Clinical trial NCT01697566
Conditions: Gynecology
Keywords: Gynecology Endometrial Cancer Chemoprevention Obese Body Mass Index (BMI) >/= 35 kg.m2 Post-menopausal Hyperinsulinemia Metformin Placebo Sugar pill Lifestyle intervention Diet Exercise
Sponsor: M.D. Anderson Cancer Center
Start date: 2013-05-30; Completion date: 2024-05-30
Enrollment: 29 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The goal of this clinical research study is to learn about the effects of metformin and/or a program called "lifestyle intervention" on the endometrium (inner lining of the uterus) in post-menopausal women who are also obese (both are risk factors for endometrial cancer). Researchers also want to learn how insulin levels will be affected by metformin, diet and/or exercise.

Metformin is designed to treat both diabetes and insulin resistance. Insulin resistance is a condition in which the body makes insulin but does not use it properly. It is often referred to as "pre-diabetes". Many people with insulin resistance have high levels of both sugar and insulin in their blood at the same time, which have been reported in patients with endometrial cancer.

In this study, metformin will be compared to a placebo. A placebo is not a drug. It looks like the study drug but is not designed to treat any disease or illness. It is designed to be compared with a study drug to learn if the study drug has any real effect.

Lifestyle intervention is made up of a series of in-person sessions where you meet with a coach to discuss strategies for losing weight and ways to increase physical activity. It also consists of materials designed to help you lose weight and will offer opportunities for supervised exercise.

Detailed description:
Study Groups:

If you are found to be eligible to take part in this study, you will be randomly assigned (as in the roll of dice) to 1 of 4 study groups:

* Metformin only.
* Placebo and lifestyle intervention.
* Metformin and lifestyle intervention.
* Placebo only.

Neither you nor the study staff will know if you are receiving metformin or placebo. However, if needed for your safety, the study staff will be able to find out what you are receiving.

In the first month you will gradually increase the dose of metformin/placebo by mouth as listed below:

* Week 1: One capsule each day
* Week 2: One capsule 2 times each day
* Week 3: One capsule 3 times each day
* Week 4: Two capsules 2 times each day

After week 4, you will continue to take 2 capsules of metformin/placebo 2 times each day.

If you are receiving the lifestyle intervention, you will have 16 lifestyle intervention sessions over 4 months. At each of these sessions, you will discuss methods for managing your weight. You will receive print materials and worksheets, measuring utensils, and a food scale. You will be instructed how to use each of these materials and how often to use them during the study. You will also be provided with opportunities for supervised exercise (such as group exercise class and group walks).

If you are traveling during a scheduled session, it can be performed over the telephone. Each call should last about 30 minutes, during which you will discuss the same goals and information that you would discuss in the in-person sessions.

Study Visits:

Before you can begin receiving the study drug/placebo:

* At the second screening visit , you will have a dual energy x-ray absorptiometry (DEXA) scan to measure your body composition. The scan requires you to lie still on a table for about 8-10 minutes while a low-energy x-ray takes an image that allows the study staff to get an image of your body fat density.
* You will be given an accelerometer (a small, portable device that measures your physical activity) and be taught how to use it at the first Screening Visit. You will wear the accelerometer at all times while you are awake for 7 days. At the second Screening Visit, you will return the accelerometer at an intervention session. If you are not able to return it at the second visit then you will mail it back to the study staff in a pre-paid envelope. You will receive the accelerometer again at Month 4 and wear it for another 7 days.
* You will receive a study diary in which you will record any and all exercise you perform each day while you are on study. It should take about 5 minutes to complete each time.
* You will complete questionnaires about diet and exercise. These questionnaires should take about 30-40 minutes to complete.
* You will also be taught to complete a questionnaire on the internet to record the food you eat in a 24 hour period. You will do this 3 times over a 2 week period. The first time, you will complete it at the clinic. The study staff will call and remind you to complete the questionnaire the other 2 times. Each time you complete this questionnaire, it should take about 40 minutes.

At Month 1, all participants will return for a study visit. The following tests and procedures will be performed:

* Blood (about 2 teaspoons) will be drawn for routine tests. If this blood draw shows that your liver or kidney function is abnormal, you will need to return for an additional blood °draw at Month 2.
* If you are having symptoms that you would describe as moderate or worse, you will have a non-pelvic physical exam including measurement of your vital signs and any unreported symptoms you may be having.

At each month, you will be asked about any symptom(s) that you may have to learn if it is related to the study. If you are called, this phone call should last about 5 minutes. If you are asked in a in-person visit, the visit can last up to an hour.

Length of Study:

You will receive the study drug/placebo and/or lifestyle intervention for up to 4 months. You will be taken off study if you have intolerable side effects or if you develop endometrial cancer or hyperplasia during the study. Your participation in the study will be over after the end-of-study visit.

Post-Treatment Visit:

At the end of the 4th month, you will have a post-treatment visit. You will repeat all procedures performed in the first and second visit:

* Your height, weight, waist, and hip circumference will be measured.
* You will have a DEXA scan.
* You will complete the questionnaires about food, diet, and exercise.
* You will be given a questionnaire about your satisfaction (based on the intervention) to complete and return at this visit. This questionnaire will take up to an hour to complete.
* You will have an endometrial biopsy.
* Blood (about 8 tablespoons) will be drawn for routine tests and for biomarker testing. You will have to fast (not eat or drink anything except water) for 12 hours before this blood draw.
* You will complete the questionnaire on the internet to record the food you eat in a 24 hour period. You will do this 3 times over a 2 week period. The first time, you will complete it at the clinic. The study staff will call and remind you to complete the questionnaire the other 2 times. Each time you complete this questionnaire, it should take about 40 minutes.

Follow-Up:

About 1 year after the screening visits (+/- 1 month), the following tests and procedures will be performed:

* Your height, weight, waist, and hip circumference will be measured.
* You will have a DEXA scan.
* Blood (about 8 tablespoons) will be drawn for routine tests and for biomarker testing. °You will have to fast (not eat or drink anything except water) for 12 hours before this blood draw.
* You will wear the accelerometer for 7 days.
* You will complete questionnaires about food, diet, and exercise.
* You will complete the questionnaire on the internet to record the food you eat in a 24 hour period. You will do this 3 times over a 2 week period. The first time, you will complete it at the clinic. The study staff will call and remind you to complete the questionnaire the other 2 times. Each time you complete this questionnaire, it should take about 40 minutes.

This is an investigational study. Metformin is FDA approved and commercially available for the treatment of diabetes and insulin resistance.

Up to 100 patients will take part in this study. All will be enrolled at MD Anderson Cancer Center.

Arms: arm 1: Participants gradually increase the dose of metformin by mouth as listed below:

Week 1: One capsule each day Week 2: One capsule 2 times each day Week 3: One capsule 3 times each day Week 4: Two capsules 2 times each day

After week 4, participant continues to take 2 capsules of metformin 2 times each day.

Each capsule is 425 mg. arm 2: Placebo taken by mouth twice daily for 4, 30 day cycles. Lifestyle intervention consists of 16 in-person sessions offered over a 4 month period. arm 3: Participants gradually increase the dose of metformin by mouth as listed below:

Week 1: One capsule each day Week 2: One capsule 2 times each day Week 3: One capsule 3 times each day Week 4: Two capsules 2 times each day

After week 4, participant continues to take 2 capsules of metformin 2 times each day.

Each capsule is 425 mg.

Lifestyle intervention consists of 16 in-person sessions offered over a 4 month period. arm 4: Placebo taken by mouth twice daily for 4, 30 day cycles.
Interventions: intervention 1: Metformin intervention 2: Placebo intervention 3: Endometrial Biopsy intervention 4: Lifestyle Intervention intervention 5: Questionnaires
Intervention descriptions: intervention 1: Participants gradually increase the dose of metformin by mouth as listed below:

Week 1: One capsule each day Week 2: One capsule 2 times each day Week 3: One capsule 3 times each day Week 4: Two capsules 2 times each day

After week 4, participant continues to take 2 capsules of metformin 2 times each day.

Each capsule is 425 mg. intervention 2: Placebo taken by mouth twice daily for 4, 30 day cycles. intervention 3: Endometrial biopsies obtained at baseline and after 4 months. intervention 4: Lifestyle intervention consists of 16 in-person sessions offered over a 4 month period. intervention 5: Completion of questionnaires about diet and exercise at baseline, during treatment and at end of study. Questionnaires take about 30-40 minutes to complete.

Protocol documents:
Prot_SAP_000.pdf: 
 
 
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Contents 
1.0  Objectives ................................................................................................................................................. 3 
1.1 Primary Objectives ............................................................................................................................... 3 
1.2 Endpoints ............................................................................................................................................... 3 
2.0 Background ............................................................................................................................................... 4 
2.1 Obesity and endometrial cancer ......................................................................................................... 4 
2.2 Metformin, mechanism of action and possible role in the prevention of cancer ........................ 5 
2.3 Diet and exercise linked to hyperinsulinemia and insulin resistance ........................................... 6 
2.4 Rationale and definition of cohort ...................................................................................................... 7 
2.5 Safety issues of metformin, as chemoprevention for endometrial cancer in a cohort of 
obese women .............................................................................................................................................. 8 
2.6 Biomarkers as surrogate endpoints ................................................................................................... 8 
2.7 Description of Proposed Involvement of Human Subjects ........................................................... 10 
3.0 Background Drug Information .............................................................................................................. 10 
3.1 Agent .................................................................................................................................................... 10 
3.2 How supplied ....................................................................................................................................... 10 
3.3 Dose groups ........................................................................................................................................ 10 
3.4 Dose and Duration of exposure ....................................................................................................... 11 
3.5 Half life ................................................................................................................................................. 11 
3.6 Administration ..................................................................................................................................... 11 
3.7 Side effects .......................................................................................................................................... 11 
3.8 Contraindications ................................................................................................................................ 11 
3.9 Use during pregnancy ....................................................................................................................... 12 
3.10 Manufacturer and Supplier ............................................................................................................. 12 
4.0 Participant Eligibility ............................................................................................................................... 12 
4.1 Inclusion Criteria ................................................................................................................................. 12 
4.2 Exclusion Criteria ................................................................................................................................ 12 
5.0 Design and Methods .............................................................................................................................. 13 
5.1 Study Design ....................................................................................................................................... 13 
5.2 Screening Visit 1 ................................................................................................................................. 14 
5.3 Screening Visit 2 ................................................................................................................................. 17 


 
 
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5.4 Randomization / Visit 3 ...................................................................................................................... 19 
5.5 Study Compensation .......................................................................................................................... 20 
5.6 Registration Process .......................................................................................................................... 20 
5.7 Participating Center ............................................................................................................................ 21 
6.0 Treatment Plan ....................................................................................................................................... 21 
6.1 Treatment ............................................................................................................................................ 21 
6.2 Lifestyle Intervention .......................................................................................................................... 22 
6.3 Supervised Exercise Session ........................................................................................................... 24 
6.4 Toxicity assessment ........................................................................................................................... 24 
7.0 Data Monitoring Plan ............................................................................................................................. 25 
7.1 Protocol compliance and assessment of toxicity data .................................................................. 26 
7.2 Data collection Plans ......................................................................................................................... 26 
8.0 Criteria for Removal from the Study .................................................................................................... 27 
9.0 Statistical Considerations ...................................................................................................................... 27 
10.0 Reporting Requirements ..................................................................................................................... 28 
10.1 Adverse Event .................................................................................................................................. 29 
10.2 Serious Adverse Event .................................................................................................................... 29 
10.3 Eliciting Adverse Event Information ............................................................................................... 29 
10.4 Grading/Rating Scale ....................................................................................................................... 29 
10.5 Non-NCI Adverse Event Grading Scale Grade/Description ...................................................... 29 
10.6 Relationship to Study Treatment .................................................................................................... 30 
11.0 Laboratory Evaluations and Procedures .......................................................................................... 30 
11.1 Laboratories ...................................................................................................................................... 30 
11.2 Collection and Handling Procedures ............................................................................................. 31 
11.3 Shipping Instructions ....................................................................................................................... 31 
11.4 Experimental Methodology and Preliminary Results .................................................................. 31 
12.0 Transcripts to be Analyzed by Q-PCR - Endometrial Biopsies ..................................................... 32 
12.1 Preparation of RNA from Endometrial Biopsies for Real- Time Q-PCR .................................. 32 
12.2 Analysis of Q- PCR Transcripts ..................................................................................................... 33 
12.3 Real-Time Q-PCR ............................................................................................................................ 34 
12.4 Logistics of Real- Time Q-PCR .......................................................................................................... 34 
12.5 Databases and Data Storage for Real- Time Q-PCR ................................................................. 35 


 
 
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12.6 Immunohistochemistry .................................................................................................................... 35 
13.0 Schedule of Events .............................................................................................................................. 35 
14.0 References ............................................................................................................................................ 37 
 
Synopsis 
 
This study will use a 2 x 2 randomized design with all 100 post- menopausal subjects 
being randomized to metformin, placebo, placebo and intensive lifestyle intervention, or 
metformin and intensive lifestyle intervention.       Treatments will last 4 months. 
Participants  will  be  assessed  at  baseline  and  end  of  treatment  with  endometrial 
sampling to see if there are changes in the biomarkers. 
 
