To investigate effects of dietary mushrooms and joint effects of mushrooms and green tea on breast cancer, a case–control study was conducted in southeast China in 2004–2005. The incident cases were 1,009 female patients aged 20–87 years with histologically confirmed breast cancer. The 1,009 age‐matched controls were healthy women randomly recruited from outpatient breast clinics. Information on frequency and quantity of dietary intake of mushrooms and tea consumption, usual diet, and lifestyle were collected by face‐to‐face interview using a validated and reliable questionnaire. Compared with nonconsumers, the Odds ratios (Ors) were 0.36 (95% CI = 0.25–0.51) and 0.53 (0.38–0.73) for daily intake of ≥10 g fresh mushrooms and ≥4 g dried mushrooms, based on multivariate logistic regression analysis adjusting for established and potential confounders. There were dose–response relationships with significant tests for trend ( p < 0.001). The inverse association was found in both pre‐ and postmenopausal women. Compared with those who consumed neither mushrooms nor green tea, the ORs were 0.11 (0.06–0.20) and 0.18 (0.11–0.29) for daily high intake of fresh and dried mushrooms combined with consuming beverages made from ≥1.05 g dried green tea leaves per day. The corresponding linear trends were statistically significant for joint effect ( p < 0.001). We conclude that higher dietary intake of mushrooms decreased breast cancer risk in pre‐ and postmenopausal Chinese women and an additional decreased risk of breast cancer from joint effect of mushrooms and green tea was observed. More research is warranted to examine the effects of dietary mushrooms and mechanism of joint effects of phytochemicals on breast cancer. © 2008 Wiley‐Liss, Inc.

Breast cancer is the most common type of cancer among women worldwide and its rate is increasing in both developed and developing countries.1 The burden is not evenly distributed and there are large variations in the incidence rates of breast cancer between different countries. The incidence rate in China is 18.7 per 100,000 women‐years, which is 4 to 5‐fold lower than rates typically found in developed countries.2 However, the rate of breast cancer increased by 50.5% from 1972 to 1994 in the now relatively affluent southeast of China.3 Mushrooms and mushroom extracts have been shown to possess anticarcinogenic properties, antimutagenic activity and to stimulate immune responsiveness in experimental models, both in vitro and in vivo.4-10 Laboratory studies have demonstrated that mushrooms inhibited aromatase activity in breast cancer cell line and suppressed growth and invasiveness of metastatic breast cancers.9, 10 Despite the demonstration of the anticarcinogenic effect of mushrooms by many experimental studies, little relevant epidemiologic evidence is available from human populations. Only a few epidemiological studies have examined the association between mushroom intake and cancer.11-15 Two case–control studies reported a decreased risk of breast cancer associated with dietary mushrooms consumption in Korean women.14, 15 Tea polyphenols, particularly green tea polyphenols, have been demonstrated to possess anticarcinogenic effects against breast cancer in experimental.16 Epidemiologic data reported that green tea was inversely associated with breast cancer risk including one of our studies.17, 18 In view of the variations in rates of breast cancer and patterns of dietary practices, as well as the separately emerging bodies of evidence on the possible cancer‐protective properties of mushrooms and green tea, it is important to investigate whether the joint effect of mushrooms and green tea is synergistic on breast cancer, which may explain the lower incidence of breast cancer in China. We conducted a case–control study in southeast China to investigate the association between breast cancer risk and dietary intake of fresh and dried mushrooms and joint effect of mushrooms and green tea drinking on the malignant disease, adjusted for other lifestyle factors.

