Identification And Validation Of Multi-omics Markers For Parathyroid Carcinoma

ObjectivesParathyroid carcinoma(PC)is a rare endocrine tumor with poor prognosis and often has difficulty making an accurate diagnose.Currently,the pathogenesis of PC remains unclear and effective markers for diagnosis and classification of PC are still lacking.The aim of the present study was to systematically screen and validate the candidate diagnostic markers of sporadic PC at genomic and transcriptomic levels,respectively,and to explore their biological functions and potential of clinical applications.MethodsWe collected the clinical parametres and samples from 23 patients with PC,41 patients with parathyroid adenoma(PA).Whole-genome sequencing was used to analyze the mutational spectrum and signature of the coding and non-coding regions of the genome.The long non-coding RNA(lncRNA),circular RNA(circRNA)and mRNA markers were screened by high-throughput expression profiling microarrays.Various bioinformatics methods were applied for prediction of biological function and co-expression analysis of the candidate markers.Then,candidate markers including lncRNA,circRNA,mRNA and microRNA(miRNA),were validated in extended samples using Sanger sequencing,immunohistochemistry staining,quantitative real-time reverse transcription polymerase chain reaction(RT-qPCR),in situ hybridization(ISH)and fluorescence ISH.Binary logistic regression analysis and receiver operating characteristic curve analysis were used to assess the efficacy of the model for differential diagnosis.ResultsUsing whole-genome sequencing,we found that the most common genetic variant in sporadic PC was CDC73(39.1%,9/23).CDC73 mutation significantly correlated with increased tumor mutational burden(P=0.026)and tumor recurrence/metastasis(P=0.007).Genetic variants in the PI3K/AKT/mTOR pathway were detected in 78.3%(18/23)of PC.PC with wild-type CDC73 harboured mutations associated with antigen presentation and autoimmune diseases.Using lncRNA expression profiling microarrays,we found that compared with PA,2641 lncRNAs and 2165 mRNAs were differentially detected in PC.Bioinformatics analysis showed that PC were mainly involved in the extracellular matrix(ECM)-receptor interaction and energy metabolism pathways,while ATF3,ID1,FOXM1,EZH2 and MITF may be crucial upstream regulators.The differentially expressed lncRNAs and mRNAs of the 'plateau' profiles were selected as the most significant patterns to build the co-expression network.Among the lncRNAs selected for validation,lncRNA PVT1 and lncRNA GLIS2-AS1 have novel diagnostic values,and yielded the area under curve(AUC)of 0.871(P<0.001)and 0.860(P<0.001),respectively.Using circRNA expression microarrays,we found that 2521 up-regulated and 2232 down-regulated circRNAs were identified in PC.Among the circRNAs selected for validation,hsa_circ₀075005 showed a good distinguishing potential with an AUC of 0.770(P=0.001).A high expression level of hsa_circ₀035563 significantly correlated with CDC73 mutations(P=0.022)and disease recurrence in PC patients(P=0.042).Using RT-qPCR,we found that miR-222 expression was significantly up-regulated in PC(P=0.041),while miR-139,miR-30b,miR-517c,and miR-126*were significantly down-regulated(P<0.05).The combination of miR-139 and miR-30b was the best diagnostic marker and yielded an AUC of 0.888.In addition,serum calcium,intact parathyroid hormone(iPTH)and alkaline phosphatase levels were negatively correlated with miR-30b level(P<0.001).ConclusionsCDC73 and PI3K/AKT/mTOR pathway components play an important pathogenic role in sporadic PC in the Chinese population and are expected to be potential diagnostic markers at the genomic level.Meanwhile,several RNA transcripts,such as lncRNA PVT 1,lncRNA GLIS2-AS1,hsa_circ₀035563,hsa_circ₀075005 and the combination of miR-139 and miR-30b,are important in the molecular mechanisms of PC initiation and are expected to be novel diagnostic markers and therapeutic targets at the transcriptomic level.