Evaluation of CD146 as Target for Radioimmunotherapy against Osteosarcoma.

BACKGROUND: Osteosarcoma is a rare form of cancer but with a substantial need for new active drugs. There is a particular need for targeted therapies to combat metastatic disease. One possible approach is to use an antibody drug conjugate or an antibody radionuclide conjugate to target the osteosarcoma metastases and circulating tumor cells. Herein we have evaluated a radiolabeled monoclonal antibody targeting CD146 both in vitro and in vivo. METHODS AND RESULTS: A murine monoclonal anti-CD146 IgG1 isotype antibody, named OI-3, was developed along with recombinant chimeric versions with human IgG1 or human IgG3 Fc sequences. Using flow cytometry, selective binding of OI-3 to human osteosarcoma cell lines OHS, KPDX and Saos-2 was confirmed. The results confirm a higher expression level of CD146 on human osteosarcoma cells than HER2 and EGFR; antigens targeted by commercially available therapeutic antibodies. The biodistribution of 125I-labeled OI-3 antibody variants was compared with 125I-labeled chimeric anti-EGFR antibody cetuximab in nude mice with subcutaneous OHS osteosarcoma xenografts. OI-3 was able to target CD146 expressing tumors in vivo and showed improved tumor to tissue targeting ratios compared with cetuximab. Subsequently, the three OI-3 variants were conjugated with p-SCN-Bn-DOTA and labeled with a more therapeutically relevant radionuclide, 177Lu, and their biodistributions were studied in the nude mouse model. The 177Lu-labeled OI-3 variants were stable and had therapeutically relevant biodistribution profiles. Dosimetry estimates showed higher absorbed radiation dose to tumor than all other tissues after administration of the chimeric IgG1 OI-3 variant. CONCLUSION: Our results indicate that CD146 can be targeted in vivo by the radiolabeled OI-3 antibodies.

Primary bone cancer in Leonbergers may be associated with a higher bodyweight during adolescence.

Weight-bearing stress may be a risk factor for both human and canine primary bone cancer. A cohort of Leonbergers (LB) was followed from birth to death and the cause of death recorded. We hypothesised that dogs dying due to primary bone cancer would be larger; measured by bodyweight (BW) and the circumference of the distal radius and ulna (CDRU) than those of the same breed that died of other causes. Information obtained from breeders, owners and veterinary surgeons were questionnaire-based. The dogs were examined by a veterinary surgeon at pre-specified "observational ages" (3, 4, 6, 12, 18, and 24 m). Data were recorded, including BW and CDRU. The study population consisted of 196 LB, 9 of which died due to primary bone cancer (6 males, 3 females). Individual growth curves, showing BW and CDRU during the first 2 years of life, were made for these 9 dogs and compared to gender-specific mean values for LB that died from other causes. These curves showed that LB succumbing to primary bone cancer generally had a higher BW during the growth period than the remaining dogs, and that this difference appeared to be largest in the male LB. Male LB that developed primary bone cancer later in life also had a larger CDRU during most part of this period, as compared to those that did not develop this disease. Logistic regression showed a statistically significant effect of BW on the odds ratio of developing primary bone cancer at 12 m and 18 m and of CDRU at 18 m, and a Poisson regression verified consistency of these results. At these ages, an increase in BW of 1 kg yielded a nearly 20% higher risk of developing primary bone cancer, while a 1 cm larger CDRU was associated with a nearly 70% increased risk. These findings support that weight-bearing stress during the period of high proliferative activity in the long bones associated with growth may increase the risk of canine primary bone cancer.

HES1, a target of Notch signaling, is elevated in canine osteosarcoma, but reduced in the most aggressive tumors.

