Loss of neuronatin expression is associated with promoter hypermethylation in pituitary adenoma.

The imprinted gene, neuronatin (NNAT), is one of the most abundant transcripts in the pituitary and is thought to be involved in the development and maturation of this gland. In a recent whole-genome approach, exploiting a pituitary tumour cell line, we identified hypermethylation associated loss of NNAT. In this report, we determined the expression pattern of NNAT in individual cell types of the normal gland and within each of the different pituitary adenoma subtypes. In addition, we determined associations between expression and CpG island methylation and used colony forming efficiency assays (CFE) to gain further insight into the tumour-suppressor function of this gene. Immunohistochemical (IHC) co-localization studies of normal pituitaries showed that each of the hormone secreting cells (GH, PRL, ACTH, FSH and TSH) expressed NNAT. However, 33 out of 47 adenomas comprising, 11 somatotrophinomas, 10 prolactinomas, 12 corticotrophinomas and 14 non-functioning tumours, irrespective of subtype failed to express either NNAT transcript or protein as determined by quantitative real-time RT-PCR and IHC respectively. In normal pituitaries and adenomas that expressed NNAT the promoter-associated CpG island showed characteristics of an imprinted gene where approximately 50% of molecules were densely methylated. However, in the majority of adenomas that showed loss or significantly reduced expression of NNAT, relative to normal pituitaries, the gene-associated CpG island showed significantly increased methylation. Induced expression of NNAT in transfected AtT-20 cells significantly reduced CFE. Collectively, these findings point to an important role for NNAT in the pituitary and perhaps tumour development in this gland.

The relationship between the systemic inflammatory response, tumour proliferative activity, T-lymphocytic and macrophage infiltration, microvessel density and survival in patients with primary operable breast cancer.

The significance of the inter-relationship between tumour and host local/systemic inflammatory responses in primary operable invasive breast cancer is limited. The inter-relationship between the systemic inflammatory response (pre-operative white cell count, C-reactive protein and albumin concentrations), standard clinicopathological factors, tumour T-lymphocytic (CD4+ and CD8+) and macrophage (CD68+) infiltration, proliferative (Ki-67) index and microvessel density (CD34+) was examined using immunohistochemistry and slide-counting techniques, and their prognostic values were examined in 168 patients with potentially curative resection of early-stage invasive breast cancer. Increased tumour grade and proliferative activity were associated with greater tumour T-lymphocyte (P<0.05) and macrophage (P<0.05) infiltration and microvessel density (P<0.01). The median follow-up of survivors was 72 months. During this period, 31 patients died; 18 died of their cancer. On univariate analysis, increased lymph-node involvement (P<0.01), negative hormonal receptor (P<0.10), lower albumin concentrations (P<0.01), increased tumour proliferation (P<0.05), increased tumour microvessel density (P<0.05), the extent of locoregional control (P<0.0001) and limited systemic treatment (P

The relationship between tumour proliferative activity, the systemic inflammatory response and survival in patients undergoing curative resection for colorectal cancer.

BACKGROUND: The aim of the present study was to examine the relationship between Ki-67, C-reactive protein and cancer-specific survival in patients undergoing resection for colorectal cancer. METHOD: One hundred and forty-seven patients undergoing potentially curative resection for colorectal cancer had preoperative C-reactive protein concentrations and tumour Ki-67 labelling index measured. RESULTS: On univariate analysis, age (P < 0.001), Dukes stage (P < 0.001), C-reactive protein (P < 0.001) and expression of Ki-67 (< 0.01) were associated with poorer cancer-specific survival. Ki-67 labelling index and C-reactive protein were correlated (r(s) = 0.172, P = 0.037). On multivariate analysis, age (HR 1.96, 95% CI 1.26-3.04, P = 0.003), Dukes stage (HR 4.38, 95% CI 2.11-9.09, P < 0.001) and C-reactive protein (HR 4.09, 95% CI 2.04-8.24, P < 0.001) retained significance. CONCLUSION: Increased tumour proliferation is associated with a systemic inflammatory response and poor cancer-specific survival in patients undergoing potentially curative surgery for colorectal cancer.

