Clinical Meanings of Quantitative PCR of Patient-Specific Immunoglobulin VDJ Regions Using Various Materials of Myeloma Patients
Authors: Sata Hiroshi , Shibayama Hirohiko , Maeda Ikuhiro , Habuchi Yoko , Nakatani Eiji , Fukushima Kentaro , Fujita Jiro , Ezoe Sachiko , Tadokoro Seiji , Maeda Tetsuo , Mizuki Masao , Kosugi Satoru , Nakagawa Masashi , Ueda Shuji , Iida Masato , Tokumine Yukihiro , Mitsui Hideki , Azenishi Yasuhiko , Oritani Kenji , Kanakura Yuzuru 
Published: October 2019

Abstract
Introduction; The PCR with patient-specific, allele-specific oligonucleotide primers for individual IgH VDJ regions (ASO-PCR) is considered the most sensitive method to detect minimal residual disease (MRD) levels in patients with multiple myeloma (MM). In this study, we quantified the ASO-PCR products by using peripheral blood mononuclear cells (PBMNCs) as well as bone marrow mononuclear cells (BMMNCs). We also quantified the ASO-PCR products in mRNAs from CD20+38- B-cells in BM to examine whether there are clonogenic cells in relatively earlier B-cell fraction as well as cell-free DNAs from the sera to examine whether there are DNA fragments from MM cells in PB.
Materials and methods; We have analyzed 30 MM cases. After an informed consent, patient-oriented PCR primers were designed from sequence information of immunoglobulin heavy chain (IgH) variable regions of myeloma cells of each patient.
Results; The median age in this cohort was 64.5 years (36-83); the ratio of men to women was 16:14; and the numbers of IgG-, IgA-, and Bence Jones protein (BJP)- type patients were 18, 7, and 5, respectively. The patient-specific ASO primers could be designed in 25 cases, but not in 3 BJP-type cases or 2 IgG-type cases. We could quantify the ASO-PCR products in 20 of 30 BMMNCs samples at diagnosis. The ASO-PCR levels (IgH/β−actin levels)in BMMNCs correlated with those in PBMNCs, but not to the percent of plasma cells in BM or the values for M-protein. However, the ASO-PCR levels were decreased after the treatment and reflected tumor burden well individually. The ASO-PCR levels in PBMNCs showed a statistically significant correlation with those in BMMNCs at diagnosis (Spearman’s ρ= 0.98, P<0.001), 6 months (Spearman’s ρ= 0.83, P = 0.020) and 12 months (Spearman’s ρ= 1.90, P = 0.001). Therefore, ASO-PCR using PBMNCs as well as BMMNCs is suitable for MRD evaluation. We could detect the patient-specific IgH DNA sequences in cell-free DNA extracted from the sera and quantify the ASO-PCR products. The sequences of the ASO-PCR product were identical to the originally designed sequence, suggesting that detection of the ASO-PCR products in cell-free DNA could reflect the persistence of myeloma cells somewhere in body. The ASO-PCR products for CD20+CD38- B-cells in BM were relatively low but were clearly detected in 17 cases at diagnosis. The ASO-PCR levels in CD20+CD38- B-cells in BM showed good correlation with both values of ASO-PCR in BMMNCs (Spearman’s ρ= 1.35, P <0.001) and in PBMNCs (Spearman’s ρ= 1.09, P <0.001). Thus, the evaluation of relatively earlier B-cell stages of myeloma cells seems to be of interst, including for the possible existence of MM clones in CD20+CD38- B-cell population in BM.
Discussion; Our findings that we could quantify the ASO-PCR products in PBMNCs, BM CD20+CD38- B-cells, cell-free DNA as well as BMMNCs strongly suggest wide sources of clinical materials to analyze. There were statistically significant correlations in values of the ASO-PCR products between BMMNCs and PBMNCs, therefore suggesting the possibility that clonogenic plasma cells or myeloma precursor cells might circulate in peripheral blood. We expect the possibility that PBMNCs will be a good source to monitor MRD. We could also detect and quantify the ASO-PCR products in cell-free DNA from the sera. To the best of our knowledge, this is the first report that IgH DNA fragments from MM cells circulate in the sera. Furthermore, we could detect and quantify the ASO-PCR products in CD20+CD38- B-cells in BM at diagnosis. These results indicated that clonogenic MM cells could exist not only in the CD20-CD38high plasma cell fraction but also in the CD20+CD38- B-cell fraction, which might include myeloma stem or initiating cells. Thus, we could consider the treatment strategies to include anti-CD20 antibodies against the clonogenic CD20+CD38- B-cell population. In conclusion, our ASO-PCR using various clinical materials is supposed to be useful for detecting MRD in the patients with MM as well as for clarifying the pathogenesis of MM.

Disclosures
Sata: Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Shibayama:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Habuchi:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Fukushima:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Fujita:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Ezoe:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Tadokoro:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Maeda:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Mizuki:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Oritani:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Kanakura:Janssen: Research Funding; Takeda: Research Funding; Celgen: Research Funding.