Number Of Clonogenic Tumor Cells Research Articles

Heuristic estimation of the α/β ratio for a cohort of Mexican patients with prostate cancer treated with external radiotherapy techniques.

The aim of the study was to Estimate and compare the radiobiological ratio α/β with the heuristic method for a cohort of Mexican patients with prostate cancer (PCa) who were treated with external radiotherapy (RT) techniques at three Hospital Institutions in Mexico City. With the Kaplan-Meier technique and the Cox proportional hazards model, the biochemical relapse-free survival (bRFS) is determined and characterized for cohorts of Mexican patients with PCa who received treatment with external RT. Using these clinical outcomes, the radiobiological parameter α/β is determined using the heuristic methodology of Pedicini et. al. The α/β is calculated from the survival curves for different treatment schemes implemented at three distinct hospitals. The Pedicini's techniques allow to determine the parameters α/β, k and N 0 when treatments are not radiobiologically equivalent, therefore, are built up of a set of curved pairs for the biologically effective dose (BED) versus the ratio α/β, where the ratio is given by the intersection for each pair of curves. Six different values of α/β were found: the first α/β = 2.46 Gy, the second α/β = 3.30 Gy, the third for α/β = 3.25 Gy, the fourth α/β = 3.24 Gy, the fifth α/β = 3.38 Gy and the last α/β = 4.08 Gy. These values can be explained as follows: a) The bRFS of the schemes presents a statistical variation; b) The absorbed doses given to the patient present uncertainties on the physical dosimetry that are not on the modeling; c) Finally, in the model for the bRFS of Eq. (3), there are parameters that have to be considered, such as: the number of clonogenic tumor cells N 0 , the overall treatment time (OTT), the kick-off time for tumor repopulation T k and the repopulation doubling time. Therefore, the mean value to α/β for all schemes has an average value of 3.29 (± 0.52) Gy. The value of is determined from cohorts of Mexican patients with PC a treated with external radiotherapy using the time-dependent LQ model, which is a higher value with respect to the "dogma" value of α/β 1.5 Gy obtained with the LQ model without temporal dependence. Therefore, there is a possibility of optimizing treatments radiobiologically and improving the results of bRFS in Mexican patients with PCa treated with external radiotherapy.

Epidermal growth factor receptor inhibitors for radiotherapy: biological rationale and preclinical results

Blocking the epidermal growth factor receptor (EGFR) represents a role model for a successful biological targeting approach to improving outcomes after radiotherapy. This review summarizes data from several local tumour control experiments in which EGFR inhibitors were combined with radiation in FaDu human squamous cell carcinomas xenografted into nude mice. BIBX1382BS is an oral bioavailable inhibitor of the intracellular tyrosine kinase domain of EGFR. It was administered in different experimental settings: concurrent with fractionated radiotherapy, following completion of irradiation, and in the period between surgery and adjuvant irradiation. Despite beneficial effects on tumour growth, in none of these experimental settings did BIBX1382BS improve local tumour control. In contrast, cetuximab (Erbitux), an IgG1 monoclonal antibody against the extracellular ligand-binding domain of EGFR, improved local tumour control when given concurrently with radiation. Results from a series of local tumour control experiments designed to elucidate the underlying mechanisms of cetuximab suggest that multiple radiobiological mechanisms might contribute to the observed effects: decreased number of clonogenic tumour cells, increased cellular radiation sensitivity, decreased repopulation and improved reoxygenation of clonogenic tumour cells during the combined treatment. In summary, the data suggest that different classes of EGFR inhibitors may have a different potential to improve local tumour control after fractionated irradiation.

Pre-treatment number of clonogenic cells and their radiosensitivity are major determinants of local tumour control after fractionated irradiation

