Genetic aberrations of c-myc and CCND1 in the development of invasive bladder cancer

Detrusor muscle invasive transitional cell carcinoma is associated with poor prognosis and is responsible for the majority of bladder cancer related deaths. Amplifications of c-myc and CCND1 are associated with detrusor-muscle-invasive transitional cell carcinoma, however, their precise role in driving disease progression is unclear. Fluorescence in situ hybridisation on archival tissue from 16 patients with primary diagnosis of ⩾pT2 transitional cell carcinoma and 15 cases with primary pTa/pT1 disease subsequently progressing to detrusor-muscle-invasion was performed, in the latter group both pre and post muscle invasive events were studied. No patients presenting with ⩾pT2 had amplification of c-myc, two out of 16 (12.5%) had CCND1 amplification. Of patients who developed ⩾pT2, two out of 15 (13.3%) had amplification of c-myc, both in ⩾pT2, five out of 15 (33.3%) had CCND1 amplification, two in pTa/pT1 tumours, three in ⩾pT2 transitional cell carcinomas. In total, two out of 31 (6.5%) of patients' ⩾pT2 TCCs were amplified for c-myc and six out of 31 (19%) were amplified for CCND1. Eighty-seven per cent (40 out of 46) of tumours were polysomic for chromosome 8 and 80% (37 out of 46) were polysomic for chromosome 11 and this reflected the high copy numbers of c-myc and CCND1 observed. In almost all cases an increase in c-myc/CCND1 copy number occurred prior to invasion and persisted in advanced disease. Amplification of CCND1 or alterations in c-myc/CCND1 early in bladder cancer may have clinical relevance in promoting and predicting progression to detrusor-muscle-invasive transitional cell carcinoma. British Journal of Cancer (2002) 87, 654–658. doi:10.1038/sj.bjc.6600531 www.bjcancer.com © 2002 Cancer Research UK

Muscle invasive transitional cell carcinoma (TCC) of the urinary bladder (characterised by invasion to the detrusor-muscle wall of the bladder or beyond) has a poor prognosis. Invasive TCC is routinely treated with radiotherapy or surgery but, despite treatment, up to 50% of patients develop metastases and die within 5 years (reviewed by van der Meijden, 1998). A large number of studies have attempted to identify which molecular markers may be driving progression, ultimately to develop targeted therapies (reviewed by Unwin et al, 1999;Reznikoff et al, 2000;Witjes et al, 2000). Cell cycle regulators such as p53 and Rb have been implicated in bladder cancer progression (reviewed by Knowles, 1999).
Previous research within this laboratory demonstrated that polysomy of chromosomes 9, 7 and 17 are associated with recurrence (Bartlett et al, 1998, Watters et al, 2000, but that these genetic events are not linked with polyploidy (Bartlett et al, 1999). A small study within this laboratory investigating the drug metabolising enzyme NAT2 (the gene is located at chromosome 8p22) in TCC recurrence or progression suggested that polysomy 8 was associated with disease progression . Further research showed an association with polysomy of one or eight in preinvasive TCCs associated with subsequent invasion (Watters et al, 2002).
Gene amplification has been postulated as a mechanism driving progression in many carcinomas. c-myc, located at 8q24, encodes for a nuclear transcription factor that regulates cell proliferation and differentiation (Visscher et al, 1997). Gene amplification or increased copy number of c-myc has been reported in breast (Visscher et al, 1997), head and neck and ovarian cancer (Sauter et al, 1995). There has been a suggestion that gain of chromosome 8q is involved in progression in bladder cancer (Sauter et al, 1995). 8q24 gains have been associated with advanced stage and grade bladder cancer (Wagner et al, 1997). Low rates of amplification (15%) at the 11q13 region have been reported in bladder cancer (reviewed by Schuuring, 1995). CCND1 maps to 11q13 (Bringuier et al, 1996), involved in the regulation of the G1/S phase transition of the cell cycle (Bringuier et al, 1996).
Following on from previous studies within this laboratory (Bartlett et al, 1998(Bartlett et al, , 1999Watters et al, 2000Watters et al, , 2001 which associated specific chromosomal aberrations with recurrence or progression, we hypothesised that alteration of key cell cycle modulators c-myc and CCND1 via gene alterations drives progression of superficial and locally invasive TCC (pTa/pT1). This study determined chromosome and gene copy number by FISH in a cohort of patients with either detrusor muscle invasive TCC at presentation or who developed detrusor muscle invasive TCC following a presentation with non-muscle invasive (pTa/pT1) TCC. from a bladder cancer database in the Department of Surgery, Glasgow Royal Infirmary. Patients were categorised into two groups: PP (progressed at presentation; patients with 5pT2 at initial diagnosis) and RP (recurrent and progressive; patients with pTa/pT1 disease at initial diagnosis who subsequently recurred and progressed with their disease). In order to attempt to understand the genetic events underlying progression from pTa or pT1 TCC, the pre and post muscle-events were studied in the RP group; the presenting lesion in the PP group. This approach was based on previous research within this laboratory that demonstrated that there was a considerable increase in genetic aberrations from patients with a primary pTa or pT1 TCC that progressed to detrusor-muscle-invasion when compared to those with non-invasive recurrences (Watters et al, 2002).
A total of 46 tumours were studied. Five micron formalin fixed paraffin processed tissue sections of each tumour were cut onto sialinised slides and baked at 568C overnight. All TCCs analysed had a representative section stained with haematoxylin and eosin and were restaged and regraded by a specialist urological pathologist (KMG) following current UICC guidelines (Sobin and Wittekind, 1995).

