Normal Parathyroid Tissue Research Articles

Hypermethylated in Cancer 1 (HIC1), a Tumor Suppressor Gene Epigenetically Deregulated in Hyperparathyroid Tumors by Histone H3 Lysine Modification

Primary hyperparathyroidism (pHPT) resulting from parathyroid tumors is a common endocrine disorder with incompletely understood etiology. In renal failure, secondary hyperparathyroidism (sHPT) occurs with multiple tumor development as a result of calcium and vitamin D regulatory disturbance. The aim of the study was to investigate whether HIC1 may act as a tumor suppressor in the parathyroid glands and whether deregulated expression involves epigenetic mechanisms. Parathyroid tumors from patients with pHPT included single adenomas, multiple tumors from the same patient, and cancer. Hyperplastic parathyroid glands from patients with sHPT and hypercalcemia and normal parathyroid tissue specimens were included in the study. Quantitative RT-PCR, bisulfite pyrosequencing, colony formation assay, chromatin immunoprecipitation, and RNA interference was used. HIC1 was generally underexpressed regardless of the hyperparathyroid disease state including multiple parathyroid tumors from the same patient, and overexpression of HIC1 led to a decrease in clonogenic survival of parathyroid tumor cells. Only the carcinomas showed a high methylation level and reduced HIC1 expression. Cell culture experiments, including use of primary parathyroid tumor cells prepared directly after operation, the general histone methyltransferase inhibitor 3-deazaneplanocin A, chromatin immunoprecipitation, and RNA interference of DNA methyltransferases and EZH2 (enhancer of zeste homolog 2), supported a role of repressive histone H3 modifications (H3K27me2/3) rather than DNA methylation in repression of HIC1. The results strongly support a growth-regulatory role of HIC1 in the parathyroid glands and suggest that perturbed expression of HIC1 may represent an early event during tumor development. Repressive histone modification H3K27me2/3 is involved in repression of HIC1 expression in hyperparathyroid tumors.

The DNA methylome of benign and malignant parathyroid tumors.

The role of DNA methylation of CpG islands in parathyroid tumorigenesis has not been analyzed in an unbiased, systematic fashion. DNA was isolated from normal and pathologic parathyroid tissues, bisulphite modified and analyzed using the Infinium HumanMethylation27 BeadChip. Distinct hierarchical clustering of genes with altered DNA methylation profiles in normal and pathologic parathyroid tissue was evident. Comparing normal parathyroid tissue with parathyroid adenomas, 367 genes were significantly altered, while 175 genes significantly differed when comparing parathyroid carcinomas and normal parathyroid tissues. A comparison between parathyroid adenomas and parathyroid carcinomas identified 263 genes with significantly distinct methylation levels. Results were confirmed for certain genes in a validation cohort of 40 parathyroid adenomas by methylation-specific PCR. Genes of known or putative importance in the development of parathyroid tumors showed significant and frequent hypermethylation. DNA hypermethylation of CDKN2B, CDKN2A, WT1, SFRP1, SFRP2, and SFRP4 was associated with reduced gene expression in both benign and malignant parathyroid tumors. Treatment with 5-aza-2'-deoxycytidine of primary cell cultures restores expression of hypermethylated genes in benign and malignant parathyroid tumors. In conclusion, the unbiased, genome-wide study of the parathyroid tumor DNA methylome identified a number of genes with altered DNA methylation patterns of putative importance to benign and malignant parathyroid tumorigenesis.

Role of Multifunctional Cell Cycle Modulators in Advanced Secondary Hyperparathyroidism

