Detection of Early Tumor Response to Axitinib in Advanced Hepatocellular Carcinoma By

1 Detection of Early Tumor Response to Axitinib in Advanced Hepatocellular

2 Carcinoma by Dynamic Contrast Enhanced Ultrasound

1,2 1 1 1 3 Glen M. Lo , MD, Hassan Al Zahrani , MD, Hyun-Jung Jang , MD, Ravi Menezes , PhD,

3 3 4, 5 1 4 John Hudson , PhD, Peter Burns , PhD, Mairéad G McNamara , MB PhD, Sonja Kandel ,

1 4 1 1 5 MD, Korosh Khalili , MD, Jennifer Knox , MD, Patrik Rogalla , MD, Tae Kyoung Kim , MD

7 Affiliations

8 1. Medical Imaging, University of Toronto, Toronto, ON, Canada.

9 2. Department of Radiology, Sir Charles Gairdner Hospital, QEII Medical Centre, Perth,

10 Western Australia.

11 3. Department of Medical Biophysics, Sunnybrook Health Sciences Centre, Toronto, ON,

12 Canada.

13 4. Division of Medical Oncology, Princess Margaret Cancer Centre, University of Toronto,

14 Toronto, ON, Canada.

15 5. Department of Medical Oncology, The Christie NHS Foundation Trust/University of

16 Manchester (Institute of Cancer Sciences), Manchester, United Kingdom.

18 Corresponding author:

19 Tae Kyoung Kim, MD

20 Joint Department of Medical Imaging, University Health Network

21 Toronto General Hospital Peter Munk Building, 1 PMB-298

1 2 22 585 University Avenue Toronto, Ontario M5G 2N2

23 Phone: 1-416- 340-3372

24 Fax: 1-416-593-0502

25 [email protected]

26 ABSTRACT

28 This study aimed to evaluate the utility of dynamic contrast-enhanced ultrasound (DCE-US) in

29 measuring early tumor response of advanced hepatocellular carcinoma (HCC) to axitinib.

30 Twenty patients were enrolled (age 18–78 years; median 65). DCE-US was performed with

31 bolus injection and infusion/disruption-replenishment. Median overall survival (OS) was 7.1

32 months (1.8-27.3) and progression free survival (PFS) was 3.6 months (1.8-17.4). Fifteen

33 patients completed infusion scans and 12 completed bolus scans at 2 weeks. Among the

34 perfusion parameters, fractional blood volume at infusion (INFBV) decreased at 2 weeks in 10/15

35 (16–81% of baseline, mean 47%) and increased in 5/15 (116–535%, mean 220%). This was not

36 significantly associated with PFS (p=0.310) or progression at 16 weeks (p=0.849) but was

37 borderline statistically significant (p=0.050) with OS, limited by a small sample size. DCE-US

38 is potentially useful in measuring early tumor response of advanced HCC to axitinib, but a

39 larger trial is needed.

41 ABSTRACT WORD COUNT: 150

43 KEYWORDS: dynamic contrast-enhanced ultrasound, hepatocellular carcinoma, liver

44 neoplasms, axitinib, tumor response, perfusion

3 4 45 INTRODUCTION

47 Novel therapeutics for the treatment of hypervascular tumors, such as hepatocellular carcinoma

48 (HCC), target tumor neoangiogenesis creating the potential for developing imaging techniques

49 that can assess early treatment response by measuring early changes in tumor perfusion, rather

50 than assess the later change in tumor size (Eisenhauer et al. 2009; Jain et al. 2009; O'Connor et

51 al. 2008; Turkbey et al. 2009).

53 HCC is a hypervascular neoplasm, with tumor vascularity lending itself to imaging of perfusion.

54 Perfusion imaging can be performed using several modalities (e.g. CT, MRI, US, PET), with

55 ultrasound being relatively accessible, cheaper and not involving ionizing radiation (Provenzale

56 2007). Several groups have published promising results of dynamic contrast-enhanced

57 ultrasound (DCE-US) monitoring in various tumors measuring response to antiangiogenics,

58 including renal cell carcinoma (Lamuraglia et al. 2006; Williams et al. 2011), advanced HCC

59 (Assunta et al. 2013; Frampas et al. 2013; Sugimoto et al. 2013) and gastrointestinal stromal

60 tumors (GIST) (Knieling et al. 2013). DCE-US is now endorsed to monitor treatment response

