Pilot Trial to Evaluate Blood and Imaging Based Biomarkers for Aromatase Inhibitor Induced Musculoskeletal Syndrome
Phase: NA
Conditions: Aromatase Inhibitor Induced Musculoskeletal Syndrome (AIMSS), Breast Cancer
Sponsor: University of Arizona

 
 
Official Title: 
Pilot Trial to Evaluate Blood and Imaging Based Biomarkers 
for Aromatase Inhibitor Induced Musculoskeletal 
Syndrome 
NCT number: 
NCT03665077 
Document Type:  
Study Protocol and Statistical Analysis Plan 
Date of the 
Document: 
AIMSS IIT Protocol v4.0 12-2-2020 
 
 

1 
Protocol Version 4 dated December 2, 2020 
 
 
STUDY TITLE: Pilot Trial to Evaluate Blood and Imaging Based Biomarkers for 
Aromatase Inhibitor Induced Musculoskeletal Syndrome  
 
Protocol Version 1 Dated October 6, 2017 
Protocol Version 2 Dated May 14, 2018 
Protocol Version 3 Dated October 28, 2019 
Protocol Version 4 Dated December 2, 2020 
 
Principal investigator: 
 
Pavani Chalasani, M.D, MPH 
Associate Professor  
The University of Arizona Cancer Center 
1515 N. Campbell Avenue, PO Box 245024 
Tucson, AZ 85724-5024 
P- (520) 626-0191 
F- (520) 626-2225 
pchalasani@uacc.arizona.edu 
 
Co-Principal Investigator: 
 
Jessica A. Martinez, PhD 
Assistant Research Professor 
Department of Nutritional Sciences 
The University of Arizona Cancer Center 
(520) 626-6326 
 
Statistician 
Denise Roe, DrPH 
Professor of Biostatistics 
Director of Biostatistics Shared Resource  
 
 
Funding: University of Arizona Cancer Center   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Protocol Version 4 dated December 2, 2020 
 
 
 
Protocol Synopsis 
 
Title of Study: Pilot Trial to Evaluate Blood and Imaging Based Biomarkers for Aromatase 
Inhibitor Induced Musculoskeletal Syndrome (AIMSS)  
 
Hypotheses:  Baseline plasma oxylipin profiles can be used to predict who is at risk for 
developing AIMSS. 
 
Primary Objectives: 
 
• 
To compare baseline oxylipin levels in women that do vs. those that do not develop 
AIMSS  
 
Secondary Objectives: 
 
• 
To correlate changes in oxylipin panels with changes in tendon stiffness  
• 
To correlate changes in levels of oxylipins with changes in pain scores through 6-
month AI treatment.  
 
Study design: This is a prospective single arm study enrolling patients (n = 35) with breast 
cancer scheduled to start adjuvant hormonal therapy. Patients would have completed all 
their primary treatments (surgery± radiation therapy) and are scheduled to start their 
adjuvant hormonal therapy. They get baseline blood drawn for oxylipins. They start 
adjuvant anastrozole and have blood drawn at 3mths and 6mths for measurement of 
oxylipins.. This is a pilot trial to evaluate blood biomarkers. Once pilot data is analyzed, 
goal is to apply for funding for a larger trial to further evaluate and validate these 
biomarkers 
 
Study Center:  Banner – University Medical Center, University of Arizona Department of 
Medical Imaging, University of Arizona Cancer Center-North Campus, University of 
Arizona Cancer Center-Orange Grove Campus, Tucson, AZ 
 
Number of Patients: We plan to accrue a total of 35 patients into this prospective single 
arm trial to have 20 evaluable patients for analysis 
 
Main Criteria for Inclusion/Exclusion:  
 
Inclusion Criteria  
 
To be eligible for this trial patient must  
 
1. Be capable of understanding the investigational nature of the study and all 
pertinent aspects of the study 
2. Be capable of signing and providing written consent in accordance with institutional 
and federal guidelines 
3. Have a histologically-confirmed diagnosis of breast cancer  
4. Be willing and able to comply with scheduled visits, treatment plan 

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Protocol Version 4 dated December 2, 2020 
 
5. Age ≥ 21 years 
6. Post-menopausal women with 1st event of ER+ early stage breast cancer (0-3) 
7. Completed definitive therapy (surgery ± radiation)  
8. Candidates for adjuvant AI therapy  
 
Exclusion criteria 
 
Patients who fulfill any of the following criteria will be excluded from the study: 
 
1. Have received adjuvant or neo-adjuvant chemotherapy 
2. Prior endocrine therapy (AI or tamoxifen) 
3. History of rheumatoid arthritis or other autoimmune arthritis 
4. Daily non-steroidal anti-inflammatory drug (NSAID) use (except for daily aspirin 
use)  
5. Current use of daily corticosteroids or immunosuppressive therapies  
 
Intervention:  
 
 
 
 
Statistical analysis 
 
Statistical Considerations:  This is a pilot study to estimate the values needed to 
design a larger study.  The primary objectives are to estimate the baseline oxylipin levels 
in patients who develop with AIMSS vs. those who do not develop AIMSS.  Analysis will 
be based on descriptive statistics and 95% confidence intervals for each group.  
Although we will not formally test for differences between the two groups, we will 
estimate the difference with a 95% confidence interval to inform the design of a larger 
study.   
 