Elevated luteinizing hormone induces expression of its receptor and promotes steroidogenesis in the adrenal cortex.

Transgenic (TG) female mice expressing bLHbeta-CTP (a chimeric protein derived from the beta-subunit of bovine luteinizing hormone [LH] and a fragment of the beta-subunit of human chorionic gonadotropin [hCG]) exhibit elevated serum LH, infertility, polycystic ovaries, and ovarian tumors. In humans, increased LH secretion also occurs in infertility and polycystic ovarian syndrome, often concomitant with adrenocortical dysfunction. We therefore investigated adrenal function in LH overexpressing bLHbeta-CTP female mice. The size of their adrenals was increased by 80% with histological signs of cortical stimulation. Furthermore, adrenal steroid production was increased, with up to 14-fold elevated serum corticosterone. Primary adrenal cells from TG and control females responded similarly to ACTH stimulation, but, surprisingly, the TG adrenals responded to hCG with significantly increased cAMP, progesterone, and corticosterone production. LH receptor (LHR) expression and activity were also elevated in adrenals from female TG mice, but gonadectomized TG females showed no increase in corticosterone, suggesting that the dysfunctional ovaries of the intact TG females promote adrenocortical hyperfunction. We suggest that, in intact TG females, enhanced ovarian estrogen synthesis causes increased secretion of prolactin (PRL), which elevates LHR expression. Chronically elevated serum LH, augmented by enhanced PRL production, induces functional LHR expression in mouse adrenal cortex, leading to elevated, LH-dependent, corticosterone production. Thus, besides polycystic ovaries, the bLHbeta-CTP mice provide a useful model for studying human disorders related to elevated LH secretion and adrenocortical hyperfunction.

TNM staging of midgut and hindgut (neuro) endocrine tumors: a consensus proposal including a grading system.

Criteria for the staging and grading of neuroendocrine tumors (NETs) of midgut and hindgut origin were established at the second Consensus Conference in Frascati (Rome) organized by the European Neuroendocrine Tumor Society (ENETS). The proposed tumor-node-metastasis (TNM) classifications are based on the recently published ENETS Guidelines for the Diagnosis and Treatment of gastroenteropancreatic NETs and follow our previous proposal for foregut tumors. The new TNM classifications for NETs of the ileum, appendix, colon, and rectum, and the grading system were designed, discussed, and consensually approved by all conference participants. These proposals need to be validated and are meant to help clinicians in the stratification, treatment and follow-up of patients.

TNM staging of foregut (neuro)endocrine tumors: a consensus proposal including a grading system.

The need for standards in the management of patients with endocrine tumors of the digestive system prompted the European Neuroendocrine Tumor Society (ENETS) to organize a first Consensus Conference, which was held in Frascati (Rome) and was based on the recently published ENETS guidelines on the diagnosis and treatment of digestive neuroendocrine tumors (NET). Here, we report the tumor-node-metastasis proposal for foregut NETs of the stomach, duodenum, and pancreas that was designed, discussed, and consensually approved at this conference. In addition, we report the proposal for a working formulation for the grading of digestive NETs based on mitotic count and Ki-67 index. This proposal, which needs to be validated, is meant to help clinicians in the stratification, treatment, and follow-up of patients.

Loss of pRb expression in pituitary adenomas is associated with methylation of the RB1 CpG island.

We recently showed loss of pRb in a proportion of pituitary tumors that was not associated with loss of heterozygosity of an RB1 intragenic marker. To further define the mechanism responsible for loss of retinoblastoma protein (pRb) expression, we have investigated the methylation status of the CpG island contained within the promoter region of the RB1 gene, together with sequence analysis of the essential promoter region and exons coding for the protein-binding pocket domain. Methylation of the CpG island within the RB1 promoter region was detected in 6 of 10 tumors that failed to express pRb. In contrast, 18 of 20 tumors and all six histologically normal postmortem pituitaries that expressed pRb were unmethylated. No inactivating mutations were found within the RB1 promoter region in the four unmethylated tumors that failed to express pRB. However, one or more exons comprising the coding region for the protein-binding pocket domain were shown to be homozygously deleted in three of four tumors available for analysis. This study describes an additional tumor type, in addition to retinoblastoma, in which methylation of the RB1 promoter is associated with loss of pRb expression. Furthermore, we show that in addition to methylation of the RB1 promoter region, deletion within the protein-binding pocket domain is associated with a loss of detectable pRb expression. The reactivation of tumor suppressor genes, silenced through methylation, represents a promising therapeutic target in sporadic pituitary adenomas.

