Characterization of cell lines originating from an invasive transitional cell carcinoma.

A reporter gene (lacZ) was introduced into a human transitional cell cancer cell line (Hu1703He) by means of liposomal transfection. The lacZ-transfected cell line induced subcutaneous tumours in nude rats and cells from one rat tumour were then established as a monolayer culture. The two lacZ-transfected cell lines both stained positive for CK7 and negative for CK14 and additionally formed spheroids in three-dimensional cultures. Insignificant genomic changes occurred in the tumour cells after incubation in nude rats, while the lacZ transfection caused alterations that probably correspond to increased invasiveness and tumourigenicity in vitro and in vivo. Most important is the observation that lacZ transfection of this human TCC cell line does not reduce its invasion potential in vitro or in vivo. The lacZ reporter gene may thus be exploited to facilitate the identification and quantification of migrating tumour cells and subsequently for studies of invasion in in vitro coculture systems. The observation that the spheroidal growth is reduced after transfection of the cell line, in contrast to increased invasion and cellular growth in monolayer, is an observation indicating that a three-dimensional arrangement mimicking the in vivo conditions offers important regulating factors to cellular growth.

Prognostic significance of p16 and CDK4 proteins in localized prostate carcinoma.

BACKGROUND: In prostate carcinoma, a very low frequency of point mutations of the tumor suppressor gene CDKN2/MTS1 (p16(INK4) ) has been reported, but deletions of 9p21 and inactivation by promoter methylation are observed more frequently. In the current study the authors evaluated the expression of p16 and CDK4 proteins and their prognostic significance in patients with clinically localized prostate carcinoma. METHODS: The levels of p16 and CDK4 proteins were quantitated by immunofluorescence flow cytometry, using paraffin embedded material, in 104 adenocarcinomas of the prostate after radical prostatectomy. These levels then were compared with 25 cases of benign prostate hyperplasia (BPH). RESULTS: In prostatic carcinoma specimens, p16 protein was elevated significantly compared with BPH, with a median fluorescence index (FI) of 15.4 versus 10.7, respectively (P = 0.010). This was not the case for CDK4 protein, although p16 protein expression correlated significantly with CDK4 protein expression in BPH (Spearman rank correlation [R(S)] = 0.63) and carcinoma (R(S) = 0.78). In univariate survival analysis of the first 5 years, high levels of p16 protein expression (FI > 11.7) (P = 0.005), tumor greatest dimension, World Health Organization (WHO) histologic grade, capsular penetration, seminal vesicle invasion, positive surgical margins, lymph node involvement, and preoperative serum prostate specific antigen > 20 ng/mL all were significant predictors of biochemical failure. In multivariate survival analysis, high p16 protein expression (P = 0.015), age, WHO histologic grade, capsular penetration, and seminal vesicle involvement remained as independent predictors of biochemical failure. CONCLUSIONS: These data suggest that increased expression of p16 protein, but not CDK4 protein, may be involved in the development of prostate carcinoma and may represent an independent predictor of biochemical failure after radical prostatectomy.

Surgical management of chylous fistula after retroperitoneal lymph node dissection.

Conservative treatment with low-fat diet, medium-chain triglyceride or total parenteral nutrition, depending on the general condition of the patient, is the mainstay in the treatment of chylous ascites. In patients with persistent chylous fistula direct surgical closure is a valid treatment option.

Lack of diagnostic-value of DNA content and p53 immunostaining in normal, hyperplastic and neoplastic parathyroid tissue.

The aim of this project was to study the diagnostic value of DNA content and p53 protein expression in normal, hyperplastic and neoplastic parathyroid lesions. Tissue samples of 74 parathyroid glands from 34 patients with primary hyperparathyroidism were studied by DNA flow cytometry and p53 immunostaining. In 9 of 23 patients (39%) with parathyroid adenoma, a nondiploid cell population was present. Some normal looking glands removed from the same patients also had a nondiploid DNA index. Multiglandular hyperplasia was found in 11 patients, and in 5 of these (45%) the histograms showed nondiploid cells. The proliferative activity was generally low and S-phase fraction did not differ in glands with hyperplasia or adenoma, when compared with normal looking glands. One single case of hyperplasia showed a weak p53 positivity in scattered nuclei, probably representing wild type p53 protein. Thus, our present results suggest that DNA content and p53 protein staining are of no value in the routine work up of parathyroid glands removed from patients with primary hyperparathyroidism.

