Nipple Angiofibromas with Loss of TSC2 Are Associated with Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous syndrome that leads to hamartoma formation in multiple organs, including the skin (Curatolo et al., 2008Curatolo P. Bombardieri R. Jozwiak S. Tuberous sclerosis.Lancet. 2008; 372: 657-668Abstract Full Text Full Text PDF PubMed Scopus (825) Google Scholar). Cutaneous hamartoma formation occurs secondary to loss of function of either the TSC1 or TSC2 gene in fibroblast-like cells and subsequent dysregulation of the mechanistic target of rapamycin complex 1 (Li et al., 2008Li S. Takeuchi F. Wang J.A. Fan Q. Komurasaki T. Billings E.M. et al.Mesenchymal-epithelial interactions involving epiregulin in tuberous sclerosis complex hamartomas.Proc Natl Acad Sci USA. 2008; 105: 3539-3544Crossref PubMed Scopus (44) Google Scholar, Tyburczy et al., 2014Tyburczy M.E. Wang J.A. Li S. Thangapazham R. Chekaluk Y. Moss J. et al.Sun exposure causes somatic second-hit mutations and angiofibroma development in tuberous sclerosis complex.Hum Mol Gen. 2014; 23: 2023-2029Crossref PubMed Scopus (69) Google Scholar). Angiofibromas are among the most well-recognized TSC-related skin hamartomas and consist of multiple pink papules on the central face (Little, 1909Little E.G. Adenoma sebaceum in a girl aged 20.Proc R Soc Med. 1909; 2: 162-163Google Scholar). Here we describe angiofibromas of the nipple-areolar complex. Between October 2012 and January 2015, 53 patients with TSC (50 women, 3 men) participated in studies of lymphangioleiomyomatosis, a TSC-associated lung disease with female predominance, at the National Institutes of Health in Bethesda, Maryland. Written informed consent was obtained according to IRB-approved protocols 00-H-0051, 95-H-0186, and/or 82-H-0032. Eleven patients with TSC (10 women, 1 man; median age 41 years [range 21–71 years]) with papules on the nipple and/or areola were identified (Table 1), eight of whom had germline mutations in TSC2. Clinical examination revealed 1 to 25, 1–3 mm, pink to red dome-shaped papules on the nipple and/or areola, affecting one or both breasts (Figure 1a). Per patient recollection, the median age of onset was 33 years (n = 7 women; range 16–38 years). One patient reported painless bleeding from her nipple papules during breastfeeding, whereas the rest remained asymptomatic.Table 1Clinical, microscopic, and mutational analysis of nipple-areolar complex angiofibromasPatientSexTSC cutaneous major featuresHistory of breastfeedingLesions on nipple (biopsied)Lesions on areola (biopsied)HistopathologyTissue for mutational analysisTSC2 mutation (af)TSC2 protein changeP25FAF, HMNo113 (1)Angiofibroma–––P27FAF, FCPYes3 (1)3Angiofibroma, associated hidrocystomaCultured fibroblastsc.4375C>T (.99)p.(Arg1459*)P28FAF, SP, FCPYes3 (1)0Angiofibroma, prominent vascular componentCultured fibroblastsc.4868_4875delCCCTGATG (.54)Out-of-frame deletionP34FAF, HM, UFNo10––––P35FAF, HM, UF, FCPNo10––––P36FAF, HM, UFYes5 (1)5 (1)AngiofibromaCultured fibroblastsc.2098-1G > A (.5)c.4858C>T (.05)Splicep.(His1620Tyr)P37FAF, HM, UFYes11 (2)14 (1)AngiofibromaWhole tissue – T1c.2098-1G > A (.5) c.138+1G > A (.05)SplicespliceWhole tissue – T2c.2098-1G > A (.5) c.3574C > T (.03)splicep.(Gln1192*)Whole tissue – T3c.2098-1G > A (.5)SpliceP43FAF, UFNo3 (1)0AngiofibromaCultured fibroblastsc.3999C > A (.5)p.(Tyr1333*)P46FAF, HM, UFNo13––––P47FAF, HM, UFYes1 (1)0Angiofibroma–––P48MAF, HM, UF–110––––Abbreviations: AF, angiofibromas; af, allelic frequency; F, female; FCP, fibrous cephalic plaque; HM, hypomelanotic macules; L, left; M, male; R, right; SP, shagreen patch; TSC, tuberous sclerosis complex; UF, ungual fibromas.Bold denotes germline mutation when documented in blood or control skin. Open table in a new tab Abbreviations: AF, angiofibromas; af, allelic frequency; F, female; FCP, fibrous cephalic plaque; HM, hypomelanotic macules; L, left; M, male; R, right; SP, shagreen patch; TSC, tuberous sclerosis complex; UF, ungual fibromas. Bold denotes germline mutation when documented in blood or control skin. Histopathological examination of 10 biopsied nipple or areolar papules from seven patients revealed increased number of dilated capillaries in the papillary dermis