Cystic Fibrosis Related Bone Disease: the Role of CFTR
Conditions: Cystic Fibrosis Related Bone Disease
Sponsor: Massachusetts General Hospital

CYSTIC FIBROSIS RELATED BONE DISEASE: THE ROLE OF CFTR 
PI: Melissa Putman MD 
8/28/2015, Version 8 
 
1. BACKGROUND AND RATIONALE 
 
1.1 Cystic Fibrosis Related Bone Disease 
Over the past several decades, life expectancy for patients with CF has increased 
significantly, from an average of 10 years of age in 1962 to more than 37 years of age in 
2009. As patients live longer, other co-morbidities related to CF have become more 
prevalent, including CF-related bone disease.1 The pathogenesis of a low BMD and 
increased fracture risk in patients with CF is currently not well understood.  
 
Multiple clinical studies have reported that BMD measured by DXA is low in patients 
with CF.2-5 For example, in a recent meta-analysis, the prevalence of T-scores between -1 
and -2.5 in patients aged 18-32 years old with CF was 38%, and the prevalence of T-
scores less than -2.5 was 23.5%, whereas the expected prevalence would be 
approximately 18% and 2.5% for people of this age.6  In addition, bone turnover may be 
uncoupled in patients with CF with elevated bone resorption and low bone formation.7,8 
 
Fracture rates in patients with CF are higher than in the healthy population.9-12  In the 
previously mentioned meta-analysis, the prevalence of vertebral and non-vertebral 
fractures in adults with CF was 14% and 19.7%, respectively.6  One study found that in 
70 patients covering 1410 patient-years of analysis, fracture rates  were twice as high in 
adolescents and young adults with CF compared to the general population.9  In addition, 
kyphosis is also commonly seen in adolescents and young adults with CF.11,13,14  
Fractures and kyphosis in patients with CF can have severe repercussions. For example, 
rib and vertebral fractures can lead to pain, chest wall deformities, reduced lung volumes, 
ineffective cough and airway clearance, and ultimately compromised lung function. In 
addition, severe bone disease may obviate lung transplantation.  Due to this high 
morbidity, CF related bone disease can significantly affect the health, well-being, and 
longevity of these patients. 
 
CF patients have multiple risk factors for low bone density, including vitamin D 
deficiency, malabsorption, physical inactivity, malnutrition with low body weight, 
glucocorticoid use, diabetes, hormonal deficiencies, delayed puberty, inflammation, and 
failure to achieve peak bone mass. 1,2,7,15-17  In addition to these acquired factors, recent 
studies have also suggested that the CF genetic mutation in itself may play a causative 
role in low bone density and fracture seen in this population.   
 
1.2 Potential Role of CFTR in CF Related Bone Disease 
Cystic fibrosis is caused by a mutation in the cftr gene, which encodes the CF 
transmembrane conductance regulator (CFTR).  CFTR traditionally serves as a chloride 
channel in epithelial cells and plays an important role in maintaining hydration required 
for mucillary clearance in pulmonary and intestinal tissue. Interestingly, recent evidence 
in mouse models suggests that CFTR is also present in bone cells and may play a role in 
bone formation and resorption. Knockout mice for cftr (cftr-/cftr-) have significantly 
decreased bone density and decreased bone formation rates compared to normal mice, 
even in the setting of normal lung and gastrointestinal function.18-20  In addition, human 

CYSTIC FIBROSIS RELATED BONE DISEASE: THE ROLE OF CFTR 
PI: Melissa Putman MD 
8/28/2015, Version 8 
osteoblasts in culture have been shown to express CFTR.21  Early data suggest that 
inhibiting CFTR expression in human osteoblasts may decrease osteoproteregin and 
prostaglandin E2, two important signaling molecules involved in bone formation and 
resorption. 22 This potential role of CFTR in bone resorption and formation is consistent 
with clinical studies in humans that have shown abnormalities in bone turnover markers 
and bone histomorphometric studies that reflect a decrease in bone formation and an 
increase in bone resorption.8,23 
 
1.3 VX-770 
VX-770 is a new medication that pharmacologically potentiates CFTR activity in patients 
with at least one copy of the G551D-CFTR mutation. Targeting CFTR systemically, VX-
770 not only improved pulmonary function in these patients during phase 2 and 3 clinical 
trials, it also resulted in extrapulmonary improvements.24,25 VX-770 was FDA approved 
in January 2012. 
 
