Abstract
Background: Mutation of the p53 gene has been implicated in the development of carcinoma in situ (CIS) to invasive solid urothelial carcinomas (UC) whereas loss of heterozygosity (LOH) at chromosome 9 has been suggested to plag part in the development of papillary UCs. Patients and Methods: The p53 mutation and LOH at chromosomes 17p13.1 and 9 were analysed in 120 UCs. Tumor and matched normal DNA were used for microsatellite allelotyping of chromosome 17p and the entire chromosome 9. Results: LOH at 17p13.1 was found in each grade and stage of the UCs, but mutation of the p53 occurred only in the highly malignant G3 tumors including papillary pT1G3 UCs. LOH were found at one or more of the seven tumor suppressor gene loci along chromosome 9 in all but two of the UCs with p53 mutation. Conclusion: Mutation of the p53 gene is not a pathway correlated genetic change, but is associated with the increased cell proliferation of G3 UCs.
Several studies have been carried out to find genetic changes of biological importance in urothelial carcinomas (UCs) (1). Applying different techniques, heterozygous or homozygous losses, gains and amplifications at several chromosomes have been described and the mutation or methylation of genes located at altered DNA sequences has been implicated in the molecular pathology of UC. Based on the most frequent alterations, several pathways indicating a step-by-step order of genetic changes from normal urothelial cell to malignant tumor have been proposed (1-5). One of the simplest models suggested that the development of papillary UCs is associated with loss of heterozygosity (LOH) at chromosome 9p followed by mutation of the p53 gene in invasive tumors, whereas flat tumors, e.g. carcinoma in situ (CIS) are initiated by mutation of the p53 gene (2). Although several studies have reported LOH at chromosome 9 not only in papillary non-invasive but also in solid invasive UCs, it remains unclear whether alteration of chromosome 9 is the “primary” step in UC development.
To study the association between inactivation of p53 and LOH at chromosome 9 120 UCs were analyzed by allelotyping at the chromosome 17p13.1 region, the exons 5-8 of the p53 gene were sequenced and subsequently the allelic changes at chromosome 9 in UCs showing a mutation of the p53 gene were established.
Patients and Methods
Tumor samples and microsatellite analysis. Fresh tumor tissues were obtained by transurethral resection or radical cystectomy at the Departments of Urology, Philipps-University of Marburg and Mannheim Clinic, Ruprecht-Karls-University of Heidelberg. The use of material for this study was approved by the Ethics Commission of the University of Heidelberg. A part of the tumor tissue was immediately snap-frozen in liquid nitrogen and stored at −80°C. All the histological slides were re-evaluated according to the WHO classification by one of the authors (GK). The distribution of the tumor grades and stages are shown in Table I.
DNA from the frozen tumor samples and matched blood lymphocytes were extracted with phenol-chloroform as previously described (6). The microsatellites D17S796, D17S1353 and D17S786 were analysed to establish the allelic changes at the p53 gene. The microsatellite markers used for allelotyping of chromosome 9 are shown in Figure 1. The primer sequences of the markers and their positions were obtained from National Cancer for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov). The matched normal/tumor DNA samples were amplified as described previously (6). Analysis was carried out on an automated DNA analysis system (ALFexpressII, Amersham/Pharmacia Biotech, Freiburg, Germany). The collected raw data were visualized using the Fragment Manager (FM 1.2) software (Amersham/Pharmacia Biotech). Allelic changes were evaluated according to the score system described previously (6). As a minimal contamination with normal DNA may occur in all DNA samples obtained from tumor tissues, and the Fragment Manager software adjusts the peaks in all cases to the same size, the small interstitial “retention of heterozygosity” was evaluated as a homozygous deletion.
Sequencing of TP53. Exons 5-8 of the p53 gene harbouring mutation hotspots were sequenced. The exon sequences were first PCR amplified with tailed primers. The amplifications were performed in a PTC200 thermal cycler (MJ Research Inc., Watertown, MA, USA). For sequencing an Excel II Thermosequenase Kit (Epicentre Biotechnologies, Madison, WI, USA) was used following the manufacturer's instructions. The forward sequencing primer was labelled with IR800 and the reverse sequencing primer with IR700 in a PCR reaction with 30 cycles of 15 sec at 94°C, 30 sec at 58°C and 1 min at 70°C. Then 6 μl loading buffer solution were added, the samples were denatured at 95°C for 2 min and immediately cooled on ice for 1 minute before loading 1 μl on a 45 cm long 6% polyacrylamide gel, which was run at 400V, 55mA and 30W in 0.8× Tris/Borate/Ethylenediaminetetraacetic acid (TBE) buffer at a constant gel temperature of 55°C on an automated DNA analysis system (LICOR DNA4200, MWG-Biotech, Ebersberg, Germany). The collected raw data were evaluated using the BaseImage IR software. The sequence was compared to the normal p53 sequence (database ID AF135120) for detection of mutations.
Results
Allelic changes at chromosome 17p and p53 gene mutations. LOH at the p53 locus at 17p13.1 was detected in 44 out of the 120 UCs analysed (37%). The frequency of allelic loss at 17p13.1 was similar in so-called superficial pTa and pT1 tumors (20% and 23%, respectively) but was found to be increased in the pT2-4 tumors (65%) (Table I). The frequency of LOH has also been found to be increasing along with the higher grade of UC, with a clear cut difference between grade1-2 (16%,17%) and grade 3 (55%) tumors.
