Infusion of 14 days Blinatumomab in Combination with Chemotherapy for 46-day MRD+ Pediatric B-ALL Patients in Intermediate/High-Risk Group Results in MRD Conversion and BiTE Induced Immune Response to Malignant Cells

Infusion of 14 days Blinatumomab in Combination with Chemotherapy for 46-day MRD+ Pediatric B-ALL Patients in Intermediate/High-Risk Group Results in MRD Conversion and BiTE Induced Immune Response to Malignant Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Infusion of 14 days Blinatumomab in Combination with Chemotherapy for 46-day MRD+ Pediatric B-ALL Patients in Intermediate/High-Risk Group Results in MRD Conversion and BiTE Induced Immune Response to Malignant Cells Changwen Xue, Chao Wu, Wenyu Yang, Yao Zou, Ye Guo, Yumei Chen, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3940188/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The conventional intensity of chemotherapy has reached its limit. Despite the efficacy of blinatumomab in B-cell acute lymphoblastic leukemia (B-ALL) adults with measurable residual disease (MRD) has been proved, studies in children with MRD positivity (MRD+) are still lacking. We conducted an exploratory trial of 14 days of blinatumomab monotherapy in combination with a B-ALL chemotherapy protocol in pediatric patients with MRD+ at the day 46 and evaluated the early efficacy and safety of this approach (Blin-14d-therapy, ChiCTR2100053318, registered on 11/18/2021). Totally 9 pediatric patients were included in Blin-14d group and 38 in non-Blin-14d group. After monotherapy with blinatumomab, all evaluable patients became MRD negative in Blin-14d group and achieved MRD conversion earlier than conventional chemotherapy group, predicting better treatment outcomes. Compared to previous investigations, the incidence of AEs was lower and MRD clearance was higher in this study. In conventional blinatumomab studies, a single course was administered for 28 days. T-cell counts continued to trend upward from day 14 to 21, whereas a downward trend was observed from day 21 to 28, suggesting that T-cell exhaustion occured. While in our regimen, blinatumomab was administered for 14 days, and T-cell levels still had an upward trend in the first week after the end of the infusion, suggesting that T-cell exhaustion at this time was not significant and anti-tumor effect prolonged. Initial data from this study demonstrate that Blin-14d-therapy with chemotherapy has encouraging clinical activity and a manageable safety profile in pediatric patients with day46 MRD+ B-ALL in IR/HR groups. B-cell acute lymphoblastic leukemia blinatumomab pediatric minimal residual disease immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction In recent years, the EFS and OS of pediatric acute lymphoblastic leukemia (ALL) have been improved to 80% and 90% respectively due to the advancement of treatment methods. The cumulative recurrence rate has been reduced to about 10% 1,2 . However, 10–20% of pediatric ALL patients will relapse. Persistence of MRD is the most important risk factor for ALL relapse 3 . Chinese Children's Cancer Group Acute Lymphoblastic Leukemia-2015 Project (CCCG-ALL-2015) data showed that regardless of the initial risk grouping, patients with positive minimal residual disease (MRD ≥ 0.01%) at the Day 46 had a higher cumulative risk of relapse (P < 0.001) 4 . Therefore, achieving MRD remission is important for delaying and even preventing ALL relapse. However, St. Jude's Total XVI Study finds the conventional intensity of chemotherapy has reached its limit 1 . Data from China and international studies also show that the treatment-related toxicity of current chemotherapy is relatively high, which not only limits the efficacy but also affects the long-term quality of life 5 . Therefore, we need to replace part of the traditional highly toxic chemotherapy with molecular targeted therapy and cellular immunotherapy to improve outcomes and patient survival quality. Blinatumomab is a bispecific T-cell engager (BiTE) targeted to CD19 and CD3, which promotes immune-mediated elimination of B-cell lymphoblasts by T cells. Studies have shown that blinatumomab is a safe and efficacious treatment for children and adults with relapsed or refractory B-lineage ALL 6–8 . At present, there are few studies on blinatumomab in patients with MRD + in newly-diagnosed pediatrics B-ALL in China, and we hope to introduce this drug into the early treatment of newly-diagnosed pediatrics B-ALL, with the aim of turning MRD negative as early as possible and further improving the prognosis of childhood ALL. It was found that after 4 weeks of continuous intravenous blinatumomab treatment in the R/R ALL patient population, most patients had complete suppression of B cells in the circulation. The massive expansion and proliferation of T cells after blinatumomab treatment occurred in the second and third weeks of continuous infusion. The number of T cells continues to rise to more than two times the baseline, which facilitates further clearance of tumor cells. Notably, continued killing of B cells by proliferating T cells was observed up to 1 week after data collection and discontinuation of dosing 9,10 . But T cells declining was also observed due to exhaustion 1 week after the end of administration 11,12 . Blinatumomab has a median treatment period of 1–2 cycles in clinical trials and real-world studies due to its unique mechanism of rapid onset of action. In the Phase II study (BLAST study) to validate the efficacy of MRD clearance, the median treatment period for blinatumomab was 1 cycle. Considering the above evidences and the high cost of blinatumomab therapy 13 , we propose to add 1 course of blinatumomab to the original treatment for MRD + patients at the day 46 of initial induction therapy, and adjust the duration of treatment to 14 days to explore the effectiveness and safety of shortening the duration of continuous infusion in MRD + children with newly diagnosed B-ALL. Methods Study Design We conducted a prospective, open-label, single-center exploratory study that included patients who initiated B-ALL chemotherapy between November 16, 2020 and November 24, 2022 at the Pediatric Blood Diseases Center of Blood Diseases Hospital, Chinese Academy of Medical Sciences. The study was approved by the Institutional Ethics Committee of Blood Diseases Hospital and conducted in accordance with the Declaration of Helsinki. Signed informed consent was obtained from the parents, guardians, or patients, as appropriate (Clinical trial information: ChiCTR2100053318). Patients The diagnosis of ALL was based on morphology and immunophenotypic and genetic features of leukemic cells. Patients with B-cell ALL(B-ALL) aged between 1 year and < 10 years, and leukocyte count 50 chromosomes, or ETV6-RUNX1 oncogene fusion and without CNS3 status, testicular leukemia, MRD < 1% on day 19 of induction, and MRD < 0.01% on day 46 of induction were classified as having low-risk disease. Patients with MRD ≥ 1% (or ≥ 5% blasts morphologically without suitable markers for MRD) in bone marrow on day 46 of induction and infants younger than 6 months with KMT2A rearrangement and leukocyte count ≥ 300 × 10 9 /L were considered to have high-risk ALL. The remaining cases were classified as intermediate-risk ALL. Therapy All patients received dexamethasone for 4–5 days as upfront window therapy, followed by remission induction with prednisone, vincristine, daunorubicin, and pegaspargase from day 5 to 28, and cyclophosphamide, mercaptopurine, and cytarabine from day 29 to 35 (Fig. 1 ). Patients with B-ALL who had MRD ≥ 1% on Day 19 of remission induction received additional early intensification therapy (EIT) with cyclophosphamide, cytarabine, mercaptopurine, vincristine, and pegaspargase (CAM plus ) between day 50 and 57. In children with MRD + on day 46, especially in combination with positive high-risk fusion-genes, blinatumomab was an option for EIT. Blinatumomab was administered by continuous intravenous infusion over 14 days, at a dose of 15µg/m 2 /d (weight ≤ 45kg) or 28µg/d (weight > 45kg) as part of EIT. The primary chemotherapy regimen was continued after blinatumomab infusion. Objectives The primary objective of the study was to assess the anti-leukemic activity of Blin-14d-therapy versus non-Blin-14d-therapy in MRD response. Secondary objectives included the safety and tolerability of Blin-14d-therapy. And the secondary efficacy objective was comparison of long-term outcomes between Blin-14d-therapy and non-Blin-14d-therapy. The exploratory objectives were measures of pharmacodynamics of Blin-14d-therapy, including change of cytokines and lymphocyte subpopulation. Outcome Measures This exploratory study evaluated MRD remission rate, and the incidence and severity of adverse events (AEs). Hematologic and MRD assessments were performed of bone marrow aspirates and biopsies obtained at screening (day 1, day 19 and day 46), and one week after the end of blinatumomab infusion. MRD was assessed using real-time quantitative polymerase chain reaction (PCR) and flow-cytometry (FCM). MRD remission was defined as no target amplification with a minimum sensitivity of 10 − 04 . Genetic abnormalities including mutated or fused genes were measured by PCR. Cytokines release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were