High-grade serous ovarian carcinoma (HGSOC) is usually diagnosed at a late stage and is associated with poor prognosis. Understanding early stage disease biology is essential in developing clinical biomarkers to detect HGSOC earlier. While recent studies indicate that HGSOCs arise from fallopian tube secretory epithelial cells, a considerable body of evidence suggests that HGSOC can also arise from ovarian surface epithelial cells (OSECs). PAX8 is overexpressed in HGSOCs and expressed in fallopian tube secretory epithelial cells, but there are conflicting reports about PAX8 expression in OSECs. The purposes of this study were to comprehensively characterize PAX8 expression in a large series of OSECs and to investigate the role of PAX8 in early HGSOC development. PAX8 protein expression was analyzed in the OSECs of 27 normal ovaries and 7 primary OSEC cultures using immunohistochemistry and immunofluorescent cytochemistry. PAX8 messenger RNA expression was quantified in 66 primary OSEC cultures. Cellular transformation was evaluated in OSECs expressing a PAX8 construct. PAX8 was expressed by 44% to 71% of OSECs. Calretinin and E-cadherin were frequently coexpressed with PAX8. Expression of PAX8 in OSECs decreased cellular migration (P = .028), but had no other effects on cellular transformation. In addition, PAX8 expression was significantly increased (P = .003) in an in vitro stepwise model of neoplastic transformation. In conclusion, PAX8 is frequently expressed by OSECs, and endogenous levels of PAX8 expression are non-transforming. These data indicate that in OSECs, PAX8 expression may represent a normal state and that OSECs may represent an origin of HGSOCs.

We report a kindred with euthyroid multi-nodular goitre (MNG) of adolescent onset. Two of the seven subjects with MNG have progressed to papillary thyroid cancer. One affected male had nodular kidney disease, and breast cancer occurred in one affected female. Genes that were candidates on the basis of the associated kidney (PAX8) and breast diseases (sodium iodide symporter (NIS)), were sequenced. No mutations were found in the coding region, intron/exon splice sites or in the promoter sequences (from -1248 relative to the translation initiation codon) of PAX8. Similar results were obtained for NIS. Subsequently, microsatellite analyses were performed on 14 informative family members. We used 2 to 3 markers per locus for 6 loci (on chromosomes 1,2,3,14,19,X) previously reported to predispose to MNG and/or familial non-medullary thyroid cancer (FNMTC). On the basis of non-significant logarithm of the odds ratio (LOD) scores or inheritance of different alleles in affected individuals, all loci have been excluded. Thyroidectomy specimens from three members of the kindred show multiple benign lesions, with papillary cancer in two. The morphological features do not resemble those seen in familial adenomatous polyposis, Cowden syndrome, or in multiple oxyphil lesions. From these findings and from the absence of any linkage to any of the known loci associated with MNG or FNMTC, we suggest that this represents a new form of inherited MNG with a significant risk of progression to papillary carcinoma.

The maintenance of thyroid hormone (TH) homeostasis is dependent on the synthesis and secretion of TH regulated by TSH. This is achieved, in turn, by the negative feedback of TH on TSH secretion and synthesis, which requires the interaction with TH receptors (TRs). Derived by alternative splicing of two gene transcription products, three TRs (TRbeta1, TRbeta2 and TRalpha1) interact with TH while another, TRalpha2, binds to DNA but not to TH. In this study we compare the results of thyroid function tests in mice with deletions of the TRalpha and TRbeta genes alone and present novel data on mice that are double homozygous and combined heterozygous. Homozygous deletions of both the TRalpha and TRbeta in the same mouse (TRalphao/o; TRbeta-/-) resulted in serum TSH values only slightly lower than those in athyreotic, Pax8 knockout mice. Whereas the absence of TRalpha alone does not cause resistance to TH, the absence of TRbeta in the presence of TRalpha results in a 205, 169, 544% increase in serum thyroxine (T(4)), triiodothyronine (T(3)) and TSH concentrations respectively. However, in the absence of TRbeta, loss of one TRalpha allele can worsen the resistance to TH with a 243 and 307% increase in T(4) and T(3) respectively. Similarly, while the heterozygous mouse with a single TRbeta allele shows no alteration in thyroid function, the concomitant deletion of TRalpha brings about mild but significant resistance to TH. Furthermore, the severity of the resistance to TH was noted to decrease with age in parallel with the decrease in serum free T(4) values also seen in wild-type mice. These results demonstrate that (1) unliganded TRalpha or TRbeta are not absolutely necessary for the upregulation of TSH; (2) TRbeta but not TRalpha is sufficient for TH-mediated downregulation of TSH; and (3) TRalpha may partially substitute for TRbeta in mediating a partial TH-dependent TSH suppression.

