We report 2 cases of bland, otherwise nondescript axillary lymph node inclusions that have the immunophenotype of endosalpingiosis in patients with concurrent invasive breast carcinomas. Neither inclusion demonstrated the classic morphology of endosalpingiosis with admixed ciliated and secretory cells. Rather, both cases were composed of nondescript cuboidal to columnar bland epithelial cells situated within the lymph node capsule. Whereas both inclusions labeled diffusely for estrogen receptor and lacked evidence of a myoepithelial component, both labeled diffusely for PAX8 and WT-1, which distinguished them from their corresponding concurrent primary mammary carcinomas. These findings suggest that a subset of otherwise nondescript axillary lymph node inclusions represent endosalpingiosis and highlight the utility of PAX8 and WT-1 immunohistochemistry in distinguishing these from metastatic well-differentiated ductal carcinoma.

We aimed to compare the genetic background of different areas in follicular variant papillary thyroid carcinomas (FVPTC) with or without classical nuclear changes. Sixteen cases of FVPTC were included in our study. All tumors were well demarcated from surrounding thyroid tissue and had both areas with nuclear features (WNF) and areas without nuclear features (WONF) of papillary carcinoma. DNA is obtained by laser microdissection from WNF and WONF areas of each case. Point mutations for NRAS codon 61, HRAS codon 61, and BRAF were investigated by direct sequencing. In 11 cases, reverse transcription PCR was performed for the presence of PAX8-PPARɣ and RET/PTC1-3 gene rearrangements. Point mutation for NRAS codon 61 was also studied in 15 colloidal nodules. Seven cases (44 %) showed at least one mutation; two cases (13 %) revealed the same mutation in both WNF and WONF areas, while in the rest only WNF areas were mutated. None of the studied 11 cases demonstrated RET/PTC1-3 gene rearrangement and in only one case PAX8-PPARɣ gene rearrangement was found. Six cases (38 %) showed NRAS codon 61 mutation, involving only WNF areas in five cases and both WNF and WONF areas in one case. Neither HRAS codon 61 nor BRAF mutations were present. Fifteen colloidal nodules were also wild type for NRAS codon 61. Our findings suggest that NRAS codon 61 point mutations and PAX8-PPARɣ gene rearrangement play a role in the FVPTC pathogenesis and may be established before the morphological/phenotypical features fully develop.

Adenylyl cyclase (AC)-stimulated cAMP plays a key role in modulating transport and channel activity along the nephron. However, the role of individual adenylyl cyclase isoforms in such regulation is largely unknown. Since adenylyl cyclase 3 (AC3) is expressed throughout nephron, we investigated its role in the physiologic regulation of renal Na(+) and water transport. Mice were generated with inducible nephron knockout of AC3 (AC3 KO) by breeding mice with loxP-flanked critical exons in the Adcy3 gene with mice expressing Pax8-rtTA and LC-1 transgenes. After doxycycline treatment at 1 month of age, nephron AC3 KO mice had 100% Adcy3 gene recombination in all renal tubule segments, but not in glomeruli. Sodium intake, urinary Na(+) excretion, glomerular filtration rate, and blood pressure were similar between nephron KO mice and the controls during normal, high, and low Na(+) diets. Plasma renin concentration was not different between the two groups during varied Na(+) intake. Moreover, there were no differences in urine volume and urine osmolality between the two genotypes during normal or restricted water intake. In conclusion, these data suggested that AC3 is not involved in the physiological regulation of nephron Na(+) and water handling.