graded according to the ASTCT consensus guideline 14 . Individual organ toxicities were graded in accordance with the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) Version 5.0. CRS was managed aggressively with corticosteroids. Other side effects were managed with standard of care. Peripheral lymphocyte subsets were evaluated by FACS analyses. Peripheral blood levels of cytokines, including IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17, tumor necrosis factor α (TNF- α ) and interferon gamma γ (IFN- γ ), were assessed by FACS-based analyses. Statistical Analyses Demographic and disease characteristics were assessed by descriptive statistics. Categorical variables were expressed as number and percentage (%). Continuous variables were expressed as mean/median. Diseases-free survival was defined as the time from study entry to relapse, death from any cause, or second cancer, whichever occurred first. Overall survival was defined as the time from study entry to death from any cause. Data were censored at the date of last contact when no event was observed. SPSS statistics version 25.0 and GraphPad Prism version 9.3.0 were used for all statistical analyses. Results Patients In November 16, 2020 and November 24, 2022, forty-seven pediatric patients were included in this study, among whom 9 received Blin-14d-therapy. The final follow-up was on July 9, 2023. Of all patients, 9 (19%) received Blin-14d-therapy as EIT constituted the Blin-14d group, while the remaining constituted the non-Blin-14d group. The baseline characteristics of the patients are shown in Table 1 . There were no significant differences in age, sex and level of MRD between the patients in Blin-14d and non-Blin-14d group. Table 1 Patient characteristics Blin-14d Non-Blin-14d Characteristics No. of pts No. of pts p-value Sex Male 6 20 0.585 Female 3 18 Age(Month) Average 84.78 101.45 0.38 Median 76 100.5 Range 18–185 19–200 Risk stratification Intermediate 8 28 < 0.001 High 1 10 MRD (%) before infusion Average 0.58 1.25 0.454 Median 0.09 0.025 Range 0.03–2.50 0.01–36.18 For Blin-14d group, the median percentage of pre-blinatumomab MRD was 0.09% (range: 0.03–2.50%). Seven subjects had risk-related genetic abnormalities. One cycle of blinatumomab treatment was 14 days (2 weeks) of continuous intravenous infusion. Patient 9 (pt9) was BCR-ABL positive and continued to receive dasatinib during the infusion, and blinatumomab was administrated in a stepwise dose escalation from 5 to 15 µg/m 2 per day after reinduction, while other patients received the full dosage (pt7 28µg and others 15 µg/m 2 ) from day 1. For the prevention of severe cytokine release syndrome (CRS) and neurologic events, patients with MRD + were treated with 10 mg/m 2 oral dexamethasone for 6 hours before the infusion and 5 mg/m 2 for 0.5 hour after the infusion of blinatumomab. Levetiracetam was administrated with 0.25g in 6 patients (pt4 to 9) for 2 days in prevention of seizure related to ICANS/blinatumomab treatment. For non-Blin-14d group, the median percentage of pre-EIT MRD was 0.025% (range: 0.01–36.80%). Two withdrawals occurred due to lost to follow-up, and 12 (32%) patients had deviations from protocol-prescribed treatment after EIT. MRD Response and Disease-free Survival MRD response is shown in Fig. 2 and Fig. 3 . Seven to ten days after the end of blinatumomab infusion, all (100%) patients had MRD negative remission and achieved molecular complete remission (mCR). While in chemotherapy group, the percentage of patients who were MRD-negative at the start of consolidation therapy was 47%, increasing to 64% at half of consolidation and to 76% before interphase therapy. Figure 3 shows the MRD response and outcomes in individual patients. Twelve (32%) patients in chemotherapy group and one (pt9, 11%) in Blin-14d group underwent treatment deviations after EIT, due to non-response, relapse, MRD or fusion gene persistence. For pt9, BCL-ABL infusion gene reoccurred during the treatment of interphase therapy, for which dasatinib was replaced by olverembatinib. The median time to firstly detected MRD-negativity in bone marrow was 89days (range, 77-124days) in Blin-14d group and 112 days (range, 56–381 days) in chemotherapy group (Fig. 4 A). For patients in non-Blin-14d group who chose other treatment regimens, follow-up continued despite the discharge from the chemotherapy group. The median duration of follow-up after initiation of induction treatment was 381 days (range, 294–442 days) in Blin-14d group and 394 days (range, 56–956 days) in non-Blin-14d group (Fig. 4 B). For Blin-14d group, all patients had response durations and survived until the last follow-up, while overall survival data were not mature at the time of data cutoff. For non-Blin-14d group, 97.4% and 92.1% had response durations of > 100 days and > 200 days, respectively; the median event-free survival was not reached, and 2-year event-free survival rate was 89.5% (95%CI: 74.3–95.9%). Toxic Effects of Blinatumomab In Blin-14d group, eight (89%) patients experienced adverse events (AEs) of any grade, with 1 experiencing grade 3 AEs (Table 2 ). No grade 4 or higher events related to blinatumomab. CRS occurred in 7 (78%) patients with 5 (56%) grade 1 CRS, 2 (22%) of grade 2, and no grade 3 or higher CRS (data not shown). Peripheral sensory neuropathy was observed in 1 (11%) patient with grade 3 ICANS. The most frequent manifestation of CRS was fever, particularly a moderate fever of > 38.3°C. The median time to CRS onset from blinatumomab infusion was 1.25 days (range: 1–4), and the median time to resolution was 2 days. All CRS and ICANS events were managed including early intervention before blinatumomab infusion and when fever of ≥ 37.3°C occurred. Table 2 Adverse events Total (N = 9) No. of pts (%) Adverse events Any * 8(89) Pyrexia 7(78) Hypotension 2( 22 ) Hypokalemia 1( 11 ) Nausea 1( 11 ) Fungal infection 1( 11 ) Neurologic symptom ** 1( 11 ) *Among all patients. Multiple events have occurred in some patients. **One patient (pt2) had a neurologic event of grade 3 with numbness and pain in the lower limbs Pharmacodynamics of Blinatumomab Totally 9 assessable patients (all of Blin-14d group) were observed. Data from pt8 was not included in curve plotting due to missing cytokine and lymphocyte subpopulation examination on day 7. Pt9 was not reexamined on day 21 due to the COVID-19 pandemic. The change of IL-6 and IL-10 levels before and after infusion were selectively shown in Fig. 5 A and 5 B. The peak levels of these two cytokines were observed when CRS occurred. The maximum cytokine levels were detected for IL-6 (1448.17 pg/ml) and IL-10 (811.64 pg/ml). The proliferative trends of CD4 + and CD8 + T cells as well as total T cells were observed in 5 of 9 (56%) evaluable patients (Fig. 5 C to 5 E). Count of lymphocyte subpopulation were calculated for the indicated number of evaluable patients per detected point and the mean count is shown for each point (Fig. 5 F). When detectable circulating B cells rapidly disappeared from peripheral blood after start of blinatumomab infusion, T cells declined rapidly. After a swift drop, these cells recovered to pre-treatment levels and expanded over baseline during the further course of the single treatment cycle. The absolute counts of CD8 + T cells increased from an average of 854.22/µL at baseline to 1762.00/µL on day 21. Discussion In this study, adding blinatumomab to standard chemotherapy backbone in newly diagnosed day46 MRD + B-ALL was feasible and appeared to be safe. Our data indicate a higher level of anti-leukemic activity than the traditional chemotherapy protocol, as reflected by good responses to MRD and improved event-free survival. Although the follow-up time was relatively short, it included the period historically defined as the period of increased risk of relapse. These outcome data are very promising, given that conventional chemotherapy has reached its limits. The design of our study was based on three hypotheses. First, because of the early events that are known to occur during therapy, we hypothesized that new interventions should be implemented early in treatment. Second, we hypothesized that higher response rates and a lower incidence of side effects would be associated with a low leukemia burden at the time of blinatumomab administration. Third, we hypothesized that T cells would still have high levels of tumor killing when blinatumomab was discontinued after 14 days of administration. Here, a new regime of blinatumomab with a 14day-therapy course was developed. In the present study, we evaluated the safety, early efficacy, B and T cell response, and cytokine release of blinatumomab for 9 children with day46 MRD + B-ALL and compared the anti-leukemic effects with conventional chemotherapy as well as long-term outcomes. After monotherapy with blinatumomab, all evaluable patients became MRD negative and even no detectable signal for leukemic cells by PCR in evaluable patients. Blin-14d-therapy achieved MRD conversion earlier than conventional chemotherapy, predicting better treatment outcomes 15 . Compared to previous investigations, the incidence of AEs was lower and MRD clearance was higher in this study 7,10 . Milder and less CRS and neurotoxicity occurred, possibly associated with low tumor load before blinatumomab