The pronephros is the first kidney type to form in vertebrate embryos. The first step of pronephrogenesis in the zebrafish is the formation of the intermediate mesoderm during gastrulation, which occurs in response to secreted morphogens such as BMPs and Nodals. Patterning of the intermediate mesoderm into proximal and distal cell fates is induced by retinoic acid signaling with downstream transcription factors including wt1a, pax2a, pax8, hnf1b, sim1a, mecom, and irx3b. In the anterior intermediate mesoderm, progenitors of the glomerular blood filter migrate and fuse at the midline and recruit a blood supply. More posteriorly localized tubule progenitors undergo epithelialization and fuse with the cloaca. The Notch signaling pathway regulates the formation of multi-ciliated cells in the tubules and these cells help propel the filtrate to the cloaca. The lumenal sheer stress caused by flow down the tubule activates anterior collective migration of the proximal tubules and induces stretching and proliferation of the more distal segments. Ultimately these processes create a simple two-nephron kidney that is capable of reabsorbing and secreting solutes and expelling excess water-processes that are critical to the homeostasis of the body fluids. The zebrafish pronephric kidney provides a simple, yet powerful, model system to better understand the conserved molecular and cellular progresses that drive nephron formation, structure, and function.

High-grade serous carcinoma (HGSC) is the most lethal ovarian cancer histotype. The fallopian tube secretory epithelial cells (FTSECs) are a proposed progenitor cell type. Genetically altered FTSECs form tumors in mice; however, a spontaneous HGSC model has not been described. Apart from a subpopulation of genetically predisposed women, most women develop ovarian cancer spontaneously, which is associated with aging and lifetime ovulations. A murine oviductal cell line (MOE(LOW)) was developed and continuously passaged in culture to mimic cellular aging (MOE(HIGH)). The MOE(HIGH) cellular model exhibited a loss of acetylated tubulin consistent with an outgrowth of secretory epithelial cells in culture. MOE(HIGH) cells proliferated significantly faster than MOE(LOW), and the MOE(HIGH) cells produced more 2D foci and 3D soft agar colonies as compared to MOE(LOW) MOE(HIGH) were xenografted into athymic female nude mice both in the subcutaneous and the intraperitoneal compartments. Only the subcutaneous grafts formed tumors that were negative for cytokeratin, but positive for oviductal markers, such as oviductal glycoprotein 1 and Pax8. These tumors were considered to be poorly differentiated carcinoma. The differential molecular profiles between MOE(HIGH) and MOE(LOW) were determined using RNA-Seq and confirmed by protein expression to uncover pathways important in transformation, like the p53 pathway, the FOXM1 pathway, WNT signaling, and splicing. MOE(HIGH) had enhanced protein expression of c-myc, Cyclin E, p53, and FOXM1 with reduced expression of p21. MOE(HIGH) were also less sensitive to cisplatin and DMBA, which induce lesions typically repaired by base-excision repair. A model of spontaneous tumorogenesis was generated starting with normal oviductal cells. Their transition to cancer involved alterations in pathways associated with high-grade serous cancer in humans.

High grade serous ovarian cancer (HGSOC) is the fifth leading cause of cancer deaths among women yet effective targeted therapies against this disease are limited. The heterogeneity of HGSOC, including few shared oncogenic drivers and origination from both the fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE), has hampered development of targeted drug therapies. PAX8 is a lineage-specific transcription factor expressed in the FTE that is also ubiquitously expressed in HGSOC where it is an important driver of proliferation, migration, and cell survival. PAX8 is not normally expressed in the OSE, but it is turned on after malignant transformation. In this study, we use proteomic and transcriptomic analysis to examine the role of PAX8 leading to increased migratory capabilities in a human ovarian cancer model, as well as in tumor models derived from the OSE and FTE. We find that PAX8 is a master regulator of migration with unique downstream transcriptional targets that are dependent on the cell's site of origin. Importantly, we show that targeting PAX8, either through CRISPR genomic alteration or through drug treatment with micelle encapsulated thiostrepton, leads to a reduction in tumor burden. These findings suggest PAX8 is a unifying protein driving metastasis in ovarian tumors that could be developed as an effective drug target to treat HGSOC derived from both the OSE and FTE.