Long-term dialysis for patients with end stage renal disease leads to an unavoidable common complication, which is secondary hyperparathyroidism. Two histological patterns (nodular and diffuse hyperplasia) are detected, indicating that continuous uremia-related stimulation promotes parathyroid cell proliferation from diffuse to nodular growth. However, the key molecular mechanism is not fully understood, which narrows the range of therapeutic options for advanced secondary hyperparathyroidism. To address this issue, we utilized surgically resected normal and hyperplastic parathyroid glands to perform immunohistochemical analysis of a multifunctional cell cycle modulator, CCAAT enhancer binding protein (C/EBP)β. In contrast to normal parathyroid tissue and diffuse hyperplasia, the intensity of C/EBPβ staining was homogeneously increased in the parathyroid cells from nodules, along with a higher cyclin D1 labeling index (108.0 ± 19.5, mean ± SEM) and Ki-67 labeling index (31.70 ± 0.49). Normal and diffuse hyperplastic parathyroid glands had far fewer cyclin D1- and Ki-67-positive cells (P < 0.001). Immunofluorescent double staining showed abundant coexpression of Th235 (mitogen-activated protein kinase [MAPK] phosphorylation site) C/EBPβ, along with upregulation of cytoplasmic Ras in nodular hyperplasia. In conclusion, hyperplastic parathyroid cells in nodules have an autonomous proliferation mechanism similar to that of cancer, in which C/EBPβ is upregulated and phosphorylated to interact with the oncogenic Ras/MAPK pathway. C/EBPβ may be a novel target molecule for blocking the growth circuit that underlies parathyroid tumorigenesis in secondary hyperparathyroidism.

Regulator of G Protein Signaling 5 Is Highly Expressed in Parathyroid Tumors and Inhibits Signaling by the Calcium-Sensing Receptor

The molecular mechanisms responsible for aberrant calcium signaling in parathyroid disease are poorly understood. The loss of appropriate calcium-responsive modulation of PTH secretion observed in parathyroid disease is commonly attributed to decreased expression of the calcium-sensing receptor (CaSR), a G protein-coupled receptor. However, CaSR expression is highly variable in parathyroid adenomas, and the lack of correlation between CaSR abundance and calcium-responsive PTH kinetics indicates that mechanisms independent of CaSR expression may contribute to aberrant calcium sensing in parathyroid disease. To gain a better understanding of parathyroid tumors and the molecular determinants that drive parathyroid adenoma development, we performed gene expression profiling on a panel of 64 normal and neoplastic parathyroid tissues. The microarray data revealed high-level expression of genes known to be involved in parathyroid biology (PTH, VDR, CGA, CaSR, and GCM2). Moreover, our screen identified regulator of G protein signaling 5 (RGS5) as a candidate inhibitor of CaSR signaling. We confirmed RGS5 to be highly expressed in parathyroid adenomas relative to matched-pair normal glands. Transient expression of RGS5 in cells stably expressing CaSR resulted in dose-dependent abrogation of calcium-stimulated inositol trisphosphate production and ERK1/2 phosphorylation. Furthermore, we found that RGS5-nullizygous mice display reduced plasma PTH levels, an outcome consistent with attenuated opposition to CaSR activity. Collectively, these data suggest that RGS5 can act as a physiological regulator of calcium sensing by CaSR in the parathyroid gland. The abnormally elevated expression of RGS5 observed in parathyroid adenomas could thus represent a novel mechanism of CaSR desensitization in patients with primary hyperparathyroidism.

Morphological evaluation of parathyroid adenomas and immunohistochemical analysis of pcna and ki-67 proliferation markers

Parathyroid adenomas are the most common cause of primary hyperparathyroidism. Biological studies have shown that parathyroid adenomas are monoclonal proliferations. Up to date, five cell types have been identified in normal parathyroid tissues; chief cells, vacuolated chief cells, dark chief cells, oxyphil cells and transitional oxyphil cells. Most parathyroid adenomas are predominantly composed of chief cells. In this study, we aimed to indicate the relationship between the predominant cell type in parathyroid adenomas and proliferating cell nuclear antigen, Ki-67 antigen, and serum parathormone levels and the gland weight. 15 cases who had a diagnosis of parathyroid adenomas were included in the study. Histopathologically, the predominant cell type was determined in all the cases. Paraffin blocks were immunohistochemically stained with proliferating cell nuclear antigen and Ki-67. The average parathormone level of the cases was 239.52 ± 36.61 pg/ml before surgery. Mean gland weight was 1.69 ± 0.49 g. Two of the cases showed atypical adenoma characteristics. The predominant cell type was vacuolated chief cell. Immunohistochemical investigation showed that the mean average Ki-67 index value was 4.26 ± 0.86%. The mean proliferating cell nuclear antigen index was 93.20± 45.72/10³. There was a meaningful relationship between gland weights and serum parathormone levels. There was no meaningful relationship between predominant cell types and serum parathormone levels, proliferating cell nuclear antigen index, and Ki-67 index. The chief cell was identified as the predominant cell type. It can be concluded that parathyroid adenomas come into existence as a result of neoplastic proliferation of chief cells, especially vacuolated chief cells.

