Genetic differences between neurocytoma and dysembryoplastic neuroepithelial tumor and oligodendroglial tumors

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1 J Neurosurg 97: , 2002 Genetic differences between neurocytoma and dysembryoplastic neuroepithelial tumor and oligodendroglial tumors HIRONORI FUJISAWA, M.D., KOHEI MARUKAWA, M.D., MITSUHIRO HASEGAWA, M.D., YASUO TOHMA, M.D., YUTAKA HAYASHI, M.D., NAOYUKI UCHIYAMA, M.D., OSAMU TACHIBANA, M.D., AND JUNKOH YAMASHITA, M.D. Department of Neurosurgery, Division of Neuroscience, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan N Object. Because of their histological similarities, it is occasionally difficult to differentiate neurocytoma and dysembryoplastic neuroepithelial tumor (DNT) from oligodendroglial tumors. This study was conducted to investigate genetic differences among these tumor types in terms of loss of heterozygosity on chromosomes 1p and 19q, and p53 gene mutation. Methods. A total of 24 tumors were analyzed, consisting of eight central neurocytomas, three DNTs, seven oligodendrogliomas, four oligoastrocytomas, and two undetermined extraventricular tumors with neurocytoma features (ETNFs). Allelic loss was determined using microsatellite markers that cover the common deletions on chromosomes 1p and 19q in oligodendrogliomas. A p53 gene mutation was identified using polymerase chain reaction single-strand conformation polymorphism analysis and subsequent direct sequencing. Immunohistochemical studies with synaptophysin and electron microscopy investigations were also conducted. Allelic loss on 1p and 19q was detected in six oligodendrogliomas (86%) and in three oligoastrocytomas (75%), but in none of the central neurocytomas or DNTs. A p53 missense mutation was detected at codon 161 (GCC ACC, Ala Thr) in only one oligoastrocytoma without allelic loss. Synaptophysin was expressed in all central neurocytomas and DNTs, in three oligodendrogliomas (43%), and in three oligoastrocytomas (75%). Of the ETNFs, one demonstrated synaptophysin expression and neural ultrastructures but lacked genetic alterations, whereas the other showed allelic loss on 1p and 19q but was negative immunohistochemically and ultrastructurally. The former was diagnosed as a potential intraparenchymal neurocytoma and the latter as an oligodendroglioma. Conclusions. Despite histological similarities, central neurocytomas and DNTs are genetically distinct from oligodendroglial tumors. Examination for allelic loss on 1p and 19q and for p53 mutation can be useful for making this distinction. KEY WORDS neurocytoma dysembryoplastic neuroepithelial tumor p53 gene mutation loss of heterozygosity chromosome arms 1p and 19q Abbreviations used in this paper: DNT = dysembryoplastic neuroepithelial tumor; ETNFs = extraventricular tumors with neurocytoma features; ID = identification; LOH = loss of heterozygosity; PCR-SSCP = polymerase chain reaction single-strand conformation polymorphism. EUROCYTOMAS and DNTs are benign neuronal tumors of the central nervous system that were identified after the 1980s. Both affect mainly adolescent and young adult patients. Histologically, neurocytomas consist of uniform neoplastic cells with a round nucleus and perinuclear halo in a fibrillary matrix, 16,17 which closely resembles morphological findings in oligodendrogliomas. Although neurocytomas characteristically arise in or adjacent to the lateral ventricles (central neurocytomas), 16,17 cases at extraventricular sites have been reported occasionally in the literature. 8,24,29 On the other hand, DNTs are usually associated with a refractory partial seizure and are characterized by the presence of specific glioneuronal element. 4,6 In some DNTs, especially in complex forms, glial components that closely resemble either oligodendroglioma, astrocytoma, or both have been described. 4,5 For confirmation of the tumors neuronal origin, immunoreactivity to synaptophysin has been most reliably and widely used, 32,36 with the addition of electron microscopic analysis when needed. 