WO2025106794A1

WO2025106794A1 - Assessment of somatic mutation burden and patient selection

Info

Publication number: WO2025106794A1
Application number: PCT/US2024/056093
Authority: WO
Inventors: Timothy Looney
Original assignee: Quest Diagnostics Investments LLC
Current assignee: Quest Diagnostics Investments LLC
Priority date: 2023-11-16
Filing date: 2024-11-15
Publication date: 2025-05-22
Anticipated expiration: 2026-05-16

Abstract

Described herein are methods of detecting somatic mutation burden in a sample, comprising obtaining one or more samples comprising skin cells from a subject with cancer, a subject suspected of having an autoimmune disease, or a subject at risk of an infectious disease, wherein the subject is a candidate for treatment with immunotherapy; sequencing DNA from the one or more samples; and detecting a level of somatic mutation burden in each of the one or more samples.

Description

ASSESSMENT OF SOMATIC MUTATION BURDEN AND PATIENT SELECTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. U.S. 63/599,906, filed November 16, 2023, and U.S. Provisional Patent Application No. U.S. 63/552,552, filed February 12, 2024, both of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

[0002] Described herein, are methods of measuring somatic mutation burden in subjects as an indicator to predict immunotherapy response and select patients for treatment.

BACKGROUND

[0003] Immunotherapy treatments are one of the most cost-effective measures available to the health care industry for the prevention and treatment of disease. However, only a subset of patients derive clinical benefit from such treatment. Therefore, there remains an urgent need to identify patients who will respond favorably to immunotherapy.

[0004] Numerous studies have demonstrated an inverse relationship between environmental UV exposure and lifetime cancer risk. Described herein is a previously unappreciated type of abscopal response to radiation, a consequence of which is that mutation burden of phenotypically normal tissue can be exploited to serve as an indicator of adaptive immune health and thus the likelihood of success of an immunotherapy regimen. This disclosure describes methods for measuring and applying somatic mutation burden to quantify adaptive immune health, thereby providing a means to predict immunotherapy response and select cancer patients for treatment. Further disclosed are applications of the method for the treatment and prevention of infectious and autoimmune disease.

SUMMARY

[0005] The present disclosure provides methods of detecting somatic mutation burden in a sample, comprising: a) obtaining one or more samples comprising skin cells from a subject with cancer that is a candidate for treatment with immunotherapy; b) sequencing DNA from the one or more samples; and c) detecting a level of somatic mutation burden in each of the one or more samples. [0006] Additionally, the present disclosure provides methods of detecting somatic mutation burden in a sample, comprising: a) obtaining one or more samples comprising skin cells from a subject having or suspected of having an autoimmune disease; b) sequencing DNA from the one or more samples; and c) detecting a level of somatic mutation burden in each of the one or more samples.

[0007] Additionally, the present disclosure provides methods of detecting somatic mutation burden in a sample, comprising: a) obtaining one or more samples comprising skin cells from a subject that has or is at risk of contracting an infectious disease; b) sequencing DNA from the one or more samples; and c) detecting a level of somatic mutation burden in each of the one or more samples.

[0008] Additionally, the present disclosure provides methods of selecting a subject for treatment, comprising: a) obtaining one or more samples from a subject; b) detecting a somatic mutation burden for each of the one or more samples; and c) selecting the subject for treatment based on the one or more somatic mutation burden values. In some embodiments, the one or more samples comprises a section of a tissue. In some embodiments, the one or more samples comprises an image of a section of a tissue. In some embodiments, the one or more somatic mutation burden values are determined by image analysis of tissue. In some embodiments, image analysis is performed using Al-assisted image analysis. In some embodiments, image analysis is performed by immunohistochemistry or FISH.

[0009] In some embodiments, obtaining the sample(s) comprises tape stripping, stickers, adhesive collection methods.

[0010] In some embodiments, rein detecting the level of somatic mutation burden in each of the one or more samples comprises detecting one or more UV-mediated dinucleotide or triplet mutations.

[0011] In some embodiments, the one or more samples comprise skin cells from one or more regions of the body subjected to UV exposure and one or more control samples, and determining the somatic mutation burden comprises: a) sequencing a first sample comprising skin cells from a first region of the body subjected to UV exposure to generate a first set of sequence reads; b) sequencing one or more control samples to generate a second set of sequence reads; and c) aligning the first and second sets of sequence reads to determine the somatic mutation burden of the first sample. [0012] In some embodiments, the foregoing methods may further comprise generating a somatic mutation burden score for the subject based on the somatic mutation burden. In some embodiments, generating a somatic mutation burden score comprises weighting the somatic mutation burden for the one or more samples according to the sample location and area of skin represented by the sample.

[0013] In some embodiments, the one or more samples comprise skin cells from one or more regions of the body subjected to chronic UV exposure, one or more regions of the body subjected to intermittent UV exposure, or a combination thereof.

[0014] In some embodiments, the one or more control samples comprises skin cells from one or more regions of the body subjected to no UV exposure, skin cells from one or more regions of the body subjected to minimal UV exposure, or a combination thereof. In some embodiments, the one or more regions of the body subjected to chronic UV exposure are selected from forehead, neck, forearms, hands, face, or a combination thereof. In some embodiments, the one or more regions of the body subjected to intermittent UV exposure are selected from torso, back, thighs, ankles, or a combination thereof. In some embodiments, the one or more regions of the body subjected to minimal or no UV exposure is selected from buttocks, armpit, inner arm adjacent to armpit, peripheral blood cells, buccal swab, or a combination thereof.

[0015] In some embodiments, the one or more samples are non-cancerous samples from the subject. In some embodiments, the one or more samples comprise skin cells. In some embodiments, the one or more samples comprise lung cells. In some embodiments, the one or more samples comprise intestinal epithelial cells. In some embodiments, the one or more samples comprise extracellular nucleic acids. In some embodiments, the one or more samples comprise cell-free DNA. In some embodiments, the extracellular nucleic acids or cell-free DNA originates from one or more of skin cell, lung cells, and intestinal epithelial cells.

[0016] In some embodiments, the one or more samples comprises a control sample. In some embodiments, the control sample comprises one or more regions of the body subjected to minimal or no UV exposure.

[0017] In some embodiments, obtaining is performed by a non-invasive method. [0018] In some embodiments, determining the somatic mutation burden comprises extracting genomic DNA (gDNA), mitochondrial DNA, or RNA from the one or more samples.

[0019] In some embodiments, determining the somatic mutation burden comprises preparing sequencing libraries from the one or more samples.

[0020] In some embodiments, determining the somatic mutation burden comprises target enrichment. In some embodiments, target enrichment comprises enrichment of target sequences by PCR, hybrid capture, on-sequencer enrichment, or a combination thereof.

[0021] In some embodiments, determining the somatic mutation burden comprises performing next-generation sequencing (NGS), high-fidelity sequencing, or digital droplet PCR.

[0022] In some embodiments, the treatment is an infectious disease treatment or prophylaxis thereof. In some embodiments, the infectious disease is HIV.

[0023] In some embodiments, the treatment is a non-infectious disease treatment or prophylaxis thereof. In some embodiments, the non-infectious disease is cancer or an autoimmune disease.

[0024] In some embodiments, the treatment is anti-aging treatment. In some embodiments, the anti-aging treatment prevents age-related cellular damage.

[0025] In some embodiments, the treatment comprises immunotherapy. In some embodiments, the immunotherapy comprises immune checkpoint inhibitors, T-cell transfer therapy, monoclonal antibodies, vaccines, and immune system modulators.

[0026] Additionally, the present disclosure provides methods for determining the prognosis of a subject diagnosed as having a disease comprising: a) obtaining one or more samples from a subject; b) detecting a somatic mutation burden for each of the one or more samples; and c) identifying the subject as having poor prognosis when the somatic mutation burden is below a threshold, or having a favorable prognosis when the somatic burden is above a threshold.

[0027] Additionally, the present disclosure provides methods of determining the risk of a disease in a subject, comprising: a) obtaining one or more samples from a subject; b) detecting a somatic mutation burden for each of the one or more samples; and c) identifying the subject as having high-risk of a disease when the somatic mutation value is below a threshold.

[0028] Additionally, the present disclosure provides methods for predicting a response to a treatment, comprising: a) obtaining one or more samples from a subject; b) detecting a somatic mutation burden for each of the one or more samples; and c) predicting the subject as having a favorable response to the treatment when the somatic mutation burden score is above a threshold.

[0029] In some embodiments, the one or more samples comprise skin cells from one or more regions of the body subjected to UV exposure and one or more control samples, and determining the somatic mutation burden comprises: a) sequencing a first sample comprising skin cells from a first region of the body subjected to UV exposure to generate a first set of sequence reads; b) sequencing a control sample to generate a second set of sequence reads; and c) aligning the first and second sets of sequence reads to determine the somatic mutation burden of the first sample.

[0030] In some embodiments, the foregoing methods may further comprise generating a somatic mutation burden score for the subject based on the somatic mutation burden. In some embodiments, generating a somatic mutation burden score comprises weighing the somatic mutation burden for the one or more samples according to the sample location and area of skin represented by the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Fig. 1 shows UV-derived skin neoantigens drive adaptive immune education. Solar UV-derived DNA damage gives rise to neoantigens in skin cells, resulting in the stimulation of antigen-specific T and B cells. Over time, such immune stimulation drives adaptive immune diversification, yielding superior protection against infectious disease and cancer, and augmenting response to immunotherapy. Immune cell network image adapted from Nestle F, et al. Nat Rev Immunol (2009). https://doi.org/10.1038/nri2622

[0032] Fig. 2 shows Non-invasive measurement of skin mutation burden (SMB) as a biomarker. Skin cells are collected from one or more sun-exposed regions of the body and optionally a sun protected region (armpit) via non-invasive dermal tape. For each sampled site, DNA is extracted, then analyzed via low pass whole genome sequencing (WGS) to quantify the frequency of UV damage-specific pyrimidine dimer mutations (COTT) at each sampled site. The mutation values from the one or more sun-exposed regions are compared with the sun-protected region, then combined to produce a mutation burden score reflecting the extent of UV-driven adaptive immune education.

[0033] Fig. 3 shows non-invasive collection of skin cells via dermal tape stickers. (A) Image of a panel of 10 D-Squame-101 14 mm diameter stickers. (B) A series of stickers are applied to a region of interest under firm pressure, then slowly removed in a constant and fluid motion. DNA extracted from the stickers is analyzed to quantify the skin mutation burden.

[0034] Fig. 4 shows a bioanalyzer trace of an exemplary NGS library prepared using DNA extracted from skin cells collected via dermal tape.

[0035] Fig. 5 shows insert lengths of mapped reads for a set of representative libraries. Each trace indicates the insert profile for a single library. Insert lengths were determined upon mapping of paired next generation sequencing reads to the human reference genome. gDNA extracted from dermal tape was subjected to enzymatic fragmentation prior to library preparation. Figure produced using MultiQC.

[0036] Fig. 6 shows the number of COTT dinucleotides detected in samples derived from dermal tape-based collection of skin cells from the forearm (sun exposed) and inner arm adjacent to armpit (sun protected) of healthy donors, and peripheral blood samples from healthy donors. gDNA was extracted from each sample, then interrogated by next-generation sequencing to determine the presence of UV damage-associated COTT dinucleotide mutations. Fitting expectation, samples derived from the sun exposed region tend to have a greater number of COTT mutations than sun protected skin samples, while blood samples have the fewest mutations.

[0037] Fig. 7 shows normalized mutation burden values for a set of samples derived from the forearm (sun exposed), inner arm adjacent to armpit (sun protected), and peripheral blood from healthy donors. gDNA was extracted from samples, then subjected to next-generation sequencing to determine the number of UV-characteristic CC>TT dinucleotides in each sample. Finally, the number of CC>TT mutations was normalized by the number of aligned sequence reads obtained for each sample. [0038] Fig. 8 shows distribution of mutation burden scores in a healthy donor cohort. The mutation burden score was defined as the number of COTT mutations detected in the sun exposed sample minus the number of mutations detected in the sun protected sample from the same donor. Horizontal lines indicate score thresholds used in classifying a donor as mutation burden low, medium, or high. The upper tertile of the cohort possesses a mutation burden score > 10. In some embodiments of the invention, such individuals are classified as skin mutation burden high and have a greater probability of responding to cancer immunotherapy, with a longer overall survival than individuals having a mutation burden score in the lowest tertile.

[0039] Fig. 9 shows exemplary bioinformatics workflow. Following sample collection and library preparation, libraries are sequencing using a next generation sequencing device. Reads are aligned to the human genome (e.g. via bwa-mem, Li H. (2013) Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv: 1303.3997v2 .), and mapped reads are filtered to select those mapping with high confidence to a single location in the genome (e.g. MQ>30). Aligned reads are further filtered to select reads mapping to high confidence regions of the genome, defined as those where mapping and sequencing errors are low. Aligned, filtered reads are further processed to tally the number of “qualified” COTT dinucleotides having a sequencing base quality score above a given threshold (e.g. phred quality score > 30 for each base). Qualified COTT mutations are further filtered to eliminate those overlapping with SNPs or blacklisted regions of the genome (e.g. sites of elevated error), then the remaining mutations are tallied. The mutation burden of the sample is determined as the number of COTT mutations normalized according to sequencing depth. Finally, the site-specific mutation burden value is optionally combined with mutation burden values from other skin sites and patient metadata to produce a mutation burden score. In some embodiments of the invention, the assay further reports the genotype of the individual.

