Cell Kinetic Measurements Research Articles

The predictive value of cell kinetic measurements in a European trial of accelerated fractionation in advanced head and neck tumors: An interim report

The value of cell kinetic measurements in head and neck tumors in predicting which patients will benefit from accelerated fractionation radiotherapy regimens is being tested in a multicenter European trial (EORTC trial 22851). This paper reports on the first analysis of the correlation of kinetics with outcome in this trial. A proportion of patients in both the accelerated arm (72 Gy in 5 weeks, 1.6Gy per fraction, 45 fractions) and the conventional arm (70–72 Gy in 7–8 weeks, 1.8–2.0 Gy per fraction, 35–40 fractions) were given an i.v. injection of 100 mg/m2 IUdR (iododeoxyuridine) before treatment, and a tumor biopsy was taken several hours later. The potential doubling time of the tumor (Tpot) was obtained from a flow cytometric analysis of tumor cell nuclei using an anti-IUdR antibody. From a total of 260 patients entered in the trial, 53 have undergone kinetic analysis. Adequate IUdR labeling was seen in 47 patients (88.7%), from which the mean value for Tpot was found to be 4.5 ± 2.5 days (±S.D.). Of the IUdR labeled patients, 30 have now been followed up for at least 1 year, 17 with conventional and 13 with accelerated radiotherapy. These patients were split into those with fast and those with slowly growing tumors, the dividing line being the median Tpot value of 4.6 days. After conventional 7-week radiotherapy, 2 of 6 patients with “fast” growing tumors obtained local control compared with 8 of 11 with “slow” growing tumors. A smaller difference in local control was seen been fast and slow tumors in the accelerated arm (59 vs. 34). These preliminary data support the hypothesis that patients with fast growing tumors do poorly with conventional radiotherapy and that pretreatment kinetic measurements can select patients at risk. The predictive power of the method must await the final analysis of trial results.

Rapid S-phase determination of non-Hodgkin's lymphomas with the use of an immunofluorescence bromodeoxyuridine labeling index procedure.

Cell kinetic measurements are currently being investigated to determine if they are useful in the clinical management of patients with non-Hodgkin's lymphomas (NHLs). Although the tritiated thymidine labeling index (TLI) is the standard method of S-phase measurement, it is difficult to perform. The authors describe a slide-based immunofluorescence labeling index (LI) method that uses 5-bromo-2-deoxyuridine (BrdUrd) as the pulsing medium and a monoclonal antibody (BU-1) to BrdUrd. The BrdUrd LI was performed on 217 NHLs and compared with routine histologic results. The authors found a median BrdUrd LI of 0.9% for low-grade NHLs; 7.5% for intermediate-grade; 10.4% for high-grade; and 2.2% for T-cell NHLs. This method provides a rapid, reliable S-phase measurement that can be easily performed in the clinical laboratory. It should replace the TLI and allow wider application of S-phase measurements in the NHL.

Measurement of cell kinetics in human tumours in vivo using bromodeoxyuridine incorporation and flow cytometry

The proliferative potential of human solid tumours, in vivo, was investigated using bromodeoxyuridine (BrdUrd) incorporation and flow cytometry (FCM). Patients with solid tumours from a variety of sites were injected with 500 mg BrdUrd, intravenously, several hours prior to biopsy or surgical excision. The labelling index (LI), duration of S-phase (Ts) and thus the potential doubling time (Tpot) could be measured within 24 h of sampling. The results show that both the LI and Ts vary greatly between tumours (Ts ranges from 5.8 to 30.7 h). However, within this study of 26 evaluable patients, tumours of the same tissue origin tended to have similar Ts values. Melanomas had the shortest Ts (8.8 h), nine patients with head and neck cancer had Ts values ranging from 5.8 to 18.8 h (median 12.5 h). The longest Ts values (24 h) were found in lung and rectum. The estimates of Tpot ranged from only 3.2 days in an oat cell carcinoma to 23.2 days in a lymphoma. The striking feature of the study was that 38% of the tumours had a potential doubling time of 5 days or less. We found no relationship between proliferation and histopathological differentiation or DNA ploidy. It should now be possible to assess the prognostic significance of pretreatment cell kinetic measurements which may, in the future, aid in the selection of treatment schedules for the individual patient.

