Spatial Genomic Heterogeneity in Multiple Myeloma: Surrogate Markers and Its Significance for Understanding the Evolutionary Processes Leading to Relapse

Introduction

Multiple Myeloma (MM) frequently presents with genomic variation between individual cancer foci, which is also termed spatial heterogeneity (SH). However, the extent of SH differs considerably between patients with some having extensive SH while others show homogeneous mutational profiles within the bone marrow (BM). Since SH poses a significant challenge to personalized therapy, surrogate markers for its extent would be useful clinically. In this respect an understanding of the spatial evolutionary patterns taking place during MM treatment and the clonal architecture they give rise to at relapse would also be of fundamental importance. To address these important points we have analyzed paired samples from multiple BM sites collected at baseline (n=52 patients) or in a longitudinal fashion during treatment (n=30).

Patients and Methods

A genomic analysis was performed on iliac crest BM and CT-guided fine needle aspirates from distinct anatomical sites from 52 newly diagnosed patients. The impact of treatment on SH was analyzed in 30 patients with a median number of 6 samples per patient (range 4-14). Plasma cells were CD138-enriched and underwent whole exome sequencing using an Illumina HiSeq 2500. Sequencing data were aligned using BWA. Mutations were identified using MuTect. Copy number aberrations were derived from Illumina HumanOmni 2.5 bead chip data using ASCAT. Subclonal reconstruction was performed using SciClone. Statistical analyses were carried out using R packages.

Results

We investigated whether the extent of SH was associated with clinical features and found that age, an adverse prognostic factor, was positively correlated with SH (P=0.01). Patients over the age of 60 had a 3-fold higher proportion of private mutations compared to younger patients (median 15 vs. 5%). Hypothetically, this could reflect the longer evolutionary time period to MM arising at older ages. The size of the biopsied FLs was also significantly associated with the proportion of private mutations (P <0.001), with large FLs (>2.5 cm) having the highest values. Of note, a linear model including age and FL size explained 49% of the variation in SH. In contrast, neither the ISS, adverse molecular markers identified at the iliac crest, the number of FLs, nor the anatomical distance between investigated sites correlated with SH. In summary, we show that SH is increased in elderly patients and/or patients with large FLs.

Next, we investigated the impact of treatment on the spatial clonal architecture. For patients who had achieved a CR, we expected relapse to be dominated by a limited number of selected clones and as a result less SH. Indeed, analyzing samples derived from multiple sites at baseline and at subsequent relapses, the predominant pattern was characterized by sweeps of advanced clones resulting in BM-wide replacement of previous clones. Of note, evidence for clonal sweeps was also seen in patients with stable disease according to IMWG criteria, which is a perplexing observation since stable serological parameters do not seem to be consistent with major sub-clonal changes.

In contrast to cases with clonal sweeps, there were also patients with multiple site-specific clones at relapse. On the one hand, "mixed responses" of FLs, which are frequently seen in medical imaging after multiple relapses, could be related to this pattern of pronounced SH. On the other hand, we found private mutations affecting the same genes or pathways in patients with site-specific subclones, indicating parallel evolution. In this context, one patient showed distinct KRAS mutations which affected the exact same amino acid (Gly12Val vs. Gly12Asp), emphasizing that treatment could select for multiple clones with similar or even identical features. This observation may explain, why individual patients still respond to MAPK pathway inhibitors despite pronounced SH.

Conclusions

We provide new insights into the biology of MM progression, which has implications for diagnostic and treatment strategies. The strong association of private mutations with age and FL size may allow for the identification of patients with increased levels of SH for whom treatment approaches can be modified. The existence of clonal sweeps of aggressive clones, which replace previously dominant clones, even in poorly responding patients, highlights the need for regular molecular testing, especially if personalized treatment is intended.