10,20,36 The findings are troublesome, however, for the following reasons: 1) immunoreactivity to synaptophysin can vary from none to strong, depending on fixing conditions; 10,20 2) oligodendrogliomas show both microscopic and ultrastructural focal neuronal differentiation; 7,23,39,40 3) no specific immunohistochemical marker for oligodendrogliomas has yet been identified; and 4) ultrastructural studies can be used for fresh samples but not for archival, paraffin-embedded ones. Such morphological and immunohistochemical limitations make it more difficult to distinguish a neurocytoma arising at an extraventricular site or a DNT lacking specific glioneuronal element from an oligodendroglioma or an oligoastrocytoma. Recently, it has been reported that oligodendrogliomas show LOH on 1p, 19q, or both (approximately 80%), 1,2,30 as well as the p53 mutation (approximately 15%). 1,2 Because LOH on 19q is detected uniquely in oligodendrogliomas among human neoplasms 28,37 and those with LOH on 1p respond well to procarbazine, lomustine, and vincristine (PCV) therapy, 2,31 LOHs on 19q and 1p are currently assumed to be genetic and chemosensitivity markers, respectively, for oligodendrogliomas. Such alterations are also demonstrated in oligoastrocytomas, but less frequently. The genetic background of neurocytomas and DNTs, on the other hand, remains largely unknown. 1350

2 Genetic analysis of neurocytoma and DNT FIG. 1. Light photomicrographs of tumor tissue sections stained with H & E and synaptophysin. A and B: The tumor designated by ID No is composed of monomorphous round cells with perinuclear halo scattered in an abundant fibrillary matrix with branching capillaries (A). The tumor designated by ID No shows a honeycomb pattern with many capillaries but lacks astrocytic differentiation (B). These two tumors were tentatively diagnosed as ETNFs. C and D: In Case 5, the recurrent tumor (ID No. 3765, central neurocytoma) shows a honeycomb pattern with abundant capillaries (C). In tissue from the recurrent tumor, synaptophysin is diffusely expressed (D), although it had been negative in the initial biopsy specimen (ID No. 3765). E and F: The tumor designated by ID No (oligodendroglioma) contains both honeycomb (left half of panel) and fibrillary areas (right half of panel) (E). Synaptophysin is positive in the right half (F). G and H: Of the ETNFs, the one designated by ID No shows diffuse intense expression of synaptophysin in a fibrillary matrix (G), but the one designated by ID No lacked such expression (H). Original magnifications 220. Because the treatment protocol and prognosis differ from tumor to tumor, neurocytomas and DNTs need to be easily and clearly distinguished from oligodendrogliomas and oligoastrocytomas. The aim of our study was to determine whether genetic examinations for LOH on 1p and 19q and for p53 mutations are valid tools for making such a distinction. Clinical Material and Methods Tumor Samples and DNA Extraction All specimens were obtained in patients who underwent surgery in the Department of Neurosurgery, Kanazawa University Hospital, except for one DNT (ID No ) obtained from an affiliated hospital. Tumors were fixed in a 3.7% formaldehyde (ID Nos. 630, 707, 1158, and 1628) or 4% paraformaldehyde solution for 1 or 2 days and embedded in paraffin. With the aid of a light microscope, the regions of oligodendroglial morphology with more than 80% neoplastic cells were marked and scraped off, followed by isolation of the tumor DNA as previously reported. 11 Control DNA was extracted from corresponding blood lymphocytes by using a DNA mini kit or from normal brain tissues on paraffin sections. Informed consent for the genetic analysis was obtained from all patients before the study was conducted. Histological Classification All tumors were stained routinely with hematoxylin eosin and the antibodies to glial fibrillary acidic protein and MIB-1, and subjected to consensus review by two independent neuropathologists. Histological typing and grading of the tumors were performed according to the World Health Organization classification system. 19 Only tumors with similar morphological features were included in this study. Central neurocytomas were diagnosed on the basis of classic features observed under light microscopy: uniform neoplastic cells with a round nucleus and perinuclear halo in a fibrillary matrix, and classic ventricular location. 