Intraoperative use of OCT in Robotic Assisted Thyroidectomy

CONCLUSION: This is the first study to systematically use OCT invivo to differentiate thyroid and parathyroid glands during robotic assisted thyroidectomy. OCT can be used to evaluate these structures, which can be then confirmed with histopathologic evaluation via frozen or permanent sections. In the future, OCT has potential for use as an intraoperative method to more efficiently differentiate parathyroid glands, and thus not only decrease the operating time, but potentially obviate the need for frozen section confirmation, which decreasing the risk of damaging the parathyroid during dissection. DISCUSSION: This study represents the first reported use of OCT during robot assisted surgery, adding to the collective knowledge of optical coherence tomography, while simultaneously evaluating trans-axillary robotic assisted surgery. Particular attention was paid to the feasibility of instrumentation during the procedure. As mentioned, an OCT catheter handle was created to direct the probe to its target tissue in addition to protecting the fiber from harm (Figure 3). Manual manipulation was found to be tedious and ineffective in positioning the probe in light contact with the tissue of interest. Utilization of the catheter allowed the consol surgeon to manipulate the probe with the bipolar graspers, providing control, while protecting the delicate technology from inadvertent destruction by the attenuated tactile sensation provided by the robot arms (Figure 4). In Figure 5, an OCT image of normal parathyroid tissue is illustrated, where the stromal fat can be observed (arrows) and clear differences can be observed when compared to fat tissue (Figure 6). METHODS: An OCT imaging system (Niris, Imalux, Cleveland, OH) was used to image parathyroid and thyroid in ten patients during robotic assisted thyroidectomy. Images were obtained by raster scanning, a single mode fiber across the interior of the probe. The imaging probe was sterilized and inserted through the incision and placed in light contact with the tissue of interest. Images were acquired at a frame rate of 1Hz and resolution of ~10 mm with a 1 mm depth penetration. Robotic assisted surgery was performed via the SI da Vinci Robot (Intuitive Surgical, Sunnyvale, CA). Due to the direction of the operative field and the depth of dissection from the axilla during robotic thyroid resection (Figure 1), novel instrumentation was created for this procedure, including the OCT probe handle, as seen in Figure 2. INTRODUCTION: Optical coherence tomography is a high resolution optical imaging modality that generates a cross-sectional image within turbid media such as living tissue with resolution approaching that of light microscopy. OCT relies upon intrinsic differences in tissue optical properties for image contrast and has been shown to accurately differentiate tissue types. Trans-axillary robotic surgery is a relatively new approach to thyroid lobectomy, parathyroidectomy, and total thyroidectomy. This technique preserves the anterior neck from potentially deforming scar, while maintaining a wide surgical access, which at times is sacrificed during minimally invasive techniques that attempt to reduced the visibility of the incision. Recent domestic and international literature and personal experience suggest its safety and efficacy in the surgical removal of the thyroid and parathyroid glands. Figure 1. Patient positioning and field of dissection.

Experience of the Reliability of Intraoperative Sampling of Tissue PTH in Parathyroid Surgery: Reply

