Hematology ISSN: (Print) 1607-8454 (Online) Journal homepage: https://www.tandfonline.com/loi/yhem20 Minimal residual disease level predicts outcome in adults with Ph-negative B-precursor acute lymphoblastic leukemia Nicola Gökbuget, Hervé Dombret, Sebastian Giebel, Monika Bruggemann, Michael Doubek, Robin Foà, Dieter Hoelzer, Christopher Kim, Giovanni Martinelli, Elena Parovichnikova, Alessandro Rambaldi, Josep-Maria Ribera, Marieke Schoonen, Julia M. Stieglmaier, Gerhard Zugmaier & Renato Bassan To cite this article: Nicola Gökbuget, Hervé Dombret, Sebastian Giebel, Monika Bruggemann, Michael Doubek, Robin Foà, Dieter Hoelzer, Christopher Kim, Giovanni Martinelli, Elena Parovichnikova, Alessandro Rambaldi, Josep-Maria Ribera, Marieke Schoonen, Julia M. Stieglmaier, Gerhard Zugmaier & Renato Bassan (2019) Minimal residual disease level predicts outcome in adults with Ph-negative B-precursor acute lymphoblastic leukemia, Hematology, 24:1, 337-348, DOI: 10.1080/16078454.2019.1567654 To link to this article: https://doi.org/10.1080/16078454.2019.1567654 © 2019 The Author(s). Published by Informa View supplementary material UK Limited, trading as Taylor & Francis Group Published online: 13 Feb 2019. Submit your article to this journal Article views: 570 View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=yhem20 HEMATOLOGY 2019, VOL. 24, NO. 1, 337–348 https://doi.org/10.1080/16078454.2019.1567654 Minimal residual disease level predicts outcome in adults with Ph-negative B- precursor acute lymphoblastic leukemia Nicola Gökbugeta, Hervé Dombretb, Sebastian Giebelc, Monika Bruggemannd, Michael Doubek e, Robin Foàf, Dieter Hoelzera, Christopher Kimg, Giovanni Martinellih, Elena Parovichnikova i, Alessandro Rambaldij, Josep-Maria Ribera k, Marieke Schoonenl, Julia M. Stieglmaierm, Gerhard Zugmaierm and Renato Bassann a Department of Medicine II, Department of Hematology/Oncology, University Hospital, Frankfurt, Germany; bHôpital Saint-Louis, University Paris Diderot, Paris, France; cMaria Sklodowska Curie Memorial Cancer Center, Gliwice, Poland; dDepartment of Hematology and Oncology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany; eDepartment of Internal Medicine, Hematology and Oncology, University Hospital, Brno, Czech Republic; f“Sapienza” University of Rome, Rome, Italy; gAmgen Inc, Thousand Oaks, CA, USA; hIstituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy; iNational Research Center for Hematology, Moscow, Russia; jDipartimento di Oncologia ed Ematologia, Università degli Studi di Milano and Ospedale Papa Giovanni XXIII, Bergamo, Italy; kICO- Hospital Germans Trias I Pujol, Josep Carreras Research Institute, Barcelona, Spain; lAmgen Ltd, London, UK; mAmgen GmBH, Munich, Germany; nOspedale dell’Angelo, Mestre-Venezia, Italy ABSTRACT KEYWORDS Objectives: Detectable minimal residual disease (MRD) after therapy for acute lymphoblastic Acute lymphoblastic leukemia (ALL) is the strongest predictor of hematologic relapse. This study evaluated leukemia; minimal residual outcomes of patients with B-cell precursor ALL with MRD of ≥10−4 disease; MRD; allogeneic Methods: Study population was from ALL study groups in Europe managed in national study stem cell transplant protocols 2000–2014. MRD was measured by polymerase chain reaction or flow cytometry. Patients were age ≥15 years at initial ALL diagnosis. Patients were excluded if exposed to blinatumomab within 18 months of baseline or prior alloHSCT. Results: Of 272 patients in CR1, baseline MRD was ≥10−1, 10−2 to <10−1, 10−3 to <10−2, and 10−4 to <10−3 in 15 (6%), 71 (26%), 109 (40%), and 77 (28%) patients, respectively. Median duration of complete remission (DoR) was 18.5 months (95% confidence interval [CI], 11.9–27.2), median relapse-free survival (RFS) was 12.4 months (95% CI, 10.0–19.0) and median overall survival (OS) was 32.5 months (95% CI, 23.6–48.0). Lower baseline MRD level (P ≤ .0003) and white blood cell count <30,000/µL at diagnosis (P ≤ .0053) were strong predictors for better RFS and DoR. Allogeneic hematopoietic stem cell transplantation (alloHSCT) was associated with longer RFS (hazard ratio [HR], 0.59; 95% CI, 0.41–0.84) and DoR (HR, 0.43; 95% CI, 0.29–0.64); the association with OS was not significant (HR, 0.72; 95% CI, 0.50–1.05). Discussion: In conclusion, RFS, DoR, and OS are relatively short in patients with MRD-positive ALL, particularly at higher MRD levels. AlloHSCT may improve survival but has limitations. Alternative approaches are needed to improve outcomes in MRD-positive ALL. Introduction chemotherapy, approximately 30–50% of adult and Outcomes for adult patients with acute lymphoblastic 10–20% of pediatric patients at CR1 exhibit minimal leukemia (ALL) have improved. However, some residual disease (MRD) [1–8]. MRD is defined as the patients remain resistant to standard chemotherapy. presence of leukemic cells in patients with hematologic Despite hematologic complete remission (CR) rates CR below 5%, which is the detection level of of 80–90% with intensive induction/consolidation