Eliane Kellen
email: eliane.kellen@uz.kuleuven.ac.be

A E Greet

Vansant Ae

Marie-Rose Christiaens

A E Patrick

Neven Ae

Erik Van Limbergen

E Van

G Vansant

M.-R Christiaens

Á P Neven

Á E Van

Lifestyle changes and breast cancer prognosis: a review

Keywords: Breast cancer, Life style, Prognosis

Objective Because of its high incidence and improving survival, breast cancer is currently the most prevalent cancer in the world. We reviewed the published literature in our search for modifiable factors valuable as an adjuvant health measure to surgery, radio and systemic therapy for breast cancer. Methods We included material which was identified from computerised searches of Pub-Med (1966 to May, 2007). Published material was restricted to prospective cohort studies and randomised clinical trials. We reviewed the literature concerning the association between physical activity, smoking, weight gain after the diagnosis, diet and prognosis. Results and conclusion We conclude that life style changes following standard breast cancer therapy are highly recommended (although scientific evidence is still lacking for some of inconsistency regarding available data). They include physical activity, weight control, high consumption of fruit and vegetables, and a reduction of dietary fat intake.

Introduction

Breast cancer is by far the most frequent cancer of women. More than half of the cases occur in industrialized countries. Although, the incidence is more modest in Eastern Europe, South America, Southern Africa, and Western Asia, it is still the most common female cancer in these geographic regions. The rates are low in most parts of Africa (with the exception of South Africa) and in most of Asia [START_REF] Parkin | Global cancer statistics, 2002[END_REF].

Most of the breast cancer risk factors described in the literature are related to a woman's life time exposure to oestrogens: early menarche and late menopause, high total numbers of menstrual cycles and use of oestro-progestons increase the risk; while it is reduced by an early first birth and early menopause [START_REF] Key | Epidemiology of breast cancer[END_REF]. Lactation protects against breast cancer while alcohol increases the risk. The relationship of body fatness with breast cancer risk is equivocal. It probable decreases the risk of premenopause cancer while it increases the risk for postmenopause cancer. Postmenopausal breast cancer seems to be positively associated with abdominal fatness and adult weight gain and negatively with physical activity [START_REF]Food, nutrition, physical activity, and the prevention of cancer: a global perspective[END_REF]. Data regarding the role of soy remains conflicting, but adolescent exposure appears to be protective [START_REF] Duffy | Implications of phytoestrogen intake for breast cancer[END_REF]. Positive or negative effects of dietary exposures may be limited to subgroups of women defined by specific genetic characteristics [START_REF] Michels | Diet and breast cancer: a review of the prospective observational studies[END_REF]. Breast cancer risk is positively associated with increased smoking during the period between menarche and first childbirth, suggesting that the sensitivity of the female breast to tobacco carcinogens is increased during adolescence and early adulthood [START_REF] Ha | Smoking cigarettes before first childbirth and risk of breast cancer[END_REF].

The large variability in the clinical outcome of women with morphological identical breast cancers receiving comparable therapeutic strategies suggest the importance of other life style changes. We therefore reviewed the available published literature in our search for modifiable life style factors that may be useful as adjuvant treatment to surgery, radio and systemic therapy for breast cancer.

Search strategy and selection criteria

Material included in this review was identified from computerised searches of PubMed (1966 to February, 2008) using the medical subject headings (MeSH) terms ''breast neoplasm '' OR ''breast cancer'', ''AND prognosis'' ''AND survival'' ''AND diet'' ''AND life style'' ''AND physical activity'' ''AND smoking'' ''AND obesity'' ''AND phytoestrogenes'' ''AND dietary fat''. Only peer-reviewed English language papers were eligible for inclusion. Additional information and studies were obtained by checking the reference lists from retrieved papers. Only prospective breast cancer cohort studies and randomised clinical trials reporting on breast cancer recurrence and survival were included. Currently, few such studies have been designed with the objective to investigate the association between different lifestyle factors and breast cancer prognosis. Most epidemiologic studies were designed to examine aetiology and do therefore not answer whether or not the studied population continued their original lifestyle or altered it after the diagnosis of breast cancer which may bias the results (Table 1).

The Life After Cancer Epidemiology (LACE) Study consists of cohort of 2,321 early stage breast cancer survivors and was designed to investigate how modifiable behavioral risk factors such as diet and physical activity affect quality of life and long-term survival [START_REF] Caan | Life After Cancer Epidemiology (LACE) Study: a cohort of early stage breast cancer survivors (United States)[END_REF].

The Health, Eating, Activity, and Lifestyle (HEAL) Study is a population-based, multicenter prospective cohort study that has enrolled 1,185 breast cancer patients to study the influence of factors such as body weight, physical activity and diet on prognosis [START_REF] Irwin | Physical activity levels before and after a diagnosis of breast carcinoma: the Health, Eating, Activity, and Lifestyle (HEAL) study[END_REF].

The Shanghai Breast Cancer Survival (SBSS) Study comprises 2,236 Chinese women with breast cancer and collects information on lifestyle related factors and diseaseand treatment related factors [START_REF] Lu | Impact of newly diagnosed breast cancer on quality of life among Chinese women[END_REF]. The Women's Healthy Eating and Living (WHEL) Study is a multisite randomized controlled trial of the effectiveness of a high-vegetable, low-fat diet, in reducing additional breast cancer events and early death in women with early-stage invasive breast cancer (within 4 years of diagnosis). The study randomly assigned 3,088 women to an intensive diet intervention or to a comparison group [START_REF] Pierce | A randomized trial of the effect of a plant-based dietary pattern on additional breast cancer events and survival: the Women's Healthy Eating and Living (WHEL) Study[END_REF].

The Women's Intervention Nutrition (WINS) Study, a randomized, prospective clinical trial of 2,437 assigned women with resected, early-stage breast cancer, receiving conventional therapy, to a dietary intervention group receiving a program of individualized instruction for reducing total fat intake or a dietary control group with minimal dietary counselling. The dietary intervention aims to reduce the percentage of calories with 15% of habitual intake while maintaining nutritional adequacy [START_REF] Chlebowski | Adherence to a dietary fat intake reduction program in postmenopausal women receiving therapy for early breast cancer. The Women's Intervention Nutrition Study[END_REF].

Results

Physical activity

A cohort study of 2,987 women in the Nurses' Health Study, diagnosed with stage I, II or III breast cancer found a protective effect of physical activity on cancer-related mortality.

The greatest benefit occurred in women who performed the equivalent of walking 3-5 h per week at an average pace and among women with hormone-responsive tumours [START_REF] Holmes | Physical activity and survival after breast cancer diagnosis[END_REF]. Similar results were found by a cohort study of 4,482 invasive breast cancer patients [START_REF] Holick | Physical activity and survival after diagnosis of invasive breast cancer[END_REF] and for two studies that investigated the relationship between breast cancer mortality and pre-diagnostic physical activity [START_REF] Abrahamson | Recreational physical activity and survival among young women with breast cancer[END_REF][START_REF] Enger | Exercise activity, body size and premenopausal breast cancer survival[END_REF]. Results from the WHEL study indicated that the combination of consuming at least five servings of vegetables-fruits and physical activity (30 min of walking at moderate pace for 6 days a week) is associated with an increase in survival in women with estrogen-positive receptor tumours [START_REF] Pierce | Greater survival after breast cancer in physically active women with high vegetable-fruit intake regardless of obesity[END_REF]. However, an Australian cohort of 451 women with breast cancer found no association. Likewise a cohort study in Canada of 603 women with incident breast cancer found no association between physical activity and breast cancer mortality [START_REF] Borugian | Insulin, macronutrient intake, and physical activity: are potential indicators of insulin resistance associated with mortality from breast cancer?[END_REF].

