The Genomic and Transcriptomic Landscape of Double-Refractory Multiple Myeloma

In Multiple myeloma (MM) no treatment has a curative potential and even complete response to novel agents such as proteasome inhibitors (PIs) and immunomodulatory agents (IMiDs) are followed by relapse over time. Next generation sequencing (NGS) has showed how MM at diagnosis is defined by several somatic mutations, but only few drivers, even fewer “druggable” mutations, and many found at a subclonal level. At relapse, targeted studies have shown occasional mutations in drug target genes but the genomic and transcriptomic determinants of chemoresistance in MM remains elusive. We selected 42 MM patients refractory to both lenalidomide and PIs. Whole exome sequencing was performed in 40 of them, and RNAseq in 27. Clinical annotation was available for all patients. Standard analysis pipelines where applied to analyze mutations, copy number alterations (CNAs), mutational signatures, gene expression and expressed mutations. Patients received a median of 3 lines of treatment, with median overall survival of 14.6 months from sampling. We found a median of 77.5 mutations per patient, which is more than what reported at diagnosis (Bolli et al, Nature Communications 2014;5:2997). 100% of samples showed evidence of subclonality, and 37% of them exhibited a higher number of subclonal than clonal variants. Therefore, even at this advanced stage the MM genome is evolving and is composed of different subclones that may display different chemosensitivity. The mutational landscape was also different. TP53 mutations were the second most common after KRAS (20% and 17.5%, respectively). Interestingly TP53 mutations all clustered in patients receiving bortezomib as the last line of treatment. Only 2 patients showed a CRBN mutation, both subclonal. Combining mutations and CNA analysis, the TP53 pathway was the most frequently inactivated (45% of patients). Altogether, mutations or deletions of genes in the CRBN E3 ubiquitin ligase complex were found in 32.5% of patients, while proteasomal subunit genes were infrequently hit. Refractory cases were also uniquely characterized by a novel signature linked to exposure to alkylating agents, whose activity was more pronounced after high-dose melphalan suggesting a mutagenic effect of the drug on residual cells at the time of transplant. Whether this has any pathogenetic role on the disease course remains to be elucidated. RNAseq analysis did not show any influence of treatment or mutational data on the clustering of samples, which was