BACKGROUND:B-cell lymphoblastic lymphoma (B-LBL) represents a rare variety of non-Hodgkin lymphoma, with limited research on its biology, progression, and management. METHODS:A retrospective analysis was performed on the clinical characteristics of 256 patients aged ≤18 years who received treatment under the China Net Childhood Lymphoma (CNCL)-NHL-2017-lymphoblastic lymphoma regimen from April 2017 to March 2023. RESULTS:Among the 256 patients, the median age at diagnosis was 5.0 years, with a slight male predominance. Subcutaneous tissues, skin, and osteolytic bone were the most common sites of the disease. More than 90% of patients exhibited disseminated disease (Stage III or IV). Approximately 19.9% of the diagnosed patients exhibited central nervous system involvement. Adverse events were observed in 33 patients (12.9%), with disease progression or relapse occurring in 10.2% of cases, particularly linked to unfavorable outcomes in instances of early relapse or progression. Salvage chemotherapy combined with immunotherapies, followed by bridging hematopoietic stem cell transplantation, significantly improved the prognosis of relapse and disease progression in children. Overall, the follow-up time was 37.6 (Q1-Q3, 28.9-38.0) months, and 3-year event-free survival rate and overall survivals were 86.3% ± 2.5% and 95.2% ± 1.5%, respectively, with a treatment-related mortality of 1.6%. Multivariate analysis showed that poor prednisone response and no complete remission on Day 33 of induction were risk factors for poor prognosis. CONCLUSION:The CNCL-NHL-2017-lymphoblastic lymphoma regimen was effective in children with B-LBL. The response to initial treatment is vital for improving prognosis in patients with B-LBL.

Objective：To analyze the clinical features of central nervous system involvement (CNS3) in pediatric anaplastic large cell lymphoma (ALCL) and evaluate the efficacy of CNCL-ALCL-2017 protocol for the treatment of CNS3. Methods：The clinical data of 215 pediatric ALCL patients ≤18 years old enrolled in the Chinese Children's Cancer Group (CNCL) between April 2017 and March 2023 were collected sequentially. All enrolled patients staging and CNS layering were according to the IPNHLSS staging system and treated with CNCL-ALCL-2017 protocol (modified BFM-ALCL99 protocol). The clinical features at diagnosis and treatment outcomes of CNS3 patients were analyzed. All patients were followed up until June 30, 2023. Data were collected using a unified information platform. Associations between patient characteristics were analyzed with Chi-squared or Fisher's exact tests. Eventfree survival (EFS) and overall survival (OS) curves were estimated according to the KaplanMeier method and compared by Logrank test. Furthermore, statistical analysis was performed using SPSS 22.0 software. P<0.05 was considered statistically significant. Results: Among the 215 ALCL pediatric patients, 17 (7.9%) had CNS3, including 13 males and 4 females with median age of 8.0 (range 1.0-14.0) years. In this patients, brain parenchyma and spinal cord involvement were found in 58.8% (10/17) patients on imaging, with space-occupying lesions in 7 cases and abnormal signals on MRI in 4 cases. Concurrent space-occupying lesions and abnormal signals on MRI was detected in 1 case. Cerebrospinal fluid (CSF) positivity was detected in 35.3% (6/17) patients, including 2 cases with positive ALK gene and 5 cases with positive flow cytometry, with 1 case positive for both. Cranial nerve deficits such as facial nerve palsy, visual or hearing impairment were observed in 23.5% (4/17) patients. Concurrent CSF, imaging and cranial nerve abnormalities were present in 5.9% (1/17), while two of this were present in 29.4% (5/17). Isolated CSF positivity without other abnormalities was detected in 23.5% (4/17). Pathological subtypes included common type (76.5%, 13/17), histiocyte-rich variant (5.9%, 1/17), small cell variant (5.9%, 1/17), others (11.8%, 2/17). Immunohistochemistry showed ALK + in 94.1% (16/17) and CD3 + in 76.5% (13/17). Concurrent ALK gene positivity in both peripheral blood and bone marrow was detected in 64.7% (11/17), with 58.8% (10/17) positive in both samples. The median follow-up of all patients was 