Urinary Bladder Cancer Patients Research Articles

Detection of H-ras Mutations in Urine Sediments by a Mutant-enriched PCR Technique

Identification of DNA mutations in urine sediments has been proposed as a noninvasive and early indicator of urinary tract cancer (1)(2)(3)(4). The sensitivity of this approach is limited by the proportion of DNA containing the mutant allele, because the resolution of the technique used to detect oncogenes or suppressor gene mutations (mainly single-strand conformational polymorphism) requires ∼10% representation of mutant product (4)(5), whereas estimates to the proportion of tumor cells in the urine of bladder cancer patients were not >7% (1). H- ras represents a single member of a family of genes coding for a 21-kDa protein involved in the regulation of cell growth and differentiation (6). Most activation events of this protooncogene to the oncogenic form found in human bladder cancer occur via a point mutation at codon 12 (7)(8). To provide a more sensitive means for detecting mutations, we have devised a sensitive, nonradioactive procedure based on two consecutive PCRs with intermediate restriction digestion, which leads to effective enrichment of mutant alleles for further amplification and elimination of wild-type alleles by digestion (5)(9)(10)(11). Wild-type DNA was prepared from the urine sediments of three healthy volunteers, and two DNA samples containing known mutations in H- ras codon 12 were mixed with an equal quantity (200 ng) of that wild-type DNA. These mutant-type samples were extracted from tumor specimens after surgical resection. The mutations were known through direct sequencing. Amplifications in the first round were carried out in a final volume of 100 μL in 1× PCR buffer containing 2 mmol/L MgCl2, 50 pmol of each primer, dNTPs at 40 μmol/L each, and 2.5 units of UlTma® DNA Polymerase (Perkin–Elmer–Cetus). After an initial denaturation step at 94 °C for 2 …

Increased concentrations of genotype-interpreted Ca 19-9 in urine of bladder cancer patients mark diffuse atypia of the urothelium

We investigated the use of genotype-interpreted measurements of the tumor marker Ca 19-9 in the urine of bladder cancer patients as a marker of the extent of urothelial disease. Ca 19-9 in urine (sialyl-Le(a)/creatinine ratio) was measured in 81 bladder cancer patients and correlated to T-category, histologic grade, and presence of urothelial dysplasia. As reference group, Ca 19-9 ratio was measured in urine from 21 apparently healthy individuals. The amount of sialyl-Le(a) expressed is influenced by the Lewis genotype and secretor status. Accordingly, secretor status was determined in urine by a novel ELISA method, and the Lewis genotypes of all of the individuals were determined by PCR cleavage methods. Ca 19-9 concentrations in urine were higher (P < 0.01) in bladder cancer patients than in healthy individuals and significantly (P = 0.02) higher in cancer patients with concomitant urothelial dysplasia than in those with normal urothelium. For individuals Lewis-genotyped as homozygous wild-type, Ca 19-9 concentrations in urine were higher, both in cancer patients (P = 0.06) and in healthy individuals (P = 0.004), than in the heterozygous individuals. Furthermore, nonsecretor cancer patients had higher (P < 0.01) Ca 19-9 concentrations in urine. Attention is drawn to the possibility of a general genotype interpretation of a result in clinical chemistry.

Microsatellite analysis and telomerase activity in archived tissue and urine samples of bladder cancer patients

We performed microsatellite analysis and tested telomerase activity in paired tissue and urine of bladder cancer patients from frozen archived samples. DNA obtained from microdissected tumor and urine sediment was analyzed and compared to peripheral lymphocytes for microsatellite alterations (loss of heterozygosity [LOH] or instability) using a panel of 20 microsatellite markers in 15 patients with transitional or squamous cell carcinoma of the urinary tract. Additionally, telomerase activity was determined in 12 microdissected tumor specimens and corresponding frozen urine pellets. Tumor cell DNA was detected by microsatellite analysis (LOH or shift) in at least one marker in 14/15 microdissected tumor specimens and in 13/15 DNA samples obtained from urine sediments. Telomerase activity was present in 11/12 tumor samples but could not be detected in any of the corresponding urine sediments. Frozen archived urine samples are useful for retrospective studies utilizing microsatellite analysis or other PCR-based approaches after DNA extraction. However, the evaluation of telomerase protein activity in stored urine samples appears to be unsuitable. Int. J. Cancer 74:625–629, 1997.© 1997 Wiley-Liss, Inc.

