Juvenile animal studies for the development of paediatric medicines: a description and conclusions from a European medicines agency workshop on juvenile animal testing for nonclinical assessors

A workshop organised by the European Medicines Agency involved assessors and experts present in a Nonclinical Working Group evaluating juvenile animal studies for Paediatric Investigation Plans in collaboration with the Paediatric Committee and the Safety Working Party of the Committee for Human Medicinal Products. The objective of the workshop was to analyse which juvenile animal studies proposals were received and agreed by the Paediatric Committee, to check consistency and how to apply the existing European guideline on juvenile animal studies. A comparison of main organ system development in man vs. animal species was presented to guide the review and to support species selection and protocol design. An analysis of juvenile animal studies included in finalised PIP's was also presented. Out of 109 paediatric investigation plans finalised between November 2008 and March 2009, 43 included one or more juvenile animal studies. In most cases the preferred species was the rat; one species only was requested to be studied (20/22), but in a minority two species were required (2/22). When deciding on the characteristics of the juvenile animal studies, such as age of animals at study start, the age of the children targeted by the medicine was considered. It is expected that the increasing experience gained by Applicants and Regulators will allow further refining the criteria for these juvenile animal studies. Further research on this topic is highly encouraged in the European Regulatory framework. Birth Defects Res (Part B) 89:467–473, 2010. © 2010 Wiley‐Liss, Inc.     

The thyroid tissue of juvenile Oncorhynchus mykiss is tubular, not follicular

The three dimensional morphology of the thyroid tissue in juvenile rainbow trout Oncorhynchus mykiss was determined using serial transverse and longitudinal sections of lower jaws stained with haematoxylin and eosin. The thyroid tissue comprised interconnecting tubular units not the follicles that are widely described in the literature. Some of these tubules were subdivided by branching, giving the appearance of follicles in the sections. The thyroid tissue was aggregated into two major concentrations of tissue, associated with the first and second branchial arteries, with the majority of the tissue located dorsal and lateral to the aorta. These observations in juvenile rainbow trout suggest that thyroid tissue of some species of fishes may not be follicular, as previously assumed. The physiological and evolutionary implications have yet to be determined.     

Update on: proteome analysis in thyroid pathology – part II: overview of technical and clinical enhancement of proteomic investigation of the thyroid lesions

ABSTRACT

Introduction: An accurate diagnostic classification of thyroid lesions remains an important clinical aspect that needs to be addressed in order to avoid ‘diagnostic’ thyroidectomies. Among the several ‘omics’ techniques, proteomics is playing a pivotal role in the search for diagnostic markers. In recent years, different approaches have been used, taking advantage of the technical improvements related to mass spectrometry that have occurred.

Areas covered: The review provides an update of the recent findings in diagnostic classification, in genetic definition and in the investigation of thyroid lesions based on different proteomics approaches and on different type of specimens: cytological, surgical and biofluid samples. A brief section will discuss how these findings can be integrated with those obtained by metabolomics investigations.

Expert commentary: Among the several proteomics approaches able to deepen our knowledge of the molecular alterations of the different thyroid lesions, MALDI-MSI is strongly emerging above all. In fact, MS-imaging has also been demonstrated to be capable of distinguishing thyroid lesions, based on their different molecular signatures, using cytological specimens. The possibility to use the material obtained by the fine needle aspiration makes MALDI-MSI a highly promising technology that could be implemented into the clinical and pathological units.     

The number of nucleoli in benign and malignant thyroid lesions: a useful diagnostic sign in cytological preparations

The slides of fine needle aspiration cytology specimens from 99 cases of cold thyroid nodules with known histology were reviewed and the number of nucleoli per nucleus counted and correlated with the different histopathological groups. Significant differences were observed between benign and malignant thyroid lesions in the number of nucleoli in the cytological material. Lower values were present in nodular goitres and follicular adenomas compared to carcinomas. In benign lesions the majority of nuclei contained one nucleolus and nuclei with two, three or more nucleoli were less frequent than in follicular, papillary, medullary and anaplastic carcinomas. Only one case of follicular adenoma had cells containing three or more nucleoli compared to more than half the cases of follicular carcinoma.     

