smarcb1/ini1 tumor suppressor gene is frequently...

SMARCB1/INI1 Tumor Suppressor Gene Is Frequently

Inactivated in Epithelioid Sarcomas

Piergiorgio Modena,1Elena Lualdi,

1Federica Facchinetti,

1Lisa Galli,

1

Manuel R. Teixeira,3Silvana Pilotti,

2and Gabriella Sozzi

1

Units of 1Molecular Cytogenetics and 2Molecular Pathology, Istituto Nazionale per lo Studio e la Cura dei Tumori,Milano, Italy; and 3Department of Genetics, Portuguese Oncology Institute, Porto, Portugal

Abstract

Epithelioid sarcoma is a rare soft tissue neoplasm of uncertainlineage that usually arises in the distal extremities of adults,presents a high rate of recurrences and metastases andfrequently poses diagnostic dilemmas. The recently reportedlarge-cell ‘‘proximal-type’’ variant is characterized by in-creased aggressiveness, deep location, preferential occurrencein proximal/axial regions of older patients, and rhabdoidfeatures. Previous cytogenetic studies indicated that the mostfrequent alterations associated with this tumor entity affectchromosome 22. In this study, combined spectral karyotyping,fluorescence in situ hybridization, and array-based compar-ative genomic hybridization analyses of two proximal-typecases harboring a rearrangement involving 10q26 and 22q11revealed that the 22q11 breakpoints were located in a 150-kbregion containing the SMARCB1/INI1 gene, and that homozy-gous deletion of the gene was present in the tumor tissue. TheSMARCB1/INI1 gene encodes for an invariant subunit of SWI/SNF chromatin remodeling complex and has been previouslyreported to act as a tumor suppressor gene frequentlyinactivated in infantile malignant rhabdoid tumors. Weanalyzed SMARCB1/INI1 gene status in nine additionalepithelioid sarcoma cases ( four proximal types and fiveconventional types) and altogether we identified deletions ofSMARCB1/INI1 gene in 5 of 11 cases, all proximal types. Weconfirmed and further extended the number of cases withSMARCB1/INI1 inactivation to 6 of 11 cases, by real-timequantitative PCR analysis of mRNA expression and bySMARCB1/INI1 immunohistochemistry. Overall, these resultspoint to SMARCB1/INI1 gene involvement in the genesis and/or progression of epithelioid sarcomas. Analysis of largerseries of epithelioid sarcomas will be necessary to highlightputative clinically relevant features related to SMARCB1/INI1inactivation. (Cancer Res 2005; 65(10): 4012-9)

Introduction

Epithelioid sarcoma is a rare and distinctive soft tissue sarcomafirst described in 1970 (1). This neoplasm typically arises in the distalextremities of young adults as a painless, slow-growing nodulewithin dermis or subcutis or, less commonly, in deep fascial ortenosynovial tissue. Morphologically, the classic, also called ‘‘distal’’,

form of epithelioid sarcoma consists of tumor cells that range fromspindle to large round or polygonal, bearing a strong similarity toepithelioid or squamous cells, and arranged in nodular aggregatesthat commonly exhibit central necrosis. Upon microscopic exam-ination, tumor cells show slight nuclear atypia, vesicular nuclei, andsmall nucleoli (2–7). Two variants of this form have been described:the ‘‘angiomatoid,’’ characterized by pseudoangiosarcomatousfeatures and the ‘‘fibroma like,’’ with a predominance of spindle cellpattern with minimal cellular pleomorphism. The immunopheno-type of epithelioid sarcoma shows positivity for vimentin and bothepithelial markers EMA and cytokeratin. A number of the cases arealso positive for CD34, muscle-specific and smooth muscle actins,neuron-specific enolase, and S100 protein (8–12).Recently, an atypical large cells variant of epithelioid sarcoma,

which is characterized by increased aggressiveness, multinodularpattern of growth, rhabdoid features and deep location, has beendescribed (13–15). It mainly but not exclusively occurs in the pelvic,perineal and genital tract as well as in the proximal extremities ofmiddle aged adults and therefore it has also been referred as‘‘proximal-type’’ epithelioid sarcoma (PES). Upon microscopicexamination, PES consists of large epithelioid carcinoma-likeand/or rhabdoid cells. Despite the differences in clinical presen-tation and behavior, conventional epithelioid sarcoma andproximal-type epithelioid sarcoma share a similar immunopheno-typic profile suggesting a common pathogenesis. It is not clear yetif the unfortunate behavior of PES is related to the prominentrhabdoid phenotype or merely to classic prognostic factors, such astumor size, depth, proximal/axial location, resectability, or vascularinvasion (2, 13, 14).Conventional cytogenetic studies of epithelioid sarcoma revealed

complex karyotypes with various chromosome deletions and gains,but the only recurrent breakpoints in structural rearrangementswere 18q11 and 22q11 in two cases each (16–25). Spectralkaryotyping (SKY) analysis of six PES revealed the presence of 11rearrangements on chromosome 22q in five of the six casesanalyzed, with two cases showing a similar t(10, 22) translocation(26). In addition, loss of heterozygosity analysis identified frequentdeletion of 22q material in epithelioid sarcoma (27). These datasuggest that a region on chromosome 22q may contain a tumorsuppressor gene involved in the development of this neoplasm.In this study, 11 epithelioid sarcoma, six proximal types and five

