Cancer Genetics 206 (2013) 387e392


Multiple EWSR1-WT1 and WT1-EWSR1 copies in two cases of desmoplastic round cell tumor Roberta La Starza a,1, Gianluca Barba a,1, Valeria Nofrini a,1, Tiziana Pierini a, Valentina Pierini a, Luca Marcomigni b, Katia Perruccio b, Caterina Matteucci a, Clelia Tiziana Storlazzi c, Giulia Daniele c, Barbara Crescenzi a, Michele Giansanti d, Paolo Giovenali e, Paola Dal Cin f, Cristina Mecucci a,* a

Hematology and Bone Marrow Transplantation Unit, University of Perugia, Perugia, Italy; b Oncology Unit, General Hospital, Perugia, Italy; c Department of Genetics and Microbiology, University of Bari, Bari, Italy; d Department of Pathology, University of Perugia, Perugia, Italy; e Diagnostic Cytology and Histology, Perugia General Hospital, Perugia, Italy; f Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts To provide new insights into the genomic profile of desmoplastic round cell tumors (DSRCT), we applied fluorescence in situ hybridization (FISH) and metaphase comparative genomic hybridization (M-CGH) to two newly diagnosed cases. FISH detected multiple subclones bearing one to three copies of der(11)t(11;22)(p13;q12) and/or der(22)t(11;22)(p13;q12) in both patients. This peculiar genomic imbalance might result from derivative chromosome duplication due to nondisjunction and/or mitotic recombination between normal and derivative chromosomes 11 and 22. Concomitant loss of normal chromosomes (i.e., 11 in patient 1 and 22 in patient 2) caused loss of the WT1 or EWSR1 wild-type allele. M-CGH identified other genomic imbalances: gain at chromosome 3 in both cases and chromosome 5 polysomy in patient 1. Common genomic events (i.e., trisomy 3 and extra EWSR1-WT1 and WT1-EWSR1 copies) probably contributed to disease pathogenesis and/or evolution of DSRCT. Our study demonstrated that an integrated molecular cytogenetic approach identified EWSR1-WT1 cooperating molecular events and genetic markers for prognosis. Thus, FISH and M-CGH might well be applied in a large series of patients to elucidate the genomic background of DSRCT. Keywords EWSR1-WT1, FISH, M-CGH ª 2013 Elsevier Inc. All rights reserved.

Desmoplastic round cell tumor (DSRCT), a rare aggressive neoplasm with distinct clinical, histological, and immunohistochemical features, most frequently affects young men. Essentially all cases of DSRCT are associated with the t(11;22)(p13;q12), which fuses the WT1 gene at 11p13 with EWSR1 at 22q12 (1). The EWSR1-WT1 chimeric product generally contains EWSR1 gene exons 1e7, which encode the amino-terminal domain, and WT1 gene exons 8, 9, and 10, which encode the last three zinc fingers of the DNAbinding domain. The fusion protein acts as an aberrant transcription factor, which transactivates genes that are

Received July 23, 2013; received in revised form October 23, 2013; accepted October 30, 2013. * Corresponding author. E-mail address: [email protected] 1 These authors contributed equally to this work. 2210-7762/$ - see front matter ª 2013 Elsevier Inc. All rights reserved.

normally regulated by WT1. First identified as a tumor suppressor gene, WT1 can also play the role of oncogene. In fact, WT1 deletions and loss-of-function mutations are typically associated with Wilms’ tumor and T-cell acute lymphoblastic leukemia, whereas WT1 overexpression is linked to acute myeloid leukemia (2e4). The EWSR1 gene participates in fusions with diverse partners in solid tumors as well as in acute leukemias (5e7). Since all fusion proteins include the EWSR1 transactivation domain and the partner DNA binding domain, the fusion protein acts as an ectopic transcription factor (8,9). EWSR1-WT1 fusion product variability has been reported, but no clinical and/or pathological significance has been attached to the variant transcripts (1). Since the genomic abnormalities that cooperate with EWSR1-WT1 in DSRCT pathogenesis are still largely unknown, we integrated fluorescence in situ hybridization (FISH) with metaphase comparative genomic hybridization (M-CGH) to investigate two cases.


