Conditions: Lung Cancer
Keywords: Sulforaphane Chemoprevention Former Smokers
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 43
Healthy volunteers: no

Brief summary:
This research study involves taking an experimental anti-cancer dietary supplement called Sulforaphane (SF) or a placebo (product without any supplement content) over a period of twelve months in order to determine if it is a useful dietary supplement for prevention of lung cancer in humans.

The main goals of this research study are:

1. To learn about the effects of giving Sulforaphane (SF) to former smokers who are still at high risk of developing cancer due to their smoking history and whether or not their condition improves, stays the same or becomes worse after Sulforaphane (SF) is given.
2. To learn whether Sulforaphane (SF) might reverse some of the lung cell changes associated with future development of lung cancer.

Detailed description:

Arms:
arm 1: Sulforaphane four tablets 2 times per day with breakfast and dinner each dose contains approximately 120 micromole of Sulforaphane arm 2: Placebo (containing no active drug) four tablets 2 times per day with breakfast and dinner

Interventions: intervention 1: Sulforaphane intervention 2: Placebo
Intervention descriptions: intervention 1: Sulforaphane (SF) is a naturally occurring substance (phytochemical) found in cruciferous vegetables. intervention 2: Inactive ingredients

Protocol document:
Prot_SAP_000.pdf: 
Phase II Clinical Trial of Lung Cancer Chemoprevention with Sulforaphane: Clinical Protocol Version 11 (09/23/2019) 
  
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CLINICAL PROTOCOL 
Study Title: Randomized Clinical Trial of Lung Cancer Chemoprevention with 
Sulforaphane in Former Smokers 
NCT03232138 
September 23, 2019 Version 11. 
PRINCIPAL INVESTIGATOR 
CO-PRINCIPAL INVESTIGATOR 
Jian-Min Yuan, M.D., Ph.D. 
Professor 
Division of Cancer Control and 
Population Sciences 
University of Pittsburgh Cancer Institute 
5150 Centre Avenue 
UPMC Cancer Pavilion, Suite 4C 
Pittsburgh, PA 15232 
(412) 864-7889 
yuanj@upmc.edu 
David O. Wilson, M.D. 
Associate Professor 
Division of Pulmonary, Allergy and Critical Care 
Medicine, School of Medicine 
University of Pittsburgh Cancer Institute 
5150 Centre Avenue 
UPMC Cancer Pavilion, Suite 4C 
Pittsburgh, PA 15232 
(412) 687-3355 
wilsondo@upmc.edu 
CO-INVESTIGATORS 
Sanja Dacic, M.D., Ph.D. 
Professor 
Department of Pathology  
School of Medicine 
UPMC-PUH, Room C608 
200 Lothrop Street 
Pittsburgh, PA15213 
(412) 647-8694 
dacics@upmc.edu 
James Herman, M.D. 
Professor 
Division of Hematology/Oncology 
University of Pittsburgh Cancer Institute 
5117 Centre Avenue 
2.18d Hillman Cancer Research Pavilion 
Pittsburgh, PA 15232 
(412) 623-7769 
hermanj3@upmc.edu 
Thomas W. Kensler, Ph.D. 
Professor 
Department of Pharmacology and 
Chemical Biology, School of Medicine 
E1352 Thomas E. Starzl BST  
200 Lothrop Street 
Pittsburgh, PA15213 
(412) 648-1942 
tkensler@pitt.edu 
 
Daniel Normolle, Ph.D. 
Associate Professor 
Department of Biostatistics 
UPMC Hillman Cancer Center Biostatistics Facility 
201 North Craig Street 
Pittsburgh, PA 15213 
(412) 383-1591 
dpn7@pitt.edu 
Shivendra Singh, Ph.D. 
Professor 
Department of Pharmacology and 
Chemical Biology, School of Medicine 
University of Pittsburgh Cancer Institute 
5117 Centre Avenue 
2.32A Hillman Cancer Research Pavilion 
Pittsburgh, PA 15232 
(412) 623-3262 
Avrum Spira, M.D., M.Sc. 
Department of Medicine, Pathology and 
Bioinformatics 
Boston University School of Medicine 
72 East Concord Street, E-631 
Boston, MA 02118-2308 
(617) 414-6980 
aspira@bu.edu 
 


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singhs@upmc.edu 
 
 
 
TABLE OF CONTENTS  .............................................................................................................2 
1. OBJECTIVES ..........................................................................................................................5 
1.1 Primary Aims .................................................................................................................5 
1.2 Secondary Aims .............................................................................................................5 
2. BACKGROUND ......................................................................................................................5 
2.1  Rationale for sulforaphane as a chemopreventive agent for lung cancer ......................5 
2.2 Prior chemoprevention studies .......................................................................................8 
3. PATIENT SELECTION .........................................................................................................9 
3.1 Eligibility Criteria ..........................................................................................................9 
3.2 Exclusion Criteria ........................................................................................................10 
3.3 Recruitment Methods...................................................................................................11 
3.4 Consent Procedure .......................................................................................................11 
3.5 Patient Payment ...........................................................................................................11 
3.6 Registration Process .....................................................................................................11 
4. STUDY AGENT INFORMATION ......................................................................................12 
4.1 Broccoli seed preparations (BSP) ................................................................................12 
4.1.1 
Summary ....................................................................................................12 
4.1.2 
Description of Origin of Sulforaphane, Purification, and Stabilization .....13 
4.1.3 
Physical, Chemical, and Pharmaceutical Properties and Formulations .....14 
4.1.4 
Non-Clinical Pharmacology.......................................................................15 
4.1.4.1 
Pharmacokinetics .....................................................................15 
4.1.4.2 
Pharmacodynamics ..................................................................15 
4.1.4.3 
Non-clinical Metabolism .........................................................16 
4.1.4.4 
Non-clinical Toxicology ..........................................................16 
4.1.4.5 
Mutagenicity ............................................................................17 
4.1.5  
Pharmacokinetics and Metabolism of Broccoli Seed Preparations ...........17 
4.1.5.1 
Broccoli Sprout Extracts ..........................................................17 
4.1.5.2 
Broccoli sprout extract without daikon ....................................19 
4.1.5.3 
Broccoli sprout extract with daikon .........................................19 
4.1.5.4 
Cruciferous Vegetables ............................................................20 
4.1.5.5 
Broccoli preparations containing active myrosinase ...............20 
4.1.6  
Safety and Efficacy ....................................................................................20 
4.1.6.1 
Broccoli Sprout ........................................................................20 
4.1.6.2 
Broccoli Sprout Preparations (BSPs) and Extracts (BSEs) .....21 
4.1.7 
Rationale for Selected Doses .....................................................................24 
4.2 Rational for Proposed Study in High-Risk Former Smokers.......................................25 
5. TREATMENT PLAN AND STUDY PROCEDURES .......................................................26 
5.1 Pre-Treatment Evaluation ............................................................................................26 
5.2 Randomization .............................................................................................................27 
5.3 Treatment Plan .............................................................................................................27 


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5.3.1 Nutraceutical Information ................................................................................27 
5.3.2 Dose .................................................................................................................27 
5.3.3 Treatment Duration ..........................................................................................27 
5.3.4 Criteria for Removal from Study .....................................................................28 
5.4 Dosing Delays/Dose Modifications .............................................................................28 
5.5 Clinical Visits...............................................................................................................28 
5.6 Withdrawal from the Protocol .....................................................................................29 
5.7 Off Study Date .............................................................................................................29 
5.8 Post-Treatment Follow-Up  .........................................................................................29 
6. STUDY CALENDAR ............................................................................................................30 
7. ADVERSE EVENTS: LIST AND REPORTING REQUIREMENTS .............................31 
7.1 Adverse Events and Risks ............................................................................................31 
7.1.1 
Adverse Events for Broccoli Sprout Preparation Avmacol® .....................31 
7.1.2 
Risks Associated with Study Procedures ...................................................33 
7.1.2.1 
Bronchoscopy ..........................................................................33 
7.1.2.2 
Blood Samples and IV Infusion During Bronchoscopy ..........33 
7.1.2.3  
Nasal and buccal brushings ......................................................33 
7.1.3 
Violation of Privacy and Loss of Confidentiality ......................................33 
7.2 Definitions of Adverse Events .....................................................................................34 
7.3 Reporting Adverse Events to the Responsible IRB .....................................................35 
7.4 DSMB ..........................................................................................................................35 
8. BIOSPECIMENS, BIOMARKER, AND SPECIAL STUDIES ........................................36 
8.1 Human Biospecimen Collection ..................................................................................36 
8.1.1 
Endobronchial Biopsies .............................................................................36 
8.1.2 
Endobronchial Brushing ............................................................................36 
8.1.3 
Bronchoalveolar Lavage ............................................................................36 
8.1.4 
Nasal Epithelia ...........................................................................................37 
8.1.5  
Blood ..........................................................................................................37 
8.1.6  
Urine ..........................................................................................................37 
8.1.7  
Creation of a Biospecimen Repository ......................................................37 
8.1.8 
Identifiers ...................................................................................................38 
8.2 Biomarkers ...................................................................................................................38 
8.2.1 Histology (atypia) ............................................................................................38 
8.2.2 Cell proliferation (Ki-67 labeling index) .........................................................38 
8.2.3 Apoptosis (TUNEL and Caspase-3) ................................................................39 
8.2.4 Gene expression markers .................................................................................39 
8.3 End of Treatment Evaluation .......................................................................................40 
8.3.1 Histological Response ......................................................................................40 
8.3.2 Cellular Biomarker Response ..........................................................................41 
8.3.3 Gene Expression Response ..............................................................................41 
8.3.4 Safety and Toxicity Assessment ......................................................................41 
9. STATISTICAL CONSIDERATIONS .................................................................................41 
9.1 Analysis plan and sample size evaluation: ...................................................................42 
9.2 Statistical Analyses for Cellular Proliferation/Apoptosis Markers ..............................42 


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9.3 Statistical Analyses for Gene Expression ....................................................................43 
9.4 Statistical Analyses for Adverse Events and Toxicity Measures ................................44 
 
10. 
GENDER AND MINORITY INCLUSION .....................................................................44 
11. 
ETHICAL AND REGULATORY CONSIDERATIONS ...............................................44 
11.1 Informed Consent .......................................................................................................45 
11.2 Basic Elements of Informed Consent .........................................................................45 
11.3 Additional Elements of Informed Consent .................................................................46 
11.4 Institutional Review ...................................................................................................46 
11.5 Drug Accountability ...................................................................................................47 
12. 
STUDY DATA MANAGEMENT .....................................................................................47 
13. 
FUNDING ...........................................................................................................................47 
14. 
REFERENCES ...................................................................................................................48 


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1. 
OBJECTIVES 
 
1.1 Primary Aims 
 
1.1.1 To recruit 72 former smokers at high risk of developing lung cancer who will be 
randomly assigned to either receiving Avmacol® or placebo tablets for 12 
months. Bronchoscopy-guided bronchial biopsy and brushing, bronchoalveolar 
lavage, nasal brushing, blood and urine samples will be collected from each 
subject at pre- and post-intervention time points. 
 
1.1.2. To determine if daily oral dose of 120 micromole sulforaphane (SF) for 12 
months can modulate the changes of bronchial dysplasia index, cell proliferation 
marker Ki-67, and apoptosis markers caspase-3 and TUNEL in bronchial biopsies 
in former smokers at high risk for lung cancer.  
 
1.2 Secondary Aims 
 
1.2.1 To explore if daily oral dose of 120 micromole SF for 12 months can modulate 
the changes of the lung cancer-related gene expression markers in bronchial 
epithelia in former smokers at high risk for lung cancer.  
 
1.2.2 To explore if daily oral dose of 120 micromole SF for 12 months can modulate 
the changes of bronchial premalignant lesions-related gene expression markers in 
former smokers at high risk for lung cancer.  
 
1.2.3. To explore if daily oral dose of 120 micromole SF for 12 months can modulate 
the similar changes of the gene expression markers in nasal epithelia as in the 
bronchial epithelia identified in Aims 1.2.1 and 1.2.2. 
 
1.1.4 To determine the safety and toxicity of daily oral dose of 120 micromole SF for 
12 months in former smokers at high risk for lung cancer. 
2. 
BACKGROUND 
 
2.1  Rationale for sulforaphane as a chemopreventive agent for lung cancer 
 
Lung cancer is leading cause of lung cancer death in both men and women worldwide 
(1.5 million deaths) and in the US (158,000 deaths) (2,3).  Cigarette smoking causes 
90% of lung cancer (4).  Smoking cessation is an obvious and practical strategy for 
lung cancer prevention for smokers.  The smoking cessation programs and 
antismoking campaign have resulted in significant reduction in prevalence of 
cigarette smoking in the US, from 60% in late 1960s to 17% in 2015; currently there 
are more former smokers than current smokers (5).  However, former smokers remain 
at high risk of lung cancer even after they quit smoking for many years (6). In fact, 
the majority of new lung cancer cases occur among former smokers (7,8). 
Unfortunately the preventive intervention for lung cancer targeting former smokers is 
lacking. 


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Epidemiological studies have shown that intake of isothiocynantes (ITCs), natural 
products formed from glucosinolates found in certain cruciferous vegetables such as 
broccoli and its seeds, are associated with a reduced risk of lung cancer.  Using a 
validated biomarker approach (9,10), we, for the first time, demonstrated that high 
urinary total ITCs, including both sulforaphane (SF) and phenethyl isothiocyanate 
(PEITC), were associated significantly with a 35% reduced risk of lung cancer (11).  
These novel findings stimulated many epidemiological and experimental studies on 
the potential lung cancer protection of dietary ITCs and cruciferous vegetables in 
many populations.  A meta-analysis including 14 studies and more than 8,000 lung 
cancer cases reported a statistically significant 20% reduced risk of lung cancer for 
subjects with high intake of cruciferous vegetables (12).  These data implicate a 
protective role of ITCs or cruciferous vegetables as a whole against the development 
of lung cancer in humans. However, epidemiological studies are inherent with 
measurement errors and potential confounding, thus the observed association between 
ITCs and lung cancer risk may not be causal. Furthermore, results from observational 
studies usually do not provide a biological underpinning. 
 
Preclinical data in rodent models is quite persuasive for prevention of lung cancer 
with ITCs.  In an experiment by Hecht et al. (13), 160 rats were assigned to 4 groups: 
1) 60 rats treated by tobacco-specific lung carcinogen NNK (2 ppm) only, 2) 60 rats 
of NNK (2 ppm) plus PEITC (3 micromole/g diet), 3) 20 rats of PEITC (3 
micromole/g diet) only, and 4) 20 rats untreated as controls. PEITC was added to diet 
at a non-toxic concentration for the entire study period (112 weeks) whereas NNK 
was added to the drinking water one week after the initial administration of PEITC.  
At the end of the study (after 112 weeks of PEITC and 111 weeks of NNK treatment), 
70% of the NNK-treated only rats developed lung adenoma and adenocarcinoma 
while only 5% of rats treated with PEITC and NNK developed lung tumors, similar to 
those in the PEITC-treated only or untreated control rats.  These results showed that 
PEITC, treated concurrently with NNK, a setting analogous to the situation of current 
smokers who are continuously exposed to tobacco carcinogens including NNK, 
completely inhibited carcinogen-induced lung tumors in rats. 
 
In another experiment by Chung and colleagues (14) with a different study design, 
mice were treated with a mixture of tobacco carcinogens: 3 micromole 
benzo(a)pyrene [B(a)P] and 3 micromole NNK, given gavage once a week for 8 
weeks first, stopped carcinogen treatment for 12 weeks, and then fed a diet containing 
different concentrations of sulforaphane (SF) (1.5 or 3.0 micromole/g diet) or 
phenethy isothiocyanate (PEITC) (1.5 or 3.0 μmole/g diet), for 22 more weeks. At the 
end of the study, the incidence of lung adenocarcinoma was reduced by a statistically 
significant 52% in mice treated with carcinogens plus SF, and by 55% in mice treated 
with carcinogens plus PEITC, compared with mice treated with carcinogens only. 
The tumor multiplicity was reduced by 70% in mice treated with carcinogens plus SF 
and by 60% in mice treated with carcinogens plus PEITC.  These results show that 
both dietary SF and PEITC can inhibit the development of lung tumors induced by 
two major tobacco lung carcinogens – B(a)P and NNK.  More importantly, the 


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chemopreventive agents SF and PEITC were given 12 weeks after the carcinogen 
treatment was stopped, a setting analogous to a situation of exposure for former 
smokers. 
 
