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

Relationship between subtype-specific minimal residual disease level and long-term prognosis in children with acute lymphoblastic leukemia | 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 Relationship between subtype-specific minimal residual disease level and long-term prognosis in children with acute lymphoblastic leukemia 筱彤 黄, Chan-Juan Wang, Chao Gao, Tian-Lin Xue, Zi-Jing Zhao, Tian-You Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3810073/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Mar, 2024 Read the published version in Annals of Hematology → Version 1 posted 7 You are reading this latest preprint version Abstract In childhood acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) risk stratification criteria specific to common genetic subtypes are unclear. Among 723 children with newly diagnosed ALL treated with the Chinese Children Leukemia Group trial CCLG-2008 protocol, at time point 1 (TP1, the end of induction) and TP2 (before consolidation for standard risk and intermediate risk group, or before the second HR-I' block for high risk group), the MRD levels of children carrying different fusion genes or with T-ALL were significantly different ( P all <0.001), and the prognostic significance of the same MRD level in the above subtypes was greatly different. For patients carrying BCR::ABL1 or KMT2A rearrangements, or ETV6::RUNX1 , or with T-ALL, we defined those with both TP1 and TP2 MRD levels <10 -2 , TP1 MRD <10 -3 and TP2 MRD-negative, TP1 MRD <10 -3 , as low MRD group respectively; the remaining children as high MRD group of each subtype. The 10-year relapse free survival (RFS) of low MRD group was significantly better than that of high MRD group. We verified the clinical value of the above MRD stratification criteria in patients treated with “Beijing Children’s Hospital BCH-ALL2003” protocol. In conclusion, subtype-specific MRD risk stratification may contribute to the precise treatment of childhood ALL. ALL MRD fusion gene relapse risk stratification Figures Figure 1 Figure 2 Introduction Acute lymphoblastic leukemia (ALL) is the most common malignant tumor in children. While the 5-year survival rate has reached over 90%, 15–20% of the patients still relapse. Relapsed ALL is associated with a poor prognosis and one of the leading causes of cancer death in children [ 1 ]. Studies have shown that although intense chemotherapy can eliminate most tumor cells, certain factors such as leukemia stem cells, clonal evolution/selection, genetic and epigenetic changes, and abnormal immune microenvironments make some leukemic cells resistant to chemotherapy, eventually leading to the recurrence of ALL. The prognosis for recurrence at different sites varies. Children with bone marrow (BM) recurrence, whether combined with extramedullary relapses or not, have a worse prognosis than those with recurrence at other sites. Following induction chemotherapy, a few leukemia cells may still be present in the body, referred to as minimal residual disease (MRD). This MRD not only contributes to leukemia recurrence but also plays a crucial role in evaluating the prognosis of children with ALL. As an important independent prognostic factor, MRD significantly affects the risk of recurrence and treatment outcome [ 2 ]. While leukemia cells in different children may exhibit varying immune phenotypes, fusion genes, and genetic mutations, children are divided into three risk groups based on the same MRD risk stratification criteria. Furthermore, recent studies have revealed that the relationship between the same MRD level and the risk of recurrence may not always be consistent among different subtypes of patients [ 3 , 4 ]. This study delves into the correlation between MRD levels and long-term prognosis in children with ALL who carry different fusion genes, using research data from BCH-2003 protocol and CCLG-2008 protocol. The study seeks to establish subtype-specific MRD stratification standards. Materials and methods Patients From February 2005 to March 2008, 415 children with newly diagnosed ALL were treated with BCH-2003 protocol (BCH-2003 group). From April 2008 to December 2012, 723 children with newly diagnosed ALL were treated with CCLG-2008 protocol (CCLG-2008 group). The patients were diagnosed and classified based on morphology, immunophenotype, cytogenetics and molecular biology [ 5 , 6 ]. Totally, 663 and 346 patients, in BCH-2003 and CCLG-2008 group respectively, were enrolled in this study. Sixty-nine and sixty children in the above two groups respectively, were excluded, due to insufficient sampling or lack of suitable markers for MRD detection. The Ethics Committee of Beijing Children's Hospital has approved this study. Treatments Upon initial diagnosis, the children underwent a thorough evaluation for relapse risk based on factors such as peripheral white blood cells, age, immunophenotype, and cytogenetic and molecular genetic abnormalities. Then the children were categorized into one of three risk groups: standard risk (SR), intermediate risk (IR), or high risk (HR). The risk evaluation was further assessed based on prednisone response on day 8 (we defined ≥ 1×10 9 /L blasts in peripheral blood on day 8 as poor prednisone response), and BM response status on day 22 (BCH-2003 protocol) or day 15 (CCLG-2008 protocol), and day 33. Additionally, MRD levels were also taken into account to adjust risk stratification in CCLG-2008 protocol. The specific standards for risk stratification and adjustment of the BCH-2003 protocol and CCLG-2008 protocol are shown in Supplementary Fig. 1 and Supplementary Fig. 2. Fusion gene testing The reverse transcription-multiple nested polymerase chain reaction (PCR) method was used to detect 29 common fusion genes in leukemia, which has been described previously [ 5 , 6 ]. MRD detection MRD levels were determined at the end of induction remission therapy (time point 1, TP1) and before consolidation therapy (time point 2, TP2). We used real-time quantitative PCR method, or multi-parameter flow cytometry four-color fluorescence direct labeling method to detect MRD levels in bone marrow. Immunoglobulin/T cell receptor (Ig/TCR) gene rearrangements were used as a molecular marker in the former method [ 7 ]. The sensitivity and quantitative range of the detection were at least 10 − 4 [ 8 ]. Statistics All children were followed up until April 30, 2023. The chi-square test was used to analyze the relationship between MRD level and treatment outcome. Relapse-free survival (RFS) was defined as the date from diagnosis to leukemia relapse. Children in continuous complete remission (CCR) were followed up to the last contact. The Kaplan-Meier method and Log-rank test were used to compare the differences in RFS between the two groups of children. SPSS 26.0 software was used for statistical analysis, and a P < 0.05 was considered a significant difference. Results 1. Clinical characteristics and treatment outcome There were 663 patients in CCLG-2008 group, including 417 boys and 246 girls. The median age was 4.5 years (0.6 – 15.5years), and the median follow-up time was 12.05 years. The 10-year RFS was 88.0%±1.3%. One hundred and four children died from relapse (43, 41.3%), infection (35, 33.6%), transplantation complications (6, 5.8%), persistent non-remission (5, 4.9%), and other causes (15, 14.4%). There was no significant difference between the patients included and excluded, except molecular subtypes. The details of the children enrolled in this study are shown in Table 1. There were 346 children with newly diagnosed ALL in BCH-2003 group, including 209 boys and 137 girls. The median age was 5 years (0.5- 16 years), and the median follow-up time was 16.06 years. The 10-year RFS was 82.2%±2.1%. There was no significant difference between the patients included and excluded, except poorer prognosis of the patients included. Table 1 Clinical characteristics and treatment effects of enrolled children BCH-2003 CCLG-2008 Inclusion (%) Exclusion (%) P Inclusion (%) Exclusion (%) P Total 346 (83.4) 69 (16.6) 663 (91.7) 60 (8.3) Sex Male 209 (60.4) 40 (58.0) 0.093 417 (62.9) 36 (60.0) 0.657 Female 137 (39.6) 29 (42.0) 246 (37.1) 24 (40.0) Age ≥ 1 & < 10 280 (80.7) 54 (78.3) 0.643 566 (85.4) 51 (85.0) 0.938 < 1 or ≥ 10 67 (19.3) 15 (21.7) 97 (14.6) 9 (15.0) Immunophenotype B cell 302 (82.5) 64 (89.8) 0.201 618 (93.2) 52 (86.7) 0.062 T cell 44 (17.5) 5 (10.2) 45 (6.8) 8 (13.3) WBC ( × 10 9 /L) <50 272 (78.6) 58 (84.1) 0.288 538 (81.1) 43 (71.7) 0.077 ≥ 50 74 (21.4) 11 (15.9) 125 (18.9) 17 (28.3) Molecular subtype ETV6::RUNX1 74 (22.2) 11 (16.4) 0.182 142 (21.4) 2 (3.3) <0.001 TCF3::PBX1 21 (6.3) 2 (3.0) 35 (5.3) 6 (10.0) BCR::ABL1 16 (4.8) 8 (11.9) 38 (5.7) 4 (6.7) KMT2A 6 (1.8) 1 (1.5) 9 (1.4) 4 (6.7) T-ALL 31 (9.3) 4 (6.0) 45 (6.8) 8 (13.3) B others 186 (55.7) 41 (61.2) 394 (59.4) 36 (60.0) 10y RFS 82.2±2.1 96.2±2.7 0.013 88.0±1.3 86.1±5.8 0.655 2. Characteristics of MRD levels in ALL patients with different molecular subtypes In CCLG-2008 group, MRD levels were measured in all 663 patients at TP1. Of those, 620 children (93.5%) underwent MRD detection at TP2. The analysis showed that at TP1 and TP2, there were significant differences in MRD levels in children with different molecular subtypes (both P <0.001, Figure 1). Children carrying BCR::ABL1 or KMT2A rearrangements still had high TP1 MRD levels. TP1 MRD level was ≥10 -4 and ≥10 -2 in 97.1% and 51.4% of children with BCR::ABL1 respectively. Similarly, TP1 MRD levels ≥10 -4 and ≥10 -2 were observed in 88.9% and 37.5% of children with KMT2A rearrangements respectively. Contrarily, ETV6::RUNX1 - and TCF3::PBX1 -positive patients had low MRD levels. At TP1, only 52.1% and 34.2% of patients had MRD levels ≥10 -4 , respectively, and no children had MRD levels ≥10 -2 . Children with T-ALL were similar to those with BCR::ABL1 and KMT2A rearrangements. Among those, 88.9% and 22.5% of T-ALL patients had TP1 MRD ≥ 10 -4 or ≥ 10 -2 respectively. At TP2, MRD level in children with BCR::ABL1 and KMT2A rearrangements was still high: MRD level ≥10 -2 was observed in 28% and 25% of the children respectively; only 56.0% and 50.0% of children had MRD levels <10 -4 . In contrast, more than 90% of ETV6::RUNX1 - and TCF3::PBX1 -positive patients had MRD levels below 10 -4 , and no patient had MRD ≥ 10 -3 . Among children with T-ALL, MRD level was between the above two situations. About 70% of children with T-ALL had MRD levels <10 -4 , and only 7.6% of the patients had MRD levels ≥10 -3 . 3. The relationship between MRD levels and recurrence in children with different molecular subtypes We compared 10-year RFS of children with different molecular subtypes and MRD levels in CCLG-2008 group. Among patients with BCR::ABL1 , the 10-year RFS of patients with MRD ≥10 -2 at both TP1 and TP2 were significantly lower than that of children with MRD<10 -2 (22.6%±11.5% vs 64.2%±14.1%, P =0.011; 30.4%±11.4% vs 65.7%±15.9%, P =0.004). Similar results appeared in children with KMT2A rearrangements, at both TPs, MRD ≥ 10 -2 was associated with lower 10-year RFS (both 0 vs 66.7%±19.2%, both P = 0.004). In fact, there was no patient carrying KMT2A rearrangements with MRD levels between 10 -3 and 10 -2 at both TPs, leading to the establishment of an MRD level cut-off value of 10 -2 for this subtype. Among patients with ETV6::RUNX1 , 10-year RFS of patients with TP1 MRD≥10 -3 and TP2 MRD-positive was significantly lower than that of patients with TP1 MRD<10 -3 and TP2 MRD-negative respectively (76.9%±11.7% vs 96.1%±1.7%, P =0.004; 82.4%±9.2% vs 95.9%±1.8%, P =0.019). It was noteworthy that patients with TCF3::PBX1 had low MRD levels at both TP1 and TP2, we did not observe significant correlation of MRD levels with patient prognosis (Table 2). For children with T-ALL, TP1 MRD≥10 -3 was associated with poor prognosis (60.7%±12.6% vs 90.9%±6.1%, P =0.033), however, there was no significant correlation between TP2 MRD level and treatment outcome (Table 2). Based on the above analysis, we divided children in each molecular subtype into two groups and compared the prognosis of the two groups respectively. For patients with BCR::ABL1 or KMT2A rearrangements, we defined patients with MRD<10 -2 at both TP1 and TP2 as low MRD group, and the others as high MRD group. The 10-year RFS of the former was significantly better than that of the latter (77.4% ± 11.5% vs 35.8% ± 14.1%, P =0.011; 76.2%±14.8% vs 0, P <0.001, respectively). For children with ETV6::RUNX1 , we defined patients with TP1 MRD<10 -3 and TP2 MRD-negative as low MRD group, and the others as high MRD group. The 10-year RFS of the two groups were 96.6% ± 1.7% and 81.8% ± 8.2% ( P = 0.005) respectively. For children with T-ALL, We defined children with TP1 MRD ≥ 10 -3 as high MRD group, and the rest as low MRD group. As expected, the 10-year RFS of the former was significantly worse (60.7 %±12.6% and 90.9%±6.1%, P = 0.033). For children with TCF3::PBX1 , since the MRD levels at TP1 and TP2 were not significantly related to prognosis, we did not conduct further analysis on patients in this subtype. We verified the above analysis results of each molecular subtype in children of BCH-2003 group. The results showed that for children carrying BCR::ABL1 and KMT2A rearrangements, the 10-year RFS of the high/low MRD group were 100% vs 35.7%±19.8%, and 100% vs 0% respectively. It is worth noting, however, that we did not observe any significant differences in both subtypes ( P =0.242 and 0.063, respectively), which may be due to the small sample size. Among children with ETV6::RUNX1 , the 10-year RFS of low MRD group was significantly better than that of high MRD group (88.2%±5.9% vs 57.9%±19.9%, P =0.013). Similarly, the 10-year RFS of children with T-ALL in low MRD group was significantly better than that in high MRD group (100% vs 66.7%±10.3%, P =0.012). It is of importance that children in B-others subgroup carry various genetic mutations that had not been determined, and thus cannot be accurately classified. As a result, we did not carry out depth analysis on this subtype. In summary, there were significant differences in the prognostic significance of the same MRD level among ALL patients carrying different fusion genes or immunephenotypes. Discussion Children with ALL carry a range of genetic aberrations, such as fusion genes. This study showed that patients carrying different fusion genes have significant differences in MRD levels at TP1 and TP2, and the same MRD level has different prognostic significance among various molecular subtypes. For TP1 MRD ≥ 10 − 4 , the 10-year RFS were only 56.5%±10.0% and 57.1%±18.7% in patients with BCR::ABL1 or KMT2A rearrangements, respectively; in contrast, it was up to 92.0%±3.1% and 84.6%±10.0% in those with ETV6::RUNX1 or TCF3::PBX1 , respectively. This suggests that it is necessary to fully take into account genetic subtypes in stratification of childhood ALL based on MRD, in order to achieve more accurate treatment and in turn better effectiveness. Next-generation sequencing technology (NGS) has gained widespread popularity as a powerful tool for analyzing variations in nucleic acids [ 9 ]. This innovative method can accurately identify specific rearrangement fragments of Ig and TCR genes [ 10 ], as well as other gene anomalies such as insertions, deletions, and fusion genes. In leukemia, NGS has shown remarkable sensitivity (about 10 − 5 ~10 − 7 ), far more sensitive than other technologies like FCM and PCR [ 10 ]. This study indicated that 15.7% and 48.6% of children with TCF3::PBX1 were MRD-negative at TP1 and TP2, respectively. However, there was no significant correlation between MRD levels and prognosis. Future study could utilize NGS to show the existence of extra low level of MRD, which could promote accurate risk stratification and prognosis evaluation [ 12 ]. This study showed that among children with BCR::ABL1 , the prognosis of low MRD group was significantly better than that of high MRD group, however, this is based on traditional intensive chemotherapy. Studies have shown that the application of small molecule tyrosine kinase inhibitors such as imatinib could significantly