Adult B-cell Precursor Acute Lymphoblastic Leukemia Research Articles

Mutually-Exclusive Pathways between IKZF1plus and RAS Mutations and TP53 double-Hit Alterations Lead Relapse in B-Other, Ph-like and Hyperdiploidy Genetic Groups in Adult B-Cell Acute Lymphoblastic Leukemia

Introduction: Adult B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a hematologic malignancy characterized by a high genetic heterogeneity and high relapse rate. Despite treatment advances have led to high cure rates in pediatric BCP-ALL (90%), improving survival in adults remains a challenge. Identification of genetic alterations driving relapse and both new prognostic and therapeutic biomarkers are important to improve survival in adults with BCP-ALL. Aim: To identify genetic alterations that lead to relapse in adult BCP-ALL patients (18-72 years) based on the tumor genetic landscape. Methods: Forty-five adult BCP-ALL patients (median age: 43 years; range 18-72 years) were studied at diagnosis and relapse by massive sequencing using a custom panel that allows detection of single nucleotide variants (SNVs) and insertions/deletions (INDELs) in 150 genes involved in BCP-ALL, in addition to copy number variations (CNVs), gene fusions and aneuploidy. Thirty-eight percent of patients belonged to the BCP-other ALL group, 16% were Philadelphia-positive (Ph+), 16% were classified as Philadelphia-like (Ph-like), 11% belonged to the high hyperdiploidy group, 8% had KMT2A rearrangements ( KMT2Ar), 4% TCF3::PBX1 fusion, 3% showed hypodiploidy, 2% low hyperdiploidy and 2% de novo MYC and BCL2 double-hit. Patients were treated according to the Spanish Adult-PETHEMA high-risk ALL protocols. Results: The most frequently deleted gene at diagnosis was IKZF1 (25/45, 56%). Among these cases, 72% (20/25) had IKZF1plus genetic profile, characterized by co-occurrence of IKZF1 deletions with at least one deletion in CDKN2A/B, PAX5 or PAR1, without ERG deletion. The IKZF1plus genetic profile was associated with specific genetic subgroups: Ph+ (5/7, 71%), Ph-like (5/7, 71%) and B-other (10/17, 59%). Moreover, it was significantly related with the presence of minimal/measurable residual disease (MRD) after induction (16/18, 89%) p < 0.001 and persistence at relapse in most patients (18/20, 90%; p < 0.001). Interestingly, among those 5 patients initially diagnosed with IKZF1 deletion but who did not meet the criteria for the IKZF1plus profile, 2 of them acquired the IKZF1plus profile at relapse. This suggests a possible cooperation of these genetic deletions contributing to therapy resistance and disease progression in these genetic subgroups. Eighty-six percent (6/7) of Ph+ patients who received imatinib treatment presented ABL1 mutations that were detected exclusively at relapse. Of these cases, 67% (4/6) were also associated with IKZF1plus at relapse (Figure 1A). Two mutually exclusive genetic patterns were identified at relapse in adult B-other and Ph-like patients. Thus, patients with IKZF1plus profile did not show RAS pathway mutations (59% and 57%, respectively), while patients with RAS pathway mutations lack in IKZF1plus profile (18% and 29%, respectively) (Figure 1B). Moreover age-related differences in the adult B-other ALL patient group were observed. The presence of mutations in RAS pathway was associated with younger age (<40 years) (3/10, 30% vs. 0/7, 0%), whereas older patients (≥40 years) predominated in the IKZF1plus profile (6/7, 86% vs. 3/10, 30%). TP53 alterations were detected at diagnosis and/or relapse in 11 patients (24%), most of which (9/11, 82%) had a heterozygous mutation plus a deletion or a homozygous mutation (double-hit). The high hyperdiploidy subgroup was enriched in TP53 mutations (4/5, 80%), most of which were already present at diagnosis (3/4, 75%). These findings indicate that genetic biomarkers usually linked to a favorable prognosis, such as high hyperdiploidy, can be associated with unfavorable genetic markers, leading to poorer outcomes in this group of patients (Figure 1C). Both the IKZF1plus profile and TP53 alterations were associated with poorer overall survival (OS) rates (P=0.034 and P=0.040, respectively) in adult BCP-ALL (Figure 2). Conclusion: Our results highlight the clinical importance of the TP53 alterations and the IKZF1plus profile as prognostic factors in adult BCP-ALL, suggesting they might help guiding more personalized therapeutic strategies to improve outcomes in this group of BCP-ALL patients. Grants: SACYL(GRS2386/A/21,GRS2385/A/21,GRS2527/A/22,GRS2506/A/22), JCyL SA118P20, FMM21/002 AP176752021, Pfizer 69383919.

Efficacy and Tolerance of Brexucabtagene Autoleucel in Adults with Relapsed/Refractory B-Cell Precursor Acute Lymphoblastic Leukemia : A Graall Study from the Descar-T Registry

Background : Brexucabtagene autoleucel (brexu-cel) was recently approved for adult patients with relapsed/refractory (R/R) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) based on the results of the ZUMA-3 phase 2 study. After a median follow-up of 39 months, brexu-cel showed a complete remission (CR)/CR with incomplete hematologic recovery (CRi) rate of 71% and a median overall survival of 26 months (Shah B, et al. ASCO 2023). In the present study, we aimed at investigating the efficacy and safety of brexu-cel in a real-life cohort of adult BCP-ALL patients included in the French early access program. Methods : In this Group for Research on Adult Acute Lymphoblastic leukemia (GRAALL) study, we performed an analysis of the DESCAR-T registry which collects real-life data of patients treated with CAR T-cells since July 2018 in France. All patients who underwent a leukapheresis with an intent to manufacture brexu-cel were included (N=80). Efficacy and safety analyses were performed in the 64/66 infused patients with available response assessment after infusion. The data cut-off was June 2023. The present study reports on best overall response, overall survival (OS), relapse-free survival (RFS), as well as immune effector cell-associated neurotoxicity syndrome (ICANS), and cytokine release syndrome (CRS) incidence and grading (according to ASTCT Consensus). Results : Between May 2019 and February 2023, 80 adults with R/R BCP-ALL from 22 French centers had a leukapheresis for brexu-cel. Among them, 14 patients (18%) were not infused because of death (8 pts), progression (1 pt), manufacturing failure (2 pts), or other causes (3 pts). Among the 64 infused patients with available response assessment, median age was 43.5 yrs (range, 22-69). A Philadelphia(Ph)-chromosome was found in 20/64 (31%). Patients had received a median of 3 (range, 1-6) prior lines of treatment including blinatumomab, inotuzumab ozogamicin and allogeneic hematopoietic stem cell transplantation (allo-HSCT) in 67%, 19% and 70% of patients respectively. Twelve patients (19%) had an active CNS disease (CNS2/3) prior to apheresis and 18/40 (45%) had bone marrow blasts ≥ 25% before lymphodepletion (LD). ECOG before infusion was ≥2 in 6/64 (9%) patients. All patients received fludarabine and cyclophosphamide as LD. Median time from apheresis to infusion was 40 days (range, 27-99). CRS and ICANS occurred in 48/64 (77%, grade 3+ in 6%) and 28/64 (45%, grade 3+ in 8%) of patients respectively and were reversible. A CR was achieved in 49/64 patients (77%) of whom 23/25 (92%) were MRD-negative. An older age, a prior allo-HSCT, and the occurrence of CRS were associated with significantly higher CR rates. The median follow-up was 13 months. Three patients were bridged to an allo-HSCT in continuous complete remission (second allo-HSCT for 2 of them). The median RFS and OS were 12.9 months and 15.6 months respectively (Figure). In patients who achieved a CR, median OS was 25.8 months (Figure). Patients with CNS involvement before leukapheresis had a significantly shorter RFS (HR 5.4, 95%CI 1.9-15.8, p=0.002) and OS (HR 3.3, 95%CI 1.4-8.2, p=0.008). Number of prior treatment lines, prior inotuzumab, prior blinatumomab, or pre-LD BM blast% were not associated with outcome. A better OS was observed in patients with prior allo-HSCT (HR .4, 95%CI 0.2-0.9, p=0.04). Conclusion : This multicenter real-life study confirms the efficacy and acceptable safety profile of brexu-cel in adult patients with R/R BCP-ALL. It also suggests that patients with CNS involvement, that were not eligible to ZUMA-3 study, have a limited benefit of brexu-cel when compared to other patients.

