Molecular Targeting with Recombinant Cytotoxins of Interleukin-13 Receptor α 2-expressing Glioma

A restricted receptor for interleukin 13 (IL-13R2) is over-expressed in high-grade astrocytoma (HGA), but not in normal organs. In order to design and examine new anti-HGA therapies, which are molecularly directed against IL-13R2, we established an IL-13R2-expressing syngeneic immunocompetent murine model of HGA. The model was obtained by transfecting G-26 murine glioma cells with IL-13R2. G-26-IL-13R2(+) cells, but not mock-transfected cells, became susceptible to IL-13 mutant-based cytotoxic proteins that kill human HGA cells. G-26-IL-13R2(+) cells maintained their tumorigenicity in immunocompetent C57BL/J6 mice and preserved their expression of IL-13R2 in vivo. These characteristics of the G-26-IL-13R2(+) tumors allowed us to test molecularly defined anti-glioma passive immunotherapy. A targeted recombinant chimera cytotoxin composed of multiply mutated IL-13 (IL-13.E13Y/R66D/S69D) and a derivative of Pseudomonas exotoxin (PE), PE1E, IL-13.E13Y/R66D/S69D-PE1E, was used in anti-tumor experiments. G-26-IL-13R2(+) cells were killed by IL-13.E13Y/R66D/S69D-PE1E in an IL-4-independent fashion. To test the cytotoxin in vivo, G-26-IL-13R2(+) tumors were established in C57BL/J6 mice and when the tumors reached a size of at least 50mm³, the mice were treated with IL-13.E13Y/R66D/S69D-PE1E. In the mice treated with the targeted fusion cytotoxin, the tumors regressed and 80% of the animals were cured. This study documents the establishment of an IL-13R2-positive model of HGA in immunocompetent rodents. Furthermore, the effectiveness and safety of the targeted IL-13-based cytotoxin against IL-13R2-expressing tumors in a more clinically relevant in vivo HGA model is promising with regard to the future clinical utility of the cytotoxin.     

Prediction of Pathology and Survival by FDG PET in Gliomas

Objectives: Despite being in use for nearly two decades, the utility of [18F]2-fluoro-2deoxy-d-glucose positron emission tomography (FDG PET) in the evaluation and treatment of brain tumors remains controversial. We retrospectively analyzed all patients with histologically proven gliomas, between the years 1990 and 2000, who underwent FDG PET studies at various stages of their treatment and who were followed till either death or for a minimum period of 1 year in an attempt to bring resolution to this controversy. Methods: All PET scans prior to 1997 were acquired on an ECAT 951/31 scanner in 2D. Scans since 1997 were obtained on a Siemens HR+ scanner in 3D mode. The majority of FDG PET scans were co-registered with the magnetic resonance imaging (MRI) scans to aid in diagnosis and therapy. Based on independent visual inspection, two board certified nuclear medicine physicians graded the highest activity level of the tumor using the metabolic grading: 0 = no uptake; 1 = uptake less or equal to normal white matter; 2 = uptake greater than normal white matter and less than gray matter; 3 = uptake equal to or greater than gray mater. The measure of association of lambda was used to measure the strength of predictive ability of FDG PET for pathological grading of the gliomas. The Cox proportional hazards regression model was used to assess the significance of grade of uptake on survival. Results: A total of 331 patients were analyzed of which 137 had a PET scan prior to histological diagnosis and therapeutic intervention (mean age = 46.5years; M:F = 1.7:1). Eighty six percent (143/166) of the patients with low uptake (metabolic scores 0,1) had low-grade gliomas (grade I,II) and 14% (23/166) high-grade gliomas (grade III,IV) on histologic examination. Ninety four percent (154/165) of the patients with high uptake (metabolic scores 2,3) on PET had high-grade gliomas and 7% (11/165) had low-grade gliomas on histologic examination. The grade of uptake had increasing significance on survival as the level increased from 'low' to 'high' (P = 0.0009). Ninety four percent (156/166) of the patients with low uptake survived for >1 year (median survival of 28 months) and 19% survived for >5 years. Only 29% (48/165) of patients with high uptake survived for >1 year, (median survival of 11 months) and none survived for >5 years. Irrespective of when the scan showed a high uptake of FDG, before or after intervention, the prognosis following that scan was poor. Conclusions: Our observations confirm the utility of FDG PET as a prognostic tool for the histological grading and survival in patients with gliomas and appears to more than complement pathological grading.     