Selenium and rubidium changes in subjects with pathologically altered thyroid

Concentrations of selenium and rubidium in groups of subjects with hyperthyroidism, carcinomas, or adenomas and in controls were determined by neutron activation analysis with coirradiated inorganic standards and IAEA reference material. Se was decreased in all pathological groups with the greatest modification in thyroids with carcinomas. Rb was elevated in all pathological groups with the greatest increase in carcinomas as well. According to the literature, Se has a protective effect on carcinogenity as well as on biochemical pathways in thyroid cells. There are no data in the literature on the effects of Rb in those cells. On the grounds of the present results, it seems possible to use the trapping of Rb for diagnostic purposes in cases of pathologically altered thyroids.     

Cytopathology of Follicular Tumours of the Thyroid With Clear Cell Change

A retrospective cytological study of nine follicular tumours of the thyroid with clear cell change was undertaken. In five clear cell adenomas and one moderately differentiated clear cell follicular carcinoma the epithelial cells occurred singly or in sheets and clusters; they sometimes assumed a trabecular or follicular pattern. The cells usually had pale diffusely vacuolated cytoplasm with ill-defined boundaries, a variable degree of anisonucleosis, nucleolar enlargement, and nuclear overlapping. Smears from a signet-ring cell adenoma contained in addition a few cells with large cytoplasmic vacuoles and compressed eccentric nuclei. In these cases a cytological diagnosis of 'follicular lesion' (or follicular neoplasia), clear cell type or signet-ring cell type, was given. A cytodiagnosis of 'carcinoma' was made only in the poorly differentiated follicular carcinoma-clear cell variant studied which showed unequivocal features of malignancy. Features suggestive of thyroid cyst, nodular goitre, Hashimoto's thyroiditis, and cell hyperactivity (marginal vacuoles, 'fire flare') were also found in the aspirated specimens of these cases of clear cell tumour of the thyroid.     

Role of fine needle aspiration cytology in evaluation of paediatric lymphadenopathy

Fine needle aspiration cytology (FNAC) in children has been slow to gain acceptance as compared with its use in adults. The aim of this retrospective study is to determine the utility of FNAC in diagnosing paediatric lymphadenopathy. Six hundred and ninety-two paediatric lymph nodes were aspirated during the period from January 1996 to June 2001. Aspirated material in 32 cases (4.6%) was deemed inadequate for cytological diagnosis. In the remaining cases, 62.2% were reactive lymphadenopathy, 25.2% tuberculous lymphadenitis, 6.3% acute suppurative lymphadenitis, nine cases revealed malignancy (seven lymphoma, two metastatic deposits) and one case had Langerhan's cell histiocytosis. No significant complications were encountered in any of the cases. FNAC emerged as a safe and reliable diagnostic procedure in paediatric age group obviating the need for excision biopsy.     

Helicobacter canis bacteraemia in a 7-month-old child

On the basis of biochemical, phenotypic and 16S rRNA analyses, Helicobacter canis was isolated and identified from an otherwise healthy 7-month-old girl with intermittent fever. Blood cultures signalled bacterial growth after 5 days that was characterized as small gram-negative spiral rods. Subculturing on Colombia plates with 5% sheep blood, chocolate agar and brucella agar, aerobically and anaerobically as well as in a microaerophilic atmosphere, showed scanty growth after an additional 4 days. Secondarily seeded with fluid from the original bottle, the paediatric blood bottles repeatedly signalled growth after one night's incubation, whereas the conventially treated bottles did not support growth after 7 days' incubation. From the secondary seeded paediatric bottles a pure culture was isolated on chocolate agar plates, and identified as H. canis. This case indicates that blood culture systems should be compared and improved for their capacity to detect Helicobacter and related pathogenic bacteria species. Further studies are also needed to determine the importance of H. canis as a primary pathogen, and the role of cats in the possible zoonotic spread of H. canis to humans.     

An alternate model of thyroid cytology: practical not perfect

A sample of 384 thyroid cytology specimens prepared by cytospin over a 2.5-year period was classified by original report into inadequate, non-neoplastic and suspicious of neoplasia or worse. This was then compared with subsequent histology. The resulting data showed an inadequacy rate of 33%, a sensitivity of 55%, a specificity of 59%, a positive predictive value of 64% and a negative predictive value of 93%. On review of the cytology, in knowledge of the subsequent histology, the maximum achievable results were determined to have a positive predictive value of 79% and a negative predictive value of 97%. No clinically significant adverse event was detected.     

