A deep ocean acoustic noise floor, 1-800 Hz.

The ocean acoustic noise floor (observed when the overhead wind is low, ships are distant, and marine life silent) has been measured on an array extending up 987 m from 5048 m depth in the eastern North Pacific, in what is one of only a few recent measurements of the vertical noise distribution near the seafloor in the deep ocean. The floor is roughly independent of depth for 1-6 Hz, and the slope (∼ f-7) is consistent with Longuet-Higgins radiation from oppositely-directed surface waves. Above 6 Hz, the acoustic floor increases with frequency due to distant shipping before falling as ∼ f-2 from 40 to 800 Hz. The noise floor just above the seafloor is only about 5 dB greater than during the 1975 CHURCH OPAL experiment (50-200 Hz), even though these measurements are not subject to the same bathymetric blockage. The floor increases up the array by roughly 15 dB for 40-500 Hz. Immediately above the seafloor, the acoustic energy is concentrated in a narrow, horizontal beam that narrows as f-1 and has a beam width at 75 Hz that is less than the array resolution. The power in the beam falls more steeply with frequency than the omnidirectional spectrum.

Surface gravity waves and their acoustic signatures, 1-30 Hz, on the mid-Pacific sea floor.

In 1999, Duennebier et al. deployed a hydrophone and geophone below the conjugate depth in the abyssal Pacific, midway between Hawaii and California. Real time data were transmitted for 3 yr over an abandoned ATT cable. These data have been analyzed in the frequency band 1 to 30 Hz. Between 1 and 6 Hz, the bottom data are interpreted as acoustic radiation from surface gravity waves, an extension to higher frequencies of a non-linear mechanism proposed by Longuet-Higgins in 1950 to explain microseisms. The inferred surface wave spectrum for wave lengths between 6 m and 17 cm is saturated (wind-independent) and roughly consistent with the traditional Phillips κ(-4) wave number spectrum. Shorter ocean waves have a strong wind dependence and a less steep wave number dependence. Similar features are found in the bottom record between 6 and 30 Hz. But this leads to an enigma: The derived surface spectrum inferred from the Longuet-Higgins mechanism with conventional assumptions for the dispersion relation is associated with mean square slopes that greatly exceed those derived from glitter. Regardless of the generation mechanism, the measured bottom intensities between 10 and 30 Hz are well below minimum noise standards reported in the literature.

Wave-wave interactions and deep ocean acoustics.

Deep ocean acoustics, in the absence of shipping and wildlife, is driven by surface processes. Best understood is the signal generated by non-linear surface wave interactions, the Longuet-Higgins mechanism, which dominates from 0.1 to 10 Hz, and may be significant for another octave. For this source, the spectral matrix of pressure and vector velocity is derived for points near the bottom of a deep ocean resting on an elastic half-space. In the absence of a bottom, the ratios of matrix elements are universal constants. Bottom effects vitiate the usual "standing wave approximation," but a weaker form of the approximation is shown to hold, and this is used for numerical calculations. In the weak standing wave approximation, the ratios of matrix elements are independent of the surface wave spectrum, but depend on frequency and the propagation environment. Data from the Hawaii-2 Observatory are in excellent accord with the theory for frequencies between 0.1 and 1 Hz, less so at higher frequencies. Insensitivity of the spectral ratios to wind, and presumably waves, is indeed observed in the data.

Bromocriptine and dopamine mediate independent and synergistic apoptotic pathways in pituitary cells.

Dopamine (DA) agonists are the primary treatment choice for prolactinoma, effectively suppressing prolactin expression and reducing tumour size. However, the intracellular pathway(s) through which either DA or its agonists impact on proliferation or lead to tumour shrinkage are incompletely understood. To identify the mediators in the apoptotic cascades after DA or DA agonist challenges we used a well-characterized model system, the rodent somatolactotroph cell line GH3. In these cells, we show that apoptosis induced by the DA agonist bromocriptine (BC), but not DA, is initiated through activation of the JNK pathway. However, both DA and BC activate the terminal effector caspase, caspase-3. Kinetic studies and chemical inhibitor co-incubation experiments support a role for JNK activation preceding caspase-9 activation in BC challenged cells, however, engagement of these mediators was not apparent in DA challenge cells. These studies suggest that apoptosis induced by BC or DA is mediated through distinct and independent pathways that converge with activation of the terminal caspase, caspase-3. These observations were further reinforced by our findings that DA and BC, in co-incubation experiments, synergistically induce apoptosis. These findings raise the possibility that drugs acting through the same pathway as DA may be clinically beneficial when combined with BC.

