Citrus consumption was associated with an increased risk of malignant melanoma in two cohorts of women and men. Nevertheless, further investigation is needed to confirm our findings and explore related health implications.

Over 24 to 26 years of follow-up, we documented 1,840 incident melanomas. After adjustment for other risk factors, the pooled multivariable hazard ratios for melanoma were 1.00 for overall citrus consumption < twice per week (reference), 1.10 (95% CI, 0.94 to 1.30) for two to four times per week, 1.26 (95% CI, 1.08 to 1.47) for five to six times per week, 1.27 (95% CI, 1.09 to 1.49) for once to 1.5 times per day, and 1.36 (95% CI, 1.14 to 1.63) for ≥ 1.6 times per day ( P trend < .001). Among individual citrus products, grapefruit showed the most apparent association with risk of melanoma, which was independent of other lifestyle and dietary factors. The pooled multivariable hazard ratio for melanoma comparing the extreme consumption categories of grapefruit (≥ three times per week v never) was 1.41 (95% CI, 1.10 to 1.82; P trend < .001).

A total of 63,810 women in the Nurses' Health Study (1984 to 2010) and 41,622 men in the Health Professionals Follow-Up Study (1986 to 2010) were included. Dietary information was repeatedly assessed every 2 to 4 years during follow-up. Incident melanoma cases were identified through self-report and confirmed by pathologic records.

Citrus products are widely consumed foods that are rich in psoralens and furocoumarins, a group of naturally occurring chemicals with potential photocarcinogenic properties. We prospectively evaluated the risk of cutaneous malignant melanoma associated with citrus consumption.

INTRODUCTION Section: Choose Top of page Abstract INTRODUCTION << METHODS RESULTS DISCUSSION REFERENCES Malignant melanoma is a potentially lethal form of cutaneous malignancy, the incidence of which has been increasing for at least 30 years in the United States.1 Citrus products are rich sources of psoralens, a group of naturally occurring furocoumarins (furanocoumarins).2–5 Photochemotherapy using oral psoralen (methoxsalen) and ultraviolet (UV) A radiation (PUVA) is a highly effective therapy for severe psoriasis.6 PUVA takes advantage of the high UV absorbance of psoralen. Psoralen is first taken orally to sensitize the skin, and the skin is then exposed to UVA light to treat the cutaneous problem therapeutically. However, photocarcinogenic properties of psoralens and furocoumarins have been demonstrated in many experimental studies.7–12 PUVA induced melanocytic tumors in a mouse model12 and can contribute to the pathogenesis of melanoma by exerting a stimulatory effect on the outgrowth of melanoma cells.13 Epidemiologic evidence also suggests that long-term PUVA therapy increases the risk of melanoma.14,15 Psoralens had been used as tanning activators until 1996, and individuals who used psoralen tanning activators had a higher risk of melanoma.16 Psoralen-containing sunscreen users also had a substantially increased risk of melanoma as compared with regular sunscreen users.17 As a result, strict regulations have been imposed on psoalen-containing suntan lotions and cosmetic products in recent years.16,18 However, whether dietary consumption of psoralen-rich foods may increase melanoma risk is unknown, and whether the public should be advised about dietary psoralens remains a question.5 In our earlier investigation of antioxidant nutrient intake and melanoma risk in the Nurses' Health Study (NHS),19 we observed an unexpected higher risk in women who consumed more orange juice and vitamin C from foods, but not from supplements. Because vitamin C has preferential toxicity for melanoma cells,20 induces apoptosis in melanoma cells via multiple pathways,21,22 and suppresses proliferation of melanoma cells,23 we hypothesized that the increased melanoma risk associated with dietary vitamin C, but not with supplemental vitamin C, was likely the effect of other components (eg, photoactive compounds) in vitamin C–rich foods. To address the hypothesis that the consumption of psoralen-rich foods (ie, citrus products) may be associated with an increased risk of cutaneous malignant melanoma, we expanded our previous analysis to comprehensively examine citrus consumption in relation to risk of melanoma with extended follow-up of 24 to 26 years in two large US cohorts of women and men.

