ETV6-NTRK3 fusion was identified in several cancers including the recently described mammary analog secretory carcinoma (MASC) of the salivary glands and a minority of papillary thyroid carcinomas. We describe three cases of primary MASC of the thyroid gland and provide a detailed clinical and pathological characterization of the tumor morphology, immunoprofile, and genetic background. Immunohistochemistry for PAX8, TTF-1, thyroglobulin, mammaglobin, GCDFP-15, S-100 protein, and p63 was used to define the tumor immunophenotype. Fluorescence in situ hybridization for ETV6 rearrangement was performed in three, and the next-generation sequencing assay MSK-IMPACT™ (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets) was performed in two cases. Primary MASC of the thyroid occurred in two women and one man, age 47-72 years. All patients presented with high T stage, infiltrative, locally aggressive tumors with extrathyroidal extension. Two cases were associated with well-differentiated papillary thyroid carcinoma. Histologically, they appeared as low-grade tumors, resembling MASC of the salivary glands and labeled positive for mammaglobin, GCDFP-15, S-100 protein, p63, weakly positive for PAX8, and negative for TTF-1 and thyroglobulin. Fluorescence in situ hybridization revealed ETV6 rearrangement in all cases. In two tested cases MSK-IMPACT™ confirmed the presence of ETV6-NTRK3 gene fusion. Two patients had at least two local recurrences, one was alive with disease, and one was alive and free of disease after 14 and 17 years, respectively. The third patient was alive and free of disease after 2 years. MASC of the thyroid is histologically, immunophenotypically, and genetically similar to its salivary gland counterpart. Thyroid MASC can be associated with a well-differentiated papillary thyroid carcinoma component, supporting follicular cell origin. Clinically, these carcinomas may show frequent recurrences but are associated with long-term survival. Patients with MASC of the thyroid may potentially benefit from Trk molecular-targeted therapy.

Ovarian cancer is the leading cause of cancer-related death in gynecologic malignancies and consists of different histologic types. Histopathologic examination and accurate subtype diagnosis has become increasingly important in guiding patient management and, as such, is the most important currently available ovarian carcinoma "biomarker." In this study, we examined the expression of PAX2 and PAX8 by immunohistochemistry in 58 cases of ovarian serous tumors and 68 cases of ovarian mucinous tumors. The results demonstrated that PAX2 and PAX8 were detected in 100% (30/30) and 77% (23/30) of serous cystadenomas, 100% (16/16) and 94% (15/16) of borderline serous cystadenomas, and 100% (12/12) and 83% (10/12) of serous carcinomas, respectively. However, PAX2 and PAX8 were detected in only 0% (0/29) and 0% (0/29) of mucinous cystadenoma, 4% (1/24) and 4% (1/24) of borderline mucinous cystadenoma, and 0% (0/15) and 7% (1/15) of mucinous carcinoma, respectively. Further, there is a linear correlation between PAX2 and PAX8 (R=0.745; P<0.0001). Overall, our data indicated that PAX2 correlated with PAX8, and these 2 proteins differentially expressed in ovarian serous tumors and ovarian mucinous tumors. Thus, PAX2 and PAX8 are useful biomarker in the differential diagnosis of ovarian serous and mucinous tumors.

Differential diagnosis of collecting duct carcinoma (CDC) from invasive upper tract urothelial carcinoma (UTUC) can be challenging. PAX8 and p63 are 2 markers often used in this setting. GATA binding protein 3 (GATA3) is a marker of urothelial differentiation. We investigated GATA3 expression in CDC and UTUC and its use in this differential. Eighteen CDC and 25 UTUC cases were used to build 2 tissue microarrays. GATA3, p63, and PAX8 nuclear expression was evaluated using standard immunohistochemistry. Staining intensity and percentage of positive cells were assessed. Sensitivity, specificity, and positive and negative predictive values of the markers and their combination were also evaluated. We found GATA3 positivity in 22 (88%) of 25 UTUCs and 1 (6%) of 18 CDCs. The median GATA3 extent of expression was higher in UTUC than in CDC (74% versus 0%, P = .00). We found p63 positivity in 23 (92%) of 25 UTUCs and 2 (11%) of 18 CDCs. PAX8 was positive in 3 (12%) of 25 UTUCs and all (100%) CDCs. GATA3 sensitivity and specificity for UTUC were 88% and 94%, respectively. p63 sensitivity and specificity for UTUC were 92% and 89%, respectively. The p63+/PAX8- profile showed higher sensitivity for UTUC than did the GATA3+/PAX8- profile (80% versus 76%). Both showed a specificity of 100% for UTUC. GATA3+ or p63+/PAX8- sensitivity and specificity for UTUC were 84% and 100%, respectively. Immunohistochemical expression of GATA3 was higher in UTUC, suggesting a potential role for distinguishing UTUC from CDC. Adding this marker to the combination panel of p63 and PAX8 might improve its performance in the diagnosis of epithelial neoplasms involving the renal sinus.