The secondary objectives are to correlate changes in oxylipin levels with changes in pain 
scores.  These analyses will be based on all 20 women, irrespective of whether they 
develop AIMSS.   
 
Feasibility Issues:  From 2015 to 2016, ~40 patients were started on adjuvant 
anastrozole therapy who meet our eligibility criteria. Thus, we anticipate accrual of 2-3 
patients per month and completion of accrual this pilot study within 8-12 months of study 
initiation. Based on previously published studies, it is expected that 50% of women 
initiating AI will develop AIMSS within one year, with most women experiencing symptoms 
by 2 months.17 Women who discontinue use of anastrozole before the 6 month visit will 
complete all measurements at the time that they switch to another AI and come off study. 
 
Dr. Chalasani and the research personnel will be conducting monthly meetings to review 
patient accrual data, address any barriers to accruals and resolve them in a timely manner. 
Dr.Martinez will be overseeing the oxylipin data analysis.  
 
 
 
 

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Protocol Version 4 dated December 2, 2020 
 
 
 
 
STUDY CALENDAR 
 
 
 
Treatment -Anastrozole 
Screening 
Pre-Treatment 
3mths 
6mths  
Informed Consent 
X a 
History and Physical Exam 
X a 
Collection of Blood for 
biomarker analysis 
X b,d 
Xc,d  
Xc,d 
 
 
 
 
 
Questionnaires 
X b 
Xc  
Xc  
A- Performed within 28 days of trial entry 
B- May be collected/performed any time prior to starting anastrozole (can be on the 
same day) 
C- Can be done within 28 day window (±) of the time point  
D- Labs have to be fasting  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Protocol Version 4 dated December 2, 2020 
 
 
TABLE OF CONTENTS 
                                                     
Page 
Protocol Synopsis 
 
2 
1. Introduction 
 
6 
2. Study Objectives 
 
10 
3. Study Design 
 
10 
4. Selection of Patients 
4.1. 
Inclusion Criteria 
4.2. 
Exclusion Criteria 
4.3. 
Study Patient Identification 
4.4. 
Study exit  
 
10 
5. Study Procedures 
5.1. 
Pretreatment 
5.2. 
Patient Registration 
5.3. 
Research blood draw 
5.4. 
Self-reported questionnaire 
5.5. 
Toxicities to be monitored 
5.6. 
Follow-up 
 
 
11 
11 
12 
12 
12 
13 
 
6. Criteria for Evaluation 
 
13 
7. Study Statistics 
7.1. 
 
13 
8. Data and Safety Monitoring Plan 
 
13 
9.  Data Submission Schedule 
 
15 
10. Ethics 
 
16 
11. Bibliography 
 
17 
12. Appendix  
12.1 BCPT-MS 
12.2 WOMAC 
12.3 PHQ-9 
 
23 
25 
26 
13. Protocol Signature Page 
27 
 
 
 
 
 
 
 

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Protocol Version 4 dated December 2, 2020 
 
1. Introduction 
 
Aromatase Inhibitors (AIs), Adherence, Musculoskeletal Symptoms and Mortality. 
Adjuvant aromatase inhibitor (AI) therapy is the recommended first-line adjuvant 
treatment for postmenopausal women with estrogen receptor positive (ER+) breast 
cancer based on improved disease-free survival (DFS) compared to tamoxifen in 
randomized control trials. More recently extended adjuvant AI therapy (for 10 years) has 
been shown to improve DFS when compared to placebo in women who completed 5 
years of standard AI therapy.1,2 However, AI therapy has been associated with 
significant, activity-limiting musculoskeletal symptoms including arthralgia, myalgia and 
joint stiffness or AI-induced musculoskeletal syndrome (AIMSS).3 AIs block estrogen 
synthesis and induce menopausal symptoms that are particularly severe in younger 
patients.4,5 Musculoskeletal complaints are among the most commonly reported and 
debilitating side effects of adjuvant AI therapy;6 findings that are supported by 
observations in aromatase knockout mice which exhibit increased pain responses.7 
Among women with arthralgia syndromes at initiation of AI therapy, half report symptom 
exacerbation. Joint-related symptoms manifest early after initiation of AI therapy with 
worsening of symptoms up to 1 year post-initiation of AI.8,9 AIMSS impairs ability to 
perform activities of daily living as well as work-related tasks as the most commonly 
affected joints are hands, knees and hips.10,11 In a survey conducted by Breast Cancer 
Action, 27% of respondents reported that they had discontinued adjuvant AI use 
because of adverse effects (AE), with AIMSS being the leading AE.12 In a cohort study in 
California, 61% of patients on AIs developed joint and/or bone pain resulting in 
discontinuation of therapy in 20% of patients.13 In a recent study, full adherence, defined 
as full duration and optimal schedule, was observed for only 49% of breast cancer (BC) 
patients prescribed any hormone therapy (HT) and was related to higher mortality in 
those who discontinued [Hazards Ratio (HR) = 1.26 (Confidence interval (CI) 1.09– 
1.46)] or were non-adherent to HT [HR = 1.49 (CI 1.23–1.81)].14 This study is concerning 
and consistent with clinical trial evidence from prior tamoxifen studies showing higher 
recurrence rates and worse outcomes with < 5 years or suboptimal HT use.15-17 Clearly, 
there is a need to maximize efficacy and improve tolerance to AIs. Approaches to study 
interventions that enhance both adherence and response rates for AIs are important 
research and clinical gaps that must be addressed to reduce death from breast 
cancer.18,19 A critical first step is to identify those who are at risk of developing 
AIMSS by developing predictive biomarkers that will provide physicians 
opportunity for early intervention, thereby improving adherence to AIs. Currently 
there are no predictive biomarkers available to identify those at risk of developing 
AIMSS. 
Mechanisms of Aromatase Inhibitor-Associated Pain. The mechanism of AIMSS is 
not well understood and is grossly understudied. Based on observational data and 
animal data, it is likely that AIMSS is directly related, at least in part, to a) loss of the 
anti-nociceptive action of estradiol - resulting in greater sensitivity to stimuli and b) 
presence of underlying inflammation and the nociceptive activity or pain-inducing 
response of PGE2. In aromatase-knockout mice, which lack endogenous estrogen 
production and serve as an animal model of AI therapy, estrogen deprivation results in a 
dominance of pro-nociceptive (pain) over anti-nociceptive response mechanisms 
following noxious stimuli such as formalin injection.20 Results from experimental models 
to determine the role of estradiol in inflammation and specifically in cyclooxygenase 
(COX)-mediated PGE2 are inconsistent and model dependent. Hunter et al 
demonstrated that ovariectomized rats exhibited greater sensitivity to a pain stimulus, as 

