The Study Of RAS/RAF Gene Mutation And Its CircRNA Expression Profile In Multiple Myeloma

Background:Oncogenic mutations in BRAF,KRAS and NRAS genes have been reported in multiple myeloma and can be explored as biomarkers for selection of targeted therapies.Methods:Unamplified DNA from CD 138+bone marrow mononuclear cells and circulating tumor DNA(ctDNA)from serum and bone marrow aspirates from patients with multiple myeloma(MM)was tested for BRAF V600E,KRAS G12/G13,NRAS G12/G13 and NRAS Q61 mutations using multiplex assays for droplet digital PCR(BioRad).Agreement among methods was evaluated and results we compared to clinical outcomes.MM cell lines were treated with pan-Raf inhibitor LY3009120 to detect whether the proliferation of MM cells will be suppressed.Results:Of 85 patients with multiple myeloma,4(5%)were found to have BRAF V600 mutation(all confirmed V600E),15(18%)KRAS G12/G13 mutation,3(4%)NRAS G12/G13 mutation and 15(18%)NRAS Q61 mutation in archival tumor tissue.Most mutations were subclonal with a median variant allelic frequency(VAF)1.4%(range,0.13%-34%).Survival data were available for 47 patients and patients with high mutation VAF had a trend to shorter survival than patients with low mutation VAF(24 months vs.56 months,P=0.38).Of these 85 patients 20(23.5%)had simultaneous serum ctDNA collection,which demonstrated BRAF V600 mutation in 7(35%;3 confirmed as V600E),KRAS G12/G13 mutation in 2(10%),NRAS G12/G13 mutation in 0(0%)and NRAS 061 mutation in 4(20%)of patients.In addition,13(15%)patients had simultaneous bone marrow serum aspirate ctDNA collection,which demonstrated BRAF V600 mutation in 1(8%;confirmed as V600E),KRAS G12/G13 mutation in 0(0%),NRAS G12/G13 mutation in 0(0%)and NRAS 061 mutation in 1(8%)of patients.Agreement rates for all the 3 samples were more than 70%.Frequent overexpression of Raf isofroms and downstream activation of p-MEK1,2/p-ERK1,2 were found in MM cell lines.The anti-MM effect of pan-Raf inhibitor did not correlated with RAS mutation status,and pan-Raf activated both RAS/RAF/MEK/ERK and PDK/Akt signal pathways.Conclusion:Common oncogenic mutations in BRAF,KRAS and NRAS genes are prevalent with relatively low VAF in multiple myeloma and can be detected with a sensitive techniques such as ddPCR.Their prognostic significance and therapeutic utility needs to be further investigated.Pan-Raf inhibitor might be a novel reasonable treatment approach for MM patients with or without RAS/RAF mutation.Background:In recent years,circular RNAs(circRNAs)have attracted considerable attention because they play a significant role in many fields of cancer biology.However,circRNA in multiple myelomaOMM)is still a blank research field.Method:Twelve MM and eight healthy people were involved in this study.Paired-end reads were harvested from lllumina HiSeq 4000 sequencer,and were quality controlled by Q30.After 3 ' adaptor-trimming and low quality reads removing by cutadapt software(vl.9.3).The high quality trimmed reads were used to analyse circRNAs.The high quality reads were aligned to the reference genome/transcriptome with STAR software(v2.5.1b)and circRNAs were detected and identified with DCC software(v0.4.4).edgeR software(v3.16.5)was used to normalized the data and perform differentially expressed circRNA analysis.GO and KEGG analysis were performed for the differentially expressed circRNA-associated genes.The circRNA-miRNA network has also been predicted.Results:In this study,we identified 105 significantly and differentially expressed circRNAs,including 84 that were up-regulated and 21 that were down-regulated.GO analysis showed that these differentially expressed circRNAs might be related to peptidyl-amino acid modification,intracellular membrane-bounded organelle,histone-lysine N-methyltransferase activity and so on.KEGG analysis showed that the most highly correlated pathways include transcriptional misregulation in cancer,thyroid hormone signaling pathway and so on.The circRNA-miRNA network predicted the potential roles of these differentially expressed circRNAs and the interaction of circRNAs with miRNAs.Conclusion:Our study broadens the horizon of research on circRNA in MM and provides novel targets for further research.These circRNA we discovery might be the new biological marker of MM.