Papillary thyroid carcinoma with desmoid-type fibromatosis or nodular fasciitis-like stroma is an extremely unusual and poorly understood subtype of papillary thyroid cancer. Although prior studies have demonstrated alterations in the Wnt/β-catenin signaling pathway in some of these tumors, controversy still exists regarding the nature of the stromal spindle component. We have studied seven cases of papillary thyroid carcinoma with prominent myofibroblastic stroma, including six men and one woman aged 20-65 years (mean age = 44). All cases displayed areas consistent with conventional papillary thyroid carcinoma embedded in abundant myofibroblastic-like stroma. The myofibroblastic stroma in six cases resembled desmoid-type fibromatosis and in one case it more closely resembled nodular fasciitis. By immunohistochemical staining, the stromal spindle component showed positivity for SMA and low MIB1 proliferation index in all cases, and there was at least patchy strong nuclear positivity for beta-catenin in six/seven cases. Stains for cytokeratin AE1/AE3 and PAX8 were positive in the epithelial elements but negative in the stromal component. Next-generation sequencing was performed on six of seven cases. CTNNB1 gene mutations were identified in six/seven cases. The epithelial component showed BRAF mutations in two cases and an NRAS mutation in one case. The case with fasciitis-like stroma was negative for beta-catenin by sequencing and immunostaining as well as negative for USP6 gene rearrangement. Our findings indicate that papillary thyroid carcinoma with prominent myofibroblastic stroma may represent more than one category of lesions.

BACKGROUND

Four members of the PAX family, PAX2, PAX4, PAX6 and PAX8 are known to be expressed in the pancreas. Accumulated evidences indicate that several pancreatic expressed PAX genes play a significant role in pancreatic development/functionality and alterations in these genes are involved in the pathogenesis of pancreatic diseases. Areas covered: In this review, we summarize the ongoing research related to pancreatic PAX genes in diabetes mellitus and pancreatic neuroendocrine tumors. We dissect the current knowledge at different levels; from mechanistic studies in cell lines performed to understand the molecular processes controlled by pancreatic PAX genes, to in vivo studies using rodent models that over-express or lack specific PAX genes. Finally, we describe human studies associating variants on pancreatic-expressed PAX genes with pancreatic diseases. Expert opinion: Based on the current literature, we propose that future interventions to treat pancreatic neuroendocrine tumors and diabetes mellitus could be developed via the modulation of PAX4 and/or PAX6 regulated pathways.

BACKGROUND

Molecular analysis for common somatic mutations in thyroid cancer can improve diagnostic accuracy of fine-needle aspiration cytology (FNAC) in the nondiagnostic or indeterminate category of thyroid nodules. In this study, we evaluated the feasibility of molecular diagnosis from residual liquid-based cytology (LBC) material after cytological diagnosis.

METHODS

This prospective study enrolled 53 patients with thyroid nodules diagnosed as nondiagnostic, atypia of undetermined significance (AUS), or follicular lesion of undetermined significance (FLUS) after FNAC. DNAs and RNAs were isolated from residual LBC materials. BRAF(V600E) and RAS point mutations, PAX8/peroxisome proliferator-activated receptor γ (PPARγ), RET/PTC1, and RET/PTC3 rearrangements were evaluated by real-time polymerase chain reaction and pyrosequencing.

RESULTS

All DNAs from 53 residual LBC samples could be analysed and point mutations were detected in 10 samples (19%). In 17 AUS nodules, seven samples (41%) had point mutations including BRAF (n=4), NRAS (n=2), and KRAS (n=1). In 20 FLUS nodules, three samples (15%) had NRAS point mutations. RNA from only one FLUS nodule could be analysed for rearrangements and there was no abnormality.

CONCLUSIONS

Molecular analysis for BRAF and RAS mutations was feasible in residual LBC materials and might be useful for diagnosis of indeterminate thyroid nodules.