61 in GIST by the European Society of Medical Oncology (ESMO / European Sarcoma Network

62 Working Group 2012).

64 DCE-US has been reported useful in early monitoring of response to sorafenib (an established

65 tyrosine kinase inhibitor) in advanced HCC, (Assunta et al. 2013; Knieling et al. 2012;

66 Shiozawa et al. 2012; Sugimoto et al. 2013). Axitinib is a novel selective tyrosine kinase

67 inhibitor (TKI) of vascular endothelial growth factor receptors (VEGFs) 1, 2, and 3 (Choueiri

68 2008) and has been investigated as a second-line treatment option in advanced HCC when first-

69 line therapy with sorafenib failed. This study is a sub-study of a phase II trial of second-line 4 5

70 axitinib following prior antiangiogenic therapy in advanced HCC (McNamara et al. 2015),

71 focusing on the utility of DCE-US as an imaging biomarker. The results reported by McNamara

72 et al and from a randomized phase II study of axitinib versus placebo plus best supportive care

73 in second-line treatment of advanced HCC (Kang et al. 2015) concluded that further study of

74 this agent should be in a selected population incorporating potential biomarkers of response. To

75 our knowledge, there has been no report on the use of DCE-US as an imaging biomarker for

76 axitinib treatment monitoring in HCC.

78 In addition, antiangiogenic therapy not only changes the perfusion of liver tumors, but also has

79 an effect on the perfusion of the liver parenchyma. Perfusion change of liver parenchyma with

80 the use of antiangiogenics has been recently reported to predict major adverse events as a result

81 of sorafenib therapy (Sugimoto et al. 2013).

83 The purpose of this study was to determine if DCE-US was useful in the early detection of

84 tumor response to axitinib in the treatment of patients with advanced HCC and to determine if

85 DCE-US can detect perfusion changes of liver parenchyma related to the use of axitinib.

87 MATERIALS AND METHODS

89 This is a sub-study of a prospective single arm, open-label phase II trial of axitinib in advanced

90 HCC (McNamara et al. 2015). The institutional Research Ethics Board approved the trial.

91 Written informed consent was obtained from all patients.

93 Subjects

5 6 94

95 The clinical trial enrolled 30 patients with advanced HCC for treatment with axitinib

96 (McNamara et al. 2015). Eligible patients had unresectable and/or metastatic HCC and were

97 Child-Pugh score of A or B7, with measurable progressive disease after prior treatment with

98 antiangiogenics. Twenty patients consented to undergo the DCE-US sub-study and were

99 prospectively enrolled from January 2011 to October 2013. Four patients were excluded

100 because of incomplete imaging (their clinical condition precluded them from attending all

101 imaging appointments; those that attended imaging tolerated imaging procedures). One patient

102 was excluded because he did not take the trial medication although he underwent all scheduled

103 DCE-US tests. Axitinib was started at 5 mg bid orally, titrated to 2-10 mg bid as tolerated (28

104 days = 1 cycle). Treatment continued until progressive disease or intolerable toxicity/patient

105 withdrawal. Tumor response was evaluated by multi-phasic contrast-enhanced CT, performed

106 at baseline and every 8 weeks as per protocol (McNamara et al. 2015).

108 Ultrasound Scanning

110 DCE-US was performed at baseline and 2 weeks after starting axitinib. Patients were scanned

111 using a Philips iU22 scanner (Philips Ultrasound, Bothell, WA) with a C5-1 probe, in dual-

112 imaging mode (contrast/grayscale), at low mechanical index (MI < 0.06) with non-linear

113 imaging (power modulated pulse inversion [PMPI] mode). Patients were scanned according to

114 trial protocol by one of three trial radiologists (HJ, KK, TK), experienced in DCE-US. A single

115 target lesion, which was most easily accessible with ultrasound, in each patient was examined in

116 either sagittal or coronal planes, so that respiratory motion was confined to the scan plane and

117 could be corrected for post-processing. Tumor sizes were measured, in three planes. DCE-US

118 was performed by using two different methods: bolus-injection and infusion with 6 7

119 disruption/replenishment. First, a single bolus injection was measured, imaging the tumor at its

120 largest visible plane. Then six disruption-replenishment acquisitions with continuous infusion of

121 the contrast agent were performed at the same location. The ultrasound contrast agent used

122 consisted of perflutren lipid microspheres (Definity; Lantheus, North Billerica, MA). For the

123 bolus injection, 0.2 mL of contrast agent was injected into an antecubital intravenous catheter,

124 followed by a 5 mL saline flush. Scanning time started at the end of the flush and was

125 continuous for 2 minutes, without a breath-hold. Infusion began after the bolus technique. An

126 infusion of 0.9 mL of perflutren lipid microspheres diluted in 54 mL of saline was administered

127 over 12 minutes by using an injection pump (Medfusion 3500; Smiths Medical, Dublin Ohio).

