WO2012040318A2 - Compositions, methods and kits for detecting melanoma and margins of melanoma - Google Patents

Abstract

Described herein are compositions, methods and kits for detection of melanoma and determination of melanoma margins. The present invention relates to a novel panel of volatile metabolic biomarkers that can be used in the diagnosis of melanoma skin cancer. A novel approach to detect melanoma is based on volatile by-products of altered cancer metabolism. This invention further provides a method for identifying molecules useful in the detection of melanoma and sets a foundation for development of a non-invasive detection technology, a biosensor, for melanoma diagnosis. Uses for this technology include a diagnostic screen that will help clinicians to assess this disease. One advantage provided by the compositions, methods and kits described herein is that metabolites are at the end phase of the genome-transciptome-proteome-metabolome cascade and thus are the most predictive of the cancer phenotype.

Description

COMPOSITIONS, METHODS AND KITS FOR DETECTING MELANOMA AND

MARGINS OF MELANOMA

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of Provisional Application Serial No. 61/385,811 filed September 23, 2010, which is herein incorporated by reference in its entirety.

FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant no. 1R21CA132046- 01A1 awarded by The National Cancer Institute and National Institutes of Health. The U.S. government has certain rights in the invention.

FIELD OF THE FNVENTION

[0003] The invention relates generally to the fields of molecular genetics, molecular biology, and medicine.

BACKGROUND

[0004] The National Cancer Institute estimated that in 2009 over 68,000 new cases will be diagnosed, and over 8,600 people will die of melanoma. Melanoma is the most serious form of skin cancer, and despite significant advances in education, diagnosis and treatment, the incidence of melanoma is increasing and mortality rates have not been significantly reduced. Given its origin, as a lesion on the surface of the skin, melanoma presents a unique opportunity for early detection (Halpern, A.C. and J.A. Lieb, Curr Opin Oncol 2007 19(2): p. 109-115). Early detection is critical for a positive outcome for patients with melanoma because the survival rate is related to the melanoma stage and depends directly on melanoma thickness. There is a general consensus of melanoma specialists that the greatest immediate impact on mortality can be achieved through early diagnosis.

[0005] In terms of melanoma diagnosis, several molecular markers of melanoma progression have been identified (Bernard et al., Cancer Res 2003 63(20): p. 6716-6725; DeRisi et al., Nat Genet 1996 14(4): p. 457-460) Many of these markers are not specific for melanoma (Nozawa et al, J Pathol 1996 178(l):p. 48-52; Skaar et al, J Steroid Biochem Mol Biol 1998 67(5-6): p. 391-402) and most of them have poor specificity (i.e. they would yield false positives as diagnostics). On the other hand, tyrosinase, a highly specific marker for melanocytes, showed very poor sensitivity (i.e. the protein is expressed at levels too low for reliable detection) (Tsao et al., Arch Dermatol 2001 137(3):p. 325-330). To date, most tumor markers are not sensitive or specific enough to be used independently for cancer screening.

[0006] Classification and staging of melanoma is currently based on a number of parameters like the ABCD system, Breslow thickness, mitotic rate, ulceration, and Clark level. Some malignant melanoma variants like spitzoid, desmoplastic, regressed, small cell, varicose melanoma, and verrucous naevoid melanoma, can mimic benign lesions and are difficult to diagnose (Blessing et al, J Clin Pathol 2000 53(8): p. 591-595). On the other hand, a small number of pigmented skin lesions, difficult to classify because of their unusual features and resemblance to melanoma, can be misdiagnosed as melanoma (e.g. Spitz naevi) (Edwards, S.L. and K. Blessing, J Clin Pathol 2000 53(6): p. 409-418; Barnhill et al, Hum Pathol 1999 30(5):p.513-520). A number of diagnostic markers can be used in order to separate difficult lesions e.g. nuclear DNA content; chromatin compactness, karyometry; transcriptional activity of nuclear organizer regions - argyrophilic staining; ΜΙΒ1-ΚΪ67, bcl-2, HBM45, PCNA, p53, cyclin dl and S100A6 immunoreactivity (Li et al., Melanoma Res 2003 13(6):p. 581-586; Dahlstrom et al, Pathology 2004 36(5): p. 452-457). However, there is no single technique that can be used for melanoma diagnosis.

[0007] Clinical evaluation of melanoma using the current standards is flawed, and a need exists for improving melanoma diagnosis. Success using traditional methods depends on the experience of the clinician and requires special training. Despite this, a number of studies have demonstrated a lack of consensus between clinicians even on the diagnosis of the same tissue sample (Farmer et al, Hum Pathol 1996 27(6): p. 528-531). Thus, all studies of clinical evaluation of melanoma rely on experienced dermatologists making the diagnosis. However, access to dermatologists is extremely limited; nationwide, there is a median wait of 6 weeks to see a dermatologist (Tsang, M.W. and J.S. Resneck, Jr., J Am Acad Dermatol 2006 55(l):p. 54- 58).

SUMMARY

[0100] Described herein are compositions, methods and kits for detection of melanoma. The present invention relates to a novel panel of volatile metabolic biomarkers (volatile compounds or metabolites) that can be used in the diagnosis of melanoma skin cancer. A novel approach to detect melanoma is based on volatile by-products of altered cancer metabolism. This invention further provides a method for identifying molecules useful in the detection of melanoma and sets a foundation for development of a non-invasive detection technology, a biosensor (e.g., one or more biosensors), for melanoma diagnosis. Uses for this technology include a diagnostic screen that will help clinicians to assess this disease. Described herein is a novel approach for detecting metabolites of melanoma and demonstration of a proof of principle that a differential metabolic signature of melanoma does indeed exist. These results support the hypothesis that volatile metabolites change as a result of the cancerous process. This altered volatile signature can be used for the development of a new diagnostic tool. It is particularly important for melanoma, since early detection of melanoma is critical for a positive outcome for patients. The results indicate that combining head space solid phase micro-extraction (HS-SPME) with gas chromatography/mass spectrometry (GC/MS) to detect volatile signatures from naevi and melanoma tissue is a valid approach. In this study, it was shown that after volatile collection, tissue can be used for histology analysis. Thus, volatile collection and analysis do not change tissue appearance and do not interfere with standard clinical procedures related to melanoma diagnosis. As described in the experiments described below, one example of a compound that can serve as a biomarker for detecting melanoma is dodecane. This simple alkane was detected in both fresh and frozen melanoma by all statistical tests used (Chi-square test, t-test and fuzzy logic). This compound is a candidate marker of melanoma in addition to 4-methyl decane, and undecane. Also reported herein is a case study, the analysis of the volatile metabolic signature of a malignant melanoma using matched, non-neoplastic skin tissue from the same patient as a control. Use of non-neoplastic skin tissue from the same patient as a control may be particularly useful, since it is well known that diet, skin type, genetic background, age, sex and environment all contribute to individual variation in the skin volatile signature. In the present study, 32 volatile compounds were identified; 9 volatile compounds were increased in melanoma when compared to normal skin and 23 volatile compounds were detected only in melanoma and not in normal skin. One advantage provided by the compositions, methods and kits described herein is that metabolites are at the end phase of the genome-transcriptome-proteome-metabolome cascade and thus are the most predictive of the cancer phenotype.

[0008] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. [0009] The terms "patient," "subject" and "individual" are used interchangeably herein, and mean a mammalian (e.g., human) subject to be treated, diagnosed and/or to obtain a biological sample from.

[0010] As used herein, "bind," "binds," or "interacts with" means that one molecule recognizes and adheres to a particular second molecule in a sample or organism, but does not substantially recognize or adhere to other structurally unrelated molecules in the sample. Generally, a first molecule that "specifically binds" a second molecule has a binding affinity greater than about 10⁸ to 10¹² moles/liter for that second molecule and involves precise "hand-in- a-glove" docking interactions that can be covalent and noncovalent (hydrogen bonding, hydrophobic, ionic, and van der waals).

[0011] As used herein, the terms "diagnostic," "diagnose" and "diagnosed" mean identifying the presence or nature of a pathologic condition (e.g., melanoma).

[0012] By the term "headspace" is meant the space above a tissue or melanoma. The tissue or melanoma can be isolated from a subject, or volatiles can be collected in the headspace above the melanoma lesion without prior tissue isolation ("in situ").

[0013] As used herein, the terms "volatile compound" and "volatile metabolite" are used interchangeably to mean any compound which has vapor pressure >0. lmmHg.

[0014] Accordingly, described herein is a method of detecting melanoma in at least one subject suspected of having or at risk of having melanoma. The method includes (a) obtaining a biological sample (e.g., a frozen or fresh punch biopsy) from the at least one subject; (b) collecting volatile compounds from the headspace of the biological sample; (c) measuring the levels of a plurality of volatile compounds collected; (d) identifying volatile compounds that are present at increased or decreased levels relative to a control sample (e.g., non-neoplastic skin tissue from the subject); and (e) correlating the presence of one or more volatile compounds that are present at increased or decreased levels relative to a control sample with the presence of melanoma in the at least one subject. In a typical method, melanoma is detected in the subject at an early stage. The volatile compounds collected include one or more of the volatile compounds listed in Tables 3, 4, and 6-12 (e.g., dodecane, 4-methyl decane, and undecane; 1-Hexadecanol, Benzene, 1,3,5-trimethyl, and dodecane; Bis(2-ethylhexyl)phthalate, decane, undecane, decane,4-methyl, ethylene oxide, isopropyl palmitate, and phthalic acid,isobutyl 4-octyl ester, etc.). Step (b) of collecting volatile compounds from the headspace of the biological sample can include Head-space Solid Phase Micro-Extraction (HS-SPME), step (c) of measuring the level of each volatile compound can include Gas Chromatography/Mass Spectrometry (GC-MS), and step (d) of correlating the presence of one or more volatile metabolites that are present at increased or decreased levels relative to a control sample with the presence of melanoma in the at least one subject can include use of a software program. In the method, the at least one subject can be a plurality of subjects suspected of having or at risk of having melanoma. The method can further include step f) of performing a histological analysis of the biological sample.

[0015] Also described herein is a method of detecting at least one margin of a melanoma on a subject. The method includes the steps of: a) providing a biosensor comprising a substrate having a plurality of different olfactory receptors bound thereto, each olfactory receptor specific for a different volatile compound selected from the group consisting of: a volatile compound listed in Table 3, a volatile compound listed in Table 4, a volatile compound listed in Table 6, a volatile compound listed in Table 7, a volatile compound listed in Table 8, a volatile compound listed in Table 9, a volatile compound listed in Table 10, a volatile compound listed in Table 11, and a volatile compound listed in Table 12 wherein each olfactory receptor is conjugated to a detectable label; (b) placing the biosensor over a plurality of areas of skin on the subject, each area an increasing distance away from the center of the melanoma; c) identifying volatile compounds collected from the headspace of each area that are present at increased or decreased levels relative to volatile compounds collected from headspace of non-neoplastic skin tissue from the subject; d) for each area, correlating the presence of one or more of the volatile compounds that are present at increased or decreased levels relative to volatile compounds collected from headspace of non-neoplastic skin tissue from the subject with the presence of melanoma cells in the area; and (e) identifying at least one area that does not include volatile compounds that are present at increased or decreased levels relative to volatile compounds collected from headspace of non-neoplastic skin tissue from the subject as at least one margin of the tissue. The volatile compounds can include one or more of the volatile compounds listed in Tables 3, 4, and 6-12 (e.g., dodecane, 4-methyl decane, and undecane). In the method, step (d) of correlating the presence of one or more of the volatile compounds that are present at increased or decreased levels relative to volatile compounds collected from headspace of non-neoplastic skin tissue from the subject with the presence of melanoma cells in the biopsy can include use of a software program. In a typical method, all margins of the melanoma are detected. [0016] Further described herein is a method of detecting at least one margin of a melanoma on a subject. The method includes the steps of: a) providing a biosensor including a substrate having a plurality of different olfactory receptors bound thereto, each olfactory receptor specific for a different volatile compound selected from the group consisting of: a volatile compound listed in Table 3, a volatile compound listed in Table 4, a volatile compound listed in Table 6, a volatile compound listed in Table 7, a volatile compound listed in Table 8, a volatile compound listed in Table 9, a volatile compound listed in Table 10, wherein each olfactory receptor is conjugated to a detectable label; (b) placing the biosensor over a plurality of areas of skin on the subject, each area an increasing distance away from the center of the melanoma; c) determining the presence or absence of a plurality of the volatile compounds listed in at least one of Tables 3, 4, and 6-12 in the headspace of each area; and d) for each area, correlating the presence or absence of the plurality of the volatile compounds listed in at least one of Tables 3, 4, and 6-12 in the headspace of each area with the presence or absence of melanoma cells in the area and determining at least one margin of the melanoma. Step c) of determining the presence or absence of a plurality of the volatile compounds listed in at least one of Tables 3, 4, and 6-12 in the headspace of each area can include determining the presence or absence of the plurality of the volatile compounds in the headspace of non-neoplastic skin tissue from the subject. The plurality of the volatile compounds can include, for example, dodecane, 4-methyl decane, and undecane.

[0017] Still further described herein is a biosensor for diagnosing melanoma in a subject. The biosensor includes a substrate having a plurality of different olfactory receptors bound thereto, each olfactory receptor specific for a different volatile compound selected from the group consisting of: a volatile compound listed in Table 3, a volatile compound listed in Table 4, a volatile compound listed in Table 6, a volatile compound listed in Table 7, a volatile compound listed in Table 8, a volatile compound listed in Table 9, a volatile compound listed in Table 10, a volatile compound listed in Table 11, and a volatile compound listed in Table 12, wherein each olfactory receptor is conjugated to a detectable label. The biosensor can further include a detector to detect the detectable label, wherein the volatile compounds include bis(2-ethylhexyl) phthalate, decane, undecane, decane-4-methyl, ethylene oxide, isopropyl palmitate, phthalic acid, isobutyl 4-octyl ester, 1-hexadecanol, benzene, 1,3,5 trimethyl, and dodecane. The detectable label can be fluorescence, the detector can be a fluorometer, and the biosensor can further include a processor to analyze the fluorescence. In one embodiment, at least 15 different volatile compounds are analyzed. The biosensor can further include at least one positive control and at least one negative control, as well as packaging and instructions for use.

