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. Author manuscript; available in PMC: 2022 Nov 19.
Published in final edited form as: Nutr Cancer. 2020 Dec 11;73(11-12):2614–2626. doi: 10.1080/01635581.2020.1842895

Pretreatment Dietary Patterns, Serum Carotenoids and Tocopherols Influence Tumor Immune Response in Head and Neck Squamous Cell Carcinoma

Ilona Argirion 1, Anna E Arthur 2,3, Katie R Zarins 1, Emily Bellile 4, Sylvia L Crowder 2, Lahin Amlani 5, Jeremy MG Taylor 4, Greg T Wolf 5, Jonathan McHugh 6, Ariane Nguyen 5, Alison M Mondul 7, Laura S Rozek 1; The University of Michigan Head and Neck SPORE Program
PMCID: PMC9675403  NIHMSID: NIHMS1760255  PMID: 33307825

Abstract

Background:

Tumor infiltrating lymphocytes (TILs) aid in informing treatment for head and neck squamous cell carcinoma (HNSCC). Nevertheless, little is known about the role of diet on TILs.

Methods:

Immunohistologic expression of CD4, CD8, CD68, CD103, CD104 and FOXP3 were assessed in tissue microarrays from 233 previously untreated HNSCC patients. Associations between these markers and pre-treatment dietary patterns were evaluated using linear regression. Associations between baseline serum carotenoids, tocopherols and TILs were assessed using logistic regression. Cox models evaluated the association between diet and TILs on overall and recurrence-free survival.

Results:

Consumption of a Western dietary pattern was associated with lower CD8+ and FOXP3+ infiltrates (p-value:0.03 and 0.02, respectively). Multivariable logistic regression models demonstrated significantly higher CD8+ (OR:2.21;p-value:0.001) and FOXP3+ (OR:4.26;p-value:<0.0001) among patients with high gamma tocopherol. Conversely, high levels of xanthophylls (OR:0.12;p-value:<0.0001), lycopene (OR:0.36;p-value:0.0001) and total carotenoids(OR:0.31;p-value: <0.0001) were associated with significantly lower CD68+. Among those with high CD4+ (HR:1.77;p-value:0.03), CD68+ (HR:2.42;p-value:0.004), CD103+ (HR:3.64;p-value:0.03) and FOXP3+ (HR:3.09;p-value:0.05), having a high Western dietary pattern increased the risk of overall mortality when compared to a low Western dietary pattern.

Conclusion:

Dietary patterns and serum carotenoids may play an important role in modifying TILs, and ultimately, outcome after diagnosis with HNSCC.

Keywords: Head and Neck Cancer, Diet, Epidemiology, Carotenoids, Micronutrients, Serum Markers, Vegetables and Fruit

Introduction

Head and neck cancer (HNC) encompasses malignant lesions found within the epithelial tissue of the oral cavity, pharynx, larynx and nasopharynx, together comprising the sixth most common cancer worldwide (1). More than 90% of neoplasms within the head and neck are reported to be squamous cell carcinoma (HNSCC), commonly associated with extensive exposure to alcohol and tobacco use(2); however, decreases in tobacco consumption in the United States (US)(3, 4) has led to the implication of high-risk human papillomavirus (HPV) as the primary etiologic factor associated with oropharyngeal carcinoma (5). Despite advances in treatment, HNC survival has remained largely stagnant, with global five-year relative survival rates ranging from 40–50% (6). In order to improve patient outcomes, further research and a better understanding of this heterogenous disease is needed.

Recent studies on the role of tumor-infiltrating lymphocytes (TILs) and cancer suggest tumorigenesis to be an immunologic disease as well as genetic one. HNC tumors are unique in that they lie within close proximity to lymphoid tissue in the Waldeyer’s ring, therefore allowing for dense infiltration of immune cells to be easily integrated into the lesion (7). Several studies assessing the role of TILs in HNC have suggested these immune markers to be strong prognostic tools to guide treatment decisions (7). HPV+ HNSCC patients commonly display nodal metastasis, yet the overall and progression-free survival rates are typically better than those among their HPV- counterparts. Due largely to reports of dense lymphocytic infiltrates in HPV+ oropharyngeal carcinoma, one proposed rationale for the observed survival benefit is that the body’s immune response against the viral antigen, particularly in the form of CD8+ and FOXP3+, may aid in driving the elimination of the tumor cells (8–13). TILs have also been repeatedly noted as an important indicator in maximizing treatment efficacy, with increased CD3+, CD4+, CD8+, CD103+ and FOXP3+ T-cell infiltrates correlated with improved survival among HNSCC patients(14–18); conversely, high levels of CD68+ myeloid derived suppressor cells, have previously been reported to be associated with poor HNSCC prognosis(14),