35.4 months (range 0.5-74.9). All CNS3 patients had stage IV disease and were treated with regimen D (with 5 g/m 2 methotrexate). The 3-year OS was 94.3%, 98.2% and 93.3% for CNS1 (144, 67.0%), CNS2 (54, 25.1%) and CNS3 (17, 7.9%) patients, respectively; the 3-year EFS was 85.6%, 90.3% and 86.7%, respectively. No significant differences were found among the three groups. The 3-year OS and EFS for the whole cohort were 95.1% and 84.7%, respectively, also with no significant differences from the CNS3 group. Of the CNS3 patients, 1 was lost to follow-up after the first course, 1 died during treatment, and the interim CR rate was 75.0% (12/16). CSF conversion was achieved in 3/6 (50%) CSF-positive patients. 6.25% (1/16) patient had CSF ALK gene re-positivity during maintenance and persistent positivity despite additional ALK inhibitor alectinib and intrathecal therapy. In the CNS1 group, 2 cases were lost to follow-up and 4 cases died. The relapse rate was 11.4% (16/140), and the rate of central nervous system relapse was 2.1% (3/140). Additionally, the rate of CNS1 relapse was slightly higher but not statistically significantly different for CNS2. Conclusions: CNS involvement is relatively rare in pediatric ALCL. Detection rate of CNS3 can be improved by CSF flow cytometry and gene screening. The prognosis of CNS3 was significantly improved with CNCL-ALCL-2017 protocol, which may be related to the increased dose of methotrexate in the protocol. The short follow-up time and the small number of cases in this group may affect the results. Furthermore, CNS relapse rate of CNS2 was not markedly higher compared to CNS1. Keywords: Anaplastic large cell lymphoma; Pediatric; Central nervous system; Clinical features; Prognosis

To analyze the clinical features of early T-cell precursor acute lymphoblastic leukemia/lymphoma (ETP-ALL/LBL) in pediatric patients and to summarize the efficacy evaluation of the 2017 protocol by the China Net Childhood Lymphoma (CNCL) Non-Hodgkin Lymphoma (NHL) 2017-LBL for ETP-LBL. Methods Clinical data of 19 children with ETP-LBL admitted to CNCL from May 2017 to August 2023 were retrospectively collected. Results The median age of onset was 10 years (4.5-13 years), including 16 males (84.2%). All patients were in stage IV and were treated with high-risk chemotherapy regimen. 11 of them (57.9%) underwent hematopoietic stem cell transplantation (HSCT). The proportion of leukemia stage in the HSCT group was significantly higher than that in the chemotherapy group (72.7% vs. 25.0%), and the central nervous system (CNS) status was CNS2/3 in the HSCT group (100% vs. 50%).The 3-year overall survival (OS) and event free survival (EFS) were (93.8±0.61) % and (88.2±0.78) %, respectively. Conclusion Over 50% of pediatric ETP-LBL patients received HSCT, with their long-term survival rates comparable to the chemotherapy group.

T-cell lymphoblastic lymphoma (T-LBL) is an aggressive lymphoma that primarily affects children and young adults, and a comprehensive understanding of its molecular features is crucial for improving patient outcomes. In this study, 552 patients were included, with targeted next-generation sequencing of 262 lymphoma-associated genes performed on tumour samples from 119 patients. The associations between mutations and survival rates, as well as relapse and other clinical factors, were analysed. The results demonstrated that pleural effusion (PE) invasion was significantly associated with adverse event-free survival (EFS) and overall survival (OS) (p < 0.05). Additionally, we identified 92 genes with recurrent mutations, among which NOTCH1 (44%), FBXW7 (28%), PHF6 (11%), KRAS (10%) and NRAS (10%) were the most frequently altered. Patients with NOTCH1 mutations exhibited improved EFS and OS (p < 0.01), whereas those carrying CREBBP, PTEN and LYST mutations exhibited worse prognosis (p < 0.05). In conclusion, NOTCH1 mutations are associated with a favourable prognosis in paediatric T-LBL, while CREBBP, PTEN and LYST mutations, as well as PE invasion, are linked to poor prognosis. This study identifies key molecular and clinical factors in paediatric T-LBL progression, aiding high-risk patient identification and personalized treatment strategies.