R. La Starza et al.

Materials and methods


Case reports

DNA was obtained from 50, 3-mm thin, paraffin embedded tissue sections. Paraffin was eliminated by two xylene incubations (5’ at 55 C) followed by 2,500 rpm per 5 minute centrifugations. After two absolute ethanol incubations (10’ at 55 C) and 2,500 rpm per 5 minute centrifugations, cells were resuspended in a paraffin lysis buffer (50 mM TrisHCl pH 8.5/ 1 mM EDTA/0.5% Tween 20) and used for a classic saltingout extraction. M-CGH was performed as already described with slight modifications (10). Chromosomal regions are over-represented when the corresponding green-to-red ratio exceeds 1.18 and under-represented when the ratio is below 0.83. High-level amplification is achieved when the ratio exceeds 1.5. These thresholds are based on control experiments using DNA from healthy male and female donors. Telomeric and centromeric regions as well as heterochromatic regions of chromosomes 1, 9, 16, and Y were excluded from analysis. Analysis was performed at 99.9% and 99% of confidence in patients 1 and 2, respectively.

Patient 1 Clinical examination of a 38-year-old man, referred for abdominal pain, detected an aching, tender hypogastric mass. Ultrasound and computerized tomography (CT) scans revealed enlarged confluent lymph nodes (the largest being 10  7  5 cm) in the right iliac fossa, in the pelvis, retroperitoneum, and splenic and hepatic hila. Histopathology showed proliferation of small undifferentiated cells in large solid nests with necrotic foci. Cells were positive for desmin, vimentin, epithelial membrane antigen (EMA), cytokeratin, and neuron specific enolase (NSE). Morphology and immunohistochemistry indicated DSRCT. Partial response was obtained with 11 cycles of chemotherapy, alternating vincristine, adriamycin, ifosfamide, cisplatinum, and etoposide. The patient underwent autologous peripheral stem cell transplantation preceded by high-dose busulfan and melphalan but died of disease progression 50 months after diagnosis. Patient 2 A 23-year-old man was referred for epigastralgia. An abdominal CT scan showed a bulky mass in the pelvis, secondary lesions in the liver, and many enlarged lymph nodes. Tumor histology was characterized by multi-nodular growth of solid nests consisting of small cells enclosed in a desmoplastic stroma (Figure 1A). Cells were immunoreactive to desmin, vimentin, EMA, cytokeratin, NSE (Figure 1BeF), and focally to WT1. DSRCT was diagnosed. At his last checkup, 22 months after diagnosis, the patient presented with disease progression.

FISH FISH was performed according to standard protocols on four micron tissue sections from primary tumor biopsy samples in both patients, applying genomic clones for WT1/11p13 (RP11-122I7 and RP11-482E3) and EWSR1/22q12 (RP1191J21 and RP11-367E7) (Vysis LSI EWSR1; Abbott Molecular Milan, Italy), which were used in double color double fusion (DCDF) or break-apart (BA) assays (Figure 2A). In patient 1, a double color (DC) experiment that combined MLL/11q23 (RP11-770J1, RP11-832A4, and RP11-861M13) with WT1/11p13 (RP1-74J1, RP11-122I7, and RP11-482E3) was conducted to precisely evaluate the number of der(11) and der(22) in diverse clones. To assess copy number variation, CEP3 (alpha satellite centromeric region of chromosome 3, D3Z1) and Vysis LSI EGR1 (5q31)/ D5S721,D5S23 (5p15.2) (Abbott Molecular) were used. RP1 and RP11 clones belong to the Roswell Park Cancer Institute libraries in Buffalo, NY, and were kindly provided by Dr. Mariano Rocchi (Department of Genetics and Microbiology, University of Bari, Bari, Italy) ( BACPAC). Clones were validated on normal metaphases obtained from healthy donors’ peripheral blood T lymphocytes to rule out non-specific binding. As positive controls, soft tissue tumors and leukemias with WT1, EWSR1, and MLL involvement were used. For each experiment, at least 100 intact non-overlapping cells were analyzed.

