Clinical Features And Whole Exosome Sequencing Of Renal Collecting Duct Carcinoma

Objective There is still a debate about the clinical,pathological and prognosis features,and treatment of collecting ducts carcinoma(CDC).The genomic profile of Chinese population with CDC remains to be uncovered.Our goal is to summarize the clinical,pathological,treatment and prognosis features of CDC;to initially construct the single nucleotide variation(SNV),copy number variation(CNV)and germline mutation spectrum for Chinese CDC patients.Methods Nineteen patients confirmed by pathological examinations by pathologists from Guangdong Second Provincial Hospital and Changhai Hospital,were retrospectively analyzed.Clinical,auxiliary,pathological,and surgical information,postoperative adjuvant treatment and follow-up data of these 19 included patients were summarized.Ten normal paired CDC patient samples included were further used to perform whole exom sequencing(WES).After quality control by Fastqc,the reads obtained were compared with the human reference genome(hg38)using BWA.MuTect2 detected both SNVs and small insertions or deletions(Indel).The identified mutations were annotated with ANNOVAR,and then validated by the Sanger sequencing.Control-FREEC was used to detect CNVs,and GISTIC was applied to detect the frequently mutated altered regions.Varscan2 was used to detect the germline mutations,which will be annotated by the Intervar database.A comparsion of genomic mutations was conducted between our study and other TCGA databases,including the kidney renal clear cell carcinoma(KIRC),kidney renal papillary cell carcinoma(KIRP)and bladder urothelial carcinoma(BLCA).The real time quantitative PCR,western blotting and immunofluorescence experiments were conducted to exam the expression of CDKN2A.Results There were 15 males and 4 females patients,with a mean age of(58.9±8.7)years.Gross hematuria and lumbago were the main first symptoms.Computed tomography showed a circular heterogeneous mass,and a "slow in and slow out"enhanced manifestation.MRI frequently indicated a low signal on the T2WI sequence.There were 7 cases on the left size and 12 cases on the right side.The maximum diameter of tumors of these patients was(6.6±2.9)cm.Regionally lymph node metastases were found to occur in 12 patients,while distant metastases were found in 4 patients.Seventeen patients received the surgical treatment,while two underwent puncture biopsy.Pathological HE staining showed that CDC tumor cells were stained as irregular glandular tubular or papillary shape microscopically,with a stroma fibrous tissue hyperplasia and inflammatory cell infiltration.Immunohistochemical staining indicated that the positive expression rates of HCK,PAX8,VIM,p63,GATA3,INI-1,OCT3/4 and FH were 100.0%,83.3%,76.5%,28.6%,16.7%,66.7%,100.0%and 100.0%,respectively.Three patients were post-operatively treated with GC chemotherapy/paclitaxel combined with PD-1.Five patients received targeted drug therapies;however,they displayed different degrees of disease progression with an unsatisfactory clinical efficacy.Fourteen patients were followed up for(23.5±16.0)months on average.The one-,three-and five-year survival rate of these patients was 89.2%,49.0%and 24.5%,respectively.Ten normal-matched whole-exome sequencing data of patients with CDC were finally included to analyze the somatic mutational landscape.The mean tumor mutation burden was 1.37 Mut/Mb.Six new recurrent somatic mutated genes were identified,including RBM14,MTUS1,GAK,DST,RNF213 and XIRP2,which were also validated by the Sanger sequencing.Compared with normal renal tissues,RBM14 and XIRP2 were highly expressed in cancer tissues,while SETD2,MTUS1,CDKN2A and TP53 were low expressed in cancer tissues(p<0.05).Next,twenty-nine amplifications and 6 deletions of recurrent focal somatic copy number variants(CNVs)were identified by GISTIC2.0,which displayed differently from KIRC,KIRP and BLCA cohorts.Of note,in terms of CNV-based genes,CDKN2A and CDKN2A-AS1 were the only overlapped genes of these four cohorts.Importantly,the CDKN2A mutation in our cohort differed from previous studies in the urinary carcinomas.CDKN2A mediated CDK4 signal pathway may be an important signal pathway affecting the development of CDC in Chinese population.Besides,the most frequently altered genomic pathway of our CDC cohort was CDKN2 A-mediated CDK4 pathway.In addition,six new nonsense putative pathogenic germline mutations were obtained from Varscan2.0 germline calling algorithm.Conclusions Renal collecting duct carcinoma tends to develop regional lymph node and distant metastases,with a rapid progress and rather poor prognosis.The gold standard for diagnosis is pathological examination.At present,the main treatment is still surgery.Chemotherapy,targeted therapy and immunotherapy could be selected as adjuvant therapies,but the curative effect is not good.Our study also first offered an initial genomic spectrum of Chinese population with CDC.The recurrently altered CDKN2A mediated CDK4 pathway might provide new insights to potential therapy targets for patients with CDC,especially for those with CDKN2A deletions.