Tumor Cell Doubling Time Research Articles

Mathematical Modeling of Preclinical Alpha-Emitter Radiopharmaceutical Therapy.

Preclinical studies, in vivo, and in vitro studies, in combination with mathematical modeling can help optimize and guide the design of clinical trials. The design and optimization of alpha-particle emitter radiopharmaceutical therapy (αRPT) is especially important as αRPT has the potential for high efficacy but also high toxicity. We have developed a mathematical model that may be used to identify trial design parameters that will have the greatest impact on outcome. The model combines Gompertzian tumor growth with antibody-mediated pharmacokinetics and radiation-induced cell killing. It was validated using preclinical experimental data of antibody-mediated 213Bi and 225Ac delivery in a metastatic transgenic breast cancer model. In modeling simulations, tumor cell doubling time, administered antibody, antibody specific-activity, and antigen-site density most impacted median survival. The model was also used to investigate treatment fractionation. Depending upon the time-interval between injections, increasing the number of injections increased survival time. For example, two administrations of 200 nCi, 225Ac-labeled antibody, separated by 30 days, resulted in a simulated 31% increase in median survival over a single 400 nCi administration. If the time interval was 7 days or less, however, there was no improvement in survival; a one-day interval between injections led to a 10% reduction in median survival. Further model development and validation including the incorporation of normal tissue toxicity is necessary to properly balance efficacy with toxicity. The current model is, however, useful in helping understand preclinical results and in guiding preclinical and clinical trial design towards approaches that have the greatest likelihood of success. SIGNIFICANCE: Modeling is used to optimize αRPT.

ANTIINTEGRIN AVG3 SAV-RGD AS COMBINANT FOR DTIC WITH HUMAN AMELANOTIC SKIN MELANOMA

Introduction. The avB3 integrin (cell adhesion molecule) plays the role of the receptor, including, metalloproteinase matrix 2, involved in melanoma metastasis. We have been characterized as an antimelanoma original anti-integrin agent SAV-RGD (lyo-SAV-RGD), specifically binding to melanoma cells by means of the Arg-Gly-Asp tripeptide. More significant inhibited effect was obtained on amelanotic human skin melanoma MeWo. Accordingly, the experimental evaluation of the effectiveness of lyo-SAV-RGD in combination with the «gold» standard of antimelanoma chemotherapy dacarbazine (DTIC) is relevant. Objective: a comparative study of the combination of DTIC+lyo-SAV-RGD on the model of pigmented melanoma of human skin MeWo. Material and methods. The model of human skin melanoma MeWo in vivo/in vitro: s.c. xenografts in Balb/c nude mice, sensitive to DTIC cell line MeWo and obtained prospectively stable subline MeWo/DTIC was used. In vitro experiments the time of cell doubling in both models was determined and the individual range of agent concentrations was studied. Therapy with DTIC+lyo-SAV-RGD were administered in simultaneously sequentially, first the single 150 mg/kg DTIC, then lyo-SAV-RGD 9-fold course in the total dose of 900 mg/kg (the first dose is doubled). To control of the group with single dose 250 mg/kg of DTIC close to the maximum tolerated dose (MTD). All experiments were performed by standard methods using significant evaluation criteria and adequate statistical processing of the results (p<0.05). Results. For MeWo and MeWo/DTIC cells with a certain prospectively identical doubling time of the doubling time of tumor cells (Tpot) at the level of 23.0±2.3 h and 23.1±1.8 h, DTIC was practically non-cytotoxic, IC50=410±4 g/ml and IC50=860±27 g/ml (IC50<1o0 g/ml). However, the absolute value of the active concentrations of DTIC was significantly less known for murine pigmented melanoma, 1200-1400 g/ ml. Lyo-SAV-RGD, on the contrary, was cytotoxic for both melanoma variants at the same level, IC50=100±3.1 g/ml, and in the range from 1,0 to 100 g/kg caused cell death in direct dependence on the concentration. On s.c. MeWo xenografts DTIC at the dose of 250 mg/kg was effective at T/ C<30% (criterion T/C<42%) with partial death of mice from toxicity. The combination of DTIC+lyo-SAV-RGD showed an identical effect regardless of dose reduction by 40%, T/C=38% (p=0.001). Conclusion. A comparative study on the model of human skin melanoma MeWo revealed in vitro the absence of significant cytotoxicity of DTIC and at the same time cytotoxicity of anti-integrin avB3 lyo-SAV-RGD for MeWo and MeWo/DTIC cells. On xenografts MeWo combined therapy DTIC+lyo-SAV-RGD with dose reduction cytostatic 40% made it possible to obtain the maximum antitumor effect (T/C=38% vs 30%) with improved tolerability. The obtained data suggest that the introduction of anti-integrin avB3 into the scheme with DTIC is advisable to increase the selectivity of cytotoxic action of cytostatics.

