Conceived and designed the experiments: CPR YB DGD JAM JPE GDD RKJ. Performed the experiments: CPR YB DGD MA JL JPE RKJ. Analyzed the data: CPR YB DGD JAM RQ MA JL GDD RKJ. Contributed reagents/materials/analysis tools: CPR YB DGD JAM RQ MA JL JPE GDD RKJ. Wrote the paper: CPR YB DGD MA GDD RKJ.
Current address: National Cancer Centre Singapore, Singapore, Singapore
Current address: Since the study: AstraZeneca PLC, Boston, Massachusetts, United States of America
¶ These authors also contributed equally to this work.
Chandrajit P. Raut is a Novartis - Honorarium. Johanna Lahdenranta has an employment/leadership position at Merrimack Pharmaceuticals. J. Paul Eder is currently employed at AstraZeneca PLC. George D. Demetri is a Consultant for Novartis, Pfizer, Ariad, Johnson & Johnson, Genentech, Infinity Pharmaceuticals, ZioPharm, Alnylam, Idera, Momenta Pharma, EMD-Serono, Glaxo Smith Kline, Amgen, Daiichi-Sankyo, ArQule, Enzon, Millenium/Takeda, PamGene (no compensation), Plexxikon, N-of-One (no compensation), Champions Biotechnology, and Kolltan Pharmaceuticals; on the Scientific Advisory Board of ZioPharm, PamGene, Plexxikon, N-of-One, Kolltan Pharmaceuticals (chair); on the Medical Advisory Board at Kolltan Pharmaceuticals (chair); an Honorarium at Novartis and Pfizer; provides research support (to Dana-Farber Cancer Institute for clinical trial) at Novartis, Pfizer, Ariad, Johnson & Johnson, Bristol-Myers Squib, Infinity Pharmaceuticals, and Daiichi-Sankyo; and has equity (minor stake, non-public) at PamGene, Plexxikon, N-of-One, Champions Biotechnology, and Kolltan Pharmaceuticals. Rakesh K. Jain has a Consultant/advisory role at Millenium, Dyax, AstraZeneca, Regeneron, Astellas-Fibrogen, MorphoSys AG, Genzyme, SynDevRx, and Noxxon; is Honoraria at Pfizer and Genzyme (honoraria for lecture); provides research funding/contracted research at Dyax, AstraZeneca and MedImmune; and has ownership interest in SynDevRx. There are no patents or products in development or marketed products to declare. This does not alter the authors′ adherence to all the PLoS ONE policies on sharing data and materials. Yves Boucher, Dan G. Duda, Jeffrey A. Morgan, Richard Quek and Marek Ancukiewicz declare no competing interest.
Sorafenib is a multi-targeted tyrosine kinase inhibitor with therapeutic efficacy in several malignancies. Sorafenib may exert its anti-neoplastic effect in part by altering vascular permeability and reducing intra-tumoral interstitial hypertension. As correlative science with a phase II study in patients with advanced soft-tissue sarcomas (STS), we evaluated the impact of this agent on intra-tumor interstitial fluid pressure (IFP), serum circulating biomarkers, and vascular density.
Patients with advanced STS with measurable disease and at least one superficial lesion amenable to biopsy received sorafenib 400 mg twice daily. Intratumoral IFP and plasma and circulating cell biomarkers were measured before and after 1–2 months of sorafenib administration. Results were analyzed in the context of the primary clinical endpoint of time-to-progression (TTP).
In 15 patients accrued, the median TTP was 45 days (range 14–228). Intra-tumoral IFP measurements obtained in 6 patients at baseline showed a direct correlation with tumor size. Two patients with stable disease at two months had post-sorafenib IFP evaluations and demonstrated a decline in IFP and vascular density. Sorafenib significantly increased plasma VEGF, PlGF, and SDF1α and decreased sVEGFR-2 levels. Increased plasma SDF1α and decreased sVEGFR-2 levels on day 28 correlated with disease progression.