Numerous epidemiologic studies have demonstrated a bi-directional effect of diet on HNSCC risk, progression and prognosis(19–28). While poor nutrition is believed to account for an estimated third of all cancer related deaths in the US (29, 30), the molecular mechanisms underlying the protective effects of bioactive dietary agents remains to be fully elucidated. Immune system modulation through dietary components that can mediate inflammation within the tumor microenvironment is an important mechanism that has been understudied in HNSCC. Although pro-inflammatory factors are essential in the biological response to trauma or infection, excessive production of cytokines, chemokines, growth factors, prostaglandins, and reactive oxygen and nitrogen species have been shown to impact tumor initiation and progression by increasing mutation and cellular proliferation rates, decreasing apoptosis and facilitating angiogenesis (31, 32). Increased consumption of fruits and vegetables have consistently been shown to improve HNSCC prognosis, possibly by counteracting many of the aforementioned inflammatory products through their antioxidant, antimutagenic and antiproliferative properties (26, 33–35). Nevertheless, no studies to date have assessed the role of diet on anti-tumor immune response in HNC.

With increasing interest in the role of tumor immune response in personalized medicine and immunotherapy, it is important to assess the potential role of diet on tumor immune response and, ultimately, outcome. The results of this study will not only impact our knowledge regarding disease pathogenesis, but also aid in facilitating a conversation about dietary recommendations for both HNSCC prevention and intervention.

Methods

Study Population

Incident cases diagnosed with HNSCC were recruited to participate in this prospective cohort study as part of the University of Michigan Head and Neck Specialized Program of Research Excellence (HN-SPORE). Upon approval from the Institutional Review Board at the University of Michigan, study participants provided signed, informed consent to participate. Exclusion criteria included being <18 years of age, pregnant, non-English speaking, diagnosed with another non-upper aerodigestive tract cancer, diagnosed with mental instability, or diagnosed with another head and neck primary within five years preceding the signed consent. In total, 1,042 subjects were enrolled into the study between November 2008 and October 2014.

At baseline, subjects were asked to complete a self-administered health questionnaire that ascertained data on demographic characteristics, tobacco and alcohol use, physical activity, comorbidities, sleep, depression and quality of life. Thirty mL peripheral blood samples were taken prior to treatment using routine venipuncture techniques and formalin-fixed paraffin-embedded (FFPE) tissue blocks were collected from diagnostic biopsies. In order to track patient status, participants were routinely followed every three to six months in clinic. Vital status was reviewed annually following diagnosis. Mortality and recurrence status were obtained by research assistants through medical record review, annual health surveys, the Social Security Death Index and LexisNexis through April 2016. Time to first recurrence was calculated as the number of days between the initial diagnosis at Michigan Medicine and the date of first recurrence. Patients who completed treatment but exhibited persistent disease (were never determined by a medical doctor to be disease-free) were given a recurrence time of 1 day.

For the purpose of this analysis, patients who had not completed their baseline food frequency questionnaire (FFQ) or were not included on a tumor microarray (TMA) were excluded. Additional exclusion criteria included patients who had withdrawn consent, had uncommon tumor sites or unknown primaries, resulting in a final sample size of 233. Finally, for the purposes of the serum carotenoid/tocopherol analysis, those who did not have available baseline serum carotenoid data were excluded, resulting in a sample size of 70.

Specimen and Data Collection

Dietary intake

Dietary intake was collected using the self-administered, semi-quantitative Harvard food frequency questionnaire (FFQ) and analyzed as pretreatment dietary patterns as described previously by Arthur et al.(26). Dietary components and their factor loadings for each of the two dietary patterns can be found in Supplementary Table 2. In brief, the FFQ was designed to evaluate respondents’ typical dietary intake from food and supplements prior to diagnosis. The questionnaire consists of 131 items, and the reproducibility and validity have both been previously reported (36, 37). The FFQ included standard portion sizes for each item (eg: 1 banana, 3–5 oz chicken), which allowed participants to select their average frequency of consumption over the past year from a list of up to 9 choices ranging from “almost never” to “≥6 times per day.” Total energy and nutrient intake were ascertained by summing intakes from each food item based on the selected portion size, reported frequency of consumption, and nutrient content of each food item(36). Estimations for daily food group servings were calculated by summing the frequency weights of each food item based on reported daily frequency of consumption. Upon exclusion based on missing values and energy intake outliers, 39 foods and food groups were isolated using previously defined methods(26, 38, 39). Dietary patterns were deduced by principal component analysis and pattern factor scores were calculated for each patient by summing the reported consumption of the factor food variables weighted by factor loading.

Serum micronutrients

The extraction of serum carotenoids and tocopherols was completed using high-performance liquid chromatography as previously described (40, 41). In summary, equal volumes of serum and ethanol containing butylated hydroxytoluene were mixed and extracted with hexane, utilizing Tocol as the internal standard. To separate carotenoids from tocopherols, a YMC C30 reverse-phase column was used (YMC Company, LTD, Kyoto, Japan) to conduct a gradual elution at 0.2 mL/min total flow on the Shimadzu LC-20AT HPLC system (Shimadzu Corperation, Kyoto, Japan). While detection for carotenoids was at 450 and 472 nm, electrochemical detection using the Coularray electrochemical detector set (Thermo Scientific, Waltham, Mass) was used for Tocol and tocopherols at 310, 390 and 470 mV.

