Abnormalities detected in metaphase chromosomes in bladder carcinoma: prognostic value and comparison with histopathological factors.

The objective of this study was to detect the incidence and prognostic value of chromosomal aberrations in metaphase chromosomes (hypodiploidy, hyperdiploidy and/or structural abnormalities) in Ta and T1 transitional cell carcinoma (TCC) of the bladder. Of 266 patients, the metaphase chromosomes of the primary tumour were studied using a direct microscopic analysis and classified into two categories: normal and abnormal. Recurrence and progression were prospectively recorded during a median follow-up period of 40 months and in a retrospective analysis compared with other prognostic factors. Chromosomal abnormalities were found in 48% of Ta tumours and in 92% of T1 tumours. In univariate analysis, chromosomal abnormalities were associated with recurrence-free survival ( P=0.03) and progression-free survival ( P=0.01). In multivariate analysis, chromosomal abnormalities (RR=1.98) and age (RR=0.64) were independent predictors of recurrence-free survival but not progression-free survival.

MIB-1 as a proliferative marker in transitional cell carcinoma of the bladder: clinical significance and comparison with other prognostic factors.

BACKGROUND: Staging and grading of transitional cell carcinoma of the bladder are generally viewed as indicators of prognosis and form the basis of therapy, but they do not predict outcome accurately. This study was designed to evaluate the value for predicting recurrence, progression, and survival of proliferation fraction in transitional cell carcinoma of the bladder determined by immunostaining of histopathologic specimens with the monoclonal antigen MIB-1. METHODS: In a prospectively followed group of 301 patients with transitional cell carcinoma of the bladder, formalin fixed tumor specimens were immunostained and the MIB-1 labeling index was determined. Crude survival, progression free survival, and recurrence free survival (for patients with Ta and T1 tumors) were assessed in univariate and multivariate analysis according to stage, grade, mitotic index of the tumor, and patient age. The median value of continuous variables was used as a cutoff point in statistical analysis. RESULTS: In univariate analysis there was a strong association between all included factors and crude survival, progression free survival, and recurrence free survival with a median follow-up period of 60 months. In multivariate analysis, crude survival and progression free survival were determined by stage (P = 0.0001) and age (P = 0.0001). Recurrence free survival for patients with Ta and T1 tumors was determined by MIB-1 labeling index (P = 0.0317), mitotic index (P = 0.0229), and age (P = 0.0001). CONCLUSIONS: MIB-1 immunostaining in transitional cell carcinoma of the bladder correlated well with grade, stage, and clinical outcome. In multivariate analysis, proliferation fraction had prognostic value in predicting recurrence free survival for patients with Ta and T1 tumors, whereas stage and age appeared to be predictors of progression free survival.

A simplified grading method of transitional cell carcinoma of the urinary bladder: reproducibility, clinical significance and comparison with other prognostic parameters.

OBJECTIVE: To determine the extent to which the biological potential of transitional cell neoplasms can be predicted by histological grading of the primary tumour in a two grade system using simple histological criteria and to evaluate the additional value of grading when combined with other prognostic factors. The inter-observer variability of the World Health Organization grading and the two grade system was tested. PATIENTS AND METHODS: The study included 311 patients with newly diagnosed transitional cell carcinoma of the urinary bladder. Two-hundred and fifty-six patients (82.3%) were men and 55 (17.7%) were women. Age ranged from 17 to 92 years with a mean of 66 years. The median follow-up was 38 months, with a maximum of 150 months (mean 46.2 months). RESULTS: A simplified grading system was developed in which only low-grade and high-grade tumours were distinguished. Reproducibility of this grading system was good to excellent with a group kappa value of 0.78. The survival of patients with low-grade tumours was significantly better than that of patients with high-grade tumours (P < 0.0001). The progression-free interval was also significantly longer in patients with low-grade tumours than in patients with high-grade tumours (P = 0.0032). Combining low-high grading, histological stage, mitotic index and age, histological stage appeared to be the most important parameter in predicting survival and progression. CONCLUSION: A reproducible and discriminating system such as this low-high grade system is an important prognostic factor when stage cannot be established with certainty.

Cytogenetic analysis in transitional cell carcinoma of the bladder.

