Chemoprevention of Squamous Cell Cancer of the Skin in High Risk Patients
Phase: PHASE2
Conditions: Squamous Cell Carcinoma of the Skin
Sponsor: University of Rochester

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Assessing the Impact of Pioglitazone on Squamous Cell Skin Cancer Incidence in High 
Risk Patients 
 
Principal Investigator – Alice P Pentland, MD 
Department of Dermatology Box 697 
University of Rochester Medical Center 
Rochester NY 14642 
 
 
1. PURPOSE OF THE STUDY AND BACKGROUND 
 
1.1. Purpose of the study 
This study is intended to determine if treatment with the oral agent pioglitazone (also called Actos, 
a drug approved to treat diabetes) can decrease the number of new squamous cell cancers in people 
who have a high risk of developing skin cancer. This patient population includes, but is not limited 
to those with immunosuppression secondary to organ transplantation. 
 
Specifically, the purposes of this pilot study are to collect preliminary data in order to plan for a 
larger study, the objective of which will be to assess whether pioglitazone will:  
 
 Decrease the number of new skin cancers patients develop while taking the drug compared 
to their stable baseline 
 Determine if skin cancer formation reoccurs when stopping drug  
 Determine if the study drug affects the phenotype of the skin cancers occurring while on 
treatment (i.e., well differentiated vs. poorly differentiated tumors) 
 Change the expression of markers influenced by PPAR or AKR1C3 (apoptosis, 
proliferation) in tumors removed from treated patients.   
   
 
1.2. Background 
Squamous cell carcinoma (SCC) is the second most common form of skin cancer, with a steadily 
increasing annual incidence of over 250,000 cases in the United States(Miller et al., 2010; 
Schmults et al., 2013). The mortality rate of these cancers remains low (~1 %), however in certain 
populations such as organ transplant recipients, rates of metastasis and mortality are considerably 
higher(Mullen et al., 2006). When patients develop metastatic disease, up to 50 % will have a 
recurrence with a mortality rate of up to 85 %(Goh et al., 2010). Beyond mortality and high 
morbidity, treating non-melanoma skin cancers is costly, accounting for 4.5 % of all Medicare 
cancer costs, a figure that increased 41 % between 1992 and 1995(Rogers et al., 2010). Clearly, 
therapies that can prevent or stop the progression of SCC are sorely needed. Recent work 
demonstrated that selective inhibition of prostaglandin E synthesis using Celebrex resulted in a 40 
– 50% decrease in squamous cell cancers over a 6 month period(Elmets et al., 2010). However, 
the utility of Celebrex and other NSAIDs is decreased because of the significantly increased risk 
of heart attacks(Schmults et al., 2013). 

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Aldo-Keto Reductase 1C3 (AKR1C3) is another 
prostaglandin-synthesizing enzyme that could be a target 
for SCC prevention(Matsuura et al., 1998). AKR1C3 
belongs to a large superfamily of NAD(P)(H)-dependent 
AKR enzymes which metabolize various substrates 
including prostaglandins and sex hormones (Jez and 
Penning, 2001). In humans, the AKR1C subfamily consists 
of 4 enzymes termed AKR1C1, AKR1C2, AKR1C3 and 
AKR1C4 which despite their extensive structural overlap, 
exhibit different substrate binding preferences (Penning et 
al., 2000).  Among these enzymes, AKR1C3 expression 
has been associated with cancer risk.  
 
AKR1C3 is overexpressed in various cancers originating 
from non-steroidogenic tissues such as kidney (Azzarello 
et al., 2009), central nervous system (Park et al., 2010), 
pancreas, gastrointestinal tract and lung (Chang et al., 
2013; Miller et al., 2012). AKR1C3 can activate 
carcinogens (Lan et al., 2004), modulate oxidative stress 
(Birtwistle et al., 2009), and its polymorphisms are 
associated with risk for childhood leukemia (Liu et al., 
2008) and bladder cancer (Figueroa et al., 2008). 
Collectively, these observations suggest a tumor promoting 
function for AKR1C3 (Azzarello et al., 2009; Miller et al., 
2012). Unlike other AKR1C members, AKR1C3 can also 
synthesize PGF2from PGH2, an arachidonic acid 
derivative synthesized by cyclooxygenase (Komoto et al., 
2006). Its role in prostaglandin metabolism also includes 
the conversion of PGD2, a pro-inflammatory lipid 
mediator, to 9,11-PGF2 (Byrns et al., 2010; Komoto et 
al., 2006; Matsuura et al., 1998). Thus, via its 
involvement in PG metabolism and its downstream 
effects on PPAR, AKR1C3 may regulate biological and 
pathological responses in hormone-independent tissues.  
Our recent work investigated AKR1C3 expression in 
human 
non-melanoma 
skin 
cancers, 
revealing 
overexpression in 7 human squamous cell carcinomas 
(SCC) examined.  Effects of AKR1C3 expression were 
then evaluated using 3 SCC cell lines (Fig. 1). AKR1C3 
was detected in all SCC cell lines and its expression was 
upregulated in response to its substrate, PGD2. Treatment 
of the SCCs with PGD2 and its dehydration metabolite, 
15d-PGJ2,decreased SCC proliferation, indicating that the 
PPAR receptor was the key effector of this profound 
inhibitory response.  SCC treatment with the PPAR 
Figure 1, above. PGD2 and 15d-PGJ2 attenuated 
SCC cell proliferation in a PPAR-dependent 
manner. (a) SCC cells were treated with the 
indicated concentration of PGD2, 15d-PGJ2, 9,11-
PGF2 or DMSO for 24 hours followed by cell 
proliferation assessment by Click-iT assay. Data 
presented is the average of 3 experiments (±SEM), 
*P<0.005, **P=0.012. To study the involvement of 
PPAR activation in the anti-proliferative effects 
mediated by 15d-PGJ2 and by its precursor PGD2, 
SCC cells were treated with 1.5M 15d-PGJ2 (b) or 
7M PGD2 (c) in the presence of the indicated 
concentration of the PPAR specific inhibitor 
GW9662 (GW). Cell proliferation was assessed by 
Click-iT assay.  n=3 *P<0.05. (d) The effects of 
Pioglitazone on SCC proliferation. Each SCC cell 
line was treated for 24h as indicated and cell 
proliferation was assessed by Click-iT assay.(n=3) 