Fluorescence in situ hybridisation (FISH)
To determine gene and chromosome copy numbers, levels of gene amplification or deletion in situ, FISH, using locus specific DNA probes for c-myc, CCND1 and chromosome enumeration probes for eight and 11 in a dual hybridisation reaction, was applied to five micron tissue sections of tumour. Included in each run was bladder tissue with normal copy number for all the probes used.
The FISH methodology was followed as outlined: Tissue sections were dewaxed and rehydrated, then subjected to pretreatments 0.2 N HCl for 20 min at room temperature, 8% sodium thiosulphate at 808C for 30 min and 0.5% pepsin in 0.2 N HCl for 26 min at 378C. Tissue sections were post-fixed in 10% neutral buffered formalin at room temperature for 10 min before dehydration in ascending grades of alcohol and air drying. These steps were carried out on a VP2000 robotic pretreatment slide processor (Vysis, UK, Ltd).
The tissue sections were assessed for extent of digestion as previously outlined (Watters et al, 2000). Tissue sections were denatured in 70% formamide, 26SSC pH 7 -8 at 728C for 5 min on the Omnislide hybridisation module (Hybaid, UK Ltd), dehydrated and air-dried. Two separate experiments were carried out on adjacent sections, with probes for the pericentromere of chromosome 8, labelled with SpectrumGreen TM and a SpectrumOrange TM labelled probe for c-myc (Vysis, UK). The alternative probes set was probes for the pericentromere of chromosome 11, labelled with SpectrumGreen TM and a SpectrumOrange TM labelled probe for CCND1 (Vysis, UK). Both combinations were applied as a double label. For each section, 1 ml of each probe was added to 7 ml hybridisation mix (50% formamide, 26SSC, 10% dextran sulphate) and 1 ml deionised water and denatured in a waterbath at 728C for 5 min, applied to the tissue sections and hybridised overnight at 378C (Vysis, Inc).
Following posthybridisation washes in 0.46SSC, 0.3% Nonidet 30, pH 7 at 728C for 2 min, the tissue sections were mounted in 0.25 mg ml 71 DAPI in antifade (Vectashield, UK) and viewed with a Leica DMLB microscope. A triple band pass filter block spanning the excitation and emission wavelengths of the Spec-trumGreen TM and SpectrumOrange TM fluors (Vysis, UK) and DAPI was used in the analysis of the hybridisation. Image capture was achieved using a digital camera and software (Leica DC200, Leica UK).

