Pilot Study of the Effect of High Doses of Radiation on Bone Metabolism and Structure in Patients Treated With Adjuvant Radiotherapy and Surgery for Sacral Tumors
Phase: NA
Conditions: Malignant Bone Tumors
Sponsor: Massachusetts General Hospital

Effect of High Doses of Radiation on Bone Structure and Metabolism 
October, 5, 2016 
Protocol Version Date: October, 5, 2016 
 
 
NCI Protocol #: N/A 
Local Protocol #: 14-208 
 
Title: Pilot Study of the Effect of High Doses of Radiation on Bone Metabolism and Structure in 
Patients Treated with Adjuvant Radiotherapy and Surgery for Sacral Tumors.  
 
Principal Investigator: Joseph H Schwab, MD, MS 
Radiation Oncology Co-PI: Yen-Lin Chen, M.D. 
Co-Investigators:  
 
Thomas F. DeLaney, M.D. 
Sjoerd Nota, MD (Massachusetts General Hospital) 
Petur Nielsen, MD (Massachusetts General Hospital) 
Martin Torriani, MD (Massachusetts General Hospital) 
Mary Bouxsein, PhD (Massachusetts General Hospital) 
Francis Hornicek, MD, PhD (Massachusetts General Hospital) 
Elaine Yu, M.D. (Massachusetts General Hospital) 
Stein Janssen, MD (Massachusetts General Hospital) 
Olivier van Wulfften Palthe, MD (Massachuesetts General Hospital) 
Miriam Bredella, MD (Massachuesetts General Hospital) 
Karen Bernstein, MD (Massachuesetts General Hospital) 
Quirina Thio, MD (Massachusetts General Hospital) 
 
 
Statistician: Beow Yeap, Sc.D. 
Massachusetts General Hospital 
 
Study Coordinator/Data Manager:  
Quirina Thio, MD (Massachusetts General Hospital) 
 
 
Responsible Research Nurse: N/A 
 
 
Agent(s):  
N/A 
 
Funding: Federal Share of Program Income earned by Massachusetts General Hospital on C06 
CA059267, Proton Therapy Research and Treatment Center, Thomas F. DeLaney, M.D., 
Principal Investigator 
 
 

Effect of High Doses of Radiation on Bone Structure and Metabolism 
October, 5, 2016 
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SCHEMA  
 
Note: 
* A window of up to +1 week is allowed for the interval between end of radiation and surgery (stage I surgery can be performed 
4 to 5 weeks after completion of radiation).   
** 6 weeks is an average interval between surgery and start of radiation.  A window of +/- 2 weeks is allowed for the interval 
between surgery and postoperative radiation to allow adequate  time for wound healing.    #3 Blood draw can take place 
whenever postoperative radiation has been completed.   

Effect of High Doses of Radiation on Bone Structure and Metabolism 
October, 5, 2016 
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TABLE OF CONTENTS 
1. 
OBJECTIVES ...................................................................................................................................................... 6 
1.1 
Study Design ................................................................................................................................................... 6 
1.2 
Primary Objectives.......................................................................................................................................... 6 
1.3 
Secondary Objectives ...................................................................................................................................... 7 
2. 
BACKGROUND .................................................................................................................................................. 7 
2.1 
Rationale ......................................................................................................................................................... 7 
2.2 
Study Disease .................................................................................................................................................. 8 
2.3 
Bone Labeling Drugs ...................................................................................................................................... 9 
2.4 
Correlative Studies Background ..................................................................................................................... 9 
3. 
PARTICIPANT SELECTION ......................................................................................................................... 14 
3.1 
Eligibility Criteria ......................................................................................................................................... 14 
3.2 
Exclusion Criteria ......................................................................................................................................... 15 
3.3 
Inclusion of Women, Minorities and Other Underrepresented Populations ................................................. 15 
4. 
REGISTRATION PROCEDURES .................................................................................................................. 16 
4.1 
General Guidelines for DF/HCC and DF/PCC Institutions .......................................................................... 16 
4.2 
Registration Process for DF/HCC and DF/PCC Institutions ..................................................................... 16 
5. 
TREATMENT PLAN ........................................................................................................................................ 16 
5.1 
Pre-treatment Criteria .................................................................................................................................. 18 
5.2 
Bone Labeling Drug Administration ............................................................................................................. 18 
5.3 
Other Studies ................................................................................................................................................. 18 
5.4 
Definition of Dose-Limiting Toxicity ............................................................................................................. 18 
5.5 
General Concomitant Medication and Supportive Care Guidelines ............................................................. 18 
5.6 
Duration of Therapy ...................................................................................................................................... 19 
5.7 
Duration of Follow Up .................................................................................................................................. 19 
5.8 
Criteria for Removal from Study ................................................................................................................... 19 
6. 
EXPECTED TOXICITIES AND DOSING DELAYS/DOSE MODIFICATIONS ..................................... 19 
6.1 
Anticipated Toxicities .................................................................................................................................... 19 
6.2 
Toxicity Management .................................................................................................................................... 22 
6.3 
Dose Modifications/Delays ........................................................................................................................... 23 
7. 
DRUG FORMULATION AND ADMINISTRATION ................................................................................... 24 
7.1 
Tetracycline ................................................................................................................................................... 24 
7.2 
Demeclocycline (Declomycin) ....................................................................................................................... 25 
8. 
CORRELATIVE/SPECIAL STUDIES ........................................................................................................... 27 

Effect of High Doses of Radiation on Bone Structure and Metabolism 
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8.1 
Pharmacokinetic Studies ............................................................................................................................... 27 
8.2 
Pharmacodynamic Studies ............................................................................................................................ 27 
8.3 
Special Studies .............................................................................................................................................. 27 
9. 
STUDY CALENDAR ........................................................................................................................................ 32 
10. 
MEASUREMENT OF EFFECT ..................................................................................................................... 33 
11. 
ADVERSE EVENT REPORTING REQUIREMENTS ............................................................................... 33 
11.1 
Definitions ................................................................................................................................................... 33 
11.2 
Procedures for AE and SAE Recording and Reporting ............................................................................... 35 
11.3 
Reporting Requirements .............................................................................................................................. 35 
11.4 
Reporting to the Study Sponsor ................................................................................................................... 36 
11.5 
Reporting to the Institutional Review Board (IRB) ..................................................................................... 37 
11.6 
Reporting to the Food and Drug Administration (FDA) N/A ...................................................................... 37 
11.7 
Reporting to the NIH Office of Biotechnology Activities (OBA) N/A .......................................................... 37 
11.8 
Reporting to the Institutional Biosafety Committee (IBC) N/A ................................................................... 37 
11.9 
Reporting to Hospital Risk Management .................................................................................................... 37 
11.10 
Monitoring of Adverse Events and Period of Observation ........................................................................ 37 
12. 
DATA AND SAFETY MONITORING ......................................................................................................... 38 
12.1 
Data Reporting ............................................................................................................................................ 38 
12.2 
Safety Meetings ........................................................................................................................................... 38 
12.3 
Monitoring .................................................................................................................................................. 39 
13. 
REGULATORY CONSIDERATIONS .......................................................................................................... 39 
13.1 
Protocol Review and Amendments .............................................................................................................. 39 
13.2 
Informed Consent ........................................................................................................................................ 39 
13.3 
Ethics and Good Clinical Practice (GCP) .................................................................................................. 40 
13.4 
Study Documentation .................................................................................................................................. 40 
13.5 
Records Retention ....................................................................................................................................... 40 
13.6 
Multi-center Guidelines .............................................................................................................................. 41 
13.7 
Cooperative Research and Development Agreement (CRADA)/Clinical Trials Agreement (CTA) ............ 41 
14. 
STATISTICAL CONSIDERATIONS ........................................................................................................... 41 
14.1 
Study Design/Endpoints .............................................................................................................................. 41 
14.2 
Sample Size/Accrual Rate ........................................................................................................................... 42 
14.3 
Stratification Factors .................................................................................................................................. 43 
14.4 
Analysis of Secondary Endpoints ................................................................................................................ 43 
14.5 
Reporting and Exclusions ........................................................................................................................... 43 

Effect of High Doses of Radiation on Bone Structure and Metabolism 
October, 5, 2016 
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15. 
PUBLICATION PLAN ................................................................................................................................... 44 
16. 
REFERENCES ................................................................................................................................................. 44 
17. 
APPENDICES .................................................................................................................................................. 47 
17.1 
Appendix 1: List of Figures ......................................................................................................................... 47 
17.2 
Appendix 2: Patient Drug Schedule And Diary .......................................................................................... 48 
17.3 
Appendix 3: Summary of Experimental Procedures ................................................................................... 51 
17.4 
Appendix 4: Quality of Life Surveys .............................................................. Error! Bookmark not defined. 

Effect of High Doses of Radiation on Bone Structure and Metabolism 
October, 5, 2016 
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6 
1. OBJECTIVES 
 
1.1 
Study Design 
 
This is a pilot study designed to characterize the effects of high energy radiation on human bone 
degradation, with a specific interest in reducing the rate of sacral fractures. 
 
Figure 1:  Phase Transition into Clinical Trials 
 
 
 
Based on the data published in animal models, we hypothesized that bone degradation after irradiation is 
associated with clinically detectable changes in serum markers, bone density, and bone architecture. This 
study is a step towards better characterizing those changes in humans, and understanding the mechanism 
of the underlying pathology. The results of this research may ultimately lead to the development of 
interventions aimed at reducing the burden of radiation-induced fractures, as outlined in Figure 1:    
 
Figure 2 provides a succinct layout of the proposed experimental plan and a summary of the data we 
expect to collect.  
 
Figure 2: Experimental Design Summary 
 
1.2 
Primary Objectives 
 
1.2.1 
To measure the effect of high doses of radiation on trabecular bone mineral density (Tb. 
TBD) in the adult sacrum using the quantitative computer tomography technique (QCT) in 
patients undergoing treatment with combination surgery and adjuvant radiotherapy, or 
radiation alone.   