A trial that randomized sedentary, obese breast cancer patients to a cardiovascular and strength training exercise intervention or to a usual care control demonstrated that exercise is associated with a decrease in insulin levels [START_REF] Ligibel | Impact of a mixed strength and endurance exercise intervention on insulin levels in breast cancer survivors[END_REF].

Biological mechanism

The literature remains controversial whether or not in postmenopausal women, increased physical activity is associated with decreased serum concentrations of oestradiol and oestrone [START_REF] Campbell | Exercise and biomarkers for cancer prevention studies[END_REF]. Nonetheless, a randomized clinical trial found that exercise may decrease serum oestrogens levels in previously sedentary, obese postmenopausal women [START_REF] Mctiernan | Effect of exercise on serum androgens in postmenopausal women: a 12-month randomized clinical trial[END_REF].

In premenopausal women, research indicates longer menstrual cycles, and lower progesterone and oestradiol levels in athletes compared with control subjects. A randomized controlled trial concluded that a 12-week aerobic exercise training intervention did not alter oestrogen metabolism in premenopausal women [START_REF] Campbell | Effects of aerobic exercise training on estrogen metabolism in premenopausal women: a randomized controlled trial[END_REF].

High circulating levels of Insulin-Like Growth factor (IGF)-1 may be positively associated with increased risk of breast cancer. The current literature is not conclusive on the relation between increasing physical activity and IGF-1 levels [START_REF] Campbell | Exercise and biomarkers for cancer prevention studies[END_REF]. However, it was demonstrated that IGF-1 is a mediator of estradiol mitogenic effects [START_REF] Adesanya | Insulin-like growth factor 1 is required for G2 progression in the estradiol-induced mitotic cycle[END_REF].

Smoking

A cohort study of 5,056 women in the Nurses' Health Study, diagnosed with breast cancer, found no association between a history of smoking and breast cancer-related mortality or an increased risk of breast cancer recurrence [START_REF] Holmes | Smoking and survival after breast cancer diagnosis[END_REF]. Similar results were concluded by two other cohort studies [START_REF] Fentiman | Smoking and prognosis in women with breast cancer[END_REF][START_REF] Sagiv | Active and passive cigarette smoke and breast cancer survival[END_REF]. In contrast, a Swedish cohort study concluded that the breast cancer mortality was the lowest in those who had never smoked, the highest in smokers, and somewhere in between in ex-smokers [START_REF] Manjer | Survival of women with breast cancer in relation to smoking[END_REF].

Biological mechanism

Nicotine is the main immunosuppressive constituent of cigarette smoke, which inhibits both the innate and adaptive immune responses [START_REF] Sopori | Effects of cigarette smoke on the immune system[END_REF]. Smoking may have an 'antioestrogenic' effect although endogenous oestrogen levels have not been found to differ significantly when comparing current smokers with former smokers or non-smokers [START_REF] Kendall | Influences on circulating oestrogens in postmenopausal women: relationship with breast cancer[END_REF]. However, it should be noted that cigarette smoking is inversely associated with the risk of endometrial cancer among postmenopausal women, which is another oestrogen dependent tumour [START_REF] Terry | Cigarette smoking and the risk of endometrial cancer[END_REF].

Obesity and weight gain after the diagnosis

The majority of the existing literature indicates a negative relationship between obesity and poor prognosis of breast cancer in both pre-and post-menopausal women [START_REF] Carmichael | Obesity and prognosis of breast cancer[END_REF]. Weight gain after diagnosis has been reported frequently, possibly through increased caloric intake, changes in physical activity and change in host metabolic status [START_REF] Chlebowski | Dietary fat intake reduction for patients with resected breast cancer[END_REF]. Women who gained weight after diagnosis are found to be at greater risk for breast cancer recurrence and death compared to women with a lower body mass index (BMI) [START_REF] Chlebowski | Weight loss in breast cancer patient management[END_REF].

Analyses from the Nurses' Health Study showed that weight and weight gain after the date of diagnosis were related to higher rates of breast cancer recurrence and mortality. These findings were especially apparent in women who never smoked, among women with earlier stage disease or those who were normal weight before diagnosis [START_REF] Kroenke | Weight, weight gain, and survival after breast cancer diagnosis[END_REF]. In contrast, a collaborative effort between the LACE study and the WHEL study, did not observe an association between post-diagnosis weight gain and recurrence risk in the first 5-7 years postdiagnosis [START_REF] Caan | Post-diagnosis weight gain and breast cancer recurrence in women with early stage breast cancer[END_REF].

If circulating estrogen levels are the linking mechanism between adiposity and breast cancer recurrence, weight loss may no longer have an impact on recurrence or survival in postmenopausal women with estrogen-positve receptors treated with the third generation aromitase inhibitors (exemestane, anastrozole, letrozole). As these drugs suppress estradiol levels in post-menopausal women by 84% or more [START_REF] Byers | A weight loss trial for breast cancer recurrence: pre-menopausal, post-menopausal, both, or neither?[END_REF].

Increasing BMI is associated with increased oestradiol levels. Hyperinsulinism has been associated with a poor breast cancer prognosis, possibly mediated by the neuroendocrine hormone leptin that is produced by adipose tissue [START_REF] Kendall | Influences on circulating oestrogens in postmenopausal women: relationship with breast cancer[END_REF]. Obese postmenopausal breast cancer survivors have increased estrone, estradiol, free estradiol, levels and decreased sex-hormone-binding globulin levels [START_REF] Mctiernan | Adiposity and sex hormones in postmenopausal breast cancer survivors[END_REF]. Furthermore, it has been suggested that obese women with breast cancer may receive reduced doses of adjuvant chemotherapy [START_REF] Griggs | Undertreatment of obese women receiving breast cancer chemotherapy[END_REF]. Higher BMI has been related to more advanced stages of breast cancer at diagnosis in terms of tumour size [START_REF] Carmichael | Obesity and breast cancer: a review of the literature[END_REF]. Obesity appears not to interfere with the efficacy of tamoxifen in women with lymph-node negative, oestrogen receptor-positive breast cancer [START_REF] Dignam | Obesity, tamoxifen use, and outcomes in women with estrogen receptor-positive early-stage breast cancer[END_REF].

Phytoestrogenes

Phytooestrogens are plant compounds that are structurally similar to mammalian oestrogens. They possess oestrogenic and antioestrogenic properties through binding on oestrogen receptors. Some data from in vitro and animal studies suggest that isoflavones, especially genistein, may stimulate the growth of oestrogen-sensitive tumours. Further research is therefore needed to determine effect of soy foods or isoflavones on breast cancer risk in high-risk women and on the survival of breast cancer patients [START_REF] Messina | Addressing the soy and breast cancer relationship: review, commentary, and workshop proceedings[END_REF]. Although, a Chinese study found no relationship between soy food intake before diagnosis and survival [START_REF] Boyapati | Soyfood intake and breast cancer survival: a followup of the Shanghai Breast Cancer Study[END_REF], it is not recommended to use soy to reduce menopausal symptoms in breast cancer patients [START_REF] Duffy | Implications of phytoestrogen intake for breast cancer[END_REF].

It has been suggested that phytooestrogenes may influence the effect of tamoxifen, a selective oestrogen receptor modulator, in breast cancer treatment either in a antagonistic or synergistic way [START_REF] Duffy | Implications of phytoestrogen intake for breast cancer[END_REF]42]. However, a crosssectional study in Asian Americans with breast cancer who were tamoxifen users found no relation between selfreported intake of soy and serum levels of isoflavones [START_REF] Wu | Tamoxifen, soy, and lifestyle factors in Asian American women with breast cancer[END_REF].

