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Distribution of p53 alterations in a case of gliomatosis cerebri
DISTRIBUTION OF P53 ALTERATIONS IN A CASE OF GLIOMATOSIS CEREBRI CHRISTIAN MAWRIN, MD, HARTMUT LINS, MD, ELMAR KIRCHES, PHD, HANS-ULRICH SCHILDHAUS, MD, CORDULA SCHERLACH, MD, DIMITRIOS KANAKIS, AND KNUT DIETZMANN, MD Gliomatosis cerebri (GC) is a rare neuroepithelial tumor characterized by diffuse infiltration of large parts of the brain. The origin of GC is unknown, and the molecular alterations underlying this tumor have not been determined. Because mutations in the p53 tumor-suppressor gene are frequent in common gliomas, we investigated the distribution of p53 alterations by immunohistochemistry and direct sequencing in a GC case with a disease involving both hemispheres and the basal ganglia. Nuclear accumulation of p53 protein was detected in a single region with features of a high-grade glioma. In the remaining 10 regions, corresponding to low-grade gliomas, no p53 accumulation was seen. In 1 low-grade tumor sample,
a pathogenic splice site mutation was detected. These findings suggest that p53 alterations occur in GC, but are no prerequisite of malignant progression. The distribution of p53 alterations demonstrates the existence of topographically different clones in 1 patient. HUM PATHOL 34:102-106. Copyright 2003, Elsevier Science (USA). All rights reserved. Key words: gliomatosis cerebri, p53, magnetic resonance imaging. Abbreviations: GC, gliomatosis cerebri; LI, labeling index; MRI, magnetic resonance imaging.
The p53 gene is among the best-studied tumor-suppressor genes. Mutated in more than 50% of human cancers, p53 is essentially involved in the control of the cell cycle, response to DNA damage, cell death, and cell differentiation.1 Mutations in the p53 gene are known to play a major role in the formation of astrocytic brain tumors, both in the formation of low-grade astrocytomas and in the progression toward secondary glioblastoma.2 In contrast, p53 alterations occur less frequently in oligodendrocytic tumors.3 Gliomatosis cerebri (GC) is a rare glial neoplasm involving extensive diffuse brain infiltration but relative preservation of the underlying architecture.4 In some cases the entire neuraxis may be involved by the neoplastic process, and varying degrees of differentiation from low-grade to high-grade gliomas may be observed in the same patient.5 In contrast to common gliomas, the underlying molecular alterations remain largely unknown. Accumulation of p53 protein has been reported,6 but the frequency and distribution of p53 mutations have not been determined. We describe a 56-year-old man suffering from GC with a distinct pattern of p53 alterations, including a pathogenic p53 mutation.
hemisphere with occasionally p53-immunopositive tumor cells. Seven months before death, the patient experienced a generalized seizure. Clinical symptoms were still unimpressive (gait slightly spastic, reduced skills of the right hand, moderate cognitive impairment). F-18-desoxyglucose positron emission tomography demonstrated reduced activity in the left temporal, parietal, and occipital lobes and in the left thalamus. One month before death, the patient was readmitted to hospital after suffering a second generalized seizure; MRI examination demonstrated diffuse tumor involvement of the left and right frontoparietal region, basal ganglia, and the thalamus on both sides (Fig 1A). The radiologic appearance was suggestive of a diffuse infiltrating low-grade tumor. After administration of gadolinium-diethylenetriamine penta-acetic acid, contrast enhancement in the left frontal lobe lesion was noted (Fig 1B). It was suggested that this lesion could represent a circumscribed tumor progression toward a malignant glioma. Single-photon emission computed tomography showed increased metabolism in the left frontal lobe. The patient became progressively somnolent and was given valproic acid and clobazam to treat the persistent seizures. Before palliative radiation therapy was started, the patient developed acute dyspnea and cardiovascular failure; resuscitation was unsuccessful. Autopsy revealed pulmonary embolism as the cause of death.