Effect of High Doses of Radiation on Bone Structure and Metabolism 
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1.3 
Secondary Objectives 
 
1.3.1 
To characterize changes in the levels of serum markers of bone turnover during the course of 
12 months following treatment with high doses of radiation  
1.3.2 
To analyze the effects of high doses of radiation on the structure and mechanical properties 
of bone by microscopic computer tomography (micro-CT), and nanoindentation    
1.3.3 
To characterize the effect of high doses of radiation on the dynamic parameters of bone 
turnover (e.g. mineral apposition rate, mineral formation rate, and mineralization lag time) 
by tetracycline quadruple labeling method  
1.3.4 
To characterize the effect of high doses of radiation on the mineralized and cellular 
components of bone architecture (e.g. volume, thickness, separation of trabecular bone, and 
osteoid surface and volume) by undecalcified bone histology 
 
2. BACKGROUND 
 
2.1 
Rationale 
 
External beam radiation plays an important role in the management of bone malignancies. In combination 
with surgical resection, high dose radiation was shown to significantly improve survival in historically 
difficult to treat tumors [4, 6, 15, 22]. The detrimental effects of radiation increase proportionally with the 
dose delivered. Even at relatively low doses of radiation (30 to 40 Gy), skeletal complications such as 
radiation-induced osteoporosis, insufficiency fractures (see  
 
 
 
 
 
 
Figure 3), physeal arrest, and non-union are common [2, 14, 21, 29, 33]. At high doses (70 Gy and 
above), the rate of bone-related complications increases dramatically. Despite the high rate of 
complications, high-dose radiation is becoming increasingly more utilized because of its positive effects 
on patient survival and prevention of recurrence. For example, combination of surgery and adjuvant 
radiation doses of greater than 70 Gy achieves meaningful control of locally aggressive tumors (like 
sacral chordomas), that otherwise have a very high rate of recurrence [6]. 
 
 
 
 
 

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Figure 3: Bilateral Sacral Fractures  
 
 
Given its growing significance in the treatment of malignant bone tumors, we propose to investigate the 
impact of high dose radiation (over 70 Gy) on human bone metabolism, architecture, and mechanical 
properties. It is presently unknown what biological mechanisms and structural changes occur in human 
bone in response to high doses of radiation. Local effects on bone may differ for different parts of the 
body. To minimize the variability in bone properties, we have chosen to focus this study on one type of 
bone - the sacrum. Still, our results are likely to be generalizable to other bones because of the common 
physiology. Sacrum is a particularly good target for this study because 1) tumors affecting the sacrum are 
often treated with high doses of radiation and 2) sacrum is one of the bones maximally affected by post-
radiation complications.  
 
 
 
 
 
 
Figure 3 shows a schematic representation of a typical insufficiency fracture that can occur in the sacrum 
after radiation treatment. Our group recently reported the rate of sacral insufficiency fractures as high as 
64% in patients treated with high dose radiation and surgery (submitted). Thus, for the purposes of this 
pilot study we will focus on patients with primary malignant tumors of the sacrum.  
 
Most MGH patients with sacral tumors are treated with combination of surgery and adjuvant radiation.  
They receive 50.4 Gy of photon/proton beam radiation before surgery, in accordance with the protocol 
summarized in Figure 9 (A). After the tumor is surgically removed, patients receive another 19.8 Gy of 
radiation, for a total dose of 70.2 Gy. We plan to enroll 20 patients treated in this fashion into the 
“Surgical Arm” of the study. It has been previously suggested that in patients treated with combination of 
surgery and radiation, the high rate of sacral fractures may be partially attributable to surgery. Yet, sacral 
fractures have been reported in patients who were treated with radiation alone. There is no data to date 
that can account for these observations. To explore this question, we plan to enroll 10 patients treated 
with radiation alone into the “Non-surgical Arm” of our study. These patients receive between 72 Gy 

Effect of High Doses of Radiation on Bone Structure and Metabolism 
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9 
 
 
(osteosarcoma and chondrosarcoma) to 77.4  Gy (for chordoma) of radiation over the course of 7 to 8.5 
weeks, as outlined in Figure 9 (B).  
 
The long term goal of this research is to improve treatment outcomes and quality of life in patients with 
tumors of any origin, who undergo treatment with radiation. We hope that the results of this translational 
research may ultimately enable the predication of patients at risk of fracture and allow intervention before 
fracture occurs. 
2.2 
Study Disease 
 
This study does not focus on a single disease. Instead, the study is concerned with effects of high dose 
radiation therapy on bone quality in general. For the pilot study, we will focus on patients with primary 
malignant bone tumors of the sacrum, but the results will most likely be generalizable to other conditions, 
including metastatic disease.  
 
2.3 
Bone Labeling Drugs 
 
2.3.1 
Tetracycline 
 
Tetracycline is an FDA-approved anti-microbial drug, available in the US in generic form. It has minimal, 
well-documented adverse effects. Tetracycline can be safely self-administered by the patient in a form of 
oral pill on outpatient basis. In this study, it will be used as a fluorescent label for dynamic bone 
histomorphometry studies, in accordance with a well-established protocol. Manufacturer: Varies 
2.3.2 
Demeclocycline (Declomycin) 
 
Demeclocycline is an FDA-approved anti-microbial drug, available in the US in generic form. It is a 
tetracycline derivative, and has a similar safety profile. All associated adverse effects are minimal and 
well-documented. Demeclocycline can be safely self-administered by the patient in a form of oral pill on 
outpatient basis. In this study, it will be used as an accessory fluorescent label (in addition to tetracycline) 
for dynamic histomorphometry studies, in accordance with a well-established protocol. Manufacturer: 
Varies 
 
2.4 
Correlative Studies Background 
 
We hypothesized that bone degradation after irradiation is associated with detectable changes in serum 
markers, bone metabolism, bone structure and mechanical properties. In order to characterize these 
changes, we designed a series of in vivo and in vitro experiments, summarized in  
 
 
 
 
 
 

Effect of High Doses of Radiation on Bone Structure and Metabolism 
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Figure 4.  
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 4: Experimental Design: Summary of the Proposed Experimental Techniques and 
Measurements to be Collected in the Study 

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2.4.1 
Serum Markers 
 
A number of circulating serum molecules are used clinically as markers of bone turnover rate and bone 
remodeling. N-Telopeptide (NTX) is a breakdown products of Type I collagen molecule, which is 
particularly abundant in bone. It has been shown to be useful in predicting the risk of fractures [32] and 
other skeletal complications in cancer patients [17]. We will als test serum tartrate-resistant acid 
phosphatase 5b (TRAP5b). TRAP 5b may reflect defferent aspects of osteoclast function; osteoclast 
adhesion, osteoclast number and degradation of non-collagenous proteins [37, 38]. We will use these 
measurements as markers of bone resorption. In addition, we will measure several markers of bone 
formation: osteocalcin (OC) and type 1 procollagen N-terminal (P1NP). 
In addition to bone-specific markers, we will measure calcium (as part of s atandard metabolic panel), 
phosphate, parathyroid hormone levels (PTH), and 25-hydroxycalciferol as indicators of systemic 
metabolism that can affect bone health. The changes in serum bone turnover markers are expect to 
predate fractures, if fractures do occur. While we expect the markers to increase with the onset of 
radiation, we will follow the trend after the radiation has b een discontinued to see if and when the 
markers begin to return to normal. 
 
 
 
2.4.2 
QCT  
 
Quantitative computer tomography analysis (QCT) is a high resolution imaging technique that can detect 
changes in bone mineralization density and trabecular bone density [20, 33]. Figure 5 (E) shows the 
difference between two bone types. Cortical bone is thick and solid, and provides the weight-bearing 
strength of bone. Trabecular bone is porous, and has a woven-like structure. Radiation causes sclerosis of 
the trabecular bone, rendering it solid and dense, like cortical bone. Despite this increase in density, 
irradiated bone becomes brittle and prone to fractures. Traditional method of measuring bone density by 
dual-energy X-ray absorptiometry (DEXA) measures an average density of cortical and trabecular bone. 
It cannot differentiate between normal bone and fragile, irradiated bone affected by sclerosis. QCT can 
measure the density of trabecular bone directly. Figure 5(A-C) shows the axial, coronal and sagittal scans 
of the lumbar vertebral body by QCT. A three-dimensional reconstruction of a slice through the vertebral 
body allows determination of the architecture of the trabecular bone, as illustrated in Figure 5(D).   
 
Figure 5: Determination of Bone Structure in the Adult Spine by Quantitative CT (QCT) 

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Bone density can be calculated from the radiopacity of the bone expressed in Hounsfield units, averaged 
over a small region of interest (ROIs) as shown in Figure 6. In the lumborosacral spine, ROIs are drawn 
through the anterior aspect of the L4, the sacral alae, and midline of S1, avoiding any areas of abnormal 
anatomy or sclerosis due to fractures or degenerative changes. The Hounsfield unit values are then 
entered into software to calculate values for the trabecular bone mineral density (Tb. BMD) in the region 
of interest. 
 
 
 
 
 
Figure 6: Definition of ROI in the Lumbar Spine 

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2.4.3 
Bone Histomorphometry 
 
Histomorphometry is broadly defined as the measurement of the shape or form of a tissue. Bone 
histomorphometry is a quantitative analysis of bone structure and bone remodeling.  It provides valuable 
information on the amount of bone and its cellular activity. Static histomorphometry allows quantitative 
evaluation of bone structure and cellular content at any particular time point. Undecalcified bone samples 
embedded in plastic resin are used for this purpose. Cellular content of is evaluated by 
immunohistochemical staining methods. Changes in bone structure and the volume of mineralized tissue 
are assessed by micro-computed tomography, quantitative back scatter electron imaging, and 
nanoindentation.  
 
Dynamic histomorphometry provides a quantitative assessment of the extent of bone formation over a 
specific period of time (usually 10-14 days). If a fluorescent label, such as tetracycline, is present, it 
becomes incorporated into the newly formed bone, leaving a clear linear record of the site where 
mineralization was occurring (see Figure 7). If a second label is given 10-14 days after the first one, one 
can calculate the mineral apposition rate (MAR, mm/day) as the distance between the midpoints of the 
two consecutive labels, divided by the time interval between the labeling periods. The bone formation 
rate (BFR/BS, mm3/mm2/day) is the amount of newly formed bone seen under the fluorescent microscope 
divided by the time interval during which the label was given. We will use a quadruple labeling 
scheduele, using two sets of double tetracycline labels witch differ in color under fluorescent light. This 
way we will be able to distinguish the first and second label set. This technique has the great advantage 
that the patient can serve as his or her own pretreatment control, eliminating problems caused by the large 
intersite variablility in histomorphometric variables [39]. 
 