Tumor infiltrating lymphocytes

Hematoxylin-eosin stained slides were created from the aforementioned FFPE tissue specimens and reviewed by an expert pathologist (JM). Upon verification of histology, those slides that expressed >70% cellularity and minimal necrosis were set aside for tissue sampling. Triplicate 0.7mm diameter cores were selected, punched and extracted for each patient to comprise six tissue microarray (TMA) blocks representative of 541 patients. The samples were processed in two batches (Supplemental Table 1)

Slides cut from TMAs were stained for CD4, CD8, CD68, CD103, CD104 and FOXP3 TILs utilizing techniques previously described (14, 17). Briefly, after overnight incubation in a 65°C oven, the sections were deparaffinized, and rehydrated using xylene, graded alcohols, as well as buffer immersion steps. Heat-induced epitope retrieval was then followed by incubation of the slides in a preheated pressure cooker with either Citrate buffer pH6 or Tris-EDTA buffer pH9 and blocked using horse serum. A DAKO autostainer, with chromogens liquid streptavidin biotin horseradish peroxidase and DAB, was then used for immunohistochemical staining. The deparaffinated sections, along with positive and negative controls, were then stained with monoclonal antibodies at the following titrations: CD4–1:250 (Abcam Ab846); CD8–1:40 (Nova Castra VP-C320); CD68 −1:100 (Dako M0814); CD103–1:500 (Abcam Ab129202); CD104 −1:50 (Beta-4 integrin, eBioscience 439–9b); and FoxP3 −1:200 (Abcam Ab20034). The stained slides were digitally imaged to be used in Aperio ImageScope v.12 software (Leica Biosystems).

A technician naive to clinical status scored the stained TMA slides. Grid software (Measure, C Thing Software 2.01) was employed to overlay each tissue core before counting TILs. CD104+ (beta-4 integrin) staining was used before counting TILs to identify the location and extent of carcinoma in each core. Cores were scored as 25%, 50%, 75%, or 100% tumor. Cores deemed to have <25% tumor parenchyma were not scored and therefore excluded from the analysis. Using 200 X magnification, TILs stained with CD4, CD8, CD68, CD103 and FoxP3 antibodies were manually counted. Only those TILs that infiltrated the tumor parenchyma were counted. The TIL count for each core was normalized by dividing each raw value by the fraction of each 0.7 mm core that was indicated as malignant through CD104 staining. Normalizing TIL counts ensured that variation in counted cells was representative of increased TIL density within the tumor parenchyma and not tumor proportion within cores. Normalized mean counts of triplicate samples for each patient were utilized for statistical analysis.

Statistical methods

Descriptive statistics reflective of demographic, clinical and serum carotenoid/tocopherol variables were compared between binary determinants of dietary pattern score. Due to the fact that participants with dietary data were generally healthier than those missing dietary data, we utilized inverse probability weighting (IPW) in order to allow our subcohort patients to better represent the larger HN-SPORE cohort (Supplementary Table 3), thereby improving generalizability(42). Propensity scores (PS) were generated using baseline demographic variables, including: age at diagnosis, sex, stage, disease site, comorbidity score, drinking and smoking status. The reciprocal of the PS was then used as the IPW in statistical analyses. To construct parsimonious statistical models, covariates believed to be relevant to the analyses were chosen by a priori knowledge, input by treating physicians, multicollinearity assessment and determined using backward selection. To assess the association between binary dietary pattern score (around the median) and each continuous TIL marker, both crude and age, sex and TMA adjusted linear regression models were utilized. Due to limited sample size and nonadherence to linear regression assumptions, logistic regression models were used to evaluate associations between TILs and binary serum carotenoids and tocopherols (both individually, and as total groupings); univariate and multivariable models (adjusted for age, sex, disease site and TMA) were compared using Bonferroni correction to account for multiple testing. Finally, Cox proportional hazard (HR) models, adjusted for age, sex, stage, disease site, HPV status and TMA, was used to assess the joint role of dietary pattern and TILs on overall and recurrence-free survival, consequently assessing the potential modification of the relationship between TILs and survival by dietary pattern. All statistical tests were conducted using SAS software 9.4 (SAS Institute, Inc.).

Results

Median follow-up for the 233 participants in this subcohort was 48 months and five year survival was 73.4%; during the course of the study, 65 (27.9%) of patients died and 56 (24.0%) recurred. The average age at diagnosis was 60.5 (SD: 11.3) years. Most of the study subjects were male (73.4%), white (95.7%), and had some post-secondary education (62.5%). The oral cavity was the most common tumor location (44.2%) and just over half of all patients were diagnosed with stage IV tumors (55.8%). Demographic and clinical characteristics stratified by binary dietary pattern score are shown in Table 1a. Individuals who consumed higher quantities of the prudent diet, characterized by high intake of fruits, vegetables, legumes, whole grains and low-fat dairy, were more likely to be older, more highly educated and former smokers, compared to their counterparts, who were more likely to be current smokers. Males were more likely to consume diets high in red and processed meats, refined grains, potatoes, French fries, high-fat dairy, condiments, desserts, snacks and sugar-sweetened beverages characterized by the Western dietary pattern.

