Analytical evaluation of the clonoSEQ Assay for establishing measurable (minimal) residual disease in acute lymphoblastic leukemia, chronic lymphocytic leukemia, and multiple myeloma

Abstract

Background: The clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines.

Methods: Sensitivity and specificity were established by defining the limit of detection (LoD), limit of quantitation (LoQ) and limit of blank (LoB) in genomic DNA (gDNA) from 66 patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and three cell lines. Healthy donor gDNA was used as a diluent to contrive samples with specific DNA masses and malignant-cell frequencies. Precision was validated using a range of samples contrived from patient gDNA, healthy donor gDNA, and 9 cell lines to generate measurable residual disease (MRD) frequencies spanning clinically relevant thresholds. Linearity was determined using samples contrived from cell line gDNA spiked into healthy gDNA to generate 11 MRD frequencies for each DNA input, then confirmed using clinical samples. Quantitation accuracy was assessed by (1) comparing clonoSEQ and multiparametric flow cytometry (mpFC) measurements of ALL and MM cell lines diluted in healthy mononuclear cells, and (2) analyzing precision study data for bias between clonoSEQ MRD results in diluted gDNA and those expected from mpFC based on original, undiluted samples. Repeatability of nucleotide base calls was assessed via the assay's ability to recover malignant clonotype sequences across several replicates, process features, and MRD levels.

Results: LoD and LoQ were estimated at 1.903 cells and 2.390 malignant cells, respectively. LoB was zero in healthy donor gDNA. Precision ranged from 18% CV (coefficient of variation) at higher DNA inputs to 68% CV near the LoD. Variance component analysis showed MRD results were robust, with expected laboratory process variations contributing ≤3% CV. Linearity and accuracy were demonstrated for each disease across orders of magnitude of clonal frequencies. Nucleotide sequence error rates were extremely low.

Conclusions: These studies validate the analytical performance of the clonoSEQ Assay and demonstrate its potential as a highly sensitive diagnostic tool for selected lymphoid malignancies.

Keywords: Acute lymphoblastic leukemia; Analytical validation; Chronic lymphocytic leukemia; Leukemia; Lymphoma; Measurable residual disease; Minimal residual disease; Multiple myeloma; Myeloma; Next-generation sequencing.

Fig. 1

The clonoSEQ Assay Processg: DNA is extracted from the patient sample, and the CDR3 regions of B- and T-cell receptors are subject to multiplexPCR to amplify their unique VDJ or VJ sequences. Amplified DNA undergoes a second round of PCR to add index sequences to prepare for NGS, which is performed via synthesis. The resulting sequences are processed by bioinformatics software to ensure accuracy of results

Fig. 2

Preparation of total gDNA input samples for precision analysis and MRD frequencies used in Linearity testing. Frequencies are presented parenthetically; sample names are presented below the boxes; pre-dilution malignant cell concentrations were determined by mpFC and/or immunohistochemistry. Abbreviations for image: BM bone marrow, BMA bone marrow aspirate, gDNA genomic DNA, mc malignant cells, OPA overall percent agreement

Fig. 3

Precision of the clonoSEQ Assay as a function of input cancer cellsThe red dashed line is at 70%, which is the total error threshold used to define the LOQ of the clonoSEQ Assay.Abbreviations for image: ALL acute lymphoblastic leukemia, CLL chronic lymphocytic leukemia, MM multiple myeloma

Fig. 4

Linearity plots for the clonoSEQ Assay by gDNA input level and disease(ALL, CLL, and MM) Abbreviations for image: ALL acute lymphoblastic leukemia, CLL chronic lymphocytic leukemia, MM multiple myeloma, MRD minimal residual disease

Fig. 5

Pairwise comparison of MRD frequency measurements from multiparametric flow cytometry (mpFC; x-axis) and the clonoSEQ Assay (y-axis) for ALL and MM. R = 0.98 Abbreviations for image: Flow, mpFC.