Material and methods Study design and subjects A hospital‐based case–control study of breast cancer risk was conducted in Hangzhou, the capital city of Zhejiang Province, between July 2004 and September 2005. All participants were Chinese women resident in Zhejiang Province and aged between 20 and 87 years. Cases were identified from medical records in 4 teaching hospitals of the School of Medicine, Zhejiang University. All the participating hospitals were public hospitals with 500–2,000 beds and received patients from all over the province. A total of 1,009 female patients, who were newly diagnosed with invasive ductal carcinomas or in situ carcinoma of the breast, were recruited to the study. All diagnoses were histopathologically confirmed after surgery. The patients were excluded if breast cancer was neither the primary nor final diagnosis. All relevant hospital and laboratory pathology reports were reviewed daily to ensure full ascertainment of the cases. The proportion of lost or nonresponding patients among the cases was 1.2%. During the same period of data collection, 1,009 healthy women were selected consecutively in the participating hospitals as controls to match each case's age within 5‐year age group using a daily update of the list of cases after they had consulted their doctors. Each control was recruited as the first in the matched age group to attend the outpatient breast clinic for a routine preventive care and who consented to participate. The proportion of selected controls who participated was 98.7%. Potential control women were excluded if they had a diagnosis of any benign or neoplastic breast disease, or another malignant disease at the recruitment. The project received ethics clearance from both the Human Research Ethics Committee of The University of Western Australia and the Chinese hospital authorities. Dietary assessment and interview Subjects were briefed regarding the general aims of the study to investigate lifestyle factors, confidentiality, and anonymity issues. An appointment for an interview was made after obtaining their consent via an initial contact. A face‐to‐face interview was then conducted by trained interviewers using a structured questionnaire in the hospital setting and usually took 30–40 minutes. The cases were interviewed in breast surgery wards, most of them (91.6%) within 3 months after diagnosis, while the controls were interviewed in the outpatient clinic of the same hospital. A validated and reliable questionnaire was used to collect the information on: (i) demographic and lifestyle characteristics, e.g., residential area, education, tea consumption, and physical activity; (ii) dietary intake assessed by a food frequency questionnaire (FFQ) including fresh and dried mushrooms and (iii) factors relevant to hormonal status, including menstrual history and menopausal status, reproductive and lactation history, oral contraceptive use, benign proliferation breast disease, and family history of breast cancer. Habitual dietary intake was assessed using a 100‐food item FFQ. This quantitative FFQ was adapted from a dietary questionnaire used in our previous studies on cancer.19 Dietary intake of mushrooms was measured by 2 items: “fresh” and “dried” mushrooms. The most common mushroom species reported by study participants was the white button mushroom (Agaricus bisporus) for fresh mushrooms and the fragrant mushroom (Lentinula edodes) for dried mushrooms. The frequency of food intake was categorized into never or hardly ever, once a month, 2–3 times a month, once a week, 2–3 times a week, 4–6 times a week, once a day, twice a day, and ≥3 times a day. Information was also sought on the usual amount of each food consumed per meal as well as cooking methods used and vitamins or mineral supplements taken. The quantitative variables were measured using the Chinese common measure liang (equivalent to 50 g). Standard containers (small 200 ml, medium 400 ml, and large 600 ml bowls, and a 350–400 ml cup), which were the typical eating utensils and teacup used by Zhejiang residents, were displayed during the interview to increase the accuracy of measurement. Food consumption was based on habitual diet and a “reference” recall period was set as 1 year prior to diagnosis for cases or interview for controls. If there was any recent change in habits, only information on the habits before the