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Lancet Oncol. Author manuscript; available in PMC 2020 December 29.
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Published in final edited form as:

Lancet Oncol. 2017 February ; 18(2): 202–211. doi:10.1016/S1470-2045(16)30648-9.

A genome-based model for adjusting radiotherapy dose (GARD):

a retrospective, cohort-based study
Jacob G Scott, Anders Berglund, Michael J Schell, Ivaylo Mihaylov, William J Fulp, Binglin
Yue, Eric Welsh, Jimmy J Caudell, Kamran Ahmed, Tobin S Strom, Eric Mellon, Puja
Venkat, Peter Johnstone, John Foekens, Jae Lee, Eduardo Moros, William S Dalton, Steven
A Eschrich, Howard McLeod, Louis B Harrison, Javier F Torres-Roca
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Department of Radiation Oncology (J G Scott MD, J J Caudell MD, K Ahmed MD, T S Strom, MD,
E Mellon MD, P Venkat MD, Prof P Johnstone MD, Prof E Moros PhD, Prof L B Harrison MD, J F
Torres-Roca MD), Department of Integrated Mathematical Oncology (J G Scott), Department of
Integrated Bioinformatics and Biostatistics (A Berglund PhD, Prof M J Schell PhD, W J Fulp MS, B
Yue MS, E Welsh PhD, Prof J Lee PhD, S A Eschrich PhD), DeBartolo Family Personalized
Medicine Institute (Prof W S Dalton MD, Prof H McLeod Pharm D), and Department of Chemical
Biology and Molecular Medicine (J F Torres-Roca), Moffitt Cancer Center and Research Institute,
Tampa, FL, USA; Department of Radiation Oncology, University of Miami, Miami, FL, USA (I
Mihaylov PhD); and Department of Medical Oncology and Cancer Genomics, Erasmus Medical
Center, Rotterdam, Netherlands (Prof J Foekens MD)

Summary
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Background—Despite its common use in cancer treatment, radiotherapy has not yet entered the
era of precision medicine, and there have been no approaches to adjust dose based on biological
differences between or within tumours. We aimed to assess whether a patient-specific molecular
signature of radiation sensitivity could be used to identify the optimum radiotherapy dose.

Methods—We used the gene-expression-based radiation-sensitivity index and the linear

quadratic model to derive the genomic-adjusted radiation dose (GARD). A high GARD value
predicts for high therapeutic effect for radiotherapy; which we postulate would relate to clinical
outcome. Using data from the prospective, observational Total Cancer Care (TCC) protocol, we
calculated GARD for primary tumours from 20 disease sites treated using standard radiotherapy
doses for each disease type. We also used multivariable Cox modelling to assess whether GARD
was independently associated with clinical outcome in five clinical cohorts: Erasmus Breast
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Correspondence to: Dr Javier F Torres-Roca, Moffitt, Cancer Center and Research, Institute, Tampa, FL 33612, USA,
[email protected].
Contributors
JGS, LBH, and JFT-R designed the study. WSD and JF collected data. AB, MJS, IM, WJF, BY, EW, JJC, KA, TJS, EM, PV, PJ, JF, JL,
SAE, HLM, and JFT-R contributed to data analysis. JGS, AB, MJS, IM, WJF, BY, EW, JL, SAE, and JFT-R designed the figures. All
authors contributed to data interpretation and writing,
Declaration of interests
JGS and JFT-R are named inventors in a patent pending for systems for providing personalised radiation therapy. SAE and JFT-R are
named inventors in patent number 8,660,801, patent number 8,665,598 and patent number 7,879,545 are related to radiosensitivity
index. PJ reports receipt of personal fees from Novocure. SAE is a cofounder of Cvergenx and serves on the board and as an officer
for the company. He holds stock and stock options in the Cvergenx. HLM serves on the board of directors for Cancer Genetics and on
an advisory board for Kew Corporation. JFT-R reports stock in Cvergenx and has a patent issued for radiation sensitivity index with
royalties paid to Cvergenx, and a patent pending for GARD.
 Scott et al. Page 2

Cancer Cohort (n=263); Karolinska Breast Cancer Cohort (n=77); Moffitt Lung Cancer Cohort
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(n=60); Moffitt Pancreas Cancer Cohort (n=40); and The Cancer Genome Atlas Glioblastoma
Patient Cohort (n=98).

Findings—We calculated GARD for 8271 tissue samples from the TCC cohort. There was a
wide range of GARD values (range 1·66–172·4) across the TCC cohort despite assignment of
uniform radiotherapy doses within disease types. Median GARD values were lowest for gliomas
and sarcomas and highest for cervical cancer and oropharyngeal head and neck cancer. There was
a wide range of GARD values within tumour type groups. GARD independently predicted clinical
outcome in breast cancer, lung cancer, glioblastoma, and pancreatic cancer. In the Erasmus Breast
Cancer Cohort, 5-year distant-metastasis-free survival was longer in patients with high GARD
values than in those with low GARD values (hazard ratio 2·11, 95% 1·13–3·94, p=0·018).

Interpretation—A GARD-based clinical model could allow the individualisation of radiotherapy

dose to tumour radiosensitivity and could provide a framework to design genomically-guided
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clinical trials in radiation oncology.