Given the possible role of CFTR in bone cells, VX-770 may also have a direct benefit on 
bone density and strength, but this has not yet been studied. This medication offers a 
unique opportunity to study the role of CFTR in human bone by assessing effects of 
pharmacologically increasing CFTR activity in subjects with G551D-CFTR mutations on 
bone micro-architecture and bone turnover markers.  Because the effects of VX-770 on 
bone are of unknown magnitude, new imaging modalities like HR-pQCT that are highly 
reproducible and capable of detecting small changes are needed to be capable of 
detecting changes in bone that might otherwise be missed with standard imaging 
techniques such as DXA and pQCT.  
 
1.4 Limitations of DXA and Standard pQCT 
Several modalities are currently available for the clinical measurement of bone density, 
including dual-energy x-ray absorptiometry (DXA) and peripheral quantitative computed 
tomography (pQCT). Each of these modalities possess inherent limitations in the 
evaluation of CF-related bone disease due to unique factors complicating their 
interpretation. 
 
At the present time, DXA is the primary method for measuring BMD and assessing 
fracture risk in both children and adults.  DXA expresses bone mineral content (in grams) 
per unit area (cm2) so that results are expressed as g/cm2. Because DXA values are 
expressed per unit area rather than per unit volume, DXA does not truly give a measure 
of bone mineral density (which would be expressed as g/cm3); rather, it gives values for 
“areal” or two-dimensional bone density. As such, DXA values are influenced by bone 
size, overestimating BMD for large bones and underestimating BMD for small bones, as 
are seen in many patients with CF. This artifact does not occur with techniques that 
provide volumetric (i.e., three-dimensional) measurements of BMD. Moreover, DXA 
cannot distinguish between trabecular and cortical bone.26,27 Most importantly, some data 
suggest that DXA may not predict fracture risk accurately in patients with CF, such that 
they have increased risk of fracture even with normal DXA BMD measurements.10 
 

CYSTIC FIBROSIS RELATED BONE DISEASE: THE ROLE OF CFTR 
PI: Melissa Putman MD 
8/28/2015, Version 8 
pQCT is  a modality that has been used as a method of evaluating BMD and some aspects 
of bone structure.   Unlike DXA, pQCT assesses true volumetric bone density and it may 
be able to distinguish cortical and trabecular bone compartments. In adolescents and 
young adults with CF, one study using pQCT reported that cortical thickness of the radius 
was reduced in both men and women.28 However, the radiation exposure from a QCT 
exam is about 100 fold higher than DXA, limiting its utility in children, particularly if 
serial measurements are planned.  Additionally, the resolution of pQCT is 0.4 mm, which 
is not ideal for assessing small differences in cortical thickness and does not allow for 
assessment of other bone micro-architecture features including cortical porosity.  
 
1.5  High Resolution Peripheral Quantitative Computed Tomography (HR-pQCT) 
HR-pQCT is a new imaging modality that has a resolution (voxel size) of 82 um, which 
allows for the assessment of the microstructure of bone (e.g., cortical thickness, 
trabecular number and thickness, trabecular spacing, and cortical porosity) in three 
dimensions and has the potential to provide a more accurate estimation of bone strength 
using microfinite element analyses. In addition, it measures true volumetric bone density, 
rather than areal bone density, of the total bone segment as well as the individual cortical 
and trabecular compartments. 29-31 HR-pQCT is highly reproducible and appears to give 
unique information that adds to the information provided by DXA.  Furthermore, HR-
pQCT is not limited by projection artifacts caused by bone size and is not affected by the 
amount or distribution of adipose tissue as can occur with DXA measurements. In 
addition, HR-pQCT also offers the advantage over standard pQCT of smaller voxel size 
(82um vs. 0.4mm), lower radiation dose, and faster scanning time. HR-pQCT is currently 
being studied as an alternative method of measuring bone density, predicting fracture, 
and assessing response to medications used to treat osteoporosis. Presently, studies using 
HR-pQCT in various disease populations, including type 2 diabetes and chronic kidney 
disease, have found significant differences in bone micro-architecture that were not 
captured by DXA BMD measurements.32-36 Because radiation exposure is very low, HR-
pQCT can be used for longitudinal studies to assess changes in volumetric bone density 
and bone micro-architecture over time.37-39 
 
1.6 Current Proposal 
We propose a prospective multiple cohort study with the goal of evaluating the skeletal 
effects of improving CFTR function in humans with CF. Subjects with at least one copy 
of the G551D mutation starting VX-770 will be evaluated using HR-pQCT at baseline 
and 12 and 24 months after initiating this medication and then compared to CF patients 
not receiving this medication as well as healthy subjects matched for age and gender.  
This study is highly innovative. Using cutting edge imaging techniques, we will assess 
the use of bone micro-architectural changes as a novel outcome in the assessment of 
medical interventions in CF patients. In addition, this study will provide important 
information regarding the pathogenesis of cystic fibrosis related bone disease and the 
underlying role of CFTR, and will help guide future management and prevention of this 
disease. These data can also serve as supportive evidence for future studies assessing the 
effects of VX-770, as well as to validate another outcome measure studying future CFTR 
modulators. 
 

CYSTIC FIBROSIS RELATED BONE DISEASE: THE ROLE OF CFTR 
PI: Melissa Putman MD 
8/28/2015, Version 8 
1.7 Preliminary Studies 
To learn more about the underlying alterations in bone micro-architecture in CF patients, 
we are currently recruiting subjects with CF for a cross sectional study at MGH (protocol 
2011P000893, SPID 978). The goal of this study is to use HR-pQCT to describe the 
micro-architectural basis for the differences in BMD in patients with cystic fibrosis, 
which will expand our understanding of the pathogenesis of the reduced bone mass seen 
in patients with this disease.  Fifty subjects (25 male and 25 female) with CF between the 
ages of 18-30 years are being recruited through the Children’s Hospital Boston (CHB) 
and the Massachusetts General Hospital (MGH) Cystic Fibrosis Centers to enroll in this 
cross-sectional study. Subjects undergo a laboratory evaluation and complete health, 
medication, and nutrition surveys at the MGH Clinical Research Center (CRC), followed 
by dual energy x-ray absorptiometry (DXA) and HRp-QCT scans at the MGH Bone 
Density Center. Five patients have currently completed this study. Once completed, data 
will be compared to healthy controls matched for age and gender. 
 
 
2. SPECIFIC AIM AND HYPOTHESES 
 
Specific Aim 1: To assess the change in bone micro-architecture, bone strength, and 
bone turnover markers over time in CF subjects treated with VX-770 (ivacaftor) 
compared to CF subjects not receiving this medication. 
Primary Hypothesis: Total, cortical, and trabecular volumetric densities, as well as 
overall bone strength (estimated failure load) will be higher in subjects in subjects treated 
with VX-770 than in those who are not receiving this medication.   
Secondary Hypothesis: Markers of bone formation will increase and markers of bone 
resorption will decrease in subjects treated with VX-770 medication compared to those 
who are not receiving this medication. 
 
Specific Aim 2: To assess the change in bone micro-architecture, bone strength, and 
bone turnover markers over time in CF subjects not treated with VX-770 compared to 
healthy subjects. 
Primary Hypothesis: Total, cortical, and trabecular volumetric densities, as well as 
overall bone strength (estimated failure load) will be lower in CF subjects and will 
decline over time compared to healthy subjects. 
Secondary Hypothesis: Markers of bone formation will be lower and markers of bone 
resorption will be higher in CF subjects compared to healthy subjects. 
 
3.  SUBJECT SELECTION 
 
3.1 Study population:  
Three subject cohorts will be recruited as follows: 
 
Cohort 1:  
30 subjects ages 6 – 75 years will be recruited in accordance with the written guidelines 
of the Children’s Hospital Boston and Massachusetts General Hospital Instritutional 
Review Boards.  Subjects will be required to meet the following criteria: 

CYSTIC FIBROSIS RELATED BONE DISEASE: THE ROLE OF CFTR 
PI: Melissa Putman MD 
8/28/2015, Version 8 
Inclusion criteria: 
 Established diagnosis of CF with at least one abnormal G551D-CFTR allele 
 Eligibility for and intent to start treatment with VX-770 or started treatment with VX-
770 within previous 6 months  
Exclusion criteria: 
 Psychiatric or mental incapacity that would preclude subject from assenting to study 
participation 
 Current pregnancy 
 History of organ transplantation 
 History of Burkholderia dolosa infection 
 
Of note, if subjects initially are enrolled as a part of Cohort 1 but then do not start 
ivacaftor for any reason, then they can remain in the study and will be moved from 
Cohort 1 to Cohort 2. 
 
