Chemoprevention of Cancer

Transcription

1 Chemoprevention of Cancer Michael B. Sporn, MD Scott M. Lippman, MD 30 There are two general approaches to the problem of control of cancer. The classic approach, followed for more than a hundred years, has been to deal with the disease once it manifests in its terminal stages, which are characterized by clinical symptoms or laboratory findings related to the phenomenon of invasiveness or metastasis. Invasion and metastasis have long been considered the true hallmarks of cancer. Once patients have been diagnosed with cancer, as defined by the above criteria, they have then been treated with the various modalities of surgery, radiation therapy, chemotherapy, immunotherapy, or other treatments directed at the eradication of detectable lesions. Despite some spectacular successes in the treatment of relatively rare cancers, this approach has not yet led to a significant decrease in cancer mortality resulting from the common forms of many metastatic epithelial cancers, such as carcinoma of the lung, breast, colon, prostate, pancreas, and other sites. 1 An alternative approach to the problem of cancer control is one that attempts first, to provide new understanding of the fundamental nature of the chronic disease process which eventually leads to invasive and metastatic carcinoma, and second, to develop new pharmacologic agents that arrest or reverse this chronic disease process in its earliest stages, well before it reaches its terminal invasive and metastatic phase. 2, 3 This alternative approach is known as chemoprevention. 2, 4 It is based on the concept that the very term cancer, widely used by clinicians, laboratory scientists, and laypersons alike, is a misnomer, in that it is inadequate to describe the pathogenesis of the disease process as it actually occurs in the patient. The term cancer denotes a static circumstance, whereas the disease in reality is an evolving chronic molecular and cellular process carcinogenesis. By focusing on end-stage invasive and metastatic disease, we lose the opportunity to intervene in the disease process at earlier stages, when it may be more amenable to the use of pharmacologic agents for its prevention. Since 20 years or more may elapse between the original mutagenic initiation of the carcinogenic process and the subsequent development of invasive or metastatic cells, 5, 6 there is a long window of opportunity in which to use preventive agents. Indeed, since there is only a stochastic probability that an early stage of carcinogenesis will progress to a later one, the intrinsic biology of the carcinogenic process suggests that preventive agents can be developed and successfully used. 5, 6 Perhaps the most significant aspect of carcinogenesis is that most people do not develop invasive and metastatic carcinoma, despite endogenous and exogenous mutational burdens and often significant preinvasive neoplastic lesions in various epithelia. The natural history of the disease process of carcinogenesis thus convincingly indicates that there are intrinsic biologic mechanisms that protect the organism against the development of clones of malignant cells. 7, 8 It would therefore seem reasonable to utilize these mechanisms to design strategies for prevention of cancer. It is paradoxical that the phenomenon that not all dysplastic, preinvasive lesions progress to invasiveness has been used as an argument against the clinical use of chemopreventive agents, with the false premise that since a patient may not yet have an invasive lesion (does not yet have cancer ), the patient is therefore healthy. 9 In this regard the oncology community lags far behind the cardiovascular community in recognizing and accepting the importance of early precursor lesions as antecedents of clinically symptomatic and life-threatening disease. In cardiovascular medicine it is well accepted that precursor lesions, such as fatty streaking and arterial plaques, have an important causal relationship to the end-stage clinical outcomes of myocardial infarction and stroke. 10 Great advances have been made in the development of new drugs that can lower cholesterol levels and that have had major impact on lowering mortality from cardiovascular disease. 11 Moreover, the fact that not all arterial precursor lesions progress to a thrombotic or embolic state has not been used as an argument against interventive chemoprevention, and it is generally accepted that patients with extensive precursor lesions in their arterial tree may be at high risk for serious, lifethreatening events. There is widespread acceptance of the concepts that patients with such lesions are not healthy from a cardiovascular perspective, even though they may not be symptomatic, and that it is appropriate to use pharmacologic agents to prevent further progression of early arterial lesions. Rather than dismissing precursor lesions of invasive cancer as biologically insignificant on the grounds that not all of these lesions progress to invasiveness, the oncology community should attempt to understand the intrinsic mechanisms in epithelia that prevent progression of these lesions, and then design new pharmacologic agents that could enhance the activity of such mechanisms. These concepts provide the basis for the ultimate use of chemoprevention to control cancer death rates. AGENTS FOR CHEMOPREVENTION AND THEIR MECHANISM OF ACTION TYPES OF CHEMOPREVENTIVE AGENTS In the broadest sense, agents for chemoprevention of cancer fall into two principal categories 4 : (a) those that prevent the mutagenic initiation of the carcinogenic process ( blocking agents) and (b) those that prevent the further promotion or progression of lesions that have already been established ( suppressing agents). Since mutation continues as part of the entire chronic process of carcinogenesis, the distinction between the two categories, at least in the dimension of time, is artifactual. However useful the concept of twostage carcinogenesis (initiation followed by promotion) was in the past to describe relatively simple experimental systems in laboratory rodents, it can no longer be accepted as a valid model for the process of carcinogenesis in human subjects, in whom mutation and continued initiation of molecular lesions play an ongoing role throughout the rest of the individual s life span. Extensive information is now available on the ability of cells, such as neutrophils and macrophages, to generate potent agents, such as superoxide, hydrogen peroxide, hydroxyl radical, and nitric oxide, all of which can damage deoxyribonucleic acid (DNA). It is now clear that endogenous metabolism as well as exposure to exogenous agents can have major influences on the process of carcinogenesis. 12 In this chapter, we discuss both types of agents, since both have found clinical application. Although various dietary constituents, such as ascorbic acid, β-carotene, folic acid, and α- tocopherol (vitamin E), have been the subject of many clinical trials to prevent cancer (reviewed in the last part of this chapter), for the most part the results with vitamins and other nutrients have been disappointing. It should be clear by now that if chemoprevention is truly to have a practical impact on the control of cancer, it will be necessary to develop a fundamentally pharmacologic approach to the problem. In the face of the intense mutagenic pressure that drives the process of carcinogenesis, it will be necessary to use