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Protocol Version 4 dated December 2, 2020 
 
measured by latency to paw withdrawal following an inflammatory insult.21 Serum levels 
of PGE2 were increased in these rats, indicating an increase in inflammation. Pain 
sensitivity was reversed with estradiol replacement but serum PGE2 levels did not 
change, indicating a separate mechanism for inflammatory response. The addition of a 
COX2 inhibitor increased latency to paw withdrawal compared to estradiol replacement 
alone, indicating a reduced pain response for these animals, and again indicating 
separate roles for inflammation and estradiol in pain response.22 Using this same model, 
ibuprofen (a non-steroidal anti-inflammatory drug, NSAID; a COX inhibitor) + estradiol 
replacement was shown to be more effective in reducing pain than either estradiol or 
ibuprofen alone.22 Collectively, these results indicate that the loss of estrogen alone 
does not increase pain by inducing PGE2; but in the presence of PGE2-mediated 
inflammation, estrogen deprivation increases sensitivity to inflammatory pain. Therefore, 
if PGE2 is elevated at baseline, this can predispose women to experience pain 
upon the depletion of estradiol with the addition of an AI.  
In healthy women, joint and muscle pain worsen with age with greatest increases 
between 50 - 59 years.23,24 New onset of rheumatoid arthritis is also highest in the 
decade following menopause. Such observations, in combination with extensive 
experimental studies in animal models, have established estradiol as having both anti-
inflammatory and anti-nociceptive properties.24 Increased inflammation with aging, 
mediated by bradykinin and the pro-inflammatory prostaglandins (PGs), including PGE2, 
has been mechanistically linked to hyperalgesia (increased sensitivity to painful stimuli) 
and allodynia (painful response to what would normally be an innocuous stimulus). Thus, 
inflammation and injury that stimulate PG release are thought to result in a heightened 
nociception and allodynia with the decline in estradiol levels with menopause.25  
Management of AIMSS in AI Patients. While all 3 third-generation AIs in routine clinical 
use cause different levels of estradiol suppression,26 toxicity profiles are similar for all of 
them.27-29 Further, many studies show that there is no correlation with degree of estrogen 
depletion and pain in women on AI, regardless of AI type.30,31 Differences between 
agents in terms of quality of life (QOL) and tolerability are very important when selecting 
the optimal therapy for individual patients. Randomized clinical trials comparing 
tolerability and QOL between letrozole and anastrozole did not show any significant 
differences between the QOL scores. They did report that subjects who were intolerant 
to one AI were nonetheless able to tolerate another agent.32,33 Other studies have also 
demonstrated that switching AIs can result in improvements of AIMSS.33,34 This is a 
significant finding given that—despite AIs having similar toxicity profiles—AIMSS can 
sometimes be managed by switching AIs.   
AIMSS management is an area of active research interest. For patients developing 
AIMSS, there is evidence that acupuncture,35,36 high dose vitamin D37 or exercise38 might 
help in alleviating their symptoms. Yet results are not always consistent and are limited 
by small numbers of participants. Recently, a randomized, placebo-controlled study 
reported a 50% decrease in AIMSS symptoms with ω-3 fatty acid supplementation; 
however, this change was not significantly different when compared to the placebo 
arm.39 Retrospective clinical data reported a significantly decreased recurrence and 
longer disease-free survival in obese women with ER+ tumors who take NSAIDs.40 Anti-
inflammatory medications like NSAIDs or COX2 inhibitors might alleviate arthralgia and 
myalgia but have gastrointestinal and cardiac side effects. Currently, for a patient who 
develops AIMSS, routine management involves i) pain control with NSAID’s or other 
pain medicines, ii) stopping and switching to a different AI or tamoxifen, iii) stopping 
adjuvant hormonal therapy completely or iv) referral to orthopedic surgeon based on 

