Characterization of polyclonal and monoclonal anti-taxol antibodies and measurement of taxol in serum.

Anti-taxol antibodies were generated in the rabbit using a taxol-bovine serum albumin conjugate prepared from 2'-succinyltaxol using a mixed anhydride procedure. Immunization with 2'-succinyltaxol-bovine serum albumin gave rise to polyclonal anti-taxol antibodies. By a radioimmunoassay using [3H]taxol, a standard curve gave a 50% inhibitory concentration of 1.0 nM. Taxol levels in human serum could be measured, with the lower limit of detection and measurement being 0.1 nM or 0.085 ng/ml. Two mouse monoclonal anti-taxol antibodies were isolated by immunizing BALB/c mice with the same antigen. One was an immunoglobulin G1 (69E4A8E) and the other was immunoglobulin M (29B7B3C). The specificity of these antibodies was determined by a competitive enzyme-linked immunosorbent assay with taxol and 10 different related derivatives and analogues. 29B7B3C had higher binding affinities for biologically active derivatives and markedly lower affinities for inactive derivatives; i.e., the specificity was consistent with the results of tubulin disassembly and cytotoxicity studies using the same taxol derivatives, making it suitable for screening for taxol or taxol-like compounds in extracts of natural products. 69E4A8E recognized the benzamidocarbamyl group at the C-3' position of taxol and had a lower affinity for other active compounds with different substitutions. Taxol levels in human serum could be detected and measured by 69E4A8E using a competitive enzyme-linked immunosorbent assay. The lower limit of measurement was about 50 nM or approximately 42 ng/ml. Similar measurements could be made by radioimmunoassay.

Taxol sensitizes human astrocytoma cells to radiation.

Taxol is a chemotherapeutic drug which acts by stabilizing microtubules, preventing normal mitosis and resulting in a block of the cell cycle at G2 and M. The drug is isolated from the yew, Taxus sp. L., and is currently being evaluated in a series of Phase II and Phase III clinical trials. Taxol blocks cells in the most radiosensitive phases of the cell cycle and thus could act as a cell cycle-specific radiosensitizer. We report the results of combined taxol-radiation exposures in the human Grade III astrocytoma cell line, G18. Taxol is a potent inhibitor of G18 cell division; a concentration of 10 nM is cytostatic for a cell population observed for at least two doubling times. Cell survival curves for G18 cells showed a significant concentration-dependent interaction between taxol and radiation. Treatment of G18 cells with a fixed taxol concentration and radiation dose showed the interaction to be dependent on the duration of taxol exposure and consequently the fraction of cells in the G2 or M phase of the cell cycle. The sensitizer enhancement ratio for 10 nM taxol at 10% survival is 1.8 and, for 1 nM taxol, it is 1.2. These results suggest that appropriate combinations of taxol have a more than additive interaction in human tissue culture and may have a role in clinical protocols.

Inhibition of the growth of glioblastomas by CGP 41251, an inhibitor of protein kinase C, and by a phorbol ester tumor promoter.

Protein kinase C (PKC) plays a central role in signal transduction pathways that mediate the action of certain growth factors, tumor promoters, and cellular oncogenes. To explore whether PKC might be an appropriate target for the chemotherapy of human brain tumors, cell lines were established from five glioblastomas, one mixed gliosarcoma and glioblastoma, two astrocytomas, and one choroid plexus carcinoma. The staurosporine derivative CGP 41251, an inhibitor of PKC, inhibited cell proliferation in all nine cell lines with an IC50 in the range of 0.4 micrometer. Drug withdrawal and clonogenicity assays showed that CGP 41251 induced an irreversible growth arrest. Three cell lines were examined in detail: two human glioblastoma cell lines, GB-1 and GB-2, and one gliosarcoma cell line, GS-1. All of these three cell lines were highly aneuploid and displayed morphologies and immunohistochemical markers characteristic of the glial lineage. The compound 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter and activator of PKC, also inhibited the growth of these cell lines. CGP 41251 in combination with TPA caused further growth inhibition. Cultures treated with CGP 41251 displayed an increase in the fraction of cells in G2-M, a decrease of cells in S phase, and no consistent effect on G0-G1. Immunohistochemical analyses demonstrated that growth inhibition by CGP 41251 was associated with the formation of giant nuclei with extensive fragmentation and apoptotic bodies. These effects of CGP 41251 were abrogated by withdrawal of serum from the medium or by exposure of these cells to aphidicolin, actinomycin D, cycloheximide, or TPA. In contrast to the effects seen with the glioblastoma cell lines, nontransformed astrocyte lines remained viable in the presence of 0.4 and 0.8 micrometer CGP 41251 and displayed only a slight increase in the fraction of giant nuclei with fragmentation. The antitumor activity of CGP 41251 was demonstrated in vivo against xenografts of the glioblastoma cell lines U87 MG and U373 MG. These findings suggest that CGP 41251 might be a useful agent for the treatment of glioblastomas.