Cohort 2:  
30 subjects will be recruited in accordance with the written guidelines of the Children’s 
Hospital Boston and Massachusetts General Hospital Institutional Review Boards. 
Subjects will be grouped by gender, age and race to match subjects in Cohort 1 within 
two years. Pubertal subjects will be matched by Tanner stage instead of age. Subjects will 
be required to meet the following criteria: 
Inclusion criteria: 
 Established diagnosis of CF 
Exclusion criteria: 
 Psychiatric or mental incapacity that would preclude subject from assenting to study 
participation 
 Current pregnancy 
 History of organ transplantation 
 History of Burkholderia dolosa infection 
 
Cohort 3: 
30 subjects will be recruited in accordance with the written guidelines of the Children’s 
Hospital Boston and Massachusetts General Hospital Institutional Review Boards. 
Subjects will be grouped by gender, age and race to match subjects in Cohort 2 within 
two years. Pubertal subjects will be matched by Tanner stage instead of age. Subjects will 
be required to meet the following criteria: 
Inclusion criteria: 
 Clinically stable, deemed able to complete the screening, baseline, and scheduled 
study visits. 
Exclusion criteria: 
 History of significant cardiac, renal, pulmonary, hepatic, or malignant disease, current 
alcohol or illicit drug abuse, or major psychiatric disorder 
 Current diagnoses known to affect bone metabolism, including cystic fibrosis, 
osteoporosis, amenorrhea >3 months (in menstruating women who are not taking oral 
contraceptives or have an IUD), hyperthyroidism, diabetes, hyperparathyroidism, 

CYSTIC FIBROSIS RELATED BONE DISEASE: THE ROLE OF CFTR 
PI: Melissa Putman MD 
8/28/2015, Version 8 
Paget’s disease, kidney stones, chronic inflammatory diseases, malabsorptive 
disorders, malnutrition, prolonged immobility, and skeletal dysplasias 
 History of a non-digital fracture in the previous 6 months, history of one pathologic 
fracture, or greater than four total lifetime non-digital fractures 
 Cumulative lifetime use of oral glucocorticoids for greater than 2 months 
 Current or prior use of medications known to affect bone metabolism including 
hormone replacement therapy, anti-estrogens, bisphosphonates, calcitonin, fluoride, 
lithium, suppressive doses of levothyroxine, or anticonvulsants. 
 Pregnancy 
 BMI less than 18.5 or greater than 30 kg/m2 in subjects 18 years and older, or BMI 
less than 5th or greater than 95th percentile in subjects under the age of 18 years. 
 Any medical or psychiatric condition or situation that would compromise subject 
safety, informed consent/assent, treatment compliance, follow-up measurements, or 
data quality 
 
3.2 Recruitment Strategy 
Subjects with CF will be recruited from the Cystic Fibrosis Centers at Children’s 
Hospital Boston and Massachusetts General Hospital. A brochure describing the study 
will be made available in each of the clinics. Potential subjects who have previously 
given their consent in writing to the CF Program granting permission to be contacted 
regarding research studies will be contacted by phone or email according to their 
previously indicated preference. In addition, potential subjects will be asked by their 
pulmonologist, pediatrician, or other health care provider during their regularly scheduled 
clinic visits if they are interested in participating in a research project. If they agree, then 
they will be approached by members of the research team either at the appointment or 
contacted by phone/email according to their preference. Potential subjects who are 
interested will be forwarded an informed consent form to review and will be invited to 
schedule a study visit. 
 
Healthy subjects will be recruited from the community using flyers, postings, and 
advertisements using IRB-approved materials. Phone screening will be used to determine 
inclusion/exclusion criteria for potential subjects. 
 
Subjects will receive $50 and reimbursement for transportation upon completion of each 
study visit. 
 
