Deletion mapping of chromosome 8 in cancers of the urinary bladder using restriction fragment length polymorphisms and microsatellite polymorphisms.

We have used a combination of restriction fragment length polymorphism (RFLP) markers and highly informative microsatellite polymorphisms to map a common region of deletion on chromosome 8p in cancers of the urinary bladder. Analysis of loss of heterozygosity (LOH) using microsatellite polymorphisms was shown to be at least as sensitive as detection of RFLPs by Southern blotting. A total of 110 tumours was analysed for loss of heterozygosity (LOH) on 8p and 8q; 109 patients were informative for at least one marker on each chromosome arm and 29 tumours (26%) showed LOH of chromosome 8 markers, 26 of which (25%) showed LOH on 8p. Sixteen tumours (14%) showed LOH on 8q. Thirteen of these also had LOH on 8p. Of the 29 tumours with LOH, five had LOH at all informative loci, indicating loss of an entire copy of chromosome 8. An association was found between high tumour grade and stage and chromosome 8 LOH. Fifty-three per cent of grade 3 muscle-invasive tumours showed LOH compared with 11% of grade 1 non-invasive tumours (0.01 < P < 0.025 and 0.025 < P < 0.05 for grade and stage respectively). Deletion mapping of tumours with chromosome 8 LOH suggests the presence of a suppressor gene(s) for urothelial cancer within a region defined by the loci NEFL and PLAT (8p21-q11.2). If there is a common target for deletions in bladder and those in hepatocellular and colorectal tumours reported previously, this defines a common region of deletion at 8p21.3.

Reduced expression of TGF beta is associated with advanced disease in transitional cell carcinoma.

The gene structure and expression of the related peptide regulatory factors TGF beta 1 and TGF beta 2 were studied in a panel of seven urothelial carcinoma cell lines and 40 transitional cell carcinomas. The latter comprised 15 grade 1, 18 grade 2 and 5 grade 3 tumours and two cases of carcinoma in situ. Control tissues included ten matched 'field' biopsies and 17 other biopsies including 11 biopsies of macroscopically normal urothelium, two of which were from patients with no history of bladder cancer. No amplification of rearrangements of either TGF beta 1 or TGF beta 2 were detected in any sample. A complex pattern of expression or the two genes was found in the urothelial cell lines. High, but variable levels of the 2.5 kb TGF beta 1 transcript were detected and lower and more variable levels of the three (4.1 kb, 5.1 kb and 6.5 kb) transcripts of TGF beta 2 were detected. Although those cell lines expressing most TGF beta 1 tended to express less TGF beta 2 transcript there was no clear-cut relationship. In comparison, no TGF beta 2 transcript was identified in any primary transitional cell carcinoma or control tissue. Markedly reduced or undetectable levels of TGF beta 1 transcript were detected in 4/15 (26%) grade 1, 5/18 (28%) grade 2 and 3/5 (60%) grade 3 tumours. There was no clear relationship to tumour stage, lymphocytic infiltration or stromal content of the tumours. Clinical review one year after the 2 year period of tumour collection showed that 6/9 (66%) of patients with tumours with reduced levels of transcript had died or had disease which was not controllable by local resection and 3/9 (33%) had developed tumour re-occurrences. In comparison, in the group with normal levels of expression of TGF beta 1, 3/18 (17%) had disease which was not controllable by local means, 9/18 (50%) had tumour re-occurrence and 6/18 (33%) had no evidence of disease. The association of reduced expression of TGF beta 1 and advanced disease was statistically significant P < 0.02 (Fisher's test). Although the sample size is small, we suggest that the loss of expression of TGF beta 1 may be a potential marker of progressive disease or prognosis in transitional cell carcinoma and warrants further study.

Loss of heterozygosity at the RB locus is frequent and correlates with muscle invasion in bladder carcinoma.