infusion 16 . Neurologic symptom in one patient was recorded during the first day after the start of blinatumomab infusion. No patients had ended treatment due to AEs. Our exploratory study demonstrated that Blin-14d-therapy showed early promising efficacy with a manageable safety profile in these patients. Probably due to prior chemotherapy, all evaluable patients in this study had barely detectable B cells in peripheral blood at baseline. The observation of low B-cell count at the end of treatment suggested the high cytotoxicity of blinatumomab by depleting new B cells 17 . Consistent with previous studies, there is an increase in absolute number of T cells, which most likely results from CD3-engaging bispecific antibodies 18 . However, in patients pt7 and pt9, T-cell proliferation levels were not as high as expected, both for CD4 + and CD8 + T cells (Fig. 5 C to 5 E). The count of CD3 + T cells was 530/µL at baseline vs. 483/µL at the end of blinatumomab treatment and 468/µL one week later in pt7. This may be related to the depletion of T cells by the CAM plus regimen used in the previous chemotherapy in pt7, as studies showed that pegaspargase has an anti-NK/T-cell lymphoma effect 19,20 . The count of T cells was 851/µL at baseline vs. 965/µL at the end of infusion in pt9. Although follow-up data on T-cell subpopulations were missing for pt9, the effect of Tyrosine kinase inhibitor (TKI) on T-cell proliferation and blinatumomab efficacy results need to be taken into account, considering that he was still receiving dasatinib during his treatment with blinatumomab 21 . Another interesting finding was that trends in proliferation changes of T-cell subpopulations did not exactly match those of conventional regimens when the treatment course was shortened by half. In a phase II clinical study in MRD + B-ALL, CD4 + and CD8 + T cell counts were measured up to day 35, one week after the end of dosing, and found that T-cell counts, particularly CD8 + T-cells, declined slightly from day 21 to 28 and decreased significantly from day 28 to 35 when the infusion had been stopped 10 . In those conventional blinatumomab studies, a single course of treatment was administered for 28 days. T-cell counts continued to trend upward from day 14 to 21, whereas a downward trend was observed from day 21 to 28, suggesting that T-cell exhaustion occured between day 14 and day 28 of infusion. Based on the above evidences, we designed this exploratory study and tried to explore when T-cell counts would decline if discontinued after 14 days of blinatumomab infusion. In our regimen, blinatumomab was administered for 14 days, and CD4 + and CD8 + T cell levels still had an upward trend in the first week after the end of the infusion, suggesting that T-cell exhaustion at this time was not significant and anti-tumor effect prolonged. In conventional Blin-28d-therapy, in order to ensure that sufficient T cells can be activated by blinatumomab in the next cycle, if any, a 2-week withdrawal period is set between treatment cycles to allow for the normalization of the reduced T cells and to allow time for CD3 target recovery and proper T cell expansion. However, for Blin-14d-therapy, given the unique pharmacokinetic profile of blinatumomab, the withdrawal period needs to be revisited, e.g., to assess the exhaustion of T-cell and its subpopulations under this regimen. To further improve the outcome of pediatric acute lymphoblastic leukemia, precise prognostic stratification and MRD surveillance are needed in addition to improving treatment. NGS results are currently less commonly used as a standard for MRD monitoring because of the need to design patient-specific panels, the high cost and complexity of the process 22,23 . This study has several limitations. Since this exploratory study was conducted in a single center, some limitations were unavoidable. The first limitation was the small sample size. Secondly, the follow-up time in Blin-14d group was relatively short. Thirdly, 13 treatment deviations were reported in our study, due to non-response, relapse, MRD or fusion gene persistence. Twelve (32%) of these cases occurred in non-Blin-14d group and one (11%) in Blin-14d group, suggesting that Blin-14d-therapy was more efficacious. Adding blinatumomab 14day-therapy to conventional chemotherapy in newly diagnosed day46 MRD + B-ALL appeared to be safe and promising efficacy in terms of earlier MRD response and toxicity. While blinatumomab therapy has shown significant efficacy for B-ALL patients, the high cost and lengthy course of blinatumomab infusion limit its broader use. In addition, its therapeutic potential in terms of depth and the duration of remission can still be further improved. Declarations Acknowledgments The authors would like to appreciate the efforts of all those who contributed data to the study. We appreciate the support from patients, families, friends, caregivers, study staff and study investigators for their support in this study. Ethics Approval and Consent to Participate The study was approved by the Institutional Ethics Committee of Blood Diseases Hospital and conducted in accordance with the Declaration of Helsinki. Signed informed consent was obtained from the parents, guardians, or patients, as appropriate (Clinical trial information: ChiCTR2100053318). Consent for Publication Not applicable. Availability of Data and Materials Deidentified individual patient data are available on request from the corresponding author, Li Zhang ( [email protected] ) and Xiaofan Zhu ( [email protected] ). Competing Interests The authors declare no conflicts of interest. Funding The project was supported in part by the National Key Research and Development Program of China (2021YFE0106900), the CAMS Innovation Fund for Medical Sciences (CIFMS 2022-I2M-1-022; 2021-I2M-1-041), the National Natural Science Foundation of China (82270144), and the Haihe Laboratory of Cell Ecosystem 418 Innovation Fund [22HHXBSS00039 (Y.Z.)]. Authors’ Contributions Li Zhang and Xiaofan Zhu designed the clinical trial. Wenyu Yang, Yao Zou, Ye Guo, Yumei Chen, Xiaojuan Chen and Yang Wan executed the research and collected the data. Changwen Xue and Chao Wu analyzed the data and drafted the original manuscript. Yueshen Ma contributed to the statistical analysis. All authors reviewed and approved the final version of this manuscript. Changwen Xue and Chao Wu contributed equally to this article. Author Contributions: Li Zhang and Xiaofan Zhu designed the clinical trial. Wenyu Yang, Yao Zou, Ye Guo, Yumei Chen, Xiaojuan Chen and Yang Wan executed the research and collected the data. Changwen Xue and Chao Wu analyzed the data and drafted the original manuscript. Yueshen Ma contributed to the statistical analysis. All authors reviewed and approved the final version of this manuscript. Changwen Xue and Chao Wu contributed equally to this article. Funding Information: The project was supported in part by the National Key Research and Development Program of China (2021YFE0106900), the CAMS Innovation Fund for Medical Sciences (CIFMS 2022-I2M-1-022; 2021-I2M-1-041), the National Natural Science Foundation of China (82270144), and the Haihe Laboratory of Cell Ecosystem 418 Innovation Fund [22HHXBSS00039 (Y.Z.)]. Conflict of Interest Disclosure: The authors declare no conflicts of interest. Data Availability Statement: Deidentified individual patient data are available on request from the corresponding author, Li Zhang ( [email protected] ) and Xiaofan Zhu ( [email protected] ). Ethics Approval Statement: The study was approved by the Institutional Ethics Committee of Blood Diseases Hospital and conducted in accordance with the Declaration of Helsinki. Patient Consent Statement: Signed informed consent was obtained from the parents, guardians, or patients, as appropriate (Clinical trial information: ChiCTR2100053318). 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Int J Environ Res Public Health. 2016;13(10):1008. Zheng W, Gao Y, Ke X, et al. PEG-L-CHOP treatment is safe and effective in adult extranodal NK/T-cell lymphoma with a low rate of clinical hypersensitivity. BMC Cancer. 2018;18(1):910. Kauer J, Märklin M, Pflügler M, et al. BCR::ABL1 tyrosine kinase inhibitors hamper the therapeutic efficacy of blinatumomab in vitro. J Cancer Res Clin Oncol. 2022;148(10):2759–71. Subhash VV, Huang L, Kamili A, et al. Whole-genome sequencing facilitates patient-specific quantitative PCR-based minimal residual disease monitoring in acute lymphoblastic leukaemia, neuroblastoma and Ewing sarcoma. Br J Cancer. 2022;126(3):482–91. Bartram J, Patel B, Fielding AK. Monitoring MRD in ALL: Methodologies, technical aspects and optimal time points for measurement. Semin Hematol. 2020;57(3):142–8. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3940188","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":272952803,"identity":"ae60c039-6fee-4fe4-aa7e-b305edf38d5a","order_by":0,"name":"Changwen Xue","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences Institute of Hematology and Blood Diseases Hospital","correspondingAuthor":false,"prefix":"","firstName":"Changwen","middleName":"","lastName":"Xue","suffix":""},{"id":272952804,"identity":"7dce6d55-f4c2-48d1-b4f5-f89ea5c3c22a","order_by":1,"name":"Chao Wu","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences Institute of Hematology and Blood Diseases Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Wu","suffix":""},{"id":272952805,"identity":"c421b25b-4fe0-47ca-b644-7c94a3f4c7db","order_by":2,"name":"Wenyu Yang","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences Institute of Hematology and Blood Diseases Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wenyu","middleName":"","lastName":"Yang","suffix":""},{"id":272952806,"identity":"033926e6-aa5b-4c54-b6da-efb832251b8d","order_by":3,"name":"Yao Zou","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences Institute of Hematology and Blood Diseases Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Zou","suffix":""},{"id":272952807,"identity":"f6954c55-d884-4934-93e0-058cac276e29","order_by":4,"name":"Ye Guo","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences Institute of Hematology and Blood Diseases Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Guo","suffix":""},{"id":272952808,"identity":"59e886ac-8c51-4886-a719-f964b2252b4f","order_by":5,"name":"Yumei Chen","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences Institute of Hematology and Blood Diseases Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yumei","middleName":"","lastName":"Chen","suffix":""},{"id":272952810,"identity":"5e1f87fc-4723-44ca-b899-6c93a3e4bd9c","order_by":6,"name":"Xiaojuan Chen","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences Institute of Hematology and Blood Diseases Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaojuan","middleName":"","lastName":"Chen","suffix":""},{"id":272952811,"identity":"0d2a412f-d3da-4311-8668-adac4ab9e663","order_by":7,"name":"Yueshen Ma","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences Institute of Hematology and Blood Diseases Hospital, Leukemia Center","correspondingAuthor":false,"prefix":"","firstName":"Yueshen","middleName":"","lastName":"Ma","suffix":""},{"id":272952812,"identity":"a4f3ec13-023a-4cf5-a908-4c77c39dfdcf","order_by":8,"name":"Yang wan","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences Institute of Hematology and Blood Diseases Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"wan","suffix":""},{"id":272952813,"identity":"8496a384-68cd-476b-96b8-ac97f5d93113","order_by":9,"name":"Xiaofan Zhu","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences Institute of Hematology and Blood Diseases Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaofan","middleName":"","lastName":"Zhu","suffix":""},{"id":272952814,"identity":"8ca4af5e-489a-4db9-a6fa-05212c908829","order_by":10,"name":"Li Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYDACCRBRIMHDz8xDkhYDCRnJZhK1MNgYHCBWi/zs5mcPvxhY8Bgf5z344WdOHQN/+wHGzwV4tDDOOWZuLGMgwWN2mC9ZsnfbYQaJMwnM0jPwaGGWSDCTlgBr4TFj4N12gIHhBgMb3qBgk0j/BtZi3Mxjxvh3Wx2DPCEtPBI5ZpIfgFoMmHnMmHm3MTMYENIiIZFTJg0MZB6JwzzG0rLbDvMYnklslsanRX5G+jbJHxV19vz9Zww/vt1WJyd3/PDBz4QCHMUZQDZjAwENQCU/CCoZBaNgFIyCEQ0ANcU6yzIFK7AAAAAASUVORK5CYII=","orcid":"","institution":"Chinese Academy of Medical Sciences Institute of Hematology and Blood Diseases Hospital","correspondingAuthor":true,"prefix":"","firstName":"Li","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-02-08 14:22:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3940188/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3940188/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51241581,"identity":"40995475-0b16-41d1-bb49-ea8c2284234f","added_by":"auto","created_at":"2024-02-16 17:51:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":228493,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment Protocol\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eBlack arrows indicate time points at which minimal residual disease (MRD) was measured: during the induction chemotherapy (Day 19 and Day 46), one week after the end of the blinatumomab infusion, before consolidation treatment. Dex, dexamethasone; VDLP consists of prednisone, vincristine, daunorubicin, and pegaspargase; CAT consists of cyclophosphamide, mercaptopurine, and cytarabine; CAT+ consists of cyclophosphamide, cytarabine, mercaptopurine, vincristine, and pegaspargase.\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3940188/v1/51a297fe9fba21befde3a8e6.jpg"},{"id":51241582,"identity":"78380b48-ee28-4cf3-af2c-fb2bc17296cf","added_by":"auto","created_at":"2024-02-16 17:51:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":312513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMRD at Time Points Throughout the Study\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe figure shows the percentages of patients who were negative for MRD (i.e., in whom MRD was undetectable) or had MRD of less than 0.01% (i.e., \u0026lt;1 leukemic cell per 10,000 normal cells) among the patients in Blin-14d group and among chemotherapy group; 95% exact confidence intervals (CIs) are shown for the attributable risk in these percentages between the two populations. A two-sided Fisher’s exact test was performed to compared the MRD conversion rate among the two population. The numbers of patients included at each time point were 37 patients before consolidation; 33 patients at half of consolidation and before interphase therapy.\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3940188/v1/fbd0fff76a202890c77e0f57.jpg"},{"id":51241134,"identity":"c3de5c29-0328-4a4e-9aaf-27d3a5977b5b","added_by":"auto","created_at":"2024-02-16 17:43:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":466655,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSwimmer Plot of MRD Response and Outcomes in Individual Patients\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe time line is synchronized for different chemotherapy blocks according to protocol time lines. The actual time of starting a block for individual patients may differ. The red square indicates the conversion of MRD. HSCT, hematopoietic stem cell transplantation; Blin, blinatumomab; CAR-T, Chimeric Antigen Receptor T-Cell Immunotherapy.\u003c/p\u003e","description":"","filename":"floatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3940188/v1/45d03dbea6f89df6fd180a8a.jpg"},{"id":51241137,"identity":"8eef6c71-9d13-4791-a651-060c47d317f1","added_by":"auto","created_at":"2024-02-16 17:43:49","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":236840,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMRD Response and Disease-free Survival\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKaplan-Meler curves for (A) \u003c/strong\u003eCumulative incidence of MRD conversion, Blin-14d group versus chemotherapy group; \u003cstrong\u003e(B)\u003c/strong\u003e Event-free survival, Blin-14d group versus non-Blin-14d group.\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3940188/v1/73c5c5c5b15d9eb626abe87b.jpg"},{"id":51241138,"identity":"d2633c06-142c-4890-93e6-54c7f170e190","added_by":"auto","created_at":"2024-02-16 17:43:50","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":624614,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmacodynamics of blinatumomab\u003c/strong\u003e.\u003cstrong\u003e A\u003c/strong\u003e The change of IL-6 or \u003cstrong\u003eB\u003c/strong\u003e IL-10 levels in peripheral blood. Green, CRS grade 0; red, CRS grade 1; brown, CRS grade 2; \u003cstrong\u003eC-F \u003c/strong\u003eThe course of CD4+ and CD8+ T cells as well as CD19+ B cells in the peripheral blood. The first data point shows the baseline value before the start of blinatumomab infusion, and the last data point shows the post-treatment value 7 days after the end of infusion.\u003c/p\u003e","description":"","filename":"floatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3940188/v1/1e829ba707ecd2d0f4f61646.jpg"},{"id":56599327,"identity":"941d378b-581c-4625-bb27-9f447a8d9f91","added_by":"auto","created_at":"2024-05-16 11:41:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2481633,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3940188/v1/44d73ea2-a114-4187-a47a-a73b1fcb0941.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Infusion of 14 days Blinatumomab in Combination with Chemotherapy for 46-day MRD+ Pediatric B-ALL Patients in Intermediate/High-Risk Group Results in MRD Conversion and BiTE Induced Immune Response to Malignant Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, the EFS and OS of pediatric acute lymphoblastic leukemia (ALL) have been improved to 80% and 90% respectively due to the advancement of treatment methods. The cumulative recurrence rate has been reduced to about 10%\u003csup\u003e1,2\u003c/sup\u003e. However, 10\u0026ndash;20% of pediatric ALL patients will relapse. Persistence of MRD is the most important risk factor for ALL relapse\u003csup\u003e3\u003c/sup\u003e. Chinese Children's Cancer Group Acute Lymphoblastic Leukemia-2015 Project (CCCG-ALL-2015) data showed that regardless of the initial risk grouping, patients with positive minimal residual disease (MRD\u0026thinsp;\u0026ge;\u0026thinsp;0.01%) at the Day 46 had a higher cumulative risk of relapse (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001)\u003csup\u003e4\u003c/sup\u003e. Therefore, achieving MRD remission is important for delaying and even preventing ALL relapse.