 
 
 
 
 
 

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Figure 7: Visualization of Tetracycline Labeled Bone by Fluorescent Microscopy 
 
 
2.4.4 
Micro-computed tomography (micro-CT) 
 
Microtomography (micro-CT) is used for in vitro bone studies. It uses a similar technique to conventional 
X-ray tomography systems (regular CT scans), but with much finer resolution.  Morphological parameters 
of any object can be analyzed with very high precision using three-dimensional reconstructions. The 
process is non-destructive and requires no special preparation of the specimen. Micro-CT can measure 
static morphological parameters of bone microscopic architecture. The bone volume fraction (BV/TV), 
trabecular thickness (Tb. Th.), trabecular separation (Tb. Sp.), trabecular number (Tb. N.), Connectivity 
density (Conn.D), structure model index (SMI), and trabecular bone pattern factor (TBPf) provide 
information about the characteristics of the trabecular bone structure. In networks of trabecular bone, 
connectivity density provides an estimation of the number of trabecular connections per unit volume. The 
structure model index determines the geometry of the intertrabecular network.  
2.4.5 
Nanoindentation 
 
Nanoindentation is a powerful method for investigating the mechanical properties of materials in small 
dimensions. This is done by pressing a hard diamond tip into the sample of the material of interest with a 
known amount of force. Traditional indentation methods measured hardness of materials from the area of 
the “footprint” left in the sample by the diamond tip. Nanoindentation creates “footprints” in the 
submicrometer range, much too small to examine without a powerful microscope. Instead, it uses 
specialized depth sensing equipment to make tiny indentations in the surface. At the same time it records 
the load and displacement of the indenter with very high accuracy and precision. A load-displacement 
curve is constructed from the recorded values (Figure 8). Experimental data can be interpreted using 
analytical models to obtain hardness, the elastic modulus, and other mechanical properties.  
 
 

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Figure 8: Schematic Representation of a Typical Load-Displacement Curve 
 
 
We will measure the elastic modulus of bone from the unloading segment of the load-displacement curve 
using the Oliver-Pharr method. The elastic modulus is a measurement of bone’s ability to resist 
mechanical stress. Healthy bone is strong and it effectively resists the outside forces exerted on it. When 
the body is in the upright position, gravity exerts a significant force on the bones of the spine and pelvis 
causing deformation. Healthy bone resists deformation: it returns to its original form when the force is 
removed. In contrast, bone weakened by osteoporosis or radiation cannot resist stress exerted by normal 
activities, like walking. Gravity causes it to deform permanently; even when the force is removed or 
reduced, the deformation remains. This is the mechanism proposed to explain the development of sacral 
insufficiency fractures after radiation. We want to measure the elastic modulus of bone in the sacrum 
before and after radiation to characterize the effect of radiation on the elastic properties. It is exceedingly 
likely that the pathogenesis of radiation-induced sacral insufficiency fractures is multifactorial. Given this 
complexity, the ability to isolate the effect of radiation on elastic property makes nanoindentation a 
uniquely valuable tool for our study. 
 
3. PARTICIPANT SELECTION 
 
3.1 
Eligibility Criteria 
 
All laboratory tests that are a part of the eligibility criteria must be completed within 14 days prior to the 
date of registration. Diagnostic tests that are a part of the eligibility criteria must be performed within 30 
days of the date of registration. Participants must meet the following criteria on screening examination to 
be eligible to participate in the study: 
3.1.1 
Study participants must have histologically confirmed primary malignant bone tumor in the 
sacrum for which surgery and radiation or radiation alone are planned.   

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3.1.2 
Age 18 years or older. In children under the age of 8, tetracycline derivatives have been 
reported to stain tooth enamel yellow color. These considerations lead us to exclude young 
persons under the age of 18 from the study. 
3.1.3 
Participants in the surgical arm must have normal organ and marrow function as defined 
below: 
 
▪ 
Total bilirubin within normal institutional limits 
▪ 
AST (SGOT)/ALT (SGPT) < 2.5 X institutional upper limit of normal 
▪ 
Creatinine within normal institutional limits or creatinine clearance > 60 
mL/min/1.73 m2 for subjects with creatinine levels about institutional normal 
limit  
3.1.4 
The effects of tetracyclines and radiation used in computer tomography on the developing 
human fetus are known to be detrimental. For this reason, women of child-bearing potential 
and men must agree to use adequate contraception (hormonal or barrier method of birth 
control; abstinence) prior to study entry and until after the last study related computer 
tomography scan . Should a woman become pregnant or suspect she is pregnant while 
participating in this study, she should inform her treating physician immediately. 
3.1.5 
Ability to understand and willingness to sign a written informed consent. 
3.2 
Exclusion Criteria 
 
Participants who exhibit any of the following conditions at screening will not be eligible for admission 
into the study. 
 
3.2.1 Participants who have had surgery, chemotherapy, or radiotherapy of the sacrum prior to 
entering the study 
3.2.2 History of allergic reactions attributed to compounds of similar chemical or biologic 
composition to tetracyclines. 
3.2.3 Pregnant or nursing 
3.2.4 
Uncontrolled inter current illness including, but not limited to ongoing or active infection, 
symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situations that would limit compliance with study requirements. 
3.3 
Inclusion of Women, Minorities and Other Underrepresented Populations 
 
We will not restrict subject selection by race, gender, or age group. The external validity of the results 
obtained in this study would benefit from population diversity. Since the study aims at improving 
outcomes for all patients with bone tumors, exclusion of any group that can be affected by the disease can 
result in the failure to collect clinically important information. 
 

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4. REGISTRATION PROCEDURES 
 
4.1 
General Guidelines for DF/HCC and DF/PCC Institutions 
 
Institutions will register eligible participants with the DF/HCC Quality Assurance Office 
for Clinical Trials (QACT) central registration system. Registration must occur prior to the 
initiation of therapy. Any participant not registered to the protocol before treatment begins 
will be considered ineligible and registration will be denied.  
 
A member of the study team will confirm eligibility criteria and complete the 
protocol-specific eligibility checklist. 
  
Following registration, participants may begin participation in the protocol. Issues that 
would cause delays should be discussed with the Principal Investigator. If a participant does 
not participate in the protocol following registration, the participant’s protocol status must be 
changed. Notify the QACT Registrar of participant status changes as soon as possible. 
 
4.2 
Registration Process for DF/HCC and DF/PCC Institutions 
 
The QACT registration staff is accessible on Monday through Friday, from 8:00 AM to 5:00 PM 
Eastern Standard Time.  
   
The registration procedures are as follows: 
 
1. 
Obtain written informed consent from the participant prior to the performance of any 
study related procedures or assessments. 
 
2. 
Complete the protocol-specific eligibility checklist using the eligibility assessment 
documented in the participant’s medical/research record. To be eligible for 
registration to the study, the participant must meet each inclusion and exclusion 
criteria listed on the eligibility checklist. 
  
Reminder: Confirm eligibility for ancillary studies at the same time as eligibility for 
the treatment study. Registration to both treatment and ancillary studies will not be 
completed if eligibility requirements are not met for all studies. 
  
3. 
Fax the eligibility checklist(s) and all pages of the consent form(s) to the QACT at 
617-632-2295. 
  
4. 
The QACT Registrar will (a) validate eligibility and (b) register the participant on the 
study. 
  
5. 
The QACT Registrar will send an email confirmation of the registration to the person 
initiating the registration immediately following the registration. 
 
5. TREATMENT PLAN 
 

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Study participants will receive treatment as determined by their treating physicians at the 
Massachusetts General Hospital for Malignant Bone Tumors of the Sacrum.  This may be radiation 
alone or pre-operative radiation therapy followed by surgery.  The treatment plan including MRI and CT 
imaging is determined by the clinical situation and not by the study.  Patients planned for surgery 
typically receive pre-operative MRI of their pelvis and/or sacrum.  In addition, patients often receive a 
pelvic or sacral computed tomographic image.  A CT of the chest is performed to evaluate for pulmonary 
metastasis. Figure 9 outlines the current standard radiation treatment options. Study participants may also 
receive radiation therapy as part of another clinical trial.  For surgical candidates, the standard treatment 
includes pre-operative radiation of 50.4 Gy, followed by a recovery period of approximately 4 to 5 weeks.  
Surgery involves removing the malignant tumor in the sacrum in one piece, preferably with a cuff of 
normal tissue around the tumor. After approximately 6 weeks of recovery, to allow the surgical incision to 
heal, the patient is treated with another 19.8 Gy up to 27 Gy of radiation postoperatively depending on the 
final margin status (higher for gross residual disease) , as outlined in Figure 9(A).  If the wound is not 
healed or there is another medical reason to delay adjuvant radiation, then radiation may begin later. The 
patients are typically followed every 3 months with an MRI of the pelvis/sacrum to evaluate for a local 
recurrence. A CT scan is often ordered if there is a concern for a fracture.  In addition, patients typically 
receive a chest x-ray at 3 months and 9 months after surgery.  A chest CT is ordered at 6 months and 12 
months after surgery.  The same protocol is used during the second year of follow up.  Non-surgical 
candidates receive 72 up to 77.4 Gy of radiation depending on the histology (72 Gy for osteosarcoma and 
chondrosarcoma and 77.4 Gy for chordoma) as outlined in Figure 9 (B).   
 
Figure 9: Radiation Treatment Flowchart
 

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5.1 
Pre-treatment Criteria 
 
Not applicable, as long as labs are within normal limits (see Section 3.1.3) 
 
5.2 
Bone Labeling Drug Administration 
 
For participants in the non-surgical arm, no bone labeling drugs are administered.  These participants will 
receive a standard radiation regimen with blood samples and imaging (QCT) only as their participation in 
this study.  For participants in the surgical arm, the bone labeling drugs are described below. 
5.2.1 
 Demeclocycline 
 
Participants in the surgical arm will self-administer the first label set; demeclocycline 150 mg PO QID 
(daily dose 600 mg) during 2 periods of 3 days, starting day 18 and day 3 prior to the start of their 
schedueled pre-operative radiotherapy. Participants will be given a drug diary (Appendix 1) to keep a 
record of the dosing. Demeclocycline comes in a form of an oral pill and does not require any special 
equipment for administration. It has a well-established safety profile with minimal adverse effects. No 
observation period or vital signs monitoring is required. Demeclocycline should be administered 1 hour 
before or 2 hours after food or milk, with adequate amounts of fluid to decrease the risk of esophageal 
irritation and ulceration. If a dose is vomited, the participant should contact the research coordinator, so 
we can provide an extra dose. Contact information will be provided in the drug diary. 
 
5.2.2 
Tetracycline 
 
Participants in the surgical arm will also self-administer the second label set; tetracycline 250mg PO QID 
(daily dose 1000 mg) during 2 periods of  3 days, starting day 24 and day 9 prior to their scheduled 
surgery. Participants will be given a drug diary (Appendix 1) to keep a record of the dosing. Tetracycline 
comes in a form of an oral capsule and does not require any special equipment for administration. It has a 
well-established safety profile with minimal adverse effects. No observation period or vital signs 
monitoring is required. Tetracycline should be administered on an empty stomach: 1 hour prior to meals, 
or 2 hours after meals. If a dose is vomited, the participant should contact the research coordinator, so we 
can provide an extra dose. Contact information will be provided in the drug diary. 
 