Objective The response of tumours to fractionated radiotherapy is determined by many factors including repopulation, reoxygenation, the number of clonogenic cells, and their intrinsic radiosensitivity. However, after single radiation doses given under conditions of clamp hypoxia, the dose to control a tumour locally is dependent only on the number of clonogenic cells and their cellular radiosensitivity. Therefore, these parameters were investigated using local control after single doses given under hypoxia, to predict the outcome of fractionated irradiation. Materials and methods Ten hSCC cell lines (FaDu, UT-SCC-15, UT-SCC-14, XF354, UT-SCC-5, UT-SCC-45, SAS, CAL-33, UT-SCC-8, and HSC-4) were transplanted subcutaneously into the right hind-leg of NMRI nude mice. At 7 mm in diameter, tumours were irradiated either with graded single doses under clamp blood flow conditions ( n = 873) or with 30 graded fractions within 6 weeks ( n = 905) under ambient conditions. Local tumour control was determined 120 days after irradiation. Radiation response was quantified in terms of TCD 50, i.e. the dose required to control 50% of tumours locally. Results Ten tumour lines investigated showed a pronounced heterogeneity in both TCD 50(30fx/6w) after fractionated irradiation and TCD 50(SDclamp) after single dose irradiation. TCD 50(30fx/6w) varied between 45 Gy for UT-SCC-45 and 127 Gy for SAS; TCD 50(SDclamp) varied between 42 Gy for UT-SCC-14 and 66 Gy for CAL-33. Two tumours were excluded from further analysis due to immunogenicity or non-defined TCD 50. Linear regression analysis revealed a significant positive correlation between TCD 50(SDclamp) and TCD 50(30fx/6w) ( R 2 = 0.82, p = 0.002). Conclusions Significant association between TCD 50(SDclamp) and TCD 50(30fx/6w) suggests that the pre-treatment number of clonogenic tumour cells and their cellular radiosensitivity have a major impact on local control after fractionated radiotherapy.

A stochastic model of tumor response to fractionated radiation: limit theorems and rate of convergence

The iterated birth and death Markov process is defined as an n-fold iteration of a birth and death Markov process describing kinetics of certain population combined with random killing of individuals in the population at moments τ 1,…, τ n with given survival probabilities s 1,…, s n . A long-standing problem of computing the distribution of the number of clonogenic tumor cells surviving an arbitrary fractionated radiation schedule is solved within the framework of iterated birth and death Markov process. It is shown that, for any initial population size i, the distribution of the size N of the population at moment t⩾ τ n is generalized negative binomial, and an explicit computationally feasible formula for the latter is found. It is shown that if i→∞ and s n →0 so that the product is 1⋯ s n tends to a finite positive limit, the distribution of random variable N converges to a probability distribution, which for t= τ n turns out to be Poisson. In the latter case, an estimate of the rate of convergence in the total variation metric similar to the classical Law of Rare Events is obtained.

Distribution of the number of clonogens surviving fractionated radiotherapy: a long-standing problem revisited

Purpose : A long-standing problem is addressed: what form of the probability distribution for the number of clonogenic tumor cells remaining after fractionated radiotherapy should be used in the analysis aimed at evaluating the efficacy of cancer treatment? Over a period of years, a lack of theoretical results leading to a closed-form analytic expression for this distribution, even under very simplistic models of cell kinetics in the course of fractionated radiotherapy, was the most critical deterrent to the development of relevant methods of data analysis. Materials and methods : Rigorous mathematical results associated with a model of fractionated irradiation of tumors based on the iterated birth and death stochastic process are discussed. Results : A formula is presented for the exact distribution of the number of clonogenic tumor cells at the end of treatment. It is shown that, under certain conditions, this distribution can be approximated by a Poisson distribution. An explicit formula for the parameter of the limiting Poisson distribution is given and sample computations aimed at evaluation of the convergence rate are reported. Another useful limit that retains a dose-response relationship in the distribution of the number of clonogens has been found. Practical implications of the key theoretical findings are discussed in the context of survival data analysis. Conclusions : This study answers some challenging theoretical questions that have been under discussion over a number of years. The results presented in this work provide mechanistic motivation for parametric regression models designed to analyze data on the efficacy of radiation therapy.

Iterated birth and death process as a model of radiation cell survival

The iterated birth and death process is defined as an n-fold iteration of a stochastic process consisting of the combination of instantaneous random killing of individuals in a certain population with a given survival probability s with a Markov birth and death process describing subsequent population dynamics. A long standing problem of computing the distribution of the number of clonogenic tumor cells surviving a fractionated radiation schedule consisting of n equal doses separated by equal time intervals τ is solved within the framework of iterated birth and death processes. For any initial tumor size i, an explicit formula for the distribution of the number M of surviving clonogens at moment τ after the end of treatment is found. It is shown that if i → ∞ and s → 0 so that is n tends to a finite positive limit, the distribution of random variable M converges to a probability distribution, and a formula for the latter is obtained. This result generalizes the classical theorem about the Poisson limit of a sequence of binomial distributions. The exact and limiting distributions are also found for the number of surviving clonogens immediately after the nth exposure. In this case, the limiting distribution turns out to be a Poisson distribution.