conventional types, were analyzed by cytogenetic and molecularapproaches. We provide evidence of genomic deletion of theSMARCB1/INI1 gene in 5 of 11 (45%) epithelioid sarcoma cases thatcorrelates with mRNA and/or protein down-regulation. SMARCB1/INI1 immunohistochemistry confirmed inactivation at the proteinlevel and extended the number of cases with SMARCB1/INI1alterations to 6 of 11 (55%), of which 5 of 6 are proximal types. Ourdata suggest the involvement of SMARCB1/INI1 alterations in asubstantial fraction of epithelioid sarcoma.

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

P. Modena and E. Lualdi contributed equally to this work.Requests for reprints: Gabriella Sozzi, Molecular Cytogenetics Unit, Istituto

Nazionale per lo Studio e la Cura dei Tumori, via Venezian 1, 20133 Milano, Italy. Phone:39-02-23902232; Fax: 39-02-23902764; E-mail: [email protected].

I2005 American Association for Cancer Research.

Cancer Res 2005; 65: (10). May 15, 2005 4012 www.aacrjournals.org

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Materials and Methods

Patients and pathology. This study included 11 epithelioid sarcoma

cases. Ten were retrieved from the files of the Department of Pathology,Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy,

comprising the period 1991 to 2002. One additional case was collected at

the Portuguese Oncology Institute, Porto, Portugal. In all cases, routine

sections were available. Six cases showed features consistent with PES, 3with rhabdoid-like and 2 with epithelioid-like appearance. In one case (case

5), the tumor exhibited a major spindle-cell component admixed with

epithelial-like features. Five cases corresponded to classic epithelioidsarcoma, distal type. Immunophenotyping was in keeping with the

morphologic diagnosis in all the 11 cases, showing positivity for low-weight

cytokeratin, epithelial membrane antigen, and vimentin. CD34 yielded

positive results in cases 4, 6, 10, and 11. S100 protein staining was positive incase 2. Patients’ clinical data, including gender, age, tumor location, and

histologic type and subtype are summarized in Table 1.

Cell lines. Previously described (26) short-term tumor-derived cell

cultures of cases 1 to 4 were cultured with Amniomax C100 (LifeTechnologies, Carlsbad, CA) supplemented with Amniomax C100 supple-

ment (Life Technologies) and harvested with 0.01 Ag/mL Colcemid

(Karyomax Colcemid, Life Technologies) overnight. Cell lines 293 and

G-401 were purchased from American Type Culture Collection (Manassas,VA) and were maintained in RPMI 1640 supplemented with 10% heat-

inactivated FCS (Life Technologies).

Fluorescence in situ hybridization analysis. BAC probes used for dual-color fluorescence in situ hybridization (FISH) were extracted using

QIAprep Midiprep (Qiagen, Valencia, CA), verified by PCR amplification of

sequence-tagged site markers and labeled with Spectrum Orange dUTP and

Spectrum Green dUTP (Vysis, Inc., Downers Grove, IL) by Nick TranslationKit (Vysis) according to the manufacturer’s instructions. All probes were

tested on normal metaphase and interphase samples to calculate probe

efficiency (i.e., number of observed signals/number of expected signals),

which was >90% in all cases. FISH analyses on metaphase cells from short-term cultures and on touch preparations of frozen tissue were carried out

following standard methods (28). To perform FISH experiments on paraffin-

embedded samples, dewaxed 3-Am paraffin sections were boiled in Tris/EDTA buffer solution (5 mmol/L Tris and 1 mmol/L EDTA), incubated with

0.4% of pepsin (Sigma, St. Louis, MO) in 0.01 N HCl at 37jC, denatured in

70% formamide-2� SSC for 12 minutes at 85jC and hybridized overnight at

37jC with the probes denatured at 80jC for 5 minutes. Post-hybridizationwashes and detections were done as described previously (28). The FISH

hybridization signals were analyzed in an Olympus BX51 microscope

coupled to a charge-coupled device camera COHU 4912 (Olympus, Milano,

Italy). The images captured were analyzed using the Mac Probe software(PowerGene Olympus, Milano, Italy). A minimum of 150 nuclei were

counted in FISH experiments from paraffin-embedded tissue and results

were adjusted taking into account the probe efficiency.

Comparative genomic hybridization. Array-based comparative ge-

nomic hybridization (array-CGH) was done on demand by Spectral Genomics

(Houston, TX) using 1 Ag of tumor and reference genomic DNA and the 2- to

4-Mb resolution KTH-1400 arrays. Briefly, two oppositely labeled experiments

were done for each sample in which tumor and reference DNAs were labeled

with different fluorochromes (Cy3-labeled test and Cy5-labeled reference

DNA, Cy3-labeled reference and Cy5-labeled test DNA) and DNA mixes were

hybridized onto duplicate microarray slides as recommended by the

manufacturer. Images were acquired using Axon 4000 scanner and analyzed

by Spectralware software (Spectral Genomics) to normalize the Cy5:Cy3

intensity ratios for each slide and to compute the normalized Cy5/Cy3

intensity ratios for both arrays, that were plotted for each chromosome. A loss

of a particular clone is manifested as the simultaneous deviation of the ratio

plots from a modal number of 1.0, with the red ratio plot showing a positive

deviation (upward) whereas the blue ratio plot shows a negative deviation

(downward). Conversely, DNA copy number gains show the opposite pattern.