China-Net Childhood Lymphoma (CNCL) group B-NHL-2017 study is a prospective multi-center study in China, with the purpose of standardizing the diagnosis and treatment of childhood lymphoma, and improving the prognosis. From May 2017 to June 2023, 20 centers participated in the diffuse large B-cell lymphoma (DLBCL) study. The clinical data were analyzed to summarize the clinical characteristics, treatment response and outcome. The primary endpoint was 5-year event-free survival (EFS). The trial is registered with the Chinese Clinical Trial Registry (ChiCTR1800020067). A total of 138 children and adolescents were enrolled, including 101 males and 37 females. The median age of disease diagnosis was 9.0 years (range: 2.3–15.5 years). The range of follow-up time was 17 d–6.0 years. A total of 12 events occurred in this study, including 7 deaths. of which 4 patients died of disease and chemotherapy comorbidities (severe infection, septic shock, etc.), 1 died of disease progression (enlargement of the primary tumor and tumor metastasis), 1 died of recurrence, and 1 died of severe pneumonia in the third year after completing all chemotherapy courses. Recurrence occurred in 6 (4.3

Supplementary Tables S1, S2, S5-S13 includes Supplementary Tables S1, S2, S5-S13. Supplementary Table S1 provides a summary of BL and DHL patient demographics for the immunohistochemistry study presented in Fig. 1, E and F. Supplementary Table S2 lists the MYC-dysregulated genes whose expression is significantly altered following depletion of eIF5A or DHPS, related to Fig. 4F. Supplementary Table S5 shows DHPS GISTIC count and survival of select TCGA PanCancer datasets, related to Fig. 7J. Supplementary Table S6 shows the genetic mouse models used in this study, related to Methods. Supplementary Table S7 shows the mouse and human cell lines used in this study, related to Methods. Supplementary Table S8 lists the antibodies used in this study, related to Methods. Supplementary Table S9 lists reagents used in this study, related to Methods. Supplementary Table S10 summarizes the plasmids used in this study, related to Methods. Supplementary Table S11 lists the sequences of the oligonucleotides used in this study, related to Methods. Supplementary Table S12 lists accession numbers and publicly deposited data from this study, related to Methods. Supplementary Table S13 lists software and algorithms used in this study, related to Methods.

Burkitt lymphoma (BL) is a B-cell malignancy with a rapid doubling time, originating in follicular germinal centers. We aimed to explore the characteristics and prognosis of childhood Burkitt leukemia. A total of 124 children with Burkitt leukemia enrolled during the 6-year period of China Net Childhood Lymphoma- mature B-cell lymphoma 2017 regimen (CNCL-B-NHL-2017) were assessed. The median age at onset was 7 years (1–15 years), with a male-to-female ratio of 4.17:1. Of the total, 50.8

Objective: To investigate the clinical-pathology characteristics, risk factors, necessary for VBL maintenance therapy，through summarize the clinical data of 221 cases of pediatric Anaplastic Large Cell Lymphoma (ALCL), treated with CNCL-ALCL-2017 witch is modify from BFM-ALCL-99 (±vincristine maintenance therapy) in China Net Childhood Lymphoma (CNCL). Methods: Data were collected on 221 children with ALCL enrolled from CNCL at time between April 2017 to March 2023, including: numbers of cases enrolled in each single center, gender and age at the time of initial diagnosis, the first initial symptom, a delay diagnosis, the misdiagnosis disease , the site of involvement, the level of blood uric acid, the level of blood lactate dehydrogenase, the bone marrow and CNS status, tumor complication, complicated with HLH, staging and treatment subgroups, pathological subtypes, CD3 expression in tumor tissue, bone marrow and peripheral blood ALK gene expression at initial diagnosis (qPCR + FISH), and treatment outcomes, treatment strategy(vincristine maintenance or not), time from initial diagnose to relapse, second-line treatment regimen after relapse, and treatment outcomes after relapsed. Statistical analysis was conducted using SPSS 21.0 software. Results: 221 cases were from 22 hospitals in China. Male = 144 cases, female = 71 cases, age range from 1-16 years (median age 8.9 years), duration from initial symptoms onset to diagnosis was 0.3-11 months (median time 1.0 months), delayed diagnosis was present in 51(23%) children (45 children were