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Background: Familial nontoxic multinodular goiter (MNG) is a rare disease. One of the associated genes is Kelch-like ECH-associated protein 1 (KEAP1), which encodes the main inhibitor of nuclear factor erythroid 2-related transcription factor 2 (Nrf2), a central mediator of antioxidant responses. The association of KEAP1 with familial MNG is based on only two loss-of-function mutations identified in two families, only one of which included proper phenotyping and adequate demonstration of co-segregation of the phenotype and the mutation. There is no experimental evidence from model organisms to support that decreased Keap1 levels can, indeed, cause goiter. This study used mice hypomorphic for Keap1 to test whether decreased Keap1 expression can cause goiter, and to characterize the activation status of Nrf2 in their thyroid. Methods: C57BL/6J Keap1 flox/flox (Keap1 knock-down [Keap1 KD ]) mice were studied at 3 and 12 months of age. Plasma and thyroid glands were harvested for evaluation of thyroid function tests and for gene and protein expression by real-time polymerase chain reaction and immunoblotting, respectively. Results: Keap1 KD mice showed diffuse goiter that began to develop in early adult life and became highly prominent and penetrant with age. The goiter was characterized by a markedly increased size of thyroid follicles, most notably of the colloid compartment, and by absence of thyroid nodules or hyperplasia. Keap1 KD mice also showed decreased T4 levels in early adult life that were eventually well compensated over time by increased thyrotropin (TSH) levels. Nrf2 was activated in the thyroid of Keap1 KD mice. Despite a known stimulatory effect of Nrf2 on thyroglobulin (Tg) gene transcription and Tg protein abundance, the expression levels were decreased in the thyroid of Keap1 KD mice. No clear patterns were observed in the expression profiles of other thyroid hormone synthesis-specific factors, with the exception of Tg-processing and Tg-degrading cathepsins, including an increase in mature forms of cathepsins D, L, and S. Conclusions: Keap1 KD mice develop age-dependent diffuse goiter with elevated TSH levels. The precise mechanism accounting for the thyroidal phenotype remains to be elucidated, but it may involve enhanced Tg solubilization and excessive lysosomal Tg degradation.

Background: Several single-nucleotide polymorphisms (SNPs) are known to increase the risk of Hashimoto's thyroiditis (HT); such SNPs reside in thyroid-specific genes or in genes related to autoimmunity, inflammation, and/or cellular defense to stress. The transcription factor Nrf2, encoded by NFE2L2, is a master regulator of the cellular antioxidant response. This study aimed to evaluate the impact of genetic variation in NFE2L2 on the risk of developing HT. Methods: In a case-control candidate gene association study, functional SNPs in the NFE2L2 promoter (rs35652124, rs6706649, and rs6721961) were examined either as independent risk factors or in combination with a previously characterized HT risk allele (rs28665122) in the gene SELENOS, encoding selenoprotein S (SelS). A total of 997 individuals from the north of Portugal (Porto) were enrolled, comprising 481 HT patients and 516 unrelated healthy controls. SELENOS and NFE2L2 SNPs were genotyped using TaqMan ® assays and Sanger sequencing, respectively. Odds ratios (ORs) were calculated using logistic regression, with adjustment for sex and age. Expression of SelS was analyzed by immunohistochemistry in thyroid tissue from HT patients and control subjects. Molecular interactions between the Nrf2 and SelS pathways were investigated in thyroid tissues from mice and in rat PCCL3 thyroid follicular cells. Results: When all three NFE2L2 SNPs were considered together, the presence of one or more minor alleles was associated with a near-significant increased risk (OR = 1.43, p = 0.072). Among subjects harboring only major NFE2L2 alleles, there was no increased HT risk associated with heterozygosity or homozygosity for the SELENOS minor allele. Conversely, in subjects heterozygous or homozygous for the SELENOS risk allele, the presence of an NFE2L2 minor allele significantly increased HT risk by 2.8-fold (p = 0.003). Immunohistochemistry showed reduced expression of SelS in thyroid follicular cells of HT patients. In Nrf2 knockout mice, there was reduced expression of SelS in thyroid follicular cells; conversely, in PCCL3 cells, reducing SelS expression caused reduced activity of Nrf2 signaling. Conclusions: The NFE2L2 promoter genotype interacts with the SELENOS promoter genotype to modulate the risk of HT in a Portuguese population. This interaction may be due to a bidirectional positive feedback between the Nrf2 and SelS pathways.

The recent update of The Bethesda System for Reporting Thyroid Cytology (TBSRTC) is a very important development in the evaluation of thyroid nodules. Clinical experience and scientific literature both show that practitioners performing thyroid fine-needle aspiration are accustomed to basing the clinical management of patients on reports using TBSRTC. Specifically, clinicians are familiar with the percent risk of malignancy corresponding to each TBSRTC diagnostic category (DC), as well as with the respective recommendation for clinical management. However, most clinicians are much less familiar with the specific considerations that lie between a given DC, on the one end, and the respective risk of malignancy and associated management recommendation, on the other end.A deeper understanding of the system can enlighten the clinician's thinking about the specific nodule under examination and can guide the decision-making process in a more meaningful way. Such an understanding can only be developed via close two-way communication between cytopathologists and clinicians. Through this type of interaction in the authors' tertiary medical center, recurring issues of particular importance for clinical practice were identified, which are reported here in the form of 16 frequently asked questions posed by the clinician to the cytopathologist.For each frequently asked question, an answer is provided based on the literature, the authors' experience, the new version of TBSRTC, and the new World Health Organization classification of tumors of endocrine organs.