and increased number of stellate and spindle-shaped fibroblasts interspersed in the dermal collagen, consistent with the diagnosis of an angiofibroma (Figure 1b). Increased numbers of factor XIIIa positive spindle to stellate shaped cells were observed in the dermis of the nipple-areolar complex papules (Figure 1c), as characteristically observed in TSC-related facial angiofibromas (Li et al., 2005Li S. Takeuchi F. Wang J.A. Fuller C. Pacheco-Rodriguez G. Moss J. et al.MCP-1 overexpressed in tuberous sclerosis lesions acts as a paracrine factor for tumor development.J Exp Med. 2005; 202: 617-624Crossref PubMed Scopus (39) Google Scholar). To detect activation of mechanistic target of rapamycin complex 1 in nipple-areolar complex papules, immunohistochemical staining against phosphorylated ribosomal protein S6 (Ser235/236) was performed on 10 samples, revealing avid positive dermal fibroblast-like cells as seen in other TSC-related skin hamartomas (Figure 1d) (Li et al., 2008Li S. Takeuchi F. Wang J.A. Fan Q. Komurasaki T. Billings E.M. et al.Mesenchymal-epithelial interactions involving epiregulin in tuberous sclerosis complex hamartomas.Proc Natl Acad Sci USA. 2008; 105: 3539-3544Crossref PubMed Scopus (44) Google Scholar). Western blot analysis of fibroblast-like cells grown from two of four nipple angiofibromas demonstrated loss or near complete loss of TSC2. All four showed increased phosphorylation of ribosomal protein S6 under serum starvation, compared with dermal fibroblasts from normal-appearing skin (Supplementary Figure S1 online), consistent with prior molecular analysis of facial angiofibromas (Tyburczy et al., 2014Tyburczy M.E. Wang J.A. Li S. Thangapazham R. Chekaluk Y. Moss J. et al.Sun exposure causes somatic second-hit mutations and angiofibroma development in tuberous sclerosis complex.Hum Mol Gen. 2014; 23: 2023-2029Crossref PubMed Scopus (69) Google Scholar). Mutational analysis of DNA extracted from papule-derived fibroblast-like cells or whole tissue biopsies was performed as described previously using targeted next generation sequencing of TSC1 and TSC2, with validation by Sanger sequencing or SNaPshot analysis (Tyburczy et al., 2014Tyburczy M.E. Wang J.A. Li S. Thangapazham R. Chekaluk Y. Moss J. et al.Sun exposure causes somatic second-hit mutations and angiofibroma development in tuberous sclerosis complex.Hum Mol Gen. 2014; 23: 2023-2029Crossref PubMed Scopus (69) Google Scholar). Seven angiofibromas from five patients were analyzed and biallelic mutations in TSC2 were identified in four samples (Table 1; Supplementary Figure S2 online). A nonsense mutation in TSC2 (p.Arg1459*) was present in one nipple angiofibroma fibroblast culture at 99% allele frequency, consistent with both a germline p.Arg1459* mutation and second hit loss of the wild-type allele (Table 1, P27; Supplementary Figure S2a). Three samples (two whole tissue, one cultured cells; Table 1, P36 and P37) had second hit point mutations in TSC2 that were seen at low allele frequency (3% to 5%), albeit undetectable in patient controls, consistent with the low prevalence of neoplastic fibroblast cells in these biopsies. All three of the low-frequency second hit mutations seen in these samples were validated by an independent method (Supplementary Figure S2e–j). The remaining samples in which neither second hit point mutations nor loss of heterozygosity was seen in TSC2 may be due to the occurrence of loss of heterozygosity, which is very difficult to detect in a sample in which the tumor cells are at low frequency. This microscopic and molecular evidence of mechanistic target of rapamycin complex 1 activation with inactivating mutations in TSC2 provides strong support for the concept that these lesions arise through two-hit inactivation of TSC1/TSC2, as we have shown previously for facial angiofibromas (Tyburczy et al., 2014Tyburczy M.E. Wang J.A. Li S. Thangapazham R. Chekaluk Y. Moss J. et al.Sun exposure causes somatic second-hit mutations and angiofibroma development in tuberous sclerosis complex.Hum Mol Gen. 2014; 23: 2023-2029Crossref PubMed Scopus (69) Google Scholar). In our study, the frequency of nipple-areolar complex angiofibromas in adult women was 20%. The median age of