microscopy. Several methods for standardized and CONTACT Nicola Gökbuget goekbuget@em.uni-frankfurt.de University Hospital, Theodor Stern Kai 7, D-60590 Frankfurt, Germany Supplemental data for this article can be accessed at https://dx.doi.org/10.1080/16078454.2019.1567654. © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 338 N. GÖKBUGET ET AL. quantitative measurement of MRD at a sensitivity of ensure a standardized and quality-controlled data col- 10−4 (i.e. 0.01%) are available, such as flow cytometry lection process. Variables included information on or real-time quantitative polymerase chain reaction demographics, disease characteristics, history of ALL (RQ-PCR) of individual clonal rearrangements of immu- treatment prior to the detection of MRD, diagnostics noglobulin [9]. and level of MRD, details of transplantation for patients MRD indicates resistance to standard chemotherapy who underwent alloHSCT after their MRD detection, and and is the most important risk factor for hematologic clinical event data including occurrence of hematologic relapse in adult and pediatric ALL [2,4,10,11–14]. Stan- relapse, date of death, or date last known to be alive. dards for a description of MRD status have been described [15]. Patients with high MRD levels after Patient eligibility induction and consolidation (i.e. MRD persistence) have a higher risk of hematologic relapse (70–100%), Patients were eligible if they had Ph/BCR-ABL–negative whereas patients with complete MRD response are BCP ALL in hematologic CR (<5% blasts in bone marrow far less likely to have a hematologic relapse (2–8%) after ≥3 intensive chemotherapy blocks) and no extra- [1,3,4–6,10,16]. Conversion from MRD negativity to medullary involvement. Other key eligibility criteria MRD positivity (i.e. MRD reappearance) is highly predic- were: MRD of ≥10−4 by RQ-PCR of clonally rearranged tive of subsequent hematologic relapse [5,7]. Time immunoglobulin or ≥10−3 by flow cytometry at a refer- from MRD positivity to hematologic relapse is also cor- ence lab; age ≥15 years at initial ALL diagnosis; initial related with MRD level [5]. ALL diagnosis in the years 2000–2014; availability of Most study groups recommend allogeneic hemato- data for history of ALL treatment (including response poietic stem cell transplant (alloHSCT) as the most inten- to first therapy and number of prior relapses); and sive treatment approach available for the management relapse status and disease follow-up after MRD detec- of MRD [17–19]. Patients who receive alloHSCT have tion. To focus on standard therapies, patients were better outcomes compared with those who do not excluded if exposed to blinatumomab within 18 [20]. However, alloHSCT is not always an option due to months of MRD detection. Patients with MRD after associated toxicities, lack of suitable donors, or other prior alloHSCT were excluded. reasons. Patients may relapse awaiting alloHSCT, par- ticularly those with higher MRD levels. Additionally, Statistical analyses patients with detectable MRD before alloHSCT may have a higher risk of relapse after alloHSCT [5,13,21]. Patients were included in all analyses regardless of The objectives of this study were to estimate the whether they received a transplant after MRD detection. duration of hematologic CR (DoR), hematologic The primary outcome, RFS, was defined as the time from relapse-free survival (RFS), and overall survival (OS) baseline MRD detection until hematologic relapse or for patients with MRD-positive B-cell precursor (BCP) death due to any cause; patients alive without relapse ALL treated with standard of care in Europe. In addition, were censored at their last disease assessment. The sec- several statistical methods were used to closely explore ondary outcome, OS, was defined as the time from how alloHSCT status may influence clinical outcomes. baseline MRD detection until death; patients without a recorded death were censored at last follow-up. The analysis of DoR was defined as the time from baseline Methods MRD detection until relapse; patients who died in CR were censored at the date of death. RFS, OS, and DoR Study design were summarized with Kaplan-Meier curves and pro- We analyzed a cohort of adults with ALL who had portions; point estimates were accompanied by 2- reached CR but had detectable MRD after the standard sided 95% confidence intervals (CI). of care treatment according to national study proto- Baseline MRD was the first measurement of MRD- cols. The study population was from ALL study positivity after CR. Baseline MRD status was defined groups in Europe (Czech Republic, France, Germany, as MRD persistence (MRD positivity without prior docu- Italy, Poland, Russia, Spain and the United Kingdom) mented negativity) or MRD reappearance (return to with protocols that included prospective MRD testing MRD positivity after achievement of complete MRD in national reference laboratories. Entry criteria, data response) [15]. In univariate Cox regression models, collection methods, and