[Analysis of 41 cases of non-metastatic Ewing's sarcoma in children]. / 儿童非转移性尤文肉瘤41例分析.

OBJECTIVES: To summarize the clinical characteristics, treatment outcomes, and prognostic factors of children with non-metastatic Ewing's sarcoma (ES). METHODS: A retrospective analysis was conducted on the clinical data of 41 children with non-metastatic ES diagnosed and treated at the Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine from January 2010 to December 2018. All patients underwent chemotherapy based on the RMS-2009 protocol of the center, and local treatment such as surgery and/or radiotherapy was performed according to risk grouping. The Kaplan-Meier method was used to calculate the overall survival (OS) and event-free survival (EFS) rates. Univariate prognostic analysis was performed using the log-rank test, and multivariate analysis was conducted with Cox regression. RESULTS: Of the 41 children, 21 were male and 20 were female. The median age at diagnosis was 7.7 years (range: 1.2-14.6 years). The median follow-up time for patients with event-free survival was 68.1 months (range: 8.1-151.7 months). As of the last follow-up, 33 patients were in complete remission, and the overall 5-year EFS and OS rates were (78±6)% and (82±6)%, respectively. Univariate analysis by the log-rank test showed that a tumor diameter ≥8 cm, time from diagnosis to start of local treatment ≥16 weeks, and incomplete surgical resection were associated with poor prognosis (P<0.05). Multivariate Cox regression analysis indicated that incomplete surgical resection (HR=8.381, 95%CI: 1.681-41.801, P=0.010) was an independent risk factor for poor prognosis in children with ES. Secondary tumors occurred in 2 cases. CONCLUSIONS: A comprehensive treatment strategy incorporating chemotherapy, surgery, and radiotherapy can improve the prognosis of children with ES. Poor prognosis is associated with an initial tumor diameter ≥8 cm, while complete surgical resection and early initiation of local treatment can improve outcomes.

Prognostic factors of childhood acute lymphoblastic leukemia with TCF3::PBX1 in CCCG-ALL-2015: A multicenter study.

BACKGROUND: Contemporary risk-directed treatment has improved the outcome of patients with acute lymphoblastic leukemia (ALL) and TCF3::PBX1 fusion. In this study, the authors seek to identify prognostic factors that can be used to further improve outcome. METHODS: The authors studied 384 patients with this genotype treated on Chinese Children's Cancer Group ALL-2015 protocol between January 1, 2015 and December 31, 2019. All patients provisionally received intensified chemotherapy in the intermediate-risk arm without prophylactic cranial irradiation; those with high minimal residual disease (MRD) ≥1% at day 46 (end) of remission induction were candidates for hematopoietic cell transplantation. RESULTS: The overall 5-year event-free survival was 84.4% (95% confidence interval [CI], 80.6-88.3) and 5-year overall survival 88.9% (95% CI, 85.5-92.4). Independent factors associated with lower 5-year event-free survival were male sex (80.4%, [95% CI, 74.8-86.4] vs. 88.9%, [95% CI, 84.1-93.9] in female, p = .03) and positive day 46 MRD (≥0.01%) (62.1%, [95% CI, 44.2-87.4] vs. 87.1%, [95% CI, 83.4-90.9] in patients with negative MRD, p < .001). The presence of testicular leukemia at diagnosis (n = 10) was associated with particularly dismal 5-year event-free survival (33.3% [95% CI, 11.6-96.1] vs. 83.0% [95% CI, 77.5-88.9] in the other 192 male patients, p < .001) and was an independent risk factor (hazard ratio [HR], 5.7; [95% CI, 2.2-14.5], p < .001). CONCLUSIONS: These data suggest that the presence of positive MRD after intensive remission induction and testicular leukemia at diagnosis are indicators for new molecular therapeutics or immunotherapy in patients with TCF3::PBX1 ALL.

Safety and efficacy of co-administration of CD19 and CD22 CAR-T cells in children with B-ALL relapse after CD19 CAR-T therapy.

