WO2007048107A2 - Profilage protéomique de tumeurs de cellule souche et procédés associés, protéines et molécules d’acide nucléique - Google Patents

Profilage protéomique de tumeurs de cellule souche et procédés associés, protéines et molécules d’acide nucléique Download PDF

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WO2007048107A2
WO2007048107A2 PCT/US2006/060073 US2006060073W WO2007048107A2 WO 2007048107 A2 WO2007048107 A2 WO 2007048107A2 US 2006060073 W US2006060073 W US 2006060073W WO 2007048107 A2 WO2007048107 A2 WO 2007048107A2
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sample
biomarker
tumor
nucleic acid
molecule
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PCT/US2006/060073
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WO2007048107A3 (fr
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Susan E. Lana
Patrick J. Gaines
Timothy Powell
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Heska Corporation
Colorado State University Research Foundation
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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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers

Definitions

  • the invention relates to the field of serum proteoraic profiling of tumors, and in particular canine mast cell tumors. More particularly, the invention relates to the utilization of mass spectroscopy to identify biological molecules (biomarkers) that can be used to identify the presence of tumors in animals. Such biomarkers may also be used to differentiate the grade and aggressiveness of tumors.
  • biomarkers biological molecules
  • the present invention also relates to biological molecules identified using such techniques, including proteins, nucleic acid molecules and antibodies raised against such biological molecules, and detection methods and kits that utilize such biological molecules for, e.g. diagnosis, determination of the stage of cancer and monitoring the treatment of cancer.
  • Cancer which is the leading cause of natural death in dogs and cats, is a disease resulting from uncontrolled cell growth. Cancer cells are different, from non- cancer (normal) cells in that cancer cells have lost Lhe usual regulatory mechanisms which control cell growth and which usually prevent the cell from dividing inappropriately. This loss of control can result from loss of intracellular regulatory mechanisms or the loss of the ability of the cell to respond to extracellular signals that govern cell growth. Either way the result is that cancer cells, in contrast to normal cells which cease to grow and divide in response to extracellular signals, ignore such regulatory signals and continue to divide and form new, abnormal cells. Such unregulated growth leads to die formation of masses of cells referred to as tumors. Such tumors are generically referred to as cancer and the terms can be, and are, used interchangeably.
  • Cancers, or tumors are classified as either benign or malignant, depending on the characteristics of the cells which form the tumor.
  • Cells in benign tumors are usually noninvasive with regard to surrounding tissue and usually do not spread to areas of the body beyond the initial tumor site.
  • cells within malignant tumors usually grow rapidly, are invasive and usually spread to other sites in the body where they begin to grow and invade other tissues.
  • Whether a tumor is benign or malignant is determined by many factors, a major one being the phenotypic characteristic and behavior of the underlying cells which make up the tumor. The characteristics and behaviors of these cells are important in that they are linked to the severity of the resulting disease and can be used to predict the patients long term, clinical outcome.
  • Tumors are classified using a system which assigns the tumor a grade based on the histological appearance of cells removed from the tumor. The grade, along with several other parameters, is then used to predict the long term response to treatment, and survival.
  • the grading of cancers is based on the physical appearance of tumor cells. While different cancer types have different grading systems, in general, the grading scale ranges from I to III or IV. In grade 1 tumors (low grade), the cells are usually well-differentiated and grow relatively slowly. In grade III or IV tumors (high grade), the cell are poorly differentiated and no longer display the characteristics of cells from the tissue of their origin. Additionally, these cells are considered aggressive in that they grow rapidly and will often invade the surrounding areas, including lymph nodes and bones. Once these cells have invaded such surrounding areas, they are transported throughout the body, seeding other sites and allowing for new tumor growth at those distant sites. Not surprisingly, higher-grade tumors have a poorer clinical outcome.
  • mast cell tumors A common tumor seen in dogs and cats is the mast cell tumor which accounts for approximately 11 to 27% of all cutaneous neoplasms in the dog and 15% in the cat.
  • MCTs mast cell tumors
  • Mast cells which are a normal component of the immune system and an important component of the inflammatory response, contain cytoplasmic granules containing biologically active substances such as heparin and histamine. The reason for the malignant transformation of these cells remains unclear.
  • mast cell tumors are histologically categorized into three grades using various grading schemes, the most common of which is the Patnaik grading scale.
  • grade I tumors contain cells which are well-differentiated with large and plentiful cytoplasmic granules and clearly defined, cytoplasmic boundaries.
  • Grade II (intermediate) tumors contain cells which are more closely packed, with indistinct cytoplasmic boundaries, and fewer cytoplasmic granules.
  • grade III tumors contain cells which have highly undifferentiated cytoplasmic boundaries and a low number of cytoplasmic granules.
  • Patients with grade I tumors have the greatest chance of survival, with 77% surviving at least 6 months following surgery to remove the tumor. The 6 month survival rate drops to 45% and 13%, respectively, for patients with grade II or grade HI tumors.
  • Such methods use arrays of nucleic acid probes to capture and display the population of mRNA's present in a population of cells at a given time or under a given set of conditions.
  • Later developments have focused on examining the expression pattern of the entire set of proteins, referred to as the proteome, present in a population of cells at a given time or under a given set of conditions.
  • proleomic profiling is referred to as proleomic profiling.
  • Several methods have been used to analyze the proteome including 2D-gel electrophoresis, parallel chromatography, antibody arrays and mass spectrometry.
  • Methods utilizing mass spectrometry for the analysis of target proteins in a sample are well known to those in the art; examples include Matrix Assisted Laser Desorption (MALDI), continuous or pulsed EIectrospray (ESI), Massive Cluster Impact (MCI), and Surfaces Enhanced for Laser Desorption/Ionization (SELDI).
  • MALDI Matrix Assisted Laser Desorption
  • ESI continuous or pulsed EIectrospray
  • MCI Massive Cluster Impact
  • SELDI Surfaces Enhanced for Laser Desorption/Ionization
  • Various detection formats are also well known to those of skill in the art, including, for example, linear or non-linear reflection Time-of-flight (TOF) and Fourier Transform Ion Cyclotron Resonance (FTICR).
  • TOF linear or non-linear reflection Time-of-flight
  • FTICR Fourier Transform Ion Cyclotron Resonance
  • one of skill in the art can compare proteins found in animals having a particular disease state with the proteins present in an apparently healthy animal and identify proteins whose expression pattern is altered when the disease state is present, i.e. the researcher can identify "biomarkers" that correlate to the disease state, see for example Petricoin et al., 2002, Lancet 359:572-577 which identified proteomic patterns associated with ovarian cancer in women. Once such biomarkers are identified, mass spectrometry may itself be used as a diagnosis technique, or the protein may be further isolated and utilized for a different diagnostic format, including raising antibodies to the protein for use in a diagnostic.
  • mast cell tumors are usually initially detected once the tumor has grown large enough for the pet owner to visually see the mass. Additionally, grading of these tumors is still performed histologically which, as described above, has variable prognostic value . Therefore, the need exists for methods which enable earlier detection of mast cell tumors, and methods which allow for more accurate grading of tumors and prediction of clinical outcome.
  • the present invention satisfies these needs and provides useful molecules related to the detection, grading and prognostic classification of mast cell tumors.
  • the present invention provides proteins that are diagnostic of cancer cells classified as grade I, grade II or grade III; nucleic acid molecules encoding such proteins; and antibodies raised against such proteins. Also described are proteomic profiles useful for classifying cancer cells as grade I 5 II or III.
  • the present invention also includes methods to obtain such proteins, proteomic profiles, and associated nucleic acid molecules, and antibodies.
  • the present invention also relates to the use of such isolated proteins, proteomic profiles, nucleic acid molecules and antibodies for the classification of tumor cells in dogs.
  • the present invention also relates to recombinant molecules, recombinant viruses and recombinant cells that include a nucleic acid molecule of the present invention.
  • the present invention also relates to methods to classify mast cell tumor cells in dogs by mass spectroscopy.
  • a biological sample from a canine mast cell tumor is obtained and a proteomic profile determined by mass spectroscopy.
  • the results are compared to the proteomic profile of cells from tumors having various outcomes. From this comparison, a prediction can be made as to the effectiveness of treatment and the survivability of the patient.
  • the present invention provides for biological molecules, referred to as biomarkers, and classification trees useful in detecting and grading canine tumors in animals.
  • the present invention is also useful in determining the prognosis of a patient known to have a tumor.
  • biomarkers biological molecules
  • classification trees useful in detecting and grading canine tumors in animals.
  • the present invention is also useful in determining the prognosis of a patient known to have a tumor.
  • One embodiment of the present invention is a method to detect a tumor comprising: (a) obtaining a sample from an animal to be tested; and
  • the presence of the biomarker may indicate the presence of absence of a tumor.
