Translocation, Deletion/Amplification, and Expression of HMGIC and MDM2 in a Carcinoma ex Pleomorphic Adenoma
2002; Elsevier BV; Volume: 160; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)64862-6
ISSN1525-2191
AutoresE. Röijer, Anders Nordkvist, Anna-Karin Ström, Walter Ryd, Margareta Behrendt, Jörn Bullerdiek, Joachim Mark, Göran Stenman,
Tópico(s)RNA modifications and cancer
ResumoCarcinoma ex pleomorphic adenoma (CexPA) is a carcinoma developing within a pre-existing benign pleomorphic adenoma (PA). Here we describe the identification and characterization of a series of genetic events leading to translocation, deletion/amplification, and overexpression of the HMGIC and MDM2 genes in a CexPA at an early stage of development. The tumor had a pseudodiploid stemline karyotype with a del(5)(q22–23q32–33) and a t(10;12)(p15;q14–15). In addition, there were several sidelines with double minute chromosomes (dmin) or homogeneously staining regions (hsr). Fluorescence in situ hybridization (FISH) mapping revealed that the 12q14–15 breakpoint was located centromeric to HMGIC and that the entire gene was juxtaposed to the der(10) chromosome. Detailed analysis of cells with dmin and hsr revealed that HMGIC and MDM2 were deleted from the der(10) and that the dmin and hsr were strongly positive for both genes. Southern blot analysis confirmed that both HMGIC and MDM2 were amplified and that no gross rearrangements of the genes had occurred. Immunostaining revealed that the HMGIC protein was highly overexpressed particularly in the large polymorphic cells within the carcinomatous part of the tumor. These findings suggest that amplification and overexpression of HMGIC and possibly MDM2 might be important genetic events that may contribute to malignant transformation of benign PA. Carcinoma ex pleomorphic adenoma (CexPA) is a carcinoma developing within a pre-existing benign pleomorphic adenoma (PA). Here we describe the identification and characterization of a series of genetic events leading to translocation, deletion/amplification, and overexpression of the HMGIC and MDM2 genes in a CexPA at an early stage of development. The tumor had a pseudodiploid stemline karyotype with a del(5)(q22–23q32–33) and a t(10;12)(p15;q14–15). In addition, there were several sidelines with double minute chromosomes (dmin) or homogeneously staining regions (hsr). Fluorescence in situ hybridization (FISH) mapping revealed that the 12q14–15 breakpoint was located centromeric to HMGIC and that the entire gene was juxtaposed to the der(10) chromosome. Detailed analysis of cells with dmin and hsr revealed that HMGIC and MDM2 were deleted from the der(10) and that the dmin and hsr were strongly positive for both genes. Southern blot analysis confirmed that both HMGIC and MDM2 were amplified and that no gross rearrangements of the genes had occurred. Immunostaining revealed that the HMGIC protein was highly overexpressed particularly in the large polymorphic cells within the carcinomatous part of the tumor. These findings suggest that amplification and overexpression of HMGIC and possibly MDM2 might be important genetic events that may contribute to malignant transformation of benign PA. The pleomorphic adenoma (PA) is the most common type of salivary gland neoplasm. It is usually a benign, slow-growing tumor originating from the minor and major salivary glands.1Waldron CA Mixed tumor (pleomorphic adenoma and myoepithelioma).in: Ellis GL Auclair PL Gnepp DR Surgical Pathology of the Salivary Glands. WB Saunders, Philadelphia1991: 165-186Google Scholar Microscopically, PA show a wide morphological spectrum with mainly epithelial and myoepithelial cells forming a variety of patterns in a mucoid, myxoid, or chondroid matrix. Occasionally, these normally benign