Characterization of bladder papilloma by two-parameter DNA-RNA flow cytometry

Two-parameter flow cytometry (FCM) studies of 0.9% NaCl solution bladder irrigation specimens were performed on 48 patients with histologically orderly or atypical papilloma of the urinary bladder in order to assess the value of RNA as a possible second parameter, along with DNA, in the detection of bladder tumors. DNA, RNA, and nuclear diameter measurements were obtained for each of 5000 cells/sample, and analyses were based on the distributions of those values. With the use of DNA content alone, 22 cases (46%) were classified positive by FCM. With RNA content as an additional parameter, 40 cases (83%) were positive. Two cases were suspicious, and 6 cases were normal by both parameters. Of 28 patients with papillomas showing histological atypia, 16 patients had positive DNA histograms, including 3 patients with aneuploid stemlines, but 24 of the 28 patients had positive RNA histograms. Of 20 patients with orderly papillomas, 6 patients had positive DNA histograms, including 3 patients with aneuploid DNA stem cell lines, but 16 of the 20 patients had positive RNA histograms. Thus, the probability of positive DNA histograms is higher in atypical papillomas (57%) than in orderly papillomas (30%), whereas elevated (positive) RNA is more characteristic of all papillomas without distinction between those that are histologically atypical (86% positive) or orderly (80% positive). For patients at risk of developing papillary bladder tumors, two-parameter DNA-RNA FCM appears to offer greater diagnostic sensitivity than does FCM based on DNA content alone.     

Check samples for laboratory self-assessment in DNA flow cytometry. The National Cancer Institute's Flow Cytometry Network experience

The National Cancer Institute's Flow Cytometry Network (NCI-FCN) is attempting to facilitate the transfer of flow cytometry (FCM) of exfoliated bladder cells from the research laboratory to the clinical laboratory. Demonstrating interinstitutional consistency in FCM analysis of replicate specimens simulating clinical barbotage specimens, fixed to allow easy transportation and storage at room temperature was one specific objective. Simulated barbotage specimens were prepared by mixing cultured aneuploid bladder carcinoma cells with normal or mitogen-stimulated peripheral blood mononuclear cells in different ratios. The samples were fixed in 10% formalin for 30 minutes, stored in buffer, and enucleated with pepsin, pH 1.5, before staining with propidium iodide for FCM DNA analysis. Preservation in ethanol or other common DNA cytochemical reagents was found to be unsatisfactory. In contrast, the formalin-fixed samples showed excellent preservation of quantitative DNA fluorescence and coefficient of variation of histogram peaks for over 2 weeks. Exchange of eight fixed specimens among five network laboratories that analyzed them as "unknowns" showed good overall agreement on histogram data and interpretation, although some noteworthy interlaboratory differences were found. This technique could be used for self-assessment surveys of clinical laboratory performance in DNA FCM of bladder barbotage specimens.     

Flow cytometric analysis of DNA content in human bladder tumors and irrigation fluids

Flow cytometry (FCM) was used to study the DNA distribution of 99 tumor biopsy specimens and 41 bladder irrigation samples from patients with transitional cell carcinoma of the bladder. For tumor biopsy and cystectomy specimens, the frequency of aneuploidy increased with advancing tumor stage and grade. All T0 tumors were diploid. Twenty-seven percent of T1, 71.4% of T2, and 75% of T3 and T4 tumors were aneuploid. All Grade I tumors were diploid. Thirty percent of Grade II and 76.9% of Grade III tumors were aneuploid. The frequency of aneuploidy of tumors in the early stages (Ta, T1) is similar to the incidence of subsequent progression by these tumors described in the literature. For irrigation fluids, the relationship between grade and stage and the frequency of aneuploidy was similar to the relationship seen with tumor specimens. All four patients with only carcinoma in situ had aneuploid cells in their irrigations. The comparison of FCM data of bladder biopsy and bladder irrigation from the same cystoscopic evaluation suggests adequate representation of tumor cells in the irrigation fluids for almost all cases. The authors conclude that DNA ploidy analysis by FCM appears useful in a clinically important group of patients with aneuploid superficial tumors of moderate or high grade. Bladder irrigation analysis appears useful in the follow-up of patients with a history of carcinoma in situ and those with aneuploid tumors.     

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 DNA analysis of hepatic tumours on ultrasound-guided fine-needle aspirates.

