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W E Farrell

Publications and source records attributed to W E Farrell.

At least 37 records · Page 2Linked to original sources
PubMed2000

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.

Adenoma↗
PubMed2000

Molecular pathogenesis of pituitary tumors.

Pituitary tumors are the result of a monoclonal outgrowth where the intrinsic genetic defects involve oncogenes, tumor suppressor genes (TSG), and most likely genes responsible for differentiation. In addition, hypothalamic and intrapituitary derived growth factors are imposed upon these aberrant cells, contributing to their growth characteristics. While histological examination will not identify those tumors likely to progress toward an invasive phenotype or those destined toward recurrence recent advances in the molecular pathology of these tumors holds significant promise for prediction of recurrence and the design of novel treatment strategies. Moreover, emerging data clearly indicate that different molecular mechanisms are involved in the pathogenesis of the various pituitary tumor subtypes. Until recently the gsp oncogene was the only oncogene significantly associated with pituitary tumors; however, emerging data have describe a role for PTTG and cyclin D1 in pituitary tumorigenesis. For known and putative TSG loci, allelic losses on the long arms of chromosomes 10, 11, and 13 are significantly associated with the transition from the noninvasive to the invasive and metastatic phenotype, while losses on chromosome 9p occur early in pituitary tumorigenesis. Studies of known TSG at these loci, including the menin gene and RB1, would suggest a limited role, if any, in pituitary tumors. However, loss of pRB is evident in a proportion of somatotropinomas but is not associated with allelic loss of an RB1 intragenic marker. The gene encoding p16/CDKN2A is neither deleted nor mutated in pituitary tumors; however, its associated CpG island is frequently methylated and is associated with a loss of p16 protein expression. Allelic losses on chromosome 9p, frequent methylation, and loss of p16 protein appear as early changes in nonfunctional tumors, whereas they are infrequent events in somatotropinomas. The functional consequence of enforced expression of p16/CDKN2A in the mouse corticotroph cell line AtT20 has shown that it is responsible for a profound reduction in cell proliferation and the mechanism is a G(1) arrest, mimicking the in vivo role of this cell cycle regulator in most tissues. The combined data from several groups show that the allelic losses reported at known TSG loci are not accompanied by mutation in the retained allele. However, since abnormal methylation patterns may precede and predispose toward genetic instability this could account for the allelic losses on these chromosomes. Equally, since DNA methylation may lead to reduced expression of a gene it might also account for the reduced expression of as yet unidentified TSGs implicated in pituitary tumorigenesis. Collectively these studies hold significant promise as markers predictive of tumor behavior and point to novel treatment strategies, which may include the reactivation of TSGs that are intact but silenced through epigenetic mechanisms.

Alleles↗
PubMed2000

Different patterns of allelic loss (loss of heterozygosity) in recurrent human pituitary tumors provide evidence for multiclonal origins.

Sporadic human pituitary tumors are benign adenomas of monoclonal origin. This implies that they arise from de novo somatic mutation(s) within a single pituitary cell. The availability of original and recurrent/regrown tumors from the same patient allowed testing of the prediction that recurrent/regrown tumors have identical genetic abnormalities as the original tumor sample. We used PCR amplification, from archival slide-extracted DNA, to allelotype microsatellite polymorphisms as an indication of clonality and confirmed this by X chromosome inactivation analysis in samples from women. Tumors from 33 of 49 (67%) patients with two or more specimens showed loss of heterozygosity (LOH) of at least one marker in at least one of their samples. Two patterns of LOH were observed. In pattern A in 14 of 33 (42%) of patients, the LOH pattern of the first tumor was preserved in the second recurrent sample, with some recurrent tumors also showing additional LOH. In these patients, the original and second tumors are presumed to arise from the same original clone with or without progressive accumulation of LOH. In pattern B [19 of 33 (58%) patients], LOH seen in the first tumor was not preserved in the second or subsequent tumors, as evidenced by retention of heterozygosity compared with the first tumor. The simplest explanation is that the second tumor, although still monoclonal, arises from another independently abnormal clone. This was confirmed by X chromosome inactivation analysis in all 11 women where this was informative. These results show that initial and recurrent tumors, of a benign tumor type, are frequently derived from separate independent clones. This suggests that either: (a) more than one abnormal clone is present from the outset though only one dominates; or (b) several clones arise independently at different times. In both scenarios, the initiating event(s) that predisposes to transformation might result in multiclonal hyperplasia, possibly as a consequence of exogenous stimulation.