tumors may undergo malignant transformation. The frequency by which this occurs varies in different series from about 2 to 23%. For example, in the AFIP series of 326 carcinoma ex pleomorphic adenoma (CexPA) cases, they represented 4.5% of all PA and 6.5% of all malignant salivary gland tumors.2Gnepp DR Wenig BM Malignant mixed Tumors.in: Ellis GL Auclair PL Gnepp DR Surgical Pathology of the Salivary Glands. WB Saunders, Philadelphia1991: 350-368Google Scholar The incidence of malignant transformation increases with the preoperative duration of the tumors.3Eneroth CM Zetterberg A Malignancy in pleomorphic adenoma: a clinical and microspectrophotometric study.Acta Otolaryngol. 1974; 77: 426-432Crossref PubMed Scopus (86) Google Scholar CexPA is usually an aggressive tumor. Almost one-half of the patients develop recurrences, and approximately one-third of the patients with parotid tumors develop metastases. Cytogenetic information about the chromosomal pattern in CexPA is scarce; only 14 cases have so far been analyzed.4Bullerdiek J Hutter KJ Brandt G Weinberg M Belge G Bartnitzke S Cytogenetic investigations on a cell line derived from a carcinoma arising in a salivary gland pleomorphic adenoma.Cancer Genet Cytogenet. 1990; 44: 253-262Abstract Full Text PDF PubMed Scopus (15) Google Scholar, 5Bullerdiek J Vollrath M Wittekind C Caselitz J Bartnitzke S Mucoepidermoid tumor of the parotid gland showing a translocation (3;8)(p21;q12) and a deletion (5)(q22) as sole chromosome abnormalities.Cancer Genet Cytogenet. 1990; 50: 161-164Abstract Full Text PDF PubMed Scopus (20) Google Scholar, 6Mark J Wedell B Dahlenfors R Stenman G Karyotypic variability and evolutionary characteristics of a polymorphous low grade adenocarcinoma in the parotid gland.Cancer Genet Cytogenet. 1991; 55: 19-29Abstract Full Text PDF PubMed Scopus (25) Google Scholar, 7Higashi K Jin Y Heim S Mandahl N Biörklund A Wennerberg J Dictor M Mitelman F Chromosome abnormalities in a carcinoma in pleomorphic adenoma of the lacrimal gland.Cancer Genet Cytogenet. 1991; 55: 125-128Abstract Full Text PDF PubMed Scopus (6) Google Scholar, 8Mark J Dahlenfors R Stenman G Bende M Melen I Cytogenetical observations in two cases of polymorphous low-grade adenocarcinoma of the salivary glands.Anticancer Res. 1992; 12: 1195-1198PubMed Google Scholar, 9Hrynchak M White V Berean K Horsman D Cytogenetic findings in seven lacrimal gland neoplasms.Cancer Genet Cytogenet. 1994; 75: 133-138Abstract Full Text PDF PubMed Scopus (39) Google Scholar, 10Jin Y Mertens F Limon J Mandahl N Wennerberg J Dictor M Heim S Mitelman F Characteristic karyotypic features in lacrimal and salivary gland carcinomas.Br J Cancer. 1994; 70: 42-47Crossref PubMed Scopus (40) Google Scholar, 11Mark HF Hanna I Gnepp DR Cytogenetic analysis of salivary gland type tumors.Oral Surg Oral Med Oral Pathol Oral Radiol. 1996; 82: 187-192Abstract Full Text PDF Scopus (36) Google Scholar, 12Martins C Fonseca I Roque L Pinto AE Soares J Malignant salivary gland neoplasms: a cytogenetic study of 19 cases.Eur J Cancer B Oral Oncol. 1996; 32: 128-132Abstract Full Text PDF Scopus (42) Google Scholar, 13Rao PH Murty VV Louie DC Chaganti RS Nonsyntenic amplification of MYC with CDK4 and MDM2 in a malignant mixed tumor of salivary gland.Cancer Genet Cytogenet. 1998; 105: 160-163Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 14Jin C Martins C Jin Y Wiegant J Wennerberg J Dictor M Gisselsson D Strömbeck B Fonseca I Mitelman F Tanke HJ Höglund M Mertens F Characterization of chromosome aberrations in salivary gland tumors by FISH, including multicolor COBRA-FISH.Genes Chromosomes Cancer. 