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Medullary thyroid cancer (MTC) is the third frequent subtype of thyroid cancer-driving from thyroid C-cells. The first-line strategy to treat MTC is surgery, but tumor recurrence and patients' mortality rate have still been demonstrated in approximately 4-10% of MTC cases. Therefore, to treat and prevent the progressive form of the disease, the early diagnosis of MTC is assumed to play a critical role. In this regard, recently, circulating biomarkers have drawn researchers' attention for their nonaggressive manners in the sample collection. In this systematic review, we aimed to focus on circulating biomarkers and their applications in MTC diagnosis, prognosis and follow-up, and we discussed their clinical application and how they can affect clinical decision making in the future. A literature search (from 2000 to 2021) was accomplished on MTC circulating biomarkers in different databases, and only English articles were evaluated. We found that calcitonin (CT) and carcinoembryonic antigen (CEA) are the most important circulating biomarkers in the MTC diagnosis. Other circulating biomarkers included pro-calcitonin (Pro-CT), pro-Gastrin releasing peptide (Pro-GRP), carbohydrate antigen 19-9 (CA 19-9) and chromogranin A (CgA). Some novel circulating biomarkers comprised vaspin and retinol-binding protein-4 (RBP4), myostatin, tumor cells (CTCs), RET M918T mutated cfDNA, circulating tumor DNA (ctDNA), miR-375 and Alu83 and Alu244 cfDNAs. Several circulating biomarkers have been identified to optimize the accuracy of diagnosis and offer new prognostic criteria, which should be verified before any clinical application. Although different circulating biomarkers contributed to MTC have been discovered, a few of them could be used in clinical diagnosis. In many cases, the application of each marker may not be useful lonely; therefore, a combination of two or more biomarkers could open a new avenue in the diagnosis, prognosis and prediction of MTC., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

GATA binding protein 3 (GATA3) belongs to a family of transcription factors comprising six members. These proteins identify G-A-T-A containing sequences in the target gene and bind to DNA target via two zinc-finger domains. The aim of this study was to evaluate the role of GATA3 in the diagnosis of tumors and its value as a prognostic marker. To perform this review, a comprehensive search was conducted through PubMed, Embase, Scopus, Cochrane and Google Scholar databases from 1985 to 2020. Articles were considered thoroughly by independent reviewers and data were extracted in predefined forms. Final synthesis was conducted by using appropriate data from included articles in each topic. Studies have shown that GATA3 has a critical role in the development of epithelial structures in both embryonic and adult tissues. The majority of studies regarding GATA3 expression in tumor evaluation focused on breast and urothelial neoplasms, whether primary or metastatic. Its sensitivity in these neoplasms has been reported to be high and made this marker more valuable than other available immunohistochemistry markers. However, GATA3 expression was not restricted to these tumors. Studies have shown that GATA3 immunostaining could be a useful tool in various tumors in kidney, salivary gland, endocrine system, hematopoietic system, and skin. GATA3 can also be used as a useful prognostic tool. Although GATA3 is a multi-specific immunohistochemical stain, it is a valuable marker in the panel for confirming many epithelial or mesenchymal neoplasms as both a diagnostic and prognostic tool., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Papillary thyroid carcinoma (PTC) represents the most common primary malignant thyroid lesion in all age groups. As diagnosis of PTC could be challenging in some cases and borderline nuclear features could be seen in some benign mimickers, several immunohistochemical markers are proposed to be helpful for making the correct diagnosis. In this study the authors investigated the diagnostic value of transmembrane glycoprotein (TROP2) for differentiating PTC from other thyroid follicular lesions. A total of 155 total thyroidectomy specimens including 84 cases of PTC and 71 cases of non-PTC were investigated. Among non-PTC group, 45 cases were follicular neoplasms and 26 cases were Hashimoto thyroiditis. TROP2 expression was observed in 82 of 84 cases of PTC group. In contrast only 5 cases of non-PTC group, all from Hashimoto thyroiditis specimens, showed positive expression. The sensitivity, specificity, positive predictive value, and negative predictive value of TROP2 for diagnosis of PTC was 98%, 93%, 94%, and 97%, respectively. The authors concluded that high sensitivity and specificity of TROP2 as well as its uniform negative reaction in follicular adenoma and carcinoma makes it a valuable immunohistochemical marker for diagnosis of PTC., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Circulating cell-free DNA (cfDNA) has been considered as a diagnostic source to track genetic and epigenetic alterations in cancer. We aimed to study mutation in addition to the methylation status in the promoter regions of RASSF1 and SLC5A8 genes in tissues and circulating free DNA samples of patients affected with papillary thyroid carcinoma (PTC) and thyroid nodules as controls. BRAF V600E mutation was studied by ARMS-scorpion real-time polymerase chain reaction method in 57 PTC and 45 thyroid nodule cases. Methylation status of RASSF1 and SLC5A8 promoter regions was analyzed by methylation-specific high-resolution melting curve analysis. BRAF V600E mutation was found in 39 (68.4%) out of 57 PTC tissue samples, while in 33 (49.1%) cases of cfDNA, this mutation was detected. The frequency of BRAF V600E mutation in cfDNA was significantly different between metastatic and nonmetastatic PTC cases (22 of 33 PTC cases vs. 5 of 34 thyroid nodule samples). Methylation levels of three promoter regions of SLC5A8 and proximal promoter region of RASSF1 was significantly different between PTC and thyroid nodule cases in both cfDNA and tissue DNA. In addition, the methylation status of these two genes in tissue DNA was reflected in methylation status observed in cfDNA. This study confirmed that BRAF V600E mutation is better for discrimination between papillary thyroid carcinoma and thyroid nodules. On the other hand, hypermethylation in the more proximal promoter regions to RASSF1 and SLC5A8 genes showed higher sensitivity and more acceptable specificity for this discrimination., (© 2020 Wiley Periodicals, Inc.)