DETAILED DESCRIPTION

[0040] Numerous studies over the past century have reported an inverse relationship between lifetime solar UV exposure and all-cancer incidence and mortality. This seemingly paradoxical observation reflects two distinct trends: (1) a negative correlation between UV exposure and the incidence and mortality of a broad range of solid tumors responsible for the great majority of human cancer, and (2) a weak positive correlation between UV exposure and the incidence and mortality of comparatively rare skin cancers. Fascinatingly, the same studies suggest that early life UV exposure results in a lifelong reduction in cancer risk, implying the action of some type of long-lived anti-cancer factor.

[0041] This relationship was hypothesized to reflect the action of photosynthesized vitamin D, spurring interest in the exploration of vitamin D pathway agonists as anti-cancer therapeutics. However, subsequent clinical trials have failed to demonstrate the expected anticancer properties of vitamin D, and to date the US Preventive Service Task Force finds insufficient evidence to warrant vitamin D supplementation for the prevention of cancer.

[0042] Rather than a consequence of vitamin D, this relationship correlation reflects the action of an immune-mediated abscopal response to radiation: UV radiation strikes the skin, damaging skin cell DNA and generating a multitude of neoantigens. These neoantigens are presented to the robust network of immune cells within the skin in a manner facilitated by the upregulation of cytokines, chemokines, and antigen presentation as part of the radiation damage response, ultimately resulting in the stimulation of antigen-specific T and B cells. Over time, this UV-mediated immune education drives immune repertoire diversification and superior adaptive immune responses to antigen challenge (Fig. 1). The response is termed abscopal given that radiation strikes the skin, but the effect is observed throughout the body.

[0043] Such UV-mediated immune education provides several advantages to the organism, foremost of which, from an evolutionary standpoint, is improved resistance to infectious disease. Infectious disease has historically been the primary cause of mortality, particularly for children and those of reproductive age. UV-mediated immune education provides a means to strengthen and diversify adaptive immunity - even within genetically homogenous, isolated populations - without the risks and limitations inherent to building immunity through natural infection. The consequence of UV-mediated immune education is lower mortality from infectious disease. This mechanism explains the strong selection for mutationenhancing light skin pigmentation in humans who settled UV-poor northerly latitude regions following the out-of-Africa migration, an occurrence which is otherwise poorly explained as an adaptation solely to facilitate vitamin D synthesis. The insight is supported by observations that phenotypically normal human skin may harbor many neoantigens in a manner dependent on underlying skin pigmentation and lifestyle, and that skin neoantigens are able to efficiently elicit systemic T cell responses, as evidenced by the efficacy of skin scarification as a vaccination modality. Beyond reducing the severity of infectious disease, UV-mediated immune education may be powerful enough to elicit sterilizing immunity against a previously unencountered pathogen in a subset of the population.

[0044] A secondary advantage of such immune education is improved anti-cancer responses. It may be considered secondary in that it likely has been a minor driver of selection for light pigmented skin, given that natural selection inefficiently acts upon traits affecting diseases that tend to occur after reproductive age, as is the case for most cancer. UV-mediated anticancer immunity may explain the persistent racial disparities in US cancer incidence and mortality, particularly the elevated incidence of breast, colon, and prostate cancer - amongst the most UV-sensitive cancers as determined by ecological studies - in Black versus White Americans. It may also explain the growing body of evidence suggesting reduced efficacy of immune checkpoint inhibition immunotherapy (ICI) in Black versus White Americans.

[0045] Taken further, those whose skin has accumulated the greatest number of mutations - and thus have the most developed anti-cancer adaptive immunity - are also those most likely to respond to cancer immunotherapy. First, cutaneous immune-related adverse events (irAEs) associate with favorable response to ICI. Cutaneous irAEs may reflect immune recognition of UV-derived neoantigens in phenotypically normal skin following loss of tolerance due to ICI, with the likelihood of a cutaneous irAE proportional to the neoantigen burden at a given site; this would explain the tendency for cutaneous irAEs to involve sun exposed parts of the body such as extremities. Second, current or previous smokers respond more favorably to immunotherapy than never-smokers, across all cancer types and treatment modalities. Analogous to UV mutation of skin, smoking generates an abundance of neoantigens in the normal lung epithelium which are presented to resident immune cells and strengthen adaptive immunity. Third, tumor mutation burden (TMB) as a predictive biomarker of ICI response has divergent predictive value across cancer types and is most predictive of response for cancers related to chronic mutagenic exposure such as non-small cell lung cancer and melanoma. For such mutagen exposure-driven cancers, TMB inadvertently reflects the mutation burden of the mutagen-exposed normal tissue, making the reading more indicative of the organism-wide extent of adaptive immune education. Lastly, individuals harboring germline loss of function mutations in DNA damage response genes such as BRCA2 tend to respond more favorably to immunotherapy; analogous to smoking, a germline mutation to a DNA damage response gene increases the organism-wide mutation burden, leading to enhanced adaptive immune education, and superior response to immunotherapy. [0046] Recognizing the role of the skin as a driver of adaptive immunity reveals new opportunities to advance human health. First, skin mutation burden (SMB) may serve as a predictive and prognostic biomarker for cancer immunotherapy, enabling first-line immunotherapy for cancers where current biomarkers fail to identify most responders. For vaccine and drug trials, measurement of SMB during patient enrollment will help to ensure that treatment and control arms are balanced, thereby eliminating a hidden and potentially confounding variable. For genome-wide association studies, methods that simultaneously capture genotype and SMB will facilitate discovery of causative variants, given that SMB values can be used to correct for an important source of environmental variation. This disclosure describes methods for measuring and applying somatic mutation burden to quantify adaptive immune health, thereby providing a means to predict immunotherapy response. Further disclosed are applications of the method to the treatment and prevention of infectious and autoimmune disease. For example, in one embodiment, the present disclosure provides methods for non-invasive, dermal tape-based measurement of SMB.

I. Definitions

[0047] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. The term “about” is used herein to mean plus or minus ten percent (10%) of a value. For example, “about 100” refers to any number between 90 and 110.

[0048] It is understood that aspects and variations of the invention described herein include “consisting” and/or “consisting essentially of’ aspects and variations.

[0049] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally or topically. Administration includes self-administration and the administration by another.

[0050] The terms “cancer” or “tumor” are used interchangeably and refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell. As used herein, the term “cancer” includes premalignant, as well as malignant cancers. Nonlimiting examples of cancers may include melanoma, renal cell carcinoma, bladder cancer, head and neck cancer, sarcoma, endometrial cancer, gastric cancer, hepatobiliary cancer, colorectal cancer, esophageal cancer, pancreatic cancer, mesothelioma, ovarian cancer, or breast cancer.

[0051] As used herein, a “control” is an alternative sample used in an experiment for comparison purpose. A control can be “positive” or “negative.” For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed. In embodiments, a control may be a sample that has not been exposed to UV radiation or has been exposed to minimal UV radiation. In embodiments, a control sample may be obtained from a region of the body that has been exposed to minimal or no UV radiation. For example, in some embodiments, a control sample may comprise epidermal cells obtained from the buttocks or armpit inner arm adjacent to armpit, peripheral blood cells, buccal swab, or a combination thereof.

[0052] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a “therapeutically effective amount” of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.

[0053] The term “mutation” herein refers to a change introduced into a reference sequence, including, but not limited to, substitutions, insertions, deletions (including truncations) relative to the reference sequence. Mutations can involve large sections of DNA (e.g., copy number variation). Mutations can involve whole chromosomes (e.g., aneuploidy). Mutations can involve small sections of DNA. Examples of mutations involving small sections of DNA include, e.g., point mutations or single nucleotide polymorphisms (SNPs), single nucleotide variants (SNVs), multiple nucleotide polymorphisms, insertions (e.g., insertion of one or more nucleotides at a locus but less than the entire locus), multiple nucleotide changes, deletions (e.g., deletion of one or more nucleotides at a locus), inversions (e.g., reversal of a sequence of one or more nucleotides), an genomic rearrangements (e.g., deletions, duplications, inversions, and translocations). In some embodiments, the mutations may include dinucleotide or triplet mutations. For example, in some embodiments, the mutation may include CC to TT mutations. In some embodiments, the reference sequence is a parental sequence. In some embodiments, the reference sequence is a reference human genome, e.g., hl9. In some embodiments, the reference sequence is derived from a non-cancer (or nontumor) sequence. In some embodiments, the mutation may refer to alterations to normal patterns of gDNA methylation (e.g., hypomethylation of a region expected to be hypermethylated in the assayed tissue type, or vis versa). Examples of types of methylation that may be assayed include 5’methyl-cytosine and 5 ’hydroxymethyl cytosine. In some embodiments, a mutation may comprise an ectopically expressed gene. For example, regarding the analysis of skin, detecting a mutation may comprise detecting a signature of transcription from a gene known to be exclusively expressed in another organ (e.g., testis, brain, liver). In some embodiments, the mutation is inherited. In some embodiments, the mutation is spontaneous or de nova. In some embodiments, the mutation is a “somatic” mutation or variant.

[0054] The term “somatic mutation burden” or “SMB” herein refers to the level, e.g., number, of an alteration (e.g., one or more alterations, e.g., one or more somatic alterations) per a preselected unit (e.g., per megabase; Mb) in a predetermined set of genomic regions in a non-tumor sample. Somatic mutation burden can be measured, e.g., on a whole genome or exome basis, or on the basis of a subset of genome or exome. In certain embodiments, the somatic mutation burden measured on the basis of a subset of genome or exome can be extrapolated to determine a whole genome or exome mutation burden.

[0055] The term “tumor mutation burden” or “TMB” herein refers to the level, e.g., number, of an alteration (e.g., one or more alterations, e.g., one or more somatic alterations) per a preselected unit (e.g., per megabase; Mb) in a predetermined set of genes (e.g., in the coding regions of the predetermined set of genes) in a tumor. Tumor mutation burden can be measured, e.g., on a whole genome or exome basis, or on the basis of a subset of genome or exome. In certain embodiments, the tumor mutation burden measured on the basis of a subset of genome or exome can be extrapolated to determine a whole genome or exome mutation burden.

[0056] In certain embodiments, the tumor mutation burden is measured in a tumor sample (e.g., a tumor sample or a sample derived from a tumor), from a subject. In certain embodiments, the tumor mutation burden is expressed as a percentile, e.g., among the mutation burden in samples from a reference population. In certain embodiments, the reference population includes patients having the same type of cancer as the subject. In other embodiments, the reference population includes patients who are receiving, or have received, the same type of therapy, as the subject. In certain embodiments, the TMB correlates with the whole genome or exome mutation load.

[0057] The term “subject”, “patient”, or individual, herein used interchangeably, refers to an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the subject, patient, or individual is a human.

[0058] The term “sample” herein refers to a biological sample obtained or derived from a source of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cellcontaining body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and/or excretions; and/or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc. In some embodiments, the sample may be a “non-invasive sample”, meaning a sample collected using a non-invasive method or device.

[0059] The term “non-invasive” herein refers to a method or device for collecting a sample that poses minimal risk to an individual. For example, a non-invasive method of collecting a biological sample may include, but is not limited to a venipuncture, a swab, a collection of fluid such as sputum or urine or a biopsy or other similar procedure. In some embodiments described herein, a non-invasive device for collecting a skin sample may include an adhesive collection device, sticker, strip, or patch.

[0060] The term “target sequence” herein refers to a selected target polynucleotide, e.g., a sequence present in a cfDNA molecule, whose presence, amount, and/or nucleotide sequence, or changes in these, are desired to be determined. Target sequences are interrogated for the presence or absence of a somatic variant. The target polynucleotide can be a region of gene associated with a disease. In some embodiments, the region is an exon. The disease can be cancer.

[0061] The terms “treat,” “treatment,” and “treating” refer to the reduction or amelioration of the progression, severity, and/or duration of a proliferative disorder e.g., cancer, or the amelioration of a proliferative disorder resulting from the administration of one or more therapies. [0062] The term “derived from” encompasses the terms “originated from,” “obtained from,”

“obtainable from,” “isolated from,” and “created from,” and generally indicates that one specified material (e.g., a biological sample) finds its origin in another specified material or individual or has features that can be described with reference to another specified material.

[0063] The term “detect” or “detection” when used with respect to a target nucleic acid sequence means the discovery or determination of its presence, absence, level or amount. The expressions “detecting the presence or absence” and related expressions include qualitative and quantitative detection.

[0064] The term “sequence read” or simply “read” herein refers to sequence information of a nucleic acid fragment obtained through a sequencing assay, such as a next generation sequencing (NGS) assay. In some embodiments, a sequence read refers to data representing a sequence of nucleotide bases that were measured using a clonal sequencing method. Clonal sequencing may produce sequence data representing single, or clones, or clusters of one original DNA molecule. A sequence read may also have associated quality score at each base position of the sequence indicating the probability that nucleotide has been called correctly.

[0065] The term “clinical decision” herein refers to any decision to take or not take an action that has an outcome that affects the health or survival of an individual. In the context of cancer diagnosis, a clinical decision may refer to a decision to start or change a treatment plan. A clinical decision may also refer to a decision to conduct further testing or to take actions to mitigate an undesirable phenotype.

[0066] The tern “risk” or “risk of a disease” herein refers to a probability of a subject or a patient to develop or arrive at a certain disease outcome.

