Biomarkers of chronic hand eczema and methods of treatment

Abstract

Provided herein are methods for diagnosis and treatment of chronic hand eczema (CHE) using skin surface biomarkers. The methods include obtaining from a subject skin samples, detecting certain biomarkers, determining the levels of the biomarkers, comparing the levels to reference levels, and determining whether the subject has CHE.

Description

Attorney Docket No.: MS-0050-01-US-NPBIOMARKERS OF CHRONIC HAND ECZEMA AND METHODS OF TREATMENT CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U. S. C. § 119(e) to U. S. Provisional Application No. 63 / 738,394, filed December 23, 2024, which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates generally to methods for diagnosing and treating chronic hand eczema (CHE) by targeting specific molecular biomarkers.BACKGROUND

[0003] Chronic hand eczema (CHE) is an inflammatory skin condition that significantly impairs the quality of life of patients (Agner et al., J Eur Acad Dermatol Venereal., 2020; Thyssen et al., Contact Dermatitis. 2010). It is the most prevalent occupational skin disease, affecting approximately 15% of individuals over their lifetime. (Scalone et al., Br J Dermatol.2015; Quaade et al. Contact Dermatitis, 2021) Typically, it starts in the early to mid-twenties, with a higher incidence among females. The clinical presentation of CHE is not uniform, and distinguishing it from other conditions, such as psoriasis and tinea, can be challenging both on clinical and histopathological levels. (Wold et al., Curr Allergy Asthma Rep. 2014; Coenraads PJ, N Engl J Med. 2012; Park et al., J Am Acad Dermatol. 2017; Jay den etal., JCutan Pathol.2008)

[0004] Treatment options for this debilitating disease are limited and about 60% of severe CHE patients are refractory to standard therapies. Given these diagnostic and therapeutic challenges, there is a growing need for enriched insight into pathophysiological mechanisms, including the identification of novel biomarkers. To date, there are only a few molecular studies available on CHE, limiting the understanding of disease pathogenesis and, consequently, the development of effective novel therapeutics. While full-thickness biopsies are the gold standard of molecular skin investigations, these are difficult to obtain in CHE. The recently emerged tape stripping method has now been established as a useful alternative for molecular skin profiling. ThisAttorney Docket No.: MS-0050-01-US-NPminimally invasive procedure involves the repeated application and removal of adhesive tape to the affected skin, effectively capturing outer skin layers up to the stratum granulosum. This method has been extensively utilized to characterize immune and epidermal barrier biomarkers in conditions such as atopic dermatitis, psoriasis, and hidradenitis suppurativa. This method is a valuable tool not only in clinical trials and longitudinal studies but also holds potential for routine clinical practice applications.

[0005] So far, tape strip data on CHE are limited. The largest transcriptomic study has been performed by Solberg et al. using tape strips from 30 Danish CHE patients and 16 controls. However, non-lesional CHE skin was collected from the upper arm, rather than the hands, and only two tapes were obtained for transcriptomics, limiting the overall sensitivity of the dataset. Another transcriptomic study by Voorberg et al. used skin biopsies from 10 Dutch hand eczema patients only with vesicular subtype, combined with real-time quantitative PCR and immunohistochemistry, revealing differences in antimicrobials and epidermal differentiation markers in lesional vs non-lesional and lesional vs healthy controls (HC) skin. These data were further used by Rosenberg et al. to compare them with atopic dermatitis (AD) from the trunk and upper extremities highlighting increased interferon signaling in vesicular CHE as compared to AD. Additional proteomic studies conducted by Solberg et al. and Quaade et al. profiled skin and serum samples, respectively, demonstrating varying levels of type-1 and type-2 inflammation among CHE patients. However, most of these studies had a small sample size, were limited to European populations, did not have anatomically matched non-lesional samples and allowed patients to use topical steroids up to 24-48 hours prior to sample collection.

[0006] Thus, there is a need to identify CHE specific biomarkers in patients that can be used to identify effective treatment regimes.SUMMARY

[0007] The present disclosure addresses these issues by revealing CHE specific biomarkers that were obtained using tape stripping from patients with and without concomitant atopic dermatitis (AD). These biomarkers provide the molecular disease landscape of CHE.

[0008] In an aspect, the disclosure provides method of treating chronic hand eczema (CHE) in a subject in need thereof, the method comprising: (a) providing a surface skin sample from the subject obtained from one or more tape-strips applied to a skin surface, wherein application of the one or more tape-strips to the skin surface obtains skin materials to the tape-strip; (b) determining a level of one or more biomarkers in the surface skin sample; (c) comparing theAttorney Docket No.: MS-0050-01-US-NPlevels of the one or more biomarkers in the surface skin sample to a reference level of the one or more biomarkers to obtain a comparative level; (d) determining that the subject has CHE based on the comparative level; and (e) administering to the subject a CHE therapy.

[0009] In an embodiment, the levels of two or more biomarkers are determined in the surface skin sample. In an embodiment, the one or more biomarkers include a gene product of any of: GNLY; CD1B; CD1E; CD40; TIGIT; ICOS; CD28; IL3RA; CD96; CCR8; FOXP3; CTLA4; IRF4; TSLPR / CRLF2; and IL12B, or combinations thereof. In an embodiment, the one or more biomarkers include a gene product of any of: OX40 / TNFRSF4; CRLF2; IL7R; CD122 / IL2RB; JAK1; JAK2; and IL13, or combinations thereof. In an embodiment, the one or more biomarkers include a gene product of any of: KRT27; KRT71; KRTAP1-5; KRTAP5-3; KRTAP12-2; SPRR3; CCL7; NOS2; NEUROG1; OTX2: GPM6A ADGRL 3; OR6S1; OR4C6; OR5H14 FLG; LORICRIN; and KRT9, or combinations thereof. In an embodiment, said determining further comprises differentiating between: (a) subjects having CHE and having AD (CHE+AD); and (b) subjects having CHE in the absence of AD (CHE- AD). In an embodiment, the one or more biomarkers include a gene product of any of: RGS1; IL19; RCOR3; MTR; NPHP3; HSPA1A; HMGN3; REV3L; FAM229B; GPR31; MACC1; ADAMDEC1; IDO1; IDO2; ZBTB43; TRIM66; OAF; EEA1; SDSL; RSRC2; SPG11; TRIM69; FURIN; TVP23A; CTNS; VMO1; RGS9; CCDC57; SIGLEC12; or ENTHD1. In an embodiment, said determining further comprises differentiating between lesional and non-lesional skin in said subjects. In an embodiment, the one or more biomarkers include a gene product of any of: DPM3; FAM114A2; FZD3; PTPDC1; or PRM1, or combinations thereof. In an embodiment, the one or more biomarkers include a gene product of any of: GNLY; CD IB; CD1E; CD40; TIGIT; ICOS; CD28; IL3RA; CD96; CCR8; FOXP3; CTLA4; IRF4; IL12B; KRT27; KRT7; KRTAP1-5; KRTAP5-3; KRTAP12-2; KRT9; SPRR3; CCL7; NOS2); NEUROG1; OTX2: GPM6A; ADGRL3; OR6S1; OR4C6; OR5H14; FLG; LORICRIN; CD3D; CD3E; CD3G; CD4; CD5; CD8A; ITK; IL7R; GZMB; CD69; CD27; CTLA4; CD28; CD48; CD80; CD86; ICOS; XCL1; XCL2; CXCR1; CXCR2; CXCR4; CCR1; CCR7; IL12RB2; IFNGR1; IFNGR2; MX1; CCL7; CCL13, CCL17; CCL20; CCL21; CCL22; CCL24; GATA3; CX3CL1; CXCL6; CXCL9; CXCL10, CXCL11; CXCL13; CXCL17; IFNA1; IFNA2; IL7; IL12; IL13; IL15; IL18; IL19; IL22; IL23; IL34; IL12B; IL17F; LCN2; PI3; LYG1; IL23R; IDO1; IDO2; FCGR3B; S100P; OASL; PI3; IFNA2; TGFB3; LAT2; RORC; CD27; CD28; CD48; CD80; CD86; IL1RAP; TRAIL / TNFSF10; CCR2; RUNX1; IL27RA; SOCS2; OX40 / TNFRSF4; ISG20; CD137 / TNFRSF9; TSLPR / CRLF2; IL7R; CD122 / IL2RB; IL15RA; IRF1, FCER1A, AHR, or a combination thereof. In an embodiment, the gene product is an RNA transcript, aAttorney Docket No.: MS-0050-01-US-NPprotein, or both. In an embodiment, said biomarker is down regulated in the subject. In an embodiment, said biomarker is up-regulated in the subject. In an embodiment, said determining uses one or more of an RNA-sequencing analysis, a PCR analysis, a qPCR analysis, a proteomic analysis, or combinations thereof. In an embodiment, said determining uses an RNA-sequencing analysis. In an embodiment, the level of the one or more biomarkers is determined by measuring mRNA levels of the biomarker. In an embodiment, said treatment comprises a topical treatment, oral medication, a biologic, or combinations thereof. In an embodiment, the treatment comprises administering a therapeutic selected from the group consisting of: rocatinlimab, telazorlimab, tezepelumab, ordesekimab, amlitelimab, AMG 714, PRV-015, delgocitinib, peficitinib, ustekinumab, tofacitnib, upadacitinib, baricitinib, roxulitinib. lebrikizumab, dupilumab, and tralokinumab.

[0010] In another aspect, the disclosure provides a method of diagnosing chronic hand eczema in a subject in need thereof, the method comprising: (a) providing a surface skin sample from the subject obtained from one or more tape-strips applied to a skin surface, wherein application of the one or more tape-strips to the skin surface obtains skin materials to the tape-strip; (b) determining a level of one or more biomarkers in the surface skin sample; (c) comparing the levels of the one or more biomarkers in the surface skin sample to a reference level of the one or more biomarkers to obtain a comparative level; (d) determining that the subject has CHE based on the comparative level.

[0011] In an embodiment, the levels of two or more biomarkers are determined in the surface skin sample. In an embodiment, the one or more biomarkers include a gene product of any of: GNLY; CD1B; CD1E; CD40; TIGIT; ICOS; CD28; IL3RA; CD96; CCR8; FOXP3; CTLA4; IRF4; TSLPR / CRLF2; and IL12B, or combinations thereof. In an embodiment, the one or more biomarkers include a gene product of any of: OX40 / TNFRSF4; CRLF2; IL7R; CD122 / IL2RB; JAK1; JAK2; and IL13, or combinations thereof. In an embodiment, the one or more biomarkers include a gene product of any of: KRT27; KRT71; KRTAP1-5; KRTAP5-3; KRTAP12-2; SPRR3; CCL7; NOS2; NEUROG1; OTX2; GPM6A; ADGRL3; OR6S1; OR4C6; OR5H14. FLG; LORICRIN; and KRT9, or combinations thereof. In an embodiment, said determining further comprises differentiating between: (a) subjects having CHE and having AD (CHE+AD); and (b) subjects having CHE in the absence of AD (CHE- AD). In an embodiment, the one or more biomarkers include a gene product of any of: RGS1; IL19; RCOR3; MTR; NPHP3; HSPA1A; HMGN3; REV3L; FAM229B; GPR31; MACC1; ADAMDEC1; IDO1; IDO2; ZBTB43; TRIM66; OAF; EEA1; SDSL; RSRC2; SPG11; TRIM69; FURIN; TVP23A; CTNS; VMO1; RGS9; CCDC57; SIGLEC12; or ENTHD1. In anAttorney Docket No.: MS-0050-01-US-NPembodiment, said determining further comprises differentiating between lesional and non-lesional skin in said subjects. In an embodiment, the one or more biomarkers include a gene product of any of: DPM3; FAM114A2; FZD3; PTPDC1; or PRM1, or combinations thereof.

[0012] In an embodiment, the one or more biomarkers include a gene product of any of: GNLY; CD1B; CD1E; CD40; TIGIT; ICOS; CD28; IL3RA; CD96; CCR8; FOXP3; CTLA4; IRF4; IL12B; KRT27; KRT7; KRTAP1-5; KRTAP5-3; KRTAP12-2; KRT9; SPRR3; CCL7; NOS2); NEUROG1; OTX2: GPM6A; ADGRL3; OR6S1; OR4C6; OR5H14; FLG; LORICRIN; CD3D; CD3E; CD3G; CD4; CD5; CD8A; ITK; IL7R; GZMB; CD69; CD27; CTLA4; CD28; CD48; CD80; CD86; ICOS; XCL1; XCL2; CXCR1; CXCR2; CXCR4; CCR1; CCR7; IL12RB2; IFNGR1; IFNGR2; MX1; CCL7; CCL13, CCL17; CCL20; CCL21; CCL22; CCL24; GATA3; CX3CL1: CXCL6; CXCL9: CXCL10, CXCL11; CXCL13; CXCL17; IFNA1; IFNA2; IL7; IL12; IL13; IL15; IL18; IL19; IL22; IL23; IL34; IL12B; IL17F; LCN2; PI3; LYG1; IL23R; IDO1; IDO2; FCGR3B; S100P; OASL; PI3; IFNA2; TGFB3; LAT2; RORC; CD27; CD28; CD48; CD80; CD86; IL1RAP; TRAIL / TNFSF10; CCR2; RUNX1; IL27RA; SOCS2; OX40 / TNFRSF4; ISG20; CD137 / TNFRSF9; TSLPR / CRLF2; IL7R; CD122 / IL2RB; IL15RA; IRF1, FCER1A, AHR. or a combination thereof. In an embodiment, the gene product is an RNA transcript, a protein, or both. In an embodiment, said biomarker is down regulated in the subject. In an embodiment, said biomarker is up-regulated in the subject. In an embodiment, said determining uses one or more of an RNA-sequencing analysis, a PCR analysis, a qPCR analysis, a proteomic analysis, or combinations thereof. In an embodiment, said determining uses an RNA-sequencing analysis. In an embodiment, the level of the one or more biomarkers is determined by measuring mRNA levels of the biomarker.

