Enhanced protein kinase B/Akt signalling in pituitary tumours.

Pituitary tumours have previously been shown to harbour several abnormalities that cause deregulation of the cell cycle, particularly down-regulation of expression of the cyclin-dependent kinase inhibitor p27. However, it has been unclear whether these are the primary initiating events, or are secondary to other more proximate alterations in signalling pathways. In other cellular systems the Akt signalling pathway has been associated with downstream modulation of cell-cycle control. The aim of the present study was to test the hypothesis that Akt signalling is enhanced in pituitary tumours, and to see if changes in Akt expression are related to previous findings on low expression levels of the nuclear cell-cycle inhibitor p27 in pituitary tumours. We examined normal and adenomatous human pituitary tissue for mRNA and protein expression of Akt1, Akt2 and p27, and the activation of Akt, as well the phosphatase involved in the inactivation of Akt, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). In pituitary adenomas Akt1 and Akt2 mRNA were found to be over-expressed compared with normal pituitary, while PTEN transcripts showed similar levels between the two tissue types. Immunohistochemical expression of phospho-Akt was found to be higher in the tumours than normal pituitaries, while the protein expression of nuclear p27 and PTEN was lower in the adenomas. However, the expression of p27 and Akt were not directly correlated. PTEN sequencing revealed no mutation in the coding region of the gene in pituitary adenomas, and thus we did not locate a cause for the increased phosphorylation of Akt. In summary, we have shown over-expression and activation of the Akt pathway in pituitary tumours, and we speculate that cell-cycle changes observed in such tumours are secondary to these more proximate alterations. Since Akt is a major downstream signalling molecule of growth factor-liganded tyrosine kinase receptors, our data are most compatible with an abnormality at this level as the primary driver of pituitary tumorigenesis.

Treatment with oral biphosphonates can increase the sensitivity of sestamibi radionuclide imaging in patients with primary hyperparathyroidism.

The sensitivity of 99mTc-sestamibi scan in detecting parathyroid disease in primary hyperparathyroidism (PHP) is almost 90%, and therefore facilitates successful parathyroidectomy. To enhance the diagnostic accuracy of the procedure, we repeated imaging with 99mTc-sestamibi in 15 patients with PHP and an initially negative (11 patients) or weakly positive (four patients) 99mTc-sestamibi scan after the administration of 10 mg of oral alendronate for 2 months. Serum calcium, phosphate and parathormone (PTH) measurements were obtained at presentation and after 1 and 2 months' treatment with alendronate. Eight patients with an initially negative 99mTc-sestamibi scan demonstrated at least one area of uptake in the repeated scan. Six of these patients underwent surgery and obtained a biochemical cure; a single adenoma was found in four and hyperplasia in the remaining two. In all four patients with an initially weakly positive 99mTc-sestamibi scan, the repeated scan demonstrated enhanced uptake and also revealed further areas of uptake. Two of these patients underwent surgery with a biochemical cure; an adenoma was found in one and hyperplasia in another. Compared with baseline there was a significant increase in PTH but not in calcium or phosphate levels during treatment with alendronate. We suggest that, in patients with PHP and a negative or weakly positive initial 99mTc-sestamibi scan, administration of oral alendronate may be associated with a positive repeated 99mTc-sestamibi scan and can thus enhance the sensitivity of the procedure.

Activating point mutations in cyclin-dependent kinase 4 are not seen in sporadic pituitary adenomas, insulinomas or Leydig cell tumours.

Cell cycle dysregulation is one of the defining features of cancer. Cyclin-dependent kinase 4 (CDK4), together with its regulatory subunit cyclin D, governs cell cycle progression through the G1 phase. Cyclin-dependent kinase inhibitors, including p16(INK4A) (encoded by CDKN2A), in turn regulate CDK4. In particular, dysregulation of the p16/CDK4/cyclin D complex has been established in a variety of types of human tumours. Dominant activating mutations affecting codon 24 of the CDK4 gene (replacement of Arg24 by Cys or His) render CDK4 insensitive to p16(INK4) inhibition and are responsible for melanoma susceptibility in some kindreds. However, 'knock-in' mice homozygous for the CDK4(R24C) mutation were noted to develop multiple neoplasia, most commonly including endocrine tumours: pituitary adenomas, insulinomas and Leydig cell testicular tumours. We therefore speculated that sporadic human endocrine tumours might also harbour such mutations. The aim of the current study was to analyze the CDK4 gene for the two characterized activating mutations, R24C and R24H, in sporadic human pituitary adenomas, insulinomas and Leydig cell tumours. We used DNA extracted from 61 pituitary adenomas, and paired tumorous and neighboring normal genomic DNA extracted from 14 insulinoma and 6 Leydig cell tumour samples. Genomic DNA from patients with familial melanoma harbouring the R24C or the R24H mutations served as positive controls. All samples were subjected to PCR, mutation-specific restriction digests and/or sequencing. Both methodologies failed to detect mutations at these two sites in any of the sporadic endocrine tumours including pituitary adenomas, benign or malignant insulinomas or Leydig cell tumours, while the positive controls showed the expected heterozygote patterns. Protein expression of CDK4 was demonstrated by immunohistochemistry and Western blotting in pituitary and pancreatic samples. These data suggest that the changes in the regulatory 'hot-spot' on the CDK4 gene, causing various endocrine tumours in CDK4(R24C/R24C )mice, are not a major factor in sporadic pituitary, insulin beta-cell or Leydig cell tumorigenesis.

Composite somatotroph--ACTH-immunoreactive pituitary adenoma with transformation of hyperplasia to adenoma.

The majority of pituitary adenomas are solitary and monohormonal, producing only one hormone. Double or multiple adenomas are rare. Plurihormonal adenomas may be monomorphous consisting of one cell type producing more than one hormones or plurimorphous composed of two or more distinct cell populations each producing different hormones. Primary pituitary hyperplasia is uncommon and transformation to adenoma has rarely been documented. We describe a unique case of somatotroph adenoma combined with ACTH-immunoreactive cell hyperplasia and focal transformation to adenoma. The 53-yr-old man was presented with a 2-yr history of headaches, enlargement of the hands and feet and coarsening of facial features. His blood GH was 17.5 ng/ml and he had absence of GH suppressional oral glucose tolerance testing. MRI demonstrated a mass with maximum diameter of 1.5 cm, on the left side of the pituitary, without invasion of surrounding tissues. Transsphenoidal surgery was performed. Morphology disclosed a mostly chromophobic tumor, immunoreactive for GH with ultrastructural characteristics of sparsely granulated somatotroph adenoma. The adenoma cell population was focally admixed with hyperplastic PAS positive and ACTH immunoreactive cells showing the electron microscopic features characteristic of corticotrophs. In these areas the acini were enlarged with distorted architecture of the reticulin pattern. Dissolution of the reticulin fiber network and transformation of hyperplastic ACTH-immunoreactive cells to adenoma was evident in small areas. The hyperplastic and adenomatous ACTH-immunoreactive cells were admixed with somatotroph adenoma cells. Due to lack of biochemically obvious cortisol hypersecretion, this ACTH-immunoreactive adenoma was classified as silent "corticotroph" subtype 1. This is an unusual case of composite pituitary adenoma consisting of somatotroph cells and hyperplastic ACTH-immunoreactive cells transforming to a frank adenoma.