D-245 MG Research Articles

Abstract 1488: In vivo magnetic resonance elastography in pediatric brain tumor models

Abstract Refined imaging strategies that could improve diagnosis and management of children with brain malignancies are urgently required. Pediatric brain malignancies possess distinct underlying biologies that discriminate them from adult tumors, even within a common neuropathological diagnosis such as glioblastoma (GBM). Magnetic resonance elastography (MRE) exploits the ability of MRI to visualize the propagation of shear waves resulting from vibrations applied to the cranium to map and quantify (kPa) brain tissue elasticity (Gd) and viscosity (Gl) in vivo. The altered viscoelastic properties of tumors, combined with the sensitivity of MRE for differences in tumor microstructure, establishes MRE as an attractive modality for the detection and differential diagnosis of brain malignancies. Here we aimed to determine whether the potential of MRE in the neuroradiological management of patients with brain malignancies could be extended to the pediatric population. The viscoelastic properties of two pediatric brain tumor models that faithfully emulate high risk childhood disease were investigated: i) Orthotopic D-212 MG pediatric supratentorial giant cell GBM (H3F3A wildtype, 11 year old patient) xenografts propagated in NCr nu/nu mice and ii) GTML/Trp53KI/KI transgenic mice that spontaneously develop aggressive, MYCN driven, p53 depleted, medulloblastomas. D-212 MG and GTML/Trp53KI/KI tumors were less elastic (lower Gd) and viscous (lower Gl), and therefore softer, than the surrounding brain tissue. Both tumor types were also significantly less elastic (D-212 MG Gd = 3.9±0.2; GTML/Trp53KI/KI Gd = 3.5±0.1) than the soft thalamic parenchyma in non-tumor-bearing mice (Gd = 5.9±0.2; p = 0.001 and p = 0.02, respectively, Mann-Whitney), in addition to being less viscous. Interestingly, GTML/Trp53KI/KI tumors demonstrated a bimodal distribution of Gd, which reflected the more marked transition between their relatively stiffer rim and the softer core compared with D-212 MG tumors. We demonstrate that two representative models of major high risk pediatric brain malignancies share the unique softness previously observed in adult brain tumor models, allowing their detection by MRE. This supports observations that pediatric GBMs are morphologically indistinguishable from adult GBMs. Although median Gd values were not sufficient to discriminate between the tumor types, the marked bimodal distribution of Gd in the GTML/Trp53KI/KI tumors was not apparent in D-212 MG GBM tumors. Whilst ongoing histopathological investigation into growth patterns, vascular, cellular and extracellular networks will aid elucidation of the pathological determinants of the bimodal signature, to date unique to GTML/Trp53KI/KI, these data reinforce the potential of MRE for the detection and differential diagnosis of pediatric brain malignancies based on their mechanical properties. Citation Format: Jin Li, Jessica K.R. Boult, Maria Vinci, Sergey Popov, Karen Barker, Zai Ahmad, Yann Jamin, Craig Cummings, Suzanne A. Eccles, Jeffrey C. Bamber, Ralph Sinkus, Louis Chesler, Chris Jones, Simon P. Robinson. In vivo magnetic resonance elastography in pediatric brain tumor models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1488. doi:10.1158/1538-7445.AM2015-1488

Abstract 3271: Novel orthotopic pediatric high grade glioma xenografts evaluated with magnetic resonance imaging mimic human disease

