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Clonality of oligoastrocytomas
Clonality of Oligoastrocytomas ZHI-QIAN DONG, MB, JESSE CHUNG-SEAN PANG, MSC, CAROL YUEN-KWAN TONG, MPHIL, LIANG-FU ZHOU, MD, AND HO-KEUNG NG, MD, FRCPATH, FRCPA Oligoastrocytomas (OA) are mixed glial tumors that show morphologic features of both oligodendrogliomas and astrocytomas. The histogenesis of these tumors remains undefined. The aim of this study was to investigate the clonality of OA on the basis of tumordependent genetic alterations and tumor-independent X-chromosome inactivation. We microdissected 11 biphasic OA and subjected the oligodendroglial and astrocytic components to allelic loss analysis of chromosomes 1p, 9p21, 10q, 13q, 17p, and 19q; TP53 immunohistochemical and mutation analyses; and X-linked HUMARA gene methylation study. On the basis of the genetic findings, we categorized these tumors into 3 groups. Group 1 consisted of 4 tumors that showed identical genetic aberrations in the 2 histologic elements, characterized by allelic loss on 1p and 19q. These results suggest that group 1 tumors are of monoclonal origin and share a precursor cell with oligodendrogliomas. Group 2 consisted of 5 tumors characterized by losses on 1p and 19q, with additional allelic losses on chromosomes 9p, 10q, 13q and/or 17p. Four of these tumors were of the anaplastic type. Thus, group 2 tumors may be regarded as advanced
variants of group 1 OA with heterogeneous genetic changes during clonal expansion. The X-chromosome inactivation analysis confirmed the monoclonality of groups 1 and 2 OA. Group 3 consisted of two tumors that showed divergent allelic loss patterns in the 2 histologic components. Mutation and overexpression of TP53 were detectable in the astrocytic components only. These findings raise the possibility that group 3 tumors have a biclonal origin. In conclusion, our results suggest that OA are predominantly of monoclonal origin but that a small subset of tumors may be derived from different precursors. HUM PATHOL 33:528-535. Copyright 2002, Elsevier Science (USA). All rights reserved. Key words: oligoastrocytoma, clonality, loss of heterozygosity, TP53, microdissection, X-chromosome inactivation. Abbreviations: OA, oligoastrocytoma; GFAP, glial fibrillary acidic protein; WHO, World Health Organization; PCR, polymerase chain reaction; LOH, loss of heterozygosity; SSCP, single-strand conformation polymorphism.
Oligoastrocytomas (OA) are glial tumors composed of 2 admixed neoplastic cell types that show morphologic features of oligodendroglioma and diffuse astrocytoma.1 The 2 histologic components of OA may be either intermingled (diffuse type) or separated into distinct areas (biphasic or compact type).1 Because of a lack of reliable immunohistochemical markers, diagnosis of OA may be subjective, with significant interobserver variation, and the delineation of neoplastic oligodendroglial cells, especially the minigemistocytes, from neoplastic astrocytes is not always easy.1,2 The differentiation of OA from astrocytomas is of clinical importance, because recent studies have indicated that the former are sensitive to chemotherapy containing procarbazine-lomustine-vincristine, whereas pure astrocytomas are largely nonresponsive to such treatment.2-4 Misdiagnosis of OA may therefore have a great impact on patient, resulting in inappropriate or delayed treatment. Recent advances in molecular genetic analysis of gliomas have greatly contributed to our understanding of the genetic events underlying the development of these tumors. Oligodendrogliomas commonly have deletions on chromosomes 1p and 19q, and additional
deletions on 9p and 10q are seen in the anaplastic variants.5-9 In contrast, diffuse astrocytomas are characterized by allelic loss of chromosome 17p, TP53 gene mutation, or both.10 Additional genetic lesions, such as losses of 10q and 19q, CDKN2A deletion, and RB1 alteration, are observed in the late stages of tumor development.5,11,12 OA are found to have genetic features characteristic of both oligodendrogliomas and astrocytomas.5-9 The histogenesis of OA remains unresolved. Investigating tumor clonality is one approach to providing clues to the origin of cells. Whether OA arise monoclonally from a single precursor cell with divergent oligodendroglial and astrocytic differentiation capability or polyclonally from simultaneous transformation and proliferation of 2 cell types within the same tumor is not known. Investigation of these alternatives has concentrated on a search for common genetic markers within the 2 components, which is suggestive of a common progenitor cell. Genetic study by Maintz et al13 suggests that there are at least two genetic subtypes of OA: 1 type showing alterations in 1p and 19q that may be genetically related to oligodendroglioma, and the other with TP53 mutations that may be genetically related to astrocytomas.13 In 1995, Kraus et al14 performed microsatellite analysis on three microdissected OA by conventional methods and detected identical allelic loss patterns on 1p and 19q in both histologic elements.1 Using an X-chromosome inactivation assay, Kattar et al15 found a monoclonal pattern in one informative anaplastic OA. These results indicate that the histologic components of OA have common molecular features and hint that OA may be monoclonal in origin.14,15
From the Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, and Department of Neurosurgery, Hua Shan Hospital, Shanghai, China. Accepted for publication December 17, 2001. Supported by a grant (CUHK4277/99M) from the Research Grant Council of Hong Kong. Address correspondence and reprint requests to Ho-Keung Ng, MD, Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong. Copyright 2002, Elsevier Science (USA). All rights reserved. 0046-8177/02/3305-0012$35.00/0 doi:10.1053/hupa.2002.124784
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FIGURE 1. Case OA8. (A) Typical oligodendroglial region. (B) Adjacent astrocytic region. (C) Region showing increased cellularity, cellular atypia and mitosis (arrow). (Hematoxylin and eosin; original magnifications, A, ⫻150; B, ⫻150, C, ⫻300.)
Although previous genetic investigations on OA have suggested that these tumors are derived from a single precursor cell, the limited numbers of genetic markers and informative samples examined might not have revealed the full spectrum of clonality patterns.14,15 The aim of the present study, therefore, was to investigate the clonality of OA using an extensive array of tumor-dependent genetic markers and tumorindependent X-chromosome inactivation analysis. Our results suggest that OA are predominantly of monoclonal origin, although a subset of tumors may be derived from different precursors.
cytes in the oligodendroglial areas (Fig 3). Synaptophysin staining, which was negative in all tumor cells, excluded any neuronal tumor that could masquerade as an oligodendroglial tumor. The MIB1 staining showed a distinct difference between the grade II and grade III tumors. The former had staining in less than 5% of the cells, whereas the latter had staining exceeding 15%. No area of any tumors stained less than 3%. All tumors were reviewed by 2 neuropathologists (H.K.N. and Z.Q.D.). There were 8 male and 3 female patients, with ages ranging from 18 to 60 (mean, 39 ⫾ 12) years. Table 1 summarizes the clinical data of the 11 patients. Matched blood samples were also obtained for all patients.
