ABL-class Fusions Research Articles

CD36 cell surface expression as a surrogate marker to identify ABL/JAK-class kinase fusions in pediatric BCP-ALL.

Genetic alterations are the cornerstone of risk stratification in B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and their accurate identification is critical for optimal treatment. Most cases with ABL-class fusion are classified as high-risk yet display good responses to tyrosine kinase inhibitors (TKIs). Current clinical protocols recommend adding a TKI to chemotherapy as soon as possible, making it mandatory to rapidly identify these alterations. We investigated here whether the identification of immunophenotypic features associated with these molecular alterations could be a valuable screening tool. CD36 expression was shown to be a characteristic feature of ABL- or JAK-class kinase fusions. The main genetic subgroups clustering in the subset with Philadelphia (Ph)-like features were also found to display specific immunophenotypic characteristics. A predictive multiparameter scoring system was generated, segregating genetic subtypes with aberrant kinase activation (PAX5/CRLF2alt, BCR::ABL1, ABL/JAK-class). The most robust markers identified were the TSLPR with CD19/22/9/38/81/304 and CD49f. As TKI adjunction is currently limited to the ABL-class kinase fusions, immunophenotypes distinguishing ABL from JAK-class were also investigated. The flow cytometry method reported here, accessible to most hematology departments, is thus a new useful tool to quickly screen for Ph-like kinase fusion with a good sensitivity (95%) and specificity (96%).

Feasibility and Outcome of Post-Induction Therapy Incorporating Dasatinib for Patients with Newly Diagnosed ABL-Class Fusion B-Lymphoblastic Leukemia (ABL-class Fusion B-ALL): Children's Oncology Group AALL1131

Background: Approximately 2-3% of B-ALL patients have Philadelphia chromosome like (Ph-like) B-ALL with an ABL-class fusion ( ABL1, ABL2, PDGFRB, or CSF1R fusions), but lacking BCR::ABL1. These patients are predicted to be sensitive to ABL-class tyrosine kinase inhibitors, such as imatinib or dasatinib. Ph-like B-ALL is associated with male sex, older age, higher initial white blood cell (WBC) count, elevated end of induction (EOI) minimal residual disease (MRD), and poor outcome. The AALL1131 Dasatinib arm was designed to evaluate response to therapy following induction of patients with ABL-class fusion B-ALL, when given dasatinib continuously on a modified Berlin-Frankfurt-Münster (MBFM) backbone. Methods: Between February 2012 and March 2019, AALL1131 enrolled patients 1-30 years with newly diagnosed high risk (HR) B-ALL. AALL1131 was amended in August 2016, to include the Dasatinib arm for patients with ABL-class fusions involving ABL1, ABL2, PDGFRB, and CSF1R. Patients with Down Syndrome were not eligible for the Dasatinib arm. ABL-class fusion B-ALL patients with a predicted TKI-sensitive mutation were screened by low density array (LDA) PCR and confirmed by additional molecular testing. Following 4-drug induction, patients with HR B-ALL and ABL-class fusions received MBFM with dasatinib (60 mg/m 2, maximum 140 mg) daily from start of consolidation through end of maintenance. Dasatinib was held only for toxicity. Results: Twenty-two evaluable patients with HR B-ALL were non-randomly assigned to the Dasatinib arm. ABL-class 3' partners included ABL1 (n=4), ABL2 (n=4), PDGFRB (n=12), and CSF1R (n=2). Compared to all patients on AALL1131, patients treated on the Dasatinib arm were older (median age at diagnosis 14 versus 10 years, p=0.008), more often male (77% versus 56%, p=0.046), and had a trend towards an increased initial WBC (median WBC at diagnosis 44,000/µL versus 19,000/µL, p=0.086). Patients with ABL-class fusions had EOI MRD > 0.01% in 20 of 22 patients and available end of consolidation MRD >0.01% in 6 of 10 patients. Only 5 of 22 (22.7%) patients completed prescribed protocol therapy. Reasons for therapy discontinuation included induction failure (n=2), relapse (n=1), alternate therapy (n=1), determined to be in the patient's best interest (n=10), death (n=1), unknown (n=2). Compared to all patients on AALL1131, four-year disease-free survival was 52.5+18.1% versus 86.8+0.7% (p<0.0001) and overall survival 79.4+13.6% versus 89.2+0.4% (p<0.0001). Dasatinib was well tolerated with no unexpected treatment related toxicities. Conclusion: Patients with ABL-class fusions were more likely male, EOI MRD+, and had poorer outcomes. Seventy-seven percent of patients enrolled on the Dasatinib arm did not complete prescribed therapy. While dasatinib was well tolerated, treatment failures occurred early, indicating alternate strategies are needed.