[0018] Additionally described herein is a method of detecting melanoma in at least one subject suspected of having or at risk of having melanoma. The method includes: (a) providing at least a first biosensor including a substrate having a plurality of different olfactory receptors bound thereto, each olfactory receptor specific for a different volatile compound selected from the group consisting of: a volatile compound listed in Table 3, a volatile compound listed in Table 4, a volatile compound listed in Table 6, a volatile compound listed in Table 7, a volatile compound listed in Table 8, a volatile compound listed in Table 9, a volatile compound listed in Table 10, a volatile compound listed in Table 11, and a volatile compound listed in Table 12, wherein each olfactory receptor is conjugated to a detectable label; (b) contacting the at least first biosensor with volatile compounds from the headspace of a melanoma or tissue isolated from the at least one subject or from the headspace of a melanoma or tissue on the at least one subject; (b) detecting binding of one or more of the olfactory receptors with one or more of the volatile compounds; and (c) correlating binding of one or more of the olfactory receptors to one or more of the volatile compounds with the presence or absence of melanoma in the at least one subject. Step (c) of correlating binding of one or more of the olfactory receptors to one or more of the volatile compounds with the presence or absence of melanoma in the at least one subject can include correlating the presence of one or more volatile compounds that are present at increased or decreased levels relative to a control sample (e.g., a non-neoplastic skin tissue from the subject) with the presence of melanoma in the at least one subject. The method can further include contacting a second biosensor with volatile compounds obtained from headspace of nonneoplastic skin tissue from the same subject, detecting binding of one or more of the olfactory receptors with one or more of the volatile compounds from the headspace of non-neoplastic skin tissue from the same subject, and comparing the bound volatile compounds detected by the first and second biosensors. The melanoma or tissue isolated from the at least one subject can be a punch biopsy (e.g., a frozen biopsy or a fresh biopsy). In a typical method, melanoma is detected in the subject at an early stage. The volatile compounds can include one or more of the volatile compounds listed in Tables 3, 4, and 6-12 (e.g., dodecane, 4-methyl decane, and undecane; 1- Hexadecanol, Benzene, 1,3,5-trimethyl, and dodecane; Bis(2-ethylhexyl)phthalate, decane, undecane, decane,4-methyl, ethylene oxide, isopropyl palmitate, and phthalic acid,isobutyl 4- octyl ester, etc.). The at least one subject can be a plurality of subjects suspected of having or at risk of having melanoma. The method can further include step d) of performing a histological analysis of the melanoma or tissue.

[0019] Yet further described herein is a kit for detecting melanoma in a subject. The kit includes (a) at least a first biosensor including a substrate having a plurality of different olfactory receptors bound thereto, each olfactory receptor specific for a different volatile compound selected from the group consisting of: a volatile compound listed in Table 3, a volatile compound listed in Table 4, a volatile compound listed in Table 6, a volatile compound listed in Table 7, a volatile compound listed in Table 8, a volatile compound listed in Table 9, a volatile compound listed in Table 10, a volatile compound listed in Table 11, and a volatile compound listed in Table 12, wherein each olfactory receptor is conjugated to a detectable label; (b) at least one reagent for detecting binding of one or more of the olfactory receptors to one or more of the volatile compounds from the headspace of a melanoma on the subject or the headspace of a melanoma or portion of a melanoma isolated from the subject; and (c) instructions for use.

[0020] Although compositions, kits, and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable compositions, kits, and methods are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The particular embodiments discussed below are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 is a schematic illustration of a method for identifying melanoma biomarkers as described herein. GC/MS metabolic profiling results in complex chromato grams. For statistical analysis, signal intensity of the each peak was transformed into the logio of absolute ion counts in the area under the deconvoluted peak. Approximately 325 unique volatile compounds were identified from naevi, melanoma and skin samples. Differential volatile compounds were statistically identified based on the amplitude of the signal and on the frequency of appearance, as well as fuzzy logic analysis.

[0022] FIG. 2 is a plot and a photograph of a melanoma showing spatial mapping of biomarkers. [0023] FIG. 3 is a series of micrographs and a schematic illustration showing that the volatile collection preserves the tissue morphology. H&E staining of naevus (A and B) and melanoma (C and D). Histological analysis of the first punch biopsy sample placed immediately in formalin (A and C). Histological analysis of the second punch biopsy, after collection of volatiles (B and D). No obvious deterioration of the tissue samples was detected by the histopathologist. E. Volatile collection by HS-SPME method. Skin, naevi or melanoma 3 mm punch biopsy sample was placed in a small capped vial. PDMS-DVB fiber (red) was exposed to the head-space above the biopsy sample for 1 hour. After volatile collection, the fiber is retracted, and injected into GC/MS.

[0024] FIG. 4 is a series of micrographs and photographs, a series of chromatograms, and a series of graphs showing from melanoma and nevus to volatile signatures. Pictures from naevus (A) and melanoma skin lesion (D), H&E staining of biopsy from naevus (B) and melanoma lesion (E), chromatograms from naevus (C) and melanoma (F). Some peaks are unique in melanoma (***), some are increased (**) and some are decreased (*) in melanoma vs. naevi. (G, J and M) Chemical structure of pyridine, 3-hexanol and 2,5 dimethyl benzenamine, and their retention time in the chromatograms (indicated by the arrow). (H, K and N) Mass spectra of the indicated peaks (extracted spectrum, above) and mass spectra from the library (library hit and identification of the compound, bellow). (I, L and O) Frequency distribution of these three compounds in blank (B), melanoma (M) and naevi (N) group, as well as their expression analysis (log of integrated signal) are presented (t-test, mean±SEM). In (O) right panel n=16 for N and n=3 for M. Dimethyl benzenamine (2,5; 2,3; 2,4 or 2,6) is a volatile compound present in 19 out of 25 nevi samples, in 4 out of 5 melanoma samples and detected in only one air sample. A peak of 2,5 dimethyl benzenamine is shown eluting at 17.8 min. This compound is common in both melanoma and naevi group.

[0025] FIG. 5 is a series of graphs showing differentially expressed volatile compounds in melanoma vs naevi. The expression level of compounds as indicated by log of integrated signal, in melanoma group (black bars, n=5) and naevi group (grey bars, n=25) are shown in panels A, B and C. Data are expressed as mean ± STDEV, *p=0.05-0.09, **p=0.005 -0.05, ***p=0.0001- 0.005. In A: 2-propanamide* is 2-propanamide, 2-methyl; benzene** is benzene, 1,3 dimethyl and phthalate*** is bis(2-ethylhexyl) phthalate. [0026] FIG. 6 is a table, graph and a heat map showing results from a Fuzzy logic analysis of frozen skin and melanoma samples. A. list of the volatile compounds, their Goodman Kruskal Lambda values, the number of selections in all (38) leave-one-out runs, and the percentage of how often they were selected. B. Receiver operating characteristic curve (ROC). C. Heat map for the frozen data. Each column represents one sample. Each row represents one compound. Red colors represent retention time (RT) values that are high above the average; blue colors represent RT -values that are low and much below average. The first row represents the category; whether the sample belongs to the skin samples (left 20 columns with blue color in the first row) or to the melanoma samples (rights 18 columns with red color in the first row). The light blue color represents a missing value. Misclassified in the leave one out method are samples 4 and 12 from skin group, and samples 14 and 18 from the melanoma group.

[0027] FIG. 7 is a Venn diagram showing the number of volatile compounds specific for each tested group as well as the numbers of overlapping volatiles between the groups (e.g. naevi has 80 volatiles not expressed in any other group).

[0028] FIG. 8 is a pair of graphs and a pair of tables showing optimization of the HS-SPME conditions. (A) Effect of different fiber coating (PDMS/Carboxen and PDMS/DVB) on total ion count (TIC). (B) Comparative analysis of two different chromatograms obtained with different fiber coating from A. (C) Effect of sample size on total ion count (S/N ratio>5) (D) Change in % of TIC for volatile compounds analyzed from the same axilla (lymph node) sample (two biopsies) within 3 hours (black) and after 24 hours of biopsy (red) (sample was kept at +4°C).

[0029] FIG. 9 shows a comparison of volatile signatures from a malignant melanoma biopsy and nearby healthy non-neoplastic matching skin biopsy. A. Histology - H&E staining of the #2 proximal punch biopsy melanoma lesion (40X magnification). B. Full chromatogram of melanoma sample. Some compounds found to be differentially expressed in melanoma vs skin are numbered and indicated in the chromatograms. Their names and structures are presented in C. D. Biopsy sites from the right forearm. E. Histology - H&E staining of healthy, nonneoplastic skin showing signs of solar elastosis. F. Full chromatogram of the non-neoplastic healthy matched skin sample.

DETAILED DESCRIPTION

[0030] Described herein are compositions, methods and kits for detecting melanoma and determining melanoma margins. Based on the experimental results described below, volatile compounds emanating from a melanoma may be used as biomarkers when analyzing the headspace of a subject's melanoma in situ, or the headspace of an isolated tissue or melanoma sample for diagnosis of melanoma. The results described herein show an increase in methylated aromatic hydrocarbons (benzenes) and alkanes in melanoma. This is the first evidence of methylation of small molecules or metabolites that has been reported in connection with melanoma. Comprehensive volatile metabolomic studies may also assist in improving melanoma classification. Finding a correlation between volatile molecular signatures and clinical parameters of melanoma can be used to complement recent genotype-phenotype studies [17, 18] and ultimately lead to an improved targeted therapy.

Identification of Melanoma Biomarkers

[0031] The identification of multiple biomarkers of melanoma (multiple metabolites) is described herein. The process of collection, separation and identification of the volatiles metabolites in melanoma is described in detail in Example 1 below. Briefly, a 3 mm punch biopsy of melanoma lesion was performed. As a control, punch biopsy of naevi (moles) from healthy volunteers were used. The sample was placed in a small capped vial. After one hour of incubation at room temperature, the HS-SPME method was performed. The fiber with collected volatile metabolites was directly injected in the injection port of gas chromatograph (GC). Volatile compounds were eluted from the column, fragmented into ions and detected by the mass spectrometer (MS). A schematic presentation of the experimental protocol is shown in FIG. 1.

Methods of Detecting Melanoma

[0032] Described herein are methods of detecting melanoma in a subject (e.g., human) using biomarkers (volatile compounds or metabolites). Typically, the subject is suspected of or at risk of having melanoma. In methods of detecting melanoma in a subject, the presence and/or level of one or more volatile compounds as described herein is analyzed. For example, a volatile metabolite signature or profile can be used as a biomarker panel for diagnosing melanoma. A method of detecting melanoma in a subject can include the steps of: obtaining a biological sample from at least one subject; collecting volatile compounds from the headspace of the biological sample; measuring the level of one more (e.g., a plurality) of the volatile compounds; identifying volatile compounds that are present at increased or decreased levels relative to a control sample (e.g., non-neoplastic skin tissue from the same subject); and correlating the presence of one or more volatile metabolites that are present at increased or decreased levels relative to a control sample with the presence of melanoma in the at least one subject. However, in some embodiments, the presence or absence of volatile compounds is analyzed, rather than levels of one or more volatile compounds. Such a method of detecting melanoma in a subject can include the steps of: obtaining a biological sample from at least one subject; collecting volatile compounds from the headspace of the biological sample; determining the presence or absence of one or more of the volatile compounds listed in Tables 3, 4 and 6-12; and correlating the presence or absence of the one or more volatile compounds listed in Tables 3, 4 and 6-12 with the presence of melanoma in the at least one subject.

[0033] The control sample can be any suitable control sample. In a typical method, the control sample is non-neoplastic skin tissue from the subject. An alternative or additional control would be a mole/nevus. The biological sample can be a punch biopsy (a frozen biopsy or a fresh biopsy). In some embodiments, a melanoma is detected in the subject at an early stage. By the phrase "early stage" is meant preclinical or subclinical, prior to clinical intervention. This is typically defined by a Breslow thickness less than 1mm. The volatile compounds collected include one or more of the volatile compounds listed in Tables 3, 4, and 6-12, e.g., dodecane, 4- methyl decane, and undecane. In another example, the volatile compounds collected include 1- Hexadecanol, Benzene, 1,3,5-trimethyl, and dodecane. In another example, volatile compounds collected include Bis(2-ethylhexyl)phthalate, decane, undecane, 4-methyl decane, ethylene oxide, isopropyl palmitate, and phthalic acid, isobutyl 4-octyl ester. In yet another example, the volatile compounds whose presence or absence or concentration levels are analyzed are: 1- Hexadecanol, Benzene, 1,3,5-trimethyl, dodecane, Bis(2-ethylhexyl)phthalate, decane, undecane, 4-methyl decane, ethylene oxide, isopropyl palmitate, phthalic acid, and isobutyl 4-octyl ester. Any suitable number of volatile compounds can be analyzed, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, etc.

[0034] Any suitable biological sample (e.g., biopsy) can be used in the methods. Examples of biological samples include fresh tissue, frozen tissue, and skin, nevi (moles) and melanoma lesions (fresh or frozen). In some embodiments, a fresh sample and a frozen sample from a particular subject may be analyzed when attempting to diagnose melanoma in the subject. The steps of the method can be performed using any suitable protocol(s) or assay(s). In the experiments described below, the levels of volatile compounds that are present at higher or lower levels in headspace from melanoma samples relative to non-melanoma tissue were measured using GC-MS. In some embodiments, however, a method of diagnosing melanoma includes detecting the presence of one or more of the volatile compounds described herein and does not require measuring the levels of the volatile compounds. In one embodiment, the presence or absence of those volatile compounds listed in Table 10 and that are not indicated by an * or # is analyzed in a method of diagnosing melanoma. Examples of additional suitable assays or protocols for detecting the presence of the volatile compounds described herein and/or measuring their levels include olfactory receptor based detectors, and other related techniques. However, any suitable method or assay can be used to detect the presence of and/or measure the level of one or more of the volatile compounds described herein from (emanating from) a biological sample (e.g., punch biopsy) from a subject or a tissue or melanoma isolated from the subject. In some embodiments, biological samples from a plurality of subjects having melanoma, suspected of having, or at risk of having melanoma can be analyzed simultaneously, e.g., in a high- throughput format.

[0035] Whether or not one or more of the volatile compounds described herein is present at increased or decreased levels in the headspace of a subject's melanoma or tissue in situ or in the headspace of an isolated tissue or melanoma relative to control levels (levels from non- melanoma tissue or other control) can be determined by comparing the level of the volatile compound(s) in the headspace of the subject to a baseline level (also known as a control level) of the volatile compound. A "baseline level" is a control level, and in some embodiments a normal level or a level not observed in subjects having melanoma. Therefore, it can be determined, based on the control or baseline level of the volatile compound, whether a headspace to be evaluated for melanoma has a measurable increase or decrease in the level of that volatile compound, as compared to the baseline level. In certain embodiments, the baseline level can be established from a previous headspace from the subject being tested, so that the disease state of the subject can be monitored over time and/or so that the efficacy of a given therapeutic protocol can be evaluated over time.

[0036] In a typical embodiment, matched, non-neoplastic skin tissue from the same patient is used as a control. Use of non-neoplastic skin tissue from the same patient as a control may be particularly useful, since it is well known that diet, skin type, genetic background, age, sex and environment all contribute to individual variation in the skin volatile signature.