change was used in data analysis. The validity and reliability of the FFQ was assessed in a preliminary study and then evaluated by a test‐retest.20 The reproducibility of the FFQ was evaluated and the intraclass correlation coefficients for the intake of total energy, fresh mushrooms, dried mushrooms, alcohol, and tea consumption were 0.84, 0.86, 0.74, 0.88, and 0.83, respectively.20 The high coefficients for test‐retest reliability suggested that the FFQ may be reliable in assessing food consumption. After interview, anthropometric measurements were requested of all participants. Data management and analysis All data were checked for completeness at the end of each interview. The data were coded and analyzed using the SPSS version 12.0. Data collected by different interviewers were compared and we confirmed that no consistent difference in recording of key variables such as passive smoking and tea consumption occurred within cases or controls. The daily intake of foods (in grams) was obtained based on the variables of frequency and quantity derived from the FFQ, accounting for the edible portion of each food, cooking methods used, seasonal factors, and market availability.21 Total energy intake was estimated using Chinese Food Composition Tables.22 Physical activity was expressed in terms of weekly metabolic equivalent task hour (weekly MET‐hour).23 MET scores 6, 4.5, and 2.5 were assigned respectively for vigorous, moderate, and walking activity based on a compendium of physical activities.24 To facilitate analysis, approximate tertiles were used as a cut‐off method to classify the controls into 3 categories based on their overall daily intake of dietary mushrooms and green tea while nonconsumers were grouped into the first category. A combination variable was generated to assess the joint effect of dietary mushrooms and green tea. The women who consumed neither dietary mushrooms nor green tea were used as the reference category. Thereafter, the medians of overall daily intake of mushrooms and green tea in controls as a cut‐off point for low and high subgroups. Demographic characteristics and potential risk factors present in cases and controls, and selected characteristics of study subjects associated with intake levels of dietary mushrooms were compared using a t‐test or one‐way ANOVA for continuous variables and Chi‐square test for categorical variables. Odds ratios (ORs), 95% confidence intervals (CIs) and p values were estimated using unconditional logistic regression. Univariate analysis was undertaken to screen potential explanatory variables for subsequent multivariate analysis. We firstly evaluated the effect of dietary intake of mushrooms and its joint effect with green tea on breast cancer risk, adjusting for age, resident area, education, BMI 5 years prior to diagnosis for cases or interview for controls (calculated as the weight in kilograms at 5‐years ago divided by square of height in meters), age at menarche, oral contraceptive use, hormone replacement therapy, breast cancer in first degree relatives, total energy intake, and menopausal status. We further assessed the effect and combined effect on breast cancer risk, by including additional lifestyle factors for alcohol consumption, tobacco smoking, passive smoking, physical activity, and tea drinking (not for the joint effect). These potential confounders were included in the models because either they emerged as significant risk factors for breast cancer in previous studies18, 25-27 or because we observed some evidence of potential confounding in our data set by comparisons of univariate and multivariate analyzes. Each quantitative measure of dietary intake of mushrooms was subjected to a linear trend test. Since the biological mechanisms for breast cancer may differ between pre‐ and postmenopausal women, separate analyzes stratified by menopausal status were also conducted. Women were defined as postmenopausal if they indicated they had ceased having periods for at least 1 year at the time of interview and (for cases) had stopped them prior to the cancer surgery. Women who had had a surgical menopause at age over 50 years were also assigned the postmenopausal group.

