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Sorafenib is an inhibitor of multiple kinases (e.g., VEGF receptors, PDGFR, FLT3, RET, BRAF, KIT) and is approved by FDA for treatment of two adult cancers. The activity of sorafenib was evaluated against the PPTP's in vitro and in vivo panels.
Sorafenib was evaluated against the PPTP in vitro panel using 96 hour exposure at concentrations ranging from 1.0 nM to 10.0 μM. It was tested against the PPTP in vivo panels at a dose of 60 mg/kg administered by oral gavage daily for 5 days per week, repeated for 6 weeks.
In vitro sorafenib demonstrated cytotoxic activity, with a median IC50 value of 4.3 μM. Twenty of 23 cell lines had IC50 values between 1.0 and 10.0 μM. A single cell line (Kasumi-1) with an activating KIT mutation had an IC50 value < 1.0 μM (IC50 = 0.02 μM). In vivo sorafenib induced significant differences in EFS distribution compared to control in 27 of 36 (75%) of the evaluable solid tumor xenografts and in 1 of 8 (12.5%) of the evaluable ALL xenografts. Sorafenib induced tumor growth inhibition meeting criteria for intermediate activity (EFS T/C) in 15 of 34 (44%) evaluable solid tumor xenografts. No xenografts achieved an objective response.
The primary in vitro activity of sorafenib was noted at concentrations above 1 μM, with the exception of a more sensitive cell line with an activating KIT mutation. The primary in vivo effect for sorafenib was tumor growth inhibition, which was observed across multiple histotypes.
Sorafenib is a small molecule inhibitor of multiple kinases that control tumor cell proliferation and angiogenesis. Sorafenib was originally developed to inhibit tumor cell growth and angiogenesis by specifically targeting Raf-1 (CRAF) serine/threonine kinase, a member of the RAF-MEK-ERK signaling pathway , and it is a potent in vitro inhibitor of this kinase . Sorafenib has also been found to inhibit at low nanomolar concentrations vascular endothelial growth factor receptors (VEGFR), platelet-derived growth factor receptors (PDGFR), RET, FLT3, and KIT . Preclinical studies of human melanoma, renal, colon, pancreatic, hepatocellular, thyroid, and ovarian and non-small cell lung carcinomas (NSCLCs) document the ability of sorafenib to inhibit tumor growth against a variety of cancers and in selected cases to induce tumor regression . Furthermore, combination studies with other drugs (gefitinib, vinorelbine, gemcitabine, and irinotecan) indicate that sorafenib has a tolerability profile that is conducive to be combined with other agents .
Sorafenib was approved by FDA for the treatment of renal cell carcinoma (RCC) in 2005 and for hepatocellular cancer (HCC) in 2007. The approval for advanced RCC was based on an improvement in progression-free survival (PFS) from 2.8 months for patients assigned to placebo to 5.5 months for patients receiving sorafenib . Partial responses were observed in 10% of patients, suggesting that the primary benefit for sorafenib resulted from tumor growth inhibition. For advanced HCC, sorafenib significantly increased median overall survival (10.7 months for sorafenib versus 7.9 months for placebo) and median time to radiologic progression (5.5 months for sorafenib versus 2.8 months for placebo). Tumor regression was uncommon, indicating that sorafenib is effective against HCC primarily by slowing the rate of disease progression . Of direct relevance in the pediatric setting, sorafenib is also being evaluated for acute myeloid leukemia (AML) in adults in combination with standard anti-leukemia agents, given its potent activity against FLT3 and KIT . On the strength of the clinical results for sorafenib and its interesting pattern of kinase inhibition, the PPTP evaluated this agent to gain insight into its utility against pediatric tumors.
In vitro testing was performed using DIMSCAN, a semiautomatic fluorescence-based digital image microscopy system that quantifies viable (using fluorescein diacetate [FDA]) cell numbers in tissue culture multiwell plates . Cells were incubated in the presence of sorafenib for 96 hours at concentrations from 1.0 nM to 10.0 μM and analyzed as previously described .
