; mixed phenotype acute leukemia (MPAL), n = 2) and 20 controls were analyzed by targeted RNA-seq panel of 507 genes developed by our lab. Of these, 16 patients were simultaneously analyzed for gene mutations at the DNA level using a next-generation sequencing panel of 51 genes. Fusion genes, CRLF2 expression, and IKZF1 intragenic deletion were also detected by reverse transcription-polymerase chain reaction (RT-PCR). Karyotype analysis was performed using the R-banding and G-banding technique on bone marrow cells after 24 hours of culture. Partial fusion genes were analyzed using fluorescence in situ hybridization (FISH). Compared with the results of Karyotype analysis, FISH, and RT-PCR, the detection rate of fusion genes by targeted RNA-seq increased from 48.3% to 58.3%, and six unexpected fusion genes were discovered, along with one rare isoform of IKZF1 intragenic deletion (IK10). The DNA sequencing analysis of 16 ALL patients revealed that 96.2% (25/26) of gene mutations identified at the DNA level were also detectable at the RNA level, except for one mutation with a low variant allele fraction. The detection of CRLF2 overexpression exhibited complete concordance between RT-PCR and RNA-seq. The utilization of RNA-seq enables the identification of clinically significant genetic abnormalities that may go undetected through conventional detection methods. Its robust analytical performance might bring great application value for clinical diagnosis, prognosis, and therapy in ALL.

Comprehensive detection of CRLF2 alterations in acute lymphoblastic leukemia: a rapid and accurate novel approach.

Introduction: Acute lymphoblastic leukemia (ALL) is a prevalent childhood cancer with high cure rate, but poses a significant medical challenge in adults and relapsed patients. Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype, with approximately half of cases characterized by CRLF2 overexpression and frequent concomitant IKZF1 deletions. Methods: To address the need for efficient, rapid, and cost-effective detection of CRLF2 alterations, we developed a novel RT-qPCR technique combining SYBR Green and highresolution melting analysis on a single plate. Results: The method successfully identified CRLF2 expression, P2RY8::CRLF2 fusions, and CRLF2 and JAK2 variants, achieving a 100% sensitivity and specificity. Application of this method across 61 samples revealed that 24.59% exhibited CRLF2 overexpression, predominantly driven by IGH::CRLF2 (73.33%). High Resolution Melting analysis unveiled concurrent CRLF2 or JAK2 variants in 8.19% of samples, as well as a dynamic nature of CRLF2 alterations during disease progression. Discussion: Overall, this approach provides an accurate identification of CRLF2 alterations, enabling improved diagnostic and facilitating therapeutic decision-making.

Hematologic Neoplasms Associated with Down Syndrome: Cellular and Molecular Heterogeneity of the Diseases.

The molecular basis of Down syndrome (DS) predisposition to leukemia is not fully understood but involves various factors such as chromosomal abnormalities, oncogenic mutations, epigenetic alterations, and changes in selection dynamics. Myeloid leukemia associated with DS (ML-DS) is preceded by a preleukemic phase called transient abnormal myelopoiesis driven by GATA1 gene mutations and progresses to ML-DS via additional mutations in cohesin genes, CTCF, RAS, or JAK/STAT pathway genes. DS-related ALL (ALL-DS) differs from non-DS ALL in terms of cytogenetic subgroups and genetic driver events, and the aberrant expression of CRLF2, JAK2 mutations, and RAS pathway-activating mutations are frequent in ALL-DS. Recent advancements in single-cell multi-omics technologies have provided unprecedented insights into the cellular and molecular heterogeneity of DS-associated hematologic neoplasms. Single-cell RNA sequencing and digital spatial profiling enable the identification of rare cell subpopulations, characterization of clonal evolution dynamics, and exploration of the tumor microenvironment's role. These approaches may help identify new druggable targets and tailor therapeutic interventions based on distinct molecular profiles, ultimately improving patient outcomes with the potential to guide personalized medicine approaches and the development of targeted therapies.

A surrogate molecular approach for the detection of Philadelphia chromosome-like B-acute lymphoblastic leukemia.

Philadelphia chromosome (Ph)-like B-acute lymphoblastic leukemia (B-ALL) is a clinically significant, high-risk genetic subtype of B-ALL cases. There are few data on the incidence, characterization, and treatment outcomes of Ph-like ALL cases from low- and middle-income countries. There is a pressing need to establish a well-organized/cost-effective approach for identifying Ph-like ALL instances. Multiplex reverse transcriptase polymerase chain reaction, nCounter NanoString, and fluorescence in situ hybridization were used to detect and characterize Ph-like ALL cases among recurrent genetic abnormalities (RGA)neg B-ALL cases. At the end of induction therapy, flow cytometry-minimal residual disease (MRD) assay was used to quantify MRD positivity in Ph-like ALL cases. Of 130 newly diagnosed B-ALL cases, 25% (BCR::ABL1), 4% (ETV6::RUNX1), 5% (TCF3::PBX1), 2% (KM2TA::AFF1), and 65% RGAneg B-ALL cases were revealed by multiplex reverse transcriptase polymerase chain reaction. Among RGAneg B-ALL cases, 24% Ph-like ALL cases using nCounter NanoString were identified, with 48% CRLF2high cases with 45% CRLF2::P2RY8 and 18% CRLF2::IGH rearrangements(∼r) revealed by fluorescence in situ hybridization. In 52% of CRLF2low cases, 17% ABL1 and JAK2∼r 8% EPOR::IGH & PDGRFB∼r were identified. Ph-like ALL cases had higher total leukocyte count (p<.05), male preponderance (p<.05), and high MRD-positivity/induction failure compared with RGAneg B-ALL cases. Furthermore, in Ph-like ALL cases, 11 significant genes using quantitative polymerase chain reaction were identified and validated. CRLF2, IGJ, CEACAM6, MUC4, SPATS2L and NRXN3 genes were overexpressed and show statistical significance (p<.05) in Ph-like ALL cases. This study showed the high incidence of Ph-like ALL cases with kinase activating alterations and treatment outcomes from low- and middle-income region. Furthermore, a surrogate cost-effective multiplex panel of 11 overexpressed genes for the prompt detection of Ph-like ALL cases is proposed. Identification of recurrent gene abnormalities (RGA)neg B-acute lymphoblastic leukemia (B-ALL) cases using multiplex-reverse transcriptase polymerase chain reaction. Identification and characterization of Philadelphia (Ph)-like ALL cases using nCounter NanoString gene expression profiling and fluorescence in situ hybridization. Furthermore, Ph-like ALL cases were characterized according to CRLF2 expression and kinase-activating genomic alterations. Minimal residual disease of Ph-like ALL cases were quantified using flow cytometry-minimal residual disease assay. A surrogate molecular approach was established to detect Ph-like ALL cases from low- and middle-income countries.

