Background: Most uterine cervical high-risk human papillomavirus (HPV) infections are transient, with only a small fraction developing into cervical cancer. Family aggregation studies and heritability estimates suggest a significant inherited genetic component. Candidate gene studies and previous genome-wide association studies (GWASs) report associations between the HLA region and cervical cancer. Adopting a genome-wide approach, we aimed to compare genetic variation in women with invasive cervical cancer and cervical intraepithelial neoplasia (CIN) grade 3 with that in healthy controls.

Methods: We did a GWAS in a cohort of unrelated European individuals using data from UK Biobank, a population-based cohort including 273 377 women aged 40-69 years at recruitment between March 13, 2006, and Oct 1, 2010. We used an additive univariate logistic regression model to analyse genetic variants associated with invasive cervical cancer or CIN3. We sought replication of candidate associations in FinnGen, a large independent dataset of 128 123 individuals. We also did a two-sample mendelian randomisation approach to explore the role of risk factors in the genetic risk of cervical cancer.

Findings: We included 4769 CIN3 and invasive cervical cancer case samples and 145 545 control samples in the GWAS. Of 9 600 464 assayed and imputed single-nucleotide polymorphisms (SNPs), six independent variants were associated with CIN3 and invasive cervical cancer. These included novel loci rs10175462 (PAX8; odds ratio [OR] 0·87, 95% CI 0·84-0·91; p=1·07 × 10^-9) and rs27069 (CLPTM1L; 0·88, 0·84-0·92; p=2·51 × 10^-9), and previously reported signals at rs9272050 (HLA-DQA1; 1·27, 1·21-1·32; p=2·51 × 10^-28), rs6938453 (MICA; 0·79, 0·75-0·83; p=1·97 × 10^-17), rs55986091 (HLA-DQB1; 0·66, 0·60-0·72; p=6·42 × 10^-28), and rs9266183 (HLA-B; 0·73, 0·64-0·83; p=1·53 × 10^-6). Three SNPs were replicated in the independent Finnish dataset of 1648 invasive cervical cancer cases: PAX8 (rs10175462; p=0·015), CLPTM1L (rs27069; p=2·54 × 10^-7), and HLA-DQA1 (rs9272050; p=7·90 × 10^-8). Mendelian randomisation further supported the complementary role of smoking (OR 2·46, 95% CI 1·64-3·69), older age at first pregnancy (0·80, 0·68-0·95), and number of sexual partners (1·95, 1·44-2·63) in the risk of developing cervical cancer.

Interpretation: Our results provide new evidence for the genetic susceptibility to cervical cancer, specifically the PAX8, CLPTM1L, and HLA genes, suggesting disruption in apoptotic and immune function pathways. Future studies integrating host and viral, genetic, and epigenetic variation, could further elucidate complex host-viral interactions.

Funding: NIHR Imperial BRC Wellcome 4i Clinician Scientist Training Programme.

The recent reclassification of a follicular variant of papillary thyroid carcinoma (FVPTC) subset as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) aims to avoid overtreatment of patients with an indolent lesion. The diagnosis of NIFTP has recently been revisited using more rigid criteria. This study presents histological and molecular findings and a long clinical follow up of 94 of FVPTC, 40 cases of follicular adenoma (FTA) and 22 cases of follicular carcinoma (FTC) that were classified before the advent of the NIFTP classification. BRAF V600E mutation was found in EFVPT samples. Slides were reviewed using these rigid criteria and analysis of numerous sections of paraffin blocks and reclassified as 7 NIFTPs, 2 encapsulated noninvasive FVPTC (EFVPTCs), 29 infiltrative FVPTC (IFVPTCs), 57 invasive EFVPTC (I-EFVPTCs), 39 FTA and 22 FTC. Remarkably, EFVPTC and NIFTP patients were all free of disease at the end of follow up and showed no BRAF mutation. Only one NIFTP sample harbored mutations, an NRAS Q61R. Nonetheless, NRAS mutation was also found in I-EFVPTC. PAX8/PPARG fusion was found in I-EFVPTCs and FTC. Although additional studies are needed to define a specific molecular profile to aid in the diagnosis of lesions with borderline morphological characteristics, we confirmed that the BRAF V600E mutation is an important tool to exclude the diagnosis of NIFTP. We also show that rigorous histopathological criteria should be strongly followed to avoid missing lesions in which more aggressive behavior is present, mainly via the analysis of capsule or vascular invasion and the presence of papillary structures.

BACKGROUND

We present a series of papillary renal cell carcinomas (PRCC) reminiscent of so-called "oncocytic variant of papillary renal cell carcinoma" (OPRCC), included in the 2016 WHO classification as a potential type 3 PRCC. OPRCC is a poorly understood entity, cytologically characterized by oncocytic cells with non-overlapping low grade nuclei. OPRCC is not genotypically distinct and the studies concerning this variant have shown an inconsistent genetic profile. The tumors presented herein demonstrated predominantly papillary/tubulopapillary architecture and differed from OPRCC by pseudostratification and grade 2-3 nuclei (Fuhrman/ISUP). Because there is a morphologic overlap between renal oncocytoma (RO) and PRCC in the cases included in this study, the most frequently affected chromosomes in RO and PRCC were analyzed.

