Pe d i a t r i c I m a g i n g O r i g i n a l R e s e a r c h

McCarville et al.
Ultrasound Assessment of Response to Antiangiogenic Therapy

Pediatric Imaging
Original Research
Use of Quantitative Dynamic
Contrast-Enhanced Ultrasound
to Assess Response to
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Antiangiogenic Therapy in Children

FOCUS ON:

and Adolescents With Solid

Malignancies: A Pilot Study
M. Beth McCarville1 OBJECTIVE. The purpose of this study was to investigate contrast-enhanced ultrasound
Jamie L. Coleman1 assessment of tumor response to antiangiogenic therapy in children and adolescents with sol-
Junyu Guo1 id malignancies.
Yimei Li2 SUBJECTS AND METHODS. Children with recurrent solid tumors who were en-
Xingyu Li2 rolled in an institutional phase 1 study of antiangiogenic therapy underwent contrast-en-
hanced ultrasound of target lesions before therapy, on therapy days 3 and 7, and at the end
Patricia J. Honnoll1
of course 1. Acoustic data from target lesion ROIs were used to measure peak enhancement,
Andrew M. Davidoff 3 time to peak, rate of enhancement, total AUC, AUC during wash-in (AUC1), and AUC dur-
Fariba Navid 4,5 ing washout (AUC2). The Cox regression model was used to assess the association between
McCarville MB, Coleman JL, Guo J, et al.
changes in parameters from baseline to follow-up time points and time to tumor progression.
Values of p 0.050 were considered significant.
RESULTS. Target lesion sites included liver (n= 3), pleura (n= 2), and supraclavicular
mass, soft-tissue component of bone metastasis, lung, retroperitoneum, peritoneum, lymph
node, muscle mass, and perineum (n= 1 each). Hazard ratios for changes from baseline to end
Keywords: antiangiogenic, contrast-enhanced
of course 1 for peak enhancement (1.17, p= 0.034), rate of enhancement (3.25, p= 0.029), and
ultrasound, pediatric, tumor response
AUC1 (1.02, p= 0.040) were significantly associated with time to progression. Greater de-
DOI:10.2214/AJR.15.15789 creases in these parameters correlated with longer time to progression.
CONCLUSION. Contrast-enhanced ultrasound measurements of tumor peak enhance-
Received November 4, 2015; accepted after revision ment, rate of enhancement, and AUC1 were early predictors of time to progression in a cohort
December 31, 2015.
of children and adolescents with recurrent solid tumors treated with antiangiogenic therapy.
Based on a presentation at the Society for Pediatric Further investigation of these findings in a larger population is warranted.
Radiology 2015 annual meeting, Bellevue, WA.
ngiogenesis plays a critical role in giogenic cancer therapy. Children are ideally

A
Supported in part by the American Lebanese Syrian
Associated Charities and Cancer Center core grant tumor growth, metastasis, and suited for ultrasound because their small size
027165. M. B. McCarville receives product support from survival, and targeted antiangio- facilitates placement of the transducer near
GE Healthcare. genic therapies are being increas- the structure of interest, thus reducing arti-
1
ingly used in cancer clinical trials. Unlike fact. Furthermore, ultrasound does not re-
Department of Diagnostic Imaging (MS 220), St. Jude
conventional cytotoxic chemotherapy, these quire sedation and, most important, does not
Childrens Research Hospital, 262 Danny Thomas Pl,
Memphis, TN 38105. Address correspondence to agents are cytostatic and may be effective entail ionizing radiation, an issue of consider-
M.B.McCarville (beth.mccarville@stjude.org). without causing tumors to shrink. Therefore, able concern in the care of children.
traditional methods that depend on changes We previously reported that tumor en-
in tumor size are not suitable for assessing re- hancement measured with CEUS as early
2
Department of Biostatistics, St. Jude Childrens
Research Hospital, Memphis, TN.
sponse to antiangiogenic agents, and tech- as 24 hours after initiation of antiangiogen-
3
Department of Surgery, St. Jude Childrens Research niques that provide quantitative measure- ic therapy is statistically significantly asso-
Hospital, Memphis, TN. ments of tumor perfusion are needed. ciated with the degree of tumor vascularity
4
Dynamic contrast-enhanced ultrasound in murine models of pediatric malignancies
Department of Oncology, St. Jude Childrens Research
(CEUS) findings are emerging as a reliable [1215]. We have also found that ultrasound
Hospital, Memphis, TN.
indicator of tumor response to antiangiogenic contrast agents are safe and well tolerated in
5
Present address: McLean, VA. therapy in adults with hepatocellular carcino- children with underlying solid malignancies
ma, renal cell carcinoma, malignant melano- and can improve visualization of tumor mar-
AJR 2016; 206:933939 ma, and gastrointestinal stromal tumor [1 gins [16, 17]. The purpose of this pilot study
11]. To our knowledge, however, there have was to build on our preclinical and clinical
0361803X/16/2065933
been no reports of the value of this modality experience and to investigate the value of
American Roentgen Ray Society in children and adolescents receiving antian- quantitative dynamic CEUS in the assess-