improve treatment outcome of children with BCR::ABL1 [ 13 ]. It is crucial, therefore, to investigate more exact MRD stratification criteria for targeted therapy combined with chemotherapy. [ 14 ]. Recent studies suggest that children in B others group may have various fusion genes or mutations, such as DUX4 rearrangements, MEF2D , ZNF384 rearrangements, NUTM1 rearrangements, TCF3::HLF , IKZF1 mutations, which were not determined in this study. Thus, we did not conduct an in-depth analysis of this subgroup of children. Similarly, T-ALL has many genetic abnormalities, such as fusion genes involving TAL1 , HOXA10 , TLX3 , TLX1 , NKX2-1 , LMO1/2 , and others. [ 15 ]. Future studies could explore the above genetic variant-specific MRD stratification criteria to facilitate the precise stratification and treatment of childhood ALL. This study draws upon clinical data from two treatment regimens, namely BCH-2003 and CCLG-2008 protocol. While the primary clinical characteristics of these two groups of patients did not exhibit significant differences, additional multi-center study is necessary to obtain more precise conclusions. This will facilitate the development of more effective risk stratification and treatment guidelines. Declarations Authors' contributions HXT analyzed the data and wrote the original draft. WCJ and ZZJ collected the clinical data. GC and XTL performed the experiments. WTY, WMY and ZRD made clinical contributions. LZG , CL and ZRD designed the research and revised the paper. All authors were involved in the final approval of the paper. Funding This work was supported by the National Natural Science Foundation of China (No. 81970135 and No. 81870114), the Beijing Natural Science Foundation of China (No. 7202044), the New Sunshine Charity Foundation, China (No. 2017002), and Funding for Reform and Development of Beijing Municipal Health Commission. Conflict of interest The authors declare no conflict of interest. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics approval statement This study was performed according to the principles of the Declaration of Helsinki, and approved by the Institutional Review Board of Beijing Children’s Hospital (2007-20). Patient consent statement Informed consent were obtained from guardians of the patients. References Hunger SP, Mullighan CG (2015) Acute Lymphoblastic Leukemia in Children. 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Biol Blood Marrow Transplant 23(4):691–696 Druker BJ (2002) Perspectives on the development of a molecularly targeted agent. Cancer Cell 1(1):31–36 Schrappe M et al (2011) Late MRD response determines relapse risk overall and in subsets of childhood T-cell ALL: results of the AIEOP-BFM-ALL 2000 study. Blood 118(8):2077–2084 Brady SW et al (2022) The genomic landscape of pediatric acute lymphoblastic leukemia. Nat Genet 54(9):1376–1389 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 18 Mar, 2024 Read the published version in Annals of Hematology → Version 1 posted Editorial decision: Revision requested 25 Jan, 2024 Reviews received at journal 08 Jan, 2024 Reviewers agreed at journal 30 Dec, 2023 Reviewers invited by journal 30 Dec, 2023 Submission checks completed at journal 28 Dec, 2023 Editor assigned by journal 28 Dec, 2023 First submitted to journal 26 Dec, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Hematology Center, Beijing Children's Hospital, Capital Medical University, National Center for Children’s Health, Beijing","correspondingAuthor":false,"prefix":"","firstName":"Rui-Dong","middleName":"","lastName":"Zhang","suffix":""},{"id":264476347,"identity":"da181112-00ab-4b6a-a541-ccbe0c681768","order_by":9,"name":"Zhi-Gang Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIie3PvQrCMBDA8SuBdDl0rehDnBQc7atEXIO7+FGl4CS46ot0binoUnfFxeos1N3BtDg4NR0F859CuB/JAZhMP5i3sBZwBfBX9rq8wIaOECgiAKwNpgCRIlxPVAXZbWVJQE/ayTIXrxmj8yO+P2W/w4Flt1PVLo1B4Ag8cLqMhhSHQ/Ux7rqy6hW0ViCcPdJF9pw4ZIogb+sJ7R06pwXx6xIxpdYWC5LUImqXKBJNlC4dwwNyptnFW9tZnr/mgttp9zoOJ17TDrJ7FfmUfJ2ZfrxoXm/MZDKZ/rM3vL9FRSj29fIAAAAASUVORK5CYII=","orcid":"","institution":"Hematologic Diseases Laboratory, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, National Center for Children’s Health, Beijing","correspondingAuthor":true,"prefix":"","firstName":"Zhi-Gang","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2023-12-27 01:59:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3810073/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3810073/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00277-024-05687-y","type":"published","date":"2024-03-18T15:01:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49133432,"identity":"df399559-676a-42ac-b61d-197c4e362829","added_by":"auto","created_at":"2024-01-03 16:43:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":181985,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of MRD levels at TP1 and TP2 in children with different molecular subtypes in CCLG-2008 group.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3810073/v1/6ca0e23e2247dac3762370bb.png"},{"id":49134261,"identity":"5ee137a5-44f4-4def-93c4-b336738963d3","added_by":"auto","created_at":"2024-01-03 16:51:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":98695,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between MRD levels and treatment outcomes in children with ALL. A: RFS at different MRD levels in children in different subtypes in CCLG-2008 group. B: RFS at different MRD levels in children in different subtypes in BCH-2003\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3810073/v1/c46d587bd31dc3c3e473272c.png"},{"id":53404239,"identity":"d2f9c2ac-8e64-4764-820f-79d2a6e3c2b6","added_by":"auto","created_at":"2024-03-25 15:16:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":400311,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3810073/v1/46ca494c-93cd-4b72-b86f-da5d33184797.pdf"},{"id":49133434,"identity":"1ed5cb16-c480-456b-aa63-e1d1427d4e4a","added_by":"auto","created_at":"2024-01-03 16:43:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":38441,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-3810073/v1/4b28842289f4dd3d6f5528d6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Relationship between subtype-specific minimal residual disease level and long-term prognosis in children with acute lymphoblastic leukemia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute lymphoblastic leukemia (ALL) is the most common malignant tumor in children. While the 5-year survival rate has reached over 90%, 15\u0026ndash;20% of the patients still relapse. Relapsed ALL is associated with a poor prognosis and one of the leading causes of cancer death in children [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Studies have shown that although intense chemotherapy can eliminate most tumor cells, certain factors such as leukemia stem cells, clonal evolution/selection, genetic and epigenetic changes, and abnormal immune microenvironments make some leukemic cells resistant to chemotherapy, eventually leading to the recurrence of ALL. The prognosis for recurrence at different sites varies. Children with bone marrow (BM) recurrence, whether combined with extramedullary relapses or not, have a worse prognosis than those with recurrence at other sites.\u003c/p\u003e \u003cp\u003eFollowing induction chemotherapy, a few leukemia cells may still be present in the body, referred to as minimal residual disease (MRD). This MRD not only contributes to leukemia recurrence but also plays a crucial role in evaluating the prognosis of children with ALL. As an important independent prognostic factor, MRD significantly affects the risk of recurrence and treatment outcome [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While leukemia cells in different children may exhibit varying immune phenotypes, fusion genes, and genetic mutations, children are divided into three risk groups based on the same MRD risk stratification criteria. Furthermore, recent studies have revealed that the relationship between the same MRD level and the risk of recurrence may not always be consistent among different subtypes of patients [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study delves into the correlation between MRD levels and long-term prognosis in children with ALL who carry different fusion genes, using research data from BCH-2003 protocol and CCLG-2008 protocol. The study seeks to establish subtype-specific MRD stratification standards.