| Objective:Ossifying fibroma(OF)and fibrous dysplasia(FD)are two fibro-osseous lesions with overlapping clinicopathological features,making diagnosis challenging.In this study,we applied Multiregional Microdissected sequencing approach to facilitate differential diagnosis via precise profiling of copy number alterations(CNAs)and transcriptome analysis using minute amounts of DNA and RNA extracted from morphologically correlated microdissected tissue samples.Methods:1.A total of 29 cases(220 samples)of OF and 28 cases(174 samples)of FD arising from the jaws,as well as their adjacent normal tissues,collected from 2008 to 2018 were obtained from the tissue bank of Peking University Hospital of Stomatology.All samples were obtained with the approval of the Ethics Committee of the Peking University Hospital of Stomatology.Records were reviewed,and the diagnosis was confirmed by three experienced pathologists according to the current clinical,histological,radiolographic criteria and for OF and FD.Once removed,all fresh tissues were snaply frozen in liquid nitrogen and immediately stored at80?.Genomic DNA from FD patients was extracted and conventional PCR and Sanger sequencing were performed to analyze the presence of GNAS mutations at exon 8 and 9.2.Spatial genomic copy number alteration and transcriptome analysis:OF and FD samples were collected via the LCM(Laser Capture Microdissection)System.To construct the whole genome and transcriptome libraries using direct lysis/Tn5 transposase protocols and SMARTseq2 magnetic bead purification.Then genomic and transcriptome differences between the two diseases were analyzed by high-throughput sequencing.3.Through the comprehensive analysis of CNA and RNA-seq,potential target genes that may cause OF were identified and further validated by qPCR.4.Comprehensive correlation analysis between clinicopathological features and CNAs was performed.Results:1.It is the first to reveal the molecular map OF copy number alteration in the OF and FD genomes through CNA.We found that the rate of recurrent CNAs in OF cases was much higher(44.8%,13 of 29,p<0.001)than that in FD cases(3.6%,1 of 28).Sixty-nine percent(9 of 13)of the CNA-containing OF cases involved segmental amplifications and deletions on Chrs 7 and 12.2.We also identified eight CNA-associated genes(HILPDA,CALD1,C1GALT1,MICALL2,PHF14,AIMP2,MDM2,and CDK4)with amplified expression,which was consistent with the copy number changes.In addition,it was found that the above 8 genes were up-regulated in OF by RNA-seq,indicating that the results of CNA and RNA-seq were consistent.3.Systematic clinicopathological analysis showed that the age of onset(p=0.043)and location(p<0.001)of OF and FD were statistically different,and there was no correlation between the occurrence of CNA and the age,sex,site and histological subtype of OF.We further confirmed a jaw lesion with a previous uncertain diagnosis due to its ambiguous morphological features and the absence of GNAS mutation as OF based on the typical Chr 12 amplification pattern in its CNA profile.Moreover,analysis of a set of longitudinal samples collected from an individual with a cellular lesion in suspicion of OF at the first surgery,recurrence and the latest malignant transformation revealed identical CNA patterns at the three timepoints.Conclusions:The results showed that the genomic copy number alteration of OF and FD was signifitly different;CNAs were more common in OF than FD.The segmental amplification and deletion of chromosomes 7 and 12 is a key molecular event with high specificity and recurrence in OF patients.Overall,CNA and RNA profiling of fibro-osseous lesions greatly improve differential diagnosis between OF and FD and help predict disease progression. |
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