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

D.J. Simpson, A.A. Fryer, A.B. Grossman1, J.A.H. Wass2, M. Pfeifer3, J.M. Kros4, R.N. Clayton and W.E. Farrell,5

Centre for Cell and Molecular Medicine, School of Postgraduate Medicine, Keele University, North Staffordshire Hospital, Stoke-on-Trent ST4 7QB,
1 Department of Endocrinology, St Bartholomew's Hospital, London,
2 Department of Endocrinology, Radcliffe Infirmary, Oxford OX2 6HE, UK,
3 Department of Endocrinology, University Medical Centre, Ljubljana, Republic of Slovenia and
4 Department of Pathology, Location JNI, Dijkizigt ziekenhuis/E.M.C.R., Postbus 2040, 3000 CA Rotterdam, The Netherlands


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The paradigm for initiation and progression of tumours is best described in colorectal cancer (1) and a similar multi-step aetiology has been proposed for endocrine tumours (2,3). Since sporadic pituitary tumours are predominantly monoclonal in origin (4), genetic alterations to a single cell may offer a growth advantage over surrounding cells allowing for tumour development and expansion. Consequently, genes that influence cell cycle control may be critical.

Aberrations in both oncogenes (5–7) and tumour suppressor genes (TSG) (8) at the G1–S cell cycle checkpoint have been shown to be frequent events in a number of malignancies. The proto-oncogene cyclin D1 (CCND1) is one of the most frequently amplified genes in human neoplasia (7,9), where inappropriate expression of its protein product may lead to unregulated cellular proliferation. In addition to amplification, another principal mechanism leading to inappropriate cyclin D1 expression is cytogenetic inversion as described in parathyroid tumours (10). Overexpression of cyclin D1 protein is associated with a lower rate and/or interval to recurrence rate in breast (11), bladder (12) and non-small cell lung cancer (5). Conversely in squamous cell carcinoma of the head and neck overexpression of cyclin D1 is associated with a decrease in disease free interval (13).

The use of current routine histological criteria cannot determine those pituitary tumours that are destined to show progression. However, our own previous studies (14,15) have shown an association between loss of heterozygosity at a number of loci and transition to an invasive phenotype. In addition Hibberts et al. (16) have shown frequent overexpression of cyclin D1 in non-invasive tumours suggesting this as an early event in pituitary tumourigenesis.

Recent reports have identified a significant association between CCND1 genotype and patient outcome (17,18). We therefore exploited the frequent A/G polymorphism, at nucleotide 870, within the exon 4/intron 4 splice region of the CCND1 gene (17) to determine the influence of CCND1 alleles and genotype distribution in sporadic pituitary tumours.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Peripheral blood samples were obtained from 294 unrelated European Caucasian patients with sporadic pituitary tumours. Patient details were obtained retrospectively and tumours were defined as non-invasive or invasive on the basis of CT and/or MRI reports prior to DNA analysis and graded according to published criteria (14,15). Grade 1 tumours were microadenomas and grade 2 tumours consisted of enclosed macroadenomas with or without suprasellar extension. Both grade 1 and 2 tumours were defined as non-invasive. Grade 3 tumours were locally invasive with evidence of bony destruction and tumour within the sphenoid and/or cavernous sinus. Grade 4 tumours demonstrate CNS/extracranial spread with or without distant metastases. Grade 3 and 4 tumours were considered to be invasive. Subtype classification was based on typical clinical phenotype and hormonal measurements. Of the 294 patient cases, 72 tumours were classified as grade 1, 100 as grade 2, 109 as grade 3 and 13 as grade 4 (Table IGo).


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Table I. CCND1 genotype distribution and allele frequencies in case and control populations
 
Within the 294 patient cases 54 had evidence of a recurrent tumour. The recurrent tumours comprised 37 non-functional adenomas (grade 1, 12; grade 2, 17; grade 3, 8), 8 somatotrophinomas (grade 1, 1; grade 2, 4; grade 3, 3), 4 corticotrophinomas (grade 1, 3; grade 3, 1) and 5 prolactinomas (grade 2, 3; grade 3, 2).