[0067] The term “prognosis” herein refers to any aspect of the course of a disease or condition either in the absence or presence of treatment. A prognosis may be determined based on information including, but not limited to, the average life expectancy of a patient, the likelihood that a patient will survive for a given amount of time (e.g., 6 months, 1 year, 5 years, etc.), the likelihood that a patient will be cured of a disease, the likelihood that a patient’s disease will respond to a particular therapy (wherein response may be defined in any of a variety of ways). [0068] A “spacer” may consist of a repeated single nucleotide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,

10, or more of the same nucleotide in a row), or a sequence of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. A spacer may comprise or consist of a specific sequence, such as a sequence that does not hybridize to any target sequence in a sample. A spacer may comprise or consist of a sequence of randomly selected nucleotides.

[0069] The phrases “substantially similar” and “substantially identical” in the context of at least two nucleic acids typically means that a polynucleotide includes a sequence that has at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% sequence identity, in comparison with a reference (e.g., wild-type) polynucleotide or polypeptide. Sequence identity may be determined using known prog such as BLAST, ALIGN, and CLUSTAL using standard parameters. (See, e.g., Altshul et al. (1990) J. Mol. Biol. 215:403- 410; Henikoff etal. (1989) Proc. Natl. Acad. Sci. 89: 10915; Karin etal. (1993) Proc. Natl. Acad. Sci. 90:5873; and Higgins et al. (1988) Gene 73:237). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. Also, databases may be searched using FASTA (Person et al. (1988) Proc. Natl. Acad. Sci. 85:2444-2448.) In some embodiments, substantially identical nucleic acid molecules hybridize to each other under stringent conditions (e.g., within a range of medium to high stringency).

11. Practical Applications of Somatic Mutation Burden

[0070] The present disclosure provides multiple novel practical applications based on assessment and/or quantitation of somatic mutation burden in samples of a subject, as well as methods of detecting somatic mutation burden in samples (e.g., skin samples, lung samples, intestinal samples) obtained from particular populations.

[0071] For example, the present disclosure provides methods of detecting somatic mutation burden in a sample, comprising: a) obtaining one or more samples comprising skin cells from a subject with cancer that is a candidate for treatment with immunotherapy; b) sequencing DNA from the one or more samples; and c) detecting a level of somatic mutation burden in each of the one or more samples. The subject may be considered a candidate for treatment with an immunotherapy because he or she has been diagnosed with a cancer, particularly a cancer that is responsive to immunotherapy. Such cancers may include, but are not limited to, cervical cancer, head and neck cancer, non-small cell lung cancer (NSCLC), melanoma, bladder cancer, kidney cancer, breast cancer, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoma, stomach cancer, brain cancer, and leukemia. A candidate for treatment with immunotherapy may have previously been treated with another treatment modality (e.g., surgery, radiation, chemotherapy, or a previous immunotherapy regimen) that was either non-responsive or after which the cancer has recurred. Alternatively, the immunotherapy may be the first-line treatment. In either case, the immunotherapy may be administered alone, concurrently, or in series with one or more additional therapeutics.

[0072] It was unexpectedly found that the level of somatic mutation burden in the skin positively correlates with adaptive immune function (i.e., is a biomarker of adaptive immunity), and therefore, a high level of somatic mutation burden is indicative of strong adaptive immune function. Candidates with high somatic mutation burden may therefore be more responsive to immunotherapy for treating a given cancer, thus making a preliminary screen for SMB in a subject’s skin a way of improving treatment outcomes by selecting the best patients for treatment.

[0073] Because a subject’s adaptive immune function can have a significant impact on the success of treatment, particularly treatment with an immunotherapy, the present disclosure also provides methods of selecting a subject for treatment, comprising: a) obtaining one or more samples from a subject; b) detecting a somatic mutation burden for each of the one or more samples; c) selecting the subject for treatment when the somatic mutation burden is above a threshold. Alternatively, if a subject’s somatic mutation burden is below a threshold, the subject may be selected for a non-immunotherapeutic treatment (e.g., chemotherapy). Suitable immunotherapies for the purposes of the disclosed methods include, but are not limited to, immune checkpoint inhibitors, such as Atezolizumab (Tecentriq), Avelumab (Bavencio), Dostarlizumab (Jemperli), Durvalumab (Imfinzi), Ipilimumab (Yervoy), Nivolumab (Opdivo), and Pembrolizumab (Keytruda); non-specific immunotherapies such as interferons, interleukins, and Bacillus Calmette-Guerin (BCG); oncolytic viruses such as Talimogene laherparepvec (Imlygic) or T-VEC; and cancer vaccines. In some embodiments, the immunotherapy may be an immune checkpoint inhibiting antibody, such as an antibody that binds to CTLA4, PD-1, or PD-L1. [0074] Other subject populations for which it is useful to understand the functioning of the subject’s adaptive immune response include subjects with an autoimmune disease and subject that have or are at risk of contracting an infectious disease.

[0075] Thus, the present disclosure provides methods of detecting somatic mutation burden in a sample, comprising: a) obtaining one or more samples comprising skin cells from a subject suspected of having an autoimmune disease; b) sequencing DNA from the one or more samples; and c) detecting a level of somatic mutation burden in each of the one or more samples. Autoimmune diseases that may be assessed for the purposes of the present disclosure include, but are not limited to, rheumatoid arthritis, ankylosing spondylitis, Crohn’s disease, ulcerative colitis, lupus, multiple sclerosis, Sjogren's syndrome, vasculitis, and celiac disease. The present disclosure likewise provides methods of detecting somatic mutation burden in a sample, comprising: a) obtaining one or more samples comprising skin cells from a subject at risk of an infectious disease; b) sequencing DNA from the one or more samples; and c) detecting a level of somatic mutation burden in each of the one or more samples. Infectious diseases that may be assessed for the purposes of the present disclosure include, but are not limited to, AIDS/HIV, hepatitis, influenza, COVID-19, and tuberculosis.

[0076] The present disclosure also provides methods for determining the prognosis of a subject diagnosed as having a disease comprising: a) obtaining one or more samples from a subject; b) detecting a somatic mutation burden for each of the one or more samples; c) identifying the subject as having poor prognosis when the somatic mutation burden is below a threshold. The disease may be a cancer, an autoimmune disease, or an infectious disease.

[0077] The present disclosure also provides methods of determining the risk of a disease in a subject, comprising: a) obtaining one or more samples from a subject; b) detecting a somatic mutation burden for each of the one or more samples; c) determining the risk of a disease in the subject based on the level of somatic mutation burden in the subject.

[0078] The present disclosure also provides methods predicting a response to a treatment, comprising: a) obtaining one or more samples from a subject; b) detecting a somatic mutation burden for each of the one or more samples; c) predicting the subject as having a favorable response to the treatment when the somatic mutation burden score is above a threshold. III. The Abscopal Effect

[0079] The abscopal effect describes tumor regression outside of an irradiated region and has been demonstrated in many different cancer types after ionizing radiation is directed at the primary tumor. Mediated by a systemic anti-tumor immune response, the effect alludes to the regression of non-irradiated metastatic lesions at sites away from the primary site of irradiation. Systemic reviews of case reports have shown that the abscopal effect following radiotherapy has been observed in a number of cancer types, including but not limited to lung cancer, renal cell carcinoma, hepatocellular carcinoma, lymphoma, and melanoma (Abuodeh et al. Curr. Probl. Cancer., 40:25-37, 2016). Both pre-clinical and clinical studies have supported that regression of tumors outside of the irradiation field is mediated by the effects of radiation on the immune system. With the introduction of immunotherapy, the understanding of immune activation by radiation treatment has further strengthened the role of radiation therapy in systemic disease, as well as demonstrated how the two can work synergistically for tumor burden control. The methods described herein leverage insight into a previously unappreciated type of immune mediated abscopal response to radiation, whereby non-ionizing UV radiation of the skin gives rise to neoantigens that elicit systemic adaptive immune responses which mediate recognition and destruction of cancer cells distal to the irradiated area. The extent of such neoantigen-driven adaptive immune stimulation, and thus the adaptive immune health of the subject, can be quantified by measuring the somatic mutation burden of phenotypically normal skin cells. The measured adaptive immune health indicates the likelihood of success of a cancer immunotherapy regimen.

IV. Methods and Devices for Obtaining Samples from a Patient

[0080] In general, for the purposes of the discloses methods, the samples utilized for the disclosed methods and kits are skin samples (e.g., skin samples obtained via non-invasive skin sampling). In some embodiments, samples are obtained from lung (e.g., via lung biopsy), mouth (e.g., via analysis of human gDNA from saliva), intestinal epithelial cells (e.g., via analysis of human gDNA from stool), bladder (e.g., via urine cfDNA analysis), blood plasma (e.g., via analysis of cfDNA from blood), or non-cancerous cells within a tumor biopsy.

[0081] A skin sample may be obtained from a subject using a collection device (such as an adhesive strip or patch). In some embodiments described herein, a skin sample is obtained from the subject by applying an adhesive strip or patch to a skin region of the subject. In some embodiments, the skin sample is not obtained with an adhesive patch. In some embodiments, the skin sample is obtained using a brush. In some embodiments, the skin sample is obtained using a swab, for example a cotton swab. In some cases, the skin sample is obtained using a probe. In some cases, the skin sample is obtained using a hook. In some embodiments, the skin sample is obtained using a medical applicator. In some embodiments, the skin sample is obtained by scraping a skin surface of the subject. In some cases, the skin sample is obtained through excision. In some embodiments, the skin sample is biopsied. In some embodiments, the skin sample is a biopsy. In some embodiments, the skin sample is obtained using one or more needles. For example, the needles may be microneedles. In some embodiments, the biopsy is a needle biopsy, or a microneedle biopsy. In some embodiments, the skin sample is obtained invasively. In some embodiments, the skin sample is obtained non-invasively. A skin sample in some embodiments is obtained iteratively from the same skin area of a subject. In some embodiments, multiple samples are obtained from a single skin area and pooled prior to analysis.

[0082] The methods provided herein may generate samples from various layers of skin. While not wishing to be bound by theory, sampling at the surface of the skin provides results differentiated from that of deeper (invasive, e.g., biopsy) sampling for skin cancer and other disease derived from external/environmental factor interactions (e.g., UV). For example, the quantity of sun exposed cells and number of mutations in some embodiments results in higher sensitivity or specificity in measuring mutation burden.

[0083] In some embodiments, methods generate samples from the top or superficial layers of skin, which have been exposed to higher levels of one or more environmental factors. In some embodiments, the skin sample comprises cells of the stratum corneum. In some embodiments, the skin sample consists of cells of the stratum corneum. In some embodiments, non-invasive sampling described herein does not fully disrupt the epidermal: dermal junction. Without being bound by theory, non-invasive sampling described herein does not trigger significant wound healing which normally results from significant damage to the epithelial barrier. In some embodiments, the skin sample comprises at least 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, or at least 99.9% of cells derived from the basal keratinocyte layer. In some embodiments, the skin sample comprises less than 10%, 5%, 3%, 2%, 1 %, 0.1 %, 0.05%, or less than 0.01 % cells derived from the basal keratinocyte layer. In some embodiments, the skin sample does not include the basal layer of the skin. In some embodiments, the skin sample comprises or consists of a skin depth of 10 pm, 50 pm, 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, or a range of skin depths defined by any two of the aforementioned skin depths. In some embodiments, the skin sample comprises or consists of a skin depth of about 10 pm, 50 pm, 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, or about 500 pm. In some embodiments, the skin sample comprises or consists of a skin depth of 50-100 pm. In some embodiments, the skin sample comprises or consists of a skin depth of 100-200 pm. In some embodiments, the skin sample comprises or consists of a skin depth of 200-300 pm. In some embodiments, the skin sample comprises or consists of a skin depth of 300-400 pm. In some embodiments, the skin sample comprises or consists of a skin depth of 400-500 pm.

[0084] Non-invasive sampling methods described herein may comprise obtaining multiple skin samples from the same area of skin on an individual using multiple collection devices (e.g., tapes). In some embodiments, each sample obtained from the same area or substantially the same area results in progressively deeper layers of skin cells. In some embodiments, multiple samples are pooled prior to analysis. The skin sample may be from one collection device or from multiple collection devices. For example, one collection device may be used to obtain an amount of cellular material described, or the skin samples from multiple collection devices may be used to obtain a given amount of cellular material. For example, skin samples from 2 or more adhesive patches may be pooled to obtain an amount of genetic cellular material sufficient for a method described herein. In some embodiments, skin samples from at least 2, 3, 4, 5, 6, 8, 10, 12, 16, or more adhesive patches are pooled to obtain an amount of genetic cellular material sufficient for a method described herein. In some embodiments, skin samples from at least 2-16, 2-12, 2-10, 2-8, 2- 6, 2-4, 4-16, 4-12, 4-8, 6- 16, or 8-20 adhesive patches are pooled to obtain an amount of genetic cellular material sufficient for a method described herein.

[0085] The skin sample may be defined by thickness, or how deep into the skin cells are obtained. In some embodiments, the skin sample is no more than 10 pm thick. In some embodiments, the skin sample is no more than 50 pm thick. In some embodiments, the skin sample is no more than 100 pm thick. In some embodiments, the skin sample is no more than 150 pm thick. In some embodiments, the skin sample is no more than 200 pm thick. In some embodiments, the skin sample is no more than 250 pm thick. In some embodiments, the skin sample is no more than 300 pm thick. In some embodiments, the skin sample is no more than 350 pm thick. In some embodiments, the skin sample is no more than 400 pm thick. In some embodiments, the skin sample is no more than 450 pm thick. In some embodiments, the skin sample is no more than 500 pm thick.