It should be noted that estrogen therapy is contraindicated after the diagnosis of breast cancer [START_REF] Von | Menopausal hormone therapy after breast cancer: the Stockholm randomized trial[END_REF][START_REF] Holmberg | HABITS (hormonal replacement therapy after breast cancer-is it safe?), a randomised comparison: trial stopped[END_REF].

Dietary fat

In women with oestrogen receptor positive breast cancer, a positive association between treatment failure and high intakes of total fat, saturated fat and polyunsaturated fatty acid has been suggested [START_REF] Holm | Treatment failure and dietary habits in women with breast cancer[END_REF]. The association between dietary fat intake and reduced survival has been confirmed in both postmenopausal and premenopausal breast cancer patients [START_REF] Zhang | Better breast cancer survival for postmenopausal women who are less overweight and eat less fat. The Iowa Women's Health Study[END_REF][START_REF] Mceligot | Dietary fat, fiber, vegetable, and micronutrients are associated with overall survival in postmenopausal women diagnosed with breast cancer[END_REF][START_REF] Hebert | The effect of dietary exposures on recurrence and mortality in early stage breast cancer[END_REF][START_REF] Saxe | Diet and risk for breast cancer recurrence and survival[END_REF][START_REF] Rohan | Dietary factors and survival from breast cancer[END_REF]. Interim results of the WINS study suggest a benefit of dietary fat reduction on relapse-free survival. Secondary analyses suggested a stronger effect for dietary fat reduction on breast cancer recurrence in women with hormone receptor-negative cancers compared with women whose cancers were hormone receptor positive [START_REF] Chlebowski | Dietary fat reduction and breast cancer outcome: interim efficacy results from the Women's Intervention Nutrition Study[END_REF]. In contrast, the Nurses' Health Study did not conclude that a low fat diet after diagnosis leads to a survival advantage [START_REF] Holmes | Dietary factors and the survival of women with breast carcinoma[END_REF]. The same conclusion was reached by a prospective study of 213 breast cancer patients in Japan; it should be remarked that Japanese women develop breast cancer at an earlier age and consume relatively small amounts of fat (mean dietary fat intake was about 23% of calories) [START_REF] Kyogoku | Diet and prognosis of breast cancer[END_REF].

Biological mechanism

Experimental studies indicate that the polyunsaturated fatty acids, rich in linoleic acid enhance the promotion and progression of mammary cancer, whereas omega-3 fatty acid-containing fish exert a protective effect [START_REF] Wynder | Breast cancer-the optimal diet[END_REF]. Laboratory and animal experimental evidence indicate that dietary fat intake influences breast cancer growth through changes in circulatory hormonal levels, changes in cell membrane structure and function, changes in prostaglandin and cytokine/monokine levels and altered regulatory gene expression [START_REF] Chlebowski | Dietary fat intake reduction for patients with resected breast cancer[END_REF]. A high-fiber, low fat diet intervention has been shown to be associated with reduced serum bioavaliable oestradiol concentration in women diagnosed with breast cancer [START_REF] Rock | Effects of a highfiber, low-fat diet intervention on serum concentrations of reproductive steroid hormones in women with a history of breast cancer[END_REF].

Other dietary macro-and micronutrients

It has been shown that in post-menopausal women diagnosed with breast cancer, reduced fat and increased fiber, vegetable and fruit intakes improves overall survival [START_REF] Mceligot | Dietary fat, fiber, vegetable, and micronutrients are associated with overall survival in postmenopausal women diagnosed with breast cancer[END_REF]. But analyses from the Nurses' Health Study found that adherence to a prudent dietary pattern (characterized by a diet high in fruits, vegetables, whole grains, legumes, poultry and fish) was not associated with breast-cancer mortality but was negatively related to mortality from other causes [START_REF] Kroenke | Dietary patterns and survival after breast cancer diagnosis[END_REF]. The WHEL randomized trial confirmed that adherence to a diet very high in vegetables, fruit and fiber and low in fat does not reduce breast cancer recurrence or mortality [START_REF] Pierce | Influence of a diet very high in vegetables, fruit, and fiber and low in fat on prognosis following treatment for breast cancer: the Women's Healthy Eating and Living (WHEL) randomized trial[END_REF]. In contrast, high consumption of b-carotene and vitamin C seems to influence survival [START_REF] Ingram | Diet and subsequent survival in women with breast cancer[END_REF]. Supplement use of vitamin C or E has been suggested to be associated with an decrease in breast cancer recurrence or breast-cancer related death [START_REF] Fleischauer | Antioxidant supplements and risk of breast cancer recurrence and breast cancer-related mortality among postmenopausal women[END_REF]. Women, assigned to the non-intervention group of the WHEL study, with high plasma-carotenoid concentration, had a significantly reduced risk for breast cancer recurrence [START_REF] Rock | Plasma carotenoids and recurrence-free survival in women with a history of breast cancer[END_REF]. Interestingly, analyses of a cohort study of 477 breast cancer patients found that the association between key dietary variables with survival may be U-shaped and not linear. Thus moderate, rather than low, fat intakes are associated with optimal survival [START_REF] Goodwin | Diet and breast cancer: evidence that extremes in diet are associated with poor survival[END_REF]. A randomized clinical trial of fenretinide (a synthetic retinoid) showed a reduction of second breast cancer, which was more apparent among premenopausal women [START_REF] Veronesi | Fifteen-year results of a randomized phase III trial of fenretinide to prevent second breast cancer[END_REF].

No evidence has been found that antioxidant vitamin supplements reduce the toxicity associated with chemotherapy [START_REF] Ladas | Antioxidants and cancer therapy: a systematic review[END_REF]. Some authors even advise against the use of antioxidant supplements during chemotherapy or radiotherapy [START_REF] D 0 Andrea | Use of antioxidants during chemotherapy and radiotherapy should be avoided[END_REF].

High alcohol consumption may be associated with an increase in breast cancer mortality [START_REF] Mcdonald | Breast cancer survival in African American women: is alcohol consumption a prognostic indicator?[END_REF].

Biological mechanism

The cancer-preventive capacities of carotenoids are attributed to their anti-oxidant capacities and their effect on gap junctional communication which play a role in the regulation of cell growth, differentiation and apoptosis [START_REF] Tapiero | The role of carotenoids in the prevention of human pathologies[END_REF]. It has been demonstrated that carotenoids influence the proliferative capacity and differentiation of mammary epithelial cells [START_REF] Rock | Carotenoids induce morphological changes in human mammary epithelial cell cultures[END_REF]. But the negative relationship between prognosis and increased fruit and vegetable consumption may partly due to the fact that a high fruit and vegetable consumption is merely a marker for low dietary fat intake. An increased estrogen concentration and a change in folate metabolism are mentioned among the mechanisms linking alcohol with breast cancer [START_REF] Boffetta | Alcohol and cancer[END_REF].

Discussion

Despite the wealth of etiologic studies on breast cancer risk and the growing number of breast cancer survivors, surprising little is known on the association between life style changes made after the diagnosis of breast cancer and the outcome. Currently, only two randomized control trials are studying the relationship between lifestyle related factors and prognosis [START_REF] Pierce | A randomized trial of the effect of a plant-based dietary pattern on additional breast cancer events and survival: the Women's Healthy Eating and Living (WHEL) Study[END_REF][START_REF] Chlebowski | Adherence to a dietary fat intake reduction program in postmenopausal women receiving therapy for early breast cancer. The Women's Intervention Nutrition Study[END_REF]. Both of them restricted inclusion to early stage breast cancer. While merely three large cohort studies of breast cancer patients are collecting data [START_REF] Caan | Life After Cancer Epidemiology (LACE) Study: a cohort of early stage breast cancer survivors (United States)[END_REF][START_REF] Irwin | Physical activity levels before and after a diagnosis of breast carcinoma: the Health, Eating, Activity, and Lifestyle (HEAL) study[END_REF][START_REF] Lu | Impact of newly diagnosed breast cancer on quality of life among Chinese women[END_REF].