Determination of CCND1 genotype

Peripheral blood samples were collected in EDTA and genomic DNA extracted from leukocytes using commercially available reagents (Nucleon 1; Scotlab, Strathclyde, UK). CCND1 genotype was identified using PCR amplification encompassing nucleotide 870 (A/G polymorphic site) at the exon 4/intron 4 splice region (17) using gene specific oligonucleotides (sense, GCAGTGCAAGGCCTGAACCT; antisense, GGGACATCACCCTCACTTAC). PCR reactions were carried out in 25 µl volumes with 1.5 mM MgCl2, 200 µM each dATP, dGTP, dTTP and dCTP, 2 pmol each primer, 200 ng template DNA and 1 U Taq DNA polymerase. PCR was carried out for 27 cycles. PCR product (5 µl) was digested with 3 U ScrfI (New England Biolabs, Herts, UK) for 3 h at 37°C. Digested products were run on 8% non-denaturing polyacrylamide gels, fixed and visualized by silver staining as previously described (15,16). CCND1 genotype was assigned after visualization of 113 (CCND1 AA), 91 (CCND1 GG) or 113 and 91 bp (CCND1 AG) fragments (Figure 1Go).



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Fig. 1. Examples of CCND1 genotypes. (A) Patient DNA was amplified using specific oligonucleotides encompassing the frequent A/G polymorphism contained within the exon 4/intron 4 splice region of the CCND1 gene. After ScrfI digestion PCR products were visualized on 8% polyacrylamide gels. CCND1 genotype was assigned when fragments of 113 (CCND1 AA), 91 (CCND1 GG) or 113 and 91 bp (CCND1 AG) were identified. Patient numbers are shown above each CCND1 genotype. The shorter 22 bp restriction fragment associated with CCND1 GG and AG genotypes is not shown. (B) CCND1 genotype, assigned using the restriction enzyme digest technique described above, was confirmed in 24 cases by direct sequence analysis of the region encompassing nucleotide 870. Representative examples of CCND1 genotype as assessed by direct sequence analysis are shown. CCND1 genotype was confirmed in all cases examined. CCND1 AA, GG and AG genotypes are shown from left to right.

 
Sequence analysis of CCND1 A/G polymorphism

The CCND1 genotype was confirmed by direct sequence analysis of nucleotide 870 within the intron 4/exon 4 splice in 24 randomly selected cases comprising eight of each (AA, AG and GG) genotype. The CCND1 A/G polymorphism, and the surrounding region, was subjected to PCR amplification using the gene specific oligonucleotides and conditions as described above. PCR amplicons were subjected to cycle sequencing reactions according to the manufacturer's protocol (BigDyeTM Terminator Cycle Sequencing; PE Applied Biosystems, Warrington, UK) using both sense and antisense PCR oligonucleotides. Cycle sequencing products were subjected to capillary electrophoresis using an automatic sequencer (ABI Prism 310 Genetic Analyser; PE Applied Biosystems). Sequencing data were analysed using Sequencing Analysis V3.0 (ABI Prism; PE Applied Biosystems).

Statistical analysis

All statistical analyses were performed using the Stata statistical package (version 5, Stata Corp., TX, USA). The Chi-squared test was used to assess homogeneity between groups (e.g. cases and controls). The Armitage trend test was used to examine the relationship between CCND1 allele frequencies and genotype distribution with tumour grade. This test is used to identify an increase or decrease in proportions over ordered categories, as distinct from the chi-squared test, which is used to determine if non-ordered categories differ in proportions. Significance was taken at the 5% level.

For those cases where peripheral bloods were available from patients with evidence of recurrence association between genotype and time to recurrence were examined by Cox's proportional hazards regression and represented graphically using Kaplan Meier analysis.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Normal control population
Three studies (17–19) have reported the CCND1 allele frequencies of separate European Caucasian control populations. Since statistical analysis showed no significant difference between the CCND1 allele frequencies or genotype distribution in these three populations (n = 414) we have used the combined allele frequencies (A = 0.43, G = 0.57) and genotype distribution (AA = 18%, AG = 50%, GG = 32%) from these reports in our present study.

