Giant Angiofibromas in Tuberous Sclerosis Complex

Giant angioﬁbromas in tuberous sclerosis complex: A possible role for localized lymphedema in their pathogenesis

Denisa Kacerovska, MD, PhD,a,b Katrin Kerl, MD,c Michal Michal, MD,a,b Hana Filipova, MSc,d Radek Vrtel, PhD,d Tomas Vanecek, PhD,a,b Hana Zelenakova, MD,e Jaroslav Kraus, MD,f Roman Kodet, MD, PhD,g and Dmitry V. Kazakov, MD, PhDa,b Pilsen,Olomouc,Tloskov,Benesov,andPrague,CzechRepublic;andZurich,Switzerland

Background: Giant angioﬁbromas in patients with tuberous sclerosis complex (TSC) are rare.

Objective: We sought to report two patients who had TSC with unusually large and disﬁguring facial angioﬁbromas and to identify underlying histopathologic changes that may possibly explain the clinical features.

Methods: We performed a clinicopathologic, immunohistochemical, and molecular biologic study using 42 lesional specimens and peripheral blood from one of the two patients. The immunohistochemical investigations were mainly focused on the vascular moiety of the lesions. TSC1 and TSC2 alterations were studied using multiplex ligation-dependent probe amplification for large deletion/duplication mutations, whereas screening for small mutations was performed using polymerase chain reaction amplification of individual coding exons and exon-intron junctions of both genes followed by an analysis on denaturation gradient gel electrophoresis.

Results: Histopathologic examination revealed, in addition to findings typical of angiofibroma, several unusual features including multinucleated giant cells containing multiple intracytoplasmic vacuoles, Touton-like cells, emperipolesis, pagetoid dyskeratosis, vacuolar alteration at the dermoepidermal junction, Civatte bodies, and melanophages in the subjacent dermis. Numerous dilated lymphatic vessels were detected indicating localized lymphostasis, probably caused by secondary lymphedema. The lymphatic nature of the vessels was confirmed by immunohistochemical study. Genetic testing for TSC1 and TSC2 gene mutations revealed a substitution on position c.2251C[T resulting in a nonsense mutation R751X in fragment 20.2.

Limitations: Histopathologic specimens and peripheral blood were available from only one patient.

Conclusion: Localized lymphedema may contribute to the formation of large disﬁguring angioﬁbromas in patients with TSC. ( J Am Acad Dermatol 2012;67:1319-26.)

Key words: adenoma sebaceum; elephantiasis; ﬁbrous papule; giant angioﬁbroma; localized lymphedema; tuberous sclerosis complex.

From Sikl’s Department of Pathology, Charles University in Prague, Presented in part at the XXX Symposium of the International Faculty of Medicine in Pilsena; Bioptical Laboratory, Pilsenb; Society of Dermatopathology, Santiago de Chile, October Department of Dermatology, University Hospital, Zurichc; 28-31, 2009. Department of Clinical Genetics and Fetal Medicine, University Reprint requests: Dmitry V. Kazakov, MD, PhD, Sikl’s Department of Hospital, Olomoucd; Diagnostic Institution of Social Care, Pathology, Charles University Medical Faculty Hospital, Alej Tloskove; Department of Otorhinolaryngology, Hospital Bene- Svobody 80, 304 60 Pilsen, Czech Republic. E-mail: kazakov@ sovf; and Institution of Pathology and Molecular Medicine, medima.cz. Second Medical Faculty and Teacher’s Hospital, Prague.g Published online May 2, 2012. Funding sources: None. 0190-9622/$36.00 Conflicts of interest: None declared. Ó 2012 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2012.03.021

