Medullary Thyroid Carcinoma Samples Research Articles

MicroRNA Profiles in Familial and Sporadic Medullary Thyroid Carcinoma: Preliminary Relationships with RET Status and Outcome

MicroRNAs (miRNAs) are involved in the pathogenesis of human cancers, including medullary thyroid carcinoma (MTC). The aim of this study was to test the hypothesis that different miRNA profiles are related to RET status and prognosis in patients with hereditary MTC (hMTC) and sporadic MTC (sMTC). We analyzed the expression of nine miRNAs (miR-21, miR-127, miR-154, miR-224, miR-323, miR-370, miR-9*, miR-183, and miR-375) by quantitative real-time-polymerase chain reaction in 34 cases of sMTC, 6 cases of hMTC, and 2 cases of C-cell hyperplasia (CCH). We also analyzed the immunohistochemical expression of PDCD4, an miR-21 gene target. sMTC (n=34) was genotyped for somatic RET and RAS mutations. Disease status was defined on the basis of the concentration of serum calcitonin at the latest follow-up and other parameters as indicated in the results. MTC and CCH were both characterized by a significant overexpression of the whole set of miRNAs (the increase being 4.2-fold for miR-21, 6.7-fold for miR-127, 8.8-fold for miR-154, 6.6-fold for miR-224, 5.8-fold for miR-323, 6.1-fold for miR-370, 13-fold for miR-9*, 6.7-fold for miR-183, and 10.1 for miR-375, p<0.0001). PDCD4 expression was significantly downregulated in MTC samples, consistent with miR-21 upregulation. Significantly lower miR-127 levels were observed in sMTC carrying somatic RET mutations in comparison to sMTC carrying a wild-type RET. In sMTC and familial MTC, the miR-224 upregulation correlated with the absence of node metastases, lower stages at diagnosis, and with biochemical cure during follow-up. miRNAs are significantly dysregulated in MTC, and this dysregulation is probably an early event in C-cell carcinogenesis. miR-224 upregulation could represent a prognostic biomarker associated with a better outcome in MTC patients.

Increased Expression of Vascular Endothelial Growth Factor and Its Receptors, VEGFR-1 and VEGFR-2, in Medullary Thyroid Carcinoma

Vascular endothelial growth factor (VEGF-A) expression is upregulated in the majority of human tumors, where it stimulates proliferation, migration, and survival of endothelial cells. Studies have suggested that VEGF inhibitors can be used as an alternative therapy in medullary thyroid carcinoma (MTC), but data about expression of VEGF-A and its receptor in this tumor are scarce. The aims of this study were to evaluate VEGF-A, VEGF receptor (VEGFR)-1, VEGFR-2, and microvessel density (MVD) expression in MTC samples and correlate it with clinical parameters. Paraffin-embedded samples from 38 MTC patients were evaluated for VEGF-A, VEGFR-1, VEGFR-2, and MVD expression by immunohistochemistry. Clinical data were retrospectively reviewed in medical records. Thirty-eight patients aged 31.8 +/- 17.1 years were enrolled. Twenty-seven patients had hereditary disease (71.1%). Twenty-five of them were found to have multiple endocrine neoplasia (MEN) 2A and two were found to have MEN 2B. VEGF-A immunohistochemical staining was detected in 95% (36/38), VEGFR-1 in 96% (36/37), and VEGFR-2 in 91% (31/34) of MTC samples. Age at surgery was positively correlated with VEGFR-2 (p = 0.003). There was no correlation between VEGF-A, VEGFR-2, and tumor stage (tumor node metastasis). Nevertheless, VEGFR-1 was found to be inversely correlated with tumor node metastasis (p = 0.034). We also observed a trend toward an association between VEGFR-1 signal intensity and cure of disease, although this did not reach statistical significance (p = 0.054). Neither VEGF-A nor VEGFR-2 was associated with disease outcome after a median follow-up period of 5 years (p = 0.882 and p = 0.236, respectively). As expected, MVD was correlated with age at surgery (p = 0.005) and tumor size (p = 0.03). Patients with the hereditary form of the disease had a stronger intensity for VEGFR-1 (p = 0.039), whereas patients with sporadic disease displayed higher MVD counts (44 [27-63] vs. 21 [9-49], p = 0.018). The VEGF-A, VEGFR-1, and VEGFR-2 immunoreactive proteins are overexpressed in MTC lesions and might be implicated in tumor progression. It is not clear, however, if expression of these molecules provides prognostic information regarding the spread or outcome of MTC.

Type 2 iodothyronine deiodinase is highly expressed in medullary thyroid carcinoma

Type II deiodinase (D2) plays a critical role in controlling intracellular T3 concentration and early studies indicated a follicular but not a parafollicular C-cell origin of D2 activity in the thyroid gland. Here, we show that D2 is highly expressed in human medullary thyroid carcinoma (MTC), a tumor that arises from the C-cells. D2 transcripts were detected in all MTC samples obtained from 12 unselected MTC patients and the levels of D2 activity were comparable to those found in surrounding normal follicular tissue (0.41+/-0.10 fmol min mg protein vs. 0.43+/-0.41 fmol min mg protein, P=0.91). Additional analysis in the TT cells, a human MTC cell line, demonstrated that the D2 expression is downregulated by thyroid hormones and enhanced by cAMP analogs and dexamethasone. The thyroid hormone receptor alpha1 and beta isoforms were also detected in all MTC samples and in TT cells, thus suggesting a potential role of T3 locally produced by D2 in this neoplastic tissue.