[0013] In another aspect, the disclosure provides a method of determining the efficacy of treatment of chronic hand eczema in a subject in need thereof, the method comprising: (a) providing a surface skin sample from the subject obtained from one or more tape-strips applied to a skin surface, wherein application of the one or more tape-strips to the skin surface obtains skin materials to the tape-strip; (b) determining a level of one or more biomarkers in the surface skin sample; (c) comparing the levels of the one or more biomarkers in the surface skin sample to a reference level of the one or more biomarkers to obtain a comparative level; (d) determining the efficacy of the CHE treatment based on the comparative level.

[0014] In an embodiment, the levels of two or more biomarkers are determined in the surface skin sample. In an embodiment, the one or more biomarkers include a gene product of any of: GNLY CD1B; CD1E; CD40; TIGIT; ICOS; CD28: IL3RA; CD96; CCR8; FOXP3; CTLA4; IRF4; TSLPR / CRLF2; and IL12B, or combinations thereof. In an embodiment, the one or moreAttorney Docket No.: MS-0050-01-US-NPbiomarkers include a gene product of any of: OX40 / TNFRSF4; CRLF2; IL7R; CD122 / IL2RB; JAK1; JAK2; and IL13, or combinations thereof. In an embodiment, the one or more biomarkers include a gene product of any of: KRT27; KRT71; KRTAP1-5; KRTAP5-3; KRTAP12-2; SPRR3; CCL7; NOS2; NEUROG1; OTX2; GPM6A; ADGRL3; OR6S1; OR4C6; OR5H14; FLG; LORICRIN; and KRT9, or combinations thereof. In an embodiment, said determining further comprises differentiating between: (a) subjects having CHE and having AD (CHE+AD); and (b) subjects having CHE in the absence of AD (CHE- AD). In an embodiment, the one or more biomarkers include a gene product of any of: RGS1; IL19; RCOR3; MTR; NPHP3; HSPA1A; HMGN3; REV3L; FAM229B; GPR31; MACC1; ADAMDEC1; IDO1; IDO2; ZBTB43; TRIM66; OAF; EEA1; SDSL; RSRC2; SPG11; TRIM69; FURIN; TVP23A; CTNS; VMO1; RGS9; CCDC57; SIGLEC12; or ENTHD1. In an embodiment, said determining further comprises differentiating between lesional and non-lesional skin in said subjects. In an embodiment, the one or more biomarkers include a gene product of any of: DPM3; FAM114A2; FZD3; PTPDC1; or PRM1, or combinations thereof. In an embodiment, the one or more biomarkers include a gene product of any of: GNLY; CD1B; CD1E; CD40; TIGIT; ICOS; CD28; IL3RA; CD96; CCR8; FOXP3; CTLA4; IRF4; IL12B; KRT27; KRT7; KRTAP1-5; KRTAP5-3; KRTAP12-2; KRT9; SPRR3; CCL7; NOS2); NEUROG1; OTX2; GPM6A; ADGRL3; OR6S1; OR4C6; OR5H14. FLG; LORICRIN; CD3D; CD3E; CD3G; CD4; CD5; CD8A; ITK; IL7R; GZMB; CD69; CD27; CTLA4; CD28; CD48; CD80; CD86; ICOS; XCL1; XCL2; CXCR1; CXCR2; CXCR4; CCR1; CCR7; IL12RB2; IFNGR1; IFNGR2; MX1; CCL7; CCL13; CCL17; CCL20; CCL21; CCL22; CCL24; GATA3; CX3CL1; CXCL6; CXCL9; CXCL10, CXCL11; CXCL13; CXCL17; IFNA1; IFNA2; IL7; IL12; IL13; IL15; IL18; IL19; IL22; IL23; IL34; IL12B; IL17F; LCN2; PI3; LYG1; IL23R; IDO1; IDO2; FCGR3B; S100P; OASL; PI3; IFNA2; TGFB3; LAT2; RORC; CD27; CD28; CD48; CD80; CD86; IL1RAP; TRAIL / TNFSF10; CCR2; RUNX1; IL27RA; SOCS2; OX40 / TNFRSF4; ISG20; CD137 / TNFRSF9; TSLPR / CRLF2; IL7R; CD122 / IL2RB; IL15RA; IRF1, FCER1A, AHR, or a combination thereof.Attorney Docket No.: MS-0050-01-US-NP

[0015] In another aspect, the disclosure provides a method of treatment of chronic hand eczema (CHE) in a subject in need thereof, the method comprising: (a) providing a first surface skin sample from the subject obtained from one or more tape-strips applied to a skin surface, wherein application of the one or more tape-strips to the skin surface obtains skin materials on the tape-strip and wherein the skin surface is from lesional skin; (b) determining a level of one or more biomarkers in the first surface skin sample; (c) providing a first surface skin sample from the subject obtained from one or more tape-strips applied to a skin surface, wherein application of the one or more tape-strips to the skin surface obtains skin materials on the tapestrip and wherein the skin surface is from non-lesional skin; (d) determining a level of one or more biomarkers in the second surface skin sample; (e) comparing the levels of the one or more biomarkers in said first surface skin sample to a reference level obtained from the one or more biomarkers in said second surface skin sample to obtain a comparative level; (f) determining that the subject has CHE based on the comparative level; and (g) administering to the subject a CHE therapy.

[0016] In an embodiment, the levels of two or more biomarkers are determined in the surface skin sample. In an embodiment, the one or more biomarkers include a gene product of any of: GNLY; CD1B; CD1E; CD40; TIGIT; ICOS; CD28; IL3RA; CD96; CCR8; FOXP3; CTLA4; IRF4; TSLPR / CRLF2; and IL12B, or combinations thereof. In an embodiment, the one or more biomarkers include a gene product of any of: OX40 / TNFRSF4; CRLF2; IL7R; CD122 / IL2RB; JAK1; JAK2; and IL13, or combinations thereof. In an embodiment, the one or more biomarkers include a gene product of any of: KRT27; KRT71; KRTAP1-5; KRTAP5-3; KRTAP12-2; SPRR3; CCL7; NOS2; NEUROG1; OTX2; GPM6A; ADGRL3; OR6S1; OR4C6; OR5H14; FLG; LORICRIN; and KRT9, or combinations thereof. In an embodiment, said determining further comprises differentiating between: (a) subjects having CHE and having AD (CHE+AD); and (b) subjects having CHE in the absence of AD (CHE- AD). In an embodiment, the one or more biomarkers include a gene product of any of: RGS1; IL19; RCOR3; MTR; NPHP3; HSPA1A; HMGN3; REV3L; FAM229B; GPR31; MACC1; ADAMDEC1; IDO1; IDO2; ZBTB43; TRIM66; OAF; EEA1; SDSL; RSRC2; SPG11; TRIM69; FURIN; TVP23A; CTNS; VMO1; RGS9; CCDC57; SIGLEC12; or ENTHD1. In an embodiment, said determining further comprises differentiating between lesional and non-lesional skin in said subjects. In an embodiment, the one or more biomarkers include a gene product of any of: DPM3; FAM114A2; FZD3; PTPDC1; or PRM1, or combinations thereof. In an embodiment, the one or more biomarkers include a gene product of any of: GNLY; CD IB; CD1E; CD40; TIGIT; ICOS; CD28; IL3RA; CD96; CCR8; FOXP3; CTLA4; IRF4; IL12B;Attorney Docket No.: MS-0050-01-US-NPKRT27; KRT7; KRTAP1-5; KRTAP5-3; KRTAP12-2; KRT9; SPRR3; CCL7; NOS2; NEUROG1; OTX2; GPM6A; ADGRL3; OR6S1; OR4C6; OR5H14; FLG; LORICRIN; CD3D; CD3E; CD3G; CD4; CD5; CD8A; ITK; IL7R; GZMB; CD69; CD27; CTLA4; CD28; CD48; CD80; CD86; ICOS; XCL1; XCL2; CXCR1; CXCR2; CXCR4; CCR1; CCR7; IL12RB2; IFNGR1; IFNGR2; MX1; CCL7; CCL13, CCL17; CCL20; CCL21; CCL22; CCL24; GATA3; CX3CL1; CXCL6; CXCL9; CXCL10, CXCL11; CXCL13; CXCL17; IFNA1; IFNA2; IL7; IL12; IL13; IL15; IL18; IL 19; IL22; IL23; IL34; IL12B; IL17F; LCN2; PI3; LYG1; IL23R; IDO1; IDO2; FCGR3B; S100P; OASL; PI3; IFNA2; TGFB3; LAT2; RORC; CD27; CD28; CD48; CD80; CD86; IL1RAP; TRAIL / TNFSF10; CCR2; RUNX1; IL27RA; SOCS2; OX40 / TNFRSF4; ISG20; CD137 / TNFRSF9; TSLPR / CRLF2; IL7R; CD122 / IL2RB; IL15RA; IRF1, FCER1A, AHR. or a combination thereof. In an embodiment, the gene product is an RNA transcript, a protein, or both. In an embodiment, said biomarker is down regulated in the subject. In an embodiment, said biomarker is up-regulated in the subject. In an embodiment, said determining uses one or more of an RNA-sequencing analysis, a PCR analysis, a qPCR analysis, a proteomic analysis, or combinations thereof. In an embodiment, said determining uses an RNA-sequencing analysis. In an embodiment, the level of the one or more biomarkers is determined by measuring mRNA levels of the biomarker. In an embodiment, said treatment comprises a topical treatment, oral medication, a biologic, or combinations thereof. In an embodiment, the treatment comprises administering a therapeutic selected from the group consisting of: rocatinlimab, telazorlimab, tezepelumab, ordesekimab. amlitelimab, AMG 714, PRV-015, delgocitinib, peficitinib, ustekinumab, tofacitnib, upadacitinib, baricitinib, roxulitinib, lebrikizumab, dupilumab, and tralokinumab.

[0017] In another aspect, the disclosure provides a method of determining the efficacy of treatment of CHE in a subject, comprising: (a) providing a first level of one or more biomarkers in a first surface skin sample from the subject; (b) administering to the subject a therapy for treating CHE; (c) providing a second level of one or more biomarkers in a second surface skin sample from the subject obtained at a later time; (d) comparing the first level of the one or more biomarkers to the second level of the one or more biomarkers to obtain a comparative level; and (e) determining the efficacy of the treatment based on the comparative level; wherein the first skin sample is obtained from a first set of one or more tape-strips applied to a skin surface, and the second skin sample is obtained from a second set of one or more tape-strips applied to a skin surface; and wherein application of the tape-strips to the skin surface obtains skin materials to the tape-strip.Attorney Docket No.: MS-0050-01-US-NP

[0018] In an embodiment, the levels of two or more biomarkers are determined in the surface skin sample. In an embodiment, the one or more biomarkers include a gene product of any of: GNLY; CD1B; CD1E; CD40; TIGIT; ICOS; CD28; IL3RA; CD96; CCR8; FOXP3; CTLA4; IRF4; TSLPR / CRLF2; and IL12B, or combinations thereof. In an embodiment, the one or more biomarkers include a gene product of any of: OX40 / TNFRSF4; CRLF2; IL7R; CD122 / IL2RB; JAK1; JAK2; and IL13, or combinations thereof. In an embodiment, the one or more biomarkers include a gene product of any of: KRT27; KRT71; KRTAP1-5; KRTAP5-3; KRTAP12-2; SPRR3; CCL7; NOS2; NEUROG1; OTX2; GPM6A; ADGRL3; OR6S1; OR4C6; OR5H14; FLG; LORICRIN; and KRT9, or combinations thereof. In an embodiment, said determining further comprises differentiating between: (a) subjects having CHE and having AD (CHE+AD); and (b) subjects having CHE in the absence of AD (CHE- AD). In an embodiment, the one or more biomarkers include a gene product of any of: RGS1; IL19; RCOR3; MTR; NPHP3; HSPA1A; HMGN3; REV3L; FAM229B; GPR31; MACC1; ADAMDEC1; IDO1; IDO2; ZBTB43; TRIM66; OAF; EEA1; SDSL; RSRC2; SPG11; TRIM69; FURIN; TVP23A; CTNS; VMO1; RGS9; CCDC57; SIGLEC12; or ENTHD1. In an embodiment, said determining further comprises differentiating between lesional and non-lesional skin in said subjects. In an embodiment, the one or more biomarkers include a gene product of any of: DPM3; FAM114A2; FZD3; PTPDC1; or PRM1, or combinations thereof. In an embodiment, the one or more biomarkers include a gene product of any of: GNLY; CD IB; CD1E; CD40; TIGIT; ICOS; CD28; IL3RA; CD96; CCR8; FOXP3; CTLA4; IRF4; IL12B; KRT27; KRT7; KRTAP1-5; KRTAP5-3; KRTAP12-2; KRT9; SPRR3; CCL7; NOS2); NEUROG1; OTX2; GPM6A; ADGRL3; OR6S1; OR4C6; OR5H14; FLG; LORICRIN; CD3D; CD3E; CD3G; CD4; CD5; CD8A; ITK; IL7R; GZMB; CD69; CD27; CTLA4; CD28; CD48; CD80; CD86; ICOS; XCL1; XCL2; CXCR1; CXCR2; CXCR4; CCR1; CCR7; IL12RB2; IFNGR1; IFNGR2; MX1; CCL7; CCL13, CCL17; CCL20; CCL21; CCL22; CCL24; GATA3; CX3CL1; CXCL6; CXCL9; CXCL10, CXCL11; CXCL13; CXCL17; IFNA1; IFNA2; IL7; IL12; IL13; IL15; IL18; IL19; IL22; IL23; IL34; IL12B; IL17F; LCN2; PI3; LYG1; IL23R; IDO1; IDO2; FCGR3B; S100P; OASL; PI3; IFNA2; TGFB3; LAT2; RORC; CD27; CD28; CD48; CD80; CD86; IL1RAP; TRAIL / TNFSF10; CCR2; RUNX1; IL27RA; SOCS2; OX40 / TNFRSF4; ISG20; CD137 / TNFRSF9; TSLPR / CRLF2; IL7R; CD122 / IL2RB; IL15RA; IRF1, FCER1A, AHR, or a combination thereof. In an embodiment, the gene product is an RNA transcript, a protein, or both. In an embodiment, said biomarker is down regulated in the subject. In an embodiment, said biomarker is up-regulated in the subject. In an embodiment, said determining uses one or more of an RNA-sequencing analysis, a PCR analysis, a qPCRAttorney Docket No.: MS-0050-01-US-NPanalysis, a proteomic analysis, or combinations thereof. In an embodiment, said determining uses an RNA-sequencing analysis. In an embodiment, the level of the one or more biomarkers is determined by measuring mRNA levels of the biomarker.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various embodiments of the invention and, together with the description, explain the principles of the invention. The drawings are not to be construed as limiting the invention, but rather as illustrative of certain embodiments thereof. At least a single-color drawing may be included in this patent's file.

