Hyperkeratotic hand eczema: Eczema or not?

Abstract Background Hyperkeratotic hand eczema (HHE) is a typical clinical hand eczema subtype with a largely unknown pathophysiology. Objective To investigate histopathology, expression of keratins (K), epidermal barrier proteins, and adhesion molecules in HHE. Methods Palmar skin biopsies (lesional and perilesional) were obtained from seven HHE patients and two healthy controls. Moreover, 135 candidate genes associated with palmoplantar keratoderma were screened for mutations. Results Immunofluorescence staining showed a significant reduction of K9 and K14 in lesional skin. Upregulation was found for K5, K6, K16, and K17 in lesional skin compared with perilesional and healthy palmar skin. Further, upregulation of involucrin and alternating loricrin staining, both in an extracellular staining pattern, was found. Filaggrin expression was similar in lesional, perilesional, and control skin. No monogenetic mutations were found. Conclusion Currently, the phenotype of HHE is included in the hand eczema classification system; however, it can be argued whether this is justified. The evident expression of filaggrin and involucrin in lesional skin does not support a pathogenesis of atopic eczema. The upregulation of K6, K16, and K17 and reduction of K9 and K14 might contribute to the underlying pathogenesis. Unfortunately, comparison with hand eczema studies is not possible yet, because similar protein expression studies are lacking.

classification system in which all clinical subtypes, including HHE, could have identifiable causes of hand eczema such as atopic dermatitis (AD), exposure to irritants, or to contact allergens in sensitized persons. 2 The pathophysiology of this well-described monomorphic subtype is largely unknown and it could be questioned whether it should be classified as eczema.
Palmar hyperkeratotis is seen in other diseases as well, such as palmar psoriasis and palmar plantar keratoderma (PPK). Palmar psoriasis resembles HHE. It was reported that it can be distinguished from psoriasis by lack of scaling typical for psoriasis, but the clinical distinction is very problematic. 6 PPK is an umbrella term for any form of persistent thickening of the epidermis at palmar and/or plantar skin and includes both genetic and acquired conditions. Literature about PPK showed that mutations in several keratin genes can result in a weakened cell cytoskeleton resulting in abnormally thickened and keratotic palmoplantar skin. 7,8 Similarly, mutations in genes encoding desmogleins (DSG-1-4), desmocollins (DSC1-3), desmoplakin (DSP), plakoglobin (PG), plakophilins (PKP1-3), and corneodesmosin are associated with palmar hyperkeratosis. 9,10 Here we present a case series of patients with a clinical phenotype of HHE. The aim of this study was to gain more insight into the etiology of this phenotype. Therefore, we have investigated the histopathology, the expression of different palmar keratins, epidermal barrier proteins, and adhesion molecules in lesional and nonlesional skin of patients with the clinical phenotype of HHE. In addition, we screened all patients for variants in genes related to PPK.

| Study design and participants
This pilot study was conducted at the Dermatology Department of the University Medical Center Groningen, a tertiary referral center for hand eczema, between August 2018 and December 2018. Adult patients with a clinical phenotype of HHE were eligible. 1,2 Exclusion criteria were a current diagnosis of AD, 11 topical corticosteroid use within the last 2 weeks, immunosuppressive/immunomodulatory treatment or ultraviolet radiation therapy within the last 4 weeks, contact allergies with clinical relevance to the hands in which exposure to allergens is not avoided, a history of psoriasis or psoriasis lesions elsewhere on the body, and other (genetic) skin diseases or infections of the hands.

| Clinical characteristics
The following data were collected: age; sex; onset of symptoms; history of AD; history of allergic asthma (regarding Global Initiative for Asthma guideline) 12 ; history of allergic rhinitis (regarding Allergic Rhinitis and its Impact on Asthma guideline) 13 ; specific inhalant immunoglobulin IgE allergens; atopy score <3 points (probability of AD is very small), 3 to 9 points (possible diagnosis of AD), and >10 points (reliable diagnosis of AD) 14 ; patch test results; occupation; workrelated exposure to irritants 15 ; and foot involvement.