NADPH oxidase 1 supports proliferation of colon cancer cells by modulating reactive oxygen species-dependent signal transduction

Reactive oxygen species (ROS) play a critical role in cell signaling and proliferation. NADPH oxidase 1 (NOX1), a membrane-bound flavin dehydrogenase that generates O2˙̄, is highly expressed in colon cancer. To investigate the role that NOX1 plays in colon cancer growth, we used shRNA to decrease NOX1 expression stably in HT-29 human colon cancer cells. The 80–90% decrease in NOX1 expression achieved by RNAi produced a significant decline in ROS production and a G1/S block that translated into a 2–3-fold increase in tumor cell doubling time without increased apoptosis. The block at the G1/S checkpoint was associated with a significant decrease in cyclin D1 expression and profound inhibition of mitogen-activated protein kinase (MAPK) signaling. Decreased steady-state MAPK phosphorylation occurred concomitant with a significant increase in protein phosphatase activity for two colon cancer cell lines in which NOX1 expression was knocked down by RNAi. Diminished NOX1 expression also contributed to decreased growth, blood vessel density, and VEGF and hypoxia-inducible factor 1α (HIF-1α) expression in HT-29 xenografts initiated from NOX1 knockdown cells. Microarray analysis, supplemented by real-time PCR and Western blotting, revealed that the expression of critical regulators of cell proliferation and angiogenesis, including c-MYC, c-MYB, and VEGF, were down-regulated in association with a decline in hypoxic HIF-1α protein expression downstream of silenced NOX1 in both colon cancer cell lines and xenografts. These studies suggest a role for NOX1 in maintaining the proliferative phenotype of some colon cancers and the potential of NOX1 as a therapeutic target in this disease.

Lifespan based pharmacokinetic-pharmacodynamic model of tumor growth inhibition by anticancer therapeutics.

Accurate prediction of tumor growth is critical in modeling the effects of anti-tumor agents. Popular models of tumor growth inhibition (TGI) generally offer empirical description of tumor growth. We propose a lifespan-based tumor growth inhibition (LS TGI) model that describes tumor growth in a xenograft mouse model, on the basis of cellular lifespan T. At the end of the lifespan, cells divide, and to account for tumor burden on growth, we introduce a cell division efficiency function that is negatively affected by tumor size. The LS TGI model capability to describe dynamic growth characteristics is similar to many empirical TGI models. Our model describes anti-cancer drug effect as a dose-dependent shift of proliferating tumor cells into a non-proliferating population that die after an altered lifespan TA. Sensitivity analysis indicated that all model parameters are identifiable. The model was validated through case studies of xenograft mouse tumor growth. Data from paclitaxel mediated tumor inhibition was well described by the LS TGI model, and model parameters were estimated with high precision. A study involving a protein casein kinase 2 inhibitor, AZ968, contained tumor growth data that only exhibited linear growth kinetics. The LS TGI model accurately described the linear growth data and estimated the potency of AZ968 that was very similar to the estimate from an established TGI model. In the case study of AZD1208, a pan-Pim inhibitor, the doubling time was not estimable from the control data. By fixing the parameter to the reported in vitro value of the tumor cell doubling time, the model was still able to fit the data well and estimated the remaining parameters with high precision. We have developed a mechanistic model that describes tumor growth based on cell division and has the flexibility to describe tumor data with diverse growth kinetics.

