Abstract
Background
Men with a family history of prostate cancer and African American men are at high risk for prostate cancer and in need of personalized risk estimates to inform screening decisions. This study evaluated genetic variants in genes encoding microRNA (miRNA) binding sites for informing of time to prostate cancer diagnosis among ethnically-diverse, high-risk men undergoing prostate cancer screening.
Methods
The Prostate Cancer Risk Assessment Program (PRAP) is a longitudinal screening program for high-risk men. Eligibility includes men ages 35-69 with a family history of prostate cancer or African descent. Participants with ≥ 1 follow-up visit were included in the analyses (n=477). Genetic variants in regions encoding miRNA binding sites in four target genes (ALOX15, IL-16, IL-18, and RAF1) previously implicated in prostate cancer development were evaluated. Genotyping methods included Taqman® SNP Genotyping Assay (Applied Biosystems) or pyrosequencing. Cox models were used to assess time to prostate cancer diagnosis by risk genotype.
Results
Among 256 African Americans with ≥ one follow-up visit, the TT genotype at rs1131445 in IL-16 was significantly associated with earlier time to prostate cancer diagnosis vs. the CC/CT genotypes (p=0.013), with a suggestive association after correction for false-discovery (p=0.065). Hazard ratio after controlling for age and PSA for TT vs. CC/CT among African Americans was 3.0 (95% CI 1.26-7.12). No association to time to diagnosis was detected among Caucasians by IL-16 genotype. No association to time to prostate cancer diagnosis was found for the other miRNA target genotypes.
Conclusions
Genetic variation in IL-16 encoding miRNA target site may be informative of time to prostate cancer diagnosis among African American men enrolled in prostate cancer risk assessment, which may inform individualized prostate cancer screening strategies in the future.
Keywords: microRNA, genetic variation, prostatic neoplasms, African American, screening
Introduction
Prostate cancer remains the second leading cause of cancer-related death in men in the United States1. Men with a family history of prostate cancer and African American men are at increased risk for prostate cancer development2-4, with some at greater risk of mortality from prostate cancer5-7. These high-risk men in particular are in need of informed decision-making regarding prostate cancer screening, especially given the controversy over the benefit of population-wide PSA-based screening for prostate cancer8,9. Such informed decision-making regarding prostate cancer screening is advocated by the American Cancer Society10, the American Society of Clinical Oncology11, and by the American Urologic Association12. However, the standard risk factors of age, race, and family history lack precision in providing individualized prostate cancer risk estimates, limiting their ability to inform decisions regarding prostate cancer screening at the individual level. Biomarkers that refine prostate cancer risk estimates are needed to inform prostate cancer screening discussions and screening strategies for men with familial prostate cancer and African American men.
Genetic variants with potential biological implications in prostate cancer development may hold promise for informing of risk. In recent years, microRNAs (miRNAs) have emerged as key regulators of cancer initiation and progression13, with specific miRNAs found to be of importance in prostate cancer biology14-21. MiRNAs bind to target regions in genes and affect gene expression by degrading the encoded messenger RNA or altering the translation of messenger RNA to protein22. If polymorphisms exist in these miRNA target gene regions, miRNA binding to their target mRNA may be abrogated, impacting gene function and thereby contributing to carcinogenesis23. This study evaluated four genetic variants in miRNA target regions of genes with prior reported relevance to prostate cancer risk and/or development. The specific genes encoding miRNA targets included in this analysis were IL-16, IL-18, ALOX15, and RAF1. IL-16 resides in a locus implicated for association to prostate cancer from a genomewide association study19, and higher IL-16 expression in prostate tumors has been associated with higher Gleason score and higher biochemical recurrence after prostatectomy21. In addition, a recent fine-mapping study revealed the association of genetic variation in IL-16 to prostate cancer risk among African American men24. IL-18 promoter polymorphisms have been reported to associate with prostate cancer risk in a Chinese population18 and the IL-18 cytokine has been reported to inhibit the growth of murine prostate carcinomas25. ALOX15 (arachidonate 15-lipooxygenase) has been shown to be down-regulated in prostate tumors compared to benign tissues26 suggesting that this gene may be involved in prostate tumor suppression. RAF1 (v-raf-1 murine leukemia viral oncogene homolog 1) is a reported oncogene which has been shown to influence progression of prostate cancer to androgen independence20. These four genes (ALOX15, IL-16, IL-18, and RAF1 ) have been previously implicated in prostate cancer risk/biology and encode miRNA target sites, supporting the study of miRNA target genetic variation in these genes for this study.