Table 1a:

Patient characteristics by dietary pattern

Prudent Diet Western Pattern
Low (N=116) High (N=117) Low (N=116) High (N=117)
Age: Mean±SD [range], y 59±11 [25–95] 62±11 [39–92] 62±12 [30–95] 59±10 [25–92]
Sex, N (%)
Male 89 (79.72) 82 (70.09) 77 (66.38) 94 (80.34)
Female 27 (23.28) 35 (29.91) 39 (33.62) 23 (19.66)
Race
White 109 (93.97) 114 (97.44) 112 (96.55) 111 (94.87)
Black 3 (2.59) 1 (0.85) 1 (0.86) 3 (2.56)
American Indian 3 (2.59) 1 (0.85) 1 (0.86) 3 (2.56)
Other 1 (0.86) 1 (0.85) 2 (1.72) 0 (0.00)
Education
Less than High School 10 (8.70) 6 (5.13) 9 (7.83) 7 (5.98)
High School/GED 46 (40.00) 25 (21.37) 36 (31.30) 35 (29.91)
Some College 42 (36.52) 45 (38.46) 40 (34.78) 47 (40.17)
4-yr Degree 10 (8.70) 15 (12.82) 11 (9.57) 14 (11.97)
More than 4-yr Degree 7 (6.09) 26 (22.22) 19 (16.52) 14 (40.17)
BMI: Mean±SD [range], kg/m2 26.93±6.02
[16.01–47.94]		 28.15±5.53
[16.44–54.36]		 27.17±5.29 [16.01–40.21] 27.92±6.26 [16.02–54.36]
Stage, N (%)
1 16 (13.79) 19 (16.24) 18 (15.52) 17 (14.53)
2 15 (12.93) 17 (14.53) 17 (14.66) 15 (12.82)
3 18 (15.52) 18 (15.38) 17 (14.66) 19 (16.24)
4 67 (57.76) 63 (53.85) 64 (55.17) 66 (56.41)
Site, N (%)
Larynx 21 (18.10) 22 (18.80) 14 (12.07) 29 (24.79)
Oral Cavity 50 (43.10) 53 (45.30) 55 (47.41) 48 (41.03)
Oropharynx 42 (36.21) 40 (34.19) 45 (38.79) 37 (31.62)
Hypopharynx 3 (2.59) 2 (1.71) 2 (1.72) 3 (2.56)
HPV Status, N (%) *
Positive 40 (34.48) 33 (28.21) 49 (42.24) 58 (49.57)
Negative 50 (43.10) 57 (48.72) 39 (33.62) 34 (29.06)
Invalid/Missing 26 (22.41) 27 (23.08) 28 (24.14) 25 (21.37)
ACE-27 Score, N (%)
None 33 (28.70) 32 (27.35) 27 (23.48) 38 (32.48)
Mild 59 (51.30) 56 (47.86) 60 (52.17) 55 (47.01)
Moderate 16 (13.91) 17 (14.53) 18 (15.65) 15 (12.82)
Severe 7 (6.09) 12 (10.26) 10 (8.70) 9 (7.69)
Drinking Status, N (%)
Never 9 (7.76) 9 (7.69) 11 (9.48) 7 (5.98)
Current 80 (68.97) 84 (71.79) 75 (64.66) 89 (76.07)
Former (quit >12 months) 27 (23.28) 24 (20.51) 30 (25.86) 21 (17.95)
Smoking, N (%)
Never 30 (25.86) 27 (23.08) 36 (31.03) 21 (17.95)
Current 55 (47.41) 38 (32.48) 41 (35.34) 52 (44.44)
Former (quit >12 months) 31 (26.72) 52 (44.44) 39 (33.62) 44 (37.61)
Treatment Modality, N (%)
Surgery Alone 28 (24.14) 30 (25.64) 29 (25.00) 29 (24.79)
Surgery + Adj rad 17 (14.66) 19 (16.24) 16 (13.79) 20 (17.09)
Surgery + Adj chemorad 14 (12.07) 14 (11.97) 16 (13.79) 12 (10.26)
Radiation Alone 10 (8.62) 6 (5.13) 7 (6.03) 9 (7.69)
Chemorad Alone 38 (32.76) 38 (32.48) 40 (34.48) 36 (30.77)
Chemo Alone 1 (0.86) 3 (2.56) 1 (0.86) 3 (2.56)
Palliative, Unknown 8 (6.90) 7 (5.98) 7 (6.03) 8 (6.84)
Deceased, N (%) 33 (28.45) 32 (27.35) 30 (25.86) 35 (29.91)
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*

one patient missing ACE-27 score

Abbreviations: ACE-27, Adult Comorbidity Evaluation-27; BMI, body mass index; HPV, human papillomavirus; SD, standard deviation.