Medullary thyroid carcinoma (MTC) is a scarce cancerous disease, originating from parafollicular C cells of the thyroid gland. MTC can be manifested as an aggressive carcinoma with metastasis, especially in sporadic forms. Mutations of the rearranged during transfection (RET) proto-oncogene occurs in all hereditary and a few somatic MTCs, so detection of RET mutations is needed for prompt and appropriate treatment. MicroRNAs (miRNAs) are noncoding regulatory RNAs. Extensive studies have done in progress or suppression of several types of cancers such as MTCs with the remarkable application as prognostic markers. Of the effective miRNAs in cancers, miR-144 and miR-34 were evaluated in our study. Blood samples of 25 RET-positive and 25 RET-negative blood samples of patients with MTC were evaluated for these miRNAs, using quantitative real-time polymerase chain reaction (RT-qPCR). Analysis of the results was performed by the 2 -ΔΔCt method, showing that miR-144 and miR-34a expression had a relative increase in patients with MTC compared with normal control samples and also in RET positives versus RET negatives. We recruited 50 out of 350 MTC plasma samples (27 female and 23 male) which were selected based on RET mutation in exon 11 (25 RET-positive and 25 RET-negative), with a mean ± SD age of 37.04 ± 1.74 years. Receiver operating characteristic (ROC) curve analysis was done to investigate the prognostic value of these miRNAs; although, they showed no significant prognostic value as MTC biomarkers in plasma samples. In conclusion, miRNAs can be used as biomarkers of cancers such as MTC; however, more studies are needed to find the best candidate miRNAs for the diagnosis of cancers., (© 2019 Wiley Periodicals, Inc.)

