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Genetics of malignant pleural mesothelioma: molecular markers and biologic targets
Thorac Surg Clin 14 (2004) 461 – 468
Genetics of malignant pleural mesothelioma: molecular markers and biologic targets Raphael Bueno, MD, Gavin J. Gordon, PhD* Department of Surgery, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA Division of Thoracic Surgery, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA Thoracic Surgery Oncology Laboratory, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA
Malignant pleural mesothelioma (MPM) is a mesodermally derived neoplastic disease that arises in the pleura and grows relentlessly into adjacent structures until it ultimately results in death of the patient. Unlike many other cancers, MPM does not metastasize until late in the course of the disease [1,2]. Asbestos is a major factor in the pathogenesis of this malignancy, with up to 80% of patients with MPM noting a history of asbestos exposure. Approximately 3000 patients are diagnosed with MPM in the United States annually, and the incidence worldwide is projected to continue to rise in the next two decades [2 – 7]. MPM is a unique malignancy. The most common cause, asbestos, is known, but the mechanisms of carcinogenesis are not fully understood, and the latency period is prolonged. Another characteristic peculiar to MPM is the multifocal location of tumor around the pleura at the time presentation, even in early disease. Unlike lung cancer, MPM respects tissue planes until a relatively advanced stage. As a consequence, a pleurectomy can be performed to remove the tumor-laden pleural envelope of vital tissues, such as the aorta, spine, and heart. Defining the genetic abnormalities that lead to MPM tumor
* Corresponding author. Division of Thoracic Surgery, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115. E-mail address: [email protected] (G.J. Gordon).
cells is important for the development of therapeutic options for this malignancy.
Diagnosis and staging of malignant pleural mesothelioma MPM has four distinct histologic subtypes: epithelial, sarcomatoid, mixed (biphasic), and transitional (undifferentiated). Most cases of MPM are epithelial, and patients with this subtype generally have a longer survival than is associated with the other subtypes. The differential diagnosis of MPM is broad and related to the histologic subtype under consideration. It includes adenocarcinoma of the lung, atypical mesothelial hyperplasia, chronic pleuritis, sarcoma, metastatic adenocarcinoma, solitary fibrous tumor of the pleura, and occasionally lymphoma, melanoma, and spindle cell carcinoma [8]. The need to distinguish MPM from these other conditions has been met partially by a combined approach using histology, immunohistochemistry, cytology, electron microscopy, cytogenetics, and clinical information. As a result of these elaborate diagnostic requirements, most specimens that are suspected as having MPM eventually find their way to a specialized pathology center, which can result in a delay of diagnosis and treatment [9]. The final pathologic diagnosis is not certain in a small but significant number of cases, particularly in differentiating early MPM from pleuritis or adenocarcinoma of the lung [7,8,10]. It is not uncommon to miss
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the diagnosis of early MPM and delay treatments that are only effective in the early stage [11,12].
Chromosomal aberrations in malignant pleural mesothelioma The pattern of gene expression in MPM has been studied extensively at the chromosomal level. Several specific patterns of chromosomal aberration have been characterized and can be used diagnostically [1,13,14]. These distinct diagnostic patterns are obtained via cytogenetics-based evaluation of tumor specimens. For this reason, cytogenetic analysis is performed whenever the diagnosis is uncertain. Unlike most other solid cancers in general, and adenocarcinoma of the lung in particular, most MPM tumors are diploid in DNA content with only intermediate-to-low proliferative rates [1].
Growth factors and proto-oncogenes in malignant pleural mesothelioma Several reports have demonstrated that several growth factors, such as the human platelet-derived growth factor B-chain and the vascular endothelial growth factor, have elevated expression in MPM cell lines [15 – 18]. The epidermal growth factor receptor is elevated in approximately 70% of cases of MPM and is elevated in MPM cell lines in response to exposure to asbestos fibers. Inhibition of this receptor in vitro recently was shown to diminish the malignant phenotype of MPM cell lines [19]. Oncogenes are proteins that are expressed aberrantly in tumors and are believed to be mechanistically responsible for malignant transformation. To date, no clear-cut oncogenes, such as H-ras, K-ras or others, have been identified for MPM. The receptor tyrosine kinase protein, MET, and the MET ligand are overexpressed in MPM compared with nonneoplastic mesothelial cells [20]. Other differentially expressed factors in MPM include c-fos, c-jun, MetAP2, c-myc, and fra-1 [21,22].
Tumor suppressor genes and growth control pathways The replication of eukaryotic cells occurs and is regulated by a specific sequence of activation and inactivation of cyclins coupled with cyclin-dependent kinase complexes. Tumor suppressors are genes whose aberrant expression can facilitate unchecked
tumor growth. Tumor suppressors often act by influencing the cell replication cycle directly, but they also can have indirect affects. MPM tumors have been extensively characterized cytogenetically, demonstrating specific deletions in large proportions of tumors. The regulator protein p16INK4a, for example, binds to its associated cyclin to regulate cell replication. This gene is deleted or methylated in a large proportion of MPMs, which results in the absence of its protein product. The Rb pathway, of which this gene is a component, seems to be inactivated in MPM [23], but not through a mutated Rb protein. Other genes that seem to be inactivated are NF2, RASSF1A, and FHIT [24,25]. Unlike many other tumors, p53 is only rarely altered or deleted in MPM [26]. Some researchers also have proposed that the simian virus 40 (SV40) T antigen may bind and functionally exclude p53 from the nucleus, which results in the loss of tumor suppression [25].
The extracellular matrix It has been reported that the expression of several different proteoglycan genes is elevated in MPM cell lines [27 – 30] and tumor tissues [31]. Proteoglycans, which are localized at the extracellular matrix (ECM) and cell membrane, function to regulate cell proliferation and differentiation under the influence of growth factors. Microscopic analysis of MPM tumors reveals the presence of substantial stromal tissue around the tumor cells. This stroma is far more common in the non-epithelial subtypes. Rihn et al [32], using microarray analysis, reported several ECM genes elevated in MPM cell lines. We also have observed this phenomenon in a large number of tumors screened with high-density oligonucleotide arrays (Gordon G.J. et al, unpublished results) [31]. Interaction between tumor cells and the ECM is believed to influence cancer progression and explain the unique characteristics of MPM [33].
Aberration in the apoptotic pathways Apoptosis refers to the process by which a cell self-destructs in response to internal or external stimuli. Cancer cells often escape apoptosis despite internal abnormalities. Aberrations in some apoptotic pathways already have been described for MPM. For example, the overexpression of anti-apoptotic proteins in MPM, including survivin, bcl-x, and the inhibitor of apoptosis protein-1 gene (IAP-1), have been reported [34 – 37]. We recently demonstrated
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that IAP-1 is overexpressed in MPM tumors and at least partially mediates the inherent resistance of MPM cell lines to cisplatin [37]. These experiments used a targeted antisense oligonucleotide strategy to demonstrate dramatic abrogation of cisplatin resistance. We then hypothesized that the MPM tumor cells prevent self-destruction by changing the internal intracellular balance of the apoptosis cascade in a manner favoring self-preservation. Antisense treatments using several other members of the apoptosis pathway as targets in MPM also have been shown to represent a successful strategy for modifying the drug resistant phenotype of certain MPM cell lines [35,37]. Whether this phenomenon is the primary step in the transformation of mesothelial cells or just an advantageous byproduct of tumorigenesis is not known currently.
