Parafibromin as a Diagnostic Instrument for Parathyroid Carcinoma-Lone Ranger or Part of the Posse?

The diagnosis of parathyroid carcinoma requires an invasive growth pattern or metastases detected at histopathological examination; unfortunately, not all carcinomas exhibit visible malignant properties at the initial assessment. Therefore, immunohistochemical markers have been sought for the recognition of parathyroid malignancy. In 2003, the Hyperparathyroidism 2 (HRPT2) gene was found mutated in the majority of sporadic parathyroid carcinomas investigated, and studies regarding the protein product parafibromin proposed loss of nuclear parafibromin as a highly sensitive marker for the detection of parathyroid carcinoma. Recent studies have not fully reproduced these findings, as subsets of carcinomas display positive parafibromin immunoreactivity, and fractions of adenomas demonstrate absent expression. Overall, parafibromin is a marker of value to the endocrine pathologist, but it cannot be recommended as a sole indicator of parathyroid carcinoma. Additional markers such as protein gene product 9.5 (PGP9.5) and adenomatous polyposis coli (APC) could complement parafibromin when assessing malignant potential of parathyroid tumours

Frequent Promoter Hypermethylation of the APC and RASSF1A Tumour Suppressors in Parathyroid Tumours

BACKGROUND: Parathyroid adenomas constitute the most common entity in primary hyperparathyroidism, and although recent advances have been made regarding the underlying genetic cause of these lesions, very little data on epigenetic alterations in this tumour type exists. In this study, we have determined the levels of promoter methylation regarding the four tumour suppressor genes APC, RASSF1A, p16(INK4A) and RAR-beta in parathyroid adenomas. In addition, the levels of global methylation were assessed by analyzing LINE-1 repeats. METHODOLOGY/PRINCIPAL FINDINGS: The sample collection consisted of 55 parathyroid tumours with known HRPT2 and/or MEN1 genotypes. Using Pyrosequencing analysis, we demonstrate APC promoter 1A and RASSF1A promoter hypermethylation in the majority of parathyroid tumours (71% and 98%, respectively). Using TaqMan qRT-PCR, all tumours analyzed displayed lower RASSF1A mRNA expression and higher levels of total APC mRNA than normal parathyroid, the latter of which was largely conferred by augmented APC 1B transcription levels. Hypermethylation of p16(INK4A) was demonstrated in a single adenoma, whereas RAR-beta hypermethylation was not observed in any sample. Moreover, based on LINE-1 analyses, parathyroid tumours exhibited global methylation levels within the range of non-neoplastic parathyroid tissues. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that APC and RASSF1A promoter hypermethylation are common events in parathyroid tumours. While RASSF1A mRNA levels were found downregulated in all tumours investigated, APC gene expression was retained through APC 1B mRNA levels. These findings suggest the involvement of the Ras signaling pathway in parathyroid tumorigenesis. Additionally, in contrast to most other human cancers, parathyroid tumours were not characterized by global hypomethylation, as parathyroid tumours exhibited LINE-1 methylation levels similar to that of normal parathyroid tissues

Clinical, genetic, and immunohistochemical characterization of 70 Ukrainian adult cases with post-Chornobyl papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) exhibits various molecular abnormalities, both when sporadic and radiation-related. PTC is still diagnosed in adult individuals who were younger than 18 years at the time of the Chornobyl accident in 1986 and lived within the contaminated area. The preoperative diagnosis of PTC is based on ultrasound-guided fine needle aspiration cytology (FNAC), which is highly informative in up to 90% of biopsies. FNAC is not informative for the discrimination of follicular thyroid carcinoma (FTC) from follicular thyroid adenoma (FTA). Moreover, FNAC is often unreliable for diagnosis of cystic PTC due to its common presentation as a mural nodule in a cystic mass. In case of cystic PTC, biopsy sometimes reveals a cystic fluid containing insufficient amount of representative cells for cytology. In this work, PTC was characterized in relation to irradiation from radioactivity at childhood. Possible preoperative diagnostic markers for discrimination between PTC and other follicular thyroid neoplasms were identified, and their validity was tested. In Study I molecular, genetic and clinical characteristics in 70 post-Chornobyl PTCs were investigated. A common BRAF 1799T>A mutation was detected in 26 cases, overrepresentation of RET/PTC1 in 20 whereas RET/PTC3 was found in 4 cases. BRAF mutation was observed 3.5 times less frequent in the PTC accompanied by chronic lymphocytic thyroiditis (PTC/CLT) as compared to PTC only (12% vs. 44%). Greater expression of cyclin A was observed in PTC ≥ 2 cm as compared to PTC < 2 cm (1.2% vs. 0.6%). In conclusion, BRAF mutation and RET/PTC1 rearrangement as well as other molecular features of adult post-Chornobyl PTC were partly overlapping with other reported PTC cohorts. In Study II the SELDI-TOF mass spectrometry method was applied for PTC, FTC, FTA and normal thyroid tissue (NT). Significant overexpression of the protein S100A6 was identified in PTC as compared to FTC, FTA and NT (p < 0.05). This result was verified both by Western blot (WB), using the same samples, and by IHC in these and additionally in the PTC samples investigated in Study I. Moreover, the presence of two post-translational modifications of S100A6 was observed and verified by LC-MS/MS. S100A6 expression is strongly associated with PTC, and can therefore be tested for discrimination between follicular thyroid tumors and PTC. In Study III a two dimensional gel electrophoresis followed by MALDI-TOF mass spectrometry for proteomic profiling of PTC, FTC and FTA was performed. 25 protein spots showing significantly different expression between studied groups were identified. Of these, 9 protein spots were selected for further analyses by WB using the initially studied samples and by IHC using these as well as samples from Study I. The findings suggest additional proteins to be deregulated in thyroid tumors, and their clinical significance can now be further studied. In Study IV preoperative diagnostic markers for PTC in cystic lesions were identified by applying LC-MS/MS method. Out of all 1581 identified proteins, annexin A3 (ANXA3), carboxymethylenebutenolidase homolog (CMBL) cytokeratin 19 (CK- 19) and S100A13 were selected for validation by IHC and WB. ANXA3 and CMBL showed overexpression in both controls and PTCs, whereas S100A13 and CK-19 were up-regulated in PTC only (p < 0.05), suggesting their possible role for discrimination between cystic PTC and benign thyroid cysts

