Mechanism-related circulating proteins as biomarkers for clinical outcome in patients with unresectable hepatocellular carcinoma receiving sunitinib

Hepatocellular carcinomas (HCCs) overexpress several angiogenic proteins, including vascular endothelial growth factor-A (VEGF-A) [1‐3], VEGF-D [4], and platelet-derived endothelial growth factor (PDGF) [2], as well as expressing receptors to these ligands (comprising VEGF receptors [VEGFRs]-1, -2 [5], and -3 [4]). Tumor expression of VEGF-A increases progressively during development of HCC from low-grade dysplastic nodules, and VEGF-A expression correlates with microvessel density during HCC development [6]. High serum levels of VEGF-A [7] and basic fibroblast growth factor [8] have been associated with poor clinical outcome in HCC [8], and VEGF-A polymorphisms have been associated with prognosis [9]. The hepatitis B virus X protein (HBx) is expressed in HBV-infected cells and enhances VEGF-A expression by stabilizing the transcription factor HIF-1α through inhibition of HIF-1α binding to VHL [10]. These and other findings strongly implicate angiogenesis in the pathophysiology of HCC (reviewed in [5]).

The development of sorafenib has set a precedent for the use of targeted antiangiogenic therapy in advanced HCC [11, 12]. Sunitinib, an oral multitargeted tyrosine kinase inhibitor with antiangiogenic activity in vivo, has been investigated in advanced HCC within several phase II trials [13‐15], and a phase III trial comparing sunitinib with sorafenib has recently been halted due to futility and an increased incidence of serious adverse events in the sunitinib versus the sorafenib arm. Sunitinib inhibits VEGFRs-1, -2, and -3, PDGFRs -α and -β, stem cell factor receptor (KIT), glial cell line-derived neurotrophic factor receptor (REarranged during Transfection; RET), colony-stimulating factor 1 receptor (CSF-1R), and FMS-like tyrosine kinase 3 (FLT3) [16‐21]. The antiangiogenic activity of sunitinib likely results from inhibition of VEGFRs on endothelial cells and PDGFR-β on stromal cells.

Biomarkers of angiogenesis and tumor proliferation are often used to demonstrate the pharmacodynamic effects of therapeutic agents, but also have the potential to play a role in predicting which patients are likely to benefit from a particular treatment. Soluble forms of proteins involved in tumor-cell proliferation (e.g. soluble stem-cell factor receptor [sKIT]) or tumor angiogenesis (such as VEGF-A, VEGF-C, soluble VEGFR-2 [sVEGFR-2], and soluble VEGFR-3 [sVEGFR-3]) can be rapidly and readily measured in serum or plasma samples by highly specific enzyme-linked immunosorbant assays (ELISAs). If sufficiently sensitive and specific, associations between biomarker levels and clinical outcome could offer practical benefits, both for refining clinical research and for clinical decision-making.

A phase II study of sunitinib 50 mg/day on Schedule 4/2 (4 weeks on treatment, followed by 2 weeks off treatment) in 37 patients with advanced HCC was recently reported by Faivre et al. [14]. Although this trial did not meet its primary endpoint based on Response Evaluation Criteria in Solid Tumors (RECIST), secondary endpoints were indicative of clinical activity in this population. Median time to tumor progression (TTP) and overall survival (OS) were 5.3 and 8.0 months, respectively. Disease control rate (partial response or stable disease > 3 months) was 37.8%. In the preliminary analyses previously reported by Faivre et al., patients with baseline VEGF-C levels above the median achieved significantly longer TTP and OS, as well as improved disease control, compared with patients with low VEGF-C levels. This trial also investigated potential correlations between clinical outcome and other soluble proteins that are directly related to the mechanism of action of sunitinib and are associated with angiogenesis or tumor proliferation (VEGF-A, sVEGFR-2, sVEGFR-3, and sKIT). Here we report a detailed exploratory analysis of the pharmacodynamics and predictive value of these sunitinib target-related plasma proteins.