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Tuberous sclerosis complex (TSC) is an autosomal evident. The patient had a history of a subependymal dominant neurocutaneous syndrome characterized giant cell astrocytoma, obstructing the foramen of by various hamartomas and tumors involving mainly Monro and resulting in raised intracranial pressure, the skin, brain, retina, kidney, heart, and lung.1-10 It is which was surgically removed at the age of 16 years. caused by alterations in either of two tumor sup- Because the facial lesions obstructed the nostrils and pressor genes: TSC1 located on chromosome 9q34 interfered with normal breathing they were removed and encoding the protein hamartin (130 kd)11; by radiofrequency ablation. and TSC2 located on chro- The patient died 14 mosome 16p13.3 and encod- months after the cutaneous CAPSULE SUMMARY ing the protein tuberin (180 surgery. A voluminous tumor kd).10,12-15 Mutations in TSC2 of the retroperitoneum caus- d Giant angiofibromas in patients with appear to generate a more ing bowel compression was tuberous sclerosis complex are severe phenotype than alter- detected by computed to- extremely rare. ations in TSC1.8,10,16-19 mography. The nature of the Cutaneous lesions in pa- d Localized lymphedema was found on tumor remained unknown as tients with TSC encompass histopathologic study of multiple permission for autopsy was facial angiofibromas, hypo- lesional specimens from one patient. denied by her family. melanotic macules, shagreen d Localized lymphostasis may contribute patches, forehead fibrous to the pathogenesis and development of Patient 2 plaques, and ungual or peri- giant angiofibromas in tuberous sclerosis This female patient pre- ungual fibromas (so-called complex. sented at the age of 6 years ‘‘Koenen tumors’’).10 Facial and was followed up by the angiofibromas show a predi- Department of Dermatology, lection for the centrofacial area. They appear usually University Hospital, Zurich, for almost 60 years (from between 2 and 6 years of age, with increased growth 1933 to 1992). Her facial angiofibromas steadily at puberty, and mostly remain 4 to 5 mm in size.20,21 increased in size causing obstruction of the left There are a few reports describing unusual giant nostril (Fig 2). Several attempts at carbon-dioxide angiofibromas in both patients with TSC and in laser ablation failed (1985 to 1991). In addition, she patients without an accompanying neurocutaneous had periungual fibromas on the toes, hypopig- syndrome.22-26 We report two patients with giant mented macules on the trunk, and confetti-like disfiguring angiofibromas associated with TSC. In hypopigmentation on both arms. She had severe one case, histopathologic study of many lesional mental retardation. She was unrelated to patient 1. specimens revealed unusual features consistent with lymphostasis, suggesting that this pathogenetic METHODS mechanism might contribute to their clinical A total of 42 lesional specimens were available for appearance. study from patient 1. No histopathologic specimens were available from the second patient. The material CASE REPORT was routinely fixed in 4% formaldehyde and embed- Patient 1 ded in paraffin. The paraffin blocks were cut into The patient was a 29-year-old woman with epi- 5-m-thick sections and stained with hematoxylin lepsy (generalized tonic-clonic seizures), advanced and eosin. In addition, periodic acideSchiff, Giemsa, mental retardation, and conspicuous cutaneous le- Verhoeff, Prussian blue (iron), Alcian blue pH 2.5, sions on the face, which appeared during childhood mucicarmine, and chloracetate esterase staining was and rapidly increased in number and size around 15 performed. years of age. There were multiple large nodules, pale Immunohistochemical studies were performed to pink, with a smooth glistening surface, 0.5 to 2.0 on 5 lesions using the antibody against cytokeratins cm in diameter, on the chin, nasolabial folds, and (clone AE1/AE3, dilution 1:1000, Neo Markers, cheeks (Fig 1, A). Similar but smaller lesions were Westinghouse), CAM 5.2 (clone CAM 5.2, dilution observed on her forehead and earlobes (Fig 1, B and 1:200, Becton Dickinson, Erembogedem), K903 C ). On the right ear, the papules were superimposed (clone 34bE12, dilution 1:300, Dako, Carpinteria, on a background of otophyma (Fig 1, B). There were CA), CK 7 (clone OV-TL12/30, dilution 1:200, Dako, pink periungual nodules on the fingers and toes (Fig Glostrup), S-100 protein (polyclonal, dilution 1:10000, 1, D). Inconspicuous but multiple hypopigmented Dako, Glostrup), leukocyte common antigen (clone macules were found on the trunk and the extremi- 2B111PD7/26, dilution 1:500, Dako, Glostrup), CD1a ties. Prominent gingival hyperplasia was also (clone MTB1, Novocastra, Newcastle), CD68 (clone JAM ACAD DERMATOL Kacerovska et al 1321 VOLUME 67, NUMBER 6

