C L I N I C A L A N D L A B O R A T O R Y I N V E S TI G A T I O N S
BJD
British Journal of Dermatology
Histopathology of drug rash with eosinophilia and systemic symptoms syndrome: a morphological and phenotypical study N. Ortonne,1,2,3 L. Valeyrie-Allanore,3,4 S. Bastuji-Garin,3,5 J. Wechsler,1 S. de Feraudy,1 T.-A. Duong,3,4 M.-H. Delfau-Larue,2,3,6 O. Chosidow,3,4 P. Wolkenstein3,4 and J.-C. Roujeau3 Assistance Publique – H^opitaux de Paris (AP-HP), H^opital Henri-Mondor, 1Departement de Pathologie, 4Service de Dermatologie, 5Service de Sante-Publique, 6 Service d’Immunologie Biologique, 94010 Creteil Cedex, France 2 INSERM U955 equipe 9, H^opital Henri-Mondor, 94010 Creteil Cedex, France 3 Universite Paris Est Creteil (UPEC), Faculte de Medecine, LIC EA4393, 94010 Creteil Cedex, France
Summary Correspondence Nicolas Ortonne. E-mail: [email protected]
Accepted for publication 25 January 2015
Funding sources None.
Conflicts of interest None declared. N.O., L.A., O.C. and P.W. contributed equally to this paper. DOI 10.1111/bjd.13683
Background The histopathological features of drug rash with eosinophilia and systemic symptoms (DRESS) syndrome remain poorly characterized. Objectives To better characterize the histopathological features of DRESS syndrome, and define the phenotype of the effector cells in the skin and compare it with maculopapular rash (MPR). Methods We conducted a retrospective study on 50 skin biopsies from patients with DRESS syndrome (n = 36). Histopathological and immunophenotypical features were studied and compared with a series of MPRs (n = 20). Results Foci of interface dermatitis, involving cutaneous adnexae, were frequently seen in cases of DRESS. Eosinophils were seen in only 20% of cases and neutrophils in 42%. Eczematous (40%), interface dermatitis (74%), acute generalized exanthematic pustulosis-like (20%) and erythema multiforme-like (24%) patterns were observed. The association of two or three of these patterns in a single biopsy was significantly more frequent in cases of DRESS than in a series of nondrug-induced dermatoses (P < 001), and appeared to be more marked in DRESS syndrome with severe cutaneous lesions (P = 001) than in less severe cases of DRESS and MPR. A higher proportion of CD8+ and granzyme B+ lymphocytes was observed in cases of DRESS with severe cutaneous eruptions (erythroderma and/or bullae). Atypical lymphocytes were found in 28% of biopsies, and expressed CD8 in most cases; a cutaneous T-cell clone was rarely found (6%). Conclusions The histopathology of DRESS syndrome highlights various associated inflammatory patterns in a single biopsy. Cutaneous effector lymphocytes comprise a high proportion of polyclonal CD8+ granzyme B+ T lymphocytes.
What’s already known about this topic?
• • •
The histological features of drug rash with eosinophilia and systemic symptoms (DRESS) vary from spongiotic dermatitis to an erythema multiforme-like aspect. In DRESS, skin biopsies often show eosinophils and apoptotic bodies. Atypical lymphocytes may be found in skin infiltrates.
What does this study add?
• • 50
The association of several inflammatory patterns in a single biopsy is suggestive for diagnosis. Cutaneous infiltrates can compromise atypical lymphocytes resembling Sezary cells.
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© 2015 British Association of Dermatologists
Histopathology of DRESS syndrome, N. Ortonne et al. 51
• •
DRESS shows a higher density of inflammatory infiltrates, more apoptosis, associated inflammatory patterns and granzyme B+ cells than maculapapular rash. Effector T cells are mainly polyclonal granzyme B+ CD8+ T cells.
The most common type of cutaneous drug reaction is the morbilliform-type or maculopapular rash (MPR), but more severe cutaneous drug eruption can occur, including drug rash with eosinophilia and systemic symptoms (DRESS) syndrome. In 1996, Bocquet and colleagues proposed this term in order to reduce confusion with hypersensitivity syndrome. DRESS syndrome is often characterized by a skin rash and multivisceral involvement, usually associated with hypereosinophilia.1,2 In some patients, DRESS syndrome can present with more aggressive cutaneous lesions, including erythroderma, or with bullae. The disease may be associated with reactivation of herpesvirus replication, especially human herpesvirus (HHV)-6 and HHV-7,3 but also Epstein–Barr virus (EBV) and cytomegalovirus.4,5 The histopathological aspect of DRESS syndrome is not well described in the literature, and is often only mentioned in passing in dermatopathology textbooks.6 To the best of our knowledge, three series of DRESS syndrome focusing on the histology have previously been published, all highlighting the polymorphous aspect of DRESS syndrome, with various inflammatory patterns.7–9 Interestingly, Walsh et al.9 suggested that the presence of apoptotic keratinocytes correlated with a more aggressive phenotype, with liver injury and an erythema multiforme (EM)-like cutaneous aspect, while most biopsies showed a spongiotic dermatitis. Chi et al.7 also found that skin biopsies of DRESS syndrome displayed various inflammatory aspects, and showed that interface dermatitis with apoptotic keratinocytes were more frequent in DRESS syndrome than in MPR. Owing, in part, to the fact that the inflammatory infiltrates of DRESS syndrome may comprise atypical lymphocytes, in cases with diffuse skin eruption or erythroderma, differential diagnosis with lymphoma, especially Sezary syndrome (SS), may be histopathologically difficult. In addition, it has been shown that T-cell clonality analyses may give positive results in DRESS syndrome.10 In contrast to toxic epidermal necrolysis (TEN), in which it has been shown that granulysin is the predominant cytokine inducing the apoptosis of epithelial cells, little attention has been paid to the cellular and molecular mechanisms involved in DRESS syndrome.11 As in other severe cutaneous drug reactions, understanding of the effectors involved in T-cell activation and organ cytotoxicity are important, and may represent the first step in the development of new, targeted therapies. It is known that, following drug exposure, DRESS syndrome is characterized by an expansion of effector CD8+ T cells and regulatory T cells (Tregs) in the blood.12,13 The aim of the present study was to better characterize the histopathological features of DRESS syndrome, and to
© 2015 British Association of Dermatologists
make a comparison with various inflammatory dermatoses and MPRs, which are less severe forms of drug reaction. We also focused on the phenotype of the effector cells in the skin.
Materials and methods Patients and material selection We retrospectively included 50 biopsy specimens over an 11year period from 36 patients [17 men and 19 women, median age 523 years (range 150–900)] who had a diagnosis of DRESS syndrome. In the majority (39%) of cases the offending drug was allopurinol, followed by carbamazepin (11%), minocycline, sulfamethoxazole + trimethoprim (5%) and sulfasalazine (5%). In each case, the diagnosis of DRESS syndrome (at least probable) was established according to previously published criteria.14 The mean delay between the onset of symptoms and skin biopsy, available in 22 cases, was 14 days (range 1–80). In all but three cases the delay ranged between 1 and 12 days and thus the biopsy was considered to have been performed during the acute phase. We compared the histopathology in patients with DRESS syndrome with severe cutaneous lesions (n = 21), presenting with erythroderma and/or bullae, with those with a less severe phenotype (n = 17), presenting only with a maculopapular eruption. The main clinical features of the patients are summarized in Table 1. We compared the biopsies from patients with DRESS syndrome with biopsies from patients with a less severe druginduced skin eruption (MPR). The latter group comprised 20 skin biopsies from 20 patients [six men and 14 women, median age 67 years (range 17–91)]. Patients were diagnosed with MPR when they presented with a drug-induced rash and met no criteria for DRESS syndrome according to Kardaun et al.14 The following clinical and laboratory data were retrospectively collected in all patients with DRESS syndrome: fever; type of eruption (erythematous rash and MPR, or erythroderma); presence of purpuric lesions on the lower limbs; presence of skin necrosis symptoms (superficial erosions, blistering or Nikolski sign); polyadenopathy; hypereosinophilia (> 700 eosinophils per mm3) and eosinophil blood count; presence of circulating hyperbasophil or ‘atypical’ lymphocytes; liver and renal dysfunction. The study was approved by the Comite de Protection des Personnes Ile de France IV (institutional review board no. 00003835).
British Journal of Dermatology (2015) 173, pp50–58
52 Histopathology of DRESS syndrome, N. Ortonne et al. Table 1 Clinical characteristics of the 36 included patients with drug rash with eosinophilia and systemic symptoms (DRESS) syndrome Female sex Mean age, years (range) Offending drug Allopurinol Carbamazepine Sulfamethoxazole+ trimethoprim Minocycline Sulfasalazine Other Dermatological manifestations Erythroderma Maculopapular rash Bullae/erosive lesions Pustules Purpuraa General and extracutaneous manifestations Fever Hyperbasophilic lymphocytes (n = 23) Polyadenopathyb Liver dysfunctionc Renal dysfunctiond Hypereosinophiliae
19 (528) 52 (15–90) 14 4 2 2 2
(39) (11) (5) (5) (5)
17 21 6 14 5
(47) (58) (17) (39) (14)
32 15 24 26 16 32
(89) (65) (67) (72) (44) (89)
Values are given as n (%) unless otherwise indicated. aThe purpura was not infiltrated; bpatients with polyadenopathy had enlarged lymph nodes (> 1 cm) in at least two different anatomical sites; cpatients with serum glutamic oxaloacetic transaminase and/or serum glutamic-pyruvic transaminase and/or gammaglutamyl transferase and/or alkaline phosphatase abnormal level(s) [over twice the normal laboratory value(s)] were considered to have liver dysfunction; drenal dysfunction was defined as an abnormal creatinine level; e700–15 000 eosinophils per mm3.