Abstract Pediatric high grade gliomas (pHGG) may arise supratentorially in the cerebral hemispheres, or in midline structures such as the thalamus or the pons (diffuse intrinsic pontine glioma, DIPG). Tumors have distinct underlying biology compared with adult disease, with genetic subtypes corresponding to anatomical location. Differential mutations in genes encoding histone H3.3/H3.1 variants occur in supratentorial and midline tumors, while mutations in the gene encoding the BMP type I receptor ACVR1 are restricted to the pons and occur in a quarter of DIPGs. Co-option of existing brain vasculature in areas of infiltrative tumor growth leaves the blood brain barrier (BBB) intact. It is imperative to accurately model these tumors in vivo for the evaluation of emerging targeted therapeutics. Assessment of such models in situ is vital and requires sensitive functional imaging. T2-weighted (T2-w) MRI of orthotopically implanted PBT7 cells (supratentorial glioblastoma (GBM), 9yr old, H3F3A wt) showed heterogeneous tumors with relatively well defined borders. Hypointensity on T2-w and fluid-attenuated inversion recovery (FLAIR) images suggests the presence of necrosis and edema. Minimal heterogenous enhancement following Gd-DTPA administration demonstrated some loss of BBB integrity. FLAIR hyperintensity is used clinically to identify infiltrative tumor. Serially xenografted D-212 MG cells (supratentorial giant-cell GBM, 11yr old, H3F3A wt) formed tumors with FLAIR hyperintensity principally at the rim and Gd-DTPA enhancement prevalent at the tumor core. Histopathological assessment of H&E staining confirmed the presence of masses with focal invasion at the periphery. Tumors established following pontine injection of HSJD-DIPG-007 cells (6yr old, H3F3A K27M, ACVR1 R206H) were identifiable by T2-w MRI hyperintensity in the pons and cerebellum. Histology confirmed a highly infiltrative tumor throughout the pons, cerebellum and leptomeninges, including the fissures of the cerebellum, which was validated by human nuclear antigen immunohistochemical staining. No extravasation of Gd-DTPA was observed indicating that the BBB remained intact. SU-DIPG-VI cells (6yr old, H3F3A K27M) grew diffusely in the pons with evidence of edema detected by T2-w MRI hyperintensity and FLAIR hypointensity. Diffusely hyperintense T2-w lesions with indistinct boundaries and limited Gd-DTPA extravasation were observed when imaging the diffusely infiltrative tumors resulting from thalamic implantation of QCTB-R059 primary thalamic GBM cells (10yr old, H3F3A K27M). In vivo propagation of primary and serially xenografted pHGG cells results in tumors that possess features of clinical disease, including infiltrative growth and necrosis. There is substantial potential for the use of these models to evaluate preclinical efficacy of novel therapeutics in a range of anatomical and biological subgroups of GBM and DIPG. Citation Format: Jessica K.R. Boult, Kathryn R. Taylor, Maria Vinci, Sergey Popov, Alexa Jury, Valeria Molinari, Marta M. Alonso, Wendy Ingram, Angel M. Caraboso, Michelle Monje, Suzanne A. Eccles, Chris Jones, Simon P. Robinson. Novel orthotopic pediatric high grade glioma xenografts evaluated with magnetic resonance imaging mimic human disease. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3271. doi:10.1158/1538-7445.AM2015-3271

Cellular redox modulator, ortho Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin, MnTnHex-2-PyP(5+) in the treatment of brain tumors.

Despite intensive efforts to improve multimodal treatment of brain tumor, survival remains limited. Current therapy consists of a combination of surgery, irradiation and chemotherapy with predisposition to long-term complications. Identifying novel targeted therapies is therefore at the forefront of brain tumor research. This study explores the utility of a manganese porphyrin in a brain tumor model. The compound used is ortho isomer, mangnese(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin, MnTnHex-2-PyP(5+). It is a powerful SOD mimic and peroxynitrite scavenger and a potent modulator of redox-based cellular transcriptional activity, able to suppress excessive immune and inflammatory responses and in turn proliferative pathways. It is further one of the most lipophilic compound among cationic Mn(III) N-alkylpyridylporphyrins, and thus accumulates predominantly in mitochondria relative to cytosol. In mitochondria, MnTnHex-2-PyP(5+) mimics our key antioxidant system, mitochondrial superoxide dismutase, MnSOD, whose overexpression has been widely shown to suppress tumor growth. Importantly, MnTnHex-2-PyP(5+) crosses blood brain barrier in sufficient amounts to demonstrate efficacy in treating CNS injuries. For those reasons we elected to test its effects in inhibiting brain tumor growth. This study is the first report of the antitumor properties of MnTnHex-2-PyP(5+) as a single agent in adult and pediatric glioblastoma multiforme (D-54 MG, D-245 MG, D-256 MG, D-456 MG) and pediatric medulloblastoma (D-341 MED), and is the first case where a redox-able metal complex has been used in glioma therapy. When given subcutaneously to mice bearing subcutaneous and intracranial xenografts, MnTnHex-2-PyP(5+) caused a significant (P ≤ 0.001) growth delay in D 245 MG, D-256 MG, D-341 MED, and D-456 MG tumors. Growth delay for mice bearing subcutaneous xenografts ranged from 3 days in D-54 MG to 34 days in D-341 MED. With mice bearing intracranial xenografts, MnTnHex-2-PyP(5+) increases median survival by 33% in adult glioblastoma multiforme (D-256 MG; p≤ 0.001) and 173% in pediatric medulloblastoma (D-341 MED, <0.001). The beneficial effects of MnTnHex-2-PyP(5+) are presumably achieved either (1) indirectly via elimination of signaling reactive oxygen and nitrogen species (in particular superoxide and peroxynitrite) which in turn would prevent activation of transcription factors; or (2) directly by coupling with cellular reductants and redox-sensitive signaling proteins. The former action is antioxidative while the latter action is presumably pro-oxidative in nature. Our findings suggest that the use of Mn porphyrin-based SOD mimics, and in particular lipophilic analogues such as MnTnHex-2-PyP(5+), is a promising approach for brain tumor therapy.