We were delighted to read Mr. James Kirkby-Bott and Professor Bruno Carnaille’s comments [1] on our recent article. Beyond that, we were more than happy to learn that their findings may confirm our results. Furthermore, their large series will surely provide additional invaluable data regarding the intraoperative application of parathyroid aspirates in determination of tissue origin. We would like to respond to four points raised in their letter. First, as far as the different technique of sampling are concerned, we tried a size 26 needle in seven patients, but we did not detect much difference in terms of parathyroid hormone (PTH) level compared to the size 22 needle that we used in the rest of the study. We were more comfortable with the latter size and therefore used it. We cannot, however, provide evidence that needle size would not influence PTH level. While it may easily do so, most studies use the size 22 needle, and that also influenced our decision to use it. What is more important, we believe, is that the technique is well standardized within the practice of an institute, and that the same size needle is used to get comparable results. Whether it is a size 22 or a size 26 is irrelevant. If intraoperative tissue aspiration becomes popular in most centers worldwide, we can expect that a standardized technique will be agreed on in the future. Second, it is always difficult to explain why a few tests in a large series do not bring the expected result, as occurred in four of the cases in Kirkby-Bott and Carnaille’s study. However, we noticed a small difference in the sampling technique, which may provide some explanation. We always rinsed the needle after aspiration with 1 ml isotonic sodium chloride solution into a test tube and then vortexed it. Third, we firmly believe that normal parathyroid tissue cannot be reliably differentiated with intraoperative aspiration from pathological parathyroid tissue. Although our data also showed a statistically significant difference, the extensive overlap in between PTH ranges prevents differentiation to be applied in everyday practice at this time. We would be curious to learn Kirkby-Bott and Carnaille’s opinion once their study has been completed. Fourth and last, the economical aspect of a new test is certainly a key issue nowadays. It is a multifactorial problem and has affected factors like the way of determining of PTH levels, the cost of pathology, etc. Because time in the operating suite is considered to be relatively expensive worldwide, high sensitivity quick intraoperative diagnostic tests are generally attractive on the medical as well as managerial level. J. Horanyi (&) R. Szlavik 1st Surgical Department, Semmelweis University, Budapest, Hungary e-mail: hj@seb1.sote.hu

Experience of the Reliability of Intraoperative Sampling of Tissue PTH in Parathyroid Surgery: Letter to the Editor

We congratulate Horanyi et al. [1] on their work using parathyroid hormone (PTH) tissue aspirates to confirm tissue as being parathyroid in origin. Theirs is the largest reported series out of the four series published [1–4]. We have also been using a similar technique over the past 15 months and have collected 248 samples that have been validated by comparing biochemical results with fixed specimen histopathology results. Our results are due to be presented at the ESES meeting in Vienna in May. Our results look very much like Horanyi et al.’s with a 100% specificity achievable. However, we had four false-negative results from an inadequate first sample; repeat sampling confirmed their true nature. Because of this, the PTH level we needed to confirm parathyroid tissue origin at a 100% specificity was substantially higher at 1168 pg/ml. We constructed a ROC curve for the test (Table 1), the results of which were very encouraging: At a PTH level of 403 pg/ml, a sensitivity of 97.62% and specificity of 98.04% were achieved. Our sampling technique was slightly different and we are not sure if this may have accounted for the difference in sampling results. We used a smaller needle (23 gauge) and diluted the tissue in the hub of the needle to 2 ml of saline before placing the sample in an EDTA serum sample bottle to send to the lab as if it were a blood sample for intact PTH analysis. We used the standard assay and not the ‘‘quick assay’’ reading. We also found a significant difference in abnormal and normal parathyroid tissue aspirates in primary hyperparathyroidism (HPT) (paired t test, p = 0.011). As many people also measure intraoperative PTH, we agree that this is an economic technique for confirming tissue origin rather than relying on frozen section.

Intrathyroidal oncocytic parathyroid adenoma: A diagnostic pitfall on fine‐needle aspiration

Oncocytic parathyroid adenoma is a rarely seen benign neoplasm of the parathyroid and intrathyroidal location of this lesion is also uncommon. It can be easily misdiagnosed as Hürthle cell thyroid neoplasm on fine-needle aspiration (FNA). Here, an intrathyroidal oncocytic parathyroid adenoma in a 32-year-old male is reported. Ultrasonography showed a nodule 2 cm in largest diameter in the left thyroid lobe. FNA revealed cellular smears containing monotonous oncocytic cells arranged in monolayered sheets, pseudopapillary structures and clusters within a rich vascular network. The case was reported as "suspicious for Hürthle cell thyroid neoplasm" on FNA. Frozen section examination showed intrathyroidal oncocytic parathyroid adenoma with a rim of normal parathyroid tissue. The diagnosis was also confirmed with immunohistochemical stains (TTF-1, PTH, thyroglobulin) performed on permanent sections. Cytopathologic features of oncocytic parathyroid adenoma and Hürthle cell thyroid neoplasm are similar. It is important to keep oncocytic parathyroid adenoma in mind in the differential diagnosis of Hürthle cell thyroid neoplasm. Prior knowledge of clinical, radiological and laboratory data will avoid wrong cytopathologic diagnosis.

