CRLF2 Rearrangements Research Articles

Philadelphia chromosome-like acute lymphoblastic leukemia with concomitant rearrangements of CRLF2 and ABL1: a pediatric case report

BackgroundBCR::ABL1-like or Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) was first reported in 2009. Ph-like ALL is characterized by gene signature similar to Philadelphia chromosome ALL, but without BCR::ABL1 fusions. Molecularly, Ph-like ALL is divided into seven categories, with CRLF2 and ABL-class rearrangements being the two most common subtypes, exhibiting alterations in distinct downstream signaling cascades.Case presentationWe report a rare case of pediatric Ph-like ALL with concomitant CRLF2 and ABL1 rearrangements. CRLF2 was fused with P2RY8, its most common fusion partner, whereas ABL1 was fused with MYO18B, a novel fusion partner that has not been previously reported. The 4-year-old female patient was treated using the national multicenter CCCG-ALL-2020 protocol with the addition of dasatinib at the end of induction when ABL1 rearrangement was confirmed by RNA-seq. Morphologically and molecularly, the patient remained in continuous remission until the last follow-up. To the best of our knowledge, this is the first case of Ph-like ALL harboring two distinct rearrangement categories.ConclusionsOur results identified that ABL1 rearrangement and CRLF2 rearrangement can coexist. The application of FISH, whole transcription sequencing, PCR can help us to have a more comprehensive understanding of ALL cytogenetics and molecular biology. Further studies are needed to explore the role of targeted therapies in such rare clinical scenarios.

CRLF2 and IKZF1 abnormalities in childhood hematological malignancies other than B-cell Acute Lymphoblastic Leukemia

Rearrangements and overexpression of CRLF2 are hallmarks of poor outcomes in BCR::ABL1-like B-ALL, and CRLF2 overexpression is a high-risk marker in T-ALL. However, CRLF2 alterations in pediatric hematologic malignancies other than B-ALL have not been reported. In this study, we analyzed the CRLF2 overexpression, rearrangements (P2RY8::CRLF2 and IGH::CRLF2), activation (pSTAT5 and pERK), and the expression of dominant-negative IKZF1 isoforms (Ik6 and Ik8), implied in CRLF2 dysregulation, in 16 pediatric patients (AML, n = 9; T-ALL, n = 3; LBL, n = 2; HL, n = 1; cytopenia, n = 1). A high frequency of CRLF2 rearrangements and overexpression was found in the 16 patients: 28.6% (4/14) showed CRLF2 overexpression, 93.8% (15/16) were positive for CRLF2 total protein (cell-surface and/or cytoplasmic), while 62.5% (10/16) were positive for P2RY8::CRLF2 and 12.6% (2/16) for IGH::CRLF2. In addition, 43.8% (7/16) expressed Ik6 and Ik8 isoforms. However, only a few patients were positive for the surrogate markers pSTAT5 (14.3%; 2/14) and pERK (21.4%; 3/14).

IKZF1PLUS alterations contribute to outcome disparities in Hispanic/Latino children with B-lymphoblastic leukemia.

Compared to other ethnicities, Hispanics/Latinos (H/L) have a high incidence of acute lymphoblastic leukemia (ALL), enrichment of unfavorable ALL genetic subtypes, and worse outcomes, even after correcting for socioeconomic factors. We previously demonstrated increased incidence of the high-risk genetic drivers IKZF1 deletion and IGH::CRLF2 rearrangement in H/L compared to non-H/L children with B-ALL. Here in an expanded pediatric cohort, we sought to identify novel genetic drivers and secondary genetic alterations in B-ALL associated with H/L ethnicity. Comprehensive clinicopathologic data from patients with B-ALL treated from 2016 to 2020 were analyzed. Subtype was determined from karyotype, fluorescence in situ hybridization (FISH), chromosome microarray (CMA), and our next-generation sequencing (NGS) panel (OncoKids). Non-driver genetic variants were also examined. p-Values less than.05 (Fisher's exact test) were considered significant. Among patients with B-ALL at diagnosis (n=273), H/L patients (189, 69.2%) were older (p=.018), more likely to present with CNS2 or CNS3 disease (p=.004), and NCI high-risk ALL (p=.014) compared to non-H/L patients. Higher incidence of IGH::CRLF2 rearrangement (B-ALL, BCR::ABL1-like, unfavorable; p=.016) and lower incidence of ETV6::RUNX1 rearrangement (favorable, p=.02) were also associated with H/L ethnicity. Among secondary (non-subtype-defining) genetic variants, B-ALL in H/L was associated with IKFZ1 deletion alone (p=.001) or with IGH::CRLF2 rearrangement (p=.003). The IKZF1PLUS profile (IKZF1 deletion plus CDKN2A/2Bdel, PAX5del, or P2RY8::CRLF2 rearrangement without DUX4 rearrangement) was identified as a novel high-risk feature enriched in H/L patients (p=.001). Our study shows enrichment of high-risk genetic variants in H/L B-ALL and raises consideration for novel therapeutic targets.

The Impact of Age and Genomics on Drug Sensitivity in 1,076 Children and Adults with B-Cell Acute Lymphoblastic Leukemia