Delayed Intensification Including Venetoclax and Bortezomib Prolongs Survival in Very High Risk Acute Lymphoblastic Leukaemia

Modern multi-drug risk-stratified response-adapted chemotherapy protocols lead to >85% cure rates in childhood acute lymphoblastic leukaemia (ALL). Between 2013-2022, 707 children (0-18 years) with acute lymphoblastic leukaemia (ALL) treated systematically on the ICiCLe-ALL-14 risk-adapted protocol achieved a 3-year event-free survival (EFS) of 73% (95% confidence interval, CI 69-77). A very high risk (VHR) subgroup (N = 107) was identified with poor outcome (3-year EFS, 38% [95% CI 25-51]). This included patients with induction failure, persistent minimal residual disease (MRD) and/or select high risk cytogenetic subtypes ( BCR::ABL1 other ABL-class fusion ALL, TCF3::HLF, low hypodiploidy, IKZF1plus with high end-induction MRD, KMT2A-rearranged infant ALL). Co-culture based high-throughput drug response profiling (DRP) was performed to identify alternate chemotherapeutic drugs for these VHR patients. Primary ALL blasts extracted by density centrifugation, were seeded on immortalized human bone marrow stromal cells and treated with a 14-drug panel: prednisolone; vincristine (VCR), daunorubicin; asparaginase (ASNase), bortezomib (BZM), venetoclax (VEN), panobinostat (PNB), mitoxantrone, cyclophosphamide, cytarabine, selinexor, dasatinib, 6-thioguanine and idarubicin. Seventy-four patients (25 VHR, 37 high-risk, 7 intermediate-risk, and 5 standard-risk,) were analysed. Venetoclax, BZM and PNB were identified as active agents (IC 50 <100nM for VEN and PNB, <10nM for BZM) in 71%, 42% and 75% of VHR samples, respectively. Based on drug-sensitivity patterns of VHR ALL, a pilot study was initiated to prospectively evaluate the addition of VEN and BZM in the frontline delayed intensification (DI) phase of VHR ALL patients (N=24) after informed consent (Figure 1). Response was assessed before and after VHR-DI in 46% (11/24) patients. Good response, i.e. decrease in MRD ≥ 1 log after VHR-DI, was noted in 82% (9/11) patients. Landmark analysis comparing survival of patients (from start of DI) treated on standard DI versus VHR-DI showed significant improvement in 2-year EFS from 54% (95% CI 40-66) to 76% (95% CI 51-89) ( p=0.037, log-rank test). Overall, VHR-DI was tolerated well. No significant difference was noted in grade 4 and 5 toxicities (Common Terminology Criteria for Adverse Events v4.0) between standard DI and VHR-DI ( p= 0.053 & p=0.988 respectively, chi-square test). Fever-neutropenia and sepsis were the most frequent toxicities with VHR-DI. To identify potential mechanisms of resistance in poor-responders, transcriptome analysis was performed on diagnostic blast cells from VHR patients with good (N=5) or poor (N=5) response to VHR-DI and compared with highly sensitive SR patients (N=5). Upregulation of oxidative phosphorylation and ubiquitin-mediated proteolysis were found in good responders while non-responders showed upregulation of amino acid metabolic and RNA splicing pathways. In conclusion, drug response profiling identifies alternative combination therapies for VHR ALL. Our pilot study provides proof-of-concept of effectiveness and tolerability of inclusion of VEN and BZM in the post-induction treatment of patients with poor-response disease.

Use of Ponatinib Alone or Combined with Other Therapies in Relapsed/Refractory Ph-like Acute Lymphoblastic Leukemia. a Campus ALL Real-Life Study

Introduction. Ponatinib is a third-generation tyrosine kinase inhibitor (TKI) employed in relapsed/refractory (R/R) and, more recently, also in newly diagnosed Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL). Ph-like ALL is a genetically heterogenous subgroup that shares a common transcriptional profile, similar to that of true Ph+ ALL, though lacking the BCR::ABL1 rearrangement. Ph-like ALL is characterized by a poor prognosis, a high relapse rate and a worse overall survival if treated with standard chemotherapy. Several molecular pathways are involved, including JAK/STAT, CRLF2 and ABL-class mutations, providing a heterogeneous genetic landscape with very limited data on the subsequent clinical outcome. Few isolated cases of successful treatment of R/R Ph-like ALL with ponatinib have been reported, including also patients with lesions different from ABL-class mutations. Currently, ponatinib prescription in Italy is not allowed for Ph-like ALL. Patients and Methods. Between January 2019 and July 2023, 17 patients were candidate to receive ponatinib on a nominate compassionate use basis; data were collected in the context of the Campus ALL network in Italy. The criteria for inclusion were a diagnosis of Ph-like ALL, with either hematological or molecular evidence of disease, and a treatment period of at least 28 consecutive days of ponatinib. The Ph-like signature was based on the BCR/ABL1 like-predictor (Chiaretti et al, BJH 2018) and, whenever possible, targeted RNA sequencing. Final data were collected for 15 patients (2 patients were excluded due to a treatment period <28 days) treated with ponatinib either as a single agent or in association with immuno and/or chemotherapy. Results. All patients had common ALL. Eight of the 15 cases were classified as high risk or very high risk ALL at diagnosis; 10 were males, the median age was 28 years (14-66) and the median white blood count at diagnosis was 25.6 x10 9/l (2.7-317 x10 9/l). Targeted RNA sequencing was carried out in 10/15 cases: 7 presented gene fusions ( ABL-class mutations in 2, JAK2 mutations in 2, and CRLF2::P2RY8, IKZF1::DDC and RB1::RCBTB2 in 1 case each); 2 additional cases had a CRLF2 rearrangement, evaluated by FISH. Copy number aberrations analysis by multiplex ligation-dependent probe amplification (MLPA) technology was performed in 12/15 cases: 5 cases had a IKZF1 plus signature, 6 had a IZKF1 loss, while 1 case was IKZF1 wild type. Ponatinib was started in first (n=2) or second (n=5) hematologic relapse in 7 patients (46%), with 3 also having an extramedullary disease; 7 additional patients (46%) were treated in first (n=5) or second (n=2) minimal residual disease (MRD) persistence/recurrence; finally, 1 patient was refractory to 3 subsequent lines of treatment. In 8/15 patients ponatinib was used as single agent or in association with steroids or intrathecal chemotherapy only, in 4 it was administered in combination with chemotherapy and in 3 with blinatumomab; 7 patients started ponatinib after having failed an allogeneic transplant (6 hematologic relapses and 1 molecular relapse). A complete hematological and molecular remission (MRD- CR) was achieved in 6 patients (40%); notably, 4 of these 6 cases carried a gene fusion (1 JAK2 rearrangement, 1 ABL-class fusion, 1 IKZF1::DDC and 1 RB1::RCBTB2); 2 further patients in hematologic relapse at the start of treatment achieved a MRD+ CR (1 patient had a ABL-class gene fusion). Thus, the overall response rate was 53%. Four patients were refractory, while 2 maintained a stable disease. After ponatinib-based treatment, 6 patients were allografted and 1 received a CAR-T cell infusion. The toxicity profile was mild: 3 patients developed a transaminitis, 1 also had a fungal pneumonia, and 1 an increase in pancreatic enzymes. At a median follow-up of 3 months (1-5), 10 patients are alive, 8 being in continuous complete remission. Conclusions. Treatment with ponatinib showed promising results in terms of CR achievement in a heavily pre-treated population of Ph-like ALL patients, with an acceptable toxicity profile. In a pre-transplant setting, ponatinib was effective as a bridge to cellular therapies in 7 out of 8 patients as intention-to-treat (ITT), thus suggesting that this strategy may represent an effective bridge to further therapies. To our knowledge, this is the largest cohort of adult Ph-like ALL cases treated with ponatinib so far reported.

