Phase I trial results of iodine-131-labeled antitenascin monoclonal antibody 81C6 treatment of patients with newly diagnosed malignant gliomas.

PURPOSE: To determine the maximum-tolerated dose (MTD) of iodine-131 ((131)I)-labeled 81C6 antitenascin monoclonal antibody (mAb) administered clinically into surgically created resection cavities (SCRCs) in malignant glioma patients and to identify any objective responses with this treatment. PATIENTS AND METHODS: In this phase I trial, newly diagnosed patients with malignant gliomas with no prior external-beam therapy or chemotherapy were treated with a single injection of (131)I-labeled 81C6 through a Rickham reservoir into the resection cavity. The initial dose was 20 mCi and escalation was in 20-mCi increments. Patients were observed for toxicity and response until death or for a minimum of 1 year after treatment. RESULTS: We treated 42 patients with (131)I-labeled 81C6 mAb in administered doses up to 180 mCi. Dose-limiting toxicity was observed at doses greater than 120 mCi and consisted of delayed neurotoxicity. None of the patients developed major hematologic toxicity. Median survival for patients with glioblastoma multiforme and for all patients was 69 and 79 weeks, respectively. CONCLUSION: The MTD for administration of (131)I-labeled 81C6 into the SCRC of newly diagnosed patients with no prior radiation therapy or chemotherapy was 120 mCi. Dose-limiting toxicity was delayed neurologic toxicity. We are encouraged by the survival and toxicity and by the low 2.5% prevalence of debulking surgery for symptomatic radiation necrosis.

Dosimetry and dose-response relationships in newly diagnosed patients with malignant gliomas treated with iodine-131-labeled anti-tenascin monoclonal antibody 81C6 therapy.

PURPOSE: The objective of this study was to perform the dosimetry and evaluate the dose-response relationships in newly diagnosed patients with malignant brain tumors treated by direct injections of (131)I-labeled 81C6 monoclonal antibody (MAb) into surgically created resection cavities (SCRCs). METHODS AND MATERIALS: Absorbed doses to the 2-cm-thick shell as measured from the margins of the resection cavity interface were estimated for 42 patients with primary brain tumors. MR images were used to assess the enhanced-rim volume as a function of time after radiolabeled MAb therapy. Biopsy samples were obtained from 15 patients and 1 autopsy. RESULTS: The average absorbed dose [range] to the 2-cm shell region was 32 [3-59] Gy. For the endpoint of minimal time to MR contrast enhancement, the optimal absorbed dose and initial dose-rate were 43 +/- 16 Gy and 0. 41 +/- 0.10 Gy/h, respectively. There was a correlation between the absorbed dose and dose rate to the shell region and biopsy outcome (tumor recurrence, radionecrosis, and tumor recurrence and/or radionecrosis). In this Phase I study, the maximum tolerated dose (MTD) was 120 mCi. At this MTD, the estimated average absorbed dose and initial dose rate to the 2-cm shell were 41 [9-89] Gy and 0.51 [0.24-1.13] Gy/h, respectively. These values are in agreement with the optimal values based on the time to MR lesion rim enhancement. CONCLUSIONS: The average absorbed dose to the 2-cm shell region varied considerably and mainly depended on cavity volume. In future clinical trials, the administered activity of (131)I-labeled 81C6 MAb may be adjusted based on cavity volume in order to deliver the optimal absorbed dose of 43 Gy rather than giving a fixed administered activity.

125I-labeled anti-epidermal growth factor receptor-vIII single-chain Fv exhibits specific and high-level targeting of glioma xenografts.