Histopathology and immunohistochemistry: techniques and parameters analysed Formalin-fixed, paraffin-embedded (FFPE) skin biopsies were retrieved from archive material of the Department of Pathology, Assistance Publique–H^ opitaux de Paris. Haematoxylin, eosin and saffron (HES) staining and immunohistochemistry were applied to 3-lm-thick sections. Immunostaining was done using monoclonal antibodies to CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD56, CD123, granzyme B (Dako, Glostrup, Denmark) and FoxP3 (236/AE7; Abcam, Cambridge, U.K.). We used a standard avidin–biotin–peroxidase method with diaminobenzidine (DAB) chromogen and the NexES immunostainer (Ventana, Tucson, AZ, U.S.A.), after antigen retrieval by heat in the appropriate buffer. Immunostaining of FoxP3 was done manually using a biotin–avidin system conjugated to horseradish peroxidase [HRP; VECTASTAINâ ABC–AP kit (Vector Laboratories, Burlingame, CA, U.S.A.)]. Doublestaining experiments (CD3/CD8) were performed using the Bond max device (Menarini Diagnostics, Rungis, France). All HES slides were reviewed with a multiheaded microscope and discussed by two dermatopathologists (N.O. and J.W.). The following morphological parameters were recorded systematically: (i) aspect of the stratum corneum – normal, British Journal of Dermatology (2015) 173, pp50–58
orthokeratosis or parakeratosis (continuous, psoriasiform or focal); (ii) aspect of granulous layer – normal or thickened; (iii) aspect of the spinous layers – acanthosis [psoriasiform (regular hyperplasia) or irregular], epidermal atrophy, spongiosis [grade 1 – diffusely enlarged intercellular spaces; grade 2 – marked confluent spongiosis (‘prevesicles’); grade 3 – constituted vesicles] or apoptotic keratinocytes, i.e. ‘Civatte bodies’; (iv) aspect of dermoepidermal junction – normal, focal interface dermatitis (apoptotic keratinocytes or vacuolized basement membrane zone) or widespread interface dermatitis; (v) adnexal lichenoid interface dermatitis lesions – follicular interface dermatitis or lichenoid interface dermatitis at the acrosyringium; (vi) characterization of intraepidermal inflammatory cells – lymphocytes, eosinophils, neutrophils and corneal/subcorneal pustules; (vii) presence or absence of atypical lymphocytes (medium-sized lymphocytes with enlarged nuclei, lymphocytes with enlarged hyperconvoluted nuclei suggesting Sezary cells or large atypical lymphocytes); (viii) dermal infiltrate – localization (superficial, superficial and deep, or deep dermis, hypodermis), architecture (bandlike, perivascular, periadnexal, interstitial, diffuse), and density (low, i.e. few scattered lymphocytes not forming aggregates; intermediate, i.e. cohesive cells forming some aggregates; or high, i.e. sheets of lymphocytes grouped in large aggregates) and components (eosinophils, neutrophils, lymphocytes and plasma cells); (ix) dermal changes: vasculitis, oedema of papillary dermis and leucocytoclastic nuclei. The presence of particular inflammatory patterns was recorded in each case. The eczematous pattern was defined as a grade 2 or 3 spongiosis with lymphocytes exocytosis; interface dermatitis as basal lymphocyte exocytosis with keratinocyte vacuolization and/or apoptosis; acute generalized exanthematic pustulosis (AGEP)-like as a multilocular subcorneal or intracorneal pustulosis; psoriasiform as an association of psoriasiform hyperplasia with continuous parakeratosis; pustular psoriasis as a psoriasis pattern with pustules; and EMlike as lymphocytic exocytosis with aggregates of apoptotic keratinocytes. We recorded in each case the number of inflammatory patterns observed in a single biopsy. As a comparative group, we analysed the inflammatory patterns of 47 skin biopsies of patients with a nondrug-induced dermatosis (21 men and 26 women, median age 504 years). This control group was established by selecting nondrug-induced dermatoses that are known to involve a dermal lymphocytic infiltrate and epidermal inflammatory changes: eczematous reaction (n = 9), psoriasis (n = 11), lichen planus (n = 10), subacute/ chronic lupus (n = 9) and SS (n = 8). We performed an immunohistochemical study in 24 biopsies from the DRESS syndrome group (CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD56, CD123, granzyme B, and FoxP3), as well as in biopsies from the SS and MPR groups (CD3, CD8, granzyme B, FoxP3). The phenotype of atypical lymphocytes, when present, was analysed. In three DRESS syndrome samples with atypical lymphocytes, we performed double stainings for CD3 and CD8. The presence of CD8+, CD56+, CD123+ and granzyme B+ cells was assessed in both dermis and epidermis. © 2015 British Association of Dermatologists
Histopathology of DRESS syndrome, N. Ortonne et al. 53
The density of positive cells was categorized as negative (no positive cell), low (0–5% positive lymphocytes), intermediate (> 5–50%) or strong (> 50%). Epstein Barr virus-specific in situ hybridization The search for EBV in skin samples was done by in situ hybridization on deparaffinized slides, using consensus probes for EBV-encoded small RNA (EBER) transcripts (Bond ISH probe; Menarini Leica, Florence, Italy) coupled with fluorescein and developed with secondary antifluorescein antibodies coupled to HRP and DAB. T-cell clonality analysis For T-cell clonality studies, DNA was extracted from either a snap frozen skin biopsy or from the FFPE samples, and analysed by DNA amplification of TCRG using consensus primers and separation of the amplimers by polymerase chain reaction denaturing gradient gel electrophoresis, as previously described.15 Statistical analysis Qualitative variables were compared across different populations using the Fisher’s exact or Kruskal–Wallis tests. All tests were two-tailed and P-values < 001 were considered statistically significant, using a Bonferroni correction. Statistical analyses were performed using STATA (version 11.0; StataCorp, College Station, TX, U.S.A.).
Results Histopathological and phenotypic study of drug rash with eosinophilia and systemic symptoms The main histopathological findings are summarized in Table 2. A diffuse parakeratotic layer (84%) and foci of lichenoid interface dermatitis (76%) were frequent. In particular, an interface dermatitis involving the adnexae was commonly observed in the acrosyringium and the infundibular and isthmic portions of pilar units. Apoptotic keratinocytes were seen in 60% of cases and neutrophil exocytosis in 12%, sometimes with subcorneal pustules (18%). The dermal infiltrates appeared to be polymorphous, with plasma cells in 18% of cases and neutrophils in 42%. While most patients presented with hypereosinophilia (89%), with an eosinophil count ranging from 700 to 15 000 per mm3 (Table 1), eosinophils were only significantly present in 20% of skin biopsies. Atypical lymphocytes, sometimes resembling Sezary cells, were present in almost one-third of cases (14 of 50; 28%) (Fig. 1c). Nuclear debris within the dermis was also often seen, but leucocytoclastic vasculitis was not observed. In most cases, the infiltrate was located only in the superficial dermis (88%), and predominated around dermal capillaries (73%). The density of inflammatory infiltrates was low (42%) or intermediate (50%) in the majority of cases. Overall, various inflammatory © 2015 British Association of Dermatologists
patterns were observed. The most frequent was an interface dermatitis (74%), followed by eczematous (40%), EM-like (24%) and AGEP-like pustulosis (20%). Interestingly, more than one pattern was frequently observed in a single biopsy. This multiplicity of inflammatory pattern was significantly more pronounced in DRESS than in nondrug-induced dermatoses (56% vs. 25% of cases, respectively; P < 001). A representative example of a DRESS biopsy with three different patterns is shown in Figure 2. The most frequently associated patterns were eczematous and interface dermatitis (20%). The second most frequently associated patterns were interface and EM-like dermatitis (14%). Lymphocyte phenotyping showed that most cases of DRESS comprised a high proportion of CD8+ lymphocytes, with numerous cytotoxic cells expressing granzyme B (Table 3; Fig. 3). No CD56+ natural killer cells, and no or very few CD123+ plasmacytoid dendritic cells and CD20+ B cells were found (< 5% in all investigated cases). FoxP3+ lymphocytes were identified in 58% of cases, but were scattered in most samples. Among 14 analysed skin biopsies from patients with DRESS, EBER expression was only present in one, in a few scattered lymphocytes. T-cell clonality results were available for 17 patients. A T-cell clone was detected in only one (6%) skin sample. None of the seven patients with atypical lymphocytes in the skin had a cutaneous T-cell clone. Two patients, including one with a cutaneous T-cell clone, had a T-cell clone in blood. Clinical pathological correlations in drug rash with eosinophilia and systemic systems No correlation could be established between the most frequent offending drug (allopurinol) and any of the morphological parameters analysed. Among the 15 patients with circulating atypical/hyperbasophilic lymphocytes, only three (20%) had atypical lymphocytes identified in the skin. Cutaneous eosinophils were identified in only nine (28%) of the 32 patients with blood hypereosinophilia. A proportion (14%) of patients had purpuric lesions, but leucoclastic or lymphocytic vasculitis was not seen. Significant red blood cell extravasation was not found, but skin biopsies were done on the lower limb in only one patient with purpuric lesions. Interestingly, biopsies from patients with a severe phenotype (erythroderma and/or bullae) more frequently showed an EM-like pattern (P < 001), while the presence of other inflammatory patterns, atypical lymphocytes, pustules and the deepness of dermal infiltration did not significantly differ between DRESS presenting with a maculopapular eruption or MPR. Patients with apoptotic keratinocytes in skin biopsies showed a tendency to have more liver (80% vs. 64%) and renal (60% vs. 43%) dysfunctions. In addition, DRESS with severe cutaneous lesions had significantly more associated patterns (P = 001) and a higher proportion of granzyme B+ lymphocytes (P = 004). The proportion of CD8+ effector T British Journal of Dermatology (2015) 173, pp50–58
54 Histopathology of DRESS syndrome, N. Ortonne et al. Table 2 Comparison of the histological features of patients with drug rash with eosinophilia and systemic symptoms (DRESS) syndrome and maculopapular rash (MPR) Histological feature
DRESS (n = 50)
Parakeratosis Acanthosis Pustules Focal ID Apoptotic keratinocytes Lymphocyte exocytosis Follicular interface dermatitis (n = 13/n = 3) Interface dermatitis of acrosyringium (n = 49/n = 6) Papillary oedema Atypical lymphocytes Dermal eosinophils Dermal neutrophils Leucocytoclasia Dermal plasma cells Mid and deep dermis infiltration Infiltrate density Low Intermediate High Inflammatory patterns None (perivascular infiltrate only) Eczematous ID EM-like AGEP-like Mean no. of existing patterns None 1 ≥2 Type of associated patterns ID + eczematous ID + eczematous + AGEP-like ID + EM-like
42 27 10 38 30 32 13 19 24 14 10 21 15 9 13
(84) (54) (20) (76) (60) (64) (100) (39) (48) (28) (20) (42) (30) (18) (26)
21 (42) 25 (50) 4 (8) 7 20 37 12 10
(14) (40) (74) (24) (20)
MPR (n = 20) 8 2 5 7 6 7 2 3 7 7 9 6 1 0 1
(40) (10) (25) (35) (30) (35) (67) (59) (35) (35) (45) (30) (5) (5)
P-value 020 < 001 075 < 001 006 003 – – 043 058 004 042 003 005 004 < 001
17 (85) 3 (15) 0 8 7 8 0 4
(40) (35) (40) (20)
6 (12) 16 (32) 28 (56)
8 (40) 7 (35) 5 (25)
10 (20) 6 (12) 7 (14)
0 2 (10) 0
– 079 001 001 1 < 001
– – –
Values are given as n (%). Using a Bonferroni correction, P-values ≤ 001 were considered significant. ID, interface dermatitis; EM, erythema multiforme; AGEP, acute generalized exanthematic pustulosis.