Higher incidence of delayed methotrexate clearance in pediatric acute lymphoblastic leukemia patients treated with imatinib

BackgroundPediatric patients with Philadelphia chromosome positive (Ph+) or ABL-class fusion positive acute lymphoblastic leukemia (ALL) are currently treated with the tyrosine kinase inhibitor (TKI) imatinib added to a chemotherapy backbone that also contains several courses of high-dose methotrexate (HDMTX). A limited number of case studies suggested delayed MTX clearance in patients concomitantly using HDMTX and imatinib. MethodsMTX clearance and serum creatinine in 24 newly diagnosed Ph+/ABL-class fusion positive pediatric ALL patients treated with imatinib (imatinib group) were compared with 281 randomly selected age-matched Ph-negative pediatric ALL patients treated without TKI (control group), receiving in total 61 and 897 HDMTX courses, respectively. ResultsMild to severe delayed MTX clearance (MTX level at T48 hours>0.4 µM) was more frequently found in the imatinib group than in the control group in all courses (55.7% vs 41.1% p = 0.036). After the first HDMTX course, 12.5% (n = 3) of the imatinib group presented with a severe delayed MTX clearance (T48 > 5 µM) versus 2.3% in the control group (p = 0.039). In two (8.3%) of these patients in the imatinib group, this was accompanied by increased creatinine, therefore meeting the criteria for Delayed MTX Elimination (DME). In the control group 12/281 (4.3%) patients presented with DME. Glucarpidase was administered in 2 (8.3%) vs 4 (1.4%) patients in the imatinib group and control group, respectively. ConclusionImatinib increased the risk of delayed MTX clearance in newly diagnosed pediatric Ph+ chromosome or ABL-class fusion positive ALL patients. We therefore recommend stringent supportive care measures and for patients with severe delayed MTX clearance resulting in severe toxicities temporary discontinuation of imatinib or a lower dose of MTX during the next HDMTX courses.

Olverembatinib Treatment in Pediatric Patients With Relapsed Philadelphia-Chromosome-Positive Acute Lymphoblastic Leukemia

Treatment outcomes for children with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) remained poor despite the use of intensive chemotherapy, imatinib or dasatinib, and consolidative allogeneic hematopoietic cell transplantation. Oleverembatinib, a third-generation ABL inhibitor, was found to be highly effective and safe in adults with chronic myeloid leukemia and in some adults with relapsed or refractory Ph+ ALL. We reviewed the efficacy and safety profile of olverembatinib treatment in 6 children with relapsed Ph+ ALL and 1 with T-ALL and ABL class fusion, all of whom had previously received dasatinib or intolerance to dasatinib. The median duration of olverembatinib treatment was 70 days (range: 4-340) and the median cumulative dose was 600 mg (range: 80-3810). Complete remission with negative minimal residual level (<0.01%) was achieved in 4 of the 5 evaluable patients, 2 of whom were treated with olvermbatinib as a single agent. Safety profile in 6 evaluable patients was excellent with grade 2 extremity pain occurred in 2 patients and grade 2 myopathy of lower extremity and grade 3 fever in 1 patient each. Olverembatinib appeared to be safe and effective in children with relapsed Ph+ ALL.

Philadelphia-Like Genetic Rearrangements in Adults With B-Cell ALL: Refractoriness to Chemotherapy and Response to Tyrosine Kinase Inhibitor in ABL Class Rearrangements.

