Clinical trial NCT04621500
Conditions: Prostate Cancer Vitamin D Deficiency Stress Reaction
Sponsor: Medical University of South Carolina
Start date: 2019-06-04; Completion date: 2022-02-04
Enrollment: 60 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
Veterans between the ages of 50-75, who are having a prostate biopsy, will be recruited for their permission to collect an extra biopsy core for RNA-sequencing. If the participants' treatment decision is Active Surveillance, they will be enrolled into the intervention phase of the study. They will receive supplementation with Vitamin D3 (4,000 IU) daily with repeat (surveillance) prostate biopsy one year later. At that time an extra prostate sample core will be collected for RNA-sequencing to determine changes over time. Measurements for allostatic load (body stress/inflammatory markers) will also be collected at the time of enrollment and at the repeat prostate biopsy visit.

Detailed description:
This is an open-label pilot study. The goal is to investigate the transcription and biological pathways in prostate cancer that are especially relevant to prostate cancer disparities between African American and Caucasian men. Also, to determine any significant differences in the molecular signature that exist in African American and Caucasian men in relation to their Vitamin D levels.

Recruitment will be at the Urology Clinic at the Ralph H. Johnson VA Medical Center in Charleston, SC. Men who are scheduled for a Standard of Care prostate biopsy will be consented for their permission to collect an extra prostate tissue core for RNA-sequencing.

After the pathology report is discussed by their Urologist, and the treatment decision is Active Surveillance, the participant is enrolled into the Intervention Phase. At that time, baseline allostatic load measurements (Blood pressure, pulse, waist/hip ratio, height, and weight for BMI) and bloodwork: (lipid panel, HgbA1c, albumin and creatinine, IL-6, CRP, and DHEA-s (inflammatory markers) plus a Vitamin D level, will be collected. Vitamin D3 soft gels (4,000 IU) daily (six months' supply) will be dispensed. A Social Determinants survey will also be dispensed for subject completion.

At the Urology Standard of Care six-month follow-up appointment, the vitamin D serum level will be collected and the next six months' supply of vitamin D3 will be dispensed.

At the Urology Standard of Care follow-up (repeat at 1 year) surveillance prostate biopsy, an extra prostate tissue core will be collected for RNA-sequencing. The allostatic load measurements and bloodwork will also be obtained.

Arms: arm 1: All enrolled subjects will receive vitamin D3 at 4,000 IU daily for approximately one year.
Interventions: intervention 1: cholecalciferol intervention 2: Standard of Care Prostate Biopsy - collection of 13th core for RNA-seq intervention 3: Standard of Care Prostate Biopsies at baseline and after one year vitamin D3 supplementation intervention 4: Allostatic Load
Intervention descriptions: intervention 1: Enrolled subjects will supplement with vitamin D3 at 4,000 IU daily for one year intervention 2: RNA-seq analysis will be used to (a) identify changes in transcriptional profiles and biological pathways in the prostate between African American and Caucasian men and (b) compare Allostatic Load results to determine stress response and (c) identify changes in the molecular signature in relation to vitamin D levels intervention 3: Pathology results of the prostate biopsies will be analyzed for changes in the (a) Gleason Score (b) the number of positive cores (0 to 12) and (c) the highest percentage of positive core involvement (0 to 100%). intervention 4: Diagnostic Test: Allostatic Load Measurements at baseline and after one year vitamin D3 supplementation Allostatic Load measurements to determine body stress compared at baseline and after one year of vitamin D3 supplementation. Also compared with RNA-seq results to determine stress response.

Other Names:

Bloodwork for HgbA1c, lipid profile, creatinine, albumin and vitamin D levels. Body measurements: Ht. Wt, BMI, waist and hip measurements for waist/hip ratio. Measurements of systolic and diastolic blood pressure and heart rate

Protocol documents:
Prot_SAP_001.pdf: 
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PROTOCOL TITLE: Vitamin D supplementation in RNA-seq profiles of single core prostate 
biopsy samples, including diversity and stress determinants, among 
Veterans. 
 
PRINCIPAL INVESTIGATOR:  Stephen J. Savage, MD 
 
1.0      Objectives / Specific Aims 
Prostate cancer is the most prevalent non-cutaneous malignancy in men, responsible for 
approximately 30,000 deaths in 2013 (1). There are considerable and persistent racial 
disparities in prostate cancer outcomes.  Prostate cancer disproportionately affects African 
American men in terms of incidence, morbidity, and mortality (1), even after adjustment for 
stage (2). African American men have a 2- to 3- times increased risk of developing prostate 
cancer and have a greater mortality rate compared to white men (2). Reduced access to health 
care services also contributes to racial disparities in prostate cancer outcomes, but even in 
equal access health care systems such as the Veterans Administration (VA), African American 
Veterans have higher serum prostate-specific antigen (PSA) values and higher-grade tumors 
than white Veterans (3, 4), even when presenting at the same stage of disease. Thus, access to 
health care is necessary but not sufficient for eliminating racial differences in prostate cancer 
outcomes. A better understanding of the biological mechanisms underlying these disparities is 
needed to develop strategies to overcome them.   
 
Racial disparities in prostate cancer outcomes mirror racial differences in vitamin D levels 
(5). Several studies have shown that about 60% of African American men have sub-optimal 
levels of circulating 25(OH)D (6,7). For this reason, vitamin D3 supplementation is likely to 
especially benefit these men. Vitamin D promotes the differentiation of prostate cancer cells and 
maintains the differentiated phenotype of prostate epithelial cells, raising the possibility that 
long-term vitamin D deficiency may contribute to the progression from subclinical prostate 
cancer to clinical disease (5), especially among African American men.  
 
We propose to investigate: (a) the transcription and biological pathways that are 
especially relevant to prostate cancer disparities between African American and Caucasian men 
and (b) any significant differences in the molecular signature that exist in African American and 
Caucasian men in relation to their vitamin D levels.  
The proposed studies will analyze transcriptional profiles and biological pathways in prostate 
tissue specimens obtained at time of prostate biopsy from these subjects, using bioinformatics 
analyses of RNA sequencing (RNA-seq) data. Implementation of the studies proposed in this 
application will allow us to test the central hypothesis that circulating levels of vitamin D 
significantly impact the transcriptional and biological profiles in prostate tissue samples from 
African American men.  
To test our hypothesis we propose the following Objectives: 
1. To utilize RNA-seq analyses to further demonstrate and identify a specific stress 
pattern signal that exists between African American and Caucasian men who are 
undergoing prostate biopsy.  We have demonstrated initial findings that transcriptional 
profiles and biological pathways in the prostate are significantly different between African 
American and Caucasian subjects; the further elucidation as a result of these studies will allow 
us to identify the molecular underpinnings of health disparities in prostate cancer.  
2. To identify changes in molecular signature that exist in African American and 
Caucasian men in relation to their vitamin D levels.  We will assess ECM-receptor 
interaction, cell adhesion molecules (CAMs) and Pi3K-Akt signaling pathway according to 
serum levels as well as differences in those on supplementation.   
3. Assess the above findings specifically as they relate to serum ancestry markers.  
We plan to assess the varying importance and relationship of specific markers of ancestry.  Our 
population has significant proportions of pure African ancestry patients which will assist in this 
process.  
 


