Differential diagnosis of thyroid nodules using fine-needle aspiration cytology and oncogene mutation screening: are we ready?

Thyroid nodules are a very common clinical finding, and although the majority of them are benign, thyroid carcinoma accounts for about 5-15% of nodules. Fine-needle aspiration cytology (FNAC) is actually used for the differential diagnosis of these lesions. Although in most cases this examination clearly distinguishes benign from malignant lesions, some fine-needle aspiration (FNA) samples fall into undetermined thyroid cytology categories, which according to the most recent classification of thyroid FNAC consist of ‘suspicious for malignancy’, ‘suspicious for follicular or Hurtle cell neoplasm’, and ‘follicular lesion of undetermined significance/atypia of undetermined significance’. Moreover, some samples are insufficient for diagnosis. Taken together, these categories account for almost 20-30% of nodules. Owing to the high risk of papillary thyroid carcinoma, patients with lesions that are ‘suspicious for malignancy’ are currently subjected to lobectomy or total thyroidectomy. On the other hand, patients with ‘atypia of undetermined significance’ undergo repeated FNAs, and patients with ‘suspicious for follicular or Hurtle cell neoplasm’ are subjected to diagnostic lobectomy and subsequently, in the case of histological diagnosis of carcinoma, total thyroidectomy. Recent studies clearly indicate that molecular analysis of thyroid nodules can significantly improve the diagnostic power of cytology and drive the appropriate clinical management of these patients

Identification and Characterization of Two Labeled Intermediates in the Biosynthesis of Rat Thyroglobulin

Abstract Two protein components related to 19 S thyroglobulin and labeled at early times during its biosynthesis have been isolated and characterized. A 3–8 S fraction was prepared from the soluble extract of rat thyroid glands incubated in vitro with radioactive amino acids ([3H]leucine and [14C]isoleucine) or carbohydrates ([3H]- or [14C]mannose and [3H]- or [14C]galactose) or both. From this fraction three components were purified. Two of them, homogeneous by ultracentrifugal and electrophoretic criteria, had sedimentation rates of 6 S and 7 S and molecular weights close to 112,000 and 186,000, respectively. The molar ratios of labeled leucine to isoleucine incorporated into the 6 S and 7 S fractions were the same as those found in 19 S thyroglobulin and in its 12 S subunit. Both the 6 S and 7 S protein units were quantitatively precipitated by anti-rat thyroglobulin antibodies. Double labeling with [3H]leucine and 14C-carbohydrates demonstrated that mannose is present in the fully assembled 19 S and 12 S proteins as well as on the slower sedimenting 6 S and 7 S units, whereas galactose is incorporated only after the formation of the fully assembled stable thyroglobulin molecule. It is concluded that the 6 S and 7 S labeled proteins, which contain only part of the carbohydrate moiety of thyroglobulin, participate in the structure of newly formed unstable 19 S molecules and may represent intermediates during the assembly process of the elementary polypeptide chains of thyroglobulin

Biosynthesis of Thyroid Iodoproteins in Vivo and in Tissue Slices

Abstract The time course of formation of the thyroid iodoprotein 19 S and its subunits (6 S, 7 S, and 12 S) has been studied after intravenous administration of [3H]leucine to normal rats and guinea pigs and after in vitro incorporation in thyroid hemilobes. The uptake of the [3H]leucine by the rat gland in vivo paralleled the disappearance of the radioactivity from the plasma (30 min): the amino acid was rapidly bound to a particulate fraction and was more slowly incorporated into proteins having solubility properties similar to that of thyroglobulin. Sucrose gradient analysis of the newly formed thyroglobulin-like labeled proteins showed the presence of a significant proportion of thyroglobulin and of its half-sized, 12 S subunit even at the earliest labeling interval (10 min). These data suggest that the assembly of the polypeptide chains of thyroglobulin occurs before the newly formed molecules are released from the intracellular membranes. The 6 S and 7 S labeled intermediates appear to be related to newly formed, unstable thyroglobulin molecules, the equilibrium between these units and the fully assembled molecules being shifted toward the latter with increasing labeling times. A similar pattern was observed after pulse labeling guinea pigs in vivo. In this species, however, there is a larger proportion of 12 S subunits derived from dissociation of newly formed and consequently poorly iodinated thyroglobulin molecules. Both in rats and guinea pigs, the sedimentation rate of newly formed thyroglobulin increased progressively from 14 to 19 S with the labeling time. After labeling thyroid hemilobes in vitro, the disappearance of the labeled intermediates 6 S, 7 S, and 12 S was both delayed and incomplete. It appears that the time-dependent disappearance of the labeled subunits 6 S, 7 S, 12 S, and of the unfolded 14 to 17 S species (prethyroglobulin) is related to later chemical modifications which increase the stability of the newly formed thyroglobulin molecules toward dissociation and unfolding. These later modifications include iodination and its oxidative side effects (oxidation of —SH to —S—S—) and addition of the carbohydrate moiety

