Response: Hodgkin lymphoma stem cells

In his letter to the editor, Dr Küppers calls attention to additional support that clonotypic B cells are present in Hodgkin lymphoma (HL) and may represent HL stem cells. We were unaware of Jansen and colleagues' prior work describing such cells in HL lymph nodes,^1,2  and we thank Dr Küppers for pointing out this further evidence. We regret the oversight; we certainly were never trying to imply that our data were the first in this area, as we cited the 1988 paper by Newcom et al³  describing these cells in an HL cell line. However, our description of these cells in the blood, and their potential as targets for CD20-directed therapy, remain novel findings.⁴ 

Dr Küppers then shifts gears and declares that “none of these [data] are convincing. First … the isolated HRS cell population surprisingly gave a polyclonal pattern … .” He is referring to Figure 5C, where the predominant clonal immunoglobulin gene rearrangement in Hodgkin and Reed-Sternberg (HRS) cells is accompanied by a polyclonal background. As Dr Küppers' group showed,⁵  HRS cells isolated by magnetic cell separation columns are highly contaminated with other blood cells.⁴  Dr Küppers further dismisses the clonality demonstrated in the microdissected HRS cells by Vκ light chain rearrangements (Figure 5D) stating that “ … the fragment length analysis of Vκ light chain rearrangements is unsuitable to demonstrate clonal identity, because the V-J joints … show little length variation; 66% of polyclonal VκJκ joints have an identical CDRIII length of 27 bp.” Length analysis of Vκ light chain rearrangements is a standard, commonly used method for the identification of B-cell clonality⁶  and may be more robust than heavy chain rearrangements.⁷  Whereas the kappa light chain V region repertoire is more limited than that of the immunoglobulin heavy chain, our laboratory and others obtain a typical polyclonal distribution of peaks for normal tissues using the methods described. Specifically, the modal peak in our normal controls falls at 148 bp, which differs significantly from the clonal peaks at 146 and 150 bp.

Dr Küppers is correct that the VκJκ sequences in supplemental Figure 3 do not cover the entire CDR3 region. The direct sequencing of the limited HRS cell material available did not provide quality sequence into the Jκ region. However, the Vκ region identified (Vκ1) is relatively infrequently found in HRS cells,⁸  making coincidental rearrangements unlikely. Dr Küppers' suggestion that intraclonal heterogeneity “nearly never” occurs is overstated. In fact, his own group⁹  and others⁸  reported intraclonal heterogeneity of the HRS cells in about 10% of cases. Notably, we sequenced small pools of microdissected HRS cells (400-500) rather than single microdissected cells; this may provide greater ability to detect intraclonal heterogeneity than single-cell microdissection.

We agree with Dr Küppers' final comments that more work on the topic of clonotypic B cells in HL is needed. We hope that our recent paper⁴  and discussions such as these letters to the editor will spark interest in a topic that has largely been ignored or dismissed despite work from several groups^1-3  (eg, before our paper, the report by Newcom et al³  had been cited by others just once in the 21 years since its publication). Better understanding the association of clonotypic B cells and HRS cells in HL may have important implications not only for HL but also perhaps more broadly to the field of cancer stem cells.