METHODS Section: Choose Top of page Abstract INTRODUCTION METHODS << RESULTS DISCUSSION REFERENCES Study Population Our study population consisted of participants from two ongoing cohorts: the NHS and Health Professionals Follow-Up Study (HPFS). The NHS was established in 1976, when 121,700 married, registered female nurses between the ages of 30 and 55 years and residing in the United States at the time of enrollment responded to a baseline questionnaire that included questions about their medical history and lifestyle risk factors. The HPFS was established in 1986, when 51,529 male health professionals between the ages of 40 and 75 years completed a baseline questionnaire. Information on medical history and lifestyle factors was collected biennially via mailed questionnaires. Dietary intake was assessed using a validated food frequency questionnaire (FFQ) at least every 4 years. The study protocol was approved by the institutional review boards of the Brigham and Women's Hospital and Harvard School of Public Health. Completion of the self-administered questionnaire was considered to imply informed consent. We observed participants for incident melanoma starting from 1984 in the NHS and 1986 in the HPFS, when the participants completed a baseline FFQ with detailed information on citrus consumption. A total of 81,685 women and 49,617 men completed the dietary questionnaire at baseline. Study participants with a baseline history of any cancer (including nonmelanoma skin cancers) were excluded. Because of low risk and small numbers, nonwhite participants were also excluded. After exclusions, 63,810 women and 41,622 men (N = 105,432) with complete dietary data at baseline remained in our analysis. Assessment of Dietary Consumption In all FFQs, participants were asked how often on average (never to ≥ six servings per day) during the previous year they had consumed grapefruit (half), oranges (one), and grapefruit and orange juices (one small glass [6 oz]). Overall citrus consumption was calculated as the sum of these individual products. Grapefruit and grapefruit juice were asked as a single item in the 2002 and 2006 FFQs. Dietary intake collected using the FFQ has been shown to be a valid estimator of relative food intake when compared with multiple diet records.24,25 The correlation coefficients ranged from 0.75 to 0.84 for the correlations between intake of individual citrus products assessed in the baseline FFQ and intake assessed in two 1-week dietary records.24 Vitamin C content in citrus products was derived from US Department of Agriculture sources.26 Information on other dietary factors, including intake of total energy, alcohol, coffee, vegetables, and other fruits and juices, was also collected during follow-up. Assessment of Covariates In the biennial follow-up questionnaires, we inquired about and updated information on anthropometric and lifestyle factors for chronic diseases, including body height and weight, cigarette smoking, physical activity, vitamin C from supplements, and menopausal status and postmenopausal hormone use among women. Data on the following host and sun exposure characteristics were also collected through questionnaires27: family history of melanoma, natural hair color, number of moles on arms, skin reaction to sun exposure for ≥ 2 hours as a child or adolescent, number of lifetime blistering sunburns, cumulative UV flux exposure since baseline, and average time spent in direct sunlight since high school. Ascertainment of Patient Cases of Melanoma Participants reported new diagnoses of melanoma biennially. We sought permission from these participants to acquire their medical and pathologic reports, which were reviewed by study physicians to validate the diagnoses. Information on tumor stage and location was also retrieved if available. In situ melanomas were defined as early-stage tumors restricted to the epidermis, and invasive melanomas were defined as those that had grown into the dermis or surrounding tissues. Melanomas were further classified into the following two subgroups according to tumor location: melanomas on body sites with higher continuous sun exposure, including head, neck, and extremities, and melanomas on body sites with lower continuous sun exposure, including truncal sites, shoulder, back, hip, abdomen, and chest. We documented 1,840 incident patient cases of cutaneous melanoma over 2 million person-years of follow-up (NHS: 1,023 patient cases and 1,290,954 person-years; HPFS: 817 patient cases and 711,479 person-years). These included 949 invasive melanomas and 891 melanomas in situ, among which 1,154 (62.1%) occurred on the head, neck, or extremities and 614 (33.1%) occurred on truncal sites. Mucosal melanomas and acral melanomas were excluded from the analysis because of potential heterogeneous etiologies. Median tumor thickness (measured as Breslow thickness) was 0.63 mm for invasive melanomas (n = 814). Invasive melanomas occurring on the head, neck, or extremities (median thickness, 0.67 mm) were slightly thicker than those occurring on truncal sites (median thickness, 0.60 mm). Statistical Analysis Dietary consumption was cumulatively updated in analyses to create the best estimates of long-term intake and minimize within-person variation. That is, at the beginning of every 2-year follow-up cycle, each dietary intake was calculated as the mean of all reported intake up to that time. Because grapefruit and grapefruit juice were asked as a single item in the 2002 and 2006 FFQs, analyses for separate grapefruit and grapefruit juice used cumulative updated intake up to 1998 for the subsequent follow-up. As a sensitivity analysis, we created a new intake variable for combined intake of grapefruit and grapefruit juice. Each participant contributed person-time from the return month of the baseline questionnaire to the date of the first diagnosis of any cancer, date of death, or end of follow-up (June 2010 for women; January 2010 for men), whichever came first. We used Cox proportional hazards models to estimate the hazard ratios (HRs), with 95% CIs, of melanoma associated with dietary consumption. Multivariable analyses were adjusted for other known melanoma risk factors and potential confounders. A detailed justification for the covariates included in the analysis is shown in the Data Supplement. Trend tests across categories of dietary consumption were performed by assigning median values for these categories and treating the new variable as a continuous term in the models. We performed several sensitivity analyses to examine the robustness of the results. To assess the influence of common medications (statins and antiarrhythmic agents) that have been reported to interact with grapefruit,28 total vitamin C, adherence to a healthy diet (as assessed by Alternate Healthy Eating Index 2010),29 and sunscreen use, we performed separate analyses with adjustment for each of these variables. A propensity score analysis was performed to reduce the confounding effects from other covariates in the evaluation of association between exposure and outcome.30 To address the concern about reverse causality between dietary assessment and cancer diagnosis as well as to identify any potential timing effect, we performed lag analyses with different intervals from 2 to 14 years between dietary assessment and cohort follow-up. To test whether the positive association with melanoma was specific to citrus products, we examined the melanoma risk associated with consumption of other fruits and juices and vegetables (39 items). We also performed subgroup analyses stratified by potential confounders. For the sex-specific, lagged and stratified analyses, we combined the two highest categories of intake to maintain statistical power. HRs among women and men were pooled using a random-effects model, given the similar associations. P values for heterogeneity were calculated using the Q statistic. We used SAS software (version 9.2; SAS Institute, Cary, NC) for all statistical analyses. All statistical tests were two tailed, and the significance level was set at P < .05.