Differences in serum carotenoid/tocopherol concentrations by levels of dietary pattern can be found in Table 1b. While beta carotene and total carotenoids were the only biomarkers found to be significantly elevated among high vs. low prudent diet consumers, high Western dietary pattern patients were significantly more likely to have lower levels of beta carotene, xanthophylls, total carotenoids, as well as alpha-, gamma- and total tocopherol when compared to low Western dietary pattern participants.

Table 1b.

Serum Micronutrient Concentrations by Dietary Pattern

Prudent Diet Western Pattern
Low (N=34) High (N=36) Low (N=25) High (N=45)
Serum Micronutrients ug/ml, Mean (SD)
Alpha Carotene 0.01 (0.02) 0.02 (0.01) 0.02 (0.01) 0.01 (0.02)
Beta Carotene 0.05 (0.07) 0.07 (0.10) 0.08 (0.11) 0.04 (0.05)
Xanthophylls 0.14 (0.10) 0.17 (0.13) 0.18 (0.14) 0.14 (0.09)
Lycopene 0.05 (0.04) 0.05 (0.04) 0.05 (0.03) 0.05 (0.04)
Total Carotenoids 0.26 (0.14) 0.30 (0.24) 0.34 (0.27) 0.25 (0.14)
Alpha tocopherol 7.98 (5.02) 10.33 (6.07) 10.59 (7.49) 8.34 (4.13)
Gamma tocopherol 1.14 (0.57) 1.18 (0.72) 1.18 (0.55) 1.13 (0.79)
Total tocopherol 9.12 (5.32) 11.50 (6.12) 11.72 (7.76) 9.52 (4.22)
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Linear regression model results, assessing the association between individual TIL counts and binary dietary pattern (high/low), can be found in Table 2. While no significant associations were found between any of the TILs and the prudent dietary pattern, high intake of the Western dietary pattern was consistently associated with lower levels of CD4+, CD8+, CD103+ and FOXP3+ infiltrates; significant reductions were noted for CD8+ and FOXP3+ markers, even after adjustment for age, sex and TMA (β: −19.99 and −12.54, respectively).

Table 2:

Linear Regression Model Assessing the Association between Tumor Infiltrating Lymphocytes and Binary Dietary Pattern

Unadjusted Adjusted*
CD4 CD8 CD68 CD103 FOXP3 CD4 CD8 CD68 CD103 FOXP3
Sample Size 228 228 130 93 232 228 228 130 93 232
Prudent Pattern β(SE)
≤ −0.20 ref ref ref ref ref ref ref ref ref ref
> −0.20 −1.47 (6.65) 4.03 (9.13) 0.86 (4.78) 19.84 (21.90) 3.39 (5.37) −1.89 (6.71) 3.48 (9.26) 1.54 (4.92) 20.82 (22.00) 4.54 (5.42)
p-value for trend 0.82 0.66 0.86 0.37 0.53 0.78 0.71 0.76 0.35 0.40
Western Pattern β(SE)
≤ −0.16 ref ref ref ref ref ref ref ref ref ref
> −0.16 −9.00 (6.63) −19.99 (9.04) 0.28 (4.78) −35.88 (22.00) −10.78 (5.33) −8.52 (6.76) −19.99 (9.27) −0.13 (4.82) −41.72 (23.16) −12.54 (5.42)
p-value for trend 0.18 0.03 0.95 0.11 0.04 0.21 0.03 0.98 0.08 0.02
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*

Adjusted for age, sex and TMA

Serum carotenoids and tocopherols were evaluated for their association with tumor immune response using logistic regression models and significance was determined based on Bonferroni correction (Table 3). Multivariable models, adjusted for age, sex, disease site and TMA, demonstrated an increased odds of having high CD8+ and FOXP3+ infiltrates among those with high levels of gamma tocopherol (OR: 2.21 [95%CI: 1.37, 3.56] and OR: 4.26 [95%CI: 2.40, 7.58], respectively). High levels of xanthophylls, lycopene, and total carotenoids were shown to decrease the odds of having high CD68+ myeloid derived suppressor cells, a TIL marker previously associated with poor HNSCC prognosis (14) (OR: 0.21 [95%CI: 0.14, 0.32]; OR: 0.53 [95%CI: 0.36, 0.78] and OR: 0.46 [95%CI: 0.31, 0.67], respectively).