CASE REPORT This 56-year-old man presented 4 years before death with slowly progressive movement disturbances in the right leg and right arm. One year later, magnetic resonance imaging (MRI) of the head revealed a tumor that was slightly hypointense in T1-weighted images and showed no contrast enhancement. The tumor diffusely infiltrated large parts of the left hemisphere, including the thalamus. On follow-up, involvement of the right thalamus was noted. Two years later, stereotactic biopsy performed in another hospital revealed a low-grade astrocytoma (World Health Organization grade II) in the left
From the Departments of Neuropathology, Neurology, Pathology, Radiology, and Psychiatry, Otto von Guericke University, Magdeburg, Germany. Accepted for publication September 26, 2002. Supported in part by a Start-up grant from Magdeburg University (to C.M.), and by a grant from the Land Sachsen-Auhalt (0032IF0000, to C.M.). Address correspondence and reprint requests to Christian Mawrin, MD, Department of Neuropathology, Otto von Guericke University, Leipziger Strasse 44, D-39120 Magdeburg, Germany. Copyright 2003, Elsevier Science (USA). All rights reserved. 0046-8177/03/3401-0017$30.00/0 doi:10.1053/hupa.2003.1
MATERIALS AND METHODS The brain and spinal cord were fixed in 10% buffered formalin for 2 weeks. After sectioning, paraffin-embedded brain tissue samples were routinely stained with hematoxylin and eosin, Nissl stain, and luxol fast-blue. Immunohistochemistry was performed on 4-m-thick deparaffinized tissue sections using a streptavidin-biotin horseradish peroxidase method (LSAB2 kit; Dako, Hamburg, Germany). Antibodies directed against glial fibrillary acidic protein (polyclonal, dilution 1:1500; Sigma, St. Louis, MO), MIB-1 (monoclonal, clone Ki-S5, 1:50; Dako), synaptophysin (monoclonal, clone SY38, 1:200; Dako), cytokeratin (monoclonal, clone MNF116, 1:50; Dako), and p53 (monoclonal, clone DO-7, 1:50; Dako) were used. For p53 and MIB-1, a labeling index (LI) was assessed by counting the number of immunopositive nuclei per 100 cells in a total of 10 high-power fields (⫻40 objective). For molecular studies, total DNA was isolated from dewaxed paraffin sections following standard protocols. Exons 5 to 8 of the p53 gene were analyzed using the polymerase chain reaction single-strand conformation polymorphism technique as recently described.5 Gels were stained using a
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FIGURE 1. Gliomatosis cerebri. (A) On T2-weighted images lesions with high signal intensity are seen in the left and right frontoparietal region, left basal ganalia, pons, and both thalami. (B) T1-weighted axial MRi scan after gadolinium administration showing signal-enhancement in the left frontal lobe.
modified silver staining protocol. PCR fragments showing mobility shifts of their single strands were sequenced on an automated fluorescence sequencer (ABI 373A; Applied Biosystems, Foster City, CA) using the dye terminator cycle sequencing protocol (Applied Biosystems).
Pathologic Findings The fresh brain weighed 1,600 g, and bilateral uncal and cerebellar tonsillar herniation were evident. After coronal sectioning, a circumscribed soft yellow-white lesion with hemorrhages (1⫻1⫻2 cm) was found in the left frontal lobe. Additionally, a diffuse white discoloration of the cerebral cortex with abolished gray-white matter border was evident in the right parietal lobe and in the left frontal, temporal, and parietal lobes. The cingulate gyri on both sides were of white color and soft consistency. The basal ganglia and the thalamus on both sides were also of white color and decreased consistency, but the anatomic structures could still be separated. The enlargement of both thalami resulted in narrowing of the lateral ventricles. In the brain stem, the tectal plate was also affected by the tumor. Microscopically, the tumor in the left frontal lobe was highly cellular and composed of polymorphic cells, sometimes with abundant cytoplasm and astrocytic processes. Mitotic figures were frequent, and vascular proliferations were seen (Fig 2A). Focal hemorrhages were present, but no necroses with pseudopalisading of tumor cells were observed. In all other affected regions, a tumor with moderately increased cellularity, composed of elongated tumor cells with oval hyperchromatic nuclei, was found (Fig 2B). Mitoses, vascular proliferations, and necroses were absent. Immunohistochemical investigations showed moderate expression of glial fibrillary acidic protein in all tumor sam-
ples. Neither synaptophysin nor cytokeratin was expressed in the tumor samples. In tumor regions corresponding to a low-grade glioma, no MIB-1–positive tumor nuclei could be detected. In contrast, the anaplastic area in the left frontal lobe showed a mean MIB-1 LI of 9.0% (Fig 3). Nuclear accumulation of p53 protein was detected in only the left frontal lobe (LI 13.0%; Fig 2C), not in any of the other tumor regions (Fig 3). Molecular studies revealed a heterozygeous G–C transition at position ⫺1 of the 3⬘ splice acceptor site of intron 7 in the p53 gene, which abolishes the splicing of intron 7 during transcription of the defective allele. This transition occurred selectively in the sample from the right cingulate gyrus (Figs 2D and 3), but was missing in all other tumor samples of this patient. The function of the tumor-suppressor p53 may be restored by the intact allele, but the mutation can be used as a marker of the tumor clone in the right cingulate gyrus.