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2.0 
Background 
There are considerable and persistent racial disparities in prostate cancer outcomes.  
Prostate cancer disproportionately affects African American men in terms of incidence, 
morbidity, and mortality (1), even after adjustment for stage (2), as African American race is 
perhaps the most consistent risk factor for prostate cancer. Reduced access to health care 
services also contributes to racial disparities in prostate cancer outcomes, but even in equal 
access health care systems such as the Veterans Administration (VA), African American 
Veterans have higher serum PSA values and higher-grade tumors than white Veterans (3,4), 
even when presenting at the same stage of disease. Thus, access to health care is necessary 
but not sufficient for eliminating racial differences in prostate cancer outcomes. A better 
understanding of the biological mechanisms underlying these disparities is needed to develop 
strategies to overcome them. Vitamin D-mediated biological and biochemical actions are of 
great interest from the standpoint of racial disparities in prostate cancer outcomes because 
differences between African American and Caucasian men mirror racial differences in vitamin D 
levels (5). 
Vitamin D and Prostate Cancer: Prostate cells express the vitamin D receptor (VDR), 
vitamin D-25-hydroxylase, 25-hydroxy-vitamin D-1-alpha-hydroxylase and the 25-
hydroxyvitamin D-24-hydroxylase (8-13). Therefore, normal prostate cells can synthesize 
25(OH)D (calcidiol) from vitamin D3 (cholecalciferol), and 1,25(OH)2 D (calcitriol) from 25(OH)D 
(calcidiol) (14, 15). 1,25(OH)2D is the hormonal, most potent form of vitamin D and in prostate 
cells it acts in a paracrine/autocrine fashion.  
Several mechanisms of vitamin D-mediated anti-cancer action have been identified (16). 
Calcitriol induces cell cycle arrest through the expression of insulin growth factor binding 
protein-3 (IGFBP-3), which increases the levels of the cell-cycle inhibitor p21 in LNCaP prostate 
cancer cells (17); calcitriol also activates expression of IGFBP-3 by direct transcriptional 
stimulation through a vitamin D response element (VDRE) located in its promoter sequence 
(18). VDRE sequences have also been demonstrated in the promoter of the tumor growth factor 
beta (TGFβ)-2 gene (19), and treatment of PC-3 prostate cancer cells with TGFβ leads to 
enhanced expression of IGFBP-3 and subsequent growth arrest and apoptosis (20). Additional 
studies have suggested a link between chronic inflammation and prostate cancer (21). Vitamin 
D suppresses the expression of cyclooxygenase-II (COX-2), the key enzyme for the synthesis of 
prostaglandins, mediators of inflammation and thought to be important for cancer progression 
(22); COX-2 expression in biopsy cores and prostate cancer surgical specimen is an 
independent predictor of recurrence (23). Furthermore, there is considerable evidence that 
calcitriol inhibits nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling, 
and decreases the levels of the angiogenic and pro-inflammatory cytokine interleukin-8 (IL-8) in 
prostate cancer cells (24). NFκB is a transcription factor that plays a central role in the control of 
inflammation and is expressed at high levels in prostate cancers with high Gleason scores (25). 
This is only a very limited list of the many molecular pathways and mechanisms affected by 
vitamin D, as it is now well established that VDR may recognize cognate VDRE present within 
the regulatory sequences of hundreds of human genes, implicating vitamin D in a vast network 
of gene regulation, and underlying its broad physiological actions (26, 27). The recent emphasis 
on non-skeletal functions of vitamin D has to do with the realization that vitamin D deficiency 
has major implications for human health in general, and cancer biology in particular (32). 
Hypovitaminosis D in African American Populations: Exposure of skin to sunlight in the 
ultraviolet B (UVB) range of the spectrum (290 to 315 nm) results in the photolytic conversion of 
7-dehydrocholesterol to previtamin D3, which is transformed to vitamin D3 (cholecalciferol) by 
thermally induced isomerization (28, 29). Vitamin D3 can be obtained from the diet; however, it is 
distributed very poorly in natural foodstuffs. Dark skin pigmentation, due to melanin, probably 
evolved in equatorial regions to protect individuals from skin cancers. Increased skin 
pigmentation, however, limits one’s ability to produce vitamin D3 (30). Vitamin D deficiency 
occurs when serum levels of 25(OH)D are at <50 nmol (<20 ng/mL); as a result, a majority of 


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African-Americans are vitamin D-deficient (4). We recently published the results of a prospective 
clinical study aimed at examining the effects of vitamin D3 supplementation at 4,000 IU per day 
for two months in male subjects who had selected surgical removal of the prostate 
(prostatectomy) as a definitive treatment for their prostate cancer. According to current standard 
of care, a two-month interval between biopsy and prostatectomy is needed to resolve the 
inflammation due to the biopsy procedure. Moreover, the initial two months of vitamin D3 
supplementation register the fastest rise in serum levels of 25(OH)D, as reported by us (31,32) . 
This prostatectomy study was aimed at investigating the effects of vitamin D3 supplementation 
(4000 IU per day for two months) on prostate tissue specimens obtained at surgery, by means of 
RNA-seq technologies, with special emphasis on differential gene expression in African 
American men compared to Caucasian men, and between supplemented and placebo groups 
(33). This paper is available in PDF format as Appendix 1. 
We set Caucasian subjects (17 samples) as the control and African American subjects (10 
samples) as the test. The results of our analyses showed that 3107 genes were differentially 
expressed using a stringent (highly significant) FDR<0.1, and 8237 genes were differentially 
expressed using a less stringent (still significant) FDR<0.4. Pathway analysis using the 3107 
differentially expressed genes highlight major differences between the two groups, with African 
American showing higher expression of genes associated with immune response and 
inflammation. It must be noted that our observations are consistent with a previous report (34) 
based on the analysis of prostate tissue-derived RNA, performed using Affymetrix microarrays. 
This analysis revealed enhanced expression - in tissue samples from African American subjects 
- of biological pathways that are linked to autoimmune diseases, allergies, and inflammatory 
diseases. Even at this preliminary stage the results of our RNA-seq studies support the 
existence of considerable biological differences within the prostate between African American 
and Caucasian men and suggest that overexpression of genes linked to the inflammatory 
process may very well contribute to the increased severity and faster progression of prostate 
cancer in African American men, even at the early stage of disease (33). 
Stress Response and Allostatic Load:  
Allostatic load defines a set of clinical and biochemical parameters that can be easily 
measured in a basic clinical setting (35). These parameters can be analyzed to gauge the level 
of stress experienced by each subject. These values reflect chronic, steady state levels of 
stress as well as failure to shut-off responses to acute stressors. To the best of our knowledge, 
there is only one published report of a correlation between allostatic load in African American 
subjects and specific biomarkers (36). This report identified the dopamine receptor gene, DRD4, 
and the serotonin transporter gene, SLC6A4, as indicators of stress, because of a non-
supportive family environment. Interestingly, both of these genes were very significantly up-
regulated (at a FDR<0.1) in prostate tissue samples from African American men. These 
considerations suggest that the prostate appears to be, even at the molecular level, a “sentinel” 
organ for chronic stress.    
The results of our RNA-seq analyses are consistent with our study objectives. These 
preliminary indications make us confident that the acquisition of additional RNA-seq data will 
allow us to strengthen the significance of our findings and help us identify specific pathways for 
possible targeted intervention.   
Genomic technologies can be easily applied to the prostate tissue samples obtained from 
eligible subjects, undergoing prostate biopsy as part of their standard medical care. The results 
of our preliminary studies have already shown significant differences in the transcription profiles 
in prostate tissue samples between African American and Caucasian men. In addition, special 
emphasis will be given to RNA/seq analyses of Hypothalmic-Pituitary-Adrenal (HPA) -related 
transcription profiles, measurements of allostatic load and comparison of these profiles by race. 
 
 
 


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4.0 
Study Endpoints 
            We will be investigating Veterans who are undergoing prostate biopsy.  If the Veterans 
are eligible and seek active surveillance as their treatment modality, they will be offered 
an opportunity to receive 4,000 IU Vitamin D supplementation as per previous protocol.  
Upon repeat biopsy (standard of care), they will have repeat RNA-seq analyses which will 
serve as further internal control. We will be measuring serum prostate specific antigen 
(PSA) as per standard of care recommendations. A blood sample for ancestry markers 
will be collected at the conclusion of the study.   
Primary clinical endpoint: We will be assessing for clinical activity based on PSA changes, 
and investigating whether or not vitamin D supplementation results in a 20% decrease 
from baseline PSA. 
 
5.0 
Inclusion and Exclusion Criteria/ Study Population 
 Males only, due to the limitation of the study to prostate tissue 
            Part 1 
Inclusion Criteria 
a) Age: 50 - 75 years of age 
b) Scheduled to undergo a standard of care prostate biopsy because of an elevated 
PSA, an abnormal DRE (digital rectal exam) or other diagnostic procedure. 
c) Permission for investigators to follow subject’s post biopsy diagnosis, treatment 
decision and follow/up care (including subsequent prostate biopsies). 
Exclusion criteria: 
Inability or unwillingness of subject to give informed consent 
Part 2 
Inclusion Criteria 
a) Prostate biopsy pathology report with diagnosis of prostate cancer 
b) Treatment recommendation and/or subject decision of active surveillance 
c) Agreement to take vitamin D3 4000 IU daily for approximately one year 
d) Standard of care repeat PSA at six months and surveillance (repeat) prostate 
biopsy after one year 
Exclusion Criteria 
a) Initial testing of prostate biopsy core sample indicates non-viable RNA for further 
sequencing. 
b) Current vitamin D supplementation > 2000 IU daily 
c) Inability or unwillingness to continue as a participant in the study 
 
 
6.0 
Number of Subjects 
            In an ongoing RNA-seq study of prostate tissue, 28 % of the enrolled subjects had  
pathology results of prostate cancer with the potential of active surveillance as the 
treatment option. Therefore, for this study, 250 African American and Caucasian males 
will enroll in Part 1 of the study with the goal of 60 African American and Caucasian 
males participating in Part 2.  There may be less than 250 subjects in Part 1 if Part 2 
accrues more quickly. 
 
7.0 
Setting 
• Subject recruitment/enrollment will be conducted at the Ralph H. Johnson VA Medical 
Center’s Urology Clinic and Research Offices/Clinics.  
• The allostatic load blood analysis will be conducted by the Ralph H. Johnson 
Laboratory Services and MUSC’s Laboratory Services 
• The Vitamin D serum analysis will be conducted by Dr. Bruce Hollis’ Laboratory at 
MUSC.  


