Outcomes in children with first-relapsed acute lymphoblastic leukemia in Japan: Results from JCCG Study JPLSG-ALL-R08.

BACKGROUND/OBJECTIVES: The Berlin-Frankfurt-Münster (BFM)-S classification is a crucial prognostic indicator in children experiencing first-relapsed acute lymphoblastic leukemia (ALL). Early molecular response to therapy, evaluated by measurable/minimal residual disease (MRD), has a significant impact on the survival of patients with childhood ALL. Applying risk stratification based on the BFM-S classification and MRD response after induction, the first nationwide prospective multicenter study, ALL-R08, was conducted in children with first-relapsed ALL in Japan. METHODS: The ALL-R08 study comprised two parts: ALL-R08-I, an observational study aimed at obtaining an overall picture of outcomes in first-relapsed childhood ALL, and ALL-R08-II, a clinical trial for the non-T-ALL S2 risk group. In ALL-R08-II, patients with an MRD level of ≥10-3 at the end of induction therapy were assigned to undergo allogeneic hematopoietic stem cell transplantation (allo-HCT), whereas those with an MRD level less than 10-3 and isolated extramedullary relapse continued to receive chemotherapy. RESULTS: In total, 163 patients were enrolled in the ALL-R08 study, and 82 and 81 patients were enrolled in the ALL-R08-I and the ALL-R08-II, respectively. In ALL-R08-I, the probability of 3-year event-free survival (EFS) for patients with S1, S2, S3, S4, and post-HCT groups was 83% ± 15%, 37% ± 11%, 28% ± 8%, 14% ± 7%, and 0%, respectively. In the ALL-R08-II trial, 3-year EFS in patients with post-induction MRD less than 10-3 and ≥10-3 was 70% ± 9% (n = 27) and 68% ± 8% (n = 31) (p = .591), respectively. CONCLUSIONS: ALL-REZ BFM-type treatment is equally effective for children with first-relapsed ALL treated according to the Japanese frontline protocols and for children with first-relapsed ALL treated according to the BFM-type frontline protocols.

Adverse prognostic impact of KIT exon 17 mutations despite negative flow cytometric measurable residual disease in pediatric acute myeloid leukemia with RUNX1::RUNX1T1.

Not available.

Landscape of driver mutations and their clinical effects on Down syndrome-related myeloid neoplasms.

ABSTRACT: Transient abnormal myelopoiesis (TAM) is a common complication in newborns with Down syndrome (DS). It commonly progresses to myeloid leukemia (ML-DS) after spontaneous regression. In contrast to the favorable prognosis of primary ML-DS, patients with refractory/relapsed ML-DS have poor outcomes. However, the molecular basis for refractoriness and relapse and the full spectrum of driver mutations in ML-DS remain largely unknown. We conducted a genomic profiling study of 143 TAM, 204 ML-DS, and 34 non-DS acute megakaryoblastic leukemia cases, including 39 ML-DS cases analyzed by exome sequencing. Sixteen novel mutational targets were identified in ML-DS samples. Of these, inactivations of IRX1 (16.2%) and ZBTB7A (13.2%) were commonly implicated in the upregulation of the MYC pathway and were potential targets for ML-DS treatment with bromodomain-containing protein 4 inhibitors. Partial tandem duplications of RUNX1 on chromosome 21 were also found, specifically in ML-DS samples (13.7%), presenting its essential role in DS leukemia progression. Finally, in 177 patients with ML-DS treated following the same ML-DS protocol (the Japanese Pediatric Leukemia and Lymphoma Study Group acute myeloid leukemia -D05/D11), CDKN2A, TP53, ZBTB7A, and JAK2 alterations were associated with a poor prognosis. Patients with CDKN2A deletions (n = 7) or TP53 mutations (n = 4) had substantially lower 3-year event-free survival (28.6% vs 90.5%; P < .001; 25.0% vs 89.5%; P < .001) than those without these mutations. These findings considerably change the mutational landscape of ML-DS, provide new insights into the mechanisms of progression from TAM to ML-DS, and help identify new therapeutic targets and strategies for ML-DS.