The bacterial lipopolysaccharide (LPS) is a biological activator that induces expression of multiple genes in several cell types. LPS has been proposed as an etiopathogenic agent in autoimmune diseases. However, whether LPS affects the expression of autoantigens has not been explored. Thyroglobulin (TG) is a key protein in thyroid hormonogenesis and one of the major thyroid autoantigens. This study aimed to analyze the action of LPS on TG gene expression in Fisher rat thyroid cell line FRTL-5 thyroid cells. We demonstrate that LPS increases the TSH-induced TG protein and mRNA level. Evidence that the effect of LPS is exerted at the transcriptional level was obtained by transfecting the minimal TG promoter. The C element of the TG promoter, which contains sequences for paired box domain transcription factor 8 (Pax8) and thyroid transcription factor (TTF)-1 binding, is essential for full TG promoter expression under TSH stimulation. The transcriptional activity of a construct containing five tandem repeats of the C site is increased by LPS, indicating a possible involvement of the C site in the LPS-induced TG gene transcription. We demonstrate that the TG promoter mutated at the Pax8 or TTF-1 binding element in the C site does not respond to LPS. In band shift assays, binding of Pax8 and TTF-1 to the C site is increased by LPS. The Pax8 and TTF-1 mRNA and protein levels are augmented by LPS. The half-lives of TG, Pax8, and TTF-1 are increased in endotoxin-treated cells. Our results reveal the ability of LPS to stimulate the expression of TG, a finding of potential pathophysiological implication.

OBJECTIVE

Follicular thyroid carcinoma (FTC) has been a diagnostic challenge for decades. The PAX8-PPARγ rearrangement has been detected in FTC and classic papillary thyroid carcinomas (PTCs). The aims of this study were to assess the presence of PAX8-PPARγ by using tissue microarrays in a large cohort of different thyroid neoplasms, and to assess its diagnostic and prognostic implications.

RESULTS

Fluorescence in-situ hybridization (FISH) analysis for PAX8-PPARγ was performed on 226 thyroid tumours, comprising FTCs (n = 59), PTCs (n = 126), poorly differentiated thyroid carcinomas (PDs; n = 34), follicular thyroid adenomas (FTAs; n = 5), and follicular tumours of unknown malignant potential (FTUMPs; n = 2). PAX8-PPARγ was detected in 12% of FTCs, 1% of PTCs, 7% of PDs, and in both cases of FTUMP. There was no correlation between the extent of capsular or vascular invasion and PAX8-PPARγ, or between lymph node or haematogenous metastasis and PAX8-PPARγ. Overall survival (OS), tumour-specific survival (TSS) and relapse-free-survival (RFS) were not influenced by PAX8-PPARγ.

CONCLUSIONS

In this study, we demonstrate for the first time the presence of PAX8-PPARγ in PDs and FTUMPs, whereas in FTCs and PTCs the prevalence of PAX8-PPARγ is lower than previously reported. PAX8-PPARγ did not correlate with invasiveness or affect prognosis in any tumour type.

Of the nine known PAX genes, only two (PAX2 and PAX8) are expressed in the developing kidney. Genetic evidence in mice and humans indicates that PAX2 plays a critical role in normal renal development and may sit atop a molecular cascade which unfolds during the transition from undifferentiated mesenchyme to the early stages of nephrogenesis. Less is known about the role of PAX8 in kidney development; although PAX8 is expressed in the S-shaped body and early proximal tubule, preliminary data suggest that renal morphogenesis is unaffected by its absence. In this review, we discuss the basic aspects of PAX gene structure and function and how mutations of PAX2 might interfere with structure of the developing nephron.

