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

High frequency of inactivating mutations in the 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 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- Both germ-line and somatic NF2 gene mutations have been gressive tumors that develop most frequently in the pleura of extensively characterized in and patients exposed to asbestos. In contrast to many other (11-15). The observed mutations represent predominantly , relatively few molecular alterations have been de- either nonsense or splice site mutations, or frameshift nucle- scribed in MMs. The most frequent numerical cytogenetic otide deletions and insertions leading to truncated forms of abnormality in MMs is loss of chromosome 22. The neurofi- merlin. In addition to these tumor types, somatic mutations bromatosis type 2 gene (NF2) is a have been identified in neoplasms seemingly unrelated to the assigned to chromosome 22q which plays an important role in NF2 disorder such as malignant (like schwannomas, the development of familial and spontaneous tumors of neu- however, this tumor type derives from tissue of neural-crest roectodermal origin. Although MMs have a different histo- origin) as well as carcinoma of the breast (12) and colon (16). genic derivation, the frequent abnormalities of chromosome However, the frequency of NF2 mutations detected in breast 22 warranted an investigation ofthe NF2 gene in these tumors. and colon cancers is significantly lower than the incidence of Both cDNAs from 15 MM cell lines and genomic DNAs from allelic losses from chromosome 22q observed in these tumors 7 matched primary tumors were analyzed for mutations (17). within theNF2 coding region. NF2 mutations predicting either Malignant mesotheliomas (MMs) are mesodermally de- interstitial in-frame deletions or truncation of the NF2- rived, primarily pleural tumors that respond poorly to current encoded protein (merlin) were detected in eight cell lines therapeutic approaches. Although MMs are relatively rare, (53%), six of which were confirmed in primary tumor DNAs. their incidence has continued to increase, and considerable In two samples that showed NF2 gene transcript alterations, interest has focused on this neoplasm because of its association no genomic DNA mutations were detected, suggesting that with asbestos exposure (18, 19). Loss of chromosome 22 is the aberrant splicing may constitute an additional mechanism single most consistent numerical cytogenetic change in MMs for merlin inactivation. These findings implicate NF2 in the (20, 21). Rearrangement and loss of chromosomes lp, 3p, 6q, oncogenesis of primary MMs and provide evidence that this and 9p are also a prominent feature of this gene can be involved in the development of tumors other than (20-24). The critically involved genes located within these nervous system neoplasms characteristic of the NF2 disorder. deleted regions are currently unknown. A putative tumor In addition, unlike NF2-related tumors, MM derives from the suppressor genep16/MTSI/CDKN2 is located in chromosome mesoderm; of this origin have not previously 9p, and we recently showed that alterations of p16 are a been associated with frequent alterations of the NF2 gene. common occurrence in MM cell lines and, to a lesser extent, in primary tumors (25). While MMs have a different histogenic Neurofibromatosis type 2 (NF2) is an autosomal dominant derivation than neuroectodermal neoplasms, the frequent disorder characterized by the development of bilateral vestib- abnormalities of chromosome 22 in MM prompted us to ular schwannomas of the eighth cranial nerve and by other investigate whether alterations of the NF2 gene are involved in brain tumors, including meningiomas and (1). these tumors. Affected individuals are also predisposed to develop spinal schwannomas and meningiomas (1-3). Genetic linkage and MATERIALS AND METHODS deletion mapping analyses of sporadic and familial NF2- associated tumors suggested that inactivation of a tumor Human Primary Tumors, Cell Lines, and Nucleic Acid suppressor gene in chromosome 22q was involved in the Extraction. Fifteen human MM cell lines were established etiology of this disorder (4-6). These studies, coupled with from primary cultures of surgically resected tumors as de- have led to the recent isolation scribed previously (25). Matched primary tumor tissues were positional cloning approaches, snap frozen and stored at -70°C. DNA was isolated from cell of the NF2 gene (7, 8). line and tissue samples by using standard methods (26). The NF2 gene is a tumor suppressor gene which encodes a Normal diploid human mesothelial cell strain LP-9 (27) was 595-amino acid protein called merlin (for moesin-ezrin- obtained from the NIA Aging Cell Repository. Total RNA was radixin-like protein) (8) or schwannomin (7). Merlin exhibits extracted from frozen tumor specimens and from cell lines by significant homology to a highly conserved family of proteins lysis in guanidinium thiocyanate and extraction with phenol/ postulated to play a role in cell surface dynamics and structure chloroform by linking the cytoskeleton to components of the plasma (28). membrane (9, 10). Abbreviations: MM, malignant mesothelioma; NF2, - tosis type 2; RT, reverse transcriptase; SSCP, single-strand confor- The publication costs of this article were defrayed in part by page charge mation polymorphism. payment. This article must therefore be hereby marked "advertisement" in tA.B.B. and S.-I.M. contributed equally to this work. accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed.