DISCUSSION GC is a rare condition, with approximately 200 cases described in the literature.4,6 The steadily increasing number of cases reported during the last few years7 has been attributed to the widespread use of modern neuroimaging techniques. Because molecular changes in GC are largely unknown, the disease is listed in the group of neuroepithelial tumors of uncertain origin in the recent World Health Organization classification of brain tumors.8 GC can be subdivided into 2 forms based on descriptive neuropathologic grounds. Type I presents as diffusely infiltrating glioma without formation of an obvious tumor mass,
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FIGURE 2. (A) Photomicrographs of the tumor in the left frontal lobe, corresponding to a high-grade glioma, with marked nuclear atypia, high cellularity, frequent mitotic figures (arrow), and vascular proliferations (H&E stain, original magnification ⫻200.) (B) Diffuse infiltration of the right cingulate gyrus by a tumor with increased cellularity and nuclear atypia corresponding to a low-grade glioma (H&E stain, original magnification ⫻400.) (C) Nuclear accumulation of p53 protein in the tumor sample from the left frontal lobe (p53 immunohistochemistry, original magnification ⫻400.) (D) The sequence shows a G-C transition in the conserved ⫺1 position of the splice acceptor site of itnron 7 of the p53 gene in the tumor sample from the right cingulate gyrus.
whereas type II gliomatosis (even called secondary GC) denotes the coexistence of diffuse infiltration with associated tumor mass formation, the latter usually showing features of a malignant glioma.4 In some cases, various degrees of differentiation, ranging from low-grade gliomas to areas with features of a glioblastoma multiforme may be found within 1 patient.5 In particular, the presentation of type II disease suggests the presence of a malignant progression comparable to that already known in the formation of the so-called “secondary glioblastoma”9 and raises the question about the mechanisms involved in tumor initiation, spreading, and malignant progression in GCs. Loss of the wild-type activity of the p53 gene has been shown to increase genomic instability, and p53 mutations are known to play a role in the tumorigenesis of low-grade gliomas.10 Although we detected a pathogenic p53 mutation in a tumor region corresponding to a low-grade glioma, it seems unlikely that this p53 mutation in the right cingulate gyrus had initiated the tumor process, because it was lacking in the
other low-grade tumor regions from the right hemisphere. Therefore, it can be supposed that the p53 mutation occurred during tumor progression as a secondary event, with minor significance for the tumor progression itself. This hypothesis is supported by the lack of any p53 alteration in the remaining tumor regions from the left hemisphere, especially in the sample corresponding to a high-grade glioma. In the concept of malignant progression in gliomas, it has been suggested that loss of p53 activity can accelerate neoplastic progression,11 and the frequency of p53 mutations is high in secondary glioblastomas. In most cases, the mutations are already present in the first biopsy with features of low-grade glioma.10 However, the tumor progression in the left hemisphere in our patient seems to be independent from p53 mutations, because they were absent in both low-grade tumor areas and the high-grade tumor region in the frontal lobe. The detection of nuclear p53 accumulation without detectable p53 mutation in our case is well known in gliomas12 and has also been reported in GCs.6 This may be because
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FIGURE 3. Regional distribution of tumor grade, p53 immunoreactivity, p53 mutation, and MIB-1 labeling index (p53 - : no p53 immunoreaction; MIB-1 - : no MIB-1 immunoreaction; LI: labeling index).