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agonist pioglitazone was then tested, showing a profound capacity to inhibit SCC 
proliferation, using concentrations achieved in patients on drug treatment (50 – 100 
M)(Christensen et al., 2005). This work suggests that AKR1C3 over-expression within the tumor 
microenvironment may protect SCC cells from the anti-proliferative effects of PGD2 and that 
PPAR agonists such as pioglitazone may be a useful treatment adjunct for those at high risk of 
SCC. There are no currently available approved selective inhibitors of AKR1C3. However, 
Pioglitazone is a currently approved oral treatment for diabetes that can be safely administered to 
non-diabetics(Bell et al., 2012).  We propose to learn in this clinical trial if its use can be helpful 
in patients who develop multiple SCCs each year. The protocol outlined here is intended to 
examine this possibility as well as address the mechanisms by which it exerts its effects.  
 
 
 
2. STUDY DESIGN 
 
2.1. Overview 
 
This is a single center open label feasibility pilot project based at the University of Rochester. 
Patients followed in the University of Rochester Dermatology Clinic who have had between 2 to 
6 squamous cell cancers treated in the past year, without contraindication for the use of 
Pioglitazone, and are on a stable drug treatment regimen will be offered participation.  The study 
has a cross-over design, so patients will be enrolled and randomized in one arm of two treatment 
protocols: 1) six months of usual care while documenting and characterizing any new tumors that 
occur followed by 6 months of Pioglitazone treatment plus usual care for the next 6 months or 2) 
six months of Pioglitazone treatment followed by 6 months off treatment receiving usual care (see 
protocol diagram, Section 5, below). The second group will offer the opportunity to assess whether 
there is any persistent beneficial effect after pioglitazone treatment ends, while tumors from the 
first group that occur during the initial 6 months of usual care will be characterized by study 
parameters for comparison to tumors arising while on treatment. At the end of the one year 
treatment period, the number of new tumors that patients develop while taking Pioglitazone will 
be counted and compared with the number that patients developed during the 6 month period they 
were not receiving treatment as well as the numbers that occurred during the 6 months after 
treatment (washout effect). This information will be used as the basis for a larger multicenter study.  
 
 

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Fig 2. Schematic for the Proposed Study (study visit day are +/- 7 or 14 days) 
 
2.2. Rationale for Study Design 
An open label study with a crossover design was selected because this is a feasibility pilot project 
funded by the Department of Dermatology, and we require a study design that can yield useful 
statistical information for sample size for a subsequent multi-center trial with the number of 
subjects we are likely able to recruit here at Rochester.  We calculate that 40 patients will be needed 
to provide the sample size required to determine a statistically significant drug effect for a larger 
study. We also expect that it may not be possible to identify and enroll more high risk patients at 
Rochester in a reasonable period of time given the incidence of multiple SCC and the eligibility 
criteria. It is possible this design may not give a long enough period of time on drug to substantially 
decrease the onset of new tumors, however we believe it will be sufficient since 2 other drugs that 
affect the frequency of SCC in susceptible patients do produce substantial effects in this time frame 
(Celebrex, Soriatane)(Elmets et al., 2010; Kadakia et al., 2012). A one year long study will permit 
completion of a feasibility pilot in a reasonable period to allow a larger, definitive study to be 
done. We hypothesize that Pioglitazone will have the capacity to reduce incidence of SCC by 
>50%, as >90% decrease in growth is seen in tissue culture of human SCC using levels of 
pioglitazone available in human serum (see preliminary data, above). This is also the effect size 
seen with Celebrex treatment, a drug that can also selectively modulate prostaglandin synthesis.  
 
