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Title:
SKIN CELL ANALYSIS
Document Type and Number:
WIPO Patent Application WO/2020/035707
Kind Code:
A1
Abstract:
The present invention describes an adhesive tape stripping transfer method for quick and non-invasive diagnosis of skin diseases by MALDI-TOF mass spectrometry analysis. Cells from the epidermis layer of a suspicious skin area of a patient are collected using adhesive tapes via at least one application of tape stripping procedure. The adhesive tapes carrying collected skin cells are analyzed directly with MALDI-TOF MS without any sample pretreatment like cell lysis or preparatory cellular component extraction. Detection and diagnosis of human and mouse melanoma are illustrated as examples in the present invention.

Inventors:
HO PING-CHIH (CH)
GIRAULT HUBERT (CH)
JOVIC MILICA (CH)
LESCH ANDREAS (IT)
PICK HORST (CH)
ZHU YINGDI (CH)
LIN TZU-EN (TW)
Application Number:
PCT/GB2019/052925
Publication Date:
February 20, 2020
Filing Date:
October 14, 2019
Export Citation:
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Assignee:
ECOLE POLYTECHNIQUE FED LAUSANNE EPFL (CH)
HANSON WILLIAM BENNETT (GB)
International Classes:
H01J49/04
Foreign References:
US20100261215A12010-10-14
US7989165B22011-08-02
US7297480B22007-11-20
US6949338B22005-09-27
US6720145B22004-04-13
US77907310A2010-05-13
Other References:
G HOCHART ET AL: "688 Human keratinocyte biomarker mapping on skin tape strips using mass spectrometry imaging", JOURNAL OF INVESTIGATIVE DERMATOLOGY, vol. 138, no. 5, 19 April 2018 (2018-04-19), pages S117, XP055661409, DOI: 10.1016/j.jid.2018.03.697
N/A: "Stratum corneum", WIKIPEDIA, 6 May 2018 (2018-05-06), XP055661424, Retrieved from the Internet [retrieved on 20200123]
YINGDI ZHU: "Mass Spectrometry Methods for Bacterial Infection Diagnosis and Cancer Analysis", 2019, Lasusanne, XP055661038, Retrieved from the Internet [retrieved on 20200123], DOI: 10.5075/epfl-thesis-9322
HOCHART GUILLAUME ET AL: "Biomarker Mapping on Skin Tape Strips Using MALDI Mass Spectrometry Imaging", JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY, ELSEVIER SCIENCE INC, US, vol. 30, no. 10, 12 August 2019 (2019-08-12), pages 2082 - 2091, XP036913253, ISSN: 1044-0305, [retrieved on 20190812], DOI: 10.1007/S13361-019-02277-5
B. E. G.ROTHBERGM. B.BRACKEND. L.RIMM: "Tissue Biomarkers for Prognosis in Cutaneous Melanoma: A Systematic Review and Meta-Analysis", J. NATL. CANCER INST., vol. 101, 2009, pages 452 - 474, XP055052687, DOI: 10.1093/jnci/djp038
V. GRAY-SCHOPFERC. WELLBROCKR. MARAIS: "Melanoma Biology and New Targeted Therapy", NATURE, vol. 445, 2007, pages 2771 - 2776
N. ABBASIH. SHAWD. RIGEL: "Early Diagnosis of Cutaneous Melanoma: Revisiting the Abed Criteria", JAMA J. MED. ASSOC., vol. 292, 2004, pages 2771 - 27776
G. C. BETHUNEA. S. L. PETTITD. VELDHUIJZEN VAN ZANTENP. J. BARNES: "Well Differentiated Invasive Breast Cancers with Equivocal Her2 Immunohistochemistry: What Is the Yield of Routine Reflex in Situ Hybridization Testing?", HISTOPATHOLOGY, vol. 70, 2017, pages 966 - 974
H. FRICKMANNA. E. ZAUTNERA. MOTERJ. KIKHNEYR. M. HAGENH. STENDERS. POPPERT: "Fluorescence in Situ Hybridization (Fish) in the Microbiological Diagnostic Routine Laboratory: A Review", CRIT. REV. MICROBIOL., vol. 7828, 2017, pages 1 - 31
N. VISHWANATHANA. BANDYOPADHYAYH. Y. FUK. C. JOHNSONN. M. SPRINGERW. S. HU: "A Comparative Genomic Hybridization Approach to Study Gene Copy Number Variations among Chinese Hamster Cell Lines", BIOTECHNOL. BIOENG., vol. 114, 2017, pages 1903 - 1908
A. A. MARGHOOBL. D. SWINDLEC. Z. M. MORICZF. A. SANCHEZ NEGRONB. SLUEA. C. HALPERNA. W. KOPF: "Instruments and New Technologies for the in Vivo Diagnosis of Melanoma", J. AM. ACAD. DERMATOL, vol. 49, 2003, pages 777 - 797
A. GADELIYA GOODSOND. GROSSMAN: "Strategies for Early Melanoma Detection: Approaches to the Patient with Nevi", J. AM. ACAD. DERMATOL., vol. 60, 2009, pages 719 - 735, XP026142046, DOI: 10.1016/j.jaad.2008.10.065
T. WADHAWANN. SITUK. LANCASTERX. YUANG. ZOURIDAKIS: "Proc. - Int. Symp. Biomed. Imaging", 2011, IEEE, article "Skinscan©: A Portable Library for Melanoma Detection on Handheld Devices", pages: 133 - 136
G. ZONIOSA. DIMOUI. BASSUKASD. GALARISA. TSOLAKIDISE. KAXIRAS: "Melanin Absorption Spectroscopy: New Method for Noninvasive Skin Investigation and Melanoma Detection", J. BIOMED. OPT., vol. 13, 2008, pages 014017
A. COHENA. EI-ANEED: "Mass Spectrometry, Review of Bascis: Electrospray, Maldi, and Commonly Used Mass Analyzers'], Banoub", APPL. SPECTROSC. REV., vol. 44, 2009, pages 210 - 230
R. M. A. HEERNK. CHUGHTAI: "Mass Spectrometric Imaging for Biological Tissue Analysis", CHEM. REV., vol. 110, 2010, pages 3237 - 3277
J. A. MOBLEYD. S. CORNETTE. C. DIASM. ANDRESSONC. L. ARTEAGAM. E. SANDERSR. M. CAPRIOLI: "A Novel Histology-Directed Strategy for Maldi-Ms Tissue Profiling That Improves Throughput and Cellular Specificity in Human Breast Cancer", MOL. CELL. PROTEOMICS, vol. 5, 2006, pages 1975 - 1983, XP002548366, DOI: 10.1074/MCP.M600119-MCP200
H. S. KANGS. C. LEEY. S. PARKY. E. JEONJ. H. LEES-Y. JUNGI. H. PARKS. H. JANGH. M. PARKC. W. YOO: "Protein and Lipid Maldi Profiles Classify Breast Cancers According to the Intrinsic Subtype", BMC CANCER, vol. 11, 2011, pages 465, XP021113121, DOI: 10.1186/1471-2407-11-465
B. FLATLEYP. MALONER. CRAMER: "Maldi Mass Spectrometry in Prostate Cancer Biomarker Discovery", BIOCHIM. BIOPHYS. ACTA, vol. 1844, 2014, pages 940 - 949, XP028845403, DOI: 10.1016/j.bbapap.2013.06.015
K. SCHWAMBORNR. C. KRIEGM. RESKAG. JAKSER. KNUECHELA. WELLMANN: "Identifying Prostate Carcinoma by Maldi-Imaging", INT. J. MOL. MED., vol. 20, 2007, pages 155 - 159, XP002548367
M. EL AYEDD. BONNELR. LONGUESPEEC. CASTELIERJ. FRANCKD. VERGARAA. DESMONSA. TASIEMSKIA. KENANID. VINATIER: "Maldi Imaging Mass Spectrometry in Ovarian Cancer for Tracking, Identifying, and Validating Biomarkers", MED. SCI. MONIT., vol. 16, 2010, pages BR233 - 245
R. LEMAIRES. A. MENGUELLETJ. STAUBERV. MARCHAUDONJ. P. LUCOTP. COLLINETM. O. FARINED. VINATIERR. DAYP. DUCOROY: "Specific Maldi Imaging and Profiling for Biomarker Hunting and Validation: Fragment of the 11s Proteasome Activator Complex, Reg Alpha Fragment, Is a New Potential Ovary Cancer Biomarker", J. PROTEOME RES., vol. 6, 2007, pages 4127 - 4134
E. BONAPARTEC. PESENTIL. FONTANAR. FALCONEL. PAGANINIA. MARZORATIS. FERREROM. NOSOTTIP. MENDOGNIC. BAREGGI: "Molecular Profiling of Lung Cancer Specimens and Liquid Biopsies Using Maldi-Tof Mass Spectrometry", DIAGN. PATHOL., vol. 13, 2018, pages 4
A. BONDARENKOY. ZHUL. QIAOF. CORTES SALAZARH. PICKH. H. GIRAULT: "Aluminium Foil as a Single-Use Substrate for Maldi-Ms Fingerprinting of Different Melanoma Cell Lines", ANALYST, vol. 141, 2016, pages 3403 - 3410
R. GURANL. VANICKOVAV. HORAKS. KRIZKOVAP. MICHALEKZ. HEGERO. ZITKAV. ADAM: "Maldi Msi of Melim Melanoma: Searching for Differences in Protein Profiles", PLOS ONE, vol. 12, 2017, pages e0189305
M. A. J. WETERMANG. N. P. VANMUIJEND. J. RUITERH. P. J. BLOEMERS: "Thymosin Beta-10 Expression in Melanoma Cell-Lines and Melanocytic Lesions - a New Progression Marker for Human Cutaneous Melanoma", INT. J. CANCER, vol. 53, 1993, pages 278 - 284, XP002041675, DOI: 10.1002/ijc.2910530218
G. WEINLICHK. EISENDLEE. HASSLERM. BALTACIP. O. FRITSCHB. ZELGER: "Metallothionein - Overexpression as A Highly Significant Prognostic Factor in Melanoma: A Prospective Study on 1270 Patients", BRIT. J. CANCER, vol. 94, 2006, pages 835 - 841, XP055086055, DOI: 10.1038/sj.bjc.6603028
P. KASKELC. BERKINGS. SANDERM. VOLKENANDTR.U. PETERG. KRAHN: "S-100 protein in Peripheral Blood: A Marker for Melanoma Metastases - A Prospective 2-center Study of 570 Patients with Melanoma", J. AM. ACAD. DERMATOL., vol. 41, 1999, pages 962 - 969
S. PETERSSONE. SHUBBARL. ENERBACKC. ENERBACK: "Expression Patterns of S100 Proteins in Melanocytes and Melanocytic Lesions", MELANOMA RES., vol. 2009, 2009, pages 215 - 225
Y. D. ZHUL. QIAOM. PRUDENTA. BONDARENKON. GASILOVAS. B. MOLLERN. LIONH. PICKT. Q. GONGZ. X. CHEN: "Sensitive and Fast Identification of Bacteria in Blood Samples by Immunoaffinity Mass Spectrometry for Quick BSI Diagnosis", CHEM. SCI., vol. 7, 2016, pages 2987 - 2995, XP055410693, DOI: 10.1039/C5SC04919A
Attorney, Agent or Firm:
HANSON, William (GB)
Download PDF:
Claims:
Claims