Pathophysiology of ageing, longevity and age related diseases

On April 18, 2007 an international meeting on Pathophysiology of Ageing, Longevity and Age-Related Diseases was held in Palermo, Italy. Several interesting topics on Cancer, Immunosenescence, Age-related inflammatory diseases and longevity were discussed. In this report we summarize the most important issues. However, ageing must be considered an unavoidable end point of the life history of each individual, nevertheless the increasing knowledge on ageing mechanisms, allows envisaging many different strategies to cope with, and delay it. So, a better understanding of pathophysiology of ageing and age-related disease is essential for giving everybody a reasonable chance for living a long and enjoyable final part of the life

Neurophysiological aspects in SARS-CoV-2–induced acute respiratory distress syndrome

Patients with coronavirus disease 2019 (COVID-19) often develop acute respiratory failure and acute respiratory distress syndrome (ARDS) that requires intensive care unit (ICU) hospitalization and invasive mechanical ventilation, associated with a high mortality rate. In addition, many patients fail early weaning attempts, further increasing ICU length of stay and mortality. COVID-19 related ARDS can be complicated by neurological involvement with mechanisms of direct central nervous system (CNS) infection and with overlapping para-infective mechanisms of the peripheral nervous system (PNS). We aimed to evaluate the possible involvement of the brainstem and PNS in patients with COVID-19 related ARDS and difficulty in weaning from mechanical ventilation. We evaluated electroencephalogram (EEG), brainstem auditory evoked potentials (BAEPs), electroneurography of the four limbs and the phrenic nerve in 10 patients with respiratory insufficiency due to SARS-CoV-2. All were admitted to intensive care unit and were facing prolonged weaning from mechanical ventilation. All ten patients showed a mild diffuse non-specific slowing of brain electrical activity on the EEG. Four patients had an acute motor axonal neuropathy with absent or reduced amplitude phrenic nerve CMAP while four patients showed impairment of the BAEPs. A patient with peripheral nerve impairment suggestive of Guillain-Barré syndrome (GBS) underwent an intravenous immunoglobulin (IVIg) cycle that led to an improvement in the weaning process and progressive motor improvement. The inclusion of a comprehensive neurological evaluation in COVID-19 patients in ICU facilitated the early identification and effective management of Nervous System involvement

Signaling through Ras is essential for ret oncogene-induced cell differentiation in PC12 cells.

Specific germline mutations of the receptor tyrosine kinase, Ret, predispose to multiple endocrine neoplasia types 2A and 2B and familial medullary thyroid carcinoma. The mechanisms by which different Ret isoforms (Ret-2A and Ret-2B) cause distinct neoplastic diseases remain largely unknown. On the other hand, forced expression of these mutated versions of Ret induces the rat pheochromocytoma cell line, PC12, to differentiate. Here we used an inducible vector encoding a dominant-negative Ras (Ras p21(N17)) to investigate the contributions of the Ras pathway to the phenotype induced in PC12 cells by the expression of either Ret-2A or Ret-2B mutants. We show that the Ret-induced molecular and morphological changes are both mediated by Ras-dependent pathways. However, even though inhibition of Ras activity was sufficient to revert Ret-induced differentiation, the kinetics of morphological reversion of the Ret-2B- was more rapid than the Ret-2A- transfected cells. Further, we show that in Ret-transfected cells the suc1- associated neurotrophic factor-induced tyrosine phosphorylation target, SNT, is chronically phosphorylated in tyrosine residues, and associates with the Sos substrate. These results indicate the activation of the Ras cascade as an essential pathway triggered by the chronic active Ret mutants in PC12 cells. Moreover, our data indicate SNT as a substrate for both Ret mutants, which might mediate the activation of this cascade