KP1, dilution 1:200, Neo Markers, Westinghouse), CD (Fig 6, A). Some specimens disclosed foci of vacuolar 117 (polyclonal, dilution 1:150, Dako, Glostrup), CD alteration at the dermoepidermal junction, Civatte 34 (clone QBEnd/10, dilution 1:800, Neo Markers, bodies, and melanophages in the subjacent dermis Westinghouse), factor XIIIa (polyclonal, dilution (Fig 6, B). One lesion manifested lipomatous meta- 1:300, Calbiochem, La Jolla, CA), vimentin (clone V9, plasia of the stroma. In several blocks there were dilution 1:1000, Neo Markers, Westinghouse), D2-40 areas of fibrosis associated with nodules of plasma (clone D2-40, dilution 1:200, Dako, Glostrup), and cells. alpha-smooth muscle actin (clone 1A4, Dako, dilution 1:1000, Glostrup). Immunohistochemical findings For molecular biologic studies, genomic DNA was Staining for D2-40 and alpha-smooth muscle actin isolated from the peripheral blood of patient 1 (after confirmed that many dilated vessels represented obtaining written informed contest) according to the lymphatics (Fig 4, B and C ). Collapsed (normal) method of Miller et al27 and underwent routine lymphatic vessels labeled with D2-40 were also seen. screening for causative mutations established in There was strong expression of CD68 and vimen- our DNA laboratory.28 Examination for large dele- tin by the multinucleated giant cells, which were tion/duplication mutations of the TSC2 gene using negative for factor XIIIa, cytokeratins (AE1/AE3, multiplex ligation-dependent probe amplification CAM 5.2, K903, CK7), S100-protein, CD34, CD117, was performed.29 The commercially available Salsa leukocyte common antigen, and CD1a. multiplex ligation-dependent probe amplification kit P046 TSC2 (MRC-Holland) was used to this end. Molecular testing for deletions at the TSC2 and Screening for small mutations was performed using PKD1 loci polymerase chain reaction amplification of individ- Examination for large deletions or duplication ual coding exons and exon-intron junctions of the mutations in the TSC2 gene using multiplex ligation- TSC1 and TSC2 genes followed by an analysis on dependent probe amplification showed a normal denaturation gradient gel electrophoresis and, in pattern, thus excluding TSC2/PKD1 contiguous gene case of denaturation gradient gel electrophoresis syndrome. The denaturation gradient gel electro- suspiciously/potentially positive results, by bidirec- phoresis screening for small mutations in TSC1 tional sequencing. disclosed no alterations, whereas screening of TSC2 gene yielded only an aberration in fragment 20.2. RESULTS Subsequent sequencing of fragment 20.2 revealed a Histopathologic features substitution on position c.2251C[T resulting in a All 42 lesional specimens (obtained from patient nonsense mutation R751X. 1) grossly appeared as nodules, some of which were pedunculated with a smooth glistening surface DISCUSSION (Fig 3, A). Giant angiofibromas, occurring either sporadi- The histopathologic features in most specimens cally or in the setting of TSC, are rarely reported. were typical of angiofibroma with spindled or stel- The reason for their large size is unknown and has not late cells associated with dilated vessels in a fibro- been studied specifically. We suggest that localized myxoid stroma (Fig 3, B and C ). Many of the dilated lymphedema may contribute to the formation of vessels possessed a thin wall, scant basement mem- these giant lesions. Healthy lymphatic vessels, being brane, and sparsely distributed endothelial cells, empty and collapsed, are not readily visualized in suggesting lymphatic vessels (Fig 4, A). Adnexae histopathologic specimens. The numerous dilated were absent or, in two lesions, were present only at lymphatic vessels we detected microscopically in the periphery of the lesions. Concentric perifollicular many of the giant angiofibroma specimens can fibrosis was not prominent. In many specimens, therefore be regarded as a sign of lymphostasis, there were multinucleated giant cells containing probably caused by secondary lymphedema. The multiple intracytoplasmic vacuoles varying greatly pendulous nature of the specimens and abnormal in size (Fig 5, A). Some of these giant cells with tissue (fibrosis and areas with lipomatous metaplasia) vacuoles showed a wreathlike arrangement of nuclei are also indicative of localized lymphedema. Thus, resembling that seen in Touton cells (Fig 5, B) and an giant angiofibromas appear to attain their great size as intracytoplasmic mucinous substance was present in a result of expansion of the mesenchymal tissues. some (Fig 5, C ). Also, occasional cells showed Increased lymphatics have been noted in earlier emperipolesis: lymphocytes and erythrocytes were histopathologic descriptions of TSC angiofi- noted within the cytoplasm (Fig 5, D). Pagetoid bromas20,30 and in immunohistochemical studies dyskeratosis was identified in 14 of the 42 lesions of angiofibromas showing vessels negative for 1322 Kacerovska et al JAM ACAD DERMATOL DECEMBER 2012

Fig 1. Patient 1. Tuberous sclerosis complex. A, Multiple large confluent nodules involving chin, nasolabial folds, and cheeks are evident. B, On right ear, papules occur on background of otophyma. C, Smaller lesions are observed on forehead. D, Pink periungual nodule representing so-called Koenen tumor, or ungual fibroma is seen on finger.

