Preliminary Study of 89Zr Labeled Bevacizumab to Detect Angiogenesis in a Pre-Clinical Model of Sarcoma

Introduction

Soft tissue sarcomas (STSs) make up <1% of adult malignancies with an estimated 12,000 new cases of STS diagnosed in the US each year, with approximately 5,000 deaths [1]. STSs are derived from mesenchymal cells with their most common location. Involving the extremities/chest wall regions of the body [1]. At the time of initial diagnosis, distant metastases are rarely present, but blood is the most common route for the disease to spread, most frequently to the lungs [2]. Current treatments for STSs include surgery, radiation, and chemotherapy with little progress being made with respect to targeted therapeutics. TNM stage histologic grade remain the most important clinicopathological indicators of prognosis with the additional factors of surgical margins, age, anatomic site, and histologic subtype [3].

Non-invasive functional imaging involving fluorodeoxyglucose (18F-FDG)-positron emission tomography (PET)/computed tomography (CT) imaging has been assessed for its utility for discriminating benign from malignant lesions, correlation with tumor grade and histological subtype and with prognosis with variable conclusions as to its utility [4, 5, 6]. FDG is a useful non-specific probe to assess metabolic activity in a wide range of cancers but is influenced by many biological factors. Imaging probes that are directed towards more specific attributes of STS may be more informative. The metastatic rate of sarcomas is dependent on the development of angiogenesis or new blood vessels formation [7, 8]. An important mediator of angiogenesis has been identified as vascular endothelial growth factor (VEGF)–A. High levels of this growth factor, in patients with STSs, correlates with higher tumor grade, increased recurrence and metastatic rate, and decreased overall survival [8].

Antibody based positron emission tomography (immuno-PET) imaging is a relatively new field which is gaining increasing attention [9]. However, the slow binding characteristics and receptor occupancy of intact antibodies requires the use of radionuclides with longer half-lives than used in traditional PET studies. 89Zirconium (89Zr) has a half- life of 78.4 hours (h) which matches the pharmacokinetics of antibodies and it has a relatively low average positron energy of 395 keV; both characteristics making it a suitable candidate for high resolution PET imaging of slow- accumulating biomolecules [10]. Bevacizumab (Bev) is an antibody that targets all splice variants of VEGF-A, some of which are partly associated with tumor blood vessels and the extracellular matrix of tumor cells. 89Zr--Bev PET/CT imaging has already been shown to be tolerable and effective for noninvasive in vivo imaging of VEGF-A in preclinical human ovarian tumor xenografts [11]. In addition, several published preliminary studies of adult human tumors in Europe including primary breast cancers, advanced, progressive neuroendocrine tumors and renal cell carcinomas [12, 13, 14] and have demonstrated safe and accurate detection of VEGF-A with 89Zr-Bev PET/CT.

To our knowledge there are no reported studies of using 89Zr-Bev PET/CT in sarcomas. In addition, 89Zr is not yet approved as a PET radiotracer in the USA. This preliminary communication was performed to establish the utility of 89Zr- Bev to image angiogenesis in a sarcoma model to support imaging correlates in a clinical trial entitled “ A phase Ib Trial of Image-Guided Preoperative Simultaneous Integrated Boost Radiation Therapy for Patients with Locally Advanced Non-Metastatic Soft Tissue Sarcomas of the Extremity/Body Wall”.

Results

89Zr-bev imaging

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Figure 1 shows the sequential PET images from HT- 1080 xenografts in both mouse strains and Figure 2 shows the KBq/cc and SUVMAX for tumor, leg muscle, leg bone and background. Tumor visualization was excellent with the SUVMAX reaching maximum values at 4 days post-injection. There was uptake in well perfused organs such as liver and spleen which declined at later time-points. Clearance of 89Zr-Bev from internal organs was more rapid in the athymic mouse strain compared to the BALB/c mice. This was evident in Table 1 where the CNR was maximal at 4 days in the athymic mice but reached a maximum at 8 days in BALB/c mice due to persistence of radioactivity in organs and muscle.

As has been observed previously, there was significant uptake of 89Zr-Bev in bone (Figure 2). This was analyzed by a VOI in the knee region of the opposite side to the flank implanted with tumor. The SUVMAX increased with time and plateaued at day 4 and remained at this level until day 11 (Figure 2B and 2D) despite the radioactivity counts declining with time (Figure 2A and 2C).

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Discussion

In this preliminary communication we have been able to demonstrate significant uptake of 89Zr-Bev in a xenograft model of human fibrosarcoma. Previous studies have established that HT-1080 cells form aggressive tumors when injected subcutaneously in the flanks of the immuno- compromised mice [17]. Histology has shown that the tumors contain several vascular channels harboring red blood cells that are indicative of high levels of angiogenesis whilst immunohistochemical analyses reveal various angiogenic markers like PECAM, VE-Cadherin, VEGF, VEGF165, NRP-1 and VEGFR-2 [17]. This emphasizes that HT-1080 cells acquire a vascular-like phenotype in vivo making it a suitable model to study a vascular imaging probe in the context of sarcoma.

As mentioned previously 89Zr has attributes that make it an ideal radionuclide for immuno-PET imaging in terms of stability, half-life and being a residualizing radionuclide which results in improved tumor retention and enhanced tumor-to-normal tissue ratios [18]. However, it also has some disadvantages which involve chelation instability. It has been shown that the 89Zr−DFO complex is partly unstable and can result in release of 89Zr from the chelator and the subsequent accumulation in bone [19]. In this study we have used a desferrioxamine (DFO) derivative, Df-Bz-NCS, which facilitated direct conjugation to bevacizumab. Interestingly, the original publication describing the chelation method did not report any accumulation of radioactivity in bone [16] but our study did show significant uptake and retention. This may be due to the fact that we are using an antibody that targets vasculature whereas the previous study conjugated antibodies against epidermal growth factor receptor (EGFR) and CD44v6 [16]. Many new chelators with improved 89Zr coordination properties are under development [19].

This preliminary study does have several limitations. First, very few mice were studied in this proof-of-principle study, more research is now required to study different sarcoma tumor models and different stages of tumor growth. Second, we were not able to study the correlation between 89Zr-Bev imaging and assessment of vascularity; this is planned for future studies.

In summary, the uptake of 89Zr-Bev in this present study was consistent with that described in previous studies [11, 12, 14] with maximum uptake and tumor to normal tissue ratios at 3 to 4 days after injection. Although the results were qualitatively similar in the two mouse strains, the athymic nude mice seemed to clear the tracer more rapidly from normal tissues than the BALB/c. Our preliminary results demonstrate that radiolabeled bevacizumab is a potential new tracer for noninvasive imaging of VEGF in sarcomas.