[0020] Figure 1 provides an overview of the patient population, applied methods, and identified biomarkers. Tape strip-based RNA sequencing of 66 chronic hand eczema patients revealed multiple differentially expressed genes, highlighting significant inflammatory and epidermal barrier alterations. Chronic hand eczema patients with atopic dermatitis background showed a more pronounced type 2 inflammation, unlike the more type 1 skewed inflammation in those without such comorbidity.

[0021] Figure 2 provides an overview of the skin tape strip transcriptome. Tape strips from lesional and non-lesional skin of 66 chronic hand eczema (CHE) patients and their matching 20 healthy controls (HC) were sequenced and analyzed. Principal component analysis was calculated on the entire study cohort and is presented in aggregate (A) as well as stratified for the presence (B) or absence (C) of AD. Values in parentheses denote the percentage of variance explained by each principal component, representing how much of the overall variation in the data is captured along that axis. Points represent samples in 2D space, color-coded by sample type; underlying ellipses represent 95% level for a multivariate t-distribution. (D) Venn diagram of overlapping and unique upregulated (red text) and downregulated (blue text) DEG counts in the non-stratified cohort, when comparing LS vs NL (red circle), LS vs HC (blue circle), and NL vs HC (green circle). Intersection areas contain the number of shared DEGs across two or three comparisons. Bottom right box shows the number of genes not differentially expressed in any comparison. DEGs were defined by criteria fold change (|FCH )>1.5 and false discovery rate (FDR)<0.05. CHE, Chronic hand Eczema; HC, Healthy controls; LS, lesional skin; NL, non-lesional skin; PC, principal component.

[0022] Figure 3 shows the top 75 upregulated and downregulated genes in the common CHE phenotype highlighting multiple inflammatory and skin barrier changes.Attorney Docket No.: MS-0050-01-US-NP(A-B) Venn diagram of overlapping and unique DEG counts across comparisons, with circle overlaps indicating shared DEGs, utilizing criteria of fold change (|FCH|)>1.5 and false discover}' rate (FDR)<0.05. Bottom right box shows the number of genes not differentially expressed in any comparison. CHE+ADsamples were compared with CHE-'0samples for their overlapping DEGs in lesional or non-lesional vs HC, respectively. (C-D) Summary' heat maps of top 75 up- and downregulated differentially expressed genes (DEGs) of the common CHE phenotype. Heat map color intensity represents the normalized gene-wise mean expression; The numeric table shows FCHs; Asterisks denote significance levels. Top 75 genes were depicted by the maximum |FCH| across all common phenoty pe comparisons; Order of genes was determined by hierarchical clustering. (E-G) Boxplots of normalized expression levels for selected genes of the common CHE phenoty pe. Dots represent individual samples; Diamonds represent means; Horizontal lines represent the medians; Lower and upper hinges correspond to the 25th and 75th percentiles, respectively; Upper and lower whisker extends from the hinge to the most extreme value no further than 1.5 times the inter-quantile range. Y axis represents log2 normalized expression levels. LS, lesional skin; NL, non-lesional skin; HC, healthy controls skin; CHE+AD, patients with concomitant atopic dermatitis; CHE'AD. patients without concomitant atopic dermatitis; CHEALL: All chronic hand eczema patients, both with and without concomitant AD. +: FDR<0.1; *; FDR<0.05; **: FDR<0.01; ***: FDR<0.001.

[0023] Figure 4 shows the inflammatory profile of chronic hand eczema shows mixed type-1- and type-2-mediated inflammation. (A) Heat map showing the immune genes that are differentially expressed in both LS CHE+ADvs HC and LS CHE'AI)vs HC, utilizing criteria of fold change (|FCH|)>1.5 and false discovery' rate (FDR)<0.05. (B) Heat map of immune genes significantly differentially expressed in lesional CHE+ADvs HC and a reciprocal nonsignificance in lesional CHE’ADysHC. (C) Heat map of immune genes significantly differentially expressed in lesional CHE‘AI)vs HC and a reciprocal non-significance in lesional CHE+ADVS HC. Heat map color intensity represents the normalized gene-wise mean expression; The numeric table shows FCHs; Asterisks denote significance levels. Order of genes was determined by hierarchical clustering. CHE '1', patients with concomitant atopic dermatitis; CHE'AD, patients without concomitant atopic dermatitis; CHEALL: All chronic hand eczema patients, both with and without concomitant AD. +; FDR<0.1; *: FDR<0.05; **; FDR<0.01; ***: FDR<0.00L

[0024] Figure 5 shows a Gene-set analysis of T-helper 1, 2, 17, 22 and epidermal barrier differentiation. Lesional and non-lesional CHE samples stratified by the presence (A) and absence (B) of comorbid atopic dermatitis status were compared by their gene-set Z-score forAttorney Docket No.: MS-0050-01-US-NPmajor T-helper related pathways and epidermal barrier differentiation related pathways. Dots represent individual samples; diamonds represent means; horizontal lines represent the medians; lower and upper hinges correspond to the 25th and 75th percentiles, respectively; upper and lower whisker extends from the hinge to the most extreme value no further than 1.5 times the inter-quantile range. Y axis represents the calculated gene-set Z-score. LS, lesional skin; NL, non-lesional skin; HC, healthy controls skin. CHE+AD, patients with concomitant atopic dermatitis; CHEAD. patients without concomitant atopic dermatitis. *: FDR<0.05; **: FDR<0.01; ***: FDRcO. OOL

[0025] Figure 6 shows a correlation plot between inflammatory DEGs and clinical activity markers in lesional samples of patients with comorbid atopic dermatitis. (A) Lesional CHE+ADnormalized gene expression levels were correlated to their respective patients’ clinical severity scores using Spearman rank correlation method, producing a correlation matrix; We depicted the top 100 dysregulated inflammatory genes (by absolute fold-change) from all lesional vs non-lesional and / or lesional vs HC comparisons. Asterisks denote statistically significant correlations. Clinical markers are colored in blue, genes that have at least one moderate correlation (|p|>0.4) are colored in red. Order of genes / clinical markers was determined by hierarchical clustering. Green box: DLQI and Pain VAS cluster; Orange box: HECSI and mTLSS cluster. (B-F) Sperman correlation between gene expression levels and clinical severity scores. Dots represent rank, the diagonal red line is the fitted linear model, with confidence intervals in gray. CHE+AD: chronic hand eczema patient with concomitant atopic dermatitis; HECSI. hand eczema severity index; mTLSS. modified total lesion symptoms score; DLQI, dermatology life quality index; VAS: visual analog scale.

[0026] Figure 7 shows a correlation plot between inflammatory DEGs and clinical activity markers in lesional samples of patients without comorbid atopic dermatitis. (A) Lesional CHE'AI)normalized gene expression levels were correlated to their respective patients’ clinical severity scores using Spearman rank correlation method, producing a correlation matrix; We depicted the top 100 dysregulated inflammatory' genes (by absolute fold-change) from all lesional vs non-lesional and / or lesional vs HC comparisons. Asterisks denote statistically significant correlations. Clinical markers are colored in blue, genes that have at least one moderate correlation (|p|>0.4) are colored in red. Order of genes / clinical markers was determined by hierarchical clustering. Green box: Clinical severity scores cluster; Orange box: Type-1 immunity cluster; Pink box: Type-2 immunity cluster. (B-D) Sperman correlation between gene expression levels and clinical severity scores. Dots represent rank, the diagonal red line is the fitted linear model, with confidence intervals in gray. CHEAD, chronic handAttorney Docket No.: MS-0050-01-US-NPeczema patient without concomitant atopic dermatitis; HECSI, hand eczema severity index; mTLSS. modified total lesion symptoms score; DLQI, dermatology life quality index; VAS: visual analog scale.

[0027] Figure 8 shows the variance of gene regulation in lesional vs non-lesional CHE skin. (A) Variances of normalized gene expression levels were calculated and compared across all genes in the entire CHE cohort between lesional and non-lesional CHE skin. Diagram shows genes that had a significantly higher variance in non-lesional samples (purple circle), lesional samples (brown circle), or a non-significant variance difference (red circle). (B) Enrichment analysis of the 4,595 genes showed a significantly higher variance in non-lesional vs lesional CHE using the EnrichR tool (Reactome database). The X axis depicts the combined enrichment score41, the Y axis displays the -log₁₀(adjusted p-value), the color gradient represents the calculated Z-score41, and circle size corresponds to the number of genes overlapping with the enrichment term; the dashed blue line represents an adjusted p-value of 0.05. Va Variance; LS, lesional skin; NL, non-lesional skin.

[0028] Figure 9 shows an enrichment analysis using the “Reactome” database demonstrates enrichment of inflammatory pathways and the under-enrichment of skin structure pathways in lesional CHE skin. Lesional CHE and HC samples were compared for differential gene expression; upregulated and downregulated DEGs w ere analyzed separately via the EnrichR tool within the databases. Enriched terms with ten or more genes overlapping with DEGs were shown as a scatterplot. X axis is the combined enrichment score, the Y axis is -log₁₀(adjusted p-value), the color gradient represents the Z-score, the size corresponds to the number of significant DEGs overlapping with the enrichment term and the dashed blue line represents p-value of 0.05. Top enriched terms were labeled, excluding those deemed redundant due to their equivalency to other labeled terms. (A) Enrichment analysis of upregulated DEGs within the “Reactome"’ database; (B) Under-enrichment analysis of downregulated DEGs within the “Reactome” database. LS, lesional skin; HC, healthy controls.

[0029] Figure 10 shows the DEGs dysregulated in lesional CHE samples stratified for the presence of comorbid AD. Summary heat map of dysregulated genes in lesional, non-lesional and healthy control skin, stratified for the presence of comorbid atopic dermatitis. DEGs were selected for significant upregulation (A) and downregulation (B) in lesional CHE+ADvs HC and a reciprocal non-significance in lesional CHE_ADvs HC. The top 75 genes were depicted by the maximum |FCH| in the respective lesional vs HC comparison; Order of genes was determined by hierarchical clustering. LS. lesional skin; NL, non-lesional skin; HC, healthyAttorney Docket No.: MS-0050-01-US-NPcontrols skin. CHE+AD, patients with concomitant atopic dermatitis; CHFAD, patients without concomitant atopic dermatitis. +: FDR<0.1; *: FDR<0.05; **: FDR<0.01; ***: FDR< 0.001.

[0030] Figure 11 shows the DEGs dysregulated in lesional CHE samples stratified for the absence of comorbid AD. Summary heat map of dysregulated genes in lesional, non-lesional and healthy control skin, stratified for the absence of comorbid atopic dermatitis. DEGs were selected for significant upregulation (A) or downregulation (B) in lesional CHE'AI)vs HC and a reciprocal non-significance in lesional CHE+ADvs HC. The top 75 genes were depicted by the maximum |FCH| in the respective lesional vs HC comparison; Order of genes was determined by hierarchical clustering. LS. lesioncd skin; NL, non-lesional skin; HC, healthy controls skin. CHE+AD, patients with concomitant atopic dermatitis; CHE'AD, patients without concomitant atopic dermatitis. +: FDR<0.1; * FDR<0.05; **: FDR< 0.01; ***; FDR< 0.001.DETAILED DESCRIPTION

[0031] Systems and methods for diagnosing and treating chronic hand eczema are provided.

[0032] The description of the various embodiments is for purposes of illustration only and is not intended to be exhaustive or limiting. Modifications and variations will be apparent to one of ordinary skill in the art in light of the teachings herein and may be made without departing from scope of the present disclosure.

