Proc. Natl. Acad. Sci. USA Vol. 92, pp. 10854-10858, November 1995 Medical Sciences High frequency of inactivating mutations in the neurofibromatosis type 2 gene (NF2) in primary malignant mesotheliomas ALBERT B. BIANCHI*t, SHIN-ICHIRO MITSUNAGAtt, JIN QUAN CHENG§, WALTER M. KLEIN§, SURESH C. JHANWARI, BERND SEIZINGER*, NIKoLAI KLEY*, ANDRES J. P. KLEIN-SZANTOt, AND JOSEPH R. TESTA§II *Department of Molecular Genetics and Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000; Departments of tPathology and of §Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA 19111; and IDepartment of Human Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 Communicated by C. C. Tan, Fudan University, Shanghai, China, August 8, 1995 ABSTRACT Malignant mesotheliomas (MMs) are ag- gressive tumors that develop most frequently in the pleura of patients exposed to asbestos. In contrast to many other cancers, relatively few molecular alterations have been de- scribed in MMs. The most frequent numerical cytogenetic abnormality in MMs is loss of chromosome 22. The neurofi- bromatosis type 2 gene (NF2) is a tumor suppressor gene assigned to chromosome 22q which plays an important role in the development of familial and spontaneous tumors of neu- roectodermal origin. Although MMs have a different histo- genic derivation, the frequent abnormalities of chromosome 22 warranted an investigation of the NF2 gene in these tumors. Both cDNAs from 15 MM cell lines and genomic DNAs from 7 matched primary tumors were analyzed for mutations within the NF2 coding region. NF2 mutations predicting either interstitial in-frame deletions or truncation of the NF2- encoded protein (merlin) were detected in eight cell lines (53%), six of which were confirmed in primary tumor DNAs. In two samples that showed NF2 gene transcript alterations, no genomic DNA mutations were detected, suggesting that aberrant splicing may constitute an additional mechanism for merlin inactivation. These findings implicate NF2 in the oncogenesis of primary MMs and provide evidence that this gene can be involved in the development of tumors other than nervous system neoplasms characteristic of the NF2 disorder. In addition, unlike NF2-related tumors, MM derives from the mesoderm; malignancies of this origin have not previously been associated with frequent alterations of the NF2 gene. Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder characterized by the development of bilateral vestib- ular schwannomas of the eighth cranial nerve and by other brain tumors, including meningiomas and ependymomas (1). Affected individuals are also predisposed to develop spinal schwannomas and meningiomas (1-3). Genetic linkage and deletion mapping analyses of sporadic and familial NF2- associated tumors suggested that inactivation of a tumor suppressor gene in chromosome 22q was involved in the etiology of this disorder (4-6). These studies, coupled with positional cloning approaches, have led to the recent isolation of the NF2 gene (7, 8). The NF2 gene is a tumor suppressor gene which encodes a 595-amino acid protein called merlin (for moesin-ezrin- radixin-like protein) (8) or schwannomin (7). Merlin exhibits significant homology to a highly conserved family of proteins postulated to play a role in cell surface dynamics and structure by linking the cytoskeleton to components of the plasma membrane (9, 10). Both germ-line and somatic NF2 gene mutations have been extensively characterized in meningiomas and schwannomas (11-15). The observed mutations represent predominantly either nonsense or splice site mutations, or frameshift nucle- otide deletions and insertions leading to truncated forms of merlin. In addition to these tumor types, somatic mutations have been identified in neoplasms seemingly unrelated to the NF2 disorder such as malignant melanoma (like schwannomas, however, this tumor type derives from tissue of neural-crest origin) as well as carcinoma of the breast (12) and colon (16). However, the frequency of NF2 mutations detected in breast and colon cancers is significantly lower than the incidence of allelic losses from chromosome 22q observed in these tumors (17). Malignant mesotheliomas (MMs) are mesodermally de- rived, primarily pleural tumors that respond poorly to current therapeutic approaches. Although MMs are relatively rare, their incidence has continued to increase, and considerable interest has focused on this neoplasm because of its association with asbestos exposure (18, 19). Loss of chromosome 22 is the single most consistent numerical cytogenetic change in MMs (20, 21). Rearrangement and loss of chromosomes lp, 3p, 6q, and 9p are also a prominent feature of this malignancy (20-24). The critically involved genes located within these deleted regions are currently unknown. A putative tumor suppressor gene p16/MTSI/CDKN2 is located in chromosome 9p, and we recently showed that alterations of p16 are a common occurrence in MM cell lines and, to a lesser extent, in primary tumors (25). While MMs have a different histogenic derivation than neuroectodermal neoplasms, the frequent abnormalities of chromosome 22 in MM prompted us to investigate whether alterations of the NF2 gene are involved in these tumors. MATERIALS AND METHODS Human Primary Tumors, Cell Lines, and Nucleic Acid Extraction. Fifteen human MM cell lines were established from primary cultures of surgically resected tumors as de- scribed previously (25). Matched primary tumor tissues were snap frozen and stored at -70°C. DNA was isolated from cell line and tissue samples by using standard methods (26). Normal diploid human mesothelial cell strain LP-9 (27) was obtained from the NIA Aging Cell Repository. Total RNA was extracted from frozen tumor specimens and from cell lines by lysis in guanidinium thiocyanate and extraction with phenol/ chloroform (28). Abbreviations: MM, malignant mesothelioma; NF2, neurofibroma- tosis type 2; RT, reverse transcriptase; SSCP, single-strand confor- mation polymorphism. tA.B.B. and S.-I.M. contributed equally to this work. ITo whom reprint requests should be addressed. 10854 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Downloaded from https://www.pnas.org by 171.243.67.90 on May 30, 2023 from IP address 171.243.67.90.
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