We have chosen tumor incidence as the primary outcome measure. Because patients with high 
tumor risk develop new tumors at a variable pace, some subjects may not have any new tumors 
during the period of the study. However, in aggregate we feel that a tumor endpoint is the best 
choice. This decision was made because of our recent experience studying the effects of Celebrex 
on skin cancer incidence. That study assessed pre-skin cancers (actinic keratoses) as the primary 
endpoint(Elmets et al., 2010). However, when data was analyzed, it was found there was no 
significant change in precursor lesions, only in actual tumor numbers. Thus, we have elected to 
focus on a high risk population that will have high numbers of new tumors. These patients are also 
the target group that could derive the most benefit from treatment. 
 

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This study will also assess secondary endpoints. First, we will examine whether the ratio of well-
differentiated to poorly differentiated SCC is influenced while subjects are on treatment. This 
endpoint is included because it is unknown whether there may be more signaling mediated by 
PPAR in one tumor morphology vs. another. Second, we will determine whether patients on 
treatment might have different numbers of “borderline” lesions biopsied; precancerous lesions are 
often suspicious enough to biopsy. It may be that there will be fewer lesions in this category, as 
well as fewer squamous cell cancers. Third, we will test tumors that are excised after they are 
processed for routine diagnostic pathology to see if markers of proliferation or apoptosis that are 
influenced by PPAR activity or the presence of AKR1C3 are altered. These endpoints would 
support the idea that any changes produced in tumor incidence while patients are on study drug 
are related to the mechanistic effect on PPAR that is proposed. 
 
2.3. Rationale for Dosage 
Subjects will be treated with Pioglitazone 30 mg po daily when on treatment, following an initial 
2 week period to assess tolerability, particularly in patients with stage 1 or stage 2 heart failure.  
Pioglitazone was selected because of our recent investigations showing its profound efficacy in 
decreasing proliferation in human SCC in vitro (see BACKGROUND). The oral dose selected 
results in serum levels of Pioglitazone that correspond to the in vitro dose tested in our 
studies(Christensen et al., 2005).  In addition to the drug itself acting as an agonist for PPAR, 
there are 2 persistent metabolites that have similar agonist effects, increasing the possibility of 
tumor growth inhibition.  Thus, this dosing regimen is likely to give optimal suppression of tumor 
growth. This is a standard dose (30 mg) taken by patients who are taking Pioglitazone for 
management of their diabetes. When given to non-diabetic patients with non-alcoholic liver 
disease or those with hepatitis C to prevent cirrhosis(Bell et al., 2012), there were no untoward 
effects on glucose metabolism.  
 
 
 
3. CHARACTERISTICS OF THE RESEARCH POPULATION 
 
3.1. Subject Characteristics 
 
a) Number of Subjects:   
Forty subjects will be recruited to participate in this protocol. It is expected that we will have 1 
screen failure for every 4 subjects consented (mostly due to onset of other complex disease 
unrelated to their skin). There are no other participating sites. 
 
 
b) Gender and Age of Subjects:   
Adult subjects of both genders will be recruited. However, high frequency of SCC is more common 
in men, and the balance of patients recruited to the study will reflect this. SCC is a disease of older 
individuals, usually 50 and up, unless they are transplant recipients or have a genodermatosis that 
predisposes them to multiple tumors. This disease is virtually unknown in pediatric patients, so 
patients < 18years will be excluded.  
 
c) Racial and Ethnic Origin:   

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Squamous cell carcinoma in skin is induced by chronic exposure to ultraviolet light. Individuals 
who have dark skin pigmentation are highly resistant to this disease, so the study population will 
be largely Caucasian. Most Hispanic individuals are also too darkly pigmented to suffer from this 
condition because of the capacity of their skin to tan.  
 
3.2. Inclusion and Exclusion Criteria 
 
a) Inclusion Criteria:   
 >18 years of age, male or female, state of health stable 
 Able understand protocol and give consent 
 Has had treatment of 2 – 6 squamous cell carcinomas of the skin during the year 
prior to enrollment, & pathology is available for verification 
 Stable treatment regimen for their skin cancer problems in place for 1 year, with 
expectation to keep medications the same during study 
 Able to keep study appointments & comply with protocol 
b) Exclusion Criteria:  
 Unwillingness or unable to complete informed consent process 
 < 18 years of age 
 Allergy to Pioglitazone 
 Taking Rifampin, Trimethaprim, Celebrex or Gemfibrozil 
 Pregnant or breastfeeding (Pregnancy Category C) 
 History of heart failure NYHA Class III or Class IV 
 Subjects with type 1 or type 2 diabetes 
 Problems with pedal edema 
 Liver disease (ETOH, viral hepatitis, drug-induced hepatitis) or elevated ALT, AST 
or total bilirubin 
 Osteoporosis with high risk of fracture 
 History of bladder cancer 
 Recent change in chronic oral medications. Participants enrolled while on a 
systemic medication for their skin cancer must remain on treatment.  
 