Microsatellite analysis and telomerase activity in archived tissue and urine samples of bladder cancer patients

We performed microsatellite analysis and tested telomerase activity in paired tissue and urine of bladder cancer patients from frozen archived samples. DNA obtained from microdissected tumor and urine sediment was analyzed and compared to peripheral lymphocytes for microsatellite alterations (loss of heterozygosity [LOH] or instability) using a panel of 20 microsatellite markers in 15 patients with transitional or squamous cell carcinoma of the urinary tract. Additionally, telomerase activity was determined in 12 microdissected tumor specimens and corresponding frozen urine pellets. Tumor cell DNA was detected by microsatellite analysis (LOH or shift) in at least one marker in 14/15 microdissected tumor specimens and in 13/15 DNA samples obtained from urine sediments. Telomerase activity was present in 11/12 tumor samples but could not be detected in any of the corresponding urine sediments. Frozen archived urine samples are useful for retrospective studies utilizing microsatellite analysis or other PCR-based approaches after DNA extraction. However, the evaluation of telomerase protein activity in stored urine samples appears to be unsuitable.

Differential production of gamma delta T cells in the urine of bladder cancer patients receiving Bacillus Calmette Guerin immunotherapy

Bacillus Calmette Guerin (BCG) intravesical therapy of bladder cancer is arguably the most effective immunotherapy for any human solid tumour. It combines a high incidence of remission induction with a low level of side effects and a low rate of recurrence. Its mechanism, however, remains poorly understood. In this study we have investigated whether gamma delta T lymphocytes, which are known to be activated by mycobacteria, are preferentially induced in patients urine following therapy. This has necessitated the development of a procedure which facilitates the preservation, enrichment and detection of small number of lymphocytes [especially of those bearing gamma delta T cell receptor (TCR)I which appear in patients urine. Here we describe in detail a method for phenotyping of a minor subpopulation of lymphocytes in patients' urine, namely, gamma delta T lymphocytes which comprised less than 0.1% of all urinary sells. Using this technique we have found gamma delta T cells in the urine of all patients. Furthermore, the patients could be separated into 2 distinct groups, with low and high numbers of gamma delta cells (0.5-5% and 5-20% respectively of the CD3 positive cells). The elevation of gamma delta T cells was observed locally but not in peripheral blood and the detected gamma delta T were almost entirely of the V delta 2 gamma 9 subset.

Prognostic value of biological markers in superficial bladder tumors

The early identification of urinary bladder cancer patients, who are at a high risk of developing recurrences and tumor progression, is of utmost importance, because they require a more aggressive therapeutic approach and close follow-up. The routine use of new prognosticators, more specific and reliable than the established clinicopathologic factors, is therefore mandatory. The present work reviews some of the most significant biological tumor markers (cell cycle proteins, blood group antigens, tumor suppressor genes, oncogenes, CD44 gene transcripts, etc.) defined immunohistochemically in bladder cancer patients. It summarizes most of the recently published results of the clinical trials, using these markers as predictors of the tumor growth, invasion and metastasis. It is also an attempt to highlight the present state of the problem, the still existing controversy and some of the hypotheses concerning the predictive potential of the recently detected tumor markers. Biomedical Reviews 1995; 4: 85-94.

Unusual retention of introns in CD44 gene transcripts in bladder cancer provides new diagnostic and clinical oncological opportunities.

Increased and disorganised expression of CD44 variant exons has been demonstrated in biopsy samples of several types of human malignancy by many groups. This abnormality can be used to detect exfoliated tumour cells in the urine of bladder cancer patients. The present report demonstrates that the deranged activity of this gene in neoplasia also results in the accumulation of immature mRNA transcripts containing non-coding sequences (introns) from this gene in cancer tissues and cells. There is simultaneous overexpression of a newly identified 437 bp exon (coding sequence) located in the variably active region of the gene and of many abnormal variant exon combinations. This is the first report describing the specific retention of introns in gene transcripts in clinical diagnostic samples of tumour tissues and cells. The phenomenon was seen repeatedly in samples from cancer patients and in a cancer cell line, and thus could form the basis for a unique new and specific method of cancer diagnosis.

Elevated Levels of the Angiogenic Peptide Basic Fibroblast Growth Factor in Urine of Bladder Cancer Patients

Elevated Levels of the Angiogenic Peptide Basic Fibroblast Growth Factor in Urine of Bladder Cancer Patients

Unconventional fractionation for palliative radiotherapy of urinary bladder cancer. A retrospective review of 94 patients.

Ninety-four urinary bladder cancer patients with locally advanced, recurrent or metastatic urinary bladder cancer were treated with external radiotherapy with the aim of palliating local disease. Conventional radical radiotherapy was inappropriate because of extensive disease, poor general condition or old age of the patients. The palliative course studied was 30 Gy (mid-point dose) in six fractions, two fractions a week. Eighty-two patients (87%) tolerated treatment as planned. Forty patients (43%) had complete palliation of initial symptoms. Thirty-eight patients (40%) had initial local control of the tumour, which was lasting in 25 cases (26%). Median disease-specific survival of the patients was 13.3 months. The estimated five-year survival was 13% and survival from bladder cancer 27%.

