Bruce F. Kimler

Selective Estrogen-Receptor Modulators for Primary Prevention of Breast Cancer

Selective estrogen receptor modulators (SERMs) impact a variety of biologic processes regulated by activated estrogen receptor (ER). Depending on the target tissue, physiologic conditions, and their structure, SERMs may exhibit either estrogen antagonist or estrogen agonist effects. SERMs have a long history in the treatment of breast cancer, based on estrogen antagonist activity in ER-positive breast cancer cells. Beginning with tamoxifen, SERMs have moved to the prevention arena, where their partial estrogen effects may provide other organ benefits, particularly for postmenopausal women. It is because of these partial estrogen agonist effects on organs such as the bone, vagina, CNS, and cardiovascular system that SERMs are likely to remain preferable to pure antiestrogens as primary breast cancer prevention agents in the context of total women’s health. The ideal SERM should function as an antiestrogen in the breast and uterus and a partial estrogen agonist in skeletal, cardiovascular, CNS, gastrointestinal tract, and vagina. Further, this ideal SERM should be devoid of procoagulant effects and should not be associated with an increase in perimenopausal symptoms. Although several generations of SERMs have been developed, the ideal SERM for prevention remains elusive. We will review progress to date for SERMs as single agents and potential for combination therapy in the future. BIOLOGY BEHIND THE MULTIFACETED ACTION OF SERMs

Prenatal irradiation : a major concern for the developing brain

Irradiation of the mammalian foetus produces a broad spectrum of congenital abnormalities, growth retardations, developmental delays, and functional deficits, depending upon the dose and the specific gestational phase of irradiation. The developing brain is particularly susceptible to production of deleterious effects, with decreased brain size, behavioural alterations, and mental retardation having been documented. Supplementing the limited human data, rodent models have been extensively used to investigate the specific processes by which relatively low doses, with correspondingly minor cellular damage to the developing neocortex, can produce dramatic postnatal consequences in brain structure and function. The effects of a variety of physical (dose, linear energy transfer, dose rate, fractionation) and biological (species, strain, gestational age, time course post-irradiation) parameters have been examined in an attempt to provide much needed information on such critical aspects as dose response, threshold doses for effect, and extrapolation to human risk estimates. Various acute cellular responses (e.g. appearance of pyknotic cells and macrophages) observed in the developing neocortex 0-24 h after in utero irradiation can be associated with postnatal effects. Moreover, it is possible to correlate thinning of specific layers of the cerebral cortex with specific behavioural aberrations, allowing prediction of brain structural changes from functional alterations, and vice versa. Thus, it is possible to speculate as to the mechanisms and targets for extremely sensitive, radiation-induced cellular damage in the developing foetal brain, that will interfere with the orderly and precisely programmed development of the mammalian brain, leading finally to postnatal expression as delays in growth and development, perturbations in behaviour, and alterations in brain structure.

Univariate and multivariate statistical analysis of high-grade gliomas: The relationship of radiation dose and other prognostic factors☆☆☆

Univariate and multivariate statistical analyses were used to examine the relationships between duration of survival and multiple variables in the presentation and treatment of 82 patients with high-grade gliomas (16 grade 3, 66 grade 4). The median survival time of the eight patients who received less than or equal to 40 Gy to the tumor bed was 16 weeks and was 17 weeks for the three who received between 40 and 50 Gy. Patients who received 50-60 Gy had a median survival time of 62 weeks, compared to 54 weeks in patients who received 60-70 Gy. These differences in median survival time were statistically significant between the extremes (p = 0.0001), as well as between the 40-50 Gy group and the 50-60 Gy group (p = 0.02). However, no significant difference could be detected between the groups receiving 50-60 Gy versus 60-70 Gy. Univariate analysis also identified preoperative performance status, age, histologic grade, extent of surgery, and seizure history as prognostic factors. Cox multivariate analysis was performed to identify variables that were significant in independently predicting duration of survival. Although contemporary studies have shown many variables to be significant in predicting survival, our analysis found that many of them were not independent predictors. The variables which independently predicted improved duration of survival were greater total radiation dose to the tumor bed (p less than 0.0001), superior preoperative performance status (p = 0.003), and grade 3 versus grade 4 (p = 0.04). Younger age at diagnosis was marginally significant (p = 0.07). In the group of 60 patients receiving greater than 50 Gy, a discriminant analysis was also performed. The patients were divided into two groups based on apparent clusters of survival times: greater than or equal to 60 weeks versus less than 60 weeks. The only variable that was found to be predictive of membership in the cluster with longer survival was the presence of seizure activity (p = 0.02). Although univariate and multivariate analyses both showed an apparent statistically significant improvement in survival with increasing total radiation dose to the tumor bed, no additional benefit could be demonstrated for doses greater than 60 Gy.