Influence of tissue preparation techniques on p53 expression in bronchial and bladder carcinomas, assessed by immunofluorescence staining and flow cytometry.

In a series of bronchial and bladder carcinomas, p53 protein expression was examined. Samples from formalin-fixed, paraffin-embedded tissue (routine-treated) were compared with parallel samples of fresh tissue and tissue fixed in paraformaldehyde and ethanol. The expression of p53 was measured by immunofluorescence staining and dual parameter flow cytometry, with simultaneous monitoring of DNA content. For each tumor, p53 fluorescence with different fixatives was expressed relative to fresh tissue. The p53 fluorescence signals were on average brighter from routine-treated tissue than from fresh tissue. The tissue fixed in paraformaldehyde showed no difference from fresh tissue. In the ethanol-fixed tissue, however, fluorescence signals were reduced by nearly 70%, and the fraction of detectable p53 positive cells in tumor tissue was reduced by more than 50%. This loss of fluorescence was probably due to a leakage of the antigen from nucleus to cytoplasm. Pepsin treatment did not influence p53 fluorescence. Within the same tumor, the S-phase fraction in p53 positive cells was significantly higher than in p53 negative cells (13.1 +/- 2.0% vs. 6.5 +/- 0.8%). This pattern was not influenced by formalin fixation or pepsin treatment. Our study demonstrates that in measuring a nuclear antigen, tissue handling may influence the results, and care should be taken to optimize the preparation procedure. Using the antibody PAb 1801, p53 expression measured in archival material is not reduced as compared to fresh tissue.

Flow cytometry of biopsy specimens from ulcerative colitis, colorectal adenomas, and carcinomas.

The cellular DNA distribution pattern in biopsy specimens from all main segments and neoplastic lesions of the large bowel, obtained from 16 patients with ulcerative colitis, 22 with adenomas, 17 with carcinoma, and 20 controls, has been studied by the flow cytometry technique. Aneuploid cell populations were demonstrated in three patients with ulcerative colitis (19%), in six with tubular adenomas (27%), and in nine with carcinoma (53%). Aneuploid cells were found in all cases of poorly differentiated adenocarcinomas. The fractional number of cells with a DNA content corresponding to the DNA synthetic and G2M phases of the cell cycle was defined as the 'proliferative index' (PI). In controls PI and its complementary G1 cell fraction varied significantly (p less than 0.005) between the segments, on the basis of analysis of variance. When compared two by two, PI was significantly higher in the sigmoid colon (p less than 0.01) and rectum (p less than 0.05) than in the ascending part. The PI of uninvolved mucosa from adenoma patients with diploid histograms was significantly higher than that in controls (p less than 0.01), ulcerative colitis (p less than 0.05), and cancer patients (p less than 0.05), when dependence on segment was taken into account.

Flow cytometric DNA studies in human gastric cancer and polyps.

Flow cytometric DNA studies were performed on cell suspensions of biopsy specimens from gastric tumors and neutral gastric mucosa in 18 patients with gastric cancer and 9 patients with gastric polyps. In cancer, aneuploidy was found in two tumors in the antral and five in the body part of the stomach (39%). The mean DNA index for aneuploid cancers was 1.57. In patients with aneuploid carcinomas three biopsy specimens from uninvolved mucosa also showed aneuploidy. In diploid carcinomas in the antral part of the stomach, the proliferative index (PI) was increased when compared with that of antral mucosa in controls (p less than 0.05). Increased PI was found in uninvolved mucosa in aneuploid carcinomas of the body part of the stomach when compared with that in diploid carcinomas (p less than 0.001). In uninvolved body mucosa in aneuploid carcinomas of the body part significantly reduced levels of acid-beta-glucuronidase (p less than 0.0001) were found when compared with diploid carcinomas. No polyps showed aneuploidy, and the PI in biopsy specimens from patients with gastric polyps did not differ from that in controls.

Flow cytometric DNA studies in normal human gastric mucosa, gastritis, and resected stomachs.