Results FISH Detailed FISH results are reported in Supplementary Table 1. The presence of a EWSR1-WT1 fusion was confirmed by DCDF experiments (Figure 2B,C and Supplementary Table 1). Cell sub-clones with diverse chromosome modal numbers were identified in both cases. In patient 1, the EWSR1 BA assay indicated that one to two normal chromosomes 22 were present, whereas WT1 BA revealed that the normal chromosome 11 was missed (Figure 2D and Supplementary Table 1). Neoplastic cells bore one to two der(22) and one to three der(11). DC experiments with MLL/11q23 and WT1/11p13 detected three main clones: the first (30.5% of cells) had a balanced ratio between der(11) and der(22), the second (57.6%) a positive der(11):der(22) ratio, and the third (11.9%) a negative der(11):der(22) ratio (Supplementary Figure 1). Vysis probe LSI EGR1/D5S721, D5S23 (Abbott Molecular) (diploidy: two green and two orange signals) showed that approximately 50% of the cells bore 5-n green/orange signals, indicating chromosome 5 polysomy. The CEP3 probe detected four subclones with, respectively, two (31% of cells), three (26%), four (26%), and five or more signals (17%). In patient 2, EWSR1 BA and WT1 BA indicated that the neoplastic cells had one to two copies of normal chromosome 11, one to two copies of both der(11) and der(22), but lacked the normal 22 (Figure 2E and Supplementary Table 1). The CEP3 probe detected four subclones with, respectively, two (8% of cells), three (15%), four (23%), and five or more signals (54%).

M-CGH M-CGH revealed imbalances in the ratio along single chromosomes but was less sensitive in detecting aneuploidies due to the presence of many clones with diverse modal numbers. In patient 1, a high instability copy number

Multiple copies of EWSR1-WT1 and WT1-EWSR1 in DSRCT


Figure 1 Histology and immunohistochemistry in patient 2. Immunostaining with (A) hematoxylin and eosin; (B) desmin; (C) vimentin; (D) epithelial membrane antigen; (E) cytokeratin; and (F) neuron specific enolase.


R. La Starza et al.

Figure 2 (A) Schema of genomic probes used to study the t(11;22)(p13;q12) in two cases of DSRCT; (B,C) Double color double fusion EWSR1 (RP11-91J21 þ RP11-367E7 green)/WT1 (RP1-74J1 þ RP11-482E3 þ RP11-122I7 orange) assay in patient 1 (B) and patient 2 (C); (D,E) FISH experiments with a Vysis LSI EWSR1 (22q12) Dual Color Break Apart Probe (Abbott, Milan, Italy) performed on four micron tissue sections taken from biopsy sample of the primary abdominal masses in both patients. (D) Patient 1: two fusion

Multiple copies of EWSR1-WT1 and WT1-EWSR1 in DSRCT variation (CNV) profile was characterized by gains of 1p21ep31, 1q25eq31, 2p16ep21, 2q22eq34, 3p12ep14, 3q13.1eq13.3, 3q23eq26.3, chromosomes 4 and 5, 6p12, 6q11eq24, 7p15ep21, 7q31, 8q12eq23, 9p21epter, 10q21, 11q14eq23, 12p12, 12q14eq21, 13q14eq32, 14q21, 18q12eq21, and 21q; and losses at 1p34.2epter, 9q34eqter, 11p14epter, 12q24.1eq24.2, 16p, chromosome 17, chromosome 19, 20q, and 22q11.1eq11.2 (Figure 2D). Polysomy 5 was confirmed by FISH. The unbalanced ratio between the number of der(11) (two to three copies) and the number of der(22) (one to two copies) in 57.6% of cells resulted in a loss at 11p14epter (telomeric to WT1) and at 22q11.1eq11.2 (centromeric to EWSR1) (Supplementary Table 1 and Supplementary Figure 1). In patient 2, M-CGH detected a gain of the entire chromosome 3, which was confirmed by FISH (Figure 2E).

Discussion DSRCT, an aggressive tumor, is characterized by transient response to high-dose chemotherapy and rapid progression. DSRCT is one of a group of small, round cell tumors, including rhabdomyosarcoma, neuroblastoma, Ewing sarcoma/primitive neuroectodermal tumor, and non-Hodgkin lymphoma, which share overlapping features. Consequently, the genetic diagnosis helps classify the disease correctly and stratify prognosis. Although the t(11;22)(p13;q12), the distinctive DSRCT genetic hallmark, generates the EWSR1-WT1 fusion gene, which is widely considered diagnostic, it could also be associated with neoplasia, which differs in clinical outcome and histopathology. For example, Alaggio et al. reported two EWSR1-WT1 positive tumors with hybrid morphological and immunohistochemical features of leiomyosarcoma and DSRCT and an unusually favorable clinical course (11). Because additional abnormalities probably contribute to the DSRCT phenotype, genomic studies may be useful to detect molecular lesions that cooperate with EWSR1-WT1 in the pathogenesis of disease. In a recent study, no mutations of 29 putative oncogenes/tumor suppressor genes were detected in 24 children with DSRCT (12). One noteworthy finding in the present study, which combined FISH and MCGH approaches in two young male patients with DSRCT, was the presence of multiple copies of derivative chromosomes involved in the t(11;22) (i.e., der(11)t(11;22) and/or der(22)t(11;22)). In patient 1, multiple copies of der(11) t(11;22)(p13;q12) replaced the normal chromosome 11, so the neoplastic cells bore multiple WT1-EWSR1 fusions but lacked the wild-type WT1 allele. In patient 2, multiple copies of either der(11)t(11;22)(p13;q12) or der(22)t(11;22) (p13;q12) were present, but cells lacked the normal chromosome 22. Consequently, gain of WT1-EWSR1 and/or EWSR1-WT1 occurred with complete loss of the EWSR1 wild-type allele. Whether absence of functionally normal WT1