In Vitro Efficacy of Anti-HER2 CAR T-Cells in HER2 + BCP-ALL

Introduction: Around 30% of adults and 15% of children with B cell precursor acute lymphoblastic leukemia (BCP-ALL) relapse after a first line of treatment. The prognosis of these patients remains poor. Recent advances have involved targeted therapies with monoclonal antibodies and chimeric antigen receptor (CAR) T-lymphocytes (anti-CD19 CAR T-cells), which have produced very encouraging results in the high risk BCP-ALL setting(Maude et al. 2018). Nevertheless, half of these patients relapse after such therapies. In a third of cases, the disease no longer expresses the antigen previously targeted. New targets are therefore needed. HER2 is expressed in above one third of adult BCP-ALL (Chevallier et al. 2004) and 10% of childhood BCP-ALL (Camuset et al. 2021). HER2 expression is associated with chemoresistance and poor prognosis (Chevallier et al. 2004) (Camuset et al. 2021). The anti-HER2 antibody trastuzumab was prospectively tested in adult patients with HER2+ BCP-ALL, but yielded disappointing results (Chevallier et al. 2012). Anti-HER2 CAR T-cells could be an interesting alternative. Indeed, they have shown encouraging preclinical efficacy in solid tumors expressing HER2/neu (Budi et al. 2022). The present work was designed to evaluate, in vitro, the sensitivity of HER2+ BCP-ALL to anti-HER2 CAR engineered Epstein Barr virus (EBV) specific T-cells. Methods: DMSO-frozen cell-samples from 6 HER2+ BCP-ALL patients were available for this study. Patient characteristics are shown in Table 1. Blast cells from patient samples were preferred to cell lines in order to be more representative of BCP-ALL biology. level of HER2 expression on BCP-ALL samples was determined by flow cytometry and compared to the HER2 expressing breast-cancer cell line BT474. Anti-EBV cytotoxic T lymphocytes (CTLs), armed with a trastuzumab-based scFv-CH2-CH3-IgG2A-FcεRIγ chimeric receptor as previously described (Clémenceau et al. 2015) were used for long-term cytoxicity assays. For 4 samples, non-transduced anti-EBV CTLs were used as controls. Cytotoxic activity was assessed at a 3-to-1 effector-to-target ratio, in a flow cytometry assay. Cells were harvested at H0, H4 and H24 and stained with a viability stain (VS780). Further staining with anti-CD22 or anti-CD24 (depending on the BCP-ALL immunophenotype) and anti-CD3 antibodies allowed to characterize blast cells and CAR T-cells respectively. The study was approved by the local review board and two alive patients provided informed consent. Results: The level of HER2 expression by the 6 HER2+ BCP-ALL samples was variable with a median relative fluorescence intensity ratio (RFI) of 9 (range 1.8 to 16). These levels of expression were lower than those of the BT474 cell line (RFI= 79). No BCP-ALL lysis was observed when cells were incubated with trastuzumab alone (not shown). When co-cultured with non-transduced anti-EBV CTLs (CAR negative) (n=4), a decrease of residual viable BCP-ALL cells was observed at variable levels (Figure 1A). The lysis observed at 24h was possibly due to allogeneic recognition since anti-EBV CTLs were not autologous to the leukemias tested. Conclusion: This study first reveals that, although CTLs with TCRs restricted to EBV antigens and therefore intrinsically low allo-specificity were used, CAR-independent lysis was observed in 24 hours co-culture with BCP-ALL cells. These results underline the need for cautious interpretation of cytotoxicity test results in an allogeneic setting. Secondly, these preliminary data suggest that an anti-HER2 CAR T-cell approach could be promising for BCP-ALL even with weak antigen expression. Anti-EBV CTLs have already been used as a platform for CAR-engineered T-cells (Rossig et al. 2017). Should the cytotoxic activity of allogeneic EBV-CTL be proven to be specifically directed against BCP-ALL, the synergy between TCR and anti-HER2-CAR might be beneficial, and adoptive transfer of anti-HER2 CAR EBV CTLs could be a promising therapeutic option for patients with HER2+ BCP-ALL. * This research is funded by the non-profit organization Etoile de Martin/SFCE.

Role of Microenvironment in Influencing the Activity of CD19 CAR T Cells in B-Cell Precursor Acute Lymphoblastic Leukemia (B-ALL)

