- Email: [email protected]
SMARCB1 deletion by a complex three-way chromosomal translocation in an extrarenal malignant rhabdoid tumor
Cancer Genetics
-
(2014)
-
BRIEF COMMUNICATION
SMARCB1 deletion by a complex three-way chromosomal translocation in an extrarenal malignant rhabdoid tumor Armita Bahrami a,*, Seungjae Lee a, Kari D. Caradine b, Susana C. Raimondi a, Andrew L. Folpe c a b
Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, USA; Department of Pathology, The University of Arkansas for Medical Sciences, Little Rock, AR, USA; c Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Rhabdoid tumors (RTs) are highly aggressive malignant neoplasms of early childhood that arise in the kidney, brain, and extrarenal sites. The disease is genetically defined by biallelic disruption of the SMARCB1/INI1/SNF5 tumor suppressor gene, a core component of the ATP-dependent chromatin remodeling SWI/SNF complex. The molecular changes leading to SMARCB1 alterations in RTs are heterogeneous, including germline or constitutional inactivating mutations, partial or total gene deletions, copy number neutral loss of heterozygosity, and, less commonly, reciprocal translocations. We report a novel three-way chromosomal rearrangement, which was identified by conventional cytogenetic and sequential fluorescence in situ hybridization studies as the underlying molecular mechanism of the loss of SMARCB1 in an extrarenal RT. This case highlights the heterogeneity of genetic events that may lead to the loss of SMARCB1 and the development of RTs. Keywords Rhabdoid tumor, SMARCB1, INI1, conventional cytogenetic, FISH, sequential G-banded FISH ª 2014 Elsevier Inc. All rights reserved.
Malignant rhabdoid tumors (RTs) are highly aggressive neoplasms that develop in infancy and early childhood. First described in the kidney, RTs have since been identified in various organs and anatomic sites, including soft tissues throughout the body and the brain, where they are referred to as atypical teratoid/rhabdoid tumor (ATRT). RTs are genetically defined by biallelic alterations in the SMARCB1/INI1/ hSNF5/BAF47 tumor suppressor gene located at chromosome 22q11.2 (1e4). Loss of function of the SMARCB1 gene is a consistent finding in RTs, with the exception of a small subset of cases that instead show alterations in the SMARCA4 gene, which is another SWI/SNF subunit (5). The underlying molecular changes that lead to SMARCB1 gene inactivation in RTs are highly variable and include partial or complete gene deletions, germline or somatic inactivating
Received July 31, 2014; received in revised form August 10, 2014; accepted August 13, 2014. * Corresponding author. E-mail address: [email protected] 2210-7762/$ - see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cancergen.2014.08.002
mutations, copy number loss of heterozygosity, and, less frequently, reciprocal translocations (6e9). We herein report an extrarenal malignant RT found by cytogenetic and sequential fluorescence in situ hybridization (FISH) studies to have homozygous loss of SMARCB1 as the result of a novel three-way chromosomal translocation in one allele and gene deletion in the second allele. This case expands our understanding of the molecular changes that may lead to alteration of SMARCB1 in RTs.
Materials and methods Histologic evaluation Formalin-fixed paraffin-embedded (FFPE) tissue sections (4m) were processed for immunohistochemistry using heatand/or enzyme-induced epitope retrieval with the Leica Refine Polymer (Leica Microsystems, Newcastle, United Kingdom), Ventana iVIEW (Ventana Medical Systems, Inc., Tucson, AZ), or Ventana ultraView detection systems. Antibodies to the following antigens were used: cytokeratins
2 (clone AE1/AE3; DAKO, Carpinteria, CA), epithelial membrane antigen (clone Mc5, Ventana), desmin (clone D33; DAKO), CD34 (QBEnd/10, Ventana Medical Systems, Inc.), and INI1 (MRQ-27; Cell Marque, Rocklin, CA).
Conventional cytogenetic studies The harvesting and preparation of chromosomes for analysis were done according to standard methods. The chromosomes were evaluated by G-banded analysis, and the karyotype was designated according to the available International System for Human Cytogenetic Nomenclature (ISCN 2013).
