Cytogenomic characterization of pediatric T-cell acute lymphoblastic leukemia reveals TCR rearrangements as predictive factors for exceptional prognosis.

BACKGROUND: T-cell acute lymphoblastic leukemia (T-ALL) represents a rare and clinically and genetically heterogeneous disease that constitutes 10-15% of newly diagnosed pediatric ALL cases. Despite improved outcomes of these children, the survival rate after relapse is extremely poor. Moreover, the survivors must also endure the acute and long-term effects of intensive therapy. Although recent studies have identified a number of recurrent genomic aberrations in pediatric T-ALL, none of the changes is known to have prognostic significance. The aim of our study was to analyze the cytogenomic changes and their various combinations in bone marrow cells of children with T-ALL and to correlate our findings with the clinical features of the subjects and their treatment responses. RESULTS: We performed a retrospective and prospective comprehensive cytogenomic analysis of consecutive cohort of 66 children (46 boys and 20 girls) with T-ALL treated according to BFM-based protocols and centrally investigated cytogenetics and immunophenotypes. Using combinations of cytogenomic methods (conventional cytogenetics, FISH, mFISH/mBAND, arrayCGH/SNP and MLPA), we identified chromosomal aberrations in vast majority of patients (91%). The most frequent findings involved the deletion of CDKN2A/CDKN2B genes (71%), T-cell receptor (TCR) loci translocations (27%), and TLX3 gene rearrangements (23%). All chromosomal changes occurred in various combinations and were rarely found as a single abnormality. Children with aberrations of TCR loci had a significantly better event free (p = 0.0034) and overall survival (p = 0.0074), all these patients are living in the first complete remission. None of the abnormalities was an independent predictor of an increased risk of relapse. CONCLUSIONS: We identified a subgroup of patients with TCR aberrations (both TRA/TRD and TRB), who had an excellent prognosis in our cohort with 5-year EFS and OS of 100%, regardless of the presence of other abnormality or the translocation partner. Our data suggest that escalation of treatment intensity, which may be considered in subsets of T-ALL is not needed for nonHR (non-high risk) patients with TCR aberrations.

Genomic DNA-based measurable residual disease monitoring in pediatric acute myeloid leukemia: unselected consecutive cohort study.

Measurable residual disease (MRD) monitoring in childhood acute myeloid leukemia (AML) is used to assess response to treatment and for early detection of imminent relapse. In childhood AML, MRD is typically evaluated using flow cytometry, or by quantitative detection of leukemia-specific aberrations at the mRNA level. Both methods, however, have significant limitations. Recently, we demonstrated the feasibility of MRD monitoring in selected subgroups of AML at the genomic DNA (gDNA) level. To evaluate the potential of gDNA-based MRD monitoring across all AML subtypes, we conducted a comprehensive analysis involving 133 consecutively diagnosed children. Integrating next-generation sequencing into the diagnostic process, we identified (presumed) primary genetic aberrations suitable as MRD targets in 97% of patients. We developed patient-specific quantification assays and monitored MRD in 122 children. The gDNA-based MRD monitoring via quantification of primary aberrations with a sensitivity of at least 10-4 was possible in 86% of patients; via quantification with sensitivity of 5 × 10-4, of secondary aberrations, or at the mRNA level in an additional 8%. Importantly, gDNA-based MRD exhibited independent prognostic value at early time-points in patients stratified to intermediate-/high-risk treatment arms. Our study demonstrates the broad applicability, feasibility, and clinical significance of gDNA-based MRD monitoring in childhood AML.

Characterization of the input material quality for the production of tisagenlecleucel by multiparameter flow cytometry and its relation to the clinical outcome.

