Tumor Potential Doubling Time Research Articles

Cell Kinetics of Renal Cell Carcinoma Studied with in Vivo Iododeoxyuridine Incorporation and Flow Cytometry

In 29 patients with renal cell carcinoma tumor cell kinetics were studied by in vivo labeling with 100mg. iododeoxyuridine given 3 to 7hours before nephrectomy. Two to 6 samples were analyzed from each tumor after fixation in ethanol. Iododeoxyuridine was analyzed by specific antibodies using flow cytometric analysis of labeled cells and parameters, such as labeling index and potential tumor doubling time, could be determined. Samples from 28 tumors were evaluable. Mean labeling index of the tumors was 2.7%, which was significantly different from the 0.35% index in kidney cortex tissue. There was no statistical difference in labeling index among the clinical stages. The mean potential tumor doubling time in the tumor cells was 20.3 days, which was significantly different compared with 137.7 days in kidney cortex tissue. There was a frequent intra-tumoral heterogeneity of potential tumor doubling times. Diploid and aneuploid samples had significantly different potential tumor doubling (mean 37.4 and 12.5 days, respectively). Potential tumor doubling had a significant impact on the survival time (p = 0.004), which also was noted in the patients with aneuploid tumors (p = 0.003). Our study shows that in vivo incorporation of iododeoxyuridine successfully can be analyzed in renal cell carcinoma. Potential tumor doubling time gives valuable prognostic information on the clinical behavior of the patients.

Quantifying the effect of dose inhomogeneity in brachytherapy: Application to permanent prostatic implant with 125I seeds

Purpose : To quantitate the influence of dose inhomogeneity on brachytherapy efficacy. Methods and Materials : A computed tomography-based system of planning, implementation and evaluation was used to generate tumor-specific dose-volume histograms of eight permanent 125I implants of prostate cancers. The radiobiological effect was then assessed, voxel by voxel, in terms of the biologically effective dose and the associated cell inactivation. The overall cell survival of the entire target volume was then computed. To evaluate the influence of inhomogeneity, the dose-volume histogram was modified in an iterative fashion, with the corresponding surviving fraction calculated after each step. Specifically, the volume in the highest dose bin was combined with that in the next bin to give a new frequency distribution from which cell survival was generated. Tumor control probability (TCP) was also used as an endpoint, using the same iterative procedure. Results : Doses 20–30% higher than D99 (the dose that covered 99% of the target volume) contributed to additional cell inactivation, but still higher doses did not further increase cell kill. With homogeneous irradiation at D 99 as a reference, we defined the inhomogeneity enhancement factor as the ratio of the biologic effective dose of the actual implant to that of the reference dose distribution. The calculated enhancement factors were inversely dependent on tumor potential doubling time (Tp), about 1.2 – 1.3 for a Tp of 30 days, and between 1.3 and 1.7 if Tp = 10 days, with higher values for implants with low D99. Dose inhomogeneity enhanced TCP. For implants with high control probabilities, doses significantly higher (> 20%) than the D99 value did not further enhance the tumor control probabilities. In contrast, for implants with relatively low tumor control and D 99 values, the control probability continued to increase with doses significantly higher than D 99, up to a dose of 2 × D 99. The underlying reasons were the incorporation of patient “population averaging” in the calculation and the saturation of tumor control dose response at about 120 Gy. Conclusion : Dose heterogeneity in implants increased tumor cell kill and local control probability, although doses > 20% higher than the prescription dose is wasted. The increase the beneficial effect of dose inhomogeneity may be greatest when most needed.

Cell Kinetics of Head and Neck Squamous Cell Carcinomas: Prognostic implications

Forty-three squamous cell carcinomas of the head and neck region were in vivo labelled with the thymidine analogue iododeoxyuridine. Combined flow cytometric (FCM) and immunohistochemical (IHC) analysis was performed, and the following parameters calculated: labelling index (LI), S-phase time (TS) and potential tumor doubling time (Tpot). Complete FCM and IHC analyses could successfully be performed in 31 cases, showing a median LI of 13.6% with FCM and 9.1% with IHC. A correlation achieved between LI/FCM and LI/IHC was due to the aneuploid cases, whereas the diploid cases showed no such correlation. Data indicated that Tpot calculated with LI from IHC (Tpot/IHC) might be a prognostic factor, in contrast to Tpot determined using LI/FCM.

