For several decades, glucocorticoids have been used empirically to treat rapid progressive GN. It is commonly assumed that glucocorticoids act primarily by dampening the immune response, but the mechanisms remain incompletely understood. In this study, we inactivated the glucocorticoid receptor (GR) specifically in kidney epithelial cells using Pax8-Cre/GRfl/fl mice. Pax8-Cre/GRfl/fl mice did not exhibit an overt spontaneous phenotype. In mice treated with nephrotoxic serum to induce crescentic nephritis (rapidly progressive GN), this genetic inactivation of the GR in kidney epithelial cells exerted renal benefits, including inhibition of albuminuria and cellular crescent formation, similar to the renal benefits observed with high-dose prednisolone in control mice. However, genetic inactivation of the GR in kidney epithelial cells did not induce the immunosuppressive effects observed with prednisolone. In vitro, prednisolone and the pharmacologic GR antagonist mifepristone each acted directly on primary cultures of parietal epithelial cells, inhibiting cellular outgrowth and proliferation. In wild-type mice, pharmacologic treatment with the GR antagonist mifepristone also attenuated disease as effectively as high-dose prednisolone without the systemic immunosuppressive effects. Collectively, these data show that glucocorticoids act directly on activated glomerular parietal epithelial cells in crescentic nephritis. Furthermore, we identified a novel therapeutic approach in crescentic nephritis, that of glucocorticoid antagonism, which was at least as effective as high-dose prednisolone with potentially fewer adverse effects.

OBJECTIVE

To examine squamous cell carcinomas (SCCs) from diverse anatomic sites and invasive urothelial carcinomas (UCs) for expression of the oncofetal antigen glypican 3 (GPC3), the paired box transcription factor PAX8, and the ΔN isoform of p63 (p40).

METHODS

Immunohistochemistry for GPC3, PAX8, and p40 was performed on whole sections of 107 SCCs from 11 anatomic sites and 49 UCs; evaluation included extent and intensity of staining.

RESULTS

GPC3 was detected in 20% of SCCs and 12% of UCs and PAX8 in 3% of SCCs, limited to the uterine cervix, and 10% of UCs. p40 Was found in 99% of SCCs and 96% of UCs.

CONCLUSIONS

GPC3 expression is frequent in SCC/UC, awareness of which should guard against an incorrect diagnosis of hepatocellular carcinoma, while PAX8, limited in distribution, may have some use in suggesting a cervical or urothelial tract origin in a metastatic squamotransitional carcinoma of unknown primary. There is no drop-off in sensitivity for the diagnoses of SCC or UC with ΔNp63-specific immunohistochemistry, and if this performance can be extended to other applications, p40 may supplant the dominant "pan-p63" antibody clone.

The differential diagnosis of renal cell neoplasms with solid or nested architecture and eosinophilic cytoplasm has become increasingly complex. Despite recent advances in classifying a number of entities exhibiting this morphology, some tumors remain in the unclassified category. Here we describe a morphologically distinct group of sporadic renal cell carcinoma (RCC) with predominantly nested architecture, eosinophilic, and remarkably vacuolated cytoplasm retrospectively identified from a cohort of previously unclassified tumors. We examined the clinicopathologic and immunohistochemical features of these tumors and investigated their mutational and copy number alterations using a targeted next-generation sequencing platform. The study included 7 patients with a mean age of 54 years (range: 40 to 68 y) and a male to female ratio of 3:4. All patients presented with a solitary renal mass and had no prior medical or family history raising concern for syndromic conditions. Tumors were well-circumscribed, unencapsulated, and comprised of nests of eosinophilic cells in a hypocellular and often edematous stroma. Tumor cells had round nuclei with prominent nucleoli and granular cytoplasm with striking vacuolization. Thick-walled vessels and calcifications were also frequently present, whereas increased mitotic activity, necrosis, foamy histiocytes or lymphocytic infiltrates were not identified. All cases were positive for PAX8, had retained expression of SDHB and FH, and exhibited a CK7-/CK20- phenotype. While cathepsin-K was positive in 5 cases, none exhibited immunoreactivity to HMB45 or Melan A, or TFE3 immunostaining. Next-generation sequencing identified somatic inactivating mutations of TSC2 (3/5 tumors tested) or activating mutations of MTOR (2/5) as the primary molecular alterations, consistent with hyperactive mTOR complex 1 signaling which was further demonstrated by phospho-S6 and phospho-4E-BP1 immunostaining. Copy number analysis revealed a loss of chromosome 1 in both cases with MTOR mutation. These tumors represent a novel subset of sporadic RCC characterized by alterations in TSC1-TSC2 complex or the mTOR complex 1 pathway. Recognition of their characteristic morphologic and immunophenotypic features will allow them to be readily identified and separated from the unclassified RCC category.