A study was performed on a nonconsecutive series of 51 patients in order to assess the feasibility, reliability, and usefulness of flow cytometric (FCM) DNA analysis of samples obtained from benign and malignant hepatic tumours by means of ultrasound-guided fine-needle aspiration (UG-FNA). Cytological and often histological confirmation of the nature of the lesion was obtained in all cases from an expert pathologist. For FCM DNA analysis in 32 cases, it was also possible to use samples obtained at surgery from the actual tumours. There were no post UG-FNA complications, either early or late. It was possible to perform FCM DNA analysis on 6/7 (85.7%) of the benign tumour aspirates and all 44 (100%) coming from the malignant tumours. All the benign tumours showed a DNA-diploid pattern, while the DNA content was aneuploid in 91% of the malignant tumours. Apart from one case, the results of the FCM DNA analysis of the samples removed at surgery were the same as those obtained from the aspirates (97%). FCM DNA analysis on UG-FNA samples from hepatic tumours is a fairly simple, reproducible, well-tolerated technique; it does not involve risks if performed by skilled operators and, since it can be easily repeated even on small tumours, it is a suitable method for monitoring hepatic metastases during chemotherapy.     

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.     

Feasibility and reliability of flow-cytometry (fcm) DNA analysis of fresh and fixed urine samples

The qualitative results of FCM DNA analysis on fresh and fixed urine specimens (28 and 97, respectively) from 68 normal subjects and 10 patients with a past history of bladder cancer were compared. FCM DNA evaluability was not significantly different in fresh and fixed samples (63% vs 73%, respectively) whereas mean CV was significantly higher (7.3% vs 5.7%, respectively; p=0.04). A double FCM analysis on fresh and fixed urine was also performed in 16 cases. In this subgroup, the percentage of evaluable histograms from fixed urine specimens was slightly higher than that from fresh specimens. Aneuploid cases were found only in the fixed urine samples but the CVs from fresh and fixed cell suspensions did not differ. The absence of inflammatory cells with cytological analysis of the same samples was associated with low percentages of FCM evaluability and higher CVs. The use of fixed samples improves the quality of FCM DNA analysis permitting its use for screening programs.     

Flow cytometry and interactive image cytometry in endometrial carcinoma. A comparative and prognostic study

Comparative DNA measurements were performed in 139 women with endometrial carcinoma using flow cytometry (FCM) and interactive image cytometry (ICM). Ploidy level and the percentage of S-phase cells were determined by FCM and ploidy level and the percentage of cells with a DNA content exceeding 2.5c, 3c, 4c and 5c, respectively, were calculated by ICM. The aim was to compare ploidy level obtained by the two methods and to evaluate the prognostic value of all the above-mentioned parameters. Recurrence or residual disease after completing treatment were used as end-points. Follow-up time was 18-48 months. An agreement was obtained in 85% of the cases as regards ploidy level, but in 15% the tumors were regarded as near-diploid by one method and as grossly aneuploid by the other. Both ploidy level (both methods) and S-phase rate (FCM) were correlated with histopathologic grade (p less than 0.001 and p less than 0.05, respectively). In univariate analysis, ploidy level (obtained either by FCM or ICM) correlated with recurrence rate, with a more favourable prognosis for near-diploid cases. When using multivariate models (Cox analysis) including clinical variables, ploidy level by FCM (but not ICM) was still significant as regards prognosis. In the multivariate analysis, S-phase fraction (as measured by FCM) also yielded independent prognostic information. In a separate analysis the proportion of cells with a DNA content greater than 5c gave independent prognostic information besides that of the S-phase fraction and ploidy level. We conclude that measurements of the percentage of cells exceeding 5c give prognostic information beyond the information obtained from flow cytometric determination of ploidy level and S-phase fraction.     

乳腺癌细胞DNA含量分析

本文应用流式细胞术(Flow Cytomotry简称FCM)测定41例乳腺癌细胞DNA含量,分析结果:DNA指数(DI)范围为0.9～2.91,其中二倍体肿瘤13例,占31.7％,异倍体肿瘤28例,占68.3％。DI和病理组织学分级呈正相关系,随着肿瘤组织学分级的增高,DI也升高。但是,在目前研究中,DI尚不反映临床分期。DI和预后的关系,共观察19例,发现二倍体肿瘤的预后好,生存期长,异倍体肿瘤的预后差,生存期短。本文应用针吸肿瘤细胞和切除标本新鲜组织块检测DNA含量,发现两种取材方法所得数值无显著差别。因此,认为针吸细胞应用FCM的DNA检查可作为术前辅助诊断方法,为治疗提供参考。     

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.     