BACKGROUND

Thyroid dysgenesis may be associated with mutations in the paired box transcription factor 8 (PAX8) gene and is characterized by congenital hypothyroidism transmitted in an autosomal dominant mode.

OBJECTIVE

The aim of this study was to identify new mutations in the PAX8 gene. Sixty congenital hypothyroidism-affected individuals with dysgenetic (agenesis, ectopia and hypoplasia) and eutopic thyroid glands were studied.

METHODS

The 12 exons of the PAX8 gene along with their exon-intron boundaries were amplified from genomic DNA and a mutational screening was performed by single-strand conformational polymorphism (SSCP) followed by direct sequencing of samples with abnormal migration patterns. The PAX8 mutations were functionally characterized by transient transfection experiments.

RESULTS

Molecular analysis of the PAX8 gene indicated that four affected individuals had four sequence differences: three novel variations [c.699C>T (p.L233L), c.1006G>A (p.G336S) and c.1317A>G (p.A439A)] and one recently reported [c.674C>T (p.T225M)], whereas the 56 remaining patients showed only wild-type alleles of PAX8. p.T225M, p.L233L and p.G336S variants were not detected in 530 chromosomes from 265 subjects randomly selected from the general population, whereas the p.A439A variant was identified in only one of the 530 chromosomes analysed. Functional analysis of the nonsynonymous substitutions showed that the p.T225M and p.G336S proteins had not lost their ability to bind a specific DNA sequence and to activate the transcription of the thyroglobulin (TG) promoter in synergy with thyroid transcription factor 1 (TTF1).

CONCLUSIONS

We report the occurrence of two nonsynonymous substitutions, one recently reported (p.T225M) and one novel (p.G336S), and two novel synonymous substitutions (p.L233L and p.A439A) in the PAX8 gene. p.T225M and p.G336S are rare sequence variants or may act by inhibiting an unknown particular function. Our study also confirms the very low prevalence of PAX8 mutations in thyroid dysgenesis.

The development of the vertebrate inner ear is a complex process that has been investigated in several model organisms. In this work, we examined genetic interactions regulating early development of otic structures in medaka. We demonstrate that misexpression of Fgf8, Dlx3b and Foxi1 during early gastrulation is sufficient to produce ectopic otic vesicles. Combined misexpression strongly increases the appearance of this phenotype. By using a heat-inducible promoter we were furthermore able to separate the regulatory interactions among Fgf8, Foxi1, Dlx3b, Pax8 and Pax2 genes, which are active during different stages of early otic development. In the preplacodal stage we suggest a central position of Foxi1 within a regulatory network of early patterning genes including Dlx3b and Pax8. Different pathways are active after the placodal stage with Dlx3b playing a central role. There Dlx3b regulates members of the Pax-Six-Eya-Dach network and also strongly affects the early dorsoventral marker genes Otx1 and Gbx2.

OBJECTIVE

Congenital hypothyroidism (CH) may be caused by defects in the thyroid or in one of the stages in the synthesis of thyroid hormones. Thyroid dysgenesis may be associated with mutation in the paired box transcription factor 8 (PAX8) gene. We attempted to screen PAX8 gene mutation in 50 CH patients with thyroid dysgenesis.

METHODS

The patients were classified in two groups as agenesis and ectopic based on biochemical and para clinical tests. By employing PCR, Single Strand Conformation Polymorphism (SSCP) and sequencing, exons 3 to 12 of PAX8 gene with their exon-intron boundaries were studied.

RESULTS

No mutation was found in these patients in any of the exons.

CONCLUSIONS

Our results, once again, indicate that the PAX8 mutation rate is very low and can only explain a minority of the cases. Therefore, it is highly needed to further investigate the genes controlling development and function of thyroid.