Tisagenlecleucel (tisa-cel) is a CD19-specific CAR-T cell product approved for the treatment of relapsed/refractory (r/r) DLBCL or B-ALL. We have followed a group of patients diagnosed with childhood B-ALL (n = 5), adult B-ALL (n = 2), and DLBCL (n = 25) who were treated with tisa-cel under non-clinical trial conditions. The goal was to determine how the intensive pretreatment of patients affects the produced CAR-T cells, their in vivo expansion, and the outcome of the therapy. Multiparametric flow cytometry was used to analyze the material used for manufacturing CAR-T cells (apheresis), the CAR-T cell product itself, and blood samples obtained at three timepoints after administration. We present the analysis of memory phenotype of CD4/CD8 CAR-T lymphocytes (CD45RA, CD62L, CD27, CD28) and the expression of inhibitory receptors (PD-1, TIGIT). In addition, we show its relation to the patients' clinical characteristics, such as tumor burden and sensitivity to prior therapies. Patients who responded to therapy had a higher percentage of CD8+CD45RA+CD27+ T cells in the apheresis, although not in the produced CAR-Ts. Patients with primary refractory aggressive B-cell lymphomas had the poorest outcomes which was characterized by undetectable CAR-T cell expansion in vivo. No clear correlation of the outcome with the immunophenotypes of CAR-Ts was observed. Our results suggest that an important parameter predicting therapy efficacy is CAR-Ts' level of expansion in vivo but not the immunophenotype. After CAR-T cells' administration, measurements at several timepoints accurately detect their proliferation intensity in vivo. The outcome of CAR-T cell therapy largely depends on biological characteristics of the tumors rather than on the immunophenotype of produced CAR-Ts.

Constitutively active Lyn kinase causes a cutaneous small vessel vasculitis and liver fibrosis syndrome.

Neutrophilic inflammation is a hallmark of many monogenic autoinflammatory diseases; pathomechanisms that regulate extravasation of damaging immune cells into surrounding tissues are poorly understood. Here we identified three unrelated boys with perinatal-onset of neutrophilic cutaneous small vessel vasculitis and systemic inflammation. Two patients developed liver fibrosis in their first year of life. Next-generation sequencing identified two de novo truncating variants in the Src-family tyrosine kinase, LYN, p.Y508*, p.Q507* and a de novo missense variant, p.Y508F, that result in constitutive activation of Lyn kinase. Functional studies revealed increased expression of ICAM-1 on induced patient-derived endothelial cells (iECs) and of ß2-integrins on patient neutrophils that increase neutrophil adhesion and vascular transendothelial migration (TEM). Treatment with TNF inhibition improved systemic inflammation; and liver fibrosis resolved on treatment with the Src kinase inhibitor dasatinib. Our findings reveal a critical role for Lyn kinase in modulating inflammatory signals, regulating microvascular permeability and neutrophil recruitment, and in promoting hepatic fibrosis.

Integrated genomic analysis reveals actionable targets in pediatric spinal cord low-grade gliomas.

Gliomas are the most common central nervous tumors in children and adolescents. However, spinal cord low-grade gliomas (sLGGs) are rare, with scarce information on tumor genomics and epigenomics. To define the molecular landscape of sLGGs, we integrated clinical data, histology, and multi-level genetic and epigenetic analyses on a consecutive cohort of 26 pediatric patients. Driver molecular alteration was found in 92% of patients (24/26). A novel variant of KIAA1549:BRAF fusion (ex10:ex9) was identified using RNA-seq in four cases. Importantly, only one-third of oncogenic drivers could be revealed using standard diagnostic methods, and two-thirds of pediatric patients with sLGGs required extensive molecular examination. The majority (23/24) of detected alterations were potentially druggable targets. Four patients in our cohort received targeted therapy with MEK or NTRK inhibitors. Three of those exhibited clinical improvement (two with trametinib, one with larotrectinib), and two patients achieved partial response. Methylation profiling was implemented to further refine the diagnosis and revealed intertumoral heterogeneity in sLGGs. Although 55% of tumors clustered with pilocytic astrocytoma, other rare entities were identified in this patient population. In particular, diffuse leptomeningeal glioneuronal tumors (n = 3) and high-grade astrocytoma with piloid features (n = 1) and pleomorphic xanthoastrocytoma (n = 1) were present. A proportion of tumors (14%) had no match with the current version of the classifier. Complex molecular genetic sLGGs characterization was invaluable to refine diagnosis, which has proven to be essential in such a rare tumor entity. Moreover, identifying a high proportion of drugable targets in sLGGs opened an opportunity for new treatment modalities.

CD38: A target in relapsed/refractory acute lymphoblastic leukemia-Limitations in treatment and diagnostics.

