OBSERVATION Expression of Wilms Tumor 1 Gene Distinguishes Vascular Malformations From Proliferative Endothelial Lesions Leslie P. Lawley, MD; Francesca Cerimele, MD, PhD; Sharon W. Weiss, MD; Paula North, MD; Cynthia Cohen, MD; Harry P. W. Kozakewich, MD; John B. Mulliken, MD; Jack L. Arbiser, MD, PhD Background: Vascular malformations and hemangio- Observations: The biochemical differences between mas, which are endothelial lesions of childhood, may re- hemangiomas, which involute, and vascular malforma- sult in considerable morbidity because they can cause dis- tions, which do not involute, are not well understood. comfort and functional impairment and have a negative We found that the transcription factor encoded by the affect on the patient’s appearance. Although vascular mal- Wilms tumor 1 (WT1) gene is expressed in the endothe- formations may initially appear very similar to heman- lium of hemangiomas but not in vascular malforma- giomas, they have distinct clinical courses. Infantile hem- tions. angiomas progress through 3 stages: proliferative, involuting, and involuted. The proliferative phase is char- Conclusions: Defects in WT1 signaling may underlie the acterized by clinical growth. Once hemangiomas reach inability of malformation endothelial cells to undergo their maximum size, they begin to regress or involute. physiologic apoptosis and remodeling. The availability Histologically, this stage is characterized by endothelial of WT1 staining in hospital laboratories may allow the apoptosis. Finally, the involuted stage of the heman- clinician to distinguish hemangiomas from vascular mal- gioma occurs when the original lesion is replaced by a formations and thus to give appropriate therapy to the connective tissue remnant. In contrast to hemangio- patient. mas, vascular malformations do not involute but con- tinue to enlarge as the patient grows. Arch Dermatol. 2005;141:1297-1300 ASCULAR MALFORMATIONS vation is a common feature of malignant can cause significant mor- transformation.4,5 We hypothesized that bidity as a result of hemor- vascular malformations may also show rhage, mass effect in the common abnormalities in signaling ow- brain, induction of connec- ing to diverse mutations. Wilms tumor 1 Vtive tissue hypertrophy and limb asym- (WT1) is a transcription factor that is ini- metry, and pain. No medical treatment is tially isolated through a reverse genetic ap- effective for vascular malformations. Sur- proach in hereditary Wilms tumor, but it gical resection, embolization, and sclero- can also be activated by a translocation in Author Affiliations: therapy may provide benefit for selected le- desmoplastic small round cell tumors or Departments of Dermatology sions, but many vascular malformations are transcriptionally elevated in leuke- (Drs Lawley, Cerimele, and unresectable or too extensive for destruc- mias.6-17 Given the diverse activities of WT1, Arbiser) and Pathology and Laboratory Medicine (Drs Weiss tive modalities. Therefore, a better under- we examined it as a candidate signaling and Cohen), Emory University standing of the signaling pathways that un- molecule in endothelial tumors. In the pre- School of Medicine, and derlie vascular malformations is needed to sent study, we found that WT1 messenger Department of Dermatology, help develop novel therapies. RNA is expressed at high levels in human Veterans Affairs Hospital We previously observed that common endothelium that is stimulated by angio- (Drs Lawley, Cerimele, and signaling abnormalities may be present in poietin 2. Also, immunohistochemical Arbiser), Atlanta, Ga; neoplasms that arise from distinct genetic analysis of human hemangiomas and vas- Department of Pathology, mechanisms.1 For example, we previ- cular malformations revealed strong endo- University of Arkansas School ously demonstrated that mitogen- thelial staining of hemangiomas but greatly of Medicine, Little Rock activated protein (MAP) kinase is univer- decreased endothelial staining in vascular (Dr North); and Department of Pathology (Dr Kozakewich) and sally activated in hemangiomas of infancy malformations. Immunohistochemical Division of Plastic Surgery but not in malignant endothelial neo- studies of WT1 may help distinguish hem- 2,3 (Dr Mulliken), Children’s plasms. In melanoma, which can arise angiomas from vascular malformations in Hospital and Harvard Medical through mutations in N-ras or B-raf or loss difficult cases and thus prevent inappro- School, Boston, Mass. of PTEN and p16ink4a, MAP kinase acti- priate therapy. (REPRINTED) ARCH DERMATOL/ VOL 141, OCT 2005 WWW.ARCHDERMATOL.COM 1297 ©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 Table. Results of Immunostaining for Wilms Tumor 1 A B C Results, No. (%) Type