Flow cytometric measurements of cellular DNA have been performed on biopsy specimens obtained by gastroscopy in patients with normal gastric mucosa (controls), superficial and atrophic gastritis, and resected stomachs. Aneuploidy was found in two biopsy specimens from a patient with superficial gastritis and in one specimen from a resected stomach. The fractional number of cells with a DNA content corresponding to the DNA synthetic and (G2 + M) phases of the cell cycle was defined as the 'proliferative index' (PI). No difference between the antral and body part of the stomach was found for the PI in controls or between controls and superficial and atrophic gastritis. PI was weakly but significantly correlated to a selection of mucosal enzymes. In resected stomachs increased PI was found when compared with that in the body part of the stomach in controls (p less than 0.01). Biopsy specimens with intestinal metaplasia (p less than 0.01) or atrophy (p less than 0.05) in the resected stomach showed significantly higher PI when compared with specimens with such findings from the body part of the stomach in atrophic gastritis.

Methylthioadenosine phosphorylase in human breast cancer.

Methylthioadenosine (MTA) phosphorylase activity was measured in 47 biopsies from primary breast cancers (n = 34) and metastatic tumors (n = 13). Most specimens were also evaluated by DNA flow cytometry and determination of estrogen and progesterone receptor contents. Median MTA phosphorylase activity was 317 pmol/mg protein/min (range 50-1312 pmol/mg protein/min), but great variations were observed. Samples from four individuals had very low MTA phosphorylase activity (less than or equal to 70 pmol/mg protein/min). No correlation with aneuploidy, receptor status, or the presence of metastases in the lymph nodes could be demonstrated. However, MTA phosphorylase activity showed a significant (p = 0.009) negative correlation with the fraction of cells in the S-phase of the cell cycle.

Characterization of pleural effusions by flow-cytometric DNA analysis.

Pleural effusions from 58 patients were studied by cytology and flow-cytometry. Relative cellular DNA content was measured, giving an estimate of ploidy as well as cell cycle distribution. Twenty-one patients had non-malignant diseases while the rest had malignant diseases. Pleural effusions from 13 patients contained malignant cells. In 2 cytologically equivocal cases and in one cytologically negative case aneuploid cells were demonstrated in the histograms. The cell cycle distribution of the diploid tumour cells did not differ from that of the diploid cells in benign effusions. The demonstration of an aneuploid histogram by flow cytometry favours a cancer diagnosis, and can provide valuable and decisive information in addition to cytological results.

Ploidy aberrations and agar cloning ability of malignant cells from human bladder carcinoma.

Nine patients with urinary bladder tumours were examined by cystoscopy. Cold cup biopsies were obtained from the tumours and histological sections made. Mapping of the bladder mucosa was performed by selectively aspirating cells from the tumours and from different areas of the bladder. Cellular DNA content was measured by flow cytometry. Additionally, tumour cells were grown in soft agar. Plating efficiency seemed to relate neither to histological grade in the original tumour, to ploidy state, nor to the proliferative fraction of cells in the tumours. Flow cytometry and morphology combined seemed to be more reliable indicators for biological properties of the tumours.

Local chemotherapeutic effects in bladder cancer demonstrated by selective sampling and flow cytometry.

A method is described by which the effect of intravesical chemotherapy can be monitored. Cytological samples obtained selectively during treatment were used for morphological and flow cytometric studies, and isoantigen (A, B and H) assessment in 2 patients with urothelial cancer. With flow cytometry even small aneuploid populations in the urothelium could be identified. From the histograms the urothelium was seen to contain 2 different cell populations: 1) diploid and 2) aneuploid. The ratio between aneuploid and diploid cells decreased significantly during treatment. Treatment was continued until no evidence of aneuploid cells could be identified in the histograms. Thus, it is demonstrated that intravesical chemotherapy for certain types of bladder cancer can eradicate the aneuploid cell population. A good correlation was found between cytological studies and flow cytometric measurements. Isoantigen assessment was done in the cell suspension used for morphological and flow cytometric studies. Isoantigen assessment also showed loss of antigens after completion of treatment, indicating that the diploid population was not normal biologically. Thus, 3 parameters can be correlated and related also to topography.

Seasonal variations of symptoms and occurrence of human bladder carcinomas.