Based on available literature, a multidisciplinary approach regarding breast cancer is recommended and should include physical activity, body weight control, high consumption of fruit and vegetables, and a reduction of dietary fat intake. Recent results from the WHEL study suggested that a healthy lifestyle that combines high levels of fruit and vegetables consumption with physical activity is associated with an increase in survival [START_REF] Pierce | Greater survival after breast cancer in physically active women with high vegetable-fruit intake regardless of obesity[END_REF]. Thus rather than one factor, a combination of lifestyle behaviors may improve breast cancer survival [START_REF] Ballard-Barbash | Is the whole larger than the sum of the parts? The promise of combining physical activity and diet to improve cancer outcomes[END_REF]. Besides the beneficial effect on recurrence, a healthy diet and regular exercise has the potential to reduce the risk of co morbidity (such as other cancers, cardiovascular disease, diabetes, etc.) and improving the quality of life (through a reduction of the risk on fatigue and depression) [START_REF] Jones | Diet, exercise, and complementary therapies after primary treatment for cancer[END_REF]. The American Cancer Society has summarized the best clinical practices related to optimal nutrition and physical activity during treatment and recovery, living after recovery from treatment and living with advanced cancer [START_REF] Doyle | Nutrition and physical activity during and after cancer treatment: an American Cancer Society guide for informed choices[END_REF]. An overview of other resources available concerning information and health promotion to cancer survivors is given elsewhere [START_REF] Jones | Diet, exercise, and complementary therapies after primary treatment for cancer[END_REF]. Although many patients with cancer report healthier lifestyles after diagnosis, these changes might be temporary. Few studies have investigated the most appropriate intervention time (at diagnosis or later), patients' preferences about delivery of interventions or the relative efficacy of different methods (clinic versus home-based, face-to-face counselling, mailed interventions, telephone counselling, etc.) [START_REF] Jones | Diet, exercise, and complementary therapies after primary treatment for cancer[END_REF]. The FRESH START trial which included newly diagnosed breast or prostate cancer patients found that tailored mailed interventions are more effective in promoting healthful life styles (which included exercise, consumption of fruit and vegetables, overall diet quality, and decreasing intake of fat and saturated fat) than standardized materials [START_REF] Demark-Wahnefried | Main outcomes of the FRESH START trial: a sequentially tailored, diet and exercise mailed print intervention among breast and prostate cancer survivors[END_REF]. More research is warranted. Unfortunately, although cancer survivors may be susceptible to health promotion, many health care providers may miss opportunities to counsel survivors about healthy behaviours [START_REF] Sabatino | Provider counseling about health behaviors among cancer survivors in the United States[END_REF]. Nevertheless, health care providers do have an important educational role concerning modifiable causes of cancer. A survey of cancer survivors' beliefs showed that the respondents underestimated the importance of behavioral factors (such as obesity and physical inactivity) known to be related to be with increased cancer risk, while overestimating the importance of stress and environmental pollution [START_REF] Wold | What do cancer survivors believe causes cancer? (United States)[END_REF].

More research is needed before conclusive advice can be given to women with breast cancer. Intervention studies should answer on what lifestyle changes may be used as adjuvant therapy or as a modifier of tumor growth and progression. Future studies on the association between breast cancer prognosis and modifiable factors need to obtain complete clinical, dietary, and lifestyle information at moment of breast cancer diagnosis and this should be updated regularly during follow up. Albeit a challenge in designing, clinical trials are needed to determine the effect of physical activity on the recurrence and mortality of breast cancer [START_REF] Irwin | Randomized controlled trials of physical activity and breast cancer prevention[END_REF]. To date, the WINS study is the first randomized trial that suggested that a dietary intervention may reduce breast cancer recurrence [START_REF] Chlebowski | Dietary fat reduction and breast cancer outcome: interim efficacy results from the Women's Intervention Nutrition Study[END_REF]. Future studies should investigate whether or not there is a threshold of dietary fat intake at which an increase in tumour recurrence is observed (Table 2).

  54 (0.27-1.04) Age at diagnosis, age at menopause, tumour stage, tamoxifen, radiation therapy use, hormone replacement therapy, smoking, exercise, and dietary intake of vitamin C or E. Rock et al. (2005) [61] 1,551 7 years Breast cancer recurrence HR: 0.57 (0.37-0.89) Age at diagnosis, clinical centre, plasma cholesterol, body mass index, tumour hormone receptors, and adjuvant chemotherapy. Veronesi et al. (2006) [63] 1,739 14.6 years Breast cancer recurrence HR: 0.62 (0.46-0.83) (premenopausal) Menopausal status at randomization, type of surgery, pT and histology. HR: 1.23 (0.63-2.40) (postmenopausal) McDonald et al. (2002) [66] 125 Breast cancer mortality HR: 2.7 (1.3-5.58) Stage of disease at diagnosis, radiation therapy, and cigarette smoking.

Table 1

 1 Overview of the prospective studies and randomized clinical studies investing the association between life style factors and breast cancer prognosis

		Country Breast cancer	Major exposures of interest
	Prospective cohort studies
	Life After Cancer Epidemiology (LACE)	US	Stage I & II invasive	Diet
	Study [7]			Physical activity
				Quality of life
	Health, Eating, and	US	In situ breast carcinoma to stage IIIa breast	Diet
	Lifestyle (HEAL) Study [8]		carcinoma	Physical activity
				Weight
	Shanghai Breast Cancer Survival (SBSS)	China	Newly diagnosed	Life-style
	Study [9]			Disease-and treatment-related
				factors
				Quality of life
	Randomized clinical trials
	Women's Healthy Eating	US	Stage I (C1 cm), II or IIIA invasive	High-vegetable, low-fat diet
	and Living (WHEL) Study [10]
	Women's Intervention Nutrition (WINS)	US	Invasive (exclusion for tumour size less than	Dietary fat reduction
	Study [11]		1 cm with negative nodes; tumour size greater
			than 5 cm with positive nodes; 10 or more nodes
			positive)

Table 2

 2 Overview of the different studies that investigated the relationship between lifestyle changes and breast cancer prognosis Age, time since diagnosis; body-mass index; energy intake, smoking, physical activity , diet missing in1986, 1990, 1994, 1998, age at menarche, oral contraceptive use , birth index, menopausal status and use of postmenopausal hormone therapy , age at menopause , tamoxifen , chemotherapy; stage at diagnosis. Tumour stage, clinic site, age at randomization, anti-oestrogen use, and oophorectomy status.