Protocol Sections 
1.0  Objectives 
 
1.1 Primary Objectives 
 
1.1.1 The primary objective of this study is to evaluate the independent effects of 
metformin and a diet and physical activity intervention on potential surrogate 
endpoint biomarkers (SEBs) of the endometrium in obese post-menopausal women.  
The SEBs includes the panel of genes listed in Table 1 in Section 2.6 and Ki-67 
biomarker. 
 
1.1.2 Other biomarkers will be examined, including 
 
1.1.2.1 A panel of genes (PCNA, IGF-IR, RALDH2, sFRP4, survivin, and 
EIG121) 
relevant 
to 
estrogen-dependent 
endometrial 
proliferation, 
hyperplasia and cancer using Q-PCR. 
 
1.1.2.2 Biomarkers specific to the effect of metformin treatment (phospho- 
AMPKa, phospho-ACC, phospho-mTOR and phospho-S6 ribosomal protein). 
 
1.2 Endpoints 
 
1.2.1 The primary endpoint biomarker that will be investigated is proliferation Ki-
67. 
 
1.2.2 Secondary endpoints include: 
 
1.2.2.1  Changes  in  serum  levels  of  estradiol,  estrone,  testosterone, 
DHEA-S, sex hormone binding globulin (SHBG), adiponectin, glucose 
(fasting), HbA1c, insulin (fasting) and insulin-like growth factor-1 (IGF-1). 


 
 
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1.2.2.2 Changes in diet, physical activity, body composition (BMI, waist and 
hip circumference, and measurements from the DEXA scan), quality of  life,  
and  psychosocial  determinants  of  behavior  change  will  be examined 
in this sample of obese, post-menopausal women taking metformin or 
receiving a diet and physical activity intervention. 
2.0 Background 
 
2.1 Obesity and endometrial cancer 
 
Numerous epidemiologic studies have demonstrated that obesity is strongly associated 
with an increased risk of endometrial cancer. While an average woman has a 3% 
lifetime risk of endometrial cancer, obese women have a 9-10% lifetime risk of 
endometrial cancer.[1] In addition, obese women account for almost 50% of all 
endometrial cancers.[1] In a recent review of diet and cancer by the American Institute 
for Cancer Research and World Cancer Research Fund (WCRF), authors stated that 
the evidence relating body mass index and cancer is strongest for endometrial cancer, 
but also is compelling for breast, colon, kidney and gallbladder cancer.[2] 
 
There are a number of ways in which obesity has been hypothesized to increase risk of 
endometrial cancer. Obese women have increased bioavailability of adrenal steroids, 
increased peripheral conversion in adipose tissues of the adrenal steroids to estrone, 
and increased bioavailability of free estrogens due to decreased sex hormone binding 
globulin. This “hyper-estrogenic state” is believed to result in increased endometrial cell 
mitosis, leading to endometrial hyperplasia, a pre- malignant lesion, and to endometrial 
cancer. However, increased levels of circulating endogenous estrogen cannot 
completely explain the association of obesity and endometrial cancer. While some 
clinical studies show a correlation between higher plasma levels of endogenous 
estrogens or androgens in women with endometrial cancer versus controls, others do 
not.[3-13] Epidemiologic studies addressing this question also yield conflicting results. 
In a large study by Potischman et al, the authors found that body mass index 
remained a significant risk factor for the development of endometrial cancer even after 
controlling for endogenous estrogen. They concluded that high levels of endogenous 
estrogens were not sufficient to explain the increased risk of obesity to endometrial 
cancer.[11] Another prospective multicenter study demonstrated that the risk of 
endometrial cancer associated with endogenous estrogen levels in postmenopausal 
women remained significant after adjusting for BMI.[13] 
 
What other mechanisms could explain the association of obesity to the development of 
endometrial cancer? Are there other mechanisms that explain why obesity 
isassociated with a number of other cancers? The American Cancer Society has 
recently highlighted the increased risk of a number of cancers in obese women. In 
particular, cancers of hormonally regulated organs, including endometrium, breast 
and ovary, have been shown in epidemiologic studies to be associated with obesity.[14]  


 
 
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Other cancers including gallbladder, colon and prostate are also seen more frequently 
in the obese.[14] Is there an underlying metabolic cause for this increased cancer risk? 
 
Hyperinsulinemia and the insulin resistant state are closely associated with obesity, and 
may contribute in a meaningful way to increase the risk of cancer in an obese person. 
Recently, adiponectin, an adipocyte- derived protein highly correlated with 
hyperinsulinemia, has been shown to be associated with endometrial cancer, 
independent of body mass index.[15, 16]   One study has demonstrated that this 
association between adiponectin and endometrial cancer may be stronger in 
postmenopausal women than premenopausal women.[16]  Studies in patients with 
diabetes have demonstrated varying effects of metformin on serum levels of 
adiponectin.[17, 18] 
 
Furthermore, since insulin has been shown to potentiate estrogen-regulated gene 
expression, obese women in particular may be at increased risk for estrogen associated 
cancers, including endometrial cancer. Epidemiologically, a number of studies have 
shown a modest association of diabetes with endometrial cancer risk.[19-24] 
Interestingly, in three studies the most significant risks were seen in women who were 
both obese and diabetic.[19, 22, 24]   A study by Troisi et al examined insulin levels in 
women with endometrial cancer compared to controls in order to determine if elevated 
insulin levels could explain the association of obesity and endometrial cancer.[25]  
While elevated serum insulin levels, as measured by C-protein, were associated with 
increased risk of endometrial cancer, elevation of serum insulin levels alone could not 
account for the association of obesity and endometrial cancer.     In this study we will 
address the hypothesis that obesity associated hyperinsulinemia and insulin resistance, 
in combination with hyperestrogenism, contributes to the increased risk of endometrial 
cancer. 
 
2.2 Metformin, mechanism of action and possible role in the prevention of cancer 
 
While metformin was first described more than 50 years ago, the exact molecular 
mechanism of action for this drug has only recently been determined.[26] Metformin 
activates AMP-activated protein kinase (AMPK) via phosphorylation by the LKB1 tumor 
suppressor.[27, 28]   AMPK is typically activated in response to metabolic stress, and 
results in inhibition of ATP-consuming pathways, such as cholesterol synthesis, fatty 
acid synthesis and protein translation.[29, 30]   Importantly, AMPK inhibits mTOR via 
phosphorylation and activation of tuberous sclerosis complex 2 (TSC2).    TSC1/TSC2 
forms the TSC complex that negatively regulates mTOR signaling.[31] 
 
Recent clinical studies suggest that metformin may be associated with a decreased risk 
of cancer. In an observational cohort study of diabetics performed by Libby et al, incident 
cancer was diagnosed among 7.3% of 4,085 metformin users compared with 11.6% 
of 4,085 comparators (p<0.001).[32]   In a recent case-control study performed at MD 
Anderson, Li et al reported that diabetic patients who had taken metformin had a 
significantly lower risk of pancreatic cancer compared to those who had not taken 
metformin.[33]  Jiralerspong et al reported that among diabetic women with breast 


 
 
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cancer undergoing neoadjuvant chemotherapy, those individuals who were on 
metformin had a higher pathologic complete response rate than those who were on 
other anti-diabetic medication.[34]     The results of these studies are provocative, and 
suggest that metformin may have a role in both the prevention and treatment of multiple 
cancers. 
 
The signaling pathways activated by metformin treatment suggest a promising role 
for  metformin  as  an  anti-cancer  agent  in  endometrial  cancer,  as  well  as  other 
cancers that have activated PIK3CA/AKT/mTOR pathways.  Metformin treatment in 
estradiol   treated   Balb/c   mice   resulted   in   significantly   decreased   density   of 
endometrial glands, decreased immunohistochemical expression of mTOR signaling 
and decreased proliferation indices as measured by PCNA expression.[35]   Ben Sahra 
et al investigated the effects of metformin on human prostate cancer cells (LNCaP) and 
in an in vivo xenograft mouse model.  Their studies identified that in LNCaP  cancer  
cells  metformin  activated  AMPK  and  inhibited  proliferation  by blocking the cell 
cycle in G0/G1 phase, which was accompanied by decreased cyclin D1, decreased 
phosphorylation of Rb and increased p27Kip1 expression.  Metformin treatment of an 
LNCaP tumor bearing xenograft model resulted in a significant reduction in tumor 
growth.[36]  Schneider et al. investigated the effect of metformin in high-fat diet fed 
hamsters; a model of pancreatic cancer.  They identified that 50% of the high-fat diet 
fed hamsters develop pancreatic tumors; whereas metformin treated high-fat diet fed 
hamsters did not develop tumors.[37]      Additionally, administration of metformin to 
LKB1 hypomorphic PTEN+/- mice increased AMPK phosphorylation  and  significantly  
delayed  tumor  onset  with  100%  of  the  control group developing tumors of the liver, 
spleen and intestine by 4 months of age and the metformin treated group developing 
tumors by 5 months of age.[38] Taken together, these studies suggest that metformin 
may prevent or significantly delay tumor progression as a consequence of AMPK 
activation. 
 
The effect of metformin on the endometrium has been examined in women with 
polycystic ovarian syndrome.   In a small, open-label study of metformin versus 
rosiglitazone, endometrial histology was evaluated at baseline and following a 3- month 
treatment period in 16 women with PCOS.     Two women with simple hyperplasia  were  
treated  with   metformin  and  experienced   regression   during treatment providing 
preliminary evidence 
that metformin may have 
beneficial effects on 
the 
endometrium.[39]   The results of this study, in addition to our studies in rodents, provide 
evidence that metformin has a direct effect on the endometrium. 
2.3 Diet and exercise linked to hyperinsulinemia and insulin resistance 
 
The landmark Diabetes Prevention Program (DPP) trial compared a lifestyle intervention 
or metformin to metformin placebo. The intensive portion of the lifestyle intervention 
was composed of 16 face-to-face sessions with a lifestyle coach in which 
participants were assisted with increasing their physical activity, reducing their caloric 
intake, and reducing weight by at least 7%.  The trial demonstrated that both a lifestyle 
intervention and metformin reduced incidence of diabetes, with larger effects for the 
lifestyle intervention [22].   Particularly notable given the relationship between 


 
 
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endometrial cancer and adiponectin was that both interventions were associated 
with increases in adiponectin, with the effects of the lifestyle intervention being 
approximately 3.5 times larger than those for the metformin group [23].  Lifestyle 
intervention and metformin were also associated with reductions in metabolic syndrome 
[24] and (C-reactive protein) CRP in women, the reduction in CRP was related to weight 
loss [25]. The effects of the lifestyle intervention on adiponection may be related 
primarily to weight loss; a small study found that reduced calorie diet with or without 
exercise increased circulating adiponectin, but that exercise alone and exercise plus 
reduced calorie diet enhanced expression of adiponectin receptors in skeletal muscle 
and adipose tissue [26]. Increases in adiponection also have been found in response to 
weight loss after bariatric surgery [27]. Interestingly, in a recent report on the association 
of bariatric surgery and cancer incidence, the disease most profoundly affected was 
uterine cancer, primarily EndoCa [28]. 
 