cells was higher in less severe DRESS, but the difference was not significant. The number of cases studied for FoxP3 was too limited to search for statistical differences between the two groups. Comparison with maculopapular rashes Interface dermatitis was observed in seven (35%) cases of MPR, which was significantly less than in DRESS syndrome (P < 001). Apoptotic keratinocytes were seen only in 30% of cases, while they were identified in 60% of instances of DRESS syndrome, but the difference was not significant. Exocytosis of neutrophils and pustulosis were seen in 25% of cases, with no significant differences compared with DRESS syndrome. Atypical activated lymphocytes were noted in seven (35%) cases. As in DRESS syndrome, vasculitis was never seen but nuclear debris was observed, although it appeared to be rarer (P = 003). The density of the inflammaBritish Journal of Dermatology (2015) 173, pp50–58
tory infiltrates was, in most cases, slight (17 of 20; 85%), with only three biopsies showing an intermediate-density infiltrate and none a high-density one. The density of dermal infiltrate was significantly lower than in DRESS syndrome (P < 001). By comparison, 58% of samples from the DRESS syndrome group showed intermediate- or high-density infiltrates (Table 2). In addition, patients with DRESS syndrome had more mid-dermis infiltration (P = 004). In two cases of MPR, there were minimal lesions, with a very subtle lymphocytic perivascular infiltrate and no epidermal injury. A significant proportion of the MPRs had mainly dermal infiltrates, with no significant inflammatory pattern (40% of MPR vs. 12% of DRESS; P < 001). The most represented patterns in MPR were interface dermatitis and eczematous changes. However, the proportion of MPRs with interface dermatitis was lower than in DRESS syndrome (P = 001), and no cases of MPR vs. 24% of cases of DRESS syndrome had an EM-like aspect (P = 001). Interestingly, fewer cases of MPR presented © 2015 British Association of Dermatologists
Histopathology of DRESS syndrome, N. Ortonne et al. 55
Fig 1. Various histopathological aspects of cutaneous infiltrates of drug rash with eosinophilia and systemic symptoms (DRESS) syndrome. (a) Spongiotic dermatitis with confluent areas of spongiosis within the epidermis associated with lymphocyte exocytosis. (b) This case of DRESS syndrome shows a marked lichenoid interface dermatitis with mild acanthosis and a heavy lymphocytic infiltrate extending from the superficial dermis to the epidermal basal layer, in which many apoptotic keratinocytes are seen. (c) This case closely resemble Sezary syndrome, with a mild perivascular infiltrate that comprises atypical lymphocytes with enlarged hyperchromatic nuclei (arrowheads and inset). (d) A large multilocular pustule is present, as usually seen in acute generalized exanthematic pustulosis.
(a)
(b)
(c)
(d)
with two or more associated inflammatory patterns (25% vs. 56%; P < 001). As in DRESS, MPRs showed infiltration of CD8+ T lymphocytes and comprised a proportion of granzyme B+ cytotoxic effector cells (Table 3). No significant differences in the proportion of FoxP3 lymphocytes were observed. Although the proportion of CD8+ T cells was not different, the proportion of granzyme B+ cells in both the epidermis (P < 001) and the dermis (P < 001) was significantly higher in DRESS syndrome than in MPR. Finally, we compared, for a selection of parameters, DRESS syndrome with severe cutaneous lesions (erythroderma and/ or bullae) with DRESS syndrome presenting with a maculopapular eruption and with MPR. Interestingly, DRESS syndrome with severe cutaneous lesions more frequently showed apoptotic keratinocytes and an EM-like pattern (P < 001), which was never seen in MPR. In addition, DRESS syndrome with severe cutaneous lesions had significantly more associated patterns (mean 195 vs. 095; P = 001) and a higher proportion of granzyme B+ lymphocytes (P = 004). © 2015 British Association of Dermatologists
Discussion DRESS syndrome is a severe systemic cutaneous drug reaction, with a potentially fatal outcome due to visceral involvement, and particularly to hepatic and cardiac injuries.16 Recently, the European Severe Cutaneous Adverse Reactions to Drugs group (EuroSCAR/RegiSCAR) proposed diagnostic criteria for the disease, allowing constitution of homogeneous groups of patients, as presented in this series. It is noteworthy that none of these criteria rely on histopathology. As for most drug reactions, except those with a particular feature, such as TEN and AGEP, the histopathological aspect of DRESS syndrome is not well described in the literature. In textbooks, the syndrome is either not described on histopathological grounds,6 or it is only stated that it may present with various histopathological features, including spongiotic dermatitis, erythema multiformis or aspects of TEN.17 In Lever’s Histopathology of the Skin, it is described as a variant of exanthemic drug reactions, where eosinophils and scattered apoptotic keratinocytes are commonly but not always seen.18 In their British Journal of Dermatology (2015) 173, pp50–58
56 Histopathology of DRESS syndrome, N. Ortonne et al.
(a)
(b)
(c)
(d)
Fig 2. Drug rash with eosinophilia and systemic symptoms (DRESS) syndrome with multiple different inflammatory patterns. (a) In this unique section of a DRESS syndrome skin biopsy, three distinct inflammatory patterns can be observed: (b) a multilocular pustule (arrowheads), (c) a spongiotic dermatitis with a vesicle containing Langerhans’ cells, and (d) foci of vacuolar or lichenoid interface dermatitis at the dermal– epidermal junction and within a follicular adnexa.
clinicopathological study, Chiou et al.8 highlighted the polymorphous histological expression of DRESS syndrome, which may present with various inflammatory patterns. More recently, in a series of 27 cases, Walsh et al.9 found that the most frequent inflammatory changes were a spongiotic dermatitis or basal cell vacuolization with apoptotic keratinocytes, the former being more frequent in patients with MPR. We found that the histopathological presentation of DRESS syndrome is highly variable, encompassing many inflammatory patterns, from a slight perivascular lymphocytic infiltrate to a pustular, AGEP-like, EM-like, eczematous or interface dermatitis, with the latter being the most frequent. What appeared to be a special feature was the association of different inflammatory patterns in a single specimen, a finding that was significantly more frequent in DRESS syndrome than in nondruginduced dermatoses and MPR. The histopathological presentation did not seem to correlate with the culprit drug, although relevant statistical analyses could only be obtained for allopurinol. Interestingly, dermal eosinophils were present in only 20% of cases and apoptotic keratinocytes in only half of cases. British Journal of Dermatology (2015) 173, pp50–58
Our results confirm that in a significant proportion of cases, atypical lymphocytes may be found in dermal infiltrates, so that differential diagnosis with cutaneous T-cell lymphoma may be challenging, especially with SS. In this context, the demonstration of a CD8+, granzyme B+ phenotype of the atypical cells may be a helpful feature, as SS neoplastic cells are CD4+. Taken together, our results further support a major role for CD8+ effector T cells and the perforin/granzyme pathway in drug reactions, especially in DRESS syndrome, as previously shown in MPR.19 Interestingly, the histological aspect of MPR, previously described in a large case series by Gerson et al.,20 shares many features with DRESS syndrome. Whether MPR and DRESS syndrome belong to the same spectrum of drug reactions, with MPR representing the less severe end of the spectrum, is therefore questionable. The main difference in our series was the intensity of the inflammatory changes, as the proportion of cases with epidermal lesions, as well as the density of inflammatory infiltrates, appeared to be lower in MPR than in DRESS syndrome, as previously shown.7 Whether the severity of skin inflammation and the extension © 2015 British Association of Dermatologists
Histopathology of DRESS syndrome, N. Ortonne et al. 57 Table 3 Phenotypical comparison between patients with drug rash with eosinophilia and systemic symptoms (DRESS) syndrome and maculopapular rash (MPR)
Proportion of CD8+ dermal cells (n = 22/n = 18) ≤ 5% 5–50% > 50% Presence of epidermotropic granzyme B+ cells (n = 20/17) Proportion of dermal granzyme B+ cells (n = 21/n = 11) Score 1: ≤ 5% Score 2: 5–50% Score 3: > 50% Proportion of dermal FoxP3+ cells (n = 12/n = 14) ≤ 5% 5–50% > 50%
DRESS (n = 24)
MPR (n = 20)
0 4 (18) 18 (82) 18 (90)
1 3 14 7
(5) (17) (78) (41)
P-value 080
< 001
4 (19) 13 (62) 4 (19)
4 (36) 3 (27) 4 (36)
< 001
8 (67) 4 (33) 0
6 (43) 7 (50) 1 (6)
060
Values are given as n (%).