To date, 13 cases of sporadic renal hemangioblastoma have been reported. In this article, we report such a case that might cause the diagnostic pitfall. A 37-year-old Japanese was found to have a renal mass by periodic medical check-up. He underwent radical nephrectomy. Macroscopically, the tumor was well-defined without fibrous capsule and the cut surface of the tumor exhibited light brown to gray-tan color without hemorrhage or necrosis. Microscopically, the tumor was made up of large polygonal to short spindle cells with eosinophilic cytoplasm with occasional vacuolization and abundant arborizing capillary network. Immunohistochemically, neoplastic cells showed diffuse positivity for inhibin-alpha, S-100 protein, vimentin, CA9, PAX2 and PAX8, but negativity for cytokeratin CAM5.2, alpha smooth muscle actin, Melanosome, Melan A, TFE3 and cathepsin K. In genetic analyses, this tumor showed no changes of VHL gene mutation, hypermethylation and loss of heterozygosity of chromosome 3p. Additionally, G-band karyotype and array comparative genomic hybridization studies showed a normal chromosome. In conclusion, the positivity for CA9, PAX2 and PAX8 in sporadic renal hemangioblastoma may cause the critical diagnostic pitfall in the differential diagnosis from clear cell renal cell carcinoma. Pathologists need to pay attention to systemic evaluation including macroscopic, microscopic and immunohistochemical findings. In some cases, molecular genetic study may be necessary.

Several genes control cell growth, differentiation and apoptosis. Any alteration in the sequence or expression of these genes can cause an uncontrolled growth of the tissue and produce a tumor. Quantitative and qualitative gene expression studies using genes as tumor markers are essential for the diagnosis and prognosis of the tumor and its behavior. Oncogenes are genes that stimulate cell growth and have an increased expression. On the contrary, tumor suppressor genes are genes that inhibit cell growth and have a decreased expression in tumor cells. To study these tumor markers we apply simple and random molecular biology techniques such as polymerase chain reaction (PCR), reverse transcription and genomic sequencing. In the case of thyroid epithelial neoplasia, tumor markers such as PTEN/MMAC1/TEP1, telomerase, RET/PTC, b-catenine, PAX8/PPAR(1, ciclooxygenase, thyroid stimulating hormonal receptor (TSHR), and thyro-globulin are being investigated. These markers are analyzed for somatic mutations in the genetic sequence, chromosomical rearrangements, alterations in the promoter zone that affect gene expression, regulation and studies of genes at mRNA level. A deeper study of these markers is deemed to help improve the accuracy of tumor diagnosis, behavior and prognosis. Hence, more effective therapeutic options will be adapted to each individual, eventually reducing hospital costs.

► The breast as a site of metastasis from primary ovarian carcinoma is uncommon. ► Distinguishing these metastases from primary breast tumors is important because the prognosis and therapeutic approach differ significantly. ► Immunohistochemical markers (e.g., PAX8) can be utilized when morphology and clinical history are insufficient to render the correct diagnosis.

Renal calyx carcinoma (RCXC) may mimic collecting duct carcinoma (CDC) or urothelial carcinoma (UC) of the renal pelvis. RCXC is distinguished from CDC and UC of the renal pelvis as having the tumor epicenter in the renal calyx, with limited involvement of the surrounding renal pelvis surface urothelium. In this study, we summarize our experience with this entity. Ten cases of RCXC, including 9 cases with urothelial differentiation (RCXC-UC) and 1 case with salivary gland-type differentiation (RCXC-SC), were identified. Ten consecutive cases of UC were selected for comparison, with extensive renal pelvis involvement and with secondary renal parenchymal invasion. Two cases of collecting duct carcinoma (CDC) were also examined. Immunohistochemistry (IHC) was performed on representative tissue blocks for PAX8, PAX2, CK5, CK7, CK20, p63, GATA3, AMACR, RCC, CD10, vimentin, S100, and MSA. The 10 cases of RCXC (M:F=4:6, ages: 62-91 years, mean: 76) presented with renal masses of 3-6cm. Ureteroscopic studies and renal pelvic washings showed atypical/malignant cells in three cases. Seven patients were treated with nephrectomy followed by radiation±chemotherapy, and all cases developed metastases to lymph nodes or liver/lung/bone. In all 7 cases with nephrectomy, there was extensive renal parenchymal involvement with infiltrating borders and diffuse spread along collecting ducts. Six RCXC-UC contained focal squamous differentiation. The RCXC-SC displayed features of adenoid cystic and basaloid features. In situ UC, with or without papillary components, was identified in the calyces in all 7 nephrectomy cases with remaining renal pelvis harboring small tumor burden in 5 cases, and no tumor in another 2 cases. Of the three cases without nephrectomy, no tumor in the renal pelvis could be visualized with endoscopy, however one case was associated with UC of the urinary bladder. Of 10 control UC cases, tumor was limited to the tip of renal papilla in 7 cases, extensive in 3 cases, and with no extension into the collecting ducts. RCXC-UC were all positive for p63, CK5, CK7, and PAX2, with all negative for RCC. PAX8 (70% positive) and GATA3 (50% positive) reactivity was variable. The 10 UC cases shared IHC properties with RCXC-UC but frequent negativity for PAX8 and positivity for GATA3. RCXC is an aggressive neoplasm with high risk of metastases. Similar to CDC, it is located in the renal papilla and rarely with clinically visible renal pelvis tumor or ureteral urine positive for tumor cells. Unlike CDC and non-calyceal UC, RCXC shows predominantly urothelial and squamous differentiation and is associated with an in situ component of adjacent renal calyces. By IHC, RCXC exhibited features intermediate between UC and CDC with decreased or negative immunoreactivity for PAX8 and GATA.