Introduction
Radiotherapy is an efficacious and cost-effective treatment that is received by up to two-
thirds of all patients with cancer in the USA. It is estimated to be responsible for 40% of all
cancer cures, yet represents only 5–10% of all cancer-related health expenditures. Despite its
therapeutic importance, it is under-represented in the national portfolio of clinical trials (eg,
only 5·5% of US National Cancer Institute trials involve radiotherapy).1,2

The sequencing of the human genome has paved the way for the era of precision medicine,
which aims for the right treatment to be delivered to the right patient at the right time. The
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US National Institutes of Health defines precision medicine as an approach to disease

prevention and treatment based on individual differences in environment, genes, and
lifestyle.3 Although the genomic era has affected the delivery of chemotherapy and targeted
biological agents,4 it has yet to affect radiotherapy, the most commonly used therapeutic
agent in oncology.5 A central principle in precision medicine is that cancer therapy should
be tailored to individual tumour biology.6 Despite this, radiotherapy dose protocols are
uniform (ie, one-size-fits-all), with the underlying assumption that every patient has the
same opportunity to benefit from radiotherapy. However, genomic studies have shown
biological heterogeneity to be a central characteristic of cancer.7

Previously, our group has focused on the identification of pan-tissue radiotherapy-specific

biomarkers. We hypothesised that the biological networks that regulate radiosensitivity and
radioresistance would be conserved across disease sites. We thus developed a gene-
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expression-based radiosensitivity index as a molecular estimate for cellular survival fraction

at 2 Gy (SF2). We did this by training a linear regression model to predict the experimental
SF2 value for 48 cancer cell lines from nine different disease sites, based on the expression
of ten specific genes extracted from an interaction network developed using a systems
biology approach.8–10

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The gene-expression-based radiosensitivity index was validated in vitro and was shown to
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predict tumour response to preoperative radiotherapy in patients with rectal cancer or

oesophageal cancer and to predict clinical outcome in patients with breast cancer, head and
neck cancer, glioblastoma, pancreatic cancer, and metastatic colorectal cancer treated with
radiotherapy.8,9,11–15 These data support the concept that clinical benefit from radiotherapy
(ie, the effect radiotherapy has on clinical outcome) is non-uniform and is highest in a
subpopulation of genomically-distinct patients (ie, a radiosensitive population). We use this
notion to derive the concept of radiation therapeutic effect.

The linear quadratic model was first proposed by Lea16 to describe the biological response
to radiation. The linear quadratic model is used to estimate different radiation fractionation
schemes with similar clinical effect, and has been successfully used in several large
randomised trials in prostate cancer comparing standard fractionation with
hypofractionation.17 Integrating individual biological differences into radiotherapy protocols
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is a key step towards realising the promise of precision medicine. We hypothesise that the
gene-expression-based radiosensitivity index, together with the linear quadratic model, in
the form of the genomic-adjusted radiation dose (GARD), can serve as the basis for
precision medicine in radiation oncology. We aimed to develop and validate the GARD
model by generating GARD scores and modelling associations between GARD and clinical
outcomes after radiotherapy.

Methods
Study design and cohort population
The Total Cancer Care (TCC) protocol is a prospective tissue collection protocol that has
been active at Moffitt Cancer Center (Tampa, FL, USA) and 17 other institutions since
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2006.18 We used primary tumour samples from patients enrolled in the TCC protocol (TCC
cohort) to calculate GARD scores and assess the range of GARD values within and between
tumour types. We used samples from 20 disease sites from patients who received standard of
care treatment at the discretion of their physician, mostly between 2010 and 2012. TCC
patients consent for their samples to be collected and profiled.

We used data from five clinical cohorts (Erasmus Breast Cancer Cohort, Karolinska Breast
Cancer Cohort, Moffitt Lung Cancer Cohort, Moffitt Pancreas Cancer Cohort, and The
Cancer Genome Atlas Glioblastoma Patient Cohort) to assess whether GARD was
associated with clinical outcomes. All cohort studies received institutional approval.

The Erasmus Breast Cancer Cohort comprises patients with T1–T3 primary tumours and no
clinical evidence of lymph node metastases (N0) treated at the Erasmus Medical Center
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(Rotterdam, Netherlands).11 We used data for 263 patients who received lumpectomy plus
whole-breast radiotherapy with or without a boost to the tumour cavity, with total doses
ranging from 40 Gy to 74 Gy, delivered at 1·8–2 Gy per fraction. The primary outcome,
early metastasis, was defined as a distant recurrence in the first 5 years following completion
of primary treatment. Raw gene expression data are publically available (GSE2034,
GSE5327).

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The Karolinska University Hospital, Radiumhemmet Cohort (Karolinska Cohort) is a

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prospective cohort of patients with breast cancer treated at the Karolinska University
Hospital (Solna, Sweden), between Jan 1, 1994, and Dec 31, 1996.19 The cohort included
patients with T1–T3 primary tumours with or without clinical evidence of lymph node
metastases (N0–N1). We used data for 77 patients who received segmentectomy or
mastectomy plus radiotherapy of 50 Gy in 25 fractions delivered to the conserved breast or
chest wall, with or without local nodes. No patient received a tumour cavity or chest-wall-
scar boost. All patients underwent axillary dissection. Follow-up data were obtained from
the Swedish Breast Cancer Registry and was supplemented with patient charts as previously
described.19 The primary endpoint was relapse-free survival (any distant, regional, or local
relapse from the end of primary treatment).