The purpose of this study was to evaluate genetic variants in regions encoding miRNA targets within ALOX15, IL-16, IL-18, and RAF1 for informing of time to prostate cancer diagnosis among high-risk men undergoing prostate cancer screening. The specific regions encoding miRNA target sites included rs916055 in ALOX15 (target site for mir-588/183), rs360727 in IL-18 (target site for mir-197/361), rs1131445 in IL-16 (target site for mir135a/135b), and rs1051208 in RAF1 (target site for mir-213)27. Time to diagnosis of prostate cancer was chosen as a primary endpoint since high-risk men have been reported to have greater rates of early-onset or younger age at diagnosis of prostate cancer3,4,6, which has been associated with poorer prognosis6,7. Thus high-risk men with genetically-predicted earlier time to prostate cancer diagnosis may opt for more intensive screening approaches versus others who may not need such frequent PSA testing or further diagnostic studies. Our study was performed among high-risk men enrolled in the Prostate Cancer Risk Assessment Program (PRAP) – a prospective longitudinal prostate cancer screening and research program for men at high-risk for prostate cancer28.
Subjects and Methods
Prostate Cancer Risk Assessment Program (PRAP)
PRAP was established in 1996 to provide prostate cancer screening and education for men at high-risk for prostate cancer28. Eligibility for PRAP include any man between ages 35-69 years without a previous diagnosis of prostate cancer with one first-degree relative with prostate cancer, two second-degree relatives with prostate cancer on the same side of the family, or any African American man regardless of family history28. Accrual to PRAP is ongoing and participants are followed longitudinally for prostate cancer screening and cancer detection. This analysis includes participants consecutively accrued to PRAP from 1996-2009. The PRAP study is approved by the Institutional Review Board at FCCC and at currently active community hospital sites that enrolled participants to PRAP.
Screening Approach in PRAP
Participants undergo annual prostate cancer screening, which includes the total PSA, digital rectal examination (DRE), and estimation of PSA velocity. Prostate cancer detection rates in PRAP and cancer characteristics have been described in detail previously28. Current biopsy criteria include: (1) PSA greater than 2.0 ng/ml, (2) PSA 1.5 to 2.0 ng/ml with fPSA 25% or less, (3) any abnormality on DRE or (4) PSAv 0.75 ng/ml/year. All biopsies are transrectal ultrasound guided 5-region biopsies with additional biopsies obtained at physician discretion29.
Body Mass Index (BMI) and smoking status
Since BMI and smoking have been reported to influence inflammatory cytokine levels (related to the miRNA target genes analyzed in this study)30-32, these two variables were included in Cox models for analysis of time to prostate cancer diagnosis. Smoking data was coded as current vs other, where other was former smoker/never smoked or was missing smoking information (n=104). BMI was calculated as weight (kg)/(height(m))2 using measurements obtained at the PRAP initial clinic visit, or by self-reported values if the clinical information was missing. BMI was categorized as normal (<25), overweight (25-30), obese (>30) and missing (n=62).
Genotyping of candidate miRNA target polymorphisms
DNA was isolated from blood samples collected from PRAP participants and stored per standard operating procedure in the Biosample Repository Facility at FCCC. Polymorphisms evaluated included rs916055 in ALOX15 (target site for mir-588/183), rs360727 in IL-18 (target site for mir-197/361), rs1131445 in IL-16 (target site for mir135a/135b), and rs1051208 in RAF1 (target site for mir-213)27. Genotyping for all variants except rs360727 in IL-18 was performed using a fluorogenic 5' nuclease allelic discrimination assay (TaqMan® SNP Genotyping Assay, Applied Biosystems). Reactions were prepared using TaqMan® Universal PCR Mastermix, No AmpErase UNG or TaqMan® Genotyping MasterMix (Applied Biosystems) according to manufacturer's instructions. Thermal cycling and analysis were performed using an ABI7900 Sequence Detection System (Applied Biosystems). Control DNA samples with known genotypes were included in each run. In addition, a no template (water) control was included to assess DNA contamination. Genotype assignment was achieved automatically with the SDS software (Applied Biosystems) using a proprietary algorithm. In addition, genotypes were confirmed on a random selection of 2% of the samples by standard sequencing with 100% concordance.