Fig 3. Phenotype of lymphocytes in drug rash with eosinophilia and systemic symptoms (DRESS) syndrome. (a) More than 50% of lymphocytes, both in the epidermis and the dermis, are CD8+ T cells. (b) Scattered FoxP3+ lymphocytes are present within the infiltrate (arrowheads). (c) DRESS syndrome biopsy showing many granzyme B+ lymphocytes both in the dermis and in the epidermis with typical cytoplasmic granular staining. (d) Double-staining shows that the atypical medium-sized lymphocytes seen in the papillary dermis and around the capillaries in this sample express CD8 (arrowheads).
(a)
(b)
(c)
(d)
to extracutaneous sites in DRESS syndrome, in contrast to MPR, are due to differences in cytotoxic effector cells and/or the infiltration of Tregs is therefore an interesting issue. FoxP3+ Tregs were recently reported to be dramatically expanded in blood during the acute phase of DRESS syndrome.13 Although both MPR and DRESS syndrome were characterized by a significant infiltration of CD8+ and cytotoxic granzyme B+ cells, we found that DRESS syndrome with a severe phenotype, presenting with an erythroderma and/or bullae, had significantly more granzyme B+ cells. These cases also presented with more inflammatory changes, as the number of associated inflammatory patterns was significantly higher than in MPR and DRESS syndrome presenting with a MPR. A potential role for skin-infiltrating Tregs in the limitation of skin lesions in fixed drug reactions, in contrast to TEN, has already been suggested, as sequential biopsies have © 2015 British Association of Dermatologists
proven that the proportion of these cells in skin infiltrates increases progressively.21 We found that FoxP3+ Tregs were not as numerous as expected within the skin infiltrates during the acute phase of DRESS syndrome. We speculate that DRESS syndrome is actually characterized by a dramatic expansion of Tregs in blood, while effector CD8+ T cells are recruited in the skin, where they can exert cytotoxic functions because of a lower proportion of Tregs recruited at the same time from blood. The fact that different cytokinic environments may coexist in blood and skin, leading to such differences, remains speculative and requires further investigation. The major role played by expanded activated T cells in DRESS syndrome has already been demonstrated. In particular, it has been shown recently that expanded CD8+ T-cell populations, found in all the involved organs, are directed against herpesviruses, especially EBV. This suggests that DRESS syndrome may also be British Journal of Dermatology (2015) 173, pp50–58
58 Histopathology of DRESS syndrome, N. Ortonne et al.
regarded as a multiorgan antiviral T-cell response.12 For this reason, we investigated whether EBV – a putative target of the CD8+ T cell – was present in the skin. With the exception of one case, we failed to demonstrate the recurrent presence of EBV in situ. We also found no significant infiltration of CD56+ and CD123+ cells. This finding further suggests that the effector phase of DRESS mostly relies on adaptive immunity, with the activation of cytotoxic T cells. In addition to lymphocyte phenotyping, T-cell clonality probably represents an important diagnostic criterion for differential diagnosis between DRESS syndrome and cutaneous T-cell lymphomas. A T-cell clone in skin or blood was only detected in two of 17 investigated cases from this series. Interestingly, none of these cases showed atypical T cells in the skin. This is in agreement with a previous study in which T-cell clones were only detected in blood of patients with DRESS syndrome.10 Of note, in most cases, the search for a cutaneous T-cell clone was performed on paraffin-embedded tissues, demonstrating that, in difficult cases, this can be done easily by dermatopathologists. In conclusion, various inflammatory patterns are observed in DRESS syndrome, and these different patterns are often seen to be associated in a single biopsy, which may represent a histopathological clue in diagnosis. Effector lymphocytes, at least in skin, comprise a high proportion of polyclonal CD8+ granzyme B+ lymphocytes. The histopathological presentations and the cutaneous effector cells in DRESS and MPR show some overlap, but DRESS is characterized by more inflammation and more granzyme B+ effector cells, especially when patients present with an erythroderma and/or bullae.
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Acknowledgments We thank Laetitia Gregoire, Unite de Recherche Clinique (URC), Henri Mondor Hospital, and Audrey Colin from the Department of Dermatology, Henri Mondor Hospital, for their help in presenting the study to the ethics committee (Comite de Protection des Personnes, Ile de France IV).
15
16 17
References 18 1 Ben m’rad M, Leclerc-Mercier S, Blanche P et al. Drug-induced hypersensitivity syndrome: clinical and biologic disease patterns in 24 patients. Medicine (Baltimore) 2009; 88:131–40. 2 Kardaun SH, Sekula P, Valeyrie-Allanore L et al. Drug reaction with eosinophilia and systemic symptoms (DRESS): an original multisystem adverse drug reaction. Results from the prospective RegiSCAR study. Br J Dermatol 2013; 169:1071–80. 3 Tohyama M, Hashimoto K, Yasukawa M et al. Association of human herpesvirus 6 reactivation with the flaring and severity of
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19 20 21
drug-induced hypersensitivity syndrome. Br J Dermatol 2007; 157:934–40. Seishima M, Yamanaka S, Fujisawa T et al. Reactivation of human herpesvirus (HHV) family members other than HHV-6 in druginduced hypersensitivity syndrome. Br J Dermatol 2006; 155:344–9. Shiohara T, Inaoka M, Kano Y. Drug-induced hypersensitivity syndrome (DIHS): a reaction induced by a complex interplay among herpesviruses and antiviral and antidrug immune responses. Allergol Int 2006; 55:1–8. Weedon D. Weedon’s Skin Pathology, 3rd edn. Edinburgh: Churchill Livingstone, 2010. Chi MH, Hui RC, Yang CH et al. Histopathological analysis and clinical correlation of drug reaction with eosinophilia and systemic symptoms (DRESS). Br J Dermatol 2014; 170:866–73. Chiou CC, Yang LC, Hung SI et al. Clinicopathological features and prognosis of drug rash with eosinophilia and systemic symptoms: a study of 30 cases in Taiwan. J Eur Acad Dermatol Venereol 2008; 22:1044–9. Walsh S, Diaz-Cano S, Higgins E et al. Drug reaction with eosinophilia and systemic symptoms (DRESS): is cutaneous phenotype a prognostic marker for outcome? A review of clinicopathological features of 27 cases. Br J Dermatol 2013; 168:391–401. Cordel N, Lenormand B, Courville P et al. Study of the clonality of cutaneous and blood lymphocytes during drug-induced hypersensitivity in 6 patients. Ann Dermatol Venereol 2004; 131:1059–61. Chung WH, Hung SI, Yang JY et al. Granulysin is a key mediator for disseminated keratinocyte death in Stevens-Johnson syndrome and toxic epidermal necrolysis. Nat Med 2008; 14:1343–50. Picard D, Janela B, Descamps V et al. Drug reaction with eosinophilia and systemic symptoms (DRESS): a multiorgan antiviral T cell response. Sci Transl Med 2010; 2:46ra62. Takahashi R, Kano Y, Yamazaki Y et al. Defective regulatory T cells in patients with severe drug eruptions: timing of the dysfunction is associated with the pathological phenotype and outcome. J Immunol 2009; 182:8071–9. Kardaun SH, Sidoroff A, Valeyrie-Allanore L et al. Variability in the clinical pattern of cutaneous side-effects of drugs with systemic symptoms: does a DRESS syndrome really exist? Br J Dermatol 2007; 156:609–11. Delfau-Larue MH, Petrella T, Lahet C et al. Value of clonality studies of cutaneous T lymphocytes in the diagnosis and follow-up of patients with mycosis fungoides. J Pathol 1998; 184:185–90. Bourgeois GP, Cafardi JA, Groysman V et al. A review of DRESSassociated myocarditis. J Am Acad Dermatol 2011; 66:e229–36. McKee PH, Calonje E, Granter SR. Pathology of the Skin with Clinical Correlations, 3rd edn, vol. 1. London: Elsevier Mosby, 2005. Elder DE. Lever’s Histopathology of the Skin, 10th edn. Philadelphia, PA: Lippincott Williams and Wilkins, 2009. Yawalkar N, Egli F, Hari Y et al. Infiltration of cytotoxic T cells in drug-induced cutaneous eruptions. Clin Exp Allergy 2000; 30:847–55. Gerson D, Sriganeshan V, Alexis JB. Cutaneous drug eruptions: a 5-year experience. J Am Acad Dermatol 2008; 59:995–9. Mizukawa Y, Yamazaki Y, Shiohara T. In vivo dynamics of intraepidermal CD8+ T cells and CD4+ T cells during the evolution of fixed drug eruption. Br J Dermatol 2008; 158:1230–8.