Medullary thyroid carcinoma (MTC) is a rare neoplasia derived from neural parafollicular C cells. MicroRNAs (miRNAs) are small regulatory RNAs with essential roles in the biology of cancers such as MTC and can be applied as diagnostic markers. According to previous studies, miR-144 and miR-34 and their two oncogenes target, mammalian target of rapamycin (mTOR) and AXL receptor tyrosine kinase (AXL), were selected for further investigations in our study. Thirty MTC samples as well as thirty adjacent normal thyroid tissues were applied in this study including 28 formalin-fixed, paraffin-embedded (FFPE) and 2 fresh-frozen MTC samples. RNA extraction and complementary DNA (cDNA) synthesis were performed for all samples. After primer pairs and probes were designed, real-time polymerase chain reaction (real-time PCR) method was used, and the results were analyzed using 2 -ΔΔCt method. Receiver operating characteristic (ROC) curve analysis was applied to assess the diagnostic value of the two miRNAs. AXL protein level was measured in all clinical samples using enzyme-linked immunosorbent assay (ELISA) method. Both miRNAs were up-regulated in all clinical samples compared to the normal tissues. AXL was up-regulated in most clinical samples while mTOR was down-regulated in most samples. Furthermore, the level of AXL protein increased. ROC curve analysis demonstrated that increased expression of miR-34a and miR-144 in MTC patients had significant predictive value. The results demonstrated that high expression of miR-144 and miR-34a can be considered as biomarkers of MTC. However, there was no statistically significant correlation between the expression of these miRNAs and target genes in MTC clinical samples., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Pheochromocytomas and paragangliomas (PPGLs) are tumors arising from the adrenal medulla and sympathetic/parasympathetic paraganglia, respectively. According to Th e Cancer Genome Atlas (TCGA), approximately 40% of PPGLs are due to germ line mutations in one of 16 susceptibility genes, and a further 30% are due to somatic alterations in at least seven main genes (VHL, EPAS1, CSDE1, MAX, HRAS, NF1, RET, and possibly KIF1B). Th e diagnosis of malignant PPGL was straight forward in most cases as it was defined as presence of PPGL in non-chromaffin tissues. Accordingly, there is an extreme need for new diagnostic marker(s) to identify tumors with malignant prospective. Th e aim of this study was to review all suggested genetic and epigenetic alterations that are remarkably different between benign and malignant PPGLs. It seems that more than two genetic mutation clusters in PPGLs and other genetic and methylation biomarkers could be targeted for malignancy discrimination in different studies.

Papillary thyroid carcinoma (PTC), including its variants and widely varying behavior, constitutes about 80% of all thyroid malignancies. Increased knowledge regarding molecular alterations has led to attempts to identify diagnostic or prognostic factors for a reliable preoperative approach to the classification of patients according to risk of recurrence. In this study, 107 cases of PTC with known histological properties, including vascular or capsular invasion, were assessed for expression of MMP2 and Caspase-3 using immunohistochemistry. Considering 10% as a cutoff to discriminate cases with invasive behavior from the non-invasive group, there was no relationship between expression of MMP2 or Caspase-3 in tumor cells and the presence of capsular invasion (p=0.45 and 0.64, respectively), as well as for the expression of Caspase-3 and vascular invasion (p=0.43). In case of MMP2, a borderline correlation was found between the positive reaction of tumor cells with the presence of vascular invasion (p=0.05). So the evaluation of MMP2 in thyroid PTC appears to be of some benefit to the prediction of tumor behavior while Caspase-3 as a marker of prediction seems to be of no use., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Purpose: Definite diagnosis of follicular thyroid carcinoma (FTC) is based on the presence of capsular or vascular invasion. To date, no reliable and practical method has been introduced to discriminate this malignant neoplasm from follicular thyroid adenoma (FTA) in fine needle aspiration biopsy material. Matrix metalloproteinase-2 (MMP-2), by degrading extracellular matrix, and caspase-3, by induction of apoptosis, have been shown to play important roles in carcinogenesis and aggressive behavior in many tumor types. The aim of this study was to examine expression of MMP-2 and caspase-3 in thyroid follicular neoplasms and to determine their usefulness for differential diagnosis., Method: Sixty FTAs and 41 FTCs were analysed immunohistochemically for MMP-2 and caspase-3., Result: MMP-2 was positive in 4 FTCs (9.8%), but in none of FTAs, with statistical significance (p=0.025). Caspase-3 was positive in 30 (50%) of FTAs and in 27 (65.9%) of FTCs., Conclusion: Our results show MMP-2 expression only in FTCs and suggest that this protein may be a useful marker to confirm diagnosis of FTC versus FTA with 100% specificity and 100% predictive value of a positive test. We failed to show any differential diagnostic value for caspase-3 in thyroid follicular neoplasms.

