Study On The Mechanism Of Sarcomatoid Components In Sarcomatoid Renal Cell Carcinoma Based On Whole Transcriptome Sequencing And Exome Sequencing

Objective To analyze the characteristics of gene changes of carcinomatous and sarcomatoid components in sarcomatoid renal cell carcinoma(SRCC)by whole transcriptome sequencing and exome sequencing.To verify the sequence information of rs3802711 status of Cadherin 23(CDH23)gene in SRCC and clear cell renal carcinoma(CCRCC)by expanding the sample size.To investigate the expression of CDH23 protein,and the association between its expression and clinicopathological features of SRCC and CCRCC.Methods 1.Pathogenic genes were screened in paired carcinomatous and sarcomatoid componets of 5 SRCC cases by high-throughput sequencing.2.Sanger sequencing was performed on 40 SRCC patients and 50 CCRCC patients to check the SNP status of CDH23 rs3802711 selected from the SNP pool with high mutation frequency based on the whole exome sequencing.These samples were detected by immunohistochemistry(IHC)to measure the expression differences of CDH23 protein.Statistical analysis was used to explore the difference of CDH23 rs3802711 status and CDH23 protein expression in SRCC and CCRCC,and the association of CDH23 gene status and protein expression with clinicopathological characteristics and the prognosis of renal cell carcinoma with or without sarcomatoid differentiation.Results 1.Analysis of transcriptome sequencing results showed 582 new genes were found,of which the function of 283 genes were annotated.A total of 15 common down-regulated genes in the sarcomatoid tissues.2.Somatic single nucleotide variants(SSNVs)in SRCC were screened in the 5 groups by whole exome sequencing.Among them,455 SSNVs and 346 SSNVs were found in sarcomatoid and carcinomatous tissues of more than one group,respectively.The two tissues shared 37.1%-61.6%SSNVs.There were 25 common genes in more than three groups of carcinomatous tissues in exon area,including 13 missense mutation genes.There were 21 genes variant in the exon region of sarcomatoid tissues with 8 missense mutation genes in more than three groups.CDH23 rs3802711 mutant genotype was found in 3 sarcomatoid tissues and 2 carcinomatous tissues simultaneously.3.CDH23 rs3802711 mutant genotype was found in 22 of 40 SRCC cases and the same mutant genotype were found in normal kidney,carcinomatous and sarcomatoid tissues.Only 10 among 50 CCRCC cases presented rs3802711 mutant genotype,with normal renal tissues and carcinomatous tissue showing the same mutant genotype.There was significant difference in CDH23 rs3802711 mutant genotype(?2=11.88,P=0.001)between 40 SRCC and 50 CCRCC tissues.No significant difference between CDH23 rs3802711 status and the clinicopathological features and prognosis of SRCC was revealed.However,Kaplan-Meier analysis showed that mutant genotype,histological types,metastasis and TNM stage were associated with overall survival in total cases(P<0.001).Cox multivariate regression analysis showed that mutant genotype and diameter were independent prognostic factors of overall survival in total samples(P<0.001).4.CDH23 protein was negatively or low expressed in most SRCC samples,and the expression of carcinomatous tissue was consistent with that of sarcomatoid tissue.CDH23 protein was highly expressed in CCRCC samples.The positive expression rates of CDH23 in SRCC and CCRCC were 42.5%(17/40)and 76%(38/50),respectively(?2=10.494,P<0.001).In SRCC,there was no significant correlation between CDH23 expression and clinicopathological features.CDH23 rs3802711 was correlated with CDH23 protein expression in total samples(r=0.59,P=0.000).Conclusion There are differences in gene variations between carcinomatous and sarcomatoid tissues at transcriptome and exome level in SRCC.However,the two components own most of the same somatic mutations,indicating that they might originate from common precursor cells.CDH23 rs3802711 mutant genotype exists in most SRCC.CDH23 gene mutation leads to the abnormal expression of CDH23 protein,further resulting in epithelial-mesenchymal transformation,which is the hallmark of sarcomatoid differentiation.CDH23 rs3802711 mutant genotype might be a critical gene in SRCC.