Introduction:For improving outcomes of patients in early treatment, diverse detections were developed for prognosis prediction. Multiple Myeloma (MM) patients should be observed during clinical therapy for better outcomes. Combined with next-generation sequencing (NGS), Immunoglobulin (Ig) repertoire has been used for predict patient's prognosis in malignant hematological diseases previously. However, in MM, the study of immune genes landscape was rare. For T cell receptor (TCR) diversity, which was previously proved to have the potential to indicate prognosis in MM patients' overall survival time (OS). In this study, we performed correlative genomic DNA (gDNA) sequencing studies on MM patients for the immuno-gene landscape description and the prognosis association. Additionally, we introduced TCR diversity as the biomarker for NDMM patients' classification and prognosis.

Methods:Bone marrow (BM) and peripheral blood (PB) samples from patients and health donor (HD) were collected. Using CD138 beads to sort malignant plasma cells in BM, and extracting gDNA to construct libraries for NGS. The DNA samples were subjected to a two-step multiplex PCR amplification process. Firstly, we employed primers that specifically targeting complementarity-determining region (CDR3) of the Ig genes as well as same regions of TCR genes. Secondly, we incorporated a unique barcode sequence and Illumina adapter to each amplified product. Subsequently, all libraries were sequenced using an Illumina NovaSeq X Plus system.

Results:Firstly, NGS of Ig repertoire was used to identify tumor clonotypes and build the Ig genes landscape, in this study, 274 dominant clonotypes were identified in 79 MM patients. Patients then were classified into CR, VGPR, and other group. It's suggested patients possessed the high proportion of IGK clones before therapy with low frequency, are more likely to have better outcomes, and vice versa. Then, we conducted analysis on patients' progression-free survival (PFS) and OS. The results showed patients would have better clinical outcomes when the number of chains of the major tumor clone is 1 or 2, compared with 3 or 4 chains of the major tumor clone. After that, we applied the TCRβ sequencing, the Shannon, HVJ, and clonality diversity were used to evaluate TCR clone. It was found that the TRB diversity in PB of MM patients differs from that of HDs. Subsequently, Shannon, HVJ, and clonality were combined into the new parameter. A multivariate regression equation was used to optimize it, resulting in the formula: diversity = 0.27clonality + 0.66HVJ - 0.31*Shannon. This parameter can effectively distinguish between MM patients and HDs, with an AUC as high as 0.988. This study preliminarily established a TCR evaluation system related to MM, scoring based on the comprehensive TCR phenotype of each sample, and then conduct correlation analysis between the scoring results of each sample and the clinical outcomes. Through the ORR evaluation and the overall OS evaluation, it was found that there was no significant difference in PFS, also no concentrated area was found in death group in OS data performance. In contrast, the overall data performance of the surviving group was below 0.35%. Therefore, we used TRB clone performance of 0.35% as the cutoff value for evaluating survival prediction. At the same time, the evaluation standard for TRB clones was proposed as: “ratio = the number of MM-related TRB sequences in the sample TRB sequence / the total number of clones in the sample”. The survival curve assessment presented that the P value of the two groups in OS was below 0.05, which indicated the risk prediction value via MM prognosis predictive algorithm of TRB established in this study.

Conclusion:In this study, we firstly established the Ig and TRB profiling of plasma cells and PB in NDMM patients respectively. And then, we identified the potential prognosis function of different chains number in Ig clone. Furthermore, we uncovered the TRB diversity potency as the biomarkers for MM diagnosis and prognosis in baseline stage, provided the possibility of TCR diversity to be regarded as the non-invasive liquid biopsy. This study established the foundation of exploring the immunological distinction in MM patients.

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2025
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