In vitro and in vivo evaluation of In-111-DTPAGlu-G-CCK8 for cholecystokinin-B receptor imaging

Regulatory peptides and their analogs are being extensively investigated as radiopharmaceuticals for cancer imaging and therapy. Receptors of the cholecystokinin family have been shown to be overexpressed in different types of neuroendocrine tumors. The purposes of this study were to evaluate the cholecystokinin octapeptide amide (CCK8) peptide tagged with a diethylenetriaminepentaacetic acid derivative (DTPAGlu) and to test whether a 111In-labeled conjugate (111In-DTPAGlu-G-CCK8, a derivative containing the chelating agent DTPAGlu bound through a glycine linker at the N-terminal end of the bioactive peptide CCK8) is suitable for cholecystokinin-B receptor (CCKBR) imaging. Methods: CCK8 was synthesized by solidphase techniques and covalently coupled to DTPAGlu through a glycine linker at its amino terminus. The compound was labeled with 111In. The radiochemical purity and stability of the compound were assessed by chromatographic methods. NIH-3T3 and A431 cells overexpressing CCKBR were used to characterize the in vitro properties of the compound. Nude mice bearing control and CCKBR-overexpressing A431 xenografts were used as an in vivo model. Results: DTPAGlu-G-CCK8 showed rapid and efficient labeling with 111In. The radiolabeled conjugate showed specific binding to both cell lines overexpressing CCKBR. Binding was saturable, with a dissociation constant of 20 nmol/L in both cell systems. Both cell lines showed internalization of the ligand after interaction with the receptor. Biodistribution studies showed rapid localization of 111In-DTPAGlu- G-CCK8 on CCKBR-overexpressing A431 xenografts that was severalfold higher than that on control tumors at all time points tested. Unbound activity showed rapid clearance of over 80% through the kidneys by 30 min after injection. The labeled peptide conjugate was very stable in serum but showed a rapid breakdown after injection. Incubation with kidney homogenates suggested that most breakdown occurred in the kidneys, favoring the clearance of unbound activity. Conclusion: Our findings indicate that the in vitro and in vivo characteristics of 111In-DTPAGlu-G-CCK8 are favorable for CCKBR imaging, as thepeptide shows high-affinity binding to the receptor, is internalized in CCKBR-expressing cells, and shows avid uptake in CCKBR-overexpressing xenografts, with rapid clearance of unbound radioactivity through the kidneys. Furthermore, the ease of synthesis, high labeling efficiency, and chemical stability of DTPAGlu make this chelating moiety an ideal candidate for widespread use in peptide radiolabeling for nuclear medicine applications

MxA mRNA quantification and disability progression in interferon beta-treated Multiple Sclerosis patients

Even though anti-interferon beta (IFNβ) antibodies are the main determinants of IFNβ bioactivity loss and Myxovirus-resistance protein A (MxA) is the most established marker of IFNβ biological activity in IFNβ-treated multiple sclerosis patients, their usefulness in the routine clinical practice is still debated. Therefore, 118 multiple sclerosis patients naïve for treatment were enrolled for a 3-year longitudinal observational study mimicking the conditions of a real-world setting. In order to evaluate the kinetics of bioactivity loss in blood samples obtained every 6 months after therapy initiation, MxA and interferon receptor isoform/subunit mRNA were quantified by real-time PCR, anti-IFNβ binding antibodies were detected by radioimmunoprecipitation, and neutralizing antibodies by cytopathic effect inhibition assay. Clinical measures of disease activity and disability progression were also obtained at all time points. We found that, at the individual-patient level, the response to IFNβ therapy was extremely heterogeneous, including patients with stable or transitory, early or late loss of IFNβ bioactivity, and patients with samples lacking MxA mRNA induction in spite of absence of antibodies. No interferon receptor isoform alterations that could explain these findings were found. At the group level, none of these biological features correlated with the measures of clinical disease activity or progression. However, when MxA mRNA was evaluated not at the single time point as a dichotomic marker (induced vs. non-induced), but as the mean of its values measured over the 6-to-24 month period, the increasing average MxA predicted a decreasing risk of short-term disability progression, independently from the presence of relapses. Therefore, a more bioactive treatment, even if unable to suppress relapses, reduces their severity by an amount that is proportional to MxA levels. Together with its feasibility in the routine laboratory setting, these data warrant the quantification of MxA mRNA as a primary tool for a routine monitoring of IFNβ therapy