The simian virus 40 hypothesis Since the early 1990s, several sources of evidence have implicated SV40 in the pathogenesis of MPM. Widespread exposure of humans to SV40 occurred between 1955 and 1963 from inadvertent contamination of most lots of poliovirus vaccine, which provided a source for potential infection and transformation [38,39]. DNA sequences and protein immunoreactivity associated with the SV40 large T antigen have been identified in a high proportion (50% – 60%) of fresh-frozen tissues and paraffinembedded tissues obtained from patients with MPM in the United States and in several European countries [25,40 – 51]. SV40 is a double-stranded DNA tumor virus that contains 5243 base pairs arranged in a circular DNA molecule. The genes in this virus can be divided into two groups: the early region, which encodes the large T antigen, small t antigen, and 17kT, and the late region, which encodes the viral coat proteins. The early region is responsible for the tumor transformation potential of this virus [43,44,50]. SV40 has been demonstrated to have three modes of cell infection. One mode of infection results in cell lysis (as in monkey cells and human fibroblasts). A second mode results in viral integration into the cellular DNA with malignant transformation (as in hamster and rodent cells). The third mode results in successful malignant transformation with minimal cell lysis and without viral integration into the chromosome because the viral DNA is maintained as an episome. The last mode of transformation has been demonstrated to be the most common in cultured human mesothelial cells [25]. Experiments in vitro show that once the SV40 virus
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infects mesothelial cells in tissue culture, the large T antigen protein impedes the action of p53 and Rb tumor suppressor proteins by means of inhibitory binding. SV40 transformation also results in inhibition of the protein phosphatase 2A (PP2A) and the tumor suppressor gene RASSF1A, the latter by hypermethylation of its promoter region. The transformation process also results in the upregulation of NOTCH-1, the c-Met oncogene, and the insulin-like growth factor 1 [25]. It has been hypothesized that asbestos facilitates transformation of mesothelial cells by SV40 directly and by impairing the local and systemic immune response of the body [43,49]. In summary, SV40 is hypothesized to facilitate the transformation of mesothelial cells in MPM in the presence or absence of asbestos. Specific mechanisms and alterations of pathways have been proposed for this transformation [25]. Despite the multitude of publications that supports a role for SV40 in the pathogenesis of MPM, several studies have cast doubt on this hypothesis. Several negative studies have failed to detect SV40 sequences in MPM by DNA or protein analysis. Some of these studies have reported geographic variability, indicating the absence of SV40 infections in certain countries, such as Finland [43]. Other negative studies were performed in the United States using patient populations similar to those used in the positive studies [38 – 40,42,52]. Most of the epidemiologic studies to date do not support the hypothesis that SV40 is causative for MPM. Nor is there evidence to date of an increased risk of MPM or an outright SV40 infection in people who were exposed to the contaminated polio virus vaccines at birth [40]. Human contacts with simian species that are infected with SV40 are common in certain geographic areas, and in these regions few SV40 infections have been reported in humans. Finally, in all of the SV40-positive MPM samples described to date, viral DNA sequences amplified via polymerase chain reaction (PCR) are identical to those in the native host species. It is highly improbable, although not inconceivable, that SV40 is capable of human transmission without involving some sort of adaptive mechanism reflected in changes of viral DNA sequences. At this point it is unclear whether SV40 plays a significant role in the causality of MPM or if the gene expression aberrations ascribed to SV40 occur as a result of this virus.
Whole genome profiling Until recently, the usual method of identifying genes responsible for a particular phenotype of a
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cancer was haphazard. Genes identified in other areas of research (or different organs) frequently were investigated to see if they had a similar or important effect in MPM. This analysis usually was based on mechanistic hypotheses. For example, p53 mutations were sought in MPM (with little success) because this gene codes for a tumor suppressor that is often inactivated in other systems. Mutations and expression patterns of other genes were examined because they were physically mapped to areas of the chromosome that were usually deleted in MPM. Many genes were studied in MPM with variable success. The most recent innovation to be applied to MPM is gene expression profiling using high-density oligonucleotide microarrays or similar. Microarrays are miniaturized devices that permit simultaneous measurement of the expression levels of thousands of genes in a single specimen. With this technology, rapid screening of the expression of most of the human genome can be accomplished to determine variation among MPM tumors and between MPM and other tissues. Rinh et al [32] published the first such analysis using MPM cell lines and microarrays, and their report described various differences between tumor and mesothelial cell lines. Using arrays that contain 6500 genes, they found fewer than 300 genes that differed in a statistically meaningful manner. Singhal et al [53] recently reported the profile of 16 MPMs using microarray filters embedded with sequences for 4132 genes and EST clones. They found upregulation of several pathways in MPM when compared with normal pleura, including metabolism, protein translation, and cytoskeletal remodeling pathways.
Development of the gene expression ratio technique We have developed a new metric to overcome the many limitations in the direct clinical application of gene profiling using microarrays. This method, the gene expression ratio technique, requires the initial comparison of two groups of tissues that differ by a single clinical parameter (eg, histology, outcome). The tissues are analyzed for differences in gene expression levels using standard supervised methods for microarray analysis. The genes with expression levels that are found to differ most significantly are identified, selected, and used to construct a set of one to five ratios that singly or in combination predict the correct parameter in the training set. The numerical value of gene ratios can be compared with a rationally chosen threshold and used to identify the
desired clinical characteristic in future samples. For example, when comparing multiple specimens of tumor A to multiple specimens of tumor B, we look for gene X that is significantly higher in expression in most or all of the specimens of tumor A (compared with tumor B) and for gene Y that is significantly higher in expression in most or all of the specimens of tumor B (compared with tumor A). The ratio created by dividing the expression level for gene X by the expression level for gene Y (both measured in a new sample) can be used to predict whether the sample tested represents tumor A or tumor B by comparing the ratio value to a threshold value of b1.Q We hypothesize that a small number of such carefully selected ratios can predict in a binary way a given clinical parameter, such as tumor type, tumor presence, and response to specific therapy (or prognosis). Accordingly, we select genes that provide a sufficient level of signal relative to noise and may have a direct mechanistic explanation for their expression patterns. By virtue of the fact that it is a ratio, the gene expression ratio method (1) negates the need for a third reference gene when determining expression levels because two variable genes are measured, (2) is independent of the platform used for data acquisition, (3) requires only small quantities of RNA (as little as 10 pg using RT-PCR), (4) does not require the coupling of transcription to translation for chosen genes, and (5) permits analysis of individual samples without reference to additional btraining samplesQ requiring data acquired from the same platform [31,54,55].
Gene ratios in the differential diagnosis of malignant pleural mesothelioma and lung adenocarcinoma MPM and adenocarcinoma of the lung are two cancers that often can present in the same clinical manner (eg, unilateral malignant pleural effusion) and are often difficult to differentiate without a surgical biopsy. To better define the differences between these two malignancies, we performed expression profiling experiments using discarded tissues after surgical resection of MPM and adenocarcinoma and searched all of the genes represented on the microarray for those with the most statistically significant differences in average expression levels [31]. Using this small number of genes, we calculated multiple expression ratios per sample by dividing the expression value of each of the genes expressed at relatively higher levels in MPM by the expression value of each of the genes expressed at relatively higher levels in
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Table 1 Accuracy of all ratio combinations in predicting tumor diagnosis in test set Calretinin VAC-b MRC OX-2 KIAA097
Claudin-7
TACSTD1
TITF-1
97% 97% 97% 97%
98% 97% 97% 95%
91% 94% 95% 94%
(145/149) (144/149) (145/149) (145/149)
(146/149) (145/149) (145/149) (142/149)
(136/149) (140/149) (142/149) (140/149)
Eight candidate diagnostic genes were identified in a training set of samples. A total of 15 possible expression ratios (column/ row intersection) were calculated where both genes used to form the ratio possessed inversely correlated average expression levels in both tumor types. The accuracy of each ratio in predicting diagnosis was examined in the 149 remaining tumor specimens not included in the training set (15 mesothelioma and 134 adenocarcinoma). Predictions are stated as the fraction diagnosed correctly.
adenocarcinoma. We then tested the diagnostic accuracy of these ratios, singly and in combinations, in 149 additional samples. We found that the ratios tested could be used to distinguish correctly between adenocarcinoma and MPM tumors with a high degree of accuracy (99%, 148/149) (Table 1). We then used real-time quantitative RT-PCR to confirm gene expression levels of diagnostic molecular markers identified in microarray-based analysis and demonstrate that the ratio-based diagnosis of MPM and lung cancer is equally accurate using data obtained from another platform.