2001; 30: 161-167Crossref PubMed Scopus (58) Google Scholar In contrast, our knowledge about the cytogenetics of benign PA is comprehensive. Karyotypic data are available for almost 500 cases.15Sandros J Stenman G Mark J Cytogenetic and molecular observations in human and experimental salivary gland tumors.Cancer Genet Cytogenet. 1990; 44: 153-167Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 16Bullerdiek J Wobst G Meyer-Bolte K Chilla R Haubrich J Thode B Bartnitzke S Cytogenetic subtyping of 220 salivary gland pleomorphic adenomas: correlation to occurrence, histological subtype, and in vitro cellular behavior.Cancer Genet Cytogenet. 1993; 65: 27-31Abstract Full Text PDF PubMed Scopus (131) Google Scholar, 17Mark J Dahlenfors R Wedell B Impact of the in vitro technique used on the cytogenetic patterns in pleomorphic adenomas.Cancer Genet Cytogenet. 1997; 95: 9-15Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 18Mitelman Database of Chromosome Aberrations in Cancer. Edited by F Mitelman, B Johansson, F Mertens.http://cgap.nci.nih.gov/Chromosomes/MitelmanDate: 2001Google Scholar About 70% of the tumors have abnormal karyotypes. Four major cytogenetic subgroups have been identified, ie, tumors with rearrangements involving 8q12 (39%), tumors with rearrangements of 12q14–15 (8%), tumors with sporadic, clonal changes not involving 8q12 or 12q14–15 (23%), and tumors with an apparently normal karyotype (30%). Recently, we identified the genes consistently rearranged in PA with 8q12 and 12q14–15 abnormalities. The target gene in 8q12 is PLAG1, a developmentally regulated zinc finger gene.19Kas K Voz ML Röijer E Åström AK Meyen E Stenman G Van de Ven WJ Promoter swapping between the genes for a novel zinc finger protein and beta-catenin in pleomorphic adenomas with t(3;8)(p21;q12) translocations.Nat Genet. 1997; 15: 170-174Crossref PubMed Scopus (297) Google Scholar, 20Voz ML Åström AK Kas K Mark J Stenman G Van de Ven WJ The recurrent translocation t(5;8)(p13;q12) in pleomorphic adenomas results in up-regulation of PLAG1 gene expression under control of the LIFR promoter.Oncogene. 1998; 16: 1409-1416Crossref PubMed Scopus (138) Google Scholar, 21Åström A-K Voz ML Kas K Röijer E Wedell B Mandahl N Van de Ven W Mark J Stenman G Conserved mechanism of PLAG1 activation in salivary gland tumors with and without chromosome 8q12 abnormalities: identification of SII as a new fusion partner gene.Cancer Res. 1999; 59: 918-923PubMed Google Scholar The translocations result in promoter swapping/substitution between PLAG1 and a ubiquitously expressed translocation partner gene (eg, CTNNB1, LIFR, or SII), leading to activation of PLAG1 expression. The breakpoints invariably occur in the 5′ non-coding regions of both the target gene and the promoter donor genes. The target gene in adenomas with rearrangements of 12q14–15 is the high mobility group protein gene, HMGIC.22Schoenmakers EF Wanschura S Mols R Bullerdiek J Van den Berghe H Van de Ven WJ Recurrent rearrangements in the high mobility group protein gene HMGI-C in benign mesenchymal tumours.Nat Genet. 1995; 10: 436-444Crossref PubMed Scopus (532) Google Scholar, 23Geurts JM Schoenmakers EF Röijer E Stenman G Van de Ven WJ Expression of reciprocal hybrid transcripts of HMGIC and FHIT in a pleomorphic adenoma of the parotid gland.Cancer Res. 1997; 57: 13-17PubMed Google Scholar, 24Geurts JM Schoenmakers EF Röijer E Åström AK Stenman G Van de Ven WJ Identification of NF1B as recurrent translocation partner gene of HMGIC in pleomorphic adenomas.Oncogene. 1998; 16: 865-872Crossref PubMed Scopus (122) Google Scholar This gene is also rearranged in a variety of mesenchymal tumors.22Schoenmakers EF Wanschura S Mols R Bullerdiek J Van den Berghe H Van de Ven WJ Recurrent rearrangements in the high mobility group protein gene HMGI-C in benign mesenchymal tumours.Nat Genet. 