Besides the inhibition of tumor incidence and multiplicity, both SF and PEITC can 
inhibit cellular proliferation markers such as Ki-67 and induction of apoptosis (15), 
hallmarks of tumorigenesis.  In the experiment by Chung and colleagues (14), dietary 
intake of SF (1.5 micromole/g diet) reduced number of lung cells expressing 
proliferative cell nuclear antigen, an alternative to Ki-67, by 61% (from 44.4 ± 1.9 to 
17.3 ± 11.7) (14).  In a double-blinded randomized clinical trial, women with 
abnormal mammograms and scheduled for breast biopsy who consumed SF precursor 
GR supplement (estimated daily urinary excretion of 9.7 μmole total metabolites of 
SF) for 2-8 weeks showed significantly reduced Ki-67 labeling indices (LI) in breast 
benign tissue (16).  Dietary SF and PEITC also induced markers of apoptosis in lung 
tissue of mice after treated with tobacco carcinogens. Compared with control animals, 
mice given SF (1.5 micromole/g diet) showed a statistically significant 6-fold 
increase (from 4.7±1.9 to 29.3±4.7%) in activated caspase-3, and a 2-fold increase 
(from 12.7±3.0 to 29.4±5.8%) in TUNEL (terminal transferase dUTP nick end 
7labelling) of lung cells (14). 
 
We recently completed two randomized, double-blinded, placebo-controlled clinical 
trials. The first trial was led by Dr. Kensler to evaluate whether intake of SF enhances 
the detoxification metabolism of environmental carcinogens and toxicants such as 
benzene and acrolein in 283 subjects (89% were non-smokers) who were exposed to 
substantial levels of airborne pollutants in China.  Intake of a broccoli sprout-derived 
beverage providing daily doses of 600 micromole GR and 40 micromole SF for 12 
weeks significantly increased the urinary excretion of the detoxification mercapturic 
acids formed from benzene, an environmental carcinogen present in both tobacco 
smoke and polluted air, by 61% (P<0.001) and acrolein by 23% (P=0.01) (17). The 
median levels of the 24-hour urinary excretion of SF metabolites with this dosing 
regimen were 54-62 micromole across the intervention period (17).  The second trial 
was led by Dr. Yuan to evaluate if intake of PEITC inhibits the metabolic activation 
of tobacco-specific carcinogen NNK and enhances the detoxification metabolism of 
tobacco-nonspecific carcinogens and toxicants such as benzene and acrolein. Overall, 
intake of PEITC 40 mg/day for 5 days significantly inhibited NNK metabolic 
activation by 8% (P = 0.023), and increased urinary detoxification mercapturic acids 
formed from benzene by 25% (P = 0.002) and acrolein by 15% (P = 0.005), in 82 
current smokers in the US.  The mean level of the 24-hour urinary excretion of 
PEITC-NAC was approximately 80 micromole. These very similar results of the two 
trials on the detoxification of environmental carcinogens and toxicants suggest that 
both SF and PEITC have a very similar biological mechanism and share the strongly 
overlapping chemopreventive properties, as shown in the animal experiment (14). 
 
These two randomized phase 2 clinical trials provided crucial data on the safe and 
effective dose and duration of treatment. The unbiased results of these trials elucidate 
the modulating effect of these ITCs on the metabolisms of tobacco-specific (NNK) 
and nonspecific carcinogens (benzene) and toxicants (acrolein) through specific 


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biological pathways. These encouraging results are very important for further 
development of these ITCs as primary chemopreventive agents against the 
development of lung cancer.  However, these results are systemic and related to 
actions at the upstream on the lung carcinogenesis pathway. Before we can launch 
primary chemoprevention studies against the development of lung cancer as an end 
point, we need to demonstrate if ITCs such as SF can modulate the changes of 
downstream biomarkers on the lung carcinogenesis pathway such as bronchial 
dysplasia, cell proliferation and apoptosis, and gene expression in bronchial biopsies 
that have been shown to be directly linked to the development of lung cancer (18). 
 
We propose to further evaluate the chemopreventive effect of SF against lung 
carcinogenesis through a randomized, double-blinded, placebo-controlled phase 2 
clinical trial, a proof-of-principal study, with a longer treatment period (12 months) 
for former smokers who are at high risk for lung cancer.  We hypothesize that intake 
of SF will improve pathological features of bronchial dysplasia, decrease cell 
proliferation and increase apoptosis in endobronchial biopsies, and improve the gene 
expression profile of lung cancer-related genes in endobronchial epithelial cells, all of 
which have been shown to predict lung cancer development and diagnosis (18,19).  
Successful outcome of this systemic analysis is crucial to further develop SF as a 
chemopreventive agent for the primary prevention of lung cancer.  Our ultimate goal 
is to validate the efficacy of this readily available, widely accessible, inexpensive, 
natural compound as a primary chemopreventive agent against the development of 
lung cancer in humans. 
 
Findings from epidemiological and animal experimental studies as well as from short-
term randomized phase 2 clinical trials have shown cancer chemoprotective 
properties of sulforaphane.  Although these results are encouraging, they are not 
sufficient and justifiable for launching large randomized phase 3 clinical trials to 
evaluate the efficacy of SF on reduction of lung cancer incidence and mortality as the 
primary endpoints, which require large amounts of resources and many years of 
intervention and many more years of post-intervention follow-up. We, the research 
community, have learned hard lessons from failed large placebo-controlled phase 3 
chemoprevention trials when their premises were primarily based on the observed 
inverse association from epidemiological studies, but not by strong biological 
mechanisms supported by pre-clinical studies in animals and early phase randomized 
clinical trials in humans. These previously failed trials included CARET (beta-
carotene and retinol) and ATBC (beta-carotene and alpha-tocopherol) (20,21)}. 
Therefore, direct evidence of a protective effect of SF on bronchial cellular and 
molecular biomarkers proposed here is crucial for further development of SF as a 
primary chemoprevention agent against the development of lung cancer in humans. 
The proposed study, if it proves our hypotheses to be true, will have significant public 
health implications on reduction of lung cancer incidence and mortality in former 
smokers. 
2.2 Prior chemoprevention studies 
 


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There have been a limited number of phase 2 chemoprevention studies in lung cancer. 
Among them were the Iloprost and Celecoxib trials. We participated in the 
multicenter double-blind, randomized, placebo-controlled phase 2 trial of oral 
iloprost, a prostacyclin analogue, in current and former smokers with sputum 
cytological atypia or endobronchial dysplasia (22). Bronchoscopy was performed at 
study entry and after completion of the six-months treatment. Within each subject, we 
calculated the average histological score of all biopsies, the worst biopsy score, and 
the dysplasia index. Compared with former smokers in the placebo group, former 
smokers treated with oral iloprost exhibited a significantly greater improvement in 
average score (0.41 units better, P = 0.010), in the worst biopsy score (1.10 units 
better, P = 0.002), and in the dysplasia index (-12.5%, 95% CI -21.0% to -3.9%, P = 
0.006). The 6-month treatment with iloprost did not show any improvement of 
histological scores in current smokers (22). Endobronchial dysplasia is a presumed 
precursor of non-small cell lung cancer (NSCLC). In a follow-up study of a high-risk 
cohort of subjects with endoscopic biopsies, persistence of bronchial dysplasia was 
associated significantly with a 7-fold increased risk of developing NSCLC (adjusted 
hazard ratio = 7.8, 95% CI 1.6-39.4). Furthermore, elevated Ki-67 LI at baseline 
endobronchial biopsies were associated significantly with increased bronchial 
dysplasia score, which predates the development of lung cancer (18). 
 
Another similar phase 2 clinical trial was the celecoxib trial conducted by Mao and 
colleagues (23). The study enrolled former smokers (age ≥45 years, ≥30 pack-years 
of smoking, ≥1 year of sustained abstinence from smoking) to evaluate the impact of 
COX-2 selective inhibitor celecoxib on cellular and molecular events associated with 
lung cancer pathogenesis. The primary endpoint was bronchial Ki-67 LI after 6 
month treatment. Celecoxib significantly reduced Ki-67 LI by an average of 34% (P = 
0.04). Furthermore the decrease in Ki-67 LI correlated with a reduction and/or 
resolution of lung nodules on CT (P = 0.008).   
 
Although both iloprost and celecoxib have shown effective on the reduced regression 
of bronchial dysplasia in former smokers (22-24), both are not available for further 
development as potential chemopreventive agents due to their toxicity (celecoxib) or 
lack of readily deliverable form such as tablets (iloprost). Sulforaphane is a promising 
chemopreventive agent, a natural compound with minimal side effects, readily 
available, widely accessible, and inexpensive for mass production.  Smaller trials with 
intermediate endpoints in high-risk subjects are needed to provide crucial data for 
further development of SF as a primary chemoprevention agent against the 
development of lung cancer in humans. The proposed study, if it proves our 
hypotheses to be true, will have significant public health implications on reduction of 
lung cancer incidence and mortality in former smokers. 
 
3. 
PATIENT SELECTION 
 
Seventy-two volunteers will be recruited for this phase II clinical trial. 
 
3.1 
Eligibility Criteria 
 


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1) 
Man or woman 55-74 years of age. 
2) 
Patients with normal endobronchial biopsy findings or pre-cancerous lesions at 
baseline will be eligible for the study. Pre-cancerous lesions include (a) reserve cell 
hyperplasia, (b) squamous metaplasia, (c) mild dysplasia, (d) moderate dysplasia, 
and (e) severe dysplasia. 
2) 
A former smoker who has a history of smoking with ≥30 pack-years, quits 
smoking within the past 10 years, and has more than 1 year sustained abstinence 
from smoking. 
3) 
Female subjects must be of non-child bearing potential or must have a negative 
urine pregnancy test at screening (within 72 hours of first dose of study 
medication) if of childbearing potential.  
4) 
Male and female subjects of childbearing potential must be willing to use 
adequate barrier methods of contraception from the time starting with the 
screening visit through 30 days after the last dose of study therapy.  
5) 
Abstinence is acceptable if this is the established and preferred contraception for 
the subject. 
6) 
Generally healthy with liver enzyme and blood count values within the ranges 
shown below on the blood sample drawn at the baseline screening visit. 
Specifically: 
White blood cells  
 
≥ 3,000/mL 
Total bilirubin 
 
 
≤ 1.5 x ULN (upper limits of normal) 
AST (SGOT)/ALT (SGPT) 
≤ 2.5 x ULN 
BUN and serum creatinine 
≤ 1.5 x ULN 
7)   
The presence of airflow obstruction on spirometry (GOLD 1-4 & FEV1/FVC<0.7 
&FEV1 <0.8) COPD; and/or any emphysema on CT scan. 
8) 
Participants must have a Southwest Oncology Group (SWOG) performance status 
of 0-2 (Appendix 10). 
9) 
Participants must be able and willing to undergo a bronchoscopy before and after 
treatment for 12 months. 
10) 
Patients must be fully informed of the investigational nature of this study and 
must sign an informed consent in accordance within institutional and regulatory 
guidelines. 
 
3.2 Exclusion Criteria 
 
Patients are not eligible if any one of the following conditions exists: 
1) 
Carcinoma in situ or invasive cancer on baseline endobronchial biopsy. 
2) 
A malignancy except for adequately treated basal cell or squamous cell skin 
cancer or in situ cervical cancer. 
3) 
Severe lung disease or inability to undergo two bronchoscopies. 
4) 
Had pneumonia or acute bronchitis within the past 2 weeks prior to the date of 
enrollment (i.e., signing the consent form). 
5) 
Had myocardial infarction (MI) or other severe heart diseases  such as ventricular 
tachycardia, multifocal premature ventricular contractions or supraventricular 
tachycardias with a rapid ventricular response within the past 6 weeks prior to 
enrollment.    
6) 
Hypoxemia (less than 90% saturation with supplemental oxygen).  


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7) 
Prior chemotherapy or thoracic radiation within the past 5 years.  
8) 
Woman who is pregnant or plan to be pregnant in next 12 months, or is breast 
feeding or plan to begin breast feeding in next 12 months. 
9) 
Life expectancy of < 12 months. 
10) 
Have a history of irritable bowel disease such as Crohn’s disease and ulcerative 
colitis.  
 
3.3 Recruitment Methods 
Patients for this trial will be recruited primarily from the participants of the Pittsburgh 
Lung Screening Study (PluSS) at the UPMC Hillman Cancer Center.  This unique 
high-risk PluSS cohort enrolled approximately 3,600 current and former smokers in 
Pittsburgh area. The initial contact with the eligible subjects for the proposed study 
will be mailed a letter, followed up by a telephone call made by PluSS staff members 
who will briefly explain to the study subjects about the clinical trial and complete an 
eligibility screening form. Alternatively  Any patients who are interested in 
participating in the study can telephone the clinical investigation office at 412-623-
3317 Monday through Friday, 9:00 am to 5:00 pm (Eastern Time). The clinical 
research assistant will conduct a telephone interview with the potential study 
participant using the Eligibility Screening Form (Appendix 1). If all the requirements 
are met, the clinic research assistant will mail the following documents for subject to 
review before she or he has a scheduled clinical visit: 
1) 
Locator Form (Appendix 2) 
1) 
The Informed Consent Form (Appendix 3). 
2) 
Baseline Medical History Questionnaire (Appendix 4). 
3) 
Questionnaire for Cruciferous Vegetables (Appendix 5) 
 
3.4 Consent Procedure 
At the screening clinical visit (stage 1-visit 1), a clinical research nurse practitioner 
and/or the project manager will go over with the study subjects about the study, 
eligibility criteria, procedures, and potential risk and benefits in participating in the 
study in a setting that is quiet and unhurried. The project manager will go over the 
written informed consent form with the study subject and answer any questions the 
patient may have. The authorization describes in detail what information, including 
the HIPPA-protected information, will be collected and for what purposes it will be 
used.  A copy of signed consent will be provided to subject. 
3.5 
Patient Payment 
Subjects will receive no health benefit from participating in this research study.  If SF 
is approved to be chemopreventive, subjects assigned to treatment arm may have 
some chemopreventive benefit from Avmacol®.  Subjects will receive $500 for each 
research bronchoscopy at baseline and at the end of the treatment. In addition, we will 
pay participants $50 for each of 6 scheduled clinical visits from stage 1-visit 1 to 
statge 2-visit 6 to cover their costs of transportation and making effort to participate 
in the study. Parking for all clinical visits will be provided free of charge to the study 


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participants. At the end of the study treatment, the subject will receive $500 as bonus 
for the compliance and completion of the study (see Appendix 6. Participant 
Compsentation Form).  
3.6 
Registration Process 
 
After being deemed eligible, participants will be assigned a patient number and be 
registered into the CTMA database. 
 
4. 
STUDY AGENT INFORMATION 
 
4.1 Broccoli seed preparations (BSP) 
 
4.1.1 
Summary 
 
Broccoli seed preparations (BSP) have been studied extensively in multiple Phase I 
and Phase II clinical trials without Grade 2 or higher adverse effects. Broccoli seeds 
contain high concentrations of glucoraphanin (GR), the glucosinolate precursor of 
sulforaphane (SF), which is converted to SF by the release of intrinsic seed 
myrosinase during chewing or by thioglucosidaes in the microbiota in the human gut 
(25-27). The isothiocyanate SF (1-isothiocyanato-4R-(methylsulfinyl)butane) was 
originally isolated from commercial broccoli, and is one of the most potent naturally-
occurring inducers of cytoprotective enzymes identified to date (28). SF activates the 
KEAP1-NRF2 signaling pathway, thereby increasing transcription of antioxidative 
and detoxication genes. For example, SF has been shown to induce cytoprotective 
enzymes in a variety of animal and human cell lines and tissues (29). 
 