Association of CCND1 allele frequencies and genotype distribution in sporadic pituitary tumours
We first assessed allele frequencies and genotype distribution in 294 cases previously reported with sporadic pituitary tumours (14–16,20,21). Figure 1AGo shows representative examples of the three CCND1 genotypes. Table IGo summarizes CCND1 allele frequencies and genotype distribution in all cases examined together with previously reported controls (17–19). Assignment of CCND1 genotype by restriction enzyme digest was confirmed in 24 randomly selected cases by direct sequence analysis (Figure 1BGo). As further confirmation of correct genotype assignment, we assessed CCND1 genotype distribution of the total patient cohort using the Hardy–Weinberg equation. No significant difference between observed and expected genotypes (P = 0.16) was observed. We next compared CCND1 allele frequency and genotype distribution in the total patient cohort compared with normal controls, no significant difference in CCND1 allele frequencies or genotype distribution between the total patient cohort and normal controls was observed (P = 0.8).

Association of CCND1 allele frequencies and genotype distribution with tumour subtype
There was no significant difference in CCND1 allele frequencies between individual tumour subtypes and normal controls (P = 0.5–0.8) (Table IGo). In addition, with one exception (somatotrophinomas), CCND1 genotype distribution showed no significant difference between individual tumour subtypes and normal controls. CCND1 genotype distribution within the somatotrophinoma cohort showed a significant difference ({chi}2 [2df] = 9.6; P = 0.008) compared with normal controls.

Association of CCND1 allele frequencies and genotype distribution with tumour grade
The relative proportions of each tumour grade varied within individual tumour subtypes, however, since tumour grades (1–4) could be assigned to ordered categories; we used Armitage trend analysis to calculate significance within patient cases. Within the total tumour cohort analysis of CCND1 allele frequencies showed a highly significant (P = 0.005) reciprocal change in the CCND1 A and G allele frequencies across the tumour grades (Figure 2AGo). The CCND1 A allele frequency significantly increased from grade 1 (0.37) through to grade 4 (0.62) tumours. Conversely, the CCND1 G allele frequency decreased from grade 1 (0.63) through to grade 4 (0.38) tumours (Figure 2AGo and Table IGo). Using trend analysis, we next analysed CCND1 genotype distributions across the tumour grades. Our analysis showed a significant progressive increase in CCND1 AA genotype distribution from grade 1 (15%) through to grade 4 (46%) tumours (AA versus GG + AG; P = 0.005). Conversely, CCND1 GG genotype distribution progressively decreased from grade 1 (41%) through to grade 4 (23%), although the decrease was not statistically significant (GG versus AA + AG; P = 0.204) (Figure 2BGo; Table IGo). No association was observed between CCND1 AG genotype and tumour grade (Figure 2BGo).



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Fig. 2. (A) Calculated CCND1 allele frequencies across the tumour grades. Using Armitage trend analysis, we compared CCND1 allele frequencies in the total patient cohort (n = 294) across the tumour grades. CCND1 A and G alleles show a highly significant reciprocal increase/decrease (respectively) in allele frequencies when compared with increasing tumour grade (P = 0.005). (B) CCND1 genotype frequencies across the tumour grades. Since tumour grades could be assigned to ordered categories we used Armitage trend analysis to compare CCND1 genotype distribution across the tumour grades. CCND1 AA genotype significantly increased from grade 1 through to grade 4 (AA versus AG + GG; P = 0.005) whilst CCND1 GG genotype progressively decreases (GG versus AG + AA; P = 0.204) with increasing tumour grade.

