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Maintenance, response status, and subclonal structure at MM relapse
dependant probe amplification (MLPA) (SALSA MLPA P425-B1 multiple myeloma probemix, MRC Holland, Amsterdam, the Netherlands) and the bioinformatics assessment tool Sequenza (v.2.1.2).33,34 Paired MLPA and Sequenza data was available for 90 of 112 (80%) tumor samples, with a consensus between MLPA and Sequenza being seen in 85 of 90 samples (94%). For the five patients for whom a mismatch was observed, Sequenza was used to call the copy number profile. Translocations were determined using MANTA (v.0.29.3).35 For 46% (51 of 112) of patient samples, translocations involving the immunoglobulin heavy chain (IGH) were also assessed using multiplexed qRT-PCR.36 A consensus between MANTA and qRT-PCR was observed in 84% (43 of 51). In the eight patients for whom a mismatch was seen, the integra- tive genomics viewer (IGV) was used to confirm or exclude the translocation. All suspected bi-allelic copy number abnormality (CNA) events were confirmed by manual interrogation of BAM files using IGV. Bi-allelic inactivation was also called in patients with evidence of a non-synonymous mutation with mono-allelic loss or a single mutation with a VAF of ≥80%. A summary of the methods, bioinformatics tools and filters used to complete this analysis is available in Online Supplementary Figure S2.
Statistical analysis was performed using GraphPad Prism v.7.01 and R v.3.2.1. P=0.05 (two-sided) was considered statistically sig- nificant. Wilcoxon matched-pairs signed rank tests were conduct- ed to determine significance between paired data sets, including the mutational load at presentation/relapse and PFS according to maintenance allocation, depth of response, and induction treat- ment. Fisher exact test was used to determine differences between
two nominal variables, including the change in mutational profile from presentation to relapse and the evolutionary mechanism leading to relapse.
Results
Relapse is characterized by a change in mutational spectrum
At presentation, genes mutated in >10% of patients were NRAS (23%, n=13), KRAS (23%, n=13), and DIS3 (13%, n=7) ((Figure 1A and B)). At relapse, TP53 was also seen in >10% of patients (11%, n=6). We examined genes that have previously been shown to be recurrently mutat- ed in myeloma, including tumor suppressor genes, epige- netic modifiers, and genes within the RAS, NFκB and apoptosis pathways, the results of which are summarized (Figure C).18,28,37-44 Non-synonymous mutations were seen in one or more of these genes in 79% (44 of 56) of patients at presentation and 80% (45 of 56) at relapse. Importantly, gain and/or loss of one or more of these lesions at relapse was seen in 37% of patients (21 of 56), with 21% (12 of 56) of patients gaining a new lesion, 11% (6 of 56) losing a lesion, and 5% (3 of 56) both gaining and losing lesions. The most common new mutations at relapse were KRAS and PRDM1, both seen in 5% of patients (3 of 56), and NRAS and TP53, each seen in 4% (2 of 56). However, mutations in KRAS and NRAS were just as likely to be lost
ABC
Figure 2. The proportion of patients with a change in the profile of mutations known to be recurrent in myeloma or important in immunomodulatory mechanism of action at relapse. (A) The proportion of all patients (n=56) with a change in the profile of recurrent mutations at relapse. The majority of complete remission (CR) series patients had a change in the mutational profile at relapse, 67% (16 of 24) versus 25% (8 of 32) of non-CR patients (Wilcoxon matched pairs, P=0.003). (B) The proportion of observation patients (n=26) with a change in the profile of recurrent mutations at relapse. Only 19% (3 of 16) of non-CR patients under observation had a change in the profile of mutations at relapse, compared to 70% (7 of 10) of CR patients under observation (P=0.02). (C) The proportion of lenalidomide main- tenance patients (n=30) with a change in the profile of recurrent mutations at relapse. The same pattern of mutational profile change was seen in the lenalidomide maintenance patients, with 64% (9 of 14) of the CR patients having a mutational profile change at relapse compared to 31% of non-CR patients (P=0.14).
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