misdiagnosed with infectious diseases). Pathological subtypes were: common sub type = 150(67.8%), small cell sub type = 19(8.5%), histiocytic variant subtype = 9(4%), ALK negative subtype = 12(5.4%).others = 31(14%). CD3 expression in tumor: negative = 119, positive = 91. ALK positive by qPCR in peripheral blood at the time of the initial diagnosis = 77, ALK positive by qPCR in bone marrow = 78, bulky disease= 21, mediastinal invasion = 84, CNS invasion = 17, skin invasion = 32. tumor related HLH = 25, normal LDH level at initial diagnosis = 133, 1-fold elevated = 45, 2-3-fold elevated = 39, 4-fold elevated = 2, >4-fold elevated = 2, stage I = 5, stage II = 24, stage III = 70, stage IV = 120 , leukemic stage = 2 . Grouping: group A = 1, group B = 21, group C = 191, group D = 8. Vincristine maintenance = 121. Median follow-up time was 35.4 months (0.5-74.9 months), OS at 3 years = 95.1±1.5% (95% CI = 90%-97.3%, Fig. 1), EFS at 3 years = 84.7±4.5% (95% CI = 79%-89.9%, Fig. 2), and there were a total of 23 patients with events, median time was7 months. There were 10 patients died, 5 of them quit to the treatment, median time was 5 months, and 13 patients who had an event but still alive after second line treatment. Univariate results of the 3 years EFS are detailed in Table 1, with statistically significant including (<0.05): tumor related HLH before treatment, LDH levels higher than 4 times normal and MDD positive at the beginning of the disease, and treated without VBL. The event patients are 23 cases ,the detailed in Table 2, which showed that the earlier the event occurs, the higher the mortality rate. Conclusion:Pediatric ALCL in China is mostly found in school-age boys, and it is easy to be diagnosed as infectious diseases at the time of initial diagnosis due to high fever and elevated CRP. 87% of patients were diagnosed as late stage or high-risk group. The application of the CNCL-ALCL-2017 protocol showed a 3-year OS 84.7% and 3-year EFS 95.1%, indicating that the efficacy was significantly better than that of various centers before the multi center cooperation. The overall survival time is significantly better than the event free survival time, indicating that most patients with progression and recurrence still have a chance of re remission after second line treatment. Adverse prognostic factors include a significant increase in LDH levels at initial diagnosis, initial onset of HLH, positive MDD before treatment, and no use of vinblastine maintenance therapy. Recurrent children: The median recurrence time is 7 months, and the prognosis of early progression and recurrence is worse than that of late recurrence. Key words: Anaplastic large cell lymphoma, Pediatric, Clinical-pathology features, Prognosis

BACKGROUND:People living with HIV (PWH) have improved life expectancy because of effective human immunodeficiency virus (HIV) therapy but still experience immune impairment (e.g., altered CD4/CD8 T cells). We hypothesized that tumors diagnosed in PWH would have distinct molecular features. METHODS:We utilized whole-exome sequencing of paired tumor and germline DNA and RNA from 229 patients with cancer enrolled into the Oncology Research Information Exchange Network to classify total tumor mutation burden (TMB), MHC class I neoantigen count, and MHC class II neoantigen count. RESULTS:Specimens from 229 patients with cancer (110 PWH and 119 without HIV) were evaluated. Average TMB for tumors diagnosed in PWH was 249, compared with 172 for those without HIV. After adjustment for age, sex, race, smoking, and cancer site, the association between HIV and TMB remained statistically significant (OR = 1.72; 95% confidence interval (CI), 1.26-2.43). We further observed an association between HIV and higher putative class I neoantigen count (OR = 1.62; 95% CI, 1.10-2.41) but no association with putative class II neoantigens. When considering cancer sites separately in unadjusted analyses, average TMB was elevated in PWH for thyroid (P < 0.01) and bladder cancers (P = 0.03) and sarcoma (P = 0.04). Similarly, putative class I neoantigen count was elevated in PWH for head and neck (P < 0.01) and thyroid (P = 0.01) cancers, as well as sarcoma (P = 0.04). CONCLUSIONS:Our findings indicate that tumors diagnosed in PWH harbor a higher TMB and a higher number of putative class I neoantigens. IMPACT:A higher TMB in PWH may portend a more favorable response to certain cancer treatment modalities such as immune checkpoint inhibitors.