A novel immunotoxin recognising the epithelial glycoprotein-2 has potent antitumoural activity on chemotherapy-resistant lung cancer.

Resistance to chemotherapy is a major cause for failure in the treatment of lung cancer. Compared to conventional cytotoxic drugs, immunotoxins act by different mechanisms and thus might be promising for the treatment of chemoresistant cancer. The monoclonal antibody MOC31 recognises the epithelial glycoprotein-2 (EGP-2), a cell-surface antigen associated with small-cell lung cancer (SCLC) and a major fraction of lung adenocarcinomas. An immunotoxin composed of MOC31 and a recombinant from of Pseudomonas exotoxin A lacking the cell-binding domain (ETA252-613) was prepared and its effect on lung cancer cell lines examined. MOC31 ETA252-613 was selectively cytotoxic to EGP-2-positive SCLC and adenocarcinoma cell lines inhibiting proliferation by 50% at concentrations ranging from 0.01 nM to 0.3 nM. Moreover, the immunotoxin reduced the number of clonogenic tumour cells from cultures by factors of 10(4) and 10(5) during a 24-h and a 3-week exposure respectively. In athymic mice, the immunotoxin, which revealed a serum half-life of approximately 4 h, caused substantial regression of small (40 mm3) chemoresistant tumour xenografts and significantly delayed the growth of larger tumours (120 mm3). This finding indicates that MOC31-ETA252-613 may be useful for the treatment of lung cancer in the setting of chemoresistant minimal residual disease.

Kinetics of depopulation, repopulation and host cell infiltration in the rhabdomyosarcoma R1H after 14 MeV neutron irradiation.

The kinetics of depopulation and repopulation of the solid transplantable rhabdomyosarcoma R1H in the rat was studied following irradiation with 5 Gy of 14 MeV neutrons. Several parameters were sequentially measured over a time period of 4 weeks after irradiation: the tumour volume was assessed by in situ caliper measurements; the numerical density of tumour cells was obtained by morphometry; the clonogenic fraction of tumour cells was derived from in vitro colony assay; and the numerical ratio of host to tumour cells was determined by flow cytometry. From these primary parameters the number of clonogenic tumour cells, non-clonogenic tumour cells, and nucleated host cells per tumour, as well as their variation with time, were derived. The results were compared with two sets of data obtained previously for the same tumour exposed to 15 Gy of 200 kVp X-rays. Survival of tumour cells was reduced to 5.5 +/- 0.5% by 5 Gy neutrons and to 4.5 +/- 0.5% by 15 Gy X-rays, i.e. an RBE of close to 3. There was a lag period before the onset of repopulation (4.9 +/- 0.4 days and 4.9 +/- 0.5 days, respectively), followed by a high initial rate of repopulation corresponding to a doubling time of 2.0 +/- 0.2 days for neutrons and 2.1 +/- 0.2 days for X-rays. The rate of depopulation was significantly different for the two treatment modalities; the halving time for the number of non-clonogenic tumour cells was 11 +/- 4 days for neutrons and 2.8 +/- 0.5 days for X-rays.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Population Kinetics of the Rhabdomyosarcoma R1H of the Rat after Single Doses of X-rays