Examination of the optimal condition on the in vitro sensitivity to telomelysin in head and neck cancer cell lines

Telomelysin (OBP-301) is a telomerase-specific replication-competent adenovirus with a human telomerase reverse transcriptase (hTERT) promoter. Telomelysin has a strong antitumor effect on a variety of cancers, including head and neck squamous cell carcinoma (HNSCC), and combining telomelysin treatment with paclitaxel or cisplatin enhances the antitumor effect on HNSCC. In the present study, we investigated the relationship between the antitumor activity of telomelysin and tumor cell doubling time(DT), S-phase fraction, and E1A expression. We also investigated whether the antitumor effects of OBP-301-resistant tumor cells are enhanced by cisplatin, paclitaxel, or streptolysin O. The tumor cell DT of 17 human HNSCC cell lines was examined. Antitumor activities of telomelysin (OBP-301) for each HNSCC cell line were examined by MTT assay. Cell cycle analysis was conducted by flowcytometry. E1A gene expressions after infection with telomelysin, hTERT, CAR (Cocksackie Adenovirus Receptor), and c-Myc were examined by quantitative PCR, and E1A expressions were examined again after pretreatment with cisplatin, paclitaxel, or streptolysin O. Correlations were analyzed by Spearman's correlation coefficient. There was a significant relationship between telomelysin sensitivity and DT, S-phase fraction and early E1A expression, and pretreatment with cisplatin, paclitaxel, and streptolysin O increased infectivity of telomelysin-resistant HNSCC cell lines. These findings are useful for advancing clinical trials, and suggest that adjuvant telomelysin treatment would be effective even in telomelysin-resistant HNSCC cell lines.

Well-posedness and stability for an elliptic-parabolic free boundary problem modeling the growth of multi-layer tumors

In this paper we study a free boundary problem modeling the growth of multi-layer tumors. This free boundary problem contains one parabolic equation and one elliptic equation, defined on an unbounded domain in R2 of the form 0 0, then the unique flat equilibrium is asymptotically stable provided that the constant c representing the ratio between the nutrient diffusion time and the tumor-cell doubling time is sufficiently small.

Dose escalation model for limited-stage small-cell lung cancer

Background and purpose To construct a model for the local dose–response relationship of limited-stage small-cell lung cancer (LS-SCLC) along with subsequent models for dose-per-fraction escalation. Materials and methods Reported doses and 2-year progression free survival ( PFS 2year ) rates from LS-SCLC studies were used to construct a dose–response model. Modeling incorporated effects of chemotherapy, incomplete repair of sublethal damage and variations in both kick-off time for rapid reproliferation and effective tumor cell doubling time. Response was modeled as a function of overall treatment duration in order to shed light on the optimum radiotherapy treatment duration. Subsequent models were constructed for more aggressive radiotherapy by means of dose-per-fraction escalation for once a day (QD) and twice a day (BID) fractionation schedules. Results Modeling response as a function of treatment duration resulted in an optimum treatment duration of 3 weeks for both fractionation schedules. Modeling for dose-per-fraction escalation resulted in an increase in the expected local PFS 2year for both fractionation schedules with possible gains in a BID schedule with lower expected late complication rates than a QD schedule yielding the same expected local PFS 2year . Conclusions This modeling suggests more aggressive treatment through escalation of dose-per-fraction, which may result in substantial gains in local PFS 2year . This points towards treatment durations of 3 weeks as being optimal with a BID schedule predicting possible gains in local PFS 2year at lower rates of expected late complications than a QD schedule.

An estimation of radiobiologic parameters from clinical outcomes for radiation treatment planning of brain tumor

To estimate a plausible set of radiobiologic parameters such as alpha, alpha/beta values, from clinical outcomes for biologically based radiation treatment planning of brain tumors. Linear-quadratic (LQ) formalism and the concept of equivalent uniform dose were used to analyze a series of published clinical data for malignant gliomas involving different forms of radiation therapy. A plausible set of LQ parameters was obtained for gliomas: alpha = 0.06 +/- 0.05 Gy(-1), alpha/beta = 10.0 +/- 15.1 Gy, the tumor cell doubling time T(d) = 50 +/- 30 days, with the repair half-time of 0.5 h. The present estimated biologic parameters can reasonably predict the effectiveness of most of the recently reported clinical results employing either single or combined radiation therapy modalities. Different LQ parameters between Grade 3 and Grade 4 astrocytomas were found, implying the radiosensitivity for different grade tumors may be different. Smaller alpha, beta from in vivo was observed, indicating lower radiosensitivity occurred in vivo as compared with in vitro. A plausible set of radiobiologic parameters for gliomas was estimated based on clinical data. These parameters can reasonably predict most of the clinical results. They may be used to design new treatment fractionation schemes and to evaluate and optimize treatment plans.