PCR-based analysis of genomic DNA. PCR amplifications were done ona 9700 Thermocycler (Applied Biosystems, Foster City, CA), using 1.5 mmol/

L MgCl2, 0.2 mmol/L deoxynucleotide triphosphates, 0.5 Amol/L of each

primer, 0.6 unit AmpliTaqGold (Applied Biosystems), 1� reaction buffer.Semiquantitative PCR amplification of SMARCB1/INI1 exon 1 was done

using primers INIex1F (5V-gcatttcgccttccggcttcg-3V) and INIex1R (5V-gatgaatggagacgcgcgct-3V), 292 bp, at 59jC in the presence of 5% DMSO.

Four replicate experiments were done using serial dilutions of 25, 5, 1, 0.2 ngof template DNA. PCR products were loaded onto ethidium bromide-

stained 2% agarose gel and quantitated by densitometric analysis using

Quantity One 4.5 (Bio-Rad, Hercules, CA). Mean intensities and SDs were

calculated from the four independent experiments. Using the sameapproach, control amplifications were done using primers D16S3121F (5V-catgttgtacatcgtgatgc-3V) and D16S3121R (5V-agcttttatttcccaggggt-3V), 90 to 98

bp, 55jC; FKHR2F (5V-aacgacacatagctgggtgtcaggc-3V) and FKHR3R (5V-agttcctgctgtcagacaatctgaagtac-3V), 558 bp, 55jC. Donor-derived genomic

DNA served as positive control, whereas genomic DNA from G401 cell line

(carrying a homozygous deletion of the entire SMARCB1/INI1 gene) was

used as negative control. A 1:3 mixture of normal/G401 genomic DNA wasincluded to assess the sensitivity of the test.

To isolate normal tissue areas as well as different tumor areas from

paraffin-embedded tissue, tissue blocks were trimmed by a pathologist using

a razor blade with the assistance of a light microscope. DNA was extracted

using QIAmp Tissue kit (Qiagen) and PCR-amplified with primer sets for

INI1 exons as well as control loci: INIex1F and INI1ex1rev (5V-ctgcgagg-gatcaggagggct-3V), 77 bp, 62jC in the presence of 5% DMSO; INI4F (5V-tgccgtgccatgctccacaacc-3V) and INIi27 (5V-cacaaggtcaaagcagagtg-3V), 176 bp,

56jC; D16S3121F and D16S3121R ; D1S2736F (5V-cctccagggtattcttgg-3V) andD1S2736R (5V-tttttgaggtgtgagagcag-3V), 122 to 134 bp, 55jC.

Real-time quantitative reverse transcription-PCR. Retrotranscriptionwas done using 1 Ag of total RNA extracted by Trizol Reagent (Invitrogen,

San Diego, CA), using random hexamers in the presence (RT+) or absence

(RT�) of Superscript Reverse Transcriptase (Invitrogen). Relative quantifi-cation of SMARCB1/INI1 expression was done using Taqman Gene

Expression Assays on Demand (Applied Biosystems) and ABI Prism 7900

HT Sequence Detection System (Applied Biosystems). A validation

experiment was done to assess that the target (SMARCB1/INI1 ,Hs00268260_m1, Applied Biosystems) and reference (hHPRT, huHPRT

PDAR, Applied Biosystems) efficiencies of amplification were equal at

different template diluitions. Reactions consisted of 1� Taqman Universal

PCR Master Mix (Applied Biosystems), 1� Assay on Demand, 1 of 40 of theretrotranscription reaction as template and were run according to

manufacturer’s universal cycling conditions. Quantification of SMARCB1/

INI1 expression was done by comparative C t method according to themanufacturer instructions, using the HPRT gene as endogenous reference

control and normal brain cDNA as calibrator. Target and reference probe

sets were amplified in triplicates, in separate wells of 96-well plates. Results

Table 1. Clinical data and tumor phenotype of the 11epithelioid sarcoma cases under study

Case Gender/age Tumor location Morphology

1 F/31 thigh Proximal ES, epithelioid like2 F/47 perineum Proximal ES, rhabdoid like

3 M/71 dorsal region Proximal ES, rhabdoid like

4 M/30 lumbar region Proximal ES, epithelioid like

5 M/36 sacral region Proximal ES, prominentspindle-cell component

6 F/66 inguinal region Proximal ES, rhabdoid like

7 M/27 arm Distal ES, classic type

8 F/55 foot Distal ES, classic type9 M/31 hand Distal ES, classic type

10 M/22 hand Distal ES, classic type

11 M/27 arm Distal ES, classic type

Abbreviation: ES, epithelioid sarcoma.