Philadelphia-like (Ph-like) B-cell ALL is a high-risk subtype of B-cell ALL that shares a gene expression profile with Ph-positive ALL, but without a BCR::ABL1 fusion. A subgroup of these patients have fusions or rearrangements involving genes such as ABL1, ABL2, PDGFRβ, JAK2, and EPOR, some of which are potentially sensitive to tyrosine kinase inhibitors (TKIs). Prompt identification of these genetic aberrations are important for prognostication and treatment decisions. We performed a retrospective review of patients with B-cell ALL treated at MD Anderson Cancer Center to identify recurrent genetic fusions commonly seen in Ph-like ALL and focus on patients treated with TKI. We identified 23 patients with recurrent genetic fusions commonly seen in Ph-like ALL; 14 had ABL class fusions (eight ABL1, one ABL2, and five PDGFRβ) and nine had JAK2 class fusions (five JAK2 and four EPOR). Notably, several of these fusions were cryptic by conventional cytogenetics and fluorescent in situ hybridization (FISH) assays and identified only by multiplex fusion assay. Thirteen of these 23 patients received a TKI as part of their treatment; this included ABL1 fusion (n = 8), PDGFRβ fusion (n = 4), and EPOR fusion (n = 1). All four patients with ABL1 fusions who received TKI with induction chemotherapy are alive in first remission. Understanding the genomics of B-cell ALL is important for disease prognostication and for precise treatment planning. Besides conventional cytogenetics and directed FISH testing, multiplex fusion assays can help identify recurrent chromosomal translocations that are seen in patients with Ph-like ALL. Early initiation of TKI appears beneficial; larger studies are required to fully understand the benefit of TKI and to design rational combination therapies for these patients.

Role of Cytogenetics and FISH in Laboratory Workup of B Cell Precursor Acute Lymphoblastic Leukemia

AbstractModern therapeutic protocols in acute leukemias risk stratify disease based on genetic characterization of the neoplastic cells and their response to treatment. Genetic characterization is routinely performed by cytogenetic testing of leukemic cells and is a standard component of modern risk-adapted therapy in acute lymphoblastic leukemia (ALL). High-throughput technologies like RNA sequencing have identified multiple novel subtypes in recent years. The cytogenetic strategy using GTG and fluorescent in-situ hybridization (FISH) has to be adapted to identify not only the primary principal chromosomal abnormalities but also the novel subtypes. In the review, we describe a systematic comprehensive cytogenetic strategy that integrates information from immunophenotyping, flow-based DNA ploidy, and karyotyping complemented by targeted FISH studies to identify more than 70% of genetic abnormalities described in B cell precursor ALL. The simplified strategy includes a four-probe FISH and flow ploidy strategy, ± karyotyping that identifies high risk (KMT2A, BCR::ABL1, hypodiploidy, iAMP21) and standard risk (ETV6::RUNX1 and high hyperdiploid) cytogenetic groups. The extended FISH panel includes probes targeting MEF2D, ZNF384, and CRLF2 rearrangements that are used intuitively on integrating the immunophenotyping features that characterize these entities. The strategy also includes a systematic approach to identify masked hypodiploidy integrating targeted FISH analysis directed toward identifying monosomies of chromosomes 7, 15, and 17 and flow cytometry-based DNA ploidy analysis. The recently described PH-like ALL is characterized by ABL class fusions and rearrangements of CRLF2 and JAK2 genes. FISH analysis using break-apart probes can be used to identify these aberrations. The cytogenetic approach also includes FISH analysis to identify intragenic and whole gene deletions of the IKZF1 genes that identify a subset of patients associated with high risk of treatment failure.

Integrative genomic analysis of childhood acute lymphoblastic leukaemia lacking a genetic biomarker in the UKALL2003 clinical trial

Incorporating genetics into risk-stratification for treatment of childhood B-progenitor acute lymphoblastic leukaemia (B-ALL) has contributed significantly to improved survival. In about 30% B-ALL (B-other-ALL) without well-established chromosomal changes, new genetic subtypes have recently emerged, yet their true prognostic relevance largely remains unclear. We integrated next generation sequencing (NGS): whole genome sequencing (WGS) (n = 157) and bespoke targeted NGS (t-NGS) (n = 175) (overlap n = 36), with existing genetic annotation in a representative cohort of 351 B-other-ALL patients from the childhood ALL trail, UKALL2003. PAX5alt was most frequently observed (n = 91), whereas PAX5 P80R mutations (n = 11) defined a distinct PAX5 subtype. DUX4-r subtype (n = 80) was defined by DUX4 rearrangements and/or ERG deletions. These patients had a low relapse rate and excellent survival. ETV6::RUNX1-like subtype (n = 21) was characterised by multiple abnormalities of ETV6 and IKZF1, with no reported relapses or deaths, indicating their excellent prognosis in this trial. An inferior outcome for patients with ABL-class fusions (n = 25) was confirmed. Integration of NGS into genomic profiling of B-other-ALL within a single childhood ALL trial, UKALL2003, has shown the added clinical value of NGS-based approaches, through improved accuracy in detection and classification into the range of risk stratifying genetic subtypes, while validating their prognostic significance.