Background: As a therapy based on immune cells, CAR T cells treatment elicits an acute response involving both innate and adaptive immunity which is followed by a process of resolution. A major interest is to identify, within the tumor microenvironment (TME), factors that influence the activity and potency of anti-CD19 CAR T cells. The B-ALL TME provides factors that protect leukemia and, conversely, suppress effector T cells. However, studies on the interaction between the bone marrow (BM) TME and CAR T cells are still limited. We hypothesized that the immunological niche of the BM reacts to CAR T-cell-mediated inflammation by modulating the immune response through the activation of inhibitory pathways and molecules. Methods: We conducted an academic phase I/II study (NCT03389035) with donor-derived anti-CD19 CAR T cells generated with Sleeping Beauty transposon (CARCIK-CD19) in B-ALL patients relapsed after alloHSCT. Hematology laboratory data, CAR T,and CD3+ cell kinetics were analyzed in 43 pediatric and adult B-ALL patients who underwent commercial and investigational anti-CD19 CAR T cell treatment. The scRNA-seq libraries of BM samples were generated using Chromium Single Cell 5′ Reagent Kit of the 10x Genomics (v3.1). Validation was performed in 20 pre-/post- CAR-T matched BM samples by a 30-color flow cytometry panel. Results: Following the peak of expansion, CAR T-cell counts decreased in the third and four-week post-infusion. A similar dynamic of expansion and contraction was observed in CD3+ T cells, total white blood cells (WBC), and monocytes, indicating that a widespread resolution of CAR T-driven inflammation occurs during the first month after infusion. Interestingly, the intensity of the response seems to differ depending on the cell product used. To elucidate the mediators involved in the resolution of CAR T-cell-mediated inflammation, the single-cell transcriptome of patients' BM cells at early time points post-CAR T-cell infusion (1-2 months) was compared to pre-treatment samples at the moment of disease relapse and the infusion products. Eighteen sequencing libraries were analyzed, 6 libraries per patient, for a total of 71407 high-quality cells at an average of 65000 reads per cell. Unbiased clustering of BM cells and infusion products was performed by UMAP embedding. Notably, extensive integration of the dataset coming from the individual patients was observed.The more representative clusters were classified into infusion product, CD4 and CD8 endogenous population, B cells, myeloid cells, pDC, NK, and NK-T cells. We observed profound changes in the composition of BM after CAR T cells infusion compared to pre-treatment samples. Complete disappearance of the B cells associated cluster was observed after treatment. CAR T-cell treatment generated a remodeling of the BM microenvironment, with an increase in the myeloid cells (specifically monocytes), NK, NK-T, and exhausted CD8+ T cell populations. After CAR T-cell infusion, myeloid cells displayed a higher resemblance to myeloid-derived suppressor cells (MDSCs). GeneSet Enrichment Analysis (GSEA) showed significant enrichment in pathways associated with immunosuppression both in the myeloid compartment and in endogenous T cells and in CAR T cells. Of note, the genes involved are also strictly correlated to the generation of terminally exhausted T cells and the emergence of MDSCs. Spectral flow cytometry-based experiments validated our observation of an increase of monocytes, MDSC-like cells, NK, NK-T, and CD8 terminal cells. Modeling intercellular communication using NicheNet suggested the induction of immunosuppressive genes in myeloid cells by both CAR T cells and endogenous T cells. Using the same tool with all niches as the sender and T cells as the receiver, we observed that myeloid cells induced significant signaling in both CAR T cells and endogenous T cells. Conclusions: Through sc-RNAseq and spectral flow cytometry, we have characterized changes in the BM TME after CAR T cell therapy. Specifically, CAR T cells-mediated myeloid activation is associated with pathways of immune dysregulation that may dampen CAR T cell expansion and antagonize the effects of the therapy. Furthermore, these data support the hypothesis that transcriptomic interrogation can infer pathways relevant to the communication between CAR T cells and the TME.

Genetic alterations and MRD refine risk assessment for KMT2A-rearranged B-cell precursor ALL in adults: a GRAALL study

AbstractKMT2A-rearranged (KMT2A-r) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is widely recognized as a high-risk leukemia in both children and adults. However, there is a paucity of data on adults treated in recent protocols, and the optimal treatment strategy for these patients is still a matter of debate. In this study, we set out to refine the prognosis of adult KMT2A-r BCP-ALL treated with modern chemotherapy regimen and investigate the prognostic impact of comutations and minimal residual disease (MRD). Of 1091 adult patients with Philadelphia-negative BCP-ALL enrolled in 3 consecutive trials from the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL), 141 (12.9%) had KMT2A-r, with 5-year cumulative incidence of relapse (CIR) and overall survival (OS) rates of 40.7% and 53.3%, respectively. Molecular profiling highlighted a low mutational burden in this subtype, reminiscent of infant BCP-ALL. However, the presence of TP53 and/or IKZF1 alterations defined a subset of patients with significantly poorer CIR (69.3% vs 36.2%; P = .001) and OS (28.1% vs 60.7%; P = .006) rates. Next, we analyzed the prognostic implication of MRD measured after induction and first consolidation, using both immunoglobulin (IG) or T-cell receptor (TR) gene rearrangements and KMT2A genomic fusion as markers. In approximately one-third of patients, IG/TR rearrangements were absent or displayed clonal evolution during the disease course, compromising MRD monitoring. In contrast, KMT2A-based MRD was highly reliable and strongly associated with outcome, with early good responders having an excellent outcome (3-year CIR, 7.1%; OS, 92.9%). Altogether, our study reveals striking heterogeneity in outcomes within adults with KMT2A-r BCP-ALL and provides new biomarkers to guide risk-based therapeutic stratification.

Clinical characteristics and outcomes of childhood B-ALL with ZNF384 and MEF2D rearrangements

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has many subtypes with diverse clinical and biological features and outcomes. Next generation sequencing has revealed several novel subtypes, including the ZNF384 and MEF2D rearrangements. The clinical characteristics and outcomes of the largest series of BCP-ALL cases with ZNF384 and MEF2D rearrangements in an international collaborative study are described here. Patients with ZNF384 rearrangements appear to express various leukemic phenotypes, including BCP-ALL (with or without abnormal expression of myeloid markers) and B/myeloid mixed phenotype acute leukemia. We provide strong evidence that among BCP-ALL patients with a ZNF384 fusion, the partner gene is associated with demographic features and influences the outcome; particularly the EP300-ZNF384 fusion is associated with a low risk of relapse. MEF2D rearrangements have been primarily described in children and young adults with BCP-ALL. Previous research has suggested that patients with MEF2D-BCL9 fusion have a high risk of relapse. Despite having the MEF2D-HNRNPUL1 fusion gene, the prognosis was favorable. Improved diagnostic genomic testing will enable future prospective studies to clarify the clinical significance of the ZNF384 and MEF2D rearrangements in childhood and young adult BCP-ALL.

The prognostic significance of CRLF2 expression at diagnosis in adult Ph-negative B-cell precursor acute lymphoblastic leukemia

The prognostic significance of cytokine receptor like factor 2 (CRLF2) expression at diagnosis in adult B-cell precursor acute lymphoblastic leukemia (BCP-ALL) needs to be clarified. A total of 357 bone marrow samples collected from consecutive adult cases with Ph-negative BCP-ALL at diagnosis retrospectively detected CRLF2 transcript levels by real-time quantitative PCR. Twenty percent was selected as the cutoff value for CRLF2 to divide patients into CRLF2_H and CRLF2_L groups. CRLF2_H was associated with higher WBC count, P2RY8–CRLF2 fusion and IKZF1 deletions (IKZF1del). In both the whole cohort and B-other patients, CRLF2_H independently predicted lower CR rates after induction. Furthermore, CRLF2_H/IKZF1del(+) patients had significantly lower CR, RFS, and OS rates and tended to have lower RFS and OS rates than others in the whole cohort and B-other patients, respectively. Therefore, coexistence of CRLF2_H and IKZF1del at diagnosis predicts poor response and outcome in adult Ph-negative BCP-ALL.

