Whole Exome Sequencing Reveals High Genetic Diversity in Relapsed Multiple Myeloma

Background: Treatments like lenalidomide and proteasome inhibitors alone or in combination with chemotherapy have dramatically improved the outcome in multiple myeloma in the past years. Although the majority of patients achieves a remission, almost all patients eventually relapse. Previous studies have found recurrent mutations in TP53 and FAM46C (Weinhold et al., Blood 2016) in relapsed multiple myeloma as well as CRBN mutations (Kortüm et al., Blood 2016) specifically in lenalidomide-treated patients.

Methods: In order to identify additional genetic aberrations that are associated with therapy resistance, we analyzed CD138-selected multiple myeloma cells in bone marrow samples obtained pretreatment (n=12) and at different time points at progression during treatment (n=17) by fluorescence in situ hybridization (FISH) and whole exome sequencing (WES). Peripheral blood was used as a germline control. The median number of therapy lines between pretreatment and progression samples was 1 (range, 1 - 4). Progression samples were obtained after treatment with lenalidomide (n=16), bortezomib (n=14), or high-dose melphalan/autologous stem cell transplantation (n=9) alone or in combination or as sequential treatment. WES was performed by paired-end sequencing (read length 100 bp) on an Illumina HiSeq platform. The mean coverage was 117x. A variant allele frequency (VAF) of 5% was set as a cut-off for mutation calling.

Results: In the pretreatment samples, the median number of cytogenetic aberrations was 2 (range, 1 - 4) and the median number of somatic variants was 39 (range, 24 - 63) per sample. There was no significant change in these numbers in posttreatment samples: median number of cytogenetic aberrations 2 (range, 0 - 4), median number of somatic variants 52 (range, 11 - 107).

The most recurrent cytogenetic alterations at diagnosis were +1q21 (n=6), +9q34.2 (n=4), and del13q14 (n=3). At relapse, 13 of the 18 cytogenetic abnormalities detected at diagnosis were still present, 5 were lost and 5 were newly acquired. The most frequently lost aberration was +9q34.2 (n=2) and the most commonly acquired aberrations were +1q21 (n=2) and del13q14 (n=2). In pretreatment samples we found recurrent mutations in NRAS (n=4), KRAS (n=3), DIS3 (n=3), and IGLL5 (n=3). The median stability of mutations between pre- and posttreatment samples was 62% (range, 13 - 81%). NRAS mutations were lost in 2 progression samples and DIS3 in 1 progression sample while KRAS mutations remained stable. We found 430 relapse-specific mutations in the 17 posttreatment samples. Mutations in TP53 (n=4) were the most frequently newly acquired genetic events in progression samples, which is consistent with previous studies. Of the newly acquired gene mutations, only eight genes were affected in more than one progression sample, all other gene mutations were non-recurrent. Among genes involved in the mechanism of lenalidomide we found a DDB1 mutation in one progression sample after lenalidomide treatment. Another patient had an IKZF3 mutation at relapse. However, the mutation was located outside the lenalidomide degron sequence and the VAF was only 5%, suggesting that it was not responsible for lenalidomide resistance in the major clone. No relapse-specific mutations were found in other genes encoding proteins involved in lenalidomide or proteasome inhibitor activity like IKZF1, IRF4, CRBN, CUL4A, CUL4B or proteasome subunits. Pathway analyses revealed that mutations in genes involved in WNT/beta catenin and KRAS signaling were enriched in the progression samples.

Conclusions: In conclusion, we found a high degree of genetic heterogeneity in relapsed multiple myeloma. There were no obvious mutations in genes involved in lenalidomide or proteasome activity that might drive drug resistance. TP53 mutations and +1q21 were the most recurrent gene aberrations identified at relapse, however, these events may cause a general robustness of multiple myeloma cells to multiple treatment modalities and might not be treatment specific. These results suggest that the genetic basis for drug resistance is heterogeneous and warrants genetic characterization of further patients by DNA and RNA sequencing as well as functional analyses of candidate genes and pathways.