The Moffitt Lung Cancer Cohort comprises archived tumours that were resected between
2000 and 2010 from patients in the TCC and Moffitt Cancer Center tissue database.20 We
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used tissue samples and data for 60 patients who had pathologically confirmed, American
Joint Committee on Cancer version 6, stage IIIA or IIIB non-small-cell lung cancer and
underwent surgical resection and post-operative radiotherapy with a mean dose to the
planning target volume of 54·8 Gy (range 43·2–70 Gy). Recurrence was assessed based upon
the determination of the treating physician in clinical source documentation. Gene
expression data were obtained from TCC. Investigators received written informed consent
for tissue acquisition and molecular profiling and follow-up.

The Moffitt Pancreas Cancer Cohort comprises patients with pancreatic cancer from the
TCC and Moffitt Cancer Centre tissue database. We used data for tissue samples available
for analysis from 40 patients who underwent upfront surgical resection for pancreatic cancer
between 2000 and 2011 and received radiotherapy with concurrent fluorouracil or
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gemcitabine chemotherapy. The median radiation dose was 50 Gy (range 43·2–54 Gy) in
180 to 200 cGy daily fractions for a median of 28 fractions (range 24–30) to the pancreatic
tumour bed and regional lymphatics. Patients were excluded if they received neoadjuvant
therapy. Gene expression data were obtained from TCC. Investigators received written
informed consent for tissue acquisition and molecular profiling and follow-up.

We used data for 98 patients from The Cancer Genome Atlas (TCGA) Glioblastoma Patient
Cohort who underwent radio therapy and concurrent temozolomide treatment. Patient data
were included for analysis if gene expression array data21 were available with a sample that
included 50% tumour or more. Patients were excluded if they received neoadjuvant
treatment or had low MGMT expression. Clinical and array-based gene expression data
(Affymetrix HT Human Genome U133 Array Plate Set level 2) was downloaded from
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TCGA.

Procedures
We assayed tumours from adult patients enrolled in the TCC protocol on Affymetrix Hu-
RSTA-2a520709 (Affymetrix; Santa Clara, CA, USA), which contains approxi mately 60
000 probesets representing 25 000 genes. Chips were normalised using iterative rank-order
normalisation.22 Batch effects were reduced using partial-least squares. We extracted from
the TCC database normalised, debatched expression values for 13 638 samples from 60 sites

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of origin and the ten radiosensitivity index genes (AR, c-Jun, STAT1, PKC-beta, RelA,
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cABL, SUMO1, PAK2, HDAC1, and IRF1). We excluded all metastatic duplicate samples
and disease sites with fewer than 25 samples. We included samples irrespective of receipt of
radiotherapy.

Gene-expression-based radiosensitivity index scores were generated previously for all

clinical cohorts except for the Moffitt Lung Cancer Cohort.11 To calculate radiosensitivity
index scores for this cohort, we normalised gene expression values from Affymetrix U133A
CEL files using the robust multiarray average (RMA) algorithm23 and did linear scaling to
avoid negative values. We ranked each of ten genes in the algorithm based on gene
expression (highest expressed gene is ranked at ten and lowest at one) and calculated the
radiosensitivity index using the predetermined algorithm (equation 1):

RSI = − 0.0098009 * AR + 0.0128283 * cJun + 0.0254552 * STAT 1 − 0 ⋅ 0017589 * PKC ‐ beta − 0.0038171
* RelA + 0 ⋅ 1070213 * cABL − 0 ⋅ 0002509 * SUMO1 − 0.0092431 * PAK2 − 0.0204469 * HDAC1
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− 0.0441683 * IRF 1

Statistical analysis
The linear quadratic model proposes that there are two parameters that impact on
radiotherapy cytotoxicity, one that is proportional to radiotherapy dose (α) and one that is
proportional to the square of the dose (β). The linear quadratic model in its simplest form is
represented by (equation 2):

S = e−nd(α + βd)

Here, e is the natural logarithm, with S representing the surviving fraction after n fractions
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of radiation, each of dose (d). α represents the linear radiosensitivity parameter and β
represents the quadratic radiosensitivity parameter. Effect (E) was calculated as (equation 3):
24

E = nd(α + βd)

Here effect represents the cytotoxic effect of radiotherapy in cell lines (a higher effect results
in higher cytotoxicity).

We derived GARD scores using the linear quadratic model, the individual gene-expression-
based radiosensitivity index, and the standard of care radiation dose and fractionation
schedule for each patient (appendix p 1). The calculation for GARD is similar to
biologically effective dose, except that patient-specific α is derived by substituting
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radiosensitivity index for survival (S) in equation 1, where dose (d) is 2 Gy, n=1, and β is a
constant (0·05/Gy2).25 A higher GARD value predicts a higher radiation therapeutic effect.
We make the assumption that radiation therapeutic effect is equivalent to clinical benefit. We
calculated GARD using a script written into Excel.