Dicer is a crucial enzyme for the maturation of miRNAs. Mutations in the Dicer gene are highly associated with Pleuro Pulmonary Blastoma-Family Dysplasia Syndrome (PPB-FDS, OMIM 601200), recently proposed to be renamed Dicer syndrome. Aside from the pulmonary phenotype (blastoma), renal nephroma and thyroid goiter are frequently part of Dicer syndrome. To investigate the renal phenotype, conditional knockout (cKO) mice for Dicer in Pax8 expressing cells were generated. Dicer cKO mice progressively develop a glomerulocystic phenotype coupled with urinary concentration impairment, proteinuria and severe renal failure. Higher cellular turnover of the parietal cells of Bowman's capsule precedes the development of the cysts and the primary cilium progressively disappears with cyst-enlargement. Upregulation of GSK3β precedes the development of the glomerulocystic phenotype. Downregulation of β-catenin in the renal cortex and its cytosolic removal in the cells lining the cysts may be associated with observed accumulation of GSK3β. Alterations of β-catenin regulating pathways could promote cystic degeneration as in other models. Thus, miRNAs are fundamental in preserving renal morphology and function. Alteration of the GSK3β/β-catenin pathway could be a crucial mechanism linking miRNA dysregulation and the development of a glomerulocystic disease.

Morphologic distinction of Müllerian carcinomas from non-Müllerian carcinomas in effusion specimens by cytomorphology alone can be diagnostically challenging. Therefore, immunohistochemical adjuncts can be useful in differentiating Müllerian from non-Müllerian metastases. In this study, we evaluated the expression of PAX8 and PAX2 in malignant effusions collected from patients with known Müllerian and non-Müllerian carcinomas. Sections from cell blocks prepared from 152 effusion specimens (54 and 98 cases representing metastases from Müllerian and non-Müllerian primaries, respectively) were immunostained with rabbit polyclonal antibodies against PAX8 and PAX2. Immunopositivity was defined as the presence of strong nuclear staining in at least 25% of the tumor cells. Fifty-two (96%) and 13 (24%) of the 54 Müllerian carcinomas were positive for PAX8 and PAX2, respectively. PAX8 positivity was seen in only four (4%) of 98 non-Müllerian carcinomas; these represented metastasis from a large cell neuroendocrine lung carcinoma, papillary thyroid carcinoma, renal cell carcinoma, and acinic cell carcinoma of the parotid gland. PAX2 positivity was not seen in any of the non-Müllerian carcinomas. The results demonstrate that both PAX8 and PAX2 are highly specific markers for metastatic Müllerian carcinomas in cell block preparations from effusion specimens (96% and 100%, respectively). PAX8, however, is more sensitive than PAX2 in identifying Müllerian carcinomas in fluids (96% versus 24%). Overall, immunohistochemistry for PAX8 and PAX2 represent diagnostically useful adjuncts in identifying a Müllerian carcinoma as a source of a malignant effusion.

Neoplasms frequently present structural chromosomal aberrations that can alter the level of expression of a protein or to the expression of an aberrant chimeric protein. In the thyroid, the PAX8-PPARG fusion is present in the neoplastic lesions that have a follicular architecture-follicular thyroid carcinoma (FTC) and follicular variant of papillary thyroid carcinoma (FVPTC), and less frequently in follicular thyroid adenoma (FTA), while the presence of RET/PTC fusions are largely restricted to papillary thyroid carcinoma (PTC). The ability to detect fusion genes is relevant for a correct diagnosis and for therapy. We have developed a new fusion gene microarray-based approach for simultaneous analysis of all known and predicted fusion gene variants. We did a comprehensive screen for 548 known and putative fusion genes in 27 samples of thyroid tumors and three positive controls-one thyroid cancer cell line (TPC-1) and two PTCs with known CCDC6-RET (alias RET/PTC1) fusion gene, using this microarray. Within the thyroid tumors tested, only well known, previously reported fusion genes in thyroid oncology were identified. Our results reinforce the pathogenic role played by RET/PTC1, RET/PTC3, and PAX8-PPARG fusion genes in thyroid tumorigenesis.

OBJECTIVE

Follicular thyroid tumours present several genetic alterations such as aneuploidy, RAS mutations and PAX8/PPARgammarearrangements. The molecular basis of aneuploidy remains undefined in the majority of human cancers. It has been proposed that mutations in RAS oncogenes could be related to chromosomal instability, although this issue remains controversial. The aim of our study was to investigate the correlation between aneuploidy, RAS mutations and PAX8/PPARgamma gene rearrangement in thyroid follicular tumours.

METHODS

Ploidy status was determined by flow cytometry in 111 thyroid lesions (42 follicular thyroid adenomas, 27 follicular thyroid carcinomas, 19 follicular variants of papillary thyroid carcinoma, 20 poorly differentiated thyroid carcinomas and 3 anaplastic thyroid carcinomas). RAS mutations and PAX8/PPARgamma fusion gene were investigated in 101 and 87 of these samples, respectively.