A Multi-Cohort Gene Expression Classifier (ALLCatchR) Identifies B-Precursor ALL Subtypes and Their Developmental Trajectories across Age Groups

The molecular landscape of B cell precursor acute lymphoblastic leukemia (BCP-ALL) describes >20 disease subtypes defined by initiating genomic alterations and corresponding gene expression signatures. Some subtypes represent phenocopies with shared gene expression signatures but distinct or absent genomic drivers. The majority of BCP-ALL subtypes have been acknowledged as diagnostic entities by the currently revised classifications of hematologic malignancies (WHO-HAEM5 / International Consensus Classification). Thus, unified diagnostic approaches are needed to systematically identify subtypes in individual BCP-ALL cases in clinical routine. Gene expression signatures represent the shared functional equivalent of heterogeneous genomic driver alterations. Therefore, we established a gene expression-based classifier to identify BCP-ALL subtypes and related clinical and biological phenotypes. We analyzed a total of n=3477 BCP-ALL gene expression profiles obtained from transcriptome sequencing of adult (n=1253) and pediatric patients (n=2224) including publicly available (n=2074; Gu Z et al. Nat. Genet. 2019; Schmidt B. et al. Blood Adv. 2022) and own data sets (n=1403) from adult and pediatric European study groups (GMALL, AIEOP-BFM, CELL). Raw read counts from 6 cohorts representing heterogeneous library preps (i.e., stranded/unstranded; mRNA/total RNA), sequencing platforms/depths, and gene count quantifications were included. All samples had been previously allocated to molecular BCP-ALL subtypes based on integration of independent genomic profiling, gene fusion calling and gene expression. For classification, data with defined subgroup allocation were randomly split into training (n=1831; 70%) and testing sets (n=785; 30%) and two hold-out studies (n=305). To define gene expression signatures of 21 BCP-ALL subtypes, we used training set samples and extracted specific gene sets employing different feature selection algorithms as well as ranking of genes in individual subtypes. Based on these definitions we established a BCP-ALL subtype classifier (ALLCatchR), which integrates machine learning models (support vector machines) and nearest-neighbors associations of single sample gene set enrichment analyses (Fig. A). ALLCatchR performed molecular subtype allocation for the testing set at high sensitivity (0.934±0.165) and specificity (0.997±0.005) on average across subtypes. A similar performance was achieved in the two hold-out cohorts despite completely independent data structures which have not been shown to the classifier (sensitivity: 0.946±0.097 / specificity: 0.996±0.007). Overall accuracies of 0.952 and 0.92 for testing and hold-out-data sets indicate that ALLCatchR reliably identifies molecular BCP-ALL subtypes in mixed adult / pediatric cohorts based on gene expression profiles alone (Fig. A, below). Among n=44/1090 (4%) samples of the combined hold-out/testing set with ambiguous subtype allocation (deemed 'unclassified'), ALLCatchR provides candidate subtype allocations (sensitivity: 0.671±0.393 / specificity: 0.978±0.036) for confirmation based on genomic driver alterations. In addition, we modified our classifier to predict baseline variables such as patient's sex (accuracy: 0.991), immunophenotype (accuracy: 0.935) or blast count (R=0.633). To define the corresponding B cell differentiation stage of origin, we compared BCP-ALL subtypes to differentiation stage-specific gene sets extracted from 7 FACS-sorted lymphopoietic subsets from healthy bone marrow donors (n=4). We observed defined BCP-ALL subtype-specific enrichment patterns which can be grouped along 4 major differentiation stages (Pro-B to Pre-B-II-Large, Fig. 1B) with a high degree of similarity in independent adult and pediatric cohorts. Thus, B cell developmental trajectories underlying BCP-ALL subtypes are dissected at high resolution by gene expression profiling and differentiation stages may favor the selection of specific leukemogenic drivers. ALLCatchR is a gene expression classifier for 21 established BCP-ALL molecular subtypes, which performs at independently validated cohorts with an accuracy of >90% and allows imputation of clinical baseline variables as well as underlying B cell developmental trajectories. It provides a reliable basis for systematic diagnostic approaches in pediatric and adult BCP-ALL patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical features and outcomes of fusion gene defined adult Ph-negative B-cell precursor acute lymphoblastic leukemia patients: A single institutional report.

More clinical studies are needed to clarify the risk stratification by the integration of all fusion genes in adult B-cell precursor acute lymphoblastic leukemia (BCP-ALL). A total of 320 consecutive adult Ph-negative BCP-ALL patients who had been tested classical fusions (KMT2A rearrangement and TCF3-PBX1) at diagnosis were further retrospectively screened novel fusion genes (Ph-like, ZNF384 and MEF2D fusions) by multiplex real-time quantitative PCR (RQ-PCR). Classical fusions were identified in 12.5% of patients, while 4.4%, 17.2% and 3.8% of patients were identified Ph-like, ZNF384 and MEF2D fusions, respectively. 1-course CR rate, relapse-free survival (RFS) and overall survival (OS) rates tended to show or showed statistically significant differences among fusion-defined subgroups (P = 0.084, 0.001 and 0.0093, respectively). Based on individual outcomes, patients with KMT2A rearrangement, TCF3-PBX1, Ph-like, and MEF2D fusions were classified into fusion-defined high-risk group (n = 66, 20.6%). High-risk group had significantly lower 3-year RFS and 3-year OS rates than standard-risk group (P 0.001 and = 0.0022), and was an independent adverse prognostic factor for RFS in the entire cohort (P 0.001). In conclusion, the spectrum of fusion genes in the current Chinese cohort was distinct from that in reports from western countries. Detection of fusion genes improved risk stratification in adult Ph-negative BCP-ALL patients.

Bone Marrow Stromal Cell Regeneration Profile in Treated B-Cell Precursor Acute Lymphoblastic Leukemia Patients: Association with MRD Status and Patient Outcome.