BACKGROUND: CD19-targeted chimeric antigen receptor T-cell (CAR-T) therapy has shown remarkable efficacy in treating relapsed or refractory pediatric B-lineage acute lymphoblastic leukemia (B-ALL). However, poor results are obtained when the same product is reused in patients who relapse after CAR-T. Therefore, there is a need to explore the safety and efficacy of co-administration of CD19- and CD22-targeted CAR-T as a salvage second CAR-T therapy (CART2) in B-ALL patients who relapse after their first CD19 CAR-T treatment (CART1). METHODS: In this study, we recruited five patients who relapsed after CD19-targeted CAR-T. CD19- and CD22-CAR lentivirus-transfected T cells were cultured separately and mixed before infusion in an approximate ratio of 1:1. The total dose range of CD19 and CD22 CAR-T was 4.3 × 106-1.5 × 107/kg. Throughout the trial, we evaluated the patients' clinical responses, side effects, and the expansion and persistence of CAR-T cells. RESULTS: After CART2, all five patients had minimal residual disease (MRD)-negative complete remission (CR). The 6- and 12-month overall survival (OS) rates were 100%. The median follow-up time was 26.3 months. Three of the five patients bridged to consolidated allogeneic hematopoietic stem cell transplantation (allo-HSCT) after CART2 and remained in MRD-negative CR at the cut-off time. In patient No. 3 (pt03), CAR-T cells were still detected in the peripheral blood (PB) at 347 days post-CART2. Cytokine release syndrome (CRS) only occurred with a grade of ≤ 2, and no patients experienced symptoms of neurologic toxicity during CART2. CONCLUSIONS: Mixed infusion of CD19- and CD22-targeted CAR-T cells is a safe and effective regimen for children with B-ALL who relapse after prior CD19-targeted CAR-T therapy. Salvage CART2 provides an opportunity for bridging to transplantation and long-term survival. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR2000032211. Retrospectively registered: April 23, 2020.

High titers of neutralizing antibodies in the blood fail to eliminate SARS-CoV-2 viral RNA in the upper respiratory tract.

Retest-positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA, as a unique phenomenon among discharged individuals, has been demonstrated to be safe in the community. Still, the underlying mechanism of viral lingering is less investigated. In this study, first, we find that the frequency of viral RNA-positive retesting differs among variants. Higher ratios of viral RNA-positive retest were more frequently observed among Delta (61.41%, 514 of 837 cases) and Omicron (39.53%, 119 of 301 cases) infections than among ancestral viral infection (7.27%, 21 of 289 cases). Second, the tissues where viral RNA reoccurred were altered. Delta RNA reoccurred mainly in the upper respiratory tract (90%), but ancestral virus RNA reoccurred mainly in the gastrointestinal tract (71%). Third, vaccination did not reduce the frequency of viral RNA-positive retests, despite high concentrations of viral-specific antibodies in the blood. Finally, 37 of 55 (67.27%) Delta-infected patients receiving neutralizing antibody therapy become viral RNA retest positive when high concentrations of neutralizing antibodies still patrol in the blood. Altogether, our findings suggest that the presentence of high titers of neutralizing antibodies in the blood is incompetent in clearing residual viral RNA in the upper respiratory tract.

Identification of functional cooperative mutations of GNAO1 in human acute lymphoblastic leukemia.

Leukemogenesis is characterized by chromosomal rearrangements with additional molecular disruptions, yet the cooperative mechanisms are still unclear. Using whole-exome sequencing of a pair of monozygotic twins who were discordant for childhood acute lymphoblastic leukemia (ALL) with ETV6-RUNX1 (E/R) gene fusion successively after birth, we identified the R209C mutation of G protein subunit α o1 (GNAO1) as a new ALL risk loci. Moreover, GNAO1 missense mutations are recurrent in ALL patients and are associated with E/R fusion. Ectopic expression of the GNAO1 R209C mutant increased its GTPase activity and promoted cell proliferation and cell neoplastic transformation. Combined with the E/R fusion, the GNAO1 R209C mutation promoted leukemogenesis through activating PI3K/Akt/mTOR signaling. Reciprocally, activated mTORC1 phosphorylated p300 acetyltransferase, which acetylated E/R and thereby enhanced the E/R transcriptional activity of GNAO1 R209C. Thus, our study provides clinical evidence of the functional cooperation of GNAO1 mutations and E/R fusion, suggesting GNAO1 as a therapeutic target in human leukemia.