The aim of this study was to evaluate the frequency (as qualitative analysis) and level (as quantitative analysis) of promoter hypermethylation of four genes, P16, TSHR, RASSF1A and RARβ2, and to assess their diagnostic or prognostic values in papillary thyroid tumors. Fifty formalin-fixed paraffin-embedded (FFPE) samples consisting of 25 malignant tumors and 25 non-malignant thyroid tumors were analyzed using COBRA method. Promoter hypermethylation of P16, TESH, RASSF1A and RARB2 genes was noted not only in 10, 7, 19 and 13 cases of malignant tumors, but also it was detected in 7, 11, 23 and 8 cases of benign tumors, respectively, limiting its diagnostic usefulness. The quantitative hypermethylation level was significantly higher in malignant tumors compared to benign tumors for P16 (P<0.004), TSHR (P<0.006) and RASSF1A (P<0.001), but the methylation level of RARβ2 (P<0.31) showed considerable overlap between the two groups. The mean levels of hypermethylation of P16, TSHR and RASSF1A genes were significantly higher in malignant papillary thyroid tumors compared to benign tumors and by choosing the appropriate cutoff for each gene, we could distinguish 9, 9 and 8 PTCs from 25 cases by P16, RASSF1A and TSHR methylation analysis, respectively. According to our results, these three genes, in combination, may be useful as molecular markers. The findings of present study imply that the P16, TSHR and RASSF1A gene promoter hypermethylation may play important roles in the pathogenesis of PTC and can be a potential biomarker for selecting patients with PTC.

Background: The molecular and cellular mechanisms linking chronic inflammation and gastrointestinal malignancy are not known with certainty., Aim: To investigate changes in potential causative factors during progression of esophagus cancer in a population living in high-risk area in Iran., Subjects: Formalin-fixed, paraffin-embedded esophageal specimens (n=87) from patients with gastroesophageal reflux disease (GERD), Barrett's metaplasia, adenocarcinoma (ADC) and squamous cells carcinoma (SCC) were collected based on their pathological diagnosis., Methods: Immunohistochemical (IHC) technique was used to study tissue accumulation of P53, P21, cyclooxygenase-2 (COX-2), glutathione S-transferase-P (GST-Pi) and nitrotyrosine (NT) in patients and controls., Results: P53 expression was not detected in esophageal tissues from normal and GERD samples, whereas it was found positive in Barrett's, ADC, and SCC samples. P21 positive sample was relatively higher in ADC patients as compared to that in SCC (ADC: 52.6%; SCC: 25%). GST-Pi expression was equally accumulated in all the samples. NT was predominantly expressed in ADC (72.7%). COX-2 expression was significantly higher in Barrett's (60.0%) and ADC (66.6%) as compared to that in GERD, SCC and normal. These data were further confirmed by detecting the scores of immunostainings in all the positive samples., Conclusion: The pathological changes in ADC and SCC samples which were associated with increasing frequency of NT and COX-2 provides further evidence for involvement of these inflammatory factors in progression of esophagus cancer.

Patients with poor prognosis in thyroid cancers resistant to currently available therapeutic modalities are often in search of a new treatment. The epidermal growth factor receptor (EGFR) vIII, a ligand-independent, constitutively active, mutated form of EGFR has been shown to play a role in the pathogenesis of some cancers. Consequently, the immunohistochemical detection of EGFRvIII with novel camel antibodies, which are valuable for their ability to interact with less antigenic epitopes in contrast to the conventional antibodies, might be worthy in diagnostic techniques of thyroid neoplasms. EGFRvIII was evaluated on paraffin-embedded tissue specimens of 40 samples of follicular carcinomas, papillary carcinomas, medullary carcinomas, follicular adenomas, and goiter of the thyroid gland by immunohistochemistry. Positive immunostaining of neoplastic tissues with camel and rabbit polyclonal, as a control, were 81.3% and 39.1%, respectively. No goiter tissue was stained with either antibody preparation. Also, the results showed that the sensitivity of camel heavy chain antibodies (65%) is higher in contrast to conventional rabbit under the same conditions (39.1%). Considering the results of this study, exploiting the smaller heavy chain antibodies of camels against EGFRvIII seems promising in the diagnosis procedures of thyroid neoplasms.