1. A method of transferring cells from the skin of a patient onto a MALDI target, for MALDI-TOF mass spectrometry, comprising:

- Collecting cells from the epidermis layer of a selected skin region, by applying an adhesive tape to the skin;

- Fixing the adhesive tape with collected cells on the MALDI target; and

- Overlaying the skin cells directly without any pretreatment with a MALDI matrix for direct MALDI-TOF MS analysis.

2. The method of claim 1 wherein the patient is a human being.

3. The method of claim 1 wherein the patient is an animal.

4. The method of claim 1, 2 or 3, including a subsequent step of carrying out MALDI-TOF analysis for the detection of at least one protein indicating malignant melanoma, basal cell carcinoma, squamous cell carcinoma or Merkel cell carcinoma.

5. The method of any preceding claim wherein the adhesive tape is single-sided.

6. The method of claim 5 wherein the adhesive tape is fixed to the MALDI target using a target tape that bears adhesive on both sides.

7. The method of any one of claims 1 to 4, wherein the adhesive tape is double- sided for direct adhesion to the MALDI target.

8. The method of any preceding claim, wherein the adhesive tape is mechanically fixed to the MALDI target with a frame.

9. The method of claim 1 wherein the adhesive tape sampling method is used to image by mass spectrometry the skin area under investigation.

10. The method of claim 1, used to collect infectious cells such as bacteria, fungi and/or viruses for mass spectrometry analysis.

Description:
SKIN CELL ANALYSIS

Field of the Invention

[0001] This invention relates to a method of analyzing skin cells.

Background to the Invention

[0002] Skin cancers, especially cutaneous malignancies like melanoma, strike millions of people worldwide. The survival rate of a melanoma patient increases with early detection when the cancer is still superficial and can be removed through minor surgery. Melanoma can be lethal in late stages. Therefore, reliable methodologies for early diagnosis and unequivocal identification of skin cancer progression stages are important for health care. 1 ' 2

[0003] Dermatologists usually examine suspicious, i.e. potentially cancerous, skin areas by visual observation (e.g. using dermoscopy) following the "ABCDE" signs or performing skin biopsies. 3 ABCDE refers to the characteristics of Asymmetry, Border irregularity, Color variegation, Diameter greater than 6 mm and Evolving (i.e. lesions that have changed over a relatively short time). However, even with dermoscopy, visual ABCDE signs might not be effective for diagnosing melanoma in its early stages. A skin biopsy is usually performed, in which a piece of skin is removed from the patient and sectioned into thin slices, rendering a microscopic diagnosis by a pathologist. Staining processes are necessary for revealing the cell morphology, nucleic acids and disease biomarkers. Commonly used tests for microscopic analysis include immunohistochemistry (IHC) 4 , fluorescence in situ hybridization (FISH) 5 , or comparative genomic hybridization (CGH) 6 . In IHC, a primary antibody for the specific binding to a target melanoma-associated antigen in the skin tissue and a secondary antibody conjugated with a "color producing" enzyme, e.g. a phosphatase or peroxidase, linking to the primary antibody are applied. The "color producing" enzyme catalyzes in solution a reaction in which the product can be detected by its color or fluorescence change or generation. FISH is a molecular cytogenetic technique that uses fluorescent probes that bind to target DNA or RNA (e.g. mRNA, lncRNA and miRNA) in cells. However, these methods require expensive antibodies and probes that can suffer from poor specificity, which leads to a high level of background staining or cross reaction with multiple analytes. Furthermore, invasive skin sampling methods such as biopsies may cause painful wounds and could result in the exposure to the risks of infection. Moreover, biopsies can hardly provide the molecular information on the skin surface, which is the main goal and that is extremely important for early cancer diagnosis.

