Relationship between subtype-specific minimal residual disease level and long-term prognosis in children with acute lymphoblastic leukemia.

Minimal residual disease (MRD) based risk stratification criteria for specific genetic subtypes remained unclear in childhood acute lymphoblastic leukemia (ALL). Among 723 children with newly diagnosed ALL treated with the Chinese Children Leukemia Group CCLG-2008 protocol, MRD was assessed at time point 1 (TP1, at the end of induction) and TP2 (before consolidation treatment) and the MRD levels significantly differed in patients with different fusion genes or immunophenotypes (P all < 0.001). Moreover, the prognostic impact of MRD varied by distinct molecular subtypes. We stratified patients in each molecular subtype into two MRD groups based on the results. For patients carrying BCR::ABL1 or KMT2A rearrangements, we classified patients with MRD < 10-2 at both TP1 and TP2 as the low MRD group and the others as the high MRD group. ETV6::RUNX1+ patients with TP1 MRD < 10-3 and TP2 MRD-negative were classified as the low MRD group and the others as the high MRD group. For T-ALL, We defined children with TP1 MRD ≥ 10-3 as the high MRD group and the others as the low MRD group. The 10-year relapse-free survival of low MRD group was significantly better than that of high MRD group. We verified the prognostic impact of the subtype-specific MRD-based stratification in patients treated with the BCH-ALL2003 protocol. In conclusion, the subtype-specific MRD risk stratification may contribute to the precise treatment of childhood ALL.

Optical Genome Mapping Reveals Novel Structural Variants in Lymphoblastic Lymphoma.

BACKGROUND: Accurate histologic and molecular genetic diagnosis is critical for the pathogenesis study of pediatric patients with lymphoblastic lymphoma (LBL). Optical genome mapping (OGM) as all-in-one process allows the detection of most major genomic risk markers, which addresses some of the limitations associated with conventional cytogenomic testing, such as low resolution and throughput, difficulty in ascertaining genomic localization, and orientation of segments in duplication, inversions, and insertions. Here, for the first time, we examined the cytogenetics of 5 children with LBL using OGM. METHODS: OGM was used to analyze 5 samples of pediatric LBL patients treated according to the modified NHL-BFM95 backbone regimen. Whole-exon Sequencing (WES) was used to confirm the existence of structural variants (SVs) identified by OGM with potentially clinical significance on MGI Tech (DNBSEQ-T7) platform. According to the fusion exon sequences revealed by WES, the HBS1L :: AHI1 fusion mRNA in case 4 was amplified by cDNA-based PCR. RESULTS: In total, OGM identified 251 rare variants (67 insertions, 129 deletions, 3 inversion, 25 duplications, 15 intrachromosomal translocations, and 12 interchromosomal translocations) and 229 copy number variants calls (203 gains and 26 losses). Besides all of the reproducible and pathologically significant genomic SVs detected by conventional cytogenetic techniques, OGM identified more SVs with definite or potential pathologic significance that were not detected by traditional methods, including 2 new fusion genes, HBS1L :: AHI1 and GRIK1::NSDHL , which were confirmed by WES and/or Reverse Transcription-Polymerase Chain Reaction. CONCLUSIONS: Our results demonstrate the feasibility of OGM to detect genomic aberrations, which may play an important role in the occurrence and development of lymphomagenesis as an important driving factor.

Clinical, biological, and outcome features of P2RY8-CRLF2 and CRLF2 over-expression in pediatric B-cell precursor acute lymphoblastic leukemia according to the CCLG-ALL 2008 and 2018 protocol.

OBJECTIVES: CRLF2 alterations are associated with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This study aimed to explore the clinical, biological, and outcome features of pediatric BCP-ALL with CRLF2 abnormalities. METHODS: This study enrolled 630 childhood BCP-ALLs treated on CCLG-ALL 2008 or 2018 protocol. P2RY8-CRLF2 was determined by Sanger sequencing and CRLF2 expression was evaluated by qRT-PCR. The correlation between clinical, biological features and outcomes with P2RY8-CRLF2 or CRLF2 over-expression were analyzed. RESULTS: P2RY8-CRLF2 and CRLF2 over-expression were found in 3.33% and 5.71% respectively. P2RY8-CRLF2 was associated with male, higher frequency of CD7 expression, high WBC and MRD before consolidation. CRLF2 over-expression showed ETV6-RUNX1- , higher frequency of CD22, CD34, CD66c, CD86 expression, hyperdiploidy and high MRD at early treatment. The lower overall survival (OS) was found in patients with P2RY8-CRLF2 and confined only in IR group. Furthermore, adverse event-free survival and OS of P2RY8-CRLF2 were discovered comparing to those without known fusions or treated on CCLG-ALL 2008 protocol. However, P2RY8-CRLF2 was not confirmed as independent prognostic factors and no prognostic impact of CRLF2 over-expression was found. CONCLUSIONS: These findings indicate P2RY8-CRLF2 identifies a subset of patients with specific features and adverse outcomes that could be improved by risk-directed treatment.