3.3. Vulnerable Subjects 
 
We will not be recruiting vulnerable subjects. 
 
3.4. Discussion of Subject Population 
Patients with diseases that are likely to be made worse by Pioglitazone are excluded (heart 
failure, liver disease, osteoporosis or bladder cancer). Patients must have had a sufficient 
number of prior tumors that their increased risk of developing tumors in the future is clear.  
 
4. SUBJECT IDENTIFICATION, RECRUITMENT AND CONSENT 
 
4.1. Method Of Subject Identification And Recruitment 
Patients receiving care at the University of Rochester Department of Dermatology for their 
squamous cell cancer who meet study inclusion criteria will be asked by Dermatologic surgeon 
Dr. Sherrif Ibrahim about their interest in study participation. Subjects will be given the consent 

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form to examine and the contact number for the study coordinator, JoAnne VanBuskirk, who is 
approved to enroll subjects. Dr. Ibrahim will have no involvement in the consenting process for 
individuals he sees as a patient.   It will be made clear to the potential subjects that their 
participation is voluntary and their care in the Department of Dermatology will not be affected 
by their decision to participate in this study.   
 
To further enhance enrollment, a “Dear Colleague” Letter may be sent to Dermatologists and 
Mohs surgeons in the Rochester area who work at sites other than Red Creek, or refer patients to 
the University of Rochester Department of Dermatology in order to recruit subjects. For those 
faculty employed full-time at the University of Rochester, who see potential study subjects in our 
Dermatology practice, a prescreening process will also be conducted by study coordinator 
JoAnne VanBuskirk. She will review the EPIC records of potential subjects coming for 
appointments  to see if they meet eligibility criteria. If a patient coming to a visit meets study 
criteria, a bright pink reminder sheet  titled “Inclusion-Exclusion Criteria Pioglitazone Pink 
Sheet” outlining the key aspects of the study and its inclusion and exclusion criteria will be 
placed with the intake form.  This visual reminder to employed faculty that the patient they are 
about to see may be eligible to participate is intended to facilitate their asking the patient about 
their interest in the study.  Faculty will then be able to let the patient know about the study and 
ask if the patient has an interest in being contacted by the study coordinator. If the potential 
subject shows interest, the study coordinator will contact them by phone to further explain the 
study. Interested subjects will be mailed the consent form and a date arranged for them to come 
to the office to formally review the consent document and give their consent. Again, it will be 
made clear to the potential subjects that their participation is voluntary and their care in the 
Department of Dermatology will not be affected by their decision to participate in this study.   
 
4.2. Process of Consent 
Dr. Pentland or the coordinator will meet with interested subjects in a private room.  The consent 
form will be read in its entirety by the subject. Time allotted to consenting will be dependent on 
the subjects’ needs, questions, and comprehension of the information in the consent form.  Each 
subject will be encouraged to read the consent form on his/her own and ask any specific questions. 
 
At the time of consent, subjects will be offered the option to provide permission to be contacted 
in the future regarding this and other dermatologic studies.  Those who provide permission for 
future contact will be added to a list of high risk squamous cell skin cancer patients that are 
interested in participating in clinical research. 
 
Subject Capacity 
We will not be recruiting subjects incapable of giving informed consent. The subject’s capacity 
to comprehend the information in the consent form will be assessed by asking questions about 
the content and encouraging the subject to describe the study procedures. 
 
Documentation of Consent 
The participant’s consent will be documented by signing the consent form by both the study subject 
and the person obtaining consent. 
 
5. METHODS AND STUDY PROCEDURES 
 

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A schedule of study visits is provided in Appendix 1. A description of the study procedures are 
also listed in appendix 1. 
 
Outlined below are the assessments that will be performed to evaluate efficacy and those to 
evaluate safety.  Tests and interventions which are considered experimental and performed 
exclusively for research purposes have been identified and differentiated from that which would 
occur regardless of the research (i.e., standard of care). 
 
5.1. Efficacy Assessments 
Administration of Pioglitazone to prevent/decrease the aggressive phenotype of skin cancer is 
experimental. NB- obtaining biopsy of lesions suspected of skin cancer is NOT experimental; it is 
required for usual patient care in these high risk patients. In order to know whether Pioglitazone is 
efficacious for preventing SCC in high risk individuals, we will count the number of skin tumors 
documented by biopsy/treatment that occur in our subjects before, during, or after Pioglitazone 
treatment. This assessment will take into account compliance, as described below. The tumor 
counts will be best interpreted in aggregate for the whole sample group, since there is variability 
in the frequency with which new tumors appear based on the predisposing condition. Since 
Pioglitazone has been on the market for some time without observation of a negative impact on 
skin or wound healing, the study focus will be determining if numbers of tumors decrease and 
whether their phenotype changes. The physician reading the pathology of the skin biopsies will be 
responsible for identifying the number of tumors that occur –i.e., some lesions thought to be tumors 
and therefore biopsied in clinical practice turn out to be hypertrophic actinic keratosis (not tumors). 
These would not be counted as skin cancers, since they are precancerous lesions. 
 