Dissecting Ph-like ALL: The Role of Genomic Lesion and Minimal Residual Disease in Refining Outcome

Cardinali D and Della Starza I contributed equally Foà R and Chiaretti S contributed equally Background. Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is a subgroup of B-lineage ALL characterized by a gene expression profile (GEP) that resembles that of Ph-positive ALL but lacks the BCR/ABL1 transcript. Ph-like ALL cases are characterized by CRLF2 overexpression, JAK-STAT pathway mutations, IKZF1 deletions and rearrangements involving cytokine receptors and tyrosine kinases. To identify Ph-like ALL cases, our group developed a predictive tool named "BCR/ABL1-like predictor", which is based on the quantification by quantitative PCR of 10 transcripts, specifically overexpressed by Ph-like ALL cases1. ALL is the first neoplasm where the assessment of early response to therapy by minimal residual disease (MRD) monitoring is pivotal for guiding therapeutic choices. Real-time-quantitative PCR (RQ-PCR) of IG/TR gene rearrangements is the most widely used molecular method for MRD assessment. The GIMEMA LAL2317 is a clinical trial designed for newly diagnosed adult B-lineage Ph-negative ALL that includes two cycles of blinatumomab in the consolidation phase. MRD is evaluated at established time points (TPs), the most important being TP2, i.e., after the first consolidation cycle with high-dose chemotherapy, and TP3, after the first cycle of blinatumomab. Aim of this study. To refine risk relapse categories of Ph-like ALL cases based on their genomic features at presentation in combination with the MRD status. Methods. We performed this sub-analysis on a cohort of Ph-like ALL cases enrolled in the GIMEMA LAL2317 protocol identified according to the BCR/ABL1-like predictor assay and that underwent a centralized comprehensive molecular screening at diagnosis by IG/TR gene rearrangements and targeted DNA/RNA sequencing. MRD status was evaluated at specific TPs, i.e., TP2 and TP3, by RQ-PCR and digital droplet PCR (ddPCR). Results. A Ph-like profile was documented in 31/109 evaluable patients (28.4%); 11 of them (35.5%) experienced a relapse. By targeted RNA sequencing, 9/11 presented a gene fusion at presentation: in 4/9 a CRLF2-P2RY8 gene fusion was identified, whereas the remaining cases had PAX5-ZCCHC7, ETV6-BCL21L4, IKZF1-DDC, JAK2-r and ABL-class fusion in 1 case each (Figure 1A). The MRD status was evaluated at TP2 and TP3 by RQ-PCR in 23/31 patients: 15/23 were negative at both TPs, while 8/23 were positive at TP2 and negative at TP3; in the remaining 8 cases, MRD was not evaluated because of refractoriness (n=3), early death (n=2) or loss to follow-up (n=3). DdPCR analysis was performed in 9 patients and compared to RQ-PCR: 6/9 were concordant by both methods, while 3/9 resulted discordant with RQ-PCR being negative at TP3 while being positive by ddPCR. On the basis of the Ph-like signature and MRD results, we defined 3 subgroups: i) Ph-like ALL cases (n=12, 39%) that did not relapse, being MRD-negative at both TP2 and TP3; 3 cases were positive for fusion genes; ii) relapsed Ph-like ALL cases (n=8, 26%) who were MRD-positive at TP2 but became MRD negative at TP3, with 7 harboring a fusion gene; iii) relapsed Ph-like ALL cases (n=3, 10%), always MRD-negative at both TPs, with 2/3 positive for fusion genes (Figure 1B). Finally, by flow cytometry analysis, available in 7 patients at relapse, 6 maintained CD19 positivity and only 1 proved CD19-negative. Conclusions. Ph-like ALL patients tend to relapse early even after blinatumomab treatment and despite becoming MRD-negative. Relapse is not related to a mechanism of CD19 escape. Based on the gene fusions at presentation and on the MRD status, we could identify 3 subgroups of Ph-like ALL. These findings suggest that Ph-like ALL cases should be followed with different markers in addition to IG/TR, particularly in cases with well-defined fusion genes, who are at a very high risk of relapse. In this sense, a refined and rapid genetic characterization at presentation of Ph-like ALL cases is warranted for a more personalized and targeted patient management. 1. Chiaretti S et al., BJH 2018 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical Characteristics and Stratified Analysis of Ph-like Acute Lymphoblastic Leukemia in Children: A Retrospective Study from China

Objective: Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (All), also known as BCR-abl1 like ALL, has a negative BCR-abl1 fusion gene, and the gene expression profile overlaps significantly with Ph+ ALL. PH like ALL contains a variety of genomic changes, including ABL class fusion (abl1, abl2, CSF1R, PDGFRB, PDGFRA); JAK pathway gene (JAK2), chemokine receptor gene (CRLF2) or MEK-ERK pathway genes, that activate kinases and cytokine receptors. Herein, we analyzed the clinical characteristics and outcome of 24 childhood Ph-like ALL cases in single center, and performed a stratified analysis. Methods: This retrospective study of Ph-like B-ALL other than Ph+ ALL was performed in patients aged 1-16 years and treated from April 2015 to October 2020 according to the CCCG-ALL-2015 protocols in our center in China. The baseline characteristics and treatment outcomes of patients were collected. The 4-year event-free survival (EFS) and 4-year overall survival (OS) were estimated by use of Kaplan-Meier methods, and the 4-year cumulative incidence of relapse was calculated by use of a competing risk model gray test. The comparison was performed between groups by use of log rank test. Results: We identified 24 pediatric patients with newly diagnosed Ph-like B-ALL, including 16 males (66.7%) and 8 females (33.3%). The median age was 7 years old (1-16 years old). Median leukocyte count was 31.8 (2.65-846.5) × 10^9/L。4(17%) of 24 patients were PDGFRB fusion-positive, 2 (8%) were ABL1 fusion-positive, 2 (8%) were ABL2 fusion-positive, 1 (4%) were PDGFRA fusion-positive, 1 (4%) were JAK2 fusion-positive, and 14 (59%) were CRLF2 fusion-positive. The 4-year EFS was 70.6% (95% CI, 60·5-79·1), the 4-year OS was 81·1% (95% CI, 68·6-87·5), and the 4-year cumulative incidence of relapse was 16.7%. Overall survival was significantly different between the groups (p=0·037), that were 100% in patients who were ABL1 fusion positive, 92.9% in those who were CRLF2 fusion-positive, 50% in those who were ABL2 fusion-positive, and 49% in those who were PDGFRB fusion-positive. Conclusion: Ph-like ALL in children are characterized by an older age and a high white blood cell count at diagnosis. Ph-like ALL is a heterogeneous group of disorders, Overall survival was significantly different between the different genomic groups. Children with abl1 positive or CRLF2 positive had better survival, while those with PDGFRB positive or abl2 positive had a poor outcome. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ALL-180 A Case of Refractory Philadelphia-Like Acute Lymphoblastic Leukemia Treated With Ruxolitinib/Blinatumomab and Ruxolitinib/Inotuzumab Ozogamicin Prior to Allogeneic Marrow Transplant

Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is a high-risk subset associated with genomic alterations. These alterations can be grouped into major subclasses that either phenocopy BCR-ABL1 or activate JAK/STAT signaling. Ongoing trials suggest that patients harboring ABL-class fusions are candidates for ABL1 inhibitors, whereas alterations activating the JAK-STAT pathway may be amenable to treatment with JAK inhibitors. We hereby describe the first case of the use of ruxolitinib in combination with blinatumomab and inotuzumab in relapsed Ph-like ALL. Case report. Tertiary referral hospital. Ph-like ALL patient harboring a CRLF2 mutation. Our patient received three cycles of first-line Hyper-CVAD: 1A/1B and 2A. BMBx after the third cycle revealed persistent disease. He then received two cycles of second-line ruxolitinib/blinatumomab prior to disease progression. He was then treated with three cycles of third-line ruxolitinib/inotuzumab followed by allogeneic transplant. Reports encouraging the use of JAK inhibitors in CRLF2 Ph-like ALL exist for children, and trials are currently ongoing for adolescents and young adults [NCT03571321]. Ruxolitinib was used at a starting dose of 15 mg BID, as determined by initial dose escalation phase trials testing the safety of ruxolitinib in myelofibrosis. Dose titration (maximum 25 mg BID/100 mg QD) was implemented according to response and platelet and neutrophil counts. Complete response (CR) and measurable residual disease (MRD). After progressing on Hyper-CVAD, our patient switched to ruxolitinib/blinatumomab and achieved CR with negative MRD after only one cycle of treatment. He progressed on this regimen after cycle 2 and moved to third-line ruxolitinib/inotuzumab. After three cycles, he achieved CR and negative MRD and underwent allogeneic transplant from a matched unrelated donor. Today, our patient is 10 months post transplant and remains in clinical and molecular remission. The combination of ruxolitinib plus blinatumomab or inotuzumab is a promising chemotherapy-free salvage regimen given outpatient for relapsed Ph-like ALL. It achieved CR in our patient prior to allo SCT with a limited side effect profile. Further clinical trials adding ruxolitinib to front-line and next-line therapies are needed.

P343: GENOMIC CHARACTERISATION OF B-OTHER ALL IN UKALL2003 PATIENTS BY NEXT GENERATION SEQUENCING

Background: Incorporating genetics into risk stratification for the treatment of childhood acute lymphoblastic leukaemia (ALL) has contributed to increased survival rates. About 30% of patients (B-other-ALL) harbour none of the known major chromosomal changes. Recently, we estimated that up to two thirds of B-other-ALL can be classified into novel subtypes using FISH and MLPA alone. We showed that ABL-class fusions and ERG deletions were linked to prognosis (Schwab et al, BJHaem, 2022). Aims: To characterise B-other-ALL in the UKALL2003 trial using next generation sequencing (NGS), to evaluate the added benefits of these techniques and validate the prognostic significance of B-other-ALL subtypes. Methods: B-other-ALL patients were tested using whole genome sequencing (WGS) (n=158) and bespoke targeted NGS (t-NGS) for the detection of abnormalities in 64 genes commonly mutated or rearranged in B-ALL (n=180), 42 patients had both WGS and t-NGS. Data were integrated with results from FISH, MLPA and cytogenetics. Results: A representative cohort of 347 B-other-ALL was classified into subgroups, including six with ≥20 cases (Figure). Among patients tested by WGS, 92% (n=146/158) were classified compared to 75% (n=104/138) tested by t-NGS. NGS-based approaches allowed for the detection of PAX5 P80R, IKZF1 N169Y and ZEB2 H1038R and fusion partner genes. Identification of DUX4-r was not possible using t-NGS. PAX5alt was the most frequently observed (n=90), including dic(9;20) (n=27), dic(9;12) (n=11), PAX5 fusions (n=21), PAX5 mutation (n=11) and PAX5-ITD (n=12). Additionally, 11 patients were observed with PAX5 P80R mutations, classified as a separate subgroup due to the associated gene expression signature. PAX5alt had an outcome similar to B-other-ALL overall (overall survival (OS) at 10yrs 82% vs 86%). There was no significant difference in outcome between PAX5 abnormalities, although an association with age was observed: dic(9;20) was more commonly observed in children aged 1-4 (p< 0.001), while both dic(9;12) and PAX5 P80R were seen in older children aged 10-15 years (p=0.005). One subgroup with DUX4 rearrangements and/or ERG deletions (DUX4-r/ERG-d) (n=78) had DUX4 involvement confirmed by WGS in 57 patients. In the remaining 21 patients ERG-d were identified by t-NGS and/or MLPA. ERG-d is exclusive to DUX4-r patients, thus can be used as a surrogate marker. In line with other studies, patients with DUX4-r had a lower relapse rate (RR) (5%) and improved OS (96%) compared to other subgroups. Patients classified as ETV6-RUNX1-like (n=21) were characterised by multiples abnormalities of ETV6, including rearrangements (n=17) and/or deletion (n=12) as well as rearrangements (n=7) and/or deletions (n=6) of IKZF1. An ETV6-RUNX1-like gene expression signature was confirmed by RNA-Seq (n=6). No relapses or deaths were reported within this subgroup. As previously reported, ABL-class fusions (n=25) were associated with an inferior outcome compared to other subgroups (RR=63% and OS=50%). Patients with rearrangements of CRLF2 (n=52) and ZNF384 (n=37) had similar outcomes to the cohort overall. Image:Summary/Conclusion: A combination of techniques has classified a representative cohort of B-other-ALL patients from UKALL2003 into clinically relevant subgroups. The identification of DUX4-r, subgroup defining mutations and fusion partner genes demonstrates the value of NGS-based approaches. We have confirmed the good and poor prognostic associations of DUX4-r and ABL-class fusions, respectively, and identified ETV6-RUNX1-like subgroup to have a good prognosis.

