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Introduction Thyroid nodules occur in 65% of population. Most of them are benign lesions. Recent large retrospective studies conducted at a high-volume centers revealed TC in 3%–15% of biopsies. Thyroid cancer (TC) accounts for more than 95% of endocrine cancers. Over the last three decades TC has become one of the most rapidly increasing cancer in the USA. Fine needle aspiration biopsy (FNAB) comprises basic method in preoperative diagnostic work-up. FNAB is charakcteryzed by high level of accuracy and enables to preliminary assessment of the lesions as benign or malignant. The results are presented according to the Bethesda system which categorizes into six groups. In the doctoral the analysis of patients with FNAB result Bethesda tier IV - suspicious for follicular neoplasm (SFN) has been performed. The malignancy rate varies between 8,2 – 19%. To date, there are no diagnostic tools allowing to exclude malignancy and indeterminate cytopathology often requires diagnostic surgery for definitive. There have been several studies appraising the potential of miRNAs as diagnostic markers for thyroid cancer. miRNAs are endogenous, single-stranded, tiny, highly conserved, noncoding molecules with lengths ranging from 18 to 25 nucleotides RNAs. miRNAs are encompassed in many biological and pathological processes as proliferation, differentiation and apoptosis. miRNAs ; alter gene expression at the post-transcriptional level of messenger RNAs (mRNAs). miRNAs are extremely stable and remain intact in tissues, whether fresh, frozen or formalin-fixed paraffin-embedded (FFPE) 37. This property of miRNAs has been exploited for the development of several commercially available miRNA-based molecular tests. To date many papers revealed association between malignant neoplasm and altering miRNA expression. In 2006 Weber et al in their study showed miRNA as a potential molecule allowing to discriminate FA and FTC. To date there are many researches emphasising mi RNA as a valuable biomarker, mostly papillary thyroid cancer. Objectives The purpose of this study was twofold: to characterize the miRNAs signature of the tumors assessed as SFN and to define circulating miRNA pattern to distinguish follicular adenoma from follicular cancer in patients with thyroid nodules verified in FNAB as SFN. Materials and Methods In this study 110 consecutive patients were included. All patients were operated in Department of General Surgery of G. narutowicz Hospital, Third Chair of General Surgery, Jagiellonian University Medical College in Cracow, between 2016 and 2020. The indication for surgery was FNAB result “suspicious for follicular neoplasm”. Inclusion criteria were: the result of fine needle aspiration biopsy “ suspicious for follicular/oxyphilic ; neoplasm”, absence of invasive neoplasm features as follows infiltration of surrounding tissue or lymph nodes/distant metastases, informed consent. Typical features of malignant process as suspicion on lymph nodes metastases or infiltration of adjacent tissues excluded from the study. In first paper the aim was to define factors correlating with risk of malignancy. Six specific categories were defined and the clinical records for patients were collected: sex, age, presence of oxyphilic cells, diameter of the tumour, presence of Hashimoto disease, aggregate amount of clinical and ultrasonographic features of malignancy according to ATA The second study assessed the impact of elective central lymph node dissection on postoperative pathological staging and early surgical complication rate. Staging of thyroid cancer was presented according to 8th edition TNM/AJCC 2017. Surgical early postoperative complications were reported and the rate was compared between the study group and the control group consisting of patients operated on in the same period for benign nodular goiter. The vocal cords mobility was assessed with video laryngoscope on the second postoperative day. Hypocalcemia was defined as calcium concentration in serum below 2,0 mmol/l. Statistical analysis of phenotype data was performed using Statistica 13.0 software. P‐values <0.05 were considered statis ; tically significant. To determine if a data set is wellmodeled by a normal distribution, the chi-square test of independence has been applied. To define statistical significance, the Mann–Whitney U test and t test were used, as appropriate. The third paper shows the results of molecular analysis performed on 10 patients with WDTC and 14 patiens with FA. Age, sex, presence of Hashimoto disease, diameter of the tumor, presence of FNAB suspicious for Hurthle cell neoplasm and US