RESULTS

Altogether, 12 of 50 (24%) diploid tumours presented RAS mutation which contrasts with 3 of 51 (5.9%; P = 0.0124) RAS mutations in the group of aneuploid tumours. The aneuploid tumours harbouring RAS mutations were two poorly differentiated carcinomas and one follicular variant of papillary thyroid carcinoma with poorly differentiated areas. None of the tumours with RAS mutations expressed the PAX8/PPARgamma fusion gene. Three of five (60%) follicular thyroid adenomas and 1 of 7 (14%) follicular thyroid carcinomas, with the PAX8/PPARgamma fusion gene, were aneuploid.

CONCLUSIONS

Our data suggest that aneuploidy and RAS mutations are mutually exclusive events in the development of well-differentiated thyroid follicular tumours.

The PAX8/PPARγ fusion protein (PPFP) occurs in 36% of human follicular thyroid carcinoma (FTC) and is associated with favorable prognosis. To elucidate the function of PPFP in FTC, we analyzed the consequences of PPFP expression in immortalized thyrocytes in vitro and in vivo via xenograft tumorigenesis. While PPFP-expressing cells exhibited oncogenic hallmarks, including increased growth and decreased apoptosis, in vitro, xenograft tumors were initiated but not sustained in vivo. PPFP xenograft tumors exhibited reduced CD31 staining and VEGF expression, suggesting that PPFP modulates neovascularization. Microarray analysis demonstrated increased expression of tissue inhibitor of metalloproteinase (TIMP-3), an inhibitor of angiogenesis, in PPFP cells and tumors, a finding confirmed by quantitative PCR and immunohistochemistry. Immunohistochemical staining of archival human thyroid tumors demonstrates a significant decrease in CD31 staining in all adenomas and carcinomas containing the PAX8/PPARγ rearrangement. Decreased angiogenesis in PPFP-containing tumors is directly correlated with our observations in the xenograft model and provides evidence for the first time that PPFP may impact FTC tumorigenesis by modulating angiogenesis in vivo.

Diagnosis of thyroid by fine needle aspiration is challenging for the "indeterminate" category and can be supported by molecular testing. We set out to identify miRNA markers that could be used in a diagnostic setting to improve the discrimination of mutation-negative indeterminate fine needle aspirations. miRNA high-throughput sequencing was performed for freshly frozen tissue samples of 19 RAS and PAX8/PPARG mutation-negative follicular thyroid carcinomas, and 23 RAS and PAX8/PPARG mutation-negative follicular adenomas. Differentially expressed miRNAs were validated by quantitative polymerase chain reaction in a set of 44 fine needle aspiration samples representing 24 follicular thyroid carcinomas and 20 follicular adenomas. Twenty-six miRNAs characterized by a significant differential expression between follicular thyroid carcinomas and follicular adenomas were identified. Nevertheless, since no single miRNA had satisfactory predictive power, classifiers comprising two differentially expressed miRNAs were designed with the aim to improve the classification. Six two-miRNA classifiers were established and quantitative polymerase chain reaction validated in fine needle aspiration samples. Four out of six classifiers were characterized by a high specificity (≥94 %). The best two-miRNA classifier (miR-484/miR-148b-3p) identified thyroid malignancy with a sensitivity of 89 % and a specificity of 87 %. The high-throughput sequencing allowed the identification of subtle differences in the miRNA expression profiles of follicular thyroid carcinomas and follicular adenomas. While none of the differentially expressed miRNAs could be used as a stand-alone malignancy marker, the validation results for two-miRNA classifiers in an independent set of fine needle aspirations are very promising. The ultimate evaluation of these classifiers for their capability of discriminating mutation-negative indeterminate fine needle aspirations will require the evaluation of a sufficiently large number of fine needle aspirations with histological confirmation.

