Eijun Nishihara

Background: Macro-thyrotropin (TSH) is a high molecular weight complex of TSH associated with apparently elevated levels of serum TSH, which can lead to an incorrect diagnosis of subclinical hypothyroidism. Given that levothyroxine (LT4) replacement is unnecessary in such cases, the accurate identification of macro-TSH based on clinical and laboratory findings is of particular importance. We aimed to determine the prevalence and clinical features of macro-TSH in patients with subclinical hypothyroidism evaluated after the introduction of TSH harmonization. Patients and Methods: Between August and November 2023, we included 1599 consecutive patients with subclinical hypothyroidism (TSH > 4.23 mIU/L with normal fT4 levels) who were either untreated or receiving LT4 replacement monotherapy. Polyethylene glycol (PEG) precipitation was performed in all cases. Patients with TSH recovery of 150 kDa) under neutral conditions that disappeared under acidic conditions, with a shift to the native molecular weight (approximately 28 kDa), indicating the initial presence of macro-TSH. The TSH complex was detected in peaks corresponding to IgG, IgA, and IgM. These findings were verified using Protein G and Jacalin precipitation assays, with no evidence of assay interference. Reevaluation of TSH recovery following PEG precipitation showed substantial variation in two patients who had abundant monomeric TSH compared with the high-molecular complex. All four patients with macro-TSH had previously received LT4 therapy, which resulted in a modest reduction in TSH levels, with three of these patients testing positive for TgAb and/or TPOAb. Conclusions: Macro-TSH complexes comprise several immunoglobulin isotypes. In cases with low antibody-TSH binding affinity and positive antithyroid autoantibodies, TSH levels may fluctuate during the clinical course, thereby complicating diagnosis and management.

Objective: Electrochemiluminescence immunoassays (ECLIAs) are widely used for thyroid function testing but may be affected by rare assay interferences that can lead to misdiagnosis. We aimed to investigate the frequency and types of assay interferences in thyrotropin (TSH), free thyroxine (FT4), and free triiodothyronine (FT3) measurements using the Roche ECLIA. Methods: Between 2019 and 2023, 124,615 patients underwent thyroid function testing. Assay interference was suspected in 283 cases based on clinical–laboratory mismatch, thyroid hormone imbalance, or discrepancies between assay methods. Further validation assessed potential interferences. Results: Of the total tests (TSH: 124,609; FT4: 116,012; and FT3: 83,044), 71 interference cases were confirmed (TSH: 11 (0.009%), FT4: 6 (0.005%), and FT3: 53 (0.064%)). FT3 interferences were mainly due to idiotype and anti-streptavidin antibodies. Macro-TSH was detected in six cases and often led to unnecessary levothyroxine treatment. Furthermore, most interferences were detected by recognizing hormonal imbalances rather than clinical symptoms. Conclusion: FT3 tests are most prone to assay interference. Awareness of inter ference patterns and close collaboration between physicians and laboratory staff are essential to prevent diagnostic errors.

In adults, cases of dyshormonogenetic goiter (DG) that go undiagnosed during childhood may necessitate thyroidectomy due to unexplained thyroid enlargement, often presenting diagnostic challenges. This study aimed to characterize the histopathological features of the thyroid in adult DG cases, and to evaluate the feasibility of predicting subtypes from the findings. Fifty adult patients with DG confirmed by the presence of gene variants were included. All resected thyroids showed diffuse goiter with microscopically altered thyroid follicles throughout the gland. All patients with SLC5A5 abnormality (iodide transport defect) showed lobulation accentuated by interlobular fibrosis. Follicular cells were enlarged, except in patients with SLC26A4 abnormality (Pendred syndrome). A scant colloid was observed in all patients with thyroglobulin (TG) gene abnormality. Thyroid peroxidase (TPO) gene abnormality and SLC26A4 abnormality were characterized by large follicles with Sanderson’s polsters. Multiple non-encapsulated nodular lesions were present except in two patients with SLC26A4 abnormality. These were mainly follicular thyroid adenoma-like nodules associated with TG gene abnormality, TPO gene abnormality, and SLC5A5 variant. With SLC26A4 abnormality, the nodules were follicular nodular disease-like. The proposed histological framework may be particularly valuable for adult patients undergoing thyroidectomy for unexplained diffuse enlargement or multinodularity, particularly when no prior diagnosis of DG is documented.