Primary ovarian mucinous tumors can be difficult to distinguish from metastatic gastrointestinal neoplasms by histology alone. The expected immunoprofile of a suspected metastatic lower gastrointestinal tumor is CK7^-/CK20⁺/CDX2⁺/PAX8^-. This study assesses the addition of a novel marker SATB2, to improve the diagnostic algorithm. A test cohort included 155 ovarian mucinous tumors (105 carcinomas and 50 borderline tumors) and 230 primary lower gastrointestinal neoplasms (123 colorectal adenocarcinomas and 107 appendiceal neoplasms). All cases were assessed for SATB2, PAX8 CK7, CK20, and CDX2 expression on tissue microarrays. Expression was scored in a 3-tier system as absent, focal (1-50% of tumor cells) and diffuse ( >50% of tumor cells) and then categorized into either absent/present or nondiffuse/diffuse. SATB2 and PAX8 expression was further evaluated in ovarian tumors from an international cohort of 2876 patients (expansion cohort, including 159 mucinous carcinomas and 46 borderline mucinous tumors). The highest accuracy of an individual marker in distinguishing lower gastrointestinal from ovarian mucinous tumors was CK7 (91.7%, nondiffuse/diffuse cut-off) followed by SATB2 (88.8%, present/absent cut-off). The most effective combination was CK7 and SATB2 with accuracy of 95.3% using the 3-tier interpretation, absent/focal/diffuse. This combination outperformed the standard clinical set of CK7, CK20 and CDX2 (87.5%). Re-evaluation of outlier cases confirmed ovarian origin for all but one case. The accuracy of SATB2 was confirmed in the expansion cohort (91.5%). SATB2 expression was also detected in 15% of ovarian endometrioid carcinoma but less than 5% of other ovarian histotypes. A simple two marker combination of CK7 and SATB2 can distinguish lower gastrointestinal from ovarian primary mucinous tumors with greater than 95% accuracy. PAX8 and CDX2 have value as second-line markers. The utility of CK20 in this setting is low and this warrants replacement of this marker with SATB2 in clinical practice.

Molecular testing for mutations activating the mitogen-associated protein kinase signaling pathway is being used to help diagnose thyroid carcinomas. However, the prevalence of these mutations in thyroid lymphomas has not been reported. Therefore, we studied the prevalence of BRAF, NRAS, HRAS, and KRAS mutations in 33 thyroid lymphomas and correlated the mutational status with the clinical, pathological, cytogenetic, and immunophenotypic findings. Eleven cases were also tested for PAX8/PPARγ translocations. The lymphomas included 25 diffuse large B-cell lymphomas, 6 extranodal marginal-zone lymphomas of mucosa-associated lymphoid tissue type, and 2 follicular lymphomas. Seventeen diffuse large B-cell lymphomas were germinal center type, six non-germinal center type, and two unclassifiable (Hans algorithm). None of the cases had an associated thyroid carcinoma. Mutations of the BRAF gene were identified in six (24%) diffuse large B-cell lymphomas (D594G in three germinal center diffuse large B-cell lymphomas, K601N in two germinal center diffuse large B-cell lymphomas, and V600E in one non-germinal center diffuse large B-cell lymphoma) and of the NRAS gene in two (8%) non-germinal center diffuse large B-cell lymphomas (Q61K and Q61H). BRAF and NRAS mutations were not found in any extranodal marginal-zone lymphomas of mucosa-associated lymphoid tissue type or follicular lymphomas. HRAS and KRAS mutations were not identified in any of the cases, nor were PAX8/PPARγ translocations found. Thus, interpretation of finding a BRAF or NRAS mutation in the thyroid, particularly in preoperative thyroid aspirates, must take into account the differential diagnosis of a lymphoma. In addition to the diagnostic importance, our data also demonstrate that alteration in the mitogen-associated protein kinase pathway may have a role in the pathogenesis of some large B-cell lymphomas of the thyroid with potential therapeutic implications.

BACKGROUND

Thyroid dysgenesis (TD) is the most common cause of congenital hypothyroidism (CH). Important genetic factors possibly contributing to TD etiologies include mutations of thyroid transcription factors and TSHR-encoding genes.

OBJECTIVE

Our objective was to determine multiplex ligation-dependent probe amplification (MLPA) utility in detecting the copy number changes in patients with CH and TD.

METHODS

The study included 45 children from southeastern Poland selected via already established neonatal screening for CH. Genomic DNA was extracted from peripheral blood samples and used in MLPA analysis. Genetic variations were analyzed within selected fragments of the PAX8, FOXE1, NKX2-1, thyroid stimulating hormone receptor (TSHR), and TPO genes.

RESULTS

Three heterozygous deletion types in probe hybridization regions were identified for the following genes: PAX8 (exon 7), TSHR (exon 2), and FOXE1 (exon 1). Monoallelic deletions were identified in 5/45 TD subjects.

CONCLUSIONS

MLPA is a useful tool for copy number changes detection and might both improve and expand genetic analysis for CH and TD.

