| Thyroid cancer is a common endocrine malignancy,and its prevalence increases year by year.The papillary carcinoma accounted for more than 80%.It has harmed people's health and increase the economic burden.It is a hot issue of concern today.At present,the methods of diagnosis and auxiliary examination commonly used in clinical methods include ultrasonic diagnostic examination,fine needle aspiration(FNA)cytology examination,serum thyroxine,etc.FNA cytological examination is the most effective method to distinguish benign and malignant thyroid nodules at present,but in clinical practice,about 10% ~ 20% of FNA cytology is difficult to make accurate diagnosis.The conventionalization of differential diagnosis and monitoring methods has always been a major clinical problem.In recent years,the use of proteomics technology has made gratifying progress in early diagnosis of many types of tumors.A series of tumor-associated proteins and potential tumor markers have been discovered and applied clinically.There have been proteomic studies of thyroid cancer at home and abroad,and some proteins associated with thyroid cancer have been found.However,these results are still at an early stage and there are no tumor markers that can be used for clinical diagnosis.Most of the studies have not conducted the differential analysis of benign and malignant tumors,and there have been few reports on thyroid cancer proteomics at home and abroad.Based on the current status of thyroid cancer,we first conducted a retrospective analysis of the incidence of thyroid cancer in our hospital in recent years.From the aspects of gender,age,lymph node status,and composition ratio of pathology,comparative analysis was conducted.Secondly,matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF/TOF MS)was used to detect serum peptides in patients with thyroid papillary carcinoma,benign tumors and normal healthy individuals,and a diagnosis and prediction model of thyroid papillary carcinoma was established.Serum peptides were isolated,identified and functionally analyzed.Finally,protein profile imaging was used to analyze in situ the expression of thyroid papillary carcinoma protein.In situ distribution of differential proteins within tissues.Part ?: A Retrospective Analysis of Clinical and Pathological Characteristics of Thyroid CarcinomaThe clinical and pathological data of 3567 patients with thyroid sarcoidosis who underwent surgical treatment in the General Hospital of Jinan Military Region from January 2011 to December 2017 were retrospectively analyzed.Postoperative paraffin pathology showed that 1392 patients were benign thyroid neoplasms,and 2175 patients were thyroid cancer patients,of whom 2,135 were thyroid papillary carcinoma.SPSS19.0 statistical software was used for analysis.All statistical results were statistically significant at P<0.05.The results showed: 1.Patients with benign thyroid tumors account for 39.02% of all patients with thyroid nodules.Nodular goiter and thyroid adenoma are the main components of benign thyroid tumors.2.The number of thyroid carcinoma increased year by year,with the most significant increase in papillary carcinoma.3.The sex ratio of male to female was 1:3.22;Between 2011 and 2017,the sex ratio of thyroid cancer patients has not changed significantly.There was no significant difference between years(P> 0.05).The age range of male and female was 30~59 years old.The average age of male was 44.83±11.91 years,and the average age of female was 45.69±10.98 years.The average age of male and female was similar(P> 0.05).4.The proportion of papillary thyroid microcarcinoma accounting for papillary carcinomas is increasing year by year(P<0.05).The total lymph node metastasis rate of thyroid carcinoma was 54.39%,and there was no significant difference between years(P>0.05).It can be seen that thyroid benign tumors account for a large proportion of thyroid diseases,while nodular goiter and thyroid adenoma are the main components of benign thyroid tumors.The number of thyroid papillary carcinomas increases year by year,and the proportion of papillary microcarcinoma increases year by year.With no significant change in the sex ratio and age of onset.Nodular goiter and thyroid adenoma are the main components of benign thyroid neoplasms.The number of papillary microcarcinomas has also been increasing.Although the overall lymph node metastasis rate is high,but there is no statistical difference between years.Therefore,in the following study,we choose the thyroid papillary and benign thyroid tumor as the research object.We establish a diagnostic predictive model for the differential diagnosis of papillary thyroid carcinoma and benign thyroid tumors and healthy people,and to screen and identify potential specific serum peptide biomarkers for the diagnosis of papillary thyroid carcinoma.Part ?: Screening of serum biomarkers for thyroid papillary carcinomaThrough the retrospective analysis of thyroid diseases from the first part and different researchers,it was found that the number of thyroid papillary carcinoma and benign nodular thyroid tumor was the main pathological type of thyroid disease.For better differential diagnosis of thyroid papillary carcinoma,we analyzed 52 cases with thyroid