METHODS

147 PRCC composed of oncocytic cells were retrieved from our registry in order to select a group of morphologically uniform tumors. 10 cases with predominantly papillary, tubulopapillary or solid architectural patterns were identified. For immunohistochemical analysis, the following antibodies were used: vimentin, antimitochondrial antigene (MIA), AMACR, PAX8, CK7, CK20, AE1-3, CAM5.2, OSCAR, Cathepsin K, HMB45, SDHB, CD10, and CD117. Enumeration changes of locus 1p36, chromosomes 7, 14, 17, X, Y and rearrangement of CCND1 were examined by FISH. For further study, only tumors showing karyotype similar to that of RO were selected. The tumors exhibiting either trisomy of chromosomes 7, 17 or gain of Y, thus abnormalities characteristic for PRCC, were excluded.

RESULTS

There were 5 males and 5 females, with patient age ranging from 56 to 79 years (mean 66.8 years). The tumor size ranged from 2 to 10 cm (mean 5.1 cm). Follow-up was available for 8/10 patients (mean 5.2 years); one patient died of the disease, while 7 of 8 are alive and well. Immunohistochemically, all cases were reactive for AMACR, vimentin, PAX8, OSCAR, CAM5.2, and MIA. SDHB was retained in all cases. 9/10 cases were positive for CD10, 7/10 cases reacted with CK7, 4/10 with Cathepsin K, and 2/10 with AE1-3. None of the cases were positive for CD117, HMB45 and CK20. All 10 cases were analyzable by FISH and showed chromosomal abnormalities similar to that usually seen in RO (i.e. loss of 1p36 gene loci, loss of chromosome Y, rearrangement of CCND1 and numerical changes of chromosome 14).

CONCLUSIONS

We analyzed a series of renal tumors combining the features of PRCC/OPRCC and RO, that included pseudostratification and mostly high grade oncocytic cells lining papillary/tubulopapillary structures, karyotype characterized by loss of 1p36, loss of chromosome Y, rearrangement of CCND1 gene and numerical changes of chromosome 14. Despite the chromosomal numerical abnormalities typical of RO, we classified these tumors as part of the spectrum of PRCC because of their predominant papillary/tubulopapillary architecture, immunoprofile that included reactivity for AMACR, vimentin and lack of reactivity for CD117, all of which is incompatible with the diagnosis of RO. This study expands the morphological spectrum of PRCC by adding a cohort of diagnostically challenging cases, which may be potentially aggressive.

ALK-rearranged renal cell carcinoma is a provisional entity in the 2016 WHO Classification of Tumors of the Urinary System and Male Genital Organs. The reported fusion partners included VCL, TPM3, EML4, STRN, and HOOK1. Herein, we present a peculiar renal cell carcinoma morphologically resembling metanephric adenoma and harboring a novel PLEKHA7-ALK fusion. Microscopically, the tumor is composed of bland epithelial cells with scant to moderate amount of amphophilic cytoplasm, round and uniform nuclei, delicate chromatin, and inconspicuous nucleoli, arranged in tightly packed small acini and angulated tubules. Papillary formation, intraluminal glomeruloid tufts, microcysts, and solid nests were focally observed. Psammomatous calcifications were evident. The tumor cells were diffusely reactive for CK7, AMACR, PAX8, and ALK, while non-reactive for WT1, BRAF V600E, CD57, carbonic anhydrase IX, TFE3, and cathepsin K. Fluorescence in situ hybridization showed breaking apart of ALK. A novel PLEKHA7exon18-ALKexon20 fusion was detected using ArcherDX FusionPlex next-generation sequencing panel and was further confirmed with reverse-transcriptase PCR. Our case demonstrates that in contrast to prior cases showing high-grade tumor cells, ALK-rearranged renal cell carcinoma may also present as a low-grade renal tumor mimicking metanephric adenoma. Immunohistochemistry and molecular testing are helpful to identify this tumor, which may be eligible for ALK inhibitor-targeted therapy.

Aims: Papillary neoplasms of the middle and inner ear are rare and poorly characterized. The current WHO classification lumped them into two major subtypes: aggressive papillary tumors (APTs) and endolymphatic sac tumors (ELSTs). We present two papillary neoplasms of the middle ear not fitting either classical APT or ELST, and compare them with 3 ELSTs.

Methods and results: Patients were a 48-year-old female and a 59-year-old male without a history of other neoplasms. Histology showed papillary-cystic growth of predominantly oncocytic (Case 1) or mucinous (Case 2) cells surrounded by p63-positive basal layer. The overall histology was reminiscent of oncocytic sinonasal papilloma (Case 1) and pancreatobiliary or salivary intraductal papillary mucinous neoplasms (Case 2). Ovarian-type stroma, invasion and malignant features were absent. Immunohistochemistry revealed expression of CK7, but not CAIX and PAX8 (except very focal PAX8 in Case 1). The TST15 gene panel (Illumina) and HRAS sequencing revealed no pathogenic mutations in BRAF, KRAS, EGFR, AKT1 or HRAS. The TruSight RNA fusion panel (Illumina) revealed a MKRN1-BRAF fusion in Case 1. No fusion was detected in Case 2. The three ELSTs showed classical features of the entity, expressed CK7, EMA, PAX8 and CAIX, and lacked a basal cell layer.

