Cancer Chemoprevention Studies Research Articles

Vitamin C-induced decomposition of lipid hydroperoxides to endogenous genotoxins.

Epidemiological data suggest that dietary antioxidants play a protective role against cancer. This has led to the proposal that dietary supplementation with antioxidants such as vitamin C (vit C) may be useful in disease prevention. However, vit C has proved to be ineffective in cancer chemoprevention studies. In addition, concerns have been raised over potentially deleterious transition metal ion-mediated pro-oxidant effects. We have now determined that vit C induces lipid hydroperoxide decomposition to the DNA-reactive bifunctional electrophiles 4-oxo-2-nonenal, 4,5-epoxy-2(E)-decenal, and 4-hydroxy-2-nonenal. The compound 4,5-Epoxy-2(E)-decenal is a precursor of etheno-2'-deoxyadenosine, a highly mutagenic lesion found in human DNA. Vitamin C-mediated formation of genotoxins from lipid hydroperoxides in the absence of transition metal ions could help explain its lack of efficacy as a cancer chemoprevention agent.

Validation of surrogate endpoint biomarkers in prostate cancer chemoprevention trials

Phase 2 cancer chemoprevention trials are designed to provide estimates of the efficacy of an agent at a specified dose, and the expected size of the risk reduction that may be achieved in a subsequent phase 3 randomized trial. To allow these trials to be rapid and efficient, the study outcome is modulation of a surrogate endpoint biomarker (SEB), that is, a molecular event assumed to be on the causal pathway between the chemopreventive agent and the desired reduction in cancer incidence. However, SEBs commonly used in prostate cancer chemoprevention studies, such as prostate-specific antigen, high grade prostatic intraepithelial neoplasia, proliferation, and apoptosis, have not been validated by documenting that changes in the SEBs ultimately translate to decreased prostate cancer risk. Because of uncertainty in the pathway from SEBs to cancer, additional considerations are necessary to permit valid inferences from phase 2 trial data. This article considers the framework underlying validation and use of SEBs in specific chemoprevention models and methodologic issues in quantifying the effect of an agent. In particular, inferences depend on whether a single pathway involving the SEBs is assumed to mediate the effect of the agent on cancer incidence. If there are competing pathways of equal or greater importance than the one involving the candidate SEB, then the estimate of chemopreventive efficacy will be biased and may greatly underestimate the magnitude of the achievable risk reduction. Strategies for validating biomarkers and minimizing the degree of bias in the risk reduction estimate are discussed. Finally, problems associated with phase 2 study designs commonly used for prostate cancer chemoprevention are discussed, along with possible solutions.

The Prostate Cancer Prevention Trial: Current status and lessons learned

The Prostate Cancer Prevention Trial is the first phase 3 prevention trial for prostate cancer in the United States. The implementation of a large, randomized trial has provided a wealth of information that will aid in future cancer chemopreventive studies in US men. The experience from the implementation of the Prostate Cancer Prevention Trial was reviewed. Lessons learned from the study include: (1) US men are willing to enroll in prevention trials; (2) participants in chemoprevention trials are well educated and healthy; (3) the successful cancer prevention trial is viewed by participants as a “men’s health trial”; (4) data management and discipline coordination at participating institutions are critical; (5) study design change is commonly required owing to changes in clinical practice over the course of the trial; and (6) training of institutional staff is essential. With proper design, robust data management, and a flexible staff, large-scale randomized chemoprevention trials can be accomplished in the United States. With the extraordinary number of potential agents, it is expected that much will be accomplished with this strategy in the near future.

