Renal sparing treatment of upper tract malignant urothelial tumours using photodynamic therapy (PDT)-three case reports.

Urothelial cancers of the upper urinary tract are usually treated by excision of the kidney, ureter and cuff of the bladder on the affected side. These three cases demonstrate the feasibility, safety and efficacy of photodynamic therapy as a renal sparing procedure for urothelial tumours.

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.

Immunocytochemical localization of c-erbB-2 protein in transitional cell carcinoma of the urinary bladder.

The level of expression and cellular localization of the c-erbB-2 gene product in transitional cell carcinoma of the urinary tract is controversial. Analysis of the c-erbB-2 gene structure and comparison of its expression in the same cells by Southern, Northern and immunoblotting, and by immunocytochemistry minimize the errors of interpretation inherent in one technique. Such a 'correlative study' has been performed on tumours from 82 patients. c-erbB-2 gene amplification was detected in 14 per cent of initial tumours and was associated with grade (P < 0.001). Raised levels of mRNA were seen in those tumours with increased gene copy number and in 13 per cent of the remainder. Immunoblotting detected the expected 185 kD immunoreactive protein and a 155 kD protein associated with high gene copy number. Immunocytochemistry localized c-erbB-2 immunoreactivity to the cell membrane and cytoplasm, and the latter predominated. Four antibodies to c-erbB-2 (AB-3, 21N, pAb 1, and NCL CB11) were compared on contiguous sections of the same tumour and showed the same pattern of immunoreactivity. Similarly, analyses carried out in three independent laboratories identified the same cellular localization. Membrane and cytoplasmic immunoreactivity was demonstrated in all tumours with gene amplification or increased mRNA levels and in 40 per cent of the remaining tumours. We showed that immunocytochemistry requires careful standardization of techniques and quantitation between different groups. However, despite variations in the intensity of immunoreactivity, the total number of positive cells remained constant. Therefore quantitation must be based on the number of positive cells and, ideally, their immunoreactive content relative to normal and positive tissue controls.

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.

Simultaneous isolation of DNA, RNA, and antigenic protein exhibiting kinase activity from small tumor samples using guanidine isothiocyanate.

Correlative studies of genes and their expression in human tumors are often hampered by the small sample size and the need to use differing and incompatible techniques to obtain DNA, RNA, and protein. We describe an extension of the established guanidine isothiocyanate method for isolation of DNA and RNA which allows the simultaneous isolation of total cellular protein. The protein obtained by this method (from solid tumors and cell lines) was comparable to protein extracted by a standard detergent solubilization method. Antigenicity was retained as demonstrated by Western blotting for epidermal growth factor receptor and actin and by immunoprecipitation of p53. Kinase activity was similar in proteins extracted by the two methods. It seems probable that most monomeric proteins can be obtained in a form suitable for Western analysis and immunoprecipitation and that these may also retain some functional activity.