\u003c/p\u003e \u003cp\u003eHowever, St. Jude's Total XVI Study finds the conventional intensity of chemotherapy has reached its limit\u003csup\u003e1\u003c/sup\u003e. Data from China and international studies also show that the treatment-related toxicity of current chemotherapy is relatively high, which not only limits the efficacy but also affects the long-term quality of life\u003csup\u003e5\u003c/sup\u003e. Therefore, we need to replace part of the traditional highly toxic chemotherapy with molecular targeted therapy and cellular immunotherapy to improve outcomes and patient survival quality.\u003c/p\u003e \u003cp\u003eBlinatumomab is a bispecific T-cell engager (BiTE) targeted to CD19 and CD3, which promotes immune-mediated elimination of B-cell lymphoblasts by T cells. Studies have shown that blinatumomab is a safe and efficacious treatment for children and adults with relapsed or refractory B-lineage ALL\u003csup\u003e6\u0026ndash;8\u003c/sup\u003e. At present, there are few studies on blinatumomab in patients with MRD\u0026thinsp;+\u0026thinsp;in newly-diagnosed pediatrics B-ALL in China, and we hope to introduce this drug into the early treatment of newly-diagnosed pediatrics B-ALL, with the aim of turning MRD negative as early as possible and further improving the prognosis of childhood ALL.\u003c/p\u003e \u003cp\u003eIt was found that after 4 weeks of continuous intravenous blinatumomab treatment in the R/R ALL patient population, most patients had complete suppression of B cells in the circulation. The massive expansion and proliferation of T cells after blinatumomab treatment occurred in the second and third weeks of continuous infusion. The number of T cells continues to rise to more than two times the baseline, which facilitates further clearance of tumor cells. Notably, continued killing of B cells by proliferating T cells was observed up to 1 week after data collection and discontinuation of dosing\u003csup\u003e9,10\u003c/sup\u003e. But T cells declining was also observed due to exhaustion 1 week after the end of administration\u003csup\u003e11,12\u003c/sup\u003e. Blinatumomab has a median treatment period of 1\u0026ndash;2 cycles in clinical trials and real-world studies due to its unique mechanism of rapid onset of action. In the Phase II study (BLAST study) to validate the efficacy of MRD clearance, the median treatment period for blinatumomab was 1 cycle.\u003c/p\u003e \u003cp\u003eConsidering the above evidences and the high cost of blinatumomab therapy\u003csup\u003e13\u003c/sup\u003e, we propose to add 1 course of blinatumomab to the original treatment for MRD\u0026thinsp;+\u0026thinsp;patients at the day 46 of initial induction therapy, and adjust the duration of treatment to 14 days to explore the effectiveness and safety of shortening the duration of continuous infusion in MRD\u0026thinsp;+\u0026thinsp;children with newly diagnosed B-ALL.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design\u003c/h2\u003e \u003cp\u003eWe conducted a prospective, open-label, single-center exploratory study that included patients who initiated B-ALL chemotherapy between November 16, 2020 and November 24, 2022 at the Pediatric Blood Diseases Center of Blood Diseases Hospital, Chinese Academy of Medical Sciences. The study was approved by the Institutional Ethics Committee of Blood Diseases Hospital and conducted in accordance with the Declaration of Helsinki. Signed informed consent was obtained from the parents, guardians, or patients, as appropriate (Clinical trial information: ChiCTR2100053318).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eThe diagnosis of ALL was based on morphology and immunophenotypic and genetic features of leukemic cells. Patients with B-cell ALL(B-ALL) aged between 1 year and \u0026lt;\u0026thinsp;10 years, and leukocyte count\u0026thinsp;\u0026lt;\u0026thinsp;50 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e/L, hyperdiploidy\u0026thinsp;\u0026gt;\u0026thinsp;50 chromosomes, or \u003cem\u003eETV6-RUNX1\u003c/em\u003e oncogene fusion and without CNS3 status, testicular leukemia, MRD\u0026thinsp;\u0026lt;\u0026thinsp;1% on day 19 of induction, and MRD\u0026thinsp;\u0026lt;\u0026thinsp;0.01% on day 46 of induction were classified as having low-risk disease. Patients with MRD\u0026thinsp;\u0026ge;\u0026thinsp;1% (or \u0026ge;\u0026thinsp;5% blasts morphologically without suitable markers for MRD) in bone marrow on day 46 of induction and infants younger than 6 months with \u003cem\u003eKMT2A\u003c/em\u003e rearrangement and leukocyte count\u0026thinsp;\u0026ge;\u0026thinsp;300 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e/L were considered to have high-risk ALL. The remaining cases were classified as intermediate-risk ALL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTherapy\u003c/h2\u003e \u003cp\u003eAll patients received dexamethasone for 4\u0026ndash;5 days as upfront window therapy, followed by remission induction with prednisone, vincristine, daunorubicin, and pegaspargase from day 5 to 28, and cyclophosphamide, mercaptopurine, and cytarabine from day 29 to 35 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Patients with B-ALL who had MRD\u0026thinsp;\u0026ge;\u0026thinsp;1% on Day 19 of remission induction received additional early intensification therapy (EIT) with cyclophosphamide, cytarabine, mercaptopurine, vincristine, and pegaspargase (CAM\u003csup\u003eplus\u003c/sup\u003e) between day 50 and 57. In children with MRD\u0026thinsp;+\u0026thinsp;on day 46, especially in combination with positive high-risk fusion-genes, blinatumomab was an option for EIT. Blinatumomab was administered by continuous intravenous infusion over 14 days, at a dose of 15\u0026micro;g/m\u003csup\u003e2\u003c/sup\u003e/d (weight\u0026thinsp;\u0026le;\u0026thinsp;45kg) or 28\u0026micro;g/d (weight\u0026thinsp;\u0026gt;\u0026thinsp;45kg) as part of EIT. The primary chemotherapy regimen was continued after blinatumomab infusion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eThe primary objective of the study was to assess the anti-leukemic activity of Blin-14d-therapy versus non-Blin-14d-therapy in MRD response. Secondary objectives included the safety and tolerability of Blin-14d-therapy. And the secondary efficacy objective was comparison of long-term outcomes between Blin-14d-therapy and non-Blin-14d-therapy. The exploratory objectives were measures of pharmacodynamics of Blin-14d-therapy, including change of cytokines and lymphocyte subpopulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eOutcome Measures\u003c/h2\u003e \u003cp\u003eThis exploratory study evaluated MRD remission rate, and the incidence and severity of adverse events (AEs). Hematologic and MRD assessments were performed of bone marrow aspirates and biopsies obtained at screening (day 1, day 19 and day 46), and one week after the end of blinatumomab infusion. MRD was assessed using real-time quantitative polymerase chain reaction (PCR) and flow-cytometry (FCM). MRD remission was defined as no target amplification with a minimum sensitivity of 10\u003csup\u003e\u0026minus;\u0026thinsp;04\u003c/sup\u003e. Genetic abnormalities including mutated or fused genes were measured by PCR. Cytokines release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were graded according to the ASTCT consensus guideline\u003csup\u003e14\u003c/sup\u003e. Individual organ toxicities were graded in accordance with the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) Version 5.0. CRS was managed aggressively with corticosteroids. Other side effects were managed with standard of care. Peripheral lymphocyte subsets were evaluated by FACS analyses. Peripheral blood levels of cytokines, including IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17, tumor necrosis factor \u003cem\u003eα\u003c/em\u003e (TNF-\u003cem\u003eα\u003c/em\u003e) and interferon gamma \u003cem\u003eγ\u003c/em\u003e (IFN-\u003cem\u003eγ\u003c/em\u003e), were assessed by FACS-based analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eDemographic and disease characteristics were assessed by descriptive statistics. Categorical variables were expressed as number and percentage (%). Continuous variables were expressed as mean/median. Diseases-free survival was defined as the time from study entry to relapse, death from any cause, or second cancer, whichever occurred first. Overall survival was defined as the time from study entry to death from any cause. Data were censored at the date of last contact when no event was observed. SPSS statistics version 25.0 and GraphPad Prism version 9.3.0 were used for all statistical analyses.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eIn November 16, 2020 and November 24, 2022, forty-seven pediatric patients were included in this study, among whom 9 received Blin-14d-therapy. The final follow-up was on July 9, 2023. Of all patients, 9 (19%) received Blin-14d-therapy as EIT constituted the Blin-14d group, while the remaining constituted the non-Blin-14d group. The baseline characteristics of the patients are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. There were no significant differences in age, sex and level of MRD between the patients in Blin-14d and non-Blin-14d group.