title: Quantitative polymerase chain reaction analysis with allele-specific oligonucleotide primers for individual IgH VDJ regions to evaluate tumor burden in myeloma patients.
abstract: Quantitative polymerase chain reaction (PCR) with patient-specific, allele-specific oligonucleotide (ASO) primers for individual immunoglobulin H VDJ region (ASO-PCR) amplification was performed using several sources of clinical material, including mRNA from peripheral blood cells (PBMNCs), whole bone marrow cells (BMMNCs), and the CD20+ CD38- B-cell population in bone marrow, as well as cell-free DNA from the sera of patients with multiple myeloma (MM). We designed the ASO primers and produced sufficient PCR fragments to evaluate tumor burden in 20 of 30 bone marrow samples at diagnosis. Polymerase chain reaction amplification efficiency depended on primer sequences because the production of ASO-PCR fragments did not correlate with serum M-protein levels. However, the ASO-PCR levels in BMMNCs showed statistically significant correlations with those in PBMNCs and CD20+ CD38- B-cells. The good association between the BMMNC and PBMNC data indicated that PBMNCs could be a suitable source for monitoring minimal residual disease (MRD). In the case of cell-free DNA, ASO-PCR levels showed a unique pattern and remained high even after treatment. Because the sequence information for each ASO-PCR product was identical to the original, the cell-free DNA might also be useful for evaluating MRD. Moreover, the ASO-PCR products were clearly detected in 17 of 22 mRNA samples from CD20+ CD38- populations, suggesting that MM clones might exist in relatively earlier stages of B cells than in plasma cells. Thus, ASO-PCR analysis using various clinical materials is useful for detecting MRD in MM patients as well as for clarifying MM pathogenesis.
authors.affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Suita, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sata
authors.affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Suita, Japan. Electronic address: hiro@bldon.med.osaka-u.ac.jp.
authors.forename: Hirohiko
authors.initials: H
authors.lastname: Shibayama
authors.affiliation: Central Laboratory for Clinical Investigation, Osaka University Hospital, Suita, Japan.
authors.forename: Ikuhiro
authors.initials: I
authors.lastname: Maeda
authors.affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Suita, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Habuchi
authors.affiliation: Department of Statistical Analysis, Translational Research Informatics Center, Kobe, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Nakatani
authors.affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Suita, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Fukushima
authors.affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Suita, Japan.
authors.forename: Jiro
authors.initials: J
authors.lastname: Fujita
authors.affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Suita, Japan.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Ezoe
authors.affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Suita, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Tadokoro
authors.affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Suita, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Maeda
authors.affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Suita, Japan.
authors.forename: Masao
authors.initials: M
authors.lastname: Mizuki
authors.affiliation: Department of Internal Medicine, Toyonaka Municipal Hospital, Toyonaka, Japan.
authors.forename: Satoru
authors.initials: S
authors.lastname: Kosugi
authors.affiliation: Department of Hematology and Oncology, Nissay Hospital, Osaka, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Nakagawa
authors.affiliation: Department of Internal Medicine, Hyogo Prefectural Nishinomiya Hospital, Nishinomiya, Japan.
authors.forename: Shuji
authors.initials: S
authors.lastname: Ueda
authors.affiliation: Department of Hematology, Kawasaki Hospital, Kobe, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Iida
authors.affiliation: Department of Hematology, Itami City Hospital, Itami, Japan.
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Tokumine
authors.affiliation: Department of Internal Medicine, Minoh City Hospital, Minoh, Japan.
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Azenishi
authors.affiliation: Department of Hematology, Otemae Hospital, Osaka, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Mitsui
authors.affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Suita, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Oritani
authors.affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Suita, Japan.
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Kanakura
mesh_terms: D051997:ADP-ribosyl Cyclase 1 / Q000378:metabolism; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000483:Alleles; D018951:Antigens, CD20 / Q000378:metabolism; D001402:B-Lymphocytes / Q000378:metabolism; D001854:Bone Marrow Cells / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics*; D005260:Female; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009101:Multiple Myeloma / Q000235:genetics* / Q000473:pathology; D018365:Neoplasm, Residual / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D047368:Tumor Burden / Q000235:genetics; D050441:VDJ Exons / Q000235:genetics*
chemical_list: D018951:Antigens, CD20; D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D009841:Oligonucleotides; D051997:ADP-ribosyl Cyclase 1