2 414 SECTION 4 / Cancer Epidemiology, Prevention and Screening agents that either are potent antimutagens or can significantly alter patterns of gene expression. Retinoids The molecules that have been most intensively studied for chemoprevention of carcinogenesis are the retinoids, which are defined as natural and synthetic analogues of retinol (vitamin A). 2, 13 More than a thousand such molecules have been made by synthetic chemistry, 14 and as knowledge of the receptors that mediate their mechanism of action increases, so also does the number of new agents that are ligands for these receptors. Of particular importance to retinoid studies has been the unification of molecular and cell biology that occurred with the discovery of the steroid receptor superfamily. 15, 16 This has been a major advance in the attempt to develop new agents for the chemoprevention of carcinogenesis, since it is now apparent that the intracellular receptors for the retinoids, vitamin D, and thyroid hormone as well as those for the classic steroids such as estrogens, progestogens, androgens, and glucocorticoids all belong to a superfamily involved in the selective regulation of transcription of specific genes that control cell differentiation and proliferation Studies on the mechanism of action of the above ligands with their respective receptors now provide the basis for rational design, development, and testing of new agents for the chemoprevention of carcinogenesis. The impact of this new knowledge has been especially important in studies of retinoids. Many years ago Wolbach and Howe demonstrated that the normal function of retinoids was essential for the proper regulation of the differentiation and proliferation of all the epithelia that are the common sites of carcinogenesis in men and women. 18 They clearly recognized that during retinoid deficiency there was a failure of stem cells to mature into appropriate differentiated cells; this was accompanied by enhanced cellular proliferation, with the formation of lesions resembling those found in malignant or premalignant tissues. It is now known that retinoids are required to maintain normal differentiation and proliferation of almost all cells, including nonepithelial cells of mesenchymal origin, during both embryogenesis and adult life. 19 Further advances related to the chemoprevention of carcinogenesis came from organ culture and cell culture studies. It was shown that retinoids could reverse the premalignant lesions induced in mouse prostate organ cultures by carcinogenic hydrocarbons such as 3-methylcholanthrene 20 and that retinoids could act directly on cells previously treated with such carcinogens to suppress the appearance of the malignant phenotype. 21 The latter cell culture studies were particularly important because they emphasized that the continuing presence of the retinoid was essential for the suppression of malignancy; removal of the retinoids from the cultures allowed the expression of the transformed state in cells that had previously been exposed to carcinogen. This phenomenon of a continued requirement for a retinoid to suppress carcinogenesis has also been seen repeatedly in many studies in intact animals and undoubtedly is clinically relevant. However, there are also some situations in which retinoids can alter the differentiation of invasive neoplastic cells and induce terminal differentiation. The most striking example of this phenomenon is the induction of terminal differentiation in many types of teratocarcinoma and leukemia cells, of both animal and human origin Several synthetic retinoids have been successfully used in a large number of studies for the prevention of carcinogenesis in experimental animals. Among those that have potential for clinical application are the following: all-transretinoic acid (tretinoin), 4-hydroxyphenyl alltrans-retinoic acid amide (fenretinide), 13-cisretinoic acid (isotretinoin), and 9-cis-retinoic acid. There are six known retinoic acid receptors (RAR) that mediate the actions of these retinoids. The first receptors to be cloned, RARα, RAR-β, and RAR-γ, bind all-trans-retinoic acid and 9-cis-retinoic acid with high affinity, 25, 26 but bind neither fenretinide nor 13-cisretinoic acid. These latter two retinoids are presumably prodrugs; the isomerization of 13-cisto all-trans-retinoic acid occurs readily. However, the enzymatic hydrolysis of fenretinide to the free acid has yet to be shown, either in cell culture or in vivo. More recently three new retinoid receptors, known as RX R-α, RXR-β, and RXR-γ, have been cloned 27 ; these retinoid x receptors (RXRs) bind only 9-cis-retinoic acid, 25, 26 and do not bind any of the other three retinoids just mentioned above. However, since all-trans-retinoic acid can be metabolized to the 9-cis derivative, it is ultimately a potential ligand for the RXRs in vivo. The importance of the RXRs is emphasized by their ability to form heterodimers with many other members of the steroid receptor superfamily, including RARs, the vitamin D receptor, and the thyroid hormone receptor. RXRs and their ligands are thus permissive systems that can modulate the activity of other ligands and receptors in the steroid receptor superfamily. With the recent demonstration that terpenoid molecules other than 9-cis-retinoic acid can act as ligands for RXRs, the functional domain of this system now appears to be even broader than anticipated. There is an extensive literature on the use of retinoids to arrest or reverse the process of carcinogenesis and to prevent the development of invasive carcinoma in experimental animals. 28 Of particular importance are the many studies that have shown efficacy of retinoids when they are administered after animals have been treated with carcinogen; this experimental design is highly relevant to human populations. Significant activity has been shown for all-transretinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid, 29 fenretinide, and many other retinoids, as reviewed by Moon and colleagues. 28 The epithelial sites studied include breast, skin, lung, bladder, pancreas, liver, oropharynx, esophagus, stomach, and prostate. In addition to efficacy as single agents, retinoids have been particularly effective when used in combination with other preventive agents, especially tamoxifen. Tamoxifen Tamoxifen is a nonsteroidal triphenylethylene derivative that binds to the estrogen receptor. 30 It has both estrogenic and antiestrogenic actions, depending on the target tissue. It is strongly antiestrogenic on mammary epithelium, hence its use in both the prevention and treatment of breast cancer; it is proestrogenic on uterine epithelium, hence the current controversy regarding its safety in cancer prevention 9, especially since an increased incidence of endometrial carcinoma has been found in women treated chronically with tamoxifen..30 It is therefore inappropriate to refer to tamoxifen simply as an antiestrogen. The term selective estrogen receptor modulator is more appropriate. Tamoxifen was originally screened in a drug development program oriented toward discovering new contraceptive agents. Although it was effective in rats, it was not a useful drug for control of fertility in women, and it was not until the early 1970s that it was shown to be useful for clinical palliation of advanced breast cancer. Subsequently, animal studies performed in rats, using both dimethylbenzanthracene (DMBA) and nitrosomethylurea as carcinogens, showed that tamoxifen was highly effective in preventing the development of experimental breast cancer 31, 32 ; these results have also been confirmed in the mouse model in which murine mammary tumor virus is the carcinogen. The mechanism of action of tamoxifen is complex. Clearly, its principal mechanism of action is mediated by its binding to the estrogen receptor and the blocking of the proliferative actions of estrogen on mammary epithelium. One suggested mechanism for this antiproliferative action is the induction by tamoxifen of the synthesis of the cytokine transforming growth factor-β (TGF-β), which acts as a negative autocrine regulatory molecule. 33 However, it has also been shown that tamoxifen can induce synthesis of TGF-β in estrogen receptor negative cells, such as fetal fibroblasts. 34 Moreover, immunohistochemical studies have shown that tamoxifen induces the synthesis of TGF-β in the stromal (mesenchymal) compartment of breast cancers, suggesting a paracrine as well as autocrine mechanism of action, independent of an interaction with the estrogen receptor. 35 Reports of some clinical efficacy of tamoxifen in the treatment of women with estrogen receptor-negative breast carcinomas would appear to be in accord with these mechanistic conclusions. 36 Other studies that are in accord with these observations are the findings that tamoxifen can lower the circulating levels of insulinlike growth factor I (IGF-I) in breast cancer patients. 37, 38 IGF-I is a potent mitogen for breast cancer cells and may act by endocrine, paracrine, and autocrine routes to stimulate their growth.