SMARCB1 Inactivation in Epithelioid Sarcoma

www.aacrjournals.org 4013 Cancer Res 2005; 65: (10). May 15, 2005



were analyzed using ABI PRISM Sequence Detection System software(version 2.1, Applied Biosystems). The use of normal muscle or normal

kidney cDNAs as calibrator provided comparable results (data not shown).

Mutation analysis.Mutation detection of SMARCB1/INI1 gene was done

by exons amplification and direct sequencing using previously describedprimers (29).

Western blot analysis. Western blot analysis was done using standard

techniques from total lysates extracted from frozen tumor tissue and cell

lines. Frozen tissue was preliminarily homogenized using Mixer Mill MM300

(Qiagen). From each sample, 40 Ag of proteins were separated on 10% SDS-

PAGE and electro-blotted onto polyvinylidene difluoride membranes

(Amersham Biosciences, Buckinghamshire, United Kingdom). Subsequently,

membranes were incubated 1 hour at room temperature in a solution of

PBS supplemented with 5% nonfat dry milk. For immunodetection, the anti-

BAF47/SNF5 antibody (BD Transduction Laboratories, Franklin Lakes, NJ)

was used. After overnight incubation at 4jC with the primary antibody

diluted 1:250, membranes were washed in TBST [10 mmol/L Tris-HCl (pH

8.0), 0.15 mol/L NaCl, and 0.05% Tween 20], followed by horseradish

peroxidase–conjugated goat-antimouse or goat-antirabbit antibody. En-

hanced chemiluminescence (Amersham Biosciences) was used for detec-

tion. Protein loading equivalence was assessed using an anti-hactinantibody (Sigma).

Immunohistochemistry. Immunophenotypic analysis was done by

autostainer instrument together with ChemMate detection kit 5001 (Dako,

Milano, Italy) using heat-induced epitope retrieval. Cytokeratin staining

required trypsin predigestion. Antibodies used were low-weight Cytokeratin

(mouse monoclonal, 1:10, BD Transduction Laboratories), EMA (mouse

monoclonal, 1:500, Dako), Vimentin (mouse monoclonal, 1:200, Dako), CD34

(mouse monoclonal, 1:50, Novocastra, Newcastle upon Tyne, United

Kingdom), and S100 (rabbit polyclonal, 1:2000, Dako).

Immunohistochemistry of SMARCB1 protein was done using the mouse

monoclonal antibody anti-BAF47/SNF5 as primary antibody. Endogenousperoxidase was blocked with 0.3% hydrogen peroxide in methanol for 30

minutes. The slides were immersed in citrate buffer solution 5 mmol/L

(pH 6) and heated in autoclave at 95jC for 6 minutes for antigen retrieval.

The slides were incubated at room temperature for 30 minutes with normalgoat serum (Dako) and with the primary antibody (1:250) for 1 hour at room

temperature. Negative controls were done using mouse immunoglobulin1:12,000 (Dako). We used Dako ChemMate detection kit 5001 in the

subsequent steps using biotinylated secondary antibody, streptavidin-

conjugated horseradish peroxidase, and diaminobenzidine as the cromogen.

Finally, the slides were counterstained with Carazi hematoxylin.

Results

Fluorescence in situ hybridization analysis of chromosome22 breakpoints in short-term tumor cell cultures. In ourprevious report of SKY analysis in six PES cases (26), we described acluster of breakpoints on 22q11 chromosome band and thepresence of a similar t(10;22) translocation in two cases. To mapthe chromosome 22 breakpoints more precisely, we performedFISH experiments using a panel of BAC clones in four of the sixcases previously analyzed by SKY. Notably, the breakpoints in cases1 and 2 were found located within a 150-kb region covered by theBAC clone RP11-71G19 and flanked by RP11-61P17 and RP11-80O7,that overlap 5V- and 3V- ends of RP11-71G19, respectively. In case 1(Fig. 1A), FISH experiments showed that the t(10;22) was balanced,with the exception of clone RP11-71G19, that showed three signalsindicating that it spans the breakpoint (Fig. 1B), whereasoverlapping clones RP11-61P17 and RP11-80O7 showed signalsonly on the der(10) and der(22), respectively (Fig. 1C). Intriguingly,BAC clone RP11-71G19 contains SMARCB1/INI1 gene, a knowntumor suppressor gene inactivated in malignant rhabdoid tumors(30, 31). FISH experiments in case 2, that harbored a complexrearrangement involving chromosomes 10, 15, and 22 (Fig. 1D),showed the loss of clone RP11-80O7 that overlaps with RP11-71G19and is flanking SMARCB1/INI1 gene, therefore suggesting a similarbreakpoint region as in case 1 (Fig. 1E-F).With the same strategy in case 3, carrying a t(12;22), the

breakpoint on chromosome 22 was found to be located in a 1-Mbregion of 22q12 (Fig. 1F). EWSR1 gene did not seem rearranged asshown by FISH and Southern blot (data not shown); furthermore,

Figure 1. SKY and FISH analyses ofmetaphase chromosomes fromtumor-derived short-term cell cultures incases 1 and 2. A and D, visualization ofnormal chromosomes 22 and aberrantchromosomes involving 22 material afterSKY analysis in cases 1 and 2,respectively. For each aberration the RGB(red-green-blue ), the inverted DAPI, andthe pseudocolor images of thechromosomes (left to right ). Numbers onthe right side of the derivativechromosomes denote the chromosomalorigin. Complete SKY results have beenpreviously published (26). B, FISH analysisof clone RP11 71G19 containingSMARCB1/INI1 gene in case 1 displayedsplitting of the signal on both derivativesof the translocation t(10;22)(q26;q11).C, double-color FISH analysis of clonesRP11-61P17 (green ) and RP11-80O7(red) flanking SMARCB1/INI1 gene in case1 displayed separated signal on der(22)and der(10), respectively. E, the samedouble-color FISH analysis in case 2displayed loss of RP11-80O7 (red signal )on der(10). F, summary of FISH results incases 1 to 4.

