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Developing a reliable and quantitative assessment of the potential virulence of a malignancy has been a long-standing goal in clinical cytometry. DNA histogram analysis provides valuable information on the cycling activity of a tumor population through S-phase estimates; it also identifies nondiploid populations, a possible indicator of genetic instability and subsequent predisposition to metastasis. Because of conflicting studies in the literature, the clinical relevance of both of these potential prognostic markers has been questioned for the management of breast cancer patients. The purposes of this study are to present a set of 10 adjustments derived from a single large study that optimizes the prognostic strength of both DNA ploidy and S-phase and to test the validity of this approach on two other large multicenter studies. Ten adjustments to both DNA ploidy and S-phase were developed from a single node-negative breast cancer database from Baylor College (n = 961 cases). Seven of the adjustments were used to reclassify histograms into low-risk and high-risk ploidy patterns based on aneuploid fraction and DNA index optimum thresholds resulting in prognostic P values changing from little (P < 0.02) or no significance to P < 0.000005. Other databases from Sweden (n = 210 cases) and France (n = 220 cases) demonstrated similar improvement of DNA ploidy prognostic significance, P < 0.02 to P < 0.0009 and P < 0.12 to P < 0.002, respectively. Three other adjustments were applied to diploid and aneuploid S-phases. These adjustments eliminated a spurious correlation between DNA ploidy and S-phase and enabled them to combine independently into a powerful prognostic model capable of stratifying patients into low, intermediate, and high-risk groups (P < 0.000005). When the Baylor prognostic model was applied to the Sweden and French databases, similar significant patient stratifications were observed (P < 0.0003 and P < 0.00001, respectively). The successful transference of the Baylor prognostic model to other studies suggests that the proposed adjustments may play an important role in standardizing this test and provide valuable prognostic information to those involved in the management of breast cancer patients.

The analysis of chromosomal imbalances in solid tumors using comparative genetic hybridization (CGH) has gained much attention. A survey of the literature suggests that CGH is more sensitive in detecting copy number aberrations than is karyotyping, although careful comparisons between CGH and cytogenetics have not been performed. Here, we compared cytogenetics and CGH in 29 invasive breast cancers after converting the karyotypes into net copy number gains and losses. We found 15 tumors (56%) with a significant agreement between the two methods and 12 tumors (44%) where the methods were in disagreement (two cases failed CGH analysis). Interestingly, in 13 of the 15 tumors where the two methods were concordant, there was also a strong correlation between chromosome index and DNA index by flow cytometry. In the opposite situation, i.e., when chromosome and DNA indices were not matching, there was disagreement between cytogenetics and CGH in 10 of the 12 tumors. Of the discordant cases, all except one had a "simple" abnormal karyotype. Unresolved chromosomal aberrations (marker chromosomes, homogeneously staining regions, double minutes) could not completely explain the differences between CGH and karyotyping. A likely explanation for the discrepancies is that the methods analyzed different cell populations. Gains and losses found by CGH represented the predominant (often aneuploid) clone, whereas the abnormal, near-diploid karyotypes represented minor cell clone(s), which, for unknown reasons, had a growth advantage in vitro.

Samples from 48 benign and 102 malignant bone and soft-tissue tumors were analyzed cytogenetically and by DNA flow cytometry. Clonal chromosome abnormalities were found in 82 tumors and normal karyotypes in 68; 61 tumors were DNA-non-diploid and 89 were diploid. The cytogenetically abnormal tumors were used for comparison between the 2 types of investigation; 45 of these tumors were DNA-diploid and 37 were DNA-non-diploid. There was, with few exceptions, good correspondence between the quantitative estimates of genomic changes by the 2 methods, indicating that the cells cytogenetically analyzed from short-term cultures are representative of the in vivo cell populations. Discrepancies were primarily found in cases with indexes above 1.5, in which the DNA index was higher than the chromosome index. The chromosome analysis suggested that skewed stemline (G0/G1) peaks in the diploid region in DNA histograms indicate the presence of cell populations with small net quantitative genomic changes, although not all such populations were detected by DNA flow cytometric analysis. The view that one of the peaks in bimodal stemline DNA histograms with narrow peaks represents a non-diploid cell population was also corroborated. On average, the cell populations giving rise to double stemlines in DNA histograms showed quantitatively larger genomic changes than those that gave rise to broad or skewed diploid G0/G1 peaks. The findings indicate that these histogram profiles are not artifactual but reflect chromosomal changes in the tumor parenchyma.