Microdissection and DNA Extraction
MATERIALS AND METHODS Tumor Specimens A total of 11 cases of oligoastrocytic tumors were retrieved from the archives of our hospitals. Diagnostic criteria were those of the World Health Organization (WHO).1 The oligodendroglial component consisted of typical honeycomb areas, sometimes with minigemistocytes. Astrocytic areas exhibited features of fibrillary, protoplasmic, or classic gemistocytic astrocytomas. All cases were of the biphasic type, as described in the WHO classification.1 The proportion of the smaller component always exceeded 20% and consisted of nodular aggregates that could be delineated and microdissected from the other component. Seven tumors were WHO grade II, and 4 tumors were anaplastic OA, WHO grade III.1 Anaplastic tumors had obvious pleomorphic and atypical features and prominent mitotic figures, sometimes combined with necrosis and endothelial proliferation (Figs 1 and 2). The archival slides and special preparations were retrieved, and in some cases, special stains were performed so that all 11 cases ultimately underwent glial fibrillary acidic protein (GFAP), vimentin, MIB1, and synaptophysin staining. The GFAP and vimentin stains decorated and highlighted fine astrocytic processes in the astrocytic areas, and GFAP also showed some entrapped astrocytes or revealed minigemisto-
The oligodendroglial and astrocytic components of the tumors were microdissected either manually or by laser capture microdissection (PixCell LCM System; Arcturus Engineering, Mountain View, CA) from formalin-fixed, paraffinembedded sections as described previously.16 Precautions were taken in microdissecting the individual histologic components, and areas in which intermingled cell types were observed were excluded. The microdissected tissues were collected into microtubes containing 100 L of digestion buffer consisting of 50 mM Tris-HCl, pH 8.9, 2 mM ethylenediaminotetraacetic acid, 0.5% Tween-10, and 500 g/mL proteinase K. Digestion was carried out overnight at 55°C, followed by inactivation of protease at 95°C for 10 minutes. The resultant aliquots containing chromosomal DNA were used for subsequent genetic analyses. DNA from tumor-matched blood samples was purified using the standard phenol/chloroform protocol and used as constitutional controls.17
Microsatellite Analysis Microsatellite analysis was performed on the microdissected OA components according to a reported protocol.16 A panel of 31 polymorphic markers that cover chromosomes 1p, 9p21.3-22.1, 10q, 13q14.11-34, 17p, and 19q were used.
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FIGURE 2. Case OA9. (A) Necrosis adjacent to area with oligodendroglial differentiation; (B) endothelial proliferation; (C) astrocytic area. (Hematoxylin and eosin; original magnifications, A, ⫻150; B, ⫻300; C, ⫻150.)
Briefly, polymerase chain reaction (PCR) was performed in a final volume of 5 L containing DNA, 10 mM Tris HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 62.5 M deoxyribonucleoside triphosphate, 0.5 unit of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA), and 2.5 pmoles of each primer, with 1 of them radioactively end-labeled with [␥-32P]-ATP. The PCR amplification commenced with an enzyme activation step at 95°C for 10 minutes, 40 cycles of 94°C for 1 minute, 55 to 60°C for 1 minute, and 72°C for 1 minute, and finalized with an extension step of 10 minutes at 72°C. The PCR products were resolved on 6% denaturing polyacrylamide gels and then subjected to autoradiography for 4 to 24 hours at room temperature. The analysis of allelic loss was based on the comparison of signal intensities of the maternal and paternal alleles with the normal blood and tumor samples. Loss of heterozygosity (LOH) was inferred by a 70%
reduction of allele signal intensity in tumor samples relative to that of the matched constitutional DNA samples.
X-chromosome Inactivation Analysis Analysis of X-chromosome inactivation detects the methylation pattern of the androgen receptor (HUMARA) gene, located on chromosome X.18 Genomic DNA was chemically modified with sodium bisulfite using the CpGenome DNA modification kit (Intergen, Purchase, NY) to convert unmethylated cytosine in DNA to uracil while leaving the unmethylated cytosine intact. PCR was then performed to amplify the polymorphic locus within the first exon of HUMARA using methylation-specific and unmethylation-specific primers. The primers used for amplifying the unmethylated allele of HUMARA were UF (5⬘-GGTTGTGAGTGTAGTATTTTTT-
FIGURE 3. (A) GFAP staining of oligodendroglial area showing mostly negative cells; minigemistocytes have a thin rim of immunopositivity. (B) GFAP staining of astrocytic area. (C) Vimentin staining of another astrocytic area. (Original magnification, A, ⫻300; B, ⫻300; C, ⫻300.)
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TABLE 1. Clinical Information of Patients having Oligoastrocytomas Case no.
Sex
Age (years)
OA-1 OA-2 OA-3 OA-4 OA-5 OA-6 OA-7 OA-8 OA-9 OA-10 OA-11
F F M M M M M F M M M
33 50 42 47 28 18 31 43 60 39 46
Histologic diagnosis
AOA AOA AOA AOA
OA OA OA OA OA OA (AO ⫹ A) (AO ⫹ A) (O ⫹ AA) (AO ⫹ A) OA
WHO grade II II II II II II III III III III II
Abbreviations: AOA, anaplastic oligoastrocytoma; O, oligodendroglial component; A, astrocytic component; AO, anaplastic oligodendroglial component; AA, anaplastic astrocytic component.
GGT-3⬘) and R (5⬘-TAAAAAAAACCATCCTCACC-3⬘), whereas those for methylated allele were MF (5⬘-CGAGCGTAGTATTTTTCGGC-3⬘) and R.18 The PCR conditions were essentially the same as those described for microsatellite analysis, except that the annealing temperature was set at 55°C and nonradioactive primers were used. The PCR products were resolved by 12% polyacrylamide gel electrophoresis, stained with ethidium bromide and visualized under ultraviolet illumination.
PCR Single-Strand Conformation Polymorphism and Direct Sequencing of TP53 Exons 4 to 8 of the TP53 gene were PCR amplified and subjected to single-strand conformation polymorphism (SSCP) analysis as previously described.19 DNA of abnormal shifts visualized in SSCP gels were eluted, PCR reamplified, and sequenced using the BigDye Terminator Cycle Sequencing kit (Applied Biosystems). Each mutation was verified at least twice independently.
Immunohistochemical Analysis of TP53 Protein Formalin-fixed, paraffin-embedded tissue sections from the same blocks used for microdissection were processed for TP53 immunostaining using the avidin-biotin-peroxidase complex method as previously described.16 The anti-human TP53 monoclonal antibody DO-7 (diluted 60-fold; Dako, Carpentiera, CA), which detects both wild-type and mutant TP53 proteins, was used. An adenocarcinoma of the colon with TP53 overexpression was used as a positive control. For a negative control, the primary antibody was omitted in the immunohistochemical process. The detection of immunostaining in the nuclei of tumor cells was considered positive for TP53 and was scored semiquantitatively as ⫹, ⫹⫹, and ⫹⫹⫹ when 5% to 25%, 26% to 50%, and ⬎50% of the cells stained positively, respectively.