For analysis of the TCC cohort, we ranked each patient’s GARD from highest to lowest. We
assigned radiotherapy dose and fractionation protocols for each disease type: subclinical (45

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Gy in 25 fractions), microscopic (60 Gy in 30 fractions), and macroscopic disease (≥70 Gy

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in 35–40 fractions). We then defined three GARD levels cor responding to the proportion of
patients within each radiotherapy dose group. We compared median GARD between disease
sites for each assigned dose group using the Fisher’s exact test. We created violin plots using
MATLAB R2016a (The MathWorks, Natick, MA, USA) and the “Violin Plots for plotting
multiple distributions (distributionPlot.m)” toolbox from MATLAB Central File).26

For each clinical cohort, we ranked GARD values from highest to lowest and grouped
patients into three radiotherapy dose groups: low, intermediate, and high. We then defined
three GARD levels (low, medium, and high), corresponding to the proportion of patients
within each dose group. We estimated distant-metastasis-free survival in the Erasmus Breast
Cancer cohort using the Kaplan-Meier method and used the log-rank test to identify
differences by high versus low GARD score, dichotomised at the 75th percentile, based on
previous gene-expression-based radiosensitivity index analyses.11 We calculated biologically
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effective dose assuming a constant αβ ratio of 2·88 for breast cancer as previously described
(BED2·88).27,28 We assessed the correlation between GARD and BED2·88 with Spearman
correlation. Proportional hazards regression analyses were used to calculate the effect of
biologically effective dose on distant-metastasis-free survival. Proportional hazards
regression analyses were also used for multivariate analysis of GARD as a continuous
variable or dichotomous variable in oestrogen-positive patients. We compared
sociodemographic and clinicopathological characteristics between patients included and
excluded from the Erasmus cohort using Fisher’s exact test for categorical variables,
including gene-expression-based radiosensitivity index, and Wilcoxon rank-sum test for
continuous variables.

For the five clinical cohorts, we used multivariable Cox proportional hazards regression to
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assess the association between GARD and the studied endpoint, adjusting for potential
confounders and using a backward elimination model with a significant level-to-stay of 0·10.
GARD cut-points for the Karolinska breast cancer (75th percentile), glioblastoma (75th
percentile), and pancreatic cancer (50th percentile) cohorts were similar to the gene-
expression-based radiosensitivity index cut-points used in previous analyses. We tested two
cut-points for the lung cancer cohort (75th percentile and 60th percentile [which was
eventually used]); Bonferroni correction was performed for multiple testing in this cohort.

Since GARD is a model combining gene-expression-based radiosensitivity index with the

linear quadratic model, we compared the performance of GARD to the radiosensitivity index
in the Erasmus Breast Cancer Cohort using backward elimination in a multivariable model
fitted with candidate variables (hormone receptor status, T stage, age, GARD, BED2·88 and
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radiosensitivity index) to calculate the likelihood ratio.

We hypothesised that there would be a threshold that separates high and low GARD
subpopulations, towards which a clinician can adjust the radiotherapy dose to increase
radiation therapeutic effect. Given the equation for GARD, it could be expected that an
increasing proportion of patients will achieve the GARD threshold with escalating
radiotherapy doses. To test this possibility, we built a model to test whether GARD could
predict benefit from radiotherapy shown in a real-life cohort. We used assumptions as

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observed in the Erasmus Breast Cancer cohort (eg, GARD threshold 75th percentile,
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radiosensitivity index distribution, hazard ratio [HR] between high GARD and low GARD
of 2·11) to develop a formula for the potential effect on distant-metastasis-free survival of
radiotherapy dose escalation in breast cancer predicted by the GARD-based model (equation
4)

a * HR + (1 − a) * 1
b * HR + (1 − b) * 1

In this equation, a and b are the estimated proportions of patients to achieve the GARD
threshold dose level at the radiotherapy doses being compared (range 50–76 Gy vs 50 Gy
reference dose). We assume an improvement in distant-metastasis-free survival in the high
GARD population similar to that observed in the Erasmus cohort (HR=2·11). We use the
GARD equation to calculate the distribution of GARD values at 66 Gy (a) and 50 Gy (b),
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assuming that the radiosensitivity index distribution of a random breast cancer population is
the same as that observed in the Erasmus Breast Cancer Cohort. We then estimated the
percentage of patients that would have reached the GARD threshold value at each
radiotherapy dose. We chose to compare 50 Gy and 66 Gy doses to simulate a completed
clinical trial in patients with early stage breast cancer (T 1–2, N 0–1) breast cancer (EORTC
22881–10882). EORTC 22881–10 882 randomly assigned 5318 patients to postoperative
whole-breast radiotherapy of 50 Gy, with a 16 Gy boost (n=2661) or without a boost dose
(n=2657).29 We generated HRs comparing distant-metastasis-free survival after radiotherapy
as calculated in our GARD-based model and compared it with the published results of
EORTC 22881–10882. The difference in outcome between high-GARD and low-GARD
populations used for this modelling experiment was derived from the multivariable analysis
of the Erasmus cohort.
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All tests were two-sided with a significance level of 0·05. We used SAS version 9.3 for all
analyses.

Role of the funding source

There was no funder for this study. The corresponding author had full access to all the data
in the study and had final responsibility for the decision to submit for publication.

Results
GARD values for the 8271 tissue samples in the TCC cohort ranged from 1·66–172·4 (figure
1, appendix pp 2–3). 2517 (30%) samples were assigned the 45 Gy dose level, 4877 (59%)
were assigned the 60 Gy dose level, and 877 (11%) were assigned the 70 Gy or more dose
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level. Using these proportions, we derived three GARD levels by percentile: low (0 to
30·40), middle (30·41 to 89·40), and high (89·41 to 100). There was a wide range of GARD
values within each uniform radiotherapy dose group (figure 1). Patients assigned a dose of
45 Gy had GARD values from 3·03 to 56·34. Patients assigned to 60 Gy had GARD values
from 1·66 to 122·38. Patients assigned to 70 Gy or more had GARD values from 9·73 to
172·4.