	Adjustment confounding variables			Age, interval between diagnosis and physical activity assessment,	smoking, BMI, menopausal status and hormone therapy use, age at	first birth and parity, oral contraceptive use, energy intake, energy-	adjusted protein intake, disease stage, radiation treatment,	chemotherapy and tamoxifen treatment.	Age at diagnosis, stage at diagnosis, state of residence, interval	between diagnosis and physical assessment, post diagnosis BMI,	post diagnosis menopausal status, post diagnosis hormone therapy	use, total energy intake year before enrolment, education level,	family history of breast cancer at diagnosis, and initial treatment	modality.	Stage and income.		Age, stage at diagnosis and BMI.		Age, total caloric intake, stage at diagnosis.				Age, alcohol, tumour hormone receptor status and time from diagnosis	to study entry.		Age, alcohol intake, energy, protein, vitamin C and b carotene intake,	estrogen receptor, progesterone receptor, body mass index, oral	contraceptive use, stage, tumour type, categories of treatment, year	of diagnosis, age at first birth and parity, menopausal status and	postmenopausal hormone use.	Stage, tumour grade, tumour size, age at diagnosis and menopausal	status.	Age at diagnosis, household income, hormone-replacement therapy,	and length of residence in interview home.	Age, stage at diagnosis.
	Relative risk/hazard ratio (95% CI) comparison	of highest quartile/quintile vs lowest		RR: 0.60 (0.40-0.89)		RR: 0.74 (0.53-1.04)		HR: 0.51 (0.29-0.89)						HR: 0.78 (0.56-1.08)		HR: 0.78 (0.45-1.34)		RR: 1.0 (0.6-1.6)				HR: 0.56 (0.31-0.98)			RR: 1.02 (0.83-1.24)		RR: 1.07(0.88-1.29)	HR: 1.08 (0.55-2.13)	HR: 1.39 (0.72-2.68)	HR: 1.04 (0.63-1.71)	RR: 2.14 (1.47-3.10)
	Outcome			Breast cancer	mortality	Recurrence		Breast cancer	mortality					Overall	mortality	Breast cancer	mortality	Breast cancer	mortality			Mortality			Breast cancer	mortality	Recurrence	Breast cancer	survival	Distant relapse-	free survival	Breast cancer	mortality	Breast cancer	mortality
	Author, year No. of Median length	(reference) cases followed	Physical activity	Holmes et al. 2,987 8 years	(2005) [12]				Holick et al. 4,482 5.6 years	(2008) [13]					Abrahamson 1,264 8-10 years	et al. (2006) [14]	Enger et al. 717 10.4 years	(2004) [15]	Borugian et al. 603 Between 7.9 (Stage	(2004) [17] II) and 8.3 years	(Stage I)	In combination with diet rich in fruit/vegetables	Pierce et al. 1,490 6.7 years	(2007) [16]	Smoking (current versus never smoker)	Holmes et al. 5,056 8.25 years	(2007) [23]	Fentiman et al. 166 11 years	(2005) [24]	Sagiv et al. 1,273 5.8 years	(2007) [25]	Manjer et al. 792 12.1 years	(2000) [26]	Obesity and weight gain after diagnosis

Breast Cancer Res Treat (2009) 114:13-22


R J Jones
email: r.jones@beatson.gla.ac.uk

Ae O Young

Ae L Renshaw

Ae V Jacobs

Ae M Fennell

Ae A Marshall

Ae T P Green

Ae P Elvin

Ae C Womack

Ae G Clack

Ae J M Dixon

O Young

Á L Renshaw

J M Dixon

V Jacobs

Á M Fennell

Á A Marshall

Á T P Green

Á P Elvin

Á C Womack

Á G Clack

Src inhibitors in early breast cancer: a methodology, feasibility and variability study

Keywords: Src kinase, AZD0530, Early breast cancer, Focal adhesion kinase, Paxillin, Immunohistochemistry, Phosphorylation

Early clinical trials of anticancer agents may be enriched by robust biomarkers of activity. Surrogate measures used in trials of cytotoxic agents, such as tumor size regression, may not be informative when investigating targeted agents that act principally to inhibit invasion or proliferation. This study aimed to determine the validity of invasion-related biomarkers of activity for AZD0530, a potent Src inhibitor currently in clinical development. Focal adhesion kinase (FAK) and paxillin are downstream phosphorylation substrates of Src and mediate tumor cell adhesion and invasiveness. These were therefore selected as biologically relevant markers of Src inhibition. Early breast cancer was chosen as a model as multiple samples can be collected during standard treatment and there is an intervening period in which experimental intervention can be applied. Tumor tissue was collected from diagnostic core biopsies and subsequent surgical tumor excision samples in 29 women with early breast cancer attending a single center. Protein levels were assessed quantitatively by Luminex Ò and qualitatively by immunohistochemistry. AZD0530 inhibited tumor growth in a manner independent of dose and inhibited phosphorylation of FAK and paxillin in a dose-dependent manner in a Calu-6 xenograft model. In the clinical study, agreement of within-visit and also of between-visit measurements was high and the estimated number of patients required to detect a drug effect would be low enough to allow use of these markers as endpoints in future dose selection studies.

Background

Rationale for a Src inhibitor in breast cancer Src kinase and its potential role in cancer development has been reviewed extensively in the literature [START_REF] Frame | Newest findings on the oldest oncogene; how activated src does it[END_REF][START_REF] Irby | Role of Src expression and activation in human cancer[END_REF]. As with many other common solid tumors, Src expression and activity is elevated in human breast cancers [START_REF] Ottenhoff-Kalff | Characterization of protein tyrosine kinases from human breast cancer: involvement of the c-src oncogene product[END_REF][START_REF] Verbeek | c-Src protein expression is increased in human breast cancer An immunohistochemical and biochemical analysis[END_REF][START_REF] Reissig | Elevated activity and expression of Src-family kinases in human breast carcinoma tissue versus matched non-tumor tissue[END_REF] with high activity being associated with poor outcome [START_REF] Ito | Activation of c-Src is inversely correlated with biological aggressiveness of breast carcinoma[END_REF]. Preclinical data support a role for Src in the regulation of breast cancer cell motility, invasion and metastasis [START_REF] Rucci | Inhibition of protein kinase c-Src reduces the incidence of breast cancer metastases and increases survival in mice: implications for therapy[END_REF][START_REF] Hiscox | Src kinase promotes adhesion-independent activation of FAK and enhances cellular migration in tamoxifen resistant breast cancer cells[END_REF][START_REF] Hiscox | Elevated Src activity promotes cellular invasion and motility in tamoxifen resistant breast cancer cells[END_REF]. Evidence also exists linking Src signaling to estrogen receptor signaling [START_REF] Hiscox | Elevated Src activity promotes cellular invasion and motility in tamoxifen resistant breast cancer cells[END_REF]. It is therefore possible that inhibition of Src could have an effect in tumors that fail to respond to hormone or growth factor therapies [START_REF] Finn | Dasatinib, an orally active small molecule inhibitor of both the src and abl kinases, selectively inhibits growth of basal-type/''triple-negative'' breast cancer cell lines growing in vitro[END_REF][START_REF] Hiscox | Src as a therapeutic target in anti-hormone/anti-growth factor-resistant breast cancer[END_REF], a group where there remains a high level of unmet clinical need.

Methodological rationale in early breast cancer

Early breast cancer is an attractive disease model for pharmacodynamic studies using tumor tissue biomarkers. In most centers, the routine diagnosis and management of breast cancer is a two-stage process. Patients undergo tumor biopsy-usually a core biopsy-to confirm the diagnosis of invasive cancer at which point samples can be taken for experimental investigation. This is followed by surgical excision of the tumor permitting the use of tissue, which is excess to diagnostic requirements, for further biomarker assessment. Clinical logistics usually dictate that there will be a window of time between the biopsy and definitive surgery during which an experimental intervention (such as drug administration) can take place without compromising the patient's routine cancer care. Hence, this approach provides an ideal model for measuring the effects of novel drugs on tissue biomarkers in patients with early, treatment-naı ¨ve cancer (Fig. 1). AZD0530 AZD0530 is a potent Src inhibitor that modulates multiple key signalling pathways in cancer. In keeping with its mechanism of action, AZD0530 has little effect on tumor growth in preclinical models, but does inhibit cancer cell invasion and metastasis in vitro and in vivo [START_REF] Green | Pre-clinical and early clinical activity of the highly selective, orally available, dual Src/Abl kinase inhibitor AZD0530[END_REF].