Activation by the Protein-Bound Polysaccharide PSK (Krestin) of Cytotoxic Lymphocytes that Act on Fresh Autologous Tumor Cells and T24 Human Urinary Bladder Transitional Carcinoma Cell Line in Patients with Urinary Bladder Cancer

PSK, a protein-bound polysaccharide Kureha, was tested for its ability to modulate the cytotoxicity of lymphocytes that act on autologous tumor cells and T24 human urinary bladder tumor cells in urinary bladder cancer patients in a 6-h 51Cr release assay. In vitro treatment of peripheral blood lymphocytes (PEL) with PSK for 18hours resulted in an augmentation or induction of cytotoxicity against relatively resistant T24 cells in previously reactive and nonreactive cases, respectively. The PSK-treated PBL were able to kill more effectively tumor cells that were freshly isolated from the same cancer patients than non-treated PBL. The effects of PSK were noted with PBL as well as tumor infiltrating lymphocytes (TIL) and with PSK at concentrations of 10 to 100μg./ml., while PSK at higher doses reduced their lytic activities. The addition of PSK to the assay at the same concentrations also enhanced the cytotoxicities. Autologous tumor killing (ATK) activities of both large granular lymphocytes (LGL) and T lymphocytes were enhanced by PSK. Treatment of PBL with PSK did not effect on the proportion of PBL binding to the tumor cells, while it augmented the cytotoxic activity. Cell-free supernatant of PSK-stimulated lymphocyte culture did not contain any detectable amounts of interferon-α (IFN-α), interferon-γ (IFN-γ) and interleukin-2 (IL-2). In addition, anti-IFN-α monoclonal antibody (MAb), anti-IFN-γ MAb and anti-IL-2 MAb did not inhibit PSK-induced agumentation of cytotoxicity against T24. Oral administration of PSK (three gm./day) to patients with urinary bladder cancer daily for seven days before operation resulted in an augmentation of the cytotoxicity against T24 cells in five out of 10 patients and no change of the cytotoxicity In the other five patients. ATK activity was also enhanced by oral administration of PSK in three out of five patients. These results indicate that the antitumor activity of PSK may be in part mediated through activation of tumor killing system independent of IFN-α, IFN-γ and IL-2.

Establishment and Characterization of a Human Urinary Bladder Carcinoma Cell Line (TSGH-8301)

A cell line derived from a well-differentiated human transitional cell carcinoma of the urinary bladder, designated TSGH-8301, was established in vitro. The cultured epithelioid cells exhibited monolayer growth and loss of contact inhibition. The tumorigenicity of TSGH-8301 had been shown by growth in soft agar and tumor induction in athymic nude mice. A reverse ratio of lactate dehydrogenase (LHD) isoenzyme in the cell line and nude mouse-grown tumors was seen predominantly with LDH-V. Chromosomal analysis revealed a heterodiploid stem line with a modal number of 50. Sera of urinary bladder cancer patients reacted with membrane antigens of the TSGH-8301 cells, suggesting the existence of tumor-associated antigens in the cells. In vitro chemosensitivity tests of these cells may provide data valuable in the selection of proper anticancer drugs for the TSGH-8301 donor patient.

Establishment and characterization of a human urinary bladder carcinoma cell line (TSGH-8301).

A cell line derived from a well-differentiated human transitional cell carcinoma of the urinary bladder, designated TSGH-8301, was established in vitro. The cultured epithelioid cells exhibited monolayer growth and loss of contact inhibition. The tumorigenicity of TSGH-8301 had been shown by growth in soft agar and tumor induction in athymic nude mice. A reverse ratio of lactate dehydrogenase (LHD) isoenzyme in the cell line and nude mouse-grown tumors was seen predominantly with LDH-V. Chromosomal analysis revealed a heterodiploid stem line with a modal number of 50. Sera of urinary bladder cancer patients reacted with membrane antigens of the TSGH-8301 cells, suggesting the existence of tumor-associated antigens in the cells. In vitro chemosensitivity tests of these cells may provide data valuable in the selection of proper anticancer drugs for the TSGH-8301 donor patient.

Tumor markers (CEA, TPA and CA 19-9) in urine of bladder cancer patients.

This study was carried out to evaluate the usefulness of determining urinary levels of carcinoembryogenic antigen (CEA), tissue-polypeptide antigen (TPA), and gastro-intestinal cancer antigen (Ca19-9) in addition to the usual diagnostic procedures for bladder cancer. Sixty-seven patients with transitional bladder cancer, 40 healthy controls and 20 patients with inflammatory diseases of the urinary tract were considered. All urine samples were obtained from patients with intact renal function and no urinary tract infection. TPA and Ca19-9 urinary levels in patients with G3 bladder tumors were significantly higher than in those with lower graded neoplasms. The sensitivity, specificity, and predictive value of a positive (PV+) or negative (PV-) test and the diagnostic accuracy were also evaluated. Ca19-9 was the best urinary marker for bladder cancer (sensitivity 71.6%, specificity 91.6%, PV+ 90.5%, PV- 74.3%, diagnostic accuracy 81%).

Study of microscopic porphyrin fluorescence

A technique of porphyrin (hematoporphyrin derivative [Hpd]) fluorescence microscopy was evaluated by studying Hpd uptake, retention, and loss in established human cancer cell lines. Hpd uptake appeared to be qualitatively identical in the three cell lines used, but the rate of loss was slowest in the renal-carcinoma-derived line, suggesting a cellular characteristic. The technique was readily applied to demonstrate porphyrin fluorescence in exfoliated cells in urine of bladder cancer patients.

Radiation therapy combined with hyperthermia in advanced cancer

Radiation therapy combined with radiofrequency (RF) hyperthermia was performed on 5 advanced cancer patients. Included were one each with urinary bladder cancer, hepatoma with left axillary node metastasis, breast cancer, tongue cancer with left cervical metastasis, and mandibular cancer. All had large tumours, which were judged to be uncontrollable by radiotherapy alone. They were treated with irradiation (Linac: 10 MV X-ray 1.8-2.0 Gy/day, 5 days/week), followed within an hour by RF hyperthermia once or twice a week. Partial response was obtained in the urinary bladder cancer patient. Surface overheating around the margin of electrodes occurred in all but no severe complications were observed.

The clinical significance of tissue polypeptide antigen (TPA) in the urine of bladder cancer patients.

A 2-stage study has been carried out to evaluate the usefulness of urinary tissue polypeptide antigen (TPA) levels in patients with bladder cancer as an adjunct to the routine procedures for detection of bladder tumours. Two-hour urine samples were collected from 83 bladder cancer patients and normal individuals for the first part of the study, 24-h samples from 54 patients and normal individuals for the second part. Urinary TPA was determined using radioimmunoassay. In 2-h samples there was no significant difference in the amounts of TPA/l in any of the groups. In contrast, the TPA results of 24-h urine samples (n = 54) were markedly different from 2-h samples and the former correlated very well with the presence or absence of bladder cancer. A reason for this difference may be circadian rhythm effects.

N-acetyltransferase phenotype and risk in urinary bladder cancer: approaches in molecular epidemiology. Preliminary results in Sweden and Denmark.

A variable but often significant proportion of urinary bladder cancer in urban areas can be attributed to occupational and cultural (cigarette smoking) situations associated with exposures to various arylamines. The variable N-acetylation of carcinogenic arylamines by human hepatic enzyme systems, the known genetic regulation and polymorphic distribution of this enzyme activity in humans, and the known enhanced susceptibility of individuals with the genetically-distinct "slow acetylator" phenotype to various arylamine toxicities, has prompted examination of possible correlations between N-acetyltransferase phenotype and urinary bladder cancer risk in rural and urban populations. In this context, N-acetylation is viewed as a component of detoxication pathways with respect to arylamine bladder carcinogenesis. In preliminary utilizations of this approach, a population of urban urinary bladder cancer patients from Copenhagen, Denmark displayed a 13% excess (p = 0.065) of individuals with the slow acetylator phenotype (46/71 = 64.8%) when compared to a Danish control population (38/74 = 51.4%). These data are consistent with the possibility that arylamines may play an etiological role in bladder cancer in this locale and that slow acetylator individuals may be at higher relative risk (1.74) than rapid acetylator individuals. As 95% of patients reported histories of smoking, it was not possible to isolate and examine smoking factors. In contrast, a population of rural urinary bladder cancer patients from Lund, Sweden, where bladder cancer incidence (20/100,000) (1971) is lower than in Copenhagen (43.8/100,000) (1968-72), no difference in slow acetylator distribution was observed between bladder cancer (80/115 = 69.6%) and Swedish control (79/118 = 66.9%) populations, indicating a relative lack of involvement of arylamines in the etiology of rural bladder cancer. Populations of "spontaneous" bladder cancer patients would be expected to contain variable portions of disease related to arylamine exposure and would be less likely to display a detectable correlation than would an industrial population with documentable arylamine exposure. Consequently, confirmation of this hypothesis is being pursued by examination of industrial populations in an effort to obtain an empirical estimate of relative risk for slow and rapid acetylator phenotypes. These studies involve exposure-matched workmen both with and without bladder cancer.