Chromosome 13q deletion mapping in pituitary tumors: infrequent loss of the retinoblastoma susceptibility gene (RB1) locus despite loss of RB1 protein product in somatotrophinomas.

Two recent studies have described allelic loss of an RB1 intragenic marker on chromosome 13q in aggressive and metastatic pituitary tumors that did not correlate with loss of pRB. The second report also showed that losses were more frequently associated with a more centromeric marker. Because both of these studies suggest the presence of another or other tumor suppressor genes (TSGs) on 13q, we carried out an allelotype analysis encompassing known and recently described TSG loci on 13q, together with immunohistochemical analysis of pRB. We analyzed 82 nonfunctional tumors and 53 somatotrophinomas subdivided into invasive and noninvasive cohorts. A significantly higher frequency of loss, at one or more of 13 markers, was evident in the invasive nonfunctional tumors (54%, 26 of 48) than in their noninvasive counterparts (29%, 10 of 34). An approximately equal frequency of loss was apparent in invasive (28%, 5 of 18) and noninvasive (31%, 11 of 35) somatotrophinomas at one or more markers. In those tumors harboring deletion, loss at two or more markers was more frequent in invasive nonfunctional tumors 65% (17 of 26) compared with 36% (4 of 11) of their noninvasive counterparts. In somatotrophinomas, 40% (2 of 5) of invasive tumors as compared with 64% (7 of 11) of noninvasive tumors had evidence of two or more deletions. In tumors showing loss at two or more loci, the majority showed large deletions; however, loss of the RB1 intragenic marker D13S153 was infrequent. In most cases, loss at individual markers was more frequent in invasive tumors than their noninvasive counterparts. A marker 3 cM telomeric to RB1 (D13S1319) showed the highest frequency of deletion in both invasive cohorts (29% of somatotrophinomas and 24% of nonfunctional tumors). Immunohistochemical analysis of pRB showed frequent loss in somatotrophinomas (27%, 9 of 33) in comparison with 4% (2 of 53) of non-functional tumors. Although loss of pRB did not correlate with loss of an intragenic marker or tumor grade, it was significantly associated with the somatotrophinoma subtype (P = 0.002). These data suggest that chromosome 13q is a frequent target for allelic deletion in pituitary tumors and point to another or other TSG loci in these regions.

Best practice no 183. Examination of parathyroid gland specimens.

The pathological examination of parathyroid glands is an essential component of the evaluation of hyperparathyroidism. Traditionally, this has involved intraoperative frozen sections during bilateral surgical exploration of the neck, to confirm removal of parathyroid tissue. With recent developments in imaging, some diseased glands can be localised preoperatively, enabling removal by minimally invasive, targetted surgery, with or without additional non-histological intraoperative procedures to confirm the removal of all hyperfunctioning parathyroid tissue. This article reviews these developments and describes the ideal approach to reporting parathyroid specimens.

Bromocriptine-responsive Cushing's disease associated with anterior pituitary corticotroph hyperplasia or normal pituitary gland.