Downstream regulatory element antagonistic modulator (DREAM) was originally identified in neuroendocrine cells as a calcium-binding protein that specifically binds to downstream regulatory elements (DRE) on DNA, and represses transcription of its target genes. To explore the possibility that DREAM may regulate the endocrine activity of the thyroid gland, we analyzed its mRNA expression in undifferentiated and differentiated thyroid cells. We demonstrated that DREAM is expressed in the normal thyroid tissue as well as in differentiated thyroid cells in culture while it is absent in FRT poorly differentiated cells. In the present work, we also show that DREAM specifically binds to DRE sites identified in the 5' untranslated region (UTR) of the thyroid-specific transcription factors Pax8 and TTF-2/FoxE1 in a calcium-dependent manner. By gel retardation assays we demonstrated that thapsigargin treatment increases the binding of DREAM to the DRE sequences present in Pax8 and TTF-2/Foxe1 5' UTRs, and this correlates with a significant reduction of the expression of these genes. Interestingly, in poorly differentiated thyroid cells overexpression of exogenous DREAM strongly inhibits Pax8 expression. Moreover, we provide evidence that a mutated form of DREAM unable to bind Ca(2+) interferes with thyroid cell proliferation. Therefore, we propose that in thyroid cells DREAM is a mediator of the calcium-signaling pathway and it is involved in the regulation of thyroid cell function.

Coordination of events leading to differentiation is mediated by the concerted action of multiple signal transduction pathways. In general, the uncoupling of mechanisms linking differentiation to cell cycle exit is a hallmark of cancer, yet the identity and regulation of molecules integrating signal transduction pathways remains largely unknown. One notable exception is DARPP-32 (dopamine and cAMP-regulated neuronal phosphoprotein, molecular mass, 32 kDa), a third messenger that integrates multiple signaling pathways in the brain. Thyroid cells represent an excellent model for understanding the coupling of signal transduction pathways leading to both proliferation and differentiation. The cooperative action of IGF-I and TSH together, but not alone, enable thyroid cells to proliferate while maintaining their differentiated state. How signaling downstream from these molecules is integrated is not known. Here we show that DARPP-32 expression is targeted by TSH and IGF-I in thyrocytes. Significantly, dedifferentiated, tumoral, or Ras-transformed thyrocytes fail to express DARPP-32 whereas short interfering RNA-mediated silencing of DARPP-32 expression in normally differentiated thyroid cells results in loss of differentiation markers such as thyroid transcription factor 1, Pax8, thyroglobulin, and the Na/I symporter. Consistently, DARPP-32 reexpression in ras-transformed cells results in reactivation of the otherwise silent thyroglobulin and thyroperoxidase promoter. Thus, DARPP-32 is critical for the maintenance of thyroid differentiation by TSH and IGF-I, and loss of DARPP-32 expression may be a characteristic of thyroid cancer. Our results also raise the possibility that DARPP-32 may play a similar role in the maintenance of differentiation of a range of other cell types.

BACKGROUND

Cystinuria is an inherited disorder of luminal reabsorptive transport for cystine and dibasic amino acids in the renal proximal tubule. Two cystinuria genes have been identified. Mutations of SLC7A9, which encodes the luminal transport channel itself, tend to be dominant and mutations of SLC3A1 (rBAT), which encodes a transporter subunit, are always recessive. Patients who inherit two recessive mutations or two dominant mutations have equally severe forms of cystinuria. Heterozygotes excrete cystine in the normal (type I), moderate (type III), or high stone-forming (type II) range.

METHODS

Infants with cystinuria were identified via the Quebec Newborn Urinary Screening Program. In a subgroup of these infants, cystinuria was severe in the first months of life, but partially resolved by 2 to 4 years postnatally. We assigned each patient a final cystinuria phenotype at 3 to 4 years. In addition, we characterized SLC3A1 gene expression in fetal and postnatal human kidney.

RESULTS

Most infants with transient neonatal cystinuria are eventually classified as type III heterozygotes. All infants with mutant cystinuria genes have exaggerated neonatal cystine excretion except those who inherit two SLC3A1 mutations (type I/I cystinuria); these children have persistent severe cystinuria, implying that wildtype SLC3A1 is required for the maturational effect. Expression of SLC3A1 mRNA was found to be tenfold higher in postnatal vs. fetal kidney; SLC3A1 expression is doubled by the proximal tubule transcription factor, PAX8. rBAT is expressed in the proximal convoluted and straight tubules in both fetal and adult kidney.

CONCLUSIONS

Maturation of SLC3A1 gene expression between midgestation and 4.5 years postnatal age may account for transient neonatal cystinuria.

OBJECTIVE

To discuss the use of molecular mutation analysis in the surgical management of pediatric thyroid nodules.

