SMARCB1/INI1 Tumor Suppressor Gene Is Frequently Inactivated in Epithelioid Sarcomas

Research Article

Piergiorgio Modena,1 Elena Lualdi,1 Federica Facchinetti,1 Lisa Galli,1 Manuel R. Teixeira,3 Silvana Pilotti,2 and Gabriella Sozzi1

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 form of epithelioid sarcoma consists of tumor cells that range from spindle to large round or polygonal, bearing a strong similarity to Epithelioid sarcoma is a rare soft tissue neoplasm of uncertain lineage that usually arises in the distal extremities of adults, epithelioid or squamous cells, and arranged in nodular aggregates presents a high rate of recurrences and metastases and that commonly exhibit central necrosis. Upon microscopic exam- frequently poses diagnostic dilemmas. The recently reported ination, tumor cells show slight nuclear atypia, vesicular nuclei, and large-cell ‘‘proximal-type’’ variant is characterized by in- small nucleoli (2–7). Two variants of this form have been described: creased aggressiveness, deep location, preferential occurrence the ‘‘angiomatoid,’’ characterized by pseudoangiosarcomatous in proximal/axial regions of older patients, and rhabdoid features and the ‘‘fibroma like,’’ with a predominance of spindle cell features. Previous cytogenetic studies indicated that the most pattern with minimal cellular pleomorphism. The immunopheno- frequent alterations associated with this tumor entity affect type of epithelioid sarcoma shows positivity for vimentin and both chromosome 22. In this study, combined spectral karyotyping, epithelial markers EMA and cytokeratin. A number of the cases are also positive for CD34, muscle-specific and smooth muscle actins, fluorescence in situ hybridization, and array-based comparative genomic hybridization analyses of two proximal-type neuron-specific enolase, and S100 protein (8–12). cases harboring a rearrangement involving 10q26 and 22q11 Recently, an atypical large cells variant of epithelioid sarcoma, which is characterized by increased aggressiveness, multinodular revealed that the 22q11 breakpoints were located in a 150-kb pattern of growth, rhabdoid features and deep location, has been region containing the SMARCB1/INI1 gene, and that homozy- described (13–15). It mainly but not exclusively occurs in the pelvic, gous deletion of the gene was present in the tumor tissue. The perineal and genital tract as well as in the proximal extremities of SMARCB1/INI1 gene encodes for an invariant subunit of SWI/ middle aged adults and therefore it has also been referred as SNF chromatin remodeling complex and has been previously ‘‘proximal-type’’ epithelioid sarcoma (PES). Upon microscopic reported to act as a tumor suppressor gene frequently examination, PES consists of large epithelioid carcinoma-like inactivated in infantile malignant rhabdoid tumors. We and/or rhabdoid cells. Despite the differences in clinical presen- analyzed SMARCB1/INI1 gene status in nine additional tation and behavior, conventional epithelioid sarcoma and epithelioid sarcoma cases (four proximal types and five proximal-type epithelioid sarcoma share a similar immunopheno- conventional types) and altogether we identified deletions of typic profile suggesting a common pathogenesis. It is not clear yet SMARCB1/INI1 gene in 5 of 11 cases, all proximal types. We if the unfortunate behavior of PES is related to the prominent confirmed and further extended the number of cases with rhabdoid phenotype or merely to classic prognostic factors, such as SMARCB1/INI1 inactivation to 6 of 11 cases, by real-time tumor size, depth, proximal/axial location, resectability, or vascular quantitative PCR analysis of mRNA expression and by invasion (2, 13, 14). SMARCB1/INI1 immunohistochemistry. Overall, these results Conventional cytogenetic studies of epithelioid sarcoma revealed point to SMARCB1/INI1 gene involvement in the genesis and/ complex karyotypes with various chromosome deletions and gains, or progression of epithelioid sarcomas. Analysis of larger but the only recurrent breakpoints in structural rearrangements series of epithelioid sarcomas will be necessary to highlight were 18q11 and 22q11 in two cases each (16–25). Spectral putative clinically relevant features related to SMARCB1/INI1 karyotyping (SKY) analysis of six PES revealed the presence of 11 inactivation. (Cancer Res 2005; 65(10): 4012-9) rearrangements on chromosome 22q in five of the six cases analyzed, with two cases showing a similar t(10, 22) translocation Introduction (26). In addition, loss of heterozygosity analysis identified frequent deletion of 22q material in epithelioid sarcoma (27). These data Epithelioid sarcoma is a rare and distinctive soft tissue sarcoma suggest that a region on chromosome 22q may contain a tumor first described in 1970 (1). This neoplasm typically arises in the distal suppressor gene involved in the development of this neoplasm. extremities of young adults as a painless, slow-growing nodule In this study, 11 epithelioid sarcoma, six proximal types and five within dermis or subcutis or, less commonly, in deep fascial or conventional types, were analyzed by cytogenetic and molecular tenosynovial tissue. Morphologically, the classic, also called ‘‘distal’’, approaches. We provide evidence of genomic deletion of the SMARCB1/INI1 gene in 5 of 11 (45%) epithelioid sarcoma cases that correlates with mRNA and/or protein down-regulation. SMARCB1/ Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). INI1 immunohistochemistry confirmed inactivation at the protein P. Modena and E. Lualdi contributed equally to this work. level and extended the number of cases with SMARCB1/INI1 Requests for reprints: Gabriella Sozzi, Molecular Cytogenetics Unit, Istituto alterations to 6 of 11 (55%), of which 5 of 6 are proximal types. Our 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]. data suggest the involvement of SMARCB1/INI1 alterations in a I2005 American Association for Cancer Research. substantial fraction of epithelioid sarcoma.