1995; 10: 436-444Crossref PubMed Scopus (532) Google Scholar, 25Ashar HR Fejzo MS Tkachenko A Zhou X Fletcher JA Weremowicz S Morton CC Chada K Disruption of the architectural factor HMGI-C: DNA-binding AT hook motifs fused in lipomas to distinct transcriptional regulatory domains.Cell. 1995; 82: 57-65Abstract Full Text PDF PubMed Scopus (412) Google Scholar HMGIC encodes an architectural transcription factor that promotes activation of gene expression by modulating the conformation of DNA.26Wolffe AP Architectural transcription factors.Science. 1994; 264: 1100-1101Crossref PubMed Scopus (172) Google Scholar The protein has three DNA-binding domains (AT-hook motifs) that bind to the minor groove of AT-rich DNA.27Reeves R Nissen MS The AT-DNA-binding domain of mammalian high mobility group I chromosomal proteins: a novel peptide motif for recognizing DNA structure.J Biol Chem. 1990; 265: 8573-8582Abstract Full Text PDF PubMed Google Scholar The majority of breakpoints in HMGIC occur within the third large intron, resulting in separation of the DNA-binding domains from the highly acidic, carboxy-terminal domain. Several translocation partner genes have been identified, including ALDH2, LPP, LHFP, RAD51B, COX6C, HEI10, FHIT, and NF1B.23Geurts JM Schoenmakers EF Röijer E Stenman G Van de Ven WJ Expression of reciprocal hybrid transcripts of HMGIC and FHIT in a pleomorphic adenoma of the parotid gland.Cancer Res. 1997; 57: 13-17PubMed Google Scholar, 24Geurts JM Schoenmakers EF Röijer E Åström AK Stenman G Van de Ven WJ Identification of NF1B as recurrent translocation partner gene of HMGIC in pleomorphic adenomas.Oncogene. 1998; 16: 865-872Crossref PubMed Scopus (122) Google Scholar, 28Kazmierczak B Hennig Y Wanschura S Rogalla P Bartnitzke S Van de Ven W Bullerdiek J Description of a novel fusion transcript between HMGI-C, a gene encoding for a member of the high mobility group proteins, and the mitochondrial aldehyde dehydrogenase gene.Cancer Res. 1995; 55: 6038-6039PubMed Google Scholar, 29Petit MMR Mols R Schoenmakers EF Mandahl N Van de Ven WJ LPP, the preferred fusion partner gene of HMGIC in lipomas, is a novel member of the LIM protein gene family.Genomics. 1996; 36: 118-129Crossref PubMed Scopus (187) Google Scholar, 30Petit MM Schoenmakers EF Huysmans C Geurts JM Mandahl N Van de Ven WJ LHFP, a novel translocation partner gene of HMGIC in a lipoma, is a member of a new family of LHFP-like genes.Genomics. 1999; 57: 438-441Crossref PubMed Scopus (78) Google Scholar, 31Schoenmakers EF Huysmans C Van de Ven WJ Allelic knockout of novel splice variants of human recombination repair gene RAD51B in t(12;14) uterine leiomyomas.Cancer Res. 1999; 59: 19-23PubMed Google Scholar, 32Kurose K Mine N Doi D Ota Y Yoneyama K Konishi H Araki T Emi M Novel gene fusion of COX6C at 8q22–23 to HMGIC at 12q15 in a uterine leiomyoma.Genes Chromosomes Cancer. 2000; 27: 303-307Crossref PubMed Scopus (36) Google Scholar, 33Mine N Kurose K Konishi H Araki T Nagai H Emi M Fusion of a sequence from HEI10 (14q11) to the HMGIC gene at 12q15 in a uterine leiomyoma.Jpn J Cancer Res. 2001; 92: 135-139Crossref PubMed Scopus (33) Google Scholar The two latter are fusion partners identified in PA with t(3;12) and ins(9;12).23Geurts JM Schoenmakers EF Röijer E Stenman G Van de Ven WJ Expression of reciprocal hybrid transcripts of HMGIC and FHIT in a pleomorphic adenoma of the parotid gland.Cancer Res. 1997; 57: 13-17PubMed Google Scholar, 24Geurts JM Schoenmakers EF Röijer E Åström AK Stenman G Van de Ven WJ Identification of NF1B as recurrent translocation partner gene of HMGIC in pleomorphic adenomas.Oncogene. 