Trial Information

Radioactive Iodide Therapy for Pediatric Graves' Disease

Primary Aims. The investigators propose to assess the safety of 131I use in children with
hyperthyroidism due to Graves' disease (GD). The investigators will measure whole body
radiation exposure following 131II therapy in children treated for GD. The investigators
will assess the effects of GD treatment on chromosome structure.

These studies will involve collaborative efforts with Dr. Patrick Zanzonico (Memorial
Sloan-Kettering Cancer Center), who is an expert in 131I dosimetry and Dr. James Tucker
(Wayne State University), who is an expert in cytogenetic effects of radiation. The studies
will involve children treated for GD at University of Florida University and Baylor
University. These studies have been designed with the help of the University of Florida
Center for Clinical Investigation, Biostatistics Support Unit, which will be involved in
data analysis.

Characteristics of study population. The investigators will recruit a total of 150 patients
diagnosed with GD younger than 18 years of age. All subjects are to be treated with 131I. In
this trial, children will not be randomized to treatment, but will be treated per physician
prescribed care. To ensure an equal distribution of age and gender between the two groups of
children, the investigators stratify enrollment by gender (male vs. female) and age (5-10
yrs, 10-15 yrs, 15-18 yrs).

Two sites will enroll patients to achieve the desired sample size: Baylor College of
Medicine and University of Florida University. These sites have been selected for the
following reasons. (1) These are large centers where radioactive iodide has been used for
decades. (2) Each site has treated large number of children with radioactive iodide. (3)
Each site has computerized patient databases and contact information for children treated
with 131I is known. (4) The investigators have working relationships with collaborators at
these sites. Based on the relative patient volumes of Baylor and University of Florida, the
investigators anticipate that 70% of patients will come from Baylor and 30% from University
of Florida. Calculations supporting the sample size are detailed in each of the two aims
below.

Patient eligibility. Eligibility criteria include the following:

1. A diagnosis of GD based on initial laboratory studies showing a suppressed Thyroid
Stimulating Hormone (TSH) (i.e. <0.01); high total triiodothyronine (T3), high total
thyroxine (T4) and/or free T4 level; an elevated thyroid stimulating immunoglobulin
(TSI) titer; increased and diffuse uptake of 123I, 131I, or 99Tc in the thyroid gland.

2. Age <18 years at the time of GD disease diagnosis.

3. Non-smoking parents. Subject enrollment. Practitioners in the University of Florida
Pediatric Thyroid Center and the Baylor Pediatric Endocrinology Division will identify
eligible individuals for study participation. Patients will be enrolled after
appropriate consent/assent procedures have been satisfied. At the time of collection,
the investigators will record age, gender, current treatment, and treatment history for
Graves' disease (i.e., antithryoid drugs (ATDs) and/or 131I therapy including dose).

These studies will only be performed on children treated with 131I as part of physician
prescribed clinical care. Children will not be treated with 131I for the sole purpose of
generating subjects for this study.

After treatment is decided upon by the physician and the patient, then the patient will be
offered participation as to provide balanced enrollment for each treatment/age/gender
category.

1. Primary aim (1) and secondary aim (i): Perform dosimetry to assess whole body and
tissue specific radiation exposure in children treated with 131I and determine
potential cancer risk from these data. At present, no data are available to assess
whole body and tissue-specific radiation exposure for children treated with 131I for
GD. Theoretical modeling has been done, but this has not been based on actual data.
Knowing the exposure of specific organs to radioactivity can be used to determine
tissue-specific risk for malignancies. The investigators thus propose to perform a
cross-sectional dosimetry study on children being treated with 131I to determine tissue
specific and whole body radiation exposure. These studies have been designed by Dr.
David Cheng (University of Florida University), Dr. Patrick Zanzonico (Memorial
Sloan-Kettering Cancer Center; NY), and Dr. James Dziura (University of Florida
University).

