Heinz Baisch

Analysis of PCP-Data to Determine the Fraction of Cells in the Various Phases of Cell Cycle*

SummaryMathematical models for the analysis of pulse-cytophotometric (PCP) data are described. With computer programs based on these models the fractions of cells in G1-, S- and(G2 + M)-phases are obtained. The methods are applied to PCP measurements of Ehrlich ascites tumor cells, human bone marrow cells and L-929-cells in culture. The results of the L-cell experiment are compared with autoradiographic results; for both methods the duration of the various phases has been calculated. Two different mathematical models for PCP-data evaluation and the autoradiographic method yielded results agreeing within statistical error. The application of the two models on different types of DNA-histograms is discussed: One model is suitable for asynchronous cell populations with a low fraction of S-phase cells, the other can be applied for partially synchronized cells and high S-phase fractions as well.

Prognostic factors in medullary thyroid carcinomas. Survival in relation to age, sex, stage, histology, immunocytochemistry, and DNA content

Patients with medullary thyroid carcinomas (MTC) were analyzed according to age, sex, and tumor stage. In addition, the MTC were screened for the predominant histologic pattern, immunocytochemical spectrum (60 tumors), and DNA content (DNA cytophotometry and DNA flow cytometry, 25 tumors). These findings were correlated with follow‐up data available for 45 of these patients. Forty‐eight percent of the tumors revealed a polygonal cell pattern, whereas 22% showed spindle‐cell predominance. All tumors contained cytokeratin, chromogranin A, and calcitonin (CT). Calcitonin gene‐related peptide (CGRP) was present in 92%, carcinoembryonic antigen (CEA) in 77%, neuron‐specific enolase (NSE) in 75%, and vimentin in 53% of cases. Positivity for neurotensin, somatostatin, neurofilaments, bombesin, and alpha human chorionic gonadotropin (a‐hCG) and serotonin ranged between 3% and 27%. All MTC were negative for substance P, adrenocorticotropic hormone (ACTH), thyroglobulin (TG), or S‐100 protein. Local recurrences and regional lymph node metastases revealed identical staining patterns as the primaries. Prognosis of MTC was found not to be related to histologic features (dominant architectural pattern, cellular shape, presence of amyloid deposits) or immunocytochemical pattern. Instead, survival was significantly correlated to age, sex, and stage of disease. The best prognosis was seen in women younger than 40 years and revealing an early stage of disease. DNA measurements added valuable information in assessing the prognosis of MTC.

A comparison of mathematical methods for the analysis of DNA histograms obtained by flow cytometry

Abstract. Twelve methods for analysing FCM‐histograms were compared using the same set of data. Some of the histograms that were analysed were simulated by computer and some were taken from experiments. Simulated data were generated assuming asynchronously growing cell populations and (i) measurement coefficients of variation (CV) from 2 to 16%; (ii) constant measurement CV or CVs increasing from G1 to G2 phase, and (iii) varying fractions of cells in each phase. Simulated data were also generated assuming synchronous cell populations in which a block in early S phase was applied and released. DNA histograms were measured for L‐929 cells at various times after mitotic selection. Labelling indices were also measured for these cells at the same time.

Tumor Cell Deoxyribonucleic Acid Content and Prognosis in Human Renal Cell Carcinoma

Flow cytometry was used to study tumor tissue from 68 patients with stages I to III renal cell carcinoma who were followed for 1 to 4 years. Results of flow cytometry correlated extremely well with the clinical course of the patients: 21 per cent of those with diploid tumor cells had metastases during the interval of observation, compared to 89 per cent of those with aneuploid tumor cells. We concluded that flow cytometry helps to identify at the time of radical nephrectomy patients who are most likely to suffer recurrent tumor and, therefore, are candidates for early chemotherapy. If nuclear grading is applied in addition to flow cytometry the rates of false positive and false negative results in respect to prediction of prognosis are reduced to 3 and 14 per cent, respectively.

Impact of overall treatment time of fractionated irradiation on local control of human FaDu squamous cell carcinoma in nude mice

A series of experiments were performed to determine the impact of overall treatment time on local control of human FaDu squamous cell carcinoma irradiated with 30 fractions under ambient conditions in nude mice. The TCD50 increased with treatment time between 15 days and 10 weeks from 43 Gy to 102 Gy. The data can be well described by a single linear function. The dose recovered per day is 1.0 Gy. However, the data can also be adequately fitted by two components with an initial delay of about 30 days followed by a steep increase at a rate of 1.5 Gy per day. Assuming that the increase of TCD50 is solely caused by repopulation of clonogenic tumor cells, and that the cellular radiation sensitivity in vitro reflects the radiation sensitivity of FaDu cells in vivo, the doubling time of clonogenic tumor cells during treatment is estimated to be approximately 1.8 days for the one-component model and, after an initial delay, approximately 1.2 days for the two-component model. Both values are shorter than the doubling time of clonogenic cells in untreated FaDu tumors and similar to the potential doubling time determined by flow cytometry after BrdUrd labelling. It is concluded that the dose necessary to control FaDu squamous cell carcinoma increases considerably with increasing time of a fractionated radiation treatment. It appears most likely that this increase is caused by repopulation of clonogenic tumor cells; however, other mechanisms such as an increasing fraction of hypoxic tumor cells can not be ruled out at present.