Neonatal screening in Macedonia detects congenital hypothyroidism (CH) with an incidence of 1 in 1,585, and more than 50% of cases exhibit a normally located gland-in-situ (GIS). Monogenic mutations causing dyshormonogenesis may underlie GIS CH; additionally, a small proportion of thyroid hypoplasia has a monogenic cause, such as TSHR and PAX8 defects. The genetic architecture of Macedonian CH cases has not previously been studied. We recruited screening-detected, non-syndromic GIS CH or thyroid hypoplasia cases (n = 40) exhibiting a spectrum of biochemical thyroid dysfunction ranging from severe permanent to mild transient CH and including 11 familial cases. Cases were born at term, with birth weight >3,000 g, and thyroid morphologies included goiter (n = 11), thyroid hypoplasia (n = 6), and apparently normal-sized thyroid. A comprehensive, phenotype-driven, Sanger sequencing approach was used to identify genetic mutations underlying CH, by sequentially screening known dyshormonogenesis-associated genes and TSHR in GIS cases and TSHR and PAX8 in cases with thyroid hypoplasia. Potentially pathogenic variants were identified in 14 cases, of which four were definitively causative; we also detected digenic variants in three cases. Seventeen variants (nine novel) were identified in TPO (n = 4), TG (n = 3), TSHR (n = 4), DUOX2 (n = 4), and PAX8 (n = 2). No mutations were detected in DUOXA2, NIS, IYD, and SLC26A7. The relatively low mutation frequency suggests that factors other than recognized monogenic causes (oligogenic variants, environmental factors, or novel genes) may contribute to GIS CH in this region. Future non-hypothesis-driven, next-generation sequencing studies are required to confirm these findings.

Keywords: congenital hypothyroidism; dyshormonogenesis; genes; goiter; iodine; screening; thyroid hypoplasia.

Purpose: Congenital primary hypothyroidism (CH) is a state of inadequate thyroid hormone production detected at birth, caused either by absent, underdeveloped or ectopic thyroid gland (dysgenesis), or by defected thyroid hormone biosynthesis (dyshormonogenesis). A genetic component has been identified in many cases of CH. This review summarizes the clinical and biochemical features of the genetic causes of primary CH.

Methods: A literature review was conducted of gene defects causing congenital hypothyroidism.

Results: Mutations in five genes have predominantly been implicated in thyroid dysgenesis (TSHR, FOXE1, NKX2-1, PAX8, and NKX2-5), the primary cause of CH (85%), and mutations in seven genes in thyroid dyshormonogenesis (SLC5A5, TPO, DUOX2, DUOXA2, SLC6A4, Tg, and DEHAL1). These genes encode for proteins that regulate genes expressed during the differentiation of the thyroid, such as TPO and Tg genes, or genes that regulate iodide organification, thyroglobulin synthesis, iodide transport, and iodotyrosine deiodination. Besides thyroid dysgenesis and dyshormonogenesis, additional causes of congenital hypothyroidism, such as iodothyronine transporter defects and resistance to thyroid hormones, have also been associated with genetic mutations.

Conclusion: The identification of the underlying genetic defects of CH is important for genetic counseling of families with an affected member, for identifying additional clinical characteristics or the risk for thyroid neoplasia and for diagnostic and management purposes.

Keywords: Congenital hypothyroidism; Genetics; Thyroid dysgenesis; Thyroid dyshormonogenesis; Thyroid malignancy.

Purpose: Mutations and phenotypic characteristics remain unclear in patients with congenital hypothyroidism (CH), and no study concerning whether the outcome of transient CH (TCH) or permanent CH (PCH) is determined by mutations has been reported.

Methods: We searched the literature up to April 2019. Eligible studies and data extraction were performed. We estimated the relationship between mutations and phenotypic characteristics in pooled patients with CH.

Results: Two hundred forty-one cases were pooled from 41 eligible studies. The thyroid morphology, classification of mutated genes, and types of mutations were different between 94 patients with TCH and 147 patients with PCH. Heterozygous missense mutations prevailed in PAX8, TSHR, FOXE1, and NKX2-5, and patients with these mutated genes had a higher risk of PCH (OR = 37.38, 95% CI 5.04-277.21, P < 0.001). TCH and PCH have equal shares in patients with mutated DUOX2 or DUOXA2. Dual-site and multisite mutations were frequently detected in DUOX2. High phenotypic heterogeneity was observed in mutated DUOX2 even in the same mutations. However, there was no relationship found between mutations and transient or permanent outcome in patients with mutated DUOX2.

Conclusion: Transient or permanent outcomes were influenced by the biological function of mutated genes instead of types of mutations among patients with CH. Patients whose mutations were related to thyroid dysgenesis (TD) were more likely to have PCH. The relationship between mutations and phenotypic characteristics is complicated, and phenotypic characteristics may be affected by mutations and other factors.