1998; 16: 865-872Crossref PubMed Scopus (122) Google Scholar Since no common functional domain so far has been identified among the translocation partners, the critical event seems to be the separation of the DNA-binding domains from the acidic carboxy-terminal tail of HMGIC.23Geurts JM Schoenmakers EF Röijer E Stenman G Van de Ven WJ Expression of reciprocal hybrid transcripts of HMGIC and FHIT in a pleomorphic adenoma of the parotid gland.Cancer Res. 1997; 57: 13-17PubMed Google Scholar We report here extensive molecular cytogenetic characterization of a CexPA at an early stage of development. Detailed analysis revealed a t(10;12)(p15;q15) translocation with a 12q breakpoint 5′ of HMGIC and translocation of the entire gene to the 10p+ marker chromosome followed by deletion/amplification of a segment containing HMGIC and MDM2 from this marker. The amplified sequences were mapped to double minute chromosomes (dmin) and homogeneously staining regions (hsr). These findings suggest that amplification of HMGIC and MDM2 might be important genetic events in the malignant transformation of benign PA. Fresh tumor tissue was obtained from a 35-year-old woman who had a several months history of a tumor in the left parotid gland. The tumor, which measured 11 × 18 × 20 mm, was removed with tumor-free margins by a superficial parotidectomi. Macroscopically, the tumor was circumscribed and had solid, gray-white cut surfaces. Microscopic examination revealed a cell-rich salivary gland tumor with occasional foci characteristic of PA with monomorphic tumor cells growing in strands and nests in a hyalinized stroma (Figure 1, A). The overall histological appearance was, however, that of a carcinoma with pronounced cellular polymorphism (Figure 1, B and C). Certain solid areas were comprised of small cells with minimal cytoplasm, others of large polymorphic, cytoplasm-rich cells. There was a pronounced cellular atypia with enlarged, polymorphic, and hyperchromatic nuclei containing prominent nucleoli (Figure 1, B and C). Serial sections of the tumor specimen revealed areas of microinvasion with growth of tumor nests in a vascularized stroma (Figure 1D). Immunostaining revealed that the polymorphic tumor cells had a strong nuclear positivity for the HMGIC oncoprotein (Figure 1E) (see Results). Occasional mitotic figures were observed in the carcinomatous areas. Focally, the tumor showed a moderate proliferative activity as judged by immunostaining of Ki-67 (Figure 1F). Immunostains for cytokeratin (CAM 5.2), vimentin and S100 were also positive in parts of the tumor. The overall morphological picture of the tumor with high cellularity, pronounced cellular polymorphism, and microinvasion together with the results of the immunostains were considered compatible with the diagnosis of a CexPA at an early stage of development. Subsequently, a total parotidectomi was performed. Histopathological examination revealed no signs of tumor growth in the resected specimens. The patient received no adjuvant treatment. Three years postoperatively there were no signs of local recurrences or metastases. Primary cultures were established from a fresh, unfixed specimen of the primary tumor as previously described.34Nordkvist A Mark J Gustafsson H Bang G Stenman G Non-random chromosome rearrangements in adenoid cystic carcinoma of the salivary glands.Genes Chromosomes Cancer. 1994; 10: 115-121Crossref PubMed Scopus (120) Google Scholar Chromosome preparations were made from exponentially growing primary cultures and these were subsequently G-banded and analyzed using standard procedures. Metaphase spreads used for FISH were prepared from cells stored in fixative at −20°C. The following probes were used: whole chromosome painting probes specific for chromosomes 5, 9, 10, 12, and 13 (Vysis, Inc., Downers Grove, IL); CEPH YACs 975B8 (SAS/CDK4); 811A7 (MDM2); 452E1 (HMGIC); the LL12NCO1-derived cosmid clones 142H1 and 27E12 (containing exons 1–2 and 4–5, respectively, of HMGIC);22Schoenmakers EF Wanschura S Mols R Bullerdiek J Van den Berghe H Van de Ven WJ Recurrent rearrangements in the high mobility group protein gene HMGI-C in benign mesenchymal tumours.Nat Genet. 