AJR:206, May 2016 933

 McCarville et al.

ment of tumor response in children and ado-
lescents with recurrent or refractory solid tu-
mors treated in an institutional phase 1 trial
of antiangiogenic therapy.
Subjects and Methods
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ment) were investigated. In this study, we report
on six parameters from a larger cohort. To be eli-
gible for CEUS, patients had to have a target le-
sion (residual primary tumor or metastasis) that
was visible with gray-scale sonography. Because
of a theoretic risk of microemboli caused by mi-
course was 21 days in duration. Tumor response
was assessed with the original Response Evalua-
tion Criteria in Solid Tumors (RECIST) with pro-
gression defined as a 20% or greater increase in
sum of diameters of target lesions compared with
the smallest sum of diameters achieved or the

Patient Population and Outcome Measures
Our cohort comprised a subset of patients 21
years old or younger at the time of diagnosis of
a solid malignancy, with recurrent or refractory
disease, and enrolled in an institutional phase 1
study of bevacizumab, sorafenib, and low-dose
cyclophosphamide (ClinicalTrials.gov identifi-
er, NCT00665990) between December 2008 and
April 2013. The study was approved by our insti-
tutional review board, was HIPAA compliant, and
was performed under U.S. Food and Drug Ad-
ministration investigational new drug application
62,852. Treatment study design, eligibility and
exclusion criteria, preliminary study results, and
initial CEUS findings in four subjects have been
reported [18]. In that report only two CEUS pa-
rameters (peak enhancement and rate of enhance-
crosphere contrast agent bypassing the pulmonary
circulation, all subjects were required to undergo
a complete history and physical examination, 12-
lead ECG, and echocardiography. Those with a
history of ultrasound contrast allergy, a right-to-
left or intracardiac shunt, pulmonary hyperten-
sion, or oxygen saturation less than 92% on room
air were not eligible for CEUS. Eligible patients
and guardians signed informed assent and consent
for CEUS at the time of enrollment in the phase
1 study. Subjects were monitored with continuous
pulse oximetry and three-lead ECG (Propaq mon-
itor, Welch Allyn) immediately before, during, and
for 30 minutes after contrast injection.
Conventional imaging was performed at base-
line, at the ends of courses 1 and 2, and after ev-
ery other course until disease progression. Each
presence of new disease [19]. Time to progression
was defined as the time in days between the on-
study date and the date of tumor progression.
Contrast-Enhanced Ultrasound Technique,
Image Analysis, and Timing
The choice of target lesion was based on
ease of visibility at gray-scale sonography and
identification of landmarks to ensure similar trans-
ducer placement during follow-up (Figs. 1A and
2A). In general, the largest tumor area was identi-
fied in the transverse or longitudinal plane, and the
transducer was held in that position throughout a
60-second recording (rate, 10 Hz) begun just before
contrast administration. All subjects received per-
flutren protein type A microsphere contrast mate-
rial (Optison, GE Healthcare). One subject was re-

Fig. 115-year-old girl with recurrent synovial sarcoma.

A, Color Doppler sonogram of largest transverse area of left supraclavicular tumor
(T) shows common carotid artery (arrow) used as landmark to ensure similar
transducer placement on follow-up contrast-enhanced ultrasound (CEUS) studies.
B, Baseline transverse CEUS image shows ROI (solid line) drawn just inside tumor
margins. Vertical line in inset time-intensity curve indicates that this image was
obtained at peak enhancement of 28.9 dB.
C, Transverse CEUS image obtained on day 7 after initiation of therapy at peak
enhancement of 2.0 dB shows 93% reduction compared with baseline. Time to
progression was 242 days. Solid line indicates ROI.
A