mutations may have occurred outside the sequenced region, although the mutational rate outside this region seems to be low. Alternatively, staining could be increased by the expression of proteins that enhance p53 transcription or stabilize p53. In addition, a fraction of human cancers with p53 mutations shows no immunopositivity for the protein. In the series described by Taylor et al,13 this phenomenon was detected in 28% of the cases with the same antibody used in our study. Most of these cases were nonsense mutations or, as in our case, splice mutations, which severely alter the protein structure. The spatial distribution of p53 alterations in our case must be further discussed in regard to the question of the clonal origin of GC. The extraordinary mode of spread has led to various assumptions about the origin of GC. The development of neoplasms from multiple embryonic rests (“blastomatous malformation”) with subsequent centrifugal infiltration, a common glial neoplasm at various stages of differentiation, or a central proliferation of Schwann’s lemmoblasts have been suggested as the cause of GC.14 Using conventional cytogenetics and fluorescence in situ hybridization, large-scale chromosomal rearrangements different from those frequently found in astrocytomas were detected in 1 case.15 The observation of 2 distinct karyotypes differing only in ploidy grade, not in chromosomal rearrangement, was compatible with a monoclonal origin in this case. Kattar et al16 investigated the clonal origins of various gliomas by analyzing X chromosome inactivation in female patients. All
gliomas were monoclonal, whereas 1 GC case included in that study was not. In our patient, the presence of at least 2 different tumor clones with p53 alterations can be assumed, although a common precursor cannot be excluded. One clone is represented by the low-grade glioma harboring the p53 mutation. The other is represented by the anaplastic region in the left frontal lobe demonstrating nuclear p53 accumulation in the absence of a detectable p53 mutation. Acknowledgment. The technical assistance of I. Schellhase, S. Hartmann, and T. Fuchs is gratefully acknowledged. Furthermore, we thank Th. Weber and A. Rowlin for their photographic help.
REFERENCES 1. Levine AJ: p53, the cellular gatekeeper for growth and division. Cell 88:323-331, 1997 2. Watanabe K, Sato K, Biernat W, et al: Incidence and timing of p53 mutations during astrocytoma progression in patients with multiple biopsies. Clin Cancer Res 3:523-530, 1997 3. Ohgaki H, Eibl RH, Wiestler OD, et al: p53 mutations in nonastrocytic human brain tumors. Cancer Res 51:6202-6205, 1991 4. Jennings MT, Frenchman M, Shehab T, et al: Gliomatosis cerebri presenting as intractable epilepsy during early childhood. J Child Neurol 10:37-45, 1995 5. Mawrin C, Aumann V, Kirches E, et al: Gliomatosis cerebri: Postmortem molecular and immunohistochemical analyses in a case treated with thalidomide. J Neurooncol 55:11-17, 2001
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6. Kim DG, Yang HJ, Park IA, et al: Gliomatosis cerebri: Clinical features, treatment, and prognosis. Acta Neurochirurg 140:755-762, 1998 7. Freund M, Hahnel S, Sommer C, et al: CT and NRI findings in gliomatosis cerebri: A neuroradiologic and neuropathologic review of diffuse infiltrating brain neoplasms. Eur Radiol 11:309-316, 2001 8. Kleihues P, Burger PC, Scheithauer BW: The new WHO classification of brain tumors. Brain Pathol 3:255-268, 1993 9. Kleihues P, Ohgaki H: Primary and secondary glioblastomas: From concept to clinical diagnosis. Neuro-oncology 1:44-51, 1999 10. Ohgaki H, Watanabe K, Peraud A, et al: A case history of glioma progression. Acta Neuropathol 97:525-532, 1999 11. Malkin D, Li FP, Strong LC, et al: Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 250:12331238, 1990
12. Louis DN, von Deimling A, Chung RY, et al: Comparative study of p53 gene and protein alterations in human astrocytic tumors. J Neuropathol Exp Neurol 52:31-38, 1993 13. Taylor D, Koch WM, Zahurak, M, et al: Immunohistochemical detection of p53 protein accumulation in head and neck cancers:Correlation with p53 gene alterations. H UM P ATHOL 30:12211225 14. Artigas J, Cervos-Navarro J, Iglesias JR, et al: Gliomatosis cerebri: Clinical and histological findings. Clin Neuropathol 4:135-148, 1985 15. Hecht BK, Turc-Carel C, Chatel M, et al: Chromosomes in gliomatosis cerebri. Genes Chromosomes Cancer 14:149-153, 1995 16. Kattar MM, Kupsky WJ, Shimoyama RK, et al: Clonal analysis of gliomas. HUM PATHOL 28:1166-1179, 1997
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a pathogenic splice site mutation was detected. These findings suggest that p53 alterations occur in GC, but are no prerequisite of malignant progression. The distribution of p53 alterations demonstrates the existence of topographically different clones in 1 patient. HUM PATHOL 34:102-106. Copyright 2003, Elsevier Science (USA). All rights reserved. Key words: gliomatosis cerebri, p53, magnetic resonance imaging. Abbreviations: GC, gliomatosis cerebri; LI, labeling index; MRI, magnetic resonance imaging.
