Analysis Of Lymph Node Metastasis And Cellular Immune Characteristics Of Papillary Thyroid Carcinoma

Objective:To explore the effect of papillary thyroid carcinoma(PTC)induced individual immune status regional immune response on its biological behavior,the relationship between age at diagnosis,the tumor size,and lymph node metastasis(LNM)was analyzed at a population level,T cell receptors(TCR)composition in tumor,regional lymph nodes and peripheral blood was compared,and the epitope of RET/PTC1 gene fusion product was predicted and validated.Methods:1.Adult surgically-treated PTC and follicular thyroid carcinoma(FTC)patients were identified from the Surveillance,Epidemiology,and End Results(SEER)database(2004-2015)to analyze the risk factors of LNM,and investigate the relationship between age at diagnosis and clinical characteristics by curve estimation.The adjusted odds ratio of age at diagnosis and LNM rate was determined using a multivariate logistic regression model adjusted for other risk factors.2.Peripheral blood mononuclear cells(PBMC),lymphocytes derived from cervical lymph nodes(LN)and the tumor tissues(T)of PTC patients were collected to performe Immune Repertoire Sequencing(IR-Seq),as well as bioinformatics and statistical analysis.3.SYFPEITHI and net MHCpan4.0 were used to predict the epitope of RET/PTC1 gene fusion product,then the corresponding pentamer was customized.We picked up PTC patients with HLA-A*11:01 genotype and RET/PTC1 rearrangement,then utilized pentamer to test their PBMC and LN for specific CD8~+T cells and cultured in vitro.Results:1.A total of 50,347 patients(PTC,48,166;FTC,2,181)(median age:45 and 50 years,respectively)from SEER database met the inclusion criteria;44.5%of PTC(21,428)patients had LNM.The distribution of age at diagnosis was different between LNM-positive and LNM-negative PTC patients(P<0.0001).The relationship between age at diagnosis,tumor size,and LNM described a quadratic curve in PTC.The mean tumor diameter and LNM rate correlated linearly with age at diagnosis in patients aged 18-59 years.LNM rate decreased with age(R~2=0.932,P<0.0001),especially in women(R~2=0.951,P<0.0001).After adjusting for other risk factors of LNM,compared with 18-year-old PTC patients,the diagnosis age is a protective factor of LNM,but the protective effect is gradually weakened.2.The analysis of the immune repertoire showed that there was no statistical difference in the total raw reads and total TCR CDR3 of the three groups(PBMC,LN and the tumor tissues).The unique CDR3 was statistically different amoung the three tissues(p=0.0002);the unique CDR3 of LN tissues was higher than the PBMC,and both were higher than the tumor tissues.At the same time,the CDR3 diversity index(Shannon diversity and D50)among the three groups were statistically different.The CDR3diversity index of the LN tissues was higher than the PBMC and the tumor tissues(T),meanwhile the PBMC and the tumor tissues(T)had no statistical difference.The amino acid level CDR3 HEC number(p=0.0003)and amino acid level TCR CDR3V-J rearrangement HEC(p=0.0013)of the three groups were statistically different;of which the tumor tissues(T)was higher than the PBMC,and the latter was higher than the LN tissues.3.SYFPEITHI and net MHCpan4.0 were used to predict the epitope of RET/PTC1 gene fusion product,and the HLA-A*11:01 restricted ASVTIEDPK was screened as a potential antigen epitope.In 9 PTC patients with HLA-A*11:01 genotype and RET/PTC1 rearrangement,flow cytometry analysis of pentamer in PBMC showed that specific CD8~+T cells accounted for 0.69%-1.64%.The PBMC of 4 patients were stimulated in vitro,and the proportion of specific CD8~+T cells increased.Conclusion:1.In some PTC patients with young and middle-age,tumor may shrink and LNM may resolve spontaneously with delayed diagnosis and management.Active surveillance of low-risk PTC is justified.2.Compared with lymph node samples,tumor samples have low CDR3 diversity,high amino acid level CDR3 HEC,low amino acid level CDR3 V-J rearrangement number,and high V-J HEC,indicating that T cells in tumor tissue may directly activate and participate in anti-tumor immunity comparing with lymph node tissue and blood.3.In PTC patients with RET/PTC1 rearrangement,HLA-A*11:01 restricted ASVTIEDPK is its epitope.