Pretreatment intra-tumoral IFP correlated with tumor size and decreased in two evaluable patients with SD on sorafenib. Sorafenib also induced changes in circulating biomarkers consistent with expected VEGF pathway blockade, despite the lack of more striking clinical activity in this small series.
ClinicalTrials.gov
The bi-aryl urea sorafenib was initially developed as an inhibitor of
Studies from our group and others have shown that the intra-tumoral interstitial fluid pressure (IFP) in human sarcomas, melanomas, and carcinomas (including colon, breast, lung, head and neck, cervix) is significantly higher than in normal tissues
Using study drug supplied by the NCI Cancer Therapy Evaluation Program (CTEP), we conducted a phase II trial of sorafenib in patients with advanced soft tissue sarcomas (STS), with the aim of exploring whether sorafenib administration is associated with mechanistically-related changes in intra-tumoral IFP and vascular density as well as circulating biomarkers of angiogenesis.
The protocol for this trial and supporting CONSORT checklist are available as supporting information; see
Evaluations included physical examination, laboratory data, documentation of ECOG performance status, CT or MRI imaging (at the discretion of the treating physicians), and electrocardiogram. Each of these evaluations was performed prior to initial sorafenib administration, every one to four weeks (depending on cycle) while on study, and one month after the last dose of sorafenib was administered. Imaging was performed every other month while on study. Adverse events and toxicities were assessed on schedule every one to four weeks (depending on cycle) and one month after the last dose of sorafenib was administered. Pharmacokinetic data were measured on days 28 and 56.
The correlative scientific endpoints of this trial included measurements of changes in circulating biomarkers and IFP, radiographic responses, toxicity, and pharmacokinetics. The primary clinical endpoint was time-to-progression (TTP), measured from date of registration to date of radiographic progression. Response and progression were evaluated using the Response Evaluation Criteria in Solid Tumors (RECIST)
Biomarker changes from baseline were tested using the exact paired Wilcoxon test
Patient and tumor characteristics are listed in
Patient characteristics | Number (%) |
Median age | 59 years (range, 30–84 years) |
Sex | |
Male | 9 (60) |
Female | 6 (40) |
ECOG |
|
0 | 8 (53.3) |
1 | 7 (46.7) |
Histology | |
Angiosarcoma | 1 (6.7) |
Desmoplastic small round cell tumor | 1 (6.7) |
Gastrointestinal stromal tumor | 1 (6.7) |
Leiomyosarcoma | 4 (26.7) |
Liposarcoma | 1 (6.7) |
Malignant diffuse-type giant cell tumor | 1 (6.7) |
Malignant peripheral nerve sheath tumor | 1 (6.7) |
Malignant phyllodes tumor | 1 (6.7) |
Myxofibrosarcoma | 2 (13.3) |
Synovial sarcoma | 2 (13.3) |
Primary site | |
Upper extremity | 1 (6.7) |
Lower extremity | 1 (6.7) |
Trunk | 12 (80.0) |
Pelvis | 1 (6.7) |
*ECOG, Eastern Cooperative Oncology Group.