 
5.3 
Other Studies 
 
As outlined in section 8.3. 
 
5.4 
Definition of Dose-Limiting Toxicity 
 
N/A 
 
5.5 
General Concomitant Medication and Supportive Care Guidelines 

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Patients may receive all concomitant therapy deemed necessary to provide adequate support for any 
standard treatment or study related events.  
5.6 
Duration of Therapy 
 
 Standard treatment will continue for study participants as long as clinically indicated until the standard 
treatment is completed.  As noted above, participants in the radiation arm will provide blood samples for 
up to 6 months after completion of radiation therapy.  Participants in the surgical arm will self administer 
the bone labeling drugs as described above and will provide blood samples for up to 6 months after 
completing radiation therapy. 
 
5.7 
Duration of Follow Up 
 
Participants will be followed for radiographic and/or clinical evidence of fracture and assessment 
of quality of life for 12 months after their surgery/radiotheraphy or until death, whichever occurs 
first. Participants removed from study for unacceptable adverse events will be followed until resolution or 
stabilization of the adverse event. 
 
5.8 
Criteria for Removal from Study 
 
Participants will be removed from study if any of the adverse events listed in section 6.2.2 or 6.1.2 occur. 
The reason for study removal and the date the participant was removed must be documented in the study-
specific case report form (CRF). Alternative care options will be discussed with the participant. 
 
In the event of unusual or life-threatening complications, participating investigators must immediately 
notify the Principal Investigator, Dr. Schwab at 617-724-8636 or MGH pager #15724 
 
6. EXPECTED TOXICITIES AND DOSING DELAYS/DOSE MODIFICATIONS 
 
If possible, symptoms will be managed symptomatically. In the case of toxicity, appropriate medical 
treatment will be used (including anti-emetics, anti-diarrheals, etc.). 
 
All adverse events experienced by participants will be collected from the time of the first dose of study 
treatment, through the study and until the final study visit. Participants continuing to experience toxicity 
at the off study visit may be contacted for additional assessments until the toxicity has resolved or is 
deemed irreversible. 
6.1 
Anticipated Toxicities 
 
All drugs in this study have been in routine clinical use for many years, and are associated with rare 
adverse effects. Additional radiation exposure is is less than the yearly natural background radiation in the 
US. A list of the adverse events and potential risks associated with the drugs administered in this study 
appear below and will determine whether dose delays and modifications will be made or whether the 
event requires expedited reporting in addition to routine reporting. 

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6.1.1 
Adverse Event List for Tetracycline 
 
Significant Adverse Reactions (Rare): 
▪ 
Cardiovascular: Pericarditis  
▪ 
Central nervous system: Bulging fontanels in infants, increased intracranial pressure, paresthesia, 
pseudotumor cerebri  
▪ 
Dermatologic: Exfoliative dermatitis, photosensitivity, pigmentation of nails, pruritus  
▪ 
Gastrointestinal: Abdominal cramps, anorexia, antibiotic-associated pseudomembranous colitis, 
diarrhea, discoloration of teeth and enamel hypoplasia (young children), esophagitis, nausea, 
pancreatitis, staphylococcal enterocolitis, vomiting  
▪ 
Hematologic: Thrombophlebitis  
▪ 
Hepatic: Hepatotoxicity  
▪ 
Renal: Acute renal failure, azotemia, renal damage  
▪ 
Miscellaneous: Anaphylaxis, candidal superinfection, hypersensitivity reactions, superinfection 
 
Contraindications: Hypersensitivity to demeclocycline, tetracyclines, or any component of the 
formulation 
 
Concerns related to adverse effects: 
▪ 
Increased blood urea nitrogen (BUN): May be associated with increases in BUN secondary to 
antianabolic effects; use caution in patients with renal impairment. 
▪ 
Photosensitivity: May cause photosensitivity; discontinue if skin erythema occurs. Use skin 
protection and avoid prolonged exposure to sunlight; do not use tanning equipment. 
▪ 
Pseudotumor cerebri: Has been (rarely) reported with tetracycline use; usually resolves with 
discontinuation. 
▪ 
Superinfection: Prolonged use may result in fungal or bacterial superinfection, including C. 
difficile-associated diarrhea (CDAD) and pseudomembranous colitis; CDAD has been observed 
>2 months postantibiotic treatment. 
 
Disease-related concerns: 
▪ 
Hepatic impairment: Use with caution in patients with hepatic impairment; dose adjustment 
and/or adjustment to interval frequency recommended. Hepatotoxicity and hepatic failure have 
been reported rarely with use. 
▪ 
Renal impairment: Use with caution in patients with renal impairment; dosage adjustment 
recommended. Nephrotoxicity has also been reported with use, particularly in the setting of 
cirrhosis. 
 
Special populations: 
 
Pediatrics: May cause tissue hyperpigmentation, enamel hypoplasia, or permanent tooth discoloration; 
use of tetracyclines should be avoided during tooth development (children <8 years of age) unless other 
drugs are not likely to be effective or are contraindicated. 
 
Pregnancy: Do not use during pregnancy or nursing. In addition to affecting tooth development, 
tetracycline use has been associated with retardation of skeletal development and reduced bone growth 
 

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6.1.2 
Adverse Event List for Demeclocycline  
 
Significant Adverse Reactions (Rare): 
▪ 
Cardiovascular: Pericarditis  
▪ 
Central nervous system: Bulging fontanels (infants), dizziness, headache, pseudotumor cerebri 
(adults)  
▪ 
Dermatologic: Angioedema, anogenital inflammatory lesions (with monilial overgrowth), 
erythema multiforme, erythematous rash, exfoliative dermatitis (rare), maculopapular rash, 
photosensitivity, pigmentation of skin, Stevens-Johnson syndrome (rare), urticaria  
▪ 
Endocrine & metabolic: Microscopic discoloration of thyroid gland (brown/black), nephrogenic 
diabetes insipidus, thyroid dysfunction (rare) 
▪ 
Gastrointestinal: Anorexia, diarrhea, dysphagia, enterocolitis, esophageal ulcerations, glossitis, 
nausea, pancreatitis, vomiting  
▪ 
Genitourinary: Balanitis  
▪ 
Hematologic: Eosinophilia, neutropenia, hemolytic anemia, thrombocytopenia  
▪ 
Hepatic: Hepatitis (rare), hepatotoxicity (rare), liver enzymes increased, liver failure (rare)  
▪ 
Neuromuscular & skeletal: Myasthenic syndrome, polyarthralgia, tooth discoloration (children <8 
years, rarely in adults)  
▪ 
Ocular: Visual disturbances  
▪ 
Otic: Tinnitus  
▪ 
Renal: Acute renal failure, BUN increased 
▪ 
Respiratory: Pulmonary infiltrates  
▪ 
Miscellaneous: Anaphylaxis, anaphylactoid purpura, fixed drug eruptions (rare), lupus-like 
syndrome, superinfection, systemic lupus erythematosus exacerbation 
 
Contraindications: Hypersensitivity to demeclocycline, tetracyclines, or any component of the 
formulation 
 
Concerns related to adverse effects: 
▪ 
Diabetes insipidus syndrome: Dose-dependent nephrogenic diabetes insipidus is common with 
use; however, this adverse event of demeclocycline has been used as a therapeutic advantage in 
the off-label use of hyponatremia associated with SIADH. 
▪ 
Increased BUN: May be associated with increases in BUN secondary to antianabolic effects; use 
caution in patients with renal impairment. 
▪ 
Photosensitivity: May cause photosensitivity; discontinue if skin erythema occurs. Use skin 
protection and avoid prolonged exposure to sunlight; do not use tanning equipment. 
▪ 
Pseudotumor cerebri: Has been (rarely) reported with tetracycline use; usually resolves with 
discontinuation. 
▪ 
Superinfection: Prolonged use may result in fungal or bacterial superinfection, including C. 
difficile-associated diarrhea (CDAD) and pseudomembranous colitis; CDAD has been observed 
>2 months postantibiotic treatment. 
 
Disease-related concerns: 
▪ 
Hepatic impairment: Use with caution in patients with hepatic impairment; dose adjustment 
and/or adjustment to interval frequency recommended. Hepatotoxicity and hepatic failure have 
been reported rarely with use. 
▪ 
Renal impairment: Use with caution in patients with renal impairment; dosage adjustment 
recommended. Nephrotoxicity has also been reported with use, particularly in the setting of 
cirrhosis. 

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23 
 
 
 
Special populations: 
 
Pediatrics: May cause tissue hyperpigmentation, enamel hypoplasia, or permanent tooth discoloration; 
use of tetracyclines should be avoided during tooth development (children <8 years of age) unless other 
drugs are not likely to be effective or are contraindicated. 
 
Pregnancy: Do not use during pregnancy. In addition to affecting tooth development, tetracycline use has 
been associated with retardation of skeletal development and reduced bone growth 
 
6.1.3 
Radiation Exposure Risks  
 
Will perform a volumetric duel energy CT of L1, L2, and S1 using a second-generation dual-source 128-
row multi-detector CT scanner (Somatom De nition Flash; Siemens Medical Solutions, Forchheim, 
Germany). Subjects will be positioned on a quantitative CTPro calibration phantom (Mindways 
Software). Helical scans will be performed at 80 and 140 kV by using 210 and 80 mAs, respectively. 
Other scanning parameters included 1-second gantry rotation time, 0.9:1 pitch, and 64 3 0.6-mm 
detector configuration with double z-sampling. The images will be reconstructed at 2-mm section 
thickness and 2-mm section interval by using the I31f reconstruction kernel with a sinogram-affirmed 
iterative reconstruction (SAFIRE; Siemens Healthcare) setting of 2. The effective dose for each DECT 
scan is about 2 mSv. For 2 scans, the total effective dose is about 4 mSv which is about the annual 
background radiation dose. 
6.2 
Toxicity Management 
 
6.2.1 
Management of Tetracycline Toxicity  
 
Renal / Hepatic Impairement: Tetracycline will not be administered to patients with pre-existing renal or 
hepatic impairment.  
 
Photosensitivity: Patients will be advised to use skin protection while taking tetracycline. In addition, 
patients will be advised against prolonged exposure to sunlight and use of tanning equipment. If skin 
erythema occurs, patients will be advised to discontinue taking the drug and contact the study team. 
 
Superinfection: Patients will be informed of this risk, and will be closely monitored for any GI symptoms. 
Patients will be advised to discontinue taking the drug and contact their primary care provider and the 
study team, if any symptoms of infection occur.  
 
Pseudotumor: Patients will be advised to discontinue taking the drug and contact the study team if 
headache, photophobia, gait disturbances, of any other neurological deficits occur.  
 
