Are minimal residual disease measurement after consolidation therapy useful in children with acute lymphoblastic leukemia?

Are minimal residual disease measurement after consolidation therapy useful 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 Article Are minimal residual disease measurement after consolidation therapy useful in children with acute lymphoblastic leukemia? janine Stutterheim, Rachella van der Waarden, Hester de Groot-Kruseman, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3411409/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Sep, 2024 Read the published version in Leukemia → Version 1 posted 9 You are reading this latest preprint version Abstract Minimal residual disease (MRD) is regularly measured at later timepoints after end of first consolidation (EOC) in children with acute lymphoblastic leukemia (ALL). The question remains whether this is useful for detecting (molecular) relapse. We investigated the clinical relevance of MRD after EOC in intermediate risk patients treated on DCOG-ALL-10 (n = 271) and DCOG-ALL-9 (n = 122), with MRD < 0.05% at EOC. EOC MRD negative patients (n = 178) had excellent outcomes, irrespective of MRD results at later timepoints; 6-years relapse free survival (6y-RFS) of 92.0% (95% CI:88–97) for those with MRD negativity at all later timepoints compared to 96.2% (95% CI:89–100) for those with one or more later timepoints being positive (p = 0.46). Patients with positive EOC MRD (n = 91) of whom the subsequent timepoints were MRD negative (n = 43), had comparable good outcomes, 6y-RFS of 93.0% (95% CI:85–100). Patients being MRD positive at EOC and MRD positivity at one or more subsequent timepoints (n = 48) had a higher risk of relapse, 6y-RFS 70.5% (95% CI:58–84), p = 0.002. These findings were confirmed in the validation cohort of ALL-9. In patients who are MRD negative at EOC, MRD measurements at later timepoints can be abandoned. For patients who are EOC MRD positive the subsequent MRD measurement might be informative for further risk stratification. Biological sciences/Cancer/Haematological cancer/Leukaemia/Acute lymphocytic leukaemia Health sciences/Medical research/Translational research Figures Figure 1 Figure 2 Introduction End of induction (EOI) and end of consolidation (EOC) are the most important time points to measure minimal residual disease (MRD) for risk stratification in current treatment protocols. 1–6 In many protocols bone marrow (BM) punctures are performed after EOC for evaluation of remission in the bone marrow at the start of the subsequent treatment courses. In the current Alltogether1 protocol, no follow up bone marrow evaluations are performed after EOI for standard risk patients (SR) and after EOC for intermediate risk (MR) patients, as these are not used for stratification. The question remains whether later time points are indeed not informative. We sought for answers for the two following questions: What is the prognostic value of MRD at later time points if 1) EOC MRD is negative and if 2) EOC MRD is positive. Pui et al. already debated if subsequent MRD measurements are warranted in patients that achieve a MRD-negative status after remission induction treatment, since in there study MRD reemerged in very few of these patients. 7 In addition they showed that in patients with MRD ≥ 0.01% EOC, sequential MRD measurements were useful to guide treatment decisions. However, there are no published data on the prognostic significance of very low positive MRD levels below the level of quantification at EOC and at time points later than EOC. In the first Dutch MRD-risk based protocol, DCOG ALL-10, bone marrow punctures were performed at designated time points after EOC. The purpose of the present study was to investigate the clinical relevance of these MRD measurements at later timepoints. We validate the results in a separate cohort of patients treated according to another protocol (DCOG ALL-9). Methods Patients and treatment protocol ALL-10 Patients were included in the present study if they were stratified as intermediate risk patient and had availability of MRD data at least at one specified follow-up time points (TP) after EOC (n = 271), see CONSORT diagram, Fig. 1 . Children with Down syndrome were excluded. The MR stratification criteria were defined as: Cytomorphologic complete remission (CR) at day 33; MRD-positivity at day 33 and/or at day 79 (< 0.05%); a good prednisone response at day 8; absence of KMT2A :: AFF1 and absence of BCR :: ABL1 . Patients were recruited in ALL-10 from November 2004 to April 2012. 3 The collected outcome data from ALL-10 were lastly updated in august 2018. This is 14 years after the first inclusion, and 4 years after the last patient completed the study protocol treatment. ALL-9 MRD and outcome data from patients treated on ALL-9 were used for validation. Since this protocol did not use MRD-based risk stratification, patients were included in the present study if they fulfilled the following criteria to match our ALL-10 cohort of the present study: MRD data available at EOC, absence of Down syndrome, absence of BCR::ABL1, EOI in CR1, EOC MRD < 0.05% and no event before TP3 (n = 122), see CONSORT diagram, supplementary Fig. 1. ALL9 patients were recruited from January 1997 to November 2004. 8 The collected outcome data from ALL-9 was lastly updated in august 2008. All patients were enrolled into ALL-10 or ALL-9 protocol. Both protocols were approved by institutional review boards. Informed consent was signed by parents and patients according to Dutch law. Samples ALL-10 Bone marrow samples were obtained at diagnosis, at end of induction (EOI, day 33, TP1; n = 271), at end of consolidation 1 (EOC, day 79, TP2; n = 269), at end of consolidation 2/start of intensification (4–5 months after diagnosis, TP3, MRG week 1; n = 252), at end of intensification, at start of maintenance therapy (8–10 months after diagnosis, TP4, MRG week 19; n = 251) or during maintenance at 12 months after diagnosis (TP5, MRG week 37; n = 263). See Supplementary Fig. 2a for MRD timepoints and treatment scheme ALL-10. Bone marrow samples were available at EOC and at least one later TP in 269 patients, see CONSORT diagram Fig. 1 . ALL-9 Bone marrow samples were obtained at diagnosis, EOI (day 42, TP1), after first consolidation with HD-MTX courses (EOC, TP2, 3 months after diagnosis, n = 122 EOC MRD < 0.05%), and at 6 months (TP3; n = 104), 9 months (TP4; n = 107) 12 months (TP5; n = 110) after diagnosis. (See Supplementary Fig. 2b and 2c, for MRD timepoints and treatment schemes ALL-9). Bone marrow samples were available at EOC and at least one later timepoint in 120 patients, see CONSORT diagram supplementary Fig. 1. Detection of MRD Immunoglobulin and/or T-cell receptor (IG/TR) gene rearrangements were used as MRD-PCR targets. IG/TR gene rearrangements were detected in the diagnostic specimens as previously described. 