Skin based test for detection of radiation exposure

Abstract

Provided herein are methods and kits for the rapid testing of biomarkers in human skin that are upregulated within 24 hours, across a range of dosages, to identify patients who have been exposed to radiation.

Description

[0001] SKIN BASED TEST FOR DETECTION OF RADIATION EXPOSURE

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial Nos. 63 / 603,037, filed on November 27, 2023, and 63 / 689,425, filed on August 30, 2024. The entire contents of the foregoing are hereby incorporated by reference.

[0004] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with Government support under Grant No. AI148306 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0006] TECHNICAL FIELD

[0007] Provided herein are methods and kits for the testing of biomarkers in human skin to identify patients who have been exposed to radiation.

[0008] BACKGROUND

[0009] In the event of a large scale radiologic or nuclear event, it will be necessary to discriminate exposed individuals from the worried well and identify individuals at risk for radiation-induced tissue injury. There are currently no FDA-approved tests to measure radiation exposure and estimate dosage received.

[0010] SUMMARY

[0011] Provided herein are methods and kits for the rapid testing of biomarkers in human skin that are upregulated within 24 hours, across a range of dosages, to identify patients who have been exposed to radiation. Skin is visually observable, accessible to non-invasive testing and easily sampled, making it amenable to a variety of testing modalities. The skin is one of the most radiosensitive organ systems, along with the bone marrow, reproductive and gastrointestinal systems, and the brain.1In the case of an external radiologic or nuclear event, the skin will receive at least as much radiation exposure as other tissues. Radiation-induced damage in skin is likely to be a useful predictor of injury at other tissue sites. Skin biomarkers that identify radiation exposure may be a rapid and effective way of identifying patients at risk for other systemic complications.

[0012] Thus, provided herein are methods comprising: (a) providing a sample comprising cells of the epidermal layer of the skin of a subject, (b) optionally eluting mRNA or protein from the sample, and (c) detecting the presence of one or more, e.g., two, three, four or more, radiation specific mRNA or protein biomarkers in the sample, e.g., in the eluate, wherein the one or more radiation specific mRNA or protein biomarkers are listed in Table 1, 2, or 3, shown in a figure, or are selected from or comprise: (i) one, two, three, or all four of KRT5, KRT14, CTSG, and FABP5; (ii) one or two of FOS or DUSP1; (iii) one, two, three, four or more of S100A8, S100A9, KRT6A / B / C, KRT16, IFI27, CD74, HLA-DRB, and STAT1; (iv) one, two, three, four or more of BST2, ISG15, MX1, IFI44L and CXCL10; (v) one or both of KRT15 and N0TCH1, or (vi) any combination thereof.

[0013] In some embodiments, the sample is obtained by tape stripping, optionally using 5-20, 5-25, or 8-12 tape strips applied to the same site.

[0014] Also provided herein are diagnostic kits for detecting radiation exposure in a patient comprising reagents, optionally antibodies, for detecting or measuring one or more, e.g., two, three, four or more, mRNAs or proteins, wherein the one or more mRNA or proteins are listed in Table 1, 2, or 3, are shown in a figure, or are selected from or comprise or consist of: (i) one, two, three, or all four of KRT5, KRT14, CTSG, and FABP5; (ii) one or two of FOS or DUSP1; (iii) one, two, three, four or more of S100A8, S100A9, KRT6A / B / C, KRT16, IFI27, CD74, HLA-DRB, and STAT1; (iv) one, two, three, four or more of BST2, ISG15, MX1, IFI44L and CXCL10; (v) one or both of KRT15 and NOTCH1, or (vi) any combination thereof.

[0015] In some embodiments, the kit comprises (a) at least one test strip, (b) elution buffer for eluting mRNA or protein from the test strip, and (c) means for determining the presence or absence of any radiation specific biomarkers in the eluate.

[0016] Additionally provided herein are methods for determining exposure to radiation in a subject. The methods comprise: (a) providing a sample of the epidermal layer of the skin of the subject, (b) optionally eluting mRNA or protein from the sample, and (c) detecting the presence or level of one or more, e.g., two, three, four or more, radiation specific mRNA or protein biomarkers in the sample, e.g., in the eluate, wherein the one or more radiation specific mRNA or protein biomarkers are listed in Table 1, 2, or 3, are shown in a figure, or are selected from or comprise or consist of: (i) one, two, three, or all four of KRT5, KRT14, CTSG, and FABP5; (ii) one or two of FOS or DUSP1; (iii) one, two, three, four or more of S100A8, S100A9, KRT6A / B / C, KRT16, IFI27, CD74, HLA-DRB, and STAT1; (iv) one, two, three, four or more of BST2, ISG15, MX1, IFI44L and CXCL10; (v) one or both of KRT15 and N0TCH1, or (vii) any combination thereof, and comparing the presence or level of the one or more biomarkers to a reference level, wherein the presence or level of the biomarkers above the reference indicates that the subject has been exposed to radiation.

[0017] In some embodiments, (i) the presence of one, two, three, or all four of KRT5, KRT14, CTSG, and FABP5 indicates that radiation exposure has occurred; (ii) the presence of one or both of FOS and DUSP1 indicates that 1 Gy radiation exposure has occurred; (iii) the presence of one, two, three, four or more of S100A8, S100A9, KRT6A / B / C, KRT16, IFI27, CD74, HLA-DRB, and STAT1 indicates that 1 Gy or 2 Gy radiation exposure has occurred; (iv) the presence of one, two, three, four or more of BST2, ISG15, MX1, IFI44L and CXCL10 indicates that 2 Gy radiation exposure has occurred; (v) the presence of one or both of KRT15 and N0TCH1 indicates that 5 Gy radiation exposure has occurred.

