Proc. Natl. Acad. Sci. USAVol. 92, pp. 10854-10858, November 1995Medical Sciences

High frequency of inactivating mutations in theneurofibromatosis type 2 gene (NF2) inprimary malignant mesotheliomasALBERT 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 oftPathology and of §Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA 19111; and IDepartment of Human Genetics, MemorialSloan-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 ofpatients exposed to asbestos. In contrast to many othercancers, relatively few molecular alterations have been de-scribed in MMs. The most frequent numerical cytogeneticabnormality in MMs is loss of chromosome 22. The neurofi-bromatosis type 2 gene (NF2) is a tumor suppressor geneassigned to chromosome 22q which plays an important role inthe development of familial and spontaneous tumors of neu-roectodermal origin. Although MMs have a different histo-genic derivation, the frequent abnormalities of chromosome22 warranted an investigation of theNF2 gene in these tumors.Both cDNAs from 15 MM cell lines and genomic DNAs from7 matched primary tumors were analyzed for mutationswithin theNF2 coding region.NF2 mutations predicting eitherinterstitial 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 thataberrant splicing may constitute an additional mechanismfor merlin inactivation. These findings implicate NF2 in theoncogenesis of primary MMs and provide evidence that thisgene can be involved in the development of tumors other thannervous system neoplasms characteristic of the NF2 disorder.In addition, unlike NF2-related tumors, MM derives from themesoderm; malignancies of this origin have not previouslybeen associated with frequent alterations of the NF2 gene.

Neurofibromatosis type 2 (NF2) is an autosomal dominantdisorder characterized by the development of bilateral vestib-ular schwannomas of the eighth cranial nerve and by otherbrain tumors, including meningiomas and ependymomas (1).Affected individuals are also predisposed to develop spinalschwannomas and meningiomas (1-3). Genetic linkage anddeletion mapping analyses of sporadic and familial NF2-associated tumors suggested that inactivation of a tumorsuppressor gene in chromosome 22q was involved in theetiology of this disorder (4-6). These studies, coupled withpositional cloning approaches, have led to the recent isolationof 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 exhibitssignificant homology to a highly conserved family of proteinspostulated to play a role in cell surface dynamics and structureby linking the cytoskeleton to components of the plasmamembrane (9, 10).

Both germ-line and somatic NF2 gene mutations have beenextensively characterized in meningiomas and schwannomas(11-15). The observed mutations represent predominantlyeither nonsense or splice site mutations, or frameshift nucle-otide deletions and insertions leading to truncated forms ofmerlin. In addition to these tumor types, somatic mutationshave been identified in neoplasms seemingly unrelated to theNF2 disorder such as malignant melanoma (like schwannomas,however, this tumor type derives from tissue of neural-crestorigin) as well as carcinoma of the breast (12) and colon (16).However, the frequency of NF2 mutations detected in breastand colon cancers is significantly lower than the incidence ofallelic losses from chromosome 22q observed in these tumors(17).

Malignant mesotheliomas (MMs) are mesodermally de-rived, primarily pleural tumors that respond poorly to currenttherapeutic approaches. Although MMs are relatively rare,their incidence has continued to increase, and considerableinterest has focused on this neoplasm because of its associationwith asbestos exposure (18, 19). Loss of chromosome 22 is thesingle 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 thesedeleted regions are currently unknown. A putative tumorsuppressor genep16/MTSI/CDKN2 is located in chromosome9p, and we recently showed that alterations of p16 are acommon occurrence in MM cell lines and, to a lesser extent,in primary tumors (25). While MMs have a different histogenicderivation than neuroectodermal neoplasms, the frequentabnormalities of chromosome 22 in MM prompted us toinvestigate whether alterations of the NF2 gene are involved inthese tumors.