OBJECTIVE

To compare the expression of genes playing a decisive role during the embryonic development of the female genital tract (WNT4, WNT5A, WNT7A, PAX8) in the peritoneum of patients with endometriosis and control patients.

METHODS

Experimental study using real-time polymerase chain reaction and in situ hybridization.

METHODS

University-based laboratory.

METHODS

Patients with and without endometriosis undergoing surgery for benign indications.

METHODS

None.

METHODS

Percentage of samples positive for gene expression by using real-time polymerase chain reaction, as well as relative expression values compared with housekeeping genes. Confirmation of results by in situ hybridization.

RESULTS

Expression of WNT7A and PAX8 was found in the normal peritoneum in approximately half of the patients with endometriosis in contrast to the controls. In patients with endometriosis WNT7A and PAX8 in histologically normal peritoneum (with no evidence of endometriosis, endosalpingiosis, or other changes) were confirmed by in situ hybridization.

CONCLUSIONS

The expression of these genes in the normal peritoneum suggests that endometriosis can arise through metaplasia and can in the process make use of the developmental steps involved in the embryonic development of the female genital tract.

BACKGROUND

Mutations in PAX8, a transcription factor gene, cause thyroid dysgenesis (TD). The extreme variability of the thyroid phenotype makes it difficult to identify individuals harboring PAX8 gene mutations. Here we describe two patients with TD and report two novel PAX8 gene mutations (S54R and R133Q). We performed in vitro studies to functionally characterize these mutations.

METHODS

Using PAX8 expression vectors, we investigated whether the PAX8 mutants localized correctly to the nucleus. To analyze the DNA-binding properties of S54R and R133Q, electrophoretic mobility shift assays were performed. Furthermore, we measured whether the mutant PAX8 proteins were able to activate the thyroglobulin (TG)- and the thyroperoxidase (TPO)-promoters.

RESULTS

S54R had an impaired binding to DNA and a negligible activity on the TG- and the TPO-promoters. The DNA-binding property of R133Q, which is located in the highly conserved terminal portion of the PAX8 DNA-binding domain, was normal. Interestingly, it also exhibited dramatically impaired activation of the TG- and TPO-promoters. However, R133Q has no dominant negative effect on the WT protein in vitro. Thus, the underlying molecular mechanism by which the function of R133Q is impaired remains to be elucidated.

CONCLUSIONS

We identified and functionally characterized two novel mutations of the PAX8 gene that lead to TD by distinct mechanisms. A structural defect of the mutant R133Q leading to a reduced capability for induced fit upon DNA interaction might explain the disparity between its apparently normal binding to DNA, but lack of promoter activation.

Adenomatoid tumors (ATs) are uncommon benign mesothelial tumors with a predilection for the genital tract. We reviewed 47 ATs diagnosed at our institutions during 10-year period. Thirty tumors (64%) originated in the female (21 uterine, 8 tubal, and 1 ovarian) and 17 (36%) in the male (9 epididymal and 8 testicular) genital tract. The median age for females and males was 47.5 and 51 years, respectively. While 83% of tumors in females were incidental findings in resections for unrelated diseases, 94% of male lesions presented as clinical masses leading to surgery. The median size was 2, 1, and 0.5 cm for uterine, epididymo-testicular, and tubo-ovarian lesions, respectively. Architecturally, the microcystic/angiomatoid pattern was the most frequent (32/47; 68%), followed by combined microcystic/trabecular (26/47; 55%) and retiform/adenoid (15/47; 32%) pattern. The trabecular/solid (6%) and macrocystic (4%) patterns were uncommon. However, 57% of cases revealed ≥2 growth patterns. Taken by anatomic site, 20 of 21 uterine cases were at least focally microcystic but none was retiform. In contrast, the retiform pattern dominated in male genital tract tumors (12/17; 71%). Immunohistochemistry showed expression of calretinin (36/36) and D2-40 (30/30) and lack of CD34 (0/30) and PAX8 (0/32). GLUT-1 was expressed in 11/11 male genital tract tumors but in none of the microcystic uterine lesions. Estrogen and progesterone receptor expression was weak and focal (two and three uterine cases, respectively). None stained for the androgen receptor. Our study illustrates the great site-specific morphological diversity of ATs emphasizing their wide site-dependent differential diagnosis.