Table 3:

Logistic Regression Analysis Assessing the Association between Tumor Infiltrating Lymphocytes and Serum Carotenoids [OR(95%CI)]

Tumor Infiltrating Lymphocytes (TILs)
Unadjusted Adjustedǂ
CD4 CD8 CD68 FOXP3 CD4 CD8 CD68 FOXP3
Sample Size 69 69 68 70 69 69 68 70
Alpha Carotene
Low (≤ 0.01 ug/ml) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref)
High (>0.01 ug/ml) 1.48 (1.01, 2.18) 2.25 (1.53, 3.30) 0.86 (0.59, 1.25) 1.54 (1.05, 2.25) 1.05 (0.64, 1.70) 1.05 (0.65, 1.71) 0.53 (0.33, 0.85) 0.44 (0.25, 0.79)
p-value 0.05 <.0001 0.42 0.03 0.85 0.84 0.01 0.01
Beta Carotene
Low (≤0.03 ug/ml) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref)
High (>0.03 ug/ml) 0.91 (0.62, 1.34) 1.51 (1.03, 2.20) 1.08 (0.74, 1.57) 1.13 (0.78, 1.65) 0.74 (0.47, 1.17) 0.95 (0.59, 1.53) 0.91 (0.58, 1.40) 0.48 (0.29, 0.82)
p-value 0.64 0.03 0.70 0.52 0.19 0.83 0.66 0.01
Xanthophylls
Low (≤0.13 ug/ml) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref)
High (>0.13 ug/ml) 1.17 (0.80, 1.71) 0.98 (0.67, 1.43) 0.21 (0.14, 0.32) 0.93 (0.64, 1.36) 0.73 (0.45, 1.18) 0.58 (0.35, 0.96) 0.12 (0.07, 0.21) 0.91 (0.55, 1.51)
p-value 0.43 0.92 <.0001 0.72 0.20 0.03 <.0001 0.71
Lycopene
Low (≤0.04 ug/ml) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref)
High (>0.04 ug/ml) 0.83 (0.56, 1.21) 1.51 (1.03, 2.20) 0.53 (0.36, 0.78) 1.18 (0.81, 1.73) 0.70 (0.43, 1.14) 0.47 (0.27, 0.81) 0.36 (0.22, 0.61) 1.02 (0.59, 1.75)
p-value 0.33 0.03 0.001 0.38 0.15 0.01 0.0001 0.95
Total*
Low (≤0.24 ug/ml) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref)
High (>0.24 ug/ml) 0.90 (0.61, 1.32) 1.23 (0.85, 1.80) 0.46 (0.31, 0.67) 0.80 (0.55, 1.17) 0.61 (0.37, 1.02) 0.44 (0.26, 0.75) 0.31 (0.19, 0.51) 0.56 (0.32, 0.97)
p-value 0.57 0.27 <.0001 0.25 0.06 0.003 <.0001 0.04
Alpha tocopherol
Low (≤8.42 ug/ml) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref)
High (>8.42 ug/ml) 2.34 (1.59, 3.46) 2.09 (1.43, 3.06) 1.30 (0.89, 1.89) 1.68 (1.15, 2.44) 1.61 (1.00, 2.60) 1.68 (1.04, 2.71) 0.94 (0.60, 1.47) 1.08 (0.65, 1.80)
p-value <.0001 0.0001 0.17 0.01 0.05 0.03 0.78 0.77
Gamma tocopherol
Low (≤1.09 ug/ml) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref)
High (>1.09 ug/ml) 0.69 (0.47, 1.02) 1.49 (1.02, 2.17) 1.54 (1.05, 2.24) 1.45 (1.00, 2.11) 0.73 (0.46, 1.15) 2.21 (1.37, 3.56) 1.89 (1.22, 2.93) 4.26 (2.40, 7.58)
p-value 0.06 0.04 0.03 0.05 0.18 0.001 0.004 <.0001
Total tocopherol ¥
Low (≤9.44 ug/ml) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref) (Ref)
High (>9.44 ug/ml) 1.79 (1.22, 2.64) 1.60 (1.10, 2.33) 1.12 (0.77, 1.62) 1.29 (0.89, 1.88) 1.25 (0.78, 1.99) 1.44 (0.90, 2.30) 0.96 (0.62, 1.49) 0.93 (0.56, 1.54)
p-value 0.003 0.01 0.56 0.18 0.35 0.13 0.86 0.78
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*

calculates as: alpha carotene+beta carotene+xanthophylls+lycopene

¥

calculated as: alpha tocopherol+gamma tocopherol

ǂ

Age, sex, disease site and TMA adjusted

Cox proportional hazard model results for the joint association between binary TILs and binary dietary pattern on overall and recurrence free survival, adjusted for age, sex, stage, disease site, HPV status and TMA, can be found in Figures 1 and 2. Surprisingly, among those with high CD4+ TIL counts, having a high prudent dietary pattern (in reference to a low prudent dietary pattern) was associated with increased risk of overall mortality (p for interaction: 0.02). Among those with high CD4+ (p for interaction: 0.03), CD68+ (p for interaction: 0.004), CD103+ (p for interaction: 0.03) and FOXP3+ (p for interaction: 0.05) infiltrates, having a high Western dietary pattern significantly increased the risk of overall mortality when compared to those with a low Western dietary pattern. Similar results were found for recurrence-free survival; among those with high CD4+ (p for interaction: <0.0001) and FOXP3+ (p for interaction 0.0002) TILs, having a high Western dietary pattern appeared to increase the risk of recurrence when compared to those with low Western diet consumption.