Mutations in exons 5-8 of the p53 gene were found in 25 (21%) of the 120 UCs. Sequence changes were detected in exons 5, 6, 7, and 8 in 3, 6, 7 and 9 UCs, respectively (Table II). Most tumors revealed a missense point mutation. All but one (a pT1,G3) UC with p53 mutation revealed also an LOH at the p53 locus. Only a CIS out of the 55 non-invasive pTa UCs showed p53 mutation, whereas mutation was seen in 27-42% of the pT1-pT4 invasive UCs (Table I). None of the G1 or G2 tumours showed alterations of exons 5-8 of the p53 gene. Summarising the results of LOH and p53 mutation analysis, LOH at chromosome 17p13.1 occurred in all stages and grades albeit at different frequencies, but mutation of the p53 gene was seen exclusively in the high grade (G3) UCs including a CIS.
Allelic changes at chromosome 9 and mutation of the p53 gene. The LOH data obtained from 39 loci in 21 of the UCs showing p53 mutations were evaluated. LOH at chromosome 9 occurred in 19 (90%) out of these 21 UCs (Table II and Figure 1). Most of the tumors showed LOH along the entire chromosome 9. Three UCs displayed LOH only at chromosome 9p, one of them showing a homozygous deletion of the cyclin-dependent kinase inhibitor 2A and 2B (CDKN2A/B) region as the only change. Three tumors displayed LOH only at chromosome 9q including two cases with LOH only at the 9q22 (pathed 1, PTCH) or 9q33 (deleted in bladder cancer 1, DBC1) region. A homozygous deletion of the CDKN2A/B region was seen in 9 cases whereas a homozygous deletion at the death-associated protein kinase 1 (DAPK1) and PTCH genes each occurred in one UC.
Discussion
Several studies have proposed that deletion and/or mutation of the p53 gene is characteristic of the CIS, pT1, muscle invasive carcinoma pathway, whereas LOH at chromosome 9p and mutation of the fibroblast growth factor receptor 3 (FGFR3) gene marks low grade papillary UCs (1, 2). In the present study LOH was found at 17p13.1 in solid and papillary UCs of all grades and stages, but mutation of the p53 gene was detected exclusively in the high grade tumors suggesting that 17p13.1 deletion is only a rate limiting step towards the biallelic inactivation of the p53 gene. Moreover, p53 mutations were found in CIS, solid growing pT1G3 and pT2-4 UCs and in papillary pT1G3 UCs as well. A high frequency (90%) of LOH at chromosome 9 including homozygous losses at the CDKN2A/B, DAKP1 and PTCH gene regions were detected in both papillary and solid growing invasive UCs displaying p53 mutations, suggesting that p53 mutation and alteration of chromosome 9 are not exlusively pathway specific genetic changes. The finding that concomitant FGFR3 and p53 mutations occur in 9% of of pT1G3 UCs indicates that papillary UCs with FGFR3 mutation may progress towards invasive tumor by acquiring p53 mutations (7).
Previously, by exploiting the mechanism of probabilistic reasoning in Bayesian Networks and reconstructing the possible flow of progression of allelic changes, we have suggested that LOH at chromosome 9 is the primary event in UC pathogenesis (5). The first alteration in the development of UC is a monosomy or uniparental isodisomy of chromosome 9 or LOH at distinct chromosome 9 regions harbouring the tumor suppressor genes protein tyrosine phosphatase, receptor type, D (PTPRD), basonuclin (BNC2), CDKN2A/B, DAPK1, PTCH, DBC1 and tuberous sclerosis 1 (TSC1). Therefore, at least seven genes controlling different cellular functions such as cell proliferation and apoptosis might be inactivated along chromosome 9 by hemi- or homozygous deletions, mutation and methylation (8-24).
LOH at chromosome 9 occurs at high frequency in urothelial hyperplasia and dysplasia (25-27), which also supports the role of chromosome 9 as a key player of pre-neoplastic and neoplastic cell proliferation. If such cells acquire an activating mutation of the FGFR3 gene, a papillary pTaG1 UC may arise. In some cases a G1 to G3 transition may take place, presumably after acquiring a p53 mutation. Mutations in the highly conserved DNA-binding domain of the p53 gene inhibit the p53-mediated cell cycle arrest, apoptosis or other responses to cell stress leading to increased cell proliferation (28). If hyperplastic or dysplastic cells with chromosome 9 alteration acquire p53 mutation, they start to grow as a group of aneuploid tumor cells, e.g. CIS, which after selection of the fittest cell clone grow as an invasive pT1G3 UC. These tumors carry only LOH at chromosome 9 and the p53 mutation, but not the FGFR3 mutation.
Several other alterations at distinct chromosomal regions and genes are involved in the genetics of UC, most of which can be observed in later stages of tumor development. These genes may modify the differentiation, morphology or growth capacity of tumor cells, may trigger the invasive growth or other biological and morphological characteristics of UCs, but they are probably secondary to the alteration of genes at chromosome 9.
In summary, based on data from this study and the literature, chromosome 9 harboring at least 7 tumor suppressor genes, is a key player in UC development. Deletional-mutational inactivation of the p53 gene and mutational activation of the FGFR3 gene mark the pathways of flat and papillary UCs whereas pT1G3 UCs are at the crossroad of the two pathways.
Acknowledgements
Dr Beothe's work in Heidelberg was supported by an ICRETT grant of the UICC.
- Received November 18, 2011.
- Revision received December 19, 2011.
- Accepted December 21, 2011.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved