S-phase Values Research Articles

Clinical Behavior of Acoustic Tumors: A Flow Cytometric Analysis

Recently, nonsurgical treatment of acoustic tumors has been advocated as an alternative to surgical resection. Because of the relatively short follow-up in reported series of radiation-treated acoustic tumors, the lack of growth of some tumors may merely reflect the variable biologic growth potential of these tumors and not the result of treatment. DNA flow cytometry has been used to predict biologic activity in other solid tumors. It is applied in this study to assess the variability of growth potential in a typical acoustic tumor population and to determine whether relationships exist between flow cytometric data and clinical characteristics of acoustic tumors. DNA flow cytometry techniques were used to evaluate formalin-fixed, paraffin-embedded tissue previously obtained from patients who were surgically treated for acoustic neuromas. Relationships between flow cytometry data and historical data were also statistically evaluated. Tissue samples were from patients of a large private otologic practice. Subjects were a convenience sample of 49 patients (26 female and 23 male) with a mean age of 59 years who had undergone surgical removal of an acoustic neuroma. None of the patients had other stigmata of neurofibromatosis or tumor recurrence. All tissue specimens were pathologically confirmed acoustic neurons, with a range in tumor size from 1 to 6 cm. The measures included DNA ploidy and S-phase fraction. Historical data included age, sex, size of tumor, presenting symptom, and symptom duration. All 49 tumors showed a diploid distribution, with S-phase values ranging from 1.07% to 20.74% (mean +/- SD, 6.30 +/- 4.24). The ploidy and S-phase data compare favorably with previously published data in which fresh tissue was used. There were no statistically significant relationships between S-phase value and historical data. The wide range of S-phase values is consistent with a large variation in tumor growth potential and suggests caution in interpreting the results of radiation treatment of acoustic tumors when follow-up relatively short.

A comparative study of proliferation‐associated parameters in b‐cell non‐hodgkin lymphoma

The prognostic value of three proliferation-associated parameters, the frequency of cells in S-phase, mitotic index (MI) and serum deoxythymidine kinase levels (S-TK), was examined in 106 primary cases of B-cell non-Hodgkin lymphomas (NHL), 76 'low grade' NHL and 30 'high grade' NHL and compared with morphology and clinical variables. All three proliferation factors displayed large differences in the mean values of the two groups 'low grade' and 'high grade' NHL, and all revealed significant prognostic information. In 'low grade' NHL, the information decreased with follow-up time. A correlation (r = 0.7) was noted between S-phase values and MI but not between S-TK and the two others. In the entire patient material and in the two prognostic groups defined by morphology, S-TK gave the best prognostic information. MI gave better prognostic information than S-phase values in the whole material and in 'low grade' NHL, whereas the reverse appeared to be true for 'high grade' NHL. In the multivariate analyses, no other parameter apart from S-TK provided any further prognostic importance in the cases of 'high grade' NHL, whereas in that of 'low grade' NHL, MI, stage, age and histology had independent importance.

Tumor Proliferative Fraction in Solid Malignant Neoplasms A Comparative Study of Ki-67 Immunostaining and Flow Cytometric Determinations

Tumor proliferative fraction (TPF) has been shown to correlate with prognosis in some malignancies. A method for its determination that is practical, accurate, and reproducible is still being sought. In this comparative study of techniques, TPF values were determined in mirror-image samples of 126 consecutive solid malignant neoplasms using flow cytometry and immunostaining with Ki-67, a monoclonal antibody that recognizes an unknown nuclear antigen expressed during the entire cell proliferation cycle but not in resting cells. The mean TPF values for all cases were 19.5 +/- 15.6% (percentage of tumor cells stained) by Ki-67 (range, 1-86%) and 15.7 +/- 9.6% (S + G2M) by flow cytometry (range, 3-60%), which correlated significantly at r = 0.53 and P = 0.005. The correlation was less strong in tumors with low S-phase values (less than or equal to 10%, r = 0.28) than in tumors with intermediate and high S-phase values (r = 0.66). Ki-67 staining percentages did not correlate with patient age, sex, or tissue origin of the tumor. Ki-67 staining appears comparable to flow cytometry determination of TPF in solid malignancies with intermediate and high S-phase values. In tumors with low S-phase values, Ki-67 immunostaining shows higher TPF values, which perhaps reflect an increase in the proportion of G1-phase cells or dilutional effect of nonneoplastic cells in the tumors with low proliferative fraction.