Measurable residual disease analysis in paediatric acute lymphoblastic leukaemia patients with ABL-class fusions

BackgroundABL-class fusions including NUP214-ABL1 and EBF1-PDGFRB occur in high risk acute lymphoblastic leukaemia (ALL) with gene expression patterns similar to BCR-ABL-positive ALL. Our aim was to evaluate new DNA-based measurable residual disease (MRD) tests detecting these fusions and IKZF1-deletions in comparison with conventional immunoglobulin/T-cell receptor (Ig/TCR) markers.MethodsPrecise genomic breakpoints were defined from targeted or whole genome next generation sequencing for ABL-fusions and BCR-ABL1. Quantitative PCR assays were designed and used to re-measure MRD in remission bone marrow samples previously tested using Ig/TCR markers. All MRD testing complied with EuroMRD guidelines.ResultsABL-class patients had 46% 5year event-free survival and 79% 5year overall survival. All had sensitive fusion tests giving high concordance between Ig/TCR and ABL-class fusion results (21 patients, n = 257 samples, r2 = 0.9786, P < 0.0001) and Ig/TCR and IKZF1-deletion results (9 patients, n = 143 samples, r2 = 0.9661, P < 0.0001). In contrast, in BCR-ABL1 patients, Ig/TCR and BCR-ABL1 tests were discordant in 32% (40 patients, n = 346 samples, r2 = 0.4703, P < 0.0001) and IKZF1-deletion results were closer to Ig/TCR (25 patients, n = 176, r2 = 0.8631, P < 0.0001).ConclusionsMRD monitoring based on patient-specific assays detecting gene fusions or recurrent assays for IKZF1-deletions is feasible and provides good alternatives to Ig/TCR tests to monitor MRD in ABL-class ALL.

MUC4 expression by immunohistochemistry is a specific marker for BCR-ABL1+ and BCR-ABL1-like B-lymphoblastic leukemia

BCR-ABL1-like B-acute lymphoblastic leukemia (B-ALL) is a genetically heterogeneous group of high-risk B-ALL that benefits from targeted tyrosine kinase inhibitor (TKI) therapy. The incidence of this high-risk B-ALL is relatively low and screening with surrogate markers will be useful to identify patients for further genetic testing. Here we demonstrate that widely available MUC4 protein immunohistochemistry (IHC) is predictive of a BCR-ABL1-like genotype for a subset of patients. Overall, MUC4 expression was observed in 36% (9/25) BCR-ABL1-like, 43% (3/7) BCR-ABL1+ and 9% (2/22) B-ALL other cases (p=.019 for BCR-ABL1 like and BCR-ABL1+ versus B-ALL others). Furthermore, 83% (5/6) of patients with ABL class fusions showed MUC4 expression when compared to 25% (4/16, p=.006) patients with JAK class fusions. Overall, the study demonstrates that MUC4 expression is highly specific (90.9%) for BCR-ABL1+ and BCR-ABL1-like B-ALL with high sensitivity for cases with ABL class fusions.

Genetic characterisation of childhood B-other-acute lymphoblastic leukaemia in UK patients by fluorescence insitu hybridisation and Multiplex Ligation-dependent Probe Amplification.