Promoter swapping of truncated PDGFRB drives Ph-like acute lymphoblastic leukemia.

Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL) is a subset of ALL that demonstrated a high treatment failure rate. One of the hallmarks of Ph-like ALL is PDGFRB gene fusion, with fusion partner proteins often harboring dimerization domains and enhancing the kinase activity of PDGFRB. We determined a novel oncogenic PDGFRB fusion gene, NRIP1::PDGFRB, from a pediatric patient with ALL, encoding a protein with the carboxy-terminal kinase domain of PDGFRB, without the partner peptide. We confirmed the oncogenic potential of NRIP1::PDGFRB in vitro and the efficacy of all ABL1-specific inhibitor generations, including imatinib, dasatinib, nilotinib, and ponatinib, in suppressing this potential. PDGFRB activation mechanism may include juxtamembrane domain truncation in the predicted peptide. In conclusion, we determined a novel fusion gene pattern in Ph-like ALL.

[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.

Nelarabine, intensive L-asparaginase, and protracted intrathecal therapy for newly diagnosed T-cell acute lymphoblastic leukaemia in children and young adults (ALL-T11): a nationwide, multicenter, phase 2 trial including randomisation in the very high-risk group.

BACKGROUND: T-cell acute lymphoblastic leukaemia has distinct biological characteristics and a poorer prognosis than B-cell precursor acute lymphoblastic leukaemia. This trial aimed to reduce the rate of radiation and haematopoietic stem-cell transplantation (HSCT) while improving outcomes by adding nelarabine, intensified L-asparaginase, and protracted intrathecal therapy in the Berlin-Frankfurt-Münster (BFM)-type treatment. METHODS: In this nationwide, multicenter, phase 2 trial, we enrolled patients with newly diagnosed T-cell acute lymphoblastic leukaemia (age <25 years at diagnosis) conducted by Japan Children's Cancer Group and Japan Adult Leukemia Study Group. Patients were stratified into standard-risk, high-risk, and very-high-risk groups according to prednisolone response, CNS status, and end-of-consolidation minimal residual disease. We used the Associazione Italiana di Ematologia Oncologia Pediatrica (AIEOP)-BFM-ALL 2000-backbone chemotherapy. Nelarabine (650 mg/m2 per day for 5 days) was given to high-risk and very high-risk patients. All patients received, until the measurement of end-of-consolidation minimal residual disease, an identical therapy schedule, which included the prednisolone pre-phase remission induction therapy with dexamethasone (10 mg/m2 per day, for 3 weeks [for patients <10 years] or for 2 weeks including a 7-day off interval [for patients ≥10 years]) instead of prednisolone, and consolidation therapy added with Escherichia coli-derived L-asparaginase. On the basis of the stratification, patients received different intensities of treatment; L-asparaginase-intensified standard BFM-type therapy for standard risk and nelarabine-added high risk BFM-type therapy for high risk. In the very high-risk group, patients were randomly assigned (1:1) to group A (BFM-based block therapy) and group B (another block therapy, including high-dose dexamethasone) stratified by hospital, age (≥18 years or <18 years), and end-of-induction bone marrow blast percentage of M1 (<5%) or M2 (≥5%, <25%)+M3 (≥25%). Cranial radiotherapy was limited to patients with overt CNS disease at diagnosis (CNS3; >5 white blood cells per µL with blasts) and patients with no evidence of CNS disease received protracted triple intrathecal therapy. Only very high-risk patients were scheduled to receive HSCT. The primary endpoint was 3-year event-free survival for the entire cohort and the proportion of patients with disappearance of minimal residual disease between randomly assigned groups A and B in the very high-risk group. Secondary endpoints were overall survival, remission induction rate, and occurrence of adverse events. 3 years after the completion of patient accrual, a primary efficacy analysis was performed in the full analysis set and the per-protocol set. This study is registered with the Japan Registry of Clinical Trials, jRCTs041180145. FINDINGS: Between Dec 1, 2011, and Nov 30, 2017, of 349 eligible patients (median age 9 years [IQR 6-13]), 238 (68%) were male, and 28 (8%) patients had CNS3 status. 168 (48%) patients were stratified as standard risk, 103 (30%) as high risk, 39 (11%) as very high risk, and 39 (11%) as no risk (patients who had off protocol treatment before risk assessment. The composite complete remission (complete remission plus complete remission in suppression) rate after remission induction therapy was 89% (298 of 335 patients). HSCT was performed in 35 (10%) of 333 patients. With a median follow-up of 5·2 years (IQR 3·6-6·7), 3-year event-free survival was 86·4% (95% CI 82·3-89·7%) and 3-year overall survival was 91·3% (87·7-93·8%). The proportion of minimal residual disease disappearance was 0·86 (12 of 14 patients; 95% CI 0·57-0·98) in group A and 0·50 (6 of 12 patients, 0·21-0·79) in group B. Grade 3 peripheral motor neuropathy was seen in 11 (3%) of 349 patients and sensory neuropathy was seen in 6 (2%) patients. The most common grade 3 or worse adverse event was febrile neutropenia (294 [84%] of 349 patients). Treatment-related death occurred in three patients due to sepsis, gastric perforation, or intracranial haemorrhage during remission induction. INTERPRETATION: The ALL-T11 protocol produced encouraging outcomes with acceptable toxicities despite limited cranial radiotherapy and HSCT use. FUNDING: Ministry of Health, Labor and Welfare of Japan, and Japan Agency for Medical Research and Development. TRANSLATION: For the Japanese translation of the abstract see Supplementary Materials section.