Taxol and radiation.

The cytotoxic effects of Taxol and/or ionizing radiation were evaluated in four human tumor cell lines. The recognized antimicrotubular effects of the drug leading to transitory accumulations of cells in the G2/M phase of the cell cycle, the most radiosensitive phase of the cycle, prompted this assessment of the potential for Taxol to function as a cell-cycle, phase-specific radiosensitizer. Taxol alone was cytotoxic to all four cell lines at low (< 25 nM) concentrations. A Taxol concentration of 10 nM for 24 hours led to 48, 15, 8, and 4.4% of cells retaining clonogenic potential for melanoma, two cervical carcinomas, and astrocytoma, respectively. There were significant Taxol concentration-time-dependent differences in response between the cell lines. Cell lines also showed significant differences in their responses to ionizing radiation. Combined treatment resulted in a demonstration of radiation sensitization with the astrocytoma and melanoma cell lines but not with the cervical carcinoma cell lines. Sensitizer enhancement ratios at the 10% cell survival level were 1.8 for 10 nM Taxol for 24 hours with the astrocytoma cells and 1.2 for 40 nM Taxol for 24 hours with the melanoma cells. The cervical carcinoma cell lines showed an additive effect for radiation and Taxol at all drug concentrations; that is, combined treatments elicit an additive or supra-additive response with, however, no simple relationship between Taxol concentration, Taxol time of treatment, and radiation dose in optimizing cytotoxic effectiveness. Combined modality treatments using relatively low concentrations of Taxol and ionizing radiation can result in an enhanced response and, at the least, an additive response, which could be advantageous in a clinical setting.

Expression of immediate early genes after treatment of human astrocytoma cells with radiation and taxol.

PURPOSE: The promising chemotherapeutic agent, taxol, has been shown to sensitize the G18 line of human astrocytoma cells to ionizing radiation. The present studies were performed to identify specific changes in gene expression associated with this altered sensitivity. METHODS AND MATERIALS: The radioresistant, grade 3 human astrocytoma cell line, G18, was exposed for varying periods of time to treatment with taxol, tetradecanoyl phorbol acetate (TPA), serum, isoproterenol, dibutyryl cyclic adenosine monophosphate, or ionizing radiation alone or in combination with taxol pretreatment. Ribonucleic acid samples from the cells were monitored for the expression of a group of immediate early genes (IEGs), including c-fos, c-jun, TIS1, TIS7, TIS8, TIS11 and TIS21, by northern blot hybridization analysis. RESULTS: Transient immediate early gene induction was observed after treatment of G18 cells with tetradecanoyl phorbol acetate, serum, isoproterenol, or ionizing radiation, but not after treatment with taxol. Of the seven immediate early genes analyzed, all but TIS7 were found to be inducible by one or more of the treatments. Only TIS8 (also known as egr-1 or zif268) was significantly inducible by radiation, and this transient induction was decreased by at least four-fold by pretreatment for 24 hr with a dose of taxol that was previously shown to block 96.5% of the cells in G2/M and enhance radiosensitivity. CONCLUSION: The products of immediate early genes, which are induced transiently in cells in response to a variety of treatments, including growth factors, neurotransmitters, and irradiation with UV light or X rays, are thought to initiate a cascade of genetic responses to alterations in cellular environment. The present results demonstrate a dramatic attenuation in one immediate early gene response in association with a treatment that enhances radiosensitivity in a refractory human brain tumor line.

Physician/patient-driven risk assignment in radiation oncology: reality or fancy?