Association of mitochondrial DNA transversion mutations with familial medullary thyroid carcinoma/multiple endocrine neoplasia type 2 syndrome

Medullary thyroid carcinoma (MTC) is a malignant tumour of the calcitonin-secreting parafollicular C cells of the thyroid, and occurs sporadically or as a component of the multiple endocrine neoplasia (MEN) type 2/familial medullary thyroid carcinoma (FMTC) syndromes. In the present study, we investigated the frequency of mtDNA mutations in 26 MTC tumour specimens (13 sporadic and 13 familial MTC) and their matched normal tissues by sequencing the entire coding regions of mitochondrial genome. Nonsynonymous mutations were detected in 20 MTC samples (76.9%): nine out of 13 sporadic MTC (69.2%) and 11 out of 13 (84.6%) familial MTC/MEN2. Both transition and transversion types of mutations were found in the samples. Interestingly, 76.2% (16/21) of transversion mutations were found in FMTC/MEN2 patients, whereas 66.7% (12/18) of transition mutations were in sporadic MTC. Synonymous mutations were found in 12 MTC samples. In total, we identified 27 transversion mutations (21 nonsynonymous and six synonymous) in MTC. Of them, 22 (81.5%) were from FMTC/MEN2, and five (18.5%) were from sporadic MTC. The association of transversion mutation with familial MTC/MEN2 was statistically significant (P = 0.0015, binomial test). Majority of the mutations were involved in the genes located in the complex I of the mitochondrial genome, and were often resulting in a change of a moderately or highly conserved amino acid of their corresponding protein. Mitochondrial respiratory function was also compromised in a TT cell line, which carries mtDNA mutation at nt 4917 and 11,720, and in peripheral lymphocytes of MTC patients with mtDNA mutations. These data suggest that mtDNA mutation may be involved in MTC tumourigenesis and progression. Given that mtDNA mutation spectra are different between sporadic and familial MTC, different mechanisms of oxidative DNA damage may occur in the disease process.

The Ret proto-oncogene in the WAG/Rij rat strain: an animal model for inherited C-cell carcinoma?

WAG/Rij rat strain has been suggested as an animal model for the study of inherited human medullary thyroid carcinoma (MTC), due to its high incidence of spontaneous C-cell thyroid tumours. Although the role of the Ret proto-oncogene mutations, as responsible for human MTC, is well established, nothing has been published concerning this putative animal model. Based upon the previously reported rat Ret sequence, exons 10, 11, 13, 14, 15, and 16, known to carry activating mutations in humans, have been analysed in the WAG/Rij rat by PCR, single strand conformational polymorphism (SSCP) and direct sequencing. Neither the germline nor MTC samples showed any Ret sequence difference in the exons when analysed in comparison to a non-MTC-susceptible rat strain. Our results indicate that Ret exons relevant in humans are not involved in WAG/Rij rat MTC, as expected, and this questions the validity of this strain as a model for the human disease, and suggests there must be additional mechanisms for the genesis and progression of rat MTC.

Allelic imbalance of the mutant and wild-type RET allele in MEN 2A-associated medullary thyroid carcinoma.

Germline mutations of the RET proto-oncogene are responsible for the familial tumor syndrome called multiple endocrine neoplasia type 2 (MEN 2) that includes medullary thyroid carcinoma (MTC). Although inherited mutations of RET lead to tumor formation in patients with MEN 2, it is not understood why only selected cells develop into tumors. We have recently shown that duplication of the mutated RET allele or loss of the wild-type allele might represent mechanisms of tumorigenesis in patients with MEN 2A-related pheochromocytoma. We now analysed 19 DNA samples of MTC (15 of which were non-microdissected, four of which were microdissected) from patients with MEN 2A. Using polymorphic marker and phosphorimage densitometry analyses, we found allelic imbalance of the mutated and wild-type RET allele in six of 19 DNA MTC samples. Of note, two of the four microdissected tumor DNA samples showed allelic imbalance of RET, whereas only four of the 15 non-microdissected MTC samples did. These results underscore the significance of microdissection in the analysis of tumor DNA. In our study, some of the non-microdissected tumor DNA samples may have failed to display allelic imbalance of RET, because of contamination of tumor DNA with nonneoplastic DNA or noninformative microsatellite marker analysis. Taken together, our results suggest allelic imbalance between mutated and wild-type RET as a possible mechanism for tumor formation in some patients with MEN 2A-related MTC.

Monoclonal antibodies to rat calcitonin: their use in antigenic mapping and immunohistochemistry.

A library of monoclonal antibodies (mAbs) to rat calcitonin (rCT) was raised from several fusions. Antibodies were screened by enzyme-linked immunosorbent assay with solid phase rCT. Affinities for rCT ranged from 10(9)-10(11) M(-1). Some mAbs reacted preferentially with solid phase rCT, but not with liquid phase, 125I-labeled rCT. Cross-reactivity with human CT (hCT) was assessed using solid phase hCT. Although there are only two amino acid differences, at least nine of the mAbs do not cross-react with solid phase hCT. Several cross-reactive mAbs were chosen for immunohistochemical studies of human medullary thyroid carcinoma samples and showed strong positive staining. A 33 x 33 matrix solid-liquid mAb inhibition assay was carried out to probe the rCT molecule. Five different clusters of mAb were distinguished and interpreted as reflecting five distinct antigenic regions on the surface of the rCT molecule.