Adverse events probably or definitely related to treatment are listed in
Toxicity | Grade 1 (%) | Grade 2 (%) | Grade 3 (%) | Grade 4 (%) | Any Grade (%) |
Hand-foot syndrome | 13/6 (40.0) | 5/2 (13.3) | 3/3 (20.0) | 0 | 21/7 (46.7) |
Rash/desquamation | 4/1 (6.7) | 2/1 (6.7) | 3/1 (6.7) | 0 | 8/1 (6.7) |
Fatigue | 5/3 (20.0) | 0 | 0 | 0 | 5/3 (20.0) |
Mucositis/stomatitis | 5/4 (26.7) | 0 | 0 | 0 | 5/4 (26.7) |
Hypertension | 2/2 (13.3) | 0 | 2/1 (6.7) | 0 | 4/3 (20.0) |
Extremity pain | 4/1 (6.7) | 0 | 0 | 0 | 4/1 (6.7) |
Erythema multiforme | 2//1 (6.7) | 1/1 (6.7) | 0 | 0 | 3/2 (13.3) |
Skin – other | 2/2 (13.3) | 0 | 1/1 (6.7) | 0 | 3/2 (13.3) |
Hemoglobin | 0 | 2/1 (6.7) | 0 | 0 | 2/1 (6.7) |
Anorexia | 2/1 (6.7) | 0 | 0 | 0 | 2/1 (6.7) |
Bilirubin | 2/1 (6.7) | 0 | 0 | 0 | 2/1 (6.7) |
Oral cavity – pain | 2/1 (6.7) | 0 | 0 | 0 | 2/1 (6.7) |
Platelets | 1/1 (6.7) | 0 | 0 | 0 | 1/1 (6.7) |
Fever without neutropenia | 1/1 (6.7) | 0 | 0 | 0 | 1/1 (6.7) |
Alopecia | 1/1 (6.7) | 0 | 0 | 0 | 1/1 (6.7) |
Pruritis | 0 | 1/1 (6.7) | 0 | 0 | 1/1 (6.7) |
Dehydration | 0 | 0 | 1/1 (6.7) | 0 | 1/1 (6.7) |
Diarrhea | 0 | 1/1 (6.7) | 0 | 0 | 1/1 (6.7) |
Alkaline phosphatase | 1/1 (6.7) | 0 | 0 | 0 | 1/1 (6.7) |
Muscle - pain | 1/1 (6.7) | 0 | 0 | 0 | 1/1 (6.7) |
As a mechanistic pharmacodynamic assessment of sorafenib administration, we measured circulating levels of angiogenic biomarkers before and after sorafenib dosing, compared baseline biomarker levels with baseline tumors characteristics, and correlated baseline biomarker levels or changes in biomarker levels with radiographic responses. Sorafenib treatment induced significant increases in plasma circulating VEGF, PlGF, IL-8, and SDF1α and decreases in sVEGFR2, but not other angiogenic and inflammatory biomarkers (bFGF, sVEGFR-1, TNF-α, IL-6, CPCs or VEGFR-2+ monocytes) (
Pre-Treatment | Day 28 | ||
Plasma Biomarker | (N = 14) | (N = 10) | |
|
140 [87,161] | 214 [154,311] | 0.002 |
|
36 [19,68] | 29 [15,86] | 0.19 |
|
22 [17,34] | 52 [40,62] | 0.002 |
|
112 [99,142] | 83 [66,93] | 0.38 |
|
6212 [5826–7207] | 4781 [3942–5484] | 0.002 |
|
2306 [2218,2582] | 2705 [2531,3472] | 0.0039 |
|
5.8 [3.9,17.2] | 12 [5,33] | 0.13 |
|
5.7 [4.3,14.5] | 7.1 [5.6,22.2] | 0.0059 |
|
9.2 [7.4,11.8] | 9.2 [7.4,14.8] | 0.11 |
|
0.050 [0.030,0.074] | 0.057 [0.029,0.075] | 0.20 |
Data are shown as medians and interquartile ranges (in square brackets) compared to baseline levels.
VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; PlGF, placental growth factor; sVEGFR-1, soluble VEGF receptor-1; sVEGFR-2, soluble VEGF receptor-2; SDF1α, stromal cell-derived factor-1-alpha; IL-6, interleukin-6; IL-8, interleukin-8; TNF-α, tumor necrosis factor-alpha, CPCs, circulating progenitor cells; PBMC, peripheral blood mononuclear cells.
Kendall's |
Pre-Treatment Size | Response (SD) | Radiographic Response |
|
0.87 [0.56,1.17] | −0.43 [−0.91,0.05] | −0.20 [−0.63,0.23] |
0.017 | 0.40 | 0.82 | |
|
0.42 [0.10,0.74] | 0.11 [−0.23,0.45] | 0.09 [−0.36,0.54] |
0.037 | 0.70 | 0.74 | |
|
0.31 [−0.03,0.65] | −0.39 [−0.64,−0.14] | −0.61 [−0.94,−0.27] |
0.12 | 0.11 | 0.0054 | |
|
N/A | 0.62 [0.37,0.87] | 0.56 [0.31,0.80] |
0.033 | 0.029 | ||
|
N/A | −0.47 [−0.77,−0.17] | −0.56 [−1.04,−0.07] |
0.17 | 0.045 |
Data are shown as Kendall's
Data are shown as Kendall's
SD, stable disease; IFP, interstitial fluid pressure; IL-6, interleukin-6; PlGF, placental growth factor; sVEGFR-2, soluble vascular endothelial growth factor receptor-2; SDF1α, stromal cell-derived factor-1-alpha.