Cushing's disease may originate from either the anterior pituitary lobe or the neurointermediate lobe, a major characteristic of the latter group being bromocriptine responsiveness. This study of two patients with Cushing's disease demonstrates that bromocriptine responsiveness also may be associated with anterior pituitary corticotroph hyperplasia or a normal pituitary gland. The two patients were a 14-yr-old boy (patient 1) and a 29-yr-old woman (patient 2); their cortisol production rates were 121 and 234 mumol/24 h (normal values, less than 80 mumol/24 h), respectively. A single oral dose of 2.5 mg bromocriptine resulted in a gradual decrease in plasma cortisol from 680 to 130 nmol/L after 6 h in patient 1 and from 640 to 170 nmol/L after 4 h in patient 2. Both patients then received medical treatment for a period of 2 yr. Whereas sodium valproate was ineffective, bromocriptine (5 mg/day) abruptly decreased the cortisol production rate to 60 mumol/24 h in patient 1 and to 138 mumol/24 h in patient 2, and both patients had a partial clinical remission. Despite an increase in bromocriptine dosage to 30 mg daily and 24 mg/day cyproheptadine, the clinical and biochemical remission was not sustained in patient 1, and no further improvement occurred in patient 2. Total hypophysectomy then was performed in both patients. Sections of the pituitary from patient 1 showed diffuse anterior pituitary corticotroph hyperplasia, with early nodule formation in some areas. The sections from patient 2 showed normal numbers and distribution of corticotrophs. We conclude that the heterogeneous nature of Cushing's disease cannot be explained on the basis simply of anterior vs. intermediate lobe origin of the disease.

Metastatic prolactinoma: effect of octreotide, cabergoline, carboplatin and etoposide; immunocytochemical analysis of proto-oncogene expression.

A 49-yr-old woman presented with an extensive prolactinoma (serum PRL > 10,000 mU/L, normal range < 450 mU/L). Over a 5-yr period following transsphenoidal surgery and pituitary irradiation, she became increasingly resistant to high doses of bromocriptine and underwent transfrontal surgery followed by stereotactic radiotherapy. In spite of these treatments, serum prolactin estimations rose progressively to > 100,000 mU/L. Magnetic resonance imaging scanning demonstrated a massive cystic tumor invading the temporal lobes, extending into the cervical and thoracic spine, with metastases to cervical lymph nodes. High-dose cabergoline administration resulted in a 30% decrease in serum PRL. Octreotide was administered as a continuous sc infusion with a profound analgesic effect on facial pain but with no effect on tumor progression. She was treated with a course of chemotherapy consisting of carboplatin and etoposide without any noticeable effect. The patient died 6 months following chemotherapy. Immunocytochemical analysis demonstrated positive nuclear staining for WAF-1, Rb protein, c-myc, and p53 both in the original and metastatic tumors. The metastases but not the primary tumor stained for c-jun. Metastatic prolactinoma remains a therapeutic challenge. It is associated with a variable proto-oncogene expression, which may be coincidental or causal. Cabergoline had no advantage over bromocriptine. Octreotide relieved facial pain but did not alter tumor progression. An effective therapy for metastatic prolactinoma remains to be identified.

Allelic deletion in pituitary adenomas reflects aggressive biological activity and has potential value as a prognostic marker.

Tumors of the pituitary gland are usually benign adenomas and account for 10% of all intracranial neoplasms. Five pituitary tumors have previously been reported to harbor multiple allelic deletions. Of these, three displayed particularly aggressive biological behavior, whereas there were no clinical details provided for the others. This study was designed to test the hypothesis that genetic deletions are a marker of invasive behavior and to identify the loci most commonly involved. Accordingly, we studied two cohorts of pituitary tumors, classified radiologically as invasive or noninvasive, for loss of heterozygosity (LOH). There is a significantly higher frequency of LOH in invasive tumors (10.8% of all loci examined) compared to noninvasive tumors (2.4%; P < 0.001). Of the 11 loci investigated, 75% of the allelic deletions identified in invasive tumors were found at 4 loci: 11q13, 13q12-14, 10q, and 1p. Twenty of 47 invasive tumors had evidence of at least 1 allelic deletion, whereas 14 of 20 had more than 1. Of the 6 tumors with only 1 deletion, 5 involved the 11q13 locus, suggesting that this is an early change in the transition from noninvasive to invasive adenoma. Comparison of invasive and noninvasive tumors demonstrates a significantly higher frequency of deletions affecting 11q13 (P < 0.001), 13q12-14 (P < 0.05), and 10q26 (P < 0.05) in invasive tumors. In addition, allelic deletion correlates with increasingly invasive behavior (modified Hardy classification), as 73% of grade 4 tumors compared to 33% of grade 3 and 9.5% of grade 1 and 2 tumors demonstrated LOH at any locus. Furthermore, in some tumors we identified a breakpoint between markers intragenic and extragenic to the retinoblastoma gene (Rb1) on chromosome 13q, suggesting that tumor suppressor genes other than or in addition to Rb1 may be involved in pituitary tumorigenesis. This was further supported by the presence of Rb protein in two of four tumors where the genetic loss extended to include the intragenic marker D13S153. Early identification of tumors with likely invasive potential by means of genetic analysis (LOH) may provide useful information on potential tumor behavior and aid tumor management in a manner that is not possible using routine histological methods. A large prospective study is required in patients without radiological evidence of invasion to assess the value of LOH in predicting outcome and for planning treatment.

