Bladder cancer diagnosis by flow cytometry. Correlation between cell samples from biopsy and bladder irrigation fluid

Results of flow cytometry (FCM) examinations of bladder irrigation specimens were compared with those of FCM examinations of cell suspensions from bladder biopsies of 44 urologic patients. The fluorescent dye, acridine orange (AO), was used to stain DNA and RNA differentially and abnormal urothelial cells were identified by their relative content of nucleic acids. Granulocytes and squamous cells could be distinguished from transitional cells in this procedure, and did not interfere with the analyses. Of 28 patients with papillary carcinoma, carcinoma in situ, and invasive carcinoma 27 were identified through FCM examination of irrigation cytology specimens; the one false-negative result was from a low-grade papillary carcinoma. Of 7 patients with papilloma, FCM examinations of irrigation specimens were positive in 4 and negative in 3. Results of FCM studies of biopsy specimens were in good but not complete agreement with those of irrigation specimens. In several cases, irrigation FCM disclosed tumor stemlines that were not identified in biopsy specimens. Discrepancies of this kind seemed most likely due to differences in sampling. Irrigation FCM seems to be a sensitive method for assessing multiple-site bladder tumors, and may be a useful technique for monitoring the course of conservatively managed bladder tumors.     

Preoperative evaluation of tumor ploidy in endometrial carcinoma: An accurate tool to identify patients at risk for extrauterine disease and recurrence.

BACKGROUND: Tumor ploidy is a strong prognostic factor in patients with endometrial carcinoma, but generally is evaluated only after surgery. The availability of a simple and reliable method to determine tumor ploidy before any treatment is initiated could be helpful in the selection of patients at high risk for advanced primary disease and subsequent recurrence, with several possible benefits. The objectives of the current study were: 1) to test the accuracy of flow cytometric determination of tumor ploidy from preoperative outpatient endometrial biopsies compared with standard postoperative evaluation from the surgical specimen and 2) to correlate this preoperative parameter with the local recurrence and extrauterine tumor spread. METHODS: Tumor ploidy from both preoperative biopsy material and the macroscopic surgical specimens was evaluated prospectively in 50 consecutive patients with endometrial carcinoma. DNA analyses were performed in a blind fashion. Patients were followed for a median of 26 months (range, 16-46 months). RESULTS: In 9 of 50 cases (18%) an aneuploid tumor was found by the standard postoperative analysis. All 9 aneuploid tumors (100%) also were identified correctly by the preoperative test on biopsy material. Occult extrauterine tumor spread was found in 10 patients (20%). The incidence rate of aneuploidy among these tumors was 50% compared with 10% in surgical International Federation of Gynecology and Obstetrics Stage I tumors (P = 0.01). The recurrence rate was 55.5% (5 of 9 tumors) in the aneuploid group and 2.4% (1 of 41 tumors) in the diploid group (P < 0.001). The disease free survival rates of patients with diploid and aneuploid tumors were 97.5% and 44.4%, respectively (P < 0.0001). CONCLUSIONS: Preoperative tumor ploidy determination based on outpatient endometrial biopsy is as accurate as the standard postoperative evaluation in patients with endometrial carcinoma. Tumor aneuploidy confirms the usefulness of this method in selecting patients at risk for occult extrauterine tumor diffusion and recurrence.     

DNA aneuploidy and cell proliferation in breast tumors

The cellular DNA content of 30 benign and 180 malignant breast tumors was analyzed by means of flow cytometry (FCM). All benign tumors exhibited a normal DNA content (diploid), whereas 65% of the malignant tumors showed an abnormal DNA content (aneuploid). The ploidy distribution of malignant tumors was bimodal with an increasing frequency near diploid DNA index (DI), and a second group had a DI ranging from triploid to tetraploid. In estimating the degree of malignancy eight independent histomorphologic and cytologic criteria were introduced. A good correlation was observed between DNA content abnormalities and the grade of differentiation of breast carcinomas. The percentage of S-phase cells of DNA aneuploid cell lines was significantly higher than in the diploid ones. The highly differentiated breast carcinomas (Grade 1) indicated lower S-phase values as compared to the undifferentiated (Grade 3) ones. S-phase values estimated by FCM were about two times higher than the 3H-thymidine labeling index (LI) obtained by an in vitro procedure. The data estimated in this study showed that DNA determinations as an adjunct to conventional histopathologic assessment may provide objective clinically relevant information with respect to the degree of malignancy and prognosis of patients with breast carcinoma.     