B C

934 AJR:206, May 2016

 Ultrasound Assessment of Response to Antiangiogenic Therapy

moved from the protocol but continued the same
treatment outside of the study and received perfl-
utren lipid microsphere contrast agent (Definitiy,
Lantheus) during follow-up because the protein
type A agent was available only for the phase 1
study participants. Subjects weighing less than 20
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ization. Tumor ROI analysis was performed online
with the CPS Auto-Tracking Contrast Quantifica-
tion system, which corrects for respiratory motion.
Because video-compressed data are known to be
less reliable than noncompressed data for quanti-
tation of tumor perfusion, the CPS system operates
line values were generated for measurement of the
AUC in arbitrary units. The AUC was divided into
wash-in (AUC1) and washout (AUC2). Because
maximal enhancement was seen approximately 10
seconds after arrival of the contrast agent, we de-
fined wash-in as the first 10 seconds and washout as

kg received 0.3 mL and those weighing 20 kg or
more received 0.50.6 mL through an indwelling
central venous line. The one exception was a patient
who preferred peripheral IV access. Bolus hand in-
jections were administered by the principal investi-
gator or radiologist coinvestigator and followed by
a 5-mL sterile saline flush.
All examinations were performed with an Acu-
son Sequoia ultrasound machine (Siemens Health-
care) with a 6C2 or 15L8 MHz transducer and the
vendors Contrast Pulse Sequencing (CPS) soft-
ware. Scanning parameters were preset by the CPS
system and included a low mechanical index of 0.2
to avoid contrast agent destruction. The focal zone,
depth, and gain were optimized for tumor visual-
with noncompressed, acoustic data [20]. The princi-
pal investigator, blinded to RECIST response, drew
an ROI on the target lesion keeping inside tumor
margins to avoid including adjacent, normal tissue
(Figs. 1B, 1C, 2B, and 2C). If the tumor did not en-
hance at least 5 dB at baseline, the contrast dose was
doubled, and the injection was repeated after a wait
of 10 minutes for the first dose to clear. If the tumor
did not enhance at least 5 dB after the second, high-
er dose, the subject was not eligible for follow-up.
The ROI time-intensity curve data were used to
measure peak enhancement (PE), time to PE, and
rate of enhancement as shown in Figure 3A. Raw
data were exported to Microsoft Excel, 2003 files,
and time-intensity curves normalized to their base-
the subsequent 10 seconds of the AUC (Fig. 3B). All
subjects underwent initial CEUS within an average
of 1 day (range, 06 days) before treatment initia-
tion. On the basis of our preclinical data, we per-
formed follow-up on days 3 and 7 (2 days) after
the start of study therapy and then at conventional
imaging response assessment time points until dis-
ease progression [1215]. To assess interobserver
variability, three reviewers (two radiologists with
11 and 5 years of CEUS experience, one sonogra-
pher with 7 years of CEUS experience) drew ROIs
on each tumor at the baseline, day 3 and 7, and end
of course 1 time points. To assess intraobserver
variability, each reviewer repeated the ROI mea-
surements for each tumor at each time point.

Fig. 221-month-old girl with recurrent rhabdoid tumor.

A, Transverse gray-scale sonogram shows peritoneal tumor (T) posterior to liver
(L) and medial to right kidney (K). Tumoral calcification (arrow) and adjacent
organs were used as landmarks for transducer placement.
B, Baseline transverse contrast-enhanced ultrasound (CEUS) image obtained at
peak enhancement of 33.5 dB shows ROI (solid line) inside tumor margins.
C, Transverse CEUS image obtained on day 7 after initiation of therapy at
peak enhancement of 30.6 dB shows 8.7% reduction compared with baseline.
Progression occurred before end of course 1, at 22 days. Solid line indicates ROI.
A

B C

AJR:206, May 2016 935

 McCarville et al.

Normalized Signal Intensity (AU)

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2 AUC1 AUC2

0
0 10 20 30 40
10 s 10 s Time (s)
1

A B
Fig. 3Use of ROI time-intensity curve data to measure peak enhancement (PE), time to PE (TTP), and rate of enhancement.
A, Screen shot shows time-intensity curve and parameters obtained from ROI analysis within tumor shown in Figure 1A. A= baseline unenhanced signal intensity in
decibels, B= maximal enhancement (PE= B A in decibels), C= time of arrival of contrast agent into ROI in seconds, D= time of maximal enhancement in seconds (TTP=
D C in seconds), E= rate of change in enhancement calculated as PE / TTP in decibels per second.
B, Graph shows normalized time-intensity curve for measurement of AUC generated offline from exported raw data for patient in Figure 1. AUC1= wash-in, AUC2=
washout, AUC= AUC1+ AUC2.