Effect of N-glycosylation on constitutive signal transduction by mutated cytokine receptor-like factor 2

BackgroundCytokine receptor-like factor 2 (CRLF2) is a subunit of the receptor for thymic stromal lymphopoietin (TSLP). A somatic mutation (insEIM) in the transmembrane domains of CRLF2 has been identified in acute lymphocytic leukemia (ALL), and Glu-Ile-Met (EIM) CRLF2 induces constitutive activation of signals. However, the signaling mechanism remains unclear. MethodsHEK293 cells were transfected with expression vectors encoding wild-type (WT), insEIM CRLF2, or their mutants which N-glycosylation site was replaced with a glutamine. Cell surface expression of CRLF2 was assessed by flow cytometry. Total CRLF2 and phosphorylated signal transducer and activator of transcription 5 (STAT5) were detected by western blotting. ResultsThree major species of CRLF2 (53-, 57- and 58-kDa) were identified. Deglycosylation analysis revealed that they were modified with complex-type and oligomannose-type glycans. The expression of both WT and EIM CRLF2 decreased in N-acetylglucosaminyltransferase (GnT)-I (MGAT1) knockout (KO) cells and slightly decreased in α1,6-fucosyltransferase (Fut8) KO cells compared to that in the control cells. In GnT-I or Fut8 KO cells, WT CRLF2 did not induce ligand-independent activation. Both WT and EIM CRLF2 contained four N-glycosylation sites. N55 of CRLF2 was required for the cell surface expression and activation by EIM CRLF2. ConclusionsWe found that N-glycosylation of CRLF2 plays crucial roles for its cell surface expression and signaling. However, N-glycan processing in the Golgi apparatus does not seem to be essential for ligand-independent activation of EIM CRLF2. General significanceOur studies provide a crucial role of glycosylation in the cell surface expression of receptors.

Classification and genetics of pediatric B-other acute lymphoblastic leukemia by targeted RNA sequencing.

Acute lymphoblastic leukemia (ALL) can be classified into different subgroups based on recurrent genetic alterations. Here, targeted RNA sequencing was used to identify the novel subgroups of ALL in 144 B-other and 40 "classical" ALL samples. The classical TCF3-PBX1, ETV6-RUNX1, KMT2A-rearranged, and BCR-ABL1, and novel P2RY8-CRLF2, ABL-, JAK2-, ZNF384-, MEF2D-, and NUTM1-fusions were easily identified by fusion transcript analysis. IGH-CRLF2 and IGH-EPOR were found by abnormally high levels of expression of CRLF2 or EPOR. DUX4-rearranged was identified by the unusual expression of DUX4 genes and an alternative exon of ERG, or by clustering analysis of gene expression. PAX5-driven ALL, including fusions, intragenic amplifications, and mutations were identified by single-nucleotide variant analysis and manual inspection using the IGV software. Exon junction analysis allowed detection of some intragenic ERG and IKZF1 deletions. CRLF2-high associated with initial white blood cell (WBC) counts of ≥50× 103/μL and GATA3 risk alleles (rs3781093 and rs3824662), whereas ABL/JAK2/EPOR-fusions associated with high WBC counts, National Cancer Institute's high-risk classification, and IKZF1del. ZNF384-fusions associated with CALLA-negativity and NUTM1-fusions in infants. In conclusion, targeted RNA sequencing further classified 66.7% (96 of 144) B-other ALL cases. All BCP-ALL subgroups, except for iAMP21, hyperdiploid and hypodiploid cases, were identified. Curiously, we observed higher frequencies of females within B-rest ALLs and males in PAX5-driven cases.

Clinical, biological, and outcome features of P2RY8-CRLF2 and CRLF2 over-expression in pediatric B-cell precursor acute lymphoblastic leukemia according to the CCLG-ALL 2008 and 2018 protocol.

CRLF2 alterations are associated with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This study aimed to explore the clinical, biological, and outcome features of pediatric BCP-ALL with CRLF2 abnormalities. This study enrolled 630 childhood BCP-ALLs treated on CCLG-ALL 2008 or 2018 protocol. P2RY8-CRLF2 was determined by Sanger sequencing and CRLF2 expression was evaluated by qRT-PCR. The correlation between clinical, biological features and outcomes with P2RY8-CRLF2 or CRLF2 over-expression were analyzed. P2RY8-CRLF2 and CRLF2 over-expression were found in 3.33% and 5.71% respectively. P2RY8-CRLF2 was associated with male, higher frequency of CD7 expression, high WBC and MRD before consolidation. CRLF2 over-expression showed ETV6-RUNX1- , higher frequency of CD22, CD34, CD66c, CD86 expression, hyperdiploidy and high MRD at early treatment. The lower overall survival (OS) was found in patients with P2RY8-CRLF2 and confined only in IR group. Furthermore, adverse event-free survival and OS of P2RY8-CRLF2 were discovered comparing to those without known fusions or treated on CCLG-ALL 2008 protocol. However, P2RY8-CRLF2 was not confirmed as independent prognostic factors and no prognostic impact of CRLF2 over-expression was found. These findings indicate P2RY8-CRLF2 identifies a subset of patients with specific features and adverse outcomes that could be improved by risk-directed treatment.