[0086] In some embodiments, the skin sample is at least 10 pm thick. In some embodiments, the skin sample is at least 50 pm thick. In some embodiments, the skin sample is at least 100 pm thick. In some embodiments, the skin sample is at least 150 pm thick. In some embodiments, the skin sample is at least 200 pm thick. In some embodiments, the skin sample is at least 250 pm thick. In some embodiments, the skin sample is at least 300 pm thick. In some embodiments, the skin sample is at least 350 pm thick. In some embodiments, the skin sample is at least 400 pm thick. In some embodiments, the skin sample is at least 450 pm thick. In some embodiments, the skin sample is at least 500 pm thick.

[0087] In some embodiments, non-invasive skin sampling is performed using a collection device including tape stripping, a sticker, patch, or an adhesive collection device. For example, in some embodiments, the non-invasive skin sampling is performed using a collection device that comprises a collection area having an adhesive matrix located on a skin facing surface. In some embodiments, the collection device additionally comprises a second area that functions as a tab, suitable for applying and removing the adhesive tape. The tab is sufficient in size so that while applying the adhesive tape or patch to a skin surface, the applicant does not come into contact with the matrix material of the collection area. In some embodiments, the collection device does not contain a tab. In some embodiments, the collection device is handled with gloves to reduce contamination of the adhesive matrix prior to use.

[0088] In some embodiments, the collection area is a polyurethane carrier film. In some embodiments, the adhesive matrix is comprised of a synthetic rubber compound. In some embodiments, the adhesive matrix is a styrene-isoprene-styrene (SIS) linear block copolymer compound. In some embodiments, the collection device does not comprise latex, silicone, or both. In some embodiments, the collection device is manufactured by applying an adhesive material as a liquid solvent mixture to the collection area and subsequently removing the solvent. In some embodiments, the adhesive matrix is configured to adhere cells from the stratum comeum of a skin sample. [0089] Generally, the matrix material is sufficiently sticky to adhere to a skin sample. The matrix material is not so sticky that it causes scarring or bleeding or is difficult to remove. In some embodiments, the matrix material is comprised of a transparent material. In some embodiments, the matrix material is biocompatible. In some embodiments, the matrix material does not leave residue on the surface of the skin after removal. In some embodiments, the matrix material is not a skin irritant.

[0090] In some embodiments, the collection device comprises a flexible material, enabling the tape to conform to the shape of the skin surface upon application. In some embodiments, at least the collection area is flexible. In some embodiments, the tab is plastic. In some embodiments, the collection device is made of a transparent material, so that the skin sampling area of the subject is visible after application of the collection device to the skin surface. The transparency ensures that the collection device is applied on the desired area of skin comprising the skin area to be sampled.

[0091] In some embodiments, the collection device is between about 5 and about 100 mm in length. In some embodiments, the collection area is between about 5 and about 40 mm in length. In some embodiments, the collection area is between about 10 and about 20 mm in length. In some embodiments the length of the collection area is configured to accommodate the area of the skin surface to be sampled, including, but not limited to, about 19 mm, about 20 mm, about 21 mm, about 22 mm, about 23 mm, about 24 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, about 50 mm, about 55 mm, about 60 mm, about 65 mm, about 70 mm, about 75 mm , about 80 mm , about 85 mm , about 90 mm , and about 100 mm . In some embodiments, the collection area is elliptical. In some embodiments, the collection area is circular. In some embodiments, the collection area is square. In some embodiments, the collection area is rectangular.

[0092] In some embodiments, the collection area is between about 1 mm² to about 50 mm². In some embodiments, the collection area is between about 5 mm² to about 40 mm². In some embodiments, the collection area is between about 10 mm² to about 30 mm². In some embodiments, the collection area is configured to accommodate the area of the skin surface to be sampled, including, but not limited to about 1 mm², about 2 mm², about 3 mm², about 5 mm², about 10 mm², about 20 mm², about 30 mm², about 40 mm², or about 40 mm². [0093] In some embodiments, the collection device removes a skin sample from the subject at a depth no greater than 10 pm. In some embodiments, the collection device removes a skin sample from the subject at a depth no greater than 50 pm. In some embodiments, the collection device removes a skin sample from the subject at a depth no greater than 100 pm. In some embodiments, the collection device removes a skin sample from the subject at a depth no greater than 150 pm. In some embodiments, the collection device removes a skin sample from the subject at a depth no greater than 200 pm. In some embodiments, the collection device removes a skin sample from the subject at a depth no greater than 250 pm. In some embodiments, the collection device removes a skin sample from the subject at a depth no greater than 300 pm. In some embodiments, the collection device removes a skin sample from the subject at a depth no greater than 350 pm. In some embodiments, the collection device removes a skin sample from the subject at a depth no greater than 400 pm. In some embodiments, the collection device removes a skin sample from the subject at a depth no greater than 450 pm. In some embodiments, the collection device removes a skin sample from the subject at a depth no greater than 500 pm.

[0094] In some embodiments, the collection device removes 1, 2, 3, 4, or 5 layers of stratum corneum from a skin surface of the subject. In some embodiments, the collection device removes a range of layers of stratum corneum from a skin surface of the subject, for example a range defined by any two of the following integers: 1, 2, 3, 4, or 5. In some embodiments, the collection device removes 1-5 layers of stratum corneum from a skin surface of the subject. In some embodiments, the collection device removes 2-3 layers of stratum corneum from a skin surface of the subject. In some embodiments, the collection device removes 2-4 layers of stratum corneum from a skin surface of the subject. In some embodiments, the collection device removes no more than the basal layer of a skin surface from the subject.

[0095] In some embodiments, skin samples are obtained from different regions of the body. In some embodiments, skin samples are obtained from sampling of one or more regions comprising skin subjected to different levels of UV exposure. In some embodiments, skin samples are obtained from sampling of one or more regions comprising skin subjected to chronic UV exposure. In some embodiments, regions subjected to chronic UV exposure include forehead, neck, forearms, hands, face. In some embodiments, skin samples are obtained from sampling of one or more regions comprising skin subjected to intermittent UV exposure. In some embodiments, regions subjected to intermittent UV exposure include torso, back, thighs, and ankles. In some embodiments, skin samples are obtained from sampling of one or more regions comprising skin subjected to limited or no UV exposure. In some embodiments, regions subjected to limited or no UV exposure include buttocks, armpit, or control samples. In some embodiments, control samples may include skin samples from regions subjected to limited or no UV exposure or other samples from the subject including blood or oral swab.

[0096] In some embodiments, the regions exposed to chronic, intermittent, limited or no UV exposure may vary depending on the subject. For example, the regions exposed to chronic, intermittent, limited or no UV exposure may vary depending on the gender, location, race, religion, or age of the subject. The regions from which skin samples are taken may be selected by the doctor or physician following a wholistic examination of the patient.

[0097] In one embodiment, the tape stripping method comprises preparing the skin sample prior to application of the adhesive tape. Preparation of the skin sample includes, but is not limited to, removing hairs on the skin surface, cleansing the skin surface and/or drying the skin surface. In some embodiments, the skin surface is cleansed with an antiseptic including, but not limited to, alcohols, quaternary ammonium compounds, peroxides, chlorhexidine, halogenated phenol derivatives and quinolone derivatives. In some embodiments, the alcohol is about 0 to about 20 %, about 20 to about 40 %, about 40 to about 60 %, about 60 to about 80 %, or about 80 to about 100 % isopropyl alcohol. In some embodiments, the antiseptic is 70 % isopropyl alcohol. In some embodiments, the antiseptic is about 0.1% benzalkonium chloride.

[0098] In some embodiments, the tape stripping method is used to collect a skin sample from the surfaces including, but not limited to, the face, head, neck, arm, chest, abdomen, back, leg, hand, or foot. In some embodiments, the skin surface is not located on a mucous membrane. In some embodiments, the skin surface is not ulcerated or bleeding. In some embodiments, the skin surface has not been previously biopsied.

[0099] In some embodiments, cellular components, including nucleic acids, are stable on a collection device when stored for a period of time or at a particular temperature. In some embodiments, the period of time is at least or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, or more than 4 weeks. In some embodiments, the period of time is about 7 days. In some embodiments, the period of time is about 10 days. In some embodiments, the temperature is at least or about -80 °C, -70 °C, -60 °C, -50 °C, -40 °C, -20 °C, -10 °C, -4 °C, 0 °C, 5 °C, 15 °C, 18 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, or more than 50 °C. The cellular components or nucleic acids on the collection device, in some embodiments, are stored for any period of time described herein and any particular temperature described herein. For example, the nucleic acids on the collection device are stored for at least or about 7 days at about 25 °C, 7 days at about 30 °C, 7 days at about 40 °C, 7 days at about 50 °C, 7 days at about 60 °C, or 7 days at about 70 °C. In some embodiments, the nucleic acids on the collection device are stored for at least or about 10 days at about -80 °C.

[0100] In further embodiments, samples (e.g., skin samples) for analysis may be obtained using noninvasive techniques or minimally invasive techniques. In some embodiments, a minimally invasive technique comprises the use of microneedles. In some embodiments, a sample such as a skin sample is collected using one or more microneedles. In some embodiments, a plurality of microneedles are used to obtain a sample. In some instance, microneedles are polymeric. In some instance, microneedles are coated with a substance (e.g., enzymes, chemical, or other substance) capable of disrupting an extracellular matrix. In some embodiments, microneedles pierce a subject's skin to obtain samples of skin cells, blood, or both. In some embodiments, microneedles are coated with probes that bind to one or more nucleic acid targets described herein.

[0101] The non-invasive sampling methods provided herein provide advantages over traditional biopsy methods, including but not limited to self-application by a patient/subject, increased signal to noise ratio of sample exposed to the skin surface (leading to higher sensitivity and/or specificity), lack of temporary or permanent scarring at the analysis site, lower change of infection, or other advantage.

[0102] Examples of subjects include but are not limited to vertebrates, animals, mammals, dogs, cats, cattle, rodents, mice, rats, primates, monkeys, and humans. In some embodiments, the subject is a vertebrate. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is an animal, a mammal, a dog, a cat, cattle, a rodent, a mouse, a rat, a primate, or a monkey. In some embodiments, the subject is a human. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject has skin previously exposed to UV light. In some embodiments, the subject has previously been diagnosed with cancer.

V. Sample Collection and Processing

[0103] In some embodiments, the methods and devices provided herein, involve applying a collection device to the skin in a manner so that an effective or sufficient amount of a tissue, such as a skin sample, adheres to the adhesive matrix of the collection device. In some cases, the skin sample adhered to the adhesive matrix comprises or consists of cells from the stratum corneum of a subject. For example, the effective or sufficient amount of a skin sample is an amount that removably adheres to a material, such as the matrix or collection device. The adhered skin sample, in certain embodiments, comprises cellular material including nucleic acids, proteins, and organelles. In some embodiments, the nucleic acid is RNA or DNA. In some embodiments, the nucleic acid is RNA (e.g., mRNA). In some embodiments, the DNA is genomic DNA (gDNA) or mitochondrial DNA (mtDNA). In some embodiments, the protein is an intracellular protein, extracellular protein, or a cell surface protein. An effective amount of a skin sample contains an amount of cellular material sufficient for performing an assay. In some embodiments, the assay is performed using the cellular material isolated from the adhered skin sample on the used adhesive patch. In some embodiments, the assay is performed on the cellular material adhered to the used adhesive patch.

[0104] In some embodiments, an effective amount of a skin sample comprises an amount of RNA sufficient to perform a genomic analysis. Sufficient amounts of RNA include, but are not limited to, picogram (pg), nanogram (ng), and microgram (pg) quantities. In some embodiments, the RNA includes mRNA. In some embodiments, the RNA includes microRNAs. In some embodiments, the RNA includes mRNA and microRNAs. In some embodiments, the RNA is a microRNA (miRNA), a premiRNA, a pri-miRNA, a mRNA, a pre-mRNA, a viral RNA, a viroid RNA, a virusoid RNA, circular RNA (circRNA), a ribosomal RNA (rRNA), a transfer RNA (tRNA), a pre-tRNA, a long non-coding RNA (IncRNA), a small nuclear RNA (snRNA), a circulating RNA, a cell-free RNA, an exosomal RNA, a vector-expressed RNA, a RNA transcript, a synthetic RNA, or combinations thereof.

[0105] In some embodiments, an effective amount of a skin sample comprises an amount of DNA sufficient to perform a genomic analysis. Sufficient amounts of DNA includes, but not limited to, pg, ng, and pg quantities. In some embodiments, the DNA includes gRNA. In some embodiments, the DNA includes mtDNA. In some embodiments, the DNA includes gDNA and mtDNA. In some embodiments, the DNA includes, but is not limited to, genomic DNA, viral DNA, mitochondrial DNA, plasmid DNA, amplified DNA, circular DNA, circulating DNA, cell-free DNA, or exosomal DNA In some embodiments, the DNA is single stranded DNA (ssDNA), double-stranded DNA, denaturing double-stranded DNA, synthetic DNA, and combinations thereof. In some embodiments, the DNA is genomic DNA. In some embodiments, the DNA is cell-free circulating DNA.