Prognostic factors for CNS control in children with acute lymphoblastic leukemia treated without cranial irradiation.

To identify the prognostic factors that are useful to improve central nervous system (CNS) control in children with acute lymphoblastic leukemia (ALL), we analyzed the outcome of 7640 consecutive patients treated on Chinese Children's Cancer Group ALL-2015 protocol between 2015 and 2019. This protocol featured prephase dexamethasone treatment before conventional remission induction and subsequent risk-directed therapy, including 16 to 22 triple intrathecal treatments, without prophylactic cranial irradiation. The 5-year event-free survival was 80.3% (95% confidence interval [CI], 78.9-81.7), and overall survival 91.1% (95% CI, 90.1-92.1). The cumulative risk of isolated CNS relapse was 1.9% (95% CI, 1.5-2.3), and any CNS relapse 2.7% (95% CI, 2.2-3.2). The isolated CNS relapse rate was significantly lower in patients with B-cell ALL (B-ALL) than in those with T-cell ALL (T-ALL) (1.6%; 95% CI, 1.2-2.0 vs 4.6%; 95% CI, 2.9-6.3; P < .001). Independent risk factors for isolated CNS relapse included male sex (hazard ratio [HR], 1.8; 95% CI, 1.1-3.0; P = .03), the presence of BCR-ABL1 fusion (HR, 3.8; 95% CI, 2.0-7.3; P < .001) in B-ALL, and presenting leukocyte count ≥50×109/L (HR, 4.3; 95% CI, 1.5-12.2; P = .007) in T-ALL. Significantly lower isolated CNS relapse was associated with the use of total intravenous anesthesia during intrathecal therapy (HR, 0.2; 95% CI, 0.04-0.7; P = .02) and flow cytometry examination of diagnostic cerebrospinal fluid (CSF) (HR, 0.2; 95% CI, 0.06-0.6; P = .006) among patients with B-ALL. Prephase dexamethasone treatment, delayed intrathecal therapy, use of total intravenous anesthesia during intrathecal therapy, and flow cytometry examination of diagnostic CSF may improve CNS control in childhood ALL. This trial was registered with the Chinese Clinical Trial Registry (ChiCTR-IPR-14005706).

Causes of death and treatment-related mortality in newly diagnosed childhood acute lymphoblastic leukemia treatment with Chinese Children's Cancer Group study ALL-2015.

To investigate the possible risk factors for death at post-treatment in children with acute lymphoblastic leukemia (ALL). A multivariate competing risk analysis was performed to retrospectively analyze the data of children with ALL who died after treatment with CCCG-ALL-2015 in China and to determine the possible risk factors for death at post-treatment in children with ALL. Age at the first diagnosis of ≥10 years; final risk level of high-risk; D19 minimal residual disease (MRD) (≥0.01%) and D46 MRD (≥0.01%); genetic abnormalities, such as KMT2A-rearrangement, c-Myc rearrangement, and PDGFRB rearrangement; and the presence of CNS3 (all P values, <0.05) were identified as independent risk factors, whereas the risk level at the first diagnosis of low-risk (LR) and ETV6::RUNX1 positivity was considered as independent protective factors of death in children with ALL. Among the 471 cases of death, 45 cases were treated with CCCG-ALL-2015 only, and 163 (34.61%) were treatment-related, with 62.42% due to severe infections. 55.83% of treatment-related mortality (TRM) occurred in the early phase of treatment (induction phase). TRM has a significant impact on the overall survival of pediatric patients with ALL. Moreover, the CCCG-ALL-2015 regimen has a better safety profile for treating children with ALL, with rates close to those in developed countries (registration number: ChiCTR-IPR-14005706; date of registration: June 4, 2014).

Extended vincristine and dexamethasone pulse therapy may not be necessary for children with TCF3-PBX1 positive acute lymphoblastic leukaemia.