THE CLINICAL SIGNIFICANCE OF DNA ANALYSIS BY FLOW CYTOMETRY IN NON-HODGKIN'S LYMPHOMA

The nuclear DNA content of paraffined-embedded surgical biopsies from 84 patients with non-Hodgkin's lymphoma was measured by flow cytometry. DNA aneuploidy was detected in 32 cases (38%) and was not related to histologic grade. The incidence of aneuploidy did not correlate with survival. S-phase fraction was significantly higher in morphologically high grade and intermediate grade than in low-grade (P < 0.05) . The percentage of S-phase cells was found to be a valuable prognostic parameter using a cut point at 18 percent. The S-phase values below 18 percent had a significantly better survival than those with S-phase values above 18 percent (P<0.05) .We considered that the flow cytometric DNA analysis revealed characteristic features in non-Hodgkin's lymphomas, and the S-phase value might be useful in predicting the clinical outcome of patients. Key words: Non-Hodgkin's lymphoma; DNA analysis, flow cytometry; paraffinedembedded samples

Is DNA ploidy and proliferative activity of prognostic value in advanced gastric carcinoma?

We analyzed the DNA content of 116 gastric carcinomas and lymph node metastasis in 53 of these using flow cytometry on archival material. DNA index and proliferative activity (% S-phase) values were obtained in 111 and 76 tumors, respectively, and survival rates were obtained for 108. Tumor classification was based on histologic type, grade, and growth pattern and stage. All were advanced gastric carcinomas. Fifty four tumors (47%) were aneuploid. In single variable analysis, survival was related to stage (muscularis propria versus serosa P = .002) and lymph node metastasis (P = .04). Infiltrative tumor growth had a slightly worse prognosis (P = .06). In multifactorial analysis, tumor stage was the only independent prognostic factor. Neither DNA ploidy nor % S-phase values had any effect on survival, although, patients with tumors with higher DNA indexes (greater than 1.7) showed a tendency toward worse survival rates. We failed to prove the prognostic value of DNA ploidy in gastric carcinoma. Before its effect is ruled out, some factors have to be considered: (1) all tumors were in advanced stage and the true effect of ploidy on survival may have been diluted, (2) quality of fixation--near diploid aneuploid populations may have been overlooked, and (3) intratumoral DNA ploidy heterogeneity can cause sample discrepancy (20% in our series).

Steroid receptors and Ki‐67 reactivity in ovarian cancer and in normal ovary: Correlation with dna flow cytometry, biochemical receptor assay, and patient survival

Steroid hormone receptors and reactivity for Ki-67 proliferation antigen were studied immunohistochemically in non-neoplastic post-menopausal human ovary and in 29 ovarian cancers. In the normal ovary, oestrogen (OR) and progesterone receptors (PR) were found in the surface epithelium and PR also in the ovarian stroma. Of the ovarian carcinomas 38 per cent (11/29) contained OR and 69 per cent (20/29) PR. Oestrogen receptor expression was confined to malignant cells, whereas PR was present occasionally also in the tumour stroma. In most cases, ORs and PRs were found only in a small population of cancer cells. The growth fractions assessed by the percentage of Ki-67-positive cells ranged from 1 to 59 per cent (mean 19.7 per cent) with a significant correlation (r = 0.74, P less than 0.0001) to S-phase values (mean 12.9 per cent, range 1.2-25.9 per cent) determined by DNA flow cytometry. High Ki-67 (greater than or equal to 15 per cent) and S-phase levels (greater than or equal to 7.5 per cent) correlated with advanced disease stage and patient survival but not with OR or PR status, suggesting that hormone-receptor pathways and proliferative activity are not related in ovarian cancer. Positive OR status, however, identified patients with a better prognosis (P = 0.02), suggesting a correlation with tumour differentiation. The independent prognostic value of oestrogen receptor status and Ki-67 remains to be determined, but the prognostic impact of Ki-67 was comparable to that of S-phase values.