Expression and association of TRPC subtypes with Orai1 and STIM1 in human parathyroid

The mechanism behind Ca(2)(+) entry into the parathyroid cells has been widely debated, and the molecular identities of the responsible ion channels have not been established yet. In this study, we show that the parathyroid cells lack voltage-operated Ca(2)(+) channels. Passive store depletion by thapsigargin, on the other hand, induces a large non-voltage-activated non-selective cation current. The increase in intracellular Ca(2)(+) caused by thapsigargin is attenuated by 2-aminoethoxydiphenyl borate, a blocker of store-operated Ca(2)(+) entry (SOCE). Candidate molecules for non-voltage-operated Ca(2)(+) signaling were investigated. These included members of the transient receptor potential canonical (TRPC) ion channel family, as well as Ca(2)(+) release-activated Ca(2)(+) modulator 1 (Orai1) and stromal interaction molecule 1 (STIM1) that are key proteins in the SOCE pathway. Using RT-PCR screening, quantitative real-time PCR, and western blot, we showed expression of TRPC1, TRPC4, and TRPC6; Orai1; and STIM1 genes and proteins in normal and adenomatous human parathyroid tissues. Furthermore, co-immunoprecipitation experiments demonstrated a ternary complex of TRPC1-Orai1-STIM1, supporting a physical interaction between these molecules in human parathyroid.

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

BackgroundParathyroid 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, p16INK4A and RAR-β in parathyroid adenomas. In addition, the levels of global methylation were assessed by analyzing LINE-1 repeats.Methodology/Principal FindingsThe 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 p16INK4A was demonstrated in a single adenoma, whereas RAR-β 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/SignificanceThe 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.

Water-Clear Cell Adenoma of Parathyroid Gland: A Case Report and Literature Review

Parathyroid adenoma of water-clear cell type is an extremely rare neoplasm, and only very few cases have been reported to date. Here we report a case of waterclear cell adenoma of the parathyroid in a 59-year old female patient who presented with hypercalcemia and high parathyroid hormone (PTH) level. Sestamibi scan showed a possible parathyroid adenoma involving the right superior parathyroid gland. The surgically excised specimen weighed 13.3 grams and contained a cystic hemorrhagic mass measuring 1 x 1 x 0.8 cm. Histologically, the tumor mass was surrounded by a thin fibrous capsule with a compressed thin rim of normal parathyroid tissue. High power examination revealed the tumor to be composed of cells with vacuolated cytoplasm, and with minimal nuclear pleomorphism. Immunohistochemical analysis demonstrated positive staining for PTH and chromogranin. Histological examination of the right inferior parathyroid gland showed unremarkable parathyroid tissue. Postoperatively, the patient’s serum calcium level returned to normal. These findings support the diagnosis of waterclear cell adenoma of the parathyroid gland. In this report, we also reviewed related literature and compared our case with similar cases reported before.

Genes up- or down-regulated by high calcium medium in parathyroid tissue explants from patients with primary hyperparathyroidism