Rationale for choice of biomarkers Multiple substrates have been described for Src. It is likely that the main mode of action of Src during cancer cell invasion is via regulation of focal adhesion complexes. Focal adhesion complexes contain transmembrane integrins that interact with the extracellular matrix (ECM) and a multi-component, intracellular complex which mediates and regulates interactions between the ECM and the intracellular signaling machinery and cytoskeleton. Src activation leads to phosphorylation of focal adhesion kinase (FAK) and paxillin, both of which are components of the focal adhesion complex [13,[START_REF] Rodina | Phosphorylation of p125FAK and paxillin focal adhesion proteins in src-transformed cells with different metastatic capacity[END_REF].

The Luminex Ò platform (Luminex Corporation, Austin, TX, USA) used in this study, presents a reproducible system for quantifying relative concentrations of analytes in tumor lysates. Immunohistochemistry (IHC) allows qualitative assessment of these analytes (and hence the ability to control for intra-biopsy heterogeneity).

Methods

Preclinical xenograft model

The preclinical study was conducted at Oncodesign (Dijon, France). Nude female rats (RH-rnu/rnu) 1 were sublethally irradiated prior to implantation using a cobalt (Co 60 ) radiation source. Treatment began 1 h prior to implantation and the four groups were given once-daily oral doses of AZD0530 5, 10 or 20 mg/kg or vehicle alone. Each animal was inoculated subcutaneously with 2 9 10 7 Calu-6 (human pulmonary carcinoma) cells. Tumor volume was measured twice-weekly using callipers and tumor growth was recorded. Dosing continued for 28 days. The animals were sacrificed 6 h after receiving the final dose and tumor tissues were collected.

Patients

Female patients with early breast cancer undergoing core biopsy and subsequent surgical excision of their breast tumors presenting to a single center were enrolled. The planned recruitment was 30 patients as this was a number considered likely to provide appropriate information about the distribution of the endpoints being investigated. Patients who had received investigational therapies within 4 weeks of study entry were excluded. All women gave signed, informed consent to participate in the study and the study was approved by the local area ethics committee.

Tissue sampling procedure

At the time of diagnostic core biopsy, following informed consent, six extra samples were taken using a 14-gauge core biopsy needle. Five further samples from each cancer were collected using a 14-gauge core biopsy needle from the excised tumor immediately after surgical removal. Two samples from the diagnostic and surgical cores were prepared for Luminex analysis, and three samples from each Fig. 1 Methodological feasibility of biomarker studies in early breast cancer time point for frozen-section IHC. The sixth sample, taken at the diagnostic core biopsy, was prepared for paraffinembedded sectioning for IHC. All samples were immersed in the respective fixative within 5 min of removal from the patient.

Tissue fixation and storage

Samples for Luminex analysis were snap frozen in liquid nitrogen. Samples for paraffin-embedded IHC were immediately immersed in 10% neutral buffered formalin for 24 h prior to paraffin-block preparation. Samples for frozen IHC were immediately immersed in liquid nitrogen-precooled isopentane. All frozen specimens were stored at -80°C, and paraffin blocks were stored at room temperature.

Lysate preparation

Samples were transported to a central laboratory on dry ice, mechanically disrupted together with a 15-fold (wt/vol) quantity of frozen lysis buffer, pH 7.4 (10 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na 4 P 2 O 7 , 2 mM Na 3 VO 4 , 1% Triton-X100, 10% glycerol, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM PMSF and 5% Sigma P2714 Protease Inhibitor Cocktail (Sigma Aldrich Co Ltd., Poole, Dorset, UK)) using a SPEX 6850 liquid nitrogen freezer mill (SPEX CertiPrep, Metuchen, NJ, USA) to produce a mixed powder of tissue and lysate solution at -80°C. Samples were thawed, mixed and centrifuged. Total protein in the supernatant was quantified using the Pierce BCA optical protein assay (Pierce and Warriner, Chester, UK). A 1.5 mg/ml lysate was prepared from cultured mouse NIH 3T3 cells overexpressing active Src in the same lysis buffer for use as an assay standard.

Rabbit polyclonal capture antibodies were coupled to Luminex beads using the Bio-Rad Amine coupling kit as described in the manufacturer's instructions (bead sets and kit: Bio-Rad Laboratories Ltd., Hemel Hempstead, Hertfordshire, UK; other reagents: Sigma Aldrich Co Ltd.).

Tyr861 on FAK and Tyr31 on paxillin were chosen as specific substrates, although other tyrosine residues are also phosphorylated in a Src-dependent manner on both proteins. As the amount of any phosphorylated protein is dependent on the total amount of that protein, total paxillin was also measured. Reliable reagents for quantification of total FAK could not be identified. The primary measure of Src activity at focal adhesions was therefore expressed as the ratio of phosphorylated paxillin to total paxillin. Antibodies were supplied without bovine serum albumin or azide. Antibody (5 lg per reaction) was used for both anti-p-FAK Y861, and anti-paxillin pY31 (BioSource International, Camarillo, CA, USA) and 10 lg total paxillin antibody per reaction (Cell Signaling Technology Inc., Beverly, MA, USA).

Luminex assay

The assay technique, reagents and controls have been described in detail [START_REF] Jones | The impact of delay in cryo-fixation on biomarkers of Src tyrosine kinase activity in human breast and bladder cancers[END_REF]. In brief, 25 ll of diluted bead antibody conjugate (at 100 beads/ll) was added to each well. Lysate (100 ll) was then added to each well. A c-Src3T3 lysate standard dilution range was prepared starting at 100 lg/ml and the test lysates were diluted to 175 lg/ml. All samples and standards were run in triplicate. Phycolink anti-phosphotyrosine (type 1) R-phycoerythrin conjugate (50 ll/well) (Prozyme via Europa Bioproducts Ltd., Ely, Cambridgeshire, UK) was added prior to being read on the Bio-Rad Bio-Plex using Bio-Plex Manager v3 for data collection and quantitation of the unknowns against the c-Src3T3 standard curves. In the total paxillin assay, the c-Src3T3 lysate concentration was 300 lg/ml and the test lysate concentration was reduced to 100 lg/ml. Detection for these plates was by incubation with 50 ll/well antipaxillin (BD Transduction Laboratories, Franklin Lakes, NJ, USA) (diluted 1 in 83) followed by further incubation with 50 ll/well diluted (1 in 100) anti-mouse IgG/ biotin (Upstate Biotechnology (UBI), Lake Placid, NY, USA) and a further incubation with 50 ll/well (diluted at 1 in 100) streptavidin-phycoerythrin conjugate (UBI). The beads used for the total phosphorylation assay were Qiagen LiquiChip Avidin Beads (Qiagen GmbH, Hilden, Germany), which were allowed to bind to Cell Signaling Technology pY100-Biotin (75 ll/well of a 1:1600 dilution).

Immunohistochemistry

For the preclinical studies immunohistochemical (IHC) analyses were conducted as described in Jones et al. [START_REF] Jones | The impact of delay in cryo-fixation on biomarkers of Src tyrosine kinase activity in human breast and bladder cancers[END_REF]. The following commercial antibodies were used in the range of 5-10 lg/ml:

-Total paxillin (Santa Cruz, SC-5574) -Phospho (p)-paxillin (BioSource, pY31 #44-720) -Total FAK (Santa Cruz, SC-932) -p-FAK (BioSource, pY861 #44-626G) For the clinical studies, sections (7 and 4 lm) were prepared from frozen and paraffin-embedded samples, respectively, and processed using the Dako EnVision Detection Kit.