RESULTS Section: Choose Top of page Abstract INTRODUCTION METHODS RESULTS << DISCUSSION REFERENCES Participants with higher citrus intake were less likely to smoke cigarettes and drink coffee, more likely to exercise, and had higher intake of individual citrus products and vitamin C (Table 1; Data Supplement). In contrast, there was no appreciable difference in sun exposure–related variables and other host risk factors over the intake categories. Citrus consumption remained relatively constant over follow-up (Data Supplement). Correlations between intake of individual citrus products were generally low (Data Supplement). Table 1. Characteristics of Person-Years According to Frequency of Overall Citrus Consumption* Characteristic Frequency (mean ± SD) < Twice per Week Two to Four Times per Week Five to Six Times per Week Once to 1.5 Times per Day ≥ 1.6 Times per Day Age, years 58.2 ± 9.8† 60.0 ± 9.9 61.2 ± 10.0 61.0 ± 10.2 61.2 ± 10.1 Family history of melanoma, % 5.6 5.9 6.2 6.2 5.7 Red or blonde hair, % 14.0 14.2 14.2 14.4 14.8 Arm with moles, % 33.9 35.3 35.7 35.8 35.4 Painful burn or blistering sun reaction as child or adolescent, % 17.5 16.7 16.7 16.2 16.7 No. of lifetime blistering sunburns 10.0 ± 9.2 10.0 ± 9.0 10.0 ± 9.1 9.8 ± 9.0 10.0 ± 9.5 Annual UV flux at residence (× 10−4 Robertson-Berger units)† 128 ± 27 126 ± 27 125 ± 26 123 ± 25 123 ± 25 Average time spent in direct sunlight since high school, hours per week 6.1 ± 4.2 6.2 ± 4.2 6.2 ± 4.2 6.2 ± 4.2 6.5 ± 4.4 Body mass index, kg/m2 26.2 ± 5.0 26.5 ± 5.0 26.4 ± 4.9 25.9 ± 4.7 25.7 ± 4.6 Physical activity, metabolic equivalents per week 19.0 ± 28.7 21.3 ± 28.9 23.1 ± 30.4 24.0 ± 31.0 29.0 ± 36.6 Current smoker, % 17.0 11.9 9.4 8.8 8.5 Dietary intake Grapefruit, servings per day 0.03 ± 0.04 0.07 ± 0.09 0.13 ± 0.14 0.16 ± 0.20 0.33 ± 0.37 Grapefruit juice, servings per day 0.01 ± 0.03 0.03 ± 0.07 0.06 ± 0.12 0.09 ± 0.18 0.22 ± 0.45 Oranges, servings per day 0.06 ± 0.05 0.14 ± 0.12 0.21 ± 0.18 0.25 ± 0.24 0.48 ± 0.48 Orange juice, servings per day 0.06 ± 0.06 0.20 ± 0.14 0.39 ± 0.24 0.71 ± 0.33 1.12 ± 0.78 Total energy, kcal per day 1,661 ± 495 1,754 ± 482 1,838 ± 487 1,913 ± 495 2,094 ± 529 Alcohol, g per day 8.0 ± 14.1 7.4 ± 12.4 7.3 ± 11.8 7.8 ± 12.0 8.0 ± 12.4 Coffee, cups per day 1.6 ± 1.6 1.5 ± 1.5 1.4 ± 1.4 1.3 ± 1.4 1.2 ± 1.4 Total vitamin C intake, mg per day 295 ± 355 332 ± 343 367 ± 338 407 ± 354 513 ± 390 From diet 91 ± 45 121 ± 41 152 ± 43 182 ± 47 258 ± 79 From supplements 204 ± 349 211 ± 338 216 ± 333 225 ± 348 255 ± 377 The association between overall citrus consumption and melanoma risk appeared in an exposure-dependent manner in the pooled analysis (P trend < .001; Table 2) and was generally consistent among women and men (Data Supplement). Separate analyses for individual citrus products found that the positive association between grapefruit consumption and melanoma risk was most apparent. Orange juice consumption also showed a significant but less apparent association with melanoma risk. Consumption of grapefruit juice and oranges was generally not associated with melanoma risk. Table 2. Pooled HRs for Incident Melanoma According to Frequency of Citrus Consumption Citrus Type Serving Category P trend < Twice per Week Two to Four Times per Week