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The Expression of WT1 in the Differentiation of Rhabdomyosarcoma from Other Pediatric Small Round Blue Cell Tumors D

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The Expression of WT1 in the Differentiation of Rhabdomyosarcoma from Other Pediatric Small Round Blue Cell Tumors D The Expression of WT1 in the Differentiation of Rhabdomyosarcoma from Other Pediatric Small Round Blue Cell Tumors D. F. Carpentieri, M.D., K. Nichols, M.D., P. M. Chou, M.D., M. Matthews, M.D., B. Pawel, M.D., D. Huff, M.D. Departments of Pathology (DFC, MM, BP, DH) and Oncology (KN), The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania; Department of Pathology (PMC), Children’s Memorial Hospital, Chicago, Illinois; Department of Pathology (DH), University of Pennsylvania, Philadelphia, Pennsylvania plasmic staining favors an RMS, and a strong The WT1 gene encodes a transcription factor impli- nuclear staining is suggestive of a Wilms’ tumor. A cated in normal and neoplastic development. The role for WT1 in the pathogenesis of rhabdomyosar- purpose of this study was to evaluate the diagnostic comas is raised. The limited sampling precludes any utility of a commercial WT1 antibody on a variety of conclusions regarding the value of tissue or periph- pediatric small round blue cell tumors (SRBCT). A eral blood analysis for WT1 mRNA in patients with mouse monoclonal antibody (clone: 6F-H2, DAKO) rhabdomyosarcoma. raised against the N-terminal amino acids 1–181 of the human WT1 protein was tested. Microscopic KEY WORDS: Immunohistochemistry, Rhabdomyo- sections from 66 specimens were stained using an sarcoma, RT-PCR, Western blot, WT1. antigen retrieval protocol with trypsin. The tumors Mod Pathol 2002;15(10):1080–1086 included peripheral neuroectodermal tumors (PNET/Ewing’s), neuroblastomas, desmoplastic The WT1 gene (1) encodes a protein with four zinc small round cell tumors (DSRCT), lymphomas, fingers of the Kruppel-type in the C-terminal region Wilms’ tumors, and rhabdomyosarcomas (RMS). that recognizes a guanidine-cytidine (GC)–rich One RMS case was investigated by Western blot “EGR1” consensus sequence (2) required in tissue analysis and RT-PCR to confirm the antibody spec- differentiation and proliferation (2–4). The ificity. A strong cytoplasmic staining was demon- N-terminal half contains a large proline-glutamine– strated in all RMS (11/11). The Western blot analysis rich domain important for inhibition of transcrip- confirmed the WT1 protein in the tissue, and the tional activation (5, 6). There are at least eight pro- RT-PCR confirmed the presence of WT1 mRNA in tein isoforms ranging between 52 and 62 kDa in the peripheral blood and tissue of one RMS patient. mammals produced by a combination of alterna- The Wilms’ tumors had a variable nuclear and/or tive splicing and RNA editing (4, 7). cytoplasmic positivity in most (17/24) cases. All The WT1 proteins are normally expressed in the PNET/Ewing’s were negative. The nuclei of two lym- nuclei of glomerular podocytes and mesothelial phoblastic lymphomas stained strongly. A weak nu- cells. It has also been demonstrated in stem cells clear or cytoplasmic staining was reported in a few bearing the CD34ϩ phenotype (8). The role of WT1 DSRCT (3/5), lymphomas (2/10), and neuroblasto- in normal human development also extends to a mas (2/8). This is a useful antibody in the differen- tiation of RMS from other SRBCTs. A strong cyto- diversity of mammalian mesodermal tissues (9), in- cluding the body-wall musculature in a 13.5-days postconception (dpc) mouse embryo (43–49 dpc human). Embryologic studies of wt1-null mice re- Copyright © 2002 by The United States and Canadian Academy of Pathology, Inc. veal a failure to develop kidney and gonads (10). VOL. 15, NO. 10, P. 1080, 2002 Printed in the U.S.A. Mutations and splicing disruptions of WT1 have Date of acceptance: June 21, 2002. The preliminary findings of this study were presented at the Society for been described in Denys-Drash (11–14), WAGR Pediatric Pathology meeting in New Orleans, Louisiana in the Spring of (15), and Frasier (13, 16) syndromes. 2000 and thus published previously in abstract form (Carpentieri et al. Mod Pathol 2000;13:6P). These proteins were first recognized as tumor Address reprint requests to: David F. Carpentieri, M.D., Pathology Depart- suppressors. However, an activator or oncogenic ment, Phoenix Children’s Hospital, 1919 East Thomas Road, Phoenix, AZ 85016; e-mail: [email protected]; fax: 602-546-1284. behavior may be acquired by missense mutations. DOI: 10.1097/01.MP.0000028646.03760.6B WT1 expression has been demonstrated in hema- 1080 tological malignancies (17–22), mesothelial-derived antibody raised against the N-terminal 1–181 neoplasms (23–27), breast cancer (28, 29), genito- amino acids of human WT1 (41). The antibody was urinary tumors (30, 31), and small round blue cell freshly diluted at 1:50 in phosphate buffered saline tumors (SRBCT; 24, 27, 32–34). Recent studies have (PBS) every time that it was used. The antigen re- evaluated the possible role of peripheral blood RNA trieval protocol required trypsin digestion for 20 (18, 19), serum