PURPOSE: Treatment plan optimization in radiation oncology entails designing multiple x-ray beams to irradiate a tumor to a dose that will achieve locoregional control while minimizing normal tissue complications. For some anatomical sites, it is possible to estimate tumor control probabilities (TCP) and normal tissue complication probabilities (NTCP) as a function of radiation dose. Thus, treatment plan optimization can be based on biologic end points rather than on dose calculations alone. Given multiple plans with different NTCPs and TCPs, a tradeoff must be made between maximizing TCP and maintaining an acceptable NTCP. How do physicians reach these decisions? Can the process be quantified? Should patients participate in the process? METHODS AND MATERIALS: Physicians and patients were asked to rank a series of treatment plans having different combinations of TCP and NTCP. Responses were parametrized into a figure of merit (FM) equation which quantifies predilections of TCP and NTCP. RESULTS: Physician-based FM equations are site- and patient-specific. Variations exist among physicians, but treatment plan selection is often conservative in accordance with the primum non nocere dictum. FM equations generated from the responses of patients suggest that some patients may be willing to accept higher treatment toxicity in exchange for increased TCP. CONCLUSION: The term "optimized treatment plan" contains inherently subjective criteria which reflect one's willingness to accept treatment morbidity in exchange for probability of cure. These criteria may differ among patients and/or physicians. A quantifiable FM may permit the design of custom-made treatment plans that include physician and patient input.

Pulsed-dose-rate brachytherapy: design of convenient (daytime-only) schedules.

PURPOSE: To design pulsed-brachytherapy (PDR) protocols that are expected to be at least as clinically efficacious (in terms of both tumor control and late sequelae) as continuous low-dose-rate (CLDR) regimens, but that involve irradiation only during extended office hours. Both interstitial and intracavitary brachytherapy protocols are considered. METHODS AND MATERIALS: The linear quadratic formalism was used in which the late normal tissue damage and tumor control for one protocol relative to another are assumed to be determined primarily by the level of cellular survival. PDR schedules were designed in which pulses are delivered during "extended office hours" (8 A.M. to 8 P.M.) with no irradiation overnight. Generally, the proposed PDR regimes last the same number of treatment days as the corresponding CLDR regimen, but the PDR treatment lasts longer on the final day (i.e., until 8 P.M.). PDR doses were calculated such as to produce a tumor control which is equivalent to standard CLDR protocols, and the corresponding predicted late complication rate was compared with that for CLDR. Ranges of plausible values for the half-times of sublethal damage repair for tumors and for late-responding normal tissues were considered. RESULTS: As has been previously shown, the efficacy of PDR relative to CLDR depends considerably on the repair rates for sublethal damage repair. Clinical and experimental evidence suggests that average repair half-times for early effects (e.g., tumor control) are less than about a half hour, and for late sequelae are more than about an hour. If these estimates are correct, daytime PDR regimes can usually be designed which take the same number of days as the corresponding CLDR regimen, but have comparable or better therapeutic ratios than CLDR. CONCLUSION: Protocols for PDR can be designed to involve irradiation only during extended office hours, that are likely to result in clinical results comparable or better than CLDR, for any expected combination of the repair half-times of early- and late-responding tissues. The suggested protocols allow all of the advantages of a computerized remote-controlled afterloader while preserving the benefits of low dose rate. In addition, the protocols could allow the patient to go home overnight, or to stay overnight in an adjacent medical inn or hospital-associated hotel, rather than in a hospital bed-which could have major economic benefits. In such an economic situation, an extra treatment day for the daytime PDR could well be considered, which would virtually guarantee an improved clinical advantage relative to CLDR.

Prostate-specific antigen density: a new prognostic indicator for prostate cancer.