10854 Downloaded by guest on September 28, 2021 Medical Sciences: Bianchi et al. Proc. Natl. Acad. Sci. USA 92 (1995) 10855 Reverse Transcriptase (RT)-PCR, Genomic PCR, and Sin- variant SSCP conformers revealed nucleotide deletions and gle-Strand Conformation Polymorphism (SSCP) Analysis. insertions and one nonsense mutation in the NF2 gene tran- cDNA synthesis was performed by denaturing total RNA at script, predicting in all cases truncated forms of the merlin 70°C for 10 min in diethyl pyrocarbonate-treated water. After protein. For 7 of the 8 MM cell lines with NF2 cDNA chilling on ice the denatured RNA was incubated in 20 mM alterations, matched primary tumor specimens were available Tris-HCl, pH 8.4/50 mM KCl/2.5 mM MgCl2/10 mM dithio- and analyzed at the genomic DNA level; NF2 mutations were threitol containing bovine serum albumin at 100 ng/,ul, each confirmed in 6 of the 7 matched primary tumors (Table 1). NF2 deoxynucleoside triphosphate at 500 ,uM, primer 3m3 or 3m6 cDNA aberrations found in cell lines 222 and 1-53 were not at 12 ng/,ul, and SuperScript (GIBCO/BRL) RT at 10 units/,ul detected at the genomic level. for 10 min at room temperature followed by 60 min at 42°C. NF2 cDNA frameshift deletions of 1 and 7 bp were found in The reaction was terminated by heating to 95°C for 2 min and cell lines 6-53 and 2-63, with a stop codon occurring 93 and 48 quenching on ice. PCR amplifications and SSCP analyses were nt, respectively, downstream of the deletion breakpoint. performed as described previously (12). Oligonucleotide prim- Genomic PCR amplification and sequencing analysis of exon ers were identical to those reported in earlier studies for 3 and exon 6 of the NF2 gene by using DNA extracted from RT-PCR assays (12) and for genomic PCR amplification of matched primary tumors confirmed intragenic deletions of 1 individual exons (13). and 7 bp in MM 6-53 (Fig. 2) and 2-63, respectively. An 18-bp Sequence Analysis. Individual bands were carefully excised in-frame deletion encompassing nt 691-708 was detected in from dried SSCP gels and placed into 100 ,ul of deionized cDNA from cell line 217 and confirmed at the genomic level water, and the DNA was allowed to elute for 4-6 hr at room by amplification of exon 8 using DNA from the corresponding temperature with gentle shaking. Ten microliters of eluted primary tumor. Sequencing of the variant conformer detected DNA was reamplified with appropriate primers as described in cDNA from cell line 1-55 (Fig. 1) revealed a nonsense above. Amplified products were subcloned in the plasmid mutation in codon 198. This point mutation was also con- vector PCR II (Invitrogen), and inserts were sequenced, using firmed in the matched primary tumor by genomic PCR am- double-stranded recombinant plasmids as template for the plification of exon 6. dideoxynucleotide chain-termination method (29). Reaction Complete skipping of exon 10 in the NF2 gene transcript was products were electrophoresed on 6% polyacrylamide/8 M found in cDNA from cell line 222 as compared with that urea/0.1 M Tris borate, pH 8.3/2 mM EDTA gels. Some of the observed in normal mesothelial cells (Figs. 1 and 3a). Analysis clones were sequenced by dideoxynucleotide terminator chem- of genomic DNA from cell line 222 did not reveal any istry in an Applied Biosystems 373-A automated DNA se- mutations that may account for the skipping of exon 10. No quencer. Double-strand sequencing of three clones was per- mutations were detected in either exon 10 or the flanking formed for each mutant sample. Sequence data were analyzed intron 9 splice donor (not shown) and acceptor sites (cag/G; by using the MACVEcTOR 4.1 software package (International Fig. 3b), or in the intron 10 splice donor site (not shown), or Biotechnologies). the branch point element (TATTAAC; consensus YN- YURAY) positioned 26 nt upstream of exon 10 (Fig. 3b), as compared with genomic DNA from mesothelial control cells. RESULTS As shown in Table 1, the remaining NF2 cDNA alterations To investigate the presence of mutations within the NF2 detected by RT-PCR/SSCP analyses of cell lines 1-53, 2-50, coding region, RT-PCR and SSCP analyses were performed and 1-51 represent frameshift nucleotide insertions of 43, 119, with RNA extracted from 15 MM cell lines. Abnormal SSCP and 55 bp, respectively, positioned at different splice junction patterns were detected in cDNAs from 8 of the 15 (53%) cell sites. Genomic PCR analysis of the intron sequences flanking lines. A representative subset of MM samples analyzed by exons 3 and 4 in DNA from cell line 2-50, and exons 1 and 2 RT-PCR/SSCP is shown in Fig. 1. Sequencing analysis of the in primary tumor 1-51, revealed that these nucleotide inser- tions actually corresponded to partial intronic sequences. Sequence comparison