[0106] Such nucleic acids in some embodiments are obtained from obtaining skin using a single collection device. In some embodiments, nucleic acids and proteins are obtained from samples pooled from multiple collection devices. In some embodiments, nucleic acids and proteins are obtained from samples from a single collection device applied to the skin multiple times (1, 2, 3, or 4 times). In additional embodiments, the adhered skin sample comprises cellular material including nucleic acids such as RNA or DNA in an amount that is at least about 1 pg. Cellular material in some embodiments is obtained from skin using a single collection device. In some embodiments, cellular material is obtained from samples pooled from multiple collection devices. In some embodiments, cellular material is obtained from samples from a single collection device applied to the skin multiple times (1, 2, 3, or 4 times). In some embodiments, an amount of cellular material described herein refers to the amount of material pooled from multiple collection devices (e.g., 1-6 devices). In some embodiments, the amount of cellular material is no more than about 1 ng. In further or additional embodiments, the amount of cellular material is no more than about 1 pg. In still further or additional embodiments, the amount of cellular material is no more than about 1 milligram (mg). In still further or additional embodiments, the amount of cellular material is no more than about 1 gram (g).

[0107] A total amount of cellular material may be obtained from a kit (e.g., a kit comprising multiple collection devices each applied to skin). In some embodiments, cellular material collected in a kit is less than 20 mg, less than 10 mg, less than 5 mg, less than 2 mg, less than 1 mg, less than 500 pg, less than 200 pg, or less than 100 pg. In some embodiments, the collection device in a kit comprises an adhesive strip or patch. In some embodiments, each adhesive strip or patch comprises 1 pg to 2000 pg, 1 pg to 1000 pg, 1 pg to 500 pg, 1 pg to 100 pg, or 1 pg to 10 pg per patch or strip of cellular material. [0108] In further or additional embodiments, the amount of cellular material is from about 1 pg to about 1 g. In further or additional embodiments, the cellular material comprises an amount that is from about 50 pg to about 1 g, from about 100 pg to about 500 pg, from about 500 pg to about 100 pg, from about 750 pg to about 1 pg, from about 1 ng to about 750 ng, or from about 1 ng to about 500 ng. In further or additional embodiments, the cellular material comprises an amount that is from about 5 pg to about 1 gram, from about 1 pg to about 500 pg, from about 1 pg to about 250 pg, from about 1 pg to about 1 pg, from about 1 ng to about 750 ng, or from about 1 ng to about 500 ng.

[0109] In further or additional embodiments, the amount of cellular material is from about 1 pg to about 1 pg. In further or additional embodiments, the amount of cellular material, including nucleic acids such as RNA or DNA, comprises an amount that is from about 50 pg to about 500 pg, from about 100 pg to about 450 pg, from about 100 pg to about 350 pg, from about 100 pg to about 300 pg, from about 120 pg to about 250 pg, from about 150 pg to about 200 pg, from about 500 ng to about 5 ng, or from about 400 ng to about 10 ng, or from about 200 ng to about 15 ng, or from about 100 ng to about 20 ng, or from about 50 ng to about 10 ng, or from about 50 ng to about 25 ng. In some cases, about 2 ng of genomic DNA is sufficient to provide robust variant detection via a detection platform such as mass spectrometry (e.g., MassARRAY) or next generation sequencing (e.g., NextSeq 2000). Some embodiments include at least about 2 ng of a cellular material such as DNA or RNA. In some cases, at least 1 ng of cellular material such as DNA or RNA is sufficient. In some cases, at least 100 pg is sufficient.

[0110] In further or additional embodiments, the amount of cellular material is from about 1 pg to about 1 mg. In further or additional embodiments, the amount of cellular material, including nucleic acids such as RNA or DNA, or protein, comprises an amount that is from about 50 mg to about 500 pg, from about 100 mg about 450 pg, from about 100 mg about 350 pg, from about 100 mg about 300 pg, from about 120 mg about 250 pg, from about 150 mg about 200 pg, from about 5 mg to about 500 mg, or from about 5 mg to about 100 mg, or from about 20 mg to about 150 mg, or from about 1 mg to about 20 mg, or from about 1 mg to about 50 mg, or from about 1 mg to about 100 mg.

[0111] In further or additional embodiments, the amount of cellular material, including nucleic acids such as RNA or DNA, or protein, is less than about 1 g, is less than about 500 pg, is less than about 490 pg, is less than about 480 pg, is less than about 470 pg, is less than about 460 microgram (pg), is less than about 450 pg, is less than about 440 pg, is less than about 430 pg, is less than about 420 pg, is less than about 410 pg, is less than about 400 pg, is less than about 390 pg, is less than about 380 pg, is less than about 370 pg, is less than about 360 pg, is less than about 350 pg, is less than about 340 pg, is less than about 330 pg, is less than about 320 pg, is less than about 310 pg, is less than about 300 pg, is less than about 290 pg, is less than about 280 pg, is less than about 270 pg, is less than about 260 pg, is less than about 250 pg, is less than about 240 pg, is less than about 230 pg, is less than about 220 pg, is less than about 210 pg, is less than about 200 pg, is less than about 190 pg, is less than about 180 pg, is less than about 170 pg, is less than about 160 pg, is less than about 150 pg, is less than about 140 pg, is less than about 130 pg, is less than about 120 pg, is less than about 110 pg, is less than about 100 pg, is less than about 90 pg, is less than about 80 pg, is less than about 70 pg, is less than about 60 pg, is less than about 50 pg, is less than about 20 pg, is less than about 10 pg, is less than about 5 pg, is less than about 1 pg, is less than about 750 ng, is less than about 500 ng, is less than about 250 ng, is less than about 150 ng, is less than about 100 ng, is less than about 50 ng, is less than about 25 ng, is less than about 15 ng, is less than about 1 ng, is less than about 750 pg, is less than about 500 pg, is less than about 250 pg, is less than about 100 pg, is less than about 50 pg, is less than about 25 pg, is less than about 15 pg, or is less than about 1 pg.

[0112] In further or additional embodiments, the amount of cellular material, including nucleic acids such as RNA or DNA, is less than about 1 g, is less than about 500 mg, is less than about 490 mg, is less than about 480 mg, is less than about 470 mg, is less than about 460 mg, is less than about 450 mg, is less than about 440 mg, is less than about 430 mg, is less than about 420 mg, is less than about 410 mg, is less than about 400 mg, is less than about 390 mg, is less than about 380 mg, is less than about 370 mg, is less than about 360 mg, is less than about 350 mg, is less than about 340 mg, is less than about 330 mg, is less than about 320 mg, is less than about 310 mg, is less than about 300 mg, is less than about 290 mg, is less than about 280 mg, is less than about 270 mg, is less than about 260 mg, is less than about 250 mg, is less than about 240 mg, is less than about 230 mg, is less than about 220 mg, is less than about 210 mg, is less than about 200 mg, is less than about 190 mg, is less than about 180 mg, is less than about 170 mg, is less than about 160 mg, is less than about 150 mg, is less than about 140 mg, is less than about 130 mg, is less than about 120 mg, is less than about 110 mg, is less than about 100 mg, is less than about 90 mg, is less than about 80 mg, is less than about 70 mg, is less than about 60 mg, is less than about 50 mg, is less than about 20 mg, is less than about 10 mg, or is less than about 5 mg.

[0113] In some embodiments, the layers of skin include epidermis, dermis, or hypodermis. The outer layer of epidermis is the stratum corneum layer, followed by stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale. In some embodiments, the skin sample is obtained from the epidermis layer. In some cases, the skin sample is obtained from the stratum corneum layer. In some embodiments, the skin sample is obtained from the dermis. In some cases, the skin sample is obtained from the stratum germinativum layer. In some cases, the skin sample is obtained from no deeper than the stratum germinativum layer.

[0114] The sample may comprise skin cells from a superficial depth of skin using the noninvasive sampling techniques described herein. In some embodiments, the sample comprises skin cells from about the superficial about 0.01, 0.02, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4 mm of skin. In some embodiments, the sample comprises skin cells from no more than the superficial about 0.01, 0.02, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4 mm of skin. In some embodiments, the sample comprises skin cells from at least the superficial about 0.01, 0.02, 0.05, 0.08, 0.1, 0.2, 0.3, or at least 0.4 mm of skin. In some embodiments, the sample comprises skin cells from the superficial about 0.01-0.1, 0.01-0.2, 0.02-0.1, 0.02-0.2 0.04-0.0.08, 0.02-0.08, 0.01- 0.08, 0.05-0.2, or 0.05-0.1 mm of skin. In some embodiments, the sample comprises skin cells from about the superficial about 0.01, 0.02, 0.05, 0.08, 0.1, 0.2, 0.3, or about 0.4 pm of skin. In some embodiments, the sample comprises skin cells from no more than the superficial about 0.01, 0.02, 0.05, 0.08, 0.1, 0.2, 0.3, or no more than 0.4 pm of skin. In some embodiments, the sample comprises skin cells from at least the superficial about 0.01, 0.02, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4 pm of skin. In some embodiments, the sample comprises skin cells from the superficial about 0.01-0.1, 0.01-0.2, 0.02-0.1, 0.02-0.2 0.04-0.0.08, 0.02-0.08, 0.01- 0.08, 0.05-0.2, or 0.05-0.1 pm of skin.

[0115] The sample may comprise skin cells a number of skin cell layers, for example the superficial cell layers. In some embodiments, the sample comprises skin cells from 1-5, 1-10, 1-20, 1-25, 1-50, 1-75, or 1-100 cell layers. In some embodiments, the sample comprises skin cells from about 1, 2, 3, 4, 5, 8, 10, 12, 15, 20, 22, 25, 30, 35, or about 50 cell layers. In some embodiments, the sample comprises skin cells from no more than 1, 2, 3, 4, 5, 8, 10, 12, 15, 20, 22, 25, 30, 35, or no more than 50 cell layers. [0116] The sample may comprise skin cells collected from a defined skin area of the subject having a surface area. In some embodiments, the sample comprises skin cells obtained from a skin surface area of 10-300 mm², 10-500 mm², 5-500 mm², 1-300 mm², 5-100 mm², 5-200 mm², or 10-100 mm². In some embodiments, the sample comprises skin cells obtained from a skin surface area of at least 5, 10, 20, 25, 30, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, or at least 350 mm². In some embodiments, the sample comprises skin cells obtained from a skin surface area of no more than 5, 10, 20, 25, 30, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, or no more than 350 mm².

[0117] The nucleic acid (DNA or RNA) may be isolated from the sample according to any methods well known to those of skill in the art. If necessary, the sample may be collected or concentrated by centrifugation and the like. The sample may be subjected to lysis, such as by treatments with enzymes, heat, surfactants, ultrasonication, or a combination thereof. The lysis treatment is performed in order to obtain a sufficient amount of nucleic acid. The sample may be subjected to liquid chromatography to partially purify the genomic nucleic acid.

[0118] Suitable nucleic acid isolation methods include phenol and chloroform extraction. See, Maniatis el al., Molecular Cloning, A Laboratory Manual, 2d, Cold Spring Harbor Laboratory Press, page 16.54 (1989). Numerous commercial kits also yield suitable DNA including, but not limited to, QIAamp™ mini blood kit, Agencourt Genfind™, Roche Cobas® Roche MagNA Pure® or phenol: chloroform extraction using Eppendorf Phase Lock Gels®.

[0119] Total DNA (e.g., genomic, mitochondrial, microbial, viral) can be purified from any biological sample using commercially available kits e.g., QIAamp DNA and QIAamp DNA Blood mini kits, Qiagen M96 robot and reagents, Qiagen Gentra robot and reagents, and Qiagen 9604 reagents (Qiagen, Valencia, Calif.). In one example, blood can be spotted on Guthrie cards. Blood spots can be punched from each card using BSD 1000 GenePunch Instrument and DNA was extracted using Qiagen BioSprint reagents.

[0120] Genomic DNA may be isolated from cells or tissues using standard methods, see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y.

[0121] In another embodiment, sample nucleic acid may be mRNA or cDNA generated from mRNA or total RNA. RNA can be isolated from cells or tissue samples using standard techniques, see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y. In addition, kits for isolating mRNA and synthesizing cDNA are commercially available e.g., RNeasy Protect Mini kit, RNeasy Protect Cell Mini kit from Qiagen.

[0122] Nucleic acid need not be extracted, but may be made available by suitable treatment of cells or tissue such as described in U.S. Pat. No. 7,521,213.

[0123] Following extraction of nucleic acids from a sample, the nucleic acids, in some embodiments, are further purified. In some embodiments, the nucleic acids are RNA. In some embodiments, the nucleic acids are DNA. In some embodiments, the RNA is human RNA. In some embodiments, the DNA is human DNA. In some embodiments, the RNA is microbial RNA. In some embodiments, the DNA is microbial DNA. In some embodiments, cDNA is generated by reverse transcription of RNA. In some embodiments, human nucleic acids and microbial nucleic acids are purified from the same biological sample. In some embodiments, nucleic acids are purified using a column or resin based nucleic acid purification scheme. In some embodiments, this technique utilizes a support comprising a surface area for binding the nucleic acids. In some embodiments, the support is made of glass, silica, latex or a polymeric material. In some embodiments, the support comprises spherical beads.

[0124] In some cases, a yield of the nucleic acid or protein products obtained using methods described herein is about 500 pg or higher, about 600 pg or higher, about 1000 pg or higher, about 2000 pg or higher, about 3000 pg or higher, about 4000 pg or higher, about 5000 pg or higher, about 6000 pg or higher, about 7000 pg or higher, about 8000 pg or higher, about 9000 pg or higher, about 10000 pg or higher, about 20000 pg or higher, about 30000 pg or higher, about 40000 pg or higher, about 50000 pg or higher, about 60000 pg or higher, about 70000 pg or higher, about 80000 pg or higher, about 90000 pg or higher, or about 100000 pg or higher.