RESULTS Microsatellite Analysis Previous investigations have shown that OA share genetic features with oligodendrogliomas and astrocy-
tomas. We hypothesized that a comparison of the genetic patterns in the oligodendroglial and astrocytic components of OA would provide insights into tumor clonality. Thus, we selected 7 critical chromosome regions known to be frequently deleted in oligodendrogliomas and astrocytomas and evaluated their LOH patterns in the microdissected components of OA. These regions were 1p, 9p21 (CDKN2A and CDKN2B loci), 10q23 (PTEN locus), 10q26.13 (DMBT1 locus), 13q (RB1 locus), 17p (TP53 locus), and 19q. Eleven biphasic OA were studied by microsatellite analysis. In each case, both oligodendroglial and astrocytic components were informative for at least 23 of the 31 microsatellite loci examined (Table 2). LOH was detected in at least 1 marker of chromosomes 1p (9 of 11 cases), 9p21-22 (5 cases), 10q22-23 (3 cases), 10q26.13 (6 cases), 13q14.11-34 (3 cases), 17p (5 cases), and 19q (9 cases). Of note is the absence of LOH on the intragenic markers of TP53 and PTEN (D10S2492) in all cases. On the basis of the genetic similarities between the oligodendroglial and astrocytic components, we divided the 11 OA into 3 groups. Group 1 consisted of 4 tumors: OA1, OA2, OA3, and OA11. These tumors displayed identical allelic loss patterns in the oligodendroglial and astrocytic components. Three cases (OA1, OA2, and OA3) showed LOH predominantly on chromosomal arms 1p and 19q. Case OA11 showed LOH for only 1 (D17S831) of the 22 informative markers examined. Five OA (OA5, OA7, OA8, OA9, and OA10) were allocated to group 2. These tumors were also characterized by concurrent LOH on chromosomes 1p and 19q, but additional allelic losses were found on chromosomes 9p, 10q, 13q, and/or 17p. At some loci, disparate LOH patterns at corresponding alleles were observed in the 2 histologic components. For example, OA5 showed concordant LOH of all informative markers on chromosomes 1p and 19q and on D10S1723 in both the oligodendroglial and astrocytic components, whereas allelic losses on markers D9S162, D10S541, and D10S587 were found in the astrocytic component only (Fig 4A). Following chromosomes 1p and 19q, the DMBT1 locus (D10S1723 and D10S587) was the next most frequently affected site in this group of OA, with 4 of 5 tumors showing allelic loss. More important, when the genetic data were correlated with tumor grade, we found that 4 of these 5 tumors were of the anaplastic type. Group 3 OA consisted of 2 tumors that showed entirely different LOH patterns in the 2 histologic components (Fig 4A). In OA4, LOH was detected in 1 marker each on chromosomes 19 (D19S596) and 10 (D10S216) specifically in the oligodendroglial element, whereas the astrocytic component showed retention of heterozygosity in all 24 informative markers that covered the 7 chromosomal arms. In OA6, 11 of 25 informative markers showed allelic loss or microsatellite instability. Notably, of the 6 markers (D1S458, D1S482, D9S162, D13S319, D17S831, and D17S799) that showed LOH in both histologic elements, the losses were found on different alleles at the corresponding
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TABLE 2. Summary of LOH, TP53 Immunohistochemical, and Mutation Analyses in Microdissected Components of Oligoastrocytomas Group 1 OA1 Microsatellite markers
Known genes
p73 TP73 D1S458 D1S482 D1S493 D1S186 D19S219 D19S412 D19S112 D19S596 D9S162 IFNA D9S736 D9S171 CDKN2A/B D9S1748 D9S1749 D10S215 D10S541 D10S2492 PTEN D10S216 D10S1723 DMBT1 D10S587 D13S267 D13S319 RB D13S158 D13S285 D17S849 D17S831 TP53 TP53 D17S1852 D17S799 D17S1303 TP53 immunohistochemistry TP53 mutation
Cytogenetic mapping 1p36.33 1p36.12 1p36.12 1p35.5 1p34.3 19q13.32 19q13.32 19q13.32 19q13.33 9p22.1 9p21.3 9p21.3 9p21.3 9p21.3 9p21.3 10q23.31 10q23.31 10q23.33 10q26.13 10q26.13 10q26.13 13q14.11 13q14.3 13q33.1 13q34 17p13.3 17p13.3 17p13.1 17p11.2 17p11.2 17p11.2
A
O
F F F F F F F E E E E E E E E E E E E E E
F F F F F F F E E E E E E E E E E E E E E
OA2 A
O
Group 2
OA3 A
OA11
O
A
O
OA5 A
O
OA7 A
O
OA8 A
O
Group 3 OA9 A
O
OA10 A
O
OA4 A
O
OA6 A
O
E E F F E E F F E E F F F F E E E E E E F F E E F F E E FL FS F F F F F F E F F F F F F F E E FL FS F F F F E E F F F F E E F F E E E E E E F F F F F F F F F F E F E E E E F F F F E E F F F F E E F F F F E E F F F F F F F F F F E E E E F F F F E E F F F F F F F F E E E E F F F F E E F F F F F F F F F F E F E E E E E E F E E E F F E E E E F L FS E E E E E E E E E E F F E E E E E E F F E E E E E E E E E E F F E E E E E E E E E E E E E E E E E E E E E E E E E E E E F F E E F F E E E E E E E E E E E E FS FL E E F E E E E F E E E E E E E E E E E E E E E E E E E E MI MI E E E E E F E E E E E E E F F E E E E E E E F F F F F F F F E E E E E E E E E E E E F E E E F F F F E E E E F E E E E E E E F F MI F E E E E E E E E E F E E E E E E F L FS E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E F F E E E E E E E E F F E E E F MI E E E F S FL E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E F E E E E E E E F S FL E E E E E E E E E F E E F F E E MI F ⫺ve ⫺ve ⫺ve ⫺ve ⫹ ⫹ ⫹ ⫹⫹ ⫺ve ⫹ ⫺ve ⫺ve ⫹ ⫹⫹ ⫹⫹ ⫹ ⫹ ⫹⫹ ⫹ ⫺ve ⫹ ⫺ve NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO E8 NO NO NO
Abbreviations: O, oligodendroglial component; A, astrocytic component; open circle, retention of heterozygosity; dark circle, loss of heterozygosity (L, longer allele; S, shorter allele); MI, microsatellite instability; ⫺, noninformative; gray boxes, genetic discrepancies between components detected; ⫺ve, immunonegative; ⫹, immunopositive cells ⬍ 25%; ⫹⫹, immunopositive cells 26% to 50%; NO, no mutation detected; E8, mutation found on exon 8, codon 273, from CGT (Arg) to TGT (Cys).
loci in each component. This tumor also exhibited microsatellite instability of 3 markers (see Table 2). X-Chromosome Inactivation Analysis Tumor clonality has been studied in female somatic cells using X-chromosome inactivation analysis based on differential methylation patterns of active (unmethylated) and inactive (methylated) alleles. The HUMARA gene contains a polymorphic short tandem repeat in the first exon, and hypermethylation of the inactive allele is associated with X-chromosome inactivation.18 The unmethylated and methylated polymorphic loci of HUMARA can be differentially detected using methylation-specific PCR. Three tumors from female patients (OA1, OA2, and OA8) were analyzed, and representative results are shown in Figure 4B. In the blood sample of OA8 (group 2), 2 PCR bands representing the 2 alleles of HUMARA were detected in both unmethylation and methylation reactions, indicating that the peripheral leukocytes were polyclonal in origin. In the astrocytic component, only 1 allele showed hypermethylation, whereas the second allele was found to be unmethylated in the unmethylation
PCR. The same methylation pattern was observed in the oligodendroglial component. Taken together, these results suggest that the histologic elements of OA8 were derived from the same precursor cell. Case OA2 of group 1 OA showed a methylation pattern similar to that of OA8. The HUMARA locus in OA1 was noninformative. Immunohistochemistry of TP53 Protein Eight cases of OA showed immunopositivity for TP53 in either the oligodendroglial and astrocytic component or both. In group 1 OA, concordant expression or nonexpression of TP53 protein was observed in both components. Four of 5 group 2 OA showed concordant overexpression of TP53 in both histologic elements. The exception was OA5, in which the TP53 protein was detectable only in the oligodendroglial component. In group 3 OA, the TP53 immunostaining pattern was different in the histologic elements. Expression of TP53 protein was detectable only in the astrocytic components, but not in the oligodendroglial portion.