[0004] In order to seek safer and more efficient methods for skin cancer diagnosis, different modalities were developed to enhance the clinical examination quality. For instance, pigmentation patterns of suspicious melanoma areas can be examined by dermoscopy with computer-based analysis, multispectral digital dermoscopy, confocal scanning laser microscopy, and optical coherence tomography 7 ' 8 . With the advancing of artificial intelligence (AI) technology, hand-held devices with high resolution cameras, such as smartphones, can be employed using software that can process the analysis algorithms for the comparison of suspicious skin moles and melanoma images based on a database and thus can help to determine malignancy. 9 Despite the versatility of these optical modalities, the pigmentation can only represent a part of the melanoma characteristics, yet they cannot provide clear molecular information 10 .

[0005] Over the years, matrix assisted laser desorption ionization mass spectrometry (MALDI MS) has become a fast and powerful technique for the analysis of biomolecules including DNA, metabolites, sugars, lipids, peptides and proteins. Samples are deposited on MALDI plates together with a MALDI matrix, typically a salt. The principle of MALDI ionization then lies in the absorption of laser energy by the MALDI matrix, which transfers the energy to the analytes and facilitates their desorption and ionization by protonation or de-protonation. The ions are then accelerated along an electric field, and then separated from each other on the basis of their mass-to-charge ratio ( m/z ). The ions are then detected and measured using different types of mass analyzers like quadrupole mass analyzers, ion trap analyzers, time of flight (TOF) analyzers, etc 11 .

[0006] MALDI-TOF MS has become a powerful screening method for protein expression patterns and identifying molecular signatures that can be associated with a disease state, from global proteome profiling to imaging of tumor biomarkers in the tissue slices 12 . The robustness of this mass spectrometry technique allows the investigation of complex samples without prior fractionation or separation of proteins and peptides, it provides high sensitivity, large tolerance for contaminants, a wide mass range, little fragmentation and label free detection. It has been used in pathology to identify the presence of tumor biomarkers, to distinguish between molecular signatures of different types of tissues, such as normal vs. tumor tissue, and to type or identify cancer cell lines including breast cancer 13 ' 14 , prostate cancer 15 ' 16 , ovarian cancer 17 ' 18 , lung cancer 19 or melanoma 20 .

[0007] Protein changes associated with the transition from normal skin cells to mediate cells and the final cancer cells can be used for skin state checking and for screening of high-risk patients by MALDI-TOF MS. Furthermore, such protein signatures or profiles can be used for skin cancer classification, stratifying patients by the risk of recurrence and providing valuable insights into prognostic evaluation 21 . The main limitation in diagnosis of melanoma using MALDI-TOF MS is the need for invasive methods, such as skin biopsies, for isolating the sample to be analyzed (typically used sample are fresh frozen, formalin-fixed, paraffin-embedded tissue samples). Furthermore, MALDI analysis of tissues is usually limited by the sample thickness and tissue sectioning process.

[0008] Recently, non-invasive sampling methods have been extensively proposed to improve the detection of skin diseases. For instance, a tape stripping procedure was used to collect DNA or messenger RNA from the outer skin layer, i.e. the stratum corneum, to perform genomic analysis. 22 - 25 The method provides accurate molecular information for analysis of skin diseases and pathological skin state. However, a polymerase chain reaction is required in the methods for amplifying the trace nucleic acid, which is time consuming, costty and labor intensive. Furthermore, non- invasive sampling was used to collect material from the skin and diagnose psoriasis using mass spectrometry 26 . However, the method required extraction of biological material collected on the adhesive tape (such as enzymatic digestion) prior to conduct the MS analysis of extracted material.

Summary of the Invention

[0009] The present invention seeks to provide an adhesive tape stripping transfer method of skin cells from the skin of a human or animal to a MALDI target for quick and non-invasive diagnosis of skin diseases by mass spectrometry without any sample pretreatment.

[0010] The present invention provides a method as set out in claim 1, the dependent claims specifying optional items. The method includes: i) an adhesive tape stripping sampling of skin cells from suspicious skin region, ii ) fixing directly the adhesive tape with collected cells on the MALDI target plate and covering the sample with a MALDI matrix, iu) MALDI-TOF MS analysis of the intact sample without any sample pretreatment like cell lysis, preparatory cellular component extraction, enzymatic digestion, etc.

[0011] Thus, the method of the invention is based on non-invasive tape stripping of skin cells and direct transfer of the tape on the MALDI target where the tape carrying intact cells are overlaid with MALDI matrix and directly analyzed by MALDI-TOF mass spectrometry without any sample pretreatment

Brief Description of the Drawings

[0012] The invention will now be described in more detail, by way of example only, with reference to the accompanying drawings, in which: [0013] Fig. 1 schematically illustrates steps in the method of an embodiment of the present invention;

[0014] Fig. 2 shows the MALDI-TOF MS pattern of a non-used adhesive tape;

[0015] Fig. 3a shows MALDI-TOF MS patterns of a normal skin region of a first person obtained with four operations of tape stripping from the same skin region using the method of Fig. 1;

[0016] Fig. 3b shows MALDI-TOF MS patterns of a mole of the first person obtained with four operations of tape stripping from the same mole;

[0017] Fig. 3c shows MALDI-TOF MS patterns of a non-used double side adhesive tape and patterns of normal skin and a mole of the first person obtained with one operation of tape stripping method using the double side adhesive tape;

[0018] Fig. 4a shows MALDI-TOF MS patterns of a normal skin region of a second person obtained with four operations of tape stripping from the same skin region;

[0019] Fig. 4b shows MALDI-TOF MS patterns of a black mole of the second person obtained with four operations of tape stripping from the same mole;

[0020] Fig. 5 shows MALDI-TOF MS patterns of a small black mole of a third person obtained with seven operations of tape stripping from the same mole;

[0021] Fig. 6 mimics the analysis of human melanoma using the tape stripping mass spectrometry method of Fig. 1;

[0022] Fig. 7 shows the comparison and cluster analysis of mass spectra from Fig. 3 to Fig. 6 (the third layer spectrum from each figure);

[0023] Figs. 8a and 8b shows four groups of marker mass spectrometric peaks for human melanoma; and

[0024] Figs. 9a to 9c show the application of the method of the invention for the diagnosis of melanoma on mice. Detailed Description of Particular Embodiments

[0025] Fig. 1 schematically illustrates the adhesive tape stripping transfer method for mass spectrometry analysis of skin cells. It includes three parts, i.e. firstly, in Steps I to III, adhesive tape stripping-based sampling of the outermost skin layer (i.e. stratum corneum), secondly, in Steps IV and V, fixing the tape on a MALDI target with the collected cells on the top side and deposition of a MALDI matrix on the cells and thirdly, in Step VI, the MALDI-TOF MS analysis of the collected sample.

[0026] In Step I, an adhesive tape 1, i.e. a thin plastic foil coated with an adhesive layer, is applied on a skin area 2. In Step II, the tape is gently pressed against the suspicious area 3 for a certain period of time to achieve a good interaction of the adhesive layer with the cells 4 from the stratum corneum. In Step III, the tape 1 is then slowly removed from the skin thereby collecting a certain amount of skin cells on the tape. In Step IV, the tape carrying collected skin cells 4 is turned downside up and then fixed onto a MALDI target 5. In Step V, a MALDI matrix 6 is deposited onto the adhesive tape to cover the skin cells. In Step VI, the target is afterwards loaded into the MALDI instrument where a high voltage 7 is applied and the sample is shot with a laser 8 to generate the mass spectra 9 of the collected skin cells.