16,17 Atypical variants were determined using an MIB-1 labeling index of more than 2% and by the presence of vascular proliferation. 21,32 The DNTs were diagnosed on the basis of characteristic histological architecture, including specific glioneuronal element. 4,6 Tumors showing a homogeneous honeycomb appearance and a chicken-wire capillary pattern with partial astrocytic differentiation were diagnosed as oligodendrogliomas. Only tumors exhibiting a clear fibrillary, protoplasmic, or gemistocytic astrocytoma, in addition to typical oligodendroglial parts, qualified separately for the diagnosis of oligoastrocytoma. Although all specimens were resected from the tumor center, two cerebral tumors (ID Nos and 3668) were difficult to diagnose. One (ID No. 3279) had developed in the right temporal lobe and was surgically removed en bloc. It contained scattered neoplastic cells with a perinuclear halo in an abundant fibrillary matrix (Fig. 1A). The other (ID No. 3668) had developed in the left frontal lobe and was partially resected. All specimens from the latter tumor showed a homogeneous honeycomb appearance with numerous capillaries but lacked astrocytic differentiation (Fig. 1B). These two lesions were therefore tentatively diagnosed as ETNFs. 12 A total of 24 tumors, including eight central neurocytomas, three DNTs, seven oligodendrogliomas, four oligoastrocytomas, and two ETNFs were examined in this study (Table 1). Immunohistochemical Procedures for Synaptophysin Immunostaining was performed on paraffin sections by using the streptavidin biotin complex with diaminobenzidine as the substrate. Unstained sections were deparaffinized and incubated overnight at 4 C with monoclonal antibody to synaptophysin (clone SY38, dilution 1:20). No antigen retrieval method was used. Normal brain tissue ob- 1351

3 H. Fujisawa, et al. TABLE 1 Genetic differences between oligodendroglial tumors and neurocytomas and DNTs* Age Neural 1p 19q Case No. (yrs), WHO Synapto- Ultra- (ID No.) Sex Grade Tumor Location physin structure central neurocytomas 1 (707) 25, M II bilat LV focal NI 2 (1158) 28, M II rt LV diffuse NI NI NI 3 (2506) 23, M II lt LV diffuse 4 (3547) 18, M II lt LV diffuse 5 (3765) 20, F II lt LV 6 (3992) 25, M II lt LV diffuse NI 7 (4033) 22, M II lt LV diffuse NI 8 (3306) 60, M atypical rt LV focal NI DNTs 9 (2987) 28, F I lt parietal diffuse NA NI NI NI 10 (3819) 17, M I lt parietal diffuse NA NI 11 (94-997) 16, M I lt frontal focal NA NI NI oligodendrogliomas 12 (630) 38, F II rt frontal NA NI NI 13 (1628) 51, M II lt frontal NA LOH NI LOH LOH 14 (2416) 54, F II rt frontal focal NA LOH LOH LOH NI 15 (2664) 42, F II rt temporal focal NA LOH LOH LOH NI LOH 16 (2904) 57, M II rt frontal NA LOH LOH LOH LOH LOH 17 (3032) 27, M II lt temporal NA LOH LOH LOH LOH LOH 18 (3595) 56, F II rt frontal focal NA LOH LOH LOH LOH LOH oligoastrocytomas 19 (3387) 39, M II rt frontotemporal NA NI NI 20 (3497) 32, M II lt parietal focal NA NI LOH LOH LOH LOH 21 (3667) 42, M II rt frontal diffuse NA NI LOH NI LOH NI 22 (3726) 32, M II rt frontotemporal focal NA LOH LOH LOH LOH LOH ETNFs 23 (3279) 16, F NA rt temporal diffuse NI NI 24 (3668) 35, M NA lt frontal LOH LOH LOH LOH NI * The only p53 mutation was in Case 19: 161 GCC ACC, Ala Thr. Abbreviations: LV = lateral ventricle; NA = not available; NI = not informative; WHO = World Health Organization; = no LOH. Previously reported by Kubota, et al. Diffuse at recurrence. tained in a patient who had undergone a frontal lobectomy (Case 22) served as positive control, and tissue without the primary antibody served as negative control. Ultrastructural Examination Fresh, representative tumor specimens were immediately fixed in 2.5% cold glutaraldehyde in cacodylate buffer, postfixed in 1% osmium tetroxide, and embedded in Epon- Araldite. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined using an electron microscope. 14,15,20 Analysis Using PCR-SSCP and Direct DNA Sequencing for p53 Mutations Prescreening for mutations in exons 5 to 8 of the p53 gene was accomplished using PCR-SSCP analysis as previously described. 