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By taking gene-expression-based radiosensitivity index into account, GARD scores show

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that a higher radiotherapy dose does not always result in a higher radiotherapeutic effect
across a population. For example, if GARD was exclusively related to radiotherapy dose it
would be expected that patients assigned to 45 Gy would be in the low GARD group.
However, although 1456 (58%) of 2517 patients assigned to 45 Gy were in the low GARD
group, 1023 (21%) of 4877 patients in the middle GARD group and 38 (4%) of 877 patients
in the high GARD group were assigned to 45 Gy (figure 1). Thus highly radiosensitive
patients (those with a low radiosensitivity index) assigned to 45 Gy had GARD values that
were similar to some patients assigned to higher radiotherapy doses (60 or 70 Gy) but who
were less radiosensitive. Similarly, although most patients assigned to 70 Gy were in the
high GARD group, 588 (11%) of 4877 patients in the middle GARD group were assigned to
70 Gy (figure 1).

For cancers usually treated with 70 Gy radiotherapy, patients with cervical cancer and
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oropharyngeal head and neck cancer had the highest median GARD values (figure 2A).
Median GARD was higher in patients with oropharyngeal head and neck cancer (39·71) than
in those with non-oropharyngeal head and neck cancer (32·56; p=0·0417), which is
consistent with the superior clinical outcomes after radiotherapy in patients with
oropharyngeal cancer.30 In the group of disease sites usually treated with 60 Gy, glioma
(median GARD 16·55) and sarcoma (17·94) had lower GARD compared with all other
disease sites at this dose level (p<0·0001; figure 2B). GARD also shows that the
radiotherapy therapeutic effect at 60 Gy is larger in non-melanoma skin cancer (median
GARD 25·80) than in melanoma (21·17; p=0·01144). Oesophageal cancer and rectal cancer
are usually treated with preoperative radiotherapy; oesophageal cancer had a higher GARD
than did rectal cancer (p=0·00032; figure 2C). Gastric and pancreatic cancer are both
commonly treated with postoperative radio therapy; GARD identified a higher predicted
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radiation therapeutic effect for gastric cancer than for pancreatic adeno carcinoma
(p=0·00171; figure 2C).

Patients included in our analysis of the Erasmus Breast Cancer Cohort did not have
significantly different sociodemographic and clinicopathological characteristics compared
with those we excluded (appendix pp 1–2). Establishing generalisability for the Erasmus
Breast Cancer Cohort population, we show the radiosensitivity index distribution between
patients in this cohort and those in the TCC cohort did not significantly differ (appendix p
11). GARD values were widely distributed in patients in this cohort (median 27·22, range
4·01–104·25; figure 3A). GARD by actual radiotherapy dose group is in the appendix (p 4).
Patients who had a GARD at or above the 75th percentile for this cohort (≥38·9; high
GARD) had longer distant-metastasis-free survival compared with those with GARD below
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the 75th percentile (low GARD; figure 3B). There was a weak but significant correlation
between GARD and BED2·88 (R=0·25, p<0·0001), with an increase in GARD generally
associated with an increase in BED2·88. In univariable analysis, BED2·88 did not predict
distant-metastasis-free survival (p=0·12; appendix p 5). In multivariable analyses, GARD
was the only independent predictor of distant-metastasis-free survival in this cohort;
hormone receptor status, T stage, age, and surgery type were not independently associated
with distant-metastasis-free survival (tables 1, 2). GARD was also an independent variable
predicting clinical outcome in oestrogen-receptor-positive patients in the Erasmus breast

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cancer cohort (appendix pp 8–9). In multivariable analysis, GARD was significantly

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associated with relapse-free survival in the Karolinska Breast Cancer Cohort, local control in
the Moffitt Lung Cancer Cohort, and overall survival in the TCGA Glioblastoma Cohort and
the Moffitt Pancreas Cancer Cohort (table 2).

In the multivariate model to compare GARD with the radiosensitivity index, the
radiosensitivity index was the last variable eliminated, yielding a final model including
GARD and BED2·88, with an overall likelihood ratio χ2 value of 10·45 (p=0·0054). Nine
patients were excluded from this analysis because of missing oestrogen and progesterone
receptor status. When these patients were included back, as is analytically preferable, the
model had an overall likelihood ratio χ2 value of 12·07 (p=0·0024). Notably, the
corresponding model excluding GARD with only radiosensitivity index and BED2·88 had a
likelihood ratio χ2 value of 6·95 (p=0·0310). The gain of 5·12 points (ie, 12·07 subtract
6·95) is substantial, demonstrating that the GARD and BED2·88 model is much better than
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the gene-expression-based radiosensitivity index.