[0125] In some cases, a yield of the nucleic acids products obtained using methods described herein is about 100 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 5 ng, 10 ng, 15 ng, 20 ng, 21 ng, 22 ng, 23 ng, 24 ng, 25 ng, 26 ng, 27 ng, 28 ng, 29 ng, 30 ng, 35 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 150 ng, 200 ng, 250 ng, 300 ng, 400 ng, 500 ng, or higher. [0126] In some cases, methods described herein provide less than less than 10%, less than 8%, less than 5%, less than 2%, less than 1 %, or less than 0.5% product yield variations between samples.

[0127] In some embodiments, a number of cells are obtained for use in a method described herein. Some embodiments include use of an adhesive patch comprising an adhesive comprising a tackiness that is based on the number of cells to be obtained. Some embodiments include use of a number of adhesive patches based on the number of cells to be obtained. Some embodiments include use of an adhesive patch sized based on the number of cells to be obtained. The size and/or tackiness may be based on the type of skin to be obtained. For example, normal looking skin generally provides less cells and RNA yield than flaky skin. In some embodiments, a skin sample is used comprising skin from a subject's temple, forehead, cheek, or nose. In some embodiments, only one patch is used. In some embodiments, only one patch is used per skin area (e.g., skin area on a subject's temple, forehead, cheek, or nose). In some embodiments, the skin sample is a non-cancerous skin sample. In some embodiments, the non-cancerous skin sample is from a subject diagnosed with cancer.

[0128] In some cases, methods described herein provide a substantially homogenous population of a nucleic acid product. In some cases, methods described herein provide less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 8%, less than 5%, less than 2%, less than 1 %, or less than 0.5% contaminants.

[0129] In some embodiments, following extraction, nucleic acids are stored. In some embodiments, the nucleic acids are stored in water, Tris buffer, or Tris-EDTA buffer before subsequent analysis. In some embodiments, this storage is less than 8° C. In some embodiments, this storage is less than 4° C. In certain embodiments, this storage is less than 0° C. In some embodiments, this storage is less than -20° C. In certain embodiments, this storage is less than -70° C. In some embodiments, the nucleic acids are stored for about 1, 2, 3, 4, 5, 6, or 7 days. In some embodiments, the nucleic acids are stored for about 1, 2, 3, or 4 weeks. In some embodiments, the nucleic acids are stored for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

[0130] In some embodiments, nucleic acids isolated using methods described herein are subjected to an amplification reaction following isolation and purification. In some embodiments, the nucleic acids to be amplified are RNA including, but not limited to, human RNA and human microbial RNA. In some embodiments, the nucleic acids to be amplified are DNA including, but not limited to, human DNA and human microbial DNA. Non-limiting amplification reactions include, but are not limited to, quantitative PCR (qPCR), selfsustained sequence replication, transcriptional amplification system, Q-Beta Replicase, rolling circle replication, or any other nucleic acid amplification known in the art. In some embodiments, the amplification reaction is PCR. In some embodiments, the amplification reaction is quantitative such as qPCR.

[0131] In some embodiments, nucleic acids isolated using methods described herein are subjected to NGS library preparation to incorporate sequencing adapters onto the nucleic acids. In some embodiments, isolated nucleic acids may be mechanically or enzymatically fragmented to generate shorter fragments. In some embodiments, the fragments may be end repaired prior to ligation of adapters. In some embodiments, sample specific barcodes may be incorporated into the nucleic acids to allow for multiplexing of multiple samples. In some embodiments, unique molecular identifiers (UMIs) may be introduced during library preparation and used to bioinformatically correct for sequencing or library preparation derived errors.

[0132] In some embodiments, the nucleic acids may be enriched for specific target sequences. In some embodiments, target enrichment may be performed before or after sequencing library preparation. In some embodiments, the samples may be enriched for a panel of target sequences specific for genomic profiling of tumor samples. In some embodiments, enrichment may be performed by amplification, on-sequencer enrichment, or hybrid capture of target sequences. In some embodiments, enrichment may be performed using commercially available kits, including but not limited to the TruSight Oncology 500 Assay from Illumina, the AVENIO Tumor Tissue Targeted Kit from Roche, Tempus, FoundationOne CDx, Guardant360 CDx, Oncomine Comprehensive Assay from Thermo Fisher, or similar assays designed for measurement of mutation burden.

[0133] In some embodiments, in order to improve the efficiency of sequencing, nucleic acids isolated using methods described herein may undergo further selection to enrich for fragments comprising a plurality of CC/GG dinucleotides. For example, in some embodiments, multiplex PCR is used to selectively amplify high CC/GG content regions of the human genome. [0134] In some embodiments, samples collected using the methods or devices described herein may be prepared for image analysis. For example, samples may comprise a section of a tissue. In some embodiments, the samples may be stained with H&E staining followed by brightfield imaging. In some embodiments, samples may be fixed. In some embodiments, mutations may be identified using staining techniques. In some embodiments, immunogenic staining techniques may be used. For example, mutation-specific antibodies may be used.

[0135] In some embodiments, one or more techniques are employed to reduce the abundance of nucleic acids derived from skin-resident bacteria or fungi when extracting nucleic acids from a skin sample. As non-limiting examples, in some embodiments, following cell capture and prior to DNA extraction, the cell lysis time is modulated to result in preferential lysis of human cells. For example, in some embodiments, the sample is incubated in a thermomixer at 56C for approximately 5, 10, 20, 30, 60 minutes, or more than 60 minutes, in the presence of proteinase K. In some embodiments, following nucleic acid extraction, human-derived nucleic acids are enriched by hybridization capture using probes specific to human nucleic acids. In some embodiments, enrichment of human nucleic acids is achieved by single-plex or multiplex PCR using human specific PCR primers. In some embodiments, enrichment of human nucleic acids is achieved by wiping the skin region of interest with a dilute solution comprising chloride, eg BKZ Antiseptic Towelette wipes (Dynarex, Cat #1331) prior to sample collection.

[0136] In some embodiments, following nucleic acids extraction, nucleic acids are subjected to a fragmentation process prior to sequencing. For example, in some embodiments, nucleic acids are subjected to mechanical shearing (eg via Covaris acoustic shearing) or enzymatic cleavage prior to next-generation sequencing library preparation.

VI. Methods of Analyzing Samples from a Subject

[0137] The samples processed using the methods described herein may be further analyzed using methods known in the art. For example, samples may be sequenced by whole genome sequencing (WGS), whole exome sequencing, next-generation sequencing (NGS), high fidelity sequencing, bottleneck sequencing, RNA-seq. In some embodiments, high fidelity sequencing may be achieved through single molecule consensus sequencing (e.g., PacBio HiFi). In some embodiments, high fidelity sequencing may be achieved through consensus error correction (e.g., Safe-SeqS, Duplex Sequencing, bottleneck sequencing). Samples may also be analyzed by amplification methods including qPCR or digital droplet PCR.

[0138] In some embodiments, the sample is a non-cancerous skin sample. In some embodiments, the non-cancerous skin sample is from a subject diagnosed with cancer. In some embodiments, the sample is a phenotypically normal sample. In some embodiments, the phenotypically normal sample is from a subject suspected of having an autoimmune disease. In some embodiments, the phenotypically normal sample is from a subject at risk of an infectious disease. In some embodiments, the infectious disease is HIV.

[0139] In some embodiments, following sequencing, sequencing reads are aligned to a reference genome, then analyzed to identify mutations with respect to reference. Bioinformatic methods for quantifying mutations in a sample are known to those familiar with the art. In some embodiments, the mutations detected in the targeted region are used to infer the genome wide mutation rate and calculate a somatic mutation burden (SMB). In some embodiments, the SMB is expressed as mutations per Mb. In some embodiments, the mutations per Mb value for a UV-exposed sample may be further refined by comparison to the mutations per Mb for a sample derived from a non-UV-exposed site from the same individual.

[0140] In some embodiments, the SMB value for each sample may be used to calculate a somatic mutation score. In some embodiments, the score is calculated as a weighted sum of the SMB values obtained from each of the samples. In some embodiments, SMB values are weighted according to the sample location and the area of skin represented by the sample. For example, in some embodiments, the score is calculated as follows:

Somatic Mutation Score = C1R1S1 + C2R2S2 + C3R3S3 + . . .

[0141] Where Ci, C2, C3 are region specific constants, Ri, R2 and R3 reflect the area of the skin region represented by the sample, and Si, S2, S3 are the SMB values for each of the samples. In some embodiments, the somatic mutation score calculation comprises a measurement of the distribution of the frequencies of mutations detected in each sample (z.e., the clonality). In some embodiments, calculating a somatic mutation score comprises estimating the number of cells represented by each clonal proliferation of cells detected in each sample. In some embodiments, the somatic mutation score excludes mutations having a frequency below a certain predefined cutoff threshold. In some embodiments, the cutoff threshold is approximately 0.01%, 0.02%, 0.1%, or 0.5%. In other embodiments the cutoff threshold is approximately 1%, 2%, 3%, or 5%.

[0142] In some embodiments, the SMB value exclusively reflects mutations consistent with damage from a mutagen of interest. For example, in some embodiments, the SMB value of a skin sample is calculated exclusively using mutations consistent with UV damage. For example, UV-specific mutations may include dinucleotide and triplet mutations or variants, such as CC to TT mutations, or single nucleotide variants, such as C to T mutations in the context of a CC dimer, or single base thymine deletions in the context of a thymine homopolymer (Wei L, et al. Ultradeep sequencing differentiates patterns of skin clonal mutations associated with sun-exposure status and skin cancer burden. DOI:

10.1126/sciadv.abd770; Catalog of Somatic Mutations in Cancer (COSMIC) signatures SBS7a or ID13. cancer.sanger.ac.uk/signatures).

[0143] In some embodiments utilizing NGS for the quantification of SMB, targeted sequencing need not be used. In some embodiments, somatic mutation burden is calculated from whole genome sequencing data. In some embodiments, the whole genome sequencing data comprises 30x, 60x, lOOx, 200x, 500x coverage depth from a high throughput NGS platform (e.g., Illumina, Ultima, Element, Singular Genomics, Ion Torrent, etc.). In some embodiments, the whole genome sequencing leverages ultra-high fidelity NGS. Examples of this include data produced by the PacBio Onso platform or sequencing error correction methods (z.e., duplex sequencing (Twin Strand Bio), linked paired-strand sequencing, CODEC, bottleneck sequencing, or similar method). In some embodiments, the ultra-high fidelity NGS achieves an error rate of <lE-4, <lE-5, <lE-6. In such cases, a lower sequencing depth may be employed. For example, in some embodiments, SMB is calculated using the PacBio Onso platform and whole genome sequencing of approximately 0.01X, 0.05X, 0.1X, 0.5x, lx, 2x, or lOx coverage. In further embodiments, high fidelity sequencing may be achieved through single molecule consensus sequencing (e.g., PacBio HiFi). In some embodiments, somatic mutation burden is calculated from RNA-Seq data. In embodiments where UV-specific dinucleotide mutations are used to calculate SMB, the SMB may be calculated using low pass WGS sequencing (e.g., 0.01X, 0.05X, 0.1X, 0.5x, lx, 2x, or lOx coverage) with a next generation sequencing device (e.g., Illumina).

[0144] In some embodiments, the mutation burden values for each sample are expressed as the number of detected mutations per number of analyzed nucleotide bases. For example, in some embodiments, the mutation burden is expressed as the number of COTT dinucleotide mutations per number of analyzed CC dinucleotides. In some embodiments, analyzed bases are those deriving from a read mapping with high confidence to a single location within a reference which is also of high sequence quality, as determined by a PHRED score. For example, in some embodiments, after alignment of reads to a human genome (e.g. via bwa- mem) the number of analyzed bases is defined as those deriving from reads mapping to the human genome with a MQ>30 and a PHRED quality score >30.

[0145] In some embodiments, to further reduce noise owing to differences in GC sequencing bias across sequencing runs, the number of analyzed bases reflects the number of CC or GG dinucleotides passing read alignment and base quality filters.

[0146] In some embodiments, SMB is calculated by image analysis of tissue. In some embodiments, the sample comprises skin tissue or a section of skin tissue, and analysis comprises treatment with H&E staining followed by brightfield imaging, with SMB inferred by comparing the tissue morphological features to morphological features of other samples that were previously assessed for SMB by sequencing. In some embodiments, the analysis makes use of IHC, FISH, tissue autofluorescence, spatial multiomics (e.g., via lOx Genomics Xenium or Nanostring Cosmix), or combination thereof. In some embodiments, morphology is analyzed by a pathologist. In other embodiments, tissue morphology analysis is performed using a computer image analysis algorithm (e.g., an Al-assisted image analysis algorithm). In some embodiments, SMB is estimated by dermatoscopy.

[0147] In some embodiments, the somatic mutation score integrates SMB measurements from multiple tissue types. For example, in some embodiments, SMB measurements are obtained from skin (e.g., via non-invasive skin sampling), lung (e.g., via lung biopsy), mouth (e.g., via analysis of human gDNA from saliva), intestinal epithelial cells (e.g., via analysis of human gDNA from stool), bladder (e.g., via urine cfDNA analysis), blood (e.g., via blood plasma cfDNA analysis). In further embodiments, the SMB score is combined with other patient characteristics to further refine a patient’s risk of disease or likelihood to respond to a given therapy. For example, in an individual with cancer, the SMB may be combined with demographic information, ECOG status, smoking status, tumor mutational profiling, gene expression profiling of the tumor microenvironment, immune repertoire sequencing of the tumor microenvironment, immune repertoire sequencing of peripheral blood, flow cytometry analysis of immune cells, tumor histopathology, Al-assisted image analysis of tumor, single cell analysis of blood or tissue, cfDNA sequencing of blood plasma or urine, MSI and dMMR status, as non-limiting examples.