While the cure rate of pediatric acute lymphoblastic leukemia (ALL) now exceeds ~90% with contemporary combination chemotherapy, the prognosis for adults with ALL remains significantly inferior with long-term overall survival ranging from 50% to 70%. Recent studies have uncovered marked differences in ALL genomics between children and adults, with some high-risk subtypes becoming more prevalent with age. However, the underlying biology of age-related disparities in ALL is not fully understood, especially with regard to differences in leukemia sensitivity to chemotherapy. To address this knowledge gap, we performed exvivo drug sensitivity profiling (i.e., pharmacotyping) of 21 anti-leukemia agents on primary B-ALL diagnostic samples from 767 pediatric (age, 0-18 years) and 309 adult (19-84 years) patients. Drug sensitivity was measured as LC 50: the concentration of drug required to kill 50% of the leukemia cells (PMID: 36604538). A total of 7,975 unique LC 50 values were experimentally determined. RNA-seq was used for subtype classification and gene expression analysis. Among 21 drugs, seven showed significant differences in overall LC 50 between children and adults ( P<0.05 after Bonferroni correction): children displayed higher sensitivity to asparaginase, prednisolone, mercaptopurine, daunorubicin, and inotuzumab, while adults showed higher sensitivity to dasatinib and nelarabine. In multivariate models adjusting for 23 ALL molecular subtypes, only mercaptopurine remained significantly associated with age ( P=1.5×10 -5), suggesting that age-related differences in drug sensitivity can be primarily attributed to the variation in ALL subtypes between children and adults. For mercaptopurine, within KMT2A, CRLF2, and DUX4 subtypes, pediatric samples consistently showed a lower LC 50 than adults carrying the same genomic abnormality ( P=0.032, 0.0045, and 0.02, respectively). To explore intra-subtype heterogeneity, we performed unsupervised clustering using gene expression data for each of these three subtypes. Remarkably, within each of these subtypes, we identified two clusters with distinct transcriptomic profiles that were also largely segregated by age group, i.e., an adult-dominated cluster (C-a) and a pediatric-dominated cluster (C-p). In the KMT2A subtype, cases in C-a exhibited an over-representation of the KMT2A:: AFF1 fusion, and resistance to mercaptopurine ( P=0.029), prednisolone ( P=0.0039), vincristine ( P=0.046) and cytarabine ( P=0.0037). Within CRLF2 ALL, cases in C-a were associated with the presence of BCR:: ABL1-like signature and IGH:: CRLF2 rearrangements, and were more resistant to mercaptopurine ( P=0.0073) and prednisolone ( P=0.00036) compared to those in C-p. For DUX4 ALL, C-a was characterized by an under-representation of ERG deletions and resistance to mercaptopurine ( P=0.0056) and prednisolone ( P=0.0031), compared to C-p within DUX4. To explore the clinical relevance of this heterogeneity, we analyzed the invivo treatment response of KMT2A (N=35), CRLF2 (N=59) and DUX4 (N=118) B-ALL enrolled in six frontline ALL trials. Compared to cases in C-p (usually drug-sensitive), those in C-a (usually drug-resistant) consistently had significantly poorer initial treatment responses as measured by persistent end-of-induction minimal residual disease (≥0.01%) in KMT2A (58% vs 9%; P=0.0063), CRLF2 (74% vs 41%; P=0.030), and DUX4 (66% vs 40%; P=0.0058) ALL. In conclusion, these studies have revealed important new insights into the pharmacogenomic basis of age-related differences in B-ALL treatment response. These results indicate that both inter- and intra-subtype heterogeneity contribute to inferior prognosis in adults with ALL, but also point to therapeutic opportunities to improve their outcomes.

Role of Allogeneic Stem Cell Transplantation in Preventing Relapse in Adult BCR::ABL1-like Acute Lymphoblastic Leukemia

Background Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is characterized by the activation of several kinase pathways that promote treatment resistance leading to poor survival of patients at all ages. Despite the use of tyrosine kinase inhibitors in the Ph-like ABL-class subtype (15% Ph-like ALL), there is not a consensus approach for treatment of most patients (JAK-class especially), and the role of allogeneic stem cell transplantation (alloSCT) has scarcely been explored so far (i.e., mandatory vs. MRD-oriented). Objective To assess the outcome of Ph-like patients enrolled in the ongoing ALL19 trial from the Programa Español de Tratamientos en Hematología (PETHEMA) for adults (18-60y) with Ph-negative ALL, and to evaluate the effectiveness of alloSCT in first complete remission (CR). Methods In the ALL19 trial, patients are allocated to alloSCT according both to the end of induction (EOI) measurable residual disease (MRD) level and to high-risk genetic markers such as low-hypodiploidy, KMT2A rearrangement, TP53 biallelic alteration and concomitant deletion of IKZF1 and CDKN2A/B. Therefore, BCR::ABL1-like subtype was not a criteria for early transplantation unless meeting either poor MRD clearance at EOI or concomitant deletion of IKZF1+CDKN2A/B. Patients with MRD≥0.01% on day+35 (EOI) and/or high-risk genetics were assigned to early alloSCT (preceded by 1 cycle of consolidation chemotherapy) while those with good MRD clearance and standard risk genetics received early and delayed consolidation (3 cycles each) and maintenance therapy. Bone marrow or peripheral blood samples were analyzed by G-banding+FISH, SNP array (750K Affymetrix, Thermo Fisher) and next generation sequencing (NGS, with a custom DNA gene panel, Illumina) at diagnosis in 4 reference laboratories. MRD was centrally assessed by next generation flow cytometry with a sensitivity up to 2x10 -6. Results Twenty-four Ph-like out of 248 adult BCP ALL (10%) were detected. Baseline characteristics were similar between Ph-like and the remaining BCP-ALL. Most Ph-like ALL patients showed the JAK-class profile (20/24 [83%]: 17/24 CRLF2 rearrangement[r], 1/24 JAK2r, 1 BLNK::DNTT and 1 JAK2 R683 mutation), 3/24 belonged to the ABL-class subtype (2 ABL2r, 1 NUP214::ABL1) and 1 patient was identified by RNAseq (gene expression profiling similar to BCR::ABL1 ALL) without identifying any rearrangement. Regarding secondary genetic alterations, 17/24 (71%) patients showed the IKZF1 plus profile, 10/23 (43%) harbored JAK2 R683 mutation, 4/23 N/KRAS mutations, 2/23 CRLF2 mutation and 1/23 showed mutations in several genes such as IKZF1, NR3C1 or FLT3, among others. There were no significant differences in the probability of achieving CR between Ph-like and the remaining BCP ALL (15/20 [75%] vs. 137/162 [85%], p=0.334). However, EOI MRD level of Ph-like individuals was significantly poorer (MRD<0.01% Ph-like 4/15 [27%] vs. other BCP-ALL 85/136 [63%], p=0.007). By intention to treat, most Ph-like patients (17/19, 89%) were allocated to alloSCT (vs. 91/153 [59%] in the other BCP-ALL, p=0.011) due to either EOI MRD≥0.01% (9/18), IKZF1+CDKN2A/B concomitant deletions (3/18) or both (6/18). The 2-y overall survival (OS) in both groups was not significantly different (57% [95% CI, 27%-78%] vs. 67% [55%-77%, p=0.304]) (Figure 1). Interestingly, most Ph-like patients died due to treatment-related toxicity (2 transplant-related mortality, 1 CART-related, 1 during induction, 1 in CR and 1 by COVID-19 infection). The 2-y cumulative incidence of relapse (CIR) was also similar (Ph-like 27% [8%-50%] vs. other BCP-ALL 35% [25%-45%], p=0.946) (Figure 1). Conclusions Despite the short follow up of this series, our results show that Ph-like ALL patients have poorer EOI MRD clearance and higher rate of alloSCT realization than the remaining BCP ALL patients, without significant differences in outcome. This suggests that early alloSCT in first CR might overcome the poor prognosis of Ph-like ALL patients. Funding Funded in part by: PI10/01417 y FIS PI19/01183 Ministerio de Salud Carlos III, JCyL_SA118P20, FMM21/002_AP176752021, “La Caixa” Foundation, Pfizer Spain, Fundación PETHEMA. This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No IMI001-07. The JU receives support from the European Union's Horizon 2020 research and innovation programme and EFPIA.