PURPOSE: Prostate specific antigen density, previously described as a ratio of serum prostate specific antigen to the volume of the prostate, has been shown to be an important factor in the discrimination of patients with occult metastatic disease and patients with benign versus malignant prostatic disease. We undertook a retrospective study to determine if prostate specific antigen density was a predictor of outcome following definitive conformal radiation therapy. METHODS AND MATERIALS: Between January 1989 and August 1991, 86 patients with localized prostate cancer (confined to the prostate, periprostatic tissue, or seminal vesicles) were treated in the Department of Radiation Oncology, Columbia-Presbyterian Medical Center with definitive radiation therapy using computed tomography-guided conformal technique. Thirteen patients were excluded on the basis of prior prostatectomy, hormonal therapy, or no pretreatment prostate specific antigen measurement. Seventy-three patients were evaluable: 19% (14/73) American Urologic Association Stage A (T1), 41% (30/73) B (T2), and 40% (29/73) C (T3). Prostate specific antigen density was defined as the ratio of the pretreatment serum prostate specific antigen to the prostate volume as determined from computed tomography treatment planning scans. Pretreatment prostate specific antigen density was calculated for each patient and ranged from 0.04-3.85 with a mean and median value of 0.66 and 0.33, respectively. Prostate specific antigen failure was defined as a rise above normal level or, for patients whose nadir was above 4 ng/ml, an increase of greater than 10% above nadir. Mean prostate specific antigen follow-up was 13 months (range 2.3-31 months) by which time 66% of patients had normal prostate specific antigen (< or = 4 ng/ml) levels. RESULTS: Nine patients experienced prostate specific antigen failure. The mean prostate specific antigen density of patients with disease-free survival versus failures was 0.53 and 1.6, respectively (p < 0.05). Kaplan-Meier analysis showed that patients with a prostate specific antigen density < or = 0.3 (n = 30) had 100% actuarial disease-free survival at 30 months compared with 62% for patients with prostate specific antigen density > 0.3 (n = 43, p < 0.01). Patients with a prostate specific antigen density < or = 0.6 (n = 52) and > 0.6 (n = 21) had an 88% and 57% actuarial disease-free survival at > 24 months (p < 0.05). CONCLUSION: Prostate specific antigen density was an excellent predictor of disease-free survival (p < 0.01) and was superior to clinical stage (p > 0.05), Gleason's score (p > 0.05), and pretreatment prostate specific antigen (p < 0.05). These results suggest that patients with low prostate specific antigen density (< or = 0.3), including those with locally advanced clinical stage, high Gleason's score, or elevated pretreatment prostate specific antigen, do well with conventional radiation therapy and should not be subjected to high risk protocols. Further follow-up will be required to determine if patients with low prostate specific antigen density will have improved overall survival.

Taxol: a novel radiation sensitizer.

The investigational antineoplastic agent, taxol, a natural product from the yew, Taxus sp. L., is currently being evaluated in a series of Phase II clinical trials. To date, the drug has shown activity against ovarian cancer, lung cancer, and melanoma. Taxol is a potent microtubule stabilizing agent that selectively blocks cells in the G2 and M phases of the cell cycle and is cytotoxic in a time-concentration dependent manner. It is well known from radiobiological principles that G2 and M are the most radiosensitive phases of the cell cycle. On the rationale that taxol could function as a cell-cycle selective radiosensitizer, we examined the consequences of combined drug-radiation exposures on the human grade 3 astrocytoma cell line, G18. Survival curve analysis shows a dramatic interaction between taxol and ionizing radiation with the degree of enhanced cell killing dependent on taxol concentration and on the fraction of cells in the G2 or M phases of the cell cycle. The sensitizer enhancement ratio (SER) for 10 nM taxol at 10% survival is approximately 1.8. These results obtained with cycling aerated radioresistant brain tumor cells indicate that significant advantage may derive from appropriate time-concentration dependent interactions in combined modality protocols.

Dosimetric and volumetric criteria for selecting a source activity and a source type ((125)I or (103)Pd) in the presence of irregular seed placement in permanent prostate implants.