1995; 10: 436-444Crossref PubMed Scopus (532) Google Scholar the microdissection library ML12q13–15 (specific for the 12q13–15 segment); and the PAC-clones PAC233 and PAC235 (PLAG1). DNAs were either amplified by InterAlu-PCR and labeled with biotin-16-dUTP (Roche Diagnostic, Basel, Switzerland) or labeled with biotin-16-dUTP (Roche Diagnostic), and subsequently cohybridized with α-satellite probes for chromosomes 8, 9, 10, 12, and 13 (Appligene Oncor, Qbiogene, Carlsbad, CA) in different combinations. Hybridization and probe detection were as previously described.35Röijer E Kas K Klawitz I Bullerdiek J Van de Ven W Stenman G Identification of a yeast artificial chromosome spanning the 8q12 translocation breakpoint in pleomorphic adenomas with t(3;8)(p21;q12).Genes Chromosomes Cancer. 1996; 17: 166-171Crossref PubMed Scopus (19) Google Scholar Chromosomes were counter-stained with 4′,6′-diamidino-2′-phenylindole dihydrochloride (DAPI). FISH analysis of formalin-fixed, paraffin-embedded tissue sections were performed using the tissue conversion kit S1337-TC and in situ hybridization kit S1340 (Appligene Oncor). The sections were counter-stained with propidium iodide. Fluorescence signals were digitalized, processed, and analyzed using the PowerGene FISH image analysis system (Applied Imaging International Ltd., Newcastle-upon-Tyne, UK). Spectral karyotype (SKY) analysis was performed using the SkyPaint probe kit which consists of a cocktail of 24 differentially labeled chromosome specific painting probes (ASI-Applied Spectral Imaging Ltd., Migdal Ha'Emek, Israel). The conditions for pretreatment, hybridization, posthybridization washes, detection, and analyses were as previously described36Sjögren H Wedell B Meis-Kindblom J Kindblom L-G Stenman G Fusion of the NH2-terminal domain of the basic helix-loop-helix protein TCF12 to TEC in extraskeletal myxoid chondrosarcoma with translocation t(9;15)(q22;q21).Cancer Res. 2000; 60: 6832-6835PubMed Google Scholar and as recommended by the manufacturer. Tissue sections were processed according to the avidin-biotin complex (ABC) method. Briefly, sections were deparaffinized, treated in a microwave oven and exposed to hydrogen peroxidase. A polyclonal HMGIC antibody was obtained by immunizing rabbits with a peptide corresponding to a sequence in the N-terminal part of the human HMGIC protein (SARGEGAGQPSTSA) (GSAB4, dilution 1:25; Innovagen AB, Lund, Sweden). The antiserum was affinity purified using the same peptide. The specificity of the antibody was confirmed by analysis of known HMGIC positive and negative PA.21Åström A-K Voz ML Kas K Röijer E Wedell B Mandahl N Van de Ven W Mark J Stenman G Conserved mechanism of PLAG1 activation in salivary gland tumors with and without chromosome 8q12 abnormalities: identification of SII as a new fusion partner gene.Cancer Res. 1999; 59: 918-923PubMed Google Scholar The MDM2 protein was detected by two mouse monoclonal antibodies; clone IF2 (dilution 4 μg/ml; CN Biosciences, Inc./Calbiochem, Darmstadt, Germany) recognizes an epitope within amino acid residues 26–169 of the human MDM2 protein and clone 1B10 (dilution 1:50; Novocastra Laboratories Ltd., Newcastle-upon-Tyne, UK) recognizes an epitope in the carboxy-terminal portion of the MDM2 protein. Other primary antibodies used for immunohistochemistry were: TP53 (DO-7, dilution 1:200; DAKO A/F, Glostrup, Denmark), Ki-67 (MIB-1, dilution 1:100; DAKO), cytokeratin (CAM 5.2, dilution 1:10; Becton Dickinson, Franklin Lakes, NJ), vimentin (dilution 1:400; DAKO), and S100 (dilution 1:1000; DAKO). Control