Evaluation of a modeling system for S-phase estimation in breast cancer by flow cytometry

Using software programs provided by Coulter Electronics, we have developed an analysis system that would address problems encountered in DNA flow cytometric analysis of heterogeneous solid tumor populations, especially where the G2-M phase of the diploid population contaminates the S-phase of the aneuploid population, causing an overestimation of cells in S phase. We used the PARA 1 and PARA 2 programs in concert and developed three analysis models: (a) for euploid tumors; (b) for hyperdiploid tumors with overlapping populations; and (c) for near-diploid aneuploid tumors. Our purpose in this paper is to determine the limits and reproducibility of this analysis system with an emphasis on tumors with overlapping populations. Aliquots of frozen, pulverized breast tumor tissue (50 to 100 mg), routinely used in the steroid receptor assay, were used for routine flow cytometric measurement of the DNA index and S-phase fraction. To determine the accuracy of the analysis when overlapping populations were present, we mixed an aneuploid breast cancer cell line with human blood lymphocytes in varying ratios. A 10% mixture of aneuploid cells, the lowest mixture tested, still allowed analysis results within 95% confidence limits. Reproducibility of the system was assessed on frozen breast tumor tissue by intra- and interassay variation studies measuring cell cycle parameters and coefficient of variation of the G0-G1 peak width. Within any sample the amount of variation (+/- 2 SD) for the G0-G1 value was +/- 4.40 for intraassay and +/- 4.60 for interassay, and the amount of variation for S phase was +/- 3.0 and +/- 3.2 for intraassay and interassay, respectively. There was no difference in the variation of estimates for G2-M (+/- 2.6 for both intra- and interassay). In this study, the coefficient of variation of the G0-G1 peak greater than 5% was defined as unacceptable for accurate analysis, with the conclusion that S-phase fractions in aneuploid tumors can be routinely analyzed in human breast tumor biopsies despite tumor cell heterogeneity.     

DNA ploidy and S-phase in recurrent astrocytomas: a retrospective study by flow cytometry of deparaffinized specimens

Summary Twenty-two patients with recurrent astrocytic tumors were treated surgically two or even three times. At the time of the first surgery 6 tumors were fibrillary astrocytomas (grade II), 9 anaplastic astrocytomas (grade III) and 7 glioblastomas (grade IV).Histopathological specimens from second surgery demonstrated in 12 cases signs of higher grades of malignancy. Flow cytometry (FCM) did not reveal any significant changes of S-phase fraction (p = 0.55). This study supports the theory that, given enough time, the histopathology of brain tumors change significantly from more benign forms to more malignant ones. The flow cytometry (FCM) could trace a weak tendency to higher S-phase fractions at the time of the second surgery. No apparent change of ploidy pattern was observed. In spite of the unequivocal histopathological changes of the recurrent astrocytomas the flow cytometry failed to indicate similar changes in terms of ploidy and S-phase fraction parameters.     

Measurement of the kinetics of cell proliferation of cancer of the oropharynx in vivo by the incorporation of bromodeoxyuridine and flow cytometry]

Forty-six tumors from patients with oropharyngeal carcinoma were analysed by flow cytometry after injection of bromodeoxyuridine (Budr) for the labelling index, the duration of S phase and the potential doubling time (Tpot). The results show large variations in Tpot (from 2.6 to 16.7 days) among these tumors from the same site and with the same histology. The variations in Tpot were not significantly related to TNM status and differentiation grade. However, aneuploid tumors had statistically significant shorter Tpot. The predictive value of Tpot regarding the response to radiotherapy is currently under investigation.     

Breast cancer cell kinetics: immunocytochemical determination of growth fractions by monoclonal antibody Ki-67 and correlation with flow cytometric S-phase and with some features of tumor aggressiveness.