Biosensor For Detection Of Skin Cancer Melanoma [0037] Described herein is a biosensor for detecting (diagnosing) melanoma in a subject. A biosensor generally includes a substrate having a plurality of different olfactory receptors bound thereto, each olfactory receptor specific for a different volatile compound as described herein, i.e., one or more (e.g., 1, 2, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, etc.) of the following: a volatile compound listed in Table 3, a volatile compound listed in Table 4, a volatile compound listed in Table 6, a volatile compound listed in Table 7, a volatile compound listed in Table 8, a volatile compound listed in Table 9, and a volatile compound listed in Table 10, a volatile compound listed in Table 11, a volatile compound listed in Table 12, wherein each olfactory receptor is conjugated to a detectable label. Typically, the biosensor is an array of olfactory receptors that are specific for the volatile compounds described herein. The biosensor can be placed over a melanoma or area of skin on a subject, or exposed to an isolated tissue or melanoma. The olfactory receptors are thus exposed to any volatile compounds emanating from the skin, tissue or melanoma. Receptor activation is measured, i.e., receptors that are activated and those that are not activated are identified. A biosensor generally includes at least one positive control and at least one negative control and is usually packaged within an appropriate packaging material and optionally accompanied by instructions for use. A biosensor can further include or be operably connected to a detector to detect the detectable label (e.g., fluorescence). In one example of a biosensor, the detectable label is fluorescence, the detector is a fluorometer, and the biosensensor includes or is operably connected to a processor to analyze the fluorescence. Any detectable label, however, can be used. Detectable labels are well known in the art. A biosensor can be conveniently used by opening the packaging, exposing the biosensor to a melanoma or tissue on a subject (i.e., in situ) by placing the biosensor over the subject's body or an isolated sample of tissue or melanoma thus exposing the array of olfactory receptors to volatile compounds emanating from the tissue or melanoma, and measuring which olfactory receptors are activated or not activated by exposure to the volatile compounds.

[0038] A biosensor (e.g., one or more biosensors) based on olfactory receptors recognition as described herein can be used as a non-invasive diagnostic tool. It also presents an opportunity to facilitate reliable staging of melanoma, defining the melanoma surgical margins, diagnosis of other skin cancers, and possibly many other skin disorders. Olfactory receptors, a fluorescent reporter assay, and a device for reading the array and interpreting the pattern of activated receptors can be used in the context of common skin cancers or any skin disorder. A typical biosensor includes three components: (1) a disposable array of known/predicted olfactory receptors which act as biosensors, (2) a means for reporting binding of ligands of interest, and (3) a method for interpreting the array.

[0039] In one example of a biosensor, each spot of the array has a unique olfactory receptor/reporter system consisting of receptor proteins embedded in a lipid bilayer on the sampling plate. In this example, the reporter system includes an intrinsic fluorescent label built into the receptor that records the presence of melanoma biomarkers as receptor activation. The interpreting system contains a method to image the reporter olfactory receptor/reporter array and a database of known responses. The image of the reporter array can be obtained with an array of photodiodes or a small camera. The interpreting system is based on a neural network algorithm trained to associate the reporter array output with known stimuli (i.e. melanoma, benign nevi, squamous cell carcinoma, etc.). After exposure to an unknown test sample, the array reports which receptors have been activated. The interpreter searches a database of known stimuli to find the best match. See FIG. 2, which shows the possibility of margin detection. With the development of a biosensor, early and better detection practices are possible, as well as greater cooperation and communication between primary care physicians and patients themselves, as to minimize detection at the late stage of the disease. In addition to diagnosing melanomas, the biosensors described herein can be used to diagnose other skin disorders such as squamous cell carcinoma, basal cell carcinoma, psoriasis, Kaposi's sarcoma.

[0040] In some embodiments, a biosensor includes or is operably connected to an interpreting system. This interpreting system involves a method to image the reporter olfactory receptor/reporter array and a database of known responses. The image of the reporter array is obtained with an array of photodiodes or a small camera. The interpreting system is trained to associate the reporter array output with known stimuli (i.e. melanoma, benign nevi, squamous cell carcinoma, etc.) in order to build a database or neural network. After exposure to an unknown test sample, the array reports which receptors have been activated. The interpreter searches a database of known stimuli to find the best match.

[0041] A software program that can compare the result from each subject and indicate if melanoma is present or absent is typically used used. The output of HS-SPME GC/MS includes a chromatogram and associated mass spectra. The chromatogram is a time series of total-ion- count. After deconvolution (with AMDIS or similar software), the amplitude of a given peak reflects the amount of that particular substance identified from the mass spectrum associated with the peak. A software program will take into account a database of healthy control samples (mole and skin) and a database of melanoma samples. These databases will reflect the identity of compounds and their relative levels in each of the groups. The identification of melanoma will then be based on: (1) a statistical comparison of the subjects healthy skin sample and the test sample (suspected melanoma) and (2) a statistical comparison between these samples and the database of known samples. Based on the presence or absence of compounds in the test sample relative to the subject's healthy sample (frequency analysis) and on the level of compounds in the test sample relative to the healthy sample (amplitude analysis), melanoma can be identified using a software analysis program.

[0042] In one embodiment, a biosensor includes or is operably connected to an electronics package or processing unit including a processor coupled to a device (e.g., fluorometer) for measuring signals produced by binding between the olfactory receptors and the volatile compounds. The processing unit characterizes the signals and displays results on a monitor, for example. In some other embodiments, the results are produced graphically, numerically, or as positive or negative answers. The results may also be presented textually.

[0043] Also described herein is a method of detecting melanoma in at least one subject suspected of having or at risk of having melanoma that includes use of a biosensor as described herein. One example of such a method includes the following steps: contacting a first biosensor as described herein with volatile compounds obtained from the headspace of a melanoma or tissue isolated from the at least one subject (e.g., a fresh or frozen punch biopsy) or from the headspace of a melanoma or tissue on the at least one subject (in situ); detecting binding of one or more of the olfactory receptors with one or more of the volatile compounds; and correlating binding of one or more of the olfactory receptors to one or more of the volatile compounds with the presence or absence of melanoma in the at least one subject. The volatile compounds can be one or more of the volatile compounds listed in Tables 3, 4, and 6-12, e.g., dodecane, 4-methyl decane, and undecane, and/or 1-Hexadecanol, Benzene, 1,3,5-trimethyl, and dodecane, and/or Bis(2-ethylhexyl)phthalate, decane, undecane, decane,4-methyl, ethylene oxide, isopropyl palmitate, and phthalic acid,isobutyl 4-octyl ester. By using this method, melanoma can be detected in the subject at an early stage. In a typical method, matched, non-neoplastic skin tissue from the same subject (e.g., patient) is used as a control. Thus, the method can further include the steps of contacting a second biosensor with volatile compounds obtained from the headspace of non-neoplastic skin tissue from the same subject, detecting binding of one or more of the olfactory receptors with one or more of the volatile compounds, and comparing the volatile compounds detected by the first and second biosensors. In a typical method and/or system, two biosensor arrays are used. One biosensor would be positioned over the suspicious lesion and another over normal (non-neoplastic) skin. A measuring device would analyze the volatiles detected by the two sensors and make a decision based on the differences in the volatile signatures between the two sites. Alternatively, a single biosensor could detect the melanoma volatile signature relative to a database of previously collected skin and nevi samples.

[0044] This method can further include performing a histological analysis of the melanoma or tissue to further substantiate a diagnosis of melanoma or a determination that the at least one subject does not have melanoma. In the method, one or more subjects can be simultaneously tested for the presence of melanoma.

Detection of Melanoma Margins

[0045] In a subject who has a melanoma or who had a melanoma that was surgically removed or excised, the volatile compounds described herein can be used to detect the margins of the melanoma. In general, volatile compounds emanating from tissue surrounding an existing melanoma or surrounding an area from which a melanoma was removed are examined and compared to control or baseline levels. A method of detecting at least one margin of a melanoma on a subject typically includes placing a biosensor as described herein over the skin of a subject at different distances from the melanoma lesion in order to detect safe margins. As with the methods described above, the volatile compounds analyzed include one or more of the volatile compounds listed in Tables 3, 4, and 6-12, e.g., dodecane, 4-methyl decane, and undecane, and/or 1-Hexadecanol, Benzene, 1,3,5-trimethyl, and dodecane; and/or Bis(2- ethylhexyl)phthalate, decane, undecane, decane,4-methyl, ethylene oxide, isopropyl palmitate, and phthalic acid,isobutyl 4-octyl ester.

Kits

[0046] Described herein are kits for detecting the presence of melanoma in a subject (e.g., human). A typical kit for detecting melanoma in a subject suspected or at risk of having melanoma includes at least a first biosensor as described herein, at least one reagent for detecting binding of one or more of the olfactory receptors to one or more of the volatile compounds obtained from the headspace of a melanoma on the subject or the headspace of a melanoma or portion of a melanoma isolated from the subject, instructions for use, and appropriate packaging. A kit can further include a second biosensor and a second reagent for the detection of binding of one or more of the olfactory receptors to one or more of the volatile compounds obtained from the headspace of a melanoma on the subject or the headspace of a melanoma or portion of a melanoma isolated from the subject. A kit may include a well plate to carry the mixture of the different reagents, as well as one or more washing buffers. Optionally, kits may also contain one or more of the following: containers which include positive controls, containers which include negative controls, photographs or images of representative examples of positive results and photographs or images of representative examples of negative results.

EXAMPLES

[0047] The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and should not be construed as limiting the scope of the invention in any way.

Example 1 - Different Volatile Signatures From Skin, Naevi and Melanoma - a Novel Approach To Detect A Pathological Process

[0048] Early detection of melanoma is of great importance to reduce mortality. Discovering new melanoma biomarkers would improve early detection and diagnosis. Here, a novel approach to detect volatile compounds from skin is presented. HS-SPME and GC/MS were used to identify volatile signatures from melanoma, naevi and skin samples. It was hypothesized that the metabolic state of tissue alters the profile of volatile compounds. Volatiles released from fresh biopsy tissue of melanoma and benign naevus were compared based on their difference in frequency distribution and their expression level. Also analyzed were volatile profiles from frozen tissue, including skin and melanoma. Three volatiles, 4-methyl decane, dodecane and undecane were preferentially expressed in both fresh and frozen melanoma, indicating that they are candidate biomarkers. Twelve candidate biomarkers evaluated by fuzzy logic analysis of frozen samples distinguished melanoma from skin with 89% sensitivity and 90% specificity. The results demonstrate proof-of-principle that there is differential expression of volatiles in melanoma. The volatile metabolomic approach will lead to a better understanding of melanoma and can enable development of new diagnostic and treatment strategies based on altered metabolism. RESULTS

[0049] The volatile collection preserves the tissue morphology. The diagnosis of melanoma is based on histological analysis of tissue biopsies and remains the primary modality of detection. Here, it is shown that the method of volatile collection used in the experiments described herein does not alter tissue morphology. Five naevi samples and three melanoma lesions big enough to obtain two parallel 3mm samples were used for both histology (H&E staining) and volatile collection analysis. The first biopsy sample from each naevus and melanoma was put straight into formalin, embedded, sectioned and stained using standard histopathological methods (Fig. 3 A and C). The second, parallel biopsy sample from each naevus and melanoma was first subjected to the volatile collection by using the HS-SPME method (Fig. 3E) and volatile analysis. Less than three hours after biopsy, collection and volatile analysis, samples were put into formalin and processed for histology (Fig. 3B and D). Because the histological samples from the two groups were indistinguishable, it was concluded that the volatile analysis of tissue biopsies performed as described herein does not alter tissue morphology. Thus, this volatile collection does not change tissue appearance and does not interfere with standard clinical procedures related to melanoma diagnosis.

[0050] A comparative analysis of volatile compounds in the fresh naevi and melanoma was performed. Demographic data for volunteers and melanoma patients used to obtain fresh tissue, as well as data obtained from the Cooperative Human Tissue Network bank (CHTN) regarding frozen tissue are summarized in the Table 1. In addition, histopathology reports for the 5 fresh melanoma biopsy samples are presented in Table 2. Analysis of the volatile molecules collected from fresh naevi (n=25) and fresh melanoma samples (n=5) revealed complex volatile chromatographic signatures (Fig. 4, C and F). Some peaks in the chromatograms were only present in melanoma group (indicated with ***), while some were increased (**) or decreased (*) in the melanoma group as compared to the naevi group. In total, using AMDIS (Automated Mass Spectral Deconvolution and Identification software) and the NIST 2.0 mass spectral library, 325 unique volatile compounds were detected and identified from naevi (N) and melanoma (M) samples with >60% confidence.

Table 1. Demographic data for volunteers and melanoma patients used in this study (W- white, B- black, IND-indian).

Nevi 25 median 40 (21-64) 10F 17M 24 W 1 IND

fresh melanoma 5 median 68 (61-98) 3F 2M 5W

frozen melanoma 18 median 55 (28-79) 7 F 11M 17W IB

frozen skin 20 median 46 (28-73) 18F 2M 16W 4B

Table 2. Demographic data and histopathology reports for melanoma patients used to obtain fresh biopsy samples.

Table 3. Volatile compounds with significant differences in frequency distribution in fresh

hydrocarbon

Benzene, 1 -ethyls- aromatic

622-96-8 4/5 6/25 0.017 13 methyl hydrocarbon

Styrene (benzene, aromatic

100-42-5 2/5 1/25 0.016 16 ethenyl-) hydrocarbon

85-68-7 Benzyl butyl phthalate ester 2/5 1/25 0.016 16

123-72-8 Butanal aldehyde 2/5 1/25 0.016 16

67-71-0 Dimethyl sulfone sulfone 3/5 1/25 0.016 16 nitrogen

120-72-9 Indole 3/5 1/25 0.016 16 heterocycle

Aziridine,2-methyl

151-56-4 amine 3/5 3/25 0.01 11

(ethyleneimine)

112-53-8 1-Dodecanol alcohol 4/5 8/25 <0.05 9

36653-82-

1-Hexadecanol alcohol 4/5 8/25 <0.05 9 4

629-76-5 1-Pentadecanol alcohol 3/5 3/25 0.016 11

112-70-9 1-Tridecanol alcohol 4/5 8/25 0.049 9

18172-67- cyclic

B-Pinene 3/5 3/25 0.016 11 3 monoterpene

Table 4. Six volatile compounds with significant increase in frequency distribution from frozen melanoma (M-melanoma, S-skin).