The kinetics of depopulation and repopulation of the solid transplantable rhabdomyosarcoma R1H of the rat following local irradiation with single subcurative X-ray doses of 7.5, 15 and 30 Gy was studied. Several parameters were sequentially measured over a time interval of 4 weeks after irradiation: the ratio of the number of tumour to host cells, and the cellular DNA content of tumour and host cells, were determined by flow cytometry; the amount of DNA per gram of tumour tissue was determined biochemically; the clonogenic fraction of tumour cells was obtained from in vitro colony assay; and the tumour volume was assessed by in situ caliper measurements. From the amount of DNA per gram and the average DNA content per cell, the total number of cells per gram of tumour tissue was obtained. From this and the other parameters measured, the number of clonogenic tumour cells, non-clonogenic tumour cells and nucleated host cells per tumour, as well as their variation with time and dose, could be derived. The results showed that there was a lag period prior to depopulation amounting to 3.8 +/- 1.4, 1.4 +/- 0.8 or 0 +/- 0.7 days for 7.5, 15 or 30 Gy, respectively. The rate of depopulation of non-clonogenic tumour cells increased with dose; the halving times of non-clonogens were 4.7 +/- 1.8, 2.6 +/- 0.7 or 2.1 +/- 0.4 days for the three doses applied. There were no indications that proliferation of doomed cells contributed significantly to tumour growth after irradiation. After lag periods that were similar in length to those prior to depopulation, a massive immigration of host cells was observed. Under certain conditions more than 97 per cent of the cells present in irradiated tumours were found to be of host origin. There was a lag period before the onset of repopulation by clonogenic tumour cells, the length of which increased from 2.7 +/- 0.7 to 5.0 +/- 0.8 or 6.3 +/- 1.0 days for 7.5, 15 or 30 Gy, respectively. The initial rate of repopulation increased with radiation dose; after the end of the lag period the doubling time of clonogenic tumour cells (in controls amounting to 3.7 +/- 0.2 days) was 3.1 +/- 0.1, 2.1 +/- 0.1 and 1.1 +/- 0.1 days for the three doses applied.(ABSTRACT TRUNCATED AT 400 WORDS)

Radiotherapy of the rhabdomyosarcoma R1H of the rat: Kinetics of cellular inactivation by fractionated irradiation

The kinetics of cellular inactivation by fractionated irradiation in the RIH rhabdomyosarcoma of the rat was studied in the dose range of 1.07 to 12.50 Gy per fraction. Regimens of 1, 3, 5, 7, and 10 fractions per week for several weeks were compared. The number of clonogenic tumor cells per tumor in the course of the different treatment schedules was determined using an in vitro colony assay. The results show that the proliferation of clonogenic tumor cells is decelerated in the course of a fractionated radiotherapy. The deceleration persists for several days after end of treatment, until accelerated repopulation is initiated. The fraction of tumor cells inactivated per week was only dependent on the total dose per week, that is the cellular response was the same whether the weekly dose was applied in 1, 3, 5, 7, or 10 fractions. Thus, the fractionation regimens were considerably more effective than expected from calculations based on single-dose in situ survival curves.

Comparison of Conventional and Split-Course Radiotherapy as Primary Treatment in Carcinoma of the Larynx

Based on our experience with conventional, daily irradiation, a split-course radiation schedule was introduced in 1978. The schedule, which was based on Cohen's models for squamous cell carcinoma and vascular damage respectively, predicted an improved tumour control and a reduced rate of late complications, e.g. late oedema, if the conventional, daily treatment was replaced by a split-course schedule. The schedule has later been abandoned, but the experience gained from split-course treatment at various dose levels has been analysed and the results compared with those obtained by conventional radiation. The data allowed construction of dose-response curves and estimation of iso-effect doses. Split-course treatment was associated with a significantly reduced therapeutic ratio because, disappointingly, it did not improve tumour control, and the severity of late complications grew. No late complications were avoided by introducing a 3-week pause in the radiation therapy regimen, nor was the tumour response improved despite a 12-Gy increase in total dose. This indicates a significant repopulation corresponding to more than 0.5 Gy/day, equivalent to an up to 100-fold increase of the number of clonogenic tumour cells during the pause--an increase that occurred despite the decrease, clinically, of the tumours during this period.

In vivo toxicity of phenothiazines to cells of a transplantable tumor.

The toxicities of three phenothiazines, promazine, chlorpromazine, and trifluoperazine, towards cells of a mouse fibrosarcoma were quantified by means of an in vitro assay of clonogenic cell survival. For all three drugs cell kill was proportional to the amount of drug injected. Following injection of equimolar (0.2 mM/kg) amounts, cell survival was progressively reduced for a period of at least 48 h. On the basis of cell survival at 48 h after administration the ranking of the drugs for cytotoxicity, in ascending order, was trifluoperazine, chlorpromazine, promazine. A period of acute hypoxia prior to processing of the tumor did not enhance the toxicity of any of the drugs, and no change in the size of the hypoxic fraction of the tumor was seen 24 h after the injection of chlorpromazine. On this basis it was concluded that there was no evidence of enhanced toxicity of drugs for either chronically or acutely hypoxic tumor cells. A reduction in the number of clonogenic tumor cells per gram of tumor was largely the result of a fall in the number of viable cells recovered from the tumor. The plating efficiency of surviving cells remained constant or was only slightly depressed.