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the presence of t(12;22)-associated fusion genes EWSR1-DDIT3 andEWSR1-ATF1 , typical of myxoid-round cell liposarcoma and clearcell sarcoma, respectively, was excluded by reverse transcription-PCR experiments (data not shown). In case 4, because of thecomplexity of the rearrangements involving chromosome 22 andinsufficient cell pellet, it has only been possible to show the loss of22q12 and that the breakpoints did not encompass the regioncovered by RP11-71G19 (Fig. 1F).We showed the in vivo occurrence of t(10;22) and 22q11

breakpoint in case 1, by double-color interphase FISH experimentsfrom frozen tumor biopsy (Supplementary Fig. A). Severalsubclones of tumor cells were present, in which either bothderivatives were retained (shown by split signals) or one/twoderivatives were lost (absence of signal).Array-based comparative genomic hybridization and fluo-

rescence in situ hybridization analysis of tumor tissue samples1 and 2. Whole genome array-CGH analysis of cases 1 and 2 at 2- to4-Mb resolution displayed a normal chromosome 22 content in case2 and a 22q heterozygous loss covering f15 Mb and comprisingSMARCB1/INI1 in case 1 (Fig. 2A-B). Because in malignant rhabdoidtumors SMARCB1/INI1 gene is homozygously deleted in a markedlyhigh proportion of the cases (29), and 22q rearrangementsfrequently seem to trigger SMARCB1/INI1 loss (32), we didinterphase FISH with clone RP11-71G19, that contains SMARCB1/INI1 gene, to directly evaluate the SMARCB1/INI1 gene dosage intumor samples 1 and 2. In case 1, double-color FISH analysis ontouch preparations of frozen tissue showed a high frequency ofclone RP11-71G19 homozygous loss and control clone RP11-91K24heterozygous loss (Fig. 2C). These two clones are separated by 3 Mb,and therefore SMARCB1/INI1 homozygous loss lies within the wideregion of heterozygous loss detected by array-CGH but is below theresolution of the array employed. Similarly, case 2 displayed noevidence of loss by array-CGH, but double-color FISH analysis onparaffin-embedded tumor tissue showed attainment of normal copynumber of clone RP11-91K24 and homozygous loss of clone RP11-71G19 (Fig. 2D), suggesting that the region of loss comprisingSMARCB1/INI1 is below the resolution of the array employed.Additional double-color interphase FISH experiments were done

to define the extent of the regions of homozygous deletion

(Supplementary Fig. B). In case 1, the results showed homozygousdeletion of clone RP11-71G19 in the majority of nuclei that retainedthe signal of the flanking clones RP11-61P17 and RP11-80O7. Thus,the combined array-CGH and FISH results allowed us to narrowdown the region of homozygous loss to less then 150-kb and tostrongly pinpoint SMARCB1/INI1 as the gene inactivated by theobserved rearrangements. Two additional known genes are presentin this region, pre-B lymphocyte gene 3 (VPREB3) involved in B-celldifferentiation and matrix metalloproteinase 11 (MMP11), a genereported overexpressed in a variety of tumors (33–35). In case 2,double-color FISH experiments were done on paraffin sections and,together with array-CGH and FISH results reported in Fig. 2B andD , indicated that the extent of the region of HD is between 450 kband 5 Mb (Supplementary Fig. B).Whereas molecular analyses (either array-CGH or FISH) of PES

tumor biopsies 1 and 2 disclosed a loss of SMARCB1/INI1 gene,cytogenetic and FISH analyses of short-term cell culture of thesame cases had revealed chromosome 22 rearrangements in thevicinity of SMARCB1/INI1 gene but no evidence of loss. Such asupposed discrepancy between results obtained on short-term cellculture and frozen biopsy suggested both that a specific tumor cellclone was selected during in vitro cell culture and that thetranslocation involving 22q preceded loss of 22q11 material. Thesehypotheses are substantiated by FISH results reported inSupplementary Figs. A and B.Fluorescence in situ hybridization analysis of SMARCB1/INI1

locus in paraffin-embedded tumor samples. We extended theanalysis of SMARCB1/INI1 locus to the 11 epithelioid sarcomacases under study (Table 1) by double-color interphase FISHwith clone RP11-71G19 that contains SMARCB1/INI1 gene, incombination with a control probe from 22q13 (RP11-262A13) onparaffin-embedded tumor tissue. With the exception of case 3,which did not display any loss by FISH, all other PES cases (cases 1,2, 4-6) revealed intratumor loss of SMARCB1/INI1 signal (Fig. 3, band b). Interestingly, it seemed evident that SMARCB1/INI1 loss didnot correlate with the rhabdoid cell compartment. In fact, case 1morphology was homogeneously epithelioid, carcinoma like(Fig. 3, 1a), and displayed a high proportion of homozygous loss(Fig. 3, 1b). Case 2 had a multinodular pattern with rhabdoid cells