[0018] In some embodiments, the methods further comprise administering a treatment for radiation exposure to a subject who has been exposed to radiation. In some embodiments, the methods comprise administering a treatment for radiation to a subject who has been exposed to at least 2 Gy of radiation. In some embodiments, the treatment comprises administration of one or more of granulocyte colony -stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or KGF (keratinocite grow factor), potassium iodide, Prussian blue, dyethilene-triamine- pentacetic acid (DTPA), e.g., Ca-DTPA or Zn-DTPA, oral calcium or aluminium phosphate solutions, antibiotics, fresh blood, platelet transfusions, plasma, antiemetics, sedation, or fluids (e.g., oral, subcutaneous, or IV fluids) and electrolytes.

[0019] In some embodiments, the subject is a human.

[0020] Also provided herein are methods of detecting radiation exposure biomarkers in a skin sample from a person comprising (a) providing a sample of the epidermal layer of the skin of the person, (b) eluting mRNA or protein from the sample, and (c) detecting the presence of one or more radiation specific mRNA or one or more protein biomarkers in the eluate.

[0021] In some embodiments, the one or more radiation specific mRNA or one or more protein biomarkers are selected from p53 / TP53, CDKNlA / p21, S100A8, S100A9, ZMAT3, TNFRSF10B, Apobec3c, TNFRSF10B, POLH, RPS27L, Krt6A, Krtl6, IFIT1, POLH, BAX, TIGAR, SPATAI 8, and MIR34A

[0022] Also provided herein are diagnostics kit for detecting radiation exposure in a patient comprising reagents for detecting or measuring one or more mRNAs or proteins selected from p53 / TP53, CDKNlA / p21, S100A8, S100A9, ZMAT3, TNFRSF10B, Apobec3c, TNFRSF10B, POLH, RPS27L, Krt6A, Krtl6, IFIT1, POLH, BAX, TIGAR, SPATAI 8, and MIR34A.

[0023] Additionally provided herein are kits for detecting exposure to radiation in a patient comprising (a) at least one test strip, (b) elution buffer for eluting mRNA or protein from the test strip, and (c) means for determining the presence or absence of any radiation specific biomarkers in the eluate.

[0024] In some embodiments, the radiation specific biomarkers are selected from p53 / TP53, CDKNlA / p21, S100A8, S100A9, ZMAT3, TNFRSF10B, Apobec3c, TNFRSF10B, POLH, RPS27L, Krt6A, Krtl6, IFIT1, POLH, BAX, TIGAR, SPATAI 8, and MIR34A.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary' skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0026] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. DESCRIPTION OF DRAWINGS

[0027] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0028] FIGs. 1A-E show aspects of the human skin grafted model used to identify biomarkers of radiation in human skin. (A) a photograph of an human adult skin grafted NSG mouse. (B, C) microscopic histologic images. H&E staining 8 weeks after grafting shows clearly defined basal, spinous, and granular layers, basket weave keratin and pigmentation of the basal layer by resident melanocytes. (B) shows the native mouse skin (left half) adjoining the human skin graft (right half) (D) Immunostaining at 8 weeks demonstrates Langerhans cells (CD la), melanocytes (MART-1), dermal mast cells (tryptase), T cells (CD3), macrophages (CD 163) and dendritic cells (CD 11c). (E) An illustration of the experimental process used to identify biomarkers of radiation exposure in human skin. Cohorts of 11 mice were grafted with skin from a single human donor. The human skin grafts were irradiated at the indicated dosages. Skin grafts were removed 24 hours after irradiation and analyzed by spatial transcriptional profiling and multiplex immunostaining.

[0029] FIGs. 2A-F. 1 Gy irradiation induced Inflammation & TGFP induced fibrosis in human skin grafted mice. (A-C) Differentially expressed genes, pathway, and histologic studies of 1 Gy irradiated skin grafts demonstrate marked inflammation 24 hours after irradiation, followed by (D-F) induction of TGFp signaling, TGFp induced gene expression and fibrosis 3 months later (Masson’s trichrome stain). 6 mice / group. Genes expressed by stressed keratinocytes are shown in open circles in panel A.

[0030] FIGs. 3A-B illustrate the experimental process used to identify genes upregulated by irradiation in superficial keratinocytes, the cell population sampled in the described test. Spatial profiling was carried out on tissue sections derived from the human skin grafts using the NanoString CosMx Spatial Molecular Imager. This instrument produces gene expression data at the single cell level. (A) is an image showing CosMx imaging data from a skin graft that received 1 Gy irradiation. (B) Is a UMAP showing the cell clusters isolated from a total of six irradiated skin grafts (2 grafts each irradiated at 0, 1, 2 or 5 Gy). Superficial keratinocytes (cluster 1) are the cell population that will be sampled by the test. FIGs. 4A-C illustrate genes and proteins useful in discriminating irradiated from non-irradiated individuals. (A) Shown are expression levels of four genes that are upregulated 24 hours after all dose levels of radiation (1, 2, 5 Gy). (B,C) Keratin 14 (KRT 14) is upregulated at both the (B) mRNA and (C) protein levels. (B) shows a spatial mapping of KRT mRNA expression by spatial imaging and (C) shows a photomicrographs of human skin grafts immunostained for KRT 14 after 0 Gy (left panel) and 1 Gy (right panel) irradiation. The white box indicates cells that will be sampled by the described test.

[0031] FIGs. 5A-C illustrate genes and proteins useful in determining the dose of radiation received. (A,B) tThis figure shows the levels of biomarker mRNA expression, as measured by spatial profiling, in superficial keratinocytes 24 hours after 0, 1, 2 or 5 Gy irradiation. (C) Photomicrographs of human skin grafts immunostained for keratin 15 (KRT 16) and S100A8 are shown, confirming upregulation of these two proteins in the superficial keratinocytes of the epidermis after 1 Gy irradiation.

[0032] FIG. 6A-B illustrate the mRNA expression levels of five biomarkers selectively upregulated in superficial keratinocytes after 2 Gy irradiation, which are useful in determining the dose of radiation received. mRNA expression was measured by spatial profiling.

[0033] FIG. 7 illustrates the mRNA expression levels of two biomarkers (KRT 15 and NOTCH1) that are selectively upregulated in superficial keratinocytes after 5 Gy irradiation, which are useful in determining the dose of radiation received. mRNA expression was measured by spatial profiling.

