Dermatology: Practical and Conceptual 28 Research | Dermatol Pract Concept 2018;8(1):6 DERMATOLOGY PRACTICAL & CONCEPTUAL www.derm101.com Immunoreactivity of Wilms tumor 1 (WT1) as an additional evidence supporting hemangiomatous rather than inflammatory origin in the etiopathogenesis of angiolymphoid hyperplasia with eosinophilia Fatma Tokat1, Julia S. Lehman2, Engin Sezer3, Emel Dikicioglu Cetin1, Umit Ince1, Emel Ozturk Durmaz3 1 Department of Pathology Acibadem University School of Medicine, Istanbul, Turkey 2 Department of Dermatopathology Mayo Clinic, Rochester, MN, USA 3 Department of Dermatology Acibadem University School of Medicine, Istanbul, Turkey Key words: angiolymphoid hyperplasia with eosinophilia, Wilms tumor 1, GLUT1, hemangioma Citation: Tokat F, Lehman JS, Sezer E, Dikicioglu Cetin E, Ince U, Ozturk Durmaz E. Immunoreactivity of Wilms tumor 1 (WT1) as an additional evidence supporting hemangiomatous rather than inflammatory origin in the etiopathogenesis of angiolymphoid hyperplasia with eosinophilia. Dermatol Pract Concept. 2018;8(1):28-32. DOI: https://doi.org/10.5826/dpc.0801a06 Received: July 18, 2017; Accepted: November 14, 2017; Published: January 31, 2018 Copyright: ©2018 Tokat et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: None. Competing interests: The authors have no conflicts of interest to disclose. All authors have contributed significantly to this publication. Corresponding author: Engin Sezer, MD, Acibadem University School of Medicine, Department of Dermatology, Buyukdere Caddesi No: 40, Istanbul, 34457, Turkey. Tel. 902123044626; Fax. 902123044440. Email: eseze@yahoo.com Background: Angiolymphoid hyperplasia with eosinophilia (ALHE) is a rare vascular proliferative disorder mainly located in the periauricular region. The etiopathogenesis of ALHE is unknown, and it is still controversial as to whether the entity represents a benign vascular neoplasm or an inflamma- tory process. Aim: Recently, the intracytoplasmic staining pattern of Wilms tumor 1 (WT1) on immunohistochem- istry has highlighted true vascular neoplasms, such as microvenular hemangioma, tufted angioma, and spindle cell hemangioma, which has made it helpful to distinguish ALHE from vascular malforma- tions, as there is a negative staining pattern in the other entities. We aimed to investigate the immu- noreactivity of ALHE specimens for WT1 as well as glucose transporter protein 1 (GLUT1) immuno- histochemistry, an important and sensitive marker for the diagnosis of infantile hemangioma, which recently has been described to label other hemangiomas, such as verrucous hemangioma. Material and methods: Clinical data and histopathological specimens from patients diagnosed with ALHE were reviewed, and immunohistochemical staining and microscopic analysis for WT-1 and GLUT1 were performed. Results: Intracytoplasmic endothelial staining of WT1 was detected in 19 of 20 ALHE specimens. GLUT1 was not detected in any ALHE specimen. Conclusions: We conclude that ALHE may represent a true hemangioma (i.e., benign vascular neopla- sia) characterized by an eosinophil- and lymphocyte-rich inflammatory component as opposed to the reactive inflammatory dermatosis with a positive intracytoplasmic staining pattern for WT1. As far as we are aware, WT1 staining for ALHE has not been described to date. ABSTRACT mailto:eseze@yahoo.com Research | Dermatol Pract Concept 2018;8(1):6 29 distilled water and tap water. The tissue was counterstained with Mayer’s hematoxylin. All slides were covered with a cover slip. The staining intensity was evaluated as negative (-), weak (+), moderate (++), and strong staining (+++) with a primary intensity score of tumor cell staining as a positive control for the immune markers. Results Clinical findings Clinical data for 20 patients with histopathologically con- firmed ALHE were evaluated. The age of the patients ranged from 9 and 93 years of age at the time of biopsy procedure with a median age of 48. A slightly increased male (n = 12) to female (n = 8) ratio was identified. Most of the lesions were located on the head and neck (n = 15), followed by upper extremities (n = 4) and in a lower