Figure 2. Array-CGH analysis at 2- to 4-Mb resolution in cases 1(A) and 2 (B ), displaying a heterozygous deletion of f15 Mb ofchromosome 22 in case 1 and normal dosage in case 2. Cloneposition is based on UCSF April 2002 release. Double-colorFISH analysis on frozen tumor tissue of case 1 (C ) and onparaffin-embedded tissue of case 2 (D ), confirmed the dosage ofclone RP11-91K24 (red signal ) as identified by array-CGH andrevealed homozygous deletion, below the resolution of thearray-CGH, of clone RP11-71G19 (green signal ) containingSMARCB1/INI1 gene.





and showed homozygous loss (Fig. 3, 2a and b); case 4 displayedSMARCB1/INI1 loss confined to a region of epithelioid morphology(Fig. 3, 4a and b). Interestingly, in case 5, SMARCB1/INI1homozygous loss was restricted to areas of spindle cell morphologywhereas epithelioid-like areas were normal (Fig. 3, 5a-b and a-b).Finally, case 6, of prominent rhabdoid morphology, displayedhomogeneous heterozygous loss (Fig. 3, 6a and b). Cases 7-11

displayed a normal SMARCB1/INI1 copy number by FISH and case11 is shown as a representative example in Fig. 3, 11a and b .SMARCB1/INI1 protein expression analysis. To circumvent

the potential limitations related to the presence of normal cellsinfiltrating the tumor and to further investigate SMARCB1/INI1inactivation at the protein level, immunohistochemistry was doneusing a monoclonal antibody against SMARCB1/INI1 protein.

Figure 3. FISH and immunohistochemistryanalysis of SMARCB1/INI1 . Numbers inthe panels refer to case numbers as inTable 1. a and a, H&E staining (100�).In case 5, (5a ) show epithelioid-likeareas while (5a ) show the spindle-cellcomponent of the tumor. b and b,double-color FISH analysis of the sameparaffin-embedded sections usingSMARCB1/INI1 clone RP11-71G19(green ) and control probe RP11-262A13(located in 22q13, red). Deletion ofSMARCB1/INI1 locus is present in cases1, 2, 4, 6 and in the spindle cellcomponent of case 5. c, representativeresults of immunohistochemistry usingmonoclonal antibody anti-BAF47/hSNF5(100�). Cases 2 and 6 display negativeimmunostaining of tumor cells whilenormal cells infiltrating the tumor displaynuclear staining. Cases 3 and 7 representpositive and focally positivecases, respectively. Both FISH andimmunohistochemistry results aretabulated (bottom ). *, use of frozen tumormaterial for FISH analysis.

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Immunohistochemistry showed absence of the protein in 6 of 11(55%) cases, including 5 of 6 PES samples and 1 of 5 conventionalepithelioid sarcoma samples. One additional conventional epithe-lioid sarcoma (case 7) displayed focal expression. In total, all (5 of5) cases displaying SMARCB1/INI1 loss at the molecular level alsodisplayed absence of the protein, and 5 of 6 cases without apparentgene alterations retained complete or focal protein expression(Fig. 3). Control normal tissues as well as internal controls (derma,lymphocytes) displayed nuclear staining. No significant correlationwas present between protein expression and age, sex, primarytumor location, and survival. Representative examples ofSMARCB1/INI1 immunohistochemistry results are reported inFig. 3, c .In selected cases for which sufficientmaterial was available, whole

cell lysates were prepared from frozen tissue and analyzed byWestern blot using a monoclonal antibody against SMARCB1/INI1(Fig. 4A). Lysates from normal kidney cell line 293 and mouse brainwere used as positive controls, whereas cell line G-401, derived froma malignant rhabdoid tumor of the kidney, was used as negativecontrol. Two protein isoforms were visible of f47 kDa, likelycorresponding to the two previously described SMARCB1/INI1splicing variants that differ for 27 bp in exon 2. Concordant withimmunohistochemistry results, minimal protein expression wassuggested in cases 1, 5, and 9, compatible with the presence ofnormal cells infiltrating the tumor, whereas protein expression wasretained in cases 3 and 8. In case 7, reduced protein expression,consistent with the focal pattern observed by immunohistochem-istry, was also suggested.Molecular analysis of SMARCB1/INI1 locus. To better

elucidate the mechanism of SMARCB1/INI1 inactivation inepithelioid sarcoma, we analyzed the gene status in more detailin each case where frozen tissue was available (cases 1-3 and 5-11).Real-time quantitative reverse transcriptase-PCR analysis of