The prognostic significance of cytokine receptor-like factor 2 expression and &lt;i&gt;JAK2&lt;/i&gt; mutation in pediatric B-cell acute lymphoblastic leukemia: A prospective cohort study

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Philadelphia (Ph)-like B-cell acute lymphoblastic leukemia (B-ALL) is defined by a gene expression profile similar to Phpositive B-ALL and shows a large number of genetic alterations in the cytokine receptor and kinasesignaling pathway genes that contribute to its aggressive phenotype and frequent disease recurrence – the main cause of death in affected children. Here, we aimed to correlate CRLF2 expression and JAK2 mutations in B-ALL patients with other prognostic factors and the patients’ outcomes as well as to evaluate their prognostic significance. The study was approved by the local institutional review board and written consents were obtained from a parent of each child before their enrolment. We included 54 newly diagnosed B-ALL pediatric patients (median age: 9.0 (2.0–18.0)) who were stratified either into a standard-risk (SR) or high-risk (HR) group and treated according to the modified BerlinFrankfurt-Münster 90 protocol (ALL-BFM 90). Fresh bone marrow samples were used to determine CRLF2 expression as well as to search for the JAK2 V617F mutation. Normal CRLF2 expression was reported in the SR patients much more often than in the HR group, while its overexpression was more common in the HR patients than in the SR ones (22 vs 6 and 18 vs 8, respectively, p &lt; 0.001). CRLF2 was also more often overexpressed in the MRD-positive cases than in the negative ones (17 vs 9, p &lt; 0.001), while normal CRLF2 expression was more common in the MRD-negative patients compared to the MRD-positive ones (24 vs 4, p &lt; 0.001) which supports the unfavorable prognostic value of CRLF2 in relation to MRD positivity at the end of the induction treatment. JAK2 mutation was detected only in 2 patients belonging to the CRLF2 overexpression group which made the assessment of the prognostic significance of this mutation impossible. Notably, none of the patients with normal CRLF2 expression ended up relapsing while 4 patients with overexpressed CRLF2 developed a relapse (p = 0.031). The study subjects were followed up for up to 24 months, and we did not find CRLF2 overexpression to negatively influence overall survival, however, it did have an adverse effect on relapse-free survival. In summary, CRLF2 overexpression was found to be an unfavorable prognostic factor in childhood ALL as it was expressed more in high-risk patients and in those with poor treatment response. The analysis of CRLF2 expression in B-ALL pediatric patients may help in risk stratification and can potentially offer new treatment options based on novel CRLF2 inhibitors.

CRLF2 overexpression defines an immature-like subgroup which is rescued through restoration of the PRC2 function in T-cell precursor acute lymphoblastic leukemia.

CRLF2 overexpression has been described as a biomarker of poor prognosis in T-cell acute lymphoblastic leukemia (T-ALL). In the present study, we aimed to unravel the genomic profile underlying CRLF2 overexpression (CRLF2-high) by analyzing RNA-seq, WES, and SNP-array data from 264 T-ALL patients and five cell lines deposited on the TARGET initiative, Cancer Cell Line Encyclopedia and Gene Expression Omnibus. These data allowed us to delineate the genomic landscape of CRLF2-high in T-ALL, which was associated with PTEN, JAK3, PHF6, EZH2, and RUNX1 mutations. We also observed an enrichment of CRLF2-high in early T-precursor (ETP)-ALL (23.08% vs. 4.02%, P=7.579e-06 ) and a very similar gene upregulation profile between these two entities. The inhibition of BET (iBET) proteins is a strategy previously demonstrated to reverse the gene upregulation pattern of ETP cells through restoration of polycomb repressive complex 2 (PRC2) activity. While CRLF2 expression was rescued by using this strategy in LOUCY (untreated vs. iBET P =0.0095, DMSO vs. iBET P=0.0286), a classical ETP-ALL cell line, PRC2 loss was not sufficient to promote CRLF2 upregulation in JURKAT, a more mature T-ALL cell line. Considering the role of IKZF1 in CRLF2 regulation and in recruitment of PCR2, we evaluated IKZF1 status according to CRLF2-expression subgroups. We identified that IKZF1 transcripts with intron retention were upregulated in the CRLF2-high subgroup. Here, we delineated the gene expression profile of CRLF2-high T-ALL samples and unraveled the crucial role of PRC2 in CRLF2 regulation in ETP-ALL.

Over-Expression of CRLF2 By Flow Cytometry (FC) Impacts on Overall Survival (OS) in Mexican Patients with Acute Lymphoblastic Leukemia (ALL)