analyses were defined in the potential association between RFS, DoR, and OS a joint protocol (clinicaltrials.gov identifier: with each of the following factors was investigated: NCT02010931). Ethical approval was obtained accord- age at diagnosis (15–34, 35–54, 55–64, or ≥65 years); ing to individual country requirements. sex (female vs. male), MRD status (persistence vs. Patient-level data were obtained from clinical data- reappearance); MRD at baseline (≥10−1, 10−2 to bases and entered by central staff of each study group <10−1, 10−3 to <10−2, or 10−4 to <10−3), and white into a study-specific electronic case report form to blood cell (WBC) count at diagnosis (≥30,000/µL vs. HEMATOLOGY 339 <30,000/µL). Using forward selection, factors significant reappearance and 1% had an unknown MRD status. at P < .10 were included in a multivariable Cox Median time from initial ALL diagnosis to MRD positiv- regression model describing RFS, DoR or OS. ity was 4.2 months. Median follow-up was 23.0 months Several methods were employed to explore the for patients included in time-to-event analyses. effect of receiving alloHSCT after detection of MRD. (1) Conditional landmark analyses of alloHSCT defined Duration of remission the landmark at 3 months, which was approximately Median DoR was 18.5 months (95% CI, 11.9–27.2) after the median time to alloHSCT from baseline (3.4 baseline MRD assessment (Figure 1(A)). At 36 months, months; interquartile range, 1.7–6.3); additionally, a 38% of patients remained in hematologic CR (95% CI, landmark at 6 months was assessed which included 32% to 45%). Median DoR was longer in patients with most patients that underwent a transplant. For each lower baseline MRD: 2.0, 10.9, 18.5, and 42.4 months analysis, patients with an event (DoR, RFS, OS) before after baseline MRD of ≥10−1, 10−2 to <10−1, 10−3 to the defined landmark were excluded, transplant <10−2, and 10−4 to <10−3, respectively (P < .0001; status was assessed at the time of the landmark (3 Figure 2(A) and Table 2). Median DoR was also longer months and 6 months), wherein a patient was con- in patients with WBC <30,000/µL vs. ≥30,000/µL at sidered to be transplanted only if the transplant was diagnosis (P = .003), younger vs. older patients (P conducted on or before the landmark time. Any = .21), and patients with MRD persistence vs. MRD patients who underwent a transplant after the land- reappearance (P = .03; Table 2 and Supplemental mark were included in the nontransplant group. (2) Figure 1). In a multivariable model, younger age (P Mantel–Byar analysis handles alloHSCT as a time- = .08), lower WBC count at diagnosis (P = .005), lower dependent covariate. A patient is attributed to the non- baseline MRD (P = .002), and MRD persistence vs. transplant group until the time of transplant, once reappearance (P = .03) were associated with longer transplanted, is considered part of the transplant DoR (Table 3). Year of diagnosis, which was not a group. No patients were excluded from this analysis significant factor for DoR in the univariate analysis as in the conditional landmark analysis. This method (P = .86), was not included in the multivariable model. accounts for time bias observed in fixed-time analyses Median DoR was slightly shorter when patients were because patients undergoing alloHSCT must survive censored at HSCT (Supplemental Figure 2). long enough to receive alloHSCT [22]. The Simon– Makuch method (with and without a 3-month land- mark) was used to graphically display results from the Relapse-free survival Mantel–Byar test [23]. (3) A multivariate Cox pro- Median RFS was 12.4 months (95% CI, 10.0–19.0) after portional hazards model, adjusting for the baseline baseline MRD assessment (Figure 1(B)). At 36 months, factors that were identified in the multivariate Cox estimated RFS was 33% (95% CI, 27–39%). Median model as described above, included alloHSCT as a RFS was longer in patients with a lower baseline time-dependent covariate. Statistical significance was MRD: 2.0, 9.7, 10.6, and 31.3 months after baseline measured at P < .05. No adjustment was considered MRD of ≥10−1, 10−2 to <10−1, 10−3 to <10−2, and for multiplicity. 10−4 to <10−3, respectively (P = .0003; Figure 2(B) and Table 2). Lower WBC count at original ALL diagnosis was also associated with longer median RFS (P = .005; Results Table 2 and Supplemental Figure 1). In a multivariable Patient population model, lower baseline MRD (P = .003), lower WBC at diagnosis (P = .007), MRD persistence vs. reappearance Data were captured from 287 patients. Analyses of (P = .11) and younger vs. older age (P = .05) were associ- baseline characteristics included 272 patients with ated with longer RFS (Table 3). As with DoR, year of quantifiable baseline MRD and excluded 15 patients diagnosis was not significant in the univariate analysis whose MRD was qualitatively reported to be ≥10−4 (P = .91) and was not included in the multivariable but had non-quantifiable MRD. Of the 272 patients, model. Median RFS was slightly shorter when patients time-to-event analyses for DoR, RFS, or OS included were censored at HSCT (Supplemental Figure 2). 