Activity of VNP40101M (Cloretazine) in the treatment of CNS tumor xenografts in athymic mice

VNP40101M, or 1,2-bis(methylsulfonyl)-1-(2-choloroethyl)-2-(methylamino)carbonylhydrazine (Cloretazine), is a bifunctional prodrug that belongs to a class of DNA-modifying agents-the sulfonylhydrazines-that has been synthesized and been shown to have activity against a wide spectrum of xenografts. The current study was designed to assess the activity of VNP40101M administered at a dose of 18 mg/kg daily for five days against a panel of human adult and pediatric CNS tumors growing subcutaneously or intracranially in athymic nude mice. The results demonstrated statistically significant (p < 0.05) growth delays of 15.0, 8.3, 51.0, 60+, 60+, and 60+ days in subcutaneous xenografts derived from childhood glioblastoma multiforme (D-456 MG), childhood ependymoma (D-528 EP and D-612 EP), childhood medulloblastoma (D-425 MED), and adult malignant glioma (D-245 MG and D-54 MG), respectively, with corresponding tumor regressions in 10 of 10, 4 of 10, 8 of 10, 9 of 10, 9 of 10, and 10 of 10 treated mice, respectively. Delayed toxicity was seen more than 60 days after treatment, with 23 deaths in 100 treated animals, despite a median weight loss of only 0.06%. In mice bearing intracranial D-245 MG xenografts, treatment with VNP40101M at a dose of 18 mg/kg daily for five days produced a 50% increase in median survival compared with controls. Additional experiments conducted against subcutaneous D-245 MG xenografts by using reduced doses of 13.5 or 9.0 mg/kg daily for five days demonstrated tumor growth delays of 82.2 and 53.5 days, with corresponding tumor regressions in 8 of 9 and 9 of 10 treated mice, respectively (all values, p < 0.001), with one toxic death. These findings suggest that VNP40101M is active in the treatment of a wide range of human central nervous system tumors and warrants translation to the clinic.

Poly(ADP-ribose) polymerase-1 inhibition reverses temozolomide resistance in a DNA mismatch repair–deficient malignant glioma xenograft

Temozolomide is a DNA-methylating agent used in the treatment of malignant gliomas. In this study, we have examined if inhibition of poly(ADP-ribose) polymerase (PARP) could increase the cytotoxicity of temozolomide, particularly in cells deficient in DNA mismatch repair. Athymic mice, transplanted with mismatch repair-proficient [D-245 MG] or deficient [D-245 MG (PR)] xenografts, were treated with a combination of temozolomide and the PARP inhibitor, INO-1001. For the tumors deficient in mismatch repair, the most effective dose of INO-1001 was found to be 150 mg/kg, given i.p. thrice at 4-hour intervals with the first injection in combination with 262.5 mg/kg temozolomide (0.75 LD(10)). This dose of temozolomide by itself induced no partial regressions and a 4-day growth delay. In two separate experiments, the combination therapy increased the growth delay by 21.6 and 9.7 days with partial regressions observed in four of eight and three of nine mice, respectively. The addition of INO-1001 had a more modest, yet statistically significant, increase in tumor growth delay in the mismatch repair-proficient xenografts. In these experiments, mice were treated with a lower amount of temozolomide (88 mg/kg), which resulted in growth delays of 43.1 and 39.2 days. When the temozolomide treatment was in combination with 200 mg/kg INO-1001, there was an increase in growth delay to 48.9 and 45.7 days, respectively. These results suggest that inhibition of PARP may increase the efficacy of temozolomide in the treatment of malignant gliomas, particularly in tumors deficient in DNA mismatch repair.