A subset of subjects who have completed the study visit of current Protocol 
2011P000893 (“Bone strength and micro-architecture in young adults with cystic 
fibrosis”) will be eligible for participation in this proposed study. The study procedures 
for the study visit of Protocol 2011P000893 are identical to the baseline visit of the 
current proposed study. Therefore, if subjects in Protocol 2011P000893 are interested in 
also participating in the current proposed study, then their completed study visit will take 
the place of the baseline visit of this proposed protocol, and they will subsequently be re-
consented for enrollment in the proposed study at Follow Up Visit 1 (12 months after the 
date of their protocol 2011P000893 study visit, or 12 months after starting VX-770 in 

CYSTIC FIBROSIS RELATED BONE DISEASE: THE ROLE OF CFTR 
PI: Melissa Putman MD 
8/28/2015, Version 8 
those in cohort 1). This will avoid any unnecessary radiation exposure and blood draws to 
subjects who desire to participate in both studies. 
 
 
4.  SUBJECT ENROLLMENT 
 
4.1 Methods of enrollment 
Potential subjects identified above will be scheduled for a research visit at the MGH CRC, 
at which time informed consent and assent will be obtained and subjects enrolled in the 
study.  
 
4.2 Informed consent 
Written informed consent will be obtained using a consent form that has been approved by 
the Partners Institutional Review Board before subjects undergo any study procedures other 
than asking simple questions to determine potential eligibility and interest in the study.  
Informed consent will be obtained by a licensed physician with the subject and parent 
and/or guardian as necessary for subjects under the age of 18 years.  All subjects will be 
informed regarding the purpose of the research, the details of the study protocol, risks and 
benefits, alternatives to participation, costs, reimbursements, their right to privacy and 
confidentiality, their right to refuse to participate or withdraw from the study at any time, 
their rights in the event of a study-induced injury, and whom to contact for questions about 
the study.  Subjects will be given a copy of their signed consent form and an additional 
copy will be kept in our research files. Children and adolescents under the age of 18 years 
will be asked to sign an assent form and their guardian will sign the consent form.  
 
4.3 Treatment assignment and randomization 
N/A 
 
 
5.  STUDY PROCEDURES 
 
5.1 Protocol 
Subjects will be seen at the MGH General Clinical Research Center (GCRC) for baseline 
visit and 12- and 24-month follow-up visits. 
 
Baseline visit: 
Baseline visit will be arranged within 6 months of initiation or anticipated initiation of VX-
770 in Cohort 1.  After obtaining informed consent, a survey will be administered to obtain 
information regarding medical history, hospitalizations in the previous 12 months, 
medication use over the previous 12 months, history of fractures, physical activity, and 
sunlight exposure. Most recent pulmonary function test results will be obtained from the 
medical record. Urine hCG will be performed for female subjects. Blood and urine will be 
collected for the tests listed below.  Subjects will then be escorted to the MGH Bone 
Densitometry Center, where they will undergo DXA measurements of the PA and lateral 
spine, hip, radius, and total body and HR-pQCT measurements of the non-dominant distal 
radius and tibia.  

CYSTIC FIBROSIS RELATED BONE DISEASE: THE ROLE OF CFTR 
PI: Melissa Putman MD 
8/28/2015, Version 8 
1. Blood Studies 
a. Routine chemistry panel (including calcium, phosphate, magnesium, 
albumin, BUN, and creatinine) 
b. C-reactive protein (CRP) 
c. Parathyroid hormone (PTH) 
d. Insulin-like growth factor-1 (IGF-1) 
e. 25-hydroxyvitamin D [25(OH)D] 
f. Osteocalcin (OC) 
g. Bone-specific alkaline phosphatase (BSAP) 
h. C-telopeptides of type 1 collagen (CTX) 
i. Total testosterone (males only) 
j. Sex hormone binding globulin (SHBG), males only 
One extra tube of blood will be collected and stored in 0.5 mL aliquots in case 
any tests need to be repeated and for measurement of additional analytes of 
interest in the future.  
In subjects less than 18 years of age, no more than 3 cc/kg body weight of 
blood will be drawn per 8 week period. 
2. Urine studies 
a. N-telopeptides of type 1 collagen (NTX) 
b. Urine HCG (females only) 
3. Bone density and body composition by dual-energy x-ray absorptiometry (DXA) 
a. Whole lumbar vertebrae (DXA in the PA projection) 
b. Lumbar vertebral bodies (DXA in the lateral projection), subjects ≥18y only 
c. Proximal femur (femoral neck and total hip) 
d. Forearm (distal radius) 
e. Total body BMD 
f. Vertebral facture assessment (VFA) in adults ≥18y only 
g. Fat and lean mass 
4. Bone micro-architecture by HR-pQCT (see below) 
a. Distal radius 
i. Standard site in adults 
ii. 7% site in children and adolescents <17y 
b. Tibia 
i. Standard site in adults 
ii. 8% site in children and adolescents <17y 
5. Anthropometric measures (weight, height, BMI) 
6. Physical Exam (pubertal children only) for Tanner staging 
7. Medical history (from survey and/or review of patient medical chart) 
a. Fracture history (with X-ray confirmation if available) 
b. Hospitalizations in the previous 12 months 
c. Medication use in the previous 12 months 
d. Vitamin and other supplement use in the previous 12 months 
e. Lifetime oral glucocorticoid use 
f. Growth chart 
g. Sun exposure (using standardized scale) 40 
h. Self identified race/ethnicity 