[0051] Thirty two compounds were present in 60% of the samples from the naevi group. Most of these compounds were also detected in both melanoma and air samples (blanks). Only pyridine and 3-hexanol appeared to be specific for naevi (Fig. 4, G and J). Pyridine is present at 60%> frequency in naevi group. It was detected only in one melanoma sample and it was not detected in air samples. 3-hexanol was detected exclusively in the naevi group. Dimethyl benzenamine (at carbons 2,3;2,4;2,5 or 2,6) was also identified as a common volatile compound present in both naevi and melanoma (Fig. 4, M, N and O). Compounds that differ only in a methyl group position (as is the case with the dimethyl benzenamine) have similar mass spectra and thus are hard to distinguish using GC/MS. [0052] Statistical analysis of melanoma differential volatiles identified from fresh biopsy samples was performed. Differential components were statistically identified based on two criteria, the amplitude or the frequency. A difference in amplitude was defined as a statistical significance of the difference in the pair-wise mean value ("Total Integrated Signal" or "area under the peak") (Student t-test) of expression between groups (naevi "N", melanoma "M", and blank "B", ambient air sample). A difference in frequency was defined as the statistical significance of the difference in the frequency of appearance (Cochran-Mantel Haenszel test). Relative frequencies of the volatiles in each group were examined and their significant difference in distribution were tested by using the odds/ratio (comparing a frequency in M versus frequency in N group) and Cochran-Mantel Haenszel test (how likely it is to see a compound in the M vs N group?); an odds ratio of >2.5 and a metabolite present in 40% or more melanoma biopsies indicated a potential biomarker or molecule of interest.

[0053] Twenty-one volatile compounds were found to be present at a significantly different frequency between the two groups (Table 3); among them alkanes (e.g. nonane), methylated alkanes, alkenes (e.g. tridecene) were abundant. Phthalate, butanal, dimethylsulfone and indole, were all 16X more likely to be detected in the melanoma then in the naevi group. The presence of longer chain alcohols were identified, from dodecanol to hexadecanols (Ci2-Ci₆). Methylated benzenes (compounds 5-10 in Table 3) were also abundant in the melanoma group.

[0054] The summary of the t-tests, where significant differences in the mean values of the compounds were compared, the associated p-values together with their structures are presented in FIG. 5A, B and C. It is interesting to note that only acetamide (FIG. 5A) and isopropyl alcohol (FIG. 5B) showed decreased levels in the melanoma group relative to the naevi group; all other compounds were found to be significantly increased. Xylene was detected in melanoma but not in naevi. o- and p- xylene have very similar mass spectra and it is difficult to distinguish them by mass spectrometry. Xylene is part of the benzene, toluene, ethylbenzene, o-, m- and p-xylene complex (BTEX), an index of environmental contamination of soil and ground water by petroleum products.

[0055] In summary, the increased presence of methylated alkanes and benzenes may indicate an increased methylation process in melanoma. The presence of secondary metabolites of membrane lipid peroxidation, e.g alkanes (nonane, decane, undecane, dodecane, tridecane), alkenes (decene, tridecene), aldehydes (propanal, butanal) may be an indicator of oxidative stress.

[0056] A statistical analysis of differential volatiles from frozen melanoma and skin samples was performed. The availability of the fresh melanoma tissue is often a limiting step in the experimental work. Thus, other sources, such as frozen tissue, need to be considered and explored. With this in mind, 18 frozen melanoma and 20 frozen skin samples obtained from the CHTN tissue bank were collected and analyzed. Similar to the work with fresh samples, analysis of frozen melanoma and frozen skin revealed differential volatile profiles in the two biopsy sets. Demographic data and histopathology reports for frozen melanoma tissue samples were summarized in the Table 5. In total, 220 compounds in the two sample sets were detected; this is less than the 325 detected in fresh samples. Out of 18 melanoma samples, only 3 were skin biopsy samples, the remaining samples were metastatic melanoma tissue from: lymph node (10), intestine (2), liver (1), lung (1) and breast (1). Compounds listed in Table 4 were present at a significantly increased frequency distribution in the melanoma group vs the skin group. Compounds listed in Table 6 are volatiles detected at a significantly higher distribution frequency in skin samples. Xylene was detected in the frozen skin samples. This compound was also detected in fresh melanoma, but not in the fresh naevi group. Volatiles with significantly increased mean value and their associated p-values in melanoma vs skin group are presented in Table 7. Two compounds, dodecane and 5-methyl dodecane, were significantly increased in melanoma using either the expression level (Table 4) or the frequency of distribution (Table 7).

Table 5. Demo ra hic data and histo atholo re orts for frozen melanoma tissue sam les

Melan-A, SI 00 and

MM, Breslow

HMB45 positive;

thickness 4mm, Clark

skin from left arm SMA and desmin M 79 W level V, ulceration

negative; CD68

present

positive

MM, ulceration MM involving

lymph node, left neck M 53 W present adipose tissue

satellite nodules with

MM, nodular type,

lympho-vascular and

level V, Breslow

skin, left shoulder perineural invasion, F 40 B thickness 4.5mm,

mitotic activity

ulceration present

15/mm2

metastatic MM,

Beslow thickness liver spindle cell neoplasm M 49 W 5mm

metastatic MM lymph node, left axillary M 63 W metastatic MM lymph node F 83 W lymph node, left superficial

metastatic MM M 76 W inguinal

Melan-A and S-100

metastatic MM lymph node positive, HMB-45 F 40 W negative

metastatic MM breast F 47 W metastatic MM soft tissue, left axilla M 28 W metastatic MM metastasis to colon F 54 W

Table 6. Volatile compounds with significant increase in frequency distribution from frozen skin.

Table 7. Volatile compounds with significantly increased mean value (log integrated signal) in the frozen melanoma rou t-test .

[0057] A Fuzzy logic-based statistical analysis of the frozen tissue bank samples was performed. Retention times from the chromatograms obtained from frozen skin and melanoma samples were used to create a table of test samples from which volatile compounds relevant for the discrimination between the skin and melanoma groups were derived. From a total of 38 samples (18 melanoma and 20 skin samples), twelve volatile compounds were identified as relevant and a fuzzy logic prediction algorithm was created. The list of these compounds with their relevant Goodman Kruskal Lambda value is presented in FIG. 6A. A higher Goodman Kruskal Lambda value indicates a higher likelihood that a volatile compound is predictive for melanoma. These potential candidate markers were evaluated by fuzzy logic analysis and demonstrated the ability to distinguish melanoma from skin with 89% sensitivity (16/18) and 90% specificity (18/20). Nonanal is a common and abundant compound found on human skin (Table 6, p<0.0001). Fuzzy logic analysis identified this volatile as one of the 12 compounds that can be used to distinguish melanoma from skin (FIG. 6). Leave -one -out cross validation with variable selection in each leave -one-out run was used to assess the predictive performance of the fuzzy logic analysis. The area under the Receiver-Operating characteristic Curve (ROC) is determined as 0.936; indicating that these 12 compounds are able to correctly classify melanoma with high sensitivity and specificity (FIG. 6B). A graphical representation of these data is presented in the form of the heat map (FIG. 6C). [0058] A comparison of fresh and frozen volatile signatures in melanoma biopsies was made. In FIG. 7, the number of volatile compounds detected in each group, as well as the number of compounds that overlap between fresh naevi, fresh melanoma, frozen skin and frozen melanoma are presented as a Venn diagram to illustrate the complex relationship between the volatile fingerprints of the different sample sets. A total of 35 compounds unique to melanoma were detected, and 3 compounds unique to and common to both fresh and frozen melanoma samples were detected. The availability of the fresh melanoma tissue for research is limited and it was thus investigated whether the frozen tissue will be suitable for this type of study. A differential expression of volatiles in frozen and fresh melanoma samples as compared to the controls was documented; however, the complete overlap in these two sets of melanoma volatile profiles was not observed. This was probably due to a difference in the type of control tissue used and in the tissue preparation (fresh or frozen).

[0059] In the case of 3 volatile compounds, tissue preparation methodology (freezing/thawing) did not have an effect. Thus, despite the differences in methodology, three compounds identified in fresh tissue were still predictive of melanoma in frozen tissue. 4-methyl decane, dodecane and undecane were detected from both fresh and frozen melanoma at a significant level compared to the control group; thus these compounds are candidate biomarkers. 4-methyl decane was present at significantly increased frequency, while dodecane and undecane were present at significantly increased both frequency and expression level. Dodecane was also one of the 12 candidate volatiles identified by fuzzy logic analysis. However, methylated benzenes, dimethyl sulfone, ethyleneimine, nonane, indole and longer chain alcohols initially detected in fresh melanoma samples, together with some aldehydes (propanal, butanal) were not found in the frozen samples, indicating that the volatile signature may have changed due to the freezing process and prolonged storage. The results from the frozen melanoma group have a different control, skin instead of naevus (used as a control in the fresh group), precluding a full comparison; however if a volatile is to be a true melanoma biomarker, its presence should be detected neither in skin, nor in nevus.

MATERIALS AND METHODS:

[0060] Tissue collection: Biopsy samples were obtained from subjects recruited in accordance with an approved University of Miami Institutional Review Board (IRB) protocol (No. 2006117) and Veteran Administration IRB protocol (No. 00762). All naevi samples were collected from the volunteers (asked not to wash 8 hours before the biopsy) and were confirmed by histology analysis using hematoxylin & eosin staining. Each naevus was removed by using a 3mm punch device (AcuPunch, Acuderm Inc). Fresh melanoma samples were collected from patients scheduled for the excisional biopsy irrespective of histotype or disease stage. No exclusion criteria were used, except that all samples were from patients over 21 years of age. The melanoma lesion was first excised and then cut with a 3mm punch device in order to obtain the same sample size as for nevi. The reason why each melanoma lesion was first excised and then cut using the punch biopsy technique is because excisional biopsy of melanoma is a preferred method to remove a malignant lesion. Immediately after the lesion was removed, a 3 mm punch biopsy on the excised tissue was performed for research purposes. It is not believed that punch biopsy of melanoma tissue immediately after excision of the tissue introduces any artifact that could account for the sampling difference. After biopsy and volatile collection, melanoma samples were analyzed and confirmed by H&E staining. Also collected and analyzed were 17 control air samples to identify air contaminants. The air was collected from the same room where biopsy of nevi samples took place and the whole procedure was identical to the one with biopsy tissue. In addition, chromatograms were obtained from the anesthetic used in the biopsies (l%Xylocaine/Epinephrine & Na-bicarbonate). Eighteen frozen melanoma and twenty frozen skin samples were obtained from the CHTN tissue bank (Cooperative Human Tissue Network). Prior to storage at the tissue bank these samples were snap frozen (in liquid N₂) within 1 hour after biopsy to quench metabolism, and kept at -70°C for not more than one year. Frozen samples were also cut with the 3mm punch device to obtain a uniform size, placed in the 1.5mL vial and thawed on ice for 1 hour and later processed in the same way as fresh nevi and melanoma samples.

[0061] Histology analysis: Hematoxylin/eosin staining was done at the Dermatopathology Laboratory Services Department of Dermatology University of Miami, Miller School of Medicine. All frozen samples after volatile collection, were put into formalin and confirmed by histology analysis done at the Department of Pathology, Jackson Memorial Hospital.

[0062] HS-SPME collection of volatiles: An HS-SPME (head space-solid phase microextraction) method was used to collect the volatiles (Zhang, Z. and J. Pawliszyn, Analytical Chemistry, 1993. 65: p. 1843-1852; Pawliszyn, J., J Chromatogr Sci, 2000. 38(7): p. 270-8; Risticevic, S., et al, Anal Bioanal Chem, 2009. 393(3): p. 781-95). This method uses a small, portable device with a coated fiber to extract and collect volatile compounds for analysis by gas chromatography. The biopsy sample was placed in a vial (Agilent, No.5182-0715, 1.5mL, with 0.3mL polyspring insert) and capped with a Teflon coated silicone septum (FIG. 3E). The sample was kept refrigerated for not more than one hour. After that the sample was left at room temperature for one hour to equilibrate. The headspace was sampled with a polydimethysiloxane-divinylbenzene fiber for one hour at room temperature (65-μιη PDMS-DVB, Cat No. 57344-U, Supelco, Belle fonte, PA, USA). Extraction selectivity depends on the type of the fiber and the coating thickness. Since an unknown complex matrix was being examined, it was decided to use a fiber with broad selectivity, non-polar PDMS/DVB. In order to achieve higher sensitivity, the sample headspace should be as small as possible (Zhang, Z. and J. Pawliszyn, Analytical Chemistry, 1993. 65: p. 1843-1852), therefore the inner tube was introduced into the 1.5mL vial. It was also decided to use a fiber of 65μιη, of medium thickness; a general rule is that a thick fiber adsorbs more compounds; however their diffusion from the fiber during thermal desorption is slower. Analysis of air samples obtained in parallel with biopsy collection (n=17) revealed 34 compounds that were present in at least 40% of the samples. Some components known to elute from the fiber itself were excluded from this list (largely siloxanes and their derivatives). Volatile analysis of the anestheticlidocaine used in the biopsy procedure of nevi samples revealed the presence of additional volatiles: oxime-,metoxy-phenyl and methylparaben. Lidocaine (5μί) was placed in the vial, and the volatiles were collected in the same fashion as described for tissue samples in the Material and Methods section. In one biopsy of melanoma, in addition to lidocaine, midazolam, propofol and fentanyl were used as anesthetics. Volatiles from these anesthetics were analyzed and benzene, 2,4 diisocyanato-1 -methyl and diphenyl ether were detected in addition to original anesthetics and the above mentioned volatiles from lidocaine. No information was available about anesthetics used to obtain frozen samples from CHTN. Compounds like propofol and methoxy-phenyl oxime (Table 7), as well as 2,4 diisocyanato-1- methyl benzene (FIG. 6A) were most likely released from anesthetics used in the biopsy procedure.