extremity (n = 1). Immunohistochemical findings Nineteen of 20 specimens were positive for WT1 intracyto- plasmic staining (Figure 1). Moderate staining intensity (n = 10) was more frequent than weak (n = 5), and strong (n = 4) Introduction Angiolymphoid hyperplasia with eosinophilia (ALHE) is an uncommon, idiopathic vascular proliferative disorder char- acterized by dermal or subcutaneous red to brown papules or nodules, commonly located in the head and neck regions. ALHE is mainly a disorder of young adulthood to middle age, although children and elderly patients with the disorder have also been described. The precise etiopathogenesis of ALHE is unknown and it is controversial whether it is a true vascular neoplasm (i.e., a hemangioma variant with overlying eosinophilic and lymphocytic infiltration related to cytokine expression) or a vascular proliferative disorder (secondary to an inflammatory tissue response). In this investigation, we assessed the immu- noreactivity for Wilms tumor 1 (WT1) in ALHE specimens, which shows a cytoplasmic staining pattern in benign neo- plasms such as microvenular hemangioma, tufted angioma, and spindle cell hemangioma, to highlight the pathogenesis of this rare entity [1,2]. In these tissues, we also assessed glucose transporter protein 1 (GLUT1) immunohistochemistry, an important and sensitive marker for the diagnosis of infantile hemangioma, which recently has been described to label some other hemangiomas such as verrucous hemangioma [3]. Material and Methods Clinical data and histopathological specimens from patients (n = 20) diagnosed with ALHE (between 2006 and 2016) were obtained from the Acibadem University School of Medicine Pathology Department and the Mayo Clinic Der- matopathology Department. Approval was obtained from the Institutional Review Board (IRB) of the Mayo Clinic regarding the ethical concerns for the study. The diagno- sis of ALHE was histopathologically confirmed with the characteristic features of epithelioid endothelial cells with cytoplasmic vacuoles and a perivascular inflammatory cell infiltrate composed of numerous eosinophils, lymphocytes, and histiocytes. Immunohistochemical staining and micro- scopic analysis for WT1 and GLUT1 were performed for all ALHE specimens. The method used for immunostaining was the streptavidin-biotin-amplified system. The slides were submitted for subsequent steps of deparaffinization and rehydration. Sections were sliced (6 µm thick) and air-dried for 30 minutes. Then the sections were fixed in cold acetone for 10 minutes. After blocking endogenous peroxidase using 0.2% sodium azide for 5 minutes, they were washed with phosphate buffered saline for 15 minutes. Subsequently, the sections were incubated with primary antibodies for 1 hour. The primary antibodies were WT-1 (Cell Marque, California, USA) and GLUT1 (Cell Marque, California, USA) with a dilu- tion of 1:100. After incubation, the sections were rinsed with Figure 1. Histopathologic panoramic view of ALHE (hematoxylin and eosin stain) (a) and positive staining pattern for WT1 (b) on low power magnification. 30 Research | Dermatol Pract Concept 2018;8(1):6 This rare condition has also been termed epithelioid heman- gioma, histiocytoid hemangioma, inflammatory angiomatous nodule, and pseudo- or atypical pyogenic granuloma [5]. The lesions commonly arise in the third and third decade of life with a slight predilection in females. Histopathologically, ALHE presents as an ill-defined, dermal, lobulated mass com- posed of numerous vascular spaces of varying luminal diam- eter lined by large rounded endothelial cells with conspicuous eosinophilic cytoplasm and cytoplasmic vacuoles representing primitive lumina. A prominent inflammatory infiltrate com- posed largely of lymphocytes, numerous eosinophils, and histiocytes surrounding the vessels are identified [6]. The etiology of ALHE is unclear, and it is still contro- versial whether the disorder represents a benign vascular neoplasm or an inflammatory process. Association with arteriovenous shunts in 43% of cases of ALHE strengthens the conclusion that the entity may represent a vascular neo- plastic proliferation [7]. ALHE has also been reported to develop within a port wine stain in a patient, and the authors highlighted the role of increased serum renin levels in the pathogenesis of this case in which the histopathology speci- men revealed expression of angiotensin converting enzyme and angiotensin II receptors [8]. In a retrospective study of 116 ALHE patients, 10 cases (9%) were found to be associated with antecedent trauma patterns. The staining pattern for WT1 was intracytoplasmic in endothelial cells, similar to that reported previously for various benign vascular neoplasms such as microvenular hemangioma and verrucous hemangioma, which revealed an intracytoplasmic staining pattern as well [1,2] (Figure 2). GLUT1 immunohistochemistry was negative for all ALHE specimens despite positive controls (Figure 3). Table 1 sum- marizes the results of the immunohistochemical stains for WT1 and GLUT1 in 20 histopathologically proven ALHE specimens. Discussion ALHE was first described by Wells and Whimster in 1969 and was originally considered to represent late-stage Kimura’s disease, but it is now widely accepted as a separate entity [4]. ALHE is a benign vascular proliferation characterized by dull, single or multiple nodules mainly located in the head and neck region. Involvement of the oral mucosa, arm, hand, shoulder, genital region, breast, parotid gland, orbit, colon, bone, and parapharyngeal spaces has also been described. Figure 2. Details of the same specimen with epithelioid endothe- lial cells showing characteristic cytoplasmic vacuoles and associated eosinophilic and lymphocytic inflammation (a) Intracytoplasmic staining pattern for WT1 is identified on high power magnification revealing a strong staining intensity (b). Figure 3. The lack of staining with GLUT1 in endothelial cells (ar- rows). Erythrocytes in blood vessels serve as the positive control. TABLE 1. Immunohistochemical results of WT1 and GLUT1 immunoreactivity. Staining Intensity WT1 (n) GLUT1 (n) Strong staining (+++) 4 0 Moderate staining (++) 10 0 Weak staining (+) 5 0 Negative staining (-) 1 20 Research | Dermatol Pract Concept 2018;8(1):6 31 suggesting that it is a vascular neoplasm rather than a vas- cular malformation [3]. In another study, WT1 was found to be focally positive in 14/74 cases of verrucous hemangioma [14]. In this study, a positive control for WT1 is omitted and a high false-negative rate may explain the discrepancy, which may also result from differences in immunohistochemical techniques and reagents. In our study, cytoplasmic staining of WT1 was detected in the vascular endothelial cells of 19/20 specimens. This result is in keeping with the context that the entity may represent a true vascular neoplasm rather than a vascular prolifera- tive response to inflammation. This concept is supported by the fact that various anti-inflammatory medications are not effective in most cases of ALHE, but that the best therapeutic response would be excision/destructive treatment modalities [15-17]. A case report of a congenital ALHE with a blaschkoid segmental distribution in the anogenital region also suggests that the vascular proliferation may be unlikely to represent a secondary phenomenon of reactive inflammatory process, which is not expected to develop during intrauterine period, but rather a true vascular neoplasm such as congenital invo- luting/noninvoluting hemangioma [18]. This phenomenon also supports our point of view regarding these study results. Rapid remission of severe pruritus related to ALHE with pulsed dye laser in a patient, who was persistent to topical, intralesional, and oral corticosteroid medications suggests that the inflammatory component of this entity would be secondary to vascular neoplastic proliferation. The prolifer- ating endothelial cells of ALHE express adhesion molecules ICAM-1, ELAM-1, and VLA-1, -3, -5, which is considered to result in an inflammatory response [19]. In a systemic review of ALHE including 416 studies representing 908 patients, treatment failure was found to be lowest for excision and pulsed dye laser compared with anti-inflammatory treatment strategies [20]. We suggest that this phenomenon