MATERIALS AND METHODSHuman Primary Tumors, Cell Lines, and Nucleic Acid

Extraction. Fifteen human MM cell lines were establishedfrom primary cultures of surgically resected tumors as de-scribed previously (25). Matched primary tumor tissues weresnap frozen and stored at -70°C. DNA was isolated from cellline and tissue samples by using standard methods (26).Normal diploid human mesothelial cell strain LP-9 (27) wasobtained from the NIA Aging Cell Repository. Total RNA wasextracted from frozen tumor specimens and from cell lines bylysis 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 chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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Reverse Transcriptase (RT)-PCR, Genomic PCR, and Sin-gle-Strand Conformation Polymorphism (SSCP) Analysis.cDNA synthesis was performed by denaturing total RNA at70°C for 10 min in diethyl pyrocarbonate-treated water. Afterchilling on ice the denatured RNA was incubated in 20 mMTris-HCl, pH 8.4/50 mM KCl/2.5 mM MgCl2/10 mM dithio-threitol containing bovine serum albumin at 100 ng/,ul, eachdeoxynucleoside triphosphate at 500 ,uM, primer 3m3 or 3m6at 12 ng/,ul, and SuperScript (GIBCO/BRL) RT at 10 units/,ulfor 10 min at room temperature followed by 60 min at 42°C.The reaction was terminated by heating to 95°C for 2 min andquenching on ice. PCR amplifications and SSCP analyses wereperformed as described previously (12). Oligonucleotide prim-ers were identical to those reported in earlier studies forRT-PCR assays (12) and for genomic PCR amplification ofindividual exons (13).

Sequence Analysis. Individual bands were carefully excisedfrom dried SSCP gels and placed into 100 ,ul of deionizedwater, and the DNA was allowed to elute for 4-6 hr at roomtemperature with gentle shaking. Ten microliters of elutedDNA was reamplified with appropriate primers as describedabove. Amplified products were subcloned in the plasmidvector PCR II (Invitrogen), and inserts were sequenced, usingdouble-stranded recombinant plasmids as template for thedideoxynucleotide chain-termination method (29). Reactionproducts were electrophoresed on 6% polyacrylamide/8 Murea/0.1 M Tris borate, pH 8.3/2mM EDTA gels. Some of theclones were sequenced by dideoxynucleotide terminator chem-istry in an Applied Biosystems 373-A automated DNA se-quencer. Double-strand sequencing of three clones was per-formed for each mutant sample. Sequence data were analyzedby using the MACVEcTOR 4.1 software package (InternationalBiotechnologies).

RESULTSTo investigate the presence of mutations within the NF2coding region, RT-PCR and SSCP analyses were performedwith RNA extracted from 15 MM cell lines. Abnormal SSCPpatterns were detected in cDNAs from 8 of the 15 (53%) celllines. A representative subset of MM samples analyzed byRT-PCR/SSCP is shown in Fig. 1. Sequencing analysis of the

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FIG. 1. SSCP analysis of RT-PCR products made by using RNAfrom normal mesothelial control cells (Co) and the MM cell linesindicated. Arrows show some of the aberrant SSCP conformerssequenced. Oligonucleotide primer pairs used in this analysis were

5ml-3ml (MM 6-53); 5m3-3m3 (MM 1-55); 5m4-3m4 (MM 222); and5m2-3m2 (MM 2-50), according to Bianchi et al. (12).

variant SSCP conformers revealed nucleotide deletions andinsertions and one nonsense mutation in the NF2 gene tran-script, predicting in all cases truncated forms of the merlinprotein. For 7 of the 8 MM cell lines with NF2 cDNAalterations, matched primary tumor specimens were availableand analyzed at the genomic DNA level; NF2 mutations wereconfirmed in 6 of the 7 matched primary tumors (Table 1).NF2cDNA aberrations found in cell lines 222 and 1-53 were notdetected at the genomic level.NF2 cDNA frameshift deletions of 1 and 7 bp were found in