Scoring criteria
Serially sectioned haematoxylin and eosin stained tissue sections were first examined microscopically to localise areas of TCC. FISH stained sections were then scanned at 6400 magnification to localise the TCC areas identified. Three distinct areas were identified and signals per nucleus in 20 nuclei per area for chromosome 8 and c-myc and chromosome 11 and CCND1 respectively were counted at 61000 magnification and the results recorded manually. Average chromosome and gene copy number per area was calculated by totalling the number of signals counted and dividing this figure by the number of nuclei assessed. Mean copy number per section was calculated by combining the results for the three areas. Where chromosomal heterogeneity was noted the most abnormal score was used in the final analysis. Control sections from normal bladder tissue obtained from post mortem normal bladder were included in the analysis. Values for disomy were derived from the analysis of these tissue sections as previously reported (Bartlett et al, 1998;Watters et al, 2000).
The ratio of gene to chromosome copy number was determined by dividing the number of signals for c-myc or CCND1 by the number of signals for chromosome 8 or 11 respectively. Gene amplification was defined as a ratio greater than two, based on values determined by Vysis Inc and incorporated in their guidelines for using the PathVysion TM system to determine amplification levels of the HER2/neu gene in breast cancer in a diagnostic setting . This was based on the normal mean copy number for the gene as being less than four signals per nucleus. In some cases with high amplification ratios (45 copies per chromosome), clusters of gene signals, analogous to homogeneous staining regions, were observed. In these cases it was not possible to accurately assess the true gene copy number, but by focusing through the cluster a reasonable estimate was achieved. In none of these cases did the presence of a cluster of signals alter the final result.

Statistics
Crosstabs (SPSS for windows release 9.0) was used to compare similarity of copy number between chromosomes 8 and 11 and c-myc and CCND1.

Patients
The average age of the PP group was 70.9 years, (range 50 to 89 years) and the RP group 68.9 years (range 43 to 84 years). The M : F ratio was 13 : 3 in the PP group, 12 : 3 in the RP group. Mean follow-up for the PP group was 42 months (range 6 -91) and for the RP group 39 months (range 3 -117). In the PP group, 13 patients were treated with radiotherapy, one died before radiotherapy commenced, one refused radiotherapy and one had a cystectomy. In the RP group, six had radiotherapy after the muscle-invasive event, five had a cystectomy, two had no treatment. One patient had palliative radiotherapy early in his disease history, subsequently recurred with pT1 and pTa TCCs and eventually, 13 years later, succumbed to his disease, and one patient had radiotherapy before his muscle-invasive TCC. Of the PP group, 11 patients died from their disease, range 6 to 59 months following diagnosis. The four patients still alive had no evidence of local or metastatic disease at last clinic appointment. Of the RP group 9 patients died of their disease, range 3 -15 months following diagnosis of invasive disease. Of the six patients still alive, two had no evidence of local or metastatic disease at last clinic appointment, two had metastatic disease, and there were no records following diagnosis with invasive TCC for the remaining two.

FISH
All hybridisations were successful apart from that of one PP with the probes for c-myc and chromosome 8. The normal range for copy number of each DNA probe is detailed in Table 1, and an example of a TCC with disomy of c-myc and chromosome 8 is shown in Figure 1A.

