Abstract 4737: Comprehensive profiling of immunoglobulin sequences using hybrid capture-based next generation sequencing in B-cell hematologic malignancies
Authors: Nahas Michelle K. , Young Lauren E. , Gardner Jeff , Abdel-Wahab Omar , He Jie , Donahue Amy L. , Knapp Kristina M. , Otto Geoff A. , Lipson Doron , Miller Vincent A. , Levine Ross L. , Stephens Philip J. 
Published: August 2015

Background:

Sequencing the genes encoding immunoglobulins is critical in detecting clonal cell populations as well as determining prognosis and therapeutic decisions in patients with lymphoid malignancies including B-cell leukemias, lymphomas and multiple myeloma. Current assays for identifying the rearranged immunoglobulin sequence in B-cell malignancies rely on sequence specific PCR based amplification of conserved immunoglobulin (IG) regions. We have developed a novel, hybrid capture-based approach to sequencing the immunoglobulin chains that enables identification of the heavy and light chain variable domains, complementarity-determining region (CDR) sequences and somatic hypermutation (SHM) status.

Method:

RNA and DNA were successfully extracted from 60 specimens, including 7 mantle cell lymphoma cell (MCL) lines and 53 clinical chronic lymphocytic leukemia (CLL) bone marrow aspirates. Adaptor-ligated DNA and cDNA sequencing libraries were captured by solution hybridization using custom bait-sets targeting the immunoglobulin variable, joining and class segments. All captured libraries were sequenced to high depth (Illumina HiSeq) in a CLIA-certified laboratory (Foundation Medicine).

Results:

The capture-based approach was validated using 7 MCL cell lines and 53 CLL samples profiled using a CLIA-certified commercial PCR-based assay (Invivoscribe). The immunoglobulin sequences derived from the 7 MCL cell lines were 100% concordant with identifying the published heavy and light chain variable domain and percent SHM. Comparison to 53 previously clinically tested samples showed 98% (52/53) concordance for identifying the presence of a clonal population, and 100% (39/39) concordance for identifying the IGHV domain. Comparison to 50 CLL samples previously tested for SHM resulted in 96% (48/50) overall concordance with 94% (31/33) concordant for no SHM and 100% (17/17) concordant for the presence of SHM. Additionally, secondary clones were identified in 11 samples.

Conclusions:

We have demonstrated that hybrid capture-based targeted DNA and RNA sequencing can be used to comprehensively characterize the immunoglobulin sequence of clonal tumor B cell populations. This capability enables quantification of SHM and identification of the variable domain, CDR3 sequence and class restriction. Integration of this methodology with comprehensive genomic profiling approaches will expand the clinical utility of such assays in patients with hematological malignancies and may provide important insights in immune oncology and response of patients to immunotherapies, including patients with solid tumors.

Citation Format: Michelle K. Nahas, Lauren E. Young, Jeff Gardner, Omar Abdel-Wahab, Jie He, Amy L. Donahue, Kristina M. Knapp, Geoff A. Otto, Doron Lipson, Vincent A. Miller, Ross L. Levine, Philip J. Stephens. Comprehensive profiling of immunoglobulin sequences using hybrid capture-based next generation sequencing in B-cell hematologic malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4737. doi:10.1158/1538-7445.AM2015-4737