BACKGROUND: Hairy and enhancer of split 1 (HES1), a basic helix-loop-helix transcriptional repressor, is a downstream target of Notch signaling. Notch signaling and HES1 expression have been linked to growth and survival in a variety of human cancer types and have been associated with increased metastasis and invasiveness in human osteosarcoma cell lines. Osteosarcoma (OSA) is an aggressive cancer demonstrating both high metastatic rate and chemotherapeutic resistance. The current study examined expression of Notch signaling mediators in primary canine OSA tumors and canine and human osteosarcoma cell lines to assess their role in OSA development and progression. RESULTS: Reverse transcriptase - quantitative PCR (RT-qPCR) was utilized to quantify HES1, HEY1, NOTCH1 and NOTCH2 gene expression in matched tumor and normal metaphyseal bone samples taken from dogs treated for appendicular OSA at the Colorado State University Veterinary Teaching Hospital. Gene expression was also assessed in tumors from dogs with a disease free interval (DFI) of <100 days compared to those with a DFI > 300 days following treatment with surgical amputation followed by standard chemotherapy. Immunohistochemistry was performed to confirm expression of HES1. Data from RT-qPCR and immunohistochemical (IHC) experiments were analyzed using REST2009 software and survival analysis based on IHC expression employed the Kaplan-Meier method and log rank analysis. Unbiased clustered images were generated from gene array analysis data for Notch/HES1 associated genes. Gene array analysis of Notch/HES1 associated genes suggested alterations in the Notch signaling pathway may contribute to the development of canine OSA. HES1 mRNA expression was elevated in tumor samples relative to normal bone, but decreased in tumor samples from dogs with a DFI < 100 days relative to those with a DFI > 300 days. NOTCH2 and HEY1 mRNA expression was also elevated in tumors relative to normal bone, but was not differentially expressed between the DFI tumor groups. Survival analysis confirmed an association between decreased HES1 immunosignal and shorter DFI. CONCLUSIONS: Our findings suggest that activation of Notch signaling occurs and may contribute to the development of canine OSA. However, association of low HES1 expression and shorter DFI suggests that mechanisms that do not alter HES1 expression may drive the most aggressive tumors.

Age-period-cohort analysis of primary bone cancer incidence rates in the United States (1976-2005).

BACKGROUND: Primary bone cancer comprises three major histologic types: osteosarcoma (OS), Ewing sarcoma (ES), and chondrosarcoma (CS). Given the limited knowledge about the etiology of primary bone cancer, we undertook an age-period-cohort (APC) analysis to determine whether incidence varied by birth cohort or calendar period. The purpose was to examine the temporal development of each bone cancer type and generate etiologic hypotheses via the observed birth cohort-related changes. METHODS: An APC model was fitted to incidence data for U.S. whites for OS, ES, and CS obtained from nine registries of the Surveillance, Epidemiology, and End Results program, which covers about 10% of the U.S. population, 1976-2005. RESULTS: The incidence of OS decreased between 1976 and 2005 among those aged over 60 years, a decline that occurred among patients with OS as their primary malignancy only. From 1986-1995 to 1996-2005, the incidence rate of CS among females of 20 to 69 years rose by about 50%, with rates increasing among consecutive cohorts born during 1935-1975. CS rates among males were stable, as were rates of ES. CONCLUSION: The risk reduction in OS as a primary malignancy at older ages could possibly be related to diminished exposure over time to bone-seeking radionuclides. The CS increase among females corresponds to birth cohorts with rising exposures to oral contraceptives and menopausal hormonal therapy. IMPACT: As the estrogen signaling pathway has been shown to stimulate proliferation of normal and malignant chondrocytes, estrogen exposure may increase the risk for CS. Further studies are warranted to clarify its possible etiological significance.

Breed-specific incidence rates of canine primary bone tumors--a population based survey of dogs in Norway.

This is one of few published population-based studies describing breed specific rates of canine primary bone tumors. Incidence rates related to dog breeds could help clarify the impact of etiological factors such as birth weight, growth rate, and adult body weight/height on development of these tumors. The study population consisted of dogs within 4 large/giant breeds; Irish wolfhound (IW), Leonberger (LB), Newfoundland (NF), and Labrador retriever (LR), born between January 1st 1989 and December 31st 1998. Questionnaires distributed to owners of randomly selected dogs--fulfilling the criteria of breed, year of birth, and registration in the Norwegian Kennel Club--constituted the basis for this retrospective, population-based survey. Of the 3748 questionnaires received by owners, 1915 were completed, giving a response rate of 51%. Forty-three dogs had been diagnosed with primary bone tumors, based upon clinical examination and x-rays. The breeds IW and LB, with 126 and 72 cases per 10 000 dog years at risk (DYAR), respectively, had significantly higher incidence rates of primary bone tumors than NF and LR (P < 0.0001). Incidence rates for the latter were 11 and 2 cases per 10 000 DYAR, respectively. Pursuing a search for risk factors other than body size/weight is supported by the significantly different risks of developing primary bone tumors between similarly statured dogs, like NF and LB, observed in this study. Defining these breed-specific incidence rates enables subsequent case control studies, ultimately aiming to identify specific etiological factors for developing primary bone tumors.