3 Raloxifene Although the risks are actually quite small, there is major concern about the safety of the use of tamoxifen as a chemopreventive agent for the prophylaxis of breast cancer, because of its estrogenic effect on uterine epithelium and the attendant increased risk for development of uterine cancer. Thus, there has been a search for new agents that would resemble tamoxifen in their overall mechanism of action, but would be inhibitory to the growth of uterine epithelium. One such molecule is raloxifene, the chemical structure of which is totally different from that of tamoxifen; tamoxifen is a triphenylethylene derivative, but raloxifene is a benzothiophene. Like tamoxifen, raloxifene binds to the estrogen receptor and has both estrogenic and antiestrogenic actions; it is another estrogen response modifier. It has been shown to have both therapeutic and preventive activity on breast tumors induced in rats by chemical carcinogens. 32 Most notably, in contrast to tamoxifen, raloxifene does not act as an estrogen agonist in the uterus and does not stimulate the growth of uterine epithelium in ovariectomized rats. 39 However, raloxifene is strongly estrogenic in its positive actions on bone and serum lipids; it is a potent agent for prevention of bone loss in the ovariectomized rat. 39 Because of this unusual spectrum of pharmacologic activity, raloxifene is an attractive agent for the prevention of bone loss in osteoporotic, postmenopausal women, which is its primary therapeutic application at present. If large numbers of women are treated chronically with raloxifene for the prevention of osteoporosis, this therapy may also provide a clinical trial of the efficacy of this agent for prevention of breast cancer. Deltanoids (Vitamin D and Its Synthetic Analogs) Another important ligand of the steroid receptor superfamily is 1,25-dihydroxycholecalciferol (1,25-DHCC), the active metabolite of dietary vitamin D. 1,25-DHCC has potent actions in controlling the expression of many genes and can induce differentiation in many tumor cells, particularly those of myeloid lineage. 40,41 However, because of its marked hypercalcemic activity, it is not a suitable agent for clinical chemoprevention. A large number of synthetic analogs of 1,25-DHCC have been made, with the goal of increasing differentiative activity and decreasing calcemic actions 42 ; we have suggested the term deltanoids, analogous to retinoids, for the entire family of natural and synthetic molecules related to 1,25-DHCC. 43 Many of the new analogs are markedly less calcemic and more active in inducing differentiation, and some have been shown to be active in the prevention of breast cancer in animal experiments. 43, 44 The clinical potential for the use of these agents is still unrealized. Finasteride Prostate carcinogenesis in both experimental animals and humans is driven by androgen, in much the same way that mammary carcinogenesis is driven by estrogen. The testosterone metabolite 5α-dihydrotestosterone (DHT) has higher binding affinity for the androgen receptor than testosterone and is believed to play a critical role in the development of the prostate gland. DHT is formed from testosterone by the action of the enzyme 5α-reductase, and several androgen analogs have been developed as antagonists of this enzyme. One of these analogs, finasteride, is now in widespread use to treat benign prostatic hyperplasia (BPH). 45 Although there are essentially no published studies on the use of finasteride to prevent prostate cancer in experimental animals, this agent is now being evaluated for chemoprevention of prostate carcinogenesis in a large clinical trial because of its known molecular mechanism of action and its known clinical efficacy in the treatment of BPH. Difluoromethylornithine Agents that can suppress cell proliferation are obvious candidates for chemoprevention if they have sufficient selectivity. One such molecule is difluoromethylornithine (DFMO), a potent irreversible inhibitor of the enzyme ornithine decarboxylase, which catalyzes the formation of putrescine, a polyamine involved in DNA synthesis. 46 There is a very extensive literature on the use of DFMO to prevent carcinogenesis in animal models of colon, bladder, breast, liver, skin, and stomach cancer. 47 The National Cancer Institute (NCI) has conducted extensive preclinical and clinical toxicologic evaluations of this drug, and further clinical trials are planned. Nonsteroidal Antiinflammatory Drugs A large number of nonsteroidal antiinflammatory drugs (NSAIDs) have shown potent chemopreventive activity in many test systems. Among the NSAIDs that have been studied at length are aspirin, ibuprofen, sulindac, and piroxicam. All of these molecules are cyclooxygenase inhibitors that block prostaglandin synthesis, and they are in widespread clinical use for the chronic treatment of various inflammatory diseases, most notably osteoarthritis or rheumatoid arthritis. There is therefore an abundance of information about the safe dosage for their long-term administration that would be required for a chemoprevention trial. All of these inhibitors of prostaglandin synthesis have been shown to be active in a multiplicity of animal models for the suppression of carcinogenesis, with particular efficacy in preventing experimental colon carcinogenesis. Most recently, attention has been focused on selective cyclooxygenase-2 (COX-2) inhibitors, such as celecoxib, since these have the potential to prevent cancer with less in the way of undesirable side effects than classical NSAIDs. 48 Based on these results, a number of clinical trials have been designed. N-Acetylcysteine and Oltipraz Glutathione in its reduced form (GSH) is a critical molecule in the chemical deactivation of many carcinogens. Since glutathione itself is not a practical agent for chemoprevention, a great deal of effort has been devoted to the development of exogenous agents that would elevate intracellular GSH levels. This principle, termed electrophile counterattack, 49 has been the basis of extensive CHAPTER 30 / Chemoprevention of Cancer 415 investigation. N-acetylcysteine and oltipraz are two of the most important such molecules that act by this mechanism. Both of these agents can block the mutagenic activity of a variety of carcinogens by preventing their binding to DNA; a substantial decrease in DNA adducts has been seen if either N-acetylcysteine or oltipraz is given to animals when they are treated with carcinogens such as aflatoxin, benzo[a]pyrene, or acetylaminofluorene. 