Glandular lesions of the endocervix can be diagnostically challenging and occasionally the differential diagnosis includes endocervical adenocarcinoma in situ (EC AIS) and well-differentiated endocervical adenocarcinoma (ECA). PAX8 and IMP3 are two markers which have not been well studied in the endocervix. Our aim was to evaluate their immunohistochemical (IHC) expression in benign and malignant endocervical glandular lesions as well as to compare them to the traditionally used panel (Ki-67, p16, CEA).

METHODS

We searched our surgical pathology files for a cohort of benign endocervical glandular lesions as well as premalignant and malignant groups including EC AIS and ECA. An IHC panel consisting of PAX8, IMP3, Ki-67, p16, and CEA was performed on all cases. Immunoreactivity was scored on a degree of positivity (S0=no immunoreactivity, S1=up to 10% cells, S2=between 10 and 50% cells, S3=>50% cells) and intensity (Int0 - absent, Int1 - mild/faint, Int2 - moderate, Int3 - strong).

RESULTS

PAX8 showed diffuse positivity (S3) with at least a moderate intensity of staining (Int2) in the benign group. PAX8 was focal (S1) in ECA and faint (Int1), compared to EC AIS, which was moderate (S2) and faint (Int1). IMP3 expression was focal in the benign group (S1), moderate (S2) in EC AIS and moderate-to-diffuse (S2-3) in ECA. IMP3 intensity was faint (Int1) in benign lesions, moderate (Int2) in EC AIS, and strong (Int3) in ECA. Significant Ki-67, p16, and CEA expression was noted in the premalignant/malignant cohort.

CONCLUSIONS

PAX8 and IMP3 can be helpful in the differential diagnosis of benign vs. malignant endocervical glandular lesions. Our study, however, shows that there is some degree of overlap of staining in both the benign and malignant group. As such, PAX8 and IMP3 should always be interpreted with caution and in combination with the histomorphology.

Transcription factors PAX2 and PAX8 are expressed in the nuclei of Müllerian glandular epithelial cells. In situ carcinomas of the cervix are exemplified by adenocarcinoma in situ (AIS) and high-grade squamous intraepithelial lesions (HSILs), both of which present histologically as hyperchromatic crowded groups of epithelial cells exhibiting loss of polarity. Herein, we sought to investigate the immunohistochemical expression of PAX8 and PAX2 in AIS and HSIL. A total of 66 and 55 cases of AIS and HSIL were examined, respectively. PAX8 positivity was observed in 64 (97%) of 66 cases of AIS. Nuclear PAX2 expression was completely lost in 59 (89%) of the 66 cases of AIS. Eleven (20%) of the 55 HSILs were positive for PAX8. The difference in PAX8 positivity rates between AIS and HSIL was statistically significant (P<0.0001). The PAX2 immunostain was completely negative in the 18 HSILs examined for PAX2 expression. PAX8 and PAX2 immunostaining patterns in benign endocervical glandular epithelium were examined for 98 and 62 cases, respectively. The benign endocervical glandular epithelium was positive for PAX8 and PAX2 expression in 100% and 97% of cases, respectively. In conclusion, immunohistochemical analysis for PAX2 is effective in discriminating AIS from benign endocervical glandular epithelium. The majority of AIS lesions and a subset of HSILs are PAX8(+). With regard to the distinction between AIS and HSIL, a PAX8(-) immunophenotype is particularly predictive of high-grade squamous dysplasia.

Immunohistochemistry for transcription factor PAX8 (paired box gene 8) has recently emerged as a powerful tool in the differential diagnosis of gynecologic malignancies, especially when encountered at a metastatic site. Previous studies have shown PAX8 expression in the majority of ovarian and endometrial carcinomas; however, data regarding PAX8 expression in cervical tumors are limited. In this study PAX8 expression was evaluated in 136 epithelial malignancies of the uterine cervix-including 103 squamous cell carcinomas (SCC), 20 adenocarcinomas of usual type, 6 endometrioid adenocarcinomas, and 7 adenosquamous carcinomas-on tissue microarray slides. PAX8 immunopositivity was defined as at least weak nuclear staining in >5% of tumor cells. The majority of SCC were PAX8 negative (92%; 95/103), whereas among the endocervical adenocarcinomas PAX8 was positive in 70% (14/20) of the usual type and in 83% (5/6) of the endometrioid-type tumors. PAX8 expression was observed in 29% (2/7) of adenosquamous carcinomas. We conclude that PAX8 immunostain is negative in most cervical SCC and is less frequently expressed in endocervical adenocarcinomas as compared with the previously reported high sensitivity for ovarian and endometrial adenocarcinomas. When evaluating possible primary sites of a metastatic lesion, a negative PAX8 immunostain does not rule out common cervical epithelial malignancies.