Comparison of PCNA/cyclin immunohistochemistry with flow cytometric S-phase fraction in breast cancer

Kinetic index determined by enumeration of neoplastic cells positive for proliferative cell nuclear antigen (PCNA) in 70 breast carcinomas (avidin-biotin immunoperoxidase technique) was compared to synthesis-phase fraction (S-phase, or SPF) values obtained by flow cytometry (FCM) using a multiparametric, 2 color method (dual-label propidium iodide/cytokeratin-FITC). The percent PCNA positive tumor cells (12.5% mean, range 1–28%) was significantly greater in aneuploid tumors (14.2% mean, N=35) compared to diploid range tumors (10.7% mean, N=35) (p<0.05), and was correlated with SPF derived from ungated DNA histograms (12.5% mean ± 5.5%, r=0.45, p<0.001). Marginally stronger statistical correlations were observed between the PCNA index and SPF values calculated from cytokeratin-gated (15.8% mean, r=0.53, p<0.001) DNA histograms or from SPF values obtained following linear baseline debris subtraction (mean=8.1%, r=0.48, p<0.001). Significant associations were identified between PCNA index and prognostically important clinicopathologic parameters including nuclear grade (p=0.014), presence of necrosis (p=0.005), and angiolymphatic invasion (p=0.003). We conclude: 1) PCNA index is comparable to FCM SPF and correlates with factors of known prognostic importance in carcinoma of the breast; 2) baseline debris and contaminating events derived from non-epithelial cells both represent significant artifacts in proliferative fraction estimates derived from FCM DNA histograms; and 3) multiparametric analysis may represent one means of improving the specificity and clinical value of FCM SPF determinations.     

Inter-laboratory comparison of DNA flow cytometric results from paraffin-embedded breast carcinomas

Summary Consecutive sections from 33 paraffin-embedded human breast carcinomas without intratumor heterogeneity were sent for flow cytometric (FCM) DNA analysis in two experienced laboratories. FCM instruments, run conditions, and tumor disaggregation procedures were different in the two laboratories. In four cases (12%) the laboratories reported a different DNA ploidy and DNA index (DI). These variations were due to analytical reasons, differences in the detection rates of near-diploid and tetraploid DIs, not due to interpretation of data or the criteria used for aneuploidy. There was a significant correlation between S-phase fractions (SPF) obtained in the two laboratories (r = 0.90, p<0.0001) if only cases with concordant DI were included. Discordant DI usually led to very different SPF values.     

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.     

骨髓涂片和流式细胞术在神经母细胞瘤骨髓转移微小病灶检测中的临床应用

目的:探讨骨髓涂片和流式细胞术在神经母细胞瘤骨髓转移微小病灶检测中的临床应用价值.方法:回顾性分析2019年01月至2020年10月我院收治的经病理确诊为神经母细胞瘤患者126例,收集患者临床资料,特别是在治疗期间同时同部位进行骨髓穿刺行骨髓涂片和流式细胞术(flow cytometry,FCM)检测,分析两种方法在神...     

Cell-cycle distribution of urothelial tumour cells as measured by flow cytometry

The fraction of cells in S + G2 + mitosis from 54 urothelial tumours was calculated by flow cytometry after acridine orange (AO) staining of cells obtained by bladder irrigation or biopsy. Fluorescence signals emitted by the AO-stained DNA and RNA of each cell were separated optically and measured for 5,000 cells per specimen. The patients were classified by the histology of their tumours and clinical data into 5 diagnostic categories: NED (no evidence of disease, but history of bladder tumour), 3; papilloma, 8; non-invasive papillary carcinoma, 8; carcinoma in situ, 17 and invasive carcinoma, 18. The fraction of cells with DNA values in S + G2 + M of the cell cycle varied between 7 and 57% of the total, with a wide range within each diagnostic category, but no statistically significant differences between the groups. The proportion of cells in S + G2 + M from an individual tumour was not correlated with histologic grade or clinical behaviour. The possibility that some tumour cells with DNA values above G1 level are quiescent cells arrested at S or G2 is discussed.     

Flow cytometric analysis of deparaffinized nuclei in urinary bladder carcinoma. Comparison with cytogenetic analysis

Both cytogenetic analysis and flow cytometry (FCM) have prognostic efficacy in the analysis of transitional cell carcinoma (TCC) of urinary bladder. To correlate results of the two methods, we studied a unique group of patients whose tumors had undergone prospective conventional cytogenetic analysis. Paraffin blocks of these tumors were processed for FMC, then analyzed for nuclear DNA content. Of the 34 tumors processed, 30 (88%) yielded interpretable DNA histograms. In 26 (87%) of these, there was good correlation between the two methods with respect to the presence or absence of a hyperdiploid cell line. Discrepancies may have resulted from sampling error or from interpretation of a tetraploid peak as a prominent G2M region. Retrospective FCM analysis of paraffinized TCC tissue correlates well with conventional, prospective cytogenetic analysis and is applicable to the majority of urinary bladder transitional cell carcinomas.     