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Mutation Analysis of TP53 Of the 11 OA studied, mutation of TP53 was detected only in the astrocytic component of a group 3 OA (case OA4). The missense mutation was localized to
exon 8, codon 273, CGT (Arg) 3 TGT (Cys) (Fig 4C), which is a hot spot for TP53 mutation in astrocytic tumors.10 DISCUSSION OA are admixed tumors containing oligodendroglial and astrocytic elements. Their precise diagnostic criteria are controversial,1 and their distinction from a diffuse infiltrating astrocytoma may be difficult.20 The cell of origin of each element has long been a clinicopathologic mystery. Two pathways whereby these tumors arise have been proposed: monoclonal or polyclonal expansion. It has even been suggested that oligodendrogliomas and OA share a uniform lineage from neuronal precursor A2B5⫹ cells.21 To investigate the clonality of OA, we examined the methylation pattern of X-chromosome inactivation; the LOH status of chromosomes 1p, 9p, 10q, 13q, 17p, and 19q, and the TP53 status in 11 microdissected tumors. On the basis of the genetic similarities between the histologic components, we divided the series into 3 groups. Group 1 OA display identical genetic alterations in the oligodendroglial and astrocytic components, supporting the notion that these 2 components are derived from the same precursor cell.14,15 The X-chromosome inactivation analysis also confirms that this group of tumors is monoclonal in origin. The precursor cells may have the ability to differentiate into oligodendroglial and astrocytic cells, leading to biphasic tumors. The latter hypothesis is supported by the finding that certain rat glial progenitor cells are capable of astrocytic and oligodendrocytic differentiation under different culture conditions.22 Three tumors (OA1, OA2, and OA3) showed allelic losses on both 1p and 19q, and 2 of these had normal TP53 status. These genetic changes resemble those of oligodendrogliomas, suggesting that OA and oligodendroglioma had a common cell of origin.14 All 5 cases of group 2 OA are characterized by allelic loss of 1p and 19q, compatible with genetic features of oligodendrogliomas. The high concordance of genetic patterns on chromosomes 1p and 19q and the methylation pattern on HUMARA suggest that these tumors, like group 1 tumors, are derived from a 4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ FIGURE 4. (A) Microsatellite analysis of microdissected components of oligoastrocytomas. OA5 shows allelic loss of D10S587 in the astrocytic component, whereas OA7 displays LOH on D17S831 in the oligodendroglial tumor portion. Genetic discrepancies between components at loci D17S799 and D17S1303 were detected in OA6. A, astrocytic component; O, oligodendroglial component; B, matched constitutional control; L, loss of heterozygosity; R, retention of heterozygosity; N, normal allelic pattern; MI, microsatellite instability. (B) X-chromosome inactivation analysis of the HUMARA gene by methylation-specific PCR. Results suggest that OA2 is monoclonal in origin. B, matched constitutional control; A, astrocytic component; O, oligodendroglial component; U, unmethylation; M, methylation. (C) Sequencing profile of the TP53 gene in antisense orientation. A missense mutation was found at exon 8, codon 273, CGT (Arg) 3 TGT (Cys) in the astrocytic component of OA4.
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single precursor cell. Combining these tumors with group 1 OA, our results indicate that most biphasic OA have a monoclonal origin. In addition, more allelic losses on chromosomes 9p, 10q, 13q, and/or 17p were found in group 2 tumors. Notably, 4 of 5 tumors in this group are classified as WHO grade III, supporting the view that advanced gliomas accumulate more genetic alterations.8,9,23 Thus, group 2 tumors may be regarded as an advanced variant of group 1 OA. We also noted occasional genetic discrepancies in the 2 histologic components at some microsatellite loci. Such dissimilarities between the components may be explained by the acquisition of different genetic changes in the 2 components during tumor development. Our data also indicate that allelic loss at the DMBT1 locus on chromosome 10q26.13 is frequently (in 4 of 5 cases) detectable in group 2 OA. Complete loss of chromosome 10 has been shown previously in high-grade oligodendrogliomas.24 Maier et al25 also reported partial deletion of chromosome 10q25-26 in oligodendrogliomas. These results suggest that DMBT1 is involved in the progression of oligodendroglial tumors. Mutation analysis of the DMBT1 gene in oligodendroglial tumors is currently in progress in our laboratory. The most intriguing finding in our study is seen in 2 OA in group 3. These tumors show divergent genetic patterns in the oligodendroglial and astrocytic components. First, the allelic loss profiles are different. Two major types of LOH patterns are observed at the microsatellite loci: LOH versus retained alleles and LOH on different alleles in each component. The loss of different alleles at the corresponding loci in each neoplastic component suggests that these loci are more susceptible to deletion. This idea is supported by the observation that these loci (e.g., D1S482, D9S162, and D17S831) are also commonly deleted in other OA. Second, aberrant TP53 status is detectable in the astrocytic component only. Taken together, these results strongly suggest that group 3 OA evolved from 2 independent precursor cells, which give rise to tumors with different genetic backgrounds. “Collision” tumors are not uncommon and have been described in a variety of tumor types.26-28 These tumors are predominantly monoclonal in origin, but some show polyclonal genetic patterns.26,28 Collision tumors with glial differentiation have also been reported and mostly involved 2 different types of gliomas that existed in close proximity in the brain.29-31 In this study, we reported for the first time the detection of polyclonality in biphasic OA. The clonality findings in biphasic OA have raised an important question: whether such clonal patterns are also seen in diffuse OA. From previous genetic studies of OA, it is believed that diffuse OA, like their biphasic counterparts, are monoclonal in origin.13-15 The study of clonality in diffuse OA is hampered by the absence of a reliable method of separating the individual elements. This barrier may now be overcome by using the marker that can identify oligodendroglial cells and the latest laser capture microdissection system that can procure tissue on a single-cell basis.32 The 2
cell types can be separately isolated and their clonality patterns deduced by genetic methods. Microsatellite instability that is the signature of mismatch repair deficiency is seen early during tumorigenesis and increases as the tumors progress.33 In our series, 5 microsatellite instabilities were detected in a total of 538 informative markers, a finding consistent with the infrequent incidence of microsatellite instability in astrocytic and oligodendroglial tumors.8,34 In conclusion, our data suggest that OA are predominantly of monoclonal origin. The concurrent loss of chromosomes 1p and 19q in OA hints that these tumors and oligodendrogliomas have a common cell of origin. A subset of OA, in which the histologic elements show dissimilar genetic patterns, may be derived from different progenitor cells.
REFERENCES 1. Reifenberger G, Kros JM, Burger PC, et al: Oligoastrocytoma, in Kleihues P, Cavenee WK (eds): Pathology and Genetics of Tumours of the Nervous System. Lyon, France, IARC Press, 2000, pp 65-69 2. Krouwer HGJ, van Duinen SG, Kamphorst W, et al: Oligoastrocytomas: A clinicopathological study of 52 cases. J Neurooncol 33:223-238, 1997 3. Glass J, Hochberg FH, Gruber ML, et al: The treatment of oligodendrogliomas and mixed oligodendroglioma-astrocytomas with PCV chemotherapy. J Neurosurg 76:741-745, 1992 4. Kim L, Hochberg FH, Thornton AF, et al: Procarbazine, lomustine, and vincristine (PCV) chemotherapy for grade III and grade IV oligoastrocytomas. J Neurosurg 85:602-607, 1996 5. von Deimling A, Louis DN, von Ammon K, et al: Evidence for a tumor suppressor gene on chromosome 19q associated with human astrocytomas, oligodendrogliomas, and mixed gliomas. Cancer Res 52:4277-4279, 1992 6. Reifenberger J, Reifenberger G, Liu L, et al: Molecular genetic analysis of oligodendroglial tumors shows preferential allelic deletions on 19q and 1p. Am J Pathol 145:1175-1190, 1994 7. Bello MJ, Vaquero J, De Campos JM: Molecular analysis of chromosome 1 abnormalities in human gliomas reveals frequent loss of 1p in oligodendroglial tumors. Int J Cancer 57:172-175, 1994 8. von Deimling A, Fimmers R, Schmidt MC, et al: Comprehensive allelotype and genetic analysis of 466 human nervous system tumors. J Neuropathol Exp Neurol 59:544-558, 2000 9. Bigner SH, Matthews MR, Rasheed BKA: Molecular genetic aspects of oligodendrogliomas including analysis by comparative genomic hybridization. Am J Pathol 155:375-386, 1999 10. von Deimling A, Eibl RH, Ohgaki H, et al: P53 mutations are associated with 17p allelic loss in grade II and grade III astrocytoma. Cancer Res 52:2987-2990, 1992 11. Fujisawa H, Kurrer M, Reis RM, et al: Acquisition of the glioblastoma phenotype during astrocytoma progression is associated with loss of heterozygosity on 10q25-qter. Am J Pathol 155:387-394, 1999 12. Ueki K, Ono Y, Henson JW, et al: CDKN2/p16 or RB alterations occur in the majority of glioblastomas and are inversely correlated. Cancer Res 56:150-153, 1996 13. Maintz D, Fiedler K, Koopmann J, et al: Molecular genetic evidence for subtypes of oligoastrocytomas. J Neuropathol Exp Neurol 56:1098-1104, 1997 14. Kraus JA, Koopmann J, Kaskel P, et al: Shared allelic losses on chromosomes 1p and 19q suggest a common origin of oligodendroglioma and oligoastrocytoma. J Neuropathol Exp Neurol 54:9195, 1995 15. Kattar MM, Kupsky WJ, Shimoyama RK, et al: Clonal analysis of gliomas. HUM PATHOL 28:1166-1179, 1997 16. Cheng Y, Ng HK, Ding M, et al: Molecular analysis of micro-