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003ePatients\u003c/p\u003e \u003cp\u003eFrom February 2005 to March 2008, 415 children with newly diagnosed ALL were treated with BCH-2003 protocol (BCH-2003 group). From April 2008 to December 2012, 723 children with newly diagnosed ALL were treated with CCLG-2008 protocol (CCLG-2008 group). The patients were diagnosed and classified based on morphology, immunophenotype, cytogenetics and molecular biology [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Totally, 663 and 346 patients, in BCH-2003 and CCLG-2008 group respectively, were enrolled in this study. Sixty-nine and sixty children in the above two groups respectively, were excluded, due to insufficient sampling or lack of suitable markers for MRD detection. The Ethics Committee of Beijing Children's Hospital has approved this study.\u003c/p\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003cp\u003eUpon initial diagnosis, the children underwent a thorough evaluation for relapse risk based on factors such as peripheral white blood cells, age, immunophenotype, and cytogenetic and molecular genetic abnormalities. Then the children were categorized into one of three risk groups: standard risk (SR), intermediate risk (IR), or high risk (HR). The risk evaluation was further assessed based on prednisone response on day 8 (we defined\u0026thinsp;\u0026ge;\u0026thinsp;1\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L blasts in peripheral blood on day 8 as poor prednisone response), and BM response status on day 22 (BCH-2003 protocol) or day 15 (CCLG-2008 protocol), and day 33. Additionally, MRD levels were also taken into account to adjust risk stratification in CCLG-2008 protocol. The specific standards for risk stratification and adjustment of the BCH-2003 protocol and CCLG-2008 protocol are shown in Supplementary Fig.\u0026nbsp;1 and Supplementary Fig.\u0026nbsp;2.\u003c/p\u003e \u003cp\u003eFusion gene testing\u003c/p\u003e \u003cp\u003eThe reverse transcription-multiple nested polymerase chain reaction (PCR) method was used to detect 29 common fusion genes in leukemia, which has been described previously [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMRD detection\u003c/p\u003e \u003cp\u003eMRD levels were determined at the end of induction remission therapy (time point 1, TP1) and before consolidation therapy (time point 2, TP2). We used real-time quantitative PCR method, or multi-parameter flow cytometry four-color fluorescence direct labeling method to detect MRD levels in bone marrow. Immunoglobulin/T cell receptor (Ig/TCR) gene rearrangements were used as a molecular marker in the former method [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The sensitivity and quantitative range of the detection were at least 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStatistics\u003c/p\u003e \u003cp\u003eAll children were followed up until April 30, 2023. The chi-square test was used to analyze the relationship between MRD level and treatment outcome. Relapse-free survival (RFS) was defined as the date from diagnosis to leukemia relapse. Children in continuous complete remission (CCR) were followed up to the last contact. The Kaplan-Meier method and Log-rank test were used to compare the differences in RFS between the two groups of children. SPSS 26.0 software was used for statistical analysis, and a \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered a significant difference.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e1. Clinical characteristics and treatment outcome\u003c/p\u003e\n\u003cp\u003eThere were 663 patients in CCLG-2008 group, including 417 boys and 246 girls. The median age was 4.5 years (0.6 \u0026ndash; 15.5years), and the median follow-up time was 12.05 years. The 10-year RFS was 88.0%\u0026plusmn;1.3%. One hundred and four children died from relapse (43, 41.3%), infection (35, 33.6%), transplantation complications (6, 5.8%), persistent non-remission (5, 4.9%), and other causes (15, 14.4%). There was no significant difference between the patients included and excluded, except molecular subtypes. The details of the children enrolled in this study are shown in Table 1.\u003c/p\u003e\n\u003cp\u003eThere were 346 children with newly diagnosed ALL in BCH-2003 group, including 209 boys and 137 girls. The median age was 5 years (0.5- 16 years), and the median follow-up time was 16.06 years. The 10-year RFS was 82.2%\u0026plusmn;2.1%. There was no significant difference between the patients included and excluded, except poorer prognosis of the patients included.\u003c/p\u003e\n\u003cp\u003eTable 1 Clinical characteristics and treatment effects of enrolled children\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"626\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.926517571884983%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.65814696485623%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eBCH-2003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.415335463258785%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eCCLG-2008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.926517571884983%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.654952076677317%\" valign=\"top\"\u003e\n \u003cp\u003eInclusion (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.175718849840255%\" valign=\"top\"\u003e\n \u003cp\u003eExclusion (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.827476038338658%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.335463258785943%\" valign=\"top\"\u003e\n \u003cp\u003eInclusion (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.97444089456869%\" valign=\"top\"\u003e\n \u003cp\u003eExclusion (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.105431309904153%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.926517571884983%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.654952076677317%\" valign=\"top\"\u003e\n \u003cp\u003e346 (83.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.175718849840255%\" valign=\"top\"\u003e\n \u003cp\u003e69 (16.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.827476038338658%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.335463258785943%\" valign=\"top\"\u003e\n \u003cp\u003e663 (91.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.97444089456869%\" valign=\"top\"\u003e\n \u003cp\u003e60 (8.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.105431309904153%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.926517571884983%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.654952076677317%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.175718849840255%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.827476038338658%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.335463258785943%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.97444089456869%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.105431309904153%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.926517571884983%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.654952076677317%\" valign=\"top\"\u003e\n \u003cp\u003e209 (60.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.175718849840255%\" valign=\"top\"\u003e\n \u003cp\u003e40 (58.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.827476038338658%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.093\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.335463258785943%\" valign=\"top\"\u003e\n \u003cp\u003e417 (62.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.97444089456869%\" valign=\"top\"\u003e\n \u003cp\u003e36 (60.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.105431309904153%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.657\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.192307692307693%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.846153846153847%\" valign=\"top\"\u003e\n \u003cp\u003e137 (39.