SU-FF-T-369: An Estimation of Radiobiological Parameters From Clinical Outcomes for Radiation Treatment Planning of Brain Tumor

Purpose: Appropriate organ‐specific radiobiological parameters are crucial for biologically‐based treatment planning. The purpose of this work is to derive a plausible set of such radiobiological parameters for malignant gliomas (MG) based on clinical outcomes. Method and Materials: Several radiobiological models, including the linear quadratic formalism with consideration of repopulation and repair, tumorcontrol probability and equivalent uniform dose, were used to analyze a series of published clinical data for MG involving different regimens of radiation therapy. The least chi‐square χ2 fitting technique was employed to estimate the LQ parameters. Results: A plausible set of LQ parameters: α = 0.08 ± 0.02 Gy−1, α/β = 11.4 ± 8.6 Gy, the tumor cell doubling time Td = 50 ± 30 days, with the repair halftime of 0.5 h was obtained for gliomas. The presently estimated biological parameters reasonably predict the effectiveness of the most of recently reported clinical results employing either single or combined RT modalities. In addiation, the radiosensitivity for grade III and VI astrocytoma was found to be: α = 0.18 ± 0.03 Gy−1, α/β = 6.0 ± 4.1 Gy and α = 0.09 ± 0.04 Gy−1, α/β = 9.0 ± 9.8 Gy respectively. For Grade III, our result agreed with the published in vitro data, while for Grade 4, the α and α/β values estimated presently based on clinical data are smaller than those from in vitro measurements, indicating lower radiosensitivity occurred in vivo as compared to in vitro. The derived α and α/β values demonstrated that GBM is quite radioresistant as known from clinical practice. Conclusion: The radiobiological parameters derived presently for MG can reasonably predict the most of the recently reported clinical results employing either single or combined RT modalities. These parameters can be potentially useful in evaluating, optimizing, and designing biological/functional image guided IMRT strategies.

Impact of tumor repopulation on radiotherapy planning

Biologic/functional imaging (e.g., fluorodeoxyglucose/3'-deoxy-3'-fluorothymidine-positron emission tomography) is promising to provide information on tumor cell repopulation. Such information is important in the design of biologically conformal radiotherapy for cancer. The questions remaining unclear are whether it is necessary to escalate the dose to the regions with rapid cell repopulation in the tumor target and, if so, by how much. The purpose of this work was to address these questions using radiobiologic modeling. The generalized linear-quadratic model, extended to account for the effect of clonogenic cell repopulation, was used to calculate the cell-killing efficiency of radiotherapy. The standard Poisson tumor control probability (TCP) model was used to bridge cell killing to treatment outcome. Prostate cancer was chosen as the example for this study. In situ measurements of prostate cancer patients have shown that the potential doubling time of tumor cells has a large variation, ranging from 15 to 170 days. On the basis of the linear-quadratic and TCP parameters (alpha = 0.14 Gy(-1), alpha/beta = 3.1 Gy, and the number of clonogens K = 10(6)-10(7) cells) determined in earlier studies, we evaluated the influence of tumor cell repopulation during protracted treatment courses on treatment outcome. The dose escalations, which can be used to combat aggressive cell repopulation in regions with different doubling times (15-170 days) and sizes (5, 10, 15, and 40 cm(3) of a 40-cm(3) tumor), were calculated for commonly practiced radiotherapy modalities. The influence of linear-quadratic parameters on this calculation was also considered. The impact of tumor cell repopulation on TCP and the corresponding dose escalation required to account for this impact were investigated for both external beam radiotherapy and permanent implantation. The results indicated that for regions with aggressive tumor cell growth, dose escalation is necessary to compensate for the repopulation effect. For example, for tumors with an effective doubling time changing from 42 days to 15 days, the prescription dose of external beam radiotherapy needs to be increased from 75.6 to 81 Gy to maintain a target TCP of 80% for intermediate-risk prostate cancer. For (125)I implants, dose escalation from 152 to 160 Gy is required for the same target TCP. These data were calculated on the basis of an alpha/beta ratio of 3.1 Gy. Greater dose escalations are required if the alpha/beta ratio is 1.5 Gy (e.g., 88 Gy for external beam radiotherapy or 180 Gy for (125)I implantation for the same treatment outcome). Our study results showed that it is important to cover the entire tumor volume, including all aggressive spots, with the desired prescription dose, especially for low-dose-rate brachytherapy. Dose escalation is necessary to offset the accelerated tumor cell repopulation during prolonged treatment courses. This study provides a preliminary estimate of the dose escalation for prostate cancer based on the in situ measurements of potential doubling time and radiobiologic models. The proposed dose prescriptions are technically feasible for clinical trials.