Cytokinetic investigation of lung tumors using the anti‐bromodeoxyuridine (BUDR) monoclonal antibody method: Comparison with dna flow cytometric data

In order to investigate the cytokinetics of malignant tumors and non-malignant lesions of the lung, tissue samples from 57 patients affected by non-small-cell carcinoma (NSCLC), small-cell carcinoma (SCLC), and benign and inflammatory lesions have been analyzed using the BUdR monoclonal antibody (MAb) method. This method is based on the preparation, at the time of surgery, of viable monocellular suspensions (using collagenase and DNase treatment) and the concomitant administration of BudR. The percentage of BudR-labelled cells was monitored by fluorescent microscopy using an FITC-labelled second antibody. In NSCLC, each histological group showed a wide range of labelling index (LI) values. On the contrary, SCLC exhibited a more homogeneous kinetic behaviour as evidenced by a narrowly distributed, higher LI. Tumors shown to be diploid by flow cytometry did not show a lower LI than aneuploid tumors. Furthermore, differences were constantly observed between the S-phase percent calculated using BUdR and that calculated using the DNA flow cytometric (FC) histogram, the latter always showing higher S-phase values. In an attempt to study the intra-tumor proliferative heterogeneity, multiple-site sampling was performed. Proliferative heterogeneity seemed to be higher inter-tumor than intra-tumor. Finally, a positive correlation (p less than 0.05) was found between LI and the actual doubling time (DT) of the primary tumor mass, evaluated using sequential radiographs. In conclusion, the present BUdR method can be considered a useful source of relevant information on in vivo cell growth, in parallel to other clinical (DT) and biological (DNA content) approaches.

Development of Ploidy and Cell Proliferation in Serial Samples of Ascites from Patients with Ovarian Carcinoma

Repeated samples of ascites were obtained from 14 ovarian carcinoma patients over a period of 10 to 360 days. The samples were studied by means of flow-cytometry and monoclonal antibodies specific for bromodeoxyuridine. Ploidy and proliferation of tumor cell populations were measured to establish the significance of these properties in the development of the tumors. Ploidy was mostly a stable feature while the proportion of S-phase values increased significantly over the passage of time. This increase is discussed in relation to the expression of selective processes towards more aggressive tumor cell lines and to the development of chemoresistance.

Comparative Flow Cytometric Deoxyribonucleic Acid Studies on Exophytic Tumor and Random Mucosal Biopsies in Untreated Carcinoma of the Bladder

In 290 patients with untreated carcinoma of the bladder the deoxyribonucleic acid properties, as measured by flow cytometry, of 3 random mucosal biopsies were studied and compared to those of the exophytic tumors. Mucosal aneuploidy was found with few exceptions in aneuploid tumors only, and in a significantly lower frequency in aneuploid tumors of grade 2 than grade 3. The individual specificity of bladder tumors is emphasized by the observation that the level of ploidy was mostly the same in aneuploid mucosal biopsies as in the exophytic tumor. This is underlined further by the occurrence of cell populations of the same ploidy in different parts of the bladder mucosa. However, S-phase values of the concomitant intraurothelial lesions were significantly lower than those of the exophytic tumors. Therefore, we concluded that the process of evolution from malignantly transformed lesions, confined to the urothelium, to an exophytic or invasive tumor is dependent on a further elevated proliferation of the urothelial lesions.

