Identification of malignant cells in multiple myeloma bone marrow with immunoglobulin VH gene probes by fluorescent in situ hybridization and flow cytometry.
Because it has been difficult to identify and separate malignant cells in human lymphoid malignancies, we have developed a flow cytometry-based fluorescent in situ hybridization (FISH) technique using immunoglobulin (Ig) heavy chain variable region (VH) gene probes. After obtaining the specific VH gene sequence expressed by the multiple myeloma IM-9 cell line and the malignant cells in five multiple myeloma patients, sense and antisense biotinylated single-stranded RNA probes were prepared by transcription from the malignant clone's VH DNA sequences. The cells from the IM-9 cell line and from the mononuclear bone marrow cells of multiple myeloma patients were fixed, hybridized with the above biotinylated RNA probes, incubated with streptavidin-phycoerythrin, and analyzed by FACS analysis. The myeloma cells stained positive with their own specific antisense VH biotinylated RNa probes, whereas sense and irrelevant antisense biotinylated probes demonstrated only background staining. Dilutional concentrations of the IM-9 cell line with normal bone marrow cells were also accurately quantitated by this procedure. The application of this technique will allow a more accurate assessment of tumor burden in patients with multiple myeloma and should permit an accurate method of tumor cell purification for clinical as well as biological studies. Furthermore, this technological advance should be equally effective at identifying specific VH gene-expressing cells in other lymphoid malignancies, as well as in nonmalignant B cell disorders.
Cao
J
J
Division of Hematology/Oncology, Department of Veterans Affairs, Los Angeles, California 90073.
Vescio
R A
RA
Hong
C H
CH
Kim
A
A
Lichtenstein
A K
AK
Berenson
J R
JR
D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D001853:Bone Marrow / Q000473:pathology*; D005434:Flow Cytometry / Q000379:methods*; D005803:Genes, Immunoglobulin / Q000235:genetics*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000473:pathology*; D010799:Phycoerythrin; D016133:Polymerase Chain Reaction; D015347:RNA Probes; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D019809:Streptavidin
D001426:Bacterial Proteins; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D015347:RNA Probes; D016372:RNA, Antisense; D012333:RNA, Messenger; D010799:Phycoerythrin; D001710:Biotin; D019809:Streptavidin