Novel Insights in TKI Resistance in BCR:: ABL1-Positive B-Cell Acute Lymphoblastic Leukemia Beyond Kinase Domain Mutations

Background and Aim Despite the introduction of tyrosine kinase inhibitors (TKIs), approximately 30% of newly diagnosed pediatric and adult BCR:: ABL1-positive B-cell acute lymphoblastic leukemia (B-ALL) patients experience relapsed or refractory disease (Biondi et al, Lancet Haematology 2018; Bassan et al, Journal of Clinical Oncology 2010). ABL1 kinase domain mutations, hindering TKI binding, explain part of the relapses and are acquired during TKI therapy but are rarely detected in newly diagnosed BCR:: ABL1-positive B-ALL patients (Baer et al, Haematologica 2022). Intrinsic TKI resistance mechanisms in BCR:: ABL1-positive B-ALL present prior to TKI treatment are less well documented. Therefore, we aimed to identify potential biomarkers for TKI sensitivity at diagnosis using primary patient samples and newly generated cell lines made from patient-derived xenografts (PDX cell lines). Patients and Methods Leukemic cells from diagnosis of BCR:: ABL1-positive B-ALL patients without ABL1-kinase domain mutations were used to measure cell survival in an ex vivo co-culture assay after imatinib (1.95-125 μM), dasatinib (1.95-125 nM), or bosutinib (0.016-2 μM) exposure. TKI sensitivity was assessed using the area under the dose-response curve (AUC), with <80 arbitrary units (AU) classified as imatinib-sensitive, 80-120 AU as intermediate, and >120 AU as resistant. Multiplex ligation-dependent probe amplification, whole exome sequencing, and RNA sequencing were performed on diagnostic material if available. ABL1 phosphorylation and expression levels were measured using (phospho)flow cytometry in patient-derived xenografts from diagnostic samples and (PDX) cell lines. Results We determined ex vivo sensitivity to imatinib, dasatinib, and bosutinib in 32 pediatric and 19 adult BCR:: ABL1-positive B-ALL patient samples. We observed a substantial variability in ex vivo imatinib response in pediatric and adult patient samples (median AUC: 98 AU; range: 16 to 281 AU). The sensitivity towards imatinib strongly correlated with the sensitivity towards second-generation TKIs dasatinib and bosutinib (rho = 0.84; p < 0.001 and rho= 0.89; p <0.001). Moreover, imatinib's AUC of samples derived from newly diagnosed patients who relapsed after TKI treatment was higher compared with those derived from patients who did not suffer from a relapse (median AUC 123 vs 84 AU; p = 0.03). We observed that an increase in ex vivo imatinib resistance was correlated with a decreased amount of phosphorylated ABL1 protein (rho=-0.92; p=0.0013) and total ABL1 protein (rho=-0.82; p=0.01) in untreated, newly diagnosed samples. Ex vivo exposure to imatinib reduced the ABL1 phosphorylation levels in both sensitive and resistant samples but resistant cells exhibited lower levels of (p)ABL1 protein, indicating reduced dependence on BCR-ABL1 signaling. The ex vivo imatinib resistance in samples carrying deletions in B-cell development genes IKZF1 and/or PAX5 was higher than in samples without these deletions (median AUC: 122 versus 80 AU; p=0.01). In addition, one imatinib-resistant patient had a p.His1038Arg mutation in ZEB2, which is recurrent in B-ALL and this gene has also been described as B-cell development gene (Studd et al, Blood Cancer Journal 2021). Interestingly, another resistant patient had a mutation in SETD2,a gene previously associated with ex vivo TKI resistance in chronic myeloid leukemia cell lines (Sheng et al, Cell Proliferation 2019). Other secondary lesions included SH2B3 inactivation lesions and CRLF2 rearrangements. These lesions were present in intermediately sensitive and resistant BCR:: ABL1-positive samples and are expected to lead to increased JAK/STAT signaling, potentially serving as a resistance mechanism for TKI. Conclusion Our study revealed that the amount of total and phosphorylated ABL1 protein was correlated with ex vivo TKI sensitivity. Newly diagnosed TKI-resistant BCR:: ABL1 samples were further characterized by somatic lesions in B-cell development genes including IKZF1. These results link the known poor prognostic value of IKZF1 deletions in BCR:: ABL1-positive B-ALL patients (van der Veer et al. Blood 2014; Fedullo et al, Haematologica 2019) with intrinsic mechanisms of TKI resistance other than tyrosine kinase domain mutations.

Outcome Disparities Among Hispanic and Non-Hispanic Patients with Acute Lymphoblastic Leukemia

Introduction: Acute lymphoblastic leukemia (ALL) is the predominant type of leukemia diagnosed in children and accounts for approximately 20% of all cases of acute leukemia in adults. In pediatric cases, ALL generally exhibits a favorable prognosis and outcome. Unfortunately, this optimistic scenario does not extend to adults, as the majority of deaths related to ALL occur in this age group. The outcomes observed in children with ALL are linked to the favorable cytogenetic profile of their leukemia. However, as individuals age, the cytogenetic profile progressively shifts towards high-risk groups. Furthermore, several epidemiologic studies have identified ethnicity, specifically Hispanic and African American backgrounds, as independent factors associated with worse outcomes. The factors contributing to these outcomes are diverse and include limited healthcare access, insurance coverage, immigration status, treatment adherence, socioeconomic status, and timeliness of treatment initiation, among others. Additionally, individuals of Hispanic ethnicity are more prone to complex cytogenetics and Central Nervous System involvement, both of which are commonly associated with a poor prognosis. Our study aimed to assess the differences in treatment outcomes among adults diagnosed with ALL by conducting a specific comparison between non-Hispanic and Hispanic individuals. Methods: In this retrospective observational study, we collected data from the electronic medical records (EMR) of patients aged 18 years and older who received a diagnosis of Acute Lymphoblastic Leukemia between February 1, 2011, and February 28, 2020 at Ascension Seton Hospitals in Texas. The collected data included sociodemographic factors such as gender, ethnicity, and age at diagnosis, as well as clinical characteristics such as cytogenetics, immunophenotype, treatment, and overall survival. Bivariate and multivariate analyses were conducted using a chi-squared test. Statistical significance was defined as a P-value less than 0.05. Results: A total of 56 patients with ALL were identified, with 45% being non-Hispanic and 55% being Hispanic. The incidence of ALL did not show significant variation based on gender or age, but it exhibited a significant difference depending on insurance status. The analysis revealed that Hispanic patients had a significantly higher likelihood of lacking insurance coverage (75%) compared to non-Hispanic patients (25%), with a statistically significant p-value of 0.001. With the exception of one non-Hispanic patient diagnosed with T-cell ALL, almost all participants in the study had B-cell ALL. Among the patients who received chemotherapy, 44% were non-Hispanic, while 56% were Hispanic. Among patients with t(9;22)/BCR-ABL1 positive ALL, non-Hispanics represented 76% and Hispanics represented 23%. Other cytogenetic abnormalities, including CRLF2-IGH fusion, IgH rearrangement, CRLF2 rearrangement, PBX1/TCF3 fusion, t(1;19) with PBX1/TCF3 fusion, ETV6 deletion, near haploidy, and 9p deletion, were more prevalent in Hispanic patients (79%) compared to non-Hispanic patients (21%). The overall survival rates at 1 year were 64% for non-Hispanic patients and 67.7% for Hispanic patients. However, at 3 years, the overall survival rates decreased to 32% for non-Hispanic patients and 25.8% for Hispanic patients. Conclusions: Our study provides important insights into the demographic and clinical characteristics of patients with ALL and highlights notable disparities between non-Hispanic and Hispanic patients. A higher proportion of Hispanic patients lacked any form of health insurance coverage at the time of diagnosis. Interestingly, the incidence of Philadelphia-positive ALL was found to be higher among non-Hispanic patients, whereas other cytogenetic abnormalities were more prevalent among Hispanic patients. Additionally, Hispanic patients experienced lower rates of overall survival at 3 years, suggesting potential disparities in outcomes. The differences observed between Hispanic and non-Hispanic patients highlight the necessity for additional investigation and tailored interventions to address the specific challenges faced by Hispanic patients, including access to healthcare, insurance coverage, and the prevalence of unfavorable cytogenetic abnormalities.