METHODS

This study is a case series with retrospective chart review performed at a tertiary children's hospital. Pediatric patients who presented to the Children's Hospital of Pittsburgh of UPMC with a thyroid nodule and had subsequent fine needle aspiration with positive molecular mutation between November 2009 and February 2012 were identified and charts were reviewed. Patient demographics, presenting signs, lab results, pathologic findings, and surgical outcomes were collected.

RESULTS

5 pediatric patients with positive molecular mutation studies on preoperative FNA were identified. FNA results were categorized as follows: suspicious for follicular neoplasm (n = 2), suspicious for malignant cells (n = 1), and positive for malignant cells (n = 2). The following molecular mutations were identified: BRAF (V600E) (n = 2), PAX8/PPARγ (n = 1), HRAS (n = 1), and RET/PTC (n = 1). A total thyroidectomy was performed on each patient. In all cases the final pathology was positive for malignancy (papillary thyroid carcinoma (PTC), n = 3; follicular variant of PTC, n = 2). Three of five patients had transient postsurgical hypocalcemia. There were no other postoperative complications.

CONCLUSIONS

This series provides evidence that preoperative FNA with reflex molecular testing in pediatric thyroid nodules can help guide surgical decision making, reduce the need for repeat surgeries, and diminish the risk of complications from a staged procedure.

Cadherin-16 was originally identified as a tissue-specific cadherin present exclusively in kidney. Only recently, Cadherin-16 has been detected also on the plasma membrane of mouse thyrocytes. This last finding prompted us to note that the expression profile of Cadherin-16 resembles that of the transcription factor Pax8, a member of the Pax (paired-box) gene family, predominantly expressed in the developing and adult kidney and thyroid. Pax8 has been extensively characterized in the thyroid and shown to be a master gene for thyroid development and differentiation. In this study, we determined the role of the transcription factor Pax8 in the regulation of Cadherin-16 expression. We demonstrate that the Cadherin-16 minimal promoter is transcriptionally active in thyroid cells as well as in kidney cells, that Pax8 is able to activate transcription from a Cadherin-16 promoter reporter construct, and more importantly, that indeed Pax8 is able to bind in vivo the Cadherin-16 promoter region. In addition, by means of Pax8 RNA interference in thyroid cells and by analyzing Pax8 null mice, we demonstrate that Pax8 regulates also in vivo the expression of Cadherin-16. Finally, we reveal that the expression of Cadherin-16 is TSH dependent in FRTL-5 thyroid cells and significantly reduced in mouse thyroid carcinomas. Therefore, we conclude that Cadherin-16 is a novel downstream target of the transcription factor Pax8, likely since the early steps of thyroid development, and that its expression is associated with the fully differentiated state of the thyroid cell.

BACKGROUND

Gene variants have been reported to be associated with congenital hypothyroidism (CH), the purpose of this study was to analyze the mutation spectrum and prevalence of 12 known causative genes (TSHR, PAX8, NKX2.1, NKX2.5, FOXE1, DUOX2, TG, TPO, GLIS3, NIS, SLC26A4 and DEHAL1) in CH in China.

METHODS

Peripheral venous blood samples were collected from the patients. Genomic DNA was extracted from peripheral blood leukocytes. All exons and their exon-intron boundary sequences of the 12 known CH associated genes in 66 CH patients were screened by next-generation sequencing (NGS).

RESULTS

NGS analysis of 12 known CH associated genes revealed that 32 patients (32/66, 48.5%) were detected to have at least one potentially functional variant. 21, 9, 1, 1, 1 and 1 patients were found to have potential pathogenic variants in DUOX2, TG, PAX8, SLC26A4, TSHR and TPO genes, respectively. Novel variants included one DUOX2 and one TPO missense variants of unknown significance (VUS).

CONCLUSIONS

Our study expands the mutation spectrum of DUOX2 and TPO genes. 48.5% CH patients had at least one potential pathogenic variant. We found relatively high frequency of DUOX2 (31.8%) and TG (13.6%) mutations in our cohort.

