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In Hodgkin's disease DNA aneuploidy is not a prognostic factor. However, the prognostic significance of DNA content in Hodgkin's disease may be missed by either intratumor DNA heterogeneity or DNA analysis of limited samples. For flow cytometry usually one section of 40-60 microns is used for the analysis. In breast cancer this proved to be insufficient. In Hodgkin's disease no data are available. Therefore, we examined if analysis of more sections does increase the yield of aneuploidy. Archival, formalin-fixed, parafin embedded tissues were used. From 13 patients four sections of 50 microns could be analysed for DNA content. In 12 of 13 patients the results were consistent in all four sections of one patient case; seven diploid, four aneuploid and one multiploid. In one case ploidy status changed: two sections were diploid and two were aneuploid. The DNA-index of the aneuploid samples ranged from 0.75 to 1.38 and varied from 0.02 to 0.14 within one case. The S-phase fraction remained constant within all evaluable cases (sd: 0.5-1.5%), except for one (sd: 4.7%). In conclusion, in Hodgkin's disease the ploidy status of the first section can be regarded to represent the whole tissue sample. Therefore, the absence of prognostic value of ploidy status is not explained by sampling errors in tissues analysed.

The analysis of the cell cycle distributions by univariate flow cytometric DNA measurement has been widely applied in the clinic to determine kinetic parameters of human malignancies. A common problem with measurements of cell cycle phase distributions in tumor biopsy material is the presence of non-malignant diploid cells. Furthermore, such a static measurement might not be accurate enough to describe the dynamic process of cell proliferation. For this purpose alternative methods have been developed to include BrdUrd incorporation or the presence of intrinsic proliferation associated markers such as PCNA or Ki67-Ag into the analysis. However, the presence of nonmalignant diploid cells will influence also these bivariate analyses, especially in case of DNA-diploidy of the tumor cells. Here we present a three parameter flow cytometric assay based on the simultaneous detection of cytokeratin, DNA and a proliferation associated marker, such as BrdUrd, PCNA or Ki67-Ag. Based on the presence of cytokeratin, epithelial cells can be selected for a detailed cell cycle analysis. This method can be applied to frozen tissue, which makes this assay useful for multicentre clinical studies.

A prognostic value of DNA content in cervix carcinoma for local recurrence following radiotherapy is suggested by both retro- and prospective studies. The purpose of this study was to confirm these data. Flow cytometry and computerized image cytometry were used to measure DNA content of tumor cells retrospectively in paraffin-embedded archive material from 98 patients with carcinoma of the uterine cervix FIGO stage IB-III. All patients were treated with combined megavoltage irradiation and brachytherapy. Minimum follow-up has been 5.1 years. Pelvic recurrence was seen in 10, 26, and 54% of patients with stages IB II, and III, respectively (P = 0.003). DNA content, expressed as DNA index (DI), was not correlated with the FIGO stage (P = 0.25), histopathologic grading (P = 0.26), or age (P = 0.63) at diagnosis in this series. Using univariate analysis pelvic disease-free survival (PDFS) was best predicted by the clinical stage (P = 0.004) and by an age greater than 55 years (P = 0.04) before treatment. PDFS tended to be better for patients with aneuploid tumors (DI > 1.2), but this difference was not statistically significant (P = 0.17). Using Cox's regression model for multivariate analysis, only stage was independently correlated to PDFS (P = 0.009). After stratification for stage, ploidy level was significantly correlated with PDFS in stage II patients (P = 0.04). Overall results suggest aneuploid tumors to be more radiosensitive than diploid tumors in this series.

Ninety-one cases of primary breast cancers and their nodal metastases were examined with DNA flow cytometry. No differences were found between the stemline distributions in the primary tumors and nodal metastases. At both sites stemlines clustered around a DNA index of 1.0 (33-40% of cases) and 1.8. The mean S-phase fractions were 7.9 in primary tumors versus 5.6% in nodal metastases (p = 0.02); this difference was also observed if the analysis was restricted to cases with DNA aneuploidy at both sites (10.2 versus 7.6%, p = 0.04). Our results indicate that axillary nodal ploidy and proliferation reflect primary tumor characteristics rather than displaying changes associated with selection during the lymphatic metastatic process. Lymph nodes may have a suppressive effect on the proliferation of tumor cells.

To assess the reliability of DNA estimation using image cytometry, deparaffinized lymph nodes from 70 patients with Hodgkin's disease were examined and the results obtained were compared with those from flow cytometry. Image analysis without discriminating between the various cell types, as found in Hodgkin's disease, revealed no separate aneuploid peak. Selecting on morphologically defined nuclear types DNA aneuploidy was detected in 20% of the cases (14/70). The aneuploid populations were limited to the population of nuclei defined as Reed-Sternberg (RS)-like or medium-sized lymphocytes. Benign lymph nodes DNA aneuploidy was not found in any of the controls. Comparison of DNA histograms obtained by image and flow cytometry showed aneuploid peaks using image cytometry in 4 of 30 diploid and 10 of 40 aneuploid flow histograms. In conclusion, image analysis using the CAS 200 system as compared to flow cytometry is more time-consuming and less sensitive to assess ploidy status, although it may provide extra information in some selected cases. Evidence is obtained that DNA aneuploidy in Hodgkin's disease is preferentially expressed by cells with the RS/H-like and medium-sized lymphocyte morphology.