Gene therapy for experimental brain tumors using a xenogenic cell line engineered to secrete hIL-2

Summary Local delivery of cytokines has been shown to have a potent anti-tumor activity against a wide range of malignant brain tumors. In this study, we examined the feasibility and efficacy of using a rat endothelial cell line (NTC-121) transfected with the human interleukin-2 (IL-2) gene in treating experimental murine CNS tumors. The NTC-121 cells were injected intracranially in C57BL/6 mice (N = 10/group) along with non-irradiated, non-transfected B16/F10 (wild type) melanoma cells. Sixty percent of mice treated with IL-2 (p<0.001 vs. control) were long-term survivors (LTS) of >120 days. Control animals that received only wild type cells had a median survival of 18 days (range 15–20). Histopathological examination of brains from animals sacrificed at different times showed no tumor growth in the non-irradiated NTC-121 group, moderate (1–2 mm) tumor growth in the irradiated group, and gross tumor invasion (>2 mm) and tissue necrosis in the control group. Moreover, animals treated with IL-2 showed an accumulation of CD8+ T cells around the site of the injected tumor. The use of a xenogenic cell line to deliver hIL-2 stimulates a strong immunologic cytotoxic anti-tumor response that leads to significant prolongation of survival in mice challenged with the B16/F10 intracranial melanoma tumor. Our findings demonstrate that the use of a xenogenic cell line can provide a potent vehicle for the delivery of gene therapy and may therefore represent a new approach for brain tumor therapy.     

Rat Brain Tumor Models to Assess the Efficacy of Boron Neutron Capture Therapy: A Critical Evaluation

Development of any therapeutic modality can be facilitated by the use of the appropriate animal models to assess its efficacy. This report primarily will focus on our studies using the F98 and 9L rat glioma models to evaluate the effectiveness of boron neutron capture therapy (BNCT) of brain tumors. Following intracerebral implantation the biological behavior of each tumor resembles that of human high grade gliomas in a number of ways. In both models, glioma cells were implanted intracerebrally into syngeneic Fischer rats and 10–14 days later BNCT was initiated at the Brookhaven National Laboratory Medical Research Reactor. Two low molecular weight (M _r < 210Da) ¹⁰B-containing drugs, boronophenylalanine (BPA) and/or sodium borocaptate (BSH) were used as capture agents, either alone or in combination with each other. The 9L gliosarcoma, which has been difficult to cure by means of either chemo- or radiotherapy alone, was readily curable by BNCT. The best survival data were obtained using BPA at a dose of 1200mg/kg (64.8mg ¹⁰B), administered intraperitoneally (i.p.), with a 100% survival rate at 8 months. In contrast, the F98 glioma has been refractory to all therapeutic modalities. Tumor bearing animals, which had received 500mg/kg (27mg ¹⁰B) of BPA, or an equivalent amount of BSH i.v., had mean survival time (MST) of 37 and 33 days, respectively, compared to 29 days for irradiated controls. The best survival data with the F98 glioma model were obtained using BPA + BSH in combination, administered intra-arterially via the internal carotid artery (i.c.) with hyperosmotic mannitol induced blood–brain barrier disruption (BBB-D). The MST was 140 days with a cure rate of 25%, compared to a MST of 73 days with a 5% cure rate without BBB-D, and 41 days following i.v. administration of both drugs. A modest but significant increase in MST also was observed in rats that received intracarotid (i.c.) BPA in combination with Cereport (RMP-7), which produced a pharmacologically mediated opening of the BBB. Studies also have been carried out with the F98 glioma to determine whether an X-ray boost could enhance the efficacy of BNCT, and it was shown that there was a significant therapeutic gain. Finally, molecular targeting of the epidermal growth factor receptor (EGFR) has been investigated using F98 glioma cells, which had been transfected with the gene encoding EGFR and, intratumoral injection of boronated EGF as the delivery agent, followed by BNCT. These studies demonstrated that there was specific targeting of EGFR and provided proof of principle for the use of high molecular weight, receptor targeting-boron delivery agents. Finally, a xenograft model for melanoma metastatic to the brain has been developed using a human melanoma (MRA27), stereotactically implanted into the brains of nude rats, and these studies demonstrated that BNCT either cured or significantly prolonged the survival of tumor-bearing rats. It remains to be determined, which, if any, of these experimental approaches will be translated into clinical studies. Be that as it may, rat brain tumor models already have made a significant contribution to the design of clinical BNCT protocols, and should continue to do so in the future.     

