NKp46-Specific Expression on Skin-Resident CD4+ Lymphocytes in Mycosis Fungoides and Sézary Syndrome

P Schneider et al.

NKp46 Expression on Skin-Resident CD4 þ Cells in CTCLs

CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS This study was supported by the Priority Medicines Rare Diseases (E-RARE) grant 113301091 from the Netherlands Organisation for Health Research and Development (ZonMw). MFJ is supported by a grant from Vlinderkind (Dutch Butterfly Child Foundation). FL is supported by grants PI11/0125 from Spanish ISCIII and P2010BMD-2359 from Comunidad de Madrid. MDR is supported by grants SAF 2010-16976 from MICINN and S2010/BMD-2420 (CELLCAM) from Comunidad de Madrid.

Antoni Gostyn´ski1,5, Sara Llames2,5, Marta Garcı´a3,4, Marı´a J Escamez3, Lucı´a Martinez-Santamaria3,4, Miranda Nijenhuis1, Alvaro Meana2, Hendri H. Pas1, Fernando Larcher3, Anna M.G. Pasmooij1, Marcel F. Jonkman1 and Marcela Del Rio3,4 1

Department of Dermatology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; 2 Tissue Engineering Laboratory, CCST-PA and Centro de Investigaciones Biome´dicas en Red de Enfermedades Raras (CIBERER U714), Oviedo, Spain; 3Regenerative Medicine Unit, CIEMAT and Centro de Investigaciones Biome´dicas en Red de Enfermedades Raras (CIBERER U714), Madrid,

Spain and 4Department of Bioengineering, Universidad Carlos III de Madrid, Madrid, Spain E-mail: [email protected] 5 These authors contributed equally to this work. SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

REFERENCES Duheron V, Hess E, Duval M et al. (2011) Receptor activator of NF-kappaB (RANK) stimulates the proliferation of epithelial cells of the epidermo-pilosebaceous unit. Proc Natl Acad Sci USA 108:5342–7

Llames S, Garcia E, Garcia V et al. (2006) Clinical results of an autologous engineered skin. Cell Tissue Bank 7:47–53 Llames SG, Del Rio M, Larcher F et al. (2004) Human plasma as a dermal scaffold for the generation of a completely autologous bioengineered skin. Transplantation 77:350–5 Pasmooij AM, Jonkman MF (2012) First symposium on natural gene therapy of the skin. Exp Dermatol 21:236–9 Pasmooij AM, Nijenhuis M, Brander R et al. (2012) Natural gene therapy may occur in all patients with generalized non-herlitz junctional epidermolysis bullosa with COL17A1 mutations. J Invest Dermatol 132:1374–83

Garcia M, Escamez MJ, Carretero M et al. (2007) Modeling normal and pathological processes through skin tissue engineering. Mol Carcinog 46:741–5

Pasmooij AM, Pas HH, Deviaene FC et al. (2005) Multiple correcting COL17A1 mutations in patients with revertant mosaicism of epidermolysis bullosa. Am J Hum Genet 77:727–40

Gostynski A, Deviaene FC, Pasmooij AM et al. (2009) Adhesive stripping to remove epidermis in junctional epidermolysis bullosa for revertant cell therapy. Br J Dermatol 161:444–7

Van den Bergh F, Eliason SL, Burmeister BT et al. (2012) Collagen XVII (BP180) modulates keratinocyte expression of the proinflammatory chemokine, IL-8. Exp Dermatol 21:605–11

Hamill KJ, Hopkinson SB, Jonkman MF et al. (2011) Type XVII collagen regulates lamellipod stability, cell motility, and signaling to Rac1 by targeting bullous pemphigoid antigen 1e to alpha6beta4 integrin. J Biol Chem 286:26768–80

Yuen WY (2012) Chapter 11. new versatile monoclonal antibodies against type XVII collagen endodomain that distinguish type XVII collagen-related epidermolysis bullosa subytpes. In: Junctional Epidermolysis Bullosa (Thesis). Groningen, The Netherlands, pp 185–202