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Materials and Methods Italy). The images captured were analyzed using the Mac Probe software (PowerGene Olympus, Milano, Italy). A minimum of 150 nuclei were Patients and pathology. This study included 11 epithelioid sarcoma counted in FISH experiments from paraffin-embedded tissue and results cases. Ten were retrieved from the files of the Department of Pathology, were adjusted taking into account the probe efficiency. Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy, Comparative genomic hybridization. Array-based comparative ge- comprising the period 1991 to 2002. One additional case was collected at nomic hybridization (array-CGH) was done on demand by Spectral Genomics the Portuguese Oncology Institute, Porto, Portugal. In all cases, routine (Houston, TX) using 1 Ag of tumor and reference genomic DNA and the 2- to sections were available. Six cases showed features consistent with PES, 3 4-Mb resolution KTH-1400 arrays. Briefly, two oppositely labeled experiments with rhabdoid-like and 2 with epithelioid-like appearance. In one case (case were done for each sample in which tumor and reference DNAs were labeled 5), the tumor exhibited a major spindle-cell component admixed with with different fluorochromes (Cy3-labeled test and Cy5-labeled reference epithelial-like features. Five cases corresponded to classic epithelioid DNA, Cy3-labeled reference and Cy5-labeled test DNA) and DNA mixes were sarcoma, distal type. Immunophenotyping was in keeping with the hybridized onto duplicate microarray slides as recommended by the morphologic diagnosis in all the 11 cases, showing positivity for low-weight manufacturer. Images were acquired using Axon 4000 scanner and analyzed cytokeratin, epithelial membrane antigen, and vimentin. CD34 yielded by Spectralware software (Spectral Genomics) to normalize the Cy5:Cy3 positive results in cases 4, 6, 10, and 11. S100 protein staining was positive in intensity ratios for each slide and to compute the normalized Cy5/Cy3 case 2. Patients’ clinical data, including gender, age, tumor location, and intensity ratios for both arrays, that were plotted for each chromosome. A loss histologic type and subtype are summarized in Table 1. of a particular clone is manifested as the simultaneous deviation of the ratio Cell lines. Previously described (26) short-term tumor-derived cell plots from a modal number of 1.0, with the red ratio plot showing a positive cultures of cases 1 to 4 were cultured with Amniomax C100 (Life deviation (upward) whereas the blue ratio plot shows a negative deviation Technologies, Carlsbad, CA) supplemented with Amniomax C100 supple- (downward). Conversely, DNA copy number gains show the opposite pattern. ment (Life Technologies) and harvested with 0.01 Ag/mL Colcemid PCR-based analysis of genomic DNA. PCR amplifications were done on (Karyomax Colcemid, Life Technologies) overnight. Cell lines 293 and a 9700 Thermocycler (Applied Biosystems, Foster City, CA), using 1.5 mmol/ G-401 were purchased from American Type Culture Collection (Manassas, L MgCl , 0.2 mmol/L deoxynucleotide triphosphates, 0.5 Amol/L of each VA) and were maintained in RPMI 1640 supplemented with 10% heat- 2 primer, 0.6 unit AmpliTaqGold (Applied Biosystems), 1Â reaction buffer. inactivated FCS (Life Technologies). Semiquantitative PCR amplification of SMARCB1/INI1 exon 1 was done Fluorescence in situ hybridization analysis. BAC probes used for dual- using primers INIex1F (5V-gcatttcgccttccggcttcg-3V) and INIex1R (5V- color fluorescence in situ hybridization (FISH) were extracted using gatgaatggagacgcgcgct-3V), 292 bp, at 59jC in the presence of 5% DMSO. QIAprep Midiprep (Qiagen, Valencia, CA), verified by PCR amplification of Four replicate experiments were done using serial dilutions of 25, 5, 1, 0.2 ng sequence-tagged site markers and labeled with Spectrum Orange dUTP and of template DNA. PCR products were loaded onto ethidium bromide- Spectrum Green dUTP (Vysis, Inc., Downers Grove, IL) by Nick Translation stained 2% agarose gel and quantitated by densitometric analysis using Kit (Vysis) according to the manufacturer’s instructions. All probes were Quantity One 4.5 (Bio-Rad, Hercules, CA). Mean intensities and SDs were tested on normal metaphase and interphase samples to calculate probe calculated from the four independent experiments. Using the same efficiency (i.e., number of observed signals/number of expected signals), approach, control amplifications were done using primers D16S3121F (5V- which was >90% in all cases. FISH analyses on metaphase cells from short- catgttgtacatcgtgatgc-3V) and D16S3121R (5V-agcttttatttcccaggggt-3V), 90 to 98 term cultures and on touch preparations of frozen tissue were carried out bp, 55jC; FKHR2F (5V-aacgacacatagctgggtgtcaggc-3V) and FKHR3R (5V- following standard methods (28). To perform FISH experiments on paraffin- agttcctgctgtcagacaatctgaagtac-3V), 558 bp, 55jC. Donor-derived genomic embedded samples, dewaxed 3-Am paraffin sections were boiled in Tris/ DNA served as positive control, whereas genomic DNA from G401 cell line EDTA buffer solution (5 mmol/L Tris and 1 mmol/L EDTA), incubated with (carrying a homozygous deletion of the entire SMARCB1/INI1 gene) was 0.4% of pepsin (Sigma, St. Louis, MO) in 0.01 N HCl at 37jC, denatured in used as negative control. A 1:3 mixture of normal/G401 genomic DNA was 70% formamide-2Â SSC for 12 minutes at 85jC and hybridized overnight at included to assess the sensitivity of the test. 37jC with the probes denatured at 80jC for 5 minutes. Post-hybridization To isolate normal tissue areas as well as different tumor areas from washes and detections were done as described previously (28). The FISH paraffin-embedded tissue, tissue blocks were trimmed by a pathologist using hybridization signals were analyzed in an Olympus BX51 microscope a razor blade with the assistance of a light microscope. DNA was extracted coupled to a charge-coupled device camera COHU 4912 (Olympus, Milano, 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- Table 1. Clinical data and tumor phenotype of the 11 tgccgtgccatgctccacaacc-3V) and INIi27 (5V-cacaaggtcaaagcagagtg-3V), 176 bp, epithelioid sarcoma cases under study 56jC; D16S3121F and D16S3121R; D1S2736F (5V-cctccagggtattcttgg-3V) and D1S2736R (5V-tttttgaggtgtgagagcag-3V), 122 to 134 bp, 55jC. Case Gender/age Tumor location Morphology Real-time quantitative reverse transcription-PCR. Retrotranscription was done using 1 Ag of total RNA extracted by Trizol Reagent (Invitrogen, 1 F/31 thigh Proximal ES, epithelioid like San Diego, CA), using random hexamers in the presence (RT+) or absence 2 F/47 perineum Proximal ES, rhabdoid like (RTÀ) of Superscript Reverse Transcriptase (Invitrogen). Relative quantifi- 3 M/71 dorsal region Proximal ES, rhabdoid like cation of SMARCB1/INI1 expression was done using Taqman Gene 4 M/30 lumbar region Proximal ES, epithelioid like Expression Assays on Demand (Applied Biosystems) and ABI Prism 7900 5 M/36 sacral region Proximal ES, prominent HT Sequence Detection System (Applied Biosystems). A validation spindle-cell component experiment was done to assess that the target (SMARCB1/INI1, 6 F/66 inguinal region Proximal ES, rhabdoid like Hs00268260_m1, Applied Biosystems) and reference (hHPRT, huHPRT 7 M/27 arm Distal ES, classic type PDAR, Applied Biosystems) efficiencies of amplification were equal at 8 F/55 foot Distal ES, classic type different template diluitions. Reactions consisted of 1Â Taqman Universal 9 M/31 hand Distal ES, classic type PCR Master Mix (Applied Biosystems), 1Â Assay on Demand, 1 of 40 of the 10 M/22 hand Distal ES, classic type retrotranscription reaction as template and were run according to 11 M/27 arm Distal ES, classic type manufacturer’s universal cycling conditions. Quantification of SMARCB1/ INI1 expression was done by comparative Ct method according to the manufacturer instructions, using the HPRT gene as endogenous reference Abbreviation: ES, epithelioid sarcoma. control and normal brain cDNA as calibrator. Target and reference probe sets were amplified in triplicates, in separate wells of 96-well plates. Results www.aacrjournals.org 4013 Cancer Res 2005; 65: (10). May 15, 2005