We  are  intrigued  by  the  possibility  that  both  pharmacologic  intervention  with 
metformin and a lifestyle behavior intervention, both of which reduce insulin resistance, 
might also have a direct chemopreventive effect of EC. Currently, options for   EC   
chemoprevention   include   use   of   the   oral   contraceptive   or   use   of progestins.[40] 
Both have limitations in an obese cohort. Oral contraceptives (OCP) are contraindicated 
in morbidly obese individuals due to an increase in rate of thrombo-embolic events. In 
addition, obese women frequently have hypertension or hypercholesterolemia, which 
are also considered contraindications to OCP use. Progestins have similar 
contraindications and compound obesity by stimulating appetite. While weight loss and 
exercise are obvious solutions to decreasing the risk of EC in obese individuals, 
exploration of alternative approaches is necessary. The novelty 
of 
these 
chemoprevention/behavioral prevention strategy is twofold. First, it proposes to treat 
an underlying metabolic syndrome. Rather than focusing on the prevention of a single 
cancer, this strategy may have not only had an overall benefit for the cardiovascular 
health of the obese woman but may also decrease the risk of endometrial, pancreatic 
and perhaps other cancers associated with obesity and insulin resistance.[33] Second, 
it exploits the newly identified function of metformin as  a  drug that demonstrates  
direct  anti-proliferative  effects on  endometrial  (and other) cells via activation of AMPK 
and inhibition of mTOR. 
2.4 Rationale and definition of cohort 
 
Endometrial cancer has well-defined risk cohorts. Epidemiologic studies have 
consistently shown that women who are overweight are at increased risk of endometrial 
cancer, and women who are very overweight are at very increased risk. Brinton et al 
conducted a large case control study and found that women who were greater than 200 
lbs. had a relative risk of 7.2 compared with women less than 125 lbs.[41]   In order to 
maximize risk for our cohort, we will include women who fulfill standard criteria for 
obesity grade I and above, which is defined as body mass index (BMI) greater than or 
equal to 30 kg/m2. An upper age limit of 65 has been set. We chose  not  to  include  
pre-menopausal  women  in  this  study  because  of  the confounding problem of 
irregular cycling and varying estrogen levels in pre- menopausal  obese,  insulin  
resistant  women  (i.e.  women  with  polycystic  ovary 


 
 
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syndrome).  We  also  chose  not  to  include  women  who  fulfill  criteria  for diabetes, 
since additional agents, other than metformin, may be necessary to control their 
diabetes. 
 
2.5 Safety issues of metformin, as chemoprevention for endometrial cancer in a cohort of 
obese women 
 
Metformin hydrochloride (Glucophage, Bristol-Myers Squibb) is a biguanide 
antihyperglycemic agent.   It corrects Type II diabetes mellitus by altering glucose 
production and absorption, in contrast to sulfonylureas, which stimulate insulin 
secretion.  Specifically, metformin  improves  insulin  sensitivity  by  increasing peripheral 
glucose and utilization, decreasing intestinal absorption of glucose and suppressing 
glucose production by the liver, thus lowering serum insulin levels. 
 
We believe that metformin is not only safe in this population, but also beneficial. 
 
2.5.1 A randomized phase III study with metformin was conducted in this same 
population i.e.: insulin resistant but non-diabetic patients (the Diabetes Prevention 
Program Randomized Trial NEJM 2002 (6)346, p393), and demonstrated that 
metformin  was  well  tolerated  in  a  population  of  insulin  resistant,  non-diabetic 
individuals (and decreased the incidence of diabetes).[42] This study demonstrates 
the safety of using this drug in this population and also suggests that we can 
potentially dovetail the cardiovascular and metabolic health benefit of the drug with 
chemoprevention. 
 
2.5.2 Metformin does not increase secretion of insulin and therefore does not cause 
hypoglycemia. 
 
2.5.3 Use of metformin in insulin resistant but non-Type II diabetics also has 
precedence in the treatment of women with polycystic ovarian syndrome, or PCOS. 
Clinicians use metformin or thiazolidinediones, insulin-sensitizing agents, in young 
PCOS  women,  whose  underlying  metabolic  problem  is  insulin-resistance. 
Interesting, treatment for their insulin resistance results in normalization of their 
pituitary/reproductive  hormone  axis  and  subsequently  results  in  ovulation  and 
regular periods.[43, 44] 
 
 
 
2.6 Biomarkers as surrogate endpoints 
 
Endpoints for chemoprevention studies can either be development of cancer, or a 
surrogate for the development of cancer. In this study we will use surrogate biomarkers 
as the primary endpoint. 
 
Identification  of  appropriate  biomarkers  is  essential  for  this  study.     An  ideal 
biomarker is a histological or molecular "red flag" that precedes the onset of cancer. 
Ideal surrogate endpoint biomarkers are quantitative and are modulated by 


 
 
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chemopreventive  agents.[45]    In  our  prior  endometrial  cancer  chemoprevention 
study of women with HNPCC, we evaluated a panel of 30 biomarkers thought to be 
relevant in endometrial carcinogenesis.   From that study, we have narrowed down 
the field to a subset of 6 validated, estrogen-regulated genes and a proliferation marker 
Ki-67.[46] 
 
The following is a summary of the analyses to be performed on the endometrial biopsies 
at baseline and following four months of hormonal therapy: 
 
• 
Proliferation index Ki-67 
• 
Histology 
• 
Quantitative PCR transcript analysis 
 
Table 1: Estrogen-dependent panel of genes for PCR transcript analysis 
 
Gene 
Marker / Pathway 
Proliferating Cell Nuclear Antigen (PCNA) 
Proliferation marker 
Insulin-like Growth Factor Receptor (IGF-IR) 
IGF Pathway 
Retinaldehyde dehydrogenase 2 (RALDH2) 
Retinoid Pathway 
Secreted Frizzled-Related Protein 4 (sFRP4) 
Wnt Pathway 
Survivin 
Apoptosis 
Estrogen-inducted gene (EIG121) 
Novel gene of unknown function 
 
 
Biomarkers associated with metformin function and downstream signaling 
includes: 
 
• phospho-AMPKa 
• phospho-ACC 
• phospho-mTOR 
• phospho-S6 ribosomal protein. 
 
We will examine pre- and post- treatment endometrial biopsies for histology and 
proliferation using Ki-67. These are standard surrogate endpoints used in cancer 
chemoprevention trials. In addition, our group has identified a set, or panel of genes 
expression assays relevant to estrogen-dependent endometrial 
proliferation, 
hyperplasia and cancer. The set of genes, listed in Table 1 above will also be examined 
using real-time PCR in the pre- and post- treatment biopsies. 
 
The Ki-67 as well as the biomarkers associated with metformin action analysis will 
be performed by the MD Anderson Core Histology lab and read by Dr. Broaddus in the 
Pathology Core. Q-PCR analyses will be performed by Dr. David Loose, PI of the 
Biomarkers core. Dr. Loose has been interested in identifying genes in human 
endometrium whose expression is altered by hormone replacement therapy (HRT). 
Over the last 5 years he has used several techniques including differential display, 
microchip array screening, and real-time quantitative PCR to find such genes. The 
tools he has developed are directly applicable to the investigation of surrogate endpoint 
biomarkers (SEBs) in HNPCC patients treated with estrogens or estrogens plus 


 
 
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progestins. 
 
2.7 Description of Proposed Involvement of Human Subjects 
 
This study will be conducted at UT-MD Anderson Cancer Center, with recruitment 
focused on 100 subjects. Women >/=50 to </=65  years old, with BMI > 30 kg/m2, 
fasting blood glucose < 126 mg/dl, and hyperinsulinemia, but not frankly diabetic are 
eligible. Those women who demonstrate fasting blood glucose greater than 126 
mg/dl will not be referred for the study, and will instead be referred to contact an 
Endocrinologist from their insurance provider for management of diabetes.  Women of 
any race or ethnicity who meet the eligibility criteria will be enrolled in the study. Based 
on our participant base, we anticipate the minority inclusion to be: 58% White and Non-
Hispanic; 30% Hispanic; and 12% African-American. 
 
Hyperinsulinemia will be determined by using the fasting insulin and fasting glucose 
levels to calculate the QUICK I, an index of insulin resistance.   QUICK I = 1/ [log 
fasting insulin (µU/ml) + log fasting glucose (mg/dL)]. Values below and equal to 
0.357 will be used to identify women who are insulin resistant. [47-49]. 
3.0 Background Drug Information 
 
Metformin hydrochloride is a biguanide antihyperglycemic agent. It corrects Type II 
diabetes mellitus by correcting insulin resistance, in contrast to sulfonylureas, which 
stimulate insulin secretion. Specifically, metformin improves insulin sensitivity by 
increasing peripheral glucose and utilization, decreases intestinal absorption of glucose 
and suppress glucose production by the liver.  Metformin does not increase secretion 
of insulin and therefore does not cause hypoglycemia. In both animal models and in 
humans, metformin decreases plasma glucose. 
 
3.1 Agent 
Metformin hydrochloride (Greenpark Pharmacy) 
 
3.2 How supplied 
Each capsule contains 425 mg of metformin hydrochloride that will be used in this 
study. 
 
3.3 Dose groups 
Metformin hydrochloride 425 mg capsule(s) vs. no treatment. See below for monthly 
dose and duration of Metformin hydrochloride. 
 


 
 
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3.4 Dose and Duration of exposure 
 
Table 2: Monthly Dosage of Metformin or Placebo capsule(s) 
 
Week 1 
Week 2 
Week 3 
Week 4 
1st 
Month 
One capsule 
daily 
One capsule 2X 
daily 
One capsule 3X 
daily 
Two capsules 2X 
daily 
2-4 
Month 
Two capsules 
2X daily 
Two capsules 2X 
daily 
Two capsules 2X 
daily 
Two capsules 2X 
daily 
 
  Orally for 120 days (plus or minus 10 days). 
  The dose of metformin hydrochloride will be in 425 mg capsule(s) by mouth, the 
dose used in The Diabetes Prevention Program Randomized Trials.[50] 
 
3.5 Half life 
Following oral administration, approximately 90% of the absorbed drug is eliminated via 
the renal route within the first 24 hours, with a plasma elimination half-life of 
approximately 6.2 hours.  In blood,  the  elimination  half-life  is approximately 17.6 
hours, suggesting that the erythrocyte mass may be a compartment of distribution. 
 
3.6 Administration 
Metformin hydrochloride is self-administered. 
 
3.7 Side effects 
Diarrhea 
Nausea/ Vomiting 
Flatulence 
Abdominal discomfort 
Asthenia Ingestion 
Weight gain Edema 
Headache Heart 
failure Lactic 
Acidosis 
 
 
3.8 Contraindications 
 
3.9.1 Renal disease or renal dysfunction (e.g., as suggested by serum creatinine 
levels ≥ 1.4 mg/dL [females] or abnormal creatinine clearance) which may also 
result from conditions such as cardiovascular collapse (shock), acute myocardial 
infarction, and septicemia. 