Oxidative stress in chemoprevention trials

Prostate cancer continues to be the most frequently diagnosed cancer in men in the United States. Despite aggressive intervention, a significant number of men with prostate cancer will not be cured of their disease and will face the possibility of metastatic disease. Thus, development of potent prevention strategies to diminish or eliminate this threat is in order. Cellular exposure to chronic oxidative stress may be 1 possible etiologic factor in the development of many cancers, including prostate cancer. Oxygen radicals can attack DNA directly and result in the accumulation of potentially promutagenic oxidized DNA bases such as 8-hydroxydeoxyguanosine. In addition, chronic oxidant stress may also result in lipid peroxidation and the subsequent generation of a range of reactive products that can damage DNA. Disruption of certain genes may result in cellular tolerance to oxidative genomic injury. GSTP1 is an enzyme that helps catalyze the conjugation reaction between potentially damaging electrophiles and glutathione. Inactivation of GSTP1 has been documented to occur in nearly 100% of human prostate cancers; it is also frequently inactivated in prostatic intraepithelial neoplasia lesions. This inactivation may leave the cell vulnerable to oxidative DNA damage and/or tolerant to accumulation of oxidized DNA base adducts. These base adducts can be measured by several quantitative methods, such as gas chromatography–mass spectrometry with selected ion monitoring. These sophisticated methods can be readily integrated into prostate cancer chemoprevention studies of new and developing prevention agents by providing quantitative assessment of oxidative DNA damage before and after administration of these candidate chemopreventive drugs. The combination of genetic information, state-of-the-art assessment tools, and novel agents will allow rational, directed prostate cancer chemoprevention studies to be performed and, together, will help determine the role of chronic oxidative stress in the carcinogenic process of prostate cancer.

New DNA-based biomarkers for oxidative stress and cancer chemoprevention studies

Etheno (ε) modified DNA bases (εdA, εdC, εdG) are generated from the carcinogens vinyl chloride and urethane, but also by reactions of DNA with products derived from lipid peroxidation (LPO) and oxidative stress via endogenous pathways. Recently developed ultrasensitive methods allowed the detection of these ε-adducts in vivo and their role to be studied in experimental and human carcinogenesis. Highly variable background levels of ε-adducts were detected in DNA from different organs of unexposed humans and rodents. Several known cancer risk factors increased the level of these DNA lesions in target organs: elevated ε-adducts were found in hepatic DNA from patients with metal storage diseases, after overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) in a mouse model, and in colonic polyps of familial adenomatous polyposis patients. A high ω-6-polyunsaturated fatty acid diet increased ε-DNA adducts in white blood cells of female subjects. In conclusion, ε-adducts were found to be elevated in cancer-prone tissues, suggesting that these promutagenic lesions could drive cells to malignancy. Therefore, exocyclic DNA adducts offer new tools in cancer aetiological research and in verifying the efficacy of chemopreventive agents.

Asian studies of cancer chemoprevention: latest clinical results

Chemoprevention trials in Asia, including those already completed and those now ongoing, are reviewed. Information was mainly collected from Japan, Korea and China. Each country features its own characteristics. Cancer chemoprevention trials targeting, from various aspects, hepatocellular carcinoma, gastric cancer and colon cancer have been, and are now being, conducted in Japan. Japan also has a long history of basic carcinogenesis research and carcinogenic research using animal experiments. In Korea, ginseng is the main focus of studies of chemopreventive agents. A large body of information has been collected and prospective studies are also ongoing. In China, the Linxian study, a cooperative study participated in by China and the NCI of the USA, is well known and the results impressive. However, we must exercise caution because, for example, the population of Linxian are chronically deficient in multiple vitamins and trace minerals. This situation may, therefore, differ from that observed in other countries. In any event, chemoprevention studies will be popular from an economical point of view even in Asia because cancer is becoming the number one cause of death in these countries.

Cellular responses of mammary carcinomas to aromatase inhibitors: effects of vorozole.

Vorozole (Vz) is a competitive non-steroidal inhibitor of aromatase, which has been used to treat breast cancer in postmenopausal women and in various chemoprevention pre-clinical studies. Recently, we assessed the inhibitory effect of Vz on MNU-induced mammary carcinogenesis (Lubet et al., 1994), as well as on the progression of mammary tumors (Lubet et al., 1998). In this study we evaluated the effects of Vz on tumor growth, serum estradiol, cell proliferation, apoptotic and non-apoptotic cell death to determine whether any of these 'surrogate' markers might reflect the efficacy of various doses of Vz. Vz at doses of 2.5 (Hi), 0.32 (Md), and 0.08 (Lo) mg/kg body weight induced complete (100%), 60%, and 20% regression of mammary tumors, respectively. Vz at Hi and Md doses caused a decrease in serum estradiol within the first two days of treatment, and the estradiol values remained low with additional treatment for 4 and 10 days. When Vz was administered to animals bearing palpable tumors a time and dose-dependent decrease in the proliferating cells (BrdU-L1) was observed. The percentage of apoptotic cells (A1) sharply increased 2 days after initiation of Vz treatment and then decreased followed by an increase in non-apoptotic dead cells. Interestingly even the Lo dose of Vz, which was only moderately effective in suppressing tumor growth, decreased cell proliferation and increased cell death in the peripheral tumor areas at 4 and 10 days after initiation of treatment. The time- and dose-dependent alterations in various cell parameters suggest two different phases of Vz-induced cellular responses: (1) an early phase (2-4 days of treatment) with a sharp increase in apoptotic cells and decrease in proliferating cells, and (2) a later phase (10 days) with disintegration of tumor parenchyma, increase in non-apoptotic dead cells, and decrease in apoptotic cells. The dose-dependent decrease in proliferating cells and increase in apoptotic and non-apoptotic cell death in Vz-treated animals suggest that these biomarkers might be used as potential surrogate endpoints for efficacy in breast cancer chemoprevention and therapy studies with aromatase inhibitors.