  • a entity or “an” entity refers to one ⁇ r more of thai entity.
  • a protein refers to one or more proteins or at least one protein.
  • the terms “a”, “an”, “one or more” and “a least one” can be used interchangeably.
  • the terms “comprising”, “including” and “having” can also be used interchangeably.
  • the terms “amount” and “level” are also interchangeable and may be used to describe a concentration ⁇ r a specific quantity.
  • the lerm “selected from the group consisting of refers to one or more members of the group in the list that follows, including mixtures (i.e. combinations) of two or more members.
  • a tumor refers to an abnormal growth of cells or tissue.
  • a mast cell tumor is a tumor arising from the abnormal growth of mast cells.
  • cancer and “tumor” can be used interchangeably. While the inventors have exemplified the use of the disclosed biomarkers and methods in relation to mast cell tumors, it should be noted that the disclosed biomarkers and methods may also be applied to other types of tumors, for example lymphomas, or solid tumors such as mammary carcinomas or osteosarcomas.
  • a sample is collected from an animal that may or may not be suspected of having a tumor. That sample is then analyzed for the presence or amount of a biomarker.
  • a sample is any specimen obtained from the animal that can be used to measure for the presence of a tumor.
  • useful samples include blood samples, saliva samples, lacrimal fluid (tears) samples and urine samples.
  • Preferred samples to use include whole blood samples, serum samples, plasma samples and any derivatives thereof.
  • the term "animal” is meant to encompass any non-human organism capable of developing a tumor.
  • Preferred animals are those that can develop mast cell tumors.
  • Suitable animals to test for the presence of a tumor include, but are not limited to companion animals (i.e. pets), food animals, work animals, or zoo animals.
  • Preferred animals include, but are not limited to, cats, dogs, horses, ferrets and other Mustelids, cattle, sheep, swine, and rodents. More preferred animals include cats, dogs, horses and other companion animals, with cats, dogs and horses being even more preferred.
  • the term "companion animal” refers to any animal which a human regards as a pet.
  • a cat refers to any member of the cat family (i.e., Felidae), including domestic cats, wild cats and zoo cats. Examples of cats include, but are not limited to, domestic cats, lions, tigers, leopards, panthers, cougars, bobcats, lynx, jaguars, cheetahs, and servals.
  • a preferred cat is a domestic cat.
  • a dog refers to any member of the family Canidae, including, but not limited to, domestic dogs, wild dogs, foxes, wolves, jackals, and coyotes and other members of the family Canidae. A preferred dog is a domestic dog.
  • a horse refers to any member of the family Equidae.
  • An equid is a hoofed mammal and includes, but is not limited to, domestic horses and wild horses, such as, horses, asses, donkeys, and zebras.
  • Preferred horses include domestic horses, including race horses.
  • a biomarker refers to any molecule which can be used to determine the presence, grade or prognostic classification of a tumor.
  • Biomarkers of the present invention may be referred to simply as “biomarkers”, “tumor biomarkers” or “tumor-related biomarkers”; these terms can be used interchangeably.
  • a MCT biomarker refers to any molecule which can be used to determine the presence, grade or prognostic classification of a MCT. According to the present invention, the amount of a biomarker is altered, or the biomarker becomes modified, as a consequence of the presence, grade or prognostic classification of a tumor.
  • the amount of a MCT biomarker present in an animal may be increased or decreased depending on whether or not a MCT is present, or as a consequence of the grade or prognostic classification of a MCT.
  • tumor biomarkers include, but are not limited to, proteins, nucleic acid molecules, lipids, carbohydrates and polysaccharides, as well us molecules which are combinations of these types of molecules (e.g., a lipoprotein). Such molecules may be referred to as biomarker molecules.
  • a MCT tumor biomarker which is a protein may be referred to as a MCT biomarker protein.
  • Tumor biomarker molecules may or may not originate from tumor cells meaning they may be produced by the tumor itself or by any other cell or tissue in the body.
  • a biomarker may or may not be a molecule (e.g., protein, lipid, etc.) which originates (e.g., is encoded) in the animal to be tested.
  • a biomarker may be a canine or feline protein produced by a dog or cat.
  • the biomarker need not be a molecule produced by the animal.
  • a biomarker may originate from a virus or a bacteria present in an animal. The origin of the biomarker is of little importance so long as the biomarker can be used to detect, grade or prognoslically classify a tumor. Examples of useful biomarkers, and methods of finding such biomarkers, are disclosed herein.
  • biomarkers of the present invention may be modified forms of native proteins, nucleic acid molecules, lipids, carbohydrates and polysaccharides, or molecules which are combinations of these types of molecules (e.g., a lipoprotein).
  • a native molecule is the only, or most prevalent, form of a molecule found in a normal patient.
  • methods of the present invention may look for molecules which have been modified, wherein the modified form of the native molecule indicates the presence, grade or prognostic classification of a tumor.
  • modifications include, but are not limited to, proteolytic processing (or the inhibition of native processing) , and /or the addition or removal of peptides, carbohydrates, sugars, lipids, phosphates, methyl groups or any other group or molecule known to be used in the modification of biological molecules.
  • modifications and the groups used to make them are known to those skilled in the art.
  • Methods of analyzing a sample for the presence, amount of, or modifications to, a biomarker are known to those skilled in the art. Any method of analysis which is capable of detecting a tumor biomarker is suitable for use in methods of the present invention.
  • Examples of such analysis methods include, but are not limited to, an enzyme-linked immunoassay, a competitive enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, an agglutination assay, a particulate-based assay (e.g., using particulates such as, but not limited to, magnetic particles or plastic polymers, such as latex or polystyrene beads), an immunoprecipitation assay, a BioCoreTM assay (e.g., using colloidal gold), an immunodot assay (e.g., CMG's Tmmu ⁇ odot System, Fribourg, Switzerland), an irnmunoblot assay (e.g., a western blot), an phosphorescence assay, a chromatography assay, a PAGe-based assay, a surface plasm
  • Eleclrospray Ionization ESI
  • Massive Cluster Impact MCI
  • SMDI Surfaces Enhanced for Laser Desorption/Ionization
  • PCR polymerase chain reaction
  • PCR polymerase chain reaction
  • one embodiment of the present invention is a method to detect a tumor comprising: (a) obtaining a sample from an animal to be tested; and
  • While the disclosed methods can be practiced by either detecting the presence or amount of one or more biomarkers, it will be appreciated by those skilled in the art that by detecting the presence of a biomarker, one inherently determines the amount. This is due to the fact that all detection methods inherently require some amount of a molecule be present in order to detect it. The amount of a biomarker that needs to be present in order for it to be detected differs depending on the detection method used. Those of skill in the art are capable of selecting detection methods capable of detecting amounts of biomarker useful for practicing methods of the instant invention.
  • the "amount" of a biomarker can refer to the absolute level of a biomarker as well as the concentration.
  • the amount may be measured in absolute units, e.g., micrograms or milligrams, or it may be measured as a concentration e.g., units/microliter, units/ milliliters, units/deciliter, etc.
  • methods of the present invention may require a comparison between the amount of biomarker in a test sample and the amount of biomarker in a normal sample, the amount of biomarker in these samples may or may not be determined at the same time.
  • the amount of biomarker present in a normal sample could be determined at any time prior to, or after, determining the amount of biomarker present in the sample from the animal to be tested. These amounts could then be compared at a later time.
  • the amount of a biomarker present in s ample from a normal animal could be determined first.
  • a test for example a lateral flow assay, could then be designed to only detect the biomarker in a patient having a tumor when the amount of biomarker in the patient sample is higher, or lower, than the amount found in a normal animal.
  • normal refers to a patient, and samples obtained therefrom, known to be free of tumors. As an example, a normal patient would be free of mast cell tumors.
  • the amount of biomarker present in a test sample may be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, or about 300% more, or less, than the amount present in a normal sample.
  • the amount of biomarker present in a test sample may be at least about I . I X (fold), at least about 1.2X, at least about 1.3X, at.
  • an assay could be designed in which the amount of biomarker in a normal sample and the amount in a patient to be tested are both expressed visually using, for example, a chromogenic substrate, a particulate substrate or a luminescent substrate.
  • the amount of each biomarker could be represented, for example, by the color, darkness or brightness of a band or spot. The two bands or spots could then be compared to determine if the amount of biomarker in the patient being tested is different from the amount present in a normal patient.
  • An example of one such an assay is described in U.S. Patent No. 6,001,658, issued December 14, 1999 to Fredrickson. Those of skill in the art will appreciate that the assay being described is merely one. example and that numerous variations and formats utilizing the disclosed principles are possible.