In operable breast cancer, cell kinetics can be utilized in the prediction of the clinical outcome of patients. The discovery of monoclonal antibodies recognizing antigens related to cell proliferation has permitted the assessment of cell kinetics by rapid and practical immunocytochemical methods. It is claimed that the Ki-67 mouse monoclonal antibody recognizes an antigen expressed in proliferating cells but not present in quiescent (G0) cells. To study the relationship between Ki-67 score and DNA flow cytometric S-Phase Fraction (SPF), the latter being one of the most widely used methods to assess cell kinetics, we compared these two techniques of measurement in 122 breast carcinomas using both for each specimen. In this series 90% of tumors were Ki-67 positive, with a median value of 7.5% (range 1% to 70%). DNA flow cytometric analysis revealed that 69 tumors (57%) were aneuploid, whereas 53 were diploid. The median SPF value was 8% for diploid and 15% for aneuploid tumors (range 2% to 32%). Ki-67 scores were significantly higher in the DNA aneuploid compared to the diploid carcinomas (p = 0.015). Overall, a good correlation was found between Ki-67 and SPF values both in diploid (r = 0.60) and in aneuploid (r = 0.38) tumors. High Ki-67 scores were associated with the presence of axillary lymph node metastases (p = 0.0023) and poor histologic differentiation (p = 0.0028). Menopausal status, tumor size and peritumoral vessel invasion were unrelated to the Ki-67 score. Over-expression of the Epidermal Growth Factor receptor (EGF-r) and the c-erbB-2 oncogene were not correlated with Ki-67 staining. In conclusion, in this study Ki-67 immunostaining correlated with other indices of cell proliferation (SPF and Grade) and with some features of tumor aggressiveness (DNA aneuploidy and lymph node metastases) but seemed to be independent of some biological markers (EGR-r and c-erbB-2). Since the major objective for assessing proliferative status in Stage I-II breast carcinoma is to determine prognosis, it will have to be evaluated whether the determination of the Growth Fraction has comparable or even greater prognostic value than other cell kinetics markers.     

Cellular DNA of human neoplastic B-cells measured by flow cytometry

Flow cytometric analysis of DNA of tumor cells rapidly provides information on cell kinetics and tumor ploidy. Human B-cell lymphomas, however, often contain high numbers of nonneoplastic cells, mainly T-lymphocytes, which may hamper the accurate measurement of cell cycle phases and ploidy level of these tumors. The neoplastic cells in each B-cell lymphoma express a single immunoglobulin light chain. Therefore, we labeled surface immunoglobulin light chains to discriminate between predominantly neoplastic B-cells and nonneoplastic cells in the same tissues. Using this label as well as antibodies against nonneoplastic T-cells, we performed multiparameter correlated flow cytometric analysis of 52 human B-cell lymphomas measuring cellular DNA in neoplastic and nonneoplastic populations from the same tissues without physical separation of cells. Comparison of cellular DNA of immunoglobulin light chain-bearing neoplastic cells with that of nonneoplastic cells from the same tumor enabled us to detect DNA changes (aneuploidy) in almost 80% of the lymphomas, an incidence higher than observed previously by conventional DNA analysis of unseparated cells. These ploidy changes were confirmed by comparing in the same tumor the DNA of normal T-cells with that of predominantly neoplastic cells. The proportion of neoplastic cells in the synthetic phase of the cell cycle (S-fraction) varied widely from tumor to tumor. Lymphomas with high neoplastic S-fractions (higher than 10%) were mostly hyperdiploid tumors and histologically corresponded to intermediate- and high-grade unfavorable lymphomas. Tumors with low neoplastic S-fractions (less than 5%) were predominantly diploid and near diploid, histologically low-grade lymphomas. Six lymphomas showed two discrete cell populations bearing the same immunoglobulin light chain but containing different amounts of DNA suggesting the presence of two neoplastic clones in the same tumor (biploidy). In two patients in whom the lymphoma relapsed at 17 and 34 months, respectively, after the initial biopsies, repeat tumor samples were obtained. Despite an increase in the neoplastic S-fraction, no change in ploidy level was observed in either case. Light scatter analysis suggested a relationship between cell size and genomic size; large cells in these tumors were mostly presynthetic aneuploid cells. The ability to measure DNA, antigens, and cell size in individual cells in a rapid, correlated manner is a unique attribute of flow cytometry.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tumour growth rates in squamous carcinoma of the head and neck measured by in vivo bromodeoxyuridine incorporation and flow cytometry.