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CLONALITY OF OLIGOASTROCYTOMAS (Dong et al) dissected de novo glioblastomas and paired astrocytic tumors. J Neuropathol Exp Neurol 58:120-128, 1999 17. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual (ed 2). Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1989 18. Uchida T, Ohashi H, Aoki E, et al: Clonality analysis by methylation-specific PCR for the human androgen-receptor gene (HUMARA-MSP). Leukemia 14:207-212, 2000 19. Tong CY, Ng HK, Pang JC, et al: Molecular genetic analysis of non-astrocytic gliomas. Histopathology 34:331-341, 1999 20. Daumas-Duport C, Varlet P, Tucker M-L, et al: Oligodendrogliomas. Part I: Patterns of growth, histological diagnosis, clinical and imaging correlations: A study of 153 cases. J Neurooncology 34:37-59, 1997 21. de la Monte SM: Uniform lineage of oligodendrogliomas. Am J Pathol 135:529-540, 1989 22. Raff MC, Miller RH, Noble M: A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium. Nature 303:390-396, 1983 23. Louis DN: A molecular genetic model of astrocytoma histopathology. Brain Pathol 7:755-764, 1997 24. Jeuken JW, Sprenger SH, Wesseling P, et al: Identification of subgroups of high-grade oligodendroglial tumors by comparative genomic hybridization. J Neuropathol Exp Neurol 58:606-612, 1999 25. Maier D, Comparone D, Taylor E, et al: New deletion in low-grade oligoastrocytoma at the glioblastoma suppressor locus on chromosome 10q25-26. Oncogene 15:997-1000, 1997
26. Fujii H, Zhu XG, Matsumoto T, et al: Genetic classification of combined hepatocellular-cholangiocarcinoma. HUM PATHOL 31: 1011-1017, 2000 27. Bovee JV, Cleton-Jansen AM, Rosenberg C, et al: Molecular genetic characterization of both components of a dedifferentiated chondrosarcoma. J Pathol 189:454-462, 1999 28. Wada H, Enomoto T, Fujita M, et al: Molecular evidence that most but not all carcinosarcomas of the uterus are combination tumors. Cancer Res 57:5379-5385, 1997 29. Perry A, Giannini C, Scheithauer BW, et al: Composite pleomorphic xanthoastroyomas and gangliogliomaa: report of four cases and review of the literature. Am J Surg Pathol 21:763771, 1997 30. Perry A, Scheithauer BW, Szczesniak DM, et al: Combined oligodendroglioma/pleomorphic: a probable collision tumor: case report. Neurosurgery 48:1358-1361, 2001 31. Molnar P, Hegedus K: Adjacent astrocytoma and ependymoma: Dependent mixed neoplasia or unusual collision tumors? Surg Neurol 22:455-460, 1984 32. Marie Y, Sanson M, Mokhtari K, et al: OLIG2 as a specific marker of oligodendroglial tumour cells. Lancet 358:298-300, 2001 33. Lengauer C, Kinzler KW, Vogelstein B: Genetic instabilities in human cancers. Nature 396:643-649, 1998 34. Zhu JJ, Santarius T, Wu X, et al: Screening for loss of heterozygosity and microsatellite instability in oligodendrogliomas. Genes Chromosomes Cancer 21:207-216, 1998
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variants of group 1 OA with heterogeneous genetic changes during clonal expansion. The X-chromosome inactivation analysis confirmed the monoclonality of groups 1 and 2 OA. Group 3 consisted of two tumors that showed divergent allelic loss patterns in the 2 histologic components. Mutation and overexpression of TP53 were detectable in the astrocytic components only. These findings raise the possibility that group 3 tumors have a biclonal origin. In conclusion, our results suggest that OA are predominantly of monoclonal origin but that a small subset of tumors may be derived from different precursors. HUM PATHOL 33:528-535. Copyright 2002, Elsevier Science (USA). All rights reserved. Key words: oligoastrocytoma, clonality, loss of heterozygosity, TP53, microdissection, X-chromosome inactivation. Abbreviations: OA, oligoastrocytoma; GFAP, glial fibrillary acidic protein; WHO, World Health Organization; PCR, polymerase chain reaction; LOH, loss of heterozygosity; SSCP, single-strand conformation polymorphism.
Oligoastrocytomas (OA) are glial tumors composed of 2 admixed neoplastic cell types that show morphologic features of oligodendroglioma and diffuse astrocytoma.1 The 2 histologic components of OA may be either intermingled (diffuse type) or separated into distinct areas (biphasic or compact type).1 Because of a lack of reliable immunohistochemical markers, diagnosis of OA may be subjective, with significant interobserver variation, and the delineation of neoplastic oligodendroglial cells, especially the minigemistocytes, from neoplastic astrocytes is not always easy.1,2 The differentiation of OA from astrocytomas is of clinical importance, because recent studies have indicated that the former are sensitive to chemotherapy containing procarbazine-lomustine-vincristine, whereas pure astrocytomas are largely nonresponsive to such treatment.2-4 Misdiagnosis of OA may therefore have a great impact on patient, resulting in inappropriate or delayed treatment. Recent advances in molecular genetic analysis of gliomas have greatly contributed to our understanding of the genetic events underlying the development of these tumors. Oligodendrogliomas commonly have deletions on chromosomes 1p and 19q, and additional
deletions on 9p and 10q are seen in the anaplastic variants.5-9 In contrast, diffuse astrocytomas are characterized by allelic loss of chromosome 17p, TP53 gene mutation, or both.10 Additional genetic lesions, such as losses of 10q and 19q, CDKN2A deletion, and RB1 alteration, are observed in the late stages of tumor development.5,11,12 OA are found to have genetic features characteristic of both oligodendrogliomas and astrocytomas.5-9 The histogenesis of OA remains unresolved. Investigating tumor clonality is one approach to providing clues to the origin of cells. Whether OA arise monoclonally from a single precursor cell with divergent oligodendroglial and astrocytic differentiation capability or polyclonally from simultaneous transformation and proliferation of 2 cell types within the same tumor is not known. Investigation of these alternatives has concentrated on a search for common genetic markers within the 2 components, which is suggestive of a common progenitor cell. Genetic study by Maintz et al13 suggests that there are at least two genetic subtypes of OA: 1 type showing alterations in 1p and 19q that may be genetically related to oligodendroglioma, and the other with TP53 mutations that may be genetically related to astrocytomas.13 In 1995, Kraus et al14 performed microsatellite analysis on three microdissected OA by conventional methods and detected identical allelic loss patterns on 1p and 19q in both histologic elements.1 Using an X-chromosome inactivation assay, Kattar et al15 found a monoclonal pattern in one informative anaplastic OA. These results indicate that the histologic components of OA have common molecular features and hint that OA may be monoclonal in origin.14,15
From the Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, and Department of Neurosurgery, Hua Shan Hospital, Shanghai, China. Accepted for publication December 17, 2001. Supported by a grant (CUHK4277/99M) from the Research Grant Council of Hong Kong. Address correspondence and reprint requests to Ho-Keung Ng, MD, Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong. Copyright 2002, Elsevier Science (USA). All rights reserved. 0046-8177/02/3305-0012$35.00/0 doi:10.1053/hupa.2002.124784
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FIGURE 1. Case OA8. (A) Typical oligodendroglial region. (B) Adjacent astrocytic region. (C) Region showing increased cellularity, cellular atypia and mitosis (arrow). (Hematoxylin and eosin; original magnifications, A, ⫻150; B, ⫻150, C, ⫻300.)