The effect of prolonged pulse therapy with vincristine and dexamethasone (VD) during maintenance therapy on the outcome of paediatric patients with TCF3-PBX1 positive acute lymphoblastic leukaemia (ALL) remains uncertain. We conducted non-inferiority analysis of 263 newly diagnosed TCF3-PBX1 positive ALL children who were stratified and randomly assigned (1:1) to receive seven additional VD pulses (the control group) or not (the experimental group) in the CCCG-ALL-2015 clinical trial from January 2015 to December 2019 (ChiCTR-IPR-14005706). There was no significant difference in baseline characteristics between the two groups. With a median follow-up of 4.2 years, the 5-year event-free survival (EFS) and 5-year overall survival (OS) in the control group were 90.1% (95% confidence interval [CI] 85.1-95.4) and 94.7% (95% CI, 90.9-98.6) comparable to those in the experimental group 89.2% (95% CI 84.1-94.7) and 95.6% (95% CI 91.8-99.6), respectively. Non-inferiority was established as a one-sided 95% upper confidence bound for the difference in probability of 5-year EFS was 0.003, and that for 5-year OS was 0.01 by as-treated analysis. Thus, omission of pulse therapy with VD beyond one year of treatment did not affect the outcome of children with TCF3-PBX1 positive ALL.

Therapy-induced mutations drive the genomic landscape of relapsed acute lymphoblastic leukemia.

To study the mechanisms of relapse in acute lymphoblastic leukemia (ALL), we performed whole-genome sequencing of 103 diagnosis-relapse-germline trios and ultra-deep sequencing of 208 serial samples in 16 patients. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2) involved in drug response. Their prevalence was 17% in very early relapse (<9 months from diagnosis), 65% in early relapse (9-36 months), and 32% in late relapse (>36 months) groups. Convergent evolution, in which multiple subclones harbor mutations in the same drug resistance gene, was observed in 6 relapses and confirmed by single-cell sequencing in 1 case. Mathematical modeling and mutational signature analysis indicated that early relapse resistance acquisition was frequently a 2-step process in which a persistent clone survived initial therapy and later acquired bona fide resistance mutations during therapy. In contrast, very early relapses arose from preexisting resistant clone(s). Two novel relapse-specific mutational signatures, one of which was caused by thiopurine treatment based on in vitro drug exposure experiments, were identified in early and late relapses but were absent from 2540 pan-cancer diagnosis samples and 129 non-ALL relapses. The novel signatures were detected in 27% of relapsed ALLs and were responsible for 46% of acquired resistance mutations in NT5C2, PRPS1, NR3C1, and TP53. These results suggest that chemotherapy-induced drug resistance mutations facilitate a subset of pediatric ALL relapses.

A prospective multicenter study investigating rituximab combined with intensive chemotherapy in newly diagnosed pediatric patients with aggressive mature B cell non-Hodgkin lymphoma (CCCG-BNHL-2015): a report from the Chinese Children's Cancer Group.

The evidence for the safety and efficacy of adding rituximab to intensive chemotherapy in pediatric patients with aggressive mature B cell non-Hodgkin lymphoma/leukemia (B-NHL/B-AL) is not yet robust. In this prospective multi-institutional trial, 419 evaluable patients ≤ 16 years of age with newly diagnosed B-NHL/B-AL were enrolled. Patients were stratified into 4 risk groups according to stage, resection status, and serum lactate dehydrogenase. Patients in group R1 received 3 therapy courses in the treatment order A-B-A. Patients in group R2 received 5 courses A-B-A-B-A. Patients in group R3 received 6 courses A-BB-AA-BB-AA-BB. For patients in group R4, rituximab was added to the chemotherapy backbone for patients in R3 (A-RBB-RAA-RBB-RAA-BB). At a median follow-up of 54 months, the 4-year event-free survival (EFS) for the entire group was 88.3 ± 1.6% (76.0 ± 4.3% in the historical study). The EFS rates according to the intention-to-treat principle were 100%, 98.6 ± 1.2%, 94.2 ± 1.8%, and 73.5 ± 3.7% for patients in treatment groups R1, R2, R3, and R4, respectively (P < 0.001). There were 9 (2.1%) toxic deaths due to infection during treatment. Regarding the toxicities of rituximab, grade 3/4 thrombocytopenia, mucositis, and infection occurred in 44.0%, 33.3%, and 64.0% after courses R-BB and grade 3/4 neutropenia, thrombocytopenia, and infection occurred in 96.3%, 77.8%, and 54.1% after courses RAA. The addition of rituximab to intensive chemotherapy is feasible even in a developing country. EFS was significantly improved when compared with the historical data. clinicals.gov identifier: NCT02405676.

