Molecular Diagnostics of Fine Needle Aspiration for the Presurgical Screening of Thyroid Nodules

“The incidence of thyroid cancer, the most common endocrine malignancy, is rising. The two most common types of thyroid cancer are papillary and follicular” thyroid carcinomas. “Fine-needle aspiration (FNA) of thyroid nodules” can permit to detect many genetic mutations and other molecular alterations, including RAS and BRAF point mutations, PAX8/peroxisome proliferator-activated receptor (PPAR)γ and “RET/PTC rearrangements, occurring in thyroid papillary and follicular carcinomas” (more than 70% of cases), which can be used successfully to improve the diagnosis “and the management of patients with thyroid nodules”. The most extensive experience has been accumulated with “the diagnostic use of BRAF mutation”, which is highly specific for malignancy. “Testing FNA samples for a panel of mutations” that typically includes RAS, BRAF, PAX8/PPARγ and RET/PTC could permit to achieve the biggest diagnostic impact. “The accuracy of cancer diagnosis in thyroid nodules could be improved significantly using these and other emerging molecular markers”.     

Alterations in Hypothalamus-Pituitary-Adrenal/Thyroid Axes and Gonadotropin-Releasing Hormone in the Patients with Primary Insomnia: A Clinical Research

The hypothalamus-pituitary-target gland axis is thought to be linked with insomnia, yet there has been a lack of further systematic studies to prove this. This study included 30 patients with primary insomnia (PI), 30 patients with depression-comorbid insomnia (DCI), and 30 healthy controls for exploring the alterations in the hypothalamus-pituitary-adrenal/thyroid axes’ hormones and gonadotropin-releasing hormone (GnRH). The Pittsburgh Sleep Quality Index was used to evaluate sleep quality in all subjects. The serum concentrations of corticotrophin-releasing hormone (CRH), thyrotrophin-releasing hormone (TRH), GnRH, adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), cortisol, total triiodothyronine (TT3), and total thyroxine (TT4) in the morning (between 0730 h and 0800 h) were detected. Compared to the controls, all hormonal levels were elevated in the insomniacs, except ACTH and TSH in the PI group. Compared to the DCI patients, the PI patients had higher levels of CRH, cortisol, TT3, and TT4 but lower levels of TRH, GnRH, and ACTH. Spearman’s correlation analysis indicated that CRH, TRH, GnRH, TSH, cortisol, TT4, and TT3 were positively correlated with the severity of insomnia. The linear regression analysis showed that only CRH, GnRH, cortisol, and TT3 were affected by the PSQI scores among all subjects, and only CRH was included in the regression model by the “stepwise” method in the insomnia patients. Our results indicated that PI patients may have over-activity of the hypothalamus-pituitary-adrenal/thyroid axes and an elevated level of GnRH in the morning.     

Thyroid Nodules and Thyroid Cancer

A review of clinical and laboratory features of thyroid cancer, designed to help in a more precise selection of patients for operation, showed that factors contributing to a high index of suspicion of cancer include previous exposure to low doses of radiation, the presence of a firm, solitary thyroid nodule clearly different from the rest of the gland, a young patient, nodules that are “cold” on scan with radioiodine, and nodules that fail to regress after an adequate trial of thyroxine therapy. Factors contributing to a low index of suspicion of thyroid cancer include soft or cystic lesions, multinodular goiters, nodules that are “hot” on ¹³¹ I scan, and those that regress during thyroxine treatment.When these factors are used to select patients for surgical operation, about 30 percent are found to have thyroid cancer.Until more precise methods for preoperative diagnosis are established, it is suggested that this type of clinical selection may be very helpful in the management of patients with thyroid nodules or nontoxic goiter.     

SCINTIGRAMS OF THE THYROID GLAND—The Diagnosis of Morphologic Abnormalities with I131

Functioning thyroid tissue containing sufficient radioiodine can be visualized by scanning the gland with a directional scintillation counter.⁴ This visual representation of the gland is called a “scintigram.” Scintigrams have been invaluable in the detection and study of both “toxic” and non-functioning nodules, diffuse enlargement in hyperthyroidism and the subsequent reduction in gland size after treatment, carcinoma, and aberrant thyroid tissue.     

The Normal Thyroidal Uptake of Iodine

The range of values for the 24-hour thyroidal accumulation of radioactive iodine in euthyroid persons varies with geographic location. In the San Bernardino Valley region of Southern California the “normal range” is 6 percent to 33 percent in euthyroid subjects. This is lower than in studies from other areas of the United States. The urinary iodide excretion and the absolute iodine uptake of the thyroid are higher than in studies from many other areas of the United States, pointing to iodine abundance as the reason for this difference. The geographic variation and the possibility of changing dietary iodine intake of normal persons point to the necessity of current and local determinations of the “normal range” of the thyroidal uptake of radioiodine if the results of this thyroid function test are to be properly interpreted.     