cell lines 6-53 and 2-63, with a stop codon occurring 93 and 48nt, respectively, downstream of the deletion breakpoint.Genomic PCR amplification and sequencing analysis of exon3 and exon 6 of the NF2 gene by using DNA extracted frommatched primary tumors confirmed intragenic deletions of 1and 7 bp in MM 6-53 (Fig. 2) and 2-63, respectively. An 18-bpin-frame deletion encompassing nt 691-708 was detected incDNA from cell line 217 and confirmed at the genomic levelby amplification of exon 8 using DNA from the correspondingprimary tumor. Sequencing of the variant conformer detectedin cDNA from cell line 1-55 (Fig. 1) revealed a nonsensemutation in codon 198. This point mutation was also con-firmed in the matched primary tumor by genomic PCR am-plification of exon 6.Complete skipping of exon 10 in theNF2 gene transcript was

found in cDNA from cell line 222 as compared with thatobserved in normal mesothelial cells (Figs. 1 and 3a). Analysisof genomic DNA from cell line 222 did not reveal anymutations that may account for the skipping of exon 10. Nomutations were detected in either exon 10 or the flankingintron 9 splice donor (not shown) and acceptor sites (cag/G;Fig. 3b), or in the intron 10 splice donor site (not shown), orthe branch point element (TATTAAC; consensus YN-YURAY) positioned 26 nt upstream of exon 10 (Fig. 3b), ascompared with genomic DNA from mesothelial control cells.As shown in Table 1, the remaining NF2 cDNA alterations

detected by RT-PCR/SSCP analyses of cell lines 1-53, 2-50,and 1-51 represent frameshift nucleotide insertions of 43, 119,and 55 bp, respectively, positioned at different splice junctionsites. Genomic PCR analysis of the intron sequences flankingexons 3 and 4 in DNA from cell line 2-50, and exons 1 and 2in primary tumor 1-51, revealed that these nucleotide inser-tions actually corresponded to partial intronic sequences.Sequence comparison with normal mesothelial cells also re-vealed point mutations in the intron 3 splice donor site(CAGgtacat CAGctacat) and the exon 1 splice donor site(GAGitaacg GAAgtaacg) in cell line 2-50 and tumor 1-51,respectively (Table 1). These mutated G nucleotides have beenshown to be conserved 100% and 80% (positions 1 and -1,respectively, relative to the cleavage site) as part of themammalian 5' splice consensus site, and mutations in this sitehave been reported to often activate nearby cryptic splice sitesin vivo, thereby causing partial intron retention in the splicedtranscript (30).The 43-bp nucleotide insertion at the junction of exons 13

and 14 detected in cDNA from cell line 1-53 was not observedat the genomicDNA level (Table 1), and the inserted sequencewas not homologous to any portion of the intron 13 flankingsegments examined.

Similar to previous findings in various tumor types, the NF2mutations reported in this study showed a uniform distributionalong the coding sequence. Whereas two of the eight NF2cDNA alterations described here would lead to interstitialin-frame deletions of the peptide sequence, six of the remain-ing mutations predict a truncation of the encoded protein atvarious positions leading to the removal of the C-terminalregion of merlin.

Interestingly, of the eight MMs with NF2 cDNA alterations,six were of the biphasic type. Among the seven tumors with

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Table 1. Analysis of NF2 mutations in human MMs

Mutation

Sample Cell-line cDNA* Tumor DNAt Predicted effectt6-53 1-bp deletion at nt 271; exon 3 Confirmed Frameshift; protein truncated at aa 122217 18-bp deletion at nt 691-708; exon 8 Confirmed Deletion of aa 231-2362-63 7-bp deletion at nt 568-574; exon 6 Confirmed Frameshift; protein truncation at aa 2081-55 Nonsense mutation at nt 592, CGA -- IGA; exon 6 Confirmed Arg -- stop; protein truncation at aa 1982-50 119-bp insertion at exon 3-exon 4 junction nt 363 Ggt -> Gft; 161-aa truncated protein altered at aa 121

(5' SS of intron 3)1-51 G -- A at nt 114; 55-bp insertion at exon 1-exon 2 junction jgt -+ Agt; 138-aa truncated protein altered at aa 38

(5' SS of intron 1)222 Deletion of exon 10 ND Deletion of aa 296-3331-53 43-bp insertion at exon 13-exon 14 junction ND§ 507-aa truncated protein altered at aa 482