Adolescent↗
PubMed1999

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.

Chromosome Deletion↗
PubMed1999

Sequence analysis and transcript expression of the MEN1 gene in sporadic pituitary tumours.

The majority of pituitary tumours are monoclonal in origin and arise sporadically or occasionally as part of multiple endocrine neoplasia type 1 (MEN1). Whilst a multi-step aetiology involving both oncogenes and tumour suppressor genes has been proposed for their development, the target(s) of these changes are less clearly defined. Both familial and sporadic pituitary tumours have been shown to harbour allelic deletion on 11q13, which is the location of the recently cloned MEN1 gene. We investigated 23 sporadic pituitary tumours previously shown to harbour allelic deletion on 11q13 with the marker PYGM centromeric and within 50 kb of the MEN1 locus. In addition, the use of intragenic polymorphisms in exon 9 and at D11S4946, and of telomeric loci at D11S4940 and D11S4936, revealed that five of 20 tumours had loss of heterozygosity (LOH) telomeric to the menin gene. However, the overall pattern of loss in informative cases was indicative of non-contiguous deletion that brackets the menin gene. Sequence analysis of all MEN1 coding exons and flanking intronic sequence, in tumours and matched patient leucocyte DNA, did not reveal mutation(s) in any of the 23 tumours studied. A benign polymorphism in exon 9 was encountered at the expected frequency, and in seven patients heterozygous for the polymorphism the tumour showed retention of both copies of the menin gene. Reverse transcription polymerase chain reaction analysis of ten evaluable tumours and four normal pituitaries revealed the presence of the menin transcript. Whilst these findings suggest that gene silencing is unlikely to be mechanistic in sporadic pituitary tumorigenesis, they do not exclude changes in the level or stability of the transcript or translation to mature protein. Our study would support and extend very recent reports of a limited role for mutations in the MEN1 gene in sporadic pituitary tumours. Alternatively, these findings may point to an, as yet, unidentified tumour suppressor gene in this region.

Chromosomes, Human, Pair 11↗
PubMed1999

Tumour suppressor genes in pituitary tumour formation.

Studies of the molecular changes that characterize pituitary tumours have gone some way towards increasing our understanding of the events responsible for their initiation and progression. Allelic deletions on chromosomes 10, 11 and 13 are significantly associated with invasive and metastatic tumours, while losses on 9p occur early in pituitary tumorigenesis. Studies of known tumour suppressor genes within these regions of loss suggest a limited role, if any, in pituitary tumours. However, a loss of pRB is evident in a proportion of somatotrophinomas. Loss of p16 protein expression is associated with methylation of this gene's CpG island and is an early change in non-functional tumours. The enforced expression of p16/CDKN2A in the AtT20 cell line has shown that it is responsible for G1 arrest, mimicking its in vivo role. Methylation may provide a unifying mechanism preceding and predisposing towards allelic loss, and in other cases leading to reduced tumour suppressor gene expression. Pharmacological interventions designed to induce the re-expression of genes silenced through this mechanism offer considerable therapeutic potential.

Chromosomes, Human, Pair 10↗
PubMed1999

Transfection of an inducible p16/CDKN2A construct mediates reversible growth inhibition and G1 arrest in the AtT20 pituitary tumor cell line.

Recent studies have shown that methylation of the CpG island within the p16/CDKN2A gene is associated with an absence of p16 protein in human pituitary tumors. However, the effect of restoration of p16 protein expression in this tumor type has not been investigated. In the absence of an available human pituitary cell line we first assessed the suitability of the mouse corticotroph cell line AtT20 as a model system. Initial experiments showed that the p16/CDKN2A gene was not expressed, whereas a transcript for RB1 was detected as assessed by RT-PCR. Further studies showed the p16/CDKN2A gene to be homozygously deleted. The absence of p16/CDKN2A and presence of RB1, the down-stream effector of p16-mediated cell cycle arrest confirmed the suitability of the AtT20 cell line as a model system. Stable transfectants were generated in which p16/CDKN2A is regulated by an inducible promoter. The regulatory effects of p16/CDKN2A expression on cell proliferation were assessed and complemented by fluorescence-activated cell sorting (FACS) analysis of cell cycle profile. Induced expression of p16/CDKN2A resulted in a profound inhibition of cell growth and G1 arrest (80-82%). Western blot analysis showed concomitant expression of p16 protein in arrested cells and a shift in the phosphorylation status of pRB toward its hypophosphorylated form. To further confirm that expression of p16/CDKN2A mimicked its in vivo role, reversibility was assessed using alternate cycles in the presence and absence of inducer (isopropyl-1-thio-beta-D-galactopyranoside). Over three cycles the absence of induced expression of p16/CDKN2A resulted in release from G1 arrest. These results show that, in a pituitary cell line model, restoration of p16 expression is indeed sufficient to arrest cells in G1 and inhibit cell proliferation and is reversible. Thus restoration of p16 expression through novel strategies, including gene therapy or demethylating agents, may offer successful therapeutic intervention in human forms of this disease.