Five to Six Times per Week Once to 1.5 Times per Day ≥ 1.6 Times per Day Overall citrus No. of person-years 404,850 376,476 471,647 491,559 257,901 No. of cases 260 323 496 488 273 Age-adjusted HR (95% CI) 1.00 1.19 (1.01 to 1.40) 1.38 (1.19 to 1.61) 1.40 (1.20 to 1.63) 1.47 (1.24 to 1.75) < .001 Multivariable-adjusted HR (95% CI)* 1.00 1.10 (0.94 to 1.30) 1.26 (1.08 to 1.47) 1.27 (1.09 to 1.49) 1.36 (1.14 to 1.63) < .001 Serving Category Never < Once per Week Once per Week Twice per Week ≥ Three Times per Week Grapefruit No. of person-years 515,163 647,023 354,732 224,690 260,825 No. of cases 317 589 353 275 306 Age-adjusted HR (95% CI) 1.00 1.30 (1.13 to 1.49) 1.46 (1.25 to 1.70) 1.53 (1.30 to 1.81) 1.61 (1.18 to 2.19) < .001 Multivariable-adjusted HR (95% CI)* 1.00 1.18 (1.03 to 1.36) 1.31 (1.12 to 1.53) 1.33 (1.12 to 1.58) 1.41 (1.09 to 1.83) < .001 Multivariable-adjusted HR (95% CI)† 1.00 1.17 (1.02 to 1.36) 1.30 (1.10 to 1.53) 1.33 (1.11 to 1.59) 1.41 (1.10 to 1.82) < .001 Grapefruit juice No. of person-years 1,107,142 482,543 167,713 111,640 133,396 No. of cases 909 508 172 126 125 Age-adjusted HR (95% CI) 1.00 1.12 (1.00 to 1.25) 1.13 (0.96 to 1.33) 1.15 (0.95 to 1.39) 1.10 (0.92 to 1.33) .13 Multivariable-adjusted HR (95% CI)* 1.00 1.05 (0.94 to 1.17) 1.07 (0.90 to 1.26) 1.08 (0.89 to 1.30) 1.07 (0.88 to 1.29) .36 Multivariable-adjusted HR (95% CI)† 1.00 0.98 (0.87 to 1.10) 0.98 (0.82 to 1.16) 0.99 (0.81 to 1.20) 0.98 (0.81 to 1.19) .79 Serving Category Never ≤ Once per Week Twice per Week Three Times per Week ≥ Four Times per Week Oranges No. of person-years 231,632 931,801 343,685 271,097 224,218 No. of cases 138 828 372 261 241 Age-adjusted HR (95% CI) 1.00 1.26 (1.05 to 1.51) 1.25 (0.94 to 1.64) 1.32 (1.07 to 1.63) 1.35 (1.09 to 1.67) .05 Multivariable-adjusted HR (95% CI)* 1.00 1.13 (0.94 to 1.36) 1.06 (0.78 to 1.44) 1.16 (0.94 to 1.43) 1.18 (0.95 to 1.47) .30 Multivariable-adjusted HR (95% CI)† 1.00 1.06 (0.88 to 1.28) 0.97 (0.71 to 1.32) 1.05 (0.85 to 1.31) 1.08 (0.86 to 1.35) .81 Serving Category < Once per Week One to Two Times per Week Three to Four Times per Week Five to Six Times per Week ≥ Once per Day Orange juice No. of person-years 505,507 456,849 401,096 314,923 324,057 No. of cases 359 434 365 377 305 Age-adjusted HR (95% CI) 1.00 1.16 (1.01 to 1.34) 1.13 (0.90 to 1.42) 1.36 (1.18 to 1.58) 1.32 (1.13 to 1.53) < .001 Multivariable-adjusted HR (95% CI)* 1.00 1.11 (0.96 to 1.28) 1.09 (0.89 to 1.33) 1.27 (1.09 to 1.47) 1.28 (1.10 to 1.50) < .001 Multivariable-adjusted HR (95% CI)† 1.00 1.07 (0.93 to 1.24) 1.04 (0.84 to 1.30) 1.22 (1.05 to 1.42) 1.25 (1.07 to 1.47) < .001 The association between citrus consumption and melanoma risk remained essentially unchanged when we adjusted for other potential confounders (Data Supplement). Lag analyses suggested associations between citrus consumption and subsequent melanoma generally similar to those from the main analyses (Fig 1; Data Supplement). The significant positive association between citrus consumption and subsequent melanoma persisted for 12 years after intake for overall citrus and 8 years after intake for orange juice; it remained constant for grapefruit over different lags (Fig 1). However, we did not find any consistent significant association