WO2020035707A1 - Analyse de cellules cutanées - Google Patents

Analyse de cellules cutanées Download PDF

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WO2020035707A1
WO2020035707A1 PCT/GB2019/052925 GB2019052925W WO2020035707A1 WO 2020035707 A1 WO2020035707 A1 WO 2020035707A1 GB 2019052925 W GB2019052925 W GB 2019052925W WO 2020035707 A1 WO2020035707 A1 WO 2020035707A1
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maldi
skin
cells
melanoma
tof
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PCT/GB2019/052925
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English (en)
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Ping-Chih HO
Hubert Girault
Milica JOVIC
Andreas Lesch
Horst Pick
Yingdi ZHU
Tzu-en LIN
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École Polytechnique Fédérale de Lausanne
HANSON, William Bennett
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Publication of WO2020035707A1 publication Critical patent/WO2020035707A1/fr

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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • H01J49/0459—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components for solid samples
    • H01J49/0463—Desorption by laser or particle beam, followed by ionisation as a separate step
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A61B10/02—Instruments for taking cell samples or for biopsy
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • H01J49/0409—Sample holders or containers
    • H01J49/0418—Sample holders or containers for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
    • H01J49/161—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission using photoionisation, e.g. by laser
    • H01J49/164—Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI]

Definitions

  • This invention relates to a method of analyzing skin cells.
  • 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).
  • 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.
  • IHC immunohistochemistry
  • FISH fluorescence in situ hybridization
  • CGH comparative genomic hybridization
  • 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.
  • target DNA or RNA e.g. mRNA, lncRNA and miRNA
  • 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.
  • invasive skin sampling methods such as biopsies may cause painful wounds and could result in the exposure to the risks of infection.
  • 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.
  • MALDI MS matrix assisted laser desorption ionization mass spectrometry
  • 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 .
  • 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.
  • 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).
  • MALDI analysis of tissues is usually limited by the sample thickness and tissue sectioning process.
  • non-invasive sampling methods have been extensively proposed to improve the detection of skin diseases.
  • 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.
  • a polymerase chain reaction is required in the methods for amplifying the trace nucleic acid, which is time consuming, costty and labor intensive.
  • non- invasive sampling was used to collect material from the skin and diagnose psoriasis using mass spectrometry 26 .
  • 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.
  • 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.
  • 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.
  • 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
  • FIG. 1 schematically illustrates steps in the method of an embodiment of the present invention
  • Fig. 2 shows the MALDI-TOF MS pattern of a non-used adhesive tape
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • Fig. 6 mimics the analysis of human melanoma using the tape stripping mass spectrometry method of Fig. 1;
  • Fig. 7 shows the comparison and cluster analysis of mass spectra from Fig. 3 to Fig. 6 (the third layer spectrum from each figure);
  • Figs. 8a and 8b shows four groups of marker mass spectrometric peaks for human melanoma
  • 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
  • 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.
  • Steps I to III adhesive tape stripping-based sampling of the outermost skin layer (i.e. stratum corneum)
  • 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
  • Step VI the MALDI-TOF MS analysis of the collected sample.
  • Step I an adhesive tape 1, i.e. a thin plastic foil coated with an adhesive layer, is applied on a skin area 2.
  • 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.
  • Step III the tape 1 is then slowly removed from the skin thereby collecting a certain amount of skin cells on the tape.
  • Step IV the tape carrying collected skin cells 4 is turned downside up and then fixed onto a MALDI target 5.
  • Step V a MALDI matrix 6 is deposited onto the adhesive tape to cover the skin cells.
  • 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.
  • the target can include, or be provided with, a frame for retaining the tape in position.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • peak 1 peak at 4,933 ⁇ 4 m/z
  • peak 2 peak group at 10,085 ⁇ 10 m/z
  • peak group at 11,500 - 11,850 m/z peak 4
  • 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.
  • 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:
  • 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
  • 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:
  • 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
  • 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):
  • 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:
  • 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
  • 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):
  • 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):
  • 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):
  • 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.
  • Fig. 9a 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.
  • the MALDI-TOF MS analysis of tape-stripped normal skin 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 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the pattern from 6 weeks of tumour formation showed the appearance of some new peaks, as marked with the black arrow in Fig. 9a.
  • 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.
  • 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.
  • 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).
  • the present invention is highly promising for routine clinical practice.
  • Example 1 Tape stripping mass spectrometry for melanoma diagnostics
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • BacteriaMS http: / / www.bacteriams.com
  • 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 :
  • 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.
  • 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.

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Abstract

La présente invention concerne un procédé de transfert par retrait de ruban adhésif pour le diagnostic rapide et non invasif de maladies cutanées par analyse par spectrométrie de masse MALDI-TOF. Des cellules provenant de la couche d'épiderme d'une zone cutanée suspecte d'un patient sont collectées à l'aide de rubans adhésifs par le biais d'au moins une application de procédure de retrait de ruban. Les rubans adhésifs portant les cellules cutanées collectées sont analysés directement par MS MALDI-TOF sans aucun prétraitement d'échantillon comme la lyse cellulaire ou l'extraction de composant cellulaire préparatoire. La détection et le diagnostic du mélanome de l'homme et de la souris sont illustrés à titre d'exemples dans la présente invention.
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US11753687B2 (en) 2008-05-14 2023-09-12 Dermtech, Inc. Diagnosis of melanoma and solar lentigo by nucleic acid analysis
US11976332B2 (en) 2018-02-14 2024-05-07 Dermtech, Inc. Gene classifiers and uses thereof in non-melanoma skin cancers
US11643689B2 (en) 2018-05-09 2023-05-09 Dermtech, Inc. Methods for diagnosing atopic dermatitis using gene classifiers
US11578373B2 (en) 2019-03-26 2023-02-14 Dermtech, Inc. Gene classifiers and uses thereof in skin cancers
WO2021189226A1 (fr) * 2020-03-24 2021-09-30 The Procter & Gamble Company Procédés de test d'échantillons de peau
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