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.26923076923077%\" valign=\"top\"\u003e\n \u003cp\u003e29 (42.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.46153846153846%\" valign=\"top\"\u003e\n 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width=\"9.105431309904153%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.926517571884983%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026ge;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;1 \u0026amp; \u0026lt; 10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.654952076677317%\" valign=\"top\"\u003e\n \u003cp\u003e280 (80.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.175718849840255%\" valign=\"top\"\u003e\n \u003cp\u003e54 (78.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.827476038338658%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.643\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.335463258785943%\" valign=\"top\"\u003e\n \u003cp\u003e566 (85.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.97444089456869%\" valign=\"top\"\u003e\n \u003cp\u003e51 (85.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.105431309904153%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.938\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.192307692307693%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt; 1 or\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026ge;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.846153846153847%\" valign=\"top\"\u003e\n \u003cp\u003e67 (19.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.26923076923077%\" valign=\"top\"\u003e\n \u003cp\u003e15 (21.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.46153846153846%\" valign=\"top\"\u003e\n \u003cp\u003e97 (14.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.23076923076923%\" valign=\"top\"\u003e\n \u003cp\u003e9 (15.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.926517571884983%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eImmunophenotype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.654952076677317%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.175718849840255%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.827476038338658%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.335463258785943%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.97444089456869%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.105431309904153%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.926517571884983%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eB cell\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.654952076677317%\" valign=\"top\"\u003e\n \u003cp\u003e302 (82.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.175718849840255%\" valign=\"top\"\u003e\n \u003cp\u003e64 (89.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.827476038338658%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.201\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.335463258785943%\" valign=\"top\"\u003e\n \u003cp\u003e618 (93.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.97444089456869%\" valign=\"top\"\u003e\n \u003cp\u003e52 (86.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.105431309904153%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.062\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.192307692307693%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eT cell\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.846153846153847%\" valign=\"top\"\u003e\n \u003cp\u003e44 (17.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.26923076923077%\" valign=\"top\"\u003e\n \u003cp\u003e5 (10.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.46153846153846%\" valign=\"top\"\u003e\n \u003cp\u003e45 (6.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.23076923076923%\" valign=\"top\"\u003e\n \u003cp\u003e8 (13.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.926517571884983%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eWBC (\u003c/strong\u003e\u003cstrong\u003e\u0026times;\u003c/strong\u003e\u003cstrong\u003e10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.654952076677317%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.175718849840255%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.827476038338658%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.335463258785943%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.97444089456869%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.105431309904153%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.926517571884983%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.654952076677317%\" valign=\"top\"\u003e\n \u003cp\u003e272 (78.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.175718849840255%\" valign=\"top\"\u003e\n \u003cp\u003e58 (84.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.827476038338658%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.288\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.335463258785943%\" valign=\"top\"\u003e\n \u003cp\u003e538 (81.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.97444089456869%\" valign=\"top\"\u003e\n \u003cp\u003e43 (71.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.105431309904153%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.077\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.192307692307693%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026ge;\u003c/strong\u003e\u003cstrong\u003e50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.846153846153847%\" valign=\"top\"\u003e\n \u003cp\u003e74 (21.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.26923076923077%\" valign=\"top\"\u003e\n \u003cp\u003e11 (15.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.46153846153846%\" valign=\"top\"\u003e\n \u003cp\u003e125 (18.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.23076923076923%\" valign=\"top\"\u003e\n \u003cp\u003e17 (28.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.926517571884983%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMolecular subtype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.654952076677317%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.175718849840255%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.827476038338658%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.335463258785943%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.97444089456869%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.105431309904153%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.926517571884983%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eETV6::RUNX1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.654952076677317%\" valign=\"top\"\u003e\n \u003cp\u003e74 (22.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.175718849840255%\" valign=\"top\"\u003e\n \u003cp\u003e11 (16.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.827476038338658%\" rowspan=\"6\" valign=\"top\"\u003e\n \u003cp\u003e0.182\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.335463258785943%\" valign=\"top\"\u003e\n \u003cp\u003e142 (21.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.97444089456869%\" valign=\"top\"\u003e\n \u003cp\u003e2 (3.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.105431309904153%\" rowspan=\"6\" valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.192307692307693%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eTCF3::PBX1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.846153846153847%\" valign=\"top\"\u003e\n \u003cp\u003e21 (6.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.26923076923077%\" valign=\"top\"\u003e\n \u003cp\u003e2 (3.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.46153846153846%\" valign=\"top\"\u003e\n \u003cp\u003e35 (5.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.23076923076923%\" valign=\"top\"\u003e\n \u003cp\u003e6 (10.