食管鳞状细胞癌DNA倍体异质性及其与患者预后关系的研究

 本研究采用多点取材的方法，应用流式细胞术对80例中晚期食管鳞状细胞癌的瘤体内DNA倍体异质性进行了研究。DNA异倍体的检出率为88.8%（71/80），DNA指数范围为0.77～1.74。80例标本中，38例瘤体内表现为DNA倍体异质现象（47.50%）。DNA倍体异质性肿瘤患者的5年生存率（34.2%）极显著地低于非异质性肿瘤患者（64.3%）（P<0.025）。文中对瘤体内DNA倍体异质性的可能意义和发生机制进行了讨论。     

Cell kinetics aspects of human malignant neuroepithelial tumors: a follow-up study

The prognostic value of proliferative activity and DNA distribution (ploidy), determined by flow cytometry (FCM), was evaluated in 38 cases of human malignant neuroepithelial tumors. No statistically significant correlation was found between flow cytometric data and clinical outcome. In particular, there was no significant difference between mean survival in cases with percentage of cells in S-phase lower and higher than 5%, respectively. In 21 cases with unimodal DNA distribution, the mean survival was 11.7 months; in 17 cases with bimodal DNA distribution, the mean survival was 12.5 months. The difference was not statistically significant. In our experience, proliferative activity and ploidy do not correlate well with the clinical course and survival of patients with malignant neuroepithelial tumors. However, application of FCM may provide, aside from histopathologic examination, additional biologic information that may be valuable in understanding the variation observed in the course of individual patients.     

Molecular genetic evaluation of fluorescence diagnosis in bladder cancer

Fluorescence diagnosis of superficial bladder cancer using 5-aminolevulinic acid (ALA) is a highly sensitive technique (95%). However, the specificity is only 60-70% due to false-positive results after histopathological examination. We hypothesized that the biopsies in fluorescence endoscopy could represent early preneoplastic lesions not detectable by histopathology. In order to evaluate the specificity of fluorescence endoscopy at the molecular genetic level we performed comparative genomic hybridization (CGH) and investigated telomerase activity of ALA-positive tissue samples. For CGH, DNA was isolated from 5-10 frozen sections. Tumor and normal (control) DNAs were amplified by DOP-PCR and labeled with biotin-dUTP and digoxigenin-dUTP, respectively. Hybridization and detection were carried out according to standard protocols. Telomerase activity was analyzed using a non-radioactive system (TRAP-assay). In 33 out of 118 bladder cancer cases (28%) detected by conventional cystoscopy, additional suspicious areas were found using ALA. CGH revealed genetic changes in 27% of samples with non-malignant histological diagnoses. Telomerase activity was found in 59% of these samples. Tumor samples showed genetic alterations in 84% and in 69% telomerase activity occurred. The type of genetic alterations in the normal biopsies was identical to the tumors. Based on these molecular data, the portion of false-positive results obtained by fluorescence diagnosis is lower than defined by histopathology alone. Genetic alterations and activation of telomerase activity are early events of tumor development in bladder cancer occurring earlier than histological features of neoplasia. The clinical importance of fluorescence diagnosis and the possible reduction of the recurrence rate have to be shown in ongoing clinical studies.     

Comparative DNA Analysis by Image Cytometry and Flow Cytometry in Non-small Cell Lung Cancer

To determine whether image cytometry (ICM) is advantageous for clinical DNA analyses of tumor cells, nuclear DNA contents measured by ICM were compared with those by flow cytometry (FCM), using 46 samples of non-small cell lung cancers. ICM was performed on smear specimens of fresh materials (f-ICM) and cell suspensions obtained from paraffin-embedded tumors (p-ICM). The same cell suspensions were also analyzed by FCM (p-FCM). Aneuploid rates/coefficient of variation (CV) of f-ICM, p-ICM, and p-FCM were 76.1/4.90, 71.7/5.01 and 60.9/5.31%, respectively. There was a high correlation in the DNA indices between p-ICM and p-FCM (r=0.80). In the comparative DNA analysis, there were seven discordant samples. Six of them were estimated as aneuploid by p-ICM, but they were miscounted as diploid or undefinablc (impossible) by p-FCM. This was caused by measuring condensed nuclei or debris. All "impossible" samples in p-FCM were squamous cell carcinoma with necrosis. In cell cycle analysis, the S and S+G2/M phase fractions in diploid samples were higher in p-ICM than those in p-FCM ( P < 0.005), because the GO/G1 phase (2N) fraction presented by FCM was composed of cancer and non-malignant cells in diploid cancers. In ICM, they can be separately measured by means of morphological selection. These findings indicated that ICM is superior to FCM, especially for the practical DNA measurement of a few cancer cells and in the evaluation of the proliferation rates.