▪Introduction:“Philadelphia like” ALL has been related to poor prognosis. CRLF2 over-expression (cytokine receptor like factor-2) has been found in up to 50% of patients with Philadelphia-like ALL and its expression can be measured by Flow Cytometry (FC). CRLF2 over-expression is more common in Hispanic population (45-68%) however, there is no current recommendation in using it as a prognostic marker.Objectives:Find the prevalence of CRLF2 overexpression measured by FC, in adult Mexican patients with treatment-naïve ALLDescribe the outcomes of the patients who over-expressed CRLF2 by Complete Response (CR), Minimal Residual Disease (MRD), Leukemia-Free Survivale (LFS) and Overall Survival (OS).Methodology:This is a retrospective cohort study in adults with newly diagnosed ALL from two reference centers in Mexico City. We measured CRLF2 expression by FC in fresh bone marrow samples from treatment-naïve patients at one location; to define over-expression, samples were first analyzed by two different experts who grouped the cases in over-expression or no overexpression using Mean Fluorescence Intensity (MFI) between two populations, blasts and controls (normal B cells). Outcomes were compared using chi-square test for binary variables and log-rank test for time-to-event variables with a p value <0.05 as significant.Results:From April 2018 to January 2020 46 patients with treatment-naïve B-cell ALL were evaluable; the median age was 29.5 years, 38 (82.6%) were Adolescents and Young Adults (AYA), 22 (47.8%) had leukocytosis, 15 (53.5%) of the evaluable karyotypes, were assigned to high-risk group. The median time of follow-up was 24.5 months and 19 (41.3%) patients were positive for CRLF2-overexpression. For the follow-up cohort all of the patients were evaluable for outcomes. CNS disease was detectable in 11(24.5%) patients which was higher in CRLF2-overexpresed patients (15.5% vs 8.9%, p=0.015). We found no difference in Complete Remission (CR) in CRLF2 status but a high tendency for R/R (Relapse/Refractory) disease (83.3% in CRLF2-overexpression vs 60% in CRLF2 negative group; p=0.09) and dead (63.2% in CRLF2-overexpression vs 37% in CRLF2 negative group; p=0.07). MRD1, 2 and 3 (1=after induction, 2= week 16 and 3= before maintenance) was significantly worse in patients with CRLF2 overexpression (1=15.8% vs 58.3%, p<0.01; 2=7.1% vs 52.6%, p<0.01; 3=0% vs 55.6%, p<0.05).Overall Survival was significantly worse in patients with CRLF2 overexpression (Median Not Reached vs 11.05 months; p=0.04) (Figure 1); Disease-Free Survival (DFS) had a tendency towards worse outcome in patients with CRLF2 overexpression (18.48 vs 5.82 months, p=0.07) (Figure 2).Conclusion:Survival in patients who have CRLF2 overexpression is significantly worse when measured by FC, this might be related to early high-risk markers as MRD. CRLF2 overexpression in this hispanic sample was higher (41%) than other reports. CRLF2 measured as a prognostic factor by FC needs to be further considered due to the high availability of this technique across Latin-America. [Display omitted] DisclosuresRangel-Patiño: Abbvie: Speakers Bureau; Bristol: Consultancy. Espinosa: Pfizer: Consultancy; Amgen: Speakers Bureau; Janssen: Consultancy. Demichelis: Novartis: Consultancy, Research Funding, Speakers Bureau; Gilead: Consultancy; Bristol/Celgene: Consultancy, Speakers Bureau; Jazz: Consultancy; ASH: Research Funding; Astellas: Consultancy; AMGEN: Consultancy, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau.

Genomic and Outcome Analyses of Philadelphia Chromosome-like (Ph-like) NCI Standard Risk B-Acute Lymphoblastic Leukemia (SR B-ALL) Patients Treated on Children's Oncology Group (COG) AALL0331

Introduction: Ph-like ALL is defined by a gene expression profile similar to Philadelphia chromosome-positive ALL but lacking the BCR - ABL1 fusion. Ph-like ALL is comprised of diverse genomic alterations activating kinase and cytokine receptor signaling, including chimeric fusions involving ABL1 and related genes (ABL2, CSF1R, PDGFRB ; ABL-class fusions), genomic rearrangements leading to CRLF2 over-expression, JAK2 gene fusions, and truncating rearrangements of EPOR . We have published several large studies describing the clinical features, treatment outcome, and genomic alterations in Ph-like ALL that focused on patients with NCI high risk (HR) ALL (age ≥10 years or WBC ≥50,000) or those with SR ALL (age 1-9.99 years and WBC <50,000) and elevated minimal residual disease (MRD) at the end of induction (EOI; Reshmi, BLOOD 2017 and Roberts, NEJM 2014). The purpose of the current study was to assess the frequency of genomic lesions in Ph-like SR ALL patients lacking HR features.Methods: We retrospectively analyzed 1023 unselected newly-diagnosed SR ALL patients enrolled on COG AALL0331 between 2006 and 2008, and compared characteristics of SR Ph-like cases to the HR patients described in our prior studies. All cases were screened using an 8-gene Taqman low-density array (LDA) PCR assay to identify the Ph-like GEP (Harvey, ASH 2013). Ph-like cases with elevated CRLF2 expression were tested for CRLF2 rearrangement (CRLF2 -R; P2RY8 - CRLF2 by Taqman PCR on the LDA card and IGH - CRLF2 by FISH). JAK mutations in CRLF2 -R cases were tested by Sanger sequencing. Ph-like cases without CRLF2 -R were tested for previously identified kinase fusions involving ABL1, ABL2, CSF1R, JAK2, NTRK3, and PDGFRB by RT-PCR. A subset of Ph-like cases without detected genomic alterations underwent RNA-sequencing using standard Illumina library preparation.Results: A total of 205 patients had LDA value ≥0.5, with 145 (14.2%) remaining after exclusion of BCR-ABL1 and ETV6-RUNX1 . Of these 145, 85 were CRLF2high (57.9%) as defined by CRLF2 expression levels on the LDA card. Among these 85, 42 (50%) had CRLF2 -R, compared to 80% in HR patients. P2RY8 - CRLF2 was seen in 36 cases, IGH - CRLF2 in 4 and 2 had CRLF2 -R with an unknown partner; 14 (32.6%) of these had a concomitant JAK mutation. Of the 60 Ph-like CRLF2low cases, five had a targetable kinase fusion identified by RT-PCR or RNA sequencing: two ABL - class fusions (ZMIZ1 - ABL1, EBF1-PDGFRB); one PAX5-JAK2 fusion; one ETV6-JAK2 fusion; and one ETV6 - NTRK3 fusion. The 145 Ph-like cases had inferior 7-year event-free survival (EFS; 83.1±3.5% vs. 90.6 ±0.1%; P=0.005) and non-significantly inferior 7-year overall survival (OS; 93.5±2.3% vs. 95.5 ±0.8%; P=0.184) compared to 869 non Ph-like cases. Ph-like ALL cases were more likely to have end induction MRD ≥ 0.01% (24.3% vs. 17.9%; P=0.049), and positive MRD was predictive of outcome for Ph-like (7-year EFS: 73.2±8.3%, vs. 86.6 ±3.7%; P=0.079; 7-year OS: 88.2 ±5.9% vs. 95.9±2.1%; P=0.162) and non Ph-like patients (7-year EFS: 79.7 ±3.7% vs. 93.3 ±1.0%; P <0.0001; 7-year OS: 90.2% SE 2.7 vs. 97.4±0.9%; P <0.0001). In multivariate analyses, Ph-like and MRD status EOI were significant predictors of EFS (p=0.016; <0.0001 respectively), while only MRD had a significant impact on OS.Conclusion: Ph-like ALL is less common in NCI SR than HR patients with significant differences in the frequency of various genomic alterations in Ph-like ALL cases based on NCI risk group. Our previous studies have shown that Ph-like ALL HR patients have a substantially increased risk of treatment failure and death as compared to non Ph-like HR patients (Mullighan, NEJM 2009 and Roberts, NEJM 2014). This cohort of SR Ph-like patients had a significantly inferior EFS and a trend to inferior OS, but the magnitude of effect was much smaller than that seen in HR Ph-like patients, with overall outcomes much better in SR as compared to HR Ph-like patients. These findings underscore the significant differences in Ph-like ALL frequency, genetic subtypes, and outcome based on age and initial WBC. [Display omitted] DisclosuresBorowitz:Beckman Coulter: Honoraria; Becton-Dickinson Biosciences: Research Funding; HTG Molecular: Honoraria. Zweidler-McKay:ImmunoGen: Employment. Mattano:HARP Pharma Consulting, LLC: Employment. Mullighan:Loxo Oncology: Research Funding; Amgen: Consultancy. Hunger:Amgen: Consultancy, Equity Ownership; Novartis: Consultancy; Erytech Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Honoraria.