38 Samples that showed mobility shift in the SSCP gels were reamplified and sequenced bidirectionally. Sequence analysis was performed with a semiautomated device. Analyses for LOH on Chromosome Arms 1p and 19q Based on previous reports by others, 2,27 two microsatellite markers (D1S508, D1S2734) and based on our previous reports, 11,22 three markers (D19S219, D19S412, and D19S596) were selected for the common deletions in oligodendrogliomas on chromosome arms 1p and 19q, respectively. The PCR and subsequent polyacrylamide gel electrophoresis were performed as previously reported, 11,22 and the bands were visualized with a silver staining kit in an automated gel stainer. The gels were then scanned and the signal intensity was analyzed using a Macintosh computer running the public domain National Institutes of Health Image program. An LOH was assumed when the signal intensity of the allele in the tumor was less than 50% of that in the reference DNA. Sources of Supplies and Equipment The QIAamp DNA mini kit was purchased from Qiagen, Tokyo, Japan. The LSAB kit used for immunostaining was obtained from Dako Corp., Carpinteria, CA. The monoclonal antibody to synaptophysin was supplied by Boehringer Mannheim Corp., Indianapolis, IN. The electron microscope (model H-600) was acquired from Hitachi Corp., Tokyo, Japan. The semiautomated sequencer (model 373) was obtained from Applied Biosystems, Foster City, CA. All chromosome markers were purchased from Research Genetics, Huntsville, AL. The silver staining kit was acquired from Pharmacia Biotech, Tokyo, Japan. The Image software program was downloaded from the National Institutes of Health website ( 1352

4 Genetic analysis of neurocytoma and DNT FIG. 2. Blots showing representative results of microsatellite analysis. Microsatellite markers are indicated on the left, and the ID No. and histological type are on the bottom of each panel. In oligodendrogliomas (OL, ID Nos and 3595), allelic loss was clearly demonstrated at the loci of D1S508 and D19S219, respectively (arrowheads). In one of the ETNFs (ID No. 3279), heterozygosity was retained at D19S412 but was not informative at D1S2734. In the other one (ID No. 3668), in contrast, LOH was clearly demonstrated at both loci (arrowheads). N = normal tissue (blood or normal brain); T = tumor. Results Synaptophysin Expression Expression of synaptophysin was detected in the fibrillary matrix in seven (88%) of eight central neurocytomas, with individual expression varying from focal to diffuse. Immunoreactivity for synaptophysin was completely negative in one neurocytoma (ID No. 3765), but the tumor that recurred after 1.5 years was diffusely positive (Fig. 1C and D). In all DNTs, synaptophysin was expressed in the neuronal processes and extracellular matrix in the area with morphological features of oligodendroglioma. On the other hand, expression of synaptophysin was detected in three (43%) of seven oligodendrogliomas (Fig. 1E and F) and three (75%) of four oligoastrocytomas. The expression was extracellular and focal in all but one oligoastrocytoma (ID No. 3667), with an expression that was diffuse but weaker than that of the central neurocytoma and DNT. Of the ETNFs, one (ID No. 3279) showed extracellular diffuse and intense expression of synaptophysin (Fig. 1G), but the other (ID No. 3668) lacked such expression completely (Fig. 1H). Mutations in the p53 Gene We performed PCR-SSCP analysis for all tumor samples, and mobility shift was detected in only one oligoastrocytoma (ID No. 3387). Direct DNA sequencing revealed a p53 missense mutation at codon 161 (GCC ACC) and its predicted amino acid change was Ala Thr (Table 1). An LOH on 1p and 19q FIG. 3. Electron micrograph of a section of the tumor designated by ID No (one of the ETNFs), showing abundant coated vesicles and microtubules. Original magnification 10,600. Using representative microsatellite markers, a total of 48 polymorphic loci on chromosome arm 1p and 72 on 19q were examined, and the analysis yielded 42 (88%) and 51 (71%) with informative results, respectively. An LOH on 1p and 19q was detected in six (86%) of seven oligodendrogliomas and in three (75%) of four oligoastrocytomas, whereas LOH was not detected in either neurocytoma or DNT (Table 