In figure 4, we identify a subset of 23% (78 of 344) of patients (radiosensitivity index 0·18–
0·35) that could achieve the GARD threshold (GARD ≥38·9) if they received radiotherapy
doses of 45–75 Gy. Using the observed distribution of high versus low GARD
subpopulations in the Erasmus Breast Cancer Cohort at each delivered radiotherapy dose to
estimate the potential benefit of GARD, we estimate that uniform and unselected
radiotherapy dose escalation would result in an overall slight improvement in distant-
metastasis-free survival (figure 4B, C). The predicted improvements, while substantial,
would not be noticed in an unselected randomised trial. For example, our model estimates
that dose escalation from 50 Gy to 66 Gy would result in an increased distant-metastasis-
free survival (HR 0·92). A trial with 80% power to detect this difference without genomic
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guidance would require 14 489 patients (appendix p 9). By contrast, a GARD-based

approach predicts that the benefit of dose escalation from 50 Gy to 66 Gy would require 230
patients if using only a subgroup of patients with gene-expression-based radiosensitivity
index scores lower than 0·31 (appendix p 10).

We compared the potential benefit calculated by GARD for dose escalation to 66 Gy with
the results of the randomised EORTC 22 881–10 882 trial of 5318 patients with breast
cancer.29 In this trial, after a median follow-up of 17·2 years, dose escalation resulted in a
lower local (ipsilateral) recurrence (HR 0·65, 99% CI 0·52–0·81; p>0·0001) and no
difference in distant metastasis (1·06, 0·92–1·24; p=0·29). The estimated distant-metastasis-
free survival for dose escalation to 66 Gy calculated by GARD (HR of 0·92) was within the
lower 99% CI of the HR reported by EORTC 22 881–10 882 for this endpoint.30 The
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estimated HR for distant-metastasis-free survival is a quarter of the observed effect on local

recurrence (HR 0·65), consistent with the 4:1 relationship between local recurrence and
breast cancer death observed in the EBCTCG meta-analysis,31 although it should be noted
that more recent analyses of radiotherapy benefit do not show the same ratio.32

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Discussion
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We show that GARD varies widely within populations and tumour types, and is associated
with outcomes in five clinical cohorts and in a model comparing predictions to real-world
results. Several threads of evidence support the clinical value of GARD. GARD is largely
based on gene-expression-based radiosensitivity index and the linear quadratic model, both
of which have extensive clinical validation. Gene-expression-based radiosensitivity index
has been validated as a predictor of outcome in multiple datasets of radiotherapy-treated
patients and the linear quadratic model has been used as the basis for dose and fractionation
in clinical radiation oncology.8,9,11–15,27,33 GARD ranges are consistent with the clinical
heterogeneity of radiotherapy therapeutic benefit. For example, the higher GARD scores for
glioma versus sarcoma, oropharyngeal versus non-oropharyngeal head and neck cancer,
oesophageal cancer versus rectal cancer, non-melanoma skin cancer versus melanoma, and
gastric cancer versus pancreatic cancer reflect better radiotherapy outcomes for these disease
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types in clinical studies.30,34–40 We also show the clinical validity of GARD in patients with
breast cancer who did not receive chemotherapy or hormonal therapy, thus limiting
confounding factors. Additionally, there was substantial variation in the radiation doses
delivered to patients in this cohort, which allowed radiotherapy dose to affect GARD.
GARD has been developed to enable adjustment of radiotherapy dose to match an individual
tumour’s radiosensitivity, with higher GARD values predicting a higher therapeutic effect
from radiotherapy. Therefore, it is reasonable to test the clinical validity of GARD by testing
whether patients with higher GARD values have better clinical outcomes.

Our analyses show that GARD is an independent predictor of a radiotherapy-specific

outcome and outperforms both gene-expression-based radiosensitivity index and BED2·88 in
patients in the Erasmus Breast Cancer cohort. Furthermore, GARD was an independent
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predictor of clinical outcome in four additional independent cohorts of patients with breast
cancer, glioblastoma, lung cancer, and pancreatic cancer. In both breast cancer cohorts, we
used endpoints chosen by the original investigators. Although local control is the classic
endpoint used to measure radiotherapy effectiveness, and radiosensitivity index has been
previously shown to predict local recurrence in breast cancer, distant-metastasis-free survival
has emerged as an appropriate endpoint for radiotherapy-based interventions30,41,42 thus
making distant-metastasis-free survival and relapse-free survival relevant endpoints for an
radiotherapy-focused analysis.42 The glioblastoma dataset was obtained from TCGA and
overall survival was the outcome reported. Since radiotherapy has been shown to impact
overall survival in glioblastoma, we think this is a relevant endpoint for GARD in this
disease.43 Data for both the pancreas and lung cancer cohorts were obtained from our
institutional database. We chose to report overall survival for the pancreas cancer cohort
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since there are data that, although controversial, support the notion of post-operative
radiotherapy having an effect on overall survival in this disease.44 Finally, we chose local
control for the lung cancer cohort since this is a classic endpoint used for the most direct
clinical effect for radiotherapy.

This work has several important implications. First, we identify genomically-distinct

populations that derive differential benefit from radiotherapy. Furthermore, we provide a
method by which to customise radiation dose to match the radiosensitivity of an individual

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 Scott et al. Page 11

patient’s tumour with existing technology. We provide, to the best of our knowledge, the first
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framework to design genomically-stratified, radiotherapy-based trials using specifically

defined genomic subpopulations. Thus, a key potential utility of this work is the use of
GARD to test genomic-based radiation dosing to improve clinical outcomes. Genomic-based
clinical trial design could greatly improve the efficiency of clinical trials in radiation
oncology and lead to a reduction in both the number of patients required to test a hypothesis
and the time to complete the trial, both of which should lead to substantial cost-savings. We
emphasise that GARD is not a predictive assay or biomarker for clinical outcome but rather
a model to adapt the prescribed radiation dose to match individual tumour radiosensitivity. It
is not trained to predict clinical outcome, but was developed as a novel genomic
radiotherapy prescription framework.