Role of Dicer1 in thyroid cell proliferation and differentiation

DICER1 plays a central role in the biogenesis of microRNAs and it is important for normal development. Altered microRNA expression and DICER1 dysregulation have been described in several types of tumors, including thyroid carcinomas. Recently, our group identified a new somatic mutation (c.5438A>G; E1813G) within DICER1 gene of an unknown function. Herein, we show that DICER1 is overexpressed, at mRNA level, in a significant-relative number of papillary (70%) and anaplastic (42%) thyroid carcinoma samples, whereas is drastically downregulated in all the analyzed human thyroid carcinoma cell lines (TPC-1, BCPAP, FRO and 8505c) in comparison with normal thyroid tissue samples. Conversely, DICER1 is downregulated, at protein level, in PTC in comparison with normal thyroid tissues. Our data also reveals that DICER1 overexpression positively regulates thyroid cell proliferation, whereas its silencing impairs thyroid cell differentiation. The expression of DICER1 gene mutation (c.5438A>G; E1813G) negatively affects the microRNA machinery and cell proliferation as well as upregulates DICER1 protein levels of thyroid cells but has no impact on thyroid differentiation. In conclusion, DICER1 protein is downregulated in papillary thyroid carcinomas and affects thyroid proliferation and differentiation, while DICER1 gene mutation (c.5438A>G; E1813G) compromises the DICER1 wild-type-mediated microRNA processing and cell proliferation.     

碘与甲状腺疾病

碘是人体必需的微量元素,是合成甲状腺激素（thyroid hormone,TH）的主要原料。人体内的碘主要从饮水及食物中获取。碘的摄入缺乏或过量,不仅对TH的合成及分泌有至关重要的影响,而且与甲状腺形态及多种甲状腺疾病的发生、发展及转归密切相关。加碘盐的推广有效预防了碘缺乏可能引起的相关疾病;而碘过量导致的甲状腺疾病谱和发病率的急剧变化,也引起各界高度重视。流行病学调查表明,碘的摄入量与甲状腺功能亢进、自身免疫性甲状腺炎、甲状腺功能减退、甲状腺肿大和甲状腺癌等疾病的发病率密切相关。针对碘的相关生物学问题,对近年来的基础研究和人群研究进行综述,希望借此抛砖引玉,引起相关部门的重视,提高人民群众对碘营养的科学认识水平。     

The impact of the non‐invasive follicular thyroid neoplasm with papillary‐like nuclear feature terminology in the routine diagnosis of thyroid tumours

Background

Due to the recent proposal of the non‐invasive follicular thyroid neoplasm with papillary‐like nuclear feature (NIFTP) category, the authors analyse the state of the art in the challenging diagnosis of follicular thyroid neoplasms in routine practice.

Methods and results

A consecutive series of 200 histological diagnoses, with complete cytological correlation, was analysed following the introduction of the NIFTP definition. The study was conducted in a general hospital with a high prevalence of thyroid benign nodules that accounted for approximately 60% of surgically‐treated nodules. The significant incidence of the new NIFTP category was 7%. Concurrently, a gradual decrease of the follicular variant of papillary thyroid carcinoma (fvPTC) was observed (3.5%). When evaluating the FNA biopsies within the NIFTP group, despite the systematic evaluation of nuclear crowding, enlargement, irregularities and clearing, the final cytological class was often indeterminate for malignancy (Thy3/III‐IV, 71%). At histology, the application of the semiquantitative NIFTP score for the evaluation of the PTC‐like nuclear features was able to discriminate benign lesions (score 0/1) from fvPTC (score 2/3). A certain degree of overlapping still persisted between NIFTP and fvPTC (score 2) or between NIFTP and benign lesions (score 1).

Conclusions

In the routine evaluation of FNA biopsies, the presence of subtle and questionable PTC‐like nuclear features still remains a controversial aspect of the diagnostic workflow. Given that the NIFTP category was introduced to stratify the low‐risk group of thyroid tumours more precisely, pathologists should force themselves to apply the nuclear score rigorously and to classify cases assigned a score of 1 as benign proliferations.