CYSTIC FIBROSIS RELATED BONE DISEASE: THE ROLE OF CFTR 
PI: Melissa Putman MD 
8/28/2015, Version 8 
i. Pubertal development 
j. Menstrual history (in females) 
k. Dietary history including vitamin D intake (24 hour food records) 
l. Physical activity survey 41 
m. Most recent pulmonary function tests (FEV1) within the preceding 4-8 
weeks in Cohorts 1 and 2 
n. Results from previous genetic testing (if performed) regarding CFTR 
mutation type in Cohorts 1 and 2 
 
Interventions 
This is a non-interventional study. All pulmonary and endocrine clinical care will 
continue as per the discretion of subjects’ primary clinical providers. VX-770 will be 
prescribed and managed by their primary pulmonologist.  
 
Follow up Visits 
Follow-up visits will take place 12 and 24 months after the baseline visit. This visit will 
include similar procedures and surveys as described in baseline visit, obtaining pertinent 
data over the previous 12 months. Because bone density changes happen very slowly and 
in order to accommodate subjects’ schedules (including their medical care needs, school, 
relocation to other parts of the country, and other issues), study visits will be scheduled at 
12 and 24 months with a window of +/- 3 months so as not to be overly prohibitive or 
coercive with scheduling. Data analysis will take into account length of time between 
follow up visits.  
1. Blood Studies 
a. Routine chemistry panel (including calcium, phosphate, magnesium, 
albumin, BUN, and creatinine) 
b. CRP 
c. PTH 
d. IGF-1 
e. 25(OH)D 
f. OC 
g. BSAP 
h. CTX 
i. Total testosterone (males only) 
j. SHBG (males only) 
One extra tube of blood will be collected and stored in 0.5 mL aliquots in case 
any tests need to be repeated and for measurement of additional analytes of 
interest in the future.    
In subjects less than 18 years of age, no more than 3 cc/kg body weight of 
blood will be drawn per 8 week period. 
2. Urine studies 
a. NTX 
b. Urine HCG (females only) 
3. Bone density and body composition by dual-energy x-ray absorptiometry (DXA) 
a. Whole lumbar vertebrae (DXA in the PA projection) 
b. Lumbar vertebral bodies (DXA in the lateral projection), subjects ≥18y only 

CYSTIC FIBROSIS RELATED BONE DISEASE: THE ROLE OF CFTR 
PI: Melissa Putman MD 
8/28/2015, Version 8 
c. Proximal femur (femoral neck and total hip) 
d. Forearm (distal radius) 
e. Total body BMD 
f. Vertebral facture assessment (VFA) in adults >17 only (at 24 month follow 
up visit only) 
g. Fat and lean mass 
4. Bone micro-architecture by HR-pQCT (see below) 
a. Distal radius, using same site as baseline visit 
b. Tibia, using same site as baseline visit 
5. Anthropometric measures (weight, height, BMI) 
6. Physical Exam (pubertal children only) for Tanner staging 
7. Medical history (from survey and/or review of patient medical chart) over past 12 
months 
a. Fracture history in previous 12 months (with X-ray confirmation if 
available) 
b. Hospitalizations in the previous 12 months 
c. Medication use, including glucocorticoids, in the previous 12 months 
d. Vitamin and other supplement use in the previous 12 months 
e. Growth chart 
f. Sun exposure (using standardized scale) 40 
g. Pubertal development 
h. Menstrual history (in females) 
i. Dietary history including vitamin D intake (24 hour food records) 
j. Physical activity survey 41 
k. Most recent pulmonary function tests (FEV1) within the preceding 4-8 
weeks in Cohorts 1 and 2 
l. Self report of compliance with VX-770 (Cohort 1 only) 
 
 
Subjects in cohorts 1 and 2 and/or their insurance companies will not be billed for any 
procedure that is not standard of care for patient with CF. Subjects in cohort 3 and/or 
their insurance companies will not be billed for any study procedures. 
 
