Clonal competition with alternating dominance in multiple myeloma

Abstract

Emerging evidence indicates that tumors can follow several evolutionary paths over a patient's disease course. With the use of serial genomic analysis of samples collected at different points during the disease course of 28 patients with multiple myeloma, we found that the genomes of standard-risk patients show few changes over time, whereas those of cytogenetically high-risk patients show significantly more changes over time. The results indicate the existence of 3 temporal tumor types, which can either be genetically stable, linearly evolving, or heterogeneous clonal mixtures with shifting predominant clones. A detailed analysis of one high-risk patient sampled at 7 time points over the entire disease course identified 2 competing subclones that alternate in a back and forth manner for dominance with therapy until one clone underwent a dramatic linear evolution. With the use of the Vk*MYC genetically engineered mouse model of myeloma we modeled this competition between subclones for predominance occurring spontaneously and with therapeutic selection.

Figure 1

Paired sample analysis identifies 3 patterns of clonal evolution. (A) Heat map showing CNAs in the 24 patients with sample pairs analyzed on the Agilent 244k exclusively (4 samples analyzed on Agilent 44k or mixture of 44k and 244k are not shown). The first 2 sequential samples for each patient are stacked, with the first sample shown on the top. Sample pairs are indicated on the y-axis and chromosome location on the x-axis. Blue shading indicates the presence of copy number loss, red indicates copy number gain, and white indicates regions with no CNA. Black, blue, and red bars indicate groups of patients with no copy number changes, with only acquired new changes, or with both losses and gains of CNA over time, respectively. The latter 2 groups are ordered from top to bottom from the least to the most changes. (B) Summarized findings from the 28 patients with a sequential sample pair. Colored bars represent theoretical CNAs perceived to exist at a particular point in the evolution of the tumor. (C) Bar graph showing the relation between copy number changes and standard (n = 18) and high (n = 8) cytogenetic risk status. Samples are ordered left to right, based on the total number of changes. The number of losses and acquired events are shown independently. There is a significant difference in the total number of changes detected between the standard and high-risk cytogenetic groups (P < .02) with a higher number of changes found in the high-risk group.

Figure 2

Clinical course of a patient with high-risk MM. The clinical course of a single patient (MC1130) studied throughout the entire disease course is shown. Red line indicates the quantitative IgA level detected and units are shown on the left y-axis. Blue line indicates the free light chain ratio detected with units shown on the right y-axis. Alternating color regions indicate type and durations of treatment received during each interval. Red arrows highlight the time points at which BM aspirates were analyzed, the green arrow indicates the time of collection of the terminal PCL sample from peripheral blood. Dx indicates diagnosis; Rem, remission: R1, relapse 1; R2, relapse 2; R3, relapse 3; and R4, relapse 4. The assays performed at each time point are indicated under their representative arrows.

Figure 3

Copy number analysis of a patient with high-risk MM. (A) A whole chromosome CGH dot plots for chromosome 8 are shown for each sample assayed. Statistically significant CNAs are indicated by light gray shading, which coincides with regions with extensive green dots (deletions) or red dots (amplifications). The 2 regions used to define subclone progenitors 1 and 2, which ebb and flow with time in this patient, are visible as large deletions on the 8p and 8q, respectively. (B) Heat map showing copy number changes in the 5 samples analyzed by Agilent 244k. Sample types are shown on the y-axis and ordered longitudinally. The chromosome location is on the x-axis. Blue shading indicates the presence of copy number loss; red, copy number gain; and white, no copy number abnormality. (C) Dendrogram showing the relation of the observed subclones. Branch length represents the number of CNAs detected by aCGH and assigned to each evolutionary step. There are 5 CNAs shared in all samples and a clear divergence of 2 subclone progenitors defined by 27 or 19 CNAs, respectively. Clones related to subclone progenitor 1 represent the major tumor population at diagnosis and relapse 3 and are differentiated by 2 and 4 CNAs, respectively. Clones related to subclone progenitor 2 represent the majority of the tumor population at relapse 1 and relapse 4/PCL, which are differentiated by 13 and 39 CNAs, respectively.

Figure 4

Clonal dynamics in a patient with high-risk MM. The summarized results of 8 different FISH assays are shown to indicate the relative abundance of each clone defined by aCGH at the 5 time points studied. Pie charts showing the relative proportions of each indicated clone are ordered clockwise starting on the left in longitudinal order. Arrow length is proportional to the time interval. The relative ploidy of the tumor population at each time point is also indicated. The cIg FISH of relapse 4 and the PCL identified clonal cells with low levels of cytoplasmic immunoglobulin (cIg-low) and larger cells with abundant cytoplasmic immunoglobulin (cIg-high) that were scored independently.

Figure 5

Clonal dynamics in a murine model of MM. (A) Serum protein electrophoresis performed at the indicated weeks on a biclonal Vk*MYC mouse shows the emergency at 80 weeks of a secondary clone that becomes dominant by 100 weeks. Arrows highlight the M-spike secreted by the dominant clone. Arrowhead points to the M-spike secreted by the competed out clone. (B) BM cells from a donor mouse (D) with a triclonal myeloma were transplanted into 3 wt congenic mice recipient mice (R). Serum protein electrophoresis shows engraftment of only 1 of the 3 clones in all 3 recipient mice. Arrows highlight the M-spike secreted by the dominant clone. Arrowheads point to the M-spikes secreted by the competed out clones. (C-E) BM cells from Vk*MYC myeloma-bearing mice (Donor) were transplanted into either wt congenic mice (wt) or recipient Vk*MYC mice with preexisting MM (R). Serum protein electrophoresis was performed at the indicated weeks after transplantation. Arrows highlight the M-spikes secreted by the donor mouse MM cells and arrowheads points to the preexisting M-spikes in the recipient mice.

Figure 6

Therapy induced clonal dynamics in a murine model of MM. (A) Three independent Vk*MYC mice with biclonal or triclonal MM were treated biweekly with escalating doses of bortezomib (0.16-1 mg/kg). SPEP performed at the indicated weeks shows clonal modulation, with arrowheads pointing to the M-spikes secreted by the competed out clones, and arrows indicating the dominant bortezomib-resistant clones. (B) SPEP performed before and after carfilzomib or pomalidomide treatment of Vk*MYC mice with oligoclonal myeloma identifies responsive (arrowheads) and refractory (arrows) clones.