  • a tumor is assigned a grade using a method comprising:
  • a tumor is assigned a grade using a method comprising:
  • the animal may or may not be suspected of having a tumor.
  • the term "grade” refers to the aggressiveness of the tumor, based on the histological appearance of tumor cells.
  • Methods and schemes for histologically grading tumors are known to those skilled in the art.
  • the most common scheme used to grade mast cell tumors is the Patnaik grading scale which grades tumors as grade I 1 grade II or grade III; however, any histological grading scale used by those skilled in the art can be used.
  • Methods of the present invention can be used to classify tumors as grade I, II or III, depending on which biomarkers are selected. If other Grading schemes are used, methods and biomarkers of the present invention can be used to classify tumors based on the scale used by the chosen scheme.
  • One embodiment of the present invention is a method to determine the prognosis of a patient having a tumor, the method comprising:
  • the prognosis of patient having a tumor is determined using a method comprising:
  • the disclosed methods are useful for any tumor of any grade, they are particularly useful for patients having a grade II tumor, and in particular, a grade II mast cell tumor. Tn such a method, the animal may or may not be suspected of having a tumor.
  • the terms "to determine the prognosis”, and the like, and “prognostic classification” can be used interchangeably. Moreover, such terms can be applied to the patient of the tumor. For example, one can used the disclosed methods to "determine the prognosis" of a patient or tumor, or to “prognostically classify” the patient or tumor.
  • a "good” prognosis is defined as a clinical outcome in which the tumor (1) remains local, meaning it does not become metastatic, and (2) is non-recurrent following surgical removal.
  • a "bad” prognosis is defined as a clinical outcome in which the tumor ( L) is metastatic and (2) is recurrent following surgical removal.
  • animals having a good prognosis have a higher 5 year survivability rate than animals having a bad prognosis.
  • methods of the present invention are useful for predicting an animal's survivability rate, in particular, their 5 year survivability rate.
  • methods of the present invention utilize one or more biomarkers, each biomarker having a mass selected from the group consisting of 8980 Da, 8446 Da, 8435 Da, 12784 Da, 42682 Da, 13898 Da, 34521 Da, 4960 Da, 11274 Da, 4686 Da, 4984 Da, 9988 Da, 7620 Da, 6287 Da and 2914 Da.
  • a biological sample from an animal to be diagnosed is analyzed by mass spectroscopy and the results are compared to results from animals known to have a tumor and, optionally, to results from animals that do not have tumors. The presence, absence or amount of biornarkers associated with disease is then determined in order to determine whether the animal being tested has a tumor.
  • Such a method is particularly suited to the grading and prognostic classification of mast cell tumors.
  • the method utilizes SELDI-TOF mass spectroscopy, as is described in greater detail in the attached examples. Disease may be indicated by either up-regulation (i.e. greater levels of protein expression) or down- regulation (i.e.
  • fraction .1-2 prepared as described in the attached Examples, is analyzed by SELDI- TOF mass spectroscopy using a weak cation exchange substrate.
  • the method uses a laser setting of 187, 195, 210 and/or 225.
  • the presence, grade or prognostic class of a tumor in an animal is indicated by the up or down regulation, and/or by modification, of one or more the following proteins: a protein having a molecular weight of about 8980 daltons (Da), a protein having a molecular weight of about 8446 Da, a protein having a molecular weight of 4960 Da, a protein having a molecular weight of about 11274 Da, a protein having a molecular weight of about 4686 Da and/or a protein having a molecular weight of 4984 Da.
  • Da a protein having a molecular weight of about 8980 daltons
  • 8446 Da a protein having a molecular weight of 4960 Da
  • a protein having a molecular weight of about 11274 Da a protein having a molecular weight of about 4686 Da and/or a protein having a molecular weight of 4984 Da.
  • the masses of biomarkers disclosed herein included masses within about 0.1 %, 0.25%, 0.5% or about 1.0% of the mass disclosed.
  • a biomarker disclosed as having a mass of "about 8980 Da” includes biomarkers having a mass within about 9 DA, about 23 DA, about 45 Da, or about 90 Da of 8980 Da.
  • One embodiment of a method to detect, grade or prognostically classify a tumor involves the use of a lateral flow assay, examples of which are described in U.S. Patent No. 5,424, 193, issued June 13, 1995, by Pronovost et al.; U.S. Patent No.
  • a lateral flow assay is an example of a single-step assay, In a single-step assay, once the sample has been obtained and made ready for testing, only a single action is necessary on the part of the user to detect the present of an analyte. For example, the sample, in whole or part, can be applied to a device which then measures analyte in the sample.
  • a sample is placed in a lateral flow apparatus that includes the following components: (a) a support structure defining a flow path; (b) a labeling reagent comprising a bead conjugated to a specific antibody, the labeling reagent being impregnated within the support structure in a labeling zone; and (c) a capture reagent.
  • Preferred antibodies include antibodies which bind to biomarkers of the present invention.
  • the capture reagent is located downstream of the labeling reagent within a capture zone fluidly connected to the labeling zone in such a manner that the labeling reagent can flow from the labeling zone into the capture zone.
  • the support structure comprises a material that does not impede the flow of the beads from the labeling zone to the capture zone.
  • Suitable materials for use as a support structure include ionic (i.e., anionic or cationic) material. Examples of such a material include, but are not limited to, nitrocellulose, PVDF, or carboxymethylcellulose.
  • the support structure defines a flow path that is lateral and is divided into zones, namely a labeling zone and a capture zone.
  • the apparatus can further include a sample receiving zone located along the flow path, preferably upstream of the labeling reagent.
  • the flow path in the support structure is created by contacting a portion of the support structure downstream of the capture zone, preferably at the end of the flow path, to an absorbent capable of absorbing excess liquid from the labeling and capture zones.
  • the assay can be inherently designed so that when specific biomarkers are present in an amount above or below a certain threshold amount, which corresponds to a particular level above or below the amount detected in a normal sample, the assay gives a result which corresponds to the presence, grade or prognostic class of a tumor. For example, if the amount of a particular biomarker is known to increase above a certain concentration when a tumor is present, then a single step assay can be designed such that the antibodies do not bind the biomarker until the amount of biomarker is present at or above the crucial concentration.
  • Such an assay can be designed using antibodies having a particular avidity for the biomarker. Using antibodies of different avidities or affinities will allow one to "tune” the assay to detect biomarkers at various levels or concentrations. Similarly, assays could be designed to detect a drop in biomarker concentration. Assays could also be designed which allow one to detect modifications (e.g., proteolytic processing, methylation, glycosylation, etc.) to biomarkers of the present invention. Such methodology can be used to design single step assays to delect and grade tumors as well as to assign them to a prognostic class. Single step assays of the present invention may utilize one or more biomarkers of the present invention in a single assay.
  • a classification tree is a decision model which uses a set of predictors, and their interactions, to optimally predict a dependent response.
  • the predictors are the individual biomarkers disclosed herein and whether or not such biomarkers are absent or present, and if present, in what amount or form.
  • the form of a biomarker refers to whether or not a biomarker is present in it's native state or if it is modified by, for example, processes including, but not limited to, truncation, proteolytic processing, and/or the addition or removal of phosphates, carbohydrates, sugars, lipids, amide groups, methyl groups, acetyl groups and the like.
  • the dependent response is the presence, absence, grade or prognostic class of a tumor. In a preferred embodiment, the dependent response is the presence, absence, grade or prognostic class of a mast cell tumor.
  • a classification tree of the present invention refers to a decision model based on the presence, absence, amount and/or form of one or more individual biomarkers of the present invention, which can be used to determine the presence of, grade, and/or the prognostic class of a tumor, for example a mast cell tumor.
  • Classification trees of the present invention are constructed using biomarkers and methods of the present invention.
  • the presence, absence, amount and/or form of the individual biomarkers of a classification tree collectively form an overall expression pattern which is referred to as a proteomic profile.
  • the dependent response is determined by comparing the overall expression pattern (proteomic profile) from a normal sample with the overall expression pattern in a sample from an animal to be tested. It is the difference in the overall expression profile which is deterministic of the presence, grade and/or prognostic class of a tumor.
  • the proteomic profile represented by Proteomic Profile 3 (the absence of Biomarker 1 and the presence Biomarker 2) is indicative of the presence of a tumor. In this case, only Proteomic Profile 3 would indicate a tumor is present. Alternatively, the presence of either biomarker could result in the same dependent outcome (e.g., a tumor being present). Therefore, both Proteomic Profile 2 and Proteomic Profile 3 would indicate the same dependent response (e.g., a tumor being present).
  • a tumor is detected using a method comprising:
  • the results of such analyses indicates the presence or absence of a tumor.
  • the result of the classification tree analysis indicates the animal has a tumor.