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.

Association of Latino Ethnicity with Cytogenetic Subtype in Pediatric Acute Lymphoblastic Leukemia: A Report from the Reducing Ethnic Disparities in Acute Leukemia Consortium

Introduction: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, and is a heterogeneous group of bone marrow diseases with multiple cytogenetic subtypes leading to distinct clinical outcomes. Despite advances in treatments that improve survival, racial and ethnic disparities continue to persist. Latino children have a higher incidence of ALL and poorer overall survival compared with non-Latino white children. Although several studies conducted have identified cytogenetic differences in Latinos, larger studies are needed to confirm and further elaborate these findings. The aim of our study was to analyze cytogenic profiles of a large, diverse cohort of children with ALL to assess the associate between cytogenic subtypes in Latino ethnicity. Methods: We analyzed the distribution of ALL subtypes and self-reported race/ethnicity in the Reducing Ethnic Disparities in Acute Leukemia (REDIAL) Consortium retrospective registry, which includes patients (age 0-24 at diagnosed) with newly diagnosed ALL from six pediatric cancer centers in the southwest region that were diagnosed between 2017 - 2021. The demographic and clinical factors included self-reported race/ethnicity, sex, age at diagnosis, immunophenotype, and cytogenetic aberration. Cytogenetic subtypes evaluated included double trisomies (DT; trisomies chromosomes 4 and 10), BCR::ABL (Ph+), ETV6::RUNX1 KMT2A ( MLL) rearrangements, hypodiploidy, intrachromosomal amplification of chromosome 21 ( iAMP21) TCF3::PBX1, IGH, and CRLF2 rearrangements. Multivariable logistic regression models were used to estimate adjusted odds ratios (aORs) and 95% confidence intervals (95% CIs), accounting for sex and age at diagnosis. Results: Of the 2,388 ALL patients evaluated, 90.2% had B-cell ALL and 64.4% self-identified as Latino and the mean age at diagnosis was 6.6 years. Compared with non-Latino white patients, the cytogenetic findings associated with favorable prognosis, DT and ETV6:: RUNX1 t(12;21), were less frequent in Latino patients (aOR: 0.80, 95% CI: 0.65-0.98 for DT and aOR: 0.67, 95% CI: 0.53-0.85 for ETV6:: RUNX1). Overall, favorable cytogenetics were less frequent among Latino children compared with non-Latino white children (aOR: 0.82, 95% CI: 0.67-0.99). The neutral cytogenetic subtype TCF3:: PBX1 was also less frequent clinically among Latino patients (aOR: 0.39, 95% CI: 0.18 - 0.85). Among unfavorable cytogenetic alterations, iAMP21 was significantly less frequent among Latino children compared with non-Latino white children (aOR: 0.57, 95% CI: 0.32-0.99). There was no significant association between other unfavorable cytogenetic subtypes and Latino ethnicity, either individually or for the overall unfavorable group. Conclusions: Overall, our findings indicate that Latino children are significantly less likely to have favorable cytogenetic subtypes, TCF3:: PBX1 and iAMP21 and prognostically neutral cytogenetics. Further evaluation of cytogenetic subtypes and race/ethnicity may elucidate their contribution to outcome disparities.

Emerging molecular subtypes and therapies in acute lymphoblastic leukemia.

Tremendous strides have been made in the molecular and cytogenetic classification of acute lymphoblastic leukemia based on gene expression profiling data, leading to an expansion of entities in the recent International Consensus Classification (ICC) of myeloid neoplasms and acute leukemias and 2022 WHO Classification of Tumours: Haematolymphoid Tumors, 5th edition. This increased diagnostic and therapeutic complexity can be overwhelming, and this review compares nomenclature differences between the ICC and WHO 5th edition publications, compiles key features of each entity, and provides a diagnostic algorithmic approach. In covering B-lymphoblastic leukemia (B-ALL), we divided the entities into established (those present in the revised 4th edition WHO) and novel (those added to either the ICC or WHO 5th edition) groups. The established B-ALL entities include B-ALL with BCR::ABL1 fusion, BCR::ABL1-like features, KMT2A rearrangement, ETV6::RUNX1 rearrangement, high hyperdiploidy, hypodiploidy (focusing on near haploid and low hypodiploid), IGH::IL3 rearrangement, TCF3::PBX1 rearrangement, and iAMP21. The novel B-ALL entities include B-ALL with MYC rearrangement; DUX4 rearrangement; MEF2D rearrangement; ZNF384 or ZNF362 rearrangement, NUTM1 rearrangement; HLF rearrangement; UBTF::ATXN7L3/PAN3,CDX2; mutated IKZF1 N159Y; mutated PAX5 P80R; ETV6::RUNX1-like features; PAX5 alteration; mutated ZEB2 (p.H1038R)/IGH::CEBPE; ZNF384 rearranged-like; KMT2A-rearranged-like; and CRLF2 rearrangement (non-Ph-like). Classification of T-ALL is complex with some variability in how the subtypes are defined in recent literature. It was classified as early T-precursor lymphoblastic leukemia/lymphoma and T-ALL, NOS in the WHO revised 4th edition and WHO 5th edition. The ICC added an entity into early T-cell precursor ALL, BCL11B-activated, and also added provisional entities subclassified based on transcription factor families that are aberrantly activated.