Anaplastic thyroid carcinoma (ATC) is a very aggressive human malignancy, having a marked degree of invasiveness and no features of thyroid differentiation. It is known that either HDAC inhibitors or PARP inhibitors have antiproliferative effects on thyroid cancer cells. Therefore, in this study the possible synergy between the two types of compounds has been investigated. The ATC-derived cell line SW1736 has been treated with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and the PARP inhibitor PJ34, alone or in combination. In terms of cell viability, the combination index value was always lower than 1 at various tested dosages, indicating, therefore, synergy in a wide range of doses for both compounds. Synergy was also observed in induction of apoptosis. In terms of thyroid-specific gene expression, synergy was observed for TSHR mRNA levels but not for NIS, TTF1, TTF2, and PAX8 mRNA levels. Altogether, these data suggest that the combined use of HDAC and PARP inhibitors may be a useful strategy for treatment of ATC.

Forkhead box E1 encodes the transcription factor FOXE1 (or TTF-2), which together with Homeobox protein NKX2-1, PAX8 and HHEX, are pivotal proteins required for thyroid gland formation, differentiation and function. Recently, genome-wide association studies have identified FOXE1 as a thyroid cancer (TC) susceptibility gene in populations of European descent. After that, a number of studies reported that the rs965513, rs1867277, and rs71369530 polymorphism in FOXE1 has been implicated in TC risk. However, the causal variants remain unknown. To derive a more precise estimation of the relationship, a meta-analysis of 9,828 TC cases and 109,995 controls from 14 case-control studies was performed. Overall, significant results were observed for rs965513 (OR=1.71, 95% CI: 1.59-1.85, P<10(-5)), rs1867277 (OR=1.64, 95% CI: 1.51-1.78, P<10(-5)) and rs71369530 (OR=2.01, 95% CI: 1.66-2.44, P<10(-5)) polymorphism. In the subgroup analysis by ethnicity, we found that rs965513 polymorphism confer high risk for Caucasians with per-allele OR of 1.80 (95% CI: 1.69-1.92, P<10(-5)) compared to East Asians of 1.35 (95% CI: 1.09-1.67, P=0.006). There was strong evidence of heterogeneity, which largely disappeared after stratification by ethnicity. In the subgroup analysis by sample size, and study design, significantly increased risks were found for the polymorphism. In conclusion, this meta-analysis demonstrated that common variations of FOXE1 are a risk factor associated with increased TC susceptibility.

Benzophenone-4 (BP-4) is frequently used as UV-absorber in cosmetics and materials protection. Despite its frequent detection in the aquatic environment potential effects on aquatic life are unknown. In this study, we evaluate the effects of BP-4 in eleuthero-embryos and in the liver, testis and brain of adult male fish on the transcriptional level by focusing on target genes involved in hormonal pathways to provide a more complete toxicological profile of this important UV-absorber. Eleuthero-embryos and males of zebrafish were exposed up to 3 days after hatching and for 14 days, respectively, to BP-4 concentrations between 30 and 3000 μg/L. In eleuthero-embryos transcripts of vtg1, vtg3, esr1, esr2b, hsd17ß3, cyp19b cyp19a, hhex and pax8 were induced at 3000 μg/L BP-4, which points to a low estrogenic activity and interference with early thyroid development, respectively. In adult males BP-4 displayed multiple effects on gene expression in different tissues. In the liver vtg1, vtg3, esr1 and esr2b were down-regulated, while in the brain, vtg1, vtg3 and cyp19b transcripts were up-regulated. In conclusion, the transcription profile revealed that BP-4 interferes with the expression of genes involved in hormonal pathways and steroidogenesis. The effects of BP-4 differ in life stages and adult tissues and point to an estrogenic activity in eleuthero-embryos and adult brain, and an antiestrogenic activity in the liver. The results indicate that BP-4 interferes with the sex hormone system of fish, which is important for the risk assessment of this UV-absorber.