[0063] Optimization of the HS-SPME conditions: the parameters such fiber coating (75 μιη PDMS/Carboxen Cat No. 57284-U and 65 μιη PDMS/DVB), sample size (2 and 3 mm) as well as post biopsy time of volatile collection were investigated (FIG. 8). PDMS/Carboxen is predominantly used for relatively small volatiles, compounds between 2-10 carbons and low MW compounds (MW=30-225), while PDMS/DVB is used for volatiles, amines, nitro -aromatic compounds, ranging from 50-300 Daltons. Two 3mm biopsy samples from the same nevus were analyzed with both fibers (TIC, total ion count, for PDMS/DVB is about 91535, while for the PDMS/Carboxen is 70127) (FIG. 8A). By comparing these two chromatograms, it was observed that the difference is mainly quantitative and not qualitative. The AMDIS postprocess comparison of these two files is presented in FIG. 8B. From the 3mm sample, about 20 times more signal was detected (Total Ion Count, TIC) than from the 2mm sample. Also, the number of identified compounds was much larger (344 vs 260) (FIG. 8C). Thus, in all subsequent experiments, a 3mm punch biopsy was performed. To examine the effect of post biopsy collection time on volatile compounds, the axilla sample from melanoma patient (lymph node tissue - not melanoma or nevi) was collected and punched twice; the first sample was analyzed within 3 hours of biopsy, while the second sample was kept refrigerated overnight and analyzed the next day. The integrated signal for each compound was divided by the sum of signals from the whole sample (TIC) and the percent of each volatile compound was calculated (FIG. 8D). Comparison of these two samples indicates loss of volatile compounds of about 30% in TIC. In the first sample, 21 compounds were identified, while in the second, 25 compounds were identified. Out of 17 compounds that were detected in both samples, eight showed an increase of >2 fold after 24 hours. These compounds were: 1-Hexanol 2-ethyl-; 3-Methylbenzothiophene; Acetic acid butyl ester; Diethyl Phthalate; Hexane 3, 3 -dimethyl-; Limonene; Oxime- methoxy- phenyl-1 and Phenol 2,4-bis(l-methylethyl)- acetate. Volatile compounds that couldn't be detected in the 24hours/second sample, and thus were lost were: Benzene 1,4-dichloro-, Dodecane, Ethanol and Hydrazine methyl-. The difference in volatile profile between these axilla samples indicate that a degradation process is indeed happening, even if the sample was kept refrigerated overnight. In addition, the volatile composition of two fresh skin melanoma samples was analyzed within three hours and after 6-8 hours post biopsy. Around 15% of compounds could not be detected after the prolonged incubation at room temperature. Disappearance of some volatiles after prolonged incubation indicates a need for a stringent control of experimental conditions, in particular volatile collection time post biopsy. Thus, in all fresh tissue samples (melanoma and nevi) reported herein, volatiles were collected and analyzed within three hours post biopsy. [0064] GC/MS: The PDMS/DVB fiber with collected volatiles was directly injected onto a 0.75mm i.d. injection port of Hewlett Packard 6890 gas chromatograph (Hewlett Packard, Avondale, PA) and chromatographed on a non-polar DB-5MS column (model No. J&W 128- 5522, 25mx0.2mm ί.ά.χ0.33μιη film) under the following temperature program: 40°C for two minutes followed by 6°C min-1 ramp to 270°C and hold for 5 minutes. Helium carrier gas flow was run in constant flow at 0.7 mL min^"1. As volatile compounds elute from the column, they were fragmented into ions (by electron ionization) and detected in the quadrupole mass spectrometer. Each compound produced a unique spectrum of molecular fragments (ions) with specific masses and a fixed relative abundance. The Agilent 5973 mass spectrometer was used in the full scan mode (30-300 amu).

[0065] Preprocessing of the data: GC/MS metabolic profiling results in complex chromatograms with huge differences in the relative abundance of different compounds and with many co- eluting peaks that have to be deconvoluted. The AMDIS deconvolution algorithm freely available at www.amdis.net (Automated Mass Spectral Deconvolution and Identification software) was used. Deconvolution finds ions whose individual abundances rise and fall together over time, indicating that they are from the same compound. AMDIS parameters were: 60% minimum matching factor, threshold-low, resolution-medium, sensitivity-high, shape requirements-medium, adjacent peak subtraction-two, low m/z 50, high m/z 300. AMDIS generates a report file, where area under each peak represents the total absolute amount of ions from each compound/metabolite present. The profiling quantifies metabolites based on their absolute mass ion intensity. This eliminates the need for internal standards and makes measurements of both known and novel metabolites possible (Tagore, R., et al, J Am Chem Soc, 2008. 130(43): p. 14111-3; Saghatelian, A., et al, Biochemistry, 2004. 43(45): p. 14332-9). All components from AMDIS analysis were searched in the NIST database (with one reported hit per compound and with a minimum match factor set to 60%, meaning that a threshold of 60% similarity was used for the spectral matching). After generating an analysis report in the text file, the annotated compound list was transferred to EXCEL for further processing. For each sample identity of the compound; CAS or NIST number; retention time and integrated signal were reported. Signal intensity represents the logio transformation of absolute ion counts in the area under the deconvoluted peak (Integrated signal), thus the signals from multiple samples were compared. [0066] Data analysis: The Student t-test and Cochran-Mantel Haenszel Chi-square test were used to detect statistical significance between the studied groups. In addition, for analysis of frozen melanoma and skin samples, fuzzy logic methodology by Interrelation Miner software from SystAim was used. The Interrelation Miner methodology analyses the relations between the measured variables/volatiles statistically and constructs fuzzy functions for every of these interrelations. With these fuzzy functions, the software creates the membership matrix for each group of samples such as the membership matrix for the group of samples with Disease Present and membership matrix for the group of samples with the Disease Not Present. Using the membership matrix of each group of samples it can be calculated how typical a sample is for each group. The prediction is simply the group with the highest fuzzy membership.

DISCUSSION

[0067] The current prevailing opinion is that many melanomas arise de novo and only a subset of melanoma arises from the naevus (Rivers, J.K., Lancet, 2004. 363(9410): p. 728-30; Michaloglou, C, et al, Nature, 2005. 436(7051): p. 720-4). In the experiments described herein, melanocytic naevi (i.e. moles) acquired from volunteers were used as a control group to which the melanoma samples were compared. Naevi are benign tumors of melanocytes with an oncogene-induced senescent phenotype. Naevi exist in a growth-arrested state predominantly induced by BRAFV600E (Michaloglou, C, et al, Nature, 2005. 436(7051): p. 720-4). There is no literature record of volatiles released from naevi, and this study is the first to address this question. Although most volatiles present in naevi were also present in the melanoma group, the level of these volatiles detected was significantly different for 21 compounds (FIG. 5). In addition, the volatile signature from naevi revealed pyridine and 3-hexanol as specific naevi compounds not found in melanoma (FIG. 4 and FIG. 7).

Example 2 - Detection of Melanoma Biomarkers

[0068] The melanoma biomarkers described herein were detected in the fresh and frozen tissue based on the differences in the mean value of the compound (t-test, the same compounds are depicted in FIG. 5 and Table 7; based on the differences in the frequency of the compound distribution (Cochran-Mantel Haenszel test, Table 3 and Table 4) and by using fuzzy logic statistical analysis (FIG. 6). The compounds identified by fuzzy logic analysis are able to distinguish melanoma from skin with 89%sensitivity and 90% specificity. A full list of the significantly different compounds is presented below in Table 8 (listed in the alphabetical order).

Table 8

[0069] Additional experimentation was performed to provide independent confirmation of the results described above and identify additional biomarkers of melanoma. These new results were obtained using an improved methodology: the volatiles collected from a melanoma biopsy and a normal skin biopsy obtained from the same patient during the same surgical procedure were compared. In Table 9, compounds are listed that were detected in both skin and melanoma, but with a higher level in melanoma. Compounds with an asterisk or pound sign were previously identified in the experiments described above as melanoma biomarkers, and are also confirmed in this study. Results presented here were obtained using a matching control (healthy skin lesion biopsy from the same melanoma patient was analyzed for volatile expression profile). Volatile compounds that showed >100% increase in melanoma compared to the healthy skin are listed below together with the %TIC (Total Ion Count, for details see our manuscript) and their ratio in Melanoma vs Skin (M/S).

Table 9

RATIO

% TIC IN

CAS COMPOUND %TIC IN M Melanoma/

SKIN

Skin

1,2-Benzenedicarboxylic acid,

117-84-0 1.03 0.61 1.68 diisooctyl ester

36653-82-4 1-Hexadecanol* 3.29 0.09 35.90

2-Ethylhexyl trans-4-

83834-59-7 1.02 0.78 1.31 methoxycinnamate

108-67-8 Benzene, 1,3,5-trimethyl** 0.12 0.03 3.41

75-43-4 Dichlorodifluoromethane 0.03 0.01 1.80

112-40-3 Dodecane 0.13 0.11 1.27

N-Morpholinomethyl-

77422-34-5 0.32 0.23 1.37 isopropyl-sulfide

17301-32-5 Undecane, 4,7-dimethyl 0.17 0.11 1.54

* this compound CAS 36653-82-4 was previously identified (see Table 3)

** compound CAS108-67-8 is 1,3,5 trimethyl benzene and compound CAS526-73-8 1,2,3 trimethyl benzene have very similar mass spectra; the latter being previously identified (see Table 3)

compound CAS 112-40-3 is dodecane, detected previously (see Table 4)

[0070] Using this analysis, an additional 21 melanoma-specific compounds were identified that were not detected in the matching skin sample (Table 10). Seven compounds were identified in the experiments described above (identified by asterisk or pound sign), as compounds that are significantly different from nevi. When compared with skin control, these compounds apprear to be melanoma specific. However, there presence is also detected in nevi previously analyzedThe remaining 14 compounds (those with no asterix or pound sign) represent melanoma specific biomarkers identified using this paired biopsy analysis approach.

Table 10: CAS COMPOUND CAS COMPOUND

2175-91-9 1,3-Cyclopentadiene, 5-(l- 6975-98-0

Decane, 2-methyl- methylethylidene)- 2847-72-5

629-96-9 1-Eicosanol Decane, 4-methyl-*

111-76-2

629-73-2 1-Hexadecene Ethanol, 2-butoxy-

2-Isopropylamino-4-

75-21-8 Ethylene oxide

methylbenzonitrile

14365-44-7 Adenosine, 5'-amino-5'-deoxy- ■ 75-12-7 Formamide

Heptane, 2,4-dimethyl-

Benzaldehyde, 4-methoxy- 2213-23-2

Bis(2-ethylhexyl) phthalate 142-91-6 Isopropyl Palmitate'

1678-91-7 Cyclohexane, ethyl- Pentane, 2,3,4-trimethyl-

112-31-2 NIST Phthalic acid, isobutyl 4-

Decanal

314847 octyl ester

### Spiro [bicy clo [2.2.1] hept-5 -

124-18-5 Decane 6572-50-5

ene-2, 1 '-cyclopropane]

1120-21-4 Undecane MM

^compound CAS 2847-72-5 is decane, 4-methyl previously detected (see Table 3, Table 4) compound CAS75-21-8 is ethylene oxide also previously detected (see FIG. 5)

*** compound CAS 117-81-7 is bis(2-ethylhexyl)phthalate previously detected (see Table 7) * compound CAS 142-91-6 is isopropyl palmitate previously detected (see Table 4)

compound NIST314847 is phthalic acid, isobutyl 4-octyl ester with very similar mass spectrao NIST 229113, bis(2-ethylhexyl) phthalate previously detected (see FIG. 5)

###

compound CAS 124-18-5 is decane previously detected (see FIG. 5)

MM

compound CAS 1120-21-4 is undecane previously detected (see FIG. 5 and Table 4 ) [0071] Those volatile compounds (8) listed in Table 9 are significantly different comparing to skin. A sign means that these compounds (3) are also different when comparing to nevi (based on previous experiments). Thus, those with pound or asterisk signs (in both Table 9) are compounds found to be different in melanoma comparing to both skin and nevi, meaning that they do appear in skin and nevi, but at much lower levels, therefore they can be biomarkers of melanoma (increase/decrease category), making them the most informative. All compounds listed in Table 10 (21) are melanoma specific comparing to skin. However, based on previous experiments, those with the signs (7) were detected in nevi, Those with signs are increased in melanoma when compared to nevi (increase/decrease) and also melanoma specific when compared to skin. Those without signs (14) are melanoma- specific volatiles (present/absent category when comparing to both skin and nevi).

Example 3 - A case report - Volatile metabolomic signature of malignant melanoma using matching skin as a control

[0072] Reported herein is a case analysis of the volatile metabolic signature of a malignant melanoma using matched, non-neoplastic skin tissue from the same patient as a control. An untargeted approach was employed, as this represents an efficient tool for early detection of disease. Head Space Solid Phase Micro-Extraction (HS-SPME) (Pawliszyn J: Theory of solid- phase microextraction. J Chromatogr Sci 2000;38:270-278) was used to collect volatile compounds from 2mm punch biopsy skin samples, and GC/MS to separate and analyze these volatiles (as described in Examples 1 and 2). In these complex samples, there are still a significant number of compounds whose identity could not be reliably identified using GC/MS. A similarity threshold of 60% was used with the NIST 2.0 mass spectral database to assign the identity of the compounds. Thus, all compounds with a similarity <60% were excluded from analysis. The total number of identified and unidentified compounds in the melanoma sample was 166 and 693, respectively. In the matched, normal skin sample we identified 132 volatile compounds and 500 were unidentified. In summary, 32 compounds were identified; 9 volatile compounds were increased in melanoma and 23 volatile compounds were detected only in melanoma and not in normal skin.

Material and Methods

[0101] Biopsy samples were obtained in accordance with an approved University of Miami Institutional Review Board (IRB) protocol (No. 2006117). Fresh melanoma and skin samples were collected with a 2-mm punch device. Volatile collection and analysis was done as described in Examples 1 and 2. Briefly, HS-SPME (head space-solid phase microextraction) method was used to collect the volatiles. The headspace was sampled with a polydimethysiloxane- divinylbenzene fiber for one hour at room temperature (65-μιη PDMS-DVB, Cat No. 57344-U, Supelco, Bellefonte, PA, USA). After volatile collection, samples were put into formalin and hematoxylin/eosin staining was done at the Department of Pathology, University of Miami. The PDMS/DVB fiber with collected volatiles was directly injected onto a 0.75 mm i.d. injection port of Hewlett Packard 6890 gas chromatograph (Hewlett Packard, Avondale, PA) and chromatographed on a non-polar DB-5MS column (model No. J&W 128-5522, 25 m x 0.2 mm i.d.x0.33 um film) under the following temperature program: 40°C for two minutes followed by 6°C min-1 ramp to 270°C and hold for 5 minutes. Helium carrier gas flow was run in constant flow at 0.7 mL min-1. As volatile compounds elute from the column, they were fragmented into ions (by electron ionization) and detected in the Agilent 5973, quadrupole mass spectrometer (in the full scan mode, 30-300 amu). The AMDIS deconvolution algorithm freely available at www.amdis.net (Automated Mass Spectral Deconvolution and Identification software) was used. All components from AMDIS analysis were searched in the NIST 2.0 database, with one reported hit per compound and with a minimum match factor set to 60%.