In our initial clinical trials, we investigated BSP in the form of re-hydrated, 
previously lyophilized broccoli sprout powders rich in either GR or SF produced by 
the Cullman Chemoprotection Center at Johns Hopkins under the supervision of Drs. 
Jed Fahey and Paul Talalay.  Broccoli sprouts (Brassica oleracea) were grown from 
specially selected BroccoSprouts™ seeds to provide a consistent yield of GR or SF. 
In our initial trial, the GR-rich powder was considered as a food by the Johns 
Hopkins IRB. It is simply the freeze-dried powder derived from a hot water extract of 
broccoli sprouts. In 2010, the US FDA made a determination that beverages 
containing SF-rich broccoli sprout powders (but not GR-rich broccoli sprout 
powders) must be considered as drugs because the SF is formed ex vivo from the 
plant by the addition of myrosinase-containing daikon seeds during the preparation of 
the SF-rich powder. Thus, subsequent trials in Qidong, China (NCT01437501) as 
well as the pilot study at the University of Pittsburgh (NCT 02023931) have been 
conducted with an IND from the FDA (#112038) held by Thomas Kensler PhD (Co-
I).   
 
There are three main problems in achieving consistent delivery and bioavailability of 
active SF by means of lyophilized broccoli sprout extracts: (1) SF is only moderately 
storage- and heat-stable over time, especially in aqueous solution.  (2) SF-rich 
broccoli sprout extract powders are extremely hygroscopic and the preparation of 


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capsules containing accurate doses by weight is challenging and very expensive. (3) 
Administration of GR as an oral precursor of SF results in highly variable 
conversions of GR to SF metabolites (dithiocarbamates) among individuals, ranging 
from 1 – 40%, but fairly consistent between individuals (17,27,30-32).  
 
In this study in healthy former smokers, BSP will be provided in the form of the 
commercially available dietary supplement, Avmacol®. Although a number of 
dietary supplements containing GR have been available in the U.S. market for several 
years now, Avmacol® is the first commercial product to contain GR plus the fully 
active enzyme myrosinase, thus yielding a higher and much more consistent SF dose 
upon ingestion. Avmacol® tablets contain only GR-rich broccoli seed extract, freeze-
dried broccoli sprouts for the myrosinase source, and the inert excipients required to 
form a tablet. They are manufactured by Nutramax Laboratories, Inc. (Edgewood, 
Maryland, USA).  Avmacol® has been sold as a nutritional supplement in the United 
States since 2013, and is manufactured under GMP standards, which has a shelf life 
of two years at ambient temperature. Based on previous human studies with both 
dietary and topical broccoli sprout extracts, and related broccoli seed preparations, 
and the absence of severe or serious risks (no Grade II toxicities have been observed 
in > 500 patients) (see Table 3 in Clincal Protocol) , we do not anticipate serious 
adverse events to be associated with the administration of Avmacol® to study 
participants in this proposed trial.  
 
4.1.2. 
Description of Origin of Sulforaphane, Purification, and Stabilization 
 
Sulforaphane (SF) and its precursor GR are naturally occurring substances 
(phytochemicals) found in cruciferous vegetables.  For many years now, the only 
reliable source of SF for clinical trials was our own broccoli sprout extract 
preparation.  Whereas glucoraphanin has in recent years been made available in the 
supplement market by a number of manufacturers, SF has continued to elude 
commercialization.  Now, with the introduction of Avmacol® to the dietary 
supplement market, a vetted, stable source of SF is available to clinical trial 
participants, and could be purchased by them or by others, even after their 
involvement in clinical studies ends.  This is something that has not been possible 
with previous SF-rich broccoli sprout extract preparations. 
 
The biologically active phytochemical in Avmacol® is glucoraphanin (GR; 4-
methylsulfinylbutyl-glucosinolate), the precursor of sulforaphane (1-isothiocyanato-
(4R)-(methylsulfinyl)butane). Sulforaphane (SF) is the aglycone breakdown product 
of the GR. The molecular formula of SF is C6H11NOS2, and its molecular weight is 
177.29 daltons. The structural formulae of GR and SF are shown in Figure 1. After 
ingestion and absorption, SF is conjugated with glutathione by glutathione-S-
transferases (GSTs), then metabolized sequentially by -glutamyltranspeptidase 
(GTP), cysteinylglycinase (Cgase), and N- acetyltransferase (NAT) to form 
mercapturic acids, the predominant metabolites of SF found in urine (Figure 2) 
(26,27,31). 
 
 
 


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Figure 1. The Structural Formulae of Glucoraphanin and Sulforaphane 
 
 
 
 
Figure 2. Metabolism of Glucoraphanin in Humans 
 
 
 
 
 
 
 
 
Sulforaphane conjugates quickly accumulate in various types of cells in culture, 
reaching intracellular concentrations in the millimolar (mM) range (33). Intracellular 
accumulation is achieved through conjugation with cellular glutathione (34), a 
reaction that is accelerated by glutathione S-transferases (35), and the glutathione 
conjugate is then exported by a transporter-mediated mechanism (36). SF activates 
the Nrf-2 pathway resulting in elevated gene transcription via the Antioxidant-
Response Element in the regulatory domain of its target genes (28). 
4.1.3 
Physical, Chemical, and Pharmaceutical Properties and Formulations 
The active ingredient in Avmacol® (as in broccoli sprout extracts) is glucoraphanin 
(GR; 4-methylsulfinylbutyl-glucosinolate).  The biologically active metabolite of this 
inert plant precursor, sulforaphane (SF; 1-isothiocyanato-4R-(methylsulfinyl)butane) 
is formed by the enzymatic action of myrosinase released when sprout tissues are 
damaged or chewed. SF conversion is maximized in broccoli 
sprout extracts through the addition of small amounts of 
myrosinase-containing daikon sprouts. Sulforaphane is an 
isothiocyanate. Its molecular formula is C6H11NOS2, and its 
molecular weight is 177.29 daltons. The structural formula of SF 
is: 
Sulforaphane 


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Avmacol® is a proprietary supplement manufactured using industry standard 
tabulating and tablet-coating methods.  In short, a broccoli seed and sprout extract 
(GR and myrosinase source) and excipients are pressed into tablets and then coated 
using industry standard technology.     
 
 
4.1.4 Non-Clinical Pharmacology 
4.1.4.1 
Pharmacokinetics 
Analysis of the pharmacokinetics of oral SF was performed using male Fischer rats 
receiving a single 50 mol oral dose of SF. The average plasma concentrations of SF 
are illustrated in Figure 3. The plasma concentration of SF increased very rapidly, 
detectable at 1 h and peaking at 20.8 M, 4 h after dosing. SF displayed fairly rapid 
absorption and an elimination half-life of 2.23 h (1). 
 
4.1.4.2 
Pharmacodynamics 
 
Effects on gene expression 
 
Hu et al. investigated the effects of two oral doses of 50 mol of SF given at 0 and 24 
hours on gene expression profiles by DNA microarray on male Fischer rat liver in 
vivo.  562 genes were found to be 2-fold up- or down-regulated at one or more time 
points (2, 4, 12, 24, 48 hours) compared to control. The most robust induction was of 
the metallothionein-like genes (MT-1/2 and MT-1a), which are considered important 
antioxidant genes, which increased up to 10-fold after SF dosing. Effects of this high 
Figure 3. Plasma concentrations of SF with respect to time after a 50 µmol oral dose of 
SF in rats with three animals per time point. Results are mean ± S.D. The curve is the fitted 
plasma concentrations to a one-compartmental model with first-order absorption using 
WinNonlin (1). 


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dose oral SF on selected cytochrome P450 genes were statistically significant with 
up-regulation of CYP 19, 3A3, 3A9, 1B1, 2B19, and 4B1 to 3.28-, 2.88-, 2.78-, 2.49-, 
2.04-, and 2.05-times baseline, respectively. Cytochrome P450 2C29 and CYP1A1 
were lowered to 20% and 72% of baseline levels, respectively (1). 
 
Comblatt et al. investigated the effects of a single oral dose of 150 micromol of SF on 
gene and protein expression in Sprague Dawley rats. A maximal 12-fold induction of 
NQO1 transcripts was observed in the mammary gland 12 h after dosing and there 
was significant induction as early as 2 h. A biphasic pattern of heme oxygenase-1 
(HO-1) transcript induction was observed, with an initial peak at 2 h followed by a 
subsequent peak at 12 h. The minimal time to statistically significant HO-1 induction 
was 1 h. A maximal 2.8-fold induction of NQO1 enzymatic activity in the mammary 
gland was observed 24 h after dosing with minimal time to statistically significant 
induction at 4 h (29). 
 
Effects on enzyme expression 
Murine studies of oral SF feeding have examined the effect on detoxication enzyme 
induction. When SF doses of 15 micromole (88.5 mg/kg/day) were administered to 
mice daily for 5 days by gavage, there was a 2.5-fold increase in hepatic NQO1 
activity (37).  Another murine study of SF administered intragastrically at a dose of 
17 micromole (150 mg/kg/day) for 4 days showed 2.6 fold induction of mammary 
gland NQO1 activity, but hepatic levels of the enzyme were not significantly elevated 
(38).  When SF (50 mg/kg/Day) was given daily for 7 Days by gavage to male 
Fischer rats the hepatic NQO1 activity increased significantly, but hepatic CYP 1A 
activity was not affected (37). Notably, no toxic effects were noted in these studies in 
which high doses of SF were administered.  
 
Yoxall et al. (39) performed studies with oral SF in male Wistar rats. Using doses of 
3 and 12 mg/kg daily for 10 days, they examined the effects of SF on cytochrome 
P450 enzymes. In these studies, SF exposure had no obvious adverse effect on the 
animals and body weight gain was unaffected. Treatment with SF elevated levels of 
CYP1A2 nearly doubling the level at the highest dose. However, the newly 
synthesized enzyme was not catalytically competent, at least partly due to SF 
metabolite binding to the enzyme rendering it metabolically inactive. Thus, the net 
effect was that activity of CYP1A2 was depressed. CYP2B expression decreased to 
85% of control only at the higher SF dose. CYP3A2 expression decreased to 80% of 
control with SF treatment only at the higher dose. CYP2E1 was unaffected by SF. 
 
4.1.4.3. Non-clinical Metabolism 
 
Kassahun et al. (40) examined the biotransformation of SF in the rat by administering 
50 mg of SF per kg to male Sprague-Dawley rats. Bile and urine were subsequently 
collected. In bile, five thiol conjugates were detected including glutathione (GSH) 
conjugates of SF, erucin (the sulfide analog of SF), the N-acetylcysteine (NAC) 
conjugates of SF, and the GSH conjugate of a desaturated metabolite of SF, 
suggesting oxidative metabolism of the parent compound. Fecal metabolites were not 
identified. NAC conjugates of SF were detected in the urine. Quantitative urine 


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determinations indicated that 60% of a single dose of SF is eliminated in 24 h as the 
NAC conjugate of SF. Thus, SF undergoes metabolism by S-oxide reduction and 
dehydrogenation. GSH conjugation is the major pathway by which the parent 
compound and its metabolites are eliminated in the rat. 
 
4.1.4.4 Non-clinical Toxicology 
 
A number of animal studies have investigated the biological effects and toxicology of 
oral SF-containing broccoli seeds and SF itself. While these investigations have 
examined a wide variety of endpoints and effects, they have demonstrated the overall 
in vivo safety and beneficial biological effects of broccoli seeds and SF. Animal 
studies are summarized below. 
 
Myzak et al. (41) treated C57BL mice with 6 micromol SF/Day for 10 weeks with no 
adverse effect on body weight, hematocrit, or spleen weight. Taking into account 
species scaling factors, human consumption of 106 g of broccoli seeds daily would 
achieve daily SF intake similar to this murine study. 
Jones et al. (42) treated male F344 rats with SF 50 mg/kg/Day (280 micromol / kg / 
Day) by gavage for five Days. The dose was selected based on reports of non-toxicity 
and efficacy in inducing cytoprotective enzymes in other model systems. During the 
study 2 of 10 animals in the SF treatment group died after feeding due to aspiration of 
the dose. On the sixth Day, after five Days of SF feeding, rats were sacrificed and 
organs examined. The SF was well tolerated by the animals and there was no 
apparent toxicity over the duration of the study. The SF feeding did not affect the 
relative weights of the prostate, kidneys, liver or bladder. Body weight was not 
affected compared to control animals, though there was an 8% decrease in body 
weight in the SF treated group compared to initial weight. The SF dose used in this 
study is >10-times greater than that achieved by the proposed BSP dosing for our 
protocol. 
Zhang et al. (43) treated female Sprague-Dawley rats with BSP to study the effect on 
tissue GST and NQO1 and the urinary excretion levels of isothiocyanate (ITC) 
metabolites. These experiments included administration of BSP providing 40, 80, and 
160 micromol isothiocyanate/kg body weight daily for 14 Days. None of the extract 
doses were associated with any sign of toxicity, with all rats in good health and body 
weight gain not significantly different among treatment and control groups. No gross 
abnormalities were detected at necropsy. No pathological changes were visible in rat 
bladder tissues when examined microscopically. BSP in the doses administered 
effectively induced GST and NQO1 enzyme expression in rat bladder, duodenum, 
and stomach in a dose-dependent fashion. Significant enzyme expression was 
induced in rat colon, kidney, and lungs at the higher dose of broccoli seed extract. 
Measurement of urinary ITC and metabolites by cyclocondensation assay showed 
extensive (70-78%) elimination of the ITC doses within 24 hours. ITCs are known to 
be metabolized in vivo mainly through the mercapturic acid pathway and to be 
excreted in the urine as NAC conjugates. 
4.1.4.5 
Mutagenicity 


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In a study in rats by Yoxall et al. conversion of 2-amino-3-methylimidazolo-[4,5-f] 
18harmacod to mutagenic intermediates (Ames test) was reduced by treatment with 
SF at both 3 and 12 mg/kg/Day (17 and 68 micromol /kg/Day) doses (39). 
4.1.5 Pharmacokinetics and Metabolism of Broccoli Seed Preparations 
4.1.5.1 
Broccoli Sprout Extracts 
Ye et al. (44) conducted a human study with broccoli sprout extracts to provide 
information on plasma, serum, and erythrocyte concentrations of isothiocyanates (ITC) 
after ingestion of metered doses of ITC. Four healthy male human subjects were fed 
single doses of a SF-rich extract made from sprouted broccoli seeds, in which the 
glucosinolates had been hydrolyzed by daikon myrosinase to their cognate 
isothiocyanates, i.e. GR to SF. Cyclocondensation assays were performed on two 
occasions to verify the dose: first at the time of dose preparation and second near the time 
of feeding, with the two analyses within <5% agreement. The four subjects were fed a 
single dose of myrosinase-hydrolyzed extract of sprouted broccoli seeds containing 200 
mol of ITC (77% SF). Maximum urinary excretion rate of ITC and dithiocarbamates 
(DTC) metabolites of ITC, was attained between 1 and 1.5 hours in 3 subjects and 
between 0.5 and 1.0 hours in 1 subject. The cumulative 8 hour urinary excretion of the 
DTCs averaged 58.3  2.8% of the dose. 72 hours after consumption, urinary excretion 
totaled 77.9  6.4 % of the dose, suggesting the isothiocyanates did not react irreversibly 
with proteins or other macromolecules, but were rapidly converted to dithiocarbamate 
metabolites. The plasma and erythrocyte concentrations and urinary excretion rates 
plotted in Figure 4 show that the pharmacokinetic behavior of the ITC was very similar 
in all 4 human subjects. ITC/DTC levels were detected in plasma/erythrocytes 15 
minutes after dosing and the concentration rose to a maximum in the 1 hr sample of all 
subjects with a mean maximum of 2.00  0.30 mol/l; range: 1.62 – 2.27 micromol /l. 
Between 2 and 6 hours after dosing, plasma concentrations of ITC/DTC declined with 
first-order kinetics with a mean half-life of 1.77  0.13 hours. The calculated renal 
clearance is 369  53 ml/min, which is more than double the glomerular filtration rate for 
the subjects, suggesting tubular secretion plays a major role in the elimination process. 
The bioavailability of SF in humans is believed to be 60-90%. From these data, an 
apparent volume of distribution is 59.9  7.0 liters which is consistent with ITC/DTC 
distribution in total body water (27,44). Despite the relatively short half-life, it is believed 
that SF has a longer duration of action since most of the induced phase 2 enzymes have 
half-lives measured in days. Thus, a high cellular concentration need not be maintained 
continuously (45). 