[0034] FIG. 8 shows keratin 14 expression in pediatric human skin 24 hours after irradiation with 1 or 2 Gy as noted.

[0035] FIGs. 9A-B show keratin 16 expression in (A) adult and (B) pediatric human skin 24 hours after irradiation with 1 or 2 Gy as noted.

[0036] FIGs. 10A-B show S100A8 expression in (A) adult and (B) pediatric human skin 24 hours after irradiation with IGy.

[0037] DETAILED DESCRIPTION

[0038] Provided herein are biomarkers of radiation exposure and a method and non- invasive rapid point-of-care test and kit to determine if a person has been exposed to radiation and, if so, to estimate the dosage of radiation. The method, test and kit make use of biomarker genes or their protein products found in the superficial aspects of the epidermal layer of skin that discriminate exposed from nonexposed individuals.

[0039] Thus, the invention is a noninvasive test for radiation exposure that detects the expression of biomarkers we have identified that are expressed in the top layer of the epidermis. The final product is a rapid, point-of-care, noninvasive test for radiation exposure.

[0040] A first set of biomarkers of radiation exposure were identified by grafting NSG mice with living, immunologically intact human skin.

[0041] These skin grafts retain viable populations of human keratinocytes, fibroblasts, and immune cells (FIGs. 1A-D), and maintain human blood vessels that anastomose with the mouse circulation.9As shown below, irradiation of these skin grafts faithfully recapitulates the acute inflammation, blood vessel injury and fibrosis observed in patients exposed to radiation (Fig. 2). Human skin grafted to immunodeficient mice can survive and grow hair for at least a year.10We utilize NSG mice grafted with healthy adult human skin or neonatal foreskin to study human cutaneous immune responses and homing of T cells to skin.9’11Human skin grafts maintained epithelial differentiation and retained resident populations of melanocytes and immune cells including Langerhans cells, mast cells, macrophages, dendritic cells and T cells for at least 8 weeks (FIG. ID).

[0042] The methods included exposing the human skin graft to ionizing radiation via a gantry mounted 220 kVp 13mA x-ray tube, and assaying gene and protein expression changes in the skin using spatial transcriptional profiling and immunostaining. These biomarkers were induced in a dose-dependent manner in the skin and evident by 24 hours after radiation exposure. A subset of these markers were expressed in the top layers of the human epidermis and can be noninvasively measured for RNA or protein expression, e g., by means of non-invasive tape stripping.

[0043] Spatial transcriptional profiling experiments were performed to directly measure gene expression in superficial keratinocytes, the population sampled by noninvasive tape stripping, 24 hours after skin irradiation. These studies found multiple biomarkers that discriminated radiated from non-irradiated skin, and selective biomarkers for 1, 2 and 5 Gy irradiation (FIGs. 3-7). Full lists of differentially expressed genes (DEG) at each radiation dose are included in Tables 1-3 and selected biomarkers are discussed in FIGs. 3-7.

[0044] Thus, biomarkers that can be used in the present methods can include those that are listed in Table 1, 2, or 3, or can be selected from or comprise:

[0045] (i) one, two, three, or all four of KRT5, KRT14, CTSG, and FABP5 (the presence of these indicates that radiation exposure has occurred);

[0046] (ii) one or both of FOS and DUSPl(the presence of these indicates that 1 Gy radiation exposure has occurred);

[0047] (iii) one or more of FOS, DUSP1, S100A8, S100A9, KRT6, KRT16, IFI27, CD74, HLA-DRB or STAT1 (the presence of these indicates that 1 or 2 Gy radiation exposure has occurred);

[0048] (iv) one or more of BST2, ISG15, MX1, IFI44L and CXCL10 (the presence of these indicates that 2 Gy radiation exposure has occurred);

[0049] (v) one or both of KRT15 and N0TCH1 (the presence of these indicates that 5 Gy radiation exposure has occurred), or

[0050] (vi) any combination thereof.

[0051] Accordingly, provided herein are methods of determining if a person has been exposed to radiation. For example, there is provided a method of detecting radiation exposure biomarkers in a skin sample from a person comprising (a) providing a sample of the epidermal layer of the skin of the person, and detecting the presence of one or more radiation specific mRNA or protein biomarkers in the sample. Optionally an epidermal sample is obtained from the person and mRNA and / or protein biomarkers are eluted from the tape strip using a buffer solution optimized to detect the biomarkers. The eluate is then tested for the presence of the biomarkers and compared to a reference; if the biomarkers are present in the solution, or are present at a level above a reference level, the person has been exposed to radiation.

[0052] The methods can be performed on a sample comprising epidermal keratinocytes from a subject. Subjects can be mammals, e.g., human or non-human veterinary subjects such as cats, dogs, horses, and cows. The subject can be an adult (for humans, 18 or older) or pediatric (for humans, under age 18) subject.

[0053] The sample can be obtained, e.g., using tape stripping, scraping, or biopsy (e.g., punch biopsy or shave biopsy); preferably tape stripping is used. (For subjects with hair or fur, the area to be sampled can be shaved first to expose the skin.) Preferably the sample is obtained within 24-48 hours of the possible exposure.

[0054] Tape Stripping

[0055] Tape strips are widely as a minimally invasive method to sample the epidermis. Hughes et al., Br J Dermatol. 2021 Jul; 185(1 ):26-35 ; Tsoi et al., J Invest Dermatol. 2022 Jun;142(6):1587-1596.e2. Tape strips are plastic discs or strips with an adhesive side that sticks to the skin surface. Components of the epidermis adhere to the tape strip when removed. Sequential application of the tape strip allows deeper levels of the stratum comeum to be accessed with each strip. Only mild discomfort is caused by the application of the tape strip. It leaves a red mark on the skin which fades; and it causes neither bleeding nor scarring. Use of tape strips requires no skin preparation. For additional information on tape strips and their use, see Hughes et al, Br J Dermatol 185(1): 26-35 (2021); Paliwal et al, Eur J Pharm Sci 50: 546-56 (2013); He et al, J Allergy Clin Immunol 147: 199-212 (2021); Bretemitz et al, Br J Dermatol 156: 231-40 (2007). In some embodiments, tape-stripping is particularly useful for detecting S100A8 proteins, which are secreted proteins that should be easily eluted from tape strips.