Conclusion: These novel cases suggest that papillary tumors of the ear represent a heterogeneous spectrum of distinct neoplasms unified by prominent papillary-cystic pattern rather than a single entity. Future studies should clarify if the MKRN1-BRAF fusion is a defining recurrent driver event, especially in those cases reported as sinonasal-type middle ear papillomas.

Keywords: BRAF; IPMN; Papillary mucinous neoplasms; Schneiderian papilloma; aggressive papillary tumor; ear; endolymphatic sac tumor; oncocytic.

Transcription factor PAX8 expression is upregulated in several types of cancers. However, little is known about the function of PAX8 in the progression of hepatoma and its regulatory mechanisms. Here, we show that PAX8 silencing inhibits the proliferation and clonogenicity of hepatoma cells and its growth in vivo. The HBV X protein (HBx) does not directly interacts, but stabilizes PAX8 by inhibiting proteasome-dependent ubiquitination and degradation. Furthermore, the E3 ubiquitin ligase complex component Skp2 through its LRR domain directly interacts with the Prd domain of PAX8 and targets PAX8 by recognizing its lysine 275 for ubiquitination and degradation in hepatoma cells. In addition, HBx directly interacts and is colocalized with Skp2 to inhibit its recognition and subsequent ubiquitination and degradation of PAX8 in hepatoma cells. Moreover, HBx upregulates the expression and phosphorylation of Aurora A, a serine-threonine kinase, which interacts with and phosphorylates PAX8 at S209 and T277, compromising the Skp2-recognized PAX8 ubiquitination and destabilization. Thus, HBx stabilizes PAX8 protein by inhibiting the Skp2 targeted PAX8 ubiquitination and enhancing the Aurora A-mediated its phosphorylation, contributing to the progression of hepatoma. Our findings suggest that PAX8 may a new target for design of therapies and uncover new insights into the pathogenesis of hepatoma.

Major differences with respect to the diagnostic performance of a "ruling in" approach in the presurgical diagnosis of indeterminate thyroid fine-needle aspirations (FNAs) have been reported. Therefore, the aim of this prospective multicenter study was to investigate the specific diagnostic impact of mutation testing using a seven-gene panel in a routine primary referral setting analyzing FNAs from endocrinology and nuclear medicine practices in Germany.

RNA and DNA was extracted from 564 routine air-dried FNA smears obtained from 64 physicians and cytologically graded by one experienced cytopathologist. PAX8/PPARG and RET/PTC rearrangements were detected by quantitative polymerase chain reaction, while BRAF and RAS mutations were detected by pyrosequencing. Molecular data were compared to histology and follow-up >1 year, which were available for 322/348 patients undergoing surgery and 33/74 patients having follow-up. Histology results were obtained from the local routine pathologists who were blinded to the molecular test results.

BRAF and RET/PTC mutations were associated with carcinoma in 98% and 100% of samples, respectively. RAS and PAX8/PPARG mutations were associated with carcinoma in 31% and 0% of samples, respectively. Thirty-six percent of the carcinomas were identified by molecular testing in the atypia of undetermined significance/follicular lesion of undetermined significance and follicular neoplasm/suspicious for a follicular neoplasm categories, with malignancy rates of 15% and 17%, respectively. Due to a low percentage of RAS mutation-positive carcinomas in combination with a rather high percentage of RAS mutation-positive benign nodules, the positive predictive values of 41% and 36% in the atypia of undetermined significance/follicular lesion of undetermined significance and follicular neoplasm/suspicious for a follicular neoplasm categories offer only limited diagnostic potential.

In conclusion, the data suggest that the application of the current seven-gene panel in a routine primary referral setting does not improve the presurgical diagnosis of thyroid FNAs. While the diagnostic relevance of RAS mutations in thyroid tumors needs further investigation, more comprehensive mutation panels with more cancer-specific mutations may improve the presurgical diagnosis of thyroid FNAs.

Background: The rete testis connects seminiferous tubules of the testis with efferent ducts having a mesonephric origin. The development of the rete testis is insufficiently studied, but there is evidence suggesting that it originates from gonadal cells. Here, the formation of the rete testis was investigated from E11.5 to E16.5 using immunofluorescent staining and 3D modelling.

Results: The rete testis became visible by SOX9 and PAX8 staining starting from E12.5. It was located in the mesonephros but connected with testis cords formed by Sertoli cells expressing SOX9, AMH, DMRT1. Between E13.5 and E14.5, AMH+ network of testis cords at the mesonephric side began to disintegrate in a gradient-dependent manner along the anterior-posterior axis of the gonad and connections between testis cords gradually lost AMH becoming a part of the rete. Cells combining features of Sertoli and rete cells (PAX8+AMH+ and DMRT1+AMH- cells) were detected starting from E14.5, suggesting that some rete cells originated from Sertoli cells. The rete ovarii, a female counterpart of the rete testis, developed in a similar way as the rete testis until E13.5.