[0033] For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, "top.” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present disclosure or the appended claims. Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1%, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1%, such as less than or equal to ± 0.05%.Attorney Docket No.: MS-0050-01-US-NP

[0034] The term “about"’ or “approximately,’" as used herein, can mean within an acceptable error range for the particular value as determined by one of ordinary’ skill in the art. which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about"’ can mean an acceptable error range for the particular value, such as 10% of the value modified by the term “about.” As used herein, the term “about,” can mean relative to the recited value, e.g., amount, dose, temperature, time, percentage, etc., ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0035] Notwithstanding that the disclosed numerical ranges and parameters are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily’ resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10.

[0036] The terms “comprising,"’ “including” and “having"’ are used interchangeably in this disclosure. The terms "comprising,” “including” and “having” mean to include, but not necessarily be limited to the things so described, and mean that there may be additional elements other than the listed elements. Wherever the terms “comprising” or “including” are used, it should be understood the disclosure also expressly contemplates and encompasses additional aspects “consisting of" the disclosed elements, in which additional elements other than the listed elements are not included. The terms “or” and “and / or,” as used herein, are to be interpreted as inclusive or meaning any one or any’ combination. Therefore, “A, B or C” or “A, B and / or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.

[0037] Any example(s) following the term “e.g.” or “for example” is not meant to be exhaustive or limiting. Unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular.

[0038] The nomenclatures used in connection with, and the laboratory procedures and techniques of biochemistry, immunology, microbiology', molecular biology, and virologyAttorney Docket No.: MS-0050-01-US-NPdescribed herein are those well-known and commonly used in the art. The phrases and terminology employed herein are for the purpose of description and should not be regarded as limiting.

[0039] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, pharmacology, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, genetics and protein and nucleic acid chemistry, described herein, are those well- known and commonly used in the art. In case of conflict, the present specification, including definitions, will control.

[0040] Unless defined otherwise herein, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.). Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991), all of which are incorporated by reference herein. As used herein, the following terms have the meanings ascribed to them below; unless specified otherwise.

[0041] The practice of the present application will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology; cell biology, biochemistry and immunology; which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al.. 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory' Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc ); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology7(F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY (2001); Ausubel et al.. Current Protocols in Molecular Biology, John Wiley &Attorney Docket No.: MS-0050-01-US-NPSons, NY (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in Protein Science, John Wiley & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999).

[0042] Further, as the present disclosure is susceptible to aspects of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present disclosure and not intended to limit the present disclosure to the specific aspects shown and described. Any one of the features of the present disclosure may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and / or the like in the description mean that the feature and / or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect.” “aspects,” and / or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present disclosure may include a variety of combinations and / or integrations of the aspects described herein.

[0043] Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present disclosure will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be encompassed by the claims.

[0044] Where aspects or embodiments are described in terms of a Markush group or other grouping of alternatives, the present application encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members. The present application also envisages the explicit exclusion of one or more of any of the group members in the Markush group or other grouping of alternatives.

[0045] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the various aspects and embodiments. The materials, methods, and examples are illustrative only and not intended to be limiting.DefinitionsAttorney Docket No.: MS-0050-01-US-NP

[0046] In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherw ise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.

[0047] As used herein, and unless otherwise specified, the terms "treat'’, “treating"’ and “treatment” and variations thereof refer to an action that occurs in a subj ect with eczema, which reduces the severity of at least one discernible symptom of eczema, or retards or slows the progression of at least one discernible symptom of eczema. In some embodiments, “treat” and its variations refers to an amelioration of at least one measurable phy sical parameter, not necessarily discernible by the patient. In some embodiments, “treat” and its variations refers to inhibiting or reducing or slowing the progression of eczema, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both, relative to an untreated control. In certain embodiments, “treat” and its variations refers to slowing the progression or reversing the progression of eczema relative to an untreated control.

[0048] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double- stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues See, e.g., Batzer et al., Nucleic Acid Res.19:5081 (1991), the disclosure of which is incorporated in its entirety herein.

[0049] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly areAttorney Docket No.: MS-0050-01-US-NPreferred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.■‘Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0050] Atopic dermatitis (AD) is a condition that causes dry, itchy and inflamed skin. It can be common in young children but can occur at any age. Atopic dermatitis can be long lasting (chronic) and can exhibit episodic recurrences. AD can be irritating but is not contagious. Symptoms of AD can include dry cracked skin: itchiness; rash on swollen skin that varies in color depending on skin color; small, raised bumps on the skin; oozing and crusting; thickened skin; darkening of the skin around the eyes; and raw, sensitive skin from scratching. AD onset can begin before age 5 and may continue into the teen and adult years. For some people, AD symptoms flare and then clear up for a time, even for several years.

[0051] “Chronic hand eczema” is an inflammatory skin disease known to clinicians. See the background section and references cited therein for an understanding of the disease.

[0052] A “reference level” of a biomarker in a control refers to the baseline amount of that biomarker in the tape-stripped normal skin sample. In an aspect, the reference level may be an average level of a biomarker obtained from subjects without hand eczema. In some respects, the reference level may correspond to the biomarker level obtained from the subject for whom a diagnosis is being made, or a treatment efficacy is being determined, but at a time prior to current. Thus, in some aspects, the reference level may comprise one or more of the levels of the at least one biomarker from a subject not suffering from hand eczema, an average of the level of the at least one biomarker from subjects not suffering from hand eczema, a previously provided reference level from the subject. The amount of the biomarker that is measured in the sample may be relative or absolute. In some embodiments the relative expression of mRNA or cDNA is measured in the test sample versus the control sample. In other embodiments, the absolute amount of protein biomarker is measured in the test sample versus the control sample.

[0053] In some embodiments, the relative expression of mRNA or cDNA is measured in the test sample versus the control sample. In other embodiments, the absolute amount of protein biomarker is measured in the test sample versus the control sample.

[0054] As used herein, the term “control” refers to a tape-stripped surface skin sample taken from a healthy subject, which skin sample does not exhibit lesions or discoloration. A “reference level” of a biomarker in a control refers to the baseline amount of that biomarker inAttorney Docket No.: MS-0050-01-US-NPthe tape-stripped normal skin sample. The amount of the biomarker that is measured in the sample may be relative or absolute. In some embodiments the relative expression of mRNA or cDNA is measured in the test sample versus the control sample.

[0055] As used herein, the term '‘expressed” or '‘expression” refers to the transcription from a gene to a ribonucleic acid (RNA) molecule at least complementary' in part to a region of one of the two nucleic acid strands of the gene. Alternatively, the term “expressed” or “expression” may refer to the translation from the RNA molecule to give a protein, a polypeptide or a portion thereof.

[0056] The level of mRNA or protein expression may “decrease” in a subject administered a hand eczema treatment. Alternatively, the level of mRNA, cDNA or protein may “increase” following hand eczema treatment. In some situations, the mRNA, cDNA or protein level may remain unchanged upon a given treatment., an mRNA, cDNA or protein level can be “downregulated”, i.e., the level of mRNA or protein may be decreased, for example, by about 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1% or less of the comparative control mRNA, cDNA or protein level. Alternatively, an mRNA, cDNA or protein level from a subject sample can be “upregulated”, i.e., the level of mRNA, cDNA or protein may be increased, for example, by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 100%, 200%, 300%, 500%, 1,000%, 5,000%, 10,000%, 15,000%, 20,000% or more of the comparative control mRNA, cDNA or protein level.

[0057]

[0058] As used herein, the term “predict” refers to determine or foresee in advance. When used to “predict” the effectiveness of chronic hand eczema treatment, for example, the term “predict” can mean that the likelihood of the outcome of the treatment can be determined at the outset, before the treatment has begun, or before the treatment period has progressed substantially.

[0059] As used herein, the term “monitor” refers to regularly observing and tracking the progress or quality7over a period of time. In the context of this disclosure, “monitoring” may refer to tracking the effectiveness in treating chronic hand eczema in a subject, or a cell or tissue obtained from a subject. Similarly, the term "monitoring," when used in connection with subject compliance, either individually, or within a clinical trial, refers to the tracking or confirming that the subject is actually following the treatment regimen being tested as prescribed.Attorney Docket No.: MS-0050-01-US-NP

[0060] As used herein, the term ‘‘discriminate” refers to the ability to distinguish chronic hand eczema from other conditions, based on the biomarker profile obtained from the surface skin samples. Suitable biomarkers and combinations thereof are provided herein.

[0061] As used herein, the term '‘skin lesion” or “lesional skin” is a part of the surface skin in a human subject that has an abnormal growth or appearance compared to the skin around it. The skin lesion may be dry, raised, red, and / or covered in silvery white scales. The lesional skin may be patchy, covered in white bumps or pustules. The lesions may appear anywhere on the body, such as the elbows, knees, scalp, lower back, armpits, groin, between the buttocks, under the breasts, etc. The lesional skin may be cracked, itchy, sore and / or bleeding.

[0062] As used herein, the term “surface skin sample” is one or more samples of surface skin obtained from a subject by a minimally invasive, non-scarring technique such as tape-stripping or equivalent methods. The skin sample may be lesional skin or non-lesional skin from a subj ect.

[0063] In the “tape-stripping” approach, serial adhesive films are used to capture the stratum comeum and the upper part of the granular layer as described previously. See Helen He et al.2020 Front. Immunol.; vol 11, Kim BE, et al. Journal of Investigative Dermatology 2019; 139:2387; DyjackN, et al. JAllergy Clin Immunol 2018; 141:1298- 309. In some instances, at least 5-25 tape-strips are obtained from the same skin surface and pooled to obtain a single sample. “Tape-stripping” or equivalent methods are described in U. S. Patent 7183057, U. S. Patent App. No. US 18 / 155,702, and Publication No. PCT / US2025 / 011337 incorporated herein in their entirety.

[0064] An “individual” or “subject,” as used interchangeably herein, is a mammal. In certain aspects, the individual or subject is a human.

[0065] The term “administration” and variants thereof (e.g., “administering” a composition) in reference to a treatment composition of the disclosure means introducing the composition or a prodrug of the composition into the system of the subject in need of treatment. When a composition of the disclosure or prodrug thereof is provided in combination with one or more other active agents (e.g., a cytotoxic agent, etc.), “administration” and its variants are each understood to include concurrent and sequential introduction of the composition or prodrug thereof and other agents. The present disclosure includes within its scope prodrugs of the compositions of this disclosure. In general, such prodrugs will be functional derivatives of the compositions of this disclosure which are readily convertible in vivo into the required composition. Thus, in the methods of treatment of the present disclosure, the term “administering” shall encompass the treatment of the various conditions described with theAttorney Docket No.: MS-0050-01-US-NPcomposition specifically disclosed or with a composition which may not be specifically disclosed, but which converts to the specified composition in vivo after administration to the patient.

[0066] The term "likelihood" generally refers to an increase in the probability of an event. The term "likelihood" when used in reference to the effectiveness of a patient response generally contemplates an increased probability that the symptoms of chronic hand eczema will be lessened or decreased.Therapies for eczema

[0067] Treatment of eczema can vary depending on the severity of the disease. Mild to moderate eczema can be treated topically with a combination of glucocorticoids and phototherapy.

[0068] In some instances, eczema treatment involves treating the disease topically with a cream, lotion, spray, moisturizer, bath salt, immunomodulator, coal tar, anthralin, and / or corticosteroid. The moisturizer may contain one or more of salicylic acid, lactic acid, petroleum jelly, and paraffin. In some instances, the eczema therapy involves treatment with a smallmolecule therapeutic or targeted biological drug. Examples of small -molecule therapeutics include but are not limited to methotrexate, cyclosporine, acitretin, fumaric acid esters, apremilast. Immunomodulators including but not limited to tacrolimus and pimecrolimus may also be used to treat eczema. The biological drug, dupilumab may also be used to treat eczema.Therapies for Atopic Dermatitis

[0069] In some instances, AD treatment involves treating the disease topically with a cream, lotion, spray, moisturizer, bath salt, or immunomodulator. In some instances, AD treatment includes at least one therapy selected from a group consisting of topical treatment, phototherapy, oral medication, and a biological drug. In some instances, the biological drug is selected from dupilumab, dupixent, tralokinumab, upadicitinib, abrocitinib, and lebrikizumab. In some instances, the topical treatment is ruxolitinib.Biomarkers for Eczema

[0070] Biomarkers can be used to distinguish chronic hand eczema (CHE)from other skin conditions such as AD, to identify subjects who would benefit from CHE therapy, or to determine if a therapy is working,. A biomarker is a characteristic that can be objectively measured and evaluated as an indicator of normal biologic processes, pathologic processes, or pharmacological responses to a therapeutic intervention. Biomarkers can be biological (e.g., small molecules, metabolites, peptides, proteins, RNA, DNA), physiological (e.g., bloodAttorney Docket No.: MS-0050-01-US-NPpressure, electromyography, respiratory function), or structural measures (e.g., ultrasound, magnetic resonance imaging, or histological assessment). The biomarkers may be prognostic biomarkers that predict a future clinical outcome; disease progression biomarkers that are indicative of the severity of disease impact; predictive biomarkers that predict a future clinical response to therapy and helps stratify therapies; pharmacodynamics biomarkers that monitor or quantify a therapeutic effect; and surrogate end point biomarkers that predict a future clinical response to therapy wherein a change in the end point is associated with a future clinical response.