Studies have confirmed that rituximab (RTX) can improve the efficacy of BL, but there is a certain effect on the level of immunoglobulin, which will lead to the verification of infection, and the previous study of our center has confirmed that reducing the dose of RTX (4 doses) can achieve a similar effect to the standard dose of RTX (6 doses), can it reduce the effect on the level of immunoglobulin? To date, few studies have concentrated on the effects of immunoglobulin (Ig) on Chinese paediatric patients. This study aimed to examine whether there is a variation in the impact of different doses of RTX on immunoglobulin levels in the high-risk group of children with BL. Clinical data of high-risk pediatric patients with BL who were treated in Beijing Children’s Hospital (Beijing, China) were retrospectively analysed. Baseline characteristics and serum Ig levels were collected at four distinct time points (t0 = pre-chemotherapy, t1 = at the end of chemotherapy, t2 = 6 months post-chemotherapy, t3 = 12 months post-chemotherapy). Ig levels were measured at various time points before and after treatment within three RTX treatment groups: R0 group (standard chemotherapy without RTX), R6 group (6 doses of RTX + chemotherapy), and R4 group (4 doses of RTX + chemotherapy). The objective was to compare whether differences existed among the three groups. The results revealed that the study enrolled 300 high-risk BL patients, including 256 boys and 44 girls, distributed across three groups based on RTX dosage: R0 group (n = 38), R6 group (n = 87), and R4 group (n = 175). Median Ig levels were assessed at four time points (t0, t1, t2, t3) for each group. In the R0 group, IgA and IgM levels significantly decreased at t1 compared with t0 (P = 0.006 and 0.002, respectively), while were gradually recovered at t2, returning to t0 levels at t3 (P = 0.073 and 0.293, respectively). IgG levels exhibited no significant difference between t0 and t1 (P = 0.89), reaching their lowest levels at t2 and returning to t0 levels at t3 (P = 0.14). In the R4 group, the minimum levels of IgA, IgM, and IgG were identified at t1 (P < 0.001, < 0.001, and < 0.001, respectively), which were gradually recovered at t2, while remained lower than t0 levels at t3 (P < 0.001, < 0.001, and = 0.005, respectively). The R6 group exhibited reduction in IgA and IgM levels at t1, with gradual recovery at t2 and t3, while remained lower than t0 levels (P = 0.003 and < 0.001, respectively). IgG levels in the R6 group decreased at t1 (P < 0.001) and did not return to t0 levels at t3 (P = 0.004). In the R4 and R6 groups, it was observed that children with hypogammaglobulinemia pre-RTX were more likely to combine with persistent hypogammaglobulinemia (PH-Ig) post-RTX. A 1:1 matched comparison between R4 and R6 groups (78 patients each) revealed consistently higher IgA, IgM, and IgG levels in the R4 group at each time point after chemotherapy. Notably, IgA and IgG levels recovered earlier in the R4 group than those in the R6 group (P < 0.05). Burkitt lymphoma in the high-risk group were more likely to complicated hypoimmunoglobulinemia after treatment, it is important to monitor Ig levels before and during treatment, and to inform replacement therapy for Ig. Compared with standard chemotherapy group, the RTX group had a longer time of low Ig and a slower recovery, reducing the dosage of rituximab can improve the recovery of IgA and IgG levels.

