Next-generation sequencing–based MRD in adults with ALL undergoing hematopoietic cell transplantation

Measurable residual disease (MRD) monitoring after induction and consolidation and before and after HCT is the standard of care in the management of acute lymphoblastic leukemia (ALL).^1-3 Detection of MRD is associated with an increased risk of relapse and death across subtypes and therapies, including before allogeneic hematopoietic cell transplant (HCT).^4,5 Traditionally, MRD has been clinically defined at a sensitivity of 10^–4 and is detected using either multiparameter flow cytometry or polymerase chain reaction (PCR) assays, with regulatory bodies historically using a conservative estimate of 10^–3 for drug approval indications.⁶ Next-generation sequencing (NGS) of unique immunoglobulin (Ig) and/or T-cell receptor (TCR) clonotypes allows for a routine sensitivity of at least 10^–6 and is now widely available⁷ and increasingly used across the United States to evaluate ALL treatment response. Studies have demonstrated that patients with ALL with MRD-negative (MRD^–) results at 10^–4 via multiparameter flow cytometry but MRD-positive via NGS have an increased risk of relapse compared with patients who test MRD^– via both methods,^8-10 highlighting the enhanced prognostic value of more sensitive NGS-based MRD. In addition, the generally close correlation between peripheral blood (PB) and bone marrow (BM) reported using NGS-based MRD is attractive for noninvasive disease monitoring.^11-13 

In adult patients with ALL undergoing HCT, the clinical significance of NGS-based MRD detected below 10^–4 remains underreported. In a small retrospective study (n = 29) using banked samples from adults with ALL who underwent HCT, Logan et al demonstrated that NGS MRD of the PB or BM at <10^–4 was highly associated with post-HCT outcomes.¹² Data from larger cohorts of children undergoing HCT provide mixed results regarding the prognostic value of MRD <10^–4. For example, a large study of 616 children with ALL who underwent HCT examined the role of quantitative PCR at levels <10^–4 and showed that post-HCT MRD (MRD_post) at any level, including <10^–4, was strongly associated with post-HCT relapse, whereas low levels of pre-HCT MRD (MRD_pre) were less prognostic.¹⁴ In contrast, in another study of 56 children with ALL, both pre- and post-HCT NGS MRD strongly correlated with post-HCT relapse.⁹ Furthermore, the prognostic significance of various clonotypes (eg, IgH vs Igκ/Igλ) detected using NGS MRD remains unknown. For example, the uniqueness of non-IgH sequences may be less unique and, therefore, less prognostic of disease relapse.

To provide additional insights into the potential value of NGS-based MRD in adult ALL, we constructed a large cohort of patients drawn from 2 large US transplant centers and evaluated the association between NGS-based MRD and HCT outcomes. We hypothesized that both detectable MRD_pre and MRD_post would be associated with worse outcomes, even for MRD <10^–4.

This retrospective study included adults (≥18 years) with B-cell or T-cell ALL who underwent HCT at Stanford University or Oregon Health & Science University (OHSU) between January 2014 and April 2021, with planned MRD monitoring using the NGS-based clonoSEQ assay (Adaptive Biotechnologies, Seattle, WA).^7,15 MRD_pre (the MRD sample directly preceding HCT without interval therapy) and MRD_post were obtained from the BM and/or PB and were assessed per the treating physician’s routine clinical practice for up to 1 year after HCT.

We followed up with patients for leukemia relapse and/or death for up to 2 years after HCT. Relapse was defined as morphologic (≥5% blasts in PB or BM) or clinical (initiation of therapy for recurrence of disease as deemed by the treating physician). Leukemia-specific mortality was defined as the time from HCT to death due to relapse or last follow-up; nonrelapse mortality was considered a competing event.

For the primary analyses, MRD positivity was defined as the presence of a detectable IgH clonotype in B-cell ALL or the presence of a detectable TCRβ or TCRγ clonotype in T-cell ALL; conversely, MRD-negativity was defined as the absence of detectable IgH (B-cell) or TCRβ/TCRγ (T-cell) clonotypes that had previously been identified as representing the leukemic blast population. Non-IgH (ie, Igκ/Igλ) clonotypes detected after HCT in B-cell ALL were evaluated only in the exploratory analyses. NGS MRD results were normalized to residual clonal cells per million nucleated cells and characterized as undetectable (0), low (<10^–4), high (≥10^–4 to ≤10^–3), or very high (>10^–3). Because the clonoSEQ B-cell ALL assay has a limit of detection to capture malignancy-associated clonotypes, we considered MRD below the limit of detection to be low-positive. Because most MRD_pre assessments were performed on BM and most MRD_post assessments were performed on PB, when patients had simultaneous samples from both sources, we used the BM result for MRD_pre and the PB result for MRD_post.

Concordance between PB and BM MRD was performed using the Pearson correlation method for patients who had samples from both sources collected within 30 days of each other. Univariable Fine-Gray subdistribution hazard models were used to estimate the effect of MRD_pre level on the cumulative incidences of relapse and leukemia-specific mortality in our cohort; death due to and not due to relapse were competing risks, respectively. Univariable cause-specific Cox proportional hazards models with time-dependent variables were used to evaluate the effect of MRD_post on relapse and leukemia-specific mortality. To analyze the association of both MRD_pre and MRD_post levels with relapse and leukemia-specific mortality, we constructed multivariable cause-specific Cox proportional hazards models, with MRD_pre as a fixed covariate and MRD_post as a time-varying covariate. A multivariable cause-specific Cox proportional hazards model with PB-based MRD_pre as a fixed covariate and PB-based MRD_post as a time-varying covariate was constructed for sensitivity analyses. Exploratory analyses limited to patients with B-cell ALL only described the outcomes of patients with detectable IgH vs non-IgH (Igκ/λ) clonotypes after HCT. Analyses were performed using SAS/STAT software version9.4 (SAS Institute Inc., Cary, NC). This study was approved by the institutional review boards of Stanford University and OHSU and was conducted in accordance with the Declaration of Helsinki.

Among 174 patients with ALL who underwent HCT and had a diagnostic sample sent to Adaptive Biotechnologies for clonality evaluation, 158 (91%) had at least 1 dominant sequence identified for tracking and MRD monitoring, 15 (9%) failed dominant sequence identification because of polyclonality, and 1 (1%) had only a dominant TCRδ clonotype, which was not tracked via the clonoSEQ assay. Of the 15 patients without a dominant sequence, 7 (47%) had B-cell ALL, and 8 (53%) had T-cell ALL. Similar to the rest of our cohort, most patients without a dominant sequence were in first complete remission at the time of the HCT and underwent myeloablative conditioning. Of the patients without a dominant sequence, only 1 patient relapsed during follow-up, 3 patients died of nonleukemia causes, and 12 patients were alive and in remission at the last follow-up.

Of the 158 patients with a trackable clonotype, 122 (77%) underwent MRD_pre assessment, and 139 (88%) underwent ≥1 MRD_post assessment; 111 (70%) underwent both MRD_pre and ≥1 MRD_post assessments and formed the analytic cohort for multivariable survival models (supplemental Figures 1 and 2). In the 11 patients who underwent MRD_pre but not MRD_post assessment, the reasons were death shortly after HCT (n = 9), relocation to a different state (n = 1), and physician decision to monitor MRD with BCR-ABL PCR instead of NGS in Ph⁺ B-cell ALL (n = 1). The source of MRD was equally distributed between PB and BM, with a greater proportion of MRD_pre assessed using BM (69%) and a greater proportion of MRD_post assessed using PB (75%) samples. In 81 patients with 120 paired PB and BM MRD samples, a high concordance was observed between PB and BM MRD (Pearson coefficient = 0.78; P < .001; supplemental Table 1).

The median age of the patients was 44 years (range, 19-70 years), 62 (56%) were male, and 95 (86%) had B-cell ALL. (Table 1). Most (83%) of patients underwent myeloablative HCT, most commonly from matched unrelated donors (39%), and 80 (72%) and 24 (24%) were in first or second complete remission at the time of HCT, respectively. The 2-year incidence of relapse, nonrelapse mortality, and leukemia-specific mortality after HCT was 21%, 12%, and 11%, respectively, and the 2-year overall survival was 77%.

The median time between the MRD_pre assessment and HCT was 24 days (range, 3-124 days). MRD_pre was undetectable, low, high, and very high in 82 (67%), 24 (20%), 11 (9%), and 5 (4%), respectively. The cumulative incidence of post-HCT relapse increased per the MRD_pre level (Figure 1A; Table 2), including an increase in the risk of relapse among patients with low (relative to undetectable) MRD_pre (hazard ratio [HR], 3.56; 95% confidence interval [CI], 1.39-9.15; P = .01). There were no significant differences in leukemia-specific mortality based on the MRD_pre level (Table 2). Given the similarity in relapse risk between patients with low and high MRD_pre, these 2 groups were subsequently combined and defined as “MRD_pre moderate” (<10^–4 to ≤10^–3) in multivariable survival models.

The median time between HCT and the first MRD_post assessment was 32 days (range, 20-337 days), with a median monitoring frequency of every 43 days over the first year after HCT (supplemental Figure 2). MRD_post was detectable via NGS at any level in 48 (33%) patients. Patients with detectable MRD_post had a significantly increased risk of post-HCT relapse (HR, 6.31; 95% CI, 4.56-8.74; P < .0001; Figure 1B) and a significantly increased risk of leukemia-specific mortality (HR, 7.71; 95% CI, 4.86-12.31; P < .0001; Table 2).

The results of the multivariable cause-specific Cox proportional hazards model using MRD_pre as a fixed covariate and MRD_post as a time-varying covariate are shown in Figure 2. We found that after adjusting for MRD_post results, moderate (HR, 1.81; 95% CI, 1.16-2.82; P = .01) and very high (HR, 3.38; 95% CI, 1.84-6.19; P < .0001) MRD_pre remained significantly associated with an increased risk of post-HCT relapse. Similarly, after adjusting for MRD_pre results, detectable MRD_post at any time point remained highly associated with subsequent post-HCT relapse (HR, 4.60; 95% CI, 3.01-7.02; P < .0001). Sensitivity analyses limited to the PB MRD results demonstrated similar associations between MRD_pre, MRD_post, and clinical relapse (supplemental Table 2). Multivariable models evaluating the effect of MRD_pre and MRD_post on leukemia-specific mortality showed a significant association only with detectable MRD_post and leukemia-specific mortality (HR, 9.85; 95% CI, 4.98-19.54; P < .0001).

In contrast to the aforementioned analyses, to explore the potential prognostic significance associated with the detection of non-IgH (ie, Igκ/Igλ) MRD clonotypes in B-cell ALL, we examined the outcomes of patients with B-cell ALL included in our cohort based on MRD_post results, including IgH and non-IgH clonotypes (Figure 3). We found that of the 29 patients with solely IgH MRD clonotypes detected after HCT, 20 (61%) relapsed and only 6 (21%) survived. However, among the 24 patients with only non-IgH (Igκ/λ) MRD clonotypes detected after HCT, only 5 (21%) relapsed and 18 (75%) survived.

In this retrospective, real-world study of 174 adult ALL HCT recipients treated across 2 large transplant centers, we demonstrated the prognostic value of MRD monitoring using highly sensitive NGS technology. We found that detectable MRD_pre at levels as low as <10^–4 were associated with an increased risk of post-HCT relapse. Conversely, patients entering HCT with undetectable MRD_pre and those whose MRD remained undetectable via NGS monitoring for the first year after HCT had a low likelihood of relapsing by 2 years after HCT.

We were particularly interested in the association between very low levels of detectable MRD_pre via NGS and the risk of post-HCT relapse because the clearance of pre-HCT MRD has growing clinical relevance, and it was previously unclear whether elimination of MRD to a level of less than 10^–4 was necessary in the pre-HCT setting. The majority of our patients underwent myeloablative HCT, suggesting that the strength of the conditioning regimen was not sufficient to overcome MRD positivity. The use of blinatumomab to reduce MRD before HCT is becoming increasingly common^3,6 and was recently shown in the ECOG-ACRIN E1910 phase 3 trial to improve overall survival in patients with MRD^– when added to consolidation chemotherapy after intensive induction chemotherapy.¹⁶ Although these results may ultimately reduce the need for HCT in adult ALL, our data suggest that the optimal pre-HCT goal should be undetectable MRD at a level of 10^–6. The use of blinatumomab to clear pretransplant MRD has not been studied in a randomized clinical trial, and it is unlikely that such a trial will ever be undertaken. Interestingly, we did not observe a significant association between detectable MRD_pre and leukemia-specific mortality after HCT. This may be due to the relatively small number of leukemia-related deaths, limited follow-up of 2 years after HCT, and recent findings that after HCT relapse in ALL (particularly B-cell ALL) is more salvageable now than in the past.¹⁷ 

Although the association between MRD_pre and relapse was significant, detectable MRD_post at any level remained highly associated with subsequent relapse and leukemia-specific mortality, regardless of the MRD_pre results. These data should further encourage close monitoring of MRD in the post-HCT setting, regardless of the patient’s MRD_pre results because MRD results are becoming increasingly actionable.¹⁷ However, which of the sequences initially identified in the malignant clone have prognostic significance in the post-HCT setting is important. To our knowledge, we have shown for the first time that the specific MRD clonotype matters: detectable IgH MRD clonotypes after HCT were highly associated with relapse in our primary results, whereas our exploratory findings demonstrated that detectable non-IgH (ie, Igκ/λ) clonotypes in the absence of detectable IgH clonotypes were not clinically significant. This may reflect the fact that Ig light chain sequences are, on average, less “unique” than Ig heavy chain sequences and are, therefore, more likely to have a background level contributed by independent nonmalignant clones in the same patient. The clonoSEQ assay accounts for these differences in clonotype uniqueness by adjusting the limit of detection threshold. Additional larger studies should be conducted to confirm this effect, and post-HCT relapse prevention trials should incorporate NGS MRD as a trigger for subsequent therapy.

Because this was an observational study in which MRD testing was performed in accordance with clinical practice parameters, our patient cohort included MRD obtained from both PB and BM. One advantage of the NGS-based MRD assay is its ability to use PB as a source for MRD monitoring. We previously demonstrated high concordance of MRD results in PB and BM using NGS-based MRD (n = 69 patients; 126 paired samples),¹¹ and, here, we show similar findings using data from patients who underwent transplant at 2 centers (n = 81 patients; 120 paired samples). In this study, most of the MRD_post samples were obtained from PB, thereby strengthening the evidence related to NGS-based PB MRD and outcomes in ALL. Monitoring MRD by using PB is clearly less invasive and more cost-effective and can allow for more frequent monitoring when necessary.

Our study cohort comprised a heterogeneous population consisting of patients with both B-cell and T-cell ALL receiving various conditioning regimens and different transplant sources. However, despite this heterogeneity, we detected strong associations between MRD levels and the risk of relapse. Although of great interest, our a priori study objectives did not include subgroup or multivariable analyses incorporating age, cytogenetics, Ph-like phenotype, immunophenotype, or conditioning regimen, which may have additional clinical utility. In addition, our limited sample size did not allow for post hoc subgroup analyses. Although we did not prospectively assign the frequency or duration of NGS-based MRD monitoring because this was a retrospective analysis, most patients underwent MRD assessment within 45 days of HCT, and the most common monitoring interval was approximately every 1 or 2 months during the first year after HCT. Although most relapses occurred within the first year after HCT, the optimal duration of MRD monitoring for patients at high risk (those with detectable MRD_pre and/or detectable MRD_post) was unknown. In the 12 patients with B-cell ALL with detectable MRD_post but who did not relapse, the reasons included early transplant-related mortality (n = 3), clearance of MRD with onset of graft-versus-host disease (n = 4), initiation of post-HCT tyrosine kinase inhibitor therapy in Ph⁺ B-cell ALL (n = 3), NGS MRD detected only below the limit of detection (n = 1), and relapse after the study cut off (n = 1).

In conclusion, we demonstrated the prognostic value of the highly sensitive NGS-based MRD in adults with ALL undergoing HCT. Our data suggest that MRD clearance to an undetectable level at the 10^–6 or lower threshold is optimal before HCT, and that detectable MRD (specifically, IgH clonotypes in B-cell ALL or TCR clonotypes in T-cell ALL) after HCT via NGS of the PB or BM should raise concerns for impending clinical relapse.

This study was funded by the National Institutes of Health, National Cancer Institute (NCI) grant P01CA049605 and is partially supported by the Biostatistics Shared Resource of the NCI-sponsored Stanford Cancer Institute (P30CA124435).

Contribution: J.T.L. and L.M. supervised and provided the concept and designed the study; E.C.L., S.E.D., J.M.G.S., S.T., A.Z., K.M., J.T.L., and L.M. were responsible for the acquisition, analysis, and interpretation of data; E.C.L. and L.M. drafted the manuscript; A.Z. and K.M. performed the statistical analysis; and all the authors critically reviewed the manuscript.

Conflict-of-interest disclosure: J.T.L. reports research funding from AbbVie and Amgen, and consultancy for Pfizer, Adaptive, Kite, and Takeda. L.M. reports research funding from Adaptive, Astellas, Jasper, Kite, and Bristol Myers Squibb; provides consulting and is a member of advisory boards for Adaptive, Amgen, CTI Biopharma, Kite, Medexus, and Pfizer; and receives honoraria from Adaptive and UpToDate. The remaining authors declare no competing financial interests.

Correspondence: Lori Muffly, 300 Pasteur Dr H0144, Stanford, CA 94305; e-mail: lmuffly@stanford.edu.