Fluorescence in situ hybridization studies An interphase, dual-color fluorescence in situ hybridization (iFISH) study was performed on a 4-m section of FFPE tissue, using laboratory developed probes. A probe set was designed with a bacterial artificial chromosome (BAC) probe CTD-2034E7, which was mapped to the SMARCB1 locus at 22q11.23 and labeled with rhodamine (red fluorescence dye), and a BAC probe RP11-350L11, which was mapped to 22q13.33 and labeled with fluorescein isothiocyanate (green fluorescence dye) as a same-arm control probe (BACPAC Resources, Oakland, CA). iFISH was performed as previously described (10). In addition, a sequential FISH procedure on G-banded metaphases was performed on cultured cells, using a similar probe set as described previously.
Results Clinical history The patient was a previously healthy 4-year-old female who presented with a rapidly growing soft tissue mass of the neck, which was biopsied. Tissue was submitted for routine histopathological evaluation and karyotyping.
A. Bahrami et al. vesicular nuclei and prominent nucleoli (Figure 1A). In some fields, scattered characteristic rhabdoid cells with eccentrically placed nuclei and intracytoplasmic eosinophilic inclusions were noted. By immunohistochemistry, the neoplastic cells were variably positive for epithelial membrane antigen and cytokeratins (AE1/AE3), and entirely negative for INI1 (Figure 1B), desmin, and CD34. With the supporting morphological and immunohistochemical findings, as well as consistent clinical features, the diagnosis of extrarenal malignant RT was established.
Cytogenetic findings Conventional cytogenetic evaluation of the cultured cells from the tumor yielded three normal and six abnormal metaphases. The abnormal cells showed the following karyotype: 46,X,t(X;22;11) (q13;q11.2;q14.2) (Figure 2A).
Fluorescence in situ hybridization studies Interphase FISH revealed homozygous deletions of SMARCB1 in the majority of cells (Figure 1C). Of 184 evaluable nuclei on iFISH, approximately 85% revealed a homozygous deletion and a minor subset (approximately 2%) showed a hemizygous deletion of SMARCB1. The G-banded metaphase plate of the karyotypically normal cells showed that both SMARCB1 signals were retained. The G-banded metaphase plate of the abnormal cells contained one normal chromosome 22 and a der(22) t(X;22;11), which resulted from a three-way translocation with chromosomes X, 22, and 11 (Figure 2A). Sequential FISH of this metaphase showed that both the normal chromosome 22 and the der(22)t(X;22;11) had lost the SMARCB1 signal, whereas the control signal on 22q had been translocated from the der(22)t(X;22;11) to the der(11) t(X;22;11) (Figure 2B and C).
Discussion Histologic findings H&E stained sections showed a diffuse infiltrate of undifferentiated-appearing, large, discohesive cells with
Recurrent biallelic alteration of SMARCB1 in RT was discovered over a decade ago, but only recently, it was shown to be the sole genetic change in these tumors (11e14). Studies by
Figure 1 (A) H&E photomicrograph of the tumor shows large, discohesive polygonal cells with vesicular nuclei and prominent nuclei. (B) Immunohistochemical assay for SMARCB1 shows the absence of protein expression in the nuclei of tumor cells, whereas the non-neoplastic cells show expression of the protein. (C) Interphase dual-color FISH for SMARCB1 reveals homozygous gene deletion. In this view, three cells show homozygous loss of SMARCB1 (red signal). The one cell showing two pairs of red and green signals is likely a non-neoplastic cell.
SMARCB1 deletion by a complex translocation in a rhabdoid tumor
3
Figure 2 (A) Karyotype of abnormal cell. The chromosome X translocated to 22q11.2, the 22q translocated to 11q14, and the 11q translocated to Xq13. (B) The G-banded metaphase plate with a normal chromosome 22 (upper circle) and a der(22)t(X;22;11) (lower circle). (C) On reverse DAPI (40 ,6-diamidino-2-phenylindole; Vector Labs, Burlingame, CA), sequential G-banded FISH shows that SMARCB1 signals (in red) are absent from the metaphase. The control (green signal) is present on the normal chromosome 22 and on the der(11)t(X;22;11). As shown, the 22q control probe (green signal) has moved to the derivative 11, which confirms the three-way translocation.