Patient's Total-Body Mass and Administered Activity. On the day of administration of
the therapeutic administered activity of 131I, the patient will be weighed. Immediately
prior to administration, the therapeutic administered activity of 131I will be measured
in a dose calibrator on the 131I setting, and the activity and the dates and times of
assay and of administration recorded. This activity will be prescribed by the treating
physicians at University of Florida or Baylor.

Gamma Camera Imaging. All 131I gamma camera whole-body scanning will be performed using
a 20% photopeak energy window (i.e. 364 keV + 10% = 328 to 400 keV) and a scan speed of
10 cm/min for all scans. The scan length will be set for each patient to include the
entire patient and the same scan length will be used for all scans of a given patient.
The exact dates and times of each whole-body scan will be recorded. The time
post-administration of each whole-body scan will be calculated as the time interval (in
hours) from the date and time of 131I administration and the date and time of
whole-body scanning.

Determination of Organ and Total-Body Activities. The determination of organ and
total-body activities will use each patient as his or her own calibration standard. The
patient will undergo a conjugate-view whole-body scan within 30 to 60 minutes after the
131I administration (i.e. nominally specified as time 0) but before the first
post-administration void or bowel movement. In addition, scans will be performed at one
day and, four days after administration of the dose. A blood sample (10cc) will also be
drawn to measure the amount of radioactive iodide in the blood at these times and for
assessment of DNA damage markers. For each patient, the net (i.e.
background-subtracted) geometric-mean count rate for the total body for this initial
scan thus corresponds to 100% of the administered activity. As noted, this scan will be
performed at 30 to 60 minutes post-administration to allow some dispersion of the
activity throughout the body, so that the effects of scatter and attenuation are
grossly the same for this initial scan as for subsequent scans of the patient. For the
time-0 and each subsequent conjugate-view whole-body scan, the posterior
(lower-detector) gamma-camera image is "mirrored" to align it with the anterior
(upper-detector) image.

The regions of interest (ROIs) will be manually drawn around the organs of interest
(the thyroid, salivary glands, liver, intestines, stomach, and urinary bladder) and the
total body. For each organ, its ROI may be drawn in the scan in which it is best
visualized and then copied and pasted onto the other whole-body scans, translating
and/or rotating the ROI as needed on these other scans to accurately superimpose it on
the organ. Note that, for each scan, a single background (BG) ROI, drawn outside of but
close to the body, may be used.

Statistical Analysis. OLINDA-based Calculation of Organ Absorbed Doses and Effective
Dose. The OLINDA dosimetry program will be used to assess absorbed doses (11, 76). In
OLINDA, the investigators will select the "Fraction and Half-times" option (in OLINDA's
"Kinetics Input Form") and enter the best-fit parameters of the respective
time-activity functions (A/100% and Ta and, if, applicable, B/100% and Tb) for the
specified source regions - the thyroid, salivary glands, liver, intestinal contents,
stomach contents, urinary bladder contents, red marrow, and total body. Note that
OLINDA requires the zero-time intercept values in fraction (not %) of the administered
activity. Click "hr" radiobutton for the "Half-life Units" and the "Biological"
radiobutton for the "Half-lives." Also in OLINDA, the investigators will select
iodine-131 (I-131") as the nuclide (in OLINDA's "Nuclide Input Form") and the anatomic
model most closely approximating the age or total-body mass of the patient as the model
(in OLINDA's "Model Input Form"). Then, select the "Main Input Form" and click the
"Doses" button to calculate the organ doses and the effective dose.

Evaluation of Radiation exposure. Distributions of the primary outcome measures for the
total body and organ-specific radiation exposure (described above) will be summarized
graphically (boxplots) and numerically (means, standard deviations, medians,
interquartile ranges).

Radiation exposure (e.g., absorbed dose of 131I in the total body and specific organs)
will be compared across specific categories of the administered dose of 131I, as well
as across age groups, and gender using Analysis of Variance (ANOVA). The investigators
will also evaluate whether there were outcome differences by study site. Should data
not comply with distributional assumptions required of the ANOVA, alternative
non-parametric techniques (i.e. Kruskal-Wallis test) will be considered. The
investigators will correlate the administered dose of 131I with the absorbed dose of
the radioactive agent, using Spearman's Rank Correlation. In all analyses, alpha of
0.05 will be used.