A single-chain antibody fragment, MR1(scFv), with specific binding to epidermal growth factor receptor-vIII (EGFRvIII), was produced, radiolabeled, and evaluated for biodistribution in human glioma-bearing athymic mice. The mutant receptor EGFRvIII has a deletion in its extracellular domain that results in the formation of a new, tumor-specific antigen found in glioblastomas, breast carcinomas, and other tumors. The scFv molecule, designed as V(H)-(Gly4-Ser)3-V(L), was expressed in Escherichia coli in inclusion body form; recovered scFv fragments were properly refolded in redox-shuffling buffer. Size-exclusion chromatography of purified scFv demonstrated a protein monomer of Mr 26,000. Labeling was performed using N-succinimidyl 5-[125I]iodo-3-pyridinecarboxylate (SIPC) or Iodogen to specific activities of 0.5-2.0 mCi/mg, with yields of 35-50% and 45-70%, respectively. The immunoreactive fraction (IRF) of the labeled MR1(scFv) was 65-80% when SIPC was used and 50-55% when Iodogen was used. The affinity (K(A)) of MRI(scFv) for EGFRvIII was 4.3 x 10(7) +/- 0.1 x 10(7) M(-1) by BIAcore analysis, and it was 1.0 x 10(8) +/- 0.1 x 10(8) M(-1) and by Scatchard analysis versus EGFRvIII-expressing cells. After incubation at 37 degrees C for 24 h, the binding affinity was maintained, and the IRF was maintained at 60-70%. The specificity of MR1(scFv) for EGFRvIII was demonstrated in vitro by incubation of radiolabeled MR1(scFv) with the EGFRvIII-expressing U87MG.deltaEGFR cell line in the presence or absence of competing unlabeled MR1(scFv) or anti-EGFRvIII MAbs L8A4 and H10. In biodistribution studies using athymic mice bearing s.c. U87MG.deltaEGFR tumor xenografts, animals received intratumoral or i.v. infusions of paired-label [125I]SIPC-MR1(scFv) and [131I]SIPC-anti-Tac(scFv) as a control. When given by the intratumoral route, MR1(scFv) retained high tumor uptakes of 85% injected dose per gram of tissue at 1 h and 16% injected dose per gram of tissue at 24 h following administration. Specific: control scFv tumor uptake ratios of more than 20:1 at 24 h demonstrated specific localization of MR1(scFv). The excellent tumor retention of MR1(scFv), combined with its rapid clearance from normal tissues, resulted in high tumor:normal organ ratios.

Iodine-131-labeled antitenascin monoclonal antibody 81C6 treatment of patients with recurrent malignant gliomas: phase I trial results.

PURPOSE: To determine the maximum-tolerated dose (MTD) of iodine 131 (131I)-labeled 81C6 monoclonal antibody (mAb) in brain tumor patients with surgically created resection cavities (SCRCs) and to identify any objective responses to this treatment. METHODS: In this phase I trial, eligible patients were treated with a single injection of 131I-labeled 81C6. Cohorts of three to six patients were treated with escalating dosages of 131I (starting dose of 20 mCi with a 20-mCi escalation in subsequent cohorts) administered through an Ommaya reservoir in the SCRC. Patients were followed up for toxicity and response until death or for a minimum of 1 year after treatment. The SCRC patients, who were previously irradiated, were followed up without additional treatment unless progressive disease was identified. RESULTS: We administered 36 treatments of 131I doses up to 120 mCi to 34 previously irradiated patients with recurrent or metastatic brain tumors. Dose-limiting toxicity was reached at 120 mCi and was limited to neurologic or hematologic toxicity. None of the patients treated with less than 120 mCi developed significant neurologic toxicity; one patient developed major hematologic toxicity (MHT). The estimated median survival for patients with glioblastoma multiforme (GBM) and for all patients was 56 and 60 weeks, respectively. CONCLUSION: The MTD for administration of 131I-labeled 81C6 into the SCRCs of previously irradiated patients with recurrent primary or metastatic brain tumors was 100 mCi. The dose-limiting toxicity was neurologic toxicity. We are encouraged by the minimal toxicity and survival in this phase I trial. Radiolabeled mAbs may improve the current therapy for brain tumor patients.

In vitro and in vivo behavior of radiolabeled chimeric anti-EGFRvIII monoclonal antibody: comparison with its murine parent.

The mutant version of the epidermal growth factor receptor EGFRvIII has been found on gliomas and other tumors, but not on normal tissues. Radioiodinated murine (mu) L8A4 monoclonal antibody (MAb) specifically targets EGFRvIII xenografts in vivo when labeled using N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC). A chimeric (ch) MAb consisting of the variable region of muL8A4 and the constant domains of human IgG2 has been developed that has an affinity and radioiodinated immunoreactive fraction comparable to muL8A4. In vitro, both MAbs were internalized and processed by EGFRvIII expressing cell lines (U87MG delta EGFR or NR6M) at similar rates (maximum intracellular retention, 35-40%). In paired-label tissue distribution studies in athymic mice bearing U87MG delta EGFR tumor xenografts, the ch:mu L8A4 uptake ratio in normal tissues rose to greater than 2:1, whereas in tumor, the ratio remained 1:1 throughout the experiment. These results indicate that chL8A4 exhibits similar binding and internalization properties as its murine parent, but suggest different intracellular processing and/or deposition of catabolites in normal tissues for chL8A4.

Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: targeting with a single chain antibody variable domain isolated by phage display.

EGFRvIII is a mutant epidermal growth factor receptor found in glioblastoma, and in carcinoma of the breast, ovary, and lung. The mutant receptor has a deletion in its extracellular domain that results in the formation of a new, tumor-specific extracellular sequence. Mice were immunized with a synthetic peptide corresponding to this sequence and purified EGFRvIII. A single chain antibody variable domain (scFv) phage display library of 8 x 10(6) members was made from the spleen of one immunized mouse. A scFv specific for EGFRvIII was isolated from this library by panning with successively decreasing amounts of synthetic peptide. This was used to make an immunotoxin by fusing the scFv DNA sequence to sequences coding for domains II and III of Pseudomonas exotoxin A. Purified immunotoxin had a Kd of 22 nM for peptide and a Kd of 11 nM for cell-surface EGFRvIII. The immunotoxin was very cytotoxic to cells expressing EGFRvIII, with an IC50 of 1 ng/ml (16 pM) on mouse fibroblasts transfected with EGFRvIII and an IC50 of 7-10 ng/ml (110-160 pM) on transfected glioblastoma cells. There was no cytotoxic activity at 1000 ng/ml on the untransfected parent glioblastoma cell line. The immunotoxin was completely stable upon incubation at 37 degrees C for 24 h in human serum. The combination of good affinity, cytotoxicity and stability make this immunotoxin a candidate for further preclinical evaluation.

Intrathecal 131I-labeled antitenascin monoclonal antibody 81C6 treatment of patients with leptomeningeal neoplasms or primary brain tumor resection cavities with subarachnoid communication: phase I trial results.

We aimed to determine the maximum tolerated dose (MTD) of 131I-labeled 81C6 in patients with leptomeningeal neoplasms or brain tumor resection cavities with subarachnoid communication and to identify any objective responses. 81C6 is a murine IgG monoclonal antibody that reacts with tenascin in gliomas/carcinomas but does not react with normal adult brain. 131I-labeled 81C6 delivers intrathecal (IT) radiation to these neoplasms. This study was a Phase I trial in which patients were treated with a single IT dose of 131I-labeled 81C6. Cohorts of three to six patients were treated with escalating doses of 131I (starting dose, 40 mCi; 20 mCi escalations) on 10 mg 81C6. MTD is defined as the highest dose resulting in serious toxicity in no more than two of six patients. Serious toxicity is defined as grade III/IV nonhematological toxicity or major hematological toxicity. We treated 31 patients (8 pediatric and 23 adult). Eighteen had glioblastoma multiforme. Patients were treated with 131I doses from 40 to 100 mCi. Hematological toxicity was dose limiting and correlated with the administered 131I dose. No grade III/IV nonhematological toxicities were encountered. A partial response occurred in 1 patient and disease stabilization occurred in 13 (42%) of 31 patients. Twelve patients are alive (median follow-up, > 320 days); five are progression free >409 days median posttreatment. The MTD of a single IT administration of 131I-labeled 81C6 in adults is 80 mCi 131I-labeled 81C6. The MTD in pediatric patients was not reached at 131I doses up to 40 mCi normalized for body surface area.

Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas.