genome-wide single nucleotide polymorphism (SNP) array, multiplex ligation-dependent probe amplification, and molecular inversion probe SNP have found genomic stability in RT with no recurrent chromosomal gains, losses, or copy neutral loss of heterozygosity, other than those affecting the SMARCB1 locus (12e14). Moreover, genome-wide exome sequencing has shown RT to have a simple genome with an extremely low mutation rate (11). These findings have suggested that loss of SMARCB1, a component of the SWI/SNF chromatin remodeling complex, plays an elemental role in the tumorigenesis of RT. Furthermore, heterozygous SMARCB1 knockout mouse models develop soft tissue and brain tumors that lack the wildtype allele, providing further support that SMARCB1 is a bona fide tumor suppressor gene in certain cell types (15e17). Loss of SMARCB1 is not specific to RT and occurs in a number of other malignant neoplasms, such as epithelioid sarcoma, renal medullary carcinoma, a subset of small cell hepatoblastoma, extraskeletal myxoid chondrosarcoma, and epithelioid malignant peripheral nerve sheath tumor (18e23). RTs and other SMARCB1-deficient tumors have in common a lack of expression of the SMARCB1 protein and a rhabdoid cell phenotype, while differing (presumably) in the genomic background and cell of origin. RT, including malignant renal and extrarenal RT and ATRT, are a distinct category of tumors, with an unknown cell of origin, usually occurring in very young children. Histologically, RTs are characterized by an infiltrate of large, primitive-appearing malignant cells with classic rhabdoid cells in some fields and a divergent immunophenotype. RT cells can express markers of epithelial, mesenchymal, and neural lineage, and are commonly positive for embryonic stem cell markers, such as SALL4 (24e27). Chromosomal translocations at breakpoint 22q11.2, which is the locus of SMARCB1, have been described in RT with various partner chromosomes, including 1p36.1, 8q12, 9p13, 9q34, 11p15.5, and 18q21 (6e8,28e32). To our
knowledge, a complex three-way chromosomal translocation has not been described as the cause of SMARCB1 loss in RT before. The absence of the SMARCB1 signal on the nontranslocated chromosome 22 in this case indicates that the second allele was likely lost by gene deletion. Approximately one third of patients with RTs are found to have a germline deletion or truncating mutation in SMARCB1, either as a sporadic event or in the setting of familial rhabdoid predisposition syndrome (33,34). Analysis of the blood sample for germline mutations or deletion of SMARCB1 was not possible in this case, but given that a number of karyotypically normal cells with retained SMARCB1 were isolated on the cell culture and a number of normal, likely stromal, cells with intact genes were found on the iFISH assay, we concluded that both losses had to be somatic events in this case. In summary, we have described, for the first time, a SMARCB1 deletion as a result of a three-way translocation t(X;22;11) (q13;q11.2;q14.2) in a patient with an extrarenal malignant RT. This case emphasizes the diverse genetic events that may result in RT tumorigenesis, and it illustrates the role of FISH studies in the elucidation of complex cytogenetic abnormalities.
Acknowledgments This work was supported in part by the American Lebanese Syrian Associated Charities. The authors thank Samantha Melton for her excellent technical support.
References 1. Rorke LB, Packer RJ, Biegel JA. Central nervous system atypical teratoid/rhabdoid tumors of infancy and childhood: definition of an entity. J Neurosurg 1996;85:56e65.