Tools for making correct decisions regarding hormone therapy. Part II. organ response and clinical applications

Objective To review existing scientific knowledge of the complicated and variable behavior and response to hormone therapy (HT) of different organs during aging, and to summarize long-term consequences on human health. Design A MEDLINE computer search was performed to identify relevant articles. Result(s) Five body organs were evaluated. [1] Physiologic aging of the bone has deleterious consequences on women's health and quality of life. Bone fractures could be attributed to the combination of estrogen depletion and osteoporosis, mechanisms of applied forces, and disturbed brain function, partially reversible by timely estrogen administration. [2] Estrogen seems to have a profound neuroprotective effect. As physiologic aging of the brain is an unhealthy phenomenon, possible intervention is justified. The therapeutic time window seems crucial. [3] The differentiation between response of a healthy or already damaged organ to sex hormones is the key factor to understanding the possible cardioprotective effects. [4] Based on doubling time of tumor cells, intracrinology, epidemiological data on breast cancer, and behavior of breast cancer survivors in response to estrogen treatment, estrogen seems to be mainly a promoter and even a protector of breast cancer survivors. [5] Colon cancer appears to be an estrogen-dependent tumor with a wide therapeutic window, as every report regardless of age and dose demonstrates protective effects. Conclusion(s) Knowledge of each organ's response to aging and sex hormone substitutions demonstrates that the organs could benefit from properly designed intervention. In the wake of the publication of the Women's Health Initiative study, which shocked the medical community, we suggest that the results be reevaluated according to the aforementioned principles, and that menopausal medicine could play an important role.

Mechanisms Linking Hemostatic Factors to Tumor Growth in Mice

A persuasive link between hemostatic factors and cancer has been developed through extensive studies of both human and animal malignancies. Coagulation-, platelet- and fibrinolytic-factors all have been tightly associatedwith tumor growth and/or dissemination. Nevertheless, the mechanisms by which these factors influence tumor growth and progression remain to be fully defined. Recent studies of mice with selected deficits in hemostatic factors have generally supported the hypothesis that hemostatic factors contribute significantly to tumor biology. However, the findings with gene-targeted mice have often been unexpected or tumor model-dependent. Particularly enigmatic was the dual finding that neither fibrin(ogen) nor plasmin(ogen) were significant determinants of Lewis lung carcinoma (LLC) or T241 fibrosarcoma tumor growth following transplantation into the dorsal subcutis of immune-compatible mice. However, follow up studies have revealed that tumor growth is critically dependent on plasminogen when LLC and T241 tumors cells are transplanted to another anatomical location, the footpad. Furthermore, the negative impact of plasminogen deficiency on footpad tumor growth was effectively relieved by genetically superimposing a deficit in fibrinogen. The likely mechanistic basis for the suppression of tumor growth in plasminogen-deficient mice was revealed through transmission electron microscopy studies of tumor tissues. Widespread microvascular thrombi were found within footpad tumors of plasminogen-deficient mice, whereas no such lesions were observed within tumors from mice that also lacked fibrinogen. These occlusive events were shown to result in a substantial lengthening of tumor cell doubling time in vivo, but increased tumor cell dropout is also likely to contribute to the diminution of footpad tumor growth in plasminogen-deficient mice. Taken together, these studies show that both plasminogen and fibrinogen can serve as critical determinants of tumor growth, but their importance is dependent on anatomical location/microenvironment. Furthermore, one target of plasmin(ogen) relevant to tumor success in vivo is intravascular fibrin.