Marker rs360727 in IL-18 was genotyped using pyrosequencing. Briefly, nested PCR amplification was performed. Primer pairs are available upon request. The forward inner primers were biotinylated to facilitate single-strand DNA template preparation for pyrosequencing. Primers were synthesized and HPLC-purified by Integrated DNA Technologies (Coralville, IA). Reactions were prepared using Choice Taq Blue Mastermix (Denville Scientific Inc.) and 20ng of genomic DNA according to manufacturer's instructions. 1μl of product from the first round of PCR with outer primers was used as template for the second PCR round with inner primers. Thermal cycling conditions are available upon request. Preparation of the single-stranded DNA template for pyrosequencing was performed utilizing the PSQ™ Vacuum Prep Tool (Biotage) according to manufacturer's instructions. Twenty μl of biotinylated PCR product was immobilized on Streptavidin-coated Sepharose™ High Performance beads (GE Healthcare, Piscataway, NJ) and processed to obtain a single-stranded DNA using the PSQ 96 Sample Preparation Kit (Biotage) according to manufacturer's instructions. The sequencing-by-synthesis reaction of the complementary strand was automatically performed using the PSQ™ 96MA instrument (Biotage) at room temperature using PyroGold reagents (Biotage). SNP assignment and quality assessment of the raw data was performed using PSQ 96 SNP Software (Biotage).
Statistical Methods
Distributions of candidate miRNA target SNPs were summarized by self-reported race and compared using chi-square tests. In addition, Hardy-Weinberg equilibrium (HWE) was tested for each allele within each self-reported race group using the Chi-Square Goodness-of-Fit Test33. Estimates of survival functions for freedom from prostate cancer diagnosis by genotype were made using the Kaplan-Meier product-limit method. The association between high risk genotype and time to prostate cancer diagnosis, within self-reported race group, was examined using Cox proportional hazards modeling, controlling for age and PSA at entry into PRAP. Men with ≥1 follow-up visit, complete genotype, and PSA data were included in the Cox models. To account for likely pre-existing cancer, men with prostate cancer diagnosed within 6 months after enrollment into PRAP were excluded. Correction for false-discovery rate (FDR) was performed using the Benjamini & Hochberg method to control for multiple comparisons within race groups. Analyses were done using SAS statistical software; FDR adjustment and Kaplan Meier plots were generated using R version 2.15.3
Results
The overall PRAP cohort at the time of this analysis included 768 PRAP participants, of whom 483 (63%) were African American and 285 (37%) were Caucasian with a family history of prostate cancer. Table 1 shows the descriptive characteristics of these 768 PRAP participants. The mean age at entry into PRAP was 49.5 years for African American participants and was 50.2 years for Caucasian participants. Mean PSA at baseline was 1.56 ng/mL for African American participants and 1.69 ng/mL for Caucasian participants. There was a significant difference in BMI (p<0.0001) and current smoking status (p=0.03) by race. More Caucasian PRAP participants underwent prostate biopsies (30.1%) compared to African American participants (22.6%) (p=0.029). There was a significant difference in follow-up by race, with a greater percentage of Caucasian PRAP participants following up compared to African American participants (p<0.0001). Differences in socio-demographic characteristics were compared by follow-up within each race group. Age at entry (p<0.0001), PSA at entry (p=0.0003), and family history of prostate cancer (p=0.004) were significantly different by follow-up among African American participants. Among Caucasian PRAP participants, only age at entry had a borderline difference by follow-up (p=0.069). No difference in rectal exam findings, education, or marital status were noted by follow-up within race groups (results available upon request). When comparing African American to Caucasian participants, no other differences were noted in clinical characteristics, particularly regarding PSA at entry into PRAP, age at prostate cancer diagnosis, PSA prior to diagnosis, or Gleason score (Table 1). Prostate cancer was diagnosed in 52 African American men (10.8% of the total African American sample set) and in 42 Caucasian participants (14.7% of Caucasian sample set).