Ploidy and proliferation in human bladder tumors as measured by flow-cytofluorometric DNA-analysis and its relations to histopathology and cytology.

Biopsies from bladder tumors of 41 patients were investigated by flow-cytofluorometric DNA analysis and compared with exfoliated cells. The degrees of ploidy and proliferation were determined. Good agreement was found between the degrees of ploidy and proliferation in the biopsies and the exfoliated cell material. Tumors Grade I-II were either euploid or aneuploid. All Grade III tumors were aneuploid. The S-phase fractions were about 6% in the diploid tumors and 17% with large variations in the aneuploid tumors. The histological grading was well correlated to the number of S-phase cells and the occurrence of aneuploidy. When the Grade II tumors were divided into two groups having lesser and more pronounced atypia, the two groups differed significantly with regard to their degrees of proliferation. In addition to aneuploidy as an important criterium for malignancy, the degree of proliferation appears to be of major biological significance.     

Predictive value of DNA measurements in bladder washings. Comparison of flow cytometry, image cytophotometry, and cytology in patients with a past history of urothelial tumors

Comparative DNA ploidy measurements were carried out by flow cytometry and by image analysis on cells in 71 bladder washing specimens from 50 patients with past histories of bladder tumors. Among the specimens classified as diploid or questionable by flow cytometry, 14 showed the presence of aneuploid DNA values documented by image analysis. In 18 of the 50 patients, recurrent tumors were observed during a relatively brief period of follow-up. In 15 of them the DNA pattern was aneuploid and in three it was questionable. In nine of the 15 patients, both methods of DNA analysis disclosed aneuploidy, but in six patients aneuploidy was detected by image analysis only. A combination of DNA aneuploidy, whether observed by flow cytometry, image analysis, or both, and of positive or suspicious urine cytology is highly predictive of recurrence of high grade bladder tumors. Image analysis of DNA content in bladder washings adds information of clinical value above and beyond that obtained by flow cytometry.     

A flow cytometric analysis of 23 carcinoid tumors

Twenty-three carcinoid tumors were investigated from paraffin-embedded tissue with flow cytometry (FCM) in order to correlate the DNA ploidy with clinical variables. DNA aneuploidy was found in ten tumors (45%) and one tumor was classified as tetraploid. Diurnal urinary excretion of 5-hydroxy indolic acetic acid (5-HIAA) was known to be elevated in seven of eight diploid tumors and in four of seven aneuploid carcinoids with distant metastases. Six (55%) of the diploid tumors had not given rise to metastases at the time of diagnosis, compared with three (30%) of the aneuploid tumors. Six of seven patients with an aneuploid tumor and three of five patients with a diploid tumor, observed for at least 10 years, died of the disease. It was concluded that, unlike in earlier studies with static DNA cytometry, DNA aneuploidy is common in human carcinoid tumors and may occur in tumors secreting biogenic amines.     

Acridine-orange flow cytometry of urinary bladder washings for the detection of transitional cell carcinoma of the bladder. The influence of prior local therapy

Analysis of cellular DNA and RNA contents of 249 bladder irrigation specimens from 129 patients with a history of transitional cell carcinoma (TCC) of the bladder was performed using acridine-orange flow cytometry (FCM). Washings from patients with prior intravesical chemotherapy or radiation therapy were compared to those from patients with no history of treatment other than tumor resection to evaluate the reliability of FCM for the detection of tumor and the influence of prior local therapy on that reliability. Five FCM patterns were defined on the basis of DNA and RNA indexes in relationship to peripheral blood lymphocytes. FCM results were compared to cytologic findings in 237 cases, cystoscopic findings in 230 cases, and histologic data in 99 cases. Presence of a single diploid stem line was associated with absence of bladder tumor in 71% of cases from patients treated with surgery alone or with radiation therapy, but there was residual tumor in 53% of patients exposed to prior local chemotherapy. An elevated RNA content in a diploid cell population did not provide additional diagnostic information. Presence of an aneuploid stem line was associated with tumor in 85% of cases, regardless of prior therapy. Aneuploidy predicted the appearance of tumor in four of six patients with a negative cystoscopy. Tetraploidy (greater than 10% of total cell population) was associated with tumor in 79% of patients treated with surgery alone, whereas no tumor was found in more than 50% of patients who had undergone prior chemotherapy or radiation therapy. This study stresses the importance of prior treatment history in evaluating the results of DNA-FCM for bladder cancer. It demonstrates the unreliability of FCM diploid and tetraploid cell populations in patients previously treated by local chemotherapy or radiation. However, it also supports prior observations that DNA-aneuploidy and DNA-tetraploidy are useful for detecting and predicting bladder cancer in patients submitted to surgery alone.     

