The mutational status of the immunoglobulin heavy-chain variable-region (IgVH) gene in the leukemic cells of patients with chronic lymphocytic leukemia (CLL) is an important prognostic marker. Lack of IgVH mutation is associated with rapid disease progression and shorter survival. The assay used to determine IgVH mutation status requires specific amplification of the mRNA of the expressed clonal IgVH gene in CLL. However, in some patients with CLL or lymphocytic lymphoma, the bulk of the disease resides in the lymph nodes or bone marrow, with few circulating leukemic cells. In addition, contamination of leukemic clonal cells by reactive polyclonal plasma cells can cause difficulty in obtaining homogenous IgVH mRNA for sequencing, and may therefore lead to failed sequencing or sequencing of the wrong IgVH mRNA. We have reported that plasma is enriched with leukemia-specific DNA, RNA, and protein because of the high turnover of leukemic cells relative to non-neoplastic cells. We thus reasoned that plasma might be a more reliable source of IgVH mRNA than cells from peripheral blood or bone marrow, where mixed populations of leukemic cells and non-neoplastic lymphocytes or plasma cells could hinder sequencing. We tested the plasma from 8 patients in whom routine (cell-based) testing for IgVH mutation status failed to yield results due to a paucity of leukemic cells (<10% of total cells). The plasma of all the 8 samples showed unique, easily defined amplification products of the IgVH mRNA. Sequencing of these products showed the presence of mutated IgVH in 5 patients and unmutated IgVH in 3. Testing paired plasma and cell samples from 19 patients with CLL showed identical mutation rates and family types of the expressed IgVH gene. In contrast, plasma from 20 normal individuals showed no IgVH amplification products that could be sequenced. This data suggest that plasma can be used as an alternative to cells for testing IgVH mutation status. More importantly, plasma is more reliable when few leukemic cells are in circulation and the bulk of the tumor is in the bone marrow or lymph nodes.

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