Localization of EGF receptors in frozen tissue sections by antibody and biotinylated EGF-based techniques.

We developed a sensitive EGF receptor detection method for frozen tissue sections using biotinylated EGF as the primary reagent. The method was directly compared with an immunohistochemical technique based on an anti-EGF receptor monoclonal antibody (MAb EGFR1) in normal human and rat tissues and in human tumors. The method was more sensitive than a previously published biotinylated EGF-based technique. In normal human tissues and in 37 of the 50 tumors, the binding pattern mirrored that of positive staining with EGFR1. Five further tumors showed weak immunoreactivity, but in these no binding of biotinylated EGF was detected. The remaining eight tumors were negative by both techniques. The discordant cases may reflect a lower level of sensitivity of the ligand-binding technique or, alternatively, abnormal receptors may have been expressed in these tissues. EGF receptors could be detected in rat liver with biotinylated EGF but not with the antibody, indicating the usefulness of the ligand-based technique in cross-species studies.

The mitogenic effects of corticotrophin-releasing factor on the anterior pituitary gland of the rat.

The immunohistochemically defined corticotroph population in the anterior pituitary gland of the adult male Sprague-Dawley rat has been quantified at 2 and 6 weeks after bilateral adrenalectomy using the stereological measurement, volume density (Vv). An approximately twofold increase in corticotroph Vv was demonstrated at 2 weeks in adrenalectomized rats compared with that in sham-operated controls and this was maintained at 6 weeks. Daily i.p. injections of ovine corticotrophin-releasing factor (CRF-41) induced a significant dose-related increase in corticotroph Vv when administered at doses of 25 and 50 micrograms/kg body weight, but this was less than the increase following adrenalectomy. Assessment of changes in mitotic activity of the total anterior lobe of adult Sprague-Dawley rats and of the corticotroph population was also made after daily i.p. injections of 50 micrograms CRF-41/kg for 2 and 7 days. There was no increase in overall mitotic index at either time. However, the numbers of mitotic corticotrophs were significantly increased in CRF-injected animals compared with those in saline-injected rats. These results indicate a role for CRF-41 in the regulation of corticotroph growth.

The corticotrophic cells of the canine pituitary gland in pituitary-dependent hyperadrenocorticism.

The distribution of specifically stained corticotrophic cells has been studied in the pituitary glands of 11 dogs with pituitary-dependent hyperadrenocorticism. The results suggest that the disease is not a single entity, and that some cases are caused by primary abnormality of the pituitary gland whereas others appear to be the result of dysfunction of the hypothalamus or central nervous system. The patterns correspond closely to those demonstrated in the human pituitary gland in Cushing's disease, and confirm that the canine disease is a useful model for the study of the pathogenesis of the variants of the condition.

The expression of inhibin/activin subunits in the human adrenal cortex and its tumours.

