DIFFERENTIAL DIAGNOSIS OF ECZEMA AND PSORIASIS

Abstract

The present invention relates to a method of diagnosing eczema and/or psoriasis, wherein said method differentiates between eczema and psoriasis, and comprises determining the expression of at least two markers in a sample taken from an individual, wherein said at least two markers are selected from CCL27, NOS2, IL36G, KLK13, SOST, NPTX1, PLA2G4D, GDA, IL36A, TGM1, CLEC4G, IL13, TCN1, TMPRSS11D and RHCG, provided that said at least two markers consist of or comprise (a) CCL27 and NOS2; (b) CCL27 and KLK13; (c) IL36G and KLK13; (d) CCL27 and IL36G; (e) NOS2 and IL36G; (f) NOS2 and KLK13; (g) SOST; or (h) NPTX1; and assessing on the basis of the expression of said at least two

Claims

1. A kit comprising means for quantifying the expression of at least two genes selected from CCL27, NOS2, IL36G, KLK13, SOST, NPTX1, PLA2G4D, GDA, IL36A, TGM1, CLEC4G, IL13, TCN1, TMPRSS11D and RHCG, provided that said at least two genes include at least one of sets (a) to (h): (a) CCL27 and NOS2; (b) CCL27 and KLK13; (c) IL36G and KLK13; (d) CCL27 and IL36G; (e) NOS2 and IL36G; (f) NOS2 and KLK13; (g) SOST; or (h) NPTX1; and wherein said means are primers that specifically bind to nucleic acids of said genes.

2. The kit of claim 1, wherein said at least two genes comprise CCL27 and NOS2.

3. The kit of claim 1, wherein said at least two genes are only CCL27 and NOS2.

4. The kit of claim 1, wherein the primers that specifically bind to nucleic acids of said genes are pairs of primers, and: the primers that specifically bind to nucleic acids of CCL27 have the nucleotide sequences of SEQ ID NOs: 1 and 2; the primers that specifically bind to nucleic acids of NOS2 have the nucleotide sequences of SEQ ID NOs: 3 and 4; the primers that specifically bind to nucleic acids of IL36G have the nucleotide sequences of SEQ ID NOs: 5 and 6; the primers that specifically bind to nucleic acids of KLK13 have the nucleotide sequences of SEQ ID NOs: 7 and 8; the primers that specifically bind to nucleic acids of SOST have the nucleotide sequences of SEQ ID NOs: 9 and 10; the primers that specifically bind to nucleic acids of NPTX1 have the nucleotide sequences of SEQ ID NOs: 11 and 12; the primers that specifically bind to nucleic acids of PLA2G4D have the nucleotide sequences of SEQ ID NOs: 13 and 14; the primers that specifically bind to nucleic acids of GDA have the nucleotide sequences of SEQ ID NOs: 15 and 16; the primers that specifically bind to nucleic acids of IL36A have the nucleotide sequences of SEQ ID NOs: 17 and 18; the primers that specifically bind to nucleic acids of TGM1 have the nucleotide sequences of SEQ ID NOs: 19 and 20; the primers that specifically bind to nucleic acids of CLEC4G have the nucleotide sequences of SEQ ID NOs: 21 and 22; the primers that specifically bind to nucleic acids of 1L13 have the nucleotide sequences of SEQ ID NOs: 23 and 24; the primers that specifically bind to nucleic acids of TCN1 have the nucleotide sequences of SEQ ID NOs: 25 and 26; the primers that specifically bind to nucleic acids of TMPRSS11D have the nucleotide sequences of SEQ ID NOs: 27 and 28; the primers that specifically bind to nucleic acids of RHCG have the nucleotide sequences of SEQ ID NOs: 29 and 30.

20. The kit of claim 2, wherein the primers that specifically bind to nucleic acids of said genes are pairs of primers, and: the primers that specifically bind to nucleic acids of CCL27 have the nucleotide sequences of SEQ ID NOs: 1 and 2; the primers that specifically bind to nucleic acids of NOS2 have the nucleotide sequences of SEQ ID NOs: 3 and 4.