Simple SummaryFor the last 20 years, measurable residual disease (MRD) has proven to be a strong prognostic factor in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the effects of therapy on the bone marrow (BM) microenvironment and their potential relationship with MRD and patient outcome still remain to be evaluated. Here, we show that mesenchymal stem cells (MSC) and endothelial cells (EC) are constantly present at relatively low frequencies in normal BM and in most follow-up BM samples from treated BCP-ALL patients. Of note, their levels are independent of the MRD status. From the prognostic point of view, an increased percentage of EC among stromal cells (EC plus MSC) at day +78 of therapy was associated with shorter disease free survival (DFS), independently of the MRD status both in childhood and in adult BCP-ALL. Thus, an abnormally high EC/MSC distribution at day +78 of therapy emerges as an adverse prognostic factor, independent of MRD in BCP-ALL.For the last two decades, measurable residual disease (MRD) has become one of the most powerful independent prognostic factors in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the effect of therapy on the bone marrow (BM) microenvironment and its potential relationship with the MRD status and disease free survival (DFS) still remain to be investigated. Here we analyzed the distribution of mesenchymal stem cells (MSC) and endothelial cells (EC) in the BM of treated BCP-ALL patients, and its relationship with the BM MRD status and patient outcome. For this purpose, the BM MRD status and EC/MSC regeneration profile were analyzed by multiparameter flow cytometry (MFC) in 16 control BM (10 children; 6 adults) and 1204 BM samples from 347 children and 100 adult BCP-ALL patients studied at diagnosis (129 children; 100 adults) and follow-up (824 childhood samples; 151 adult samples). Patients were grouped into a discovery cohort (116 pediatric BCP-ALL patients; 338 samples) and two validation cohorts (74 pediatric BCP-ALL, 211 samples; and 74 adult BCP-ALL patients; 134 samples). Stromal cells (i.e., EC and MSC) were detected at relatively low frequencies in all control BM (16/16; 100%) and in most BCP-ALL follow-up samples (874/975; 90%), while they were undetected in BCP-ALL BM at diagnosis. In control BM samples, the overall percentage of EC plus MSC was higher in children than adults (p = 0.011), but with a similar EC/MSC ratio in both groups. According to the MRD status similar frequencies of both types of BM stromal cells were detected in BCP-ALL BM studied at different time points during the follow-up. Univariate analysis (including all relevant prognostic factors together with the percentage of stromal cells) performed in the discovery cohort was used to select covariates for a multivariate Cox regression model for predicting patient DFS. Of note, an increased percentage of EC (>32%) within the BCP-ALL BM stromal cell compartment at day +78 of therapy emerged as an independent unfavorable prognostic factor for DFS in childhood BCP-ALL in the discovery cohort—hazard ratio (95% confidence interval) of 2.50 (1–9.66); p = 0.05—together with the BM MRD status (p = 0.031). Further investigation of the predictive value of the combination of these two variables (%EC within stromal cells and MRD status at day +78) allowed classification of BCP-ALL into three risk groups with median DFS of: 3.9, 3.1 and 1.1 years, respectively (p = 0.001). These results were confirmed in two validation cohorts of childhood BCP-ALL (n = 74) (p = 0.001) and adult BCP-ALL (n = 40) (p = 0.004) treated at different centers. In summary, our findings suggest that an imbalanced EC/MSC ratio in BM at day +78 of therapy is associated with a shorter DFS of BCP-ALL patients, independently of their MRD status. Further prospective studies are needed to better understand the pathogenic mechanisms involved.

Immunophenotypic characteristics of ZNF384 rearrangement compared with BCR-ABL1, KMT2A rearrangement, and other adult B-cell precursor acute lymphoblastic leukemia.

ZNF384 rearrangement has been recently identified as a new subtype of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, comprehensive studies clarifying immunophenotypic features and discriminating them from non-ZNF384 in adult BCP-ALL remain scarce to date. Flow cytometric assessments were retrospectively performed in 43 patients with ZNF384 rearrangement, 45 with BCR-ABL1, 29 with KMT2A rearrangement and 44 with other BCP-ALL in the analysis cohort. CD33- and CD13-positive frequencies were significantly higher in patients with ZNF384 rearrangement than in those with non-ZNF384; however, no significant difference was observed in CD10- and CD123-positive frequencies. Analysis of antigen-positive cell proportion and median fluorescence intensity (MFI) further indicated that patients with ZNF384 rearrangement had significantly lower CD10 and higher CD33, CD13, and CD123 proportion and MFI. However, compared with KMT2A rearrangement, the CD10 expression in patients with ZNF384 rearrangement was higher, with the median percentage and MFI of 36.16 (3.63-94.79)% versus 4.53 (0.03-21.00)%, and 4.50 (0.86-32.26) versus 2.06 (0.87-4.04), respectively (p < 0.0001). Furthermore, compared with BCR-ABL1 and other BCP-ALL, ZNF384 rearrangement had significantly higher CD33 and CD13 proportion and MFI (p < 0.0001 and p < 0.05, respectively). In addition, higher CD123 proportion and MFI in ZNF384 rearrangement than those in the other three groups were reported for the first time (p < 0.01). A flow cytometry scoring system, including CD10%, CD33MFI, CD13%, and CD123MFI, was proposed and verified to predict ZNF384 rearrangement with high sensitivity and specificity, that is, 76.74% and 91.53% in the analysis and 87.50% and 91.30% in the validation cohort. The multiparameter immunophenotypic scoring system could suggest ZNF384 rearrangement.

Characterization of Immunophenotypic Aberrancies with Respect to Common Fusion Transcripts in B-Cell Precursor Acute Lymphoblastic Leukemia: A Report of 986 Indian Patients

Objective:Based on the immunophenotype, acute lymphoblastic leukemia (ALL) can be categorized into B-cell or T-cell lineages. B-cell precursor ALL (BCP-ALL) cases show various genetic/molecular abnormalities, and varying frequencies of chimeric fusion transcripts in BCP-ALL cases are reported from different parts of the world. We studied the immunophenotypic aberrancy profiles of a large number of BCP-ALL cases with respect to various common chimeric fusion transcripts.Materials and Methods:Flow cytometric immunophenotyping and multiplex reverse-transcription polymerase chain reaction assays were performed for 986 BCP-ALL cases.Results:Among 986 BCP-ALL cases, the incidence of various fusion transcripts was 38.36% in adult cases and 20.68% in pediatric cases. Adult BCP-ALL patients with t(9;22)(BCR-ABL1) fusion transcripts and expression of aberrant myeloid markers were significantly older at presentation (p=0.0218) with male preponderance (p=0.0246) compared to those without aberrant myeloid expression. In pediatric patients with the t(12;21)(ETV6-RUNX1) chimeric fusion transcript, aberrant expression of CD13 was observed in 39.13%, CD33 in 36.95%, and CD117 in 8.69% of patients, respectively. Pediatric BCP-ALL patients with the ETV6-RUNX1 fusion transcript and expression of aberrant myeloid markers were not significantly different compared to those without with respect to demographic and clinical/hematological characteristics (p=0.5955). Aberrant myeloid markers were rarely or never expressed in pediatric and adult BCP-ALL patients with the t(4;11)(KTM2A-AF4) and t(1;19)(TCF3-PBX1) fusion transcripts.Conclusion:Aberrant myeloid markers were frequently expressed among BCP-ALL patients with the t(9;22)(BCR-ABL1) and t(12;21)(ETV6-RUNX1) fusion transcripts. However, BCP-ALL patients with the t(4;11)(KTM2A-AF4) and t(1;19)(TCF3-PBX1) fusion transcripts rarely or never expressed aberrant myeloid markers. Aberrant myeloid CD markers can be used in predicting chimeric fusion transcripts at baseline so as to plan appropriate tyrosine kinase inhibitor therapy in cases of BCP-ALL with specific chimeric fusion transcripts. This study has delineated the relationship of chimeric fusion transcripts with the aberrant expression of myeloid markers in a large cohort of BCP-ALL cases.

Cryptic TCF3 fusions in childhood leukemia: Detection by RNA sequencing.