Update from Asia. Asian studies on cancer chemoprevention.

In Asia, nontoxic dietary products are considered desirable primary prevention vehicles for conquering cancer. As early as 1978, investigators in Korea carried out extensive long-term anticarcinogenicity experiments using the mouse lung tumor model and observed an anticarcinogenic effect of Panax ginseng C.A. Meyer extract in 1980. The results showed that natural products can provide hope for human cancer prevention. A newly established nine-week medium-term model using mouse lung tumors (Yun's model) could confirm the anticarcinogenicity of ginseng that varies according to its type and age. Subsequently, the ginseng was shown by epidemiological studies to be a nonorgan-specific cancer preventive agent associated with a dose-response relationship. The anticarcinogenic effects of vegetarian foods common at every dining table in Korea and some synthetics were also studied using Yun's nine-week model. In brief, ascorbic acid, soybean lecithin, capsaicin, biochanin A, Ganoderma lucidum, caffeine, and a novel synthetic 2-(allylthio)pyrazine decrease the incidence of mouse lung tumors, whereas fresh ginseng (4 years old), carrot, spinach, Sesamum indicum, beta-carotene, and 13-cis retinoic acid do not. This result regarding beta-carotene is consistent with the ineffective findings of the ATBC trial, the CARET trial, and the Physicians' Health Study. In 1983, a cancer chemoprevention study group was first established in Japan. Subsequently, (-)-epigallocatechin gallate, cryptoporic acid E, and sarcophytol A from natural products, and synthetic acyclic retinoid and canventol were shown to be anticarcinogenic or chemopreventive in human subjects. Despite the frequent consumption of tea wordwide as a beverage and current experimental evidence of anticarcinogenesis, including controversial results of epidemiological studies, more systematic clinical trials for confirmation of preventive activity of tea against cancer are needed. Placebo-controlled intervention trials of dietary fiber are under study in Japan. In the past decade, new triterpenoids were isolated from various natural sources, and its biological activities were investigated in Asia. In the late 1970s a comprehensive chemoprevention program was established at the Institute of Materia Medica, Chinese Academy of Medical Sciences. Since then, many retinoid compounds have been synthesized and screened in the search for chemopreventive cancer agents. The National Cancer Institute (USA) and China are jointly engaged in the two-nutrition intervention in Linxian, China. The results of joint study of the general population and of dysplasia in China should stimulate further research to clarify the potential benefits of micronutrient supplements. We need to clarify if there is a connection between the lower rates of cancer mortality in Korea and the frequent consumption of anticarcinogenic vegetables or traditional foods, including ginseng and Ganoderma lucidum. The constituents of the nontoxic stable dietary products promise to be the future hope for conquering cancers in the coming years.

4-(hydroxyphenyl)retinamide selectively inhibits the development and progression of ductal hyperplastic lesions and carcinoma in situ in mammary gland.