Statistical Analysis
Statistical analyses were performed with SAS
software (version 9.3, SAS Institute). We used
the Cox regression model on the first ROI mea-
surements made by reviewer 1 to assess the as-
sociation between each of the CEUS parame-
ters (PE, time to PE, rate of enhancement, AUC
(AUC1+ AUC2), AUC1, and AUC2) at baseline
and changes in these parameters from baseline
to early follow-up time points (days 3 and 7, end
of course 1) and time to progression separately.
We also investigated the association between the
time, in days, between the maximal and minimal
values of each parameter, caliber of change be-
tween baseline and maximal change, and cali-
ber of change between the maximal and minimal
values for each parameter at any time point with
time to progression separately.
Because of the small sample size, multiple regres-
sion models were not explored. Subjects who had
progression on day 42 or sooner (end of course 2)
were considered nonresponders, and those who had
progression after 42 days were considered respond-
ers. We compared the baseline values and percent-
age change of each parameter at the early follow-up
time points for responders versus nonresponders.
Values of p 0.050 were considered significant.
The variability of ROI measurements was in-
vestigated with an ANOVA model to obtain in-
terobserver and intraobserver reliability coef-
ficients that characterize the consistency and
reproducibility of measurements made by differ-
ent observers and by the same observer at review
1 or 2. Reliability coefficients vary between 0 and
1, higher values indicating greater consistency and
reproducibility in repeated measurements.
Results
Twenty-five patients underwent CEUS
screening. Four of them had lesions that were
not adequately visualized with gray-scale ul-
trasound (skull metastasis, middle medias-
tinal mass, pleural nodule, and orbital apex
nodule), two had visible lesions but declined
participation, one had a cystic mass that was
not suitable for CEUS, and one signed con-
sent for CEUS but became ineligible before
starting therapy because of clinical deteriora-
tion. Four of the remaining 17 consenting sub-
jects were excluded: two because they had no
baseline study (one, missed appointment; one,
equipment failure), one because of oxygen sat-
uration less than 92% on room air, and one
because the target lesion enhanced less than 5
dB even after the contrast dose was doubled.

TABLE 1: Values of Contrast-Enhanced Ultrasound Parameters at Baseline and Changes in Parameters at Various
Time Points During and at the End of Course 1
Parameter
Peak Rate of
Time Point n Enhancement (dB) Enhancement (dB/s) Time to Peak (s) AUC (AU) AUC1 (AU) AUC2 (AU)
Baseline 13 17.30 1.54 11.21 175.99 90.15 80.89
(4.47 to 33.4) (0.20 to 10.26) (3.21 to 33.59) (55.13 to 447.60) (18.69 to 290.12) (30.71 to 275.13)
Day 3 11 2.08 0.23 1.03 6.62 5.58 11.14
(12.37 to 5.88) (4.14 to 1.54) (16.9 to 2.97) (184.9 to 86.31) (97.96 to 26.93) (147.83 to 59.38)
Day 7 12 4.59 0.48 2.11 49.25 32.69 30.45
(26.83 to 3.84) (6.64 to 1.49) (7.37 to 12.73) (427.42 to 52.86) (161.72 to 15.46) (265.70 to 37.41)
End of course 1 12 3.24 0.85 0.65 31.81 5.26 13.62
(24.15 to 10.64) (2.73 to 2.31) (16.08 to 19.38) (385.21 to 124.7) (147.10 to 54.30) (238.11 to 70.41)
NoteData are median with range in parentheses. AU= arbitrary units.

936 AJR:206, May 2016

 TABLE 2: Hazard Ratios for Risk of Progression Associated With Changes in Contrast-Enhanced Ultrasound
Ultrasound
Parameters at Baseline Assessment
and Early of Response
Follow-Up to Antiangiogenic Therapy
Time Points
Parameter
Peak Rate of
Time Point Enhancement p Enhancement p Time to Peak p AUC p AUC1 p AUC2 p
Baseline 0.96 0.38 1.18 0.47 1.03 0.53 0.995 0.18 1.00 0.96 0.99 0.17
(0.871.05) (0.761.83) (0.941.13) (0.991.0) (0.991.01) (0.981.0)
Day 3 1.26 0.051 0.97 0.92 0.99 0.87 1.01 0.12 1.03 0.15 1.02 0.15
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(0.991.59) (0.491.89) (0.831.16) (1.01.03) (0.991.07) (0.991.05)

Day 7 1.24 0.072 1.23 0.56 0.99 0.83 1.01 0.14 1.03 0.065 1.02 0.17
(0.981.57) (0.612.46) (0.901.09) (1.01.03) (1.01.06) (0.991.04)
End of course 1 1.17 0.034 3.25 0.029 0.95 0.28 1.00 0.06 1.02 0.04 1.02 0.059
(1.011.35) (1.139.35) (0.861.04) (0.991.02) (1.01.04) (1.01.03)
NoteValues in parentheses are 95% CI.

Of the 13 evaluable subjects, four were on days 3 and 7, change in AUC1 on day 7, The baseline values of parameters, time be-
girls and nine were boys (mean age, 13 years; and changes in AUC and AUC2 at the end of tween the maximal and minimal values of
range, 1.819.8 years). The primary diagno- course 1 approached statistical significance. each parameter, caliber of change between
ses were rhabdomyosarcoma (n= 2), rhab-
doid tumor (n= 2), Wilms tumor (n= 2), and TABLE 3: Median Baseline Contrast-Enhanced Ultrasound Parameter Values
renal cell carcinoma, hepatocellular carci- and Percentage Change in Median Values at Early Follow-Up Time
noma, osteosarcoma, synovial sarcoma, ep- Points in Responders and Nonresponders
ithelioid sarcoma, and Ewing sarcoma (n=
Parameter Nonresponders (n= 4) Responders (n= 9)
1 each). Target lesion sites included liver
(n= 3), pleura (n= 2), and supraclavicular Baseline value
mass, soft-tissue component of a bone me- Peak enhancement (dB) 16.78 17.30
tastasis, lung, retroperitoneum, peritoneum,
Rate of enhancement (dB/s) 2.82 1.54
lymph node, muscle mass, and perineum
(n= 1 each). A total of 74 CEUS examina- Time to peak enhancement (s) 8.50 11.21
tions were performed at the following time AUC (AU) 175.99 188.92
points: baseline (n= 13), day 3 (n= 11), day AUC1 (AU) 102.96 53.81
7 (n= 12), end of course 1 (n= 12), end of
AUC2 (AU) 80.89 116.94
course 2 (n= 8), end of course 4 (n= 8), end
of course 6 (n= 5), end of course 8 (n= 3), Percentage change day 3
and end of courses 10 and 12 (n= 1 each). Peak enhancement 9.63 40.17
End of course 1 AUC and AUC2 data were Rate of enhancement 53.86 29.93
excluded for one nonresponder and one re-
Time to peak enhancement 16.95 25.42
sponder because of inadequate AUC2 data.
One subject reported brief taste alteration AUC 3.51 41.78
after contrast injection during three of five AUC1 15.89 27.58
examinations. There were no other adverse AUC2 60.93 37.28
events. There were nine responders and four
Percentage change day 7
nonresponders. The median time to progres-
sion for all 13 subjects was 95 days (range, Peak enhancement 10.34 59.97
22242 days), for responders was 142 days Rate of enhancement 24.25 54.37
(range, 61242 days), and for nonresponders Time to peak enhancement 10.30 35.33
was 23 days (range, 2227 days).
AUC 36.54 65.42
Table 1 summarizes descriptive data for
CEUS parameters at baseline and changes AUC1 20.68 39.15
from baseline to each early follow-up time AUC2 7.19 66.04
point. Table 2 summarizes results with the Percentage change end of course 1
Cox regression model, showing hazard ratios
Peak enhancement 8.49 49.31
for the association between time to progres-
sion and changes in parameters from base- Rate of enhancement 1.37 55.16
line to early follow-up time points. Nota- Time to peak enhancement 1.47 8.03
bly, changes in PE, rate of enhancement, and AUC 15.99 35.49
AUC1 at the end of course 1 were significant-
AUC1 5.60 28.28
ly associated with time to progression; great-
er decreases were predictive of longer time AUC2 21.58 35.08
to progression (Figs. 1 and 2). Changes in PE NoteAU= arbitrary units.

AJR:206, May 2016 937


baseline and maximal change, and caliber of
change between the maximal and minimal
values at any time point were not predictive
of time to progression, with the exception of
caliber change between maximal and mini-
mal PE (p = 0.018), such that subjects with
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larger differences had longer time to progres-
sion. There were too few subjects to reach sta-
tistical power for a comparison of respond-
ers with nonresponders. However, the median
baseline values for each group were similar
(with the exception of AUC1 and AUC2), al-
though responders had substantially greater
percentage reductions in all parameters by
day 3, day 7, or the end of course 1 compared
with nonresponders (Table 3). PE, AUC,
AUC1, and AUC2 had consistently greater re-
ductions in responders than in nonresponders
at all early time points. Interobserver and in-
traobserver reliability was generally high for
all parameters at all time points with a few
exceptions. The parameter with the best re-
liability was PE; interobserver reliability
ranged from 0.87 to 0.99 and intraobserver
reliability from 0.91 to 1.0.
Discussion
With the increasing number of molecular-
ly targeted agents being evaluated in clini-
cal trials, there is a growing need for imag-
ing methods that go beyond an assessment
of anatomic change yet yield quantitative
measurements of tumor response. Numerous
functional and metabolic imaging modalities
are being incorporated into clinical trials,
and each has unique indications, merits, and
limitations. In addition to dynamic CEUS,
promising methods of assessing tumor vas-
cularity include dynamic contrast-enhanced
CT and MRI and 15O-labeled water PET [21
24]. Dynamic CT and PET expose patients
to ionizing radiation and therefore are less
attractive for the pediatric population. Dy-
namic contrast-enhanced MRI avoids expo-
sure to radiation but is technically challeng-
ing. Because MRI contrast agents diffuse
freely across the vascular membrane, com-
plex pharmacokinetic models are needed to
analyze gadolinium concentration time-in-
tensity curves [23]. Furthermore, sedation is
often required for young patients who can-
not lie still for lengthy MRI acquisitions. In
contrast, dynamic CEUS is an ideal modal-
ity for children because it is well tolerated,
does not require sedation, and has the impor-
tant attribute of not exposing this vulnerable
population to radiation. Ultrasound contrast
agents are composed of microspheres that re-
938 AJR:206, May 2016
McCarville et al.
main within the vascular space and, because
they contain a gas, are highly reflective at ul-
trasound imaging, allowing detection at the
capillary level [25]. These contrast agents
therefore are ideal surrogate markers of tu-
mor blood flow and can be quantitated with
modern CEUS software. Dynamic quantita-
tive CEUS is becoming an important meth-
od of monitoring the effects of antiangiogen-
ic therapies in adults. The current challenge
is to define which CEUS parameters are pre-
dictive of tumor response and patient out-
come, the ideal timing for assessment, and
with regard to children, which malignancies
are most suitable for this technique.
Our findings show that CEUS depicts
changes in blood flow in a variety of solid ma-
lignancies in children very early in the course
of antiangiogenic therapy and that these
changes can be predictive of time to progres-
sion. Importantly, this imaging modality may
help identify poor responders before conven-
tional RECIST values do, thus affording the
opportunity for early intervention and tailored
management. This approach could avoid use
of toxic and ineffective maintenance therapy
for weeks or months before conventional im-
aging shows morphologic changes that indi-
cate tumor progression. In our study, subjects
with greater decreases in PE, rate of enhance-
ment, and AUC1 at the end of course 1 had
significantly longer times to progression. In
addition, reductions in PE on day 3, PE and
AUC1 on day 7, and AUC and AUC2 at the
end of course 1 were marginally significant.
Although our cohort was too small to at-
tain statistical power, there were substantial
differences in percentage changes in all pa-
rameters at early time points in responders
compared with nonresponders. Among the
six parameters that we investigated, PE ap-
pears to be the most robust indicator of tu-
mor response. We found that the early per-
centage decreases in PE relative to baseline
were 56 times greater in responders than in
nonresponders. This is perhaps not surpris-
ing because PE depends on tumor perfusion,
and tumors that respond well to antiangio-
genic therapies will have substantially re-
duced blood flow and contrast enhancement.
Our findings agree with those of Lassau et
al. [4], who investigated the value of CEUS
in 24 adults with gastrointestinal stromal tu-
