Detection Of Classical Hodgkin Lymphoma In Peripheral Blood Using High-Throughput Sequencing Assay

It was only a little more than a decade ago that the origin of the pathognomonic Reed Sternberg cells of Classical Hodgkin lymphoma (CHL) was established as B-cell malignancy characterized by a clonal expansion (Marafioti et al. Blood 2000). A previous report suggested that clonotypic B cells may be present in the blood of newly diagnosed CHL patients by virtue of identifying some light chain restricted populations by flow cytometry (Jones et al. Blood 2009). However, definitive investigation to assess whether CHL clones are detectable in the blood has been lacking. We developed the LymphoSIGHT™ platform, a high-throughput sequencing-based method, to detect evidence of lymphoid malignancies in peripheral blood samples, as this could potentially be used for detection of minimal residual disease (MRD) after treatment (Faham et al. Blood 2012). This sequencing platform has a sensitivity to detect one lymphoma cell per million leukocytes in peripheral blood. We conducted a pilot study to assess the ability of this platform to detect the lymphoma clone in peripheral blood samples from 13 CHL patients at the time of diagnosis or disease recurrence.

This study was IRB-approved and consent was obtained from all patients. Using universal primer sets, we amplified immunoglobulin heavy chain (IGH) variable, diversity, and joining gene segments from genomic DNA in tumor biopsy and peripheral blood samples (plasma and peripheral blood mononuclear cell [PBMC] compartments) collected at initial diagnosis or at the time of recurrence. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire in the lymph node biopsy sample. The presence of the tumor-specific clonotype was then quantitated in plasma and PBMC compartments from a blood sample obtained around the time of primary tissue biopsy. A quantitative and standardized measure of clone level among all leukocytes in the sample was determined using internal reference DNA.

Clinical and histopathological characteristics are summarized in Table 1. We detected a high-frequency IGH clonal rearrangement in 8 of 13 (62%) lymph node biopsy samples. We observed a trend for a low rate of lymphoma clonotype identification in untreated patients with early stage disease (50%, 4 of 8 patients), and a higher rate of identification in patients with relapsed disease (80%, 4 of 5). In the 8 patients with an identified lymphoma specific clonotype in the biopsy sample, the clonotype was also detected in the plasma and/or PBMC compartment in 7 (88%) patients (Table 1). We detected a lymphoma clonotype more frequently in plasma (88%, 7 of 8) than in PBMC (33%, 2 of 6).

Our data is the first to show that circulating clonal tumor cells can be detected in the blood of patients with CHL, providing new opportunities to explore novel methods to detect minimal residual disease. Additional cases are currently being analyzed to determine the sensitivity and specificity in early stage versus advanced stage disease, and to determine whether the detection of MRD post therapy would correlate with clinical relapse.

Carlton:Sequenta, Inc: Employment, Equity Ownership. Kong:sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.