[0027] As an alternative to adhering the tape to the target, the target can include, or be provided with, a frame for retaining the tape in position.

[0028] The adhesive tape is composed of a thin flexible plastic film, coated on one side with an adhesive layer to collect skin cells and coated on the opposite side with another adhesive layer to be fixed to the MALDI target. The opposite side can alternatively or additionally be fixed to the MALDI target by applying a standard double-sided adhesive Scotch tape.

[0029] Fig. 2 shows a MALDI-TOF MS pattern of a commercial adhesive patch (DermTech, La Jolla, California, USA) covered with a MALDI matrix. The patch was then fixed onto a MALDI target plate using a double-sided Scotch sticky tape (3M, product No. 34-8509-3289-7, St. Paul, MN 55144-1000). The MALDI matrix used for this measurement was sinapinic acid (20 mg mL -1 in 50/49.9/0.1% acetonitrile/ water/ trifluoroacetic acid). The mass range used for the measurement was 2,000-20,000 m/z.

[0030] Fig. 3, Fig. 4 and Fig. 5 show MALDI-TOF MS patterns collected from the normal skin region (i.e. skin without melanoma, moles or other localized variations) and a mole (Fig. 3a and 3b, respectively) on the right forearm of Person A; a normal skin region and a mole (Fig. 4a and 4b, respectively) on the right-side waist of Person B; and a small mole on the right forearm of Person C (Fig. 5), respectively. Person A was a male volunteer in his 30's. Person B was a female volunteer in her late 20' s and Person C was a female volunteer in her early 30's. Adhesive tapes were used to collect cells from related skin regions via the tape stripping sampling procedure. Figures contain four mass spectrometric patterns, which came from four successive tape stripping (each time a new piece of adhesive tape was used on exactly the same area). Fig. 5 contains seven mass spectra, which are from seven times of tape stripping applied on the same mole of Person C with a new piece of adhesive tape used for each time of tape stripping. In both Fig. 3a and Fig. 4a, the normal skin area being analyzed was ~10 mm x 10 mm. The mole from Person A analyzed in Fig. 3b is ~5 mm in diameter, and the mole from Person B analyzed in Fig. 4b was ~6 mm in diameter. The mole from Person C analyzed in Fig. 5 was ~1.5 mm in diameter. The patterns in Fig. 3c were obtained from the double-sided adhesive tape where one side of the tape was used to collect the skin cells and other side to fix on the MALDI target.

[0031] Fig. 6 shows MALDI-TOF MS patterns of tape-stripped human melanoma skin region where the collected samples where spiked with melanoma cells to mimic real human melanoma tape stripping sampling. Melanoma cells, previously cultured in Petri dishes, were harvested and deposited on the adhesive tapes covered with collected normal skin cells from the right forearm of Person A (four times of tape stripping sampling). Three different melanoma cell lines were applied: primary melanoma SBcl2 (radial growth phase) (Fig. 6a), primary melanoma WM115 (vertical growth phase) (Fig. 6b) and metastatic melanoma WM239 (Fig. 6c). [0032] Fig. 7 a shows the visual comparison of mass spectra from the tests in Fig. 3 to Fig. 6. The spectrum from the third tape stripping sampling was selected from each figure for comparison. Pattern matching of eight spectra in Fig. 7a by a cluster analysis procedure was performed, with the result shown in the form of a heatmap (Fig. 7b) and in the form of dendrogram (Fig. 7c). The pattern matching was conducted via the open access software BacteriaMS (http: / / www.bacteriams.com), which has been developed for bacteria identification using a MALDI-TOF MS profiling method. With this software, similarity scores between each two spectra are calculated using a cosine correlation algorithm and the samples are divided into different groups (the so-called cluster analysis) accordingly. With the cosine correlation algorithm, the maximum similarity score is 1, meaning that the two spectra compared are exactly the same. The heatmap in Fig. 7b shows the grouping of the eight samples according to the similarity scores between each two spectra. Fig. 7c shows a dendrogram of the grouping result with a relative distance value between each two groups. The maximum relative distance value is 1.000, meaning that the two groups are totally different from each other and they can be well separated.

[0033] Fig. 8a indicates four groups of mass spectrometric peaks from Fig. 7a, i.e. peak at 4,933 ± 4 m/z (peak 1), peak group at 6,000 - 6,100 m/z (peak 2), peak at 10,085 ± 10 m/z (peak 3) and peak group at 11,500 - 11,850 m/z (peak 4). These peaks appear only in human melanoma samples with high intensity, but do not appear in normal skin samples or are below the detection limit. It is presumed that proteins corresponding to these four groups of peaks could be human melanoma marker proteins. In order to identify these proteins, both a bottom-up and a top-down proteomic approach were used. For a bottom-up approach, the cultured SBcl2 cells were harvested and lysed in sodium dodecyl sulphate loading buffer, and the extracted protein mixtures are separated by sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE). The gel bands at 5 kDa, 6 kDa and 10-12 kDa are excised (Fig. 8b), digested in trypsin followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. To further confirm the identification result, the SBcl2 cells were also analysed with a top-down proteomic approach. The cultured SBcl2 cells were harvested and lysed in sodium dodecyl sulphate loading buffer. The extracted protein mixtures were then filtered through a centrifugal filter to remove proteins heavier than 30 kDa and precipitated using a mixture solution of methanol/ chloroform/ pure water. The precipitated proteins were then dissolved in an injection buffer (pure water/ acetonitrile/ formic acid 95/5/0.2%) for LC-MS/MS measurement. The identification results from this top-down LC-MS/MS procedure coincide with the bottom-up SDS-PAGE&LC- MS/MS procedure, and the precursor mass ( m/z ) of the four groups of proteins observed with top-down LC-MS/MS match well the observed mass in the MALDI- TOF MS patterns. Details about the identified proteins are as follows:

[0034] Peak 1 in Fig. 8a: measured mass with MALDI-TOF MS 4,933 ± 4 m/z ; observed precursor mass with top-down LC-MS/MS 4,933 m/z ; gel band analyzed with bottom-up procedure 5 kDa; identified as thymosin beta-10; Uniprot accession P63313; 100% identification probability; theoretical molecular weight 5,026.0 Da; protein sequence (sequence measured with the bottom-up procedure is highlighted with underline): MADKPDMGEIASFDKAKLKKTETQEKNTLPTKETIEQEKRSEIS

[0035] Peak 2 in Fig. 8a (a group of preaks): measured mass range with MALDI- TOF MS 6,000 - 6,100 m/z; observed precursor mass with top-down LC-MS/MS 6,014, 6,039, 6,042, 6,067 m/z; gel band analyzed with bottom-up procedure 6 kDa; identified as metallothioneins:

[0036] Metallothionein-IE; Uniprot accession P04732; 100% identification probability; theoretical molecular weight 6,013.4 Da; protein sequence (sequence measured with the bottom-up procedure is highlighted with underline): MDPNCSCATGGSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAOGCVCKGA SEKCSCCA

[0037] Metallothionein-IH; Uniprot accession P80294; 100% identification probability; theoretical molecular weight 6,038.8 Da; protein sequence (sequence measured with the bottom-up procedure is highlighted with underline): MDPNCSCEAGGSCACAGSCKCKKCKCTSCKKSCCSCCPLGCAKCAOGCICKGAS

EKCSCCA

[0038] Metallothionein-2; Uniprot accession P02795; 100% identification probability; theoretical molecular weight 6,041.6 Da; protein sequence (sequence measured with the bottom-up procedure is highlighted with underline):

MDPNCSCAAGDSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAOGCICKGAS

DKCSCCA

[0039] Metallothionein-lX; Uniprot accession P80297; 100% identification probability; theoretical molecular weight 6,067.6 Da; protein sequence (sequence measured with the bottom-up procedure is highlighted with underline): MDPNCSCSPVGSCACAGSCKCKECKCTSCKKSCCSCCPVGCAKCAOGCICKGTS DKCSCCA

[0040] Peak 3 in Fig. 8a: measured mass with MALDI-TOF MS 10,085 ± 10 m/z) observed precursor mass with top-down LC-MS/MS 10,084 m/z ; gel band analyzed with bottom-up procedure 10-12 kDa; identified as protein S100-A6; Uniprot accession P06703; 100% identification probability; theoretical molecular weight 10,180.4 Da; protein sequence (sequence measured with the bottom-up procedure is highlighted with underline):

MACPLDOAIGLLVAIFHKYSGREGDKHTLSKKELKELIOKELTIGSKLQDAEIARL

MEDLDRNKDOEVNFQEYVTFLGALALIYNEALKG

[0041] Peak 4 in Fig. 8a (a group of peaks): measured mass range with MAFDI-TOF MS 11,550 - 11,850 m/z ; observed mass with top-down FC-MS/MS 11,610, 11,631, 11,643, 11,703 m/z ; gel band analyzed with bottom-up procedure 10-12 kDa; identified as S100 proteins:

[0042] Protein S100-A3; Uniprot accession P33764; 100% identification probability; theoretical molecular weight 11,713.3 Da; protein sequence (sequence measured with the bottom-up procedure is highlighted with underline): MARPLEOAVAAIVCTFOEYAGRCGDKYKLCOAELKELLOKELATWTPTEFRECD

YNKFMSVLDTNKDCEVDFVEYVRSLACLCLYCHEYFKDCPSEPPCSO

[0043] Protein S100-A4; Uniprot accession P26447; 100% identification probability; theoretical molecular weight 11,729.0 Da; protein sequence (sequence measured with the bottom-up procedure is highlighted with underline):

MACPLEKALDVMVSTFHKYSGKEGDKFKLNKSELKELLTRELPSFLGKRTDEAAF

OKFMSNFDSNRDNEVDFOEYCVFFSCIAMMCNEFFEGFPDKOPRKK

[0044] Protein S100-A11; Uniprot accession P31949; 100% identification probability; theoretical molecular weight 11,741.1 Da; protein sequence (sequence measured with the bottom-up procedure is highlighted with underline):

MAKISSPTETERCIESLIAVFQKYAGKDGYNYTLSKTEFLSFMNTELAAFTKNOKD

PGVEDRMMKKEDTNSDGOEDFSEFENEIGGEAMACHDSFEKAVPSOKRT

[0045] Protein S100-A16; Uniprot accession Q96FQ6; 100% identification probability; theoretical molecular weight 11,801.9 Da; protein sequence (sequence measured with the bottom-up procedure is highlighted with underline):

MSDCYTEEEKAVIVEVENFYKYVSKYSEVKNKISKSSFREMEOKEENHMESDTGN

RKAADKEIONEDANHDGRISFDEYWTEIGGITGPIAKEIHEOEOOSSS

[0046] Fig. 9 shows the application of the method of the present invention for the diagnosis of melanoma on living mice. Mice carrying melanoma tumor were induced by genetic approach to introduce a driver mutation BrafV 600E (a common mutation in more than 40% of human melanoma patients) and deletion of PTEN to mimic pathophysiology and tumorigenesis in human melanomas. Mice induced for 4.5 weeks and 6 weeks of tumor formation were provided by the Eudwig Center for Cancer Research in Epalinges, Switzerland. Adhesive patches (DermTech, La Jolla, California, USA) were used to collect skin cells from the melanoma region and normal non-melanoma. Sampling was applied four times on each skin region, and the tapes from the fourth sampling were covered with sinapinic acid matrix for the MALDI-TOF MS measurement. The generated mass spectra together with a mouse melanoma reference mass spectrum (collected from cultured mouse melanoma cell line B16, a genetically irrelevant melanoma cell line) are shown in Fig. 9a. The four spectra in Fig. 9a were analyzed using the pattern matching method in the BacteriaMS software, with a cluster analysis heatmap displayed in Fig. 9b and a dendrogram displayed in Fig. 9c.

Results

[0047] Before using adhesive tapes to collect skin cells, MALDI-TOF MS analysis of a piece of clean, non-used single-sided or double-sided adhesive tape was performed. As shown in Fig. 2 and Fig. 3c, no mass spectrometric peaks were generated within the 2,000-20,000 m/z mass range indicating that the adhesive tape itself will not bring interferences to the MALDI-TOF MS analysis. Furthermore, single-sided adhesive tapes could be fixed on the MALDI target using additional thin adhesive, while double-sided adhesive tapes can be directly fixed on the target (one adhesive side to collect the skin cells, the other adhesive side to fix on the target).

[0048] The MALDI-TOF MS analysis of tape-stripped normal skin (Fig. 3a) shows good quality mass spectra with well-shaped mass spectrometric peaks being detected within the mass range of 2,000-20,000 m/z. All of these peaks originate solely from the collected skin cells. This result indicates that the tape stripping sampling and transfer procedure is highly compatible with the MALDI-TOF MS measurement without requiring any sample treatment. The four mass spectra obtained from normal skin (displayed in Fig. 3a) are similar to each other, especially the spectra from the third and fourth tape stripping of the same skin region, i.e. the third and fourth layers. The same phenomena were observed in Fig. 3b, Fig. 4a-4b and Fig. 5 (third to seventh layers). The reproducible mass spectrometric patterns clearly show that the present invention is highly reliable for quick, non-invasive analysis of human skin, and that at least three operations of tape stripping are recommended for the sampling of a single skin region. [0049] Fig. 6 shows that four generated patterns from human-mimicking melanoma tape stripping samples were similar for each of three melanoma cell lines. As a large number of melanoma cells (~ 1 c 10 4 cells per 3 mm diameter spot area) were deposited onto collected normal skin cells, the detected mass spectrometric peaks from the melanoma cells were more pronounced than from the normal cells.

[0050] To visually compare the mass spectra generated from all the above tests, the third layer spectrum was taken from each figure (i.e. Fig. 3 to Fig. 6) and displayed together in Fig. 7a. It can be seen that the mass spectra generated from normal skin regions or skin moles share many of the high intensity peaks, meaning that the collected cells share many of their high abundant cellular proteins. The three human- mimicking melanoma samples also show similar patterns, while the patterns from normal skin regions or skin moles were different from those of melanoma samples. All of the eight patterns in Fig. 7a were then analysed with the pattern matching method, i.e. pattern comparison using mathematic algorithms. The heatmap in Fig. 7b shows that the eight patterns can be divided into two groups (clusters), i.e. a group of human normal skin or skin mole (shown as 1 - 5 in Fig. 7b) and a group of human melanomas (shown as 6 - 8 in Fig. 7b). Three melanoma patterns show high similarity scores (0.65 - 0.89) between each other defining one group (=cluster). At the same time, five normal skin and skin mole samples were similar to each other, with the lowest similarity score higher than 0.2, forming a second group. The pattern similarity scores between each two group-crossing samples were low, mostly lower than 0.05, meaning that melanoma cells (developing from melanocytes) have a different cellular protein expression compared to normal skin cells (mainly corenocytes) or skin mole cells (mostly melanocytes). The dendrogram in Fig. 7c shows that the relative distance between the above two groups was large with a relative distance value of 0.999. This high value indicates that human melanoma can be distinguished from normal skin or skin moles. Within the latter group, the two normal skin patterns (from Person A and Person B, respectively) were closest to each other, with a relative distance as low as 0.113. These two patterns were close to the small mole region of Person C with a relative distance of 0.285, followed by the mole of Person B with a relative distance of 0.365, and then the mole of Person A with relative distance of 0.696. These results suggest that the present tape stripping and transfer method for mass spectrometry measurements, combined with a mathematical pattern matching analysis, can be used for rapid melanoma cell identification, especially for the measurements of protein profiles that can be used to detect skin cancers. Additionally, the three human melanoma patterns are 11 - 35% different from each other with the observation of characteristic peaks, respectively. This is due to the expression of different proteins at different melanoma stages. Likewise, different types of skin cancers should also express different proteins. Thus, the present method could be used for the identification of skin cancer types and cancer stages.

