A METHOD FOR CHARACTERIZING MELANOCYTIC LESIONS

Abstract

The present disclosure pertains to the identification of novel biomarkers for the characterization of melanocytic lesions as can be found in skin diseases such as melanomas and psoriasis. In particular, the present invention is directed to a method for characterizing melanocytic lesions and their use in an automated or semi-automated device for the diagnosis and/or monitoring of benign, pre-malignant, and malignant melanocytic lesions, as further defined in the claims.

Claims

1-15. (canceled)

16. A method for characterizing melanocytic lesions, comprising determining the expression level of one or more biomarkers of keratinocytes surrounding melanocytes in a biopsy sample obtained from a subject, wherein the one or more biomarker is selected from the group consisting of ADAM10, Notch1, p27.sup.KIP1, CD63, PPAR, TAP73, and SPPL3; wherein ADAM10, Notch1, and p27.sup.KIP1 is present in healthy keratinocytes and when a pre-malignant melanocytic lesion develops, but disappears when a malignant melanocytic lesion develops; and wherein CD63, PPAR, TAP73, and SPPL3 is not present in healthy keratinocytes and appears when a pre-malignant or malignant melanocytic lesion develops, wherein the expression level of said biomarker is stronger when a malignant melanocytic lesion develops as compared to when a pre-malignant melanocytic lesion develops; thereby characterizing the melanocytic lesion as a pre-malignant or malignant melanocytic lesion.

17. The method of claim 16, wherein the expression level of one or more biomarker selected from the group consisting of ADAM10, Notch1, and p27.sup.KIP1 is determined, and the expression level of one or more biomarker selected from the group consisting of CD63, PPAR, TAP73, and SPPL3 is determined.

18. The method of claim 16, wherein the expression level of at least two biomarkers selected from the group consisting of ADAM10, Notch1, and p27.sup.KIP1 is determined.

19. The method of claim 18, wherein the expression level of all biomarkers selected from the group consisting of ADAM10, Notch1, and p27.sup.KIP1 is determined.

20. The method of claim 16 , wherein the expression level of at least two biomarkers selected from the group consisting of CD63, PPAR, TAP73, and SPPL3 is determined.

21. The method of claim 20, wherein the expression level of at least three biomarkers selected from the group consisting of CD63, PPAR, TAP73, and SPPL3 is determined.

22. The method of claim 21, wherein the expression level of all biomarkers from the group consisting of CD63, PPAR, TAP73, and SPPL3 is determined.

23. The method of claim 16, wherein the expression level of at least two of said biomarkers, the expression level of at least three of said biomarkers, the expression level of at least four of said biomarkers, the expression level of at least five of said biomarkers, the expression level of at least six of said biomarkers, or the expression level of all seven biomarkers is determined.

24. The method of claim 16, wherein further the expression level of APBB1, CD71, or both APPB1 and CD71 is determined, wherein APPB1 and CD71 is not present in healthy keratinocytes and appears when a pre-malignant or malignant melanocytic lesion develops, wherein the expression level of said biomarker is stronger when a malignant melanocytic lesion develops as compared to when a pre-malignant melanocytic lesion develops.

25. The method of claim 16, wherein further the expression level of CD66abce is determined, wherein CD66abce is not present in healthy keratinocytes and pre-malignant melanocytic lesion, but appears when a malignant melanocytic lesion develops.

26. The method of claim 16, wherein further the expression level of IFN- is determined, wherein IFN- is not present in healthy keratinocytes, but is present when a benign melanocytic lesion develops.

27. The method of claim 16, wherein the expression level of the one or more biomarker is determined by immunostaining, in particular by multi-epitope-ligand-cartography (MELC); or wherein the expression level of the one or more biomarker is determined by PCR or real-time PCR, on RNA isolated from keratinocytes which have been isolated from said biopsy sample, which RNA has been reversely transcribed prior to PCR.

28. The method of claim 27, wherein the expression of SSPL3 is primarily perinuclear when a malignant melanocytic lesion develops and primarily cytosolic when a premalignant melanocytic lesion develops.

29. The method of claim 16, wherein said biopsy sample is a skin tissue biopsy sample.

30. The method of claim 16, wherein said biopsy sample is a lymph node biopsy sample.

31. The method of claim 16, wherein the subject is a mammal.

32. The method of claim 16, wherein the subject is a human.

33. The method of claim 16, wherein the subject has or is suspected to have a skin disease involving melanocytes.

34. The method of claim 33, wherein the skin disease involving melanocytes is malignant melanoma.

35. The method of claim 33, wherein the skin disease involving melanocytes is psoriasis.

36. The method of claim 16, wherein the evaluation of the determined expression levels is automated by a software-supported manner.

37. The method of claim 16, wherein the method is carried out in an automated or semi-automated device.

Description

DESCRIPTION OF THE FIGURES

[0063] FIG. 1: Malignant, premalignat and benign melanocytic skin lesions have a similar PEP that does not allow a discrimination of these cells.

