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© 2003 American Society for Clinical Oncology
Topoisomerase-II
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXAUTHORS’ DISCLOSURES OF...REFERENCES |
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Materials and Methods: Biopsies and clinical data from 51 patients with MPNST were included in this study. Based on our previous research implicating chromosome arm 17q amplification in MPNST, we performed gene expression analyses of 14 MPNSTs using chromosome 17–specific cDNA microarrays. Copy numbers of selected gene probes and centromere probes were then determined by interphase fluorescence in situ hybridization in 16 MPNSTs. Finally, we generated a tissue microarray containing 79 samples from 44 MPNSTs, on which in situ protein expressions of candidate genes were examined and related to clinical end points.
Results: Among several deregulated genes found by cDNA microarray analyses, topoisomerase II
(TOP2A) was the most overexpressed gene in MPNSTs compared with benign neurofibromas. Excess copies of the TOP2A were also seen at the DNA level in 10 of 16 cases, and high expression of the TOP2A protein was seen in 83% of the tumors on the tissue microarray. The TOP2A-expressing tumors were associated with poor cancer-specific survival and presence of metastases.
Conclusion: We have identified TOP2A as a target gene in MPNST, using a focused gene expression profiling followed by a DNA copy number evaluation and clinical validation of the encoded protein using a tissue microarray. This study is the first to suggest that TOP2A expression may be a predictive factor for adverse outcome in MPNST.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXAUTHORS’ DISCLOSURES OF...REFERENCES |
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The NF1 gene maps to 17q11.2 and encodes a protein called neurofibromin.7–9 One of its known functions is to downregulate p21-RAS by accelerating the conversion of the active GTP-bound form into its inactive GDP-bound form. Inactivation of NF1 in tumors leads to increased RAS signaling and increased cell proliferation. Potential therapies include inactivation of RAS by preventing posttranslational modifications like farnesylation, or to block downstream targets of the RAS mitogenic signaling pathway.4
Neurofibromin is expressed in the Schwann cells, from which the MPNSTs are believed to have their origin.1,10 About two-thirds of all MPNSTs develop through a neurofibroma stage, often of the plexiform type, and in the setting of NF1.1 The fact that MPNST is frequently found in NF1 patients carrying a germline mutation of NF1 suggested that a second hit leading to a nonfunctional gene would contribute to tumor development. Indeed, complete inactivation of NF1 has been found in benign neurofibromas, thereby demonstrating that NF1 inactivation is not sufficient for malignant transformation.11 Mice heterozygous for mutations in both Nf1 and Tp53 develop MPNSTs, suggesting the contribution of an altered TP53 pathway also in development of human MPNST.12,13 Alterations in the TP53 gene have been found in some MPNSTs,14–17 but biallelic inactivation of TP53 is rare.18 Other central components of the cell cycle have been studied, and CDKN2A is deleted or rearranged in a large subgroup of these tumors.19–21
MPNSTs have been estimated to account for approximately 5% of all soft tissue sarcomas. In contrast to several other soft tissue sarcomas, the MPNSTs do not have a disease-associated chromosome translocation, and their karyotypes are often complex.22,23 We and others have, by a molecular cytogenetic approach, found that gain of distal 17q sequences is frequently found in MPNST.24–26 Schmidt et al25 further showed that a gain of 17q was associated with poor disease outcome. In order to identify the relevant genes on chromosome 17, we present a focused expression profiling of a series of MPNSTs using a chromosome 17–specific cDNA microarray. To validate the resulting candidate genes, interphase fluorescence in situ hybridization (FISH) was used to evaluate the DNA copy numbers within specific loci, and clinical associations were assessed by in situ protein expression analyses on a tissue microarray.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXAUTHORS’ DISCLOSURES OF...REFERENCES |
