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.     

Tissue-specific markers in flow cytometry of urological cancers. II. Cytokeratin and vimentin in renal-cell tumors

Nine primary human renal-cell tumors (RCT), one lymph-node metastasis, 4 human xenografts of a RCT in nude mice and a rat RCT line were analyzed by flow cytometry (FCM) using propidium iodide for DNA analysis and antibodies to cytokeratin and vimentin in the indirect immunofluorescence technique for labelling of specific tumor-cell populations. By means of 2-dimensional FCM analysis, vimentin- and cytokeratin-positive (tumor) cells were compared and their DNA content and proliferative fraction analyzed separately from those of cytokeratin-negative stromal and inflammatory cells. In primary human RCT, 2 subpopulations of cells were detected and analyzed separately. Small numbers of tumor cells with an abnormal DNA stemline were also detected. In addition, co-expression of intermediate filament proteins of both the cytokeratin and the vimentin types was detected in the aneuploid cell population. Comparison of 2 model systems of RCT with primary human RCT revealed a similar pattern of tumor-cell subfractions within these tumors. The 2-parameter FCM analysis permits the detection of subpopulations in complex cell suspensions and the quantification of these fractions, as well as analysis of their cellular DNA content.     

Cytogenetic abnormalities in benign lymphoid hyperplasia: a dual-parameter study using chromosome analysis and flow cytometry

This is a prospective study of lymphoid tissue showing benign reactive hyperplasia (18 lymph nodes and 2 tonsils), using cytogenetic analysis of cells stimulated with T- or B-cell mitogens. The reason for this study was the detection of an abnormal chromosomal population in cells from an enlarged lymph node excised from a 7-year-old female who on further investigation was found to be clinically well and after one year's close follow-up had not developed any further signs or symptoms of malignancy. In addition, DNA content was measured by flow cytometry (FCM) on fresh cell suspensions in 17 cases and fixed cell suspensions in 3 cases. Structural and numerical clonal chromosome abnormalities were found in 9 of the 20 samples, but no common specific defect was identified. FCM showed an abnormal DNA content in 10 of the 20 samples studied; 3 of these showed clonal chromosome abnormalities. Surface membrane immunoglobulin studies were carried out on 15 of the 20 samples using cell suspensions and frozen tissue sections. In 5 of the 15 cases, monoclonal surface immunoglobulin was detected. There was no direct correlation between the surface membrane immunoglobulin studies and the chromosome and FCM analyses. We conclude that aneuploidy is a common feature in reactive lymphoid tissue, but both cytogenetics and FCM are needed to identify it.     

Numerical aberrations of chromosomes 8, 9, 11, and 17 in squamous cell carcinoma of the pharynx and larynx: a fluorescence in situ hybridization and DNA flow cytometric analysis of 50 cases

Squamous cell carcinoma of the pharynx and larynx (SCCPL) is a genetically complex disease and is frequently associated with nonrandom chromosomal alterations. Fifty primary SCC of the pharynx (oropharynx, n=11): see and hypopharynx, n=11) and larynx ( n=28) were examined for numerical aberrations of chromosomes 8, 9, 11, and 17 with a panel of chromosome-specific repetitive DNA probes by fluorescence in situ hybridization (FISH). DNA ploidy analysis was also performed by flow cytometry (FCM). Aneusomic copy numbers of chromosomes 8, 9, 11, and 17 were discovered in 66%, 68%, 68% and 78% of tumors, respectively. FCM showed abnormal DNA content in 74% of cases (mean DNA index=1.69). Polysomy was the main finding in both DNA-aneuploid and DNA-diploid tumors (64.5% of cases). Numerical aberrations of chromosomes 8 and 11 correlated to DNA ploidy by FCM (P< 0.05). Aneusomy was present in 69.23% of DNA-diploid tumors. Marked intratumoral and intertumoral chromosomal heterogeneity was noted between individual tumors, suggesting a notable heterogeneity in aneuploid and diploid cell populations. Interphase FISH can be used to study important cytogenetic changes which occur during the development of SCC of the pharynx and larynx.     