6. A method for treating psoriasis or eczema in a subject comprising: i) quantitating in a sample taken from the subject the expression of at least two genes selected from CCL27, NOS2, IL36G, KLK13, SOST, NPTX1, PLA2G4D, GDA, IL36A, TGM1, CLEC4G, IL13, TCN1, TMPRSS11D and RHCG, provided that said at least two genes include at least one of sets (a) to (h): (a) CCL27 and NOS2; (b) CCL27 and KLK13; (c) IL36G and KLK13; (d) CCL27 and IL36G; (e) NOS2 and IL36G; (f) NOS2 and KLK13; (g) SOST; or (h) NPTX1; wherein said means are primers that specifically bind to nucleic acids of said genes; and ii) administering to the subject a treatment for psoriasis if the level of expression in the sample compared to the level of expression in a sample from a healthy subject is: at least 1.584 for NOS2; at least 2.304 for TMPRSS11D; at least 1.364 for RHCG; at least 0.939 for SOST; at least 2.398 for TCN1; at least 0.999 for GDA; at least 2.868 for IL36A; at least 1.219 for IL36G; at least 1.05 for KLK13; at least 1.081 for PLAG4D; at least 0.8832 for TGM1; at least 0.7974 for IL13; at least 0.2029 for CLEC4G; less than 0.3983 for CCL27; less than 0.4779 for NPTX1; or iii) administering to the subject a treatment for eczema to the subject if the level of expression in the sample from the subject compared to the level of expression in a sample from a healthy subject is: less than 1.584 for NOS2; less than 2.304 for TMPRSS11D; less than 1.364 for RHCG; less than 0.939 for SOST; less than 2.398 for TCN1; less than 0.999 for GDA; less than 2.868 for IL36A; less than 1.219 for IL36G; less than 1.05 for KLK13; less than 1.081 for PLAG4D; less than 0.8832 for TGM1; less than 0.7974 for IL13; less than 0.2029 for CLEC4G; at least 0.3983 for CCL27; at least 0.4779 for NPTX1.

7. The method of treatment of claim 6, wherein said at least two genes comprise NOS2 and CCL27.

8. The method of treatment of claim 6, wherein said at least two genes are only NOS2 and CCL27.

9. The method of treatment of claim 6, wherein an anti-TNF? medicament or a medicament comprising IL-4 is used for treatment of psoriasis and an anti-IL-4 receptor antibody is used for treatment of eczema.

10. The method of treatment of claim 7, wherein an anti-TNF? medicament or a medicament comprising IL-4 is used for treatment of psoriasis and an anti-IL-4 receptor antibody is used for treatment of eczema.

11. The method of treatment of claim 8, wherein an anti-TNF? medicament or a medicament comprising IL-4 is used for treatment of psoriasis and an anti-IL-4 receptor antibody is used for treatment of eczema.

Description

THE FIGURES SHOW

[0066] FIG. 1

[0067] A Real-time PCR validation of 15 genes significantly different between psoriasis (n=9) and eczema (n=10). The curve shows the cut-off values between the two groups for each gene. Frames indicate the two genes chosen to build up the disease classifier. *=p<0.05, **p<0.01, ***p<0.0001, no star p<0.1 (Bonferroni corrected for multiple testing. B A disease classifier consisting of NOS2 (y-axis) and CCL27 (x-axis) accurately separates psoriasis and eczema patients in a training set consisting of 19 patients (9 psoriasis, 10 eczema). Shown are data samples of the training set after log transformation and scaling. The crosses indicate the support vectors, the circles indicate the remaining data samples of the training set. C Performance of the disease classifier in an independent test cohort (16 psoriasis patients, 18 eczema patients). The square shows one initially mis-classified patient, the squares with discontinued contour a clinically and histologically unclear patient illustrated in D. Scale bars indicate 100 ?m in overview and 50 ?m in islets, respectively.

[0068] FIG. 2

[0069] Immunofluorescence stainings for DAPI, NOS2, and CCL27 of eczema and psoriasis skin.

[0070] FIG. 3

[0071] CCL27 staining pattern distinguishes psoriasis from eczema. The Examples illustrate the invention.

Example 1

Classifier Build Up

[0072] 15 marker genes were selected according to the following criteria: genes with most predominant difference between psoriasis and eczema; and functional annotation as epidermal, immune related, and metabolic genes.

[0073] For training the classifier on the real-time PCR data of an independent disease cohort (n=19), the R package e1071 (http://CRAN.R-project.org/package=e1071) was applied using support vector machines (SVMs). To get normally distributed data the measurements of the selected 15 genes were transformed using the logarithm to the base 10. Then, a two-sample, two-sided Welch's t-test followed by a Bonferroni p-value correction was used to test for differential expression. The two genes which were most significantly down-regulated (CCL27, adjusted p-value=5.31*10{circumflex over ()}(?4)) , or up-regulated (NOS2, adjusted p-value=1.53*10{circumflex over ()}(?6)) were selected for the preferred classifier. The scaled and log transformed data of the two genes were used as a training set for a C-classification using a linear kernel function with C=1. To train the classifier a 10-fold cross-validation was used. Then, the classifier was tested on log-transformed data samples of a third independent cohort (n=34) by predicting the disease class and computing probability predictions based on the trained model.