391 or EWSR1 alleles contributed to DSRCT phenotype in these cases remains to be established. Duplication of derivative chromosomes with loss of the normal counterpart was observed in myelodysplastic syndromes (MDS), acute leukemias, and chronic myeloid leukemia (CML). It might result from a duplication of the derivative chromosomes, due to non-disjunction and/or mitotic recombination between normal chromosomes 11 and/ or 22 and derivatives (13e16). Interestingly, mitotic recombination was demonstrated in other translocations in human leukemias such as the t(11;21)(q23.3;q11.2) in MDS and the t(9;22)(q34;q11)/BCR-ABL in CML (15,16). Whatever the mechanism, gains of EWSR1-WT1 and/or WT1-EWSR1 fusions were present in our cases. Likewise, multiple copies of EWSR1-CREB1 were reported in a case of angiomatoid fibrous histiocytoma (17). Whether a gene dosage effect influences disease phenotypes in these cases remains to be determined. Our most striking M-CGH findings were recurrent gains at chromosome 3 in both cases and high-level chromosome 5 amplification in patient 1, who displayed other chromosome gains and losses. In DSRCT with complex karyotypes, recurrent numerical changes are trisomy of chromosomes 1, 5, and 18 ( Indeed, trisomy 5 is one of the most frequent numerical abnormalities in many malignant and benign soft tissue tumors; focal chromosome 5 amplifications, involving a variable DNA segment at the short or long arm, were also found in clear renal cell and lung carcinomas ( Chromosomes/Mitelman). Thus, identification of chromosome 5 critical regions and putative oncogenes might contribute to understanding the genetic background of DSRCT, other soft tissue tumors, and epithelial tumors such as renal and lung carcinoma. In conclusion, in our two patients, FISH reliably detected EWSR1-WT1, the diagnostic hallmark of DSRCT, and revealed for the first time multiple copies of EWSR1-WT1 and/or WT1-EWSR1 in diverse neoplastic cell subclones. These findings suggest that a dosage effect of both fusions deriving from the t(11;22)(p13;q12) can contribute to disease pathogenesis and/or progression. Neoplastic cell instability might favor acquisition of genomic imbalances, which then contribute to the aggressiveness of disease. An integrated molecular cytogenetic approach might usefully be applied in a large series of patients with DSRCT to identify molecular events cooperating with EWSR1-WT1 and genetic markers that influence prognosis.

Acknowledgment The authors thank Fondazione Cassa di Risparmio di Perugia (grant no. 2011.0159.021), Associazione Daniele Chianelli, Perugia, Italy, and Fondazione Sergio Luciani, Fabriano, Italy. Tiziana Pierini was supported by Progetto

signals correspond to normal chromosomes 22, three green signals to der(11)t(11;22)(p13;q12), and one orange signal to der(22) t(11;22)(p13;q12). (E) Patient 2: two green and two orange signals correspond to der(11)t(11;22)(p13;q12) and der(22) t(11;22)(p13;q12), respectively. Absence of a fusion signal indicates the normal chromosome 22 was missed. (F) M-CGH in patient 1 shows losses (red bars) and gains (green bars) of diverse genomic regions, including 11p14epter and 22q11.1eq11.2. (G) M-CGH in patient 2 shows a gain of the entire chromosome 3 (green bar).

392 Regione POR Umbria FSE 2007e2013 Asse IV Capitale Umano. We also thank Dr. Geraldine Boyd for help in editing the manuscript.

Supplementary data Supplementary data related to this article can be found at

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Multiple EWSR1-WT1 and WT1-EWSR1 copies in two cases of desmoplastic round cell tumor.

To provide new insights into the genomic profile of desmoplastic round cell tumors (DSRCT), we applied fluorescence in situ hybridization (FISH) and m...
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