2. Primary aim (2) and secondary aim (ii): Assess chromosomal translocations in children
treated with 131I and evaluate chromosomal translocations as related to patient's age
and 131I exposure. Low-level, whole body irradiation is a risk factor for cancer 58 .
The prolonged use of certain medications is associated with the risk of cancer in some
circumstances as well. Current 131I therapy for Graves' disease in children and adults
aims for ablation sufficient amounts of thyroid gland to result in a hypothyroid state.
This treatment, though, will also be associated with low-level whole body
irradiation11. Studies of adults, who have been treated with 131I, have revealed small
increases in rates of stomach and breast cancer. Although it has been suggested that
children are more prone to carcinogenic risks of low level irradiation58, there have
not been any studies with a sufficient sample size to assess long-term cancer risk in
children treated with 131I.

Recent data convincingly show that chromosome translocations are associated with long-term
cancer risks. Chromosome translocations are a molecular sign of ionizing radiation exposure.
Importantly, translocations persist for decades after radiation exposure22. This persistence
makes chromosomal translocations the gold-standard aberration type for performing radiation
dosimetry when there is a lag between the time of exposure and assessment. Normative data
for chromosomal translocations are available, as related to age and gender20.

The investigators therefore propose to perform an observational cohort study of children
treated for Graves' disease to assess chromosomal translocation. These studies will be
performed on the children in which dosimetry is performed, as detailed above. The
investigators will stratify enrollment by gender and age to ensure a comparable distribution
of these characteristics. The chromosome translocation studies, at baseline and at the 12
month-follow-up.

Treatment with 131I. Patients will be treated with 131I as detailed above. Sample
Collection. Blood will be obtained for chromosome translocation analysis at baseline and at
12 months after treatment with 131I, or after receiving surgery or ATDs. For blood
collection, a heparinized vacutainer will be used to collect 5 ml of blood. Blood will be
obtained at the time of routine phlebotomy for assessment of thyroid hormone levels.

1. Sample size calculation. The investigators will test the hypothesis that translocation
frequencies are higher in subjects receiving 131I compared to subjects receiving
alternative treatment (ATDs or surgery only) for GD. Since there is a low level of
chromosomal breaks in healthy children20, if there is an increase in chromosomal
translocation it should be possible to detect significant increases with a relatively
small sample size. Our estimates of sample size are based on rates of translocation
described by Sigurdson who observed rates of 0.2 translocations per 100 cell
equivalents in children under 20 y. Given these baseline rates and using the PASS 2005
module for Poisson regression, the investigators estimated that a sample size of 135
children treated with 131I and 135 treated with ATDs or surgery will provide 80% power
at the two-sided 0.05 significance level to detect a doubling of the chromosomal
translocation rate between the two groups of patients at 12 months after tre. The
investigators will aim for 1/3 of the children being in each of the following age
groups: 5-10 yrs, 10-15 yrs, 15-18 yrs. The investigators will enroll 150 subjects in
each group to accommodate a potential 10% loss to follow-up.

2. FISH assay for chromosome aberrations. Personnel in Dr. Tucker's laboratory will
determine the frequency of chromosome translocations using Fluorescence In Situ
Hybridization (FISH) whole chromosome painting probes. All samples will be coded so
that the Tucker laboratory will not know the radiation exposure history of the
subjects. Cell cultures will be initiated 24-48 hr after phlebotomy in Dr. Tucker's
laboratory and processed according to routine cytogenetic methods. Approximately 1,800
metaphase cells will be evaluated per subject, and this will be equivalent to 1,800 x
0.56 = 1,000 metaphase cells (define as cell equivalents; CEs) as if the full genome
had been scored. All translocations in cells will be enumerated and the frequency of
translocations per 100 CEs will be used as the dependent variable in the statistical
analyses.

