RESUMO
Mutational characterisation utilising plasma (PL)-derived circulating tumour DNA (ctDNA) in multiple myeloma (MM) has been recently described. Mutational analyses of paired bone marrow (BM) MM cell DNA and ctDNA from 76 patients (n = 24, new diagnosis (ND), n = 52, relapsed/refractory (RR)) for (ras/raf signaling pathway) and tumour protein p53 (TP53) mutations using the OnTarget™ Mutation Detection (OMD) platform was performed. The total number and proportions of mutations in each of the compartments (BM-specific, PL-specific or shared) was significantly higher in RR patients compared to ND patients (p = 0.0002 and p < 0.0001, respectively). Patients with > 2 mutations or > 1% fractional abundance (FA) in the PL had significantly shorter overall survival (OS) (p = 0.04 and p = 0.0006, respectively). Patients with PL-specific TP53 mutations had significantly shorter OS compared to patients with no PL-TP53 mutations (p = 0.003), while no differences were observed in patients with (K-ras) KRAS mutations. Targeted deep amplicon sequencing (TAS) of matched PL and BM samples from 36 MM patients for DNA-repair and RAS-RAF pathway genes found that DNA-repair genes were present at significantly higher levels in the PL when compared to RAS-RAF mutations (p = 0.0095). We conclude that ctDNA analysis identifies a higher prevalence of potentially actionable DNA-repair gene mutated subclones than BM analysis.
RESUMO
In this study, we evaluated the utility of extracellular RNA (exRNA) derived from the plasma of multiple myeloma (MM) patients for whole transcriptome characterization. exRNA from 10 healthy controls (HC), five newly diagnosed (NDMM), and 12 relapsed and refractory (RRMM) MM patients were analyzed and compared. We showed that ~45% of the exRNA genes were protein-coding genes and ~85% of the identified genes were covered >70%. Compared to HC, we identified 632 differentially expressed genes (DEGs) in MM patients, of which 26 were common to NDMM and RRMM. We further identified 54 and 191 genes specific to NDMM and RRMM, respectively, and these included potential biomarkers such as LINC00863, MIR6754, CHRNE, ITPKA, and RGS18 in NDMM, and LINC00462, PPBP, RPL5, IER3, and MIR425 in RRMM, that were subsequently validated using droplet digital PCR. Moreover, single nucleotide polymorphisms and small indels were identified in the exRNA, including mucin family genes that demonstrated different rates of mutations between NDMM and RRMM. This is the first whole transcriptome study of exRNA in hematological malignancy and has provided the basis for the utilization of exRNA to enhance our understanding of the MM biology and to identify potential biomarkers relevant to the diagnosis and prognosis of MM patients.
RESUMO
Monitoring tumour burden and therapeutic response through analyses of circulating cell-free tumour DNA (ctDNA) and extracellular RNA (exRNA) in multiple myeloma (MM) patients were performed in a Phase Ib trial of 24 relapsed/refractory patients receiving oral azacitidine in combination with lenalidomide and dexamethasone. Mutational characterisation of paired BM and PL samples at study entry identified that patients with a higher number of mutations or a higher mutational fractional abundance in PL had significantly shorter overall survival (OS) (p = 0.005 and p = 0.018, respectively). A decrease in ctDNA levels at day 5 of cycle 1 of treatment (C1D5) correlated with superior progression-free survival (PFS) (p = 0.017). Evaluation of exRNA transcripts of candidate biomarkers indicated that high CRBN levels coupled with low levels of SPARC at baseline were associated with shorter OS (p = 0.000003). IKZF1 fold-change <0.05 at C1D5 was associated with shorter PFS (p = 0.0051) and OS (p = 0.0001). Furthermore, patients with high baseline CRBN coupled with low fold-change at C1D5 were at the highest risk of progression (p = 0.0001). In conclusion, this exploratory analysis has provided the first demonstration in MM of ctDNA for predicting disease outcome and of the utility of exRNA as a biomarker of therapeutic response.