128 After waiting 2 minutes for the infusion to reach steady state, six disruptions (high-mechanical

129 index 8-frame flash) were performed, and each corresponding replenishment sequence was

130 measured over 30 seconds, with breath-hold (Williams et al. 2011).

132 Image Analysis

134 Ultrasound data were transferred from the scanner via DVD to a separate workstation for

135 analysis. Raw data was saved, to allow linearization. If the clip from either bolus or infusion

136 measurements demonstrated severe respiratory motion which makes it difficult to place the

137 regions of interest (ROI), a motion correction region-of-interest was drawn on the image,

138 encompassing as much tumor and liver visible, and the software’s motion compensation

139 algorithm applied (Williams et al. 2011). Regions of interest (ROI) were drawn: the first within

140 the tumor (T) and the second in adjacent liver parenchyma (L). For tumor, a free-hand ROI was

141 drawn on grayscale image as close to the tumor margin as possible, to ensure inclusion of

142 peripheral tumor vascularity (Figures 1, 2). For the liver, a free-hand ROI was drawn at the

7 8 143 same depth and size as the tumor ROI. Not all sonographic windows enabled adequate

144 visibility of adjacent liver for a liver ROI analysis.

146 Contrast Enhancement Models

148 Our study implemented two different methods of DCE-US with their correspondingly

149 appropriate perfusion models (below). The first method relies on a single bolus of contrast

150 injected intravenously in a peripheral vein. The strengths of this technique include its

151 widespread use among DCE-US centers permitting a more direct comparison of results, and its

152 high signal to noise ratio. In a practical setting, the method is limited to a single measurement

153 per study. Reproducibility is also affected by the unknown influence of cardio-pulmonary on

154 the local arterial/portal input function. Implementing the infusion method requires additional

155 equipment (infusion pump), but permits multiple measurements to made in quick succession

156 and has direct control over the arterial/portal input function (Hudson et al. 2015). For each

157 DCE-US study, in the single bolus injection clip and six infusion clips, the observed perfusion

158 in the selected region-of-interest was plotted as a time-intensity curve by the computer software

159 fitting data to an appropriate model (Hudson et al. 2011), allowing for quantitative

160 measurements of perfusion parameters as follows.

162 Bolus measurements were fitted to a model using a local-density random-walk equation (Figure

163 1). For the bolus injection, seven perfusion parameters were recorded:

164 1. Total area under the curve (BOLAUC).

165 2. Peak signal intensity (BOLPEAK).

166 3. Time to peak signal intensity (BOLTTP).

167 4. Area under the curve wash-in (area up to peak, BOLAUC-in).

168 5. Area under the curve wash-out (area after peak, BOLAUC-out).

169 6. Mean transit time (BOLMTT).

170 7. Inflow slope (BOLSLOPE).

172 Infusion disruption-replenishment measurements were fitted to a lognormal perfusion model,

173 using in-house software (Matlab; The Mathworks Inc., Natick, MA). The resulting curve’s

174 plateau reflects tumor fractional blood volume (INFBV) and its slope reflects tumor mean flow

175 velocity (INFVM) (Hudson et al. 2011). The average of six (or more) measurements was used.

176 For infusion or disruption/replenishment technique, two perfusion parameters were recorded:

177 1. Peak

178 intensity, which reflects fractional blood volume (INFBV).

179 2. Slope, which reflects flow velocity (INFVM).

181 Standard Endpoints

183 Tumor response evaluation was performed according to RECIST 1.1 at CT scan (Eisenhauer et

184 al. 2009). The primary endpoint of tumor control rate in the clinical study was assessed by CT

185 scan at 16 weeks by RECIST 1.1 criteria (Edeline et al. 2012). Secondary clinical endpoints

186 included progression free survival (PFS) and overall survival (OS). Perfusion parameters for

187 each patient were examined as a percentage change observed at the 2 week DCE-US scan,

188 compared to baseline. Patients were then grouped for each perfusion parameter according to

189 whether an increase or decrease in perfusion was observed.