Conclusions: A part of the rete testis originates from connections between testis cords. Evidence that Sertoli cells contribute to rete cells is provided. This article is protected by copyright. All rights reserved.

Keywords: 3D models; Gonad development; Reproductive tract; Sertoli cell.

Twelve cases of thymomas with prominent glandular differentiation are presented. The patients were 7 men and 5 women aged between 45 and 68 years (average, 56.5 years). Clinically, the patients presented with nonspecific symptoms of chest pain, cough, and fatigue. None of the patients had a history of myasthenia gravis or other autoimmune syndrome. Thymectomy was performed in all patients. The tumor size ranged from 4 to 7 cm in greatest diameter. Macroscopically, the tumors were described as firm and light tan without areas of necrosis, hemorrhage, or cystic change. Histologically, 7 tumors were classified as spindle cell (World Health Organization type A), 2 as mixed spindle cell and conventional (A+B1), 2 as conventional (B1), and 1 as atypical thymoma (B3). In 4 cases, the tumors showed invasion into periadipose thymic tissue. All cases showed the typical growth patterns of their particular subtypes. In addition, a distinct glandular component was present in all cases showing mucinous differentiation in 4 of them. Immunohistochemical studies showed tumor cells positive for CAM5.2, cytokeratin 5/6, and Pax8 and negative for carcinoembryonic antigen, thyroid transcription factor 1, and epithelial membrane antigen. Calretinin showed focal weak staining in the nonmucinous glandular components in 3 cases. Follow-up information obtained in 8 patients showed that all were alive and well in a period ranging from 2 to 5 years. The possibility of a glandular component in thymomas should be kept in mind in the assessment of mediastinoscopic biopsies to avoid misdiagnosis for other neoplasms that may require different treatment modalities.

OBJECTIVE

The purpose of this study was to compare the immunohistochemical staining profiles of PAX8-polyclonal, PAX8-monoclonal, PAX5-monoclonal and PAX6-monoclonal antibodies in several histological types of primary thoracic and thyroid tumours. In addition, we analysed PAX8 mRNA expression by using in-situ hybridization.

RESULTS

We compared polyclonal PAX8 and monoclonal PAX8, PAX5 and PAX6 antibodies in 962 samples (687 lung carcinomas, 40 malignant pleural mesotheliomas, 138 thymic tumours and 97 thyroid tumours) using the tissue microarray technique. Among thyroid tumours, the monoclonal and polyclonal PAX8 antibodies showed a high positive rate (98.0%). Of 167 polyclonal PAX8 antibody-positive tumours, except for thyroid tumours, 54 cases tested positive for PAX5 and/or PAX6 (31 lung carcinomas and 23 thymic tumours). No PAX8 mRNA expression was detected using RNAscope (in-situ hybridization technique) other than in thyroid tumours. A portion of polyclonal PAX8 antibody-positive tumours showed cross-reactivity for PAX5 or PAX6 protein.

CONCLUSIONS

Monoclonal PAX8 antibody showed high specificity to thyroid tumours and was superior to the polyclonal antibody.

Clear-cell carcinoma of the thyroid has been regarded as a variant of follicular (FTC) or papillary (PTC) thyroid carcinoma. Twenty-one primary thyroid carcinomas with clear-cell features, diagnosed in 20 patients (12 female) were identified between 1992 and 2012 (0.5% of in-house thyroid carcinomas).

Hematoxylin and eosin slides were reviewed. SNaPshot multigene mutational analysis and a translocation panel were successfully performed on 15 of these cases.

Twelve (57%) were FTC, five were conventional PTC, two were follicular variant of PTC, and two were poorly differentiated thyroid carcinomas. Five cases had RAS mutation (four FTC and one PTC); two had PAX8-PPARgamma translocations (both FTC, one with concurrent p53 mutation); one had an EML4-ALK translocation (PTC); and one had a TFG-MET translocation (follicular variant of PTC). Five carcinomas were metastatic to regional lymph nodes (three FTC and two PTC), and two were metastatic to bone (both FTC). Disease confined to the thyroid (67%) and rates of regional lymph node metastasis (24%) and distant metastasis (10%) were near the national averages (68%, 25%, and 5%, respectively). One patient with a poorly differentiated thyroid carcinoma died one year after diagnosis, and a patient with metastatic FTC died two years after diagnosis. Overall mortality was 10%.

Clear-cell change in thyroid carcinoma is rare, is more common in FTC than it is in PTC, is found focally or multifocally within a given lesion, and is frequently associated with RAS mutations (33%). Clear-cell change in thyroid neoplasia should raise the possibility of follicular carcinoma, and should not be treated differently from other carcinomas of similar grade and stage.