Jonkman MF, Pasmooij AM (2009) Revertant mosaicism–patchwork in the skin. N Engl J Med 360:1680–2 Jonkman MF, Scheffer H, Stulp R et al. (1997) Revertant mosaicism in epidermolysis bullosa caused by mitotic gene conversion. Cell 88:543–51

Yuspa SH, Kilkenny AE, Steinert PM et al. (1989) Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro. J Cell Biol 109:1207–17

NKp46-Specific Expression on Skin-Resident CD4 þ Lymphocytes in Mycosis Fungoides and Se´zary Syndrome Journal of Investigative Dermatology (2014) 134, 574–578; doi:10.1038/jid.2013.321; published online 29 August 2013

TO THE EDITOR Se´zary syndrome (SS), mycosis fungoides (MF), and transformed mycosis fungoides (TMF; Olsen et al., 2007) belong to the heterogeneous group of cutaneous T-cell lymphoma (CTCL). They are characterized as extranodal non-Hodgkin lymphoma from malignant, mature T lymphocytes that home to the skin and persist there (Girardi et al., 2004). SS and MF are the most

common CTCL types. Recent genetic and phenotypic studies suggest that MF and SS, previously considered as different stages of the same disease, arise from two distinct T-cell subsets (van Doorn et al., 2009; Campbell et al., 2010). Pathological diagnosis is often hampered by the small proportion of malignant cells and by the morphological similarity to inflammatory skin diseases (ISDs). Thus, a reliable cuta-

Abbreviations: CBCL, cutaneous B-cell lymphoma; CTCL, cutaneous T-cell lymphoma; ISD, inflammatory skin disease; LM, laser microdissection; NCR, natural cytotoxicity receptor; RT-PCR, real-time PCR; SEM, standard error of the mean; SS, Se´zary syndrome; MF, mycosis fungoides; TMF, transformed mycosis fungoides Accepted article preview online 30 July 2013; published online 29 August 2013

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neous biomarker able to distinguish malignant cells from reactive benign T cells in the infiltrate of CTCL is still needed. Most previous studies have focused on blood malignant T lymphocytes in patients with leukemic disease (Vonderheid et al., 2001; Ferenczi et al., 2002; Su et al., 2003; Bensussan et al., 2011). When skin samples were considered, a specific expression of EphA4, Twist, TOX, PDCD1, or CDK158k/ KIR3DL2 gene was found in MF or SS patients. NKp46 has been shown on blood malignant CD4 þ T lymphocytes in SS but not in MF or in TMF (Bensussan et al., 2011). NKp46 belongs to natural

P Schneider et al.

NKp46 Expression on Skin-Resident CD4 þ Cells in CTCLs

CD8

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400

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TMF

MF

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300 200 100 0

SS

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400 300 200 100 0

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Relative Nkp46 expression whole tissue sections

10 8 6 4 2 0 SS

MF

ISD

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CBCL Normal Jurkat skin

* * * * Figure 1. Transcriptional expression of NKp46 in epidermotropic T-cell lymphoma skin. (a) CD4, CD8, and CD56 immunostainings in skin biopsies from CTCLs and ISDs. SS/MF/TMF pictures show massive dermal infiltration of CD4 þ cells with epidermotropism, sparse CD8 þ cells, and no CD 56 þ cells. Bar ¼ 300 mm. Histograms represent CD4 þ , CD8 þ , and CD56 þ cell counts in each CTCL and ISD groups. Bars represent mean±SD. (b) qRT-PCR of NKp46 in CTCLs, CBCLs, ISDs, and normal skin samples. Reference gene used for normalization was transcription factor IID/TATA-binding protein (TBP). The 2  DDCTmethod was used for mRNA quantification. mRNA from a Se´zary patient expressing NKp46 gene was used as the calibrator. Middle bar represents the mean of the group. All data points represent the average of triplicate tests. NKp46 overexpression in CTCL, but not in ISDs, was also found when NKp46 expression was correlated to CD4 expression. CBCL, cutaneous B-cell lymphoma; CTCL, cutaneous T-cell lymphoma; ISD, inflammatory skin disease; MF, mycosis fungoides; qRT-PCR, quantitative real-time PCR; SS, Se´zary syndrome; TMF, transformed mycosis fungoides.

