Prospective Investigation of Genetic Alterations in Osteolytic Lesions Compared to Paired Random Aspirates

Background:

Bone disease is the most frequent clinical manifestation of multiple myeloma. In this prospective study we ask whether osteolytic lesions (OL) are driven by myeloma cells showing a different background of genetic alterations in terms of chromosomal aberrations and expressed single nucleotide variants (SNVs) compared to random aspirates (RA) from diffuse myeloma cell infiltration at the iliac crest (spatial genetic heterogeneity).

Material and Methods:

Consecutive sample-pairs (n=41) were prospectively obtained by CT-guided biopsies of OLs as well as simultaneous random bone marrow aspirates of the iliac crest, the latter undergoing CD138-purification of myeloma cells, in transplant eligible patients with previously untreated symptomatic multiple myeloma, after written informed consent. Peripheral blood mononuclear cells were used as germline control. Plasma cell infiltration in biopsies was quantified histologically. Samples pairs (n=8) were subjected to RNA-sequencing (Illumina HiSeq2000), gene expression profiling using DNA-microarrays (Affymetrix U133 2.0), whole exome sequencing (Illumina NextSeq 500), and arrayCGH (Affymetrix cytoscan array).

Results and Discussion:

Expressed single nucleotide variants.The spectrum of mutated genes in our samples comprises two of the most frequently mutated in symptomatic myeloma, i.e. KRAS and FAM46C, alongside those implicated in myeloma pathophysiology, e.g. mutations in IRF4, FGFR3, and CD200. In total, 1-10 clonal expressed non-synonymous SNVs were exclusively found in OL compared to RA, comprising e.g. WHSC1, FAM46C, and ROCK1P1. In 2/8 patients (25%), no expressed clonal differences between RA and OL were present.

Single nucleotide variants.In investigated samples, 77-1569 non-synonymous SNVs appear with an allele frequency of ≥10% in OL and RA, clustering in 4-5 groups. The clonal constitution can vary, but subclones are detectable in both. Subclonal complexity is maintained (subclones remain present) in OL compared to RA, and the vast majority of subclonal changes is present in both, especially for expressed non-synonymous SNVs, incompatible with an "osteolytic clonal variant" driving OL in the majority of patients.

Copy number alterations and loss of heterozygosity.Subtle differences in copy number between OL and RA are present. However, only 1/8 patients (12.5%) showed further "gained" aberrations in OL compared to RA, i.e. deletions on chromosome 7p, 8p, and 11p as well as 19p gain. Loss of heterozygosity was observed in 3/8 patients (37.5%) with a shared pattern between OL and RA in all of them.

Conclusions:

In our prospective study, the majority of alterations is shared between RA and OL. Spatial heterogeneity is present, but nature and frequency of alterations detectable exclusively in OL make them unlikely candidates in most myeloma patients for being causative for generation of OL.