1). In two oligodendrogliomas (ID Nos and 2416), a breakpoint was identified on chromosome arm 1p between D1S508 and D1S2734. One of the ETNFs (ID No. 3668) demonstrated LOH on 1p and 19q, but the other (ID No. 3279) retained heterozygosity (Fig. 2). Ultrastructural Findings Results of electron microscopic examination were available in 10 tumors (eight central neurocytomas and two ETNFs), but for the remaining lesions no adequate tissue sample was obtained. Neural ultrastructures, including coated vesicles, microtubules, and mature synapses, were recognized in all of the central neurocytomas and one of the ETNFs (ID No. 3279, Fig. 3), but not in the other (ID No. 3668). The findings for two of the central neurocytomas (ID Nos. 707 and 1158) have been reported previously. 20 Discussion Treatment for Central Neurocytoma and DNT Central neurocytoma can be cured by complete excision, and even when this cannot be achieved, the residual tumor usually follows a benign clinical course because of a low proliferation rate 32 and rare craniospinal dissemination. 9 Generally, radiotherapy is used for the residual tumor but chemotherapy is not indicated. Similarly, DNT has been reported to recur or disseminate rarely, even if it is partially excised, except in one case. 13 Therefore, neither radiotherapy nor chemotherapy is indicated for residual tumors. Because these tumors contain a component closely resembling oligodendroglioma but the prognosis is different for each type, accurate differential diagnosis is essential in clinical practice. Limitations of Morphological Studies in Differential Diagnosis In several large series of oligodendrogliomas, 7,23,39,40 synaptophysin expression was detected in up to 54% of cas- 1353

5 H. Fujisawa, et al. es. In the majority, the staining pattern was focal, and it was observed in less than 10% of tumor cells. 23 On electron microscopy examination, microtubules, neurosecretory granules, synapses, and synapse-like structures have been detected in neoplastic oligodendrocytes in five (62.5%) of eight oligodendrogliomas. 23 In our study, synaptophysin was completely negative in the initial sample in Case 5, a central neurocytoma (ID No. 3765), but it was diffusely positive at recurrence (Fig. 1C and D, Table 1). The initial sample was fixed for 7 days by mistake and this is assumed to account for the negative staining. Consistent with the previous reports, synaptophysin expression was frequently detected in this study in oligodendroglioma (in three [43%] of seven) and oligoastrocytoma (in three [75%] of four), but unfortunately no ultrastructural study was available because of a lack of adequate samples. Together with these findings, the authors have confirmed recent evidence of the instability of synaptophysin immunostaining and neuronal differentiation in oligodendroglial tumors. Recently, it has been reported that neurocytomas in extraventricular sites 8,24,29 and those with atypical histological findings 18 are apt to recur and that they have a poor prognosis in comparison with central neurocytomas. 35 It remains to be determined, however, whether they are really neurocytomas, because most of them have been diagnosed on the basis of immunoreactivity to synaptophysin but not on the basis of ultrastructural control. A representative case has been presented by Vallat-Decouvelaere, et al., 35 who first identified central neurocytoma, in which an oligodendroglioma was misdiagnosed as a supratentorial intraparenchymal neurocytoma only because of the presence of immunoreactivity to synaptophysin (the diagnosis was corrected after 2 years). Cerebral neurocytomas, originally reported by Nishio, et al., 24 are now regarded as DNTs. 12,17,35 The histopathological and clinical features of intraparenchymal neurocytoma remain unclear. Genetic Alterations in Oligodendroglial Tumor, Central Neurocytoma, and DNT In this study, LOH on 1p and 19q was coupled and identified in six (86%) of seven oligodendrogliomas and in three (75%) of four oligoastrocytomas, and a p53 missense mutation was detected in the remaining oligoastrocytoma. In oligoastrocytomas, LOH and p53 mutation were mutually exclusive. All of these findings are consistent with those previously reported. 