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatient characteristics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBlin-14d\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNon-Blin-14d\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. of pts\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. of pts\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.585\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge(Month)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e101.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRange\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u0026ndash;185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19\u0026ndash;200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRisk stratification\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntermediate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMRD (%) before infusion\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.454\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.025\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRange\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.03\u0026ndash;2.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.01\u0026ndash;36.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFor Blin-14d group, the median percentage of pre-blinatumomab MRD was 0.09% (range: 0.03\u0026ndash;2.50%). Seven subjects had risk-related genetic abnormalities. One cycle of blinatumomab treatment was 14 days (2 weeks) of continuous intravenous infusion. Patient 9 (pt9) was \u003cem\u003eBCR-ABL\u003c/em\u003e positive and continued to receive dasatinib during the infusion, and blinatumomab was administrated in a stepwise dose escalation from 5 to 15 \u0026micro;g/m\u003csup\u003e2\u003c/sup\u003e per day after reinduction, while other patients received the full dosage (pt7 28\u0026micro;g and others 15 \u0026micro;g/m\u003csup\u003e2\u003c/sup\u003e) from day 1. For the prevention of severe cytokine release syndrome (CRS) and neurologic events, patients with MRD\u0026thinsp;+\u0026thinsp;were treated with 10 mg/m\u003csup\u003e2\u003c/sup\u003e oral dexamethasone for 6 hours before the infusion and 5 mg/m\u003csup\u003e2\u003c/sup\u003e for 0.5 hour after the infusion of blinatumomab. Levetiracetam was administrated with 0.25g in 6 patients (pt4 to 9) for 2 days in prevention of seizure related to ICANS/blinatumomab treatment. For non-Blin-14d group, the median percentage of pre-EIT MRD was 0.025% (range: 0.01\u0026ndash;36.80%). Two withdrawals occurred due to lost to follow-up, and 12 (32%) patients had deviations from protocol-prescribed treatment after EIT.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMRD Response and Disease-free Survival\u003c/h2\u003e \u003cp\u003eMRD response is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Seven to ten days after the end of blinatumomab infusion, all (100%) patients had MRD negative remission and achieved molecular complete remission (mCR). While in chemotherapy group, the percentage of patients who were MRD-negative at the start of consolidation therapy was 47%, increasing to 64% at half of consolidation and to 76% before interphase therapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the MRD response and outcomes in individual patients. Twelve (32%) patients in chemotherapy group and one (pt9, 11%) in Blin-14d group underwent treatment deviations after EIT, due to non-response, relapse, MRD or fusion gene persistence. For pt9, BCL-ABL infusion gene reoccurred during the treatment of interphase therapy, for which dasatinib was replaced by olverembatinib.\u003c/p\u003e \u003cp\u003eThe median time to firstly detected MRD-negativity in bone marrow was 89days (range, 77-124days) in Blin-14d group and 112 days (range, 56\u0026ndash;381 days) in chemotherapy group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). For patients in non-Blin-14d group who chose other treatment regimens, follow-up continued despite the discharge from the chemotherapy group. The median duration of follow-up after initiation of induction treatment was 381 days (range, 294\u0026ndash;442 days) in Blin-14d group and 394 days (range, 56\u0026ndash;956 days) in non-Blin-14d group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). For Blin-14d group, all patients had response durations and survived until the last follow-up, while overall survival data were not mature at the time of data cutoff. For non-Blin-14d group, 97.4% and 92.1% had response durations of \u0026gt;\u0026thinsp;100 days and \u0026gt;\u0026thinsp;200 days, respectively; the median event-free survival was not reached, and 2-year event-free survival rate was 89.5% (95%CI: 74.3\u0026ndash;95.9%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eToxic Effects of Blinatumomab\u003c/h2\u003e \u003cp\u003eIn Blin-14d group, eight (89%) patients experienced adverse events (AEs) of any grade, with 1 experiencing grade 3 AEs (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). No grade 4 or higher events related to blinatumomab. CRS occurred in 7 (78%) patients with 5 (56%) grade 1 CRS, 2 (22%) of grade 2, and no grade 3 or higher CRS (data not shown). Peripheral sensory neuropathy was observed in 1 (11%) patient with grade 3 ICANS. The most frequent manifestation of CRS was fever, particularly a moderate fever of \u0026gt;\u0026thinsp;38.3\u0026deg;C. The median time to CRS onset from blinatumomab infusion was 1.25 days (range: 1\u0026ndash;4), and the median time to resolution was 2 days. All CRS and ICANS events were managed including early intervention before blinatumomab infusion and when fever of \u0026ge;\u0026thinsp;37.3\u0026deg;C occurred.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAdverse events\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal (N\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. of pts (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdverse events\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAny\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8(89)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePyrexia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7(78)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypotension\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypokalemia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNausea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFungal infection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeurologic symptom\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e*Among all patients. Multiple events have occurred in some patients.\u003c/p\u003e \u003cp\u003e**One patient (pt2) had a neurologic event of grade 3 with numbness and pain in the lower limbs\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePharmacodynamics of Blinatumomab\u003c/h2\u003e \u003cp\u003eTotally 9 assessable patients (all of Blin-14d group) were observed. Data from pt8 was not included in curve plotting due to missing cytokine and lymphocyte subpopulation examination on day 7. Pt9 was not reexamined on day 21 due to the COVID-19 pandemic. The change of IL-6 and IL-10 levels before and after infusion were selectively shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. The peak levels of these two cytokines were observed when CRS occurred. The maximum cytokine levels were detected for IL-6 (1448.17 pg/ml) and IL-10 (811.64 pg/ml). The proliferative trends of CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells as well as total T cells were observed in 5 of 9 (56%) evaluable patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC to \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Count of lymphocyte subpopulation were calculated for the indicated number of evaluable patients per detected point and the mean count is shown for each point (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). When detectable circulating B cells rapidly disappeared from peripheral blood after start of blinatumomab infusion, T cells declined rapidly. After a swift drop, these cells recovered to pre-treatment levels and expanded over baseline during the further course of the single treatment cycle. The absolute counts of CD8\u0026thinsp;+\u0026thinsp;T cells increased from an average of 854.22/\u0026micro;L at baseline to 1762.00/\u0026micro;L on day 21.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, adding blinatumomab to standard chemotherapy backbone in newly diagnosed day46 MRD\u0026thinsp;+\u0026thinsp;B-ALL was feasible and appeared to be safe. Our data indicate a higher level of anti-leukemic activity than the traditional chemotherapy protocol, as reflected by good responses to MRD and improved event-free survival. Although the follow-up time was relatively short, it included the period historically defined as the period of increased risk of relapse. These outcome data are very promising, given that conventional chemotherapy has reached its limits.\u003c/p\u003e \u003cp\u003eThe design of our study was based on three hypotheses. First, because of the early events that are known to occur during therapy, we hypothesized that new interventions should be implemented early in treatment. Second, we hypothesized that higher response rates and a lower incidence of side effects would be associated with a low leukemia burden at the time of blinatumomab administration. Third, we hypothesized that T cells would still have high levels of tumor killing when blinatumomab was discontinued after 14 days of administration. Here, a new regime of blinatumomab with a 14day-therapy course was developed. In the present study, we evaluated the safety, early efficacy, B and T cell response, and cytokine release of blinatumomab for 9 children with day46 MRD\u0026thinsp;+\u0026thinsp;B-ALL and compared the anti-leukemic effects with conventional chemotherapy as well as long-term outcomes.