9,10 On the basis of the junctional region of the identified genomic rearrangement, patient-specific primers were designed and tested for quantitative range (QR) and sensitivity, at the two national reference laboratories. Real-time quantitative PCR data were analyzed according to the EuroMRD guidelines. 9 MRD results were classified as negative, positive not quantifiable (“positive, <QR”), and positive quantifiable (≥ QR), following guidelines for therapy reduction. 9 For MRD analyses in ALL-10 for TP3, TP4 and TP5, also guidelines for therapy intensification were applied for comparison of the MRD results applying both guidelines. In short, a sample is considered positive if the CT value of at least one of the three replicates is ≥1.0 CT (therapy reduction) or ≥3.0 CT (therapy intensification) lower than the lowest CT of background, and the CT value of at least one of the three replicates is within 4.0 CT from the highest CT value of the ‘sensitivity’ (fulfilling all ‘sensitivity’ criteria). 9 Statistical Analysis Chi square tests were performed to check if there was no selection bias between patients with and without MRD evaluations with respect to known prognostic features. Relapse free survival (RFS) was used as endpoint since we wanted to know if positive MRD was associated with relapse rather than with any other event. RFS was defined as time from diagnosis until first relapse; this was not corrected for competing events (death before relapse). Patients that did not adhere to the MR protocol due to major treatment modifications were not excluded based on an intention-to-treat analysis. RFS curves were estimated using the Kaplan–Meier (KM) methodology. Estimates of RFS at 6 years along with the corresponding 95% confidence intervals (CIs) were reported for different time points. The Log-rank test was used to compare relapse free survival (RFS) for the different time points. The analyses were performed using SPSS IBM version 26 and R version 4.2.2. 11 Results ALL-10 Patients characteristics Out of 464 MR patients enrolled in the ALL-10 protocol, MRD data at later timepoints than EOC were available for 271 (see the CONSORT diagram, Figure 1). The mean follow-up time was 10.0 years (range 6.1-13.5 years). No significant differences in sex, age, WBC, immunophenotype (B-lineage versus T-lineage) and CNS status at diagnosis were detected between patients with and without available MRD data (Table 1). The percentage of patients with MRD positivity at EOC did not differ significantly between patients with and without MRD data at later time points, nor did the 6-year RFS (SE) differ (89.2 (95% CI: 86-93) versus 92.1% (95% CI:88-96) respectively, (p = 0.29)). Thirty out of 271 patients (11.1%) relapsed (24 isolated BM, 4 combined CNS and BM, 2 isolated CNS), 3 (1.1%) died in remission and 1 (0.4%) developed a second malignant neoplasm. Prognostic significance of MRD at the end of consolidation 1 (EOC) and subsequent time points At EOC (TP2) all patients had MRD < 0.05% as per protocol definition for intermediate risk patients. At EOC 178 out of 269 (66.2%) patients were MRD negative (undetectable MRD) and 91 (33.8%) MRD positive. Of the MRD positive patients, 80 (29.7%) had positive not quantifiable MRD and 11 (4.1%) had positive quantifiable MRD levels (0.01% < 0.05%). EOC MRD negativity was associated with a better RFS, with a 6 years RFS of 93.1% (89-97) for MRD- negative patients, and 80.3% (72-89) for MRD positive patients (p <0.01), supplementary figure 3a. We analyzed the 3 timepoints after consolidation1. For each timepoint, patients with negative MRD had a better outcome than patients with positive MRD (supplementary figures 3 b,c,d). Value of MRD at later timepoints for MRD negative patients at EOC From the 178 EOC MRD negative patients, 152 remained MRD negative at later timepoints and 26 patients showed positive MRD results at one or more later timepoints (TP3, TP4, and/or TP5). MRD positivity at later timepoints had no prognostic relevance as both groups had comparable outcomes; 6-y RFS of 92.0% (95% CI:88-97) and 96.2% (95% CI:89-100) for those remaining negative at later timepoints versus those who did not respectively, see figure 2a. When applying guidelines for therapy intensification, 12 of 26 (46%) patients that showed positive MRD results at one or more later timepoints, were now defined as MRD negative at all subsequent timepoints. 2) Value of MRD at later timepoints for MRD positive patients at EOC From the 91 patients with positive EOC MRD, 43 were MRD negative at all subsequent timepoints and 48 had a positive MRD result at one or more of the subsequent timepoints (TP3, TP4 and/or TP5). Patients that were MRD negative at all subsequent timepoints had a 6-y RFS of 93% (95% CI:85-100), which is comparable to that of patients being EOC MRD negative mentioned in the paragraph above. However, patients that were MRD positive at later timepoints had a significantly worse 6y RFS (70.5%; 95% CI:58-84, p =0.002 see figure 2a). When applying guidelines for therapy intensification, 5 of 48 (10%) patients that showed positive MRD results at one or more later timepoints, were now defined as MRD negative at all subsequent timepoints. The RFS for these 43 patients was comparable, 6-y RFS 69.4% (95% CI: 56-83), see supplementary figure 4. All patients with quantifiable disease at TP2 (EOC, n=11), were MRD positive at TP3; 3 with quantifiable disease and 8 with positive not quantifiable disease. At TP3 7/46 MRD positive patients had positive quantifiable disease. Two of these patients also had positive quantifiable disease at TP4. No other patients had quantifiable disease at TP4 and at TP5 none of the patients had positive quantifiable disease. Validation cohort ALL-9 MRD data were available for 122 patients of the ALL-9 study who had EOC (TP2) MRD < 0.05% (see the CONSORT diagram, Supplementary Figure 1). Seventy eight out of these 122 (64.0%) patients were MRD negative (undetectable MRD) and 44 (36.0%) MRD positive. Of the EOC MRD positive patients, 37 (30.3%) had positive not quantifiable MRD and 7 (5.7%) had positive quantifiable MRD levels (0.01% < 0.05%). Patients with MRD negativity at EOC had a better 6-years RFS (93.6%; 95% CI:88-99) than patients with positive MRD at EOC (83.5%; 72-0.95) (p = 0.029), supplementary figure 5. Of 120 patients also MRD results at later timepoints were available; 77 patients were EOC MRD negative; and 43 patients were EOC MRD positive. As in ALL-10, we divided the EOC MRD negative group in two groups; patients that remained MRD negative (n=69), and patients that had any positive MRD results at TP3, TP4, and/or TP5 (n=8). MRD positivity at later time points had no prognostic value as both groups had comparable outcomes; 6-y RFS of 94.2% (88-99) and 87.5% (67-100) respectively (p=0.27), see figure 2b. Also for the EOC MRD positive patients (n=43) we observed the same results as in ALL-10; patients that became MRD negative at all later timepoints (n=17) had an RFS that was comparable to that of patients that were EOC MRD negative (6-y EFS 94.1 (83-100)). However, patients that remained MRD positive at later time points (n=26) had a worse 