Radiolabeled pertuzumab for imaging of human epidermal growth factor receptor 2 expression in ovarian cancer

Abstract

Purpose: Human epidermal growth factor receptor 2 (HER2) is over-expressed in over 30% of ovarian cancer cases, playing an essential role in tumorigenesis and metastasis. Non-invasive imaging of HER2 is of great interest for physicians as a mean to better detect and monitor the progression of ovarian cancer. In this study, HER2 was assessed as a biomarker for ovarian cancer imaging using ⁶⁴Cu-labeled pertuzumab for immunoPET imaging.

Methods: HER2 expression and binding were examined in three ovarian cancer cell lines (SKOV3, OVCAR3, Caov3) using in vitro techniques, including western blot and saturation binding assays. PET imaging and biodistribution studies in subcutaneous models of ovarian cancer were performed for non-invasive in vivo evaluation of HER2 expression. Additionally, orthotopic models were employed to further validate the imaging capability of ⁶⁴Cu-NOTA-pertuzumab.

Results: HER2 expression was highest in SKOV3 cells, while OVCAR3 and Caov3 displayed lower HER2 expression. ⁶⁴Cu-NOTA-pertuzumab showed high specificity for HER2 (K_a = 3.1 ± 0.6 nM) in SKOV3. In subcutaneous tumors, PET imaging revealed tumor uptake of 41.8 ± 3.8, 10.5 ± 3.9, and 12.1 ± 2.3%ID/g at 48 h post-injection for SKOV3, OVCAR3, and Caov3, respectively (n = 3). In orthotopic models, PET imaging with ⁶⁴Cu-NOTA-pertuzumab allowed for rapid and clear delineation of both primary and small peritoneal metastases in HER2-expressing ovarian cancer.

Conclusions: ⁶⁴Cu-NOTA-pertuzumab is an effective PET tracer for the non-invasive imaging of HER2 expression in vivo, rendering it a potential tracer for treatment monitoring and improved patient stratification.

Keywords: Human epidermal growth factor receptor 2 (HER2); Molecular imaging; Ovarian cancer; Pertuzumab; Positron emission tomography (PET).

Fig. 1

Evaluation of binding affinity of pertuzumab to HER2. (A) Western blot analysis of HER2 expression in three ovarian cancer cell lines showed high expression in SKOV3 cells and low expression in OVCAR3 and Caov3 cells. (B) HER2 expression was examined using flow cytometry, revealing high HER2 expression of SKOV3 cells and low levels of HER2 expression by OVCAR3 and Caov3 cells. (C) Saturation binding assays showed that ⁶⁴Cu-NOTA-pertuzumab displayed specific binding to SKOV3 cells, with a receptor density of 1.6 million HER2 receptors per SKOV3 cell.

Fig. 2

PET imaging and quantitative analysis of HER2 expression with ⁶⁴Cu-NOTA-pertuzumab in subcutaneous ovarian cancer models. (A) PET maximum intensity projection (MIP) images at 3, 24, and 48 h after injection of ⁶⁴Cu-NOTA-pertuzumab. Arrow: tumor, *: blood pool, †: liver (B) Time-activity curves of region-of-interest (tumor, blood, liver, and spleen) after intravenous injection of ⁶⁴Cu-NOTA-pertuzumab in three subcutaneous ovarian cancer models. ⁶⁴Cu-NOTA-pertuzumab kinetics in the blood pool, liver, and spleen showed no significant difference between the three groups. **: P < 0.01

Fig. 3

Immunofluorescence staining of tumor tissue sections. Pertuzumab and FITC-labeled anti-human secondary antibody were used for HER2 staining (green), CD31 was stained to expose locations of the vasculature (red), and DAPI was used for the visualization of cell nuclei locations (blue). Scale bar = 20 μm

Fig. 4

PET/CT imaging of HER2 expression in orthotopic models of ovarian cancer. (A) PET maximum intensity projection (MIP) images at 3, 24, and 48 h after injection of ⁶⁴Cu-NOTA-pertuzumab in SKOV3 and OVCAR3 orthotopic ovarian models, where tumors are indicated by arrows. (B) Representative transverse PET/CT images of SKOV3 and OVCAR3 tumor-bearing mice at 48 h after injection of ⁶⁴Cu-NOTA-pertuzumab (n = 4, for each group).

Fig. 5

Radiological-surgical correlation of SKOV3 orthotopic ovarian cancer model. (A) In PET/CT imaging, a primary tumor with strong uptake was found in the right ovary area (yellow arrow), and there were many focal uptakes in the peritoneal space (white arrow). (B) Surgical exploration was done in the same animal after terminal PET/CT imaging. The primary tumor mass was found in the right ovary (yellow arrow), and there was white nodular tissue (white arrow) at the location of focal uptake on the PET/CT imaging, suggesting peritoneal implants. (C) Ex vivo PET imaging of excised peritoneal implants was performed. The small peritoneal implants showed high PET uptake values.

Fig. 6. Ex vivo

biodistribution studies for the validation of PET/CT results. Biodistribution of ⁶⁴Cu-NOTA-pertuzumab in SKOV3 and OVCAR3 orthotopic ovarian cancer models at 48 h after injection (n = 4). SKOV3 tumors showed a significantly higher tracer accumulation comparing to OVCAR3 tumors, while normal ovaries in both groups displayed the low levels of ⁶⁴Cu-NOTA-pertuzumab uptake.