Assessment of long-term radiotoxicity after treatment with the low-dose-rate alpha-particle-emitting radioimmunoconjugate (227)Th-rituximab.

PURPOSE: The anti-CD20 antibody rituximab labelled with the alpha-particle-emitting radionuclide (227)Th is of interest as a radiotherapeutic agent for treatment of lymphoma. Complete regression of human lymphoma Raji xenografts in 60% of mice treated with 200 kBq/kg (227)Th-rituximab has been observed. To evaluate possible late side effects of (227)Th-rituximab, the long-term radiotoxicity of this potential radiopharmaceutical was investigated. METHODS: BALB/c mice were injected with saline, cold rituximab or 50, 200 or 1,000 kBq/kg (227)Th-rituximab and followed for up to 1 year. In addition, nude mice with Raji xenografts treated with various doses of (227)Th-rituximab were also included in the study. Toxicity was evaluated by measurements of mouse body weight, white blood cell (WBC) and platelet counts, serum clinical chemistry parameters and histological examination of tissues. RESULTS: Only the 1,000 kBq/kg dosage resulted in decreased body weight of the BALB/c mice. There was a significant but temporary decrease in WBC and platelet count in mice treated with 400 and 1,000 kBq/kg (227)Th-rituximab. Therefore, the no-observed-adverse-effect level (NOAEL) was 200 kBq/kg. The maximum tolerated activity was between 600 and 1,000 kBq/kg. No significant signs of toxicity were observed in histological sections in any examined tissue. There were significantly (p < 0.05), but transiently, higher concentrations of serum bile acids and aspartate aminotransferase in mice treated with either (227)Th-rituximab or non-labelled antibody when compared with control mice. The maximum tolerated dose to bone marrow was between 2.1 and 3.5 Gy. CONCLUSION: Therapeutically relevant dose levels of (227)Th-rituximab were well tolerated in mice. Bone marrow suppression, as indicated by decrease in WBC count, was the dose-limiting radiotoxicity. These toxicity data together with anti-tumour activity data in a CD20-positive xenograft mouse model indicate that therapeutic effects could be obtained with relatively safe dosage levels of the radioimmunoconjugate.

Targeted cancer therapy with a novel low-dose rate alpha-emitting radioimmunoconjugate.

Alpha-emitting radionuclides are highly cytotoxic and are of considerable interest in the treatment of cancer. A particularly interesting approach is in radioimmunotherapy. However, alpha-emitting antibody conjugates have been difficult to exploit clinically due to the short half-life of the radionuclides, low production capability, or limited source materials. We have developed a novel technology based on the low-dose rate alpha-particle-emitting nuclide (227)Th, exemplified here using the monoclonal antibody rituximab. In vitro, this radioimmunoconjugate killed lymphoma cells at Becquerel per milliliter (Bq/mL) levels. A single injection of (227)Th-rituximab induced complete tumor regression in up to 60% of nude mice bearing macroscopic (32-256 mm(3)) human B-lymphoma xenografts at Becquerel per gram (Bq/g) levels without apparent toxicity. Therapy with (227)Th-rituximab was significantly more effective than the control radioimmunoconjugate (227)Th-trastuzumab and the standard beta-emitting radioimmunoconjugate for CD20(+) lymphoma(90)Y-tiuxetan-ibritumomab. Thorium-227 based constructs may provide a novel approach for targeted therapy against a wide variety of cancers.