CB17SC-M scid−/− female mice (Taconic Farms, Germantown NY), were used to propagate subcutaneously implanted kidney/rhabdoid tumors, sarcomas (Ewing, osteosarcoma, rhabdomyosarcoma), neuroblastoma, and non-glioblastoma brain tumors, while BALB/c nu/nu mice were used for glioma models, as previously described . Human leukemia cells were propagated by intravenous inoculation in female non-obese diabetic (NOD)/scid−/− mice as described previously . Female mice were used irrespective of the patient gender from which the original tumor was derived. All mice were maintained under barrier conditions and experiments were conducted using protocols and conditions approved by the institutional animal care and use committee of the appropriate consortium member. Ten mice (solid tumors) or 8 mice (leukemia models) were used in each control or treatment group. Tumor volumes (cm3) [solid tumor xenografts] or percentages of human CD45-positive [hCD45] cells [ALL xenografts] were determined as previously described  and responses were determined using three activity measures as previously described . An in-depth description of the analysis methods is included in the Supplemental Response Definitions section.
The exact log-rank test, as implemented using Proc StatXact for SAS®, was used to compare event-free survival distributions between treatment and control groups. P-values were two-sided and were not adjusted for multiple comparisons given the exploratory nature of the studies. The Mann–Whitney test was used to test the difference between VEGFA expression level between groups of xenografts with greater versus lesser tumor growth inhibition (EFS T/C ≥ 2 versus < 2).
Sorafenib was provided to the PPTP by Bayer HealthCare Pharmaceuticals Inc., Wayne, NJ, through the Cancer Therapy Evaluation Program (NCI). Sorafenib (as the tosylate salt) was formulated in CremophorEL/ethanol/water (12.5/12.5/75), warmed and sonicated (20-30 min), and the suspension was administered at a dose of 60 mg/kg by oral gavage daily for 5 days per week, repeated for 6 consecutive weeks.
Sorafenib demonstrated an activity pattern consistent with a cytotoxic effect, with T/C% values approaching 0% for many cell lines at the highest concentration tested. Twenty of 23 cell lines of the PPTP showed IC50 values between 1.0 and 10.0 μM, Table I. The ratio of the median IC50 of the entire panel to that of each cell line is presented in Table I and Figure 1A. Higher ratios are indicative of greater sensitivity to sorafenib and are shown by bars to the right of the midpoint line. For most of the cell lines there was little variation in their in vitro response to sorafenib, with 20 of the 23 cell lines having IC50 values within 50% (above or below) the median for the entire panel (4.3 μM). The cell line with the lowest IC50 value, Kasumi-1, was derived from a patient with core binding factor AML with t(8;21) and is known to have a gain-of-function KIT mutation (Asn822Lys) . The IC50 value for Kasumi-1 (0.02 μM, Figure 1B) was more than 200-fold lower than the median IC50 for the PPTP in vitro panel, and is contrasted with BT-12 a rhabdoid cell line with an IC50 of 3.5 μM (Figure 1C).
Sorafenib was evaluated in 44 xenograft models. Nine of 840 mice died during the study (1.1%), with 4 of 419 in the control arm (1.0%) and 5 of 421 in the sorafenib treatment arm (1.2%). No tumor lines were excluded from analysis due to toxicity greater than 25 percent. A complete summary of results is provided in Supplemental Table I, including total numbers of mice, number of mice that died (or were otherwise excluded), numbers of mice with events and average times to event, tumor growth delay, as well as numbers of responses and T/C values.
Sorafenib induced significant differences in event-free survival (EFS) distribution compared to control in 27 of 36 (75%) of the solid tumor xenografts and in 1 of 8 (12.5%) of the ALL xenografts. For those xenografts with a significant difference in EFS distribution between treated and control groups, the EFS T/C activity measure additionally requires an EFS T/C value of > 2.0 for intermediate activity and indicates a substantial agent effect in slowing tumor growth. High activity further requires a reduction in final tumor volume compared to the starting tumor volume. Sorafenib induced tumor growth inhibition meeting criteria for intermediate activity in 15 of 34 (44%) evaluable solid tumor xenografts, but no solid tumor xenografts met criteria for high EFS T/C activity. Intermediate activity for the EFS T/C metric occurred most frequently in the osteosarcoma (4 of 5), neuroblastoma (4 of 5), and glioblastoma (3 of 4) panels. Intermediate EFS T/C activity was not observed for any rhabdomyosarcoma xenografts (0 of 6) and was observed in only a single ALL xenograft (1 of 8). Examples of tumors demonstrating criteria for intermediate activity are shown in Figure 2.
