Glypican-3 targeted positron emission tomography detects sub-centimeter tumors in a xenograft model of hepatocellular carcinoma.

BACKGROUND: Early intrahepatic recurrence is common after surgical resection of hepatocellular carcinoma (HCC) and leads to increased morbidity and mortality. Insensitive and nonspecific diagnostic imaging contributes to EIR and results in missed treatment opportunities. In addition, novel modalities are needed to identify targets amenable for targeted molecular therapy. In this study, we evaluated a zirconium-89 radiolabeled glypican-3 (GPC3) targeting antibody conjugate (89Zr-αGPC3) for use in positron emission tomography (PET) for detection of small, GPC3+ HCC in an orthotopic murine model. Athymic nu/J mice received hepG2, a GPC3+ human HCC cell line, into the hepatic subcapsular space. Tumor-bearing mice were imaged by PET/computerized tomography (CT) 4 days after tail vein injection of 89Zr-αGPC3. Livers were then excised for the tumors to be identified, measured, bisected, and then serially sectioned at 500 µm increments. Sensitivity and specificity of PET/CT for 89Zr-αGPC3-avid tumors were assessed using tumor confirmation on histologic sections as the gold standard. RESULTS: In tumor-bearing mice, 89Zr-αGPC3 avidly accumulated in the tumor within four hours of injection with ongoing accumulation over time. There was minimal off-target deposition and rapid bloodstream clearance. Thirty-eight of 43 animals had an identifiable tumor on histologic analysis. 89Zr-αGPC3 immuno-PET detected all 38 histologically confirmed tumors with a sensitivity of 100%, with the smallest tumor detected measuring 330 µm in diameter. Tumor-to-liver ratios of 89Zr-αGPC3 uptake were high, creating excellent spatial resolution for ease of tumor detection on PET/CT. Two of five tumors that were observed on PET/CT were not identified on histologic analysis, yielding a specificity of 60%. CONCLUSIONS: 89Zr-αGPC3 avidly accumulated in GPC3+ tumors with minimal off-target sequestration. 89Zr-αGPC3 immuno-PET yielded a sensitivity of 100% and detected sub-millimeter tumors. This technology may improve diagnostic sensitivity of small HCC and select GPC3+ tumors for targeted therapy. Human trials are warranted to assess its impact.

Glypican-3-Targeted 227Th α-Therapy Reduces Tumor Burden in an Orthotopic Xenograft Murine Model of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a significant cause of morbidity and mortality worldwide, with limited therapeutic options for advanced disease. Targeted α-therapy is an emerging class of targeted cancer therapy in which α-particle-emitting radionuclides, such as 227Th, are delivered specifically to cancer tissue. Glypican-3 (GPC3) is a cell surface glycoprotein highly expressed on HCC. In this study, we describe the development and in vivo efficacy of a 227Th-labeled GPC3-targeting antibody conjugate (227Th-octapa-αGPC3) for treatment of HCC in an orthotopic murine model. Methods: The chelator p-SCN-Bn-H4octapa-NCS (octapa) was conjugated to a GPC3-targeting antibody (αGPC3) for subsequent 227Th radiolabeling (octapa-αGPC3). Conditions were varied to optimize radiolabeling of 227Th. In vitro stability was evaluated by measuring the percentage of protein-bound 227Th by γ-ray spectroscopy. An orthotopic athymic Nu/J murine model using HepG2-Red-FLuc cells was developed. Biodistribution and blood clearance of 227Th-octapa-αGPC3 were evaluated in tumor-bearing mice. The efficacy of 227Th-octapa-αGPC3 was assessed in tumor-bearing animals with serial measurement of serum α-fetoprotein at 23 d after injection. Results: Octapa-conjugated αGPC3 provided up to 70% 227Th labeling yield in 2 h at room temperature. In the presence of ascorbate, at least 97.8% of 227Th was bound to αGPC3-octapa after 14 d in phosphate-buffered saline. In HepG2-Red-FLuc tumor-bearing mice, highly specific GPC3 targeting was observed, with significant 227Th-octapa-αGPC3 accumulation in the tumor over time and minimal accumulation in normal tissue. Twenty-three days after treatment, a significant reduction in tumor burden was observed in mice receiving a 500 kBq/kg dose of 227Th-octapa-αGPC3 by tail-vein injection. No acute off-target toxicity was observed, and no animals died before termination of the study. Conclusion:227Th-octapa-αGPC3 was observed to be stable in vitro; maintain high specificity for GPC3, with favorable biodistribution in vivo; and result in significant antitumor activity without significant acute off-target toxicity in an orthotopic murine model of HCC.