9 10 191 Perfusion parameters were analyzed for associations with response (responders demonstrating

192 stable disease or treatment response, and non-responders having progressive disease on the 16

193 week CT), OS and PFS. For each parameter, patients were grouped as having increased or

194 decreased perfusion and correlated with response, PFS or OS using Kaplan-Meier analysis.

195 Differences between perfusion parameter subgroups (increased or decreased perfusion) were

196 examined using the log-rank test. Analysis was made for associations between infusion

197 parameters at the initial 2-week US scan and LFTs at 16 weeks, using Spearman’s rho

198 correlation coefficients and the Mann-Whitney test.

200 RESULTS

201 Fifteen patients were included in final analysis (10 men and 5 women, mean age 57 years, range

202 18 to 78 years). Causes of hepatic cirrhosis included alcohol (n = 2), hepatitis B (n = 6),

203 hepatitis C (n = 3) and other (n = 4). (Table. 1.)

205 Among the 15 patients, all had complete infusion data but only 12 had complete bolus-injection

206 data at 2 weeks, due to corrupted data storage on the single bolus-injection run, discovered at

207 post-processing (the patients underwent the scanning). Mean tumor size at baseline was 5.2 cm

208 (range, 1.6–9.3 cm). At 2 weeks, mean tumor size was 102% of baseline (100% of baseline

209 being no change, standard deviation 22 %). Median PFS was 3.6 months (range, 1.8–17.4

210 months; 95% CI: 2.085, 5.115) based on RECIST 1.1 (Edeline et al. 2012). Median OS was 7.1

211 months (range, 1.8–27.3 months; 95% CI: 0, 14.270). At the time of analysis, five patients were

212 still alive, and OS was assigned as the current duration of therapy.

214 DCE-US Infusion Method

216 Reduction of tumor fractional blood volume (INFBV) using the infusion method at 2 weeks

217 occurred in 10/15 (16–81% of baseline, mean 47%) while 5/15 showed an increase (116–535%,

218 mean, 220%). (Figure 3.) The Kaplan-Meier analyses for INFBV at 2 weeks and PFS showed no

219 statistically significant difference (p = 0.310) but for OS the p-value was borderline (p = 0.050,

220 Figure 5). For the clinical outcome of tumor response at 16 weeks, Kaplan-Meier analysis and

221 log-rank test also showed no association between INFBV at 2 weeks and progression (p = 0.849).

222 (Table 2.)

224 Reduction of infusion tumor blood velocity (INFVM) at 2 weeks occurred in 5/15 (42-98% of

225 baseline, mean 83%) while 8/15 showed an increase (1–25% increase from baseline, mean, 9%)

226 and there was no change in 2/15 measured. An increase or decrease in INFVM at 2 weeks did not

227 correlate to the clinical outcomes of PFS, OS, or treatment response at 16 weeks (analyses not

228 shown).

230 DCE-US Bolus Method

232 Reduction of BOLAUC at 2 weeks occurred in 5/12 (11–68% of baseline, mean, 38%) while 7/12

233 showed an increase (2–72% increase, mean, 32%). (Figure 4.) The Kaplan-Meier analyses for

234 BOLAUC at 2 weeks and PFS or OS showed no statistically significant difference between

235 patients that had an increase or decrease in measured tumor perfusion (p = 0.357 and p = 0.711,

236 respectively, Figure 5). For the clinical outcome of tumor response at 16 weeks, Kaplan-Meier

237 analysis and log-rank test showed no association between BOLAUC at 2 weeks and progression

238 (p = 0.369).

11 12 239

240 The remaining six bolus method perfusion parameters, including BOLPEAK, BOLTTP, BOLAUC-in,

241 BOLAUC-out, BOLMTT, and BOLSLOPE, also did not correlate with the clinical outcomes of PFS, OS,

242 and tumor response at 16 weeks (analyses not shown).

245 Liver Perfusion

247 Eight patients had measureable liver bolus data and 11 patients had measureable liver infusion

248 data at baseline and at 2 weeks. (Table 3.) Not all patients had visible liver parenchyma in the

249 images recorded to evaluate the tumor mass at each time point. Liver function test results were

250 available for all patients at 16 weeks. Six of 8 bolus and 10 of 11 infusion patients had

251 measurable decrease in BOLAUC or INFBV in liver parenchyma at two weeks (BOLAUC median

252 -44%, range -94% to +574%; INFBV median -47%, range -67% to +99%). No patients were

253 recorded to have liver adverse events (rise of LFTs, jaundice, or encephalopathy). No patients

254 were recorded to have treatment-related liver adverse events. One patient with liver

255 decompensation had tumor progression and dehydration and was palliated.