Extinction of the weakest

Purpose: To examine whether changes in the effective doubling time of tumor cells during irradiation of head-and-neck cancer are linked to accumulating dose.Methods and Materials: Optimal fitting of the results of four apparently iso-effective regimens in three recently reported randomized controlled trials (continuous hyperfractionated accelerated radiation therapy [CHART], Radiation Therapy Oncology Group [RTOG] 90-03, and Trans-Tasman Radiation Oncology Group [TROG] 91.01) was attempted using two different types of model of the change in effective doubling time that may occur during treatment. The first involved the traditional approach where doubling time changes at specific times after the start of treatment regardless of fractionation used (“fixed response time” models). The second is where changes in doubling time are linked to accumulating biologic dose (“cellular depletion” models).Results: Changes in effective doubling time occur during radiotherapy for head-and-neck cancer. Data from the three trials can be fitted successfully by functions that imply a continuous reduction in effective doubling time. Models linking the reductions in effective doubling time to the cellular depletion that occurs in the tumor during radiotherapy fit the data satisfactorily. Effective doubling time ultimately reduces to 2 days, or slightly less, during high-dose radiotherapy regimens designed to cure squamous head-and-neck cancer.Conclusions: If the assumption of iso-effectivity is justified then this study indicates that the “repopulation phenomenon” may be best described by a function that depicts a reduction in effective tumor cell doubling time that decreases continuously during treatment down to a certain minimum value. Furthermore, this reduction may be linked to cellular depletion.

Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97.

To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms. Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97, and biological characteristics of the target clones selected by in vivo screening were studied. Two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potential were isolated from the parent cell line. Compared with MHCC97-L, MHCC97-H had smaller cell size (average cell diameter 43 microm vs 50 microm) and faster in vitro and in vivo growth rate (tumor cell doubling time was 34.2h vs 60.0h). The main ranges of chromosomes were 55-58 in MHCC97-H and 57-62 in MHCC97-L. Boyden chamber in vitro invasion assay demonstrated that the number of penetrating cells through the artificial basement membrane was (37.5 +/- 11.0) cells/field for MHCC97-H vs (17.7 +/- 6.3)/field for MHCC97-L. The proportions of cells in G0-G1 phase, S phase, and G2-M phase for MHCC97-H/MHCC97-L were 0.56/0.65, 0.28/0.25 and 0.16/0.10, respectively, as measured by flow cytometry. The serum AFP levels in nude mice 5wk after orthotopic implantation of tumor tissue were (246 +/- 66) microg.L(-1) for MHCC97-H and (91 +/- 66) microg.L(-1) for MHCC97-L. The pulmonary metastatic rate was 100% (10/10) vs 40% (4/10). Two clones of the same genetic background but with different biological behaviors were established, which could be valuable models for investigation on HCC metastasis.

Prognostic relevance of AgNORs in tumor pathology

The importance of the analysis of the silver-stained nucleolar organizer regions (AgNORs) for prognostic purposes in tumor pathology has been reviewed. Current available data from the literature demonstrate that the evaluation of the quantity of interphase AgNORs is an independent prognostic factor in several types of human tumors. Results of our investigations indicate that AgNORs are the most powerful variable predicting survival in patients with pharyngeal carcinoma, multiple myeloma, male breast and prostate carcinoma. The combination of AgNOR counts and histologic pattern allows the stratification of patients with multiple myeloma, pharyngeal and prostate carcinoma into low- and high-risk groups, which could benefit from different therapy. Moreover, AgNOR analysis predicts response to treatment in adult patients with acute myelogenous leukemia, and appears as an independent prognostic factor in a prospective study on renal cell carcinoma. Therefore, AgNOR analysis is a really important prognostic factor for several human neoplasias. The experimental and theoretical justifications for AgNORs as a prognostic factor are also reviewed, in particular the strict correlation between AgNOR quantity and tumor cell doubling time. Lastly, the lack of prognostic significance of AgNOR analysis in some circumstances is critically discussed.