Expression ratios designed to predict treatment-related outcome for malignant pleural mesothelioma We performed expression profiling of tumors collected at extrapleural pneumonectomy from patients with early stage MPM who underwent standard preoperative staging and postoperative chemotherapy and radiation therapy [54]. Our goal was to identify and evaluate prognostic genes using the gene ratio method. We used samples from patients in the training set whose survival was within the twenty-fifth percentile of both survival extremes to form two groups: relatively good outcome (survival 17 months) and relatively poor outcome (survival 6 months). We identified the genes represented on the microarray with a statistically significant difference in average expression levels between good outcome and poor outcome tumors. In this analysis, we identified a total of 46 prognostic genes [54]. We then chose the genes most significantly overexpressed in each outcome group and calculated multiple expression ratios as before to assess classification accuracy. We found that we could identify tumor samples with 100% accuracy in the training set. We tested the ability of expression ratios to predict outcome in an independent cohort of MPM tumor
samples not subjected to microarray analysis using quantitative RT-PCR for data acquisition. The estimated Kaplan-Meier median survival for the good outcome group (36 months) was more than fivefold higher than that for the poor outcome group (7 months). We also found that a three-ratio combination predicted and correlated significantly (P = 0.0035) with the outcome from therapy (Fig. 1). Of note, we found that when using multivariate survival analysis, this technique correctly predicted outcome independently of the histologic subtype and stage of the tumor in the new set of samples, which suggested that the ratio method may be a better prognostic tool [54]. We identified and validated a set of genes that correlate with outcome in MPM and a specific prognostic test related to trimodality therapy.
Fig. 1. Overall survival in the new set of samples for good outcome (top line, median survival = 36 months) and poor outcome (bottom line, median survival = 7 months) groups as defined by the four-gene expression ratio model that used only reverse transcription-polymerase chain reaction for data acquisition. (From Gordon GJ, Jensen RV, Hsiao L-L, Gullans SR, Blumenstock JE, Richards WG, et al. Using gene expression ratios to predict outcome among patients with mesothelioma. J Natl Cancer Inst 2003;95:600; with permission.)
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Summary MPM is a poorly understood lethal malignancy. Although the pathobiology of MPM is not completely elucidated, new genomic technology is likely to help shed light on the mechanisms of carcinogenesis through genome-wide screening of tumorspecific gene expression. Related efforts to identify the molecular markers of mesothelioma are pursued with the aim of refining current diagnostic capabilities, predicting prognosis, and designing appropriate trimodality programs. These new genomic tools also will assist efforts to tailor current adjuvant and neoadjuvant therapies, optimizing their effect and furthering research that may lead to new therapeutic options.
[11]
[12]
[13]
[14]
References [1] Huncharek M. Genetic factors in the aetiology of malignant mesothelioma. Eur J Cancer 1995;31A: 1741 – 7. [2] Aisner J. Diagnosis, staging, and natural history of pleural mesothelioma. In: Aisner J, Arriagada R, Green MR, Martini N, Perry MC, editors. Comprehensive textbook of thoracic oncology. Baltimore7 Williams and Wilkins; 1996. p. 785 – 99. [3] Sugarbaker DJ, Liptay MJ. Therapeutic approaches in malignant mesothelioma. In: Aisner J, Arriagada R, Green MR, Martini N, Perry MC, editors. Comprehensive textbook of thoracic oncology. Baltimore7 Williams and Wilkins; 1996. p. 786 – 98. [4] Sugarbaker D, Norberto J, Bueno R. Current therapy for mesothelioma. Cancer Control 1997;4:326 – 34. [5] Peto J, Hodgson JT, Matthews FE, Jones JR. Continuing increase in mesothelioma mortality in Britain. Lancet 1995;345:535 – 9. [6] Pass HI, Robinson BW, Testa JR, Carbone M. Emerging translational therapies for mesothelioma. Chest 1999;116:455S – 60S. [7] Ong S-T, Vogelsang N. Current therapeutic approaches to unresectable (primary and recurrent) disease. In: Aisner J, Arriagada R, Green M, Martini N, Perry M, editors. Comprehensive textbook of thoracic oncology. Baltimore7 Williams and Wilkins; 1996. p. 799 – 814. [8] Corson JM, Renshaw AA. Pathology of mesothelioma. In: Aisner J, Arriagada R, Green MR, Martini N, Perry MC, editors. Comprehensive textbook of thoracic oncology. Baltimore7 Williams and Wilkins; 1996. p. 757 – 8. [9] Nash G, Otis CN. Protocol for the examination of specimens from patients with malignant pleural mesothelioma: a basis for checklists. Arch Pathol Lab Med 1999;123:39 – 44. [10] Ordonez NG. The value of antibodies 44 – 36A, SM3,
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
HBME-1, and thrombomodulin in differentiating epithelial pleural mesothelioma from lung adenocarcinoma. Am J Surg Pathol 1997;21:1399 – 408. Cury PM, Butcher DN, Corrin B, Nicholson AG. The use of histological and immunohistochemical markers to distinguish pleural malignant mesothelioma and in situ mesothelioma from reactive mesothelial hyperplasia and reactive pleural fibrosis. J Pathol 1999; 189:251 – 7. Scurry J, Duggan MA. Malignant mesothelioma eight years after a diagnosis of atypical mesothelial hyperplasia. J Clin Pathol 1999;52:535 – 7. Balsara BR, Bell DW, Sonoda G, De Rienzo A, du Manoir S, Jhanwar SC, et al. Comparative genomic hybridization and loss of heterozygosity analyses identify a common region of deletion at 15q11.1 – 15 in human malignant mesothelioma. Cancer Res 1999; 59:450 – 4. Xiao S, Daizong L, Vijg J, Sugarbaker DJ, Corson JM, Fletcher JA. Codeletion of p15 and p16 in primary malignant mesothelioma. Oncogene 1995;11:511 – 5. Ohta Y, Shridhar V, Bright RK, Kalemkerian GP, Du W, Carbone M, et al. VEGF and VEGF type C play an important role in angiogenesis and lymphangiogenesis in human malignant mesothelioma tumours. Br J Cancer 1999;81:54 – 61. Galffy G, Mohammed KA, Dowling PA, Nasreen N, Ward MJ, Antony VB. Interleukin 8: an autocrine growth factor for malignant mesothelioma. Cancer Res 1999;59:367 – 71. Versnel MA, Hagemeijer A, Bouts MJ, van der Kwast TH, Hoogsteden HC. Expression of c-sis (PDGF B-chain) and PDGF A-chain genes in ten human malignant mesothelioma cell lines derived from primary and metastatic tumors. Oncogene 1988;2: 601 – 5. Kumar-Singh S, Weyler J, Martin MJH, Vermeulen PB, Van Marck E. Angiogenic cytokines in mesothelioma: a study of VEGF, FGF-1 and -2, and TGFb expression. J Pathol 1999;189:72 – 8. Janne PA, Freidlin B, Saxman S, Johnson DH, Livingston RB, Shepherd FA, et al. Twenty-five years of clinical research for patients with limited-stage small cell lung carcinoma in North America. Cancer 2002; 95:1528 – 38. Klominek J, Baskin B, Liu Z, Hauzenberger D. Hepatocyte growth factor/scatter factor stimulates chemotaxis and growth of malignant mesothelioma cells through c-met receptor. Int J Cancer 1998;76: 240 – 9. Sandhu H, Dehnen W, Roller M, Abel J, Unfried K. mRNA expression patterns in different stages of asbestos-induced carcinogenesis in rats. Carcinogenesis 2000;21:1023 – 9. Cao XX, Mohuiddin I, Ece F, McConkey DJ, Smythe WR. Histone deacetylase inhibitor downregulation of bcl-xl gene expression leads to apoptotic cell death in mesothelioma. Am J Respir Cell Mol Biol 2001; 25:562 – 8.