In most previous chemoprevention studies on inhibition of mammary carcinogenesis, the formation of palpable tumors has been used as an end-point. Little is known about whether chemopreventive agents may similarly or selectively suppress hyperplastic and premalignant stages of the neoplastic process. In this study, we evaluated the effect of 4-(hydroxyphenyl)retinamide (4-HPR) on the development and progression of hyperplastic lesions and carcinoma in situ (CIS) in the N-methyl-N-nitrosourea (MNU) mammary carcinogenesis model in rats. 4-HPR was used as the chemopreventive agent because of its proven inhibitory effect on both the early and late phases of mammary carcinogenesis. Treatment with 4-HPR (2.0 mM/kg diet), beginning 2 days after MNU administration and administered continuously for 10 weeks, suppressed all mammary gland lesions (hyperplasia, CIS and invasive carcinoma) in 35% of animals. In the remaining 65%, 4-HPR allowed the development of hyperplastic lesions, alone or combined with CIS, and/or invasive carcinomas (CA). 4-HPR also increased by 2-fold the ratio between CIS and CA (0.75 per animal in control versus 1.5 in 4-HPR-treated animals), suggesting that it may also suppress the transition of CIS into CA. 4-HPR, when administered beginning 4 weeks after MNU administration [when hyperplastic and premalignant (CIS) lesions are present in the mammary gland], inhibited the frequency of terminal end bud hyperplasia (TEBH) and CA but did not significantly suppress ductal hyperplasia, ductal alveolar hyperplasia, alveolar hyperplasia and CIS. In these animals, 4-HPR induced partial disintegration of mostly peripheral areas of lesions, including carcinomas. Taken together, our data indicate that 4-HPR selectively suppresses the development and progression of hyperplastic lesions and CIS in TEBs. Furthermore, it appears that, in addition to mammary carcinomas, TEBH and CIS could also be used as end-point biomarkers in breast cancer chemoprevention studies.

Recruitment experience from phase II cancer chemoprevention studies

Recruitment experience from phase II cancer chemoprevention studies

Experimental models of gene-environment interaction for cancer chemoprevention studies.

The recent development of mouse strains with cancer-related genes overexpressed or inactivated has provided investigators with new models for testing chemoprevention strategies to offset specific genetic susceptibilities to cancer. This review focuses on the three genetically altered mouse models that have been the most widely used in chemoprevention studies: Min mice, which carry a mutation in the adenomatous polyposis coli (APC) gene; APC-knockout mice; and p53-knockout mice. Studies with the Min and APC-knockout mice provide the strongest evidence to date that the enzyme cyclooxygenase-2 plays a major role in colon carcinogenesis, and that nonsteroidal anti-inflammatory drugs that target cyclooxygenase-2 have great potential as colon cancer chemopreventive agents. In addition, chemoprevention studies in mice deficient of the p53 tumor-suppressor gene, the most commonly altered gene in human cancer, suggest that the increased susceptibility to cancer resulting from the loss of p53 function may be offset by preventive approaches. Other recently developed transgenic and knockout models of potential interest for chemoprevention studies will also be discussed.

Retinoids and their nuclear receptors in the development, prevention, and treatment of upper airways epithelial cancers

Retinoids and their nuclear receptors in the development, prevention, and treatment of upper airways epithelial cancers

Cancer biomarkers in the field of tea.

Suitable intermediate end-point biomarkers are needed in order to reduce the time and expense associated with cancer chemoprevention studies. Because of the multi-step nature of cancer intermediate biomarkers reflect a continuum of events associated with different stages of disease development, and range from genetic damage linked to cancer initiation, to tumor growth and expansion during disease progression. With tea, experimental evidence points to potential protection at several stages of carcinogenesis including endogenous carcinogen formation, carcinogen activation and detoxification, DNA damage and destabilisation, cell proliferation, neoplastic growth and metastatic spread. If used effectively in tea research, biomarkers should contribute to a better understanding of the possible extent of cancer protection afforded by tea, as well as the putative mechanisms involved and the principal components of tea with prophylactic potential.

G1 phase-specific suppression of the Cdk2 activity by ginsenoside Rh2 in cultured murine cells.

Ginsenoside Rh2, a plant glycoside with a dammarane skeleton resembling a steroid skeleton as an aglycone, has anticancer potentials in vitro or in vivo. To elucidate the molecular mechanisms of the effects of Rh2, we have examined the Cyclin-dependent kinase-2 (Cdk2) activity in G1 arrested B16 melanoma cells and in S phase-arrested Meth-A sarcoma cells, that have been treated with Rh2. The kinase activity was suppressed in B16 cells but not in Meth-A cells. In addition, Rh2 was found to induce G1 arrest and concomitantly suppress the Cdk2 activity in carcinogen-susceptible BALB/c 3T3 A31-1-1 and A31-1-13 cell lines. Thus, Rh2 has a G1 phase-specific suppressive effect on the Cdk2 activity, supporting further evaluation of Rh2 and its related compounds in cancer chemoprevention studies.

