Supplementary Material

MATERIALS AND METHODS

Study PopulationThe Health Professionals Follow-up Study (HPFS) is a prospective cohort study of

51,529 US male health professionals aged 40-75 at baseline in 1986 (Kenfield et al. 2014). Participants reported medical diagnoses, medications, and lifestyle factors at baseline and every two years thereafter. Participants also completed a validated semi-quantitative food frequency questionnaire (FFQ) at baseline and every four years thereafter. The average questionnaire response rate is over 90%. Men with implausible energy intakes (<800 or >4,200 kcal/day), or who did not report nut intake or had more than 70 food items missing from the baseline FFQ were excluded from the current analysis. Men diagnosed with cancer (except for non-melanoma skin cancer) prior to baseline were also excluded. The final incidence analysis included 47,299 men, followed until January 2012. For the case-only survival analysis, PCa patients with clinical stage of T3b or higher (n=303) and those without information on clinical stage (n=1,002) were excluded. Men without any post-diagnosis nut data were also excluded, leaving 4,346 men for follow-up of survival outcomes through January 2012.

Dietary AssessmentDietary intake was measured using FFQs (Kenfield et al. 2014). In each FFQ, participants

were asked how often they had consumed a serving (serving size, 28 g [1 oz]) of peanuts and other nuts during the preceding year: never or almost never, one to three times a month, once a week, two to four times a week, five to six times a week, once a day, two to three times a day, four to five times a day, six or more times a day. Total nut consumption was defined as the sum of peanut and other nut consumption. A validation study of the FFQ indicated that nuts were reported reasonably accurately: the correlation coefficients between nut intakes assessed on the baseline dietary questionnaire and four one-week diet records were 0.75 (Salvini et al. 1989).

To calculate the Mediterranean diet score for each participant, food items were sorted into nine categories of the traditional Mediterranean Diet (Trichopoulou et al. 2003). These nine categories included vegetables, fruits and nuts, legumes, grains, fish and seafood, fat, alcohol, red and processed meat products, and dairy products. For the score, each participant received 1 point each for being below the median in dairy and meat intake, 1 point for alcohol intake between 10 and 50g/day, 1 point each for being above the median intake of vegetables, legumes, fruits and nuts, grains, fish and the ratio of polyunsaturated to saturated lipids. The total score ranged from 0 to 9.

Assessment of Non-dietary FactorsAt baseline and in all subsequent questionnaires, men were asked about their smoking

status, physical activity, and history of diabetes, high blood pressure and elevated cholesterol. Information was also collected on race and family history of chronic diseases such as PCa, diabetes mellitus, myocardial infarction, and cancer. Body mass index (BMI) was calculated from height reported at baseline and weight, which was reported at baseline and updated every 2 years. Prostate-specific antigen (PSA) screening history was first assessed in 1994 and updated every 2 years.

Ascertainment of Prostate Cancer

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After a PCa diagnosis was reported, medical records and pathology reports were obtained to confirm the diagnosis. Data on diagnosis date, clinical T-stage, grade (Gleason score), PSA at diagnosis, metastasis, and treatments were extracted. Since 2000, biennial questionnaires were sent to participants with confirmed PCa and their physicians to update information on treatments, disease progression, and metastases. Family reports and National Death Index searches were used to identify deaths. Over 98% of deaths were ascertained (Rich-Edwards et al. 1994). Medical records were reviewed by study physicians to confirm causes of death; death certificates were used if medical records were not available. We examined risk of the following categories of PCa: total (excluding T1a cancers, which are discovered incidentally during treatment for benign prostatic hypertrophy), advanced, lethal, fatal, low-grade (Gleason score 2-6) and high-grade (Gleason score > 7). Advanced PCa was defined as stage T3b, T4, N1, or M1 at diagnosis, or lymph node metastases, distant metastases, or PCa death during follow-up. Lethal cancer, the primary focus of our study, was defined as cases that metastasized to distant organs at diagnosis or over follow-up, or that caused PCa death. Fatal cancers were defined as those that caused PCa death. We also investigated post-diagnostic nut intake in relation to risk of lethal PCa, fatal PCa, and all-cause mortality among men diagnosed with localized or regional PCa (clinical stage T1-T3a).

Statistical AnalysisAll analyses were two sided and carried out using SAS 9.2 (SAS Institute, Inc., Cary,

NC). Results with a p-value <0.05 were considered statistically significant.

Prostate Cancer Incidence Analysis Cox proportional hazards models were used to estimate hazard ratios (HR) and 95%

confidence intervals (CIs) for the association of pre-diagnostic total nuts, peanuts and other nuts with risk of PCa incidence. Men were followed from return of baseline questionnaire until diagnosis, death, or end of follow-up (January 31, 2012).