© 2015 British Association of Dermatologists
BJD
British Journal of Dermatology
Histopathology of drug rash with eosinophilia and systemic symptoms syndrome: a morphological and phenotypical study N. Ortonne,1,2,3 L. Valeyrie-Allanore,3,4 S. Bastuji-Garin,3,5 J. Wechsler,1 S. de Feraudy,1 T.-A. Duong,3,4 M.-H. Delfau-Larue,2,3,6 O. Chosidow,3,4 P. Wolkenstein3,4 and J.-C. Roujeau3 Assistance Publique – H^opitaux de Paris (AP-HP), H^opital Henri-Mondor, 1Departement de Pathologie, 4Service de Dermatologie, 5Service de Sante-Publique, 6 Service d’Immunologie Biologique, 94010 Creteil Cedex, France 2 INSERM U955 equipe 9, H^opital Henri-Mondor, 94010 Creteil Cedex, France 3 Universite Paris Est Creteil (UPEC), Faculte de Medecine, LIC EA4393, 94010 Creteil Cedex, France
Summary Correspondence Nicolas Ortonne. E-mail: [email protected]
Accepted for publication 25 January 2015
Funding sources None.
Conflicts of interest None declared. N.O., L.A., O.C. and P.W. contributed equally to this paper. DOI 10.1111/bjd.13683
Background The histopathological features of drug rash with eosinophilia and systemic symptoms (DRESS) syndrome remain poorly characterized. Objectives To better characterize the histopathological features of DRESS syndrome, and define the phenotype of the effector cells in the skin and compare it with maculopapular rash (MPR). Methods We conducted a retrospective study on 50 skin biopsies from patients with DRESS syndrome (n = 36). Histopathological and immunophenotypical features were studied and compared with a series of MPRs (n = 20). Results Foci of interface dermatitis, involving cutaneous adnexae, were frequently seen in cases of DRESS. Eosinophils were seen in only 20% of cases and neutrophils in 42%. Eczematous (40%), interface dermatitis (74%), acute generalized exanthematic pustulosis-like (20%) and erythema multiforme-like (24%) patterns were observed. The association of two or three of these patterns in a single biopsy was significantly more frequent in cases of DRESS than in a series of nondrug-induced dermatoses (P < 001), and appeared to be more marked in DRESS syndrome with severe cutaneous lesions (P = 001) than in less severe cases of DRESS and MPR. A higher proportion of CD8+ and granzyme B+ lymphocytes was observed in cases of DRESS with severe cutaneous eruptions (erythroderma and/or bullae). Atypical lymphocytes were found in 28% of biopsies, and expressed CD8 in most cases; a cutaneous T-cell clone was rarely found (6%). Conclusions The histopathology of DRESS syndrome highlights various associated inflammatory patterns in a single biopsy. Cutaneous effector lymphocytes comprise a high proportion of polyclonal CD8+ granzyme B+ T lymphocytes.
What’s already known about this topic?
• • •
The histological features of drug rash with eosinophilia and systemic symptoms (DRESS) vary from spongiotic dermatitis to an erythema multiforme-like aspect. In DRESS, skin biopsies often show eosinophils and apoptotic bodies. Atypical lymphocytes may be found in skin infiltrates.
What does this study add?
• • 50
The association of several inflammatory patterns in a single biopsy is suggestive for diagnosis. Cutaneous infiltrates can compromise atypical lymphocytes resembling Sezary cells.
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© 2015 British Association of Dermatologists
Histopathology of DRESS syndrome, N. Ortonne et al. 51
• •
DRESS shows a higher density of inflammatory infiltrates, more apoptosis, associated inflammatory patterns and granzyme B+ cells than maculapapular rash. Effector T cells are mainly polyclonal granzyme B+ CD8+ T cells.
The most common type of cutaneous drug reaction is the morbilliform-type or maculopapular rash (MPR), but more severe cutaneous drug eruption can occur, including drug rash with eosinophilia and systemic symptoms (DRESS) syndrome. In 1996, Bocquet and colleagues proposed this term in order to reduce confusion with hypersensitivity syndrome. DRESS syndrome is often characterized by a skin rash and multivisceral involvement, usually associated with hypereosinophilia.1,2 In some patients, DRESS syndrome can present with more aggressive cutaneous lesions, including erythroderma, or with bullae. The disease may be associated with reactivation of herpesvirus replication, especially human herpesvirus (HHV)-6 and HHV-7,3 but also Epstein–Barr virus (EBV) and cytomegalovirus.4,5 The histopathological aspect of DRESS syndrome is not well described in the literature, and is often only mentioned in passing in dermatopathology textbooks.6 To the best of our knowledge, three series of DRESS syndrome focusing on the histology have previously been published, all highlighting the polymorphous aspect of DRESS syndrome, with various inflammatory patterns.7–9 Interestingly, Walsh et al.9 suggested that the presence of apoptotic keratinocytes correlated with a more aggressive phenotype, with liver injury and an erythema multiforme (EM)-like cutaneous aspect, while most biopsies showed a spongiotic dermatitis. Chi et al.7 also found that skin biopsies of DRESS syndrome displayed various inflammatory aspects, and showed that interface dermatitis with apoptotic keratinocytes were more frequent in DRESS syndrome than in MPR. Owing, in part, to the fact that the inflammatory infiltrates of DRESS syndrome may comprise atypical lymphocytes, in cases with diffuse skin eruption or erythroderma, differential diagnosis with lymphoma, especially Sezary syndrome (SS), may be histopathologically difficult. In addition, it has been shown that T-cell clonality analyses may give positive results in DRESS syndrome.10 In contrast to toxic epidermal necrolysis (TEN), in which it has been shown that granulysin is the predominant cytokine inducing the apoptosis of epithelial cells, little attention has been paid to the cellular and molecular mechanisms involved in DRESS syndrome.11 As in other severe cutaneous drug reactions, understanding of the effectors involved in T-cell activation and organ cytotoxicity are important, and may represent the first step in the development of new, targeted therapies. It is known that, following drug exposure, DRESS syndrome is characterized by an expansion of effector CD8+ T cells and regulatory T cells (Tregs) in the blood.12,13 The aim of the present study was to better characterize the histopathological features of DRESS syndrome, and to
© 2015 British Association of Dermatologists
make a comparison with various inflammatory dermatoses and MPRs, which are less severe forms of drug reaction. We also focused on the phenotype of the effector cells in the skin.
Materials and methods Patients and material selection We retrospectively included 50 biopsy specimens over an 11year period from 36 patients [17 men and 19 women, median age 523 years (range 150–900)] who had a diagnosis of DRESS syndrome. In the majority (39%) of cases the offending drug was allopurinol, followed by carbamazepin (11%), minocycline, sulfamethoxazole + trimethoprim (5%) and sulfasalazine (5%). In each case, the diagnosis of DRESS syndrome (at least probable) was established according to previously published criteria.14 The mean delay between the onset of symptoms and skin biopsy, available in 22 cases, was 14 days (range 1–80). In all but three cases the delay ranged between 1 and 12 days and thus the biopsy was considered to have been performed during the acute phase. We compared the histopathology in patients with DRESS syndrome with severe cutaneous lesions (n = 21), presenting with erythroderma and/or bullae, with those with a less severe phenotype (n = 17), presenting only with a maculopapular eruption. The main clinical features of the patients are summarized in Table 1. We compared the biopsies from patients with DRESS syndrome with biopsies from patients with a less severe druginduced skin eruption (MPR). The latter group comprised 20 skin biopsies from 20 patients [six men and 14 women, median age 67 years (range 17–91)]. Patients were diagnosed with MPR when they presented with a drug-induced rash and met no criteria for DRESS syndrome according to Kardaun et al.14 The following clinical and laboratory data were retrospectively collected in all patients with DRESS syndrome: fever; type of eruption (erythematous rash and MPR, or erythroderma); presence of purpuric lesions on the lower limbs; presence of skin necrosis symptoms (superficial erosions, blistering or Nikolski sign); polyadenopathy; hypereosinophilia (> 700 eosinophils per mm3) and eosinophil blood count; presence of circulating hyperbasophil or ‘atypical’ lymphocytes; liver and renal dysfunction. The study was approved by the Comite de Protection des Personnes Ile de France IV (institutional review board no. 00003835).
British Journal of Dermatology (2015) 173, pp50–58
52 Histopathology of DRESS syndrome, N. Ortonne et al. Table 1 Clinical characteristics of the 36 included patients with drug rash with eosinophilia and systemic symptoms (DRESS) syndrome Female sex Mean age, years (range) Offending drug Allopurinol Carbamazepine Sulfamethoxazole+ trimethoprim Minocycline Sulfasalazine Other Dermatological manifestations Erythroderma Maculopapular rash Bullae/erosive lesions Pustules Purpuraa General and extracutaneous manifestations Fever Hyperbasophilic lymphocytes (n = 23) Polyadenopathyb Liver dysfunctionc Renal dysfunctiond Hypereosinophiliae
19 (528) 52 (15–90) 14 4 2 2 2
(39) (11) (5) (5) (5)
17 21 6 14 5
(47) (58) (17) (39) (14)
32 15 24 26 16 32
(89) (65) (67) (72) (44) (89)
Values are given as n (%) unless otherwise indicated. aThe purpura was not infiltrated; bpatients with polyadenopathy had enlarged lymph nodes (> 1 cm) in at least two different anatomical sites; cpatients with serum glutamic oxaloacetic transaminase and/or serum glutamic-pyruvic transaminase and/or gammaglutamyl transferase and/or alkaline phosphatase abnormal level(s) [over twice the normal laboratory value(s)] were considered to have liver dysfunction; drenal dysfunction was defined as an abnormal creatinine level; e700–15 000 eosinophils per mm3.