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.

Correlation of the surface expression of thymic stromal lymphopoietin receptor with the presence of CRLF2 gene rearrangements in children with B-lineage acute lymphoblastic leukemia.

In this study, we aimed to compare the immunophenotype of tumor cells in children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) harboring rearrangements of the CRLF2 gene with that in children without such aberrations with a specific focus on the surface expression of the related protein thymic stromal lymphopoietin receptor (TSLPR). We examined bone marrow samples from 46 patients with primary BCP-ALL who had CRLF2 rearrangements detected by FISH (CRLF2(+) cohort). A total of 140 consecutive patients with intact CRLF2 were included in a control CRLF2(-) cohort. TSLPR expression was studied by flow cytometry. The majority of CRLF2(+) patients were conventionally positive (≥20% positive cells) for TSLPR (33 of 46, 71.7%). Among the remaining children in this group, two were completely TSLPR-negative, seven had less than 10% TSLPR-positive cells, and four had between 10% and 20% TSLPR-positive cells. By contrast, the majority of CRLF2(-) patients had no TSLPR-positive cells (119 of 140, 85.0%), while in 15 cases (10.7%), the percentage of TSLPR-positive cells was below 10%, and in six cases (4.3%), it was between 10% and 20%. Receiver operator characteristic analysis revealed a threshold of only 1.6% TSLPR-positive cells for the effective prediction of the presence of CRLF2 rearrangement. Moreover, this threshold retained its predictive value when only children with low TSLPR positivity were studied. When surface TSLPR is detected at the diagnosis of BCP-ALL, close attention should be given to the search for chromosomal aberrations involving CRLF2 at any level of expression.

Outcomes of Patients with B-ALL with Concomitant BCR::ABL1 and CRLF2 Rearrangements

Background: Outcomes of patients with Philadelphia chromosome positive (Ph+) ALL have improved significantly with the introduction of tyrosine kinase inhibitors (TKI). Ph-like ALL is a high-risk subtype of B-ALL with gene expression profile similar to Ph+ ALL, but without the BCR-ABL translocation. About 50% of the patients with Ph-like ALL have CRLF2 rearrangement (CRLF2-R) and are associated with poor outcomes with conventional chemoimmunotherapy. The co-occurrence of both Ph+ and CRLF2-R is rare (Jain, Haematologica 2017) and long-term outcomes unknown. Methods: We retrospectively analyzed the Leukemia and Pathology databases for patients who presented to the MD Anderson Cancer Center (MDACC) and were noted to have concomitant Ph+ and CRLF2-R. Ph+ was assessed by conventional cytogenetics / FISH / PCR for BCR-ABL1. CRLF2-R was assessed by flow cytometry / FISH. Outcomes assessed were best response (complete remission (CR), relapse, minimal residual disease (MRD), event-free survival (EFS) and overall survival (OS). Results: We identified 8 patients (5 newly diagnosed and 3 R/R B-ALL) who had concurrent Ph+ and CRLF2-R rearrangement (Table 1). The median age was 29 (range, 14-57), 6 (75%) were of Hispanic ethnicity, and 6 (75%) were men. The median clone size of CRLF2-R and Ph+ were 73% (56-92%) and 52% (4-96%), respectively. The most frequent mutated gene was JAK2 in 3/8 (38%) patients. All patients were treated with chemoimmunotherapy or immunotherapy + TKI (ponatinib, n=7; dasatinib, n=1). The median follow-up was 23 months. The best response achieved was CR in 6/8 (75%). The median EFS was 8.6 months; 12-month OS was 75% (Figure 1). A total of 3/6 (50%) responders had a subsequent relapse with 2/3 relapses occurring in the extramedullary compartment (one in central nervous system and one intraperitoneal); both with CRLF2-R clone without Ph+. Of the 5 patients who presented to MDACC as newly diagnosed B-ALL, two were treated with frontline with Hyper-CVAD + ponatinib/dasatinib, two with blinatumomab + ponatinib and the one with a pediatric regimen + dasatinib. Best response was CR with negative MRD in 5/5 (100%). All 5 patients cleared both rearrangements (Ph+ and CRLF2) at the time of remission; 2/5 patients showed CRLF2-R during relapse (without the Ph+). The 12-month OS and EFS were 80% and 60%, respectively. Three patients presented to MDACC with R/R B-ALL. They received HCVAD + ponatinib/dasatinib +/-ruxolitinib. Best response was CR in 1/3 (33%) patients. All 3 patients cleared Ph+, but CRLF2-R persisted in 2 patients. Conclusions: Patients who harbor concomitant Ph+ and CRLF2-R represent a rare but high-risk subgroup of B-ALL. High rate of relapse was noted despite treatment with chemotherapy + TKI +/- ruxolitinib; several patients cleared the Ph+ clone, but CRLF2-R clone persisted suggesting novel interventions against CRLF2-R B-ALL are warranted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Novel Drugs to Target JAK2 Rearrangements in Pediatric Acute Lymphoblastic Leukemia