[0064] Representative analysis of 6 markers by MELC (B) in healthy melanocytes (hlt. skin), a compound nevus and a SSM (stained by Melan-A) shown in (A). Tissue areas depicted in (A) by squares and capital letters were analyzed in (B). The scale bar represents 100 m (A) and 10 m (B). Note: all panels in one horizontal row in (B) depict the same tissue section.

[0065] FIG. 2: Melanoma- but not nevi-associated keratinocytes have an altered PEP.

[0066] A compound nevus and an early SSM were stained for the indicated factors by MELC. The antibody directed against APBB1 recognizes the amyloid precursor protein. The scale bar represents 100 m.

[0067] FIG. 3: Keratinocytes in association with melanoma cells change their PEP

[0068] (A) A tissue section representing the transition from healthy epidermis (p27.sup.KIP1, green) to melanoma (CD63, white) was stained for the indicated markers. Individual cells show an all or nothing staining phenotype for specific markers (right panel; see also Table 1). The scale bar represents 50 m. (B) Co-culture of melanoma cells (Mel.) with Keratinocytes (Ker.), showing how melanoma cells establish contact with keratinocytes. The scale bar represents 50 m.

[0069] FIG. 4: The keratinocyte PEP reflects the transformation process from benign nevi to invasive melanoma.

[0070] Keratinocyte tissue areas from healthy skin, different nevi and melanomas (left panels) were systematically analyzed for keratinocyte-specific markers using MELC. The most prominent as well as representative results are shown and otherwise summarized in FIG. 5. Markers either increased in expression (CD63, SPPL3, TAP73, PPAR, CD71, APBB1, IFN), lost expression entirely (Notch1, ADAM10, p27.sup.KIP1), extended their expression into different epidermal layers (-catenin, p-EBF3, Nestin), or changed their subcellular localization (SPPL3, LAMp1, C/EBP). The scale bar represents 50 m.

[0071] FIG. 5: Quantification of the keratinocyte PEP in healthy skin, different nevi and SSM as demonstrated in FIG. 4.

[0072] Relative expression levels of proteins were determined in distinct melanocytic lesions (healthy skin, nevi, SSM) by assessing the grey value intensity. Expression levels for a given antigen were determined by calculating the mean from 6 different tissues per lesion. The highest value of these 6 expression levels were set to 10. Error bars represent standard deviations of the mean.

EXAMPLE

[0073] Using a systemic approach with the multi-epitope-ligand-cartography (MELC)-technology, we analyzed protein expression profiles (PEP) in nevi and BRAFV600E+ superficial spreading melanomas (SSM) for key transformation events.

[0074] To obtain antibodies applicable in the MELC-technology, 814 randomly selected hybridoma supernatants from the antibody production facility of the Helmholtz-Centre in Munich, and 173 commercially available antibodies were subjected to a screening algorithm to obtain those antibodies giving a specific staining in tissue (epidermis and dermis) for melanoma cells (n=57). We reasoned that key factors of the transformation process would appear in melanomas but not in nevi. We also selected antibodies that were specific for melanoma-associated keratinocytes (n=7) or for melanomas and keratinocytes (n=12). Single tissue sections of 6 BRAFV600E+ SSM, 6 junctional and compound nevi (3/3), and 6 samples of healthy skin were stained by the whole antibody set. For each antigen the average relative expression level was determined using an equation integrating grey value intensities of the background and 6 to 30 different staining areas for each sample.

[0075] Surprisingly, almost all antibodies gave a positive staining with melanocytes, nevi and melanoma tissue. There were only few exceptions, as for example CD63 was detected only in nevi and melanoma, and CD36 was only found in melanocytes and nevi (FIG. 1). However, the relative expression level of selected antigens differed significantly. Nevi showed a >2-fold increase in 6/57 antigens over melanocytes, and melanoma cells further increased expression levels in 10/57 antigens, including those increased in nevi, like CD63 and CD71. A number of proteins were also reduced in concentration >2-fold in nevi compared to melanocytes (12/57), and in melanoma cells compared to nevi (6/57). Thus protein expression levels of key proteins changed rather uniformly across different samples and potentially had a role in the transformation process.

[0076] To substantiate this assumption we compared PEPs in three different layers of the SSMs, namely in the basal (BL), apical (AL) and dermal layer (DL), as DL cells have a more aggressive growth behavior. However, these results did not point to crucial events of the transformation process.