Human/murine chimeric 81C6 F(ab′) 2 fragment: preclinical evaluation of a potential construct for the targeted radiotherapy of malignant glioma

We have obtained encouraging responses in recent Phase I studies evaluating 131I-labeled human/murine chimeric 81C6 anti-tenascin monoclonal antibody (ch81C6) administered into surgically-created tumor resection cavities in brain tumor patients. However, because the blood clearance is slow, hematologic toxicity has been higher than seen with murine 81C6 (mu81C6). In the current study, a series of paired-label experiments were performed in athymic mice bearing subcutaneous D-245 MG human glioma xenografts to compare the biodistribution of the fragment ch81C6 F(ab′) 2 labeled using Iodogen to a) intact ch81C6, b) mu81C6, and c) ch81C6 F(ab′) 2 labeled using N-succinimidyl 3-[ 131I]iodobenzoate. Tumor retention of radioiodine activity for the F(ab′) 2 fragment was comparable to that for intact ch81C6 for the first 24 h and to that for mu81C6 for the first 48 h; as expected, blood and other normal tissue levels declined faster for ch81C6 F(ab′) 2. Radiation dosimetry calculations suggest that 131I-labeled ch81C6 F(ab′) 2 may warrant further evaluation as a targeted radiotherapeutic for the treatment of brain tumors.

Therapeutic activity of 7-[(2-trimethylsilyl)ethyl)]-20 (S)-camptothecin against central nervous system tumor-derived xenografts in athymic mice.

Camptothecins have emerged as an important new class of antitumor drugs. Camptothecin derivatives such as CPT-11 and topotecan are commercially available and approved for the treatment of colorectal (CPT-11) and ovarian and small-cell lung cancer (topotecan). This study was designed to test the efficacy of karenitecin, a novel highly lipophilic camptothecin derivative, against a panel of human tumor xenografts derived from adult and pediatric central nervous system malignancies growing in athymic nude mice. Xenografts evaluated were derived from childhood high-grade gliomas (D-212 MG, D-456 MG), adult high-grade gliomas (D-54 MG, D-245 MG), medulloblastomas (D-341 MED, D-487 MED), and ependymomas (D-528 EP, D-612 EP), as well as sublines with demonstrated resistance to procarbazine (D-245 MG (PR)) and busulfan (D-456 (BR)). In replicate experiments, karenitecin was given at 1.0 mg/kg per dose via intraperitoneal injection for a period of 10 consecutive days, which is the dosage lethal to 10% of treated animals. Karenitecin produced statistically significant (P < or = 0.001) growth delays in all subcutaneous xenografts tested, including the sublines resistant to procarbazine and busulfan. Growth delays ranged from 12.1 days in D-456 MG (BR) to 90+ days in D-212 MG and D-341 MED. Karenitecin also produced statistically significant (P < or = 0.001) increases in survival of animals bearing D-341 MED intracranial xenografts (69% increase) and those bearing D-456 MG xenografts (62% increase). These preclinical studies confirm that karenitecin is active against human central nervous system xenografts and should undergo clinical evaluation in patients with malignant central nervous system tumors.

O 6 -Benzylguanine-mediated enhancement of nitrosourea activity in Mer − central nervous system tumor xenografts - implications for clinical trials

To evaluate the role of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) plus O6-benzylguanine (O6-BG) in the treatment of both Mer+ and Mer- tumors. The effect of pretreatment with O6-BG on the activity of BCNU against Mer- human central nervous tumor xenografts D-54 MG and D-245 MG was evaluated in athymic nude mice. BCNU (1.0 LD10; dose lethal to 10% of treated animals) produced growth delays of 8.9 days and 7.5 days and tumor regressions in six of ten and one of nine animals against D-54 MG, which was derived from a human malignant glioma xenograft. Dose reduction of BCNU to 0.38 LD10 eliminated antitumor activity. The combination of BCNU (0.38 LD10) plus O6-BG produced growth delays of 8.8 days and 7.9 days, with tumor regressions in four of ten and two of nine animals, respectively. BCNU (1.0 LD10) produced a growth delay of 49.8 days and ten of ten tumor regressions against D-245 MG, which was derived from a glioblastoma multiforme. BCNU (0.38 LD10) produced a growth delay of 19.4 days, with nine of ten tumor regressions. The combination of BCNU (0.38 LD10) plus O6-BG produced a growth delay of 65.7 days and seven of eight tumor regressions. These results suggest that the combination of BCNU plus O6-BG may be a rational intervention for both Mer+ as well as Mer- tumors.

Intrathecal busulfan treatment of human neoplastic meningitis in athymic nude rats.

