121212ournal ofNeurology, Neurosurgery, and Psychiatry 1994;57:1212-1215 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.57.10.1212 on 1 October 1994. Downloaded from Tenascin in cerebrospinal fluid is a useful biomarker for the diagnosis of brain tumour J Yoshida, T Wakabayashi, S Okamoto, S Kimura, K Washizu, K Kiyosawa, K Mokuno Abstract Subsequently, Erikson and Taylor showed Tenascin, an extracellular matrix glyco- that this was biochemically identical with protein, has been reported to be tenascin.6 Within the CNS the use of peroxi- expressed predominantly on glioma tis- dase antiperoxidase immunohistology with sue in the CNS, both in a cell associated 81 C6 MCA showed that the antigen was and an excreted form. Recently, a highly expressed in glioblastomas but not in astrocy- sensitive sandwich type enzyme tomas or in normal adult brain.7 immunoassay for quantitative determi- In 1991, Herlyn et al found that most nation of tenascin was developed. In the melanoma cells secreted tenascin in vitro. present study, the amount of tenascin in They also reported that tenascin was present CSF was measured. An increase of in the serum of patients with melanoma and tenascin in CSF (>100 nglml) was found that the concentration of tenascin in serum in patients with an astrocytic tumour. was a tumour marker for advanced The concentration was significantly melanomas because they found lower concen- higher (>300 nglrn) in high grade astro- trations in patients with low tumour burden cytoma (anaplastic astrocytoma and and in normal donors.8 Recently, we devel- glioblastoma) and a further increase oped a sandwich type enzyme immunoassay (>1000 ng/ml) was found in cases of CSF for tenascin9 and showed, with this assay sys- dissemination ofhigh grade astrocytoma. tem, that not only human melanoma, but also On the other hand, tenascin concentra- human lung cancer and glioma cells secreted tions were less than 100 nglml in non- 1-14 jug/ml of tenascin in vitro (data not astrocytic tumours and non-neoplastic shown). In the present study, serum and CSF neurological diseases, except meningeal were obtained from patients with various neo- dissemination of tumour cells, meningeal plastic and non-neoplastic neurological dis- stimulation by infection, and subarach- eases, and the amount of tenascin was noid haemorrhage. In cases of treated quantitatively determined. It was expected astrocytomas in remission, tenascin was that an increased level of expression of this negligible (<100 ng/ml) in the CSF. The extracellular matrix glycoprotein would be measurement oftenascin in CSF is useful found in neoplasms compared with other dis- for differential diagnosis of brain eases of the CNS, and that the level of expres- tumours and monitoring of astrocytic sion would be useful in indicating the type of http://jnnp.bmj.com/ Department of tumours. brain tumour. Neurosurgery, Nagoya University School of Medicine, Nagoya, (7 Neurol Neurosurg Psychiatry 1994;57:1212-1215) Japan Patients and methods J Yoshida SANDWICH TYPE ENZYME IMMUNOASSAY T Wakabayashi S Okamoto Tenascin is an extracellular matrix glycopro- Details of the methodology of the sandwich Department of tein involved in tissue interaction during fetal type enzyme immunoassay for tenascin has on September 25, 2021 by guest. Protected copyright. Research and development and oncogenesis. Chiquet- been reported elsewhere.9 Briefly, an anti- Development, Amano Ehrismann et al reported that tenascin was serum to human tenascin was raised in rab- Pharmaceutical Company, Aichi, present in the mesenchyme of the fetal rat bits. Two antihuman tenascin monoclonal Japan mammary gland but not in the adult mam- antibodies, 8C9 (RCBl)'0 and 9B2, were sup- S Kimura mary gland. The protein was, however, pre- plied by Amano Pharmaceutical Co, Aichi, K Washizu dominantly re-expressed in carcinogen Japan and F(ab)'2 fragments of both antibod- Department of induced mammary tumours.1 This extracellu- ies, prepared from lgG by pepsin digestion, Neurology, Nagoya University School of lar matrix glycoprotein was discovered inde- were used. The RCB1 fragment was immo- Medicine, Nagoya, pendently by several laboratories, each bilised non-covalently on polystyrene beads Japan showing a different aspect of its biology, and and the 9B2 fragment was coupled to ,B3D- K Kiyosawa K Mokuno each giving it a different name: myotendinous galactosidase. Polystyrene beads with anti- were with various amounts Correspondence to: antigen,2 glioma mesenchymal extracellular bodies incubated Dr Jun Yoshida, matrix antigen,'3 Jl glycoprotein,4 cytotactin,5 of purified tenascin or samples of serum and Departmnent of ml Neurosurgery, Nagoya hexabrachion,6 and tenascin.1 Bourdor et al CSF (10 Ml) in a final volume of 051 with University School of reported a glioma mesenchymal extracellular sodium phosphate