between consumption of other fruits and juices and vegetables and melanoma risk (data not shown). Subgroup analyses suggested that the association between grapefruit consumption and melanoma risk was generally independent of age, lifestyle, and dietary confounders (Table 3) and known melanoma risk factors (P > .10 for all interaction tests; Data Supplement). Nevertheless, the positive association seemed to be more apparent among those who had higher sunburn susceptibility as a child or adolescent, those with a higher number of blistering sunburns, those who spent more time in direct sunlight, and those with higher annual UV flux at residence. Additional subgroup analyses revealed that the association between grapefruit and melanoma was also independent of consumption of other fruits and juices and vegetables and was not affected by physical screening status during the previous 2 years (indicator of health awareness; Data Supplement). Table 3. Pooled Multivariable HRs for Incident Melanoma According to Frequency of Grapefruit Consumption in Subgroups of Potential Confounders* Potential Confounder Grapefruit Serving Category P trend P interaction † Never < Once per Week Once per Wek ≥ Twice per Week Age group, years < 65 No. of person-years 386,342 443,482 237,215 272,430 No. of cases 180 314 167 219 Multivariable-adjusted HR (95% CI) 1.00 1.23 (1.01 to 1.50) 1.28 (1.02 to 1.62) 1.38 (1.10 to 1.73) .01 .82 ≥ 65 No. of person-years 128,820 203,543 117,517 213,084 No. of cases 137 275 186 362 Multivariable-adjusted HR (95% CI) 1.00 1.10 (0.89 to 1.37) 1.31 (1.03 to 1.66) 1.33 (1.07 to 1.66) .002 Body mass index, kg/m2 < 30 No. of person-years 423,239 529,285 293,553 402,710 No. of cases 276 487 300 490 Multivariable-adjusted HR (95% CI) 1.00 1.10 (0.94 to 1.29) 1.23 (1.03 to 1.47) 1.28 (1.08 to 1.52) .001 .39 ≥ 30 No. of person-years 87,025 114,189 59,136 80,721 No. of cases 41 100 53 89 Multivariable-adjusted HR (95% CI) 1.00 1.64 (1.11 to 2.43) 1.71 (1.08 to 2.69) 1.88 (1.22 to 2.88) .02 Physical activity < Median No. of person-years 283,031 317,106 156,235 195,401 No. of cases 155 259 123 213 Multivariable-adjusted HR (95% CI) 1.00 1.20 (0.74 to 1.94) 1.16 (0.68 to 1.98) 1.46 (1.03 to 2.05) .008 .90 > Median No. of person-years 206,457 313,570 189,110 281,741 No. of cases 157 329 228 363 Multivariable-adjusted HR (95% CI) 1.00 1.17 (0.83 to 1.65) 1.39 (0.86 to 2.24) 1.33 (1.07 to 1.64) .003 Smoking status Never No. of person-years 205,283 293,329 166,494 221,425 No. of cases 132 279 166 267 Multivariable-adjusted HR (95% CI) 1.00 1.22 (0.91 to 1.62) 1.31 (1.02 to 1.68) 1.39 (1.07 to 1.82) .01 .72 Ever No. of person-years 269,419 323,488 172,901 243,694 No. of cases 172 289 178 304 Multivariable-adjusted HR (95% CI) 1.00 1.11 (0.91 to 1.36) 1.29 (1.03 to 1.62) 1.38 (1.11 to 1.71) .001 Alcohol intake < Median No. of person-years 275,932 321,995 166,834 228,432 No. of cases 155 268 136 239 Multivariable-adjusted HR (95% CI) 1.00 1.22 (0.99 to 1.50) 1.20 (0.93 to 1.54) 1.37 (1.09 to 1.73) .02 .64 > Median No. of person-years 239,231 325,029 187,898 257,081 No. of cases 162 321 217 342 Multivariable-adjusted HR (95% CI) 1.00 1.14 (0.94 to 1.40) 1.38 (1.11 to 1.73) 1.37 (1.11 to 1.70) .001 Coffee intake, cups per day Low (< 1.0) No. of