Paired box (PAX) genes are transcription factors that play important roles in embryonic development. Although the PAX gene family occurs in animals only, it is widely distributed. Among the vertebrates, its 9 genes appear to be the product of complete duplication of an original set of 4 genes, followed by an additional partial duplication. Although some studies of PAX genes have been conducted, no comprehensive survey of these genes across the entire taxonomic unit has yet been attempted. In this study, we conducted a detailed comparison of PAX sequences from 188 chordates, which revealed restricted variation. The absence of PAX4 and PAX8 among some species of reptiles and birds was notable; however, all 9 genes were present in all 74 mammalian genomes investigated. A search for signatures of selection indicated that all genes are subject to purifying selection, with a possible constraint relaxation in PAX4, PAX7, and PAX8. This result indicates asymmetric evolution of PAX family genes, which can be associated with the emergence of adaptive novelties in the chordate evolutionary trajectory.

A 36-year-old male was found to have a 7.0 cm left upper pole renal mass on renal ultrasound. Following nephrectomy, the mass was grossly ill-demarcated, friable and red-brown, invading renal parenchyma, hilar fat and the renal vein. Microscopically, the tumor had a nested and papillary architecture. The cells demonstrated abundant clear and eosinophilic cytoplasm and focal intracytoplasmic melanin pigment. Nucleoli were prominent. By immunohistochemistry, the tumor was positive for TFE3; HMB-45 stained approximately 5% of tumor cells corresponding to the histologic melanin pigment, which was confirmed with Fontana-Masson stain with bleach. Immunostains for PAX8, CD10, MiTF, and CAIX were negative; keratins Cam 5.2 and AE1/AE3 were focally positive. Targeted next-generation sequencing revealed an ARID1B-TFE3 gene fusion. Melanotic Xp11 renal cell carcinoma is a rare, pigment containing translocation variant demonstrating overlapping features with melanoma and is usually associated with an SFPQ-TFE3 gene fusion. The patient is alive and without evidence of disease 7 years after his diagnosis. The combination of high grade histopathology, the presence of melanin, absent PAX8, keratin positivity, and relatively indolent clinical behavior with a unique translocation may warrant recognition as a distinct renal cell carcinoma translocation subtype.

Beside the classical flat parietal epithelial cells (PECs), we investigated proximal tubular epithelial-like cells, a neglected subgroup of PECs. These cells, termed cuboidal PECs, make up the most proximal part of the proximal tubule and may also line parts of Bowman's capsule. Additionally, a third intermediate PEC subgroup was identified at the junction between the flat and cuboidal PEC subgroups at the tubular orifice. The transgenic mouse line PEC-rtTA labeled all three PEC subgroups. Here we show that the inducible Pax8-rtTA mouse line specifically labeled only cuboidal and intermediate PECs, but not flat PECs. In aging Pax8-rtTA mice, cell fate mapping showed no evidence for significant transdifferentiation from flat PECs to cuboidal or intermediate PECs or vice versa. In murine glomerular disease models of crescentic glomerulonephritis, and focal segmental glomerulosclerosis (FSGS), intermediate PECs became more numerous. These intermediate PECs preferentially expressed activation markers CD44 and Ki-67, suggesting that this subgroup of PECs was activated more easily than the classical flat PECs. In mice with FSGS, cuboidal and intermediate PECs formed sclerotic lesions. In patients with FSGS, cells forming the tip lesions expressed markers of intermediate PECs. These novel PEC subgroups form sclerotic lesions and were more prone to cellular activation compared to the classical flat PECs in disease. Thus, colonization of Bowman's capsule by cuboidal PECs may predispose to lesion formation and chronic kidney disease. We propose that tip lesions originate from this novel subgroup of PECs in patients with FSGS.

OBJECTIVE

This study examined the potential of amniotic fluid mesenchymal stem cells (AF-MSCs) to generate lung precursor cells in vitro and on a xenologous three-dimensional de-cellularized lung scaffold.

METHODS

AF-MSCs were isolated from human amniotic fluid obtained from 17-37 weeks gestation. Lung differentiation was induced on Matrigel or on de-cellularized rat lungs intra-tracheally injected with AF-MSCs by culturing with a modification of small airway growth medium (mSAGM) lacking retinoic acid (RA) and triodothyronine (T3) with addition of fibroblast growth factor-10 (FGF10). Cells and scaffolds were characterized by immunofluorescence and RT-PCR for markers of viability, proliferation, and lung distal airway differentiation (TTF-1(+) and SPC(+)) in the absence of markers of brain (TuJ1(-)) and thyroid (Pax8(-)).

