Genomic Profile And Clinical Implications In Primary Immunoglobulin Light Chain Amyloidosis

Backgroud and ObjectivesPrimary immunoglobulin light chain amyloidosis(pAL)is a clonal,nonproliferative plasma cell disorder in which fragments of immunoglobulin light chain are deposited in tissues.Clinical features depend on organs involved but can include restrictive cardiomyopathy,nephrotic syndrome,hepatic failure,peripheral/autonomic neuropathy.It is known that pAL is mainly caused by conformational changes in subunit proteins,but the mechanism of conformational changes remains unknown.Next generation sequencing technology has been widely used in clinical research due to its high throughput,high efficiency and low cost.For hematological malignancies,especially rare diseases,finding specific gene mutation for diagnosis and treatment,has become an important research tool in this field.However,there is a lack of systematic research on pAL genomic profile and the combination of gene mutation and clinical implications.Methods and MaterialsPatients with pAL seen at Peking Union Medical College Hospital between December 2015 and May 2017,were included in the current study.Clinical information,serum and bone marrow samples were collected with the Institutional approval.For each bone marrow samples,CD 138 positive cells were sorted using monoclonal antibody(anti-CD 13 8)-tagged magnetic microbeads,and DNA was extracted from the CD 138 positive plasma cells and peripheral blood mononuclear cells using DNA Kit.Moreover,whole-exome sequencing(WES)was performed in pAL cases with their peripheral blood as normal control.After screening for significantly mutated genes(SMGs)and known driving genes,targeted gene sequencing was further conducted in a large group of pAL bone marrow plasma cells.The association between pAL genomic profile and clinical implications was explored by comparing the number of gene mutations and the mutations of different gene.Statistical analyses were conducted using SPSS 22.0,and a p<0.05 was considered as significant.ResultsIn this study,ten patients were enrolled in WES.A total of 3307 SNVs were detected,and the exon coding region was 14.3%,with non-synonymous mutations accounting for 69.5%(missense mutation 63.6%,stopgain 5.5%,stoploss 0.4%).The number of non-synonymous mutations per million base was 1.22(0.15-3.08)/Mb.Twelve SMGs were screened with FDR values of<0.2.And four of them were associated with NF-κB signaling pathways,including DUSP2,NFKBIA,KRAS,BIRC3,proving the important role of NF-κB signaling pathway in the pathogenesis of pAL.Finaly,fifty-two SMGs and 17 known driving genes were screened out,and 29 reported genes were screened by reviewing the literature.Futhermore,98 genes in 48 pAL patients were subjected to targeted gene sequencing,resulting in 133 single nucleotide variants(SNVs)and 20 insertions/deletions(InDel).The overall mutation frequency was low,with an average of 3(0-13)mutations per patient.Of the 48 patients,41(85%)had at least one SNVs or InDel.The overall mutation frequency is low and the average number of mutations per patient is three.A total of 70 mutant genes were detected.IGLL5 was the most common mutation(17%)in the study,followed by ASCC3(15%).There were 14 patients(29.2%)with mutations in NF-κB signaling pathway-related genes.The number of mean gene mutations in the high-risk group(Mayo2012 stage 3 and 4)was significantly higher than that in the low-risk group(Mayo2012 stage 1 and 2)(3.8±0.5 vs 2.1κ0.5,p=0.021).The overall survival(OS)was negatively correlated with the number gene mutations,with a predicted 1-year survival rate of 52.1%(p=0.022)for patients with four or more mutations and 90.4%for patients with less than four mutations(p=0.005).When compared with Mayo 2012 staging(Area Under the Curve=0.870),the prognostic model of the combined sequencing results increased the predictive potential(AUC=0.915).The study also found four recurrent mutations in the pAL:ASB15(c.844 C>T),ASCC3(c.1595A>G),HIST1H1E(c.311 C>T)and KRAS(c.35 G>A).Survival analysis showed poor prognosis in patients with ASB15 or HIST1H1E mutations(p<0.05).ConclusionThe overall mutation frequency of clonal plasma cells in pAL is low.The number of gene mutations was negatively correlated with the prognosis of the patients.When the number of gene mutations was more than 4,the prognosis was significantly worse.And incorporation of the mutation data showed an increase in the prognostic potential.There are four significantly mutated genes related to NF-κB signaling pathway(DUSP2,NFKBIA,KRAS,BIRC3),suggesting that activation of NF-κB signaling pathway may be related to the pathogenesis of pAL.Four recurrent mutations were found in pAL:ASB15,ASCC3,HISTIHIE,KRAS,and there was a poor prognosis in patients with ASB15 or HIST1H1E mutations.