person-years 227,043 282,630 157,097 225,217 No. of cases 144 257 173 285 Multivariable-adjusted HR (95% CI) 1.00 1.13 (0.91 to 1.40) 1.40 (1.10 to 1.78) 1.37 (1.09 to 1.72) .002 .64 High (≥ 1.0) No. of person-years 288,120 364,394 197,635 260,298 No. of cases 173 332 180 296 Multivariable-adjusted HR (95% CI) 1.00 1.20 (0.99 to 1.46) 1.23 (0.98 to 1.54) 1.38 (1.12 to 1.71) .008 Vitamin C from supplements < Median No. of person-years 283,462 324,903 169,728 214,162 No. of cases 152 253 135 223 Multivariable-adjusted HR (95% CI) 1.00 1.16 (0.94 to 1.44) 1.21 (0.94 to 1.56) 1.40 (1.11 to 1.77) .01 .97 > Median No. of person-years 231,702 322,120 185,005 271,353 No. of cases 165 336 218 358 Multivariable-adjusted HR (95% CI) 1.00 1.19 (0.97 to 1.44) 1.36 (1.10 to 1.70) 1.36 (1.11 to 1.68) .002 The positive association of melanoma with grapefruit consumption differed between site-specific melanomas (Table 4). There were significant associations between grapefruit consumption and melanomas on the body sites with higher continuous sun exposure (P trend < .001 for overall melanoma). In contrast, associations with melanomas on the body sites with lower continuous sun exposure were not apparent (P trend = .22 for overall melanoma). Further analysis stratified by Breslow thickness for invasive melanoma revealed an essentially similar association for tumors with thickness > and < the median of 0.63 mm (data not shown). Table 4. Pooled Multivariable HRs for Incident Melanoma by Subgroup According to Frequency of Grapefruit Consumption* Risk Grapefruit Serving Category P trend Never < Once per Week Once per Week ≥ Twice per Week No. of person-years 515,163 647,023 354,732 485,515 Risk of overall melanoma on body sites with higher continuous sun exposure (head, neck, extremities) No. of cases 175 374 224 381 Multivariable-adjusted HR (95% CI) 1.00 1.36 (1.12 to 1.65) 1.45 (1.16 to 1.81) 1.58 (1.28 to 1.95) < .001 Risk of overall melanoma on body sites with lower continuous sun exposure (truncal sites) No. of cases 121 198 118 177 Multivariable-adjusted HR (95% CI) 1.00 1.04 (0.78 to 1.38) 1.13 (0.76 to 1.69) 1.14 (0.88 to 1.49) .22 Risk of melanoma in situ on body sites with higher continuous sun exposure (head, neck, extremities) No. of cases 87 187 117 206 Multivariable-adjusted HR (95% CI) 1.00 1.32 (1.00 to 1.75) 1.50 (1.07 to 2.11) 1.61 (1.13 to 2.30) .03 Risk of melanoma in situ on body sites with lower continuous sun exposure (truncal sites) No. of cases 46 93 47 79 Multivariable-adjusted HR (95% CI) 1.00 1.13 (0.78 to 1.63) 1.14 (0.74 to 1.75) 1.16 (0.78 to 1.74) .59 Risk of invasive melanoma on body sites with higher continuous sun exposure (head, neck, extremities) No. of cases 88 187 107 175 Multivariable-adjusted HR (95% CI) 1.00 1.40 (1.06 to 1.84) 1.37 (1.00 to 1.88) 1.53 (1.13 to 2.06) .03 Risk of invasive melanoma on body sites with lower continuous sun exposure (truncal sites) No. of cases 75 105 71 98 Multivariable-adjusted HR (95% CI) 1.00 0.96 (0.52 to 1.77) 1.15 (0.69 to 1.91) 1.10 (0.62 to 1.95) .33 Finally, we also investigated the risks of other major nonskin cancers (eg, breast cancer and prostate cancer) associated with citrus consumption and did not find any similar positive associations (data not shown).