Measurement of cell kinetics in cervical tumours using bromodeoxyuridine.

The pre-treatment cell kinetics of 120 cervical tumours were assessed following the in vivo labelling with the thymidine analogue Bromodeoxyuridine (BrdUrd). In 89% both static and temporal kinetic parameters could be measured. Through the analysis of multiple biopsies from each tumour marked intra tumour heterogeneity was demonstrated. The median values for the most highly labelled sample analysed for each tumour were; S-phase duration (Ts) 12.1 h, BrdUrd labelling index (CLI) 9.5% and potential tumour doubling time 4.4 days. There was a significant elevation in CLI, but no difference in Ts, between tumour and non-neoplastic cervical tissue. There was a significant elevation in CLI, advanced stage and large size tumours. Although a significant elevation in CLI was found in aneuploid tumours this is likely to represent the systemic bias of the calculation methods, with no difference being seen between aneuploid and diploid tumours when BrdUrd labelling was measured with-out reference to the nuclei DNA content. The majority of these patients were treated with radiotherapy and cell kinetic data will be correlated with treatment response when adequate follow up has been achieved.

Growth fraction of human tumors: Assays and complications

T he fraction of cells actively proliferating in human tumors is expected to be useful in predicting tumor response to therapy. 1,2 There are several excellent reviews 3-5 and a comprehensive monograph 6 describing both static and flow cytometry assays developed to measure growth fraction. This article introduces a new method, and in describing our results using clinical samples, provides a basis for understanding why this information is useful or necessary and identifies factors that may limit our ability to define growth fraction accurately for different tumor types. The doubling time of a tumor has long been known to be a valuable prognostic indicator. As an obvious generalization, patient survival is shorter when the tumor grows more rapidly, although tumor size and doubling time can vary independently] The recent application of accelerated radiotherapy to overcome rapid tumor cell repopulation a is in large part responsible for the current interest in categorizing tumors into slowly and rapidly proliferating. 9 There are three major kinetic factors contributing to tumor doubling time: growth fraction (GF), cell cycle time, and cell loss rate. l~ Although no method is able to accurately determine cell loss rate in human tumors, the two remaining factors are used to describe potential tumor doubling time (Tpot), which is proportional to the S phase duration (T~) divided by the labeling index (LI), or alternatively, the cell cycle duration divided by the GF. II It has been argued that Tpo t is more informative than GF alone in predicting tumor response to accelerated radiotherapy because Tpo~ includes a measure of rate of cell proliferation (Ts) as well as state of proliferation (LI). However, when analyzing the components that determine Tpo, it is common for tumors of the same tissue of origin

Single biopsy, tumor kinetic analyses: A comparison of methods and an extension to shorter sampling intervals

There is emerging and established clinical and laboratory evidence that proliferation of tumor clonogens during radiation therapy can impair local tumor control. The pre-treatment, tumor potential doubling time, T pot, estimated with in situ bromodeoxyuridine (BrdUrd) labeling, followed by a single biopsy and flow cytometry, may be a predictor of a given tumor's ability to undergo such intra-treatment proliferation. Recent studies have found a strong similarity between T pot's determined in this fashion and the effective doubling times of surviving tumor cells during radiotherapy, as estimated from tumor control versus treatment duration data. Furthermore, several preliminary clinical studies have indicated that T pot may be a predictor of outcome, with faster tumors doing worse. Accelerated fractionation might overcome such proliferation, but is more acutely toxic and is unlikely to benefit patients with slowly proliferating tumors. Thus, the BrdUrd/single biopsy method may offer the possibility of selecting between accelerated and conventional or hyperfractionated treatment. Several approaches to the analysis of data generated by this method have been described, but there has been little documentation of the validity of methods in experimental systems, particularly in human experimental tumors. This study explores various analytic methods employed with the BrdUrd, delayed, single-biopsy technique used in determining the potential doubling time, T pot, of tumors. It compares methods of analysis in three experimental systems and in 40 in situ-labeled human tumors, and proposes a method for shortening the required labeling-biopsy interval to a clinically more convenient range of 3 to 4 hours.

Kinetic studies on a murine sarcoma and an analysis of apoptosis.