 
 
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3.9.2 Congestive heart failure requiring pharmacologic treatment. 
 
3.9.3 Known hypersensitivity to Metformin hydrochloride. 
 
3.9.4 Acute or chronic metabolic acidosis, including diabetic ketoacidosis, with or 
without coma.  Diabetic ketoacidosis should be treated with insulin. This will be 
assessed based on symptoms reported and reviewed with research staff during 
monthly toxicity screening visits. 
 
3.9.5 Metformin will require a two day hold if given certain contrast medicines 
before X-rays, CT scans, MRI, and other procedures. 
 
3.9 Use during pregnancy 
Studies have shown that the drug does not exert teratogenic or embryocidal effects 
on animals, but there are no controlled studies in women. 
 
3.10 Manufacturer and Supplier 
Greenpark Pharmacy 
4.0 Participant Eligibility 
4.1 Inclusion Criteria 
 
4.1.1 For this study, only women will be enrolled 
4.1.2 Body Mass Index (BMI) > 30 kg/m2 
4.1.3 Not frankly diabetic, as measured by a fasting blood glucose <126 
mg/dL. 
4.1.4 Demonstrate hyperinsulinemia with a QUICK I value </=0.357. 
4.1.5 Age >/= 50 and </= 65 
4.1.6 Zubrod Performance Scale 0-1 
4.1.7 Hemoglobin >/= 10 g/dl 
4.1.8 TSH 0.27 – 4.20 µlU/mL   
4.1.9 Menopause as defined as no menses for 1 year and /  o r  
FSH > 25.8 mlU/ml. 
4.1.10 Must be able to read, write, and speak English. 
4.1.11 Must have a Primary Care Provider (PCP). 
4.2 Exclusion Criteria 
 
4.2.1 Prior hysterectomy or endometrial ablation. 
4.2.2 ALT > 2.0x Upper Limit of Normal (ULN) 
4.2.3 Serum creatinine > 1.4 mg/dl 


 
 
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4.2.4 Triglycerides (fasting) > 400 
4.2.5 Known inability to participate in the ongoing appointments for 
the four months of the study and scheduled follow-up tests. 
4.2.6 Significant medical or psychiatric history which would make the 
participant a poor protocol candidate, in the opinion of the principal 
investigator, for any aspect of study participation including 
metformin, unsupervised exercise program or dietary behavior 
change. 
4.2.7 Participant reported history of congestive heart failure 
4.2.8 Prior treatment with Metformin 
4.2.9 Currently being treated for diabetes or meeting criteria for new diagnosis of 
diabetes. 
4.2.10 Known allergy to Metformin or other biguanide (Proguanil). 
4.2.11 Use of Aromatose Inhibitors, GNRH-agonists i.e.Lupron, 
Zoladex within the last 6 months 
4.2.12 Use of SERMS (selective estrogen receptor modulators) in the past 
6 months, including Tamoxifen and Raloxifen 
4.2.13 Hormone replacement therapy within the last 6 months 
4.2.14 Women who have been treated with chemotherapy for prior 
malignant disease or currently have an untreated malignancy 
other than non-melanoma skin cancer 
4.2.15 Participants who have had prior radiation to the pelvis 
5.0 Design and Methods 
 
5.1 Study Design 
 
For the proposed study, post- menopausal women with a BMI ≥ 30 kg/m2 who 
demonstrate insulin resistance will be recruited from the Harris Health Hospital 
(Lyndon B. Johnson General Hospital - LBJ), community, and from the employee 
pool at MD Anderson Cancer Center using various outlets as advertisement. Project 
LEAP fliers will be posted at the LBJ for recruitment purposes only.  All consenting, 
study visits, and study procedures will be done at the MD Anderson Cancer Center. 
 
Participants will be assessed for their eligibility, first during a Screening Call, in 
which interested women will call the contact number and be screened using the 
Screening Script. 
 
  Screening Call 
 
o Participants are screened using screening script.   Once preliminary 
eligibility has been determined, then their information is forwarded to 
the study research nurse. 
 
o Study research nurse will: 
 Obtain reported medical history and medication history and 
schedule for Screening Visit 1. 


 
 
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• Explain the informed consent process.   Participant may review 
the informed consent information electronically by email, by 
telephone, or in-person. 
 
• Instruct participant on fasting and explain the blood tests to be 
collected on Visit 1 (See Table 5: Study Parameters for the list 
of lab tests to be collected on Screening Visit 1.) 
 
• Provide driving directions to MD Anderson Cancer Center. 
 
• Answer any relevant questions or concerns of the study. 
 
• Initiate a New Patient (participant) Intake form by phone and 
send to Mays Clinic Gynecologic Oncology Business Center. 
Instruct participant that she will register in-person at the Mays 
Clinic  Gynecologic  Oncology  Business  Center  on  Screening 
Visit 1 day to obtain Medical Record Number (MRN). 
 
5.2 Screening Visit 1 
 
  Screening Visit 1 includes: 
 
a.  Registration  at  Mays  Clinic  Gynecologic  Oncology  Business  Center 
(MCGOBC) to obtain a Medical Record Number (MRN). 
 
b.  Confirm that participant has fasted that day.  Review previous information 
discussed by phone, and answer questions as needed. 
 
c.  Participant  to  meet  with  study  Gynecologist  briefly  at  MCGOBC  for 
obtaining  consent  and  discussing  any  concerns  of  the  study.    Once 
consent is attained then participant will be scheduled for laboratory tests to 
be collected during the same day (see Table 5: Study Parameters for the list 
of laboratory test to be collected for Screening Visit 1). 
 
d.  Pre-registration  completed  in  CORe  (See  section  5.6  for  Registration 
Process). 
 
e.  Provide participant education: 
 
i. Endometrial biopsy (education packet that lists pre- and post- procedure 
instructions and contact information).    See Appendix L for Patient 
Education Packet on Endometrial Biopsy (EMB). 
 
ii.  Taking NSAIDs (Non-Steroidal Anti-inflammatory Drugs) orally such as 
ibuprofen or Motrin or Advil if no prior allergies and available over 
the counter. 
 
1.  Pre-biopsy: 600 mg of NSAIDs once approximately 1 hour 
before EMB. 
 
2.  Post-biopsy: 600 mg of NSAIDs every 6 hours as needed for 
cramps or pain. 
 


 
 
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iii.  Participants who are unable to take ibuprofen / Motrin / Advil and have 
no allergies can also take acetaminophen (Tylenol) over the counter as 
an alternative NSAID. 
 
1.  Pre-biopsy: 325 mg acetaminophen take once approximately 
1 hour before EMB 
 
2.  Post-biopsy: 325 mg of acetaminophen every 6 hours as 
needed for cramps or pain 
 
At the Screening Visit 1, participants will be asked to complete one 24-hour 
dietary recall during the screening visit, using NCI’s online ASA-24 system. Anytime 
between the Screening Visit 2 and 7 days post biopsy, the participant will complete 
two additional 24-hour dietary recalls (one on a weekday and 1 on a weekend). These 
additional recalls can be completed by phone or using their own home computer. 
Participants will also be given the survey packet to be completed before they return 
for the Screening Visit 2.   Both the 24-hour diet recall and survey packet are 
described in detail below. All participants will be given an accelerometer (to obtain 
objective measure of physical activity), shown how to use it, and asked to wear it for 
one week between the screening visit and 
7 days post-biopsy. They will return the accelerometer at the Screening Visit 2. If they 
are not able to return it at the second visit, they may return it by mail (prepaid  
mailer  will  be  provided)  or  at  their  first  intervention  session  (if randomized to 
receive the lifestyle intervention). 
 
 
5.2.1 24-hour Dietary Recall 
 
The  Automated  Self-administered  24-hour  Dietary  Recall  (ASA24) will  be used 
to document the participant's food intake for a total of 24 hours. Three recalls will be 
obtained at baseline and at 4 months, at least one of which will be for a weekend 
day. 
The 
overview 
of 
this 
questionnaire 
can 
be 
located 
at 
http://riskfactor.cancer.gov/tools/instruments/asa24. The participants will be given a 
designated url which will provide them access to the online questionnaire.  If the 
participant does not have computer or internet access, the  recall  will  be  
performed  over  the  phone  with  the  assistance  of  the Research Coordinator or 
Research Dietitian.     To assist participants, the ASA24 utilizes graphics, pictures 
(to help approximate portion size), an animated guide, and audio cues and 
languages, which aid populations with low literacy [60]. The foods selected by the 
participants are from the USDA's Food and Nutrient Database for Dietary Studies' 
(FNDDS) most up-to-date database.            Participants will begin completing these 
assessments approximately one week before their Screening Visit  2 and at 
four-month BRTC  visits,  with  the  third  assessment  completed  within  1  week  of  
the second visit/4-month visits. 
 
5.2.2 Godin Leisure Time Physical Activity 
 
A 3-item modified version of the Godin Leisure Time Physical Activity Questionnaire 


 
 
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will be used to measure the participants' usual leisure-time exercise habits. It will be 
administered as part of the survey packet and will be given to the participant during 
the Screening Visit and the Post-Treatment 
visit. It is easy to administer and has good test-retest reliability (.81 for total score), 
and significant correlations with VO2max. The responses from this survey at baseline 
will be used to determine if the participant is meeting guidelines for physical activity, 
as discussed in section 7, and used for randomization. 
 
5.2.3 Accelerometers 
 
At baseline and month 4, participants will wear accelerometers (Actigraph GT3X+) 
for 7 days to provide an objective measure of physical activity. Before the16  week  
assessment  staff  will  mail  the  accelerometers  to  participants along with the 
materials needed for the 24 hour recall interviews.     The accelerometer, which will 
be worn while the participant is awake, is small (1.5 x 2 inches) and can either be 
clipped to the belt or waistband or placed in a pouch attached to a belt that can 
be worn under clothing. Cutpoints have been established for light, moderate, hard, 
and very hard activity [63-65]. Participants also will complete a paper-and-pencil 
exercise diary each day during the week they wear the accelerometer. The diary 
will ask participants to indicate what type of exercise was performed, duration of 
the exercise in minutes, and the effort level during the exercise. 
 
 
5.2.4 Behavioral determinants 
 
Guide to Health Survey 
 
The Guide to Health survey, developed by Anderson-Bill et al,  will be used to 
measure Social Cognitive Theory-based behavioral determinants (social support, 
self-efficacy, outcome expectations, and self-regulatory behavior) related to healthy 
eating patterns and physical.   The subscales have very good internal consistency 
(with the exception of one subscale with an alpha of 
.66, all are between .77 and .95); they were largely responsive to an online 
intervention targeting physical activity and healthy eating, and were related to 
behavior  change  [66,  67].  The  diet  self-efficacy  scales  measure  healthy eating 
patterns (e.g., eating more fruits and vegetables, less fat). 
 
 
 
Weight Efficacy Life-style (WEL) 
 
The weight efficacy life-style (WEL) questionnaire will be used to assess the 
participant’s confidence that they can regulate their eating. This scale has been 
shown to have high internal consistency reliability and is responsive to cognitive-
behavioral weight loss interventions [68]; it also partially mediated the effect of a 
group weight loss intervention for older adults [69]. Also, the Guide to Health 
scales measure self-efficacy for maintaining an exercise program over time and in 
a range of situations, but in our study of exercise with endometrial cancer survivors 
we found that self-efficacy for being able to physically  do  the  exercise  (task  self-
efficacy)  was  also  important  [70]. Therefore we will use the exercise self-efficacy 


 
 
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scale from that study (internal consistency reliability = .96), which was based on 
measures by McAuley [71]. 
 