50, 51 Both agents are active in animal test systems for the prevention of cancer, and both are in clinical trial. CHEMOPREVENTION TRIALS More than 80 randomized chemoprevention trials involving over 190,000 subjects have been reported to date (Table 30 1). These include premalignancy (Phase II) studies in the head and neck, lung, colon, skin, esophagus, bladder, and cervix, and cancer incidence (Phase III) trials for cancer of the head and neck, lung, colon, skin, breast, esophagus, and stomach. Chemoprevention only recently entered the realm of standard clinical practice, when, following unblinding of the Breast Cancer Prevention Trial (BCPT), the Food and Drug Administration (FDA) approved tamoxifen for breast-cancer risk reduction in high-risk women. Several ongoing Phase II and Phase III trials also show promise for helping to introduce other chemopreventive agents into standard practice in certain settings. 52, 53 HEAD AND NECK Oral Premalignancy Oral leukoplakia is a premalignant lesion that manifests with a white patch unclassifiable as any other disorder. 54 Current therapy is excisional. Chemoprevention may become standard systemic therapy in certain cases, such as those involving extensive multiple lesions or field carcinogenesis, that is, carcinogenic exposure of the field extending from the oral cavity to the lungs. 55 These cases cannot be controlled by local therapy. Oral leukoplakia is an excellent model system for clinical testing of chemopreventive agents with potential activity throughout the aerodigestive tract. This lesion is related to tobacco use and associated with squamous cell carcinoma. It is easily monitored clinically, cytologically, and histologically and is related to carcinogenesis in other aerodigestive tract sites. 54, 55 The oral leukoplakia system has been used for clinical laboratory translational studies of agent effects on histopathologic and other intermediate end-point biomarkers of carcinogenesis Systemic retinoid, β-carotene, vitamin E, and selenium intervention have been used in nonrandomized chemoprevention trials in oral leukoplakia. 52, 54, 58 Only the retinoids have had their preliminary activity confirmed in randomized trials In the only other randomized drug intervention trial, significant activity was reported with topical bleomycin. 65 Although interesting, the potential value of this local approach is limited by its inability to treat the diffuse aerodigestive epithelial field at risk.

4 416 SECTION 4 / Cancer Epidemiology, Prevention and Screening Table 30-1 Randomized Chemoprevention Trials a Study Setting/ Author (Year) End Point Design Number Intervention Primary Outcome Head and Neck Hong et al. (1986) 59 Oral leukoplakia Phase II 44 Isotretinoin (1 2 mg/kg/d) Positive Stich et al. (1988) 62 Oral leukoplakia Phase II 65 Vit A (200,000 IU/wk) Positive Han et al. (1990) 63 Oral leukoplakia Phase II 61 Retinamide (40 mg/d) Positive Lippman et al. (1993) 60 Oral leukoplakia Phase II 70 Isotretinoin (0.5 mg/kg/d) Positive Chiesa et al. (1993) 64 Oral leukoplakia Phase II 80 Fenretinide (200 mg/d) Positive Epstein et al. (1994) 65 Oral leukoplakia Phase II 22 Topical bleomycin (1%) Positive Hong et al. (1990) 69 Prior SCC Phase III 103 Isotretinoin (100 mg/m 2 /d) Positive (SPT) Bolla et al. (1994) 72 Prior SCC Phase III 316 Etretinate (50, 25 mg/d) Negative Lung Heimburger et al. (1988) 77 Metaplasia Phase II 73 Vit B 12 (500 µg/d), folic acid (10 mg/d) Positive Arnold et al. (1992) 74 Metaplasia Phase II 150 Etretinate (25 mg/d) Negative Van Poppel et al. (1995) 78 Micronuclei Phase II 114 βc (20 mg/d) Positive Lee et al. (1994) 75 Metaplasia Phase II 87 Isotretinoin (1 mg/kg/d) Negative Van Poppel et al. (1995) 79 8-oxodG Phase II 122 βc (20 mg/d) Negative McLarty et al. (1995) 80 Metaplasia Phase II 755 βc (50 mg/d), Vit A (25,000 IU q.o.d.) Negative Kurie et al. (1999) 76 Metaplasia Phase II 68 Fenretinide (200 mg/d) Negative ATBC (1994) 83 Lung Cancer Phase III 29,133 βc (20 mg/d); Vit E (50 mg/d) Negative (2x2 factorial) CARET (1996) 87 Lung cancer Phase III 18,314 βc (30 mg/d); Vit A (25,000 IU/d) Negative Pastorino et al. (1993) 88 Prior NSCLC Phase III 307 Vit A (300,000 IU/d) Positive (SPT) van Zandwijk et al. (1999) 89 Prior HNC, Phase III 2,592 Vit A (300,000/150,000 IU/d); NAC Negative NSCLC (2x2 factorial) (600 mg/d) Colon Bussey et al. (1982) 111 FAP Phase II 36 Vit C (3 g/d) Positive (polyp) McKeown-Eyssen et al.(1988) 112 Adenoma Phase II 137 Vits C (400 mg/d) and E (400 mg/d) Negative DeCosse et al. (1989) 113 FAP Phase II 58 Vits C (4 g/d), E (400 mg/d), fiber Positive (polyp) (22.5 g/d) Gregoire et al. (1989) 105 Prior cancer Phase II 30 Calcium (1,200 mg/d) Negative (LI) Stern et al. (1990) 104 Prior FAP Phase II 31 Calcium (1,200 mg/d) Negative (LI) Labayle et al. (1991) 102 FAP Phase II 10 Sulindac (300 mg/d) Positive (polyps) Kikendall et al. (1991) 110 Adenoma Phase II 257 βc (15 mg/d) Negative Barsoum et al. (1992) 106 Adenoma Phase II 14 Calcium (1.25 g/d) Positive (LI) Wargovich et al. (1992) 103 Adenoma Phase II 20 Calcium (2,000 mg/d) Positive (LI) Paganelli et al. (1992) 116 Adenoma Phase II 41 Vits A (30,000 IU/d), E (70 mg/d), Negative (LI) C (1 g/d) Alberts et al. (1992) 115 Adenoma Phase II 100 WBF (2.0 or 13.5 g/d), Calcium (250 Negative (LI) or 1,500 mg/d) Giardiello et al. (1993) 91 FAP Phase II 22 Sulindac (300 mg/d) Positive (polyps) MacLennan et al. (1995) 114 Adenoma Phase II 395 Fat (< 25% of calories), WBF (11 g/d), Negative (polyps) βc (20 mg/d) Bostic et al. (1993) 107 Adenoma Phase II 21 Calcium (1,200 mg/d) Negative (LI) Ronucci et al. (1993) 117 Adenoma Phase II 209 Vits A (30,000 IU/d), C (1 g/d), Positive E (70 mg/d); lactulose (20 g/d) Greenberg et al. (1994) 118 Adenoma Phase II 751 βc (25 mg/d); Vits E (400 mg/d), Negative C (1 g/d) Cats et al. (1995) 108 HNPCC Phase II 30 Calcium (1,500 mg/d) Negative (LI) Baron et al. (1995) 96 Adenoma Phase II 333 Calcium (3,000 mg/d) Negative (LI) Baron et al. (1999) 109 Adenoma Phase II 913 Calcium (3,000 mg/d) Positive Skin Moriarity et al. (1982) 120 AK Phase II 50 Etretinate (75 mg/d) Positive Watson (1986) 121 AK Phase II 15 Etretinate (75 mg/d) Positive Kligman & Thorne (1991) 119 AK Phase II 527 Topical tretinoin (0.05%) Negative Kligman & Thorne (1991) 119 AK Phase II 455 Topical tretinoin (0.10%) Positive Levine et al. (1997) 127 Prior BCC/SCC Phase III 524 Isotretinoin (5 10 mg/d); Vit A Negative (25,000 IU/d) Greenberg et al. (1990) 124 Prior BCC/SCC Phase III 1,805 βc (50 mg/d) Negative Tangrea et al. (1992) 125 Prior BCC Phase III 981 Isotretinoin (10 mg/d) Negative Moon et al. (1997) 126 AK Phase III 2,298 Vit A (25,000 IU/d) Positive Bouwes Bavinck et al.(1995) 123 Renal transplant Phase III 38 Acitretin (30 mg/d) Positive Clark et al. (1996) 128 Prior BCC/SCC Phase III 1,312 Selenium (200 µg/d) Negative Breast Fisher et al. (1998) 130 High risk/bc Phase III 13,388 Tamoxifen (20 mg/d) Positive Veronesi et al. (1998) 134 BC Phase III 5,408 Tamoxifen (20 mg/d) Negative Powles et al. (1998) 135 High risk/bc Phase III 2,471 Tamoxifen (20 mg/d) Negative Fisher et al. (1999) 132 DCIS/BC Phase III 1,804 Tamoxifen (20 mg/d) Positive Veronesi et al. (1999) 136 CBC Phase III 2,849 Fenretinide (200 mg/d) Positive (Continued)