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NKp46 Expression on Skin-Resident CD4 þ Cells in CTCLs

cytotoxicity receptors (NCRs), which induce natural killer (NK)-cell activation upon binding to non-major histocompatibility complex ligands. Expressed on NK cells, regardless of their state of activation, NKp46 is directly involved in target-cell recognition and killing (Sivori et al., 1997). We analyzed here NKp46 gene expression in skin-resident CD4 þ T lymphocytes from epidermotropic CTCL, cutaneous diffuse large B-cell lymphoma, leg-type (CBCL), and ISDs. The Institutional Ethics Committee approved this study. Written informed consent was provided according to the

Declaration of Helsinki Principles. According to the French Law of August 2004–800 revised in 2011–814, each patient was informed that the part of their sample remaining after the diagnosis had been established could be used for this research and that they could oppose to this. No patients opposed to the research use of their remaining sample. Included in this study were 15 CTCLs: five MF, at stage IA with only patches and plaques; five TMF, at stage IIA (n ¼ 2) and IIB (n ¼ 3); and five SS: at stage IVa with less than 1 year evolution (male/female ¼ 10/5; median

age 63 years, range 35–81). Controls were 10 ISDs (five eczema, four psoriasis, one drug eruption; male/female ¼ 6/4; median age 57 years, range 31–85), five CBCLs (male/female ¼ 3/2; median age 74 years, range 71–83), and five normal skin samples (male/ female ¼ 3/2; median age 53 years, range 43–56). All biopsies were performed before any treatment, because steroids or methotrexate impairs NK-cell phenotype including NKp46 expression, or they suppress NK-cell proliferation (Chiossone et al., 2007; Dauguet et al., 2010). Diagnoses were reviewed NKp46

CD4

128 bp

ISD ISD

CBCL

TMF TMF

MF

SS

SNK

CBCL

ISD

TMF

MF

SS

PBMC

86 bp

CD56

CD8

58 bp

CBCL

MF

SS

SNK

CBCL

ISD

TMF

MF

SS

PBMC

58 bp

SS MF TMF

*

*

ISD CBCL 0

20

40

60

80

Relative Nkp46 expression in CD4+ laser-microdissected cells Figure 2. Transcriptional expression of NKp46 in cutaneous CD4+ laser-microdissected cells in epidermotropic T cell lymphomas. (a) PCR products of lasermicrodissected cells on agarose gel electrophoresis. CD4 and NKp46 mRNA are detected in SS, MF, and TMF, with a bright distinctive band at 128 and 86 bp, respectively, but CD8 mRNA and CD56 mRNA are not detected. In ISDs and CBCLs, only CD4 mRNA is detected. (b) qRT-PCR of NKp46 in CD4 þ lasermicrodissected cells in CTCLs, ISDs, and CBCLs. The median level of NKp46 mRNA in CD4 þ laser-microdissected cells is higher in the nine pooled CTCLs when compared with five CBCLs (Po0.05) or five ISDs (Po0.05). The reference gene used for normalization was the transcription factor IID/TATA-binding protein (TBP). The 2-DDCT method was used for mRNA quantification. mRNA from a Se´zary patient expressing the NKp46 gene was used as the calibrator. Bars represents mean±SD. CBCL, cutaneous B-cell lymphoma; CTCL, cutaneous T-cell lymphoma; ISD, inflammatory skin disease; MF, mycosis fungoides; PBMC, peripheral blood mononuclear cell; qRT-PCR, quantitative real-time PCR; SNK, NK lymphoma cell lines; SS, Se´zary syndrome; TMF, transformed mycosis fungoides.

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P Schneider et al.