Daratumumab, an anti-CD38 antibody, is used experimentally in the treatment of relapsed acute lymphoblastic leukemia (ALL). We treated five patients suffering from relapsed ALL with daratumumab. Four patients had T ALL, three of whom achieved complete remission (CR) after treatment and underwent stem cell transplant (SCT). Two of them had a second relapse and died 6 and 8 months after SCT, respectively. One transplanted T ALL patient remained in CR2 15 months after relapse. In the remaining T-ALL patient, the disease progressed under daratumumab treatment, and the patient died early after the first relapse. The B-cell precursor ALL patient with a second CD19-negative relapse, whose disease turned out to be resistant to the combination of daratumumab with chemotherapy, later achieved CR3 with inotuzumab ozogamicin, underwent SCT and remained in CR3. Leukemia burden should be monitored after daratumumab, and care should be taken not to misclassify leukemic cells with false negativity of surface CD38; using an antibody reacting with nondaratumumab epitopes is advantageous.

Other (Non-CNS/Testicular) Extramedullary Localizations of Childhood Relapsed Acute Lymphoblastic Leukemia and Lymphoblastic Lymphoma-A Report from the ALL-REZ Study Group.

Children with other extramedullary relapse of acute lymphoblastic leukemia are currently poorly characterized. We aim to assess the prevalence and the clinical, therapeutic and prognostic features of extramedullary localizations other than central nervous system or testis in children with relapse of acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL) treated on a relapsed ALL protocol. PATIENTS AND METHODS: Patients with relapse of ALL and LBL, treated according to the multicentric ALL-REZ BFM trials between 1983 and 2015, were analyzed for other extramedullary relapse (OEMR) of the disease regarding clinical features, treatment and outcome. Local treatment/irradiation has been recommended on an individual basis and performed only in a minority of patients. RESULTS: A total of 132 out of 2323 (5.6%) patients with ALL relapse presented with an OEMR (combined bone marrow relapse n = 78; isolated extramedullary relapse n = 54). Compared to the non-OEMR group, patients with OEMR had a higher rate of T-immunophenotype (p < 0.001), a higher rate of LBL (p < 0.001) and a significantly different distribution of time to relapse, i.e., more very early and late relapses compared to the non-OEMR group (p = 0.01). Ten-year probabilities of event-free survival (pEFS) and overall survival (pOS) in non-OEMR vs. OEMR were 0.38 ± 0.01 and 0.32 ± 0.04 (p = 0.0204) vs. 0.45 ± 0.01 and 0.37 ± 0.04 (p = 0.0112), respectively. OEMRs have been classified into five subgroups according to the main affected compartment: lymphatic organs (n = 32, 10y-pEFS 0.50 ± 0.09), mediastinum (n = 35, 10y-pEFS 0.11 ± 0.05), bone (n = 12, 0.17 ± 0.11), skin and glands (n = 21, 0.32 ± 0.11) and other localizations (n = 32, 0.41 ± 0.09). Patients with OEMR and T-lineage ALL/LBL showed a significantly worse 10y-pEFS (0.15 ± 0.04) than those with B-Precursor-ALL (0.49 ± 0.06, p < 0.001). Stratified into standard risk (SR) and high risk (HR) groups, pEFS and pOS of OEMR subgroups were in the expected range whereas the mediastinal subgroup had a significantly worse outcome. Subsequent relapses involved more frequently the bone marrow (58.4%) than isolated extramedullary compartments (41.7%). In multivariate Cox regression, OEMR confers an independent prognostic factor for inferior pEFS and pOS. CONCLUSION: OEMR is adversely related to prognosis. However, the established risk classification can be applied for all subgroups except mediastinal relapses requiring treatment intensification. Generally, isolated OEMR of T-cell-origin needs an intensified treatment including allogeneic stem cell transplantation (HSCT) as a curative approach independent from time to relapse. Local therapy such as surgery and irradiation may be of benefit in selected cases. The indication needs to be clarified in further investigations.

A novel class of ZNF384 aberrations in acute leukemia.