Effects of SLCO1B1 on elimination and toxicities of high-dose methotrexate in pediatric acute lymphoblastic leukemia.

Aim: To evaluate the association between SLCO1B1 polymorphisms and elimination/toxicities of high-dose methotrexate (MTX). Methods: SLCO1B1 rs11045879 and rs4149056 polymorphisms were retrospectively genotyped in 301 children with newly diagnosed acute lymphoblastic leukemia. MTX concentration, doses of leucovorin rescue and toxicities were recorded. Results: SLCO1B1 rs11045879C carriers (CC + CT) had higher plasma MTX levels at 96 hr, and longer MTX elimination time. The number of leucovorin rescue doses in rs4149056C carriers (CC + CT) was more than those in TT ones. Moreover, SLCO1B1 polymorphisms were associated with HDMTX toxicities including thrombocytopenia, renal toxicity and anal mucositis, but not associated with MTX level at other time points or delayed elimination. Conclusions: Our data demonstrate that genotyping of SLCO1B1 might be useful to optimize MTX therapy.

Prognostic significance of NOTCH1/FBXW7 mutations in pediatric T cell acute lymphoblastic leukemia: a study of minimal residual disease risk-directed CCLG-ALL 2008 treatment protocol.

NOTCH1/FBXW7 mutation is common in T-cell acute lymphoblastic leukemia (T-ALL), but controversy looms on its prognostic significance. We screened 98 pediatric T-ALL patients treated on minimal residual disease (MRD) risk-directed CCLG-ALL 2008 protocol. NOTCH1/FBXW7 mutations were analyzed by Sanger sequencing, and MRD was evaluated by flow cytometry. In overall, 51.02 and 8.75% of patients harbored NOTCH1 and FBXW7 mutations respectively. More favorable 10-year overall survival (OS), event-free survival (EFS), and disease-free survival (DFS) were seen in NOTCH1mut patients (NOTCH1mutvs. NOTCH1wt, OS, 82.7 ± 5.6% vs. 62.4 ± 7.4%, p = .020; EFS, 80.9 ± 5.8 vs. 48.4 ± 7.8%, p = .001; DFS, 82.9 ± 5.6 vs. 52.9 ± 7.7%, p = .001). NOTCH1 gene status and MRD post-induction were identified as independent prognostic factors. A combination of NOTCH1 gene status and MRD could distinguish patients with NOTCH1 mutations and MRD < 1 × 10-4 with 100% OS, EFS, and DFS. These results indicated NOTCH1 mutation predicted a favorable outcome in pediatric T-ALL and may be considered a risk stratification factor.

Optical Genome Mapping for Comprehensive Assessment of Chromosomal Aberrations and Discovery of New Fusion Genes in Pediatric B-Acute Lymphoblastic Leukemia.

PURPOSE: To assess the potential added value of Optical Genomic Mapping (OGM) for identifying chromosomal aberrations. METHODS: We utilized Optical Genomic Mapping (OGM) to determine chromosomal aberrations in 46 children with B-cell Acute lymphoblastic leukemia ALL (B-ALL) and compared the results of OGM with conventional technologies. Partial detection results were verified by WGS and PCR. RESULTS: OGM showed a good concordance with conventional cytogenetic techniques in identifying the reproducible and pathologically significant genomic SVs. Two new fusion genes (LMNB1::PPP2R2B and TMEM272::KDM4B) were identified by OGM and verified by WGS and RT-PCR for the first time. OGM has a greater ability to detect complex chromosomal aberrations, refine complicated karyotypes, and identify more SVs. Several novel fusion genes and single-gene alterations, associated with definite or potential pathologic significance that had not been detected by traditional methods, were also identified. CONCLUSION: OGM addresses some of the limitations associated with conventional cytogenomic testing. This all-in-one process allows the detection of most major genomic risk markers in one test, which may have important meanings for the development of leukemia pathogenesis and targeted drugs.

HLA-DRB1*16:02 is associated with PEG-asparaginase hypersensitivity.

Aim: To evaluate the associations between human leukocyte antigen (HLA)-DRB1 variants and the rs6021191 variant in nuclear factor of activated T cells 2 (NFATC2) with PEG-asparaginase hypersensitivity in children with acute lymphoblastic leukemia (ALL) treated according to the Chinese Children Leukemia Group (CCLG) ALL 2018 protocol. Methods:HLA-DRB1 genotyping was performed using a PCR sequence-based typing (SBT) method. NFATC2 rs6021191 was genotyped applying TaqMan Genotyping Assay. Results: T-ALL and higher risk groups were at higher risk for PEG-asparaginase hypersensitivity. No association was found between NFATC2 rs6021191 and PEG-asparaginase hypersensitivity. HLA-DRB1*16:02 variant was associated with PEG-asparaginase allergy both in univariate and multivariate logistic regression analysis. Conclusion: Our results confirm that variations in HLA-DRB1 might influence the development of asparaginase hypersensitivity.