Pituitary tumours: all silent on the epigenetics front.

Investigation of the epigenome of sporadic pituitary tumours is providing a more detailed understanding of aberrations that characterise this tumour type. Early studies, in this and other tumour types adopted candidate-gene approaches to characterise CpG island methylation as a mechanism responsible for or associated with gene silencing. However, more recently, investigators have adopted approaches that do not require a priori knowledge of the gene and transcript, as example differential display techniques, and also genome-wide, array-based approaches, to 'uncover' or 'unmask' silenced genes. Furthermore, through use of chromatin immunoprecipitation as a selective enrichment technique; we are now beginning to identify modifications that target the underlying histones themselves and that have roles in gene-silencing events. Collectively, these studies provided convincing evidence that change to the tumour epigenome are not simply epiphenomena but have functional consequences in the context of pituitary tumour evolution. Our ability to perform these types of studies has been and is increasingly reliant upon technological advances in the genomics and epigenomics arena. In this context, other more recent advances and developing technologies, and, in particular, next generation or flow cell re-sequencing techniques offer exciting opportunities for our future studies of this tumour type.

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.

Epigenetic control of fetal gene expression.

Each differentiated cell type has its own epigenetic signature, which reflects its genotype, developmental history, and environmental influences, and is ultimately reflected in the phenotype of the cell and organism. Some cells undergo major epigenetic 'reprogramming' during fetal development. The proper, or improper, handling of these highly sensitive periods may have significant short-term and long-term effects on the newborn and his/her progeny. This review highlights the impact of environmental and nutritional factors on the epigenome and the potential effect of epigenetic dysregulation on maternal and fetal pregnancy outcomes, as well as possible long-term implications.

A novel apoptosis gene identified in the pituitary gland.

Although multiple different cancers have been described, it is likely that these tumour types share a small, and common, number of newly acquired functional capabilities. Tumours that arise within the pituitary gland are no exception with respect to these new functional capabilities. Although compelling evidence for self-sufficiency in growth signals is presented, loss of functional tumour suppressor genes by classic mechanisms has not been clearly established. However, and in this context, methylation-mediated or -associated gene silencing, in particular of tumour suppressor genes, has been reported by numerous investigators in this tumour type. More recently, a search for novel genes on the basis of their inappropriate methylation has led to identification of a novel pro-apoptotic gene. Its pituitary tumour derivation and role in drug-induced apoptosis resulted in the acronym PTAG (pituitary tumour apoptosis gene) being assigned to this gene. In a model pituitary tumour cell line, AtT20, expression of PTAG per se had no discernible effects on proliferation, cell cycle profile or viability. However, enforced expression was associated with a significantly increased sensitivity to the apoptotic effects induced by bromocriptine challenge. Apoptosis was mediated through caspase activation and associated with DNA fragmentation as determined by TUNEL labelling. Similar findings are also evident in the rodent pituitary cell line, GH3 and our data shows that drugs other than bromocriptine, and that engage characterized receptors, elicit a PTAG-augmented apoptotic response. The isolation of novel genes, on the basis of their methylation status, offers a significant advantage with respect to our understanding of tumorigenesis in both the pituitary and other tumour types. The reversal of apparent gene silencing may lead to tumour cell 'sensitisation' to chemo- and radiotherapeutic treatment strategies.

Pituitary tumours: findings from whole genome analyses.