Carcinoma showing thymus-like elements (CASTLE) is a rare malignant neoplasm of the thyroid gland, morphologically and immunohistologically similar to a thymic carcinoma, whose histogenesis is still debated. Hypotheses include an origin from ectopic thymic tissue, vestige of the thymopharyngeal duct, or branchial pouch remnants from which solid cell nests (SC-nests) originate. The diagnosis of CASTLE may be treacherous due to its rarity and its propensity to mimic other poorly differentiated tumors such as squamous cell carcinoma.We present a case of CASTLE in a 58-year-old man initially diagnosed as a poorly differentiated squamous cell carcinoma both on fine-needle aspiration cytology (FNAC) and on biopsy, arising in close association with SC-nests. A thorough literature review, with special emphasis on its diagnosis and histogenesis of CASTLE, was also conducted.Magnetic resonance images revealed a 4.0-cm cervical mass on the left side of the trachea, involving the lateral middle/inferior portion of the left lobe of the thyroid gland. FNAC was performed with a diagnosis of "malignant cells, consistent with squamous cell carcinoma." A histological evaluation of the resected specimen revealed a malignant proliferation of cells, focally exhibiting a squamoid appearance, which were immunopositive for CD5 and p63. A diagnosis of CASTLE was made. The tumor was located in direct continuity with SC-Nests, and the cell morphology of both the SC-nests and CASTLE was very similar with merging. Moreover, the immunohistochemical expression profiles of most markers useful in the diagnosis of CASTLE were identical in the SC-nests.The inclusion of CASTLE in the differential diagnosis of poorly differentiated tumors of the thyroid region and the use of ancillary studies are essential to diagnose this rare entity associated with a relatively favorable prognosis. The close association of CASTLE with SC-nests opens the way to a new scenario for studies of its histogenesis.

Phosphatase and tensin homolog deleted on chromosome ten (PTEN)-hamartoma tumor syndrome (PHTS) is a complex disorder caused by germline inactivating mutations of the PTEN tumor suppressor gene. PHTS includes Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome (BRRS), and Proteus-like syndromes. Affected individuals develop both benign and malignant tumors in a variety of tissues, including the thyroid. This study is to better characterize and describe the thyroid pathology within the different entities of this syndrome, and examine whether there is an association between specific thyroid findings and different PTEN mutations.Twenty patients with known PTEN mutations, and/or clinical diagnosis of PHTS, and thyroid pathology were identified: 14 with CS and 6 with BRRS.Thyroid pathology findings were as follows: multiple adenomatous nodules in a background of lymphocytic thyroiditis (LT) in 75%, papillary carcinoma in 60%, LT alone in 55%, follicular carcinoma in 45%, C-cell hyperplasia in 55%, and follicular adenomas in 25%. Within the papillary carcinoma group, there were 6 microcarcinomas, 5 follicular variants, and 1 classical type.There were no morphologic differences between the thyroid findings in CS and BRRS. Also, there was no correlation between specific PTEN germline mutations (exons 5, 6, and 8) and pathologic findings. Distinctive and characteristic findings in PHTS include multiple unique adenomatous nodules in a background of LT, and C-cell hyperplasia; it is vital that pathologists recognize the classical histologic features of this syndrome to alert clinicians to the possibility of this syndrome in their patients.

Riedel's thyroiditis (RT) is a very rare chronic fibrosing disorder of unknown etiology that is often associated with multifocal fibrosclerosis (MFS). Immunoglobulin (Ig) G4-related sclerosing disease (IgG4-RSD), a new clinico-pathological entity also associated with MFS, is characterized by IgG4+ plasma cell infiltration and fibrosis in one or more organs. Although the association of RT and IgG4-RSD has been suggested, it has seldom been studied or reported. We report a classical case of RT with serological (IgG4 levels) and immunohistochemical (IgG and IgG4) assessment, in search of an underlying IgG4-RSD.The patient was a 57-year-old woman who underwent a subtotal thyroidectomy for a long-standing goiter with a rapidly enlarging isthmic nodule.Histopathological examination of the surgical specimen revealed all of the morphological features of RT and IgG4-RSD, including partial fibrosis of the thyroid gland with destruction of the thyroid follicular architecture; obliterative phlebitis; and a mixed infiltrate composed of lymphocytes, eosinophils, and plasma cells. The fibro-inflammatory process extended beyond the thyroid capsule into the surrounding tissues. Immunohistochemical examination revealed approximately 70 IgG4+ plasma cells per high power field (HPF) with an IgG4/IgG ratio of 35%. Although serum levels of IgG4 were normal (20 mg/dL), total IgG levels were slightly elevated (1370 mg/dL). There was no evidence of involvement of other organs at the time of RT diagnosis.The morphological similarities between RT and IgG4-RSD suggest that these entities are closely related. Therefore, RT with increased IgG4+ plasma cells, with or without elevated IgG4 serum levels, may represent the first clinical manifestation of an underlying IgG4-RSD. However, due to the rarity of both conditions and the limited specificity and sensitivity of both IgG4 serum levels and IgG/IgG4 immunohistochemistry in the diagnosis of IgG4-RSD, further studies are needed to verify this hypothesis.