[0071] In an aspect, a suitable biomarker is a gene product, for example an RNA or a protein. In an aspect, a suitable biomarker may be discerned using the methods provided in the examples herein. Examples of suitable biomarkers may include any of the following: granulysin (GNLY); (dendritic cell markers: CD1B, CD1E, and CD40); (T-cell exhaustion / activation markers: TIGIT, ICOS, CD28, IL3RA, and CD96); chemokine receptor 8 (CCR8); forkhead box P3 (FOXP3); Cytotoxic T-Lymphocyte Antigen 4 (CTLA4); interferon regulatory factor 4 (IRF4); IL12B (keratin-associated protein genes: KRT27, KRT71, KRTAP1-5, KRTAP5-3, KRTAP12-2, KRTAP9, the epidermal differentiation complex component SPRR3; CC chemokine ligand 7 (CCL7); inducible nitric oxide synthase (NOS2); (neuronal development markers: NEUROG1, OTX2, GPM6A, ADGRL3); (olfactory receptor genes: OR6S1, OR4C6, and OR5H14); (terminal differentiation markers: FLG and LORICRIN); (T-cell markers: CD3D, CD3E, CD3G, CD4, CD5, CD8A, ITK, IL7R, GZMB, and CD69); (immune checkpoint genes: CD27 and CTLA4); (T-cell costimulatory genes: CD28, CD48, CD80, CD86, and ICOS); (chemokines: XCL1 and XCL2); (chemokine receptors: CXCR1, CXCR2, CXCR4 and CCR1, CCR7); interleukin receptor beta 2 (IL12RB2); interferon-gamma receptors 1 (IFNGR1) and 2 (IFNGR2); myxovirus resistance 1 (MX1) (CC motif chemokine ligands: CCL7, CCL13, CCL17, CCL20, CCL21, CCL22, CCL24); GATA binding protein 3 (GATA3); CX3CL1; CXCL6; CXCL9; CXCL10, CXCL11; CXCL13; CXCL17; (interferons: IFNA1 and IFNA2); (interleukins: IL7, IL12, IL13, IL15, IL18, IL19, IL22, IL23, IL34, IL12B, IL17F), lipocalin 2 (LCN2); PI3; LYG1, interleukin 23 receptor (IL23R) interferon-γ-induced indoleamine-pyrrole-2,3-dioxygenases: IDO1 and IDO2), FCGR3B; S100P; OASL; PI3; IFNA2; TGFB3; LAT2; RORC; indoleamine 2,3-dioxygenase 2 (IDO2); CD27; CD28; CD48; CD80; CD86; IL1RAP; TRAIL / TNFSF10; CCR2; RUNX1; IL27RA; SOCS2; OX40 / TNFRSF4; ISG20; CD137 / TNFRSF9; TSLPR / CRLF2; IL7R; CD122 / IL2RB; IL15RA; IRF1, FCER1A, AHR.

[0072] Dysregulation of levels of gene products linked to any of the biomarkers provided herein may be used in the methods disclosed herein.Attorney Docket No.: MS-0050-01-US-NP

[0073] In some embodiments, the biomarker is any of: OX40 / TNFRSF4; CRLF2,' IL7R; CD122 / IL2RB; IL15RA; JAK1; JAK2; IL13. alone or in combination.

[0074] In some embodiments, the biomarker is any of: CXCL6; CXCR1; and CXCR2. alone or in combination.

[0075] In some embodiments, the biomarker is any of: OX40 / TNFRSF4; CRLF2; IL7R; IL12B; IL12RB2; IL13 and CD122 / IL2RB, alone or in combination.

[0076] In some embodiments, the presence of any of: IL13; CCL17.' IL1RAP; TRAIL / TNFSF10; CCR2; RUNX1; IL27RA; and SOCS2 above a threshold in a surface skin sample is indicative of CHE. In some instances, the presence of any of: OX40 / TNFRSF4; CRLF2; IL7R; CD122 / IL2RB; JAK1; JAK2; and IL13 above a threshold level in a surface skin sample is indicative of CHE. In some instances, the presence of any of: IL12RB2; IFNGR1; IFNGR2; MX1: CCL22; CCL24; OX40 / TNFRSF4; TSLPR / CRLF2; and GATA3 above a threshold level in a surface skin sample is indicative of CHE, regardless of AD status. In some instances, the presence of FLG or LORICRIN below a threshold in a surface skin sample is indicative of CHE, regardless of AD status. In some instances, the presence of IL15RA or CXCL9 above a threshold level in a surface skin sample is indicative of CHE, in patients without AD. In some instances, the presence of IL13 or CCL17 above a threshold level in a surface skin sample is indicative of CHE, in patients with AD.

[0077] In some embodiments, the presence of any of: RGS1; IL19; RCOR3; MTR; NPHP3; HSPA1A; HMGN3; REV3L; FAM229B; GPR31; MACC1; ADAMDEC1; IDO1; IDO2; ZBTB43; TRIM66; OAF; EEA1; SDSL; RSRC2; SPG11; TRIM69; FURIN; TVP23A; CTNS; VMO1; RGS9; CCDC57; SIGLEC12; and ENTHD1, alone or in combination, can be used to differentiate between: subjects having CHE and having atopic dermatitis (CHE+AD); and subjects having CHE in the absence of atopic dermatitis (CHE- AD).

[0078] In some embodiments, the presence of any of: DPM3; FAM114A2; FZD3; PTPDC1; or PRM1, alone or in combination, can be used to differentiate between lesional and non-lesional skin in said subjects.Determining Expression Levels or Concentrations of Biomarkers

[0079] In an embodiment, gene expression can be detected as RNA or protein expression of one or more target genes (gene product). The presence or expression level (amount) of a gene product can be determined by detecting and / or measuring the level of mRNA or protein expression of the gene. In some aspects, gene expression can be detected as the activity of the gene product.Attorney Docket No.: MS-0050-01-US-NP

[0080] In another embodiment, the expression level of a gene product of interest is determined by measuring RNA levels. A variety of suitable methods can be employed to detect and / or measure the level of mRNA expression of a gene. For example, mRNA expression can be determined using Northern blot or dot blot analysis, reverse transcriptase-PCR (RT-PCR, e.g., quantitative RT-PCR), in situ hybridization (e.g., quantitative in situ hybridization) or nucleic acid array (e.g., oligonucleotide arrays, gene chips) analysis, advanced transcriptomics methods such as RNA sequencing (RNA-seq). Details of such methods are described below and in, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual Second Edition vol. 1, 2 and 3. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, USA, Nov.1989; Gibson et al. (1999) Genome Res., 6(10):995-1001; and Zhang et al. (2005) Environ. Sci. Technol., 39(8):2777-2785; U. S. Publication No.2004086915; European Patent No.0543942; and U. S. Patent No. 7,101,663; the disclosures of each of which are incorporated herein by reference in their entirety.

[0081] In an embodiment, the expression level of a gene product of interest is determined by measuring RNA levels. A variety of suitable methods can be employed to detect and / or measure the level of mRNA expression of a gene. For example, mRNA expression can be determined using Northern blot or dot blot analysis, reverse transcriptase-PCR (RT-PCR, e.g., quantitative RT-PCR), in situ hybridization (e.g., quantitative in situ hybridization) or nucleic acid array (e.g., oligonucleotide arrays, gene chips) analysis, advanced transcriptomics methods such as RNA sequencing (RNA-seq). Details of such methods are described below and in, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual Second Edition vol. 1, 2 and 3. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, USA, Nov. 1989; Gibson et al. (1999) Genome Res., 6(10):995-1001; and Zhang et al. (2005) Environ. Sci. Technol., 39(8):2777-2785; U. S. Publication No.2004086915; European Patent No. 0543942; and U. S. Patent No. 7,101,663; the disclosures of each of which are incorporated herein by reference in their entirety.

[0082] In another example, the presence or amount of discrete populations of mRNA (e.g., mRNA for biomarkers provided herein) in a surface skin sample can be -25- 101054975 PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center determined using nucleic acid (or oligonucleotide) arrays. For example, isolated mRNA from a surface skin sample can be amplified using RT-PCR with, e.g., random hexamer or oligo(dT)-p rimer mediated first strand synthesis. The amplicons can be fragmented into shorter segments. The RT-PCR step can be used to detectably-label the amplicons, or. optionally, the amplicons can be detectably labeled subsequent to the RT-PCR step. For example, the detectable-label can beAttorney Docket No.: MS-0050-01-US-NPenzymatically (e.g., by nick-translation or kinase such as T4 polynucleotide kinase) or chemically conjugated to the amplicons using any of a variety of suitable techniques (see, e.g., Sambrook et al., supra). The detectably-labeled-amplicons are then contacted with a plurality of polynucleotide probe sets, each set containing one or more of a polynucleotide (e.g., an oligonucleotide) probe specific for (and capable of binding to) a corresponding amplicon, and where the plurality contains many probe sets each corresponding to a different amplicon. Generally, the probe sets are bound to a solid support, and the position of each probe set is predetermined on the solid support. The binding of a detectably labeled amplicon to a corresponding probe of a probe set indicates the presence or amount of a target mRNA in the surface skin sample. Additional methods for detecting mRNA expression using nucleic acid arrays are described in, e.g., U. S. Patent Nos. 5,445,934; 6,027,880; 6,057,100; 6,156,501; 6,261,776; and 6,576,424; the disclosures of each of which are incorporated herein by reference in their entirety.

[0083] In an aspect, expression of more than one nucleic acid molecule can be detected. As illustrated in the examples, expression of about multiple genes is altered in the skin of an eczema patient. Therefore, changes of skin state from normal to a state corresponding to eczema, are accompanied by changes in at least these gene products. Therefore, in certain examples, methods provided herein characterize skin by analyzing expression of 2 or more, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, or all of the biomarkers provided herein.

[0084] In another embodiment, the present disclosure provides a method for identifying an expression profile indicative of CHE in a subject, including applying an adhesive tape to an area of skin afflicted with the disease or pathological state in a manner sufficient to isolate an epidermal sample adhering to the adhesive tape, wherein the epidermal sample includes nucleic acid molecules, and applying the nucleic acid molecules to a microarray or a related advanced sequencing technique (for example, RNAseq). Nucleic acid molecules can optionally be isolated from the epidermal sample before being applied to the microarray. Expression levels of at least 10 genes is then determined using the microarray; wherein an altered expression level for at least 2, 3, 4. 5, 6, 7, 8, 9. or each of the at least 10 genes as compared with expression in an epidermal sample from a normal skin sample identifies a subject is afflicted with MAA or SAA, thereby identifying the expression profile indicative of the disease.

[0085] In embodiments where expression of more than 1 gene is analyzed, the detection can be performed using a microarray. For example, the microarray can include an array of probes, for example, directed to 2 or more, 10 or more, 25 or more, 50 or more, 100 or more, 500 orAttorney Docket No.: MS-0050-01-US-NPmore, 1000 or more genes. The microarrays form another embodiment of the disclosure. For microarray expression analysis, approximately 0.1 to 1 milligram, typically 1 to 10 nanograms of RNA are isolated from an epidermal sample, for example obtained using a tape stripping method disclosed herein. Isolated RNA is then amplified and used for hybridization to probes on a biochip. Amplification typically results in a total of at least 1 microgram, and more typically at least 20 micrograms of amplified nucleic acid. For example, amplification can be performed using a MessageAMp™ RNA kit (Ambion Inc.). Isolated RNA can be biotin labeled before contacting the biochip such that binding to the target array can be detected using streptavidin.

[0086] Methods of detecting and / or for quantify ing a detectable label depend on the nature of the label. The products of reactions catalyzed by appropriate enzymes (where the detectable label is an enzyme; see above) can be, without limitation, fluorescent, luminescent, or radioactive or they may absorb visible or ultraviolet light. Examples of detectors suitable for detecting such detectable labels include, without limitation, x-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers, and densitometers.

[0087] Methods for detecting or measuring gene expression (e.g., protein or mRNA expression) can optionally be performed in formats that allow for rapid preparation, processing, and analysis of multiple samples. This can be, for example, in multi -welled assay plates (e.g., 96 wells or 386 wells) or arrays (e.g., nucleic acid chips or protein chips). Stock solutions for various reagents can be provided manually or robotically, and subsequent sample preparation (e.g., RT-PCR, labeling, or cell fixation), pipetting, diluting, mixing, distribution, washing, incubating (e.g., hybridization), sample readout, data collection (optical data) and / or analysis (computer aided image analysis) can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting the signal generated from the assay. Examples of such detectors include, but are not limited to, spectrophotometers, luminometers, fluorimeters, and devices that measure radioisotope decay. Exemplary high-throughput cell-based assays (e.g.. detecting the presence or level of a target protein in a cell) can utilize Array Scan® VTI HCS Reader or KineticScan® HCS Reader technology (Cellomics Inc., Pittsburg, PA).

[0088] In some embodiments, the expression level of the biomarkers of this disclosure can be assessed and / or measured. To aid in detecting the presence or level of expression of the biomarker genes of interest, any part of the nucleic acid sequence of the genes can be used, e.g., as hybridization polynucleotide probes or primers (e.g., for amplification or reverseAttorney Docket No.: MS-0050-01-US-NPtranscription). The probes and primers can be oligonucleotides of sufficient length to provide specific hybridization to an RNA, DNA, cDNA, or fragments thereof isolated from a surface skin sample. Depending on the specific application, varying hybridization conditions can be employed to achieve varying degrees of selectivity of a probe or primer towards target sequence. The primers and probes can be detectably labeled with reagents that facilitate detection (e.g., fluorescent labels, chemical labels (see, e.g., U. S. Patent Nos.4, 582, 789 and 4,563,417). or modified bases).