257 DISCUSSION

259 Emerging use of antiangiogenic chemotherapeutics necessitates measurement of treatment

260 response by imaging of tumor perfusion, to detect changes in tumor vascularity, rather than

261 assess the later change in tumor size (Eisenhauer et al. 2009; Jain et al. 2009; O'Connor et al.

262 2008; Turkbey et al. 2009). Axitinib is a novel antiangiogenic agent and is both a potent and

263 selective tyrosine kinase inhibitor (TKI). It acts through interrupting pathways for tumor 12 13

264 neoangiogenesis, blocking signaling at vascular endothelial cell growth factor receptor

265 (VEGFR), platelet-derived growth factor receptor-β (PDGFR-β) and colony stimulating factor-1

266 receptor (CSF-1) (Choueiri 2008; Kelly and Rixe 2010; Rugo et al. 2005). It has been studied

267 in the treatment of hematologic and solid-organ cancers, including cholangiocarcinoma,

268 pancreatic cancer, breast cancer, acute myeloid leukemia and renal cell carcinoma (Rugo et al.

269 2005; Takahashi et al. 2014; Wilmes et al. 2007).

271 Our study is the first to report infusion technique parameters utilizing DCE-US for early

272 detection of treatment response to axitinib in patients with advanced HCC. The included patient

273 cohort was 2/3 male with the cause of cirrhosis predominantly relating to hepatitis viral

274 infection, which is representative of patients referred to a tertiary centre in Canada. Concordant

275 with other published studies examining DCE-US perfusion parameters in early detection of

276 HCC response to antiangiogenics, we have demonstrated measureable differences in tumor

277 perfusion at 2 weeks, a time frame similar to other studies. Our results demonstrate

278 measureable changes in HCC tumor perfusion at 2 weeks of therapy with axitinib. Tumor sizes

279 remained stable at this early time point (according to RECIST 1.1) but increases or decreases in

280 perfusion parameters were measurable. Early detection of response could potentially reassure

281 the patient and clinician to persist with therapy. Early detection of non-response could trigger an

282 escalation of dose in the absence of concerning toxicity, or discontinue or change therapy in a

283 declining patient with troublesome side effects. Treatment-related adverse events were

284 commonly seen on this study but usually manageable and some patients could tolerate dose

285 escalation (McNamara et al. 2015). There is a growing body of evidence in tyrosine kinase

286 inhibitor research that support a more dynamic and individualized patient dosing strategy of

287 drug based on biomarkers such as toxicity and imaging (Bjarnason et al. 2015).

13 14 288

289 In our study, the most promising perfusion parameter on DCE-US to detect response to axitinib

290 was INFBV, an initial reduction in tumor perfusion at two weeks seen in patients with increased

291 PFS and OS. In our data, tumor blood volume (INFBV) with infusion technique decreased at 2

292 weeks in 10/15 (16 - 81% of baseline, mean, 47%) and increased in 5/15 (116 - 535%, mean,

293 220%). The association was not significantly correlated with PFS (p = 0.310) or progression at

294 16 weeks (p = 0.849) but was borderline statistically significant (P = 0.050) for OS, with the

295 small sample size being a limitation. Further studies with a larger patient population are

296 required to confirm the utility of this technique in evaluating the usefulness of perfusion

297 parameters in DCE-US. Measurable changes in tumor INFBV by the infusion technique have

298 been reported in RCC: Williams et al compared bolus and infusion techniques in 17 patients

299 with RCC, treated with sunitinib (Williams et al. 2011). DCE-US was performed at 2 weeks and

300 compared to imaging using RECIST criteria at 6 weeks, and PFS. The infusion technique

301 demonstrated a reduction in median tumor fractional blood volume of 73% at this early time

302 point, before a change in perfusion by the bolus method was detected. However, this did not

303 predict PFS or RECIST response at 6 weeks.