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Cancer Research were analyzed using ABI PRISM Sequence Detection System software temperature. Negative controls were done using mouse immunoglobulin (version 2.1, Applied Biosystems). The use of normal muscle or normal 1:12,000 (Dako). We used Dako ChemMate detection kit 5001 in the kidney cDNAs as calibrator provided comparable results (data not shown). subsequent steps using biotinylated secondary antibody, streptavidin- Mutation analysis. Mutation detection of SMARCB1/INI1 gene was done conjugated horseradish peroxidase, and diaminobenzidine as the cromogen. by exons amplification and direct sequencing using previously described Finally, the slides were counterstained with Carazi hematoxylin. primers (29). Western blot analysis. Western blot analysis was done using standard Results techniques from total lysates extracted from frozen tumor tissue and cell lines. Frozen tissue was preliminarily homogenized using Mixer Mill MM300 Fluorescence in situ hybridization analysis of chromosome (Qiagen). From each sample, 40 Ag of proteins were separated on 10% SDS- 22 breakpoints in short-term tumor cell cultures. In our PAGE and electro-blotted onto polyvinylidene difluoride membranes previous report of SKY analysis in six PES cases (26), we described a (Amersham Biosciences, Buckinghamshire, United Kingdom). Subsequently, cluster of breakpoints on 22q11 chromosome band and the membranes were incubated 1 hour at room temperature in a solution of presence of a similar t(10;22) translocation in two cases. To map PBS supplemented with 5% nonfat dry milk. For immunodetection, the anti- the chromosome 22 breakpoints more precisely, we performed BAF47/SNF5 antibody (BD Transduction Laboratories, Franklin Lakes, NJ) FISH experiments using a panel of BAC clones in four of the six j was used. After overnight incubation at 4 C with the primary antibody cases previously analyzed by SKY. Notably, the breakpoints in cases diluted 1:250, membranes were washed in TBST [10 mmol/L Tris-HCl (pH 1 and 2 were found located within a 150-kb region covered by the 8.0), 0.15 mol/L NaCl, and 0.05% Tween 20], followed by horseradish BAC clone RP11-71G19 and flanked by RP11-61P17 and RP11-80O7, peroxidase–conjugated goat-antimouse or goat-antirabbit antibody. En- that overlap 5V- and 3V- ends of RP11-71G19, respectively. In case 1 hanced chemiluminescence (Amersham Biosciences) was used for detection. Protein loading equivalence was assessed using an anti-hactin (Fig. 1A), FISH experiments showed that the t(10;22) was balanced, antibody (Sigma). with the exception of clone RP11-71G19, that showed three signals Immunohistochemistry. Immunophenotypic analysis was done by indicating that it spans the breakpoint (Fig. 1B), whereas autostainer instrument together with ChemMate detection kit 5001 (Dako, overlapping clones RP11-61P17 and RP11-80O7 showed signals Milano, Italy) using heat-induced epitope retrieval. Cytokeratin staining only on the der(10) and der(22), respectively (Fig. 1C). Intriguingly, required trypsin predigestion. Antibodies used were low-weight Cytokeratin BAC clone RP11-71G19 contains SMARCB1/INI1 gene, a known (mouse monoclonal, 1:10, BD Transduction Laboratories), EMA (mouse tumor suppressor gene inactivated in malignant rhabdoid tumors monoclonal, 1:500, Dako), Vimentin (mouse monoclonal, 1:200, Dako), CD34 (30, 31). FISH experiments in case 2, that harbored a complex (mouse monoclonal, 1:50, Novocastra, Newcastle upon Tyne, United rearrangement involving chromosomes 10, 15, and 22 (Fig. 1D), Kingdom), and S100 (rabbit polyclonal, 1:2000, Dako). showed the loss of clone RP11-80O7 that overlaps with RP11-71G19 Immunohistochemistry of SMARCB1 protein was done using the mouse and is flanking SMARCB1/INI1 gene, therefore suggesting a similar monoclonal antibody anti-BAF47/SNF5 as primary antibody. Endogenous breakpoint region as in case 1 (Fig. 1E-F). peroxidase was blocked with 0.3% hydrogen peroxide in methanol for 30 minutes. The slides were immersed in citrate buffer solution 5 mmol/L With the same strategy in case 3, carrying a t(12;22), the (pH 6) and heated in autoclave at 95jC for 6 minutes for antigen retrieval. breakpoint on chromosome 22 was found to be located in a 1-Mb The slides were incubated at room temperature for 30 minutes with normal region of 22q12 (Fig. 1F). EWSR1 gene did not seem rearranged as goat serum (Dako) and with the primary antibody (1:250) for 1 hour at room shown by FISH and Southern blot (data not shown); furthermore,