c-myc and chromosome 8
Fourteen out of 15 (93%) PP tumours had increased copy number of c-myc and chromosome 8, between 2.15 and 6.35 for c-myc and between 2.03 and 7.73 for chromosome 8 ( Figure 1B). None of the tumours exhibited gene amplification (Table 2).
Twenty-seven out of 30 (90%) RP tumours had increased copy number for c-myc of between 2.03 and 5.53. Of these 14 out of 15 (93%) were abnormal in the pTa and pT1 TCCs, the TCC was pTa and 13 out of 15 (87%) were abnormal in the 5pT2 TCC (Table 2). Twenty-seven out of 30 (90%) RP tumours had polysomy 8, (range 2.05 to 4.89). Of these 13 out of 15 (87%) were polysomic in the pTa or pT1 TCCs , both normal TCCs were pTa and 14 out of 15 (93%) were polysomic in the pT2+ TCC. Two out of 30 (6.7%) tumours had gene amplification, and both were 5pT2.
There was a close correlation between c-myc and chromosome 8 copy number, P=0.001.
Twenty-four out of 30 (80%) RP tumours had an increase in CCND1 (range 2.11 to 9.00, Table 2). Of these 12 out of 15 (80%) were abnormal in the pTa or pT1 TCCs; two of the normal tumours were pTa, one was pT1, and 12 out of 15 (80%) were abnormal in the pT2+ TCC. Twenty-three out of 30 (77%) RP tumours were polysomic for chromosome 11, copy number between 2.13 and 4.57. Of these 11 out of 15 (73%) were polysomic in the pTa or pT1 TCC; three of the normal TCCs were pTa, one was pT1, and 12 out of 15 (80%) were polysomic in the pT2+ TCC. Six out of 30 (20%) tumours had amplification of CCND1, four of which were pT2 or above, the remaining two were pT1G3 and pTaG3. Of these one patient had amplification in their pre and post muscle invasive tumours. There was a close correlation with CCND1 and chromosome 11 copy number, P50.001.
In total six out of 31 patients (19%) had amplification of CCND1 in their detrusor muscle invasive tumours, compared to two out of 31 (6.5%) patients with amplification of c-myc, N/S. Both patients with amplification of c-myc had amplification of CCND1.
Data from the results of the assessment of gene copy number in the non-muscle invasive (pTa or pT1) TCCs is depicted in Table 3. This shows similar rates of increased copy number in pTa or pT1 tumours.

