General MM

Detection of MM-associated translocations and mutations in IGLL5

Cancer is now commonly thought of as a genetic disease, caused by mutations which are often acquired over time. Premalignant monoclonal gammopathy of undetermined significance (MGUS) cells display genetic alterations that are likely to initiate progression to multiple myeloma (MM). Prognostic accuracy can be dramatically improved when mutation and expression data are paired with data defining the international staging system (ISS).

Brian S. White, from the Department of Medicine, Washington University School of Medicine, St. Louis, USA, and colleagues, developed a capture-based sequencing platform, specific to MM, in order to detect novel copy number variations (CNVs), single nucleotide variants (SNVs) and translocations. The technology was described in a manuscript published in Blood Cancer Journal in March 2018.

Key highlights:
  • MM-specific sequencing platform capable of detecting CNVs, SNVs, and translocations, targeted 465 genes
  • Sequencing of 95 primary tumor (CD138-purified cells isolated from bone marrow aspirates) and paired normal (blood) samples
  • Oligonucleotide probes were complementary to both the exons, untranslated regions and splice sites of 465 genes, and exonic regions of canonical IGH translocation partners (CCND1, CCND3, FGFR3, MAF, MAFB, WHSC1, and WWOX) and exonic and intronic region of the MYC locus
  • Mean sequencing depth achieved = 104× (tumor samples) and 107× (normal samples)
  • Detection of alterations in copy number associated with poor prognosis:
    • Amp(1q), del(1p), del(13q) and del(17p)
  • Detection of IGH translocations
    • Frequency: t(11;14) = 14%; 4(4;14) = 13%; t(8;14) = 7%; t(4;14) = 2% and t(14;20) = 2%
    • Occurred mainly within the IGH constant region, also telomeric of the IGHM switch region and at times within the D and J regions
  • Detection of IGLL5 mutation (both point substitutions and translocations)
    • Validated t(14;22) translocation: breakpoints located within the IGH and IGLL5 loci
  • Upregulation of DERL3
  • Identification of MYC translocations:
    • Five intra-chromosomal and two non-IGH inter-chromosomal
  • Detection of non-silent SNVs in tumor samples:
    • ≥ 1 somatic mutation detected in all tumor samples
    • Mean no. of mutation (per tumor sample) = 20
    • Mutations in ≥ 1 tumor sample: non-synonymous mutations = 443 genes; any mutation = 581 genes
    • Detection of a cancer-related gene = 24 cases
  • Deeper sequencing yielded few additional variants
    • Original sequencing study (mean depth = 92×) = 57 variants
    • Additional sequencing study (mean depth = 1,169×) = 67 variants
  • Targeted sequencing identifies co-occurrence and mutual exclusivity across mutation types:
    • Mutation co-occurrence within CNVs (del(6q) with del(16q) and amp(1q) and of del(13q) with amp(1q) and del(14q)); within CNVs and translocations (del(14q) with t(4;14)
    • Mutual exclusivity between hyperdiploidy and t(11;14); RAS and del(6q); FAM46C and del(6q); IGLL5 and RAS (P = 0.006)
    • Mutations in IGLL5 were mutually exclusive of RAS mutations (p = 0.006), with a trend towards mutual exclusivity with KRAS (p = 0.054), NRAS (p = 0.111), and FAM46C (p = 0.113), independently 
  • IGLL5 was frequently mutated in the dataset (18%), with a median variant allele frequency (VAF) of 58%
  • Association of IGLL5 SNVs with disease progression (HR = 1.46,95% CI; 1.03–2.08; P = 0.03, log-rank test)

The use of MM-specific capture-based sequencing enabled the identification of several genetic alterations which included MYC translocations, an IGLL5 mutation and the co-occurrence and mutual exclusivity across mutation types. One key finding was the mutual exclusivity of IGLL5 with RAS mutations and its link with disease progression. It is proposed that this single platform could facilitate genomic sequencing for stratification of MM patients.

References
  1. White BS. et al. A multiple myeloma-specific capture sequencing platform discovers novel translocations and frequent, risk-associated point mutations in IGLL5. Blood Cancer Journal. March 2018. DOI: 10.1038/s41408-018-0062-y
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