mors treated with imatinib. Those investi-
gators found a strong correlation (p < 10 4)
between changes in percentage of contrast
uptake within tumors from baseline to days
7 and 14 and at 2 months in patients with a
good response, but they found no signifi-
cant change in patients with a poor response.
However, our findings differ slightly from a
subsequent study by Lassau and colleagues
[2] that assessed 42 adults with hepatocellular
carcinoma treated with bevacizumab. In that
study, changes in AUC, AUC1, AUC2, time
to PE, and mean transit time on day 3 trended
toward significant correlation with RECIST
response, but there was no correlation be-
tween PE and RECIST response or patient
survival. The reason for these differences is
not clear but could be related to differences
in the perfusion pattern of liver lesions com-
pared with other tumor sites and differences
in biologic behavior of the tumor types.
We found a high degree of interobserver
and intraobserver reliability for measurement
of all parameters at most time points. Impor-
tantly, this was especially true of PE, which
had excellent reproducibility both within a
single reviewer and between reviewers. These
findings further support the suitability of PE
as a meaningful parameter for assessing tu-
mor response to antiangiogenic therapy.
Our study had several limitations. Our
sample size was small, precluding statisti-
cal comparison of responders and nonre-
sponders. Although our findings suggest that
CEUS can be predictive of time to progres-
sion very early in the course of antiangiogen-
ic treatment, they should be interpreted with
caution. Our cohort comprised patients with
a wide variety of solid malignancies, and
CEUS may be more valuable in some than in
others. In our phase 1 study, all patients had
recurrent or refractory disease, and many
had numerous metastatic sites. Because ul-
trasound contrast agents are not U.S. Food
and Drug Administration approved for pe-
diatric use, we limited our analysis to a sin-
gle target lesion to minimize the cumulative
contrast dose. It is possible that performing
multiple contrast injections to assess several
target lesions would better represent total tu-
mor burden and overall response to therapy.
In addition, because of limitations in current
software, we imaged only a single slice of
tumor. Because tumor composition is often
heterogeneous, it may be preferable to assess
the entire tumor volume or numerous slices
throughout the tumor mass [26, 27].
Conclusion
The results of our pilot study suggest that
several quantitative CEUS parameters ob-
tained very early in the course of antiangio-
genic therapy are statistically significant pre-
 Ultrasound Assessment of Response to Antiangiogenic Therapy
dictors of time to progression in children and
adolescents with solid malignancies. Although
our cohort was too small to achieve statistical
power, we found substantial differences in per-
centage reductions in all parameters as early
as days 3 and 7 in responders versus nonre-
Downloaded from www.ajronline.org by 121.58.230.163 on 05/18/17 from IP address 121.58.230.163. Copyright ARRS. For personal use only; all rights reserved
sponders. In this patient population PE appears
to be the most robust and reproducible param-
eter. Our findings require validation in larger
clinical trials with closer-to-homogeneous pa-
tient populations. We are currently investigat-
ing the value of CEUS in children with Ewing
sarcoma who are being treated at our institu-
tion with a mammalian target of rapamycin
(mTOR) inhibitor in combination with cyto-
toxic chemotherapy. Because tumor microen-
vironments involve complex interactions be-
tween angiogenesis, tissue hypoxia, altered
metabolism, cell proliferation, and apoptosis,
the best approach to assessing tumor response
will likely require a multimodality and mul-
tiparametric approach that includes imaging
technologies that can quantify these various
processes [24]. Dynamic CEUS appears to
be an ideal modality for assessing solid tumor
vascularity in pediatric patients.
Acknowledgments
We thank Stacey Glass for technical as-
sistance in performing CEUS, Kim Johnson
for data management, the sedation nursing
team at our institution for patient monitor-
ing, Erika Thompson and Adrianne Mat-
thews for administrative assistance, and
GE Healthcare for product support.
References
1. Lassau N, Chebil M, Chami L, Bidault S, Girard
E, Roche A. Dynamic contrast-enhanced ultraso-
nography (DCE-US): a new tool for the early
evaluation of antiangiogenic treatment. Target
Oncol 2010; 5:5358
2. Lassau N, Koscielny S, Chami L, et al. Advanced
hepatocellular carcinoma: early evaluation of re-
sponse to bevacizumab therapy at dynamic con-
trast-enhanced US with quantificationprelimi-
nary results. Radiology 2011; 258:291300
3. Lamuraglia M, Escudier B, Chami L, et al. To pre-
dict progression-free survival and overall survival