Targeted RNA-Seq Identify a Subset of Adolescent and Adult Patients with Acute Lymphoblastic Leukemia with BCR-ABL1-like Characteristics

Background: BCR-ABL1-like (Ph-like) B-precursor acute lymphoblastic leukemia (ALL) shows a gene expression profile closely related to B-precursor ALL with Philadelphia (Ph) chromosome rearrangement. In addition, Ph-like ALL patients are characterized by common genetic alterations and inferior prognosis, as has been shown in pediatric and adult studies. The aim of this study was the genetic and clinical characterization of Ph-like ALL by RNAseq in a series of adolescent and adult ALL patients included in risk-adapted trials from the Spanish PETHEMA group.Methods:Fourty-nine adolescents and adults with Ph-negative B-ALL (median age 38 years, range 11-74) were included in this study. RNA-Seq custom panel of 42 genes related to the Ph-like signature (AmpliSeq, Thermo Fisher) was carried out in a S5 sequencer (Thermo Fisher). The total reads were employed to build a hierarchical clusterized heatmap with Morpheus software (Broad Institute). Mutations related to the Ph-like genotype were studied after sequencing 31 patients with a custom panel formed by 35 genes. All samples were sequenced in S5 Ion Torrent equipment. CRLF2 overexpression was analyzed by real time PCR.Contingency tables were used to analyze associations between categorical variables (χ2). Median test was used to compare medians of continuous variables between groups. Complete remission (CR) duration, disease-free survival (DFS) and overall survival (OS) curves were estimated using the Kaplan-Meier method and compared between patients using the log-rank test.Results: Thirteen out of 49 patients were classified as Ph-like (26%) based on heatmap aggrupation. There were no significant differences in baseline characteristics like age, gender, ALL sub-type, and cytogenetics, in comparison with B-ALL other subgroup (n=6, 74%). Additionally, most patients with high CRLF2 expression were classified into the Ph-like ALL subgroup (11/13, 85% vs 7/36, 19% of no Ph-like. Complete remission (CR) was attained in 42 patients (86%). At 5 years, the Ph-like ALL subgroup showed a trend to lower probability of CR duration compared to non Ph-like group (95% CI): 27% (0-58%) vs. 58% (38-78%), p=0.086. No significant differences were observed in OS (95% CI): 44% (16-72%) vs. 52% (35-69%), p=0.197), but Ph-like patients showed significantly shorter DFS probability at 5 years (95% CI): 20% (0-45%) vs. 54% (35-73%), p=0.047, (Figures 1a and 1b). A summary of the main characteristics and outcomes of the patients is presented in the Table 1. Mutations of IKZF1, KRAS, JAK2, CRLF2, NRAS, JAK1, TYK2 and PAX5 genes related to Ph-like genotype represented 7/11 Ph-like patients (64%) vs 3/20 no Ph-like (15%). Data of a validation cohort of patients will be presented.Conclusion: Targeted RNA-Seq could identify a Ph-like B-ALL subgroup within adolescents and adults with Ph-negative B-ALL homogeneously treated with risk-adapted protocols. This Ph-like B-ALL subgroup showed lower DFS probability than the remaining Ph-negative B-ALL patients. [Display omitted] DisclosuresRibera:Gilead: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Incyte: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Amgen Inc.: Research Funding, Speakers Bureau; Roche: Honoraria; ARIAD: Research Funding, Speakers Bureau.

Effects of different aberrations in the CRLF2 gene on the biologicalcharacteristics and drug sensitivities of Nalm6 cells.

