Lymphocyte predominance Hodgkin's disease: lineage and clonality determination using a single-cell assay

Abstract

Lymphocyte predominance Hodgkin's disease (LPHD) is a clinically indolent condition. Although there is evidence that the putative neoplastic cell in this disease, the “L&H” cell, is of B-cell lineage, there is conflicting data concerning the clonality of these cells. Our study was aimed at clarifying the issue of lineage and clonality of the L&H cells of LPHD using a single-cell assay. Four cases of LPHD were studied. To circumvent the difficulties of obtaining fresh tissue and to be able to study representative cases, a new method was developed to obtain single-cell suspensions of L&H cells from archival formalin- fixed paraffin-embedded tissue. Single L&H cells were identified by morphology and immunostaining for epithelial membrane antigen, isolated using a micropipette, and subjected to polymerase chain reaction (PCR) amplification of the complematarity determining region 3 (CDR3) of the Ig heavy chain (IgH) gene, which is B-cell clone-specific. The PCR products were size-fractionated by polyacrylamide gel electrophoresis and representative products were directly sequenced. Single T cells and small B cells were also isolated from the tissues and used as negative and positive controls, respectively. In all four cases of LPHD, the IgH CDR3 of single L&H cells could be amplified. Within each case, the IgH CDR3 of single L&H cells was found to be of different length or of different sequence. Therefore, our results provide strong evidence for the B-cell origin of the L&H cells and the polyclonal nature of LPHD.

Polymerase chain reaction on cerebrospinal fluid cells in the detection of leptomeningeal involvement by B‐cell lymphoma and leukaemia: a novel strategy and its implications

“…Detection of a positive signal became stochastic at a concentration of < 10 pg/PCR sample. These results were in agreement with the findings that using this technique we and the others have been capable of amplifying a single copy of an IgH rearranged gene from one B-cell as is the case in single-cell PCR methodology (Delabie et al, 1994;and unpublished observations).…”

“…In our study the widely used snPCR assay in haemolymphatic pathology for the detection of IgH gene rearrangement (Ramasamy et al, 1992;Pan et al, 1994;Al Saati et al, 1997) was applied to CSF samples. This technique has been revealed to be sufficiently sensitive to produce a detectable band from a single B cell (Delabie et al, 1994;present study). Thereby, in the absence of a polyclonal cell population, a band from a single B lymphocyte could be misinterpreted as a clonal population.…”

Primary low‐grade MALT lymphoma of the gallbladder

“…The primers for IgH were: VHa 5¢-CTGTCGACACGGCCGTGT ATTACTG-3¢, JHa 5¢-ACCTGAGGAGACGGAGAC-3¢, and JHb 5¢-ACCACGGGTCCCTTGGCCCCA-3¢, which have been described previously. 15 Nested PCR was assayed twice; first using the primer set of VHa and JHa and second using the primer set of VHa and JHb.…”

Nodular Lymphocyte Predominant Hodgkin Lymphoma

“…Single-cell polymerase chain reaction assays demonstrate that LP cells typically contain rearranged immunoglobulin (Ig) genes and variably express Ig mRNA [13,[43][44][45][46][47][48][49]. The Ig heavy chains show evidence of somatic hypermutation consistent with the cells' GC derivation [43].…”