Flow cytometric measurement of ploidy and proliferation in effusions of ovarian carcinoma and their possible prognostic significance

The cellular DNA pattern of ascites and pleural effusions from 81 patients with advanced ovarian carcinoma was prospectively studied by means of flow cytometric DNA analysis. The degree of ploidy and the proportion of S-phase values were correlated to histological differentiation, to status, and to the development of disease. According to DNA indices, the cell populations distributed in the diploid to peridiploid and in the tri-to tetraploid range. Aneuploidy was more frequently associated with poor degree of differentiation, with progressive disease, and with higher proportion of cells in S phase. Thus, although patients with stable disease had a significantly larger proportion of tumors with diploid DNA content, all except four patients were dead within a median survival period of 12 months. No correlation was observed between total survival period and ploidy; however a significantly shorter survival time was noted in patients whose ascites comprised cell populations with S-phase values exceeding 15%.

Nucleoside Transport and Proliferative Rate in Human Thymocytes and Lymphocytes

The thymus is a site of active T-lymphoid cell proliferation and DNA synthesis. In this study, the capacity of human thymocytes for nucleoside transport was assessed both by cytosine arabinoside influx and by equilibrium binding of nitrobenzylmercaptopurine riboside (NBMPR), a specific ligand for the equilibrative nucleoside transporter of leukocytes. The proportion of freshly isolated thymocytes synthesizing DNA was 8.6% +/- 2.1% (n = 12) by 3H-thymidine labeling index and 7.8% +/- 2.9% (n = 4) S-phase cells by flow cytometric analysis of DNA content. In comparison, both methods gave proliferation S-phase values less than 1% for peripheral blood lymphocytes (PBLs). Thymocytes expressed a high density of specific NBMPR binding sites (26,068 +/- 8,776 sites per cell, n = 12) as compared with PBLs (1,123 +/- 553 sites per cell, n = 8). The initial influx of cytosine arabinoside into thymocytes was 14-fold greater than into PBLs, and in both cell types the influx of nucleoside was totally inhibited by 0.5 mumol/L NBMPR, which is known to inhibit the major equilibrative nucleoside transporter in white blood cells. Depletion of mature CD3+ cells from the thymocyte preparation by anti-CD3 antibody left a residual population with both increased labeling index and up to twofold greater density of NBMPR binding sites. When PBLs were cultured for 48 hours with the T-cell mitogen phytohemagglutinin, a 40-fold increase in labeling index was observed, together with a 30-fold increase in the density of specific NBMPR binding sites. Thus, fresh thymocytes from human thymus are actively proliferating and express high densities of a functional nucleoside transporter. The more immature cells in the thymocyte population which are proliferating more actively have a greater density of nucleoside transporters than the whole population. In contrast, mitotically inactive PBLs-have few nucleoside transporters, but after mitogenic stimulation PBLs express large numbers of this transmembrane molecule.

Nucleoside transport and proliferative rate in human thymocytes and lymphocytes

The thymus is a site of active T-lymphoid cell proliferation and DNA synthesis. In this study, the capacity of human thymocytes for nucleoside transport was assessed both by cytosine arabinoside influx and by equilibrium binding of nitrobenzylmercaptopurine riboside (NBMPR), a specific ligand for the equilibrative nucleoside transporter of leukocytes. The proportion of freshly isolated thymocytes synthesizing DNA was 8.6% +/- 2.1% (n = 12) by 3H-thymidine labeling index and 7.8% +/- 2.9% (n = 4) S-phase cells by flow cytometric analysis of DNA content. In comparison, both methods gave proliferation S-phase values less than 1% for peripheral blood lymphocytes (PBLs). Thymocytes expressed a high density of specific NBMPR binding sites (26,068 +/- 8,776 sites per cell, n = 12) as compared with PBLs (1,123 +/- 553 sites per cell, n = 8). The initial influx of cytosine arabinoside into thymocytes was 14-fold greater than into PBLs, and in both cell types the influx of nucleoside was totally inhibited by 0.5 mumol/L NBMPR, which is known to inhibit the major equilibrative nucleoside transporter in white blood cells. Depletion of mature CD3+ cells from the thymocyte preparation by anti-CD3 antibody left a residual population with both increased labeling index and up to twofold greater density of NBMPR binding sites. When PBLs were cultured for 48 hours with the T-cell mitogen phytohemagglutinin, a 40-fold increase in labeling index was observed, together with a 30-fold increase in the density of specific NBMPR binding sites. Thus, fresh thymocytes from human thymus are actively proliferating and express high densities of a functional nucleoside transporter. The more immature cells in the thymocyte population which are proliferating more actively have a greater density of nucleoside transporters than the whole population. In contrast, mitotically inactive PBLs-have few nucleoside transporters, but after mitogenic stimulation PBLs express large numbers of this transmembrane molecule.