Flow cytometric assessments of DNA ploidy status and the S-phase fraction (SPF) have been shown to yield prognostic information in breast cancer. The aim of the present investigation was to elucidate the reproducibility of results with regard to tumor DNA heterogeneity, and to ascertain whether the prognostic value of DNA measurements might be enhanced by analyzing two pieces of a tumor instead of one. Agreement with regard to ploidy status (diploid versus non-diploid) was obtained in 90% of cases (71/79) when two adjacent sections of the tumor were investigated, and in 77% of cases (10/13) when four biopsies from different quadrants of the tumor specimen were investigated. The corresponding figures for agreement in SPF (divided into three categories, less than 7.0%, 7.0-11.9%, and greater than or equal to 12%) were 75% (59/79; 2-sample series) and 55% (7/13; 4-biopsy series). The main reason for variance in ploidy results was the difficulties in distinguishing near diploid cell populations. Discrepancies in SPF categories could be explained by minor fluctuations in SPF values near the cut-off levels, or by variance in ploidy status, the fraction of non-diploid nuclei, and background noise due to cell debris. There was a stepwise increase in recurrence rate (RR) among patients with increasing SPF category (RR: 20%, 41%, and 53%). Patients whose SPF categories varied, from low or intermediate in one part of the tumor to high in another, seemed to have a poor prognosis (RR = 57%).(ABSTRACT TRUNCATED AT 250 WORDS)

In this study, two different methods of estimating cell proliferation were compared: cell loss and potential growth rate of xenografted head and neck cancer grown in nude mice based on the detection of DNA incorporation of bromodeoxyuridine (BrdUrd) in one method, and [3H]thymidine ([3H]TdR) in the other. The 21-d-old xenografts were labelled in vivo, either with BrdUrd or [3H]TdR and excised at intervals during 65.5 h. In tumors containing BrdUrd, the percent labelling was measured in mid-S and mid-G1 phase windows of cytograms from bivariate DNA flow cytometry (FCM). In [3H]TdR-labelled tumors, the percent labelled mitoses (PLM) was determined by light microscopy evaluation of autoradiographs. With a computer program based on a theoretical model, the percent labelling versus time after injection was used to analyze cell cycle time, cell loss, tumor growth fraction, and potential doubling time. The values calculated from DNA incorporation with BrdUrd agreed well with those obtained from labelling with [3H]TdR, i.e., cell cycle time 2.3 vs. 2.4 d, and growth fraction 67 vs. 70%. The estimated potential doubling time was 3.1 d and cell loss factor 40% by both methods. Flow cytometry analysis of BrdUrd-labelling is considerably faster than the evaluation of [3H]TdR-labelling, and the present results provide further support for the BrdUrd labelling method as a promising alternative to the PLM method in cell cycle studies designed to evaluate the relevance of cell proliferative properties in relation to biological behavior in xenografted head and neck cancer.

Measurements of DNA ploidy can be performed either with image cytometry (ICM) or flow cytometry (FCM); both methods provide independent prognostic information in primary breast cancer. The aim of the present investigation was to compare the two methods and to relate the findings to prognosis (median follow-up 42 months). Concordance in ploidy status (diploid, tetraploid, aneuploid) was obtained in 76% of the samples (168/222). When the fraction of S-phase cells (SPF) from FCM analysis was also taken into consideration, four different groups of samples were obtained (Flow I-IV), which were considered to correspond to the Auer classification (Auer I-IV) of DNA histograms obtained from image cytometry. Complete concordance between the two techniques now was 70% (155/222). Samples classified as Flow I (diploid or near-diploid with low SPF) and Auer I had a distant metastasis rate of 3/60 (5%), as compared to 62/154 (40%) for all other combinations of the Flow and Auer classifications taken together. Thus, the only findings of prognostic importance were that some samples were Flow I but not Auer I, or vice versa. These two groups represent 17 (7.7%) and 14 (6.3%), respectively, of the total number of samples, and had frequencies of distant metastasis similar to those of the other high-risk groups, namely, 7/17 and 5/14, respectively. In a multivariate analysis, flow cytometric S-phase value was a stronger prognostic factor than either the Flow and Auer classification. We conclude that when routine FCM DNA analysis is used, diploid or near-diploid samples with a low S-phase value should be reanalyzed with ICM.