1,2,30 In contrast with oligodendroglial tumors, only a limited number of genetic studies of central neurocytomas have been published, and to our knowledge no genetic study of DNTs has been reported. Therefore, the molecular mechanisms that can explain the pathogenesis of central neurocytomas and DNTs remain largely unknown. Cerda-Nicolas, et al., 3 reported on a single case of central neurocytoma that showed loss of chromosome 17; however, subsequent studies have demonstrated no p53 mutation in 15 central neurocytomas. 25,26 Taruscio, et al., 33 demonstrated a gain of chromosome 7 in three (33%) of nine central neurocytomas by using conventional cytogenetic analysis and fluorescence in situ hybridization, whereas the other investigators reported no amplification of the epidermal growth factor receptor gene in nine central neurocytomas. 34 This receptor is located on chromosome 7p and is commonly amplified in glioblastomas. Results of LOH on 1p and 19q in central neurocytomas have been published in two recent reports. Those authors used two different methods, comparative genomic hybridization 41 and microsatellite analysis. 34 In the first study, there was no chromosomal loss on either 1p or 19q in 10 cases analyzed; on the contrary, there was a single case with entire gain on chromosome 19. In the second study, LOH on 1p was found in six (67%) and LOH on 19q in five (56%) of nine central neurocytomas analyzed; however, the majority ( 80%) of informative loci tested were retained and there was no common deletion on either arm. Such frequent but random deletions were probably found because the authors cutoff limit for signal loss in the tumor DNA was lower ( 30%) than ours ( 50%). In our study, LOH on 1p and 19q and the p53 mutation were not detected in central neurocytomas or DNTs. These findings indicate that these neuronal lesions are genetically different from oligodendroglial tumors, although they show close morphological similarities. Molecular Analysis of ETNFs Our series contained two histologically undetermined tumors that were located at extraventricular sites but showed close morphological similarities to neurocytoma. In the tumor designated ID No there was no genetic alteration, but diffuse and strong expression of synaptophysin was detected, as well as neural ultrastructures. Apart from the lower cellularity, these findings indicate a potential intraparenchymal neurocytoma, although it is still controversial whether that tumor entity really exists, and therefore further genetic studies are needed. In the tumor designated ID No. 3668, on the other hand, LOH on both 1p and 19q was identified but no synaptophysin or neural ultrastructures were seen. In the absence of astrocytic differentiation, the tumor was diagnosed as an oligodendroglioma. The results of genetic analysis correlated with those of the ultrastructural examination. Conclusions In this study, the authors have confirmed frequent LOH on chromosome arms 1p and 19q and relatively infrequent p53 gene mutation in oligodendroglial tumors, and that these two findings were mutually exclusive. On the other hand, central neurocytomas and DNTs were all negative for such genetic alterations. Because synaptophysin immunostaining is occasionally negative in central neurocytomas and oligodendroglial tumors frequently show focal neuronal differentiation, analysis of LOH on 1p and 19q and of p53 mutation appears to be an indispensable diagnostic tool for differentiating these tumors. Acknowledgments The authors are grateful to Mrs. Akiko Imamura for her excellent technical assistance and to Dr. Narihito Yamaguchi of the Department of Neurosurgery, Asanogawa General Hospital, for a surgical specimen. References 1. Binger SH, Matthews MR, Rasheed BKA, et al: Molecular genetic aspects of oligodendrogliomas including analysis by comparative genomic hybridization. Am J Pathol 155: ,

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Address reprint requests to: Hironori Fujisawa, M.D., Department of Neurosurgery, Division of Neuroscience, Graduate School of Medical Science, Kanazawa University, 13-1 Takaramachi, Kanazawa , Japan. fujisawa@ns.m.kanazawa-u.ac.jp. 1355