The Effect of a Mutation in the Thyroid Stimulating Hormone Receptor (TSHR) on Development,Behaviour and TH Levels in Domesticated Chickens

The thyroid stimulating hormone receptor (TSHR) has been suggested to be a “domestication locus” in the chicken, due to a strong selective sweep over the gene found in domesticated chickens, differentiating them from their wild ancestor the Red Junglefowl (RJF). We investigated the effect of the mutation on development (incubation time), behaviour and thyroid hormone levels in intercross chickens homozygous for the mutation (d/d), wild type homozygotes (w/w) or heterozygotes (d/w). This allowed an assessment of the effect of genotype at this locus against a random mix of RJF and WL genotypes throughout the rest of the genome, controlling for family effects. The d/d genotype showed a longer incubation time, less fearful behaviours, lower number of aggressive behaviours and decreased levels of the thyroid hormone T4, in comparison to the w/w genotype. The difference between TSHR genotypes (d/d vs. w/w) in these respects mirrors the differences in development and behaviour between pure domesticated White Leghorns and pure RJF chickens. Higher individual T3 and T4 levels were associated with more aggression. Our study indicates that the TSHR mutation affects typical domestication traits, possibly through modifying plasma levels of thyroid hormones, and may therefore have been important during the evolution of the domestic chicken.     

Q-Elastography in the Presurgical Diagnosis of Thyroid Nodules with Indeterminate Cytology

Quantitative ultrasound (US) elastography (Q-USE), able to evaluate tissue stiffness has been indicated as a new diagnostic tool to differentiate benign from malignant thyroid lesions. Aim of this prospective study, conducted at the Department of Surgical Sciences, of the “Sapienza” University of Rome, was to evaluate the diagnostic accuracy of Q-USE, compared with US parameters, in thyroid nodules with indeterminate cytology (Thy3).The case study included 140 nodules from 140 consecutive patients. Patient’s thyroid nodules were evaluated by Q-USE, measuring the strain ratio (SR) of stiffness between nodular and surrounding normal thyroid tissue, and conventional US parameters prior fine-needle aspiration cytology. Those with Thy3 diagnosis were included in the study. Forty of the nodules analyzed harbored a malignant lesion. Q-USE demonstrated that malignant nodules have a significant higher stiffness with respect to benign one and an optimun SR cut-off value of 2.05 was individuated following ROC analysis. Univariate analysis showed that hypoechogenicity, irregular margins and SR >2.05 associated with malignancy, with an accuracy of 67.2%, 81,0% and 89.8%, respectively. Data were unaffected by nodule size or thyroiditis. These findings were confirmed in multivariate analysis demonstrating a significant association of the SR and the irregular margins with thyroid nodule’s malignancy. In conclusion, we demonstrated the diagnostic utility of Q-USE in the differential diagnosis of thyroid nodules with indeterminate cytology that, if confirmed, could be of major clinical utility in patients’ presurgical selection.     

Modelling of Thyroid Peroxidase Reveals Insights into Its Enzyme Function and Autoantigenicity

Thyroid peroxidase (TPO) catalyses the biosynthesis of thyroid hormones and is a major autoantigen in Hashimoto’s disease—the most common organ-specific autoimmune disease. Epitope mapping studies have shown that the autoimmune response to TPO is directed mainly at two surface regions on the molecule: immunodominant regions A and B (IDR-A, and IDR-B). TPO has been a major target for structural studies for over 20 years; however, to date, the structure of TPO remains to be determined. We have used a molecular modelling approach to investigate plausible modes of TPO structure and dimer organisation. Sequence features of the C-terminus are consistent with a coiled-coil dimerization motif that most likely anchors the TPO dimer in the apical membrane of thyroid follicular cells. Two contrasting models of TPO were produced, differing in the orientation and exposure of their active sites relative to the membrane. Both models are equally plausible based upon the known enzymatic function of TPO. The “trans” model places IDR-B on the membrane-facing side of the myeloperoxidase (MPO)-like domain, potentially hindering access of autoantibodies, necessitating considerable conformational change, and perhaps even dissociation of the dimer into monomers. IDR-A spans MPO- and CCP-like domains and is relatively fragmented compared to IDR-B, therefore most likely requiring domain rearrangements in order to coalesce into one compact epitope. Less epitope fragmentation and higher solvent accessibility of the “cis” model favours it slightly over the “trans” model. Here, IDR-B clusters towards the surface of the MPO-like domain facing the thyroid follicular lumen preventing steric hindrance of autoantibodies. However, conformational rearrangements may still be necessary to allow full engagement with autoantibodies, with IDR-B on both models being close to the dimer interface. Taken together, the modelling highlights the need to consider the oligomeric state of TPO, its conformational properties, and its proximity to the membrane, when interpreting epitope-mapping data.     