A comparative study of DNA ploidy in 115 fresh-frozen breast carcinomas by image analysis versus flow cytometry.

BACKGROUND. Qualitative and quantitative analysis of cellular DNA content may be clinically useful in the prognostic evaluation of certain types of malignant tumors, including breast carcinoma. Flow cytometric (FCM) analysis has been the most frequently used procedure for DNA analysis, but it requires a reasonably large tissue sample. Computer-based image analysis (IA) now allows imprint, cytospin, and needle aspiration smear preparations and other small tissue samples to be used. METHODS. To resolve concern about the diagnostic efficacy of small tissue samples in the use of IA, the authors performed a comparative study of FCM analysis and IA using 115 fresh-frozen breast carcinomas. Feulgen-stained imprint preparations for IA and single-cell suspensions from the same fresh-frozen tissue for FCM analysis were used, and the respective histograms were compared. RESULTS. The results were concordant in 90.4% (104 of 115) of the cases, but 11 specimens yielded discordant data. IA provided histograms with a somewhat lower resolution and a relatively high coefficient of variation for the G0/G1 peak, thus rendering occasional tumors, which were near-diploid aneuploid by FCM analysis (four cases), not amenable to diagnosis by aneuploid characterization. In three additional cases, FCM analysis showed aneuploid hyperdiploid (two cases) and multiploid (one case) histograms, but IA only demonstrated a diploid peak. Conversely, in four other cases, aneuploid peaks were recognized only by IA. CONCLUSIONS. Computerized IA has significant advantages over FCM analysis, including lower cost, the ability to analyze very small specimens, the capability of detecting rare high ploidy cells, the capacity to classify cellular populations according to specific morphologic type, and the fact that no destructive enzyme or chemical digestion is required for specimen preparation, thereby preserving the integrity of fragile cells.     

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.     

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.     

Molecular serological detection of DNA alterations in transitional cell carcinoma is highly sensitive and stage independent.

PURPOSE: To evaluate the efficacy of fluorescent microsatellite analysis (MSA) for the serological diagnosis of transitional cell carcinoma (TCC) of the urinary tract analyzing free tumor DNA in the serum of cancer patients. EXPERIMENTAL DESIGN: We applied fluorescent MSA to detect serum-DNA alterations in patients suffering from bladder and upper urinary tract TCC and prospectively collected fresh tumor, peripheral blood, and serum specimens from 61 consecutive patients to obtain the corresponding DNA. Fluorescent MSA was performed with a total of 17 polymorphic markers from the chromosomal regions 5q, 8p, 9p, 9q, 13q, 14q, 17p, 17q, and 20q in the 61 cancer patients, as well as in 20 healthy controls. RESULTS: Molecular serological analysis led to tumor-specific diagnosis of TCC in 80.3% (49 of 61) of cases. Four healthy controls displayed serum-DNA artifacts rendering a specificity of 80%. The highest frequency of serum-DNA alterations was detected for chromosomal region 8p with 36%. Chromosomes 5q, 9p, and 20q showed serum-DNA alterations in 18 to 21%. The identification of serum-DNA alterations was not statistically associated with underlying local tumor stage (P = 0.29) but was more frequent in high-grade tumors (P = 0.08). CONCLUSIONS: MSA offers a highly sensitive method for serological diagnosis of TCC. To optimize specificity, simultaneous analysis of tumor DNA is advised to rule out artifacts resembling allelic imbalance in MSA of serum DNA.     