Animals↗
PubMed1999

Methylation mechanisms in pituitary tumorigenesis.

Methylation is essential for embryonic development, however aberrant methylation of CpG islands associated with the tumour suppressor genes (TSGs) and leading to gene silencing is found in numerous tumour types. The TSG p16/CDKN2A is involved in the genesis of many tumour types and frequent methylation of the CpG island of the p16/CDKN2A gene is associated with loss of protein expression in pituitary tumours. In addition, CpG sites are mutational hotspots and abnormal methylation patterns have been shown to lead to genetic instability, predisposing to, and preceding allelic loss. Although several studies of pituitary tumours have shown loss of genetic material at known and putative TSGs loci, studies of the retained alleles have revealed infrequent mutation. Equally, for several other TSGs no mechanisms have been described for their reduced expression. Methylation may represent a unifying theme, responsible in some cases for an absence or reduced expression and in other cases predisposing to allelic loss that may or may not encompass a TSG. In several tumour types treatment of tumours or their cognate cell lines with demethylating agents induces expression of previously methylated genes. Using the mouse corticotroph cell line AtT20 as a model system, transfection studies showed restoration of growth control through induction of ectopically expressed p16/CDKN2A. These effects were reversed by prior in vitro methylation of the constructs' CpG sites within the coding region of this gene. Methylation of an otherwise unmethylated CpG island renders a gene transcriptionally incompetent and clinically these genes represent attractive therapeutic targets since the gene is neither lost nor mutated, but may be reactivated. Future studies will no doubt describe more efficacious pharmacological interventions and identify the mechanisms responsible for the abnormal methylation patterns seen in tumours including those of pituitary origin.

Cyclin-Dependent Kinase Inhibitor p16↗
PubMed1999

Hypermethylation of the p16/CDKN2A/MTSI gene and loss of protein expression is associated with nonfunctional pituitary adenomas but not somatotrophinomas.

The cyclin-dependent kinase inhibitor 2A/multiple tumor suppressor gene 1 (CDKN2A/MTS//p16) plays an important role in the control of progression from G to S-phase of the cell cycle through the inhibition of CDK4-mediated RBI phosphorylation. In this study we investigated 46 nonfunctional pituitary tumors and 21 somatotrophinomas for aberrant methylation of the CpG island contained within the CDKN2A gene as an alternative mechanism of gene silencing. We demonstrate methylation in 32/46 (70%) of nonfunctioning tumors, in contrast to 2/21 (9.5%) somatotrophinomas and 0/15 histologically normal postmortem pituitaries. Methylation in noninvasive and invasive nonfunctional tumors was approximately equal at 15/20 (75%) and 17/26 (65%), respectively. Immunohistochemical analysis showed an absence of CDKN2A protein in 25/32 (78%) methylated nonfunctioning tumors, demonstrating a highly significant overall correlation (P = 0.00007) between hypermethylation of the gene and absence of the p 16 protein. The association between hypermethylation and absence of CDKN2A protein remained when the cohort of nonfunctional tumors was further subdivided into noninvasive 12/15 (80%; P = 0.004) and invasive 13/17 (76%; P = 0.01), suggesting this to be an early event in pituitary tumorigenesis. In contrast, a single invasive methylated somatotrophinoma failed to express the CDKN2A protein. These data show that hypermethylation of the CpG island within exon 1, but not exon 2, of the CDKN2A gene is frequently associated with loss of protein expression in nonfunctional pituitary tumors, but not somatotrophinomas, suggesting different tumorigenic pathways.