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.192307692307693%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eBCR::ABL1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.846153846153847%\" valign=\"top\"\u003e\n \u003cp\u003e16 (4.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.26923076923077%\" valign=\"top\"\u003e\n \u003cp\u003e8 (11.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.46153846153846%\" valign=\"top\"\u003e\n \u003cp\u003e38 (5.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.23076923076923%\" valign=\"top\"\u003e\n \u003cp\u003e4 (6.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.192307692307693%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eKMT2A\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.846153846153847%\" valign=\"top\"\u003e\n \u003cp\u003e6 (1.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.26923076923077%\" valign=\"top\"\u003e\n \u003cp\u003e1 (1.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.46153846153846%\" valign=\"top\"\u003e\n \u003cp\u003e9 (1.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.23076923076923%\" valign=\"top\"\u003e\n \u003cp\u003e4 (6.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.192307692307693%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eT-ALL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.846153846153847%\" valign=\"top\"\u003e\n \u003cp\u003e31 (9.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.26923076923077%\" valign=\"top\"\u003e\n \u003cp\u003e4 (6.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.46153846153846%\" valign=\"top\"\u003e\n \u003cp\u003e45 (6.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.23076923076923%\" valign=\"top\"\u003e\n \u003cp\u003e8 (13.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.192307692307693%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eB others\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.846153846153847%\" valign=\"top\"\u003e\n \u003cp\u003e186 (55.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.26923076923077%\" valign=\"top\"\u003e\n \u003cp\u003e41 (61.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.46153846153846%\" valign=\"top\"\u003e\n \u003cp\u003e394 (59.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.23076923076923%\" valign=\"top\"\u003e\n \u003cp\u003e36 (60.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.926517571884983%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e10y RFS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.654952076677317%\" valign=\"top\"\u003e\n \u003cp\u003e82.2\u0026plusmn;2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.175718849840255%\" valign=\"top\"\u003e\n \u003cp\u003e96.2\u0026plusmn;2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.827476038338658%\" valign=\"top\"\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.335463258785943%\" valign=\"top\"\u003e\n \u003cp\u003e88.0\u0026plusmn;1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.97444089456869%\" valign=\"top\"\u003e\n \u003cp\u003e86.1\u0026plusmn;5.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.105431309904153%\" valign=\"top\"\u003e\n \u003cp\u003e0.655\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2. Characteristics of MRD levels in ALL patients with different molecular subtypes\u003c/p\u003e\n\u003cp\u003eIn CCLG-2008 group, MRD levels were measured in all 663 patients at TP1. Of those, 620 children (93.5%) underwent MRD detection at TP2. The analysis showed that at TP1 and TP2, there were significant differences in MRD levels in children with different molecular subtypes (both\u003cem\u003e\u0026nbsp;P\u003c/em\u003e \u0026lt;0.001, Figure 1).\u003c/p\u003e\n\u003cp\u003eChildren carrying \u003cem\u003eBCR::ABL1\u003c/em\u003e or \u003cem\u003eKMT2A\u003c/em\u003e rearrangements still had high TP1 MRD levels. TP1 MRD level was \u0026ge;10\u003csup\u003e-4\u003c/sup\u003e and \u0026ge;10\u003csup\u003e-2\u0026nbsp;\u003c/sup\u003ein 97.1% and 51.4% of children with \u003cem\u003eBCR::ABL1\u003c/em\u003e respectively. Similarly, TP1 MRD levels \u0026nbsp;\u0026ge;10\u003csup\u003e-4\u003c/sup\u003e and \u0026ge;10\u003csup\u003e-2\u003c/sup\u003e were observed in 88.9% and 37.5% of children with \u003cem\u003eKMT2A\u003c/em\u003e rearrangements respectively. Contrarily, \u003cem\u003eETV6::RUNX1\u003c/em\u003e- and \u003cem\u003eTCF3::PBX1\u003c/em\u003e-positive patients had low MRD levels. At TP1, only 52.1% and 34.2% of patients had MRD levels\u0026nbsp;\u0026ge;10\u003csup\u003e-4\u003c/sup\u003e, respectively, and no children had MRD levels\u0026nbsp;\u0026ge;10\u003csup\u003e-2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eChildren with T-ALL were similar to those with \u003cem\u003eBCR::ABL1\u003c/em\u003e and \u003cem\u003eKMT2A\u003c/em\u003e rearrangements. Among those, 88.9% and 22.5% of T-ALL patients had TP1 MRD \u0026ge; 10\u003csup\u003e-4\u003c/sup\u003e or \u0026ge; 10\u003csup\u003e-2\u003c/sup\u003e respectively.\u003c/p\u003e\n\u003cp\u003eAt TP2, MRD level in children with \u003cem\u003eBCR::ABL1\u003c/em\u003e and \u003cem\u003eKMT2A\u003c/em\u003e rearrangements was still high: MRD level \u0026ge;10\u003csup\u003e-2\u003c/sup\u003e was observed in 28% and 25% of the children respectively; only 56.0% and 50.0% of children had MRD levels \u0026lt;10\u003csup\u003e-4\u003c/sup\u003e. In contrast, more than 90% of \u003cem\u003eETV6::RUNX1\u003c/em\u003e- and \u003cem\u003eTCF3::PBX1\u003c/em\u003e-positive patients had MRD levels below 10\u003csup\u003e-4\u003c/sup\u003e, and no patient had MRD\u0026nbsp;\u0026ge;\u0026nbsp;10\u003csup\u003e-3\u003c/sup\u003e. Among children with T-ALL, MRD level was between the above two situations. About 70% of children with T-ALL had MRD levels \u0026lt;10\u003csup\u003e-4\u003c/sup\u003e, and only 7.6% of the patients had MRD levels\u0026nbsp;\u0026ge;10\u003csup\u003e-3\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e3. The relationship between MRD levels and recurrence in children with different molecular subtypes\u003c/p\u003e\n\u003cp\u003eWe compared 10-year RFS of children with different molecular subtypes and MRD levels in CCLG-2008 group. Among patients with \u003cem\u003eBCR::ABL1\u003c/em\u003e, the 10-year RFS of patients with MRD\u0026nbsp;\u0026ge;10\u003csup\u003e-2\u003c/sup\u003e at both TP1 and TP2 were significantly lower than that of children with MRD\u0026lt;10\u003csup\u003e-2\u003c/sup\u003e (22.6%\u0026plusmn;11.5% vs 64.2%\u0026plusmn;14.1%, \u003cem\u003eP\u003c/em\u003e=0.011; 30.4%\u0026plusmn;11.4% vs 65.7%\u0026plusmn;15.9%, \u003cem\u003eP\u003c/em\u003e=0.004). Similar results appeared in children with \u003cem\u003eKMT2A\u003c/em\u003e rearrangements, at both TPs, MRD \u0026ge; 10\u003csup\u003e-2\u003c/sup\u003e was associated with lower 10-year RFS (both 0 vs 66.7%\u0026plusmn;19.2%, both \u003cem\u003eP\u003c/em\u003e = 0.004). In fact, there was no patient carrying \u003cem\u003eKMT2A\u003c/em\u003e rearrangements with MRD levels between 10\u003csup\u003e-3\u003c/sup\u003e and 10\u003csup\u003e-2\u003c/sup\u003e at both TPs, leading to the establishment of an MRD level cut-off value of 10\u003csup\u003e-2\u003c/sup\u003e for this subtype.\u003c/p\u003e\n\u003cp\u003eAmong patients with \u003cem\u003eETV6::RUNX1\u003c/em\u003e, 10-year RFS of patients with TP1 MRD\u0026ge;10\u003csup\u003e-3\u003c/sup\u003e and TP2 MRD-positive was significantly lower than that of patients with TP1 MRD\u0026lt;10\u003csup\u003e-3\u003c/sup\u003e and TP2 MRD-negative respectively (76.9%\u0026plusmn;11.7% vs 96.1%\u0026plusmn;1.7%, \u003cem\u003eP\u003c/em\u003e=0.004; 82.4%\u0026plusmn;9.2% vs 95.9%\u0026plusmn;1.8%, \u003cem\u003eP\u003c/em\u003e=0.019).\u003c/p\u003e\n\u003cp\u003eIt was noteworthy that patients with\u003cem\u003e\u0026nbsp;TCF3::PBX1\u003c/em\u003e had low MRD levels at both TP1 and TP2, we did not observe significant correlation of MRD levels with patient prognosis (Table 2). For children with T-ALL, TP1 MRD\u0026ge;10\u003csup\u003e-3\u003c/sup\u003e was associated with poor prognosis (60.7%\u0026plusmn;12.6% vs 90.9%\u0026plusmn;6.1%, \u003cem\u003eP\u003c/em\u003e=0.033), however, there was no significant correlation between TP2 MRD level and treatment outcome (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBased on the above analysis, we divided children in each molecular subtype into two groups and compared the prognosis of the two groups respectively.