5.2. Safety Assessments 
The main safety issue associated with this trial is whether an adverse effect will occur in 
patients due to the administration of pioglitazone. Clinical endpoints to monitor for safety are: 
weight, pedal edema, dyspnea, heart failure, headache, myalgia, fracture, liver injury and 
vision changes related to macular edema, blood in the urine or other changes that are out of the 
ordinary. The study related blood monitoring is to ensure there are no preexisting conditions 
that make participants susceptible to drug-related side effects. For instance, individuals with 
preexisting liver disease may have hepatotoxicity when taking pioglitazone, hence they must 
be excluded from participation. However, since participants could develop new diseases while 
participating, blood will also be taken from subjects taking pioglitazone to screen for such 
concerns. At a study visit approximately 60 days after drug initiation, blood work will be 
repeated to detect changes suggesting drug related side effects. Monitoring of blood for glucose 
levels, altered liver function, or decreased Hemoglobin could indicate the patient should stop 
taking the study drug. Tests to be followed will be CBC and CMP at baseline, approximately 
60 days after starting drug and at the end of treatment. Blood test results will be interpreted in 
the context of the clinical exam. The patient population being studied has co-morbidities that 
require them to regularly give blood to monitor their health as part of their routine care, so 
study related blood draws may not be necessary if these tests are obtained by their providers at 
appropriate intervals; it will not be necessary to repeat them. The risk of bladder cancer will 
be monitored by urinalysis. Taking Pioglitazone for > 1 year increases the risk of bladder 
cancer from 7 in 10,000 to 10 in 10,000. Female patients of child bearing age will have urine 
pregnancy testing at baseline and at subsequent study visits. Stop Criteria: Subjects will be 

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dropped from the protocol if clinical or laboratory assessment reveals grade II abnormalities 
as defined by Common terminology Criteria for adverse events V.3. Subjects will also be 
dropped from the protocol if they are unable to tolerate 30 mg Pioglitazone treatment when the 
dosage is increased to 30 mg daily after the initial 2 week treatment with 15 mg daily. 
 
 
5.3. Assessment of Subject Compliance 
The study coordinator will assess the subject’s compliance with the protocol by counting the pills 
remaining in the prescription obtained for the study. The date the prescription was filled relative 
to the study visit date will also be noted. 
 
5.4. Data & Specimen Banking for Future Research Use 
This will not be done. 
 
5.5. Genetic/Genomic Research Activities 
No genetic/genomic research activities are planned. 
 
5.6. Costs to the Subject 
Subjects will be prescribed Pioglitazone, a drug that costs $12 per month. The cost of drug will be 
paid by the Department of Dermatology. Costs of blood tests specifically related to the study to 
monitor safety of drug administration are also covered by the department. The costs related to 
subject’s cancer treatment (standard care) will be charged to insurance as is routine. There is only 
Department of Dermatology clinical grant support for this project for coverage of medication costs. 
 
5.7. Payment for Participation 
There is no payment for participation. 
 
5.8. Return of Individual Research Results 
Any side effects suspected to be related to the study drug will be reported to the subject at the time 
of their detection. Subjects who have significant side effects attributable to study drug will be 
dropped from the study, and the side effects reported to their physician. Since the study is not 
blinded, subjects will be directly aware of whether there is a detectable difference in the incidence 
of their skin cancers.  
 
6. CONCOMITANT AND DISALLOWED MEDICATIONS 
Subjects will be asked to continue their usual medications. Potential subjects with type 1 or type 2 
diabetes will not be enrolled. 
 
7. SUBJECT WITHDRAWALS 
Subjects will be advised in the written informed consent provided that they have the right to 
withdraw from the study at any time without prejudice, as well as being notified of this by the 
study coordinator. Subjects may be withdrawn by the investigator without their consent for non-
compliance, intercurrent illness, or pregnancy (Pioglitazone is pregnancy category C) for 
suspected drug related side effects. Stop criteria to be used are clinical or laboratory assessments 
revealing grade II abnormalities as defined by Common terminology Criteria for adverse event 
V.3.  No additional study activities will be completed by the subject prior to withdrawal from the 

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study.  Subjects withdrawn from the study will be replaced. 
 
8. STUDY DRUG/DEVICE/BIOLOGIC ADMINISTRATION/ASSIGNMENT 
Note:  The package insert is included in appendix 2. 
 
8.1. Study Drug/Device/Biologic 
Pioglitazone is a currently FDA approved drug. A formal request to conduct the study with the 
product to obtain an IND is in process.  Patients will be provided drug once they consent to 
participate and are found to be eligible. 
 