*Mutations detected by RT-PCR/SSCP analysis of cDNA from MM cell lines. Nucleotide (nt) numbers correspond to those reported by Rouleauet al. (7).tMutations detected by PCR of genomic DNA from the primary tumors from which cell lines were derived; confirmed, detection of the samemutation as the one reported in the cell line cDNA; lowercase letters represent introns; SS, splice site; ND, not detected.tAmino acid (aa) numbers according to Rouleau et al. (7).§DNA from the primary tumor was not available. Genomic DNA from the cell line was analyzed instead.

normal NF2 cDNA status, six were of either epithelioid orsarcomatous type, and only one was biphasic.

DISCUSSIONTo determine whether the high frequency of loss of chromo-some 22 previously reported in MM is associated with alter-ation of the NF2 tumor suppressor gene, we screened a set ofMM cell lines and matched primary tumors for mutationswithin the NF2 coding region. Alterations in the NF2 cDNAwere detected in 8 of the 15 cell lines analyzed. Whereas 6 ofthe 8 NF2 gene transcript alterations were confirmed at thegenomic level in primary tumors, the NF2 cDNA aberrationsfound in cell lines 222 and 1-53 were not detected in thecorresponding genomic DNAs.Our findings clearly implicate NF2 in MM tumorigenesis and

provide evidence that this gene can be involved in the devel-opment of malignancies other than the nervous system neo-plasms characteristic of the NF2 disorder. Upon completion ofour studies, Sekido et al. (31) reported NF2 mutations in 6 of14 (43%) MM cell lines. Three of their cell lines derived frommatched tumor samples had a deletion not detected by PCRanalysis in the tumor specimen, presumably because of thepresence of normal cells admixed with tumor cells in the freshsample. Thus, the investigators were unable to rule out thepossibility that these alterations occurred during cell culture.However, they did identify an NF2 mutation in an unmatchedprimary tumor specimen. We have confirmed their cell linefindings, and in addition we demonstrate the frequent involve-ment of NF2 mutations in the pathogenesis of primary MMs.Our data indicating a mutation incidence of at least 40% inprimary MMs rule out the possibility that these alterationsoccurred as a result of in vitro culture of the tumors, as has beensuggested in the case of p16 (32).

TUMOR 6-53

The fact that MMs are not associated with the NF2 disorderis also reminiscent of other hereditary cancer syndromes suchas familial retinoblastoma and Li-Fraumeni syndrome, inwhich the corresponding tumor suppressor gene has beenfound to be somatically mutated in seemingly unrelated ma-lignancies (33).

Similar to the retinoblastoma and adenomatous polyposiscoli tumor suppressor genes which are commonly inactivatedby nonsense mutations, the NF2 gene alterations found in MMin this study consisted mainly of frameshift nucleotide dele-tions, insertions, and nonsense mutations, with absence ofmissense mutations, typically leading to a truncation of the Cterminus of the merlin protein. This mutation spectrum closelyresembles that found in other tumor types, including schwan-nomas and meningiomas, and indicates that in most casesmajor structural alterations are necessary to impair merlinfunction.One exon-skipping alteration found in this study corre-

sponds to the in-frame deletion of exon 10 in MM cell line 222,as indicated by the variant SSCP conformer shown in Fig. 1.Interestingly, analysis of genomic DNA from MM cell line 222revealed no mutations in the flanking intron and exon se-quences analyzed, including the splice donor, splice acceptor,and lariat branchpoint elements (Fig. 3) that may account forthe generation of a truncated transcript lacking exon 10(NF2-A10 mRNA). Previously, the NF2-A10 transcript wasshown by RT-PCR to be expressed at very low levels in RNAisolated from normal leptomeningeal tissue and other sources(34). However, this transcript form was not detected in any ofthe other tumor samples or normal mesothelial control studiedhere, or in previously analyzed tissues, including cDNA fromthe eighth cranial nerve (12). Therefore, the presence of lowexpression levels of alternatively spliced transcripts previouslydetected in normal cells (34) might reflect background levels

CONTROL

S' ICIAAGGAG TCACCACT 3' 5'TTCAAAIcAIr 3'

FIG. 2. Intragenic 1-bp deletion in DNA from MM cell line 6-53. Mutant (Left) and normal mesothelial control (Right) sequences are shownabove density tracings of the sequencing gels. The arrow indicates the position of the nucleotide deleted, C-271.