Medication therapy of high-dose methotrexate: An evidence-based practice guideline of the Division of Therapeutic Drug Monitoring, Chinese Pharmacological Society.

AIMS: A lot of medication risks related to high-dose methotrexate (HDMTX) therapy still remain to be identified and standardized. This study aims to establish an evidence-based practice guideline for individualized medication of HDMTX. METHODS: The practice guideline was launched by the Division of Therapeutic Drug Monitoring, Chinese Pharmacological Society. The guideline was developed following the WHO handbook for guideline development and the methodology of evidence-based medicine (EBM). The guideline was initially registered in the International Practice Guidelines Registry Platform (IPGRP-2017CN021). Systematic reviews were conducted to synthesize available evidence. A multicentre cross-sectional study was conducted using questionnaires to evaluate patients' perception and willingness concerning individualized medication of HDMTX. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach was used to rate the quality of evidence and to grade the strength of recommendations. RESULTS: Multidisciplinary working groups were included in this guideline, including clinicians, pharmacists, methodologists, pharmacologists and pharmacoeconomic specialists. A total of 124 patients were involved to integrate patient values and preferences. Finally, the guideline presents 28 recommendations, regarding evaluation prior to administration (renal function, liver function, pleural effusion, comedications, genetic testing), pre-treatment and routine dosing regimen, therapeutic drug monitoring (necessity, method, timing, target concentration), leucovorin rescue (initial timing, dosage regimen and optimization), and management of toxicities. Of these, 12 are strong recommendations. CONCLUSIONS: We developed an evidence-based practice guideline with respect to HDMTX medication using a rigorous and multidisciplinary approach. This guideline provides comprehensive and practical recommendations involving the whole process of HDMTX administration to health care providers.

MYCN protein stability is a better prognostic indicator in neuroblastoma.

OBJECTIVE: MYCN oncogene amplification is associated with treatment failure and poor prognosis in neuroblastoma. To date, most detection methods of MYCN focus on DNA copy numbers instead of protein expression, which is the real one performing biological function, for poor antibodies. The current investigation was to explore a fast and reliable way to detect MYCN protein expression and evaluate its performance in predicting prognosis. METHODS: Several MYCN antibodies were used to detect MYCN protein expression by immunohistochemistry (IHC), and one was chosen for further study. We correlated the IHC results of MYCN from 53 patients with MYCN fluorescence in situ hybridization (FISH) and identified the sensitivity and specificity of IHC. The relationship between patient prognosis and MYCN protein expression was detected from this foundation. RESULTS: MYCN amplification status detected by FISH was most valuable for INSS stage 3 patients. In the cohort of 53 samples, IHC test demonstrated 80.0-85.7% concordance with FISH results. Further analyzing those cases with inconsistent results, we found that patients with MYCN amplification but low protein expression tumors always had a favorable prognosis. In contrast, if patients with MYCN non-amplified tumors were positive for MYCN protein, they had a poor prognosis. CONCLUSION: MYCN protein level is better than MYCN amplification status in predicting the prognosis of neuroblastoma patients. Joint of FISH and IHC could confirm MYCN protein stability and achieve better prediction effect than the singular method.

Pulse therapy with vincristine and dexamethasone for childhood acute lymphoblastic leukaemia (CCCG-ALL-2015): an open-label, multicentre, randomised, phase 3, non-inferiority trial.