In the present study we have investigated the plasma pharmacodynamics of a number of sunitinib target-related soluble proteins and investigated potential relationships between these proteins and measures of clinical outcome, as part of a phase II study of 37 patients with advanced, unresectable HCC [14]. Potentially the most clinically useful finding from this exploratory analysis is the strong correlation between high plasma concentrations of VEGF-C at baseline and improved clinical outcome, as determined by objective response (RECIST), TTP, and OS, with baseline VEGF-C remaining an independent predictor of TTP by multivariate analysis. VEGF-C and VEGF-D are members of the VEGF family of ligands that bind to and activate VEGFR-3 [33]. Mature forms of these ligands also bind to VEGFR-2 [33], and in vivo angiogenic activity has been demonstrated for VEGF-C in the mouse corneal pocket assay [34]. The correlative findings for VEGF-C presented here raise the possibility that the VEGF-C/VEGFR-3 pathway may play a role in HCC disease progression, and that inhibition of this receptor may result in improved clinical outcome in a subset of patients with this disease, following treatment with sunitinib.

In support of the proposed role for the VEGFR-3 pathway in HCC progression, Thelen et al. [4] observed high levels of tumor cell VEGF-D expression in biopsies from HCC patients but not in specimens from cirrhotic or normal livers. VEGFR-3 was expressed in both tumor endothelium and lymphatics, suggesting that both hemangiogenesis and lymphangiogenesis may be regulated by this receptor in HCC [4]. Similar findings have been reported for VEGFR-3 expression in a number of other tumor types [35‐38], and the biology of this receptor no longer appears to be restricted to lymph vessel production. When the human hepatoma cell line SKHep1, which does not express VEGF-D, was stably transfected with VEGF-D cDNA and then implanted subcutaneously in mice, larger and more metastatic tumors were formed compared with those from mock-transfected cells [4]. Interestingly, co-expression of the soluble VEGFR-3 domain in these cells blocked VEGF-D-induced tumor growth and metastatic spread.

A relationship was seen in this study between circulating VEGF-C levels prior to sunitinib dosing and the pharmacodynamics of VEGF-C and VEGF-A, but not of the soluble receptors studied. Plasma VEGF-C levels declined markedly at all time points in patients with high VEGF-C concentrations at baseline, with little change in patients with low baseline VEGF-C. This finding is consistent with the positive associations between clinical outcome and both elevated VEGF-C levels at baseline and greater reductions in VEGF-C. In contrast, sunitinib-induced increases in VEGF-A were reduced in patients with high baseline VEGF-C at some time points, suggesting an attenuated hypoxic response in this patient subset.

This is the first report in any tumor type of an association between elevated plasma levels of VEGF-C at baseline and improved clinical outcome following treatment with sunitinib. In contrast to the present finding for subjects with advanced HCC who had received no prior systemic therapy, results from a phase II study of sunitinib in patients with metastatic renal cell carcinoma (RCC) indicated that relatively low (< median) levels of VEGF-C at baseline were associated with achievement of response (RECIST) and with longer progression-free survival [39]. However, patients enrolled in this RCC study had previously progressed on bevacizumab therapy, raising the possibility that the observed biomarker correlations reflected the development of resistance to VEGF-A pathway inhibition, and no such association was seen in a phase I/II study in which patients with metastatic RCC were treated with sunitinib in combination with gefitinib [40]. It should be noted that RCC and HCC are distinct diseases that respond differently to sunitinib and that available correlative data for circulating VEGF-C in both tumors are limited, indicating a need for further research on this protein as a possible predictive biomarker in these and other tumor types.