Since we propose GARD as an approach for individualised radiotherapy dose to match

tumour radiosensitivity, a reasonable question is what is the clinical opportunity for patient-
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specific dose optimisation? Radiotherapy doses in clinical practice today have been
empirically optimised, resulting in reasonable disease control and toxicity. However, our
findings suggest that current uniform radiotherapy dose protocols can be further optimised
with tumour-specific genomic data. GARD could provide a scientific framework to adjust
radiotherapy doses that have already shown to be safe, both in terms of increasing tumour
control (increasing dose to more resistant tumours) and decreasing complication risks
(lowering the dose to more sensitive tumours), although this concept requires further
validation. Finally, it should be emphasised that GARD only accounts for tumour
radiosensitivity and that additional biological tumour parameters (ie, proliferation, hypoxia,
and DNA repair) as well as patient parameters (ie, normal tissue toxicity) would further
improve the ability of clinicians to optimise radiotherapy dose. Normal tissue toxicity is one
parameter of central importance and approaches to better estimate individual risk are in
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development.45 However, in their absence, a reasonable approach for GARD-based radiation

dose optimisation could involve isotoxic dose escalation.46

Although our modelling framework is simple and based on classically accepted principles,
we have made several assumptions to complete our analyses. Specifically, we assume that
the risk of recurrence and gene-expression-based radiosensitivity index distribution in the
Erasmus cohort are similar to a normal lymph node-negative breast cancer population. This
assumption seems reasonable in light of the observation that the gene-expression-based
radio sensitivity index distribution between the Erasmus Breast Cancer cohort and the TCC
are not significantly different. Furthermore, it is possible that since patients in the Erasmus
Breast Cancer cohort were not treated with systemic hormonal or chemotherapy, our model
is overestimating the benefit of radiotherapy. We have also made the assumption that the
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quadratic component of radiation response, β, is constant. Because we do not attempt to

model different ranges of fractional (daily) dose, this assumption should not qualitatively
affect our conclusions. Additionally, we acknowledge that our model does not address
normal tissue radiosensitivity, which could further allow the personalisation of radiation
dose. Finally, while we use the gene-expression-based radiosensitivity index as a backbone
for our analyses, the calculation of GARD could use other measures of radiosensitivity or be
expanded to include other biological parameters including hypoxia, DNA repair,
proliferation, and the immune system.

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In conclusion, a central requirement for precision medicine in radiation oncology is the

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ability to inform radiation dose parameters to match individual tumour biology, thus
delivering the right radiation dose for the right patient. GARD provides, to the best of our
knowledge, the first opportunity to genomically inform radiation dose and our findings
suggest it is a feasible approach to precision radiation oncology.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
JFT-R received grants from National Institutes of Health (R21CA101355/R21CA135620), US Army Medical
Research and Materiel Command, National Functional Genomics Center (170220051), and Bankhead-Coley
Foundation (09BB-22). Total Cancer Care is supported by DeBartolo Personalized Medicine Institute, and the
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Merck-Moffitt Research Partnership. The Merck-Moffitt Research Partnership directed the collection of samples for
the Total Cancer Care protocol. We thank David Brenner for helpful insights and discussion.

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Research in context
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Evidence before this study

We searched PubMed for articles published up to Sept 15, 2016, using the terms
“precision medicine AND radiation therapy” as well as “precision medicine AND
radiation therapy AND genomics”. Before this study, the term precision radiation therapy
generally referred to anatomic and geometric precision in the delivery of a given radiation
dose to a defined tumour target, using clinical factors, such as tumour size, tumour
response, and imaging features. There are several groups that have shown that there are
biological differences that determine both tumour and normal tissue response to
radiation, but to date no clinical strategy has been developed to integrate these biological
differences into customised radiation in clinical practice.

Added value of the study

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In this study, we develop and validate the genomic-adjusted radiation dose (GARD), a
clinical model for genomic radiation dosing that could allow the individualisation of
radiotherapy dose to tumour radiosensitivity and provide a framework to design
genomically-guided clinical trials in radiation oncology. To assess the usefulness of
GARD in a clinical setting, we generated GARD values in primary solid tumours from 20
disease sites, tested GARD as a predictor of clinical outcome in five independent clinical
datasets, and developed a GARD-based prediction model that accurately predicted the
observed impact of increasing the radiotherapy dose in a clinical trial.

Implications of all the available evidence

Precision medicine encompasses all therapeutic applications for patient care. With
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multidisciplinary care becoming standard for most patients with cancer, it is crucial that
precision medicine is expanded beyond medical oncology. GARD potentially provides a
clinically actionable framework that could allow the integration of biological differences
into radiotherapy dose. GARD represents, to the best of our knowledge, the first
opportunity to depart from the uniform application of radiotherapy and design efficient,
genomically-guided clinical trials in radiation oncology.
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Figure 1: A framework for genomic-adjusted radiation dose (GARD)

(A) The left plot shows the proportion of patients in each radiotherapy dose group. On the
right plot, GARD values for each individual patient are presented ranked from the highest to
lowest value; each line represents an individual patient; colour relates to dose assigned. Nine
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patients in the cohort had a GARD higher than 100; these patients were assigned a GARD of
100. Pie charts show dose assignments for patients in GARD score groups: (B) high (89·41–
100 percentile); (C) middle (30·41–89·4 percentile); and (D) low (0–30·4 percentile).
GARD=genomic-adjusted radiation dose.

