WO2012040318A2 - Compositions, procédés et nécessaires de détection de mélanome et de contours de mélanome - Google Patents

Compositions, procédés et nécessaires de détection de mélanome et de contours de mélanome Download PDF

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WO2012040318A2
WO2012040318A2 PCT/US2011/052538 US2011052538W WO2012040318A2 WO 2012040318 A2 WO2012040318 A2 WO 2012040318A2 US 2011052538 W US2011052538 W US 2011052538W WO 2012040318 A2 WO2012040318 A2 WO 2012040318A2
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methyl
benzene
melanoma
trimethyl
decane
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WO2012040318A3 (fr
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Tatjana Abaffy
Richard Anthony Defazio
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University of Miami
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University of Miami
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5751—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the skin, e.g. melanoma

Definitions

  • the invention relates generally to the fields of molecular genetics, molecular biology, and medicine.
  • 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.
  • melanoma 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).
  • compositions, methods and kits for detection of melanoma 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.
  • a biosensor e.g., one or more biosensors
  • 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.
  • HS-SPME head space solid phase micro-extraction
  • GC/MS gas chromatography/mass spectrometry
  • patient 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]
  • patient 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.
  • 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.
  • bind 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.
  • a first molecule that "specifically binds" a second molecule has a binding affinity greater than about 10 8 to 10 12 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).
  • diagnosis means identifying the presence or nature of a pathologic condition (e.g., melanoma).
  • 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").
  • volatile compound and “volatile metabolite” are used interchangeably to mean any compound which has vapor pressure >0. lmmHg.
  • a method of detecting melanoma in at least one subject suspected of having or at risk of having melanoma 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.
  • a biological sample e.g., a frozen or fresh punch biopsy
  • 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.
  • 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.
  • a method of detecting at least one margin of a melanoma on a subject 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-
  • 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).
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • a control sample e.g., a non-neoplastic skin tissue from the 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.
  • kits for detecting melanoma in a subject 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)
  • 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.
  • 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.
  • 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.
  • FIG. 2 is a plot and a photograph of a melanoma showing spatial mapping of biomarkers.
  • 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.
  • 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.
  • Some peaks are unique in melanoma (***), some are increased (**) and some are decreased (*) in melanoma vs. naevi.
  • 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.
  • FIG. 5 is a series of graphs showing differentially expressed volatile compounds in melanoma vs naevi.
  • 2-propanamide* is 2-propanamide, 2-methyl
  • benzene** is benzene, 1,3 dimethyl
  • phthalate*** is bis(2-ethylhexyl) phthalate.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • compositions, methods and kits for detecting melanoma and determining melanoma margins are described herein. 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.
  • melanoma a subject (e.g., human) using biomarkers (volatile compounds or metabolites).
  • biomarkers volatile compounds or metabolites
  • the subject is suspected of or at risk of having melanoma.
  • the presence and/or level of one or more volatile compounds as described herein is analyzed.
  • 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.
  • 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.
  • the control sample can be any suitable control sample.
  • 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).
  • a melanoma is detected in the subject at an early stage.
  • 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.
  • volatile compounds collected include 1- Hexadecanol, Benzene, 1,3,5-trimethyl, and dodecane.
  • volatile compounds collected include Bis(2-ethylhexyl)phthalate, decane, undecane, 4-methyl decane, ethylene oxide, isopropyl palmitate, and phthalic acid, isobutyl 4-octyl ester.
  • 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.
  • Any suitable biological sample e.g., biopsy
  • biological samples include fresh tissue, frozen tissue, and skin, nevi (moles) and melanoma lesions (fresh or frozen).
  • 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).
  • 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.
  • 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.
  • 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.
  • a biological sample e.g., punch biopsy
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the detectable label is fluorescence
  • the detector is a fluorometer
  • 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.
  • 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.
  • 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.