270 patients and excluded 2 patients with quantifiable baseline MRD missing the date for MRD. Overall survival Patient characteristics are shown in Table 1 and Sup- plemental Table 1. The median age at diagnosis was 33 Median OS was 32.5 months (95% CI, 23.6–48.0) after years (IQR, 24–45). Slightly more than half of the baseline MRD assessment (Figure 1(C)), with 48% of patients were male (58%) and aged <35 years (56%). patients alive at 36 months (95% CI, 41–54%). Among MRD was assessed by RQ-PCR in most patients (79%) 272 patients with quantifiable baseline MRD, 155 and the baseline MRD was ≥10−3 in 72%. Most patients (57%) patients died, including 23 (8%) without docu- (80%) had MRD persistence; 19% had MRD mented relapse (Supplemental Table 2). Median OS 340 N. GÖKBUGET ET AL. Table 1. Patient demographics, disease characteristics, and MRD status. Received All Patients In CR1 alloHSCT (N = 272) (N = 270) (N = 110) Follow-up, months, median (Q1–Q3) 22.5 (9.6–40.6) 23.0 (10.0–50.7) 30.2 (14.8–52.4) Sex, n (%) Male 159 (58) 157 (58) 69 (63) Female 113 (42) 113 (42) 41 (37) Age at diagnosis, years Median (range) 33 (15–65) 33 (15–65) 31.5 (16–63) Age group at diagnosis, n (%) 15–34 years 151 (56) 149 (55) 70 (64) 35–54 years 86 (32) 86 (32) 32 (29) 55–64 years 34 (13) 34 (13) 8 (7) ≥65 years 1 (<1) 1 (<1) 0 (0) Time from diagnosis to baseline, monthsa Median (Q1–Q3) 4.2 (3.4–5.3) 4.2 (3.4–5.3) 4.0 (3.3–4.9) <6 months, n (%) 217 (80) 216 (80) 95 (86) 6 months, n (%) 53 (19) 52 (19) 15 (14) Year of diagnosis, n (%) 2000–2004 99 (36) 99 (37) 31 (28) 2005–2010 149 (55) 148 (55) 67 (61) 2011 or later 24 (9) 23 (9) 12 (11) WBC count at diagnosisa, n (%) <30,000/µL 198 (73) 198 (73) 74 (67) ≥30,000/µL 72 (26) 71 (26) 35 (32) Cytogenetic/molecular aberrationsa, n (%) t(4;11) KMT2A/AFF1 19 (7) 18 (7) 11 (10) Other 36 (13) 36 (13) 12 (11) No 216 (79) 215 (80) 87 (79) Baseline MRD statusa, n (%) Persistence 217 (80) 217 (80) 91 (83) Reappearance 51 (19) 51 (19) 16 (15) Methodology, n (%) RQ-PCR 215 (79) 214 (79) 86 (78) Flow cytometry 57 (21) 56 (21) 24 (22) Baseline MRD levela Median (range) 0.0027 0.0027 0.0020 (0.000–2.400) (0.000–2.400) (0.000–0.055) −1 ≥10 , n (%) 15 (6) 15 (6) 4 (4) ≥10−2 to <10−1, n (%) 71 (26) 70 (26) 30 (27) ≥10−3 to <10−2, n (%) 109 (40) 109 (40) 42 (38) ≥10−4 to <10−3, n (%) 77 (28) 76 (28) 34 (31) Note: Q indicates quartile. a Does not include patients with missing data (n = 2 for time from diagnosis to baseline MRD status, n = 3 for WBC count at diagnosis, n = 1 for cytogenetic aberrations, n = 4 for baseline MRD status, and n = 15 for baseline MRD level). Patients with missing baseline MRD level had documented MRD but no exact level was given. was longer in patients with a lower baseline MRD (15.5, patient outcomes. Age, sex, baseline MRD, and trans- 21.5, 31.2, and 50.7 months after baseline MRD of plant status were not significantly different between ≥10−1, 10−2 to <10−1, 10−3 to <10−2, and 10−4 to patients with higher (≥30,000/µL) or lower (<30,000/ <10−3, respectively) but the trend was not statistically µL) WBC at diagnosis (Supplemental Table 3). significant (P = .14; Figure 2(C) and Table 2). When However, patients with a year of diagnosis after 2010 patients with baseline MRD ≥10−3 or <10−3 were com- had higher WBC count than those diagnosed in pared, those with higher baseline MRD had signifi- 2004–2010 (P = .02). cantly worse OS (50.7 vs. 26.5 months; P = .03; Table 2). Younger age (P = .002) and lower WBC at diagnosis Transplant outcomes (P = .02) were associated with longer median OS (Sup- plemental Figure 1). In a multivariable model, lower A total of 110 patients (40%) received alloHSCT in WBC at diagnosis (P = .002), younger age (P = .0005), ongoing hematologic CR and 162 (60%) did not (Sup- and later year of diagnosis (P = .06) were associated plemental Table 4). Patients undergoing transplant with longer median OS (Table 3). Median OS was were slightly younger at diagnosis compared to non- slightly shorter when patients were censored at HSCT transplanted patients: 64% vs. 50% were age <35 (Supplemental Figure 2). years (P = .06) and median age was 31.5 vs. 33.0 years. Median time from baseline MRD assessment to alloHSCT was 3.4 months (range, 0.1–39.8). From year 2005 onward, proportionally more patients Other analyses underwent transplant compared to patients diag- WBC at diagnosis, which was prognostic for RFS, DoR, nosed in the years 2000 to 2004 (45% [67/149] vs. and OS, was cross-tabulated with other predictors for 31% [31/97]; P = .06). HEMATOLOGY 341 Figure 1. Outcomes among patients with MRD. (A) DoR. (B) RFS. (C) OS. Note: Censored patients are indicated by vertical bars. Transplant status was associated with different 59% (95% CI, 48–69%). In transplanted patients with causes of death. At last follow-up, 58% of transplanted baseline MRD of ≥10−1, 10−2 to <10−1, 10−3 to <10−2, patients and 33% of nontransplanted patients