Table 1.
Characteristics of 768 PRAP Participants by Self-reported Race
| African American (n=483) | Caucasian (n=285) | |||||||
|---|---|---|---|---|---|---|---|---|
| n | Mean | Range | n | Mean | Range | p-value* | ||
| Overall PRAP Cohort Characteristics | ||||||||
| Age at entry (years) | 483 | 49.5 | 35-69 | 285 | 50.2 | 35-69 | 0.28 | |
| PSA at baseline (ng/mL) | 480 | 1.56 | 0.1-27.2 | 284 | 1.69 | 0.1-22.5 | 0.47 | |
| DRE at baseline, n (%) | ||||||||
| Normal/BPH** | 460 (96.0%) | 269 (96.1%) | 0.98 | |||||
| Abnormal | 19 (4.0%) | 11 (3.9%) | ||||||
| BMI | ||||||||
| Normal | 61 (12.6%) | 85 (29.8%) | <0.0001 | |||||
| Overweight | 201 (41.6%) | 128 (44.9%) | ||||||
| Obese | 167 (34.6%) | 64 (22.5%) | ||||||
| Unknown | 54 (11.2%) | 8 (2.8%) | ||||||
| Smoking Status | ||||||||
| Current Smoker | 70 (14.5%) | 26 (9.1%) | 0.030 | |||||
| Other (former/never/missing) | 413 (85.5%) | 259 (90.9%) | ||||||
| Number of Biopsies, n (%) | ||||||||
| Zero | 374 (77.4%) | 197 (69.1%) | 0.029 | |||||
| One | 50 (10.4%) | 45 (15.8%) | ||||||
| Two or more | 59 (12.2%) | 43 (15.1%) | ||||||
| ≥ 1 Follow-up visit, n(%) | 288 (59.6%) | 231 (81.1%) | <0.0001 | |||||
| Duration of follow-up (months) | 288 | 56.3 | 0.3-178.2 | 231 | 60.2 | 0.3-188.6 | 0.36 | |
| Characteristics of Prostate Cancer Cases | ||||||||
| Prostate cancer diagnosis, n (%) | 52 (10.8%) | 42 (14.7%) | 0.10 | |||||
| Age at diagnosis (years) | 52 | 56.6 | 38-74 | 42 | 58.0 | 43-70 | 0.38 | |
| Last PSA prior to diagnosis (ng/mL) | 52 | 4.21 | 0.9-31.6 | 42 | 4.01 | 1.1-22.5 | 0.82 | |
| Gleason Score, median (range) | 6 (5-8) | 6 (6-8) | 0.87 | |||||
| miRNA Target SNP Genotypes, n(%) | ||||||||
| rs1131445 (IL-16) | CC | 40 (8.5%) | 23 (8.2%) | 0.10 | ||||
| CT | 193 (40.9%) | 136 (48.8%) | ||||||
| TT | 239 (50.6%) | 120 (43.0%) | ||||||
| rs360727 (IL-18) | CC | 390 (81.3%) | 263 (92.6%) | <0.0001 | ||||
| CT | 83 (17.3%) | 17 (6.0%) | ||||||
| TT | 7 (1.5%) | 4 (1.4%) | ||||||
| rs916055 (ALOX15) | AA | 313 (66.5%) | 93 (33.7%) | <0.0001 | ||||
| AG | 139 (29.5%) | 147 (53.3%) | ||||||
| GG | 19 (4%) | 36 (13.0%) | ||||||
| rs1051208 (RAF1) | AA | 27 (5.7%) | 6 (2.2%) | <0.0001 | ||||
| AG | 201 (42.5%) | 67 (24.0%) | ||||||
| GG | 245 (51.8%) | 206 (73.8%) | ||||||
pvalue from t-test for age, PSA and duration comparisons; p-value from Chi-square test for other comparisons
BPH=Benign Prostatic Hyperplasia/Hypertrophy
MiRNA target SNP genotypes were available for the following PRAP sample numbers: ALOX15 (n=785), RAF1 (n=752), IL-16 (n=751), and IL-18 (n=764) (Table 1). There was a significant difference in distribution of miRNA target SNP genotypes by race for ALOX15 (p<0.0001), RAF1 (p<0.0001), and IL-18 (p<0.0001). No differences were detected in HWE for IL-16, ALOX15 and RAF1 genotypes. There was deviation noted from HWE for IL-18 genotype among Caucasian men (p<0.001), and therefore IL-18 was not included in the time to event analysis.