  • 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.).
  • the array 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.
  • 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.
  • a software program that can compare the result from each subject and indicate if melanoma is present or absent is typically 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.
  • 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.
  • melanoma 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.
  • 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.
  • 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.
  • melanoma can be detected in the subject at an early stage.
  • matched, non-neoplastic skin tissue from the same subject is used as a control.
  • 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.
  • 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.
  • a single biosensor could detect the melanoma volatile signature relative to a database of previously collected skin and nevi samples.
  • 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.
  • one or more subjects can be simultaneously tested for the presence of melanoma.
  • the volatile compounds described herein can be used to detect the margins of the melanoma.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • Example 1 Different Volatile Signatures From Skin, Naevi and Melanoma - a Novel Approach To Detect A Pathological Process
  • 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.
  • 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.
  • 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.
  • FIG. 5A, B and C 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.
  • 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.
  • lymph node, left neck M 53 W present adipose tissue
  • metastatic MM lymph node left axillary M 63 W metastatic MM lymph node F 83 W lymph node, left superficial
  • 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.
  • FIG. 6C A graphical representation of these data is presented in the form of the heat map (FIG. 6C).
  • 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.
  • tissue preparation methodology did not have an effect.
  • 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.
  • 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.
  • 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.
  • melanoma samples were analyzed and confirmed by H&E staining. Also collected and analyzed were 17 control air samples to identify air contaminants.
  • 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- ⁇ PDMS-DVB, Cat No. 57344-U, Supelco, Belle fonte, PA, USA). Extraction selectivity depends on the type of the fiber and the coating thickness.
  • the axilla sample from melanoma patient 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.
  • Hewlett Packard 6890 gas chromatograph Hewlett Packard, Avondale, PA
  • a non-polar DB-5MS column model No. J&W 128- 5522, 25mx0.2mm ⁇ . ⁇ . ⁇ 0.33 ⁇ film
  • Helium carrier gas flow was run in constant flow at 0.7 mL min "1 .
  • 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).
  • 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).
  • 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.
  • 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.
  • Naevi exist in a growth-arrested state predominantly induced by BRAFV600E (Michaloglou, C, et al, Nature, 2005. 436(7051): p. 720-4).
  • BRAFV600E a growth-arrested state predominantly induced by BRAFV600E
  • the volatile signature from naevi revealed pyridine and 3-hexanol as specific naevi compounds not found in melanoma (FIG. 4 and FIG. 7).
  • 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).
  • compound CAS 112-40-3 is dodecane, detected previously (see Table 4)
  • 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)
  • Example 3 A case report - Volatile metabolomic signature of malignant melanoma using matching skin as a control
  • HS-SPME Head Space Solid Phase Micro-Extraction
  • 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.
  • 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.
  • 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- ⁇ PDMS-DVB, Cat No. 57344-U, Supelco, Bellefonte, PA, USA).
  • HS-SPME head space-solid phase microextraction
  • 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%.
  • 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.
  • FAS fatty acid synthase
  • 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.
  • 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.
  • 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.
  • 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.
  • 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

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Abstract

L'invention concerne des compositions, des procédés et des nécessaires de détection d'un mélanome et de détermination de contours de mélanome. La présente invention concerne un nouveau groupe de marqueurs biologiques, métaboliques et volatils qui peuvent être utilisés dans le diagnostic d'un cancer de la peau avec présence de mélanome. Une nouvelle approche pour détecter un mélanome est fondée sur des sous-produits volatils de métabolisme de cancer altéré. La présente invention concerne en outre un procédé d'identification de molécules utiles dans la détection de mélanome et établit une fondation pour le développement d'une technologie de détection non invasive, d'un biocapteur, pour le diagnostic de mélanome. Les utilisations pour cette technologie comprennent un écran de diagnostic qui aidera les cliniciens à évaluer cette maladie. Un avantage fourni par les compositions, les procédés et les nécessaires décrits par les présentes, est que les métabolites sont à la phase finale de la cascade génome-transcriptome-protéome-métabolome et sont ainsi les plus prédictifs du phénotype de cancer.
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