TP53 and RB1 alterations characterize poor prognostic subgroups in pediatric acute myeloid leukemia.

Pediatric acute myeloid leukemia (AML) is a poor prognostic subtype of pediatric leukemia. However, the detailed characteristics of many genetic abnormalities are yet to be established in this disease. Although TP53 and RB1 are established as representative tumor suppressor genes in various cancers, alterations of these two genes, especially RB1, have not been characterized in pediatric AML. We performed next-generation sequencing in 328 pediatric AML patients from the Japanese AML-05 trial to ascertain TP53 and RB1 alterations, and their prognostic implications. We identified seven patients with TP53 alterations (2.1%) and six patients with RB1 alterations (1.8%). These alterations were found in only patients without RUNX1::RUNX1T1, CBFB::MYH11, or KMT2A rearrangements. TP53 and RB1 were frequently co-deleted with their neighboring genes PRPF8 and ELF1, respectively. Patients with TP53 alterations had significantly lower 5-year overall survival (OS; 14.3% vs. 71.4%, p < 0.001) and lower 5-year event-free survival (EFS; 0% vs. 56.3%, p < 0.001); similarly, patients with RB1 had significantly lower 5-year OS (0% vs. 71.8%, p < 0.001) and lower 5-year EFS (0% vs. 56.0%, p < 0.001) when compared to patients without these alterations. In gene expression analyses, oxidative phosphorylation, glycolysis, and protein secretion were upregulated in patients with TP53 and/or RB1 alterations. Additionally, Kaplan-Meier analysis revealed that high expressions of SLC2A5, KCNAB2, and CD300LF were related to poor OS of non-core-binding factor AML patients (p < 0.001, p = 0.001, and p = 0.021, respectively). This study will contribute to the development of risk-stratified therapy and precision medicine in pediatric AML.

Prevalence of pathogenic variants in cancer-predisposing genes in second cancer after childhood solid cancers.