BACKGROUND: Vincristine plus dexamethasone pulses are generally used throughout maintenance treatment for childhood acute lymphoblastic leukaemia. However, previous studies remain inconclusive about the benefit of this maintenance therapy and the absence of randomised, controlled trials in patients with low-risk or high-risk acute lymphoblastic leukaemia provides uncertainty. We therefore aimed to determine if this therapy could be safely omitted beyond 1 year of treatment without leading to an inferior outcome in any risk subgroup of childhood acute lymphoblastic leukaemia. METHODS: This open-label, multicentre, randomised, phase 3, non-inferiority trial involved 20 major medical centres across China. We enrolled patients who were aged 0-18 years with newly diagnosed acute lymphoblastic leukaemia that was subsequently in continuous remission for 1 year after initial treatment. Patients with secondary malignancy or primary immunodeficiency were excluded. Eligible patients were classified as having low-risk, intermediate-risk, or high-risk acute lymphoblastic leukaemia based on minimal residual disease and immunophenotypic and genetic features of leukaemic cells. Randomisation and analyses were done separately for the low-risk and intermediate-to-high-risk cohorts. Randomisation was generated by the study biostatistician with a block size of six. Stratification factors included participating centre, sex, and age at diagnosis; the low-risk cohort was additionally stratified for ETV6-RUNX1 status, and the intermediate-to-high-risk cohort for cell lineage. Patients in each risk cohort were randomly assigned (1:1) to either receive (ie, the control group) or not receive (ie, the experimental group) seven pulses of intravenous vincristine (1·5 mg/m2) plus oral dexamethasone (6 mg/m2 per day for 7 days) during the second year of treatment. The primary endpoint was difference in 5-year event-free survival between the experimental group and the control group for both the low-risk and intermediate-to-high-risk cohorts, with a non-inferiority margin of 0·05 (5%). The analysis was by intention to treat. This trial is registered with the Chinese Clinical Trial Registry, ChiCTR-IPR-14005706. FINDINGS: Between Jan 1, 2015, and Feb 20, 2020, 6141 paediatric patients with newly diagnosed acute lymphoblastic leukaemia were registered to this study. Approximately 1 year after diagnosis and treatment, 5054 patients in continuous remission were randomly assigned, including 2923 (1442 in the control group and 1481 in the experimental group) with low-risk acute lymphoblastic leukaemia and 2131 (1071 control, 1060 experimental) with intermediate-to-high risk acute lymphoblastic leukaemia. Median follow-up for patients who were alive at the time of analysis was 3·7 years (IQR 2·8-4·7). Among patients with low-risk acute lymphoblastic leukaemia, no difference was observed in 5-year event-free survival between the control group and the experimental group (90·3% [95% CI 88·4-92·2] vs 90·2% [88·2-92·2]; p=0·90). The one-sided 95% upper confidence bound for the difference in 5-year event-free survival probability was 0·024, establishing non-inferiority. Among patients with intermediate-to-high-risk acute lymphoblastic leukaemia, no difference was observed in 5-year event-free survival between the control group and the experimental group (82·8% [95% CI 80·0-85·7] vs 80·8% [77·7-84·0]; p=0·90), but the one-sided 95% upper confidence bound for the difference in 5-year event-free survival probability was 0·055, giving a borderline inferior result for those in the experimental group. In the low-risk cohort, we found no differences in the rates of infections, symptomatic osteonecrosis, or other complications during the second year of maintenance treatment between patients in the control and experimental groups. Patients with intermediate-to-high-risk acute lymphoblastic leukaemia in the control group were more likely to develop grade 3-4 pneumonia (26 [2·4%] of 1071 vs ten [0·9%] of 1060) and vincristine-related peripheral neuropathy (17 [1·6%] vs six [0·6%]) compared with the experimental group. Incidence of grade 5 fatal infection was similar between the control group and the experimental group in both the low-risk cohort (two [0·1%] of 1442 vs five [0·3%] of 1481) and intermediate-to-high risk cohort (six [0·6%] of 1071 vs five [0·5%] of 1060). INTERPRETATION: Vincristine plus dexamethasone pulses might be omitted beyond 1 year of treatment for children with low-risk acute lymphoblastic leukaemia. Additional studies are needed for intermediate-to-high-risk acute lymphoblastic leukaemia. FUNDING: VIVA China Children's Cancer Foundation, the National Natural Science Foundation of China, the China fourth round of Three-Year Public Health Action Plan (2015-2017), Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences, US National Cancer Institute, St Baldrick's Foundation, and the American Lebanese Syrian Associated Charities. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Identification of fusion genes and characterization of transcriptome features in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a clonal malignancy of immature T cells. Recently, the next-generation sequencing approach has allowed systematic identification of molecular features in pediatric T-ALL. Here, by performing RNA-sequencing and other genomewide analysis, we investigated the genomic landscape in 61 adult and 69 pediatric T-ALL cases. Thirty-six distinct gene fusion transcripts were identified, with SET-NUP214 being highly related to adult cases. Among 18 previously unknown fusions, ZBTB16-ABL1, TRA-SALL2, and involvement of NKX2-1 were recurrent events. ZBTB16-ABL1 functioned as a leukemogenic driver and responded to the effect of tyrosine kinase inhibitors. Among 48 genes with mutation rates >3%, 6 were newly found in T-ALL. An aberrantly overexpressed short mRNA transcript of the SLC17A9 gene was revealed in most cases with overexpressed TAL1, which predicted a poor prognosis in the adult group. Up-regulation of HOXA, MEF2C, and LYL1 was often present in adult cases, while TAL1 overexpression was detected mainly in the pediatric group. Although most gene fusions were mutually exclusive, they coexisted with gene mutations. These genetic abnormalities were correlated with deregulated gene expression markers in three subgroups. This study may further enrich the current knowledge of T-ALL molecular pathogenesis.