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• RNA purification from biopsy tissue specimens will be performed at the Hollings 
Cancer Center Biorepository and Tissue Analysis Shared Resource. The RNA-seq 
will be performed by Gerard Hardiman, PhD Genomics Core Laboratory, Queens 
University Belfast, Ireland. 
8.0 
Recruitment Methods 
• Potential subjects will be identified by the VAMC Urology Clinic schedule for prostate 
biopsies. A Recruitment letter from the Principal Investigator may be mailed to those 
potential subjects that may qualify for the study. 
• Potential subjects may be identified by the Urology Provider when they have their 
preliminary Urology Clinic visit and the prostate biopsy is scheduled. Study 
introduction may be given to the potential subject at that time.  
• If a progress note by the Urology Provider has been entered in the electronic record 
that the study has been introduced and then forwarded to the Research Coordinator, 
the Coordinator will then contact the potential subject before the biopsy date to  
answer any questions concerning the research study. 
• The study may also be introduced to potential subjects by their Urology Provider just 
prior to the scheduled prostate biopsy. If the potential subject states he is interested in 
participating, he will be introduced to the Research Coordinator by the Urology 
Provider for further information and potential enrollment. 
• The Research Coordinator(s) will meet the potential subject in the Urology Clinic or 
assigned research offices/space before their scheduled prostate biopsy to first obtain 
consent and then initiate study procedures. 
 
9.0 
Consent Process 
             Part 1 
• Only patients who are not cognitively impaired will be asked to participate. 
• When possible, the individual will be introduced to the study by their Urology Provider 
when the prostate biopsy is recommended and scheduled. 
• The Health Care Provider will then indicate in the Progress Note that the patient has 
been introduced to the study and the Research Coordinator will receive a message to 
contact the patient. 
• The patient will be contacted by the Research Coordinator to explain further the 
research requirements and answer any questions. The patient can then indicate 
whether he is willing to participate in the study. 
• The study may also be introduced to potential subjects by their Urology Provider just 
prior to the scheduled prostate biopsy. If the potential subject states he is interested in 
participating, he will be introduced to the Research Coordinator by the Urology 
Provider for further information and potential enrollment. 
• The consent process (time to read/review the consent and HIPAA and ask questions) 
will be conducted in a private exam room in the Urology Clinic or in assigned research 
offices/space. 
• The consents will be signed only when the patient states that he understands the 
study procedures, has no questions, and is willing to participate. 
Part 2 
• Once a subject has (a) had a follow-up appointment with the Urology Provider to  
review the prostate biopsy pathology report, (b) received a diagnosis of prostate 
cancer, (c) reviewed the treatment options and (d) decided on active surveillance, the 
Research Coordinator will contact the subject for participation in Part 2. At that time, 
the subject will be asked if he wishes to continue with the study. If yes, Part 2 
procedures will be initiated. If no, the subject will be released from further 
participation. 


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10.0 
Study Design / Methods 
Although some understanding of the race-specific and vitamin D-driven biochemical 
mechanisms and pathways affecting prostate cancer (37) have been demonstrated, the main 
objective of this proposal is to fill existing gaps in knowledge by identifying those mechanisms 
and pathways that are especially relevant to explain vitamin D effects on prostatic cells and 
tissues, as well as on prostate cancer disparities between African American and Caucasian 
men. The proposed studies will employ advanced technologies to analyze transcriptional 
profiles and biological pathways in prostate tissue specimens from male subjects undergoing 
prostate biopsy, and will involve studies of gene expression profiles, derived from RNA-seq 
data. Comparison of these transcriptional and biological profiles between African American and 
Caucasian subjects will allow us to identify the critical transcription profiles in the prostate 
affected by race. Additionally the potential variations related to vitamin D levels (before and after 
supplementation) as well as serum ancestry markers will be explored. 
 
Objective 1. To utilize RNA-seq analyses to further demonstrate and identify a 
specific stress pattern signal that exists between black and white men who are 
undergoing prostate biopsy.  We have demonstrated that transcriptional profiles and 
biological pathways in the prostate are significantly different between African American and 
Caucasian subjects. The results of these studies will allow us to further elucidate the molecular 
underpinnings of health disparities in prostate cancer, including identification of specific stress 
patterns. These studies will involve purification of ribonucleic acid (RNA) from prostate tissue 
specimens obtained at prostate biopsy, RNA sequencing (RNA/seq), analyses of transcription 
profiles, and comparison of these profiles across race by advanced bioinformatics tools. 
 
Patient Cohort: As discussed in the Backround section, a substantial amount of information 
has already been collected through the pilot prostatectomy study as well as the initial aspects of 
this study. We have invested considerable resources to procure and analyze clinical samples 
obtained through our ongoing as well as previous research. This proposed study will leverage 
these investments and continue procuring and analyzing new samples that are annotated and 
linked with social determinants and allostatic load variables.  
Male subjects (up to 250 in Part 1) will be enrolled in this study when they have their 
scheduled prostate biopsy. Pathology results at enrollment will be unknown. However, they will 
be followed to obtain data as to the biopsy results, treatment options, if recommended, for 
definitive treatment and/or management. For this project, in Part 2, sixty eligible subjects will be 
enrolled. Therefore, a total of 60 prostate tissue samples will be analyzed at baseline and again 
at study exit (repeat prostate biopsy). We anticipate that approximately 20 African American 
subjects will be enrolled in the study, since approximately 40% of the patients seen in the 
Urology Clinics of the Ralph H. Johnson VA Medical Center in Charleston are African American.  
As shown under Preliminary Studies, major differences in transcription within the prostate were 
observed between African American and Caucasian subjects enrolled in the pilot study (33).  
 
Tissue Sample Procurement: The PI will oversee the procurement of prostate tissue core 
specimens. Specifically, he will orchestrate the single added core sample from a peripheral area 
of the prostate, during the enrolled subjects’ prostate biopsy. This will ensure that the excision of 
tissue samples does not interfere with the diagnostic priorities.  
 
RNA Purification, Sequencing, and Analysis of Transcription Profiles: This project will rely on 
several facilities. The Hollings Cancer Center Biorepository and Tissue Analysis Shared 
Resource will purify total RNA from each de-identified tissue sample using standard procedures. 
RNA integrity will be verified using RNA 6000 Nano Assay chips run in Agilent 2100 Bioanalyzer 


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(Agilent Technologies, Palo Alto, CA). High-quality RNA will be divided into two de-identified-
coded samples and forwarded for storage to the Principal Investigator’s Laboratory at the 
Charleston VAMC. Only one of the two split samples will be bulk shipped to Rick Kittles, PhD 
Genomics Core Laboratory at City of Hope Comprehensive Cancer Center in Duarte, CA. for 
RNA-sequencing. 100-200 ng of total RNA will be used to prepare RNA-Seq libraries using the 
TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA) following the protocol described by the 
manufacturer. Paired end cluster generation will be performed on the cBot as described by the 
manufacturer (Illumina, San Diego, CA). Clustered RNA-seq libraries will be paired end 
sequenced with 250 cycles on a HiSeq2500 (Illumina, San Diego, CA). This new instrument 
acquired recently by MUSC generates up to 1 Tb per run, with data yields typically containing 
80% greater than Q30, 2 x 100 bp read length. The “paired end” approach will be employed for 
the RNA-seq experiments, wherein a single long read is sequenced for 100 nucleotides at both 
ends. In addition to providing digital mRNA profiling, this strategy will permit tracking alternate 
splice junctions, insertions and deletions, and facilitate transcriptome assembly in these prostate 
cells and experimental groups.  
Each sample will be sequenced to a depth of 100 million reads. Adapters will be trimmed by 
Trimmomatic (38). Alignment and annotation will be performed using DEseq2 (39), Edge R (40, 
41) and the Tuxedo Suite comprising Bowtie2, Tophat2 and Cufflinks2 (42). As we are 
interested in applying RNA-seq as a discovery tool, the Tuxedo Suite will facilitate the 
identification of alternative splicing events, allele-specific expression, and rare and novel 
transcripts in these mRNA data sets. 
 
Sample Size Estimation: The sample size of this study is 250 (Part 1) and 60 (Part 2) based 
on practical considerations and reasonable statistical power. Our inferences will be based on 
the determination of threshold on p-values to achieve a false discovery rate (FDR) of 10%.  We 
anticipate that the large majority of genes will not be significant, but do not know the p-value 
threshold; we expect it to be between 0.001 and 0.01. 
 
The primary endpoint is to estimate PSA reduction between pre and post treatment. We expect 
20% PSA reduction post-treatment with the standard deviation of 40%. With 60 subjects , the 
95% confidence interval for the estimated PSA reduction is 10% to 30%.   
 
Data Analytic Approaches: Fold-change (FC) estimation and hypothesis testing for 
differential expression will be performed using the DESeq2 Bioconducter library (43, 44, 39). 
DESeq2 models read counts as arising from a negative binomial distribution using a generalized 
linear model (GLM) approach that facilitates effect estimation of experimental design factors. 
DESeq2 conducts gene-specific comparisons between groups based on model-based linear 
contrasts, with inference based on associated two-sided Wald tests. For each gene, DESeq2 
reports estimated FC, a p-value, and a q-value (45-47) equivalent to the smallest false 
discovery rate (FDR) incurred when declaring that test significant. We will control the FDR at 
10%, but will consider less conservative FDR thresholds (e.g. 15% or 20%) if the number of 
significant genes is too small (e.g. <20). We will construct volcano plots – scatterplots of –
log10(p-value) versus log2(FC) – to identify genes strongly associated with race identification or 
circulating levels of Vitamin D with high statistical significance. Additionally, we will analyze read 
counts using edgeR (41) to confirm DESeq2 results. Finally, we will utilize Cufflinks to assemble 
the alignments into a parsimonious set of transcripts (42). This analysis will complement the 
approach described above and provide further validation of differentially expressed mRNAs.  
 