NKp46 Expression on Skin-Resident CD4 þ Cells in CTCLs

according to the WHO-EORTC classification (Olsen et al., 2007). Phenotypic analyses, performed on formalin-fixed paraffin-embedded sections, showed that CD3 þ lymphocytes were prominent in dermal infiltrates of all CTCLs. CD4 þ was the major T lymphocyte subtype with median cell count/field of 95 (range 29–132) in SS, 94 (range 49–98) in MF, and 310 (range 101–352) in TMF. The CD4/CD8 ratios were over 4:1 in all CTCLs (median 53, range 7–372). CD56 cells were rare, with median cell count/field of 0.1 (range 0–0.2) in SS, 0.2 (range 0–0.2) in MF, and 0.1 in TMF (range 0–0.1; Figure 1a). When compared with ISDs, there was no significant difference, because in ISDs the CD4 þ median cell count/field was 102 (range 70–130), CD4/CD8 ratio was 1.5, and CD56 median cell count/field was 3 (range 1–5). NKp46 gene expression level was assessed in whole tissue sections using real-time (RT)-PCR. No significant difference was found between SS, MF, and TMF. However, we found a significant overexpression of NKp46 in the 15 CTCLs, with a median expression level of 3.1 (range 0.56–8.17) compared with ISDs (Po0.05), CBCLs (Po0.05), or normal skin (Po0.05) (Figure 1b). On agarose gel electrophoresis, a distinct bright band, only found in the CTCL group, confirmed the RT-PCR data. By using RT-PCR, we found that T-plastin transcripts were detected at similar levels in CTCL and ISD groups. To ascertain whether NKp46 mRNA expression level was linked to CD4 þ T lymphocytes, we laser-microdissected immunostained CD4 þ cells from frozen skin sections (Supplementary Figure S1a online). We checked the laser-microdissected cells using endpoint reverse transcription PCR (CD3, CD4 positive, CD8, CD56 negative) and agarose gel electrophoresis (Figure 2a). On performing quantitative RT-PCR (qRT-PCR), the median NKp46 gene expression level was found to be 7.3 (range 3.7–86.18) in the 15 CTCLs, without significant difference across MF–TMF–SS. In addition, NKp46 gene expression levels in CD4 þ laser-microdissected cells were significantly higher when CTCLs were compared

with CBCLs (Po0.05) and with ISDs (Po0.05)(Figure 2b). T-plastin mRNA was not expressed in CD4 þ T lymphocytes from CTCL patients, although it was reported in leukemic T cells (Su et al., 2003). Laser microdissection combined with qRT-PCR enabled us to demonstrate that NKp46 was overexpressed in CTCLs only, and that NKp46 expression was specifically found in laser-microdissected CD4 þ T lymphocytes from SS, MF, and TMF. An aberrant NKp46 expression had been reported on circulating Se´zary cells but not in circulating cells of MF or TMF (Bensussan et al., 2011). If, early in the evolution of MF and TMF, malignant cell distribution is restricted to the skin, NKp46 would only be detected in skin-resident CD4 þ T lymphocytes and not in circulating blood cells. Our results imply that NKp46 specificity is not limited to SS but extends to other epidermotropic CTCLs, even at an early stage. This aberrant NKp46 expression recently reported in TMF skin, together with other NK-specific receptors (Ortonne et al., 2012), could be linked, in the three types of CTCLs studied here, to lymphocyte reprogramming toward a NK phenotype (Meresse et al., 2006), an hypothesis supported by expression of cytotoxic proteins as granzyme B and T cell–restricted intracellular antigen in CTCL cells (Vermeer et al., 1999). However, no alteration of the KIRs and NKp46 gene locus on 19q13.4 was found using genomic microarrays or array-based comparative genomic hybridization. The transcriptional signature of this reprogramming could result from induction of receptors and adaptors normally restricted to the NK lineage (Meresse et al., 2006). Another cause for this aberrant expression of NK markers in CTCL could be deregulation of the transcription factor-E2A implicated in T- and NK-cell lineage development (Steininger et al., 2011). Laser microdissection combined with qPCR enabled us to identify NKp46 overexpression in skin-resident CD4 þ T lymphocytes from 15 CTCLs. In the same patients, RT-PCR on whole tissue sections enabled us to characterize a significant NKp46 overexpression in CTCLs and to distinguish them from