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Figure 2: GARD score distribution and density within 70 Gy (A), 60 Gy (B), and 45 Gy (C) dose
levels, by disease site
The red dot represents the median GARD value for each disease site at assigned dose levels.
Colours in the plot correlate with the sample density.
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GARD=genomic-adjusted radiation dose. SCC=squamous cell carcinoma.

OPX=oropharyngeal. IDC=invasive ductal carcinoma. TCC=transitional cell carcinoma.
NMSC=non-melanoma skin cancer. RCC=renal cell carcinoma.

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Figure 3: GARD and distant-metastasis-free survival in the Erasmus Breast Cancer Cohort
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(A) GARD values for each individual patient are presented ranked from the highest to lowest
value; each line represents an individual patient and colour relates to radiotherapy dose
received. The number of patients in each group and the GARD ranges are online (appendix p
4). (B) Kaplan-Meier plot for distant-metastasis-free survival comparing patients with high
GARD (≥38·9; the 75th percentile) with patients with low GARD (<38·9). HR is from
univariable analysis. If no event occurred, then cases were censored at 5 years
GARD=genomic-adjusted radiation dose. HR=hazard ratio.
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Figure 4: A model for genomic-informed radiation dose in breast cancer

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(A) The red line shows the physical radiotherapy dose required to meet the GARD threshold
(GARD≥38·9) with increasing RSI. This curve is based on the radiotherapy effect calculated
for distant metastasis (not local control) using the Erasmus Breast Cancer Cohort. (B) The
probability of patients reaching the GARD threshold (GARD>38·9) in an unselected
population as a function of radiotherapy dose. (C) Estimates of the potential therapeutic
effect (HR for high versus low GARD subpopulations) of increased radiotherapy dose (the
reference dose is 50 Gy). GARD=genomic-adjusted radiation dose. RSI=radiosensitivity
index. HR=hazard ratio.
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Table 1:

Multivariable analysis of the Erasmus Breast Cancer Cohort for distant-metastasis-free survival
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Hazard ratio (95% Cl) p value

GARD score <75th percentile vs GARD score (>75th percentile) 2·11 (1·13–3·94) 0·018

Hormone receptor status*

ER+ and PR− or ER−and PR+ vs ER+ and PR+ 1·28 (0·76–2·17) 0·35
ER−and PR− vs ER+ and PR+ 0·98 (0·57–1·68) 0·93
T stage2,3,or4 vs T stage 1 1·36 (0·86–2·16) 0·18
Age (years)
41–55 vs<40 years 0·78 (0·42–1·43) 0·41
56–70 vs <40 years 0·84 (0·44–1·59) 0·59
71–83 vs <40 years 0·37 (0·12–1·13) 0·08
Lumpectomy vs surgery (mastectomy) 1·97 (0·88–4·43) 0·10
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GARD=genomic-adjusted radiation dose. ER=oestrogen receptor.

PR=progesterone receptor.
*
Nine patients did not have hormone receptor status and were excluded.
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Table 2:

Clinical cohort description and multivariate analysis for the effect of GARD on selected endpoints

Median Events Radiotherapy dose GARD Endpoint HR from multivariable p value Adjustment factors
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follow-up range (Gy) range analysis (95% Cl)

(months)
Erasmus Breast Cancer Cohort 60 23 40–74 4·01–104·25 Distant-metastasis- 2·11 (1·1–3·9) 0·018 Oestrogen and progesterone
(n=263) free survival* receptor status, age, surgery (vs
lumpectomy), and T-stage
Karolinska Breast Cancer 87 19 50 8–60 Relapse-free 7·4 (1·4–1·38) 0·014 Hormonal therapy, chemotherapy,
Cohort (n=77) survival† and oestrogen and progesterone
receptor status
Moffitt Lung Cancer Cohort 37 23 45–70 15–125 Local control‡ 3·4 (1·3–9·1) 0·016 Surgery, stage, histology,
(n=60) lymphovascular invasion
TCGAGIioblastoma Cohort 11 76 12·6–97·0 0·4–46·0 Overall survival§ 1·9 (1·1–3·3) 0·019 Age, performance status
(n=98)
Moffitt Pancreas Cancer Cohort 68 27 45–54 16–40 Overall survival§ 2·6 (1·1–6·0) 0·029 CA 19·9, margin lymph nodes
(n=40)

TCGA=The Cancer Genome Atlas.

*
Primary endpoint defined as distant recurrence in the first 5 years following completion of primary treatment.
†
Primary endpoint defined as any relapse distant, regional, or local from the end of primary treatment.
‡
Defined in this study as time from surgical resection to cancer recurrence within the irradiated field. If no event occurred, then cases were censored at the date of last clinical evaluation. Cases in which
more than 4 months elapsed without a clinical evaluation were censored at the date of antecedent clinical evaluation.22
§
Defined in this study as the interval from surgery to date of death.

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