8.2. Dosage of Study Drug/Biologic 
Subjects will be treated with 15 mg Pioglitazone for the first 2 weeks. If tolerated, the dose will 
then be increased to 30 mg Pioglitazone daily while on protocol for drug treatment. Subjects unable 
to tolerate 30 mg Pioglitazone will be dropped from the study. 
 
8.3. Subject Enrollment/Randomization 
Subjects will be randomized to one of two study groups: initial observation or initial drug treatment 
followed by washout. 
 
8.4. Accountability of Investigational Supplies 
The study drug is ordered through the investigational drug service. The study drug will be 
dispensed to the Study Coordinator n transported to the Red Creek clinic site of Dr. Ibrahim.  It 
will be stored in a locked cabinet at room temperature until the supply is given to the patient for 
use. Once patients give consent, the study drug will be dispensed when patients enter the active 
treatment arm of the protocol. On return appointment, patients will bring their study drug bottle 
with them. A pill count will be done at each study visit, and the date of drug dispensing will be 
noted. At the visit prior to completion of the active treatment arm, the exact number of pills 
required for the planned period until the next study visit will be dispensed to the patient. This will 
facilitate having patients stop taking drug at the same time as their last “on treatment” study  visit 
. 
 
8.5. Subject Withdrawal of Study Drug 
If study subjects stop drug treatment (other than as indicated in the protocol) they will be dropped 
from the study. Compliance will be considered taking the drug appropriately >80% of the time. 
 
8.6. Emergency Drug Disclosure   
 
Not applicable 
 
9. SAFETY AND REPORTABLE EVENTS 
 
9.1. Adverse Event Definition 
An adverse event is any symptom, sign, illness, or experience which develops or worsens during 
the course of the study, whether or not the event is considered related to study drug. 
 
9.2. Serious Adverse Event 
A serious adverse event is defined as any adverse medical experience that results in any of the 
following outcomes: 

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• 
death; 
• 
is life-threatening; 
• 
requires inpatient hospitalization or prolongation of existing hospitalization; 
• 
results in persistent or significant disability/incapacity; 
• 
is a congenital anomaly/birth defect; or 
• 
requires medical or surgical intervention to prevent permanent impairment or damage. 
 
9.3. CBC, CMP will be monitored at baseline and at the study visit occurring 60 days after Drug 
treatment begins. Urinalysis will be checked for Blood at each study visit.  
At each subject visit the study staff will assess adverse events by recording all voluntary 
complaints of the subject and by assessment of clinical and laboratory features.  At each study 
visit, the subject will be questioned directly regarding the occurrence of any adverse experience 
since his/her last visit.   
 
All adverse events, whether observed by the Investigator, elicited from or volunteered by the 
subject, will be documented.  Each adverse event will include a brief description of the 
experience, the date of onset, the date of resolution, the duration and type of experience, the 
severity, the relationship to taking Pioglitazone, contributing factors, and any action taken with 
respect to Pioglitazone. 
 
Recording of adverse events will occur during the time that the patient is on study drug and for the 
next study visit after finishing drug. Follow up of adverse experiences will be until the event is 
resolved or stabilized or for 6 months.  
 
9.4. Responsibilities for Reporting Serious Adverse Events 
 
The Investigator will record all serious adverse experiences that occur during the study period in 
the appropriate source documents and/or AE log as applicable.  The study period for reporting 
serious adverse events will be from the time of signing consent to final study visit.  
 
As sponsor of the relevant IND, the PI is responsible for compliance with the FDCA (21 USC §§ 
301 e. seq.) as well as the implementing regulations [Tittle 21 of the CRF].  These responsibilities 
include reporting any unexpected fatal or life-threatening suspected adverse reactions to the 
FDA/CDER no later than 7 calendar days after initial receipt of the information.   
 
Additionally, the PI will comply with all federal regulations and RSRB policies regarding the 
reporting of adverse events. 
 
 
10. RISK/BENEFIT ASSESSMENT 
 
10.1. Potential Risks 
Exacerbation of heart failure symptoms, fluid retention, pedal edema and weight gain are the 
primary risks of concern in this study. Similarly, fluid retention may cause anemia by diluting the 
blood. Liver problems have been reported in some patients receiving pioglitazone in the presence 
of active liver disease. Recent evidence links prolonged pioglitazone treatment (>1 year) in 
diabetics with increased risk of bladder cancer (from 7/10,000 to 10/10,000). Our subjects will be 

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treated for a maximum of 6 months. Additionally, by excluding subjects with a history of bladder 
malignancy we are following the FDA advice to not begin this medication in subjects with a past 
or present history of bladder cancer. Similarly, our subjects will not be on pioglitazone long enough 
to increase their risk of bone fractures. While subjects with diabetes have experienced macular 
edema and low blood sugar, these potential adverse effects are markedly less common in 
nondiabetics. Women with polycystic ovarian syndrome may have their ovulation cycles 
normalize. 
As with other drugs, some patients may develop an allergic reaction to pioglitazone. Minor risks 
associated with pioglitazone treatment included headache, sinus infection, muscle pain, sore throat, 
tooth disorders and respiratory tract infections. 
For full details, please see the attached package insert.  
 