Clinical studies in head and neck cancer chemoprevention.

Secondary neoplasms represent a major threat for patients with head and neck cancer. The prevention of secondary neoplasms has been a major goal of head and neck cancer chemoprevention efforts. In order to help develop effective strategies, reversal of oral premalignancy has been used as a model for chemoprevention. There is now sufficient data to show the chemopreventive effect in premalignant lesions of some natural compounds and their derivatives. Retinoids are the most studied chemopreventive agents for the treatment of oral leukoplakia. Other compounds with chemopreventive activity are carotenoids, Vitamin E derivatives and Selenium. There are two large prospective, randomized, chemoprevention clinical trials, one in Europe and the other in North America, using prevention of secondary malignancy as the primary study end-point. Until these trials are completed, the use of chemoprevention in head and neck cancer should be limited to clinical trials.

P53 and Ha-ras mutations in chemically induced hamster buccal pouch carcinomas.

Well-differentiated squamous cell carcinomas were induced in hamster buccal pouch epithelium by twice weekly topical applications of N-methyl-N-benzylnitrosamine (MBN) or 7,12-dimethylbenz[a]anthracene (DMBA) over a period of 15 weeks. Each of the 22 tumors induced (14 MBN and eight DMBA) were evaluated by single-strand conformation polymorphism and DNA sequencing to identify mutations in conserved exons (E5-E8) of the p53 tumor suppressor gene and codons 12/13 and 61 of Ha-ras. In addition, Northern blot analysis of 10 MBN tumors and five DMBA tumors was performed to determine whether the mdm-2 gene was overexpressed, p53 mutations were detected in five of 14 (35%) MBN-induced carcinomas and in two of eight (25%) DMBA-induced carcinomas. Ha-ras mutations were detected in three of 14 (21%) MBN-induced carcinomas and in three of eight (37%) DMBA-induced carcinomas. One MBN-induced carcinoma exhibited a mutation in both the p53 and Ha-ras genes. The majority (five of seven of p53/Ha-ras mutations induced by MBN were G->A transitions and two of these occurred at hamster p53 codon 248, which corresponds to human p53 codon 245, a known mutational tumor 'hot spot'. A->T transversion at Ha-ras codon 61 accounted for three of five (60%) DMBA-induced mutations. There was no evidence of mdm-2 overexpression in any of the tumors evaluated. Overall, the results provide additional support for the validity of the hamster buccal pouch model of oral carcinogenesis, as applied to sequential cellular and molecular analysis and cancer chemoprevention studies.

The role of prostate-specific antigen in the chemoprevention of prostate cancer

An understanding of the natural history of changes in prostate-specific antigen (PSA) may be valuable as a surrogate view of prostate dynamics, as a method to differentiate between benign and malignant growth, and as a means to assess the use of PSA as a tool for monitoring activity of chemoprevention agents. Although PSA appears to be useful as a noninvasive marker of prostatic growth, PSA changes should not be confused with a direct measure of tumor growth. Serum PSA levels are a function of tumor volume but are also influenced by the volume of benign epithelium, grade of carcinoma (if any), inflammation, androgen levels, growth factors, and the extracellular matrix. The biological functions of PSA in the prostate and in its secretions need to be more completely elucidated in order that PSA measurements may more accurately describe prostate dynamics. The expression of PSA is androgen-regulated. It is one of the most abundant prostate-derived proteins in the seminal fluid. Seminogelin, a major protein in seminal fluid, is cleaved by PSA, and this cleavage is important in the liquefaction of semen. Less is known about other PSA substrates. Current PSA studies indicate that cancer cases exhibit an early slow linear PSA phase followed by a rapid exponential phase, and that PSA levels begin to increase exponentially approximately 7-9 years before diagnosis. The establishment of age-specific PSA reference ranges (ASRR) and of PSA velocity (PSAV) rates provide elements of a baseline from which prediction models could measure malignant potential of a prostatic carcinoma. Moreover, recent discoveries of different molecular forms of PSA in serum may allow a much more accurate differentiation of benign and malignant growth as well as a more potent measure of the impact of chemoprevention agents. If PSA doubling time is approximately 2.4-3.0 years and accurately reflects tumor doubling time, and if the average man has less than 0.5 ml of latent prostatic tumor tissue and the average stage T2 cancer is approximately 4 ml when detected, then the available PSA data suggest that the 3 doublings necessary to change from 0.5-4.0 ml, would take 7-12 years for a typical small volume tumor to reach the size of most stage T2 tumors. The findings that histologic cancers appear at much younger ages than previously known is disturbing. It indicates that disease initiation may begin sooner than ever thought likely. "Normal" PSA levels for younger men (< 40 years of age) may need to be studied, and an emphasis upon premalignant lesions in this age group may be necessary. Younger men may represent the most appropriate population and premalignant lesions the most relevant clinical factor for prostate cancer chemoprevention studies and trials. The molecular composition and molecular changes of PSA derived from premalignant lesions have yet to be elucidated, but such investigations may lead to a more complete understanding of the possible progression or transformation of normal prostate cells to premalignancy and subsequently to carcinoma. High grade prostatic intraepithelial neoplasia (PIN) in and of itself does not account for elevated serum PSA levels, but subtle changes in the molecular dynamics of PSA may reveal the influence of androgens and the impact of chemopreventive agents.