[0051] Some mass spectrometric peaks appear only in the three human melanoma samples with high intensity, but do not appear in normal skin samples, as marked in Fig. 8a, indicating that these peaks could be used as marker peaks for human melanoma. To confirm this surmise, proteins corresponding to these peaks are identified with both a bottom-up procedure (SDS-PAGE & LC-MS/MS with SDS- PAGE gel running lane shown in Fig. 8b) and a top-down procedure (LC-MS/MS). Both procedures demonstrate that these proteins indeed are typical human melanoma marker proteins, i.e. Peak 1 from the protein of thymosin beta-10, Peak 2 from metallothioneins, Peak 3 and Peak 4 from S100 proteins. All of these proteins have been widely used as marker proteins for clinical diagnosis of human melanoma. 27 · 29 Peak 4 also appears in the mass spectra of Person A's skin mole, marked in Fig. 8a with an arrow. A mole is a skin region accumulating melanocytes, which also could have the expression of some S100 proteins. 30 All these results indicate that the observation of some marker peaks from the generated MALDI-TOF MS patterns could be used to further confirm the diagnosis results from pattern matching analysis. [0052] The transfer method of the invention was then applied for the analysis of melanoma in mice. Fig. 9a shows the obtained mass spectra, generated from; a normal mouse skin region, a mouse melanoma skin region with 4.5 weeks of tumour formation, a mouse melanoma skin region with 6 weeks of tumour formation, and a reference mass spectrum of mouse melanoma (in vitro cultured melanoma cell line B16). The first three spectra were collected from the fourth application of tape stripping sampling of each skin region, which was shaved prior to tape stripping. The reference spectrum was collected from cultured pure intact mouse melanoma cells B16 deposited on a clean adhesive tape. Visual inspection of the patterns in Fig. 9a indicates the difference between the normal mouse skin region and the mouse melanoma regions. The four patterns were processed with software-assisted cluster analysis based on cosine correlation pattern matching. With this software, the four patterns were automatically divided into two groups, i.e. a group of normal mouse skin (shown as 1 in Fig. 9b) and a group of mouse melanoma (shown as 2, 3, 4 in Fig. 9b). The relative distance between the two groups was 0.992 (Fig. 9c). As displayed in Fig. 9b, the similarity score between the mouse normal skin pattern (1) and the mouse melanoma reference pattern (4) was low (0.008), while patterns from 4.5 weeks or 6 weeks of tumour formation region (2, 3) were similar to the reference pattern (4) with the similarity score as high as 0.806 and 0.815, respectively. Compared to the pattern from the mouse melanoma with 4.5 weeks of tumour formation, the pattern from 6 weeks of tumour formation showed the appearance of some new peaks, as marked with the black arrow in Fig. 9a.

Advantages of the present invention

[0053] Fast, reliable and non-invasive diagnosis of skin diseases remains a challenge in clinical medicine. The present invention is based on a direct transfer method of intact skin cells from the epidermis layer of skin to the target plate for MALDI-TOF MS analysis. Compared to the invasive skin biopsy methods, the present invention relies on a non-invasive sampling procedure, i.e. tape stripping of cells from epidermis layer of the skin, which protects the patients from the suffering of painful surgical wounds and which lowers the risk of surgical site infections to a minimum. Compared to other non-invasive methods including the tape stripping genotypic and proteomic methods 22 26 the present method is based on a direct MALDI-TOF MS analysis of the intact skin cells directly from the adhesive tape without the need of any sample treatment step, and thus is fast, convenient and low-cost.

[0054] The present invention allows a single diagnosis to be completed within 30 minutes for only a few US dollars. The whole diagnosis process includes: i) tape stripping sampling from suspicious skin region (~ 10 min, few US dollars), //) adhesive tapes fixation onto a MALDI target plate and MALDI matrix deposition (~ 5 min, < 1 US dollar), iii ) MALDI-TOF MS analysis (< 10 min).

[0055] With the above advantages, the present invention is highly promising for routine clinical practice.

[0056] Examples

Example 1 - Tape stripping mass spectrometry for melanoma diagnostics

Tape stripping sampling of human skin

[0057] Normal skin regions and skin moles were selected from three volunteers (Persons A, B, C) for analysis. The selected regions were firstly rinsed with water and then cleaned with 70% ethanol aqueous solution with powderless tissues to remove dusters, lipids or other materials adherent on skin surface. The single-sided adhesive patches, i.e. with an adhesive layer stuck to a thin inert plastics film, used for tape stripping sampling, were provided by DermTech, La Jolla, California, USA (commercial skin cell collecting adhesive tape). Another skin cell collecting tape was made in-house by manually adhering 467MP-200MP adhesive, provided by 3M, Minnesota, USA, to a thin plastics film. In the former case, the adhesive patch was stuck to the MALDI target using double-sided adhesive Scotch tape, while in the latter case additional 467MP-200MP adhesive on the backside of the patch was applied for the fixation on the MALDI target. The effective sampling area on each tape was a region with ~20 mm diameter, and the thickness of each patch is ~ 0.1 mm. During the sampling, one adhesive patch was adhered onto a selected skin region with the adhesive side and pressed for a certain amount of time to guarantee good adhesion of the adhesive layer with the skin cells to be collected. The region to be measured is outlined with a marking pen on the plastic topside of the patch. The patch is then slowly stripped from the skin surface, and skin cells are thus collected on the adhesive bottom side. At least four operations of tape stripping sampling were applied to each skin region, and a new clean adhesive patch was used for each sampling. The patches with collected skin cells were transferred to a MALDI target and measured immediately with MALDI-TOF MS or stored at 4 °C in a refrigerator for at least four weeks.

Human-mimicking melanoma tape stripping samples

[0058] Three different human melanoma cell lines (SBcl2, WM115, WM239) were cultured in 25 cm 2 T-flasks (TPP, Trasadingen, Switzerland) with Dulbecco's modified Eagle's medium (Gibco Life Technologies, Basel, Switzerland) supplemented with 10% volume of fetal calf serum and 1% volume of antibiotic stock (10,000 units/mL of penicillin and 10,000 pg ml· 1 of streptomycin, Gibco Life Technologies, Basel, Switzerland). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2 for three to four days until their confluence reached ~ 90%. The confluent cells were detached from the bottom of flasks using 0.05% Trypsin- EDTA (Gibco Life Technologies, Basel, Switzerland). The cell suspensions were transferred into centrifuge tubes followed by centrifuge at 1,200 x g for 4 min. After carefully removing the supernatant, the cell pellets were suspended in deionized water to reach a final concentration of 2.5 c 10 3 cells -pL -1 . The pure intact cells were deposed onto the adhesive side of tapes stripped from Person A's normal skin region. On each piece of tape, 4 pL of each type of cells was deposited to form a human-mimicking melanoma spot with the size of ~3 mm in diameter. Three such sample spots corresponding to three types of melanoma cells were prepared on each tape.