BACKGROUND: Second malignant neoplasms (SMNs) are one of the most severe late complications after pediatric cancer treatment. However, the effect of genetic variation on SMNs remains unclear. In this study, we revealed germline genetic factors that contribute to the development of SMNs after treatment of pediatric solid tumors. METHODS: We performed whole-exome sequencing in 14 pediatric patients with SMNs, including three brain tumors. RESULTS: Our analysis revealed that five of 14 (35.7%) patients had pathogenic germline variants in cancer-predisposing genes (CPGs), which was significantly higher than in the control cohort (p < 0.01). The identified genes with variants were TP53 (n = 2), DICER1 (n = 1), PMS2 (n = 1), and PTCH1 (n = 1). In terms of the type of subsequent cancer, leukemia and multiple episodes of SMN had an exceptionally high rate of CPG pathogenic variants. None of the patients with germline variants had a family history of SMN development. Mutational signature analysis showed that platinum drugs contributed to the development of SMN in three cases, which suggests the role of platinum agents in SMN development. CONCLUSIONS: We highlight that overlapping effects of genetic background and primary cancer treatment contribute to the development of second cancers after treatment of pediatric solid tumors. A comprehensive analysis of germline and tumor samples may be useful to predict the risk of secondary cancers.

Minimal residual disease detection by mutation-specific droplet digital PCR for leukemia/lymphoma.

Minimal residual disease (MRD) is usually defined as the small number of cancer cells that remain in the body after treatment. The clinical significance of MRD kinetics is well recognized in treatment of hematologic malignancies, particularly acute lymphoblastic leukemia (ALL). Real time quantitative PCR targeting immunoglobulin (Ig) or T-cell receptor (TCR) rearrangement (PCR-MRD), as well as multiparametric flow cytometric analysis targeting antigen expression, are widely used in MRD detection. In this study, we devised an alternative method to detect MRD using droplet digital PCR (ddPCR), targeting somatic single nucleotide variants (SNVs). This ddPCR-based method (ddPCR-MRD) had sensitivity up to 1E-4. We assessed ddPCR-MRD at 26 time points from eight T-ALL patients, and compared it to the results of PCR-MRD. Almost all results were concordant between the two methods, but ddPCR-MRD detected micro-residual disease that was missed by PCR-MRD in one patient. We also measured MRD in stored ovarian tissue of four pediatric cancer patients, and detected 1E-2 of submicroscopic infiltration. Considering the universality of ddPCR-MRD, the methods can be used as a complement for not only ALL, but also other malignant diseases regardless of tumor-specific Ig/TCR or surface antigen patterns.

Hypermethylation of RASSF1A gene in pediatric rhabdoid tumor of the kidney and clear cell sarcoma of the kidney.

BACKGROUND: Among pediatric renal tumors, rhabdoid tumor of the kidney (RTK) and clear cell sarcoma of the kidney (CCSK) are rare and associated with an unfavorable prognosis, while congenital mesoblastic nephroma (CMN) is associated with a good prognosis. Methylation of the Ras association domain-containing protein 1 isoform A (RASSF1A) promoter has been reported to correlate with a poor prognosis in patients with Wilms tumors, while its methylation status is unclear in other types of pediatric renal tumors. METHOD: DNA methylation of the RASSF1A promoter in several pediatric renal tumors was analyzed with pyrosequencing. In order to clarify the correlation between expression of RASSF1A and DNA methylation of its promoter, the RTK cell line was treated with 5-Aza-2'-deoxycytidine (5-Aza-dC). RASSF1A was overexpressed in the RTK cell line to evaluate its functional effects. RESULTS: Quantitative methylation analysis demonstrated hypermethylation in the RASSF1A promoter region in RTK and CCSK, but not CMN. The 5-Aza-dC treatment induced demethylation of the RASSF1A promoter as well as increased RASSF1A mRNA expression. The transduction of RASSF1A has an effect on the suppression of viability and proliferation of RTK cells. CONCLUSION: DNA methylation-mediated deficiency of RASSF1A might be involved in the development and aggressiveness of some pediatric renal tumors and correlated with a poor prognosis.

UBTF-internal tandem duplication as a novel poor prognostic factor in pediatric acute myeloid leukemia.

