Validation of liquid Biopsy using circulating tumor DNA to monitor MM

Multiple Myeloma (MM) is both diagnosed and genotyped using purified plasma cells (PCs) from the bone marrow (BM) aspirate. These cells are also used for the identification of disease molecular markers, but this type of sampling is not only invasive for patients but might fail to provide a true representation of the spatial heterogeneity of MM. In order to overcome this, there is now a move towards the use of liquid biopsies, which rely on the detection of circulating cell-free DNA (cfDNA) shed from the tumor cells.

In order to validate such an approach for MM, Bernhard Gerber, from the Division of Hematology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland, and colleagues, compared the mutational profile of cfDNA with tumor genomic DNA (gDNA) from purified PCs from BM aspirates. Patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma and symptomatic MM were included and the results published in Haematologica in February 2018.

Key Findings:

• N = 28 patients (pts); MGUS = 2, smoldering MM (SMM) = 5, and symptomatic MM = 21 (Newly Diagnosed (ND) = 25, Relapsed and Refractory (RR) = 3)
• Samples used:
  • cfDNA isolated from plasma 
  • Tumor gDNA from CD138+ purified BM PCs
  • Germline gDNA extracted from peripheral blood (PB) granulocytes
• Use of ultra-deep Next-Generation Sequencing (NGS) on MiSeq (Illumina) using the CAPP-seq library preparation strategy (NimbleGen)
• Higher amounts of cfDNA in pts with international staging (ISS) stage 3 vs pts with MGUS/SMM and MM at ISS stages 1–2 (P = 0.01)
• Pts with ≥1 non-synonymous somatic mutation detectable in cfDNA = 18/28 pts (64%)
• N = 28 of total variants identified (range: 1–4 mutations/pt)
• Somatic mutations identified in: NRAS (25%), KRAS (14%), TP53 (11%), TRAF3 (11%), FAM46C (11%), CYLD (7%), DIS3 (7%), BRAF (4%) and IRF4 (4%)
• NRAS, KRAS and BRAF gene variants were mutually exclusive and occurred in 43% of pts
• TP53 mutations were associated with loss of the remaining allele (P = 0.02, Fisher-exact test)
• The mutations identified were similar to those identified in previously published genomic analyses, supporting the purity of the tumor cfDNA used
• To validate the mutational data from the cfDNA, PC tumor gDNA was sequenced and revealed 39 somatic mutations in 20/28 pts (71.4%)
• cfDNA genotyping identified 28 of these 39 mutations (4 in 2 SMM pts, 24 in 16 MM cases)
• The variant allele frequencies in PCs correlated significantly with that of tumor biopsies and with the degree of BM involvement
• Mutations confirmed with BM PCs but not with cfDNA (due to low representation in the tumor) = 11 (Median allelic frequency: 2.5%; range: 1.1–4.96%)
• cfDNA genotyping correlates best with tumor PC when mutations occur in ≥ 5% of the alleles of tumor PCs; 100% of biopsy-confirmed mutations detected above this threshold
• Detection of 10/21 of low-abundance mutations in tumor PC (i.e. allelic frequency <20%) using cfDNA genotyping
• cfDNA genotyping only identified mutations that were detected in the BM PCs, suggesting this was representative of the tumor genetic spectrum

This study confirmed that cfDNA genotyping can successfully identify mutations represented in tumor PCs. This is therefore an effective, manageable and non-invasive method to identify mutations in asymptomatic or symptomatic MM patients, and could be used both in clinical trials and in clinical practice. In particular, those patients at an early stage of the disease will benefit from the non-invasive approach, as it will allow them to be observed for the accumulation of high-risk features, and/or for targeted therapy to potentially begin earlier.