Adenocarcinoma of the rete testis is rare and its etiological and pathologic characteristics are not well studied. We therefore investigated the clinical, morphologic, and immunohistochemical features of 6 cases diagnosed at our institution and conducted a detailed review of the literature. The mean age was 64 years. All patients presented with testicular masses; 4 were right-sided. On gross examination, all tumors were centered in the hilum and had solid and cystic cut surfaces. Microscopically, all had intrarete and invasive growth and showed multiple patterns, with a variable proportion of papillary, solid and glandular morphology, the latter varying from slit-like lumens to well-formed glands and tubules. Less common patterns included corded/trabecular (n=3), cribriform (n=3), glomeruloid (n=3), nested (n=2), and micropapillary (n=2). Discrete nests of eosinophilic and clear cells were a distinctive feature in 3 cases. Geographic necrosis occurred in 3 cases. All showed at least moderate nuclear pleomorphism with ovoid nuclei. Transition from benign to malignant rete epithelium was seen in all cases. The stroma was hyalinized to partially fibrotic. On immunohistochemical study, the tumor cells were positive for CK7 (5/5), AE1/AE3 cytokeratin (5/5), EMA (5/5), vimentin (5/5), EpCAM (detected by BerEP4 anitbody) (4/5), CK5/6 (4/5), nuclear Wilms Tumor-1 (4/5), epithelial specific antigen (detected by MOC31 antibody) (3/4), PAX8 (3/5), and calretinin (2/5). OCT3/4, SALL4, CD30, NKX3.1, PSA, α-inhibin, CK20, and S100 protein were negative. Ki-67 proliferative index ranged from 5% to 60% (mean: 40, median: 43). At presentation, 5 patients had retroperitoneal lymph node metastasis and one of these also had pulmonary metastases. The sixth patient developed pulmonary metastasis within 15 months of diagnosis. Three died within 4 years of diagnosis. In summary, adenocarcinoma of the rete testis is a rare malignant tumor with poor survival and a high propensity for retroperitoneal lymph node metastasis that must be distinguished from other testicular neoplasms and metastasis to the testis. Hilar localization, transition from benign to malignant rete epithelium, and supportive immunostains aid its accurate diagnosis.

SIX1 and SIX4 genes play critical roles in kidney development. We evaluated the effect of these genes on pig kidney development by generating SIX1^-/- and SIX1^-/- /SIX4^-/- pig foetuses using CRISPR/Cas9 and somatic cell nuclear transfer. We obtained 3 SIX1^-/- foetuses and 16 SIX1^-/- /SIX4^-/- foetuses at different developmental stages. The SIX1^-/- foetuses showed a migration block of the left kidney and a smaller size for both kidneys. The ureteric bud failed to form the normal branching and collecting system. Abnormal expressions of kidney development-related genes (downregulation of PAX2, PAX8, and BMP4 and upregulation of EYA1 and SALL1) were also observed in SIX1^-/- foetal kidneys and confirmed in vitro in porcine kidney epithelial cells (PK15) following SIX1 gene deletion. The SIX1^-/- /SIX4^-/- foetuses exhibited more severe phenotypes, with most foetuses showing retarded development at early stages of gestation. The kidney developed only to the initial stage of metanephros formation. These results demonstrated that SIX1 and SIX4 are key genes for porcine metanephros development. The creation of kidney-deficient porcine foetuses provides a platform for generating human kidneys inside pigs using blastocyst complementation.

The differentiation program for human thyroid follicular cells (TFCs) relies on the interplay between sequence-specific transcription factors and transcriptional co-regulators. Transcriptional co-activator with PDZ-binding motif (TAZ) is a co-activator that regulates several transcription factors, including PAX8 and NKX2-1, which play a central role in thyroid-specific gene transcription. TAZ and PAX8/NKX2-1 are co-expressed in the nuclei of thyroid cells, and TAZ interacts directly with both PAX8 and NKX2-1, leading to their enhanced transcriptional activity on the thyroglobulin (TG) promoter and additional genes.

The use of a small molecule, ethacridine, recently identified as a TAZ activator, in the differentiation of thyroid cells from human embryonic stem (hES) cells was studied. First, endodermal cells were derived from hES cells using Activin A, followed by induction of differentiation into thyroid cells directed by ethacridine and thyrotropin (TSH).

The expression of TAZ was increased in the Activin A-derived endodermal cells by ethacridine in a dose-dependent manner and followed by increases in PAX8 and NKX2-1 when assessed by both quantitative polymerase chain reaction and immunostaining. Following further differentiation with the combination of ethacridine and TSH, the thyroid-specific genes TG, TPO, TSHR, and NIS were all induced in the differentiated hES cells. When these cells were cultured with extracellular matrix-coated dishes, thyroid follicle formation and abundant TG protein expression were observed. Furthermore, such hES cell-derived thyroid follicles showed a marked TSH-induced and dose-dependent increase in radioiodine uptake and protein-bound iodine accumulation.

These data show that fully functional human thyroid cells can be derived from hES cells using ethacridine, a TAZ activator, which induces thyroid-specific gene expression and promotes thyroid cell differentiation from the hES cells. These studies again demonstrate the importance of transcriptional regulation in thyroid cell development. This approach also yields functional human thyrocytes, without any gene transfection or complex culture conditions, by directly manipulating the transcriptional machinery without interfering with intermediate signaling events.