RESULTS

After culture in mSAGM on either Matrigel or lung scaffolds, there were TTF-1(+)/TuJ1(-)/Pax8(-) cells, indicating a lung precursor phenotype. In addition, SPC(+) cells also evolved suggesting a more mature lung phenotype.

CONCLUSIONS

We demonstrate that mid- to late-trimester AF-MSCs can be induced to develop into lung precursor cells when cultured on the appropriate extracellular matrix (ECM), making them a viable source for use in cell therapy or development of an ex vivo tissue engineered lung.

Adrenocortical carcinomas (ACCs) with myxoid features are rare neoplasms. We identified 7 cases of myxoid ACC and studied the clinicopathologic and immunohistochemical features of these neoplasms. The patients were 5 men and 2 women with a mean age of 45 years. Histologically, the tumors contained alcian blue-positive myxoid areas ranging from 10% to 50% of the tissue examined. One case showed lipomatous metaplasia. Areas of conventional ACC were present in all cases. Immunohistochemically, the tumors were positive for steroid receptor cofactor 1, inhibin, melan A, calretinin, and synaptophysin but negative for high-molecular-weight cytokeratin, CAM5.2, and Pax8. Clinical follow-up information for 4 patients demonstrated that all patients had died of their disease 11 to 69 months after diagnosis. Myxoid ACCs are rare tumors that expand the differential diagnosis of myxoid neoplasms involving the retroperitoneum. Contrary to previous reports proposing that the biologic behavior is similar to conventional ACC, our series seems to indicate that myxoid morphology is associated with more aggressive behavior.

Distinguishing malignant peritoneal mesothelioma (MPM) from serous carcinoma involving the peritoneum remains a diagnostic challenge, particularly in small biopsy and cytology specimens. In this distinction, PAX8 expression has been regarded as a specific marker of serous carcinoma. In addition, BAP1 loss is reportedly specific to MPM, in the distinction from both benign mesothelial lesions and ovarian serous tumors (OSTs). Using immunohistochemistry, we examined PAX8 and BAP1 expression in 27 MPMs, 25 cases of benign mesothelium, and 45 OSTs. Five MPMs were PAX8 (5/27, 18%), while 8 cases of benign mesothelium expressed PAX8 (8/25, 32%). PAX8 expression in mesothelium was significantly more common in women than in men (P=0.01). Sixteen MPMs exhibited BAP1 loss (16/25, 64%), while BAP1 was retained in all benign mesothelium and all OSTs. All cases of PAX8 mesothelium were negative for expression of estrogen receptor. These data show that PAX8 is expressed in both benign and malignant mesothelium, and that BAP1 loss is highly specific for MPM, in the differential with both benign mesothelial proliferations and OTSs. These results also have implications for primary diagnosis and for pathologic staging of OST. Caution should be applied when PAX8 expression is used to distinguish mesothelial and serous proliferations, and BAP1 loss may be confirmatory in cases where mesothelioma is favored.

Distinguishing between peritoneal epithelioid mesotheliomas and papillary serous carcinomas involving the peritoneum can be difficult on routine histological preparations, but this differential diagnosis can be facilitated by the use of immunohistochemistry. Recent investigations have indicated that PAX8, PAX2, claudin-4, and h-caldesmon are immunohistochemical markers that can assist in distinguishing between these two malignancies; however, much of the information published on the value of these markers is either insufficient or contradictory. The purpose of this study is to resolve some of the existing controversies and to fully determine the practical value of these markers for assisting in the differential diagnosis between peritoneal mesotheliomas and serous carcinomas. In order to do so, a total of 40 peritoneal epithelioid mesotheliomas and 45 serous carcinomas (15 primary, 30 metastatic to the peritoneum) were investigated. PAX8 and PAX2 nuclear positivity was demonstrated in 42 (93%) and 25 (56%) of the serous carcinomas, respectively, whereas none of the mesotheliomas expressed either marker. Forty-four (98%) of the serous carcinomas exhibited claudin-4 reactivity along the cell membrane, whereas none of the mesotheliomas were positive for this marker. All of the serous carcinomas and mesotheliomas were negative for h-caldesmon. Based on these results, it is concluded that PAX8 and claudin-4 have a higher sensitivity and specificity for assisting in discriminating between peritoneal epithelioid mesotheliomas and serous carcinomas when compared with all of the other positive carcinoma markers that are, at present, recommended to be included in the immunohistochemical panels used in this differential diagnosis. Even though it is highly specific, PAX2 has little practical value in the diagnosis of peritoneal epithelioid mesotheliomas as its sensitivity is low. The h-caldesmon is not useful.