 
Participant-Reported   Outcomes   Measurement   Information   System 
(PROMIS) 
 
The Participant-Reported Outcomes Measurement Information System (PROMIS) 
global short form will be used to evaluate the effect of the interventions on 
participant quality of life.     This questionnaire assesses physical  function,  pain,  
fatigue,  emotional  distress,  and  social  healthy domains. The PROMIS global 
short form has been validated and shown to have  similar  correlations  for  the  
global  physical  health  (GPH)  and  global mental health (GMH) items as similar 
items from the SF-36 (36-item short- form health survey) and other measures of 
health related quality of life [72]. The reliabilities for GPH and GMH were 0.81 and 
0.86 respectively which compared to SF-36 at 0.88-0.93 for their physical and 
mental health items [73]. The advantage to using the PROMIS short form versus 
the SF-36 is that it takes much less time for the participants to complete. 
 
Self-Administered Comorbidity Questionnaire 
 
Because   we   will   not   have   medical   records   readily  available   for  our 
participants, the Self-Administered Comorbidity Questionnaire will be included in the 
survey packet and be used to assess comorbidity of the participants. The 
questionnaire has been validated and has shown moderately strong correlation with 
the Charleston Index, which was used as a standard [74]. It displayed a Spearman 
correlation coefficient of 0.55 and kappas exceeding 
0.46 for almost every item [74]. This questionnaire should serve as a suitable 
substitute for the medical record. 
 
Demographic Questionnaire 
 
A basic questionnaire will be used to gather demographic information from each 
participant and will include questions on race, education, employment, and age. 
 
5.3 Screening Visit 2 
Once participant eligibility is confirmed based on the blood test results from Visit 
1, participant will be scheduled for Visit 2 in the following order: 
 
a) Appointment with MD Anderson’s Behavioral Research and Treatment Center 
(BRTC) for anthropometric assessments.   The details of these measurements 
are described below in section 5.3.2. 
b) Laboratory test collection (See Table 5: Study Parameters for the list of 
laboratory test to be collected for Visit 2). 
c)  Appointment in Mays Clinic Gynecologic Oncology for history, physical, and 


 
 
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endometrial biopsy (EMB). 
 
 
5.3.1 Endometrial biopsy 
An endometrial pipelle biopsy will be performed at the Screening Visit 2 and at 
the 4th month on days 115-125 (plus or minus 10 days) into treatment in all 
participants from (all four arms) both groups. 
 
In  addition  to  histologic  examination,  each  endometrial  biopsy  will  be 
examined for proliferation markers as well as other biomarkers. The second biopsy 
in the no treatment group is important in order to determine variability in the 
biomarkers due to sampling. In preliminary data from the Wyeth-Ayerst Study, we 
did see variability in certain transcript levels at the 3 and 6 month time points even 
in the placebo control group. Therefore, we feel strongly in this study that women in 
both groups undergo a 4 month EMB. 
 
Risks of office endometrial biopsy are small, and while there are no published data 
on risk of perforation, we believe that the risk is less than 1/1000. Even in those 
participants in whom uterine perforation occurs, observation alone is usually 
sufficient. The severe perforations requiring laparotomy and emergent hysterectomy 
are not seen in office endometrial biopsies, rather in Dilatation and Curettage (D & 
C) procedures performed in the operating room. 
 
Participants who have no allergies will be instructed to take orally Non- Steroidal 
Anti-inflammatory Drugs (NSAIDs) before and after the biopsies to reduce pain 
and cramping.  The NSAIDs medications are available over the counter (See 
Section 5.2 for the recommended dose). 
 
In the second screening visit, if the patient is experiencing discomfort while the 
provider is attempting to obtain the endometrial biopsy, we may administer topical 
lidocaine gel to the cervix. This is supported by prior studies which have 
demonstrated that patients may experience less pain with tenaculum placement and 
cervical dilation when topical lidocaine is used (Hassan et al, 2005; Davies et al, 
1997). Occasionally, some patients may have cervical stenosis, which makes it 
difficult to dilate the cervix, insert the endometrial suction curette, and obtain an 
endometrial biopsy.  In these instances, prior studies have suggested that the use of 
vaginal misoprostol may soften the cervix and increase the likelihood that the cervix 
can be dilated so that the procedure can be performed (Bastu et al, 2013; Ghosh et 
al; 2013).  If cervical stenosis is encountered during the second screening visit, and 
the study gynecologist is unable to pass the curette or dilator through the cervix, the 
participant will be given a prescription for misoprostol 200mcg #2 tablets and re-
scheduled to return to the clinic for a repeat attempt at performing the endometrial 
biopsy (within 10 days). The participant will be given instructions to self-administer 1 
tablet intravaginally the night before the procedure and one tablet the morning of the 
procedure. Both lidocaine jelly and misoprostol will be administered as needed.  
Research Nurse will counsel the participant on the common symptoms of these 
agents prior to the second screening visit.   


 
 
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5.3.2 Anthropometric assessments 
 
BRTC staff will measure the participants’ height, weight, and waist circumference at 
baseline and 4th month. Height will be measured by having the participant stand 
evenly without shoes on a flat surface at a right angle to the movable vertical board 
or cap that is pulled down to lightly touch the most superior point of the head. Weight 
will be measured using either a standard beam scale or an electronic digital scale.   
The scale should be on a level floor, and participants should be gowned or 
wearing light clothing and no 
shoes. Waist circumference will be measured with the participant standing relaxed 
with hands hanging at her sides on the exhale. The tension-controlled measurement 
tape will be placed directly on the skin, horizontally around the narrowest part of the 
torso. If unable to locate the narrowest part of the waist, the participant will be 
measured 2-3 inches above the navel. The hip circumference will be measured with 
the participant standing relaxed with hands   hanging   at   her   sides   on   the   
exhale.   The   tension-controlled measurement tape will be placed directly on the 
skin or over the underwear, horizontally around the widest part of the hip/buttocks 
area. 
 
The   participant   will   undergo   a   whole   body  DEXA   to   measure   body 
composition. The DEXA, routinely used to determine bone mineral density, has 
gained support in research application for assessing fat and lean body mass in whole 
body and in specific body regions [23, 24, 25]. Data from each scan will be as follows: 
percent body fat and % lean tissue, fat and lean mass for total body region as well 
as defined body zones (trunk, leg, and arms) expressed in grams. 
 
 
5.4 Randomization / Visit 3 
Participants, who are eligible based on results of the biopsy from Visit 2, will be called 
to schedule their third visit for randomization. 
 
This study will use a 2 x 2 randomized design with all 100 subjects being randomized: 
25 will receive both lifestyle intervention and metformin, 25 will receive  the  lifestyle  
intervention  plus  metformin  placebo,  25  will  receive metformin   only,   and   25   
will   receive   metformin   placebo   but   no   lifestyle intervention. The participants 
randomized to the lifestyle change portion of the study will also have their first 
intervention session schedule for the same day as well. Treatments will last 4 months. 
 
At Randomization / Visit 3, the participant will be: 
 
a)  randomized to one of the four intervention groups using the Department of 
Biostatistics Clinical Trial Conduct Website. See section 7.0 for details. 
b) given prescription to fill the study medication at the pharmacy at MD Anderson 
Cancer Center. 
 
c) participant will be provided education on recording and monitoring daily intake  
of  study  medication  into  the  Pill  Diary.    See  Appendix  M  for Pill Diary. 
 


 
 
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20 
 
d)  review and report side effects of the study medication. 
 
 
 
Once the participant is randomized, the research nurse will notify the pharmacist to 
inform them of the randomization code in order to keep study staff blinded to assignment 
to metformin vs. placebo. 
 
In-person toxicity assessments will be performed at the end of the first, second, third, 
and fourth month (plus or minus 10 days).   Phone assessments will be done 
periodically as needed to assess for adverse effects.   At the end of the fourth 
month of the study, the participant will return for the Post-Treatment Evaluation to 
repeat all measurements (all blood tests, anthropometric assessments, second biopsy, 
and survey packet) taken at the Screening Visits (1& 2).  The participant will also be 
given a Satisfaction Questionnaire, based on their intervention, in advance to complete 
and return at this visit.   
 
One year after baseline (+/- 1 month), participants will be contacted to complete a 1 Year 
Follow-Up Evaluation. This evaluation will include anthropometrics assessments, 
including a DEXA scan, the survey packet, and blood tests collected from the 4th month 
visit (see Table 5: Study Parameters for the list of laboratory test to be collected for 1 
year Follow-up Evaluation).  During this visit, the participant will also be compensated 
with a parking voucher to cover the cost of parking during the visit. 
 
5.5 Study Compensation 
A  monetary  compensation   will   be   provided   in   the  form   of  a   gift  card 
commensurate with the amount of time, inconvenience, and discomfort experienced by 
each participant while participating in the obtaining of the endometrial biopsies. The total 
possible compensation is $125.00 ($50 for baseline and $75 for the 4 month 
assessment).   Participants who are eligible based on the results of Screening Visit 2 
tests will receive the first gift card of 
$50.00 on the Randomization / Visit 3.  Participants found to be ineligible based on 
baseline EMB results will be contacted by phone and gift card will be mailed to them.     
Second gift card of $75.00 will be given after completion of all measurements on Post-
Treatment Evaluation visit on the 4th month.  For all study related assessment visits to 
MD Anderson each participant will receive parking validation. 
 
5.6 Registration Process 
This is a phase II, four-armed, randomized chemoprevention trial. Registration will 
be a two-step pre-enrollment to enrollment process. 
 
A. New Patient (participant) Intake form will be initiated by study research nurse 
during phone interview with potential participant.    Once form is completed, 
it will be sent to the MCGOBC to schedule participant for in-person 
registration. 


 
 
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21 
 
 
B. At the first screening visit, participant will register in-person as a new 
participant at MCGOBC and will be assigned a Medical Record Number 
(MRN).  After they have received an MRN they will meet momentarily with 
one of the study Gynecologist to obtain consent. 
 
C. Consented participants will be registered in Clinical Oncology Research 
(CORe) System and will be assigned a unique identifying number at entry 
into the trial on the first screening visit.  This unique subject number will 
not change during the study. 
 
D. Only participants who meet all eligibility criteria and have successfully 
completed  all  laboratory  tests  and  EMB  will  be  randomized  to  CTC 
website on the third visit (see section 7.0 for details on randomization). 
 
5.7 Participating Center 
The Screening Visits (1 & 2), Randomization / Visit 3, Lifestyle intervention sessions, 
In-person toxicity assessments, and   Post-Treatment Evaluation will occur at the 
University of Texas M. D. Anderson Cancer Center. 
 
6.0 Treatment Plan 
 
We have chosen to treat for a 4 month period. This period is based on studies 
demonstrating  that  metformin  will  reach  full  potential  in  the  reduction  of 
hyperinsulinemia and insulin resistance by four months. 
 
6.1 Treatment 
 
Prior to beginning therapy, participants will have a baseline endometrial biopsy 
performed. If the baseline endometrial biopsy reveals endometrial hyperplasia or cancer 
or if an office endometrial biopsy cannot be performed the participant will be removed 
from study. Women with abnormal biopsy results will be referred to contact their  
Gynecologist  from  their  insurance  provider  for  further  treatment  for  these results. 
 
The study is partially blinded in that investigator and participants will be blinded to 
the treatment of Metformin versus placebo, but will be un-blinded in regard to the lifestyle 
intervention. All participants will be followed for at least one month after their last dose 
of study medication.   All treatment-related toxicities will be followed by phone until 
resolution. All other participants will be randomized to one of four arms: 
 
 
6.1.1 Metformin and Lifestyle Intervention 
  These participants will have a baseline endometrial biopsy. 


 
 
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22 
 
  See Table 2 in section 3.4 for Metformin Monthly dose and duration. 
  Second EMB and post study labs will be done between days 115-125 
(plus or minus 10 days). 
  For the lifestyle intervention, all participants will receive the intervention 
described below in section 6.2. 
 