5 CHAPTER 30 / Chemoprevention of Cancer 417 Table 30-1 Randomized Chemoprevention Trials a (Continued) Study Setting/ Author (Year) End Point Design Number Intervention Primary Outcome Esophagus/Stomach Munoz et al. (1985) 137 Geographic risk Phase II 610 Vit A (50,000 IU/wk), riboflavin Negative (200 mg/wk), zinc (50 mg/wk) Zaridze et al. (1993) 140 Geographic risk Phase II 532 Riboflavin (80 mg/wk); Vits A (100,000 Negative IU/wk), E (80 mg/wk), βc (40 mg/d) Blot et al. (1993) 141 Geographic risk Phase III 29,584 Multiple vitamins/minerals Negative Li et al. (1993) 142 Geographic risk Phase III 3,318 Multiple vitamins/minerals Negative Buiatti et al. (1994) 143 Geographic risk Phase II 222 Bismuth 120 mg q.i.d.), amoxicillin Negative (500 mg q.i.d) (H.pylori) Bladder Alfthan et al. (1983) 145 SBT Phase II 32 Etretinate (25 50 mg/d) Positive Pederson et al. (1984) 146 SBT Phase II 73 Etretinate (50 mg/d) Negative Studer et al. (1984) 147 SBT Phase II 86 Etretinate (25 50 mg/d) Positive Lamm et al. (1994) 148 SBT Phase II 65 Megadose vitamins Positive Cervix Byrne et al. (1986) 150 CIN 2, 3 Phase II 26 HLI ( IU/wk) Negative Yliskoski et al. (1990) 151 CIN 1, 2 Phase II 20 HLI ( IU/d) Negative Frost et al. (1990) 152 CIN 2 Phase II 10 IFN-α2b ( IU/wk) Negative Dunham et al. (1990) 153 CIN 1-3 Phase II 14 IFN-α2b ( IU/wk) Negative de Vet et al. (1991) 156 CIN 1-3 Phase II 278 βc (10 mg/d) Negative Butterworth et al. (1992) 154 CIN 1,2 Phase II 235 Folic acid (10 mg/d) Negative Meyskens et al. (1994) 157 CIN 2,3 Phase II 301 Topical tretinoin (0.372%) Positive (CIN 2) Childers et al. (1995) 155 CIN 1, 2 Phase II 331 Folic acid (5 mg/d) Negative All Cancer Hennekens et al. (1996) 160 Healthy men Phase III 22,071 βc (50 mg q.o.d.) Negative Lee et al. (1999) 161 Healthy women Phase III 39,876 βc (50 mg q.o.d.) Negative AK = actinic keratoses; βc = beta-carotene; BC = breast cancer; BCC = basal cell carcinoma; CBC = contralateral breast cancer; CIN = cervical intraepithelial neoplasia; CIN 1 = mild dysplasia; CIN 2 = moderate dysplasia; CIN 3 = severe dysplasia;dcis = ductal carcinoma in situ; FAP = familial adenomatous polyposis; HLI = human leukocyte interferon; HNPCC = hereditary nonpolyposis colon cancer; IFN = interferon; LI = labeling index; NAC = N-acetylcysteine; NSCLC = non small cell lung carcinoma; q.o.d. = every other day; SCC = squamous cell carcinoma; SBT = superficial bladder tumors; SPT = second primary tumors; Vit = vitamin; WBF = wheat bran fiber. a Modified and updated from Table 2 of Lippman et al. 52 Five randomized retinoid trials in oral premalignancy have been reported. The first of these, reported in 1986, was a short-term, placebo-controlled, double-blind study of high-dose isotretinoin. 59 For three months, forty-four subjects received either 1 to 2 mg/kg/d of isotretinoin or placebo. Major clinical responses occurred in 67% (16/24) of isotretinoin recipients and in 10% (2/20) of placebo recipients (p =.0002). Histologic major responses (reversal of atypia) occurred in 54% (13/24) of isotretinoin recipients and 10% (2/20) of placebo recipients (p =.01). Although isotretinoin was active, over half of responders recurred or developed new lesions within 3 months of stopping it. Also, the high-dose isotretinoin regimen was unacceptably toxic for long-term use. A second randomized trial was conducted to solve the toxicity and relapse problems encountered in the first. 60, 61 In an induction phase, 70 patients received high-dose isotretinoin therapy (1.5 mg/kg/d for 3 months). In a subsequent maintenance phase, stable and responding patients were randomized to 9 months of lowdose isotretinoin (0.5 mg/kg/d) or β-carotene (30 mg/d). Fifty-three subjects qualified for full evaluation. Treatment failure (disease progression or new lesion development) during or after maintenance therapy was 8% (2/24) and 55% (16/29) in the isotretinoin and β-carotene groups, respectively (p <.001). The toxic effects of low-dose isotretinoin therapy were generally mild, although significantly greater than those of β-carotene. The maintenance isotretinoin dose produced tolerable and reversible mucocutaneous dryness and hypertriglyceridemia. The three other randomized trials reported significant retinoid activity in oral premalignancy. Natural vitamin A had significant activity in a 6-month placebo-controlled trial in 54 Asian betel nut chewers. 62 The synthetic retinamide, N- 4-(hydroxycarbophenyl) retinamide had significant activity in a 4-month placebo-controlled trial. 63 A maintenance trial tested fenretinide (versus no treatment) in preventing relapse or new lesion development after complete laser resection of premalignant oral lesions. 64 Interim results on 137 randomized patients who received no treatment or fenretinide at a dose of 200 mg/d for 52 weeks and with 1-year follow-up, indicate a significantly lower failure rate in the retinoid arm. There were 11 treatment failures (9 recurrences, 2 new premalignant oral lesions, 0 cancers) in the fenretinide arm and 21 treatment failures (8 recurrences, 12 new lesions, and 1 cancer) in the no-treatment control arm. 64 Adjunctive laboratory studies of retinoic acid receptors, p53 and loss of heterozygosity (LOH) have been integrated into recent clinical retinoid trials in oral premalignancy. Earlier in vitro and in vivo studies show that expression of the RAR-β messenger ribonucleic acid (mrna) is sequentially lost with carcinogenic progression to dysplasia and cancer in the head and neck, and that RAR-β expression can be upregulated by retinoic acid in vitro in cancer cell lines. In a prospective isotretinoin trial, RAR-β mrna was detected via in situ hybridization with antisense RNA in only 21 (40%) of 52 premalignant oral lesions (p = 0.003). 