[0089] Hybridization can be used to assess homology between two nucleic acid sequences. A nucleic acid sequence described herein, or a fragment thereof, can be used as a hybridization probe according to standard hybridization techniques. The hybridization of a probe of interest (e.g., a probe containing a portion of a nucleotide sequence described herein or its complement) to DNA, RNA, cDNA, or fragments thereof from a test source is an indication of the presence of DNA or RNA corresponding to the probe in the test source. Hybridization conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y, 6.3.1-6.3.6, 1991. Moderate hybridization conditions are defined as hybridization in 2X sodium chloride / sodium citrate (SSC) at 30°C, followed by a wash in 1 X SSC, 0.1% SDS at 50°C. Highly stringent conditions are defined as hybridization in 6X SSC at 45°C, followed by a wash in 0.2 X SSC, 0.1% SDS at 65°C.

[0090] Primers can be used in a variety of PCR-type methods. For example, polymerase chain reaction (PCR) techniques can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. The PCR primers are designed to flank the region that one is interested in amplifying. Primers can be located near the 5' end, the 3' end or anywhere within the nucleotide sequence that is to be amplified. The amplicon length is dictated by the experimental goals. For qPCR, the target length is closer to 100 base pairs and for standard PCR, it is near 500 base pairs. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. PCR primers can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3?to 5’ direction using phosphorami dite technology) or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNAAttorney Docket No.: MS-0050-01-US-NPpolymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair.

[0091] In addition, the nucleic acid sequences or fragments thereof (e.g., oligonucleotide probes) can be used in nucleic acid arrays for detection and / or quantitation of gene expression.

[0092] In one embodiment, the expression of a gene can be determined by detecting and / or measuring expression or concentration of a protein encoded by the gene. Methods of determining protein expression / concentration are well known in the art. A generally used method involves the use of antibodies specific for the target protein of interest. For example, methods of determining protein expression include, but are not limited to, western blot or dot blot analysis, immunohistochemistry (e.g., quantitative immunohistochemistry), immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISPOT; Coligan, J. E., et al., eds. (1995) Current Protocols in Immunology. Wiley, New York), radioimmunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, latex turbidimetric immunoassay, latex photometric immunoassay, immuno- chromatographic assay, and antibody array analysis (see, e.g., U. S. Publication Nos. 2003 / 0013208 and 2004 / 171068, the disclosures of each of which are incorporated herein by reference in their entirety). Further description of many of the methods above and additional methods for detecting protein expression can be found in, e.g., Sambrook et al. (supra).

[0093] Standard stringency conditions are described by Sambrook, et al. (supra) and Haymes, et al. Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D. C. (1985). In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular hybridization conditions (e.g., solvent and salt concentrations) employed.

[0094] In one example, the presence or amount of a protein gene product can be determined using a western blotting technique. For example, a lysate can be prepared from a surface skin sample, or the surface skin sample itself, can be contacted with Laemmli buffer and subjected to sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE-resolved proteins, separated by size, can then be transferred to a filter membrane (e.g., nitrocellulose) and subjected to immunoblotting techniques using a detectably labeled antibody specific to the protein of interest. The presence or amount of bound detectably labeled antibody indicates the presence or amount of protein in the surface skin sample.

[0095] In another example, an immunoassay can be used for detecting and / or measuring the protein expression of a biomarker disclosed herein, including but not restricted to OLINKAttorney Docket No.: MS-0050-01-US-NPProteomics, Luminex, Elisa, Singulex Erenna or other proteomic platforms. As above, for the purposes of detection, an immunoassay can be performed with an antibody that bears a detection moiety (e.g., a fluorescent agent or enzyme). Proteins from a surface skin sample can be conjugated directly to a solid-phase matrix (e.g., a multi-well assay plate, nitrocellulose, agarose, sepharose, encoded particles, or magnetic beads) or it can be conjugated to a first member of a specific binding pair (e.g., biotin or streptavidin) that attaches to a solid-phase matrix upon binding to a second member of the specific binding pair (e.g., streptavidin or biotin). Such attachment to a solid-phase matrix allows the proteins to be purified away from other interfering or irrelevant components of the surface skin sample prior to contact with the detection antibody and also allows for subsequent washing of unbound antibody. Here, as above, the presence or amount of bound detectably labeled antibody indicates the presence or amount of protein in the surface skin sample.

[0096] There is no particular restriction as to the form of the antibody and the present disclosure includes polyclonal antibodies, as well as monoclonal antibodies. The antiserum obtained by immunizing animals, such as rabbits with a protein or fragment thereof of the disclosure, as well polyclonal and monoclonal antibodies of all classes, human antibodies, and humanized antibodies produced by genetic recombination, are also included.

[0097] The antibodies may be conjugated to various molecules, such as fluorescent substances, radioactive substances, and luminescent substances. Methods to attach such moieties to an antibody are already established and conventional in the field (see. e.g., US 5,057,313 and 5,156,840).

[0098] Examples of methods that assay the antigen-binding activity of the antibodies include, for example, measurement of absorbance, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and / or immunofluorescence, and or Proximity Extension Assay (PEA) technology that is used by OLINK platform. For example, when using ELISA, a protein encoded by a biomarker of the disclosure is added to a plate coated with the antibodies of the present disclosure, and then, the antibody sample, for example, culture supernatants of antibody-producing cells, or purified antibodies are added. Then, secondary antibody recognizing the primary antibody, which is labeled by alkaline phosphatase and such enzymes, is added, the plate is incubated and washed, and the absorbance is measured to evaluate the antigen-binding activity after adding an enzy me substrate such as p-nitrophenyl phosphate. As the protein, a protein fragment, for example, a fragment comprising a C-terminus, or a fragment comprising an N-terminus may be used. To evaluate the activity of the antibody of the disclosure, BIAcore (Pharmacia) may be used.Attorney Docket No.: MS-0050-01-US-NP

[0099] By using these methods, the antibody and a sample presumed to contain a protein of the disclosure are contacted, and the protein encoded by a biomarker of the disclosure is detected or assayed by detecting or assaying the immune complex formed between the above-mentioned antibody and the protein.

[0100] Mass spectrometry -based quantitation assay methods, for example, but not limited to, multiple reaction monitoring (MRM)-based approaches in combination with stable-isotope labeled internal standards, are an alternative to immunoassays for quantitative measurement of proteins. These approaches do not require the use of antibodies and so the analysis can be performed in a cost- and time- efficient manner (see, for example, Addona et al., Nat. Biotechnol., 27:633-641, 2009; Kuzyk et al., Mol. Cell Proteomics, 8:1860-1877, 2009; Paulovich et al., Proteomics Clin. Appl., 2: 1386-1402, 2008). In addition, MRM offers superior multiplexing capabilities, allowing for the simultaneous quantification of numerous proteins in parallel. The basic theory of these methods has been well-established and widely utilized for drug metabolism and pharmacokinetics analysis of small molecules.Reference and comparative levels

[0101] The current disclosure also encompasses companng the levels of the one or more biomarker in the surface skin sample of a subject to the reference levels of the one or more biomarker. The reference level for use herein will depend on the application and can be determined by a person of ordinary' skill in the art. The reference level may be the level of the corresponding biomarker in a person not suffering from CHE. The reference level may be an average or other statistical aggregate of the level of the corresponding biomarker or biomarkers in a population of healthy individuals without any history of atopic conditions, including allergic asthma, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, food allergies, and / or eosinophilic esophagitis. Healthy is defined in relation to these aforementioned conditions and cannot be confined to an absolute evaluation or status. Healthy individuals may therefore present with other diseases, including other respirator}' conditions. The reference level may also be the subject's own biomarker level at a time other than the current level.Methods of treatment

[0102] The methods disclosed herein enable the diagnosis of chronic hand eczema based on biomarker levels in a subject’s surface skin, followed by treatment of said subject with an appropriate therapy. The methods disclosed herein enable the assessment whether or not a subject having or suspected of having CHE is likely to respond to a therapy. A subject having or suspected of having CHE who is likely to respond to the therapy can be administered theAttorney Docket No.: MS-0050-01-US-NPtherapy. Conversely, a subject having or suspected of having CHE who is not likely to respond to a therapy can be administered a different therapy that is suitable for treatment of CHE.

[0103] The methods of this disclosure also enable the stratification of subjects having or suspected of having CHE into groups of subjects that are more likely to benefit, and groups of subjects that are less likely to benefit, from treatment comprising an eczema therapy. The ability to select such subjects from a pool of subjects who are being considered for treatment with an eczema therapy is beneficial for administering an effective treatment to the subject. For example, the biomarkers may be used to classify patients based the severity of CHE and a treatment regimen can be decided accordingly.

[0104] If the subject having CHE is more likely to respond to a therapy (based on levels of biomarkers of this disclosure), the subject can then be administered an effective amount of the therapy. An effective amount of the compound can suitably be determined by a health care practitioner taking into account, for example, the characteristics of the patient (age, sex, weight, race, etc.), the progression of the disease, and prior exposure to the drug. If the subject is less likely to respond to one eczema therapy, the subject can then be optionally administered a different therapy.

[0105] In addition to the prediction of the likelihood of treatment effectiveness in a patient with CHE, the progress of a treatment modality can be followed by monitoring the levels of the biomarkers described above. In some embodiments, a method of assessing or monitoring the effectiveness of a treatment in a subject is provided. A surface skin sample is obtained from the subject, and the levels of one or more of the above-described biomarkers, are measured to determine whether the biomarker levels are increased or decreased compared to the levels prior to the initiation of the treatment.

[0106] In one embodiment, provided herein is a method of monitoring subject response to an eczema treatment comprising: obtaining a surface skin sample from the subject; measuring the level of any of the biomarkers or combinations thereof as listed herein, in the first sample; administering a treatment compound to the subject; thereafter obtaining a second surface skin sample from the subject; measuring the level of any of the biomarkers or combinations thereof as listed herein, in the second sample; and comparing the levels of the biomarkers obtained from first and second samples; wherein a decreased level of the biomarkers in the second sample indicates an effective response. In one embodiment, provided herein is a method of monitoring subject response to an eczema treatment comprising: obtaining a surface skin sample from the subject; measuring the level of any of the biomarkers or combinations thereof as listed herein, in the first sample; administering a treatment compound to the subject;Attorney Docket No.: MS-0050-01-US-NPthereafter obtaining a second surface skin sample from the subject; measuring the level of any of the biomarkers or combinations thereof as listed herein, in the second sample; and comparing the levels of the biomarkers obtained from first and second samples; wherein an increased level of the biomarkers in the second sample indicates an effective response.

[0107] The biomarkers can also be used to track and adjust individual patient treatment effectiveness. The biomarkers can be used to gather information needed to make adjustments in a patient's treatment, increasing or decreasing the dose of an agent as needed. For example, a patient receiving a treatment compound can be tested using a biomarker to see if the dosage is becoming effective, or if a more aggressive treatment plan may be needed.

[0108] Subjects of all ages can be affected by eczema. Therefore, a surface skin sample used in a method described herein can be obtained from a human subject of any age, including a fetus, an infant, a child, an adolescent, or an adult, such as an adult having, or suspected of having, eczema.

[0109] The methods can also be applied to individuals at risk of developing CHE treatable by an eczema therapy. Such individuals include those who have (i) a family history of (a genetic predisposition for) such disorders or (ii) one or more risk factors for developing such disorders.

[0110] After stratifying or selecting a subject based on whether the subject w ill be more likely or less likely to respond to a therapy, a medical practitioner (e.g., a doctor) can administer the appropriate therapeutic modality to the subject. Methods of administering eczema therapies are known in the art.Kits

[0111] This disclosure also provides kits. In certain embodiments, the kit can include one or more means for obtaining skin samples. In some embodiments, the kit includes probes that can be used to identify or detect any of the biomarkers disclosed herein. In some embodiments, the kit includes any of the nucleic acid arrays. In some embodiments, the kit includes probes and antibodies that can be used to identify or detect any of the biomarkers disclosed herein or their expression or expression levels. The kits can, optionally, contain instructions for detecting and / or measuring the concentration of one or more proteins or the levels of mRNA in a surface skin sample.

[0112] In some embodiments, the kits can include one or more reagents for processing a surface skin sample (e.g., calibration reagents, buffers, diluents, color reagents, reagents to stop a reaction). For example, a kit can include reagents for isolating nucleic acid or protein from a surface skin sample and / or reagents for detecting the presence and / or amount of one or more biomarkers in a surface skin sample (e.g., probes for detection of a biomarker RNA, anAttorney Docket No.: MS-0050-01-US-NPantibody that binds to the protein that is the subject of the detection assay and / or an antibody that binds the antibody that binds to the protein).

[0113] The kits can optionally include, e.g., a control (e.g., a concentration standard for the protein being assessed) or control labeled amplicon set containing known amounts of one or more amplicons recognized by nucleic acid probes of the array. In some instances, the control can be an insert (e.g., a paper insert or electronic medium such as a CD, DVD, or floppy disk) containing an expression level or expression level ranges of one or more proteins or RNAs predictive of CHE, or of responsiveness to an eczema therapy.

[0114] In certain embodiments, the kit includes at least one microplate (e.g., a 96 well plate, i.e., 12 strips of 8 wells). The microplate can be provided with its corresponding plate cover. The microplate can be polystyrene or of any other suitable material. The microplate can have the antibody that is used to identify the presence of a particular biomarker coated inside each well. The antibody may be conjugated to a detectable label. The kit may also include at least one adhesive strip.

[0115] In some embodiments, the kits can include a software package for analyzing the results of, e.g.. expression profile or a microarray analysis.