PURPOSE: The dosimetric merit of a permanent prostate implant relies on two factors: the quality of the plan itself, and the fidelity of its implementation. The former factor depends on source type and on source strength, while the latter is a combination of skill and experience. The purpose of this study is to offer criteria by which to select a source type ((125)I or (103)Pd) and activity. METHODS AND MATERIALS: Given a prescription dose and potential seed positions along needles, treatment plans were designed for a number of seed types and activities, specifically for (125)I with activities ranging from 0.3 to 0.7 mCi, and for (103)Pd with activities in the range of 0.8 to 1.6 mCi. To avoid human planner bias, an automated computerized planning system based on integer programming was used to obtain optimal seed configurations for each seed type and activity. To simulate the effect of seed-placement inaccuracies, random seed-displacement "errors" were generated for all plans. The displacement errors were assumed to be uniformly distributed within a cube with side equal to 2sigma. The resulting treatment plans were assessed using two volumetric and two dosimetric indices. RESULTS: For (125)I implants a coverage index (CI) of 98.5% or higher can be achieved for all activities (CI is the fraction of the target volume receiving the prescribed or larger dose). The external volume index (EI) (i.e., the amount of healthy tissue, as percentage of the target volume, receiving the prescribed or larger dose) increases from 13.9% to 20% as the activity increases from 0.3 to 0.7 mCi. For implants using (103)Pd, the external volume index increases from 10. 2% to 13.9% whenever CI exceeds 98.5%. Volumetric and dosimetric indices (coverage index, external volume index, D90, and D80) are all sensitive to seed displacement, although the activity dependence of these indices is more pronounced for (125)I than for (103)Pd implants. CONCLUSIONS: For both isotopes, the lower activities studied systematically result in lower EIs. If seeds can be placed within approximately 0.5 cm of their intended position (103)Pd should be preferred because its EI is lower than that of (125)I. For all activities the coverage indices and D90 are within the required range. If seed placement uncertainties are larger than 0.5 cm, (125)I provides slightly better target coverage; however, in terms of external volume (healthy tissue) covered, (103)Pd is superior to (125)I.

Dosimetric considerations for catheter-based beta and gamma emitters in the therapy of neointimal hyperplasia in human coronary arteries.

PURPOSE: Recent data indicate that intraluminal irradiation of coronary arteries following balloon angioplasty reduces proliferation of smooth muscle cells, neointima formation, and restenosis. We present calculations for various isotopes and geometries in an attempt to identify suitable source designs for such treatments. METHODS AND MATERIALS: Analytical calculations of dose distributions and dose rates are presented for 192Ir, 125I, 103Pd, 32P, and 90Sr for use in intracoronary irradiation. The effects of source geometry and positioning accuracy are studied. RESULTS: Accurate source centering, high dose rate, well-defined treatment volume, and radiation safety are all of concern; 15-20 Gy are required to a length of 2-3 cm of vessel wall (2-4 mm diameter). Dose must be confined to the region of the angioplasty, with reduced doses to normal tissues. Beta emitters have radiation safety advantages, but may not have suitable ranges for treating large diameter vessels. Gamma emitters deliver larger doses to normal tissues and to staff. Low energy x-ray emitters such as 125I and 103Pd reduce these risks but are not available at high enough activities. The feasibility of injecting a radioactive liquid directly into the angioplasty balloon is also explored. CONCLUSIONS: Accurate source centering is found to be of great importance. If this can be accomplished, then high energy beta emitters such as 90Sr would be ideal sources. Otherwise, gamma emitters such as 192Ir may be optimal. A liquid beta source would have optimal geometry and dose distribution, but available sources, such as 32P are unsafe for use with available balloon catheters.

Which patients with newly diagnosed prostate cancer need a radionuclide bone scan? An analysis based on 631 patients.

PURPOSE: Although radionuclide bone scans are frequently recommended as part of the staging evaluation for newly diagnosed prostate cancer, most scans are negative for metastases. We hypothesized that Gleason score, prostate-specific antigen (PSA), and clinical stage could predict for a positive bone scan (BS), and that a low-risk group of patients could be identified in whom BS might be omitted. METHODS: All patients who had both pathologic review of their prostate cancer biopsies and radionuclide BS at our institution between 1/90 and 5/96 were studied. Gleason score, PSA, and clinical stage (AJCC, 4th edition) were evaluated by univariate and multivariate analyses for their ability to predict a positive BS. Groups analyzed were Gleason of 2-6 vs. 7 vs. 8-10; PSA of 0-15 vs. greater than 15-50 vs. greater than 50; and clinical stage of T1a-T2b vs. T2c-T4. Univariate analysis using chi(2) and multivariate analysis using logistic regression were performed. RESULTS: Of the 631 consecutive patients, 88 (14%) had positive BS. Multivariate analysis (64 excluded due to missing PSA and/or clinical stage) showed Gleason score, PSA, and clinical stage to be significant independent predictors for positive BS (p < 0.002, p < 0.001, p < 0.001, respectively). The odds ratios were 5.25 (confidence interval [CI], 3.43-8.04) for PSA > 50 vs. 0-15; 2.25 (CI, 1.43-3.54) for Gleason of 8-10 vs. 2-6; 2.15 (CI, 1.54-2.99) for clinical stage T2c-T4 vs. T2b or less. Three of 308 (1%) had a positive BS in patients with Gleason 2-7, PSA of 50 or less, and clinical stage of T2b or less. In the subset of the same risk group with PSA of 15 or less, all 237 had negative bone scans. In patients with PSA greater than 50, 49/99(49.5%) had positive BS. CONCLUSION: Gleason score, PSA, and clinical stage were independent predictors for a positive radionuclide BS in newly diagnosed prostate cancer patients. PSA is the major predictor for positive BS. About one-half of the patients analyzed were in the low-risk group (Gleason 2-7, PSA < or = 50, clinical stage < or = T2b) and elimination of BS in these patients would result in considerable economic savings.