[0148] In some embodiments, the SMB value may be used to determine whether to administer a therapy for a disease. In some embodiments, a decision whether to administer a therapy for a disease is made based on the SMB value. In some embodiments, a decision whether to administer a therapy for a disease is made when the SMB is above a threshold. For example, in some embodiments, the SMB is used to determine whether to administer an immunotherapy regimen for cancer. In such embodiments, the SMB value for the individual is compared to the distribution of SMB values of individuals having the same cancer type known to have responded to the immunotherapy using an established response quantification system such as irRECIST, and compared again to the distribution of SMB values of individuals having the same cancer type known to not have responded to the immunotherapy by the same quantification system. The individual is decided to receive the immunotherapy if the SMB value obtained from the individual is more similar to the SMB values of the responding population than the non-responding population. In other embodiments, the SMB value obtained from an individual is compared to the values obtained from analysis of demographically matched members of the general population. If the value exceeds a predetermined threshold within the upper portion of the distribution of values (e.g., the upper quintile, the upper quartile, the upper tertile, as non-limiting examples), the individual is selected to receive the immunotherapy, while individuals having an SMB value below the predetermined threshold are selected to receive an alternative to immunotherapy (e.g., chemotherapy).

[0149] In some embodiments, the SMB score of an individual may integrate patient demographic, medical, and lifestyle metadata. For example, in some embodiments, a SMB value from analysis of a skin sample is combined with categorical data indicating whether the subject has a history of UV-exposure associated skin neoplasms such as basal cell carcinoma, actinic keratosis or squamous cell carcinoma, where the presence of a UV-exposure associated neoplasm increases the mutation burden score of the subject. In some embodiments, a skin mutation burden score may integrate information related to self-reported sunscreen usage, Fitzpatrick skin phototype, environmental UV intensity at region of current or childhood residence, suntanning history, and time spend outdoors, as non-limiting examples. [0150] In some embodiments, an individual identified as having an SMB value below the predetermined threshold is prescribed UV radiation therapy to increase the skin SMB level above the threshold, after which the individual undergoes a confirmatory SMB test and subsequently receives the immunotherapy. In other embodiments, the SMB is used to determine whether to apply a therapy for an infectious disease. For example, individuals with a low SMB value may be at greater risk of death from an infectious agent, and should be treated and monitored more aggressively than individuals with a high SMB value. In yet other embodiments, the SMB is used to determine whether to apply a therapy for an autoimmune disease. In some embodiments, the SMB is used to determine a patient’s risk of developing a disease. In further embodiments, the patient’s estimated risk of developing a disease is used to determine whether to administer a prophylactic. In some cases, the prophylactic is an immunotherapy (e.g., an immune checkpoint inhibitor). In other cases, the prophylactic immunotherapy comprises treatment with UV (e.g., via a UV bed). In yet other cases, the immunotherapy comprises treatment of phenotypically normal, sun exposed skin with a topical immunomodulatory agent to stimulate skin-resident disease antigen-specific immune cells (e.g., imiquimod).

VII. Additional Embodiments a. Method of reducing infectious disease in livestock

[0151] Infectious disease in livestock may cause widespread economic loss, food insecurity, animal suffering, and facilitate the transmission of zoonotic disease to humans. Vaccination of livestock with disease-associated antigens or live attenuated virus is a leading method for preventing infectious disease, though it is costly, technically challenging, requires lengthy development, and cannot protect against unknown or newly-emergent viruses or bacteria. Highlighting the limitations of traditional vaccine development, African Swine Flu has caused widespread damage to the global swine industry, yet despite years of research an effective vaccine remains unavailable.

[0152] This disclosure provides novel methods to reduce infectious disease in livestock through stimulation of adaptive immunity. The methods leverage the insight that neoantigens within the skin may stimulate adaptive immunity in a manner that elicits protection against a wide range of infectious disease agents, including those that have not yet been encountered. [0153] The dual role of skin as both a protective barrier and a driver of adaptive immunity has led to the diversity of skin pigmentation present in humans and pigs. In these species skin neoantigens largely arise as a product of errors during the repair of DNA damage arising from solar UV-A and UV-B radiation. The immune-stimulatory benefit of DNA-damage- derived neoantigens - which reduce mortality from infectious disease - is counterbalanced by a detrimental, though temporary, reduction in fertility caused by the consumption of folate during the DNA damage repair process, the depletion of which reduces fertility (Jablonski and Chaplin, PNAS 107(2): 8962-8968, 2010). Owing to these opposing evolutionary forces, the level of skin pigmentation yielding the highest fitness in a given population is determined by the population-specific level of solar UV radiation exposure and disease pressure (e.g., local disease pressure). Illustrating this trend, the domestication and use of pigs in high- density farming, where disease pressure is elevated, has led to the emergence of pinkskinned, largely hairless pigs, while their wild counterparts, which live at low density and correspondingly lower disease pressure, tend to have dark colored skin and longer hair favoring fertility over disease resistance.

[0154] With this understanding, the present disclosure provides method for reducing disease in a farmed animal comprises contacting the skin of the animal with a mutagen, thereby generating a plurality of immune-stimulatory neoantigens in the skin. In some embodiments, the mutagen comprises solar UV-A radiation, solar UV-B radiation, or UV-A or UV-B radiation derived from an artificial source.

[0155] Recognizing the divergent consequences of DNA damage - increasing adaptive immunity and temporarily reducing fertility - and the needs of a livestock farmer allows one to identify advantageous embodiments of the method. A livestock farmer aims to maximize the number of animals brought to market. To do so the farmer must maximize animal fertility while also minimizing animal loss due to infectious disease. To satisfy both demands, in one embodiment, the method comprises contacting the skin of the farmed animal with a mutagen before the animal is of reproductive age, or when reproduction or maximizing fertility of a given animal is not an objective (e.g., applying UV radiation to adult male pigs that will not be used for breeding, while sparing the breeding sows).

[0156] Methods may be used to facilitate the generation of solar UV radiation derived skin neoantigens. As non-limiting examples, for animals with significant body hair, the hair may be shaved or sheared before the animals are exposed to sunlight; a farm may be designed to place reproductively immature animals e.g., piglets) outdoors for a significant portion of the time, while reproductive age adults are maintained indoors to minimize solar exposure and maximize fertility; the indoor pens of reproductively immature animals may exclusively receive artificial UV light.

[0157] The optimal timing of the UV exposure(s) and amount of UV applied per exposure may be determined empirically for each breed of animal. As a non-limiting example, for a breed of pig, in some embodiments, the optimal amount of UV applied per day is defined as the maximal amount that may be tolerated by a pig of that breed without sign of significant discomfort (e.g. reduced appetite, skin blistering). In other embodiments, the optimal amount of UV applied per day is determined by exposing groups of pigs of that breed to different amounts of UV per day, then assessing the level of adaptive immune stimulation of each group via one or more biomarkers. For example, in some embodiments, the optimal level of UV exposure for a pig breed is determined by exposing pigs of that breed to varying amounts of UV per day, inoculating the pigs against an antigen, then measuring the level of antibodies raised against that antigen. In some embodiments, the inoculation comprises a vaccine against African Swine Flu, and the immune response is determined by challenge with the virulent parental virus, e.g. as described in the USDA research project number #445017.

[0158] In principle, any mutagen that gives rise to neoantigens in skin may be used to stimulate adaptive immunity. For example, in some embodiments, the mutagen could comprise a chemical mutagen such as 4-nitroquinoline-l -oxide (Wang et al., Journal of Investigative Dermatology 127(1): 196-205, 2007). In practice, the use of a chemical mutagen is often impractical owing to the increased hazard to humans and complexity of application. Thus, in preferred embodiments, the mutagen comprises UV radiation. b. Method of providing cancer immunotherapy by disrupting CLA function

[0159] There is an outstanding need for safe and effective methods to treat cancer. Recently, immunotherapy has emerged as a promising treatment modality, though only a subset of individuals responds favorably to existing therapies, highlighting the need for novel therapeutic strategies.

[0160] The present disclosure describes a novel method for eliciting anti-cancer responses through disruption of T cell homing to the skin. The method leverages the surprising finding that the skin is an important source of anti-cancer T cells. [0161] A subset of T cells expresses cutaneous lymphocyte antigen (CLA), which is responsible for T cell homing to the skin (Fuhlbrigge et al., Nature 389(6654): 978-981, 1997). The homing activity of CLA is believed to prevent the migration of skin resident CLA+ T cells to other sites in the body, resulting in a population of so-called permanently skin resident T cells. This CLA+ permanently skin resident T cell population may harbor T cells capable of recognizing cancer arising elsewhere in the body. By disrupting the homing activity of CLA, such cells are freed to traverse the body and destroy cancer cells in individuals with cancer.

[0162] Methods for disrupting the function of a protein of interest are known to those familiar in the art, and may include application of monoclonal or polyclonal antibodies, small molecules, aptamers, or compounds which react with and disrupt a target protein. Other methods may indirectly disrupt the function of a protein through downregulation of the gene encoding the protein or a regulator of that gene. When the objective is to disrupt the function of a receptor-ligand, agents may be engineered to target a protein receptor of interest or its cognate ligand (e.g., PD-1/PD-L1). When the agent is intended for human use, care must be taken to minimize off-target effects or toxicity. Methods to achieve this objective may include antibody humanization or application of an agent locally rather than systemically. For example, if the objective is to modulate the activity of skin resident T cells, it may be advantageous to apply an agent topically to skin rather than systemically through oral administration.

[0163] Methods for targeting CLA in an in vitro research setting are known in the art. For example, the monoclonal rat antibody, HECA-452 has been used extensively in in vitro studies to bind and disrupt the activity of human CLA. (e.g., see, Berg et al, J. Exp. Med. 174(6): 1461-1466, 1991, and the HECA-452 monoclonal antibody from ThermoFisher, Catalog # 14-9857-82).

[0164] Vitamin D pathway agonists such as Calciferol are known to downregulate CLA expression. (Yamanaka et al., J. Allergy Clin. Immunol. 121(1): 148-148-157, 2007). Calciferol and other vitamin D pathway agonists may be applied topically to treat T cell mediated skin lesions such as psoriasis, where they are believed to prevent excess skin cell proliferation, reduce immune cell activity within the lesion, and impede infiltration of the lesion by pathogenic T cells. Non-limiting examples of vitamin D pathway agonists include Calcitriol 1,25(OH)2D, EB1089, MC903, 22-oxacalcitriol, BGP-13, R024-2637, 19-nor-14- epi-23-yne-l,25(OH)2D3 (TX 522, inecalcitol) and 19-nor-14,20-bisepi-23-yne- 1,25(OH)2D3 (TX 527) (Trump, Bone Reports 9: 110-119, 2018).

[0165] Separately, the diverse hypothesized anti-cancer mechanisms of vitamin D pathway agonists (Trump, Bone Reports 9: 110-119, 2018; Deeb et al., Nature Reviews Cancer 7: 684- 700, 2007) have driven exploration of their utility when applied systemically (via oral administration) for the treatment of cancer, though to date numerous clinical trials have failed to demonstrate clinical benefit. Importantly, owing to the dosage and mode of delivery, the vitamin D bioavailability achieved by these studies was insufficient to downregulate CLA on skin resident T cells. By contrast, topical application of the vitamin D agonist may achieve a far greater bioavailability of the agonist in the skin and achieve a dosage sufficient to downregulate the expression of CLA on skin-resident T cells. Thus, in some embodiments, disruption of CLA on skin resident T cells is achieved by topical application of a vitamin D agonist to healthy skin of an individual with cancer.

[0166] In one aspect, the present disclosure provides a monoclonal antibody or antigenbinding fragment thereof, which binds to and disrupts the activity of human CLA. In some embodiments, the antibody comprises the CDR1, CDR2, CDR3 regions of the antibody HEVA-452 within a humanized heavy and light chain backbone. In some embodiments, the antibody is expressed as IgG4. In one aspect, the present disclosure provides a small molecule which binds to and disrupts the activity of CLA. In another aspect, the present disclosure provides a method of administering an agent comprising an antibody or antigenbinding fragment thereof, which binds to and disrupts the activity of human CLA, or a small molecule which binds to and disrupts the activity of CLA to a subject. In some embodiments, the method comprises topical application of the agent to skin. In another aspect, the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof which downregulates the expression of CLA. In another aspect, the present disclosure provides a small molecule which downregulates the expression of human cutaneous lymphocyte antigen (CLA). In some embodiments, the antibody or small molecule is a vitamin D pathway agonist applied topically to healthy skin for the treatment of cancer. In some embodiments, the cancer is a solid tumor or a hematologic malignancy. c. Method for improved consumer genetics health test

[0167] Consumer genetics health profiling tests (e.g., AncestryHealth) aim to identify disease dispositions to enable proactive management of health. Current tests incorporate patient reported data with genome analysis to generate a disease risk profile. Current tests fail to capture and incorporate the adaptive immune health of an individual into the risk profile.