[0116] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art can develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

[0117] For the avoidance of doubt, it is intended herein that particular features (for example integers, characteristics, values, uses, diseases, formulae, compounds or groups) described in conjunction with a particular aspect, embodiment or example of the disclosure are to be understood as applicable to any other aspect, embodiment or example described herein unless incompatible therewith. Thus, such features may be used where appropriate in conjunction with any of the definition, claims or embodiments defined herein. All the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of the features and / or steps are mutually exclusive. The disclosure is not restricted to any details of any disclosed embodiments. The disclosure extends to any novel one, or novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.Attorney Docket No.: MS-0050-01-US-NPEXAMPLESExample 1: Tape-Strips to Detect Distinct Biomarkers in Patients with Chronic Hand Eczema

[0118] An analysis of the biomarkers expressed in CHE patients was performed using the following methods.Study Population and Characteristics

[0119] A total of 66 patients with untreated CHE were recruited via a clinical trial (NCT03728504) from multiple sites in the US and Canada, and baseline tape strips before treatment were tested. Twenty age, sex, race, and location-matched healthy volunteers were included for comparison (Table 1). The inclusion criteria comprised adult subjects (aged 18-75) moderate to severe CHE (Physician Global Assessment score of 3 or 4) for at least six months that was refractory to conventional topical therapy. Exclusion criteria included BM1 >35 kg / m2, pregnancy, concomitant AD covering >15% of BSA (excluding hands and feet), recent (<4 weeks) AD flare, active skin infection, history of psoriasis, eczema herpeticum or other severe medical conditions. Further exclusion criteria comprised the use of investigatory biological agents or dupilumab during previous 12 weeks, non-biological investigatory agents, systemic retinoids, immunomodulatory agents, or phototherapy during previous 4 weeks, and topical agents (except for urea- and salicylic acid-free emollients), during previous two weeks. Concomitant atopic dermatitis was diagnosed using Hanifin and Rajka criteria. Twenty healthy subjects without hand eczema were also recruited to serve as control (NCT02270411). Before study inclusion, participants provided written informed consent for a research protocol that had been approved by Mount Sinai IRB (14-00436) and Advarra (Pro00030659). All data was recorded via methods that comply with HIPAA guidelines.TABLE 1Chronic Hand Eczema (CHE)Healthy n=66ControlsP- with concomitant without historyHand skin value AD of AD(HC) (CHE+AD) (CHE A”)Attorney Docket No.: MS-0050-01-US-NPNumber of patients 33 33 20 NAAge (y), mean (SD) 41 (18) 47 (15) 41 (H) 0.14Sex (male / female) 9 / 24 9 / 24 11 / 9 0.08Duration (y), mean (range) 9 (0.7-39.4) 14 (0.6-68.8) NA 0.11Race / EthnicityWhite 24 (73%) 30 (90%) 17 (85%)Black 5 (15%) 2 (7%) 3 (15%)0.41 Asian 3 (9%) 1 (3%) 0 (0%)Other 1 (3%) 0 (0%) 0 (0%)Severity scores, mean (range)MTLSS 13 (5-20) 12 (6-19) 0.24HECSI 69 (13-149) 56 (10-141) 0.16DLQI 13 (2-26) 11 (1-30) 0.27Pain VAS 5 (0-10) 4.4 (0-9.4) 0.4NA PGA Hand 3.7 (3-4) 3.6 (3-4) 0.3BSA of AD, excluding hand3.2 (0-14) NA NA & feetvIGA of AD,2 (0-4) NA NA excluding hand & feetAttorney Docket No.: MS-0050-01-US-NPMTLSS, Modified Total Lesion Symptom Score; HECSI, The hand eczema severity index; DLQI, Dermatology Life Quality Index; VAS, Visual Analogue Scale; PGA, Physician's global assessment; BSA, body surface area; vIGA, Validated Investigator Global Assessment

[0120] Data collection. Patients’ demographics, including age, sex, and race, were collected, along with the following scores: Modified Total Lesion Symptom Score (mTLSS), the Hand Eczema Severity Index (HECSI), Dermatology Life Quality Index (DLQI), and pain levels via a Visual Analogue Scale (Pain VAS). Additionally, patients with concomitant AD had their body surface area (BSA) involvement measured, as w ell as their Validated Investigator Global Assessment of AD (vIGA-AD) score. All data w as recorded via methods that comply w ith HIPAA guidelines.

[0121] Tape strip acquisition. Twenty successive D-Squame™ (CuDerm, Dallas, Texas) tape strips were collected from lesional CHE skin of the most affected location (palmar or dorsal hand) and from adjacent non-lesional skin. Each tape strip was applied to the skin, and a D500 D-Squame™ pressure instrument was used for even application for ~5 seconds. After collection, tapes were transferred to -80°C in storage cards until processing.

[0122] RNA isolation and sequencing. Isolation of RNA was performed as previously described. Briefly, total RNA was isolated from tape strips with miRNeasy™ Mini Kits (Qiagen, Hilden, Germany) and assessed for quantity7and quality7using the Agilent Tapestation™ 4200 (Agilent, Santa Clara, CA). RNA-seq libraries were constructed using the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (ThermoFisher, Waltham. MA) following the manufacturer's protocol. The libraries were then quantified, pooled, and sequenced on Ion S550 chips using the Ion S5™ XL System Sequencer (ThermoFisher).

[0123] Statistical and bioinformatics analysis. Statistical analyses were conducted using R Software version 4.3.2 (wwyg^ and RStudio version 2023.12.1(https.^ along with tools from Bioconductor (www.bioconductor.or ). The quality of the samples was evaluated using FastQC (ndrews S. FastQC: A Quality7Control Tool for High Throughput Sequence Data. 2010) and MultiQC3algorithms. The alignment of samples to the human reference genome was executed using STAR. The mapped sequencing reads were categorized into genomic features via the featureCounts function. All tape strips but one could be used for analyses. Overall, 11,999 genes were analyzed after removing low-expressed signatures. The counts underwent a log2 transformation with voom using quantile normalization and sample weights and were corrected for age and sex. Fold-change (FCH) calculation and hypothesisAttorney Docket No.: MS-0050-01-US-NPtesting were implemented under a general linear mixed models framework using the limma package. P-values were corrected for multiple hypothesis testing using the Benjamini-Hochberg method to calculate false discovery rate (FDR). Differentially expressed genes (DEGs) were defined with criteria of |FCH| >1.5 and FDR <0.05.

[0124] Gene-set analysis was done using the z-score approach with the GSVA package. Pathway enrichment assessments were conducted with EnrichR using the " Reactome" database. The relationship between inflammatory markers and clinical severity scores was examined using Spearman correlation analyses using the voom-normalized gene expression levels in lesional skin. For demographic data, categorical values were compared using Fisher test, numeric values were compared using Student t-test for two groups and one-way ANOVA for three groups. Variances were compared using the Levene test. All statistical tests were two-sided.ResultsBoth lesional and non-lesional CFIE samples demonstrated a strong inflammatory phenoty pewith impaired epidermal differentiation.

[0125] Principal Component Analysis (PCA) was performed to assess sample heterogeneity and congruency between groups (Figure 2). While lesional and HC tissues showed minimal overlap and formed tight, separate clusters (orange and green circles in Figure 2A), non-lesional samples showed greater variance, forming an in-between phenotype, overlapping with both lesional and HC tissues (blue circle in Figure 2A). The high variance of non-lesional CHE samples was also reflected in an analysis comparing the variability of individual gene expression, which was higher in non-lesional than lesional CHE (Figure 8A). The 4,595 high-variance genes from non-lesional CHE (Figure 8A) were assessed for pathway enrichment using the ■Reactome" database via the EnrichR tool (Figure 9B). Immune-related terms (immune system, adaptive immune system) were among the most significantly enriched pathways, suggesting that immune mediators are major contributors to this wide variability of gene expression within non-lesional CHE skin. Overall, PCA results were comparable when considering CHE patients with or without AD (Figure 2B-C). Utilizing the criteria of |FCH|>1.5 and FDR<0.05, differentially expressed genes (DEGs) were calculated for the entire CHE cohort (Figure 2D). Consistent with the PCA analysis, there was a strong overlap between lesional vs HC and non-lesional vs HC comparisons with a high number of shared DEGs (Figure 2D), suggesting substantial transcriptomic dysregulation already in non-lesional, healthy -appearing skin.Attorney Docket No.: MS-0050-01-US-NP

[0126] Stratification of the differential gene expression analysis based on the presence or absence of comorbid AD, led to the detection of a total of 4,808 and 4,842 upregulated DEGs in CHE+ADand CHE'AI)lesional skin, respectively, in comparison to HC samples, with as many as 4,417 of these DEGs overlapping in both subgroups, forming the common transcriptomic phenoty pe of both groups (Figure 3 A). Similarly, 2,905 downregulated DEGs were detected in CHE+ADlesional vs HC and 3,120 down regulated DEGs in CHE-ADlesional vs HC comparisons, with an overlap of 2,772 of these DEGs (Figure 3A). Both subgroups also exhibited significant dysregulation in non-lesional vs HC comparisons, with a similarly strong overlap between CHE with or without comorbid AD (Figure 3B).

[0127] When visualizing the top dysregulated DEGs common to both CHE+ADand CIIE’AD(“CHEALL”), the highest level of dysregulation is observed in the lesional vs HC skin comparisons in both up and down-regulated DEGs (Figures 2C-D). Similarly, non-lesional CHEA, Lalso showed considerable dysregulation compared to HC skin (Figure 2C-D). In line with the inflammatory nature of CHE, increases in multiple immune mediators were observed in lesional and non-lesional CHEALLcompared to HC. The top upregulated markers (Figure 2C) included the cytotoxic marker granulysin / G' / V / .f. markers of dendritic cells (CD IB, CD1E, CD40), T cell exhaustion / activation (TIG IT. IGOS, CD28, IL3RA, CD96), memory (CCR8) and regulatory T cells (FOXP3, CTLA4), and as well as markers of type 1 (IRF4 type 2 (TSLPR / C7? / . / '2) and type f / 17 (IL12B) immune axes (Figure 2C).

[0128] This mixed immune activation pattern was consistent with results from an EnrichR analysis of all significantly upregulated DEGs (FCH>1.5, FDR<0.05) and conoborated the strong inflammatory tone in lesional vs HC samples, as reflected by the enrichment of “Innate-” and “Adaptive Immune System’" terms from the “Reactome” database (Figure 9A).

[0129] Among top downregulated genes of CHEALL, several keratins (KRTT) and keratin-associated protein genes (KRTAPs) were identified, such as KRT27, KRT71, KRTAP1-5, KRTAP5-3, and KRTAP12-2 (Figure 3D), consistent with previous reports in CHE. Other top downregulated DEGs included the epidermal differentiation complex component SPRR3, the psoriasis-associated markers CCL7 an NOS2, as well as the neuronal developmental markers NEUROG1, OTX2. GPM6A. and A GRL3 that have previously been suggested to be involved in skin pigmentation. Multiple ectopic olfactory receptor genes (e g., OR6S1, OR4C6, OR5H14\ so far associated with the detection of chemosignals in the skin, w ere also among the top downregulated DEGs in both lesional and non-lesional skin of C 11 EAi r(Figure 2B). Consistently, an EnrichR analysis of all genes significantly downregulated in lesional CHEALLvs HC, showed enrichment for “Olfactory Signaling” and “Sensory Perception” pathways.Attorney Docket No.: MS-0050-01-US-NPalong with “Keratinization” and “Formation of Cornified Envelope” (Figure 9B). Downregulation was observed of the key terminal differentiation markers filaggrin / FZG and \or\crin I. ORICRIN both in non-lesional and lesional skin when compared to HC (Figure 3E-F), suggesting that barrier impairment already exists at the non-lesional CHE level. Keratin gene KRT9, expressed in the terminally differentiated epidermis of palms and soles, was also downregulated in both lesional and non-lesional CHEALLwhen compared to HC skin (Figure 9G). These data demonstrate that independent of comorbid AD. CHE lesions show a common disease phenotype characterized by both immune and barrier abnormalities.CHE showed a mixed immune phenotype enriched for type- 1 and h pe-2 T-cell axes.

[0130] Given the strong immune-related dysregulation captured in CHE samples, an assessment was performed of a previously published list of immune genes (Figure 4). Lesional CHE^'1' vs HC skin demonstrated upregulation of T-cell markers (e.g., CD 3D, CD3E, CD3G, CD4, CD5. CD8A, ITK, IL7R GZMB), both tissue residency (CD69. CXCR4) and central memory markers (CC?7), the immune checkpoint genes CD27 and CTLA4, and the costimulatory genes CD28, CD48. CD80. CD86 and ICOS (Figure 4A). Additionally, lesional skin showed upregulation of chemokines XCL1 and XCL2, and the chemokine receptors CCR7 and CXCR4. Moreover, markers associated with type-1 (e.g., IL12RB2, IFNGR1, IFNGR2, MXI), type-2 (e.g., CCI.22. CCL24, 0X40 / TNFRSF4, TSLPR, CRLF2, GATA3), and Th22 (AHR) immunity were also upregulated in this common immune phenotype (Figure 4A).

[0131] Conversely, IL34. a negative regulator of inflammation, was among the top downregulated genes, as well as the IL-10-family member IL19. Downregulation was observed of the CXCR4 inhibitor CXCL17, which was previously associated with anti-inflammatory properties in a psoriatic model via attraction of myeloid-derived suppressor cells. However, decreased levels were observed of certain proinflammatory cytokines such as type-1 interferons JFNA1, IFNA2) as well as IL7, IL15. and IL18 (Figure 4A). Some type-17-associated immune mediators (IL17F, LCN2, PI3) also showed reduced expression levels in lesional and non-lesional CHEA, J' compared to HC (Figure 4A). These data demonstrate considerable immune dysregulation in the common disease phenotype in CHE, consisting of upregulation mainly in type 1 and type 2 mediators, and to a lesser extent Th22, along with downregulated antiinflammatory components as well as Th 17 mediators.CHE with and without comorbid AD harbor distinct immune profiles.