[0056] Tape strips have been used to detect microbes on the surface of the skin, lipids and ceramides and superficially expressed proteases and cytokines. They have also been used to access structural proteins of the stratum comeum such as filaggrin and secreted cytokines. The most abundant non-oxidized RNA and lipids are found in tape strips 11-20, ie, the second set of ten strips applied to the same portion of the person’s skin. In the present methods, 5-20, 5-15, or 8-12 tape strips can be used. They have been used to distinguish psoriasis from atopic dermatitis, see He, id. above, to quantify stratum comeum protein; initially by weighing individual strips, and then by spectroscopy, which allows more accurate quantification of total protein. Other detection methods use SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining, and antibody-based immunofluorescence, which allows detection of many other proteins.

[0057] Tape strips are obtainable from several suppliers, for example, from CuDerm, Dallas, Texas (clinicalandderm.com / dl01-d-squame-stripping-discs).

[0058] To obtain a sample of skin from a person suspected of being exposed to radiation, one can employ a methodology set forth below. Skin sampling: A tape stnp is applied with pressure to an area of the person’s skin suspected of being exposed to radiation. After 5-10 seconds, the strip is removed and tested for mRNA and / or protein expression of one or more of the biomarkers detailed below. Sequential application of tape strips (e.g., 5-20, 5-15, or 8-12 tape strips) to the same site (which can be marked by circling it with a pen on initial application to the skin) allows deeper levels of the stratum comeum to be accessed with each strip.

[0059] Biomarker detection

[0060] The methods can include detecting biomarkers, optionally after eluting (or isolating) proteins or mRNA from the sample, e.g., from the tape strip.

[0061] Protein: Protein biomarkers can be either detected directly on the tape strip by immunostaining with biomarker specific antibodies, or can be eluted from the tape strip using a protein extraction buffer for the isolation of the selected protein biomarker(s) set forth below (e.g., a solution containing phosphate buffered saline, polypropylene glycol, sodium dodecyl sulfate and proteinase inhibitors)2. Eluted proteins can then be detected by a number of methods, including for example a lateral flow test strip that is impregnated with antibodies specific for the protein biomarker conjugated to detector particles that allow binding to be visualized if the antigen is detected.

[0062] The presence and / or level of a protein can be evaluated using methods known in the art, e.g., using standard electrophoretic and quantitative immunoassay methods for proteins, including but not limited to, Western blot; enzyme linked immunosorbent assay (ELISA); biotin / avidin type assays; protein array detection; radio-immunoassay; immunohistochemistry (IHC); immune-precipitation assay; FACS (fluorescent activated cell sorting); mass spectrometry (Kim (2010) Am J Clin Pathol 134: 157-162; Yasun (2012) Anal Chem 84(14):6008-6015; Brody (2010) Expert Rev Mol Diagn 10(8): 1013-1022; Philips (2014) PLOS One 9(3):e90226; Pfaffe (2011) Clin Chem 57(5): 675-687). The methods typically include revealing labels such as fluorescent, chemiluminescent, radioactive, and enzymatic or dye molecules that provide a signal either directly or indirectly. As used herein, the term “label” refers to the coupling (i.e. physically linkage) of a detectable substance, such as a radioactive agent or fluorophore (e.g. phycoerythrin (PE) or indocyanine (Cy5), to an antibody or probe, as well as indirect labeling of the probe or antibody (e.g. horseradish peroxidase, HRP) by reactivity with a detectable substance.

[0063] In some embodiments, an ELISA method may be used, wherein the wells of a mictrotiter plate are coated with an antibody against which the protein is to be tested. The sample containing or suspected of containing the biological marker is then applied to the wells. After a sufficient amount of time, during which antibody-antigen complexes would have formed, the plate is washed to remove any unbound moieties, and a detectably labelled molecule is added. Again, after a sufficient period of incubation, the plate is washed to remove any excess, unbound molecules, and the presence of the labeled molecule is determined using methods known in the art. Variations of the ELISA method, such as the competitive ELISA or competition assay, and sandwich ELISA, may also be used, as these are well-known to those skilled in the art.

[0064] In some embodiments, an IHC method may be used. IHC provides a method of detecting a biological marker in situ. The presence and exact cellular location of the biological marker can be detected. Typically a sample is fixed with formalin or paraformaldehyde, embedded in paraffin, and cut into sections for staining and subsequent inspection by confocal microscopy. Current methods of IHC use either direct or indirect labelling. The sample may also be inspected by fluorescent microscopy when immunofluorescence (IF) is performed, as a variation to IHC. mRNA: mRNA can be eluted from the tape strip using a buffer and gene expression is assessed by rapid quantitative PCR methods or via an isothermal and nonenzymatic signal amplification system (catalytic hairpin assembly (CHA) reaction) coupled with a lateral flow immunoassay (LFIA) strip-based detection method.3See also Tsoi et al., J Invest Dermatol. 2022 Jun;142(6):1587-1596.e2.