and 10−4 to <10−3, median DoR was not reached, remained alive. Death without documented relapse 33.1 months, not reached, and 75 months, respectively. was reported for 14% of transplanted patients and Median DoR in all nontransplanted patients was 7.4 5% of nontransplanted patients, while 28% of trans- months (95% CI, 4.8–10.9) and 36-month DoR was planted patients and 62% of nontransplanted patients 22% (95% CI, 15–28%). died after relapse (P < .0001). Median RFS from the date of alloHSCT in all trans- Median DoR in all transplanted patients was 68.4 planted patients was 34.4 months (95% CI, 21.8 to months (95% CI, 34.1 to NE) and 36-month DoR was NE) and 36-month RFS was 49% (95% CI, 39–60%). In 342 N. GÖKBUGET ET AL. Figure 2. Kaplan–Meier plots of outcomes by baseline MRD level. (A) DoR by all MRD levels. (B) RFS by all MRD levels. (C) OS by all MRD levels. (D) DoR by MRD ≥10−3 or <10−3. (E) RFS by MRD ≥10−3 or <10−3. (F) OS by MRD ≥10−3 or <10−3. Note: Censored patients are indicated by vertical bars. transplanted patients with baseline MRD of ≥10−1, nontransplanted patients, median OS was 20.4 10−2 to <10−1, 10−3 to <10−2, and 10−4 to <10−3, months overall (95% CI, 15.3–31.7) and 36-month OS median RFS from date of alloHSCT was not reached, was 38% (95% CI, 30–46%). 27.7, 47.9, and 75.4 months, respectively. In nontrans- Mantel–Byar analysis indicated improved outcomes planted patients, median RFS was 6.7 months overall among transplanted patients for DoR (odds ratio [OR], (95% CI, 4.6–9.2) and 36-month RFS was 20% (95% CI, 0.49; 95% CI, 0.33–0.73; P = .0004), RFS (OR, 0.65; 95% 13–26%). CI, 0.47–0.92; P = .02), and OS (OR, 0.72; 95% CI, 0.51– Median OS from the date of alloHSCT in trans- 1.01; P = .06; Table 4). Mantel–Byar test results were planted patients was 76.1 months overall (95% CI, visualized with Simon–Makuch plots, illustrating 41.1 to NE) and 36-month OS was 61% (95% CI, 51– longer DoR, RFS, and OS among transplanted patients 71%). In transplanted patients with baseline MRD of (Figure 3), similar results were observed with a 3- ≥10−1, 10−2 to <10−1, 10−3 to <10−2, and 10−4 to month landmark (Supplemental Figure 3). Conditional <10−3, median OS from date of alloHSCT was not landmark analyses which considered patients under- reached, 33.0, 54.4, and 75.4 months, respectively. In going alloHSCT up to month 3 suggested longer DoR, HEMATOLOGY 343 Table 2. Median DoR, RFS, and OS according to different patient factors. DoR, Months RFS, Months OS, Months Variable n Median (95% CI) Median (95% CI) Median (95% CI) Age group at diagnosis 15–34 years 151 21.3 (11.9–34.1) 15.9 (10.6–25.7) 54.4 (34.7–98.6) 35–54 years 85 22.1 (9.3–43.9) 11.8 (7.3–25.7) 20.0 (11.5–32.5) ≥55 years 34 10.7 (5.2–19.7) 10.0 (5.2–19.5) 14.7 (10.4–30.8) P .21 .20 .0024 Sex Male 159 24.4 (12.8–33.1) 17.5 (9.3–27.2) 45.9 (26.5–75.4) Female 111 12.3 (10.0–19.7) 11.3 (11.3–13.6) 24.5 (16.8–37.4) .26 .38 .16 WBC count at diagnosis <30,000/µL 196 25.7 (16.6–36.8) 17.5 (11.7–26.5) 39.0 (27.8–58.9) ≥30,000/µL 72 9.2 (4.4–14.9) 8.6 (4.3–11.8) 16.1 (12.4–31.9) P .0033 .0053 .016 Year of diagnosis 2000–2004 97 21.3 (12.3–32.9) 12.6 (10.3–22.1) 26.5 (17.5–34.7) 2005–2010 149 16.6 (9.7–29.7) 11.0 (7.3–19.7) 45.9 (24.3–82.2) 2011 or later 24 NE (5.2–NE) NE (5.2–NE) NE (15.7–NE) P .86 .92 .31 Baseline MRD level (vs. ≥10−1) ≥10−1 15 2.0 (1.4–4.8) 2.0 (1.4–4.8) 15.5 (7.1–NE) ≥10−2 to <10−1 71 10.9 (7.3–21.2) 9.7 (6.4–17.3) 21.5 (13.7–33.0) ≥10−3 to <10−2 108 18.5 (9.9–30.3) 10.6 (7.3–19.7) 31.2 (18.0–58.9) ≥10−4 to <10−3 76 42.4 (23.6–NE) 31.3 (13.6–75.4) 50.7 (31.7–NE) P <.0001 .0003 .14 Baseline MRD level (vs. ≥10−2) ≥10−2 86 9.2 (4.8–12.6) 8.2 (4.8–11.8) 21.5 (13.6–34.7) ≥10−3 to < 10−2 108 18.5 (9.9–30.3) 10.6 (7.3–19.7) 31.2 (18.0–58.9) ≥10−4 to < 10−3 76 42.4 (23.6–NE) 31.3 (13.6–75.4) 50.7 (31.7–NE) P .0003 .0006 .069 Baseline MRD level (vs. ≥10−3) ≥10−3 194 11.7 (8.7–19.5) 9.7 (7.0–12.6) 26.5 (16.9–35.4) ≥10−4 to < 10−3 76 42.4 (23.6–NE) 31.3 (13.6–75.4) 50.7 (31.7–NE) P .0008 .0008 .031 Baseline MRD status Persistence 215 22.1 (12.9–30.3) 13.0 (10.7–22.2) 30.8 (21.5–45.9) Reappearance 51 5.6 (2.8–19.0) 8.5 (2.7–17.3) 43.6 (23.6–NE) P .028 .11 .49 Note: NE indicates not estimable. RFS, and OS for patients that underwent alloHSCT. 10−4 and 10−3 had much longer DoR. This finding However, no difference was observed in transplanted underlines that MRD detection is a surrogate for patients up to month 6 (Supplemental Figure 4). In a disease burden and is consistent with previous time-dependent Cox model adjusting for baseline research showing that progressively higher MRD are factors, similar improvement after alloHSCT was associated with higher relapse rates [5,10]. In patients observed for DoR (HR, 0.43; 95% CI, 0.29–0.64; P with MRD above 10−2, the median time until hematolo- < .0001; Table 4) and RFS (HR, 0.59; 95% CI, 0.41–0.84; gic relapse was weeks (1.6–3.9 months) in this study, P = .003). Transplanted patients also appeared to have indicating that the time window to change treatment