Analysis of Cumulative Molecular Data: The cumulative data will be analyzed to identify 
race-associated differences between tissue samples from African American and Caucasian 
subjects. Using the list of differentially expressed genes from Objective 1, regression 
coefficients, t-statistics and statistical significance (p-values) will be saved and volcano plots will 


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be generated. Initially, unsupervised clustering (through hierarchal clustering) will be performed 
to identify sets of genes and proteins that tend to cluster by race and treatment as an 
exploratory approach. Furthermore, supervised machine learning techniques (random forests) 
will be used to identify genes/proteins that are associated with differential patterns by race to 
complement the analysis described above and allow us to interrogate pathways in a systematic 
way. These results can be combined with RNA-seq results using gene symbols, and additional 
enrichment analysis (e.g., KEGG, gene set enrichment analysis - GSEA) of each set separately, 
and combined. This will be accomplished through a combination of Ingenuity Pathway Analysis 
(Qiagen) and the ToppGene Suite (48).    
 
Anticipated Results and Alternative Approaches: Prostate tissue specimens, collected 
prospectively, will be processed for RNA-seq. The methods to accomplish this are already 
established. The breadth of data to be generated will be large and unprecedented for Vitamin D 
and health disparity research studies. We have established data analysis methods, and these 
will continue to evolve as more samples are analyzed. We expect that there will be significant 
differences in gene expression by race as demonstrated by the results of our preliminary studies 
(33). Linking differences to specific transcription profiles, in order to identify molecular correlates 
of health disparities, is expected to require a merged analysis of all observed changes. This will 
no doubt require continued refinement of approaches to effectively interpret these data.  
 
Objective 2. To identify changes in molecular signature that exist in African American 
and Caucasian men in relation to their vitamin D levels.  This study will involve RNA/seq 
analyses of prostate tissue in African American and Caucasian Veterans.  Previous studies 
have demonstrated varying molecular signatures that have existed both in relation to race and 
vitamin D levels.  We will further explore this relationship to strengthen and refine the findings.   
 
Precision medicine can play an important role in reducing racial disparities in morbidity and 
mortality among minority men because these efforts are designed to individualize health care 
based on biological, behavioral, and social factors that contribute to disease risks and enhance 
health outcomes. However, the development and implementation of approaches for precision 
medicine is still limited among minority men because empirical data are lacking on the ways in 
which risk factors and protective variables work on this underserved population that often relies 
for its health care needs more on small outpatient clinics than advanced teaching hospitals. This 
represents a significant gap that prevents this underserved population from taking advantage of 
what precision medicine can offer to minority men.  
 
Allostatic Load Studies: These studies attempt to address this significant gap by proposing a 
research approach that allows for the extension of precision medicine benefits to an important 
segment of African American men: those diagnosed with prostate cancer. Stress response is 
often used to describe the body’s attempt to adjust physically and psychologically to challenging 
conditions that are often perceived as adverse. The proposed studies will correlate relevant 
health parameters of African American subjects undergoing prostate biopsy with the molecular 
profiles obtained from prostate tissue samples. This objective is consistent with increasing 
evidence that pro-inflammatory mechanisms and the HPA axis are key mediators of the 
“allostatic load” and the results of our preliminary studies emphasize the transcriptional up-
regulation of many genes associated with immune response and inflammation in prostate tissue 
samples of African American men.  
Allostatic load parameters will be measured when subjects progress to Part 2 of the study. 
Allostatic load parameters selected for this study are the following: 
• 
Systolic and diastolic blood pressure (indices of cardiovascular health) 
• 
Waist-hip ratio (index of adipose tissue deposition associated with increased 
glucocorticoid activity) 


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• 
Serum high-density lipoproteins (HDL) lipid panel (index of atherosclerosis) 
• 
Blood plasma levels of glycosylated hemoglobin (HbA1C, index of glucose 
metabolism over time) 
• 
Serum dihydroepiandrosterone sulfate (DHEA-S) a functional HPA axis antagonist 
• 
C-Reactive Protein and IL-6 (indices of inflammation) 
Collections of overnight urinary output for measuring cortisol, epinephrine, and 
norepinephrine excretion levels will not be included in this study, since the outcome of the 
prostate biopsy would obviously interfere with the second collection. 
For each of the chosen parameters, subjects will be classified into quartiles, based on the 
distribution of scores. Allostatic load will be measured by summing the number of parameters 
for which the subject will fall into the highest risk quartile, with the exception of HDL and DHEA-
s values, for which the lowest quartile corresponds to the highest risk (35). 
We anticipate that we will observe significant differences in allostatic load across races. We 
expect that these differences will correlate with differences in transcriptional profiles from 
prostate tissue sample from African American men compared to prostate tissue sample from 
Caucasian men.  
 
    Objective 3.  To assess changes in RNA-seq profiles as they pertain to serum ancestry.  
Increasing evidence has shown the variability in genetic predisposition and permissiveness to a 
variety of illnesses and cancers.  Our initial findings exhibit significant variations as they relate to 
race.  Given the genetic heterogeneity that can exist in race, we seek to further explore these 
differences as they relate to genetic ancestry as determined through serum profiles.  Our patient 
populations includes the Sea Island population which typically has higher percentages of pure 
African ancestry which can help to investigate these possibilities.  
 
 
11.0  Study Related Procedures/Specimens  and Risk Prevention 
Part 1  
 
1) The collection of an extra prostate tissue (punch) core from the peripheral area of the 
prostate 
• 
Will be collected during the Standard of Care prostate biopsy by the Urologist 
• 
Will be collected only with the subject’s permission (signed Informed Consent) 
• 
Will not significantly create more risk to the subject than the standard 12 (punch) 
core collection incurred during the routine procedure 
• 
The extra collection will not interfere with the routine diagnostic process 
• 
Subjects will receive standard post-procedure instructions to minimize discomfort 
and risk as standard of care. 
2) Electronic medical chart review will be performed by a Research Coordinator 
• To verify demographics: age, race, ethnicity 
• To verify pathology results from the prostate biopsy 
• To ensure that the subject receives follow-up care in the Urology Clinic by alerting 
Urology Clinic personnel if a subject does not attend scheduled follow-up appointments. 
• To follow subject’s post-biopsy diagnosis, treatment decision and follow/up care 
(including potential collection of prostate tissue during subsequent prostate biopsies). 
• To determine if subject is eligible to proceed to Part 2 of the study 
• All data collected will be entered into the study database located on the VA secure 
server (V:) by a Research Coordinator and will be with the assigned codes. 
Part 2 
1) Blood for measuring serum concentration of 25(OH)D3.  
• 
Obtained with other required bloodwork by an experienced phlebotomist or R.N. 
using sterile supplies and clean technique. 


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• 
Will be processed, labeled with an assigned code, and stored in -20º freezer located 
in the VA Mental Health Research Building – Lab DD159 for approximately 1-4 
weeks before it is transported (refrigerated) by the Research Coordinator to Bruce 
Hollis’ Laboratory at MUSC. 
• 
Any remaining serum will be destroyed at the end of the study. 
• 
Shared results will be with the assigned codes. 
2) Blood for allostatic load (lipid panel, HgA1C, DHEA-S, C-Reactive Protein and IL-6) 
• 
Obtained before the biopsy with other study required bloodwork by an experienced 
phlebotomist or R.N. using sterile supplies and clean technique 
• 
Must be labeled with the subject’s name and VA registration number, as per VA 
Laboratory Services regulations. Results will be processed by the VA Laboratory 
Services and entered into the VA CPRS (electronic record).  
• 
After analysis, the VA Laboratory will not store any remaining blood samples. 
• 
Results will be entered into the research study data base with the assigned code by 
a Research Coordinator.  
• 
Shared results will be with the assigned codes. 
3) Allostatic load measurements: SBP/DBP (systolic and diastolic blood pressure), hart rate 
and waist/hip ratio are non-invasive measurements 
• 
SBP/DBP and heart rate will be measured in the Urology Clinic prior to the prostate 
biopsy by either the Research Coordinator or Urology Clinic staff, and recorded in 
the VA CPRS electronic record as per Urology Clinic protocol prior to this Standard 
of Care procedure. 
• 
The waist/hip ratio will be obtained using a designated tape measure by the 
Research Coordinator and recorded. 
• 
These data will be entered in the study spreadsheet with the assigned codes.  
4)  A survey (Social Determinants) will be given to each subject with an instruction sheet and 
stamped, addressed return envelope to be completed at home after their prostate biopsy.  
• 
The survey will be completed by subjects at home after their biopsy.  
• 
The survey will be identified by each subject’s assigned code. 
• 
The survey will be mailed back to Dr. Chanita Hughes-Halbert, VA HEROIC 
Investigator, for analysis and storage.  
• 
Within 21 days if the survey has not been returned to the designated research staff, 
a research assistant will contact the subject to assess if they have completed the 
survey, or need additional assistance. 
• 
Subjects will also be given the option to have the survey administered by telephone 
by a trained research assistant if they are having issues with self-administering the 
survey. 
• 
Contact information for mailing the compensation gift card or verifying that the 
compensation has been received, will be destroyed once receipt of the gift card is 
documented. 
• 
At the end of the study, these surveys will be boxed, labelled and sent to storage per 
VA Office of Research and Development’s (ORD) Records Control Schedule. 
5) Subjects will begin vitamin D3 supplementation of 4000 IU daily. 
• 
Previous studies have demonstrated no safety concern with this dose of vitamin D3 
supplementation. 
• 
According to the Endocrine Society and Institute of Medicine standards, a vitamin D 
blood level between 30-50 ng/mL is sufficient. Taking 4,000 IU of vitamin D3 daily will 
not exceed that level in the blood. Therefore, this research study amount of 4,000 IU 
daily should not produce any risk.   
• 
Preliminary studies demonstrated evidence of decreased positive biopsy cores after 
one year of supplementation at 4000 IU daily 