buffer for three hours. Medicine, 65 Tsurumai- cho, Showa-ku, Nagoya, matrix antigen defined by monoclonal anti- After washing twice with buffer, the beads 466 Japan. body 81 C6, which binds an extracellular were left standing overnight with 1 mU of ,B Received 19 March 1993 matrix antigen shared by glioma and mes- D-galatosidase labelled antibodies (mixed 0 5 and in final revised form 28 January 1994. enchymal cell lines but not carcinoma, mU of 8C9 and 9B2) in 02 ml of buffer solu- Accepted 1 March 1994 myeloid, or lymphoid cell lines.7 tion at 4°C. Beads were again washed twice Tenascin in cerebrospinalfluid is a useful biomarkerfor the diagnosis of brain tumour 1213 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.57.10.1212 on 1 October 1994. Downloaded from Table 1 Concentrations of tenascin in CSF (nglml) Table 2 Numbers ofpatients with non-neoplastic diseases Negligible Low Medium High Non-neoplastic disease n Disease n (<100) (100-300) (300-1000) (>1000) Cerebral infarction 9 Intracerebral haemorrhage 1 Non-neoplastic disease 57 54 3§ 0 0 Subarachnoid haemorrhage 3 Pituitary adenoma 10 10 0 0 0 Meningitis or encephalitis 9 Craniopharyngioma 2 2 0 0 0 Cervical or lumbar spondylosis 9 Neurinoma 2 2 0 0 0 Multiple sclerosis 4 Meningioma 6 5 1* 0 0 Spinocerebellar degeneration 6 Germ cell tumour 4 4 0 0 0 Amyotrophic lateral sclerosis Pineoblastoma 1 1 0 0 0 Parkinson's disease l CNS lymphoma 1 0 1 0 0 Huntington's disease Oligodendroglioma 1 1 0 0 0 Bulbospinal muscular atrophy Ependymoma 1 1 0 0 0 Neuropathy 3 Medulloblastoma 2 1 0 1* 0 Dementia 1 Astrocytoma 5 it 4 0 0 Involuntary movement 1 Anaplastic astrocytoma 5 it 0 3 1* Normal pressure hydrocephalus Glioblastoma 9 2t 3 2 2* Aneurysm 2 Brainstem glioma 6 3t 1 2 0 Radiation necrosis l Metastatic brain tumour 3 1 1 1 0 Arachnoid cyst Tuberculosis 1 *Associated with CSF dissemination. Head injury 1 tPostoperative state after radiotherapy or chemotherapy. *Meningeal sarcoma. SMeningitis, subarachnoid haemorrhage, normal pressure hydrocephalus. with chilled buffer and the galactosidase activ- astrocytoma or glioblastoma (n = 14). The ity bound was measured after incubation at concentrations of tenascin were statistically 37°C for 30 minutes with 0-15 mmol different between the groups. (fig 2) In the 4-methylumbelliferyl f,-D-galactoside (Sigma non-astrocytic group, tenascin in CSF was St Louis, MO, USA) as a substrate. less than 100 ng/ml except for three tumours (10%) each with CSF dissemination. More SAMPLES than 100 ng/ml of tenascin was detected in 15 Serum samples were obtained from two cases (79 0%) in the astrocytic tumour group. healthy donors and 10 patients with brain In three metastatic tumours, the level was tumours. Samples of CSF were taken from 249-7 ng/ml in meningeal carcinomatosis of patients with various neurological diseases by gastric cancer, 771-7 ng/ml in metastatic pari- spinal tap or puncture of an Ommaya reser- etal tumour of lung cancer, but only 45-5 voir connected to the lateral ventricle. Fifty ng/ml in an extradural skull base undifferenti- eight patients had brain tumours. (table 1) ated carcinoma. We classified the concentra- Samples of CSF from four patients were taken tion of tenascin in CSF into four groups; serially and the concentrations of tenascin negligible if less than 100 ng/ml, low if were compared with the tumour progression 100-300 ng/ml, medium when 300-1000 and regression. Fifty seven patients had non- ng/ml, and high for more than 1000 ng/ml neoplastic neurological disease (table 2). (table 1). In low grade astrocytoma, 80% of Brain tumour cystic fluid was also obtained the tumours expressed low concentrations of from eight patients at surgery or by puncture tenascin and, none of them had medium or of an Ommaya reservoir connected to the high concentrations, whereas more than 50% cyst. of high grade astrocytomas (anaplastic astro- http://jnnp.bmj.com/ cytoma or glioblastoma) expressed medium or high concentrations. Furthermore, all patients Results with high expression of tenascin showed CSF STANDARD CURVE Figure 1 shows a standard curve for our sand- wich type enzyme immunoassay of tenascin. * The minimum detectable amount of tenascin 700 on September 25, 2021 by guest. Protected copyright. 4)C was 0o 1 ng per assay tube or 10 ng/ml. A linear dose response of the absorbance v tenascin - 600 0) was at between 0 1 to 30 ng 40 added obtained -E C per assay tube. The results for diluted CSF S 500 .'' 0) samples were parallel with the standard curve. LL :: aoC.) 0) 3400 aJ0 CEREBROSPINAL FLUID Table 1 shows the concentrations of tenascin C1u 300 *; in CSF obtained from patients with various Ci) neurological diseases. For non-neoplastic dis- R 200 ease, 54 patients (94 8%) had tenascin con- 3 30 300 3000 centrations of less than 100 ng/ml and most 100 i Tenascin were less than 50 ng/ml.