The p53 gene is among the best-studied tumor-suppressor genes. Mutated in more than 50% of human cancers, p53 is essentially involved in the control of the cell cycle, response to DNA damage, cell death, and cell differentiation.1 Mutations in the p53 gene are known to play a major role in the formation of astrocytic brain tumors, both in the formation of low-grade astrocytomas and in the progression toward secondary glioblastoma.2 In contrast, p53 alterations occur less frequently in oligodendrocytic tumors.3 Gliomatosis cerebri (GC) is a rare glial neoplasm involving extensive diffuse brain infiltration but relative preservation of the underlying architecture.4 In some cases the entire neuraxis may be involved by the neoplastic process, and varying degrees of differentiation from low-grade to high-grade gliomas may be observed in the same patient.5 In contrast to common gliomas, the underlying molecular alterations remain largely unknown. Accumulation of p53 protein has been reported,6 but the frequency and distribution of p53 mutations have not been determined. We describe a 56-year-old man suffering from GC with a distinct pattern of p53 alterations, including a pathogenic p53 mutation.
hemisphere with occasionally p53-immunopositive tumor cells. Seven months before death, the patient experienced a generalized seizure. Clinical symptoms were still unimpressive (gait slightly spastic, reduced skills of the right hand, moderate cognitive impairment). F-18-desoxyglucose positron emission tomography demonstrated reduced activity in the left temporal, parietal, and occipital lobes and in the left thalamus. One month before death, the patient was readmitted to hospital after suffering a second generalized seizure; MRI examination demonstrated diffuse tumor involvement of the left and right frontoparietal region, basal ganglia, and the thalamus on both sides (Fig 1A). The radiologic appearance was suggestive of a diffuse infiltrating low-grade tumor. After administration of gadolinium-diethylenetriamine penta-acetic acid, contrast enhancement in the left frontal lobe lesion was noted (Fig 1B). It was suggested that this lesion could represent a circumscribed tumor progression toward a malignant glioma. Single-photon emission computed tomography showed increased metabolism in the left frontal lobe. The patient became progressively somnolent and was given valproic acid and clobazam to treat the persistent seizures. Before palliative radiation therapy was started, the patient developed acute dyspnea and cardiovascular failure; resuscitation was unsuccessful. Autopsy revealed pulmonary embolism as the cause of death.