New sensitivity test using flow cytometry

Summary Flow cytometry (FCM) has been used to evaluate not only the malignancy of tumor cells but also the effects of chemotherapy. Here a new application of FCM for selecting the best antineoplastic agent in the chemotherapy for brain tumors is reported.Through our preliminary study using established brain tumor cell lines, the system for this sensitivity test was developed. Antineoplastic agents were placed in contact with monolayer-cultured cells; then cell viability and changes in the DNA histogram were analyzed by FCM. Cell viabilities were measured with the fluorescein diacetate (FDA) staining method, and the DNA histogram was analyzed by the propidium iodide (PI) staining method. The best antineoplastic agent was determined based on changes in cell viability and cell cycle. In other words, when markedly decreased viability as compared with that of the control, is measured by FCM, then the agents can be considered to have had a cytocydal effect on the tumor cells, and thus the sensitivity of the agents is able to be evaluated. If the viability of the tumor cell is observed to be similar to that of the control, the cytostatic effects of the agents are able to be evaluated only if a marked change is observed in the DNA histogram.After the preliminary study, this system was applied clinically to malignant brain tumor cases, resulting in success in selecting the best antineoplastic agent for each individual case. Our sensitivity test using this FCM established in vitro system has much potential value for clinical use.     

Benign metastasizing giant cell tumors of bone. A DNA flow cytometric study

Approximately 2% of histologically benign giant cell tumors (BGCT) of bone are complicated by lung metastases, which can progress despite their benign histologic appearance. Almost all BGCT studied by DNA flow cytometry (FCM) have been reported to be diploid. However, the very few cases with lung metastases previously analyzed were all aneuploid. To assess the usefulness of DNA FCM in predicting the metastatic potential of BGCT, seven metastasizing BGCT were studied by DNA FCM using paraffin-embedded tissue. Five were purely diploid, one was tetraploid, and one was aneuploid. The primary and the metastasis showed the same DNA distribution in all but the tetraploid case, in which the metastasis was purely diploid. A single patient, who was in the diploid group, had unresectable tumor in the lungs; she remains alive with stable disease at 30 months. The other six patients, who underwent complete resections of their lung metastases, are free of disease. These results suggest that DNA FCM is not a sensitive method for predicting the metastatic potential of BGCT since most metastasizing cases appear to be diploid.     

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.     

Prognostic significance of DNA content in large bowel carcinoma: a retrospective flow cytometric study

Flow cytometric (FCM) determination of DNA content was performed on surgical specimens from 44 patients with previously untreated colonic carcinoma. For each tumor, cell suspensions were prepared from 2-4 40-microns thick sections obtained from formalin-fixed and paraffin embedded tissue samples. Aneuploidy was found in 47.2% of all the tumors and the aneuploid clone had a median DNA index of 1.49 (range: 1.24-1.93). Aneuploidy was found in 26.7% of highly differentiated tumors (WHO histologic classification), in 53.8% of moderately differentiated tumors and in 100% of poorly differentiated tumors (P = 0.04). The 33.3% of stages 1 + 2 (TNM) and the 70.6% of stages 3 + 4 tumors were aneuploid (P = 0.002). Median survival from surgery was 46.4 months for all patients. It was 18.8 months for patients with aneuploid tumors and 85.7 months for those with diploid tumors (P = 0.0002). FCM determination of DNA in colon carcinomas can easily be performed on archival histological material and provides prognostic information.     

Molecular biologic characteristics of seven new cell lines of squamous cell carcinomas of the head and neck and comparison to fresh tumor tissue

BACKGROUND: Squamous cell carcinoma (SCC) is the most frequent malignant tumor of the upper aerodigestive tract. Cell lines of these tumors facilitate the investigation of various tumor biological parameters. This study was conducted to compare molecular biologic characteristics between cell lines and fresh tumor tissue. METHODS: In seven SCC-derived cell lines, cytokeratin 5/6 and cytokeratin 19 expression, DNA content, chromosome aberrations and tumorigenicity were assessed in nude rats. Unbalanced numerical and structural chromosomal aberrations were investigated by comparative genomic hybridization (CGH), and results were compared to those obtained in fresh tumor tissues of the same patients. RESULTS: All cell lines expressed cytokeratins 5/6 and 19, indicating their epidermoid origin. Tumor growth after transplantation into nude rats occurred in five of seven cell lines. Routine histology and immunohistochemical examinations confirmed SCC. Aneuploidy was detected in all cell lines, with a 2c deviation index ranging from 1.9 through 9.5 and a 5c exceeding rate ranging from 2.6 through 36.7%. The most frequent chromosomal aberrations in cell lines were overrepresentations of chromosomal material on chromosomes 15q, 7p (5 cases each), 3q, 5p (4 cases each), and 11q and 17q (3 cases each) and losses of chromosomal material on chromosomes 3p, 18q (3 cases each), and 19p and 7q (2 cases each). Comparing these results to CGH analysis of fresh tumor tissue from the same patients, overrepresentations of chromosomal material on 10q, 20q and 21q, along with loss of chromosomal material on 4q was detected more frequently in primary tumors, whereas overrepresentations on 7p and loss of chromosomal material on 7q were more frequently detected in cell lines. Nevertheless, there was a high degree of similarity of chromosomal alterations in cell lines and corresponding fresh tumor tissue. CONCLUSION: The data suggest a high degree of genetic similarity between tumor cells of cell lines and the tumors from which they were derived. Therefore, these cell lines can serve as an accurate model to investigate cell biology of SCC in vitro.     