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• 
RNA-seq analysis has demonstrated improvement in inflammatory profiles in 
prostate tissue. 
6) Electronic medical chart review will be performed by a Research Coordinator 
• 
To verify demographics: age, race, ethnicity 
• 
To verify serum levels of PSA (prostate specific antigen) and pathology results from 
the prostate biopsies 
• 
To collect results of the study’s allostatic load measurements and vitamin D serum 
levels 
• 
To ensure that the subject receives follow-up care in the Urology Clinic by 
scheduling the standard of care six month repeat PSA clinic visit and one year 
repeat PSA and prostate biopsy. 
• 
All data collected will be entered into the study database located on the VA secure 
server (V:) by a Research Coordinator and will be with the assigned codes. 
7) Collection of Ancestry Markers serum sample at the final visit of the study 
• 
Obtained with other required bloodwork by an experienced phlebotomist or R.N. 
using sterile supplies and clean technique. 
• 
Will be labeled with an assigned code. 
• 
Will be stored in assigned research space in the VA Mental Health Research 
Building for batch shipment to City of Hope Comprehensive Cancer Center in 
Duarte, CA for analysis 
• 
Subjects will not receive results from this analysis 
• 
Any remaining serum will be destroyed at the end of the study. 
• 
Shared results will be with the assigned codes. 
 
8) The collection of an extra prostate tissue (punch) core from the peripheral area of the 
prostate during the repeat prostate biopsy  
• 
Will be collected during the Standard of Care prostate biopsy by the Urologist 
• 
Will be collected only with the subject’s permission (signed Informed Consent) 
• 
Will not significantly create more risk to the subject than the standard 12 (punch) 
core collection incurred during the routine procedure 
• 
The extra collection will not interfere with the routine diagnostic process 
• 
Subjects will receive standard post-procedure instructions to minimize discomfort 
and risk as standard of care. 
 
12.0 
Data Management  
The study database spreadsheets 
• 
The databases will be maintained in the VAMC Charleston secure (V:) drive and 
accessed only by the Research Coordinator(s) and the PI. 
• 
All data entered will be with the assigned codes. 
• 
All shared data will be with the assigned codes. 
 
Sample Size Estimation: The sample size of this study is 250 (Part 1) and 60 (Part 2) based on 
practical considerations and reasonable statistical power. Our inferences will be based on the 
determination of threshold on p-values to achieve a false discovery rate (FDR) of 10%.  We 
anticipate that the large majority of genes will not be significant, but do not know the p-value 
threshold; we expect it to be between 0.001 and 0.01. This sample size estimation is consistent 
with the data analytic approaches discussed under Study Design. 
 
The primary endpoint is to estimate PSA reduction between pre and post treatment. We expect 
20% PSA reduction posttreatment with the standard deviation of 40%. With 60 patients, the 
95% confidence interval for the estimated PSA reduction is 10% to 30%. 


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Statistical Analysis  
All statistical analyses will be performed using SAS v. 9.4 or R statistical analysis packages. 
The overall PSA reduction rate and the corresponding 95% confidence interval and percentage 
of each core from 12 cores will be estimated. Basic descriptive statistics will be estimated for 
demographic at pretreatment and clinical variables (blood pressure, weight, hips and waist 
circumference, vitamin D level, blood sugar, cholesterol, kidney function and stress hormones)  
at pre and post treatment. 
 
13.0 
Provisions to Monitor the Data to Ensure the Safety of Subjects 
This study requires an initial visit (Part 1): consent process plus collection of an extra prostate 
biopsy tissue core for study analysis. 
If the pathology report indicates prostate cancer and the subject’s treatment decision is active 
surveillance, he may continue participation to Part 2. Data collected in this phase will be: 
demographics, blood pressure, heart rate, hip/waist ratio, allostatic load blood results, one 
survey, a repeat prostate biopsy core collection, vitamin D levels, PSAs, RNA-seq of prostate 
core samples, and serum ancestry markers. 
All samples will be labeled with the assigned de-identified code (except for samples analyzed by 
the VAMC laboratory services which require name and last four of the social security 
number).VAMC laboratory results will be entered into the research database with the assigned 
code. All shared data will be under the assigned codes. 
 
During each study visit, an adverse or unanticipated study-related event is unlikely, but may 
occur. Therefore, if an adverse event occurs, it will be reported as required to the Institutional 
IRB, VA R&D, and all other oversight committees and organizations. The Attending Urologist in 
the VA Urology Clinic at the time of the event will evaluate the subject and the event and 
schedule follow-up medical intervention as needed. 
 
If, upon review of the study required blood results, an abnormal result is reported by one of the 
laboratories, the subject’s primary health care provider will be notified via the VA CPRS 
electronic records system for evaluation, possible treatment and follow-up. 
 
14.0 
Withdrawal of Subjects  
Participation in Part 1 of this study may be a single study visit, or if the subject agrees to another 
prostate core sample obtained as standard of care approximately 12 to 18 months after his 
initial prostate biopsy, participation will be two study visits. Participation in Part 2 will include an 
initial visit (when inclusion criteria are met), at six months for PSA and vitamin D level and a final 
visit for repeat prostate biopsy, allostatic load measurements, vitamin D level and ancestry 
marker. Subjects may withdraw their decision to participate at any time after signing the consent 
forms. This includes the use of any specimens that are not already exhausted by laboratory 
analysis. 
 
15.0 
Risks to Subjects 
1. Venipuncture: the risks of drawing blood include temporary discomfort from the needle stick 
and bruising, fainting could occur. 
2. Collection of an extra prostate tissue (punch) core from the peripheral area of the prostate 
will not significantly create more health risk to the subject than the standard 12 (punch) core 
collection incurred during the routine procedure. Prostate biopsies are done under local 
anesthesia, and the extra prostate biopsy may cause 7% more discomfort as a result. The 
collection from this area will not interfere with the Pathologist’s analysis and diagnostic 
process. 


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3. Loss of Confidentiality of protected health information (unlikely side effect). A Master List 
with contact information is required as Good Clinical Practice for dissemination of new 
information and follow-up care. This Master List will be maintained by the Research 
Coordinator(s) on the VA secure (V:) drive. Only the Research Coordinator(s) and the PI will 
have access to this list. The Master List will also serve as the link in identifying each subject 
to his assigned code. 
4. Taking vitamin D3 supplementation at 4000 IU daily poses no risk to the subjects. This dose 
is well within the recommended doses by the Endocrinology Society and Institute of 
Medicine. 
5. Some questions in the Social Determinants survey may make subjects uncomfortable or 
upset. Subjects will be instructed to skip any questions that they do not wish to answer. 
6. Genetic Research. The RNA-sequencing will analyze transcriptionaI profiles and biological 
pathways on the molecular level in prostate tissue. It is not identifying specific genes and an 
individual’s predisposition to a particular diagnosis or disease process. The ancestry 
markers will be analyzed for racial origin/significance only. No DNA analysis for disease 
identifiers will be performed. 
 
16.0 
Potential Benefits to Subjects or Others 
A direct benefit may not be gained by the participants. However, if measuring gene 
expression in prostate tissue specimens provides molecular biomarker(s) for assessing the 
effects of circulating levels of vitamin D on cancer, it will be extremely important in 
determining treatment options for future cases of prostate cancer at time of diagnosis. 
Preliminary studies have demonstrated evidence of decreased positive biopsy cores after 
one year of supplementation at 4000 IU daily. Also, RNA-seq analysis has demonstrated 
improvement in inflammatory profiles in prostate tissue in men supplemented with vitamin 
D3. 
 