A longitudinal study of transitional cell carcinomas of the urinary bladder has been performed in a region of Western Norway. Hematuria and infection were the first symptoms of neoplasia of the urinary bladder, showing marked and reproducible seasonal variations. Most of the tumours showed the first symptom at the end of the year. They also had a higher histological grade (WHO) than those occurring at other times of the year. This indicates that environmental factors dependent on the season may be of importance for the manifestation of malignant growth of the urinary bladder.

DNA aberrations of bladder mucosa in patients with transitional cell carcinomas.

DNA aberrations in bladder mucosa have been investigated in altogether 26 patients with aneuploid WHO grade 2 and 3 tumours (transitional cell carcinomas; TCC). In about 1/4 of the patients aneuploid cells were found only in the tumour. Hypotetraploid tumours showed the lowest frequency of involvement of the normal appearing mucosa of the trigone (43%). In hypertriploid tumours the corresponding value was 71%. In altogether 14 of the patients, the same type of aneuploid cells was found in the normal appearing mucosa as in the tumour. This indicates frequent involvement of the whole bladder mucosa in the tumour disease. In about 1/4 of the cases other types of ploidy aberrations were found in the normal appearing mucosa than in the tumour. This is indicative of preneoplastic changes in the mucosa, of which only one type of aberrations is associated with tumour growth. In some cases with multiple tumours of the bladder, all the tumours had the same aneuploid stemline, while in other cases the tumours were of different aneuploid stemlines. This is conformal with other reports of atypia in the surrounding mucosa in TCC and with the concept that recurrence of high grade tumours is the consequence of neoplastic involvement of the whole bladder.

Ploidy disturbance of normal-appearing bladder mucosa in patients with urothelial cancer: relationship to morphology.

A total of 4 patients with transitional cell carcinoma of the bladder underwent complete mapping of the mucosa and tumors with combined cytologic, histologic and flow cytometric evaluation of the extent of involvement of the neoplastic process. Flow cytometric measurement of the cellular deoxyribonucleic acid content in multiple cell samples taken at cystoscopy showed similar changes in the normal mucosa as in the tumors. These changes consisted of an increased fraction of cells with S-phase deoxyribonucleic acid content in 2 patients with grades 1 and 2 tumors, and the presence of extensive aneuploidy in 2 patients with World Health Organization grades 2 and 3 tumors. While grade 1 and some grade 2 tumors (World Health Organization) are composed only of diploid cells, some of the grade 2 and all grade 3 tumors consist of a mixture of diploid and aneuploid populations. Such aneuploid clones could be identified in normal-appearing mucosa and, thus, be a source of new occurrences. The impression of heterogeneity in histograms from different tumors within the same bladder is assumed to be caused by a variation in the ratio between aneuploid and diploid populations (high ratio in tumor and low in normal-appearing mucosa). This phenomenon may be the reason for variation in grading based on histological studies.

Mapping of cell cycle distribution in normal human urinary bladder epithelium.

A method is described by which urothelial cells from well defined areas in the urinary bladder mucosa can be obtained. At cystoscopy epithelial cells are aspirated by means of a ureteral catheter. The cells obtained by this selective sampling technique are well suited for morphology and for quantitative single cell measurements by flow cytometry (FCM). In the present study the cell cycle distribution has been measured by FCM. Twenty-five patients (13 males and 12 females) had urothelial cells collected from well defined areas in the mucosa and the DNA content measured from different sites separately. The distribution of bladder mucosa cells in the different parts of the cell cycle (G1, S-phase and G2 + M) is reported. No significant differences were found with regard to sex and age of the patients. Also, a low regional variation was found in the bladder mucosa indicating that the method should enable the discrimination of even small abnormal cell populations in tumour disease. No evidence of polyploidy in the human urothelium was found.

The effect of 13-cis-retinoic acid on dibutylnitrosamine induced polyploidy changes in mouse urothelium.

The epithelium in the normal urinary bladder contains cells with diploid to octoploid DNA-content. The carcinogen dibutylnitrosamine (DBN), given subcutaneously in repeated doses causes a loss of polyploidy prior to cancer development. In this study the changes in polyploidy caused by DBN was followed by use of flow cytometry. 13-cis-retinoic acid did not prevent this loss of polyploidy, and did not affect the polyploidy in the normal urothelium.