BACKGROUND

The molecular mechanisms leading to a fully differentiated thyrocite are still object of intense study even if it is well known that thyroglobulin, thyroperoxidase, NIS and TSHr are the marker genes of thyroid differentiation. It is also well known that Pax8, TTF-1, Foxe1 and Hhex are the thyroid-enriched transcription factors responsible for the expression of the above genes, thus are responsible for the differentiated thyroid phenotype. In particular, the role of Pax8 in the fully developed thyroid gland was studied in depth and it was established that it plays a key role in thyroid development and differentiation. However, to date the bases for the thyroid-enriched expression of this transcription factor have not been unraveled yet. Here, we report the identification and characterization of a functional thyroid-specific enhancer element located far upstream of the Pax8 gene.

RESULTS

We hypothesized that regulatory cis-acting elements are conserved among mammalian genes. Comparison of a genomic region extending for about 100 kb at the 5'-flanking region of the mouse and human Pax8 gene revealed several conserved regions that were tested for enhancer activity in thyroid and non-thyroid cells. Using this approach we identified one putative thyroid-specific regulatory element located 84.6 kb upstream of the Pax8 transcription start site. The in silico data were verified by promoter-reporter assays in thyroid and non-thyroid cells. Interestingly, the identified far upstream element manifested a very high transcriptional activity in the thyroid cell line PC Cl3, but showed no activity in HeLa cells. In addition, the data here reported indicate that the thyroid-enriched transcription factor TTF-1 is able to bind in vitro and in vivo the Pax8 far upstream element, and is capable to activate transcription from it.

CONCLUSIONS

Results of this study reveal the presence of a thyroid-specific regulatory element in the 5' upstream region of the Pax8 gene. The identification of this regulatory element represents the first step in the investigation of upstream regulatory mechanisms that control Pax8 transcription during thyroid differentiation and are relevant to further studies on Pax8 as a candidate gene for thyroid dysgenesis.

Ovarian cancer is a disease of older women. However, the molecular mechanisms of ovarian aging and their contribution to the pathogenesis of ovarian cancer are currently unclear. mTOR signalling is a major regulator of aging as suppression of this pathway extends lifespan in model organisms. Overactive mTOR signalling is present in up to 80% of ovarian cancer samples and is associated with poor prognosis. This study examined the role of mTOR signalling in age-associated changes in ovarian surface epithelium (OSE). Histological examination of ovaries from both aged mice and women revealed OSE cell hyperplasia, papillary growth and inclusion cysts. These pathological lesions expressed bonafide markers of ovarian cancer precursor lesions, Pax8 and Stathmin 1, and were presented with elevated mTOR signalling. To understand whether overactive mTOR signalling is responsible for the development of these pathological changes, we analysed ovaries of the Pten trangenic mice and found significant reduction in OSE lesions compared to controls. Furthermore, pharmacological suppression of mTOR signalling significantly decreased OSE hyperplasia in aged mice. Treatment with mTOR inhibitors reduced human ovarian cancer cell viability, proliferation and colony forming ability. Collectively, we have established the role of mTOR signalling in age-related OSE pathologies and initiation of ovarian cancer.

Lung adenocarcinoma and papillary thyroid carcinoma (PTC) share a number of microscopic and immunophenotypical features. Thus, patients presenting with thyroid and lung synchronous neoplasms may be difficult on fine-needle aspiration (FNA) samples to define the site of origin of the malignancy. In the case reported here, inherent to a 57-years-old man presenting with a right lung mass and a large (44 mm) thyroid nodule, an integrated cytological, immunocytochemical and molecular approach enabled to clarify the primary nature of the neoplasm. FNA cytology showed in both sites papillary structures and nuclear changes reminiscent of PTC. The lung origin of the neoplasm was suggested on cell-block immunocytochemistry showing thyroid transcription factor-1 positive and PAX8 and TGB negative neoplastic cells. Next generation sequencing performed on the Ion Torrent platforms by the Ion Ampliseq Colon and Lung Cancer panel showed a similar genomic profile in both neoplastic sites with a concurrent KRAS G12C mutation. An integrated approach on FNA biospecimen is safe, cost effective, and may be coupled effectively with modern ancillary molecular techniques that may be useful, besides their predictive value, as a adjunctive diagnostic tool when the synchronous occurrence of lesions featuring overlapping morphologies challenge the cytopathologist.