10.2. Protection Against Risks 
Patients will be advised of possible drug side effects by study personnel and encouraged to contact 
the study coordinator if they suspect side effects are occurring. Risks will be minimized by 1) good 
case selection 2) education and 3) stopping drug treatment when significant side effects are 
identified. Appropriate screening of subjects for subclinical side effects of Pioglitazone will also 
minimize risk. Since the drug’s side effects are not abrupt in their onset, the followup included in 
the study design should be effective.  Should side effects be noticed, patients will be treated by 
study personnel and counseled, or referred to their provider.  
 
10.3. Potential Benefits to Subjects 
Subjects may benefit from reduced numbers of tumors if drug treatment works as expected. 
 
10.4. Alternatives to Participation 
No research alternatives are available to the subject, and they can pursue usual care. 
 
11. CONFIDENTIALIAY OF DATA AND INFORMATION STORAGE  
Confidentiality of protected health information and samples is ensured through the following 
measures:  
 Only authorized persons will be granted access; 
 Only authorized persons may enter and view study data;   
 Passwords and system IDs will not be shared;  
 Physical security of the workstations/files will be maintained;  
 Adequate back-up plan is in effect;  
 Staff trained on data entry system and importance of security procedures;  
 Workstations with databases will not be left unattended while in use, and will be left 
locked and password protected.  
 Only study team members will have access to subject identifiers. Samples and data will 
be labeled with unique, random codes.  
 Codes will not be generated from existing identifiers, such as initials or enrollment date.  
 A link will be maintained indefinitely between subject identifiers and the codes.  
 The link will be stored on a password-protected computer, accessible only to study team 
members. 
 
 

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12. RESEARCH INFORMATION IN MEDICAL RECORDS 
Blood test results will be in the patients’ medical record for medical reasons unrelated to 
Pioglitazone treatment, as will results of biopsies as needed for normal patient care. No other study 
information will be in the medical record. 
 
13. DATA ANALYSIS AND MONITORING 
 
13.1. Sample Size Determination 
 
This pilot study will be designed as a 2-by-2 crossover trial. We refer to Section 2.2 for a 
justification of the study design.  Patients will be enrolled and randomized in one arm of two 
treatment protocols.  Each protocol will include two treatments administered during two 
consecutive time periods: 
 
- 
Treatment arm # 1: Patients enrolled in the first arm will first receive six months of usual care 
(Period 1); next, they will be treated with Pioglitazone combined with usual care for the next 
6 months (Period 2). Patients will take study drug until they come to their final “on Treatment” 
visit, which will occur 168 days +/- 14 days of their “on Treatment” date. 
 
- 
Treatment arm # 2: Patients enrolled in the second arm will first receive six months of 
Pioglitazone treatment combined with usual care (Period 1); next, they will receive usual care 
alone during the following 6 months (Period 2).  Patients will take study drug until they come 
to their final “on Treatment” visit, which will occur 168 days +/- 14 days of their “on 
Treatment” date. 
A flow diagram describing the study design is presented in Section 2.  
 
The primary endpoint will be the total number of skin tumors counted during each treatment 
period. Based on clinical evidence, we expect the total number of skin tumors to range between 0 
and 3 per patient and per treatment period (or 0 to 6 per patient per year) and to average around 
1.5.  We would consider Pioglitazone to be clinically effective if it reduces tumor counts by 50% 
(that is, reduce the average number of tumors from 1.5 to 0.75 per patient and per treatment period). 
Previous work by this investigator has shown that such reductions in tumor numbers can occur 
when prostaglandin metabolism is affected by drug treatment (Elmets et al., 2010).   
 
A two-sided t-test achieves 96% power to detect a reduction in the number of skin tumors due to  
Pioglitazone  when the total sample size of a 2-by-2  crossover design is 40 (20 patients per group).  
The calculations assumed that the actual mean difference is 0.75 (corresponding to a 50% 
reduction in the mean number of skin tumors due to treatment), the square root of the within mean 
square error is 1, and the significance level is 5%.  The figure below displays the power of the 
study as a function of the sample size (N) for three values of the square root of the within mean 
square error (SW). 