Special populations: pregnant and lactating women and children will be excluded from the study due to 
the concerns related to tetracycline use in these populations (outlined in Section 6.1.1 Adverse Effects of 
Tetracycline 6.1.2).  
 
 
 

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6.2.2 
Management of Demeclocycline Toxicity  
 
Diabetes Insipidus: Patients will be advised to discontinue taking demeclocycline and contact their PCP 
and the study team,  if they experience excessive thirst, urination, headache, dizziness, nausea/vomiting, 
or any other concerning symptoms.  
 
Renal / Hepatic Impairement: Demeclocycline will not be administered to patients with pre-existing renal 
or hepatic impairment.  
 
Photosensitivity: Patients will be advised to use skin protection while taking demeclocycline. In addition, 
patients will be advised against prolonged exposure to sunlight and use of tanning equipment. If skin 
erythema occurs, patients will be advised to discontinue taking the drug and contact the ,study team.  
 
Superinfection: Patients will be informed of this risk, and will be closely monitored for any GI symptoms. 
Patients will be advised to discontinue taking the drug and contact their primary care provider and the PI 
study team, if any symptoms of infection occur.  
 
 
Pseudotumor: Patients will be advised to discontinue taking the drug and contact the study team if 
headache, photophobia, gait disturbances, of any other neurological deficits occur.  
 
Special populations: pregnant and lactating women and children will be excluded from the study due to 
the concerns related to tetracycline use in these populations (outlined in Section 6.1.2 Adverse Effects of 
Demeclocycline6.1.2).  
 
6.2.3 
Management of Radiation Toxicity  
 
Additional radiation dose is approximately 4 mSv. This amount of radiation is less than the yearly natural 
background radiation in the US..We do not anticipate any toxic effects of this dose of radiation to the 
patients. However, patients will be advised that radiation is a potential teratogen. For this reason, women 
of child-bearing potential and men must agree to use adequate contraception (hormonal or barrier method 
of birth control; abstinence) prior to study entry and for the duration of study participation. Should a 
woman become pregnant or suspect she is pregnant while participating in this study, she should inform 
her treating physician immediately. 
 
6.3 
Dose Modifications/Delays 
 
 
 
N/A 
 

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7. DRUG FORMULATION AND ADMINISTRATION 
7.1 
Tetracycline 
 
7.1.1 
Description 
 
The chemical name is: (4S,6S,12aS)-4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12a-
pentahydroxy-6-methyl-1,11-dioxonaphthacene-2-carboxamide. The molecular formula is 
C22H24N2O8The Molecular weight is 444.435 g/mol 
 
 
7.1.2 
Form 
 
Capsule, oral, as hydrochloride: 250 mg, 500 mg 
7.1.3 
Storage and Stability 
 
All pharmaceuticals will be stored at the Massachusetts General Hospital Oncology Clinical Trials 
Pharmacy (Yawkey-7).  
7.1.4 
Compatibility 
 
N/A 
7.1.5 
Handling 
 
Qualified personnel, familiar with procedures that minimize undue exposure to themselves and the 
environment, should undertake the preparation, handling, and safe disposal of the antimicrobial drug 
 in a self-contained and protective environment. 
7.1.6 
Availability 
 
All pharmaceuticals used in this study will be obtained from the in-patient pharmacy at MGH. Dispense 
of the bone labeling drugs will happen during the last day of the radiotherapy. This will be approximately 
4 days prior to the start of the labeleling cycle. The total amount of pills for the labelset (24 tablets) will 
be devided over 2 bottles, each containing 12 pills. The bottles will be labeled ‘Period 1’ and ‘Period 2’, 
indicating one of the 3 day periods during wich the pills are self-administered.  

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7.1.7 Preparation 
 
All pharmaceuticals will be prepared at the Massachusetts General Hospital Clinical Trials Pharmacy. 
The bone labeling drugs will be devided in 2 bottles with 12 tablets per bottle. One bottle will contain the 
exact amount of pills that will be self-administred during one of the 3 day period. There will be 2 of these 
3 day periods. 
7.1.8 
Administration 
 
Participants in the surgical arm will self-administer tetracycline 250mg PO QID (daily dose 1000 mg) 
during 2 periods of  3 days with a 12 day interval, i.e. days 24 ,23 and 22 and days 9, 8 and 7 prior to the 
1st stage of their scheduled surgery. Tetracycline should be administered on an empty stomach (ie, 1 hour 
prior to, or 2 hours after meals) to increase total absorption. Patients will be asked not to take aluminum 
containing antacids two hours before and after the medication has been given as it can interfere with bone 
binding. Patients will be instructed to take the bone labeling drug no more than 3 consecutive days.  
7.1.9 
Ordering 
 
All pharmaceuticals will be ordered through the Massachusetts General Hospital Clinical Trials 
Pharmacy. The designated pharmacist for this study is Lalit Joshi at 617-643-1812. 
 
7.1.10 Accountability 
 
The investigator, or a responsible party designated by the investigator, must maintain a careful record 
of the inventory and disposition of the bone labeling drugs (investigational or free of charge) using the 
NCI Drug Accountability Record or another comparable drug accountability form 
7.1.11 Destruction and Return 
 
At the end of the study, unused supplies of tetracycline and demeclocycline will be disposed of according 
to institutional policies. Destruction will be documented in the Drug Accountability Record Form. 
 
7.2 
Demeclocycline (Declomycin) 
 
7.2.1 
Description 
 
The chemical name is: 2E,4S,4aS,5aS,6S,12aS)-2-[amino(hydroxy)methylidene]-7-chloro-4-
(dimethylamino)-6,10,11,12a-tetrahydroxy-1,2,3,4,4a,5,5a,6,12,12a-decahydrotetracene-1,3,12-trione. 
The molecular formula isC21H21ClN2O8  The Molecular weight is 464.853 g/mol 
 

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7.2.2 
Form 
 
Tablet, oral, as hydrochloride: 150 mg, 300 mg 
7.2.3 
Storage and Stability 
 
All pharmaceuticals will be stored at the Massachusetts General Hospital Oncology Clinical Trials 
Pharmacy (Yawkey-7).  
 
7.2.4 
Compatibility 
 
N/A 
 
7.2.5 
Handling 
 
Qualified personnel, familiar with procedures that minimize undue exposure to themselves and the 
environment, should undertake the preparation, handling, and safe disposal of the antimicrobial drug in a 
self-contained and protective environment. 
 
7.2.6 
Availability 
 
All pharmaceuticals used in this study will be obtained from the in-patient pharmacy at MGH. Dispense 
of the bone labeling drugs will happen during the last clinical visit before the start of the radiotherapy. 
This will be approximately 3-4 weeks prior to the start of the labeling cycle. The total amount of pills for 
the labelset (24 tablets) will be devided over 2 bottles, each containing 12 pills. The bottles will be labeled 
‘Period 1’ and ‘Period 2’, indicating one of the 3 day periods during wich the pills are self-administered. 
 
 
7.2.7 Preparation 
 
All pharmaceuticals will be prepared at the Massachusetts General Hospital Clinical Trials Pharmacy. 
The bone labeling drugs will be devided in 2 bottles with 12 tablets per bottle. One bottle will contain the 
exact amount of pills that will be self-administred during one of the 3 day period. There will be 2 of these 
3 day periods.. 

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7.2.8 
Administration 
 
Participants in the surgical arm will self-administer demeclocycline 150 mg PO QID (daily dose 600 mg) 
during 2 periods of 3 days with a 12 day interval, i.e. day 18, 17 and 16 and day 3, 2 and 1 day prior to the 
start of their scheduled radiotheraphy. Demeclocycline will be administered 1 hour before or 2 hours after 
food or milk. Administer with adequate amounts of fluid to decrease the risk of esophageal irritation and 
ulceration. Patients will be instructed to take the bone labeling drugs no more than 3 consecutive days. 
 
7.2.9 
Ordering 
 
All pharmaceuticals will be ordered through the Massachusetts General Hospital Clinical Trials 
Pharmacy. The designated pharmacist for this study is Lalit Joshi at 617-643-1812 
 
7.2.10 Accountability 
 
The investigator, or a responsible party designated by the investigator, must maintain a careful record 
of the inventory and disposition of the bone labeling drugs (investigational or free of charge) using the 
NCI Drug Accountability Record or another comparable drug accountability form. 
 
7.2.11 Destruction and Return 
 
At the end of the study, unused supplies of tetracycline and demeclocycline will be disposed of according 
to institutional policies. Destruction will be documented in the Drug Accountability Record Form. 
 
8. CORRELATIVE/SPECIAL STUDIES 
8.1 
Pharmacokinetic Studies 
 
N/A 
8.2 
Pharmacodynamic Studies 
 
N/A 
 
8.3 
Special Studies 
8.3.1 
Blood Draws 
 
We will follow the trends in serological markers over a 6 month period beginning prior to the first dose or 
radiation received by the patient. The study Schema outlines specific time points when we plan to draw 

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the blood samples. After all studies have been prefromed we will label the samples with a code. The key 
to the code will connect the sample with the subjects health information. The key to the code will be kept 
in a password protected computer of the prinicipal investigator. Samples will be stored at the BioBank 
and Research Laboratory Services fo the Maine Medical Center Research Institute. 
 
Figure 10 
What is measured: 
 
▪ 
N-Telopeptide (NTX) 
▪ 
Serum tartrate-resistant acid phosphatase 5b (TRAP5b) 
▪ 
Osteocalcin (OC) 
▪ 
Type 1 procollagen N-terminal (P1NP). 
▪ 
PTH 
▪ 
Calcium as part of a standard metabolic panel 
▪ 
Phosphate 
 
8.3.2 
Quantitative CT Scans 
 
Patients in both arms of the study will undergo two QCT scans. First QCT will be done at Day 1, prior to 
initiation of radiation treatment. Patients in the Non-Surgical Arm will undergo a second QCT at six 
months after their first radiation treatment. In contrast, patients in the Surgical Arm will undergo their 
second QCT immediately prior to surgery, 10 weeks after the first scan. Once the spinal instrumentation 
is introduced during the surgery, it renders the analysis impossible due to artifact. Scans will be 
performed by the MGH Musculoskeletal Imaging Research Core (MIRC) under the direction of Dr. 
Martin Torriani (Radiology). We will use the QCT Pro protocol, detailed in Section 6.1.3 – Radiation 
Exposure Risks. Volumetric QCT of L1, L2 and S1 vertebrae will be obtained. Images will be saved in 
standard DICOM format and transferred to a PACS workstation for analysis.  
 