To investigate genes modulated in the parathyroid glands by calcium, expression levels of mRNA for all genes expressed in parathyroid tissue explants (PTEs) obtained from patients with primary hyperparathyroidism (I degrees -HPT) were analyzed by oligo-DNA microarray. PTEs obtained from 4 patients with I degrees -HPT were precultured in normocalcemic medium (Ca(++) 1.0-1.1 mM) for 7 days and then cultured in hypocalcemic medium (Ca(++) 0.60 mM) or hypercalcemic (Ca(++) 1.60 mM) medium containing 4 mg/dl phosphate for an additional 7 days. As expected, expression levels of mRNA for PTH and chromogranin A were decreased to less than 50% in the hypercalcemic medium when compared with those in the hypocalcemic medium. Furthermore, oligo-DNA microarray analyses revealed that 7 genes were up-regulated by more than 2-fold and more than 30 genes were down-regulated by more than 1/2 in PTEs. Interestingly, 9 of these genes (up-regulated genes: chemokine ligand 8, multiple C2 domain and transmembrane region protein 1; down-regulated genes: matrix metallopeptidase-9, B-box and SPRY domain-containing protein, nitric oxide synthase 2A, PTH, cartilage acidic protein 1, chromogranin A, and fibrin 1) were involved in calcium metabolism or calcium-signaling pathways in the parathyroid tissue. However, the expression level of mRNA for alpha-klotho was variable, and it was not constantly decreased in hypercalcemic medium under the present experimental conditions. Although it was not possible to use normal parathyroid tissue, this is the first reported study to have investigated the expression levels of mRNA for all genes in human parathyroid adenomas that are modulated by high calcium concentration in organ culture.

Differential expression of microRNAs in human parathyroid carcinomas compared with normal parathyroid tissue

Parathyroid carcinoma (PaC) is a rare cause of primary hyperparathyroidism. Though the loss of the oncosuppressor CDC73/HRPT2 gene product, parafibromin, has been involved in the hyperparathyroidism-jaw tumor syndrome and in a consistent set of sporadic PaCs, parathyroid carcinogenesis remains obscure. MicroRNAs are a new class of small, non-coding RNAs implicated in development of cancer, since their deregulation can induce aberrant expression of several target genes. The aim of the present study was to identify differentially expressed microRNAs in parathyroid cancers compared with normal tissues. We performed a TaqMan low-density array profiling of four parathyroid cancers harboring CDC73 inactivating mutations and negative for parafibromin immunostaining. Their microRNA profiling was compared with that of two normal parathyroid biopsies. Out of 362 human microRNAs assayed, 279 (77%) were successfully amplified. Fourteen and three microRNAs were significantly down- and over-expressed in parathyroid cancers respectively. Of these, miR-296 and miR-139 were down-regulated, and miR-503 and miR-222 were over-expressed with a null false discovery rate. Carcinomas could be discriminated from parathyroid adenomas by a computed score based on the expression levels of miR-296, miR-222, and miR-503 as miR-139 was similarly down-regulated in both cancers and adenomas. Finally, miR-296 and miR-222 levels negatively correlated with mRNA levels of the hepatocyte growth factor receptor-regulated tyrosine kinase substrate and p27/kip1 levels respectively. These results suggest the existence of an altered microRNA expression pattern in PaCs together with a potential role of miR-296 as novel oncosuppressor gene in these neoplasia.

β-catenin activation is not involved in sporadic parathyroid carcinomas and adenomas

Aberrant accumulation of beta-catenin has been found in various types of human tumors. The aim of this study was to evaluate whether Wnt/beta-catenin signaling is activated in parathyroid carcinomas and adenomas. We studied 154 parathyroid tumors (18 carcinomas (13 with distant metastases), six atypical adenomas, and 130 adenomas). Three normal parathyroid tissues were used as control. Direct sequencing of exon 3 of the CTNNB1 gene showed absence of stabilizing mutations in all the tumors. Immunostaining of beta-catenin was performed in all carcinomas and in 66 adenomas (including three atypical). Normal parathyroid showed a homogeneous distinct outer cell membrane staining in the majority of cells and no nuclear staining. A weak cytoplasmic staining was observed in one case. All tumors showed negative nuclear staining. With the exception of one carcinoma, which had a negative membrane staining, all other samples showed a membrane staining which was similar to that of the normal parathyroid. beta-Catenin expression was heterogeneous with a range of positive cells between 5 and 80%, independently of tumor type. Our results suggest that the Wnt/beta-catenin signaling pathway is not involved in the development of parathyroid carcinomas and adenomas.

Immunohistochemical studies on specificity and affinity of the BB5-antibody against human parathyroid tissue.