6y-RFS of 75.4% (60-94), (p =0.008, figure 2b). Discussion This is the first study that has investigated the prognostic significance of MRD detection after EOC in a MRD-based pediatric ALL protocol. Often low, not quantifiable levels of MRD (< 0.05%) are found of which the prognostic and clinical significance is not clear. First, our data show that in patients who are MRD negative at EOC, MRD positivity at later timepoints did not adversely affect outcome. So no additional MRD measurements are needed in patients that are EOC MRD negative. Second, patients that are EOC MRD positive the subsequent MRD time points have additional prognostic relevance and could help to further stratify these intermediate risk patients. The data were obtained in our ALL-10 study and validated in the ALL-9 cohort. The results were in line with a previous study by Pui et al 7 , which showed that MRD re-emerged in EOC MRD negative patients in a very small proportion of patients. However, they found that the few patients that experienced re-emergence of MRD, defined as ≥ 0.01%, carried a poor prognosis. In our cohort, all positive MRD results in patient being MRD negative, were at the level of positive not quantifiable, and these patients had an excellent outcome. A likely explanation for MRD positive results in patients that were EOC MRD negative is that these very low not quantifiable levels are false positive results. This was illustrated by the fact that 46% of patients that were EOC MRD negative and showed positive MRD results at subsequent times points, were MRD negative at all subsequent when applying guidelines for therapy intensification. 9 In addition false positive MRD results can be caused by B-lymphocyte regeneration. 12 More specific techniques such as targeted next generation sequencing (NGS) could overcome this problem. 13,14 In patients who are EOC MRD positive, subsequent time points seem to be of prognostic importance. Here we show that very low, not quantifiable MRD levels detected at EOC and at subsequent time point(s) are prognostic for relapse and probably identify slow responders. These slow responders have a higher risk to develop a relapse. All relapses involved the bone marrow. Identifications of these slow responders should be the purpose of follow-up measurements and may lead to improved risk stratification with subsequent therapeutic interventions such as blinatumomab, inotuzumab and CART cell therapy. We conclude from this study that in patients with MRD based intermediate risk ALL who are MRD negative at end of first consolidation (EOC), MRD measurement at later timepoints can be abandoned. For patients who are EOC MRD positive the subsequent MRD measurements might be informative for further risk stratification and therapeutic intervention. Declarations Acknowledgments None Authorship Contributions The corresponding author confirms that she had full access to the data in the study and final responsibility for the decision to submit for publication. All authors, meet all of the following criteria: Conceived and/or designed the work that led to the submission, acquired data, and/or played an important role in interpreting the results. Drafted or revised the manuscript. Approved the final version. Agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. J.S., designed research, analyzed and interpreted data, performed statistical analysis, drafted the manuscript, Approved the final version. R.vd.W., designed research, analyzed and interpreted data, performed statistical analysis, revised the manuscript, Approved the final version. H. deG-K., analyzed and interpreted data, revised the manuscript, Approved the final version E.S., performed research, collected data, revised the manuscript, Approved the final version V.de H. performed research, collected data, analyzed and interpreted data, revised the manuscript, Approved the final version R.D. performed statistical analysis, revised the manuscript, Approved the final version E.vd S. analyzed and interpreted data, collected data, revised the manuscript, Approved the final version V.vd V., performed research, collected data, analyzed and interpreted data, revised the manuscript, Approved the final version R.P., analyzed and interpreted data, drafted the manuscript, Approved the final version Competing interest For all authors there are no competing financial interests in relation to the work described Data Availability Statement: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. 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Blood 2023; 141 : 529–533. Tables Table 1 is available in the Supplementary Files section. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files Table1MRDafterEOC782023.docx Table 1 SupplementaryfiguresandtablesMRDmaintenance1892023.pdf Cite Share Download PDF Status: Published Journal Publication published 10 Sep, 2024 Read the published version in Leukemia → Version 1 posted Editorial decision: revise 06 Dec, 2023 Review # 2 received at journal 05 Dec, 2023 Reviewer # 2 agreed at journal 08 Nov, 2023 Review # 1 received at journal 16 Oct, 2023 Reviewer # 1 agreed at journal 07 Oct, 2023 Reviewers invited by journal 05 Oct, 2023 Editor assigned by journal 05 Oct, 2023 Submission checks completed at journal 05 Oct, 2023 First submitted to journal 04 Oct, 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. 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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-3411409","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":238105405,"identity":"512d6e63-90ea-45dd-b3e2-e74f28562b30","order_by":0,"name":"janine Stutterheim","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-9828-0834","institution":"Princess Maxima Center for Pediatric Oncology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"janine","middleName":"","lastName":"Stutterheim","suffix":""},{"id":238105406,"identity":"bfca0d29-13bb-4ff6-a0db-c0b94e357f84","order_by":1,"name":"Rachella van der Waarden","email":"","orcid":"","institution":"Princess Maxima Center for Pediatric Oncology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rachella","middleName":"van der","lastName":"Waarden","suffix":""},{"id":238105407,"identity":"b4bc32b8-e3a4-497f-b28a-39ccdfea1eaa","order_by":2,"name":"Hester de Groot-Kruseman","email":"","orcid":"","institution":"Princess Máxima Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hester","middleName":"","lastName":"de Groot-Kruseman","suffix":""},{"id":238105408,"identity":"900d94aa-d996-4c4b-b342-416a01707eea","order_by":3,"name":"Edwin Sonneveld","email":"","orcid":"","institution":"Princess Máxima Center for Pediatric Oncology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Edwin","middleName":"","lastName":"Sonneveld","suffix":""},{"id":238105409,"identity":"590e8486-e8bf-4587-85f9-55e3762ee485","order_by":4,"name":"Valerie Haas","email":"","orcid":"https://orcid.org/0000-0003-0339-6816","institution":"Prinses Maxima Center for Pediatric