in metastatic renal cancer treated with sorafenib:
pilot study using dynamic contrast-enhanced Dop-
pler ultrasound. EurJ Cancer 2006; 42:24722479
AJR:206, May 2016 939
4. Lassau N, Lamuraglia M, Chami L, et al. Gastro-
intestinal stromal tumors treated with imatinib:
monitoring response with contrast-enhanced so-
nography. AJR 2006; 187:12671273
5. Escudier B, Lassau N, Angevin E, et al. Phase I
trial of sorafenib in combination with IFN alpha-
2a in patients with unresectable and/or metastatic
renal cell carcinoma or malignant melanoma. Clin
Cancer Res 2007; 13:18011809
6. De Giorgi U, Aliberti C, Benea G, Conti M,
Marangolo M. Effect of angiosonography to mon-
itor response during imatinib treatment in patients
with metastatic gastrointestinal stromal tumors.
Clin Cancer Res 2005; 11:61716176
7. Lassau N, Lamuraglia M, Vanel D, et al. Doppler
US with perfusion software and contrast medium
injection in the early evaluation of isolated limb
perfusion of limb sarcomas: prospective study of
49 cases. Ann Oncol 2005; 16:10541060
8. Goetti R, Reiner CS, Knuth A, et al. Quantitative
perfusion analysis of malignant liver tumors: dy-
namic computed tomography and contrast-en-
hanced ultrasound. Invest Radiol 2012; 47:1824
9. Averkiou M, Lampaskis M, Kyriakopoulou K, et
al. Quantification of tumor microvascularity with
respiratory gated contrast enhanced ultrasound
for monitoring therapy. Ultrasound Med Biol
2010; 36:6877
10. Frhlich E, Muller R, Cui XW, Schreiber-Dietrich
D, Dietrich CF. Dynamic contrast-enhanced ultra-
sound for quantification of tissue perfusion.
JUltrasound Med 2015; 34:179196
11. Lassau N, Chami L, Benatsou B, Peronneau P,
Roche A. Dynamic contrast-enhanced ultraso-
nography (DCE-US) with quantification of tumor
perfusion: a new diagnostic tool to evaluate the
early effects of antiangiogenic treatment. Eur
Radiol 2007; 17(suppl 6):F89F98
12. Sims TL, McGee M, Williams RF, et al. IFN-beta
restricts tumor growth and sensitizes alveolar
rhabdomyosarcoma to ionizing radiation. Mol
Cancer Ther 2010; 9:761771
13. Dickson PV, Hamner JB, Sims T, et al. Bevaci-
zumab-induced transient remodeling of the vascu-
lature in neuroblastoma xenografts results in im-
proved delivery and efficacy of systemically
administered chemotherapy. Clin Cancer Res
2007; 13:39423950
14. Dickson PV, Hamner JB, Streck CJ, et al. Continu-
ous delivery of IFN-beta promotes sustained
maturation of intratumoral vasculature. Mol
Cancer Res 2007; 5:531542
15. McCarville MB, Streck CJ, Dickson PV, Li CS,
Nathwani AC, Davidoff AM. Angiogenesis inhibitors
in a murine neuroblastoma model: quantitative assess-
ment of intratumoral blood flow with contrast-en-
hanced gray-scale US. Radiology 2006; 240:7381
16. McCarville MB, Kaste SC, Hoffer FA, et al. Contrast-
enhanced sonography of malignant pediatric abdomi-
nal and pelvic solid tumors: preliminary safety and
feasibility data. Pediatr Radiol 2012; 42:824833
17. Coleman JL, Navid F, Furman WL, McCarville
MB. Safety of ultrasound contrast agents in the
pediatric oncologic population: a single-institu-
tion experience. AJR 2014; 202:966970
18. Navid F, Baker SD, McCarville MB, et al. Phase I
and clinical pharmacology study of bevacizumab,
sorafenib, and low-dose cyclophosphamide in chil-
dren and young adults with refractory/recurrent
solid tumors. Clin Cancer Res 2013; 19:236246
19. Therasse P, Arbuck SG, Eisenhauer EA, et al. New
guidelines to evaluate the response to treatment in
solid tumors. European Organization for Research
and Treatment of Cancer, National Cancer Insti-
tute of the United States, National Cancer Institute
of Canada. JNatl Cancer Inst 2000; 92:205216
20. Peronneau P, Lassau N, Leguerney I, Roche A, Cos-
grove D. Contrast ultrasonography: necessity of lin-
ear data processing for the quantification of tumor
vascularization. Ultraschall Med 2010; 31:370378
21. Specht JM, Kurland BF, Montgomery SK, et al.
Tumor metabolism and blood flow as assessed by
positron emission tomography varies by tumor
subtype in locally advanced breast cancer. Clin
Cancer Res 2010; 16:28032810
22. Eary JF, Link JM, Muzi M, et al. Multiagent PET
for risk characterization in sarcoma. JNucl Med
2011; 52:541546
23. Winfield JM, Payne GS, deSouza NM. Functional
MRI and CT biomarkers in oncology. EurJ Nucl
Med Mol Imaging 2015; 42:562578
24. Padhani AR, Miles KA. Multiparametric imaging
of tumor response to therapy. Radiology 2010;
256:348364
25. Greis C. Quantitative evaluation of microvascular
blood flow by contrast-enhanced ultrasound (CEUS).
Clin Hemorheol Microcirc 2011; 49:137149
26. Miles KA, Ganeshan B, Hayball MP. CT texture
analysis using the filtration-histogram method:
what do the measurements mean? Cancer Imaging
2013; 13:400406
27. Hoyt K, Sorace A, Saini R. Quantitative mapping of
tumor vascularity using volumetric contrast-en-
hanced ultrasound. Invest Radiol 2012; 47:167174