Minimal residual disease level predicts outcome in adults with Ph-negative B-precursor acute lymphoblastic leukemia.

ABSTRACT Objectives: Detectable minimal residual disease (MRD) after therapy for acute lymphoblastic leukemia (ALL) is the strongest predictor of hematologic relapse. This study evaluated outcomes of patients with B-cell precursor ALL with MRD of ≥10−4 Methods: Study population was from ALL study groups in Europe managed in national study 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. GRAPHICAL ABSTRACT


Introduction
Outcomes for adult patients with acute lymphoblastic leukemia (ALL) have improved. However, some patients remain resistant to standard chemotherapy. Despite hematologic complete remission (CR) rates of 80-90% with intensive induction/consolidation chemotherapy, approximately 30-50% of adult and 10-20% of pediatric patients at CR1 exhibit minimal residual disease (MRD) [1][2][3][4][5][6][7][8]. MRD is defined as the presence of leukemic cells in patients with hematologic CR below 5%, which is the detection level of microscopy. Several methods for standardized and quantitative measurement of MRD at a sensitivity of 10 −4 (i.e. 0.01%) are available, such as flow cytometry or real-time quantitative polymerase chain reaction (RQ-PCR) of individual clonal rearrangements of immunoglobulin [9].
Most study groups recommend allogeneic hematopoietic stem cell transplant (alloHSCT) as the most intensive treatment approach available for the management of MRD [17][18][19]. Patients who receive alloHSCT have better outcomes compared with those who do not [20]. However, alloHSCT is not always an option due to associated toxicities, lack of suitable donors, or other reasons. Patients may relapse awaiting alloHSCT, particularly those with higher MRD levels. Additionally, patients with detectable MRD before alloHSCT may have a higher risk of relapse after alloHSCT [5,13,21].
The objectives of this study were to estimate the duration of hematologic CR (DoR), hematologic relapse-free survival (RFS), and overall survival (OS) for patients with MRD-positive B-cell precursor (BCP) ALL treated with standard of care in Europe. In addition, several statistical methods were used to closely explore how alloHSCT status may influence clinical outcomes.

Study design
We analyzed a cohort of adults with ALL who had reached CR but had detectable MRD after the standard of care treatment according to national study protocols. The study population was from ALL study groups in Europe (Czech Republic, France, Germany, Italy, Poland, Russia, Spain and the United Kingdom) with protocols that included prospective MRD testing in national reference laboratories. Entry criteria, data collection methods, and analyses were defined in a joint protocol (clinicaltrials.gov identifier: NCT02010931). Ethical approval was obtained according to individual country requirements.
Patient-level data were obtained from clinical databases and entered by central staff of each study group into a study-specific electronic case report form to ensure a standardized and quality-controlled data collection process. Variables included information on demographics, disease characteristics, history of ALL treatment prior to the detection of MRD, diagnostics and level of MRD, details of transplantation for patients who underwent alloHSCT after their MRD detection, and clinical event data including occurrence of hematologic relapse, date of death, or date last known to be alive.

Patient eligibility
Patients were eligible if they had Ph/BCR-ABL-negative BCP ALL in hematologic CR (<5% blasts in bone marrow after ≥3 intensive chemotherapy blocks) and no extramedullary involvement. Other key eligibility criteria were: MRD of ≥10 −4 by RQ-PCR of clonally rearranged immunoglobulin or ≥10 −3 by flow cytometry at a reference lab; age ≥15 years at initial ALL diagnosis; initial ALL diagnosis in the years 2000-2014; availability of data for history of ALL treatment (including response to first therapy and number of prior relapses); and relapse status and disease follow-up after MRD detection. To focus on standard therapies, patients were excluded if exposed to blinatumomab within 18 months of MRD detection. Patients with MRD after prior alloHSCT were excluded.