Flow cytometric analysis of DNA and cell proliferation in ovarian tumors

DNA content in tumor cells from 50 patients with ovarian tumors was analysed by flow cytometry (FCM). Solid tissue samples were processed to obtain monodispersed cells. Staining for DNA analysis was achieved with ethidium bromide and mithramycin. Peripheral blood lymphocytes were used as reference diploid cell population. All benign ovarian tumors exhibited only diploid cells. DNA aneuploid cell lines were found 66.6% of serous carcinomas and in 80% of malignant granulosa cell tumors. The S-phase fraction of DNA diploid cells in benign ovarian tumors (S = 2.4 +/- 1.2%) was smaller than those of malignant tumors (S = 8.2 +/- 5.2%). DNA aneuploid cell populations in serous carcinomas display a higher S-phase fraction (S = 19.2 +/- 9.3%) than DNA diploid cells (S = 11.7 +/- 3.2%). No major differences were obtained between primary ovarian tumors and their metastases, as far as degree of aneuploidy and S-phase fraction are concerned. A high degree of correlation was established between the grade of differentiation of ovarian tumors and the DNA ploidy abnormalities.     

Flow cytometry for detection and evaluation of urinary bladder carcinoma.

Flow Cytometry (FCM) DNA assays of bladder irrigation specimens are now recognized as a clinically useful and reliable means of detecting and monitoring carcinoma of the bladder. This technique, which identifies carcinoma by the presence of an aneuploid population of cells, can be carried out on specimens obtained in an outpatient or hospital setting and is easily performed in any medium-sized laboratory. It is most sensitive to superficial and high grade tumors. Overall, nearly 80% of superficial carcinomas of bladder will have positive flow cytometry, comparing very favorably with conventional cytology. Until now, the widest clinical application of FCM has been in monitoring the conservative treatment of stage 0-1 flat and papillary carcinomas, but newly developed dual parameter measurements are capable of quantifying proliferative activity, oncogene expression, growth factor receptors, and other cellular features that may better characterize the biologic potential of these tumors and can be expected to aid in the selection and timing of treatment.     

Intratumoral heterogeneity in DNA ploidy of bladder carcinomas.

In order to investigate whether DNA ploidy in a single area of a tumor could be representative of the entire tumor, flow-cytometric DNA analysis was performed for multiple samples (an average of 7 samples) obtained from the same tumor in 9 cases of transitional cell carcinoma of the urinary bladder. The incidence of DNA aneuploidy was 88.9% (8 out of 9 cases) and, furthermore, in 7 of these, intratumoral regional heterogeneity in DNA ploidy was evident. With high frequency, bladder carcinoma consisted of heterogeneous cell populations with different ploidy. There were 2 cases in which the tumor consisted of mixed subpopulations of diploid and aneuploid clones which were located in different parts of the same tumor. In such cases, an aneuploid line could not have been detected in a single specimen from the tumors.     

Relationship between chromosomal instability and intratumoral regional DNA ploidy heterogeneity in primary gastric cancers.

The purpose of this study was to elucidate the relationship between intratumoral regional heterogeneity in DNA ploidy and chromosomal instability (CIN) in primary gastric adenocarcinomas. In 45 sporadic gastric adenocarcinomas, we measured DNA ploidy and numerical aberrations for chromosomes 7, 11, 17, and 18 by laser scanning cytometry and fluorescence in situ hybridization, respectively, in small tissue specimens taken from 2 to 6 (on the average 4) different portions of the same tumor. A total of 231 specimens including 45 normal control specimens were examined. All 98 tumor specimens with DNA aneuploidy (DNA index > or = 1.2) showed large intercellular variations in chromosome copy number, indicating CIN. In contrast, 85 tumor specimens with (near) diploidy (1.0 < or = DNA index < 1.2) exhibited much small intercellular variations in chromosome copy number as compared with aneuploid specimens (P < 0.0001). The relationship between DNA ploidy and intercellular variation in chromosome copy number was true for tumors consisting of a mixture of (near) diploid and aneuploid subpopulations. These data indicate that DNA aneuploidy is associated with CIN but that (near) diploidy is not. Intratumoral regional DNA ploidy heterogeneity was conspicuous in 33 (92%) of 36 tumors with regions of DNA aneuploidy, and all aneuploid specimens showed great intercellular variation in chromosome copy number. Diploid regions were predominant in early stage cancers (intramucosal and submucosal cancers), and five of eight early cancers contained only diploid population. In contrast, all tumors without (near) diploid regions were advanced cancers. These observations suggest that CIN is a necessary prerequisite for developing intratumoral DNA ploidy heterogeneity with DNA aneuploidy.     