6.1.2 Metformin Placebo and Lifestyle Intervention 
  These participants will have a baseline EMB at study entry. 
  See Table 2 in section 3.4 for Metformin Placebo Monthly dose and 
duration. 
   The second EMB, as well as post- study labs will be done between 115- 
125 days (plus or minus 10 days). 
  For the lifestyle intervention, all participants will receive the intervention 
described below in section 6.2. 
 
6.1.3 Metformin and No Lifestyle Intervention 
  These participants will have a baseline endometrial biopsy. 
  A total of four 30 day cycles (plus or minus 10 days) will be given (120 
days). 
  See Table 2 in section 3.4 for Metformin Monthly dose and duration. 
  The second EMB, as well as post study labs will be done between 115- 
125 days (plus or minus 10 days). 
 
6.1.4 Metformin Placebo and No Lifestyle Intervention 
  These participants will have a baseline EMB. 
  A total of four 30 day cycles (plus or minus 10 days) will be given (120 
days). 
  See Table 2 in section 3.4 for Metformin Placebo Monthly dose and 
duration. 
  The second EMB, as well as post study labs will be done between 115- 
125 days (plus or minus 10 days). 
 
 
 
Participants  randomized  to  receive  no  lifestyle  intervention  (control)  will  be 
asked to continue their current eating and physical activity patterns for the duration 
of the study. Participants randomized to the no-lifestyle condition will be given the 
option of receiving all study materials and up to two meetings with the dietitian after 
their final assessment and up to one month after. 
 
6.2 Lifestyle Intervention 
 
The  lifestyle  intervention  will  use  the  4  month  intervention  developed  for  the 
Diabetes Prevention Trial (DPT).  This intervention has been found to be feasible, 
result in weight loss and prevention of diabetes, and produce changes in diet and 
physical activity. The goals of the intervention are for the participants to lose 7% of their 
body weight, decrease fat consumption, and increase their energy expenditure by 700 


 
 
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kilocalories per week (equivalent to approximately 2.5 hours per week of moderate 
intensity walking). Participants can choose whether they want to start with dietary or 
activity changes first (see Table 3 for schedule of topics), but both goal areas will be 
addressed. 
 
Strategies to encourage weight loss include self-monitoring of weight and reduction 
of fat intake (goal: approximately 25% of calories from fat).  Individuals who are not 
able to lose weight with these two strategies will be given a calorie goal during the 
seventh or eighth week of the intervention sessions in the session titled “Tip the Calorie 
Balance”.   Strategies to increase physical activity include self-monitoring, use of a 
pedometer to increase activity, and provision of opportunities for exercise. 
 
The intervention is delivered as a 16 in-person sessions offered over a 4 month period, 
once each week. If a participant is travelling when a session is scheduled, or has some 
other reason why she is not able to attend, the session can be done by telephone. 
Participants will receive print material, worksheets, measuring utensils, a food scale, 
and a pedometer. 
 
All print materials have been developed by the DPT and are available for use; they 
will require only minor modifications for this study.  Print material and worksheets to 
be given to participants are provided in Appendix G .  The intervention also involves 
providing opportunities for supervised exercise (e.g., group exercise class, group walks) 
at least twice a week for participants. The 16 interventions sessions will be delivered 
by a master’s level Research Coordinator or Research Dietician who will be trained 
in a manner consistent with the requirements of the DPT.   For quality control, 10% of 
the sessions he/she conducts will be audio-recorded and reviewed by Dr. Basen-
Engquist or a Senior Research Coordinator or Research Manager. 
 
 
Table 3: Schedule of intervention sessions. 
 
Session 
1 
Welcome to the Group Lifestyle Balance Program 
2 
Be a Fat and Calorie Detective 
3 
Healthy Eating 
4 
Move Those Muscles 
5 
Tip the Calorie Balance 
6 
Take Charge of What's Around You 
7 
Problem Solving 
8 
Four Keys to Healthy Eating Out 
9 
The Slippery Slope of Lifestyle Change 
10 
Jump Start Your Activity Plan 
Make Social Cues Work for You 
11 
Ways to Stay Motivated 


 
 
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12 
Preparing for Long-Term Self-Management 
More Volume, Fewer Calories 
13 
Balance Your Thoughts for Long-Term Self-Management 
Strengthen Your Exercise Program 
14 
Mindful Eating 
Stress and Time Management 
15 
Standing Up for Your Health 
Heart Health 
16 
Stretching: The Truth about Flexibility 
Looking Back and Looking Forward 
 
 
 
6.3 Supervised Exercise Session 
 
Two opportunities for supervised exercise per week will be offered for participants to 
increase their level of physical activity. Any other form of exercise will be arranged by 
the participant herself outside the institution. 
 
6.4 Toxicity assessment 
 
The summary of toxicity assessment for in-person and phone is listed below. 
 
A. In-person assessment include: 
o physical exam (only on 1st month) 
o weight 
o review of study medication side effects (as listed in consent and instruct 
participant  to  call  if  they  experience  symptoms  of  abdominal  pain, 
anorexia, nausea, jaundice, and change in urine color) 
o ALT and creatinine levels. 
o Dispense Pill Diary and prescription. 
 
B. Phone assessment include reported symptoms of: 
o gastrointestinal (abdominal pain and severe diarrhea) 
o liver (jaundice) and review ALT lab results 
o kidney (changes in urine color and amount) and review creatinine lab 
results 
o heart (CHF) 
o rare side effects listed in consent 
 
 
The in-person toxicity assessment will be at the end of each month and will be a month 
from Randomization / Visit 3.   All participants must agree to keep a drug administration 
diary (Pill Diary).  This diary will be signed by both the participant and research nurse 
and reviewed during each clinic visit to verify compliance. The medication administration 
record will be filed in the participant's chart. Any discrepancies will be noted along with 


 
 
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the reason, if known. Any remaining pills will also be returned at each clinic visit and 
documented. 
 
Participants with mild elevations in ALT (3x ULN), will continue study drug at same dose, 
and have another ALT drawn 30 days later.  If ALT levels are >/= 4x ULN, the study 
drug will be stopped, and participant removed from the study.   Participants with an 
elevation in creatinine of >/= 2.5x ULN will be taken off of the drug and removed from 
the study.  The study drug will be stopped and participant removed from study if the 
participant reports symptoms of congestive heart failure (CHF) such as:  fatigue,  
tightness  or  congestion  in  the  chest,  edema  to  ankles  or  lower extremities, and 
shortness of breath. 
 
Telephone contacts will be made periodically as needed to assess on the first, second, 
third, and fourth month (plus or minus 10 days) to encourage compliance and assess 
for the presence of toxic or adverse effects.  Participants experiencing toxic or adverse 
effects will be referred to MD Anderson health providers for medical care however 
medical care will be billed to the participant or participant’s insurance provider.  All 
participants will be contacted by telephone 4 weeks after completion of the study for 
follow-up and toxicity assessment. 
 
7.0 Data Monitoring Plan 
 
Participants will be randomized to one of the four arms: metformin, placebo, and 
metformin / placebo and lifestyle intervention using the Department of Biostatistics 
Clinical Trial Conduct Website (CTC website).   Participants who do not meet these 
criteria will be considered nonevaluable, removed from study and will not be counted 
toward the actual accrual goal. We plan to accrue 100 evaluable participants. 
 
We will use a form of adaptive randomization called minimization, which is similar to 
stratification in that participant characteristics are used to assign them to the treatment 
conditions [75, 76].     Minimization results in better group balance with respect to 
participant characteristics.   In minimization, before a p articipant is   assigned   to   
a treatment group, the total numbers of participants in each group with similar covariate 
characteristics are totaled. The totals are based on the marginal sums of the covariates 
so that each covariate is considered separately. The treatment assignment of a 
participant is determined based on which group would provide the best overall balance 
with respect to those covariates. Covariates that will be used in randomization of 
participants in this study are BMI, age, and whether the participant meets current 
guidelines for exercise recommended by the Physical Activity Guidelines for Americans. 
These guidelines recommend that adults engage in moderate physical activity at least 
150 minutes per week or in vigorous activity 75 minutes per week [77]. The baseline 
level of activity for each participant will be measured when they complete the Godin 
questionnaire, which is part of their survey packet, which will be completed before their 
Screening Visit 2 appointment. The Research Dietitian/Research Coordinator will 
determine if the participant is currently meeting these recommendations and provide 


 
 
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this data to the Research Nurse for randomization. 
 
Randomization will be performed by the research nurse using the website created 
by the Department of Biostatistics. The research nurse will then inform the pharmacist 
of randomization group (A, B, C, or D) for study drug administration. The Research 
Dietitian/Research   Coordinator   who   is   in   charge   of   administering   the   lifestyle 
intervention will be given access to the randomization website and informed of lifestyle 
intervention randomization code to determine whether the participant has been 
randomized to the lifestyle intervention. 
 
 
7.1 Protocol compliance and assessment of toxicity data 
 
The attending physician must see each participant prior to beginning study drug. All 
required interim and pretreatment data should be available to evaluate toxicities, assign 
grade, and make a determination regarding continuing treatment. 
 
 
7.2 Data collection Plans 
 
Protocol data will be entered into the following plans: Clinical Oncology Research 
System (CORe) / Protocol Data Management System (PDMS), Access Database, ASA 
server, MD Anderson Cancer Center (MDACC) Behavioral Science server, and excel 
spreadsheet after each course of therapy.  A brief explanation for required but missing 
data should be recorded as a comment. See below for the list of the specific data stored 
in PDMS and CORe: 
 
 
PDMS 
 
Monthly medication administration dates 
 
Doses taken 
 
Laboratory results 
 
Clinic visits 
 
Diagnostic test (EMB) 
 
CORe 
 
Pre-registration 
 
Adverse events entry 
 
All behavioral and anthropometric data components, including height, weight, waist and 
hip circumference, exercise diary, and survey packet data will be collected and kept in 
an Access Database. The ASA24 data is stored on the ASA site’s server until we 
request the data for analysis. The data is sent to us within 48 hours and then the file is 
stored on our server.  The Accelerometer data will also be stored on our server at 
MDACC (Department of Behavioral Science) until the statistician retrieves the data 
for analysis. The DEXA scan data is downloaded into a file such as excel 
(Department of Behavioral Science) and then is stored on the server as well. 


 
 
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8.0 Criteria for Removal from the Study 
8.1  Subjects are free to leave the study at any time. 
8.2   Participants may be removed from the study at the discretion of the investigator 
if there is sufficient reason to suspect that the participant is not compliant with 
the study requirements. 
8.3  If at any time during the study, the participant experiences symptoms of congestive 
heart failure or renal failure, drug will be stopped and the participant taken off 
study. 
8.4  Abnormal baseline endometrial biopsy or the endometrial biopsy is unable to be 
performed. 
8.5   Non-compliance: Non-compliance will be defined as a participant missing more 
than 4 consecutive doses per month or a total of more than 10 doses during the 
4 month period. 
8.6   Lost to follow-up: Diligent attempts must be made by telephone and letter to 
determine the circumstances for loss to follow-up, since loss may be related to 
study drug. 
9.0 Statistical Considerations 
 
This is a partially blinded, randomized study to evaluate the effects of intervention upon 
Ki-67 and other biomarkers.  Participants and researchers will be blinded with regard to 
assignment of study drug (placebo vs. metformin) but they will not be blinded 
with regard to assignment of lifestyle intervention.   We will use a form of adaptive 
randomization called minimization, which is similar to stratification in that participant 
characteristics are used to assign them to the treatment conditions to achieve better 
group balance.   Groups will be balanced with regard to age (50-65 years ), BMI (Class 
I and II: 30.0-39.9 and Class III or greater: ≥40), and whether they meet current 
exercise 
guidelines 
as 
recommended 
by 
the 
Physical 
Activity Guidelines 
forAmericans. 
 