66 RAR-β mrna expression increased significantly in response to high-dose isotretinoin (from 40% to 90%, p <.001), in direct association with clinical response (p =.04). These translational data conform with strong preclinical data indicating that RAR-β is the nuclear receptor most highly regulated by retinoids. Studies of p53 are another major area of translational research within head and neck cancer chemoprevention trials. Frequent alterations of the P53 gene and its protein product occur in head and neck cancer and in adjacent normalappearing and premalignant tissue. 55 A prospective study revealed a lack of p53 modulation by isotretinoin, and a significant correlation between lesion resistance to isotretinoin and levels of p53 accumulation (p =.006). 67 Aneuploidy and LOH (at 3p and 9p) have been documented in oral premalignant lesions, providing evidence of clonal expansion in preinvasive head and neck lesions, and found to be significant predictors of cancer development in this setting. LOH also has been used to monitor molecular response to chemoprevention. 68

6 418 SECTION 4 / Cancer Epidemiology, Prevention and Screening Prevention of Second Primary Tumors Compelling factors support the testing of retinoids in the adjuvant setting of second primary tumor prevention after definitive therapy of primary head and neck cancer Oral premalignancy, which is linked to second primary tumor development through field carcinogenesis and other shared etiologic and biologic features, responds to retinoids. Second primary tumors occur in a diffuse pattern throughout the aerodigestive tract and bladder, making them beyond the control of local therapies. Second primary tumors are a major cause of death following cure of head and neck cancer and are the leading cancerrelated cause of death after resection of earlystage disease. Second primary tumors develop at a constant rate of approximately 6% per year. 52, 54 The first Phase III adjuvant trial to prevent these second primary tumors involved high-dose isotretinoin. 69 Following definitive local therapy of primary head and neck tumors, 103 patients received either isotretinoin (100 mg/m 2 /d) or placebo for 1 year. This isotretinoin regimen was intolerable, and protocol doses were reduced to 50 mg/m 2 /d after the first 44 enrolled patients. At a median follow-up for all patients of 32 months, the rate of second primary tumors was significantly lower in the retinoid group (4%) than in the placebo group (24%) (p =.005). The retinoid, however, had no significant effect on disease recurrence, disease-free survival, or overall survival. A follow-up analysis of this trial after a median of 55 months was recently reported. 70 Isotretinoin s protective effect against all second primary tumors had decreased since the 32- month follow-up but remained statistically significant (p =.04). In the subset of only tobaccorelated second primary tumors, the retinoid s protective effect remained at a similar level of statistical significance (p =.008). Reversible retinoid effects in all second primary tumors and other carcinogenic settings are the norm. The strong long-term retinoid effect, without treatment, against these tobacco-related second primary tumors is unprecedented. Stringent prospective methods employed to effect surgical salvage of second primary tumor patients, most of whom came from the placebo group, may have masked the retinoid s ability to improve survival. The study population included a large percentage of patients with stages III and IV disease, who frequently experience early treatment failure and death from primary disease recurrence. Therefore, this trial could not indicate whether isotretinoin can improve survival in patients with early-stage disease. Isotretinoin s significant activity in this initial second primary tumor prevention trial led to a multicenter, Phase III trial of isotretinoin that was designed to prevent second primary tumors associated with stage I and II head and neck cancer. 71 This trial s design called for low-dose isotretinoin to solve the toxicity problems encountered in the first trial. Investigators in France assessed the efficacy of the synthetic retinoid etretinate in preventing second primary tumors following definitive therapy of stage I-III squamous cell carcinomas of the oral cavity and oral pharynx. 72 By random assignment, patients received either etretinate or placebo at doses of 50 mg/d for 1 month, followed by 25 mg/d for 2 years. Second primary tumor rates in the two study arms did not differ significantly. Interpretation of this trial was clouded by insufficient details on study compliance and second primary tumor diagnostic criteria. Still, certain valuable data were reported. The French trial confirmed earlier prospective data on the high rate of second primary tumors associated with head and neck cancer. After 41 months median follow-up, 24% of placebo recipients had developed second primary tumors. Also, data from this trial support the field carcinogenesis theory concerning aerodigestive tract cancers: approximately 80% of second primary tumors developed in the head and neck, lungs, or esophagus. THE LUNG Premalignancy Premalignant conditions of the lung have been the arena for several clinical and translational chemoprevention trials. 55,73 Four randomized trials of retinoids in smokers with metaplasia have been reported The first of these trials was a placebo-controlled trial of etretinate for the reversal of metaplasia appearing in sputum samples. 74 Therapy lasted 6 months. Metaplasia was reversed in the sputum of 32% of etretinate subjects and 30% of placebo subjects. The second trial was a placebo-controlled trial of isotretinoin. 75 This trial evaluated metaplasia in bronchial biopsy specimens. By random assignment, 87 subjects received either placebo or isotretinoin for 6 months. Both study groups experienced a substantial reduction in metaplasia index: 54% in the isotretinoin arm and 60% in the placebo arm. Smoking cessation was closely correlated with a significant reduction in the index of metaplasia. A more recent study involved fenretinide. 