What is measured: 
 
▪ 
Trabecular bone mineral density (Tb. BMD) 
 
8.3.3 
Bone Biopsies  
 
Bone biopsies will be performed on patients enrolled in the Surgical Arm of the study. Each patient will 
undergo two biopsies. At the time of each biopsy we plan to collect two bone samples. Figure 10 shows 
the location of the planned biopsy sites. The first biopsy (the control) will be taken from the iliac wing of 
the pelvis. This area lies outside of the radiated field, and will serve as a normal bone control. The second 
biopsy will be taken from the irradiated part of the sacrum. Bone biopsies will be evaluated for the end 
points of dynamic histomorphometry, and possible radiation-induced woven bone and bone 
mineralization abnormalities. Micro-CT will be performed on half of the bone samples prior to 
embedding to evaluate 3D trabecular microstructure. Biopsies that will be used for dynamic 
histomorphometry will be stored in 10% buffered formalin at room temperature in a dark space. We will 
send the biopsies to dr. Dempster at Colombia university for analysis. Tranfers will be done in batches 
using FedEx. After all studies have been performed we will label the biopsies with a code. The key to the 
code will connect the biopsy with the subjects health information. The key to the code will be kept a in 
password protected computer of the prinicipal investigator. Biopsies will be stored either at room 

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30 
 
 
temperature (embedded specimen) or frozen (fresh specimen) at the Endocrine Unit Research Laboratory 
of the Massachusetts General Hospital. 
 
Figure 10 shows the location of two biopsy sites.  
 
Figure 10: Location of the Planned Bone Biopsy Sites.  
 
 
Biopsies will be done at the time of the scheduled tumor surgery. Sacral tumors are removed in stages. 
The first stage is done via anterior exposure, allowing access to the iliac crest. We will collect these 
samples first. Using a 7,5mm Rochester trephine two bone samples will be removed from the superior 
apex of the iliac wing in the midline (as defined as a point midway between the anterior superior and 
posterior superior iliac crests. In most cases, the biopsy site will be accessible through the incision made 
for the anterior approach. However, in those rare cases when the biopsy site is not accessible via the 
incision made as part of the standard of care, then a separate small incision (at most 3 cm long) will have 
to be made.  
 
The second stage is done via posterior approach, providing access to the sacrum. We will collect the 
second biopsy samples during the second stage procedure. Using the same 7,5mm Rochester trephine, 
two bone samples will be removed from bone within one centimeter of the resection margin. To avoid 
unnecessary damage to healthy bone, we will attempt to collect the bone samples from the part of sacrum 
removed in surgery. After the tumor has been resected, it is normally brought to surgical pathology by the 
surgeon.  There the specimen is examined by the pathologist. If there is enough normal bone remaining 
on the resected specimen, and the pathologist feels that using the normal bone will not interfere with his 

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31 
 
 
ability to assess the margin of resection, then the biopsy will be obtained from the surgical specimen 
rather than from bone remaining on the sacrum still attached to the patient’s spine. Figure 11 illustrates 
the two possible sites of the sacral biopsy.  
 
Figure 11: Location of Sacral Biopsy Site 
 
8.3.4 
Undecalcified Histology 
 
One control and one experimental sample from the biopsies will be embedded in plastic resin 
(MMA/dibutylpthalate) then split in half using a fine handsaw (0.3mm kerf). So as to not alter the 
mechanical properties of bone, one half will be scanned by micro-CT then processed for thin section 
histology. The other half will be sectioned at 1 mm intervals for quantitative back scatter electron imaging 
followed by nano indentation.  
8.3.5 
Immunohistochemistry 
 

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Cellular content of bone can be evaluated by immunohistochemical staining methods. Osteoblasts will be 
counted on toluidine blue stained slides as blue/grey cuboidal cells in clusters at the bone surface. 
Osteoclasts will be counted on slides assayed for TRAP activity as TRAP-positive multinucleated cells.  
 
What is measured: 
 
▪ 
Osteoblast number (N.Ob/BPm, mm-1)  
▪ 
Osteoblast surface (Ob. S/BS, %) 
▪ 
Osteoclast number (N.Oc/BPm, mm-1)  
▪ 
Osteoclast surface (Oc.S/BS, %) 
 
8.3.6 
Micro-CT 
 
Undecalcified biopsies will be scanned with a micro-CT system (X-Tek HMXST 225 microfocus X-ray 
system, Nikon Metrology, Tring,UK).  
 
What is measured: 
 
▪ 
Bone volume fraction (BV/TV)  
▪ 
Trabecular thickness (Tb.Th.)  
▪ 
Trabecular separation (Tb.Sp.) 
▪ 
Trabecular number (Tb.N.) 
▪ 
Connectivity density (Conn.D) 
▪ 
Structure model index (SMI)  
▪ 
Trabecular bone pattern factor (TBPf) 
 
8.3.7 
Nanoindentation 
 
Experimental design, data collection and analysis for nanoindentation will be done by our collaborator Dr. 
Mary Bouxsein, who has the expert knowledge and experience with this technology. Bone samples for 
nanoindendation cannot be embedded in paraffin because it would alter the elastic properties. The portion 
of the biopsy specimen for nanoindentation will be collected and stored in a small amount of normal 
saline solution at 4°C. Bone can remain stored in this fashion for several weeks without compromising the 
quality of the data. Measurements will be done with a nanohardness tester (Agilent G200 Nanoindenter, 
Santa ClaraCA) equipped with a diamond tip of Berkovich. The indentation will be performed with a load 
of 5 mN.  
 
What is measured: 
 
▪ 
Elastic modulus  
 
8.3.8 
Tetracycline Quadruple Labeling 
 
We will use a quadruple labeling scheduele, using two sets of double tetracycline labels witch differ in 
color under fluorescent light (ie tetracycline and demeclocycline). Figure 12 shows the schedule for the 
administration of the bone labeling drug. This way we will be able to distinguish the first and second label 

Effect of High Doses of Radiation on Bone Structure and Metabolism 
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33 
 
 
set. This technique has the great advantage that the patient can serve as his or her own pretreatment 
control, eliminating problems caused by the large intersite variablility in histomorphometric variables  
 
What is measured: 
 
▪ 
Mineral apposition rate 
▪ 
Mineral formation rate  
▪ 
Mineralization lag time 
 
Figure 12: Tetracycline Administration Schedule  
 
 
 
 
 
9. STUDY CALENDAR 
9.1.1 
Non- Surgical Arm Study Calendar 
 
 
 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Final visit 
Time point 
Screening 
Start RT 
Baseline 
 
Halfway RT 
~3.5 Weeks 
End of RT 
~7 Weeks 
~3Months 
after RT 
~6 
Months 
after RT 
Medical History 
X 
 
 
 
 
 
Study Blood test 
 
X 
X 
X 
X 
X 
CT scan 
 
X 
 
 
X 
 
Pregnancy Test 
X 
 
 
 
 
 
RT = radiotherapy 
* Study visit are dependent on treatment events like start/end of pre- or post-op radiotherapy and surgery. 
Therefore treatment delay will result in delay of study visits.  
 
 
 
 
 
 
 
 
 

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34 
 
 
9.1.2 
Surgical Arm Study Calendar 
 
 
 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
Visit 7 
Visit 8 
Final 
Visit 
Before Surgery 
Surgery Week 
After 
post-op 
RT 
Follow-Up  
 
Screening 
Start RT 
Baseline 
End RT 
~5.5 
Weeks 
3-5 
days 
before 
surgery 
Surgery 
stage 1 
Surgery 
stage 2 
End post-
op RT 
1 
month 
after 
post-op 
RT 
4 
months 
after 
post-op 
RT 
Medical 
History 
X 
 
 
 
 
 
 
 
 
Eligibility 
blood test 
X 
 
 
 
 
 
 
 
 
CT scan 
 
X 
 
X 
 
 
 
 
 
Study 
Blood test 
 
X 
X 
 
 
 
X 
X 
X 
Bone 
Biopsy 
 
 
 
 
X 
X 
 
 
 
Dispense 
drugs 
X 
 
X 
 
 
 
 
 
 
Pregnancy 
Test 
X 
 
 
 
 
 
 
 
 
RT = radiotherapy 
 
* Cycle 1 drugs will be taken between visit 1 and 2. Cycle 2 drugs will be taken between visit 3 and 4* 
Study visit are dependent on treatment events like start/end of pre- or post-op radiotherapy and surgery. 
Therefore treatment delay will result in delay of study visits.  
10. MEASUREMENT OF EFFECT 
 
N/A for this corellative study 
 
 
11. ADVERSE EVENT REPORTING REQUIREMENTS 
 
11.1 Definitions 
11.1.1 Adverse Event (AE) 
 
An adverse event (AE) is any undesirable sign, symptom or medical condition or 
experience that develops or worsens in severity after starting any study procedure 
specified in the protocol. 
 

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35 
 
 
Abnormal laboratory values or diagnostic test results constitute adverse events only if 
they induce clinical signs or symptoms or require treatment or further diagnostic tests.  
 
 
 
11.1.2 Serious adverse event (SAE) 
 
A serious adverse event (SAE) is any adverse event, occurring as a result of one of the 
study procedures:  
• 
Results in death 
• 
Is life-threatening. Life-threatening means that the person was at immediate risk 
of death from the reaction as it occurred, i.e., it does not include a reaction which 
hypothetically might have caused death had it occurred in a more sever form. 
• 
Requires or prolongs inpatient hospitalization (i.e., the event required at least a 
24-hour hospitalization or prolonged a hospitalization beyond the expected 
length of stay). Hospitalization admissions and/or surgical operations scheduled 
to occur during the study period, but planned prior to study entry are not 
considered SAEs if the illness or disease existed before the person was enrolled 
in the trial, provided that it did not deteriorate in an unexpected manner during 
the trial (e.g., surgery performed earlier than planned). 
• 
Results in persistent or significant disability/incapacity. Disability is defined as a 
substantial disruption of a person’s ability to conduct normal life functions. 
• 
Is a congenital anomaly or birth defect; or 
• 
Is an important medical event when, based upon appropriate medical judgment, it 
may jeopardize the participant and require medical or surgical intervention to 
prevent one of the outcomes listed above. Examples of such medical events 
include allergic bronchospasm requiring intensive treatment in an emergency 
room or at home; blood dyscrasias or convulsions that do not result in inpatient 
hospitalization, or the development of drug dependency or drug abuse. 
 
Events not considered to be serious adverse events are hospitalizations for: 
• 
routine treatment or monitoring of the studied indication, not associated with any 
deterioration in condition, or for elective procedures 
• 
elective or pre-planned treatment for a pre-existing condition that did not worsen 
• 
emergency outpatient treatment for an event not fulfilling the serious criteria 
outlined above and not resulting in inpatient admission 
• 
respite care 
 
11.1.3 Expectedness 
 
Adverse events can be 'Expected' or 'Unexpected.'  
11.1.3.1 Expected adverse event 
 
Expected adverse events are those that have been previously identified as 
resulting from administration of the bone labeling drug. For the purposes of this 

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36 
 
 
study, an adverse event is considered expected when it appears in the current 
adverse event list, the Investigator’s Brochure, the package insert or is included 
in the informed consent document as a potential risk.   
 