longer OS than nontransplanted patients, although strategy (eg, alloHSCT) based on MRD is limited. With these findings were not statistically significant (HR, higher levels of MRD, the outcome even with alloHSCT 0.72; 95% CI, 0.50–1.05; P = .08). is not nearly as beneficial compared to lower levels of MRD or undetectable MRD [5,24]. Patients with MRD reappearance in our study popu- Discussion lation had worse outcomes compared to those with This was an analysis of a large dataset of patients with MRD persistence. Detecting MRD reappearance Ph-negative BCP-ALL and measurable MRD who strongly depends on the frequency of follow-up MRD received standard of care therapy at European aca- testing in patients with complete MRD response. demic study group protocols or at major centers. A Follow-up evaluation to detect MRD reappearance is broad cross-section of patients were included which not routine in many countries and explains the rela- increased robustness and generalizability of these tively low number of patients with MRD reappearance study results. Baseline MRD level appeared to in the studied population. The management of MRD influence DoR, RFS, and OS, demonstrating the prog- reappearance may differ from MRD persistence. nostic impact of MRD and highlighting the importance Whereas MRD persistence is often detected early of incorporating MRD assessment into routine care. after consolidation and patients usually receive contin- Patients with very high baseline MRD (≥10−1) had ued intensive treatment, this is often not the case in very short DoR, whereas those with MRD between MRD reappearance, when patients are off treatment 344 N. GÖKBUGET ET AL. Table 4. Analysis of outcomes for transplanted vs. .0005 .0017 .072 .060 – P nontransplanted patients. Test of alloHSCT DoRa RFSa OSb vs. (Events = (Events = (Events = non-alloHSCT Statistic 164c) 187c) 154c) – – Mantel–Byar Odds ratio 0.49 0.65 0.72 Reference Reference Reference 95% CI 0.30–0.78 0.52–1.45 0.39–0.80 0.61–2.84 0.54–2.38 0.34–1.58 0.90–1.00 (95% CI) (0.33–0.73) (0.47–0.92) (0.51–1.01) P .0004 .017 .059 OS, Months Cox proportional Hazard 0.43 0.59 0.72 – – hazards ratio (95% CI) (0.29–0.64) (0.41–0.84) (0.50–1.05) 0.56 P <.0001 .0031 .085 0.48 0.87 1.31 1.13 0.74 HR a .95 DoR and RFS were adjusted for age, WBC count at diagnosis, and MRD – – 1 1 1 level. b OS was adjusted for age, WBC count at diagnosis, and year of diagnosis. c N = 270 patients were evaluable for events. Events/N 107/196 154/270 75/151 61/108 55/85 24/34 46/72 46/71 38/76 – – 9/15 or in the maintenance phase, which may contribute to the high risk of immediate hematologic relapse [5,11]. Other factors that were associated with outcomes, .0548 .0034 .0068 .0998 included WBC and age at initial ALL diagnosis. High – P WBC at diagnosis is an established factor predicting poorer prognosis [25]. Although the biologic effect behind this clinical factor is not described thus far, it Reference Reference Reference Reference 0.39–0.96 0.50–1.30 0.46–0.88 0.32–1.14 0.29–0.98 0.18–0.65 0.50–1.06 – seems that the adverse impact of elevated WBC 95% CI count at diagnosis is maintained at the time of MRD RFS, Months measurement, as it confers more aggressive disease. – Similarly, in relapsed/refractory ALL, the WBC count at first diagnosis maintains an impact on survival [26]. 0.61 0.81 0.63 0.60 0.53 0.34 0.73 The youngest patients had the longest OS, whereas HR – 1 1 1 1 the oldest patients had the shortest OS, consistent with published literature [10]. Possible explanations are decreased transplant realization rate and lower- Events/N 131/196 150/215 99/151 74/108 61/85 27/34 55/72 13/15 55/71 45/76 34/51 intensity chemotherapy in older patients and increased – Only factors that fulfilled the criteria (P <0.10) were included in the multivariable analysis for each outcome. alloHSCT-related mortality in the middle age group. With no recognized standard of care for MRD-posi- Table 3 . Multivariable analysisa of baseline factors associated with RFS, DoR, and OS. tive ALL during the study period, DoR and RFS .0052 .0018 remained relatively stable over the evaluated time .077 .030 P periods from 2000 to 2014. However, more recently diagnosed patients (2011 or later) appeared to have slightly longer OS compared to patients diagnosed in Reference Reference Reference Reference – 0.37–0.94 0.43–1.17 0.43–0.86 0.28–1.03 0.24–0.84 0.15–0.58 0.44–0.96 earlier years. An increasing proportion of patients 95% CI underwent alloHSCT in general [27] and in this study. DoR, Months This may be due to the fact that the negative impact – of MRD was acknowledged to an increasing extent, leading to a clear indication for alloHSCT, which is 0.59 0.71 0.61 0.54 0.45 0.30 0.65 now part of different guidelines and first-line protocols HR – 1 1 1 1 [18]. Improvements in alloHSCT may have reduced risk of non-relapse mortality for younger patients [28]. It is possible that with more follow-up data, the effect of Events/N 112/196 128/215 89/151 63/108 50/85 25/34 50/72 13/15 50/71 38/76 