Imatinib inhibits various types of activating mutant kit found in gastrointestinal stromal tumors

Mutations of proto-oncogene c-KIT in gastrointestinal stromal tumors (GISTs) are considered to cause a constitutive activation of KIT responsible for their oncogenesis. Imatinib has therapeutic potential for GISTs because of its inhibitory effect on KIT kinase activity. To investigate the effect of Imatinib on various c-KIT mutations found in GISTs, we examined kinase activity of KIT, cell proliferation and tumorigenicity of transfectants with various c-KIT mutations. Murine lymphoid Ba/F3 cells transfected with one of the three types of mutants (KIT(del559-560), KIT(642Glu), and KIT(820Tyr)) or wild-type KIT were used for the experiments. Phosphorylation of KIT, mitogen-activated protein (MAP) and Akt was studied by immunoblotting with or without immunoprecipitation. In vitro studies on cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylcetrazolium bromide colorimetric assay and in vivo tumorigenicity assay using nude mice were also carried out. Imatinib could inhibit the KIT, MAP and Akt phosphorylation of all the transfectants but had a weaker effect on KIT(820Tyr). Imatinib potently inhibited the proliferation of cells transfected with KIT(820Tyr) at the concentration of 10 microM whereas it inhibited the other 3 types at 1 microM. Moreover, Imatinib could inhibit the tumor formation in nude mice transplanted with transfectants. In various types of activating mutant KIT, Imatinib could inhibit the constitutive activation of KIT signal transduction and cell proliferation both in vitro and in vivo although the effect of Imatinib on KIT(820Tyr) was weaker than that on KIT(del559-560) or KIT(642Glu).     

Body weight considerations in the B6C3F1 mouse and the use of dietary control to standardize background tumor incidence in chronic bioassays

In B6C3F1 mice, the rate of body growth influences susceptibility to liver neoplasia and large variations in body weight can complicate the interpretation of bioassay data. The relationship between body weight and liver tumor incidence was calculated for historical control populations of male and female ad libitum-fed mice (approx. 2,750 and 2,300 animals, respectively) and in populations of male and female mice which had been subjected to forced body weight reduction due to either dietary restriction or exposure to noncarcinogenic chemicals (approx. 1,600 and 1,700, respectively). Resulting tumor risk data were then used to construct idealized weight curves for male and female B6C3F1 mice; these curves predict a terminal background liver tumor incidence of 15-20%. Use of dietary control to manipulate body growth of male B6C3F1 mice to fit the idealized weight curve was evaluated in a 2-year bioassay of chloral hydrate. Cohorts of mice were successfully maintained at weights approximating their idealized target weights throughout the study. These mice exhibited less body weight variation than their ad libitum-fed counterparts (e.g., standard deviations of body weight were 1.4 and 3.4 g for respective control groups at 36 weeks). Historical control body weight and tumor risk data from the two male mouse populations were utilized to predict background liver tumor rates for each experimental group of the chloral hydrate study. The predicted background tumor rates closely matched the observed rates for both the dietary controlled and ad libitum-fed chloral hydrate control groups when each mouse was evaluated according to either its weekly food consumption or its weekly change in body weight.     

Expression of somatostatin receptor subtypes 1 to 5 in tumor tissue and intratumoral vessels in malignant endocrine pancreatic tumors

Somatostatin analogs are well established in the treatment of malignant endocrine pancreatic tumors (EPTs). Our goal is to individualize their treatment using receptor-subtype-specific analogs and, therefore, exploring the receptor expression is highly important. We have examined the expression of somatostatin receptor (sst) subtypes 1–5 on tumor cells and in intratumoral vessels in 28 tumor tissues from malignant EPTs with immunohistochemistry using sst-subtype-specific polyclonal antibodies. We found that sst₂ and sst₄ stained positive in 90% and sst₁ in 70% of the tumor tissues, whereas sst₃ and sst₅ stained positive in only 50% of the tumor tissues. Sst expression in intratumoral vessels was high for sst₂ and sst₄ (80%), moderate for sst₁ (40%), and low for sst₃ and sst₅ (10%). The ssts were evenly distributed among the different tumor subtypes. However, tumors belonging to the same subgroup of EPTs showed a variable expression of receptor subtypes. No differences in receptor-subtype expression could be seen between poorly and well-differentiated tumors, nor between primary tumors and metastases. Prior medical treatment did not influence sst expression pattern. In conclusion, sst₂ and sst₄ were expressed in most tumor tissues and intratumoral vessels from EPTs. However, sst₃ and sst₅ were lacking in half of the tumor tissues and in most of the intratumoral vessels. These differences indicate the importance of determining each tumor’s subset of receptors before treatment with receptor-subtype-specific analogs is initiated. The importance of sst expression in intratumoral vessels is not yet known.     