Figure 1:

Figure 1:

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Cox Proportional Hazard Models Assessing the Role of Tumor Infiltrating Lymphocytes and Diet on Recurrence Free Survival

Figure 2:

Figure 2:

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Cox Proportional Hazard Models Assessing the Role of Tumor Infiltrating Lymphocytes and Diet on Overall Survival

Discussion

This study is the first of its kind to report that both pretreatment dietary patterns, as well as serum carotenoids and tocopherols, are not only associated with tumor immune response, but can also modify the association between TILs and survival. Consuming a high Western dietary pattern was consistently associated with lower levels of infiltrates within the tumor, with significant reductions noted for CD8+ and FOXP3+ in multivariable models. Additionally, we found that high serum concentrations of xanthophylls, lycopene and total carotenoids were associated with a lower number of detrimental CD68+ cells in HNSCC tumors, while gamma tocopherol appeared to be associated with significantly higher CD8+ and FOXP3+ lymphocytes. Finally, we demonstrated that the effect the Western dietary pattern exhibits on the tumor microenvironment may have significant negative implications on overall and recurrence-free survival among HNSCC patients.

While numerous studies have analyzed the association between dietary factors and HNSCC (26, 43–46), there have been no studies, to our knowledge, that have assessed the role of dietary patterns or serum carotenoids/tocopherols on tumor immune response at a molecular level. Overall, dietary studies have largely provided evidence that poor nutritional status, often characterized by low consumption of fruits and vegetables, increases the risk of HNSCC(47), while high consumption of nutrient rich foods have the opposite effect(45, 46, 48). Smoking and alcohol consumption, two primary risk factors in HNC development, are believed to play an integral role in counteracting the protective effects conferred by dietary nutrients. Tobacco products, combustible and smokeless, have been shown to produce free radicals and reactive oxygen molecules, which have the ability to react with the lipid bilayer, denature proteins, induce DNA damage, and trigger chronic inflammation (31, 49); high alcohol consumption can lead to decreased folate absorption and increased oxidative stress through ethanol metabolism (50, 51). Nutrients found abundantly in produce have been shown to reduce inflammation and oxidative stress, maintain proper function of cellular processes, and inhibit angiogenesis, theoretically defending high-risk individuals against the carcinogenic effects of tobacco and alcohol use(52, 53). Preclinical and clinical studies have previously elucidated several bioactive agents believed to affect cell-mediated immune response (54, 55). While the prudent diet did not appear to significantly affect TILs in our cohort, we did find several significant associations between serum carotenoids and tocopherols on tumor immune response.

Serum carotenoids and tocopherols have been established by our group and others as valid biomarkers of fruit and vegetable intake (40, 56). Carotenoids, found abundantly in fruits and vegetables, represent a diverse group of natural polyene pigments and can be classified into xanthophylls (b-cryptoxanthin, lutein, and zeaxanthin) and carotenes (a-carotene, b-carotene, and lycopene) (57, 58). Both classes of carotenoids act as antioxidants, efficiently quenching singlet oxygen, reducing damage associated with reactive oxygen species and inhibiting lipid peroxidation (59–61). In this study, we found significantly lower levels of the negative prognostic marker CD68+ associated with higher consumption of xanthophylls, lycopene and total carotenoids. These findings coincide with previous reports that higher pretreatment serum carotenoids are associated with progression-free survival in HNSCC patients(62), as well as the overall survival benefit associated with high serum xanthophyll(35) and lycopene concentrations (63). Tocopherols, commonly known as vitamin E, have long been used clinically for a variety of oxidative stress-induced conditions(64) due to their functionality in trapping free radicals and protecting lipids in the cell membranes from oxidation(65, 66). In our HNC cohort, we found increased levels of serum gamma-tocopherol to significantly increase CD8+ and FOXP3+ infiltrates in adjusted models. Gamma-tocopherol has previously been shown to be a stronger regulator of inflammation than alpha-tocopherol, namely through inhibition of cyclooxygenase (COX) and modulation of reactive oxygen species(67, 68). Several studies have also reported vitamin E supplementation to enhance CD4+ lymphocyte proliferation, and improve CD4+/CD8+ ratios(69, 70). Moreover, gamma-tocopherol, but not alpha-tocopherol, has recently been reported to exhibit anti-proliferative and pro-apoptotic effects selectively on cancer, but not normal epithelial cells (71).