Genetic characterization of large parathyroid adenomas

In this study, we genetically characterized parathyroid adenomas with large glandular weights, for which independent observations suggest pronounced clinical manifestations. Large parathyroid adenomas (LPTAs) were defined as the 5% largest sporadic parathyroid adenomas identified among the 590 cases operated in our institution during 2005–2009. The LPTA group showed a higher relative number of male cases and significantly higher levels of total plasma and ionized serum calcium (P<0.001). Further analysis of 21 LPTAs revealed low MIB1 proliferation index (0.1–1.5%), MEN1 mutations in five cases, and one HRPT2 (CDC73) mutation. Total or partial loss of parafibromin expression was observed in ten tumors, two of which also showed loss of APC expression. Using array CGH, we demonstrated recurrent copy number alterations most frequently involving loss in 1p (29%), gain in 5 (38%), and loss in 11q (33%). Totally, 21 minimal overlapping regions were defined for losses in 1p, 7q, 9p, 11, and 15q and gains in 3q, 5, 7p, 8p, 16q, 17p, and 19q. In addition, 12 tumors showed gross alterations of entire or almost entire chromosomes most frequently gain of 5 and loss of chromosome 11. While gain of 5 was the most frequent alteration observed in LPTAs, it was only detected in a small proportion (4/58 cases, 7%) of parathyroid adenomas. A significant positive correlation was observed between parathyroid hormone level and total copy number gain (r=0.48, P=0.031). These results support that LPTAs represent a group of patients with pronounced parathyroid hyperfunction and associated with specific genomic features

Prolactin Receptor in Primary Hyperparathyroidism – Expression, Functionality and Clinical Correlations

<div><h3>Background</h3><p>Primary hyperparathyroidism (PHPT) is an endocrine disorder most commonly affecting women, suggesting a role for female hormones and/or their receptors in parathyroid adenomas. We here investigated the prolactin receptor (PRLr) which is associated with tumours of the breast and other organs.</p> <h3>Methodology/Principal Findings</h3><p>PRLr expression was investigated in a panel of 37 patients with sporadic parathyroid tumours and its functionality in cultured parathyroid tumour cells. In comparison with other tissues and breast cancer cells, high levels of prolactin receptor gene (<em>PRLR</em>) transcripts were demonstrated in parathyroid tissues. PRLr products of 60/70 kDa were highly expressed in all parathyroid tumours. In addition varying levels of the 80 kDa PRLr isoform, with known proliferative activity, were demonstrated. In parathyroid tumours, PRLr immunoreactivity was observed in the cytoplasm (in all cases, n = 36), cytoplasmic granulae (n = 16), the plasma membrane (n = 12) or enlarged lysosomes (n = 4). In normal parathyroid rim (n = 28), PRLr was uniformly expressed in the cytoplasm and granulae. In <em>in vitro</em> studies of short-term cultured human parathyroid tumour cells, prolactin stimulation was associated with significant transcriptional changes in JAK/STAT, RIG-I like receptor and type II interferon signalling pathways as documented by gene expression profiling. Moreover, <em>PRLR</em> gene expression in parathyroid tumours was inversely correlated with the patients’ plasma calcium levels.</p> <h3>Conclusions</h3><p>We demonstrate that the prolactin receptor is highly abundant in human parathyroid tissues and that PRLr isoforms expression and PRLr subcellular localisation are altered in parathyroid tumours. Responsiveness of PRLr to physiological levels of prolactin was observed in the form of increased PTH secretion and altered gene transcription with significant increase of RIG-I like receptor, JAK-STAT and Type II interferon signalling pathways. These data suggest a role of the prolactin receptor in parathyroid adenomas.</p> </div