Changes in biochemical disease-free survival rates as a result of adoption of the consensus conference definition in patients with clinically localized prostate cancer treated with external-beam radiotherapy.

PURPOSE: The optimal definition of biochemical recurrence of prostate cancer after definitive radiotherapy remains elusive. Different institutions have developed their own definitions, and a consensus conference (CC) sponsored by the American Society for Therapeutic Radiology and Oncology has recently proposed another definition. This study compares the definition previously used at our institution with the definition proposed by the CC. METHODS: Two hundred and eight patients were treated for localized prostate cancer with conformal external-beam radiotherapy between 1989-1993 at our institution and followed for at least 24 months. Patients were categorized as failures according to our institutional definition and the CC definition. Our definition (CPMC) required two increases in serum prostate specific antigen (PSA) over at least a 3-month period with a final value of at least 1 ng/ml or a single value resulting in clinical intervention. The CC definition required three consecutive increases in PSA. This was modified to also consider those patients with one or two increases leading to clinical intervention as failures. Differences in the failure rates between the two definitions were evaluated and factors influencing these differences were explored. In an additional analysis, CC was modified such that patients with one or two PSA increases were censored at the time of the PSA prior to the increases (CC-II), rather than at the last PSA (CC). The median follow-up time was 31 months. RESULTS: There were 36 fewer failures according to CC (n = 96) compared with CPMC (n = 132) (p < 0.001). Twenty cases called failures by CPMC subsequently had a decrease in PSA ("false failures"). The other 16 patients have had two increases in PSA, but are awaiting their next follow-up visit to obtain a third PSA ("pending failures"). Analysis of factors predicting "pending failures" showed Gleason score to be the sole predictor of this change in status in multivariate analysis (p = 0.03) with patients with lower-grade tumors being more likely to change status (Gleason 2-6: 15% vs. Gleason 7-10: 1%). On the other hand, "false failures," compared to true failures, had a lower mean PSA nadir (1.7 ng/ml vs. 7.0 ng/ml, p < 0.001) and significantly smaller mean increases in PSA (1st increase: 0.6 ng/ml vs. 3.4 ng/ml, p = 0.006; 2nd increase: 0.4 ng/ml vs. 4.8 ng/ml, p = 0.002). In 85% (17 of 20) of these patients, at least one of the increases was < or = 0.3 ng/ml compared with 44% (42 of 96) of the true failures (p = 0.0008). CC-II resulted in a small decrease in BDFS rates compared with CC, but did not affect the overall difference between CC and CPMC. A modified definition that defines failure as two consecutive increases in PSA over 3 months, with a final value greater than 1.0 ng/ml and each increase being at least 0.3 ng/ml, or three consecutive increases would result in a "false" failure rate of only 3% (3 of 99) and identify 56% (54 of 96) of the true failures after only two PSA increases. CONCLUSION: The CPMC definition of two PSA increases can falsely identify patients as failures, particularly if the increases in PSA are small (i.e., < or = 0.3 ng/ml). The CC definition requiring three increases in PSA can falsely identify patients as disease-free when the time to failure is long relative to the follow-up time. We propose a that a definition that combines aspects of both definitions (two consecutive increases in PSA over 3 months, with a final value greater than 1.0 ng/ml and each increase being at least 0.3 ng/ml, or three consecutive increases) may be a better definition of biochemical failure.

Re-examining the role of prostate-specific antigen density in predicting outcome for clinically localized prostate cancer.