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Protocol Version 4 dated December 2, 2020 
 
symptom severity. It is important to realize not all patients who take AIs develop 
AIMSS. It is crucial to determine which patients are at risk so that preventative strategies 
can be implemented early on to improve adherence to AI therapy and eventually 
decrease breast cancer relapse and death. Identifying at-risk subjects by developing 
predictive biomarkers is a critical unmet need in AIMSS.  
Pain and Oxylipin Cascade. While PGE2 is the best-studied metabolite of arachidonic 
acid metabolism, arachidonic acid and other polyunsaturated fatty acids (i.e., ω-3 and ω-
6) are also metabolized by the lipoxygenases (LOX) and cytochrome P450 (CYP) 
enzymes, yielding distinct sets of metabolites with their own biological activities. 
Collectively, these COX, LOX and CYP metabolites are called oxylipins. The oxylipin 
metabolites of ω-3 and ω-6 fatty acids comprise over 100 metabolites, each of which 
exhibit potent biological effects, including acting as modulators of inflammation (pro- and 
anti-) as well as activators of angiogenesis and mediators of hyperalgesia and 
allodynia.41-43 Recently, it has been demonstrated that there is cross-talk between the 
estrogen receptor and LOX-mediated oxylipins,44 and that AIs can upregulate the 
CYP450 pathway and downstream epoxyeicosatrienoic acids in vitro.45 It is well 
established that downregulation of PGE2 production through inhibition of COX2 
alleviates inflammatory pain. Preliminary evidence implicates both the CYP450 and LOX 
pathways in the development of tendinopathy.46,47 Two earlier clinical trials showed that 
12-hydroxyeicosatetraenoic acid (12-HETE) was significantly correlated with pain in 
menstruating women,48,49 suggesting interplay between 12-HETE and hormonal 
changes. Our preliminary data in AI-treated women participating in an intervention with 
sulindac (a non-selective COX2 inhibitor) suggests involvement of 12-HETE and other 
LOX metabolites in AIMSS (see Approach). If we successfully show that oxylipins are 
pathogenic in the development of AIMSS, there are already pharmacologic agents 
available (COX and LOX inhibitors) or in development (CYP pathway inhibitors) that can 
be implemented to target these pathways50 and alleviate symptoms, with the ultimate 
goal of improving AI adherence in clinical practice. As cited above, interventional clinical 
trials have been inconsistent, and there have been no studies that have determined 
predictive blood-based biomarkers for AIMSS in order to guide optimal subject selection 
to study appropriate interventions.  
 
Preliminary Data: 
Oxylipins:  
Dr. Chalasani is institutional PI of an ongoing 
clinical trial to determine the effect of sulindac (an 
NSAID) on breast density and pain in women that 
have been stable on AI for 6 months. Participants 
receive sulindac (150 mg BID) for 12 months. 
Table 
2: 
Relationship 
between 
oxylipins and pain in 10 breast cancer 
patients on AIa  
Enzyme 
Oxylipin 
How much pain 
interferes with 
walking ability 
R2 
P-value 
COX 
PGE2c 
0.11d 
0.36 
LOX 
12-
HETEc 
0.44 
0.04 
LOX 
15-HETE 
0.25 
0.15 
LOX 
11-HETE 
0.21 
0.18 
LOX 
9-HETE 
0.25 
0.14 
LOX 
8-HETE 
0.21 
0.19 
LOX 
5-HETE 
0.09 
0.41 
aOxylipins quantified in plasma drawn 
after a 3 month observation period 
bQuestion from the Brief Pain Inventory 
Short Form (BPI-SF) on a scale of 1-10. 
cTarget 
oxylipins: 
Prostaglandin 
E2 
(PGE2) and 12-Hydroxyeicosatetraenoic 
acid (12-HETE) 
dAll R2 values are positive correlations 
between indicated oxylipin level in 
plasma and score from BPI questionnaire 
Table 1: Plasma levels of target oxylipins 
in 10 breast cancer patients on AIa 
 
Plasma (nM)b 
PGE2c 
0.35 (±0.47) 
12-HETEd 
65.2 (±53.4) 
aOxylipins measured in plasma collected 
after a 3-month observation period 
bData presented as: mean (±SD) 
cPGE2: prostaglandin E2  
d12-HETE: 12-Hydroxyeicosatetraenoic acid 

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Protocol Version 4 dated December 2, 2020 
 