Anti-tumor promotion with food phytochemicals: a strategy for cancer chemoprevention.

Cancer chemoprevention is currently regarded as a promising avenue for cancer control. In particular, the inhibition of tumor promotion (anti-tumor promotion) in multistage carcinogenesis is expected to be an efficient strategy, because tumor promotion is experimentally accomplished through the long-term, repetitive exposures of rodents to a tumor promoter, and premalignant lesions caused by a tumor promoter regress, at least in their earlier stages. In this review, we first describe the background of cancer chemoprevention studies as well as recent results of clinical trials. Subsequently, some hypothetical biological and cellular pathways in tumor promotion are explored. In addition, the anti-tumor promoting properties of vegetables, fruits, and edible marine algae, together with their active constituents and action mechanisms thus far known, are also described. Anti-tumor promotion with food phytochemicals may be characterized as an efficient and reliable strategy for cancer chemoprevention.

986 Chemoprevention of (pre)malignancy of the upper aerodigestive tract with retinoids

Of the various possible level of risk at which chemoprevention can be applied 1) premalignant conditions (in the oral cavity: leukoplakia) and 2) patient who have been treated for cancer, but who are at risk to develop a new cancer, are for the upper aerodigestive tract the most important. Studies in oral leukoplakia as well as in the second primary tumors group will be discussed, with emphasis on EUROSCAN. EUROSCAN is an European Organization of Research and Treatment of Cancer (EORTC) chemoprevention study in curatively treated patients with oral cancer, laryngeal cancer and lung cancer which started in 1988; the end of the accrual phase was in 1994. The end of the intervention period is in 1996.

985 Breast cancer chemoprevention with retinoids and tamoxifen

In March 1987, a breast cancer chemoprevention study with Fenretinide (4-HPR) started at the Milan Cancer Institute with the aim of evaluating the effectiveness of this retinoid in reducing the incidence of contralateral breast primaries in breast cancer patients. Subjects take 4-HPR at the daily dose of 200mg for 5 years vs no treatment. Accrual is closed with 2,972 randomized patients. No difference is shown at present between the two groups but some preliminary data seem to suggest that the agent might be effective in inhibiting the growth of hormone independent tumours. Other aims are to contribute to a better understanding of the pharmacokinetics and mechanisms of action of this drug, as well as to determine its effectiveness in prevention or protection of women against ovarian cancer. Another breast cancer chemoprevention trial using Tamoxifen (TAM) began in September 1992 from an original idea of U. Veronesi and C. Maltoni. Presently, more than 3,500 hysterectomized women aged 35–70 are participating in the study. According to a double blind design, the subjects take either 20mg/day of TAM or placebo for 5 years. The decision to include only hysterectomized women was made to avoid the unjustified risk of endometrial cancer in those taking TAM. The study involves 43 Italian centres and 3 associated centres in South America. The Data Centre and the National Coordinating Centre are in Milan, at the European Institute of Oncology. Most randomized patients (89%) are between 45 and 65 years of age (median age: 51); 56% have had minor side effects (mainly menopausal symptoms).