The current study was designed to evaluate the toxicity and activity of Spartaject Busulfan, a microcrystalline preparation of busulfan, following its intrathecal administration into a nude rat model of human neoplastic meningitis. Animals were treated through permanent indwelling subarachnoid catheters. Human glioma D-456 MG growing in the subarachnoid space was treated with 8.1 micromol of intrathecal Spartaject Busulfan. Single-dose therapy was also subsequently compared with 4 doses of 8.1 and 2.0 micromol busulfan, respectively, against D-456 MG neoplastic meningitis. Additional experiments evaluated a saline control versus 8.1 micromol x 1, 6.2 micromol x 4 and 4.1 micromol x 4, respectively, against D-456 MG. A single dose of 8.1 micromol of intrathecal Spartaject Busulfan resulted in an increase in median survival of 61.7% compared with the saline control. In experiment 2, all busulfan treatments showed increases in median survival of 142.8% (8.1 micromol x 1), 52.3% (2.0 micromol x 4), and 23% (8.1 micromol x 4) (p < 0.001 for all groups) compared with the saline control. These results suggest that a narrow therapeutic dose range for both toxicity and activity has been defined for intrathecal busulfan in the treatment of human neoplastic meningitis in athymic nude rats. Although busulfan has only limited activity against solid tumors, the high doses achievable in the CSF following intrathecal administration coupled with the steep dose-response relationships of alkylating agents, provide rationale for further evaluation of this agent.

Therapeutic efficacy of vinorelbine against pediatric and adult central nervous system tumors.

The activity of vinorellbine, a new semisynthetic vinca alkaloid, was evaluated against a battery of human tumor xenografts derived from adult and pediatric CNS malignancies. Tumors included adult high-grade gliomas (D-54 MG, D-245 MG), childhood high-grade gliomas (D-212 MG, D-456 MG), medulloblastomas (D-341 MED, D-487 MED), ependymomas (D-612 EP, D-528 EP), and a mismatch repair-deficient procarbazine-resistant glioma [D-245 MG (PR)]. Tumors were grown subcutaneously in athymic nude mice and vinorelbine was administered at a dose of 11 mg/kg on days 1, 5, and 9. Additionally, vinorelbine was also administered in combination with BCNU against D-54 MG. Vinorelbine produced statistically significant growth delays in D-456 MG, D-245 MG, and D-245 MG (PR). No statistically significant growth delays were observed in D-54 MG, D-487 MED, D-212 MG, D-528 EP, D-341 MED or D-612 EP. The antitumor effects of the vinorelbine/BCNU combination were additive. Growth delays observed in the procarbazine-resistant line [D-245 MG (PR)] were greater than twofold the delays seen in the parent line (D-245 MG). Vincristine was equally potent against D-245 MG and D-245 MG (PR). Taxol demonstrated little activity against D-245 MG but produced 32- and 18-day growth delays in D245 MG (PR). These studies indicate that vinorelbine possesses antitumor activity against several glioma tumor xenografts with marked activity in a mismatch repair deficient-tumor.

Enhancement of irinotecan (CPT-11) activity against central nervous system tumor xenografts by alkylating agents.

Two major obstacles in the treatment of patients with central nervous system malignancies are drug resistance and host toxicity. The goal of combination chemotherapy is to achieve therapeutic effects that are more favorable than using a single drug alone, but without an increase in normal organ toxicity. The study reported here examined the combination of a topoisomerase I inhibitor, irinotecan (CPT-11), with three different alkylating agents: 1,3-bis(2-chloroethyl)-1-nitrosourea, busulfan, and cyclophosphamide. We evaluated the antitumor effects of these three combinations against a panel of human tumor xenografts derived from central nervous system malignancies, including adult high-grade gliomas (D-54 MG, D-245 MG) and a childhood ependymoma (D-612 EP). In replicate experiments, the alkylating agents were given on day 1 in doses varying from 10% to 75% of the dose lethal to 10% of the animals, and CPT-11 was given on days 1-5 and 8-12 in doses varying from 10% to 100% of the dose lethal to 10% of the animals. The antitumor effects of the various combinations ranged from less than additive (7.61 days below additive with 0.5 CPT-11 + 0.75 cyclophosphamide in D-54 MG) to statistically significant (P < 0.001) supraadditive effects (18.80 days above additive with 0.5 CPT-11 + 0.5 1,3-bis(2-chloroethyl)-1-nitrosourea in D-54 MG). These studies show that the combination of the topoisomerase inhibitor CPT-11 and alkylating agents may increase the antitumor effect in some cases well above additive with no increase in host toxicity (0/10 deaths in both experiments cited above) and should be considered for combination chemotherapy of central nervous system malignancies.

Characterization of the mechanisms of busulfan resistance in a human glioblastoma multiforme xenograft.