Nut consumption was presented as a cumulative average from 1986 to date of diagnosis, death, or end of follow-up (January 31, 2012), to better represent long-term dietary habits and reduce within-person variability. For example, the average nut intake in 1986, 1990, and 1994 was applied to the person-time contributed between 1994 and 1998. Men were divided into 5 groups according to their frequency of nut consumption: less than once a month (the reference group), 1 to 3 times a month, once a week, 2 to 4 times a week, and 5 or more times a week.

In the age-adjusted model, we controlled for age (months), time period (2-yr interval), and total energy intake (kcal per day, quintiles). In the primary multivariable model, we additionally adjusted for BMI (< 25, 25-29.9, ≥30 kg/m2), vigorous physical activity (<1, 1 to <3, ≥ 3 h/wk), smoking status (current, former, never), and PSA screening history (yes, no, unknown). In the secondary multivariable model, in addition to the variables in the primary multivariable model, we further adjusted for family history of PCa (yes, no), ethnicity (Caucasian, African, Asian), height (inches, quintiles), history of diabetes (yes, no), current multivitamin use (yes, no), current vitamin E and selenium supplement use (yes, no), tomato sauce (servings/week, quartiles), coffee intake (servings/day, tertiles), and Mediterranean diet (score range 0-9). Tomato sauce was associated with reduced PCa risk in the HPFS (Giovannucci et al. 2002) and coffee intake has been linked with reduced risk of PCa in many cohort studies (Liu et al. 2015). Mediterranean diet has been shown to be associated with lower overall mortality after PCa diagnosis (Kenfield et al. 2014). P values for trend were calculated by the Wald test of a score variable that contained median values of intake categories.

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In secondary analyses, we assessed interactions by ethnicity (white vs. non-white), age (<70 vs ≥ 70 yr), and BMI (<25 vs ≥ 25 kg/m2) using the Wald test of the cross-product terms of total nut consumption with those stratification variables. Because occult disease onset in the years preceding diagnosis may influence diet, we conducted an additional analysis wherein a 2-year lag period was added between nut-intake assessment and each follow-up period (e.g., the nut intake from the 1986 questionnaire was used for the follow-up period of 1988-1990). In additional to cumulative average of nut intake, we also examined baseline nut intake in relation to PCa risk.

Case-only survival analysis Cox proportional hazards models were used to estimate hazard ratios (HR) and 95%

confidence intervals (CIs) for the association of post-diagnostic total nuts, peanuts and other nuts with risk of lethal and fatal PCa and overall mortality among non-metastatic PCa patients. For lethal PCa, men were followed from PCa diagnosis to metastases. For fatal PCa, men were followed from PCa diagnosis to death from PCa. For all-cause mortality, men were followed from PCa diagnosis until death by any cause or end of follow-up (January 31, 2012), whichever came first.

Nut consumption was presented as a cumulative average from the date of diagnosis until the end of follow-up. The FFQ immediately preceding diagnosis was used to classify the participants’ diet from the diagnosis date until the next available FFQ, because it would better capture diet at the time of diagnosis without the diagnosis having affected diet.

In the age-adjusted model, we controlled for age (months), time period (2-yr interval), and total energy intake (kcal per day, quintiles). In the primary multivariable model, we included additional terms for BMI (< 25, 25-29.9, and ≥ 30 kg/m2), vigorous physical activity (<1, 1 to <3, and ≥ 3 h/wk), smoking status (current, former, never), Gleason score (<7, 7, >7), clinical T stage (T1, T2, T3), and primary treatment (radical prostatectomy, radiation, hormonal therapy, active surveillance, other). For lethal/fatal PCa, the secondary multivariable model additionally included PSA screening history (yes, no, unknown), family history of PCa (yes, no), ethnicity (Caucasian, African, Asian), height (inches, quintiles), history of diabetes (yes, no), current multivitamin use (yes, no), current vitamin E and selenium use (yes, no), tomato sauce (servings/week, quartiles), coffee intake (servings/day, tertiles), and Mediterranean diet (score range 0-9). For all-cause mortality, the secondary multivariable model included further adjustment for family history of diabetes mellitus, of myocardial infarction, of cancer, and history of high blood pressure and elevated cholesterol (all defined as yes or no).

In a sensitivity analysis, we included pre-diagnostic nut consumption from the 1986 FFQ in the multivariable models to mitigate the influence of pre-diagnostic diet. To examine whether the associations of interest were modified by other factors, we stratified analyses by ethnicity (white vs. non-white), age (<70 vs ≥ 70 yr), and BMI (<25 vs ≥ 25 kg/m2). We also conducted a secondary analysis wherein a 2-year lag period was added between nut-intake assessment and each follow-up period. Finally, we plotted the Kaplan-Meier survival curves and performed the log-rank test across categories of nut intake for all-cause mortality among the PCa cases.