Results The proportion of in situ carcinoma was only 3% of all breast cancer patients in the study, which made it impracticable to study them separately. Therefore, the patients with in situ carcinomas and invasive breast cancer were combined to form a single case group in analyzes. We report the results from all of the cases combined below. Table I contrasts demographic characteristics and lifestyle factors between cases with breast cancer and controls. Breast cancer cases had less education, oral contraceptives use and hormone replacement therapy, and a lower intake of tea and fresh mushrooms. More of cases were exposed to passive smoking, menarche age before 13 years, breast cancer in a first degree relative, and had higher BMI at 5‐years ago and energy intake. There was no difference between cases and controls in locality (rural area residence), menopausal status, age at first full‐term pregnancy, number of children breastfed, alcohol and dried mushrooms intake, smoking, and physical activity in terms of weekly MET‐hours. Table I. Selected Characteristics of Cases with Breast Cancer and Controls Cases subjects (n = 1,009) Controls subjects (n = 1,009) p1 Age at interview (yr) 48.4 ± 10.3 48.4 ± 10.3 NA Education <0.01 No formal education 183 (18.1) 151 (15.0) Primary 290 (28.7) 289 (28.6) Secondary 395 (39.2) 376 (37.3) Tertiary 141 (14.0) 193 (19.1) Resident in rural area 471 (46.7) 475 (47.1) 0.86 Body mass index (kg/m2, 5‐yr ago) 22.9 ± 3.1 22.4 ± 2.9 0.01 Physical activity (weekly MET‐hr) 58.3 ± 71.2 60.1 ± 59.1 0.54 Age at menarche <13 yr 21 (2.1) 9 (0.9) 0.03 Age at first full‐term delivery (yr) 27.3 ± 13.4 27.9 ± 14.1 0.32 Menopausal women 337 (33.4) 338 (33.5) 0.96 No. of children breastfed 1.61 ± 1.1 1.67 ± 1.2 0.24 Oral contraceptive use (ever) 267 (26.5) 325 (32.2) <0.01 Hormone replacement therapy (ever) 21 (2.1) 41 (4.1) 0.01 Breast cancer in first‐degree relatives 35 (3.5) 17 (1.7) 0.01 Alcohol consumption 236 (23.4) 264 (26.2) 0.15 Tobacco smoking 20 (2.0) 14 (1.4) 0.30 Passive smoking 694 (68.8) 482 (47.8) <0.001 Tea consumption 451 (44.7) 661 (65.5) <0.001 Fresh mushrooms intake 460 (45.6) 569 (56.4) <0.001 Dried mushrooms intake 718 (71.2) 721 (71.5) 0.88 Energy intake (kcal/day) 2184.0 ± 569.3 2084.3 ± 506.3 <0.001 Table II reports the associations between selected characteristics of subjects and dietary intake of fresh mushrooms and green tea by 4 intake levels. Younger age, higher education, higher alcohol consumption, and less likelihood of residence in a rural area were associated with higher intake of both fresh mushrooms and green tea. Table II. Association Between Selected Characteristics of Subjects by Levels of Fresh Mushrooms Intake and Green Tea Consumption Levels of fresh mushroom intake Levels of green tea leaves consumed 1st (n = 989) 2nd (n = 378) 3rd (n = 375) 4th (n = 276) p1 1st (n = 906) 2nd (n = 364) 3rd (n = 468) 4th (n = 208) p1 Median (g/day) 0 0.82 4.11 14.38 0 0.26 1.04 3.10 Age (yr) 50.0 47.5 46.3 46.7 <0.001 48.6 46.1 49.1 49.6 <0.001 Education (yr) 6.7 7.9 10.0 10.6 <0.001 7.3 8.6 8.5 9.1 <0.001 BMI (kg/m2 5‐yr ago) 22.9 22.7 22.2 22.0 <0.001 22.7 22.3 22.6 22.8 0.18 Resident in rural area (%) 56.3 51.6 32.5 26.1 <0.001 52.2 44.2 43.6 38.6 <0.001 Alcohol intake (%) 36.2 43.9 45.1 48.9 <0.001 32.1 46.2 45.7 55.4 <0.001 Tobacco smoking (%) 2.0 2.1 1.1 0.7 0.32 1.5 1.1 1.1 3.9 0.02 Passive smoking (%) 58.2 61.4 58.4 54.0 0.31 58.2 61.8 56.8 56.4 0.45 Physical activity (MET‐hr/week) 66.4 61.5 48.1 55.2 <0.001 58.5 58.6 66.4 60.0 0.17 The adjusted odds ratios of breast cancer at different levels of dietary mushroom intakes are shown in Table III. The risk of breast cancer significantly declined with the highest intake of dietary mushrooms. In the final models, compared with nonconsumers of mushrooms, the adjusted ORs were 0.36 (95% CI = 0.25–0.51) and 0.53 (0.38–0.73) for daily intake of ≥10 g of fresh mushrooms and ≥4 g of dried mushrooms in all women, The inverse association was observed in both pre‐ and postmenopausal women. In all instances, tests for trend were statistically significant. Table III. Associations Between Breast Cancer and Dietary Intakes of Mushrooms No. cases/controls OR (95% CI)1 OR (95% CI)2 Fresh mushrooms (g/day) All women 0 231/200 1.0 (referent) 1.0 (referent) <2 191/187 0.86 (0.65–1.15) 0.88 (0.66–1.19) 2–<10 185/190 0.78 (0.58–1.06) 0.81 (0.59–1.10) ≥10 84/192 0.34 (0.24–0.48) 0.36 (0.25–0.51) P trend 3 <0.001 <0.001 Premenopausal women 0 151/114 1.0 (referent) 1.0 (referent) <2 139/129 0.82 (0.58–1.16) 0.86 (0.59–1.23) 2–<10 126/136 0.69 (0.47–0.99) 0.71 (0.48–1.01) ≥10 63/140 0.33 (0.22–0.50) 0.34 (0.22–0.52) P trend 3 <0.001 <0.001 Postmenopausal women 0 80/86 1.0 (referent) 1.0 (referent) <2 52/58 1.00 (0.60–1.67) 0.97 (0.57–1.65) 2–<10 59/54 0.98 (0.57–1.69) 0.99 (0.56–1.75) ≥10 21/52 0.34 (0.17–0.67) 0.35 (0.17–0.70) P trend 3 0.01 <0.01 Dried mushrooms (g/day) All women 0 231/200 1.0 (referent) 1.0 (referent) <1 358/283 1.10 (0.85–1.42) 1.15 (0.88–1.49) 1–<4 220/221 0.87 (0.65–1.15) 0.90 (0.67–1.21) ≥4 140/217 0.50 (0.37–0.68) 0.53 (0.38–0.73) P trend 3 <0.001 <0.001 Premenopausal women 0 151/114 1.0 (referent) 1.0 (referent) <1 242/194 0.99 (0.72–1.35) 1.03 (0.74–1.45) 1–<4 139/151 0.75 (0.53–1.08) 0.76 (0.53–1.11) ≥4 95/142 0.47 (0.32–0.69) 0.51 (0.34–0.75) P trend 3 <0.001 <0.001 Postmenopausal women 0 80/86 1.0 (referent) 1.0 (referent) <1 116/89 1.30 (0.85–1.99) 1.35 (0.87–2.10) 1–<4 81/70 1.10 (0.68–1.77) 1.17 (0.71–1.93) ≥4 45/75 0.53 (0.31–0.92) 0.53 (0.30–0.93) P trend 3 <0.01 <0.01 The joint effects of dietary intakes of mushrooms and green tea on breast cancer risk are reported in Table IV. An additional decreased risk of joint effect of mushrooms with green tea was associated to breast cancer. Compared with the women who consumed neither dietary mushrooms nor green tea, the adjusted ORs were 0.11 (0.06–0.20) and 0.18 (0.11–0.29) for those who daily consumed ≥7 g of fresh mushrooms and ≥2 g of dried mushrooms, when combined with consuming beverages made from ≥1.05 g dried green tea leaves per day. The linear trends of interaction were statistically significant (p < 0.001). Table IV. Combined Effect of Dietary Mushrooms and Green Tea on Breast Cancer Risk Dietary mushrooms (g/day) Green tea leaves (g/day) No. cases/controls OR (95% CI)1 p2 Fresh mushrooms None None 141/67 1.0 (referent) <0.001 Low <1.05 180/106 0.75 (0.50–1.13) High ≥1.05 65/84 0.32 (0.20–0.51) Low <7 None 49/55 0.43 (0.25–0.71) Low <1.05 88/90 0.43 (0.28–0.68) High ≥1.05 27/79 0.13 (0.07–0.24) High ≥7 None 29/60 0.22 (0.13–0.39) Low <1.05 53/98 0.22 (0.14–0.36) High ≥1.05 21/76 0.11 (0.06–0.20) Dried mushrooms None None 141/67 1.0 (referent) <0.001 Low <1.05 210/117 0.88 (0.59–1.30) High ≥1.05 174/128 0.62 (0.42–0.93) Low <2 None 49/55 0.43 (0.26–0.72) Low <1.05 98/81 0.57 (0.37–0.89) High ≥1.05 90/133 0.31 (0.20–0.48) High ≥2 None 29/60 0.22 (0.13–0.39) Low <1.05 58/100 0.28 (0.18–0.45) High ≥1.05 47/116 0.18 (0.11–0.29)

Discussion This case–control study conducted in southeast China was specifically designed to investigate whether dietary intake of mushrooms reduces the risk of breast cancer and to evaluate the joint effects of dietary mushrooms and green tea on the cancer. We have observed that higher intake of dietary fresh and dried mushrooms were associated with a decreased risk of breast cancer in both pre‐ and postmenopausal women. We also find that an additional decreased risk of dietary mushrooms combined with green tea was associated to breast cancer, which may partially explain a lower incidence of breast cancer in Chinese women in terms of consumption of dietary mushrooms and green tea different in China from developed countries. To our knowledge, this is the first human study to evaluate the joint effect of mushrooms and green tea on the breast cancer. Mushrooms are widely consumed in different parts of the world.28 A number of bioactive phytochemicals have been identified in many mushrooms species.29 Compared with the consistent evidence on anticarcinogenic properties of mushrooms from experimental studies, limited epidemiologic evidence is available and results have been inconclusive.11-15 Our study provides epidemiologic evidence that dietary mushrooms and its association with green tea decrease breast cancer risk, a finding that is consistent with the laboratory evidence.9, 10 Our results are also consistent with those of 2 case–control studies in Korean women.14, 15 One Korean study with 189 pairs of cases and controls reported that higher frequency consumption (≥1/week) of mushrooms decreased risk of breast cancer (OR = 0.4, 95% CI = 0.3–0.7) compared with lower frequency intake (<1/week), although the authors did not define “lower” and “higher” intake in quantitative terms.14 Another study with 362 pairs of cases and controls matched by case's age and menopausal status found that a higher frequency and quantity of dietary mushrooms may decrease breast