The efficacy and safety of a homoharringtonine-based protocol for children with acute myeloid leukemia: A retrospective study in China.

The standard chemotherapy for acute myeloid leukemia (AML) is usually composed of anthracyclines and cytarabine. We previously reported that homoharringtonine (HHT) was incorporated into regimens for pediatric AML with acceptable efficacy and tolerable toxicity. We treated newly diagnosed AML patients aged 0-18 years on the AML-SCMC-2009 protocol. A total of 102 de novo newly diagnosed AML patients aged 0-18 years were enrolled. All patients were treated with ten courses of chemotherapy including double induction, high dose cytarabine consolidation, and maintenance. The cumulative dose of HHT was 165 mg/m2 and the daunorubicin dose was 120 mg/m2. Complete remission (CR), overall survival (OS) rate, event free survival (EFS) rate, adverse effect response and prognosis factors were retrospectively evaluated to investigate the long-term outcome and safety of this protocol. Eighty-two patients (80.4%) achieved complete remission with the first induction. The 5-year overall survival and event-free survival rates were 65.0% (SE, 4.9%) and 53.3% (SE, 5.2%), respectively. A first induction failure, age older than 2 years, and BCR-ABL translocation were associated with a significantly worse outcome (p < 0.05). No significant drug-related cardiac toxicity was observed. AML-SCMC-2009 protocol which use HHT as backbone drug is efficacious and tolerated. HHT could partially replace anthracycline to be an effective option for childhood AML.

CD200 is overexpressed in neuroblastoma and regulates tumor immune microenvironment.

Patients with pediatric cancers such as neuroblastoma (NB) are often unresponsive to checkpoint blockade immunotherapy. One major factor in pediatric tumor resistance to immunotherapy is considered to be the low mutation rate of pediatric tumors. Another factor may be the overexpression of additional inhibitory pathways. While analyzing the RNA-sequencing database TARGET, we found that human NB tumors overexpress immune checkpoint molecule CD200. To determine its significance and impact on tumor immune microenvironment, we analyzed 49 cases of previously untreated, surgically removed NB tumors using immunohistochemistry and multi-color flow cytometry (FACS). We found that CD200 is overexpressed in more than 90% of NB tumors. In the tumor microenvironment of NB, CD200 is mainly overexpressed in CD45- NB tumor cells, while its cognate receptor (CD200R) is mainly expressed in HLA-DR+CD14+ myeloid cells and CD11c+ dendritic cells. Low-level expression of CD200R is also observed in tumor-infiltrating CD4+ and CD8+ T cells. In NB tumors with higher CD200 expression (CD200high), we observed lower numbers of HLA-DR+CD14+ myeloid cells and less tumor-infiltrating CD4+ and CD8+ T cells. Moreover, we found that CD4+ and CD8+ T cells produced less IFN-γ and/or TNF-α in CD200high NB tumors. Thus, CD200-CD200R pathway appears to downregulate anti-tumor immunity in the tumor microenvironment of NB tumors, and blockade of this pathway may be beneficial for NB patients.

ARHGEF12 regulates erythropoiesis and is involved in erythroid regeneration after chemotherapy in acute lymphoblastic leukemia patients.

