Abstract 2455: Molecular targeted imaging of vascular endothelial growth factor receptor (VEGFR)-2 and anti-NMP autoantibodies detect ovarian tumor at early stage

Abstract

Background: Although several serum markers of ovarian cancer (OVCA) have been suggested, none can detect OVCA at early stage specifically because of the lack of a corresponding marker in the ovary. Ovarian malignant cellular transformation is an early event followed by tumor-associated neoangiogenesis (TAN). Malignant cellular transformation results in shedding of nuclear matrix proteins (NMPs) in circulation and anti-NMP autoantibody production. VEGFR-2, an ovarian TAN marker, may also be an imaging marker for early OVCA detection. Thus VEGFR-2 in association with anti-NMP antibodies may constitute an early detection test for OVCA. Objectives: The goal of this study was to examine (i) whether VEGFR-2 targeted ultrasound imaging detects ovarian TAN vessels and (ii) whether the frequency of TAN vessels is associated with serum anti-NMP antibodies at early stage of OVCA in laying hens model of human OVCA. Materials and Methods: Mature White Leghorn laying hens with normal or low egg laying rates or no egg laying were scanned by transvaginal ultrasound before and after intravenous injection with VEGFR-2 targeted microbubbles. All images were archived and analyzed offline. Serum samples were collected, hens were euthanized, ovarian tissues were processed for paraffin or frozen sections and NMP extraction. Ovarian tumors were confirmed by gross morphology and routine histology. Sera were tested for anti-NMP antibodies and VEGFR-2 levels by immunoassay and 1- and 2D-Western blot (WB). The frequencies of VEGFR-2 expressing TAN vessels were determined by immunohistochemistry (IHC). Results: VEGFR-2 targeted microbubbles bounded with ovarian tumors and appeared as a ring of irregular shape on the ovarian surface. Compared with non-targeted, VEGFR-2 targeted imaging increased the visualization of TAN vessels remarkably. VEGFR-2 expressing TAN vessels confirmed targeted ultrasound imaging predictions. Serum levels of VEGFR-2 were higher in OVCA hens than in normal hens and associated with the frequencies of ovarian TAN vessels. None of the hens with normal egg laying rates was positive for anti-NMP antibodies. Immunoreactive tumor antigens (NMP) of 50-60kDa were detected by 2D-WB. The frequencies of VEGFR-2 expressing TAN vessels were higher in hens with serum anti-NMP antibodies than in normal hens. Conclusion: VEGFR-2 targeted ultrasound imaging enhanced the visualization of ovarian TAN vessels remarkably. The intensity of targeted imaging was associated with serum VEGFR-2 levels and anti-NMP antibodies. Thus, VEGFR-2 targeted imaging together in association with serum anti-NMP antibodies may improve OVCA detection at early stage. These results will form a foundation for a clinical study. Support: DOD-(OC0100192), Elmer and Sylvia Sramek Foundation, NCI-POCROC (P50 CA83636). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2455. doi:1538-7445.AM2012-2455

Early Detection of Ovarian Cancer by Tumor Epithelium-Targeted Molecular Ultrasound

“…Serum prevalence of anti-NMP antibodies were determined by immunoassay and confirmed by 2-dimenssional Western blotting (2D-WB). We are the first to show the prevalence of serum anti-NMP antibodies in OVCA hens [3] reported earlier.…”

“…Normal or tumor ovarian NMPs from healthy hens with low egg laying rates or OVCA hens were extracted as reported previously [3,4]. ELISA (96-well) plates were coated with extracted NMPs and the immunereactivity of normal or tumor serum samples against or tumor NMPs were determined as reported earlier [15].…”

“…During malignant nuclear transformation, the shape and sizes of the nucleus undergo profound changes together with the rearrangement of nuclear matrix proteins (NMP) leading to the shedding of NMPs into the circulation. Anti-NMP antibodies are produced in response to shed NMPs [3,4]. On the other hand, expression of IL-16 by the tumor epithelium and its serum levels increase in association with ovarian tumor development [5].…”

Early Detection of Ovarian Cancer by Molecular Targeted Ultrasound Imaging Together with Serum Markers of Tumor-Associated Nuclear Change and Angiogenesis

“…Immunoassay and Immunoblotting: Serum prevalence of anti-NMP antibodies and IL-16 levels were determined by ELISA and confirmed by Western blotting as reported earlier [11,12].…”

“…Immunohistochemistry: Paraffin and frozen sections of ovarian tumors were examined for the detection of VEGFR-2-and IL-16-expressing cells using anti-chicken VEGFR-2 and anti-IL-16 primary antibodies as reported earlier [11,12]. The frequencies of VEGFR-2-expressing microvessels or IL-16 expressing cells were counted and analyzed as reported previously [11,12] Figure 1.…”