Histopathology and immunohistochemistry: techniques and parameters analysed Formalin-fixed, paraffin-embedded (FFPE) skin biopsies were retrieved from archive material of the Department of Pathology, Assistance Publique–H^ opitaux de Paris. Haematoxylin, eosin and saffron (HES) staining and immunohistochemistry were applied to 3-lm-thick sections. Immunostaining was done using monoclonal antibodies to CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD56, CD123, granzyme B (Dako, Glostrup, Denmark) and FoxP3 (236/AE7; Abcam, Cambridge, U.K.). We used a standard avidin–biotin–peroxidase method with diaminobenzidine (DAB) chromogen and the NexES immunostainer (Ventana, Tucson, AZ, U.S.A.), after antigen retrieval by heat in the appropriate buffer. Immunostaining of FoxP3 was done manually using a biotin–avidin system conjugated to horseradish peroxidase [HRP; VECTASTAINâ ABC–AP kit (Vector Laboratories, Burlingame, CA, U.S.A.)]. Doublestaining experiments (CD3/CD8) were performed using the Bond max device (Menarini Diagnostics, Rungis, France). All HES slides were reviewed with a multiheaded microscope and discussed by two dermatopathologists (N.O. and J.W.). The following morphological parameters were recorded systematically: (i) aspect of the stratum corneum – normal, British Journal of Dermatology (2015) 173, pp50–58
orthokeratosis or parakeratosis (continuous, psoriasiform or focal); (ii) aspect of granulous layer – normal or thickened; (iii) aspect of the spinous layers – acanthosis [psoriasiform (regular hyperplasia) or irregular], epidermal atrophy, spongiosis [grade 1 – diffusely enlarged intercellular spaces; grade 2 – marked confluent spongiosis (‘prevesicles’); grade 3 – constituted vesicles] or apoptotic keratinocytes, i.e. ‘Civatte bodies’; (iv) aspect of dermoepidermal junction – normal, focal interface dermatitis (apoptotic keratinocytes or vacuolized basement membrane zone) or widespread interface dermatitis; (v) adnexal lichenoid interface dermatitis lesions – follicular interface dermatitis or lichenoid interface dermatitis at the acrosyringium; (vi) characterization of intraepidermal inflammatory cells – lymphocytes, eosinophils, neutrophils and corneal/subcorneal pustules; (vii) presence or absence of atypical lymphocytes (medium-sized lymphocytes with enlarged nuclei, lymphocytes with enlarged hyperconvoluted nuclei suggesting Sezary cells or large atypical lymphocytes); (viii) dermal infiltrate – localization (superficial, superficial and deep, or deep dermis, hypodermis), architecture (bandlike, perivascular, periadnexal, interstitial, diffuse), and density (low, i.e. few scattered lymphocytes not forming aggregates; intermediate, i.e. cohesive cells forming some aggregates; or high, i.e. sheets of lymphocytes grouped in large aggregates) and components (eosinophils, neutrophils, lymphocytes and plasma cells); (ix) dermal changes: vasculitis, oedema of papillary dermis and leucocytoclastic nuclei. The presence of particular inflammatory patterns was recorded in each case. The eczematous pattern was defined as a grade 2 or 3 spongiosis with lymphocytes exocytosis; interface dermatitis as basal lymphocyte exocytosis with keratinocyte vacuolization and/or apoptosis; acute generalized exanthematic pustulosis (AGEP)-like as a multilocular subcorneal or intracorneal pustulosis; psoriasiform as an association of psoriasiform hyperplasia with continuous parakeratosis; pustular psoriasis as a psoriasis pattern with pustules; and EMlike as lymphocytic exocytosis with aggregates of apoptotic keratinocytes. We recorded in each case the number of inflammatory patterns observed in a single biopsy. As a comparative group, we analysed the inflammatory patterns of 47 skin biopsies of patients with a nondrug-induced dermatosis (21 men and 26 women, median age 504 years). This control group was established by selecting nondrug-induced dermatoses that are known to involve a dermal lymphocytic infiltrate and epidermal inflammatory changes: eczematous reaction (n = 9), psoriasis (n = 11), lichen planus (n = 10), subacute/ chronic lupus (n = 9) and SS (n = 8). We performed an immunohistochemical study in 24 biopsies from the DRESS syndrome group (CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD56, CD123, granzyme B, and FoxP3), as well as in biopsies from the SS and MPR groups (CD3, CD8, granzyme B, FoxP3). The phenotype of atypical lymphocytes, when present, was analysed. In three DRESS syndrome samples with atypical lymphocytes, we performed double stainings for CD3 and CD8. The presence of CD8+, CD56+, CD123+ and granzyme B+ cells was assessed in both dermis and epidermis. © 2015 British Association of Dermatologists
Histopathology of DRESS syndrome, N. Ortonne et al. 53
The density of positive cells was categorized as negative (no positive cell), low (0–5% positive lymphocytes), intermediate (> 5–50%) or strong (> 50%). Epstein Barr virus-specific in situ hybridization The search for EBV in skin samples was done by in situ hybridization on deparaffinized slides, using consensus probes for EBV-encoded small RNA (EBER) transcripts (Bond ISH probe; Menarini Leica, Florence, Italy) coupled with fluorescein and developed with secondary antifluorescein antibodies coupled to HRP and DAB. T-cell clonality analysis For T-cell clonality studies, DNA was extracted from either a snap frozen skin biopsy or from the FFPE samples, and analysed by DNA amplification of TCRG using consensus primers and separation of the amplimers by polymerase chain reaction denaturing gradient gel electrophoresis, as previously described.15 Statistical analysis Qualitative variables were compared across different populations using the Fisher’s exact or Kruskal–Wallis tests. All tests were two-tailed and P-values < 001 were considered statistically significant, using a Bonferroni correction. Statistical analyses were performed using STATA (version 11.0; StataCorp, College Station, TX, U.S.A.).
Results Histopathological and phenotypic study of drug rash with eosinophilia and systemic symptoms The main histopathological findings are summarized in Table 2. A diffuse parakeratotic layer (84%) and foci of lichenoid interface dermatitis (76%) were frequent. In particular, an interface dermatitis involving the adnexae was commonly observed in the acrosyringium and the infundibular and isthmic portions of pilar units. Apoptotic keratinocytes were seen in 60% of cases and neutrophil exocytosis in 12%, sometimes with subcorneal pustules (18%). The dermal infiltrates appeared to be polymorphous, with plasma cells in 18% of cases and neutrophils in 42%. While most patients presented with hypereosinophilia (89%), with an eosinophil count ranging from 700 to 15 000 per mm3 (Table 1), eosinophils were only significantly present in 20% of skin biopsies. Atypical lymphocytes, sometimes resembling Sezary cells, were present in almost one-third of cases (14 of 50; 28%) (Fig. 1c). Nuclear debris within the dermis was also often seen, but leucocytoclastic vasculitis was not observed. In most cases, the infiltrate was located only in the superficial dermis (88%), and predominated around dermal capillaries (73%). The density of inflammatory infiltrates was low (42%) or intermediate (50%) in the majority of cases. Overall, various inflammatory © 2015 British Association of Dermatologists
patterns were observed. The most frequent was an interface dermatitis (74%), followed by eczematous (40%), EM-like (24%) and AGEP-like pustulosis (20%). Interestingly, more than one pattern was frequently observed in a single biopsy. This multiplicity of inflammatory pattern was significantly more pronounced in DRESS than in nondrug-induced dermatoses (56% vs. 25% of cases, respectively; P < 001). A representative example of a DRESS biopsy with three different patterns is shown in Figure 2. The most frequently associated patterns were eczematous and interface dermatitis (20%). The second most frequently associated patterns were interface and EM-like dermatitis (14%). Lymphocyte phenotyping showed that most cases of DRESS comprised a high proportion of CD8+ lymphocytes, with numerous cytotoxic cells expressing granzyme B (Table 3; Fig. 3). No CD56+ natural killer cells, and no or very few CD123+ plasmacytoid dendritic cells and CD20+ B cells were found (< 5% in all investigated cases). FoxP3+ lymphocytes were identified in 58% of cases, but were scattered in most samples. Among 14 analysed skin biopsies from patients with DRESS, EBER expression was only present in one, in a few scattered lymphocytes. T-cell clonality results were available for 17 patients. A T-cell clone was detected in only one (6%) skin sample. None of the seven patients with atypical lymphocytes in the skin had a cutaneous T-cell clone. Two patients, including one with a cutaneous T-cell clone, had a T-cell clone in blood. Clinical pathological correlations in drug rash with eosinophilia and systemic systems No correlation could be established between the most frequent offending drug (allopurinol) and any of the morphological parameters analysed. Among the 15 patients with circulating atypical/hyperbasophilic lymphocytes, only three (20%) had atypical lymphocytes identified in the skin. Cutaneous eosinophils were identified in only nine (28%) of the 32 patients with blood hypereosinophilia. A proportion (14%) of patients had purpuric lesions, but leucoclastic or lymphocytic vasculitis was not seen. Significant red blood cell extravasation was not found, but skin biopsies were done on the lower limb in only one patient with purpuric lesions. Interestingly, biopsies from patients with a severe phenotype (erythroderma and/or bullae) more frequently showed an EM-like pattern (P < 001), while the presence of other inflammatory patterns, atypical lymphocytes, pustules and the deepness of dermal infiltration did not significantly differ between DRESS presenting with a maculopapular eruption or MPR. Patients with apoptotic keratinocytes in skin biopsies showed a tendency to have more liver (80% vs. 64%) and renal (60% vs. 43%) dysfunctions. In addition, DRESS with severe cutaneous lesions had significantly more associated patterns (P = 001) and a higher proportion of granzyme B+ lymphocytes (P = 004). The proportion of CD8+ effector T British Journal of Dermatology (2015) 173, pp50–58
54 Histopathology of DRESS syndrome, N. Ortonne et al. Table 2 Comparison of the histological features of patients with drug rash with eosinophilia and systemic symptoms (DRESS) syndrome and maculopapular rash (MPR) Histological feature
DRESS (n = 50)
Parakeratosis Acanthosis Pustules Focal ID Apoptotic keratinocytes Lymphocyte exocytosis Follicular interface dermatitis (n = 13/n = 3) Interface dermatitis of acrosyringium (n = 49/n = 6) Papillary oedema Atypical lymphocytes Dermal eosinophils Dermal neutrophils Leucocytoclasia Dermal plasma cells Mid and deep dermis infiltration Infiltrate density Low Intermediate High Inflammatory patterns None (perivascular infiltrate only) Eczematous ID EM-like AGEP-like Mean no. of existing patterns None 1 ≥2 Type of associated patterns ID + eczematous ID + eczematous + AGEP-like ID + EM-like
42 27 10 38 30 32 13 19 24 14 10 21 15 9 13
(84) (54) (20) (76) (60) (64) (100) (39) (48) (28) (20) (42) (30) (18) (26)
21 (42) 25 (50) 4 (8) 7 20 37 12 10
(14) (40) (74) (24) (20)
MPR (n = 20) 8 2 5 7 6 7 2 3 7 7 9 6 1 0 1
(40) (10) (25) (35) (30) (35) (67) (59) (35) (35) (45) (30) (5) (5)
P-value 020 < 001 075 < 001 006 003 – – 043 058 004 042 003 005 004 < 001
17 (85) 3 (15) 0 8 7 8 0 4
(40) (35) (40) (20)
6 (12) 16 (32) 28 (56)
8 (40) 7 (35) 5 (25)
10 (20) 6 (12) 7 (14)
0 2 (10) 0
– 079 001 001 1 < 001
– – –
Values are given as n (%). Using a Bonferroni correction, P-values ≤ 001 were considered significant. ID, interface dermatitis; EM, erythema multiforme; AGEP, acute generalized exanthematic pustulosis.