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4.1.5.2  Broccoli sprout extract without daikon  
 
We conducted a randomized, Phase Iia human study enrolling 200 subjects in 
Qidong, China. Healthy subjects were randomized to receive an extract of sprouted 
broccoli seeds (“tea”) containing 400 mol GR or placebo nightly for 2 weeks in an 
attempt to modulate carcinogen markers. Subjects were followed clinically for 
compliance and adverse events. Serum ALT and urinary dithiocarbamate (DTC) 
levels were assessed. DTC elimination varied up to 3-fold between participants but 
was consistent between doses within an individual, suggesting bioavailability of 
active isothiocyanates (SF) was variable among the population. Importantly, the GR 
in the extract was not treated with myrosinase and thus relied upon enteric bacterial 
flora for conversion to SF. Thus, the investigators speculate that differences in 
individual gastrointestinal flora may account for the variability. The broccoli seed tea 
was well tolerated.  All 199 subjects completed all doses in the study without 
reporting adverse events. There was a strong inverse association between DTC 
excretion levels and excreted carcinogen biomarkers for aflatoxin and polyaromatic 
hydrocarbons, suggesting that ingestion of extract from sprouted broccoli seeds may 
favorably influence the metabolism pathway of foodborne and airborne toxicants 
(32). 
 
4.1.5.3  Broccoli sprout extract with daikon  
 
Egner et al. conducted a cross-over clinical trial in Qidong, China in 2009 to compare 
the bioavailability and tolerability of SF from beverages made with two sprouted 
broccoli seed powders: one glucoraphanin-rich (GR-rich) powder and the other 
sulforaphane-rich (SF-rich) powder. SF was either generated in vivo from the GR 
contained in the GR-rich powder by gut microflora or formed by ex vivo treatment of 
GR-rich powder with myrosinase from daikon (Raphanus sativus) seeds to generate 
SF-rich powder. Fifty healthy, eligible participants were requested to refrain from 
crucifer consumption and randomized into two treatment arms. The study design was 
Figure 4. Dithiocarbamate 
Concentration Changes in 
Plasma, Erythrocytes, and 
Urinary Excretion Rates 
Over Time.  
Semi-logarithmic plot of the 
time course of changes in 
the concentrations of 
dithiocarbamates in the 
plasma (upper panel) and 
erythrocytes (middle), as 
well as the rates of urinary 
excretion (lower) of four 
volunteers who received 
197-210 umol of broccoli


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as follows: 5-Day run-in period, 7-Day administration of beverages, 5-Day washout 
period, and 7-Day administration of the opposite intervention. Isotope dilution mass 
spectrometry was used to measure levels of GR, SF and SF thiol conjugates in urine 
samples collected daily throughout the study. Bioavailability, as measured by urinary 
excretion of SF and its metabolites (in approximately 12 hour collections after 
dosing), was substantially greater with the SF (mean = 70%) than with GR-rich 
(mean = 5%) beverages. The primary urinary metabolite of following administration 
of either GR-rich or SF-rich powder was the acetylcysteine conjugate of SF.  Inter-
individual variability in excretion was considerably lower with the SF-rich than GR-
rich beverage. Elimination rates were considerably slower with GR-rich beverages 
allowing for achievement of steady state dosing as opposed to bolus dosing with SF-
rich beverages. Results from this trial led us to consider blends of SF and GR as SF-
rich and GR-rich mixtures to achieve peak concentrations for activation of some 
targets and prolonged inhibition of others implicated in the protective actions of SF. 
The Avmacol® to be administered during this trial follows this pharmacokinetic 
principle, as the bioactive ingredients include broccoli seed powder, containing both 
GR and myrosinase which is responsible for consistent in vivo delivery of 40% of the 
GR content as SF (46). 
 
4.1.5.4 
Cruciferous Vegetables 
 
Shapiro et al. conducted human studies on DTC excretion after cruciferous vegetable 
ingestion. Four healthy male volunteers were fed 250 grams of a cruciferous 
vegetable. Isothiocyanate levels were measured in each vegetable administered and 
contained 0.3 – 0.8 mol/g fresh weight. Thus ITC dosing ranged from 75 – 200 
micromol per vegetable dose. All 4 volunteers demonstrated a brisk rise in DTC 
urinary excretion after ingestion of crucifer doses. Excretion generally reached a peak 
in the first 8-h collection, was  80% complete in 24 h and returned to baseline by 72 
h after dosing (27). 
 
Lastly, Fowke et al. conducted an interventional trial in women over the age of 45 
years. Participants attended a series of classes designed to facilitate the addition of 
cruciferous vegetables to the daily diet. Urine collection for ITC concentration was 
performed at baseline and during the 4-week intervention phase. The amount (grams) 
of cruciferous vegetables ingested was recorded during the baseline and intervention 
period. During the 4-week intervention phase, subjects consumed an average of 196 
grams of cruciferous vegetables per Day (range 53-371 g). Group-average ITC 
excretion levels in urine followed the trend of vegetable intake with significant 
increases from Baseline to Intervention (P < 0.01) and a decrease from the 
Intervention to Post-intervention phase of the study (P <0.01). No adverse reactions 
related to the dietary intervention were reported (47). 
 
4.1.5.5 
Broccoli preparations containing active myrosinase. 
 
The bioavailability of a simple powder made by lyophilizing a boiling water extract 
of GR-rich broccoli sprouts (broccoli sprout extract; myrosinase de-activated by 
processing methodology), was compared with a variety of preparations containing 


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active myrosinase (BSdP - broccoli seed powder; BsdE - commercially prepared 
broccoli seed boiling water extracts that are spray-dried; FDBS - freeze-dried 
broccoli sprouts).  Multiple sample matrices and modes of delivery showed that 
regardless of delivery method, providing active myrosinase as part of the preparation 
enhanced the bioavailability of SF and reduced the variability of conversion (GR to 
SF) which can be ascribed solely to the gut microbiota.  Mean bioavailability of GR 
preparations lacking active myrosinase, as we have shown in previous studies, was 
roughly 10% of dose, whereas when active myrosinase was included in the dose, 
bioavailability increased to about 40%, see Table 1.  Both within-subject and 
between subject variability was also reduced, and when hydrolysis was accomplished 
ex-vivo, prior to dosing, bioavailability was closer to 90% (48).  
 
 


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Table 1. Mean bioavailability of sulforaphane from broccoli sprout and seed preparations 
rich in glucoraphanin, as affected by matrix, mode of delivery, and activity of myrosinase 
on its substrate (48) 
 
No. Cohort Matrix 
Mode of 
delivery 
Dose 
(µmol 
GR) 
Mean 
availability 
(as % of 
dose) 
Mean 
availability 
(as % of 
SF) 
No. 
subjects 
enrolledf 
No. 
subjects 
assessed 
1 
1 
BSEa 
Dissolved 
in water 
50 
9.4 
̶ 
5 
5 
2 
2 
BSE 
In gelcaps 
69 
12.8 
̶ 
20 
16 
3 
2 
BSE 
In gelcaps 
230 
8.3 
̶ 
20 
18 
4 
3 
BSdEb 
In gelcaps 
69 
11.2 
̶ 
20 
17 
5 
3 
BSdE 
In gelcaps 
230 
9.7 
̶ 
20 
19 
6 
4 
BSdPc 
In standard 
gelcaps 
100 
32.6 
̶ 
5 
4 
7 
4 
BSdP 
100 
31.7 
̶ 
5 
4 
8 
4 
BSdP 
100 
44.1 
̶ 
5 
4 
9 
5 
FDBSd 
100 
35.1 
̶ 
5 
5 
10 
5 
FDBS 
In acid-
resistant 
gelcaps 
100 
32.7 
̶ 
5 
5 
11 
6 
FDBS 
Pre-
hydrolyzede 
in 
pineapple-
lime juice 
50 
33.6 
n.d. 
5 
5 
12 
6 
FDBS 
50 
48.4 
97.6 
5 
5 
13 
6 
FDBS 
50 
40.2 
91.4 
5 
4 
14 
6 
FDBS 
100 
41.8 
84.3 
5 
4 
15 
6 
FDBS 
200 
40.9 
83.9 
5 
4 
 
a Broccoli Sprout Extract; b Broccoli Seed Extract produced commercially as OncoPLEXTM 
(from Xymogen); c Broccoli Seed Powder (with active myrosinase); d Freeze-Dried Broccoli 
Sprouts (with active myrosinase); e Myrosinase-converted for 10’ at room temperature in juice; f 
A subject pool of 5 volunteers participated in most tests described (numbers 1-15 above). They 
were augmented with another 15 volunteers for tests numbered 2-5 above. 
 
4.1.6 Safety and Efficacy  
 
Cruciferous vegetables, including broccoli seeds and broccoli seed sprouts, are 
generally regarded as safe and are regular dietary components in many regions of the 
world. Previous estimates of the daily dietary intake of cruciferous vegetables vary 
regionally, averaging 40 g/Day in Singapore (49), 11 g/Day in the United States (50), 
16 g/Day in Canada (51), 30 g/Day in the UK (52), and 112 g/Day in Japan (53).  
 
4.1.6.1 
Broccoli Sprout  
 
A number of small pilot clinical studies have evaluated the effect of broccoli sprouts 
on antioxidant endpoints:   
 


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Murashima and colleagues reported in 2004 on the elevation of multiple biomarkers 
of oxidative stress following a one-week course (6 young male and 6 young female 
smokers) of 100 g/d of fresh broccoli sprouts (54). Plasma markers were measured 
before and after treatment. Treatment produced decreases in serum total cholesterol, 
LDL cholesterol, coenzyme Q10, plasma 23 harmaco and phosphatidylcholine 
hydroperoxide, and urinary 8-isoprostane, and 8-OhdG. Increases in CoQ10H2/CoQ10 
ratio, and HDL cholesterol were observed.  Blood lymphocyte markers, natural killer 
cell activity, triacylglycerol, urea nitrogen, uric acid, AST, ALT, γ-GPT, and plasma 
amino acids were also measured and there were no before- to after-treatment 
differences in any of these biomarkers. 
 
In 2009, a group at UCLA reported on the ability of orally administered broccoli 
sprout homogenates (BSH) to increase phase 2 antioxidant enzymes in the upper 
airway (55). After feeding 57 subjects doses of BSH ranging from 25 to 200 grams on 
3 separate days, followed on the 4th day by blood and nasal lavage collection, there 
was a dramatic and dose-dependent increase in phase 2 enzyme expression (mRNA 
for GSTM1, GSTP1, HO-1, and NQO1). Induction of individual phase 2 enzymes in 
nasal lavage cells was strongly correlated. No serious adverse events were reported, 
and dose intolerance or side effects of broccoli sprouts were not observed, and mild, 
digestive effects are presented in the report.   
 
Christiansen et al. (56) report that ingestion of broccoli sprouts does not improve 
endothelial function in human beings with hypertension (n = 40 hypertensive, non-
diabetic subjects with cholesterol in the normal range). Subjects were fed 10 g of 
dried broccoli sprouts for 4 weeks, and their blood pressure, endothelial function 
(measured by flow-mediated dilation), and blood samples were obtained every other 
week. Glucoraphanin content of the sprouts was measured, and equated to a dose of 
259 µmol GR/day – a reasonable level, but in light of our findings perhaps not high 
enough to expect a measurable effect due to low conversion of GR to SF by the 
intestinal microflora of individuals. 
 
4.1.6.2 
Broccoli Sprout Preparations (BSPs) and Extracts (BSEs) 
 
Shapiro et al. (31) conducted a double-blind, placebo controlled, randomized, Phase I 
study of two BSPs, beverages containing SF-rich or GR-rich powder made from 
sprouted broccoli seeds, in healthy individuals to determine the safety and tolerance 
of repeated oral administration. Twelve healthy human volunteers received doses of 
BSP every 8 hours for 7 Days (total 21 doses) while undergoing clinical evaluation 
and a battery of laboratory tests. Doses used in 3 cohorts of 3 subjects and 1 control 
were 25 mol glucosinolates, 100 mol glucosinolates, and 25 mol isothiocyanates, 
respectively. Thus subjects received 75 – 300 µmol glucosinolates daily, equivalent 
to 12 - 50 g of fresh broccoli seeds, or 75 µmol isothiocyanate. No clinical adverse 
events were reported. With regard to laboratory testing, samples were obtained 6 
times during the 19 Day study and included evaluation of the blood for the following: 
CBC with differential, reticulocyte count, PT, PTT, BUN, creatinine, Na, K, CO2, Cl, 
glucose, albumin, direct/total bilirubin, alkaline phosphatase, AST, ALT, GGT, T3, 
T4, TSH. Urinalysis was performed for urine creatinine, and urine dithiocarbamates 


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(DTC). Two of 12 individuals (both receiving active preparations) showed an 
increase in plasma ALT exceeding the upper limit of normal with one meeting 
criteria for a Grade 1 toxicity. Notably, ALT levels rose for all subjects during the 
course of the study including placebo-treated subjects. Plasma AST levels rose above 
normal on Day 19 for 2 of 12 subjects. They were released from the inpatient portion 
of the study on Day 17 and their post-discharge activities (i.e., possible alcohol 
intake) could not be ascertained. Monitoring of TSH levels demonstrated that in 3 of 
12 subjects (2 active treatment, 1 placebo), TSH levels exceeded the upper limit of 
normal during or after the dosing period. Notably, TSH levels rose for 11 of 12 
subjects during the first 6 Days of hospitalization before broccoli seed administration 
was begun. TSH increases were not associated with any clinical symptoms or 
abnormalities of T3 or T4. Evaluation by 2 independent endocrinologists determined 
that the changes in TSH were mild and reversible and did not pose an obstacle to 
further studies with administration of BSPs. No other significant laboratory 
abnormalities occurred. Thus, this Phase I safety study in healthy volunteers revealed 
no evidence of systematic, clinically significant adverse effects that could be 
attributed to the administration of repeated doses of broccoli sprout extracts 
containing SF or GR (31). 
 
Cornblatt et al. (29) conducted a study to assess the bioavailability of SF-rich 
broccoli sprout extract in human breast tissue. In this proof-of-principle study, 8 
women undergoing elective mammoplasty consumed a preparation containing 200 
µmol of SF approximately 50 minutes prior to surgery. The extract was well-tolerated 
without any adverse events or complications. SF metabolite levels measured by 
cyclocondensation reaction as dithiocarbamates were used to determine SF 
distribution. Mean post-dose plasma dithiocarbamate (DTC) level was 0.92  0.72 
M, and mean epithelial/stromal enriched breast tissue DTC concentration was 1.45 
 1.12 and 2.00  1.95 picomol/mg tissue for right and left breast respectively. In 
addition, the investigators were able to measure NQO1 and HO-1 transcripts in the 
human breast tissue, demonstrating the feasibility of assessing a 24harmacodynamics 
action of SF in these tissues (29). 
 
In an ongoing randomized clinical trial at Johns Hopkins, 21 women completed a 10 
day intervention of broccoli sprout extract or placebo (mango juice). There were only 
three grade 1 mild gastrointestinal adverse events reported and no significant changes 
(1.5 times or greater) in bloods (comprehensive metabolic panel, full blood count, 
coagulation panel and thyroid tests), taken pre and post intervention (personal 
communication K. Visvanathan). 
 