Figure 1. SKY and FISH analyses of metaphase chromosomes from tumor-derived short-term cell cultures in cases 1 and 2. A and D, visualization of normal chromosomes 22 and aberrant chromosomes involving 22 material after SKY analysis in cases 1 and 2, respectively. For each aberration the RGB (red-green-blue), the inverted DAPI, and the pseudocolor images of the chromosomes (left to right). Numbers on the right side of the derivative chromosomes denote the chromosomal origin. Complete SKY results have been previously published (26). B, FISH analysis of clone RP11 71G19 containing SMARCB1/INI1 gene in case 1 displayed splitting of the signal on both derivatives of the translocation t(10;22)(q26;q11). C, double-color FISH analysis of clones RP11-61P17 (green) and RP11-80O7 (red) flanking SMARCB1/INI1 gene in case 1 displayed separated signal on der(22) and der(10), respectively. E, the same double-color FISH analysis in case 2 displayed loss of RP11-80O7 (red signal) on der(10). F, summary of FISH results in cases 1 to 4.

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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 of chromosome 22 in case 1 and normal dosage in case 2. Clone position is based on UCSF April 2002 release. Double-color FISH analysis on frozen tumor tissue of case 1 (C) and on paraffin-embedded tissue of case 2 (D), confirmed the dosage of clone RP11-91K24 (red signal) as identified by array-CGH and revealed homozygous deletion, below the resolution of the array-CGH, of clone RP11-71G19 (green signal) containing SMARCB1/INI1 gene.

the presence of t(12;22)-associated fusion genes EWSR1-DDIT3 and (Supplementary Fig. B). In case 1, the results showed homozygous EWSR1-ATF1, typical of myxoid-round cell liposarcoma and clear deletion of clone RP11-71G19 in the majority of nuclei that retained cell sarcoma, respectively, was excluded by reverse transcription- the signal of the flanking clones RP11-61P17 and RP11-80O7. Thus, PCR experiments (data not shown). In case 4, because of the the combined array-CGH and FISH results allowed us to narrow complexity of the rearrangements involving chromosome 22 and down the region of homozygous loss to less then 150-kb and to insufficient cell pellet, it has only been possible to show the loss of strongly pinpoint SMARCB1/INI1 as the gene inactivated by the 22q12 and that the breakpoints did not encompass the region observed rearrangements. Two additional known genes are present covered by RP11-71G19 (Fig. 1F). in this region, pre-B lymphocyte gene 3 (VPREB3) involved in B-cell We showed the in vivo occurrence of t(10;22) and 22q11 differentiation and matrix metalloproteinase 11 (MMP11), a gene breakpoint in case 1, by double-color interphase FISH experiments reported overexpressed in a variety of tumors (33–35). In case 2, from frozen tumor biopsy (Supplementary Fig. A). Several double-color FISH experiments were done on paraffin sections and, subclones of tumor cells were present, in which either both together with array-CGH and FISH results reported in Fig. 2B and derivatives were retained (shown by split signals) or one/two D, indicated that the extent of the region of HD is between 450 kb derivatives were lost (absence of signal). and 5 Mb (Supplementary Fig. B). Array-based comparative genomic hybridization and fluo- Whereas molecular analyses (either array-CGH or FISH) of PES rescence in situ hybridization analysis of tumor tissue samples tumor biopsies 1 and 2 disclosed a loss of SMARCB1/INI1 gene, 1 and 2. Whole genome array-CGH analysis of cases 1 and 2 at 2- to cytogenetic and FISH analyses of short-term cell culture of the 4-Mb resolution displayed a normal chromosome 22 content in case same cases had revealed chromosome 22 rearrangements in the 2 and a 22q heterozygous loss covering f15 Mb and comprising vicinity of SMARCB1/INI1 gene but no evidence of loss. Such a SMARCB1/INI1 in case 1 (Fig. 2A-B). Because in malignant rhabdoid supposed discrepancy between results obtained on short-term cell tumors SMARCB1/INI1 gene is homozygously deleted in a markedly culture and frozen biopsy suggested both that a specific tumor cell high proportion of the cases (29), and 22q rearrangements clone was selected during in vitro cell culture and that the frequently seem to trigger SMARCB1/INI1 loss (32), we did translocation involving 22q preceded loss of 22q11 material. These interphase FISH with clone RP11-71G19, that contains SMARCB1/ hypotheses are substantiated by FISH results reported in INI1 gene, to directly evaluate the SMARCB1/INI1 gene dosage in Supplementary Figs. A and B. tumor samples 1 and 2. In case 1, double-color FISH analysis on Fluorescence in situ hybridization analysis of SMARCB1/INI1 touch preparations of frozen tissue showed a high frequency of locus in paraffin-embedded tumor samples. We extended the clone RP11-71G19 homozygous loss and control clone RP11-91K24 analysis of SMARCB1/INI1 locus to the 11 epithelioid sarcoma heterozygous loss (Fig. 2C). These two clones are separated by 3 Mb, cases under study (Table 1) by double-color interphase FISH and therefore SMARCB1/INI1 homozygous loss lies within the wide with clone RP11-71G19 that contains SMARCB1/INI1 gene, in region of heterozygous loss detected by array-CGH but is below the combination with a control probe from 22q13 (RP11-262A13) on resolution of the array employed. Similarly, case 2 displayed no paraffin-embedded tumor tissue. With the exception of case 3, evidence of loss by array-CGH, but double-color FISH analysis on which did not display any loss by FISH, all other PES cases (cases 1, paraffin-embedded tumor tissue showed attainment of normal copy 2, 4-6) revealed intratumor loss of SMARCB1/INI1 signal (Fig. 3, b number of clone RP11-91K24 and homozygous loss of clone RP11- and b). Interestingly, it seemed evident that SMARCB1/INI1 loss did 71G19 (Fig. 2D), suggesting that the region of loss comprising not correlate with the rhabdoid cell compartment. In fact, case 1 SMARCB1/INI1 is below the resolution of the array employed. morphology was homogeneously epithelioid, carcinoma like Additional double-color interphase FISH experiments were done (Fig. 3, 1a), and displayed a high proportion of homozygous loss to define the extent of the regions of homozygous deletion (Fig. 3, 1b). Case 2 had a multinodular pattern with rhabdoid cells www.aacrjournals.org 4015 Cancer Res 2005; 65: (10). May 15, 2005