of Lesion Positive Negative Benign or malignant vascular tumors Hemangioma (n = 9) 8 (89) 1 (11) Pyogenic granuloma (n = 2) 2 (100) 0 Angiosarcoma (n = 9) 9 (100) 0 Epithelioid hemangioendothelioma (n = 1) 1 (100) 0 Hobnail hemangioendothelioma (n = 1) 1 (100) 0 Malignant hemangioendothelioma (n = 1) 0 1 (100) Figure 1. Immunohistochemical features of a hemangioma (A), Vascular malformations angiosarcoma (B), and vascular malformation (C) stained for Wilms tumor 1 Port-wine stain* (n = 2) 0 2 (100) (WT1). Note the presence of reactivity for WT1 in the proliferative lesions Venous malformation (n = 10)† 0 10 (100) (A and B) but the lack of reactivity in the vascular malformation (C). Lymphatic malformation (n = 8) 0 8 (100) *Marked as hemangioma–port-wine stain, but results in the hemangioma of vascular endothelial growth factor (20 ng/mL) and portion were positive and those in the port-wine stain were negative. harvested for reverse transcriptase polymerase chain reaction †Includes venous and cavernous hemangiomas. (RT-PCR) analysis. RNA was isolated from human endothe- lial cells, and RT-PCR analysis was performed with denatur- ation for 1 minute at 94°C, followed by 1 minute at 48.5°C, and then 1 minute at 72°C for 35 cycles. Primers were based METHODS on the sequence of human WT1 and were amplified using 5Ј-GCATCTGAAACCAGTGAGAA-3Ј (sense) and IMMUNOHISTOCHEMICAL ANALYSIS 5Ј-TTTCTCTGATGCATGTTG-3Ј (antisense). The identity of the RT-PCR product was confirmed by sequencing. Sections of formalin-fixed, paraffin-embedded tissue (5 µm) were stained for WT1 using a 2-step horseradish peroxidase– labeled polymer system (Envision System; Dako Corp, Carpin- STATISTICS teria, Calif) and heat-induced antigen retrieval. The horserad- ish peroxidase–labeled polymer, which is conjugated with The total number of lesions with positive endothelial staining secondary antibodies, was used in combination with an auto- was divided by the total number of positive- and negative- mated staining system (Autostainer; Dako Corp). Hematoxy- staining lesions with the same diagnosis. lin was used as the counterstain. Negative controls were gen- erated by substituting the primary antibody with buffer- RESULTS specific antibody adsorbed with antigen. Sections were deparaffinized in xylene and grades of alcohol Hemangiomas revealed endothelial cytoplasmic immu- and rehydrated in water. Antigen retrieval was performed by plac- ing the sections in citrate buffer (pH, 6) inside an electric pres- nopositivity for WT1 in 8 (89%) of 9 samples (Table) sure cooker for 3 minutes at 120°C and then cooling them for (Figure 1). Some of the slides that were positive for WT1 10 minutes before immunostaining. The sections were next ex- in tumor cells also exhibited background blood vessel posed to 3% hydrogen peroxide for 5 minutes, primary antibody staining (capillaries, venules, or arterioles). Only 1 he- for 30 minutes, horseradish peroxidase–labeled polymer for 30 mangioma sample (11%) did not stain for WT1 at all. minutes, diaminobenzidine as chromogen for 5 minutes, and he- Other vascular tumors that showed positive staining for matoxylin as a counterstain for 15 minutes. The incubations were WT1 included pyogenic granulomas (100%), angiosar- performed at room temperature. Between incubations, the sec- comas (100%), an epithelioid hemangioendothelioma tions were washed with Tris-buffered saline and coverslipped (100%), and a hobnail hemangioendothelioma (100%). (Tissue-Tek SCA; Sakura Finetek USA, Inc, Torrance, Calif). The malignant hemangioendothelioma was negative Paraffin blocks or sections for WT1 antigen staining of be- nign and malignant vascular tumors and vascular malformations for WT1. Of note, additional samples of hemangiomas were obtained from the pathology departments of Emory Uni- revealed staining of normal background blood vessels versity, Atlanta, Ga, the University of Arkansas, Little Rock, and (capillaries, venules, or arterioles). Also, the pyogenic Children’s Hospital, Boston, Mass. The vascular tumors included granulomas, 1 angiosarcoma, the epithelioid hemangio- 9 hemangiomas, 2 pyogenic granulomas, 9 angiosarcomas, 1 epi- endothelioma, and the hobnail hemangioendothelioma thelioid hemangioendothelioma, 1 hobnail hemangioendothe- exhibited normal background blood vessel staining. lioma, and 1 malignant hemangioendothelioma. The vascular The vascular malformations in our study did not show malformations included 2 port-wine stains, 10 venous malfor- any positive staining of endothelium (Figure 1). Two port- mations, and 8 lymphatic malformations. The slides were reviewed wine stains (100%), 10 venous malformations (100%), for diagnosis and positive staining with WT1. Mesothelioma sec- and 8 lymphatic