Primer Design and Real-Time PCR

[0074] Primers amplifying genes of interest were designed using the publicly accessible Primer3 software (http://frodo.wi.mit.edu/primer3/). The used primers are given in the sequence listing.

[0075] Real time PCR reactions were performed in 384-well plates using the Fast Start SYBR Green Master mix (Roche Applied Science) and the ViiA7 Real Time PCR machine (Applied Biosystems). The expression of transcripts was normalized to expression of 18S ribosomal RNA as housekeeping gene. Data were expressed as mRNA fold change, relative to non-involved skin as calibrator. Relative quantification was determined according to the formula: (RQ)=.sup.2-?Ct.

Example 2

Function of Uniquely Regulated Genes

[0076] Concerning the immune system, differences between psoriasis and eczema were observed: exclusively in psoriatic skin, cytokines belonging to the IL-10 family such as IL-19 and IL-20 as well as IL-36A and IL-36G were significantly up-regulated. A non-significant trend for a higher induction of Th17 associated cytokines IL-17A, IL-17F, IL-21, and IL-22 was also observed in psoriatic plaques. Cytokines that were exclusively induced in eczematous lesions were IL-6 and the Th2 cytokine IL-13, with a trend for a higher induction of other Th2 cytokines IL-4, IL-5, and IL-10 in eczematous as compared to psoriatic skin.

[0077] The chemokines CCL17 and CCL18 were up-regulated in eczematous lesions to a higher degree than in psoriatic skin. CCL27 was down-regulated significantly in psoriatic skin. On the other hand, CXCL1 and CXCL8 (IL-8) showed a stronger up-regulation in psoriatic plaques, with CXCL8 being exclusively regulated in psoriatic skin.

[0078] Bridging the immune system and the epidermal component, numerous antimicrobial peptides (AMPs) were found to be up-regulated in both psoriatic and eczematous skin as compared to non-involved skin, respectively. The defensin members DEFB4 and DEFB103B as well as the S100 proteins S100A7A, S100A7, S100A8, S100A9, and S100A12 were significantly up-regulated in both diseases. However, induction of all detected AMPs was much higher in psoriatic than in eczematous skin. Accordingly, the IL-20 induced Kallikrein-related peptidases KLK6, KLK9, and KLK13, demonstrated to induce AMPs, were exclusively up-regulated in psoriatic, but not in eczematous skin.

[0079] Differences were also observed regarding early differentiation markers of the small proline-rich protein (SPRR) family and the late cornified envelope (LCE) family. SPRR1A, SPRR1B, SPRR2A, SPRR2B, SPRR2C, SPRR2D, LCE3C, and cornifelin were exclusively up-regulated in psoriatic plaques. LCE3A, LCE3D, and LCE3E were up-regulated in both psoriasis and eczema skin, but to a higher degree in psoriasis. In contrast, LCE1B, and LCE5A were down-regulated both in psoriatic and eczematous skin, but to a higher degree in eczema. A heterogeneous picture was observed regarding keratin regulation, with KRT6A, KRT6B, KRT6C, KRT16 and KRT75 highly up-regulated in psoriatic skin and KRT2, KRT19 as well as KRT77 down-regulated more in psoriatic than in eczematous skin. The late differentiation genes of the filaggrin family FLG and FLG2 were also down-regulated in both psoriatic and eczematous skin. hornerin was up-regulated more in psoriatic than in eczematous as compared to non-involved skin.

[0080] Numerous genes involved in glucose, lipid, and amino acid metabolism were exclusively regulated in psoriatic, but not in eczematous skin. Namely, regulation of the phospholipase PLA2G4D, nitric oxide synthase 2 (iNOS or NOS2), ATP-binding cassettes ABCG4, the serine proteases PRSS22, PRSS27, PRSS53, kynureninase, transcobalamin, and the Wnt signaling inhibitor sclerostin were up-regulated exclusively in psoriatic plaques. The aldo-keto reductase family members AKR1B15, AKR1B10 and the peptidase inhibitor P13 were up-regulated in both psoriatic and eczematous skin, but more in psoriasis.