The present exploratory analysis also showed that sunitinib dosing significantly reduced plasma sKIT from baseline levels, with no rebound during the off-treatment period. Low sKIT ratios to baseline at cycle 1 day 14 were associated with prolonged TTP and reduced tumor density, as well as with a trend towards prolonged OS. These findings support the association between sKIT reduction and improved clinical outcome reported by Zhu et al. in a phase II study of sunitinib in HCC [13], and suggest that inhibition of KIT signaling may contribute to sunitinib antitumor activity. The lack of early separation in the sKIT TTP and OS Kaplan-Meier curves (Figures 5A and 5B, respectively) suggests that two subsets of patients with a low sKIT ratio might exist: one that has markedly prolonged TTP and OS, and another subset with no difference. However, the relatively small sample size and higher level of censoring in the low sKIT group should be taken into consideration.

In the study by Zhu et al. [13], patients with HCC were treated with sunitinib at a dose of 37.5 mg/day on Schedule 4/2. The pharmacodynamics of VEGF-A, sVEGFR-2, and sVEGFR-3 were similar to those seen in the present analysis, but levels of sKIT and VEGF-C did not change significantly from baseline over 4 cycles of sunitinib treatment, in contrast to the present findings. Nonetheless, delayed tumor progression was associated with an early (day 14) decrease in circulating sKIT, consistent with the findings presented here. The possible role of KIT (CD117) in HCC is unclear. A retrospective study of archival tumor specimens from patients with histologically confirmed HCC suggested that KIT is not significantly overexpressed in this tumor type [41]. However, KIT blockade by imatinib mesylate inhibited HCC development in mice with chronic liver injury, via antiproliferative effects on KIT-expressing liver progenitor cells [42].

A number of limitations apply to the biomarker investigation reported here. Statistical analyses were not strongly powered, with plasma samples from 37 patients at baseline and declining sample sizes over time due to treatment discontinuations. Analysis of plasma proteins in relation to objective response was further limited by the proportion of patients (27.0%) not evaluable by RECIST. As this was a single-arm sunitinib study, it was not possible to determine whether biomarker associations with clinical outcome were predictive or prognostic in nature (or perhaps both). Thus, high plasma VEGF-C at baseline may represent a predictive factor for patients with HCC treated with sunitinib, consistent with potent inhibition of VEGFR-2 and -3 by this tyrosine kinase inhibitor. Alternatively, plasma VEGF-C may represent a positive prognostic factor in HCC, independent of treatment modality, as has been shown for the absence of cirrhosis in some HCC studies (reviewed in [43]). However, there are data to support high tumor VEGF-C expression as a negative prognostic factor, independent of other variables, in non-small cell lung cancer [44], esophageal cancer [45], and gastric cancer [46], while high plasma levels of VEGF-C served as an independent negative prognostic factor in colorectal cancer [47]. These findings from correlative studies in other tumor types suggest that the positive association for plasma VEGF-C in HCC reported here may be predictive rather than prognostic in nature, but further research is necessary to address this issue. The present study was limited to a small group of circulating proteins closely linked to known molecular targets of sunitinib. However, other angiogenesis-related proteins, such as basic fibroblast growth factor, as well as markers of other processes with an important role in tumor biology, such as inflammation [13], may have value in identifying patients with HCC who have inherent or acquired resistance to sunitinib therapy.

The findings reported here for selected plasma biomarkers may have value in the design of future phase III clinical trials using sunitinib in patients with HCC. In particular, a patient selection strategy that includes baseline VEGF-C concentrations above a specified value may increase the likelihood of demonstrating clinical improvement, and conversely may prevent unnecessary drug exposure in patients unlikely to benefit. Data from a phase III trial comparing sunitinib with sorafenib (NCT00699374) will soon be presented showing no advantage for sunitinib in an unselected patient population. However, identification of a subset of patients with HCC who benefit from sunitinib treatment remains an important objective of biomarker research. Furthermore, results from the present study may have relevance to the prediction of efficacy in HCC trials of drugs with a similar mechanism of action to sunitinib.

In conclusion, high plasma levels of VEGF-C at baseline were strongly associated with improved clinical outcome in patients with HCC who received sunitinib, and plasma VEGF-C was an independent positive predictor of TTP by multivariate analysis. A more complete assessment of the potential clinical utility of these and other correlative findings obtained in this exploratory phase II study will require additional research.