The “next-generation” knowledge of papillary thyroid carcinoma

The application of Next-Generation Sequencing for studying the genetics of papillary thyroid carcinomas (PTC) has recently revealed new somatic mutations and gene fusions as potential new tumor-initiating events in patients without any known driver lesion. Gene and miRNA expression analyses defined clinically relevant subclasses correlated to tumor progression. In addition, it has been shown that tumor driver mutations in BRAF, and RET rearrangements - altogether termed “BRAF-like” carcinomas - have a very similar expression pattern and constitute a distinct category. Conversely, “RAS-like” carcinomas have a different genomic, epigenomic, and proteomic profile. These findings justify the need to reconsider PTC classification schemes.     

Light- and electron microscopic investigations on the median eminence of the pigeon after TSH and PTU treatment

Summary 1. The effects of TSH and PTU treatment with thyroid stimulating hormone and propylthiouracil on the structure of median eminence of the pigeon have been studied by light- and electron microscopy.2. By light microscopy it has been found that the aldehydefuchsin positive material of the external layers of the anterior median eminence differs functionally from that of the neural lobe.3. The light- and electron microscopic investigations revealed that the anterior and posterior median eminence differ both structurally and functionally. Specifically after the experimental interventions the considerable reaction can be observed only in the anterior median eminence.4. Electron microscopically it appears possible that the aldehyde-fuchsin positive material of the anterior median eminence may be associated with the 1200–1500 Å—size granules.5. The quantitative changes observed in the granular composition after the treatments show a close correlation with thyroid function, namely after treatment with thyroid stimulating hormone the relative quantity of the 1200–1500 Å—size granules increases markedly, while administration of propylthiouracil has the opposite effect and increases the relative quantity of the 1000 Å—size dense-core vesicles. They presumably may play a role in the control of TSH-secretion.Abbreviations TSH thyroide stimulating hormone - PTU propylthiouracil - ME median eminence - AME anterior median eminence - PME posterior median eminence - AF+ aldehyde-fuchsin positive - AF– aldehyde-fuchsin negative     

Menopause Analytical Hormonal Correlate Outcome Study (MAHCOS) and the Association to Brain Electrophysiology (P300) in a Clinical Setting

Various studies have demonstrated that increased leptin levels and obesity are inversely related to cognitive decline in menopausal women. It is hypothesized that adiposity is inversely correlated with cognitive decline, as women with increased weight are less vulnerable to diminishing cognition. However, it is increasingly observed that menopausal women, even with increased adiposity, experience significant cognitive decline. Positron emission tomography (PET) has been used to analyze cognitive function and processing in menopausal women. Evoked potentials (P300) and neurophysiologic tests have validated brain metabolism in cognitively impaired patients. Post-hoc analyses of 796 female patients entering PATH Medical Clinic, between January 4, 2009 and February 24, 2013, were performed as part of the “Menopause Analytical Hormonal Correlate Outcome Study” (MAHCOS). Patient age range was 39–76 years (46.7±0.2). P300 latency and amplitude correlated with a number of hormones: follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol, estrone, estriol, DHEA, pregnenolone, progesterone, free and total testosterone, thyroid stimulating hormone (TSH), Vitamins D 1.25 and D 25OH, leptin, and insulin-like growth factor-binding protein 3 (IGF-BP3). Corrected statistics did not reveal significant associations with P300 latency or amplitude for these hormones except for leptin plasma levels. However, factor analysis showed that FSH and LH clustered together with Vitamin D1.25 and Vitamin D25OH, P300 latency (not amplitude), and log leptin were found to be associated in the same cluster. Utilizing regression analysis, once age adjusted, leptin was the only significant predictor for latency or speed (p = 0.03) with an effect size of 0.23. Higher plasma leptin levels were associated with abnormal P300 speed (OR = 0.98). Our findings show a significant relationship of higher plasma leptin levels, potentially due to leptin resistance, and prolonged P300 latency. This suggests leptin resistance may delay electrophysiological processing of memory and attention, which appears to be the first of such an association.     