 
Study Procedure Schematic 
 
 
StudyVisit 1 
StudyVisit 2 
Study Visit 3 
Month 
0 
12 
24 
Consent 
X 
X  
(in participants 
of protocol 
2011P000893 
only) 
 
Medical history, Tanner staging (pubertal children 
only) 
X 
X 
X 
Fracture history, most recent FEV1, medication use, 
physical activity survey 
X 
X 
X 

CYSTIC FIBROSIS RELATED BONE DISEASE: THE ROLE OF CFTR 
PI: Melissa Putman MD 
8/28/2015, Version 8 
Height, weight 
X 
X 
X 
Ca and vitamin D intake questionnaire 
X 
X 
X 
Labs: 25OHD, Ca, alb, creatinine, Mg, PTH, IGF-1, 
CRP, testosterone+SHBG (males), bone turnover 
markers, HCG (females) 
X 
X 
X 
 
 
 
 
DXA scan (total body, hip, spine, wrist)  
X 
X 
X 
DXA VFA (subjects ≥ 18 years only) 
X 
 
X 
HR-pQCT radius and tibia 
X 
X 
X 
 
 
5.1.1 Additional information on high resolution peripheral computed tomography 
(Xtreme CT)  
XtremeCT (Scanco Medical AG, Basserdorf, Switzerland) is a new technology capable 
of assessing volumetric bone density as well as trabecular and cortical micro-architecture 
at the distal radius and distal tibia, with a nominal isotropic voxel size of 82 µm. The 
XtremeCT system (Figure 1) employs a two-dimensional detector array in combination 
with a small-angle cone beam X-ray source (0.07 mm spot focal size), enabling the 
simultaneous acquisition of a stack of parallel CT slices with a nominal resolution (voxel 
size) of 82 µm (100 µm at 10% MTF). The X-ray tube employs an effective energy of 60 
kVp, with a current of 95 mAs.  The field of view is 130 mm and maximum scan length 
is 150 mm. The short-term reproducibility of measurements with this device is excellent, 
with precision errors of 0.7 to 1.5% for total, trabecular and cortical bone densities and 
2.5 to 4.4% for trabecular architecture measures.  
 
Scan acquisition: During scan acquisition, the arm or leg of the patient is immobilized in an 
anatomically formed carbon fibre shell. An antero-posterior scout view is used to define the 
measurement region. At each skeletal site, 110 CT slices are acquired, thus delivering a 3D 
representation of approximately 9 mm in the axial direction. In standard analyses, a 
reference line is manually placed at the endplate of the radius and tibia, as shown in Figure 
2 (middle and right). The first CT slice is acquired 9.5 mm and 22.5 mm proximal to the 
reference line for the distal radius and distal tibia, respectively (Figure 2). In subjects under 
17 years of age, the reference line will be set at the 8% site at the radius and 7% site at the 
tibia in order to avoid radiation to the growth plate, based on previously published data 
using Xtreme CT in children and adolescents.42-43 
Figure 2. (left) Photo of XtremeCT clinical imaging device, for assessment of trabecular and cortical bone 
microarchitecture at the distal radius and distal tibia.  Image courtesy of Scanco Medical, AG. The middle and right 
images are standard antero-posterior scout views of the distal tibia (middle) and distal radius (right), showing the 
placement of a reference line (solid line) and the volume of interest where CT slices are acquired (between dashed 
lines).  

CYSTIC FIBROSIS RELATED BONE DISEASE: THE ROLE OF CFTR 
PI: Melissa Putman MD 
8/28/2015, Version 8 
 
The following data will be obtained at both sites: total bone area, cortical area, trabecular 
area, total volumetric bone density, cortical volumetric bone density, cortical thickness, 
trabecular volumetric bone density, trabecular bone volume fraction (BV/TV), trabecular 
number, trabecular separation, trabecular thickness, cortical porosity, and estimated bone 
strength (calculated microfinite element). 
 