Oncology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Valerie","middleName":"","lastName":"Haas","suffix":""},{"id":238105410,"identity":"a4c51ccc-5a5c-44dd-803b-7e6fdf26efd3","order_by":5,"name":"Rana Dandis","email":"","orcid":"","institution":"Princess Maxima Centre","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rana","middleName":"","lastName":"Dandis","suffix":""},{"id":238105411,"identity":"10317972-a6d4-4c82-a21d-502703d09616","order_by":6,"name":"C. Ellen van der Schoot","email":"","orcid":"","institution":"Sanquin","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"C.","middleName":"Ellen van der","lastName":"Schoot","suffix":""},{"id":238105412,"identity":"b2e678d3-e5ea-4370-a364-a9a98dc80ddb","order_by":7,"name":"Vincent van der Velden","email":"","orcid":"https://orcid.org/0000-0001-9457-3763","institution":"Erasmus Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Vincent","middleName":"van der","lastName":"Velden","suffix":""},{"id":238105413,"identity":"b70873d0-87b0-423b-aa75-2c00cf801cca","order_by":8,"name":"Rob Pieters","email":"","orcid":"https://orcid.org/0000-0003-2997-3570","institution":"Princess Máxima Center for Pediatric Oncology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rob","middleName":"","lastName":"Pieters","suffix":""}],"badges":[],"createdAt":"2023-10-04 19:25:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3411409/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3411409/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41375-024-02386-5","type":"published","date":"2024-09-10T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":44363799,"identity":"60699b57-abf2-4a12-a8f1-ab3809f3cc91","added_by":"auto","created_at":"2023-10-10 14:04:33","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":44769,"visible":true,"origin":"","legend":"\u003cp\u003eConsort diagram ALL-10 cohort\u003c/p\u003e\n\u003cp\u003eAbbreviations: MRD, minimal residual disease; TP, time point.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3411409/v1/00652cb5f9b30c9dbbb17947.jpg"},{"id":44363800,"identity":"836e5911-a827-4e98-9fc3-6433dc8668de","added_by":"auto","created_at":"2023-10-10 14:04:33","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":100653,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Prognostic impact of minimal residual disease (MRD) levels at end of consolidation (EOC) in combination with later time points as shown by Kaplan-Meier estimates of relapse-free survival (ALL10) as shown by Kaplan-Meier estimates of relapse-free survival (RFS)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003e Prognostic impact of minimal residual disease (MRD) levels at end of consolidation (EOC) in combination with later time points as shown by Kaplan-Meier estimates of relapse-free survival (ALL-9) as shown by Kaplan-Meier estimates of relapse-free survival (RFS)\u003c/p\u003e\n\u003cp\u003eNeg/Neg, MRD-negative at EOC and all later time points; Neg/Pos, MRD-negative at EOC and MRD-positive at any later timepoint; Pos/Neg, MRD-positive at EOC and MRD-negative at all later time points; Pos/Pos, MRD-positive at EOC and EOC at any later timepoint\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3411409/v1/6fe60b3419abaf980ca6d6ed.jpg"},{"id":64278596,"identity":"f492d244-3bee-499f-a470-ecec33031db6","added_by":"auto","created_at":"2024-09-11 07:07:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":468732,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3411409/v1/8459b7a4-61d3-4828-b570-568e78b7de93.pdf"},{"id":44363801,"identity":"58601c50-54da-4e92-84b3-014b4a7777c0","added_by":"auto","created_at":"2023-10-10 14:04:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":96937,"visible":true,"origin":"","legend":"\u003cp\u003eTable 1\u003c/p\u003e","description":"","filename":"Table1MRDafterEOC782023.docx","url":"https://assets-eu.researchsquare.com/files/rs-3411409/v1/bedaf18ba3c7db4c1fa9c02f.docx"},{"id":44363802,"identity":"331ef2c2-6942-4da9-b426-15179750ecc7","added_by":"auto","created_at":"2023-10-10 14:04:33","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":251465,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryfiguresandtablesMRDmaintenance1892023.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3411409/v1/bddd2c00dcf21808756922f5.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Are minimal residual disease measurement after consolidation therapy useful in children with acute lymphoblastic leukemia?","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEnd of induction (EOI) and end of consolidation (EOC) are the most important time points to measure minimal residual disease (MRD) for risk stratification in current treatment protocols.\u003csup\u003e1\u0026ndash;6\u003c/sup\u003e In many protocols bone marrow (BM) punctures are performed after EOC for evaluation of remission in the bone marrow at the start of the subsequent treatment courses. In the current Alltogether1 protocol, no follow up bone marrow evaluations are performed after EOI for standard risk patients (SR) and after EOC for intermediate risk (MR) patients, as these are not used for stratification. The question remains whether later time points are indeed not informative. We sought for answers for the two following questions: What is the prognostic value of MRD at later time points if 1) EOC MRD is negative and if 2) EOC MRD is positive.\u003c/p\u003e \u003cp\u003ePui et al. already debated if subsequent MRD measurements are warranted in patients that achieve a MRD-negative status after remission induction treatment, since in there study MRD reemerged in very few of these patients.\u003csup\u003e7\u003c/sup\u003e In addition they showed that in patients with MRD\u0026thinsp;\u0026ge;\u0026thinsp;0.01% EOC, sequential MRD measurements were useful to guide treatment decisions. However, there are no published data on the prognostic significance of very low positive MRD levels below the level of quantification at EOC and at time points later than EOC.\u003c/p\u003e \u003cp\u003eIn the first Dutch MRD-risk based protocol, DCOG ALL-10, bone marrow punctures were performed at designated time points after EOC. The purpose of the present study was to investigate the clinical relevance of these MRD measurements at later timepoints. We validate the results in a separate cohort of patients treated according to another protocol (DCOG ALL-9).\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and treatment protocol\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eALL-10\u003c/h2\u003e \u003cp\u003ePatients were included in the present study if they were stratified as intermediate risk patient and had availability of MRD data at least at one specified follow-up time points (TP) after EOC (n\u0026thinsp;=\u0026thinsp;271), see CONSORT diagram, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Children with Down syndrome were excluded. The MR stratification criteria were defined as: Cytomorphologic complete remission (CR) at day 33; MRD-positivity at day 33 and/or at day 79 (\u0026lt;\u0026thinsp;0.05%); a good prednisone response at day 8; absence of \u003cem\u003eKMT2A\u003c/em\u003e::\u003cem\u003eAFF1\u003c/em\u003e and absence of \u003cem\u003eBCR\u003c/em\u003e::\u003cem\u003eABL1\u003c/em\u003e.