ISDs, CBCLs, or normal skin, with a sensitivity and a specificity of 89% and 100%, respectively, and with a positive predictive value of 100%. Therefore, as long as a specific antibody is not available, RT-PCR on whole tissue sections could be helpful for day-to-day diagnosis, particularly in the early stages of epidermotropic CTCL. Further study on large patient sample series will be necessary to assess the value of NKp46 as a biomarker of epidermotropic CTCL. CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS This study was supported by grants from Universite´ Paris VII, INCa, PHRC, and PS is supported by ‘‘Anne´e Recherche’’ APHP, Paris. The authors thank L Michel, S Meyer, L Franc¸oise, N Martin, G Gapihan and M El Bouchtaoui, and the Tumorothe`que of Hoˆpital Saint-Louis. A Swaine reviewed the English language.

Pierre Schneider1,2,3, Louis-Franc¸ois Plassa4, Philippe Ratajczak1,2, Christophe Leboeuf1,2, Laurence Verneuil2, Maxime Battistella1,2,5, Armand Bensussan1,6, Martine Bagot1,3,6 and Anne Janin1,2,5 1

Laboratoire de Pathologie, UMR-S 728, Universite´ Paris Diderot, Sorbonne Paris Cite´, Paris, France; 2INSERM, U728-Paris, Paris, France; 3Service de Dermatologie, Hoˆpital Saint-Louis, AP-HP, Paris, France; 4Laboratoire de Biochimie, Hoˆpital Saint-Louis, AP-HP, Paris, France; 5Laboratoire de Pathologie, Hoˆpital Saint-Louis, AP-HP, Paris, France and 6 INSERM, U976-Paris, Paris, France E-mail: [email protected] The work was conducted in Paris, UMR-S U728 Inserm/Universite´ Paris 7, Paris, France. SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

REFERENCES Bensussan A, Remtoula N, Sivori S et al. (2011) Expression and function of the natural cytotoxicity receptor NKp46 on circulating malignant CD4 þ T lymphocytes of Sezary syndrome patients. J Invest Dermatol 131:969–76 Campbell JJ, Clark RA, Watanabe R et al. (2010) Sezary syndrome and mycosis fungoides arise from distinct T-cell subsets: a biologic rationale for their distinct clinical behaviors. Blood 116:767–71 Chiossone L, Vitale C, Cottalasso F et al. (2007) Molecular analysis of the methylprednisolone-mediated inhibition of NK-cell function:

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evidence for different susceptibility of IL-2versus IL-15-activated NK cells. Blood 109:3767–75 Dauguet N, Fournie JJ, Poupot R et al. (2010) Lenalidomide down regulates the production of interferon-gamma and the expression of inhibitory cytotoxic receptors of human Natural Killer cells. Cell Immunol 264:163–70 Ferenczi K, Fuhlbrigge RC, Pinkus J et al. (2002) Increased CCR4 expression in cutaneous T cell lymphoma. J Invest Dermatol 119:1405–10 Girardi M, Heald PW, Wilson LD (2004) The pathogenesis of mycosis fungoides. N Engl J Med 350:1978–88 Meresse B, Curran SA, Ciszewski C et al. (2006) Reprogramming of CTLs into natural killerlike cells in celiac disease. J Exp Med 203: 1343–55

Olsen E, Vonderheid E, Pimpinelli N et al. (2007) Revisions to the staging and classification of mycosis fungoides and Sezary syndrome: a proposal of the International Society for Cutaneous Lymphomas (ISCL) and the cutaneous lymphoma task force of the European Organization of Research and Treatment of Cancer (EORTC). Blood 110:1713–22 Ortonne N, Le Gouvello S, Tabak R et al. (2012) CD158k/KIR3DL2 and NKp46 are frequently expressed in transformed mycosis fungoides. Exp Dermatol 21:461–3 Sivori S, Vitale M, Morelli L et al. (1997) p46, a novel natural killer cell-specific surface molecule that mediates cell activation. J Exp Med 186:1129–36 Steininger A, Mobs M, Ullmann R et al. (2011) Genomic loss of the putative tumor