Refer to Section 6.1 for a listing of expected adverse events associated with 
the bone labeling drugs. 
11.1.3.2 Unexpected adverse event 
 
 
For the purposes of this study, an adverse event is considered unexpected when 
it varies in nature, intensity or frequency from information provided in the 
current adverse event list, the Investigator’s Brochure, the package insert or 
when it is not included in the informed consent document as a potential risk.   
11.1.4 Attribution 
 
Attribution is the relationship between an adverse event or serious adverse event and the 
study treatment. Attribution will be assigned as follows: 
 
• Definite – The AE is clearly related to the study treatment. 
• Probable – The AE is likely related to the study treatment. 
• Possible – The AE may be related to the study treatment.  
• Unlikely - The AE is doubtfully related to the study treatment. 
• Unrelated - The AE is clearly NOT related to the study treatment. 
11.2 Procedures for AE and SAE Recording and Reporting 
 
Participating investigators will assess the occurrence of AEs and SAEs at all participant 
evaluation time points during the study.  
 
All AEs and SAEs whether reported by the participant, discovered during questioning, directly 
observed, or detected by physical examination, laboratory test or other means, will be recorded 
in the participant’s medical record and on the appropriate study-specific case report forms.  
 
The descriptions and grading scales found in the revised NCI Common Terminology Criteria 
for Adverse Events (CTCAE) version 4.0 will be utilized for AE reporting. All appropriate 
treatment areas should have access to a copy of the CTCAE version 4.0. 
A copy of the CTCAE version 4.0 can be downloaded from the CTEP website at: 
 
 http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm.  
 
11.3 Reporting Requirements 
 
For multi-site trials where a DF/HCC investigator is serving as the principal investigator, each 
participating investigator is required to abide by the reporting requirements set by the DF/HCC. 
The study must be conducted in compliance with FDA regulations, local safety reporting 
requirements, and reporting requirements of the principal investigator.  

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37 
 
 
 
Each investigative site will be responsible to report SAEs that occur at that institution to their 
respective IRB. It is the responsibility of each participating investigator to report serious 
adverse events to the study sponsor and/or others as described below.  
11.4 Reporting to the Study Sponsor 
11.4.1 Serious Adverse Event Reporting 
 
All serious adverse events that occur after the initial dose of study treatment, during 
treatment, or within 30 days of the last dose of treatment must be reported to the DF/HCC 
Overall Principal Investigator on the local institutional SAE form. This includes events 
meeting the criteria outlined in Section 11.1.2, as well as the following: 
 
• 
Grade 2 (moderate) and Grade 3 (severe) Events – Only events that are 
unexpected and possibly, probably or definitely related/associated with the 
intervention. 
 
• 
All Grade 4 (life-threatening or disabling) Events – Unless expected AND 
specifically listed in the protocol as not requiring reporting. 
 
• 
All Grade 5 (fatal) Events – When the participant is enrolled and actively 
participating in the trial OR when the event occurs within 30 days of the last 
study intervention.  
 
Note: If the participant is in long term follow up, report the death at the time of 
continuing review.  
 
Participating investigators must report each serious adverse event to the DF/HCC Overall 
Principal Investigator within 24 hours of learning of the occurrence. In the event that the 
participating investigator does not become aware of the serious adverse event 
immediately (e.g., participant sought treatment elsewhere), the participating investigator 
is to report the event within 24 hours after learning of it and document the time of his or 
her first awareness of the adverse event. Report serious adverse events by telephone, 
email or facsimile to: 
 
 
 
Joseph Schwab, M.D. 
 
 
Office phone: 617-643-2483 
 
 
Email: jhschwab@partners.org 
 
 
Fax: 617-726-7587 
 
Within the following 24-48 hours, the participating investigator must provide follow-up 
information on the serious adverse event. Follow-up information should describe whether 
the event has resolved or continues, if and how the event was treated, and whether the 
participant will continue or discontinue study participation. 
11.4.2 Non-Serious Adverse Event Reporting  
 

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38 
 
 
Non-serious adverse events will be reported to the DF/HCC Overall Principal 
Investigator on the toxicity Case Report Forms.   
11.5 Reporting to the Institutional Review Board (IRB) 
 
Investigative sites within DF/HCC will report all serious adverse events directly to the DFCI 
Office for Human Research Studies (OHRS). 
 
Other investigative sites should report serious adverse events to their respective IRB according 
to the local IRB’s policies and procedures in reporting adverse events. A copy of the submitted 
institutional SAE form should be forwarded to: 
 
 
 
Joseph Schwab, M.D. 
 
 
Office phone: 617-643-2483 
 
 
Email: jhschwab@partners.org 
 
 
Fax: 617-726-7587 
 
 
The DF/HCC Principal Investigator will submit SAE reports from outside institutions to the 
DFCI Office for Human Research Studies (OHRS) according to DFCI IRB policies and 
procedures in reporting adverse events.  
11.6 Reporting to the Food and Drug Administration (FDA) N/A  
11.7 Reporting to the NIH Office of Biotechnology Activities (OBA) N/A  
11.8 Reporting to the Institutional Biosafety Committee (IBC) N/A 
 
11.9 Reporting to Hospital Risk Management 
 
Participating investigators will report to their local Risk Management office any subject safety 
reports or sentinel events that require reporting according to institutional policy. 
11.10 Monitoring of Adverse Events and Period of Observation 
 
All adverse events, both serious and non-serious, and deaths that are encountered from 
initiation of study intervention, throughout the study, and within 30 days of the last study 
intervention should be followed to their resolution, or until the participating investigator 
assesses them as stable, or the participating investigator determines the event to be irreversible, 
or the participant is lost to follow-up. The presence and resolution of AEs and SAEs (with 
dates) should be documented on the appropriate case report form and recorded in the 
participant’s medical record to facilitate source data verification.  
 
For some SAEs, the study sponsor or designee may follow-up by telephone, fax, and/or 
monitoring visit to obtain additional case details deemed necessary to appropriately evaluate the 
SAE report (e.g., hospital discharge summary, consultant report, or autopsy report).  

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39 
 
 
 
Participants should be instructed to report any serious post-study event(s) that might reasonably 
be related to participation in this study. Participating investigators should notify the DF/HCC 
Overall Principal Investigator and their respective IRB of any unanticipated death or adverse 
event occurring after a participant has discontinued or terminated study participation that may 
reasonably be related to the study.   
 
 
 
12. DATA AND SAFETY MONITORING 
 
12.1 Data Reporting 
 
12.1.1 Method  
 
The QACT will collect, manage, and monitor data for this study.  
 
12.1.2 Data Submission  
 
The schedule for completion and submission of case report forms (paper or electronic) to the QACT is as 
follows: 
 
Form 
Submission Timeline 
Eligibility Checklist 
Complete prior to registration with QACT 
On Study Form 
Within 14 days of registration 
Baseline Assessment Form 
Within 14 days of registration 
Treatment Form 
Within 30 days of the last radiation treatment 
Toxicity Report Form 
Weekly during radiation treatment; Within 30 days 
of  protocol defined follow up 
 
 
Off Treatment/Off Study Form 
Within 14 days of completing treatment or being 
taken off study for any reason 
Follow up/Survival Form 
Within 14 days of the protocol defined follow up 
visit date or call 
 
12.2 Safety Meetings 
 
The DF/HCC Data and Safety Monitoring Committee (DSMC) will review and monitor 
toxicity and accrual data from this trial as needed. The committee is composed of clinical 
specialists with experience in oncology and who have no direct relationship with the study. 
Information that raises any questions about participant safety will be addressed with the 
Principal Investigator and study team. 

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40 
 
 
 
The DSMC will meet as required to review toxicity and accrual data.  Information to be 
provided to the committee may include:  up-to-date participant accrual; current dose level 
information; DLT information; all grade 2 or higher unexpected adverse events that have been 
reported; summary of all deaths occurring within 30 days for Phase I or II protocols; for gene 
transfer protocols, summary of all deaths while being treated and during active follow-up; any 
response information; audit results, and a summary provided by the study team. Other 
information (e.g. scans, laboratory values) will be provided upon request.  
 
12.3 Monitoring 
 
Involvement in this study as a participating investigator implies acceptance of potential audits or 
inspections, including source data verification, by representatives designated by the Principal Investigator. 
The purpose of these audits or inspections is to examine study-related activities and documents to 
determine whether these activities were conducted and data were recorded, analyzed, and accurately 
reported in accordance with the protocol, institutional policy, Good Clinical Practice (GCP), and any 
applicable regulatory requirements. 
 
All data will be monitored for timeliness of submission, completeness, and adherence to protocol 
requirements. Monitoring will begin at the time of participant registration and will continue during 
protocol performance and completion.  
 
13. REGULATORY CONSIDERATIONS 
 
13.1 Protocol Review and Amendments 
 
This protocol, the proposed informed consent and all forms of participant information related to the study 
(e.g., advertisements used to recruit participants) and any other necessary documents must be submitted, 
reviewed and approved by a properly constituted IRB governing each study location.  
Any changes made to the protocol must be submitted as amendments and must be approved by the IRB 
prior to implementation. Any changes in study conduct must be reported to the IRB. The DF/HCC 
Overall Principal Investigator (or Protocol Chair) will disseminate protocol amendment information to all 
participating investigators.  
 
All decisions of the IRB concerning the conduct of the study must be made in writing. 
 
13.2 Informed Consent 
 
All participants must be provided a consent form describing this study and providing sufficient 
information for participants to make an informed decision about their participation in this study. The 
formal consent of a participant, using the IRB approved consent form, must be obtained before the 
participant is involved in any study-related procedure. The consent form must be signed and dated by the 
participant or the participant’s legally authorized representative, and by the person obtaining the 

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41 
 
 
consent. The participant must be given a copy of the signed and dated consent document. The original 
signed copy of the consent document must be retained in the medical record or research file.  
 