Pituitary tumours are common intracranial neoplasms that cause significant morbidity through mass effects and/or the inappropriate secretion of pituitary hormones. Despite a considerable literature detailing potential pathogenic changes in these tumours, their aetiology remains largely unresolved. Recent studies have employed genome-wide profiling towards the identification of novel genes and pathways that are inappropriately expressed or regulated in this tumour type. The techniques employed vary in their complexity and interpretation; however, many of the findings from these types of studies have identified novel genes with potential and, in some cases, proven roles in pituitary tumorigenesis. These studies include comparative genomic hybridization, whole genome-wide allelotyping and methodologies for identification of novel genes associated with epigenetic silencing. In addition, differential display methodologies have been instrumental in the identification of transcripts inappropriately expressed including, pituitary tumour transforming gene, growth arrest and DNA damage-inducible protein (GADD)45gamma and a maternal expressed gene 3 isoform, which in some cases have proven roles in pituitary tumorigenesis. Although few studies of whole genome transcript analysis, as determined by microarray or gene-chip technologies, are reported, these studies of human pituitary, in some cases combined with proteomics, are yielding useful data. In addition, these types of investigation have been applied to several animal models of pituitary tumorigenesis, and in these cases novel genes are highlighted as showing significant change. The identification of the initiating events responsible for the transformation of a normal pituitary cell into one with unrestrained proliferative capacity has so far eluded us. No doubt, these new technologies allowing an essentially unbiased genome-wide analysis, perhaps in combination with animal models that display a preceding hyperplasia, will allow us to identify genes critical to tumour evolution and progression.

Epigenetic mechanisms of tumorigenesis.

In the majority of human cancers, heritable loss of gene function through cell division may be mediated as often by epigenetic as by genetic abnormalities. Epigenetic modification occurs through a process of interrelated changes in CpG island methylation and histone modifications. Candidate gene approaches of cell cycle, growth regulatory and apoptotic genes have shown epigenetic modification associated with loss of cognate proteins in sporadic pituitary tumors. A search for novel genes on the basis of their differential methylation has led to the isolation and functional characterization of a pro-apoptotic mediator--a pituitary tumor apoptosis gene ( PTAG). Although PTAG expression is significantly underexpressed in most pituitary adenomas, mechanisms in addition to methylation most likely account for its loss. The GNAS gene is imprinted in normal pituitary, and activating mutations within Gsalpha, referred to as the gsp oncogene, are almost invariably associated with the maternal expressed allele in somatotrophic adenomas. In addition, epigenetic modification, manifesting as relaxation of imprinting, leads to biallelic expression of Gsalpha irrespective of gsp status. Pituitary tumors as components of familial syndromes represent a rare entity, and the role of epigenetic modification in their evolution and outgrowth is not known. Although speculative, these studies might provide new insight since methylation-associated gene silencing is a feature of other familial tumor types.

Pituitary tumour clonality revisited.

Allelotype analysis and X chromosome inactivation analysis in women enables the assessment of tissue clonality, and has demonstrated that the majority of sporadic human pituitary adenomas are monoclonal. This implies that these tumours arise from de novo somatic genetic change(s) in a single pituitary cell. However, clonality within any given tumour may be multiple or single, multiple tumours arising on the background of hyperplasia may be of identical or different clonality, multiple 'sporadic' tumours in a tissue may be of differing clonal origin, and finally morphology cannot predict genetic makeup. These general principles may also apply to the pituitary so it is simplistic to assume that monoclonality is inevitable and that pituitary tumours cannot be multiclonal in origin. Indeed, these observations would be entirely compatible with the initiating stimulus resulting in hyperplasia of specific cell subtypes in the pituitary giving rise to a number of different clones each with variable potential to develop into a discrete tumour depending on their rate of cell division/rate of apotosis. Stimuli might include pituitary-specific oncogenes, intrapituitary growth factors, or extrapituitary trophic factors (e.g. hypothalamic releasing hormones).

Prediction of recurrence of nonfunctioning pituitary tumours by loss of heterozygosity analysis.