supports the conclusion that eosinophil and lymphocytic inflammatory component would be related to vascular endothelial cells expressing proinflammatory cytokines such as ICAM-1. The efficacy of imiquimod, an immune response modifier that induces interferon-alpha, in ALHE, as well as other vascular neoplasms such as retiform hemangioendothelioma, infantile hemangioma and proliferating hemangioma of infancy also supports this point of view [21,22]. GLUT1 is an erythrocyte-type glucose transporter pro- tein expressed in juvenile hemangiomas. It is a member of the facilitative cell-surface glucose transporter family, which includes five other isoforms originally identified in human erythrocyte membranes. GLUT1 is also expressed in brain capillary endothelium, where it plays a critical role in the transport of glucose across the blood-brain barrier. GLUT1 staining has been identified in a variety of normal cell types (frostbite, surgery, laceration, frictional trauma, and other unspecified) with a time course between injury and the onset of lesions ranging from 7 months to 20 years (median, 30 months) [8]. This phenomenon has also been described in pyogenic granulomas, another vascular proliferative disor- der, which developed after thermal burn, lightning, or mine injury [9]. In one patient, multiple ALHE lesions on the volar surfaces on both wrists and antecubital fossae, situated over a superficial vein and corresponding to a site of recent veni- puncture, also supports the role of trauma in the etiopatho- genesis of this entity [10]. ALHE that arises during pregnancy suggests that hor- monal changes may also take place in the etiopathogenesis of this condition, a phenomenon also described in other vascular neoplasms such as hobnail hemangioma, pyogenic granu- loma, liver, pancreatic, and spinal epidural hemangiomas [11]. Immunohistochemistry for WT1 antigen permits differen- tiation of vascular neoplasms and malformations; the former show cytoplasmic staining pattern, as in our study, whereas the latter is negative for this marker. In a large study (n = 167), cytoplasmic WT1 was detected in 117 various vascular neo- plasms, and was not detected in 50 cases of capillary, venous and lymphatic malformations [1]. In a true vascular neoplasm such as microvenular hem- angioma, immunoreactivity for WT1 was found in 9/10 tumors, which is similar to the results described herein [12]. Despite the detailed dermatopathological evaluation of the WT1 negative case, the diagnosis was consistent with ALHE in our study. We suggest that WT1 expression could be lost in a subset of hemangiomas such as ALHE, microvenular hemangioma and verrucous hemangioma reminding the phenomenon that MART-1 (melanoma antigen recognized by T cells 1) antibody, which is a highly sensitive marker, is lost in some melanoma specimens. In another study, WT1 was detected in 9/9 cases of microvenular hemangioma [2]. In a comparative study of WT1 expression in 23 vascular tumors and 20 vascular malformations, cytoplasmic stain- ing of WT1 was detected in cases of infantile hemangiomas (8/9), angiosarcomas (9/9), pyogenic granulomas (2/2), and epithelioid hemangioendothelioma (1/1), whereas com- plete vascular malformations consisting of port wine stains, venous, and lymphatic malformations were negative for WT1. Cytoplasmic WT1 staining pattern has been described in complete cases with tufted angioma (n = 8) and a single case of spindle cell hemangioma [1]. WT1 reactivity was also shown in extracutaneous hemangiomas such as anastomosing hemangioma of the kidney, histopathologically characterized by irregular fenestrated vascular spaces and reminiscent of the splenic red pulp with tightly packed, capillary-sized vessels with small lumen [13]. In a recent study, cytoplasmic immunoreactivity of WT1 was detected in complete cases with verrucous hemangioma 32 Research | Dermatol Pract Concept 2018;8(1):6 8. Manton RN, Itinteang T, de Jong S, Brasch HD, Tan ST. Angio- lymphoid hyperplasia with eosinophilia developing within a port wine stain. J Cutan Pathol. 2016;43(1):53-56. 9. Bakan V, Aliagaoglu C, Yildiz A, Emsen A. Multiple pyogenic granulomas on the face after landmine injury. Pediatr Dermatol. 