Flow cytometric analysis of nuclear DNA content in ovarian tumors. Association of ploidy with tumor type, histologic grade, and clinical stage.

The authors undertook a prospective, flow cytometric study of nuclear DNA ploidy in 140 fresh ovarian tumors. There were 43 benign tumors, 27 borderline tumors, and 70 malignant tumors. Results of DNA ploidy analysis were compared to age at diagnosis, menopausal status, tumor size, histologic type, grade, and International Federation of Gynecology and Obstetrics (FIGO) stage. Although the majority of benign tumors were diploid, 19% were aneuploid. Among the benign tumors, DNA ploidy was significantly associated with tumor type and tumor size. All borderline tumors were diploid. Of the 70 malignant tumors, 64% were aneuploid. In the malignant tumors, DNA aneuploidy had significant univariate associations with histologic type, grade, and FIGO stage. By multivariate analysis, DNA aneuploidy remained significantly associated with stage and grade, both known predictors of survival in ovarian cancer. These results indicate that DNA ploidy varies with the aggressive potential of an ovarian cancer and may, at the time of initial diagnosis, provide additional information about tumor prognosis.     

Centrosomal abnormality is common in and a potential biomarker for bladder cancer

Centrosomal abnormalities have been implicated in chromosomal segregation aberrations that result from the formation of multipolar mitotic spindles and lead to aneuploidy. Aneuploidy is a characteristic of neoplasia and underlies the development and progression of bladder cancer. Therefore, centrosomal abnormality may play a key role in urothelial tumor transformation. The purpose of our investigation was to determine whether centrosomal abnormalities are present in malignant urothelial cells, define the relationship between centrosomal abnormalities and aneuploidy and determine whether the presence of centrosomal abnormalities might be a potential diagnostic marker for bladder cancer. Bladder wash specimens obtained from patients with and without a history of urothelial carcinoma were analyzed for centrosomal abnormalities using an immunoassay with a gamma-tubulin antibody. FISH with centromeric probes for chromosomes 4 and 9 and DNA ploidy image analysis were performed to detect aneuploidy. Defective centrosomes were found in 40 of 45 bladder wash specimens from patients with bladder cancer but in none of the 10 samples from patients without it. A large percentage (69%) of grade 1 tumors were positive for centrosomal abnormalities, and these abnormalities were increasing in numbers and size in grade 2 (93%) and grade 3 (100%) specimens. Centrosomal abnormalities and numerical chromosomal aberrations frequently appeared concomitantly in the same malignant cells. All of the specimens showing aneuploidy also exhibited centrosomal abnormalities: centrosomal defects and aneuploidy occurred together in 80% of malignant bladder tumors, with an especially high percentage in higher-grade tumors. The overall positivity of centrosomal abnormalities was higher than that of aneuploidy (88% vs. 80%), especially in grade 1 tumors (69% vs. 46%), whereas aneuploidy was strongly associated with grade 2 and grade 3 tumors. Centrosomal abnormalities are common in bladder cancer, even in low-grade tumors, and strongly associated with cancer grade and aneuploidy, especially in high-grade neoplasms. Centrosomal abnormalities appear to be intrinsic to aneuploidy and tumorigenesis and may be potential markers for early detection of bladder cancer.     

Flow cytometric DNA ploidy analysis of squamous cell carcinoma of the oral cavity. Comparison with clinical staging and histologic grading

The clonal DNA content of tumor biopsy specimens of 110 patients with primary and untreated squamous cell carcinoma of the oral cavity was determined by flow cytometric study. The ploidy status was compared with tumor size and histologic grading. Thirty tumors (27.3%) were diploid; in 80 cases (72.7%) cell lines with abnormal DNA content were detected. The portion of aneuploid tumors increased with decreasing degree of histologic differentiation (P less than 0.001) from G1 (38.1%) to G2 (76.6%) and G3 (92.0%). Only one of 13 T1 carcinomas (7.6%) showed abnormal clonal DNA content, but 76.9% of T2 and 90.6% T3 tumors (P less than 0.001) did. The emergence of aneuploid clones obviously represents a marker of malignancy progression in oral carcinoma.     