BACKGROUND

Metastases from ovarian neoplasms are commonly encountered in peritoneal fluids. In addition, reactive mesothelial cells in effusion specimens can mimic ovarian serous carcinoma, making the diagnosis difficult. Calretinin has been recognized as a reliable immunohistochemical marker for mesothelial cells, whereas WT1 has proven useful in the diagnosis of ovarian serous carcinoma. This can present a diagnostic pitfall in effusion cytology, because mesothelial cells can demonstrate immunoreactivity for WT1. Recently, paired box gene 8 (PAX8) has been used in distinguishing ovarian from mammary carcinoma. To the authors' knowledge, no studies using PAX8 have been performed on peritoneal cytology specimens to date, and its expression in metastatic ovarian serous carcinoma has not been studied.

METHODS

These markers, along with BerEP4 and MOC-31, were evaluated in cytology cell block preparations from 30 fluid cytology specimens and 11 fine-needle aspiration specimens.

RESULTS

PAX8 was found to be positive in 37 of 41 (90%) ovarian carcinoma cases studied, and was a sensitive (90%) and specific (100%) marker for the detection of metastatic ovarian carcinoma. In addition, calretinin was found to be useful for identifying mesothelial cells in fluid cytology. Furthermore, although PAX8 and WT1 have demonstrated comparable sensitivity (90% and 93%, respectively) in diagnosing metastatic ovarian carcinoma, PAX8 appears to have superior specificity because staining is not observed in mesothelial cells. BerEP4 and MOC-31 were found to have a lower sensitivity and specificity compared with PAX8.

CONCLUSIONS

PAX8-positive, calretinin-negative staining appears to be highly specific and sensitive for detecting metastatic ovarian serous carcinoma in cytologic preparations and can be useful in distinguishing it from mesothelial cells in fluid cytology.

The Bethesda system for standardized reporting of thyroid fine needle aspiration (FNA) cytology has positively affected the clarity of communication of results and management of patients evaluated for thyroid nodules. Problematic areas still exist in the triage of some of these samples, particularly those in the categories of "follicular lesion with atypia of uncertain significance" and "follicular lesion." The literature on molecular and genetic abnormalities in thyroid lesions is reviewed. Potentially useful markers for distinguishing currently problematic categories of FNA cytologic samples, especially nondiagnostic samples, atypia of uncertain significance, and follicular lesions, are discussed. The predictive value of the respective molecular analyses in these settings is examined. Evaluation of FNA samples with negative or suboptimal follicular cytology for Ras mutations may be useful in detecting potentially significant follicular lesions (carcinomas) but is quite low in overall yield. Cytologic samples with atypia of uncertain significance, which may include the possibility of papillary carcinomas, may be fruitfully evaluated using a panel of molecular tests for BRAF, RET/PTC, PAX8/PPARG1, and Ras. Other markers also have potential utility in the workup of thyroid lesions. An era of combined modality testing in thyroid cytology is emerging in which classical cytologic findings can be coupled with molecular data to increase the predictive power of diagnostic interpretations; however, there remains a group of atypical cytologic samples negative for known molecular markers in which the risk of malignancy is too high to simply follow expectantly.

The occurrence of the PAX8-PPARgamma fusion gene is thought to be restricted to follicular tumors (adenomas and carcinomas) of the thyroid (FTA and FTC). Using interphase fluorescent in situ hybridization (FISH), together with recombinant tissue-type polymerase chain reaction (RT-PCR) and immunohistochemistry, we detected the PAX8-PPARgamma translocation in 4 of 8 cases of the follicular variant of papillary thyroid carcinoma (FVPTC) exclusively or almost exclusively (>95%) composed of follicles. The 4 tumors with the translocation were larger and apparently more invasive than the remaining tumors, but the series is too small to allow a statistically meaningful comparison of the data. Our findings show that follicular thyroid carcinoma (PTC) may also harbor the PAX8-PPARgamma fusion gene and indicate that a subset of FVPTC shares some molecular features of FTA and FTC.