17.0 
Sharing of Results with Subjects 
• 
The allostatic load laboratory results analyzed in the VAMC laboratory will automatically 
be entered into the VA electronic record and can be viewed by each participant’s Health 
Care Provider and shared as needed with the subject. 
• 
Vitamin D serum levels (baseline, six months and exit) will be reported to the subject. 
The subject’s Primary Health Care Provider will be notified for follow-up as needed at the 
end of Part 2. 
• 
The blood pressure, heart rate and waist/hip measurements will be shared with each 
subject when they are obtained. 
• 
The survey is self-completed and each subject will know the information his contains. 
• 
The RNA-seq analyses will be published at the completion of the study. All results will 
remain de-identified. 
• 
The ancestry markers will not be shared with the subjects. 
 
18.0 
Drugs – vitamin D supplementation 
FDA IND# 77,839 was originally granted to Dr. Sebastiano Gattoni-Celli in 2007 for an open-label 
clinical study designed to assess the effect of vitamin D3 on subjects with early-stage prostate cancer. 
This clinical trial will be conducted under the same IND, which has been transferred to the PI, Dr. 
Stephen Savage. The vitamin D3 (cholecalciferol) is manufactured by JR Carlson Laboratories, Inc., 15 
College Ave., Arlington Heights, IL 60004-1985. JR Carlson Laboratories will manufacture soft gel 
capsules containing 4000 IU of vitamin D3 for the study. The study medication will be shipped to the 
research pharmacy of the Ralph H. Johnson VA Medical Center where it will be stored and dispensed 
as per standard procedure.  
 
 


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Appendix 1 
 
Flowchart of Study Procedures and Timeline 
 
Procedure 
Initial 
Prostate 
Biopsy 
Clinic Visit 
Baseline 
Six 
Months 
Approximately 
1 year 
Comments 
Part 1: 
 
 
 
 
Standard of Care prostate 
biopsy 
Consent 
process 
X 
 
 
 
 
13th core 
prostate tissue 
sample 
 
X 
 
 
 
X 
PSA is routinely determined 
before a prostate biopsy is 
scheduled 
 
 
Part 2: 
 
 
 
 
Subject has had follow-up 
appointment with Urology 
Provider and has decided on 
active surveillance as treatment 
option and meets inclusion 
criteria 
Reaffirm 
Consent 
 
X 
 
 
 
Allostatic load 
measurements! 
 
 
X 
 
 
X 
 
Allostatic load 
serum samples2 
 
 
X 
 
 
X 
 
Vitamin D level 
 
 
X 
 
X 
 
X 
 
PSA 
 
 
 
 
X 
 
X 
PSA not collected at baseline. 
PSA will be elevated due to the 
recent biopsy procedure. 
Ancestry 
markers 
 
 
 
 
X 
 
Dispense 
vitamin D3 4000 
IU bottle 
 
 
X 
 
X 
 
 
Collect vitamin 
D3 
bottle 
 
 
 
X 
 
X 
 
13th core 
prostate tissue 
sample 
 
 
 
 
X 
 
 
1 Blood pressure. heart rate, waist/hip ratio 
2 Lipid panel, HgbA1c, Comprehensive Metabolic Panel, CRP, IL-6 and DHEA-S 
 

ICF_000.pdf: 
 
Department of Veterans Affairs 
Ralph H. Johnson VA Medical Center 
Research Consent Form 
Version Date: 09/20/2022 
Page 1 of 10
Subject’s Name:   
Date:   
Principal Investigator:  Stephen J. Savage, MD 
Study Title: Vitamin D supplementation in RNA-seq profiles of single core prostate biopsy samples, 
including diversity and stress determinants, among Veterans.                                  (IRB # 85140)
 
IRB Number:85140 
Date Approved «ApprovalDate» 
 
  VA FORM
MAR 2006 10-1086 
SUMMARY: 
 
You are being asked to volunteer for a research study. Research studies are voluntary and include 
only people who choose to take part. This is a research study to find out if taking vitamin D3 for a year 
has any effect on the molecular make-up of prostate tissue over time. Participants will agree to have 
an extra prostate tissue core collected during a routine prostate biopsy for the molecular analysis.  
Your pathology report will be reviewed by your Urology physician. If it indicates prostate cancer and 
you decide, after discussion with your Urologist, that you will follow the plan of “active surveillance” 
(no immediate active treatment), you will be asked to participate in the second phase of the study. 
This includes three study visits that will be scheduled six months apart. Activities at each visit are 
listed in the Procedure section below and include blood samples for measuring body stress and 
vitamin D level, answering a survey and taking vitamin D3 as a supplement every day for a year.  
Routinely, after a year, you will have a repeat prostate biopsy in order to determine if the prostate 
cancer has progressed. At that time, as part of the research study, you will have blood samples 
collected for measuring body stress and vitamin D level and an extra prostate tissue core collected 
during your prostate biopsy. 
You may or may not benefit from participating in this study. There is a risk of loss of confidentiality, 
but the researchers will code the samples and research information to protect privacy. Taking vitamin 
D3 at 4,000 IU daily poses no risk to you. 
If you are interested in learning more about this study, please continue to read below: 
 
A. PURPOSE OF THE RESEARCH 
This study is being sponsored by the National Institute of Minority Health of Minority Health and 
Health Disparities and is being done at this VA only.  The purpose of this study is to use the available 
technology of RNA-sequencing (RiboNucleic Acid) to determine the molecular pathways utilized by 
prostate tissue and how these pathways (a) differ between African Americans and Caucasians; (b) 
change after supplementing with vitamin D for one year; (c) are affected by body stress and (d) are 
affected by ancestry. You are being asked to participate because you are scheduled to have a 
prostate biopsy. The investigator in charge of this study is Dr. Stephen J. Savage.  Approximately 250 
volunteers will be asked to give a prostate tissue sample for Part 1 of this study. From those 250 
volunteers, 60 will be invited to participate in Part 2. 
 
 


 
Department of Veterans Affairs 
Ralph H. Johnson VA Medical Center 
Research Consent Form 
Version Date: 09/20/2022 
Page 2 of 10
Subject’s Name:   
Date:   
Principal Investigator:  Stephen J. Savage, MD 
Study Title: Vitamin D supplementation in RNA-seq profiles of single core prostate biopsy samples, 
including diversity and stress determinants, among Veterans.                                  (IRB # 85140)
 
IRB Number:85140 
Date Approved «ApprovalDate» 
 
  VA FORM
MAR 2006 10-1086 
B. PROCEDURES 
If you agree to be in this study, the following will happen: 
PART 1 
a. You will be asked to sign this consent form when you understand all the procedures and study 
visit timelines required to participate. 
b. During your scheduled prostate biopsy, in addition to the 12 biopsy core samples that are 
taken as part of routine standard of care, a  13th core sample will be obtained for research 
purposes only. 
c. 
Your prostate tissue may have its RNA analyzed. RNA is considered genetic material and has 
a major role in making proteins, which are the building blocks of your body, cells and organs.  
d. Your prostate tissue sample for RNA sequencing will be labeled with your assigned code that 
has no personal identifiers and only study personnel can link it back to you. Researchers 
outside the Veterans Affairs (VA) will not be given the link between the code and your name or 
other identifying information. 
e. The pathology results from your biopsy will be reviewed by the research team as well as your 
Urology physician. 
f. 
You will have a follow-up appointment in the VA Urology Clinic to review your biopsy report 
with the Urology physician. You will  review and discuss your treatment options, depending on 
your diagnosis. 
g. If you choose a treatment plan of active surveillance [standard of care recommends: repeat 
PSA (prostate specific antigen) at six and twelve months and repeat prostate biopsy at twelve 
months] and you are willing to continue your participation in this study, you will be advanced to 
Part 2. 
PART 2 
a) Researchers will check your medical records to gather information about your general health 
status, which will include laboratory results related to body stress. They will also follow your 
records from the Urology Clinic. 
  
b) Your blood pressure, heart rate and weight will be measured. You will also have your hips and 
waist circumference measured with a tape measure. 
 
c) You will have blood collected for vitamin D level, blood sugar, cholesterol, kidney function and 
stress hormones (2 tablespoons or 30mL).  
 
d) You will be given a bottle of vitamin D softgels each containing 4,000 IU. You will take one 
softgel every day.  If you already take vitamin D daily, please let your study team know. Taking 


 
Department of Veterans Affairs 
Ralph H. Johnson VA Medical Center 
Research Consent Form 
Version Date: 09/20/2022 
Page 3 of 10
Subject’s Name:   
Date:   
Principal Investigator:  Stephen J. Savage, MD 
Study Title: Vitamin D supplementation in RNA-seq profiles of single core prostate biopsy samples, 
including diversity and stress determinants, among Veterans.                                  (IRB # 85140)
 
IRB Number:85140 
Date Approved «ApprovalDate» 
 
  VA FORM
MAR 2006 10-1086 
vitamin D for prostate cancer is considered experimental and has been cleared by the FDA 
(Federal Drug Administration) for studies involving prostate cancer and vitamin D 
supplementation. 
 
e) You will be asked to complete a “Social Determinant” survey. You may finish the surveys in the 
clinic or complete them at home and mail them back in. If you have not returned the survey, a 
member of the study staff will contact you by phone. You will then have the option of 
completing the survey over the phone with a member of the study team. 
 
