Comparison of High-Throughput Sequencing and Flow Cytometry for Measuring Minimal Residual Disease in Pediatric Acute Lymphoblastic Leukemia: A Children's Oncology Group Cohort

Measurement of minimal residual disease (MRD) during and after induction therapy has emerged as the most important predictor of outcome in pediatric acute lymphoblastic leukemia (ALL). Despite this, over 1/3 of relapses occur in patients who are MRD negative. In addition, ∼50% of children that have detectable MRD do not relapse. The Children Oncology Group (COG) trials use flow cytometry (FC) with a sensitivity of 10⁻⁴ for MRD detection and subsequent intensification of therapy in MRD+ patients. A more sensitive tool for monitoring MRD could lead to the identification of more patients who are likely to relapse, while a more specific assay could prevent unwarranted therapy intensification. To this end, we are employing the LymphoSIGHT platform developed by Sequenta Inc., which utilizes high-throughput sequencing for identification of clonal gene rearrangements in the B-cell repertoire and subsequent MRD measurement. In this blinded pilot study (COG AALL12B1), we compared the ability of the sequencing assay to measure MRD to that of FC in diagnostic and post-induction samples from 6 ALL patients.

Using universal primer sets, we amplified immunoglobulin heavy chain (IgH@) variable (V), diversity (D), and joining (J) gene segments from genomic DNA in diagnostic and follow-up bone marrow samples from 6 ALL patients. Amplified products were sequenced to obtain >1 million reads per sample and were analyzed using algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire in the diagnostic sample. The presence of the tumor-specific clonotype was then monitored in post-induction samples. Absolute quantification was performed by normalizing the patient's reads to internal reference DNA. We then analyzed concordance between MRD results obtained by sequencing and FC.

We detected a high-frequency IgH clonal rearrangement in 5/6 diagnostic ALL samples. MRD was assessed in the 5 post-induction samples from these patients (Table 1). Deep coverage of all MRD samples was obtained, with each original IgH molecule generating ∼20 sequencing reads, ensuring the detection of a single leukemic cell if present in the sample. Leukemic clones were detected in 4/5 follow-up samples (Table 1). In the positive samples, the number of detected leukemic molecules ranged from 12 to over 6,000 and the MRD level ranged from 0.008% to 0.3%. MRD results were concordant with FC in 3 of 5 patients and were consistent with the patient's clinical courses. In one patient we detected MRD at 0.008%, a level below the sensitivity of FC, which was negative. In another sample, FC detected MRD of 0.01–0.1%, but no leukemic clones were detected by the sequencing assay despite the fact that the sample contained sufficient cell input (almost 2 million cells). The patient remained in continuous remission. Evaluation of additional paired diagnostic and post-induction samples and their association with clinical outcomes is ongoing.

We show the application of a high-throughput sequencing method for MRD detection in childhood ALL. IgH clonal rearrangements were detected in 5/6 (83%) of samples using the sequencing assay. The absence of a clonal rearrangement in 1/6 of patients was anticipated and is likely to be mitigated by the presence of a clonal rearrangement in another immunoglobulin or T cell receptor gene. Experiments are ongoing to assess the presence of clonal rearrangements in these receptors (i.e., IgH D-J, IgK, TRB@, TRD@ or TRG@) in the diagnostic samples. In 3/5 patients there was concordance between FC and sequencing-based MRD detection. In one patient, sequencing detected MRD at a level below the threshold of FC. The last patient was negative by sequencing but positive by FC and has not relapsed. Further analysis of the sensitivity and specificity of the sequencing platform compared to FC using additional paired diagnostic and post-induction samples is ongoing.

Faham:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Zheng:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.