Background: Thyroid follicular cells are characterized by the expression of a specific set of genes necessary for the synthesis and secretion of thyroid hormones, which are in turn regulated by the transcription factors Nkx2-1, Pax8, and Foxe1. Thyroid differentiation is finely tuned by the balance between positive regulatory signals, including thyrotropin (TSH), and by negative regulatory signals, such as transforming growth factor beta (TGF-β), which counteracts the action of TSH. A role for Foxe1 as a mediator of hormonal and growth-factor control of thyroid differentiation has been previously suggested. Therefore, the aim of this work was to study the mechanisms governing Foxe1 expression to define the ligands and signals that regulate one of the important factors in thyroid differentiation. Methods: Expression of Foxe1 was evaluated in rat PCCl3 thyroid follicular cells under different treatments. The mouse Foxe1 promoter was cloned, and site-directed mutagenesis was undertaken to study its transcriptional regulation and to identify response elements. Protein/DNA binding assays were performed to evaluate the binding of different transcription factors, and gene-silencing approaches were used to elucidate their functional roles. Results:In silico analysis of the Foxe1 promoter identified binding sites for Nkx2-1, Pax8, Foxe1, and Smad proteins, as well as cAMP-response element (CRE) sites. It was found that both CRE-binding protein and CRE modulator were necessary for the TSH-mediated induction of Foxe1 expression via the cAMP/PKA signaling pathway. Moreover, transcription of Foxe1 was regulated by Nkx2-1 and Pax8 and by itself, suggesting an autoregulatory mechanism of activation and an important role for thyroid transcription factors. Finally, TGF-β, through Smad proteins, inhibited the TSH-induced Foxe1 expression. Conclusions: This study shows that Foxe1 is the final target of TSH/cAMP and TGF-β regulation that mediates expression of thyroid differentiation genes, and provides evidence of an interplay between CRE-binding proteins, thyroid transcription factors, and Smad proteins in its regulation. Thus, Foxe1 plays an important role in the complex transcriptional network that regulates thyroid follicular cell differentiation.

Nanoparticle drug delivery has many advantages over small molecule therapeutics, including reducing off-target side effects and increasing drug potency. However, many nanoparticles are administered parenterally, which is challenging for chronic diseases such as polycystic kidney disease (PKD), the most common hereditary disease worldwide in which patients need continuous treatment over decades. To address this clinical need, we present the development of nanoparticles synthesized from chitosan, a widely available polymer chosen for its ability to improve oral bioavailability. Specifically, we optimized the synthesis parameters of chitosan nanoparticles and demonstrate mucoadhesion and permeation across an intestinal barrier model in vitro. Furthermore, when administered orally to mice, ex vivo imaging of rhodamine-loaded chitosan nanoparticles showed significantly higher accumulation in the intestines compared to the free model drug, as well as 1.3 times higher serum area under the curve (AUC), demonstrating controlled release and improved serum delivery over 24 h. To test its utility for chronic diseases such as PKD, we loaded the candidate PKD drug, metformin, into chitosan nanoparticles, and upon oral administration to a PKD murine model (Pkd1^fl/fl;Pax8-rtTA;Tet-O cre), a lower cyst burden was observed compared to free metformin, and was well tolerated upon repeated dosages. Blood urea nitrogen (BUN) and creatinine levels were similar to untreated mice, demonstrating kidney and biocompatibility health. Our study builds upon previous chitosan-based drug delivery approaches, and demonstrates a novel, oral nanoformulation for PKD.

Keywords: Chitosan; Metformin; Nanoparticle; Oral delivery; Polycystic kidney disease.

Water-clear cell adenoma (WCCA) of the parathyroid gland is an exceedingly rare neoplasm. To date, 17 cases have been reported in the literature, with only one of them being intrathyroidal. Here we report a case of a 34-year-old woman who presented for evaluation of a goiter and was found to have a thyroid nodule and abnormal thyroid function tests (TFT). Fine needle aspiration biopsy of the nodule revealed thyroid follicular cells without atypia and subsequent Afirma® Gene Expression Classifier (GEC) testing results were suspicious for malignancy. As a result, the patient underwent a right thyroid lobectomy and isthmusectomy. Histological sections revealed an intrathyroidal nodule consistent with a clear cell neoplasm of parathyroid origin. The histologic appearance together with the immune profile was diagnostic of WCCA, with diffuse positivity for GATA3, focal weak positivity for parathyroid hormone, and negativity for PAX8, thyroglobulin, TTF1, synaptophysin, chromogranin, and S100p. Our study focuses on the clinical presentation, current management strategies, and review of the available literature surrounding this rare diagnosis. The ultimate goal is to help endocrinologists and surgeons establish a foundational treatment plan for intrathyroidal clear cell tumor cases.