13.3 Ethics and Good Clinical Practice (GCP) 
 
This study is to be conducted according to the following considerations, which represent good and sound 
research practice: 
• 
E6 Good Clinical Practice: Consolidated 
Guidancewww.fda.gov/downloads/RegulatoryInformation/Guidances/UCM129515.
pdf 
• 
US Code of Federal Regulations (CFR) governing clinical study conduct and ethical 
principles that have their origin in the Declaration of Helsinki 
o Title 21 Part 11 – Electronic Records; Electronic Signatures 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr11_02.html 
o Title 21 Part 50 – Protection of Human Subjects 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr50_02.html 
o Title 21 Part 54 – Financial Disclosure by Clinical Investigators 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr54_02.html 
o Title 21 Part 56 – Institutional Review Boards 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr56_02.html 
o Title 21 Part 312 – Investigational New Drug Application 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr312_02.html 
• 
State laws 
• 
DF/HCC research policies and procedures http://www.dfhcc.harvard.edu/clinical-
research-support/clinical-research-unit-cru/policies-and-procedures/ 
It is understood that deviations from the protocol should be avoided, except when necessary to eliminate 
an immediate hazard to a research participant. In such case, the deviation must be reported to the IRB 
according to the local reporting policy.  
 
13.4 Study Documentation 
 
The investigator must prepare and maintain adequate and accurate case histories designed to record all 
observations and other data pertinent to the study for each research participant. This information enables 
the study to be fully documented and the study data to be subsequently verified. 
 
Original source documents supporting entries in the case report forms include but are not limited to 
hospital records, clinical charts, laboratory and pharmacy records, recorded data from automated 
instruments, microfiches, photographic negatives, microfilm or magnetic media, and/or x-rays.  
 
13.5 Records Retention 
 
All study-related documents must be retained for the maximum period required by applicable federal 
regulations and guidelines or institutional policies.  
 

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42 
 
 
13.6 Multi-center Guidelines 
 
N/A 
 
13.7 Cooperative Research and Development Agreement (CRADA)/Clinical Trials 
Agreement (CTA) 
 
N/A 
 
 
14. STATISTICAL CONSIDERATIONS 
 
14.1 Study Design/Endpoints 
 
We will have two groups of patients, those treated with radiation alone, and combination of surgery and 
radiation. Figure 13 summarizes the proposed enrollment groups for this study, depending on the type of 
treatment patients are undergoing.  
 
Figure 13: Proposed Subject Enrollment Plan  
 
 
14.1.1 Primary Endpoint 
 
▪ 
Outcome of interest: change in trabecular bone mineral density (Tb. BMD) measured by QCT of 
the lumbar spine at the start and at the end of radiation treatment 
 
 
14.1.2 Exploratory Endpoints 
 
▪ 
Longitudinal measurement of serum markers of bone turnover rate over 6 months 
▪ 
Measurement of static and dynamic bone parameters summarized in Table 1.  
 
Table 1: Exploratory Endpoints of the Study 
Technique 
Measurement 
Immunohistochemistry:  
 
Osteoblast number and surface  
Osteoclast number and surface 

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43 
 
 
Micro-CT : 
  
Bone volume fraction  
Trabecular number 
Trabecular thickness and separation 
Connectivity density  
Structure model index  
Trabecular bone pattern factor 
Nanoindentation : 
 
Elastic modulus 
Tetracycline quadruple 
labeling : 
  
Mineralization lag time  
Mineral apposition rate 
Mineral formation rate 
 
14.2 Sample Size/Accrual Rate 
 
Berthold and Haras published a range of normal reference values for the trabecular bone mineral density 
measured by quantitative CT of the lumbar spine in young adults. In healthy males and females the mean 
trabecular bone mineral density was 150mg /mL with a standard deviation of 20mg/mL at the start of 
puberty. In contrast, we expect our patients to be considerably older, to have one or many systemic 
illnesses, and to have tumors in the sacrum that can compromise bone quality. We expect that mean bone 
density at the start of the treatment in our population will be considerably lower. Assuming that it’s at 
least 1 standard deviation less that of a normal young adult, we get an estimate of 130mg/dL at the start of 
treatment in all study patients. It is likely that our population will exhibit significant differences by sex 
owing to bone density being more affected by age in females than in males. In absence of prior studies, it 
is very difficult to estimate this difference. In fact, the purpose of this pilot study is in part to provide 
some numerical data to help guide future studies. At present, we must carry our sample size calculations 
keeping in mind these potential pitfalls. 
 
The “event of interest” in this study is a change in trabecular bone density. Our hypothesis is that 
radiation induces loss of trabecular bone density in surgical and non-surgical patients.  We would like to 
be able to detect a change in the average bone density of 10 mg/mL (approximately 5%) before and after 
radiation treatment. Assuming the standard deviation of the change we hope to observe is 15mg/dL, we 
get the following estimates.  
 
Size = 30, Power = 0.942, alpha = 0.05 
Size = 20, Power = 0.807, alpha = 0.05  
Size = 10, Power = 0.469, alpha = 0.05 
 
The main treatment arm in our patient population is the surgical patients. We plan to enroll 20 surgical 
patients given the results of the power analysis. The purpose of the non-surgical arm is to serve as a 
control for any confounding effects due to surgery. We plan to enroll 10 non-surgical patients. Smaller 
sample size significantly reduces the power of our analysis in the non-surgical arm. However, given that 
the vast majority of our patients receive surgery, the time it would take to enroll 20 patients into the non-
surgical arm would undermine the purpose of doing an exploratory study to begin with.    
 
The Kolmogorov–Smirnov test on the normality of the distribution of bone mineral density and trabecular 
bone mineral density values will be carried out in each group. To evaluate the effects of radiation, we will 

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44 
 
 
use the Student t-test and Mann–Whitney test to compare the results from the iliac crest and the sacrum. 
We will use the Cox stratified model to identify confounding factors.    
 
We plan to enroll 1-2 subjects per month. Subject enrollment is limited by patient availability. 
 
The estimated study accrual by gender, race and ethnicity is shown in the table below. 
 
Accrual Targets 
Ethnic Category 
Sex/Gender 
Females 
 
Males 
 
Total 
Hispanic or Latino 
1 
+ 
0 
= 
1 
Not Hispanic or Latino 
14 
+ 
15 
= 
29 
Ethnic Category: Total of all subjects 
15 
+ 
15 
= 
30 
Racial Category 
 
American Indian or Alaskan Native 
0 
+ 
0 
= 
0 
Asian 
0 
+ 
0 
= 
0 
Black or African American 
1 
+ 
1 
= 
2 
Native Hawaiian or other Pacific 
Islander 
0 
+ 
0 
= 
0 
White 
14 
+ 
14 
= 
28 
Racial Category: Total of all subjects 
15 
+ 
15 
= 
30 
 
 
 
 
 
 
 
14.3 Stratification Factors 
 
Patients will be stratified by the type of treatment they are undergoing (surgical vs. non-surgical). 
 
14.4 Analysis of Secondary Endpoints 
 
Paired t-test and Wilcoxon signed-rank test will be performed on the differences between the two groups 
in the average values of PROMIS T scores. A P-value of 0.05 will be considered to be statistically 
signiﬁcant. Spearman and Kendall rank correlations will be used to measure the strength of dependence 
between the bone mineral density and exploratory variables whenever possible.  
 
14.5 Reporting and Exclusions 
14.5.1 Evaluation of toxicity 
 
N/A 
 

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45 
 
 
14.5.2 Evaluation of response 
 
N/A 
 
15. PUBLICATION PLAN 
 
 
The results of the study will be made public within 24 months of the end of data collection. Our group 
plans to publish a report in a peer-reviewed journal. As an initial release, we plan to publish and present 
an abstract that meets the requirements of the International Committee of Medical Journal Editors at the 
Annual Meeting of the Sacral Tumor Research Group. A full report of the outcomes will be made public 
no later than three (3) years after the end of data collection.  
 
 
 
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7. Dudziak ME, Saadeh PB, Mehrara BJ, Steinbrech DS, Greenwald JA, Gittes GK, LongakerMT.The 
effects of ionizing radiation on osteoblast-like cells in vitro.PlastReconstr Surg. 2000;5:1049-1061. 
8. Fuchs B, Dickey ID, Yaszemski MJ, Inwards CY, Sim FH. Operative management of sacral chordoma. 
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17. APPENDICES 
17.1 Appendix 1: List of Figures 
Figure 1:  Phase Transition into Clinical Trials ............................................................................................ 6 
Figure 2: Experimental Design Summary ..................................................................................................... 6 
Figure 3: Bilateral Sacral Fractures .............................................................................................................. 7 
Figure 4: Experimental Design: Summary of the Proposed Experimental Techniques and Measurements 
to be Collected in the Study ........................................................................................................................ 10 
Figure 5: Determination of Bone Structure in the Adult Spine by Quantitative CT (QCT) ....................... 11 
Figure 6: Definition of ROI in the Lumbar Spine ....................................................................................... 12 
Figure 7: Visualization of Tetracycline Labeled Bone by Fluorescent Microscopy ................................... 13 
Figure 8: Schematic Representation of a Typical Load-Displacement Curve ............................................ 14 
Figure 9: Radiation Treatment Flowchart ................................................................................................... 17 
Figure 10: Location of the Planned Bone Biopsy Sites. ............................................................................. 29 
Figure 11: Location of Sacral Biopsy Site .................................................................................................. 30 
Figure 12: Tetracycline Administration Schedule ...................................................................................... 32 
Figure 13: Proposed Subject Enrollment Plan ............................................................................................ 41 
 
 
 
 
 
 

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17.2 Appendix 2: Patient Drug Schedule And Diary 
 
 
 
 
 
 
 
 
 
 

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17.3 Appendix 3: Summary of Experimental Procedures 
In vivo studies (serum labs and imaging) 
Experiment 
Measurement 
QCT 
Trabecular bone mineral density (Tb.BMD) 
Serum markers 
N-Telopeptide (NTX) 
Tartrate-resistant acid phosphatase (TRAP) 5b 
 Osteocalcin (OC) 
Total procollagen type 1 N-terminal propeptide 
(P1NP) 
 PTH 
Ca (as part of a standard metabolic panel) 
 
 
In vitro studies (on bone biopsy samples) 
Experiment 
Measurement 
Immunohistochemistry 
 
Osteoblast number (N.Ob/BPm, mm-1)  
Osteoblast surface (Ob. S/BS, %) 
Osteoclast number (N.Oc/BPm, mm-1)  
Osteoclast surface (Oc.S/BS, %) 
Micro-CT 
 
Bone volume fraction (BV/TV)  
Trabecular thickness (Tb.Th.)  
Trabecular separation (Tb.Sp.) 
Trabecular number (Tb.N.) 
Connectivity density (Conn.D) 
Structure model index (SMI)  
Trabecular bone pattern factor (TBPf) 
Quantitative back scatter electron imaging (qBEI)  
Bone mineral density (BMD) 
Nanoindentation 
Elastic modulus of bone 
Tetracycline quadruple labeling 
Mineral apposition rate 
Mineral formation rate  
Mineralization lag time 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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