Adenoma↗
PubMed1999

Analysis of cyclin D1 (CCND1) allelic imbalance and overexpression in sporadic human pituitary tumors.

Cyclin D1 plays an important role in the regulation of cell progression through G1 of the cell cycle and has been demonstrated to have oncogenic properties. Using RFLP-PCR, an A/G polymorphism within the cyclin D1 (CCND1) gene was analyzed in 151 sporadic human pituitary tumors, of which 60 were informative at this locus. Further analysis showed that in 15 of 60 (25%) tumors, there was evidence of allelic imbalance, which is indicative of gene amplification. Allelic imbalance was observed more frequently in invasive tumors (11 of 29 tumors; 38%) than in their noninvasive counterparts (4 of 31 tumors; 13%; P = 0.02). Forty-six of the tumors informative for the polymorphism were available for immunohistochemical analysis. Cyclin D1 expression (nuclear and/or cytoplasmic) was detected in 25 of 46 (54%) tumors. Of these cases, expression of nuclear cyclin D1 was detected in 9 of 46 (20%) tumors, whereas 16 of 46 (35%) tumors showed cyclin D1 staining exclusively confined to the cytoplasm. Neither nuclear staining nor cytoplasmic staining was observed in any of the normal pituitaries or in the negative control. Expression of cyclin D1 was observed in significantly more nonfunctional tumors (18 of 27 tumors; 67%) than in somatotrophinomas (7 of 19 tumors; 37%; P = 0.046). Nuclear cyclin D1 expression was observed more frequently in nonfunctional tumors (8 of 27 tumors; 30%) than in somatotrophinomas (1 of 19 tumors; 5%; P = 0.04). There was no correlation between cyclin D1 expression and tumor grade or between allelic imbalance of CCND1 and cyclin D1 expression. We conclude that amplification of CCND1 occurs in pituitary tumors and that the overexpression of cyclin D1 may be an early event in tumorigenesis. Cyclin D1 overexpression occurring in the absence of CCND1 allelic imbalance suggests that additional mechanisms responsible for deregulated cyclin D1 expression are involved in human pituitary tumorigenesis.

Adenoma↗
PubMed1998

Susceptibility and outcome in oral cancer: preliminary data showing an association with polymorphism in cytochrome P450 CYP2D6.

Members of the cytochrome P450 and glutathione S-transferase supergene families are candidates for susceptibility and outcome in oral squamous cell cancer. We determined GSTM1, GSTM3, GSTT1, CYP1A1 and CYP2D6 genotypes in 100 Caucasian cases and 467 control individuals. The frequency of homozygosity for mutant CYP2D6 alleles was higher in the cases (P = 0.001, OR = 3.2, 95% CI = 1.6-6.5) than control individuals. In the cases, the frequency of homozygosity for mutant alleles was greater and that of homozygosity for wild-type CYP2D6 alleles was lower in those diagnosed at > or = 65 years (P = 0.009) than in those diagnosed at < or = 64 years. The older cases included relatively more women and patients who did not consume tobacco or alcohol. The association of CYP2D6 with outcome was assessed using the Cox's proportional hazards model. The time to first cervical node metastasis was shorter in heterozygotes and homozygotes for mutant CYP2D6 alleles compared with homozygotes for wild-type alleles after correction for age at diagnosis, gender, alcohol and tobacco consumption and tumour differentiation (P = 0.04, hazard ratio 3.6, 95% CI 1.1-12.5). The mechanism for the association of CYP2D6 alleles with susceptibility and outcome is unclear though the data are compatible with the view that homozygosity for mutant alleles confers impaired detoxication of an unknown carcinogen. No associations between GSTM1, GSTM3, GSTT1 or CYP1A1 genotypes and susceptibility or, time to node metastases were identified. We previously showed that CYP2D6 genotypes were not associated with susceptibility to squamous cell cancer in the pharynx or larynx. Therefore, the data presented suggest that susceptibility to squamous cell cancer in the various parts of the upper aerodigestive tract is associated with different genes and allelic variants.

Adult↗
PubMed1998

Human adrenocorticotropin-secreting pituitary adenomas show frequent loss of heterozygosity at the glucocorticoid receptor gene locus.