  • the classification tree compares the amount of one or more biomarker in the sample to be tested with the amount of biomarker present in a sample from a normal animal. The selection of classification tree and associated biomarkers is made based on the dependent response one wishes to test for.
  • Useful classification trees comprise biomarkers of the present invention. Useful classification trees can be constructed using methods disclosed herein.
  • the method uses one or more proteins having masses selected from the group consisting of 8980 Da, 8446 Da, 8435 Da, 12784 Da, 42682 Da, 13898 Da, 34521 Da 1 4960 Da, ] 1274 Da, 4686 Da, 4984 Da 1 9988 Da, 7620 Da, 6287 Da and 2914 Da.
  • One embodiment of the present invention is a method that utilizes a classification tree comprising one or more protein having a mass selected from the group consisting of 8435 Da, 12784 Da, 42682 Da, 13898 Da and 34521 Da.
  • the method utilizes a classification tree comprising proteins having masses of 8435 Da, 12784 Da, 42682 Da, 13898 Da and 34521 Da. In one embodiment, the method is used to assign a grade to a mast cell tumor. In one embodiment, the method assigns a grade of I, II or II in accordance with the Patnaik grading scale. In one embodiment, the method utilizes a classification tree comprising one or more protein having a mass selected from the group consisting of 4684 Da and 4984 Da. In one embodiment, the method utilizes a classification tree comprising proteins having a mass selected from 4684 Da and 4984 Da.
  • a tumor is detected using a method comprising; (a) obtaining a sample from an animal to be tested; and
  • the prognosis of an animal having a tumor is determined using a method comprising:
  • the classification tree compares the amount of one or more biomarker in the sample to be tested with the amount of biomarker present in a sample from a normal animal.
  • the method uses one or more proteins having masses selected from the group consisting of 8980 Da, 8446 Da 1 8435 Da, 12784 Da 1 42682 Da, 13898 Da, 34521 Da, 4960 Da, 1 1274 Da, 4686 Da, 4984 Da 5 9988 Da, 7620 Da 1 6287 Da and 2914 Da.
  • the method utilizes a classification tree comprising one or more proteins having a mass selected from the group consisting of 4684 Da and 4984 Da.
  • the method utilizes a classification tree comprising proteins having a mass selected from 4684 Da and 4984 Da. Methods using such biomarkers are particularly suited for determining the prognosis of patients having a mast cell tumor.
  • the prognosis of an animal having a tumor is determined using a method comprising:
  • the outcome of such analysis is indicative of a good or bad prognosis.
  • the outcome of the classification tree analysis indicates the animal has a good prognosis.
  • the outcome of the classification tree analysis indicates the animal has a bad prognosis.
  • Such a method is also useful for predicting the 5 year survivability rate of an animal having a tumor. While methods which utilize classification tree analysis can be applied to any lype and grade of tumor, such methods are particularly suited for mast cell tumors. Moreover, methods which utilize classification tree analysis to determine a prognosis, while applicable to any grade and type of tumor, are particularly applicable to grade II tumors, and more specifically, grade II mast cell tumors.
  • biomarker useful for performing methods of the present invention.
  • useful biomarkers include, but are not limited to, proteins, nucleic acid molecules, lipids, carbohydrates and polysaccharides, as well as molecules which are combinations of these types of molecules (e.g., a lipoprotein) and modified forms of such molecules.
  • a biomarker is an isolated protein. Such a protein may be referred to as a biomarker protein.
  • Preferred biomarker proteins are MCT biomarker proteins.
  • MCT biomarker proteins are proteins, present in the serum of a patient, that are related to the presence, grade or prognostic classification of a MCT.
  • a MCT tumor biomarker of the present invention may be used to detect a MCT in a patient.
  • an isolated, or biologically pure, molecule is one that has been removed from its natural milieu.
  • isolated and biologically pure do not necessarily reflect the extent to which the protein has been purified.
  • An isolated protein of the present invention can be obtained from its natural source, can be produced using recombinant DNA technology, or can be produced by chemical synthesis.
  • One embodiment of the present invention is an isolated protein having a molecular weight of about 8980 daltons (Da), a protein having a molecular weight of about 8446 Da, a protein having a molecular weight of 4960 Da, a protein having a molecular weight of about 1 1274 Da, a protein having a molecular weight of about 4686 Da and/or a protein having a molecular weight of 4984 Da.
  • a biomarker of the present invention is a protein having a mass selected from the group consisting of 8980 Da, 8446 Da, 8435 Da, 12784 Da, 42682 Da, 13898 Da, 34521 Da, 4960 Da, 11274 Da, 4686 Da, 4984 Da, 9988 Da, 7620 Da, 6287 Da and 2914 Da.
  • the mass of a protein of the present invention can be determined using methods disclosed herein. In one embodiment, the mass of a protein is determined by mass spectroscopy using a strong anion substrate, a metal affinity substrate, a weak cation substrate or a reversed phase substrate.
  • mass spectroscopy is performed using a substrate chip selected form the group consisting of a Ql 0-strong anion exchanger, a IMAC30- metal affinity substrate, a CMiO-weak anion exchanger and a H50-reversed phase substrate.
  • a substrate chip selected form the group consisting of a Ql 0-strong anion exchanger, a IMAC30- metal affinity substrate, a CMiO-weak anion exchanger and a H50-reversed phase substrate.
  • Isolated biomarker proteins of the present invention can be full-length proteins or any homologue of such proteins.
  • biomarker homologue proteins include biomarker proteins in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homologue includes at least one epitope capable of eliciting an immune response against a biomarker protein, and/or of binding to an antibody directed against a biomarker protein.
  • the animal when the homologue is administered to an animal as an immunogen, using techniques known to those skilled in the art, the animal will produce an immune response against at least one epitope of a natural biomarker protein.
  • the ability of a protein to affect an immune response can be measured using techniques known to those skilled in the art.
  • epitope refers to the smallest portion of a protein or other antigen capable of selectively binding to lhe antigen binding site of an antibody or a T cell receptor. It is well accepted by those skilled in the art that the minimal size of a protein epitope is about four to six amino acids.
  • an epitope can include amino acids that naturally are contiguous to each other as well as non-contiguous amino acids that, due to the tertiary structure of the natural protein, are in sufficiently close proximity to form an epitope.
  • an epitope includes a portion of a protein comprising at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 10 amino acids, at least 35 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids or at least 50 amino acids in length.
  • useful biomarkers of the present invention can be at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids or at least 50 amino acids in length.
  • biomarker homologue protein exhibits an activity similar to its natural counterpart. Methods to detect and measure such activities vary by biomarker, but can be determined by those of skill in the art.
  • Biomarker homologue proteins can be the result of natural allelic variation or natural mutation.
  • Biomarker protein homologues of the present invention can also be produced using techniques known in the art including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
  • protein sequences can be minimally modified so as to retain the activity, immunological or otherwise, of the parent from which the homologue is derived. By minimally modified is meant the substitution of a small percent of the amino acids, or codons encoding them, within a protein.
  • a homologue has a sequence at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 92% identical, at least about 95% identical, at least about 98% identical or at least about 100% identical with the parent biomarker protein from which the homologue is derived.
  • Biomarker proteins of the present invention are encoded by biomarker nucleic acid molecules.
  • biomarker nucleic acid molecules include nucleic acid sequences related to natural bioxnarker genes found in canids, and, preferably, to Canis familiaris genes.
  • dog and canid may be used interchangeably and include domestic dogs, wild dogs and zoo dogs.
  • biomarker genes include all regions such as regulatory regions that control production of biomarker proteins encoded by such genes (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself, and any introns or non-translated coding regions.
  • nucleic acid molecule that "includes” or “comprises” a sequence may include that sequence in one contiguous array, or may include the sequence as fragmented exons such as is often found for a dog gene.
  • coding region refers to a continuous linear array of nucleotides that translates into a protein.
  • a full-length coding region is that coding region that is translated into a full-length, i.e., a complete protein as would be initially translated in its natural milieu, prior to any post-translational modifications.
  • isolated biomarker proteins are encoded by nucleic acid molecules that hybridize under stringent hybridization conditions to genes or other nucleic acid molecules encoding biomarker proteins, respectively.
  • the minimal size of such biomarker proteins of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid (i.e., hybridizing under stringent hybridization conditions) with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protein.
  • the size of a nucleic acid molecule encoding such a protein is dependent on the nucleic acid composition and the percent homology between the biomarker nucleic acid molecule and the complementary nucleic acid sequence.
  • the extent of homology required to form a stable hybrid under stringent conditions can vary depending on whether the homologous sequences are interspersed throughout a given nucleic acid molecule or are clustered (i.e., localized) in distinct regions on a given nucleic acid molecule.