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Cancer Research and showed homozygous loss (Fig. 3, 2a and b); case 4 displayed displayed a normal SMARCB1/INI1 copy number by FISH and case SMARCB1/INI1 loss confined to a region of epithelioid morphology 11 is shown as a representative example in Fig. 3, 11a and b. (Fig. 3, 4a and b). Interestingly, in case 5, SMARCB1/INI1 SMARCB1/INI1 protein expression analysis. To circumvent homozygous loss was restricted to areas of spindle cell morphology the potential limitations related to the presence of normal cells whereas epithelioid-like areas were normal (Fig. 3, 5a-b and a-b). infiltrating the tumor and to further investigate SMARCB1/INI1 Finally, case 6, of prominent rhabdoid morphology, displayed inactivation at the protein level, immunohistochemistry was done homogeneous heterozygous loss (Fig. 3, 6a and b). Cases 7-11 using a monoclonal antibody against SMARCB1/INI1 protein.

Figure 3. FISH and immunohistochemistry analysis of SMARCB1/INI1. Numbers in the panels refer to case numbers as in Table 1. a and a, H&E staining (100Â). In case 5, (5a) show epithelioid-like areas while (5a) show the spindle-cell component of the tumor. b and b, double-color FISH analysis of the same paraffin-embedded sections using SMARCB1/INI1 clone RP11-71G19 (green) and control probe RP11-262A13 (located in 22q13, red). Deletion of SMARCB1/INI1 locus is present in cases 1, 2, 4, 6 and in the spindle cell component of case 5. c, representative results of immunohistochemistry using monoclonal antibody anti-BAF47/hSNF5 (100Â). Cases 2 and 6 display negative immunostaining of tumor cells while normal cells infiltrating the tumor display nuclear staining. Cases 3 and 7 represent positive and focally positive cases, respectively. Both FISH and immunohistochemistry results are tabulated (bottom). *, use of frozen tumor material for FISH analysis.

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Immunohistochemistry showed absence of the protein in 6 of 11 immunohistochemistry (Fig. 4B). The mRNA down-regulation was (55%) cases, including 5 of 6 PES samples and 1 of 5 conventional not evident in sample 5 that displayed loss of protein expression in epithelioid sarcoma samples. One additional conventional epithe- the spindle-cell component of the tumor, either because of the lioid sarcoma (case 7) displayed focal expression. In total, all (5 of confounding effect of the tumor component that retained 5) cases displaying SMARCB1/INI1 loss at the molecular level also SMARCB1/INI1 expression, or because of the presence of post- displayed absence of the protein, and 5 of 6 cases without apparent transcriptional mechanisms of inactivation. Similarly, case 7 that gene alterations retained complete or focal protein expression displayed focal protein expression by immunohistochemistry (Fig. 3). Control normal tissues as well as internal controls (derma, retained a SMARCB1/INI1 transcript level comparable to lymphocytes) displayed nuclear staining. No significant correlation SMARCB1/INI1-positive tumor samples. was present between protein expression and age, sex, primary Southern blot analysis using a complete coding sequence tumor location, and survival. Representative examples of SMARCB1/INI1 cDNA as probe revealed an underrepresentation of SMARCB1/INI1 immunohistochemistry results are reported in Fig. 3, c. In selected cases for which sufficient material was available, whole cell lysates were prepared from frozen tissue and analyzed by Western blot using a monoclonal antibody against SMARCB1/INI1 (Fig. 4A). Lysates from normal kidney cell line 293 and mouse brain were used as positive controls, whereas cell line G-401, derived from a malignant rhabdoid tumor of the kidney, was used as negative control. Two protein isoforms were visible of f47 kDa, likely corresponding to the two previously described SMARCB1/INI1 splicing variants that differ for 27 bp in exon 2. Concordant with immunohistochemistry results, minimal protein expression was suggested in cases 1, 5, and 9, compatible with the presence of normal cells infiltrating the tumor, whereas protein expression was retained in cases 3 and 8. In case 7, reduced protein expression, consistent with the focal pattern observed by immunohistochemistry, was also suggested. Molecular analysis of SMARCB1/INI1 locus. To better elucidate the mechanism of SMARCB1/INI1 inactivation in epithelioid sarcoma, we analyzed the gene status in more detail in 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 level was down-regulated in four samples (cases 1, 2, 6, and 9) that displayed loss of SMARCB1/INI1 protein expression, compared with samples that retained at least focal protein expression by