[0132] To better understand unique disease signatures depending on AD comorbidity, the inventors assessed immune markers uniquely up- or downregulated in CHE+ADand CHE-ADsubsets (Figures 4B-C, 10, and 11). Besides the general type 2 inflammatory tone in the entireAttorney Docket No.: MS-0050-01-US-NPCHE cohort, CHEADlesions were characterized by upregulation of additional Th2 hallmark mediators, namely IL13 and CCL17 (Figure 4B). Upregulation was observed of the innate immune marker IL1RAP, the cytotoxic mediator TRAIL / TNFSF10, the chemokine receptor CCR2, as well as RUNXJ in CHE+ADlesions (Figure 10 A), a hematopoietic differentiation marker that has previously also been associated with enhanced IL22 expression. Besides these innate and adaptive immune markers, increases were observed in genes associated with immune regulation in CHE+AD, such as the IL-27 receptor component IL27RA. and the suppressor of cytokine signaling SOCS2 (Figures 4B and 10A), which might indicate compensatory or counterregulatory mechanisms in these lesions. In contrast to enhanced type 2 immunity, downregulation was observed of certain type-1 CXCL1155, CX3CL156, I. YGF ). and type-17 (IL23R) associated genes in CHE+ADlesions (Figures 4B and 10B). Conversely, CHE'AI)lesions (Figures 4C and 11) harbored a prominent activation pattern of type- 1 (IL15RA, CXCL9, ISG20) immune mediators, as well as increases in the type-17-associated chemokine CCL20. In line with this observation, CHE-ADlesions harbored upregulation of the costimulatory marker CD137 / TNFRSF9, previously shown to drive type-1 inflammation, the interferon-y-induced indoleamine-pyrrole-2,3-dioxygenases 1DO1 and IDO2, as well as the B lymphocyte chemoattractant CXCL13 (Figures 4C). Among genes differentially downregulated in CHE-AD, the inventors observed the neutrophilic chemoattractant CXCL6 and its receptors CXCR1 and CXCR259as well as the type 2-associated prostaglandin receptor PTGDR2 (Figure 11B).

[0133] To integrate these immune pathways, the inventors performed gene set variation analyses, which corroborated the notion that within the core CHE immune phenotype, Thl, Th2, and Th22 signals are enhanced in lesional CHE skin when compared to non-lesional CHE or HC skin (Figures 5A-B). However, there was a greater Thl skewing in CHE’AI). and higher Th2 pathway activation in CHE+ADlesional vs non-lesional comparisons (Figure 5A-B). Conversely, epidermal barrier signatures were decreased in lesional and non-lesional CHE skin in both groups (Figure 5A-B). Thl 7 signatures showed significant downregulation in non-lesional CHE skin but not in lesional skin, independent of AD comorbidity. Overall, these data demonstrate that lesional CHE+ADharbors a stronger type-2 immune skewing while lesional CHE-ADshowed greater type-1 responses.Correlation of immune gene expression with clinical severity scores.

[0134] The inventors next evaluated associations of different disease severity measures (HECSI, mTLSS, DLQI, Pain VAS) with the top 100 dysregulated inflammatory biomarkers in lesional skin. Positive (red) and negative (blue) correlations are presented as heat mapsAttorney Docket No.: MS-0050-01-US-NP(Figures 6-7). When analyzing the lesional CHE+ADsubgroup (Figure 6A), DLQI and Pain VAS clustered with a broad group of inflammatory markers (TRAIL / 77VFSF7ft FCGR3B, CCR1, S100P, IL19, OASL, CXC1.6. PI3, IFNA2. CCL7, CCL21, TGFB3, 1. AT2. RORC) (green box, Figure 6A), while HECSI and mTLSS tended to cluster along with more type-2 immune markers (e.g., IL13, CCL17, OXAQ / TNFRSF4, CCL13) (orange box, Figure 6A). Overall, 40 statistically significant correlations were found between disease severity scores and inflammatory mediators in CHE+ADlesions, with 22 of these biomarkers having at least moderate correlation (|p|>0.4, red labels. Figure 6A). Among the strongest positive correlations, the inventors found CCL17 with mTLSS (p=0.46, p<0.01, Figure 6B), and CCL24 with HECSI (p=0.44, p<0.05, Figure 6C). Other highly significant correlations included the interferon-related IRF1 with HECSI (p=0.41, p<0.05. Figure 6D) and XCL2 with mTLSS (p=0.57, p<0.001. Figure 6E).

[0135] Among the lesional CHE-ADsubgroup (Figure 7A), several type-2 (O AQ / TNFRSF4, GATA3, FCER1A, IL13, CCL17) and type 22 (AHR); (pink box, Figure 7A), as well as type-1 biomarkers (IL12B, IL15, CXCL9, CXCL10, CXCL11, CX3CL1: orange box. Figure 7A) clustered together. However, the four clinical severity scores clustered separately (green box. Figure 7A). In line, only 13 statistically significant correlations were found between disease severity scores and inflammatory biomarkers in CHE'Wlesions, wi th six of them having at least moderate correlation (|p|>0.4, red labels, Figure 7A). Still, some correlations between clinical severity and gene expression levels were significant: The negative regulator SOCS2, a suppressor of STAT activation, correlated with mTLSS (p=0.44, p<0.0I, Figure 7B), and IL34 negatively correlated with DLQI and mTLSS (p= -0.43, p<0.05, Figures 6C-D).

Claims

Attorney Docket No.: MS-0050-01-US-NPCLAIMSWhat is claimed is:

1. A method of treatment of chronic hand eczema (CHE) in a subject in need thereof, comprising:(a) providing a surface skin sample from the subject obtained from one or more tapestrips applied to a skin surface of the subject, wherein application of the one or more tapestrips to the skin surface obtains skin materials to the tape-strip;(b) determining a level of one or more biomarkers in the surface skin sample;(c) comparing the level of the one or more biomarkers in the surface skin sample to a reference level of the one or more biomarkers to obtain a comparative level;(d) determining that the subject has CHE based on the comparative level; and (e) administering to the subject a CHE therapy.

2. A method of diagnosing chronic hand eczema in a subject in need thereof, comprising:(a) providing a surface skin sample from the subject obtained from one or more tapestrips applied to a skin surface of the subject, wherein application of the one or more tapestrips to the skin surface obtains skin materials to the tape-strip;(b) determining a level of one or more biomarkers in the surface skin sample;(c) comparing the levels of the one or more biomarkers in the surface skin sample to a reference level of the one or more biomarkers to obtain a comparative level; and(d) determining that the subject has CHE based on the comparative level.

3. A method of detecting the efficacy of treatment of chronic hand eczema in a subject in need thereof, comprising:(a) providing a surface skin sample from the subject obtained from one or more tapestrips applied to a skin surface of the subject, wherein application of the one or more tapestrips to the skin surface obtains skin materials to the tape-strip;(b) determining a level of one or more biomarkers in the surface skin sample;(c) comparing the levels of the one or more biomarkers in the surface skin sample to a reference level of the one or more biomarkers to obtain a comparative level; and(d) determining the efficacy of the CHE treatment based on the comparative level.

4. The method of any one of claims 1-3, comprising determining the levels of two or more biomarkers in the surface skin sample.Attorney Docket No.: MS-0050-01-US-NP5. The method of claim 1, wherein the one or more biomarkers comprises a gene product of any of: GNLY; CD1B; CD1E CD40; TIGIT; ICOS; CD28; IL3RA; CD96; CCR8;FOXP3; CTLA4; IRF4; TSLPR / CRLF2; and IL12B, or combinations thereof.

6. The method of any one of claims 2-3, wherein the one or more biomarkers comprises a gene product of any of: GNLY; CD IB; CD IE; CD40; TIGIT; ICOS; CD28; IL3RA;CD96; CCR8; FOXP3; CTLA4; IRF4; TSLPR / CRLF2; and IL12B, or combinations thereof.

7. The method of claim 1, wherein said one or more biomarkers comprises a gene product of any of: OX40 / TNFRSF4; CRLF2; IL7R; CD122 / IL2RB; JAK1; JAK2; and IL 13, or combinations thereof.

8. The method of any one of claims 2-3, wherein said one or more biomarkers comprises a gene product of any of: OX40 / TNFRSF4; CRLF2; IL7R; CD122 / IL2RB; JAK1; JAK2; and IL 13, or combinations thereof.

9. The method of claim 1, wherein said one or more biomarkers comprises a gene product of any of: KRT27; KRT71; KRTAP1-5; KRTAP5-3; KRTAP12-2; SPRR3; CCL7; NOS2; NEUROG1; OTX2; GPM6A; ADGRL3; OR6S1; OR4C6; OR5H14; FLG; LORICRIN; and KRT9, or combinations thereof.

10. The method of any one of claims 2-3, wherein said one or more biomarkers comprises a gene product of any of: KRT27; KRT71; KRTAP1-5; KRTAP5-3; KRTAP12-2; SPRR3; CCL7; NOS2; NEUROG1; OTX2; GPM6A; ADGRL3; OR6S1; OR4C6; OR5H14; FLG; LORICRIN; and KRT9, or combinations thereof.

11. The method of any of claims 1-3, wherein said subject does not have atopic dermatitis (AD).

12. The method of any of claims 1-3, wherein said subject has AD.

13. The method of any of claims 1-3, wherein said determining further comprises differentiating between: (a) subjects having CHE and having AD (CHE+AD); and (b) subjects having CHE in the absence of AD (CHE- AD).

14. The method of claim 13, wherein said one or more biomarkers comprises a gene product of any of: RGS1; IL19; RCOR3; MTR; NPHP3: HSPA1A; HMGN3; REV3L; FAM229B; GPR31; MACC1; ADAMDEC1; IDO1; IDO2; ZBTB43; TRIM66; OAF; EEA1; SDSL; RSRC2; SPG11; TRIM69; FURIN; TVP23A; CTNS; VMO1; RGS9; CCDC57;SIGLEC12; or ENTHD1, or combinations thereof.

15. The method of any one of claims 1-3, wherein the skin surface is non-lesioned skin.Attorney Docket No.: MS-0050-01-US-NP16. The method of any of claims 1-3, wherein said determining further comprises differentiating between lesional and non-lesional skin in said subjects.

17. The method of claim 16, wherein said one or more biomarkers comprises a gene product of any of: DPM3; FAM114A2; FZD3; PTPDC1; or PRM1, or combinations thereof.

18. The method of claim 1, wherein said treatment comprises a topical treatment, oral medication, a biologic, or combinations thereof.

19. The method of claim 1 comprising administering a therapeutic selected from the group consisting of: rocatinlimab, telazorlimab, tezepelumab, ordesekimab, amlitelimab, AMG 714, PRV-015, delgocitinib, peficitinib, ustekinumab, tofacitnib, upadacitinib, baricitinib, roxulitinib, lebrikizumab, dupilumab, and tralokinumab.

20. The method of any of claims 5-17. wherein said biomarker is down regulated in the subject.

21. The method of any of claims 5-17, wherein said biomarker is up-regulated in the subject.

22. The method of any one of claims 5-17, wherein the gene product is an RNA transcript, a protein, or both.

23. The method of any one of claims 1-3, wherein said determining uses one or more of an RNA-sequencing analysis, a PCR analysis, a qPCR analysis, a proteomic analysis, or combinations thereof.

24. The method of claim 26, wherein said determining uses an RNA-sequencing analysis.

25. The method of any one of claims 1-3, wherein the level of the one or more biomarkers is determined by measuring mRNA levels of the biomarker.

26. The method of any one of claims 1-3, wherein said level of the one or more biomarkers is determined by measuring cDNA levels of the biomarker.

27. A method of treatment of chronic hand eczema (CHE) in a subject in need thereof, comprising:(a) providing a first surface skin sample from the subject obtained from one or more tape-strips applied to a first skin surface of the subject, wherein application of the one or more tape-strips to the skin surface obtains skin materials on the tape-strip and wherein the first skin surface is from lesional skin;(b) determining a level of one or more biomarkers in the first surface skin sample; (c) providing a second surface skin sample from the subject obtained from one or more tape-strips applied to a second skin surface of the subject, wherein application of the one or more tape-strips to the skin surface obtains skin materials on the tape-strip and wherein the second skin surface is from non-lesional skin;Attorney Docket No.: MS-0050-01-US-NP(d) determining a level of one or more biomarkers in the second surface skin sample; (e) comparing the levels of the one or more biomarkers in said first surface skin sample to a reference level obtained from the corresponding one or more biomarkers in said second surface skin sample to obtain a comparative level;(f) determining that the subject has CHE based on the comparative level; and (g) after the determining, administering to the subject a CHE therapy.

28. A method of determining the efficacy of treatment of CHE in a subject, comprising:(a) providing a first level of one or more biomarkers in a first surface skin sample from the subject;(b) administering to the subject a therapy for treating CHE;(c) providing a second level of one or more biomarkers in a second surface skin sample from the subject obtained at a later time than the first surface skin sample and wherein the second surface skin sample is obtained after administering the therapy;(d) comparing the first level of the one or more biomarkers to the second level of the one or more biomarkers to obtain a comparative level; and(e) determining the efficacy of the treatment based on the comparative level; wherein the first skin sample is obtained from a first set of one or more tape-strips applied to a skin surface, and the second skin sample is obtained from a second set of one or more tape-strips applied to a skin surface; and wherein application of the tape-strips to the skin surface obtains skin materials to the tape-strip.

Images