The prognosis of pediatric acute myeloid leukemia (AML) has improved via stratification therapy. However, relapse or death occurs in 30%-40% of cases. Novel genetic factors for pediatric AML need to be elucidated to improve prognosis. We detected recurrent internal tandem duplication in upstream binding transcription factor (UBTF-ITD) in 1.2% (6/503) of Japanese pediatric patients with de novo AML. No UBTF-ITD was detected in 175 adult patients with AML or in 65 cell lines that included 15 AML, 39 acute lymphoblastic leukemia, five chronic myeloid leukemia, and six neuroblastoma cell lines. All UBTF-ITDs were found in exon 13 and shared a duplicated region. UBTF-ITD was more frequently detected in patients with trisomy 8, FLT3-ITD, WT1 mutation, and/or high PRDM16 expression (trisomy 8, 3/6; FLT3-ITD, 5/6; WT1 mutation, 2/6; and high PRDM16 expression, 6/6). Gene expression patterns of patients with UBTF-ITD were similar to those of patients with NUP98::NSD1 or FUS::ERG. Survival analysis of the AML-05 cohort revealed that patients with UBTF-ITD had worse outcomes than those without UBTF-ITD (3-year event-free survival, 20% vs. 55%; 3-year overall survival, 40% vs. 74%). Moreover, among the 27 patients with trisomy 8, all three patients with UBTF -ITD had a poor prognosis resulting in early events (relapse or non-complete remission) within 1 year. Our findings suggest that UBTF-ITD may be a novel and significant prognostic factor for pediatric patients with AML.

Multi-omics analysis defines highly refractory RAS burdened immature subgroup of infant acute lymphoblastic leukemia.

KMT2A-rearranged infant acute lymphoblastic leukemia (ALL) represents the most refractory type of childhood leukemia. To uncover the molecular heterogeneity of this disease, we perform RNA sequencing, methylation array analysis, whole exome and targeted deep sequencing on 84 infants with KMT2A-rearranged leukemia. Our multi-omics clustering followed by single-sample and single-cell inference of hematopoietic differentiation establishes five robust integrative clusters (ICs) with different master transcription factors, fusion partners and corresponding stages of B-lymphopoietic and early hemato-endothelial development: IRX-type differentiated (IC1), IRX-type undifferentiated (IC2), HOXA-type MLLT1 (IC3), HOXA-type MLLT3 (IC4), and HOXA-type AFF1 (IC5). Importantly, our deep mutational analysis reveals that the number of RAS pathway mutations predicts prognosis and that the most refractory subgroup of IC2 possesses 100% frequency and the heaviest burden of RAS pathway mutations. Our findings highlight the previously under-appreciated intra- and inter-patient heterogeneity of KMT2A-rearranged infant ALL and provide a rationale for the future development of genomics-guided risk stratification and individualized therapy.

A common deletion at BAK1 reduces enhancer activity and confers risk of intracranial germ cell tumors.

Intracranial germ cell tumors (IGCTs) are rare brain neoplasms that mainly occur in children and adolescents with a particularly high incidence in East Asian populations. Here, we conduct a genome-wide association study (GWAS) of 133 patients with IGCTs and 762 controls of Japanese ancestry. A common 4-bp deletion polymorphism in an enhancer adjacent to BAK1 is significantly associated with the disease risk (rs3831846; P = 2.4 × 10-9, odds ratio = 2.46 [95% CI: 1.83-3.31], minor allele frequency = 0.43). Rs3831846 is in strong linkage disequilibrium with a testicular GCTs susceptibility variant rs210138. In-vitro reporter assays reveal rs3831846 to be a functional variant attenuating the enhancer activity, suggesting its contribution to IGCTs predisposition through altering BAK1 expression. Risk alleles of testicular GCTs derived from the European GWAS show significant positive correlations in the effect sizes with the Japanese IGCTs GWAS (P = 1.3 × 10-4, Spearman's ρ = 0.48). These results suggest the shared genetic susceptibility of GCTs beyond ethnicity and primary sites.

