Sushil Tripathi

VPAC1 Targeted 64Cu-TP3805 Positron Emission Tomography Imaging of Prostate Cancer: Preliminary Evaluation in Man

OBJECTIVE To evaluate (64)Cu-TP3805 as a novel biomolecule, to positron emission tomography (PET) image prostate cancer (PC), at the onset of which VPAC1, the superfamily of G protein-coupled receptors, is expressed in high density on PC cells, but not on normal cells. MATERIALS AND METHODS Twenty-five patients undergoing radical prostatectomy were PET/X-ray computerized tomography imaged preoperatively with (64)Cu-TP3805. Standardized maximum uptake (SUVmax) values were determined and malignant lesions (standardized uptake value > 1.0) counted, and compared with histologic findings. Whole-mount pathology slides from 6 VPAC1 PET imaged patients, 3 benign prostatic hyperplasia patients, 1 malignant and 1 benign lymph node underwent digital autoradiography (DAR) after (64)Cu-TP3805 incubation and were compared to hematoxylin- and eosin-stained slides. RESULTS In 25 patients who underwent PET imaging, 212 prostate gland lesions had SUVmax > 1.0 vs 127 lesions identified by histology of biopsy tissues. The status of the additional 85 PET identified prostate lesions remains to be determined. In 68 histologic slides from 6 PET imaged patients, DAR identified 105 of 107 PC foci, 19 of 19 high-grade prostatic intraepithelial neoplasias, and ejaculatory ducts and verumontanum involved with cancer. Additionally, DAR found 9 PC lesions not previously identified histologically. The positive and negative lymph nodes were correctly identified, and in 3 of 3 benign prostatic hyperplasia patients and 5 of 5 cysts, DAR was negative. CONCLUSION This feasibility study demonstrated that (64)Cu-TP3805 delineates PC in vivo and ex vivo, provided normal images for benign masses, and is worthy of further studies.

Evaluation of a PACAP Peptide Analogue Labeled with (68)Ga Using Two Different Chelating Agents.

OBJECTIVE The authors have conjugated chelating agents (DOTA and NODAGA) with a peptide (pituitary adenylate cyclase-activating peptide [PACAP] analogue) that has a high affinity for VPAC1 receptors expressed on cancer cells. To determine a suitable chelating agent for labeling with (68)Ga, they have compared the labeling kinetics and stability of these peptide conjugates. METHODS For labeling, (68)GaCl3 was eluted in 0.1 M HCl from a [(68)Ge-(68)Ga] generator. The influences of peptide concentration, pH, and temperature on the radiolabeling efficiency were studied. The stability was evaluated in saline, human serum, DTPA, transferrin, and metallic ions (FeCl3, CaCl2, and ZnCl2). Cell binding assay was performed using human breast cancer cells (T47D). Tissue biodistribution was studied in normal athymic nude mice. RESULTS Optimal radiolabeling (>95.0%) of the DOTA-peptide conjugates required a higher (50°C-90°C) temperature and 10 minutes of incubation at pH 2-5. The NODAGA-peptide conjugate needed incubation only at 25°C for 10 minutes. Both radiocomplexes were stable in saline, serum, as well as against transchelation and transmetallation. Cell binding at 37°C for 15 minutes of incubation with (68)Ga-NODAGA-peptide was 34.0% compared to 24.5% for (68)Ga-DOTA-peptide. Tissue biodistribution at 1 hour postinjection of both (68)Ga-labeled peptide conjugates showed clearance through the kidneys. CONCLUSIONS NODAGA-peptide showed more convenient radiolabeling features than that of DOTA-peptide.

Development of a voided urine assay for detecting prostate cancer non-invasively: a pilot study

To validate a hypothesis that prostate cancer can be detected non‐invasively by a simple and reliable assay by targeting genomic VPAC receptors expressed on malignant prostate cancer cells shed in voided urine.