\u003c/p\u003e \u003cp\u003eAfter monotherapy with blinatumomab, all evaluable patients became MRD negative and even no detectable signal for leukemic cells by PCR in evaluable patients. Blin-14d-therapy achieved MRD conversion earlier than conventional chemotherapy, predicting better treatment outcomes\u003csup\u003e15\u003c/sup\u003e. Compared to previous investigations, the incidence of AEs was lower and MRD clearance was higher in this study\u003csup\u003e7,10\u003c/sup\u003e. Milder and less CRS and neurotoxicity occurred, possibly associated with low tumor load before blinatumomab infusion\u003csup\u003e16\u003c/sup\u003e. Neurologic symptom in one patient was recorded during the first day after the start of blinatumomab infusion. No patients had ended treatment due to AEs. Our exploratory study demonstrated that Blin-14d-therapy showed early promising efficacy with a manageable safety profile in these patients.\u003c/p\u003e \u003cp\u003eProbably due to prior chemotherapy, all evaluable patients in this study had barely detectable B cells in peripheral blood at baseline. The observation of low B-cell count at the end of treatment suggested the high cytotoxicity of blinatumomab by depleting new B cells\u003csup\u003e17\u003c/sup\u003e. Consistent with previous studies, there is an increase in absolute number of T cells, which most likely results from CD3-engaging bispecific antibodies\u003csup\u003e18\u003c/sup\u003e. However, in patients pt7 and pt9, T-cell proliferation levels were not as high as expected, both for CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC to \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The count of CD3\u0026thinsp;+\u0026thinsp;T cells was 530/\u0026micro;L at baseline vs. 483/\u0026micro;L at the end of blinatumomab treatment and 468/\u0026micro;L one week later in pt7. This may be related to the depletion of T cells by the CAM\u003csup\u003eplus\u003c/sup\u003e regimen used in the previous chemotherapy in pt7, as studies showed that pegaspargase has an anti-NK/T-cell lymphoma effect\u003csup\u003e19,20\u003c/sup\u003e. The count of T cells was 851/\u0026micro;L at baseline vs. 965/\u0026micro;L at the end of infusion in pt9. Although follow-up data on T-cell subpopulations were missing for pt9, the effect of Tyrosine kinase inhibitor (TKI) on T-cell proliferation and blinatumomab efficacy results need to be taken into account, considering that he was still receiving dasatinib during his treatment with blinatumomab\u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAnother interesting finding was that trends in proliferation changes of T-cell subpopulations did not exactly match those of conventional regimens when the treatment course was shortened by half. In a phase II clinical study in MRD\u0026thinsp;+\u0026thinsp;B-ALL, CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cell counts were measured up to day 35, one week after the end of dosing, and found that T-cell counts, particularly CD8\u0026thinsp;+\u0026thinsp;T-cells, declined slightly from day 21 to 28 and decreased significantly from day 28 to 35 when the infusion had been stopped\u003csup\u003e10\u003c/sup\u003e. In those conventional blinatumomab studies, a single course of treatment was administered for 28 days. T-cell counts continued to trend upward from day 14 to 21, whereas a downward trend was observed from day 21 to 28, suggesting that T-cell exhaustion occured between day 14 and day 28 of infusion. Based on the above evidences, we designed this exploratory study and tried to explore when T-cell counts would decline if discontinued after 14 days of blinatumomab infusion. In our regimen, blinatumomab was administered for 14 days, and CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cell levels still had an upward trend in the first week after the end of the infusion, suggesting that T-cell exhaustion at this time was not significant and anti-tumor effect prolonged.\u003c/p\u003e \u003cp\u003eIn conventional Blin-28d-therapy, in order to ensure that sufficient T cells can be activated by blinatumomab in the next cycle, if any, a 2-week withdrawal period is set between treatment cycles to allow for the normalization of the reduced T cells and to allow time for CD3 target recovery and proper T cell expansion. However, for Blin-14d-therapy, given the unique pharmacokinetic profile of blinatumomab, the withdrawal period needs to be revisited, e.g., to assess the exhaustion of T-cell and its subpopulations under this regimen.\u003c/p\u003e \u003cp\u003eTo further improve the outcome of pediatric acute lymphoblastic leukemia, precise prognostic stratification and MRD surveillance are needed in addition to improving treatment. NGS results are currently less commonly used as a standard for MRD monitoring because of the need to design patient-specific panels, the high cost and complexity of the process\u003csup\u003e22,23\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis study has several limitations. Since this exploratory study was conducted in a single center, some limitations were unavoidable. The first limitation was the small sample size. Secondly, the follow-up time in Blin-14d group was relatively short. Thirdly, 13 treatment deviations were reported in our study, due to non-response, relapse, MRD or fusion gene persistence. Twelve (32%) of these cases occurred in non-Blin-14d group and one (11%) in Blin-14d group, suggesting that Blin-14d-therapy was more efficacious.\u003c/p\u003e \u003cp\u003eAdding blinatumomab 14day-therapy to conventional chemotherapy in newly diagnosed day46 MRD\u0026thinsp;+\u0026thinsp;B-ALL appeared to be safe and promising efficacy in terms of earlier MRD response and toxicity. While blinatumomab therapy has shown significant efficacy for B-ALL patients, the high cost and lengthy course of blinatumomab infusion limit its broader use. In addition, its therapeutic potential in terms of depth and the duration of remission can still be further improved.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eThe authors would like to appreciate the efforts of all those who contributed data to the study. We appreciate the support from patients, families, friends, caregivers, study staff and study investigators for their support in this study.\u003c/p\u003e\n\u003ch3\u003eEthics Approval and Consent to Participate\u003c/h3\u003e\n\u003cp\u003eThe study was approved by the Institutional Ethics Committee of Blood Diseases Hospital and conducted in accordance with the Declaration of Helsinki. Signed informed consent was obtained from the parents, guardians, or patients, as appropriate (Clinical trial information: ChiCTR2100053318).\u003c/p\u003e\n\u003ch3\u003eConsent for Publication\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch3\u003eAvailability of Data and Materials\u003c/h3\u003e\n\u003cp\u003eDeidentified individual patient data are available on request from the corresponding author, Li Zhang ([email protected]) and Xiaofan Zhu ([email protected]).\u003c/p\u003e\n\u003ch3\u003eCompeting Interests\u003c/h3\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eThe project was supported in part by the National Key Research and Development Program of China (2021YFE0106900), the CAMS Innovation Fund for Medical Sciences (CIFMS 2022-I2M-1-022; 2021-I2M-1-041), the National Natural Science Foundation of China (82270144), and the Haihe Laboratory of Cell Ecosystem 418 Innovation Fund [22HHXBSS00039 (Y.Z.)].\u003c/p\u003e\n\u003ch3\u003eAuthors\u0026rsquo; Contributions\u003c/h3\u003e\n\u003cp\u003eLi Zhang and Xiaofan Zhu designed the clinical trial. Wenyu Yang, Yao Zou, Ye Guo, Yumei Chen, Xiaojuan Chen and Yang Wan executed the research and collected the data. Changwen Xue and Chao Wu analyzed the data and drafted the original manuscript. Yueshen Ma contributed to the statistical analysis. All authors reviewed and approved the final version of this manuscript. Changwen Xue and Chao Wu contributed equally to this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Li Zhang and Xiaofan Zhu designed the clinical trial. Wenyu Yang, Yao Zou, Ye Guo, Yumei Chen, Xiaojuan Chen and Yang Wan executed the research and collected the data. Changwen Xue and Chao Wu analyzed the data and drafted the original manuscript. Yueshen Ma contributed to the statistical analysis. All authors reviewed and approved the final version of this manuscript. Changwen Xue and Chao Wu contributed equally to this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Information:\u003c/strong\u003e The project was supported in part by the National Key Research and Development Program of China (2021YFE0106900), the CAMS Innovation Fund for Medical Sciences (CIFMS 2022-I2M-1-022; 2021-I2M-1-041), the National Natural Science Foundation of China (82270144), and the Haihe Laboratory of Cell Ecosystem 418 Innovation Fund [22HHXBSS00039 (Y.Z.)].