| Research background and aim: It is worth noting that the incidence rate of thyroid carcinoma has been on a continuous rise in China and other countries in recent years,which brings a huge burden to both the patients and the healthcare systems.Papillary thyroid carcinoma(PTC)is the most common subtype,accounting for approximately 80% to 85% of all thyroid carcinoma.Papillary thyroid carcinoma is reported to have a high rate of multifocality and that indicates multiple cancer foci are present simultaneously within one thyroid gland.However,there is currently no satisfying research method as to the determination of the clonal relationship among these foci despite the common occurrence of multifocality.The invention and application of the next-generation sequencing(NGS)technologies has promoted the understanding the progression of cancer,drug resistance and the prognosis management of cancer patients to an unprecedented extend.As a typical exemplification of NGS,whole genome sequencing(WGS)has become an affordable choice for research and clinical purposes thanks to the technological optimization and the sharp dropping of the cost.This study aimed at(1)characterizing the m PTC patients and its clinical features by analyzing the patient profiles in a 2 year period from the Department of Thyroid and Breast Surgery of the first affiliated hospital to our university,(2)determining the clonal relationship among different cancer foci using single nucleotide variant,insertion/deletion,copy number variation and structural variation derived from the sequencing data of m PTC patients' cancer foci.Methods: The electronic medical records of the newly diagnosed papillary thyroid carcinoma between January 2013 and May 2015 in the Department of Thyroid and Breast Surgery of the first affiliated hospital to our university were collected.The data of age,gender,number of cancer foci,primary tumor size,foci location,coexistence of complications,metastasis and other indexes were used for statistics.The clinical features were statistically tested separately in male and female to distinguish the unique feature of m PTC.After obtaining the approval from the patients,the preoperative peripheral blood was collected along with the surgical resected thyroid cancer foci and adjacent non-tumor tissue available.The genomic DNA was isolated and subjected to library construction and qualitycontrol after pathological confirmation.The whole genome sequencing was conducted on Hiseq X Ten.After establishing the bioinformatic handling pipeline of WGS data,the sequencing data from each cancer foci underwent the analysis of quality control,alignment,filtering,somatic mutation discoveries,driver mutation identification,intratumor heterogeneity and clonal evolution inference by bioinformatics tools.The clonal relationship of each cancer foci with their counterpart was investigated.What's more,Sanger sequencing and immunohistochemistry analysis of BRAF V600 E were completed for confirmation.Results: The clinical data from a total number of 920 papillary thyroid carcinoma between January 2013 and May 2015 were involved in the analysis,contributed by 636 female patients and 284 male patients.The average diagnostic age was 45.69±12.68.Among these 920 patients,323(35.1%)patients were diagnosed as multifocal PTC patients with at least two cancer foci found in the thyroid gland.The rate of lymph node metastasis was higher in m PTC than single PTC for both genders(Male: 47.8% vs.25.8% with P<0.001;Female: 31.3% vs.22.1% with P=0.01).The primary tumor size of m PTC was larger than that of single PTC in female patients(P<0.001).Besides,the number of non-cancerous lesion concurrence rate were higher in single PTC than m PTC in the female group(P<0.001).In total,8 cancer foci and 3 matched germline control from 3 m PTC patients(designated as m PTC_P1-P3)were sequenced by whole genome sequencing after pathological confirmation(Targeted sequencing depth was set for 50 X and 30 X for tumor and germline sample respectively).The sequencing experiments produced an entire amount data of 1897 Gbases.According to the comparison of the mutation sites and frequencies of SNVs across the whole genome,the clonal relationship among the cancer foci in the m PTC_P1 and m PTC_P2 was determined as clonal independent(Different cancer foci developed separately)while m PTC_P3 had clonal derived pattern for the three cancer foci(The cancer foci were formed by gland spreading).The results of WGS,Sanger sequencing and immunohistochemistry consistently showed that all the 8 cancer foci shared the BRAF V600 E.Compared to the primary cancer foci,the lymph node metastasis of m PTC_P3 had 3 exonic SNVs in common and also 2 private exonic mutations.In addition,the three cancer foci of m PTC_P3 shared a copy number loss of 22 q.The mutational signature analysis revealed the presence of signature 13 and signature 2 associated with the activity of AID/APOBEC in two cancer foci of m PTC_P3.Based on the clonal SNVs,5 of 8 foci showed intratumor heterogeneity.Clonal evolution analysis indicatedthat the lymph node metastasis was an early event for m PTC_P3.Conclusions:(1)Papillary thyroid carcinoma commonly appeared in a multifocal way.The percentage of multifocal PTC reached 35.1%(323/920)in our cohort.(2)Multifocal PTC patients were found to have unique clinical features including a higher lymph node metastasis rate than that of s PTC(Male: 47.8% vs.25.8% with P<0.001;Female: 31.3% vs.22.1% with P=0.01)and a larger primary tumor diameter in female patients(P<0.001).In addition,the non-cancerous lesion concurrence rate were higher for s PTC than m PTC(P<0.001).(3)The clonal relationship of the 8 cancer foci were successfully resolved,in which 5 cancer foci had a clonal independent model and the other 3 had clonal derived model.(4)The non-exonic mutations could offer valuable information for the clonal relationship analysis.(5)Whole genome sequencing technology would surpass the traditional methods in clonal relationship analysis due to wider applicability and higher reliability.Besides,it could provide vital messages including tumor heterogeneity,clonal evolution process and the molecular genetic characteristics of cancer. |
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