R. Bueno, G.J. Gordon / Thorac Surg Clin 14 (2004) 461 – 468 [23] Hirao T, Bueno R, Chen CJ, Gordon GJ, Heilig E, Kelsey KT. Alterations of the p16(INK4) locus in human malignant mesothelial tumors. Carcinogenesis 2002;23:1127 – 30. [24] Schipper H, Papp T, Johnen G, Pemsel H, Bastrop R, Muller KM, et al. Mutational analysis of the nf2 tumour suppressor gene in three subtypes of primary human malignant mesotheliomas. Int J Oncol 2003; 22:1009 – 17. [25] Gazdar AF, Butel JS, Carbone M. SV40 and human tumours: myth, association or causality? Nat Rev Cancer 2002;2:957 – 64. [26] Ni Z, Liu Y, Keshava N, Zhou G, Whong W, Ong T. Analysis of K-ras and p53 mutations in mesotheliomas from humans and rats exposed to asbestos. Mutat Res 2000;468:87 – 92. [27] Syrokou A, Tzanakakis GN, Hjerpe A, Karamanos NK. Proteoglycans in human malignant mesothelioma. Stimulation of their synthesis induced by epidermal, insulin, and platelet-derived growth factors involves receptors with tyrosine kinase activity. Biochimie 1999;81:733 – 44. [28] Kumar-Singh S, Jacobs W, Dhaene K, Weyn B, Bogers J, Weyler J, et al. Syndecan-1 expression in malignant mesothelioma: correlation with cell differentiation, WT1 expression, and clinical outcome. J Pathol 1998; 186:300 – 5. [29] Peralta Soler A, Knudsen KA, Jaurand MC, Johnson KR, Wheelock MJ, Klien-Szanto AJ, et al. The differential expression of N-cadherin and E-cadherin distinguishes pleural mesotheliomas from lung adenocarcinomas. Hum Pathol 1995;26:1363 – 9. [30] Teder P, Versnel MA, Heldin P. Stimulatory effects of pleural fluids from mesothelioma patients on CD44 expression, hyaluronan production, and cell proliferation in primary cultures of normal mesothelial and transformed cells. Int J Cancer 1996;67:393 – 8. [31] Gordon GJ, Jensen RV, Hsiao LL, Gullans SR, Blumenstock JE, Ramaswamy S, et al. Translation of microarray data into clinically relevant cancer diagnostic tests using gene expression ratios in lung cancer and mesothelioma. Cancer Res 2002;62:4963 – 7. [32] Rihn BH, Mohr S, McDowell SA, Binet S, Loubinoux J, Galateau F, et al. Differential gene expression in mesothelioma. FEBS Lett 2000;480:95 – 100. [33] Clark EA, Golub TR, Lander ES, Hynes RO. Genomic analysis of metastasis reveals an essential role for RhoC. Nature 2000;406:532 – 5. [34] Xia C, Xu Z, Yuan X, Uematsu K, You L, Li K, et al. Induction of apoptosis in mesothelioma cells by antisurvivin oligonucleotides. Mol Cancer Ther 2002;1: 687 – 94. [35] Smythe WR, Mohuiddin I, Ozveran M, Cao XX. Antisense therapy for malignant mesothelioma with oligonucleotides targeting the bcl-xl gene product. J Thorac Cardiovasc Surg 2002;123:1191 – 8. [36] Hopkins-Donaldson S, Cathomas R, Simoes-Wust AP, Kurtz S, Belyanskaya L, Stahel RA, et al. Induction of apoptosis and chemosensitization of mesothelioma
[37]
[38]
[39]
[40]
[41]
[42]
[43] [44]
[45]
[46]
[47]
[48]
[49] [50]
[51]
[52]
467
cells by Bcl-2 and Bcl-xL antisense treatment. Int J Cancer 2003;106:160 – 6. Gordon GJ, Appasani K, Parcells JP, Mukhopadhyay NK, Jaklitsch MT, Richards WG, et al. Inhibitor of apoptosis protein-1 promotes tumor cell survival in mesothelioma. Carcinogenesis 2002;23:1017 – 24. Strickler HD, Rosenberg PS, Devesa SS, Fraumeni Jr JF, Goedert JJ. Contamination of poliovirus vaccine with SV40 and the incidence of medulloblastoma. Med Pediatr Oncol 1999;32:77 – 8. Strickler HD, Rosenberg PS, Devesa SS, Hertel J, Fraumeni Jr JF, Goedert JJ. Contamination of poliovirus vaccines with simian virus 40 (1955 – 1963) and subsequent cancer rates. JAMA 1998;279:292 – 5. Strickler HD. The International SVWG: a multicenter evaluation of assays for detection of SV40 DNA and results in masked mesothelioma specimens. Cancer Epidemiol Biomarkers Prev 2001;10:523 – 32. Pepper C, Jasani B, Navabi H, Wynford-Thomas D, Gibbs AR. Simian virus 40 large T-antigen (SV40LTAg) primer specific DNA amplification in human pleural mesothelioma tissue. Thorax 1996;51: 1074 – 6. Strickler HD, Goedert JJ, Fleming M, et al. Simian virus 40 and pleural mesothelioma in humans. Cancer Epidemiol Biomarkers Prev 1996;5:473 – 5. Carbone M, Kratzke RA, Testa JR. The pathogenesis of mesothelioma. Semin Oncol 2002;29:2 – 17. Carbone M, Pass HI. Debate on the link between SV40 and human cancer continues [comment]. J Natl Cancer Inst 2002;94:229 – 30. Carbone M, Rizzo P, Grimley PM, Procopio A, Mew DJ, Shridhar V, et al. Simian virus-40 large-T antigen binds p53 in human mesothelioma. Nat Med 1997;3: 908 – 12. Carbone M, Pass HI, Rizzo P, Marinetti M, Di Muzio M, Mew DJ, et al. Simian virus-like DNA sequences in human pleural mesothelioma. Oncogene 1994;9: 1781 – 90. Carbone M, Rizzo P, Pass HI. Simian virus 40, poliovaccines and human tumors: a review of recent developments. Oncogene 1997;15:1877 – 88. Carbone M, Fisher S, Powers A, Pass HI, Rizzo P. New molecular and epidemiological issues in mesothelioma: role of SV40. J Cell Physiol 1999;180:167 – 72. Carbone M. Introduction. Semin Cancer Biol 2001; 11:1 – 3. Carbone M. Simian virus 40 and human tumors: it is time to study mechanisms. J Cell Biochem 1999;76: 189 – 93. Capone RB, Pai SI, Koch WM, Gillison ML, Danish HN, Westra WH, et al. Detection and quantitation of human papillomavirus (HPV) DNA in the sera of patients with HPV-associated head and neck squamous cell carcinoma. Clin Cancer Res 2000;6:4171 – 5. Strickler HD, Goedert JJ. Exposure to SV40-contaminated poliovirus vaccine and the risk of cancer: a review of the epidemiological evidence. Dev Biol tand 1998;94:235 – 44.
468
R. Bueno, G.J. Gordon / Thorac Surg Clin 14 (2004) 461 – 468
[53] Singhal SWR, Malden LD, Amin KM, Matzie K, Friedberg J, Kucharczuk JC, et al. Gene expression profiling of malignant mesothelioma. Clin Cancer Res 2003;9:3080 – 97. [54] Gordon GJ, Jensen RV, Hsiao LL, Gullans SR, Blumenstock JE, Richards WG, et al. Using gene ex-
pression ratios to predict outcome among patients with mesothelioma. J Natl Cancer Inst 2003;95:598 – 605. [55] Gordon GJ, Richards WG, Sugarbaker DJ, Jaklitsch MT, Bueno R. A prognostic test for adenocarcinoma of the lung from gene expression profiling data. Cancer Epidemiol Biomarkers Prev 2003;12:905 – 10.