DISCUSSION
The observation that amplification of oncogenes predominates in later stages of cancer progression (Ried et al, 1999;Reznikoff et al, 2000) and that the majority of genetic alterations in TCC occur prior to progression (Tsao et al, 2000) implies that genetic instability of this type may influence progression. The aim of this study was to determine if changes of the oncogenes c-myc and CCND1 in preinvasive tumours drive later progression. Abnormal regula-tion of c-myc can result in phenotypic transformation, aberrant cell cycle control and genomic instability. Disruption of CCND1, that encodes for cyclin D1, involved in the regulation of the G1/ S transition of the cell cycle (Bringuier et al, 1996), may form the basis of genetic instability (Reznikoff et al, 2000).
The small numbers of tumours exhibiting gene amplification of c-myc observed here is in accordance with previous studies (Sauter et al, 1995). However, rates of amplification of CCND1 were 19%, marginally higher than a previous study of bladder cancer by Southern blotting which reported amplification rates of 11q13 of 11% (Bringuier et al, 1996). The candidate oncogenes in the region 11q13 in bladder cancer postulated by Bringuier et al (1996) are CCND1 and EMS1 (a gene involved in regulating the interactions between components in the adherens junctions, Schuuring (1995)).
Both the 8q24 (that contains c-myc) and 11q13 regions are frequently involved in translocations in lymphomas (Arber, 2000). Burkitt's lymphoma is usually associated with a translocation of c-myc and CCND1 has been suggested as a marker for mantle cell lymphoma (Arber, 2000). Although translocations are not uncommon in bladder cancer, translocation as a sole cytogenetic event has not been described (Sandberg and Berger, 1994). However, the chromosomal regions that contain c-myc and CCND1 are genetically unstable, which may be of significance in the development of muscle-invasive bladder cancer.
Previously, research from this laboratory has shown an observed increase in aneusomy from pTa to pT1 TCCs, but a lack of association with stage and recurrence or progression, suggesting that it is aneusomy and not pathological classification which is driving progression in pTa or pT1 tumours (Watters et al, , 2002Edwards et al, 2001). Furthermore, it is of interest to note that the rate of genetic abnormalities is similar when comparing the data from patients who progress from nonmuscle-invasion and those who present with muscle invasion. In this study, and previously, we have shown that there are higher rates of aneusomy in patients who progress to muscle invasion compared to those who recur with pTa/pT1 disease (Watters et al, 2000(Watters et al, , 2002. Muscle-invasive TCCs are characterised by a large number of cytogenetic alterations including overrepresentation of 8q and 11q13 (Tomovska et al, 2001). Thus this study would support the finding by Tsao et al (2000) that the majority of genetic alterations in TCC occur prior to progression. They showed that there was an increase of only 10% of genetic aberrations in metastases compared to primary cell lines from two patients reported in their study, and they suggest that most allelic losses occur before progression. It is widely recognised that genetic instability increases from precursor, or in the case of TCC, preinvasive lesions, to invasion (Ried et al, 1999). In our study we also observed that there was a very high rate of abnormalities in preinvasive lesions, regardless of stage.
A gain of chromosome 8q may contribute to bladder cancer progression (Sauter et al, 1995). The gain of a large fragment of 8q also known as an isochrome 8q has been observed in bladder Genetics and Genomics Values for normal copy number were first established by evaluating normal bladder tissue. The normal range for disomy was defined as the mean+36s.d. (99% CI).
c-myc and CCND1 in bladder cancer AD Watters et al cancer (Sauter et al, 1995). The stronger increase in 8q24 gains from pT1 to pT2 -4 than from pTa to pT1 could suggest that 8q gains occur later in bladder cancer progression than 8p deletions (Wagner et al, 1997). In a study of chromosomal aberrations by comparative genomic hybridisation, there was a very small percentage of gains of 11q13 in pTa tumours. However, in pT1 tumours, loss of chromosome 11 was observed with the exception of 11q13 which was often gained or amplified (Simon et al, 1998). In this study, c-myc and CCND1 may be dysfunctional due to the observed increased copy number, and it is possible that Genetics and Genomics  PP=progressed at presentation, patients with 5pT2 at initial diagnosis; RP=recurrer progressors, patients with pTa/pT1 disease at initial diagnosis who subsequently recurred and progressed with their disease; amp=gene amplification. There was a high percentage of tumours with elevated gene copy number but no significant differences between the patient groups. Of those with elevated gene copy number, a small proportion also had gene amplification. This table illustrates the rates of anuesomy and amplification in patients with pTa or pT1 TCCs that represented with detrusor-muscle-invasion (5pT2). Rates were similar for either pTa or pT1 TCCs.
c-myc and CCND1 in bladder cancer AD Watters et al protein expression is increased as a consequence which may be a route to muscle-invasion. When the results from this study were compared to a previous study in this laboratory that examined HER2/neu and chromosome 17 levels in TCC (unpublished data), very similar results were obtained i.e high levels of polysomy and much lower levels of amplification. This suggests that tumours that progress to pT2 and above, or present with that phenotype, have high levels of polysomy of many chromosomes, and that they may exhibit polyploidy. However, we have investigated, by the use of multiple FISH probes, the rate of polysomy in this cohort and have found that this cannot explain the findings in this study (Bartlett et al, 1999). Therefore we conclude that the alterations in these chromosomes represent events which are closely associated with disease progression and independent of polyploidisation. Ried et al (1999) noted that the number of chromosomal copy alterations increased significantly in the transition from precursor lesions to invasive carcinomas, reflecting an overall genetic instability which we have also observed here.
Further studies of gene alterations in non-invasive non progressive patient groups and investigations of protein expression are required to clarify the effects of genetic abnormalities of c-myc and CCND1 on progression of TCC. Nevertheless, this study has added to the growing evidence that genetic alterations prior to detrusor muscle invasion delineate those cancers which will progress and by extension these changes are associated with genes which therefore drive progression.