Corticotropinomas are characterized by a relative resistance to the negative feedback action of cortisol on ACTH secretion. In this respect there is a similarity with the clinical syndrome of cortisol resistance. As cortisol resistance can be caused by genetic abnormalities in the glucocorticoid receptor (GR) gene, we investigated whether the insensitivity of corticotropinomas to cortisol is also caused by de novo mutations in the GR gene. We screened for the GR gene in leukocyte and tumor DNA from 22 patients with Cushing's disease for mutations using PCR/single strand conformation polymorphism analysis. In a previous study, we identified 5 polymorphisms in the GR gene in a normal population. These polymorphisms were used as markers for the possible occurrence of loss of heterozygosity (LOH) at the GR gene locus. Except for 1 silent point mutation, we did not identify novel mutations in the GR gene in leukocytes or corticotropinomas from these patients. Of the 22 patients, 18 were heterozygous for at least 1 of the polymorphisms. In 6 of these patients, LOH had occurred in the tumor DNA. Of 21 patients examined for LOH on chromosome 11q13, only 1, with a corticotroph carcinoma, showed allelic deletion. As controls we studied 28 pituitary tumors of other subtypes (11 clinically nonfunctioning, 8 prolactinomas, and 9 GH-producing adenomas) and found evidence for LOH in only 1 prolactinoma. In six patients LOH was found at the GR gene locus (chromosome 5) in DNA derived from adenoma cells. Our observations indicate for the first time that LOH at the GR gene locus is a relatively frequent phenomenon in pituitary adenomas of patients with Cushing's disease. This might explain the relative resistance of the adenoma cells to the inhibitory feedback action of cortisol on ACTH secretion. The specificity of the GR LOH to corticotropinomas supports this concept. Somatic mutations of the GR are not a frequent cause of relative cortisol resistance in these cells.

Adenoma↗
PubMed1998

Molecular biology of human pituitary adenomas.

While the majority of pituitary tumours will remain benign, a proportion will show invasive behaviour and a still smaller proportion will become malignant. Recent studies at both the biochemical and molecular level are now defining the changes associated with pituitary tumour initiation and progression. In particular, the use of microsatellite analysis in determining regions of gene deletion has considerably advanced our understanding of pituitary tumourigenesis. Bringing together the data of several groups now allows a tentative map to be drawn showing loss of heterozygosity at several chromosomal loci to be associated with the transition to the invasive and malignant phenotype, while changes associated with chromosome 9p and silencing, through methylation, of the tumour suppressor gene p16 appear to occur early in pituitary tumourigenesis. At the biochemical level, immunohistochemical studies have defined changes in key regulatory proteins along this multistep pathway. To determine whether these changes are truly predictive of tumour behaviour awaits carefully designed prospective studies. These future studies may well aid decision-making regarding management in a manner not possible using current histological assessment.

Adenoma↗
PubMed1997

Chromosome 9p deletions in invasive and noninvasive nonfunctional pituitary adenomas: the deleted region involves markers outside of the MTS1 and MTS2 genes.

We have screened 57 cases of primary, nonfunctional, pituitary adenomas for loss of heterozygosity of markers on chromosome 9p. Using a panel of 11 microsatellite markers, we found hemizygous deletion with at least one of the markers in 18 tumors (31.5%). The frequency of loss was similar in both noninvasive (8 of 26; 31%) and invasive tumors (10 of 31; 32%), suggesting that loss on this chromosome might be an early event in pituitary tumorigenesis. Two discrete areas of loss were punctuated by a region of retention of heterozygosity between the markers D9S171 and IFNA, indicative of homozygous deletion. However, multiplex PCR analysis (MTS1 and MTS2) and the presence of a 3' untranslated region polymorphism in MTS1 suggested that neither of these tumor suppressor genes was homozygously deleted. In 6 of the 18 tumors showing LOH, sufficient DNA was also available for Southern blot analysis and, in all cases, showed retention of MTS1. Cell mixing experiments of tumor cell DNA homozygously deleted for MTS1 with DNA in which neither copy of the gene was deleted only gave rise to a signal at contamination levels greater than 30% and could discriminate homozygous and hemizygous loss. These studies support the recent findings that mechanisms other than hemi- and homozygous deletion are most likely responsible for the loss of MTS1 gene product in pituitary tumors (M. Woloschak et al., Cancer Res., 56: 2493-2486, 1996.). These data show that losses on either side of 9p21-22, both or either of which may be deleted, are involved in pituitary tumorigenesis and provide evidence for distinct suppressor gene loci, in addition to MTS1, on chromosome 9p.