The primary objective of this study is to evaluate the effect of intervention (metformin 
and/or lifestyle intervention) on Ki-67.  Secondary endpoints include examination of 
intervention on: 
 
(a) genes relevant to estrogen dependent endometrial proliferation, hyperplasia 
and cancer using Q-PCR (PCNA, sFRP4, RALDH2, IGF-1R, survivin, and 
EIG121), 
 
(b)  biomarkers   specific   to   the   effect   of   metformin   treatment   (phospho- 
AMPKa,phospho-ACC, phospho-mTOR and phospho-S6 ribosomal protein. 
 
Additionally,   we   will   examine   changes   in   serum   levels   of   estradiol,   estrone, 
testosterone, DHEA-S, sex hormone binding globulin (SHBG), adiponectin, glucose, 


 
 
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HbA1c, insulin and insulin-like growth factor-1 (IGF-1) and all biobehavioral outcomes, 
such as changes in body composition, physical activity, quality of life (QoL), diet, etc. 
 
We will analyze differences in Ki-67 and other outcomes using 2x2 ANOVA models.  
If lifestyle intervention is found to affect change in Ki-67 expression or other outcomes, 
we will use the methods described by Baron and Kenney [78] to determine whether 
these changes were mediated by weight loss, body composition changes, physical 
activity changes, and  dietary changes.    Differences  between  arms  with  regard  to  
adverse events and other toxicities will be examined using Fisher’s exact tests.  
Changes from baseline lab values will be examined 2x2 ANOVA models. 
A study of Ki-67 change in participants at high risk for endometrial cancer (Lynch 
patients) indicated we could expect a mean decrease in Ki-67 of 3.5 (after log- 
transformation) in the Metformin alone group with a standard deviation of 0.8.  For 
the purposes of estimating power, we assumed that the lifestyle intervention alone 
group would have the same mean and standard deviation decrease, but we halved 
mean decrease for our calculations to take into account the non-compliance we might 
observe in this group.  Finally, we estimated power for a moderate interaction effect – 
half of the metformin intervention effect in addition to the additive effect of metformin 
and lifestyle intervention.  Specifically, we calculated power assuming that the mean 
decreases in the control arm, metformin alone arm, lifestyle intervention alone arm 
and metformin plus lifestyle intervention arm were 0, 3.5, 1.75 and 7, respectively.  
Using a 2-sided test with 5% statistical significance, we will have over 95% power to 
detect these differences when the common standard deviation is 0.8.  Additionally, we 
will still have adequate power to detect much smaller differences.   For example, we 
will have 80% power to detect a lifestyle alone main effect of 0.3 and still have over 
95% power to detect a metformin main effect and an interaction effect of 0.6 and 1.2, 
respectively, when the common standard deviation is 0.8.   Power was calculated using 
PASS 2005 (copyright 
© 
2005, 
Jerry 
Hintze). 
 
All testing will be 2-sided with 5% statistical significance.  The primary efficacy analysis 
will be intent-to-treat.  Patients who discontinue early from the study will be included 
in the analysis as not having a change from baseline values.  Additionally, we will use 
the methods described by Benjamini and Hochberg [51] to control the false discovery 
rate in the secondary efficacy analyses. 
10.0 Reporting Requirements 
 
A data safety monitoring board (DSMB) at MD Anderson will monitor the trial annually 
for safety. 


 
 
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Criteria for response and toxicity: 
10.1 Adverse Event 
Any unfavorable or unintended symptom, sign, or disease (including abnormal lab) 
temporally associated with the use of a medical treatment or procedure that may or 
may not be considered related to the medical treatment or procedure. Such effects can 
be intervention related, dose related, route related, participant related, and caused by 
an interaction with another drug. 
 
10.2 Serious Adverse Event 
Any  adverse  drug  experience  occurring  at  any  dose  that results  in  any  of  the 
following outcomes: 
• Death 
• A life-threatening adverse drug experience – any adverse experience that 
places the participant, in the view of the initial reporter, at immediate risk of 
death from the adverse experience as it occurred. It does not include an 
adverse experience that, had it occurred in a more severe form, might have 
caused death. 
• Inpatient hospitalization or prolongation of existing hospitalization 
• A persistent or significant disability/incapacity – a substantial disruption of a 
person’s ability to conduct normal life functions. 
• A congenital anomaly/birth defect. 
 
10.3 Eliciting Adverse Event Information 
Adverse events will be elicited at each clinic visit during participation in the study. 
Participants will be given frequent toxicity screening (in-person and phone) to review 
the occurrence of adverse events monthly and throughout the study. All adverse events 
which are directly observed and all adverse events which are spontaneously reported 
by the participant are to be documented by the investigator. 
 
10.4 Grading/Rating Scale 
All adverse events reported during the study will be evaluated and graded on a scale 
of 0-5. The CTCAE version 4.0 will be used to determine the grade for all toxicities. 
For any adverse events which are not listed in the CTCAE version 4.0 the following 
rating system will be used as listed below in section 10.5. 
 
10.5 Non-NCI Adverse Event Grading Scale Grade/Description 
0  No adverse event (absent) or within normal limits 
1 Mild adverse event (minor, no specific medical intervention, asymptomatic laboratory 


 
 
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findings only, radiographic findings only, marginal clinical relevance) 
2  Moderate adverse event (minimal intervention, local intervention, non-invasive 
intervention [packing, cautery]) 
3 Severe 
and 
undesirable 
adverse 
event 
(significant 
symptoms 
requiring 
hospitalization or invasive intervention; 
transfusion; 
elective interventional 
radiological procedure; therapeutic endoscopy or operation 
4  Life  threatening  or  disabling  adverse  event  (complicated  by  acute,  life 
threatening metabolic or cardiovascular complications such as circulatory failure, 
hemorrhage, sepsis. Life-threatening physiologic consequences; need for intensive 
care or emergent invasive procedure; emergent interventional radiological 
procedure therapeutic endoscopy or operation 
5  Death related to adverse event 
 
10.6 Relationship to Study Treatment 
The investigator will use the following definitions to assess the relationship of the 
adverse event to the study treatment: 
 
Definite: Adverse event is clearly related to the study treatment. 
Probable: Adverse event is likely related to the study treatment. 
Possible: Adverse event may be related to the study treatment. 
Unlikely: Adverse event is doubtfully related to the study treatment. 
Not Related: Adverse event is clearly not related to the study treatment. 
 
11.0 Laboratory Evaluations and Procedures 
 
11.1 Laboratories 
 
11.1.1  Serum assays will be performed at the MD Anderson Cancer Center Clinical 
Laboratory (estradiol, estrone, testosterone, DHEA-S, SHBG, adiponectin, 
glucose, hemoglobin, FSH, A1c, insulin and IGF-1). 
 
11.1.2    The endometrial biopsy slides will be reviewed centrally by the study's 
co-PI, Russell Broaddus, M.D., Ph.D. at MD Anderson Cancer Center 
and PI of the Pathology core.  A pathology report will be generated and 
forwarded to the participant's study gynecologist. 
 
Cases  with  hyperplasia  or  adenocarcinoma  will  be  confirmed  by  a 
second review by Elvio Silva, M.D., a senior gynecological pathologist at 
MDACC.  Dr. Silva is a Professor with more than 25 years of experience 
and is regarded as an international expert in gynecologic pathology. 
 
11.1.3 Ki-67 immunohistochemical assays, as well as IHC for metformin associated 


 
 
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phosphoproteins, will be performed in the Histology Core Lab at MD 
Anderson. 
 
11.1.4  Q-PCR for estrogen-regulated genes will be performed in the laboratory 
of David   Loose-Mitchell,   Ph.D.,   U.T.   -Houston   Medical   School 
(Biomarkers core). 
 
11.2 Collection and Handling Procedures 
 
An endometrial biopsy using 4 mm pipelle will be used to obtain endometrial tissue. Half 
of the sample will be placed in formalin, and half of the sample will be placed in a 
cryovial and immediately snap frozen in liquid nitrogen.  The sample in formalin will 
undergo routine histological-processing and paraffin-embedding.  One H&E slide will be 
cut per block.   Following central pathologic review, the pathology report will be 
forwarded to the participant's study gynecologist. 
 
11.3 Shipping Instructions 
Snap frozen tissue will be batched and shipped periodically on dry ice to Dr. David 
Loose-Mitchell at the University of Texas - Houston Medical School, PI of the 
Biomarkers Core. 
 
Snap frozen tumor will be shipped in liquid nitrogen to M. D. Anderson Gynecologic 
Oncology Translational Research Tumor Bank. 
 
 
11.4 Experimental Methodology and Preliminary Results 
 
11.4.1 Handling of Endometrial Biopsies 
Once the endometrial biopsy has been performed, half of the biopsy will be flash- 
frozen in liquid nitrogen, and the remaining half will be fixed in formalin.  RNA will be 
extracted from the frozen portion for the Q-PCR assays.  The formalin-fixed portion 
will undergo routine histological processing and paraffin embedding.  One H&E 
stained slide will be prepared for histological diagnosis.  Additional paraffin 
embedded tissue will be used for immunohistochemical Ki-67 assay and metformin-
associated biomarkers (phopho-IHC). 
 
11.4.2 Pathological Examination of Biopsies 
A gynecologic pathologist (Russell Broaddus, M.D., Ph.D.,Co-PI and Pathology 
Core  PI)  will  review  all  of  the  endometrial  biopsies.    Difficult  cases  will  be 
reviewed with a senior consultant (Elvio Silva, M.D., leader of the Gynecologic 
Pathology  Group,  Department  of  Pathology,  M.D.  Anderson  Cancer  Center). 
The  biopsies  will  be  placed  into  diagnostic  categories,  such  as  proliferative 
phase, secretory phase, atrophic, anovulatory, hyperplasia, hyperplasia with atypia, 
or carcinoma, according to standard pathological principles. 


 
 
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11.4.3 Frozen Tissue Experiments - Quantitative PCR analysis of Transcript in 
Endometrial Tissues 
The  Q-PCR  analyses  will  be  performed  in  the  Quantitative  Genomics  Core 
Facility at the University of Texas Medical School at Houston.  This portion of the 
project will be under the direction of David Loose-Mitchell, Ph.D. (Biomarkers 
core PI) and Greg Shipley, Ph.D.     This laboratory has been interested in identifying  
genes  in  human  endometrium  whose  expression  is  altered  by hormone  
replacement therapy (HRT).    Over the  last  3  years  we  have  used several 
techniques including differential display, microchip array screening, and real-time 
quantitative PCR to find such genes.  The tools we have developed are directly 
applicable to the investigation of surrogate endpoint biomarkers (SEBs). 
Identification of appropriate biomarkers is essential for this study.   An ideal 
biomarker is a histological or molecular "red flag" that precedes the onset of cancer. 
Ideal surrogate endpoint biomarkers are quantitative and are modulated by 
chemopreventive agents. 
 
