Ulla Møller

DNA ploidy-characteristics of human malignant melanoma analysed by flow cytometry and compared with histology and clinical course.

SummaryFlow cytometric analysis of nuclear DNA content is valuable for indicating ploidy- and proliferation abnormalities in surgically removed human malignant melanomas. In 35 primary cutaneous melanomas, 20 metastases of melanoma in skin and lymph nodes, and 16 nevi the DNA distribution was analyzed by flow cytometry and compared with a variety of histological parameters and the subsequent clinical course.Heteroploid DNA distributions with increased polyploid or aneuploid fractions were found in 26 primary melanomas (74%), 14 metastases (70%), and 4 nevi (25%) indicating tumor clones with an abnormal nuclear DNA content. Three or more cell clones in a single biopsy was found in 10 primary melanomas, 2 metastases, and 1 nevus.The frequency of heteroploidy was significantly higher in primary and secondary melanomas than in nevi (p< 0.001) and was correlated significantly with a high mitotic acitivity (p<0.002), marked nuclear pleomorphism (p<0.01), large nucleoli (p<0.01) and a thickness of the primary melanoma of more than 2.25 mm (p<0.02). Such histologic findings in malignant melanomas have been shown previously to be correlated with a bad prognosis. No significant correlation was found between heteroploidy and the histologic type of melanoma or the level of invasion. A 2-year clinical follow-up showed that more patients died from melanoma if the DNA distribution in the primary or secondary melanoma was heteroploid (6/26; 23% and 8/13; 62% respectively) than if it was diploid (0/9; 0% and 2/5; 40% respectively). However, the differences were not statistically significant.It is concluded that heteroploidy 1) is not an absolute criterion of malignancy, 2) is significantly correlated with histologic features indicating marked cellular anaplasia, and 3) is apparently correlated with a bad prognosis.

THE INFLUENCE OF INJECTED TRITIATED THYMIDINE ON THE MITOTIC CIRCADIAN RHYTHM IN THE EPITHELIUM OF THE HAMSTER CHEEK POUCH

The initial effect of an injection of TdR‐5‐3H (1 μCi/g body weight; 6 Ci/mmol) in the cheek pouch epithelium of the Syrian hamster is an increase in the mitotic index. The increase is observed 1–5 hr after injection, depending upon the time of day when the injection is given, and is followed by compensatory variations in mitotic index. This deviation from the normal circadian rhythm in the mitotic index appears to depend on the fraction of G2‐cells at the time of injection. The main effect is a shortening of tg2. No effect is observed after injection of non‐radioactive TdR or isotonic saline. The results of the present experiment emphasize that unexpected results may be obtained when using mitotic indices from animals labelled with 3H‐TdR, as well as the risks of using the PLM‐method in a partially synchronized system.

The Circadian Variations In the Eithelial Growth of the Hamster Cheek Pouch: Quantitative Analysis of Dna Distributions*

The pronounced diurnal rhythm in DNA distribution of the hamster check pouch epithelium both in the S fraction and in the (G2+ M) fraction was compared with previous studies of the changes in tritiated thymidine labelling index and mitotic activity. the DNA distributions were obtained by flow cytometry after ultrasonic disaggregation of the isolated epithelium into a suspension of single nuclei. the DNA distributions were analysed with the computer program of J. Fried (1976) and by planimetry. the S fraction was higher than the autoradiographic labelling index during the whole 24 hr period. Only the computer fitted S fraction and the labelling index had the same difference between maximal and minimal values, and maxima at the same time of day. the DNA distributions showed a diurnal release of G1 cells into S phase proceeding through (G2+ M) phase and returning to G1 phase within a 24 hr period.