CASE REPORT This 56-year-old man presented 4 years before death with slowly progressive movement disturbances in the right leg and right arm. One year later, magnetic resonance imaging (MRI) of the head revealed a tumor that was slightly hypointense in T1-weighted images and showed no contrast enhancement. The tumor diffusely infiltrated large parts of the left hemisphere, including the thalamus. On follow-up, involvement of the right thalamus was noted. Two years later, stereotactic biopsy performed in another hospital revealed a low-grade astrocytoma (World Health Organization grade II) in the left
From the Departments of Neuropathology, Neurology, Pathology, Radiology, and Psychiatry, Otto von Guericke University, Magdeburg, Germany. Accepted for publication September 26, 2002. Supported in part by a Start-up grant from Magdeburg University (to C.M.), and by a grant from the Land Sachsen-Auhalt (0032IF0000, to C.M.). Address correspondence and reprint requests to Christian Mawrin, MD, Department of Neuropathology, Otto von Guericke University, Leipziger Strasse 44, D-39120 Magdeburg, Germany. Copyright 2003, Elsevier Science (USA). All rights reserved. 0046-8177/03/3401-0017$30.00/0 doi:10.1053/hupa.2003.1
MATERIALS AND METHODS The brain and spinal cord were fixed in 10% buffered formalin for 2 weeks. After sectioning, paraffin-embedded brain tissue samples were routinely stained with hematoxylin and eosin, Nissl stain, and luxol fast-blue. Immunohistochemistry was performed on 4-m-thick deparaffinized tissue sections using a streptavidin-biotin horseradish peroxidase method (LSAB2 kit; Dako, Hamburg, Germany). Antibodies directed against glial fibrillary acidic protein (polyclonal, dilution 1:1500; Sigma, St. Louis, MO), MIB-1 (monoclonal, clone Ki-S5, 1:50; Dako), synaptophysin (monoclonal, clone SY38, 1:200; Dako), cytokeratin (monoclonal, clone MNF116, 1:50; Dako), and p53 (monoclonal, clone DO-7, 1:50; Dako) were used. For p53 and MIB-1, a labeling index (LI) was assessed by counting the number of immunopositive nuclei per 100 cells in a total of 10 high-power fields (⫻40 objective). For molecular studies, total DNA was isolated from dewaxed paraffin sections following standard protocols. Exons 5 to 8 of the p53 gene were analyzed using the polymerase chain reaction single-strand conformation polymorphism technique as recently described.5 Gels were stained using a
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FIGURE 1. Gliomatosis cerebri. (A) On T2-weighted images lesions with high signal intensity are seen in the left and right frontoparietal region, left basal ganalia, pons, and both thalami. (B) T1-weighted axial MRi scan after gadolinium administration showing signal-enhancement in the left frontal lobe.
modified silver staining protocol. PCR fragments showing mobility shifts of their single strands were sequenced on an automated fluorescence sequencer (ABI 373A; Applied Biosystems, Foster City, CA) using the dye terminator cycle sequencing protocol (Applied Biosystems).
Pathologic Findings The fresh brain weighed 1,600 g, and bilateral uncal and cerebellar tonsillar herniation were evident. After coronal sectioning, a circumscribed soft yellow-white lesion with hemorrhages (1⫻1⫻2 cm) was found in the left frontal lobe. Additionally, a diffuse white discoloration of the cerebral cortex with abolished gray-white matter border was evident in the right parietal lobe and in the left frontal, temporal, and parietal lobes. The cingulate gyri on both sides were of white color and soft consistency. The basal ganglia and the thalamus on both sides were also of white color and decreased consistency, but the anatomic structures could still be separated. The enlargement of both thalami resulted in narrowing of the lateral ventricles. In the brain stem, the tectal plate was also affected by the tumor. Microscopically, the tumor in the left frontal lobe was highly cellular and composed of polymorphic cells, sometimes with abundant cytoplasm and astrocytic processes. Mitotic figures were frequent, and vascular proliferations were seen (Fig 2A). Focal hemorrhages were present, but no necroses with pseudopalisading of tumor cells were observed. In all other affected regions, a tumor with moderately increased cellularity, composed of elongated tumor cells with oval hyperchromatic nuclei, was found (Fig 2B). Mitoses, vascular proliferations, and necroses were absent. Immunohistochemical investigations showed moderate expression of glial fibrillary acidic protein in all tumor sam-
ples. Neither synaptophysin nor cytokeratin was expressed in the tumor samples. In tumor regions corresponding to a low-grade glioma, no MIB-1–positive tumor nuclei could be detected. In contrast, the anaplastic area in the left frontal lobe showed a mean MIB-1 LI of 9.0% (Fig 3). Nuclear accumulation of p53 protein was detected in only the left frontal lobe (LI 13.0%; Fig 2C), not in any of the other tumor regions (Fig 3). Molecular studies revealed a heterozygeous G–C transition at position ⫺1 of the 3⬘ splice acceptor site of intron 7 in the p53 gene, which abolishes the splicing of intron 7 during transcription of the defective allele. This transition occurred selectively in the sample from the right cingulate gyrus (Figs 2D and 3), but was missing in all other tumor samples of this patient. The function of the tumor-suppressor p53 may be restored by the intact allele, but the mutation can be used as a marker of the tumor clone in the right cingulate gyrus.