cancer risk only in postmenopausal Korean women.15 In this study of 1,009 case–control pairs, we sought detailed information on frequency and quantity of dietary intake of fresh and dried mushrooms and found higher intakes of fresh and dried mushrooms to be significantly associated with a decreased risk of breast cancer in both pre‐ and postmenopausal women. The potential for confounding of the effects dietary mushroom consumption by other factors warrants elaboration. We found that higher dietary intake of mushrooms was associated with demographic and lifestyle characteristics in the subjects; e.g., younger age, further education, lower BMI, less physical activity, higher alcohol consumption, and lower exposures to a rural residential area. Thus any residual confounding from these lifestyle factors, beyond what we could adjust for in the analysis, has affected the apparent protective effect of dietary intake of mushrooms. It is also possible the observed protective effects were biased by unknown confounders. Because the finding that dietary intake of mushrooms and their effect combined with green tea have additional protective effects on breast cancer has important implications, it is necessary to consider our study's limitations and strengths. One of the study's strengths was that detailed and extensive information was obtained on mushroom intake, tea consumption, as well as other diet lifestyle and hormonal factors. A validated instrument specifically for Chinese women was used to collect the information by face‐to‐face interviews conducted by trained interviewers. Nevertheless, the case–control design may have introduced certain sources of bias. The breast cancer cases were identified from medical records and interviewed in breast surgery wards, whereas the controls were randomly selected from outpatient breast clinics in the same hospitals by matching on the age of cases. The majority of patients are self‐referred in China and except for acute trauma associations between patients and particular hospitals tend to be maintained.30 Under the recruitment procedure, the cases and controls arose from the same populations of women using each hospital, and thus, if a control was later to develop breast cancer it was highly likely that she would have been treated in the same hospital. Control selection bias was also minimized by recruitment procedures for controls that were unrelated to convenience or clinician contact. In assessing the likelihood of any information bias, which we consider to have been low, it was relevant that no mention occurred in the popular media of any association between dietary intake of mushrooms and breast cancer prior to and at the time of the research, although extracts of the mushroom Ganoderma lucidum are used in the treatment of cancer patients by practitioners of traditional Chinese medicine.10 Exposure of cases to risk factors may change due to disease status. However, over 90% of the cases were newly diagnosed and interviewed within 3 months. It appears unlikely that disease status materially affected the interview responses and the notion that cases would have either recalled more dietary intake of mushrooms or dramatically increased their dietary consumption of mushrooms is implausible. In conclusion, the study suggests that higher dietary intakes of fresh and dried mushrooms are associated with a reduced breast cancer risk with a dose–response relationship in both pre‐ and postmenopausal Chinese women. The combination of dietary intake of mushrooms and green tea drinking decreased breast cancer risk with an additional reduced effect on the malignance. These findings have biological plausibility, being supported by strong evidence from in vitro and in vivo experiments of anticarcinogenic effects of mushrooms. Breast cancer is the most common type of cancer affecting women worldwide. Our findings, if confirmed consistently in other research, have potential implications for protection against breast cancer development using an inexpensive dietary intervention.

Acknowledgements The authors acknowledge with gratitude the participation of the Chinese women as subjects. The authors are grateful for the collaboration received from the participating hospitals and their staff. Dr. Min Zhang is supported through a postdoctoral fellowship from the National Health and Medical Research Council (Australia).