Busulfan is an alkylating agent commonly used in the treatment of chronic myelogenous leukemia and in combination with cyclophosphamide in preparation for allogeneic bone marrow transplantation. Serial treatment of a childhood high-grade glioma xenograft (D-456 MG) with busulfan resulted in a busulfan-resistant xenograft, D-456 MG(BR). Cross-resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea was seen but not resistance to cyclophosphamide or CPT-11. Cytoplasmic levels of glutathione in D-456 MG(BR) were approximately one-half those found in D-456 MG. This depletion could not be explained by levels of glutathione-S-transferase, or by amplification, rearrangement, or increased levels of transcript of gamma-glutamylcysteine synthetase. Furthermore, depletion of glutathione in D-456 MG did not alter busulfan activity. Quantitation of busulfan levels in D-456 MG and D-456 MG(BR) xenografts following treatment of mice at the dose lethal to 10% of the animals demonstrated that significantly lower levels of drug were achieved in D-456 MG(BR). These studies suggest that alterations in drug transport or metabolism of busulfan may play a role in the resistance of D-456 MG(BR) to this alkylator.

Intraarterial O6-benzylguanine enables the specific therapy of nitrosourea-resistant intracranial human glioma xenografts in athymic rats with 1,3-bis(2-chloroethyl)-1-nitrosourea.

The prognosis for patients with malignant gliomas continues to be dismal. The high degree of resistance of gliomas to nitrosourea-based chemotherapy is one major factor in poor treatment outcome. The identification of O6-alkylguanine-DNA alkyltransferase (AGAT) as a major determinant of nitrosourea resistance has resulted in the development of several agents to inactivate this repair protein and counteract tumor cell resistance. However, a major problem in preclinical trials has been the marked nitrosourea dose limitations imposed by the prior administration of AGAT-depleting agents. We investigated the AGAT depletion and selective enhancement of BCNU activity of intraarterial (i.a.) O6-benzylguanine (O6BG) in the human malignant glioma xenograft D-456 MG growing intracranially (i.e.) in athymic rats. Whereas i.a. O6BG at 2.5 mg/kg produced 100% inhibition of D-456 MG AGAT i.e. activity 8 h after administration, intraperitoneal (i.p.) O6BG at this dose produced only 40% inhibition, requiring dose escalation to 10 mg/kg to produce 100% AGAT depletion. Prior administration of i.p. O6BG (10 mg/kg) and i.a. O6BG (2.5 mg/kg) limited maximum tolerated intravenous (i.v.) BCNU doses (37.5 mg/kg when given alone) to 6.25 and 25 mg/kg, respectively. Higher doses of BCNU alone or in combination with O6BG produced histopathologic evidence of cerebral and hepatic toxicity. Therapy experiments revealed a significantly improved median survival for rats treated with O6BG i.a. (2.5 mg/kg) plus BCNU i.v. (25 mg/kg, days 61 and 59 in duplicate experiments) compared with saline (day 21. P = 0.001). O6BG i.a. or i.p. (days 22 and 23, P = 0.001), BCNU i.v. (37.5 mg/kg, day 29, P = 0.001), and O6BG i.p. (10 mg/kg), plus BCNU i.v. (6.25 mg/kg, day 37, P < 0.001). Therefore, O6BG i.a., by virtue of rapid AGAT depletion and selective uptake into i.c. tumors, offers significant potential for regional chemomodulation of AGAT-mediated nitrosourea resistance in malignant human gliomas with concomitant reduction of systemic toxicity.

Therapeutic efficacy of the topoisomerase I inhibitor 7-ethyl-10-(4-[1-piperidino]-1-piperidino)-carbonyloxy-camptothecin against pediatric and adult central nervous system tumor xenografts.