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Fourteen MPNST samples and three benign neurofibromas from patients with MPNST (15 patients altogether) were included in the cDNA microarray analysis. The mRNA from the neurofibromas was pooled and analyzed together on one microarray to obtain an average expression profile of benign lesions. Seven of the MPNSTs were from NF1 patients, and seven were tumors from sporadic cases. The MPNSTs had previously been analyzed by comparative genomic hybridization (CGH), and there were seven with, and seven without gain involving the previously reported common region of gain at 17q (Fig 1B
; Lothe et al, unpublished data).24 A pool of two breast cancer cell lines, HBL100 and MDA-436 (ATCC, Manassas, VA), was used as a common reference in all cDNA microarray experiments. These cell lines were selected based on the fact that they show no increase in copy number at 17q and the knowledge that they express most genes on the cDNA microarray to some extent.27–29
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The tissue microarray was constructed of 44 primary MPNSTs of 41 patients admitted to the Norwegian Radium Hospital during the years 1980 to 2000. Histologic malignancy grade was available for 42 tumors, of which 10 were low grade, and 32 were high grade. Fifteen patients were in complete remission, four were alive with disease, 21 were dead of sarcoma, and one died of causes unrelated to sarcoma. The surviving patients were followed up for a median of 97 months (range, 30 to 225 months). Seven of the patients had developed metastases at the time of diagnosis, whereas, altogether, 18 patients (20 tumors) had metastases detected at diagnosis and/or during follow-up. All primary tumors were surgically removed, and 20 patients received radiotherapy. Thirteen patients received chemotherapy including the topoisomerase II inhibitors etoposide and/or doxorubicin. Selection criteria for chemotherapy were local recurrence and/or distal metastases (n = 8), primary inoperable tumor (n = 2), or adjuvant treatment for patients at high risk of developing metastases (n = 3). The median age at diagnosis was 26 years (range, 15 to 71 years) for the patients diagnosed with preceding NF1 (n = 19), and 50 years (range, 20 to 86 years) for the sporadic cases (n = 22).
cDNA Microarray Experiments
Construction of the cDNA microarray with comprehensive coverage of chromosome 17 has been previously described.28,29 The microarray consisted of printed polymerase chain reaction products from 636 sequence-verified IMAGE cDNA clones (Research Genetics, Huntsville, AL), including 201 known genes from the entire chromosome 17, and 435 expressed sequence tags from the 17q arm. An additional 88 housekeeping genes were placed on the array and were used for calibration among the different experiments.30
Preparation and printing of the cDNA clones on glass slides, probe preparations, hybridizations, and image generation and analyses were performed as described previously.31 In brief, mRNA was isolated from the test samples using the Trizol reagent (Life Technologies, Rockville, MD) and oligo(dT)25 dynabeads (Dynal Biotech, Oslo, Norway) according to the manufacturers’ specifications. From the reference cell lines, mRNA was isolated directly by using FastTrack 2.0 mRNA isolation kit (Invitrogen, Carlsbad, CA). Labeled cDNA was synthesized from 1 to 3 µg or 5 µg mRNA (test or reference, respectively) in an oligo(dT)-primed polymerization with SuperScript II reverse transcriptase (Life Technologies) in the presence of either Cy3- (test) or Cy5- (reference) labeled dUTP (Amersham Pharmacia, Piscataway, NJ). Cy3-labeled test cDNA from each of 14 MPNSTs and from a cDNA pool of three neurofibromas were cohybridized with Cy5-labeled reference cDNA onto the cDNA microarray. The fluorescence intensities at the targets were detected by a laser-confocal scanner (Agilent Technologies, Palo Alto, CA).