Hematopoiesis is a finely regulated process in vertebrates under both homeostatic and stress conditions. By whole exome sequencing, we studied the genomics of acute lymphoblastic leukemia (ALL) patients who needed multiple red blood cell (RBC) transfusions after intensive chemotherapy treatment. ARHGEF12, encoding a RhoA guanine nucleotide exchange factor, was found to be associated with chemotherapy-induced anemia by genome-wide association study analyses. A single nucleotide polymorphism (SNP) of ARHGEF12 located in an intron predicted to be a GATA1 binding site, rs10892563, is significantly associated with patients who need RBC transfusion (P=3.469E-03, odds ratio 5.864). A luciferase reporter assay revealed that this SNP impairs GATA1-mediated trans-regulation of ARHGEF12, and quantitative polymerase chain reaction studies confirmed that the homozygotes status is associated with an approximately 61% reduction in ARHGEF12 expression (P=0.0088). Consequently, erythropoiesis was affected at the pro-erythroblast phases. The role of ARHGEF12 and its homologs in erythroid differentiation was confirmed in human K562 cells, mouse 32D cells and primary murine bone marrow cells. We further demonstrated in zebrafish by morpholino-mediated knockdown and CRISPR/Cas9-mediated knockout of arhgef12 that its reduction resulted in erythropoiesis defects. The p38 kinase pathway was affected by the ARHGEF12-RhoA signaling in K562 cells, and consistently, the Arhgef12-RhoA-p38 pathway was also shown to be important for erythroid differentiation in zebrafish as active RhoA or p38 readily rescued the impaired erythropoiesis caused by arhgef12 knockdown. Finally, ARHGEF12-mediated p38 activity also appeared to be involved in phenotypes of patients of the rs10892563 homozygous genotype. Our findings present a novel SNP of ARHGEF12 that may involve ARHGEF12-RhoA-p38 signaling in erythroid regeneration in ALL patients after chemotherapy.

A novel fusion gene PLEKHA6-NTRK3 in langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is the most common histiocytosis with constitutive activation of the RAS-RAF-MEK-ERK (MAPKinase) cell signaling pathway. We analyzed 89 cases of BRAF and MAP2K1 mutations by Sanger sequencing, of which 18 cases showed that these two gene mutations are negative. Whole genome sequencing of suitable specimens in these negative cases revealed a translocation from the 3 intron of PLEKHA6 to the 13 intron of NTRK3 in one case. We identified that this translocation could cause a novel fusion mutation, PLEKHA6-NTRK3. Overexpression of the PLEKHA6-NTRK3 mutant in NIH 3T3 cells enhanced MAPKinase pathway activation, promote cell growth. Our result suggested that a new mutation need be included in LCH molecular screening panel to better define its prevalence in LCH.

Current Treatment Patterns of Aplastic Anemia in China: A Prospective Cohort Registry Study.

Aplastic anemia (AA) is a hematologic disease characterized by pancytopenia and hypocellular bone marrow, potentially leading to chronic anemia, hemorrhage, and infection. The China Aplastic Anemia Committee and British Committee for Standards in Haematology guidelines recommend hematopoietic stem-cell transplantation (HSCT) or immunosuppressive therapy (IST) comprising antithymocyte globulin (ATG) with cyclosporine (CsA) as initial treatment for AA patients. With limited epidemiological data on the clinical management of AA in Asia, a prospective cohort registry study involving 22 AA treatment centers in China was conducted to describe the disease characteristics of newly diagnosed AA patients and investigate real-world treatment patterns and patient outcomes. Of 340 AA patients, 72.9, 12.6, and 3.5% were receiving IST, traditional Chinese medicine, and HSCT, respectively, at baseline; only 22.2% of IST-treated patients received guideline-recommended ATG with CsA initially. Almost all patients received supportive care (95.6%) as blood transfusion (97.8%), antibiotics (63.7%), and/or hematopoietic growth factors (58.2%). Overall, 64.8% achieved a partial or complete response, and 0.9% experienced relapse. No new safety concerns were identified; serious adverse events were largely unrelated to the treatment regimen. These results demonstrate the need to identify and minimize treatment barriers to standardize and align AA management in China with treatment guideline recommendations and further improve patient outcomes.