Proton NMR relaxation times of experimental Lewis lung carcinoma after irradiation

NMR proton spin-lattice ( T 1) and spin-spin ( T 2) relaxation times were measured ex vivo on Lewis lung carcinoma after in vivo single irradiation with an absorbed dose of 4 Gy. The results were compared to tumoural volume evolution, pathological examinations, and cell kinetic measurements. Tumour growth decreased between the third and the sixth day after irradiation while relaxation times, especially T 1, is increased 2 days before the clinical recurrence of the tumour is observed. Pathological morphometric measurements tempted to show that necrosis is less extended after irradiation. Cell cycle analysis demonstrated the G2/M phase blockade by radiation after one day, and its release 4 days later. These phenomena could be important for in vivo radiotherapy follow-up using determination of relaxation times by magnetic resonance imaging (MRI).

Clinical response and tissue effects of etretinate treatment of patients with solar keratoses and basal cell carcinoma

Patients with basal cell carcinoma and solar keratoses were treated with etretinate. Substantial and prolonged clinical improvement was seen. All patients with solar keratoses showed a decrease in the mean area and number of lesions and eight patients demonstrated complete healing clinically. Two patients experienced recurrence at 9 months after completion of treatment. Histometric and cell kinetic measurements on the epidermis of skin samples from these patients were performed. They revealed epidermal thickening and increased deoxyribonucleic acid (DNA) synthesis both in lesions and in clinically uninvolved skin following treatment. Assessments were also made of enzyme activities in lesions and uninvolved skin with the use of established quantitative cytochemical techniques. Significant reduction in levels of succinic dehydrogenase activity following etretinate treatment was detected in solar keratoses and in basal cell carcinomas. This was also the case for uninvolved skin of patients with solar keratoses. Glucose-6-phosphate dehydrogenase (G6PD) activity was significantly reduced following etretinate treatment in solar keratoses and in basal cell carcinomas, but uninvolved skin did not exhibit significant changes. These changes are in contrast to those previously reported in normal subjects, where the activity increased, but are similar to those observed in patients with ichthyotic disorders. The alterations in the cytochemical profile following administration of etretinate in the lesions of patients reported here are consistent with the view that the drug promotes a more normal pattern of epidermal differentiation. We favor the view that etretinate's antineoplastic action is exerted by preferentially allowing differentiation of normal epidermal cells and inhibiting dysplastic cells.

Cell kinetic measurements in in vivo human cancers—The effect of surgical debulking and chemotherapy

Cell kinetic measurements in in vivo human cancers—The effect of surgical debulking and chemotherapy

The effects of an aromatic retinoid (etretinate) on epidermal cell production and metabolism in normal and ichthyotic patients.

Normal subjects and ichthyotic patients were biopsied before and after etretinate (Tigason) therapy. Histometric and cell kinetic measurements showed minimal changes following treatment. The metabolic activity across the epidermis was measured by a novel approach using the Quantimet 720 image analyser, and was significantly increased in the normal subjects as demonstrated by glucose-6-phosphate dehydrogenase (G6PDH), succinic dehydrogenase (SDH) and non-specific esterase (NSE) activities. In the ichthyotics studied the levels of G6PDH and SDH activity were significantly lowered following etretinate therapy, to levels similar to those demonstrated in normal subjects. In contrast, the activity of NSE was increased in the ichthyotics after etretinate therapy. It is suggested that the effect of retinoids is to 'normalize' the process of epidermal differentiation when it is abnormal.

A monoclonal antibody reactive with 5-bromo-2-deoxyuridine that does not require DNA denaturation.

We describe a mouse monoclonal antibody (BU-1) reactive with 5-bromo-2-deoxyuridine (BrdUrd). The antibody is different from previously described BrdUrd monoclonal antibodies in that BU-1 does not require pretreatment of cells with strong DNA denaturants in order for the antibody to react with BrdUrd incorporated in the DNA. The antibody can be used in immunocytochemical and indirect immunofluorescent assays and can be used to identify cells that have incorporated BrdUrd. Double staining with BU-1 antibody and propidium iodide has been used to confirm S-phase measurements with the BU-1 antibody. Immunocytochemical stains using the BU-1 antibody do not destroy cell morphology and allow cell identification to be performed simultaneously with S-phase measurements. Flow cytometer two-color fluorescence analysis allows the simultaneous identification of cell surface or cytoplasmic markers and S-phase quantitation. The BU-1 antibody should broaden the application of cell kinetic measurements to individual elements of cell populations that are heterogeneous with respect to morphology, surface marker, and other biological features.