BACKGROUND: Postsurgical regrowth or recurrence of nonfunctioning pituitary adenomas (NFAs) is not uncommon and often requires further surgery or radiotherapy (DXT). Routine postoperative DXT increases the incidence of hypopituitarism, which is associated with increased morbidity and mortality. Identification of genetic abnormalities in the tumour tissue, which can predict recurrence, may allow targeting DXT to the most appropriate patients. DESIGN AND METHODS: We have performed loss of heterozygosity (LOH) analysis on 96 NFAs of which 43 (45%) were recurrent and 53 (55%) were nonrecurrent tumours. Analysis of all tumours was performed on the surgical specimen obtained at the time of first surgery. All tumours underwent allelotyping across nine highly informative microsatellite markers selected on the basis of high LOH frequency in an earlier study involving genome-wide allelotyping. LOH frequency across all microsatellite markers as well as across individual markers was compared between the two cohorts of tumours. RESULTS: LOH frequency in tumours that subsequently recurred was significantly higher across all microsatellite markers as compared to tumours that did not recur (P < 0.05). Allelic loss across one or more microsatellite marker was significantly higher in recurrent tumours (30/43) as compared to their nonrecurrent counterparts (17/53) (P < 0.01). On Poisson regression analysis, the higher LOH frequency in recurrent tumours was independent of the invasiveness of tumours determined radiologically. In addition, LOH at the microsatellite markers D1S215 and D1S459 was significantly higher in tumours that recurred as compared to tumours that did not (32%vs. 3% and 27%vs. 2%, respectively; P < 0.01 for both). No significant difference in LOH frequency between the two tumour groups was evident at the other markers. No association could be demonstrated between the frequency and pattern of LOH and the time to manifest recurrence. CONCLUSIONS: We have shown that it may be possible to predict recurrence of NFAs by LOH analysis of the initial tumour specimen at predefined microsatellite markers, especially on chromosome 1q. This merits further prospective study.

Epigenetic change in pituitary tumorigenesis.

Throughout the genome CpG dinucleotides are found at one-fifth of their expected frequency and their rarity is further marked by the fact that 70% are methylated. In contrast, CpG islands (CGI), found associated with the promoters of many genes, have maintained their expected frequency of this dinucleotide, and remain unmethylated. Inappropriate methylation of CGIs is associated with histone deacetylation and gene silencing, while methylation of CpGs outside of CGIs is associated with significantly higher mutation rates. Methylation of CGIs is a frequent event in numerous tumour types including those that arise within the pituitary gland. Several studies now show highly frequent methylation of the p16 gene that is significantly associated with loss of cognate protein and that appears to be an early change in pituitary tumorigenesis. Collectively, studies show that somatotrophinomas are an infrequent target for p16 CGI methylation. However, in this pituitary tumour subtype, loss of pRb is associated with either CGI methylation or micro-deletion within the protein-pocket binding domain. As in other tumour types loss of p16 or RB1 appear to be mutually exclusive events in non-functional adenomas and somatotrophinomas respectively. Investigation of the Death Associated Protein Kinase gene shows that loss of its protein (DAPK), a pro-apoptotic molecule, in pituitary tumours is also associated with either methylation or deletion within its associated CGI. In the case of DAPK, however, these changes segregate with invasive pituitary tumours irrespective of tumour subtype. Methylation represents a positive signal that can be detected with exquisite sensitivity; in addition, this change targets multiple genes that show tumour type specificity. Taken together, the detection of DNA methylation changes, using either a panel of predefined marker-islands, or CGI arrays, provides the opportunity to generate "methylation profiles". This new knowledge will increase our understanding of tumour biology and could ultimately aid medical management in these different tumour types, including those of pituitary origin.

Genome-wide amplification and allelotyping of sporadic pituitary adenomas identify novel regions of genetic loss.

Through the use of a candidate gene approach, several previous studies have identified loss of heterozygosity (LOH) at putative tumor-suppressor gene (TSG) loci in sporadic pituitary tumors. This study reports a genome-wide allelotyping by use of 122 microsatellite markers in a large cohort of tumors, consisting of somatotrophinomas and non-functioning adenomas. Samples were first subject to prior whole genome amplification by primer extension pre-amplification (PEP) to circumvent limitations imposed by insufficient DNA for whole-genome analysis with this number of microsatellite markers. The overall mean frequency of loss in invasive tumors was significantly higher than that in their non-invasive counterparts (7 vs. 3% somatotrophinomas; 6 vs. 3% non-functioning adenomas, respectively). Analysis of the mean frequency of LOH, across all markers to individual chromosomal arms, identified 13 chromosomal arms in somatotrophinomas and 10 in non-functioning tumors, with LOH greater than the 99% upper confidence interval calculated for the rate of overall random allelic loss. In the majority of cases, these losses were more frequent in invasive tumors than in their non-invasive counterparts, suggesting these to be markers of tumor progression. Other regions showed similar frequencies of LOH in both invasive and non-invasive tumors, implying these to be early changes in pituitary tumorigenesis. This genome-wide study also revealed chromosomal regions where losses were frequently associated with an individual marker, for example, chromosome arm 1q (LOH > 30%). In some cases, these losses were subtype-specific and were found at a higher frequency in invasive tumors than in their non-invasive counterparts. Identification of these regions of loss provides the first preliminary evidence for the location of novel putative TSGs involved in pituitary tumorigenesis that are, in some cases, subtype-specific. This investigation provides an unbiased estimate of global aberrations in sporadic pituitary tumors as assessed by LOH analysis. The identification of multiple "hotspots" throughout the genome may be a reflection of an unstable chromatin structure that is susceptible to a deletion or epigenetic-mediated gene-silencing events.