Glypican-3 targeted delivery of 89Zr and 90Y as a theranostic radionuclide platform for hepatocellular carcinoma.

Glypican-3 (GPC3) is a tumor associated antigen expressed by hepatocellular carcinoma (HCC) cells. This preclinical study evaluated the efficacy of a theranostic platform using a GPC3-targeting antibody αGPC3 conjugated to zirconium-89 (89Zr) and yttrium-90 (90Y) to identify, treat, and assess treatment response in a murine model of HCC. A murine orthotopic xenograft model of HCC was generated. Animals were injected with 89Zr-labeled αGPC3 and imaged with a small-animal positron emission/computerized tomography (PET/CT) imaging system (immuno-PET) before and 30 days after radioimmunotherapy (RIT) with 90Y-labeled αGPC3. Serum alpha fetoprotein (AFP), a marker of tumor burden, was measured. Gross tumor volume (GTV) and SUVmax by immuno-PET was measured using fixed intensity threshold and manual segmentation methods. Immuno-PET GTV measurements reliably quantified tumor burden prior to RIT, strongly correlating with serum AFP (R2 = 0.90). Serum AFP was significantly lower 30 days after RIT in 90Y-αGPC3 treated animals compared to those untreated (p = 0.01) or treated with non-radiolabeled αGPC3 (p = 0.02). Immuno-PET GTV measurements strongly correlated with tumor burden after RIT (R2 = 0.87), and GTV of animals treated with 90Y-αGPC3 was lower than in animals who did not receive treatment or were treated with non-radiolabeled αGPC3, although this only trended toward statistical significance. A theranostic platform utilizing GPC3 targeted 89Zr and 90Y effectively imaged, treated, and assessed response after radioimmunotherapy in a GPC3-expressing HCC xenograft model.

Yttrium-90-Labeled Anti-Glypican 3 Radioimmunotherapy Halts Tumor Growth in an Orthotopic Xenograft Model of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the second most lethal malignancy globally and is increasing in incidence in the United States. Unfortunately, there are few effective systemic treatment options, particularly for disseminated disease. Glypican-3 (GPC3) is a proteoglycan cell surface receptor overexpressed in most HCCs and provides a unique target for molecular therapies. We have previously demonstrated that PET imaging using a 89Zr-conjugated monoclonal anti-GPC3 antibody (αGPC3) can bind to minute tumors and allow imaging with high sensitivity and specificity in an orthotopic xenograft mouse model of HCC and that serum alpha-fetoprotein (AFP) levels are highly correlated with tumor size in this model. In the present study, we conjugated 90Y, a high-energy beta-particle-emitting radionuclide, to our αGPC3 antibody to develop a novel antibody-directed radiotherapeutic approach for HCC. Luciferase-expressing HepG2 human hepatoblastoma cells were orthotopically implanted in the livers of athymic nude mice, and tumor establishment was verified at 6 weeks after implantation by bioluminescent imaging and serum AFP concentration. Tumor burden by bioluminescence and serum AFP concentration was highly correlated in our model. Yttrium-90 was conjugated to αGPC3 using the chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and injected via the tail vein into the experimental mice at a dose of 200 µCi/mouse or 300 µCi/mouse. Control mice received DOTA-αGPC3 without radionuclide. At 30 days after a single dose of the radioimmunotherapy agent, mean serum AFP levels in control animals increased dramatically, while animals treated with 200 µCi only experienced a minor increase, indicating cessation of tumor growth, and animals treated with 300 µCi experienced a reduction in serum AFP concentration, indicating tumor shrinkage. Mean tumor-bearing liver weight in control animals was also significantly greater than that in animals that received either dose of 90Y-αGPC3. These results were achieved without significant toxicity as measured by body condition scoring and body weight. The results of this preclinical pilot demonstrate that GPC3 can be used as a target for radioimmunotherapy in an orthotopic mouse model of HCC and may be a target of clinical significance, particularly for disseminated HCC.

Neuroblastoma-derived secretory protein is a novel secreted factor overexpressed in neuroblastoma.