Although previous genetic investigations on OA have suggested that these tumors are derived from a single precursor cell, the limited numbers of genetic markers and informative samples examined might not have revealed the full spectrum of clonality patterns.14,15 The aim of the present study, therefore, was to investigate the clonality of OA using an extensive array of tumor-dependent genetic markers and tumorindependent X-chromosome inactivation analysis. Our results suggest that OA are predominantly of monoclonal origin, although a subset of tumors may be derived from different precursors.
cytes in the oligodendroglial areas (Fig 3). Synaptophysin staining, which was negative in all tumor cells, excluded any neuronal tumor that could masquerade as an oligodendroglial tumor. The MIB1 staining showed a distinct difference between the grade II and grade III tumors. The former had staining in less than 5% of the cells, whereas the latter had staining exceeding 15%. No area of any tumors stained less than 3%. All tumors were reviewed by 2 neuropathologists (H.K.N. and Z.Q.D.). There were 8 male and 3 female patients, with ages ranging from 18 to 60 (mean, 39 ⫾ 12) years. Table 1 summarizes the clinical data of the 11 patients. Matched blood samples were also obtained for all patients.
Microdissection and DNA Extraction
MATERIALS AND METHODS Tumor Specimens A total of 11 cases of oligoastrocytic tumors were retrieved from the archives of our hospitals. Diagnostic criteria were those of the World Health Organization (WHO).1 The oligodendroglial component consisted of typical honeycomb areas, sometimes with minigemistocytes. Astrocytic areas exhibited features of fibrillary, protoplasmic, or classic gemistocytic astrocytomas. All cases were of the biphasic type, as described in the WHO classification.1 The proportion of the smaller component always exceeded 20% and consisted of nodular aggregates that could be delineated and microdissected from the other component. Seven tumors were WHO grade II, and 4 tumors were anaplastic OA, WHO grade III.1 Anaplastic tumors had obvious pleomorphic and atypical features and prominent mitotic figures, sometimes combined with necrosis and endothelial proliferation (Figs 1 and 2). The archival slides and special preparations were retrieved, and in some cases, special stains were performed so that all 11 cases ultimately underwent glial fibrillary acidic protein (GFAP), vimentin, MIB1, and synaptophysin staining. The GFAP and vimentin stains decorated and highlighted fine astrocytic processes in the astrocytic areas, and GFAP also showed some entrapped astrocytes or revealed minigemisto-
The oligodendroglial and astrocytic components of the tumors were microdissected either manually or by laser capture microdissection (PixCell LCM System; Arcturus Engineering, Mountain View, CA) from formalin-fixed, paraffinembedded sections as described previously.16 Precautions were taken in microdissecting the individual histologic components, and areas in which intermingled cell types were observed were excluded. The microdissected tissues were collected into microtubes containing 100 L of digestion buffer consisting of 50 mM Tris-HCl, pH 8.9, 2 mM ethylenediaminotetraacetic acid, 0.5% Tween-10, and 500 g/mL proteinase K. Digestion was carried out overnight at 55°C, followed by inactivation of protease at 95°C for 10 minutes. The resultant aliquots containing chromosomal DNA were used for subsequent genetic analyses. DNA from tumor-matched blood samples was purified using the standard phenol/chloroform protocol and used as constitutional controls.17
Microsatellite Analysis Microsatellite analysis was performed on the microdissected OA components according to a reported protocol.16 A panel of 31 polymorphic markers that cover chromosomes 1p, 9p21.3-22.1, 10q, 13q14.11-34, 17p, and 19q were used.
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FIGURE 2. Case OA9. (A) Necrosis adjacent to area with oligodendroglial differentiation; (B) endothelial proliferation; (C) astrocytic area. (Hematoxylin and eosin; original magnifications, A, ⫻150; B, ⫻300; C, ⫻150.)
Briefly, polymerase chain reaction (PCR) was performed in a final volume of 5 L containing DNA, 10 mM Tris HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 62.5 M deoxyribonucleoside triphosphate, 0.5 unit of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA), and 2.5 pmoles of each primer, with 1 of them radioactively end-labeled with [␥-32P]-ATP. The PCR amplification commenced with an enzyme activation step at 95°C for 10 minutes, 40 cycles of 94°C for 1 minute, 55 to 60°C for 1 minute, and 72°C for 1 minute, and finalized with an extension step of 10 minutes at 72°C. The PCR products were resolved on 6% denaturing polyacrylamide gels and then subjected to autoradiography for 4 to 24 hours at room temperature. The analysis of allelic loss was based on the comparison of signal intensities of the maternal and paternal alleles with the normal blood and tumor samples. Loss of heterozygosity (LOH) was inferred by a 70%
reduction of allele signal intensity in tumor samples relative to that of the matched constitutional DNA samples.
X-chromosome Inactivation Analysis Analysis of X-chromosome inactivation detects the methylation pattern of the androgen receptor (HUMARA) gene, located on chromosome X.18 Genomic DNA was chemically modified with sodium bisulfite using the CpGenome DNA modification kit (Intergen, Purchase, NY) to convert unmethylated cytosine in DNA to uracil while leaving the unmethylated cytosine intact. PCR was then performed to amplify the polymorphic locus within the first exon of HUMARA using methylation-specific and unmethylation-specific primers. The primers used for amplifying the unmethylated allele of HUMARA were UF (5⬘-GGTTGTGAGTGTAGTATTTTTT-
FIGURE 3. (A) GFAP staining of oligodendroglial area showing mostly negative cells; minigemistocytes have a thin rim of immunopositivity. (B) GFAP staining of astrocytic area. (C) Vimentin staining of another astrocytic area. (Original magnification, A, ⫻300; B, ⫻300; C, ⫻300.)
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TABLE 1. Clinical Information of Patients having Oligoastrocytomas Case no.
Sex
Age (years)
OA-1 OA-2 OA-3 OA-4 OA-5 OA-6 OA-7 OA-8 OA-9 OA-10 OA-11
F F M M M M M F M M M
33 50 42 47 28 18 31 43 60 39 46
Histologic diagnosis
AOA AOA AOA AOA
OA OA OA OA OA OA (AO ⫹ A) (AO ⫹ A) (O ⫹ AA) (AO ⫹ A) OA
WHO grade II II II II II II III III III III II
Abbreviations: AOA, anaplastic oligoastrocytoma; O, oligodendroglial component; A, astrocytic component; AO, anaplastic oligodendroglial component; AA, anaplastic astrocytic component.
GGT-3⬘) and R (5⬘-TAAAAAAAACCATCCTCACC-3⬘), whereas those for methylated allele were MF (5⬘-CGAGCGTAGTATTTTTCGGC-3⬘) and R.18 The PCR conditions were essentially the same as those described for microsatellite analysis, except that the annealing temperature was set at 55°C and nonradioactive primers were used. The PCR products were resolved by 12% polyacrylamide gel electrophoresis, stained with ethidium bromide and visualized under ultraviolet illumination.
PCR Single-Strand Conformation Polymorphism and Direct Sequencing of TP53 Exons 4 to 8 of the TP53 gene were PCR amplified and subjected to single-strand conformation polymorphism (SSCP) analysis as previously described.19 DNA of abnormal shifts visualized in SSCP gels were eluted, PCR reamplified, and sequenced using the BigDye Terminator Cycle Sequencing kit (Applied Biosystems). Each mutation was verified at least twice independently.