Background: Although antithyroid drugs (ATDs) are widely used as the first-line therapy for Graves’ disease, relapse after treatment discontinuation remains common. While a prolonged low-dose ATD therapy has been associated with improved remission rates, the impact of the minimal maintenance dose before discontinuation on relapse risk remains unclear. Method: We conducted a retrospective cohort study using electronic medical records from a thyroid specialty hospital in Japan. Patients newly diagnosed with Graves’ disease between 2008 and 2024 who had discontinued methimazole (MMI) after receiving a minimal maintenance dose (≤2.5 mg/day) were included. Patients were categorized into 4 groups based on their final maintenance dose before discontinuation: 2.5 mg/day, >1.25 to ≤2.5 mg/day, 1.25 mg/day, and <1.25 mg/day. We evaluated the association between the minimal MMI dose before discontinuation and the 1-year risk of relapse using multivariable regression and propensity score–matched analyses. Results: Among 4352 eligible patients, multivariable regression showed that, compared with the 2.5 mg/day group, the 1.25 mg/day group had a significantly lower risk of relapse within 1 year [risk ratio (RR) 0.46, 95% confidence interval (CI) 0.28-0.75], and the <1.25 mg/day group had the lowest risk (RR: 0.18, 95% CI: 0.05-0.73). The propensity score-matched analysis, consistent with the multivariable regression, showed that the 1.25 mg/day group had a lower risk of relapse compared with the 2.5 mg/day group (RR: 0.44, 95% CI: 0.23-0.85; 172 matched pairs). Conclusion: Lower minimum maintenance doses of MMI before discontinuation, particularly doses <2.5 mg/day, may be associated with a reduced risk of relapse in patients with Graves’ disease. Clinicians should recognize the clinical relevance of the minimum maintenance dose in the treatment of Graves’ disease.

High-mobility group AT-hook 2 (HMGA2) is a nuclear protein involved in the differentiation and proliferation of epithelial-derived tumors and also considered to be involved in the growth and differentiation of various malignant tumors, including thyroid cancer. Immunohistochemistry (IHC) for HMGA2 has been reported to show diffuse positivity in several follicular thyroid carcinoma (FTC) cases. This study aimed to investigate whether positive immunohistochemical staining for HMGA2 in primary tumors can be used to predict the prognosis and detect prognostic factors in malignant thyroid tumors associated with metastatic recurrence in FTC. Formalin-fixed, paraffin-embedded (FFPE) resected specimens used for the IHC for HMGA2. The association of positive HMGA2 staining with metastasis and recurrence, along with the potential of HMGA2 as a prognostic marker of metastatic recurrence, was statistically determined. HMGA2 staining was positive in most malignant tissues, whereas benign tissues were unstained. HMGA2 staining of the marginal and invasive regions was observed in FTC tissues. The association of HMGA2 staining with metastasis and recurrence was significant (p = 0.018). Kaplan-Meier curves showed an association of negative HMGA2 staining with metastasis and disease-free survival (p = 0.090). Tumor size (>4 cm) and wide invasion were also significant factors (p = 0.043, p < 0.001). The risk ratio without HMGA2 was significantly reduced by 30% compared to that with HMGA2. In primary tumors, positive HMGA2 staining can be used to predict prognosis in malignant thyroid tumors associated with metastatic recurrence in FTC and negative HMGA2 staining may indicate longer disease-free survival after surgery.

Context: Kelch-like ECH-associated protein 1 (KEAP1) is associated with nuclear factor erythroid-2–related factor 2 (NRF2) and promotes NRF2 degradation in normal conditions. Genetic abnormality in KEAP1 is a rare disease and presents with familial multinodular goiter. Objective: This study assessed the clinical and molecular findings concerning nodular formation in the thyroid gland of patients harboring KEAP1 germline mutations. Methods: Next-generation sequencing analysis targeting goiter-associated genes was performed on 39 patients with familial multinodular goiter. The expression of NRF2-targeted genes from surgical thyroid specimens of patients with KEAP1 mutations were analyzed using a whole-transcript expression array and immunohistochemistry. Results: We found 5 probands with pathogenic heterozygous mutations in KEAP1 (p.Q86*, p.L136P, p.V411fs, p.R415C, and p.R483H) that had no meaningful concomitance with mutations of other goiter-associated genes at germline and somatic levels. Their common histopathological features showed multinodular goiters in the entire thyroid gland with few degenerative lesions or complications of malignancy and slow proliferation indicating less than 1% at the Ki-67 labeling index. Among 42 NRF2-targeted genes, antioxidant genes were most frequently upregulated (11/12) in the nodule, followed by detoxification genes (6/11). Immunohistochemical analysis showed relatively high expression of glutathione peroxidase 2 and NAD(P)H quinone oxidoreductase 1 (representative NRF2-targeted genes) in the nodules of various patients harboring KEAP1 mutations. Conclusion: KEAP1 germline heterozygous mutations exert excessive NRF2 activity in the thyroid gland and may confer cytoprotective effects even under abundant reactive oxygen species associated with thyroid hormone production, resulting in thyroid hyperplasia with scarce degradation.

Familial nonautoimmune hyperthyroidism (NAH) is a rare type of autosomal dominant hyperthyroidism caused by constitutively active pathogenic variants of the thyrotropin receptor (TSHR) gene. Although affected family members present with varied levels of hyperthyroid features, even when the same pathogenic variant is present, total thyroidectomy followed by radioiodine therapy is recommended for long-term management. Herein, we present the case of an 18-year-old proband and her family members with NAH (TSHR-I640V), who presented with diverse thyroid dysfunctions: fluctuations between euthyroid and subclinical hyperthyroidism, mild hyperthyroidism, and overt hyperthyroidism. Almost all affected adult family members, except the proband, showed no progression of hyperthyroidism or thyroid enlargement. A family history of thyrotropin receptor antibodies (TRAb)-negative hyperthyroidism is important for the identification of NAH in adults before TSHR genetic testing can be performed. Ablative therapy is not necessary when familial NAH presents with late-onset mild hyperthyroidism without coexisting diseases.