Nevirapine has been proved to be effective in inducing re-differentiation and suppressing tumor growth in several tumor cells. This study aims to investigate the therapeutic potential of nevirapine in dedifferentiated thyroid cancer (DeTC), which refractory to radioiodine treatment and the underlying mechanisms. The results indicated that nevirapine significantly inhibited the proliferation and increased the expressions of thyroid differentiation-related genes, thyroid stimulating hormone receptor (TSHR), sodium/iodide symporter (NIS), thyroid peroxidase (TPO), and transcriptional factor paired box 8 (PAX8) in dedifferentiated thyroid cancer cells (WRO 82-1 and dFTC-133). Furthermore, nevirapine also enhanced radioiodide uptake significantly both in vitro and in vivo, and inhibited the growth of xenograft tumors. Nevirapine might improve radioiodine sensitivity via the activation of TSHR/cAMP/CREB/PAX8 signaling pathway. This study demonstrates that nevirapine could be potentially used to improve radioiodine therapeutic efficacy in dedifferentiated thyroid cancer patients.

In cases of extragenital endometriosis or microscopic endometriosis lesions, pathological diagnosis can be challenging because endometriotic stroma and glands represent only a minor component of fibrotic endometriotic lesions. For better accuracy of diagnosis, the development of a sensitive and specific epithelial marker is beneficial. Previous studies showed that PAX8 is a highly sensitive and specific marker for primary and metastatic Mullerian epithelial tumors. Therefore, we sought to examine whether PAX8 is a highly sensitive marker for glands in extragenital endometriosis. Eight and 47 samples of ovarian endometrioma and extragenital endometriosis, respectively, were evaluated in this study. We calculated the percentage of samples positively immunostained for PAX8, CD10, estrogen receptor (ER), and progesterone receptor (PR). PAX8 was positive for endometriotic epithelial cells in 95.7% (45/47) of extragenital endometrioses and in 100% (8/8) of ovarian endometrioses. CD10 was positive for endometriotic stromal cells in 97.9% (46/47) of extragenital endometrioses. PAX8 was strongly positive for glands, even in a CD10-negative case. The expression of PAX8, CD10, and PR was not affected by preoperative hormonal therapy, and the positive rate of ER staining was significantly reduced by preoperative hormonal therapy. In conclusion, PAX8 is a highly sensitive epithelial marker for extragenital endometriosis. This specific expression was maintained under hormonal therapy. It is noteworthy that extragenital endometriosis maintains the expression of this lineage marker, although it occurs at various sites, and its cause and mechanism of development might be different. PAX8 nuclear expression can be useful in detecting extragenital endometriosis in clinical practice.

Keywords: CD10; PAX8; extragenital endometriosis; marker.

BACKGROUND

Thyroid dysgenesis (TD) is the most frequent cause of congenital hypothyroidism (CH), but its pathogenesis remains unclear. As a thyroid transcription factor, paired box transcription factor 8 (PAX8) is essential for thyroid organogenesis and development.

