Abstract 846: Improving T-cell receptor clonotyping of T-cell lymphomas using hybrid-Capture and next-generation sequencing
Authors: Mahe Etienne , Mulder David , Dowar Mark , Sukhai Mahadeo , Nguyen Linh , Ohashi Pamela , Delabie Jan , Stockley Tracy L. , Pugh Trevor , Kamel-Reid Suzanne 
Published: July 2016

Abstract
Background
T-cell clonality assays (TCAs) support a variety of clinical and research interests including T-cell malignancy clone identification, minimal-residual disease (MRD) testing and T-cell receptor (TCR) repertoire characterization for the purposes of immunotherapy. Unfortunately, many TCAs are unable to interrogate alpha, beta, gamma, and delta TCR loci simultaneously. Aiming to overcome these limitations, we designed a T-cell clonality assay (the “NTRA”), capable of identifying T-cell gene rearrangements from all four TCR loci, using hybrid-capture followed by deep next-generation sequencing (NGS). A novel informatics package exploiting the Burrows Wheeler Alignment algorithm and finding of CDR3 seed sequences was also deployed. We then validated the assay using orthogonal methods and in a series of clinical T-cell malignancy specimens.
Methods
We used DNA probes for hybrid-capture of all V and J segments in the TCR loci, followed by NGS on the Illumina NextSeq 500 platform. Analytical validation used a series of 10 specimens, including 6 flow-cytometry characterized T-cell specimens of variable degrees of immunophenotypic uniformity and 4 cell-lines with known TCR rearrangements. PCR/gel electrophoresis and Sanger sequencing were used for orthogonal validation, using primer sets designed to test each specimen for all 90th-centile V-J configurations. Subsequently, 61 clinical T-cell lymphoma specimens were tested, each previously assessed for T-cell clonality using the clinical gold standard (i.e. PCR/ electrophoresis using the BIOMED-2 consensus primers for the TCR-beta (TRB) and TCR-gamma (TRG) loci).
Results
PCR confirmed the NTRA-identified V-J configurations with an area-under-the-curve (AUC) by receiver-operator characteristic (ROC) analysis of 0.91. Relative to single-strand Sanger sequencing, the NTRA CDR3 sequence results showed a ROC AUC of 0.83. In a DNA dilution series employing the results of a “clonal” specimen (a Jurkat cell line) spiked into a “polyclonal” specimen (a mononuclear peripheral blood specimen), we could identify “clonal” specimen-specific V-J combinations and error-corrected CDR3 sequences down to less than 10⁁-5. In the final clinical validation, the NTRA performed with a ROC AUC of 0.82 relative to the current TRB & TRG BIOMED-2 clinical assay.
Conclusions
Our novel assay can overcome the current TCR locus data-yield limitations resulting from primer-based TCAs in a single-tube. The NTRA shows comparable sensitivity and specificity relative to current standard assays and excellent performance relative to current MRD techniques when applied to clinical T-cell lymphoma specimens.
Citation Format: Etienne Mahe, David Mulder, Mark Dowar, Mahadeo Sukhai, Linh Nguyen, Pamela Ohashi, Jan Delabie, Tracy L. Stockley, Trevor Pugh, Suzanne Kamel-Reid. Improving T-cell receptor clonotyping of T-cell lymphomas using hybrid-Capture and next-generation sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 846.