The cell kinetics of 82 squamous cell carcinomas of the head and neck were studied by in vivo administration of the thymidine analogue, bromodeoxyuridine (BrdUrd). Ploidy, BrdUrd labelling index (LI), duration of S-phase (Ts), potential doubling time (Tpot) and S-phase fraction (SPF) were measured by flow cytometry on 50 microns paraffin embedded sections. The range of values obtained compared well with other in vivo cell kinetic studies of head and neck cancer. Aneuploid tumours had a significantly higher BrdUrd labelling index and SPF, and a short Tpot than diploid tumours. To validate the use of 50 microns sections for measuring cell kinetic parameters by flow cytometry a comparison of values obtained by 50 microns sections and small blocks of tissue was made. No significant difference was found between the two methods. Reproducibility of values between two consecutive thick sections was also good. We conclude that reproducible cell kinetic measurements can be made in tumour samples using 50 microns sections of BrdUrd labelled tissue.     

Uptake of adriamycin by human leukemic cells as measured by flow cytometry

Leukemic cells were obtained from 12 patients with acute nonlymphocytic leukemia and 2 patients with acute lymphocytic leukemia. The specimens were incubated with a range of concentrations of adriamycin (Adr) for different periods of time and then analyzed by flow cytometry. For incubations of up to 5 h, only cells exposed to 5 or more μg of Adr ml^?1 became fluorescent while cells incubated with 1 μg ml^?1 for 24 h became fluorescent. Flourescence decreased when the cells were washed and further decreased when the cells were incubated in Adr-free media. The cells from different patients exhibited a wide range of Adr uptakes and retentions. Simultaneous analysis of right angle light scatter and fluorescence intensity demonstrated that there may be subpopulations of cells whose Adr uptake and/or retention differ from the weighted mean population values. With this method rare cells which fail to take up Adr can be recognized.     

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.     

Measurements of cellular proliferation by H-3-thymidine-labeling index and flow cytometry in breast cancer patients

In 50 node negative breast cancer patients, tumor S-phase fraction (SPF) was determined by H-3-thymidine labeling index ((HTLI)-H-3) or flow cytometry (FC). Forty-five patients had tumor cell kinetics measured by both techniques. Twenty-three patients were classified as having high proliferative activity and 22 low by (HTLI)-H-3, while 32 as highly proliferating and 13 low proliferating by FC. In 30 patients only, both indices agreed on identifying high or low proliferative activity. These results suggest the need of more careful attention to standardization and quality control of cell kinetic data before carrying out clinical trials based on these parameters.     

Prediction of node-negative breast cancer outcome by histologic grading and S-phase analysis by flow cytometry: an Eastern Cooperative Oncology Group Study (2192)