Time to prostate cancer diagnosis was evaluated among 477 PRAP participants with ≥1 follow-up visit. Men with prostate cancer diagnosed within 6 months of enrollment into PRAP (n=42) were excluded from this analysis in an attempt to limit preexisting prostate cancer. The characteristics of 477 PRAP participants with follow-up data are shown in Table 2. Among clinical characteristics, only distribution of BMI was significantly different by race (p<0.0001). There was a significant difference in miRNA SNP genotype distribution by race for ALOX15 (p<0.0001) and RAF1 (p<0.0001).
Table 2.
Characteristics of 477 PRAP Participants* with >=1 Follow-up Visit by Self-reported Race
| African American (n=264) | Caucasian (n=213) | |||||||
|---|---|---|---|---|---|---|---|---|
| n | Mean | Range | n | Mean | Range | p-value* | ||
| Age at entry (years) | 264 | 50.8 | 35-70 | 213 | 50.0 | 35-69 | 0.30 | |
| PSA at baseline (ng/mL) | 262 | 1.62 | 0.1-27.2 | 213 | 1.48 | 0.2-9.8 | 0.45 | |
| DRE at baseline, n (%) | ||||||||
| Normal/BPH** | 254 (96.2%) | 205 (97.2%) | 0.57 | |||||
| Abnormal | 10 (3.8%) | 6 (2.8%) | ||||||
| BMI | ||||||||
| Normal | 38 (14.4%) | 65 (30.5%) | <0.0001 | |||||
| Overweight | 109 (41.3%) | 95 (44.6%) | ||||||
| Obese | 92 (34.9%) | 48 (22.5%) | ||||||
| Unknown | 25 (9.5%) | 5 (2.4%) | ||||||
| Smoking Status | ||||||||
| Current Smoker | 34 (12.9%) | 18 (8.5%) | 0.13 | |||||
| Other (Former/Never/Missing) | 230 (87.1%) | 195 (91.6%) | ||||||
| Number of Biopsies, n (%) | ||||||||
| Zero | 179 (67.8%) | 143 (67.1%) | 0.57 | |||||
| One | 39 (14.8%) | 38 (17.8%) | ||||||
| Two or more | 46 (17.4%) | 32 (15.0%) | ||||||
| Prostate cancer diagnoses, n (%) | 28 (10.6%) | 24 (11.3%) | 0.82 | |||||
| miRNA Target SNP Genotypes, n(%) | ||||||||
| rs1131445 (IL-16) | CC | 17 (6.6%) | 16 (7.7%) | 0.26 | ||||
| CT | 111 (43.2%) | 103 (49.8%) | ||||||
| TT | 129 (50.2%) | 88 (42.5%) | ||||||
| rs916055 (ALOX15) | AA | 175 (38.4%) | 68 (33.3%) | <0.0001 | ||||
| AG | 73 (28.5%) | 108 (52.9%) | ||||||
| GG | 8 (3.1%) | 28 (13.7%) | ||||||
| rs1051208 (RAF1) | AA | 18 (7.0%) | 3 (1.4%) | <0.0001 | ||||
| AG | 119 (43.1%) | 54 (26.1%) | ||||||
| GG | 121 (46.9%) | 150 (72.5%) | ||||||
excludes participants with diagnoses of prostate cancer within 6 months of enrollment into PRAP: 24 African American men and 18 Caucasian men.
BPH=Benign Prostatic Hyperplasia
Among men with follow-up, IL-16 genotype was found to be an independent predictor of earlier time to prostate cancer diagnosis among 256 African American PRAP participants with complete genotype and covariate data, after controlling for age, PSA, BMI, and smoking status (p=0.013) (Table 3). Hazard ratio for TT vs. CC/CT among African Americans was 3.0 (95% CI 1.26-7.12). After correction for FDR, there remained a suggestive association of the TT genotype at the IL-16 miRNA target site with time to diagnosis among African Americans (p=0.065). Kaplan-Meier plot depicting this earlier time to prostate cancer diagnosis by IL-16 genotype among African American participants is shown in Fig. 1A. No association was seen among 207 Caucasian participants by IL-16 miRNA target site genotype (Fig. 1B). No significant association was found for the other miRNA target SNP genotypes to time to prostate cancer diagnosis in either race group. No association to Gleason grade was found by IL-16 genotype, likely due to the small number of cases.