Figure 4. Analysis of SMARCB1/INI1 status in epithelioid sarcoma. Numbers above the lanes are case numbers of Table 1. A, Western blot analysis of SMARCB1/INI1 protein expression from frozen tumor tissue lysates, normal samples (293 kidney cell line and mouse brain lysates) and negative control (G-401 cell line derived from a malignant rhabdoid tumor). Concordant with immunohistochemistry results (see Fig. 3), the weak protein expression suggested in cases 1, 5, 7, and 9 is compatible with the presence of normal cells infiltrating the tumor or with focal expression. Anti–h-actin was used as a control. B, real-time quantitative reverse transcription-PCR for relative quantification of SMARCB1/INI1 expression in epithelioid sarcoma samples. Numbers adjacent to the features correspond to case numbers. The amount of SMARCB1/INI1 target was normalized to HPRT endogenous reference and relative to the normal brain cDNA used as calibrator and was plotted (y-axis). Bars, 2 SD of the replicate experiments. Cases were grouped according to immunohistochemistry results from Fig. 3 (À, negative; +/À, focally positive; +, positive staining). Cases 1, 2, 6, and 9 that displayed negative SMARCB1/INI1 immunostaining showed SMARCB1/INI1 mRNA down-regulation, compared with cases displaying positive or focally positive immunostaining. C, semiquantitative PCR-based DNA analysis of exon 1 of SMARCB1/INI1 gene (see Materials and Methods) displayed concordant results, suggesting loss of copy number in cases 1, 2, 5, and 6. Representative example of four replicate experiments done using serial dilutions of template DNA. D, PCR amplification of SMARCB1/INI1 exons from normal- and tumor-dissected areas of paraffin-embedded tissue, displaying gene loss in case 1 and homozygous deletion of exon 1 in case 5. In case 6, which displayed homogeneous heterozygous loss of SMARCB1/INI1 locus by FISH (Fig. 3, 6b), the data indicated an additional underrepresentation of exon 4 compared with exon 1 of SMARCB1/INI1, suggesting an exon 4 homozygous deletion. The residual band in the tumor lane likely originated from normal infiltrating cells that were left after macrodissection. The same tumor sections were analyzed by FISH and immunoistochemistry as in Fig. 3. www.aacrjournals.org 4017 Cancer Res 2005; 65: (10). May 15, 2005