Alterations and Mechanism of Gut Microbiota in Graves’ Disease and Hashimoto’s Thyroiditis

To explore the role of gut microbiota in Graves’ disease (GD) and Hashimoto’s thyroiditis (HT). Seventy fecal samples were collected, including 27 patients with GD, 27 with HT, and 16 samples from healthy volunteers. Chemiluminescence was used to detect thyroid function and autoantibodies (FT3, FT4, TSH, TRAb, TGAb, and TPOAb); thyroid ultrasound and 16S sequencing were used to analyze the bacteria in fecal samples; KEGG (Kyoto Encyclopedia of Genes and Genomes) and COG (Clusters of Orthologous Groups) were used to analyze the functional prediction and pathogenesis. The overall structure of gut microbiota in the GD and HT groups was significantly different from the healthy control group. Proteobacteria and Actinobacteria contents were the highest in the HT group. Compared to the control group, the GD and HT groups had a higher abundance of Erysipelotrichia, Cyanobacteria, and Ruminococcus_2 and lower levels of Bacillaceae and Megamonas. Further analysis of KEGG found that the “ABC transporter” metabolic pathway was highly correlated with the occurrence of GD and HT. COG analysis showed that the GD and HT groups were enriched in carbohydrate transport and metabolism compared to the healthy control group but not in amino acid transport and metabolism. Our data suggested that Bacillus, Blautia, and Ornithinimicrobium could be used as potential markers to distinguish GD and HT from the healthy population and that “ABC transporter” metabolic pathway may be involved in the pathogenesis of GD and HT.     

Hyperglobulinaemic Renal Tubular Acidosis: A Report of Nine Cases

Of nine women with hyperglobulinaemic renal tubular acidosis four presented with acidosis and five had the “incomplete” form of the disorder. Seven patients had nephrogenic diabetes insipidus, but none had the Fanconi syndrome. Investigation showed abnormal immunoglobulins and autoantibodies in all nine patients. Diseases coexisting with renal tubular acidosis were Sjögren''s syndrome, hyperglobulinaemic purpura, autoimmune liver and thyroid disease, diffuse pulmonary fibrosis, and a peripheral neuropathy. It is suggested that this type of renal tubular acidosis might be due to an autoimmune process.     

Does Large Needle Aspiration Biopsy Add Pain to the Thyroid Nodule Evaluation?

Thyroid large needle aspiration biopsy is disregarded because it is thought to be associated with pain. This is in contrast with our 32 years long experience. We surveyed reports of pain in patients examined with fine needle aspiration biopsy (78, 87.2% women, mean age 59 years) or FNAB+large needle aspiration biopsy (48, 87.5% women, mean age 60 years). Each patient was questioned regarding a) no unpleasant sensation (score “0”); b) unpleasant sensation (“1”); c) mild pain (no analgesic used; “2”); or d) pain (analgesic used; “3”). The mean size of the needle used was for FNAB 22.3±0.7 or 20.8±1 gauge in the fine needle aspiration or fine needle aspiration plus large needle aspiration biopsy group, respectively (p<.0001). The number of percutaneous punctures was higher in the fine needle aspiration plus large needle aspiration biopsy group. However, the pain score in the fine needle aspiration biopsy or fine needle aspiration biopsy plus large needle aspiration biopsy group was not significantly different. Large needle aspiration biopsy after fine needle aspiration biopsy does not add any discomfort or pain and therefore in light of the demonstrable benefits, should be included in clinical algorithms for the evaluation of thyroid nodules.     

A sustained passion for intracellular trafficking

I am honored to be the first recipient of the Women in Cell Biology Sustained Excellence in Research Award. Since my graduate school days, I have enjoyed being part of a stimulating scientific community the American Society for Cell Biology embodies. Having found myself largely by accident in a career that I find deeply enjoyable and fulfilling, I hope here to convey a sense that one need not have a “grand plan” to have a successful life in science. Simply following one''s interests and passions can sustain a career, even though it may involve some migration.     

Blocking activity to action of thyroid stimulating hormone in serum from patients with primary hypothyroidism.

Spontaneous primary hypothyroidism in adults is usually associated with autoimmune thyroiditis. The hypothesis that hypothyroidism may result from the presence in serum of a factor that blocks stimulation of the thyroid by thyroid stimulating hormone was examined. Serum samples were collected from 28 patients with recently diagnosed primary hypothyroidism. After removal of endogenous thyroid stimulating hormone the effect of the serum on secretion of triiodothyronine induced by thyroid stimulating hormone or thyroid stimulating antibodies was examined in thyroid slices incubated in vitro. Serum samples from six of the patients demonstrated significant blocking of the stimulation by bovine thyroid stimulating hormone. Inhibition of the stimulatory action of thyroid stimulating antibodies was also exhibited by serum samples with blocking activity. It is concluded that in some patients with primary hypothyroidism a serum factor, which is probably an IgG, exists that can block the thyroid response to thyroid stimulating hormone and thyroid stimulating antibodies; it may represent an important mechanism in the pathogenesis of hypothyroidism.     