305 Our results did not confirm the utility of BOLAUC as a parameter predictive of tumor response

306 reported in other studies, but we only had 12 patients with complete bolus data. In contrast,

307 there are several prior studies that have reported BOLAUC as a promising imaging parameter to

308 predict the patient’s outcome. These studies involved different antiangiogenic agents, using

309 different ultrasound contrast agents and equipment. For example, Lassau et al. measured tumor

310 response to bevacizumab using the bolus technique in 42 patients with HCC, at days 3, 7, 14

311 and 60 of therapy (Lassau et al. 2011). They reported results that trended towards statistical

312 significance for prediction of tumor response by BOLAUC, as well as BOLAUC-in and BOLAUC-out 14 15

313 and TTP. Frampas et al measured tumor response to sorafenib in 19 patients with HCC using

314 the bolus method at one month post commencement of treatment. A decrease of > 40% in

315 BOLAUC was reported to predict non-progression by RECIST criteria at two months (Frampas et

316 al. 2013). Assunta et al measured tumor response to sorafenib in 28 patients with HCC using the

317 bolus method at 15 and 30 days post commencement of treatment, and demonstrated that

318 BOLAUC at 15 days predicted tumor response at two months (Assunta et al. 2013).

320 Most patients with measurable liver perfusion demonstrated a decrease in background liver

321 perfusion at 2 weeks, measured by both bolus and infusion methods: 6/8 BOLAUC and 11/12

322 INFBV; however, this did not correlate to the hepatic adverse events (increased LFT, jaundice, or

323 encephalopathy). Sugimoto et al measured tumor response to sorafenib using the bolus method

324 in 37 patients with HCC, at days 7, 14 and 28 of treatment (Sugimoto et al. 2013). The BOLAUC

325 at day 7 of liver parenchyma predicted adverse events presumed related to drug toxicity on the

326 liver. We demonstrated measureable changes in liver parenchyma perfusion parameters with

327 both bolus and infusion techniques, but report no adverse events of presumed liver drug

328 toxicity.

330 Our study had a number of limitations. This sub-study was limited by a small sample size with

331 inability to measure lesions at differing timepoints in a number of patients. Larger study

332 population numbers are needed to confirm expected utility for DCE-US in measuring tumor

333 response to the novel antiangiogenic agent, axitinib. Also, the clinical endpoint of OS may not

334 be the ideal outcome measure to correlate with DEC-US tumor response to axitinib in this study

335 population as there are competing causes for mortality, such as liver failure relating to

336 background cirrhosis. In addition, as part of this Phase II clinical trial, the enrolment was of a

15 16 337 patient group who had failed previous therapy, and whose option was salvage therapy, therefore

338 results may compromised.

340 In summary, INFBV measurement using the DCE-US infusion technique is potentially useful as

341 an imaging biomarker to predict OS in patients with advanced HCC who are treated with

342 axitinib. Early perfusion reduction of HCC tumors was detected by DCE-US (INFBV) 2 weeks

343 after treatment with axitinib in most patients, when tumor size had not changed. A larger study

344 is needed to investigate further.

346 FUNDING SUPPORT 347 This was an investigator-initiated study and was funded in part by 348 Pfizer Canada. Dr. Jennifer J. Knox acquired this funding.

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441 FIGURE LEGENDS

443 Figure 1. DCE-US with Bolus Method and Time-Intensity Curve

444 The region-of-interest (ROI) outlines the tumor on simultaneous contrast-ultrasound image

445 (left) and B-mode image (right). The intensity of contrast bubbles is plotted against time, with

446 the fit curve superimposed. 1. Peak signal intensity (BOLPEAK). 2. Time to peak signal intensity

447 (BOLTTP). 3. Area under the curve wash-in (area up to peak, BOLAUC-in). 4. Area under the curve

448 wash-out (area after peak, BOLAUC-out). 5. Total area under the curve (BOLAUC) = 3 + 4.

450 Figure 2. DCE-US with Infusion Method and Time-Intensity Curve

451 The region-of-interest (ROI) outlines the tumor on simultaneous contrast-ultrasound image

452 (left) and B-mode image (right). The intensity of contrast bubbles is plotted against time, with

453 the fit curve superimposed. 1. Peak intensity, which reflects fractional blood volume (INFBV).

454 2. Slope, which reflects flow velocity (INFVM).