onset was 33 years (range 16–38 years), nearly three decades after the onset of facial angiofibromas in the same female patients (median age 4 years; range 3–13 years). This indicates that nipple-areolar complex angiofibromas are an adult manifestation of TSC, occurring later than any cutaneous feature currently described. In addition, the presence of nipple and areolar angiofibromas in a man from our cohort suggests that unlike lymphangioleiomyomatosis, these hamartomas are not limited to women. The differential diagnosis of nipple-areolar complex papules in a patient with TSC should include several benign tumors, such as nipple adenomas/florid papillomatosis, leiomyomas, fibromas, and folliculosebaceous cystic hamartomas (Badr and Lakshmiah, 2009Badr A. Lakshmiah G.R. Folliculosebaceous cystic hamartoma of the nipple: a case report.J Cutan Pathol. 2009; 36: 597-600Crossref PubMed Scopus (12) Google Scholar, Spyropoulou et al., 2014Spyropoulou G.A. Pavlidis L. Trakatelli M. Athanasiou E. Pazarli E. Sotiriadis D. et al.Rare benign tumours of the nipple.J Eur Acad Dermatol Venereol. 2014; 29: 7-13Crossref Scopus (24) Google Scholar). For patients without known TSC, consideration should also be given to nipple papules and nodules related to other tumor syndromes, including myxomas and neurofibromas (Bongiorno et al., 2010Bongiorno M.R. Doukaki S. Arico M. Neurofibromatosis of the nipple-areolar area: a case series.J Med Case Reps. 2010; 4: 22Crossref PubMed Scopus (12) Google Scholar, Carney et al., 1986Carney J.A. Headington J.T. Su W.P. Cutaneous myxomas. A major component of the complex of myxomas, spotty pigmentation, and endocrine overactivity.Arch Dermatol. 1986; 122: 790-798Crossref PubMed Google Scholar). Biopsy and pathologic examination are valuable to confirm the diagnosis of angiofibroma, both for diagnostic purposes and for reassurance in regard to natural history. Treatment may not be necessary; unlike facial angiofibromas, angiofibromas of the nipple and areola do not bleed spontaneously. Nipple-areolar complex angiofibromas were less common than facial angiofibromas in our cohort, affecting 20% versus 100% of female patients, respectively. Less frequent occurrence of angiofibromas on the nipple or areola may be explained by protection from sun exposure as our group previously demonstrated that signature second hit UV mutations promote facial angiofibroma formation (Tyburczy et al., 2014Tyburczy M.E. Wang J.A. Li S. Thangapazham R. Chekaluk Y. Moss J. et al.Sun exposure causes somatic second-hit mutations and angiofibroma development in tuberous sclerosis complex.Hum Mol Gen. 2014; 23: 2023-2029Crossref PubMed Scopus (69) Google Scholar). We propose that repetitive trauma from breastfeeding may stimulate hamartoma development in female patients, just as shoe wear compression may stimulate ungual fibroma pathogenesis (Aldrich et al., 2010Aldrich C.S. Hong C.H. Groves L. Olsen C. Moss J. Darling T.N. Acral lesions in tuberous sclerosis complex: insights into pathogenesis.J Am Acad Dermatol. 2010; 63: 244-251Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). One patient reported onset of a nipple papule in the weeks after initiation of breastfeeding and two additional patients reported onset of papules after discontinuing breastfeeding. Thus, both somatic mutations and trauma may drive TSC-related skin tumor pathogenesis. In summary, we report nipple-areolar complex angiofibromas, a cutaneous manifestation of TSC. Similar to facial angiofibromas, they occur after second hit loss of the wild-type allele of TSC2, but manifest much later in life. They have a benign clinical course, with symptomatic manifestations only associated with local trauma. When verified by histologic analysis, they may provide additional information in favor of TSC diagnosis. This work was supported by NIH R01AR062080 and 1P01CA120964, the Sulzberger Dermatological Research and Education Endowment, the Doris Duke Charitable Foundation #2014088, European Commission 602391-2, and the Intramural Research Program of the National Institutes of Health, National Heart, Lung, and Blood Institute. Download .pdf (1.0 MB) Help with pdf files Supplementary Figures S1 and S2 and Supplementary Reference