Although limited, several studies have suggested an increased risk of HNSCC incidence and mortality associated with intake of red and processed meat, eggs and dairy (43, 72–74). In this study, the Western dietary pattern, characterized by a high consumption of red and processed meats, refined grains, potatoes, French fries, high-fat dairy, condiments, desserts, snacks and sugar-sweetened beverages, was found to be associated with lower levels of all TILs except CD68+, with significant findings for CD8+ and FOXP3+ in multivariable models. Furthermore, we found that among those individuals who had both dietary and carotenoid data, participants who consumed a high Western dietary pattern had significantly lower levels of beta carotene, xanthophylls, total carotenoids, as well as alpha-, gamma- and total tocopherols. These results suggest that study participants who consumed a high Western dietary pattern were not only consuming unhealthier food but were also eating much fewer fruits and vegetables than those in the low Western dietary pattern group, thereby foregoing the potential protective effects of these nutrients. High intake of red and preserved meats have previously been implicated in other cancers due to the production of genotoxic heterocyclic amines (HCA) and aromatic hydrocarbons (AH) during cooking, as well as nitrates and nitrites for preservation(75–77). The International Agency for Research on Cancer (IARC) has classified the consumption of processed meat as “carcinogenic to humans” (Group 1), and red meat as “probably carcinogenic to humans” (Group 2A) through mechanisms linked to DNA damage and promotion of inflammation (78, 79). Based on the current evidence, it is reasonable to believe that there may be synergistic effects associated with tobacco, alcohol and meat consumption on HNSCC that warrant further investigation.

TILs are of particular importance in HNSCC due to the close proximity of these tumors to lymphoid tissue in the Waldeyer’s ring, as well as the infectious etiology associated with oropharyngeal and nasopharyngeal carcinoma. Albeit being more aggressive, HPV and EBV related malignancies are associated with improved response to treatment, believed to be attributable to the presence of an increased immune response against the viral antigens. Nevertheless, irrespective of infection, TILs have consistently been shown to be an important prognostic tool in guiding HNSCC treatment. In a large retrospective study, increased CD4+ helper T and CD8+ cytotoxic/suppressor T cell levels significantly improved both overall and recurrence free survival in HNC patients; similar findings were reported between these infiltrates and oral cavity(80), laryngeal(81, 82), oro- and hypopharyngeal(12, 13, 16) carcinoma, particularly among patients receiving chemotherapy. FOXP3+ regulatory T cells(14, 83, 84), and more recently CD103+ infiltrates(17), or αEβ7 integrin, have also been shown to be positive prognostic markers in HNSCC, while CD68+ myeloid-derived suppressor cells have been associated with poor prognosis (14). Accounting for the effect of diet on tumor immune response, we found that among those with high CD4+, CD68+, CD103+ and FOXP3+ markers, consumption of a high Western dietary pattern was associated with significantly worse overall survival when compared to those with a low Western dietary pattern; similar findings were noted between high CD4+ and FOXP3+ infiltrates and high Western dietary pattern on recurrence-free survival. These results are particularly interesting because previous studies conducted by our group did not find differential survival among those consuming various levels of the Western diet(26). Given the reduction of all but CD68+ TILs found to be associated with increased Western dietary pattern, and the significant interaction terms between the aforementioned TILs and Western diet, it is reasonable to believe that the protective effect conferred by TILs may be modified by diet. In order to validate these results, future research should be conducted among HNSCC patients.

The results of this study should be interpreted in light of several limitations. Due to the fact that the FFQ was designed to assess dietary intake over the course of the year preceding HNC diagnosis, the effect of lifetime dietary exposure could not be assessed with respect to immune response. In addition, our sample size precluded us from being able to assess the associations of interest in stratified models, particularly evaluating differences in associations by disease site, smoking status or treatment modality. Nevertheless, smoking and alcohol status were not found to be significant confounders when using backward selection, and sensitivity analyses integrating smoking and alcohol consumption into multivariable models did not alter the overall results of the study; treatment modality was found to be highly colinear with disease site, therefore only site was selected as a covariate in adjusted models. While TMAs are significantly more cost and time effective than staining and assessing entire slides, they do not fully capture the heterogeneity present within tumors, thereby limiting our ability to draw conclusions on the overall tumor microenvironment. In an attempt to minimize any potential confounding introduced by using TMAs, we adjusted the TIL counts by the percentage of tumor parenchyma present in each core, ran the samples in triplicate to best assess the overall sample, had a technician blinded to clinical status count the TILs and adjusted for batch effects (by TMA) in statistical models.

In conclusion, dietary patterns, serum carotenoids and tocopherols may play a role in modifying TILs, and ultimately, outcome after diagnosis with HNSCC. As tumor immune response continues to emerge as an important prognostic tool in risk and treatment stratification, particularly with the advent of immunotherapy and personalized medicine, it is important to account for and educate patients on the role of modifiable lifestyle factors in these contexts. While further research is needed in order to confirm these results and elucidate some of the underlying biological mechanisms between Western dietary patterns, inflammation and immune response, the results of this study could inform dietary interventions among high risk individuals in an attempt to curb risk and improve HNSCC prognosis.

Supplementary Material

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Funding:

National Institute of Environmental Health Sciences: T32 ES007062

Academy of Nutrition and Dietetics/Colgate Palmolive Fellowship in Nutrition and Oral Health USDA-NIFA Hatch Project 1011487

Footnotes

Conflict of Interest:

None to declare

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