OBJECTIVES: To evaluate the prognostic significance of prostate-specific antigen density (PSAD) in clinically localized prostate cancer and determine whether this index is independent of or superior to prostate-specific antigen (PSA) in predicting outcome of patients treated with external beam radiotherapy. METHODS: Between January 1989 and December 1993, 175 evaluable patients with clinically localized prostate cancer received definitive radiotherapy using computed tomography (CT)-guided conformal techniques. PSAD was defined as the ratio of the pretreatment serum PSA to the prostate volume measured from CT treatment planning scans by one investigator. All PSA values were determined using the Hybritech assay. Biochemical failure was defined as two consecutive elevations in PSA separated by at least 3 months and a final PSA value greater than 1 ng/mL. RESULTS: Multivariate analysis including PSA and Gleason score revealed both to be statistically significant predictors of biochemical disease-free survival (P = 0.048 and P < 0.001, respectively). PSAD did not achieve significance on regression analysis. A direct multivariate analysis including PSA and PSAD required dichotomization in order to reduce high correlation. This analysis demonstrated a relative risk (RR) for failure of 1.27 (NS) for high PSA versus low PSA compared with a RR of 1.20 (NS) for high PSAD versus low PSAD. A regression model containing all three variables indicated only the Gleason score as significant in predicting biochemical failure. CONCLUSIONS: These data do not suggest that PSAD is either an independent prognostic factor or a stronger discriminant of outcome than PSA in patients with clinically localized prostate cancer treated with definitive external beam radiotherapy. Larger patient numbers with longer follow-up data, use of a clinical end point, or an analysis restricted to the appropriate subgroup may demonstrate the utility of PSAD in the future.

Biologic classification as an alternative to anatomic staging for clinically localized prostate cancer: a proposal based on patients treated with external beam radiotherapy.

OBJECTIVES: The prognostic significance of clinical stage in patients with prostate cancer who are treated with external beam radiotherapy is unclear. This study evaluates multiple pretreatment factors, including clinical stage, to determine which are the best prognostic factors, and develops a classification system based on these factors. METHODS: All 249 evaluable patients with clinically localized adenocarcinoma of the prostate treated with definitive conformal external beam radiotherapy without androgen deprivation at our institution between 1989 and 1993 were analyzed. Clinical stage, serum PSA level, Gleason score, race, and history of transurethral resection of the prostate (TURP) were evaluated for their ability to predict biochemical disease-free survival (BDFS). Factors predictive of BDFS were then used to construct a classification system. The classification system was then analyzed for its ability to predict BDFS, distant metastases, local recurrence, and clinical disease free survival in univariate and multivariate analyses. Median follow-up was 27 months. RESULTS: Gleason score and PSA predicted BDFS in multivariate analysis (both P <0.0001), whereas clinical stage, race, and history of a TURP did not. These two biologic factors were combined into a four-level classification system. This classification system was analyzed together with Gleason score and PSA and was found to be the only predictor of BDFS on multivariate analysis (P <0.0001). In addition, this classification system was the only predictor of distant metastases in multivariate analysis (P <0.0001). CONCLUSIONS: The classification system derived herein based on the biologic factors of Gleason score and serum PSA levels is the sole predictor of distant metastases and biochemical recurrence for patients treated with definitive conformal external beam radiotherapy for clinically localized prostate cancer. This classification system may be useful when comparing competing therapies and stratifying patients in clinical trials, but requires validation from other institutions and other therapies prior to its widespread use.

The lack of effectiveness of (-)-epicatechin against alloxan induced diabetes in Wistar rats.

(-)-Epicatechin, a naturally occurring flavonoid, has been reported to protect pancreatic beta cells from alloxan-induced diabetes (1) and to stimulate beta cell regeneration when given after alloxan administration (2,3). However, in the present study, we have not been able to confirm these findings. Administration of (-)-epicatechin (100 mg/kg/day for 15 days) beginning three days after alloxan administration (140 mg/kg) had no significant effect on blood glucose levels when compared to alloxan control animals. Another study which failed to demonstrate an antidiabetic effect of (-)-epicatechin (4) has been criticized for not using fresh solutions of (-)-epicatechin because of its instability in aqueous solution (5). We have found, however, that (-)-epicatechin is stable for at least five days based on thin-layer chromatographic analysis and optical rotation measurements.

Treatment of human glioblastoma cells with the staurosporine derivative CGP 41251 inhibits CDC2 and CDK2 kinase activity and increases radiation sensitivity.