Loss of ACTH expression in cultured human corticotroph macroadenoma cells is consistent with loss of the POMC gene signal sequence.

The proopiomelanocortin (POMC) gene is highly expressed in the pituitary gland where the resulting mRNA of 1200 base pairs (bp) gives rise to a full-length protein sequence. In peripheral tissues however both shorter and longer POMC variants have been described, these include for example placental tissue which contain 800 (truncated at the 5' end) and 1500 as well as the 1200 bp transcripts. The importance of the 800 bp transcript is unclear as the lack of a signal sequence renders the molecule to be non-functional. This transcript has not been previously demonstrated in the pituitary gland. In this report we show evidence of a 5' truncated POMC gene in human pituitary corticotroph macroadenoma cells (JE) maintained in primary culture for >1 year. The original tumour tissue and the derived cells during early passage (up to passage 4-5) immunostained for ACTH and in situ hybridisation confirmed the presence of the POMC gene in the cultured cells. These cells also secreted 15-40 pg/10(5) cells/24 h ACTH. In addition, as expected RT-PCR demonstrated the presence of all three POMC gene exons and is thus indicative of a full-length POMC gene. In late culture passages (passages 8-15) JE cells ceased to express ACTH and cell growth became very slow due presumably to cells reaching their Hayflick limit. ACTH immunostaining in these cells was undetectable and ACTH secretion was also at the detection limits of the assay and no greater than 10 pg/10(5) cells/24 h. ACTH precursor molecules were also undetectable. RT-PCR for the POMC gene in these late passage cells showed that only exon 3 was detectable, in contrast to early passage cells where all three exons were present. In summary we isolated in culture, human pituitary cells that possessed initially all three exons of the POMC gene and immunostained for ACTH. On further passaging these cells showed a loss of exons 1 and 2 in the POMC gene and a loss of ACTH immunostaining and secretion. We would like to suggest that the loss of ACTH peptide expression in these late passage cells is in part due to the loss of the POMC signal sequence. An alternative explanation for our findings is that there were originally two populations of corticotrophs in the cultures, one of which possessed the full-length POMC gene and the other only the 5' truncated POMC transcript and it is these latter cells which survived in culture. In either scenario this is the first report of the 5' truncated POMC gene occurring in pituitary cells.

Pituitary tumours are multiclonal from the outset: evidence from a case with dural metastases.

In 1992 a 54-year-old man underwent transsphenoidal adenomectomy to remove a clinically nonfunctioning pituitary adenoma during which there was a transient cerebrospinal fluid (CSF) leak. He received radiotherapy to a small residual remnant. Follow-up magnetic resonance imaging (MRI) scan in 1997 showed an increase in the tumour in the pituitary stalk region and an additional intradural lesion at C1 level. In the absence of neurological symptoms and signs, an observational policy was followed. By 1999 the cervical dural lesion had enlarged and laminectomy was performed, during which three intradural lesions were removed. Histology and immunohistochemistry of the metastases were identical to those of the initial pituitary adenoma. Follow-up MRI scan showed extension of the pituitary remnant above the chiasma, requiring transfrontal surgery. Operation was complicated by secondary brain haemorrhage from which the patient died. Autopsy revealed a small amount of residual tumour at the top of the stalk and several small intradural tumour nodules at the level of the foramen magnum. Genetic analysis of the initial pituitary tumour identified significant allelic losses in keeping with its invasive nature, while that of the metastases indicated a separate clone as shown by retention of alleles lost in the primary tumour. The regrown pituitary tumour also appeared to be of a different clone to the initial tumour and the same as two of three of the first metastases (C1 level). The foramen magnum metastasis showed the same loss of heterozygosity (LOH) pattern as one of the original C1 metastases and the pituitary tumour tissue obtained at autopsy. We speculate that at the initial pituitary surgery, cells seeded into the CSF and grew in the dura. These cells were from a different clone, implying that the original pituitary tumour contained at least two clones, possibly three, providing evidence for the contemporaneous oligoclonality of the original pituitary tumour.