Despite molecular biological advances in understanding human cancers, translation into therapy has been less forthcoming; targeting neoplastic cells still requires that tumor-specific markers, preferably those on the cell surface, be identified. The epidermal growth factor receptor (EGFR) exists in a deletion-mutant form, EGFRvIII, which has been identified by genetic and immunological means in a subset of gliomas and non-small cell lung carcinomas. Specific polyvalent antisera to the extracellular portion of the variant were readily induced, but immunization using a synthetic linear peptide representing the unique EGFRvIII primary sequence has been unsuccessful in mice or macaques. We report here five specific monoclonal antibodies (mAbs) developed through long-term immunization protocols using the EGFRvIII-specific synthetic peptide and the intact variant in different formats that maintained secondary and tertiary conformation. These mAbs identify the EGFRvIII on the cell surface with relatively high affinity (KA range, 0.13 to 2.5 x 10(9) M-1) by live cell Scatchard analysis. These mAbs are specific for EGFRvIII as determined by RIA, ELISA, Western blot, analytical flow cytometry, autophosphorylation, and immunohistochemistry. Isolating specific mAbs enabled us to analyze normal and neoplastic human tissue and establish that EGFRvIII is truly tumor specific for subsets of breast carcinomas and for previously reported non-small cell lung carcinomas and gliomas. Also, this receptor is not expressed by any normal human tissues thus far examined, including elements of the peripheral, central nervous, and lymphoid systems. With mAbs, we identified a higher incidence of EGFRvIII positivity in gliomas than previously described and identified an EGFRvIII-positive subset of breast tumors; also, we observed that the EGFRvIII epitope is not expressed in normal tissues, and we demonstrated the localizing and therapeutic potential of the mAbs for tumors expressing this epitope. Our observations strongly warrant development of this mAb-antigen system as therapy for breast, lung, and central nervous system tumors.

Phase I studies of treatment of malignant gliomas and neoplastic meningitis with 131I-radiolabeled monoclonal antibodies anti-tenascin 81C6 and anti-chondroitin proteoglycan sulfate Me1-14 F (ab')2--a preliminary report.

The advent of monoclonal antibody (MAb) technology has made Ehrlich's postulate of the 'magic bullet' an attainable goal. Although specific localization of polyvalent antibodies to human gliomas was demonstrated in the 1960s, the lack of specific, high affinity antibody populations and of defined target antigens of sufficient density precluded therapeutic applications. Not until the identification of operationally specific tumor-associated antigens (present in tumor tissue but not normal central nervous system tissue); production of homogeneous, high affinity MAbs to such antigens; and the use of compartmental administration (intrathecal or intracystic), has the promise of passive immunotherapy of primary and metastatic central nervous system neoplasms been recognized. We report here preliminary data from Phase I studies of the compartmental administration of the anti-tenascin MAb 81C6 and F(ab2)2 fragments of MAb Me1-14, which recognizes the proteoglycan chondroitin sulfate-associated protein of gliomas and melanomas, to patients with primary central nervous system tumors or tumors metastatic to the central nervous system. Phase I dose escalation studies of intracystically administered 131I-labeled anti-tenascin MAb 81C6 to either spontaneous cysts of recurrent gliomas or surgically created cystic resection cavities have resulted in striking responses. Of five patients with recurrent cystic gliomas treated, four had partial responses, clinically or radiographically. Similarly, in patients with surgically created resection cavities, a partial response at the treatment site and extended stable disease status has been obtained following intracystic administration of 131I-labeled 81C6. No evidence of hematologic or neurologic toxicity has been observed in either patient population, with the exception of transient exacerbation of a pre-existing seizure disorder in a single patient. Dosimetry calculations indicated high intracystic retention for four to six weeks with little or no systemic dissemination; estimated total doses intracystically ranged from 12,700-70,290 rad. Intrathecal administration of labeled MAbs to patients with neoplastic meningitis is more difficult to assess in terms of clinical responsiveness. Of patients so treated with either 131I-labeled 81C6 or 131I-labeled Me1-14 (F(ab)2, cerebrospinal fluid and radiographic responses have been achieved, and survival prolongation through maintenance of stable disease has been observed in several cases. Initial results from pHase I dose escalation trials are encouraging in terms of the proportion of cases of disease stabilization and partial and complete responses obtained. Importantly, neurotoxicity has been virtually nonexistent, and hematologic toxicity rare and rapidly responsive to treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Mouse/human chimeric Me1-14 antibody: genomic cloning of the variable region genes, linkage to human constant region genes, expression, and characterization.