Genetics of malignant pleural mesothelioma: molecular markers and biologic targets Raphael Bueno, MD, Gavin J. Gordon, PhD* Department of Surgery, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA Division of Thoracic Surgery, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA Thoracic Surgery Oncology Laboratory, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA
Malignant pleural mesothelioma (MPM) is a mesodermally derived neoplastic disease that arises in the pleura and grows relentlessly into adjacent structures until it ultimately results in death of the patient. Unlike many other cancers, MPM does not metastasize until late in the course of the disease [1,2]. Asbestos is a major factor in the pathogenesis of this malignancy, with up to 80% of patients with MPM noting a history of asbestos exposure. Approximately 3000 patients are diagnosed with MPM in the United States annually, and the incidence worldwide is projected to continue to rise in the next two decades [2 – 7]. MPM is a unique malignancy. The most common cause, asbestos, is known, but the mechanisms of carcinogenesis are not fully understood, and the latency period is prolonged. Another characteristic peculiar to MPM is the multifocal location of tumor around the pleura at the time presentation, even in early disease. Unlike lung cancer, MPM respects tissue planes until a relatively advanced stage. As a consequence, a pleurectomy can be performed to remove the tumor-laden pleural envelope of vital tissues, such as the aorta, spine, and heart. Defining the genetic abnormalities that lead to MPM tumor
* Corresponding author. Division of Thoracic Surgery, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115. E-mail address: [email protected] (G.J. Gordon).
cells is important for the development of therapeutic options for this malignancy.
Diagnosis and staging of malignant pleural mesothelioma MPM has four distinct histologic subtypes: epithelial, sarcomatoid, mixed (biphasic), and transitional (undifferentiated). Most cases of MPM are epithelial, and patients with this subtype generally have a longer survival than is associated with the other subtypes. The differential diagnosis of MPM is broad and related to the histologic subtype under consideration. It includes adenocarcinoma of the lung, atypical mesothelial hyperplasia, chronic pleuritis, sarcoma, metastatic adenocarcinoma, solitary fibrous tumor of the pleura, and occasionally lymphoma, melanoma, and spindle cell carcinoma [8]. The need to distinguish MPM from these other conditions has been met partially by a combined approach using histology, immunohistochemistry, cytology, electron microscopy, cytogenetics, and clinical information. As a result of these elaborate diagnostic requirements, most specimens that are suspected as having MPM eventually find their way to a specialized pathology center, which can result in a delay of diagnosis and treatment [9]. The final pathologic diagnosis is not certain in a small but significant number of cases, particularly in differentiating early MPM from pleuritis or adenocarcinoma of the lung [7,8,10]. It is not uncommon to miss
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the diagnosis of early MPM and delay treatments that are only effective in the early stage [11,12].
Chromosomal aberrations in malignant pleural mesothelioma The pattern of gene expression in MPM has been studied extensively at the chromosomal level. Several specific patterns of chromosomal aberration have been characterized and can be used diagnostically [1,13,14]. These distinct diagnostic patterns are obtained via cytogenetics-based evaluation of tumor specimens. For this reason, cytogenetic analysis is performed whenever the diagnosis is uncertain. Unlike most other solid cancers in general, and adenocarcinoma of the lung in particular, most MPM tumors are diploid in DNA content with only intermediate-to-low proliferative rates [1].
Growth factors and proto-oncogenes in malignant pleural mesothelioma Several reports have demonstrated that several growth factors, such as the human platelet-derived growth factor B-chain and the vascular endothelial growth factor, have elevated expression in MPM cell lines [15 – 18]. The epidermal growth factor receptor is elevated in approximately 70% of cases of MPM and is elevated in MPM cell lines in response to exposure to asbestos fibers. Inhibition of this receptor in vitro recently was shown to diminish the malignant phenotype of MPM cell lines [19]. Oncogenes are proteins that are expressed aberrantly in tumors and are believed to be mechanistically responsible for malignant transformation. To date, no clear-cut oncogenes, such as H-ras, K-ras or others, have been identified for MPM. The receptor tyrosine kinase protein, MET, and the MET ligand are overexpressed in MPM compared with nonneoplastic mesothelial cells [20]. Other differentially expressed factors in MPM include c-fos, c-jun, MetAP2, c-myc, and fra-1 [21,22].
Tumor suppressor genes and growth control pathways The replication of eukaryotic cells occurs and is regulated by a specific sequence of activation and inactivation of cyclins coupled with cyclin-dependent kinase complexes. Tumor suppressors are genes whose aberrant expression can facilitate unchecked
tumor growth. Tumor suppressors often act by influencing the cell replication cycle directly, but they also can have indirect affects. MPM tumors have been extensively characterized cytogenetically, demonstrating specific deletions in large proportions of tumors. The regulator protein p16INK4a, for example, binds to its associated cyclin to regulate cell replication. This gene is deleted or methylated in a large proportion of MPMs, which results in the absence of its protein product. The Rb pathway, of which this gene is a component, seems to be inactivated in MPM [23], but not through a mutated Rb protein. Other genes that seem to be inactivated are NF2, RASSF1A, and FHIT [24,25]. Unlike many other tumors, p53 is only rarely altered or deleted in MPM [26]. Some researchers also have proposed that the simian virus 40 (SV40) T antigen may bind and functionally exclude p53 from the nucleus, which results in the loss of tumor suppression [25].
The extracellular matrix It has been reported that the expression of several different proteoglycan genes is elevated in MPM cell lines [27 – 30] and tumor tissues [31]. Proteoglycans, which are localized at the extracellular matrix (ECM) and cell membrane, function to regulate cell proliferation and differentiation under the influence of growth factors. Microscopic analysis of MPM tumors reveals the presence of substantial stromal tissue around the tumor cells. This stroma is far more common in the non-epithelial subtypes. Rihn et al [32], using microarray analysis, reported several ECM genes elevated in MPM cell lines. We also have observed this phenomenon in a large number of tumors screened with high-density oligonucleotide arrays (Gordon G.J. et al, unpublished results) [31]. Interaction between tumor cells and the ECM is believed to influence cancer progression and explain the unique characteristics of MPM [33].
Aberration in the apoptotic pathways Apoptosis refers to the process by which a cell self-destructs in response to internal or external stimuli. Cancer cells often escape apoptosis despite internal abnormalities. Aberrations in some apoptotic pathways already have been described for MPM. For example, the overexpression of anti-apoptotic proteins in MPM, including survivin, bcl-x, and the inhibitor of apoptosis protein-1 gene (IAP-1), have been reported [34 – 37]. We recently demonstrated
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that IAP-1 is overexpressed in MPM tumors and at least partially mediates the inherent resistance of MPM cell lines to cisplatin [37]. These experiments used a targeted antisense oligonucleotide strategy to demonstrate dramatic abrogation of cisplatin resistance. We then hypothesized that the MPM tumor cells prevent self-destruction by changing the internal intracellular balance of the apoptosis cascade in a manner favoring self-preservation. Antisense treatments using several other members of the apoptosis pathway as targets in MPM also have been shown to represent a successful strategy for modifying the drug resistant phenotype of certain MPM cell lines [35,37]. Whether this phenomenon is the primary step in the transformation of mesothelial cells or just an advantageous byproduct of tumorigenesis is not known currently.
The simian virus 40 hypothesis Since the early 1990s, several sources of evidence have implicated SV40 in the pathogenesis of MPM. Widespread exposure of humans to SV40 occurred between 1955 and 1963 from inadvertent contamination of most lots of poliovirus vaccine, which provided a source for potential infection and transformation [38,39]. DNA sequences and protein immunoreactivity associated with the SV40 large T antigen have been identified in a high proportion (50% – 60%) of fresh-frozen tissues and paraffinembedded tissues obtained from patients with MPM in the United States and in several European countries [25,40 – 51]. SV40 is a double-stranded DNA tumor virus that contains 5243 base pairs arranged in a circular DNA molecule. The genes in this virus can be divided into two groups: the early region, which encodes the large T antigen, small t antigen, and 17kT, and the late region, which encodes the viral coat proteins. The early region is responsible for the tumor transformation potential of this virus [43,44,50]. SV40 has been demonstrated to have three modes of cell infection. One mode of infection results in cell lysis (as in monkey cells and human fibroblasts). A second mode results in viral integration into the cellular DNA with malignant transformation (as in hamster and rodent cells). The third mode results in successful malignant transformation with minimal cell lysis and without viral integration into the chromosome because the viral DNA is maintained as an episome. The last mode of transformation has been demonstrated to be the most common in cultured human mesothelial cells [25]. Experiments in vitro show that once the SV40 virus
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infects mesothelial cells in tissue culture, the large T antigen protein impedes the action of p53 and Rb tumor suppressor proteins by means of inhibitory binding. SV40 transformation also results in inhibition of the protein phosphatase 2A (PP2A) and the tumor suppressor gene RASSF1A, the latter by hypermethylation of its promoter region. The transformation process also results in the upregulation of NOTCH-1, the c-Met oncogene, and the insulin-like growth factor 1 [25]. It has been hypothesized that asbestos facilitates transformation of mesothelial cells by SV40 directly and by impairing the local and systemic immune response of the body [43,49]. In summary, SV40 is hypothesized to facilitate the transformation of mesothelial cells in MPM in the presence or absence of asbestos. Specific mechanisms and alterations of pathways have been proposed for this transformation [25]. Despite the multitude of publications that supports a role for SV40 in the pathogenesis of MPM, several studies have cast doubt on this hypothesis. Several negative studies have failed to detect SV40 sequences in MPM by DNA or protein analysis. Some of these studies have reported geographic variability, indicating the absence of SV40 infections in certain countries, such as Finland [43]. Other negative studies were performed in the United States using patient populations similar to those used in the positive studies [38 – 40,42,52]. Most of the epidemiologic studies to date do not support the hypothesis that SV40 is causative for MPM. Nor is there evidence to date of an increased risk of MPM or an outright SV40 infection in people who were exposed to the contaminated polio virus vaccines at birth [40]. Human contacts with simian species that are infected with SV40 are common in certain geographic areas, and in these regions few SV40 infections have been reported in humans. Finally, in all of the SV40-positive MPM samples described to date, viral DNA sequences amplified via polymerase chain reaction (PCR) are identical to those in the native host species. It is highly improbable, although not inconceivable, that SV40 is capable of human transmission without involving some sort of adaptive mechanism reflected in changes of viral DNA sequences. At this point it is unclear whether SV40 plays a significant role in the causality of MPM or if the gene expression aberrations ascribed to SV40 occur as a result of this virus.
Whole genome profiling Until recently, the usual method of identifying genes responsible for a particular phenotype of a
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cancer was haphazard. Genes identified in other areas of research (or different organs) frequently were investigated to see if they had a similar or important effect in MPM. This analysis usually was based on mechanistic hypotheses. For example, p53 mutations were sought in MPM (with little success) because this gene codes for a tumor suppressor that is often inactivated in other systems. Mutations and expression patterns of other genes were examined because they were physically mapped to areas of the chromosome that were usually deleted in MPM. Many genes were studied in MPM with variable success. The most recent innovation to be applied to MPM is gene expression profiling using high-density oligonucleotide microarrays or similar. Microarrays are miniaturized devices that permit simultaneous measurement of the expression levels of thousands of genes in a single specimen. With this technology, rapid screening of the expression of most of the human genome can be accomplished to determine variation among MPM tumors and between MPM and other tissues. Rinh et al [32] published the first such analysis using MPM cell lines and microarrays, and their report described various differences between tumor and mesothelial cell lines. Using arrays that contain 6500 genes, they found fewer than 300 genes that differed in a statistically meaningful manner. Singhal et al [53] recently reported the profile of 16 MPMs using microarray filters embedded with sequences for 4132 genes and EST clones. They found upregulation of several pathways in MPM when compared with normal pleura, including metabolism, protein translation, and cytoskeletal remodeling pathways.
Development of the gene expression ratio technique We have developed a new metric to overcome the many limitations in the direct clinical application of gene profiling using microarrays. This method, the gene expression ratio technique, requires the initial comparison of two groups of tissues that differ by a single clinical parameter (eg, histology, outcome). The tissues are analyzed for differences in gene expression levels using standard supervised methods for microarray analysis. The genes with expression levels that are found to differ most significantly are identified, selected, and used to construct a set of one to five ratios that singly or in combination predict the correct parameter in the training set. The numerical value of gene ratios can be compared with a rationally chosen threshold and used to identify the
desired clinical characteristic in future samples. For example, when comparing multiple specimens of tumor A to multiple specimens of tumor B, we look for gene X that is significantly higher in expression in most or all of the specimens of tumor A (compared with tumor B) and for gene Y that is significantly higher in expression in most or all of the specimens of tumor B (compared with tumor A). The ratio created by dividing the expression level for gene X by the expression level for gene Y (both measured in a new sample) can be used to predict whether the sample tested represents tumor A or tumor B by comparing the ratio value to a threshold value of b1.Q We hypothesize that a small number of such carefully selected ratios can predict in a binary way a given clinical parameter, such as tumor type, tumor presence, and response to specific therapy (or prognosis). Accordingly, we select genes that provide a sufficient level of signal relative to noise and may have a direct mechanistic explanation for their expression patterns. By virtue of the fact that it is a ratio, the gene expression ratio method (1) negates the need for a third reference gene when determining expression levels because two variable genes are measured, (2) is independent of the platform used for data acquisition, (3) requires only small quantities of RNA (as little as 10 pg using RT-PCR), (4) does not require the coupling of transcription to translation for chosen genes, and (5) permits analysis of individual samples without reference to additional btraining samplesQ requiring data acquired from the same platform [31,54,55].
Gene ratios in the differential diagnosis of malignant pleural mesothelioma and lung adenocarcinoma MPM and adenocarcinoma of the lung are two cancers that often can present in the same clinical manner (eg, unilateral malignant pleural effusion) and are often difficult to differentiate without a surgical biopsy. To better define the differences between these two malignancies, we performed expression profiling experiments using discarded tissues after surgical resection of MPM and adenocarcinoma and searched all of the genes represented on the microarray for those with the most statistically significant differences in average expression levels [31]. Using this small number of genes, we calculated multiple expression ratios per sample by dividing the expression value of each of the genes expressed at relatively higher levels in MPM by the expression value of each of the genes expressed at relatively higher levels in
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Table 1 Accuracy of all ratio combinations in predicting tumor diagnosis in test set Calretinin VAC-b MRC OX-2 KIAA097
Claudin-7
TACSTD1
TITF-1
97% 97% 97% 97%
98% 97% 97% 95%
91% 94% 95% 94%
(145/149) (144/149) (145/149) (145/149)
(146/149) (145/149) (145/149) (142/149)
(136/149) (140/149) (142/149) (140/149)
Eight candidate diagnostic genes were identified in a training set of samples. A total of 15 possible expression ratios (column/ row intersection) were calculated where both genes used to form the ratio possessed inversely correlated average expression levels in both tumor types. The accuracy of each ratio in predicting diagnosis was examined in the 149 remaining tumor specimens not included in the training set (15 mesothelioma and 134 adenocarcinoma). Predictions are stated as the fraction diagnosed correctly.
adenocarcinoma. We then tested the diagnostic accuracy of these ratios, singly and in combinations, in 149 additional samples. We found that the ratios tested could be used to distinguish correctly between adenocarcinoma and MPM tumors with a high degree of accuracy (99%, 148/149) (Table 1). We then used real-time quantitative RT-PCR to confirm gene expression levels of diagnostic molecular markers identified in microarray-based analysis and demonstrate that the ratio-based diagnosis of MPM and lung cancer is equally accurate using data obtained from another platform.