Table 3.
Cox model results for time to prostate cancer diagnosis by miRNA binding region variants among PRAP Participants with ≥ 1 Follow-Up Visit
| African American participants | Caucasian participants | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| miRNA Binding site variants | Genotype comparisons | Sample Size* | HR** | 95% CI | Unadjusted p-value | FDR corrected p-value*** | Sample Size* | HR** | 95% CI | Unadjusted p-value | FDR corrected p-value*** |
| IL-16 (rs1131445) | TT vs CC/CT | 256 | 3.00 | 1.26-7.12 | 0.013 | 0.065 | 207 | 0.74 | 0.31-1.78 | 0.503 | 0.838 |
| CC vs TT/CT | 256 | 0.0+ | -- | 0.988 | 0.988 | 207 | 0.0+ | -- | 0.992 | 0.992 | |
| ALOX15 (rs916055) | AA vs GG/AG | 255 | 1.29 | 0.55-3.05 | 0.559 | 0.932 | 204 | 0.54 | 0.19-1.49 | 0.233 | 0.583 |
| GG vs AA/AG | MNR# | 204 | 1.25 | 0.40-3.90 | 0.695 | 0.869 | |||||
| RAF1 (rs1051208) | AA vs GG/AG | 257 | 0.97 | 0.22-4.34 | 0.970 | 0.988 | MNR# | ||||
| GG vs AA/AG | 257 | 0.75 | 0.33-1.68 | 0.478 | 0.932 | 207 | 2.86 | 0.82-10.03 | 0.100 | 0.500 | |
Sample sizes vary by number with complete genotype and PSA data available.
HR = Hazard ratio after controlling for age, PSA, BMI, and smoking.
False Discovery Rated p-value, adjusted for multiple comparisons within race group using Benjamini-Hochberg method.
No prostate cancers with CC genotype
MNR = model not run, subgroup < 10 patients
Fig. 1A.
Time to Prostate Cancer Diagnosis Among 256 African American PRAP Participants with >=1 Follow-up Visit by IL-16 miRNA target site genotype
Fig. 1B.
Time to Prostate Cancer Diagnosis Among 207 Caucasian PRAP Participants with >=1 Follow-up Visit by IL-16 miRNA target site genotype
Discussion
Men with a family history of prostate cancer and African American men are considered to be at increased risk for prostate cancer as a group, with subsets at increased risk for aggressive disease2-7. However, many high-risk men undergo screening and diagnostic evaluations (such as prostate biopsies) with no cancer detected, subjecting many men to unnecessary risk. Therefore, healthcare providers are recommended to have detailed discussions regarding the risks and benefits of PSA-based screening for prostate cancer, particularly for high-risk men10-12. Biomarkers that predict aspects of prostate cancer development would aid in these discussions and inform individualized decisions for prostate cancer screening.