Immunohistochemical Analysis of TP53 Protein Formalin-fixed, paraffin-embedded tissue sections from the same blocks used for microdissection were processed for TP53 immunostaining using the avidin-biotin-peroxidase complex method as previously described.16 The anti-human TP53 monoclonal antibody DO-7 (diluted 60-fold; Dako, Carpentiera, CA), which detects both wild-type and mutant TP53 proteins, was used. An adenocarcinoma of the colon with TP53 overexpression was used as a positive control. For a negative control, the primary antibody was omitted in the immunohistochemical process. The detection of immunostaining in the nuclei of tumor cells was considered positive for TP53 and was scored semiquantitatively as ⫹, ⫹⫹, and ⫹⫹⫹ when 5% to 25%, 26% to 50%, and ⬎50% of the cells stained positively, respectively.
RESULTS Microsatellite Analysis Previous investigations have shown that OA share genetic features with oligodendrogliomas and astrocy-
tomas. We hypothesized that a comparison of the genetic patterns in the oligodendroglial and astrocytic components of OA would provide insights into tumor clonality. Thus, we selected 7 critical chromosome regions known to be frequently deleted in oligodendrogliomas and astrocytomas and evaluated their LOH patterns in the microdissected components of OA. These regions were 1p, 9p21 (CDKN2A and CDKN2B loci), 10q23 (PTEN locus), 10q26.13 (DMBT1 locus), 13q (RB1 locus), 17p (TP53 locus), and 19q. Eleven biphasic OA were studied by microsatellite analysis. In each case, both oligodendroglial and astrocytic components were informative for at least 23 of the 31 microsatellite loci examined (Table 2). LOH was detected in at least 1 marker of chromosomes 1p (9 of 11 cases), 9p21-22 (5 cases), 10q22-23 (3 cases), 10q26.13 (6 cases), 13q14.11-34 (3 cases), 17p (5 cases), and 19q (9 cases). Of note is the absence of LOH on the intragenic markers of TP53 and PTEN (D10S2492) in all cases. On the basis of the genetic similarities between the oligodendroglial and astrocytic components, we divided the 11 OA into 3 groups. Group 1 consisted of 4 tumors: OA1, OA2, OA3, and OA11. These tumors displayed identical allelic loss patterns in the oligodendroglial and astrocytic components. Three cases (OA1, OA2, and OA3) showed LOH predominantly on chromosomal arms 1p and 19q. Case OA11 showed LOH for only 1 (D17S831) of the 22 informative markers examined. Five OA (OA5, OA7, OA8, OA9, and OA10) were allocated to group 2. These tumors were also characterized by concurrent LOH on chromosomes 1p and 19q, but additional allelic losses were found on chromosomes 9p, 10q, 13q, and/or 17p. At some loci, disparate LOH patterns at corresponding alleles were observed in the 2 histologic components. For example, OA5 showed concordant LOH of all informative markers on chromosomes 1p and 19q and on D10S1723 in both the oligodendroglial and astrocytic components, whereas allelic losses on markers D9S162, D10S541, and D10S587 were found in the astrocytic component only (Fig 4A). Following chromosomes 1p and 19q, the DMBT1 locus (D10S1723 and D10S587) was the next most frequently affected site in this group of OA, with 4 of 5 tumors showing allelic loss. More important, when the genetic data were correlated with tumor grade, we found that 4 of these 5 tumors were of the anaplastic type. Group 3 OA consisted of 2 tumors that showed entirely different LOH patterns in the 2 histologic components (Fig 4A). In OA4, LOH was detected in 1 marker each on chromosomes 19 (D19S596) and 10 (D10S216) specifically in the oligodendroglial element, whereas the astrocytic component showed retention of heterozygosity in all 24 informative markers that covered the 7 chromosomal arms. In OA6, 11 of 25 informative markers showed allelic loss or microsatellite instability. Notably, of the 6 markers (D1S458, D1S482, D9S162, D13S319, D17S831, and D17S799) that showed LOH in both histologic elements, the losses were found on different alleles at the corresponding
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TABLE 2. Summary of LOH, TP53 Immunohistochemical, and Mutation Analyses in Microdissected Components of Oligoastrocytomas Group 1 OA1 Microsatellite markers
Known genes
p73 TP73 D1S458 D1S482 D1S493 D1S186 D19S219 D19S412 D19S112 D19S596 D9S162 IFNA D9S736 D9S171 CDKN2A/B D9S1748 D9S1749 D10S215 D10S541 D10S2492 PTEN D10S216 D10S1723 DMBT1 D10S587 D13S267 D13S319 RB D13S158 D13S285 D17S849 D17S831 TP53 TP53 D17S1852 D17S799 D17S1303 TP53 immunohistochemistry TP53 mutation
Cytogenetic mapping 1p36.33 1p36.12 1p36.12 1p35.5 1p34.3 19q13.32 19q13.32 19q13.32 19q13.33 9p22.1 9p21.3 9p21.3 9p21.3 9p21.3 9p21.3 10q23.31 10q23.31 10q23.33 10q26.13 10q26.13 10q26.13 13q14.11 13q14.3 13q33.1 13q34 17p13.3 17p13.3 17p13.1 17p11.2 17p11.2 17p11.2
A
O
F F F F F F F E E E E E E E E E E E E E E
F F F F F F F E E E E E E E E E E E E E E
OA2 A
O
Group 2
OA3 A
OA11
O
A
O
OA5 A
O
OA7 A
O
OA8 A
O
Group 3 OA9 A
O
OA10 A
O
OA4 A
O
OA6 A
O
E E F F E E F F E E F F F F E E E E E E F F E E F F E E FL FS F F F F F F E F F F F F F F E E FL FS F F F F E E F F F F E E F F E E E E E E F F F F F F F F F F E F E E E E F F F F E E F F F F E E F F F F E E F F F F F F F F F F E E E E F F F F E E F F F F F F F F E E E E F F F F E E F F F F F F F F F F E F E E E E E E F E E E F F E E E E F L FS E E E E E E E E E E F F E E E E E E F F E E E E E E E E E E F F E E E E E E E E E E E E E E E E E E E E E E E E E E E E F F E E F F E E E E E E E E E E E E FS FL E E F E E E E F E E E E E E E E E E E E E E E E E E E E MI MI E E E E E F E E E E E E E F F E E E E E E E F F F F F F F F E E E E E E E E E E E E F E E E F F F F E E E E F E E E E E E E F F MI F E E E E E E E E E F E E E E E E F L FS E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E F F E E E E E E E E F F E E E F MI E E E F S FL E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E F E E E E E E E F S FL E E E E E E E E E F E E F F E E MI F ⫺ve ⫺ve ⫺ve ⫺ve ⫹ ⫹ ⫹ ⫹⫹ ⫺ve ⫹ ⫺ve ⫺ve ⫹ ⫹⫹ ⫹⫹ ⫹ ⫹ ⫹⫹ ⫹ ⫺ve ⫹ ⫺ve NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO E8 NO NO NO
Abbreviations: O, oligodendroglial component; A, astrocytic component; open circle, retention of heterozygosity; dark circle, loss of heterozygosity (L, longer allele; S, shorter allele); MI, microsatellite instability; ⫺, noninformative; gray boxes, genetic discrepancies between components detected; ⫺ve, immunonegative; ⫹, immunopositive cells ⬍ 25%; ⫹⫹, immunopositive cells 26% to 50%; NO, no mutation detected; E8, mutation found on exon 8, codon 273, from CGT (Arg) to TGT (Cys).