Most types of thyroid carcinomas express PAX8 transcription factor; however, whether thyroid squamous cell carcinoma (SCC) also expresses PAX8, currently remains unknown. We herein examined the immunoreactivity of PAX8 in SCC of thyroidal and extrathyroidal origin, and discussed the diagnostic significance of PAX8. We immunohistochemically examined specimens from 11 SCC, 22 papillary thyroid carcinoma (PTC), 8 anaplastic thyroid carcinoma (ATC), and 2 mucoepidermoid carcinoma (MEC) cases as well as 5 uterine cervical SCC, 5 esophageal SCC, and 5 pulmonary SCC cases. The rates of PAX8-positive SCC, PTC, ATC, and MEC were 90.9%, 90.9%, 75.0%, and 100%, respectively. Two PAX8-negative PTC cases were cribriform variants. No uterine cervical, esophageal, or pulmonary SCC specimen reacted with PAX8 antibody. Thyroid transcription factor-1 (TTF-1) was positive in 9.1% and 95.5% of SCC and PTC cases, respectively, but negative in all ATC and MEC cases. These results demonstrate that PAX8 staining is useful for distinguishing between primary thyroid SCC and invasion or metastasis from extrathyroidal SCC. We recommend using an immunohistochemical panel of antibodies to PAX8 and TTF-1 to confirm a diagnosis of primary thyroid carcinoma.

The thyroid transcription factors TTF-1 and Pax8 cooperate in the transcriptional activation of thyroid-specific genes such as thyroglobulin (Tg), thyroperoxidase (TPO), and sodium/iodide symporter (NIS).

METHODS

Dog TTF-1 (dTTF-1) and the human Pax8 (hPax8) gene were transfected in Morris hepatoma (MH3924A) cells to investigate (a) the possible visualization of functional protein-protein interaction and (b) the induction of thyroid-specific gene expression. In MH3924A cell lines expressing dTTF-1, hPax8, or both, the activation of human Tg (hTg), human TPO (hTPO), or rat NIS (rNIS) promoter/enhancer was measured using firefly luciferase reporter constructs. Furthermore, the possible induction of thyroid-specific genes was investigated in iodide uptake and reverse transcription polymerase chain reaction (RT-PCR) experiments.

RESULTS

Low transcriptional activation of these constructs was observed in cells expressing either hPax8 or dTTF-1 alone. In contrast, the hTg and hTPO and, to a lesser extent, the rNIS regulatory region were significantly activated in cell lines expressing both transcription factors. Imaging the transcriptional activation of the thyroid-specific regulatory regions by Pax8 and TTF-1 was possible in nude mice implanted with MHhPax8dTTF-1 cells using a cooled charge-coupled device camera. Na(125)I uptake experiments and RT-PCR showed no effect of hPax8 and dTTF-1 on endogenous thyroid-specific gene expression in genetically modified cells.

CONCLUSIONS

The activation of thyroid-specific promoter/enhancer elements in Morris hepatoma cells depends on the functional interaction of hPax8 and dTTF-1. The cooperation of these 2 transcription factors can be visualized in vitro as well as in vivo. With regard to a possible application for radioiodine therapy, further modifications are required.

The regulation of the human Na(+)/I(-) symporter (NIS) gene is of considerable interest for both the diagnosis and therapy of thyroid pathologies. We investigated the influence of the thyroid-specific transcription factors TTF-1 and Pax8 on the NIS promoter and its 5'-flanking sequence. Reporter genes containing the corresponding genomic fragments coupled to a luciferase reporter gene were cotransfected with expression vectors carrying the the cDNA's for TTF-1 and Pax8. Transient transfection assays were performed in HeLa and COS-7 cells, which do not express endogenously these transcription factors. The experiments showed, that TTF-1 had no influence on the NIS promoter. Pax8, on the other hand, had a moderate stimulating effect (threefold) on the proximal NIS promoter. ABCD assays indicated an interaction of in vitro-translated Pax8 with the NIS promoter. However, DNase I footprinting experiments with bacterially expressed Pax8 were negative, suggesting an indirect mode of action with the participation of other proteins. A putative NIS upstream enhancer (NUE) 9000 bp upstream of the NIS gene, which was cloned based on its sequence homology to the rat NUE, was not transactivated by either Pax8 or TTF-1. The present data demonstrate, that the combination of Pax8 and TTF-1 is less important for NIS gene transcription than for other thyroid-specific genes. This is presumably related to the fact, that NIS is expressed also in other tissues such as mammary and salivary gland.