The potential use of numerical chromosomal abnormalities as predictive factors for the clinical behaviour of transitional cell carcinoma (TCC) was investigated. The effects on survival and progression-free survival were measured in 91 patients with TCC treated by transurethral resection. The survival rate of patients having tumours with a diploid chromosomal modal number was significantly better than that of patients having tumours with a hyperdiploid chromosomal modal number. The survival rate of patients having TCC with diploid cells only was also significantly better than that of patients having TCC with both diploid and hyperdiploid cells. Progression-free survival was significantly higher in patients having TCC with a diploid modal number of chromosomes than in patients with a hyperdiploid modal number. Simultaneous evaluation of the modal chromosome number or chromosomal range, histological grade, category and mitotic index of the tumour, and the patient's age and sex as prognostic factors in multivariate analyses showed that the category of bladder carcinomas was the most important factor in predicting the survival rate. In patients with superficial tumours (category Ta and T1) the modal chromosome number was the most important factor in predicting survival. For progression-free survival, the modal chromosome number appeared to be the most important factor. It was concluded that the modal chromosome number in TCC was useful in predicting survival in patients with superficial tumours and in predicting progression-free survival in patients with tumours of all categories.

Image cytometric DNA analysis in transitional cell carcinoma of the bladder.

BACKGROUND: The current study was initiated to investigate measurable objective and reproducible characteristics that might have prognostic significance in bladder cancer. METHODS: Tumor samples from 91 patients with primary transitional cell carcinoma (TCC) of the urinary bladder were studied by DNA image cytometry and cytogenetic analysis. Image cytometry is a more sensitive method of determining ploidy than flow cytometry, especially in tumors with a low number of aneuploid cells. RESULTS: There was a significant difference in survival between DNA image cytometry-determined diploid and nondiploid cases. The presence of nuclei with a high DNA content indicated poor prognosis. The 2C deviation index (2CDI) also was an indicator of survival. Image cytometry-determined factors also were found to be strong predictors of progression-free survival. In multivariate analysis, 2CDI was the only cytometric parameter with an independent but weak correlation with survival. In multivariate analysis, none of the cytometric parameters had an important contribution to prediction of progression-free survival. In superficial tumors (Ta and T1), 2CDI appeared to be the most important independent predictor of survival. With respect to progression-free survival, tumors with a high mitotic index proved to have a worse prognosis. CONCLUSIONS: Parameters determined by DNA image cytometry appear to be valuable in predicting survival and progression-free survival and may be useful in addition to the classic parameters of stage and grade, especially in superficial TCC.

Structural chromosome 1 aberrations in transitional cell carcinoma of the bladder: interphase cytogenetics combining a centromeric, telomeric, and library DNA probe.

Fluorescence in situ hybridization (FISH) was used to study numerical and structural chromosome 1 aberrations in interphase nuclei of transitional cell carcinomas (TCCs) of the urinary bladder. One of the characteristic numerical aberrations, as detected previously in low-grade noninvasive TCCs, included trisomy for chromosome 1 (A. H. N. Hopman et al., Cancer Res., 51: 644-651, 1991). We examined in more detail 22 cases with a centromeric (1q12) and a telomeric associated (1p36) DNA probe and with a library DNA probe from sorted human chromosome 1 in single- and double-target FISH procedures. All flow cytometrically determined DNA diploid TCCs (13 cases), which showed three spots for 1q12 (6 cases), had two spots for 1p36. Since the library DNA probe showed three separate domains in the nuclei of these cases, the additional copy for 1q12 could be explained as an extra chromosome 1p-, containing the 1q12 target. In the flow cytometrically determined DNA tetraploid/aneuploid tumors, the results were more complex. In 6 of 9 cases, we observed an overrepresentation of 1q12 as compared to 1p36, also suggesting the presence of extra copies of 1p- chromosomes. The results of the present study demonstrate the utility of the FISH method to assess structural chromosome aberrations in interphase nuclei of solid tumors.

Numerical chromosome 1, 7, 9, and 11 aberrations in bladder cancer detected by in situ hybridization.