loci in each component. This tumor also exhibited microsatellite instability of 3 markers (see Table 2). X-Chromosome Inactivation Analysis Tumor clonality has been studied in female somatic cells using X-chromosome inactivation analysis based on differential methylation patterns of active (unmethylated) and inactive (methylated) alleles. The HUMARA gene contains a polymorphic short tandem repeat in the first exon, and hypermethylation of the inactive allele is associated with X-chromosome inactivation.18 The unmethylated and methylated polymorphic loci of HUMARA can be differentially detected using methylation-specific PCR. Three tumors from female patients (OA1, OA2, and OA8) were analyzed, and representative results are shown in Figure 4B. In the blood sample of OA8 (group 2), 2 PCR bands representing the 2 alleles of HUMARA were detected in both unmethylation and methylation reactions, indicating that the peripheral leukocytes were polyclonal in origin. In the astrocytic component, only 1 allele showed hypermethylation, whereas the second allele was found to be unmethylated in the unmethylation
PCR. The same methylation pattern was observed in the oligodendroglial component. Taken together, these results suggest that the histologic elements of OA8 were derived from the same precursor cell. Case OA2 of group 1 OA showed a methylation pattern similar to that of OA8. The HUMARA locus in OA1 was noninformative. Immunohistochemistry of TP53 Protein Eight cases of OA showed immunopositivity for TP53 in either the oligodendroglial and astrocytic component or both. In group 1 OA, concordant expression or nonexpression of TP53 protein was observed in both components. Four of 5 group 2 OA showed concordant overexpression of TP53 in both histologic elements. The exception was OA5, in which the TP53 protein was detectable only in the oligodendroglial component. In group 3 OA, the TP53 immunostaining pattern was different in the histologic elements. Expression of TP53 protein was detectable only in the astrocytic components, but not in the oligodendroglial portion.
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Mutation Analysis of TP53 Of the 11 OA studied, mutation of TP53 was detected only in the astrocytic component of a group 3 OA (case OA4). The missense mutation was localized to
exon 8, codon 273, CGT (Arg) 3 TGT (Cys) (Fig 4C), which is a hot spot for TP53 mutation in astrocytic tumors.10 DISCUSSION OA are admixed tumors containing oligodendroglial and astrocytic elements. Their precise diagnostic criteria are controversial,1 and their distinction from a diffuse infiltrating astrocytoma may be difficult.20 The cell of origin of each element has long been a clinicopathologic mystery. Two pathways whereby these tumors arise have been proposed: monoclonal or polyclonal expansion. It has even been suggested that oligodendrogliomas and OA share a uniform lineage from neuronal precursor A2B5⫹ cells.21 To investigate the clonality of OA, we examined the methylation pattern of X-chromosome inactivation; the LOH status of chromosomes 1p, 9p, 10q, 13q, 17p, and 19q, and the TP53 status in 11 microdissected tumors. On the basis of the genetic similarities between the histologic components, we divided the series into 3 groups. Group 1 OA display identical genetic alterations in the oligodendroglial and astrocytic components, supporting the notion that these 2 components are derived from the same precursor cell.14,15 The X-chromosome inactivation analysis also confirms that this group of tumors is monoclonal in origin. The precursor cells may have the ability to differentiate into oligodendroglial and astrocytic cells, leading to biphasic tumors. The latter hypothesis is supported by the finding that certain rat glial progenitor cells are capable of astrocytic and oligodendrocytic differentiation under different culture conditions.22 Three tumors (OA1, OA2, and OA3) showed allelic losses on both 1p and 19q, and 2 of these had normal TP53 status. These genetic changes resemble those of oligodendrogliomas, suggesting that OA and oligodendroglioma had a common cell of origin.14 All 5 cases of group 2 OA are characterized by allelic loss of 1p and 19q, compatible with genetic features of oligodendrogliomas. The high concordance of genetic patterns on chromosomes 1p and 19q and the methylation pattern on HUMARA suggest that these tumors, like group 1 tumors, are derived from a 4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ FIGURE 4. (A) Microsatellite analysis of microdissected components of oligoastrocytomas. OA5 shows allelic loss of D10S587 in the astrocytic component, whereas OA7 displays LOH on D17S831 in the oligodendroglial tumor portion. Genetic discrepancies between components at loci D17S799 and D17S1303 were detected in OA6. A, astrocytic component; O, oligodendroglial component; B, matched constitutional control; L, loss of heterozygosity; R, retention of heterozygosity; N, normal allelic pattern; MI, microsatellite instability. (B) X-chromosome inactivation analysis of the HUMARA gene by methylation-specific PCR. Results suggest that OA2 is monoclonal in origin. B, matched constitutional control; A, astrocytic component; O, oligodendroglial component; U, unmethylation; M, methylation. (C) Sequencing profile of the TP53 gene in antisense orientation. A missense mutation was found at exon 8, codon 273, CGT (Arg) 3 TGT (Cys) in the astrocytic component of OA4.
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single precursor cell. Combining these tumors with group 1 OA, our results indicate that most biphasic OA have a monoclonal origin. In addition, more allelic losses on chromosomes 9p, 10q, 13q, and/or 17p were found in group 2 tumors. Notably, 4 of 5 tumors in this group are classified as WHO grade III, supporting the view that advanced gliomas accumulate more genetic alterations.8,9,23 Thus, group 2 tumors may be regarded as an advanced variant of group 1 OA. We also noted occasional genetic discrepancies in the 2 histologic components at some microsatellite loci. Such dissimilarities between the components may be explained by the acquisition of different genetic changes in the 2 components during tumor development. Our data also indicate that allelic loss at the DMBT1 locus on chromosome 10q26.13 is frequently (in 4 of 5 cases) detectable in group 2 OA. Complete loss of chromosome 10 has been shown previously in high-grade oligodendrogliomas.24 Maier et al25 also reported partial deletion of chromosome 10q25-26 in oligodendrogliomas. These results suggest that DMBT1 is involved in the progression of oligodendroglial tumors. Mutation analysis of the DMBT1 gene in oligodendroglial tumors is currently in progress in our laboratory. The most intriguing finding in our study is seen in 2 OA in group 3. These tumors show divergent genetic patterns in the oligodendroglial and astrocytic components. First, the allelic loss profiles are different. Two major types of LOH patterns are observed at the microsatellite loci: LOH versus retained alleles and LOH on different alleles in each component. The loss of different alleles at the corresponding loci in each neoplastic component suggests that these loci are more susceptible to deletion. This idea is supported by the observation that these loci (e.g., D1S482, D9S162, and D17S831) are also commonly deleted in other OA. Second, aberrant TP53 status is detectable in the astrocytic component only. Taken together, these results strongly suggest that group 3 OA evolved from 2 independent precursor cells, which give rise to tumors with different genetic backgrounds. “Collision” tumors are not uncommon and have been described in a variety of tumor types.26-28 These tumors are predominantly monoclonal in origin, but some show polyclonal genetic patterns.26,28 Collision tumors with glial differentiation have also been reported and mostly involved 2 different types of gliomas that existed in close proximity in the brain.29-31 In this study, we reported for the first time the detection of polyclonality in biphasic OA. The clonality findings in biphasic OA have raised an important question: whether such clonal patterns are also seen in diffuse OA. From previous genetic studies of OA, it is believed that diffuse OA, like their biphasic counterparts, are monoclonal in origin.13-15 The study of clonality in diffuse OA is hampered by the absence of a reliable method of separating the individual elements. This barrier may now be overcome by using the marker that can identify oligodendroglial cells and the latest laser capture microdissection system that can procure tissue on a single-cell basis.32 The 2
cell types can be separately isolated and their clonality patterns deduced by genetic methods. Microsatellite instability that is the signature of mismatch repair deficiency is seen early during tumorigenesis and increases as the tumors progress.33 In our series, 5 microsatellite instabilities were detected in a total of 538 informative markers, a finding consistent with the infrequent incidence of microsatellite instability in astrocytic and oligodendroglial tumors.8,34 In conclusion, our data suggest that OA are predominantly of monoclonal origin. The concurrent loss of chromosomes 1p and 19q in OA hints that these tumors and oligodendrogliomas have a common cell of origin. A subset of OA, in which the histologic elements show dissimilar genetic patterns, may be derived from different progenitor cells.
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