Fusion of the ZNF384 gene as the 3' partner to several different 5' partner genes occurs recurrently in B-cell precursor acute lymphoblastic and mixed phenotype B/myeloid leukemia. These canonical fusions (ZNF384r) contain the complete ZNF384 coding sequence and are associated with a specific gene expression signature. Cases with this signature, but without canonical ZNF384 fusions (ZNF384r-like cases), have been described previously. Although some have been shown to harbor ZNF362 fusions, the primary aberrations remain unknown in a major proportion. We studied 3 patients with the ZNF384r signature and unknown primary genetic background and identified a previously unknown class of genetic aberration affecting the last exon of ZNF384 and resulting in disruption of the C-terminal portion of the ZNF384 protein. Importantly, in 2 cases, the ZNF384 aberration, indel, was missed during the bioinformatic analysis but revealed by the manual, targeted reanalysis. Two cases with the novel aberrations had a mixed (B/myeloid) immunophenotype commonly associated with canonical ZNF384 fusions. In conclusion, we present leukemia cases with a novel class of ZNF384 aberrations that phenocopy leukemia with ZNF384r. Therefore, we show that part of the so-called ZNF384r-like cases represent the same genetic subtype as leukemia with canonical ZNF384 fusions.

Survival Following Relapse in Children with Acute Myeloid Leukemia: A Report from AML-BFM and COG.

Post-relapse therapy remains critical for survival in children with acute myeloid leukemia (AML). We evaluated survival, response and prognostic variables following relapse in independent cooperative group studies conducted by COG and the population-based AML-BFM study group. BFM included 197 patients who relapsed after closure of the last I-BFM relapse trial until 2017, while COG included 852 patients who relapsed on the last Phase 3 trials (AAML0531, AAML1031). Overall survival at 5 years (OS) was 42 ± 4% (BFM) and 35 ± 2% (COG). Initial high-risk features (BFM 32 ± 6%, COG 26 ± 4%) and short time to relapse (BFM 29 ± 4%, COG 25 ± 2%) predicted diminished survival. In the BFM dataset, there was no difference in OS for patients who had a complete remission with full hematopoietic recovery (CR) following post-relapse re-induction compared to those with partial neutrophil and platelet recovery (CRp and CRi) only (52 ± 7% vs. 63 ± 10%, p = 0.39). Among 90 patients alive at last follow-up, 87 had received a post-relapse hematopoietic stem cell transplant (HSCT). OS for patients with post-relapse HSCT was 54 ± 4%. In conclusion, initial high-risk features and early relapse remain prognostic. Response assessment with full hematopoietic recovery following initial relapse therapy does not predict survival. These data indicate the need for post-relapse risk stratification in future studies of relapse therapies.

Risk factors and outcomes in children with high-risk B-cell precursor and T-cell relapsed acute lymphoblastic leukaemia: combined analysis of ALLR3 and ALL-REZ BFM 2002 clinical trials.

AIM: Outcomes of children with high-risk (HR) relapsed acute lymphoblastic leukaemia (ALL) (N = 393), recruited to ALLR3 and ALL-REZ BFM 2002 trials, were analysed. Minimal residual disease (MRD) was assessed after induction and at predetermined time points until haematopoietic stem cell transplantation (SCT). METHODS: Genetic analyses included karyotype, copy-number alterations and mutation analyses. Ten-year survivals were analysed using Kaplan-Meier and Cox models for multivariable analyses. RESULTS: Outcomes of patients were comparable in ALLR3 and ALL-REZ BFM 2002. The event-free survival of B-cell precursor (BCP) and T-cell ALL (T-ALL) was 22.6% and 26.2% (P = 0.94), respectively, and the overall survival (OS) was 32.6% and 28.2% (P = 0.11), respectively. Induction failures (38%) were associated with deletions of NR3C1 (P = 0.002) and BTG1 (P = 0.03) in BCP-ALL. The disease-free survival (DFS) and OS in patients with good vs poor MRD responses were 57.4% vs 22.6% (P < 0.0001) and 57.8% vs 32.0% (P = 0.0004), respectively. For BCP- and T-ALL, the post-SCT DFS and OS were 42.1% and 56.8% (P = 0.26) and 51.6% and 55.4% (P = 0.67), respectively. The cumulative incidences of post-SCT relapse for BCP- and T-ALL were 36.9% and 17.8% (P = 0.012) and of death were 10.7% and 25.5% (P = 0.013), respectively. Determinants of outcomes after SCT were acute graft versus host disease, pre-SCT MRD (≥10-3), HR cytogenetics and TP53 alterations in BCP-ALL. CONCLUSION: Improvements in outcomes for HR ALL relapses require novel compounds in induction therapy to improve remission rates and immune targeted therapy after induction to maintain remission after SCT. TRIAL REGISTRATION: ALLR3: NCT00967057; ALL REZ-BFM 2002: NCT00114348.