Murine MPDZ-Linked Hydrocephalus is Caused by Hyperpermeability of the Choroid Plexus

Though congenital hydrocephalus is heritable, it has been linked only to eight genes, one of which is MPDZ. Humans and mice that carry a truncated version of MPDZ incur severe hydrocephalus resulting in acute morbidity and lethality. We show by magnetic resonance imaging that contrast-medium penetrates into the brain ventricles of mice carrying a Mpdz loss-of-function mutation, whereas none is detected in the ventricles of normal mice, implying that the permeability of the choroid plexus epithelial cell monolayer is abnormally high. Comparative proteomic analysis of the cerebrospinal fluid of normal and hydrocephalic mice revealed up to a 53-fold increase in protein concentration, suggesting that transcytosis through the choroid plexus epithelial cells of Mpdz KO mice is substantially higher than in normal mice. These conclusions are supported by ultrastructural evidence, and by immunohistochemistry and cytology data. Our results provide a straight-forward and concise explanation for the pathophysiology of Mpdz-linked hydrocephalus.

Prostate cancer sheds the αvβ3 integrin in vivo through exosomes

The αvβ3 integrin has been shown to promote aggressive phenotypes in many types of cancers, including prostate cancer. We show that GFP-labeled αvβ3 derived from cancer cells circulates in the blood and is detected in distant lesions in NOD scid gamma (NSG) mice. We, therefore, hypothesized that αvβ3 travels through exosomes and tested its levels in pools of vesicles, which we designate extracellular vesicles highly enriched in exosomes (ExVs), and in exosomes isolated from the plasma of prostate cancer patients. Here, we show that the αvβ3 integrin is found in patient blood exosomes purified by sucrose or iodixanol density gradients. In addition, we provide evidence that the αvβ3 integrin is transferred through ExVs isolated from prostate cancer patient plasma to β3-negative recipient cells. We also demonstrate the intracellular localization of β3-GFP transferred via cancer cell-derived ExVs. We show that the ExVs present in plasma from prostate cancer patients contain higher levels of αvβ3 and CD9 as compared to plasma ExVs from age-matched subjects who are not affected by cancer. Furthermore, using PSMA antibody-bead mediated immunocapture, we show that the αvβ3 integrin is expressed in a subset of exosomes characterized by PSMA, CD9, CD63, and an epithelial-specific marker, Trop-2. Finally, we present evidence that the levels of αvβ3, CD63, and CD9 remain unaltered in ExVs isolated from the blood of prostate cancer patients treated with enzalutamide. Our results suggest that detecting exosomal αvβ3 integrin in prostate cancer patients could be a clinically useful and non-invasive biomarker to follow prostate cancer progression. Moreover, the ability of αvβ3 integrin to be transferred from ExVs to recipient cells provides a strong rationale for further investigating the role of αvβ3 integrin in the pathogenesis of prostate cancer and as a potential therapeutic target.