While next-generation sequencing technologies provide excellent strategies to screen for newly defined genetic abnormalities of prognostic or therapeutic significance in patients with B-other-acute lymphoblastic leukaemia (ALL), they are not widely available. We used a dual screening approach, incorporating fluorescence insitu hybridisation (FISH) and Multiplex Ligation-dependent Probe Amplification (MLPA), to establish the frequency and long-term outcome of a representative cohort of specific subgroups of B-other-ALL recruited to the childhood ALL trial, UKALL2003. We focussed on abnormalities of known prognostic significance, including ABL-class fusions and ERG deletions, as a surrogate marker for DUX4-rearranged ALL. ABL-class fusions accounted for ~4% of B-other-ALL and were associated with high levels of minimal residual disease (MRD; 14/23 with MRD >5%) and a high relapse rate (55·7%) following treatment without tyrosine kinase inhibitor (TKI), confirming the importance of prospective screening with a view to incorporating TKI into therapy. Patients with deletions of ERG (~10% of B-other-ALL) had a 10-year event-free-survival of 97·2%, validating previous reports of their excellent outcome. Rearrangements of ZNF384, MEF2D and NUTM1 were observed at low frequencies. Here, we estimate that approximately one third of B-other-ALL patients can be reliably classified into one of the known genetic subgroups using our dual screening method. This approach is rapid, accurate and readily incorporated into routine testing.

Prognostic impact of chromosomal abnormalities and copy number alterations in adult B-cell precursor acute lymphoblastic leukaemia: a UKALL14 study

Chromosomal abnormalities are established prognostic markers in adult ALL. We assessed the prognostic impact of established chromosomal abnormalities and key copy number alterations (CNA) among 652 patients with B-cell precursor ALL treated on a modern MRD driven protocol. Patients with KMT2A-AFF1, complex karyotype (CK) and low hypodiploidy/near-triploidy (HoTr) had high relapse rates 50%, 60% & 53% and correspondingly poor survival. Patients with BCR-ABL1 had an outcome similar to other patients. JAK-STAT abnormalities (CRLF2, JAK2) occurred in 6% patients and were associated with a high relapse rate (56%). Patients with ABL-class fusions were rare (1%). A small group of patients with ZNF384 fusions (n = 12) had very good survival. CNA affecting IKZF1, CDKN2A/B, PAX5, BTG1, ETV6, EBF1, RB1 and PAR1 were assessed in 436 patients. None of the individual deletions or profiles were associated with survival, either in the cohort overall or within key subgroups. Collectively these data indicate that primary genetic abnormalities are stronger prognostic markers than secondary deletions. We propose a revised UKALL genetic risk classification based on key established chromosomal abnormalities: (1) very high risk: CK, HoTr or JAK-STAT abnormalities; (2) high risk: KMT2A fusions; (3) Tyrosine kinase activating: BCR-ABL1 and ABL-class fusions; (4) standard risk: all other patients.

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.

Low incidence of ABL-class and JAK-STAT signaling pathway alterations in uniformly treated pediatric and adult B-cell acute lymphoblastic leukemia patients using MRD risk-directed approach \u2013 a population-based study

BackgroundABL-class and JAK-STAT signaling pathway activating alterations have been associated with both a poor post-induction minimal residual disease (MRD) response and an inferior outcome in B-cell acute lymphoblastic leukemia (B-ALL). However, in most of the studies patients received non-uniform treatment.MethodsWe performed a population-based analysis of 160 (122 pediatric and 38 adult) Lithuanian BCR-ABL1-negative B-ALL patients who had been uniformly treated according to MRD-directed NOPHO ALL-2008 protocol. Targeted RNA sequencing and FISH analysis were performed in cases without canonical B-ALL genomic alterations (high hyperdiploids and low hypodiploids included).ResultsWe identified ABL-class fusions in 3/160 (1.9%) B-ALL patients, and exclusively in adults (p = 0.003). JAK-STAT pathway fusions were present in 4/160 (2.5%) cases. Of note, P2RY8-CRLF2 fusion was absent in both pediatric and adult B-ALL cases. Patients with ABL-class or JAK-STAT pathway fusions had a poor MRD response and were assigned to the higher risk groups, and had an inferior event-free survival (EFS) / overall survival (OS) compared to patients without these fusions. In a multivariate analysis, positivity for ABL-class and JAK-STAT fusions was a risk factor for worse EFS (p = 0.046) but not for OS (p = 0.278) in adults.ConclusionsWe report a low overall frequency of ABL-class and JAK-STAT fusions and the absence of P2RY8-CRLF2 gene fusion in the Lithuanian BCR-ABL1 negative B-ALL cohort. Future (larger) studies are warranted to confirm an inferior event-free survival of ABL-class/JAK-STAT fusion-positive adult patients in MRD-directed protocols.

Assessment of the leukemogenic mechanism of Ph-like acute lymphocytic leukemia for the identification of a novel therapeutic target

Ph-like acute lymphocytic leukemia (ALL) is a subtype of Ph-negative B precursor ALL, and its gene expression profile is similar to that of Ph+ALL. In recent decades, comprehensive genomic analyses have revealed that Ph-like ALL has two types. The first type is associated with the ABL-class tyrosine kinase fusion gene, and the second type with fusion genes involving cytokine receptors or molecules, including CRLF2, which are correlated with the activation of the JAK/STAT pathway. Based on these findings, tyrosine kinase or JAK inhibitors were found to be effective for Ph-like ALL. Genetic abnormalities identified in Ph-like ALL, except for CRLF2 rearrangement, are quite rare. Thus, functional studies regarding each genomic abnormality are relevant for establishing targeted therapies for Ph-like ALL. To develop a targeted molecular therapy, a functional study of NCOR1-LYN, which is a novel ABL-class fusion gene, was conducted on pediatric patients with Ph-like ALL.