A phase III clinical trial evaluating efficacy and safety of minimal residual disease-based risk stratification for children with acute myeloid leukemia, incorporating a randomized study of gemtuzumab ozogamicin in combination with post-induction chemotherapy for non-low-risk patients (JPLSG-AML-20).

The purpose of this study is to establish a treatment with appropriate intensity for children (<16 years old at diagnosis) with de novo acute myeloid leukemia (excluding acute promyelocytic leukemia and myeloid leukemia associated with Down syndrome) according to a risk stratification based on recurrent leukemic cytogenetic abnormalities and flow-cytometric minimal residual disease at end of initial induction chemotherapy and to validate the safety and efficacy of gemtuzumab ozogamicin (GO)-combined post-induction chemotherapy for the non-low-risk (non-LR) patients. The primary endpoint of this phase III study is three-year disease-free survival rate, which will be compared between the GO and non-GO arms of the non-LR (intermediate-risk and high-risk [HR]) patients. All HR patients will be allocated to allogeneic hematopoietic stem cell transplantation in first remission. This trial has been registered at the Japan Registry of Clinical Trials (jRCTs041210015).

Low NUDT15 expression levels due to biallelic NUDT15 variants and 6-mercaptopurine intolerance.

6-Mercaptopurine (6-MP) is widely used for the treatment of paediatric leukaemia and lymphoma. Recently, germline variants in the NUDT15 gene have been identified as one of the major genetic causes for 6-MP-associated adverse effects such as myelosuppression. Patients with hypomorphic NUDT15 variants accumulate excessive levels of DNA-incorporated thioguanine in white blood cells, resulting in severe myelosuppression. Although preclinical studies suggest that these variants may influence the protein stability of NUDT15, this has not been directly characterised in patients. In this study, we report the development of a series of novel monoclonal antibodies against NUDT15, using which we quantitatively assessed NUDT15 protein levels in 37 patients with acute lymphoblastic leukaemia treated with 6-MP, using sandwich enzyme-linked immunosorbent assay (ELISA). The NUDT15 genotype was highly correlated with its protein levels (p < 0.0001), with homozygous and compound heterozygous patients showing exceedingly low NUDT15 expression. There was a positive correlation between NUDT15 protein level and 6-MP tolerance (r = 0.631, p < 0.0001). In conclusion, our results point to low NUDT15 protein abundance as the biochemical basis for NUDT15-mediated 6-MP intolerance, thus providing a phenotypic readout of inherited NUDT15 deficiency.

Quantitative assessment of copy number alterations by liquid biopsy for neuroblastoma.

Liquid biopsy, a method of detecting genomic alterations using blood specimens, has recently attracted attention as a noninvasive alternative to surgical tissue biopsy. We attempted quantitative analysis to detect amplification of MYCN (MYCNamp) and loss of heterozygosity at 11q (11qLOH), which are clinical requisites as prognostic factors of neuroblastoma (NB). In this study, cell-free DNA (cfDNA) was extracted from plasma samples from 24 NB patients at diagnosis. Copy numbers of MYCN and NAGK genes were quantitatively analyzed by droplet digital PCR (ddPCR). 11qLOH was also assessed by detecting allelic imbalances of heterozygous single nucleotide polymorphisms in the 11q region. The results obtained were compared to those of specimens from tumor tissues. The correlation coefficient of MYCN copy number of cfDNA and tumor DNA was 0.88 (p < 0.00001). 11qLOH was also accurately detected from cfDNA, except for one case with localized NB. Given the high accuracy of liquid biopsy, to investigate components of cfDNA, the proportion of tumor-derived DNA was estimated by examining the variant allele frequency of tumor-specific mutations in cfDNA. The proportion of tumor-derived DNA in cfDNA was 42.5% (range, 16.9%-55.9%), suggesting sufficient sensitivity of liquid biopsy for NB. In conclusion, MYCN copy number and 11qLOH could be quantitatively analyzed in plasma cfDNA by ddPCR assay. These results suggest that plasma cfDNA can be substituted for tumor DNA and can also be applied for comprehensive genomic profiling analysis.