Statistical analyses
Patients were included in all analyses regardless of whether they received a transplant after MRD detection. The primary outcome, RFS, was defined as the time from baseline MRD detection until hematologic relapse or death due to any cause; patients alive without relapse were censored at their last disease assessment. The secondary outcome, OS, was defined as the time from 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 MRD detection until relapse; patients who died in CR were censored at the date of death. RFS, OS, and DoR were summarized with Kaplan-Meier curves and proportions; point estimates were accompanied by 2sided 95% confidence intervals (CI).
Several methods were employed to explore the effect of receiving alloHSCT after detection of MRD.
(1) Conditional landmark analyses of alloHSCT defined the landmark at 3 months, which was approximately the median time to alloHSCT from baseline (3.4 months; interquartile range, 1.7-6.3); additionally, a landmark at 6 months was assessed which included most patients that underwent a transplant. For each analysis, patients with an event (DoR, RFS, OS) before the defined landmark were excluded, transplant status was assessed at the time of the landmark (3 months and 6 months), wherein a patient was considered to be transplanted only if the transplant was conducted on or before the landmark time. Any patients who underwent a transplant after the landmark were included in the nontransplant group. (2) Mantel-Byar analysis handles alloHSCT as a timedependent covariate. A patient is attributed to the nontransplant group until the time of transplant, once transplanted, is considered part of the transplant group. No patients were excluded from this analysis as in the conditional landmark analysis. This method accounts for time bias observed in fixed-time analyses because patients undergoing alloHSCT must survive long enough to receive alloHSCT [22]. The Simon-Makuch method (with and without a 3-month landmark) was used to graphically display results from the Mantel-Byar test [23]. (3) A multivariate Cox proportional hazards model, adjusting for the baseline factors that were identified in the multivariate Cox model as described above, included alloHSCT as a time-dependent covariate. Statistical significance was measured at P < .05. No adjustment was considered for multiplicity.

Patient population
Data were captured from 287 patients. Analyses of baseline characteristics included 272 patients with quantifiable baseline MRD and excluded 15 patients whose MRD was qualitatively reported to be ≥10 −4 but had non-quantifiable MRD. Of the 272 patients, time-to-event analyses for DoR, RFS, or OS included 270 patients and excluded 2 patients with quantifiable baseline MRD missing the date for MRD.
Patient characteristics are shown in Table 1 and Supplemental Table 1. The median age at diagnosis was 33 years (IQR, . Slightly more than half of the patients were male (58%) and aged <35 years (56%). MRD was assessed by RQ-PCR in most patients (79%) and the baseline MRD was ≥10 −3 in 72%. Most patients (80%) had MRD persistence; 19% had MRD reappearance and 1% had an unknown MRD status. Median time from initial ALL diagnosis to MRD positivity was 4.2 months. Median follow-up was 23.0 months for patients included in time-to-event analyses.

Other analyses
WBC at diagnosis, which was prognostic for RFS, DoR, and OS, was cross-tabulated with other predictors for patient outcomes. Age, sex, baseline MRD, and transplant status were not significantly different between patients with higher (≥30,000/µL) or lower (<30,000/ µL) WBC at diagnosis (Supplemental Table 3). However, patients with a year of diagnosis after 2010 had higher WBC count than those diagnosed in 2004-2010 (P = .02).

Discussion
This was an analysis of a large dataset of patients with Ph-negative BCP-ALL and measurable MRD who received standard of care therapy at European academic study group protocols or at major centers. A broad cross-section of patients were included which increased robustness and generalizability of these study results. Baseline MRD level appeared to influence DoR, RFS, and OS, demonstrating the prognostic impact of MRD and highlighting the importance of incorporating MRD assessment into routine care. Patients with very high baseline MRD (≥10 −1 ) had very short DoR, whereas those with MRD between 10 −4 and 10 −3 had much longer DoR. This finding underlines that MRD detection is a surrogate for disease burden and is consistent with previous research showing that progressively higher MRD are associated with higher relapse rates [5,10]. In patients with MRD above 10 −2 , the median time until hematologic relapse was weeks (1.6-3.9 months) in this study, indicating that the time window to change treatment strategy (eg, alloHSCT) based on MRD is limited. With higher levels of MRD, the outcome even with alloHSCT is not nearly as beneficial compared to lower levels of MRD or undetectable MRD [5,24].
Patients with MRD reappearance in our study population had worse outcomes compared to those with MRD persistence. Detecting MRD reappearance strongly depends on the frequency of follow-up MRD testing in patients with complete MRD response. Follow-up evaluation to detect MRD reappearance is not routine in many countries and explains the relatively low number of patients with MRD reappearance in the studied population. The management of MRD reappearance may differ from MRD persistence. Whereas MRD persistence is often detected early after consolidation and patients usually receive continued intensive treatment, this is often not the case in MRD reappearance, when patients are off treatment 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, included WBC and age at initial ALL diagnosis. High 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 seems that the adverse impact of elevated WBC count at diagnosis is maintained at the time of MRD measurement, as it confers more aggressive disease. Similarly, in relapsed/refractory ALL, the WBC count at first diagnosis maintains an impact on survival [26]. The youngest patients had the longest OS, whereas the oldest patients had the shortest OS, consistent with published literature [10]. Possible explanations are decreased transplant realization rate and lowerintensity chemotherapy in older patients and increased alloHSCT-related mortality in the middle age group.
With no recognized standard of care for MRD-positive ALL during the study period, DoR and RFS remained relatively stable over the evaluated time periods from 2000 to 2014. However, more recently diagnosed patients (2011 or later) appeared to have slightly longer OS compared to patients diagnosed in earlier years. An increasing proportion of patients underwent alloHSCT in general [27] and in this study. 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 now part of different guidelines and first-line protocols [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 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 showed a trend toward better outcomes with alloHSCT. However, these analyses did not adjust for potential bias such as differential patient characteristics for transplant (e.g. younger age). In time-varying Cox models, Mantel-Byar analysis, and Simon-Makuch plots, alloHSCT was a time-dependent covariate and reduced     Overall, patients with persistent or recurrent MRD who underwent alloHSCT achieved a better outcome in terms of DoR and RFS and was assessed by multiple methods. Patients receiving alloHSCT also had longer OS, but was not statistically significant. A possibility is that some non-transplanted patients survived after relapse accounting for the smaller difference in OS. But these events were few and relapse events were captured in the DoR and RFS analyses. This study did not collect detailed information on transplant donor type or non-relapse related mortality. However, the patient population receiving a transplant was relatively young and mortality in CR was limited (though higher than that in nontransplanted patients). Also, risk of hematologic relapse was higher in nontransplanted patients. Overall benefits of DoR, RFS, and OS favored patients undergoing a transplant.
MRD has become a routine part of disease monitoring; in prognostic models, MRD is an important factor in guiding the development of new protocols/therapies. Current guidelines from National Comprehensive Cancer Network [19] and European Society for Medical Oncology [18] mandate MRD testing for postinduction follow-up and risk stratification. AlloHSCT is often recommended to improve outcomes of patients with MRD [1,2,5,29]. MRD detection between flow cytometry or RQ-PCR are highly concordant [30], so the most readily available standardized assay should be used. Persistent MRD has been reported as the only risk factor predictive of a significant effect of alloHSCT in first CR [20]. MRD status immediately before subsequent alloHSCT was not documented in our study. Others have reported, however, that less MRD prior to transplant was associated with improved OS and RFS [13,21,24]. In addition, many MRD-positive patients develop overt hematologic relapse despite continued chemotherapy while alloHSCT is prepared. This is also evident from the high relapse rate in MRD-positive patients in our study. Currently, many new trials are targeting MRD negativity as the primary endpoint.
Altogether, these data indicate the need for improved treatments for patients with MRD-positive ALL. Since patients with persistent MRD demonstrate resistance to chemotherapy, new drugs with different mechanisms of action are of interest. A recent trial with the bispecific antibody blinatumomab in MRDpositive BCP-ALL included patients with MRD in CR1 but in one-third of the patients in CR2/CR3. The median age was 45 years and the MRD level was above 10 −3 . In this unfavorable patient population, blinatumomab yielded conversion to MRD-negative among 78% of patients and median survival of 36.5 months. Additionally, longer survival was observed for patients with complete MRD response compared to patients who did not respond [31]. Blinatumomab was recently approved by the US Food and Drug Administration and European Medicines Agency for the treatment of >10 −3 MRD in ALL. Trials of blinatumomab and inotuzumab in relapsed/refractory ALL demonstrated that CR often was accompanied by MRD response [32,33].
The primary limitation of this study is that it was retrospective. Each country or study group followed their protocol, which specified when to conduct MRD assessments. Treatments and procedures for relapse were not captured or analyzed in this study and data collection around alloHSCT was limited. Conditioning regimen, donor type or match can be associated with survival [34], but these details were not available. Lack of these data likely biased study results to non-significance rather than causing the observed effects. The use of multiple statistical techniques was necessary because no single method is best for analyzing timevarying covariates such as alloHSCT. There may be some uncertainty on the size of the effect of alloHSCT, but not on the directional improvement of outcomes. This study included patients over a 14-year span. Changes in alloHSCT may not be fully realized in this study, as the effect of alloHSCT on outcomes was averaged over that time span. The major effect was probably the higher realization rate of alloHSCT over the years. Finally, the study sample size was modest, which limited the power to detect differences with Pvalues <.05.
In conclusion, MRD is a strong prognostic factor for clinical outcomes. This data provides a reference for outcomes of adults with MRD-positive BCP-ALL who receive standard therapies including alloHSCT. Achievement of MRD negativity is a goal of the modern treatment strategy, even patients allocated to alloHSCT consolidation. Use of alloHSCT may improve outcomes in these patients, but there is room for further improvement, possibly use of targeted therapies to enable more MRD-negative alloHSCT. Although the patient population in this study was relatively young, these conclusions may be even more relevant for older patients with persistent MRD who have the worst prognoses, likely due to not having the option to undergo alloHSCT or further intensification of chemotherapy.