Prognosis of renal cell carcinoma related to nuclear grade, DNA content and Robson stage.

In 135 renal cell carcinomas (RCCs), nuclear grade, Robson stage and DNA content, determined by flow cytometry, were related to the 3-year survival rates of the respective patients. All three parameters showed prognostic value, nuclear grading being most effective. Combined evaluation of the parameters showed that staging and also DNA determination add supplementary prognostic information. As the four nuclear grades clearly uncover the biological potential of RCC, they are regarded as the most significant criteria for the prognostic assessment of RCC.

Transplantation of Human Renal Cell Carcinoma Into NMRI NU/NU Mice. I. Reliability of an Experimental Tumor Model

Human renal cell carcinomas of 20 consecutive patients were obtained by radical nephrectomy and were transplanted into nu/nu mice (NMRI). All tumors that were not pretreated were accepted without selection and were identical with the original tumor in morphology and chromosomal modes and usually in DNA content per cell. The growth rate of these tumors after transplantation correlated well with the clinical course of the corresponding patients and was strongly influenced by pretreatment with radiation therapy. This proves the reliability of such an experimental tumor model to study adjuvant therapy in renal cell carcinoma.

DNA synthesis and cell cycle progression of synchronized L-cells after irradiation in various phases of the cell cycle.

SummaryThe varying sensitivity to radiation in the different phases of the cell cycle was investigated using L-929 cells of the mouse. The cells were synchronized by mechanical selection of mitotic cells. The synchronous populations were X-irradiated with a single dose of 10 Gy in the middle of the G1-phase, at the G1/S-transition or in the middle of the S-phase, respectively. The radiation effect was determined in 2 h intervals a) by14C-TdR incorporation (IT) into the DNA, b) by autoradiography (AR), c) by flow cytometry (FCM). The incorporation rate decreased in all three cases, but the reasons appeared to be different, as can be derived from FCM and AR data: After irradiation in G1, a fraction of cells was prevented from entering S-phase, after irradiation at G1/S a proportion of cells was blocked in the S-phase, and after irradiation in S, DNA synthesis rate was reduced. As a consequence of these effects, the mean transition time through S-phase increased. The G2 blocks, obtained after irradiation at the three stages of the cycle were also different: Cells irradiated in G1 are partly released from the block after 10 h. Irradiation at G1/S caused a persisting accumulation of 50% of the cells in G2, and for irradiation in S more than 80% of the cells were arrested in G2.

Nuclear DNA content in 27 pancreatic endocrine tumours: Correlation with malignancy, survival and expression of glycoprotein hormone alpha chain

Paraffin-embedded tissue from resection specimens of 14 functioning and 13 nonfunctioning pancreatic endocrine tumours (PET) was analysed for nuclear DNA content by image cytometry. Data on follow-up (mean 5.5 years) were available in all patients. DNA histograms with a diploid pattern were found in 13 (48%) tumours, while an aneuploid pattern was seen in the remaining 14 tumours (52%). Six (40%) of the diploid tumours and 9 (60%) of the aneuploid tumours were malignant. Survival was shorter in patients with malignant and aneuploid PET (mean 3.5 years, range 0.5–7) than in those with malignant and diploid PET (mean 5.7 years, range 3–8). Human chorionic gonadotropin-alpha was expressed in 3 of 12 benign PET, with 1 being aneuploid, and 6 of 15 malignant PET, with 4 being aneuploid. We conclude from these results that the ploidy pattern of PET allows no discrimination between benign and malignant tumours but may provide prognostic information on the aggressiveness of malignant PET.

Intratumoral heterogeneity of S phase transition in solid tumours determined by bromodeoxyuridine labelling and flow cytometry.

Abstract. Cell kinetics of human renal cell carcinomas xenotransplanted into nu/nu mice were analysed using the bromodeoxyuridine (BrdUrd) labelling method. Tumours were removed 0.5–14 h after injection of the BrdUrd solution. The tumour cells were stained with fluorescein isothiocyanate conjugated anti‐BrdUrd antibodies and propidium iodide (DNA content). From the flow cytometry data the relative movement was calculated. Relative movement data of variable intervals after BrdUrd labelling were subjected to a fit procedure using log‐normal distributions for S phase transition (Ts). The log‐normal distributions were modified by inflation factors in order to get extremely asymmetric distributions. The best fits to the experimental data were obtained using wide asymmetric Ts distributions, indicating that progression through S phase in solid human tumours is considerably heterogeneous. This implies that the potential doubling time (Tpot) is longer than calculated from a single measured relative movement value obtained a few hours after BrdUrd labelling.