OBJECTIVE

To screen PAX8 mutations and characterize the features of these mutations in Chinese TD patients.

METHODS

Blood samples were collected from 63 TD patients in Shandong Province, China, and genomic DNA was extracted from peripheral blood leukocytes. Exon 3~4 of PAX8 were analyzed by PCR and direct sequencing.

RESULTS

Direct sequencing of PAX8 revealed a heterozygous missense mutation (c.155G/C, P.Arg52Pro) in one child with agenesis. Genetic screening of the child's family revealed that the clinically unaffected parents do not carry the mutation, suggesting that the identified sequence change is a de novo mutation.

CONCLUSIONS

We report a heterozygous missense de novo mutation in PAX8 in one out of 63 unrelated Chinese TD patients, showing that the PAX8 mutation rate is very low in TD patients in China. However, de novo mutation and epigenetic mechanisms need to be considered in the future study.

Thyroid-specific transcription factor PAX8 has an indispensable role in the thyroid gland development, which is evidenced by the facts that PAX8/Pax8 mutations cause congenital hypothyroidism in humans and mice. More than 90% of known PAX8 mutations were located in the paired domain, suggesting the central role of the domain in exerting the molecular function. Structure-function relationships of PAX8, as well as other PAX family transcription factors, have never been investigated in a systematic manner. Here, we conducted the first alanine scanning mutagenesis study, in which 132 alanine variants located in the paired domain of PAX8 were created and systematically evaluated in vitro. We found that 76 alanine variants (55%) were loss of function (LOF) variants (defined by <30% activity as compared with wild type PAX8). Importantly, the distribution of LOF variants were skewed, with more frequently observed in the N-subdomain (65% of the alanine variants in the N-subdomain) than in the C-subdomain (45%). Twelve out of 13 alanine variants in residues that have been affected in patients with congenital hypothyroidism were actually LOF, suggesting that the alanine scanning data can be used to evaluate the functional importance of mutated residues. Using our in vitro data, we tested the accuracy of seven computational algorithms for pathogenicity prediction, showing that they are sensitive but not specific to evaluate on the paired domain alanine variants. Collectively, our experiment-based data would help better understand the structure-function relationships of the paired domain, and would provide a unique resource for pathogenicity prediction of future PAX8 variants.

Pax8 is a transcription factor that plays an important role in the regulation of genes that are exclusively expressed in differentiated thyroid cells. In the thyroid cell environment, evidence exists that Pax8 is part of a multiprotein complex in which its transcriptional activity may be modulated by specific co-factors. In an attempt to identify proteins that interact with Pax8, we performed pull-down experiments challenging the GST-Pax8 fusion protein with protein extracts prepared from the thyroid differentiated cell line PC Cl3. By this approach, we isolated a 113-kDa protein that is able to associate with Pax8, which was further identified by mass fingerprint experiments as poly(ADP-ribose) polymerase 1 (PARP1). To further confirm this interaction, we also showed that PARP1 can be co-immunoprecipitated with Pax8 in vivo from a thyroid cell extract. Gel shifts experiments demonstrated that PARP1 binding to Pax8 significantly inhibits Pax8 binding to DNA. Accordingly, we provide evidence that the functional outcome of such an interaction is a significant downregulation of Pax8 transcriptional activity. In the context of thyroid-specific gene transcription, our results suggest that PARP1 behaves as an important negative co-factor involved in the regulation of Pax8-dependent gene expression.

Primary retroperitoneal serous adenocarcinoma (PRSA) is a rare malignancy of which only seven cases have been reported in the literature. The clinical features and outcomes of PRSA are not well understood. We herein report a case of PRSA with liver metastasis in a 74-year-old woman who was treated with surgical excision. The tumor cells were positive for estrogen receptor, Wilms tumor 1, PAX8, p53, and cytokeratin AE1/AE3. The final diagnosis was PRSA and liver metastasis. The pathological features of PRSA resemble those of ovarian serous carcinoma, which suggests that a combination of surgical excision with adjuvant chemotherapy may be the best option.