cells was higher in less severe DRESS, but the difference was not significant. The number of cases studied for FoxP3 was too limited to search for statistical differences between the two groups. Comparison with maculopapular rashes Interface dermatitis was observed in seven (35%) cases of MPR, which was significantly less than in DRESS syndrome (P < 001). Apoptotic keratinocytes were seen only in 30% of cases, while they were identified in 60% of instances of DRESS syndrome, but the difference was not significant. Exocytosis of neutrophils and pustulosis were seen in 25% of cases, with no significant differences compared with DRESS syndrome. Atypical activated lymphocytes were noted in seven (35%) cases. As in DRESS syndrome, vasculitis was never seen but nuclear debris was observed, although it appeared to be rarer (P = 003). The density of the inflammaBritish Journal of Dermatology (2015) 173, pp50–58
tory infiltrates was, in most cases, slight (17 of 20; 85%), with only three biopsies showing an intermediate-density infiltrate and none a high-density one. The density of dermal infiltrate was significantly lower than in DRESS syndrome (P < 001). By comparison, 58% of samples from the DRESS syndrome group showed intermediate- or high-density infiltrates (Table 2). In addition, patients with DRESS syndrome had more mid-dermis infiltration (P = 004). In two cases of MPR, there were minimal lesions, with a very subtle lymphocytic perivascular infiltrate and no epidermal injury. A significant proportion of the MPRs had mainly dermal infiltrates, with no significant inflammatory pattern (40% of MPR vs. 12% of DRESS; P < 001). The most represented patterns in MPR were interface dermatitis and eczematous changes. However, the proportion of MPRs with interface dermatitis was lower than in DRESS syndrome (P = 001), and no cases of MPR vs. 24% of cases of DRESS syndrome had an EM-like aspect (P = 001). Interestingly, fewer cases of MPR presented © 2015 British Association of Dermatologists
Histopathology of DRESS syndrome, N. Ortonne et al. 55
Fig 1. Various histopathological aspects of cutaneous infiltrates of drug rash with eosinophilia and systemic symptoms (DRESS) syndrome. (a) Spongiotic dermatitis with confluent areas of spongiosis within the epidermis associated with lymphocyte exocytosis. (b) This case of DRESS syndrome shows a marked lichenoid interface dermatitis with mild acanthosis and a heavy lymphocytic infiltrate extending from the superficial dermis to the epidermal basal layer, in which many apoptotic keratinocytes are seen. (c) This case closely resemble Sezary syndrome, with a mild perivascular infiltrate that comprises atypical lymphocytes with enlarged hyperchromatic nuclei (arrowheads and inset). (d) A large multilocular pustule is present, as usually seen in acute generalized exanthematic pustulosis.
(a)
(b)
(c)
(d)
with two or more associated inflammatory patterns (25% vs. 56%; P < 001). As in DRESS, MPRs showed infiltration of CD8+ T lymphocytes and comprised a proportion of granzyme B+ cytotoxic effector cells (Table 3). No significant differences in the proportion of FoxP3 lymphocytes were observed. Although the proportion of CD8+ T cells was not different, the proportion of granzyme B+ cells in both the epidermis (P < 001) and the dermis (P < 001) was significantly higher in DRESS syndrome than in MPR. Finally, we compared, for a selection of parameters, DRESS syndrome with severe cutaneous lesions (erythroderma and/ or bullae) with DRESS syndrome presenting with a maculopapular eruption and with MPR. Interestingly, DRESS syndrome with severe cutaneous lesions more frequently showed apoptotic keratinocytes and an EM-like pattern (P < 001), which was never seen in MPR. In addition, DRESS syndrome with severe cutaneous lesions had significantly more associated patterns (mean 195 vs. 095; P = 001) and a higher proportion of granzyme B+ lymphocytes (P = 004). © 2015 British Association of Dermatologists
Discussion DRESS syndrome is a severe systemic cutaneous drug reaction, with a potentially fatal outcome due to visceral involvement, and particularly to hepatic and cardiac injuries.16 Recently, the European Severe Cutaneous Adverse Reactions to Drugs group (EuroSCAR/RegiSCAR) proposed diagnostic criteria for the disease, allowing constitution of homogeneous groups of patients, as presented in this series. It is noteworthy that none of these criteria rely on histopathology. As for most drug reactions, except those with a particular feature, such as TEN and AGEP, the histopathological aspect of DRESS syndrome is not well described in the literature. In textbooks, the syndrome is either not described on histopathological grounds,6 or it is only stated that it may present with various histopathological features, including spongiotic dermatitis, erythema multiformis or aspects of TEN.17 In Lever’s Histopathology of the Skin, it is described as a variant of exanthemic drug reactions, where eosinophils and scattered apoptotic keratinocytes are commonly but not always seen.18 In their British Journal of Dermatology (2015) 173, pp50–58
56 Histopathology of DRESS syndrome, N. Ortonne et al.
(a)
(b)
(c)
(d)
Fig 2. Drug rash with eosinophilia and systemic symptoms (DRESS) syndrome with multiple different inflammatory patterns. (a) In this unique section of a DRESS syndrome skin biopsy, three distinct inflammatory patterns can be observed: (b) a multilocular pustule (arrowheads), (c) a spongiotic dermatitis with a vesicle containing Langerhans’ cells, and (d) foci of vacuolar or lichenoid interface dermatitis at the dermal– epidermal junction and within a follicular adnexa.
clinicopathological study, Chiou et al.8 highlighted the polymorphous histological expression of DRESS syndrome, which may present with various inflammatory patterns. More recently, in a series of 27 cases, Walsh et al.9 found that the most frequent inflammatory changes were a spongiotic dermatitis or basal cell vacuolization with apoptotic keratinocytes, the former being more frequent in patients with MPR. We found that the histopathological presentation of DRESS syndrome is highly variable, encompassing many inflammatory patterns, from a slight perivascular lymphocytic infiltrate to a pustular, AGEP-like, EM-like, eczematous or interface dermatitis, with the latter being the most frequent. What appeared to be a special feature was the association of different inflammatory patterns in a single specimen, a finding that was significantly more frequent in DRESS syndrome than in nondruginduced dermatoses and MPR. The histopathological presentation did not seem to correlate with the culprit drug, although relevant statistical analyses could only be obtained for allopurinol. Interestingly, dermal eosinophils were present in only 20% of cases and apoptotic keratinocytes in only half of cases. British Journal of Dermatology (2015) 173, pp50–58
Our results confirm that in a significant proportion of cases, atypical lymphocytes may be found in dermal infiltrates, so that differential diagnosis with cutaneous T-cell lymphoma may be challenging, especially with SS. In this context, the demonstration of a CD8+, granzyme B+ phenotype of the atypical cells may be a helpful feature, as SS neoplastic cells are CD4+. Taken together, our results further support a major role for CD8+ effector T cells and the perforin/granzyme pathway in drug reactions, especially in DRESS syndrome, as previously shown in MPR.19 Interestingly, the histological aspect of MPR, previously described in a large case series by Gerson et al.,20 shares many features with DRESS syndrome. Whether MPR and DRESS syndrome belong to the same spectrum of drug reactions, with MPR representing the less severe end of the spectrum, is therefore questionable. The main difference in our series was the intensity of the inflammatory changes, as the proportion of cases with epidermal lesions, as well as the density of inflammatory infiltrates, appeared to be lower in MPR than in DRESS syndrome, as previously shown.7 Whether the severity of skin inflammation and the extension © 2015 British Association of Dermatologists
Histopathology of DRESS syndrome, N. Ortonne et al. 57 Table 3 Phenotypical comparison between patients with drug rash with eosinophilia and systemic symptoms (DRESS) syndrome and maculopapular rash (MPR)
Proportion of CD8+ dermal cells (n = 22/n = 18) ≤ 5% 5–50% > 50% Presence of epidermotropic granzyme B+ cells (n = 20/17) Proportion of dermal granzyme B+ cells (n = 21/n = 11) Score 1: ≤ 5% Score 2: 5–50% Score 3: > 50% Proportion of dermal FoxP3+ cells (n = 12/n = 14) ≤ 5% 5–50% > 50%
DRESS (n = 24)
MPR (n = 20)
0 4 (18) 18 (82) 18 (90)
1 3 14 7
(5) (17) (78) (41)
P-value 080
< 001
4 (19) 13 (62) 4 (19)
4 (36) 3 (27) 4 (36)
< 001
8 (67) 4 (33) 0
6 (43) 7 (50) 1 (6)
060
Values are given as n (%).