PURPOSE:To analyze the prognosis of children with central nervous system 3 lymphoblastic lymphoma (LBL) treated with increased four courses of high-dose methotrexate and the times of intrathecal injection, without prophylactic cranial irradiation. PATIENTS AND METHODS: From April 2017 to September 2023, 883 Chinese children with LBL were registered in the China Net-Childhood Lymphoma-NHL-2017-LBL program in the multicenter hospitals or medical centers of China and were treated with risk-adjusted therapy. Patients with CNS3 were given increased 2 times intrathecal injection in the induction phase and 2 times intrathecal injection in the reintensification phase. Their clinical outcome were evaluated. This Time to event analyses were conducted to evaluate event-free survival and to identify risk factors for treatment failure. RESULITS: CNS involvement was diagnosed in 113(12.7%) of 883 patients. 55 patients were CNS positive by flow cytometry and conventional cytology (CC) of cerebrospinal fluid (CSF), 13 patients had intracerebral mass(IM), 15 patients werer cranial nerve palsy, 11 were epidural tumors with bone lesions, and 2 paitents were epidural tumors with CSF+. The 5-year event-free survival rate was 75.7% (95% confidence interval [CI], 66.3-86.4%), and the overall survival rate was 85.0% (95% CI, 77.0-93.8%). The cumulative risk of any central nervous system relapse is 3.17% (95% CI, 0 - 6.64%). The event-free survival rate for patients older than ten years was significantly lower than for those ten years old or younger (57.4%; 95% CI, 41.0-80.4% vs. 85.8%; 95% CI, 76.8-95.9%; P < 0.005). Male patients also had a significantly lower event-free survival rate compared to female patients (69.9%; 95% CI, 58.4-83.7% vs. 86.7%; 95% CI, 72.5-100%; P < 0.05). The presence of a large tumor significantly decreased the 5-year event-free survival rate in pediatric patients. Among CNS3 pediatric patients treated with regimen for non-Hodgkin lymphoma, there were no significant differences in the 5-year event-free survival and overall survival rates between high-risk and intermediate-risk groups, as well as between B-cell LBL (B-LBL) and T-cell LBL(T-LBL). Hazard Ratios (HR) showed higher risk for males and patients older than ten years, but failed to retain independent significance in multivariate analysis. From the literature, the overall survival and event-free survival associated with the addition of cranial irradiation ranged from 70-90%. CONCLUSION: Among 113 pediatric patients with CNS3, the application of chemotherapy and increased times of intrathecal injection as an alternative to cranial irradiation in childhood lymphoblastic leukemia resulted in comparable overall survival and event-free survival rates, without increasing the risk of central nervous system relapse. This suggests that prophylactic cranial irradiation can be safely omitted.