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in childhood and adolescence. In more than 60% of cases of this heterogeneous disease, a genetic marker is identified via cytogenetic or molecular analyses. TCF3 gene fusions occur in 5%-11% of ALL patients. In < 1%, the TCF3 alteration in ALL leads to a TCF3-HLF fusion gene. Even though this is a very rare event, the detection of a TCF3-HLF fusion gene is associated with a very poor prognosis with incurable relapses in almost all patients. The frequent TCF3-PBX1 fusion gene, which is detectable in 5%-10% of childhood B-cell precursor ALLs and ~3.8% of adult B-cell precursor ALLs, is associated with a rather good prognosis, that is, an observed event-free 5-year survival of approximately 85%. Thus, the distinction of the different partner genes fused to TCF3 is essential for risk assessment. To verify RNA sequencing as a tool for detection of known and unknown fusion genes, we screened 200 cases of pediatric B-cell precursor ALL with "targeted" RNA sequencing in a pilot project in comparison to classical cytogenetic analyses (chromosome R-banding analysis), fluorescence in situ hybridization, and PCR. We observed a TCF3 fusion gene in 6.5% (13/200) of the patients. Ten (5%) patients displayed a TCF3-PBX1 fusion gene, two (1%) patients a TCF3-FLI1 fusion gene, and one (0.5%) patient a TCF3-HLF fusion gene. For the TCF3 fusions, we obtained discrepant results with the different methods, which are described in the article. Taken together, translocations leading to TCF3 fusion genes might appear cryptic and may remain undetected by a single method.

High PDGFRA Expression Does Not Serve As Effective Therapeutic Target in ERG-Deleted B-Cell Precursor Acute Lymphoblastic Leukemia

Background and aimsSeveral oncogenic aberrations of receptor tyrosine kinases occur in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here, we describe a subgroup of pediatric patients lacking common chromosomal aberrations but with high gene expression of platelet-derived growth factor receptor alpha (PDGFRA). Oncogenic fusions involving the tyrosine kinase domain of PDGFRA are recurrent in eosinophilic leukemia and are responsive to kinase inhibitors (Cools et al. N Engl J Med 2003). One case with a FIP1L1-PDGFRA fusion was reported in adult BCP-ALL (Roberts et al. J Clin Oncol 2016). Therefore, we tested for genomic aberrations underlying the overexpression of PDGFRA and evaluated the inhibition of PDGFRα as treatment option in pediatric BCP-ALL.MethodsExpression profiling was performed using Affymetrix U133 plus 2.0 arrays. BCR-ABL1 -like cases were identified using the 110 probe set expression signature (Den Boer et al. Lancet Oncol 2009). High PDGFRA was defined as log2 expression of probe set 203131_at >5.9. FISH was performed using Cytocell FIP1L1/CHIC2/PDGFRA probes. Targeted locus amplification (de Vree et al. Nat Biotechnol 2014) was performed using custom-designed primers. Cells were treated for 10 minutes with 10 ng/mL PDGF-BB with or without 10 µM imatinib or 1 µM CP673451 and analyzed by western blot. Cell viability after 4 day exposure to imatinib (0.2µM - 1.6µM - 12.5µM) or CP673451 (0.02µM - 0.16µM - 1.25µM) was assessed by flow cytometry.ResultsGene expression analysis of 654 pediatric ALL cases identified high expression of PDGFRA in 26 of 574 BCP-ALL cases (4.5%). High PDGFRA expression was exclusively found in cases belonging to the B-other (n=22) or BCR-ABL1 -like (n=4) subtypes, and not present in ETV6-RUNX1, high hyperdiploid, TCF3-PBX1, BCR-ABL1 and KMT2A -rearranged BCP-ALL subtypes or T-ALL. PDGFRA expression was confirmed by RT-qPCR, and also found in the BCP-ALL cell line Nalm6. Using RT-PCR we did not detect any of the six reported PDGFRA fusions, which involve FIP1L1, STRN, BCR, CDK5RAP2, ETV6, and KIF5B, nor did we detect novel structural aberrations affecting PDGFRA by FISH. We studied the PDGFRA locus at high resolution by targeted locus amplification. Following DNA cross-linking, digestion, and proximity-based re-ligation, targeted amplification was performed from viewpoints in intron 2 and exon 15 of PDGFRA. In the eosinophilic cell line EOL1 we confirmed an interstitial deletion on chromosome 4 which rearranges FIP1L1 intron 12 to PDGFRA exon 12. The results obtained from Nalm6 and primary BCP-ALL samples with high PDGFRA expression did not indicate genomic aberrations of PDGFRA.ERG deletions were present in 10 of 16 PDGFRA -high cases (63%), compared with 0% in PDGFRA -low BCP-ALL cases (n=71; p<0.001). Vice versa, all ERG-deleted cases expressed high levels of PDGFRA . Recently, a novel BCP-ALL subtype was described with rearrangements of the transcription factor DUX4 and deletion of ERG (Lilljebjörn et al. Nat Commun 2016; Yasuda et al. Nat Genet 2016; Zhang et al. Nat Genet 2016). Furthermore, the high PDGFRA-expressing cell line Nalm6 carries a DUX4 rearrangement (Yasuda et al. Nat Genet 2016) and PDGFRA is highly expressed in DUX4 BCP-ALL and its transcription start site directly bound by DUX4 (Zhang et al. Nat Genet 2016). Therefore, we regard it as highly likely that the PDGFRA-high cases represent this novel DUX4- rearranged subtype.Because transcription factors are difficult to target with small molecule inhibitors, PDGFRα inhibition could represent a treatment option. Exposure to the PDGFR inhibitors imatinib and CP673451 reduced phosphorylation of PDGFRα in Nalm6 and primary BCP-ALL cells. However, ex vivo drug exposure revealed no cytotoxic effect of the PDGFR inhibitors on PDGFRA-high BCP-ALL cells, either in the presence or absence of the ligand PDGF-BB. Interestingly, we found a marked ex vivo sensitivity towards prednisolone in PDGFRA-high cases, in line with the good prognosis reported for ERG -deleted/DUX4-rearranged BCP-ALL.ConclusionsWe found high PDGFRA expression, in the absence of genomic aberrations of the PDGFRA locus, in a subgroup of BCP-ALL patients with frequent ERG deletions. PDGFR inhibition caused abrogation of PDGFRα phosphorylation but was not cytotoxic in PDGFRA-high expressing BCP-ALL cells. Therefore, high PDGFRA expression does not serve as effective therapeutic target in ERG -deleted BCP-ALL. DisclosuresNo relevant conflicts of interest to declare.

The Prognostic Significance of ZNF384 Fusions in Adult Ph-Negative B-Cell Precursor Acute Lymphoblastic Leukemia: A Comprehensive Cohort Study From a Single Chinese Center.