Example 3

Establishing a Disease Classifier to Distinguish Psoriasis and Eczema

[0081] Since differences between psoriasis and eczema were observed at both single gene and signaling pathway level, we sought to translate these basic results into a disease classifier that enables to distinguish psoriasis from eczema. The 15 genes of the invention (Table 1, FIG. 1A) were included in a validation cohort with 19 patients (9 for psoriasis, 10 for eczema) to train a classifier. Expression of the selected 15 genes was detected using real-time PCR in all 19 patients and a two-sample, two-sided Welch's t-test on the log-transformed measurements followed by a Bonferroni p-value correction was used to assign each of the 15 genes with a p-value. The primer sequences used for real-time PCR are given in table S4. CCL27 and NOS2 were the genes with lowest adjusted p-values (for significantly up and down regulation, respectively, of psoriasis versus eczema). Based on these two genes a classifier was trained using a 10-fold cross-validation and support vector machines (SVMs). An average accuracy of 100% was achieved (FIG. 1B). With an independent third cohort (34 patients in total; 16 psoriasis patients and 18 eczema patients), the classifier was tested and could classify 33 out of 34 patients as predicted from clinical and histological evaluation (kappa=0.94; FIG. 10).

Example 4

Performance of the Classifier in Special Cases

[0082] One patient was classified as eczema with a probability of 0.85, although the given diagnosis was psoriasis. Back-tracing clinical and histological features of this patient revealed that the initial diagnosis psoriasis was most likely not correct. The 54 year old patient presented with disseminated, demarcated eczema-like skin lesions with centri-pedal desquamation that had erupted two months before. Histological evaluation revealed neutrophil microabscesses, spongiosis, single cell necrosis in the epidermis, and an epidermotropism of immune cells. Other hallmarks for psoriasis such as acanthosis and epidermal thinning above dermal papillae containing dilated and tortuous capillaries were not observed. In line with that observation, the patient did not respond well to dithranol. Furthermore, skin lesions did not relapse after remission. Retrospectively, other diagnoses like pityriasis rosea, eczema or pityriasis lichenoides chronica are clearly to be favored in this patient.

[0083] Besides patients with a given diagnosis from clinical and histological evaluation, one patient was tested where the gold standard methods could not distinguish between psoriasis and eczema (FIG. 1D). The female 53 year-old patient suffered from inflammatory skin lesions since years. Eczema could have been favored because she suffered from allergic asthma, her IgE was mildly elevated (108 IU/ml), and the lesions were itchy; the positive family history for psoriasis and the very stationary plaques in predilection areas were typical rather for psoriasis. Also the histological evaluation was conflicting, with a plump acanthosis, partially missing stratum granulosum and parakeratosis accounting for psoriasis. On the other hand, T cell epidermotropism and very few neutrophils with missing microabscesses were more typical for eczema. When this patient was tested in the classifier, the two biopsies were classified as eczema with a probability above 99%, indicating the classifier might be useful even in cases where established gold standard diagnostic tools fail.

Example 5

Immunofluorescence

[0084] The proposed disease classifier of psoriasis and eczema based on the markers NOS2 and CCL27 is valid at the level of immunofluorescence. This has been confirmed using data from 118 patients. Paraffin-embedded skin biopsies from psoriasis, eczema, and clinically as well as histologically unclear skin lesions were stained for DAPI (nuclear staining), NOS2, and CCL27. In line with the results obtained from PCR analysis at RNA level, psoriasis sections stained strongly positive for NOS2, but hardly for CCL27. In contrast, eczema sections were positive for CCL27, but NOS2 staining was very weak to absent (FIG. 2). In conclusion, the proposed classifier to distinguish psoriasis from eczema is valid at protein expression level, as demonstrated using immunofluorescence double stainings.

Example 6

Image analysis

[0085] The proposed markers NOS2 and CCL27 allow to distinguish psoriasis from eczema based on their protein expression pattern. In particular, CCL27 distributes homogenously over the cytoplasm in psoriasis sections, while eczema samples are characterized by a clear nuclear staining pattern (FIG. 3).

[0086] An image analysis programme was established using Python programming language: Both color channels of each image (blue channel for nuclear stain DAPI and red channel for CCL27 stain) were analysed separately and compared amongst each other using two different methods simultaneously.

[0087] The first method is based on Fourier transformation which decomposes an image into its frequencies in a two-dimensional space (sine and cosine components). Sharp edges which can be seen in the DAPI channel as well as in the CCL27 channel of eczemawhere clear borders between positive nuclei and rather negative cytoplasm are foundare represented by high frequencies, whereas ubiquitous cytoplasmatic occurrence pf CCL27 as seen in psoriasis is represented by low edges and as such by low frequencies. Similar frequency power densities between the two channels are resulting in quotients close to 1, distinct frequency power densities in quotients close to 0.

[0088] The second method is based on convolution. Here, both color channels of each part of the image are sequentially compared with each other in windows of 32?32 pixel. The higher the congruencewhich is the case of CCL27 distribution in eczema and DAPI but not between CCL27 distribution in psoriasis and DAPIthe higher the calculated values (maximal value 1, minimal value 0). For each image both values are represented by one dot in a 2 dimensional coordinate system. A line is plotted at maximal separation of both groups.