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Disclosure:\u003c/strong\u003e The authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e Deidentified individual patient data are available on request from the corresponding author, Li Zhang ([email protected]) and Xiaofan Zhu ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval Statement:\u003c/strong\u003e The study was approved by the Institutional Ethics Committee of Blood Diseases Hospital and conducted in accordance with the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient Consent Statement:\u003c/strong\u003e Signed informed consent was obtained from the parents, guardians, or patients, as appropriate (Clinical trial information: ChiCTR2100053318).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePermission to Reproduce Material from Other Sources:\u0026nbsp;\u003c/strong\u003eNo other sources were used in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJeha S, Pei D, Choi J, et al. Improved CNS Control of Childhood Acute Lymphoblastic Leukemia Without Cranial Irradiation: St Jude Total Therapy Study 16. J Clin Oncol. 2019;37(35):3377\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang J, Yu J, Cai J, et al. Prognostic factors for CNS control in children with acute lymphoblastic leukemia treated without cranial irradiation. Blood. 2021;138(4):331\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerry DA, Zhou S, Higley H, et al. Association of Minimal Residual Disease With Clinical Outcome in Pediatric and Adult Acute Lymphoblastic Leukemia: A Meta-analysis. JAMA Oncol. 2017;3(7):e170580.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang J, Chinese Children\u0026prime;s Cancer Group Acute Lymphoblastic Leukemia 2015 Study Group. [Report of Chinese Children's Cancer Group acute lymphoblastic leukemia 2015 multicenter study]. Chin J Pediatr 2022, 60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWinters A, Gore L, Hematology. 2019;2019(1):209\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTopp MS, G\u0026ouml;kbuget N, Zugmaier G, et al. Long-term follow-up of hematologic relapse-free survival in a phase 2 study of blinatumomab in patients with MRD in B-lineage ALL. Blood. 2012;120(26):5185\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG\u0026ouml;kbuget N, Dombret H, Bonifacio M, et al. Blinatumomab for minimal residual disease in adults with B-cell precursor acute lymphoblastic leukemia. Blood. 2018;131(14):1522\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown PA, Ji L, Xu X, et al. Effect of Postreinduction Therapy Consolidation With Blinatumomab vs Chemotherapy on Disease-Free Survival in Children, Adolescents, and Young Adults With First Relapse of B-Cell Acute Lymphoblastic Leukemia: A Randomized Clinical Trial. JAMA. 2021;325(9):833.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlinger M, Brandl C, Zugmaier G, et al. Immunopharmacologic response of patients with B-lineage acute lymphoblastic leukemia to continuous infusion of T cell\u0026ndash;engaging CD19/CD3-bispecific BiTE antibody blinatumomab. Blood. 2012;119(26):6226\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTopp MS, Kufer P, G\u0026ouml;kbuget N, et al. Targeted Therapy With the T-Cell\u0026ndash;Engaging Antibody Blinatumomab of Chemotherapy-Refractory Minimal Residual Disease in B-Lineage Acute Lymphoblastic Leukemia Patients Results in High Response Rate and Prolonged Leukemia-Free Survival. J Clin Oncol. 2011;29(18):2493\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhilipp N, Kazerani M, Nicholls A, et al. T-cell exhaustion induced by continuous bispecific molecule exposure is ameliorated by treatment-free intervals. Blood. 2022;140(10):1104\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Y, Aldoss I, Qu C, et al. Tumor-intrinsic and -extrinsic determinants of response to blinatumomab in adults with B-ALL. Blood. 2021;137(4):471\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuffy C, Santana V, Inaba H, et al. Evaluating blinatumomab implementation in low- and middle-income countries: a study protocol. Implement Sci Commun. 2022;3(1):62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee DW, Santomasso BD, Locke FL, et al. ASTCT Consensus Grading for Cytokine Release Syndrome and Neurologic Toxicity Associated with Immune Effector Cells. Biol Blood Marrow Transpl. 2019;25(4):625\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z, Wang J, Zhang G, Wen Z, et al. Relationship of Minimal Residual Disease in Children with ALL in Early Stage after Chemotherapy with CCCG-ALL 2015 Regimen with the Prognosis. Med Pharm J Chin PLA. 2022;34:22\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaumeister SHC, Mohan GS, Elhaddad A, Lehmann L. Cytokine Release Syndrome and Associated Acute Toxicities in Pediatric Patients Undergoing Immune Effector Cell Therapy or Hematopoietic Cell Transplantation. Front Oncol. 2022;12:841117.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaas C, Krinner E, Brischwein K et al. Mode of cytotoxic action of T cell-engaging BiTE antibody MT110. Immunobiology. 2009;214(6):441\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuell J, Dittrich M, Bedke T, et al. Frequency of regulatory T cells determines the outcome of the T-cell-engaging antibody blinatumomab in patients with B-precursor ALL. Leukemia. 2017;31(10):2181\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohamed S, Elshal M, Kumosani T, et al. L-Asparaginase Isolated from Phaseolus vulgaris Seeds Exhibited Potent Anti-Acute Lymphoblastic Leukemia Effects In-Vitro and Low Immunogenic Properties In-Vivo. Int J Environ Res Public Health. 2016;13(10):1008.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng W, Gao Y, Ke X, et al. PEG-L-CHOP treatment is safe and effective in adult extranodal NK/T-cell lymphoma with a low rate of clinical hypersensitivity. BMC Cancer. 2018;18(1):910.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKauer J, M\u0026auml;rklin M, Pfl\u0026uuml;gler M, et al. BCR::ABL1 tyrosine kinase inhibitors hamper the therapeutic efficacy of blinatumomab in vitro. J Cancer Res Clin Oncol. 2022;148(10):2759\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSubhash VV, Huang L, Kamili A, et al. Whole-genome sequencing facilitates patient-specific quantitative PCR-based minimal residual disease monitoring in acute lymphoblastic leukaemia, neuroblastoma and Ewing sarcoma. Br J Cancer. 2022;126(3):482\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBartram J, Patel B, Fielding AK. Monitoring MRD in ALL: Methodologies, technical aspects and optimal time points for measurement. Semin Hematol. 2020;57(3):142\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"B-cell acute lymphoblastic leukemia, blinatumomab, pediatric, minimal residual disease, immunotherapy ","lastPublishedDoi":"10.21203/rs.3.rs-3940188/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3940188/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe conventional intensity of chemotherapy has reached its limit. Despite the efficacy of blinatumomab in B-cell acute lymphoblastic leukemia (B-ALL) adults with measurable residual disease (MRD) has been proved, studies in children with MRD positivity (MRD+) are still lacking. We conducted an exploratory trial of 14 days of blinatumomab monotherapy in combination with a B-ALL chemotherapy protocol in pediatric patients with MRD+ at the day 46 and evaluated the early efficacy and safety of this approach (Blin-14d-therapy, ChiCTR2100053318, registered on 11/18/2021). Totally 9 pediatric patients were included in Blin-14d group and 38 in non-Blin-14d group. After monotherapy with blinatumomab, all evaluable patients became MRD negative in Blin-14d group and achieved MRD conversion earlier than conventional chemotherapy group, predicting better treatment outcomes. Compared to previous investigations, the incidence of AEs was lower and MRD clearance was higher in this study. In conventional blinatumomab studies, a single course was administered for 28 days. T-cell counts continued to trend upward from day 14 to 21, whereas a downward trend was observed from day 21 to 28, suggesting that T-cell exhaustion occured. While in our regimen, blinatumomab was administered for 14 days, and T-cell levels still had an upward trend in the first week after the end of the infusion, suggesting that T-cell exhaustion at this time was not significant and anti-tumor effect prolonged. Initial data from this study demonstrate that Blin-14d-therapy with chemotherapy has encouraging clinical activity and a manageable safety profile in pediatric patients with day46 MRD+ B-ALL in IR/HR groups.\u003c/p\u003e","manuscriptTitle":"Infusion of 14 days Blinatumomab in Combination with Chemotherapy for 46-day MRD+ Pediatric B-ALL Patients in Intermediate/High-Risk Group Results in MRD Conversion and BiTE Induced Immune Response to Malignant Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-16 17:43:45","doi":"10.21203/rs.3.rs-3940188/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"af170502-f5c1-476c-9b01-dfebf2f4525a","owner":[],"postedDate":"February 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-16T11:33:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-16 17:43:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3940188","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3940188","identity":"rs-3940188","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}