To investigate the effects and mechanism of action of upregulated CRLF2 expression resulting from different aberrations in the CRLF2 gene (CRLF2, CRLF2+IK6, P2RY8-CRLF2 and CRLF2 F232C) in the B cell ALL cell line Nalm6. Cell proliferation was measured using cell counting kit-8. Transcriptome sequencing technology (RNA-seq) was used to compare changes in gene expression resulting from different aberrations in CRLF2. High-throughput drug sensitivity testing was used to determine the drug sensitivity of cells. All four aberrations in CRLF2 upregulated CRLF2 expression and promoted the proliferation of Nalm6 cells. The RNA-seq results showed the upregulation of genes in the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and the downregulation of genes in the cell cycle pathway in the CRLF2 F232C-overexpressing cells. Western blotting showed that the expression of p-STAT5 protein was significantly higher in the CRLF2 F232C-overexpressing cells. Cells with aberrations in CRLF2 were more resistant to cyclophosphamide and drugs commonly used during treatment than cells in the vector group. The half-maximal inhibitory concentration (IC50 or GI50 ) of dexamethasone was significantly higher in the CRLF2 F232C-overexpressing cell line. The overexpression of CRLF2, CRLF2+IK6, P2RY8-CRLF2 and CRLF2 F232C promotes the proliferation of Nalm6 cells, activates the JAK/STAT signalling pathway and leads to a reduction in sensitivity towards various chemotherapeutic drugs.

Combined analysis of IKZF1 deletions and CRLF2 expression on prognostic impact in pediatric B-cell precursor acute lymphoblastic leukemia

This study aimed to investigate the combined impact of IKZF1 deletions/high expression of CRLF2 on the prognosis of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). IKZF1 deletions and CRLF2 expression were assessed in bone marrow samples from 117 children with newly diagnosed BCP-ALL. Sixteen (13.7%) patients were found to harbor IKZF1 deletions, which was associated with inferior outcomes. The event-free survival (EFS) for patients with high -CRLF2 expression was significantly worse than that for low -CRLF2 expression. Moreover, combined modeling of IKZF1+ /CRLF2 high identified 7.8% of cases as the highest risk subgroup (7-year EFS 33.3 ± 15.7%). In a multivariate analysis, IKZF1+ /CRLF2 high remained a strong independent prognostic factor for EFS (HR: 14.263, p = 0.019). IKZF1 deletions and high -CRLF2 expression were associated with inferior outcomes, and the coexistence of IKZF1+ /CRLF2 high had a significant impact on an integrated prognostic model for high-risk BCP-ALL.

Detection of activating mutations in RAS/RAF/MEK/ERK and JAK/STAT signaling pathways

The study of activating mutations (NRAS,KRAS,FLT3,JAK2,CRLF2genes) of RAS/RAF/MEK/ERK and JAK/STAT signaling pathways in B-cell acute lymphoblastic leukemia (B-ALL) in adult patients which are included in Russian multicenter clinical trials. Within the multicenter study there were 119 adult patients included withde novoB-ALL. The study was considered as prospective and retrospective. The group withBCR-ABL1-negative B-ALL consisted of up to 93 patients (45 male and 48 female, at the age of 17 to 59, the median age 31), they were treated according to the protocols ALL-2009, ALL-2016. The median follow-up lasted for 19 months (1119). The group withBCR-ABL1-positive B-ALL with up to 26 patients (10 male and 16 female, at the age of 23 to 78, the median age 34 years) was included in the study as well. The treatment was carried out according to the protocols ALL-2009 and ALL-2012 in combination with tyrosine kinase inhibitors. The median follow-up lasted for 23 months (4120). The molecular analysis of activating mutations inNRAS,KRASgenes (RAS/RAF/MEK/ERK signaling pathway) andJAK2,CRLF2genes (JAK/STAT signaling cascade) was performed via Sanger sequencing. The internal tandem duplications (ITDs) inFLT3gene were studied by fragment analysis. The evaluation of CRLF2 expression was fulfilled via flow cytometry. Activating mutations inNRAS,KRAS,FLT3genes were found in 22 (23.6%) patients withBCR-ABL1-negative B-ALL. In total, 23 mutations were revealed in theNRAS(n=9),KRAS(n=12), andFLT3(n=2) genes, according to statistics that was significantly more frequent than withBCR-ABL1-positive B-ALL, these genes mutations were not identified in patients (p=0.007). The frequency of mutations detection inKRASandNRASgenes in patients withBCR-ABL1-negative B-ALL was comparable as 12.9% (12 of 93) to 9.7% (9 of 93), respectively (p=0.488). One patient was simultaneously revealed 2 mutations in theKRASgene (in codons 13 and 61).FLT3-ITD mutations were detected in 3.5% (2 of 57) cases ofBCR-ABL1-negative B-ALL. In patients withBCR-ABL1-positive B-ALLFLT3-ITD mutations were not assessed. Violations in the JAK/STAT signaling cascade were detected in 4 (4.3%) patients withBCR-ABL1-negative B-ALL. They were represented by the missense mutations ofJAK2gene (n=3) and the overexpression of CRLF2 (n=2); in one patient were detected the overexpression of CRLF2 and a mutation inJAK2gene simultaneously. No mutations were found inCRLF2gene. In patients withBCR-ABL1-positive B-ALL noJAK2mutations were detected. As long as analyzing demographic and clinical laboratory parameters between groups of patients with and without mutations, there were no statistically significant differences obtained. In the analyzed groups of patients, long-term therapy results did not differentiate according to the mutations presence inNRAS,KRAS,FLT3,JAK2genes. Also, substantive differences were not shown in the rate of the negative status achievement of the minimum residual disease between patients with and without activating mutations in the control points of the protocol (on the 70th, 133rd and 190th days). NRAS,KRAS,FLT3,JAK2activating mutations do not affect the long-term results of the therapy and the rate of the negative status achievement of the minimum residual disease in patients withBCR-ABL1-negative B-ALL treated by the Russian multicenter clinical trials.

High incidence of RAS pathway mutations among sentinel genetic lesions of Korean pediatric BCR-ABL1-like acute lymphoblastic leukemia.