4 2. Tomlinson GE, Breslow NE, Dome J, et al. Rhabdoid tumor of the kidney in the National Wilms’ Tumor Study: age at diagnosis as a prognostic factor. J Clin Oncol 2005;23:7641e7645. 3. Biegel JA. Molecular genetics of atypical teratoid/rhabdoid tumor. Neurosurg Focus 2006;20:E11. venet N, Lange J, et al. Truncating mutations of 4. Versteege I, Se hSNF5/INI1 in aggressive paediatric cancer. Nature 1998;394: 203e206. 5. Hasselblatt M, Gesk S, Oyen F, et al. Nonsense mutation and inactivation of SMARCA4 (BRG1) in an atypical teratoid/rhabdoid tumor showing retained SMARCB1 (INI1) expression. Am J Surg Pathol 2011;35:933e935. 6. Rosty C, Peter M, Zucman J, et al. Cytogenetic and molecular analysis of a t(1;22)(p36;q11.2) in a rhabdoid tumor with a putative homozygous deletion of chromosome 22. Genes Chromosomes Cancer 1998;21:82e89. 7. Newsham I, Daub D, Besnard-Guerin C, et al. Molecular sublocalization and characterization of the 11;22 translocation breakpoint in a malignant rhabdoid tumor. Genomics 1994;19: 433e440. 8. Misawa A, Hosoi H, Imoto I, et al. Translocation (1;22)(p36;q11.2) with concurrent del(22)(q11.2) resulted in homozygous deletion of SNF5/INI1 in a newly established cell line derived from extrarenal rhabdoid tumor. J Hum Genet 2004;49:586e589. venet N, Lellouch-Tubiana A, Schofield D, et al. Spectrum of 9. Se hSNF5/INI1 somatic mutations in human cancer and genotypephenotype correlations. Hum Mol Genet 1999;8:2359e2368. 10. Bahrami A, Dalton JD, Krane JF, et al. A subset of cutaneous and soft tissue mixed tumors are genetically linked to their salivary gland counterpart. Genes Chromosomes Cancer 2012; 51:140e148. 11. Lee RS, Stewart C, Carter SL, et al. A remarkably simple genome underlies highly malignant pediatric rhabdoid cancers. J Clin Invest 2012;122:2983e2988. 12. Hasselblatt M, Isken S, Linge A, et al. High-resolution genomic analysis suggests the absence of recurrent genomic alterations other than SMARCB1 aberrations in atypical teratoid/rhabdoid tumors. Genes Chromosomes Cancer 2013;52:185e190. 13. McKenna ES, Sansam CG, Cho YJ, et al. Loss of the epigenetic tumor suppressor SNF5 leads to cancer without genomic instability. Mol Cell Biol 2008;28:6223e6233. 14. Jackson EM, Sievert AJ, Gai X, et al. Genomic analysis using high-density single nucleotide polymorphism-based oligonucleotide arrays and multiplex ligation-dependent probe amplification provides a comprehensive analysis of INI1/SMARCB1 in malignant rhabdoid tumors. Clin Cancer Res 2009;15:1923e1930. 15. Guidi CJ, Sands AT, Zambrowicz BP, et al. Disruption of Ini1 leads to peri-implantation lethality and tumorigenesis in mice. Mol Cell Biol 2001;21:3598e3603. 16. Klochendler-Yeivin A, Fiette L, Barra J, et al. The murine SNF5/INI1 chromatin remodeling factor is essential for embryonic development and tumor suppression. EMBO Rep 2000;1: 500e506. 17. Sullivan LM, Folpe AL, Pawel BR, et al. Epithelioid sarcoma is associated with a high percentage of SMARCB1 deletions. Mod Pathol 2013;26:385e392.
A. Bahrami et al. 18. Russo P, Biegel JA. SMARCB1/INI1 alterations and hepatoblastoma: another extrarenal rhabdoid tumor revealed? Pediatr Blood Cancer 2009;52:312e313. 19. Kohashi K, Oda Y, Yamamoto H, et al. SMARCB1/INI1 protein expression in round cell soft tissue sarcomas associated with chromosomal translocations involving EWS: a special reference to SMARCB1/INI1 negative variant extraskeletal myxoid chondrosarcoma. Am J Surg Pathol 2008;32:1168e1174. 20. Cheng JX, Tretiakova M, Gong C, et al. Renal medullary carcinoma: rhabdoid features and the absence of INI1 expression as markers of aggressive behavior. Mod Pathol 2008;21:647e652. neaux P, Brisse H, et al. SMARCB1 deficiency in 21. Rizzo D, Fre tumors from the peripheral nervous system: a link between schwannomas and rhabdoid tumors? Am J Surg Pathol 2012;36: 964e972. 22. Mobley BC, McKenney JK, Bangs CD, et al. Loss of SMARCB1/INI1 expression in poorly differentiated chordomas. Acta Neuropathol 2010;120:745e753. 23. Modena P, Lualdi E, Facchinetti F, et al. SMARCB1/INI1 tumor suppressor gene is frequently inactivated in epithelioid sarcomas. Cancer Res 2005;65:4012e4019. 24. Venneti S, Le P, Martinez D, et al. Malignant rhabdoid tumors express stem cell factors, which relate to the expression of EZH2 and Id proteins. Am J Surg Pathol 2011;35:1463e1472. 25. Tsokos M, Kouraklis G, Chandra RS, et al. Malignant rhabdoid tumor of the kidney and soft tissues. Evidence for a diverse morphological and immunocytochemical phenotype. Arch Pathol Lab Med 1989;113:115e120. 26. Fanburg-Smith JC, Hengge M, Hengge UR, et al. Extrarenal rhabdoid tumors of soft tissue: a clinicopathologic and immunohistochemical study of 18 cases. Ann Diagn Pathol 1998;2: 351e362. 27. Deisch J, Raisanen J, Rakheja D. Immunohistochemical expression of embryonic stem cell markers in malignant rhabdoid tumors. Pediatr Dev Pathol 2011;14:353e359. 