Patients with cervical cancer have abnormal cell proliferation and invasion after many years of latency. However, the precise mechanisms remain unclear. Mitogen- and stress-activated kinase 2 (MSK2) is a serine/threonine kinase which displays a phenotype that promotes tumor growth and metastasis in many different types of tumors. The aim of the present study was to determine the effects of MSK2 on the proliferation of cervical cancer cells and elucidate the signaling pathways through which MSK2 exerts its effects in the pathogenesis of squamous cell carcinoma (SCC). Our results confirmed that MSK2 expression was significantly upregulated in cervical cancer cells both in vivo and in vitro. We further found that the expression patterns of paired-box gene 8 (PAX8) and MSK2 were positively correlated in cervical cancer specimens. Moreover, MSK2 knockdown inhibited the phosphorylation of PAX8 and retinoblastoma protein (RB), and suppressed the sequential expressions of cell proliferation factors E2F1 and cyclin A2, resulting in the inhibition of SCC cell proliferation and tumor formation. Thus, this study demonstrates that MSK2 has oncogenic effects in the formation and development of SCC via the PAX8/RB-E2F1/cyclin A2 axis.

We analyzed the clinicopathological features of renal-type clear cell carcinoma (RTCCC) in the prostate and its diagnosis according to the example in our hospital and review of the literature. Clinicopathological features of RTCCC in the prostate were observed in a patient from our hospital combining with a review of the literature. Microscopically, the tumor was composed of cells with abundant and translucent cytoplasm, arranged in the form of the vesicular nest or glandular structure. Therefore, it was necessary to distinguish between metastatic clear cell renal cell carcinoma and primary RTCCC in the prostate. Immunohistochemistry (IHC) of this case showed tumor cells were positive expression for cytokeratin (CKpan), low-molecular weight cytokeratin, epithelial membrane antigen, and prostate-specific antigen (PSA), P504S, prostate-specific membrane antigen and partial positive expression for vimentin and CD10. The tumor cells displayed negative expression of high molecular weight cytokeratin, cytokeratin 7 (CK7), CK34, PAX8, and renal cell carcinoma. The morphological and immunohistochemical features of this tumor were in correspondence with RTCCC of the prostate. This tumor is a rare variant of the prostate carcinomas. To the best of our knowledge, this type of extrarenal tumor has only been reported in six previous studies. Combination of histology, IHC, imaging, and serum PSA is needed to perform a suitable diagnosis.

Nephrogenic adenoma is a benign lesion of the urinary tract, particularly the urinary bladder. It is a gross and microscopic mimicker of urothelial neoplasm or metastatic carcinoma. Several histological patterns (tubular, tubulocystic, polypoid, papillary, fibromyxoid) have been recognized, but a flat pattern has not been described. Histologically, nephrogenic adenoma consists of tubules, cysts or papillae lined by flat to polygonal cells with frequent hobnail appearance. The stroma is often edematous or has a granulation tissue-like appearance with acute or chronic inflammation. By immunohistochemistry, nephrogenic adenomas are positive for renal epithelial markers CK7, CD10 and alpha-methylacyl-coenzyme A racemase, and negative for bladder urothelium or prostate markers. Recent studies have shown that nephrogenic adenomas are positive for PAX2 and PAX8. We encountered an interesting case of tubular nephrogenic adenoma with adjacent areas suspicious of flat urothelial atypia. Immunohistochemistry for PAX2 and PAX8 were positive in these areas, unveiling a flat pattern of nephrogenic adenoma. This case prompted us to study 15 cases of nephrogenic adenoma to determine additional instances of flat pattern and to assess the value of PAX2 and PAX8 immunoreactivity to diagnose nephrogenic adenoma. PAX2 and PAX8 immunostaining was positive in 14/15 and 15/15 cases, respectively. The flat pattern was present at least focally adjacent to tubular, polypoid and papillary areas, in 8/15 cases of nephrogenic adenoma. In conclusion, the flat pattern is a common finding in nephrogenic adenomas, but easily under recognized by morphologic examination and may be confused with flat urothelial lesions with atypia. Immunostains for PAX2 and PAX8 are useful in the detection of nephrogenic adenomas and particularly unveil those nephrogenic adenomas with flat pattern.