Flow cytometric DNA ploidy and recurrence development in squamous cell carcinoma of the oral cavity

This prospective study on 348 patients with oral squamous cell carcinoma who underwent radical surgery established a close association between the DNA ploidy status of the primary tumor and the risk of local recurrence development. Nine percent of patients with flow cytometrically diploid tumors developed a recurrence compared to 46% of those with aneuploid tumors. This correlation held true even if evaluated with respect to tumor stage or histological grade. Thirteen percent of the diploid and 59% of the aneuploid group showed lymph node metastasis. These results provide substantial evidence that cytogenetic events that underlie aneuploidy formation from initially diploid progenitor cells are functionally linked to the development of tumor cell populations that have the capability to establish independently growing colonies in foreign tissues.     

Prognostic significance of thymidine kinase activity in bladder carcinoma

BACKGROUND: Thymidine kinase (TK) plays a key role in the complimentary or alternative salvage pathway of pyrimidine synthesis. Little is known about the significance of TK activity in bladder carcinoma. The authors examined the activity of TK in 55 patients with bladder carcinoma to determine the prognostic significance of TK activity. METHODS: TK activity in nonfixed, fresh-frozen specimens of bladder carcinoma and normal bladder tissue was determined by using the diethylaminoethanol cellulose disc method. RESULTS: The activity of TK was approximately two-fold higher in bladder carcinoma specimens compared with normal bladder tissues. The TK activity in muscle-invasive bladder carcinoma was two-fold higher compared with the activity in superficial bladder carcinoma (Ta and T1). In addition, the activity of TK in T1 tumors was two-fold higher compared with the TK activity in Ta tumors. The level of TK activity in Grade 3 bladder tumors was two-fold higher compared with the activity in Grade 1 and Grade 2 tumors. Patients with Ta and T1 bladder carcinoma who had low TK activity had a longer postoperative tumor free period compared with the patients who had high TK activity during the 2 years of follow-up. TK activity was correlated with the activity of thymidine synthase, which is a key enzyme for pyrimidine synthesis in the de novo pathway. CONCLUSIONS: This study is the first to demonstrate that the level of TK activity is correlated with both disease stage and tumor grade in patients with bladder carcinoma and that elevated TK activity predicts early recurrence in patients with Ta and T1 disease. These results suggest that the level of TK activity may be used as a prognostic parameter and that TK may be a molecular therapeutic target in patients with bladder carcinoma.     

Progression of diploid tumor cells in aneuploid head and neck squamous cell carcinomas

The development of aneuploid clones from diploid progenitor cells is a regular characteristic of head and neck squamous cell carcinoma progression. While the significance of aneuploidy formation for the acquisition of invasive and metastatic behavior is well documented, little is known about the contribution of diploid tumor cells after aneuploid clones have emerged. To distinguish diploid cells of epithelial origin from benign cellular components, we applied multiparameter flow cytometry of DNA content and cytokeratin (CK) expression to 36 primary tumors. Twenty-seven carcinomas accommodated aneuploid cell lines that stained positive for CK. All diploid cell populations obtained from aneuploid carcinomas contained CK-positive subpopulations as did all of nine tumors that consisted exclusively of diploid cells. The proportions of CK-positive diploid cells ranged between 6% and 80%, independent of whether they were achieved from entirely diploid or from aneuploid carcinomas. CK-gated diploid and aneuploid cell populations showed largely identical S-phase fractions. These results emphasize that diploid tumor cells regularly persist after the development of aneuploid clones and significantly contribute to local tumor progression. Despite the presence of diploid epithelial cells in aneuploid primary tumors, exclusively the aneuploid clones of eight corresponding lymph node metastases were CK-positive. This provides further evidence of a largely reduced metastatic potential of diploid tumor cells.     

Urothelial dysplasia and carcinoma in situ of the bladder

Urothelial cells were pepsin-extracted from paraffin-embedded specimens taken from human nontumorous bladder mucosa, dysplasia, and carcinoma in situ. After Feulgen staining for DNA, nuclei were measured with an integrating microdensitometer. The measurements show that normal urothelium consists mostly of diploid nuclei. Dysplasia means that there is a predominance of tetraploid DNA values, whereas carcinoma in situ is characterized by a high percentage of aneuploid cells. In both dysplasia and carcinoma in situ there is a considerable percentage of diploid nuclei. Thus, DNA cytophotometry can be used for standardization of preneoplastic and early stages of tumor development in bladder cancer.