Fig 3. Phenotype of lymphocytes in drug rash with eosinophilia and systemic symptoms (DRESS) syndrome. (a) More than 50% of lymphocytes, both in the epidermis and the dermis, are CD8+ T cells. (b) Scattered FoxP3+ lymphocytes are present within the infiltrate (arrowheads). (c) DRESS syndrome biopsy showing many granzyme B+ lymphocytes both in the dermis and in the epidermis with typical cytoplasmic granular staining. (d) Double-staining shows that the atypical medium-sized lymphocytes seen in the papillary dermis and around the capillaries in this sample express CD8 (arrowheads).
(a)
(b)
(c)
(d)
to extracutaneous sites in DRESS syndrome, in contrast to MPR, are due to differences in cytotoxic effector cells and/or the infiltration of Tregs is therefore an interesting issue. FoxP3+ Tregs were recently reported to be dramatically expanded in blood during the acute phase of DRESS syndrome.13 Although both MPR and DRESS syndrome were characterized by a significant infiltration of CD8+ and cytotoxic granzyme B+ cells, we found that DRESS syndrome with a severe phenotype, presenting with an erythroderma and/or bullae, had significantly more granzyme B+ cells. These cases also presented with more inflammatory changes, as the number of associated inflammatory patterns was significantly higher than in MPR and DRESS syndrome presenting with a MPR. A potential role for skin-infiltrating Tregs in the limitation of skin lesions in fixed drug reactions, in contrast to TEN, has already been suggested, as sequential biopsies have © 2015 British Association of Dermatologists
proven that the proportion of these cells in skin infiltrates increases progressively.21 We found that FoxP3+ Tregs were not as numerous as expected within the skin infiltrates during the acute phase of DRESS syndrome. We speculate that DRESS syndrome is actually characterized by a dramatic expansion of Tregs in blood, while effector CD8+ T cells are recruited in the skin, where they can exert cytotoxic functions because of a lower proportion of Tregs recruited at the same time from blood. The fact that different cytokinic environments may coexist in blood and skin, leading to such differences, remains speculative and requires further investigation. The major role played by expanded activated T cells in DRESS syndrome has already been demonstrated. In particular, it has been shown recently that expanded CD8+ T-cell populations, found in all the involved organs, are directed against herpesviruses, especially EBV. This suggests that DRESS syndrome may also be British Journal of Dermatology (2015) 173, pp50–58
58 Histopathology of DRESS syndrome, N. Ortonne et al.
regarded as a multiorgan antiviral T-cell response.12 For this reason, we investigated whether EBV – a putative target of the CD8+ T cell – was present in the skin. With the exception of one case, we failed to demonstrate the recurrent presence of EBV in situ. We also found no significant infiltration of CD56+ and CD123+ cells. This finding further suggests that the effector phase of DRESS mostly relies on adaptive immunity, with the activation of cytotoxic T cells. In addition to lymphocyte phenotyping, T-cell clonality probably represents an important diagnostic criterion for differential diagnosis between DRESS syndrome and cutaneous T-cell lymphomas. A T-cell clone in skin or blood was only detected in two of 17 investigated cases from this series. Interestingly, none of these cases showed atypical T cells in the skin. This is in agreement with a previous study in which T-cell clones were only detected in blood of patients with DRESS syndrome.10 Of note, in most cases, the search for a cutaneous T-cell clone was performed on paraffin-embedded tissues, demonstrating that, in difficult cases, this can be done easily by dermatopathologists. In conclusion, various inflammatory patterns are observed in DRESS syndrome, and these different patterns are often seen to be associated in a single biopsy, which may represent a histopathological clue in diagnosis. Effector lymphocytes, at least in skin, comprise a high proportion of polyclonal CD8+ granzyme B+ lymphocytes. The histopathological presentations and the cutaneous effector cells in DRESS and MPR show some overlap, but DRESS is characterized by more inflammation and more granzyme B+ effector cells, especially when patients present with an erythroderma and/or bullae.
4
5
6 7
8
9
10
11
12
13
14
Acknowledgments We thank Laetitia Gregoire, Unite de Recherche Clinique (URC), Henri Mondor Hospital, and Audrey Colin from the Department of Dermatology, Henri Mondor Hospital, for their help in presenting the study to the ethics committee (Comite de Protection des Personnes, Ile de France IV).
15
16 17
References 18 1 Ben m’rad M, Leclerc-Mercier S, Blanche P et al. Drug-induced hypersensitivity syndrome: clinical and biologic disease patterns in 24 patients. Medicine (Baltimore) 2009; 88:131–40. 2 Kardaun SH, Sekula P, Valeyrie-Allanore L et al. Drug reaction with eosinophilia and systemic symptoms (DRESS): an original multisystem adverse drug reaction. Results from the prospective RegiSCAR study. Br J Dermatol 2013; 169:1071–80. 3 Tohyama M, Hashimoto K, Yasukawa M et al. Association of human herpesvirus 6 reactivation with the flaring and severity of
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drug-induced hypersensitivity syndrome. Br J Dermatol 2007; 157:934–40. Seishima M, Yamanaka S, Fujisawa T et al. Reactivation of human herpesvirus (HHV) family members other than HHV-6 in druginduced hypersensitivity syndrome. Br J Dermatol 2006; 155:344–9. Shiohara T, Inaoka M, Kano Y. Drug-induced hypersensitivity syndrome (DIHS): a reaction induced by a complex interplay among herpesviruses and antiviral and antidrug immune responses. Allergol Int 2006; 55:1–8. Weedon D. Weedon’s Skin Pathology, 3rd edn. Edinburgh: Churchill Livingstone, 2010. Chi MH, Hui RC, Yang CH et al. Histopathological analysis and clinical correlation of drug reaction with eosinophilia and systemic symptoms (DRESS). Br J Dermatol 2014; 170:866–73. Chiou CC, Yang LC, Hung SI et al. Clinicopathological features and prognosis of drug rash with eosinophilia and systemic symptoms: a study of 30 cases in Taiwan. J Eur Acad Dermatol Venereol 2008; 22:1044–9. Walsh S, Diaz-Cano S, Higgins E et al. Drug reaction with eosinophilia and systemic symptoms (DRESS): is cutaneous phenotype a prognostic marker for outcome? A review of clinicopathological features of 27 cases. Br J Dermatol 2013; 168:391–401. Cordel N, Lenormand B, Courville P et al. Study of the clonality of cutaneous and blood lymphocytes during drug-induced hypersensitivity in 6 patients. Ann Dermatol Venereol 2004; 131:1059–61. Chung WH, Hung SI, Yang JY et al. Granulysin is a key mediator for disseminated keratinocyte death in Stevens-Johnson syndrome and toxic epidermal necrolysis. Nat Med 2008; 14:1343–50. Picard D, Janela B, Descamps V et al. Drug reaction with eosinophilia and systemic symptoms (DRESS): a multiorgan antiviral T cell response. Sci Transl Med 2010; 2:46ra62. Takahashi R, Kano Y, Yamazaki Y et al. Defective regulatory T cells in patients with severe drug eruptions: timing of the dysfunction is associated with the pathological phenotype and outcome. J Immunol 2009; 182:8071–9. Kardaun SH, Sidoroff A, Valeyrie-Allanore L et al. Variability in the clinical pattern of cutaneous side-effects of drugs with systemic symptoms: does a DRESS syndrome really exist? Br J Dermatol 2007; 156:609–11. Delfau-Larue MH, Petrella T, Lahet C et al. Value of clonality studies of cutaneous T lymphocytes in the diagnosis and follow-up of patients with mycosis fungoides. J Pathol 1998; 184:185–90. Bourgeois GP, Cafardi JA, Groysman V et al. A review of DRESSassociated myocarditis. J Am Acad Dermatol 2011; 66:e229–36. McKee PH, Calonje E, Granter SR. Pathology of the Skin with Clinical Correlations, 3rd edn, vol. 1. London: Elsevier Mosby, 2005. Elder DE. Lever’s Histopathology of the Skin, 10th edn. Philadelphia, PA: Lippincott Williams and Wilkins, 2009. Yawalkar N, Egli F, Hari Y et al. Infiltration of cytotoxic T cells in drug-induced cutaneous eruptions. Clin Exp Allergy 2000; 30:847–55. Gerson D, Sriganeshan V, Alexis JB. Cutaneous drug eruptions: a 5-year experience. J Am Acad Dermatol 2008; 59:995–9. Mizukawa Y, Yamazaki Y, Shiohara T. In vivo dynamics of intraepidermal CD8+ T cells and CD4+ T cells during the evolution of fixed drug eruption. Br J Dermatol 2008; 158:1230–8.
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