NGS-Based Immunoglobulin Gene Sequencing for Diagnosis and MRD Monitoring of Multiple Myeloma
Authors: Ren Yuyue , Liu Ming , Fang Jiancheng , Wang Lianjie , Yu Liqian , Xue Yanming , Zhao Weiwei , Liu Juan , Jin Yinglan , Tian Yaoyao , Chen Liyan , Cao Panxiang , Wang Fang , Liu Hongxing , Wang Wei 
Published: December 2024

Multiple myeloma (MM) is a malignant plasma cell disorder characterized by the clonal proliferation of malignant plasma cells in the bone marrow, leading to bone lesions, anemia, hypercalcemia, renal insufficiency, and immunodeficiency. Accurate diagnosis and minimal residual disease (MRD) monitoring are essential for personalized treatment strategies and long-term patient management.

In this study, we aimed to evaluate the application of NGS-based immunoglobulin(IG) gene sequencing for the diagnosis and MRD monitoring of MM patients. We sequenced the IG gene heavy and light chain genes (IGH and IGK/IGL) using RNA-seq and targeted IGH-CDR3-DNA-NGS methods, and performed immunome analysis on the sequencing data using the MiXCR software. A total of 35 newly diagnosed or relapsed MM patients were included in this study, peripheral blood(PB) and bone marrow(BM) samples were collected at the time of diagnosis and during follow-up for NGS-based analysis, a total of 125 samples were analyzed.

Our study results indicate that NGS-based IG gene sequencing offers a high level of sensitivity and specificity in identifying clonal IG gene rearrangements associated with MM. The RNA-seq method, which uses RNA as the substrate, detected clonal IG gene sequences in BM samples with a 100% detection rate (BM 33/33) and in PB samples with a 90.63% detection rate (29/32). In the 28 cases which clonal IGH gene sequences were detected using the RNA-seq method, the targeted IGH-NGS method using DNA as the substrate was able to detect clonal IGH gene sequences in 14/27 (53.85%) BM samples and in 10/25 (40%) PB samples, with sequence information consistent with the RNA-seq results.

Next, we further compared whether there is a difference in the somatic hypermutation(SHM) rate among patients who can detect clonal sequences using the targeted IGH-CDR3-DNA-NGS method. By comparing the measured sequences with the IMGT human germline V gene using the IgBlast tool, we found that the average SHM rate of the IGH V region genes in patients with detectable clonal sequences by targeted IGH-CDR3-DNA-NGS was 7.15%, while the average SHM rate for the IGH V region in negative patients was 9.02%. Our results indicate that the RNA-seq sequencing method using RNA as the substrate has a much higher sensitivity in detecting clonal IG gene sequences than the targeted NGS method using DNA as the substrate.

Additionally, we conducted further analysis of the clonal IG gene sequences expression in MM patients, B-ALL patients, as well as MM cell lines, B-ALL cell lines, and healthy controls. By conducting statistical analysis on the TPM of target genes among several groups, we found that the expression levels of clonal IG target genes in the BM and PB of patients with multiple myeloma were significantly elevated, much higher than those in B-ALL patients and healthy controls. Furthermore, there was a correlation between the expression levels of target sequences in the BM and PB of MM patients.

The reasons for the superior use of the RNA-seq method to monitor the MRD status in MM may include: During the maturation of B cells into plasma cells, they undergo SHM and affinity maturation of antibodies. The IG gene sequences of mature plasma cells exhibit reduced homology with germline genes, leading to a decreased binding capacity for primers of targeted NGS. On the other hand, within plasma cells, the synthesis of IG are extremely active, with the quantity of IG mRNA greatly exceeding that of DNA. As demonstrated by our data, in MM patients, the TPM of clonal IG genes are approximately 10 times higher than those in B-ALL patients. In MM cell lines, the TPM of clonal IG genes are 3 times higher than those in B-ALL cell lines (P&lt;0.05), indicates that the clonal IG RNA quantity of malignant plasma cells is significantly higher than malignant B lymphocytes.

In summary, the use of NGS-based IG gene sequencing in MM patients can provide comprehensive genetic information, which aids in identifying the specific gene expression patterns of tumor cells, thereby facilitating more accurate disease diagnosis and MRD monitoring. The study also highlighted the potential of the RNA-seq method for monitoring the MRD status of MM, which is more sensitive than methods that use DNA as the substrate. The integration of NGS-based IG gene sequencing into routine clinical practice has the potential to transform the management of MM patients, leading to improved outcomes and better quality of life.