With the recent elucidation of gastric-type lesions in the female genital tract (especially in the cervix), occasional cases of endometrial adenocarcinoma displaying gastric (gastrointestinal) differentiation have been reported, but they are currently not recognized as a distinct pathologic entity. We report 9 cases of endometrial mucinous lesions which exhibit gastric (gastrointestinal)-type features by morphology and immunohistochemistry, including 4 adenocarcinomas and 5 benign mucinous lesions, in patients aged 32 to 85. The adenocarcinomas showed gastric-type morphology in all 4 cases and goblet cells in 1, with a component of benign gastric-type mucinous glands in 1 case. Immunohistochemically, the adenocarcinomas were positive for CK7 (4/4), CEA (4/4), MUC6 (3/3), PAX8 (3/4), CK20 (2/4), CDX2 (2/4), and estrogen receptor (1/4). They were negative for Napsin A (0/3), with mutation-type p53 staining in 2/4 cases, block-type p16 positivity in 1/4, and scattered chromogranin-positive cells in 1/2. Targeted next-generation sequencing revealed nonsense mutation in RB1 gene for the case with block-positive p16. Follow-up was available in all adenocarcinoma cases and indicated aggressive behavior; 2 patients were dead of disease at follow-up of 7 months to 3 years, 1 was alive with progression at 9 months, and 1 was alive without disease at 7 months. The benign mucinous lesions (including the benign component in 1 adenocarcinoma) exhibited gastric-type morphologic features in 5/6 cases, goblet cells in 5/6, and Paneth-like neuroendocrine cells in 1/6. These benign mucinous lesions were associated with an endometrial polyp in 5/6 cases. Cytologic atypia was present in 2/6 cases and a lobular architecture resembling cervical lobular endocervical glandular hyperplasia in 4/6. Immunohistochemically, the benign mucinous lesions were positive for CK7 (5/5), CDX2 (5/6), estrogen receptor (4/5), MUC6 (4/5), CK20 (3/5), PAX8 (3/5), and CEA (2/4), with scattered chromogranin-positive cells in 4/4 cases; in all cases tested Napsin A was negative, p53 was wild-type and p16 was negative. We propose the term "endometrial gastric (gastrointestinal)-type adenocarcinoma" for this distinctive group of rare aggressive endometrial carcinomas. We believe that benign or atypical gastric (gastrointestinal)-type mucinous lesions are putative precursors for these adenocarcinomas, comparable to recognized premalignant gastric-type lesions in the cervix and the vagina. Future recognition and reporting of these gastric-type endometrial mucinous lesions will help delineate their pathogenesis and clinical significance.

Background: Endometrial undifferentiated/dedifferentiated carcinoma (UDC/DDC) is a recently described aggressive variant of endometrial carcinoma, which shows mismatch repair (MMR) deficiency in about half of cases.

Aim: To assess whether MMR-deficient UDC/DDC have distinct clinico-pathological features.

Materials and methods: A systematic review and meta-analysis was performed by searching 4 electronic databases from their inception to October 2020 for all studies reporting clinicopathological characteristics of UDC/DDC series. Student t-test (for continuous variables), Cox regression analysis (for overall survival) and odds ratio (OR, for dichotomous variables) were used with a significant p-value < 0.05; data were pooled by using a random effect model.

Results: Twelve studies were included. MMR-deficiency was significantly associated with older age (p = 0.024), p53-wild-type (p = 0.005), ARID1A loss (p = 0.001) and PD-L1 expression (p = 0.019), but not with overall survival (p = 0.307), extension beyond corpus (p = 0.787) or beyond uterus (p = 0.403), presence of a differentiated component (p = 0.461), loss of expression of cytokeratins (p = 0.698), EMA (p = 0.309), estrogen receptor (p = 0.605), PAX8 (p = 0.959), SMARCA4/BRG1 (p = 0.321), SMARCB1/INI1 (p = 0.225) or claudin-4 (p = 0.094), or POLE exonuclease domain mutation p = (0.773).

Conclusions: In UDC/DDC, MMR-deficiency appears associated with older age, p53-wild type and ARID1A loss, suggesting the possibility of a distinct pathway underlying dedifferentiation; the association with PD-L1 expression is attributable to the high mutational load and may have therapeutic implications. On the other hand, MMR-deficiency appears not to be associated with prognosis, stage, loss of differentiation markers or POLE mutation.

Keywords: Immunohistochemistry; Microsatellite instability; Molecular; Risk assessment; Treatment.

Cutaneous clear cell hidradenoma is an uncommon benign adnexal tumor which is not supposed to metastasize, contrary to its rare malignant counterpart, hidradenocarcinoma. We report the case of a 49-year-old man, who had had a stable inguinal lymph node enlargement for 6 years. An excision was performed and revealed an intra-nodal tumor, made of large clear cells with abundant cytoplasm and round nuclei without atypia or mitosis. The immunohistochemical staining showed diffuse positivity for keratin AE1/AE3, keratin 5/6 and p63, and focal staining with keratin 7, epithelial membrane antigen (EMA) and carcinous epithelial antigen (CEA), which underlined some ductular structures. Tumor cells were negative for renal markers PAX8 and CD10. Ki67 stained less than 1% of tumor cells. A translocation involving MAML2 gene was evidenced by fluorescence in situ hybridization (FISH) analysis. No primary cutaneous tumor was found after extensive examination. Altogether, these results are in favor of an isolated nodal hidradenoma, for which we discuss two hypothesis: a primary nodal lesion, or a 'benign metastasis' of a cutaneous tumor. Cases of morphologically benign hidradenoma with lymph node involvement are exceptional. Our case, similar to every other reported case, was associated with an excellent prognosis, supporting the idea that these patients should not be overtreated.