Pax genes encode transcription factors that control key developmental decisions in various animal phyla. The Pax2/5/8 subfamily plays a key role in specification and/or maintenance of vertebrate mid-hindbrain boundary (MHB) region by directly regulating expression of other genes, most notably En2. In the invertebrate chordate amphioxus, expression of AmphiPax2/5/8 is found in many sites that are homologous to the regions of the vertebrate embryo expressing orthologous genes Pax2, Pax5 or Pax8. However, no co-expression of AmphiPax2/5/8 and AmphiEn is detected in the region of the neural tube that might correspond to the vertebrate MHB. Based on this observation and the absence of AmphiWnt expression in this region it appears that amphioxus does not have a MHB. Here we investigated the possibility that the AmphiPax2/5/8, as a key component of MHB development, has lost some of the properties of its vertebrate counterparts. We have analyzed both the DNA-binding and transactivation properties of AmphiPax2/5/8 as well as its ability to interact with the groucho co-repressor. In all these assays AmphiPax2/5/8 is indistinguishable from the human Pax5. In addition, we found two alternatively spliced AmphiPax2/5/8 isoforms that function similarly to the alternatively spliced isoforms of human Pax8. Analysis of the AmphiEn regulatory region provided no evidence for AmphiPax2/5/8 binding and transactivation. Therefore, in amphioxus, AmphiPax2/5/8, although capable of performing all the necessary functions has not been recruited for a developmental mechanism which usually sets up MHB development in vertebrates.

Aldosterone is the main mineralocorticoid hormone controlling sodium balance, fluid homeostasis, and blood pressure by regulating sodium reabsorption in the aldosterone-sensitive distal nephron (ASDN). Germline loss-of-function mutations of the mineralocorticoid receptor (MR) in humans and in mice lead to the "renal" form of type 1 pseudohypoaldosteronism (PHA-1), a case of aldosterone resistance characterized by salt wasting, dehydration, failure to thrive, hyperkalemia, and metabolic acidosis. To investigate the importance of MR in adult epithelial cells, we generated nephron-specific MR knockout mice (MR(Pax8/LC1)) using a doxycycline-inducible system. Under standard diet, MR(Pax8/LC1) mice exhibit inability to gain weight and significant weight loss compared to control mice. Interestingly, despite failure to thrive, MR(Pax8/LC1) mice survive but develop a severe PHA-1 phenotype with higher urinary Na(+) levels, decreased plasma Na(+), hyperkalemia, and higher levels of plasma aldosterone. This phenotype further worsens and becomes lethal under a sodium-deficient diet. Na(+)/Cl(-) co-transporter (NCC) protein expression and its phosphorylated form are downregulated in the MR(Pax8/LC1) knockouts, as well as the αENaC protein expression level, whereas the expression of glucocorticoid receptor (GR) is increased. A diet rich in Na(+) and low in K(+) does not restore plasma aldosterone to control levels but is sufficient to restore body weight, plasma, and urinary electrolytes. In conclusion, MR deletion along the nephron fully recapitulates the features of severe human PHA-1. ENaC protein expression is dependent on MR activity. Suppression of NCC under hyperkalemia predominates in a hypovolemic state.