Humans and mice have cyclical regeneration of the endometrial epithelium. It is expected that such regeneration is ensured by tissue stem cells. However, their location and hierarchy remain debatable. A number of recent studies have suggested the presence of stem cells in the mouse endometrial epithelium. At the same time, it has been reported this tissue can be regenerated by stem cells of stromal/mesenchymal or bone marrow cell origin. Here we describe a single-cell transcriptomic atlas of the main cell types of the mouse uterus and epithelial subset transcriptome, and evaluate contribution of epithelial cells expressing transcription factor PAX8 to the homeostatic regeneration and malignant transformation of adult endometrial epithelium. According to lineage tracing, PAX8 positive (PAX8+) epithelial cells are responsible for long-term maintenance of both luminal and glandular epithelium. Furthermore, multicolor tracing shows that individual glands and contiguous areas of luminal epithelium are formed by clonal cell expansion. Inactivation of tumor suppressor genes Trp53 and Rb1 in PAX8+ cells but not FOXJ1+ cells leads to formation of neoplasms with features of serous endometrial carcinoma, one of the most aggressive type of human endometrial malignancy. Taken together, our results show that a progeny of single PAX8+ cells represent the main regeneration source of the adult endometrial epithelium. They also provide direct experimental genetic evidence for the key roles of the P53 and RB pathways in the pathogenesis of serous endometrial carcinoma, and suggest that PAX8+ cells represent the cell-of-origin of this neoplasm.

Keywords: Endometrial cancer; Mouse models; Single cell transcriptome; Stem cells.

Monocarboxylate transporter 8 (MCT8) transports thyroid hormone (TH) across the plasma membrane. Mutations in MCT8 result in the Allan-Herndon-Dudley syndrome, comprising severe psychomotor retardation and elevated serum T3 levels. Because the neurological symptoms are most likely caused by a lack of TH transport into the central nervous system, the administration of a TH analog that does not require MCT8 for cellular uptake may represent a therapeutic strategy. Here, we investigated the therapeutic potential of the biologically active T3 metabolite Triac (TA3) by studying TA3 transport, metabolism, and action both in vitro and in vivo. Incubation of SH-SY5Y neuroblastoma cells and MO3.13 oligodendrocytes with labeled substrates showed a time-dependent uptake of T3 and TA3. In intact SH-SY5Y cells, both T3 and TA3 were degraded by endogenous type 3 deiodinase, and they influenced gene expression to a similar extent. Fibroblasts from MCT8 patients showed an impaired T3 uptake compared with controls, whereas TA3 uptake was similar in patient and control fibroblasts. In transfected cells, TA3 did not show significant transport by MCT8. Most importantly, treatment of athyroid Pax8-knockout mice and Mct8/Oatp1c1-double knockout mice between postnatal days 1 and 12 with TA3 restored T3-dependent neural differentiation in the cerebral and cerebellar cortex, indicating that TA3 can replace T3 in promoting brain development. In conclusion, we demonstrated uptake of TA3 in neuronal cells and in fibroblasts of MCT8 patients and similar gene responses to T3 and TA3. This indicates that TA3 bypasses MCT8 and may be used to improve the neural status of MCT8 patients.

Anaplastic carcinoma (AC) is a rare but highly aggressive form of thyroid cancer. It mostly arises on a background of pre-existing well-differentiated cancer (WDC); however, whether it evolves directly from a WDC or originates as a second independent neoplasm is still to be defined. To obtain further insights into these mechanisms, we performed morphological, immunohistochemical, and next-generation sequencing analyses to compare AC and its associated WDC in a subset of 13 surgically resected specimens. Histologically, most WDC were of aggressive subtypes. Papillary carcinomas (8 cases; 62%) were tall cell (4/8), columnar (1/8), classic with hobnail features (1/8), classic and follicular variant in the remaining 2 cases; Hürthle cell and follicular carcinomas were present in 5 (38%) and in 1 (8%) patient, respectively. One patient harbored both a PTC, follicular variant, and a Hürthle cell carcinoma. We did not find any correlation between a histotype of WDC and a specific anaplastic growth pattern. Immunohistochemically, ACs retained pankeratin/PAX8 expression but with significantly lower levels than WDCs, and they tended to lose TTF1 expression, as can be expected within a dedifferentiation process. In addition, AC showed a more frequent expression of p63 and/or SMA, a mutated pattern of p53, and an abnormal expression of p16. Genetic analysis showed that the number of mutations was higher in AC than in the associated WDC, confirming a role of the progressive accumulation of genetic damage in this transition. We observed that mutations found in the WDCs were consistently identified in the anaplastic counterparts, further supporting the hypothesis of a developmental link.