Severe forms of congenital hypothyroidism lead to serious clinical symptoms if thyroid hormone replacement therapy is not instituted immediately after birth. In this study, Pax8(-/-) mice that are born without a thyroid gland were used as an animal model to study the consequences of congenital hypothyroidism. As expected, adequate treatment of these animals with thyroxine restored the general deficits of congenital hypothyroidism; however, Pax8-deficient male mice were infertile. We report here that in these mice, the efferent ducts and epididymides are either absent or the efferent ducts exhibit a reduced lumen and extensive connective tissue, which appears to impair testicular drainage and subsequently leads to complete absence of spermatozoa from the epididymis. The results suggest that, starting with the onset of pubertal testicular fluid secretion, a backpressure is created in the testis by the absence of efferent ducts or constriction of their tubule lumen when present. This subsequently leads to secondary disorganization of the seminiferous epithelium that increases with age, resulting in mixed atrophy of the testis in the adult. Serum testosterone levels as well as mRNA expression of anterior pituitary hormones are in the normal range, indicating that the observed infertility is not due to hormonal imbalance, but rather to a developmental defect of the efferent ducts. The demonstration of Pax8 expression in the epithelia of the epididymis and the efferent ducts suggests a direct morphogenic role of Pax8 in the development of these organs. It remains to be elucidated whether congenital hypothyroid male patients with mutations in the Pax8 gene are similarly affected.

OBJECTIVE

Mouse mesenchymal stem cells (MSCs) can generate sensory neurons and produce inner ear hair cell-like cells. An equivalent source from humans is highly desirable, given their potential application in patient-specific regenerative therapies for deafness. In this study, we explored the ability of human MSCs (hMSCs) to differentiate into otic lineages.

METHODS

hMSCs were exposed to culture media conditioned by human fetal auditory stem cells.

RESULTS

Conditioned media induced the expression of otic progenitor markers PAX8, PAX2, GATA3 and SOX2. After 4 weeks, cells coexpressed ATOH1, MYO7A and POU4F3 (indicators of hair cell lineage) or neuronal markers NEUROG1, POU4F1 and NEFH. Inhibition of WNT signaling prevented differentiation into otic progenitors, while WNT activation partially phenocopied results seen with the conditioned media.

CONCLUSIONS

This study demonstrates that hMSCs can be driven to express key genes found in the otic lineages and thereby promotes their status as candidates for regenerative therapies for deafness.

The International Endocervical Adenocarcinoma Criteria and Classification was developed to separate endocervical adenocarcinomas (ECAs) into 2 main categories on the basis of morphology such as human papilloma virus-associated (HPVA) and non-human papilloma virus-associated adenocarcinomas. We aimed to improve the diagnostic accuracy of International Endocervical Adenocarcinoma Criteria and Classification by performing a comprehensive immunohistochemical evaluation and constructing objective immunohistochemical-based algorithms for the classification of these tumors. Tissue microarrays were constructed from 297 of 409 cases used to develop the original classification. Immunostains included p16, p53, estrogen receptor (ER), progesterone receptor, androgen receptor, Vimentin, CK7, CK20, HER2, HIK1083, MUC6, CA-IX, SATB2, HNF-1beta, napsin A, PAX8, CDX2, GATA3, p63, p40, and TTF-1. High-risk human papilloma virus (HR-HPV) was detected by in situ hybridization (ISH) using probes against E6 and E7 mRNA expressed in 18 different virus types. Vimentin, ER, and progesterone receptor were expressed in a significant minority of ECAs, mostly HPVAs, limiting their use in differential diagnosis of endometrioid carcinoma when unaccompanied by HPV-ISH or p16. HR-HPV ISH had superior sensitivity, specificity, and negative and positive predictive values compared with p16, as published previously. HNF-1beta did not have the anticipated discriminatory power for clear cell carcinoma, nor did MUC6 or CA-IX for gastric-type carcinoma. HNF-1beta and napsin A were variably expressed in clear cell carcinoma, with HNF-1beta demonstrating less specificity, as it was ubiquitously expressed in gastric-type carcinoma and in the majority of HPV-associated mucinous (predominantly intestinal-type and invasive ECA resembling stratified mucin-producing intraepithelial lesion [iSMILE]) and usual-type carcinomas. HIK1083 was expressed in nearly half of gastric-type carcinomas, but not in the vast majority of other subtypes. GATA3 was positive in 10% of usual-type adenocarcinomas and in single examples of other subtypes. Rare gastric-type and HPVA mucinous carcinomas displayed HER2 overexpression. Androgen receptor was positive in 6% of usual-type adenocarcinomas. Aberrant p53 expression was found in only 3.6% of usual-type HPVA carcinomas, but it was more prevalent in mucinous (intestinal type and iSMILE) HPVAs and non-human papilloma virus-associates (particularly in gastric-type carcinoma, >50% of cases). The following diagnostic classification algorithms were developed with the above data. Carcinomas without overt cytoplasmic mucin (endometrioid, usual-type endocervical, clear cell, and mesonephric carcinomas) can be subclassified using HR-HPV ISH, ER, and GATA3, whereas carcinomas with easily appreciated cytoplasmic mucin (endometrioid carcinoma with mucinous features, HPVA mucinous, and gastric-type carcinomas) can be subclassified with HR-HPV ISH and ER.