Novel recurrent fusion gene types such as zinc finger protein 384 (ZNF384) fusions have been identified in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with the application of next-generation sequencing technologies. However, the comprehensive large-scale clinical cohort study for clarifying their prognostic significance remains scarce to date. A total of 242 consecutive adult Ph-negative BCP-ALL patients treated in our institute were retrospectively screened ZNF384 fusions at diagnosis by multiplex real time quantitative PCR. ZNF384 fusions were identified in 47 patients (19.4%) and all belonged to B-other ALL (having no high hyperdiploid karyotype, BCR-ABL1, TCF3-PBX1, ETV6-RUNX1, or MLL rearrangement). In the whole cohort, patients with ZNF384 fusions had significantly higher 3-year relapse-free-survival (RFS) and tended to have a higher 3-year overall survival (OS) than those with no ZNF384 fusions (80.1% vs. 52.5%, P = 0.013; 67.6% vs. 54.0%, P = 0.10). For patients receiving chemotherapy alone and received allogeneic-hematologic stem cell transplantation (allo-HSCT) were censored at the time of transplantation, patients with ZNF384 fusions had both similar RFS and similar OS to B-other ALL patients with no ZNF384 fusions (RFS: P =0.94 and 0.30; OS: P =0.94 and 0.51). For patients receiving transplantation, those with ZNF384 fusions had significantly higher 3-year RFS than B-other ALL patients with no ZNF384 fusions and their OS were similar (P = 0.022 and 0.24). Only two of 31 patients with ZNF384 fusions and receiving allo-HSCT relapsed, individually occurred 66.8 and 69.8 months after transplantation. Therefore, ZNF384 fusion is common in adult BCP-ALL, which may define a new group from BCP-ALL containing no classical fusion transcript with better prognosis through receiving allo-HSCT.

Single-Cell Transcriptome Analysis Identifies Ligand-Receptor Pairs Associated With BCP-ALL Prognosis.

B cell precursor acute lymphoblastic leukemia (BCP-ALL) is a blood cancer that originates from the abnormal proliferation of B-lymphoid progenitors. Cell population components and cell–cell interaction in the bone marrow microenvironment are significant factors for progression, relapse, and therapy resistance of BCP-ALL. In this study, we identified specifically expressed genes in B cells and myeloid cells by analyzing single-cell RNA sequencing data for seven BCP-ALL samples and four healthy samples obtained from a public database. Integrating 1356 bulk RNA sequencing samples from a public database and our previous study, we found a total of 57 significant ligand–receptor pairs (24 upregulated and 33 downregulated) in the autocrine crosstalk network of B cells. Via assessment of the communication between B cells and myeloid cells, another 29 ligand–receptor pairs were discovered, some of which notably affected survival outcomes. A score-based model was constructed with least absolute shrinkage and selection operator (LASSO) using these ligand–receptor pairs. Patients with higher scores had poorer prognoses. This model can be applied to create predictions for both pediatric and adult BCP-ALL patients.

Evaluation of serum levels of selected cytokine receptors in adult B-cell precursor acute lymphoblastic leukemia and their association with prognostic factors and survival.

To evaluate serum levels of selected cytokine receptors in B-cell precursor acute lymphoblastic leukemia (B-ALL) and their association with acknowledged prognostic factors, relapse-free survival (RFS) and overall survival (OS). A total of 42de novo adult B-ALL patients, 19BCR/ABL positive, were included in this study. Soluble receptor α for IL-2 (sIL-2Rα), soluble receptor for IL-6 (sIL-6R), soluble receptor for TNF-α type I and II (sTNFR-1, sTNFR-2) and matrix metalloproteinase-9 (MMP-9) were measured by biochip array technology at diagnosis and in complete remission (CR). At diagnosis of B-ALL, we found significantly higher levels of sIL-2Rα, sIL-6R, sTNFR-1, sTNFR-2and significantly lower levels MMP-9in comparison with CR (p< 0.001in all cases). BCR/ABL positive patients had higher levels of sIL-2Rα at diagnosis (r= 0.484; p= 0.014). Serum levels of evaluated cytokines were not associated with achievement of CR after one cycle of induction therapy, RFS or OS. Serum levels of all evaluated cytokines are significantly altered in newly diagnosed B-ALL reflecting activity of the disease. No significant correlations with response to first induction therapy, RFS or OS were found. Further studies with a longer follow-up will be needed.

Molecular Subtypes with Distinct Clinical Phenotypes and Actionable Targets in Adult B Cell Precursor ALL Treatment According to GMALL Protocols

Targeted interventions and new risk stratification approaches for the improvement of outcomes in B cell precursor acute lymphoblastic leukemia (BCP-ALL) require an understanding of high-risk driver subtypes, their clinical phenotypes and actionable targets. Currently, over 20 BCP-ALL subtypes have been identified based on genomic driver alterations and corresponding gene expression signatures, in particular in pediatric ALL. This complex molecular landscape is so far only partially defined in adult BCP-ALL patients. To characterize driver subtypes and their clinical phenotypes we performed transcriptome sequencing (Illumina) on Ph-negative adult BCP-ALL patients (n=376) treated on subsequent pediatric inspired protocols of the German Acute Lymphoblastic Leukemia Study Group (GMALL). Using random forest analyses of a well-defined patient subset with available WES/gene panel and DNA-methylation data, we established a 300-gene classifier which together with fusion calling and hotspot mutation calling reliably allocated samples to driver subtypes. Subgroup allocation was feasible for n=267 / 308 Ph-negative patients (86.7%) with Ph-like ALL (27.7%) being the most frequently observed subtype followed by DUX4 (14.6%), KMT2A (10.1%), PAX5-plus (9.7%), ZNF384 (9.0%), Near haploid - High hyperdiploid (NH-HeH, 6.0%), Low haploid - Near triploid (LH-NT, 6.0%) and TCF3-PBX1 (5.6%). With frequencies below 5% were observed: PAX5-alt, MEF2D, BCL2/MYC, ETV6-RUNX1, CEBP family member fusions and TCF3-HLF. The age distribution of these driver subtypes varied across our cohort (median age: 37 years, range: 16 - 81; left panel). Adult patients harboring ETV6-RUNX1 fusions (n=4) were 25 years or younger. NH-HeH, PAX5-plus and DUX4 ALL were also observed predominantly in younger patients (median ages: 24, 24, 30 years respectively; p&amp;lt;0.03) Conversely, LH-NT and KMT2A rearranged ALL frequencies increased in older patients (median ages: 47, 46 years respectively; p&amp;lt;0.04). As expected, male patients were slightly more represented in the cohort (55% male). However, significant differences were observed in the sex-distribution of molecular subgroups with a predominance of male patients in Ph-like (65.4%; p=0.045) and NH-HeH (82.4% p=0.024) ALL, while female patients were more frequent in DUX4 (60%, p=0.046) or KMT2A (75.5%; p=0.031) ALL. These data implicate that sex-specific host factors favor the selection of ALL driver alterations in adult ALL patients. We measured MRD by qRT-PCR quantification of patient specific immune gene rearrangement markers in our GMALL central reference lab. Data on MRD status after consolidation I which is the most relevant time-point for identification of high-risk patients in the GMALL protocol, were available in 132 patients (aged 18-55 years; right panel). Ph-like and KMT2A patients showed an unfavorable response to intensive induction with Molecular CR achieved in 60.6% and 61.1% respectively. In ZNF384 ALL patients, only n=5/13 achieved Molecular CR, while n=4 each were MRD-positive or had MRD below the quantitative detection range, suggesting that ZNF384 ALL might be a novel high-risk subgroup in adult BCP-ALL. Gene set enrichment analyses showed comparable patterns of JAK/STAT activation in ZNF384 and Ph-like cases as possible functional underpinning of a shared poor-response phenotype. Actionable genomic alterations were identified in n=13/74 Ph-like patients (17.6%). Out of these, ABL-class fusions as targets of ABL-TKI were identified in n=14 patients (PDGFRB: n=5; ABL1: n=4; CSF1R n=2) and one patient harbored an ETV6-NTRK3 fusion amenable to specific inhibitors. Our data extent the in-depth characterization of BCP-ALL molecular landscape in adult patients. We observe that sex-specific host factors favor the selection of leukemogenic drivers (male predominance: Ph-like, NH-HeH, female predominance: DUX4, KMT2A). ZNF384 ALL seems to be associated with unfavorable therapy response in adults and shares JAK/STAT activation patterns with Ph-like ALL. Figure Disclosures Fiedler: Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accomodations; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: support in medical writing; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accomodations, support in medical writing, Research Funding; Daiichi Sankyo Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accomodations; BerGenBio ASA: Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: support in medical writing; BMS: Honoraria; Gilead: Honoraria; Ariad/Incyte: Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria, Other; Morphosys: Membership on an entity's Board of Directors or advisory committees. Viardot:Novartis: Honoraria, Other: advisory board; Kite/Gilead: Honoraria, Other: advisory board; Roche: Honoraria, Other: advisory board; Amgen: Honoraria, Other: advisory board. Topp:Amgen, Boehringer Ingelheim, KITE, Regeneron, Roche: Research Funding; Amgen, KITE, Novartis, Regeneron, Roche: Consultancy. Goekbuget:Servier: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Erytech: Consultancy; Kite: Consultancy; Gilead: Consultancy. Brüggemann:Affimed: Research Funding; Incyte: Consultancy; Celgene: Consultancy; Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Roche: Consultancy; Regeneron: Research Funding.