High-molecular-weight insulin-like growth factor 2 : Immunohistochemical, ultrastructural and biochemical findings in insulin cells under normal, diabetic, and neoplastic conditions, and its role in non-islet-cell-tumour hypoglycaemia

High-Molecular-Weight Insulin-Like Growth Factor 2 Immunohistochemical, Ultrastructural, and Biochemical Findings in Insulin Cells under Normal, Diabetic, and Neoplastic Conditions, and Its Role in Non-lslet-Cell-Tumour Hypoglycaemia by Anders Höög Department of Oncology and Pathology, Unit of Tumour Pathology, Karolinska Institute and Hospital, Stockholm, Sweden. IGF-2 immunoreactive (IR) cells were identified in the normal adult pancreas of man and rat and occurred exclusively in insulin-producing cells. When frozen human pancreatic tissue was extracted and gel-size-separated, a high-molecular-weight (HMW) IGF-2 peptide could be identified. By using radioimmunoassay (RIA) the HMW IGF-2 was found to be in other fractions than those of insulin/pro-insulin. In order to study the ultrastructural localization of the IGF-2 in normal rats, a pre-embedding technique and immuno-gold-silver staining was used. The IGF-2 immunoreactivity was found to be situated in the halo of a subset of the insulin IR B-granules in normal rats. Insulin immunoreactivity occurred more frequently in the B-granules. The role of HMW IGF-2 in dysplastic insulin cells was studied in the endocrine pancreas of an animal model of non-insulin-dependent diabetes mellitus, namely the Goto-Kakizaki (GK) rat. At 2 months of age, these rats have both islets of normal structure and 'starfish-shaped' islets. They were characterized by an abundance of collagen fibers and great variations in their numbers of B-granules in the insulin cells. The most striking ultrastructural feature of these cells was the precense of large, usually electron-translucent vesicles. Some of them were well demarcated and contained electron-dense material resembling crinophagic bodies, indicating intracellular degradation of their B-granules. As expected, IGF-2 was found in the insulin cells of both GK and control rats, but RIA revealed a considerably larger amount of HMW IGF-2 peptide in the 2- and 6-month-old GK rats than in the 1-month-old GK ones and in the control rats. In situ hybridization of 3-month-old GK rat pancreas showed increased IGF-2 mRNA expression in the "starfish-shaped" islets compared to in the islets of normal structure, both in diabetic and non-diabetic rats. Then, the presence of HMW IGF-2 in neoplastic insulin and other islet cells was investigated using pancreatic tumours of both endocrine and exocrine types. IGF-2 IR cells were found in 13 out of 14 insulin-producing tumours and in 5 out of 20 other endocrine neoplasms. Biochemical techniques revealed that the amounts of the IGF-2 IR peptide and its molecular weight forms differed from case to case. By using the Northern blot technique, a 5 kb IGF-2 mRNA transcript was found in an insulinoma metastasis. Two other insulinomas, one with few, the other with an abundance of IGF-2 IR cells, showed intense IGF-2 mRNA signal in the majority of the tumour cells by means of in situ hybridization. In 17 specimens of ductal pancreatic adenocarcinomas, only a few scattered cells were IGF-2 IR but they were interpreted as non-neoplastic. No IGF-2 peptide was found in tumour extracts of the adenocarcinomas. Lastly, HMW IGF-2 was studied in a patient suffering from hypoglycaemia caused by a large, haemangiopericytoma with recurrencies for more than a decade. High concentrations of HMW IGF 2 peptide were found in serum samples and IGF-2 IR tumour cells were detected in biopsy specimens of the neoplasm. A surprisingly finding was, however, that no such IGF-2 IR cells were observed in the specimens from the first operation. When the in situ hybridization technique was used it could be shown that IGF-2 mRNA expression actually occured also in the original tumour cells, and that it was increased already 6 years before the onset of the hypoglycaemic symptoms. These observations show that HMW IGF-2 is a clinically important, biologically active member of the insulin family, closely related to normal, dysplastic and neoplastic insulin cells. Key words: Insulin-like growth factor-2, IGF-2, islets of Langerhans, B-granules, Diabetes Mellitus type 2, insulinoma, Goto-Kakizaki rat, haemangiopericytoma, ultrastructural immunohistochemistry, gene expression. ISBN: 91-628-2579-