SMARCB1/INI1 mRNA expression revealed that the transcript levelwas down-regulated in four samples (cases 1, 2, 6, and 9) thatdisplayed loss of SMARCB1/INI1 protein expression, comparedwith samples that retained at least focal protein expression by

immunohistochemistry (Fig. 4B). The mRNA down-regulation wasnot evident in sample 5 that displayed loss of protein expression inthe spindle-cell component of the tumor, either because of theconfounding effect of the tumor component that retainedSMARCB1/INI1 expression, or because of the presence of post-transcriptional mechanisms of inactivation. Similarly, case 7 thatdisplayed focal protein expression by immunohistochemistryretained a SMARCB1/INI1 transcript level comparable toSMARCB1/INI1-positive tumor samples.Southern blot analysis using a complete coding sequence

SMARCB1/INI1 cDNA as probe revealed an underrepresentation of

Figure 4. Analysis of SMARCB1/INI1 status in epithelioid sarcoma. Numbersabove the lanes are case numbers of Table 1. A, Western blot analysis ofSMARCB1/INI1 protein expression from frozen tumor tissue lysates, normalsamples (293 kidney cell line and mouse brain lysates) and negative control(G-401 cell line derived from a malignant rhabdoid tumor). Concordant withimmunohistochemistry results (see Fig. 3), the weak protein expressionsuggested in cases 1, 5, 7, and 9 is compatible with the presence of normal cellsinfiltrating the tumor or with focal expression. Anti–h-actin was used as a control.B, real-time quantitative reverse transcription-PCR for relative quantification ofSMARCB1/INI1 expression in epithelioid sarcoma samples. Numbers adjacent tothe features correspond to case numbers. The amount of SMARCB1/INI1 targetwas normalized to HPRT endogenous reference and relative to the normal braincDNA used as calibrator and was plotted (y-axis ). Bars, 2 SD of the replicateexperiments. Cases were grouped according to immunohistochemistry resultsfrom Fig. 3 (�, negative; +/�, focally positive; +, positive staining). Cases 1, 2, 6,and 9 that displayed negative SMARCB1/INI1 immunostaining showedSMARCB1/INI1 mRNA down-regulation, compared with cases displaying positiveor focally positive immunostaining. C, semiquantitative PCR-based DNA analysisof exon 1 of SMARCB1/INI1 gene (see Materials and Methods) displayedconcordant results, suggesting loss of copy number in cases 1, 2, 5, and 6.Representative example of four replicate experiments done using serial dilutionsof template DNA. D, PCR amplification of SMARCB1/INI1 exons from normal- andtumor-dissected areas of paraffin-embedded tissue, displaying gene loss in case 1and homozygous deletion of exon 1 in case 5. In case 6, which displayedhomogeneous heterozygous loss of SMARCB1/INI1 locus by FISH (Fig. 3, 6b ),the data indicated an additional underrepresentation of exon 4 compared withexon 1 of SMARCB1/INI1 , suggesting an exon 4 homozygous deletion. Theresidual band in the tumor lane likely originated from normal infiltrating cells thatwere left after macrodissection. The same tumor sections were analyzed by FISHand immunoistochemistry as in Fig. 3.





the gene, compared with an EWSR1 control probe located on thesame chromosome, in cases 1, 2, 5, and 6, and absence of abnormalfragments (data not shown), suggesting that in cases 1 and 2 thepreviously described chromosome 22 breakpoints likely did notinterrupt SMARCB1/INI1 gene.Semiquantitative PCR-based DNA analysis of exon 1 of

SMARCB1/INI1 gene on the same samples provided concordantresults, displaying evidence of gene loss in four of nine samples(cases 1, 2, 5, and 6; Fig. 4C). Donor-derived genomic DNA served aspositive control, whereas genomic DNA from G-401 cell line(carrying a homozygous deletion of the entire SMARCB1/INI1 gene)was used as negative control.To circumvent the limitations due to the presence of normal

cells infiltrating the tumor, in some cases with distinct morphologiesthe same paraffin-embedded sections analyzed by FISH weredissected to isolate normal tissue areas as well as tumor areas withdifferent morphology (Fig. 4D). DNA was extracted and PCRamplified for SMARCB1/INI1 as well as control primer sets. Case 1confirmed the presence of loss of the entire gene in epithelioid,carcinoma-like areas. Case 5 displayed homozygous loss of exon 1 inareas of spindle cell morphology and case 6, which carried ahomogeneous heterozygous loss of SMARCB1/INI1 locus by FISH(Fig. 3, 6b), displayed suggestive indication of complete loss of exon 4in areas of rhabdoid morphology.All epithelioid sarcoma cases were analyzed for mutations by

exons amplification and direct sequencing, and no mutations werefound. Therefore, despite the reduced or absent protein expression,no genetic alterations were identified in cases 7 and 9, respectively.In case 9, however, the down-regulation of SMARCB1/INI1 mRNAwas clearly shown by real-time PCR (Fig. 4B). Similarly, f10% to20% of malignant rhabdoid tumors are reported not to display anymutation of SMARCB1/INI1 exons. It will be of interest to analyze indetail these cases, to search for alternative mutational events thathave not been examined in the present report, such as epigeneticsilencing, intronic or promoter mutations, or RNA editing.