[0065] The presence and / or level of a nucleic acid can be evaluated using methods known in the art, e.g., using polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative or semi-quantitative real-time RT- PCR, digital PCR i.e. BEAMing ((Beads, Emulsion, Amplification, Magnetics) Diehl (2006) Nat Methods 3:551-559) ; RNAse protection assay; Northern blot; various types of nucleic acid sequencing (Sanger, pyrosequencing, NextGeneration Sequencing); fluorescent in-situ hybridization (FISH); or gene array / chips) (Lehninger Biochemistry (Worth Publishers, Inc., current addition; Sambrook, et al, Molecular Cloning: A Laboratory Manual (3. Sup.rd Edition, 2001); Bernard (2002) Clin Chem 48(8): 1178-1185; Miranda (2010) Kidney International 78: 191-199; Bianchi (2011) EMBO Mol Med 3:495-503; Taylor (2013) Front. Genet. 4: 142; Yang (2014) PLOS One 9(11 ): e 110641); Nordstrom (2000) Biotechnol. Appl. Biochem. 31(2): 107-112; Ahmadian (2000) Anal Biochem 280: 103-110. In some embodiments, high throughput methods, e.g., protein or gene chips as are known in the art (see, e.g., Ch. 12, Genomics, in Griffiths et al., Eds. Modem genetic Analysis, 1999,W. H. Freeman and Company; Ekins and Chu, Trends in Biotechnology, 1999, 17:217-218; MacBeath and Schreiber, Science 2000, 289(5485): 1760-1763; Simpson, Proteins and Proteomics: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 2002; Hardiman, Microarrays Methods and Applications: Nuts & Bolts, DNA Press, 2003), can be used to detect the presence and / or level of biomarkers described herein.

[0066] RT-PCR can be used to determine the expression profiles of biomarkers (U.S. Patent No. 2005 / 0048542A1). The first step in expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction (Ausubel et al (1997) Cunent Protocols of Molecular Biology, John Wiley and Sons). To minimize errors and the effects of sample-to- sample variation, RT-PCR is usually performed using an internal standard, which is expressed at constant level among tissues, and is unaffected by the experimental treatment. Housekeeping genes, such as Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Beta actin (ACTB), are most commonly used.

[0067] Gene arrays can be used, prepared by selecting probes which comprise a polynucleotide sequence, and then immobilizing such probes to a solid support or surface. For example, the probes may comprise DNA sequences, RNA sequences, copolymer sequences of DNA and RNA, DNA and / or RNA analogues, or combinations thereof. The probe sequences can be synthesized either enzymatically in vivo, enzymatically in vitro (e.g. by PCR), or non-enzymatically in vitro.

[0068] Detecting Radiation Exposure

[0069] In some embodiments, the presence and / or level of a biomarker described herein is comparable to the presence and / or level of the biomarker in the exposed reference, then the subject can be identified as having been exposed to radiation. In some embodiments, the subject has no overt signs or symptoms of radiation exposure, the subject is suspected of having been exposed and / or has one or more symptoms associated with radiation exposure.

[0070] Suitable reference values can be determined using methods known in the art, e.g., using standard clinical trial methodology and statistical analysis. The reference values can have any relevant form. In some cases, the reference comprises a predetermined value for a meaningful level of a biomarker, e.g., a control reference level that represents a normal level of a biomarker, e.g., a level in an unexposed subject, and / or an exposure reference that represents a level of the biomarker associated with radiation exposure, e.g., a level in a subject having been exposed to 1 Gy, 2 Gy, 3 Gy, or 5 Gy. A control biomarker will also be measured (Keratin 10). Keratin 10 is expressed at high and constant levels by all superficial keratinocytes and is not changed by radiation exposure. This gene will be used to normalize biomarker expression levels for the total amount of keratinocyte mRNA or protein sampled.

[0071] The predetermined level can be a single cut-off (threshold) value, such as a median or mean, or a level that defines the boundaries of an upper or lower quartile, tertile, or other segment of a clinical trial population that is determined to be statistically different from the other segments. It can be a range of cut-off (or threshold) values, such as a confidence interval. It can be established based upon comparative groups, such as where association with risk of developing disease or presence of disease in one defined group is a fold higher, or lower, (e.g., approximately 2-fold, 4-fold, 8-fold, 16-fold or more) than the risk or presence of disease in another defined group. It can be a range, for example, where a population of subjects (e.g., control subjects) is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group, or into quartiles, the lowest quartile being subjects with the lowest risk and the highest quartile being subjects with the highest risk, or into n-quantiles (i.e., n regularly spaced intervals) the lowest of the n-quantiles being subjects with the lowest risk and the highest of the n- quantiles being subjects with the highest risk.

[0072] In some embodiments, the predetermined level is a level or occurrence in the same subject, e.g., at a different time point, e.g., an earlier time point.

[0073] Subjects associated with predetermined values are typically referred to as reference subjects. For example, in some embodiments, a control reference subject has not had a radiation exposure. An exposure reference subject is one who has had a radiation exposure.

[0074] Thus, in some cases the level of a biomarker in a subject being less than or equal to a reference level of the biomarker is indicative of a clinical status (e.g., no radiation exposure). In other cases the level of a biomarker in a subject being greater than or equal to the reference level of the biomarker is indicative of a radiation exposure. In some embodiments, the amount by which the level in the subject is the less than the reference level is sufficient to distinguish a subject from a control subject, and optionally is a statistically significantly less than the level in a control subject. In cases where the level of the biomarker in a subject being equal to the reference level of the biomarker, the “being equal” refers to being approximately equal (e.g., not statistically different).

[0075] The predetermined value can depend upon the particular population of subjects (e.g., human subjects) selected. For example, an apparently healthy population will have a different ‘normal’ range of levels of a biomarker than will a population of subjects which have, are likely to have, or are at greater risk to have, a disorder described herein. Accordingly, the predetermined values selected may take into account the category (e.g., sex, age, health, risk, presence of other diseases) in which a subject (e.g., human subject) falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art.

[0076] Treatment

[0077] In some embodiments, once it has been determined that a person has had a radiation exposure, e.g., an exposure of 1 Gy or 2Gy or higher, then a treatment, e.g., as known in the art or as described herein, can be administered.