DISCUSSION Section: Choose Top of page Abstract INTRODUCTION METHODS RESULTS DISCUSSION << REFERENCES In these two large prospective cohorts, we found that citrus consumption was associated with an increased risk of cutaneous malignant melanoma among women and men after adjusting for other known melanoma risk factors and potential confounders. Those who consumed overall citrus items ≥ 1.6 times per day had a 36% higher risk compared with those who consumed < twice per week. Among individual citrus products, grapefruit showed the most apparent positive association with melanoma risk, followed by orange juice. Citrus products are dietary sources rich in psoralens,2–5 which have been identified as a group of carcinogens for decades.7,9 Photocarcinogenic properties of these chemicals have been well demonstrated in experimental studies.7–12 Animal experiments have found a correlation between epidermal and serum concentrations of psoralens after oral administration, and the appearance of phototoxicity is associated with serum concentrations of psoralens.31 It has been hypothesized that the actions of psoralens in the skin are a result of their ability to form DNA adducts after UVA light irradiation,32,33 whereas they may also mediate phototoxicity through binding to other sites in mammalian cells.34 A previous study demonstrated that PUVA therapy increased the number of human melanocytes as well as the size of the melanosomes, and these abnormalities were also present in PUVA-treated skin that was examined 15 months after treatment had been stopped.35 Oranges and grapefruit are the two most commonly consumed citrus fruits in the United States, accounting for > 90% of citrus market shares.36 Interestingly, fresh grapefruit showed the most apparent association with melanoma among individual citrus products, which may be explained by its higher levels of psoralens and furocoumarins when compared with oranges.2,3 The significant but less apparent association between orange juice with melanoma risk may be partly explained by its much higher consumption levels, which contributed to > 50% of overall citrus consumption, whereas the null association of grapefruit juice with melanoma risk may be a result of its much lower consumption levels and a large number of nonconsumers, as compared with the other individual citrus products (Data Supplement). In addition, industrial processing (eg, pasteurization treatment) for fresh fruits may reduce the contents of furocoumarins in processed juices because of heat.37 The industrial processing effect may help further explain the null association for grapefruit juice, given its low intake levels in the study population. In contrast to orange juice, the association between orange consumption and melanoma risk did not reach statistical significance in the main analyses, which may be explained by the much lower consumption levels when compared with those for orange juice. Nevertheless, there were also indicative positive associations between oranges and melanoma in lag analyses (Fig 1; Data Supplement). Previous experimental studies have revealed that PUVA could induce melanocytic tumors in a mouse model12 and stimulate more apparent in vivo outgrowth of melanoma cells than psoralen or UVA alone.13 Similarly, exposure to psoralens or UVA alone was not tumorigenic in mice, whereas exposure to psoralens plus UVA substantially increased the number of mice with skin tumors.10,11 In our study, we found that the positive association between grapefruit consumption and melanoma risk seemed to be more apparent among those who had higher sunburn susceptibility and higher exposure to UV radiation and also seemed to be stronger for melanomas on body sites with higher continuous sun exposure (eg, head, neck, and extremities) than for melanomas on body sites with lower continuous sun exposure (eg, truncal sites), which may suggest a potential synergistic effect between dietary consumption and solar UV radiation. It is likely that consumption of psoralen-rich foods may have a stronger photocarcinogenic effect in the presence of higher UV radiation as compared with that in the presence of lower UV radiation. The strengths of this study include its prospective design, large sample size, long-term follow-up over 24 to 26 years, repeated assessment of dietary and lifestyle factors, and ability to include a number of potential confounders. However, our study also has several limitations. The two cohorts mostly comprised white, educated US health professionals, which potentially limits the generalizability of the findings. However, restricting the sample to health professionals also reduces potential residual confounding from socioeconomic status. Nevertheless, future studies are needed to confirm this association in populations of other ethnicities. The dietary data were self-reported and may be subject to misclassification. However, we used cumulatively averaged intake from multiple time points to minimize the measurement error. Furthermore, misclassification was likely to be random, given that dietary information was collected prospectively, and thus may have resulted in an underestimation of the association. In conclusion, our analysis based on two large cohorts of health professionals showed that citrus consumption was associated with an increased risk of incident cutaneous malignant melanoma. Among the citrus products examined in the study, grapefruit showed the most apparent association with melanoma risk, independent of other lifestyle and dietary factors. These findings provide evidence for the potential photocarcinogenic effect of psoralen-rich foods. However, previous studies have also suggested that fruit intake may have potential beneficial effects for the prevention of chronic diseases, such as breast cancer and type 2 diabetes.38,39 Although our findings are consistent with evidence from animal experiments, which revealed a potential synergistic effect between psoralens and UV radiation,10–13 further investigation is needed to confirm our findings and guide sun exposure behaviors among individuals with high citrus consumption.