Forty transitional cell carcinomas of the human urinary bladder (TCCs) were examined for numerical aberrations of chromosomes 1, 7, 9, and 11 by in situ hybridization using chromosome-specific probes. Our interphase cytogenetic study of 24 low-grade, noninvasive TCCs, which were near-diploid by flow cytometry, showed a numerical aberration for at least 1 of these chromosomes in 14 of these cases. Most strikingly, a monosomy for chromosome 9 was found in 9 of 24 low-grade TCCs. A trisomy for chromosomes 1, 7, and 11 was detected in 5, 2, and 1 case(s), respectively. In 1 case a monosomy for chromosome 1 was detected by in situ hybridization. Monosomy for chromosome 9 was the only detected numerical change in 5 low-grade TCC cases. Examination of 16 invasive TCCs showed extra copies for chromosomes 1 and 7 in 7 flow cytometrically diploid cases with numerical chromosome aberrations; also, loss of chromosome 9 was detected. In 5 invasive and 2 noninvasive aneuploid/tetraploid TCCs a profound imbalance between the different chromosomes was found. In 5 of these cases an evident underrepresentation of chromosome 9 in comparison to chromosomes 1, 7, and 11 was detected. This underrepresentation of chromosome 9 in diploid, as well as aneuploid, TCCs, and in some cases the constant ratio between this chromosome and the other chromosomes, may be explained by a process of tetraploidization. Therefore, loss of chromosome 9 may be one of the primary genetic events in TCC oncogenesis, with secondary events, such as tetraploidization, correlated to tumor progression. Our results show that in situ hybridization can be routinely used to study important cytogenetic changes which occur during the development of a malignant disease.

Prognostic significance of type IV collagen and laminin immunoreactivity in urothelial carcinomas of the bladder.

Invasion of a carcinoma involves the degradation and penetration of the subepithelial basement membrane (BM). This phenomenon might be used for histopathologic evaluation of neoplasms of the bladder. The authors studied the clinicopathologic data and tissue specimens of 125 cases of urothelial carcinomas collected prospectively. Penetration of the BM was evaluated by immunohistochemical staining of the BM components laminin and type IV collagen. The use of this parameter as a prognostic indicator in bladder cancer was assessed. The 5-year survival rate of patients having tumors with an interrupted or absent BM was significantly lower than that of patients having tumors with an intact BM. The rate of progression was greater in tumors with an interrupted or absent BM than in tumors with an intact BM. No association was found between BM status and recurrence. However, a significant correlation between tumor stage and BM staining was found. A correlation was also found between ploidy and BM staining as well as between histologic grade and BM staining pattern. When evaluating histologic grade, stage, ploidy, age, and BM score as prognostic parameters, the stage of bladder carcinomas turned out to be the most important factor in predicting the survival rate and the progression-free survival. However, BM staining was found to be of value for early identification of microinvasion and is helpful for correct staging of urothelial carcinomas.

Detection of numerical chromosome aberrations in bladder cancer by in situ hybridization.

The nuclear DNA content of 53 transitional cell carcinomas (TCCs) of the urinary bladder, as determined by flow cytometry (FCM), was compared with chromosome ploidy as detected by nonradioactive in situ hybridization (ISH). For this purpose, probes for repetitive DNA targets in the (peri) centromeric region of chromosomes 1 and 18 were used. Hybridization results with both probes of 35 TCCs, which had a DNA index of approximately 1.0 as concluded from FCM, showed evident chromosome 1 aberrations in approximately 25% of the tumors, and in a few cases an aberration for chromosome 18 was detected. Comparison of the ISH spot numbers for both chromosomes showed in most cases a higher number for chromosome 1 than for chromosome 18. ISH on 18 cases of TCCs, which showed a single peak in FCM with a DNA-index of 1.2 to 3.2, exhibited a profound heterogeneity. In these TCCs the ratio between chromosomes 1 and 18 varied over a wide range, resulting in cases showing more hybridization signals for chromosome 1 than for chromosome 18 or the opposite. Furthermore, using ISH minor cell populations showing polyploidization and giant cells containing numerous ISH signals could occasionally be detected. Results showed that interphase cytogenetics by ISH enable a fast screening of numerical chromosome aberrations and detection of different cell populations within one tumor, which was apparently homogeneous according to FCM.

Blood group isoantigen deletion and chromosomal abnormalities in bladder cancer.

The presence or absence of blood group isoantigens in 78 patients with transitional cell carcinoma of the bladder was correlated with tumor stage and grade, and results of chromosomal analysis. For blood group isoantigen detection the indirect immunoperoxidase method with monoclonal antibodies to A, B and H antigen was used. In 51 per cent of the 59 superficial tumors blood group isoantigens were demonstrable, whereas all deeper infiltrating and higher grade tumors were negative. However in superficial tumors the mode of blood group isoantigen expression did not correlate significantly with tumor recurrence and progression. A consistent correlation was demonstrated among chromosomal numbers, tumor grade and clinical course. The chromosomal abnormalities found and mode of blood group isoantigen expression, even in combination, had no prognostic value additional to the grading criteria used.

Grading in superficial bladder cancer. (1). Morphological criteria.