76 This study s overall design (except for the drug) and negative primary result were similar to those of the earlier isotretinoin study. 75 Despite the generally negative data (see Table 30-1), encouraging Phase-IIb data have emerged from recent studies of 9-cisretinoic acid 81 and anethole dithiolthione, 82 especially in former smokers. Prevention of Primary Lung Cancer The NCI-sponsored Alpha-Tocopherol, Beta- Carotene (ATBC) Cancer Prevention Study was a Phase III trial of α-tocopherol and β-carotene to prevent primary lung cancer. The ATBC study involved 29,133 male smokers between 50 and 69 years of age who had smoked an average of one pack of cigarettes a day for approximately 36 years. 83 This trial s 2 x 2 factorial design called for α-tocopherol (50 mg/d) and β-carotene (20 mg/d) to be given in a randomized, double-blind, placebo-controlled fashion. The factorial design allowed the study scientists to assess the individual effects of each agent. Significant increases in lung cancer incidence (18% increase, p =.01) and total mortality (8%, p =.02) occurred in the β-carotene-treated subjects after 6.1 years median follow-up. α-tocopherol had no significant impact on the lung cancer mortality rate, and there was no evidence of an interaction between α-tocopherol and β-carotene. The β- carotene results from this large-scale Phase III trial are consistent with experimental lung carcinogenesis studies. 84, 85 These laboratory data and definitive clinical results contradict the epidemiologic data on β-carotene and underscore the need to confirm data of this type 53 before public health recommendations are made. The α- tocopherol group had a long-term nonsignificant trend of reduced lung cancer incidence and a significant positive secondary analysis in prostate cancer, a 32% decrease in incidence and 41% decrease in mortality. 86 The Beta-Carotene and Retinol Efficacy Trial (CARET) is the other major NCI Phase III lung cancer chemoprevention trial. This trial tested the combination of β-carotene (30 mg/d) plus retinyl palmitate (25,000 IU/d) in 17,000 smokers and asbestos workers. 87 It confirmed the major finding of the ATBC study with its primary finding that the β-carotene combination increased lung cancer risk in this high-risk population. There was no evidence from either the ATBC study or the CARET that β-carotene increased lung cancer risk in non-smokers, former, or moderate (less than one pack a day) smokers. Prevention of Second Primary Tumors Second primary tumors in lung cancer are related to those associated with head and neck cancer through etiology, region, and biology. An adjuvant trial of the natural vitamin A ester retinyl palmitate achieved a significant reduction in lung second primary tumors. 88 Patients were randomly assigned to receive either 300,000 IU of retinyl palmitate daily (150 patients) or no treatment (157 patients) for 12 months following complete resection of stage I non small cell lung cancer. Compliance was over 80%, and toxic effects were minimal. At a median follow-up of 46 months, second primary tumors occurred in 29 (8%) of control patients and in 18 (12%) of retinyl palmitate recipients. Thirteen patients in the retinoid group and 25 in the control group developed tobacco-related second primary tumors, whose development time was significantly shorter in the control arm than in the retinoid arm (p =.045). Recurrence and disease-free and overall survival rates were not significantly different between the retinoid and placebo groups. These encouraging results with second primary tumors and retinoid activity in related carcinogenic systems led to two large-scale Phase III retinoid trials in the setting of second primary tumor prevention; one was in Europe and the other in the United States. The European trial, called EUROSCAN, 89 was an open-label multicenter trial of 2 years of retinyl palmitate and N- acetyl-l-cysteine (NAC)in a 2 x 2 factorial design. Its aim was to prevent second primary tumors following definitive therapy of earlystage head and neck cancer (larynx T is, T 1-3, N 0-1 ;

7 oral cavity T 1-2, N 0-1 ) and non-small cell lung cancer (pt 1-2, N 0-1, and T 3 N 0 ). Involving 2,592 patients, Euroscan found that retinyl palmitate and/or NAC produced no improvement in eventfree survival, survival, or incidence of second primary tumors. The US multicenter phase III Lung Intergroup Trial (LIT) (NCI I ) involved low-dose isotretinoin to prevent second primary tumors after definitive therapy of stage I non small cell lung cancer. 90 Although LIT results were neutral overall, a provocative secondary finding of a drug interaction with smoking status was reported. Lung-cancer recurrence and mortality were potentially increased in current smokers and decreased in non- and former smokers. COLON AND RECTUM Colorectal trial designs have primarily employed the intermediate end points of adenomatous polyp development and response and hyperproliferation markers. Several NSAIDs, calcium salts, fiber, and vitaminmicronutrient combinations have been studied in the prevention of colon cancer. 52, 53, NSAIDs ability to inhibit colon carcinogenesis was suggested by preclinical and epidemiologic studies 52, 53 and is an active area of mechanism research. 101 Sulindac and celecoxib can effectively treat (but not prevent) adenomas in familial adenomatous polyposis FAP High-dose celecoxib (800 mg/d) reduced large-bowel polyposis by 28% 92 and duodenal polyposis, which is difficult to resect, by 14% 93 (versus placebo). The risk of sporadic adenomas was reduced 19% by aspirin (80 mg/d) 95 and 15% by calcium (1,200 mg/d) 96 overall and even greater in later-stage disease 95,97 (versus placebo). Recent randomized cancer epidemiology trials have established a link between dietary factors and cancer prevention. This link led to clinical trials of low-fat, high-fiber and high-fruit-and-vegetable diets in patients with sporadic adenomas Although the clinical trials did not result in reduced shortterm adenoma rates, the randomized epidemiologic studies still underscore the evolution of cancer epidemiology in designing better cancerprevention studies. Trials of vitamins alone in the colon have achieved largely negative results (see Table 30-1). The largest randomized trial of β-carotene or combined vitamins C and E given in a placebocontrolled, 2 x 2 factorial fashion was recently reported. 