Therapy of patients with malignant central nervous system tumors is frequently unsuccessful, reflecting limitations of current surgical, radiotherapeutic, and pharmacotherapeutic treatments. The camptothecin derivative irinotecan (CPT-11) has been shown to possess antitumor activity in phase II trials for patients with carcinoma of the lung, cervix, ovary, colon, or rectum and for patients with non-Hodgkin's lymphoma. The current study was designed to test the efficacy of the drug against a panel of human tumor xenografts derived from adult and pediatric central nervous system malignancies. Tumors included childhood high-grade gliomas (D-212 MG, D-456 MG), adult high-grade gliomas (D-54 MG, D-245 MG), medulloblastomas (D341 Med, D487 Med), ependymomas (D528 EP, D612 EP), and a rhabdomyosarcoma (TE-671), as well as sublines with demonstrated resistance to busulfan (D-456 MG (BR)), cyclophosphamide (TE-671 CR), procarbazine (D-245 MG (PR)) or melphalan (TE-671 MR), growing subcutaneously and intracranially in athymic nude mice. In replicate experiments, CPT-11 was given at a dosage of 40 mg/kg per dose via intraperitoneal injection in 10% dimethylsulfoxide on days 1-5 and 8-12, which is the dosage lethal to 10% of treated animals. CPT-11 produced statistically significant (P < 0.001) growth delays in all subcutaneous xenografts tested, including those resistant to busulfan, cyclophosphamide, procarbazine, and melphalan, with growth delays ranging from 21.3 days in D487 Med to 90+ days in several tumor lines. Further, tumor regression was evident in every treated animal bearing a subcutaneous tumor, with some xenografts yielding complete tumor regression. Statistically significant (P < 0.001) increases in survival were demonstrated in the two intracranial xenografts-D341 EP (73.0% increase) and D-456 MG (114.2% increase)-treated with CPT-11. These studies demonstrate that, of over 40 drugs evaluated in this laboratory, CPT-11 is the most active against central nervous system xenografts and should be advanced to clinical trial as soon as possible.

Activity of 9-dimethylaminomethyl-10-hydroxycamptothecin against pediatric and adult central nervous system tumor xenografts

The activity of dimethylaminomethyl-10-hydroxycamptothecin (topotecan) was evaluated against a panel of xenografts derived from ependymomas (D528 EP, D612 EP), childhood high-grade gliomas (D-456 MG, D-212 MG), adult high-grade gliomas (D-245 MG, D-54 MG), and medulloblastomas (D425 Med) growing s.c. and i.c. (intracranially) in athymic nude mice. Topotecan was given at a dose of 1.9 mg/kg by i.p. injection in 0.9% saline using a volume of 90 ml/m2 on days 1-5 and 8-12, which represents the dose lethal to 10% of treated animals. Topotecan was active in the therapy of all s.c. xenografts tested, with growth delays ranging from 6.3 days in D-54 MG to 55.7 days in D528 EP. Topotecan produced statistically significant tumor regressions in D425 Med, D-456 MG, D-245 MG, D528 EP, and D612 EP. No tumor regression was seen in any control animal. Statistically significant increases in median survival were seen in the two i.c. xenografts--D-456 MG (28.6% increase) and D-54 MG (39% increase)--treated with topotecan. These studies suggest that topotecan may be an important new addition to the therapy of central nervous system tumors.

Busulfan therapy of central nervous system xenografts in athymic mice

We evaluated the antitumor activity of busulfan against a panel of tumor cell lines and xenografts in athymic nude mice derived from childhood high-grade glioma, adult high-grade glioma, ependymoma, and medulloblastoma. Busulfan displayed similar activity against a panel of four medulloblastoma cell lines (D283 Med, Daoy, D341 Med, and D425 Med) and four corresponding sublines with laboratory-generated or clinically acquired resistance to 4-hydroperoxycyclophosphamide [D283 Med (4-HCR), Daoy (4-HCR), D341 Med (4-HCR), and D458 Med] and cross-resistance to melphalan. This is consistent with a nearly total lack of cross-resistance of busulfan to 4-hydroperoxycyclophosphamide. Busulfan was active in the therapy of all but one of the subcutaneous xenografts tested, with growth delays ranging from 14.3 days in D612 EP to 58.4 days in D528 EP. Busulfan produced statistically significant increases in the median survival of mice bearing intracranial D456 MG (66%-90%), D612 EP (18%-33%), and D528 EP (89%) xenografts. These studies suggest that busulfan may be active against medulloblastomas, high-grade gliomas, and ependymomas as well as against cyclophosphamide-resistant neoplasms.

Enhancement of nitrosourea activity in medulloblastoma and glioblastoma multiforme.