2008;25(3):397-398. 10. Stewart N, Zagarella S, Mann S. Angiolymphoid hyperplasia with eosinophilia occurring after venipuncture trauma. J Dermatol. 2013;40(5):393-395. 11. Hollo P, Marschalko M, Sikos G, Harsing J, Horwart A. Angio- lymphoid hyperplasia with eosinophilia in pregnancy. J Eur Acad Dermatol Venereol. 2005;19(5):645-646. 12. Napekoski KM, Fernandez AP, Billings SD. Microvenular heman- gioma: a clinicopathological review of 13 cases. J Cutan Pathol. 2014;41(11):816-822. 13. Chou S, Subramanian V, Lau HM, Achan A. Renal anastomosing hemangiomas with a diverse spectrum: report of two cases and review of the literature. Int J Surgi Pathol. 2014; 22(4):369-373. 14. Wang L, Gao T, Wang G. Verrucous hemangioma: a clinicopatho- logical and immunohistochemical analysis of 74 cases. J Cutan Pathol. 2014;41(11):823-830. 15. Ali FR, Madan V. Facial angiolymphoid hyperplasia with eosino- philia: sustained remission following treatment with carbondiox- ide laser. Clin Exp Dermatol. 2016;41(1):96-98. 16. Garrido-Rios AA, Sanz-Munoz C, Torrero-Anton MV, Martinez- Garcia Miranda-Romero A. Angiolymphoid hyperplasia with eosinophilia on the tongue. Clin Exp Dermatol. 2009;34: (8):729- 731. 17. Sotiriou E, Apalla Z, Patsatsi A, Panagiotidou DD, Ioannides D. Angiolymphoid hyperplasia with eosinophilia: good response to photodynamic therapy. Clin Exp Dermatol. 2009;34(8):629-631. 18. Su HH, Shan SJ, Elston DM, Guo Y, Men JL. Congenital blasch- koid angiolymphoid hyperplasia with eosinophilia of the anogeni- tal region. Am J Dermatopathol. 2016;38(4):305-306. 19. Sebok B, Batai I, Anga B, Schneider I. Angiolymphoid hyperplasia with eosinophilia. Orv Hetil. 1998;139(12):1-15. 20. Adler BL, Krausz AE, Minuti A, Silverberg JI, Lew-Tov H. Epi- demiology and treatment of angiolymphoid hyperplasia with eosinophilia (ALHE): a systemic review. J Am Acad Dermatol. 2016;74(3):506-512. 21. Isohisa T, Masuda K, Nakai N, Takenaka H, Katoh N. Angio- lymphoid hyperplasia with eosinophilia treated successfully with imiquimod. Int J Dermatol. 2014;53(1):43-44. 22. Nobeyama Y, Ishiuji Y, Nakagawa H. Retiform hemangioendo- thelioma treated with conservative therapy: report of a case and review of the literature. Int J Dermatol. 2016;55(2): 238-243. 23. Ahrens WA, Ridenour RV, Caron BL, Caron BL, Miller DV, Folpe AL. GLUT1 expression in mesenchymal tumors: an immunohis- tochemical study of 247 soft tissue and bone neoplasms. Human Pathol. 2008;39(10):1519-1526. 24. Wang L, Liu L, Gao T. Congenital disseminated tufted angioma. J Cutan Pathol. 2013; 40(4):40-48. 25. van Vugt LJ, van der Vleuten CJM, Flucke U, Blokx WAM. The utility of GLUT1 as a diagnostic marker in cutaneous anomalies: A review of literature and recommendations for daily practice. Pathol Res Pract. 2017;213(6):591-597. 26. Johann AC, Salla JT, Gomez RS, et al. GLUT-1 in oral benign vascular lesions. Oral Dis. 2007;13:51-55. including placental trophoblasts and perineural cells as well as in a subset of mesenchymal neoplasms such as epithelioid sarcoma, leiomyosarcoma, and undifferentiated pleomorphic sarcoma [23]. GLUT1 is a useful marker for differentiation of infantile hemangiomas and other vascular neoplasms such as congeni- tal hemangioma, tufted angioma and kaposiform hemangio- endothelioma, being positive in the infantile hemangiomas and negative in the other entities [24]. A recent research highlighted that complete cases of microvenular hemangioma (n = 9) and congenital hemangiomas (n = 16) were found to be devoid of GLUT1 expression [25]. In a study, the positivity of GLUT1 was described in complete specimens with verru- cous hemangioma, which was negative in our study [3]. As far as we are aware, staining findings regarding this marker for ALHE have not been described to date. GLUT1 was not detected in various benign oral vascular lesions including; 19 hemangiomas, 9 varices, 48 pyogenic granulomas, and 17 vascular malformations, which suggests that most of benign vascular proliferations (other than infan- tile hemangiomas, such as ALHE) are negative for this marker [26]. 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