Plasma as a source of mRNA for determining IgV<sub>H</sub> mutation status in patients with chronic lymphocytic leukaemia (2006)

The mutation status of the immunoglobulin heavy-chain variable-region (IgVH) gene in the affected cells of patients with chronic lymphocytic leukaemia (CLL) is an important prognostic marker (Damle et al, 1999; Hamblin et al, 1999). Mutated IgVH gene is associated with stable disease and long-survival time, while the unmutated gene is associated with rapid disease progression and short-survival time (Keating et al, 2003). The conventional, cell-based, method of determining IgVH gene mutation status involves extraction, amplification and sequencing of the mRNA expressed by the clonal IgVH gene. In some CLL patients, however (those whose disease resides primarily in the lymph nodes or bone marrow and those who have received therapy), few circulating leucocytes are available for mRNA analysis. In addition, in some cases, the presence of reactive polyclonal plasma cells, which express high levels of IgVH mRNA, makes it difficult to isolate pure clonal IgVH mRNA. We previously demonstrated that, as a result of the high turnover of tumour cells relative to non-neoplastic cells, the blood plasma of patients with haematological malignancies is enriched with tumour-specific RNA, DNA (Rogers et al, 2004) and protein (Manshouri et al, 2003), suggesting that plasma might be a reliable source of tumour-specific IgVH mRNA as compared with cells from peripheral blood or bone marrow, where mixed populations of leukaemic and non-leukaemic lymphocytes or plasma cells could hinder sequencing. Here, we investigated that possibility. We collected EDTA-peripheral blood from 49 CLL patients and 20 healthy volunteers <30 years of age. The age limit was imposed to avoid inadvertently including anyone with smouldering CLL. Forty-one of the patients were selected randomly and eight were selected because mRNA extracted from their circulating white cells failed to yield a discrete IgVH band (<10% of the cells were leukaemic). The randomly selected patients had substantial disease with >40% leukaemic cells. Blood samples were collected according to institutional review board-approved protocols. After separating plasma and cells, we extracted total nucleic acid from 0·5 ml of plasma and mRNA from the white cells in 2 ml of blood. IgVH gene mutation status was analysed by performing one-step reverse transcription-polymerase chain reaction (RT-PCR), purifying, sequencing and analysing the PCR product, and then aligning it with the NCBI IgBlast database (http://ncbi.nlm/igblast/). We defined mutation as 2% deviation from the germline sequence. Paired plasma and peripheral blood samples from each of the 41 randomly selected CLL patients yielded identical sequence results (Fig 1A), and 22 pairs (54%) showed mutations. The plasma from the eight patients whose circulating cells failed to yield a discrete IgVH product band due to paucity of leukaemic cells showed discrete bands, and 5 (68%) showed mutations. Plasma and cell mRNA samples from the randomly selected patients showed a discrete IgVH amplification product band while the control plasma samples showed only smeared IgVH bands (Fig 1B). (Smeared bands represent reactive, rather than clonal, IgVH products). (A) Sequence analysis of mRNA extracted from plasma and circulating white cells from the same patient (example 1) yielded identical results. In contrast, the second example (example 2) showed fully readable sequence from plasma, but unreadable sequence from cells. (B) Amplification of IgVH mRNA yielded similar products for plasma (P) and peripheral blood cells (C) from two patients (patient 1 and patient 2) with CLL. Plasma samples from the control subjects (CS) yielded no IgVH amplification products. The bottom panel shows significant IgVH band from plasma, but not from the peripheral blood sample of patient 3 (M, size marker). These data suggest that plasma is a reliable source of tumour-specific RNA in patients with CLL and can be used as an alternative to cells for determining IgVH gene mutation status. This technique would be especially important for patients with few circulating leukaemic cells.