Although chemotherapy offers promise of increased survival for children with medulloblastoma and glioblastoma multiforme, drug resistance occurs frequently, resulting in tumor progression and death. Resistance to nitrosoureas and methylating agents, which damage DNA, can be mediated by a DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGAT). Depletion of this protein with alkylguanines or methylating agents, however, restores tumor cell sensitivity to the cytotoxicity of chloroethylnitrosoureas (e.g., carmustine [BCNU]). This study was designed to determine whether resistance to the activity of nitrosourea (the drug BCNU) in BCNU-resistant human medulloblastoma (D341 Med) and human glioblastoma multiforme (D-456 MG) can be reversed by the methylating agent streptozocin and the O6-substituted guanines O6-methylguanine and O6-benzylguanine. Xenografts were grown subcutaneously in athymic BALB/c mice. BCNU was administered as a single intraperitoneal injection at doses of 100 mg/m2, 75 mg/m2, or 38 mg/m2--i.e., 1.0, 0.75, or 0.38, respectively, of the dose lethal to 10% of treated animals (LD10). Mice were treated intraperitoneally with a single dose of O6-benzylguanine or O6-methylguanine (240 mg/m2) or with streptozocin (600 mg/m2) daily for 4 days. Response was assessed by tumor growth delay and tumor regression. AGAT activity in the xenografts was measured at 1 and 6 hours after pretreatment, at the time tumors were excised. Pretreatment with O6-benzylguanine, O6-methylguanine, or streptozocin reduced AGAT activity to 4%, 25%, and 95% of control values, respectively, in D341 Med and 0%, 0%, and 25% of control values, respectively, in D-456 MG 1 hour after injection. After 6 hours, levels changed to 7%, 61%, and 116% of control values in D341 Med and 0%, 79%, and 21% of control values in D-456 MG, respectively. Both D341 Med and D-456 MG xenografts were completely resistant to BCNU at its LD10. Pretreatment with O6-benzylguanine increased BCNU sensitivity in both types of xenograft. In contrast, treatment with BCNU plus O6-methylguanine or streptozocin did not produce growth delays substantially different from those produced by BCNU alone, reflecting the more efficient depletion of AGAT by O6-benzylguanine. Following therapy with BCNU plus O6-benzylguanine at 0.38 LD10, tumor regressions were seen in eight of 10 D341 Med and in all 10 D-456 MG xenografts. We recommend comprehensive clinical toxicologic evaluation of combination therapy with O6-benzylguanine plus BCNU, which would allow subsequent design of phase I clinical trials.

Kinetics and glial fibrillary acidic (GFA) protein production in a transplantable human giant cell glioblastoma (D‐212 MG) of near haploid karyotype maintained in an organ culture system. An immunohistochemistry study

A transplantable subcutaneous tumour (designated D-212 MG), sequentially passaged in athymic nude mice and originally derived from a human giant cell glioblastoma, was maintained in an organ culture (matrix) system and studied immunohistochemically after in vitro pulse-labelling with bromodeoxyuridine (BrdU) and for the presence of glial fibrillary acidic (GFA) protein, after 1, 2 and 3 weeks in culture. The histological characteristics of the tumour, showing two cell populations of giant multinucleated cells and small cells, were preserved in the explants. An increased percentage of multinucleated giant cells was found after 3 weeks in vitro. A small but constant fraction (4-6%) of these cells continued to synthesize DNA. The labelling index of the small cells was somewhat higher, but decreased slightly although significantly over the 3-week period in vitro (from approximately 10.5 to 8%). The percentage of small cells that were positive for GFA protein was in the region of 75% and that of the giant multinucleated cells was in the region of 45%; it did not change significantly during the 3 weeks in vitro. The in vitro results confirm the astrocytic nature of both the small cells and the giant multinucleated cells in this tumour, the capacity of both cell populations to synthesize DNA in culture and to demonstrate invasiveness, and suggest the possibility that some of the giant multinucleated cells may have originated from the conversion of a number of small tumour cells.

Specific chromosomal abnormalities characterize four established cell lines derived from malignant human gliomas.

The four permanent human glioma-derived cell lines reported here are the first such lines for which the karyotypes have been followed from the original biopsies through the establishment of the lines in culture. Although ploidy changes were seen, each line retained either distinctive marker chromosomes or the overall original chromosomal distribution allowing the origin of each line to be established with certainty. D-263 MG expresses glial fibrillary acidic protein, all lines except D-245 MG are tumorigenic in athymic mice, and each line displays a unique pattern with respect to in vitro growth parameters and expression of biochemically defined markers, oncofetal antigens and lymphoid-associated markers. D-245 MG and D-259 MG are able to grow in the absence of supplemental glutamine; glutamine synthetase was detected in these cell lines both by immunocytochemistry and by direct assay. Thus, the four permanent human glioma-derived cell lines described here are representative of glioma lines in their general characteristics. D-259 MG retains numerous double minute chromosomes (DMs), D-263 MG contains two marker chromosomes with breaks in 9p, and D-247 MG and D-245 MG with stemlines containing 96 and 89 chromosomes contain eight and six normal copies (respectively) of chromosome No. 7. The retention in these four cell lines of the most common chromosomal abnormalities seen in biopsies of malignant human gliomas provides the opportunity to investigate the meaning of these specific chromosomal changes.