sections were incubated identically, except for the primary antibodies, which were replaced by bovine serum in TBS. Four μg of normal and tumor DNAs were digested with Hind III, electrophoresed in a 0.8% agarose gel in 0.5X tris-borate-ethylenediamine-tetra-acetic acid (TBE) buffer, and transferred to a Hybond N+ membrane. The MDM2 probe used was a 600-bp fragment corresponding to nucleotides 53 to 653 of the human cDNA (GenBank accession number Z12020). Two HMGIC probes were used: an 83-bp fragment derived from the 5′ non-translated region which corresponds to nucleotides 1–83 of the HMGIC cDNA (GenBank accession number Z31595) and a 225-bp fragment derived from the 3′ flanking region corresponding to nucleotides 18–242 of STS 12-RM133 (GenBank accession number U27137). As control for equal loading of DNA a 506-bp probe corresponding to the entire coding region of the CHOP gene in 12q13 (nucleotides 75–581; GenBank accession number X67083) was used. Probes were labeled with α-[32P]dCTP by random priming or by specific primers. Genomic DNA was isolated from tumor cells using standard methods. Exons 4–9 of the TP53 gene were amplified as previously described.37Nordkvist A Röijer E Bang G Gustafsson H Behrendt M Ryd W Thoresen S Donath K Stenman G Expression and mutation patterns of p53 in benign and malignant salivary gland tumors.Int J Oncol. 2000; 16: 477-483PubMed Google Scholar For DNA sequence analysis, 40 μl of the PCR products were denatured and the strands were separated using streptavidin-coated magnetic beads (Dynabeads M-280, Dynal, Norway). Solid support sequencing was performed using the Sequenase Version 2.0 (US Biochemical, Cleveland, OH). Samples were run on 6% denaturating polyacrylamide gels for 1.5 to 4.5 hours and subsequently exposed to x-ray films. Cytogenetic analysis of short-term cultured cells revealed that the tumor had the stemline karyotype 46, XX, del(5)(q22–23q32–33), t(10;12)(p15;q15)[11] (Figure 2, A). There were also four closely related sidelines with the karyotypes 46, XX, del(5)(q22–23q32–33), t(10;12)(p15;q15),1–34dmin[13]/46, XX, del(5)(q22–23q32–33), t(10;12)(p15;q15), hsr(13)(q14)[5]/46, XX, t(X;6)(p11.2;q27), del(5)(q22–23q32–33), t(10;12)(p15;q15)[5]/46, XX, del(5)(q22–23q32–33), hsr(9)(p22–24), t(10;12)(p15;q15)[3] (Figure 2, B and C). In addition, there were seven cells with a normal female karyotype. To confirm the presence of the del(5), t(10;12), and t(X;6) and to search for possible cryptic rearrangements, we also performed SKY analysis. Detailed analysis of the SKY and DAPI-band images from 5 metaphases corroborated the cytogenetic observations. No cryptic rearrangements were detected. Analysis of one cell with dmin suggested that the dmin contained chromosome 12 sequences. FISH analysis using painting probes for chromosomes 5, 9, 10, 12, and 13 confirmed that both the dmin and the hsr(9) and hsr(13) were derived from chromosome 12. To further map the chromosome 12q15 breakpoint in relation to HMGIC, SAS, CDK4, and MDM2 (all located at 12q14–15) we used the microdissection library ML12q13–15 as well as YAC and cosmid clones containing these genes. Detailed mapping revealed that the 12q15 breakpoint was located proximal to HMGIC, but distal to the SAS and CDK4 genes, resulting in translocation of the entire HMGIC gene to the der(10) marker (Figure 2D). Hybridization with cosmid clones corresponding to the 5′- and 3′-parts of HMGIC confirmed that the gene was not split by the translocation. The dmin and hsr were strongly positive for HMGIC (both 5′ and 3′ sequences) (Figure 2E) and MDM2 (located distal to HMGIC) (Figure 2F) , but not for SAS or CDK4, indicating that HMGIC and MDM2 are coamplified in the dmin and hsr. Interestingly, in the metaphases containing dmin or hsr, no signals