Plasma and urine samples are collected before and after a 3-month observation period 
(months -3 and 0), and after 3, 6, 9, and 12 months on sulindac.  
Participant demographics. This study is currently conducted in post-menopausal women 
diagnosed with ER+ breast cancer. Inclusion and exclusion criteria for this study 
regarding breast cancer history are the similar as the proposed work except that 
participants in the Sulindac trial must have been stable on AI for 6 months and exclusion 
criteria for this trial also includes active smokers, history of diabetes mellitus, 
uncontrolled hypertension, GI ulcers and bleeding diathesis . 
Oxylipins in women on AI. We have conducted oxylipin profiling in the plasma and urine 
in 10 women after the 3 month observation period (selected based on availability of 
samples). We quantified 92 total oxylipins in plasma and 87 in urine with good 
reproducibility. PGE2 levels were similar to the ranges previously reported in plasma of 
other breast cancer patients.86-88 Levels of target oxylipins after a 3-month observation 
period (PGE2 and 12-HETE) in women on AI are presented in Table 1.  
Pain and oxylipins. How much pain interferes with daily activities such as walking ability 
was evaluated using the Brief Pain Inventory Short Form (BPI-SF). Oxylipin levels after a 
3 month observation period were correlated with questionnaire items at the same time-
point. Table 2 presents the results from linear regression comparing how much pain 
interferes with walking ability and selected plasma oxylipin levels in 10 women stable on 
AI.  PGE2 levels were not correlated with how much pain interfered with walking ability. 
However, plasma levels of the LOX metabolite, 12-HETE, were significantly correlated. 
While not statistically significant, a similar pattern was observed across multiple LOX 
metabolites. This trend could still be observed in only 10 participants with overall low 
scores for how much pain interferes with walking ability (mean ± SD; 2.48 ± 2.45), which 
suggests a role for the LOX arm of the oxylipin pathway in the development of AIMSS. 
There was no correlation (positive or negative) with CYP450-derived metabolites and 
pain scores (data not shown).  
While these results are promising and suggest that the LOX pathway may be related to 
AIMSS, samples from this clinical trial have several limitations. First, participants in this 
study were stable on AI for at least 6 months. Therefore, there is no true “baseline” to 
quantify oxylipins levels. Second, hand-specific symptoms, common in AIMSS, were not 
evaluated. Third, there is no BPI-SF score that specifically addresses AIMSS 
development. Fourth, while we have presented our preliminary data from the baseline 
(post-NSAID washout and 3 month observation period) only, women in this study initiate 
intervention with sulindac (a non-specific COX2 inhibitor). Thus, it is likely that the 
oxylipin cascade will be modulated by the intervention, and stored samples from this trial 
will not be appropriate to determine changes in oxylipins related to the natural history of 
AIMSS. Despite these limitations, these data demonstrate that oxylipins are quantifiable 
in the plasma of women currently taking an AI, and that they are present in a wide 
enough range of levels to relate to pain scores. 
 
 
 
 
 
 
 

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Protocol Version 4 dated December 2, 2020 
 
2. Study Objectives 
 
Primary Objectives: 
 
• 
To compare baseline oxylipin levels in women that do vs. those that do not develop 
AIMSS  
 
Secondary Objectives: 
 
• 
To correlate changes in levels of oxylipins with changes in pain scores through 6-
month AI treatment.  
 
 
3. Study design:  
 
This is a prospective single arm pilot study enrolling patients (n = 35) with breast cancer 
scheduled to start adjuvant hormonal therapy. Patients would have completed all their 
primary treatments (surgery±radiation therapy) and are scheduled to start their adjuvant 
hormonal therapy. At baseline, patients will undergo a blood draw. They will start adjuvant 
anastrozole and have blood drawn at 3 and 6 months for oxylipins. This is a pilot trial to 
evaluate oxylipins as blood biomarkers of AIMSS. 
 
4. Selection of patients 
 
Number of Patients: We plan to accrue a total of 35 patients with breast cancer into this 
prospective single arm pilot trial with the goal of having data from 20 evaluable patients. 
 
Main Criteria for Inclusion/Exclusion:  
 
4.1 Inclusion criteria 
 
To be eligible for this trial patient must  
 
1. Be capable of understanding the investigational nature of the study and all 
pertinent aspects of the study 
2. Be capable of signing and providing written consent in accordance with institutional 
and federal guidelines 
3. Have a histologically-confirmed diagnosis of breast cancer  
4. Be willing and able to comply with scheduled visits, treatment plan 
5. Age ≥ 21 years 
6. Post-menopausal women with 1st event of ER+ early stage breast cancer (0-3) 
7. Completed definitive therapy (surgery ± radiation)  
8. Candidates for adjuvant AI therapy  
 
4.2 Exclusion criteria 
 
Patients who fulfill any of the following criteria will be excluded from the study 
 
1. Have received adjuvant or neo-adjuvant chemotherapy 

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Protocol Version 4 dated December 2, 2020 
 
2. Prior endocrine therapy (AI or tamoxifen) 
3. History of rheumatoid arthritis or other autoimmune arthritis 
4. Daily NSAID use (except daily aspirin use)  
5. Current use of daily corticosteroids or immunosuppressive therapies  
 
4.3 Patient identification 
 
Patients who have been consented and are undergoing study screening will be identified 
on study-related documentation and forms by their initials (first/middle and last name 
initials). All patients treated on this trial will be identified by their study initials and a unique 
study identification number. A unique study number will only be assigned to patients who 
meet the eligibility requirements and have completed the screening visit and are registered 
for treatment. The unique number will begin with the following prefix: AI. The prefix will be 
followed by the patient identification number beginning with # 001 in each cohort. These 
numbers will be issued to patients sequentially and no patient identification numbers will 
be re-assigned in the event that the subject withdraws from protocol treatment. 
 
4.4 Study exit  
 
Patients will exit the study upon completion of the last collection of blood for biomarkers 
and completing study questionnaires. Patients will not be followed further for purposes of 
this study. 
 