Objective: To identify the genetic etiologies of congenital primary hypothyroidism (CH) in Thai patients.

Design and methods: CH patients were enrolled. Clinical characteristics including age, signs and symptoms of CH, pedigree, family history, screened thyroid-stimulating hormone results, thyroid function tests, thyroid imaging, clinical course and treatment of CH were collected. Clinical exome sequencing by next-generation sequencing was performed. In-house gene list which covered 62 potential candidate genes related to CH and thyroid disorders was developed for targeted sequencing. Sanger sequencing was performed to validate the candidate variants. Thyroid function tests were determined in the heterozygous parents who carried the same DUOX2 or DUOXA2 variants as their offsprings.

Results: There were 118 patients (63 males) included. Mean (SD) age at enrollment was 12.4 (7.9) years. Forty-five of 118 patients (38%) had disease-causing variants. Of 45 variants, 7 genes were involved (DUOX2, DUOXA2, TG, TPO, SLC5A5, PAX8 and TSHR). DUOX2, a gene causing thyroid dyshormonogenesis, was the most common defective gene (25/45, 56%). The most common DUOX2 variant found in this study was c.1588A>T. TG and TPO variants were less common. Fourteen novel variants were found. Thyroid function tests of most parents with heterozygous state of DUOX2 and DUOXA2 variants were normal.

Conclusions: DUOX2 variants were most common among Thai CH patients, while TG and TPO variants were less common. The c.1588A>T in DUOX2 gene was highly frequent in this population.

Keywords: DUOX2; congenital hypothyroidism; goiter; next generation sequencing; thyroid dysgenesis; thyroid dyshormonogenesis.

Anaplastic thyroid carcinoma (ATC) is an aggressive malignant tumor composed of undifferentiated thyroid follicular cells. Pathological diagnosis of ATC can be challenging as the tumor may show morphological overlap with other neoplasms with anaplastic morphology. Immunohistochemical demonstration of thyroid origin facilitates the diagnosis of ATC. Previous studies using the polyclonal anti-PAX8 antibody 10336-1-AP suggested that PAX8 was the most sensitive marker, expressed in up to 80% of ATC. According to a 2018 NordiQC report, the monoclonal anti-PAX8 antibody MRQ-50 has become the most commonly used anti-PAX8 antibody worldwide. However, validation of this antibody in ATC is lacking. In this study, we recruited 182 ATC cases from seven institutions. Pathology slides were subjected to histology review. PAX8 immunohistochemistry using the MRQ-50 antibody was performed in whole tissue slides (n = 147) or tissue microarray sections (n = 35). We found PAX8 expression in 54.4% of the cases, which was significantly lower than those reported in prior studies with the polyclonal antibody. PAX8 expression was positively correlated with the presence of an epithelial pattern (63.6% vs 37.5%, p = 0.0008) and a coexisting differentiated thyroid carcinoma component (71.6% vs 44.3%, p = 0.0004), but was not associated with age, gender, specimen type, or presence of giant cell and sarcomatoid patterns. In conclusion, we demonstrated PAX8 expression using the monoclonal antibody MRQ-50 in only half of the cases in a large ATC series. Pathologists should be aware that PAX8 expression in ATC is less than those reported in early studies to avoid misdiagnosis.

Pediatric mesotheliomas are rare and their pathogenesis remains undefined. In this study, we report 5 cases of malignant mesothelioma in children, characterized by fusions involving the anaplastic lymphoma kinase (ALK) gene. Four cases occurred in females involving the abdominal cavity and were characterized by a pure epithelioid morphology. The fifth arose in the tunica vaginalis of a 15-year-old male and displayed a biphasic epithelioid-sarcomatoid phenotype. All cases demonstrated the classic morphologic and immunohistochemical features of malignant mesothelioma, including tubulopapillary architecture and cuboidal epithelioid cells with eosinophilic cytoplasm and uniform nuclei with vesicular chromatin. Immunohistochemically, all cases showed labeling for ALK, cytokeratins, WT1, and calretinin, while lacking expression of adenocarcinoma immunomarkers. Four cases demonstrated weak-moderate labeling for PAX8 protein, which resulted in diagnostic challenges with primary peritoneal serous carcinoma. The ALK genetic abnormalities were investigated by a combination of molecular methods. Archer FusionPlex was performed in 2 cases, showing fusions between ALK with either STRN or TPM1 genes, resulting in a transcript that retained the ALK kinase domain. One case was further studied by DNA targeted sequencing, but no additional genetic alterations were observed. In 1 case, cytogenetic analysis showed the presence of a t(2;15)(p23;q22) and fluorescence in situ hybridization confirmed the ALK gene break-apart. In the remaining 2 cases, ALK gene rearrangements were demonstrated by fluorescence in situ hybridization. Unlike adult mesotheliomas, which are tightly linked to asbestos exposure, often show loss of BAP1 expression and have complex karyotypes, ALK-rearranged mesothelioma appears to be similar to other fusion-positive mesotheliomas, such as those harboring EWSR1/FUS-ATF1 fusions, sharing significant morphologic overlap, occurring in young patients and displaying a simple, translocation-driven genetic profile.