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Supplementary Table 1. Age-standardizeda characteristics of the Health Professionals Follow-Up Study population at baseline in 1986, according to total nut consumption

Frequency of total nut consumption (28g serving)<Once/month <Once/week Once/week 2-4 times/week ≥5 times/week

Nut intake, servings/d 0-0.033 0.034-0.10 0.11-0.20 0.21-0.60 >0.60Participants (n) 11993 8382 10931 11116 4877Age, y, mean (SD) 54.3(10.0) 53.7(9.8) 53.2(9.5) 53.6(9.6) 55.3(9.4)Caucasian, % 90.3 90.4 91.0 91.5 91.7History of PSA screening, % b 44.4 46.5 46.2 47.5 48.1Current smokers, % 10.1 9.8 9.8 9.8 9.6BMI, kg/m2, mean (SD) 25.5(3.2) 25.5(3.2) 25.6(3.1) 25.4(3.1) 25.3(3.1)Height, inches, mean (SD) 70.0(2.6) 70.1(2.7) 70.2(2.7) 70.3(2.6) 70.3(2.7)Diabetes, % 3.2 3.0 2.7 3.3 3.6Family history of prostate cancer, % 5.3 5.2 5.4 6.1 6.0Vigorous exercise, h/wk, mean (SD) 1.6(3.0) 1.6(3.0) 1.7(3.3) 1.7(3.2) 1.9(3.4)Current multivitamin use, % 39.6 39.8 41.7 43.4 46.4Current Vitamin E use, % 16.5 17.3 19.1 21.1 23.9Mediterranean diet score (SD) 4.1(2.0) 4.1(2.0) 4.2(2.0) 4.4(2.0) 4.9(1.9)Dietary intake, mean (SD)

Alcohol, g/d 9.8(14.8) 10.4(14.7) 11.4(15.0) 12.5(16.0) 13.9(16.9)Vegetables, servings/d 3.3(2.2) 3.2(2.0) 3.3(1.9) 3.2(1.8) 3.1(1.8)Fruit, servings/d 2.5(1.8) 2.4(1.6) 2.5(1.5) 2.4(1.5) 2.4(1.5)Cereal, servings/d 2.5(1.4) 2.4(1.3) 2.3(1.3) 2.2(1.2) 2.1(1.1)Whole grains, servings/d 1.4(1.3) 1.3(1.2) 1.3(1.1) 1.3(1.1) 1.2(1.1)Fish, servings/d 0.4(0.4) 0.4(0.4) 0.4(0.4) 0.4(0.3) 0.4(0.3)Red/processed meat, servings/d 1.0(0.7) 1.0(0.6) 1.0(0.6) 0.9(0.6) 0.8(0.6)Dairy, servings/d 2.1(1.4) 2.0(1.3) 2.0(1.2) 1.9(1.2) 1.8(1.1)Olive oil, servings/weekc 1.1(2.3) 1.1(2.1) 1.1(2.3) 1.2(2.2) 1.4(2.5)Tomatoes, servings/week 4.0(3.7) 4.0(3.5) 4.1(3.4) 4.3(3.4) 4.6(3.7)Tomato sauce, servings/week 0.9(1.3) 0.9(1.2) 1.0(1.1) 1.0(1.1) 1.0(1.1)

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Coffee, servings/d 1.9(1.8) 1.9(1.8) 1.9(1.7) 1.9(1.8) 2.0(1.9)Ratio of polyunsaturated to saturated fat, g/d 0.5(0.2) 0.5(0.2) 0.6(0.2) 0.6(0.2) 0.7(0.2)Ratio of monounsaturated to saturated fat, g/d 1.1(0.2) 1.1(0.2) 1.1(0.2) 1.2(0.2) 1.3(0.2)

BMI=body mass index; PSA=prostate-specific antigenaAge standardized to the age distribution of the study population in 1986.bCalculated based on the study population in 1994 when history of PSA test was first assessed.cIncludes olive oil added to food, bread, or used in salad dressing and not that used in cooking.