5. Study Procedures 
 
5.1 Pretreatment 
 
Patients will be consented and evaluated for participation based on the following 
procedures before being registered and starting trial: 
• History and physical exam (should be performed within 28 days of trial entry) 
• Eligibility as determined by inclusion and exclusion criteria 
 
5.2 Patient Registration 
 
Patients must be registered prior to initiation of treatment. Patients will be registered 
through a Breast Team Clinical Research Coordinator (CRC) from 8:00 a.m. to 5:00 p.m., 
Mountain Standard Time, Monday through Friday, (excluding holidays) 
 
Registration Guidelines 
 
Before a subject participates in the trial, the investigator or delegate is responsible for 
obtaining written informed consent after adequate explanation of the aims, methods, 
anticipated benefits, subject responsibilities and potential hazards of the study and before 
any protocol-specific screening or study medications are administered. All patients must 
be registered before the start of treatment.  
 
  
 
 

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Protocol Version 4 dated December 2, 2020 
 
5.3 Research Blood Draw 
 
Currently there are no blood based biomarkers evaluating AIMSS. We propose to 
prospectively collect blood for this pilot study for initial analysis of oxylipins.  
Fasting blood will be drawn at the pre-determined time points (figure 1) and sent to Dr. 
Martinez’s laboratory at the University of Arizona Cancer Center for further processing.  
 
5.4 Self-reported questionnaires 
 
The following questionnaires will be administered for the study to look for potential 
confounders like depression which can influence reporting of pain. The pain 
questionnaires will help us classify AIMSS in an objective way.  
 
Pain Assessment. Breast Cancer Prevention Trial–Musculoskeletal Symptom (BCPT-
MS) subscale and The Western Ontario and McMaster Osteoarthritis Index (WOMAC) 
have been validated as the most responsive tools for evaluating the development of 
AIMSS.62 These tools will be administered by the study nurse. 
BCPT-MS: Individuals with a subscale score of ≥1.5 on the BCPT-MS are considered to 
have developed AIMSS, and it is expected that 50% of our sample will develop 
AIMSS.62,63 Our primary study endpoints will be correlated to three items on the BCPT-
MS: general aches and pains, joint pain, and muscle stiffness.64,65 The BCPT-MS was 
derived from the original BCPT Symptom Check-list, a 18-item questionnaire validated in 
breast cancer survivors.66 The subscale score consists of the mean of responses to 
three questions addressing general aches and pains, joint pain, and muscle 
stiffness(questions 8,9,10 of the attached subscale). Scores range from 0–4, with higher 
scores representing worse symptoms. Changes in the BCPT-MS subscale have been 
shown to be the most responsive to changes in AIMSS with 54% of women developing 
AIMSS within 6 months.62   
WOMAC: The WOMAC is a 24-item instrument developed to assess pain, stiffness, and 
physical function in participants with hip and/or knee osteoarthritis.68 This instrument is 
well validated and has been modified from its original form, and used extensively to 
examine specific joint pain changes following treatment, including drug-based (NSAID) 
interventions for back pain, rheumatoid arthritis, and fibromyalgia.69-78  For our study, we 
will use the 5-point Likert format. 
Depression Questionnaire. It has been reported that up to 50% of newly diagnosed 
breast cancer patients have symptoms of depression or anxiety79 and perception of pain 
may be altered in individuals with symptoms of depression.80 Therefore, we will also 
administer the Patient Health Questionnaire (PHQ)-9 questionnaire, which is a validated 
multipurpose tool used for screening, diagnosing, monitoring, and measuring severity of 
depression.81 Additionally, the PHQ-9 has been successfully used in breast cancer 
patients to evaluate psychometric properties.82,83 
 
 
5.5 Toxicities to be monitored 
 
Monitoring for side effects from anastrozole will be done per their standard of care by 
patient’s treating physician.  
  

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Protocol Version 4 dated December 2, 2020 
 
5.6 Follow Up 
 
Patients will not be followed for this study after completion of the 6 mths blood draw, study 
questionnaires. However, they will continue to follow-up with their treating physician per 
standard of care. 
 
6. Criteria for Evaluation 
 
The primary endpoint is comparing baseline oxylipin levels for patients who develop 
AIMSS vs. those who do not develop AIMSS. For this pilot trial of 20 evaluable patients 
we expect 10 patients to develop AIMSS and 10 who do not develop AIMSS. The goal is 
to prospectively evaluate for any baseline levels in both groups and for changes in oxylipin 
panels.  
 
 
7. Statistical Considerations:  
 
This is a pilot study to estimate the values needed to design a larger study.  The primary 
objectives are to estimate the baseline oxylipin levels in patients who develop AIMSS vs. 
those who do not develop AIMSS.  Descriptive statistics and 95% confidence intervals 
will be computed for two baseline oxylipin levels (PGE2 and 12-HETE) for each group.  
Based on clinical observation, it is expected that 50% of the women will develop AIMSS.  
Ten patients in each group will allow us to estimate the 95% confidence interval with 
width 1.4 times the observed standard deviation.  Although we will not formally test for 
differences between the two groups, we will estimate the difference between the means 
with a 95% confidence interval, to inform the sample size justification for a larger study. 
 