#Yang li and Ling Jin contribute equally to this article. *Corresponding to both Qinlong Zheng (zhengql@gobroadhealthcare.com) and Yonghong Zhang (zhangyongh@gobroadhealthcare.com). Background: B-cell lymphoblastic lymphoma (B-LBL) is a hematologic malignancy that originates from immature precursor B-cells and is less common in pediatric lymphoblastic lymphoma than T-cell lymphoblastic lymphoma. Currently, patients are stratified based on disease stage, as biological risk-factors have not yet been identified. A comprehensive understanding of B-LBL molecular status may help to improve the clinical management of these patients. Aims: The purpose of this study was to analyze the genetic features of Chinese pediatric B-LBL patients and to examine their associations with patient outcomes and clinical indicators. Methods: A total of 280 Chinese children with B-LBL treated at multiple clinical centers of the China Network for Childhood Lymphomas (CNCL) from 2017 to 2023 were included in this retrospective study. The patients were treated with a CNCL-NHL-2017-LBL treatment protocol. Targeted next-generation sequencing (t-NGS) with a panel spanning 262 lymphoma-associated genes was performed on tumor samples from 51 T-LBL patients. The associations of molecular variation with survival rates as well as with relapse and other clinical factors were analyzed. Results: This study enrolled a cohort of 280 pediatric patients diagnosed with B-LBL, with a median age of 5.8 years (range: 1-16 years) and a male-to-female ratio of 1.3:1. The median follow-up duration for our cohort was 43.7 months (95% CI: 38.2-49.1 months). We analyzed the mutational landscapes, and a total of 53 driver genes with somatic mutations were identified in 51 B-LBLs. The most frequently mutated genes were NRAS (12%), followed by FLT3 (10%), IKZF1(10%), KRAS (10%), ASXL2 (8%), CREBBP (6%) and KMT2D (6%). The recurrent mutated genes in pediatric B-LBL were mainly involved in RAS signaling pathway. Survival analysis revealed that patients with KMT2D mutations had shorter OS than patients without these mutations (log-rank p=0.008). In subsequent analysis, we collected fusion genes from 70 patients, of which the higher percentage fusion genes were TEL::AML1 (n=16, 23%), E2A::PBX1 (n=16, 23%), MLL rearranged (MLL-r, n=16, 23%), TCF3::PBX1 (n=5, 7%) and P2RY8::CRLF2 (n=5, 7%). Survival outcomes for patients with different fusion genes were analyzed, with the EFS of MLL-r positive patients being significantly worse than that of MLL-r negative patients (log-rank p<0.001). Further analysis of the distribution of gene mutations in patients with different clinical characteristics revealed that mutations in RAS pathway-related genes, including NRAS and KRAS, were detected only in patients with clinical stage IV. A high frequency of IKZF1 mutations was observed in patients with craniofacial, central nervous system (CNS), and bone marrow invasion. Additionally, the presence of P2RY8::CRLF2 in craniofacial invasion (13% vs. 3%) and E2A::PBX1 in CNS invasion (35% vs. 19%) was greater in patients with these clinical features than in those without these features. These findings suggest a correlation between genetic alterations and the severity of clinical symptoms, warranting further investigation. Conclusion: This study presents the first comprehensive molecular characterization of B-LBL in Chinese pediatric patients. There is a high incidence of mutations in genes involved in RAS signaling pathway in pediatric B-LBL. LYST mutations and MLL rearranged might be associated with poor outcomes in children with B-LBL.These results increase our understanding of the genetic heterogeneity of B-LBL in children and may pave the way for molecular risk adapted treatment protocols for B-LBL patients.

Background : T-cell lymphoblastic lymphoma (T-LBL) developed from immature precursor T cells, occurs mostly in children and adolescents. It is generally confined to a tissue lesion and may progress rapidly or relapse if not properly treated. Previous T-LBL genetic studies have shown high frequency of NOTCH1/FBXW7 and PI3K-AKT signaling gene mutations somewhat similar to T-ALL. Additionally, recent reports with limited cases suggested NOTCH1 gene fusion (NOTCH1r) was another kind of exclusive oncogenic driver event of T-LBL accompanied with higher relapse rate. However, large scale verification, further discovery of mechanisms, novel molecular features, and prognostic biomarkers are still urgently needed. Method: Retrospective analyses were performed for target sequencing of 203 T-LBL cases tested in our lab between April 2019 and July 2024. Among those, 90 patients were refractory or relapsed (R/R) including