The following primary antibodies (1-10 lg/ml) were used in analyses of frozen sections:

-Activated Src-mouse monoclonal clone 28 (supplied by Dr. Hisaaki Kawakatsu, Dean Sheppard Laboratory, San Francisco, CA, USA), stock concentration 1.9 mg/ml, assay concentration 1 lg/ml. This antibody targets the C-terminal tail of Src, which is only detectable when Src is in its active conformation [START_REF] Kawakatsu | A new monoclonal antibody which selectively recognizes the active form of Src tyrosine kinase[END_REF] Frozen sections were scored in a blinded fashion according to the intensity of staining of carcinoma cells on a scale of -, +/-, +, ++ or +++ by one observer (VJ) and confirmed by an independent reviewer (CW).

For paraffin sections, staining in nuclear, cytoplasmic and membrane subcompartments was scored on a scale of -(0), + (1), ++ (2) or +++ (3), and then multiplied by the percentage of cells stained in each subcompartment to give a H score out of 300. The H scores for each section were then summed to give a total score out of 900 (final H score).

Statistics

Previous experience indicated that the Luminex data conformed to a log-normal distribution; therefore, data were logarithmically transformed prior to analysis. Two separate analysis of variance (ANOVA) models were fitted to the Luminex data for each parameter. The first used individual results from the two separate samples available from each patient at each visit. This model included visit as a fixed effect and random effects for patient and the visit by patient interaction. This model gives an estimate of the variability between individual patients, between samples from the same patient at the same visit, and between samples from the same patient at different visits. Data were also analyzed in terms of mean value for each patient at each visit, again using an ANOVA model this time with visit fitted as a fixed effect and patient fitted as a random effect. This model gives an estimate of variability between individual patients and between visits for individual patients.

Results

In vivo tumor growth and biomarker expression

Growth of tumors in Calu-6 xenograft bearing rats was shown to be significantly decreased following treatment with AZD0530 relative to vehicle (AZD0530 5 and 20 mg/kg, P \ 0.0001; AZD0530 10 mg/kg, P = 0.0002) and was similar with all AZD0530 doses (Fig. 2a). Expression of phosphorylated Src substrates, FAK and paxillin, was also markedly reduced in these tumors following treatment with AZD0530 for 28 days as shown by IHC (Fig. 2b) and Luminex assay (Fig. 2c). These effects were dosedependent.

Patients

Thirty patients entered the study. One patient was found to have metastatic disease prior to surgery and so was excluded from the analysis.

Quantitative analysis using Luminex

Luminex results from diagnosis and surgery samples were obtained for 29, 28, 28 and 26 patients for p-paxillin, total paxillin, total p-tyrosine and p-FAK, respectively. The p-paxillin:total paxillin ratio was chosen as the primary endpoint of this study as this was considered the best reflection of Src activity. In addition, by correcting for total paxillin, this reduces inter-sample variability due to tissue heterogeneity. Twenty-eight p-paxillin:paxillin values were obtained in this study.

Intrapatient variability

For all analytes there was a wide range of values among different patients. Agreement between samples taken simultaneously from one patient was generally good (Fig. 3).

Furthermore, average readings from core biopsies had a high level of agreement with average readings at definitive surgery (Fig. 4).

The estimated components of variation for each of these endpoints are illustrated for individual values at each visit in Table 1 and for the mean values at each visit in Table 2.

The total between-patient and within-patient variability is reduced for all variables compared to the model using the individual values.

Semi-quantitative analysis by immunohistochemistry

Frozen samples

Of samples from 29 patients: 19, 24, 14, 25 and 20 provided evaluable IHC images using antibodies against activated Src, total paxillin, p-paxillin, total FAK and p-FAK, respectively. Discrimination was low between different levels of IHC staining in the frozen tissue sections, particularly for activated Src. All samples analyzed expressed active Src, with one classified as +, five as ++, and 13 as +++ at diagnosis. Representative sections are shown in Fig. 5. Agreement between visits was high: 15 assessments remained the same, and four changed by one category. The generally poor morphology of the frozen tissue sections, despite the use of a rigorous preparation protocol, meant that it was not possible to discriminate subcellular localization of antibody staining.

Paraffin-embedded samples

In contrast to the generally poor quality of the frozen tissue sections, the excellent morphological preservation observed in the paraffin-embedded tissues meant that subcellular distribution of p-paxillin and p-FAK could be visualized in sections from paraffin-embedded tissue (Figs. 6 and7).

H-score analyses provided information on the subcellular distribution of p-paxillin and p-FAK (Tables 3 and4).

Discussion

Rational drug development and pharmacodynamic biomarkers

Most oncology drugs entering Phase I trials never attain marketing approval. One common reason for failure is lack of efficacy in Phase II or III clinical trials [START_REF] Marchetti | The impact of FDA and EMEA guidelines on drug development in relation to Phase 0 trials[END_REF][START_REF] Booth | Oncology's trials[END_REF]. This has wide-ranging consequences. For example, patients and volunteers are exposed to toxic, sometimes life-threatening, side effects from drugs, which subsequently prove no clinical benefit. Furthermore, valuable resources (patients, skilled staff and funds) are consumed at the expense of other research needs. It is also probable that some drugs, which are tolerable and effective, are failing clinical development because efficacy testing is conducted using the incorrect dose, schedule or patient population. The consequence is that effective new treatments may be discarded, often late in clinical development.

The challenges facing clinical drug development have become more intense in recent years for several reasons: i. The focus has shifted from cytotoxic drugs to biological agents targeting specific oncogenic pathways; thus, therapeutic hypotheses are dependent upon specific molecular interventions. This leads to the reasonable assertion that if a drug can be robustly demonstrated to have failed to inhibit its target, then the therapeutic hypothesis will also fail. ii. New drugs often target oncogenic processes other than tumor growth. Because of this, tumor shrinkage (response rate) is a less valid surrogate for early clinical proof-of-concept testing than it is for cytotoxic agents. More relevant endpoints (such as disease stabilization, time to progression or overall survival) demand that larger numbers of patients be tested before concluding that an inactive drug is, indeed, inactive. iii. As the size of the predicted incremental benefits from additional new treatments declines, the number of patients required to demonstrate these benefits increases. This demands more resource and increases the cost of failure.

One response to these challenges is to find new ways to discover more information about a drug, in particular about its pharmacodynamic effects, at the very earliest stages of clinical development. This information can then be used for efficient decision making (for example, to terminate development prior to Phase II) and to guide later development (for example to optimize dose and schedule).

We have described part of the pre-Phase I programme for AZD0530, with a view to its potential application in early breast cancer. We have demonstrated that AZD0530 can reduce tumor growth, and modulate tumor FAK and paxillin phosphorylation in a rat xenograft model. In the clinical setting, we have shown that, in the majority of cases, it is feasible to obtain adequate amounts of tumor tissue from women undergoing routine care of early breast cancer on two separate occasions. Furthermore we have demonstrated that samples taken simultaneously and at a relevant time interval show good levels of agreement for the biomarkers in question. These data should enable a clinical trial in which these markers can be measured to assess the pharmacodynamic response to AZD0530. This approach could be used to explore a range of safe and tolerable doses so that the optimum biological dose is chosen for further development in early breast cancer. Furthermore, by estimating the variability of the proposed primary pharmacodynamic endpoint, we can predict the likely number of patients that would be required to demonstrate a true effect of a Src inhibitor such as AZD0530 in early breast cancer (Table 5). In this case, the number of patients required is sufficiently small to permit exploration of more than one dose in such a study. Tyrosine phosphorylation levels (such as those used in the present study) are, by their nature, labile, being dependent on the balance of activity of both phosphatases and kinases, themselves dependent upon upstream regulators and the availability of ATP. As ATP production is itself dependent upon oxidative metabolism, it was considered important to limit the delay between biopsy and tissue fixation. In a previous study, we have demonstrated that minor variation in this short delay is unlikely to have a significant impact on the results of Luminex analysis [START_REF] Jones | The impact of delay in cryo-fixation on biomarkers of Src tyrosine kinase activity in human breast and bladder cancers[END_REF]. One limitation of the Luminex assay described here is that it is not possible to control for inter-biopsy tissue heterogeneity. However, we have demonstrated that by averaging data from two biopsies at each patient assessment, the overall variability is sufficiently small to permit the use of these endpoints in pharmacodynamic studies.