Paraffin-embedded lymph nodes from patients with Hodgkin's disease were examined for flow cytometric DNA content. In order to increase the sensitivity of the assay we tried to enrich for the neoplastic cells by bivariate analysis using a polyclonal anti-nucleolar antibody (AN-AB) and the forward scatter (FSC). DNA aneuploidy was found to be present in 67 of all 137 cases (49%), in 24 cases only demonstrable by dual-parameter analysis. The DNA index varied from 0.69 to 1.89 with a total of 22 hypo-diploid cases. The number of aneuploid nuclei exceeded the expected frequency of Reed-Sternberg (RS) and Hodgkin (H) cells in most of the analysed specimens. In conclusion, flow cytometry in Hodgkin's disease appears to give useful information regarding the ploidy status and evidence has been provided that the malignant cell population in Hodgkin's disease is not limited to the classical RS/H cells.

Background: Individual assessment of the prognosis of patients with breast cancer is crucial for the selection of risk-adapted adjuvant therapy and in follow-up. Parameters from DNA flow-cytometry have been shown to provide significant prognostic information, but published results are in conflict and there are only a few investigations with long-term follow-up. The aim of this study is to clarify the impact of tumor DNA data on the clinical course of stage I and stage II breast cancer patients., Patients and Methods: Several flow-cytometry DNA analyses were performed on tumor samples derived from 191 breast cancer patients entered in a controlled clinical trial after a median follow-up of more than 10 years. In addition to DNA index (DNI) and the percentage of cells in S phase (SPF), an index, designated aneuploidy fraction (AF), was determined. It ascertains the percentage of aneuploid cells out of all cells in the DNA flow-cytometry histogram, and its reproducibility has been tested by measurements of AF in two different samples of the same tumor. Univariate analyses and, in the 122 patients for whom complete information was available, a Cox model, were performed to investigate the individual prognostic impact of flow-cytometry parameters compared with established clinical factors., Results: AF proved to be a very valuable prognostic indicator both in univariate and multivariate analyses, whereas DNI and SPF failed to provide independent prognostic information. The combination of AF and lymph node status clearly identifies different prognostic subgroups in operable breast cancer., Conclusions: Routine evaluation of patients with breast cancer should include tumor DNA flow-cytometry. Aneuploidy fraction is a valuable tool in assessing an individual patient's prognosis and thus can help in the choice of the appropriate adjuvant treatment strategy. Whether it, rather than DNI and SPF should be used, as we found, needs to be validated in a larger prospective investigation.

We evaluated breast cancer specimens from 241 patients of a controlled clinical trial by means of DNA flow cytometry. We report the correlations between DNA index (DNI) and fraction of cells in S-phase (SPF) and other prognostic parameters. Both univariately and in a Cox model, the predictive power of these factors is evaluated after a follow-up of more than 10 years. There are strong correlations between DNI and SPF (P = 0.0001) and between flow cytometry parameters and clinical and histopathological factors such as axillary lymph node involvement, tumour size and histological grade. In univariate analysis DNI fails to provide prognostic information, whereas SPF turns out to be able to differentiate between patients at high and low risk for relapse and death (P = 0.002). In the multivariate Cox model, too, SPF is an important prognostic parameter with respect to patient survival (relative risk: +86%), only surpassed by nodal involvement. DNI, however, turns out to be an independent predictor of relapse free survival and distant recurrence free survival. By combination of DNI and SPF, patients can be divided into three prognostic subgroups. We conclude that data from DNA flow cytometry can be of great importance for the decision on the level of aggressiveness of adjuvant therapy for an individual patient and therefore may help to avoid overtreatment and toxicity.

Investigations regarding the prognostic value of DNA content (ploidy) and proliferative characteristics [percentage of cells in S-phase or S-phase fraction (SPF)] have been greatly facilitated by the application of flow cytometry (FCM) using nuclei isolated from paraffin-embedded tissue. We have applied this technique to tumor sections from patients presenting with non-small-cell lung cancer (NSCLC) in 1980 and 1981. From 67 out of 115 patients material of sufficient quantity and quality was obtained to perform DNA-FCM. A multivariate analysis including stage of disease (UICC), age, tumor histology and treatment modality was performed to examine the prognostic significance of DNA-FCM in NSCLC. Aneuploidy was found in 65% of cases. In our study, the DNA content was not related to histology, stage of disease or treatment modality, nor to the length of survival (log rank test P = 0.62). Calculation of SPF was possible in 49/67 cases. The SPF was not related to histology, stage of disease or treatment modality, but a significant prognostic value was found for survival; patients with a high SPF died earlier (P = 0.04) and this was especially true for squamous cell carcinoma (P = 0.02). This study demonstrates the prognostic importance of DNA-FCM-derived information in NSCLC using a multivariate analysis; however further prospective studies in larger patient populations are needed.