Histologic evaluation and reporting of invasive breast cancer has effectively used Nottingham combined histologic grade (NCHG). This approach to predict outcome in invasive breast cancer has not been tested in multicenter cooperative trials. Histologic slides from selected breast cancer cases entered on node-negative Eastern Cooperative Oncology Group trials were assigned grades. Two pathologists evaluated cases for NCHG defined from differentiation, mitotic index, and nuclear grade. The study population consisted of separate samples from low- and high-risk strata, where low risk was estrogen receptor positive with a tumor size of less than 3 cm and high risk was estrogen receptor negative or tumor size greater than or equal to 3 cm. The rate of agreement was generally good, with 80% of cases classified the same for mitotic count and 76% of the cases classified the same for combined grade. There were no cases disagreeing from the lowest to the highest of the three categories. The median follow-up is 11.6 years, but for analysis of survival, this was truncated at 5 years. Mitotic index and combined grade as assessed by both pathologists showed significant associations with survival. High combined histologic grade was predictive for response to cyclophosphamide/methotrexate/5-fluorouracil (CMF) with survival differences at 5 years of 30% in the treated high-grade patients over the untreated patients. Overall, it is clear that pathologists can have close agreement in assignment of combined histologic grades, with highly significant prediction in univariate and borderline significance in multivariate analysis in prognostication of time to recurrence as well as survival. Thus, stratification used in these trials was highly prognostic as hoped, leaving a role for histologic grading in these relatively large tumors, more powerful than S-phase analysis in this series. In the subgroups of high-risk patients randomized between CMF and observation, there was a suggestion that the high-combined-grade group was predictive of treatment efficacy. We conclude that a combined histologic grade with defined criteria may be reliably assigned by practiced pathologists using readily available criteria, and that the measure may be of use in prognostication and prediction of therapeutic responsiveness when done in a technically ideal fashion.     

DNA/RNA content and proliferative fractions of colorectal carcinomas: a five-year prospective study relating flow cytometry to survival

In our prospective study, flow cytometric analysis of cellular DNA and RNA content was performed on unfixed fresh specimens of colorectal adenocarcinoma taken from 176 patients. Of the 176 tumors, 113 (64%) were aneuploid. There was no correlation between aneuploidy and tumor stage, grade, location, or size. After a median follow-up of 5.6 years, no correlation between DNA or RNA content and patient survival was found. DNA content alone was not an independent prognostic factor when the colorectal carcinomas were segregated by curable and incurable stages. However, normal mucosa, diploid tumors, and aneuploid tumors showed progressively higher proliferation and higher RNA and DNA indices. Proliferative fraction--defined as the percentage of cells in S + G2 and M phases of the cell cycle--was significantly related to ploidy and to Dukes' stage. Despite these correlations, we did not detect a significant influence of proliferative fraction on survival when patients were segregated above or below the mean proliferative fraction for all tumors. More accurate methods of identifying the proliferative fraction of tumor cells are currently being pursued. While the role of flow cytometry in the evaluation and management of patients with colorectal carcinoma is still undefined for a number of other cellular parameters, it seems unlikely that DNA index, RNA index, or the proliferative fractions calculated from the DNA histogram, will, of themselves, represent independent prognostic factors.     

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.     

Prognostic value of DNA flow cytometry in stomach cancer: a 5-year prospective study

The role of DNA flow cytometry in the prediction of prognosis for patients with stomach cancer remains to be defined. Thus we studied prospectively the role of DNA flow cytometry as a prognosis indicator in stomach cancer patients in a high-incidence area. Between November 1990 and December 1992, primary stomach cancer tissues were obtained from the surgical specimens from 217 patients (148 male, 69 female). DNA flow cytometric analyses of DNA ploidy and S-phase fraction were performed and the results were correlated with patient survival. The median age of the patients was 55 years (range 24-78). Aneuploid cell population was found in 114 of 217 samples (53%). Tumour S-phase fraction was obtained in 96 of 103 diploid tumours (93%) and 61 of 114 aneuploid tumours (54%). After median follow-up of 66.1 months, the patients with tumours with an S-phase fraction over 17% had significantly worse survival rates than patients with tumours with S-phase fractions of lower than 8% or 8-17% (45% vs 59% and 63% of patients surviving, P = 0.007). Tumour ploidy status did not correlate with patient survival. Multivariate analyses showed that the TNM stage remained the most important prognostic indicator. The tumour S-phase fraction was also an independent prognostic indicator (relative risk 2.300, 95% CI, 1.252-4.223). Tumour S-phase fraction obtained by DNA flow cytometry is an independent prognostic indicator for the survival of the patients with stomach cancer.     