Background: Risk-based treatment is curative for 85% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), however relapse remains a leading cause of mortality, urging the need of novel molecular targets. JAK/STAT alterations represent about 7% of the 'Philadelphia-like' cases. JAK2 gene encodes for a non- receptor tyrosine kinase fundamental for hematopoiesis and its mutations have been widely studied, whereas JAK2 fusion genes are still poorly characterized. Aim: This study aims to identify JAK2 fusion genes among BCP-ALL pediatric patients, developing a target strategy in preclinical models. Methods: RNA Next Generation Sequencing was applied to find JAK2 fusions in a cohort of high risk BCP-ALL pediatric patients. In vivo expansion of patients' cells has been carried out in NSG mice. A high throughput ex-vivo screening of about 174 FDA-approved drugs, through a luminescence assay (CellTiter glo) was performed. After ex-vivo and in-vivo treatments with single selected drugs, phosphoflow and apoptosis assays were done. Results: We identified 11 pediatric cases carrying a JAK2 fusion with different partners, where PAX5 gene was the only recurrent. We expanded cells from 3 cases in mice, carrying PAX5::JAK2, ATF7IP::JAK2 and ZEB2::JAK2. We demonstrated the basal activation of pY1007-1008-JAK2 compared both to wild type JAK2 (+70%, two-tailed P value 0.03) and to P2RY8::CRLF2 rearrangements as well as to JAK2 mutation (+40%, two-tailed P value 0.16). JAK2 downstream effectors pS727-STAT3 and pY694-STAT5 were also activated. CHZ868, a new class-II tyrosine kinase inhibitor (TKI), was used to target JAK2, appreciating a mean inhibition of -62% of pY1007-1008 JAK2 in PAX5::JAK2, -22% in ATF7IP::JAK2 and -35% in ZEB2::JAK2 after 30 minutes till 48h. Moreover, we observed a decrease of pS727-STAT3(-35-50%) and pY694-STAT5 (-15-50%) and reduction of phosphorylation on PI3K pathway, downregulating PDPK1, AKT, 4pEBP1 and pS6. After 48h monotherapy treatment by CHZ868, we detected decreased cell viability (20-75% at IC50), which increased in the combination with dexamethasone. In PAX5::JAK2, we also performed treatments with BIBF1120/Nintedanib, LCK inhibitor (activated downstream to PAX5 fusions) evaluating a 20% reduction of cell viability. Importantly, combination of BIBF1120 and CHZ868 showed a synergistic effect (-45%, at IC50). Moreover, ruxolitinib caused autophagy with higher levels of LC3- II compared to untreated cells (+45%, p<0.01) and reduction of apoptosis. Indeed, active caspase 3 increased after ruxolitinib and chloroquine (autophagy inhibitor) combination (+20% vs ruxolitinib alone, p<0.01). Instead, CHZ868 alone or with chloroquine did not induce autophagy, with no effect on LC3-II and active caspase 3 levels. Finally, we demonstrated the in vivo efficacy of CHZ868 in PAX5::JAK2, ATF7IP::JAK2 and ZEB2::JAK2 patient-derived- xenografts. After two weeks of 30mg/Kg daily treatment of CHZ868, we observed a significant reduction of leukemic CD10+/CD19+ cells both in BM (-43-85%), spleen (-72-89%), CNS (-13-62%) and PB (-46-80%). Moreover, CHZ868 significantly reduced the phosphorylation of pJAK2 (-18-46%), pSTAT5 (-23-71%) and pAKT(Ser473) (-18- 34%). As CHZ868 in-vivo targeting showed not complete eradication of leukemia, with low effect on CNS, we decided to apply a high-throughput ex-vivo screening of a library of 174 FDA-approved drugs in early to late clinical trials, on 4 PDXs samples carrying JAK2 fusions (PAX5::JAK2, ATF7IP::JAK2, ZEB2::JAK2 and the recently expanded novel fusion GIT2::JAK2). We confirmed that Ruxolitinib was not effective on JAK2 cohort at the 6 different dosages that were tested (5nM-25uM), neither on MUTZ5, and five Lymphoblastoid cell lines (LCLs) used as healthy controls. Instead, AT9283 (p<0.001 vs LCLs), Fedratinib (p<0.01 vs LCLs) and Gandotinib (p<0.05 vs LCLs) were found to be potent, specific, and non-toxic JAK2 inhibitors. Additionally, this extended screening led us to identify drugs, not belonging to JAK inhibitors, specific and non-toxic for rearranged JAK2 cohort, such as Birinapant (Smac mimetic), JQ1 (BRD4 inhibitor), Fludarabine (Chemotherapy) among the others. Summary/Conclusion: CHZ868 is a promising drug for the treatment of JAK2 fusions. Further studies will focus on effective and specific novel drugs found to be highly effective and specific on JAK2 rearrangements in BCP-ALL.

Targeting Hyperactivated Lck in PAX5 Rearranged Pediatric B-Cell Precursors Acute Lymphoblastic Leukemia

Despite intensive risk-based treatment protocols, 15% of pediatric patients with B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) experience relapse. There is urgent need of novel strategies to target poor prognosis subgroups. We considered 135 B-other cases consecutively enrolled in Italy in the AIEOP-BFM ALL2000/R2006 protocols without recurrent genetic rearrangements and excluding Down syndrome and High Hyper Diploid cases. By integrating gene expression, copy number analyses and fusion genes discovery by target-capture NGS, we identified 59 patients (43·7%) having a Ph-like signature; in addition to ERG-related (26%), High Hyperdiploid-like (17%), ETV6::RUNX1-like (8·9%), MEF2D-rearranged (2·2%) or KMT2A-like (1·5%). CRLF2 rearrangements were almost exclusively found in the Ph-like group, and also the IKZF1-plus profile was prevalent in Ph-like cases (8/9) and associated with a poor outcome (5-year EFS (SE) 37·5% (17·1) vs. 65·5% (7·0) for non-IKZF1-plus cases). Interestingly, PAX5 gene lesions were mostly associated with the Ph-like signature, with CNAs and translocations in 43/131 B-other patients (32·8%), with 31/57 (54·3%) in Ph-like and 12/74 (16%) in non-Ph-like cases. Notably, all the 7 PAX5t were found in the Ph-like group. EFS (p<0·001) and OS (p=0·009) analyses according to the fusion classes reveal statistical significance in Ph-like cases, classified accordingly to different class fusions, such as ABL/JAK-class, PAX5-class, other fusions and negative cases after NGS analysis. In fact, the PAX5t cases have a poor EFS, similar to ABL/JAK-class patients (53% and 50%, respectively) and lower than Ph-like cases negative for any fusion gene (78%). Moreover, PAX5t OS was inferior compared to ABL/JAK-class and negative cases (about 53% vs. 83·3% and 88·9%). These features of PAX5t cases prompted us to explore the feasibility of targeting this aberration. By applying the NetBID2 data-driven network interference algorithms, we constructed the BCP-ALL interactome and identified the hub drivers' activity (DA) based on network gene interaction on either PAX5 translocated patients versus all BCP-ALL excluding Ph-like or versus Ph-like. We also assessed a Differential Expression (DE) of genes belonging to these pathways. Interestingly, in the top NetBID2 drivers we identified the LCK signaling factor showing a significantly higher activity in PAX5t compared to other BCP-ALL subgroups. Furthermore, analysis of LCK activity in BCP-ALL patients with known PAX5 status revealed that it is an exclusive signature of PAX5t cases. Moreover, 22 genes resulted with a positive driver activity in PAX5t vs. Ph-like (p<0.05) and bioinformatic analysis to identify drug approved interactions found 7 targetable genes, including LCK. As previously showed by us, we demonstrated the efficacy of the LCK-inhibitor Nintedanib, as single agent or in combination with conventional chemotherapy, both ex-vivo and in a patient-derived xenograft model, showing a synergistic effect with dexamethasone. We herewith applied a wider ex-vivo drug screening with 174 FDA-approved drugs, by comparative cellular viability of different sub-groups of leukemia PAX5t samples and control-B-lymphoblastoid cell lines (CTR-B-LCSs) in addition to NALL-1 cell lines (carrying PAX5/ETV6 fusion), measured by ATP-Glo based luminescent based assay after exposure of the depicted drugs, whereas Staurosporin was taken as a positive control. Drug sensitivity scores (DSS) are plotted as a clustered heat map, followed by unsupervised hierarchical clustering. From the drug screening, we selected Dasatinib, Bosutinib and Foretinib, known to be among the top 10 most potent LCK ligands. Here we demonstrated their efficacy in PAX5t cases by evaluating the differential DSS, i.e., by avoiding toxicity in CTR-B-LCSs. This study provides new insights in the pathogenic mechanisms of poor risk Ph-like leukemia and identifies a potential novel therapy for targeting the PAX5t group. Moreover, in general this study opens the scenario to target LCK kinase activity in further BCP-ALL subgroups.