Keywords: Anaplastic carcinoma; Immunohistochemistry; Molecular biology; Thyroid; Well-differentiated carcinoma.

Bisphenol S (BPS) is widely used as a raw material in industry, resulting in its ubiquitous distribution in natural environment, including the aqueous environment. However, the effect of BPS on the thyroid endocrine system is largely unknown. In this study, zebrafish (Danio rerio) embryos were exposed to BPS at 1, 3, 10, and 30 μg/L, from 2 h post-fertilization (hpf) to 168hpf. Bioconcentration of BPS and whole-body thyroid hormones (THs), thyroid-stimulating hormone (TSH) concentrations as well as transcriptional profiling of key genes related to the hypothalamic-pituitary-thyroid (HPT) axis were examined. Chemical analysis indicated that BPS was accumulated in zebrafish larvae. Thyroxine (T4) and triiodothyronine (T3) levels were significantly decreased at ≥ 10 and 30 μg/L of BPS, respectively. However, TSH concentration was significantly induced in the 10 and 30 μg/L BPS-treated groups. After exposure to BPS, the mRNA expression of corticotrophin releasing hormone (crh) and thyroglobulin (tg) genes were up-regulated at ≥10 μg/L of BPS, in a dose-response manner. The transcription of genes involved in thyroid development (pax8) and synthesis (sodium/iodide symporter, slc5a5) were also significantly increased in the 30 μg/L of BPS treatment group. Moreover, exposure to 10 μg/L or higher concentration of BPS significantly up-regulated genes related to thyroid hormone metabolism (deiodinases, dio1, dio2 and uridinediphosphate glucoronosyltransferases, ugt1ab), which might be responsible for the altered THs levels. However, the transcript of transthyretin (ttr) was significantly down-regulated at ≥ 3 μg/L of BPS, while the mRNA levels of thyroid hormone receptors (trα and trβ) and dio3 remained unchanged. All the results indicated that exposure to BPS altered the whole-body THs and TSH concentrations and changed the expression profiling of key genes related to HPT axis, thus triggering thyroid endocrine disruption.

Biphenotypic sinonasal sarcoma (BSNS) is a distinctive, anatomically restricted, low-grade spindle cell sarcoma that shows considerable histologic overlap with other cellular spindle cell neoplasms. This tumor type shows both myogenic and neural differentiation, which can be demonstrated by immunohistochemistry; however, the available diagnostic markers are relatively nonspecific. BSNS is characterized by PAX3 rearrangements, with MAML3 as the most common fusion partner. Our aim was to determine whether immunohistochemistry using a monoclonal PAX3 antibody could distinguish BSNS from potential histologic mimics, as well as to evaluate a widely available polyclonal PAX8 antibody, which is known to cross-react with other paired box transcription factor family members. Immunohistochemistry for PAX3 and PAX8 was performed on whole sections of 15 BSNS (10 with confirmed PAX3 rearrangement) and 10 cases each of the following histologic mimics: malignant peripheral nerve sheath tumor, monophasic synovial sarcoma, spindle cell rhabdomyosarcoma (RMS), solitary fibrous tumor, sinonasal hemangiopericytoma, and cellular schwannoma, as well as alveolar RMS (which harbors PAX3 or PAX7 gene rearrangements). BSNS showed consistent expression of PAX3 (15/15), all multifocal-to-diffuse and most with moderate-to-strong intensity of staining. One single case of spindle cell RMS showed PAX3 expression (1/10), and all other histologic mimics were completely PAX3-negative. In contrast, nuclear staining for PAX8 was present in all 15 BSNS, 7/10 malignant peripheral nerve sheath tumor, 3/10 cellular schwannomas, 2/10 sinonasal hemangiopericytomas, 1/10 synovial sarcoma, 1 spindle cell RMS, and 1 solitary fibrous tumor. All cases of alveolar RMS were positive for PAX8, and most were also positive for PAX3 (8/10). Immunohistochemical expression of PAX3 is highly sensitive (100%) and specific (98%) for BSNS. A polyclonal PAX8 antibody also stains BSNS (likely due to cross-reactivity with PAX3) but has much lower specificity (75%), with frequent expression in numerous mimics.

