High-Throughput Sequencing of T-Cell Receptor Gene Loci for Minimal Residual Disease Monitoring in T Lymphoblastic Leukemia

Abstract 2545

There is increasing evidence for the utility of minimal residual disease (MRD) assessment in predicting clinical outcomes of patients with T cell lymphoblastic leukemia (T-ALL). Evaluation of MRD by PCR-based analysis of T-cell receptor (TCR) genes has a sensitivity of 10⁻⁵, but requires the use of individualized patient-specific primers, which is laborious, expensive and difficult to implement for real-time, clinical decision-making. Multi-parametric flow cytometry is currently limited to a sensitivity of 10⁻⁴, requires viable cells, and is poorly standardized. High-throughput DNA sequencing offers the potential to equal or surpass the higher sensitivity of PCR-based MRD testing with reduced cost, improved turn-around time, and better standardization.

Paired samples of pediatric T-ALL from 14 patients enrolled on Children's Oncology Group AALL0434 were obtained at diagnosis and at day 29 post-induction therapy. The complementarity determining regions (CDR3) regions of TCRB and TCRG were sequenced for all 28 specimens using an Illumina GA2 platform as previously described (see Blood, 114(19):4099–4107, 2009 and Sci Transl Med. 3(90):90ra61, 2011). Pre-treatment samples were used to obtain unique TCR sequences for the leukemic clone, and post-treatment samples were assessed for the frequency of each TCR sequence as a percentage of the total. The frequency of each sequence was also enumerated in post-treatment samples from all other patients to evaluate specificity. These results were compared to MRD results obtained by 9-color flow cytometry per trial protocol.

Eleven of 14 pre-treatment samples (78.6%) had a detectable clonal population based on TCRG sequence analysis, and 10 of these also had a clonal TCRB sequence. Five samples exhibited an additional unique TCRG sequence, consistent either with rearrangement of both TCRG loci or the presence of two clonal subpopulations. Two of 3 cases without a detectable clonal TCR gene sequence had the immunophenotype of early thymic precursor (ETP) T-ALL and would be expected to have germline TCRB and TCRG genes. No other cases were ETP. Clones were found in all 5 informative post-treatment samples positive for MRD by flow cytometry, as well as at a low level in 3 additional patients without MRD by flow cytometry, suggesting superior sensitivity for sequencing. The background sequence frequencies were very low (0–10⁻⁵) in other patient post-treatment samples, being slightly higher for TCRG than for TCRB, consistent with germline sequence diversity.

We demonstrate the potential of high-throughput sequencing for analysis of MRD in pediatric T-ALL. The number of cases in which the assay is informative (78.6%) is similar to that seen with standard PCR MRD methods, but evaluation of more cases is needed. MRD by sequencing appears to have a higher sensitivity than current flow cytometric methods, although direct comparison of MRD frequencies from the two techniques is problematic and will require normalization. The strong association of ETP status and lack of clonal TCR sequence identification at diagnosis suggests utility in identifying this poor outcome subset of T-ALL.