3. Data Analysis. Data analysis will be conducted in collaboration with the Biostatistics
Unit of the University of Florida Center for Clinical Investigation. All analyses will
be performed using SAS v9.2 (SAS Institute, Cary, NC) with a two-sided 0.05 type I
error used to evaluate statistical significance. Frequency distributions and
missingness will be examined for each variable. The investigators will omit from
consideration in further analyses variables with homogenous distributions or with a
high degree of missingness and collapse categorical variables with underrepresented
levels. Associations between independent variables will be examined using Spearman
correlation coefficients, principal components and hierarchical clustering (PROC
VARCLUS).

Demographic (age, gender and race of child and primary caretaker), socioeconomic
(parental education and income), and clinical variables (e.g., duration of Graves
disease, ATD treatment, and 131I dose) will be compared between the two treatment
groups at baseline using t-tests for continuous variables and chi-square tests for
categorical variables. Meaningful clinical differences will be reported and adjusted
for in the multivariate analysis of chromosomal translocation described below.

(ii) Comparison of Chromosomal Translocation Frequencies. The number of chromosomal
translocations at baseline and at 12 months post treatment will be determined using
means and confidence intervals. These data will also be compared to our data for
healthy children.

The investigators will compare the number of chromosomal translocations at baseline and
at 12 months between groups in a multivariate model, using zero-inflated Poisson mixed
model analysis69. The zero-inflated Poisson model accommodates the increased variance
that is typical of count data with a large proportion of zeroes. Furthermore, through
the inclusion of random effects, the mixed model analysis will allow for the
correlation from repeated observations. The mixed model also accommodates individuals
with incomplete observations (i.e. lost to follow-up) under the assumption that given
the observed data the missing data is not dependent on unobserved values.

The following fixed effects will be used: treatment group (131I treatment vs. surgery
or ATD group), selected covariates (e.g., child age and gender, 131I dose, parental
education or income), time (baseline and at 12months), and time by treatment group
interaction. A random effect will be used to account for possible correlation between
the number of chromosomal translocations at baseline and 12 months for the same
subject. The investigators will also explore whether the effect of treatment on
chromosomal translocations is modified by the age of child and the received radiation
dose, by including three-way interaction terms between treatment, age and time or
treatment, radiation dose and time.

Several strategies will be imposed to accommodate the likelihood that missing data will
occur during this study. Prevention is the most obvious and effective manner to control
bias and loss of power from missing data. Postcard and telephone visit reminders will
be delivered to participants prior to protocol specified collection times. Alternative
contacts will be identified on entry into the study to minimize loss-to follow-up.
Timely data entry combined with weekly missing data reports will trigger protocols for
tracking and obtaining missing data items or outcome assessments. Despite these
prevention efforts, it is reasonable to assume missing data will occur. The primary
analysis method will use a likelihood-based mixed model which accommodates incomplete
observations and operates under the assumption that the missing data is missing at
random (MAR)69. Missing data patterns and reasons for dropout will be compared between
the treatment groups. T-tests, cross-tabulations and logistic regression will be used
to evaluate whether withdrawal is dependent on any observed variables.

4. Statistical Considerations: Describe the statistical analyses that support the study
design.

The investigators used Power Analysis and Sample Size software (PASS 2005) to estimate
precision around a mean of radiation exposure (e.g., expressed as mean percent of
administered activity in an organ/total body or Olinda-based mean organ absorbed dose and
mean effective dose). A sample of 150 subjects produces a 95% confidence interval equal to a
mean plus or minus 0.16 standard deviations. From a previously published study of children
with Graves disease (ages 7-18 years)42, the investigators estimated that our sample of 150
patients can be stratified into patients who will receive 150-200 Gy and 200-300 Gy. Given
these proportions, the investigators will be able to estimate stratum-specific 95%
confidence intervals around the means with a precision of 0.32 standard deviations for the
two smaller strata and 0.16 standard deviations for the larger strata.