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.

Growth potential of acoustic neuromas.

Tumor samples from 32 patients with acoustic neuroma operated with translabyrinthine approach were analyzed with flow cytometric technique with respect to ploidy and cell cycle phase distribution. In all cases the diagnosis of acoustic neuroma was verified histologically, two patients had Recklinghausen's disease. None of the tumors showed aneuploid cell lines. Median S-phase fraction was 6.8% (range 1.4 to 19.9%). The spread of S-phase values had a greater range for the smaller tumors (volume less than or equal to 2 cm3) of the material. There was no correlation neither between the fraction of cells in S-phase and age of the patient, nor between the fraction of cells in S-phase and tumor size. The present finding with a great variability in S-phase values does not support an expectant attitude regarding surgical intervention in patients with acoustic neuromas.

Fraction of S‐phase cells in blood mononuclear cells in non‐Hodgkin's lymphomas—Correlation with clinical features and prognosis

A consecutive material of 111 untreated patients with non-Hodgkin's lymphoma was studied with respect to fraction of S-phase cells in blood mononuclear cells in relation to presence of monoclonal B cells in blood (MBCB). Fraction of S-phase cells was determined by flow cytometry and estimation of MBCB was performed by kappa:lambda analysis. The fraction of S-phase cells was significantly higher (p less than 0.001) in MBCB-positive cases (median 1.2%) than in the MBCB-negative (median 0.7%). MBCB-positive patients with S-phase values greater than or equal to 1.5% had a less favourable prognosis compared to those with less than 1.5% cells in S-phase (p = 0.01). In a Cox multiparameter analysis, advanced clinical stage, high-grade morphology and high fraction of S-phase cells in blood in MBCB-positive cases were independent, statistically significant, negative prognostic indicators. The results indicate that an elevated S-phase value in blood in non-Hodgkin's lymphoma constitutes a negative prognostic factor, probably reflecting proliferating tumour cells in blood.

Flow cytometric DNA analysis: A prognostic tool in non-hodgkin's lymphoma

Surgical biopsies from 234 untreated patients with non-Hodgkin's lymphoma (NHL), classified according to the Kiel nomenclature, were analysed with respect to proliferative activity (S-phase) and DNA content by flow cytofluorometric (FCF-DNA) analysis. The percentage of cells in S-phase was significantly higher in lymphomas of high compared to low grade NHL (p less than 0.001). Patients with lymphomas of high grade histology and low S-phase values (less than 5.6%) achieved complete remission (CR) more often (p less than 0.05) and survived significantly longer than those with high S-phase values (p less than 0.05). In the low grade NHL group the S-phase value did not correlate to response. S-phase correlated to survival for patients with the lymphocytic (CLL & IC) (p less than 0.05) and follicle center cell (FCC) derived (p less than 0.01) but not in blastic (LB, IB, Burkitt) NHL. DNA-aneuploidy was associated with poor response to therapy and shorter CR duration in low grade NHL (p less than 0.05 for both). However, the degree of DNA-ploidy (neardiploid or aneuploid) did not correlate to survival in any of the NHL groups analysed (high- or low grade, lymphocytic, FCC derived or blastic). The Cox regression analysis indicated that the S-phase value was a stronger predictor of survival than histopathology, stage or age, especially in low grade NHL. These results suggest that S-phase analysis should be included in the clinical evaluation of NHL patients as a prognostic indicator.

DNA flow cytometry and prognostic factors in 1331 frozen breast cancer specimens.