DISCUSSION GC is a rare condition, with approximately 200 cases described in the literature.4,6 The steadily increasing number of cases reported during the last few years7 has been attributed to the widespread use of modern neuroimaging techniques. Because molecular changes in GC are largely unknown, the disease is listed in the group of neuroepithelial tumors of uncertain origin in the recent World Health Organization classification of brain tumors.8 GC can be subdivided into 2 forms based on descriptive neuropathologic grounds. Type I presents as diffusely infiltrating glioma without formation of an obvious tumor mass,
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FIGURE 2. (A) Photomicrographs of the tumor in the left frontal lobe, corresponding to a high-grade glioma, with marked nuclear atypia, high cellularity, frequent mitotic figures (arrow), and vascular proliferations (H&E stain, original magnification ⫻200.) (B) Diffuse infiltration of the right cingulate gyrus by a tumor with increased cellularity and nuclear atypia corresponding to a low-grade glioma (H&E stain, original magnification ⫻400.) (C) Nuclear accumulation of p53 protein in the tumor sample from the left frontal lobe (p53 immunohistochemistry, original magnification ⫻400.) (D) The sequence shows a G-C transition in the conserved ⫺1 position of the splice acceptor site of itnron 7 of the p53 gene in the tumor sample from the right cingulate gyrus.
whereas type II gliomatosis (even called secondary GC) denotes the coexistence of diffuse infiltration with associated tumor mass formation, the latter usually showing features of a malignant glioma.4 In some cases, various degrees of differentiation, ranging from low-grade gliomas to areas with features of a glioblastoma multiforme may be found within 1 patient.5 In particular, the presentation of type II disease suggests the presence of a malignant progression comparable to that already known in the formation of the so-called “secondary glioblastoma”9 and raises the question about the mechanisms involved in tumor initiation, spreading, and malignant progression in GCs. Loss of the wild-type activity of the p53 gene has been shown to increase genomic instability, and p53 mutations are known to play a role in the tumorigenesis of low-grade gliomas.10 Although we detected a pathogenic p53 mutation in a tumor region corresponding to a low-grade glioma, it seems unlikely that this p53 mutation in the right cingulate gyrus had initiated the tumor process, because it was lacking in the
other low-grade tumor regions from the right hemisphere. Therefore, it can be supposed that the p53 mutation occurred during tumor progression as a secondary event, with minor significance for the tumor progression itself. This hypothesis is supported by the lack of any p53 alteration in the remaining tumor regions from the left hemisphere, especially in the sample corresponding to a high-grade glioma. In the concept of malignant progression in gliomas, it has been suggested that loss of p53 activity can accelerate neoplastic progression,11 and the frequency of p53 mutations is high in secondary glioblastomas. In most cases, the mutations are already present in the first biopsy with features of low-grade glioma.10 However, the tumor progression in the left hemisphere in our patient seems to be independent from p53 mutations, because they were absent in both low-grade tumor areas and the high-grade tumor region in the frontal lobe. The detection of nuclear p53 accumulation without detectable p53 mutation in our case is well known in gliomas12 and has also been reported in GCs.6 This may be because
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FIGURE 3. Regional distribution of tumor grade, p53 immunoreactivity, p53 mutation, and MIB-1 labeling index (p53 - : no p53 immunoreaction; MIB-1 - : no MIB-1 immunoreaction; LI: labeling index).