Murine monoclonal antibody Me1-14, which recognizes an epitope on chondroitin proteoglycan sulfate expressed in malignant glioma and melanoma, has been used for radioimmunolocalization and therapy both in animal models and in patients. Here, we report the generation, characterization, and in vivo biodistribution of mouse/human chimeric Me1-14. Rearranged immunoglobulin genes from the Me1-14 hybridoma were identified by Southern blot analysis. Putative rearranged light- and heavy-chain genes were cloned from Lambda-ZapII Me1-14 genomic libraries and sequenced for nucleotide analysis. One of the putative heavy-chain Eco RI fragments (3.5 kb) had all the features of an intact variable region, including a functional leader sequence, in-frame V-D and D-J junctions, and cysteines 22 and 92. The deduced amino acid sequence from the heavy-chain variable region gene showed considerable homology with the invariant protein sequence of the mouse heavy-chain subgroup IIIB. Like the heavy-chain gene, one of the putative rearranged kappa-chain Hind III fragments (4 kb) had all of the characteristics of the functional variable region, and the deduced amino acid sequence showed homology to the invariant sequence of kappa-chain group V. The variable region genes for heavy- and light-chains were linked to human constant region exons in the expression vectors at the unique sites and cotransfected into mouse SP2/0 cells. The production level of chimeric Me1-14 from ascites in the highest expressing transfectoma was 1.8 mg/ml. The chimeric Me1-14 antibody exhibited the same specificity and similar affinity as that of parent Me1-14. Direct comparison of radioiodinated chimeric and murine Me1-14 in paired-label biodistribution analysis in subcutaneous xenograft-bearing mice showed higher tumor-to-normal organ ratios for chimeric Me1-14 IgG2, suggesting that this chimeric Me1-14 may be potentially useful in vivo for diagnostic and therapeutic purposes in patients.

Investigation of a synthetic peptide as immunogen for a variant epidermal growth factor receptor associated with gliomas.

We have previously demonstrated antibody production to a glioma-associated variant form of the human epidermal growth factor receptor in rabbits that had received a synthetic peptide mimicking the unique primary structure of the variant protein as immunogen. We report here the response of mice, rabbits, goats, and macaques immunized by various protocols to this peptide. Titers to both peptide- and cell-elaborated variant receptor were measured, and the capacity to recognize the variant receptor in human tumor samples was determined. Within the range of species and strains investigated, we demonstrated a variable species-associated response to the peptide (rabbits > mice > goats > rats > macaques). Rabbits and a single goat produced specific, high titer antibody activity to the variant receptor protein following immunization with peptide alone. Murine titers to the parent protein were not appreciable following peptide immunization alone; additional immunization with variant receptor as expressed on cell membranes was used to boost this response.

Identification of nitrosourea-resistant human rhabdomyosarcomas by in situ immunostaining of O6-methylguanine-DNA methyltransferase.

Cellular levels of O6-methylguanine-DNA methyltransferase (MGMT) correlate strongly with cellular resistance to carcinogenic and chemotherapeutic agents that produce adducts at the O6-position of guanine in DNA. Although biochemical and molecular assays can indicate the average MGMT content of tissues or tumors, they cannot distinguish mixed populations of cells, such as those that exist in tumor biopsy samples. We have determined MGMT at the cellular level in a panel of pediatric rhabdomyosarcoma xenografts by in situ immunostaining with a human MGMT-specific antibody employing a very sensitive procedure that involves biotin-avidin coupled horseradish peroxidase with silver-enhanced diaminobenzidine-nickel staining. Two xenograft tumor lines known to be MGMT-deficient were not stained, whereas the nuclei in three MGMT-expressing lines were clearly stained. This is the first demonstration of an in situ procedure that discriminates drug-sensitive MGMT-deficient tumors from drug-resistant MGMT expressing tumors. This procedure should prove useful, therefore, for predicting the susceptibility of tissues and tumors to O6-guanine alkylating agents.

Production and characterization of antipeptide antibodies against human O6-methylguanine-DNA methyltransferase.

Four synthetic peptides from the sequence of human O6-methylguanine-DNA methyltransferase (MGMT), three corresponding to different hydrophilic regions and one corresponding to the sequence containing the alkyl acceptor residue cysteine 145, were used to immunize rabbits. The antibody against Peptide III (residues 171-184) was highly specific, and MGMT protein could be detected on Western blots of soluble protein extracts containing as little as 1 fmol of active MGMT. Antibodies against all of the peptides were able to immunoprecipitate denatured MGMT, while only the antibody against Peptide III was able to react with active enzyme. The antibody against Peptide III did not cross-react with methyltransferase from mice. The use of synthetic peptides has led to the production of a highly sensitive, specific antibody that recognizes native and denatured human MGMT. This antibody should prove useful in studies involving the detection, purification, and characterization of this enzyme.