 
The secondary objectives are to correlate changes in oxylipin levels with changes in pain 
scores.  These analyses will be based on all 20 women, irrespective of whether they 
develop AIMSS.    We expect that only a small number of women may switch to another 
AI due to the development of AIMSS.  For these women, we will use the values obtained 
at the time of the switch.  Linear regression will be used to estimate the change in pain 
scores (as measured by the BCPT-MS) versus changes in PGE2, 12-HETE.  
Additionally, we will control for changes in depression levels as measured by the PHQ-9. 
This model will allow us to estimate the effects of each of the oxylipin measures adjusted 
for depression in a single model.   
8.Data and Safety Monitoring Plan 
 
The University of Arizona Cancer Center Data and Safety Monitoring Board (DSMB) has 
determined that this study falls into the low risk category with regards to DSMB oversight 
or QA/QC Program monitoring, per the current University of Arizona Cancer Center Data 
and Safety Monitoring Board (DSMB) Charter 
 
This trial will undergo monitoring as indicated below: 
 
Identification of the DSMB obligated for oversight responsibilities: 
The University of Arizona Cancer Center Data and Safety Monitoring Board (DSMB) will 
provide ongoing oversight for this trial.  This study has been assigned a Low Risk level by 
the DSMB. 

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Identification of the entity obligated for routine monitoring duties: 
Routine monitoring will be provided by the Quality Assurance/Quality Control (QA/QC) 
Program to ensure that the investigation is conducted according to protocol design and 
regulatory requirements.  
 
This trial will also undergo real-time monitoring by the PI and study team, including 
documentation of real-time monitoring of any new or ongoing safety issues. PI will review 
with the study team on a monthly basis regarding any routine safety or adverse issues. 
Adverse events and serious adverse events will be dealt right away as per DSMB/IRB 
requirements. The study team will inform the PI via email regarding any AE’s and 
subsequent notification forms for the AE’s will be completed and filed.     
 
 
Monitoring progress and data review process: 
Routine monitoring of subject data will be conducted at least quarterly.   
The first routine monitoring visit will include at a minimum: 
• 
Informed consent – 50% of cases enrolled;  
• 
Subject eligibility – 10% of cases, up to two subjects; 
• 
Data review – 10% of cases, up to two subjects. 
 
All subsequent monitoring visits will consist of randomly selected subject cases based on 
current enrollment and include continuing review of previously selected cases, as 
applicable. 
 
A monitoring visit report and follow-up letter will be completed approximately two weeks 
after the routine monitoring visit; a copy will be maintained in the study file. The monitor 
will request additional source documentation, clarification, information, or corrections to 
the CRF and/or regulatory records from the Clinical Research Coordinator (CRC) or other 
applicable staff responsible for the study and resolution of queries/findings.  
Documentation of such a request will be maintained with a copy of the monitor’s visit report 
for follow-up at the next monitoring visit. Electronic records will be available in the 
institutional database or provided by the QA/QC Program staff.  
 
The Principal Investigator will ensure the accuracy, completeness, legibility and timeliness 
of the data reported in the Case Report Form (CRF), or other acceptable data formats. 
Source documentation supporting the study data should indicate the subject’s 
participation in the trial and should document the dates and details of study procedures, 
adverse events, and patient status.  
 
Case report forms, which include the inclusion/exclusion criteria form, adverse event forms 
and serious adverse event forms, etc. should be completed via the institution database or 
other acceptable data formats. Trials using paper CRFs will have the data entered with a 
black ball-point pen or typed. Corrections to the forms should not obscure the original entry 
and should be made by striking the incorrect information with a single line. Each strike 
should be accompanied by the initials of the corrector and the correction date. All subject 
forms and study files will be stored in a secure area limited to authorized staff. 
 
Note: Routine monitoring of regulatory documents will be conducted at least annually. 
 

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Protocol Version 4 dated December 2, 2020 
 
Process to implement study closure when significant risks or benefits are 
identified: 
 
This study may be prematurely terminated, if in the opinion of the principal investigator 
there is sufficient reasonable cause.   
Circumstances that may warrant termination include, but are not limited to: 
* 
Determination of unexpected, significant, or unacceptable risk to patients 
* 
Failure to enter patients at an acceptable rate 
* 
Insufficient adherence to protocol requirements 
* 
Insufficient complete and/or evaluable data                                                          
 
Description of adverse events and reporting procedures:  
This study evaluates changes in oxylipins when patients with breast cancer are treated 
with anastrozole. Side effects from anastrozole will be managed per standard of care and 
will not be captured as adverse events for the purpose of this study. Since the other study 
procedures are blood draws and questionnaires, we do not anticipate to have AE or SAEs.  
 
Plan for assuring data accuracy and protocol compliance: 
Routine study activity and safety information will be reported to the DSMB on a quarterly 
basis, or more frequently if requested. These reports will include: 
• 
Study activity, cumulative and for the period under review; 
• 
Safety (narrative description on non-serious and serious adverse events, 
protocol pre-determined early stopping rules for safety or treatment-emergent 
adverse events); 
• 
Predetermined protocol early stopping rules for efficacy/futility;  
• 
Status of study in relationship to stopping rules;   
• 
Current dose level of study agent;  
• 
Routine monitoring and protocol compliance (describe the monitoring process 
and identify the status of the monitoring); 
• 
Comments;  
• 
Attachments (AE data reviewed by the PI to compile the report, SAE letters 
and reports, results of any review(s), applicable correspondence with the IRB 
or other regulatory agencies 
 
Data, safety and study progress will be reported to: 
• 
Human Subjects Protection Program (IRB) at least annually; 
• 
Sponsor (if applicable) at least annually. 
 