The nasal lesions in our second patient clinically resembled rhinophyma, in which features of localized lymphedema, including lymphatic vessel dila- tation, stromal fibrosis with lipocytes, and stromal mucin deposits, are well recognized.33 Identical changes are seen in other phymas (swelling, masses, or bulbs) considered the end stage of rosacea and rarely involving parts other than the nose, namely the eyelids (blepharophyma), ears (otophyma), cheek (metophyma), or chin (gnatophyma).34 Multifocal presentation with involvement of 2 or 3 of the above sites has also been reported.35,36 In rosacea, lymphedema is secondary to chronic inflam- Fig 2. Patient 2. Tuberous sclerosis complex. Large con- mation, whereas in patients with TSC, lymphatic fluent disfiguring nodules located in nasolabial area. drainage is probably impaired by multiple closely apposed and confluent angiofibromas. Of note, CD34.31 One could perhaps argue that lymphan- otophyma was clinically evident in patient 1 (Fig 1, B). giogenesis may also be a potential contributing Localized lymphedema appears to be an under- factor. Angiogenesis has been used to explain recognized histopathologic phenomenon. Lu et al37 the increased blood vessels in angiofibromas32 identified histopathologic features of localized and lymphangiogenesis has been found to be lymphedema in a variety of conditions, with variable important in other TSC-related tumors, particularly factors including trauma, surgery, chronic inflamma- lymphangioleiomyomatosis. tory disease, and bacterial cellulitis. Paul and JAM ACAD DERMATOL Kacerovska et al 1323 VOLUME 67, NUMBER 6

Fig 3. Tuberous sclerosis complex, giant angioﬁbromas. A, Appearance of facial nodules coming from patient 1, some of them pedunculated with smooth glistening surface. B and C, Whole-mount sections revealing typical Fig 4. Tuberous sclerosis complex, giant angioﬁbromas. picture of angiofibroma, namely fibromyxoid stroma with Close-up view of vascular component. A, Many dilated spindled or stellate cells associated with dilated vessels. vessels possess thin wall, scant basement membrane, and (Hematoxylin-eosin stain, scan.) sparsely distributed endothelial cells, strongly suggesting that these are lymphatic vessels. B, Immunostaining con-

38 firms that many dilated vessels represent lymphatics. Carlson found that lymphangiectases are common C, Compared with other stained areas antibody highlight- underlying warts and in normal peritumoral skin. ing collapsed (normal) lymphatics. (A, Hematoxylin-eosin The same group has recently posited that localized stain; original magnification: 3200. B and C, D2-40 stain; lymphedema is an essential pathogenic factor in original magnifications: 3100.) verruciform xanthomas.39 The multinucleated floret-like giant cells with lymphedema and can also be a feature of intracytoplasmic vacuoles are unusual. We could elephantiasis.40,41 not find any description of such cells in the litera- In a third of lesions, pagetoid dyskeratosis was ture concerning angiofibromas but were able to find noted. This feature is often seen in histopathologic a single picture illustrating similar elements without specimens obtained from the intertriginous areas, a specific comment.21 We also retrieved and re- the perianal zone, vulva, and penis and is considered viewed all angiofibromas associated with TSC or to represent a reactive process in which a small part solitary angiofibromas stored in our files and did not of the normal population of keratinocytes is induced find similar cells in any of the specimens. to proliferate, possibly by friction.42-44 Rubbing of Noteworthy, multinucleated floret-like giant cells the lesions because of their large size is a likely have been described in lesions of massive localized explanation in our case. 1324 Kacerovska et al JAM ACAD DERMATOL DECEMBER 2012

Fig 5. Tuberous sclerosis complex, giant angioﬁbromas. A, Multinucleated giant cells containing multiple variably sized intracytoplasmic vacuoles. B and C, Note wreathlike arrangement of nuclei resembling that seen in Touton cells (B) and intracytoplasmic mucinous substance (C). D, Emperipolesis in giant cells: lymphocyte can be recognized in cytoplasm of giant cell. (A, B, and D, Hematoxylin-eosin stain; original magnifications: 3400. C, Alcian blue pH 2.5 stain; original magnification: 3400.)

It is known that patients with TSC2 alterations manifest a more severe phenotype than patients with TSC1. In our first patient, peripheral blood was available for molecular genetic studies and a nonsense mutation R751X was identified in the TSC2 gene. This mutation has been reported in several patients.17,18,45-48 In conclusion, we describe two patients with TSC who presented with unusually large and disfiguring facial angiofibromas. We suggest that localized lymphedema contributes to the appearance of these lesions. This factor should perhaps be considered when planning treatment. The main limitation of the study is that lesional specimens, albeit numerous, were available from only one patient; therefore, further study of these rare cases is needed to substantiate our findings.

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