Studies of second, non-ocular tumours in surviving retinoblastoma patients and their families have reported a higher than expected incidence and lower age at diagnosis of bladder tumours. This suggests that RB mutations may predispose to bladder cancer. To determine whether this gene is involved in the development of sporadic bladder tumours we have examined 162 bladder tumours for evidence of structural alterations to the RB gene. Ninety-four patients were informative with one or more intragenic RB probes, and 28 of these (29%) showed loss of heterozygosity (LOH). Of these, two tumours showed homozygous deletions with the 5' intragenic probe p123M1.8. The probe p68RS2.0, which recognizes a variable number of tandem repeats site in intron 17 of the RB gene, detected new alleles in 5 of 162 tumours, one of which also showed LOH at another polymorphic site within the gene. The 28 tumours with RB LOH were screened with the RB cDNA probes pR3.8 and pR0.9, which revealed two homozygous deletions and one rearrangement. The tumours with RB LOH were also screened for loss of three markers which flank RB, D13S1 which maps proximal and D13S2 and D13S3 which map distal to RB on chromosome 13q. Two tumours showed retention of heterozygosity for flanking markers on one side of RB and another for markers on both sides. These results suggest that RB is the target gene on 13q in these bladder tumours. When RB loss was compared with tumour grade and stage, an association between high tumour grade and RB loss (0.005 greater than P greater than 0.001) and between muscle invasion and RB loss (P greater than 0.001) was found. Twenty-six of the 28 tumours with LOH were muscle-invasive. This represents 56% of invasive tumours. Only 2/48 (4%) superficial tumours showed RB allele loss, and one of these has progressed rapidly to invasive disease. These results show that LOH at the RB locus is a frequent genetic event in bladder tumours and may identify a subset of more aggressive tumours.

Amplification at chromosome 11q13 in transitional cell tumours of the bladder.

Amplification of several markers which map to chromosome 11q13 was detected by Southern blotting in transitional cell tumours of the urinary bladder. The oncogenes INT2 and HST and the BCL1 locus were co-amplified in 20/97 (20.6%) tumours and the locus-specific minisatellite probe pMS51 (D11S97) detected amplification in 17/97 (17.5%) tumours. The high frequency of heterozygosity (greater than 70%) detected by this latter probe on HaeIII-digested DNAs provided a sensitive means to measure low levels of gene amplification (2-fold) by comparing signals obtained from each allele. A number of probes which map to 11q were used in an attempt to map the region of amplification more precisely. PGA, PGR, STMY, D11Z1 and D11S149 were not amplified in any tumours studied. SEA was amplified in 1/59 tumours and D11S146 in 12/89 tumours. A comparison of the patterns of co-amplification of individual markers in this series of tumours revealed that of the 23 tumours with amplification at this site, 11 had co-amplification of D11S97, D11S146, BCL1, INT2 and HST, 3 had co-amplification of D11S97, BCL1, INT2 and HST, 6 had co-amplification of BCL1, INT2 and HST, 1 had co-amplification of D11S97 and D11S146 and 2 had amplification of D11S97 alone. Based on available linkage data for these markers, this suggests that a putative target gene within this amplicon lies centromeric to BCL1. Amplification at 11q13 showed no correlation with tumour grade or with HER2 amplification.

Amplification and over-expression of c-erbB-2 in transitional cell carcinoma of the urinary bladder.

The structure and expression of the proto-oncogene c-erbB-2 was studied in 86 patients with transitional cell carcinoma. Initial tissue samples comprised 37 grade 1, 32 grade 2 and 13 grade 3 tumours and four cases of carcinoma in situ. At the time of this first tumour sample, amplification of the c-erbB-2 gene was demonstrated by Southern blotting in 1/37 grade 1, 5/32 grade 2 and 6/13 grade 3 tumours (0.005 less than P less than 0.01). Tumour 're-occurrences' were obtained from 23 of these patients on one or more occasions. Amplification was detected in re-occurrences from seven of these 23, none of whom showed amplification in the first tumour sample. DNA was also extracted from exfoliated cells in urine collected from five cases of carcinoma in situ and c-erbB-2 amplification was demonstrated in one of these. No gene amplification was identified in patients' lymphocytes, ten biopsies of normal urothelium and 22 various intravesical pathologies. Increased expression of c-erbB-2 mRNA correlated with amplification of the gene. In addition, raised levels of mRNA were seen in the absence of gene amplification in six tumours. Immunoblotting using the polyclonal antibody 21N, raised against the c-terminus of the c-erbB-2 protein demonstrated increased amounts of a 185 kD immunoreactive protein in tumours with increased c-erbB-2 gene copy number compared with control tissues. In some tumours with high c-erbB-2 gene copy number, a 155 kD immunoreactive protein not detected in controls was expressed at higher level than the 185 kD protein. Immunocytochemistry using a monoclonal antibody AB-3, raised against the c-terminus of the c-erbB-2 protein, showed a positive reaction in the cytoplasm and cell membrane of tumours with gene amplification and in 40% of tumours with no amplification. An association was found between c-erbB-2 amplification and over-expression and the development of tumour re-occurrences. We suggest that c-erbB-2 amplification and over-expression may provide a useful molecular marker in transitional cell carcinoma of the bladder and merits further investigation as a potential prognostic indicator.