Expression ratios designed to predict treatment-related outcome for malignant pleural mesothelioma We performed expression profiling of tumors collected at extrapleural pneumonectomy from patients with early stage MPM who underwent standard preoperative staging and postoperative chemotherapy and radiation therapy [54]. Our goal was to identify and evaluate prognostic genes using the gene ratio method. We used samples from patients in the training set whose survival was within the twenty-fifth percentile of both survival extremes to form two groups: relatively good outcome (survival 17 months) and relatively poor outcome (survival 6 months). We identified the genes represented on the microarray with a statistically significant difference in average expression levels between good outcome and poor outcome tumors. In this analysis, we identified a total of 46 prognostic genes [54]. We then chose the genes most significantly overexpressed in each outcome group and calculated multiple expression ratios as before to assess classification accuracy. We found that we could identify tumor samples with 100% accuracy in the training set. We tested the ability of expression ratios to predict outcome in an independent cohort of MPM tumor
samples not subjected to microarray analysis using quantitative RT-PCR for data acquisition. The estimated Kaplan-Meier median survival for the good outcome group (36 months) was more than fivefold higher than that for the poor outcome group (7 months). We also found that a three-ratio combination predicted and correlated significantly (P = 0.0035) with the outcome from therapy (Fig. 1). Of note, we found that when using multivariate survival analysis, this technique correctly predicted outcome independently of the histologic subtype and stage of the tumor in the new set of samples, which suggested that the ratio method may be a better prognostic tool [54]. We identified and validated a set of genes that correlate with outcome in MPM and a specific prognostic test related to trimodality therapy.
Fig. 1. Overall survival in the new set of samples for good outcome (top line, median survival = 36 months) and poor outcome (bottom line, median survival = 7 months) groups as defined by the four-gene expression ratio model that used only reverse transcription-polymerase chain reaction for data acquisition. (From Gordon GJ, Jensen RV, Hsiao L-L, Gullans SR, Blumenstock JE, Richards WG, et al. Using gene expression ratios to predict outcome among patients with mesothelioma. J Natl Cancer Inst 2003;95:600; with permission.)
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Summary MPM is a poorly understood lethal malignancy. Although the pathobiology of MPM is not completely elucidated, new genomic technology is likely to help shed light on the mechanisms of carcinogenesis through genome-wide screening of tumorspecific gene expression. Related efforts to identify the molecular markers of mesothelioma are pursued with the aim of refining current diagnostic capabilities, predicting prognosis, and designing appropriate trimodality programs. These new genomic tools also will assist efforts to tailor current adjuvant and neoadjuvant therapies, optimizing their effect and furthering research that may lead to new therapeutic options.
[11]
[12]
[13]
[14]
References [1] Huncharek M. Genetic factors in the aetiology of malignant mesothelioma. Eur J Cancer 1995;31A: 1741 – 7. [2] Aisner J. Diagnosis, staging, and natural history of pleural mesothelioma. In: Aisner J, Arriagada R, Green MR, Martini N, Perry MC, editors. Comprehensive textbook of thoracic oncology. Baltimore7 Williams and Wilkins; 1996. p. 785 – 99. [3] Sugarbaker DJ, Liptay MJ. Therapeutic approaches in malignant mesothelioma. In: Aisner J, Arriagada R, Green MR, Martini N, Perry MC, editors. Comprehensive textbook of thoracic oncology. Baltimore7 Williams and Wilkins; 1996. p. 786 – 98. [4] Sugarbaker D, Norberto J, Bueno R. Current therapy for mesothelioma. Cancer Control 1997;4:326 – 34. [5] Peto J, Hodgson JT, Matthews FE, Jones JR. Continuing increase in mesothelioma mortality in Britain. Lancet 1995;345:535 – 9. [6] Pass HI, Robinson BW, Testa JR, Carbone M. Emerging translational therapies for mesothelioma. Chest 1999;116:455S – 60S. [7] Ong S-T, Vogelsang N. Current therapeutic approaches to unresectable (primary and recurrent) disease. In: Aisner J, Arriagada R, Green M, Martini N, Perry M, editors. Comprehensive textbook of thoracic oncology. Baltimore7 Williams and Wilkins; 1996. p. 799 – 814. [8] Corson JM, Renshaw AA. Pathology of mesothelioma. In: Aisner J, Arriagada R, Green MR, Martini N, Perry MC, editors. Comprehensive textbook of thoracic oncology. Baltimore7 Williams and Wilkins; 1996. p. 757 – 8. [9] Nash G, Otis CN. Protocol for the examination of specimens from patients with malignant pleural mesothelioma: a basis for checklists. Arch Pathol Lab Med 1999;123:39 – 44. [10] Ordonez NG. The value of antibodies 44 – 36A, SM3,
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
HBME-1, and thrombomodulin in differentiating epithelial pleural mesothelioma from lung adenocarcinoma. Am J Surg Pathol 1997;21:1399 – 408. Cury PM, Butcher DN, Corrin B, Nicholson AG. The use of histological and immunohistochemical markers to distinguish pleural malignant mesothelioma and in situ mesothelioma from reactive mesothelial hyperplasia and reactive pleural fibrosis. J Pathol 1999; 189:251 – 7. Scurry J, Duggan MA. Malignant mesothelioma eight years after a diagnosis of atypical mesothelial hyperplasia. J Clin Pathol 1999;52:535 – 7. Balsara BR, Bell DW, Sonoda G, De Rienzo A, du Manoir S, Jhanwar SC, et al. Comparative genomic hybridization and loss of heterozygosity analyses identify a common region of deletion at 15q11.1 – 15 in human malignant mesothelioma. Cancer Res 1999; 59:450 – 4. Xiao S, Daizong L, Vijg J, Sugarbaker DJ, Corson JM, Fletcher JA. Codeletion of p15 and p16 in primary malignant mesothelioma. Oncogene 1995;11:511 – 5. Ohta Y, Shridhar V, Bright RK, Kalemkerian GP, Du W, Carbone M, et al. VEGF and VEGF type C play an important role in angiogenesis and lymphangiogenesis in human malignant mesothelioma tumours. Br J Cancer 1999;81:54 – 61. Galffy G, Mohammed KA, Dowling PA, Nasreen N, Ward MJ, Antony VB. Interleukin 8: an autocrine growth factor for malignant mesothelioma. Cancer Res 1999;59:367 – 71. Versnel MA, Hagemeijer A, Bouts MJ, van der Kwast TH, Hoogsteden HC. Expression of c-sis (PDGF B-chain) and PDGF A-chain genes in ten human malignant mesothelioma cell lines derived from primary and metastatic tumors. Oncogene 1988;2: 601 – 5. Kumar-Singh S, Weyler J, Martin MJH, Vermeulen PB, Van Marck E. Angiogenic cytokines in mesothelioma: a study of VEGF, FGF-1 and -2, and TGFb expression. J Pathol 1999;189:72 – 8. Janne PA, Freidlin B, Saxman S, Johnson DH, Livingston RB, Shepherd FA, et al. Twenty-five years of clinical research for patients with limited-stage small cell lung carcinoma in North America. Cancer 2002; 95:1528 – 38. Klominek J, Baskin B, Liu Z, Hauzenberger D. Hepatocyte growth factor/scatter factor stimulates chemotaxis and growth of malignant mesothelioma cells through c-met receptor. Int J Cancer 1998;76: 240 – 9. Sandhu H, Dehnen W, Roller M, Abel J, Unfried K. mRNA expression patterns in different stages of asbestos-induced carcinogenesis in rats. Carcinogenesis 2000;21:1023 – 9. Cao XX, Mohuiddin I, Ece F, McConkey DJ, Smythe WR. Histone deacetylase inhibitor downregulation of bcl-xl gene expression leads to apoptotic cell death in mesothelioma. Am J Respir Cell Mol Biol 2001; 25:562 – 8.