First in vivo evaluation of liposome-encapsulated 223Ra as a potential alpha-particle-emitting cancer therapeutic agent.

BACKGROUND: Liposomes carrying chemotherapeutics have had some success in cancer treatment and may also be suitable carriers for therapeutic radionuclides. This study was designed to evaluate the biodistribution and to estimate the radiation doses of the alpha emitter 223Ra loaded into pegylated liposomes in selected tissues. MATERIALS AND METHODS: 223Ra was encapsulated in pegylated liposomal doxorubicin (PLD) by ionophore-mediated loading. The biodistribution of liposomal 223Ra was compared to free cationic 223Ra in Balb/C mice. RESULTS: Liposomal 223Ra circulated in the blood with an initial half-life in excess of 24 hours, which agreed well with that reported for PLD in rodents, while the blood half-life of cationic 223Ra was considerably less than an hour. When liposomal 223Ra was catabolized, the released 223Ra was either excreted or taken up in the skeleton. This skeletal uptake increased up to 14 days after treatment, but did not reach the level seen with free 223Ra. Pre-treatment with non-radioactive PLD 4 days in advance lessened the liver uptake of liposomal 223Ra. Dose estimates showed that the spleen, followed by bone surfaces, received the highest absorbed doses. CONCLUSION: Liposomal 223Ra was relatively stable in vivo and may have potential for radionuclide therapy and combination therapy with chemotherapeutic agents.

Radiotoxicity of the alpha-emitting bone-seeker 223Ra injected intravenously into mice: histology, clinical chemistry and hematology.

BACKGROUND: The alpha-emitter 223Ra, which localizes in osteoblastic active zones, including on skeletal surfaces and in osteoblastic metastases, has recently been introduced as a potential therapeutic agent against skeletal metastases. Here, the adverse effects of high dosages in animals were investigated. MATERIALS AND METHODS: Balb/c mice received intravenously (i.v.) either 1250, 2500, or 3750 kBq/kg of dissolved 223RaCl2 and were followed in the initial toxicity phase. At the 4-week end-point, the animals were sacrificed and blood samples were collected to study the effects on clinical chemistry and hematological parameters. Selected organs were weighed and tissue samples examined by microscopy. RESULTS: Treatment with 223Ra caused a dose-related minimal to moderate depletion of osteocytes and osteoblasts in the bones. Furthermore, a dose-related minimal to marked depletion of the hematopoietic cells in the bone marrow, and a minimal to slight extramedullary hematopoiesis in the spleen and in the mandibular and mesenteric lymph nodes were observed. The LD50 for acute toxicity, defined as death within 4 weeks of receiving the substance, was not reached. CONCLUSION: This study demonstrated that high doses of the bone-seeker 223Ra did not completely inactivate the blood-producing cells. The relatively high tolerance to skeletal alpha doses was probably caused by the surviving pockets of red bone marrow cells beyond the range of alpha particles from the bone surfaces, and the recruitment of peripheral stems cells.

A population-based familial aggregation analysis indicates genetic contribution in a majority of renal cell carcinomas.

The etiology of RCC is incompletely understood and the inherited genetic contribution uncertain. Although there are rare mendelian forms of RCC stemming from inherited mutations, most cases are thought to be sporadic. We sought to determine the extent of familial aggregation among Icelandic RCC patients in general. Medical and pathologic records for all patients diagnosed with RCC in Iceland between 1955 and 1999 were reviewed. This included a total of 1,078 RCC cases, 660 males and 418 females. With the use of an extensive computerized database containing genealogic information on 630,000 people in Iceland during the past 11 centuries, several analyses were conducted to determine whether the patients were more related to each other than members drawn at random from the population. Patients with RCC were significantly more related to each other than were subjects in matched groups of controls. This relatedness extended beyond the nuclear family. RRs were significantly greater than 1.0 for siblings, parents and cousins of probands. RRs were 2-3 for first-degree relatives and 1.6 for third-degree relatives. The risk of RCC is significantly higher for members of the extended family of an affected individual, as well as the nuclear family. Our results indicate that germline mutations are significantly involved in what has been defined as sporadic RCC.