A Phase I Study of Single-Agent Inotuzumab Ozogamicin in Pediatric CD22-Positive Relapsed/Refractory Acute Lymphoblastic Leukemia: Preliminary Results of the ITCC-059 Study

A Phase I Study of Single-Agent Inotuzumab Ozogamicin in Pediatric CD22-Positive Relapsed/Refractory Acute Lymphoblastic Leukemia: Preliminary Results of the ITCC-059 Study

Acute Lymphoblastic Leukemia with Zinc-Finger Protein 384 (ZNF384)-Related Rearrangements: A Retrospective Analysis from the Ponte Di Legno Childhood ALL Working Group

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous disease that can be subdivided according to primary recurrent genetic abnormalities that are strongly associated with characteristic biological and clinical features. The first few cases with ZNF384-related rearrangements were described as early as 2002. The leukemic phenotype of these cases was not only BCP-ALL but also mixed phenotype acute leukemia (MPAL) and acute myeloid leukemia (AML) switched from ALL. The number of patients was small because this type of leukemia is rare and many of the fusions are cytogenetically cryptic. RNA sequencing revealed that 1% to 6% of childhood BCP-ALL cases, 5% to 15% of adult BCP-ALL cases and 48% of B/Myeloid MPAL cases harbored ZNF384 rearrangements. Of note, ZNF384 has a variety of partner genes such as TCF3, EP300 and TAF15. Their biological characteristics showed distinct expression profiles, and the cell origin might arise from primitive hematopoietic cells. The clinical features associated with ZNF384-related rearrangements have not been analyzed in a large cohort of patients. To identify the clinical characteristics of ZNF384-related rearrangements in childhood BCP-ALL (MPAL was excluded), we studied a total with 226 cases of ZNF384-related rearrangements from 15 international consortia who participate in the Ponte di Legno study group registered between 1987 and 2018. We analyzed the impact of outcome in association with clinical and biological characteristics. ZNF384-related rearrangements were detected by fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR) and/or next generation sequencing (NGS), according to local selection policies, or because of poor response to therapy. Additional genetic abnormalities were detected by multiplex ligation-dependent probe amplification (MLPA), single nucleotide polymorphism array (SNP array) and/or NGS. The median age of presentation was 9 years old (range, 1 - 25 years old). The female and male ratio was 1:1. Immunophenotypic characteristics were classified as BCP-ALL exclusively In addition, 33% were CD10 negative cases (cutoff 20%); 71% were CD13 positive; and 86% were CD33 positive. Complete hematological remission was achieved in 99% of cases. One third (31%) of patients were treated as high risk and one quarter (23%) of patients received a stem cell transplant in first remission. After a median follow-up of 5.3 years, the 5-year event free survival (EFS) rate was 84% (95%CI, 77 to 89%), and the 5-year overall survival (OS) rate was 91% (95% CI, 85 to 94%). There was no difference in survival rate by treatment era or by country or region of origin. The proportion of partner genes with ZNF384 was as follows: EP300 (37%, n=84), TCF3 (27%, n=60), TAF15 (8%, n=17), CREBBP (7%, n=16), others (8%, n=18) of identifiable partners, and unknown (14%, n=31), although a prospective unselected analysis is needed for an appropriate estimate of the partners distribution. Patients with an EP300-ZNF384 fusion had a significantly lower relapse rate at 5 years compared with the remaining patients: 5% (95% CI 2-14) versus 20% (12-32), hazard ratio 4.58 (1.56-13.45), p=0.006), respectively. The corresponding EFS and OS rates were 91% (81-96) vs. 76% (64-85), p=0.024 and 92% (81-96) vs. 90% (80-95), p=0.3, suggesting that the non-EP300 relapses were salvageable (Figure). Multivariate analysis adjusting for sex, age, WBC and treatment era did not alter these results. Of note, in cases of TCF3 and TAF15, relapse occurred very late even after 5 years from diagnosis. Additional genetic abnormalities such as IKZF1, PAX5, CDKN2A/2B deletions were also analyzed. The distribution of deletions by partner genes was different between fusion partners but were not significant as prognostic factors. We confirm that ZNF384 rearrangement is a biologically and clinically distinct subtype of BCP-ALL. Immunophenotype abnormalities imply that ZNF384 rearrangements arise from primitive hematopoietic cells. Even considering a potential selection bias for the retrospective nature of the study, the OS was excellent in this subtype, although, relapse events did not reach a plateau among patients with TCF3-ZNF384 and TAF15-ZNF384. On the other hand, EP300-ZNF384 showed good prognosis with a low relapse rate. The biological background in each fusion partner warrants further investigation. Disclosures Loh: Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees.