Peter Cole, MD, Rutgers Cancer Institute of New Jersey Embrace Kids Foundation Endowed Chair in Pediatric Hematology/Oncology and Chief of the Division of Pediatric Hematology/Oncology, also affiliated with Robert Wood Johnson University Hospital speaks about the ASH 2021 Abstract – 3485 Performance of Next Generation Sequencing for Minimal Residual Disease Detection for Pediatric Patients with Acute Lymphoblastic Leukemia: Results from the Prospective Clinical Trial DFCI 16-001.
Link to Abstract:
https://ash.confex.com/ash/2021/webprogram/Paper152369.html
Overview:
In juvenile acute lymphoblastic leukemia, a sensitive and early assessment of minimal residual disease (MRD) is critical for risk classification (ALL). To detect MRD at the level of 1 leukemic cell in a million cells, next generation sequencing (NGS) tests use unique genomic sequences formed by VDJ rearrangements in leukemia cells (Wood et al., 2018). We present our findings from the Dana Farber Cancer Institute (DFCI) ALL Consortium Protocol 16-001, in which we used NGS MRD to assign risk groups to children and adolescents with newly diagnosed ALL.
Methodologies:
Patients with B- or T-ALL aged 1 to 21 years were eligible for enrollment from 8 locations across the United States and Canada. Age, presenting leukocyte count, central nervous system (CNS) leukemia status, immunophenotype, and disease biology were used to determine the initial risk status (Table 1). All patients had their bone marrow assessed at the time of diagnosis and again four weeks later (Induction 1a, timepoint 1 (TP1)), with samples analyzed by flow cytometry (FCM) and next-generation sequencing (NGS). NGS was primarily utilized to determine MRD-based risk, with FCM serving as a backup test. Patients with a high TP1 MRD (10-4) received more aggressive treatment and had additional MRD examinations at 10 and 20 weeks.
For seven of the eight sites, multiparametric FCM was carried out in compliance with local CLIA approved lab practices. FCM was centralized at one of the sites. The commercially available assay ClonoSEQ® was used to test NGS MRD at Adaptive Biotechnologies Corporation in Seattle, WA. Clonality was assessed using the maximum sequence for MRD determination at the immunoglobulin (Ig) heavy and light chain (IgH and IgL) and T cell receptor beta and gamma (TCR-B and TCR-G) loci.
Findings:
MRD can be assessed using next-generation sequencing (NGS).
This study comprised 317 patients who were enrolled on 16-001 between 2017 and 2020. NGS identified unique trackable sequences in 98 percent of the participants (N=310). 57 percent of the 7 pts lacking trackable sequences had early T precursor (ETP) T-ALL (36 percent of all ETP pts tested). All non-ETP T-ALL pts (N=40) and 99 percent of B-ALL pts (N=263) had trackable sequences discovered by NGS.
The locus that was used to determine MRD was
A median of 5 trackable sequences (range 0-14) were found in B-ALL patients, with 92 percent having at least one IgH and 64 percent having at least one TCR-G. In 44 percent (N=115) of B-ALL patients, the IGH locus determined the highest MRD value at TP1, while TCR-G determined the highest MRD value in 41 percent (N=109). In 15% of the cases (N=39), the IgL or TCR-B locus produced the highest TP1 MRD value. T-ALL patients, on the other hand, had fewer trackable sequences, with a median of three (range 0-8). While 28 percent (N=13) had at least one Ig sequence, virtually all (98 percent, N=46) employed the TCR locus for MRD identification, with 94 percent employing TCR-G.
NGS and FCM MRD findings compared
Figures 1a-d show NGS and FCM MRD values for 309 pts with results from both tests at TP1. For patients with detectable illness by both NGS and FCM, the correlation between the two modalities was high (Pearson r=0.87, p0.0001). MRD in the range of 10-6 to 10-4 was also found by NGS in 160 individuals with FCM undetectable disease at TP1, accounting for 50% of our population. Fifty-one points (17%) showed a high NGS MRD (104), but a low (8%) or undetectable (92%) FCM MRD (104), accounting for 50% of the pts classed as high MRD at TP1.
When FCM was low (10-4, N=4) or undetectable (N=26), 43 percent (N=30) of B-ALL pts with high MRD (N=70) had high NGS (10-4) when FCM was low (10-4, N=4) or undetectable (N=26), with 90 percent of differences at the NGS level of 10-4. (Figure 1a-b). In contrast, 75 percent (N=21) of T-ALL pts with high TP1 MRD (N=28) were high by NGS alone, all with undetectable FCM. Sixty-seven percent (N=14) of these patients had NGS MRD of 10-4, whereas the remaining 33 percent (N=7) had NGS MRD of 10-3 to 10-1. (Figure 1c-d).
When FCM was above the threshold of 10-4, eight points, all with B-ALL, had low NGS MRD. One patient had undetectable NGS MRD, whereas the other seven exhibited NGS MRD ranging from 10-6 to 10-4.
Outcomes:
The use of an NGS MRD assay to deliver risk-adapted therapy for newly diagnosed pediatric patients with ALL is possible, with evaluable MRD for 98 percent of children in our cohort. NGS found more cases with high MRD than FCM, with the bulk of discrepant cases barely beyond the FCM detection limit (10-4). For both B-ALL and T-ALL, NGS exhibited better resolution in the region of 10-6 to 10-4. Longer follow-up is needed to determine the prognostic significance of these low MRD levels.