The Mutational Landscape of Relapse in High Risk Myeloma Is Significantly Impacted By the Depth of Response but Not Maintenance Lenalidomide

Introduction:

The evolutionary processes underlying disease progression in multiple myeloma (MM) are critical to the effective use of maintenance and can be better understood by determining global genetic change using next-generation sequencing. Previous studies comparing presentation and relapse samples lacked uniform treatment and response rates making interpretation difficult. In this study we used whole exome sequencing (WES) to study paired presentation and relapse samples from 56 newly diagnosed high-risk patients, 30 who received lenalidomide maintenance and 26 who did not, within the Myeloma XI clinical study.

Methods:

For all 56 patients, synonymous and non-synonymous (NS) mutations, copy number abnormalities, structural change and clonal structure at presentation and relapse were determined. All patients had relapsed within 30 months of maintenance randomization, with a median PFS of 19 months (range 8-51)

DNA was isolated from CD138-positive plasma cells with control DNA from peripheral blood samples. Whole exome-sequencing (WES) libraries were prepared using the SureSelectQXT sample prep kit and the SureSelect Clinical Research Exome kit (Agilent), with additional baits covering the immunoglobulin (Ig) and MYC loci. Paired-end sequencing was performed to a median sequencing depth of 122x for tumour samples and 58x for controls using a HiSeq2500 (Illumina). All variants were called using MuTect. The distribution of mutant alleles determined by the variant allele frequency was mapped using the R package SciClone. Translocations were determined using MANTA. Determination of clonal structure was undertaken using copy number data, MuTect, SciClone clustering and kernel density estimation plots for all mutations.

Results:

At relapse there was a significantly higher number of NS mutations, 44 compared to 39 for the whole series (p=0.005) with the most marked differences being seen in complete/near complete responders (CR/nCR), where the median mutational load at relapse had increased from 40 to 59 (p=<0.001). The same pattern in mutational load was noted in patients receiving lenalidomide maintenance and those not. Mutations in genes known to be significant in myeloma were seen in 84% (47/56) of patients and these were both lost and gained at relapse irrespective of the maintenance treatment used. The proportion of patients with bi-allelic inactivation of important tumour suppressor genes (TSG), including TP53 increased from 14% (8/56) at presentation to 18% (10/56) at relapse. Translocations involving MYC were also more frequent at relapse, seen in 27% (15/56) of patients compared to 21% (12/15) at presentation. Mutations in genes known to be important in immunomodulatory mechanism of action were seen in 13% (7/56) of patients although these were not confined to patients exposed to lenalidomide maintenance or always conserved at relapse.

We identified 4 evolutionary patterns at relapse, stable progression, stable progression with clonal loss, branching and linear. Branching evolution, characterised by gain and loss of mutational clusters at relapse was the predominant pattern, seen in 77% (23/30) of lenalidomide and 54% (14/26) of observation patients. Linear evolution, characterised by gain of mutations at relapse was seen in 13% (4/30) lenalidomide and 27% (7/26) observation patients. In this series, therefore, dynamic processes characterised by the gain and loss of mutational clusters was the predominant mechanism underlying progression, seen in 86% (48/56) of cases overall. Of the patients with branching evolution 35% (n=13/37) gained or lost one or more TSG at relapse, in contrast to 18% (2/11) of linear evolution patients and 0% (0/8) patients with stable disease or clonal loss. New bi-allelic inactivation events were only seen in patients with branching evolution.

The depth of response was an important determinant of the pattern of evolution seen at relapse. Stable progression and clonal loss were only noted in the poor responders, seen in 56% (5/9) of PR and 13% (3/23) of VGPR patients. No patients who achieved a CR/nCR (0/24) had evidence of stable progression with all patients showing branching or linear evolution (p=0.002).

Conclusion:

We show that lenalidomide maintenance does not affect the number of mutations or evolutionary pathways to relapse but the depth of response is a key determinant.