Localisation of parathyroid tissue in hyperparathyroidism may be difficult with standard methods. Immunoscintigraphy, using radiolabeled antibodies against epitopes of human parathyroid cells, could be a promising alternative. Therefore, we studied the necessary preconditions, whether the so-called BB5-antibody, directed against parathyroid cell membranes possesses sufficient specificity and affinity to be employed in immunoscintigraphy. Specificity was tested immunohistochemically with APAAP-staining of 39 different human tissues. Additionally, an immunoscore-based quantitative comparison was performed to test the affinity of BB5-antibody for normal and pathologic parathyroid tissue. Specificity was proven by the fact that of all 39 tested tissue-types only the parathyroid tissue was BB5-positive. Normal parathyroid tissue showed a significantly higher affinity to the BB5-antibody than pathologic parathyroid tissue (p < 0.03). However, all tissue samples from primary or secondary hyperparathyroidism exhibited sufficient staining. We conclude that the BB5-antibody fulfills the necessary conditions to be tried for immunoscintigraphical localisation of the parathyroid glands.

Adenomatous Human Parathyroid Cells Exhibit Impaired Sensitivity tol-Amino Acids

Primary hyperparathyroidism, which occurs most commonly in patients with adenomatous disease of a single parathyroid gland, arises as a result of impaired extracellular Ca(2+) (Ca(2+)(o))-dependent feedback on PTH secretion, a process mediated by the calcium-sensing receptor (CaR). Because the Ca(2+)(o) sensitivity of the CaR is positively modulated by L-amino acids, we decided to investigate whether the impaired feedback of PTH secretion in adenomatous parathyroid cells might arise from decreased sensitivity to L-amino acids. Samples of normal and adenomatous human parathyroid cells were prepared by collagenase treatment and then exposed in vitro to various concentrations of Ca(2+)(o) or the CaR-active amino acid, L-phenylalanine (L-Phe). Excess normal parathyroid tissue was obtained from parathyroid autotransplants at the time of thyroid surgery. Samples of adenomatous tissue were obtained from histologically confirmed parathyroid adenomas. The primary measure was sensitivity of Ca(2+)(o)-dependent PTH secretion to the amino acid L-Phe. The secondary measure was sensitivity of Ca(2+)(o)-dependent intracellular Ca(2+) mobilization to L-Phe. Parathyroid adenomas exhibited reduced sensitivity to the CaR-active amino acid L-Phe, which affected both Ca(2+)(o)-dependent PTH secretion and Ca(2+)(o)-dependent intracellular Ca(2+) mobilization as a measure of CaR-dependent signaling in parathyroid cells. Impaired L-amino acid sensing by calcium-sensing receptors in adenomatous parathyroid cells contributes to the loss of feedback control of PTH secretion in primary hyperparathyroidism. The CaR's amino acid binding site may be exploited as a target in the medical treatment of primary and perhaps other forms of hyperparathyroidism.

Remedial Operation for Primary Hyperparathyroidism

Remedial surgery for patients with persistent or recurrent primary hyperparathyroidism (1 degrees HPT) remains a significant challenge. Cervical reexploration is technically difficult; reoperative neck anatomy is distorted by fibrosis and, as a result, remedial 1 degrees HPT patients carry an increased risk of injury to the recurrent (RLN) and superior laryngeal nerve(s) as well as to normal residual parathyroid tissue. Causative hyperfunctioning parathyroid tissue is also more frequently ectopic in the remedial setting and can thus be difficult to localize. This report assimilates the current data underlying preoperative, intraoperative and postoperative remedial 1 degrees HPT management and presents an evidence-based algorithm for the management of remedial parathyroid disease. Recommendations are graded according to the quality of supporting data using the system initially developed by Sackett (Chest 95:2S-4S, 1989) and subsequently modified by Heinrich et al. (Ann Surg 243:154-168, 2006). Recent advances in preoperative localization and intraoperative adjuncts have lead to substantial improvements in outcomes after remedial surgery. Preoperative localization techniques, including sestamibi scintigraphy (MIBI), high resolution ultrasound (US), US-guided fine needle aspiration (FNA) and selective venous sampling (SVS), coupled with intraoperative adjuncts such as the rapid parathyroid hormone (PTH) assay have lead to reoperative cure rates as high as 96 percent. Nonetheless, management of remedial 1 degrees HPT varies significantly between surgeons and no formal recommendations standardizing the care of these patients have been published. Despite the significant challenges associated with remedial surgery for 1 degrees HPT, excellent outcomes can be reproducibly achieved when proper pre-, intra-, and postoperative management is employed.