\u003c/p\u003e \u003cp\u003ePatients were recruited in ALL-10 from November 2004 to April 2012.\u003csup\u003e3\u003c/sup\u003e The collected outcome data from ALL-10 were lastly updated in august 2018. This is 14 years after the first inclusion, and 4 years after the last patient completed the study protocol treatment.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eALL-9\u003c/h2\u003e \u003cp\u003eMRD and outcome data from patients treated on ALL-9 were used for validation. Since this protocol did not use MRD-based risk stratification, patients were included in the present study if they fulfilled the following criteria to match our ALL-10 cohort of the present study: MRD data available at EOC, absence of Down syndrome, absence of BCR::ABL1, EOI in CR1, EOC MRD\u0026thinsp;\u0026lt;\u0026thinsp;0.05% and no event before TP3 (n\u0026thinsp;=\u0026thinsp;122), see CONSORT diagram, supplementary Fig.\u0026nbsp;1. ALL9 patients were recruited from January 1997 to November 2004.\u003csup\u003e8\u003c/sup\u003e The collected outcome data from ALL-9 was lastly updated in august 2008.\u003c/p\u003e \u003cp\u003eAll patients were enrolled into ALL-10 or ALL-9 protocol. Both protocols were approved by institutional review boards. Informed consent was signed by parents and patients according to Dutch law.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSamples\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eALL-10\u003c/h2\u003e \u003cp\u003eBone marrow samples were obtained at diagnosis, at end of induction (EOI, day 33, TP1; n\u0026thinsp;=\u0026thinsp;271), at end of consolidation 1 (EOC, day 79, TP2; n\u0026thinsp;=\u0026thinsp;269), at end of consolidation 2/start of intensification (4\u0026ndash;5 months after diagnosis, TP3, MRG week 1; n\u0026thinsp;=\u0026thinsp;252), at end of intensification, at start of maintenance therapy (8\u0026ndash;10 months after diagnosis, TP4, MRG week 19; n\u0026thinsp;=\u0026thinsp;251) or during maintenance at 12 months after diagnosis (TP5, MRG week 37; n\u0026thinsp;=\u0026thinsp;263). See Supplementary Fig.\u0026nbsp;2a for MRD timepoints and treatment scheme ALL-10. Bone marrow samples were available at EOC and at least one later TP in 269 patients, see CONSORT diagram Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eALL-9\u003c/h2\u003e \u003cp\u003eBone marrow samples were obtained at diagnosis, EOI (day 42, TP1), after first consolidation with HD-MTX courses (EOC, TP2, 3 months after diagnosis, n\u0026thinsp;=\u0026thinsp;122 EOC MRD\u0026thinsp;\u0026lt;\u0026thinsp;0.05%), and at 6 months (TP3; n\u0026thinsp;=\u0026thinsp;104), 9 months (TP4; n\u0026thinsp;=\u0026thinsp;107) 12 months (TP5; n\u0026thinsp;=\u0026thinsp;110) after diagnosis. (See Supplementary Fig.\u0026nbsp;2b and 2c, for MRD timepoints and treatment schemes ALL-9). Bone marrow samples were available at EOC and at least one later timepoint in 120 patients, see CONSORT diagram supplementary Fig.\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDetection of MRD\u003c/h2\u003e \u003cp\u003eImmunoglobulin and/or T-cell receptor (IG/TR) gene rearrangements were used as MRD-PCR targets. IG/TR gene rearrangements were detected in the diagnostic specimens as previously described.\u003csup\u003e9,10\u003c/sup\u003e On the basis of the junctional region of the identified genomic rearrangement, patient-specific primers were designed and tested for quantitative range (QR) and sensitivity, at the two national reference laboratories. Real-time quantitative PCR data were analyzed according to the EuroMRD guidelines.\u003csup\u003e9\u003c/sup\u003e MRD results were classified as negative, positive not quantifiable (\u0026ldquo;positive, \u0026lt;QR\u0026rdquo;), and positive quantifiable (\u0026ge;\u0026thinsp;QR), following guidelines for therapy reduction.\u003csup\u003e9\u003c/sup\u003e For MRD analyses in ALL-10 for TP3, TP4 and TP5, also guidelines for therapy intensification were applied for comparison of the MRD results applying both guidelines. In short, a sample is considered positive if the CT value of at least one of the three replicates is \u0026ge;1.0 CT (therapy reduction) or \u0026ge;3.0 CT (therapy intensification) lower than the lowest CT of background, and the CT value of at least one of the three replicates is within 4.0 CT from the highest CT value of the \u0026lsquo;sensitivity\u0026rsquo; (fulfilling all \u0026lsquo;sensitivity\u0026rsquo; criteria).\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eChi square tests were performed to check if there was no selection bias between patients with and without MRD evaluations with respect to known prognostic features. Relapse free survival (RFS) was used as endpoint since we wanted to know if positive MRD was associated with relapse rather than with any other event. RFS was defined as time from diagnosis until first relapse; this was not corrected for competing events (death before relapse). Patients that did not adhere to the MR protocol due to major treatment modifications were not excluded based on an intention-to-treat analysis. RFS curves were estimated using the Kaplan\u0026ndash;Meier (KM) methodology. Estimates of RFS at 6 years along with the corresponding 95% confidence intervals (CIs) were reported for different time points. The Log-rank test was used to compare relapse free survival (RFS) for the different time points. The analyses were performed using SPSS IBM version 26 and R version 4.2.2.\u003csup\u003e11\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eALL-10\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003ePatients characteristics\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eOut of 464 MR patients enrolled in the ALL-10 protocol, MRD data at later timepoints than EOC were available for 271 (see the CONSORT diagram, Figure 1). \u0026nbsp;The mean follow-up time was 10.0 years (range 6.1-13.5 years). No significant differences in sex, age, WBC, immunophenotype (B-lineage versus T-lineage) and CNS status at diagnosis were detected between patients with and without available MRD data (Table 1). The percentage of patients with MRD positivity at EOC did not differ significantly between patients with and without MRD data at later time points, nor did the 6-year RFS (SE) differ (89.2 (95% CI: 86-93) versus 92.1% (95% CI:88-96) respectively, (p = 0.29)).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThirty out of 271 patients (11.1%) relapsed (24 isolated BM, 4 combined CNS and BM, 2 isolated CNS), 3 (1.1%) died in remission and 1 (0.4%) developed a second malignant neoplasm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003ePrognostic significance of MRD at the end of consolidation 1 (EOC) and subsequent time points\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eAt EOC (TP2) all patients had MRD \u0026lt; 0.05% as per protocol definition for intermediate risk patients. At EOC 178 out of 269 (66.2%) patients were MRD negative (undetectable MRD) and 91 (33.8%) MRD positive. Of the MRD positive patients, 80 (29.7%) had positive not quantifiable MRD and 11 (4.1%) had positive quantifiable MRD levels (0.01% \u0026lt; 0.05%). EOC MRD negativity was associated with a better RFS, with a 6 years RFS of 93.1% (89-97) for MRD- negative patients, and 80.3% (72-89) for MRD positive patients (p \u0026lt;0.01), supplementary figure 3a.