A recent western study reports that t(2; 3)(q13; p25) translocation resulting in the expression of the Pax8-PPAR gamma fusion gene in patients with thyroid follicular carcinoma (FTC) occurs with high incidence (63%). Furthermore, the products of the fusion gene were shown to suppress the function of PPAR gamma in a predominantly negative manner, conferring them with an oncogenic potential. We examined the expression of this fusion gene in FTC in Japanese patients. From 1989 to 2000, six cases with FTC were surgically treated at our institute. In these carcinoma samples, the expression of mRNAs for the Pax8-PPAR gamma fusion product was analyzed by nested RT-PCR. Their expression was also studied in other thyroid nodules (12 adenomatous goiters, 12 follicular adenomas, 12 papillary carcinomas and 12 normal thyroid tissues) obtained at surgery during the same period. Pax8-PPAR gamma fusion mRNA was not detected in any FTC samples nor in the other samples. Furthermore, none of the 6 FTCs, one follicular adenoma or one normal thyroid analyzed by fluorescence in situ hybridization (FISH) exhibited Pax8-PPAR gamma gene fusion. These findings are in contrast to previous reports and indicate that ethnic background may affect the translocation.

Overexpression of the embryonic transcription factor, Wilms' tumour protein 1 (WT1), is common in acute myeloid leukaemias (AML). Mutations of Wilms' tumour gene 1 (WT1) in AML are rare and WT1 expression may be increased by other transcription factors. PAX2, PAX8 and GATA-1 are known physiological regulators of WT1. In the present study, we analysed either bone marrow or blood samples of 43 AML patients for the expression levels of WT1, PAX2, PAX8 and GATA-1 by real-time reverse transcription polymerase chain reaction (LightCycler). Bone marrow samples of patients without haematological malignancies and stem cell preparation samples from healthy donors and lymphoma patients served as controls. PAX2 expression was found in 11 of 43 AML samples, with a clear correlation of PAX2 with WT1 expression levels observed. PAX8 expression was found in two additional samples. GATA-1 expression was detectable in 41 of 43 AML samples and also in all control samples; no significant differences between these groups were observed and no correlation of GATA-1 expression with WT1 expression levels was apparent. In conclusion, PAX2, and possibly PAX8, appears to be a candidate for the upregulation of WT1 in a proportion of AML, whereas GATA-1 expression cannot be explained as an inducer of WT1. In two-thirds of leukaemias from our series, the basis of WT1 upregulation cannot be explained by the simple upregulation of the known WT1 activators.

Hox genes encode transcription factors that play essential roles during embryo morphogenesis and organogenesis. Expression of several Hox members persists at the adult age, indicating a wide spectrum of action from embryonic to postnatal life. In the present study, we reported that in adult mice, the Hoxa5 gene shows a dynamic expression profile in the ovary that depends on the estrous cycle, the gestational status, and the age of the female, suggesting that Hoxa5 may have distinct physiological functions in the ovary. Consistent with a role for Hoxa5 in ovarian function, Hoxa5(-/-) nulliparous females exhibit precocious puberty and an early onset of estrous acyclicity. They show a prolonged estrous cycle with increased metestrus-diestrus length, a phenotype that worsens with age. Older mutant females also develop ovarian epithelial inclusion cysts reminiscent of human endosalpingiosis. Immunolabeling studies suggest that these cysts originate from the ovarian surface epithelium, a source of epithelial ovarian carcinomas. Staining of the Hoxa5(-/-) ovarian cysts by the ovarian cancer markers paired box gene 8 (PAX8) and Wilms' tumor 1 (WT1) further strengthens the notion that these cysts may constitute preneoplastic lesions. Moreover, the deregulation of the estrous cycle and the presence of ovarian epithelial cysts in Hoxa5(-/-) older females correlate with a reduced expression of specific epidermal growth factor receptor signaling components, namely Egfr, Areg, and Btc. Altogether, our data unveil that Hoxa5, a stroma-specific gene, plays a significant role in ovarian biology and may be involved in ovarian cancer predisposition.