There is currently no known clinical benefit in administering AZD0530 to patients with early breast cancer. Clinical trials in which the primary endpoint is not known to relate directly to clinical benefit are relatively commonplace, although they are usually conducted in healthy volunteers who are financially compensated for their inconvenience. In addition to the risks of administering drugs with very little prior exposure in man, a clinical trial of a novel agent in women with early breast cancer carries the additional risk that unforeseen toxicity could prevent or delay the implementation of conventional curative treatment. In particular, experience of the novel agent in association with general anesthesia and surgical wounding in man is absent. Ethically, it may be reasonable to conduct such studies in patients provided that fully informed consent is given, including information about the predicted absence of clinical benefit, and provided that toxicity is low and the risks of compromising the outcome of conventional cancer treatment are minimized.

From an ethical perspective, a trial such as that proposed in this model could only be justified if there were a reasonable degree of confidence that the result would be statistically valid. Therefore methodology studies, such as this one, are vital before such a trial could proceed in order that the trial could be sized with confidence.

Rationale for a Src inhibitor in early disease Src family members have been implicated in the regulation of multiple oncogenic processes, but the weight of a Insufficient evaluable material preclinical evidence suggests that their predominant role may be in the regulation of cancer cell invasion [START_REF] Frame | Src in cancer: deregulation and consequences for cell behaviour[END_REF]. The clinical development of drugs that target invasion presents a particular challenge, as we predict that these drugs may be of little benefit to patients with established metastatic disease. Rather they may be effective in preventing progression from early invasive disease to advanced metastatic disease. If this prediction is correct, then efficacy testing would be necessary in patients with early disease stages, using stage progression/disease-free survival as the primary endpoints. Prior to such studies it will be necessary to identify the dose which inhibits Src in vivo and to demonstrate that such a dose is safe for long-term use. AZD0530 is currently in clinical trials in patients with advanced solid malignancy.

Fig. 2

 2 Fig. 2 (a) Calu-6 tumor growth, (b) Immunohistochemistry detection of p-paxillin (pY31) and (c) Luminex detection of total paxillin and FAK, total phosphotyrosine levels and p-paxillin (Y31) and p-FAK (Y861) in rats bearing Calu-6 xenografts following treatment with AZD0530 5, 10 or 20 mg/kg for 28 days

Fig. 3

 3 Fig. 3 Correspondence between Luminex values for separate core samples taken at same visit. Values are expressed in c-Src3T3 lysate equivalent lg/ml

Fig. 4

 4 Fig.4Correspondence between diagnosis and surgery visit Luminex values. Footnote for total p-tyrosine: one patient is not plotted as the baseline value was zero. Value at visit 2 was 179.92

Fig. 5 FinalFig. 6 6 ScoreFig. 7

 5667 Fig. 5 Selected frozen tumor biopsy sections stained for active Src. (a) Patient 24, graded '+' intensity. (b) Patient 4, graded '+++' intensity

Table 1

 1

	Components of variability of individual values	Endpoint	Between patient Within patient, between visit Within patient, within visit
	for Luminex analysis	p-Paxillin/total paxillin 0.3049	0.0000	0.5064
		p-Paxillin	0.2114	0.1094	0.3545
		p-FAK Y861	0.3365	0.0984	0.1255
		Total p-tyrosine	0.6306	0.5373	0.3798
		Total paxillin	0.0343	0.0437	0.4549

Table 2

 2 Components of variability of mean values for Luminex analysis

	Endpoint	Between patient	Within patient
	p-Paxillin/total paxillin	0.2910	0.3329
	p-Paxillin	0.2493	0.1845
	p-FAK Y861	0.3625	0.1518
	Total p-tyrosine	0.6011	0.6432
	Total paxillin	0.0881	0.1588

Table 3

 3 Composite H scores for p-paxillin staining of paraffin-embedded tissue

	Patient	Nuclear			Sub	Cytoplasmic		Sub total	Membrane		Sub	Final
	number	+	++	+++	total	+	++	+++		+	++	+++	total	H score
	1	10			10	10			10	40	40		120	140
	2				0			80	240				0	240
	3				0				0				0	0
	4				0	10			10				0	10
	5				0	50	20		90		70		140	230
	6	1 0			1 0	2 0			2 0				0	3 0
	7				0		90		180				0	180
	8				0	40	40		120		40		80	200
	9	5			5	50			50		50		100	155
	10 a
	11				0	50	10		70		20	50	190	260
	1 2				0	2 0	2 0		6 0		1 0		2 0	8 0
	1 3				0	4 0			4 0	3 0			3 0	7 0
	14	30			30	30	30	10	120			30	90	240
	15	10			10	70			70	10			10	90
	16	10			10	30	30		90				0	100
	17				0	20			20	10	50		110	130
	18	10			10	10			10	10			10	30
	19	10			10	70			70	40			40	120
	20	10			10		50		100		50		100	210
	21	10			10	50	20		90		50		100	200
	22	10			10	10	40	10	120	30			30	160
	23	10			10	10		40	130		20		40	180
	24	10			10	80			80	40	40		120	210
	25	20			20	70	20		110	50	30		110	240
	26	10			10	10			10	10			10	30
	27	10			10	10			10				0	20
	28				0				0				0	0
	29	10			10	20			20				0	30

Table 4

 4 Composite H scores for p-FAK staining of paraffin-embedded tissue

	Patient	Nuclear		Sub	Cytoplasmic		Sub	Membrane	Sub	Final
	number			total				total			total	H score
		+	++	+++	+	++	+++		+	++	+++
	1			0		30		60		70	140	200
	2			0	50	50		150		50	100	250
	3			0		60		120			0	120
	4			0		60		120			0	120
	5			0		80		160		50	100	260
	6			0		70	5	155		5	10	165
	7			0		70		140		5	10	150
	8			0		90		180		70	140	320
	9			0		90		180		40	40	200	380
	10			0		100		200			50	150	350
	11			0	70	30		130			70	210	340
	12			0		40	60	260			50	150	410
	13			0	100			100	50	50	150	250
	14			0		100		200	30	30	30	180	380
	15			0	50	50		150		50	100	250
	16			0	30	20	10	100		10	20	120
	17			0				0		50	100	100
	18			0	10	40		90		50	100	190
	19			0		50	50	250		60	20	180	430
	20			0		40	40	200			50	150	350
	21			0		100		200		50	100	300
	22			0		100		200	30	30	90	290
	23			0	80	20		120		50	100	220
	24			0	50	50		150	50	10	70	220
	25			0	30	50	20	190	10	80	170	360
	26			0	90		10	120		10	20	140
	27			0	90		10	120			10	30	150
	28			0	100			100			0	100
	29			0	90	10		110			0	110

Table 5

 5 Sample size estimates a

	Endpoint	Estimate	Number of patients
		of SD	per group
	p-Paxillin/total paxillin	0.790	8
	p-Paxillin	0.659	6
	p-FAK Y861	0.717	7
	Total p-tyrosine	1.115	15
	a Sample size estimates are based on a 50% treatment difference, 1
	sided 20% significance level and 80% power

Calu-6 nude female rats were generated by Oncodesign (Dijon, France).

Breast Cancer Res Treat (2009) 114:211-221