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a Tumor 2m

.xo IIown 9

snaGLA ca r

Control

exon 10 exon 9

3'ATAOCTAAA CAGCTTATTAACA '

b ron 9 exon IO

Tumfo 222

Contl

FIG. 3. (a) Sequence of the RT-PCR-amplified product from MM 222 and normal mesothelial control cDNAs. Sequences of the junctions ofexon 9 to exon 10 or 11 are shown. The sequence of the 3' end of exon 9 is joined directly to the 5' end of exon 11 in the tumor cDNA (exon 10is skipped) as compared with the 5' end of exon 10 in the control. (b) Sequence of the intron 9/exon 10 boundary in genomic DNAs from MMcell line 222 and normal mesothelial control. No mutations were detected in exon 10, intron 9 (partial sequences shown), or intron 10 (not shown)including the splice recognition sites (3'SS, 3' splice site of intron 9: CAG/A), branchpoint sequence (BPS; TATTAAC), and polypyrimidine tract(Py), marked by solid lines.

of exon skipping, the physiological relevance of which remainsto be established. Although we cannot rule out the possibilitythat the NF2-A1O truncated transcript was generated by amutation in a region other than the sequences analyzed, whichincluded the critical splicing consensus elements, our datasuggest that this form might have resulted from aberrantsplicing of the NF2 gene transcript. Whether this' is due tospecific defects in the splicing machinery regulating the pro-cessing of the NF2 transcript remains to be established. Asimilar mechanism of tumor suppressor inactivation by abnor-mal splicing in the absence of gene mutation has previouslybeen advanced for the Wilms tumor 1 (WT1) tumor suppressorgene (35). In addition, aberrant splicing without gene mutationhas also been proposed as a possible mechanism for disruptionof the transcription factor IRF1 in human myelodysplasia/leukemia (36).

Interestingly, in a study of 75 lung carcinomas, no NF2 genemutations were detected despite the presence of chromosome

22 losses in many cases (31). This suggests that other tumorsuppressor gene(s) may be involved in some of the malignan-cies associated with chromosome 22 allelic losses in which NF2mutations were either not detected or found at very lowfrequencies,- including pheochromocytoma, astrocytoma,ependymoma, as well as ovarian, colon, and breast carcinomas(12, 16, 37, 38).The pattern of frequent chromosomal losses inMM suggests

that multiple tumor suppressor genes may be involved in thepathogenesis of this neoplasm. However, there have been fewreports demonstrating recurrent involvement of such genes inMM. We observed alterations ofp16 in 5 of 23 (22%) primarytumor specimens from MM patients (25). Mutations of othertumor suppressor genes such as TP53 and WTJ have also beenreported in a few cases ofMM (39, 40). The frequent involve-ment of NF2 alterations in both MM cell lines and primarytumors suggests that this gene could play an important role inthe pathogenesis of a large subset of MMs. These data further

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demonstrate that the NF2 gene has a more widespread role intumorigenesis than in the brain tumors typically associatedwith the NF2 syndrome. Moreover, the availability of NF2-mutant and null cell lines should provide a useful model systemfor the functional analysis of merlin.

We thank Xavier Villareal and Larry Gelbert for their collaborationin the sequencing analysis of the samples reported here. This researchwas supported by National Cancer Institute Grants CA-45745 andCA-06927, by the International Association of Heat and Frost Insu-lators and Asbestos Workers Fund, by the Lucille P. Markey Chari-table Trust, and by an appropriation from the Commonwealth ofPennsylvania. J.Q.C. is a Special Fellow of the Leukemia Society ofAmerica.

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