papillary carcinoma and 48 cases of cancer control(18 cases of benign nodular goiter patients and 30 cases of healthy physical examination)serum peptides expression using MALDI-TOF/TOF MS after weak cations extracting peptides/proteins in serum.The serum peptide/protein profile was analyzed by ClinProTools 3.0 MS software using Quick Classifier(QC)to establish a diagnostic predictive model for PTC and non-cancer controls.An independent validation group(36 PTC patients and 19 benign nodular goiter patients and 13 healthy examiners)was used to blindly verify the diagnostic ability of the diagnostic model.The results showed that recognition ability(RC)and cross validation(CV)were 90.95% and 82.90%,respectively.The model has 84.38% sensitivity and 78.13% specificity through independent sample validation.At the same time,there were 97 different peptide peaks in PTC patients and the control group,among which 6 peptides were significantly different(P<0.05)and the area under the ROC curve is not less than 0.75.4 peaks m/z 5905.22,2671.17,1466.68,1738.92 in the two groups were the biggest difference.Therefore,the diagnostic prediction model of thyroid papillary carcinoma based on MALDI-TOF MS technology has high sensitivity and specificity.Six peptides with higher diagnostic capabilities have the potential to be differentially diagnosed PTC biomarkers.Part ?: Identification serum biomarkers for thyroid papillary carcinomaIn order to further understand the sequence of these differential peptides and the properties and functions of the corresponding proteins and their roles in the pathogenesis of thyroid papillary carcinoma,we isolated and identified the 4 most distinct peptides using the nano-LC/ESI-MS/MS system and functional analysis.Thirty samples with high differential expression were taken and peptides were enriched by MB-WCX weak cation.The enriched polypeptide solution was collected and isolated and identified by nano-LC-MS/MS.The data analysis software Bioworks Browser 3.3.1 was used to perform the SequestTM(IPI Human(3.45)search library.The results showed that the peak m/z 2671.17 and 1464.68 analysis was determined the amino acid sequence as "K.SYKMADEAGSEADHEGTHSTKRGHA.K" and "A.DSGEGDFLAEGGGVR.G" and were were identified a fragment of Fibrinogen alpha chain by Database searching with the program IPI Human(3.45).The amino acid sequence of m/z1738.92 was "R.NGFKSHALQLNNRQI.R” and was identified a fragment of complement C4A/B;while the peak m/z 5905.22 was not identified,A fragment of the blood fibrinogen a chain by searching the literature.but it was also found in the literature to be a fragment of fibrinogen a chain.Therefore,the peptide segments of the blood fibrinogen a chain and complement C4A/B are potential serum specific molecular markers of thyroid papillary carcinoma and are expected to be used in the detection of serology.Part ?: The proteomic profiles of thyroid tissues were analyzed by MALDI IMS in situIn this study,we imaged 17 cases of thyroid papillary carcinoma tissue,15 cases of normal tissue adjacent to carcinoma and 5 cases of adenoma tissue by MALDI-TOF/TOF mass spectrometer to obtain Protein profiles of different tissues.MALDI MSI protein profiles were input to SCiLS Lab(2014b)software(mass spectrum image analysis software)and compared the protein profiles from different tissues.The protein profiles between cancerous tissues,adenoma tissues and adjacent normal tissues were statistically analyzed by Kruskal-wallis tests.The protein molecules at m/z 9278.369,4945.61,6847.11 and 4614.838 showed significantly different expressions in different tissues(P<0.001).The m/z 9278.369 and 4946.61 protein molecules were enriched in the adjacent normal tissues;the m/z 46148.38 protein molecule was most abundant in the adenoma tissue region,while the m/z 6847.11 protein molecule was specifically expressed in the cancer tissue region.Using these four differentially expressed proteins made into images.As can be seen from the image,the distribution of protein molecules in the thyroid tissue is significantly different.This study shows the clinical application of MALDI MSI at the molecular level to analyze frozen tissue sections.The protein abundances and regions in different thyroid tissue are significantly different in different mass-to-charge ratios.So,these protein molecules that are differentially expressed in specific tissues are expected to become potential markers for differentiating papillary thyroid carcinomas,adenomas,and adjacent normal tissues.In addition,differently expressed protein ions can be visualized in different tissue regions.The boundary of the tumor can be clearly found,and the progress of the tumor can be clearly defined.All these provided a theoretical basis and a practical basis for clinical pathological diagnosis.At the same time,it can establish a systematic diagnostic method that complements each other with histopathology and mass spectrometry image to solve the problems in clinical pathological diagnosis.In addition,screening for tumor-specific markers can provide new directions for the study of tumors,thereby elucidating the mechanism of tumorigenesis and identifying therapeutic targets. |
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