Preferential loss of Death Associated Protein kinase expression in invasive pituitary tumours is associated with either CpG island methylation or homozygous deletion.

Death Associated Protein kinase (DAP kinase) a novel calmodulin-dependent serine/threonine kinase was first identified as a positive mediator of programmed cell death. Loss of DAP kinase expression was first demonstrated in highly metastatic cells, whilst re-expression of the protein resulted in delayed local tumour growth and a decreased incidence of metastasis. Although loss of DAP kinase expression has been reported in several cell lines derived from human malignancies the mechanisms responsible have not been defined. In this study we have examined 32 sporadic pituitary tumours for expression of the DAP kinase protein and transcript. In addition, we examined the methylation and deletion status of the DAP kinase CpG island as possible mechanisms for the inactivation of the DAP kinase gene. Eleven of 32 (34%) tumours had undetectable DAP kinase expression, by Western blot and/or RT-PCR analysis. Loss of DAP kinase expression was significantly (P=0.004) associated with invasive tumours (10 of 17; 59%) compared to their non-invasive (1 of 15; 7%) counterparts. Of 11 tumours that failed to express DAP kinase, five (45%) showed de novo methylation of the CpG island contained within the promoter region, while four (36%) had evidence of homozygous deletion of this region. Statistical analysis showed that loss of DAP kinase expression was significantly (P=<0.001) associated with methylation or deletion of the DAP kinase CpG island. With two exceptions, none of the remaining tumours or five histologically normal post-mortem pituitaries examined had evidence of methylation or deletion within this region. To our knowledge this is the first report that describes two mutually exclusive mechanisms associated with loss of DAP kinase gene expression. In addition, we also show that loss of the DAP kinase protein and associated genetic aberrations preferentially segregates with tumours that show an invasive phenotype.

Cyclin D1 (CCND1) genotype is associated with tumour grade in sporadic pituitary adenomas.

The cyclin D1 (CCND1) gene contains a frequent A/G polymorphism within the splice donor region of exon 4/intron 4. CCND1 genotype is associated with clinical outcome in a number of malignancies although prognostic significance varies with tumour type. We examined CCND1 allele frequencies and genotype distribution in 294 patients with sporadic pituitary adenomas of various histologies. CCND1 allele frequencies and distribution of genotypes were similar in the 294 cases compared with previously reported control populations. Analysis according to tumour subtype showed no statistical difference in allele frequencies compared with controls. However, CCND1 genotype distribution in the somatotrophinomas showed a significant difference compared with normal controls (P = 0.008). We next examined CCND1 allele frequencies and genotype distribution across the tumour grades. Within the total tumour cohort the CCND1 allele frequencies showed a significant inverse relationship across the tumour grades (P = 0.005). The CCND1 A allele progressively increased from grade 1 (0.37) through to grade 4 (0.62) tumours, whilst the CCND1 G allele frequency progressively decreased from grade 1 (0.63) through to grade 4 (0.38) tumours. Trend analysis of CCND1 genotypes showed a significant progressive increase in AA frequency from grade 1 (15%) through to grade 4 (46%) tumours (P = 0.005). The CCND1 GG genotype progressively decreased from grade 1 (41%) through to grade 4 (23%) tumours (P = 0.204). No statistical significance was observed between CCND1 AG genotype and tumour grades. While the functional significance of the observed segregation of the CCND1 A/G polymorphism and tumour grade is unclear, our data suggest that CCND1 allele frequencies and genotype distributions show significant differences between tumour grades in sporadic pituitary adenomas. Since CCND1 genotype may be determined by analysis of peripheral blood samples it may provide a useful predictive marker for those tumours likely to show invasive behaviour. This may be clinically useful in indicating which tumours should receive adjunctive treatment (e.g. radiotherapy) immediately after surgical resection.