Among the approaches used to provide a functional inactivation of a target protein, we have chosen the recently described oligomerization chain reaction (OCR) strategy to functionally inactivate the transcription factor Pax8, a member of the Pax gene family expressed in thyroid cells. The OCR strategy is based on the fusion of the self-associating coiled-coil (CC) domain of the nuclear factor promyelocytic leukemia (PML) to target proteins that are able to self-associate naturally or that form heterocomplexes. In the thyroid tissue, the transcription factor Pax8 is involved in the morphogenesis of the gland and in the transcriptional regulation of thyroid-expressed genes. We have recently demonstrated that in thyroid cells Pax8 interacts biochemically and functionally with the transcription factor TTF-1 (thyroid transcription factor 1), and that such interaction leads to the synergistic activation of thyroglobulin (Tg) gene expression. Fusion of the CC domain to Pax8 leads to the formation of aberrant, nonfunctional high-molecular mass complexes to which TTF-1 is also recruited. The CC-Pax8 chimera inhibits the transcriptional activity of Pax8 and of TTF-1 on both synthetic and physiological promoters and prevents the synergistic activation of the Tg promoter mediated by these two transcription factors. Furthermore, the expression of the CC-Pax8 chimera in differentiated thyroid cells leads to the down-regulation of the endogenous expression of several differentiation markers such as Tg, sodium/iodide symporter, Foxe1, TTF-1, and thyroid oxidase 2. These results demonstrate that the OCR is a useful tool to functionally inactivate a transcription factor. Moreover, by this approach, we identified Foxe1, TTF-1, and thyroid oxidase 2 as new direct targets of Pax8 or TTF-1.

Anaplastic thyroid cancer (ATC) is an aggressive, fatal disease unresponsive to traditional therapies, generating a need to develop effective therapies. The PAX8/PPARγ fusion protein (PPFP) has been shown to favorably modulate tumor growth in follicular thyroid cancer, prompting our evaluation of its efficacy to inhibit ATC cell and tumor growth in vitro and in vivo. PPFP was constitutively expressed in five ATC cell lines: BHT-101, FRO, C-643, KTC-2 and KTC-3, and inhibited cell growth in four of five cell lines and xenograft tumor growth in four of four cell lines. PPFP-mediated growth inhibition involved multiple mechanisms, including upregulation of miR-122 and miR-375, associated with decreased angiogenesis and AKT pathway inactivation, respectively. Also, PPFP expression resulted in marked increase of thyroid-specific marker transcripts, including PAX8, thyroid peroxidase (TPO), sodium iodide symporter (NIS) and thyroglobulin, to varying degrees by activating their respective promoters, suggesting that PPFP induced cellular redifferentiation. Functional studies demonstrate that increased NIS messenger RNA is not associated with increased 125I uptake. However, ectopic expression of wild-type NIS-induced perchlorate-sensitive iodine uptake, suggesting that endogenous NIS in ATC cell lines is defective. As current treatment for ATC is only palliative, overexpression of PPFP may offer a novel therapeutic strategy for the treatment of ATC.

The term thyroid tumours of uncertain malignant potential (TT-UMP) has been proposed for a subgroup of follicular-patterned thyroid tumours for which benignancy or malignancy cannot be assessed with certainty. The frequency, diagnostic reproducibility, immunohistochemistry and molecular genetic profiling of such tumours have been poorly explored. We, therefore, investigated (1) the frequency of TT-UMP diagnosed in a single institution (Nice, France: 2004-2008), (2) the observer variation among four pathologists, (3) whether immunohistochemical and molecular genetic profiling of TT-UMP provide additional information concerning such lesions. A series of 31 diagnosed TT-UMP (2.9%) out of 1,078 consecutive thyroidectomies were analysed. It comprised 15 follicular thyroid tumours of UMP (FT-UMP) and 16 well-differentiated tumours of UMP (WDT-UMP). Observer concordance was 70% for all TT-UMP. More than 50% of FT-UMP expressed galectin-3 and CK19, whereas more than 50% of WDT-UMP expressed HBME-1. Five cases of TT-UMP showed N-RAS mutations, while one showed H-RAS mutation and another PAX8/PPARgamma rearrangement. In conclusion, the frequency of TT-UMP is low in our institution. Diagnostic reproducibility is within the same range as other published data on follicular-patterned thyroid tumours. The ancillary methods have a low impact on aiding diagnosis of such lesions.

BACKGROUND

Thyroid neoplasias with oncocytic features represent a specific phenotype in non-medullary thyroid cancer, reflecting the unique biological phenomenon of mitochondrial hyperplasia in the cytoplasm. Oncocytic thyroid cells are characterized by a prominent eosinophilia (or oxyphilia) caused by mitochondrial abundance. Although disruptive mutations in the mitochondrial DNA (mtDNA) are the most significant hallmark of such tumors, oncocytomas may be envisioned as heterogeneous neoplasms, characterized by multiple nuclear and mitochondrial gene lesions. We investigated the nuclear mutational profile of oncocytic tumors to pinpoint the mutations that may trigger the early oncogenic hit.