  • the minimal size of a nucleic acid molecule capable of forming a stable hybrid with a gene encoding a biomarker protein of the present invention is at least about 12 to about 15 nucleotides in length if the nucleic acid molecule is GC-rich and at least about 15 to about 17 bases in length if it is AT-rich.
  • the minimal size of a nucleic acid molecule used to encode a biomarker protein homologue. of the present invention is from about 12 to about 18 nucleotides in length.
  • the minimal size of biomarker protein homologues of the present invention is from about 4 to about 6 amino acids in length.
  • nucleic acid molecule of the present invention can include a portion of a gene or cDNA or RNA, an entire gene or cDNA or RNA, or multiple genes or cDNA or RNA.
  • the preferred size of a protein encoded by a nucleic acid molecule of the present invention depends on whether a full-length, fusion, multivalent, or functional portion of such a protein is desired.
  • Stringent hybridization conditions are determined based on defined physical properties of the biomarker nucleic acid molecule to which the nucleic acid molecule is being hybridized, and can be defined mathematically. Stringent hybridization conditions are those experimental parameters that allow an individual skilled in the art to identify significant similarities between heterologous nucleic acid molecules. These conditions are well known to those skilled in the art. See, for example, Sambrook, et «/., 1989, Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Labs Press, and Meinkoth, et ⁇ /., 1984, Anal. Biochem. 138, 267-284.
  • the determination of hybridization conditions involves the manipulation of a set of variables including the ionic strength (M, in moles/liter), the hybridization temperature ("C), the concentration of nucleic acid helix destabilizing agents (such as formamide), the average length of the shortest hybrid duplex (n), and the percent G + C composition of the fragment to which an unknown nucleic acid molecule is being hybridized.
  • these variables are inserted into a standard mathematical formula to calculate the melting temperature, or T n ,, of a given nucleic acid molecule.
  • T m is the temperature at which two complementary nucleic acid molecule strands will disassociate, assuming 100% complementarity between the two strands:
  • T 1n 81.5 0 C + 16.6 log M + 0.41 (%G + C) - 500/n - 0.63 (%formamide).
  • T d dissociation temperature
  • T d 4(G + C) + 2(A + T).
  • a temperature of 5°C below Tj is used to detect hybridization between perfectly matched molecules.
  • T m decreases about 1°C for each 1 % of mismatched base pairs for hybrids greater than about 150 bp
  • T 4 decreases about 5°C for each mismatched base pair for hybrids below about 50 bp.
  • Conditions for hybrids between about 50 and about 150 base pairs can be determined empirically and without undue experimentation using standard laboratory procedures well known to those skilled in the art. These simple procedures allow one skilled in the art to set the hybridization conditions (by altering, for example, the salt concentration, the formamide concentration or the temperature) so that only nucleic acid hybrids with greater than a specified % base pair mismatch will hybridize. Because one skilled in the art can easily determine whether a given nucleic acid molecule to be tested is less than or greater than about 50 nucleotides, and can therefore choose the appropriate formula for determining hybridization conditions, he or she can determine whether the nucleic acid molecule will hybridize with a given gene under conditions designed to allow a desired amount of base pair mismatch.
  • Hybridization reactions are often carried out by attaching the nucleic acid molecule to be hybridized to a solid support such as a membrane, and then hybridizing with a labeled nucleic acid molecule, typically referred to as a probe, suspended in a hybridization solution.
  • a labeled nucleic acid molecule typically referred to as a probe
  • Examples of common hybridization reaction techniques include, but are not limited to, the well-known southern and northern blotting procedures.
  • the actual hybridization reaction is done under non-stringent conditions, i.e., at a lower temperature and/or a higher salt concentration, and then high stringency is achieved by washing the membrane in a solution with a higher temperature and/or lower salt concentration in order to achieve the desired stringency.
  • nucleic acid molecule that hybridizes under conditions that would allow less than or equal to 30% pair mismatch with a biomarker nucleic acid molecule of the present invention of about 150 bp in length or greater
  • the following conditions could preferably be used.
  • the average G + C content of dog DNA is about 51 %, as calculated from known dog nucleic acid sequences.
  • the unknown nucleic acid molecules would be attached to a support membrane, and the .150 bp probe would be labeled, e.g. with a radioactive tag.
  • the hybridization reaction could be carried out in a solution comprising 2X SSC in the absence of nucleic acid helix destabilizing compounds, at a temperature of about 37"C (low stringency conditions).
  • Solutions of differing concentrations of SSC can be made by one of skill in the art by diluting a stock solution of 2OX SSC (175.3 gram NaCl and about 88.2 gram sodium citrate in ] liter of water, pH 7) to obtain the desired concentration of SSC.
  • the skilled artisan would calculate the washing conditions required to allow up to 20% base pair mismatch. For example, in a wash solution comprising IX SSC in the absence of nucleic acid helix destabilizing compounds, the T 1n of perfect hybrids would be about 85.4°C:
  • hybridization washes would be carried out at a temperature of less than or equal to 65.4 U C. It is thus within the skill of one in the art to calculate additional hybridization temperatures based on the desired percentage base pair mismatch, formulae and G/C content disclosed herein.
  • the T m for a hybridization reaction allowing up to 20% base pair mismatch will not vary significantly from 65.4 0 C.
  • hybridization washes would be carried out at a temperature of less than or equal to 75.4 0 C and to achieve hybridization with nucleic acid molecules having about 5% base pair mismatch, hybridization washes would be carried out at a temperature of less than or equal to 80.4 ⁇ C.
  • DNAsis software programs represent a collection of algorithms paired with a graphical user interface for using the algorithms.
  • the DNAsis and SeqLab software employ a particular algorithm, the Needleman-Wunsch algorithm to perform pair-wise comparisons between two sequences to yield a percentage identity score, see Needleman, S.B. and Wunsch, CD., 1970, /. MoL Biol. 48, 443.
  • a preferred method to determine percent, identity among amino acid sequences and also among nucleic acid sequences includes using the Needleman-Wunsch algorithm, available in the SeqLab software, using the Pairwise Comparison/Gap function with the nwsgapdna.cmp scoring matrix, the gap creation penalty and the gap extension penalties set at default values, and the gap shift limits set at maximum (hereinafter referred to as "SeqLab default parameters")-
  • An additional preferred method to determine percent identity among amino acid sequences and also among nucleic acid sequences includes using the Higgins-Sharp algorithm, available in the DNAsis software (hereinafter "DNAsis”), with the gap penalty set at 5, the number of top diagonals set at 5, the fixed gap penalty set at 10, the k-tupJe set at 2, the window size set at 5, and the floating gap penalty set at 10.
  • a particularly preferred method to determine percent identity among amino acid sequence includes using the Needleman-Wunsch algorithm, available in the SeqLab software, using the Pairwise Comparison/Gap function with
  • a preferred biomarker protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to 30% base pair mismatch, preferably under conditions that allow less than or equal to 20% base pair mismatch, preferably under conditions that allow less than or equal to 10% base pair mismatch, preferably under conditions that allow less than or equal to 8% base pair mismatch, preferably under conditions that allow less than or equal to 5% base pair mismatch or preferably under conditions that allow less than or equal to 2% base pair mismatch with a nucleic acid molecule of the present invention.
  • Another embodiment of the present invention includes a biomarker protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising IX SSC in the absence of nucleic acid helix destabilizing compounds, at a temperature of 37°C and (b) washing in a solution comprising IX SSC in the absence of nucleic acid helix destabilizing compounds, at a temperature of 65.4°C, to an isolated nucleic acid molecule of the present invention.
  • Another preferred biomarker protein of the present invention includes a protein lhat is encoded by a nucleic acid molecule that is preferably at least 70%, preferably at least 80%, preferably at least 90% identical, preferably at least 92% identical, preferably at least 95% identical or preferably at least 98% identical to a nucleic acid molecule of the present invention; also contemplated are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least 25, at least 30, at least 40, at least 50, at least 60, at least 75, at least .100 nucleotides. Percent identity as used herein is determined using the Needleman-Wunsch algorithm, available in the SeqLab software using default parameters.
  • nucleic acid molecule comprising a biomarker nucleic acid molecule, i.e. a nucleic acid molecule that can be isolated from a dog cDNA library.
  • a biomarker nucleic acid molecule i.e. a nucleic acid molecule that can be isolated from a dog cDNA library.
  • the identifying characteristics of such nucleic acid molecules are heretofore described.
  • a nucleic acid molecule of the present invention can include an isolated natural biomarker gene or a homologue thereof, the latter of which is described in more detail below.
  • a nucleic acid molecule of the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof.
  • the minimal size of a nucleic acid molecule of the present invention is a size sufficient to allow the formation of a stable hybrid (i.e., hybridization under stringent hybridization conditions) with the complementary sequence of another nucleic acid molecule.