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.     

Sensitive detection of transitional cell carcinoma of the bladder by microsatellite analysis of cells exfoliated in urine.

Transitional cell carcinoma (TCC) is the most common bladder tumor. Urine cytology can identify most high-grade tumors but sensitivity is lower if one includes lesions of all grades. Microsatellite marker alterations have been found in many tumor types including bladder cancer and have been used to detect cancer cells in body fluids including urine. The aim of our study is to further evaluate feasibility and sensitivity of microsatellite analysis to detect bladder cancer cells in urine. We studied 55 individuals: 21 with symptoms suggestive of bladder cancer, 23 patients with previous history of TCC and 11 healthy subjects. Genomic DNA was extracted from blood lymphocytes, urine sediment, bladder washings and tumor or normal bladder mucosa. Twenty highly informative microsatellite markers were analyzed for loss of heterozigosity (LOH) and microsatellite instability (MIN) by polymerase chain reaction. Microsatellite analysis of urine identified 33 of 34 (97%) patients with either primary or tumor recurrence, whereas urine cytology identified 27 of 34 (79%) patients (p = 0.0001). Detection of microsatellite abnormalities improved the sensitivity of detecting low-grade and/or stage bladder tumor: from 75-95% for grades G1-G2 and from 75-100% for pTis-pTa tumors. Bladder washings from 25 patients were also analyzed, and in all cases results were identical to those obtained from voided urine. None of the 16 patients without evidence of TCC showed LOH and/or MIN in urine samples or bladder washings. Interestingly, in a patient with persistent bladder mucosa abnormalities, microsatellite alterations were demonstrated 8 months before the histopathologic diagnosis of tumor recurrence. These results further indicate that microsatellite marker analysis is more sensitive than conventional urine cytology in detecting bladder cancer cells in urine and represents a potential clinical tool for monitoring patients with low-grade/stage TCC.     

Thymoma. The prognostic significance of flow cytometric DNA analysis.

The clinical course of patients with thymoma varies widely despite its histologically benign appearance. Treatment decisions are based on local invasion and the extent of resection. Because some patients have more aggressive tumors, the prognostic significance of flow cytometric (FCM) analysis of nuclear DNA content was examined. Adequate tissue from paraffin-embedded blocks was available for 25 patients. Using FCM, the percentage of cells in S-phase (%S) and the ploidy, based on the DNA index (DI), were determined. The mean patient age was 52 years, with a female-to-male ratio of 1.3:1 and a median follow-up of 64 months. Seventeen patients underwent total tumor resections, and 12 also received radiation therapy. Eight patients underwent subtotal resections, with five receiving radiation therapy (with or without chemotherapy) and three receiving chemotherapy alone. Based on invasion and intrathoracic dissemination, the tumors were classified into four stages. The mean %S was 5.6. There was no relationship observed between %S and patient outcome. The 5-year disease-free survival rate was 85% for the 16 patients with diploid (DI = 1) tumors and 33% for the 9 patients with aneuploid (DI more than 1) tumors (P less than 0.002). Similar significant differences were observed by stage and extent of surgery. For those who had total resection (n = 17), the disease-free survival rate was 89% when DI equaled 1 and 50% when DI was more than 1 (P = 0.01). Although the numbers studied were small, when stage, histologic findings, and type of surgery were subdivided by DI, a higher incidence of relapse was associated consistently with aneuploidy. The DI appears to be a useful prognostic parameter for identifying patients at high risk of relapse.     