OBJECTIVE

To study the expression of carbonic anhydrase IX (CAIX), PAX2 and PAX8 in different types of renal epithelial tumor and their association with clinicopathologic characteristics.

METHODS

Immunohistochemical study by EnVision method was performed in order to assess the expression of CAIX, PAX2 and PAX8 in 155 cases of renal cell carcinoma and 4 cases of metastatic clear cell renal cell carcinoma (CCRCC). Ninety-six cases of non-neoplastic renal parenchymal tissue adjacent to CCRCC, 8 cases of clear cell hepatocellular carcinoma and 2 cases of clear cell hidradenoma were used as controls.

RESULTS

CAIX was commonly expressed in CCRCC (94.0%, 63/67), of which 77.8% (49/63) showed strong positivity. CAIX was focally positive in papillary renal cell carcinoma, collecting duct carcinoma and urothelial carcinoma of renal pelvis. It was negative in chromophobe renal cell carcinoma, oncocytoma and adjacent non-neoplastic renal tissue. CAIX was also strongly expressed in the 4 cases of metastatic CCRCC. Focal expression of CAIX was demonstrated in the 8 cases of clear cell hepatocellular carcinoma and 2 cases of clear cell hidradenoma. The expression of CAIX in CCRCC did not correlate with tumor grading, clinical staging and presence of distal metastasis. On the other hand, PAX2 showed positive expression in different types of renal epithelial tumor, clear cell hepatocellular carcinoma and clear cell hidradenoma in various degrees. In contrast, PAX8 was commonly expressed in all types of renal epithelial tumor, with the exception of urothelial carcinoma of renal pelvis. PAX8 was not expressed in clear cell hepatocellular carcinoma and clear cell hidradenoma. Regarding diagnosis of CCRCC, CAIX demonstrated high sensitivity and specificity. PAX2 showed high specificity but low sensitivity. PAX8 was sensitive and specific in the diagnosis of renal epithelial tumor.

CONCLUSIONS

CAIX is a useful immunohistochemical marker with high specificity and sensitivity in distinguishing CCRCC from other types of renal epithelial tumor and clear cell tumors of non-renal origin. PAX2 is a marker with high sensitivity and low specificity for diagnosis of renal epithelial tumors. PAX8 is typically expressed in renal epithelial tumors. The combined detection of CAIX, PAX2 and PAX8 is useful in the diagnosis and differential diagnosis of renal epithelial tumors.

Follicular cell-derived thyroid carcinomas, including thyroid squamous cell carcinomas (SCCs) and anaplastic carcinomas, are immunoreactive for paired-box gene 8 (PAX8), while non-follicular cell-derived thyroid carcinomas stain negative for the PAX8 antibody. Intrathyroid thymic carcinoma (ITTC) arising from the intrathyroidal ectopic thymus exhibits moderate-to-strong nuclear reactivity for polyclonal PAX8. This is difficult to understand given that PAX8 is not associated with embryonic thymic development. We aimed to determine the diagnostic significance of monoclonal PAX8 antibody in distinguishing ITTCs from follicular cell-derived thyroid carcinomas. Ten ITTCs, 14 poorly differentiated thyroid carcinomas (PDTCs), 14 thyroid SCCs, 7 thymic tissue specimens, 7 thymomas, and 1 thymic carcinoma were analyzed using antibodies against polyclonal and monoclonal PAX8, thyroid transcription factor-1, p63, and CD5. Four ITTCs (40.0%) stained positive for polyclonal PAX8; none stained positive for monoclonal PAX8. All PDTCs and 92.9% of SCCs were immunoreactive for both polyclonal and monoclonal PAX8. All PDTCs, 46.2% of SCCs, and none of the ITTCs were immunoreactive for thyroid transcription factor-1. Eight ITTCs (80.0%), but none of the PDTCs and SCCs, were immunoreactive for CD5. We are the first to show that ITTCs stain negative for monoclonal PAX8. Monoclonal PAX8 is a more reliable marker than polyclonal PAX8 for determining follicular cell origin. We conclude that monoclonal PAX8 is a useful marker for distinguishing ITTCs from PDTCs and SCCs. Monoclonal PAX8 negativity is additional evidence in support of ITTCs not being follicular cell-derived thyroid carcinomas, but having a thymic origin.