[0077] We noticed that the PEP of keratinocytes adjacent to SSM, but not to benign nevi, changed visibly, and followed an all or nothing expression phenotype for numerous factors (FIG. 3). To confirm this finding we cultured different primary melanoma cells (ML01, -03, -05 , -07, -11) characterized previously (Lee to al. Mol. Cell 49, 668 (2013)) with keratinocytes (HaCaT cells), and noticed that all melanoma cells formed dendritic connections. We placed melanoma cells on one side of a keratinocyte colony and analyzed their PEP by MELC after 72 h. In a gradient type fashion, the keratinocytes changed their PEP and the subcellular localization of selected factors. These changes were very similar to those seen between nevi and SSM in tissue sections. A systematic analysis of melanoma cell- and SSM-associated keratinocytes (n=6 each condition) confirmed their overall similar PEP. Thus the transfer of certain effectors to keratinocytes was a conserved function of melanoma cells.

[0078] In view of these results we speculated that melanocyte transformation occurred in discernable steps, potentially mirrored by the keratinocyte PEP. Thus we systematically compared keratinocyte PEPs in healthy skin, junctional-, compound-, halo- and dysplastic nevi, as well as in BRAFV600E+ SSMs with horizontal, vertical and invasive growth patterns. Representative results of each tissue are shown in FIG. 4, revealing a progressive change of the keratinocyte PEP from healthy skin to melanoma. This change was characterized by factors that seemingly disappeared (Notch1, ADAM10, p27.sup.KIP1), gradually appeared and increased their expression (CD63, SPPL3, TAP73, PPAR, CD71, APBB1, IFN), or changed their subcellular localization (SPPL3). While changes started at the level of benign nevi with low expression levels of IFN-, CD63 and SPPL3, major differences were seen between the dysplastic nevi and the early SSM. This included the seemingly complete loss of p27.sup.KIP1, ADAM10 and its substrate Notch1. Changes in expression levels is also shown in FIG. 5, and can be summarized as follows:

[0079] 1) Malignant Lesions

[0080] Markers that are present in healthy keratinocytes and disappear when a malignant lesion develops:

[0081] ADAM10 (antibody recognizing the N-terminus of ADAM10), Notch1, p27.sup.KIP1

[0082] Markers that are not present in healthy keratinocytes and appear upon malignant transformation:

[0083] APBB1 (Amyloid precursor protein), CD63, CD66abce, CD71, PPAR TAP73, SPPL3 (perinuclear region)

[0084] Biomarker Summary Malignant Transformation:

[0085] ADAM10 (N-terminus) , Notch1 , p27.sup.KIP1, APBB1 , CD63 , CD66abce , CD71 , PPAR.Math., TAP73 , SPPL3 (perinuclear) .

[0086] Minimal Biomarker Combination in Keratinocytes for Malignant Lesions:

[0087] ADAM10 (N-terminus) , Notch1 , p27.sup.KIP1 , CD63 , PPARg , TAP73 , SPPL3 (perinuclear) .

[0088] 2) Pre-Malignant Lesions

[0089] Markers that are present in healthy keratinocytes and disappear in premalignant melanocytic lesions:

[0090] None.

[0091] Markers that are not present in healthy keratinocytes and appear in premalignant lesions:

[0092] APBB1 , CD71 , PPAR.Math., TAP73 , SPPL3 (cytoplasmic) .

[0093] Biomarker Summary Premalignant Melanocytic Lesion:

[0094] CD66abce , CD63 , ADAM10 (N-terminus) , Notch1 , p27.sup.KIP1 , SPPL3 (cytoplasmic) , APBB1 , CD71 , PPAR.Math., TAP73 .

[0095] Minimal Biomarker Combination in Keratinocytes for Malignant Lesions

[0096] CD63 , ADAM10 (N-terminus) , Notch1 , p27.sup.KIP1 , SPPL3 (cytoplasmic) , PPAR.Math., TAP73 .

[0097] 3) Benign Melanocytic Lesion

[0098] Markers that are present in healthy keratinocytes and disappear in benign melanocytic lesions:

[0099] None.

[0100] Markers that are not present in healthy keratinocytes and appear in benign melanocytic lesions:

[0101] IFN-.

[0102] Biomarker summary benign melanocytic lesion:

[0103] IFN-.

[0104] Surprisingly we found that more variables were involved in melanocyte transformation than appreciated so far, including protein expression levels, their subcellular localization and potentially the early keratinocyte environment. Keratinocytes with an altered PEP may stimulate transforming melanocytes, similar as they stimulate melanin production after sun exposure. The reciprocal transfer of new effectors may occur through cell-cell contact, cell dendrites as shown here, or alternatively through endosomal secretion or transcytosis. While we have no direct proof for such a reciprocal cell interaction, such a scenario would be consistent with the often slow changes seen in premalignant melanocytic lesions.

[0105] The here reported insights into the early melanoma transformation events were obtained through a multi-antigen assessment in tissue. The topographical allocation of PEPs was a key in our approach and a major difference to other multi-antigen approaches as for example mass spectrometry. The protein markers demonstrated in this study in melanoma-associated keratinocytes, could be used for the diagnosis of early melanomas and the discrimination of dysplastic lesions from truly transformed melanocytes.