Adenoma↗
PubMed1997

Genomic sequence analysis of a key residue (Arg183) in human G alpha q in invasive non-functional pituitary adenomas.

OBJECTIVE: With isolated exceptions the only oncogene significantly associated with pituitary tumours is a constitutively active Gs protein (G alpha s). The recent cloning of the cDNA of human G alpha q has facilitated the study of this activator of the phospholipase C beta/Ca2+/ protein kinase C pathway. Since, with isolated exceptions, non-functional tumours are responsive in vitro to TRH and GnRH which activate Gq, we have investigated the genomic sequence of G alpha q, in non-functional (NF) invasive pituitary adenomas, at a residue corresponding to the one most frequently mutated in G alpha s. PATIENTS AND MEASUREMENTS: We studied 27 invasive NF pituitary tumours by direct sequencing of DNA derived from archival slide extracted tumour cells. Primers were designed to encompass Arg183 (corresponding to Arg201 of G alpha s) which when mutated has been shown to have oncogenic potential when transfected into cultural rat fibroblasts. In a previous study we have described allelic loss at tumour suppressor gene loci (TSG) in 7 of these 27 tumours. RESULTS: We successfully amplified genomic DNA with primers designed from the cDNA sequence of G alpha q with specific exclusion of a processed pseudogene. No mutations were found at Arg183, in either the tumours showing allelic losses at specific TSG loci, or in the 20 remaining tumours in which we found no losses at the TSG loci investigated. CONCLUSIONS: Mutations at this key residue in G alpha q occur infrequently, if at all, in invasive non-functional pituitary tumours. However we cannot exclude the possibility of mutation(s) at the other key residue of G alpha q, Gin209, implicated in GTP hydrolysis, or in other components of this pathway.

Adenoma↗
PubMed1997

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.

Adenoma↗
PubMed1996

Epithelial ovarian cancer: influence of polymorphism at the glutathione S-transferase GSTM1 and GSTT1 loci on p53 expression.

The importance of polymorphism in the glutathione S-transferase GSTM1, GSTT1 and, cytochrome P450, CYP2D6 loci in the pathogenesis of epithelial ovarian cancer has been assessed in two studies; firstly, a case-control study designed to determine the influence of these genes on susceptibility to this cancer, and secondly, the putative role of these genes in the protection of host cell DNA has been studied by comparing p53 expression in patients with different GSTM1, GSTT1 and CYP2D6 genotypes. The frequencies of GSTM1, GSTT1 and CYP2D6 genotypes in 84 cases and 325 controls were not different. Immunohistochemistry was used to detect p53 expression in 63 of these tumours. Expression was found in 23 tumours. Of the patients demonstrating immunopositivity, 20 (87%) were GSTM1 null. The frequency distributions of GSTM1 genotypes in p53-positive and -negative samples were significantly different (P = 0.002) and those for GSTT1 genotypes approached significance (exact P = 0.057). The proportion of patients with both GSTM1 null and GSTT1 null was also significantly greater in the immunopositive (4/22) than in the immunonegative group (1/40) (P = 0.0493). Single-strand conformational polymorphism (SSCP) analysis was used to detect mutations in the 23 tumour samples demonstrating p53 positivity. A shift in electrophoretic mobility of amplified fragments was found in 11 patients (exons 5, 6, 7 and 8) and these exons were sequenced. In eight samples a mutation was found. No SCCP variants were identified in the other 12 immunopositive patients. Sequencing of exons 4-9 of p53 from these tumours resulted in the detection of mutations in two patients (exons 5 and 7). Thus, in 23 patients who demonstrated immunopositivity, p53 mutations were found in nine patients with GSTM1 null (90.0%). In the 13 patients in whom no mutations were identified, 11 were GSTM1 null (84.6%). The data show that overexpression of p53 is associated with the GSTM1 null genotype. We propose the data are compatible with the view that GSTM1 and GSTT1 are critical in the detoxification of the products of oxidative stress produced during the repair of the ovarian epithelium. Thus, failure to detoxify products of this stress may result in damage to various genes in the host cell, including to p53, resulting in persistent expression of mutant protein. In other patients, oxidative stress effects damage to various genes, but not including p53, resulting in overexpression of wild-type p53.

Case-Control Studies↗