Use of rhodamine 123 to investigate alterations in mitochondrial activity in isolated mouse liver mitochondria

The fluorescent dye Rhodamine-123, which selectively stains mitochondria depending on the mitochondrial membrane potential, was used with flow cytometry to evaluate alterations in activity of mitochondria isolated from mouse liver. Under in vitro conditions, with succinate and ADP present in the buffer, mitochondrial activity was affected by a variety of metabolic inhibitors that modify membrane potential. These results demonstrate clearly that flow cytometric techniques using Rhodamine-123 can be employed to study activity in isolated mitochondria.

5-Fluorouracil modulation of radiosensitivity in cultured human carcinoma cells

We evaluated conventional pulse exposure versus continuous exposure models of 5-fluorouracil (5-FU) radiosensitization in HT-29 (human colon adenocarcinoma) and DU-145 (human prostate cancer adenocarcinoma) cell lines. Cell survival following treatment with drug and/or radiation was determined by colony formation assays. Radiation was delivered either by itself, approximately midway through a 1-hr exposure to 5-FU (10 micrograms/ml), or at various times following initiation of exposure to 5-FU (0.5 microgram/ml) present throughout the entire period of incubation. Drug concentrations were selected to approximate those achieved in vivo in humans. HT-29 cells showed a plating efficiency of 87% and similar cytotoxicity (survival reduced to 0.57-0.71) for all 5-FU conditions. The Dos of the radiation survival curves were not different for 1 hr of 5-FU exposure versus radiation alone. However, continuous exposure conditions demonstrated statistically significantly different Dos from radiation alone and pulse 5-FU exposure. DU-145 cells displayed a plating efficiency of 17% and cytotoxicities of 0.10-0.91 for the 5-FU conditions. DU-145 cells showed different radiation 5-FU interactions: 5-FU produced statistically significant changes in Do well as the differences between cell lines insofar as their radiosensitization by 5-FU underscore the caution required in extrapolating these radiobiologic models to the clinical setting.

Breast cancer chemoprevention phase I evaluation of biomarker modulation by arzoxifene, a third generation selective estrogen receptor modulator

Purpose: Arzoxifene, a new selective estrogen receptor modulator with strong breast antiestrogen activity and absence of uterine agonist activity, was explored as a potential chemoprevention agent. We performed a multi-institutional evaluation of arzoxifene in women with newly diagnosed ductal carcinoma in situ or T1/T2 invasive cancer. Experimental Design: In a Phase IA trial, 50 pre- or postmenopausal women were randomized to 10, 20, or 50 mg of arzoxifene daily in the interval between biopsy and re-excision or were enrolled as no-treatment controls. In a Phase IB trial, 76 postmenopausal women were randomized to 20 mg of arzoxifene versus matched placebo. Serum specimens collected at entry and at re-excision were assayed for various hormones and growth factors. Tissue from biopsies (estrogen receptor + and/or progesterone receptor +) and re-excision specimens was evaluated immunohistochemically for proliferation (Ki-67 by MIB-1 and proliferating cell nuclear antigen) and other biomarkers. Results: In both trials, increases in serum sex hormone binding globulin were noted, as were decreases in insulin-like growth factor (IGF)-I and the IGF-I:IGF binding protein-3 ratio (P < 0.007 versus control/placebo). For 45 evaluable women in Phase IA, decreases in proliferation indices were more prevalent for arzoxifene (particularly 20 mg) than for controls. For 58 evaluable women in Phase IB, a decrease in estrogen receptor expression for arzoxifene was observed compared with no change with placebo (P = 0.0068). However, decreases in proliferation indices for arzoxifene were not statistically different from placebo, perhaps due to a confounding effect of stopping hormone replacement therapy before entry. Conclusion: Given the favorable side effect profile and the biomarker modulations reported here, arzoxifene remains a reasonable candidate for additional study as a breast cancer chemoprevention agent.