FISH
In order to examine the copy number at the gene level, FISH was performed as previously described.32 In brief, freshly frozen tumor samples were minced, fixed in methanol and acetic acid (3:1), and further treated with 60% acetic acid to dissociate single nuclei. FISH was performed with probes targeting the topoisomerase-II (TOP2A) (17q21.2) and ERBB2 (17q12) loci, and each locus-specific probe was cohybridized with a chromosome 17 centromere-specific probe (all probes: Vysis Inc, Downers Grove, IL). The nuclei and probes were denatured, and were subsequently hybridized for 16 hours at 37°C. The slides were then washed, dried, and counterstained with 1.5 µg/mL 4',6-diamino-2-phenylindole in Vectashield antifade (Vector Laboratories, Burlingame, CA). Fluorescent signals in approximately 100 nuclei per tumor were counted, and amplification was scored when the locus-specific probe had at least twice the number of spots as the centromere probe in more than 10% of the tumor cell nuclei. A control experiment with normal lymphocyte nuclei was performed, from which only one and five of 100 nuclei had more than the expected two copies of TOP2A and ERBB2, respectively.
Immunohistochemistry on Tissue Microarray
A tissue microarray33 was built up by transferring 79 cylindrical tissue cores (0.6 mm in diameter) from 44 formalin-fixed and paraffin-embedded MPNSTs using an in-house–made robotic tissue microarrayer. Sections 5 µm in thickness were transferred onto slides using the Instrumedics paraffin tape-transfer system (Instrumedics, Hackensack, NJ). Tissue microarray sections for immunohistochemistry were stained using the biotin–streptavidin-peroxidase method (Supersensitive Immunodetection System, LP000-UL; Biogenex, San Raman, CA) and OptiMax Plus Automated Cell Staining System (BioGenex). One tissue microarray section for each antibody was deparaffinized and rehydrated, and high-temperature antigen unmasking was applied for the TOP2A and Ki-67 antibodies (10 minutes boiling in a pressure cooker with 10 mmol/L citrate buffer [pH, 6.0] for TOP2A, and 5 x 5 minutes boiling in a microwave oven with 10 mmol/L citrate buffer [pH, 6.0] for Ki-67). The slides were then incubated with 1% hydrogen peroxide (H2O2) for 10 minutes to block the endogenous peroxidase activity before 30 minutes incubation at room temperature with the primary antibodies (1:20 dilution of mouse monoclonal anti-TOP2A, clone 3F6 [Novocastra Laboratories Ltd, Newcastle, UK]; and 1:25 mouse monoclonal anti-Ki-67, clone Ki-S5 [DAKO, Glostrup, Denmark]). The sections were then incubated for 20 minutes with multilink biotinylated anti-immunoglobulins (1:30; BioGenex), and 20 minutes with streptavidin peroxidase (1:30; BioGenex), followed by 5 minutes staining with 0.05% of the peroxidase substrate 3'3-diaminobenzidine tetrahydrochloride, freshly prepared in 0.05M Tris–HCl buffer at pH 7.6, containing 0.01% H2O2. Finally, the sections were counterstained with hematoxylin, dehydrated, and mounted.
For both TOP2A and Ki-67, tumors with nuclear staining in more than 5% of the neoplastic cells were classified as positive, while those with less than 5% were considered negative. Most tumors had replicate tissue cores on the tissue microarray, and a tumor was considered positive when one or more of its tumor tissue cores were positive.
Statistical Analysis
For each array element, a ratio between the relative fluorescence intensities of the test and reference was calculated. This ratio was divided by the average expressions of the 88 housekeeping genes, giving a calibrated ratio, which was then normalized, by dividing it by the average calibrated ratio of the pooled neurofibroma samples. Thus, this normalized ratio reflects relative upregulated or downregulated gene expression from benign to malignant tissues. Genes with a difference in transcript levels between groups of experiments were assessed by Significance Analysis of Microarrays (SAM).34 One hundred permutations were made, and the delta value giving the lowest false discovery rate was chosen to set the cutoff for significantly altered genes. Microarray spots with less than 100 area units, or fluorescence intensities weaker than 200 fluorescence units, were regarded as missing data. For the SAM analyses, missing data from the cDNA microarray analyses were imputed by the k-nearest neighbor average in the gene space (k = 10).