Immunoaffinity purification of human O6-alkylguanine-DNA alkyltransferase using newly developed monoclonal antibodies.

As well as repairing mutagenic lesions induced by simple methylating agents, O6-alkylguanine-DNA alkyltransferase repairs precursors of cytotoxic interstrand cross-links induced by chloroethylating anticancer drugs. Moreover, levels of the transferase correlate with cellular resistance to these agents. Thus far, the human transferase has not been highly purified. In our quest to obtain the homogeneous protein we have produced four stable cloned hybridomas that secrete monoclonal antibodies against the alkyltransferase from human lymphoblasts. The specificity of these monoclonals was established by immunoblot analysis and immunoprecipitation. All these antibodies recognized the alkyltransferase only after its denaturation by sodium dodecyl sulfate. One of them, designated 19.2, was used in immunoaffinity chromatography to obtain the pure protein.

The immunohistochemical application of three anti-GFAP monoclonal antibodies to formalin-fixed, paraffin-embedded, normal and neoplastic brain tissues.

The intermediate filament, glial fibrillary acidic protein (GFAP) has proven to be an important glial marker in diagnostic neuropathology. We report the histochemical application of three monoclonal antibodies (Mab) produced in this laboratory, 1B4, 2E1, and 4A11, which are monospecific to GFAP by radioimmunoassay, immunoblot electrophoresis, and immunoperoxidase histochemistry. The goal of this study was to compare the specificity and sensitivity of these Mab to GFAP on surgical brain biopsy specimens which had been routinely processed for diagnostic neuropathology with that of a high titer, highly specific, reference polyvalent anti-GFAP antiserum. The Mab stained astrocytes specifically in normal brain. When combined in a "cocktail" preparation, the quality of the immunoperoxidase detection of GFAP by these Mab closely approached that of the reference serum in 71 intracranial and intraspinal neoplasms. As these three Mab represent a continuous supply of a well defined, monospecific reagent, the monoclonal "cocktail" represents a standard reagent for large multi-institutional studies and for studies extending over a period of time.

Monoclonal antibodies reactive with epitopes restricted to glial fibrillary acidic proteins of several species.

The highly reproducible histochemical localization of glial fibrillary acidic protein (GFAP)+ qualifies it as an important marker of astrocytes in both research and clinical applications. The primary objective of this study was to produce monoclonal antibodies having the advantage of invariant specificity, affinity, and titer to GFAP-specific epitopes of wide species distribution. We report here the characterization of four monoclonal antibodies that recognize the same or spatially close epitopes specific to GFAP. The epitope(s) detected has been phylogenetically conserved; human, bovine, ovine, canine, porcine, rabbit, guinea pig, rat, murine, and chicken brain homogenates all specifically absorb monoclonal antibody activity. Of importance to the routine application of these new anti-GFAP monoclonal antibodies is the demonstration here of the stability of the antigen-antibody interaction in normal, reactive, and neoplastic astrocytes of both rat and human origin following various methods of fixation.

Human fetal brain antigen expression common to tumors of neuroectodermal tissue origin.