 
Influence of grade 4 adenomas on statistical analysis
Since the grade 4 tumours represented a considerably smaller number of tumours than those comprising the other grades, we further confirmed the conclusions reached by Armitage trend analysis by excluding this group from the analysis. Trend analysis of the total tumour cohort across grades 1, 2 and 3 showed a significant inverse relationship (P = 0.011) between the CCND1 A and G alleles (increasing and decreasing, respectively). A similar analysis of genotype distribution showed a significant increase in the CCND1 AA genotype frequency with increasing tumour grade (P = 0.015).

Association of CCND1 genotype distribution and allele frequencies with tumour grade within a subtype
To investigate the possibility that the observed changes in genotype distribution might segregate with a particular tumour subtype we analysed CCND1 genotype distribution across the grades within each tumour subtype. Genotype distribution in prolactinomas (Figure 3AGo; Table IIGo) showed a progressive increase in CCND1 AA from grade 1 (11%) through to grade 4 (66%) (P = 0.061), whilst the CCND1 GG genotype progressively decreased from grade 1 (43%) through to grade 4 (0%) (P = 0.112). Similar analysis of CCND1 allele frequencies showed a significant inverse relationship between CCND1 A and G alleles across the tumour grades in the prolactinomas cohort (P = 0.007).



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Fig. 3. CCND1 genotype distribution and allele frequencies across tumour grades within pituitary tumour subtypes. (A) Analysis of CCND1 genotype distribution and allele frequency across the tumour grades in prolactinomas. CCND1 AA increased from grade 1 (11%) through to grade 4 (66%) (P = 0.061), while CCND1 GG genotype decreased from grade 1 (43%) through to grade 4 (0%) (P = 0.112) (Table IIGo). Similar analysis of CCND1 allele frequencies showed a significant inverse relationship between CCND1 A and G alleles across the tumour grades in the prolactinomas cohort (P = 0.007). (B) CCND1 allele frequencies across the tumour grades in somatotrophinomas, corticotrophinomas and non-functional adenomas. CCND1 A allele progressively increased whilst the CCND1 G allele frequency decreased in all tumour subtypes with the exception of the non-functional adenomas. However, tumours within the non-functional adenomas were predominantly grade 2 and 3 tumours. Grade 4 non-functional adenomas and somatotrophinomas were under represented and have been excluded (Table IIGo).

 

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Table II. CCND1 genotype distribution and allele frequencies in non-functional adenoma, somatotrophinomas, corticotrophinomas and prolactinomas
 
Though the same trend of increasing frequency of the CCND1 A allele and decreasing frequency of the CCND1 G allele with increasing tumour grade was also observed in the somatotrophinomas and corticotrophinomas (Figure 3BGo; Table IIGo), the association did not achieve statistical significance. This trend was not evident across tumour grades in non-functional tumours (Figure 3BGo); however, within this tumour subtype this may reflect the grade distribution since there were few grade 1 and 4 tumours in this cohort (Table IIGo).

Association of CCND1 genotype distribution with time to recurrence
Within the total cohort 54 patients had been treated for recurrent disease. No significant association between genotype and time to recurrence was found (AA versus AG + GG, P = 0.108; GG versus AG + AA, P = 0.662) (Figure 4Go).



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Fig. 4. Kaplan Meier plot of the association between CCND1 genotype and time to recurrence. Time to tumour recurrence in 54 patients with evidence of a first and recurrent pituitary tumour. No association between genotype and time to recurrence was evident (AA versus AG + GG, P = 0.108; GG versus AG + AA, P = 0.662).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Numerous studies have suggested that overexpression of cyclin D1 protein and/or amplification/rearrangement of this locus is important in different tumour types (6,10,12,13,16,19,22,23). In addition, the influence of the A/G polymorphism within the exon 4/intron 4 splice region of CCND1 has been shown to be significantly associated with susceptibility and outcome in squamous cell carcinoma of the head and neck (SSCHN) (18) and non-small cell lung cancer (NSCLC) (5). In our present study, we have examined CCND1 allele frequencies and genotype distributions in a large cohort of patients with sporadic pituitary adenomas.