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13.2. Planned Statistical Analysis 
 
The main objective of the statistical analysis will be to estimate the effect of Pioglitazone on the 
occurrence of skin tumors when used in combination with usual care compared to usual care alone. 
The primary endpoint will be the total number of skin tumors counted during each treatment 
period. We will have two observations per patient, one for each treatment period of the 2-by-2 
cross-over design (either baseline to 6 months, or 6 months to 12 months post-baseline). We will 
first compute the empirical mean (and its standard error), standard deviation and median for the 
number of skin tumors within each treatment arm and each treatment period. We will also construct 
histograms for tumor counts. We expect tumor counts to range between 0 and 6 per six months, 
such that parametric mixed analysis of variance (ANOVA) may not be the most appropriate 
approach to assess treatment effects.  As an alternative, we will describe the number of skin tumors 
using log-linear Poisson regression models with random effects.  The developed models will 
include treatment arm, period, and their interaction as covariates while adjusting for potential intra-
subject dependencies (or between subject variation) using random intercepts.  We will use this 
model to investigate the presence of washout effect (secondary aim).   
 
In additional, exploratory analyses, we will assess if the phenotype of skin tumors changes during 
treatment with Pioglitazone. We will also determine if Pioglitazone changes the expression of 
markers influenced by PPAR or AKR1C3 (apoptosis, proliferation) in tumors removed from 
treated patients.  We will have an apoptosis and a proliferation score for every skin tumor that 
arises, both treated as continuous variables.  The analyses will be conducted using mixed ANOVA 
modeling the apoptosis and the proliferation scores of each tumor as a function of treatment arm, 

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period, and their interaction. Random intercepts will also be included in the models to account for 
subject to subject variability.  We will develop separate models for the apoptosis and for the 
proliferation scores. We will investigate modeling assumptions using plots of residuals and 
conditional residuals.  In the event modeling assumptions appear violated, we will consider 
applying suitable transformations (e.g., logarithmic, square root) to normalize the data. As an 
alternative, we will consider performing statistical inference using a generalized estimating 
equations (GEE) approach. 
All tests and construction of confidence intervals will be two-sided and conducted at the 5% 
significance level.  
 
 
13.3. Data and Safety Monitoring 
Drs. Pentland and Ibrahim will conduct continuous review of study data and patient safety.  The 
PI and Study Coordinator are responsible for continuously monitoring the conduct of the trial 
and are the primary individuals charged with identification and reporting of all AE and SAE 
occurrences.  The review will include the number of patients, significant Pioglitazone toxicities 
as described in the protocol, and responses observed.  The Investigator will submit summaries of 
this data at least annually to the Data Safety Monitor (DSM; see below) for review.  
Additionally, a copy of the completed RSRB Annual Progress report will be submitted to DSM.   
 
The Data Safety Monitor will be assigned oversight of safety monitoring and study progress.  
The DSM will review will review accrual and adverse events at least annually and following 
study completion.  The DSM, Dr. Leway Chen, is a board-certified and practicing cardiologist 
who has experience with clinical trials. 
 
All serious adverse events that the PI classifies as related or possibly related to therapy will be 
submitted to DSM for determination as to whether further action is required within 48 hours.  
 
14. INVESTIGATOR RESPONSIBILITIES 
Adequate training and supervision of the study team will be ensured by the Principal Investigator as 
described below.  
The PI will 
 
Provide copies of the protocol, consent and any other study related materials (including all 
other information relating to the preclinical and prior clinical experience) to all study personnel 
with delegated responsibilities for this study. 
 
Discuss all protocol materials and information with study staff to assure that they are 
adequately informed regarding Pioglitazone and conduct of the study. 
 
Assure that all study staff members are qualified by education, experience and training to 
perform their specific responsibilities.  
 
The Investigator will ensure appropriate documentation of compliance to the protocol, 
informed consent process and obtaining verbal or written consent on an ongoing basis, with 
written progress reporting quarterly.   
 
The Investigator (and study staff) will identify, report and manage potential conflicts of interest 
biannually.   
 
A delegation log to document activities that have been authorized by the Investigator to study 
staff and a training log to document necessary protocol training activities will be completed 
will  be utilized as part of this process. 
 

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15. REFERENCES 
 
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squamous cell carcinoma of the skin (North Central Cancer Treatment Group Study 969251). 

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Komoto J, Yamada T, Watanabe K, Woodward DF, Takusagawa F (2006) Prostaglandin F2alpha 
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of a principal mRNA species for human 3alpha-hydroxysteroid dehydrogenase isoform 
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Miller SJ, Alam M, Andersen J, Berg D, Bichakjian CK, Bowen G, et al. (2010) Basal cell and 
squamous cell skin cancers. J Natl Compr Canc Netw 8:836-64. 
 
Miller VL, Lin HK, Murugan P, Fan M, Penning TM, Brame LS, et al. (2012) Aldo-keto 
reductase family 1 member C3 (AKR1C3) is expressed in adenocarcinoma and squamous cell 
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Mullen JT, Feng L, Xing Y, Mansfield PF, Gershenwald JE, Lee JE, et al. (2006) Invasive 
Squamous Cell Carcinoma of the Skin: Defining a High-Risk Group. Ann Surg Oncol. 
 
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hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto reductase 
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