mutations may have occurred outside the sequenced region, although the mutational rate outside this region seems to be low. Alternatively, staining could be increased by the expression of proteins that enhance p53 transcription or stabilize p53. In addition, a fraction of human cancers with p53 mutations shows no immunopositivity for the protein. In the series described by Taylor et al,13 this phenomenon was detected in 28% of the cases with the same antibody used in our study. Most of these cases were nonsense mutations or, as in our case, splice mutations, which severely alter the protein structure. The spatial distribution of p53 alterations in our case must be further discussed in regard to the question of the clonal origin of GC. The extraordinary mode of spread has led to various assumptions about the origin of GC. The development of neoplasms from multiple embryonic rests (“blastomatous malformation”) with subsequent centrifugal infiltration, a common glial neoplasm at various stages of differentiation, or a central proliferation of Schwann’s lemmoblasts have been suggested as the cause of GC.14 Using conventional cytogenetics and fluorescence in situ hybridization, large-scale chromosomal rearrangements different from those frequently found in astrocytomas were detected in 1 case.15 The observation of 2 distinct karyotypes differing only in ploidy grade, not in chromosomal rearrangement, was compatible with a monoclonal origin in this case. Kattar et al16 investigated the clonal origins of various gliomas by analyzing X chromosome inactivation in female patients. All
gliomas were monoclonal, whereas 1 GC case included in that study was not. In our patient, the presence of at least 2 different tumor clones with p53 alterations can be assumed, although a common precursor cannot be excluded. One clone is represented by the low-grade glioma harboring the p53 mutation. The other is represented by the anaplastic region in the left frontal lobe demonstrating nuclear p53 accumulation in the absence of a detectable p53 mutation. Acknowledgment. The technical assistance of I. Schellhase, S. Hartmann, and T. Fuchs is gratefully acknowledged. Furthermore, we thank Th. Weber and A. Rowlin for their photographic help.
REFERENCES 1. Levine AJ: p53, the cellular gatekeeper for growth and division. Cell 88:323-331, 1997 2. Watanabe K, Sato K, Biernat W, et al: Incidence and timing of p53 mutations during astrocytoma progression in patients with multiple biopsies. Clin Cancer Res 3:523-530, 1997 3. Ohgaki H, Eibl RH, Wiestler OD, et al: p53 mutations in nonastrocytic human brain tumors. Cancer Res 51:6202-6205, 1991 4. Jennings MT, Frenchman M, Shehab T, et al: Gliomatosis cerebri presenting as intractable epilepsy during early childhood. J Child Neurol 10:37-45, 1995 5. Mawrin C, Aumann V, Kirches E, et al: Gliomatosis cerebri: Postmortem molecular and immunohistochemical analyses in a case treated with thalidomide. J Neurooncol 55:11-17, 2001
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6. Kim DG, Yang HJ, Park IA, et al: Gliomatosis cerebri: Clinical features, treatment, and prognosis. Acta Neurochirurg 140:755-762, 1998 7. Freund M, Hahnel S, Sommer C, et al: CT and NRI findings in gliomatosis cerebri: A neuroradiologic and neuropathologic review of diffuse infiltrating brain neoplasms. Eur Radiol 11:309-316, 2001 8. Kleihues P, Burger PC, Scheithauer BW: The new WHO classification of brain tumors. Brain Pathol 3:255-268, 1993 9. Kleihues P, Ohgaki H: Primary and secondary glioblastomas: From concept to clinical diagnosis. Neuro-oncology 1:44-51, 1999 10. Ohgaki H, Watanabe K, Peraud A, et al: A case history of glioma progression. Acta Neuropathol 97:525-532, 1999 11. Malkin D, Li FP, Strong LC, et al: Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 250:12331238, 1990
12. Louis DN, von Deimling A, Chung RY, et al: Comparative study of p53 gene and protein alterations in human astrocytic tumors. J Neuropathol Exp Neurol 52:31-38, 1993 13. Taylor D, Koch WM, Zahurak, M, et al: Immunohistochemical detection of p53 protein accumulation in head and neck cancers:Correlation with p53 gene alterations. H UM P ATHOL 30:12211225 14. Artigas J, Cervos-Navarro J, Iglesias JR, et al: Gliomatosis cerebri: Clinical and histological findings. Clin Neuropathol 4:135-148, 1985 15. Hecht BK, Turc-Carel C, Chatel M, et al: Chromosomes in gliomatosis cerebri. Genes Chromosomes Cancer 14:149-153, 1995 16. Kattar MM, Kupsky WJ, Shimoyama RK, et al: Clonal analysis of gliomas. HUM PATHOL 28:1166-1179, 1997
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