  • the minimal size of a biomarker nucleic acid molecule of the present invention is from 12 to 18 nucleotides in length. Theoretically, there is no maximum size to a biomarker nucleic acid molecule of the present invention.
  • Biomarker nucleic acid molecules may be about 20, about 25, about 0, about 35, about 40, about.45, about 50, about 60, about 70, about 80, about 90 or about 100 nucleotides, or more, in length.
  • an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subjected to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA.
  • isolated does not reflect the extent to which the nucleic acid molecule has been purified.
  • Isolated biomarker nucleic acid molecules of the present invention, or homologues thereof can be isolated from a natural source or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification or cloning) or chemical synthesis.
  • PCR polymerase chain reaction
  • Isolated biomarker nucleic acid molecules, and homologues thereof can include, for example, natural allelic variants and nucleic acid molecules modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode a biomarker protein of the present invention.
  • Biomarker nucleic acid molecules of the present invention can be isolated from a dog or prepared recombinantly or synthetically.
  • Biomarker nucleic acid molecules of the present invention can be RNA or DNA, or modified forms thereof, and can be double-stranded or single-stranded; examples of nucleic acid molecules include, but are not limited to, complementary DNA (cDNA) molecules, genomic DNA molecules, synthetic DNA molecules, DNA molecules which are specific tags for messenger RNA, corresponding r ⁇ RNA molecules, and fragments of all such molecules.
  • cDNA complementary DNA
  • genomic DNA molecules genomic DNA molecules
  • synthetic DNA molecules DNA molecules which are specific tags for messenger RNA, corresponding r ⁇ RNA molecules, and fragments of all such molecules.
  • a biomarker nucleic acid molecule of the present invention is not intended to refer to an entire chromosome within which such a nucleic acid molecule is contained, however, a biomarker nucleic acid molecule of the present invention may include all regions such as regulatory regions that control production of biomarker proteins encoded by such a nucleic acid molecule (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself, and any introns or non-translated coding regions.
  • the present invention also provides for biomarker DNA molecules that are specific tags for messenger RNA molecules.
  • DNA molecules can correspond to an entire or partial sequence of a messenger RNA, and therefore, a DNA molecule corresponding to such a messenger RNA molecule (i.e. a cDNA molecule), can encode a full-length or partial-length protein.
  • a nucleic acid molecule encoding a partial- length protein can be used directly as a probe or indirectly to generate primers to identify and/or isolate a cDNA nucleic acid molecule encoding a corresponding, or structurally related, full-length protein.
  • a cDNA encoding a partial-length biomarker protein can also be used in a similar manner to identify a genomic nucleic acid molecule, such as a nucleic acid molecule that contains the complete gene including regulatory regions, exons and introns.
  • a biomarker nucleic acid molecule homologue of the present invention can be produced using a number of methods known to those skilled in the art, see, for example, Sambrook et al., ibid.
  • nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis and recombinant DNA techniques such as site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments, PCR amplification, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules, and combinations thereof.
  • Nucleic acid molecule homologues can be selected by hybridization with biomarker nucleic acid molecules or by screening the function of a protein encoded by the nucleic acid molecule (e.g., ability to elicit an immune response against at least ⁇ ne epitope of a biomarker protein or to effect biomarker protein activity).
  • An isolated biomarker nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one biomarker protein of the present invention respectively, examples of such proteins being disclosed herein.
  • nucleic acid molecule primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a biomarker protein.
  • nucleic acid sequences of certain biomarker nucleic acid molecules of the present invention allows one skilled in the art to, for example, (a) make copies of those nucleic acid molecules, (b) obtain nucleic acid molecules including at least a portion of such nucleic acid molecules (e.g., nucleic acid molecules including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions), and (c) obtain other biomarker nucleic acid molecules.
  • nucleic acid molecules can be obtained in a variety of ways including screening appropriate expression libraries with antibodies of the present invention; traditional cloning techniques using oligonucleotide probes of the present invention to screen appropriate libraries; and PCR amplification of appropriate libraries or DNA using oligonucleotide primers of the present invention.
  • Preferred libraries to screen or from which to amplify nucleic acid molecules include cDNA libraries as well as genomic DNA libraries.
  • preferred DNA sources to screen or from which to amplify nucleic acid molecules include cDNA and genomic DNA. Techniques to clone and amplify genes are disclosed, for example, in Sambrook et al., ibid.
  • One embodiment of the present invention includes a recombinant vector, which includes at least one isolated nucleic acid molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host cell.
  • a vector contains heterologous nucleic acid sequences that are nucleic acid sequences not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule are derived.
  • the vector can be either RNA or DNA, either prokaryotic or eukaryotic and typically is a virus or a plasmid.
  • Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of " biomarker nucleic acid molecules of the present invention.
  • a recombinant molecule comprises a nucleic acid molecule of the present invention operatively linked to an expression vector.
  • the phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
  • an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule.
  • the expression vector is also capable of replicating within the host cell.
  • Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids.
  • Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, parasite, insect, other animal, and plant cells.
  • Preferred expression vectors of the present invention can direct gene expression in bacterial, yeast, insect and mammalian cells, and more preferably in the cell types disclosed herein.
  • expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention.
  • recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences that control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art.
  • Preferred transcription control sequences include those that function in bacterial, yeast, or insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as lambda p L and lambda p R and fusions that include such promoters), bacteriophage T7, ⁇ llac, bacteriophage T3, bacteriophage SP6, bacteriophage SPOl , metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoter, antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as immediate early promoter), simian virus 40, retrovirus, actin, retroviral long terminal repeat,
  • transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
  • Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with dogs, such as Canis familiaris transcription control sequences.
  • Suitable and preferred nucleic acid molecules to include in recombinant vectors of the present invention are as disclosed herein.
  • Recombinant molecules of the present invention may also (a) contain secretory signals (i.e., signal segment nucieic acid sequences) to enable an expressed biomarker protein of the present invention to be secreted from the cell that produces the protein and/or (b) contain fusion sequences which lead to the expression of nucleic acid molecules of the present invention as fusion proteins.
  • suitable signal segments include any signal segment capable of directing the secretion of a protein of the present invention.
  • Preferred signal segments include, but are not limited to, tissue plasminogen activator (t-PA), interferon, interleukin, growth hormone, histocompatibility and viral envelope glycoprotein signal segments.
  • t-PA tissue plasminogen activator
  • Suitable fusion segments encoded by fusion segment nucleic acids are disclosed herein.
  • a nucleic acid molecule of the present invention can be joined to a fusion segment that directs the encoded protein to the proteosome, such as a ubiquitin fusion segment.
  • Eukaryotic recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the present invention.
  • Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, Kpofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. It is to be noted that a cell line refers to any recombinant cell of the present invention that is not a transgenic animal.
  • Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
  • Preferred nucleic acid molecules with which to transform a cell include biomarker nucleic acid molecules disclosed herein.
  • Suitable host cells to transform include any cell that can be transformed with a nucleic acid molecule of the present invention.
  • Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule (e.g., nucleic acid molecules encoding one or more proteins of the present invention).
  • Host cells of the present invention either can be etidogenously (i.e., naturally) capable of producing biomarker proteins of the present invention or can be capable of producing such proteins after being transformed with at least one nucleic acid molecule of the present invention.
  • Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal (including yeast), parasite (including helminth, protozoa and ectoparasite), other insect, other animal and plant cells.
  • Preferred host cells include bacterial, mycobacterial, yeast, insect and mammalian cells.
  • More preferred host cells include Drosophila melanogaster S2 cells, Salmonella, Escherichia, Bacillus, Caulobacter, Listeria, Saccharomyces, Pichia, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells (Madin-Darby canine kidney cell line), CRFK cells (Crandell feline kidney cell line), CV-I cells (African monkey kidney cell line used, for example, to culture raccoon poxvirus), COS (e.g., COS-7) cells, and Vero cells.
  • Particularly preferred host cells are Escherichia coli, including E.
  • colt K-12 derivatives Salmonella typhi; Salmonella typkimurium, including attenuated strains such as UK-I ⁇ 3987 and SR-I I ⁇ 4072; Caulobacter; Pichia; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CV-I cells; COS cells; Vero cells; and non-tumorigenic mouse myoblast GS cells (e.g., ATCC CRL 1246).
  • Caulobacter Pichia; Spodoptera frugiperda
  • Trichoplusia ni BHK cells
  • MDCK cells CRFK cells
  • CV-I cells COS cells
  • Vero cells Vero cells
  • non-tumorigenic mouse myoblast GS cells e.g., ATCC CRL 1246.
  • Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK 31 cells and/or HeLa cells.