456 Figure 3. Percentage Change of BOLAUC at Two Weeks From Baseline by Patient

458 Figure 4. Percentage Change of INFBV at Two Weeks From Baseline by Patient

460 Figure 5. Kaplan-Meier Analyses for PFS and OS and Patients with Increased or

461 Decreased BOLAUC or INFBV at Two Weeks

21 22 463 TABLES

464 Table 1. Patient Demographics, Tumor Size, Standard Clinical Outcomes

r Size

Tumo 2 PFS OS

r Size weeks (mont (mont

Sex Age Cause CP (mm) (mm) hs) hs) 1 M 36 Other A5 34 25 2.5 7.1 2 F 22 Hep B A5 58 52 13.2 22.4 3 F 65 Other A5 37 29 12.6 27.3 4 F 18 Hep B A5 36 33 3.6 6.6 5 M 57 Hep C A5 26 43 1.9 6.3 6 F 52 Hep C B7 62 67 12.3 13.7 7 M 75 Hep C B7 45 51 1.8 1.8 Alcoh

8 M 69 ol A5 79 72 17.4 17.4 9 M 78 Hep B A5 16 28 4.6 4.6 10 M 50 Hep B A5 37 36 3.4 6.7 11 M 66 Hep B A5 66 68 10.8 10.8 12 M 76 Other A6 91 98 2.6 3.1 13 M 66 Hep B A5 57 67 3.5 8.1 14 F 69 Other A5 47 40 5.9 7.1 Alcoh

15 M 55 ol A6 93 97 3.4 3.4 *CP= Childs-Pugh score. PFS = Progression-Free Survival. OS = Overall

Survival. 465

467 Table 2. Changes of DCE-US Perfusion Parameters at Two Weeks Compared with Baseline and Clinical Outcomes

TUMOR TUMOR TUMOR TUMOR TUMOR TUMOR TUMOR TUMOR TUMOR PFS OS

BOLAUC BOLPEAK BOLAUC-IN BOLAUC-OUT BOLTTP BOLMTT BOLSLOPE INFBV INFVM (months) (months) 1 NA NA NA NA NA NA NA -47% 14% 2.5 7.1 2 NA NA NA NA NA NA NA -69% 19% 13.2 22.4 3 2% -14% 544% -69% 575% -11% -84% -29% -4% 12.6 27.3 4 -89% -88% -87% -89% 15% -2% -90% -80% -2% 3.6 6.6 5 17% -39% 241% -7% 461% 165% -80% 49% -14% 1.9 6.3 6 57% 127% 15% 62% -48% -53% 298% -37% 25% 12.3 13.7 7 -45% -31% -58% -42% -32% -29% -31% -51% 0% 1.8 1.8 8 36% 33% 39% 34% -2% 1% 13% -44% 3% 17.4 17.4 9 NA NA NA NA NA NA NA 16% 4% 4.6 4.6 10 -32% -12% 10280% -38% 3650% -3% -88% -19% 0% 3.4 6.7 11 -88% -84% -91% -87% -41% -48% -72% -84% 5% 10.8 10.8 12 7% -8% 10% 6% 17% 30% -11% 436% -10% 2.6 3.1 13 -60% -45% -68% -58% -40% -46% -13% -70% 1% 3.5 8.1 14 72% -56% 202% 66% 517% 803% -90% 75% -58% 5.9 7.1 15 35% 14% 202% 16% 143% -7% -42% 23% 2% 3.4 3.4 NA = Not Available. Values expressed as percentage change from baseline. i.e. no change = 0%, decrease = a negative % and

increase = a positive %.

PFS = Progression-Free Survival. OS = Overall Survival.

468 Table 3. Changes of DCE-US Perfusion Parameters at Two Weeks Compared with Baseline

469 and Clinical Outcomes

LIVER LIVER LIVER PFS (mon

Pt BOLAUC INFBV INFVM (months) ths) 1 NA -35% 5% 2.5 7.1 2 NA NA NA 13.2 22.4 3 NA NA NA 12.6 27.3 4 -93% -59% 33% 3.6 6.6 5 -22% -16% -23% 1.9 6.3 6 574% 99% 7% 12.3 13.7 7 -33% -55% -7% 1.8 1.8 8 -54% -64% 12% 17.4 17.4 9 NA -47% -20% 4.6 4.6 10 NA -4% 1% 3.4 6.7 11 -86% -67% -15% 10.8 10.8 12 NA NA NA 2.6 3.1 13 -68% -57% -2% 3.5 8.1 14 8% -16% 0% 5.9 7.1 15 NA NA NA 3.4 3.4 NA = Not Available. Values expressed as percentage

change from baseline. i.e. no change = 0%, decrease = a

negative % and increase = a positive %. PFS =

Progression-Free Survival. OS = Overall Survival. 470 471