To identify blood vessels and perivascular cells in tumor sections, we performed a double immunostaining procedure with antibodies against CD31 and α-SMA, respectively. In the biopsies of 2 patients, the decrease in vessel density was 59% and 83%, respectively, after sorafenib treatment (
Immunostaining of CD31-positive (brown) or CD31 and α-SMA-positive (brown and pink) tumor vessels before (A) and 28 days after (B) the initiation of sorafenib treatment. Sections were counterstained with hematoxylin. Note the reduced vessel density and cellular content in the sorafenib-treated lesion.
Patient | Vessels/field | CD31+/α-SMA+ | CD31+/α-SMA− |
vessels per field | vessels per field | ||
Pt#1/Day 0 | 6.9 | 3.3 | 3.6 |
Pt#1/Day 28 | 1.2 | 0.8 | 0.4 |
Pt#5/Day 0 | 15.9 | 10.1 | 5.8 |
Pt#5/Day 56 | 6.5 | 4.4 | 2.1 |
Pt#13/Day 0 | 1.2 | 0.7 | 0.5 |
Pt#13/Day 28 | 1.9 | 1.7 | 0.2 |
IFP measurements were obtained in 6 patients at baseline. The IFP in the 6 lesions varied between 2.5 and 21.0 mm Hg and showed a direct correlation with tumor size (Kendall's tau = 0.87, p = 0.017,
Studying the physiologic and pharmacodynamic impact of mechanistically-targeted drugs is a key aspect of rational therapeutic development and optimization. This study was designed to assess several mechanism-based correlative studies along with standard clinical outcomes. In this cohort of patients with multi-drug refractory STS of varied histologies, sorafenib administration was associated with modest radiographic effects, with a median TTP of 45 days. In a recent study of 145 patients with recurrent or metastatic sarcoma of various histologies treated with sorafenib, RECIST complete or partial responses were observed in five patients with angiosarcoma and one with leiomyosarcoma
While radiographic response criteria have been recently refined
Interestingly, we also found significant associations between cytokines that may mediate resistance to anti-VEGF therapy and response: a lower baseline plasma PlGF concentration correlated with a better radiographic response after treatment at day 28, whereas an increase in SDF1α by day 28, correlated with a worse radiographic response after treatment at day 28. The risk of false positive correlations is high given the multiple comparisons and the small sample size. However, it is notable that the same correlations have been seen with other anti-VEGF agents in patients with brain, rectal, and liver cancer (for plasma SDF1α), and in patients with brain, rectal and ovarian cancer (for plasma PlGF)
The sorafenib-induced stabilization of tumor growth in human carcinoma xenografts in mice is associated with a decrease in vascular density
Sorafenib shows modest clinical activity in patients with advanced refractory STS. Biomarker changes were consistent with inhibition of angiogenesis by sorafenib, including a mechanism-based decrease in the baseline high levels of intra-tumoral IFP. Preliminary circulating biomarker data from this study suggest a potential biomarker value for sVEGFR-2, PlGF, and SDF1α. Tumor IFP and vessel density appear to decrease when response is maintained. The findings of this hypothesis-generating study should be validated in large prospective trials of sorafenib, alone or in combination with other agents, in sarcoma and other cancers.
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The authors thank the Cancer Therapeutics Evaluation Program for their support, C. Koppel, K. Kinzel, and S. Roberge for expert technical support for biomarker analyses, Q. Wang for statistical support regarding clinical data, and nurses and physicians at our institutions for their assistance.
Data were presented at the American Society of Clinical Oncology Annual Meeting, June 4–8, 2010, Chicago, IL.