Immunoglobulin and T cell receptor gene high-throughput sequencing quantifies minimal residual disease in acute lymphoblastic leukemia and predicts post-transplantation relapse and survival

Abstract

Minimal residual disease (MRD) quantification is an important predictor of outcome after treatment for acute lymphoblastic leukemia (ALL). Bone marrow ALL burden ≥ 10(-4) after induction predicts subsequent relapse. Likewise, MRD ≥ 10(-4) in bone marrow before initiation of conditioning for allogeneic (allo) hematopoietic cell transplantation (HCT) predicts transplantation failure. Current methods for MRD quantification in ALL are not sufficiently sensitive for use with peripheral blood specimens and have not been broadly implemented in the management of adults with ALL. Consensus-primed immunoglobulin (Ig), T cell receptor (TCR) amplification and high-throughput sequencing (HTS) permit use of a standardized algorithm for all patients and can detect leukemia at 10(-6) or lower. We applied the LymphoSIGHT HTS platform (Sequenta Inc., South San Francisco, CA) to quantification of MRD in 237 samples from 29 adult B cell ALL patients before and after allo-HCT. Using primers for the IGH-VDJ, IGH-DJ, IGK, TCRB, TCRD, and TCRG loci, MRD could be quantified in 93% of patients. Leukemia-associated clonotypes at these loci were identified in 52%, 28%, 10%, 35%, 28%, and 41% of patients, respectively. MRD ≥ 10(-4) before HCT conditioning predicted post-HCT relapse (hazard ratio [HR], 7.7; 95% confidence interval [CI], 2.0 to 30; P = .003). In post-HCT blood samples, MRD ≥10(-6) had 100% positive predictive value for relapse with median lead time of 89 days (HR, 14; 95% CI, 4.7 to 44, P < .0001). The use of HTS-based MRD quantification in adults with ALL offers a standardized approach with sufficient sensitivity to quantify leukemia MRD in peripheral blood. Use of this approach may identify a window for clinical intervention before overt relapse.

Keywords: Acute lymphoblastic leukemia; Allogeneic hematopoietic cell transplantation; High-throughput sequencing; Minimal residual disease; Next-generation sequencing.

Figure 1. Frequency of disease clonotype in diagnostic samples

At least one immunoreceptor clonotype suitable for use in MRD quantification was identified in 93% of patients with ALL. The number of leukemia-associated immunoreceptor locus rearrangements per patient are shown (A). For each of the immunoreceptors tested, the frequency of dominant clonotypes in diagnostic specimens is shown (B). Clonotype frequency greater than 5% (dotted line) was required to identify disease-associated immunoreceptor rearrangements.

Figure 2. Comparison of molecular MRD sensitivity in bone marrow aspirates and peripheral blood

In 37 paired bone marrow (BM) aspirate and peripheral blood (PB) samples, MRD quantification was concordantly negative (0) in 18 (49%). MRD quantification was discordantly positive in BM while negative in PB in 4 (11%), and the median disease burden in these BM samples was 5.5×10⁻⁶ (range 4.5×10⁻⁶ – 1.7×10⁻⁵). When MRD was detected in both sample sources, disease burden was higher (median 8.4-fold) in BM than PB (A). MRD quantification in all samples in this study is demonstrated (B).

Figure 3. Disease-free survival impaired by pre- or post-transplant MRD

ALL disease burden ≥10⁻⁴ during the 30 days prior to transplant strongly predicts likelihood of DFS after transplant (HR 7.7, 95% CI 2.0–30, p=0.003) (A). MRD positivity (≥10⁻⁶) at any time through day +100 post-transplant predicts subsequent relapse (HR 14; 95% CI 4.7 – 44, p<0.0001) (B).

Figure 4. Overall survival and time to event analysis

Patients with persistent MRD negativity experienced significantly higher overall survival after hematopoietic cell transplantation (HCT) than patients with transient MRD negativity and failure to achieve MRD negativity (p<0.0001) (A). The Kaplan-Meier estimate of time from molecular progression to clinical relapse and death for patients who relapsed is shown (B). The median time from molecular progression to relapse was 89 days.