| BackgroundEsophageal cancer,which has a high degree of malignancy and a poor prognosis,is a common malignant tumor of digestive tract worldwide.The mortality rate ranks sixth among all cancers in the world and fourth in China.Most patients with esophageal cancer are already in the locally advanced stage at the time of diagnosis due to the insidious symptom,the overall 5-year survival rate after surgery alone is low.After decades of exploration,neoadjuvant chemotherapy has been proven to downstage tumors,eliminate micrometastasis,and prolong the long-term survival of patients.Neoadjuvant chemotherapy has gradually become one of the main treatments for esophageal cancer,especially for the locally advanced patients.According to the results of multiple clinical studies,the 5-year survival rate of patients with effective neoadjuvant chemotherapy was significantly improved.However,there was no significant survival benefit for nearly 40% of patients who did not respond to neoadjuvant chemotherapy.For these ineffective patients,the related genes and mechanisms have not been clarified,which is an urgent clinical problem to be solved.Therefore,so as to reveal the genetic changes of esophageal cancer and the gene mutation that are ineffective in neoadjuvant chemotherapy and their related drug resistance mechanisms after neoadjuvant chemotherapy,this study intends to perform next-generation sequencing of esophageal cancer sample after neoadjuvant chemotherapy,and perform conformational analysis of related gene mutations to try to find out Genes and resistance mechanisms related to neoadjuvant chemotherapy resistance in esophageal cancer.Methods Retrospectively collect the clinical data of 13 consecutive patients with esophageal cancer undergoing neoadjuvant chemotherapy who were admitted to the Department of Thoracic Surgery of Zhengzhou University Cancer Hospital from 2014 to 2015.Neoadjuvant chemotherapy regimen includes a three-week two-cycle regimen of paclitaxel combined with nedaplatin.Patients underwent surgery within 4 to 8 weeks of completing chemotherapy.The tumor samples of the patients were extracted in the sample library and white blood cell sample were collected from the peripheral venous blood samples before treatment.The tumor DNA were collected from the esophageal cancer tissue in the center of the primary tumor,and the white blood cell DNA were treated with leukocyte suspension.After the purification and extraction of DNA,all samples were sequenced by ONCOSCREEN PANEL,and the tumor tissue sequencing data and leukocyte sequencing data were matched and qualitatively controlled.After successful pairing and quality control,the gene mutations detected by leukocyte sequencing were considered as germline mutations,and mutations other than germline mutations detected by tumor tissue sequencing were considered as somatic mutations.The gastroscope specimens of 4 patients with different chemotherapy responses were followed up for the next-generation sequencing,and the sequencing results after neoadjuvant chemotherapy were compared to find out the changes of gene mutation before and after treatment and to find out the genes related to neoadjuvant chemotherapy drug resistance.Finally,the protein model of transcriptional translation after mutation was constructed by software to study the protein configuration changes caused by different mutation sites of the gene.Results The 13 patients in the group included 2 patients with complete response,6 patients with partial response and 5 patients with stable disease.The tumor samples of 11 patients were successfully matched with leukocyte samples,and 2 patients failed for the first time,and successfully matched with leukocyte samples after re-collecting tumor tissues.The median sequencing depth of all tumor samples and leukocyte samples was more than 500,and the length of insertion fragments was more than 170.In all patients,no somatic mutation was detected in 2 tumor samples whose chemotherapy response was complete response;in all patients,44 somatic mutations(81.5%)were found in 23 genes,and 9 germline mutations(18.5%)were found in 9 genes.Among them,missense mutations(28,63.6%)were more common.The median tumor mutational burden of all tumor samples was 4.13/Mb.Among the somatic mutations,TP53 gene(11 sites,25%)and NOTCH1 gene(10 sites,22.7%)were high frequency mutations in this sequencing,in which NOTCH1 gene mutations were concentrated in the binding sites of NOTCH1 receptors and ligands,and there were gene mutations at the same site(NOTCH1:c.1348 G > A)in two patients.The sequencing results of 4 patients before and after neoadjuvant chemotherapy showed that 22 somatic mutations were detected only before chemotherapy,6 somatic mutations were detected before and after chemotherapy,but the allele frequency of gene mutations after chemotherapy was significantly lower than that before chemotherapy,and 5 somatic mutations were detected only after chemotherapy,including NOTCH1:c.1348 G > A.Considering the correlation between NOTCH1 gene and the efficacy of neoadjuvant chemotherapy for esophageal cancer,five missense mutations at the binding site of receptor protein and ligand encoded by the gene were selected for protein conformational analysis.It was found that NOTCH1:c.1365 G > T(p.Glu455Asp)and NOTCH1:c.1366 T > A(p.Cys456Ser)could not bind NOTCH1 receptor to ligand,and NOTCH1:c.1348 G > A(p.Glu450Lys)would lead to abnormal binding of NOTCH1 receptor to ligand.NOTCH1 gene was considered to be related to neoadjuvant chemotherapy resistance.ConclusionsSomatic mutations are common in patients with residual tumors after neoadjuvant chemotherapy.Neoadjuvant chemotherapy can change the composition of somatic mutations and effectively reduce the abundance of tumor mutations in patients with esophageal cancer.NOTCH1 gene mutation may be associated with drug resistance of neoadjuvant. |
PDF Full Text Request