Deep-sequencing approach for minimal residual disease detection in acute lymphoblastic leukemia

Abstract

The persistence of minimal residual disease (MRD) during therapy is the strongest adverse prognostic factor in acute lymphoblastic leukemia (ALL). We developed a high-throughput sequencing method that universally amplifies antigen-receptor gene segments and identifies all clonal gene rearrangements (ie, leukemia-specific sequences) at diagnosis, allowing monitoring of disease progression and clonal evolution during therapy. In the present study, the assay specifically detected 1 leukemic cell among greater than 1 million leukocytes in spike-in experiments. We compared this method with the gold-standard MRD assays multiparameter flow cytometry and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) using diagnostic and follow-up samples from 106 patients with ALL. Sequencing detected MRD in all 28 samples shown to be positive by flow cytometry and in 35 of the 36 shown to be positive by ASO-PCR and revealed MRD in 10 and 3 additional samples that were negative by flow cytometry and ASO-PCR, respectively. We conclude that this new method allows monitoring of treatment response in ALL and other lymphoid malignancies with great sensitivity and precision. The www.clinicaltrials.gov identifier number for the Total XV study is NCT00137111.

Figures

Figure 1

Overview of the LymphoSIGHT method. (A) Schematic of the PCR primer strategy and sequencing assay. (B) Schematic of the MRD quantitation scheme.

Figure 2

Technical performance of immune repertoire sequencing assay. Diagnostic samples from 12 ALL patients containing 13 leukemic IgH clonotypes were used for technical performance studies. Serial dilutions of leukemic cells in normal PBMCs, ranging from < 1 in 1 million to > 1 in 1000 tumor cells, were prepared and analyzed in duplicate. The 7 duplicated dilutions were then subjected to amplification and sequencing in 2 replicate experiments. The expected and observed frequencies were compared on a logarithmic scale. Replicate 1 is represented by circles and replicate 2 is represented by crosses in all panels. Panel G shows the results from 2 cancer clones that were present in this tumor.

Figure 3

Clonal evolution mechanisms at the IgH gene locus. Diagnostic samples containing a clonal gene rearrangement, as determined using the VDJ (n = 106) assay were categorized into 5 groups based on the number of evolved clones present in the diagnostic sample. The fraction of all samples having a given number of evolved clones is shown.

Figure 4

Comparison of MRD results obtained by sequencing, flow cytometry, and ASO-PCR. MRD results obtained using the sequencing method were compared with flow cytometry results for 105 ALL patients (left) and with ASO-PCR results for 106 ALL patients (right). In each panel, the numbers of concordant measurements are shown in the lower left and upper right and the number of discordant measurements are shown in the upper left and lower right.

Figure 5

Schematic diagram of sequencing analysis and work flow for prospective sample collection.