Breast cancer proliferative capacity as determined by the DNA thymidine labeling index, along with estrogen and progesterone receptor status, is highly predictive for risk of relapse and overall survival. Recently, DNA ploidy and proliferative capacity (S-phase fraction [SPF]) as determined by flow cytometry have also shown significant prognostic value. The authors have developed a technique which allows a 50 to 100 mg aliquot of the same frozen breast tumor specimen routinely employed in steroid receptor assays, to be assayed for both DNA ploidy and SPF by flow cytometry. Of the 1331 tumors examined, DNA histograms were evaluable for ploidy in 89% (1184) of specimens examined; 57% of these were aneuploid. Adapting a trapezoidal model to estimate SPF in both diploid and aneuploid tumors, the authors found 81% (1084) to be evaluable for SPF, with a median SPF of 5.8% for the entire population. The median SPF was significantly lower in diploid tumors (2.6%) than in aneuploid tumors (10.3%, P less than 0.0001). Both aneuploidy and high SPF were strongly associated with absence of steroid receptors. Aneuploid tumors showed more striking differences in the frequency of high S-phase values with respect to receptor status and age or menopausal status, whereas diploid but not aneuploid tumors showed lower SPF in node-negative versus node-positive patients. Because it is particularly important to identify the high-risk minority of node-negative patients, the authors examined the node-negative group separately. High SPF subgroups appeared in each category of receptor status and age or menopausal status within the node-negative group, suggesting that SPF will be an independent prognostic factor. With the DNA flow cytometric methods used here, it is now practical to determine ploidy and SPF for nearly every breast cancer patient. These factors, which show associations with established prognostic factors, such as receptor status can now be fully evaluated for their prognostic significance in broad patient populations.

Prognostic value of DNA content in colorectal carcinoma. A flow cytometric study with some methodologic aspects.

The DNA content of 37 colorectal carcinomas was studied prospectively by flow cytometry. The DNA content from the same tumors was also studied in disintegrated paraffin sections. The methods gave similar information on the DNA content and the correlation was 0.94. In eight patients tumor imprints were prepared and the DNA content was analyzed with static cytometry. A correlation coefficient of 0.83 between flow cytometry on fresh tumor tissue and static cytometry was found. Sixty-two percent of the tumors were aneuploid and had significantly higher S-phase values than diploid tumors. Histologic grade was not related to ploidy level, whereas Dukes' C tumors often were aneuploid. When the clinical course of patients was analyzed by log rank test (mean follow-up, 30.4 months), the patients with diploid tumors showed a clear advantage in terms of survival; only two patients in this group developed progressive disease. It is concluded that the DNA content is an independent prognostic predictor of colorectal carcinoma and that DNA can be analyzed by several methods. Of these, the method using disintegrated paraffin sections appears most attractive.

Cell Kinetics of the Human Anagen Hair Follicle

Anagen hair follicles obtained from both healthy (n = 7) and uninvolved psoriatic (n = 4) scalps were segmentally analyzed for proliferative activity using DNA flow cytometry. Hair follicle kinetics were almost equal in either group except for the infundibular portion which exhibited significant increase of S-phase values in psoriatic patients. Maximum proliferation was disclosed within the bulbar segment. This study confirms that cell kinetics behavior of hair follicles from uninvolved scalp of psoriatics compared with those from healthy scalps is altered in the infundibular portion only.

DNA measurement--an objective predictor of response to irradiation? A review of 24 squamous cell carcinomas of the oral cavity.

DNA measurements on biopsy material from 24 squamous cell carcinomas of the oral cavity given preoperative radiotherapy indicate that DNA aneuploid tumours respond better to radiotherapy than do diploid and polyploid tumours. The mean S-phase value was higher (16.1%) for 8 tumours that were eradicated by preoperative radiotherapy than for 13 that did not respond (8.1%). These factors correlated better with the response than did histological and clinical (T) classifications. DNA-ploidy and S-phase estimation can complement the histological diagnosis, and may prove valuable when planning treatment.