Flow cytometry of keratinocytes

A prerequisite for using flow cytometry (FCM) is the availability of isolated single cells. Procedures for separation and isolation of keratinocytes from animals and man are available, and the resulting single cell suspensions have been subjected to FCM measurements. The major advantage of the method is the accuracy and speed with which a variety of cellular constituents can be quantified. FCM of keratinocytes has, hitherto, been mainly confined to measurements of nuclear DNA for estimation of cell-cycle distributions and for ploidy studies. In mouse epidermis, cell-cycle distributions were estimated from sequentially obtained DNA histograms and evaluated with other cell kinetic measurements, resulting in new information about epidermal cell-cycle progression, not achievable by any of the methods alone. The best way, therefore, to increase our knowledge of keratinocyte proliferation, is the combined use of DNA FCM and other cell kinetic methods. DNA FCM has also been applied to healthy and diseased human epidermis, and may add valuable information to the classification of skin disease in selected cases. It is believed that further progress in the characterization of keratinocyte growth and development will depend on parameters other than DNA alone.

Cell kinetic measurements of human tumors

Cell kinetic measurements of human tumors

Proliferation controls in a linear growth system: Theoretical studies of cell division in the cartilage growth plate

The columnar arrangement of dividing cells in the epiphyseal cartilage plates of growing bones provides a model of a linear proliferation system. One factor which determines the rate of cell production, and hence the rate of growth, is the size of the proliferating population. In this one dimensional system this size is equal to the length of the proliferation zone. Two possible mechanisms for a differentiation control that sets a limit to the length of this zone have been tested in computer simulations. While a diffusion gradient control is consistent with cell kinetic measurements a division limit based on an inheritable growth substance is shown to require further development before the model fits experimental data. Cell division in the columns produces linear clones of cells. If the final length of a bone is set by a limit on the number of divisions that the cartilage stem cells can make, then the number of cells per clone is crucial in determining overall bone growth. The parameters that affect linear clone size have been investigated in computer simulations. Clone size depends largely on the relative division rate of stem cells to proliferation zone cells — but the data on stem cell division rates are generally unreliable. The analysis could be applied to other linear proliferating systems.

Human colonic tumor cell kinetics.Potential for therapy

Using new in vitro techniques developed at the Cancer Research Unit, cell kinetic measurements were obtained in primary and metastatic human colonic tumors, polyps and normal bowel that did not require in vivo 3HTdR and required only single samples of tissue. These techniques included the measurement of the number of cells in DNA synthesis (LI), an estimate of the DNA synthesis time (Ts) and the growth fraction of tissues by means of the primer-available DNA-dependent DNA polymerase assay (PDP). From these data, the potential doubling time and the cell cycle time (Tc) of the tumors were calculated. Early preliminary data on human colonic specimens presented in Tables 1 and 2 indicate that there is an increase in LI from the low polyps to higher adenocarcinomas. There is little difference between primary and metastatic tumor cell kinetics. Growth fraction estimates (PDP) of the various colonic tissue types are also not significantly different and except for villous adenomas, DNA synthesis times are constant. The median 3HTdR labeling indices of 7% primary adenocarcinomas include a number of samples (approximately 20% of all samples) with high labeling indices (in the 10--20% range). These high labeling tumors may be those that show objective response to S-phase active drugs, e.g., 5-FU.

Selective inhibition of thymidine transport at low doses of the alkylating agents triethyleneiminobenzoquinone (Trenimon)

The alkylating antitumor agent triethyleneiminobenzoquinone (Trenimon) causes a rapid decrease in the incorporation of labeled thymidine into the DNA of Yoshida or Ehrlich ascites tumor cells. The effect is expressed 4 h after administration of 6 × 10 −8 moles/kg of the drug to mice bearing Yoshida ascites tumors or of 6 × 10 −7 moles/kg to Ehrlich ascites tumor-bearing animals, respectively. The reduced incorporation of labeled thymidine which is observed under these conditions is not due to an inhibition of DNA synthesis. DNA synthesis was measured by an isotope dilution assay after pulse-labeling with 3H-thymidine and by monitoring the increase in the total amount of DNA of the cell populations. The data demonstrate that DNA synthesis is not affected during the first 8 h after exposure to the drug. This conclusion is supported by cell kinetic measurements which indicate that the alkylating agent does not interfere with the progression of cells into the S phase, but exerts a block at the G 2 stage of the cell cycle. The reduced incorporation of thymidine into DNA is explained by a decreased transport of the nucleoside into the cells.