Kinetics of cellular inactivation by fractionated and hyperfractionated irradiation in lewis lung carcinoma

The kinetics of cellular inactivation by fractionated irradiation in Lewis lung carcinoma was studied in the dose range of 2.3 to 6.5 Gy per fraction. Regimens of one and two fractions per day for 10 days were compared. The number of clonogenic tumor cells was determined using an in vitro soft-agar colony assay. Under the experimental conditions used, the fraction of tumor cells inactivated per day was only dependent on the total dose per day, i.e., the cellular response was the same whether the daily dose was given in one or two fractions. Thus, the two fraction per day regimen was more effective than expected from calculations based on acute in vivo radiation dose-survival curves. This result could be explained if the clonogenic tumor cells were less hypoxic during the two fractions per day regimen than the one fraction per day schedule.

Misonidazole Cytotoxicity in Vivo: A Comparison of Large Single Doses with Smaller Doses and Extended Contact of the Drug with Tumor Cells

Experiments were performed to determine the kinetics and magnitude of misonidazole cytotoxicity in EMT6/Ro tumors using an in vivo-in vitro clonogenicity assay. A comparison was made between the cytotoxic effects of large single doses with smaller doses of misonidazole administered ip and those produced on extended contact of the drug with tumor cells using a continuous iv drug infusion system. After a single ip dose of 1 mg/g, cytotoxicity was maximum at 18 to 24 h; by 72 h the clonogenic cells per tumor had returned to control levels. The maximum cytotoxicity was greater (a decrease of 10 times) if the animals were kept at 37/sup 0/C compared with ambient conditions (a decrease of 4.5 times) where the body temperature would decrease due to the drug. A dose-response curve performed with the animals at 37/sup 0/C showed no significant cytotoxicity at 18 h after single ip doses of 0.5 mg/g or less. Other experiments were carried out at 37/sup 0/C using a drug continuous infusion system. Two profiles were studied: (a) continuous constant rate infusion over 3 days of constant serum and tumor levels of both 100 and 200 ..mu..g/ml and (b) continuous variable rate infusion where the maximum serummore » levels reached 80 or 200 ..mu..g/ml after 2 to 4 h and decayed with a half-life of 12 h as in humans. Significant cytotoxicity was obtained under both of these conditions. Maximum cytotoxicity occurred at about 24 h in both types of experiments and amounted to decreases of clonogenic tumor cells of 4.5 and 7 times for 100 and 200 ..mu..g/ml, respectively, after constant rate infusion and 2 to 4 times for 80 and 200 ..mu..g/ml, respectively, after variable rate infusion. Because of the relatively rapid recovery in the number of clonogenic tumor cells by 72 h, the cytotoxic effects were not reflected as changes in tumor size even when the animals were maintained at 37/sup 0/C.« less

The growth of the EMT6 tumour in the lungs of Balb C mice following intravenous inoculation of tumour cells from culture.

ABSTRACTThe growth of the EMT6 tumour in the lungs of Balb C mice has been studied following intravenous inoculation of different numbers of tumour cells taken from culture. At various times after injection of cells into mice, cell suspensions have been prepared from pairs of lungs and the number of in vitro colony forming cells assayed by plating into petri dishes. Following intravenous injection of 105 cells, the time required for doubling of the number of clonogenic tumour cells appearing in the cell suspension is around 17 hr until such time that the total tumour cell population per set of lungs reaches 108 cells (at 10–12 days). This doubling time has to be corrected for changes in ability to extract cells from the lungs into the cell suspension at various times and also for possible changes in plating efficiency in vitro. When these correction factors are applied, the most likely value for the doubling time of clonogenic tumour cells in the lungs is in the range 20–24 hr. This is a similar figure to that previously deduced for the EMT6 flank tumour during its microscopic period of growth.After reaching a total size of 108 tumour cells, the time for doubling of the number of clonogenic tumour cells in the lung increases. During the later stages of tumour growth a good correlation is seen between total lung tumour weight and the number of clonogenic cells present.For the final 3–4 days of the initial period of rapid tumour growth, it is possible to carry out a haemocytometer count of tumour cells in the lung suspension and hence surviving fraction experiments may be carried out after various forms of treatment. In this way the response to treatment of microscopic tumour foci may be determined.