 
6. BIOSTATISTICAL ANALYSIS 
6.1 Study Outcomes/Endpoints 
The primary outcome of specific aim 1 will be the difference in the mean change in 
cortical volumetric density of the radius and tibia as measured by HR-pQCT over 24 
months between cohorts 1 and 2. The primary outcome of specific aim 2 will be the 
difference in the mean change in cortical volumetric density of the radius and tibia as 
measured by HR-pQCT over 24 months between cohorts 2 and 3.  Secondary endpoints 
will include change in total and trabecular volumetric density, as well as other HR-pQCT 
parameters (trabecular number, trabecular spacing, cortical porosity, and microfinite 
element of the radius and tibia), DXA values (BMD of spine, hip, radius, and total body), 
and bone turnover markers. 
 
6.2 Statistical Methods 
Statistical analysis will include descriptive statistics, with determination of means and SD 
of change in each primary and secondary outcome variable from baseline to 12 and 24 
months for all three cohorts. For specific aim 1, mean change in primary outcome in 
cohorts 1 and 2 will be compared with independent-sample t-tests (or Wilcoxon rank sum 
if non-normality is detected). For specific aim 2, mean change in primary outcome in 
cohorts 1 and 2 will be compared with independent-sample t-tests (or Wilcoxon rank sum 
if non-normality is detected). Longitudinal linear mixed models will be applied to 
evaluate longitudinal changes in BMD, bone microarchitectural parameters, and bone 
turnover markers over the 2-year period in each group. Length of time between follow up 
visits will be accounted for in statistical analyses. Statistical analysis will be performed in 
consultation with a study statistician.  
 
6.3 Sample Size Considerations:  
Sample-size calculations were performed after consultation with our biostatistician using 
data from 103 healthy adult controls previously scanned using HR-pQCT at the MGH 
Bone Density Center as well as previously published reproducibility data.31 A sample 
size of Cohort 1 n=30 and Cohort 2 n=30 would provide 80% power (Type II error = 0.2) 
to detect an effect on the primary outcome variable (mean change in cortical volumetric 
density of the radius and tibia over 24 months) of 2.3-3.1% using a pre-post correlation of 
0.7. Based on numbers of CF patients both with and without G551D mutations treated at 
the MGH and CHB CF Centers, we will have adequate access to subjects to meet this 
sample size requirement. 

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7.  Potential pitfalls 
Although a randomized control trial would be the ideal study design to evaluate the 
effects of VX-770 on bone, we believe it would be unethical to withhold this medication 
from subjects who qualify for it given its previously established efficacy and the nature of 
this ultimately fatal disease. Therefore, we propose a prospective multiple cohort study, 
using subjects with CF who are not eligible for this medication (i.e. have CF mutations 
other than G551D) and healthy subjects without CF.  Although this will necessitate 
inherent genetic differences between CF cohorts, there are no animal studies that confirm 
phenotypic bone differences between G551D in particular and other genotypes. One 
study by King et al suggested that adult CF patients with DF508 mutations have lower 
bone density than those without this mutation, though this was limited by its cross 
sectional design and did not specifically compare subjects with G551D mutations. 
Further studies on genotype-phenotype bone differences are required. 
 
Because of this study design, confounders may exist between cohorts. In addition, if lung 
and other organ function improve with VX-770, this may independently affect bone 
density apart from the direct effects of the drug on bone. Therefore, we will plan to 
collect data regarding physical activity, sun exposure, medication use (including 
glucocorticoids), gonadal function (menstrual history in women, morning testosterone 
levels in men), inflammatory markers, PTH/vitamin D, BMI, and fat/lean body mass by 
DXA. We will adjust for potential confounders that are significantly difference between 
cohorts in our multivariate analysis. 
 

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Data have not confirmed that VX-770 will affect CFTR function in bone cells to the 
extent that it affects epithelial cells, or that it is absorbed in bone to an appreciable effect. 
To test this hypothesis, we have proposed a separate study to add bone turnover makers 
to saved sera from the previous VX-770 phase 3 clinical trial25 to determine if changes in 
bone resorption and formation occurred over the course of the year-long study. If 
significant changes in bone turnover are found, this will add further support to the present 
proposal. 
 
 
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