Results

[0102] The patient, male, aged 49, was admitted to the hospital for excisional biopsy of melanoma lesion, one month after the diagnosis of melanoma in the primary care clinic was obtained. The initial diagnosis of Nodular Melanoma (0.98mm Breslow thickness, <1 mitosis/mm²) was based on a biopsy from the #1 distal lesion on the right forearm (Figure 9D). The patient was scheduled for additional excision of the #2 proximal lesion (see Figure 9D). After informed consent was obtained for this study, a melanoma tissue sample was obtained from the #2 proximal lesion. After volatile collection and analysis, this sample was sent for histologic analysis. It was positive for malignant melanoma (H&E staining, Figure 9A, 0.78mm Breslow thickness, <1 mitosis/mm², stage Tla, N0M0). A second punch biopsy of nearby healthy skin was performed at the site indicated in Figure 9D. Chromatograms obtained from these two sites are presented in Figure 9B and F. The peaks in the chromatograms indicated the relative abundance of compounds eluting at the time indicated in the x-axis. Compounds unique to the melanoma samples, with respect to the healthy skin control, are shown in Table 11. Compounds enriched in melanoma, with respect to the healthy skin control, are shown in Table 12. This analysis shows a marked 35-fold increase in 1-Hexadecanol in melanoma, as compared to the matching skin sample (Table 12), suggesting 1-Hexadecanol as a potential biomarker of melanoma. 1-Hexadecanol has been previously detected in skin emanations (Yun et al., Science 2009;325: 1555-1559) and a high temperature GC/MS study of skin surface predominant lipids and fatty acids identified hexadecenoic acid (C16: l) and hexadecanoic acid (C16:0) (Michael- Jubeli et al., J Lipid Res 2011;52: 143-151). The greatly increased level of 1-hexadecanol in the melanoma sample may reflect increased de novo fatty acid (FA) synthesis, a crucial metabolic alteration of cancer cells required for synthesis of new plasma membranes. Increased activity of fatty acid synthase (FAS, an enzyme responsible for the synthesis of fatty acids), has emerged as a phenotype common to most tumors associated with poor outcome. FAS over-expression in melanoma has been correlated with Breslow thickness and overall poor survival. In addition, lipids impact cancer cell growth and invasion. For example, lysophosphatidic acid acts through its receptors to stimulate cancer cell proliferation and survival. Oncogene induced FA synthesis facilitates the formation of palmitate and palmitoleate. In the melanoma case, chemical reduction of abundant hexadecanoic acid results in increased levels of 1-hexadecanol and indicates the importance of palmitic acid (C16:0) and free fatty acid alcohol (1-hexadecanol). Also detected were 1-hexadecene and isopropyl palmitate, derivatives of palmitic acid. Taken together, these results implicate increased fatty acid synthesis and metabolism in the progression of melanoma. Methyled benzene (Benzene, 1,3,5-trimethyl) is also increased in the melanoma sample compared to normal skin sample (Table 2). This volatile has been previously found in skin (Bernier et al., Anal Chem 2000;72:747-756) and as described in Examples 1 and 2, the increased frequency of methylated benzenes in melanoma vs nevi was detected.

[0103] The amide formamide was detected only in the melanoma sample (Table 11). Very few amides are found to be released from human skin and to date, there are no reports of formamide detected from human skin. However, methane, ethylene and ethane were detected on human skin (Nose et al., Anal Sci 2005;21 :625-628). Biological oxidation of methane to C0₂ proceeds sequentially through methanol, formaldehyde and formic acid. As described in Examples 1 and 2, a significant increase in formic acid in melanoma was detected relative to the nevi group. The Human Metabolome Database lists formic acid as an intermediate in the metabolism of one-carbon compounds. It is a product of fatty acid oxidation and it can also be produced by bacterial fermentation via heterofermenters, microorganisms that ferment glucose to multiple end products, such as acetic acid, ethanol, formic acid and C0₂. Formic acid has also been detected in cerebrospinal fluid and urine. Since ammonia is known to be one component of human odor and sweat, one can imagine the generation of formamide from the reaction of ammonia with formic acid. This may represent the underlying biochemical pathway for the production of formamide in melanoma. In addition, the ammonia necessary for formamide synthesis may have come from increased glutaminolysis known to be a characteristic of cancer cells. Large quantities of glutamine are taken up by cancer cells, resulting in secretion of alanine and ammonium into the extracellular matrix. Increased glutaminase activity (the enzyme that catabolizes glutamine for ATP through the TCA cycle) is documented in melanoma. Ethylene oxide was detected in melanoma (Table 11 and Figure 9B and 1C). The metabolic precursor for ethylene oxide is ethylene, a natural body constituent. Ethylene was previously detected on human skin. However, there are no data on endogenous ethylene formation in humans or animals. A human enzyme potentially responsible for ethylene synthesis is 1- aminocyclopropane-l-carboxylate synthase (ACC synthase) (E.C. 4.4.1.14); however it is reportedly inactive. The mutations of two amino-acid residues within the binding site of the enzyme are responsible for the lack of activity in the human ACC synthase (Koch et al, The human cDNA for a homologue of the plant enzyme 1-aminocyclopropane-l-carboxylate synthase encodes a protein lacking that activity. Gene 2001;272:75-84). Thus, it seems unlikely that the ethylene oxide in melanoma samples is a primary metabolite.

[0104] 1-eicosanol (Table 11 and Figure 9C), an alcohol containing 20 carbons (eicosa), was identified as a volatile compound specific for melanoma. Eicosanoids are bioactive lipid metabolites derived from the metabolism of polyunsaturated fatty acids by cyclooxygenases, lipoxygenases, cytochrome P450 and nonenzymatic pathways. They are synthesized by activated inflammatory cells. 12-(S) Hydroxy-5,8,11,13-eicosatetraenoic acid (12-(S)-HETE ) and 15-(S)- HETE) are monohydroxylated lipoxygenase derivatives of arachidonic acid. When [1-¹⁴C] 15- (S)-HETE was incubated with Caco-2 cells (enterocytes derived from a human adenocarcinoma), about 10% was directly esterified into cellular lipids but most was beta-oxidized to ketone bodies, C0₂, and acetate (55%). The resulting acetate was then incorporated into the synthesis of new fatty acids, primarily [¹⁴C]palmitate. Thus, it is possible that the derivative of palmitate, isopropyl palmitate (Figure 9C and Table 11), in the melanoma case also originated from the increased metabolism of arachidonic acid in cancer cells (in addition to the oncogene induced de novo FA synthesis from glucose proposed earlier).

[0105] Increased nonanal in melanoma (Table 12) was detected. As described in Examples 1 and 2, nonanal was identified as a potential melanoma biomarker. In this study, 1-iodo nonane was detected only in melanoma (Table 11). Also as described in Examples 1 and 2, nonane was identified at a significantly increased frequency in melanoma when compared to the nevi. This analysis also identified undecane only in the melanoma sample (Table 11), while methylated undecane was increased in melanoma compared to the skin sample (Table 12).

[0106] In conclusion, out of 32 volatile compounds listed in Table 11 and Table 12, 10 compounds (in bold) were detected that were shown to be significantly different from control fresh nevi (*) and control frozen normal skin (#) as described in Examples 1 and 2. These volatile compounds are candidate volatile biomarkers of melanoma.

[0107] Table 11 - List of 23 volatile compounds found only in melanoma. In bold are volatiles identified as described in Examples 1 and 2 as differentially expressed in respect to nevi

(*) or normal skin (#).

CAS/NIST COMPOUNDS FOUND ONLY IN

MELANOMA

2175-91-9 1,3-Cyclopentadiene, 5-(l- methylethylidene)-

629-96-9 1-Eicosanol

629-73-2 1-Hexadecene

106-74-1 2-Ethoxyethyl acrylate

28195-00-8 2-Isopropylamino-4- methylbenzonitrile

14365-44-7 Adenosine, 5'-amino-5'-deoxy-

123-11-5 Benzaldehyde, 4-methoxy-

117-81-7 Bis(2-ethylhexyl) phthalate#*

1678-91-7 Cyclohexane, ethyl-

112-31-2 Decanal

124-18-5 Decane*

6975-98-0 Decane, 2-methyl-

2847-72-5 Decane, 4-methyl-*

111-76-2 Ethanol, 2-butoxy-

75-21-8 Ethylene oxide*

75-12-7 Formamide

2213-23-2 Heptane, 2,4-dimethyl-

142-91-6 Isopropyl Palmitate#

4282-42-2 Nonane, 1-iodo-

565-75-3 Pentane, 2,3,4-trimethyl- 314-84-7 Phthalic acid, isobutyl 4-octyl ester

6572-50-5 Spiro[bicyclo[2.2.1]hept-5-ene-2,r- cyclopropane]

1120-21-4 Undecane*#

[0108] Table 12 - Compounds that showed >100% enrichment in melanoma compared to the healthy matched skin sample are listed together with the % Total Ion Count, (%TIC) and their ratio in Melanoma (M) vs Skin (S) - M/S.

CAS/NIST COMPOUND %TIC in M %TIC in S RATIO M/S

117-84-0 1,2- 1.03 0.61 1.68

Benzenedicarboxyli

c acid, diisooctyl

ester

36653-82-4 1-Hexadecanol* 3.29 0.09 35.90

83834-59-7 2-Ethylhexyl trans- 1.02 0.78 1.31

Λ

methoxycinnamate

108-67-8 Benzene, 1,3,5- 0.12 0.03 3.41

trimethyl*

75-43-4 Dichlorodifluoromet 0.03 0.01 1.80 hane

112-40-3 Dodecane* 0.13 0.11 1.27

77422-34-5 N- 0.32 0.23 1.37

Morpholinomethyl- isopropyl-sulfide

124-19-6 Nonanal# 0.30 0.11 2.79

17301-32-5 Undecane, 4,7- 0.17 0.11 1.54 dimethyl

[0109] Referring to Table 12, in order to compensate for differences, the signal intensity of each volatile compound was normalized to the TIC of all volatiles detected in the particular sample. Compounds in bold are significantly different in melanoma when compared to nevi (*) or to skin (#) control as described in Examples 1 and 2.

Other Embodiments

[0110] Any improvement may be made in part or all of the compositions, kits, and method steps. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended to illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. Any statement herein as to the nature or benefits of the invention or of the preferred embodiments is not intended to be limiting, and the appended claims should not be deemed to be limited by such statements. More generally, no language in the specification should be construed as indicating any non-claimed element as being essential to the practice of the invention. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contraindicated by context.

What is claimed is:

Claims

1. A method of detecting melanoma in at least one subject suspected of having or at risk of having melanoma comprising:

(a) obtaining a biological sample from the at least one subject;

(b) collecting volatile compounds from the headspace of the biological sample;

(c) measuring the levels of a plurality of volatile compounds collected;

(d) identifying volatile compounds that are present at increased or decreased levels relative to a control sample; and

(e) correlating the presence of one or more volatile compounds that are present at increased or decreased levels relative to a control sample with the presence of melanoma in the at least one subject.

2. The method of claim 1, wherein the biological sample is a frozen or fresh punch biopsy.

3. The method of claim 1, wherein the control sample is non-neoplastic skin tissue from the subject.

4. The method of claim 1, wherein melanoma is detected in the subject at an early stage.

5. The method of claim 1, wherein the volatile compounds collected comprise one or more of the volatile compounds selected from the group consisting of: Nonane; Decane, 4-methyl; Cyclohexene, 3-methyl-6-(l-methylethenyl)-, (3R-trans)- (E-isolimonene); Benzene( 1- methylethyl); Benzene, 1,2,3 trimethyl; Benzene, 1,2,4 trimethyl; Benzene, lethyl-2-methyl (ethyltoluene); Benzene, 1 -ethyl-3methyl; Benzene, l-ethyl-4-methyl; Styrene (benzene, ethenyl); Benzyl butyl phthalate; Butanal; Dimethyl sulfone; Indole; Aziridine,2-methyl (ethyleneimine); 1-Dodecanol; 1-Hexadecanol; 1-Pentadecanol; 1-Tridecanol; B-Pinene;

Decane,4-methyl; Dodecane; Dodecane, 5-methyl; Isopropyl Palmitate; Pentadecane; Undecane; l-Methyl-bis(l,2,4)-triazole-5,l'; Benzoic acid, 4-ethoxy-, ethyl ester; D-Limonene; Decanal; Ethylbenzene; Heptanal; Hexanal; Nonanal; Octanal; P-Xylene; l-Hexanol,2-ethyl; 2-Propen-l- ol; 2H-Benzimidazol-2-one, l,3-dihydro-5-methyl-; 3,4-Hexanedione, 2, 2, 5 -trimethyl-; Bis(2- ethylhexyl) phthalate; Oxime-,methoxy-phenyl; Propofol; Propylene Glycol; Pyrrole-3- carbonitrile, 5-formyl-2,4-dimethyl-; o-Hydroxybiphenyl; 1-Tridecene; 2H-Benzimidazol-2-one, l,3-dihydro-5-methyl-; 2-Propanamide -2 -methyl; Acetamide, 2 fiuoro-; Acetone; Benzaldehyde, 2, 5 -bis [(trimethyl); Benzene, 1,3-dimethyl-; Benzene, 2,4-diisocyanato-l -methyl; Camphene; Decane; Dimethyl sulfone; E-Isolimonene; Ethanol; Ethylene oxide; Formic acid; Heptane,2,5,5- trimethyl-; Hydrazine, methyl; Isopropyl Alcohol; Limonene; Octane; o-Xylene; Propanal;

Propylene Oxide; Tridecane; 1,2-Benzenedicarboxylic acid, diisooctyl ester; 2-Ethylhexyl trans- 4-methoxycinnamate; Benzene, 1,3,5-trimethyl; Dichlorodifluoromethane; N- Morpholinomethyl-isopropyl-sulfide; Undecane, 4,7-dimethyl; 1 ,3-Cyclopentadiene, 5-(l- methylethylidene)-; 1-Eicosanol; 1-Hexadecene; 2-Isopropylamino-4-methylbenzonitrile;

Adenosine, 5'-amino-5'-deoxy-; Benzaldehyde, 4-methoxy-; Cyclohexane, ethyl-; Decane, 2- methyl-; Ethanol, 2-butoxy-; Formamide; Heptane, 2,4-dimethyl-; Pentane, 2,3,4-trimethyl-; Phthalic acid, isobutyl 4-octyl ester; Spiro[bicyclo[2.2.1]hept-5-ene-2,l'-cyclopropane]; 2- Ethoxyethyl acrylate; and Nonane, 1-iodo-.

6. The method of claim 5, wherein the volatile compounds collected comprise dodecane, 4-methyl decane, and undecane.

7. The method of claim 5, wherein the volatile compounds collected comprise 1- Hexadecanol, Benzene, 1,3,5-trimethyl, and dodecane .

8. The method of claim 5, wherein the volatile compounds collected comprise Bis(2- ethylhexyl)phthalate, decane, undecane, decane,4-methyl, ethylene oxide, isopropyl palmitate, and phthalic acid,isobutyl 4-octyl ester.

9. The method of claim 1, wherein step (b) of collecting volatile compounds from the headspace of the biological sample comprises Head-space Solid Phase Micro-Extraction (HS-SPME), step (c) of measuring the level of each volatile compound comprises Gas

Chromatography/Mass Spectrometry (GC-MS), and step (d) of correlating the presence of one or more volatile metabolites that are present at increased or decreased levels relative to a control sample with the presence of melanoma in the at least one subject comprises use of a software program.