Discussion

SMARCB1/INI1 protein was first identified as an interactorprotein of HIV Integrase (36) and is supposed to facilitate both theprovirus integration into trascriptionally active regions of the hostgenome and the virus assembly release (37). Additionally, in therecent years, SMARCB1/INI1 gene has been defined as a tumorsuppressor gene involved in the development of a rare, aggressivepediatric cancer termed malignant rhabdoid tumor (MRT), that is infact characterized by biallelic inactivation of SMARCB1/INI1 gene(30, 31). Peculiarly, the spectrum of SMARCB1/INI1 somaticmutations includes a high rate, f40% of the cases, of homozygousdeletions, that are frequently associated with chromosomal trans-locations and other rearrangements of 22q11 (29, 32, 38).Furthermore, constitutional mutations have also been describedcausing the highly penetrant hereditary cancer-predisposing syn-drome termed ‘‘rhabdoid predisposition syndrome’’ associated withrenal and extrarenal MRT and brain tumors (39).Recent experimental evidence showed that SMARCB1/INI1

protein constitutes an invariant subunit of SWI/SNF-relatedchromatin remodeling complexes, 2-MDa complexes that regulatetranscription by modulating nucleosomal structures in an ATP-dependent manner. Several observations linked the SWI/SNF-related complexes to the pathogenesis of human malignances,including the direct interaction of single components of SWI/SNFcomplex with pRb and BRCA1 (40–42) and the increased tumor

incidence in heterozygous mouse mutants for subunit BRG1 (43).Functional characterization of SMARCB1/INI1 provided insightsinto its involvement in tumorigenesis: reintroduction of SMARCB1/INI1 gene in MRT cell lines blocked cell proliferation, inducedcellular senescence via CDKN2Ap16/pRb pathway and modulatedactin cytoskeleton organization (44, 45). Furthermore, it was shownthat SMARCB1/INI1 protein directly interacts with MYC and isnecessary for MYC-mediated transactivation through SWI/SNFcomplex (46). Interaction of SMARCB1/INI1 with both ENL andAF10, two fusion partners of acute leukemia-associated HRX/MLLprotein, as well as with GADD45, a DNA damage-inducible factorwhose function is repressed by HRX/MLL fusion proteins, has beenreported (47, 48). Therefore, SMARCB1/INI1 tumor suppressor geneis involved in key biochemical pathways implicated in cell growthand development. Additional evidence of the tumor suppressor roleof SMARCB1/INI1 was provided by the recently reported mousemodels (49–52). Despite the fact that SMARCB1/INI1 knocked out(KO) mice are not vital, both heterozygous and conditional KO micedevelop tumors, including MRTs of the central nervous system,lymphomas and, notably, undifferentiated soft tissue tumors.The identification of inactivating mutations of SMARCB1/INI1

gene in the vast majority of both renal and extrarenal MRTs andthe absence of mutations detected in many other pediatric cancers(53), provided strong evidence of the existence of this pediatricneoplasm as a distinctive entity. Nonetheless, it is widely acceptedthat the histologic hallmark of MRTs (i.e., the rhabdoid features)still represents a nonspecific morphology in rare cases of otherpediatric as well as adult neoplasms (13, 54).The identification of SMARCB1/INI1 inactivation in epithelioid

sarcoma extends the spectrum of tumors harboring SMARCB1/INI1mutations to a morphologically peculiar sarcoma of young adults.Based on the present results it could be argued that epithelioidsarcoma might represent an additional entity among the extrarenalMRT spectrum. However, several lines of evidence, in addition tothe differences in clinical features, distinguish epithelioid sarcomafrom MRT: (i) the gamut of morphologic features presenting inepithelioid sarcoma, which encompasses epithelioid, rhabdoid andspindle cell growth, in contrast to the homogeneous rhabdoidgrowth pattern shown by MRT; (ii) the spectrum of SMARCB1/INI1mutations thus far identified in epithelioid sarcoma, which seemsrestricted to deletions and not to include the point mutationsfrequently observed in MRT. Unexpectedly, these alterations seemto affect various morphologic tumor components rather then to beconfined to rhabdoid morphology; (iii) the heterogeneous distri-bution of the deletions observed thus far in epithelioid sarcoma,which suggests that, contrary to MRT, at least in some cases theyrepresent relevant secondary tumor–associated changes and notthe primary event causing the tumor onset.Analysis of large, unselected series of epithelioid sarcomas will be

necessary to provide further insights into the relationship betweenSMARCB1/INI1 mutations and clinicopathologic features.

Acknowledgments

Received 8/23/2004; revised 2/13/2005; accepted 3/1/2005.Grant support: Italian Ministry of Health (Ricerca Finalizzata), Italian Association

for Cancer Research, Italian National Research Council, Fondo per gli Investimentidella Ricerca di Base (Ministero dell’Istruzione, dell’Universita e della Ricerca), andEuropean Union COST Action B19.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Francesca Andriani and Dr. Luca Roz for useful support and comments.

Cancer Research




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2005;65:4012-4019. Cancer Res Piergiorgio Modena, Elena Lualdi, Federica Facchinetti, et al. Inactivated in Epithelioid Sarcomas

Tumor Suppressor Gene Is FrequentlySMARCB1/INI1

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