VPAC1 Targeted 64Cu-TP3805 kit preparation and its evaluation

INTRODUCTION Previously, our laboratory has shown that 64Cu-TP3805 can specifically target VPAC1 receptors and be used for positron emission tomography (PET) imaging of breast (BC) and prostate cancer (PC) in humans. Present work is aimed at the formulation of a freeze-dried diaminedithiol-peptide (N2S2-TP3805) kit and its evaluation for the preparation of 64Cu labeled TP3805. Parameters such as pH, temperature and incubation time were examined that influenced the radiolabeling efficiency and stability of the product. METHODS Kits were prepared under different conditions and radiolabeling efficiency of TP3805 kit was evaluated for a range of pH3.5-8.5, after addition of 64Cu in 30μl, 0.1M HCl. Incubation temperature (37-90°C) and time (30-120min.) were also investigated. Kits were stored at -10°C and their long term stability was determined as a function of their radiolabeling efficiency. Further, stability of 64Cu-TP3805 complex was evaluated in presence of fetal bovine serum and bovine serum albumin by using SDS polyacrylamide gel electrophoresis. Kits were then used for PET imaging of BC and PC following eIND (101550) and institutional approvals. Specificity of 64Cu-TP3805 for VPAC1 was examined with digital autoradiography (DAR) of prostate tissues obtained after prostatectomy, benign prostatic hyperplasia (BPH) tissue, and benign and malignant lymph nodes. Results were compared with corresponding tissue histology. RESULTS Radiolabeling efficiency was ≥95% at final pH ~7.2 when incubated at 50°C for 90min. Kits were stable up to 18months when stored at -10°C, and 64Cu-TP3805 complex exhibited excellent stability for up to 4h at room temperature. 64Cu-TP3805 complex did not show any transchelation even after 2h incubation at 37°C in 10% FBS as well as in BSA as determined by SDS PAGE analysis. DAR identified ≥95% of malignant lesions 11 new PC lesions, 20 high grade prostatic intraepithelial neoplasia, 2/2 ejaculatory ducts and 5/5 urethra verumontanum not previously identified The malignant lymph nodes were correctly identified by DAR and for 3/3 BPH patients, and 5/5 cysts, DAR was negative. In human BC (n=19) and PC (n=26) were imaged with 100% sensitivity. CONCLUSION Availability of ready to use N2S2-peptide kits for 64Cu labeling is convenient and eliminates possible day to day variation during its routine preparation for clinical use.

PD4-09 VASOACTIVE INTESTINAL PEPTIDE AND PITUITARY ADENYLATE CYCLASE ACTIVATING PEPTIDE RECEPTOR 1 (VPAC1) TARGETED IMAGING OF PROSTATE CANCER: A PILOT STUDY

INTRODUCTION AND OBJECTIVES: Prostate cancer (CaP) overexpresses VPAC1, representing a highly suitable target for imaging and treatment. VPAC1 is over-expressed at the onset of the malignancy, which may be prior to elevation of PSA, and before cell morphology is altered. We have successfully used VPAC1 receptor-specific peptide constructs to image breast cancer in experimental animal models, and in humans. We hypothesized that VPAC1 expressed in high density on PC can be targeted for detection of intraprostatic tumor foci on whole mount radical prostatectomy specimens, using TP3805, a VPAC1 specific biomolecule labeled with Cu-64 a PET imaging radionuclide. METHODS: As part of a PET imaging protocol targeting VPAC1, men (N1⁄425) with prostate cancer undergoing radical prostatectomy were imaged preoperatively, and compared to whole mount radical prostatectomy pathologic analysis. De-paraffinized slides from whole mount pathology from two patients who participated the protocol, as well as one malignant lymph node and one benign lymph node specimens and 3 BPH negative controls underwent digital autoradiography with Cu-64, and then were H&E stained. Autoradiography images were compared with prostate H&E staining in which tumor foci were delineated. RESULTS: Prostate cancer foci (n1⁄430/31) were identified by autoradiography imaging (Figure 1). Autoradiography missed one malignant lesion due to technical artifact. Additionally, 6 small cancerous lesions were identified by autoradiography that were not previously noted by histologic examinations. A total of 7 additional lesions seen by autoradiography corresponded with HGPIN. The positive lymph node and the benign lymph node were correctly identified by auto radiography (Fig.2). For the 3 BPH patients, no cancer foci were noted by autoradiography. CONCLUSIONS: VPAC1 peptide analog constructs accurately identified foci of prostate cancer on whole mount radical prostatectomy specimens. Several additional lesions were also identified. Detection of HGPIN is consistent with the early expression of VPAC1 prior to the modulations in cell morphology. The PPV (97%) and NPV(100%) were excellent, validating VPAC1 as a potential theragnostic target for prostate cancer imaging and treatment.