  • the proteins may be expressed as heterologous proteins in myeloma cell lines employing immunoglobulin promoters.
  • a recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences, examples of which are disclosed herein.
  • the phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
  • a recombinant cell of the present invention includes any cell transformed with at least one of any nucleic acid molecule of the present invention. Suitable and preferred nucleic acid molecules as well as suitable and preferred recombinant molecules with which to transfer cells are disclosed herein. Recombinant cells of the present invention can also be co-transformed with one or more recombinant molecules including biomarker nucleic acid molecules encoding one or more proteins of the present invention and one or more other nucleic acid molecules encoding other compounds.
  • Recombinant DNA technologies can be used to improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications.
  • Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgamo sequences), modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant enzyme production during fermentation.
  • the activity of an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing nucleic acid molecules encoding such a protein.
  • Mast cell tumor biomarker proteins of the present invention can be produced in a variety of ways, including production and recovery of natural proteins, production and recovery of recombinant proteins, and chemical synthesis of the proteins.
  • an isolated protein of the present invention is produced by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein.
  • a preferred celt to culture is a recombinant cell of the present invention.
  • Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production.
  • An effective, medium refers to any medium in which a cell is cultured to produce a biomarker protein of the present invention.
  • Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • Cells of the present invention can be cultured in conventional fermentation bi ⁇ reactors, shake flasks, test tubes, microliter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
  • resultant proteins of the present invention may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or be retained on the outer surface of a cell or viral membrane.
  • Proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. Proteins of the present invention are preferably retrieved in "substantially pure” form. As used herein, “substantially pure” refers to a purity that allows for the effective use of the protein as a therapeutic composition or diagnostic. A therapeutic composition for animals, for example, should exhibit no substantial toxicity and preferably should be capable of stimulating the production of antibodies in a treated animal.
  • the present invention also includes isolated (i.e., removed from their natural milieu) antibodies that selectively bind to a biomarker protein of the present invention, referred to as biomarker antibodies.
  • Preferred antibodies are MCT biomarker antibodies.
  • the term "selectively binds to" a biomarker protein refers to the ability of biomarker antibodies of the present invention to preferentially bind to specified biomarker proteins of the present invention.
  • Such antibodies do not react with non-biomarker components in a sample, but can show avidity, affinity or selective binding for homologous biomarker proteins from other species.
  • Such reactivity is also referred to as cross reactivity as a canine biomarker antibody may react with biomarkers from another animal specie.
  • a canine MCT antibody of the present invention can, but need not, bind a canine MCT biomarker with an avidity or affinity more than about 2 times (i.e., 2X), more than about 3X, more than about 4X, more than about 5X, more than about 6X, more than about 7X, more than about SX, more than about 9X or more than about 1OX greater than the compound's avidity or affinity for the same MCT biomarker protein from other species.
  • Such avidities or cross-reactivities can be determined by those skilled in the art and methods to do so include, but are not limited to ELISA assays, equilibrium dialysis and Scatchard analysis.
  • the degree of cross reactivity possessed by a biomarker antibody may be represented as indicated above (e.g. 2X) or, for example, as a ratio or a percentage.
  • Binding can be measured using a variety of methods standard in the art including enzyme immunoassays, immunoblot assays, and the like.; see, for example, Sambrook et al., ibid., and Harlow, et a!., 1988, Antibodies, a Laboratory Manual, Cold Spring Harbor Labs Press; Harlow et al., ibid. Terms used to represent cross-reactivity values are understood by and known to those skilled in the art.
  • Isolated antibodies of the present invention can include antibodies in serum, or antibodies that have been purified to varying degrees.
  • Antibodies of the present invention can be polyclonal or monoclonal, or can be functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies or chimeric antibodies that can bind to one or more epitopes.
  • the disclosed methods of detecting a tumor may be used to detect minimal residual disease, i.e. the presence of disease that was not eliminated through surgery and/or chemotherapy, in order to measure the effectiveness of chemotherapy.
  • the methods of the present invention may also be used for determining the stage of disease.
  • biological molecules of the present invention are further isolated and the isolated molecule is used for the detection of disease using other diagnostic formats known to those of skill in the art.
  • a preferred format includes binding assays, including assays that utilize antibodies raised against biological molecules of the present invention, including an ELISA format, or other formats, such as a lateral flow or flow-through diagnostic format.
  • kits for practicing the present invention contain components useful for practicing methods of the present. Such components include, but are not limited to, biomarkers, antibodies, primers, and the like. Kits may also comprise, for example, other reagents such as, but not limited to, buffers, enzymes, and the like, as well as bottles, tubes, and instructions describing the use of kit components.
  • Example 1 is provided for the purposes of illustration and are not intended to limit the scope of the present invention.
  • Example 1 is provided for the purposes of illustration and are not intended to limit the scope of the present invention.
  • This example describes the collection, preparation and SELDI-TOF mass spectroscopy of serum samples for the detection of biomarkers related to mast cell tumors.
  • Canine serum samples were collected as follows: 3-4 ml of blood was collected from a vein of the dog. The blood was placed into a red top-blood tube and allowed to clot at room temperature for 30 minutes, after which the sample was ce ⁇ lrifuged at 2000 x g for 10 minutes at 4 "centigrade ( 0 C). The supernatant was removed and stored at -80 0 C until use. 2. Serum Fractionation
  • ion exchange resin kit (Cipheragen [ K100-0007]) following the manufacturer's instructions. Briefly, Q HyperD F (Anion Exchange or "AE") plates were rehydrated using 50 mM 2-amino-2- (hydroxymethyl)- 1 ,3-propanedioI hydrochloride (Tris-HCl), pH 9.0 (Rehydration Buffer) for 2 hours on an orbital shaker at RT. Every 15 minutes, the plate is tapped upside down on the counter top to help mix the resin. Serum samples were thawed at room temperature (RT) after which they were centrifuged at 14,000 tpm at 4°C for 10 minutes.
  • RT room temperature
  • the AE plates were washed by the addition of 200 ⁇ l of Rehydration Buffer to each well, after which the Rehydration Buffer was removed by vacuum. This process was repeated three times for a total of 4 washes.
  • the wells of the AE plate were then washed by the addition of 200 ⁇ ! of IM urea, 0.2% CHAPS, 50 mM Tris-HCl, pH 9.0 (Ul Buffer); the Ul Buffer was then removed by vacuum. This process was repeated twice for a total of three washes. After the last wash, the denatured sample was added to the AE plate, one sample per well, followed by the addition of 50 ⁇ l of Ul buffer per well. The plates were then incubated at 4°C for 30 minutes on an orbital shaker (375rpm).
  • Fraction 1 consisted of the flow through portion combined with the 1 st wash, which consisted of 100 ⁇ l of wash buffer 1.
  • the wash buffer 1 was incubated in the wells for 10 minutes on the orbital shaker at RT prior to collection.
  • the remaining fractions were collected by the addition of 100 ⁇ l of the appropriate Buffer (listed below), followed by 10 minutes of incubation at RT on the orbital shaker, followed by collection of the Fraction using vacuum.
  • the Buffers used were as follows;
  • wash buffer 1 50 mM Tris-HCl with 0.1% n-octyl glucopyranoside (OGP), pH 9)-20 ml
  • Wash buffer 2 50 mM Hepes with 0.1 % OGP, pH 7)-30 ml 3.
  • Wash buffer 3 100 mM NaAcetate with 0.1 % OGP, pH 5)-30 ml
  • wash buffer 4 (100 mM NaAcetate with 0.1 % OGP, pH 4)-30 ml
  • wash buffer 5 50 mM NaCitrate with 0.1 % OGP, pH 3)-30 ml
  • wash buffer 6 33.3% isopropanol/ 16.7 % acetonitrile/ 0.1 % trifluoracetic acid), 30 ml
  • Ciphergen Freemont, CA
  • CM 10-Weak Anion Exchanger (Ciphergen part number C573-0075)
  • the chips Prior to the addition of serum, the chips were equilibrated as follows: The IMAC30 chip was placed into a 96-well bioprocessor (C503-0008), available from Ciphergen (Fremont, CA), such that each spot on the chips formed the bottom of each well of the bioprocessor. To each spot was added 50 ⁇ l of 100 mM NiSO 4 (nickel sulfate). The chip was incubated for 10 minutes and then blotted dry. 100 ⁇ l of water was then added to each spot, the chip shaken for 2 minutes and then blotted dry. To each spot was added 100 ⁇ l of 50 mM C 2 HsNaO 2 (sodium acetate), pH4 and the chip shaken for 5 minutes.