No xenografts achieved an objective response, with the best response in the solid tumor panel being PD2 (progressive disease with growth delay), which was observed in 21 of 36 (56%) xenografts. A single ALL xenograft showed a PD2 response. The in vivo testing results for the objective response measure of activity are presented in Figure 3 in a ‘heat-map’ format as well as a ‘COMPARE’-like format, based on the scoring criteria described the Supplemental Response Definitions section. The latter analysis demonstrates relative tumor sensitivities around the midpoint score of 5 (stable disease).
The heat map representation of VEGFA expression as measured by Affymetrix U133 Plus 2.0 arrays for the PPTP xenografts and cell lines shows that highest VEGFA expression occurs for selected xenografts in the osteosarcoma panel and for xenografts in the brain tumor panels (ependymoma and glioblastoma) (Figure 4). By comparison, the ALL xenografts and cell lines generally have very low VEGFA expression. There was a trend for xenografts with greater growth inhibition (EFS T/C ≥ 2 versus < 2) to have higher baseline VEGFA expression levels. This trend was noted when using combined VEGFA probe set expression data as well as when using the expression data for the most 3′ probe set (201512_s_at) (p=0.08 and 0.06, respectively).
The in vitro activity pattern observed for the PPTP cell lines is similar to that described for sorafenib in previous reports. Cell lines that have activating mutations or translocations involving selected receptor tyrosine kinases show IC50 values in the low nanomolar range, including cell lines with FLT3 [3,15-17], KIT , RET [19,20], and PDGFR [3,21] alterations. These genes are not known to show activating mutations at clinically relevant frequencies in common childhood cancers, with the exception of FLT3 and KIT for pediatric AML [22,23]. Sorafenib also inhibits VEGFR2 signaling, with IC50 values below 100 nM . The PPTP in vitro panel contained a single cell line, Kasumi-1, with IC50 values in the low nM range, and this cell line has an activating KIT mutation .
At low micromolar concentrations, sorafenib has broad activity against the PPTP cell lines. Similar results have been described for a range of adult and pediatric cancer cell lines, including those for hepatocellular carcinoma , papillary thyroid cancer with BRAF mutations , breast cancer , lung carcinoma , medulloblastoma , and various types of leukemias . This broader activity at micromolar concentrations has been associated with inhibition of a range of cellular processes, including reduced phosphorylation levels of eIF4E [28,29], reduced levels of Mcl-1 [26,28,30], STAT3 activation , and reduced levels of MAP kinase signaling as measured by reduced phospho-ERK levels . The effect of sorafenib at these higher concentrations is clearly cytotoxic for many of the PPTP cell lines, consistent with data from adult cancer cell lines, as the T/C% values approach 0% for many cell lines. There is no histotype specificity in the activity of sorafenib at micromolar concentrations, as almost all cell lines, regardless of diagnosis, are sensitive. This micromolar level activity is unlikely to have clinical relevance, as sorafenib shows high protein binding (99.5%), resulting in more than 100-fold higher concentrations being required to achieve IC50 concentrations in plasma compared to the concentration required using standard low-serum in vitro testing conditions . The steady state sorafenib blood levels observed in patients are only in the low micromolar range and are therefore are therefore unlikely to be sufficient to inhibit tumors with sensitivity to sorafenib that is comparable to those cell lines showing micromolar level IC50 values under standard in vitro testing conditions. Furthermore, in vitro micromolar concentrations exceed by 100-fold or greater those at which sorafenib inhibits cell lines with activating alterations in RET, KIT, PDGFR, and FLT3, and this activity at high concentrations likely reflects non-specific effects on multiple other kinases .