10. The method of claim 1, wherein the at least one subject comprises a plurality of subjects suspected of having or at risk of having melanoma.

11. The method of claim 1 , further comprising step f) of performing a histological analysis of the biological sample.

12. A method of detecting at least one margin of a melanoma on a subject comprising the steps of:

a) providing a biosensor comprising a substrate having a plurality of different olfactory receptors bound thereto, each olfactory receptor specific for a different volatile compound selected from the group consisting of: Nonane; Decane, 4-methyl; Cyclohexene, 3- methyl-6-(l-methylethenyl)-, (3R-trans)- (E-isolimonene); Benzene( 1-methylethyl); Benzene, 1,2,3 trimethyl; Benzene, 1,2,4 trimethyl; Benzene, lethyl-2 -methyl (ethyltoluene); Benzene, 1- ethyl-3methyl; Benzene, l-ethyl-4-methyl; Styrene (benzene, ethenyl-); Benzyl butyl phthalate; Butanal; Dimethyl sulfone; Indole; Aziridine,2-methyl (ethyleneimine); 1-Dodecanol; 1- Hexadecanol; 1-Pentadecanol; 1-Tridecanol; B-Pinene; Decane,4-methyl; Dodecane; Dodecane, 5-methyl; Isopropyl Palmitate; Pentadecane; Undecane; l-Methyl-bis(l,2,4)-triazole-5,l';

Benzoic acid, 4-ethoxy-, ethyl ester; D-Limonene; Decanal; Ethylbenzene; Heptanal; Hexanal; Nonanal; Octanal; P-Xylene; l-Hexanol,2-ethyl; 2-Propen-l-ol; 2H-Benzimidazol-2-one, 1,3- dihydro-5 -methyl-; 3,4-Hexanedione, 2, 2, 5 -trimethyl-; Bis(2-ethylhexyl) phthalate; Oxime- ,methoxy-phenyl; Propofol; Propylene Glycol; Pyrrole-3-carbonitrile, 5-formyl-2,4-dimethyl-; o- Hydroxybiphenyl; 1-Tridecene; 2H-Benzimidazol-2-one, l,3-dihydro-5-methyl-; 2- Propanamide-2-methyl; Acetamide, 2 fluoro-; Acetone; Benzaldehyde, 2, 5 -bis [(trimethyl);

Benzene, 1,3 -dimethyl-; Benzene, 2,4-diisocyanato-l -methyl; Camphene; Decane; Dimethyl sulfone; E-Isolimonene; Ethanol; Ethylene oxide; Formic acid; Heptane,2,5,5-trimethyl-;

Hydrazine, methyl; Isopropyl Alcohol; Limonene; Octane; o-Xylene; Propanal; Propylene Oxide; Tridecane; 1 ,2-Benzenedicarboxylic acid, diisooctyl ester; 2-Ethylhexyl trans-4- methoxycinnamate; Benzene, 1,3,5-trimethyl; Dichlorodifluoromethane; N-Morpholinomethyl- isopropyl-sulfide; Undecane, 4,7-dimethyl; 1,3-Cyclopentadiene, 5-(l-methylethylidene)-; 1- Eicosanol; 1-Hexadecene; 2-Isopropylamino-4-methylbenzonitrile; Adenosine, 5'-amino-5'- deoxy-; Benzaldehyde, 4-methoxy-; Cyclohexane, ethyl-; Decane, 2-methyl-; Ethanol, 2-butoxy; Formamide; Heptane, 2,4-dimethyl-; Pentane, 2,3,4-trimethyl-; Phthalic acid, isobutyl 4-octyl ester; Spiro[bicyclo[2.2.1]hept-5-ene-2,l'-cyclopropane]; 2-Ethoxyethyl acrylate; and Nonane, 1- iodo-;

wherein each olfactory receptor is conjugated to a detectable label;

(b) placing the biosensor over a plurality of areas of skin on the subject, each area an increasing distance away from the center of the melanoma;

c) identifying volatile compounds collected from the headspace of each area that are present at increased or decreased levels relative to volatile compounds collected from headspace of non-neoplastic skin tissue from the subject;

d) for each area, correlating the presence of one or more of the volatile compounds that are present at increased or decreased levels relative to volatile compounds collected from headspace of non-neoplastic skin tissue from the subject with the presence of melanoma cells in the area; and

(e) identifying at least one area that does not include volatile compounds that are present at increased or decreased levels relative to volatile compounds collected from headspace of non-neoplastic skin tissue from the subject as at least one margin of the tissue.

13. The method of claim 12, wherein the volatile compounds comprise fifteen or more of the volatile compounds selected from the group consisting of: Nonane; Decane, 4-methyl;

Cyclohexene, 3-methyl-6-(l-methylethenyl)-, (3R-trans)- (E-isolimonene); Benzene( 1- methylethyl); Benzene, 1,2,3 trimethyl; Benzene, 1,2,4 trimethyl; Benzene, lethyl-2-methyl (ethyltoluene); Benzene, 1 -ethyl-3methyl; Benzene, l-ethyl-4-methyl; Styrene (benzene, ethenyl); Benzyl butyl phthalate; Butanal; Dimethyl sulfone; Indole; Aziridine,2-methyl (ethyleneimine); 1-Dodecanol; 1-Hexadecanol; 1-Pentadecanol; 1-Tridecanol; B-Pinene;

Decane,4-methyl; Dodecane; Dodecane, 5-methyl; Isopropyl Palmitate; Pentadecane; Undecane; l-Methyl-bis(l,2,4)-triazole-5,l'; Benzoic acid, 4-ethoxy-, ethyl ester; D-Limonene; Decanal; Ethylbenzene; Heptanal; Hexanal; Nonanal; Octanal; P-Xylene; l-Hexanol,2-ethyl; 2-Propen-l- ol; 2H-Benzimidazol-2-one, l,3-dihydro-5-methyl-; 3,4-Hexanedione, 2, 2, 5 -trimethyl-; Bis(2- ethylhexyl) phthalate; Oxime-,methoxy-phenyl; Propofol; Propylene Glycol; Pyrrole-3- carbonitrile, 5-formyl-2,4-dimethyl-; o-Hydroxybiphenyl; 1-Tridecene; 2H-Benzimidazol-2-one, l,3-dihydro-5-methyl-; 2-Propanamide -2 -methyl; Acetamide, 2 fluoro-; Acetone; Benzaldehyde, 2, 5 -bis [(trimethyl); Benzene, 1,3-dimethyl-; Benzene, 2,4-diisocyanato-l -methyl; Camphene; Decane; Dimethyl sulfone; E-Isolimonene; Ethanol; Ethylene oxide; Formic acid; Heptane,2,5,5- trimethyl-; Hydrazine, methyl; Isopropyl Alcohol; Limonene; Octane; o-Xylene; Propanal;

Propylene Oxide; Tridecane; 1,2-Benzenedicarboxylic acid, diisooctyl ester; 2-Ethylhexyl trans- 4-methoxycinnamate; Benzene, 1,3,5-trimethyl; Dichlorodifluoromethane; N- Morpholinomethyl-isopropyl-sulfide; Undecane, 4,7-dimethyl; 1 ,3-Cyclopentadiene, 5-(l- methylethylidene)-; 1-Eicosanol; 1-Hexadecene; 2-Isopropylamino-4-methylbenzonitrile;

Adenosine, 5'-amino-5'-deoxy-; Benzaldehyde, 4-methoxy-; Cyclohexane, ethyl-; Decane, 2- methyl-; Ethanol, 2-butoxy-; Formamide; Heptane, 2,4-dimethyl-; Pentane, 2,3,4-trimethyl-; Phthalic acid, isobutyl 4-octyl ester; Spiro[bicyclo[2.2.1]hept-5-ene-2,l'-cyclopropane]; 2- Ethoxyethyl acrylate; and Nonane, 1-iodo-.

14. The method of claim 13, wherein the volatile compounds comprise dodecane, 4- methyl decane, and undecane.

15. The method of claim 12, wherein step (d) of correlating the presence of one or more of the volatile compounds that are present at increased or decreased levels relative to volatile compounds collected from headspace of non-neoplastic skin tissue from the subject with the presence of melanoma cells in the biopsy comprises use of a software program.

16. The method of claim 12, wherein all margins of the melanoma are detected.

17. A method of detecting at least one margin of a melanoma on a subject comprising the steps of:

a) providing a biosensor comprising a substrate having a plurality of different olfactory receptors bound thereto, each olfactory receptor specific for a different volatile compound selected from the group consisting of: Nonane; Decane, 4-methyl; Cyclohexene, 3- methyl-6-(l-methylethenyl)-, (3R-trans)- (E-isolimonene); Benzene( 1-methylethyl); Benzene, 1,2,3 trimethyl; Benzene, 1,2,4 trimethyl; Benzene, lethyl-2 -methyl (ethyltoluene); Benzene, 1- ethyl-3methyl; Benzene, l-ethyl-4-methyl; Styrene (benzene, ethenyl-); Benzyl butyl phthalate; Butanal; Dimethyl sulfone; Indole; Aziridine,2-methyl (ethyleneimine); 1-Dodecanol; 1- Hexadecanol; 1-Pentadecanol; 1-Tridecanol; B-Pinene; Decane,4-methyl; Dodecane; Dodecane, 5-methyl; Isopropyl Palmitate; Pentadecane; Undecane; l-Methyl-bis(l,2,4)-triazole-5,l';

Benzoic acid, 4-ethoxy-, ethyl ester; D-Limonene; Decanal; Ethylbenzene; Heptanal; Hexanal; Nonanal; Octanal; P-Xylene; l-Hexanol,2-ethyl; 2-Propen-l-ol; 2H-Benzimidazol-2-one, 1,3- dihydro-5 -methyl-; 3,4-Hexanedione, 2,2,5-trimethyl-; Bis(2-ethylhexyl) phthalate; Oxime- ,methoxy-phenyl; Propofol; Propylene Glycol; Pyrrole-3-carbonitrile, 5-formyl-2,4-dimethyl-; o- Hydroxybiphenyl; 1-Tridecene; 2H-Benzimidazol-2-one, l,3-dihydro-5-methyl-; 2- Propanamide-2-methyl; Acetamide, 2 fluoro-; Acetone; Benzaldehyde, 2,5-bis[(trimethyl);

Benzene, 1,3 -dimethyl-; Benzene, 2,4-diisocyanato-l -methyl; Camphene; Decane; Dimethyl sulfone; E-Isolimonene; Ethanol; Ethylene oxide; Formic acid; Heptane,2,5,5-trimethyl-;

Hydrazine, methyl; Isopropyl Alcohol; Limonene; Octane; o-Xylene; Propanal; Propylene Oxide; Tridecane; 1 ,2-Benzenedicarboxylic acid, diisooctyl ester; 2-Ethylhexyl trans-4- methoxycinnamate; Benzene, 1,3,5-trimethyl; Dichlorodifluoromethane; N-Morpholinomethyl- isopropyl-sulfide; Undecane, 4, 7-dimethyl; 1,3-Cyclopentadiene, 5-(l-methylethylidene)-; 1- Eicosanol; 1-Hexadecene; 2-Isopropylamino-4-methylbenzonitrile; Adenosine, 5'-amino-5'- deoxy-; Benzaldehyde, 4-methoxy-; Cyclohexane, ethyl-; Decane, 2-methyl-; Ethanol, 2-butoxy; Formamide; Heptane, 2,4-dimethyl-; Pentane, 2,3,4-trimethyl-; Phthalic acid, isobutyl 4-octyl ester; Spiro[bicyclo[2.2.1]hept-5-ene-2,l'-cyclopropane]; 2-Ethoxyethyl acrylate; and Nonane, 1- iodo-;

wherein each olfactory receptor is conjugated to a detectable label;

(b) placing the biosensor over a plurality of areas of skin on the subject, each area an increasing distance away from the center of the melanoma;

c) determining the presence or absence of a plurality of the volatile compounds of step a) in the headspace of each area; and d) for each area, correlating the presence or absence of the plurality of the volatile compounds of step a) in the headspace of each area with the presence or absence of melanoma cells in the area and determining at least one margin of the melanoma.

18. The method of claim 17, wherein step c) further comprises determining the presence or absence of the plurality of the volatile compounds in the headspace of non-neoplastic skin tissue from the subject.

19. The method of claim 17, wherein the plurality of the volatile compounds comprise dodecane, 4-methyl decane, and undecane.

20. A biosensor for diagnosing melanoma in a subject, the biosensor comprising a substrate having a plurality of different olfactory receptors bound thereto, each olfactory receptor specific for a different volatile compound selected from the group consisting of: Nonane;

Decane, 4-methyl; Cyclohexene, 3-methyl-6-(l-methylethenyl)-, (3R-trans)- (E-isolimonene); Benzene( 1-methylethyl); Benzene, 1,2,3 trimethyl; Benzene, 1,2,4 trimethyl; Benzene, 1 ethyls- methyl (ethyltoluene); Benzene, l-ethyl-3methyl; Benzene, l-ethyl-4-methyl; Styrene (benzene, ethenyl-); Benzyl butyl phthalate; Butanal; Dimethyl sulfone; Indole; Aziridine,2-methyl (ethyleneimine); 1-Dodecanol; 1-Hexadecanol; 1-Pentadecanol; 1-Tridecanol; B-Pinene;

Decane,4-methyl; Dodecane; Dodecane, 5-methyl; Isopropyl Palmitate; Pentadecane; Undecane; l-Methyl-bis(l,2,4)-triazole-5,l'; Benzoic acid, 4-ethoxy-, ethyl ester; D-Limonene; Decanal; Ethylbenzene; Heptanal; Hexanal; Nonanal; Octanal; P-Xylene; l-Hexanol,2-ethyl; 2-Propen-l- ol; 2H-Benzimidazol-2-one, l,3-dihydro-5-methyl-; 3,4-Hexanedione, 2, 2, 5 -trimethyl-; Bis(2- ethylhexyl) phthalate; Oxime-,methoxy-phenyl; Propofol; Propylene Glycol; Pyrrole-3- carbonitrile, 5-formyl-2,4-dimethyl-; o-Hydroxybiphenyl; 1-Tridecene; 2H-Benzimidazol-2-one, l,3-dihydro-5-methyl-; 2-Propanamide -2 -methyl; Acetamide, 2 fluoro-; Acetone; Benzaldehyde, 2,5-bis[(trimethyl); Benzene, 1,3-dimethyl-; Benzene, 2,4-diisocyanato-l -methyl; Camphene; Decane; Dimethyl sulfone; E-Isolimonene; Ethanol; Ethylene oxide; Formic acid; Heptane,2,5,5- trimethyl-; Hydrazine, methyl; Isopropyl Alcohol; Limonene; Octane; o-Xylene; Propanal;

Propylene Oxide; Tridecane; 1,2-Benzenedicarboxylic acid, diisooctyl ester; 2-Ethylhexyl trans- 4-methoxycinnamate; Benzene, 1,3,5-trimethyl; Dichlorodifluoromethane; N- Morpholinomethyl-isopropyl-sulfide; Undecane, 4,7-dimethyl; 1 ,3-Cyclopentadiene, 5-(l- methylethylidene)-; 1-Eicosanol; 1-Hexadecene; 2-Isopropylamino-4-methylbenzonitrile;

Adenosine, 5'-amino-5'-deoxy-; Benzaldehyde, 4-methoxy-; Cyclohexane, ethyl-; Decane, 2- methyl-; Ethanol, 2-butoxy-; Formamide; Heptane, 2,4-dimethyl-; Pentane, 2,3,4-trimethyl-; Phthalic acid, isobutyl 4-octyl ester; Spiro[bicyclo[2.2.1]hept-5-ene-2,l'-cyclopropane]; 2- Ethoxyethyl acrylate; and Nonane, 1-iodo-;

wherein each olfactory receptor is conjugated to a detectable label.