In the cross over trial conducted by Egner et al  (46), 2 of the fifty participants 
randomized to receive the SR-rich beverage (150 µmol/day) during either the first or 
second wave complained of nausea or bitter taste and dropped out of the study. 
Serum chemistry studies conducted on samples obtained after the last of seven daily 
doses of SF did not present any abnormal values. This study suggests that 150 µmol 
SF/day, approximates the maximum tolerated dose. 
 


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A very recent report from Iran, describes the effect of oral broccoli sprouts on a 
variety of oxidative stress biomarkers in a double-blind, placebo-controlled, 
randomized controlled trial, in type 2 diabetes patients.  Eighty-one patients were 
randomly assigned to one of three treatment groups for 4 weeks. They received either 
10 g/day, or 5 g/day broccoli sprout powder (BSP), or a placebo of cornstarch and 
chlorophyll (57). The authors report that consumption of BSP resulted in a significant 
decrease in malondialdehyde, oxidized LDL cholesterol, and “oxidative stress index”, 
and a significant increase in total serum antioxidant capacity. They found no 
significant effect on “total antioxidant status” or fasting blood glucose.  Since this 
study employed an over-the-counter broccoli sprout supplement and the authors did 
not make independent determinations of its SF content, and since the fasting blood 
glucose values for the three groups were not closely matched, there is substantial 
concern about the robustness of reported results, although there do appear to be clear 
trends for difference. 
 
In a very recent study of men with recurrent prostate cancer at the Knight Cancer 
Institute in Portland, Oregon, treatment with 200 µmol of sulforaphane per day for 20 
weeks produced no major side effects and adverse events were mostly grade 1 
gastrointestinal (58). 
 
Dr. Kensler and his colleagues conducted a randomized phase II trial from October 
2011 to January 2012, in which 267 healthy volunteers in Qidong City, China (a 
region with extremely high liver cancer prevalence) received 84 consecutive daily 
doses of a broccoli sprout beverage containing 600 µmol of glucoraphanin and 40 
µmol of sulforaphane, or an indistinguishable placebo (17). The study participants 
comprised 136 treated with broccoli sprout beverage and 131 placebo controls. Of the 
267 participants completing the study, 50% consumed all assigned doses, and the 
remaining participants consumed at least 80 of the 84 doses. Extensive blood 
chemistries at the termination of the study showed that the means of 13 analytes were 
identical between treated and control groups, and that specifically no abnormalities 
were detected in BUN, creatinine, and transaminases (ALT and AST). Three 
individuals, 2 in the placebo arm and 1 in the treated arm, had slightly elevated total 
bilirubin (1.1, 1.6, 1.8 mg/dL) and two individuals, 1 each in placebo and treated 
arms, had slightly elevated direct bilirubin (0.5, 0.6 mg/dL levels).  
4.1.7 
Rationale for Selected Doses 
We will use doses of Avmacol® that will provide equivalent concentrations of SF as 
found in our most recent trials of various BSP preparations. Four Avmacol® tablets 
twice daily will provide an internal dose of 120 micromole SF per day.  The dose 
selection is based on the results of our previous clinical trials (NCT02023931) with 
the maximum tolerable dose and minimal toxicity effect to maximize the biological 
effect (17,59). 
This dose level represents bioactive and tolerable doses in healthy volunteer studies. 
This dose was found to be tolerable and without safety compromise with no grade 2 
or higher toxicities reported.  Although bad taste was the main complaint by study 
subjects consuming beverage made from SF-rich sprouted broccoli seed powder at 
150 μmol/day, the encapsulation of Avmacol® is expected to mask any issues with 


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taste. This dose is expected to achieve a target level of NRF2 pathway activation 
based on a previous healthy volunteer study (59).  The twice-daily dosing schedule 
will provide a steady level of in vivo SF over the 24 hr a day, as shown in a recent 
report (60).   
4.2 
Rational for Proposed Study in High-Risk Former Smokers 
It is critical to carefully define entry criteria for any lung cancer prevention trial.  
Study subjects must be of high enough risk that either lung cancer or intermediate 
endpoints will be found as frequently as possible.  We have created a unique high-risk 
cohort of approximately 3,600 current and former smokers, the Pittsburgh Lung 
Screening Study (PluSS).  At baseline, we administered a risk factor questionnaire, 
tested pulmonary function, assessed low-dose computed tomography (LDCT) for 
emphysema severity, created a biorepository (serum, plasma, and  DNA), and 
implemented procedures to ascertain and characterize new lung cancer diagnoses.  At 
enrollment, PluSS participants had a mean age of 60 years. Among the participants, 
49% were women; 93% whites, 5.5% blacks, and 1.7% other race/ethnicity groups; 
60% current smokers and 40% former smokers with a median 47 pack-years of 
smoking history (61). The PluSS participants resembled the national representative 
sample of current and former smokers who are at high risk for lung cancer. For 
example, the National Lung Screening Trial (NLST) enrolled more than 53,000 
persons at high risk for lung cancer at 33 medical centers across the US.  The mean 
age was 61 years. Forty-one % were women; 91% whites, 4% blacks, and 5% other 
race/ethnicity groups; 48% current smokers and 52% former smokers who quit 
smoking within the previous 15 years; a median 48 pack-years of smoking history 
(62). 
This unique cohort has contributed many scientific discoveries in risk biomarkers of 
lung cancer (61,63-72). For example, the presence of chronic obstructive pulmonary 
disease (COPD) significantly increased lung cancer risk (61,66). Compared with 
smokers without COPD, smokers with COPD measured as the Global Initiative for 
Chronic Obstructive Lung Disease (GOLD) II or higher or Forced Expiratory Volume  
in the first second (FEV1) < 80 % had more than doubled risk of developing lung 
cancer after controlling for smoking intensity and duration (Table 2). 
Table 2. Hazard ratios (HRs) of lung cancer associated with baseline airflow obstruction 
and radiographic emphysema in the Pittsburgh Lung Screening Study 
 
Cases 
Noncases 
Adj. HR (95% CI)* 
Airflow obstruction 
 
None 
32 
2053 
1.00 
GOLD I 
16 
477 
1.66 (0.89-3.11) 
GOLD II 
36 
792 
2.11 (1.27-3.49) 
GOLD III-IV 
15 
217 
2.86 (1.48-5.53) 
P for trend 
 
 
0.005 
Radiographic emphysema 
 
 
None 
24 
2068 
1.00 
Trace 
22 
663 
2.58 (1.43-4.66) 
Mild 
37 
493 
5.04 (2.94-8.62) 
Moderate/severe 
16 
315 
3.20 (1.65-6.23) 
P for trend 
 
 
<0.0001 
* Adjusted for age, sex, years of cigarette smoking, and number of cigarettes per day. 
 


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The presence of mild, moderate or severe radiographic emphysema on the LDCT 
scans at baseline was significantly associated with a 3- to 5-fold increased risk of 
developing lung cancer compared to smokers without emphysema (61). 
 
We will recruit subjects for the present study from the existing PluSS participants 
who had mild, moderate or severe emphysema or GOLD II-IV COPD. Among the 
original PluSS participants who, as of 12/31/2015, were <75 years old, alive, free of 
cancer, and had quit smoking within the past 15 years, 356 had GOLD II-IV COPD 
(with or without emphysema), and additional 167 with mild, moderate or severe 
emphysema (without COPD). All these 523 subjects are potentially eligible for the 
proposed study. Based on our previous experience in the Iloprost study (22), which 
used a very similar protocol as the one proposed here, there was a 20% recruitment 
rate. Thus, we expect to fulfill our recruitment goal of at least 72 former smokers into 
the study within the first 3 years of the proposed study.  With a 20% dropout rate 
based on our previous experience (22), we expect to have 60 study subjects who will 
complete the entire course of the trial.  
 
If the proposed chemopreventive agent is effective, it will bring beneficial effect for 
study participants on reducing their risk of lung cancer. After the end of the study, all 
study participants can conduct their oral intake of Avmacol® since this dietary 
supplement is relatively cheap and commercially available. 
 
5. 
TREATMENT PLAN AND STUDY PROCEDURES 
5.1 
Pre-Treatment Evaluation 
Pretreatment tests should be performed within 90 days prior to the randomization. 
1)   Signed informed consent 
2) Questionnaire data collection on patient’s characteristics and  smoking history.  
3) Physical exam including height, weight, vitals and SWOG performance status. 
4)  FEV1/FVC 
5) Blood oxygen saturation 
6) Expired Carbon Monoxide 
7) Laboratory analysis including  CBC  and serum chemistries such as total 
bilirubin, AST, ALT,   BUN, creatinine, . 
8) Baseline bronchoscopy: Prior to randomization, all participants will undergo a 
bronchoscopy at the Endoscopy Suite at the UPMC Shadyside Hospital. The 
procedure will be carried out by Dr. Wilson (Co-PI) using Narrow Band Imaging 
(NBI) white-light bronchoscopy.  The patient’s nose, throat, vocal cords and 
windpipe are sprayed with Lidocaine (numbing medicine) to help keep patient 
from coughing and to numb patient’s airways.  During the procedure, Dr. Wilson 
will perform a complete airway examination including visualization of the vocal 
cords, trachea, main carina, and orifices of the sub-segmental bronchi to the 
extent that it is visible without causing trauma to the bronchial wall.  Biopsy 
samples will be collected at the pre-determined sites. Some of these biopsies will 
be used for the pathological examination. Any patients with lung cancer or 
carcinoma in situ will be ineligible for the present study. We will inform subjects 
the abnormal  biopsy results and provide appropriate counseling from qualified 
medical personnel.  


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Since the clinical significance of moderate or severe dysplasia is unknown and no 
effective treatment available, we will not conduct additional clinical procedure or 
intervention on these patients beyond routine follow-up and care. The patients 
with dysplasia will be monitored at each clinical visit (once every three months 
during the treatment period) according to the study protocol.  At the end of study 
period, another biopsy at the same site of initial locations that show dysplasia will 
be performed to evaluate the change (progress, regress or no change) of these 
lesions detected at baseline biopsy. This is one of the primary outcome measures 
for the proposed study.  After the completion of the study, the patients will 
continue to receive routine medical care including annual CT screening if eligible. 
5.2 
Randomization 
Eligible subjects will be randomly assigned to either Avmacol® or placebo arm with a 
50%-50% chance. Each subject will be given three bottles of tablets at month 0 and at 
trimonthly clinical visits. Each bottle contains 250 tablets that will be sufficient for one 
month supply.  
5.3 
Treatment Plan 
5.3.1 Nutraceutical Information 
Please see the Avmacol® Nutraceutical Information Guide for detailed information 
on manufacturing and quality assurance (Appendix 11). 
For this study, capsules will be shipped from Nutramax to the University of 
Pittsburgh as follows:  
c/o Brian M. Miller, PharmD 
Director, Investigational Drug Service 
UPMC Hillman Cancer Center 
Ground Floor, AG40.3 
5115 Centre Avenue 
Pittsburgh, PA 15232 
Avmacol® capsules will be stored at room temperature in the Investigational Drug 
Pharmacy at the UPMC Hillman Cancer Center. After dispensing, they may be stored 
at room temperature at the participant’s home. 
5.3.2 Dose  
Following randomization, subjects will begin to take four study tablets (Avmacol or 
placebo) in the morning with breakfast and four tablets in the evening with dinner. 
Each tablet contains 15 mg of glucoraphanin (GR), but contains no SF. Thus the daily 
ingested dose is 120 mg or 274.29 micromole GR (molar mass 437.49). Based on our 
most recently completed bioavailability studies in humans (48), the internal 
conversion rate of GR to SF in standard gelcaps (the same formula for the present 
study) was 44% (see Table 1 – Cohort 5 under Section 5.5.1 Pharmacokinetics and 
Metabolism in Humans on page 23).  Thus the 8 Avmacol tablets provide a daily 
internal dose of approxi 120 micromole SF (274,29 x 0.44 = 120.68 micromole SF). 
5.3.3 Treatment Duration 
The Avmacol tablets will be administered to study participants for a total duration of 
12 months.  Data regarding the duration of SF administration on altering cellular 


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markers in human bronchial tissues are lacking. In a recent phase 2 clinical trial 
lasting 2-8 weeks, Atwell et al. evaluated the effect of SF on level of Ki-67 LI in 
breast benign tissue in women who underwent diagnostic biopsy and surgical 
treatment for breast cancer.  The daily dose of 513 micromole gluroraphanin (SF 
precursor) significantly reduced Ki-67 expression in the benign breast tissue (P<0.01) 
(16).  We note the conversion rate for this formulation of glucoraphanin to SF was not 
determined by the authors (16). It is likely lower than our Avmacol formulation: 
typically 5% in the absence of myrosinase. Our Avmacol tablets provide optimal 
internal dose of SF on apoptosis/proliferation markers, by pushing the duration of SF 
administration to 12 months. 
5.3.4    Recording of Daily Intake Dose  
To track the daily intake of study tablets, study participant will be required to keep a 
diary to record the number of tablet taken and the times of the day when the test 
tablets are taken (See Appendix 7. Log Form for Daily Intake of Test Tablets).   
5.3.4 Criteria for Removal from Study 
Given the absence of dose-limiting toxicities described in prior clinical trials, 
clinically significant toxicity is not expected from exposure to Avmacol® tablets.  
However, if a participant shows symptoms or signs of Grade ≥2 toxicity attributable 
to Avmacol®, the participant will be withdrawn from the trial and the study 
supplement will be discontinued.  Follow-up will be maintained until the abnormality 
is resolved.  
Participants maintain the right to leave the trial of their own volition at any point.  
5.4 
Dosing Delays/Dose Modifications 
There are no plans for a dose modification in the study.  
5.5 
Clinical Visits 
Besides two clinical visits in stage 1 of the study for eligibility screening, all 
eligibility study participants will begin taking the study tablets at visit 1 of stage 2 of 
the study and  return to the study clinic at the end of months 3, 6, 9, and 12 (the end 
of treatment period).  At each clinical visit, the clinical research nurse/assistant will 
conduct the following: 
1) Exam the subject’s diary for dosing and timing of intake of test tablets, and keep 
the diary in subject’s folder. 
2) Count and record the remaining tablets given. 
3) Provide new study tablets that will supply the next 3 months. 
4) Study participant will turn in the self-administered questionnaire for recording 
his/her intake of cruciferous vegetables in the past 3 months using a dietary 
frequency questionnaire (Appendix 5).  
5) Collect blood, spot urine and nasal brushing samples 
6)  Conduct pulmonary function test: FEV1/FVC 
7)  Collect safety and toxicity measures at each clinical visit (once every 3 months) 
during the 12-month treatment period:  
a) Collect adverse events (AEs) using the AE Recording Form (Appendix 8) 


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b) Conduct physical examination including body weigh, height, and vitals by a 
registered nurse and record the results in the patient’s study file 
c) Laboratory evaluation: CBC with platelets and differential, chemistry panel 
including liver function testing.  
8) Only at the end of treatment period, bronchoscopy will be performed on the 
patient using the same procedure as described above in 5.1(8) and bronchial 
biopsies and lavage samples collected (stage 2- visit 5). 
 
5.6 
Withdrawal from the Protocol 
 
1) Unacceptable toxicity  
 
2) Development of cancer 
 
3) Patient refusal 
 
4) Noncompliance 
 
5) Death 
 
5.7 
Off Study Date 
 
The “Off Study” date will be the date of the last clinical visit, i.e., stage 2 – visit 6 
after the second bronchoscopy.  If the patient withdraws from the study earlier, but 
agrees to the post-treatment follow-up, they will be contacted via telephone one 
month after the original off-study date.  
 
5.8 
Post-Treatment Follow-Up  
 
1) Study participants will be contacted by telephone at the end of first month post-treatment for 
general health 
 
2) All study participants will have routine follow-up as we do for the PluSS study participants 
through mails on an annual basis, to fill out a follow-up questionnaire for the diagnosis of 
lung cancer or any other cancer or vital status (filled out by the next-of-the-kin). 
 