METHODS

Total DNA was extracted from paraffin-embedded tissues from 45 biopsies of oncocytic tumors. High-resolution melting was used for mutation screening of mitochondrial complex I subunits genes. Specific nuclear rearrangements were investigated by RT-PCR (RET/PTC) or on isolated nuclei by interphase FISH (PAX8/PPARγ). Recurrent point mutations were analyzed by direct sequencing.

RESULTS

In our oncocytic tumor samples, we identified rare TP53 mutations. The series of analyzed cases did not include poorly- or undifferentiated thyroid carcinomas, and none of the TP53 mutated cases had significant mitotic activity or high-grade features. Thus, the presence of disruptive TP53 mutations was completely unexpected. In addition, novel mutations in nuclear-encoded complex I genes were identified.

CONCLUSIONS

These findings suggest that nuclear genetic lesions altering the bioenergetics competence of thyroid cells may give rise to an aberrant mitochondria-centered compensatory mechanism and ultimately to the oncocytic phenotype.

Napsin A is a sensitive and specific marker for pulmonary adenocarcinoma versus squamous cell carcinoma. However, studies have shown that napsin A is also positive in approximately 5% of papillary thyroid carcinomas. The prevalence of napsin A in more aggressive types of thyroid carcinoma is unknown. Napsin A positivity in metastatic thyroid carcinoma, especially in conjunction with thyroid transcription factor-1 (TTF-1), could be misdiagnosed as lung adenocarcinoma. We investigated napsin A, TTF-1, and PAX8 expression in 26 anaplastic, 16 poorly differentiated, and 2 micropapillary pattern thyroid carcinomas. A focal micropapillary component was also present in 3 poorly differentiated and 3 anaplastic thyroid carcinomas. Four of 26 (15%) anaplastic, 2/16 (13%) poorly differentiated, and 2/2 (100%) micropapillary pattern thyroid carcinomas were napsin A positive. Three of the 6 cases (50%) with a focal micropapillary component were napsin A positive (1 of these 3 cases was positive only in the micropapillary component). All napsin A-positive cases were also positive for TTF-1, and all but 1 micropapillary pattern carcinoma were also PAX8 positive. In 1 case, napsin A was positive in the micropapillary component, but PAX8 was only positive in the adjacent poorly differentiated carcinoma. In summary, a minority of anaplastic and poorly differentiated thyroid carcinomas are napsin A positive. More importantly, napsin A expression is more common in carcinomas with a micropapillary component, a pattern shared in common with some lung adenocarcinomas. PAX8 may be diagnostically useful to distinguish these napsin A-positive thyroid carcinomas from lung adenocarcinomas, which are PAX8 negative.

Correct diagnosis of pulmonary tumors is essential for treatment decision and often rely on immunohistochemical markers. We stained tissue microarrays from resected primary lung cancer (n=665) and pulmonary metastases (n=425) for CK7, CK20, CDX2, CK5, p40, p63, TTF-1, napsin A, GATA3 and PAX8 to systematically assess the diagnostic value of these markers. Primary lung adenocarcinomas expressed TTF-1 in 90% and napsin A in 84% of the cases, while 10% were positive for p63, 7% for CDX2, 2% for CK20 and 2% for GATA3. Only 68% of the lung adenocarcinomas were positive for CK7, TTF-1 and napsin A and negative for all other markers. Primary lung squamous cell carcinomas expressed CK5, p40 and p63 in 94-97% of cases, while 44% were positive for CK7, 20% for GATA3, 7% for CDX2 and 3% for TTF-1. Rare cases expressed PAX8, CK20 or napsin A. Pulmonary metastases of colorectal cancer were positive for CK20 in 83% and CDX2 in 99% of the cases. Rare cases expressed CK7, p63 or PAX8, while 4% expressed TTF-1. Pulmonary metastases of renal cell carcinomas were positive for PAX8 in 74%, napsin A in 7% and CK7 in 7% of the cases. Pulmonary metastases of breast cancer were positive for GATA3 in 93% and CK7 in 78% of the cases, while 15% expressed CK5. Information on expression and patterns of immunohistochemical markers facilitates histopathological diagnostics. Evidently, unusual immune profiles occur and may lead to incorrect diagnosis.