with normal mesothelial cells also re- 0 '0 vealed point mutations in the intron 3 splice donor site tps~ (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 been shown to be conserved 100% and 80% (positions 1 and -1, f...... I:... respectively, relative to the cleavage site) as part of the mammalian 5' splice consensus site, and mutations in this site have been reported to often activate nearby cryptic splice sites in vivo, thereby causing partial intron retention in the spliced J L:: transcript (30). The 43-bp nucleotide insertion at the junction of exons 13 m "A and 14 detected in cDNA from cell line 1-53 was not observed at the genomic DNA level (Table 1), and the inserted sequence was not homologous to any portion of the intron 13 flanking mw4So segments examined. Similar to previous findings in various tumor types, the NF2 mutations reported in this study showed a uniform distribution the Whereas two of the NF2 .ml along coding sequence. eight cDNA alterations described here would lead to interstitial in-frame deletions of the peptide sequence, six of the remain- FIG. 1. SSCP analysis of RT-PCR products made by using RNA a the encoded at from normal mesothelial control cells (Co) and the MM cell lines ing mutations predict truncation of protein indicated. Arrows show some of the aberrant SSCP conformers various positions leading to the removal of the C-terminal sequenced. Oligonucleotide primer pairs used in this analysis were region of merlin. 5ml-3ml (MM 6-53); 5m3-3m3 (MM 1-55); 5m4-3m4 (MM 222); and Interestingly, of the eight MMs with NF2 cDNA alterations, 5m2-3m2 (MM 2-50), according to Bianchi et al. (12). six were of the biphasic type. Among the seven tumors with Downloaded by guest on September 28, 2021 10856 Medical Sciences: Bianchi et al. Proc. Natl. Acad. Sci. USA 92 (1995)

Table 1. Analysis of NF2 mutations in human MMs Mutation Sample Cell-line cDNA* Tumor DNAt Predicted effectt 6-53 1-bp deletion at nt 271; exon 3 Confirmed Frameshift; protein truncated at aa 122 217 18-bp deletion at nt 691-708; exon 8 Confirmed Deletion of aa 231-236 2-63 7-bp deletion at nt 568-574; exon 6 Confirmed Frameshift; protein truncation at aa 208 1-55 Nonsense mutation at nt 592, CGA -- IGA; exon 6 Confirmed Arg -- stop; protein truncation at aa 198 2-50 119-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 296-333 1-53 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 Rouleau et al. (7). tMutations detected by PCR of genomic DNA from the primary tumors from which cell lines were derived; confirmed, detection of the same mutation 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 or The fact that MMs are not associated with the NF2 disorder sarcomatous type, and only one was biphasic. is also reminiscent of other hereditary cancer syndromes such as familial retinoblastoma and Li-Fraumeni syndrome, in which the corresponding tumor suppressor gene has been DISCUSSION found to be somatically mutated in seemingly unrelated ma- To determine whether the high frequency of loss of chromo- lignancies (33). some 22 previously reported in MM is associated with alter- Similar to the retinoblastoma and adenomatous polyposis ation of the NF2 tumor suppressor gene, we screened a set of coli tumor suppressor genes which are commonly inactivated MM cell lines and matched primary tumors for mutations by nonsense mutations, the NF2 gene alterations found in MM within the NF2 coding region. Alterations in the NF2 cDNA in this study consisted mainly of frameshift nucleotide dele- were detected in 8 of the 15 cell lines analyzed. Whereas 6 of tions, insertions, and nonsense mutations, with absence of the 8 NF2 gene transcript alterations were confirmed at the missense mutations, typically leading to a truncation of the C genomic level in primary tumors, the NF2 cDNA aberrations terminus of the merlin protein. This mutation spectrum closely found in cell lines 222 and 1-53 were not detected in the resembles that found in other tumor types, including schwan- corresponding genomic DNAs. nomas and meningiomas, and indicates that in most cases Our findings clearly implicate NF2 in MM tumorigenesis and major structural alterations are necessary to impair merlin provide evidence that this gene can be involved in the devel- function. opment of malignancies other than the nervous system neo- One exon-skipping alteration found in this study corre- plasms characteristic of the NF2 disorder. Upon completion of sponds to the in-frame deletion of exon 10 in MM cell line 222, our studies, Sekido et al. (31) reported NF2 mutations in 6 of as indicated by the variant SSCP conformer shown in Fig. 1. 