The goal of this analysis was to determine if variants in genetic regions encoding miRNA target sites in candidate genes of biologic relevance to prostate cancer may inform of prostate cancer risk specifically among high-risk men to ultimately inform decision-making regarding screening and prevention. Out of four miRNA target site SNPs evaluated in genes of biologic relevance to prostate cancer (ALOX15, RAF1, IL-16, and IL-18), our analysis revealed the strongest association of time to prostate cancer diagnosis among African American men by IL-16 genotype at rs1131445, which is in the region encoding mRNA with the binding site for miRNAs mir-135a and mir-135b. The TT genotype in IL-16 was associated with a 3-fold greater risk for earlier time to prostate cancer diagnosis among African American men undergoing screening, and did not predict time to diagnosis among Caucasian participants. One hypothesis potentially explaining the association of rs1131445 genotype to time to prostate cancer diagnosis is that genetic variation encoding the binding site for mir-135a/b may impact binding of these miRNAs to the IL-16 mRNA, affecting IL-16's chemoattractant activity for CD4+ immune cells leading to greater inflammation in the prostate and subsequent potential development of prostate cancer. Rs1131445 is located in the region of IL-16 that encodes the 3’-untranslated region (3’ UTR) of its mRNA. The 3’ UTRs have been reported to be key regions of mRNA-miRNA binding, and polymorphisms in genes impacting coding of 3’ UTRs may attenuate this mRNA-miRNA interaction23,34. Our findings also highlight the importance of studying genetic variation in diverse populations. While other studies have found an association of rs1131445 in IL-16 to prostate cancer risk in Caucasian men19,21, the association in our analysis of time to diagnosis of prostate cancer was only seen in African American participants. This may have been due to a smaller sample size of Caucasian participants, or to a different endpoint chosen in this study (time to diagnosis) compared to other studies that found an association among Caucasians. The biologic implications of genetic variation in IL-16 may also be important in understanding the racial disparity seen in our findings. Higher IL-16 expression has been associated with greater biochemical recurrence after prostatectomy in a prior study21. Our findings of earlier time to diagnosis by IL-16 genotype among African American men are complimentary to the knowledge that African American men have higher rates of advanced disease and prostate cancer specific-mortality than Caucasian men. Thus the biologic consequences of genetic variation in IL-16 are important areas of further study.
The implications of our data after further confirmation are that African American men may have miRNA target site genetic variation data included in their discussions to inform decisions and strategies for prostate cancer screening. For example, men carrying the TT genotype at IL-16 may be recommended to begin prostate cancer screening and perhaps screen more frequently (yearly PSA tests) compared to men not carrying the TT genotype, and thus make individualized decisions regarding screening. Adding genetic predictions for time to diagnosis has potential to advance personalized prostate cancer risk assessment. This clinical scenario requires further study and confirmation.
There are some limitations to be noted when interpreting our findings. There may be clinical utility from the other markers in this study which were not detected due to our sample size, and that an association to Gleason score was not detected. This may be due to the relatively small number of prostate cancer cases, and our results need further confirmation and validation in larger sample sets. Among African American men, approximately 60% returned for follow-up. While no significant differences were noted in socio-demographic characteristics by follow-up among Caucasian participants, there were differences noted by follow-up among African American participants regarding age at entry, PSA at entry, and family history of prostate cancer. The primary analysis of the association of time to prostate cancer diagnosis among participants with follow-up was controlled for age at entry, PSA, BMI, and smoking status. Therefore, while this analysis represents one of the largest prospectively followed high-risk cohorts with majority of African American men undergoing prostate cancer screening, confirmation of our findings is needed. In addition, prostate biopsies are not performed in all PRAP participants, only for those who meet biopsy criteria. However, the association of IL-16 miRNA target site genotype with time to prostate cancer diagnosis remained significant after controlling for PSA. It is noted that a prior fine-mapping study of genetic variation in IL-16 and association to prostate cancer risk among African American men did not reveal an association to prostate cancer for rs1131445.24 This may have been due a slightly different endpoint from our study, being longitudinal time to prostate cancer diagnosis, which may have accounted for the discrepant findings. Confirmation of our findings is required in larger longitudinal screening studies.
Overall, our study finds that genetic variation in IL-16 encoding a miRNA target site may inform of time to prostate cancer diagnosis among African American men, thereby having potential future clinical relevance in informed decision-making for prostate cancer screening for these high-risk men. Further study and confirmation of our findings is warranted.
Acknowledgements
We are grateful to the participants of the Prostate Cancer Risk Assessment Program.
Funding: Keystone Grant for Personalized Risk and Prevention (Grant number 72002-10, Fox Chase Cancer Center Institutional Funds)(VNG); VNG is supported by the Department of Defense Physician Research Training Award (W81XWH-09-1-0302) (VNG); P30 CA006927 from the National Cancer Institute (Cancer Center Support Grant). PRAP has been supported by Pennsylvania Department of Health Grants (98-PADOH-ME-98155) and (#4100042732).
Footnotes
Conflicts of Interest: No conflicts of interest to disclose for any of the authors relevant to this manuscript.
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