antibodies (35), and immunother- minutes in a 37° C oven. The antibody was detected apy (36, 37) in the diagnosis, monitoring, and treat- by a standard avidin–biotin method. Normal kid- ment of WT1-positive tumors. ney tissue (Fig. 1A) and an 8-week-old human fetus The microscopic evaluation of an SRBCT can be (Fig. 1,B–D) were used as controls. The results were difficult, especially in a small biopsy specimen. The graded (Table 1) as 0 if negative or as weak (ϩ1) or diagnosis often relies on clinical history, laboratory strong (ϩ2) if positive in a nuclear (N) or cytoplas- analysis, and ancillary studies on the tissue (38–40). mic (C) pattern. Immunohistochemistry has become one of the most important techniques in the interpretation of these biopsies, and the diagnostic work-up may Western Blot Analysis include antibodies against mesenchymal, epithe- The cell line 293T composed of transformed kid- lial, neuronal, lymphoid, and myogenic antigens. ney epithelial cells were transiently transfected with The WT1 antibodies have become recently avail- an expression construct encoding human WT-1, able for immunohistochemical use on paraffin- isoform B (pCDNA3-WT1B, kindly provided by Dr. embedded tissues, and a few reports have ad- Daniel Haber, Massachusetts General Hospital dressed the diagnostic utility in the differentiation Cancer Center) using calcium phosphate precipita- of tumors (23, 24, 27, 30, 32). It has been our expe- tion. After 48–72 hours, cells were rinsed one time rience that the WT1 N-terminal antibody (clone with cold PBS and harvested by scraping. Whole- 6F-H2) produces a different pattern of staining in cell lysates were prepared from 293T cells, a Wilms’ SRBCT when compared with previous studies using tumor, and a rhabdomyosarcoma sample using ly- the C-terminal antibody (24, 27, 32). This observa- sis buffer (1% NP-40, 150 mM NaCL, 50 mM Tris, pH tion led us to evaluate the utility of the WT1 7.4) containing protease inhibitors. Proteins were N-terminal antibody (6F-H2) in a cohort of pediat- separated by sodium dodecyl sulfate–polyacrylam- ric patients diagnosed with SRBCT. ide gel electrophoresis and transferred to nitrocel- lulose. After blocking in 5% bovine serum albumin– MATERIALS AND METHODS TBST for 1 hour at room temperature, Western blot analysis was performed using either anti-WT1 Specimens (clone 6F-H2; DAKO, Carpinteria, CA) or anti- Sixty-six formalin-fixed and paraffin-embedded tubulin (Santa Cruz Biotechnology, Santa Cruz, CA) tumor biopsy specimens were retrieved from the antibodies, according to standard protocols. files at the Children’s Hospital of Philadelphia. These specimens included 8 peripheral neuroecto- Reverse Transcription Polymerase dermal tumors (PNET/Ewing’s), 8 neuroblastomas, Chain Reaction 5 desmoplastic small round blue cell tumors (DSRCTs), 10 lymphomas, 24 Wilms’ tumors, and Tumor tissue and peripheral blood from a patient with RMS was snap-frozen in liquid nitrogen and 11 rhabdomyosarcomas (RMS). The lymphomas in- Ϫ cluded seven lymphoblastic, three Burkitt’s, and stored at 70° C until the time of testing. The tissue two large cell (one anaplastic). Five Wilms’ tumors was homogenized and washed twice in PBS. Ap- contained areas of heterologous differentiation proximately 10 million cells were lysed with 1 mL of (muscle or bone). The rhabdomyosarcomas in- Trizol reagent, and RNA was isolated according to cluded four embryonal, six alveolar, and one parat- the manufacturer’s protocol. One microgram of total esticular spindle cell tumor. Supporting immuno- RNA from each sample was reverse transcribed into histochemical stains included the following: MIC2, cDNA according to a standard protocol (Perkin Elmer O13, NSE, CD56, AE1, AE3, desmin, LCA, CD20, Biosystems, Santa Clara, CA). Reverse transcription UCHL-1, CD30, ALK-1, EMA, and muscle-specific (RT) polymerase chain reaction (PCR) amplification actin. Molecular translocation studies (PAX3/PAX7- of WT1 was performed using the following primers: FKHR, EWS-FLI1/ERG, and EWS-WT1) were per- 5'- GGCATCTGAGACCAG TGAGAA-3' (outer sense), formed as indicated to confirm the diagnosis. 5' GAGAGTCAG ACTTGAAAGCAGT-3' (outer anti- sense), 5'-GCT GTCCCACTTACAGATGCA-3'(inner sense), and 5'TCAAAGCGCCAGCTGGAGTTT-3' (in- Immunohistochemistry ner antisense). The first round of PCR was carried out The WT1 antibody was a mouse monoclonal with 30 amplification cycles, followed by a second (clone 6F-H2; DAKO Corporation, Carpinteria, CA) round of 30 cycles with a DNA thermal cycler (R480, Expression of WT1 in the Differentiation of Rhabdomyosarcoma (D.F. Carpentieri et al.) 1081 TABLE 1. Grading of WT1 Antibody (6F-H2) Stain specificity was confirmed by Western blot analysis Grading Pattern (Fig. 2). A band of 52 kDa from the RMS cell lysate 0 No staining comigrated with the transfected WT-1 (293/WT-1B) ϩ1 Weak (focal or multifocal) and the endogenous WT-1 (293, Wilms’ tumor). ϩ 2 Strong (multifocal or diffuse) Also, the RT-PCR (Fig. 3) from the RMS tissue (1:50 and 1:100 dilutions) and from the nondiluted pe- ripheral blood (1:1) obtained from the same patient Perkin Elmer).
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