[0168] In one aspect, the present disclosure provides methods for an improved consumer genetics health profiling test that incorporates genotype, patient reported data, and adaptive immune health through measurement of skin mutation burden. In one aspect, the methods comprise steps of obtaining the skin mutation burden of an individual; determining the genotype of the individual at one or more locations within the genome; and analyzing the genotype and skin mutation burden values to generate a health profile. In some embodiments, the skin mutation burden and genotype are determined by next-generation sequencing. In some embodiments, the genotype is determined by imputation. In some embodiments, the genotype, skin mutation burden, and patient reported data is analyzed to generate a health profile. d. Method for improved genome wide association study

[0169] In some embodiments, Genome Wide Association Studies (GWAS) aim to identify genetic variations associated with phenotypes such as disease. A challenge in GWAS is the influence of environmental factors, which create noise or variation in the data that may obfuscate signal from causal variants. Inter-individual differences in skin mutation burden are an unappreciated and important source of environmental noise, particularly for studies aiming to identify variants related to immunity and cancer, given that skin mutation burden is a driver of adaptive immunity.

[0170] In one aspect, the present disclosure provides methods for improving the sensitivity of GWAS by detecting and compensating for the skin mutation burden value of an individual. In one aspect, the methods for identifying genomic variants associated with a trait, comprise steps of obtaining the skin mutation burden of each member of a group of individuals; obtaining the genotype of each member of the group of individuals; and performing a genome wide associated study for a trait of interest. In some embodiments, the skin mutation burden value of each individual is incorporated as a covariate. In some embodiments, the skin mutation burden value and genotype values are obtained by next-generation sequencing. In some embodiments, one or more genotypes are obtained through imputation. e. Method for improved patient selection for clinical trials

[0171] To provide meaningful results, a clinical trial must ensure that patients selected for treatment and control arms do not systematically differ with respect to relevant attributes. The methods provided in the present disclosure stem from the discovery that skin mutation burden (SMB) is an important driver of adaptive immunity and thus a potentially confounding variable.

[0172] In one aspect, the present disclosure provides methods to ensure that treatment and control arms are matched with respect to SMB, enabling more accurate and sensitive clinical trials. In one aspect, the methods comprise steps of determining the skin mutation burden of one or more candidate clinical trial participants and assigning the one or more patients to a treatment or control arm of a clinical trial based on their skin mutation burden value.

EXAMPLES

[0173] Example 1 : Non-invasive measurement of skin mutation burden via dermal tape

[0174] An embodiment of the disclosed method was applied to analyze the skin mutation burden of a 12-person cohort of healthy donors. Dermal tape disks (CuDerm D-Squame D101 Stripping Discs, clinicalandderm.com/dl01-d-squame-stripping-discs/) were used to collect skin cells from a sun exposed region (forearm or back of hand) and sun protected region (inner arm adjacent to armpit) from each donor. First, the skin region of interest was cleaned using an alcohol pad, as were the hands of the individual collecting the skin sample. For each region sampled, five 14mm diameter D-Squame D101 stripping discs were smoothed over the test region, then held in place firmly under moderate pressure for 2 minutes. (Fig. 3) Next, the discs were slowly removed from the skin by gripping and pulling the sticker via the non-adhesive white flanking portion of the disc in a fluent movement under constant force using a tweezer. After removal, the stickers were placed in a 15ml Falcon conical tube, then stored at -20°C prior to further processing.

[0175] Next, DNA was extracted from the discs using the QIAamp DNA micro kit (Qiagen, 56304). Cell lysis was performed by incubating the sample with proteinase K in a thermomixer for 60 minutes at 56 °C at 900 rpm. DNA extracted from up to 5 stickers from each region was pooled and quantified using the Qubit lx dsDNA HS Assay Kit (Thermo Fisher, Q33231), then approximately 2-20 ng of extracted DNA from each region was used to prepare NGS libraries via the IDT xGen DNA Library Preparation EZ UNI kit (IDT, cat #10009822). The resultant NGS libraries had a mode size of ~400bp, as determined by analysis via the Agilent Bioanalyzer 2100 (Fig.4). Next, libraries were pooled and sequenced using the Illumina NovaSeq 6000 with 2xl50bp reads. Following sequencing, reads were aligned to the human genome using the Illumina DRAGEN aligner. Aligned reads had a mode insert size of ~180bp, Fig.5. For each sample, reads mapping with mapping quality <30 were removed (DRAGEN parameter: — vc-min-read-qual 30) and the number of phased COTT mutations were identified via the DRAGEN germline variant caller (DRAGEN parameter -vc-combine-phased-variants-distance=2). Putative COTT mutations were further filtered to eliminate those overlapping with common SNPs, as determined by the dbSNP database (Kitts A, Phan L, Ward M, et al. The Database of Short Genetic Variation (dbSNP) 2013 Jun 30 [Updated 2014 Apr 3], In: The NCBI Handbook [Internet], 2nd edition. Bethesda (MD): National Center for Biotechnology Information), those overlapping with blacklisted regions of the genome, defined as regions of the genome of low mappability, or regions where spurious CC>TT dinucleotides were recurrently observed following sequencing of negative control samples such as peripheral blood. Following filtering, the raw number of CC>TT mutations per sample was tallied. Next, the raw number of detected COTT dinucleotide mutations per sample was normalized by the total number of high- quality mapped reads per sample to yield the normalized mutation burden per sample. As an additional negative control, the same analysis pipeline was applied to calculate the mutation burden of five healthy donor peripheral blood gDNA samples. Fitting expectation, sun exposed samples tend to have a higher CC>TT mutation burden than sun protected samples, which have a mutation burden similar to peripheral blood samples. Fig.6 indicates the raw number of CC>TT mutations detected per sample, while Fig.7 indicates the normalized mutation burden per sample, calculated as the number of detected CC>TT mutations divided by the number of aligned reads.

[0176] Finally, a mutation burden score was calculated for each donor, defined as the mutation burden of the sun exposed region minus the mutation burden of the sun protected region from the same donor. In the scenario where a sun protected region had a higher mutation burden score than the paired sun exposed region (e.g. owing to sampling noise in an individual having minimal skin UV damage), the donor was assigned a mutation burden score of 0. In the healthy donor cohort, mutation burden score values ranged from 0 to 20, with one third of the cohort having a mutation burden score of 0-5 (mutation burden low), one third having a mutation burden score from 6-10 (mutation burden medium), and one third having a mutation burden score >10 (mutation burden high), Fig.8. An overview of the bioinformatics analysis pipeline is presented in Fig.9. In some embodiments of the invention, individuals classified as mutation burden high have a greater probability of responding favorably to cancer immunotherapy, with a longer overall survival time, than individuals classified as mutation burden score low.

[0177] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Therefore, the description should not be construed as limiting the scope of the invention.

[0178] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entireties for all purposes and to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be so incorporated by reference.

Claims

WHAT IS CLAIMED IS

1. A method of detecting somatic mutation burden in a sample, comprising: a) obtaining one or more samples comprising skin cells from a subject with cancer that is a candidate for treatment with immunotherapy; b) sequencing DNA from the one or more samples; and c) detecting a level of somatic mutation burden in each of the one or more samples.

2. A method of detecting somatic mutation burden in a sample, comprising: a) obtaining one or more samples comprising skin cells from a subject having or suspected of having an autoimmune disease; b) sequencing DNA from the one or more samples; and c) detecting a level of somatic mutation burden in each of the one or more samples.

3. A method of detecting somatic mutation burden in a sample, comprising: a) obtaining one or more samples comprising skin cells from a subject that has or is at risk of contracting an infectious disease; b) sequencing DNA from the one or more samples; and c) detecting a level of somatic mutation burden in each of the one or more samples.

4. A method of selecting a subject for treatment, comprising: a) obtaining one or more samples from a subject; b) detecting a somatic mutation burden for each of the one or more samples; and c) selecting the subject for treatment based on the one or more somatic mutation burden values.

5. The method of any of claims 1-4, wherein obtaining comprises tape stripping, stickers, adhesive collection methods.

6. The method of any of claims 1-5, wherein detecting the level of somatic mutation burden in each of the one or more samples comprises detecting one or more UV-mediated dinucleotide or triplet mutations.

7. The method of any of claims 1-6, wherein the one or more samples comprise skin cells from one or more regions of the body subjected to UV exposure and one or more control samples, and determining the somatic mutation burden comprises: a) sequencing a first sample comprising skin cells from a first region of the body subjected to UV exposure to generate a first set of sequence reads; b) sequencing one or more control samples to generate a second set of sequence reads; and c) aligning the first and second sets of sequence reads to determine the somatic mutation burden of the first sample.

8. The method of any of claims 1-7, further comprising generating a somatic mutation burden score for the subject based on the somatic mutation burden.

9. The method of claim 8, wherein generating a somatic mutation burden score comprises weighing the somatic mutation burden for the one or more samples according to the sample location and area of skin represented by the sample.

10. The method of claim 1-9, wherein the one or more samples comprise skin cells from one or more regions of the body subjected to chronic UV exposure, one or more regions of the body subjected to intermittent UV exposure, or a combination thereof.

11. The method of any of claims 7-10, wherein the one or more control samples comprises skin cells from one or more regions of the body subjected to no UV exposure, skin cells from one or more regions of the body subjected to minimal UV exposure, or a combination thereof.

12. The method of claim 10 or 11, wherein the one or more regions of the body subjected to chronic UV exposure are selected from forehead, neck, forearms, hands, face, or a combination thereof.

13. The method of any of claims 10-12, wherein the one or more regions of the body subjected to intermittent UV exposure are selected from torso, back, thighs, ankles, or a combination thereof.

14. The method of any of claims 10-13, wherein the one or more regions of the body subjected to minimal or no UV exposure is selected from buttocks, armpit, or a combination thereof.

15. The method of any of claims 1-14, wherein the one or more samples are non- cancerous samples from the subject.

16. The method of any of claims 1-15, wherein the one or more samples comprise skin cells.

17. The method of claim 1-16, wherein the one or more samples comprise lung cells.

18. The method of claim 1-17, wherein the one or more samples comprise intestinal epithelial cells.

19. The method of any of claims 1-18, wherein the one or more samples comprise extracellular nucleic acids.

20. The method of any of claims 1-19, wherein the one or more samples comprises cell- free DNA.

21. The method of claim 19 or 20, wherein the extracellular nucleic acids or cell-free DNA originates from one or more of skin cell, lung cells, and intestinal epithelial cells.

22. The method of any of claims 1-6, wherein the one or more samples comprises a control sample.

23. The method of claim 22, wherein the control sample comprises one or more regions of the body subjected to minimal or no UV exposure.

24. The method of any of claims 1-22, wherein obtaining is performed by a non-invasive method.

25. The method of any of claims 1-24, wherein determining the somatic mutation burden comprises extracting genomic DNA (gDNA), mitochondrial DNA, or RNA from the one or more samples.

26. The method of any of claims 1-25, wherein determining the somatic mutation burden comprises preparing sequencing libraries from the one or more samples.

27. The method of any of claims 1-26, wherein determining the somatic mutation burden comprises target enrichment.

28. The method of claim 27, wherein target enrichment comprises enrichment of target sequences by PCR, hybrid capture, on-sequencer enrichment, or a combination thereof.

29. The method of any of claims 1-28, wherein determining the somatic mutation burden comprises performing next-generation sequencing (NGS), high-fidelity sequencing, or digital droplet PCR.

30. The method of claim 4, wherein the one or more samples comprises a section of a tissue.

31. The method of claim 4, wherein one or more samples comprises an image of a section of a tissue.

32. The method of claim 30 or 31, wherein the one or more somatic mutation burden values are determined by image analysis of tissue.

33. The method of claim 32, wherein image analysis is performed using Al-assisted image analysis.

34. The method of claim 32, where image analysis is performed by immunohistochemistry or FISH.

35. The method of any of claims 4-34, wherein the treatment is an infectious disease treatment or prophylaxis thereof.

36. The method of claim 35, wherein the infectious disease is HIV.

37. The method of any of claims 4-34, wherein the treatment is a non-infectious disease treatment or prophylaxis thereof.

38. The method of claim 37, wherein the non-infectious disease is cancer or an autoimmune disease.

39. The method of any of claims 4-34, wherein the treatment is anti-aging treatment.

40. The method of claim 39, wherein the anti-aging treatment prevents age-related cellular damage.

41. The method of any of claims 1-40, wherein the treatment comprises immunotherapy.

42. The method of claim 41, wherein the immunotherapy comprises immune checkpoint inhibitors, T-cell transfer therapy, monoclonal antibodies, vaccines, and immune system modulators.

43. A method for determining the prognosis of a subject diagnosed as having a disease comprising: a) obtaining one or more samples from a subject; b) detecting a somatic mutation burden for each of the one or more samples; c) identifying the subject as having poor prognosis when the somatic mutation burden is below a threshold.

44. A method of determining the risk of a disease in a subject, comprising: a) obtaining one or more samples from a subject; b) detecting a somatic mutation burden for each of the one or more samples; c) identifying the subject as having high-risk of a disease when the somatic mutation burden is below a threshold.

45. A method for predicting a response to a treatment, comprising: a) obtaining one or more samples from a subject; b) detecting a somatic mutation burden for each of the one or more samples; c) predicting the subject as having a favorable response to the treatment when the somatic mutation burden score is above a threshold.

46. The method of any of claims 39-41, wherein the one or more samples comprise skin cells from one or more regions of the body subjected to UV exposure and one or more control samples, and determining the somatic mutation burden comprises: a) sequencing a first sample comprising skin cells from a first region of the body subjected to UV exposure to generate a first set of sequence reads; b) sequencing a control sample to generate a second set of sequence reads; and c) aligning the first and second sets of sequence reads to determine the somatic mutation burden of the first sample.

47. The method of any of claims 43-46, further comprising generating a somatic mutation burden score for the subject based on the somatic mutation burden.

48. The method of claim 47, wherein generating a somatic mutation burden score comprises weighing the somatic mutation burden for the one or more samples according to the sample location and area of skin represented by the sample.