118 Lasting 4 years, this study included 864 patients having at least one adenoma within 3 months of study entry, and excluded patients with familial polyposis or a history of colorectal cancer. Neither β-carotene nor the vitamin combination was active. THE SKIN Chemoprevention trials in reversing premalignant skin lesions and in preventing skin cancer have been conducted. Topical application of the retinoid tretinoin has exhibited doserelated activity against actinic keratosis in nonrandomized and randomized trials. 119 Systemic retinoid therapy has achieved significant activity against actinic keratosis in two placebo-controlled trials. 120, 121 The NCI has sponsored a series of small trials of isotretinoin that achieved reductions in skin tumor incidence. One of these trials was conducted in five xeroderma pigmentosum patients at extremely high risk for developing nonmelanoma skin cancer. 122 This trial achieved a significant reduction in the number of skin cancers during the 2 years of high-dose isotretinoin (2 mg/kg/d) treatment (p =.02). Subsequent studies from these investigators have shown that this chemopreventive effect was dose related. The beneficial effect of this approach was lost after cessation of treatment. A randomized placebo-controlled trial tested the retinoid acitretin in 38 renal transplant recipients. 123 This study showed significant reductions in premalignant lesions and skin cancers. The chemopreventive effects were reversible after the 6-month retinoid intervention. Five large-scale, long-term Phase III chemoprevention trials have been conducted in subjects at much lower risk of developing skin cancer Only one of these trials was positive. In this trial, retinol (25,000 IU/d) significantly reduced the incidence of primary squamous cell (but not basal cell) skin cancer in patients with actinic keratosis. 126 The other three trials, all negative, involved β-carotene, retinol, very low-dose isotretinoin and selenium in patients with previous skin cancers. The study of selenium (200 µg/day in brewer s yeast) was negative in its primary end point of preventing squamous cell and basal cell carcinomas of the skin in 1,312 patients having histories of non-melanoma skin cancer and living in low-selenium-intake regions of the United States. 128 This trial, however, produced provocative significant positive secondary analyses of selenium effects on prostate, lung, and colon cancer incidence and on total cancer incidence and mortality. A recent novel randomized trial of a topical DNA-repair enzyme in xeroderma pigmentosum resulted in a reduction in the rates of actinic keratosis and basal cell carcinoma. 129 CHAPTER 30 / Chemoprevention of Cancer 419 THE BREAST Based on highly significant positive results of the Breast Cancer Prevention Trial, tamoxifen became the first chemopreventive agent to earn FDA approval. Conducted by the National Surgical Adjuvant Breast and Bowel Project (NSABP), the BCPT compared tamoxifen against placebo in preventing breast cancer in 13,388 women at high risk of this disease. 130 The major high-risk eligibility criteria were age > 60 years old and history of lobular carcinoma in situ (LCIS), or women from 35 to 59 years old with 5-year breast-cancer risk of 1.66% based on the Gail model. The actual overall average, 5- year baseline, breast-cancer risk was 3.2%. At a median follow-up of 55 months, primary invasive breast-cancer findings for the tamoxifen and placebo groups were 89 versus 175, respectively, for a 49% relative reduction (p <.00001). The relative breast cancer risk reduction was similar for all age and risk groups and was limited to ER-positive tumors. Relative risk reductions of invasive breast cancer were 56% and 86% for women with a history of LCIS and atypical hyperplasia, respectively. Tamoxifen achieved a 50% reduction (35 versus 69 cases) in noninvasive breast cancers. Tamoxifen nonsignificantly reduced overall and breast-cancer mortality. Beneficial secondary findings included 19% fewer fractures in the tamoxifen group. Secondary adverse findings associated with tamoxifen were increased endometrial cancers, vascular events and cataracts. Neutral secondary findings included coronary heart disease and depression of mental function. Although the BCPT successfully completed testing its primary hypothesis, it also raised several key unresolved issues, such as effects on mortality, optimal tamoxifen duration, generalizability of results, and the issue of prevention versus treatment. The FDA recently approved tamoxifen for breast-cancer risk reduction in high-risk women. 53 The FDA recommendation is 20 mg/d for 5 years for high-risk women and warns of tamoxifen-associated risks. The FDA also approved and recommended tamoxifen for reducing the incidence of contralateral breast cancers, based on consistent secondary adjuvant data. 131 Adverse tamoxifen effects make this agent s use a complex, highly individualized decision. In general, an improved tamoxifen risk-to-benefit ratio applies in (1) higher breast-cancer risk at any age, (2) any breast-cancer risk at lower age, and (3) hysterectomy at any breast-cancer risk of women > 50 years old. The NSABP B-24 study tested 5 years of tamoxifen (20 mg/d) versus placebo as adjuvant therapy after resection and radiation in 1,804 patients with ductal carcinoma in situ. 132 At 74 months median follow-up, 5-year incidences of all breast cancer events (invasive and noninvasive) were 8.2% and 13.4% in the tamoxifen and placebo groups, respectively, representing a 43% relative risk reduction (p = ). The cumulative incidence at 5 years of all invasive breastcancer events in the tamoxifen group was 4.1% versus 7.2% in the placebo group (p = 0.004). The current NSABP prevention trial is the Study of Tamoxifen and Raloxifene (STAR). Secondary analyses from osteoporosis trials provided the strong rationale supporting raloxifene in the STAR. 131, 133 Two smaller European (Italian and British) tamoxifen breast cancer prevention trials, however, were negative. 134, 135 The major way both trials differed from the BCPT was in having much lower study powers. With 13,388 women and 368 breast cancer events, the BCPT had about two-times the sample size and three times the number of events of both European trials combined. Each trial also had individual differences from the BCPT. The key British-trial differences were a population of younger age, stronger family history, and concurrent use of hormone replacement therapy (26%). The key