For the TOP2A and Ki-67 immunohistochemistry results, comparisons of different groups were tested with a two-sided Fisher’s exact test. Sarcoma-specific survival analyses were performed by the Kaplan-Meier method, and the differences were assessed using the log-rank test. Patients who died of causes unrelated to sarcoma were censored at the time of death. One patient experienced both a TOP2A-negative MPNST and a TOP2A-positive MPNST (6 years later), and was thus excluded from the survival analyses.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXAUTHORS’ DISCLOSURES OF...REFERENCES |
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and Supplementary Table [Appendix]). To identify significantly overexpressed genes in the MPNSTs compared with the neurofibromas, one-class SAM analysis was applied to the data set. Here, TOP2A and fifteen additional genes were identified as significantly overexpressed in the MPNSTs compared with the neurofibromas (Table 1). By using two-class SAM analyses, no genes were detected as having significantly different transcript levels in NF1-associated versus sporadic MPNSTs, or between tumors with and without 17q gain as seen by CGH. Expression of the NF1 gene was comparable in the MPNSTs and in the pool of neurofibromas.
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). Among the tumors with amplification, there were, on average, 32% of the nuclei that had four or more TOP2A copies. Six tumors had excess ERBB2 copies, all of which also had TOP2A amplification. Finally, one additional tumor revealed a trisomic pattern for chromosome 17, inasmuch as TOP2A, ERBB2, and centromere 17 were each present in three copies in more than 80% of the nuclei.
The immunohistochemical analysis of TOP2A protein expression on the tissue microarray revealed 35 of the 42 successfully scored tumors as positive (83%; Fig 1E). Protein expression of the proliferation marker Ki-67 showed positivity in 58% of the samples (23 of 40). Although there was a significant association between TOP2A and Ki-67 expression (P = .001), 10 TOP2A-positive tumors were negative for Ki-67. Among those 10, six developed metastases, and in fact, all 20 tumors that metastasized were TOP2A-positive, compared with 71% of those with no metastases (P = .01). For Ki-67, these percentages were 68% and 48% (P = .21).
The positive immunoreactivities of both TOP2A and Ki-67 showed association trends to poor sarcoma-specific survival (both log-rank P values = .08; Fig 1F). There was a 45% difference in projected sarcoma-specific survival at 5 years between patients with TOP2A-positive tumors versus TOP2A-negative tumors (38% v 83%). Similarly, there was a 28% difference between patients with Ki-67–positive tumors versus patients with Ki-67–negative tumors (36% v 64%). Twelve of the 13 patients who received chemotherapy with a topoisomerase inhibitor had TOP2A-positive tumors. To date, two patients are alive and in complete remission, including the patient with a TOP2A-negative tumor. For the 10 patients who had measurable disease at the time of chemotherapy, four obtained radiological response, and three obtained a good histological response to treatment. The association trend between TOP2A expression and overall patient survival was retained even after 11 patients who received chemotherapy containing a TOP2A inhibitor were excluded from the survival analysis (P = .17).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXAUTHORS’ DISCLOSURES OF...REFERENCES |
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The current expression profiling of chromosome 17–specific genes in MPNST demonstrated several genes as altered, of which TOP2A was identified as the most highly overexpressed. TOP2A was also investigated in the genome-wide expression profiling of other soft tissue sarcomas,35 and variation in expression was seen across the data set. Because these data were not compared with a benign precursor lesion (or normal counterpart) we cannot directly compare the extent of gene expression levels with the those of this study.
A FISH analysis targeting TOP2A demonstrated that most of the tumors also have excess DNA copies of the gene, but even MPNSTs without the genomic amplification showed overexpression of TOP2A, indicating that this normally well-regulated gene is upregulated also by other mechanisms. Within the genomic region of TOP2A, ERBB2 is another well-characterized gene, often targeted by genome amplification in cancer.36 However, this gene was not overexpressed in these tumors, and the fact that ERBB2 was only amplified along with TOP2A, whereas four tumors showed amplification of TOP2A alone, further strengthens the latter as an important gene in development of MPNST.
Immunohistochemical analysis by use of a tissue microarray was performed to validate the involvement of TOP2A in MPNST development. Eighty-three percent of the tumors were positive for TOP2A protein expression. Interestingly, TOP2A expression showed a trend to be associated with increased cellular proliferation, primary metastatic disease, and poor outcome. The reduced survival rate seen for patients with TOP2A-positive tumors is in accordance with the association between increased copy number of chromosome arm 17q and survival, which has been both previously published25 and confirmed by us in an unpublished series of CGH results on 38 MPNSTs. In our initial CGH study of 10 MPNSTs, one tumor limited the smallest overlapping region to 17q24-ter.24 However, in the larger series (N = 38), the CGH data set revealed that the gains on 17q generally include the 17q21 chromosome band, and thus encompass the TOP2A locus.
As TOP2A expression is coupled to the cell cycle, and reaches a peak in late S phase, its expression level is generally highest in proliferating tissues.37,38 Therefore, we investigated whether the TOP2A-positive and TOP2A-negative groups could be distinguished by the Ki-67 proliferation marker, which was recently reported as an indicator of poor prognosis in MPNST.39 Although the association did not reach statistical significance, Ki-67 immunoreactivity seemed to predict poor clinical outcome also in the present study (28% survival rate difference; P = .08). Furthermore, we observed a correlation between Ki-67 and TOP2A expression. However, a distinct group of 10 TOP2A-positive but Ki-67–negative tumors was identified, indicating that the TOP2A expression does not merely reflect the proliferative status of the tumor. Moreover, when looking at the relation to metastasis, TOP2A immunopositivity was a stronger predictor than Ki-67.
The TOP2A enzyme is the primary cellular target for many of the most widely used and effective antineoplastic agents, including etoposide and doxorubicin.40 The TOP2A enzyme resolves knots and tangles in DNA by passing an intact helix through a transient double-stranded break that it generates in a separate helix. TOP2A inhibitors increase the steady-state level of cytotoxic intermediates, causing DNA double-strand breaks and inducing apoptosis.40 It has been shown in vitro that the cellular TOP2A level is related to chemosensitivity to certain TOP2A inhibitors.38,41–43 Despite high expression of TOP2A, chemotherapy seemed not to affect survival, as only two of the 13 patients receiving chemotherapy containing a TOP2A inhibitor are, to date, alive. However, all patients given chemotherapy had one or several risk factors for poor outcome, and firm conclusions regarding the efficacy of TOP2A inhibitors in MPNST cannot be made in this limited series. Coexisting drug resistance mechanisms may also contribute to chemotherapy resistance. P-glycoprotein, a transmembrane efflux pump which eliminates TOP2A inhibitors from their target sites, is generally expressed in soft tissue sarcomas,44,45 and may hence be an explanation.
In conclusion, a detailed and focused gene expression profiling of a common genomic aberration in MPNST pinpointed TOP2A as a strong candidate gene in MPNST progression. The expression of TOP2A was further evaluated on the protein level by immunohistochemistry on a tissue microarray, confirming its overexpression and revealing associations with proliferation, metastatic disease, and poor outcome. As TOP2A is the molecular target of several well-established chemotherapeutic agents, these findings highlight the possible predictive impact of TOP2A expression in MPNST, which should be evaluated in future prospective clinical trials.
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APPENDIX |
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AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENT |
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s for expert assistance on the molecular cytogenetics. |
NOTES |
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REFERENCES |
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Submitted July 10, 2003; accepted October 6, 2003.
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Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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Is Upregulated in Malignant Peripheral Nerve Sheath Tumors and Associated With Clinical Outcome