Mutation detection in samples from thyroid cancer with the addition of BRAF mutation, and also the detection of RAS, RET/PTC, and PAX8/PPARγ mutations, may also contribute to cancer diagnosis. On the other hand, the MAPK/ERK (mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway) and PI3K/Akt (lipid kinase phoshoinositid-3-kinase signaling pathway) play an important role in the transmission of cell signals. The genes, coding the signaling cascade proteins (RET, RAS, BRAF, PI3K, PTEN, AKT), are mutated or aberrantly expressed in thyroid cancer derived from follicular thyroid cells. Genetic and epigenetic alternations, concerning MAPK/ERK and PI3K/Akt signaling pathways, contribute to their activation and interaction as a consequence of malignant follicular cell transformation. The understanding of this molecular mechanism provides access to novel molecular prognostic and therapeutic strategies for inhibiting the oncogenic activity of the signaling pathways. This ability to investigate tumour biology allows for the selection of different drugs. Nowadays the most relevant are treatments directed to tyrosine kinase receptors that bind for a wide variety of ligands and are frequently mutated and induce a constitutive activation such that a chimerical protein expression takes place in follicular cells in the domain of RET, as well as in other receptors. Many molecules such as: motesanib, sorafenib, vandetanib, sunitinib, XL-184, imatinib, axitinib, pazopanib, lenvatinib, combretastatin, gefitinib, cetuximab, bortezomib and thiazoldonedione have been developed. Some of them also can act in receptors of vascular endothelial growth factor and epidermal growth factor receptors. Information obtained through cytological or biopsy samples permits the study of complex metabolic or genetic pathways, thus providing researchers with a high throughput tool for elucidating changes in the global expression patterns seen in tumour cells and allowing for different therapeutic strategies in thyroid cancer which take into account the predominant altered pathways observed in these samples.

Melatonin is an indoleamine with multiple functions in both plant and animal species. In addition to data in literature describing many other important roles for melatonin, such as antioxidant, circadian rhythm controlling, anti-aging, antiproliferative or immunomodulatory activities, our group recently reported that thyroid C-cells synthesize melatonin and suggested a paracrine role for this molecule in the regulation of thyroid activity. To discern the role played by melatonin at thyroid level and its involvement in the hypothalamic-pituitary-thyroid axis, in the present study we have analyzed the effect of thyrotropin in the regulation of the enzymatic machinery for melatonin biosynthesis in C cells as well as the effect of melatonin in the regulation of thyroid hormone biosynthesis in thyrocytes. Our results show that the key enzymes for melatonin biosynthesis (AANAT and ASMT) are regulated by thyroid-stimulating hormone. Furthermore, exogenous melatonin increases thyroglobulin expression at mRNA and protein levels on cultured thyrocytes and this effect is not strictly mediated by the upregulation of TTF1 or, noteworthy, PAX8 transcription factors. The present data show that thyroid C-cells synthesize melatonin under thyroid-stimulating hormone control and, consistently with previous data, support the hypothesis of a paracrine role for C-cell-synthesised melatonin within the thyroid gland. Additionally, in the present study we show evidence for the involvement of melatonin in thyroid function by directly-regulating thyroglobulin gene expression in follicular cells.

Rapid ("warm") autopsies of patients with advanced metastatic cancer provide invaluable insight into the natural history, pathobiology, and morphology of advanced and treatment-resistant tumors. Here, we report a rapid autopsy case of a hereditary leiomyomatosis and renal cell carcinoma (HLRCC) patient with advanced metastatic renal cell carcinoma (RCC)-the first such case described for either a primary renal tumor or HLRCC-related cancer. Mutations in the fumarate hydratase (FH) gene underlie HLRCC, a rare syndrome involving cutaneous and uterine leiomyomata and aggressive kidney tumors. Loss of heterozygosity at the wild-type FH gene locus results in profound cellular metabolic derangement, "pseudohypoxic" upregulation of hypoxia-inducible factor 1α (HIF-1α)-dependent transcription, and aberrant protein succination; these molecular changes drive oncogenesis of kidney tumors in HLRCC patients. The current index patient had a high-grade RCC with classic morphologic features of HLRCC, including large nuclei with prominent eosinophilic nucleoli and perinucleolar clearing. In addition, this patient's RCC demonstrated extensive sarcomatoid and rhabdoid features-morphologies not previously well described in HLRCC-associated kidney tumors. Here, we report the extent of metastatic dissemination and supplement this unique tumor morphology with mitochondrial enzyme histochemistry and extended immunohistochemical analysis. Tumor cells strongly expressed PAX8, vimentin, CD10, and the HIF target GLUT1 and showed increased nuclear p53 accumulation; the expression of other RCC markers was negative. We also detail microscopic tubular epithelial changes in the grossly uninvolved ipsilateral renal parenchyma and demonstrate sporadic, aberrant upregulation of the HIF targets GLUT1 and CAIX in dysplastic peritumoral tubules.