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.

Damage to the sensory hair cells and the spiral ganglion neurons of the cochlea leads to deafness. Induced pluripotent stem cells (iPSCs) are a promising tool to regenerate the cells in the inner ear that have been affected by pathology or have been lost. To facilitate the clinical application of iPSCs, the reprogramming process should minimize the risk of introducing undesired genetic alterations while conferring the cells the capacity to differentiate into the desired cell type. Currently, reprogramming induced by synthetic mRNAs is considered to be one of the safest ways of inducing pluripotency, as the transgenes are transiently delivered into the cells without integrating into the genome. In this study, we explore the ability of integration-free human-induced pluripotent cell lines that were reprogrammed by mRNAs, to differentiate into otic progenitors and, subsequently, into hair cell and neuronal lineages. hiPSC lines were induced to differentiate by culturing them in the presence of fibroblast growth factors 3 and 10 (FGF3 and FGF10). Progenitors were identified by quantitative microscopy, based on the coexpression of otic markers PAX8, PAX2, FOXG1, and SOX2. Otic epithelial progenitors (OEPs) and otic neuroprogenitors (ONPs) were purified and allowed to differentiate further into hair cell-like cells and neurons. Lineages were characterised by immunocytochemistry and electrophysiology. Neuronal cells showed inward Na⁺ (I_Na) currents and outward (I_k) and inward K⁺ (I_K1) currents while hair cell-like cells had inward I_K1 and outward delayed rectifier K⁺ currents, characteristic of developing hair cells. We conclude that human-induced pluripotent cell lines that have been reprogrammed using nonintegrating mRNAs are capable to differentiate into otic cell types.

The human fallopian tube harbors the cell of origin for the majority of high-grade serous "ovarian" cancers (HGSCs), but its cellular composition, particularly the epithelial component, is poorly characterized. We perform single-cell transcriptomic profiling of around 53,000 individual cells from 12 primary fallopian specimens to map their major cell types. We identify 10 epithelial subpopulations with diverse transcriptional programs. Based on transcriptional signatures, we reconstruct a trajectory whereby secretory cells differentiate into ciliated cells via a RUNX3^high intermediate. Computational deconvolution of advanced HGSCs identifies the "early secretory" population as a likely precursor state for the majority of HGSCs. Its signature comprises both epithelial and mesenchymal features and is enriched in mesenchymal-type HGSCs (p = 6.7 × 10^-27), a group known to have particularly poor prognoses. This cellular and molecular compendium of the human fallopian tube in cancer-free women is expected to advance our understanding of the earliest stages of fallopian epithelial neoplasia.

Keywords: PAX8; RUNX3; SOX17; ciliated epithelial cells; fallopian tube; microenvironment; ovarian cancer; scRNA-seq; secretory epithelial cells; transcription factor.

The noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) is a strictly defined thyroid lesion, reclassified in 2016, in order to more accurately reflect the biological behavior of the tumor and thus, modify the way the lesion is clinically approached and perceived both by practitioners and patients. Additionally, this newly specified designation also allows for more uniformity in reporting for general pathologists less comfortable to exclude overt malignancy with certain nuclear features. In recent years, increasing molecular analyses correlated with longitudinal clinical outcomes have fostered improved diagnostic and treatment paradigms. Important revisions made to the definition of NIFTP in 2018 include the prohibition of any true papillae formation and the exclusion of lesions harboring the BRAF V600E mutation and other high-risk genetic abnormalities. These changes reflect the imperfection of the current criteria in outcome prediction and the global efforts for improvement. NIFTP are lesions with a wide range of size and cytomorphology. Although not addressed in the original series, large (≥4 cm) and oncocytic NIFTP have recently been shown to incur no recurrence or metastatic risk. Molecularly, NIFTP have a similar mutational profile as other follicular thyroid neoplasms, with frequent RAS family mutations and PAX8-PPARɤ fusions. However, the transcriptomic landscape is highly heterogenous, adding difficulty to gene expression-based cytopathologic classification. This review summarizes the evolution of the NIFTP concept and important advances in recent literature.

Keywords: BRAF; Lobectomy; Noninvasive follicular thyroid neoplasm with papillary-like nuclear features; Oncocytic; Radioactive iodine; TERT.