33/51 – alloHSCT on survival among MRD-positive patients could be even greater than observed. Several different statistical analysis techniques were used to examine the potential role of alloHSCT and WBC count at diagnosis showed a trend toward better outcomes with alloHSCT. Age group at diagnosis Baseline MRD status Year of diagnosis ≥10−2 to < 10−1 ≥10−3 to < 10−2 However, these analyses did not adjust for potential bias Baseline MRD level ≥10−4 to <10−3 Reappearance such as differential patient characteristics for transplant 15–34 years 35–54 years ≥30,000/µL <30,000/µL Persistence ≥55 years (e.g. younger age). In time-varying Cox models, ≥10−1 Variable Mantel–Byar analysis, and Simon–Makuch plots, alloHSCT was a time-dependent covariate and reduced a HEMATOLOGY 345 Figure 3. Simon–Makuch plots of outcomes by alloHSCT status (A) DoR. (B) RFS. (C) OS. bias introduced by different on-study time to alloHSCT. However, this is not the usual situation in clinical prac- Conditional landmark analyses suggested benefit for tice where transplantation is performed earlier. Limit- patients surviving without event until month three. A ations of this method are the definition of an later landmark at month six didn’t show an advantage adequate landmark as patients with an event before for transplanted patients. Patients surviving without the landmark are excluded from the analysis, reducing transplantation until this later time-point may represent total evaluable patients. Furthermore, patients receiving a positive selected patient population, which would a transplant after the landmark are considered nontrans- include patients who do not require a transplant. planted, further attenuating the results. 346 N. GÖKBUGET ET AL. Overall, patients with persistent or recurrent MRD the treatment of >10−3 MRD in ALL. Trials of blinatumo- who underwent alloHSCT achieved a better outcome mab and inotuzumab in relapsed/refractory ALL in terms of DoR and RFS and was assessed by multiple demonstrated that CR often was accompanied by methods. Patients receiving alloHSCT also had longer MRD response [32,33]. OS, but was not statistically significant. A possibility is The primary limitation of this study is that it was ret- that some non-transplanted patients survived after rospective. Each country or study group followed their relapse accounting for the smaller difference in OS. protocol, which specified when to conduct MRD assess- But these events were few and relapse events were ments. Treatments and procedures for relapse were not captured in the DoR and RFS analyses. This study did captured or analyzed in this study and data collection not collect detailed information on transplant donor around alloHSCT was limited. Conditioning regimen, type or non-relapse related mortality. However, the donor type or match can be associated with survival patient population receiving a transplant was relatively [34], but these details were not available. Lack of young and mortality in CR was limited (though higher these data likely biased study results to non-signifi- than that in nontransplanted patients). Also, risk of cance rather than causing the observed effects. The hematologic relapse was higher in nontransplanted use of multiple statistical techniques was necessary patients. Overall benefits of DoR, RFS, and OS favored because no single method is best for analyzing time- patients undergoing a transplant. varying covariates such as alloHSCT. There may be MRD has become a routine part of disease monitor- some uncertainty on the size of the effect of alloHSCT, ing; in prognostic models, MRD is an important factor in but not on the directional improvement of outcomes. guiding the development of new protocols/therapies. This study included patients over a 14-year span. Current guidelines from National Comprehensive Changes in alloHSCT may not be fully realized in this Cancer Network [19] and European Society for study, as the effect of alloHSCT on outcomes was aver- Medical Oncology [18] mandate MRD testing for post- aged over that time span. The major effect was prob- induction follow-up and risk stratification. AlloHSCT is ably the higher realization rate of alloHSCT over the often recommended to improve outcomes of patients years. Finally, the study sample size was modest, with MRD [1,2,5,29]. MRD detection between flow cyto- which limited the power to detect differences with P- metry or RQ-PCR are highly concordant [30], so the values <.05. most readily available standardized assay should be In conclusion, MRD is a strong prognostic factor for used. Persistent MRD has been reported as the only clinical outcomes. This data provides a reference for risk factor predictive of a significant effect of alloHSCT outcomes of adults with MRD-positive BCP-ALL who in first CR [20]. MRD status immediately before sub- receive standard therapies including alloHSCT. sequent alloHSCT was not documented in our study. Achievement of MRD negativity is a goal of the Others have reported, however, that less MRD prior