\u0026nbsp;\u003c/p\u003e\n\u003col style=\"list-style-type: lower-roman;\"\u003e\n \u003cli\u003eFor patients with \u003cem\u003eBCR::ABL1\u003c/em\u003e or \u003cem\u003eKMT2A\u003c/em\u003e rearrangements, we defined patients with MRD\u0026lt;10\u003csup\u003e-2\u003c/sup\u003e at both TP1 and TP2 as low MRD group, and the others as high MRD group. The 10-year RFS of the former was significantly better than that of the latter (77.4% \u0026plusmn; 11.5% vs 35.8% \u0026plusmn; 14.1%, \u003cem\u003eP\u003c/em\u003e=0.011; 76.2%\u0026plusmn;14.8% vs 0, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, respectively).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eFor children with \u003cem\u003eETV6::RUNX1\u003c/em\u003e, we defined patients with TP1 MRD\u0026lt;10\u003csup\u003e-3\u003c/sup\u003e and TP2 MRD-negative as low MRD group, and the others as high MRD group. The 10-year RFS of the two groups were 96.6% \u0026plusmn; 1.7% and 81.8% \u0026plusmn; 8.2% (\u003cem\u003eP\u003c/em\u003e = 0.005) respectively.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eFor children with T-ALL, We defined children with TP1 MRD\u0026nbsp;\u0026ge;\u0026nbsp;10\u003csup\u003e-3\u003c/sup\u003e as high MRD group, and the rest as low MRD group. As expected, the 10-year RFS of the former was significantly worse (60.7 %\u0026plusmn;12.6% and 90.9%\u0026plusmn;6.1%, \u003cem\u003eP\u003c/em\u003e = 0.033).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eFor children with \u003cem\u003eTCF3::PBX1\u003c/em\u003e, since the MRD levels at TP1 and TP2 were not significantly related to prognosis, we did not conduct further analysis on patients in this subtype.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eWe verified the above analysis results of each molecular subtype in children of BCH-2003 group. The results showed that for children carrying \u003cem\u003eBCR::ABL1\u003c/em\u003e and \u003cem\u003eKMT2A\u003c/em\u003e rearrangements, the 10-year RFS of the high/low MRD group were 100% vs 35.7%\u0026plusmn;19.8%, and 100% vs 0% respectively. It is worth noting, however, that we did not observe any significant differences in both subtypes (\u003cem\u003eP\u003c/em\u003e=0.242 and 0.063, respectively), which may be due to the small sample size.\u003c/p\u003e\n\u003cp\u003eAmong children with \u003cem\u003eETV6::RUNX1\u003c/em\u003e, the 10-year RFS of low MRD group was significantly better than that of high MRD group (88.2%\u0026plusmn;5.9% vs 57.9%\u0026plusmn;19.9%, \u003cem\u003eP\u003c/em\u003e=0.013). Similarly, the 10-year RFS of children with T-ALL in low MRD group was significantly better than that in high MRD group (100% vs 66.7%\u0026plusmn;10.3%, \u003cem\u003eP\u003c/em\u003e=0.012).\u003c/p\u003e\n\u003cp\u003eIt is of importance that children in B-others subgroup carry various genetic mutations that had not been determined, and thus cannot be accurately classified. As a result, we did not carry out depth analysis on this subtype.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn summary, there were significant differences in the prognostic significance of the same MRD level among ALL patients carrying different fusion genes or immunephenotypes.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eChildren with ALL carry a range of genetic aberrations, such as fusion genes. This study showed that patients carrying different fusion genes have significant differences in MRD levels at TP1 and TP2, and the same MRD level has different prognostic significance among various molecular subtypes. For TP1 MRD\u0026thinsp;\u0026ge;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, the 10-year RFS were only 56.5%\u0026plusmn;10.0% and 57.1%\u0026plusmn;18.7% in patients with \u003cem\u003eBCR::ABL1\u003c/em\u003e or \u003cem\u003eKMT2A\u003c/em\u003e rearrangements, respectively; in contrast, it was up to 92.0%\u0026plusmn;3.1% and 84.6%\u0026plusmn;10.0% in those with \u003cem\u003eETV6::RUNX1\u003c/em\u003e or \u003cem\u003eTCF3::PBX1\u003c/em\u003e, respectively. This suggests that it is necessary to fully take into account genetic subtypes in stratification of childhood ALL based on MRD, in order to achieve more accurate treatment and in turn better effectiveness.\u003c/p\u003e \u003cp\u003eNext-generation sequencing technology (NGS) has gained widespread popularity as a powerful tool for analyzing variations in nucleic acids [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This innovative method can accurately identify specific rearrangement fragments of \u003cem\u003eIg\u003c/em\u003e and \u003cem\u003eTCR\u003c/em\u003e genes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], as well as other gene anomalies such as insertions, deletions, and fusion genes. In leukemia, NGS has shown remarkable sensitivity (about 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e~10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), far more sensitive than other technologies like FCM and PCR [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This study indicated that 15.7% and 48.6% of children with \u003cem\u003eTCF3::PBX1\u003c/em\u003e were MRD-negative at TP1 and TP2, respectively. However, there was no significant correlation between MRD levels and prognosis. Future study could utilize NGS to show the existence of extra low level of MRD, which could promote accurate risk stratification and prognosis evaluation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study showed that among children with \u003cem\u003eBCR::ABL1\u003c/em\u003e, the prognosis of low MRD group was significantly better than that of high MRD group, however, this is based on traditional intensive chemotherapy. Studies have shown that the application of small molecule tyrosine kinase inhibitors such as imatinib could significantly improve treatment outcome of children with \u003cem\u003eBCR::ABL1\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It is crucial, therefore, to investigate more exact MRD stratification criteria for targeted therapy combined with chemotherapy. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies suggest that children in B others group may have various fusion genes or mutations, such as \u003cem\u003eDUX4\u003c/em\u003e rearrangements, \u003cem\u003eMEF2D\u003c/em\u003e, \u003cem\u003eZNF384\u003c/em\u003e rearrangements, \u003cem\u003eNUTM1\u003c/em\u003e rearrangements, \u003cem\u003eTCF3::HLF\u003c/em\u003e, \u003cem\u003eIKZF1\u003c/em\u003e mutations, which were not determined in this study. Thus, we did not conduct an in-depth analysis of this subgroup of children. Similarly, T-ALL has many genetic abnormalities, such as fusion genes involving \u003cem\u003eTAL1\u003c/em\u003e, \u003cem\u003eHOXA10\u003c/em\u003e, \u003cem\u003eTLX3\u003c/em\u003e, \u003cem\u003eTLX1\u003c/em\u003e, \u003cem\u003eNKX2-1\u003c/em\u003e, \u003cem\u003eLMO1/2\u003c/em\u003e, and others. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Future studies could explore the above genetic variant-specific MRD stratification criteria to facilitate the precise stratification and treatment of childhood ALL.\u003c/p\u003e \u003cp\u003eThis study draws upon clinical data from two treatment regimens, namely BCH-2003 and CCLG-2008 protocol. While the primary clinical characteristics of these two groups of patients did not exhibit significant differences, additional multi-center study is necessary to obtain more precise conclusions. This will facilitate the development of more effective risk stratification and treatment guidelines.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHXT analyzed the data and wrote the original draft. WCJ and ZZJ collected the clinical data.\u0026nbsp;GC and XTL performed the experiments.