Abstract 1485: Pilot study of VPAC1 targeted PET imaging of prostate cancer

Introduction: Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase Activating Peptide Receptor 1 (VPAC1) is over-expressed in prostate cancer (PC), representing a highly suitable target for imaging and treatment. VPAC1 expression occurs at the onset of the malignancy, before alterations of cell morphology, which may be prior to elevation of serum PSA. We have successfully used VPAC1 receptor-specific peptide constructs to image breast cancer in experimental animal models, and in humans. We hypothesized that VPAC1 expressed in high density on PC can be targeted for detection of intraprostatic tumor foci, as correlated with whole mount radical prostatectomy specimens, using TP3805, a VPAC1 specific biomolecule labeled with Cu-64 a PET imaging radionuclide. Methods: Twenty five men with prostate cancer undergoing radical prostatectomy were imaged preoperatively as part of a PET imaging protocol targeting VPAC1. The PET images were compared to whole mount radical prostatectomy pathologic analysis. De-paraffinized whole mount pathology slides from two patients who participated in VPAC1 PET imaging protocol, as well as slides from 3 BPH patients, one malignant lymph node and one benign lymph node were incubated with Cu-64-TP3805, washed thoroughly with PBS, dried and subjected to 15 second digital autoradiography. Slides were then HE 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1485. doi:10.1158/1538-7445.AM2015-1485

Abstract B55: Hypoxanthine wobble base multimutant KRAS2 mRNA PET imaging agent in G12D mice

Activating mutations in KRAS2 oncogene lead to constitutive K-Ras-GTP signals regardless of EGFR signaling, enabling resistance to EGFR-targeted therapies. Some cancers are activated by mutation of the KRAS2 12th codon from the wild type GGU that encodes glycine (G) to a mutant GAU that encodes aspartate (D) or a mutant GUU that encodes valine (V). For this reason, we hypothesize that determining multiple KRAS2 mutations in vivo by external mRNA PET imaging will enable physicians to decide on alternatives to EGFR-directed therapy. We previously pioneered tumor mRNA PET imaging in pancreatic cancer and breast cancer xenografts [Paudyal, et al. (2013) Nuclear Medicine and Biology40(8):994-9] with peptide nucleic acid (PNA) 12-mers coupled to receptor-targeting peptides and imaging reporter moieties, showing single mismatch specificity. Thus, we designed and synthesized a 12-mer PNA hybridization agent with a hypoxanthine wobble base opposite the middle base of the KRAS2 mRNA 12th codon. At the C-terminus, we included a cyclized tetrapeptide based on insulin-like growth factor 1 (IGF1) to enable IGF1R-mediated cellular uptake. We included an N-terminal N2S2 chelator in order to bind 64Cu. Molecular dynamics and circular dichroism analysis of the wobble base PNA H-bonded to KRAS2 RNA showed preference for mutant A or U over wild type G [Sanders, et al. (2013) Journal of Physical Chemistry B 117(39):11584-95]. We then administered 200 μCi of the [64Cu]PNA wobble base agent, or the complementary G12D agent, or the wild type agent, by tail vein into 3-month-old G12D transgenic mice that developed spontaneous lung tumors. 4 hr and 24 hr later, mice were imaged in a Siemens Inveon microPET/CT scanner. The PET standard uptake value (SUV) with the G12D agent was 1.4, while SUV was 1.3 with the G12DVA wobble base agent, vs. 0.04 with the G12 wild type sequence. The wobble base approach enables detection of three different KRAS2 mutations, G12D, G12V, or G12A, with a single agent. mRNA PET imaging also enables external monitoring of therapeutic efficacy. Supported by NIH CA148565; IP owned by EW/MLT, licensed to MTTI. Citation Format: Eric Wickstrom, Chang-Po Chen, Matthew E. Wampole, Kaijun Zhang, Bishnuhari Paudyal, Antican Wang, Sushil Tripathi, Pardeep Kumar, Edith P. Mitchell, Bo Lu, Mathew L. Thakur, Brian D. Gray, Jeffrey A. Mattis. Hypoxanthine wobble base multimutant KRAS2 mRNA PET imaging agent in G12D mice. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B55. doi: 10.1158/1557-3125.RASONC14-B55