 


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6. 
STUDY CALENDAR 
* Day 0 is when the subject is given a treatment assignment number.  
† Study subject ID is assigned right after the consent form is signed by a potential eligible 
subject. This number will be used for the entire study and all forms and sample collection 
sheets. 
 
 
Stage 1 
Stage 2 (after randomization) 
Post-treatment 
Clinical visit 
Pre-clinical 
#1 
#2 
#1 
#2  
#3 
#4 
#5 
#6 
Phone 
Mail 
Targeted Date 
 
 
 
Day 
0* 
Day 
90±
14 
Day 
180
±14 
Day 
270±14 
Day 
360±
14 
Day 
367±
10 
1 mo. 
after 
1 yr. 
after 
Eligibility Screening Form 
Phone 
Interview  
 
 
 
 
 
 
 
 
 
 
MAIL OUT FORMS 
 
 
 
 
 
 
 
 
 
 
 
Locator Form 
(mail) 
(Rec) 
 
 
 
 
 
 
 
 
 
Baseline Questionnaire 
(mail) 
(Rec) 
 
 
 
 
(mail) 
 
(Rec) 
 
 
Cruciferous Vegetable 
intake form (compliance) 
 
 
 
X 
X 
X 
X 
 
X 
 
 
SIGN CONSENT FORM 
(PluSS PID) 
X 
 
 
 
 
 
 
 
 
 
Assign subject ID 
(SF-Seq #)† 
 
SF-001 
SF-002 
… 
 
 
 
 
 
 
 
 
 
PHYSICAL EXAM 
 
 
 
 
 
 
 
 
 
 
 
Vitals, weight & height 
 
X 
 
X 
X 
X 
X 
 
X 
 
 
CO test 
 
X 
 
X 
X 
X 
X 
 
X 
 
 
O2 test 
 
X 
 
 
 
 
 
 
 
 
 
Spirometry (PFT) 
 
X 
 
X 
X 
X 
X 
 
X 
 
 
Urinary Pregnancy Test 
 
X 
 
 
 
 
 
 
 
 
 
BLOOD (for Screening or 
AE Monitoring) 
 
 
 
 
 
 
 
 
 
 
 
CBC/platelets 
 
X 
 
 
X 
X 
X 
 
X 
 
 
Chemistry panel & LFT 
 
X 
 
 
X 
X 
X 
 
X 
 
 
BRONCHOSCOPY  
 
 
 
 
 
 
 
 
 
 
 
Bronchial biopsy 
 
 
X 
 
 
 
 
X 
 
 
 
Bronchial brushing 
 
 
X 
 
 
 
 
X 
 
 
 
Bronchoalveolar lavage 
 
 
X 
 
 
 
 
X 
 
 
 
INCLUSION/EXCLUSION 
FORM 
(X) 
(X) 
(X) 
 
 
 
 
 
 
 
 
TREATMENT 
ASSIGNMENT (TA-Seq #)‡ 
 
 
 
TA-001 
TA-002 
… 
 
 
 
 
 
 
 
Provide study tablets (for 
3 months) 
 
 
 
X 
X 
X 
X 
 
 
 
 
Pill count in returned 
bottles 
 
 
 
 
X 
X 
X 
 
X 
 
 
RESEARCH SAMPLES 
 
 
 
 
 
 
 
 
 
 
 
Blood 
 
 
 
X 
X 
X 
X 
 
X 
 
 
Nasal brushing 
 
 
 
X 
X 
X 
X 
 
X 
 
 
Spot urine 
 
 
 
X 
X 
X 
X 
 
X 
 
 
AE ASSESSMENT FORM 
 
 
 
X 
X 
X 
X 
 
X 
 
 
COMPENSATION 
 
$50 
$500 
$50 
$50 $50 
$50 
$500 
$500 
 
 
Provide parking ticket  
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
FOLLOW-UP 
 
 
 
 
 
 
 
 
 
 
 
General health 
 
 
 
 
 
 
 
 
 
X 
X 
Lung cancer 
 
 
 
 
 
 
 
 
 
X 
X 
Any cancer 
 
 
 
 
 
 
 
 
 
X 
X 


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‡ Treatment assignment number is TA-sequential # of subjects entering into stage 2 of the 
study. the TA # randomly determines the subject’s treatment arm.. 


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7. 
ADVERSE EVENTS: LIST AND REPORTING REQUIREMENTS 
 
7.1 
Adverse Events and Risks 
 
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial. 
The safety reports will be coded using current versions of Medical Dictionary for 
Regulatory Activities (MedDRA). Toxicity severity will be reported using National 
Cancer Institute’s Common Terminology Criteria for Adverse Events (CTCAE) term 
and grade: The descriptions and grading scales found in the revised NCI CTCAE 
version 4.0 will be utilized for adverse event reporting.  All appropriate treatment 
areas should have access to a copy of the CTCAE version 4.0.  A copy of the CTCAE 
version 4.0 can be downloaded from the CTEP web site 
(http://ctep.cancer.gov/protocolDevelopment/adverse_effects.htm). 
 
7.1.1 
Adverse Events for Broccoli Sprout Preparation Avmacol® 
There is no recognized risk from administration of Avmacol® tablets. The previous 
studies using broccoli sprout beverages in humans reported that the most common 
adverse events (AEs) or complaint by study participants was bad taste. As a tablet 
formulation, Avmacol will overcome the bad taste. Other expected AEs in 
gastrointenstinal system are nausea and flatulence.  Table 3 summarizes reported AEs 
in previous studies involved more than 500 study subjects. 
 
There may be adverse events or side effects that are currently unknown. Everyone 
taking part in the study will be monitored  carefully for any side effects, including 
routine physical examinations, complete blood count (CBC), blood chemistry 
including liver function test, and food intolerances.  All AEs and toxicity measures 
will be evaluated as one of the objective of the present study. 


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Table 3. Summary of Reported Adverse Events (AEs) in Studies Using Sulforphane or Sulforaphane Precursor Glucoraphanin in Humans 
Study 
(reference)* 
Design 
Subjects 
Dose* 
Treatment 
duration 
AEs 
Kensler et 
al(32) 
Randomized  placebo-
controlled phase 2  study 
200 healthy volunteers  
400 µmol glucoraphanin 
(GR)/day in hot water 
infusion of broccoli sprouts 
14 days 
No reported AEs 
Shapiro et 
al(31) 
Randomized placebo 
controlled phase I study  
12 healthy volunteers 
75-100 µmol GR/day 
7 days 
No clinical AEs; No abnormal chemistry test results.  
2/12 subjects had ALT >1x ULN (grade 1 toxicity). 
Note: ALT level rose for all subjects including placebo-
treated ones 
Corblatt et 
al(29) 
Single arm study 
8 women undergoing 
elective mammoplasty  
150 µmol SF 
Single dose  
Well tolerated, no any AEs or complications 
Egner et al(46) 
Randomized placebo-
controlled phase 2  study  
50 healthy volunteers 
Broccoli sprout extract 
(BSE) beverage:  800 µmol 
GR/day 
7 days 
No AEs observed 
Egner et al(17) 
Randomized placebo-
controlled phase 2  study 
267 healthy volunteers 
BSE beverage:  600 µmol 
GR + 40 µmol SF per day 
84 days 
10 participants (6 in BSE and 4 in placebo arm) reported 
grade 1 AEs: bad taste and mild stomach discomfort. 
One subject (in BSE) reported vomiting.   Clinical 
values of 13 analytes of blood chemistry and liver 
function test for all participants at the last day of the 
intervention were all normal.  
Atwell et al(16) 
Randomized double-
blind placebo-controlled 
phase 2  study 
54 women with 
abnormal 
mammograms and 
scheduled for breast 
biopsy  
BSE BroccoMax: 180 mg [= 
518 µmol] GR/day 
2-8 weeks 
No difference in AEs between two groups 
AEs 
BroccoMax 
Placebo 
Bloating  
5/27 
5/27 
Gas/Flatulence 
1/27 
4/27 
Diarrhea 
1/27 
2/27 
Others 
5/27 
7/27 
All 
8/27 
9/27 
Bauman et 
al(59) 
Singe arm study 
10 healthy volunteers 
BSE beverage:  
R1: oral 600 µmol GR/day 
R2: oral 150 µmol SF/day 
R3: oral rinse 150 µmol SF 
for 5 minutes, and 
expectorated  
Three 5-day 
intervention  
No AEs reported 


 
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7.1.2 Risks Associated with Study Procedures  
 
7.1.2.1 Bronchoscopy  
 
Bronchoscopy is very safe, but no procedure is entirely free of potential risk.  The 
following is a description of the risks of the procedure.  In recent publications, the 
incidence of serious complications was less than 0.05% (5 in 10,000) in over 23,000 
bronchoscopies.  Local discomfort (coughing, gagging and soreness of your nose and 
throat) are frequently unavoidable but can be greatly diminished with adequate 
anesthesia.  Bronchitis is common after bronchoscopy and may resolve spontaneously 
or with antibiotic treatment.  Nosebleed, wheezing (about 1% chance), a decrease in 
blood oxygen content of approximately 10% for approximately one half hour, low 
grade fever (less than a 5% chance), and lung infection (pneumonia) in less than 0.1% 
and slight expectoration of small flecks of blood for 24 hours after the procedure (as a 
result of minor trauma to the bronchial lining) may occur.  More serious 
complications such as major bleeding, lung collapse, vocal cord and bronchial spasm, 
and cardiac irregularity have been reported but are very rare.  Since the patient will be 
medicated for this procedure they will need to make arrangements for someone to 
drive them home. 
A Bronchoscopy Patient Communication Log Form is attached (Appendix 9) to 
follow-up the patients after the bronchoscopy is performed.  Patients are called within 
24 hrs., 72 hrs., and instructed to call within 30 days, if febrile, or with increased 
sputum production, or increasing shortness of breath or any perceived deterioration of 
respiratory condition. 
7.1.2.2 IV Infusion During Bronchoscopy 
 
An intravenous needle will be placed in the subject’s arm for the infusion of fluids.  
The subject can expect to experience some pain at the moment the needle goes into 
the arm.  In addition to this momentary pain, there will be the minor discomfort of 
having the needle taped in the arm.  In about 10 percent of cases small amount of 
bleeding under the skin will produce a bruise (hematoma).  The risk of temporary 
clotting of the vein is about 1 percent, while the risk of infection of a hematoma or 
significant external blood loss is less than one in 1,000.  In order to minimize these 
discomforts, only trained doctors and technicians will be permitted to place the 
intravenous line. 
 
7.1.2.3 Venous Blood Collection  
 
An intravenous needle will be used to collect blood samples from the subject’s arm. 
The collected blood samples will be used for clinical blood tests (CBC and chemistry) 
for subject’s general health and for research use. The subject can expect to experience 
some pain at the moment the needle goes into the arm.  In about 1-2 percent of cases 
small amount of bleeding under the skin will produce a bruise (hematoma).  The risk 
of temporary clotting of the vein is about less than one percent, while the risk of 
infection of a hematoma or significant external blood loss is less than one in 1,000.  


 
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In order to minimize these discomforts, only trained clinical research nurses or 
technicians will be permitted to place the intravenous line. 
 
7.1.2.4  
Nasal brushing 
 
Nasal  cell scraping may be associated with pressure, pain or minor bleeding. The 
patient may feel some discomfort and nasal sore after nasal brushings. Nasal 
bleedings are minor and can be stopped after holding the nose for a few minutes.  
 
 
7.1.3 
Violation of Privacy and Loss of Confidentiality 
 
Violation of privacy and loss of confidentiality are both risks to which research 
participants are exposed.  The possibility of these risks increases when protected 
health information is collected. 
 
7.2 
Definitions of Adverse Events 
 
Adverse event: Any untoward medical occurrence associated with the use of a drug in 
humans, whether or not considered drug related. 
 
Life-threatening adverse event or life-threatening suspected adverse reaction: An 
adverse event or suspected adverse reaction is considered “life-threatening” if, in the 
view of either the investigator or sponsor, its occurrence places the patient or subject 
at immediate risk of death. It does not include an adverse event or suspected adverse 
reaction that, had it occurred in a more severe form, might have caused death. 
 
Serious adverse event or serious suspected adverse reaction: An adverse event or 
suspected adverse reaction is considered “serious” if, in the view of either the 
investigator or sponsor, it results in any of the following outcomes: death, a life-
threatening adverse event, inpatient hospitalization or prolongation of existing 
hospitalization, a persistent or significant incapacity or substantial disruption of the 
ability to conduct normal life functions, or a congenital anomaly/birth defect. 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical 
judgment, they may jeopardize the patient or subject and may require medical or 
surgical intervention to prevent one of the outcomes listed in this definition. 
Examples of such medical events include allergic bronchospasm requiring intensive 
treatment in an emergency room or at home, blood dyscrasias or convulsions that do 
not result in inpatient hospitalization, or the development of drug dependency or drug 
abuse. 
 
Suspected adverse reaction: Any adverse event for which there is a reasonable 
possibility that the drug caused the adverse event. For the purposes of IND safety 
reporting, “reasonable possibility” means there is evidence to suggest a causal 
relationship between the drug and the adverse event. Suspected adverse reaction 
implies a lesser degree of certainty about causality than adverse reaction, which 
means any adverse event caused by a drug. 


 
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Unexpected adverse event or unexpected suspected adverse reaction: An adverse 
event or suspected adverse reaction is considered “unexpected” if it is not listed in the 
investigator brochure or is not listed at the specificity or severity that has been 
observed; or, if an investigator brochure is not required or available, is not consistent 
with the risk information described in the general investigational plan or elsewhere in 
the current application, as amended. For example, under this definition, hepatic 
necrosis would be unexpected (by virtue of greater severity) if the investigator 
brochure referred only to elevated hepatic enzymes or hepatitis. Similarly, cerebral 
thromboembolism and cerebral vasculitis would be unexpected (by virtue of greater 
specificity) if the investigator brochure listed only cerebral vascular accidents. 
“Unexpected,” as used in this definition, also refers to adverse events or suspected 
adverse reactions that are mentioned in the investigator brochure as occurring with a 
class of drugs or as anticipated from the pharmacological properties of the drug, but 
are not specifically mentioned as occurring with the particular drug under 
investigation. 
 
7.3 
Reporting Adverse Events to the Responsible IRB 
 
In accordance with applicable policies of the University of Pittsburgh Institutional 
Review Board (IRB), the Sponsor-Investigator will report, to the IRB, any observed 
or volunteered adverse event that is determined to be 1) associated with the 
investigational drug or study treatment(s); 2) serious; and 3) unexpected. Adverse 
event reports will be submitted to the IRB in accordance with the respective IRB 
procedures. 
 
Applicable adverse events will be reported to the IRB as soon as possible and, in no 
event, later than 10 calendar days following the sponsor-investigator’s receipt of the 
respective information. Adverse events which are 1) associated with the 
investigational drug or study treatment(s); 2) fatal or life-threatening; and 3) 
unexpected will be reported to the IRB within 24 hours of the Sponsor-Investigator’s 
receipt of the respective information. 
 
Follow-up information to a reported adverse event will be submitted to the IRB as 
soon as the relevant information is available. If the results of the Sponsor-
Investigator’s follow-up investigation show that an adverse event that was initially 
determined to not require reporting to the IRB does, in fact, meet the requirements for 
reporting; the Sponsor-Investigator will report the adverse event to the IRB as soon as 
possible, but in no event later than 10 calendar days, after the determination was 
made. 
7.4 
Data Safety and Monitoring Board (DSMB) 
We will create an institutional internal independent DSMB committee to review the 
safety and toxicity data that is submitted by the study statistician every 12 months. 
The definitions of adverse events are described in section 7.3. Specifically we will 
collect expected and unexpected adverse events in Adverse Events Recording Form 


 
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(Appendix 8). The DSMB will review the frequency, grade and relationship to the 
study agent in the treatment group compared with the placebo group. 
 