CGP, 41251, a staurosporine derivative, is a potent inhibitor of protein kinase C (PKC). In recent studies we found that this compound causes growth inhibition and induces apoptosis in human glioblastoma cell lines and also inhibits the growth of xenografts of a human astrocytoma. In this study we investigate its effects on cell cycle control. Treatment of glioblastoma or gliosarcoma cells with CGP 41251 lead to a time and dose dependent increase of the percentage of cells in the G2-M phase of the cell cycle. This correlated with a decrease of CDC2- and CDK2-associated histone H1 kinase activities as well as a decrease in the cellular level of the CDC2 protein. The decrease of CDC2- associated histone H1 kinase activity was detected within 5 hours, and there was complete inhibition after 24 hours. Assays of mixtures of cell extracts obtained from cultures treated with CGP 41251, the inactive analog CGP 42700, or untreated cultures indicated that this decrease was due to a decrease in the CDC2 kinase itself rather than the accumulation of an inhibitor of this kinase. In vitro assays in which CGP 41251 was added directly to the in vitro assay system revealed marked inhibition of both CDC2- and CDK2-associated kinase activity at about 1 microM. Thus CGP 41251 inhibits CDC2- and CDK2-associated kinase activities both in vivo and in vitro. Its biologic effects may, therefore, not be due simply to inhibition of PKC. Since cells in the G2-M phase of the cell cycle are relatively more sensitive to killing by gamma- radiation than cells in other phases of the cell cycle, we carried out radiosensitization studies. We found that CGP 41251 was a radiation sensitizer in two glioblastoma cell lines. Therefore, this compound may be useful in the treatment of glioblastomas, possibly in combination with radiation therapy.

Growth inhibition induced by Ro 31-8220 and calphostin C in human glioblastoma cell lines is associated with apoptosis and inhibition of CDC2 kinase.

Protein kinase C (PKC) is a central component in signal transduction and growth control and might be an appropriate target for the chemotherapy of human brain tumors. This study demonstrates that the staurosporine derivative Ro 31-8220, a potent PKC inhibitor, inhibited the growth of 7 human brain tumor cell lines with an IC50 of about 2 microM. Calphostin C, a structurally unrelated PKC inhibitor, inhibited the growth of two of these cell lines with an IC50 of about 100 to 300 nM. Drug withdrawal and clonogenicity assays indicated that the growth inhibition by both of these compounds was irreversible. Morphologic studies, DNA fragmentation studies and flow cytometric assays showed that the treated glioblastoma cells underwent apoptosis. Treatment of glioblastoma cells with Ro 31-8220 lead to a rapid decline in the level of the anti-apoptosis protein bcl-2. At least three of the glioblastoma cell lines carried mutant p53 alleles with missense mutations in the DNA binding domain of p53. Therefore, the induction of apoptosis in these cell lines occurred through a p53-independent mechanism. Furthermore treatment of these glioblastoma cell lines with Ro 31-8220 or calphostin C led to an increase of cells in the G2-M phase of the cell cycle. This correlated with a decrease in CDC2-associated histone H1 kinase activity, as well as a decrease in the level of the CDC2 protein as shown by immunoblotting. When added to subcellular assays Ro 31-8220 markedly inhibited CDC2 histone H1 kinase activity with an IC50 of 100 nM, but calphostin C directly inhibited this kinase activity only at very high concentrations (above 100 microM). Thus these compounds inhibit the growth of glioblastoma cells through novel mechanisms. Ro 31-8220, in particular, might be a useful agent for the treatment of human brain tumors.

Chemical constituents of Echites hirsuta (Apocynaceae).

A phytochemical investigation of an ethanolic extract of the whole plant of Echites hirsuta (Apocynaceae) resulted in the isolation and identification of the flavonoids naringenin, aromadendrin (dihydrokaempferol), and kaempferol; the coumarin fraxetin; the triterpene ursolic acid; and the sterol glycoside sitosteryl glucoside.

Chemical constituents of fruit of Cocculus carolinus D.C. (Menispermaceae).

A phytochemical investigation of an ethanolic extract of the fruit of Cocculus carolinus resulted in the isolation and characterization of the alkaloids, cocculolidine and cocculine. The cyccculine, magnoflorine, and palmatine, previously reported in the stems and leaves of this species, were also isolated and identified.