Inhibins and activins are dimeric proteins of the transforming growth factor-beta superfamily which have been shown to be expressed in the adrenal cortex. Recent studies have suggested a role for these peptides in the pathogenesis and/or function of adrenal tumours. To investigate further their physiological and pathological roles, we have documented immunoreactivity for inhibin alpha, betaA and betaB subunits in normal adult and fetal human adrenals, in hyperplastic adrenals and in adrenal tumours. In the normal and hyperplastic adult gland, diffuse immunopositivity was demonstrated for beta subunits, suggesting that activins (beta beta dimers) can be expressed in all zones. Inhibin alpha was limited to the zona reticularis and the innermost zona fasciculata in the normal gland, extending centripetally into the zona fasciculata in hyperplasia, supporting a role for ACTH in the regulation of expression, and suggesting that expression of inhibins (alpha beta dimers) is restricted. Immunopositivity for all three subunits was seen in both fetal and definitive zones of the fetal cortex, indicating that both inhibins and activins could be expressed in both. Immunopositivity for all three subunits was seen in most adrenocortical tumours. Loss of immunopositivity for inhibin alpha in a subgroup of carcinomas might indicate a role in tumour progression. The greater intensity of staining for inhibin alpha in tumours associated with Cushing's syndrome again suggests a link with cortisol production.

Vasopressin stimulation of cell proliferation in the rat pituitary gland in vitro.

Proliferative activity was measured in rat anterior pituitary cells in short-term culture by calculating the labelling index (LI), based on the immunohistochemical detection of cells incorporating the thymidine analogue bromodeoxyuridine. Basal LI was reproducible in the test system. Arginine vasopressin (AVP) induced a dose-related increase in LI up to 20 ng/ml. Corticotrophin-releasing factor-41 (CRF-41) had no effect at doses up to 20 ng/ml. However, in the presence of 10 ng CRF-41/ml, AVP induced a greater increase in LI at lower doses than did AVP alone. Fibroblast growth factor also induced a significant increase in LI. In the system used, epidermal growth factor and insulin had no effect on proliferation.

Age-related variation in circadian rhythms of mitosis in the adrenal cortex of the male rat.

Using a metaphase arrest technique, mitotic activity was quantified in the adrenal cortex over a 24-h period in 14-day-old male Sprague-Dawley rats before functional rhythmicity of the hypothalamic pituitary-adrenal (HPA) axis is established, and after its onset, in 6- to 7-week-old rats. At all times, proliferative activity was greater in the younger animals, as previously reported. A significant circadian rhythm was identified in both groups, but the timing of the peak differed, lying between 17.00 and 21.00 h at 14 days and 11.00 and 15.00 h at 6-7 weeks. These results raise the possibility that functional rhythmicity of the HPA axis may alter an inherent proliferative rhythm.

Localization of alpha-interferon in the human feto-placental unit.

The distribution of alpha-interferon in human placental tissue was investigated by immunocytochemical study of paraffin wax-embedded tissue sections using a sheep alpha-interferon antiserum. Fifty-eight placentas of gestational ages from 8 to 40 weeks were examined. alpha-Interferon was present in the syncytiotrophoblast of the chorionic villi of all placentas and was also in macrophages in 28 cases. The appearances suggest production of interferon in human placental trophoblast and, in view of its diverse biological effects, support the concept of a role for alpha-interferon in the complex series of events required for successful gestation.

Effects of trimethoprim and sulphonamide preparations on the pituitary-thyroid axis of rodents.

The effects on pituitary-thyroid function of the commonly prescribed anti-bacterial preparations co-trimoxazole and co-trifamole, and their component drugs, have been studied in the rat and compared to the changes caused by propylthiouracil. Co-trimoxazole and co-trifamole, in doses 20-fold in excess of a pharmacological dose administered for 10 days, produced marked changes in hormone levels consistent with blocking of hyperplastic goitre formation, were also demonstrated. Propylthiouracil produced less marked changes of thyroid hormone levels but higher levels of thyroid-stimulating hormone. Pharmacological doses of co-trimoxazole and co-trifamole and sulphamoxole, the sulphonamide component of co-trifamole, caused significant changes in thyroid hormone levels consistent with anti-thyroidal activity. In contrast, there was no evidence that trimethoprim, which is common to both preparations, or sulphamethoxazole, the sulphonamide component of co-trimoxazole, had an anti-thyroidal action, indeed, serum thyroxine levels were significantly increased at pharmacological dosage. We have concluded that the new commonly prescribed combination preparations retain the goitrogenic properties of the earlier sulphonamides.