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe analyzed the 3 timepoints after consolidation1. For each timepoint, patients with negative MRD had a better outcome than patients with positive MRD (supplementary figures 3 b,c,d).\u0026nbsp;\u003c/p\u003e\n\u003col start=\"2\"\u003e\n \u003cli\u003e\u003cem\u003eValue of MRD at later timepoints for MRD negative patients at EOC\u003c/em\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eFrom the 178 EOC MRD negative patients, 152 remained MRD negative at later timepoints and 26 patients showed positive MRD results at one or more later timepoints (TP3, TP4, and/or TP5). MRD positivity at later timepoints had no prognostic relevance as both groups had comparable outcomes; 6-y RFS of 92.0% (95% CI:88-97) and 96.2% (95% CI:89-100) for those remaining negative at later timepoints versus those who did not respectively, see figure 2a.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhen applying guidelines for therapy intensification, 12 of 26 (46%) patients that showed positive MRD results at one or more later timepoints, were now defined as MRD negative at all subsequent timepoints.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2) Value of MRD at later timepoints for MRD positive patients at EOC\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFrom the 91 patients with positive EOC MRD, 43 were MRD negative at all subsequent timepoints and 48 had a positive MRD result at one or more of the subsequent timepoints (TP3, TP4 and/or TP5). Patients that were MRD negative at all subsequent timepoints had a 6-y RFS of 93% (95% CI:85-100), which is comparable to that of patients being EOC MRD negative mentioned in the paragraph above. However, patients that were MRD positive at later timepoints had a significantly worse 6y RFS (70.5%; 95% CI:58-84, p =0.002 see figure 2a). When applying guidelines for therapy intensification, 5 of 48 (10%) patients that showed positive MRD results at one or more later timepoints, were now defined as MRD negative at all subsequent timepoints. The RFS for these 43 patients was comparable, 6-y RFS 69.4% (95% CI: 56-83), see supplementary figure 4.\u003c/p\u003e\n\u003cp\u003eAll patients with quantifiable disease at TP2 (EOC, n=11), were MRD positive at TP3; 3 with quantifiable disease and 8 with positive not quantifiable disease. At TP3 7/46 MRD positive patients had positive quantifiable disease. Two of these patients also had positive quantifiable disease at TP4. No other patients had quantifiable disease at TP4 and at TP5 none of the patients had positive quantifiable disease.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eValidation cohort ALL-9\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eMRD data were available for 122 patients of the ALL-9 study who had EOC (TP2) MRD \u0026lt; 0.05% (see the CONSORT diagram, Supplementary Figure 1). \u0026nbsp;Seventy eight out of these 122 (64.0%) patients were MRD negative (undetectable MRD) and 44 (36.0%) MRD positive. Of the EOC MRD positive patients, 37 (30.3%) had positive not quantifiable MRD and 7 (5.7%) had positive quantifiable MRD levels (0.01% \u0026lt; 0.05%). Patients with MRD negativity at EOC had a better 6-years RFS (93.6%; 95% CI:88-99) \u0026nbsp;than patients with positive MRD at EOC (83.5%; 72-0.95) (p = 0.029), supplementary figure 5. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOf 120 patients also MRD results at later timepoints were available; 77 patients were EOC MRD negative; and 43 patients were EOC MRD positive. As in ALL-10, we divided the EOC MRD negative group in two groups; patients that remained MRD negative (n=69), and patients that had any positive MRD results at TP3, TP4, and/or TP5 (n=8). MRD positivity at later time points had no prognostic value as both groups had comparable outcomes; 6-y RFS of 94.2% (88-99) and 87.5% (67-100) respectively (p=0.27), see figure 2b.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlso for the EOC MRD positive patients (n=43) we observed the same results as in ALL-10; patients that became MRD negative at all later timepoints (n=17) had an RFS that was comparable to that of patients that were EOC MRD negative (6-y EFS 94.1 (83-100)). However, patients that remained MRD positive at later time points (n=26) had a worse 6y-RFS of 75.4% (60-94), (p =0.008, figure 2b).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis is the first study that has investigated the prognostic significance of MRD detection after EOC in a MRD-based pediatric ALL protocol. Often low, not quantifiable levels of MRD (\u0026lt;\u0026thinsp;0.05%) are found of which the prognostic and clinical significance is not clear. First, our data show that in patients who are MRD negative at EOC, MRD positivity at later timepoints did not adversely affect outcome. So no additional MRD measurements are needed in patients that are EOC MRD negative. Second, patients that are EOC MRD positive the subsequent MRD time points have additional prognostic relevance and could help to further stratify these intermediate risk patients.\u003c/p\u003e \u003cp\u003eThe data were obtained in our ALL-10 study and validated in the ALL-9 cohort. The results were in line with a previous study by Pui et al\u003csup\u003e7\u003c/sup\u003e, which showed that MRD re-emerged in EOC MRD negative patients in a very small proportion of patients. However, they found that the few patients that experienced re-emergence of MRD, defined as \u0026ge;\u0026thinsp;0.01%, carried a poor prognosis. In our cohort, all positive MRD results in patient being MRD negative, were at the level of positive not quantifiable, and these patients had an excellent outcome.\u003c/p\u003e \u003cp\u003eA likely explanation for MRD positive results in patients that were EOC MRD negative is that these very low not quantifiable levels are false positive results. This was illustrated by the fact that 46% of patients that were EOC MRD negative and showed positive MRD results at subsequent times points, were MRD negative at all subsequent when applying guidelines for therapy intensification.\u003csup\u003e9\u003c/sup\u003e In addition false positive MRD results can be caused by B-lymphocyte regeneration.\u003csup\u003e12\u003c/sup\u003e More specific techniques such as targeted next generation sequencing (NGS) could overcome this problem.