The molecular etiology of follicular thyroid tumors is largely unknown, rendering the diagnostics of these tumors challenging. The somatic alterations present in these tumors apart from RAS gene mutations and PAX8/PPARG translocations are not well described. To evaluate the profile of somatic alteration in follicular thyroid tumors, a total of 82 thyroid tissue samples derived from 48 patients were subjected to targeted Illumina HiSeq next generation sequencing of 372 cancer-related genes. New somatic alterations were identified in oncogenes (MDM2, FLI1), transcription factors and repressors (MITF, FLI1, ZNF331), epigenetic enzymes (KMT2A, NSD1, NCOA1, NCOA2), and protein kinases (JAK3, CHEK2, ALK). Single nucleotide and large structural variants were most and least frequently identified, respectively. A novel translocation in DERL/COX6C was detected. Many somatic alterations in non-coding gene regions with high penetrance were observed. Thus, follicular thyroid tumor somatic alterations exhibit complex patterns. Most tumors contained distinct somatic alterations, suggesting previously unreported heterogeneity.

The bcl-2 proto-oncogene suppresses apoptosis in a variety of cell types and is essential for normal renal development. PAX8 is a member of the paired box class of transcription factors and is developmentally regulated. bcl-2 and PAX8 are both expressed in the kidney during development and throughout life, demonstrating a nearly identical pattern of expression. We used transient transfection reporter assays and electrophoretic mobility shift assays to test PAX8 transcriptional regulation of bcl-2. PAX8 transcriptionally activates the bcl-2 promoter and binds to promoter sequences in vitro. These findings establish that PAX8 can transcriptionally regulate bcl-2 and suggest that suppression of apoptosis is an important event in the development and maintenance of renal tubular epithelial structures.

BACKGROUND

Neoadjuvant chemotherapy followed by cytoreduction surgery has been used where an accurate cytologic or pathologic diagnosis is usually required before the initiation of neoadjuvant chemotherapy. However, it is difficult to make definitive diagnosis of presence of cancer cells, particularly gynecologic versus non-gynecologic origin, from those ascites specimens due to the absence of specific biomarkers of gynecologic cancers. In the present study, we evaluated if, in addition to the routine morphologic diagnosis, the biomarker PAX8 could be useful in recognition of ovarian epithelial cancer cells prior to the neoadjuvant chemotherapy.

METHODS

Two hundred and two cytology specimens including 120 pretreatment ovarian cancer samples, 60 benign controls, and 22 malignant non-gynecologic cases were studied. All cytology slides were morphologically reviewed in a blinded fashion without knowing corresponding pathology diagnosis, if present. A total of 168 cytology specimens with a cell block were stained with PAX8 and Calretinin. These included patients with potential for ovarian cancer neoadjuvant chemotherapy (n=96), metastatic cancers (n=22), and benign controls (n=50).

RESULTS

Among the 96 ascitic samples prior to neoadjuvant chemotherapy, 76 (79%) showing morphologic features consistent with cancers of ovarian primary were all PAX+/Calretinin-. The remaining 20 (21%) cases were positive for adenocarcinoma, but morphologically unable to be further classified. Among the 22 metastatic cancers into the pelvis, one case with PAX8+/Calretinin- represented a renal cell carcinoma and the remaining 21 PAX8-/Calretinin- metastatic cancers were either breast metastasis (n=4) and the metastasis from gastrointestinal tract (n=17). Among the 50 benign control pelvic washing cases, 5 PAX8+/Calretinin-cases represented endosalpingiosis (n=4) and endometriosis (n=1), 25 PAX8-/Calretinin+cases showed reactive mesothelial cells, and the remaining 20 specimens with PAX8-/Calretinin- phenotype typically contained inflammatory or blood cells without noticeable diagnostic epithelia.

CONCLUSIONS

PAX8 identifies all Müllerian derived benign or malignant epithelia. When combining with Calretinin, PAX8 is a sensitive marker to diagnose the carcinomas of ovarian origin, which will be ideal to be used for those patients with a possible advanced ovarian cancer prior to receiving neoadjuvant chemotherapy.