could be observed from the HMGIC and MDM2 containing YACs on the der(10) marker (Figure 2F). This indicates that both genes were deleted from this marker and that the deleted segment originally was not eliminated but retained as dmin or as an hsr. FISH analysis of the PLAG1 locus at 8q12 revealed signals only on the two chromosome 8 homologues, indicating that PLAG1 is not rearranged. Southern blot analysis of tumor DNA showed that both HMGIC and MDM2 were amplified compared to normal control DNA (Figure 3). Hybridization with probes corresponding to the 5′ and 3′ parts of HMGIC, respectively, revealed that the entire gene was amplified. No rearrangement of the HMGIC or MDM2 genes was observed. Control hybridization with a CHOP probe showed that CHOP, which is located centromeric to HMGIC in band 12q13, was not amplified. FISH analysis of tissue sections from the tumor using HMGIC and MDM2 specific YACs revealed strong hybridization signals in about 25 to 50% of the tumor cells (Figure 2G). To study whether the amplified genes were expressed we used immunohistochemistry and HMGIC and MDM2 specific antibodies. Nuclear expression of the HMGIC protein was detected in about 25 to 50% of the tumor cells (Figure 1E). About half of these cells expressed high levels of HMGIC. The HMGIC protein localized to granular, nuclear structures similar in size and appearance to the so-called PML nuclear bodies.38Zhong S Salomoni P Pandolfi PP The transcriptional role of PML and the nuclear body.Nat Cell Biol. 2000; 2: E85-E90Crossref PubMed Scopus (492) Google Scholar A similar, but less pronounced pattern, could also be seen in the other HMGIC positive cells. The location of the HMGIC positive cells largely coincided with the location of the cells with strong FISH signals for HMGIC and MDM2. The strongest HMGIC staining was observed in large, polymorphic atypical nuclei (Figure 1E). Control staining of tissue sections from a PA with known overexpression of an HMGIC-NF1B fusion transcript due to an ins(9;12)(p23;q12q15)24Geurts JM Schoenmakers EF Röijer E Åström AK Stenman G Van de Ven WJ Identification of NF1B as recurrent translocation partner gene of HMGIC in pleomorphic adenomas.Oncogene. 1998; 16: 865-872Crossref PubMed Scopus (122) Google Scholar revealed an evenly distributed nuclear expression of the protein in the majority of the tumor cells (not shown). No cells with very high expression levels were observed. Staining of sections from a PA without rearrangements of HMGIC revealed no nuclear staining in any of the tumor cells. Immunostaining for MDM2 revealed a few scattered positive cells. Similar results were obtained with both antibodies used. Immunostaining for TP53 protein was negative. Nucleotide sequence analysis of the TP53 gene revealed no mutations in exons 4 to 9. In this communication we describe the identification and characterization of a series of genetic events leading to translocation, deletion/amplification, and overexpression of the HMGIC gene in a case of CexPA. The carcinoma, which was at a relatively early stage of development, had a stemline karyotype with a del(5)(q22–23q32–33) and a t(10;12)(p15;q15) as the sole cytogenetic abnormalities. Since translocations with breakpoints in 12q14–15 are characteristic of a subgroup of PA,15Sandros J Stenman G Mark J Cytogenetic and molecular observations in human and experimental salivary gland tumors.Cancer Genet Cytogenet. 1990; 44: 153-167Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 16Bullerdiek J Wobst G Meyer-Bolte K Chilla R Haubrich J Thode B Bartnitzke S Cytogenetic subtyping of 220 salivary gland pleomorphic adenomas: correlation to occurrence, histological subtype, and in vitro cellular behavior.Can
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