21. The biosensor of claim 20, further comprising: a detector to detect the detectable label, wherein the volatile compounds comprise bis(2-ethylhexyl) phthalate, decane, undecane, decane-4-methyl, ethylene oxide, isopropyl palmitate, phthalic acid, isobutyl 4-octyl ester, 1- hexadecanol, benzene, 1,3,5 trimethyl, and dodecane.

22. The biosensor of claim 21, wherein the detectable label is fluorescence, the detector is a fluorometer, and the biosensor further comprises a processor to analyze the fluorescence.

23. The biosensor of claim 20, wherein at least 15 different volatile compounds are analyzed.

24. The biosensor of claim 21, further comprising at least one positive control and at least one negative control.

25. The biosensor of claim 24, further comprising packaging and instructions for use.

26. A method of detecting melanoma in at least one subject suspected of having or at risk of having melanoma comprising:

(a) providing at least a first biosensor comprising a substrate having a plurality of different olfactory receptors bound thereto, each olfactory receptor specific for a different volatile compound selected from the group consisting of: Nonane; Decane, 4-methyl;

Cyclohexene, 3-methyl-6-(l-methylethenyl)-, (3R-trans)- (E-isolimonene); Benzene( 1- methylethyl); Benzene, 1,2,3 trimethyl; Benzene, 1,2,4 trimethyl; Benzene, lethyl-2-methyl (ethyltoluene); Benzene, 1 -ethyl-3methyl; Benzene, l-ethyl-4-methyl; Styrene (benzene, ethenyl); Benzyl butyl phthalate; Butanal; Dimethyl sulfone; Indole; Aziridine,2-methyl

(ethyleneimine); 1-Dodecanol; 1-Hexadecanol; 1-Pentadecanol; 1-Tridecanol; B-Pinene;

Decane,4-methyl; Dodecane; Dodecane, 5-methyl; Isopropyl Palmitate; Pentadecane; Undecane; l-Methyl-bis(l,2,4)-triazole-5,l'; Benzoic acid, 4-ethoxy-, ethyl ester; D-Limonene; Decanal; Ethylbenzene; Heptanal; Hexanal; Nonanal; Octanal; P-Xylene; l-Hexanol,2-ethyl; 2-Propen-l- ol; 2H-Benzimidazol-2-one, l,3-dihydro-5-methyl-; 3,4-Hexanedione, 2, 2, 5 -trimethyl-; Bis(2- ethylhexyl) phthalate; Oxime-,methoxy-phenyl; Propofol; Propylene Glycol; Pyrrole-3- carbonitrile, 5-formyl-2,4-dimethyl-; o-Hydroxybiphenyl; 1-Tridecene; 2H-Benzimidazol-2-one, l,3-dihydro-5-methyl-; 2-Propanamide -2 -methyl; Acetamide, 2 fluoro-; Acetone; Benzaldehyde, 2,5-bis[(trimethyl); Benzene, 1,3-dimethyl-; Benzene, 2,4-diisocyanato-l -methyl; Camphene; Decane; Dimethyl sulfone; E-Isolimonene; Ethanol; Ethylene oxide; Formic acid; Heptane,2,5,5- trimethyl-; Hydrazine, methyl; Isopropyl Alcohol; Limonene; Octane; o-Xylene; Propanal;

Propylene Oxide; Tridecane; 1,2-Benzenedicarboxylic acid, diisooctyl ester; 2-Ethylhexyl trans- 4-methoxycinnamate; Benzene, 1,3,5-trimethyl; Dichlorodifluoromethane; N- Morpholinomethyl-isopropyl-sulfide; Undecane, 4,7-dimethyl; 1 ,3-Cyclopentadiene, 5-(l- methylethylidene)-; 1-Eicosanol; 1-Hexadecene; 2-Isopropylamino-4-methylbenzonitrile;

Adenosine, 5'-amino-5'-deoxy-; Benzaldehyde, 4-methoxy-; Cyclohexane, ethyl-; Decane, 2- methyl-; Ethanol, 2-butoxy-; Formamide; Heptane, 2,4-dimethyl-; Pentane, 2,3,4-trimethyl-; Phthalic acid, isobutyl 4-octyl ester; Spiro[bicyclo[2.2.1]hept-5-ene-2,l'-cyclopropane]; 2- Ethoxyethyl acrylate; and Nonane, 1-iodo-;

wherein each olfactory receptor is conjugated to a detectable label;

(b) contacting the at least first biosensor with volatile compounds from the headspace of a melanoma or tissue isolated from the at least one subject or from the headspace of a melanoma or tissue on the at least one subject;

(b) detecting binding of one or more of the olfactory receptors with one or more of the volatile compounds; and

(c) correlating binding of one or more of the olfactory receptors to one or more of the volatile compounds with the presence or absence of melanoma in the at least one subject.

27. The method of claim 26, wherein step (c) of correlating binding of one or more of the olfactory receptors to one or more of the volatile compounds with the presence or absence of melanoma in the at least one subject comprises correlating the presence of one or more volatile compounds that are present at increased or decreased levels relative to a control sample with the presence of melanoma in the at least one subject.

28. The method of claim 27, wherein the control sample is non-neoplastic skin tissue from the subject.

29. The method of claim 26, further comprising contacting a second biosensor with volatile compounds obtained from headspace of non-neoplastic skin tissue from the same subject, detecting binding of one or more of the olfactory receptors with one or more of the volatile compounds from the headspace of non-neoplastic skin tissue from the same subject, and comparing the bound volatile compounds detected by the first and second biosensors.

30. The method of claim 26, wherein the melanoma or tissue isolated from the at least one subject is a punch biopsy.

31. The method of claim 30, wherein the punch biopsy is a frozen biopsy or a fresh biopsy.

32. The method of claim 26, wherein melanoma is detected in the subject at an early stage.

33. The method of claim 26, wherein the volatile compounds comprise one or more of the volatile compounds selected from the group consisting of: Nonane; Decane, 4-methyl;

Cyclohexene, 3-methyl-6-(l-methylethenyl)-, (3R-trans)- (E-isolimonene); Benzene( 1- methylethyl); Benzene, 1,2,3 trimethyl; Benzene, 1,2,4 trimethyl; Benzene, lethyl-2-methyl (ethyltoluene); Benzene, 1 -ethyl-3methyl; Benzene, l-ethyl-4-methyl; Styrene (benzene, ethenyl); Benzyl butyl phthalate; Butanal; Dimethyl sulfone; Indole; Aziridine,2-methyl (ethyleneimine); 1-Dodecanol; 1-Hexadecanol; 1-Pentadecanol; 1-Tridecanol; B-Pinene;

Decane,4-methyl; Dodecane; Dodecane, 5-methyl; Isopropyl Palmitate; Pentadecane; Undecane; l-Methyl-bis(l,2,4)-triazole-5,l'; Benzoic acid, 4-ethoxy-, ethyl ester; D-Limonene; Decanal; Ethylbenzene; Heptanal; Hexanal; Nonanal; Octanal; P-Xylene; l-Hexanol,2-ethyl; 2-Propen-l- ol; 2H-Benzimidazol-2-one, l,3-dihydro-5-methyl-; 3,4-Hexanedione, 2, 2, 5 -trimethyl-; Bis(2- ethylhexyl) phthalate; Oxime-,methoxy-phenyl; Propofol; Propylene Glycol; Pyrrole-3- carbonitrile, 5-formyl-2,4-dimethyl-; o-Hydroxybiphenyl; 1-Tridecene; 2H-Benzimidazol-2-one, l,3-dihydro-5-methyl-; 2-Propanamide -2 -methyl; Acetamide, 2 fluoro-; Acetone; Benzaldehyde, 2, 5 -bis [(trimethyl); Benzene, 1,3-dimethyl-; Benzene, 2,4-diisocyanato-l -methyl; Camphene; Decane; Dimethyl sulfone; E-Isolimonene; Ethanol; Ethylene oxide; Formic acid; Heptane,2,5,5- trimethyl-; Hydrazine, methyl; Isopropyl Alcohol; Limonene; Octane; o-Xylene; Propanal;

Propylene Oxide; Tridecane; 1,2-Benzenedicarboxylic acid, diisooctyl ester; 2-Ethylhexyl trans- 4-methoxycinnamate; Benzene, 1,3,5-trimethyl; Dichlorodifluoromethane; N- Morpholinomethyl-isopropyl-sulfide; Undecane, 4,7-dimethyl; 1 ,3-Cyclopentadiene, 5-(l- methylethylidene)-; 1-Eicosanol; 1-Hexadecene; 2-Isopropylamino-4-methylbenzonitrile;

Adenosine, 5'-amino-5'-deoxy-; Benzaldehyde, 4-methoxy-; Cyclohexane, ethyl-; Decane, 2- methyl-; Ethanol, 2-butoxy-; Formamide; Heptane, 2,4-dimethyl-; Pentane, 2,3,4-trimethyl-; Phthalic acid, isobutyl 4-octyl ester; Spiro[bicyclo[2.2.1]hept-5-ene-2,l'-cyclopropane]; 2- Ethoxy ethyl acrylate; and Nonane, 1-iodo-.

34. The method of claim 33, wherein the volatile compounds comprise dodecane, 4- methyl decane, and undecane.

35. The method of claim 33, wherein the volatile compounds comprise 1-Hexadecanol, Benzene, 1,3,5-trimethyl, and dodecane.

36. The method of claim 33, wherein the volatile compounds comprise Bis(2- ethylhexyl)phthalate, decane, undecane, decane,4-methyl, ethylene oxide, isopropyl palmitate, and phthalic acid,isobutyl 4-octyl ester.

37. The method of claim 26, wherein the at least one subject comprises a plurality of subjects suspected of having or at risk of having melanoma.

38. The method of claim 26, further comprising step d) of performing a histological analysis of the melanoma or tissue.

39. A kit for detecting melanoma in a subject, the kit comprising:

(a) at least a first biosensor comprising a substrate having a plurality of different olfactory receptors bound thereto, each olfactory receptor specific for a different volatile compound selected from the group consisting of: Nonane; Decane, 4-methyl; Cyclohexene, 3- methyl-6-(l-methylethenyl)-, (3R-trans)- (E-isolimonene); Benzene( 1-methylethyl); Benzene, 1,2,3 trimethyl; Benzene, 1,2,4 trimethyl; Benzene, lethyl-2 -methyl (ethyltoluene); Benzene, 1- ethyl-3methyl; Benzene, l-ethyl-4-methyl; Styrene (benzene, ethenyl-); Benzyl butyl phthalate; Butanal; Dimethyl sulfone; Indole; Aziridine,2-methyl (ethyleneimine); 1-Dodecanol; 1- Hexadecanol; 1-Pentadecanol; 1-Tridecanol; B-Pinene; Decane,4-methyl; Dodecane; Dodecane, 5-methyl; Isopropyl Palmitate; Pentadecane; Undecane; l-Methyl-bis(l,2,4)-triazole-5,l';

Benzoic acid, 4-ethoxy-, ethyl ester; D-Limonene; Decanal; Ethylbenzene; Heptanal; Hexanal; Nonanal; Octanal; P-Xylene; l-Hexanol,2-ethyl; 2-Propen-l-ol; 2H-Benzimidazol-2-one, 1,3- dihydro-5 -methyl-; 3,4-Hexanedione, 2, 2, 5 -trimethyl-; Bis(2-ethylhexyl) phthalate; Oxime- ,methoxy-phenyl; Propofol; Propylene Glycol; Pyrrole-3-carbonitrile, 5-formyl-2,4-dimethyl-; o- Hydroxybiphenyl; 1-Tridecene; 2H-Benzimidazol-2-one, l,3-dihydro-5-methyl-; 2- Propanamide-2-methyl; Acetamide, 2 fluoro-; Acetone; Benzaldehyde, 2, 5 -bis [(trimethyl);

Benzene, 1,3 -dimethyl-; Benzene, 2,4-diisocyanato-l -methyl; Camphene; Decane; Dimethyl sulfone; E-Isolimonene; Ethanol; Ethylene oxide; Formic acid; Heptane,2,5,5-trimethyl-;

Hydrazine, methyl; Isopropyl Alcohol; Limonene; Octane; o-Xylene; Propanal; Propylene Oxide; Tridecane; 1 ,2-Benzenedicarboxylic acid, diisooctyl ester; 2-Ethylhexyl trans-4- methoxycinnamate; Benzene, 1,3 ,5 -trimethyl; Dichlorodifluoromethane; N-Morpholinomethyl- isopropyl-sulfide; Undecane, 4, 7-dimethyl; 1,3-Cyclopentadiene, 5-(l-methylethylidene)-; 1- Eicosanol; 1-Hexadecene; 2-Isopropylamino-4-methylbenzonitrile; Adenosine, 5'-amino-5'- deoxy-; Benzaldehyde, 4-methoxy-; Cyclohexane, ethyl-; Decane, 2-methyl-; Ethanol, 2-butoxy; Formamide; Heptane, 2,4-dimethyl-; Pentane, 2,3,4-trimethyl-; Phthalic acid, isobutyl 4-octyl ester; Spiro[bicyclo[2.2.1]hept-5-ene-2,l'-cyclopropane]; 2-Ethoxyethyl acrylate; and Nonane, 1- iodo-;

wherein each olfactory receptor is conjugated to a detectable label;

b) at least one reagent for detecting binding of one or more of the olfactory receptors to one or more of the volatile compounds from the headspace of a melanoma on the subject or the headspace of a melanoma or portion of a melanoma isolated from the subject; and

(c) instructions for use.