[0078] For example, a treatment can include administration of granulocyte colony- stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GM- CSF), or KGF (keratinocite grow factor), e.g., filgrastim (G-CSF, Neupogen), sargramostim (GM-CSF, Leukine), or pegfilgrastim (peg-CSF, Neulasta) can be administered to a subject who has been exposed to a myelosuppressive dose (e.g., 2 Gy or above). Potassium iodide can be given to block thyroid uptake in subjects exposed to radioiodine. Prussian blue can be administered in subjects who have been exposed to radioactive cesium-137, rubidium-82, or thallium-201. The chelating agent dyethilene-triamine-pentacetic acid (DTP A), e.g., Ca-DTPA and Zn-DTPA, can be administered to promote the excretion of elements such as plutonium-239, yttrium-90, americium, and curium. Oral calcium or aluminium phosphate solutions can be used after exposure to strontium. Antibiotics, fresh blood, platelet transfusions, plasma, antiemetics, sedation, fluids (e.g., oral, subcutaneous, or IV fluids) and electrolytes, can also be administered. Hematopoietic stem cell transplant can be used in patients with doses over 4 or 5 Gy with severe marrow aplasia and no endogenous hematopoiesis. See, e.g., Lopez and Martin, Rep Pract Oncol Radiother. 2011 Jul; 16(4): 138-146.

[0079] Kits

[0080] Provided herein are kits, e.g., diagnostic kit for detecting radiation exposure in a patient comprising reagents for detecting or measuring one or more mRNAs or proteins. An exemplary kit for detecting exposure to radiation in a patient can include

[0081] (a) at least one test tape strip (i.e., a skin tape stripping tape strip as described herein),

[0082] (b) elution buffer for eluting mRNA or protein from the test strip, and (c) means for determining the presence or absence of any radiation specific biomarkers in the eluate.

[0083] In these methods and kits a control, for example, a protein like a keratin that is normally expressed in the epidermis can be included to control for the total amount of protein or MRNA obtained for the sample.

[0084] EXAMPLES

[0085] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0086] EXAMPLE 1 : Identification and validation of radiation biomarkers in human skin grafted mice using spatial profiling and immunostaining

[0087] Human adult abdominal skin samples were grafted onto the backs of non- obese diabetic / severe combined immunodeficient / IL-2 receptor y chain null mice (NSG mice from Jackson Laboratory, Bar Harbor, ME). These skin grafts retain viable populations of human keratinocytes, fibroblasts, and immune cells (FIGs. 1A- D), and maintain human blood vessels that anastomose with the mouse circulation. Once the grafted mice were fully healed, they were transferred to the small animal radiation research platform (SARRP) facility for irradiation. All experiments were approved by the Brigham and Women’s Hospital and the Dana Farber Cancer Institute Institutional Animal Care and Use Committees (IACUC protocol numbers: 10-069 (SARRP) and 2016N000591 (BWH)).

[0088] For irradiation of the skin grafts, the animals were anesthetized via isoflurane inhalation (1-3%), transferred to the imaging stage of the SARRP, and positioned on a robotic rotational platform of the SARRP instrument for the duration of each treatment. Radiotherapy was delivered by a small animal irradiator (Xstrahl) at 0, 1, 2, or 5 Gy per skin graft to 3mm depth according to the guidelines and approval by the DFCI IACUC. Animals that served as untreated controls also underwent the same procedure of placement on the irradiation platform. The short-term cohort skin grafts were harvested 24 hours post-irradiation. Also, to evaluate kinetics of potential biomarker upregulation for the purpose of validating the potential biomarker(s), an additional cohort was set up, whereby the skin grafts were irradiated at 5 Gy and were harvested at different time points from 16 hours to 7 days post-irradiation.

[0089] At indicated times after irradiation, the human skin grafts were harvested. Skin samples were fixed in formalin and paraffin embedded. Tissue sections were cut and spatial profiling was carried out using the NanoString CosMx spatial transcriptional profiler. Data were processed using Seurat v. 4.9.9.9045. Data from all sections were integrated with Harmony. Principal component analysis and nonlinear dimensional reduction were carried out using Uniform Manifold Approximation and Projection (UMAP).

[0090] Human skin grafted mice faithfully recapitulated known biologic events following radiation induced skin injury. Bulk RNA sequencing of human skin grafts 24 hours after 1 Gy irradiation demonstrated marked inflammation, including activation of antigen presenting cells (APC), NK cells and T cells (FIGs. 2A, B). T cells proliferated and migrated into the irradiated epidermis (FIG. 2C). Ingenuity Pathway Analysis (IP A) of grafts 3 months after 1 Gy irradiation demonstrated upregulation of TGFP signaling and three genes associated with TGFP-driven fibrosis (Fig. 2D, E). Fibrosis was evident 3 months after irradiation (Fig. 2F). These findings demonstrate that human skin grafted mice faithfully recapitulate both early and late events observed after radiation in both animal models and in the limited studies available in humans.12

[0091] Markers for each cluster were identified and cell types were assigned to clusters. Biomarkers differentially expressed in superficial keratinocytes between radiation dosage groups were identified using DEseq2; biomarkers with a Padj<0.05 were considered significant. The upregulated genes are listed in Tables 1-3 and example biomarkers are shown in FIGs 3-7. Table 1. Upregulated genes 1 Gy vs. 0 Gy (Log2FC>1.0)

[0092] Table 2. Upregulated genes 2 Gy vs. 0 Gy (Log2FC>1.0)

[0093]

[0094] Table 3. Upregulated genes 5 Gy vs. 0 Gy (Log2FC>0.5)

[0095] Protein expression of candidate genes was then measured by immunostaining as follows. To perform immunofluorescence staining, all tissue samples were fixed in formalin, embedded in paraffin, and sectioned and stored at room temperature until further use. Briefly, slides were deparaffinized by baking at 58°C for 20 min, cooled to room temperature, washed in xylene for 30 min, and rehydrated in 100%, 95%, 75% ethanol and deionized water. For antigen retrieval, slides were pressure cooked for 15 min at 110° C in citrate buffer, pH 6.0. Slides were then allowed to cool to room temperature, washed in PBS, blocked with protein block (Dako), and stained with primary antibodies diluted in protein blocking buffer at 4°C overnight and secondary antibodies for 30 min at room temperature, with two three-minute wash steps between antibodies. Slides were washed in TBS-tween and deionized water and mounted in ProLong Gold antifade reagent with DAPI (Invitrogen #P36935). Tissues were mounted and imaged immediately on a Mantra Quantitative Pathology Workstation using Mantra Snap 1.0 imaging software and analyzed with InForm image analysis software (PerkinElmer).