14 (43%) MM cell lines. Three of their cell lines derived from Interestingly, analysis of genomic DNA from MM cell line 222 matched tumor samples had a deletion not detected by PCR revealed no mutations in the flanking intron and exon se- analysis in the tumor specimen, presumably because of the quences analyzed, including the splice donor, splice acceptor, presence of normal cells admixed with tumor cells in the fresh and lariat branchpoint elements (Fig. 3) that may account for sample. Thus, the investigators were unable to rule out the the generation of a truncated transcript lacking exon 10 possibility that these alterations occurred during cell culture. (NF2-A10 mRNA). Previously, the NF2-A10 transcript was However, they did identify an NF2 mutation in an unmatched shown by RT-PCR to be expressed at very low levels in RNA primary tumor specimen. We have confirmed their cell line isolated from normal leptomeningeal tissue and other sources findings, and in addition we demonstrate the frequent involve- (34). However, this transcript form was not detected in any of ment of NF2 mutations in the pathogenesis of primary MMs. the other tumor samples or normal mesothelial control studied Our data indicating a mutation incidence of at least 40% in here, or in previously analyzed tissues, including cDNA from primary MMs rule out the possibility that these alterations the eighth cranial nerve (12). Therefore, the presence of low occurred as a result ofin vitro culture of the tumors, as has been expression levels of alternatively spliced transcripts previously suggested in the case of p16 (32). detected in normal cells (34) might reflect background levels TUMOR 6-53 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 shown above density tracings of the sequencing gels. The arrow indicates the position of the nucleotide deleted, C-271. Downloaded by guest on September 28, 2021 Medical Sciences: Bianchi et al. Proc. Natl. Acad. Sci. USA 92 (1995) 10857

a Tumor 2m Control

.xo IIown 9 exon 10 exon 9

snaGLA ca r 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 of exon 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 10 is 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 MM cell 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 remains 22 losses in many cases (31). This suggests that other tumor to be established. Although we cannot rule out the possibility suppressor gene(s) may be involved in some of the malignan- that the NF2-A1O truncated transcript was generated by a cies associated with chromosome 22 allelic losses in which NF2 mutation in a region other than the sequences analyzed, which mutations were either not detected or found at very low included the critical splicing consensus elements, our data frequencies,- including pheochromocytoma, , suggest that this form might have resulted from aberrant , as well as ovarian, colon, and breast carcinomas splicing of the NF2 gene transcript. Whether this' is due to (12, 16, 37, 38). specific defects in the splicing machinery regulating the pro- The pattern of frequent chromosomal losses in MM suggests cessing of the NF2 transcript remains to be established. A that multiple tumor suppressor genes may be involved in the similar mechanism of tumor suppressor inactivation by abnor- pathogenesis of this neoplasm. However, there have been few mal splicing in the absence of gene mutation has previously reports demonstrating recurrent involvement of such genes in been advanced for the Wilms tumor 1 (WT1) tumor suppressor MM. We observed alterations ofp16 in 5 of 23 (22%) primary gene (35). In addition, aberrant splicing without gene mutation tumor specimens from MM patients (25). Mutations of other has also been proposed as a possible mechanism for disruption tumor suppressor genes such as TP53 and WTJ have also been of the IRF1 in human myelodysplasia/ reported in a few cases of MM (39, 40). The frequent involve- leukemia (36). ment of NF2 alterations in both MM cell lines and primary Interestingly, in a study of 75 lung carcinomas, no NF2 gene tumors suggests that this gene could play an important role in mutations were detected despite the presence of chromosome the pathogenesis of a large subset of MMs. These data further Downloaded by guest on September 28, 2021 10858 Medical Sciences: Bianchi et al. Proc. Natl. Acad. Sci. USA 92 (1995) demonstrate that the NF2 gene has a more widespread role in Mitelman, F., Louis, D., Menon, A., Newsham, I., Decker, J., tumorigenesis than in the brain tumors typically associated Kaelbling, M., Henry, I. & Deimling, A. V. (1991) Cytogenet. -C-l with the NF2 syndrome. Moreover, the availability of NF2- Genet. 58, 1080-1096. mutant and null cell lines should provide a useful model system 18. Selikoff, I. J., Churg, J. & Hammond, F. C. (1965) N. Engl. J. for the functional analysis of merlin. Med. 272, 560-565. 19. Ruffie, P. A. 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