Outcomes in Hispanic Patients with Philadelphia-Negative and Philadelphia-like Acute Lymphoblastic Leukemia with the Use of Blinatumomab

Introduction: In the United States, Hispanics have the highest incidence rate of B-cell acute lymphoblastic leukemia (ALL) amongst all minority groups. Hispanic adults with ALL have the lowest rates of progression-free survival and overall survival. Hispanic adults, for example, have an increased frequency of Philadelphia chromosome (Ph)-like ALL, a sub-type of ALL associated with significantly inferior survival outcomes In the last decade, use of Blinatumomab and other novel targeted immunotherapies were approved for treatment of relapsed or minimal residual disease positive disease (MRD +) B-cell ALL. Pivotal trial showing efficacy of blinatumomab in relapsed or MRD + setting had very limited number of Hispanic patients. Our aim is to evaluate survival outcomes and the efficacy of blinatumomab usage in Hispanic B-ALL including Philadelphia-negative (Ph negative) and Philadelphia-like (Ph-Like) patients treated at LAC-USC (Los Angeles County Hospital-University of Southern California). We report Hispanic B-cell ALL patients treated with blinatumomab outside of clinical trials Method: This is a retrospective review which included adults aged ≥18 years with newly diagnosed Philadelphia negative ALL (2016-2020) treated at the USC/Norris Comprehensive Cancer Centerand the Los Angeles County Hospital (LAC). Primary objectives was to investigate overall survival in Hispanic patients who were treated with blinatumomab. We stratified patient population into Philadelphia negative and Philadelphia-like B-cell ALL and assessed survival separately for blinatumomab administered for minimal residual positivity and for relapse disease. Most of Ph-Like patients were identified with CRLF2 rearrangement per institutional protocol. Results: 67 patients were B cell ALL, which included Ph-negative B cell ALL and Ph-like B cell ALL, 43 and 24 patients respectively. 50.7% were men and median age was 39 years. 38 patients were considered unfavorable risk due to cytogenetics risk factors and 44 patients received peg-asparaginase based induction. 30 (48%) of patients received blinatumomab, 15 in each ph-negative and ph-like group respectively. More patients received blinatumomab for relapsed disease as compared to MRD positive disease, 23 vs. 8 patients respectively. Mean overall survival for our patients was 68 months (SE=3.3). In ph-negative group, when blinatumomab was used for relapsed disease then 3 years survival probability was 0% compared to 91.5% for patients who did not receive blinatumomab (p-value=0.0001). On the other hand, for MRD positive disease, 3 years OS was 91.7% vs. 79.6% for patients who received blinatumomab compared to no blinatumomab (p-value=0.6). For ph-like group, when blinatumomab was used for relapsed disease then 3 years survival probability was 100% compared to 40% for patients who did not receive blinatumomab (p-value=0.2). For MRD positive disease, median OS was not reached for either group (p-value=0.6). Conclusion: Both relapse disease and MRD+ disease are considered poor prognostic indicator. Our study highlights that blinatumomab can decrease survival gap for MRD + disease and relapse disease for ph-like Hispanic patients and decrease survival gap in MRD + ph-negative Hispanic patients when compared to patients who did not need blinatumomab

139. Feasibility of comprehensive whole genome profiling in hematological malignancies

Whole genome sequencing (WGS) has been recently proposed as a replacement for routine karyotype and FISH analyses of myeloid and lymphoid neoplasms. WGS allows for the unbiased and comprehensive detection of multiple types of genomic aberrations, from single nucleotide to structural variants, which could streamline current genomic testing algorithms and clinical laboratory workflows for hematologic malignancies. In this study, we explored the feasibility of whole genome profiling of myeloid and lymphoid neoplasms by sequencing eleven AML, MPN, MDS, B-ALL, and T-ALL genomes with an average depth of sequencing coverage of ∼40X, and comparing genome analyses data to their available cytogenetic and molecular panel results. 81% of clinically relevant SNVs were detected by WGS, with false negatives being mostly variants at low frequencies (VAF <10%). Nine new variants were detected by WGS in an AML, B-ALL and T-ALL samples, eight of which were classified as VUS, and one (FLT3 p.Tyr572Cys) in B-ALL as likely pathogenic. 95% of clinically relevant copy-number variants (CNVs) were detected by WGS, with false negatives being mostly small (<150Kb) or mosaic CNVs present in <15% of the sample. The identification of the clinically relevant translocations in these samples (such as ETV6/RUNX1 and CRLF2 rearrangements) using WGS data is currently being investigated. This feasibility study demonstrated the potential of using WGS as the replacement of molecular panels and chromosome microarray in hematologic malignancies, particularly at the time of initial diagnosis.

P1421: BIMODAL TARGETING OF CYTOKINE RECEPTOR-LIKE FACTOR 2 (CRLF2) WITH JAK INHIBITION AND CHIMERIC ANTIGEN RECEPTOR T CELL IMMUNOTHERAPY IN DOWN SYNDROME ACUTE LYMPHOBLASTIC LEUKEMIA