The effect of EBV-conversion of two EBV-negative lymphoma lines (Ramos and BJAB) on agarose clonability and tumorigenicity in nude mice was explored. The cloning frequency was increased in all 9 sublines investigated, between 1.1 and 4.9 times compared to the original "parental" lines. Tumorigenicity was increased in one out of 2 BJAB-derived, EBV-positive lines and in 3 of 6 Ramos-derived lines, while it was decreased in 3 others. A strong positive correlation between the number of genomes/cell and the amount of EBNA/cell was detected, and in 7 of 9 converted lines the cloning frequency also correlated to the number of EBV genomes. On the other hand, no relation was established between the number of EBV-genomes/cell, the amount of EBNA/cell and tumorigenicity.

Flow-cytometric DNA analyses of single cell nuclei were performed on nuclear suspensions prepared from biopsies of primary breast carcinomas in 638 patients. Propidium-iodide-stained cell nuclei were analysed in an Ortho 50-H Cytofluorograph. The patients were staged by the TMN classification. Sixty percent of all the patients had aneuploid primary breast carcinomas; of size T1, T2, T3, and T4 tumors, 51%, 63%, 67%, and 75% were aneuploid, respectively. The proportions of aneuploid tumors in each of the tumor stages SI, SII, SIII, and SIV were 47%, 62%, 67%, and 69%, respectively. This trend to increasing aneuploidy proportions with more advanced disease was significant in contrast to the degree of aneuploidy found in relation to axillary nodal tumor involvement. Multiple aneuploid cell populations were found in 109 (17%) tumors. With a mean follow-up time of 16 months, 92 patients have relapsed out of 540 completely staged patients with unilateral breast cancer with no distant metastases at the time of initial treatment. When the influence of various treatments and tumor stage are not considered, the recurrence rate was twice as high among patients with aneuploid primary tumors than among patients with euploid tumors. The differences in relapsing rates among patients with euploid and aneuploid primary tumors decreased with more advanced disease. Out of 170 patients with T1 tumors, 17 relapsed and 16 of these were aneuploid. No such difference in relapse rate in relation to ploidy was, however, found in patients with more advanced primary disease.

Cell kinetic studies are of interest for clarifying the concepts of chemotherapeutic strategy in the multimodality therapy of advanced squamous-cell carcinoma of the head and neck. A poorly-differentiated squamous-cell carcinoma of the head and neck heterotransplanted to nude mice was used for analyses of chemotherapeutically induced cell cycle perturbations. The heterotransplanted tumour, in its 15th or later passages in nude mice, was treated with either Mitomycin C or cisplatin. After determination of dose-response relationships and toxicity, treated tumours were biopsied at different times and cell cycle distribution pattern, 3HTdR incorporation into DNA, histology and tumour volume were recorded. Mitomycin C and cisplatin gave the same pattern of cell cycle perturbations, although the changes induced by cisplatin were more profound. There was an initial increase of the fraction of cells in the S phase, concomitant with a reduction of the fraction of cells in G0 + G1 phase. When these perturbations were normalized a transient increase of the fraction of cells in G2 + M phase was observed. However, while cisplatin caused an initial transient depression of DNA synthesis, the Mitomycin-C-treated tumours exhibited a short-lasting increase of DNA-synthesis. The maxima of the induced changes in cell cycle phase distribution and DNA-synthesis lasted for only 24-48 h, which may be of importance for scheduling combinations of drugs. Though both drugs induced profound changes in tumour volume growth and cell kinetics, there was no change in the histopathological picture of the treated tumours. Routine histopathological examination is thus not likely to be of value evaluating the effect of chemotherapy.