modern treatment strategy, even patients allocated to transplant was associated with improved OS and to alloHSCT consolidation. Use of alloHSCT may RFS [13,21,24]. In addition, many MRD-positive patients improve outcomes in these patients, but there is develop overt hematologic relapse despite continued room for further improvement, possibly use of targeted chemotherapy while alloHSCT is prepared. This is also therapies to enable more MRD-negative alloHSCT. evident from the high relapse rate in MRD-positive Although the patient population in this study was rela- patients in our study. Currently, many new trials are tar- tively young, these conclusions may be even more rel- geting MRD negativity as the primary endpoint. evant for older patients with persistent MRD who have Altogether, these data indicate the need for the worst prognoses, likely due to not having the improved treatments for patients with MRD-positive option to undergo alloHSCT or further intensification ALL. Since patients with persistent MRD demonstrate of chemotherapy. resistance to chemotherapy, new drugs with different Registered at www.clinicaltrials.gov as #NCT02010 mechanisms of action are of interest. A recent trial 931. with the bispecific antibody blinatumomab in MRD- positive BCP-ALL included patients with MRD in CR1 but in one-third of the patients in CR2/CR3. The Disclosure statement median age was 45 years and the MRD level was N.G. has received research funds and honoraria and has above 10−3. In this unfavorable patient population, bli- served on advisory boards, for Amgen, Pfizer, Celgene and natumomab yielded conversion to MRD-negative Novartis. H.D. has received honoraria and/or research among 78% of patients and median survival of 36.5 funding from Amgen, Agios, Seattle Genetics, Celgene, months. Additionally, longer survival was observed Sunesis, Roche, Pfizer, Ambit-Daiichi Sankyo, Shire-Baxalta, Ariad-Incyte, Karyopharm, Abbvie, Novartis, Kite, Otsuka, for patients with complete MRD response compared Celator-Jazz, Astellas, Menarini, Cellectis, Janssen, Immuno- to patients who did not respond [31]. Blinatumomab Gen, and Servier. S.G. has served on advisory boards and was recently approved by the US Food and Drug received honoraria from Amgen. M.B. has served as an Administration and European Medicines Agency for advisor for Amgen and Incyte and on speakers’ bureaus for HEMATOLOGY 347 Amgen, Pfizer and Roche, and received research funding treatment: data from the GMALL 06/99 and 07/03 from Amgen, Affimed, and Regeneron. R.F. has been on advi- trials. Blood. 2007;109(3):910–915. sory boards and speakers bureaus for Amgen, Pfizer, Janssen, [8] van Dongen JJ, Seriu T, Panzer-Grumayer ER, et al. Novartis, AbbVie, Celgene, Sandoz, Celltrion, Roche, Incyte. Prognostic value of minimal residual disease in acute C.K., M.S., J.M.S, and G.Z. are employees and stockholders of lymphoblastic leukaemia in childhood. Lancet. Amgen. G.M. has served as an advisor to Amgen, Ariad/ 1998;352(9142):1731–1738. Incyte, Pfizer, Roche, Celgene, Janssen, and Jazz Pharmaceu- [9] van Dongen JJ, van der Velden VH, Bruggemann M, et al. ticals, on speakers’ bureaus for Novartis, Pfizer, and Minimal residual disease diagnostics in acute lympho- Celgene, and received travel compensation from Daiichi blastic leukemia: need for sensitive, fast, and standar- Sankyo, Roche, and Shire. A.R. received support from dized technologies. Blood. 2015;125(26):3996–4009. Amgen, Pfizer, Novartis, and Celgene for travel, sponsored [10] Berry DA, Zhou S, Higley H, et al. Association of minimal lectures, and advisory board meetings. J.-M.R. has received residual disease with clinical outcome in pediatric and research funds and honoraria, and served on advisory adult acute lymphoblastic leukemia: a meta-analysis. boards, for Amgen, Pfizer, Shire, and Ariad. R.B. received hon- JAMA Oncol. 2017;3(7):e170580. oraria for participation in advisory boards for Amgen, Pfizer, [11] Borowitz MJ, Wood BL, Devidas M, et al. Prognostic sig- Shire, and Incyte. E.P. received support for advisory board nificance of minimal residual disease in high risk B-ALL: meetings from Amgen, Roche and Celgene. M.D., D.H. a report from children’s Oncology group study declare no conflicts of interest. AALL0232. Blood. 2015;126(8):964–971. [12] Bruggemann M, Raff T, Kneba M. Has MRD monitoring superseded other prognostic factors in adult ALL? Funding Blood. 2012;120(23):4470–4481. [13] Sutton R, Shaw PJ, Venn NC, et al. Persistent MRD before Amgen provided funding for this study. and after allogeneic BMT predicts relapse in children with acute lymphoblastic leukaemia. Br J Haematol. 2015;168(3):395–404. ORCID [14] Van der Velden VH, Corral L, Valsecchi MG, et al. Michael Doubek http://orcid.org/0000-0002-1269-6282 Prognostic significance of minimal residual disease in Elena Parovichnikova http://orcid.org/0000-0001-6177- infants with acute lymphoblastic leukemia treated 3566 within the Interfant-99 protocol. 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