Background:CRLF2 (cytokine receptor-like factor 2; encoding the thymic stromal lymphopoietin receptor [TSLPR]) rearrangements occur in 50% of children with Down Syndrome-associated B-acute lymphoblastic leukemia (DS-ALL). These genetic alterations induce constitutive JAK/STAT and other kinase signaling that may be targetable by the JAK1/2 inhibitor ruxolitinib. Patients with DS-ALL experience substantial toxicity with conventional chemotherapy and particularly with hematopoietic stem cell transplantation (HSCT). The Children’s Oncology Group AALL1521 clinical trial (NCT02723994) is investigating the potential superior efficacy of ruxolitinib addition to chemotherapy for pediatric patients with CRLF2-rearranged/JAK pathway-mutant BCR-ABL1-like ALL, but children with DS-ALL are not eligible to participate in this study Successful development of precision medicine approaches that can increase cure, reduce treatment toxicity, and avoid HSCT remain a priority for children with D-ALL. Aims: We hypothesized that dual CRLF2 targeting with ruxolitinib and cellular immunotherapy could improve anti-leukemia activity against CRLF2-rearranged (R) DS-ALL. Methods: We previously reported preclinical activity of TSLPR-redirected chimeric antigen T cells with 4-1BB/CD3z costimulatory domains [TSLPRCART] against CRLF2-overexpressing ALL cells (Qin Blood 2015). In the present studies, we investigated the effects of TSLPRCART +/- ruxolitinib co-treatment in vitro and in vivo against CRLF2-R human ALL cell lines and childhood DS-ALL patient-derived xenograft (PDX) models (n=4). Results:In vitro incubation of CD28/CD3 bead-activated normal human T cells or TSLPRCART with ruxolitinib significantly diminished IL-2 and IFN-ɣ cytokine production, although did not induce overt apoptosis of normal or engineered T cells. As expected, treatment of CRLF2-R DS-ALL PDX models with TSLPRCART potently inhibited in vivo leukemia proliferation and induced long-term ‘cure’ of animals. However, simultaneous co-treatment of engrafted mice with ruxolitinib and TSLPRCART led to markedly diminished in vivo T cell proliferation and persistence, blunted cytokine production, and facilitated eventual leukemia relapse (representative data in Figure 1). These deleterious effects could be abrogated by 14-day delayed sequencing of ruxolitinib after TSLPRCART activation and expansion and were further fully reversible after ruxolitinib withdrawal, resulting in renewed TSLPCART functionality over time and upon leukemia rechallenge. Image:Summary/Conclusion: TSLPRCART is a promising cellular immunotherapeutic approach for DS-ALL and for BCR-ABL1-like ALL given shared features of CRLF2 rearrangement and cell surface protein overexpression. In the present studies, we demonstrate that ruxolitinib co-administration in close proximity impaired in vivo TSLPRCART-induced DS-ALL cell killing but was also beneficial in protection against life-threatening hyperinflammation/cytokine release syndrome (CRS) in dual-treated animals. These results suggest potential for both anti-ALL efficacy and CRS dampening when JAK inhibition and TSLPRCART are properly time-sequenced and may have broader applicability for toxicity mitigation with other cellular immunotherapies. Studies of CAR T cell immunotherapy and other kinase inhibitors in additional CRLF2-rearranged and non-rearranged DS-ALL models are ongoing.

P367: LONG TERM OUTCOMES OF NEWLY DIAGNOSED CRLF2 REARRANGED B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

Background: CRLF2 rearranged B cell acute lymphoblastic leukemia (B-ALL), a subtype of Philadelphia (Ph) -like ALL, constitutes a high-risk subset of B-ALL with poor outcomes with chemotherapy. Targeted therapies such as blinatumomab (blina) or inotuzumab (ino) may improve treatment outcomes for these patients (pts). Aims: To study the outcomes of newly diagnosed CRLF2 rearranged B-ALL treated with hyper CVAD based regimens and the impact of targeted therapies like inotuzumab and blinatumomab Methods: We retrospectively analyzed pts with newly diagnosed B-ALL (diagnosed between 01/2001 and 12/2021) at our center who had documented CRLF2 overexpression. Initial therapy, including use of ino and blina in CR1 were noted. Outcomes measures included CR/CRi, MRD response, relapse free survival (RFS) and overall survival (OS). RFS was censored at allogeneic stem cell transplantation (ASCT). Results: A total of 76 pts with a median age of 38 years (yrs) (range, 18-80) were identified, of which 70% were males and 81% were of Hispanic ethnicity. All pts had overexpression of CRLF2 documented by flow cytometry or gene expression profile. A subset of pts (n=37) had a concomitant CRLF2 FISH performed with all confirming CRLF2 rearrangement. Baseline disease parameters, treatment and outcomes are detailed in Table 1. Sixty-five pts (85%) received Hyper-CVAD based induction therapy [HCVAD-based (n=51); mini-CVD-based (n=14)] and 11 (15%) received augmented BFM. We focus on the outcomes of pts treated with HCVAD/mini-HCVD (n=65); among these pts, 24/65 (37%) received blina in CR1 during consolidation at a median of 3.6 months after starting induction therapy, 22/65 (34%) received ino in CR1 (in C1 as part of mini-HCVD-ino, n=14; in C2 as part of HCVAD, n=8). A total of 14/65 (22%) received both ino and blina in CR1; 32/65 (49%) received ino and/or blina. The median follow-up was 39 months (mos). CR/CRi rate after C1 was 52/65 (80%) with 25/47 (53%) MRD evaluable pts and 25/65 (38%) overall achieving MRD-neg post C1. The median RFS and OS was 17.6 and 26.6 mos, respectively (Fig. 1). Among the 32 pts who received ino and/or blina, the median RFS and OS was 17.6 mos and 38.8 mos respectively. CR/CRp rate after C1 among pts who received mini-HCVD-ino in C1 was 100% (14/14) with 79% MRD-neg. On landmark analysis to the time to blina initiation, blina treated pts had similar RFS and trended for improved OS. A total of 19/65 (29%) had ASCT in CR1; all were MRD-neg prior to ASCT. Landmark analysis for OS based on the time of ASCT (6 mos) in CR1 favored ASCT in CR1 (47.2 vs. 17.6 mos, p=0.04). Table 1: - Disease and treatment parameters Parameters N (%) or median [range] (N=76) Age, yrs 38 [18-80] Gender, male 53 (70) Ethnicity Hispanic 62 (81) Baseline parameters WBC (x 109/L) 17 [1-602] Platelets (x 109/L) 39 [3-195] PB blasts (%) 72 [0-98] BM blasts (%) 89 [29-98] CNS positive 10 (80) Cytogenetics and molecular Diploid karyotype 28 (37) CRLF2 mutation (n=31) 4 (13) JAK2 mutation (n=67) 25 (37) JAK1 mutation (n=31) 9 (29) Frontline regimen Augmented BFM 11 (15) HCVAD 51 (67) Chemotherapy alone 33/51 (65) with ino alone 2/51 (4) with blina alone 10/51 (20) with ino + blina 6/51 (12) Mini-HCVD-ino 14 (18) with ino alone 6 (43) with ino + blina 8 (57) Image:Summary/Conclusion: Despite improvements in treatment options, CRLF2 overexpressed B-ALL continue to have inferior outcomes. Earlier initiation of targeted therapies might improve outcomes.

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.