In agreement with other studies, we found no significant difference in the frequency of CCND1 alleles (17) or the distribution of genotypes between the total tumour cohort and previously reported control populations (17,19). However, allele frequencies showed a highly significant reciprocal relationship, where the CCND1 A allele frequency progressively increased and CCND1 G allele frequency decreased with increasing tumour grade. Since these data suggest that CCND1 alleles may influence or reflect the biological behaviour of these tumours, and as such could be important as a prognostic indicator, we then analysed CCND1 genotype distribution. With the exception of the somatotrophinomas subtype, no difference in CCND1 genotype distribution was observed compared with the normal control population. However, in the somatotrophinomas this difference in genotype distribution may simply reflect tumour grade proportions within this tumour subtype. We therefore analysed the association between CCND1 genotype and tumour grade. Across the total tumour cohort we observed a progressive increase in the CCND1 AA and decrease in the CCND1 GG genotype distributions from grade 1 through to grade 4 adenomas. Since there was no significant association of the CCND1 AG genotype and tumour grade, these data suggest that the influence of CCND1 A and G alleles as CCND1 AA or GG are associated with, or influence pituitary tumour progression. In a recent study, Matthias et al. (18) demonstrated a significant correlation between the homozygous CCND1 GG genotype and poor patient prognosis in SCCHN. However, a study of NSCLC (17) has shown tumours of a lower rate of recurrence to be associated with the CCND1 GG genotype. The conflicting data between these two studies suggest that the influence of CCND1 polymorphism is perhaps dependent on tumour type.

The underlying mechanism by which CCND1 genotypes are associated with pituitary tumour grade is not clear. CCND1 mRNA is alternatively spliced to form two transcripts a and b. Transcript b has no C-terminal PEST rich region (destruction box), suggesting that the half-life of this protein may be longer. Splicing of CCND1 mRNA appears to be influenced by the exon 4/intron 4 A/G polymorphism. Since tissues heterozygous for the polymorphism have increased expression of transcript b from the CCND1 A allele (17), it would be expected that tissues with a homozygous CCND1 AA genotype would favour expression of transcript b compared with the homozygous CCND1 GG genotype.

In a previous study, we showed inappropriate immunohistochemical expression (IHC) of cyclin D1 that was associated with the non-functional tumour subtype, but not with tumour grade or an observed allelic imbalance (16). Since peripheral blood from these tumours was included in the present investigation, it allowed us to examine the association between genotype and expression; however, no association was seen (data not shown). Recent data from McKay et al. (19), in this case of colorectal tumours, also found that CCND1 genotype was not related to inappropriate expression of cyclin D1 as determined by IHC.

In common with other tumour types, the growth of pituitary tumours is thought to reflect a biological continuum where low-grade tumours would, if left untreated, progress toward a higher grade. Therefore our findings of an association between tumour grade and allele frequencies/genotype distribution are not readily explainable. One possible explanation that might account for our findings and perhaps reconcile this apparent paradox may be as follows. The CCND1 AA genotype may simply reflect or be associated with a tumour that is biologically more aggressive and faster growing. Thus, at the point of tumour initiation allele frequencies and genotype distribution within a total tumour cohort will be similar to that seen in a normal population. However, subsequent growth rate may be influenced by the individual tumour CCND1 genotype. If this is the case, then the faster growing or more aggressive tumours would most likely be associated with the CCND1 A allele and the CCND1 AA genotype. However, no association was found between CCND1 genotype and time to recurrence. This may perhaps not be surprising since, to our knowledge, no association between tumour grade and time to recurrence have been described.

In conclusion, our data show that CCND1 allele frequencies and genotype distribution show significant differences according to tumour grade in sporadic pituitary adenomas and may therefore have prognostic significance. Since CCND1 genotype may be directly determined by analysis of a peripheral blood sample, its identification may provide a useful predictive marker for the clinical management of sporadic pituitary tumours.


    Notes
 
5 To whom correspondence should be addressed w.e.farrell{at}keele.ac.uk Back


    Acknowledgments
 
We are grateful to the Medical Research Council for financial support.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 14, 2001; revised July 11, 2001; accepted July 23, 2001.