Reproducibility of measurements of potential doubling time of tumour cells in the multicentre National Cancer Institute protocol T92-0045

We compared the flow cytometric measurement and analysis of the potential doubling time (Tpot) between three centres involved in the National Cancer Institute (NCI) protocol T92-0045. The primary purpose was to understand and minimize the variation within the measurement. A total of 102 specimens were selected at random from patients entered into the trial. Samples were prepared, stained, run and analysed in each centre and a single set of data analysed by all three centres. Analysis of the disc data set revealed that the measurement of labelling index (LI) was robust and reproducible. The estimation of duration of S-phase (Ts) was subject to errors of profile interpretation, particularly DNA ploidy status, and analysis. The LI dominated the variation in Tpot such that the level of final agreement, after removal of outliers and ploidy agreement, reached correlation coefficients of 0.9. The sample data showed poor agreement within each of the components of the measurement. There was some improvement when ploidy was in agreement, but correlation coefficients failed to exceed values of 0.5 for Tpot. The data suggest that observer-associated analysis of Ts and tissue processing and tumour heterogeneity were the major causes of variability in the Tpot measurement. The first two aspects can be standardized and minimized, but heterogeneity will remain a problem with biopsy techniques. © 1999 Cancer Research Campaign

The relationship between tumor oxygenation and cell proliferation in human soft tissue sarcomas

In malignant tumors the oxygenation status and tumor cell proliferation are known to influence local tumor control after radiotherapy. However, the relationship between oxygenation status and tumor cell kinetics in human tumors has not yet been described. Newly developed clinically applicable techniques such as oxygen electrode measurements and assessment of tumor cell proliferation rates have been suggested as promising predictive assays. The purpose of the present study was to characterize tumor oxygenation status in soft tissue sarcomas and to compare this with tumor cell kinetics and clinical parameters. Pretreatment tumor oxygenation status was measured by polarographic oxygen needle electrodes and evaluated as the median pO2 and the percentage of pO2 values < or = 5 mmHg and < or = 2.5 mmHg in 22 patients with primary soft tissue sarcomas. All tumors were characterized by histology, grade of malignancy, the level of microscopic necrosis, the level of effective hemoglobin, and magnetic resonance imaging estimation of tumor volume. The tumor cell potential doubling time and labeling index were measured by flow cytometric and immunohistochemical analysis of tumor biopsy specimens after in vivo incorporation of iododeoxyuridine. There was a significant correlation between the median pO2 and the tumor cell potential doubling time (p = 0.041), whereas no correlation was found between the level of hypoxia expressed by the percentage of pO2 values < or = 2.5 and < or = 5 mmHg, respectively, and tumor cell potential doubling time. Furthermore, no correlation was found between either of the three tumor oxygenation parameters and labeling index. The material represented large intertumor heterogeneity in oxygenation status, cell kinetics, and tumor volume, and no correlation was found between oxygenation status and either volume, histopathology, grade of malignancy, or effective hemoglobin. This report is the first to suggest a correlation between tumor oxygenation and tumor cell doubling time, as the fastest proliferating tumor cells were found in the poorest oxygenated soft tissue sarcomas. More data are needed to clarify if this relation is really a true biological phenomenon. Furthermore, tumor oxygenation status of soft tissue sarcomas was heterogeneous and independent of clinical and histopathological parameters.

Cellular Characterization and Retroviral Transduction of Short-Term Breast Cancer Cells

The effort to develop cell lines from surgical specimens has been a difficult goal for years. We derived short-term primary human mammary carcinoma cell lines from breast tumors in 20 of 23 patients. Morphologically, cultured cells showed small cells with high nuclear cytoplasmic ratio and larger cells with abundant dense cytoplasm. The nuclei were round to oval with one to four nucleoli. Immunocytochemically, the cells stained positive for keratin. Tumor cells showed phenotypic overexpression of the breast tumor-associated antigen DF3 compared with normal mammary epithelial cells. The doubling time of tumor cells in vitro ranged from 2.6 to 3.6 days. The cultured cells were characterized as mammary carcinoma cells by their tumorigenicity in nude mice. Of 14 of the 23 short-term cell lines tested, 5 grew in nude mice and eventually regressed, 3 grew progressively in nude mice, and the remaining 6 did not grow within 3 months. To examine the feasibility of cytokine gene transfer into human mammary carcinoma cells, we introduced the cDNA for human tumor necrosis factor-alpha (TNF-alpha) into short-term cell lines with a retroviral vector. In our short-term primary breast cancer cell lines derived from breast tumors, TNF-alpha secretion ranged between 89 ng/10(6) cells/48 h and 479 ng/10(6) cells/24 h. These findings indicate that short-term primary human mammary carcinoma cell lines can be grown consistently from breast tumors, and that retroviral mediated-cytokine gene transfer into short-term human mammary carcinoma cells is feasible and may be of potential use in immunotherapy trials.

