Intraclonal heterogeneity is a critical early event in the development of myeloma and precedes the development of clinical symptoms

Abstract

The mechanisms involved in the progression from monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) to malignant multiple myeloma (MM) and plasma cell leukemia (PCL) are poorly understood but believed to involve the sequential acquisition of genetic hits. We performed exome and whole-genome sequencing on a series of MGUS (n=4), high-risk (HR)SMM (n=4), MM (n=26) and PCL (n=2) samples, including four cases who transformed from HR-SMM to MM, to determine the genetic factors that drive progression of disease. The pattern and number of non-synonymous mutations show that the MGUS disease stage is less genetically complex than MM, and HR-SMM is similar to presenting MM. Intraclonal heterogeneity is present at all stages and using cases of HR-SMM, which transformed to MM, we show that intraclonal heterogeneity is a typical feature of the disease. At the HR-SMM stage of disease, the majority of the genetic changes necessary to give rise to MM are already present. These data suggest that clonal progression is the key feature of transformation of HR-SMM to MM and as such the invasive clinically predominant clone typical of MM is already present at the SMM stage and would be amenable to therapeutic intervention at that stage.

Figure 1. The number of acquired mutations increases as disease progresses

A, a schematic of disease progression from MGUS through to PCL. B, Boxplot showing the median number of acquired NS-SNVs increases as the disease progresses from MGUS through HR-SMM and MM to PCL.

Figure 2. Clonal heterogeneity is present in all disease states

Gaussian kernel density plots indicating the frequency of cells carrying all acquired exonic mutations. Frequency is calculated by adjusting mutant allele burden by copy number of the loci mutated. Top row, MGUS samples; Middle rows, matched HR-SMM/MM samples; Bottom row, PCL samples.

Figure 3. Clonal evolution of paired HR-SMM and MM samples from an untreated (patient 5; A-C) and a treated patient (patient 4; D-F)

A and D: Kernel density plots of the proportion of cells containing each variant from whole genome sequencing in HR-SMM (blue) and MM (red); B and E: Comparison of proportion of cells containing each variant in HR-SMM and MM samples. Positive and negative vertical deviations from the main diagonal (marked at a dashed line) indicate an increase or decrease in the variant from the HR-SMM to MM stage respectively. C and F: After estimating noise using a nearest-neighbor based classifier, an EM based clustering strategy was employed to define clusters of variants. Most of the variants in HR-SMM occur at low frequency, but a distinct increase in frequency for a large set of variants is seen in the MM sample.

Figure 4. Circos plots of translocations detected in SMM-MM paired samples

A, patient 1. B, patient 2. C, patient 5. Red lines indicate translocations present in both SMM and MM samples, orange in only the MM sample, and green in only the SMM sample. No translocations were identified in patient 4.