Part 2: 6 Month Visit  
a) You will be asked to come in 6 months after you start Vitamin D. You will be given a new bottle 
of Vitamin D and asked to return your old bottle to the research clinic. Please return the bottle 
even if there are softgels remaining in the bottle. 
b) You will have blood collected for vitamin D as part of the research study. You will also have a 
PSA (prostate specific antigen) drawn as standard of care recommends.  
(1 tablespoon or 15mL). 
Part 2: 1 Year- Final Study Visit and Repeat Prostate Biopsy 
a) Your blood pressure, heart rate and weight will be measured. You will also have your hips and 
waist circumference measured with a tape measure. 
b) You will have blood collected for vitamin D, PSA, blood sugar, cholesterol, kidney function and 
stress hormones and ancestry markers (2 ½  tablespoons or 45mL).  
c) You will be asked to return your bottle of Vitamin D. Please return the bottle even if there are 
softgels remaining or it is empty. 
d) During your scheduled prostate biopsy, in an addition to the 12 biopsy core samples that are 
taken as part of routine standard of care, a 13th core sample will be obtained for research 
purposes only. 
e) Your prostate tissue samples for RNA sequencing will be labeled with your assigned code and 
analyzed at  the Genomics Core Laboratory at Queens University Belfast, in Ireland. 
While participating in this research study, do not take part in any other research project without 
approval from the investigators. This is to protect you from possible injury from things such as extra 
blood drawing, extra x-rays, or potential medication interactions. Taking part in other research studies 
without first discussing it with the investigators of this study may invalidate the results of this study, as 
well as that of the other studies. 


 
Department of Veterans Affairs 
Ralph H. Johnson VA Medical Center 
Research Consent Form 
Version Date: 09/20/2022 
Page 4 of 10
Subject’s Name:   
Date:   
Principal Investigator:  Stephen J. Savage, MD 
Study Title: Vitamin D supplementation in RNA-seq profiles of single core prostate biopsy samples, 
including diversity and stress determinants, among Veterans.                                  (IRB # 85140)
 
IRB Number:85140 
Date Approved «ApprovalDate» 
 
  VA FORM
MAR 2006 10-1086 
C. DURATION 
Participation in Part 1 of this study is one visit on the day of your prostate biopsy. Participation in Part 
2 of this study is approximately 1 year with three visits. Each visit will take approximately 30 minutes 
to complete. 
 
D. RISKS AND DISCOMFORTS 
Participating in this study has the following risks: 
1) 
Genetic Risks: Genetic research studies may present unique risks to human subjects and their 
relatives. These involve medical, psychosocial and economic risks, such as the possible loss of 
confidentiality (private information), loss of insurability and employability, paternity, and social 
stigmas. Knowledge of one’s genetic make-up may also affect one’s knowledge of the disease risk 
status of family members.  Genetic research raises difficult questions about informing you and other 
subjects of any results, or of future results. Some people feel anxious about the possibility of having a 
defective gene that would place them or their children at risk. Some people want to know what is 
found out about them; others do not. The risks of knowing include anxiety and other psychological 
distress. The risks of not knowing what is found include not being aware if there is treatment for the 
problem being studied. But these risks can change depending on whether there is a treatment or cure 
for a particular disease and on how clear the results are. If there is a medical reason to seek specific 
information from you, your doctor will tell you this. A process called “genetic counseling” is often 
appropriate in such cases; you should ask your doctor or nurse about this if you have any questions. 
South Carolina law, mandates that your genetic information obtained from any test or from this 
research, be kept confidential. Our state law prohibits an insurer using this information in a 
discriminatory manner against your or any of your family in issuing or renewing insurance coverage 
for you or your family. Our state law further prohibits our sharing your genetic information with anyone 
except in a few narrow circumstances, one of these being a research project of this type, approved by 
the Institutional Review Board and then we must take all steps to protect your identity. You will still be 
responsible for paying for health care, however. The Ralph H. Johnson will not be responsible for 
such costs, even if care is needed for a condition revealed during research or clinical testing. 
  A federal law, called the Genetic Information Nondiscrimination Act (GINA), generally makes it illegal 
for health insurance companies, group health plans, and most employers to discriminate against you 
based on your genetic information. This law generally will protect you in the following ways: 
• Health insurance companies and group health plans may not request your genetic information that 
we get from this research. 
• Health insurance companies and group health plans may not use your genetic information when 


 
Department of Veterans Affairs 
Ralph H. Johnson VA Medical Center 
Research Consent Form 
Version Date: 09/20/2022 
Page 5 of 10
Subject’s Name:   
Date:   
Principal Investigator:  Stephen J. Savage, MD 
Study Title: Vitamin D supplementation in RNA-seq profiles of single core prostate biopsy samples, 
including diversity and stress determinants, among Veterans.                                  (IRB # 85140)
 
IRB Number:85140 
Date Approved «ApprovalDate» 
 
  VA FORM
MAR 2006 10-1086 
making decisions regarding your eligibility or premiums. 
• Employers with 15 or more employees may not use your genetic information that we get from this 
research when making a decision to hire, promote, or fire you or when setting the terms of your 
employment. 
Be aware that this new federal law does not protect you against genetic discrimination by companies 
that sell life insurance, disability insurance, or long-term care insurance, nor does it protect you 
against genetic discrimination by all employers. 
 
For this study, The RNA-sequencing will analyze profiles and biological pathways on the molecular 
level in prostate tissue. It is not identifying specific genes and an individual’s predisposition to a 
particular diagnosis or disease process. The ancestry markers will be analyzed for racial 
origin/significance only. No DNA analysis for disease identifiers will be performed. 
2) Drawing blood: drawing blood from your arm includes a momentary discomfort and/or bruising. 
Infection, excess bleeding, clotting or fainting is possible, although unlikely. 
 
3)  Loss of Confidentiality: There is a risk of loss of confidentiality of your personal information that is 
used in this study. However, an assigned code, using no personal identifying information, will be 
assigned to you and used for all data that is shared. No names or personal identifiers will be used in 
any scientific presentations or publications. 
 
4) Taking vitamin D supplementation. To date, hundreds of volunteers have taken 4,000 IU of 
vitamin D during our previous research studies with no signs of toxicity. According to the Endocrine 
Society and Institute of Medicine standards, a vitamin D blood level between 30-50 ng/mL is 
sufficient. Taking 4,000 IU of vitamin D3 daily will not exceed that level in your blood. Therefore, this 
research study amount of 4,000 IU daily should not produce any risk. 
 
5) Completing the Survey: Some of the questions the survey asks may be upsetting, or you may 
feel uncomfortable answering them. If you do not wish to answer a question, you can skip it and go to 
the next question. 
6) Collecting an extra prostate tissue core: Collection of an extra prostate tissue (punch) core from 
the outer area of the prostate will not significantly create more risk than the standard 12 (punch) core 
collection incurred during the routine procedure. The collection from this area will not interfere with 
the Pathologist’s analysis of your prostate tissue and the follow-up diagnosis. 
 


 
Department of Veterans Affairs 
Ralph H. Johnson VA Medical Center 
Research Consent Form 
Version Date: 09/20/2022 
Page 6 of 10
Subject’s Name:   
Date:   
Principal Investigator:  Stephen J. Savage, MD 
Study Title: Vitamin D supplementation in RNA-seq profiles of single core prostate biopsy samples, 
including diversity and stress determinants, among Veterans.                                  (IRB # 85140)
 
IRB Number:85140 
Date Approved «ApprovalDate» 
 
  VA FORM
MAR 2006 10-1086 
There is always a chance that any procedure can harm you. The procedures in this study are no 
different. In addition to the risks described above, you may experience a previously unknown risk or 
side effect. Risks of the usual care you receive are not risks of this study. Those risks are not 
included in this consent form. You should talk with your healthcare providers if you have any 
questions about the risks of usual care. 
 
E.  MEDICAL RECORDS and/or CERTIFICATE OF CONFIDENTIALITY 
This research is covered by a Certificate of Confidentiality from the Federal government. This means 
that the researchers may not disclose information or biospecimens that may identify you, even by a 
court subpoena, in any federal, state, or local civil, criminal, administrative, legislative, or other 
proceedings, nor can the information or biospecimens be used as evidence, unless you have 
consented to this disclosure. Information or biospecimens protected by this Certificate cannot be 
disclosed to anyone else who is not connected with the research except if you have consented to the 
disclosure, including for your medical treatment; or if it is used for other scientific research, as allowed 
by federal regulations protecting research subjects. 
A Certificate of Confidentiality does not prevent you from voluntarily releasing information about 
yourself or your involvement in this research. If you are a VA patient you have a VA medical record. If 
you have never been a VA patient, a VA medical record will be created for the purposes of this study. 
Results of research tests or procedures will be included in your VA medical record. All information 
within your medical record can be viewed by individuals authorized to access the record. We will 
make every effort to keep confidential all research information in the medical record that identify you 
to the extent allowed by law; however, there is the possibility that your research information will be 
disclosed. 
The Certificate of Confidentiality will not be used to prevent disclosure as required by federal, state, or 
local law. Examples of required disclosure include: child abuse and neglect, or harm to self or others 
Finally, a Certificate may not be used to withhold information from the Federal government needed for 
auditing or evaluating Federally funded projects or information needed by the FDA. 
 