  • C503-0008 96-well bioprocessor
  • fractionated samples were spun briefly. For each fraction to be tested, 16.7 ⁇ l of the fraction was mixed wiLh 33.3 ⁇ l of Binding Buffer appropriate for the type of chip to be used. After mixing, the diluted sample was then placed into a well on the appropriate chip. This process was repeated for each sample. After all samples had been added to the chip, the chip was sealed and incubated for one hour at RT on an orbital shaker.
  • the chips containing the serum samples had incubated for one hour, they were blotted dry and spun upside down at 300 x g for 5 seconds to remove residual liquid.
  • To each spot was then added 150 ⁇ l of the appropriate Binding Buffer, after which the chips were incubated on an orbital shaker for 5 minutes and then blotted dry. This step was repeated once for a total of two washes. Following the second wash, 150 ⁇ l of 1 MM HEPES, pH7, was added to each spot and the chip incubated for 3 minutes with shaking. The chip was blotted dry and then spun upside down at 300 x g for 5 seconds to remove any residual liquid.
  • the reference sample spot is used to optimize the setting prior to reading the rest of the spots.
  • the low laser setting is the highest setting in which no peak exceeds a height of 100 at any time during the spot protocol run.
  • An example of a low laser energy setting is 187 Set starting detector sensitivity to 9.
  • the optimization range in step 2 also changes, and. the Seldi acquisition parameters (the positions on the spot that the laser hits) also changes. This is to avoid having the laser hit the same positions on the chip with each spot protocol, and thus removing many of the proteins in those positions.
  • chip protocol runs each of the spot protocols on each spot on the chip.
  • the chip protocol select the 8 spot lettered A-H chip configuration with 3 total chip runs.
  • the chips are then placed into the Ciphergen ProteinChip Reader model PBS EC and the data collected by running the chip protocol, using the Peaks software (Ciphergen). For each spot, three spectra are generated - one each for the low, medium, and high laser energy settings. A separate experiment file is created for each laser setting, and the appropriate spectrum copied and pasted into that file. Each spectrum is given a sample name and a sample group. The sample name refers to the patient from whom the sample was collected and the sample group refers to how the sample is classified for purposes of comparison (for example, mast cell tumor).
  • the normalization factor for each spectrum is then checked to see if any of the spots failed. Any spectra that have normalization factors greater than 2.5 or less than 0.5 are considered to have failed and are deleted from the experiment.
  • An external mass calibration equation is applied to the spectra in each experiment. This mass calibration equation is generated using the "All in one protein standards" (product Number Cl 00-0004(Ciphergen).
  • the standards contain 7 proteins that are bound to an NP20 chip, read on the PBS HC unit, and the peaks selected using the manufacturer's protocol.
  • a calibration equation for the low molecular weight range is then generated using only the 4 proteins with the lowest molecular weights. This equation is then applied to the three different laser energy experiments.
  • the peaks in each spectra are manually selected using the centroid function of the Peaks software (Ciphergen).
  • the centroid function selects the highest point in a given mass range for all of the spectra. This effectively selects corresponding peaks in all of the spectra and groups them into a peak cluster.
  • the Biomarker Wizard software (Ciphergen) is run to compare the peak heighLs within each peak cluster between spectra from different sample groups.
  • the Biomarker Wizard software produces two kinds of data tiles that are analyzed separately. The first is the Sample Group Statistics, which is in spreadsheet format. This file lists all of the peak clusters, P- values, mean peak heights, and standards of deviation for each sample group.
  • the second data file is called a cluster file.
  • the cluster file can be opened by another software package called Biomarker Patterns Software (Ciphergen), or BPS.
  • BPS uses the information in the cluster file to build classification trees that sort the spectra by sample group. For determining the best individual biomarkers, the biomarkers with the lowest P- values (i.e., those listed at or near the top of the variable importance list in BPS, and those used by BPS in the best classification tree) are analyzed individually.
  • BPS calculates the percent sensitivity and specificity for classifying the spectra in two ways. The first is using all of the samples in the data set to build the best tree.
  • the second way is to use 90% of the data to build the tree, and 100% of the data to test the tree. This process is repeated nine (9) times for a total of ten (10) times, such that each sample is excluded one time from the 90% used to build the tree. This is called the "test” result, and the process for testing the samples is called cross validation.
  • Test ⁇ o accuracy This is generated by running a single peak through EPS and using cross validation to estimate the total number of spectra correctly classified, which is then divided by the total number of spectra and multiplied by 100.
  • This Example describes the proteomic profiling of serum samples from normal dogs and dogs known to have grade Il and grade III mast cell tumors.
  • Serum samples were collected from 39 dogs and fractionated as described in Example IA. The serum fractions were then analyzed in duplicate along with a reference sample on selected chip types. The reference sample was not included in the data analysis. Three different sample groups were represented, including grade 2 MCT (MCT2), grade 3 MCT (MCT3), and normal (N). In addition, the MCT2 sample group was divided into two subgroups according to the outcome of the disease. The "good” and “bad” outcome subgroups were classified based on metastasis, response to therapy, and whether the dog died of mast cell disease or not. Information on each sample is provided below in Table III:
  • Proteomic profiles obtained from serum samples from normal dogs were compared to proteomic profiles obtained from serum samples from dogs known to have mast cell tumors.
  • Table VI lists the best classification tree identified using the procedures described above. Table V.
  • This Example demonstrates biomarkers and a classification tree useful for the identification of animals having mast cell tumors.
  • Proteomic profiles produced from serum samples obtained from dogs known to have grade 2 mast cell tumors were compared to proteomic profiles produced from serum samples obtained from dogs known to have grade 3 mast cell tumors.
  • Table VIII lists the best classification tree identified using the procedures described above.
  • This Example demonstrates biomarkers and a classification tree useful for differentiating grade 2 from grade 3 mast cell tumors.
  • proteomic profiles produced from serum samples obtained from dogs having a grade 2 MCT with a good outcome were compared with the proteomic profiles produced from serum samples obtained from dogs having a grade 2 MCT with a bad outcome.
  • Table IX below lists the top two biomarkers identified using the procedures described above.
  • Table X lists the best classification tree identified using the procedures described above.
  • This Example demonstrates biomarkers and classification trees useful in predicting the prognosis of animals having grade 2 mast cell tumors.

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Abstract

L’invention concerne l’utilisation du profilage protéomique pour détecter et classer les tumeurs chez les chiens, en particulier l’utilisation de la spectroscopie de masse pour identifier les molécules biologiques indicatives du degré ou de la présence de tumeurs de cellule souche chez les chiens. L’invention concerne également l’utilisation d’un tel profilage comme outil de pronostic. La présente invention concerne également des molécules biologiques identifiées en utilisant de telles techniques, y compris des protéines, des molécules d’acide nucléique et des anticorps sollicités contre de telles molécules biologiques, de même que des procédés et des kits de détection utilisant de telles molécules biologiques.
PCT/US2006/060073 2005-10-19 2006-10-19 Profilage protéomique de tumeurs de cellule souche et procédés associés, protéines et molécules d’acide nucléique WO2007048107A2 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
US20020015964A1 (en) * 1999-03-01 2002-02-07 University Of Mississippi Medical Center Method of diagnosing and monitoring malignant breast carcinomas
US20030017515A1 (en) * 2001-06-08 2003-01-23 The Brigham And Women's Hospital, Inc. Detection of ovarian cancer based upon alpha-haptoglobin levels
US20030165887A1 (en) * 1999-09-01 2003-09-04 Reed John C. Methods for determining the prognosis for cancer patients using tucan
US20040097460A1 (en) * 2002-11-12 2004-05-20 Becton, Dickinson And Company Diagnosis of sepsis or SIRS using biomarker profiles

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Publication number Priority date Publication date Assignee Title
US20020015964A1 (en) * 1999-03-01 2002-02-07 University Of Mississippi Medical Center Method of diagnosing and monitoring malignant breast carcinomas
US20030165887A1 (en) * 1999-09-01 2003-09-04 Reed John C. Methods for determining the prognosis for cancer patients using tucan
US20030017515A1 (en) * 2001-06-08 2003-01-23 The Brigham And Women's Hospital, Inc. Detection of ovarian cancer based upon alpha-haptoglobin levels
US20040097460A1 (en) * 2002-11-12 2004-05-20 Becton, Dickinson And Company Diagnosis of sepsis or SIRS using biomarker profiles

Non-Patent Citations (2)

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Title
GRIZZLE ET AL.: 'The Use of Biomarker Expression to Characterize Neoplastic Processes' BIOTECH. & HISTOCHEMISTRY vol. 72, no. 2, 1997, *
REGUERA ET AL.: 'Canine Mast Cell Tumors Express Stem Cell Factor Receptor' AMERICAN JOURNAL OF DERMATOPATHOLOGY vol. 22, no. 1, February 2000, *

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