those relapsed after HSCT, and they were admitted to Beijing GoBroad Boren hospital for further therapy. NOTCH1 gene fusions and hotspot gene mutations were analyzed and annotated with an in-house workflow. Clinical and molecular features of these R/R T-LBL cases were then further characterized and analyzed. We also performed survival analyses for the NOTCH1r patients to consolidate the recent clinical findings. Results: NOTCH1 r were frequently observed with a much higher incidence of 25.6% (23/90) in our R/R as compared to 10.6% (12/113) in other patients without detailed clinical information (p < 0.01). The median age of the 9 female (39.1%) and 14 male (60.9%) NOTCH1r R/R T-LBL patients was 15.0 (IQR: 9.0-31.0) years, while the median age of the 12 female (17.9%) and 55 male (82.1%) non-NOTCH1r R/R cases was 12.5 (IQR: 9.3-14.0) years. Female seem to have a higher NOTCH1r incidence than male (p < 0.01), while the disease onset age between NOTCH1r and non-NOTCH1r group was not significantly different (p = 0.08). The bone marrow involvement was rare in NOTCH1r R/R cases (1/11, 9.21%) than that of non-NOTCH1r (28/42, 66.7%, p < 0.01), similar to previous report. A total of five NOTCH1 fusion partner genes, i.e., IKZF2 (9/23, 39.1%), TRB (5/23, 21.7%), TSPOAP1-AS1(4/23, 17.4%), IKZF1(3/23, 13.0%), and TRA (2/23, 8.7%) were discovered. All the fusions of NOTCH1 with these genes were previously reported as driver event in sporadic cases. The hotspot IKZF2/1 genes fused to NOTCH1 with a breakpoint in either exon 27 or 28 (NM_017617.5), result in a chimeric transcription factor protein retaining the IKZF2/1's DNA-binding domain and the NOTCH1's transmembrane and intracellular subunit. The other three genes fused to NOTCH1 with wide-range breakpoints located between intron 24 and exon 34, which may play a divergent pathogenic mechanism. Only 1 NOTCH1r patient (4.3%) harbored a NOTCH1/FBXW7 gene mutation, while 32 of 67 (47.8%) non-NOTCH1r patients harbored NOTCH1/FBXW7 gene mutation, indicating the mutual exclusivity of the two types (p < 0.01). Additionally, 9 of 23 NOTCH1r patients (39.1%) carried PI3K-AKT pathway gene mutations, which was much higher than 7.5% of non-NOTCH1r patients (5/67, p < 0.01). Median follow-up time for all the 23 R/R NOTCH1r patient was 24.5 (95% CI：15.9-24.7) months. The one year overall survival (OS) rate was 94.7% (95% CI: 85.2-100%), and the mOS was 30.4 (95% CI: 30.4-NA) months. The one year cumulative incidence of relapse rate (CIR) was 29.9% (95% CI: 11.0-49.4%), which tends to be much higher than the overall five year CIR of 12.2% as show in the EURO-LB02 trial study, further consolidating the recent findings of NOTCH1r's T-LBL. Conclusion: NOTCH1 gene fusion with divergent partner genes occurred frequently in R/R T-LBL. These oncogenic driver events were mutually exclusive with NOTCH1/FBXW7 gene mutations. However, these fusions accompanied frequently with PI3K-AKT pathway alteration, which were also regarded as worse prognostic factors. The one year CIR of 29.9% was much higher than the reported overall five year CIR of 12.2%.

Click to increase image sizeClick to decrease image size AcknowledgmentsWe thank the medical staffs of Hematology Center, Beijing Children’s Hospital, Capital Medical University, and all participated patients and their families.Authors’ contributionsDYL and GHX participated in study design and data interpretation. The manuscript was prepared by GHX, and DYL was a major contributor to review and edit the manuscript. ZCJ is responsible for tissue examination and diagnosis. JL and YJ participated in the statistical analysis and table production. ZNN is responsible for imaging diagnosis. HS, ZM, and YXL were responsible for the collection of clinical specimens and pathological data. ZYH and WTY were responsible for study design and supervision. All authors read and approved the final manuscript. All authors read and approved the final manuscript.Ethical approval and consent to participateThis study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (IRB) of Beijing Children’s Hospital, Capital Medical University. All parents signed informed consent forms.Disclosure statementNo potential conflict of interest was reported by the author(s).Informed consentwas obtained from all subjects or their legal guardian(s)/parents (for children’s below 16 years old). This study is accordance with the relevant guidelines and regulation.Consent for publicationNot applicable.Data availability statementRaw data are available from the corresponding author upon reasonable request.Additional informationFundingThe author(s) reported there is no funding associated with the work featured in this article.