The profile of ErbB/Her family genes copy number assessed by real-time PCR in parathyroid adenoma and hyperplasia associated with sporadic primary hyperparathyroidism.

Hyperparathyroidism (pHPT) is a relatively frequent endocrinopathy, however, the molecular mechanisms of its etiology remain poorly understood. This disorder is mainly associated with benign tumours (adenoma) and hyperplasia of the parathyroid, hence, the focus is directed also to genes that are likely to be involved in carcinogenesis. Among such genes are ErbB/Her family genes already used in diagnosis of other tumours (e.g., breast carcinoma) and reported also to play a role in development of endocrine lesions. So far, ErbB-1/Her-1/EGFR expression has been detected in pHPT-associated adenomas and hyperplasia as opposed to no expression in normal parathyroid tissue. Moreover, losses or gains of the fragments of chromosomes where ErbB/Her genes are located have been reported. In this study, the gene dosage of ErbB/Her family genes were determined for the first time in parathyroid adenomas, hyperplasia and morphologically unchanged tissue in order to establish their putative role in the development of the disease. Genomic DNA was isolated from 33 patients with sporadic hyperparathyroidism and the gene copy numbers were assessed using real-time PCR. The ErbB/Her genes' profile was unaltered in most of the examined samples. Two low-level amplifications of ErbB-1/Her-1/EGFR gene, two deletions of ErbB-2/Her-2, and six deletions of ErbB-4/Her-4 were found. The ErbB-3/Her-3 gene remained unaffected. No correlation with clinical parameters was found for any gene. Both the low number of alterations and a lack of their associations with clinical parameters exclude the prognostic value of the ErbB/Her genes family in parathyroid tumourigenesis. Nevertheless, the ErbB-4/Her-4 deletions seem to be interesting for further investigations, especially in the context of PTH secretion.

Parathyroid Lipoadenomas and Lipohyperplasias

Parathyroid lipoadenomas and lipohyperplasias are rare histologic variants with both an increase in stromal fat and parenchyma. We report the most comprehensive single institution series of lipoadenomas and lipohyperplasias to date and review the literature. Eight lipoadenomas and 3 lipohyperplasias (27 y period) were reviewed. The mean age was 60.3 years (range: 50 to 77 y) with a female predilection (1.75:1). The most common symptoms on presentation were fatigue (55.6%) and bone/joint pain (44.4%). Only 1 patient was euparathyroid. Ultrasound localized lipoadenomas in 50% of tested cases whereas sestamibi was successful in 71.4%. Despite increased stromal fat (median: 50%), the weight and the appearance of large, occasionally nodular expansions of parathyroid parenchyma within the fatty stroma distinguished lipoadenomas and lipohyperplasias from normal parathyroid tissue; none of the cases were misclassified as normal on frozen section. Mean weight for lipoadenomas was 1553 mg (range: 173 to 4587 mg), whereas the mean weight for lipohyperplasia glands was 389.1 mg. Variant morphologies included follicular patterned, oxyphil predominant, and thymic elements (thymolipoadenoma). In 1 lipohyperplasia case, not all glands were involved. Oil Red O stains showed decreased intracytoplasmic lipid in most cases. Median follow-up was 9.2 months (range: <1 to 51 mo). Only 1 lipohyperplasia patient had persistent hypercalcemia, but was asymptomatic. Lipoadenomas and lipohyperplasias are clinically similar and as histologically diverse as their conventional counterparts. Lipoadenomas are more difficult to localize preoperatively by imaging. Despite the potential difficulty at frozen section, accurate weight documentation and recognition of key histologic features diminish this challenge.