The antigenic relationship between human tumors of neuroectodermal origin and fetal brain were further investigated by characterization of two hybridoma antibodies derived from a fusion of P3-NS1/1-Ag 4-1 (NSI) myeloma cells and splenocytes hyperimmunized to second trimester human fetal brain homogenate. Monoclonal antibodies (MAs) 1H8cl 2 and 1H8cl 3 were analyzed by cell surface radioimmunoassay (CS-RIA), quantitative absorption, indirect immunofluorescence, and peroxidase-antiperoxidase (PAP) immunohistology. MA 1H8cl 3 is the more broadly reactive, binding to 9/14 glioblastoma (GBM), 2/3 neuroblastoma, 1/2 melanoma, and 1 medulloblastoma cell line(s) by CS-RIA analysis, and to 12/15 GBM, fetal brain, spleen, and liver, and adult spleen by PAP analysis. MA 1H8cl 2 is more restricted, binding to 7/14 GBM, 2/3 neuroblastoma, 1 medulloblastoma, and 2/3 fetal skin fibroblast cell line(s) by CS-RIA, and to 9/15 GBM and fetal brain and spleen by PAP analysis. Control non-central nervous system tumors and normal adult tissue including brain, thymus, lymph node, liver, kidney, lung, skin, and pancreas, were unreactive with both 1H8cl 2 and 1H8cl 3 by CS-RIA, PAP, and absorption analysis. The data presented here establish the unique nature of the detected antigenic specificities as compared to previously described oncofetal and onconeural antigens, and define two immune reagents which are operationally specific for tumors of neuroectodermal origin within the adult central nervous system.

Relationship of in vitro morphologic and growth characteristics of established human glioma-derived cell lines to their tumorigenicity in athymic nude mice.

Fifteen permanent cell lines derived from human gliomas which are individually distinct by immunologic and biochemical criteria were evaluated to determine if morphologic or cell biologic parameters distinguished the 4 lines which were tumorigenic in athymic nude mice. By subjective morphologic appraisal, the 4 tumorigenic lines were considered "malignant" or "borderline," but 4 of the non-tumorigenic lines were also classified in this way. By objective criteria, these 15 lines varied markedly in percentage of piled-up cells, chromatin pattern, pleomorphism, nuclear to cytoplasmic ratio, number of bizarre multinucleate giant cells, presence of abnormal mitotic figures, percentage of colony formation in soft agar, saturation density, population doubling time, and absolute plating efficiency. Among these criteria, percentage of colony formation in soft agar had the highest correlation coefficiency with tumorigenicity, and when this parameter was held constant the only additional characteristic which correlated significantly (p less than .05) was the number of bizarre multinucleate giant cells. When the 11 non-tumorigenic lines were ranked by these 2 criteria, 1 non-tumorigenic line (U-251 MGsp) had greater than .95 predicted probability of tumorigenicity. Although further tumorigenicity testing may increase the number of tumorigenic lines, the lines with few "malignant" characteristics may correspond to the population resembling cells of low grade astrocytomas seen within glioblastomas. The histologic pleomorphism of human gliomas is reflected in their morphologic and cell biologic diversity in culture.

Tumorigenic cell culture lines from a spontaneous VM/Dk murine astrocytoma (SMA).

One of the few spontaneous gliomas in inbred animals, the VM/Dk spontaneous murine astrocytoma (SMA), has seen limited use. Previously restricted to an in vivo system, the SMA was only transplantable intracerebrally (IC) using nonquantifiable suspensions of normal brain and tumor tissue. Prior attempts at establishing permanent tumorigenic SMA cell lines have not succeeded; tumorigenicity was lost during serial in vitro passage. We have established three different cell culture lines from a serially IC-transplanted SMA and two from tumors that arose from intraperitoneal (IP) injection of the IC-transplanted SMA. In contrast to previous cell cultures and transplantable lines of SMA, all five cell lines are not only tumorigenic IC but subcutaneously (SC) as well. Astrocytic feature are present in three of five lines to varying degrees, evidenced by in vitro and in vivo morphology, response to dibutyryl cyclic AMP (db-cAMP), and the presence of neuroglial fibers: None of the lines express CNPase, S-100, or GFA proteins in significant amounts. P560, highly tumorigenic and possessing the most astrocytic features of the five lines, extends the use of the spontaneous astrocytoma system of the inbred VM/Dk mouse strain by allowing quantitative in vivo and in vitro experiments.

Cytopathologic characteristics of permanent cell lines derived from anaplastic human gliomas.

Five permanent cell lines derived from human gliomas were examined at 24-hour intervals to determine optimal phases of their population growth curves for comparative morphologic studies. In early exponential growth phase, individual cytologic criteria were well displayed, while the increased cellular density of late exponential and plateau phase growth was advantageous for evaluating growth pattern, bizarre multinucleate giant cells and abnormal mitoses. These morphologic characteristics of permanent cell lines derived from human gliomas are potentially useful for correlation with their biologic, immunologic, biochemical and cell biologic parameters.