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Supplementary Table 2. Age-standardizeda characteristics of 4,346 men diagnosed with non-metastatic prostate cancer in the Health Professionals Follow-Up Study, according to nut consumption from the most recent food frequency questionnaire (FFQ) preceding diagnosis

Frequency of total nut consumption (28g serving)<Once/month <Once/week Once/week 2-4 times/week ≥5 times/week

Nut intake, servings/d 0-0.033 0.034-0.10 0.11-0.20 0.21-0.60 >0.60Participants (n) 1060 765 793 1130 598Age at diagnosis, year, mean (SD) 69.4(7.2) 69.6(7.3) 69.0(7.1) 69.8(7.0) 69.9(6.7)Clinical stage, %

T1 57.0 59.2 60.1 63.0 64.0T2 39.0 38.0 36.1 34.1 33.7T3 (excluding T3b) 4.0 2.8 3.8 2.8 2.3

Gleason score, %< 7 59.0 56.6 58.4 56.0 55.67 20.9 23.2 22.6 26.9 27.8> 7 7.8 10.8 6.8 8.3 8.7

Primary treatment, %Radical prostatectomy 43.8 48.4 45.0 46.2 45.8EBRT or brachytherapy 38.9 32.3 35.1 35.5 36.4Hormones 4.5 5.2 5.1 4.5 4.4Watchful waiting 6.1 7.7 8.5 8.0 8.1Other 1.3 1.7 1.5 2.1 1.2

History of PSA screening, % 52.8 59.6 56.5 67.5 73.0Caucasian, % 91.7 92.3 93.7 93.6 93.2Current smokers, % 4.2 4.8 3.4 3.4 3.9BMI, kg/m2, mean (SD) 25.5(4.5) 25.4(4.6) 25.3(5.2) 25.4(5.4) 24.9(4.8)Height, inches, mean(SD) 69.9(2.8) 70.0(3.4) 70.1(2.7) 70.2(2.7) 70.3(2.6)Diabetes, % 5.4 5.1 8.1 8.0 8.6Family history of prostate cancer, % 11.1 11.8 10.0 10.4 11.3Family history of myocardial infraction, % 32.8 32.8 31.6 32.7 35.2History of high blood pressure, % 23.6 20.7 23.9 20.7 19.9

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Vigorous exercise, hr/wk, mean (SD) 1.4(2.5) 1.4(2.6) 1.3(2.8) 1.2(2.5) 1.0(2.2)Current multivitamin use, % 55.1 53.2 52.6 53.8 56.6Current Vitamin E supplement use, % 37.8 38.3 38.0 36.8 41.3Mediterranean diet score (SD) 4.5(1.9) 4.5(1.9) 4.5(1.9) 4.6(2.0) 5.3(1.9)Dietary intake, mean (SD)

Alcohol, g/d 10.3(14.2) 11.8(14.7) 12.2(15.5) 13.9(16.8) 14.0(15.7)Vegetables, servings/d 3.6(2.2) 3.5(1.9) 3.5(1.9) 3.6(1.9) 3.6(1.9)Fruit, servings/d 3.1(1.7) 3.0(1.6) 3.1(1.6) 3.2(1.5) 4.1(1.6)Cereal, servings/d 3.8(6.1) 3.3(3.3) 3.3(4.1) 3.0(2.9) 3.1(3.2)Whole grains, servings/d 2.3(2.6) 2.1(2.0) 2.2(2.2) 2.0(2.0) 2.0(2.0)Fish, servings/d 0.4(0.3) 0.4(0.3) 0.4(0.3) 0.4(0.3) 0.3(0.3)Red/processed meat, servings/d 0.8(0.6) 0.9(0.6) 0.9(0.6) 0.9(0.6) 0.7(0.5)Dairy, servings/d 2.3(1.5) 2.1(1.4) 2.2(1.4) 2.1(1.3) 1.9(1.1)Olive oil, servings/weekb 1.7(3.3) 1.7(3.7) 1.8(3.5) 2.6(4.4) 3.3(5.6)Tomatoes, servings/week 3.6(3.8) 3.7(3.2) 3.8(3.7) 4.3(4.1) 5.0(4.2)Tomato sauce, servings/week 0.9(1.7) 0.9(1.1) 1.0(1.2) 1.0(1.2) 1.2(1.2)Coffee, servings/d 1.7(1.6) 1.7(1.6) 1.7(1.5) 1.6(1.5) 1.7(1.6)Ratio of polyunsaturated to saturated fat, g/d 0.6(0.2) 0.6(0.2) 0.6(0.2) 0.7(0.2) 0.9(0.4)Ratio of monounsaturated to saturated fat, g/d 1.2(0.3) 1.2(0.3) 1.2(0.3) 1.3(0.3) 1.5(0.4)

BMI=body mass index; PSA=prostate-specific antigena Age-standardized to the age distribution of the study population at prostate cancer diagnosis.b Includes olive oil added to food, bread, or used in salad dressing and not that used in cooking.

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Supplementary Figure 1. Kaplan-Meier survival curves for all-cause mortality (P<0.0004, log-rank test) among prostate cancer cases, by nut consumption.

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