IntroductionRecent advances in genetic analysis have led to the discovery of novel genetic subtypes of precursor B‐cell acute lymphoblastic leukemia (B‐ALL) with prognostic relevance. In this study, we studied a cohort of pediatric B‐ALL patients to retrospectively determine the incidence of patients harboring novel genetic subtypes, as well as their outcome.MethodsB‐ALL patients (N = 190) diagnosed in a single Korean hospital were included in the study. Patients' medical records were reviewed for data on established genetic abnormalities and outcome. CRLF2 expression was analyzed by quantitative RT‐PCR. Anchored multiplex PCR‐based enrichment was used to detect fusions and point mutations in 81 ALL‐related genes.ResultsIncidence of established recurrent genetic subtypes was as follows: high hyperdiploidy (21.6%), ETV6‐RUNX1 (21.6%), BCR‐ABL1 (7.9%), KMT2A rearrangement (7.4%) TCF3‐PBX1/TCF3‐HLF (7.4%), and hypodiploidy (1.1%). Incidence of new genetic subtypes was as follows: BCR‐ABL1‐like (13.2%), ETV6‐RUNX1‐like (2.1%), EWSR1‐ZNF384 (1.1%), and iAMP21 (1.1%). Median age at diagnosis of BCR‐ABL1‐like ALL was 6.8 years. According to type of genetic abnormality, BCR‐ABL1‐like ALL was divided into ABL class (12%), CRLF2 class (8%), JAK‐STAT class (12%), and RAS class (68%). The 5‐year event‐free survival (EFS) of BCR‐ABL1‐like patients was significantly inferior to non‐BCR‐ABL1‐like low‐ and standard‐risk patients (71.5 ± 9.1% vs 92.5 ± 3.2%, P = .001) and comparable to non‐BCR‐ABL1‐like high (75.2 ± 6.2%) and very high‐risk patients (56.8 ± 7.4%). All four ETV6‐RUNX1‐like patients survived event‐free.ConclusionAnalogous to previous studies, incidence of BCR‐ABL1‐like ALL in our cohort was 13.2% with outcome comparable to high and very high‐risk patients. A significantly high number of RAS class mutations was a distinct feature of our BCR‐ABL1‐like ALL group.

Clinical characterization and prognosis of T cell acute lymphoblastic leukemia with high CRLF2 gene expression in children.

It has been reported that overexpression of the CRLF2 gene is associated with poor outcomes in pediatric B cell acute lymphoblastic leukemia (B-ALL), but the incidence rates, clinical characteristics and outcomes of CRLF2 gene overexpression in pediatric T cell ALL (T-ALL) have not been systematically analyzed. In this study, CRLF2 mRNA expression levels and clinical and laboratory parameters in 63 pediatric T-ALL patients were detected at the Children's Hospital of Chongqing Medical University and Children’s Hospital of Xianyang between February 2015 and June 2018. The patients were treated according to the modified St. Jude TXV ALL protocol, and early treatment responses (bone marrow smear and MRD level) and prognoses in the enrolled patients were assessed. CRLF2 overexpression was detected in 21/63 (33.33%) patients. Statistical differences were not found for clinical or laboratory parameters (including sex, age, initial WBC count, incidence mediastinal involvement, abnormal karyotype and fusion genes) between patients with high CRLF2 expression and patients with low expression of CRLF2 (P>0.05). One patient died of tumor lysis syndrome and renal failure, and the treatment response was monitored on day 19 (TP1) of remission in 62 patients. One patient quit treatment because of family decisions, and 61 patients underwent treatment response evaluation on day 46 (TP2) of remission. Significant differences were not found between patients with high CRLF2 expression and patients with low CRLF2 expression in terms of the treatment responses at TP1 or TP2 (P>0.05). Following October 2018, 12 patients among the 61 evaluable patients relapsed (relapse rate: 19.67%), 3 patients died from chemotherapy, and the treatment-related mortality (TRM) rate was 4.92%. Secondary tumors occurred in 1 patient. The 3-year prospective EFS rate was 54.1±11.2% and 77.7±6.6% for the 61 evaluable patients and 58 patients without TRM. Patients with low CRLF2 expression had longer EFS durations than patients with high CRLF2 expression (61 evaluable patients: 35.91± 2.38 months vs 23.43± 2.57 months; 58 patients without TRM: 37.86± 2.08 months vs 24.55±2.43 months, P<0.05). CRLF2 expression levels were also monitored in 13 patients at TP1 and TP2, and the MRD level did not vary with the CRLF2 expression level. Our data suggest that clinical features, laboratory findings and treatment responses in the pediatric T-ALL population do not vary based on the overexpression of CRLF2 but that CRLF2 overexpression can contribute to a high risk of relapse in pediatric T-ALL. Thus, CRLF2 expression levels should not be used as biomarkers for monitoring MRD.