Secreted proteins such as growth factors, cytokines, and chemokines play important roles in tumor development. Through expression microarray and bioinformatic analysis, we discovered a novel secreted protein, neuroblastoma-derived secretory protein (NDSP). The NDSP gene is found on chromosome 1q25.2 and encodes a 167 amino acid protein with a putative signal peptide. Using real-time PCR and immunoblotting, we find that NDSP is specifically overexpressed in neuroblastoma at much higher levels than other adult and pediatric malignancies and normal tissues. NDSP is an 18-kDa protein that can be secreted by NDSP-transfected HEK-293T cells, as well as, neuroblastoma cell lines endogenously expressing NDSP. Inhibiting NDSP expression in neuroblastoma cell lines with retrovirally transduced NDSP small hairpin interfering RNA, sh-NDSP, results in decreased cellular proliferation and colony formation. We also find inhibited extracellular signal-regulated kinase (ERK)1/2 phosphorylation in the sh-NDSP cell line. Treating the parental cell line with MAP/ERK kinase 1/2 inhibitors, which diminish ERK1/2 phosphorylation, results in decreased cell proliferation. Culturing these transduced cells with recombinant NDSP, reintroducing NDSP overexpression in the knockdown cell line, or inducing Ras oncogene overexpression for constitutive ERK1/2 activation results in a reversal of the growth-inhibited phenotype and proliferation rates similar to the control cells. In addition, reintroduction of NDSP overexpression in the sh-NDSP cell line results in ERK1/2 phosphorylation similar to control. We conclude that NDSP is specifically overexpressed in neuroblastoma and actively secreted from tumor cells. Furthermore, NDSP serves as a growth factor for neuroblastoma tumor cells through activation of the ERK-mediated proliferation pathway.

Recent advances in the diagnosis and treatment of pheochromocytoma in children.

BACKGROUND: The purpose of this study was to analyze the effects of changes in the diagnosis and treatment of pheochromocytoma in a pediatric population. METHODS: We reviewed the medical records of all children who had resection of pheochromocytoma or paraganglioma at a major children's hospital since 1968. RESULTS: Fifteen children underwent surgery at 11.9 +/- 4.2 years of age. Presenting symptoms included headache, hypertension, and sweating. Three children had a mutation of the succinate dehydrogenase enzyme, and 1 child had nonsyndromic, familial pheochromocytoma. The most sensitive diagnostic modalities included 24-hour urinary and plasma norepinephrine and 24-hour urinary total metanephrines, magnetic resonance imaging, and 123I-meta-iodobenzylguanidine scintigraphy. Laparoscopic cortical-sparing adrenalectomy was performed in 3 patients with von Hippel-Lindau disease. Compared with those with open procedures (n = 7), patients who had laparoscopic resection (n = 5) had a statistically shorter hospital length of stay, and time to eating ambulation. CONCLUSIONS: The addition of 123I-meta-iodobenzylguanidine scanning, genetic testing, and laparoscopic surgery has changed the diagnosis and treatment of pheochromocytoma in children. Laparoscopic cortical-sparing adrenalectomy can be accomplished safely and is the preferred treatment for children at risk for multifocal disease.

MYCN-directed centrosome amplification requires MDM2-mediated suppression of p53 activity in neuroblastoma cells.

The MYC family oncogenes cause transformation and tumor progression by corrupting multiple cellular pathways, altering cell cycle progression, apoptosis, and genomic instability. Several recent studies show that MYCC (c-Myc) expression alters DNA repair mechanisms, cell cycle checkpoints, and karyotypic stability, and this is likely partially due to alterations in centrosome replication control. In neuroblastoma cell lines, MYCN (N-Myc) expression induces centrosome amplification in response to ionizing radiation. Centrosomes are cytoplasmic domains that critically regulate cytokinesis, and aberrations in their number or structure are linked to mitotic defects and karyotypic instability. Whereas centrosome replication is linked to p53 and Rb/E2F-mediated cell cycle progression, the mechanisms downstream of MYCN that generate centrosome amplification are incompletely characterized. We hypothesized that MDM2, a direct transcriptional target of MYCN with central inhibitory effects on p53, plays a role in MYC-mediated genomic instability by altering p53 responses to DNA damage, facilitating centrosome amplification. Herein we show that MYCN mediates centrosome amplification in a p53-dependent manner. Accordingly, inhibition of the p53-MDM2 interaction with Nutlin 3A (which activates p53) completely ablates the MYCN-dependent contribution to centrosome amplification after ionizing radiation. We further show that modulating MDM2 expression levels by overexpression or RNA interference-mediated posttranscriptional inhibition dramatically affects centrosome amplification in MYCN-induced cells, indicating that MDM2 is a necessary and sufficient mediator of MYCN-mediated centrosome amplification. Finally, we show a significant correlation between centrosome amplification and MYCN amplification in primary neuroblastoma tumors. These data support the hypothesis that elevated MDM2 levels contribute to MYCN-induced genomic instability through altered regulation of centrosome replication in neuroblastoma.