suppressor gene E2A in human lymphoma. J Exp Med 208:1585–93 Su MW, Dorocicz I, Dragowska WH et al. (2003) Aberrant expression of T-plastin in Sezary cells. Cancer Res 63:7122–7 van Doorn R, van Kester MS, Dijkman R et al. (2009) Oncogenomic analysis of mycosis fungoides reveals major differences with Sezary syndrome. Blood 113:127–36 Vermeer MH, Geelen FA, Kummer JA et al. (1999) Expression of cytotoxic proteins by neoplastic T cells in mycosis fungoides increases with progression from plaque stage to tumor stage disease. Am J Pathol 154:1203–10 Vonderheid EC, Bigler RD, Kotecha A et al. (2001) Variable CD7 expression on T cells in the leukemic phase of cutaneous T cell lymphoma (Sezary syndrome). J Invest Dermatol 117:654–62

Generalized Pustular Psoriasis Triggered by Amoxicillin in Monozygotic Twins with Compound Heterozygous IL36RN Mutations: Comment on the Article by Navarini et al. Journal of Investigative Dermatology (2014) 134, 578–579; doi:10.1038/jid.2013.354; published online 19 September 2013

TO THE EDITOR We read with great interest the recent report regarding IL36RN mutations in acute generalized exanthematous pustulosis (AGEP) by Navarini et al. (2013). They analyzed IL36RN mutations in 96 cases with AGEP and found only one homozygous mutation (p.Leu27Pro) and three heterozygous mutations. p.Leu27Pro is a founder mutation causing generalized pustular psoriasis (GPP) homozygously in African populations, and the AGEP patient described by Navarini et al. (2013) with the homozygous mutation p.Leu27Pro is an African woman (Marrakchi et al., 2011; Navarini et al., 2013). She had a previous history of drug-induced type-unknown skin reaction triggered by uncertain antibiotics. She showed pustules covering 85% of her body surface, including the mouth, and a 39.4 1C fever triggered by amoxicillin.

A positive patch test to amoxicillin was noted. Treatment of the patient was not described. No recurrence was observed during the 18-month follow-up. In a recent report, we analyzed 11 patients with GPP not accompanied by psoriasis vulgaris (PV) (GPP-alone; Sugiura et al., 2013). Among the 11 GPP-alone patients, there was a monozygotic twin with the compound heterozygous mutations p.Arg10X and p.Arg10ArgfsX1 in IL36RN whose pustules and high fever had primarily been triggered by amoxicillin. The patients were 6-year-old Japanese male identical twins. At the age of 2 years, they had erythema with pustules on the whole body and fever over 38 1C after amoxicillin intake. In one of the twins, blood examination revealed a white blood cell count of 24,300 ml  1 and a C-reactive protein concentration of 1.17 mg dl  1. Bacterial culture for

Abbreviations: AGEP, acute generalized exanthematous pustulosis; DITRA, deficiency of IL-36 receptor antagonist; GPP, generalized pustular psoriasis; IL36RN, IL-36 receptor antagonist; PV, psoriasis vulgaris Accepted article preview online 20 August 2013; published online 19 September 2013

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the pustules was negative. A skin biopsy from a pustular eruption on the trunk revealed a spongiform pustule of Kogoj in the epidermis (Figure 1c). The results of these examinations were similar to those of the other twin. Patch tests were conducted on one of the twins, and it was found to be positive for amoxicillin. Skin eruptions including pustules of the twins were improved with a dosage of 6 mg kg  1 oral cyclosporine. After the first episode, each twin has episodic systemic pustules unrelated to amoxicillin intake (Figure 1a and b). Neither twin has ever showed typical scaly erythema corresponding to PV. The two patients, monozygotic twins, had the compound heterozygous mutations p.Arg10X and p.Arg10ArgfsX1 in IL36RN (Sugiura et al., 2013). Thus, they were diagnosed as having GPP-alone with IL36RN mutations that had once been triggered by amoxicillin. Amoxicillin- or penicillin-related druginduced GPP has been reported for a long time, although the pathomechanisms of how penicillin induces GPP have never been addressed (Ryan and Baker,

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