Nephroplex: a kidney-focused NGS panel highlights the challenges of PKD1 sequencing and identifies a founder BBS4 mutation

Background: Genetic testing of patients with inherited kidney diseases has emerged as a tool of clinical utility by improving the patients' diagnosis, prognosis, surveillance and therapy. Methods: The present study applied a Next Generation Sequencing (NGS)-based panel, named NephroPlex, testing 115 genes causing renal diseases, to 119 individuals, including 107 probands and 12 relatives. Thirty-five (poly)cystic and 72 non (poly)cystic individuals were enrolled. The latter subgroup of patients included Bardet-Biedl syndrome (BBS) patients, as major components. Results: Disease-causing mutations were identified in 51.5 and 40% of polycystic and non-polycystic individuals, respectively. Autosomal dominant polycystic kidney disease (ADPKD) patients with truncating PKD1 variants showed a trend towards a greater slope of the age-estimated glomerular filtration rate (eGFR) regression line than patients with (i) missense variants, (ii) any PKD2 mutations and (iii) no detected mutations, according to previous findings. The analysis of BBS individuals showed a similar frequency of BBS4,9,10 and 12 mutations. Of note, all BBS4-mutated patients harbored the novel c.332+1G>GTT variant, which was absent in public databases, however, in our internal database, an additional heterozygote carrier was found. All BBS4-mutated individuals originated from the same geographical area encompassing the coastal provinces of Naples. Discussion: In conclusion, these findings indicate the potential for a genetic panel to provide useful information at both clinical and epidemiological levels

2-deoxy-2[F-18] fluoro-D-glucose positron emission tomography Deauville scale and core-needle biopsy to determine successful management after six doxorubicin, bleomycin, vinblastine and dacarbazine cycles in advanced-stage Hodgkin lymphoma.

Abstract Background The clinical impact of the positivity of the Deauville scale (DS) of positron emission tomography (PET) performed at the end of doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD) in patients with advanced Hodgkin lymphoma (HL), in terms of providing rationale to shift poor responders onto a more intensive regimen, remain to be validated by histopathology. Patients and methods This prospective trial involved patients with stage IIB/IV HL who after six ABVD cycles underwent PET (PET6) and core-needle cutting biopsy (CNCB) of 2-deoxy-2[F-18] fluoro- d -glucose (FDG)-avid lymph nodes. Patients received high-dose chemotherapy/autologous haematopoietic stem cell rescue (HDCT/AHSCR) if CNCB was positive for HL, alternatively, if CNCB or PET was negative, received observation or consolidation radiotherapy (cRT) on residual nodal masses, as initially planned. The end-point was 5-year progression-free survival (PFS). Results In all, 43 of the 169 (25%) evaluable patients were PET6 positive (DS 4, 32; DS 5, 11). Among them, histology showed malignancy (HL) in 100% of DS 5 scores and in 12.5% of DS 4 scores. Fifteen patients with positive biopsy received HDCT/AHSCR, whereas 28 patients with negative biopsy, as well as 126 patients with negative PET6, continued the original plan (cRT, 78 patients; observation, 76 patients). The 5-year PFS in the negative PET6 group, negative biopsy group and positive biopsy group was 95.4%, 100% and 52.5%, respectively. Conclusion DS positivity of end-of-ABVD PET in advanced HL carried a certain number of CNCB-proven non-malignant FDG-uptakes. The DS 4 scores which were found to have negative histology appeared to benefit from continuing the original non-intensive therapeutic plane as indicated by the successful outcome in more than 95% of them by obtaining similar 5-year PFS to the PET6-negative group. By contrast, the DS 5 score had consistently positive histology and was associated with unsuccessful conventional therapy, promptly requiring treatment intensification or innovative therapeutic approaches