\u0026nbsp;WTY, WMY and ZRD made clinical contributions. LZG , CL and ZRD designed the research and revised the paper. All authors were involved in the final approval of the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China\u0026nbsp;(No. 81970135 and No. 81870114), the Beijing Natural Science Foundation of China (No. 7202044), the New\u0026nbsp;Sunshine Charity Foundation, China (No. 2017002),\u0026nbsp;and Funding for Reform and Development of Beijing Municipal Health Commission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed according to the principles of the Declaration of Helsinki, and approved by the Institutional Review Board of Beijing Children\u0026rsquo;s Hospital (2007-20).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent were obtained from guardians of the patients.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHunger SP, Mullighan CG (2015) Acute Lymphoblastic Leukemia in Children. N Engl J Med 373(16):1541\u0026ndash;1552\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCampana D, Pui CH (2017) Minimal residual disease-guided therapy in childhood acute lymphoblastic leukemia. Blood 129(14):1913\u0026ndash;1918\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO'Connor D et al (2018) Genotype-Specific Minimal Residual Disease Interpretation Improves Stratification in Pediatric Acute Lymphoblastic Leukemia. J Clin Oncol 36(1):34\u0026ndash;43\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePui CH et al (2017) Clinical impact of minimal residual disease in children with different subtypes of acute lymphoblastic leukemia treated with Response-Adapted therapy. Leukemia 31(2):333\u0026ndash;339\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao C et al (2012) Clinical features, early treatment responses, and outcomes of pediatric acute lymphoblastic leukemia in China with or without specific fusion transcripts: a single institutional study of 1,004 patients. Am J Hematol 87(11):1022\u0026ndash;1027\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui L et al (2018) Outcome of children with newly diagnosed acute lymphoblastic leukemia treated with CCLG-ALL 2008: The first nation-wide prospective multicenter study in China. Am J Hematol 93(7):913\u0026ndash;920\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui L et al (2010) Combined analysis of minimal residual disease at two time points and its value for risk stratification in childhood B-lineage acute lymphoblastic leukemia. Leuk Res 34(10):1314\u0026ndash;1319\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan der Velden VH et al (2007) Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data. Leukemia 21(4):604\u0026ndash;611\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFaham M et al (2012) Deep-sequencing approach for minimal residual disease detection in acute lymphoblastic leukemia. Blood 120(26):5173\u0026ndash;5180\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJia MZ et al (2023) Tracing back of relapse clones by Ig/TCR gene rearrangements reveals complex patterns of recurrence in pediatric acute lymphoblastic leukemia. Int J Lab Hematol 45(5):717\u0026ndash;725\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePierce E et al (2022) MRD in ALL: Optimization and Innovations. Curr Hematol Malig Rep 17(4):69\u0026ndash;81\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSala Torra O et al (2017) Next-Generation Sequencing in Adult B Cell Acute Lymphoblastic Leukemia Patients. Biol Blood Marrow Transplant 23(4):691\u0026ndash;696\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDruker BJ (2002) Perspectives on the development of a molecularly targeted agent. Cancer Cell 1(1):31\u0026ndash;36\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchrappe M et al (2011) Late MRD response determines relapse risk overall and in subsets of childhood T-cell ALL: results of the AIEOP-BFM-ALL 2000 study. Blood 118(8):2077\u0026ndash;2084\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrady SW et al (2022) The genomic landscape of pediatric acute lymphoblastic leukemia. Nat Genet 54(9):1376\u0026ndash;1389\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"annals-of-hematology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aohe","sideBox":"Learn more about [Annals of Hematology](http://link.springer.com/journal/277)","snPcode":"277","submissionUrl":"https://submission.nature.com/new-submission/277/3","title":"Annals of Hematology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ALL, MRD, fusion gene, relapse, risk stratification","lastPublishedDoi":"10.21203/rs.3.rs-3810073/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3810073/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn childhood acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) risk stratification criteria specific to common genetic subtypes are unclear. Among 723 children with newly diagnosed ALL treated with the Chinese Children Leukemia Group trial CCLG-2008 protocol, at time point 1 (TP1, the end of induction) and TP2 (before consolidation for standard risk and intermediate risk group, or before the second HR-I' block for high risk group), the MRD levels of children carrying different fusion genes or with T-ALL were significantly different (\u003cem\u003eP\u003c/em\u003e all \u0026lt;0.001), and the prognostic significance of the same MRD level in the above subtypes was greatly different. For patients carrying \u003cem\u003eBCR::ABL1\u003c/em\u003e or \u003cem\u003eKMT2A\u003c/em\u003e rearrangements, or \u003cem\u003eETV6::RUNX1\u003c/em\u003e, or with T-ALL, we defined those with both TP1 and TP2 MRD levels \u0026lt;10\u003csup\u003e-2\u003c/sup\u003e, TP1 MRD \u0026lt;10\u003csup\u003e-3\u003c/sup\u003e and TP2 MRD-negative, TP1 MRD \u0026lt;10\u003csup\u003e-3\u003c/sup\u003e, as low MRD group respectively; the remaining children as high MRD group of each subtype. The 10-year relapse free survival (RFS) of low MRD group was significantly better than that of high MRD group. We verified the clinical value of the above MRD stratification criteria in patients treated with “Beijing Children’s Hospital BCH-ALL2003” protocol. In conclusion, subtype-specific MRD risk stratification may contribute to the precise treatment of childhood ALL.\u003c/p\u003e","manuscriptTitle":"Relationship between subtype-specific minimal residual disease level and long-term prognosis in children with acute lymphoblastic leukemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-03 16:43:25","doi":"10.21203/rs.3.rs-3810073/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-01-25T06:47:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-01-09T02:07:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"081ced2a-b3b4-414a-8820-162604d77f99","date":"2023-12-30T09:05:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-12-30T07:09:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-12-28T11:49:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-12-28T11:49:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Annals of Hematology","date":"2023-12-27T01:44:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"annals-of-hematology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aohe","sideBox":"Learn more about [Annals of Hematology](http://link.springer.com/journal/277)","snPcode":"277","submissionUrl":"https://submission.nature.com/new-submission/277/3","title":"Annals of Hematology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d466326f-52b1-41ca-b708-136a30d5843d","owner":[],"postedDate":"January 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-03-25T15:13:41+00:00","versionOfRecord":{"articleIdentity":"rs-3810073","link":"https://doi.org/10.1007/s00277-024-05687-y","journal":{"identity":"annals-of-hematology","isVorOnly":false,"title":"Annals of Hematology"},"publishedOn":"2024-03-18 15:01:22","publishedOnDateReadable":"March 18th, 2024"},"versionCreatedAt":"2024-01-03 16:43:25","video":"","vorDoi":"10.1007/s00277-024-05687-y","vorDoiUrl":"https://doi.org/10.1007/s00277-024-05687-y","workflowStages":[]},"version":"v1","identity":"rs-3810073","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3810073","identity":"rs-3810073","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}