Intercomparisons of new and old schedules in fractionated radiotherapy

I n this review the author will compare the schedules mentioned in this issue, and a few other well-known ones, using a standard computed diagram for each, approximately in the order in which they appear in this issue. The emphasis is on whether the high total doses obtained by hyperfractionation can compensate for the rather long overall times of 7 weeks that leave the tumor vulnerable to rapid proliferation. Thames et al ~ pointed out in 1983 that accelerated fractionation (AFX) would in general be better than hyperfractionation (HFX) only for tumor cell populations that doubled more rapidly than every 5 days. The present modeling does not contradict this, but displays many variations, depending on the various schedules. Since then, flow cytometry has made it possible to measure the potential doubling times in human tumor biopsies before the start of treatment. The median value ofTpo , in many types of tumor is in fact about 5 days, with a large spread of 2 to 30 days within each type. Individual values of Tpo ~ are found to correlate well with local control in radiotherapy of head and neck tumors, and also with the outcome of long (7-week) or short (5-week) schedules (see the articles by Begg et al and Awwad in this issue). We do not know whether T~, the actual doubling time of tumor cells during treatment, remains the same as T,~,. It might even speed up, becoming closer to To, the cell cycle time of 1 or 2 days. Reasons for To,. being similar to T~,o, measured before the start of treatment are discussed in the article Brief Summary of Radiobiological Principles earlier in this issue, as well as definitions of terms used in this review.

Tumour cell repopulation during fractionated radiotherapy: Correlation between flow cytometric and radiobiological data in three murine tumours

This study tested whether the potential doubling time of tumour cells measured before or during treatment could predict the repopulation rate of surviving clonogens during fractionated radiotherapy. Tumours used for the study were a fibrosarcoma (SSK 2), an adenocarcinoma (AT 7) and a squamous cell carcinoma (AT 478), all grown subcutaneously in the C3H mouse. Potential doubling times (Tpot) were measured using the thymidine nanalogue iododeoxyuridine (IUdR) and flow cytometry. Results were compared with previous radiobiological studies on these tumours in which repopulation rates during radiotherapy were estimated using the tumour growth delay and tumour cure assays. Fractionated treatments consisted of daily doses of 4 or 8 Gy to clamped (hypoxic) tumours, 6 days per week for 1–3 weeks. Tpot values increased markedly during therapy for two of the tumours (SSK 2 and AT 478), by a factor of more than 10 for AT 478 in the third treatment week. Tpot remained approximately constant for the third tumour (AT 7). In no case was there evidence from the labelling studies of a shortening of Tpot which would suggest accelerated repopulation. From the radiobiological data, effective clonogen doubling times during radiotherapy were calculated from the doses required to produce a given effect in short and long treatment schedules. In the second week of treatment, effective clonogen doubling times in two tumours were approximately equal to the pretreatment Tpot, and shorter than the pretreatment Tpot in the third tumour. At some time during treatment, the surviving clonogens in these tumours therefore proliferated at the same rate or faster than before treatment. The difference between the labelling and radiobiological measurements was ascribed to the fact that, shortly after the start of a fractionated treatment, the IUdR labelling technique measures primarily doomed cells. These results show that kinetic measurements using DNA labelling techniques made during fractionated radiotherapy in most cases do not reflect the proliferation status of the surviving cells which are responsible for treatment outcome. Pretreatment Tpot measurements give a much better indication of the proliferation rate of surviving cells but in some cases may underestimate repopulation during radiotherapy.