Tape stripping sampling of mouse skin

[0059] Two mice carrying melanoma tumours were provided by the Ludwig Center for Cancer Research in Epalinges, Switzerland. The tumour formation time for the two mice was 4.5 weeks and 6 weeks, respectively. The tumour region of each mouse was shaved carefully to remove hairs. The hair removal region was then cleaned gently with 70% ethanol and pasted with a clean adhesive tape under slide pressure for approx. 30s for tape stripping sampling. The tape was then stripped slowly from the mouse to collect the skin cells. For each region the tape was applied four times, with a new tape used for each time of sampling. The tapes with collected skin cells were transferred to a MALDI target and measured immediately with MALDI-TOF MS, or were stored at 4 °C for at least four weeks.

Mouse melanoma reference samples

[0060] Murin melanoma cell line B16, an often-used melanoma model, was cultured in 25 cm 2 T-flasks (TPP, Trasadingen, Switzerland) with Dulbecco's modified Eagle's medium (Gibco Life Technologies, Basel, Switzerland) supplemented with 10% volume of fetal calf serum and 1% volume of antibiotic stock (10,000 units/ mL of penicillin and 10,000 .ug mL 1 of streptomycin, Gibco Life Technologies, Basel, Switzerland). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2 for three to four days until their confluence reached ~90% . The confluent cells were detached from the bottom of flasks using 0.05% Trypsin-EDTA (Gibco Life Technologies, Basel, Switzerland). The cell suspensions were transferred into centrifuge tubes followed by centrifuge at 1,200 x g for 4 min. After carefully removing the supernatant, the cell pellets were suspended in deionized water to reach a final concentration of 2.5 c 10 3 cells -pL -1 . The pure intact cells were deposited onto the adhesive side of a clean tape to form the mouse melanoma reference sample. For each sample spot 4 pL of cells were deposited to form a circular region of ~3 mm diameter, and three such sample spots were prepared on the tape.

MALDI-TOF MS analysis

[0061] The adhesive tapes carrying collected human or mouse skin cells were fixed onto an electrically conductive MALDI target plate using either a double-sided Scotch sticky tape (3M, product No. 34-8509-3289-7, St. Paul, MN 55144-1000, < 0.1 mm thickness) or adhesive (3M, product No. 467MP-200MP) applied to the backside of the tapes. A proper volume (< 10 pL) of MALDI matrix was deposited onto the adhesive tape to cover the skin cells and dried at room temperature (~5 min) to form cells/ matrix co-crystals. The MALDI matrix used for the measurement in the present invention was sinapinic acid (20 mg mL -1 dissolved in 50/49.9/0.1% acetonitrile/ water/ trifluoroacetic acid).

[0062] At the beginning of each measurement, mass calibration was conducted with a calibration mixture containing 1 mg mL of cytochrome c (m/z[M+2H] 2+ =6181.05000, m/z[M+H] + =12360.97000), 1 mg mL· 1 of myoglobin (m/z[ M+2H] 2+ = 8476.66000, m/z[M+H]÷=16952.31000) and 1 mg mL 4 of protein A (m/z[M+2H] 2+ =22307.00000, m/z[M+H] + =44613.00000). On each piece of tape sample 4 pL of the calibration mixture was dropped in a region nearby the sample spots. After drying at room temperature, the calibration mixture was then overlaid with 1 pL of sinapinic matrix to form a calibration spot.

[0063] Thereafter, the target plate was loaded into a MALDI-TOF MS instrument for measurement (Bruker MicroFlex LRF MALDI-TOF MS, Bremen, Germany). The measurement was conducted under linear positive mode with 20 kV accelerating voltage. Instrumental parameters were set as: mass range 2,000-20,000 m/z, laser intensity 70 %, laser attenuator with 30 % offset and 40 % range, 500 laser shots accumulation for each spot, 20.0 Hz laser frequency, 20x detector gain, suppress up to 1000 Da, 350 ns pulsed ion extraction. The generated mass spectra provided the protein expression profiles of the recovered skin cells.

Pattern Matching Analysis ofMALDI-TOF MS patterns

[0064] The pattern matching analysis was conducted via open access software BacteriaMS (http: / / www.bacteriams.com), which has been developed for bacteria identification using MALDI-TOF MS profiling method. The generated mass spectra in the form of .txt files were uploaded into the software, and a cosine correlation algorithm was chosen to calculate the similarity scores between each two spectra. The similarity score between two mass spectra (i and j) was defined as 31 :

where y is the normalized intensity of a peak appearing in both spectra i and / (identical peak), 1 is the number of identical peaks in the two spectra, Y is the normalized intensity of a peak appearing in a spectrum, n is the number of peaks in a spectrum. Only peaks with S/N ³ 3 were taken into account. Peaks appearing in different spectra with A(m/z)/ ( m/z) £ 1000 ppm were considered as identical peaks. This 1000 ppm tolerance was chosen according to the resolving power of linear mode TOF analysis. This algorithm gives the maximum similarity score of 1.000, meaning the two spectra being compared are exactly the same. For cell typing (or identification of cell types), if the similarity score between the generated mass spectrum and the reference spectrum is higher than 0.62, it is considered as a reliable identification. The value of 0.62 was obtained from the analysis of a large number of samples.

[0065] If more than two mass spectra are to be compared simultaneously, cluster analysis included into the BacteriaMS software can be used. The cluster analysis will divide those mass spectra into different groups (clusters) according to the similarity score between each two of them. The grouping result is shown in the form of heatmap and dendrogram. The heatmap uses the darkness of colour to display the value of similarity scores. Darker colour means higher similarity score. The dendrogram shows the relative distance value between each two groups. The maximum relative distance value is 1.000, meaning that the two groups are totally different from each other and they can be well separated.

References

1. B. E. G. Roth berg, M. B. Bracken, and D. L.Rimm, 'Tissue Biomarkers for Prognosis in Cutaneous Melanoma: A Systematic Review and Meta- Analysis', J. Natl. Cancer Inst., 101 (2009), 452-474.

2. V. Gray-Schopfer, C. Wellbrock, and R. Marais, 'Melanoma Biology and New Targeted Therapy', Nature , 445 (2007), 2771-2776.

3. N. Abbasi, H. Shaw, and D. Rigel, 'Early Diagnosis of Cutaneous Melanoma:

Revisiting the Abed Criteria', JAMA J. Med. Assoc., 292 (2004), 2771-27776.

4. G. C. Bethune, A. S. L. Pettit, D. Veldhuijzen van Zanten, and P. J. Barnes, 'Well Differentiated Invasive Breast Cancers with Equivocal Her2 Immunohistochemistry: What Is the Yield of Routine Reflex in Situ Hybridization Testing?', Histopathology, 70 (2017), 966-974.

5. H. Frickmann, A. E. Zautner, A. Moter, J. Kikhney, R. M. Hagen, H. Stender, and S.

Poppert, 'Fluorescence in Situ Hybridization (Fish) in the Microbiological Diagnostic Routine Laboratory: A Review', Crit. Rev. Microbiol. , 7828 (2017), 1-31.