The in vivo antitumor activity observed for sorafenib against the PPTP childhood cancer models consisted exclusively of tumor growth inhibition. The growth inhibitory effect was particularly notable for two of the four tested glioblastoma xenografts and for four of the five osteosarcoma xenografts. In selected adult cancer preclinical models, sorafenib induces complete growth inhibition and in some cases regressions. For example, in the renal cell carcinoma xenograft 788-O, near complete growth inhibition was observed at a sorafenib dose of 60 mg/kg administered daily . Tumor growth inhibition in renal cancer models has been associated with a profound reduction in microvessel density at higher sorafenib doses. Sorafenib induced partial tumor regressions against the hepatocellular carcinoma xenograft PLC/PRF/5 at doses of 30 mg/kg and 100 mg/kg administered daily . MAPK pathway activation is a common feature of hepatocellular carcinoma. Mutant FLT3 AML preclinical models are responsive to sorafenib in vivo at doses as low as 3 mg/kg, indicating that these doses achieve sufficient levels to have a strong anti-leukemia effect against cells that have low nanomolar IC50 values . Papillary thyroid carcinoma xenografts harboring a RET translocation are more responsive to sorafenib than are xenografts with BRAF mutations , and a dose of 60 mg/kg administered daily is able to induce regressions in xenografts with an activating RET mutation . In a number of other adult cancer xenograft models, the in vivo activity is more similar to the activity pattern observed for the PPTP xenografts (i.e., tumor growth inhibition), and convincing evidence for sorafenib in vivo anti-angiogenic activity has been presented for these models [24,25,33]. The pattern of tumor growth inhibitory activity observed by the PPTP for sorafenib is consistent with an antiangiogenic mechanism of action for this agent against selected PPTP solid tumor xenografts, and it is similar to the pattern of activity previously described by the PPTP for sunitinib and for cediranib, both of which also inhibit VEGFR2 signaling [34,35]. The relevance of VEGFR2 inhibition to sorafenib's activity against PPTP xenografts is supported by the trend for an association between greater tumor growth inhibition and higher baseline VEGFA expression (Figure 4).
Activating mutations of RAS genes and BRAF are not commonly found in most pediatric cancers [27,28,30,36-38]. Pilocytic astrocytomas have MAPK pathway activation through BRAF activating point mutations and through a tandem duplication that results in an in-frame fusion between the 5′ end of the KIAA1549 gene and the 3′ end of the BRAF gene producing an oncogenic fusion protein [39-41]. However, none of the cell lines in the PPTP in vitro panel or the in vivo panel against which sorafenib was tested are known to have activating BRAF mutations. Sorafenib appears to show greater in vitro and in vivo activity against preclinical models with activated receptor tyrosine kinases (e.g., RET) compared to models with activating BRAF mutations , and it shows no preferential activity against BRAF mutant cell lines . Clinically, sorafenib has shown limited tumor regressing activity against melanoma (a tumor with a high proportion of BRAF mutations) . Thus, it is not clear that sorafenib should be prioritized for evaluation against BRAF-mutated pediatric tumors.
Plans for the clinical development of sorafenib in the pediatric population are under discussion, and the pediatric phase 1 trial of sorafenib has been completed . The results presented here suggest that sorafenib will primarily have tumor growth inhibitory activity rather than tumor regression inducing activity against tumors lacking those mutation-activated receptor tyrosine kinases against which sorafenib shows potent inhibitory activity. Studies of sorafenib in the pediatric AML setting for children with FLT3 internal tandem duplication (ITD) mutations are of particular interest given the potent activity of sorafenib against AML cell lines with FLT3-IT, the in vivo regression-inducing activity observed for sorafenib at low doses against FLT3-ITD xenografts, and the clinical activity of sorafenib in AML patients with FLT3-ITD [15-17,45,46]. Sorafenib, like several other VEGFR inhibitors may have relatively little single agent activity against most solid tumors, but may have value when combined with cytotoxic agents or other signaling inhibitors. Thus, this agent may have value in treatment of pediatric cancers, and future preclinical studies should include evaluation of sorafenib with standard cytotoxic agents and with other cell signaling inhibitors.
This work was supported by NO1-CM-42216, CA21765, and CA108786 from the National Cancer Institute and used sorafenib supplied by Bayer HealthCare Pharmaceuticals, Inc. In addition to the authors this paper represents work contributed by the following: Sherry Ansher, Catherine A. Billups, Joshua Courtright, Edward Favours, Henry S. Friedman, Danuta Gasinski , Debbie Payne-Turner, Chandra Tucker, , Jianrong Wu, Joe Zeidner, Ellen Zhang, and Jian Zhang. Children's Cancer Institute Australia for Medical Research is affiliated with the University of New South Wales and Sydney Children's Hospital.
CONFLICT OF INTEREST STATEMENT: The authors consider that there are no actual or perceived conflicts of interest.