To review the characteristics of newborn screening of congenital hypothyroidism (CH), we reviewed the newborn screening data, including the levels of blood spot thyroid-stimulating hormone (TSH), and serum TSH and free thyroxine (FT4), of all newborn infants who accepted the newborn screening program during the last 14 years. In total, 437,342 newborn infants underwent CH screening and 192 infants were diagnosed with CH and the incidence of CH was 1:2278. The positive rate of the initial screening was 0.96%, and the positive predictive value was 4.8%. We also designed a target sequencing panel including 13 causative genes: DUOX2, TG, TPO, TSHR, TTF1, TTF2, PAX8, NKX2-5, GNAS, THRA, TSHB, IYD and SLC5A5, to identify the spectrum and prevalence of disease-causing gene mutations in Chinese CH patients. CH-causing genes were detected by targeted next-generation sequencing in 106 CH infants. A total of 132 mutations were identified in 69 cases (65.1%). Of these 132 mutations, 92 (69.70%), 28 (21.21%), and 12 (9.09%) were related to thyroid dyshormonogenesis, thyroid dysgenesis, and thyrotropin resistance, respectively. Mutations in CH-causing genes were found mainly in DUOX2, TG and TSHR, and DUOX2 is the most gene mutation in Chinese CH patients.

Congenital hypothyroidism (CH) is a neonatal endocrine disorder that might occur as itself or be associated to congenital extra-thyroidal defects. About 85% of affected subjects experience thyroid dysgenesis (TD), characterized by defect in thyroid gland development. In vivo experiments on null mice paved the way for the identification of genes involved thyroid morphogenesis and development, whose mutation has been strongly associated to TD. Most of them are thyroid-specific transcription factors expressed during early thyroid development. Despite the arduous effort in unraveling the genetics of TD in animal models, up to now these data have been discontinuously confirmed in humans and only 5% of TD have associated with known null mice-related mutations (mainly PAX8 and TSHR). Notwithstanding, the advance in genetic testing represented by the next-generation sequencing (NGS) approach is steadily increasing the list of genes whose highly penetrant mutation predisposes to TD. In this review we intend to outline the molecular bases of TD, summarizing the current knowledge on thyroid development in both mice and humans and delineating the genetic features of its monogenetic forms. We will also highlight current strategies to enhance the insight into the non-Mendelian mechanisms of abnormal thyroid development.

Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder, affecting one in 3000 to 4000 newborns. Since the introduction of a newborn screening program in 1988, more than 300 cases have been identified. The underlying genetic defects have not been systematically studied.

To identify the mutation spectrum of CH-causing genes.

Fifty-five patients from 47 families were studied by next-generation exome sequencing.

Mutations were identified in 52.7% of patients (29 of 55) in the following 11 genes: TG, TPO, DUOX2, SLC26A4, SLC26A7, TSHB, TSHR, NKX2-1, PAX8, CDCA8, and HOXB3. Among 30 patients with thyroid dyshormonogenesis, biallelic TG mutations were found in 12 patients (40%), followed by biallelic mutations in TPO (6.7%), SLC26A7 (6.7%), and DUOX2 (3.3%). Monoallelic SLC26A4 mutations were found in two patients, one of them coexisting with two tandem biallelic deletions in SLC26A7. In 25 patients with thyroid dysgenesis, biallelic mutations in TSHR were found in six patients (24%). Biallelic mutations in TSHB, PAX 8, NKX2-1, or HOXB3 were found once in four different patients. A monoallelic CDCA8 mutation was found in one patient. Most mutations were novel, including three TG, two TSHR, and one each in DUOX2, TPO, SLC26A7, TSHB, NKX2-1, PAX8, CDCA8, and HOXB3. SLC26A7 and HOXB3 were novel genes associated with thyroid dyshormonogenesis and dysgenesis, respectively.

TG and TSHR mutations are the most common genetic defects in Saudi patients with CH. The prevalence of other disease-causing mutations is low, reflecting the consanguineous nature of the population. SLC26A7 mutations appear to be associated with thyroid dyshormonogenesis.