Next-Generation Sequencing of Immunoglobulin Heavy Chain Variable Region in Diagnostic Samples of Pediatric Acute Lymphoblastic Leukemia Identifies Hundreds of Clonal Subpopulations with Multiple Immunophenotypes

The use of flow cytometry or real-time PCR-based methods to detect minimal residual disease (MRD) in children with Acute Lymphoblastic Leukemia (ALL) is a powerful tool for risk-adapted therapy stratification. However, current protocols for MRD detection incorrectly anticipate leukemia-free survival in 20–30% of low and intermediate risk patients. In this study, we present a new method for MRD detection using next-generation sequencing of the variable region of the immunoglobulin heavy chain (IgH) gene that can overcome two important limitations of current approaches as: (1) it detects lower levels of leukemia cells and (2) it identifies multiple evolved clones.

To capture IgH sequences, we developed a set of multiplexed primers that allow the amplification of all known alleles of each V and J segment. We optimized the protocol to minimize amplification bias between primers. The products were then sequenced using the Illumina platform to obtain >1 million reads per sample. Using a streamlined algorithm, the data were used to calculate the frequency of clonotypes in each sample in a very sensitive and specific manner. Serial dilution experiments have also shown that this technology has a sensitivity of 0.0001%, or about 2 orders of magnitude better than flow cytometry.

To establish the ability to identify the leukemic clone, as well as the frequency of evolved clonotypes at diagnosis, we performed a pilot study with 24 diagnostic bone marrow samples (standard risk n=17, high risk n=6, very high risk n=1) from children with ALL. In these samples, single high-frequency clones were identified in 6 samples and multiple high-frequency clones were detected in 12 patients. Thus, we identified high-frequency clones in 75% (18/24) of samples. This is in agreement with previously published reports of PCR methods for VJ amplification. Next we compared the sequences of samples with multiple high-frequency clonotypes and found that, in all cases, the evolution was consistent with the previously described mechanism of V replacement. The evolved clones shared the same J segment allele, the number of bases deleted in the J segment, and at least part of the NDN sequence. Interestingly, in one patient there were 6 clonotypes with a frequency >1%. We then assessed whether the unique sequences present in these 6 clonal populations appeared in other lower frequency clonotypes from the same patient. We identified hundreds of distinct but related clones, consistent with active ongoing evolution of the leukemia. To further validate that the identified clonotypes in this patient were indeed leukemic, we sorted out the normal and malignant B cells, followed by repeat IgH sequencing. All the sequences suspected to have arisen by V replacement were enriched in the leukemic population and virtually absent from normal B cells. Finally, we sought to assess whether the evolved clones could have variable expression of surface markers used for MRD detection. In one patient, the different evolved clonotypes had variable CD38 expression based on IgH sequencing of blasts sorted by CD38. Further characterization of these clonotypes may reveal distinct underlying biology, as well as differing propensities for relapse. To further assess the associations between IgH sequence and immunophenotype, we are performing flow cytometry and sequencing of sorted subpopulations in additional diagnostic ALL samples (n=50).

These findings suggest that this new technology may offer superior sensitivity and specificity for MRD detection, as well as more accurate stratification for risk-adapted therapies in children with ALL.

Faham:Sequenta Inc: Employment, Equity Ownership. Willis:Sequenta Inc: Employment, Equity Ownership. Moorhead:Sequenta Inc.: Employment. Carlton:Sequenta Inc.: Employment. Zheng:Sequenta Inc.: Employment. Klinger:Sequenta Inc.: Employment.