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Cancer Research the gene, compared with an EWSR1 control probe located on the incidence in heterozygous mouse mutants for subunit BRG1 (43). same chromosome, in cases 1, 2, 5, and 6, and absence of abnormal Functional characterization of SMARCB1/INI1 provided insights fragments (data not shown), suggesting that in cases 1 and 2 the into its involvement in tumorigenesis: reintroduction of SMARCB1/ previously described chromosome 22 breakpoints likely did not INI1 gene in MRT cell lines blocked cell proliferation, induced interrupt SMARCB1/INI1 gene. cellular senescence via CDKN2Ap16/pRb pathway and modulated Semiquantitative PCR-based DNA analysis of exon 1 of actin cytoskeleton organization (44, 45). Furthermore, it was shown SMARCB1/INI1 gene on the same samples provided concordant that SMARCB1/INI1 protein directly interacts with MYC and is results, displaying evidence of gene loss in four of nine samples necessary for MYC-mediated transactivation through SWI/SNF (cases 1, 2, 5, and 6; Fig. 4C). Donor-derived genomic DNA served as complex (46). Interaction of SMARCB1/INI1 with both ENL and positive control, whereas genomic DNA from G-401 cell line AF10, two fusion partners of acute leukemia-associated HRX/MLL (carrying a homozygous deletion of the entire SMARCB1/INI1 gene) protein, as well as with GADD45, a DNA damage-inducible factor was used as negative control. whose function is repressed by HRX/MLL fusion proteins, has been To circumvent the limitations due to the presence of normal reported (47, 48). Therefore, SMARCB1/INI1 tumor suppressor gene cells infiltrating the tumor, in some cases with distinct morphologies is involved in key biochemical pathways implicated in cell growth the same paraffin-embedded sections analyzed by FISH were and development. Additional evidence of the tumor suppressor role dissected to isolate normal tissue areas as well as tumor areas with of SMARCB1/INI1 was provided by the recently reported mouse different morphology (Fig. 4D). DNA was extracted and PCR models (49–52). Despite the fact that SMARCB1/INI1 knocked out amplified for SMARCB1/INI1 as well as control primer sets. Case 1 (KO) mice are not vital, both heterozygous and conditional KO mice confirmed the presence of loss of the entire gene in epithelioid, develop tumors, including MRTs of the central nervous system, carcinoma-like areas. Case 5 displayed homozygous loss of exon 1 in lymphomas and, notably, undifferentiated soft tissue tumors. areas of spindle cell morphology and case 6, which carried a The identification of inactivating mutations of SMARCB1/INI1 homogeneous heterozygous loss of SMARCB1/INI1 locus by FISH gene in the vast majority of both renal and extrarenal MRTs and (Fig. 3, 6b), displayed suggestive indication of complete loss of exon 4 the absence of mutations detected in many other pediatric cancers in areas of rhabdoid morphology. (53), provided strong evidence of the existence of this pediatric All epithelioid sarcoma cases were analyzed for mutations by neoplasm as a distinctive entity. Nonetheless, it is widely accepted exons amplification and direct sequencing, and no mutations were that the histologic hallmark of MRTs (i.e., the rhabdoid features) found. Therefore, despite the reduced or absent protein expression, still represents a nonspecific morphology in rare cases of other no genetic alterations were identified in cases 7 and 9, respectively. pediatric as well as adult neoplasms (13, 54). In case 9, however, the down-regulation of SMARCB1/INI1 mRNA The identification of SMARCB1/INI1 inactivation in epithelioid was clearly shown by real-time PCR (Fig. 4B). Similarly, f10% to sarcoma extends the spectrum of tumors harboring SMARCB1/INI1 20% of malignant rhabdoid tumors are reported not to display any mutations to a morphologically peculiar sarcoma of young adults. mutation of SMARCB1/INI1 exons. It will be of interest to analyze in Based on the present results it could be argued that epithelioid detail these cases, to search for alternative mutational events that sarcoma might represent an additional entity among the extrarenal have not been examined in the present report, such as epigenetic MRT spectrum. However, several lines of evidence, in addition to silencing, intronic or promoter mutations, or RNA editing. the differences in clinical features, distinguish epithelioid sarcoma from MRT: (i) the gamut of morphologic features presenting in Discussion epithelioid sarcoma, which encompasses epithelioid, rhabdoid and SMARCB1/INI1 protein was first identified as an interactor spindle cell growth, in contrast to the homogeneous rhabdoid protein of HIV Integrase (36) and is supposed to facilitate both the growth pattern shown by MRT; (ii) the spectrum of SMARCB1/INI1 provirus integration into trascriptionally active regions of the host mutations thus far identified in epithelioid sarcoma, which seems genome and the virus assembly release (37). Additionally, in the restricted to deletions and not to include the point mutations recent years, SMARCB1/INI1 gene has been defined as a tumor frequently observed in MRT. Unexpectedly, these alterations seem suppressor gene involved in the development of a rare, aggressive to affect various morphologic tumor components rather then to be pediatric cancer termed malignant rhabdoid tumor (MRT), that is in confined to rhabdoid morphology; (iii) the heterogeneous distri- fact characterized by biallelic inactivation of SMARCB1/INI1 gene bution of the deletions observed thus far in epithelioid sarcoma, (30, 31). Peculiarly, the spectrum of SMARCB1/INI1 somatic which suggests that, contrary to MRT, at least in some cases they mutations includes a high rate, f40% of the cases, of homozygous represent relevant secondary tumor–associated changes and not deletions, that are frequently associated with chromosomal trans- the primary event causing the tumor onset. locations and other rearrangements of 22q11 (29, 32, 38). Analysis of large, unselected series of epithelioid sarcomas will be Furthermore, constitutional mutations have also been described necessary to provide further insights into the relationship between causing the highly penetrant hereditary cancer-predisposing syn- SMARCB1/INI1 mutations and clinicopathologic features. drome termed ‘‘rhabdoid predisposition syndrome’’ associated with renal and extrarenal MRT and brain tumors (39). Recent experimental evidence showed that SMARCB1/INI1 Acknowledgments protein constitutes an invariant subunit of SWI/SNF-related Received 8/23/2004; revised 2/13/2005; accepted 3/1/2005. chromatin remodeling complexes, 2-MDa complexes that regulate Grant support: Italian Ministry of Health (Ricerca Finalizzata), Italian Association for Cancer Research, Italian National Research Council, Fondo per gli Investimenti transcription by modulating nucleosomal structures in an ATP- della Ricerca di Base (Ministero dell’Istruzione, dell’Universita` e della Ricerca), and dependent manner. Several observations linked the SWI/SNF- European Union COST Action B19. related complexes to the pathogenesis of human malignances, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance including the direct interaction of single components of SWI/SNF with 18 U.S.C. Section 1734 solely to indicate this fact. complex with pRb and BRCA1 (40–42) and the increased tumor We thank Dr. Francesca Andriani and Dr. Luca Roz for useful support and comments.

Cancer Res 2005; 65: (10). May 15, 2005 4018 www.aacrjournals.org

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Piergiorgio Modena, Elena Lualdi, Federica Facchinetti, et al.

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