 


 
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8. 
BIOSPECIMENS, BIOMARKER, AND SPECIAL STUDIES 
8.1 
Human Biospecimen Collection 
8.1.1 
Endobronchial Biopsies 
Endobronchial biopsy samples will be collected during bronchoscopies.  The 
procedure will be carried out by Dr. Wilson (Co-PI) using Narrow Band Imaging 
(NBI) white-light bronchoscopy.  Up to 3 biopsies will be taken from each of six 
predetermined sites: upper lobe orifices and superior segment orifices (left and right), 
carina between left upper lobe division and lingular orifices and right middle lobe 
orifice.  In addition, biopsies will be taken from any sites that appear suspicious. On 
the post-treatment bronchoscopy, sites that had a mild or greater grade of dysplasia at 
baseline will be re-biopsied.   
Of the 3 tissue blocks per biopsy site. All biopsy samples will be put in an ice-cooled 
box and sent to Dr. Dacic (Co-I) for processing.  Bronchial biopsies will be fixed in 
10% neutral-buffered formalin for 6 to 24 hours, embedded in paraffin and routinely 
processed in the CLIA/CAP certified histology laboratory at the Department of 
Anatomic Pathology. One section per tissue block will be processed for routine 
hematoxylin-eosin (H&E) staining for histological evaluation and 10 unstained slides 
will be cut for immunoperoxidase studies.  
8.1.2 
Endobronchial Brushing  
Endobronchial brushing samples will be collected from study participants during their 
bronchoscopy procedure using an endoscopic cytobrush (Cellebrity Endoscopic 
Cytobrush; Boston Scientific, Boston, MA) to collect normal appearing bronchial 
epithelial cells by rubbing against the bronchial wall. The bronchial brushings will be 
targeted to normal appearing epithelial tissue of the right main-stem bronchus and 
avoid sampling tumor tissue or dysplastic cells. If any abnormalities are observed on 
the right bronchus, the bronchial brushing samples will be collected from the left 
main-stem bronchus.  Up to 4 bronchial brushing samples will be collected from each 
patient pre and post treatment, respectively. All brushing samples will be stored in an 
RNA preservative (RNAprotect; Qiagen) and sent to the test laboratory.  
8.1.3 
Bronchoalveolar Lavage  
After the completion of bronchial biopsies and brushing, the bronchoscope will be 
wedged in the right middle lobe or lingular orifice.  Up to 150  ml sterile saline will 
be instilled, retrieved by gentle suction. Percent recovery will be recorded. Recovered 
fluid will be passed through a micron sterile nylon filter (Becton Dickinson, New 
Jersey) to remove mucus and particulates, and pooled, and centrifuged. The BAL 
fluid will be harvested, aliquoted, and stored at -80oC for analysis later.  
 
8.1.4 
Nasal Epithelia   
Nasal brushings will be collected during clinical visits at month 0 3, 6, 9 and 12 after 
randomization. A nasal speculum (Bionox, Toledo, OH) then spread the nare while a 
standard cytology brush is inserted underneath the inferior nasal turbinate. The brush 
is rotated in place for 3 seconds, removed, and immediately placed in 1 ml RNA 
Later (Qiagen).  Brushings will be obtained from the right and left inferior turbinates 
as previously described (73).  


 
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8.1.5 Blood  
Besides a blood sample for eligibility screening or toxicity testing (10 ml), an 
additional  blood sample  (70 ml)   will be collected from study participants at months 
0, 3, 6, 9, and 12 after randomization.  Different blood components (serum, plasma, 
buffy coat, RNA) in blood samples will be separated and properly stored. 
8.1.6 Urine   
Spot urine samples will be collected at initial screening visit (for pregnancy test for 
women younger than 60 years). Similarly, sport urine samples will be collected as at 
months 0, 3, 6, 9, and 12 after randomization.  Aliquots of collected urine samples 
will be stored at –80oC. The urine samples will be used for quantification of total ITC 
(dietary intake of cruciferous vegetables) and SF-NAC (for monitoring intake of SF 
tablets).  
8.1.7 Creation of a Biospecimen Repository    
Blood and urine are collected for future research that may include genetic analyses in 
the study.   The Tissue Distribution Committee of the SF trial study is responsible for 
authorizing the distribution of stored DNA, serum, and urine and the determination of 
appropriateness of the proposed research which may include genetic analyses.  This 
committee will review requests for the use of stored DNA, serum and urine, and 
provide samples to investigators if the project is scientifically and ethically 
acceptable.  Patient samples will be provided to investigators with only the study 
identification number so that investigators will not know the patient’s identity; thus 
enhancing patient confidentiality.  Inadvertent discovery of non-parentage is not 
possible as we are only planning to collect blood from participants and not their 
family members.  Examples of the types of genetic testing include polymorphisms in 
metabolizing enzymes such as GST genes.  None of these genetic “susceptibility” 
factors would be sufficient to cause lung cancer, but they may interact with 
environmental exposures (such as tobacco smoke).  Since this genetic testing will be 
performed at an unspecified time in the future, it is not possible to say that we will 
provide patients with any information regarding their genetic background.  In the 
unlikely scenario that we do discover a significant gene that is clinically meaningful, 
we would make every attempt to contact patients with this information. 
 
8.1.8 
Identifiers 
The participant’s unique study ID number will be the only identifier on every 
specimen.  Specimens will be stored in a locked -80°C freezer in the research 
laboratory at the UPMC Hillman Cancer Center.  
8.2 
Biomarkers 
8.2.1 Histology (atypia) 
It has long been established that abnormal bronchial histology (atypia) is associated 
with cigarette smoking and increased risk for lung cancer. In a follow-up study of a 
high-risk cohort of subjects with endoscopic biopsies, persistence of bronchial 
dysplasia was associated significantly with a 7-fold increased risk of developing 
NSCLC (adjusted hazard ratio = 7.8, 95% CI 1.6-39.4) (18). All bronchial biopsies 


 
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for histopathological evaluation will be formalin fixed, paraffin embedded, and H&E 
stained for subsequent morphologic evaluation and classification.  Biopsies will be 
classified into 1 of 8 categories as defined by the World Health Organization (WHO) 
classification (74) and assigned a score according to the following system: 1= normal; 
2= reserve cell hyperplasia; 3 = squamous metaplasia; 4 = mild dysplasia; 5 = 
moderate dysplasia; 6 = severe dysplasia; 7 = carcinoma in situ; and 8 = invasive 
carcinoma. Subjects with baseline score 7 or 8 are ineligible for the proposed study.  
All biopsies will be graded by the study pathologist (Dr. Dacic) in a blinded fashion 
as to treatment group.  
Endobronchial histology scores will be first summarized within each bronchoscopy 
by using 3 separate endpoint measures pre and post treatment respectively: (1) The 
first endpoint measure is dysplasia index (DI), that solely examines changes in 
dysplasia pre and post-treatment.  The number of biopsies with dysplasia (with WHO 
histology classification score: 4-6) are divided by the total number of biopsies and 
multiplied by 100. A positive change of 10% or greater indicates progression while a 
negative change of 10% or greater is regarded as a positive response.  Scores in-
between are coded as stable. (2) The second endpoint measure is the mean 
histological scores. It is calculated by adding the scores from all the biopsy sites and 
determining the mean value pre and post-treatment. A positive change in the score by 
1 or more points is defined as progressive disease, a negative change in the score by 1 
point or more points is defined as a partial or complete response and scores in-
between are defined as stable disease. (3) The third model is called the “worst score”. 
It identifies the worst histological grade pre- and post- treatment. A positive change in 
the score by 1 or more points is defined as progressive disease, a negative change in 
the score by 1 point or more points is defined as a partial or complete response and 
scores in-between are defined as stable disease.   The final response determination for 
a patient requires the same response determination in 2 of the 3 measures.  For the 
rare patient with a different response for each category (progression, stable and 
response) the patient will be deemed stable.  These summary scores will be created by 
the study statistician with the input of all investigators, in particular Dr. Dacic.  
8.2.2 Cell proliferation (Ki-67 labeling index) 
A common endpoint of pathways affected by sulforaphane is cell proliferation.  Ki-67 
labeling is a validated indicator of proliferative index and can be readily detected in 
bronchial biopsy material (75-77).  Ki-67 labeling index (Ki-67 LI) is  positively 
associated with increased grade of bronchial dysplasia; a mean of 4% in normal and 
hyperplastic samples increased to 38% in biopsies with moderate or severe dysplasia 
(Table 4). When a serial set of matched biopsies were examined for change over time 
there was correlation between histological change and Ki-67 labeling scores.  Ki-67 
thus is an easily performed proliferative index that will be a primary endpoint in this 
study. 
 
Table 4.  Histology and Biomarkers in Bronchial Epithelium of 
High Risk Subjects 
Study group 
Histology 
Ki-67 (mean %) 
All subjects 
Grade 1-2 (N=14 biopsies) 
        4% 
 
Grade 3-4 (N=16 biopsies) 
      20% 


 
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Grade 5-6 (N=39 biopsies) 
      38% 
Serial Biopsies: 
 
 
   No change (N=7)   
Mean grade change 0 
     -17% 
   Progression (N=10) 
Mean grade change +2 
    +17% 
   Improvement (N=4) 
Mean grade change –2.5 
     -21% 
 
8.2.3 Apoptosis (TUNEL and Caspase-3) 
 
Like the proliferative index, the apoptotic index will provide a measure of a final 
common pathway.  Dietary SF significantly induced markers of apoptosis in lung 
tissue of mice after treated with tobacco carcinogens. Compared with control animals, 
mice given SF showed a statistically significant 6-fold increase (from 4.7±1.9 to 
29.3±4.7%) in activated caspase-3, and a 2-fold increase (from 12.7±3.0 to 
29.4±5.8%) in TUNEL (terminal transferase dUTP nick end labeling) of lung cells. 
Activated caspase-3 and TUNEL are representative markers for apoptosis (14).  Thus 
we will perform TUNEL and caspas-3 assays on sections of bronchial mucosa from 
histologically normal and abnormal sites pre and post SF treatment. 
 
8.2.4 Gene expression markers 
 
Gene expression in the airway epithelium, which can be sampled by bronchoscopic 
and nasal brushings, represents an intermediate biological endpoint between 
underlying genetic or epigenetic alterations driving carcinogenesis and the clinical 
diagnosis of lung cancer. Dr. Spira (Co-I) and colleagues (19) recently validated that 
a 23-gene expression marker in epithelial cells collected from the normal-appearing 
main-stem bronchus significantly improved the diagnostic performance of 
bronchoscopy for detection of lung cancer. The combination of the 23-gene 
expression marker plus bronchoscopy had a sensitivity of 96% (95% CI, 93% to 98%) 
in the first cohort of patients and 98% (95% CI, 96% to 99%) in the second cohort of 
patients, independent of lesion size and location. In patients with a nondiagnostic 
bronchoscopic examination, the gene-expression classifier accurately identified 
cancer in 89%. The 23-gene expression marker is the first genomic classifier to 
improve the diagnostic performance of bronchoscopy for the detection of lung cancer. 
In the proposed study, we will evaluate if the SF treatment alters the gene expression 
of these 23 genes in the bronchial epithelium, and decreases the classifier score 
towards that seen with lung benign disease (78). 
 
Using mRNA sequencing (mRNA-Seq), we profiled cytologically normal airway 
epithelial cells collected during auto-fluorescence bronchoscopy from high-risk 
smokers with (n=50) and without (n=25) bronchial premalignant lesions (PMLs). 
Using surrogate variable analysis, we identified 280 genes significantly differentially 
expressed between subjects with and without PMLs at false discovery rate 
(FDR)<0.002. The 280-gene signature has a significant concordant relationship to 
gene expression changes identified in PMLs adjacent to non-small cell lung cancer 
(NSCLC), in NSCLC tumors, and in the field of individuals with lung cancer 
(FDR<0.05). These data suggest that the change in certain gene expressions in 
bronchial epithelium takes place at premalignant stage. In the proposed study, we will 


 
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explore if the SF treatment alters the gene expression profile of these 280 PMLs-
related genes in bronchial epithelium of the study participants. 
 
The unified airway field of injury concept suggests that cigarette smoking renders 
injury throughout the entire respiratory tract. Studies have shown that tobacco-related 
gene expression alterations in the extra-thoracic epithelium of the nose and mouth 
similarly reflect the host response to cigarette smoke in the distal airway (79).  Both 
nasal and bronchial epithelial cells demonstrate similar patterns of gene expression 
changes in response to cigarette smoking (73), suggesting that the nasal epithelium 
may be a surrogate biospecimen for monitoring the individual response to smoking 
and potentially the risk for lung cancer. More recently, we have demonstrated that the 
cancer-specific gene-expression changes in the bronchial epithelium are enriched 
among gene-expression alterations observed in the nasal epithelium from 
approximately 500 smokers with matched nasal and bronchial samples. These data 
suggest that genomic profiling of these relatively accessible airway cells may be an 
intermediate endpoint of chemoprevention for lung cancer. In the proposed study, we 
will explore whether SF-induced gene expression changes in bronchial epithelium 
mirror those in the nasal epithelium. 
8.3 
End of Treatment Evaluation 
8.3.1 Histological Response 
Histology on bronchial biopsies pre-treatment and post-treatment will be compared. 
All biopsies will be graded according to the WHO classification for bronchial 
epithelium (74): 
WHO Classification 
Grade 
Normal 
 
1.0 
Reserve Cell Hyperplasia 
2.0 
Metaplasia  
3.0 
Mild Dysplasia 
4.0 
Moderate Dysplasia 
5.0 
Severe Dysplasia 
6.0 
Carcinoma in Situ 
7.0 
Carcinoma  
8.0 
Response will then be determined by three scoring methods: 
a) The worst histology grade per patient pre and post treatment. 
Worse =  1 point higher post treatment 
Better =  1 point lower post treatment 
Same = same point score pre and post treatment 
 
b) Average histology (all biopsies scores/number of biopsies) pre and post treatment 
Worse =  1 point higher post treatment 
Better =  1 point lower post treatment 
Same = same point score pre and post treatment 
 


 
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c) Dysplasia index (number of biopsies with dysplasia/total number of biopsies x 
100) pre and post treatment. 
Worse = > 10% higher post treatment 
Better = > 10% lower post treatment 
Same = < 10% difference post treatment 
The final response determination will require the same response parameter 
(worse, better or the same) for 2 of the 3 methods.   
8.3.2 Cellular Biomarker Response 
A secondary endpoint is to determine if SF can modulate a proliferative intermediate 
marker Ki-67. Based on prior studies, a response will be considered as a decrease of 
> 10 in the labeling index, progression will be an increase of > 10 in the labeling 
index and no change will signify an increase or decrease of < 10. 
We will also evaluate other biomarkers including apoptotic biomarkers TUNEL and 
caspase-3.  
8.3.3 Gene Expression Response 
The exact mechanisms for the observed SF-mediated  44 hemoprotection remain to be 
elucidated. The upregulation of NQO1 transcript suggests the activation of Nfr2 
pathway (59).  The alteration of lung cancer associated gene expression in BAL is an 
indication of SF’s impact on lung cancer risk in the study population. 
8.3.4  Safety and Toxicity Assessment 
 
The safety and toxicity will be assessed by the study physician at the research clinic 
once every three months. We plan to closely monitor all study patients for potential 
SF-related adverse events.  This includes both expected (based on previous clinical 
experience with oral SF) and unexpected events. The expected adverse events (AEs) 
are gastrointestinal including taste alternation (bad taste), dry mouth, belching, 
nausea, vomiting, and diarrhea. The frequencies of AEs were low and most were  at 
moderate by grade (see Table 3). 
 
