The Association Of Copy Number Alterations And Hotspot Mutations With Clinicopathological Factors In Esophageal Squamous Cell Carcinoma

Part 1Genome wide copy number analyses of superficial esophageal squamous cell carcinoma with and without metastasisAims:Superficial esophageal squamous cell carcinoma(ESCC)is generally considered a subtype of less invasive ESCC.Endoscopic resection remains the most widely used treatment option for superficial ESCCs because it is safe and esophaguspreserving.Yet a subset of these superficial ESCC would have metastasis after endoscopic resection and lead to poor prognosis.Thus identifying biomarkers that can assess the metastatic risk in superficial ESCC is of particular importance.The objective of this study is to determine biomarkers that can identify such subset of superficial ESCC that would have metastasis after surgery using genome wide copy number alteration(CNA)analyses.Methods:Thirty eight superficial ESCC patients,at stage T1N0M0,were collected from Cancer Hospital,Chinese Academy of Medical Sciences.The CNAs of 38 cases of superficial ESCCs originated from radical surgery,including 19 without metastasis and 19 with metastasis within 5 years' post-surgery,were analyzed using Affymetrix OncoScanTM FFPE Assay.Real Time PCR was used to validate CNAs of genes.Results:1.A 39-gene signature was identified which characterized the subset of superficial ESCC with high risk of metastasis after surgery(Mann-Whitney U-test,p<0.05).Hierarchical clustering was performed on the CNA profiles of these 39 genes and two groups were obtained,including metastasis and non-metastasis groups.In general,good separated performance of metastasis and non-metastasis was obtained with a small amount of misclassification(two metastasis misclassified and five non-metastasis misclassified).The 39 genes included FGF4,MRAS,CHECK 1 and other genes.2.We found 28 significantly recurrent focal CNAs,including 16 amplifications and 12 deletions in all 38 superficial ESCCs.The most common CNAs were amplifications of llql3.3(FGF4),3q28(TP63),14q21.1(FOXA1),8q24.21(MYC),and deletions of 9p21.3(CDKN2A),22q11.23(GSTT1),3p13(MITF)and 2q22.1(LRP1B).3.In metastasis group,eight recurrent focal CNAs were found,including 4 amplifications and 4 deletions.In non-metastasis group,fourteen recurrent focal CNAs were found,including 12 amplifications and 2 deletions.Notably amplifications of 3p26.33(SOX20T),8q24.21(MYC),14q21.1(FOXA1)and deletion of 3p12.1(GBE1)were only found to be recurrent in metastaic superficial ESCCs.Conclusioins:Using CNAs analyses,we identify a 39-gene signature which characterizes the high risk metastatic superficial ESCCs and discover several recurrent CNAs that might be the driver alterations in metastasis among superficial ESCCs.Part 2Targeted sequencing of 44 frequently mutated genes in esophageal squamous cell carcinomaAim:Targeted cancer therapies are more therapeutically beneficial than traditional chemotherapy.The identification of appropriate therapeutically targets depends on identification of the molecular changes and understanding underlying biological mechanism in cancer.In order to improve understanding of the genetic basis of esophageal squamous cell carcinoma(ESCC),several large scale genomic next generation of sequencing(NGS)analyses have been implemented.Although these analyses revealed mutational profiles of ESCC,the significance and potential application of the genes to be prognostic and potentially therapeutic biomarkers for ESCC patients are still limited.Methods:This study include 119 ESCC patients,who underwent surgical resection at Cancer Hospital,Chinese Academy of Medical Sciences between 2004 and 2009.According to published studies and Catalogue of Somatic Mutations in Cancer(COSMIC)database,we initially established a targeted NGS panel,which consisted of 44 frequently mutated genes,including genes of cell cycle,histone regulator,and NOTCH pathway and others.Ion Torrent Personal Genome Machine was used to perform targeted sequencing on the 119 ESCC formalin-fixed and paraffin-embedded tissues from patients with 5-year follow-up data.Results:1.One hundred and nineteen ESCC patients' specimens were assessed by targeted sequencing in the study.The average read depth per sample was 1036.9×(range from 521.2× to 3155.O×).The average uniformity and percentage of reads on target regions were 94.89%(range from 87.84%to 97.11%)and 96.83%(range from 90.15%to 98.73%),respectively.2.The average number of mutations was 3(range from 0 to 13)per sample.The predominant substitution subtype across all ESCC samples on target regions were C>T transitions which occurred mainly in CpG dinucleotide.3.A total of 389 somatic mutations were identified,including 21 recurrent mutations and 255 mutations occurred only once.The most frequently mutated gene was TP53(54.6%),followed by KMT2C(40.3%),NOTCH1(35.3%),PIK3CA(10.9%),FAT1(10.1%),CSMD3(9.24%),FAT2(9.24%),KMD2T(9.24%)and other genes.4.There were 112 oncogenic or likely oncogenic variants,160 unknown significant variants variants in the study.Among the 119 ESCC patients,14 patients had potential therapeutic targets,including EGFR p.L858R(2/119),BRCA2 p.R2842H(2/119),PIK3CA p.E542K(4/119),PIK3CA p.E545K(4/119)and PIK3CA p.H1047R(2/119)which mutated recurrently in these patients.5.Histologic grade,KMT2C and ZNF750 gene mutations were significantly correlated with overall survival in ESCC patients(p<0.05).In addition,the three features were also applied to construct a nomogram with concordance index 0.673 and a random survival forest model with prediction error 0.3484.Conclusions:The targeted sequencing results indicated that there were 11.8%(14/119)of ESCC patients might be qualified to receive targeted cancer therapies.KMT2C and ZNF750 gene mutations might be as the prognostic biomarkers for ESCC patients.We initially developed a targeted NGS panel which might be used for future clinical application for ESCC patients to provide prognostic and potentially therapeutic biomarkers.