A Novel Role for HMGN1 in Down Syndrome Acute Lymphoblastic Leukemia

Background Down Syndrome (DS) Acute Lymphoblastic Leukemia (ALL) patients have extremely poor outcomes with mortality rates four times greater than non-DS ALL patients within their first two years of diagnosis. They are more suspectible to treatment related toxicities and experience higher relapse rates compared to other ALL patients. Approximately 60% of DS-ALL patients harbor rearrangement of cytokine receptor like factor 2 (CRLF2r), specifically P2RY8-CRLF2, and/or the CRLF2 F232C activating mutation. These lesions are considered poor risk and currently no targeted therapy exist. How increased chromosome 21 gene dosage affect disease phenotype is not yet fully elucidated. However, the high mobility group nucleosome-binding domain-containing protein 1 (HMGN1) on chromosome 21, which competes with histone H1 to bind the nucleosome and results in gene activation may be a candidate for targeted therapy in DS-ALL. Methods We aimed to determine the role of HMGN1 in CRLF2r DS-ALL. To model CRLF2r DS-ALL, the trisomy 21 cell line, SET-2, was transduced with a retroviral vector encoding the CRLF2 F232C activating mutation. Gene knockdown of HMGN1 using CRISPR/Cas9 was performed in the SET-2 CRLF2r line and the non-trisomy-21, non-CRLF2 expressing Jurkat line. Individual knockdowns of another two genes on chromosome 21, DYRK1A and ERG were also performed. Knockdown of JAK2 was used as a control as it is critical for CRLF2 signaling. CellTiter-Glo was used to investigate proliferation of knockdown lines to test the hypothesis that HMGN1 is essential for CRLF2r DS-ALL cell proliferation. Lentiviral vectors encoding the P2RY8-CRLF2 fusion gene, CRLF2 F232C activating mutation or an overexpression construct of HMGN1 were transduced into BaF3 cells individually or in combination to test the hypothesis that overexpressing HMGN1 is associated with activation of CRLF2. Quantitative PCR (qRTPCR) for CRLF2 and flow cytometry for the CRLF2/IL7Rα receptor (TSLPR) were used to determine the effect of increased HMGN1 on CRLF2 expression. AnnexinV/7-AAD cell death assays were performed to determine if the effects of HMGN1 could be reduced by the demethylase inhibitor GSK-J4. Results Knockdown of HMGN1 resulted in an 80-90% decrease in HMGN1 protein expression in SET-2 CRLF2 and Jurkat lines compared to the Cas9 controls. While knockdowns of DYRK1A and ERG did not impair the proliferation of SET-2 CRLF2 cells, HMGN1 and JAK2 knockdowns led to a complete proliferation arrest over a period of 120hrs (p=&amp;lt;0.001, n=3), demonstrating their effect on cell division. However, no change in proliferation was observed in the Jurkat knockdown lines. Overexpression of either HMGN1 or P2RY8-CRLF2 alone in BaF3 cells did not result in cytokine independent transformation. However, cytokine independence was triggered in BaF3 cells when HMGN1 and P2RY8-CRLF2 were co-expressed (p=&amp;lt;0.001, n=3); demonstrating a role for HMGN1 in leukemic transformation. Importantly, the overexpression of HMGN1 in the BaF3 P2RY8-CRLF2 line increased the mRNA expression of CRLF2 by 5.8-fold compared to the BaF3 P2RY8-CRLF2 line without HMGN1 (p=0.034, n=3) and increased the mean fluorescence intensity of TSLPR by flow cytometry from 42 to 308 (p=0.008, n=3) (figure 1.a-c) indicating a novel role for HMGN1 in P2RY8-CRLF2 activation. While there are no pharmacological inhibitors for HMGN1, Lane et al. (2014) have shown that the restoration of H3K27 methylation using the demethylase inhibitor GSK-J4 was able to prevent DS-ALL cells from repassaging. Therefore, we have employed the inhibitor GSK-J4 to determine if it can reduce cell survival in HMGN1 overexpressed BaF3 cells. Specific inhibition of BaF3 P2RY8-CRLF2 HMGN1 cells was evident by decreased cell viability at a concentration of 3.8µM compared to BaF3 P2RY8-CRLF2 or BaF3 HMGN1 lines (p=&amp;lt;0.001, n=3) (figure 4.d). Thus, demonstrating a role for HMGN1 in the modification of the P2RY8-CRLF2 methylome and suggesting HMGN1 as a potential therapeutic target. Conclusion These data support the hypotheses that HMGN1 has a significant role in DS-ALL cell proliferation and that overexpression of HMGN1 results in activation of P2RY8-CRLF2. This is the first report of a novel role for HMGN1 in P2RY8-CRLF2 activation and leukemic transformation in CRLF2r DS-ALL. Additionally, we show that HMGN1 is a potential candidate for the development of a pharmacological inhibitor for CRLF2r DS-ALL. Disclosures Yeung: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria. White:BMS: Honoraria, Research Funding; AMGEN: Honoraria, Speakers Bureau.

Phase II Study of the Hyper-CVAD Regimen in Combination with Ofatumumab (HCVAD-O) As Frontline Therapy for Adult Patients (pts) with CD20-Positive B-Cell Acute Lymphoblastic Leukemia (B-ALL)

Phase II Study of the Hyper-CVAD Regimen in Combination with Ofatumumab (HCVAD-O) As Frontline Therapy for Adult Patients (pts) with CD20-Positive B-Cell Acute Lymphoblastic Leukemia (B-ALL)

Clinical Significance of CRLF2 High Expression in Bone Marrow Mononuclear Cells from Children with Acute Lymphoblastic Leukemia

To detect the expression of CRLF2 in bone marrow mononuclear cells from children with newly diagnosed acute lymphoblastic leukemia(ALL) and to explore its clinical significance in pediatric ALL. A total of 218 children with newly diagnosed ALL who achieveal the complete remission and had the complete follow-up information were selected, and the expression level of CRLF2 in bone marrow mononuclear cells of these children was detected by real-time fluorescent quantitative PCR, and the significance of CRLF2 expression level in clinical prognosis of ALL children was analyzed by using statistical method. 28 cases in 218 children with complete data showed high expression of CRLF2. The cumulative recurrence rate in the CRLF2 high expression group was significantly higher than that in the low expression group (53.6% vs 12.6%) (P＜0.01). The predicted 5-year recurrence-free survival rate (RFS) of ALL children with CRLF2 high expression was significantly higher than that of low expression group (P＜0.01). There was no significant difference in the predicted 5-year RFS between ALL children with CRLF2 low and high expression in the standard-risk(SR) group (P＞0.05). The predicted 5-year RFS of ALL children with CRLF2 low expression was higher than that of ALL children with CRLF2 high expression in the intermediate-risk (IR) and high-risk (HR) groups. (P＜0.05). Cox analysis showed that CRLF2 high expression is an independent risk factor for the relapse of children with ALL. The recurrence rate of pediatric ALL with CRLF2 high expression is high, and CRLF2 high expression is an important prognostic factor for high risk of relapse in ALL children with IR and HR. It is necessary to use CRLF2 expression as an indicator of risk stratification in pediatric ALL.