Radioprotection of mice by dietary squalene

Male C3H mice were fed a diet containing 2% squalene for 14 d prior to and 30 d subsequent to exposure to 6, 7 or 8 Gy of whole body γ-irradiation (Cesium-137). After 14 d on squalene-supplemented diet, plasma and jejunal tissue squalene levels were 2X and 15X that of controls. Seven days after irradiation, total white cell counts and total lymphocyte counts were substantially depressed in a radiation dose-dependent manner. Although counts in the squalene group were consistently (18–119%) higher than those in the corresponding dietary control group, the differences between dietary groups at any single dose were not significant. Nuclear area of villus cells in the jejunum of both dietary groups was significantly reduced (20%) by day 11 post-irradiation but the nuclear area in squalene-fed mice was significantly greater (15%) than in controls, before and after irradiation. There were no differences in body weight as a function of either diet or radiation dose prior to the first observations of animal lethality. Animal survival was decreased from 100 to 0% at 30 d post-irradiation by radiation doses of 6–8 Gy, with the greatest difference between dietary groups being observed at 7 Gy (median survival times of 12 and 16 d for control and squalene groups, respectively). Overall, survival of squalene-fed mice was significantly prolonged compared with control-fed mice (P=0.0054 by censored multiple regression analysis). It is concluded that squalene conferred some cellular and systemic radioprotection to mice receiving these lethal whole-body radiation doses.

Omega-3 fatty acids for breast cancer prevention and survivorship.

Women with evidence of high intake ratios of the marine omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) relative to the omega-6 arachidonic acid have been found to have a reduced risk of breast cancer compared with those with low ratios in some but not all case–control and cohort studies. If increasing EPA and DHA relative to arachidonic acid is effective in reducing breast cancer risk, likely mechanisms include reduction in proinflammatory lipid derivatives, inhibition of nuclear factor-κB-induced cytokine production, and decreased growth factor receptor signaling as a result of alteration in membrane lipid rafts. Primary prevention trials with either risk biomarkers or cancer incidence as endpoints are underway but final results of these trials are currently unavailable. EPA and DHA supplementation is also being explored in an effort to help prevent or alleviate common problems after a breast cancer diagnosis, including cardiac and cognitive dysfunction and chemotherapy-induced peripheral neuropathy. The insulin-sensitizing and anabolic properties of EPA and DHA also suggest supplementation studies to determine whether these omega-3 fatty acids might reduce chemotherapy-associated loss of muscle mass and weight gain. We will briefly review relevant omega-3 fatty acid metabolism, and early investigations in breast cancer prevention and survivorship.

Release of interleukin-1 by human alveolar macrophages after in vitro irradiation.

Therapeutic thoracic irradiation may induce two late pulmonary injury syndromes: radiation pneumonitis and subsequent pulmonary fibrosis. The alveolar macrophage has been considered a radioresistant cell and not a target cell involved in the pathogenesis of either type of radiation-induced lung injury. Alveolar macrophage-derived cytokines, including interleukin-1 (IL-1) and tumor necrosis factor (TNF), have been demonstrated to participate in inflammatory and fibrotic responses in the lung after various other types of lung injury. To evaluate whether the release of cytokines by alveolar macrophages is induced by radiation doses used clinically, alveolar macrophages recovered from nonsmoking volunteers were exposed in vitro to a single dose of 2 Gy and then maintained in culture for 18 h. Culture supernatants and cell lysates were then recovered and analyzed for IL-1 alpha and IL-1 beta by radioimmunoassay. Supernatants of irradiated alveolar macrophages contained significantly increased amounts of IL-1 alpha (P < 0.04) and IL-1 beta (P < 0.02) as well as total IL-1 (IL-1 alpha and IL-1 beta) (P < 0.02) compared to nonirradiated alveolar macrophages. Cell lysates of irradiated alveolar macrophages also contained increased amounts of IL-1 alpha and IL-1 beta, although differences from controls were not significant. The finding of increased release of IL-1 by alveolar macrophages after exposure to a single, clinically relevant dose of radiation suggests that the function of human alveolar macrophages is likely altered during therapeutic use of thoracic irradiation. Whether this release of IL-1 by alveolar macrophages contributes to early lung inflammation induced by thoracic irradiation is unclear.

MODELS FOR EARLY CHEMOPREVENTION TRIALS IN BREAST CANCER

Several models are being explored for use in the phase I and phase II evaluation of breast cancer chemoprevention agents. The short-term DCIS/small invasive cancer model is probably best used in late phase I trials in conjunction with agents likely to have activity in the progression phase of neoplastic development in addition to activity in earlier phases. The core biopsy or FNA hyperplasia models may be best used with drugs that are likely to have activity primarily in the promotion phase of neoplastic development and that are suitable for longer duration trials lasting several months to years. Morphology currently is the key surrogate endpoint biomarker for assessing efficacy in phase II trials. Other biomarkers that may undergo modulation will have to be validated, in that modulation will have to be shown to be directly related to decreased cancer risk in subsequent phase III trials. Only then can they be considered as validated surrogate endpoint biomarkers and used as stand-alone efficacy markers in phase II trials. Despite accrual challenges and technologic hurdles, interest in phase I and phase II chemoprevention trials is high.