Idiotypic oligonucleotide probes to detect myeloma cells by mRNA in situ hybridization

Authors


Dr Ross Brown, Haematology Department, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia.

Abstract

An mRNA in situ hybridization (ISH) method which used non-radioactive idiotypic oligonucleotide probes has been used to detect malignant cells in the bone marrow and peripheral blood of patients with multiple myeloma. For each of two patients with multiple myeloma a pair of biotinylated antisense oligonucleotide probes (18–22mer) was prepared from non-germline sequences of the rearranged immunoglobulin heavy chain (IgH) gene. These oligonucleotide sequences were not homologous with any previously published sequence. The probes from each patient were specific as shown by a failure to hybridize to any cells from six other myeloma patients and four normal individuals. Specific staining of IgH gene mRNA occurred only when the myeloma cells and the sequence of the probe used were from the same patient. Using simultaneous fluorescent immunocytochemistry it was shown that more than 95% of the ISH-positive cells expressed the malignant light chain in their cytoplasm. ISH positive cells were found in 1–4% of the peripheral blood mononuclear fraction of these two patients. These studies show that idiotypic oligonucleotide IgH gene probes can be used to identify individual cells belonging to the malignant clone and offer the possibility of developing innovative tumour-specific therapeutic procedures using antisense technology for patients with myeloma.

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