DNA FLOW CYTOMETRY OF ISOLATED KERATINIZED EPITHELIA: A METHODOLOGICAL STUDY BASED ON ULTRASONIC TISSUE DISAGGREGATION

Ultrasonication of keratinized, stratified, squamous epithelium, which had been separated from underlying tissue by means of acetic acid, resulted in disaggregation of all cellular layers in the epithelium, giving a suspension of single nuclei with mitoses preserved. This suspension was treated with RNAse and ethidium bromide for analysis by flow cytometry. From the resulting DNA histogram the G1, S and G2+ M fractions were estimated using the computer program of Fried (1976). Treatment with dithiothreitol before sonication increased the yield of nuclei in suspension and decreased the amount of debris and clumps, thereby suppressing overestimation of small S fractions.

Clonal heterogeneity in curetted human epidermal cancers and precancers analysed by flow cytometry and compared with histology.

DNA frequency distributions analysed by single nuclei flow cytometry were studied in sixty‐five curetted human epidermal tumours, i.e. five actinic keratoses (AK), seven Bowens diseases (BO), nine squamous cell carcinomas (SCC), forty‐three basal cell carcinomas (BCC) and one baso‐squamous carcinoma (BSC). Seventy‐five per cent (16/21) of the samples with squamous cell differentiation (AK, BO and SCC) showed features suggestive of more than one stem cell population, against 24% of the pure BCC samples (11/43).

DNA Flow cytometry of human epidermal tumours. Intra- and intertumour variability in ploidy and proliferative characteristics

SummaryTumour ploidy and proliferative characteristics can be estimated by flow cytometric measurements of the nuclear DNA content. This study considers the question whether human epidermal tumours are intrinsically homogeneous with regard to these properties, i.e. whether a single biopsy analysed by flow cytometry is representative of the entire tumour. Analyses of multiple biopsies from ten human epidermal tumours — two kerato-acanthomas (KA), two basal cell carcinomas (BCC), two basosquamous carcinomas (BSC), one Bowen’s disease (BO) and three squamous cell carcinomas (SCC) — indicated that both ploidy and proliferation characteristics were reproducible and specific for theperipheries of the different tumours regardless of the histopathologic diagnosis. The tumourcentres, however, may deviate considerably from the corresponding periphery. None of the ten tumour peripheries contained more than one cell clone, but six of the ten clones were aneuploid. Both the BO and the KA’s were hypodiploid, while one SCC, one BSC and one BCC were hyperdiploid as assessed in their peripheries. The remaining BSC was diploid in its periphery, while both a hypodiploid and a hypotetraploid cell clone were found in the corresponding centre.

Circadian variations in influx and efflux of the S phase in a partially synchronized cell system Double‐labelling with [3H]thymidine in the epithelium of the hamster cheek pouch

Abstract. By means of a double‐labelling experiment, circadian variations in the kinetic parameters of the S phase of the hamster cheek pouch epithelium were studied. The evaluation of the experiment included a recently developed correction for deviations from the strict pulse interpretation of the labelling technique.

MITOTIC INDEX, INFLUX AND MEAN TRANSIT TIME IN THE HAMSTER CHEEK POUCH EPITHELIUM, A PARTIALLY SYNCHRONIZED CELL SYSTEM*†

Mitotic activity was followed in the epithelium of the hamster cheek pouch for about 12 hr in two experiments under different conditions of noise and light intensity.

Subclassification of cells in S phase in a partially synchronized cell system

Abstract. A circadian dependent delay in the incorporation of [3H]TdR into DNA, presumably due to variations in the intracellular pool of [3H]TdR derivatives, was found. It seems reasonable to relate this effect to a circadianally varying age distribution of cells in S phase.

DNA flow cytometry on human epidermis. The effect of serial biopsy sampling at various times

The effect of serial biopsy sampling on S‐ and G2+M‐fractions in lower abdominal skin from leg ulcer patients was studied by single‐nuclei DNA flow cytometry. Significant differences in both measurements were found in four biopsies taken at equal intervals within 24 h, but not in four biopsies taken at noon on four consecutive days. The variation in S‐fractions was closely related to the injury from previous biopsy sampling(s) while the G2+M‐fraction variation tended to follow the time of the day. The study indicated local as well as systemic effects from previous biopsy sampling.