This study attempted to investigate how clonal structure evolves under the pressure of multiline therapy in relapsed/refractory multiple myeloma (RRMM). 

Eight whole exome sequencing (WES) and one single cell RNA sequencing (scRNA-seq) were performed to uncover the dynamic genomic changes in temporal consecutive samples of one RRMM patient from new diagnosis to death (about 37 months). The 63-year-old female patient who suffered from MM (P1) had disease progression (PD) nine times from July 2017 (new diagnosis (ND)) to Aug 2020 (death). In the end-stage of MM (PD9), malignant PCs appeared in her peripheral blood (PB), the proportion of which reached 10%. The patient herself refused to undergo bone marrow aspiration again. Therefore, for PD9, CD138 positive PCs from PB were used for WES, PB mononuclear cells (PBMC) were used for scRNA-seq. 

It was found that the force to drive branching-pattern evolution of malignant PCs was sustained. Moreover, the mutant-allele tumor heterogeneity (MATH) and tumor mutation burden (TMB) trends gradually presented downward at first and then upward throughout the course of the disease. Additionally, various somatic single nucleotide variants (SNVs) that had disappeared after the previous treatment reappeared in later stages, indicating that agents that were effective against these recurring mutations in the earlier stages may be re-administrated. Furthermore, chromosomal instability (CIN) and homologous recombination deficiency (HRD) scores were observed to be increased during periods of all progression. Finally, in combination with WES and scRNA-seq of P1-PD9 (the nineth PD), the data highlighted the heterogeneity and distinct evolutional subpopulations among MM cells.

This study demonstrated that temporal consecutive samples from one RRMM patient revealed the diversity of sub-clones as well as the clonal evolution trajectory driven by multi-line therapies. Moreover, distribution of driver mutations and passenger mutations were observed to be scattered in the trunk and non-trunk of the branching-pattern phylogenetic tree. Personal high-throughput sequencing that reveals specific genetic compositions and molecular phenotypes may contribute to more robust personal biomarkers and serve as guidance for personalized therapy. Furthermore, adaptive and recycle therapy based on evolutionary law may prevent a rise in resistant PCs population and provide a potential strategy for RRMM treatment.