Second Relapse of Pediatric Patients with Acute Myeloid Leukemia: A Report on Current Treatment Strategies and Outcome of the AML-BFM Study Group.

Successful management of relapse is critical to improve outcomes of children with acute myeloid leukemia (AML). We evaluated response, survival and prognostic factors after a second relapse of AML. Among 1222 pediatric patients of the population-based AML-Berlin-Frankfurt-Munster (BFM) study group (2004 until 2017), 73 patients met the quality parameters for inclusion in this study. Central review of source documentation warranted the accuracy of reported data. Treatment approaches included palliation in 17 patients (23%), intensive therapy with curative intent (n = 46, 63%) and other regimens (n = 10). Twenty-five patients (35%) received hematopoietic stem cell transplantation (HSCT), 21 of whom (88%) had a prior HSCT. Survival was poor, with a five-year probability of overall survival (pOS) of 15 ± 4% and 31 ± 9% following HSCT (n = 25). Early second relapse (within one year after first relapse) was associated with dismal outcome (pOS 2 ± 2%, n = 44 vs. 33 ± 9%, n = 29; p < 0.0001). A third complete remission (CR) is required for survival: 31% (n = 14) of patients with intensive treatment achieved a third CR with a pOS of 36 ± 13%, while 28 patients (62%) were non-responders (pOS 7 ± 5%). In conclusion, survival is poor but possible, particularly after a late second relapse and an intensive chemotherapy followed by HSCT. This analysis provides a baseline for future treatment planning.

A phase 1 study of inotuzumab ozogamicin in pediatric relapsed/refractory acute lymphoblastic leukemia (ITCC-059 study).

This phase 1 study investigated the recommended phase 2 dose (RP2D) of inotuzumab ozogamicin (InO), a CD22-directed antibody-drug conjugate, in pediatric patients with multiple relapsed/refractory (R/R) CD22+ acute lymphoblastic leukemia (ALL). Patients (age ≥1 year or <18 years) received 3 doses of InO (days 1, 8, and 15) per course. Dose escalation was based on dose-limiting toxicities (DLTs) during course 1. Dose level 1 (DL1) was 1.4 mg/m2 (0.6, 0.4, 0.4 mg/m2) and DL2 was 1.8 mg/m2 (0.8, 0.5, 0.5 mg/m2). Secondary end points included safety, antileukemic activity, and pharmacokinetics. Twenty-five patients (23 evaluable for DLTs) were enrolled. In course 1, the first cohort had 1 of 6 (DL1) and 2 of 5 (DL2) patients who experienced DLTs; subsequent review considered DL2 DLTs to be non-dose-limiting. Dose was de-escalated to DL1 while awaiting protocol amendment to re-evaluate DL2 in a second cohort, in which 0 of 6 (DL1) and 1 of 6 (DL2) patients had a DLT. Twenty-three patients experienced grade 3 to 4 adverse events; hepatic sinusoidal obstruction syndrome was reported in 2 patients after subsequent chemotherapy. Overall response rate after course 1 was 80% (95% confidence interval [CI], 59% to 93%) (20 of 25 patients; DL1: 75% [95% CI, 43% to 95%], DL2: 85% [95% CI, 55% to 98%]). Of the responders, 84% (95% CI, 60% to 97%) achieved minimal residual disease (MRD)-negative complete response, and 12-month overall survival was 40% (95% CI, 25% to 66%). Nine patients received hematopoietic stem cell transplantation or chimeric antigen receptor T cells after InO. InO median maximum concentrations were comparable to simulated adult concentrations. InO was well tolerated, demonstrating antileukemic activity in heavily pretreated children with CD22+ R/R ALL. RP2D was established as 1.8 mg/m2 per course, as in adults. This trial was registered at https://www.clinicaltrialsregister.eu as EUDRA-CT 2016-000227-71.