In a prospective study on the grading of superficial papillary neoplasms of the bladder a distinction was made between tumours showing only increased cellularity without appreciable cellular and nuclear deviation (grade 1), tumours showing slight cellular variation (grade 2a), and tumours showing clear cytological deviation and a tendency to lose normal polarity (grade 2b). Ninety-one patients with a superficial tumour were followed up for a mean of 24 months. Grade 2a tumours recurred later and in fewer patients than grade 2b tumours. Progression was seen in 4% of grade 2a tumours and in 33% of grade 2b carcinomas. Adapting our results to the WHO grading system, we suggest that all tumours in this study defined as grades 1 and 2a should be classified as low grade and tumours defined as grade 2b should be classified as intermediate grade.

Grading in superficial bladder cancer. (2). Cytogenetic classification.

Cytogenic analysis was performed in 92 newly diagnosed transitional cell bladder carcinomas and the results were correlated with the clinical course in superficial tumours. Low grade tumours appeared to have hypodiploid or peridiploid chromosomal numbers. Intermediate grade tumours were characterised by chromosomal counts up to the hyperdiploid range but could have a peridiploid modal number. In high grade tumours the modal number was hyperdiploid. The range in chromosomal counts appeared to be more reliable than the modal number as a predictor of the course of superficial tumours. The significant difference in chromosome numbers between low, intermediate and high grade tumours indicates that grading reflects major cytogenetic changes in the tumour cells.

Tissue-specific markers in flow cytometry of urological cancers. III. Comparing chromosomal and flow cytometric DNA analysis of bladder tumors.

Thirty-seven transitional-cell carcinomas (TCC) of the urinary bladder were analyzed by DNA flow cytometry (FCM). After labelling of the cell suspensions with antibodies to cytokeratin, the cytokeratin-positive cells and the non-epithelial cytokeratin-negative cells could be analyzed separately. After estimation of S- and G2M phase, 3/17 cases (18%) with a normal DNA index showed elevated proliferative levels, among cytokeratin-labelled suspensions only. Of these 17 cases, 14 showed chromosomal abnormalities. The remaining 20 cases were abnormal, irrespective of the technique used. Although immuno-labeling of tumor cells for cytokeratin in FCM increases the sensitivity of this method in detecting aneuploid tumors or tumors with high proliferation fractions, the discriminating power of chromosomal analysis of TCC is greater than FCM.

Comparison of tissue disaggregation techniques of transitional cell bladder carcinomas for flow cytometry and chromosomal analysis.

DNA index (DI) measurements and chromosomal analysis of 42 transitional cell carcinomas were done after mechanical and enzymatical disaggregation of the tumor specimens. The results obtained with these different disaggregation techniques were compared in the 33 cases (79%) that showed recognizable chromosomes. The enzymatically obtained cell suspensions could not be used for chromosomal analysis after short-term culture of 24 hours. In four cases, the DI after enzymatical treatment could not be estimated. In most cases, the DI obtained from the tumor cells was similar for both aggregation techniques, with the exception of four cases of enzymatically treated cell suspensions in which the DI could not be estimated. The average DI of the aneuploid tumors was 13% higher than the corresponding chromosome count. In 19% of the aneuploid tumors the proportion of aneuploid cells could not be measured after enzymatical treatment. In the remaining suspensions the proportion of diploid cells was higher after enzymatical disaggregation than after mechanical treatment. It is concluded that for flow cytometric and direct chromosomal analysis of bladder tumors, the mechanical disaggregation technique is most suitable.

Tissue-specific markers in flow cytometry of urological cancers: cytokeratins in bladder carcinoma.

Thirty-eight transitional-cell carcinomas (TCC) were analyzed by flow cytometry (FCM) using propidium iodide for DNA analysis and antibodies to cytokeratin by indirect immunofluorescence. By means of two-dimensional FCM analysis, cytokeratin-positive tumor cells could be analysed separately from cytokeratin-negative stromal and inflammatory cells. This resulted in an 18% increase in sensitivity of FCM detection of aneuploidy (10/38 samples with one-parameter DNA analysis versus 15/38 samples with two-parameter DNA and cytokeratin analysis). In addition, S-phase could be determined in the 15 aneuploid samples by means of two-parameter analysis where this was not possible using only DNA content because of the overlap of diploid and aneuploid populations. FCM analysis allowed quantification of the percentage of tumor cells expressing cytokeratin 18 which has previously been shown to correlate quantitatively with higher grade, higher stage TCC. The quantitative measurement of tumor-cell expression of cytokeratin 18 by FCM analysis appears to provide additional information of potential prognostic value, independent of tumor-cell ploidy and proliferative fractions.