9. 
STATISTICAL AND SAMPLE SIZE CONSIDERATIONS 
 
This is a randomized, double blind, placebo-controlled study to evaluate the efficacy of 
SF as a chemopreventive agent.   Eligible subjects are former smokers with 30 pack-
years smoking history who quit smoking <15 years, and are considered at high risk for 
developing lung cancer.  The proposed duration of therapy will be 12 months for each 
subject.  The primary objective of this trial is to assess whether oral SF supplementation 
elicits an increased histologic response when compared to placebo in this population over 
a 12-month treatment period.   The planned sample size is 72 subjects (36 per treatment 
arm).  A block randomization scheme will be used to ensure that both treatment and 
placebo group have the same proportion of men and women, respectively. The 
randomization will be done at the UPMC Hillman Cancer Center. 
9.1 
Statistical Analysis for histological responses 


 
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Endobronchial histology scores will be first summarized within each bronchoscopy by 
using 3 separate endpoint measures pre and post treatment respectively: 1) dysplasia 
index (DI, number of biopsies with dysplasia/total number of biopsies x 100), 2) average 
of all histological scores (all biopsies scores/number of biopsies), and 3) worst biopsy 
score.  The 3 summary end points will be analyzed within 4 different biopsy site 
groupings: all biopsies, biopsies from the 6 standard endobronchial sites only, site-
matched biopsies from both bronchoscopies, and site-matched biopsies where the 
baseline biopsy is non-normal.  For each patient, the mean of each of endpoints 
separately for each of biopsy site groupings pre and post treatment are then calculated.   
The primary endpoint is the dysplasia index (DI), which has a binomial distribution with 
at least 6 “trials” (i.e., 6 predetermined biopsy sites), and will be analyzed by generalized 
estimating equations (GEE) that constrains average baseline DI to be the same for both 
treatment and placebo groups, and tests whether average follow-up DI differs between 
the SF treatment and placebo groups.  All other endpoints and biopsy site groupings are 
secondary endpoints.  The primary analysis for the average and worst dysplasia scores 
will be a linear regression that assesses the difference in post-treatment scores by the two 
treatment groups, controlling for the same scores at pre-treatment.  This analysis of 
covariance (ANCOVA) approach has optimal efficiency in a study design to assess the 
pre- and post-treatment measures with high within-subject correlation due to repeated 
measures (80).  No interim analyses for futility or superiority will be performed, other 
than ongoing safety monitoring. 
Sample size consideration.  In the iloprost trial, 57 former smokers were randomized to 
receive oral iloprost or placebo for 6 months.  Treatment effects (adjusted for baseline 
levels) were statistically significant (P≤0.01) for all 3 endpoints (dysplasia index, average 
dysplasia score, worst dysplasia score). Anticipating a 20% dropout rate over 12 months, 
we will randomize at least 72 study subjects to ensure that 60 subjects complete our SF 
study.  Assuming an average baseline dysplasia index of 22% and post-therapy 11% for 
subjects receiving SF, a simulation with 10,000 trials with a sample size of 60 subjects 
has yielded an 80% statistical power to detect a statistically significant (two-sided 
p<0.05) treatment effect, using generalized linear models (binomial family, 6 biopsies) 
after controlling for baseline levels of DI.  Calculations are similar for other endpoints, 
and actual power should be greater than the DI measure because they are continuous 
measures and have observed within-subject correlation (i.e., repeated measures). 
 
9.2 
Statistical Analysis for Cellular Proliferation/Apoptosis Markers 
Ki-67, caspase-3 and TUNEL will be quantified as % positive cells in two slides of each 
tissue block. First we calculate the average values of each of the 3 IHC markers over 6 
tissue blocks within each bronchoscopy per patient separately for pre- and post-treatment.  
As described above for bronchial dysplasia measures, the primary analysis for these 
continuous measures will be a linear regression predicting post-treatment score by 
treatment group, controlling for pre-treatment score.  We hypothesize that SF treated 
subjects will exhibit lower expression of Ki-67 and higher expression of caspase-3 and 
TUNEL relative to subjects in the placebo arm. 
Sample Size Consideration. Using estimates of the mean and standard deviation 
(12.7±8.2) of Ki-67 LI in the para-basal layer of bronchial epithelia of smokers (15) and 
the logit function, we back calculated plausible estimates of the mean (2.3676) and 


 
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variance (0.3480) of log-transformed baseline Ki-67 LI (81).  Assuming these log-
transformed Ki-67 LI values are normally distributed, we simulated 10,000 sets of data 
(n=60 each) and found an 84% statistical power with a two-sided P<0.05 to detect a 
treatment effect size of 37% for change in Ki-67 LI after 12 months of SF treatment (i.e., 
median Ki-67 LI of 10.7% at baseline and 6.7% at follow-up on SF). This detectable 
effect size is smaller than 61% reduction in cell proliferation marker in the SF treatment 
group (14).  Furthermore, a recent randomized, double-blind, placebo-controlled phase 2 
clinical trial involving 58 women found that oral intake of SF glucosinolate for 2-8 weeks 
resulted in a statistically significant reduction of mean Ki-67 LI (P = 0.01) (16). 
9.3 
Statistical Analysis for Gene Expression 
A summary gene expression score will be derived from previously determined gene 
expression features of 23 genes (19,82): score = 𝑒𝑦 /(1 + 𝑒𝑦), where y = 𝑏0 + ∑𝑏𝑗∗𝑥𝑗, 
where b0 is the intercept, 𝑏𝑗 is the coefficient, and 𝑥𝑗 is the feature of the gene j 
expression [see details in (82)]. We will calculate the gene expression score (0~1) for 
each subject pre and post treatment separately. A linear mixed effect model will be used 
to assess the effect of SF on post-treatment gene expression scores, similar as described 
in Section 4.2.1. A decreased gene expression score post-treatment towards those seen in 
bronchial benign disease is indicative of the chemopreventive effect of SF on the lung 
cancer-gene expression.  
We will use the similar approach to calculate a summary gene expression score of the 
280-gene signature in bronchial premalignant lesions (PMLs) in identified our 
preliminary study. Similarly a linear mixed effect model will be used to assess the post-
treatment gene expression scores. If SF causes a significant change of the score towards 
the regression of PMLs based on histological dysplasia index score, the gene-level 
expression changes will be examined to estimate the SF-treatment effects on individual 
gene or a cluster of genes. 
To assess the similarity and difference in the gene expressions between the bronchial 
airway and nasal epithelia, we will use our similar methodologies described previously 
(73). For each gene identified in the analysis above, we will use a linear mixed effects 
model that includes site (bronchus or nose) and treatment (SF or placebo) as fixed effect. 
The regression model is given: Gij ~ µ + βs•Xs + βt•Xt + βst•Xs •Xt + εij, where Gij is the 
log2 expression value for gene i in patient j; the parameter µ is the overall mean of log2 
fold change in gene expression, βs and βt for the respective site- and treatment-specific 
effect, and βst for the interaction between site and treatment; and Xs for site (1=nose, 
0=bronchus) and Xt for treatment (1=SF, 0=placebo). 
  
Sample Size Consideration.  The Affymetrix Human Gene 1.0 ST Array contains 
~36,000 gene transcripts. The proposed study with 30 subjects per group will have a 85% 
power to detect a 2-fold gene expression difference pre and post treatment (long2 |d|=1.0, 
σd = 0.7) for 23 identified cancer-related genes at a false discovery rate (FDR)<0.05 using 
the method of Liu and Hwang  (83), assuming 99.9% non-differentially expressed genes 
(𝜋0 = 0.999). Similarly the study will have a 96% power for 280 PMLs-related genes 
(i.e., 𝜋0 = 0.990), and a 99% power for 5% differentially expressed genes (i.e., 𝜋0 =
0.950) in exploratory analysis.  
 


 
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9.4 
Statistical Analysis for Adverse Events and Toxicity Measures 
The frequencies of adverse events (AEs) and toxicity measures (e.g., CBC, liver function 
test, body weight) will be compared between the SF treatment and placebo arm.  The 
analysis will be conducted for total AEs and specific AEs, expected AEs and unexpected 
AEs, AEs by grade and attribution grouping, respectively, at each clinical visit as well as 
the summed total over 4 time points of the entire treatment period.  We will repeat the 
same analysis on a patient level.  Chi-square test will be used for comparing the 
frequencies between the two treatment groups. 
 
Sample Size Consideration.  As described in Table 3 above, the frequencies of AEs 
reported were low in previous studies. Based on the frequencies repored by Atwell et al, 
the frequency of total AE in placebo was 33% (9 out of 27 participants), the proposed 
study with a total of 72 subjects will have an 80% statistical power to detect a doubled 
frequency (67%) of total AEs in the SF-treatment group.  The proposed study can detect a 
less excess AE rate between the two treatment groups if all AEs over different time 
points are combined or all AEs are counted independently.  
 
10. 
GENDER AND MINORITY INCLUSION 
The University of Pittsburgh and UPMC are committed to providing access to cancer 
clinical trials, both therapeutic and non-therapeutic, for all citizens of their state and 
region, including their under-served populations. Every effort is made to provide access 
to clinical trials for all individuals and special emphasis is placed on retaining those who 
are enrolled.  
At enrollment, PluSS participants had a mean age of 60 years. Among the participants, 
49% were women; 93% whites, 5.5% blacks, and 1.7% other race/ethnicity groups; 60% 
current smokers and 40% former smokers with a median 47 pack-years of smoking 
history (61). The PluSS participants resembled the national representative sample of 
current and former smokers who are at high risk for lung cancer. For example, the 
National Lung Screening Trial (NLST) enrolled more than 53,000 persons at high risk for 
lung cancer at 33 medical centers across the US.  The mean age was 61 years. Forty-one 
% were women; 91% whites, 4% blacks, and 5% other race/ethnicity groups; 48% 
current smokers and 52% former smokers who quit smoking within the previous 15 
years; a median 48 pack-years of smoking history (62). 
11. 
ETHICAL AND REGULATORY CONSIDERATIONS 
The following must be observed to comply with Food and Drug Administration (FDA) 
regulations for the conduct and monitoring of clinical investigations; they also represent 
sound research practice: 
11.1  Informed Consent 
Patient consent is solicited in the clinic by Co-Principal Investigators or the 
designated trained study staff.  Obtaining subject informed consent will be done in a 
setting that facilitates information transfer that must minimally include, unhurried 
time between consent and the initiation of study procedures and a quiet setting.  The 
subject will be provided the written Consent Form (see a copy at the end of the 
Clinical Protocol) with time to read it.  The subject will be asked questions explaining 


 
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the study in their own words to insure understanding.  A copy of the signed consent 
will be provided to subject. 
The principles of informed consent are described by Federal Regulatory Guidelines 
(Federal Register Vol. 46, No. 17, January 27, 1981, part 50).  They must be followed 
to comply with FDA regulations for the conduct and monitoring of clinical 
investigations.  In seeking informed consent, the following information shall be 
provided in a language understandable to the subject. 
11.2 
Basic Elements of Informed Consent 
The following are the basic elements of informed consent which should be provided 
to each subject: 
a) 
A statement that the study involves research, an explanation of the 
purposes of the research, and the expected duration of the subject’s 
participation, a description of the procedures to be followed, and the 
identification of any procedures which are experimental. 
b) 
A description of any reasonably foreseeable risks or discomforts to the 
subject. 
c) 
A description of any benefits to the subject or to others which may 
reasonably be expected from research. 
d) 
A disclosure of appropriate alternative procedures or courses of treatment, 
if any, that might be advantageous to the subject.  
e) 
A statement describing the extent, if any, to which confidentiality of 
records identifying the subject will be maintained and that notes the 
possibility that the FDA may inspect the records. 
f) 
For research involving more than minimal risk, an explanation as to 
whether any compensation and an explanation as to whether any medical 
treatments are available if injury occurs and, if so, what they consist of, or 
where further information may be obtained. 
g) 
An explanation of whom to contact for answers for pertinent questions 
about the research and research subjects’ rights, and whom to contact in 
the event of a research-related injury to the subject. 
 
h) 
A statement that participation is voluntary, that refusal to participate will 
involve no penalty or loss of benefits to which the subject is otherwise 
entitled, and that the subject may discontinue participation at any time 
without penalty or loss of benefits to which the subject is otherwise 
entitled. 
11.3 
Additional Elements of Informed Consent 
When appropriate, one or more of the following elements of information shall 
also be provided to each subject: 
a) 
A statement that the particular treatment or procedure may involve risks to 
the subject which are currently foreseeable. 


 
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b) 
Anticipated circumstances under which the subject’s participation may be 
terminated by the investigator without regard to the subject’s consent. 
c) 
Any additional costs to the subject that may result from participation in the 
research 
d) 
The consequences of the subject’s decision to withdraw from the research 
and procedures for orderly termination of participation by subject. 
e) 
A statement that significant new findings developed during the course of 
the research which may relate to the subject’s willingness to continue 
participation will be provided to the subject. 
f) 
The approximate number of subjects involved in the study. 
A subject must give his/her written consent to participate in the study.  This 
consent must be witnessed and dated and retained by the investigator as part of 
the study records. 
If Experimental Subject’s Bill of Rights is applicable in your state, this form must 
also be prepared and signed by each subject and retained as part of the required 
study records. 
A copy of the proposed consent form must be submitted to the Institutional 
Review Board together with the protocol for approval.  Each subject’s signed 
informed consent form must be kept of file by the investigator for FDA inspection 
at any time. 
11.4 
Institutional Review 
This study must be approved by an appropriate institutional review committee as 
defined by Federal Regulatory Guidelines (Ref. Federal register Vol. 46, No. 17, 
January 27, 1981, part 56). 
The protocol and informed consent form for this study must be approved in writing 
by the appropriate Institutional Review Board (IRB).  The IRB must be from an 
institution which has a valid Multiple Project Assurance, Single Project Assurance, 
and Cooperative Oncology Group Assurance on file with the Office for Protection 
from Research Risks, National Institutes of Health.  The institution must be in 
compliance with regulations of the FDA and the Department of Health and Human 
Services. 
Significant changes to the protocol, as well as a change of principal investigators, 
must also be approved by the IRB and documentation of the approval provided to the 
study monitor.  Records of the IRB review and approval of all documents pertaining 
to this study must be kept on file by the investigator and are subject to FDA 
inspection at any time during this study.  Periodic status reports must be submitted to 
the IRB at least yearly, as well as notification of completion of the study and a final 
report within 3 months of study completion or termination.  The investigator must 
maintain an accurate and complete record of all submissions made to the IRB 
including a list of all reports and documents submitted. 
11.5 
Drug Accountability 


 
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For each drug supplied for a study, an accountability ledger containing current and 
accurate inventory records covering receipt, dispensing, and the return of study drug 
supplies must be maintained. Drug supplies must be kept in a secure, limited access 
storage area under the recommended storage conditions. During the course of the 
study, the following information must be noted on the accountability ledger; the 
identification code of the subject to whom drug is dispensed, the date(s) and quantity 
of drug dispensed to the subject, and the date(s) and quantity of drug returned by the 
subject; subjects should return empty containers to the investigator, with the return 
noted on the ledger. These Accountability Forms must be readily available for 
inspection and are open to FDA inspection at any time.  
 
12. 
STUDY DATA MANAGEMENT 
 
All case report forms (CRFs) and specimens for the UPMC Hillman Cancer Center 
(Hillman) and the multi-institutional centers will be kept and reviewed at Hillman. 
Patient confidentiality is maintained on study CRFs by use of assigned Study ID#.  
Records for this study will be maintained in locked cabinets in the study coordinator 
office in the secured file room that can be accessed only by the investigators and staff 
on the research team. Tissue, blood and urine specimens are given a specimen ID 
number that is linked to the study ID#.  Patient names do not appear in the pathology 
database.  Specimens are provided to investigators using specimen ID# as the only 
identification on the sample.  The Principal Investigator oversees access to the study 
database with only authorized investigators receiving access.  Data sets provided to 
the investigators do not contain patients names with subjects being identified by study 
ID# only.  Access to the main database where patient identification is available is 
secured using password access from identified net locations only.  Delinking of 
subject ID with specimen information occurs prior to the sharing of specimens with 
other institutions.  At the end of the study, completed records are locked in the 
Clinical Investigations storeroom. 
 
13. 
FUNDING 
 
This study has been funded by NIH from 6/14/2017 through 5/31/2022  (R01 
CA213123, PI Jian-Min Yuan). 


 
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