[0096] Primary antibodies used include: Keratin 6A (Proteintech &10590-1-AP, 1 :500), Keratin 16 (LS Bio #LS-c352434, 1 :200), Calprotectin / S100A9 (Abeam #ab22506, 1: 1000). Secondary antibodies used include goat anti-mouse IgGl-AF488, 555, IgG2a-AF594, IgG2b-AF555, and goat anti-rat IgG-AF594 antibodies, goat antimouse IgGM-AF594, goat anti-rabbit IgG-AF488, 555, 594, and 647 (Life Technologies, 1:2000). Immunostaining results from example biomarkers are shown in FIGs. 4-5. EXAMPLE 2: Biomarkers that discriminate irradiated from non-irradiated skin

[0097] Our studies identified four genes expressed by superficial keratinocytes that were upregulated by all (1, 2, 5 Gy) irradiation doses (FIGs. 4A-C): KRT5, KRT14, CTSG, and FABP5. Keratins 5 and 14 (KRT5, KRT14) are usually only expressed by basal (bottom layer) keratinocytes but can be induced in upper keratinocyte layers in inflammatory diseases such as psoriasis.4Immunostaining demonstrated that keratin 14 protein is induced and present at high levels in the superficial epidermis 24 hours after 1 Gy irradiation (FIG. 4C).

[0098] EXAMPLE 3: 1 Gy specific and 1 Gy / 2 Gy shared biomarkers

[0099] We identified two biomarkers selectively upregulated after 1 Gy irradiation (FOS, DUSP1), and eight biomarkers upregulated by both 1 and 2 Gy irradiation (FIGs. 5A-C)(S100A8, S100A9, KRT6A / B / C, KRT16, IFI27, CD74, HLA-DRB, and STAT1). Keratins 6 and 16 are not normally expressed in human skin but can be induced in the skin lesions of inflammatory diseases such as psoriasis.5We observed upregulation of both KRT6 and KRT16 RNA 24 hours after irradiation in superficial keratinocytes. Immunostaining for keratin 16 demonstrated that this protein is expressed throughout the epidermis, including by superficial keratinocytes, 24 hours after 1 Gy skin irradiation (FIG. 5C). Protein expression of keratin 16 is therefore a rapid biomarker of radiation exposure. SI 00 proteins are involved in danger signaling, and also thought to play key roles in initiating tissue fibrosis.6Immunostaining demonstrated high protein expression of S100A8 in superficial epidermal cells 24 hours after 1 Gy irradiation (FIG. 5C). S100 proteins are small, secreted molecules that should be easily eluted from tape strips and measurable by antibody-based methods.

[0100] EXAMPLE 4: 2 Gy and 5 Gy specific biomarkers

[0101] Spatial transcriptional profiling also identified five biomarkers selectively upregulated in superficial keratinocytes 24 hours after 2 Gy radiation (BST2, ISG15, MX1, IFI44L, and CXCL10; FIGs. 6A-B) and two biomarkers specifically upregulated after 5 Gy irradiation (KRT15 and NOTCHl, FIG. 7). The 2 Gy specific biomarkers are related to type I interferon and inflammatory signaling events; these are well studied molecules with commercially available antibodies that can be used to demonstrate their presence in superficial epidermal cells by tape stripping. Keratin 15 (KRT15) is a marker of epithelial stem cells that can be induced in superficial keratinocytes following severe DNA damage.7N0TCH1 is an epithelial signaling molecule involved in keratinocyte cell proliferation, differentiation, migration and apoptosis.8

[0102] EXAMPLE 5. Pediatric biomarkers

[0103] To determine whether some of the biomarkers described herein are applicable to pediatric subjects, experiments were conducted in which NSG mice were grafted with human neonatal foreskin. The grafts were selectively irradiated as described above, and then harvested at 24 hours. The results showed upregulation of Keratin 14 (FIG. 8), Keratin 16 (FIGs. 9A-B), and S100A8 (FIGs. 10A-B). Keratin 5 and 14 are basal layer keratin, normally never observed in the upper layers of skin. Keratins 6 and 16 are expressed in the lip and oral mucosae, but not in normal skin.

[0104] Keratin proteins are highly stable, cross-linked molecules, and once expressed within the skin, will be present at the protein level for 5-6 weeks as the affected skin grows out.13It is believed that this injury-associated protein expression should persist at the top layers of skin for 5-6 weeks after radiation.

[0105] References Cited:

[0106] 1 Hall, E. J. & Giaccia, A. J. Radiobiology for the radiologist. 6th edn, (Lippincott Williams & Wilkins, 2006).

[0107] 2 Messner, F. et al. Noninvasive evaluation of intragraft immune responses in upper extremity transplantation. Transplant international : official journal of the European Society for Organ Transplantation 34, 894-905, doi:l 0.1111 / tri.l 3854 (2021).

[0108] 3 Zou, M. et al. Rapid point-of-care testing for SARS-CoV-2 virus nucleic acid detection by an isothermal and nonenzymatic Signal amplification system coupled with a lateral flow immunoassay strip. Sens Actuators B Chem 342, 129899, doi: 10.1016 / j.snb.202L 129899 (2021).

[0109] 4. Thewes, M., Stadler, R., Korge, B. & Mischke, D. Normal psoriatic epidermis expression of hyperproliferati on-associated keratins. Archives of Dermatological Research 283, 465-471 (1991). 5. Zhang, X., Yin, M. & Zhang, L.-j. Keratin 6, 16 and 17 — Critical Barrier Alarmin Molecules in Skin Wounds and Psoriasis. Cells 8, 807 (2019).