\u003csup\u003e13,14\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn patients who are EOC MRD positive, subsequent time points seem to be of prognostic importance. Here we show that very low, not quantifiable MRD levels detected at EOC and at subsequent time point(s) are prognostic for relapse and probably identify slow responders. These slow responders have a higher risk to develop a relapse. All relapses involved the bone marrow. Identifications of these slow responders should be the purpose of follow-up measurements and may lead to improved risk stratification with subsequent therapeutic interventions such as blinatumomab, inotuzumab and CART cell therapy.\u003c/p\u003e \u003cp\u003eWe conclude from this study that in patients with MRD based intermediate risk ALL who are MRD negative at end of first consolidation (EOC), MRD measurement at later timepoints can be abandoned. For patients who are EOC MRD positive the subsequent MRD measurements might be informative for further risk stratification and therapeutic intervention.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eAcknowledgments\u003c/em\u003e\u003cbr\u003e\u0026nbsp;None\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthorship Contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe corresponding author confirms that \u0026nbsp;she had full access to the data in the study and final responsibility for the decision to submit for publication. All authors, meet all of the following criteria:\u003c/p\u003e\n\u003cp\u003eConceived and/or designed the work that led to the submission, acquired data, and/or played an important role in interpreting the results.\u003c/p\u003e\n\u003cp\u003eDrafted or revised the manuscript.\u003c/p\u003e\n\u003cp\u003eApproved the final version.\u003c/p\u003e\n\u003cp\u003eAgreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.\u003c/p\u003e\n\u003cp\u003eJ.S., designed research, analyzed and interpreted data, performed statistical analysis, drafted the manuscript,\u0026nbsp;Approved the final version.\u003c/p\u003e\n\u003cp\u003eR.vd.W., designed research, analyzed and interpreted data, performed statistical analysis,\u0026nbsp;revised the manuscript, Approved the final version.\u003c/p\u003e\n\u003cp\u003eH. deG-K.,\u0026nbsp;analyzed and interpreted data,\u0026nbsp;revised the manuscript, Approved the final version\u003c/p\u003e\n\u003cp\u003eE.S., performed research, collected data,\u0026nbsp;revised the manuscript, Approved the final version\u003c/p\u003e\n\u003cp\u003eV.de H. performed research, collected data, analyzed and interpreted data,\u0026nbsp;revised the manuscript, Approved the final version\u003c/p\u003e\n\u003cp\u003eR.D. performed statistical analysis,\u0026nbsp;revised the manuscript, Approved the final version\u003c/p\u003e\n\u003cp\u003eE.vd S.\u0026nbsp;analyzed and interpreted data, collected data,\u0026nbsp;revised the manuscript, Approved the final version\u003c/p\u003e\n\u003cp\u003eV.vd V., performed research, collected data, analyzed and interpreted data,\u0026nbsp;revised the manuscript, Approved the final version\u003c/p\u003e\n\u003cp\u003eR.P., analyzed and interpreted data, drafted the manuscript, Approved the final version\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interest\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFor all\u0026nbsp;\u003c/em\u003eauthors there are no competing financial interests in relation to the work described\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData Availability Statement:\u003c/em\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eVan Dongen, J. J. M., Seriu, T., Panzer-Gr\u0026uuml;mayer, E. R., Biondi, A., Pongers-Willemse, M. J., Corral, L., Stolz, F., Schrappe, M., Masera, G., Kamps, W. A., Gadner, H., Van Wering, E. R., Ludwig, W. D., Basso, G., De Bruijn, M. A. C., Cazzaniga, G., Hettinger, K., Van Der Does-Van Den Berg, A., Hop, W. C. J., Riehm, H., and Bartram, C. R. (1998) Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood. \u003cem\u003eLancet\u003c/em\u003e \u003cem\u003e352\u003c/em\u003e, 1731\u0026ndash;1738.\u003c/li\u003e\n \u003cli\u003eFlohr, T., Schrauder, A., Cazzaniga, G., Panzer-Gr\u0026uuml;mayer, R., van der Velden, V., Fischer, S., Stanulla, M., Basso, G., Niggli, F. K., Sch\u0026auml;fer, B. W., Sutton, R., Koehler, R., Zimmermann, M., Valsecchi, M. G., Gadner, H., Masera, G., Schrappe, M., van Dongen, J. J. M., Biondi, A., and Bartram, C. R. (2008) Minimal residual disease-directed risk stratification using real-time quantitative PCR analysis of immunoglobulin and T-cell receptor gene rearrangements in the international multicenter trial AIEOP-BFM ALL 2000 for childhood acute lymphoblastic leukemia. \u003cem\u003eLeukemia\u003c/em\u003e \u003cem\u003e22\u003c/em\u003e, 771\u0026ndash;782.\u003c/li\u003e\n \u003cli\u003ePieters, R., De Groot-Kruseman, H., Van Der Velden, V., Fiocco, M., Van Den Berg, H., De Bont, E., Egeler, R. M., Hoogerbrugge, P., Kaspers, G., Van Der Schoot, E., De Haas, V., and Van Dongen, J. (2016) Successful therapy reduction and intensification for childhood acute lymphoblastic leukemia based on minimal residual disease monitoring: Study ALL10 from the Dutch Childhood Oncology Group. \u003cem\u003eJ. Clin. Oncol.\u003c/em\u003e \u003cem\u003e34\u003c/em\u003e, 2591\u0026ndash;2601.\u003c/li\u003e\n \u003cli\u003eConter, V., Bartram, C. R., Valsecchi, M. G., Schrauder, A., Panzer-Gr\u0026uuml;mayer, R., M\u0026ouml;ricke, A., Aric\u0026ograve;, M., Zimmermann, M., Mann, G., De Rossi, G., Stanulla, M., Locatelli, F., Basso, G., Niggli, F., Barisone, E., Henze, G., Ludwig, W. D., Haas, O. A., Cazzaniga, G., Koehler, R., Silvestri, D., Bradtke, J., Parasole, R., Beier, R., Van Dongen, J. J. M., Biondi, A., and Schrappe, M. (2010) Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute lymphoblastic leukemia: Results in 3184 patients of the AIEOP-BFMALL 2000 study. \u003cem\u003eBlood\u003c/em\u003e \u003cem\u003e115\u003c/em\u003e, 3206\u0026ndash;3214.\u003c/li\u003e\n \u003cli\u003eSchrappe, M., Bleckmann, K., Zimmermann, M., Biondi, A., M\u0026ouml;ricke, A., Locatelli, F., Cario, G., Rizzari, C., Attarbaschi, A., Valsecchi, M. G., Bartram, C. R., Barisone, E., Niggli, F., Niemeyer, C., Testi, A. M., Mann, G., Ziino, O., Sch\u0026auml;fer, B., Panzer-Gr\u0026uuml;mayer, R., Beier, R., Parasole, R., G\u0026ouml;hring, G., Ludwig, W. D., Casale, F., Schlegel, P. G., Basso, G., and Conter, V. (2018) Reduced-Intensity delayed intensification in standard-Risk pediatric acute lymphoblastic leukemia defined by undetectable minimal residual disease: Results of an international randomized trial (AIEOP-BFM ALL 2000). \u003cem\u003eJ. Clin. Oncol.\u003c/em\u003e \u003cem\u003e36\u003c/em\u003e, 244\u0026ndash;253.\u003c/li\u003e\n \u003cli\u003eVora, A., Goulden, N., Wade, R., Mitchell, C., Hancock, J., Hough, R., Rowntree, C., and Richards, S. (2013) Treatment reduction for children and young adults with low-risk acute lymphoblastic leukaemia defined by minimal residual disease (UKALL 2003): A randomised controlled trial. \u003cem\u003eLancet Oncol.\u003c/em\u003e \u003cem\u003e14\u003c/em\u003e, 199\u0026ndash;209.