Insufficient thyroid hormone production in newborns is referred to as congenital hypothyroidism. Multinodular goiter (MNG), characterized by an enlarged thyroid gland with multiple nodules, is usually seen in adults and is recognized as a separate disorder from congenital hypothyroidism. Here we performed a linkage analysis of a family with both nongoitrous congenital hypothyroidism and MNG and identified a signal at 15q26.1. Follow-up analyses with whole-genome sequencing and genetic screening in congenital hypothyroidism and MNG cohorts showed that changes in a noncoding TTTG microsatellite on 15q26.1 were frequently observed in congenital hypothyroidism (137 in 989) and MNG (3 in 33) compared with controls (3 in 38,722). Characterization of the noncoding variants with epigenomic data and in vitro experiments suggested that the microsatellite is located in a thyroid-specific transcriptional repressor, and its activity is disrupted by the variants. Collectively, we presented genetic evidence linking nongoitrous congenital hypothyroidism and MNG, providing unique insights into thyroid abnormalities.

Context Telomerase reverse transcriptase promoter (TERT-p) mutations, which upregulate TERT expression, are strongly associated with tumor aggressiveness and worse prognosis in papillary thyroid carcinomas (PTCs). TERT expression is also observed in a proportion of PTCs without TERT-p mutations, but such tumors show less aggressiveness and better prognosis compared with TERT-p mutation-positive tumors. Objective TERT has multiple splicing variants whose relationships with the TERT-p status and clinicopathological characteristics remain poorly understood. We examined the relationship between the TERT-p mutational status, the TERT splicing pattern, and clinicopathological features. Methods We investigated the expression of two major variants, α deletion (dA) and β deletion (dB), in a series of 207 PTCs operated between November 2001 and March 2020 in Nagasaki University Hospital and Kuma Hospital. Results The TERT-p mutations were found in 33 cases, and among 174 mutation-negative cases, 24 showed TERT expression. All cases were classified into three groups: the TERT-p mutation-negative/expression-negative group (mut-/exp-), the TERT-p mutation-negative/expression-positive group (mut-/exp+), and the TERT-p mutation-positive group (mut+/exp+). The +A + B/dB ratio in mut+/exp + was significantly higher than that in mut-/exp + PTCs. Analysis with clinicopathological data revealed that +A + B expression was associated with higher PTC aggressiveness, whereas dB expression counteracted this effect. Functional in vitro study demonstrated that dB strongly inhibited cell growth, migration, and clonogenicity, suggesting its tumor suppressive role. Conclusion These results provide evidence that the TERT-p mutations alter the expression of different TERT splice variants, which, in turn, associates with different tumor aggressiveness.

Subclinical hyperthyroidism (SHyper) is defined as normal levels of free thyroxine (fT4) and free triiodothyronine (fT3) with suppressed levels of TSH. Previous studies have reported the individual pathophysiology of endogenous SHyper patients and athyreotic patients receiving TSH suppression therapy with levothyroxine; however, apparently no studies have compared the two conditions. Five-hundred-forty untreated endogenous SHyper patients and 1,024 patients receiving TSH suppression therapy who underwent total thyroidectomy for papillary thyroid carcinoma were sampled. Thyroid hormone profiles and peripheral indices related to thyrotoxicosis were investigated in endogenous SHyper patients, athyreotic patients receiving TSH suppression therapy, and healthy participants. Endogenous SHyper patients showed significantly higher thyroid hormone levels (fT4 [p < 0.001] and fT3 [p < 0.001]), and peripheral indices showed a significant tendency towards thyrotoxicosis (strong TSH suppression: alkaline phosphatase [ALP, p < 0.001], creatinine [Cre, p < 0.001], pulse rate [p < 0.05]; and mild TSH suppression: Cre [p < 0.05]) than healthy participants. In contrast, athyreotic patients receiving TSH suppression therapy showed a significant tendency towards thyrotoxicosis than healthy participants only when TSH was strongly suppressed (fT3 [p < 0.001] and Cre [p < 0.001]). Endogenous SHyper patients showed significantly higher fT3 levels (p < 0.001) than athyreotic patients receiving TSH suppression therapy; however, there was a significant tendency towards thyrotoxicosis only when TSH was strongly suppressed (ALP [p < 0.05] and pulse rate [p < 0.05]). The effects of endogenous SHyper and TSH suppression therapy on target organ function are different. Although the serum thyroid hormone profile is similar to that of the thyrotoxic state, athyreotic patients receiving TSH suppression therapy with mildly suppressed serum TSH levels are not thyrotoxic.