R. Bueno, G.J. Gordon / Thorac Surg Clin 14 (2004) 461 – 468 [23] Hirao T, Bueno R, Chen CJ, Gordon GJ, Heilig E, Kelsey KT. Alterations of the p16(INK4) locus in human malignant mesothelial tumors. Carcinogenesis 2002;23:1127 – 30. [24] Schipper H, Papp T, Johnen G, Pemsel H, Bastrop R, Muller KM, et al. Mutational analysis of the nf2 tumour suppressor gene in three subtypes of primary human malignant mesotheliomas. Int J Oncol 2003; 22:1009 – 17. [25] Gazdar AF, Butel JS, Carbone M. SV40 and human tumours: myth, association or causality? Nat Rev Cancer 2002;2:957 – 64. [26] Ni Z, Liu Y, Keshava N, Zhou G, Whong W, Ong T. Analysis of K-ras and p53 mutations in mesotheliomas from humans and rats exposed to asbestos. Mutat Res 2000;468:87 – 92. [27] Syrokou A, Tzanakakis GN, Hjerpe A, Karamanos NK. Proteoglycans in human malignant mesothelioma. Stimulation of their synthesis induced by epidermal, insulin, and platelet-derived growth factors involves receptors with tyrosine kinase activity. Biochimie 1999;81:733 – 44. [28] Kumar-Singh S, Jacobs W, Dhaene K, Weyn B, Bogers J, Weyler J, et al. Syndecan-1 expression in malignant mesothelioma: correlation with cell differentiation, WT1 expression, and clinical outcome. J Pathol 1998; 186:300 – 5. [29] Peralta Soler A, Knudsen KA, Jaurand MC, Johnson KR, Wheelock MJ, Klien-Szanto AJ, et al. The differential expression of N-cadherin and E-cadherin distinguishes pleural mesotheliomas from lung adenocarcinomas. Hum Pathol 1995;26:1363 – 9. [30] Teder P, Versnel MA, Heldin P. Stimulatory effects of pleural fluids from mesothelioma patients on CD44 expression, hyaluronan production, and cell proliferation in primary cultures of normal mesothelial and transformed cells. Int J Cancer 1996;67:393 – 8. [31] Gordon GJ, Jensen RV, Hsiao LL, Gullans SR, Blumenstock JE, Ramaswamy S, et al. Translation of microarray data into clinically relevant cancer diagnostic tests using gene expression ratios in lung cancer and mesothelioma. Cancer Res 2002;62:4963 – 7. [32] Rihn BH, Mohr S, McDowell SA, Binet S, Loubinoux J, Galateau F, et al. Differential gene expression in mesothelioma. FEBS Lett 2000;480:95 – 100. [33] Clark EA, Golub TR, Lander ES, Hynes RO. Genomic analysis of metastasis reveals an essential role for RhoC. Nature 2000;406:532 – 5. [34] Xia C, Xu Z, Yuan X, Uematsu K, You L, Li K, et al. Induction of apoptosis in mesothelioma cells by antisurvivin oligonucleotides. Mol Cancer Ther 2002;1: 687 – 94. [35] Smythe WR, Mohuiddin I, Ozveran M, Cao XX. Antisense therapy for malignant mesothelioma with oligonucleotides targeting the bcl-xl gene product. J Thorac Cardiovasc Surg 2002;123:1191 – 8. [36] Hopkins-Donaldson S, Cathomas R, Simoes-Wust AP, Kurtz S, Belyanskaya L, Stahel RA, et al. Induction of apoptosis and chemosensitization of mesothelioma
[37]
[38]
[39]
[40]
[41]
[42]
[43] [44]
[45]
[46]
[47]
[48]
[49] [50]
[51]
[52]
467
cells by Bcl-2 and Bcl-xL antisense treatment. Int J Cancer 2003;106:160 – 6. Gordon GJ, Appasani K, Parcells JP, Mukhopadhyay NK, Jaklitsch MT, Richards WG, et al. Inhibitor of apoptosis protein-1 promotes tumor cell survival in mesothelioma. Carcinogenesis 2002;23:1017 – 24. Strickler HD, Rosenberg PS, Devesa SS, Fraumeni Jr JF, Goedert JJ. Contamination of poliovirus vaccine with SV40 and the incidence of medulloblastoma. Med Pediatr Oncol 1999;32:77 – 8. Strickler HD, Rosenberg PS, Devesa SS, Hertel J, Fraumeni Jr JF, Goedert JJ. Contamination of poliovirus vaccines with simian virus 40 (1955 – 1963) and subsequent cancer rates. JAMA 1998;279:292 – 5. Strickler HD. The International SVWG: a multicenter evaluation of assays for detection of SV40 DNA and results in masked mesothelioma specimens. Cancer Epidemiol Biomarkers Prev 2001;10:523 – 32. Pepper C, Jasani B, Navabi H, Wynford-Thomas D, Gibbs AR. Simian virus 40 large T-antigen (SV40LTAg) primer specific DNA amplification in human pleural mesothelioma tissue. Thorax 1996;51: 1074 – 6. Strickler HD, Goedert JJ, Fleming M, et al. Simian virus 40 and pleural mesothelioma in humans. Cancer Epidemiol Biomarkers Prev 1996;5:473 – 5. Carbone M, Kratzke RA, Testa JR. The pathogenesis of mesothelioma. Semin Oncol 2002;29:2 – 17. Carbone M, Pass HI. Debate on the link between SV40 and human cancer continues [comment]. J Natl Cancer Inst 2002;94:229 – 30. Carbone M, Rizzo P, Grimley PM, Procopio A, Mew DJ, Shridhar V, et al. Simian virus-40 large-T antigen binds p53 in human mesothelioma. Nat Med 1997;3: 908 – 12. Carbone M, Pass HI, Rizzo P, Marinetti M, Di Muzio M, Mew DJ, et al. Simian virus-like DNA sequences in human pleural mesothelioma. Oncogene 1994;9: 1781 – 90. Carbone M, Rizzo P, Pass HI. Simian virus 40, poliovaccines and human tumors: a review of recent developments. Oncogene 1997;15:1877 – 88. Carbone M, Fisher S, Powers A, Pass HI, Rizzo P. New molecular and epidemiological issues in mesothelioma: role of SV40. J Cell Physiol 1999;180:167 – 72. Carbone M. Introduction. Semin Cancer Biol 2001; 11:1 – 3. Carbone M. Simian virus 40 and human tumors: it is time to study mechanisms. J Cell Biochem 1999;76: 189 – 93. Capone RB, Pai SI, Koch WM, Gillison ML, Danish HN, Westra WH, et al. Detection and quantitation of human papillomavirus (HPV) DNA in the sera of patients with HPV-associated head and neck squamous cell carcinoma. Clin Cancer Res 2000;6:4171 – 5. Strickler HD, Goedert JJ. Exposure to SV40-contaminated poliovirus vaccine and the risk of cancer: a review of the epidemiological evidence. Dev Biol tand 1998;94:235 – 44.
468
R. Bueno, G.J. Gordon / Thorac Surg Clin 14 (2004) 461 – 468
[53] Singhal SWR, Malden LD, Amin KM, Matzie K, Friedberg J, Kucharczuk JC, et al. Gene expression profiling of malignant mesothelioma. Clin Cancer Res 2003;9:3080 – 97. [54] Gordon GJ, Jensen RV, Hsiao LL, Gullans SR, Blumenstock JE, Richards WG, et al. Using gene ex-
pression ratios to predict outcome among patients with mesothelioma. J Natl Cancer Inst 2003;95:598 – 605. [55] Gordon GJ, Richards WG, Sugarbaker DJ, Jaklitsch MT, Bueno R. A prognostic test for adenocarcinoma of the lung from gene expression profiling data. Cancer Epidemiol Biomarkers Prev 2003;12:905 – 10.