肺癌患者外周血循环中癌细胞的流式细胞仪分析

目的：用流式细仪分析肺癌患者外周血循环中的癌细胞,方法：外周血经Ficoll梯度离心分离单核细胞组分,后者用CD45、细胞角蛋白（CK）及肺癌特异性抗体（2F／S5A）染色后,应用流式细胞仪检测CD45^-CK^ /S5A^ 细胞。结果检测了165例肺癌患者外周血,发现50例（30．3％）患者外周血癌细胞呈阳性。其中非小细胞肺癌阳性率为30．67％（45／150）,小细胞肺癌为33．33％,阳性率为与患者的病理分期有显著相关性（P＜0．05）。结论：利用流式细胞仪分析检测癌患者外周血循环中的癌细胞不驻有助于临床病理分期,而且对预测肺癌转移潜能具有应用价值。     

Flow cytometric analysis of DNA in bone and soft-tissue tumors using nuclear suspensions

Ninety-four bone and soft tissue tumors were analyzed for their DNA content using flow cytometry (FCM). A simple, rapid method for preparing isolated nuclear suspensions was used. Tissues, minced in a hypotonic solution containing detergent and propidium iodide as a fluorescent probe for DNA, provided in most instances high nuclear yields from only 0.02 to 0.03 g of solid tumor. Whereas all nonneoplastic samples had a diploid DNA content, various degrees of abnormal DNA distributions were detected in 90% of the neoplastic samples and were present in benign as well as malignant tumors. Our findings demonstrate that FCM DNA analysis is practical in most musculoskeletal tumors and support the observations of others that abnormal DNA content may serve as a general neoplastic marker in these tumors.     

DNA image cytometry on sections compared with flow cytometry in human bone metastases

The DNA analysis of tumor cells discloses characteristic features from which their biological behavior with respect to both the intensity and regulation of growth can be deduced. The objective of this study was to comparatively assess the results of image cytometry (ICM) and flow cytometry (FCM) in human bone metastases as standard methods of DNA analysis and evaluate their possible importance. The nuclear DNA content of surgically removed tumors of bone tissue was determined using ICM and FCM, and the percentage of tumor cells in various cell cycle phases and ploidy status in each case were determined based on the DNA distribution pattern. Comparable results were determined by ICM and FCM with respect to the ploidy status in about 58% of examined tumor samples. When tissue samples from various regions of a tumor were examined, it was found that DNA-euploid and -aneuploid tumor areas were present within the tumors. The DNA aneuploidy was detected in 90% of these tumors with ICM. The percentage cell-cycle phase distribution varied widely with ICM and FCM. Based on our results, the use of ICM in addition to FCM is mandatory under certain conditions for the assessment of the DNA analysis of bone metastases and necessary for the critical assessment of the obtained findings.     

DNA content and genetic evolution of human colorectal adenocarcinoma. A study by flow cytometry and cytogenetic analysis

We have conducted in parallel DNA flow cytometry (FCM) and cytogenetic (CG) analysis of a series of surgical specimens from 35 human colorectal adenocarcinomas. An excellent quantitative correlation was observed (r = 0.99) between modal peak values of FCM histograms and chromosome counts. This observation confirms that aneuploidy, as defined by FCM, accurately reflects the deviation from diploidy of the genomic DNA. FCM-derived DNA patterns have been analyzed in the context of the clonal chromosomal evolution determined by CG analysis. In the metaphases of a given tumor, even if karyotypes of different ploidy exist, the presence of identical marker chromosomes suggests a common origin for the multiple populations observed by FCM. Thus, heterogeneity in DNA content within a tumor, including the polyploidization step, would be indicative of genetic evolution.