See accompanying editorial on page 2487 Supported in part by National Cancer Institute Grants No. UM1 CA186107, P01 CA87969, UM1 CA167552, and R01 CA137365. Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org. Certain data used in this report were obtained from the Connecticut Department of Public Health, the Human Investigations Committee of which approved this study. Authors' disclosures of potential conflicts of interest are found in the article online at www.jco.org. Author contributions are found at the end of this article.

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST Disclosures provided by the authors are available with this article at www.jco.org.

AUTHOR CONTRIBUTIONS Conception and design: Shaowei Wu, Jiali Han, Eunyoung Cho, Meir J. Stampfer, Abrar A. Qureshi Financial support: Abrar A. Qureshi Administrative support: Jiali Han, Abrar A. Qureshi Provision of study materials or patients: Walter C. Willett Collection and assembly of data: Shaowei Wu, Jiali Han, Diane Feskanich, Eunyoung Cho, Meir J. Stampfer, Abrar A. Qureshi Data analysis and interpretation: All authors Manuscript writing: All authors Final approval of manuscript: All authors

REFERENCES Section: Choose Top of page Abstract INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES <<

Breslow's tumor thickness: the microstage of malignant melanoma is defined by Breslow's and Clark's classifications. The Breslow classification defines the absolute vertical thickness in mm of the primary tumor in the skin. This microstaging more accurately predicts subsequent behavior of cutaneous melanoma. confounding variables: extraneous variables in a statistical model that are associated/correlated with both the independent and dependent variables but are not on the causal pathway between independent and dependent variables. When confounding variables are present, crude (unadjusted) statistical models describing the association between independent and dependent variables are biased (ie, wrong) as the risk estimate includes the effect of the confounding variable as well (type 1 error). As a result, to properly describe the relationship between independent and dependent variables, a multivariable model that includes both the independent variable and all relevant confounding variables as predictors must be executed. Cox proportional hazards regression model: a statistical model for regression analysis of censored survival data, examining the relationship of censored survival distribution to one or more covariates. This model produces a baseline survival curve, covariate coefficient estimates with their standard errors, risk ratios, 95% CIs, and significance levels.

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST Citrus Consumption and Risk of Cutaneous Malignant Melanoma The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or jco.ascopubs.org/site/ifc. Shaowei Wu No relationship to disclose Jiali Han No relationship to disclose Diane Feskanich No relationship to disclose Eunyoung Cho No relationship to disclose Meir J. Stampfer No relationship to disclose Walter C. Willett No relationship to disclose Abrar A. Qureshi Employment: University Dermatology Leadership: University Dermatology Consulting or Advisory Role: Abbvie, Novartis, Janssen Pharmaceuticals, Pfizer Research Funding: Regeneron (Inst)

Acknowledgment We thank the participants and staff of the Nurses' Health Study and Health Professionals Follow-Up Study for their valuable contributions as well as the state cancer registries in the following states for their help: Alabama, Arizona, Arkansas, California, Colorado, Connecticut, Delaware, Florida, Georgia, Idaho, Illinois, Indiana, Iowa, Kentucky, Louisiana, Maine, Maryland, Massachusetts, Minnesota, Nebraska, New Hampshire, New Jersey, New York, North Carolina, North Dakota, Ohio, Oklahoma, Oregon, Pennsylvania, Rhode Island, South Carolina, Tennessee, Texas, Virginia, Washington, and Wyoming.

© 2015 by American Society of Clinical Oncology