28. Shashi V, Lovell MA, von Kap-herr C, et al. Malignant rhabdoid tumor of the kidney: involvement of chromosome 22. Genes Chromosomes Cancer 1994;10:49e54. 29. Biegel JA, Burk CD, Parmiter AH, et al. Molecular analysis of a partial deletion of 22q in a central nervous system rhabdoid tumor. Genes Chromosomes Cancer 1992;5:104e108. 30. Handgretinger R, Kimmig A, Koscielnak E, et al. Establishment and characterization of a cell line (Wa-2) derived from an extrarenal rhabdoid tumor. Cancer Res 1990;50:2177e2182. 31. Biegel JA, Allen CS, Kawasaki K, et al. Narrowing the critical region for a rhabdoid tumor locus in 22q11. Genes Chromosomes Cancer 1996;16:94e105. 32. Ota S, Crabbe DC, Tran TN, et al. Malignant rhabdoid tumor. A study with two established cell lines. Cancer 1993;71: 2862e2872. 33. Eaton KW, Tooke LS, Wainwright LM, et al. Spectrum of SMARCB1/INI1 mutations in familial and sporadic rhabdoid tumors. Pediatr Blood Cancer 2011;56:7e15. venet N, Sheridan E, Amram D, et al. Constitutional mutations 34. Se of the hSNF5/INI1 gene predispose to a variety of cancers. Am J Hum Genet 1999;65:1342e1348.
-
(2014)
-
BRIEF COMMUNICATION
SMARCB1 deletion by a complex three-way chromosomal translocation in an extrarenal malignant rhabdoid tumor Armita Bahrami a,*, Seungjae Lee a, Kari D. Caradine b, Susana C. Raimondi a, Andrew L. Folpe c a b
Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, USA; Department of Pathology, The University of Arkansas for Medical Sciences, Little Rock, AR, USA; c Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Rhabdoid tumors (RTs) are highly aggressive malignant neoplasms of early childhood that arise in the kidney, brain, and extrarenal sites. The disease is genetically defined by biallelic disruption of the SMARCB1/INI1/SNF5 tumor suppressor gene, a core component of the ATP-dependent chromatin remodeling SWI/SNF complex. The molecular changes leading to SMARCB1 alterations in RTs are heterogeneous, including germline or constitutional inactivating mutations, partial or total gene deletions, copy number neutral loss of heterozygosity, and, less commonly, reciprocal translocations. We report a novel three-way chromosomal rearrangement, which was identified by conventional cytogenetic and sequential fluorescence in situ hybridization studies as the underlying molecular mechanism of the loss of SMARCB1 in an extrarenal RT. This case highlights the heterogeneity of genetic events that may lead to the loss of SMARCB1 and the development of RTs. Keywords Rhabdoid tumor, SMARCB1, INI1, conventional cytogenetic, FISH, sequential G-banded FISH ª 2014 Elsevier Inc. All rights reserved.
Malignant rhabdoid tumors (RTs) are highly aggressive neoplasms that develop in infancy and early childhood. First described in the kidney, RTs have since been identified in various organs and anatomic sites, including soft tissues throughout the body and the brain, where they are referred to as atypical teratoid/rhabdoid tumor (ATRT). RTs are genetically defined by biallelic alterations in the SMARCB1/INI1/ hSNF5/BAF47 tumor suppressor gene located at chromosome 22q11.2 (1e4). Loss of function of the SMARCB1 gene is a consistent finding in RTs, with the exception of a small subset of cases that instead show alterations in the SMARCA4 gene, which is another SWI/SNF subunit (5). The underlying molecular changes that lead to SMARCB1 gene inactivation in RTs are highly variable and include partial or complete gene deletions, germline or somatic inactivating
Received July 31, 2014; received in revised form August 10, 2014; accepted August 13, 2014. * Corresponding author. E-mail address: [email protected] 2210-7762/$ - see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cancergen.2014.08.002
mutations, copy number loss of heterozygosity, and, less frequently, reciprocal translocations (6e9). We herein report an extrarenal malignant RT found by cytogenetic and sequential fluorescence in situ hybridization (FISH) studies to have homozygous loss of SMARCB1 as the result of a novel three-way chromosomal translocation in one allele and gene deletion in the second allele. This case expands our understanding of the molecular changes that may lead to alteration of SMARCB1 in RTs.