F.  BENEFITS 
There may be no direct benefit to you from participating in this study. However, it is hoped that the 
information gained from the study will help the researcher learn more about prostate cancer 
 


 
Department of Veterans Affairs 
Ralph H. Johnson VA Medical Center 
Research Consent Form 
Version Date: 09/20/2022 
Page 7 of 10
Subject’s Name:   
Date:   
Principal Investigator:  Stephen J. Savage, MD 
Study Title: Vitamin D supplementation in RNA-seq profiles of single core prostate biopsy samples, 
including diversity and stress determinants, among Veterans.                                  (IRB # 85140)
 
IRB Number:85140 
Date Approved «ApprovalDate» 
 
  VA FORM
MAR 2006 10-1086 
G. COSTS  
You will not be charged for any treatments or procedures that are part of this study. If you usually pay 
co-payments for VA care and medications, you will still pay these co-payments for VA care and 
medications that are not part of this study. 
 
H. PAYMENT TO PARTICIPANTS 
Part 1: In return for your extra time, effort and travel expenses, you will be paid $50 in cash or will 
receive a $50 Visa card for participation. You will receive your payment at the time of your prostate 
biopsy. 
Part 2: If you complete the survey and return it in the provided stamped envelope, you will receive a 
$15 gift card by mail. At your final study visit, when you have your scheduled repeat prostate biopsy, 
you will receive $50 cash or a $50 Visa card for your extra time, effort and travel expenses. 
 
The IRS requires a tax form be filed if your compensation exceeds $600.00/year. However, if the 
payment for participation will be made through Austin Financial Services Center, it may require the 
use of our social security number and may generate IRS Form 1099 automatically, regardless of the 
amount.  
 
I. ALTERNATIVES 
If you choose not to participate in this study, you will receive standard of care prostate  
biopsies (without the extra core sample for research purposes) and standard treatment  
following your prostate biopsy results. 
J. DATA SHARING 
No information about you that is collected as part of this research (whether or not it is identifiable) will 
be used or distributed for future research studies under any circumstances. 
 
K.  DISCLOSURE OF RESULTS 
Part 1: Your results from your prostate biopsy will be shared with you by your Urology physician at 
your follow up visit. 
Part 2: Your laboratory results (cholesterol and blood glucose) will automatically be entered into the 
VA electronic medical record and can be viewed by your Health Care Provider and reported to you. 
Your blood pressure, heart rate and waist/hip measurements will be shared with you when they are 
obtained. 
 


 
Department of Veterans Affairs 
Ralph H. Johnson VA Medical Center 
Research Consent Form 
Version Date: 09/20/2022 
Page 8 of 10
Subject’s Name:   
Date:   
Principal Investigator:  Stephen J. Savage, MD 
Study Title: Vitamin D supplementation in RNA-seq profiles of single core prostate biopsy samples, 
including diversity and stress determinants, among Veterans.                                  (IRB # 85140)
 
IRB Number:85140 
Date Approved «ApprovalDate» 
 
  VA FORM
MAR 2006 10-1086 
The RNA prostate tissue analysis and the blood ancestry analysis will be completed only if you 
participate in the second part of the study. These are very complicated procedures and require 
extremely long, coded reports. These results will not be reported to you. However, the overall findings 
for all the participants will be published at the completion of the study. All results will remain de-
identified (containing no personal information). 
 
 
L. SIGNIFICANT NEW FINDINGS 
If there are significant new findings during the course of your participation in this study, you will be 
notified. 
 
M.  EMPLOYEE PARTICIPATION 
Your participation or discontinuance will not constitute an element of your job performance or 
evaluation nor will it be a part of your personnel record at this Institution. 
N.   CLINICAL TRIALS.GOV  
A description of this clinical trial will be available on http://www.ClinicalTrials.gov as required by U.S. 
Law. This website will not include information that can identify you. At most, the website will include a 
summary of the results. You can search this website at any time. 
 
 
O.  COLLECTION OF SPECIMENS 
 
Your blood samples for PSA, cholesterol, blood sugar and kidney function will be evaluated by 
the VA laboratory. These results will be automatically entered into your VA electronic medical 
record and can be reviewed with your primary health provider. 
 
Your blood sample for vitamin D level and several of the blood chemistries measuring stress will 
be labeled with your assigned code and measured by laboratories at MUSC.  
Because these samples are coded, you will not receive these results. 
 
The sample for ancestry markers will also be labeled with your assigned code and will be 
analyzed at a specialized lab in California. You will not receive the results. 
 
The extra core prostate tissue sample will be labeled with your assigned code. Because of the 
complexity of the RNA-sequencing, you will not receive results. 
 
No samples will be stored for future research and any remaining blood or tissue samples will be 
destroyed by the end of the study.  
 
 


 
Department of Veterans Affairs 
Ralph H. Johnson VA Medical Center 
Research Consent Form 
Version Date: 09/20/2022 
Page 9 of 10
Subject’s Name:   
Date:   
Principal Investigator:  Stephen J. Savage, MD 
Study Title: Vitamin D supplementation in RNA-seq profiles of single core prostate biopsy samples, 
including diversity and stress determinants, among Veterans.                                  (IRB # 85140)
 
IRB Number:85140 
Date Approved «ApprovalDate» 
 
  VA FORM
MAR 2006 10-1086 
P.  FUTURE CONTACT 
In the future, we may want to contact you again to see if you’d be interested in allowing another 
prostate tissue specimen to be collected at the time of a regularly scheduled prostate biopsy. You will 
be asked to sign another consent if you agree. By initialing below, you have only agreed to allow us to 
contact you again in the future. If we may contact you again, please indicate below. 
 
YES_______ (initial)  you may contact me again for future research 
 
NO ________ (initial) please do not contact me again for future research 
 
 
CONSENT 
 
Results of this research will be used for the purposes described in this study. This information may be 
published, but you will not be identified. Information that is obtained concerning this research that can 
be identified with you will remain confidential to the extent possible within State and Federal law.  
 
The investigators associated with this study, the sponsor, and the Food and Drug Administration 
(FDA) will receive copies of the research records. The investigators associated with this study, 
employees of the sponsor, the FDA, and the MUSC Institutional Board for Human Research will have 
access to identifying information. All records in South Carolina are subject to subpoena by a court of 
law. 
The VA will provide necessary medical treatment to a research subject injured by participation in a 
research project.  This requirement does not apply to treatment for injuries that result from non-
compliance by a research subject with study procedures. If you sustain an injury as a direct result of 
your study participation, medical care will be provided by this VA Medical Center.  Financial 
compensation is not available for such things a lost wages, disability or discomfort due to an injury. 
Your participation in this study is voluntary. You may refuse to take part in or stop taking part in this 
study at any time. You should call the investigator in charge of this study if you decide to do this. Your 
decision not to take part in the study will not affect your current or future medical care or any benefits 
to which you are entitled. 
 
The investigators and/or the sponsor may stop your participation in this study at any time if they 
decide it is in your best interest. They may also do this if you do not follow the investigator’s 
instructions.  


 
Department of Veterans Affairs 
Ralph H. Johnson VA Medical Center 
Research Consent Form 
Version Date: 09/20/2022 
Page 10 of 10
Subject’s Name:   
Date:   
Principal Investigator:  Stephen J. Savage, MD 
Study Title: Vitamin D supplementation in RNA-seq profiles of single core prostate biopsy samples, 
including diversity and stress determinants, among Veterans.                                  (IRB # 85140)
 
IRB Number:85140 
Date Approved «ApprovalDate» 
 
  VA FORM
MAR 2006 10-1086 
VOLUNTEER STATEMENT 
 
Dr. Savage or a member of his research team has explained the research study to me. I have been 
told of the risks or discomforts and possible benefits of the study. I have been given a chance to ask 
questions and obtain answers. 
  
If I have more questions about my participation in this study or study related injury, or if I have 
comments, concerns or complaints, I may contact: Stephen J. Savage, MD (843.789.7816).  
 
If I have questions about my rights as a study participant, or I want to make sure this is a valid VA 
study, I may contact the Medical University of South Carolina’s Institutional Review Board (IRB). This 
is the board that is responsible for overseeing the safety of human participants in this study. I may call 
the MUSC IRB (843) 792-4148, or the Ralph H. Johnson VA Medical Center’s Research Compliance 
Officer at (843) 789-7399, if I have questions, complaints, or concerns about the study or if I would 
like to obtain information or offer input. 
 
By signing this document below, I voluntarily consent to participate in this study. I also confirm that I 
have read this consent, or it has been read to me. I will receive a copy of this consent after I sign it.  
[A copy of this signed consent will also be scanned into  your medical record]. 
      
 
                                                                      
 
I agree to participate in this research study as has been explained in this document. 
 
 
 
_________________________
Participant’s Name
____________________________ 
Participant’s Signature
___________ 
Date
_________________________
Name of person obtaining 
consent
_____________________________ 
Signature of person obtaining 
consent
____________ 
Date