[0110] 6. Gonzalez, L.L., Garrie, K. & Turner, M.D. Role of S100 proteins in health and disease. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1867, 118677 (2020).

[0111] 7. Bose, A., Teh, M.T., Mackenzie, I.C. & Waseem, A. Keratin kl 5 as a biomarker of epidermal stem cells. Int J Mol Sci 14, 19385-19398 (2013).

[0112] 8. Gratton, R., et al. Pleiotropic Role of Notch Signaling in Human Skin Diseases. Int J Mol Sci 21(2020).

[0113] 9. Watanabe, R., et al. Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells. Science Translational Medicine 7, 279ra239 (2015).

[0114] 10. Kyoizumi, S., Suzuki, T., Teraoka, S. & Seyama, T. Radiation sensitivity of human hair follicles in SCID-hu mice. Radiation research 149, 11-18 (1998).

[0115] 11. Gehad, A., et al. A primary role for human central memory cells in tissue immunosurveillance. Blood Adv 2, 292-298 (2018).

[0116] 12. Martin, M., Lefaix, J. & Delanian, S. TGF-betal and radiation fibrosis: a master switch and a specific therapeutic target? International journal of radiation oncology, biology, physics 47, 277-290 (2000).

[0117] 13. Weinstein, G.D., McCullough, J.L. & Ross, P. Cell proliferation in normal epidermis. The Journal of investigative dermatology 82, 623-628 (1984).

[0118] OTHER EMBODIMENTS

[0119] It is to be understood that while the invention has been descnbed in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method comprising:(a) providing a sample comprising cells of the epidermal layer of the skin of a subject,(b) optionally eluting mRNA or protein from the sample, and(c) detecting the presence of one or more, optionally two, three, four or more, radiation specific mRNA or protein biomarkers in the sample, optionally in the eluate, wherein the one or more radiation specific mRNA or protein biomarkers are listed in Table 1, 2, or 3, or are selected from or comprise:(i) one, two, three, or all four of KRT5, KRT14, CTSG, and FABP5;(ii) one or two of FOS or DUSP1;(iii) one, two, three, four or more of S100A8, S100A9, KRT6A / B / C, KRT16, IFI27, CD74, HLA-DRB, and STAT1;(iv) one, two, three, four or more of BST2, ISG15, MX1, IFI44L and CXCL10;(v) one or both of KRT15 and N0TCH1, or(vi) any combination thereof.

2. The method of claim 1, wherein the sample is obtained by tape stripping.

3. A diagnostic kit for detecting radiation exposure in a patient comprising reagents, optionally antibodies, for detecting or measuring one or more, optionally two, three, four or more, mRNAs or proteins, wherein the one or more mRNA or proteins are listed in Table 1, 2, or 3, or are selected from or comprise:(i) one, two, three, or all four of KRT5, KRT14, CTSG, and FABP5;(ii) one or two of FOS or DUSP1;(iii) one, two, three, four or more of S100A8, S100A9, KRT6A / B / C, KRT16, IFI27, CD74, HLA-DRB, and STAT1;(iv) one, two, three, four or more of BST2, ISG15, MX1, IFI44L and CXCL10;(v) one or both of KRT15 and N0TCH1, or(vi) any combination thereof.

4. The kit of claim 3, comprising (a) at least one test strip, (b) elution buffer for eluting mRNA or protein from the test strip, and (c) means for determining the presence or absence of any radiation specific biomarkers in the eluate.A method for determining exposure to radiation in a subject, the method comprising:(a) providing a sample of the epidermal layer of the skin of the subject,(b) optionally eluting mRNA or protein from the sample, and(c) detecting the presence or level of one or more, optionally two, three, four or more, radiation specific mRNA or protein biomarkers in the sample, optionally in the eluate, wherein the one or more radiation specific mRNA or protein biomarkers are listed in Table 1, 2, or 3, or are selected from or comprise:(i) one, two, three, or all four of KRT5, KRT14, CTSG, and FABP5;(ii) one or two of FOS or DUSP1;(iii) one, two, three, four or more of S100A8, S100A9, KRT6A / B / C, KRT16, IFI27, CD74, HLA-DRB, and STAT1;(iv) one, two, three, four or more of BST2, ISG15, MX1, IFI44L and CXCL10;(v) one or both of KRT15 and N0TCH1, or(vii) any combination thereof, and comparing the presence or level of the one or more biomarkers to a reference level, wherein the presence or level of the biomarkers above the reference indicates that the subject has been exposed to radiation.

5. The method of claim 5, wherein:(i) the presence of one, two, three, or all four of KRT5, KRT14, CTSG, and FABP5 indicates that radiation exposure has occurred;(ii) the presence of one or both of FOS and DUSP1 indicates that 1 Gy radiation exposure has occurred;(iii) the presence of one, two, three, four or more of S100A8, S100A9, KRT6A / B / C, KRT16, IFI27, CD74, HLA-DRB, and STAT1 indicates that 1 Gy or 2 Gy radiation exposure has occurred;(iv) the presence of one, two, three, four or more of BST2, ISG15, MX1, IFI44L and CXCL10 indicates that 2 Gy radiation exposure has occurred;(v) the presence of one or both of KRT15 and N0TCH1 indicates that 5 Gy radiation exposure has occurred.

6. The method of claim 6, further comprising administering a treatment for radiation exposure to a subject who has been exposed to radiation.

7. The method of claim 7, comprising administering a treatment for radiation to a subject who has been exposed to at least 2 Gy of radiation.

8. The method of claim 8, wherein the treatment comprises administration of one or more of granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or KGF (keratinocite grow factor), potassium iodide, Prussian blue, dyethilene-triamine-pentacetic acid (DTPA), optionally Ca-DTPA or Zn-DTPA, oral calcium or aluminium phosphate solutions, antibiotics, fresh blood, platelet transfusions, plasma, antiemetics, sedation, or fluids (optionally oral, subcutaneous, or IV fluids) and electrolytes.

9. The method of any of the preceding claims, wherein the subject is a human.

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