Materials and methods Histologic evaluation Formalin-fixed paraffin-embedded (FFPE) tissue sections (4m) were processed for immunohistochemistry using heatand/or enzyme-induced epitope retrieval with the Leica Refine Polymer (Leica Microsystems, Newcastle, United Kingdom), Ventana iVIEW (Ventana Medical Systems, Inc., Tucson, AZ), or Ventana ultraView detection systems. Antibodies to the following antigens were used: cytokeratins
2 (clone AE1/AE3; DAKO, Carpinteria, CA), epithelial membrane antigen (clone Mc5, Ventana), desmin (clone D33; DAKO), CD34 (QBEnd/10, Ventana Medical Systems, Inc.), and INI1 (MRQ-27; Cell Marque, Rocklin, CA).
Conventional cytogenetic studies The harvesting and preparation of chromosomes for analysis were done according to standard methods. The chromosomes were evaluated by G-banded analysis, and the karyotype was designated according to the available International System for Human Cytogenetic Nomenclature (ISCN 2013).
Fluorescence in situ hybridization studies An interphase, dual-color fluorescence in situ hybridization (iFISH) study was performed on a 4-m section of FFPE tissue, using laboratory developed probes. A probe set was designed with a bacterial artificial chromosome (BAC) probe CTD-2034E7, which was mapped to the SMARCB1 locus at 22q11.23 and labeled with rhodamine (red fluorescence dye), and a BAC probe RP11-350L11, which was mapped to 22q13.33 and labeled with fluorescein isothiocyanate (green fluorescence dye) as a same-arm control probe (BACPAC Resources, Oakland, CA). iFISH was performed as previously described (10). In addition, a sequential FISH procedure on G-banded metaphases was performed on cultured cells, using a similar probe set as described previously.
Results Clinical history The patient was a previously healthy 4-year-old female who presented with a rapidly growing soft tissue mass of the neck, which was biopsied. Tissue was submitted for routine histopathological evaluation and karyotyping.
A. Bahrami et al. vesicular nuclei and prominent nucleoli (Figure 1A). In some fields, scattered characteristic rhabdoid cells with eccentrically placed nuclei and intracytoplasmic eosinophilic inclusions were noted. By immunohistochemistry, the neoplastic cells were variably positive for epithelial membrane antigen and cytokeratins (AE1/AE3), and entirely negative for INI1 (Figure 1B), desmin, and CD34. With the supporting morphological and immunohistochemical findings, as well as consistent clinical features, the diagnosis of extrarenal malignant RT was established.
Cytogenetic findings Conventional cytogenetic evaluation of the cultured cells from the tumor yielded three normal and six abnormal metaphases. The abnormal cells showed the following karyotype: 46,X,t(X;22;11) (q13;q11.2;q14.2) (Figure 2A).
Fluorescence in situ hybridization studies Interphase FISH revealed homozygous deletions of SMARCB1 in the majority of cells (Figure 1C). Of 184 evaluable nuclei on iFISH, approximately 85% revealed a homozygous deletion and a minor subset (approximately 2%) showed a hemizygous deletion of SMARCB1. The G-banded metaphase plate of the karyotypically normal cells showed that both SMARCB1 signals were retained. The G-banded metaphase plate of the abnormal cells contained one normal chromosome 22 and a der(22) t(X;22;11), which resulted from a three-way translocation with chromosomes X, 22, and 11 (Figure 2A). Sequential FISH of this metaphase showed that both the normal chromosome 22 and the der(22)t(X;22;11) had lost the SMARCB1 signal, whereas the control signal on 22q had been translocated from the der(22)t(X;22;11) to the der(11) t(X;22;11) (Figure 2B and C).
Discussion Histologic findings H&E stained sections showed a diffuse infiltrate of undifferentiated-appearing, large, discohesive cells with
Recurrent biallelic alteration of SMARCB1 in RT was discovered over a decade ago, but only recently, it was shown to be the sole genetic change in these tumors (11e14). Studies by
Figure 1 (A) H&E photomicrograph of the tumor shows large, discohesive polygonal cells with vesicular nuclei and prominent nuclei. (B) Immunohistochemical assay for SMARCB1 shows the absence of protein expression in the nuclei of tumor cells, whereas the non-neoplastic cells show expression of the protein. (C) Interphase dual-color FISH for SMARCB1 reveals homozygous gene deletion. In this view, three cells show homozygous loss of SMARCB1 (red signal). The one cell showing two pairs of red and green signals is likely a non-neoplastic cell.