\u003c/li\u003e\n \u003cli\u003ePui, C. H., Pei, D., Coustan-Smith, E., Jeha, S., Cheng, C., Bowman, W. P., Sandlund, J. T., Ribeiro, R. C., Rubnitz, J. E., Inaba, H., Bhojwani, D., Gruber, T. A., Leung, W. H., Downing, J. R., Evans, W. E., Relling, M. V., and Campana, D. (2015) Clinical utility of sequential minimal residual disease measurements in the context of risk-based therapy in childhood acute lymphoblastic leukaemia: A prospective study. \u003cem\u003eLancet Oncol.\u003c/em\u003e \u003cem\u003e16\u003c/em\u003e, 465\u0026ndash;474.\u003c/li\u003e\n \u003cli\u003eVeerman, A. J., Kamps, W. A., van den Berg, H., van den Berg, E., B\u0026ouml;kkerink, J. P., Bruin, M. C., van den Heuvel-Eibrink, M. M., Korbijn, C. M., Korthof, E. T., van der Pal, K., Stijnen, T., van Weel Sipman, M. H., van Weerden, J. F., van Wering, E. R., and van der Does-van den Berg, A. (2009) Dexamethasone-based therapy for childhood acute lymphoblastic leukaemia: results of the prospective Dutch Childhood Oncology Group (DCOG) protocol ALL-9 (1997-2004). \u003cem\u003eLancet Oncol.\u003c/em\u003e \u003cem\u003e10\u003c/em\u003e, 957\u0026ndash;966.\u003c/li\u003e\n \u003cli\u003evan der Velden, V. H. J., Cazzaniga, G., Schrauder, A., Hancock, J., Bader, P., Panzer-Grumayer, E. R., Flohr, T., Sutton, R., Cave, H., Madsen, H. O., Cayuela, J. M., Trka, J., Eckert, C., Foroni, L., zur Stadt, U., Beldjord, K., Raff, T., van der Schoot, C. E., and van Dongen, J. J. M. (2007) Analysis of minimal residual disease by Ig/TCR gene rearrangements: Guidelines for interpretation of real-time quantitative PCR data. \u003cem\u003eLeukemia\u003c/em\u003e \u003cem\u003e21\u003c/em\u003e, 604\u0026ndash;611.\u003c/li\u003e\n \u003cli\u003evan der Velden, V. H. J., and van Dongen, J. J. M. (2009) MRD Detection in Acute Lymphoblastic Leukemia Patients Using Ig/TCR Gene Rearrangements as Targets for Real-Time Quantitative PCR, in \u003cem\u003eLeukemia: Methods and Protocols\u003c/em\u003e (Eric So, C. W., Ed.), pp 115\u0026ndash;150. Humana Press, Totowa, NJ.\u003c/li\u003e\n \u003cli\u003eR. Core Team. (2022) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL: https://www.r-project.org/\u003c/li\u003e\n \u003cli\u003evan der Velden, V. H. J., Wijkhuijs, J. M., and van Dongen, J. J. M. (2008) Non-specific amplification of patient-specific Ig/TCR gene rearrangements depends on the time point during therapy: Implications for minimal residual disease monitoring [5]. \u003cem\u003eLeukemia\u003c/em\u003e \u003cem\u003e22\u003c/em\u003e, 641\u0026ndash;644.\u003c/li\u003e\n \u003cli\u003eKotrova, M., Van Der Velden, V. H. J., Van Dongen, J. J. M., Formankova, R., Sedlacek, P., Br\u0026uuml;ggemann, M., Zuna, J., Stary, J., Trka, J., and Fronkova, E. (2017) Next-generation sequencing indicates false-positive MRD results and better predicts prognosis after SCT in patients with childhood ALL. \u003cem\u003eBone Marrow Transplant.\u003c/em\u003e \u003cem\u003e52\u003c/em\u003e, 962\u0026ndash;968.\u003c/li\u003e\n \u003cli\u003eSvaton M, Skotnicova A, Reznickova L, Rennerova A, Valova T, Kotrova M \u003cem\u003eet al.\u003c/em\u003e NGS better discriminates true MRD positivity for the risk stratification of childhood ALL treated on an MRD-based protocol. \u003cem\u003eBlood\u003c/em\u003e 2023; \u003cstrong\u003e141\u003c/strong\u003e: 529\u0026ndash;533.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\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":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3411409/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3411409/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMinimal residual disease (MRD) is regularly measured at later timepoints after end of first consolidation (EOC) in children with acute lymphoblastic leukemia (ALL). The question remains whether this is useful for detecting (molecular) relapse. We investigated the clinical relevance of MRD after EOC in intermediate risk patients treated on DCOG-ALL-10 (n\u0026thinsp;=\u0026thinsp;271) and DCOG-ALL-9 (n\u0026thinsp;=\u0026thinsp;122), with MRD\u0026thinsp;\u0026lt;\u0026thinsp;0.05% at EOC. EOC MRD negative patients (n\u0026thinsp;=\u0026thinsp;178) had excellent outcomes, irrespective of MRD results at later timepoints; 6-years relapse free survival (6y-RFS) of 92.0% (95% CI:88\u0026ndash;97) for those with MRD negativity at all later timepoints compared to 96.2% (95% CI:89\u0026ndash;100) for those with one or more later timepoints being positive (p\u0026thinsp;=\u0026thinsp;0.46). Patients with positive EOC MRD (n\u0026thinsp;=\u0026thinsp;91) of whom the subsequent timepoints were MRD negative (n\u0026thinsp;=\u0026thinsp;43), had comparable good outcomes, 6y-RFS of 93.0% (95% CI:85\u0026ndash;100). Patients being MRD positive at EOC and MRD positivity at one or more subsequent timepoints (n\u0026thinsp;=\u0026thinsp;48) had a higher risk of relapse, 6y-RFS 70.5% (95% CI:58\u0026ndash;84), p\u0026thinsp;=\u0026thinsp;0.002. These findings were confirmed in the validation cohort of ALL-9. In patients who are MRD negative at EOC, MRD measurements at later timepoints can be abandoned. For patients who are EOC MRD positive the subsequent MRD measurement might be informative for further risk stratification.\u003c/p\u003e","manuscriptTitle":"Are minimal residual disease measurement after consolidation therapy useful in children with acute lymphoblastic leukemia?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-10 14:04:28","doi":"10.21203/rs.3.rs-3411409/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2023-12-06T12:02:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-12-05T06:22:21+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-11-08T08:11:55+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-10-16T08:25:28+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-10-07T05:37:27+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2023-10-05T14:07:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-10-05T09:13:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-10-05T09:13:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Leukemia","date":"2023-10-04T19:21:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7b423711-05db-4f5d-9138-de354ebfdb00","owner":[],"postedDate":"October 10th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":25194598,"name":"Biological sciences/Cancer/Haematological cancer/Leukaemia/Acute lymphocytic leukaemia"},{"id":25194599,"name":"Health sciences/Medical research/Translational research"}],"tags":[],"updatedAt":"2024-09-11T07:06:55+00:00","versionOfRecord":{"articleIdentity":"rs-3411409","link":"https://doi.org/10.1038/s41375-024-02386-5","journal":{"identity":"leukemia","isVorOnly":false,"title":"Leukemia"},"publishedOn":"2024-09-10 04:00:00","publishedOnDateReadable":"September 10th, 2024"},"versionCreatedAt":"2023-10-10 14:04:28","video":"","vorDoi":"10.1038/s41375-024-02386-5","vorDoiUrl":"https://doi.org/10.1038/s41375-024-02386-5","workflowStages":[]},"version":"v1","identity":"rs-3411409","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3411409","identity":"rs-3411409","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}