| Skin samples
Four biopsies (3 mm) were obtained, two for histopathology and two for immunofluorescence (IF) microscopy from lesional and perilesional (noninflamed) palmar skin of seven patients. In addition, 3-mm samples from the hypothenar region of the palms of two healthy controls (HCs) were taken for comparison. HC were age and sex matched. The study was ethically approved by the local review board (M18.228998).

| Immunofluorescence staining
Skin biopsies for IF staining were snap frozen in liquid nitrogen . For IF   staining antibodies against K1, K2e, K5, K6, K9, K10, K14, K15, K16,   K17, DSG-1, DSG-3, plakophilin-1, plakoglobin, desmoplakin (rod), filaggrin, loricrin, involucrin, and corneodesmosin were used (Table S1). The immunostaining pattern and intensity in the epidermis were examined by three independent observers (MB, HP, and KP). The intensity was scored semiquantitatively using a global assessment on a continuous scale from 0 (negative) to 4 (1+, 2+, 3+, or 4+). The mean scores of the observers were used for the final conclusion.  16,17 Because no pathogenic or likely pathogenic mutations were identified in the 135 PPK-related genes that could be associated with the HHE phenotype, a second analysis was performed. Because all seven patients shared a strikingly similar clinical phenotype, we hypothesized that all patients might carry a variant in the same, as yet unknown, gene. To test this hypothesis an open-exome cohort analysis was performed. Gene variant analysis was performed using data analysis pipeline to filter out late-onset genes (eg, BRCA1).

| Clinical characteristics
All seven patients were male. Patients had a mean age of onset of 50.7 years (range 38-61 years) and a mean disease duration of 6.9 years (range 1.5-20 years; Table 1