Identification of the sponsor or funding agency, as applicable: 
The PI will immediately notify; in writing, the funding agency, if applicable, any action 
resulting in a temporary or permanent suspension of the study.  
A copy of this correspondence will also be forwarded to the DSMB and the SRC.  
 
9.Data Submission Schedule 
 
Data forms must be completed for all subjects registered to the study. 
Electronic case report forms will be completed in the OnCore system. 
 
 
 

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Protocol Version 4 dated December 2, 2020 
 
10.Ethics 
 
The trial will be conducted in accordance with the Declaration of Helsinki for biomedical 
research involving human subjects and local regulatory requirements.  
 
Ethical Principles  
 
This study will be conducted in accordance with Title 21 of the Code of Federal 
Regulations (CFR).  Specifically, this study will be conducted under a protocol reviewed 
by an Institutional Review Board; the study is to be conducted by scientifically and 
medically qualified persons; the benefits of the study are in proportion to the risks; the 
rights and welfare of the patients will be respected; the physicians conducting the study 
will ensure that the hazards do not outweigh the potential benefits; the results to be 
reported will be accurate; patients will give their informed consent and will be competent 
to do so and not under duress; and the study will comply with the ethical principles in Title 
21 of the CFR.  
 
Informed Consent  
 
This study will be conducted in full compliance with the informed consent regulations in 21 
CFR 50. The Sponsor-Investigator is responsible for ensuring that written informed 
consent from potential patients is obtained prior to performing any trial tests or 
assessments required by the protocol.  
 
A copy of the fully executed informed consent form (PHI authorization form and ancillary 
consent forms if applicable), is given to the subject. One copy is placed in the subject’s 
medical record, another copy is placed in the research chart, and the originals are filed by 
the protocol number in room 2111 at UACC North Campus. 
 
Institutional Review Board  
 
This study will be conducted in full compliance with the Institutional Review Board (IRB) 
regulations in 21 CFR 56, in accordance with the Declaration of Helsinki. This protocol will 
not be initiated unless it and the informed consent form have been reviewed and approved 
by, and remains open to continuing review by, an IRB meeting the requirements of 21 
CFR 56. The IRB shall review and have the authority to approve, require modification in 
(to secure approval), or disapprove the protocol. The IRB shall notify the Investigator and 
the institution in writing of its decision. The IRB shall require that the information given to 
patients as part of the informed consent is in accordance with 21 CFR 50.25. The IRB 
shall conduct continuing reviews of the protocol at intervals appropriate to the degree of 
risk, but not less than once per year. At the completion or early termination of the trial, a 
final report should be sent to the IRB by the Investigator. The Investigator is obligated to 
maintain an IRB correspondence file.  
 
Confidentiality of Patient Data 
 
The investigator must ensure that patient confidentiality will be maintained. Patients will 
be identified by initials and a protocol-assigned patient number as described in section 
4.5. Permission for direct access to patient data will be sought in writing for the patient by 
the investigator as part of the informed consent procedure. The patient will be informed 

17 
Protocol Version 4 dated December 2, 2020 
 
that all clinical information is confidential, but that the IRB, and regulatory authorities may 
inspect these records. 
 
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2006;107(12):2924-2931. 
 
 
 

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12. Appendix 
 
12.1 BCPT-MS 
 
 
 
 

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25 
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12.2  WOMAC 
 
 
 
 

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Protocol Version 4 dated December 2, 2020 
 
12.3  PHQ-9 
 
 
 
 

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13. Protocol Signature Page 
 
 
Investigator Agreement  
 
STUDY TITLE: Pilot Trial to Evaluate Blood and Imaging Based Biomarkers 
for Aromatase Inhibitor Induced Musculoskeletal Syndrome 
 
By signing below I agree:  
 
1) That my staff and I have read, understand and will adhere to the protocol as written, 
and that any changes to the protocol will be agreed to and approved by the Principal 
Investigator and the Institutional Review Board (IRB) 
 
2) To abide by all obligations stated on the FDA Form 1572 and other documents 
required by regulation;  
 
3) To conduct this study in accordance with the current International Conference on 
Harmonization (ICH) guidance, the Good Clinical Practices (GCP) guidance, the 
Declaration of Helsinki, US FDA regulations and local IRB and legal requirements;  
 
4) To obtain IRB approval of the protocol, any amendments to the protocol, and periodic 
re-approval as required, and to keep the IRB informed of adverse events as required by 
their guidelines report the status of the study to them;  
 
5) To ensure that each individual enrolled into the trial, or legally authorized 
representative, has read, understands, and has signed the Informed Consent form;  
 
6) To ensure that I and all persons assisting me with the study are adequately informed 
and trained about the study and the possible adverse events associated with the study 
required medication 
 
7) To make prompt reports of SAEs and deaths to the FDA according to the regulations;  
 
8) To prepare and maintain adequate and accurate case histories to document all 
observations and other data pertinent to the study for each individual enrolled in the 
clinical trial.  
 
 
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Investigator Signature  
 
 
 
 
 
Date  
 
 
Investigator Name (Print) 
 
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