Papillary thyroid carcinomas are characterized in 70% of cases by the presence of either a RET/PTC rearrangement, or an activating point mutation of RAS or BRAF genes that induce a constitutive activation of the MAP kinase pathway. Follicular carcinomas are characterized by the presence of a RAS mutation or of a PAX8-PPARgamma rearrangement. Inactivating mutations of the p53 gene are found only in anaplastic thyroid carcinomas.

Background: The success in rescuing thyroid deficiency in mice using thyroid cells derived from embryonic stem (ES) cells, together with the discovery of human induced pluripotent stem cells (iPSCs) from somatic cells, has raised the possibility of patient-specific thyroid cell replacement. In this study we demonstrate that human thyroid follicular cells can be derived from human iPSCs and show the ability of highly purified and differentiated cells to secrete thyroid hormone. Research Design and Methods: Human iPSCs were derived from adult skin fibroblasts using RNA reprogramming and differentiated in vitro into thyroid follicular cells by exposure to activin A, ethacridine and TSH as we have previously described for human ES cells. The resulting thyroid cells were then highly purified using double antibody cell sorting. Results: The iPSCs derived from human dermal fibroblasts showed stem cell-like morphologic changes and expressed pluripotent stem cell markers as assessed using qPCR, immunofluorescence staining, and FACS analysis. These cells retained their pluripotential characteristics as shown by teratoma formation after murine transplantation. Definitive endoderm cells were induced with activin A and the transcription factor TAZ was significantly induced on ethacridine treatment and translocated to the nucleus. Thyroid transcription factors NKX2-1 and PAX8 were also highly expressed in activin A derived endoderm cells and further induced by ethacridine. Following terminal differentiation with TSH, there was enhanced thyroid follicle formation, high expression of the thyroid specific genes-TG, TPO, TSHR and NIS, and secretion of thyroid hormone (T4) in vitro. Furthermore, we were able to achieve a 97% purification of TSHR+/NIS+ expressing cells after differentiation using a single purification procedure. Conclusions: These findings demonstrate that mature adult dermal fibroblasts can be matured into human iPSCs which have the potential to form functional thyroid follicular cells. This lays the groundwork for future person-specific thyroid regenerative therapy.

Keywords: NIS; NKX2-1; PAX8; TAZ; TSH receptor (TSHR); induced pluripotent stem cells (iPSCs); thyroid differentiation.

Medullary thyroid carcinoma (MTC) is a malignant tumor of the parafollicular cells, which produce calcitonin. As a result, calcitonin is an important tool for diagnosing MTC. When there is focal or no calcitonin staining, diagnosis of MTC can be challenging as this may suggest differences in cell origin and in prognosis and recurrence of the disease. The prognosis and guidelines for management and follow up for MTC remain inconclusive. Here, we present a case of primary neuroendocrine tumor of the thyroid gland with only focal calcitonin staining on immunohistochemistry and low serum calcitonin level despite a tumor size of almost 4 cm. The assumption of C-cell origin was made from positive staining for carcinoembryonic agent (CEA), thyroid transcription factor 1 (TTF-1) and paired box 8 (PAX8) without expression of thyroglobulin (Tg). The patient underwent thyroid lobectomy and follow-up monitoring with ultrasound and measurements of calcitonin and CEA levels, without any further surgical intervention. Few cases of neuroendocrine tumors with limited calcitonin expression have been published and the etiology and prognosis are still unknown. Our case suggests that limited calcitonin expression cannot completely exclude the diagnosis of MTC. This rare type of MTC should be differentiated from the typical presentation of MTC. Treatment and post-operative surveillance should be tailored based on the individual.