| Gene analysis
Analysis of the 135 PPK-related genes in the seven patients did not identify any pathogenic or likely pathogenic variants that could be associated with the HHE phenotype. In total, three pathogenic mutations and four variants of unknown significance (VOUS) were In general, the pathogenesis of hand eczema remains largely unclear and is seen as a complex interaction between endogenous and exogenous factors. An exogenous factor for developing hand eczema is repeated contact with irritating agents and factors (friction). 1 Irritants induce a disruption of the skin barrier, and lead to an inflammatory reaction, mainly mediated by the innate immunity. 18 K6/16/17 are early barrier alarmins and upregulation of these keratins is seen in both wounding and exposure to irritants, which results in keratinocyte hyperproliferation and hyperkeratosis. 19 Irritants induce also an early expression of involucrin. 20,21 Similar results were found in our study. However, this could not sufficiently cover the whole etiology because not all patients with HHE had exposure to irritants.
Probably, HHE patients have a pre-existent skin barrier problem where exposure to irritating agents and factors eventually leads to secondary dysregulation of the immune system. 22 The skin barrier problem is an underlying etiology in AD. 22,23 A two-to four-fold increased risk to develop hand eczema was found in patients with a previous or current history of AD. 24 However, in the current study only one patient had a history of AD in childhood. Three patients had a possible atopic skin diathesis based on the atopy score. 14 Four patients had mucosal atopy, although previous studies showed no association between mucosal atopy and an increased risk of developing hand eczema. [25][26][27] The histopathology of all lesional skin biopsies in this study presented confluent parakeratosis, plasma in the parakeratotic foci, presence of the stratum granulosum, epidermal hyperplasia, and spongiosis. These morphological features were described in HHE before. 28 However, Park et al described the histological changes in biopsies of 25 HHE patients and 16 patients with palmar psoriasis and found no significant differences. 29 It is therefore questionable if differentiation based on histopathology is possible. Summarizing, both histopathology and contributing etiological factors, such as exposure to irritants and a history of atopic eczema, could not completely explain the pathogenesis of this clinical phenotype.
Circumscribed hyperkeratosis of the palms is also seen in palmar psoriasis. The clinical distinction between HHE and palmoplantar psoriasis is difficult when plaques are only located on the palms. [29][30][31] Only few studies have investigated both diseases. 29,30,32,33 Lillis et al investigated interleukin-23 (IL-23), which stimulates T helper 17 (Th17) cell survival and proliferation, and found a significant upregulation of IL-23 which did not significantly differ between HHE and (palmar) psoriasis. 30 Possibly, the inflammation in the clinical phenotype of HHE is, as psoriasis, more Th17 driven. This is also supported by the strong lesional K17 staining in our study which plays an important role in the pathogenesis of psoriasis. 19,34,35 K17 is a stimulator of psoriatic T cells (Th-17) and producer of specific cytokines (IL-17) which in turn stimulate K17, again leading to an "autoimmune feedback loop." 36,37 This "feedback loop" might explain the persistent palmar hyperkeratosis at the same location of this clinical phenotype. The persistent plaques were also described by Hersle and Mobacken, who noticed after a follow-up of 10 years an almost identical clinical monomorphic picture. 31 By contrast, other clinical subtypes of hand eczema, as well as AD, show more polymorphic skin lesions which tend to vary in appearance and location over time. 1,11,38 Future studies should evaluate whether the phenotype of HHE is a variant of (localized) psoriasis.
Furthermore, a common denominator in the pathophysiology of chronic inflammatory skin diseases such as AD, hand eczema, and psoriasis seems to be skin barrier impairment. [39][40][41] We studied involucrin, loricrin, and filaggrin because these proteins have a major role in forming the cornified envelope. Involucrin provides a scaffold to which other proteins, such as loricrin, subsequently cross-link to form the cornified envelope. 42 Filaggrin contributes herein by, among others, aggregating keratin filaments and organizing lipid bilayers. 43 Mutations in FLG are known to be a risk factor for the development of AD. An independent association of FLG mutations and hand eczema has not been found. 44 involucrin. 41 In our study involucrin was strongly presented in an ECS staining pattern through the complete spinous layer. Loricrin was downregulated and alternately presented in an ECS pattern. Perilesional and HC skin showed unaltered cytoplasmic staining for both in the upper stratum spinous and granular layers. The early expression of involucrin was described in other hyperproliferative diseases, such as psoriasis and PPK, but an ECS staining pattern for both proteins has not been described before. 21,51,52 We hypothesize that the ECS staining pattern of involucrin and loricrin might be explained by an increased keratinocyte proliferation, whereby the cornified envelope is prematurely formed and involucrin and loricrin are earlier attached underneath the plasma membrane instead of the cytoplasm. Another remarkable finding in our study was the upregulation of involucrin, which has not been described in hand eczema before, although sufficient studies are lacking. The opposite, a downregulation of involucrin, is described in AD. Therefore, possibly other cytokine profiles result in palmar hyperkeratosis in HHE. 40,41,48,50,53 Hereditary PPK is another group of skin diseases with palmar hyperkeratosis, which provides more insight about the association of different keratins, desmosomal proteins, and epidermal barrier proteins with palmoplantar hyperkeratosis. [8][9][10][54][55][56][57][58] In this study no monogenetic mutations related to palmar hyperkeratosis were found. DSG-3 has a strong basal distribution associated with the proliferating cells, whereas DSG-1 expression increases from the basal to the granular layer. However, DSG-1 and DSG-3 staining in this study were both panepidermally expressed in lesional skin and showed remarkable granular staining for DSG-1. Desmogleins are in continuous turnover in the desmosome and are discarded by cellular uptake to be destroyed or recycled. 61 Perhaps the abnormal differentiation and disturbed forming of the cornified envelope results in a higher DSG-1 turnover and uptake, whereby DSG-1 is not only expressed in the membrane but also in the cytoplasm. Further research should elucidate whether the abnormal desmosomal and keratin staining represent a primary defect in the pathophysiology of HHE, or are of secondary nature.
One of the limitations of this study is the relatively small study sample, though clinical presentations were very uniform. Another limitation is that IF staining is a strong diagnostic method in case of evident differences, while smaller differences can be missed.
Furthermore, it is difficult to compare our IF data with previous literature on other palmar skin diseases, as most studies focused on nonridged skin which differs from ridged skin we assessed. 62 In conclusion, the phenotype of HHE is currently included in