In our prior endometrial cancer chemoprevention study of women with HNPCC, we 
evaluated   a   panel   of   30   biomarkers   thought   to   be   relevant   in   endometrial 
carcinogenesis.  From that study, we have narrowed down the field to a subset of 5 
validated, estrogen-regulated genes and a proliferation marker[46]. 
12.0 Transcripts to be Analyzed by Q-PCR - Endometrial Biopsies 
 
Table 4: Transcripts to be Analyzed and the Rationale. 
Transcript 
Rationale 
PCNA 
Marker of proliferation 
IGF-IR 
The major receptor for IGF-I, an immediate growth regulator of 
estradiol 
RALDH 2 
Retinaldehyde  dehydrogenase 2 is  rate-limiting  enzyme for 
retinoic acid production; induced by E2. 
SFRP4 
Wnt antagonist; highly induced by E2 in human endometrium 
survivin 
Inhibitor  of apoptosis, modestly  induced by E2 in human 
endometrium 
EIG121 
Novel estrogen induced gene of unknown function. 
Normalizers 
18s ribosomal essential for quantitating RNA yield 
cyclophilin 
mRNA not regulated by E2 in the endometrium 
b-actin 
another mRNA not regulated by E2 in the endometrium 
 
 
12.1 Preparation of RNA from Endometrial Biopsies for Real- Time Q-PCR 
 
Total RNA will be isolated from endometrial samples using a Qiagen RNeasy Kit 
(Qiagen, Inc., Chatsworth, CA) and a modification of the manufacturer's protocol. 


 
 
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Endometrial samples containing 5-20ng of tissue will be placed in 1.0 ml freshly 
prepared  lysis  buffer  containing  10mM  b-mercaptoethanol.  The  tissue  is 
homogenized with a Brinkman PT3000 (Brinkmann Instruments, Westbury NY) set 
at 30,000 rpm using a small diameter probe (PTDR3007). 
 
The lysate will be centrifuged at 1000xg for 5 minutes to reduce foam and remove 
debris. The supernatant will be placed in a 1.5ml microfuge tube and the lysate spun at 
full speed for 3 minutes.  An equal volume of 70% ethanol is added to the lysate and 
mixed thoroughly. The Qiagen columns are loaded (x3) with 700 ml of the lysate/ethanol 
mixture and centrifuged at 10,000 rpm for 15 s. The RNA is bound to the column matrix 
and the flow through discarded. After all of the lysate mixture is loaded onto the 
column, it is washed with 700 ml of wash buffer RW1. The column is then centrifuged 
at 10,000 rpm for 15 s. and a new receiving tube is attached. The second wash is with 
500 ml was buffer RPE at 13,200 rpm for 60 s. The RNA is eluted from the column 
into a new microfuge tube by applying 55 ml DEPC-treated H20 treated directly to the 
column and centrifuging at 10,000 rpm for 60 s. RNA is quantitated by A260.  In our 
previous studies on 290 post-menopausal women, this procedure yielded an average 
of 16.7 mg per sample (min 0.11, max 117 mg). In that  same  study,  230  samples  
were  from  HRT  treated  women  and  yielded  an average of 37.3 mg of total RNA. 
 
We typically measure transcripts in 10-20 ng of total RNA.   Some of the more abundant 
RNAs can be measured in as little as 1 ng; in fact, we measure 18S rRNA from a dilution 
that contains 0.025 ng. If we assume we will need an average of 20 ng per sample 
per transcript times 4 per quadruplicate determinations, then we will require 80 ng per 
transcript.  To measure the 8 transcripts will require 640ng of RNA. This is less than 1/5 
of the average amount of RNA recovered from untreated post- menopausal women. In 
our recent study, this was obtained in 87% of our post- menopausal, untreated women 
and in 100% of the samples from treated women. We should be able to recover 
sufficient RNA from nearly all samples to perform a complete analysis; in that small 
percentage where RNA becomes limiting we will prioritize our assays as described 
later in this section. 
 
12.2 Analysis of Q- PCR Transcripts 
 
Transcripts for the 7 genes will be assayed in RNA prepared as described above. 
For each transcript, compatible PCR primers and a fluorescent hybridization probe 
(TaqMan probe) will be designed using Primer Express (PE Biosystems). PCR 
conditions will be optimized for primer and probe concentration and Mg2+ 
concentrations to give assays with a slope of the sRNA standard curve of -3.1 - -3.5 
(PCR cycles per log template molecules) and a lower detection limit of <102 molecules 
input template. Assays will be assembled in a 96-well plate in which 12 wells are 
allocated to sRNA standards (102 - 107 sRNA molecules), 2 wells are assigned to 
controls (no amplification and no template controls) and 80 wells are allocated to RNA 
samples. We have found that optimal data is achieved when RT- PCR analyses of 
unknown RNA's are run in triplicate, and we always include a 4th aliquot of each sample 


 
 
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34 
 
without added reverse transcriptase (to detect amplification of residual genomic DNA). 
Using this format of 20 RNA samples are analyzed per 96-well plate. Plates are 
assembled robotically and the RT and "real-time" Q-PCR runs will be carried out in 
the ABI 7700.     The level of input RNA in unknown samples is calculated by 
interpolation of the Ct value  of the  unknown on  a  sRNA standard  curve  run in 
parallel with the unknown samples. The results of the assay are reported as the number 
of template molecules in the input unknown RNA.   Because of occasional 
inaccuracies in RNA quantitation using spectrophotometric analyses, we will determine  
the level of three  normalizer transcripts  (b-actin,  cyclophilin, and  18S rRNA) in 
each sample.  Results are then normalized to these three transcripts and are 
expressed as % normalizer mRNA. 
 
12.3 Real-Time Q-PCR 
 
96- well plates will be assembled robotically using Tecan and Biomek workstations. 
Aliquots  of  the  RNA  (10-20  ng)  will  be  reverse  transcribed  with  SuperScript  II 
reverse  transcriptase  (1  U/ml;  Boeringher  Mannheim)  in  a  10  ml  reaction  that 
includes the appropriate transcript-specific reverse primer (300 nM), 1X RT buffer 
(Gibco-RL), 4 mM MgCl2, 10 mM DTT, and 500 mM dNTPs. The reaction will be 
incubated at 50°C for 30 minutes and then terminated by incubation at 72°C for 5 
minutes. Following reverse transcription of 40 ml of "real-time" PCR reaction mix is 
added to give a final volume of 50 ml.  PCR is performed in a 7700 Prizm Sequence 
Detector (Perkin Elmer) with preheating at 95°C for 1 minute followed by 40 cycles 
of  annealing  and  extension  (60°C  for  60  s.)  and  melting  (95°  C  for  12  s.). 
 
Dequenching of the fluorescent hybridization probe is continuously monitored and 
the  Ct  (PCR  cycles  to  threshold)  calculated  using  Sequencing  Detector  1.6.3 
software (Perkin Elmer).  Unknown RNA samples will be analyzed in triplicate with a 
control that lacked reverse transcriptase (-RT control). Samples are run in parallel 
with sRNA standards (0.8 pg - 80 pg) to generate an internal standard curve (Ct versus 
sRNA template molecules). The number of template molecules in the sample is 
calculated by interpolation of Ct against the sRNA standard curve. 
 
Amplicon specific sRNAs are generated from appropriate plasmid templates by first 
synthesizing the target amplicon using the same PCR primers used in the "real-time" 
PCR reaction. The amplicon is then cloned into the pCRIItopo plasmid (Invitrogen) 
that includes a T7 site flanking the cloning cassette. sRNA is synthesized from the 
T7-tailed amplicon template using T7 Mega-script Polymerase (Ambion). sRNA is 
quantitated by 32P-UTP incorporation and checked for purity by gel electrophoresis and 
analyzed on a phosphorimager (Molecular Dynamics). Aliquots of sRNA containing 
known numbers of template molecules are reverse transcribed and subjected to real-
time PCR in parallel with unknown RNA samples to quantify transcript abundance. 
12.4 Logistics of Real- Time Q-PCR 
 


 
 
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May 13, 2013 
35 
 
The studies outlined in this proposal that use frozen specimens total 7 transcripts in 
40 women at 2 time points.   This will require 30 Q-PCR runs on a 7700.   We 
currently operate 2 ABI 7700 instruments and average throughput is 3 runs per machine 
per day.  Therefore we estimate that the proposed studies can be done in 5 machine 
days. 
 
12.5 Databases and Data Storage for Real- Time Q-PCR 
 
Data is exported electronically from the 7700 and initial calculations (means of triplicate 
samples) are performed on a custom spreadsheet.  The raw 7700 data and the 
preliminary calculations are archived on CD-ROM.     These data are then electronically 
transferred to a custom Filemaker Pro database which performs all of the final 
calculations.  Changes to the data contained in the Filemaker database are all logged 
to a separate, password-protected audit database.     The Filemaker database is 
archived to magnetic tape. 
12.6 Immunohistochemistry 
 
Ki-67 immunohistochemistry and phospho-AMPKa, phospho-ACC, phospho-mTOR 
and phospho-S6 ribosomal protein will be performed in the Histology Core Lab, 
using paraffin-embedded tissue sections and commercially available antibodies. The 
percentage cells positive will be recorded, as well as the compartment stained (epithelial 
cell vs. stromal cell); nucleus vs. cytoplasm vs. cell membrane). Dr. Broaddus will 
evaluate the immunohistochemistry slides. 
 
13.0 Schedule of Events 
 
Table 5: Study Parameters (see next page) 


 
 
2011-0739 
 
 
September 12, 2013 
 
 
 
36 
 
 
a)  Turn Around Time 
e) As needed 
b)  Plus or minus 10 days. 
f) Only if ALT levels elevated as listed in CTCAE version 4.0. 
c)  Research blood for future biomarker analysis. 
d)  1 ASA-24 completed at Screening Assessment. Other 2 ASA-24s complete randomly between screening assessment and 
7 days post-biopsy. 
Study Parameters 
TATa 
Tube 
Top 
Screenin
g 
Screening Randomi
zation 
1st 
Month 
2nd 
Month 
3rd 
Month 
4th 
Month 
 1 yr.  
 
Color 
Visit 1 
Visit 2 
Visit 3 
b 
b 
b 
b 
Follow-Up 
Informed Consent 
X 
History 
X 
Physical 
X 
X 
Research Red Tubec 
Red 
X 
X 
X 
Research Lavender Tubec 
Lavender 
X 
X 
X 
Insulin (fasting) 
4 days 
Red 
X 
X 
X 
Glucose (fasting) 
4 hrs 
Mint 
X 
X 
X 
FSH (Spec Chem Test) 
24 hrs 
Gold 
X 
X 
X 
Hemoglobin 
4 hrs Lavender 
X 
X 
X 
Estradiol 
5 days 
Red 
X 
X 
X 
Estrone 
5 days 
Red 
X 
X 
X 
Testosterone (Chem Test) 
24 hrs 
Gold 
X 
X 
X 
DHEA-S 
5 days 
Red 
X 
X 
X  
Sex Hormone Binding 
Globulin (SHBG) 
5 days 
Red 
X 
X 
X 
Adiponectin 
7 days 
Red 
X 
X 
X 
A1C (Spec Chem Test) 
24 hrs Lavender 
X 
X 
X 
IGF-1 
7 days 
Red 
X 
X 
X 
Serum Creatinine 
4 hrs 
Mint 
X 
X 
X 
X 
ALT 
4 hrs 
Mint 
X 
X 
Xf 
Xf 
X 
X 
Triglycerides (fasting) 
4 hrs 
Mint 
X 
X 
X 
TSH 
24 hrs 
Gold 
X 
X 
X 
Dispense 
Medication & Pill 
X 
X 
X 
X 
Pill Count 
X 
X 
X 
X 
 
Pre-treatment EMB 
72 hrs 
X 
Toxicity and  Compliance 
Assessment: Phonee 
X 
X 
X 
X 
 
Toxicity Assessment: In- 
person 
X 
X 
X 
X 
 
Post-treatment EMB 
72 hrs 
X 
 
DEXA 
X 
X 
X 
waist circumference 
X 
X 
X 
hip circumference 
X 
X 
X 
BMI 
X 
X 
 
ASA-24 (3)d 
X 
X 
 
Accelerometer 
X 
X 
 
Survey Packet 
X 
X 
X 


 
 
2011-0739 
 
 
September 12, 2013 
 
 
 
37 
 
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