risk factors according to EU-TIRADS were compared between both groups. The specimens of tumor and macroscopiclly normal thyroid tissue were excised in the operating theatre by experienced pathologist. The specimens were placed immediately into RNAlater® and stored at −80°C until they were ready for RNA extraction All blood samples were taken in the operating theatre, before a skin incision was done. Tumor’s RNA was extracted from 50–100 mg of disrupted tissue in TissueLyser LT (Qiagen, Hilden, Germany). RNA from 0.5ml plasma was extracted using total RNA protocol of RNAzol Small RNA was used to generated NGS library. For quality purpose we remove all reads with length below 15 nucleotides, and without adapter. As a next step new generation sequencing has been performed on NextSeq (llumina, San Diego, CA, USA). Results have been saved on the local server. Quality of reads has been assessed u ; sing FastQC software. The raw counts were normalized for library size and represented as counts per million (CPM) with miRDeep2 software. For multidimensional scaling (MDS) miRNA read count normalized for library size have been used. Statistical and exploratory analysis of miRNOME data was performed in R, limma package . The same volume of plasma from each patients for miRNA isolation had been taken. For the analysis of miRNA expression t-test was applied as appropriate. Results In the first paper, including 110 consequtive patients operateted on because of SFN of thyroid, there were no significant differences between age (53,1 vs. 55,4years, p=0,42), gender (16,6% vs. 13,1%, p=0,31), tumour diameter (14,5 mm vs. 19,4 mm, p=0,15), aggregate amount of clinical and ultrasonografic factors (1,4 vs. 1,29, p=0,71), presence of Hashimoto disease (33,3% vs. 31,5%, p=0,55) and fine needle aspiration biopsy result suspicious for Hurthle cell neoplasm (55,6%% vs. 45,7%, p=0,075) in both groups. The second study, performed on 80 patients, revealed 129 lymph nodes dissected (mean 1.6 lymph node per 1 patient), all lymph nodes were clear of cancer cells. In 26 patients there were no lymph nodes in postoperative preparation. In group of patients with WDTC lymph nodes were detected more often ( 90% vs 64%, p=0,1) and mean amount of lymph nodes was greater (2,2 vs 1,5, p=0,13) ; in postoperative specimen in compared with the group of patients with FA. No significant differences were identified in prevalence of early postoperative complications among the study group and the control group patients: unilateral recurrent laryngeal nerve (RLN) palsy 3.38% vs. 1.49%; p= 0,08), hypocalcemia (5% vs. 5.4%; p=0.86), postoperative hemorrhage (1.25% vs. 0.44; p=0.29). The analysis of complication frequency based on range of surgery revealed the postoperative hemorrhage and RLN palsy occurred in patients after total thyroidectomy. The third study shows the results of molecular analysis – differential expression of miRNA between FA and WDTC. We found that the expression level of two miRNAs, miR- 146b-5p (p=0,031) and miR-146b-3p (p= 0,034) were significantly higher, whereas miR-195- 3p (p=0,032) occurred to be significantly lower in patients with WDTC than in patients with FA. The results have been validated using external TCGA data cohort. Overall Jagiellonian University Medical College signature of WDTC has 90% overlap (18 from 20 miRNAs) with the signature of TCGA. The presence of this miRNA in plasma have been confirmed. Altered expression of miRNAs was tested by qPCR. The study revealed higher expression level of circulating miR-195-3p in patients with WDTC in compared to patients with FA, in contrary to results of miRNA expression in tissues. ; The difference was statistically significant (P =0,039). Conclusion In patients with FNAB results classified as Bethesda tier IV there are no reliable clinical features associated with low risk of malignancy and surgery should be consider in every case of tumor size above 2 cm as most appropriate manner to exclude thyroid cancer. Elective central lymph node dissection at experienced surgical hands does not improve postoperative pathological staging and is not associated with higher risk of early postoperative complications. MiRNA may be useful in the diagnostic process of patients “suspicious for follicular neoplasm”. Differential expression of miRNAs 146a, 146b and 195 has a potential of discriminating between WDTC and FA. Additionally miR-195-3p could be isolated from serum and be applied as a valuable tool which excludes thyroid cancer in preoperative workup.
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Rada Dyscypliny Nauki medyczne
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