Radiofrequency-assisted liver resection

Radiofrequency (RF)-assisted thermal ablation has been used with increasing frequency for unresectable hepatic tumors. This new approach employs RF energy to coagulate the liver at the hepatic resection line after which hepatic resection is performed with the use of a common scalpel. This procedure was used in three patients with hepatocellular carcinoma and in five patients with colorectal metastasis to the liver. These eight patients underwent a total of two left bisegmentectomies, three segmentectomies, and seven wedge resections. Mean operative time was 220 minutes. A mean of 78 sessions of RF-assisted ablation were required for these resections. Mean blood loss was 46 ml; no device other than RF ablation was required to obtain hemostasis. None of the patients needed a blood transfusion. Preoperative hemoglobin was 12.8 gm/dl and postoperative hemoglobin was 11.3 gm/dl. There were no perioperative deaths. Postoperative complications occurred in two patients: a liver abscess in one and heart failure in the other. The mean hospital stay was 9.4 days. This new approach, integrated with other techniques, reduces blood loss and coagulates the margins of resection during liver surgery. This new technique has two limitations: (1) it cannot be applied near main portal pedicles, and (2) it requires a long operative time. The best indication for this technique is when segmentectomy is required in patients with cirrhosis. Its role in major hepatic resections has yet to be determined. Further progress in the development of thermal ablation techniques and experience gained during the learning curve should help reduce the operative time, thereby improving the safety and efficacy of this procedure. Presented at the Third International Meeting, "Hepatocellular Carcinoma: Eastern and Western Experience," Lodi, Italy, November 21–22, 2002.     

Expression of the extra domain B of fibronectin,a marker of angiogenesis,in head and neck tumors

OBJECTIVES/HYPOTHESIS: The extra domain B (ED-B) of fibronectin, a naturally occurring marker of tissue remodeling and angiogenesis, is expressed in the majority of aggressive solid human tumors, whereas it is not detectable in normal vessels and tissues. STUDY DESIGN: In view of the diagnostic and therapeutic clinical applications of the L19 antibody, which is specific for the ED-B domain of fibronectin, a prospective immunohistochemical analysis of different head and neck tumors was performed. METHODS: In all, 82 head and neck tissue biopsy specimens were immunohistochemically analyzed using the L19 antibody. They consisted of 53 different malignant tumors, 8 benign tumors, 10 nontumoral lesions, and 11 normal control tissues. RESULTS: A strong positive staining with the L19 antibody could be observed in 87% of the investigated malignant tumors, in only 38% of the benign tumors, and in 20% of the nontumoral lesions (P <.0001). The extra domain B was completely absent in the normal control tissue samples. CONCLUSIONS: The results show that ED-B is abundantly expressed around the neovasculature and in the stroma of the majority of malignant tumors of the head and neck but is undetectable in normal tissues. The ED-B domain of fibronectin is a good-quality tumor-stroma-associated antigen that warrants clinical trials with antibody-based pharmaceuticals, including immunoscintigraphic investigations and radioimmunoguided surgery with the radiolabeled L19 antibody.     

Prognostic value of cystocopically pseudotumoral lesions (inflammation/granuloma) in primary stage T1 grade 3 bladder tumors treated with BCG

Objective:The aim of this study is to find out whether the pseudotumoral lesions (inflammation/granuloma) seen at the follow-up cystoscopy performed three to six months after transurethral resection of primary stage T1 grade 3 bladder tumor and instillations of BCG therapy might have some prognostic value as far as recurrence and/or long term progression are concerned.Material and methods:From the first group of one hundred and thirteen patients with primary stage of T1 grade 3 bladder tumor treated with 81 mg of BCG Connaught (weekly/during six weeks), those with recurrent tumor at the 3rd and 6th month were excluded, so we evaluated 99 patients. We identified 13 patients with cystoscopically pseudotumoral lesions. Results:of the 13 cystoscopically pseudotumoral lesions, we observed recurrence in two cases (15%), while among the rest of the 86 patients, we observed 22 recurrences (26%) (p = 0.9; not significant). Concerning progression, eight cases were reported out of 86 patients (9%) within the cistocopically normal group. No cases of progression were reported among the 13 patients with cystoscopically pseudotumoral lesions. This difference was not statistically significant (p = 0.5).Conclusions:The patients with cystoscopically pseudotumoral lesions (inflammation/granuloma) are a reduced group (13%) with less tendency to recurrence and without progression, even though this relationship is not significant.