Response to Vitamin B12 and Folic Acid in Myalgic Encephalomyelitis and Fibromyalgia

Background

Patients with myalgic encephalomyelitis (ME, also called chronic fatigue syndrome) may respond most favorably to frequent vitamin B12 injections, in vital combination with oral folic acid. However, there is no established algorithm for individualized optimal dosages, and rate of improvement may differ considerably between responders.

Objective

To evaluate clinical data from patients with ME, with or without fibromyalgia, who had been on B12 injections at least once a week for six months and up to several years.

Methods

38 patients were included in a cross-sectional survey. Based on a validated observer’s rating scale, they were divided into Good (n = 15) and Mild (n = 23) responders, and the two groups were compared from various clinical aspects.

Results

Good responders had used significantly more frequent injections (p<0.03) and higher doses of B12 (p<0.03) for a longer time (p<0.0005), higher daily amounts of oral folic acid (p<0.003) in good relation with the individual MTHFR genotype, more often thyroid hormones (p<0.02), and no strong analgesics at all, while 70% of Mild responders (p<0.0005) used analgesics such as opioids, duloxetine or pregabalin on a daily basis. In addition to ME, the higher number of patients with fibromyalgia among Mild responders was bordering on significance (p<0.09). Good responders rated themselves as “very much” or “much” improved, while Mild responders rated “much” or “minimally” improved.

Conclusions

Dose-response relationship and long-lasting effects of B12/folic acid support a true positive response in the studied group of patients with ME/fibromyalgia. It’s important to be alert on co-existing thyroid dysfunction, and we suspect a risk of counteracting interference between B12/folic acid and certain opioid analgesics and other drugs that have to be demethylated as part of their metabolism. These issues should be considered when controlled trials for ME and fibromyalgia are to be designed.     

Achilles Tendon Reflex in Accidental Hypothermia and Hypothermic Myxoedema

The photomotogram (P.M.G.) of the Achilles tendon reflex was studied in 26 patients with hypothermia (rectal temperature 33·3°C or less), 10 of whom also had myxoedema (serum protein bound iodine 2·8 μg/100 ml or less). No reflex could be elicited in eight (31%) of these patients, including three of those with myxoedema. Hypothermia increases both the contraction and the relaxation times of the reflex, the relaxation phase being particularly prolonged in those with myxoedema. In those patients from whom the reflex was elicited the ratio of the contraction time to the “half-relaxation time” in the P.M.G. was less than unity in six of the seven with myxoedema, and considerably greater than unity in eight of the 11 (73%) who were euthyroid. Thus, analysis of the Achilles tendon reflex P.M.G. correctly predicted the thyroid status in 14 of the 18 hypothermic patients in whom the Achilles tendon reflex was present (78%). The wider use of this rapid test of thyroid function would allow a more rational use of thyroid hormones in hypothermic patients and so lead to a better assessment of their value.     

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the “angio-follicular” units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.     

Thyroid function in lung cancer

Thyroid function was assessed at the time of initial diagnosis in 204 patients with lung cancer and compared with that of age and sex-matched patients with non-malignant lung disease. Abnormalities in thyroid function were found in 67 patients (33%). The most prevalent abnormality was a low T3 concentration; this was not associated with other clinical or biochemical evidence of hypothyroidism, but the short-term prognosis of these patients was worse than that of matched patients with lung cancer having normal T3 concentrations. Primary hypothyroidism occurred in three patients, low T4 concentrations and free thyroxine index (FTI) with normal thyrotrophin (TSH) concentrations in four patients, and moderately raised TSH with normal thyroid hormone concentrations in six patients; nine patients had a raised FTI with or without raised T4 concentration as the sole abnormality.Overall, the pattern of thyroid hormone metabolism in lung cancer was a tendency towards reduced T3 concentrations with significantly increased T4/T3 ratios and modestly increased 3,3′,5′-triiodothyronine (rT3) concentrations. The altered T4/T3 ratio was particularly noticeable in patients with anaplastic tumours of small (“oat cell”) and large cell types, but was not apparently related to detectable extrathoracic metastases.These data suggest that thyroid hormone metabolism is altered in patients with lung cancer by decreased 5′-monodeiodination of T4. The resulting low T3 concentrations and altered T4/T3 ratio may be partly responsible for the reduced ratio of androsterone to aetiocholanolone observed in lung cancer, which is known to be a poor prognostic sign.