A stathmokinetic method has been used to determine the cell cycle parameters, particularly the potential tumour doubling time, of a murine fat pad sarcoma. Additional information has been obtained by determining the percentage of labelled mitoses (PLM). A technique which simultaneously demonstrates autoradiographically labelled S phase nuclei and histochemically localized acid phosphatase activity has also been used at light microscope level to compare these parameters: acid phosphatase activity was demonstrated in tumour cells and macrophages. Single cell deletion by apoptosis has been investigated as distinct from necrosis. Condensed, dying apoptotic cells, have been found in proliferative areas of tumour that are not under physiological stress. The analysis of apoptosis indicated a previously unsuspected variation in apoptotic activity with tumour weight. Cell death by apoptosis initially rose as the tumour grew, but after the tumour reached a threshold weight it declined dramatically, and finally remained stable. This may reflect an initial attempt at autoregulation of tumour size which ultimately fails. Apoptosis was estimated to account for an average of 7% of the total cell loss rate in this tumour.

Proliferation kinetics of malignant non-Hodgkin's lymphomas related to histopathology of lymph node biopsies.

Since lymphomas of high and low malignancy have been differentiated by the Kiel-classification, the cell kinetics of malignant lymphomas have become an interesting subject. In 20 non-Hodgkin's lymphomas we have determined the initial labelling index, the mitosis index, the proliferation rate and the durations of DNA-synthesis and mitosis. The kinetic variables have been estimated by using an in vitro-incubation of fresh biopsies and double-labelling with radioactive DNA-precursors. in 8 lymphomas of low malignancy we found an initial labelling index of 7.8%, a mitosis index of 0.3% and a potential tumour doubling time of 100.2 h for a cell production rate without regard to cell loss. On the other hand, lymphomas of high malignancy showed a labelling index of 16.7%, a mitosis index of 0.7% and a potential tumour doubling time of 40.5 h. All these values could be differentiated with high statistical confidence. DNA-synthesis time and mitosis time of both lymphoma groups did not show any significant differences. From these findings we obtained confirmation of the cyto-morphological principles used in classifying lymphomas.

The prediction of tumor regression in human squamous carcinoma by means of the potential tumor doubling time.

Thirty-three patients with locally advanced epidermoid cancer of the mouth were investigated by means of the potential tumor doubling time prior to each of four weekly infusions of methotrexate. The agent used to induce metaphase arrest was vincristine sulphate 2 mg intravenous push and the methotrexate was given in a dose of 100 mg/M2 body surface. In twenty-one patients the investigation was completed. It was found that in those patients where the P.T.D.T. was maintained at its original level there was a greater than 50% reduction in the size of the lesion, whereas when the P.T.D.T. became greatly prolonged there was no such reduction. It is suggested that the P.T.D.T. provides a simple same day method of evaluating one parameter in the cell kinetics of accessible tumors and that this information can be used to individualize chemotherapy schedules.

Cell proliferation in human melanoma.

The cytokinetics of subcutaneous metastases in five patients with melanoma was studied. Multiple simultaneous biopsies following pulse labeling with tritiated thymidine were performed in one patient. There was relatively uniform labeling and mitotic indices among these. Within the individual tumors, there was some variation in the labeling index with small clusters of tumor cells having significantly higher labeling indices than more sparsely infiltrating tumor cells. Repetitive biopsies following pulse labeling were performed in two patients. The per cent labeled mitosis curves were similar in the two patients. By computer analysis a median G(2) period of 5.3 hr and an S period of 21 hr were obtained. The generation time (T(e)) was highly variable with a median of 3 days. This T(e) was consistent with that calculated from grain count studies in these patients. Two patients received either intermittent or continuous tritiated thymidine over a 10-20 day period. Analysis of the labeling index curve by computer fitting indicated a growth fraction of 20-30%. The growth fraction calculated by other indirect methods was consistent with the computer analysis. The potential tumor doubling time as calculated from the T(e) and growth fraction was much shorter than the actual doubling time indicating that cell loss was approximately 70% of the rate of cell production.

In vitro studies of cell proliferation in tumours—I : Critical appraisal of methods and theoretical considerations

Cell proliferation in human tumours is being investigated by in vitro labelling with H 3-Thymidine. This is carried out under high-pressure oxygen in order to preserve DNA synthesis of cells deep within the excised specimen. Thymidine labelling indices determined in this way are used to predict the cell production rate within the tumour and thus a potential tumour doubling time. The assumptions involved in this procedure are discussed, in particular the rôle of cell loss.