SMARCB1 deletion by a complex translocation in a rhabdoid tumor
3
Figure 2 (A) Karyotype of abnormal cell. The chromosome X translocated to 22q11.2, the 22q translocated to 11q14, and the 11q translocated to Xq13. (B) The G-banded metaphase plate with a normal chromosome 22 (upper circle) and a der(22)t(X;22;11) (lower circle). (C) On reverse DAPI (40 ,6-diamidino-2-phenylindole; Vector Labs, Burlingame, CA), sequential G-banded FISH shows that SMARCB1 signals (in red) are absent from the metaphase. The control (green signal) is present on the normal chromosome 22 and on the der(11)t(X;22;11). As shown, the 22q control probe (green signal) has moved to the derivative 11, which confirms the three-way translocation.
genome-wide single nucleotide polymorphism (SNP) array, multiplex ligation-dependent probe amplification, and molecular inversion probe SNP have found genomic stability in RT with no recurrent chromosomal gains, losses, or copy neutral loss of heterozygosity, other than those affecting the SMARCB1 locus (12e14). Moreover, genome-wide exome sequencing has shown RT to have a simple genome with an extremely low mutation rate (11). These findings have suggested that loss of SMARCB1, a component of the SWI/SNF chromatin remodeling complex, plays an elemental role in the tumorigenesis of RT. Furthermore, heterozygous SMARCB1 knockout mouse models develop soft tissue and brain tumors that lack the wildtype allele, providing further support that SMARCB1 is a bona fide tumor suppressor gene in certain cell types (15e17). Loss of SMARCB1 is not specific to RT and occurs in a number of other malignant neoplasms, such as epithelioid sarcoma, renal medullary carcinoma, a subset of small cell hepatoblastoma, extraskeletal myxoid chondrosarcoma, and epithelioid malignant peripheral nerve sheath tumor (18e23). RTs and other SMARCB1-deficient tumors have in common a lack of expression of the SMARCB1 protein and a rhabdoid cell phenotype, while differing (presumably) in the genomic background and cell of origin. RT, including malignant renal and extrarenal RT and ATRT, are a distinct category of tumors, with an unknown cell of origin, usually occurring in very young children. Histologically, RTs are characterized by an infiltrate of large, primitive-appearing malignant cells with classic rhabdoid cells in some fields and a divergent immunophenotype. RT cells can express markers of epithelial, mesenchymal, and neural lineage, and are commonly positive for embryonic stem cell markers, such as SALL4 (24e27). Chromosomal translocations at breakpoint 22q11.2, which is the locus of SMARCB1, have been described in RT with various partner chromosomes, including 1p36.1, 8q12, 9p13, 9q34, 11p15.5, and 18q21 (6e8,28e32). To our
knowledge, a complex three-way chromosomal translocation has not been described as the cause of SMARCB1 loss in RT before. The absence of the SMARCB1 signal on the nontranslocated chromosome 22 in this case indicates that the second allele was likely lost by gene deletion. Approximately one third of patients with RTs are found to have a germline deletion or truncating mutation in SMARCB1, either as a sporadic event or in the setting of familial rhabdoid predisposition syndrome (33,34). Analysis of the blood sample for germline mutations or deletion of SMARCB1 was not possible in this case, but given that a number of karyotypically normal cells with retained SMARCB1 were isolated on the cell culture and a number of normal, likely stromal, cells with intact genes were found on the iFISH assay, we concluded that both losses had to be somatic events in this case. In summary, we have described, for the first time, a SMARCB1 deletion as a result of a three-way translocation t(X;22;11) (q13;q11.2;q14.2) in a patient with an extrarenal malignant RT. This case emphasizes the diverse genetic events that may result in RT tumorigenesis, and it illustrates the role of FISH studies in the elucidation of complex cytogenetic abnormalities.
Acknowledgments This work was supported in part by the American Lebanese Syrian Associated Charities. The authors thank Samantha Melton for her excellent technical support.
References 1. Rorke LB, Packer RJ, Biegel JA. Central nervous system atypical teratoid/rhabdoid tumors of infancy and childhood: definition of an entity. J Neurosurg 1996;85:56e65.
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