6. N. Vishwanathan, A. Bandyopadhyay, H. Y. Fu, K. C. Johnson, N. M. Springer, and W. S. Hu, Ά Comparative Genomic Hybridization Approach to Study Gene Copy Number Variations among Chinese Hamster Cell Lines', Biotechnol. Bioeng., 114 (2017), 1903-1908.

7. A. A. Marghoob, L. D. Swindle, C. Z. M. Moricz, F. A. Sanchez Negron, B. Slue, A. C.

Halpern, and A. W. Kopf, 'Instruments and New Technologies for the in Vivo Diagnosis of Melanoma', J. Am. Acad. Dermatol. , 49 (2003), 777-797.

8. A. Gadeliya Goodson, and D. Grossman, 'Strategies for Early Melanoma Detection:

Approaches to the Patient with Nevi', J. Am. Acad. Dermatol. , 60 (2009), 719-735.

9. T. Wadhawan, N. Situ, K. Lancaster, X. Yuan, and G. Zouridakis, 'Skinscan©: A Portable Library for Melanoma Detection on Handheld Devices', Proc. - Int. Symp. Biomed. Imaging, IEEE (2011), 133-136.

10. G. Zonios, A. Dimou, I. Bassukas, D. Galaris, A. Tsolakidis, and E. Kaxiras, 'Melanin Absorption Spectroscopy: New Method for Noninvasive Skin Investigation and Melanoma Detection', /. Biomed. Opt., 13 (2008):014017. A. Cohen A. EI-Aneed, 'Mass Spectrometry, Review of Bascis: Electrospray, Maldi, and Commonly Used Mass Analyzers'] Banoub 1 , Appl. Spectrosc. Rev., 44 (2009), 210- 230.

R. M. A. Heem K. Chughtai, 'Mass Spectrometric Imaging for Biological Tissue Analysis', Chem. Rev., 110 (2010), 3237-3277.

J. A. Mobley D. S. Cornett, E. C. Dias, M. Andresson, C. L. Arteaga, M. E. Sanders, R. M. Caprioli, Ά Novel Histology-Directed Strategy for Maldi-Ms Tissue Profiling That Improves Throughput and Cellular Specificity in Human Breast Cancer', Mol. Cell. Proteomics, 5(2006), 1975-1983.

H. S. Kang, S. C. Lee, Y. S. Park, Y. E. Jeon, J. H. Lee, S-Y. Jung, I. H. Park, S. H. Jang, H. M. Park, C. W. Yoo, S. H. Park, S. Y. Han, K. P. Kim, Y. H. Kim, J. Ro, and H. K. Kim, 'Protein and Lipid Maldi Profiles Classify Breast Cancers According to the Intrinsic Subtype', BMC Cancer, 11 (2011): 465.

B. Flatley, P. Malone, and R. Cramer, 'Maldi Mass Spectrometry in Prostate Cancer Biomarker Discovery', Biochim. Biophys. Acta. 1844 (2014), 940-949.

K. Schwambom, R. C. Krieg, M. Reska, G. Jakse, R. Knuechel, and A. Wellmann, 'Identifying Prostate Carcinoma by Maldi-Imaging', Int. ]. Mol. Med., 20 (2007), 155- 159.

M. El Ayed, D. Bonnel, R. Longuespee, C. Castelier, J. Franck, D. Vergara, A. Desmons, A. Tasiemski, A. Kenani, D. Vinatier, R. Day, I. Fournier, and M. Salzet, 'Maldi Imaging Mass Spectrometry in Ovarian Cancer for Tracking, Identifying, and Validating Biomarkers', Med. Sci. Monit., 16 (2010), BR233-245.

R. Lemaire, S. A. Menguellet, J. Stauber, V. Marchaudon, J. P. Lucot, P. Collinet, M. O. Farine, D. Vinatier, R. Day, P. Ducoroy, M. Salzet, and I. Fournier, 'Specific Maldi Imaging and Profiling for Biomarker Hunting and Validabon: Fragment of the 11s Proteasome Acbvator Complex, Reg Alpha Fragment, Is a New Potential Ovary Cancer Biomarker', ]. Proteome Res., 6 (2007), 4127-4134.

E. Bonaparte, C. Pesenti, L. Fontana, R. Falcone, L. Paganini, A. Marzorah, S. Ferrero, M. Nosoth, P. Mendogni, C. Bareggi, S. M. Sirchia, S. Tabano, S. Bosari, and M. Miozzo, 'Molecular Profiling of Lung Cancer Specimens and Liquid Biopsies Using Maldi-Tof Mass Spectrometry', Diagn. Pathol., 13 (2018):4. A. Bondarenko, Y. Zhu, L. Qiao, F. Cortes Salazar, H. Pick, H. H. Girault, 'Aluminium Foil as a Single-Use Substrate for Maldi-Ms Fingerprinting of Different Melanoma Cell Lines', Analyst , 141 (2016), 3403-3410.

R. Guran, L. Vanickova, V. Horak, S. Krizkova, P. Michalek, Z. Heger, O. Zitka, and V. Adam, 'Maldi Msi of Melim Melanoma: Searching for Differences in Protein Profiles', PLos ONE , 12 (2017), e0189305.

N. R. Benson, 'Tape Stripping Methods for Analysis of Skin Desease and Pathological Skin State', U.S. Patent Application No. 7,989,165 B2 (2011).

T. Vogt, 'Method for Detection of Melanoma', U.S. Patent No.7, 297, 480 B2 (2007). L. A. Rheins, V. B. Morhenn, 'Methods and Kits for Obtaining and Analyzing Skin Samples for the Detection of Nucleic Acids', U.S. Patent No.6, 949, 338 B2 (2005). L. A. Rheins, V. B. Morhenn, 'Method for Detection of Biological Factors in Epidermis', U.S. Patent No.6. 720, 145 B2 (2004).

B. Mehul, G. Laffet, L. Russo, 'Non-Invasive Method for Collecting Biological Data for Establishing A Diagnosis of A Cutaneous Pathology', U.S. Patent Application No.12, 779, 073 (2010).

M. A. J. Weterman, G. N. P. Vanmuijen, D. J. Ruiter, H. P. J. Bloemers, 'Thymosin Beta-10 Expression in Melanoma Cell-Lines and Melanocytic Lesions - a New Progression Marker for Human Cutaneous Melanoma', Int. ]. Cancer, 53 (1993), 278- 284.

G. Weinlich, K. Eisendle, E. Hassler, M. Baltaci, P. O. Fritsch, B. Zelger, 'Metallothionein - Overexpression as A Highly Significant Prognostic Factor in Melanoma: A Prospective Study on 1270 Patients', Brit. ]. Cancer, 94 (2006), 835-841. P. Kaskel, C. Berking, S. Sander, M. Volkenandt, R.U. Peter, G. Krahn, 'S-100 protein in Peripheral Blood: A Marker for Melanoma Metastases - A Prospective 2-center Study of 570 Patients with Melanoma', J. Am. Acad. Dermatol., 41 (1999), 962-969. S. Petersson, E. Shubbar, L. Enerback, C. Enerback, 'Expression Patterns of S100 Proteins in Melanocytes and Melanocytic Lesions', Melanoma Res., 2009, (2009), 215- 225.

Y. D. Zhu, L. Qiao, M. Prudent, A. Bondarenko, N. Gasilova, S. B. Moller, N. Lion, H. Pick, T. Q. Gong, Z. X. Chen, P. Y. Yang, L. T. Loveyf and H. H. Girault, 'Sensitive and Fast Identification of Bacteria in Blood Samples by Immunoaffinity Mass Spectrometry for Quick BSI Diagnosis, Chem. Sci., 7(2016), 2987-2995.