S-phase DNA content and aneuploidy of immunophenotypic defined subpopulations in acute myeloid leukemia determined by multi-parameter flow cytometry

One of the major limitations of DNA flow cytometry (FCM) in hematologic malignancies is the lack of information about the proliferation activity of subpopulations of the heterogeneous bone marrow (BM) compartment. We studied the S-phase DNA content of immunophenotypically defined BM subpopulations (CD2+; CD19+; CD2/CD19+; glycophorin-A+; CD14+; CD13+; CD33+ and CD13/CD33+) in 18 patients with acute myeloid leukemia (AML), including three patients with M6 AML. The results were compared with the findings in twelve normal BM aspirates. The measurements were performed using a special protocol for bivariate FCM of DNA content and surface immunofluorescence (s-IF). In patients with AML the proportion of BM cells expressing the myelomonocytic and monocytic markers (M1-M5 AML) or erythroid marker (M6 AML) was expanded. However, in many patients other subpopulations were 4% or higher permitting the calculation of their S-phase DNA. No essential differences in median S-phase DNA percentages of the distinct subpopulations were observed between normal and leukemic bone marrow though the ranges in AML patients were much wider. These data suggest that AML is not characterized by an increased nor a decreased proliferation activity, but rather by a situation of cell growth independent to the normal regulatory mechanisms. Additional information was obtained upon DNA aneuploidy using CD2+ or CD2/CD19+ cells as an intrinsic DNA standard which allowed us to define differences in the DNA index as small as 2% as aneuploid. This approach appeared suitable for detecting small-degree numerical chromosomal aberrencies, as found by cytogenetics, in 4/6 cases.     

流式细胞仪动态检测成人急性白血病细胞动力学的临床意义

应用流式细胞仪（ＦＣＭ）动态检测３４例成人急性白血病患者１４６份骨髓标本的细胞动力学参数，并以１０例正常人骨髓标本为对照。结果显示：（１）成人急性白血病患者骨髓标本Ｓ％在不同临床阶段存在一定差异。（２）白血病患者完全缓解时Ｓ％与正常组差异不显著；而巩固强化可使Ｓ％一过性降低。（３）典型病例动态检测表明不同化疗方案对Ｓ％的影响不同。说明动态观察Ｓ％的变化可为诱导和巩固强化的化疗方案个体化提供客观的细胞动力学参数。     

Node-negative breast cancer: prognostic subgroups defined by tumor size and flow cytometry

Adjuvant systemic therapy for women with node-negative breast cancer is most easily justified for those patients at highest risk of relapse. We have examined the impact of tumor size, histologic grade, estrogen receptor (ER) status, tumor ploidy, and S-phase fraction (SPF) on relapse-free survival (RFS) for 169 patients with node-negative breast cancer in order to identify groups of patients at high and low risk of relapse. Patients with small tumors (less than or equal to 1.0 cm) had a significantly better RFS than those with larger tumors (P = .005), with 96% remaining relapse-free at 5 years. Patients with tumors less than or equal to 1.0 cm were thus excluded from analysis when attempting to define a group with a poor prognosis. Within the group of patients with tumors greater than 1.0 cm, tumor ploidy (P = .63), ER status (P = .3), or progesterone receptor (PgR) status (P = .24) did not predict for RFS. Patients with grade 1 or 2 infiltrating ductal tumors had a significantly better prognosis than those with grade 3 tumors (P = .04). The prognostic factor that gave the widest separation between subgroups, however, was SPF. Patients whose tumors were greater than 1.0 cm with an SPF less than or equal to 10% had a 5-year RFS of 78% compared with a 5-year RFS of 52% for those with an SPF greater than 10% (P = .006). We have combined tumor size and SPF to identify three prognostic groups: (1) tumor less than or equal to 1.0 cm, 5-year RFS 96%; (2) tumor greater than 1.0 cm plus SPF less than or equal to 10%, 5-year RFS 78%; 3) tumor greater than 1.0 cm plus SPF greater than 10%, 5-year RFS 52%. These prognostic groupings may help identify patients most suitable for adjuvant therapy.